ocr document page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 ocr document page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 1 images image 1 page 2 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 inside front cover-24-1.pmd editorial board editor-in-chief marco nemesio e. montaño, phd associate editors ma. patricia v. azanza, phd food science & nutrition jose maria p. balmaceda, phd mathematics zubaida u. basiao, phd biology carlos primo c. david, phd earth sciences joel joseph s. marciano, jr., phd computer science, engineering giovanni a. tapang, phd physics irene m. villaseñor, phd chemistry managing editor violeda a. umali, phd editorial assistant dercylis g. mararac science diliman (issn 0115-7809) is published bi-annually by the university of the philippines diliman through the office of the vicechancellor for research and development (ovcrd). address all communications to the editor-in-chief, science diliman, research dissemination and utilization office, office of the vice-chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p500.00/year (two issues), inclusive of postage us$50.00/year (two issues), inclusive of postage tel. no: (632) 981-85-00 loc. 4048 (632) 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph rduo.ovcrd2012@gmail.com website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences editorial advisors rigoberto c. advincula, phd department of chemistry university of houston radvincula@uh.edu alfonso m. albano, phd department of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu kenneth buckel, phd food science and technology group school of chemical sciences and engineering the university of new south wales, sydney, australia k.buckle@unsw.edu.au jose b. cruz, phd department of electrical and computer engineering ohio state university cruz@ece.osu.edu. flor crisanta f. galvez, phd quality assurance & technical manager kerry ingredients and flavours (americas region) 7989-82nd st., delta, vc v4g 1l7, canada ffgalvez1@yahoo.com victor c. gavino, phd department of nutrition university of montreal, canada victor.gavino@umontreal.ca kelvin s. rodolfo, phd department of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu rudolf a. roemer, phd centre for scientific computing and department of physics university of warwick r.roemer@warwick.ac.uk luis g. sison, phd electrical and electronics engineering institute university of the philippines diliman luis.sison@up.edu.ph raul k. suarez, phd department of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu abalone are herbivorous marine gastropods and are nocturnal – that is, they hide during the day amongst rocks and at night time, they go out in search of food. its fishery provides a significant source of employment and income to coastal people. from a socioeconomic perspective, the long-term sustainability of abalone fisheries is of great importance. unfortunately, abalone stocks have been overfished in many areas where they are found as a result of ever-increasing market demand, uncontrolled exploitation and/or inadequate or lack of fisheries management. one possible option for resource management is to educate local fishers to develop a corps of local resource managers who would promote sustainable fishing practices and conservation of this valuable resource (see capinpin, this issue). photo courtesy of e.c. capinpin, jr. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. cover photo from the editor in less than six months, it will be two years since the first few cases of infection from severe acute respiratory syndrome coronavirus 2 (sars-cov-2) had been reported in wuhan, china. since then, a number of sars-cov-2 variants have come and gone, but we are still dealing with the threat posed by the delta variant, which has severely affected many countries including the philippines in recent months. thanks to science, a number of vaccines against the coronavirus disease 2019 (covid-19) have been developed and proven effective in preventing and reducing the number of severe cases, hospitalizations, and deaths. in the philippines alone, as of this writing, 10 vaccines have been approved for use and around 24 million (representing around 22% of the population) have been fully vaccinated. gradually, mobility restrictions are being eased, and hopefully there will be no more new variants of concern that will arise so that we can all go back to normalcy again. in this issue, we feature three research articles. the first research article is from the group of dr. lemnuel aragones of the institute of environmental science and meteorology, university of the philippines diliman (upd). aragones and his team simulated explosions from dynamite fishing in order to analyze the underwater noise that is generated and infer its possible effects on marine mammals. these marine mammals include dolphins and whales. although dynamite fishing, also known as blast fishing, is illegal, fishermen in some localities in the philippines stealthily resort to this destructive fishing method, which kills not only large fishes but also small ones, including larvae, juveniles and other organisms that are close to the blast site. if done near coral reefs, dynamite fishing may kill corals and splinter coral colonies. the underwater noise produced by dynamite fishing also negatively affects marine mammals that make use of underwater sound for their foraging behavior, communication, and orientation. aragones et al. inferred from the results of their study that marine mammals within 150 m of the explosion may suffer injuries such as acoustic trauma and disorientation even from a single pulse. these results highlight the importance of instilling into local fishermen the negative effects of dynamite fishing. efforts to achieve this can be initiated by appropriate government agencies and non-governmental organizations involved in environmental and animal protection, particularly of marine mammals. the second research article is from the group of john vincent pleto of the institute of biological sciences, university of the philippines los baños. pleto et al. studied the effects of probiotics on the water quality and on the growth performance and health of milkfish that are grown in ponds that are supplied with polluted water from the marilao-meycauayan-obando river system. the application of probiotics is an established practice in aquaculture. this practice is aimed at improving gut health and water quality, which would eventually result in improved growth performance and higher yield. pleto and his co-workers used commercially available probiotics that consist of a strain of the bacterium bacillus. their analysis also showed the presence of bacteria belonging to the genera chlorobium and chlorobaculum. although they failed to detect these bacteria in the guts of milkfish sampled at two months and at four months of culture, they found that ponds treated with probiotics generally had better water quality and that milkfish grown in these ponds had higher survival rates and better feed conversion efficiencies. the third article is by lu kevin ong from the institute of mathematics, upd. ong used probability distributions known as recombination models to describe binary interactions in a particle system. in his paper, ong introduced some definitions and ideas to prove propagation of chaos for this particle system. jonas p. quilang, ph.d. editor-in-chief v from the editor in this issue, we feature four research articles. the first is from the group of dr. emmanuel ryan c. de chavez of the institute of biological sciences, university of the philippines los baños (uplb). de chavez and his team studied the diversity patterns of freshwater and land mollusks along dakil river at the university of the philippines laguna land grant (upllg) in paete, laguna, philippines. upllg is a naturally grown secondary forest, which was included as an important biodiversity area (iba) and a key conservation site in the philippines because of the many threatened and endemic birds that have been recorded in the area. the authors identified six gastropods and one bivalve species from six families of freshwater mollusks. as for land snails, seven species belonging to three families were identified. river velocity was found to be the most significant predictor for species richness of freshwater mollusks, while temperature and canopy cover were found to affect abundance. for land snails, altitude was found to be the most significant predictor for species richness, while canopy cover was found to significantly affect abundance. the second research article is from the group of dr. gil penuliar of the institute of biology (ib), university of the philippines diliman (upd). penuliar and his student, renz joseph artezuela, isolated fructophilic lactic acid bacteria (flab) from flowers of 14 plant species growing in the grounds of ib. flab are lactic acid bacteria that preferentially use fructose as their main source of carbohydrate under anaerobic conditions. previous studies have shown that flab can be isolated from flowers and fruits. penuliar and artezuela were the first ones to conduct such a study using plant specimens from the philippines. the authors were able to screen eight presumptive flab isolates for antimicrobial activity against test bacteria that are commonly associated with intestinal diseases. four of these isolates were found to inhibit the activity of at least two test bacteria. analysis of the dna sequences of two of these promising isolates revealed that they could be novel species. the authors suggest that further studies be done on these two isolates for possible applications in food and medicine. the third research article is from the group of dr. pablito magdalita of the institute of crop science and institute of plant breeding in uplb. magdalita and his team evaluated the morphological responses of papayas to drought based on tree and fruit characters. in the philippines, papayas are among the 10 leading fruit crops. information from this study is important for the selection of drought-tolerant lines of papaya. these selected lines can be used to produce drought-tolerant papaya varieties especially in the face of climate change. magdalita and his team found that drought-affected papaya trees have significantly reduced fruit weight, length, width, flesh thickness, and seed weight; however, these fruits were found to be sweeter than those harvested during normal conditions. trees that were tall, with thick stems and wide crowns, and still produced marketable yield even after exposure to drought were selected and marked as drought-tolerant. selected drought-tolerant lines can be used for further breeding. the fourth research article is from the group of dr. rheo lamorena-lim of the institute of chemistry, upd. lamorena-lim and his team used a uv-vis spectrophotometer with relative specular reflectance accessory in their effort to develop undergraduate laboratory experiments for the qualitative characterization of colored materials and quantitative determination of surface modification. the authors observed a differentiation of reflectance peaks in the spectra of offsetprinted commercial colored paper and acrylic paints that were sprayed onto glass cover slips. differences were also observed in the reflectance spectra of unsprayed and acrylic spray-painted surfaces of aluminum foil, plastic, and a mirror. for the calculation of band gap energy (bge), the authors applied the tauc plot method on the uv-vis absorbance data of electrodeposited zinc oxide and zinc sulfide films. bge can be used to determine whether a material is a metal, semiconductor, or insulator. i thank the authors of these four research articles for their contribution to this journal. since this is the last issue to be released with me as the editor-in-chief (eic) of this journal, i would also like to thank the editorial board, the two managing editors, the editorial assistant, the layout artists, and the copyeditors for all their valuable contributionover the last three years. our task was made more challenging during the last two years by the still ongoing covid-19 pandemic, but still we were able to overcome these challenges. i welcome and wish all the best to the incoming eic and the new members of the editorial board. with the infusion of new blood in the editorial board, i am confident that the journal will scale new heights in the coming years. jonas p. quilang, ph.d. editor-in-chief 20_hermosa 93 micro-holograms science diliman (january-june 2003) 15:1, 93-96 micro-holograms in a methyl red-doped polymer-dispersed liquid crystal (e48:pvp) n.p. hermosa ii* and m.r.h. daza national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: nhermosa@nip.upd.edu.ph abstract feasibility of a holographic point-by-point storage in a methyl red-doped polymer-dispersed liquid crystal (pdlc) is determined. micro-holograms (gratings) are recorded next to each other. smallest grating diameter obtained is 69.9 mm, with minimum grating distance of 80 mm. recording of adjacent grating reduces the diffraction efficiency of existing grating by 17% (average). introduction research on high-density storage has been fueled by the need for larger storage capacity and parallel data acquisition (ichioka et al., 1996). commercially available technologies have almost reached their full potential with these parameters. with the recent advances in materials and photo detection research, several groups have proposed techniques to overcome the storage density limit and fast retrieval of information (ueki et al., 1996). these techniques are based on optical storage system. among the most promising optical storage techniques are the multi-layered bit storage and volume holographic storage. in multi-layered bit storage, small refractive index change is recorded in different layers of three dimensional non-linear materials. the density of bit storage depends on the size of the bit datum, which is determined by the size of the beam spot (tanaka & kawata, 1996). the smaller and the more local the refractive index change, the higher the density. recently, groups working on bit-recording have succeeded in recording up to 30 layers of data at axialseparation of 7 mm and a dot separation of 2 mm for photopolymers, and three layers of data at axialseparation of 24 mm and dot separation of 4 mm for li:nbo 3 . however, a confocal phase contrast microscope is needed to read the stored information. this makes the set-up impractical. on the other hand, volume holographic techniques use several multiplexing schemes in recording information. multiplexing is possible because of strict bragg selectivity in several non-linear materials (adibi et al., 1999). these schemes include: (a) shift-multiplexing (markov et al., 1999; curtis et al., 1994); (b) peristropic-multiplexing (curtis et al., 1994); (c) angle multiplexing (chuang & psaltis, 1997; yu & psaltis, 1994); and (d) wavelength multiplexing (yu & psaltis, 1994; guerrero, 2000). shift-multiplexing makes use of spherical or speckle reference wave. this scheme is based on the features of selectivity of a non-planar reference-wave hologram. physical rotation of the holographic material after an exposure is called “peristrophic multiplexing”. the control of the read-out information is dependent on* corresponding author 94 hermosa ii and daza the angle at which the holograms are recorded. angular multiplexing uses different angles between the reference waves and the data to be recorded. one angle corresponds to a hologram. upon reconstruction, only the hologram recorded at that angle with the reference beam is reconstructed. however, cross-talk between hologram is the main issue with this scheme. both techniques are mechanically bulky. wavelength multiplexing is done by recording different holograms with different wavelengths. this requires a tunable coherent light source, which is economically costly. combination of these multiplexing schemes enables recording of up to 1,000 holograms (chuang & psaltis, 1997). in this paper, a technique for data storage in a dyedoped pdlc is presented. this scheme is different from the ones discussed above since holograms are not multiplexed, but are stored much like a bit datum–in the smallest space possible. experiments our material is a polymer dispersed liquid crystal (e48:pvp) doped with a small amount of methyl red dye. the experimental set-up is an off-axis holographic set-up, as shown in fig. 1. a beam from an ar-ion laser is collimated. the diameter is controlled by an iris before being split into two. these beams are focused with a lens to limit the size of the diameter as they intersect the sample. recording is done for 15 minutes. after recording, the sample attached to a translation stage is moved by 100 mm. the translation stage has a 10 mm smallest travel, with straightness of ±2 mm. recording is again done with the new position. change in the diffraction efficiency of the previous grating is noted. the diffraction efficiency h is the ratio of the first order diffraction and the sum of all the outgoing beams given by the equation (1) results and discussion table 1 shows the summary of the diffraction efficiencies before and after recording of another set of grating. all the second gratings were written 100 mm from the first grating. the highest change observed was 26% while the lowest was 8.5%. the average change in diffraction efficiency is 17.25 %. the small change in the diffraction efficiency is due to the isolation made by the polymer matrix. three samples of holograms written side-by-side is shown in fig. 2. in fig. 2a, the holograms were separated by 72 mm from their tips, with a center-tocenter distance of 508 mm. the bigger hologram is 533.4 mm wide while the smaller one has a diameter of 330 mm. it is interesting to note that there is a noticeable boundary between the holograms and the sample matrix. this is a visual confirmation of the small change in the diffraction efficiency and the role of the sample matrix as an isolator. another set of holographic grating is shown in fig. 2b. the region encircled is an overlap between two holograms. considerable damage is done on both holograms at this region. however, outside this overlap, the distinctive gratings in both holograms can still be seen. this indicates that grating beyond the overlap is not damaged. fig. 2c is a photomicrograph of the smallest diameter hologram possible with the current set-up. a diameter of 69.6 mm is measured. eighty-seven micrometers away from this hologram is another hologram with a first order total i i η −= fig. 1. the experiment set-up. pdlc travel direction collimated ar-ion beam (514. 5 nm)bsmirror lens 95 micro-holograms table 1. comparison of diffraction efficiencies before and after recording of another grating. sample grating diffraction efficiency % change before after 1 2 3 4 5.36 1.84 4.79 1.64 4.39 1.36 3.95 1.57 18.1 26.0 17.5 8.5 (c) (b) (a) fig. 2. micro-holograms in a pdlc (polymer-dispersed liquid crystal). diameter of 109 mm. their distance is 139.5 mm center to center. the implication of this is astounding. if an alternating grating and space is recorded with the 70 mm grating diameter and a 90 mm spacing, 62 holograms can be placed in a centimeter. in one cubic centimeter, more than three thousand holograms can be stored. conclusion micro-holograms are stored in a methyl red-doped pdlc. the smallest hologram recorded is 69.6 mm. another hologram with a diameter of 109 mm is recorded near this hologram. the distance between these holograms is 87 mm. noticeable is the clear boundaries between the grating and the sample matrix. it was observed that an average of 17% of the grating efficiency is lost. a maximum and a minimum loss of 26% and 8.5 %, respectively, reported. the current set-up limits the recording of smaller holographic grating. an improved set-up is needed to enable recording of smaller holograms. acknowledgments all samples are made by mr. albert francia at the liquid crystal laboratory, national institute of physics, university of the philippines diliman. the authors thank the philippine council for advanced science and technology research and development of the department of science and technology (pcastrd-dost) for the equipment grant. references adibi, a., k. buse, & d. psaltis, 1999. multiplexing holograms in linbo 3 :fe:mn crystals. opt. lett. 24(10): 652654. barbastathis, g. & d. psaltis, 1996. shift-multiplexed holographic memory using the two-lambda method. opt. lett. 21(6): 432-434. 96 hermosa ii and daza chuang, e. & d. psaltis, 1997. storage of 1,000 holograms with the use of a dual-wavelength method. appl. opt. 36(32): 8445-8454. curtis, k., a. pu, & d. psaltis, 1994. method for holographic storage using peristrophic multiplexing. opt. lett. 19(13): 993-994. guerrero, r., 2000. optical storage in li:nbo 3 . ms thesis. university of the philippines diliman, quezon city. ichioka, y., t. iwaki, & k. matsuoka, 1996. optical information processing and beyond. proc. of the ieee. 84(5): 694-719. markov, v., et al., 1999. multilayer volume holographic memory. opt. lett. 24(4): 265-267. tanaka, t. & s. kawata, 1996. comparison of recording densities in three-dimensional optical storage systems: multilayered bit recording versus angular multiplexed holographic grating. j. opt. soc. am. a. 13(5): 935-943. ueki, h., y. kawata, & s. kawata, 1996. three-dimensional optical bit-memory recording and reading with a photorefractive crystal: analysis and experiment. appl. opt. 35(14): 2457-2465. yu, s. & d. psaltis, 1994. three-dimensional holographic disks. appl. opt. 33(10): 3764-3774. 14_high high-resolution thermography 57 high-resolution differential thermography of semiconductor edifices vera marie sastine*, vernon julius cemine, carlo mar blanca, and caesar saloma national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: vsastine@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 57–60 *corresponding author we develop a cost-effective, high-resolution, and noninvasive imaging technique for thermal mapping of semiconductor edifices in integrated circuits. initial implementation was done using a power-stabilized optical feedback laser system that detects changes in the optical beam-induced current when the package temperature of the device is increased. the linear change in detected current can be translated to a thermal gradient, which can reveal semiconductor “hotspots”—localized sites with anomalous thermal activity. these locales are possible fault sites or areas susceptible to defects, which are the best jump-off points for failure analysis. introduction to perform successful failure analysis on a semiconductor device, precise knowledge of the exact fail site location is important. one of the most rapid ways to locate a fault zone is to uncover “hotspots”— edifices with temperatures above the accepted operating range of the device. various hotspot isolation techniques have been in existence since the earliest days of failure analysis and have been satisfactory to varying degrees for the location of suspected damage sites on different integrated circuits. thermal imaging as a failure analysis tool has been implemented using infrared cameras (rogalski, 2003) or array detectors (christofferson et al., 2001). however, the high thermal discrimination and simple operation of these detectors are offset by the high purchase cost. other detection techniques exist such as liquid-crystal microscopy (lee & pabbisetty, 1993) and scanning microscopy (shi et al., 2003), but they are too invasive and less sensitive. aside from being expensive, all these methods solely detect remnants of thermal conduction bleeding through the surface of the integrated circuit. because of their poor resolution in the axial direction, they cannot map out thermal propagation of an embedded defect. the challenge, therefore, is to develop a cost-effective, noninvasive thermographic technique that would enable us to localize and map out areas of high thermal gradient. the method we propose will utilize a custom-built optical feedback microscope operating in the near-ir range (cemine et al., 2004). the thermal response of the individual ic sections is then monitored and analyzed by measuring the optical beam-induced current (obic) as the package temperature is increased. because longer wavelengths λ suffer less scattering (~1/λ4), the method can be used to noninvasively tunnel through thick layers of semiconductor die in contrast to the surface-limited probes of the scanning electron microscope (sem) (shi et al., 2004). high-resolution sastine et al. 58 sample dissection can then be done as a verification procedure. since there is no downtime for sample preparation, the analysis is simple, fast, and does not require a vacuum environment so essential to sem imaging. this ease of maintenance is further supported by the system’s simplification of the fault identification process: both defect localization and identification can be done in the same setup, cutting cost and experimental hours. methodology a schematic diagram of the microscope setup is shown in fig. 1. a semiconductor laser (sl) (sharp lt024md, λ = 793 nm at laser case temperature = 25°c) was used as a light source. it has a built-in photodetector (pd) that is used to detect the confocal reflectance signal. the sl is controlled by a laser diode controller that is connected to the computer. the diverging elliptical illumination beam was collimated by a collimating lens (c) (melles griot 06glc003, numerical aperture = 0.276, focal length = 14.5 mm). mounted anamorphic prisms (ap) (melles griot 06gpa004, magnification = 3x) were used to circularize the beam. an iris diaphragm (d) was inserted to lessen the effect of astigmatism. an objective lens (o) (olympus uplan fl, nominal na = 0.5, working distance = 1.7 mm) was used to focus the beam on the sample. the current produced upon sample irradiation is the one-photon optical beaminduced current (1p-obic) signal. the light reflected from the sample forms the confocal reflectance signal. the sample is a photodiode array (mn8090 matsushita 5017) biased at 3.56 v and is mounted on a triaxis servomotor stage (s) (thorlabs pt3-z6) that is controlled by a motion-controller card inside the computer. the sample is heated using a thermoelectric cooler (tec) controlled by a current controller (cc), while the temperature is monitored using a multimeter. confocal reflectance and obic signals are detected using a 12-bit data-acquisition board (national instruments 6024e, conversion rate = 200 khz) inside the computer. stage-scanning and image-acquisition protocols are coded using labview 7.0 (national instruments). confocal and obic images were acquired for six sample temperatures: 25.1 (room temperature), 29, 38.2, 48.9, 61.6, and 72.1°c. the effect of the sample temperature on the obic signal generation is determined. obic thermal-gradient mapping of the sample image is undertaken to reveal die defects and sites that are susceptible to failure. results and discussion figure 2 shows the 190 mm x 190 mm 1p-obic and confocal reflectance images taken at the focus (z = 0) for three sample temperatures: (a) 25.1, (b) 48.9, and (c) 72.1°c. for each measurement the object’s axial location was monitored and adjusted to compensate for any package expansion that may have occurred. identical confocal images mean that we are always investigating the same focal plane. notice the significant decrease in intensity on the obic image as temperature increases. this signifies that as the sample temperature increases, the current generated by the beam (the 1p-obic signal) at each point on the fig. 1. optical setup. confocal and obic tandem microscope using an optical feedback laser system (sl). collimating optics (c, ap) expand the beam which is focused by an objective (o) on the sample. the ic specimen is mounted on a thermoelectric cooler (tec) to increase the package temperature by adjusting the current controller (cc). ic sample pd sl c ap d cc o gnd s z axis tec high-resolution thermography 59 observation area decreases. we operated the diode laser in power-stabilized mode ensuring a constant illumination power. the decrease in obic signal is then caused by the increase in resistance of the sample as the temperature of the sample increases. quantification of this observed phenomenon will be presented later. we determine the semiconductor and metal parts of our observation area by multiplying the obic and confocal images, and obic complement and confocal images, respectively. this method is already an established procedure for semiconductor and metal discrimination (daria et al., 2002; miranda & saloma, 2003). as inputs, we use the obic and confocal images at room temperature (t =25.1°c). figure 3 shows the confocal and obic images at room temperature and their corresponding semiconductor and metal sites. the portions that are low in intensity on both the semiconductor and metal images are either the dielectric or the substrate. five different portions (each of area 20 mm x 20 mm) on the obic images are selected to quantitatively determine the obic signal decrease as the temperature increases [figs. 4(a) and 4(b)]. figure 4(c) shows the plots of the obic average intensities versus temperature for the five portions. their respective slopes, which represent the obic signal rate of change with temperature, are also shown. fig. 2. the 190 mm x 190 mm confocal and 1p-obic images at (a) 25.1, (b) 48.9, and (c) 72.1ºc. obic intensity decreases as sample temperature increases. obic confocal (a) (b) (c) confocal obic semicon metal, etc. fig. 3. confocal and 1p-obic images for room temperature and their corresponding semiconductor and metal sites. temperature (oc) a ve . o b ic in te ns it y (a .u .) (c) semicon obic (a) (b) fig. 4. [(a) and (b)] semiconductor and obic images. (c) average obic intensity of the five monitored sites decreases versus the temperature of the sample. the numbers are the slopes of the curves. image size is 190 x 190 mm. sastine et al. 60 for the five portions, obic linearly decreases with temperature. however, the rate of decrease of the obic signal at these five regions are different, as exhibited by their differing slope values. this implies that some portions of the integrated circuit (ic) heat up faster than other areas. those regions that heat up faster reflect a faster obic signal decrease or a steeper slope, while those that heat up slower exhibit a gradual decrease in obic signal. this inhomogeneity in heating rate can reveal the locations of those sites that are either defective or highly susceptible to failures when the ic is operated at extreme conditions. we can map those defective and failure-susceptible sites by getting the thermal gradient map using the obic images for the six temperature levels. figure 5 shows the thermal-gradient map of the observation area. notice the thermal-gradient inhomogeneity on the observation area as exhibited by the different colors on the thermal-gradient map. areas that heat up fast are reflected as red on the map (pointed by arrows), while areas that heat up slow are blue. from this observation, imaging and localization of those sites that are either defective or susceptible to defect, is realized. conclusion we have demonstrated the performance of our newly developed noninvasive imaging technique for thermal mapping of semiconductor edifices in integrated circuits. the technique detects thermal-gradient inhomogeneity on the ic observation area. those sites that heat up fast are well discriminated from those sites that heat up at a slower rate. with this capability, our technique cannot only act as a failure analysis tool but can be used for precursor or predictive analysis as well. possible defect sites can be mapped out even before failure happens. in such eventuality, the onset and evolution of a failure can be recorded and investigated. semiconductor architecture can also possibly benefit from the technique by deriving thermal accumulation points in a particular semiconductor design. the technique can provide such basic tests to expose weaknesses in prototype integrated circuits under thermal stress. references cemine, v.j., b. buenaobra, c.m. blanca, & c. saloma, (in press). high-contrast microscopy of semiconductor and metal sites in integrated circuits by optical feedback detection. opt. lett. 29: 2479-2481. christofferson, j., et al., 2001. high-resolution noncontact thermal characterization of semiconductor devices. proc. spie. 4275: 119–125. daria, v.r., j. miranda, & c. saloma, 2002. high-contrast images of semiconductor sites via one-photon optical beaminduced current imaging and confocal reflectance microscopy. appl. opt. 41: 4157–4161. lee, t.w. & s.v. pabbisetty (eds.), 1993. microelectronics failure analysis. asm international, ohio: chap. 5. miranda, j.j. & c. saloma, 2003. four-dimensional microscopy of defects in integrated circuits. appl. opt. 42: 6520–6524. rogalski, a., 2003. infrared detectors: status and trends. prog. quantum electron. 27: 59–210. shi, l., et al., 2003. nanoscale thermal and thermoelectric mapping of semiconductor devices and interconnects. aip conf. proc. 683: 462–468. figure 5. semiconductor image and thermal gradient map of the sample. red portions (pointed by arrows) indicate highest rate of change in temperature while blue portions indicate lowest rate of change in temperature. 09_dela cruz 41 thermally-activated vortex motion science diliman (january-june 2003) 15:1, 41-44 thermally-activated vortex motion and electrical dissipation in a bi 2 sr 2 cacu 2 oδ thin film c.r. de la cruz*, a.p.c. dela cruz, l.j.d. guerra, and r.v. sarmago condensed matter physics laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: clar@nip.upd.edu.ph abstract the magnetoresistance, obtained from resistivity measurements with external magnetic fields up to 0.5t, was used to directly measure and investigate the electrical dissipation properties of a c-axis oriented bi 2 sr 2 cacu 2 o 8+δ thin film. an activation-related “peaked” profile below tc was observed in the magnetoresistance. in increasing applied magnetic field, the peak shifts to lower temperatures, broadens, and becomes more asymmetric. the analysis, made based on an arrhenius-type activation mechanism, shows that the activation energy decreased with increasing applied magnetic field, as predicted by the anderson-kim thermally-activated flux creep theory. therefore, in these low magnetic fields and temperatures, the vortex motion predominant in the films is thermally activated and contributes largely to the dissipation in these films. introduction transport properties of high-temperature superconductors (htsc), particularly of thin films, have been intensively studied to derive valuable information on critical currents, vortex pinning, electrical dissipation mechanisms, i-v characteristics and phase transitions, to name a few. in particular, the study of electrical dissipation in htscs proves to be of great significance for its envisioned technological application. in this paper, the behavior of the electrical dissipation due to an applied transport current in varying applied magnetic field and temperature regimes is studied. electrical dissipation because of the presence of an applied magnetic field and transport current is referred to as the magnetoresistance due to flux motion (poole et al., 1995). flux motion results from the lorentz force induced by the applied transport current and is the mechanism leading to power dissipation and flux-flow resistance in the superconducting mixed state. aside from the lorentz force, thermal activation or fluctuations may also cause flux motion (tamegai et al., 1998; zhang et al., 2001). several models have been proposed to explain the dissipative behavior of htscs in a magnetic field (tamegai et al., 1998). one of these models is the theory of thermally activated flux creep developed by p.w. anderson. the theory assumes that flux creep occurs by bundles of flux lines jumping between adjacent pinning points and that the movement of these flux lines is an activation process directed by thermal energy (anderson & kim, 1964; anderson, 1962). thermally-activated flux motion causes the broadening of the transition region of the resistance curves when a* corresponding author 42 dela cruz et al. magnetic field is applied (kucera et al., 1992). the smooth power law behavior of the voltage-current (vi) curves is also attributed to this motion of magnetic flux lines. methodology the sample used was a bi 2 sr 2 cacu 2 oδ thin film grown via liquid phase epitaxy in the condensed matter physics laboratory of the national institute of physics. x-ray diffraction (xrd) shows a preferential growth along the c-axis. transport measurements were made using the in-line contact configuration. four contacts were placed on the surface of the film using annealed silver pastes and gold wires. a current of 1 ma was supplied to the outer two contacts while voltage measurement was done across the inner two. the film was attached to a cold head that could be cooled down to approximately 10 k. for magnetoresistivity measurement, a magnetic field was applied along the film’s c-axis. the temperature dependence of the resistance was then determined, with and without applied magnetic fields. magnetic fields used were less than 0.6t and the magnetoresistance profile against varying temperature was obtained using the equation: (1) results and discussion from the resistivity measurements, the critical temperature of the sample was found to be ~87 k. in the presence of an increasing magnetic field applied along the film’s c-axis, it is observed that the transition region broadens (fig. 1). this is due to the increasing number of thermally activated mobile vortices (anderson & kim, 1964). an activation-related peaked behavior was observed in the magnetoresistance profile shown in fig. 2. thus, the behavior was analyzed using the arrhenius relation so that a magnetic field-dependent energy scale may be correlated to the activation mechanism behind the “peaked” nature of the magnetoresistance profile. the magneto resistance may then be expressed as: (2) where e a is a magnetic field-dependent energy scale. increasing applied b field 0.8 0.6 0.4 0.2 60 90 120 temperature (k) r e s is ta n c e ( ωωωω ω ) fig. 1. the broadening of the transition region with increasing applied magnetic field is attributed to thermally-activated flux flow. 120 80 40 1 0 0.60.3 fig. 2. normalized magnetoresistance (r m ) profile at applied magnetic fields from 0t to 0.6t. tempe rature (k) b (t) r m (ωωωωω ) ( ) ( ) ( ) ( ) 0 0 0 ⎡ ⎤−∆ = = ⎢ ⎥ ⎢ ⎥⎣ ⎦ m r b rr r r r ( ) max exp − = am m b e r t r k t 43 thermally-activated vortex motion this activation-related model of the magnetoresistance profile is comparable in form to the thermally activated behavior of the vortex creep velocity from the anderson-kim flux creep theory, given by the equation (anderson & kim, 1964; anderson, 1962): (3) where f(t) is a temperature-dependent energy scale. this suggests that the two are correlated and have an activation type behavior. this is supported by the fact that the drift velocity of the vortices induces an electric field with a component that retards the applied transport place so that the mobility of the flux lines decreases. hence, dr mmax /db app is smaller. further increasing b app leads to an enhanced cooperative motion of the vortices, leading to a high dr mmax /db app . fig. 4 shows a plot of the temperature position of the magnetoresistance peak as the applied field is varied. the figure shows that the peak of the magnetoresistance profile shifts to lower temperature as b app is increased. this peak shift is related to the decrease in the activation energy as b app is increased. note that as b app is increased, more vortices are introduced into the system. at temperature t = t peak , all these vortices must be fully thermally activated. therefore, as b app is increased, thermal activation of the vortices happens more readily. hence, the peak (where complete thermal activation occurs) will shift to lower temperatures. it can be observed from fig. 2 that the r m profile is inherently asymmetric even at extremely low fields. fig. 5 emphasizes this increase in the peak asymmetry as b app is increased. the shifting of the low-temperature edge of the magnetoresistance profile to lower temperatures causes the asymmetry. note that the lowtemperature edge marks the melting point when the phase transition from the vortex solid state to the vortex liquid state takes place. with a consequent decrease in activation energy as b app increases, the flux melting current and leads to electrical dissipation or magnetoresistance (yeshurun et al., 1996). from the arrhenius plot of the normalized resistance, the activation energy was observed to decrease nonlinearly with increasing applied magnetic field. this typical result is explained by the fact that vortices increase in number as the applied magnetic field increases. in turn, the interaction between vortices increases in magnitude as well, so that the propensity for these vortices to move increases too, leading to a decrease in the activation energy and an increase in the measured resistance of the system. fig. 3 shows that the r mmax changes as the applied field (b app ) is increased. from the plot, three separate regimes can be discerned. these regimes are differentiated by the rate at which the peak height increases with increasing b app (dr mmax /db app ). this rate at extremely low fields (b app < 0.1t) and at relatively high fields (b app > 0.45t) is greater than at intermediate values of the applied magnetic field (0.1t < b app < 0.45t). at low fields, only a small number of vortices exist. thus, in this regime, dr mmax /db app is high because the vortices are more mobile. increasing the field increases the number of vortices in the system. at intermediate field strengths, an ordering in the vortex system takes 0.2 0.4 2 1 0 b (t) p e a k h e ig h t ( ωωωω ω ) fig. 3. the non-linear increase in the peak height of the magnetoresistance profile as b app is increased. ( ) exp ⎛ − ⎞ = ⎜ ⎟ ⎝ ⎠ o b f t k t ν ν 44 dela cruz et al. effectively shifts to lower temperatures. this shifts the edge to lower temperatures, thus increasing the asymmetry. conclusions the magnetoresistance profile for a bi-2212 thin film was obtained for applied magnetic fields less than 0.6t. the magnetoresistance profile was found to have an activation-related peaked behavior. in the temperature range of t peak < t < t c , the expression for the magnetoresistance profile has the same form as the vortex creep velocity predicted by the anderson-kim flux creep theory. therefore, the mechanism of electrical dissipation must be via the thermally-activated motion of the vortices. also, further analyses of the behavior of the magnetoresistance profile show: (i) a non-linear increase in the r m peak height; (ii) a shift to lower temperatures of the r m peak; and (iii) an increasing peak asymmetry of the r m profile as the applied magnetic field was increased. these were explained in terms of flux ordering and energy considerations. references anderson, p.w., 1962. theory of flux creep in hard superconductors. phys. rev. lett. 9: 309-311. anderson, p.w. & y.b. kim, 1964. hard superconductivity: theory of the motion of abrikosov flux lines. rev. mod. phys. 36: 39-43. kucera, j.t., t.p. orlando, g. virshup, & j.n. eckstein, 1992. magnetic field and temperature dependence of the thermally activated dissipation in thin films of bi 2 sr 2 cacu 2 o 8+δ. phys. rev. b. 46: 11004-11013. poole, c.p., et al., 1995. superconductivity. san diego, academic press. tamegai, t., h. enriquez, y. de wilde, & n. bontemps, 1998. vortex-lattice melting, phase slips, and decoupling studied by microwave dissipation in bi 2 sr 2 cacu 2 o 8+δ. phys. rev. b. 58: r14745-r14748. yeshurun, y., a.p. malozemoff, & a. shaulov, 1996. magnetic relaxation in high-temperature superconductors. rev. mod. phys. 68: 911. zhang, y.h., h. luo, x.f. wu, & s.y. ding, 2001. effect of flux creep on ic measurement of electric transport. supercond. sci. technol. 14(6): 346-352. b (t) p e a k p o s it io n ( k ) 0.2 0.4 77 76 75 74 73 fig. 4. variation of r m peak position with applied magnetic field. the peak is observed to shift to lower temperatures with increasing applied magnetic field. the dotted line indicates the general trend of the decrease. fig. 5. increasing asymmetry with increasing applied magnetic field. 0.0 0.2 0.4 90 60 b (t) p e a k a s y m m e tr y ( k ) right/hi-temp edge left/lo-temp edge 20_filter oblefias, soriano, and saloma 84 effect of filter arrangement in the estimation accuracy of an imaging spectrometer wilma r. oblefias1, maricor n. soriano2, and caesar a. saloma3 national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: 1woblefias@nip.upd.edu.ph; 2msoriano@nip.upd.edu.ph; 3csaloma@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 84-88 we investigate the reason why increasing the number of basis spectra in a spectral imaging device does not always improve the estimation merit. a particular filter is not the cause of this observation but the components of the inverse of the transformation matrix which map the coefficient of the basis spectra to the color of the sample. we found out that the large magnitude of the components of the inverse of the transformation matrix results in error in the calculation of the coefficients. this error leads to a drop in the spectral estimation merit even when the number of basis spectra is increased. therefore, it is not enough that the filter used in an imaging spectrometer is not a linear multiple of other filters and nonzero to any of the wavelengths in the range of interest. filters must also be arranged in a sequence such that the inverse of the transformation matrix will have components with small magnitude. introduction microscopic spectral imaging techniques have been developed, but they require special optics to be attached to the microscope (schrock et al., 1996; youvan et al., 1997). recently, simpler and more compact imaging spectrometers have been introduced (saloma et al., 2004; connah et al., 2001; kasari et al., 1999; andres et al., 2004) which use lesser number of images by employing dimension-reduction algorithms. dimension-reduction algorithms such as singular-value decomposition (svd) (soriano et al., 2002) and principal component analysis (pca) (imai et al., 2000) calculate basis spectra so that the spectrum of a sample may be represented by a weighted superposition of the basis spectra. theoretically, as the number of images and basis spectra are increased, the accuracy of spectral estimation of the sample should also increase. however, this is not always the case. for example, connah et al. (2001) report anomalous result in using six basis spectra. kasari et al. (1999) observed that using four bases is better than using five or six, while andres et al. (2004) found that the estimation error does not consistently fall with increasing number of bases. all of them were unable to explain why this deviation from the theoretical prediction happens. using our developed spectral imaging method, we have found an explanation why increasing the number of images does not always increase the amount of information in the spectrum of the sample. the result of this investigation is not applicable only to our developed method, but also to other spectral imaging techniques such as that of connah, kasari, andres, and others which utilize filter and dimension-reduction algorithms. methods svd rotates the component axes wherein the data are most widely spread. if we assume an existence of a effect of filter arrangement 85 spectral library (ensemble of spectra of different samples), the axes represent the basis spectra. the basis spectra are then arranged into decreasing eigenvalue; that is, the first basis spectrum has a greater eigenvalue than the second basis, and so on. after obtaining the basis spectra ei(l), the estimated spectrum cest(l; x, y) of the reference spectrum c(l; x, y) can be expressed in terms of the first few significant ei(l): , (1) where an is the coefficient of the nth e(l). the summation is taken from n = 1 to n = n, where n is the number of ei(l) that is utilized for estimation. the basis spectra are already known after the application of a dimension-reduction algorithm. thus, n can be easily adjusted. relating the estimated spectrum to the output of the camera, we obtain , (2) where q is an m-column vector containing the channel output of a color camera (pixel color) and t is m×n transformation matrix that maps the expansion coefficients in a to the image space colors q (soriano et al., 2002). the elements {tnm} of t is described by , (3) where en(l) is the nth basis spectra and sm(l) is the mth camera sensitivity given by , (4) where η may be 1 (red channel), 2 (green channel), or 3 (blue channel). f(l) is the transmission of the lightly colored filter used. since ei(l) are immediately known after applying svd and the sensitivity of the camera and the transmission of the filter used are either obtained from the manufacturer or measured independently, tnm is just a constant for a set of basis spectra, camera sensitivity, and filter transmittance. in spectral imaging, the image output channels {qm(x, y)} and the spectral library {ck(l)}are known and the immediate task is to determine the component values of a. after a is known, one uses eq. (1), to solve for the corresponding spectral estimation cest(l; x, y) which describes the optical spectrum at the location (x, y) of the two-dimensional (2d) image of the fluorescing sample. for a colored image, the values for the different qm(x, y)’s for every pixel location (x, y) of the twodimensional image are taken from the red (r), green (g), and blue (b) channel outputs (m = 3) of the camera. the unknown coefficients {an(x, y)} are determined via , (5) where t–1 is the inverse of t. the inversion matrix t–1 is defined only if t is a square matrix because the size of t is equal to m×n, i.e., t–1 exists only if n = m. to increase the number m of color channels, the sample is image-captured with a lightly colored transmission filter placed before the camera. with the insertion of a filter, the fluorescent sample is imaged under three more independent channels (m = 6), in addition to the original three (for r, g, and b) provided by the 3ccd camera in the absence of a filter (null filter). in this study, five filters are used—null, pale lavender, pale apricot, lime, and pale yellow green. the accuracy of estimation was measured using fidelity f: , (6) where 〈·〉 is the average value and c is the theoretical spectrum. fidelity describes the general similarity between theoretical and estimated spectrum. perfect estimation occurs when f = 1. results and discussions increasing the number of basis spectra increases the cumulative eigenvalue. thus, the information that can oblefias, soriano, and saloma 86 estimate the spectrum of the sample are also increased. figure 1 shows the camera sensitivities of the 3ccd camera used with a null filter. the transmission of the lightly colored filters is shown in fig. 2. figures 3 and 4 illustrate the average fidelity of estimation of each spectrum in the spectral library (with 423 emission spectra of fluorescent dyes and microspheres) using the same set of filters but of a different sequence. for example, a null filter is used to generate the first three sensitivities of the camera and pale lavender for the next fourth to sixth sensitivities. as the number of basis spectra increases, fidelity approaches unity. standard deviation also decreases both for the two cases. this means that with the increased number of bases spectral estimation becomes more accurate and more precise. however, a prominent drop in fidelity and a sudden increase in standard deviation are observed using 11 and 9 basis spectra for figs. 3 and 4, respectively. this contradicts our prediction. the 11th basis spectrum in fig. 3 corresponds to the green channel of the camera with lime filter. if this filter causes the anomalous observation, we will expect that the effect must also be present to more set of basis spectra. however, this is not observed in using 12–15 bases. it may be hypothesized that the anomalous effect of the lime filter cancels out as the number of basis spectra is further increased. thus, the effect is present only in the 11th camera channel. if that is the case, if we rearrange the first three filters and make the last two filters fixed, we will expect to observe anomalous estimation merit again in using 11 basis spectra. however, the effect is seen in using nine basis spectra, effect of filter arrangement 87 as shown in fig. 4. pale apricot is the filter that corresponds to the ninth camera channel and the lime filter has no contribution yet since it is used in generating the 10th, 11th, and 12th camera channels. therefore, the lime filter is not the cause of the deviation from the expected result. from eq. (1), spectral estimation is done using basis spectra and coefficients. for a given spectral library, the basis spectra are fixed values. coefficients are calculated using the inverse of the transformation matrix and the pixel color as shown in eq. (5). for a given sample, pixel color is fixed for a particular channel of the camera. thus, the only freedom we have is choosing the filter to increase the number of camera channel. the sensitivity of the camera channel affects the calculation of the coefficient through the inverse of the transformation matrix. tables 1 and 2 show the components of the inverse of the transformation matrix of figs. 3 and 4, respectively, using nine basis spectra. comparing the two tables, table 2 has components with larger magnitude, with a difference of one order indicated by the shaded cell, compared with table 1. an anomalous result in using nine bases is observed only using the inverse of the transformation matrix of table 2. this observation is also seen (not shown) in comparing the inverse of transformation matrices of figs. 3 and 4 with 11 basis spectra. hence, the unexpected drop in fidelity as the number of basis spectra is increased is attributed to the large magnitude of the components of the inverse of the transformation matrix. this large magnitude leads to the instability of the equation resulting in a large calculation error of the coefficients. in practice, camera output is taken from the average of the gray value level of several pixels depending on the signal-to-noise ratio (snr) of the sample. any noise or deviation in color values will propagate when multiplying with the inverse of the transformation matrix. thus, an increase in error is observed. on the other hand, using a transformation matrix with components of smaller magnitude gives a smaller error since standard deviation is less amplified. thus, it becomes more robust to fluctuation, noise, and quantization errors of the digitizer. for a limited set of filters an anomalous result may be avoided by choosing the best arrangement that will give small components of the inverse of the transformation matrix. another way is by finding the optimum filter that will minimize the magnitude of each component for any number of basis spectra. this result implies that the conditions that (1) the filter is not correlated with other filters used and that (2) in the wavelength range considered the filter transmittance must be nonzero (soriano et al., 2002; imai et al., 2000) are not enough considerations in choosing the filter for spectral imaging. we add another constraint; that is, that (3) the arrangement of the generated camera sensitivity from the filter should make the components of the inverse of the transformation matrix stable. oblefias, soriano, and saloma 88 summary using our developed technique in spectral imaging, we illustrate why an increase in number of basis spectra is not always accompanied by an increase in the spectral estimation merit. it is shown that the cause of this is the large magnitude of the components of the inverse of the transformation matrix. we present that the cause is not a particular filter, but the arrangement of the filters. references andres, j., j. nieves, e. valero, & j. romero, 2004. spectral daylight recovery by use of only a few sensors. josa a. 21: 13–23. connah, d., s. westland, & m. thomson, 2001. recovering spectral information using digital camera systems. coloration technology. 117: 309–312. imai, f., r. berns, & d. tzeng, 2000. a comparative analysis of spectral reflectance estimated in various spaces using a trichromatic camera system. j. imaging sci. technol. 44: 280–287. kasari, m., k. miyazawa, s. toyooka, & j. parkkinen, 1999. spectral vision system for measuring color images. josa a. 16: 2352–2362. saloma, c., w. oblefias, & m. soriano, 2004. spectral microscopy of live luminescent samples. in nanophotonics: integrating photochemistry, optics, and nano/bio materials studies. elsevier: chap. 30. schrock, e., s. du manoir, t. veldman, b. schoell, j. wienberg, m. ferguson-smith, y. ning, d. ledbetter, i. bar-am, d. soenksen, y. garini, & t. ried, 1996. multicolor spectral karyotyping of human chromosomes. science. 273: 494–497. soriano, m., w. oblefias, & c. saloma, 2002. fluorescence spectrum recovery from image color and non-negativity constraint. opt. express. 10: 1458–1464. youvan, d., w. coleman, c. silva, j. petersen, e. bylina, & m. yang, 1997. fluorescence imaging microspectrophotometer (fims). biotechnology. 1: 1–16. 19_deposition effect of deposition time 79 the effect of deposition time on textured magnesium diboride thick films fabricated by electrophoretic deposition w. g. mutia, m. s. romano, and r. v. sarmago materials science and engineering program, university of the philippines, diliman, quezon city 1101 abstract science diliman (july–december 2004) 16:2, 79-83 mgb2 powders suspended in ethanol were electrophoretically deposited on high-purity molybdenum substrates having dimensions of 1 x 0.3 x 0.01 cm. the said substrate was set as the cathode and was placed 0.5 cm away from a graphite rod anode. a current density of ~0.02 ma/cm2 and a voltage of 600 v were applied. the effect of deposition time was studied by varying it as follows: 15 s, 30 s, 1 min, and 2 min. heat treatment at 950 oc for 3 h was done after deposition. mgb2 thick films were successfully fabricated for the deposition carried out for 2 min. deposition times less than 2 min resulted in insufficient deposited powder; hence formation of mgb2 was not facilitated. films deposited at 15 and 30 s have good surface characteristics, wherein no microcracks were present. x-ray diffraction and surface image analysis reveal that the deposited films have a preferred orientation along the (10l) direction. introduction the discovery of superconductivity with tc at 39 k in magnesium diboride (mgb2) was announced in the early part of 2001 by akimitsu et al. (takano et al., 2001). this stimulated an interest in mgb2 as a new family of high-temperature superconductors because it introduced a new, simple (three atoms per unit cell) binary intermetallic superconductor with a record high (by almost a factor of 2) superconducting transition temperature for a non-oxide compound (bodoardo et al., 2004). mgb2 has the hexagonal aluminum diboride (alb2) type crystal structure wherein hexagonal close-packed layers of mg atoms separate graphite-like sheets of boron atoms (kortus et al., 2001). the low anisotropy, lowcost potential, large coherence lengths, and transparency of grain boundaries to current flow in comparison to cuprates are reasons for great interest in this material (buzea & yamashita, 2001). mgb 2 has been synthesized in many forms: thin films, wires, tapes, bulk, and single crystal. one method of synthesizing mgb 2 film is by electrophoresis. electrophoretic deposition (epd) is a colloidal processing technique in which ceramic particles suspended in a liquid medium migrate in an electric field and deposit on an electrode. solvents usually used are polar aqueous dispersing liquids such as ethanol, acetone, and methanol. binding agents may also be incorporated to enhance mechanical binding of particles (gani, 1994). epd allows the shaping of freestanding objects and also allows deposition of thin films and coatings on substrates. this technique is being developed for the shaping of thin films and coatings, laminates, and graded and textured materials (windes et al., 2002). resulting films have high density and uniformity and conforms to the shape of the substrate used. the mutia, romano, and sarmago 80 thickness of the deposited film can be easily controlled and microlamination is also possible (sarkar et al., 1994). due to the use of an electric field, epd is suited for the formation of uniform films on substrates of complicated shapes, deposition on selected areas, and impregnation of porous substrates (sarkar & nicholson, 1996). sausaging and bubbling which are common in casting techniques do not occur during epd (huang & huges, 1999). rigid control of the deposition rate is also possible (zhitomirsky, 1998). one problem encountered during fabrication of superconducting films using epd is crack formation in the films. this can be remedied by reducing the particle size of the starting material (sato et al., 2001). the investigation of the epd technique as a means of fabricating mgb2 thick films was recently done by bodoardo et al. (2004). their study, which involved a two-step preparation method, showed that it is possible to produce thick films suitable for practical applications with the use of epd. here is presented the investigation of the effect of deposition temperature on the fabrication of mgb2 thick films by epd. the starting powders were deposited on molybdenum (mo) substrates. previous studies by the group showed that deposition of mgb2 on silver substrates results in microcracks on the film as a result of the difference in coefficient of thermal expansion of the substrate and film. methodology x-ray diffraction (xrd) patterns of commercially available mgb2 powder revealed that it was of high purity, hence it was selected as the starting material for this study. fabrication of the films was done using a two-step method: (1) electrophoretic deposition on mo substrates and (2) sintering of the as-deposited films. characterization of the film was then done by xrd and scanning electron microscopy (sem). mgb2 powder was ground using a mortar and pestle for 1 h, after which 0.15 g of powder was then mixed in 30 ml ethanol to obtain a well-dispersed suspension in a nonoxidizing medium. proper dispersion of the powder in suspension is ensured by ultrasonicating the suspension for 10 min. a mo substrate having dimensions of 1 x 0.3 x 0.01 cm was used as the cathodic terminal, while a high-purity graphite rod was the anode. the electrodes were spaced 0.5 cm apart. deposition was done under a current density of ~0.02 ma/cm2 and a voltage of 600 v, with deposition time varied as follows: 15 s, 30 s, 1 min, and 2 min. the as-deposited films were then heat treated in order to densify and attain effective homogeneity of the film. a sintering temperature of 950 oc and soak time of 3 h was done, after which the films were furnace cooled to room temperature. the surface morphology of the produced film was studied using sem. material composition of the sample was analyzed using xrd. results and discussion the use of mo substrates allowed the fabrication of a good quality film. previous experimental runs using silver substrates resulted in cracked films, even in films having very thin deposits of mgb2. the cracking of the film is attributed to the difference in coefficient of thermal expansion (cte) of the ag substrates and mgb2 deposit. ag has a cte value of 2.0x10 -5 mm/ mm/°c and is mismatched with the cte value of the mgb2 film which is assumed to have a very low cte value as other ceramic materials (0.5x10-5 mm/mm/°c and below) (lucas-milhaupt, inc.). the sem image in fig. 1 shows that cracks observed in mgb2 film deposited on ag substrate is obvious. due to cracking, the film peeled off from the substrate leaving a thin layer. upon analysis of the sem images of the thick mgb2 layer and the remnant (left after peeling), it was found out that both regions have the same structure. from the result of the sem analysis, it concludes that the setup is capable of depositing mgb2, however, the film peeled off due to cracks induced by the difference in ctes. in order to minimize cracking, which is theoretically induced by a large difference in cte values between effect of deposition time 81 ag substrate and mgb2 films, mo, which has a lower cte than ag, was selected. mo has a cte value of 0.6x10-5 mm/mm/°c (lucas-milhaupt, inc.), which is three times lower than that of ag and closer to the cte of ceramic materials (0.5x10-5 mm/mm/°c and below) (lucas-milhaupt, inc.). upon using mo substrates, cracking was successfully reduced. though cracks are unlikely to be observed in thinner film deposits, microcracks begin to emerge as the thickness of the film is increased. sem images of samples deposited at different periods of time as shown in fig. 2 shows that after 1 min microcracks started to appear and are more apparent in the film deposited for 2 min, as observed at 400x magnification. it is evident that thinner films deposited for about less than 1 min have good surface characteristics; wherein the surface was evenly covered and shows no signs of cracks. the x-ray diffraction patterns of the mgb2 films deposited on mo substrates by electrophoresis at various times are shown in fig. 3. analysis of the xrd patterns of the mgb2 films revealed that only peaks having an index of (10l) were present. this indicates that the grains have a preferred orientation along the (10l) direction as shown in fig. 4. it can be seen that the peak intensities of the films deposited for 15 and 30 s are quite low compared with films deposited for 1 and 2 min. it must be noted that the only peaks present in the xrd pattern of the film fig. 1. mgb2 film deposited on silver substrate. fig. 2. deposition surface of mgb2 films (400x). fig. 3. xrd patterns of mgb2 deposited on mo by epd. 2 θθθθθ r el at iv e in te ns it y (a rb u ni ts ) 20 30 40 50 60 70 mutia, romano, and sarmago 82 deposited for 15 s are those of mo. based on the absence of mgb2 peaks it can be said that trace amounts of the said material was formed. the peak intensities of mgb2 increase at longer deposition times, wherein they are depicted clearly in the xrd pattern of the film deposited for 2 min. according to blum & hogaboom (1949) the mass of deposits is given by the equation , (1) where m is the expected weight in grams (g), i is the total current flowing from the anode to the cathode in amperes (a), n is the valency of the atom involved, f is faraday’s constant, and t is the deposition time in seconds. notice that in the equation the mass deposited is directly proportional to the deposition time. hence the difference in peak intensities for the various deposition times can be attributed to the varying mass of mgb2 powder deposited, wherein depositions carried out at longer durations resulted in greater mass deposited. looking at the sem images depicted in fig. 5 we can see that spherical-like aggregates are formed at deposition times of 15 s, 30 s, and 1 min. figure 6 shows the sem image of the film deposited for 2 min at a greater magnification. it can clearly be seen that triangular aggregates are formed at this deposition time and that textured mgb2 is clearly present. the microstructure shown in fig. 6 is clearly not that of untextured mgb2, which is hexagonal. the angular structure is evidence of the preferred orientation of the grains along the (10l) direction. the layer of powder deposited on the a2 a3 a1 10l fig. 4. (10l) direction. substrate at times less than 2 min is insufficient to facilitate the formation of mgb2 films upon heat treatment resulting in low or absence of peaks in the xrd spectra. conclusion textured mgb 2 thick films were successfully fabricated using the electrophoretic deposition method fig. 5. sem images of mgb2 films at 8000x. fig. 6. enlarged sem image of mgb2 film deposited for 2 min. effect of deposition time 83 for a deposition time of 2 min. deposition times of less than 2 min resulted in insufficient deposited powder, hence formation of mgb2 was not facilitated. films deposited at 15 and 30 s have good surface characteristics, wherein no microcracks were present. x-ray diffraction and surface image analysis reveal that the deposited films have a preferred orientation along the (10l) direction. references blum, w. & g. hogaboom, 1949. principles of electroplating and electroreforming. mcgraw-hill book co., new york, 1949. bodoardo, s., et al., 2004. production by solid/liquid reaction and characterization of high purity mgb2 powders and thick films for superconducting application. j. eur. ceram. soc. 24 (2004); bodoardo s., et al., 2002. solid state mater. sci. 6: 251–260. buzea, c. & t. yamashita, 2001. review of superconducting properties of mgb2. supercond. sci. technol. 14: r115. chart of coefficient of thermal expansion by lucas-milhaupt inc., 5656 s. pennsylvania ave., cudahy, wi 53110, usa. gani, m.s.j., 1994. electrophoretic deposition: a review. industrial ceram. 14: 163-174. huang, s. & d. dew-hughes, 1999. physica c. 319: 104-126. kortus, j., i.i. mazin, k.d. belashchenko, v.p. antrpov, & l.l boyer, 2001. superconductivity of metallic boron in mgb2. phys. rev. lett. 86: 4656. sarkar, p., o. prakash, g. wang and p.s. nicholson, 1994. ceram. eng. soc. proc. 15. sarkar, p. & p.s. nicholson, 1996. electrophoretic deposition (epd): mechanisms, kinetics, and applications to ceramics. j. am. ceram. soc. 79: 1897-2002. sato, n., et al., 2001. effect of particle size reduction on crack formation in electrophoretically deposited ybco films. physica c. 357-360: 1019-1022. takano, y., h. takeya, h. fujii, h. kumakura, t. hatano, k. togano, h. kito and h. ihara, 2001. superconducting properties of mgb2 bulk materials prepared by high pressure sintering. applied physics letters. 78: 2914-2916. windes, w.e., et al., 2002. electrophoretic deposition applied to thick metal–ceramic coatings. surf. coatings technol. 157: 267–273. zhitomirsky, i., 1998. electrophoretic and electrolytic deposition of ceramic coatings on carbon fibers. j. eur. ceram. soc. 18 15_chan 71 study of the perturbation science diliman (january-june 2003) 15:1, 71-74 study of the perturbation to a bose-einstein gas l. chan national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: lchan@nip.upd.edu.ph abstract we developed a new approach to the perturbation theory for the effective hamiltonian of condensate particles in fock space. using this new theory, we can easily analyze the effect of including a somewhat problematic term in the work of ezawa et al. we thus showed that indeed, the inclusion of this term in the perturbation potential is justified. introduction the phenomenon of the bose-einstein condensation, first observed (anderson et al., 1995) for 57rb at 170 k, followed by the cases (davis et al., 1996; bradley et al., 1997) of 23na, 7li, and 1h, has excited experimental and theoretical interests on different aspects of this quantum effect. in particular, ezawa et al. (1998) studied the fluctuation of the condensate by modifying the bogoliubov prescription (bogoliubov, 1947) in replacing a 0 by 0n with (ezawa & luban, 1967; ezawa, 1965) (1) where n 0 is the number of condensate particles and a 0 , the annihilation operator. this work was done with the modified oppenheimer approach to perturbation theory in fock space to obtain the effective hamiltonian for the condensate, using as the strength parameter. it was then shown that the fluctuations are much less than n 0 , thus justifying bogoliubov’s prescription. in this work, a term which is of zero-th order in was included in the perturbation, and the perturbation was carried out to second order in . this is unusual in perturbation work. we shall therefore consider the contributions of these terms to higher orders to seek justification for this work. the system hamiltonian the hamiltonian for a bose-einstein gas in a trap is (3) where v(x) is the trap potential and v(x) = v(-x), the interaction. the former, which varies much more slowly than the latter, is the chemical potential. in terms of the new field a 0 ', the field operator takes the form (4) where ( ) ( )n n n x a u xφ = ∑ . the operator a0 shall henceforth be taken to mean a 0 '. ' 0 0 0 .a n a→ + ( ) ( )(0) † † 0 0 bc no n n n n h j a a a a ≠ = + +∑ ( ) ( ) ( ) 2 † 3: 2 a ah x x x d x m φ ν µ φ ⎧ ⎫ = − ∆ + −⎨ ⎬ ⎩ ⎭ ∫ h ( ) ( ) ( ) ( ) ( )† † 3 31 ' ' ' ' 2 a a a ax x v x x x x d xd xφ φ φ φ+ −∫ ( ) ( ) ( )0a x n u x xφ φ= + (2) ( ) 10nλ − = λ λ 72 chan terms linear in (x) will arise in h and can be eliminated by adding and subtracting the hartree potential (5) so that (6) the functions u n (x) are chosen to be real eigenfunctions of (7) in view of the short range of the interaction as compared to the wavelength of the atoms, people take the deltafunction approximation and (8) where a is the scattering length of the atoms, so that eq. (7) simplifies as (9) although we have a set of non-linear eigenvalue equations, it is easy to reflect that we still have a complete orthonormal set of eigenfunctions. the hamiltonian of the system can now be written as where and with j mn = l oomn and k lmn = l 0lmn . ezawa’s perturbation approach ezawa’s perturbation theory is formulated to solve for the effective hamiltonian (12) in (13) where (14) with a n being an operator in the hilbert space h b h c of the total system, and is an operator in h c . the perturbation problem is formulated by dividing h into three parts: h c , which acts only on the condensate; h b , which acts only on the out-ofcondensate particles; and h bc , which involves the interaction between condensate and out-of-condensate particles. the unperturbed hamiltonian is then taken to be the terms down to the zero-th order term in h b and h c , given the names h b and h c . the rest of the terms are taken as the perturbation. we note that h bc contains a zero-order term in . in the lowest order, ezawa et al. (1998) got and (15) where ( )†0 0 01 . 2 x a a= + higher order terms are obtained after diagonalizing h b , a process which involves only an orthogonal transformation to handle mutual interaction between out-of-condensate particles. using the perturbation formula , ( ) ( ) ( )2 30 0: ' 'h x n v x x u x d xν = −∫ ( ) ( ) ( ) ( ) 2 † 3 2 a h ah x x x x d x m φ ν ν µ φ ⎧ ⎫ = − + + −⎨ ⎬ ⎩ ⎭ ∫ h ( ) ( ) ( ) ( ) ( )† † 3 31 ' ' ' ' 2 a a a ax x v x x x x d xd xφ φ φ φ+ −∫ ( ) ( ) ( )† 3a h ax x x d xφ ν φ−∫ ( ) ( ) 2 . 2 hh x x m ν ν= − ∆ + + h ( ) ( )' 'x x g x xν δ− = − 24 a g m π = h ( ) ( ) 2 2 0 0 2 h x gn u x m ν= − ∆ + + h ( ) ( ) ( )† †0 0n n n n n n n h e a a n a aε µ ε µ= + − + − +∑ ( ) ( )† † , 1 2 mn m m n n m n j a a a a+ + +∑ ( )† † † , , lmn l m n l m n l m n k a a a a a aλ+ +∑ ( ) ( ) ( ) ( ) 30klmn k l m nl gn u x u x u x u x d x= ∫ 1 ( 1) (0) (1) 2 ( 2 ) ...n n n n nλ λ λ − −λ = λ + λ + λ + λ + ,n n nhψ ψ= λ ( )(1) 2 ( 2)1 ...n n n na n a a nψ λ λ= = + + + ( 1) 0 ,−λ = ( )(0) † 20 00 0 0 0 00 01 , 2 w j a a w j xλ = + + = + ( 2) (1) (1). . 0 0 , 0 .ch p va h m a⎡ ⎤λ = + ⎣ ⎦(11) (10) nλ λ 2 † † , , , klmn k l m n k l m n l a a a aλ+ ∑ ( )2 4 30 0 0 0 0 1 1 2 2 nn n e n n g u x d x jε= − − ∑∫ 73 study of the perturbation the result obtained was (16) where new perturbation method to investigate the higher order terms due to h bc (0), we shall assume that the perturbation consists of only this term v = h bc (0), so that (17) the perturbation approach is obtained by writing where (18) p is the projector to the condensate factor. we now define (19) the effective hamiltonian can now be replaced by (20) which satisfies the equation (21) from this new eigenvalue equation, we break it up into two parts by projecting it with respect to p and giving and (23) this means that we can simplify the problem by finding a perturbation operator k = qkp satisfying corresponding operator equation (24) and (25) the p equation can be simplified into (26) which allows to be solved for once k is found. furthermore, qkp can be left multiplied into this equation to give (27) from which the term containing can be eliminated with eq. (25), yielding (28) the role of this equation is to determine k perturbatively, whether eqs. (26) and (28) are the working equations of this perturbation approach. results of perturbation using this new approach to perturbation, we get (29) the first order result of ezawa et al., (30) ( ) ( )2 212 0 0 0 0 4 0 0 0 0 ,l x p p x l x p p x+ + + + ( )†0 0 01 . 2 p i a a= − − 2 4 ( 2) 3 40 0 3 0 4 0 2 42 2 p p k x k x m m λ = + + + .b ch h h v= + + ( )1n i i n l n i lψ ≠ = + + ∑ ( ) ( )1 1l p n k l n= + + + ( ) 1' 1 .i i i n i n k n i l i l l − ≠ ≠ = = +∑ ∑ ( ) 1' 1 'n n i i n l n i lψ ψ − ≠ = + = + ∑ .p n k n= + ( ) ( ) 1' 1 1n nl l − λ = + λ + ' ' ' .n n nhψ ψ= λ 1 ,q p= − ( ) ( ) ( ) ' . b c nq h h p k n qkp n qv p k n + + = λ − + ( ) ( ) ( ) ( ) ' n c np w h p k n p p k n pv p k n + + = + λ − + (22) ( ) ( ) ( ) ( )'n c np w h p k p p k pv p k+ + = + λ − + ( ) ( )'b c nq h h k qkp qv p k+ = λ − + ( ) ( )'n c np w h p p pv p k+ = λ − + ( ) ( )'n c nqkp w h p qkp qkpv p k+ = λ − + ( ) [ ] ( ) ( ),n cq h w k q h k p k q v p k− + = − + ( ) ( )† †0 0 0 0 n n n n v j a a a a ≠ = + +∑ (1) 0 . b q k vp h w = − − 'nλ 'nλ 74 chan in fact we see that k will be a polynomial in so that the term q[h c ,k]p vanishes to all orders, and the equation that determines k simplifies to (31) which is similar in form to the results of regular perturbation theory in operator form developed by speisman (1957), and, therefore, we immediately get (32) and (33) finally, the new hamiltonian is given by substituting these expressions for k into eq. (26). explicitly, is proportional to x 0 2 and proportional to x 0 4. since x 0 was estimated to be a small quantity in ezawa’s work, we see that indeed, we have explicitly verified that the procedure to include h bc (0) in v is justified. acknowledgment the author wishes to express his gratitude to professor hiroshi ezawa for exposing him to this problem. references anderson, m.h., j.r. einsher, m.r. matthew, c.e. wieman, & e.a. cornell, 1995. observation of bose-einstein condensation in a dilute atomic vapor. science. 269: 198-201. bradley, c.c., c.a. sackett, & r.g. hulet, 1997. bose-einstein condensation of lithium: observation of limited condensate number. phys. rev. lett. 78: 985-989. bradley, c.c., c.a. sackett, j.j. tollet, & r.g. hulet, 1995. evidence of bose-einstein condensation in an atomic gas with attractive interactions. phys. rev. lett. 75: 1687-1690. bogoliubov, n.n., 1947. on the theory of superfluidity. j. phys. 11: 23. davis, k.b., m.o. mewes, m.r. andrews, n.j. van druten, d.s. durfee, d.m. kurn, & w. ketterle, 1995. bose-einstein condensation in a gas of sodium atoms. phys. rev. lett. 75: 3969-3973. ezawa, h., 1965. vestigial effects of singular potentials in diffusion theory and quantum mechanics. j. math. phys. 6: 380. ezawa, h., k. nakamura, k. watanabe, & y. yamanaka, 1998. fluctuation of the bose-einstein condensate in a trap. (unpublished). ezawa, h. & m. luban, 1967. onset of odlro and the phase transition of an ideal bose gas. j. math. phys. 8: 1285. mewes, m.o., m.r. andrews, n.j. van druten, d.m. kurn, d.s. durfee, & w. ketterle, 1996. bose-einstein condensation in a tightly confirming dc magnetic trap. phys. rev. lett. 77: 416-419. speisman, g., 1957. convergent schrödinger perturbation theory. phys. rev. 107: 1180-1192. ( )†0 0 02a a x+ = ( ) ( ) 0 c q k q k v p k w h = − + − ( 2) (1) (1) 0 b q k vk k v w h ⎡ ⎤= −⎣ ⎦− ( 2) 0 0b b q q k v vp w h w h = − − (3) ( 2) ( 2) (1) (1) 0 b q k vk k vp k vk w h ⎡ ⎤= − −⎣ ⎦− 0 0 0 0 0 b b b b b q q q v v vp w h w h w h q q vpv vp w h w h ⎡ = ⎢− − −⎣ ⎤ − ⎥− − ⎦ ( )4λ ( )2λ 01_somintac 1 growth of gaas-based vcsel/rce structures growth of gaas-based vcsel/rce structures for optoelectronic applications via molecular beam epitaxy a.s. somintac*, e. estacio, m.f. bailon, and a.a. salvador condensed matter physics laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines email: somintac@nip.upd.edu.ph abstract high intensity and sharp emission peaks, at light-hole (842 nm) and heavy-hole (857 nm) excitonic transitions for a 90 å gaas quantum well (qw) were observed for vertical-cavity surface-emitting laser (vcsel) structure. excellent wavelength selectivity and sensitivity were demonstrated by resonant cavity enhanced (rce) photodetector at 859 nm, corresponding to the energy level of a 95 å gaas quantum well. introduction the success of generating coherent emission from gallium arsenide (gaas) junction by a group in the massachussets institute of technology (mit) laboratories (quist et al., 1962) for the use of transmitting information over considerable distances in the early 1960s paved way for the slow, but continuous scientific and engineering development of emitters and photodetectors with better capabilities like efficiency, speed, and miniaturization, which started the current trends in optoelectronics technology (hall et al., 1962; holonyak jr. & bevacqua, 1962; nathan et al., 1962). presently, with the ever-increasing influence of the internet, telecommunications providers around the world are replacing old copper-based telephone networks with optical fiber networks in order to satisfy the consumer demand for higher bandwidth. fiber-optics communication essentially uses light to carry the information through a fiber medium. the most basic requirements of this setup include an emitter to generate the light from an electrical signal, an optical fiber as the transmission medium, and a photodetector to convert the transmitted light back into electrical signal. fiberoptics offer multi-mode (synchronous) data transfer, which allows high data transmission density using different wavelengths. this entails a rigid criterion for both emitters and photodetectors. an emitter must have high intensity and pseudomonochromatic emission while a photodetector must be wavelength selective in order to distinguish the different wavelengths transmitted through the fiber. alongside the development of various device structures are the improved fabricating technologies, which include molecular beam epitaxy (mbe). it is a deposition technique carried out in ultra-high vacuum wherein the sources (i.e., gallium and arsenic) are evaporated and epitaxially grown (one atomic layer per second) on a relatively cooler substrate. mbe provides an excellent degree of control in the composition and smoothness of the deposited layers, which greatly affect device performance. science diliman (january-june 2003) 15:1, 1-5 * corresponding author 2 somintac et al. theory gallium arsenide (gaas) belongs to the iii-v semiconductor compounds. it is a direct band gap material (1.424 ev) which permits highly efficient generation and detection of light. this property, and the existence of its other higher energy gap alloy like aluminum-gallium arsenide (algaas) and aluminum arsenide (alas), makes it the most studied and highly regarded for optoelectronics applications. the basic structure of a light-emitting diode (led) is the p-n junction, consisting of two layers of the same band gap material (homo-junction) but with different dopants. this structure, under forward bias, emits light, and under reverse bias, detects light. perhaps one of the noteworthy innovations in this field is the development of the heterojunction led, wherein a thin layer of undoped lower gap material is deposited between the p-layer and the n-layer (hayashi et al., 1970). this provides both optical and electrical confinement of photons and carriers, respectively. incorporation of low dimensional structures in the undoped region, like quantum wells (qw), was found to greatly enhance the sharpness and intensity of the emission. quantum wells also provide design flexibility, since its energy level (thus, the emission wavelength) can be engineered simply by varying the well width. though the qw p-in structure has been successful to the point of commercialization, researchers continue to explore different device geometries. the most significant geometry is the vertical-cavity surface-emitting laser (vcsel) or resonant cavity enhanced (rce) photodetector. these vcsel/rce structures outperform other existing similar optoelectronic devices, and have only been commercially realized recently. the vcsel structure retains the basic p-i-n configuration, but the pand the n-layers were replaced with highly reflecting p-type and n-type distributed bragg reflectors (dbr). with the gaas qw at the center, the undoped region defines the emission wavelength λ. the dbr consists of alternating layers of higher gap semiconductors, algaas and alas, both having a thickness corresponding to λ/4. the undoped region (including the qws) has a thickness of (multiple of) λ. this forms a fabry-perot resonant cavity, which is responsible for the amplification of the gain in the active region (chow et al., 1997; choquette & hou, 1997; iga et al., 1984; unlu et al., 1991). experimental method all the samples were deposited via riber 32-p mbe apparatus located at the national institute of physics, college of science, university of the philippines diliman. the samples were grown on n-type (001) gaas substrates. sample p035 has a typical p-i-n structure. a 1.0 µm silicon-doped (n-type) gaas buffer layer was grown first over the substrate, with 0.5 µm grown at gaas surface reconstruction temperature of 580oc, and the other half ramped to 630oc. deposition was maintained at this temperature throughout the remainder of the process. a 1.5 µm si-doped (n-layer) algaas was grown over the buffer layer, with intended doping concentration of 1.0 x 1018 cm-3. the undoped region consists of 100 å algaas layer (barrier) and 90 å gaas (quantum well). a 1.5 µm beryllium-doped (player) algaas was grown with doping concentration of 1.0 x 1018 cm-3. finally, a 100 å be-doped gaas (cap layer) was deposited to serve as protective layer. be-doped algaas 1.5 µm 90a gaas qw si-doped algaas 1.5 µm p038 p-i-n be-doped top algaas/alas dbr gaas qw si-doped bottom algaas/alas br p038 and p041 vcsel/rce fig.1. the structures of sample p035 (p-i-n) and the samples p038 and p041 (vcsel/rce). n-type p-type substrate p-type substrate n-type 3 growth of gaas-based vcsel/rce structures two samples, p038 and p041 with vcsel/rce structure, were also grown (fig. 1). the 1.0 µm n-type gaas buffer layer was grown at the same temperature as sample p035 (p-i-n). for p038, the dbr consists of alas and algaas layers with thickness of 715 å and 605 å, respectively. the undoped region consists of three pairs of 60 å algaas (barrier) layer, with 90 å gaas (qw) and with total cavity width of 2,525 å. for sample p041, its dbr consists of 710 å alas and 623 å algaas. the undoped region consists of three pairs of 60 å algaas (barrier) and 95 å gaas (qw), with total cavity width of 2,440 å. for both p038 and p041, 100 å be-doped gaas cap layer was grown last. all samples were characterized using electroluminescence (el) spectroscopy for emission spectra and photocurrent (pc) spectroscopy for absorption. additional reflectivity experiments were done to the vcsel/rce samples in order to measure the reflectance of the dbrs. fig. 1 shows the structures of samples p035 (p-i-n) and p038 and p041 (vcsel/ rce). results and discussion in fig.2, the emission and the photocurrent spectrum of the sample p035 (p-i-n) is shown. the absorption spectra of p035 reveal a sharp onset of absorption at 862 nm, corresponding to the energy level of a 90 å gaas quantum well. at the wavelength, the emission spectra show a sharp peak with full width at half maximum (fwhm) of 110 å. the sharpness of the rise of the absorption spectra and the high intensity emission peak is characteristic of a qw based p-i-n heterojunction. for vcsel, its reflectivity spectra will show fabryperot resonant modes and a range of peak reflectance. to maximize the resonance effect, the wavelength specified by the quantum well energy level must be within the range of maximum reflectance. fig. 3 shows the fabry-perot modes and maximum reflectance from 734 nm to 795 nm in the reflectivity spectra. the emission peaks are at 842 nm and at 857 nm, both falling outside the maximum reflectance. even if this was the case, the emission peaks were still amplified by fabry-perot maxima, resulting in the resolution of two distinct peaks corresponding to the light-hole (842 nm) and heavy-hole (857 nm) excitonic transitions for a 90 å gaas quantum well. their fwhms are 60 å and 80 å, respectively. high-intensity sharp emission peak is an important requirement for light sources of fiber optic technology. photocurrent emission wavelength (angstrom) 6000 7000 8000 9000 fig. 2. the photocurrent spectra and the emission spectra of sample p035 (p-i-n). reflectivity fabry-perot modes emission 6000 7000 8000 9000 10000 wavelength (angstrom) fig. 3. the emission spectra and the reflectivity spectra of sample p038 (vcsel) showing peak amplification by fabry-perot modes. 4 somintac et al. fig. 4 shows the reflectivity and the photocurrent spectra of sample p041. the photocurrent spectra reveal a very sharp absorption peak at 859 nm, and fwhm of only 50 a. this is a clear indication that the grown rce structure is highly wavelength-selective, and the high photocurrent intensity translates to high sensitivity to the specified wavelength. for comparison purposes, the emission spectra of sample p035 (p-i-n), p038 (vcsel), and a commercially available algaas led were plotted together in fig. 5. the emission peaks are normalized, with the commercially algaas led showing a broad emission and has 25 times less intensity. conclusion in conclusion, we report the results of our efforts to design and fabricate optoelectronic devices suitable for fiber optic applications. the vcsel (p038) exhibited sharp emission peaks corresponding to the two excitonic peaks of a 90-å gaas quantum well, while the rce (p041) demonstrated high sensitivity and excellent wavelength selectivity. acknowledgment the authors would like to thank the dost-esep for its continued support in this research project. references choquette, k.d. & h.q. hou, 1997. vertical-cavity surfaceemitting laser: moving from research to manufacturing. proc. ieee. 85: 1730-1739. chow, w.w., k.d. choquette, m.h. crawford, k.l. lear, & g.r. hadley, 1997. design, fabrication, and performance of infrared and visible vertical-cavity surface-emitting lasers. ieee. j. quant. electr. 33: 1810-1824. hall, r.n., g.e. fenner, j.d. kingsley, t.j. soltys, & r.o. carlson, 1962. coherent light emission from gaas junctions. phys. rev. lett. 9: 366. photocurrent reflectivity 6000 7000 8000 9000 10000 wavelength (angstrom) fig. 4. the photocurrent spectra and the reflectivity spectra of sample p041 (rce) showing peak amplification at maximum reflectance. fig. 5. the normalized emission spectra of a commercially available algaas led, the p035 (p-i-n), and the p038 (vcsel). the spectrum of the algaas led was scaled (x25), as well as the spectrum of the p041 (x0.8), for presentation purposes. the vcsel sample exhibited the best performance in terms of intensity and sharpness of peaks. commercial led p038 vcsel p035 (p-i-n) 6000 7000 8000 9000 10000 wavelength (angstrom) 5 growth of gaas-based vcsel/rce structures hayashi, i., m.b. panish, p.w. foy, & s. sumski, 1970. junction lasers that operate continuously at room temperature. appl. phys. lett. 17: 109. holonyak jr., n. & s.f. bevacqua, 1962. coherent (visible) light emission from ga(as1-xpx) junctions. appl. phys. lett. 1: 82. iga, k., et al., 1984. room-temperature pulsed oscillation of gaalas/gaas surface-emitting injection laser. appl. phys. lett. 45: 348-350. nathan, m.i., w.p. dumke, g. burns, f.h. dill jr., & g. lasher, 1962. stimulated emission of radiation from gaas p-n junctions. appl. phys. lett. 1: 62. quist, t.m., r.h. rediker, r.j. keyes, w.e. krag, b. lax, a.l. mcwhorter, & h.j. zeiger, 1962. semiconductor maser of gaas. appl. phys. lett. 1: 91. unlu, m.s., s. strite, a. salvador, a. demirel, & h. morkoc, 1991. ieee photon. lett. 3: 1126. 05_liwag 22 liwag, sicam, and karremans science diliman (january-june 2003) 15:1, 22-26 measurement of the temperature of rubidium atoms in a magneto-optical trap j.w.f. liwag*, v.a. sicam, and k. karremans optoelectronics laboratory, department of physics, university of san carlos 6000 cebu city, philippines email: jwfliwag@cnms.net abstract we have performed measurements that can be used to determine the temperature of rubidium atoms in a magneto-optical trap. the expansion of the atomic cloud after switching off the current through the anti-helmholtz coils was recorded with a ccd camera. analysis of the measurements revealed that the cloud of atoms in optical molasses expands at a velocity of 4 cm/s. introduction laser cooling and trapping of neutral atoms is a rapidly expanding field of research. the theory for laser cooling and trapping was firmly established in 1975 (hänsch & schawlow, 1975). however, it was only in 1985 that the very first laser-cooled and trapped atoms were experimentally observed (chu, 1998). the magnetooptical trap (mot) was first realized in 1987 (raab et al., 1987). in 1997, the nobel prize in physics was awarded to chu, cohen-tannoudji, and phillips for pioneering research in the field of laser cooling and trapping of neutral atoms (phillips, 1998). at the optoelectronics laboratory of the department of physics, university of san carlos in cebu city, an mot for rubidium atoms in a vapor cell using diode lasers has been in operation since 1998 (liwag, 1999). so far, we have exerted considerable efforts to obtain high vacuum in the trapping cell to maximize the number of trapped atoms, and to increase the lifetime of the atoms in the mot. at a pressure of 10-8 mbar, we measured up to 108 trapped atoms with lifetime as long as 3 seconds. these experiments were necessary to pave the way for bose-einstein condensation (bec), which remains the ultimate goal of this research (ketterle, 1999). the measurement of the temperature of trapped atoms in an mot is one of the most exciting and challenging experiments in the field of laser cooling. we performed experiments using the time-of-flight (tof) method for measuring temperature wherein we measured the arrival times of the atoms on a probe laser beam passing directly under the atomic cloud when the mot is switched off. because the time scales involved here require sub-millisecond accuracy, we set up a computerbased system to block the trapping laser beams using an electronically-controlled relay switch (liwag et al., 2000). however, small timing problems in switching off the current and the laser beams might be the reasons we failed to record the tof signal. in this paper, we present the results of our experiments in determining temperature by measuring the expansion velocity of the atomic cloud in optical molasses. in molasses, the light-atom interaction is used to create a strong damping force which reduces the average velocity considerably. due to these lower velocities and the corresponding longer time scales, tof measurements in molasses are less difficult. the temperature, an important parameter in the process of realizing bec, is the only parameter lacking in the* corresponding author 23 measurement of the temperature of rb atoms characterization of our mot. from this point, we hope to proceed towards the realization of bec in the future. laser cooling and trapping the interaction between light (photons) and matter (atoms) involves a transfer of momentum from photons to atoms. the net momentum transfer from the beam of photons to the atoms results in an optical force which can be used to decelerate the atoms. since the kinetic energy of atoms is a direct measure of their temperature, a reduction of this kinetic energy means a reduction of the atomic temperature, or “cooling” of the atoms. the optical force can be used to create optical molasses, which can be understood using a one-dimensional description shown in fig. 1. consider an atom with a resonance frequency ω 0 moving with a velocity v along a trajectory of two counterpropagating laser beams of frequency ω l and wave vector k. due to the doppler effect, the moving atom will “see” each of the lasers to be shifted in frequency ω a given by (petra, 1998) (1) where is the doppler shift. since both lasers are red-detuned (i.e., tuned to a frequency slightly below resonance (ω l < ω 0 )), the atom moving towards the laser beam will “see” a frequency closer to resonance, while an atom moving in the same direction will experience a frequency farther from resonance. therefore, the atom absorbs more photons from the counterpropagating laser beam. as a result, there is a net force on the atom causing it to slow down. this force is proportional to the atom’s velocity and causes a viscous damping of the atom, as if the atom were moving in a thick fluid. a l kω ω ν= − ⋅ k ν⋅ optical molasses in three dimensions were observed in 1985 and was achieved using three pairs of counterpropagating laser beams along the three mutually orthogonal axes. an atom situated in the mutual intersection of the six laser beams experiences strong three-dimensional viscous damping, and its average velocity is rapidly reduced close to zero. the atoms do not actually come to rest, but execute a random walk motion which leads to atoms disappearing from the optical molasses. the minimum temperature in optical molasses is given by the doppler limit (cohen-tannoudji, 1998) (2) where h is planck’s constant, ∆ν n is the natural linewidth (in frequency units) of the atomic transition, and k b is boltzmann’s constant. for rb, the doppler limit is 144 µk. optical molasses, or 3-d doppler cooling, is not an optical trap for atoms. the force that is required to trap a doppler-cooled atom must be a position-dependent force which pushes the atom to a defined center of the optical trap. the optical trap is constructed by superimposing a quadrupole magnetic field on an optical molasses formed by using circularly polarized laser beams. the magnetic field induces spatially dependent zeeman shifts of the atomic energy levels, while the circular polarizations stimulate transitions in these zeeman-shifted levels. this produces a restoring force on the atom that is proportional to the its position from the origin of the trap defined by the magnetic field. the restoring force f in the mot is given by the relation (liwag, 1999) (3) where g f is the landé factor for the atomic transition, µ b is the bohr magneton, γ is the natural linewidth of n d b2 h t k ν∆ = 0 2 2 ( ) 4 2 1 f b s s i f z kg b z i i i µ ⎛ ⎞∆⎜ ⎟ ⎜ ⎟γ= ⎜ ⎟⎛ ⎞∆⎛ ⎞ ⎜ ⎟+ +⎜ ⎟⎜ ⎟⎜ ⎟γ⎝ ⎠ ⎝ ⎠⎝ ⎠ fig. 1. the formation of optical molasses in one dimension using two laser beams. to account for the doppler shift of resonance frequency, red-detuned laser beams are used. large force force = 0 vv atom 24 liwag, sicam, and karremans the transition, ∆ = ω l – ω 0 is the detuning of the laser, i is the intensity of the laser beam, and i s is the saturation intensity. for a “red” detuning of the laser (∆ < 0), this force is opposite the sign of the z-coordinate. an inhomogeneous quadrupole magnetic field is produced by reversing the current in one of the two coils, which are separated by a distance equal to their radii, known as helmholtz coils. the characteristic of this magnetic field is that it is zero at the center of the coil separation and increases linearly along the x-, y-, and z-axes. hence, the term b 0 z in eq. (3) represents the magnetic field strength at position z. this optical trap is commonly referred to as the magneto-optical trap or the mot. experimental setup the mot setup consists of a glass cell maintained at low pressure by an ion pump. rb atoms are loaded into the cell by heating the rb reservoir in the system. the laser used for trapping the atoms is tuned to a frequency slightly lower than the resonance frequency. the rest of this beam is shaped into a circular beam and divided into three pairs of counterpropagating beams, which intersect the center of the mot in three mutually orthogonal directions. because of the hyperfine splitting of the ground states of rb, a second diode laser is needed. this so-called “repumper” laser is tuned to the hyperfine transition of 87rb to insure continuous absorption-spontaneous emission cycles. it pumps electrons which end up in the lower (f = 1) hyperfine ground state into the f = 2 ground state, via the f = 1 → f’ = 2 transition. the “repumper” laser is simply directed into the center of the mot to overlap the center of the six trapping laser beams. a pair of anti-helmholtz coils is set up such that the center of the coils coincides with the center of the glass cell. the experimental setup of the mot is shown in fig. 2. temperature of trapped atoms in the mot the time-of-flight (tof) method is a standard experiment for measuring atomic temperatures (phillips, 1998). using this method, a probe laser beam is sent through the trapping cell at a distance of 1 cm below the atomic cloud. the probe beam enters a photodetector connected via an image acquisition (ni-imaq™) card to a computer. the expansion of the cloud when the trapping laser beams and the current are switched off simultaneously was monitored. since this simultaneous switching requires sub-millisecond accuracy, a fast relay switch with a razor blade was designed to block the trapping laser beam electronically using labview™. a program that switches on the relay to block the trapping beams and at the same time switches off the current through the anti-helmholtz coils was designed. switching times of less than 1 ms have been obtained for our relay, which makes it an inexpensive alternative to an optomechanical shutter. however, we have not achieved a temperature measurement using a photodetector. instead of measuring the tof signal after blocking the trapping laser beams, we measured the expansion in optical molasses of the atomic cloud. in this method, the expansion of the cloud is monitored when the current, through the anti-helmholtz coils, is switched off while fig. 2. experimental setup for laser cooling and trapping of rb atoms. the upper right part shows the saturation spectroscopy setup for the trapping laser. a current running through a coil wrapped around the rb vapor cell produces a magnetic field to induce a zeeman shift of the cooling transition. the upper left part shows the saturation spectroscopy setup for the repumper laser. the lower part shows the trapping cell with the anti-helmholtz coils. ion pump helmholtz coils rb source m mm m m m m m m m mm m pd rb l1 l1 l1 l l l pdrb l4 l4 l4 gp gp gp 25 measurement of the temperature of rb atoms the trapping and repumper lasers are kept on. when the atomic cloud expands in optical molasses, the velocities decrease due to damping. therefore, the time scales involved are much longer than for standard tof signals. the longer time scales make it possible to use a ccd camera for our experiment. images of the expanding cloud were captured by a ccd camera connected to a computer with the video capture software vidcap™. using a labview program to electronically switch off the current through the antihelmholtz coils, it was observed that the cloud proceeded in a direction to the upper left side of the ccd camera’s field of view. this upward motion has been attributed to a residual magnetic field, including that of the earth’s magnetic field. to compensate for this residual magnetic field, three pairs of helmholtz coils were set up around the mot, with their minimum separations limited by the mot dimensions. for each helmholtz pair, the current flows in the same direction to produce a 1-d magnetic field that is uniform at the center of the mot. the current through each of the three pairs was adjusted until the optimum compensation, which shows the atomic cloud expanding uniformly in all directions, was observed. the experimental setup for the measurement of temperature is shown in fig. 3. a typical set of images of the cloud expansion was obtained at a capture rate of 26 frames/second, and covered a total of 6 frames as shown in fig. 4. the total time elapsed since the current was switched off until the atomic cloud disappeared completely in the molasses was about 230 ms, from which a maximum velocity of 4 cm/s was calculated. the temperature t of the atoms is given by the relation (petra, 1998) (4) where m = 144 x 10-27 kg is the mass of a rb atom and v is the average thermal velocity of the atoms. using eq. (4), a temperature of 8 µk would be obtained for rb atoms–a value that is way below the doppler limit of 144 µk for 87rb. this shows that we cannot use this simple model to measure the temperature in molasses. in this case, the properties of our molasses have to be known in order to calculate the atomic velocity without damping. we are currently working on a model which will enable us to describe the expansion of an atomic cloud in optical molasses. we expect that from this analysis we can determine the value of the temperature. conclusions we calculated a maximum velocity of 4 cm/s for our rb atoms in optical molasses–more than 4 orders of magnitude lower than the most probable velocity of 21 2 bm k tν = compensating helmholtz coils anti-helmholtz coil trapping laser beams to pc with vidcaptm anti-helmholtz coil atomic cloud ccd camera fig. 3. experimental setup for the measurement of temperature for atoms expanding in optical molasses when the current through the anti-helmholtz coils is switched off. fig. 4. frame-by-frame image at 26 frames/ second of the cloud of trapped rb atoms after switching off the current in the anti-helmholtz coils, with compensation for residual magnetic fields. 26 liwag, sicam, and karremans 237 m/s for rb atoms at room temperature. the calculated temperature of 8 µk is way below the doppler limit for rb, and therefore we need to complete our analysis of the cloud expansion in optical molasses to come up with a more realistic value of the temperature. we hope that our analysis will yield a temperature that is reasonably of the order of magnitude of the doppler limit for rb. we now look forward to the realization of bec in our mot. references cohen-tannoudji, c.n., 1998. manipulating atoms with photons. rev. mod. phys. 70: 707-719. chu, s., 1998. the manipulation of neutral particles. rev. mod. phys. 70: 685-706. hänsch, t. & a. schawlow, 1975. cooling of gases by laser radiation. opt. comm. 13: 68-69. ketterle, w., 1999. experimental studies of bose-einstein condensation. phys. today. 30-35. liwag, j.w.f., et al., 2000. characterization of the magnetooptical trap for rb atoms. proc. 18th spp congress. 214-216. liwag, j.w.f., 1999. cooling and trapping of 87rb atoms in a magneto-optical trap using low-power diode lasers. m.s. thesis. university of the philippines diliman, quezon city. petra, s.j.h., 1998. development of frequency-stabilized laser diodes for building a magneto-optical trap. m.s. thesis. universiteit van amsterdam, the netherlands. phillips, w.d., 1998. laser cooling and trapping of neutral atoms. rev. mod. phys. 70: 721-741. raab, e.l., et al., 1987. trapping of neutral sodium atoms with radiation pressure. phys. rev. lett. 59: 2631-2634. 11_effect effect of dopant ions 41 effect of dopant ions on the electrical conductivity and microstructure of polyaniline (emeraldine salt) m. d. catedral, a. k. g. tapia*, and r. v. sarmago materials science and engineering program, university of the philippines, diliman, quezon city 1101 e-mail: akgtapia@yahoo.com j. p. tamayo and e. j. del rosario institute of chemistry, university of the philippines, los baños, laguna abstract science diliman (july-december 2004) 16:2, 41–46 *corresponding author samples of polyaniline (emeraldine salt) were prepared with different protonic acid dopants, namely, hydrochloric acid (hcl), nitric acid (hno3), perchloric acid (hclo4), sulfuric acid (h2so4), and hydroiodic acid (hi). using the two-point probe method, it was found that the samples had ohmic behaviors in which high linear coefficients were found in the range 0.9686–0.9997. on the other hand, the electrical conductivities were measured using the van der pauw method. the undoped sample had a conductivity of 5x10–4 s/cm. the highest conductivity of 109.04 s/cm was observed for the hclo4-doped sample, while the lowest value (0.02 s/cm) was obtained for the hi-doped sample. these conductivities were compared with the computed energy gap between the highest occupied molecular orbital (homo) and lowest unoccupied molecular orbital (lumo) where it was found that they are inversely proportional to each other. scanning electron microscopy revealed significant differences among the samples in terms of shapes and morphologies. introduction conducting organic polymers are highly conjugated pelectron systems that display unusual electronic properties such as low ionization potentials and high electron affinities; they are also called “synthetic metals.” many researches are now focused on studying conducting polymers such as polyacetylene, polythiophene, polypyrrole, and polyaniline among others (kroschwitz, 1998). polyaniline (pani) exists in a variety of forms that differ in chemical and physical properties. the most common protonated emeraldine has a conductivity at the semiconductor level of the order of 100 s cm–1, which is many orders of magnitude higher than that of common polymers (<10–9 s cm–1) but lower than that of typical metals (>104 s cm–1) (stetjskal et al., 1996; stetjskal & gilbert, 2002). the various electron states in conducting polymers are in terms of the gap between highest occupied molecular orbital (homo) and lowest unoccupied molecular orbital (lumo) or the homolumo gap, energy gap (vardeny & wei, 1998). as shown in fig. 1, the emeraldine base (pani-eb) can be made conducting by adding a protonic acid dopant. after protonation, there will be dissociation of bipolaron to form two polarons, where a bipolaron form will be achieved. it is believed that for the sarmago, catedral, and tapia 42 nondegenerate conducting polymers, the bipolarons are the charge carriers. in the polaron form, there will be delocalization of polarons after which polarons will be delocalized yielding a green conducting emeraldine salt (pani-es) (stetjskal & gibert, 2002; gorman & grubbs, 1991; trchova et al., 1998). polyaniline is not charge-conjugation symmetric, where the valence band and conduction bands are asymmetric to a great extent (ghos et al., 2001). the efficient polymerization of aniline is achieved only in acidic medium, where aniline exists as anilinium cation. a variety of inorganic and organic acids at different concentrations have been used in the syntheses of pani; the resulting pani-es, protonated with various acids, differs in solubility, conductivity, and stability (trivedi, 1997). the changes in physicochemical properties of pani that occur in response to various external stimuli are useful for various applications (levi, 2000). many of these applications are based on the electroactive properties of pani such as bioelectronics, polymer-modified electrodes, and polymer-based amperometric biosensors (lyons, 1994). the combination of electrical properties typical of semiconductors with parameters characteristic of polymers have resulted in the development of “plastic” microelectronics (hamers, 2001), electrochromic devices (rosseinsky & mortimer, 2001), tailor-made composite systems (prokes et al., 2000), and “smart” fabrics (el-sherif et al., 2000). the choice of physical properties of pani suitable for specific applications is thus of fundamental importance (stetjskal & gilbert, 2002). the main objective of the present study is to determine the effects of dopant ions on the conductivity and microstructure of pani-es. methodology chemical synthesis of polyaniline (emeraldine salt) the pani-es samples were chemically synthesized using reagent-grade aniline, ammonium peroxydisulfate (aps), and the appropriate dopant (hcl, hno3, hclo4, h2so4, or hi). the standard procedure for the polymerization of aniline was followed (stetjskal & gilbert, 2002). a 0.25 m aqueous solution of aps was mixed with 0.2 m of aniline in 1 m of the acid dopant. the mixture was stirred and maintained at 4°c in an ice bath. the green precipitate formed was filtered, washed with distilled water and acetone, and dried in a vacuum oven for 3 h at 40°c. characterization of polyaniline before characterization, the samples were pelletized at 7 tons for 10 min using a carver hydraulic press. sufficiently thin samples of ~1 mm were derived. all the prepared polyaniline samples were characterized in terms of electrical conductivity and microstructure using a scanning electron microscope (sem). for conductivity and electrical properties, silver paste was used to attach the gold wires as contacts. then, current-voltage (i-v) measurements were done using the two-point probe method and resistivity measurements were derived using the van der pauw method in an in-line configuration (ghos et al., 2001). before taking the sem images, the samples were coated with gold for 5 min for usually sufficient coating using an ion sputtering system to increase the source of electrons (ordillas). sem images of the polymeric fig. 1. protonic acid doping of polyaniline (emeraldine base) (trchova et al., 1998). n n nh nh nh nhnh a– + nh a– + nh a– + nh a– + nh a– + nh a– + nh protonation with ha emeraldine base dissociation of bipolaron to form two polarons delocalization of polaron delocalized polaron nh nh bipolaron form nh polaron form effect of dopant ions 43 samples were taken using philips xl30 sem (hussain & kumar, 2003). the samples were studied at different magnifications for topographic comparisons. results and discussion electrical properties the polyaniline samples were synthesized using different dopants. the final product was a green conducting emeraldine salt pani-es (stetjskal & gilbert, 2002; trivedi, 1997). the current-voltage (i-v) curves of the undoped and hclo4-doped samples are shown in figs. 2 and 3, respectively, which show ohmic behavior from –1.0 to 1.0 ma. this range of current was used to check the symmetry of the curves. these were derived using a two-point probe i-v measurement with gold wires attached by silver paste as contacts. inconsistent readings of the voltage near zero current was due to the difficulty of measuring small voltages. conductors generally show ohmic behavior wherein the voltage across the material varies linearly with the current and the i-v plot has a constant slope, which is the resistance of a material. this means that the current density linearly increases with the applied electric field. shown also in the graph are the correlations of current with voltage. it can be observed that the slope of the graph is the resistance of the samples since the y axis is the voltage (v) and the x axis is the current (i). inconsistent portions in some of the i-v plots, e.g., undoped and hcl-doped pani-es, may be due to the irregular structures of the samples. it is therefore necessary to ensure good pelletization in order to have good measurements from these synthesized materials. this is consistent with the observation that the electrical properties of conducting polymers are strongly influenced by the effect of disorder, since conducting polymers are partially crystalline and partially amorphous (long et al., 2003). very high correlation coefficients in the range of 0.9686–0.9997 were observed for the i-v plots of the pani samples. this indicates that all samples behave as ohmic conductors. shown in table 1 are values of the electrical conductivity of undoped and doped polyaniline samples. the differences in conductivity values are due to the effect of dopant ions. as can be seen, the undoped sample has conductivities in the range of 10–4, which makes the sample highly insulating typical of a normal polymer. the highest conductivity (109.04 s/cm) was obtained for the hclo4-doped sample; the lowest conductivity (0.08 s/cm) was exhibited by the hi-doped sample. these experimental values fit within in the range of conductivity for pani specified (trchova et al., 1998).fig. 2. i-v curve of pani-es with hclo4 dopant showing symmetry from –1 to 1 ma. current (a) vo lta ge ( v ) fig. 3. scaled plots of current density versus electric field for pani-es with different dopants showing the slopes as the conductivity in s/cm. electric field (e) c ur re nt d en si ty ( j) sarmago, catedral, and tapia 44 figure 4 is the plot of current density versus electric field of the samples. the figures were scaled to fit the slopes of different samples which are the conductivity values. the higher the slope, the higher the conductivity. from figs. 2 and 3, it is manifested that the current density linearly increases with the applied electric field. the conductivities are presented in table 1 together with computed homo-lumo energy gap data. as shown in table 1, the experimental values of conductivity were found to exhibit roughly an inverse correlation with the computed values of the energy gap between the homo and lumo; these values have been reported by other researchers (atienza et al., 2004; pascual, 2003). a plot of the conductivity versus homo-lumo gap is shown in fig. 4. thus, the smallest homo-lumo gap was observed for the hclo4-doped sample, while the biggest energy gaps were obtained for hcland hi-doped samples. the homo-lumo gap is the molecular counterpart of the band gap for the macroscopic polymeric solid. microstructure of polyaniline as shown in the sem images of fig. 5 the pani-es samples with varying dopants exhibit varying microstructures. variation in microstructure leads to different conductivities of the samples. the addition of acid dopants alters the polymer lattice, which leads to the ionization of sites in the chains. the defects in the chain due to the dopant ions provide the mobility of the charge carriers on which conduction depends (kroschwitz, 1988). the conductivity is also dependent on the number of charge carriers. the undoped sample in fig. 5(a) displays globular but irregular morphology. figure 5(b) shows the hcldoped pani showing fine threadlike structures fused together. the hno3-doped sample has a fine and grainy structure fused together but with void spaces [fig. 5(c)], while the hclo4-doped sample [fig. 5(d)] has corallike structures with elongated bodies. figure 5(e) shows a porous pattern of globular microstructures for the h2so4-doped sample. lastly, fig. 5(f) shows the loose fig. 4. conductivity versus homo-lumo gap plot of the pani-es samples with different dopants. homo-lumo gap (energy gap), ev c on du ct iv it y (s /c m ) table 1. conductivity and computed energy gap of the undoped and doped polyaniline samples. afor beta orbital. batienza et al., 2004. cpascual, 2003. dbased on hso4as dopant ion. none (eb) h+ hcl hno3 hclo4 h2so4 hi 5.00x10-4 2.43 71.90 109.04 92.13 0.02 2.61 2.44c 3.37 2.79 2.45 2.65d 3.02 dopant conductivity(s/cm) homo-lumo energy gapa,b, (ev) fig. 5. sem images at ~26000x magnification of (a) undoped pani, (b) hcl-doped, (c) hno3-doped, (d) hclo4-doped, (e) h2so4-doped, and (f) hi-doped polyaniline samples showing distinct structures pointed by arrows. effect of dopant ions 45 flaky structures of the hi-doped sample. the low conductivity of the hi-doped sample may be due to these loose flaky structures and air spaces that are present. the orientation of structures and morphology of samples at the macroscopic level affects the mobility of charge carriers and, thus, influences conductivity (kroschwitz, 1988). conclusion polyaniline salts (pani-es were found to exhibit varying electrical conductivities. all prepared samples showed ohmic behavior. the doped pani samples showed a dramatic increase in conductivities compared with undoped pani. the hclo4-doped sample gave the highest conductivity (109.04 s/cm) which is 2x105 times greater than that of the undoped sample, while hidoped pani gave a conductivity of 0.02 s/cm. the experimental conductivity values generally showed an inverse correlation with the computed values of the homo-lumo energy gap reported by other researchers (atienza et al., 2004; pascual, 2003). the scanning electron microscope (sem) pictures of the doped pani samples showed varying microstructures, e.g., the hclo4-doped sample exhibited a corallike structure, while hi-doped pani showed a loose flaky structure. further investigation of the morphology of samples will give a better understanding of bulk conductivity. references atienza, c.h., et al., 2004. effect of dopant ions on some computed molecular properties affecting the electrical conductivity of polyaniline. spp 2004. el-sherif, m.a., j. yuan, & a. g. macdiarmid, 2000. j. intelligent mater. syst. struct. 11: 407. ghos, m., a.k. meikap, s.k. chattopadhyay, & s. chatterjee, 2001. j. phys. chem. solids. 62: 475. gorman, c.b. & r.h. grubbs, 1991. conjugated polymers. kluwer academic publishers, dordretch: 1–48. hamers, r.j., 2001. nature. 412: 489-490. hussain, a.m.p. & a. kumar, 2003. electrochemical synthesis and characterization of chloride-doped polyaniline. indian academy of sciences. bull. mater. sci. 26:329-334. kroschwitz, j.i., 1988. electrical and electronic properties of polymers: a state-of-the-art compendium. wiley, new york. levi, b.g., 2000. phys. today. 53(12): 19. long, y., et al., 2003. electrical conductivity of a single conducting polyaniline nanotube. appl. phys. lett. 83: 18631865. lyons, m.e., 1994. electroactive polymer electrochemistry part 2: methods and applications. plenum press, new york. ordillas, m.u. a laboratory manual in scanning electron microscopy. department of mining, metallurgical, and materials engineering, college of engineering, university of the philippines, diliman. pascual, a.a., 2003. semi-empirical computation of molecular properties affecting the electrical conductivity of polyaniline and polypyrrole as influenced by doping (undergraduate thesis), university of the philippines, los baños. prokes, j., i. krivka, e. tobolkova, & j. stejskal, 2000. polym. degrad. stab. 68: 261. rosseinsky, d.r. & r.j. mortimer, 2001. adv. mater. 13: 783. stejskal, j. & r.g. gilbert, 2002. polyaniline-iupac: technical report. pure appl. chem. 74(5): 857–867. stejskal, j., et al., 1996. polymer. 37: 367. trchova, m., j. zemek, & j. stejskal, 1998. spectroscopy of conjugated polymers: polyaniline. macromolecules. 31: 2218–2222. trivedi, d.c., 1997. in nalwa, h.s. (ed.) handbook of organic conductive molecules and polymers. wiley, chichester: 2: 505–572. sarmago, catedral, and tapia 46 vardeny, z.v. & x. wei, 1998. optical probes of photoexcitation in conducting polymers: handbook of conduction polymers. 2nd ed. dekker, inc., new york. ocr document page 1 images image 1 23_temporal temporal coherence behavior 95 temporal coherence behavior of a nd:yag pumped waveguide raman shifter marilou m. cadatal*, ma. leilani y. torres, and wilson o. garcia national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: marilou.cadatal@up.edu.ph abstract science diliman (july–december 2004) 16:2, 95–100 *corresponding author we study the behavior and report the control of the temporal coherence length zc of a 355/532 nm nd:yag pumped h2 raman shifter with and without capillary waveguide (cwg). depending on the application of the raman-shifter light source, zc could be tuned rapidly or slowly by varying the h2 pressure p or the input power pin. a more dynamic zc behavior is observed for a waveguide raman shifter. introduction stimulated raman scattering (srs) in h2 is an efficient technique of extending the tuning range of lasers into the vacuum ultraviolet and far infrared (mannik & brown, 1986; berry et al., 1982; torres et al., 2003; palero et al., 2001; palero et al., 2000; papayanis et al., 1998). it is simple, economical, robust, and capable of high conversion efficiency (torres et al., 2003; palero et al., 2001; palero et al., 2000). recently, the threshold energy required for srs has been lowered and the conversion efficiency was improved by using a capillary waveguide (cwg) (mannik & brown, 1986; berry et al., 1982; torres et al., 2003). on the other hand, laser sources with controllable temporal coherence properties are valuable in spectroscopy and optical microscopy. with its highly directional beam outputs, these sources with low coherence can be efficient illuminators in conventional optical microscopes for generating images with acceptable levels of optical noise caused by speckles, edge ringing and shifting, and point diffractions (garcia et al., 2001). conversely, sources with longer coherence lengths are useful in interferometric applications. this work analyzes the temporal coherence properties of a h2 raman shifter with and without a cwg and demonstrates the control of its associated temporal coherence length using the h2 pressure (p) and input power (pin). experimental setup the schematic diagram of the experimental setup is shown in fig. 1. the fundamental 1064 nm output of a q-switched linearly polarized neodynium-doped yttrium-aluminum-garnet (nd:yag) laser (spectra physics gcr-230) is converted to 532 nm (second harmonic) and 355 nm (third harmonic) via kdp crystals. the nd:yag is operating at 10 hz repetition rate with a pulsewidth of 5–6 ns (fwhm). the telescope expands the beam before it passes through the aperture, a. lens l1 (f = 290 mm) focuses the beam onto the entrance of a 50-cm-long capillary tube (cwg) placed inside the raman shifter. the capillary tube has a 1 mm bore diameter. h2 is used as the raman-active medium since it has the largest raman frequency shift cadatal, torres, and garcia 96 (4155 cm–1). lens l2 collimates the raman lines to the pellin-broca (pb) prism, which disperses the raman output into the rayleigh (r), stokes (s), and anti-stokes (as) components. the time-averaged optical spectra s(l) are obtained using a grating spectrometer (spex model 1000m, 3 s integration time, and 0.001 nm resolution). the power of each raman line is measured using a power meter. this is used to normalize the spectral lines obtained from the spectrometer. determination of z c since the raman shifter is a pulsed light source, its corresponding temporal coherence length zc is extremely difficult to measure directly with a scanning michelson interferometer. a better technique is to employ a nonscanning multichannel fourier transform interferometer in which the interferogram points are formed simultaneously in space, instead of through time. this requires a very fast detector array with a large number of pixels and a uniform response over a wide spectral range for a uniform sampling of the interferogram. but such a photodiode array is expensive (garcia et al., 2001). the associated zc of s(l) is determined from the following procedure proposed by garcia et al. (2001): step 1: the sampled spectrum, {s(lm)}, is converted into its equivalent k representation, {s(km)}, where k = 1/l; m = 1,…,m; and km (cm –1) = 50,000. the 355/532 nm r line is included in {s(lm)}. step 2: an even (2m+1) data sequence, {s(n)}, is derived from {s(km)}, where s(n) = s(km) = s(–n), and n = –m,–m+1,…,m. step 3: the inverse fourier transform of s(n); f– 1[{s(n)}] = i(z) is then obtained. here, z is a distance variable in the range –mkm<z<mkm. i(z) may be interpreted as the average interferogram generated by a raman-shifter light source in a michelson interferometer. step 4: finally, zc is solved with respect to z = 0 using: (1) equation (1) is derived from the definition of coherence time tc = zc/c, where c is the speed of light in vacuum. conventionally, the envelope of the interferogram i(z) is first obtained and zc is determined as the distance from z = 0 up to where the value of the envelope has reduced to 1/e (daza et al., 1999; born & wolf, 1999). using eq. (1) permits the determination of zc of a pulsed light source with an arbitrarily profiled emission spectrum {s(lm)}. hence, the need to first establish the best-fit envelope of i(z), which is a likely source of error, to obtain zc is eliminated. z c behavior for the 355 nm pump the s and as wavelengths generated with the 355 nm pump wavelength are summarized in table 1. fig. 1. setup for the generation of raman lines from a waveguide raman shifter. m–mirror, l–lens, a–aperture, and pb–pellin-broca prism. for the conventional raman shifter, the cwg is removed. table 1. generated raman lines (lpump=355 nm). raman line wavelength (nm) raman line wavelength (nm) s1 s2 s3 415.9 502.9 635.9 as1 as2 as3 309.0 273.8 245.8 samples of the s(k) obtained with and without a cwg for different p at maximum pin (26.5 mw) are shown in fig. 2. their corresponding interferograms i(z) determined from step 3 are also shown. at 150 psi with a cwg, r and s1 lines dominate the spectrum. as the p is increased to 600 psi, s2 and s1 dominate and the r line is in turn depleted. moreover, at a higher p, four-wave mixing, which is the main mechanism for as generation, is suppressed. this leads to the depletion of the as1 and suppression of the as2 line. temporal coherence behavior 97 hence, the bandwidth is larger and the i(z) envelope is narrower at 150 psi compared with that at 600 psi. without the cwg, only the r line dominates the spectrum at 150 psi resulting in a broad interferogram envelope. at 600 psi, although the r line still dominates, s1 is intensified and s2 is generated. thus, the bandwidth of the raman shifter is increased and the interferogram envelope narrows. the dependence of zc on h2 p for different pin are shown in figs. 3(a) and 3(b) for a waveguide raman shifter and a conventional raman shifter, respectively. the zc values were determined from eq. (1). for a waveguide raman shifter, zc exhibits an inverted humplike dependence on p for all pin. at pin = 9.4 mw, zc could be varied from 1.97 to 4.40 mm, while at pin = 26.4 mw, zc ranges from 1.84 to 4.67 mm. for pin > 9.4 mw, a stable zc minimum is achieved at around 150 psi. at lower pin (7.5 and 5.6 mw), the minimum zc value is observed to shift to higher p. rapid control of zc is possible for p < 150 psi, while a slower variation of zc is possible for p > 150 psi. the zc starts to saturate at p > 300 psi for pin > 9.4 mw. however, zc is not observed to saturate for pin < 9.4 mw since at lower pin, the r line dominates the spectrum. without the cwg [fig. 3(b)], zc exhibits a less pronounced inverted humplike behavior with the stable zc minimum shifting to a higher p (300 psi) for pin > 9.4 mw. for pin < 9.4 mw, the minimum zc value is again observed to shift to higher p. at pin = 9.4 mw, zc ranges from 2.99 to 4.68 mm, while at pin = 26.4 mw, zc could be tuned from 2.74 to 4.68 mm. fig. 2. on the left are the s(k) for (i) p=150 psi and (ii) 600 psi (a) with and (b) without cwg. on the right are the corresponding interferograms of s(k). n or m al iz ed i nt en si ty ( a. u. ) wave number (x103 cm–1) path length (µµµµµm) n or m al iz ed i nt en si ty ( a. u. ) fig. 3. zc measurements as a function of h2 pressure for pin = 5.6, 7.5, 9.4, 13.3, 18.5, and 26.4 mw (a) with and (b) without cwg. pressure (psi) c o h er en t le n g th ( µµµµ µ m ) 50 250 450 650 1.5 2.5 3.5 4.5 5.5 pressure (psi) c o h er en t le n g th ( µµµµ µ m ) 50 250 450 650 1.5 2.5 3.5 4.5 5.5 (a) (b) cadatal, torres, and garcia 98 compared with that with a cwg, a slower control of zc is possible for p < 300 psi before reaching its minimum. for p > 300 psi, the value of zc starts to saturate. moreover, a lower zc minimum is obtained in the presence of a cwg since more lines are produced as shown in fig. 2. the stokes intensity is a function of pin and the h2 p as described by (torres et al., 2003; palero et al., 2000) (2) where ip and is(0) are the pump laser intensity and the initial stokes intensity, respectively, l is the interaction length between the raman-active medium, and gr is the steady-state raman gain coefficient. the gr is proportional to the raman-active gas p (torres et al., 2003; palero et al., 2000). without the cwg (lower l), a higher p is needed to achieve the same is(l) at a constant ip. however, an analysis of gr as a function of p shows that gr saturates as p is increased (palero et al., 2001). hence, even if the p is increased at a particular pin, the number of stokes lines is not increased. this leads to the saturation of the zc value at higher p. figures 4(a) and 4(b) present the dependence of zc with pin with and without a cwg. in the presence of a cwg [fig. 4(a)], the zc value is robust against variations of pin > 10.1 mw for a particular p. for instance, at 300 psi, zc is maintained at about 3.21 mm. for pin < 10.1 mw, zc can be controlled by varying the pin at a given p or by varying the p at a given pin. without the cwg, higher zc values are obtained since lesser raman lines are produced. at 100 psi, zc is maintained at 4.69 mm since the spectrum is still dominated by the r line. at p < 150 psi (with cwg) and p < 200 psi (without cwg), zc decreases with increasing pin since at low gas p, increasing pin leads to the generation of more raman lines. however, this does not work under high pressures where increasing pin only results in s2 (with cwg) and s1 (without cwg) dominating over the raman lines (fig. 2). zc behavior for the 532 nm pump the wavelengths of the generated raman lines in h2 are summarized in table 2. c o h er en t le n g th ( µµµµ µ m ) (b) c o h er en t le n g th ( µµµµ µ m ) (a) input power (mw) 5 10 15 20 25 1.5 2.5 3.5 4.5 5.5 input power (mw) 5 10 15 20 25 1.5 2.5 3.5 4.5 5.5 fig. 4. zc measurements as a function of pin for h2 pressure = 100, 150, 200, 300, 450, and 600 psi (a) with and (b) without cwg. table 2. generated raman lines (lpump= 532 nm). raman line wavelength (nm) raman line wavelength (nm) s1 as1 as2 683.0 435.7 368.9 as3 as4 319.9 282.3 figure 5 shows the dependence of zc on h2 p for different pin (a) with and (b) without a cwg. as in the 355 nm pump wavelength, a rapid control of zc is possible at certain p while a slow variation is possible for other p. a noticeable shift in the minimum zc value temporal coherence behavior 99 fig. 5. zc measurements as a function of h2 pressure for pin =15.4, 28.3, 32, 42.2, and 46.46 mw (a) with and (b) without cwg. pressure (psi) c o h er en ce l en g th ( µµµµ µ m ) 0 200 400 600 2.0 3.0 4.0 5.0 6.0 (b) pressure (psi) c o h er en ce l en g th ( µµµµ µ m ) 0 200 400 600 2.0 3.0 4.0 5.0 6.0 (a) 7.0 7.0 p in ( m w ) pressure (psi) 60 0 40 0 20 0 10 0 80 60 42.2 32.0 28.3 21.0 15.4 46.46 (a) p in ( m w ) pressure (psi) 60 0 40 0 20 0 10 0 80 60 42.2 32.0 28.3 21.0 15.4 46.46 (b) fig. 6. bandwidth as a function of h2 pressure for pin =15.4, 21, 28.3, 32, 42.2, and 46.46 mw (a) with and (b) without cwg. could be observed for both waveguide and conventional raman shifters. the shift is due to the change in the spectral bandwidth of the raman shifter as a function of p (fig. 6). as pin decreases, more srs lines are generated at higher p, thereby broadening the spectral bandwidth of the raman shifter at higher p. consequently, as pin decreases, zc minimum shifts to a higher p. while the shifting is observed for all pin with a 532 nm pump wavelength, the minimum zc is observed to shift to higher pressures only at low pin for a 355 nm pump (fig. 3). this is because the cumulative gain coefficient is inversely proportional to the pump and stokes wavelengths (palero et al., 2001) so that higher pump wavelengths lead to lower stokes conversion efficiencies and higher threshold energies. the dependence of zc as a function of pin is presented in fig. 7. for the waveguide raman shifter operating at 200 psi, zc decreases for pin < 21 mw due to the generation of raman lines. however, further increase in pin only results in s1 dominating over the other raman lines. hence, zc starts to increase for pin > 21 mw. at higher p (> 200 psi), zc increases as pin is increased since an increase in pin only results to s1 dominating the spectrum. similarly, without the cwg, zc decreases at pin where raman lines are generated. as s1 dominates over the spectrum, zc increases. conclusion the zc of a waveguide raman shifter can be varied over a wide range via an appropriate selection of h2 p and pin. for both 355 and 532 nm pumped waveguide raman shifter, zc exhibited an inverted hump behavior. for the 355 nm pumped waveguide raman shifter, a stable zc minimum is obtained at around 150 psi for pin > 9.4 mw. if pumped with 532 nm, zc minimum shifts to higher pressures for decreasing pin. lower zc values could be obtained by a waveguide raman shifter. cadatal, torres, and garcia 100 fig. 7. zc measurements as a function of pin for h2 pressure =200, 300, 400, 500, and 600 psi (a) with and (b) without cwg. c oh er en ce l en gt h ( µµµµ µ m ) (a) input power (mw) 10 20 30 40 50 2.5 3.0 3.5 4.0 4.5 5.0 c oh er en t le ng th ( µµµµ µ m ) (b) input power (mw) 10 20 30 40 50 2.5 3.0 3.5 4.0 4.5 5.0 acknowledgments the authors gratefully acknowledge the instrumentation physics laboratory of the national institute of physics for lending the detector used in this work and the philippine department of science and technology (dost) through the engineering and science education project (esep) for the equipment grant. this project is supported by the university of the philippines and the philippine council for advanced science and technology research and development (pcastrd). references berry, a.j., d.c. hanna, & d.b. hearn, 1982. low threshold operation of a waveguide h2 raman laser. opt. commun. 43: 229–232. born, m. & e. wolf, 1999. principles of optics, 7th ed. cambridge university press, cambridge. daza, m.r., a. tarun, k. fujita, & c. saloma, 1999. temporal coherence behavior of a semiconductor laser under strong optical feedback. opt. commun. 161: 123–131. garcia, w., j. palero, & c. saloma, 2001. temporal coherence control of nd:yag pumped raman shifter. opt. commun. 197: 109–114. mannik, l. & s.k. brown, 1986. tunable infrared generation using third stokes output from a waveguide raman shifter. opt. commun. 57: 360–364. palero, j.a., j.o.s. amistoso, m.f. baclayon, & w.o. garcia, 2000. generation of uv, vis, and nir laser light by stimulated raman scattering in hydrogen with a pulsed 355 nm nd:yag laser. proceedings of the 18th spp philippine physics congress. palero, j.a., r.s. ibarreta, & w.o. garcia, 2001. frequency conversion of a 532 nm nd:yag laser using a hydrogen raman shifter. proceedings of the 19th spp philippine physics congress. papayanis, a.d., g.n. trikrikas, & a.a. serafetinides, 1998. generation of uv and vis laser light by stimulated raman scattering in h2, d2, and h2/he using a pulsed nd:yag laser at 355 nm. appl. phys. b. 67: 563–568. torres, m.l., m. cadatal, & w. garcia, 2003. stimulated raman scattering in a 532 nm nd:yag laser pump hydrogen raman shifter with a capillary waveguide. proceedings of the 21st spp philippine physics congress. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 the role of temperature on morphological properties of gallium nanowires: a kinetic monte carlo study darwin b. putungan*, marvin a. albao1 computation materials research group, computational physics laboratory physics division, institute of mathematical sciences and physics university of the philippines, los banos college, laguna 4031 philippines 1present address: national energy technology laboratory, us department of energy pittsburgh, pa 15236 usa *corresponding author email: darwinbputungan@gmail.com abstract we have investigated the effects of temperature on surface morphology during deposition of ga on si(100) at room-temperature (rt) under ultra-high vacuum (uhv) conditions , using kinetic monte carlo (kmc) simulations. specifically, we are interested on the impact of temperature on key quantifiable quantities such as the ratio of homogeneous to heterogeneously nucleated islands , r, as well as the mean island size. in this study, the relevant energetic and kinetic parameters were first calculated using density functional theory (dft) which were then used as input to the simulation model. our simulations unequivocally show that as temperature increases the population of homogeneously nucleated islands grows at the expense of their heterogeneous counterparts. this suggests that the thermally driven increase in adatom surface mobility may have some asymmetric effect on the respective rates of homogeneous and heterogeneous nucleation. interestingly, the observed shift in the balance between the two types of islands with increasing temperature were accompanied by a corresponding decrease in the mean island size. the latter observation is somewhat surprising vis-a-vis prediction from conventional mean-field nucleation theory that longer (or bigger) islands should dominate at higher temperatures as a result of more efficient surface equilibration. keywords:density functional theory, kinetic monte carlo, self-assembly introduction since the early nineties, studies concerning the growth of novel nanostructures known as nanowires have evolved from a mere curiosity to a major scientific undertaking that we know of today. arguably, the impetus to these pioneering studies was the wide availability and rapidly increasing resolution of scanning tunneling microscope (stm) and similar imaging instruments providing researchers a detailed glimpse of their structure and surface characteristics. on the theoretical front, parallel advances in computational techniques such as first-principles calculations, as well as more coarse-grained approaches such as kinetic monte carlo (kmc) simulations, made it possible to probe lingering questions that have long baffled experimentalists concerning these nanostructures. one realization of nanowires is via roomtemperature (rt) deposition under ultrahigh vacuum (uhv) conditions of select group iii and group iv atoms on low-index si surfaces, specifically the si(100) and si(111). early stm studies (itoh, et al., 1993; baski et al., 1991) reveal, among others, that the lateral dimension of these wires approaches one, i.e., they are in fact 1-d atomic wires. (in this study, the terms “nanowires”, science diliman (january-june 2010) 22:1, 27-31 27 putungan, albao “atomic wires”, “1-d wires”, “1-d islands”, or simply “islands” will be used interchangeably.) additionally, an effective repulsion between two neighboring nanowires ensures that their minimum lateral separation is two lattice constants. the interest on nanowires reflects the relentless drive to reach the limit of miniaturization in integrated circuits (ic), an important component in the rapidly expanding microelectronics industry. as a practical matter, manipulation of ic components built on top of si-based substrates demands utmost precision and control, which in turn cannot be achieved without a good understanding of the underlying microscopic surface processes. it is in this regard that computer modeling techniques such as molecular dynamics or kmc simulations have become essential, as they have been successfully used to study systems as diverse as quantum dots to polymers (kratzer, et al., 2002; j. huang, et al., 2005). in the present study, of interest are the 1-d islands formed by ga atoms on si(100) surface during deposition under uhv conditions. specifically, these islands could be either homogeneously nucleated, such as the case when two diffusing ga atoms meet, or heterogeneously nucleated, such as when these adatoms are pinned by defects and other impurities. it should be stressed that although defects on si(100) typically include missing si monomers, dimers, or even trimers, these types show little reactivity with diffusing adatoms (albao, et al., 2005; albao, et al., 2010). instead, a distinct class of defects known as c-defects, believed to be trapped water molecules, have been known to pin these adatoms more effectively, acting as nucleation centers (javorsky, et al., 2008). from a practical standpoint, heterogeneously nucleated islands are preferred over homogeneously nucleated ones because the former's nucleation centers can be predicted based on the knowledge of the defect distribution. since these nanowires typically grow on one side of a defect, leaving the other side unreactive to diffusing adatoms (kocan, et al., 2008), one can in principle control not only the direction of growth, but also their mean island size. in this work, “heterogeneous island” and “homogenous island” will be used interchangeably with “heterogeneously nucleated island” and “homogeneously nucleated island” for brevity. methodology the si(100) surface was modeled as a n × n square lattice of adsorption sites, with each site corresponds to the physical binding site situated between the trenches of si dimer rows. initially, the surface was seeded with c-defects, with the choice of the defect-density consistent with experimentally known values, i.e. 10-3 defect/site (javorsky, et al., 2008). furthermore, the atomic wire's growth pattern follows the so-called parallel dimer model (nogami, 1997) in which it grows along a direction orthogonal to that of the si dimer rows. in the model, the growth direction was conveniently chosen to be the horizontal direction. next, rate constants pertaining to the relevant microscopic processes were calculated using the arrhenius relation, h = ν exp (-eact/kbt), which is generally appropriate for thermally activated surface processes. in the above, eact is the activation energy, ν is the prefactor, typically of the order of 1013 s-1 and ultimately related to the vibrational frequency for atoms or molecules trapped in potential wells, while t is the substrate temperature. below we list the key elementary processes included in our growth model and deemed operative during the course of the initial adsorption and subsequent growth of the atomic wires: : (1) adsorption. potential sites available for adsorption are selected randomly and tested for availability. sites already occupied by a ga adatom, as well as those laterally adjacent to an atomic wire or another adatom are excluded. in addition, such sites should not be labeled as defect sites. the rate of deposition is typically of the order 10-4 ml/s and reflects typical experimental flux rate. (2) detachment. terminal datoms atoms found at the end of an island and not chained to a defect as well as newly deposited adatoms, can detach and reattach at end sites. 28 science diliman (january-june 2010) 22:1, 27-31 the role of temperature on morphological properties of gallium nanowires (3) adatom diffusion and nucleation. an adatom diffuses freely on the surface until it (i) meets another adatom (homogeneous nucleation), (ii) hop onto a site adjacent to a defect (heterogeneous nucleation), and (iii) reaches a site next to a terminal adatom, in which case it gets incorporated into the existing island (aggregation). both nucleation mechanisms described herein are reversible, that is, a newly formed island can decay, mimicking the inherent atomic wire instabilities as was observed (albao, et al, 2010; kocan, et al, 2007). having described the fundamental features of the simulation model, we now comment briefly on the bortz-kalos-lebowitz (bkl) algorithm employed in the simulations (bortz, 1975). very briefly, in the bkl algorithm one compiles a comprehensive catalogue of all the microscopic elementary processes on the surface including a list of species capable of executing them. these lists are dynamic – they are updated every kmc step. unlike the metropolis algorithm where processes are executed with certain probability that depends on whether the impending move would bring the system closer to equilibrium or not, the bkl algorithm is inherently reject-free and is thus deemed more efficient. the latter algorithm ensures that at each iteration, the system advances in configurational space and thus no move is ever wasted. finally, we make a brief comment on the relatively narrow temperature range (300-500 k) in which such simulation data are collected. prior experiments suggest that at temperatures higher than 500 k, 1-d islands may begin to coexist with 2-d islands (adams, et al, 1997). as our model does not explicitly incorporate adsorbate lateral interactions, it is not our aim to simulate 2d-island growth. thus, we confine this study to the temperature regime where the technologically interesting 1d-islands dominate. results and discussions figure 1 presents snapshots of typical surface morphologies of ga/si(100) system for all the three temperatures considered in this study. in addition, table 1 shows the homogeneous to heterogeneous island ratio r for each temperature. as can be seen in figure 1, both homogeneous and heterogeneous islands are present on the surface at all temperatures. a more detailed analysis of the plots reveals that as the temperature rises, so does the number of homogeneous islands, while the number of heterogeneous islands remains roughly unchanged. this trend is evident in table 1 where the homogeneous to heterogeneous island ratio, r, is provided for all three temperatures. it is worth noting that r significantly climbed from 0.76 at 300 k to 12.58 at 500 k, signifying that homogeneous nucleation is favored at higher temperatures. on hindsight, this observation does not seem surprising since heterogeneous nucleation rate is controlled by the number of c-defects which remains constant even as the temperature increases. we note however, that at the relatively low coverage of 0.1 ml (where 1 monolayer (ml) is defined as 1 ga adatom per site) some of these c-defects remain unpaired with adatoms even at temperatures as high as 500 k, signifying that heterogeneous nucleation is somewhat inhibited. the source of the apparent suppression in heterogeneous nucleation becomes clear when one realizes that diffusion is confined mainly along the horizontal direction or parallel to the orientation of the islands. also, it should be pointed out that c-defect density is extremely low, of the order of 10-4 defect/site, ensuring that heterogeneous nucleation is a rare event. in contrast, a similar suppression in homogeneous nucleation was not observed. since the flux rate, and thus the adatom density, is independent of temperature it is reasonable to ask why there is a higher incidence of homogeneous nucleation at higher temperatures, causing r to jump 17-fold from its simulated value at 300 k to its final value at 500 k. in the simulations, we monitored incidence of collisions and observed that the frequency of such collisions increases in proportion with the number of islands. with this observation a clearer picture emerges. at higher temperatures, a surfeit in thermal energy translates into enhanced adatom mobility, therefore increasing the incidence of collisions and homogeneous nucleation. in contrast, c-defects, whose concentration provides the natural limit to the density of heterogeneous island and is very small to begin with, do not increase with temperature. additionally, a highly anisotropic diffusion ensures that the incidence of adatoms being pinned by cdefects (leading to heterogeneous nucleation) is minimal even at higher temperatures. science diliman (january-june 2010) 22:1, 27-31 29 putungan, albao (a) (b) (c) figure 1. simulated surface morphology of deposited ga on si(100) at (a) 300 k, (b) 400 k, and (c) 500 k temperature values. green lines denote homogeneous islands while the blue ones indicate their heterogeneous counterparts. unfilled black circles denote c-defects where heterogeneous nucleation starts. all lattices are 100x100 in size. a quick look at figure 2, on the other hand, conveys a slightly different picture. the plot reveals that aggregation, or growth, in general is suppressed in favor of nucleation, as signified by the marked decrease in mean island size with increasing temperature. in conventional homogeneous and heterogeneous systems, mean-field nucleation theory predicts exactly the opposite, i.e, islands tend to be longer at higher temperatures, indicative that aggregation becomes the dominant mechanism in that regime. it should be mentioned that similar anomalous behavior has been observed for ga/si(100) by albao and co-workers (albao, et. al., 2005) in a related study that does not explicitly take into account the presence of c-defects. in that kmc paper, the authors found a monotonically decreasing form for the scaled island size distribution rather than the expected monomodal shape. extensive analysis revealed that, just like in the present study, restricted aggregation the fact that growth was confined to two ends of the atomic wires was responsible for the system's rather unconventional behavior. thus we can only conclude that with or without c-defects (and heterogeneous nucleation) – scaled island size distribution on ga/si(100) is monotonically decreasing rather than monomodal in shape . table 1. homogeneous to heterogeneous island ratio, r, for different temperatures. temperature (k) r 300 0.76 400 2.71 500 12.58 figure 2. plot of the mean island size, sav vs coverage for various temperatures. conclusion it was shown in this work that temperature impacts the balance between homogeneous and heterogeneous islands. on the one hand, increased adatom mobility at higher temperatures translates into frequent adatom-adatom collisions, which in turn greatly boosts homogenous nucleation. on the 30 science diliman (january-june 2010) 22:1, 27-31 the role of temperature on morphological properties of gallium nanowires other hand, the corresponding increase in heterogeneous nucleation with increasing temperature was not observed due to the extremely low c-defect density which remains roughly constant throughout the narrow temperature (300 – 500 k). furthermore, the highly anisotropic nature of adatom diffusion on ga/si(100) (i.e., along the direction parallel to the island's orientation) restricts contact between a c-defect and an adatom, a necessary step toward heterogeneous nucleation. in addition, contrary to prediction of nucleation theory for conventional homogeneous systems, the mean island size decreases with temperature, a result mirrored in previous studies where the effect of cdefect was not explicitly taken into account. therefore, the present work confirms that the presence of c-defects has no impact on this unconventional trend. acknowledgement dbp would like to acknowledge the help of dr. feng-chuan chuang and the computational materials research lab of the national sun yat-sen university, taiwan. references adams, d.p., t.m. mayer, b.s. swartzentruber, 1998. influence of interfacial hydrogen on al thin film nucleation on si. j. appl. phys. 83(9):4690-4694. albao, m.a., m.m.r. evans, j. nogami, d. zorn, m.s. gordon, and j.w. evans, 2005. monotonically decreasing size distributions for one-dimensional ga rows on si(100). phys. rev. b 72(3):035426-035434. albao, ma, c.h. hsu, d.b. putungan, and f.c. chuang, 2010. room-temperature deposition of group iii metals on si(100): a comparative study of nucleation behavior, surface science. 604(3-4): 396-403 baski a.a. and c.f. quate, 1991. tin-induced reconstructions of the si(100) surface, phys. rev. b 44: 11167-11177 bortz, a.b., m.h. kalos, j.l. lebowitz, 1975. a new algorithm for monte carlo simulation of ising spin systems. j. comp. phys. 17(1):10-18. huang, j., z. mao and c. qian, 2006. dynamic monte carlo study of the polymer chain in random media filled by nanoparticles. polymer 47(8):2928-2932. itoh, h., j. itoh, a. schmid and t. ichinokawa, 1993. structures of low-coverage phases of al on the si(100) surface observed by scanning tunneling microscopy, phys. rev. b .48:14663-14666. javorsky, j., m. setvín, i. ošťádal, and p. sobotík, m. kotrla, 2008. stm and kmc study of indium growth on si(100). wds’08 proceedings of contributed papers, part iii, 95-100. kocán, p., p. sobotík, i. ošt'ádal, j. javorský, m. setvín, 2007. stability of in rows on si(100) during stm observation. surf. sci.601(18):4506-4509 kocan, p., l. jurczyszyn, p. sobotík, i. ošt'ádal, 2008. defects on the si(100)-(2×1) surface: anchoring sites of the surface polymerization reaction of in atoms. phys. rev. b 77:113301-113303. kratzer p., e. penev, m. scheffler, 2002. first-principles studies of kinetics in epitaxial growth of iii-v semiconductors. appl. phys. a 75(1): 79-88. nogami j., 1997. self-assembled single atom wide metal lines on si(100) surfaces. nato advanced research workshop series: atomic and molecular wires, ed. c. joachi, 341:11-22. science diliman (january-june 2010) 22:1, 27-31 31 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 ocr document page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 page 13 images image 1 page 14 images image 1 page 15 images image 1 tracking the dynamic variations in a social network formed through shared interests gerold pedemonte and may lim* national institute of physics, university of the philippines, diliman, 1101 quezon city *corresponding author: may@nip.upd.edu.ph received: 3 december 2009; revised: 12 december 2009; accepted: 14 december 2009 abstract we tracked the dynamics of a social network formed by a shared interest in movies. users-, movie ratings-, and rental date-data from the netflix prize dataset were used to construct a series of datefiltered social networks, wherein viewers were linked when they rented the same movie and gave the same rating. we obtained a nearly constant high clustering coefficient (0.60 – 0.85), and a low average path length (1.4 – 2.3) indicating a static 'small-world' network despite the dynamic behavior of the borrowers. keywords: networks, time series analysis, complex systems, small-world network introduction social network analysis helps us in understanding the nature of interactions between individuals in a society. studies on social networks provide a useful framework for analyzing social events such as opinion dynamics (amblard and deffuant 2004), failure analysis (motter and lai 2002) and information spread (mossa et al. 2002). social network dynamics is routinely described as a static network with dynamical aspects, or temporal changes, occurring on the network (barrat, barthélemy, and vespignani 2008). in a social context, this is analogous to constructing a network of people with given connectivities, i.e. friends, enemies, acquaintances, etc. which are defined by the weights between the links and actions occurring within the boundaries of the said network. for example, a disease will more likely spread from a patient who has a higher number of interactions (or links) with others. as such, it is the dynamics of the spreading of the disease that is modeled over a static network framework (pastor-satorras and vespignani 2001). compared to static networks (albert and barabasi 2002), a longitudinal study provides a more realistic picture of real world networks which are mostly dynamic in nature (kossinets and watts 2006; braha and bar-yam 2006). we can think of a dynamic network as a time-series of static networks. these series of networks can be reconstructed as a single static network if we take the sampling time interval to be equal to the total observation time. at different times, nodes continuously change connections to other nodes or even connect and disconnect from the network. the main difficulty with such an analysis is the dearth of available data describing social networks with such high time resolution. this has been recently addressed with the release of the netflix prize dataset. the dataset was first made available as the data input to the $1m netflix prize (netflix), a contest to beat the accuracy of the recommendation engine used by netflix in advising its customers on what movies to rent. in this paper, we tracked the changes in the overall structure of the shared-interest network as we varied the observation period and the degree of shared inclination. science diliman 21(1):37-43 37 mailto:may@nip.upd.edu.ph pedemonte, lim materials and methods social network of movie viewers the netflix prize dataset (netflix 2006) provides an account of movie rentals, with rental dates and corresponding user ratings culled from the information database of netflix, an online movie rental service available only in the united states. users select the movies they wish to rent online, and the physical disks are delivered to them by the us postal service. while no other information (e.g., geographical location) about the users was released, we note that the online aspect of the service presents an additional minimum requirement on the end-user (i.e., internet access) which may link the users even more than just by a shared interest in movies. the entire dataset has over 2 million users and 17,770 movies for the period october 1998 december 2005. integer ratings (highest = 5, lowest = 1) are associated with each rental. we constrained our analysis to a subset of the data with 21,324 viewers and 6,582 movies covering the period january 2000 december 2001. network construction in constructing the network of viewers, we assumed that a shared interest is reflected in the user ratings. a pair of viewers were connected if they watched the same movie and gave the same rating. we note that our definition for shared-interest evenly considers shared-like (rating = 5) or shared-dislike (rating = 1) for a particular movie. since monthly networks were constructed based on the date of rating, and not the release date of the movie, this does not discount the effect of a surge in rental requests for new releases. furthermore, ratings subnetworks were also constructed from each monthly network to see if the dynamics would change for shared-like and shared-dislike networks. our network construction method creates a fullyconnected cluster for a group of viewers who watched the same movie and gave it a common rating. network analysis we analyzed the time evolution of the constructed monthly networks from january 2000 to december 2001, as well as the time evolution of each of the five user-ratings values. we described the overall structure or topology of a network using the degree distribution, the average clustering coefficient, and the average path length. the degree ki of a node i is the number of direct connections to it. in a sparse network, a high degree is indicative of the importance of a node in maintaining network connectivity. the clustering coefficient ci measures the connectedness of neighbors of node i relative to a fully-connected idealization (eq. 1), c i=2 ei/k i ki−1 (1) where ki is the degree and ei is the number of links between the neighbors of i. the denominator in eq. (1) represents the total number of possible connections ki(ki – 1)/2 of the ki neighbors. if the average clustering coefficient <c> over all nodes is close to unity, then most of the nodes are directly connected to one another. lastly, the shortest path length measures the minimum number of hops to reach another node from a reference node. the average shortest path length is taken over all possible node pairs. when the average shortest path length is low, a network is tagged as a 'small-world' network because any two nodes are separated by just a few hops. results and discussion popularity dynamics from january 2000 to december 2001, the population of viewers n(t) actively renting movies (fig. 1a) from netflix generally increased with time. on the other hand, the popularity of a movie pm(t) defined as the fraction of viewers nm(t) / n(t) renting said movie during the specified time interval, where the subscript m is the movie index, showed fluctuations in demand (fig. 1b shows the top five movies). though some movies are very popular, the range of pm(t) for all movies is 0.0-0.565 (mean = 0.001535, stdev = 0.009236). in the absence of the giant clusters of viewers linked by the blockbuster movies, the network linkages would suddenly become sparse. while sustaining a high pm(t) is difficult to achieve for a single movie, there is always a movie which we call a 'hub movie' that is able to fulfill said role (fig. 1c), whether via the controlled movie release date or seasonal demand. on the average, the most popular movie is watched 38 science diliman tracking the dynamic variations in a social network by 10% of the active renters. overall, the top ten movies were seen by half of the active renters during the entire observation period. since the sequence of movies that the viewers will check out is not predictable, the corresponding fluctuation in fig. 1b is unpredictable as well, sans the expected surge in popularity of new releases. popularity, however, is not equivalent to satisfaction which is reflected in the ratings given by the viewers. we constructed the five rating networks constructed for each monthly network from january 2000 to december 2001. the ratings frequency were: [1 (hated it): 169,490; 2 (didn't like it): 380,887; 3 (liked it): 849,534; 4 (really liked it): 838,414; 5 (loved it): 455,149]. as expected, borrowing dynamics was not random; viewers borrowed 3.9 times more of the movies they liked over those they didn't like. since movies are available for rent at the earliest only months after the first release, reviews are already widely available by the time a viewer makes a decision to borrow. we propose that shared interest is most reflected in extreme opinions; a rating of 5 (or 1) would be more indicative of interest (or dislike) than an average rating of 3. thus, aside from a monthly network description, we also looked into the disaggregated (by ratings) description of the monthly networks. though beyond the scope of this work, we note that the rate of ratings bias science diliman 39 figure 1. (a) monthly population of users, (b) popularity of the five most watched movies, (c) monthly blockbuster movies (defined as hub movies); and (d) the top ten movies of 2000-2001. pedemonte, lim accumulation may hint on the rate of information dissemination. degree distribution from the degree distribution, we gain a picture of the current direct connectivity of viewers who share the same interest. there are several possibilities through which a viewer would have a high degree: a) by watching a single hub movie, taking a value of k equal to the number of other viewers of that hub movie, b) by watching several movies m, each with its own unique following f such that k ~ mf, or a combination of both. figures 2a and 2b illustrate a decreasing frequency (on a logarithmic scale) of viewers with increasing k. majority of the viewers are able to watch only a few hub movies in each month. the rate at which p(k) drops off gives us a picture of the relative homogeneity of the network. a sharper drop-off reflects a less diverse set of viewer interest. that is, there is a high concentration of viewers watching the hub movies (relatively low k). the range of k reflects not just the increasing population, but also the monthly dynamics and diversity of interests. months which are known to have more holidays (and, consequently, downtime to allow movie viewing) have a larger range for k. by taking the average degree distribution <k> on a monthly basis (fig. 2d) and comparing it with the viewer population (fig. 1a), we note that <k> is correlated with the population size, but is not the only determinant of its value. 40 science diliman figure 2. degree distribution of monthly aggregated ratings network for year a) 2000 and b) 2001. (c) degree distribution of rating networks for the month of december 2001. (d) mean degree of the monthly aggregated rating networks of 20002001. tracking the dynamic variations in a social network figure 3. (a) clustering coefficient for each rating network per month from 2000-2001. (b) clustering spectrum of rating network 5 for the month of january 2000. clustering coefficient and the average shortest path within the uncertainty bounds, the average clustering coefficient <c> remains constant with time and ratings (fig. 3a). since seeing a common movie fully connects all the viewers, the value of <c> would be close to unity if all the viewers watched at least one popular movie each month. that <c> is within (0.6, 0.8) implies that a significant number of viewers saw non-hub movies, thus pulling down <c>. but since the movies being watched change with time, the nearly constant value of <c> for the monthly viewers networks imply that the underlying movie hierarchy of popularity (blockbusters to unknown) remain unchanged in time. furthermore, the prevailing dynamics is robust to the addition of new viewers. the clustering spectrum c(k) shows the average clustering coefficient of nodes with the same degree k (fig. 3b). in a network where there is a lack of correlations among nodes, the value of c(k) would remain constant with k (vazquez, pastor-satorras, and vespignani 2002). that c(k) decreases with k implies a correlation. the large variance for small k implies a large variation in the structure of viewers with small k, some belong solely to hub movie networks (c = 1.0) while others watch rarelyviewed movies along with the hub movie. on the other hand, the drop in <c> coupled with a small variance at large k implies the existence of a few viewers borrowing an expanded list of titles that make them the links between many small clusters. such borrowers represent either multiple viewers borrowing under the same account, or viewers with such a wide range of interest that would make them poor predictors for a recommendation engine. figure 4. mean shortest path per month for all rating networks for year 2000. science diliman 41 pedemonte, lim figure 5. (a) estimating the maximum degree kmax = 0.56n – 63 (r2 = 0.85) as a function of the number of nodes n, (b) weight distribution of the edges, (c) movie viewers distribution, and (d) probability that a movie is connected to a blockbuster movie as a function of view count (p ≈ 1 for more than 100 views). the constant trend in the average shortest path length (fig. 4) confirms the constancy of the underlying hierarchical movie structure; there will always be movies (not necessarily the same movie) which persistently draw the majority of viewers at any given time. thus, nodes are reachable in small hops, leading to the 'small-world' effect in movie viewership. it is telling, though, that viewers whorate a movie highly have a closer affinity to each other (slightly shorter <l>) than those who commonly hate a movie (rating = 1). that <l> is, within the error bounds, constant confirms the notion that negative ratings are just as good as positive ratings in determining shared interest. extremal behavior, weights and small-world effect using the five ratings network per month for the 24month period (total of 120 data points), we determined the maximum number of connections a particular monthly rating network can have given its number of nodes. this allows us to estimate the largest possible connection with the recruitment of more viewers. the maximum value of k grows linearly with the number of nodes n; the coefficient 0.56 implies that the viewer with the widest interest is able to share the interest of 56% of the entire network. from the perspective of advertising, such a viewer would be easy to target. such a linear response is a signature of random networks, 42 science diliman tracking the dynamic variations in a social network implying that there is no change in the prevailing dynamics of the network; the topological structure is largely the same even with more viewers. the 'small world' effect is a characteristic of random networks, yet the clustering spectrum in fig. 3b points to an underlying correlation. we calculated a proxy for the “shared interest” between all pairs of viewers by taking the number of common movies for which each pair gave exactly the same ratings for the entire two-year dataset, and plotted the histogram in fig. 5b. the histogram plotted on a log-log scale, spanning two orders of magnitude on the “shared interest” axis, shows a steep (and monotonic) decline in the number of viewer pairs with a “shared interest”; there is no characteristic value for “shared interest” which reflects a truly heterogenous population. finally, we looked into the variety of movies present in the netflix database and showed the histogram of monthly movie viewership in fig. 5c. most movies in the netflix database are not known to many (low view count) and a few are considered blockbusters (which we operationally defined as the movie having the top view count in each month, fig. 1c). figure 5d shows the likelihood that a viewer of a movie with the corresponding view count will also see a blockbuster movie. the probability p is described by a sigmoidal function, p = tanh(0.08 view count), which is indicative of a sharp (threshold) response. though a movie may be viewed a mere 23 times (roughly 3% relative popularity with respect to the top draw), 19 out of 20 of those viewers (p = 0.95) would have also seen a blockbuster movie, illustrating the role of hub (or blockbuster) movies in linking diverse viewer interests. conclusion the diversity of interests of the movie-watching population effectively creates a social network with a nearly constant high clustering coefficient (0.60 – 0.85), and a low average path length (1.4 – 2.3) when analyzed on a monthly basis. even if the movies that link the viewers change rapidly from month to month, the underlying network structure remains unchanged and promotes a view that a hidden shared interest persists between viewers. the detection of this community structure would aid in targeted advertisements and messages, and can be effective in minimizing the cost of information dissemination. acknowledgements this work received financial support from the university of the philippines office of the vice chancellor for research and development through project no. 080805pnse. references albert, r., barabasi, a.l. 2002. statistical mechanics of complex networks. reviews of modern physics 74(1): 47. doi:10.1103/ revmodphys.74.47. amblard, f., deffuant, g. 2004. the role of network topology on extremism propagation with the relative agreement opinion dynamics. physica a: statistical mechanics and its applications 343:725-738. doi:10.1016/j.physa.2004.06.102. barrat, a., barthélemy, m., vespignani, a. 2008. dynamical processes on complex networks. 1st ed. cambridge university press. braha, d., bar-yam, y. 2006. from centrality to temporary fame: dynamic centrality in complex networks. complexity 12(2): 59-63. kossinets, g., watts, d. 2006. empirical analysis of an evolving social network. science 311(5757): 88-90. doi:10.1126/science.1116869. mossa, s., barthelemy, m., stanley, h.e., nunes amaral, l.a. 2002. truncation of power law behavior in "scalefree" network models due to information filtering. physical review letters 88(13): 138701. doi:10.1103/physrevlett.88.138701. motter, a.e., lai, y.c. 2002. cascade-based attacks on complex networks. physical review e 66(6): 065102. doi:10.1103/physreve.66.065102. netflix. netflix prize: home. http://netflixprize.com/. ———. 2006. uci machine learning repository: netflix prize data set. http://archive.ics.uci.edu/ml/ datasets/netflix+prize. pastor-satorras, r., vespignani, a. 2001. epidemic spreading in scale-free networks. physical review letters 86(14): 3200. doi:10.1103/ physrevlett.86.3200. vazquez, a., pastor-satorras, r., vespignani, a. 2002. large-scale topological and dynamical properties of the internet. physical review e 65(6): 066130. doi:10.1103/physreve.65.066130. science diliman 43 what does it take-.pmd what does it take to finish your graduate degree? 1science diliman (january-june 2011) 23:1, 1-7 introduction the percentage of a country’s population having advanced degrees of learning, particularly in science and engineering, is often equated with the country’s ability to sustain technological development and economic prosperity (huang et al., 2009, cisco, 2007). this as opposed to developing conventional sources of production, i.e. capital and labor (pakes and sokoloff, 1996). the philippines has long recognized the s&t factor. it has embarked on several programs in the past to improve the capability of degree-granting institutions to provide graduate programs (faculty development, research and equipment grants). numerous scholarships were also made available through the department of science and technology (dost, nsta, esep, dost-sei, bcda fund, etc.) to encourage students to pursue their master’s and doctorate studies. partnerships (e.g., national science consortium, moas with foreign universities, sucs, etc.) were built between institutions to encourage research collaboration and sharing of facilities. together with these programs, it can be argued that attention should also be given to increasing the productivity of current researchers (lacanilao, unpublished report). ultimately, these strategies are aimed to increase the philippines’ ~7,500 s&t workforce and their output. the university of the philippines diliman college of science (cs) has recognized one other problem in our quest to produce more ms and phds: the low rate of graduation among our graduate students. cs reported that for the period 2000-2008, a success rate of 50% and 34% is computed for phd and ms students for finishing their degrees, respectively. however, table 1 shows a more dismal picture. cs produces an average what does it take to finish your graduate degree?: issues, challenges and recommendations to the up college of science graduate program carlos primo david national institute of geological sciences college of science, university of the philippines diliman table 1. cs student record data for 2000-2010 source: cs graduate office david, c.p. 2 of 13 phd and 42 ms graduates each year out of 177 and 559 students, respectively. statistics from 54 u.s. and canadian universities suggest a higher phd success rate of 55-64% for the physical and life sciences (council of graduate schools, unpublished report). an earlier study of ten prominent u.s. universities reported a 58% success rate in their phd programs (nerad and cerny, 1991). there are no definitive studies for european universities but a 2230% attrition rate for phd students is what is often quoted (park, 2005). there are also no statistics on ms students’ success rate but most likely this is higher than that of the phd programs. science diliman (january-june 2011) 23:1, 1-7 figures 1a and 1b. data on actual number of years that students take to finish their degrees (average for 2000-2008) (cs graduate office data). 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 m s  p h ys ic s m s  m et eo ro lo gy m s  m se m s  g eo lo gy m s  m a th em a ti cs m s  m a ri n e  sc ie n ce m s  c h em is tr y m s  m b b m s  a p p li ed   m a th m s  b io lo gy m s  en vi  s ci en ce m s  m ic ro b io lo gy 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 p h d  p h ys ic s p h d   m a th e m at ic s p h d  m b b p h d  m a ri n e  sc ie n ce p h d  c h e m is tr y p h d   m e te o ro lo gy p h d  e n vi  s ci en ce p h d  b io lo gy what does it take to finish your graduate degree? 3science diliman (january-june 2011) 23:1, 1-7 the time it takes for students to finish their degree in the college of science is also a recognized problem. most u.k. universities report a phd completion time of 3.5-4 years (swain, 2008) and is most likely the same with the rest of europe, australia and japan. the u.s. average is 6.7 years for physical sciences and 7.0 for life sciences (national research council, 1995). however, one reason for this is that the first two years of the u.s. phd program is entirely devoted to course work. therefore, the dissertation research proper itself is 4-5 years. again, no data is available for masteral programs but the norm for international universities would be 1-3 years. in cs, students complete their phd program anywhere between 5-10 years. graduate students finish their ms degrees in 3.5-7 years (figure 1a and 1b). several studies have been undertaken in the u.s. to study the attrition rate and length of graduate programs (nsf, 2005). it should be noted that attrition per se in a graduate program may not automatically be viewed as a negative trait. examples cited in the nsf study include students who transfer programs, students who gain employment during his/her graduate training and other reasons that increased the productivity of an individual despite not completing his/her studies. therefore, what is important is to look into specific reasons or factors (herein called attrition/delay factors) why graduate students are not able to finish and/or take a long time to finish. the main objective of the present study is to uncover institutional, programmatic and personal factors that represent hurdles in successfully finishing a graduate degree. a questionnaire for cs faculty and graduate students was distributed in 2010. a total of 83 respondents (faculty and students) participated in the survey. the questionnaire contained inquiries on the respondent’s details (institute/department, years of teaching/years of graduate studies) and four questions: • what do you think are the main reasons why it takes graduate students more than the prescribed number of years (ms 2 yrs, phd 4yrs) to graduate? • how do you define an ms thesis? • how do you define a phd dissertation? what’s different from an ms thesis? • can we use any quantifiable indicator to know when a student has finished an ms thesis/phd dissertation? two of the questions explore one previously identified attrition/delay factor which is how the faculty as adviser, reader or thesis examiner gauges the quality of student research. to complement the survey, interviews were conducted with faculty members from several cs institutes who have consistently guided students in finishing their degrees. results and discussion all answers (coming from both faculty and students) to the first question pertaining to the delay factor are combined and summarized below. it is recognized that these factors are overlapping and interrelated; however, discussions will be separated for convenience into the following headings: student, adviser, field of study and system factors. student factor • poor focus among students • social obligations of students are prioritized over their studies • graduate students are often employed full-time • university-employed students (as research assistants) often do research for their supervisors that is not part of their thesis research • students coming from other schools and/or from an undergraduate course that is different from their graduate course will take a longer time to finish (need for more coursework and longer training) most faculty respondents noted that the main reason for attrition/delay is that graduate studies are not the main priority of students. students have other commitments (full-time jobs, family and other social obligations) which hinder them from taking more course work each semester and starting/sustaining their research. even those employed by the university also argue that their workload prohibits them from focusing david, c.p. 4 on their graduate research. this is one glaring difference with major universities abroad wherein there are more full-time students than part-time students. one other factor that was cited by respondents is the lack of preparedness of incoming students to conduct research. in particular, nip respondents cite the generally faster finishing time for physics students who did their undergrad in up because more often than not, these students have already been exposed to research groups (and may have even started their graduate research) during their undergraduate years. this as opposed to students coming from different bs programs or from other universities and therefore more training is required. adviser factor • mentoring skills are not present • considers advising too much workload with little reward and therefore tends to limit either the number of students or the amount of time they devote to students • retains students for an extend period of time to have people work on their research/consultancy work • sets research standards too high arnold et al. (1986) in their survey on graduate student attrition in the u.s. cites as the primary source of dissatisfaction expressed by students is their perception that the faculty was not approachable. this is a genuine concern of students but obviously being “unapproachable” needs to be further defined. graduate research can be regarded as a partnership between student and professor. this relationship can vary from having the student work on a specific topic as his thesis within the professor’s current research (much like having an employee-employer relationship) to a totally new topic but within the general scope of the professor’s field of expertise (equal partnership or independent research with some guidance). the type of relationship leads to how much involvement the faculty will have in the student’s research activities. however, what should be common in the relationship (regardless of type or extent) should be a set of responsibilities that determine expectations of both parties. this includes regular meetings or consultations and a time-bound schedule that each party should follow. mentoring students in u.p. is also regarded as an arduous task with very little benefit to the faculty. the current cs merit promotion system only puts 15 points per phd (8 pts for ms) student graduated. in comparison, an isi publication is given 90 pts. one promotional step is about 80 pts. cs is trying to correct this by giving more weight to successful mentorship (1 full step per phd mentored; ¼ step per ms mentored) in the latest proposed cs merit system. students employed as university research assistants (uras) enjoy the full benefit of working in the lab where they will also be doing the analysis for their own work. it also provides an environment that makes daily interaction with their adviser possible. two respondents believe that a conflict of interest may exist in that a professor/supervisor would tend to retain good uras in their lab to do other research/consultancy work at the expense of these researchers not finishing their degrees on time. lastly, a common response of faculty and students is that the standards set for an ms thesis or a phd dissertation is often set too high that finishing within the prescribed period is almost impossible. both faculty and student realize that both can be guilty of setting out a target research output that is too much. moreover, the final research scope and depth are determined by a panel during the thesis proposal defense. in all stages of research formulation, the qualitative evaluation of a study’s merit as to whether it is “enough” for an ms or phd research can be very arbitrary. on the other end of the spectrum, this uncertainty can also lead to phd dissertations which can be doable within four years but may be lacking in substance. the solution to this dilemma is to set out clear guidelines on research scope and depth. this is discussed in the next section. field of study factor • no facilities to use • no funding • discipline-specific attrition/delay factors science diliman (january-june 2011) 23:1, 1-7 what does it take to finish your graduate degree? 5 in general, advanced research in physical and life sciences will require sophisticated analytical instrumentation which may not be available to students for various reasons. this needs to be corrected at the stage of research formulation wherein the procedures and techniques to be considered are only those that available facilities can perform. a related issue to this is funding and that the scope of a study must somehow be limited with the available budget. moreover, while many respondents cited that funding is a major factor in completing their graduate research, surprisingly, funding is not an issue across all cs institutes. each discipline cited an attrition/delay factor that is specific to its own field of study. test organisms in the life sciences include culture time, organism mortality and other biological processes that make research run longer. laboratory-based research is also thought to run longer when results are contrary to what was initially expected. setting up of new runs and repeated calibrations contribute to delays. lastly, field-based studies take a long time as these are seasonally scheduled. some sampling programs also need to be conducted over two to three years. system factor • no clear guiding system for students (no advising during early years, no progress tracking system for latter years) • maximum residency rule (mrr) is not enforced • change in thesis/dissertation adviser and/or topic • difficult process/requirement in the program • scope of work in ms or phd thesis is poorly defined system factors are attrition/delay causes due to the lack of institutional rules in place. this includes the absence of a time-bound guiding system for students. under this is the practice of assigning a program adviser for first and second year students. a program adviser is tasked to only advise the student on what courses to take, effectively allowing the student to postpone any research work that could have been started in his first year. the practice of defending a research proposal after data gathering has been done also contributes to delays. graduate students wait till their 3rd or 4th year before defending their proposal. in msi, proposal defense is apparently one of the most difficult requirements in the program such that students wait until their 4th year to do this. time-bound deliverables should be adhered to (e.g. proposal defense on the student’s 2nd year). a guiding system in place should also effectively match students with advisers to decrease the possibility of changing topics and mentors midstream. lastly, as what was mentioned in the preceding discussions, the scope of an ms or phd research should be properly defined; if possible not by standard institute guidelines. recommendations three recommendations are herein forwarded: 1. create a mentoring program 2. level-off on the scope of a masteral thesis/doctorate dissertation 3. provide indicators of “being done” mentoring program a mentoring program starts with the preparation of students even before they enter a graduate program. the practice of nip in immersing their 4th and 5th year undergraduates definitely helps the students focus on their research interests early on. this should be adopted by other institutes. for institutes without the luxury of having an undergraduate program, it is proposed that lab immersion for students be done during the summer prior to their official entry to the program. the key is to have students start their research as soon as possible. more importantly, the institutes’ policy for accepting students should be revisited. it is recommended that cs units only allow entry of students with an accepting research adviser (as what is common practice in international universities). each unit must then set a minimum (and maximum) number of students being advised by each faculty at any given time. the process of accepting students would then include meetings with potential advisers before june. lastly, this requirement also means that students must have a clear idea of at least a field of study to be able to be assigned to an adviser. science diliman (january-june 2011) 23:1, 1-7 david, c.p. 6 second, every institute must include a student tracking system wherein semestral milestones are established. this is regularly checked by the unit and that students/ advisers are warned for not complying with time-bound requirements. this includes the adherence to mrr rules. training through workshops and instituted cs classes are recommended. cs can develop a workshop on mentoring for new cs faculty. for graduate students, it is proposed to have a cs-wide course on graduate research guidelines, research techniques, oral and writing skills. this will also serve as an opportunity for students to meet other students and faculty across cs which may lead to collaborations and interdisciplinary studies later on. scope and depth of research defining what is “enough” for a masteral or doctorate research is extremely difficult. the up faculty manual (2003) defines a masteral thesis as “original and significant research or creative work”, while a doctoral dissertation is closely defined as “original, significant, independent scientific research or creative work”. note that the word “independent” is the only difference between the two definitions. furthermore, according to the faculty manual both ms and phd research should also: 1. show the student’s capacity to make a critical evaluation of previous work done in his/her chosen research topic; and, 2. demonstrate his/her ability to present research findings in a clear, systematic, and scholarly manner. obviously, this definition does not help us much in defining significant research. a rubric in defining the components and their scope and depth is given in table 3. this is drafted to apply to all natural/applied sciences but each institute may further refine this to cater to their own specific fields. what this rubric aims to do is to standardize our evaluation of research significance. it can be used as a guide during research formulation and during the thesis/dissertation proposal defense. provide indicators of “being done” all research studies conducted appropriately should inevitably lead to more questions that need to be answered and provide insights on where new findings put forth can be applied to. this very nature of scientific research makes it a continuing process. therefore, it is important to set indicators of when a research done as an ms or phd requirement can be stopped and succeeding investigations are considered beyond the scope of the degree to be granted. in many international universities, the main indicator of “being done” is tied up to the publication of a student’s work. publication through a refereed evaluation process is a tangible indicator of a research that has attained some level of significance. it is therefore proposed to institute such milestone as a prerequisite to a student’s graduation (table 3). a publication requirement will not only fix a tangible goal for students/advisers but also provide some form of quality assurance to the research we produce. this will also partly solve the earlier argument of increasing our s&t productivity. lastly, this should also encourage more mentoring because any publication done by an adviser’s student will likewise be credited as part of his own research output. complimentary to the ms publication requirement should be the strengthening of university-based (e.g. science diliman) and national scientific publications. efforts to increase the number of issues and an efficient review process (within 6 weeks of article receipt) should make local journals an attractive publication alternative. acknowledgment this research could not have been done without the help of then dean caesar saloma, ms. gigi of the cs graduate office and all those who participated in the faculty interviews and student/faculty survey. science diliman (january-june 2011) 23:1, 1-7 what does it take to finish your graduate degree? 7science diliman (january-june 2011) 23:1, 1-7 references: arnold, l., mares, k. and calkins, e.a. 1986 exit interviews reveal why students leave a ba-md degree program prematurely. college and university 62:34-47. association of american universities (aau). institutional policies to improve doctoral education, a policy statement of the association of american universities and the association of graduate schools in the aau (washington, dc 1990) cisco publication. education and economic growth: from the 19th to the 21st century. 2007. college of science national science complex report. c. saloma (quezon city 2010) national science foundation, division of science resources studies, summary of workshop on graduate student attrition, nsf 99-314, project officer, alan i. rapoport (arlington, va 1998). nerad, m. and cerny, j. 1991. from facts to action: expanding the education role of the graduate division. in increasing graduate student retention and degree attainment, leonard l baird (ed) new directions for institutional research no 80, winter 1993, jossey-bass. huang, f., jin, l., sun, x. 2009. relationship between scale of higher education and economic growth in china asian social science 5, 55-60. pakes, a. and sokoloff, k. 1996. science, technology and economic growth. proceedings of the national academy of sciences 93, 12655-12657. park, c. 2005. new variant phd: the changing nature of the doctorate in the uk. journal of higher education policy and management 27, 189-207. up faculty manual. 2003. table 2. matrix of proposed graduate research scope and depth table 3. proposed presentation and publication requirement ocr document inside front cover-23-1.pmd editorial board editors-in-chief marco nemesio e. montaño, ph.d. maricor n. soriano, ph.d. associate editors jose maria p. balmaceda, ph.d. mathematics zubaida u. basiao, ph.d. biology joel joseph s. marciano, jr., ph.d. computer science, engineering irene m. villaseñor, ph.d. chemistry violeda a. umali, ph.d. director, rduo dercylis g. mararac editorial assistant editorial advisors rigoberto c. advincula, phd. dept. of chemistry university of houston radvincula@uh.edu alfonso m. albano, phd. dept. of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu emeritus professor kenneth buckle food science and technology group school of chemical engineering chemical sciences building (room 811) the university of new south wales unsw, sydney nsw 2052, australia k.buckle@unsw.edu.au jose b. cruz, phd. dept. of electrical and computer engineering ohio state university cruz@ece.osu.edu. dr. flor crisanta f. galvez quality assurance & technical manager kerry ingredients and flavours (americas region) 7989 82nd st. delta, bc v4g 1l7 canada ffgalvez1@yahoo.com victor c. gavino, phd. dept. of nutrition university of montreal, canada victor.gavino@umontreal.ca science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p300.00/year (two issues), exclusive of postage us$ 25.00/year (two issues), exclusive of postage tel. no: (632) 981-85-00 loc. 4048 (632) 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences cover photo “finite element modeling of a landslide box experiment showing predicted conditions within a slope during failure. finite element mesh (top left), deformed mesh and displacements (bottom left), shear strain profile (top right) and pore pressures (bottom right). figure was taken from catane et al. p. 17-30 of this issue.” contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. kelvin s. rodolfo, phd. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu rudolf a. roemer, phd. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk luis g. sison, phd. electrical and electronics engineering institute university of the philippines, diliman luis.sison@up.edu.ph raul k. suarez, phd. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu milkfish-art.8 milkfish (chanos chanos) fry concession system 59 introduction milkfish (bangus) is an important domestic foodfish and aquaculture commodity in the philippines. among the foodfish, milkfish ranks third in demand among local science diliman (july december 2000) 12:2, 59-66 abstract milkfish (chanos chanos) fry concession system in bolinao, pangasinan: implications to coastal resources management severino g. salmo iii, marie antonette r. juinio-meñez, and porfirio m. aliño marine science institute, college of science university of the philippines, diliman, quezon city 1101 philippines tel. no.: (632) 922-3921; (632) 920-5301 to 99 loc. 7428; telfax no.: (632) 924-7678; e-mail: jon@upmsi.ph the ecological and socioeconomic implications of the concession system on milkfish (chanos chanos forssk.) fry in bolinao, pangasinan were evaluated from 1996 to 1999. monitoring of landed catch from 1996 to 1998 showed that the seasonal trend and annual volume of catch varied widely during the threeyear period. the fry season in 1996 and 1997 lasted seven months, starting from the second week of april to the second week of october. however, during the 1998 season, fry were available for eight months starting in the second week of march and ending in november. the peak period also varied considerably during the three-year period. in 1996, peak abundance of fry was observed in the last week of july while in 1997 and 1998, the peak was during the second week of may. the volume of total catch for the entire season also varied widely, from as low as ~400,000 fry (1997) to as high as 2,400,000 fry (1996). the concessionaire “postor” has the sole right to buy all fry caught within the municipal waters. thus, s/he dictates the buying price. moreover, the existing concession system has no mechanism to regulate harvest of milkfish fry gathering. this arrangement allows the concessionaire to enjoy huge economic benefits while the fry gatherers only get a minimal share in the income. to promote sustainable and equitable harvest of milkfish fry, a new access arrangement through a permit system was proposed by the fry gatherers. the proposed permit system will promote a sustainable harvest of milkfish fry through the implementation of a closed period during the fry season. compared to the present concession system, the permit system is believed to be more equitable because of the abolition of the 1/3 cut levied by the concessionaire on the landed catch. the permit system also facilitates a mechanism that provides for transparency on the selling/buying price. more importantly, fry gatherers will have the opportunity to sell to buyers offering a relatively higher buying price. in addition, fry gatherers may also opt to grow out milkfish fry to fingerlings which may potentially give them higher economic returns for their catch. keywords: milkfish fry, concession system, economic benefits, sustainable/equitable resource use, permit system consumers after roundscad and tuna. several economic activities are related to the milkfish culture which include: fry gathering, hatchery, and nursery operations, processing, marketing, and other services, such as ice making and fish transport (guerrero 1981). although salmo iii et al. 60 reforms in the concession system. unfortunately, the designated milkfish fry reservation is not strategic because the area is not within the primary milkfish fry gathering area (fig. 1). effectively, the only real management scheme employed by the municipal government is the concession system. under this concession system, the concessionaire always aims to attain maximum harvest, thus, resource extraction is intensified with less regard for harvest controls. more importantly, the present system is perceived to be inequitable as the concessionaire always has the “lion’s share” in terms of economic returns. because of this situation, an alternative management scheme was proposed by the milkfish fry gatherers in order to promote sustainable harvest and equitable sharing of benefits from milkfish fry activity. bolinao 16.45 16.40 16.35 16.30 16.25 16.20 119.80 119.85 119.90 119.95 120.00 120.05 patar ilog malino estanza balingasay arnedo binabalia binabalia victory pilar fig. 1. map of bolinao showing the milkfish fry ground and the milkfish fry reservation area (inset: map of lingayen gulf commercial production of milkfish was introduced more than a century ago, significant growth of the industry was realized only in the last four decades. however, the industry is now declining, mainly as a result of the dwindling supply of milkfish fry. contributory to this decline is the destruction of natural habitats brought about by the extensive conversion of mangrove areas to fishponds, destructive fishing methods, and environmental quality degradation (e.g., high pesticide load), among others (villacorta 1994). while imported and local commercially produced milkfish fry are available, growers generally prefer wild fry over hatched fry (garcia 1997). thus, sustainable harvest of wild fry remains a critical issue in ensuring the sustainable development of the milkfish industry. in bolinao, pangasinan, the major milkfish fry gathering area is along the coastline of barangay balingasay (fig. 1). five other neighboring barangays and some parts of santiago island are also benefiting from the milkfish fry gathering industry. compared to other barangays, gatherers from balingasay are more dependent on the milkfish fry resource. almost 60% of the barangay is economically dependent on the fry gathering activity, especially during the peak season from april to july. the abundance of milkfish fry in the area may be attributed to the presence of an ecologically rich estuarine ecosystem that provides adequate riverine nutrient-rich inputs and relatively calm entrainment features that protect the fry ground from strong wave actions and current flushing (bagarinao 1984). as a traditional fishing industry, the municipal government managed the milkfish fry through: (1) the delineation of a milkfish fry reservation area (municipal ordinance #1, s1988); and (2) the implementation of milkfish (chanos chanos) fry concession system 61 the objectives of this study are: (1) to evaluate the ecological and socioeconomic implications of the milkfish fry concession system in bolinao; and (2) to evaluate the proposed alternative management scheme in terms of how it promotes sustainable utilization and equitable sharing of the milkfish fry harvest. methodology the study was conducted over a four-year period (19961999) in brgy. balingasay, the most significant source of milkfish fry in bolinao. parameters analyzed in this case study were evaluated based on sustainability, equitability, and legal-institutional considerations of the milkfish fry concession system. for sustainability, data on total landed catch were gathered from the concessionaires during the respective periods, e.g., sammabal (samahan ng mga mangingisda at mamamayan ng balingasay, inc.), as co-concessionaire in 1996, and babfga (balingasay bangus fry gatherers association) in 1997 and 1998. for equitability, data on the number of milkfish fry gatherers and beneficiaries, and buying and selling prices were derived from the records of the concessionaires. pertinent socioeconomic parameters (e.g., average catch, fishing gear use) were gathered through interviews and personal records in brgy. balingasay where the number of fry gatherers was highest. meanwhile, legal-institutional analyses were conducted by reviewing existing municipal policies/ordinances and relevant national laws. the evaluation of the proposed alternative management scheme was based on the minutes of the consultation meeting. comparative evaluation based on potential impact on sustainability of the resource and equitability of returns using projected income analysis was undertaken. results and discussion legal-institutional considerations there is no historical record as to when the milkfish fry concession system started in the municipality of bolinao or the entire province of pangasinan. however, according to some officials of the department of agriculture-bureau of fisheries and aquatic resources (da-bfar) in region i, significant harvesting of milkfish fry in the ilocos region was initiated in the 1920s. in the philippines, there were reports that milkfish culture started in the late 1920s (herre and mendoza, 1929) and intensified in the early 1930s (adams and others 1932). meanwhile, there were local anecdotal reports that massive conversion of mangrove forest into fishpond (primarily for milkfish culture) started in the 1960s. the national government’s attempts to manage milkfish fry are reflected in national laws and policies passed (smith and panayotou, 1984). the first national law on fisheries was r.a. 4003 or the fisheries decree of 1932 which provided for the allocation and designation of specific areas for exclusive uses (i.e., erecting fish corrals and oyster beds, operating fish ponds, catching of milkfish fry, etc.). this was later reinforced by the enactment of p.d. 704 or the fisheries code of 1975 which delineated the extent of municipal waters (i.e., 10 km. from the shoreline) and gave the municipality the power to award fishery privileges in the form of grants or leases. in 1991, r.a. 7160 or the local government code was enacted. this code devolved certain functions to coastal municipalities and legally empowered them to regulate resource-use activities, such as milkfish fry gathering within the municipal waters (pimentel 1993). the law provided that fry grounds be subjected to a fishery privilege to an individual or a group, i.e., the right of first purchase of all gathered fry. the difference between p.d. 704 and r.a. 7160 is that the latter specified that coastal municipalities may give preferential rights to marginal fisher’s groups in the granting of fishery privileges. recently, the passage of r.a. 8550 or the philippine fisheries code of 1998 further strengthened r.a. 7160 by granting the municipal government the power to delineates zone of specific uses. it also recognized the direct role of marginal fisher’s groups in fishery privileges. the process of granting the concession system in bolinao is interesting. legally, the municipal council (sangguniang bayan), as the legislative branch, should have the authority to grant the concession (sec. 149b, r.a. 7160). the sangguniang bayan mandated a committee to take charge of the granting of salmo iii et al. 62 concessions. the committee was composed of a representative of the mayor, three members from the sangguniang bayan, and the municipal treasurer. the process of granting the concession would start with the posting of a notice on the bulletin board of the municipal hall and/or in other strategic places for a period of two weeks, usually during the third week of october. the notice would indicate the date and time when the bids could be filed with the municipal treasurer. at the time and place designated, the committee would open all bids (usually during the first week of november). usually, all bidders would present their cash upon stating their bid. the committee would then award the lease to the highest bidder. upon being granted the rights to the concession, the winning bidder would pay the total amount of the bid, which represented a one-year rent, from january 1 to december 31 (rodriguez 1997). it has been a policy that the starting bidding price should at least be the same as the winning bid of the previous year. with this system, fry gatherers observed that only the influential and the elite in the municipality could acquire the concession. the highest bidding price was recorded in 1995 at p400,000.00. without amending the municipal policies, the usual process of granting the concession system (e.g., bidding) was not followed since 1996 and was replaced by the granting of a negotiated contract. in a negotiated contract, the municipal government may directly award the concession to an individual or an organization. in the past three years, the municipal government opted to award the concession to people’s organizations. it was perceived that such development was made because of the decrease in the number of participants in the bidding, which was attributed to the annually increasing concession fee. it is also believed that the intervention was made in consonance with the preferential right of fisher’s organizations as provided for by sec. 149b, r.a. 7160. since then, the awarding of the concession system was based on the discretion of the municipal government. however, it was observed that only those organizations closely connected to the municipal government had access to the concession. for instance, sammabal and babfga who were known to have close connections to the municipal government acquired the concession, i.e., sammabal as co-concessionaire in 1996 and babfga as concessionaire in 1997 and 1998. meanwhile, except for the granting process, other procedures traditionally imposed by the concessionaire were maintained, such as the 1/3 cut on catch. sustainability the total landed catch of milkfish fry is presented in fig. 2. there were wide intraand inter-annual variation in landed catch in the area during the fry gathering season. fry gathering usually lasted for seven months, from the second week of april until the second week of october (1996 and 1997 seasons). however, in 1998, fry gathering lasted for eight months, from the third week of march until the second week of november. volume of the total landed catch varied from ~400,000 fry in 1997 to 2.6 and 2.2 million fry in the 1996 and 1998 seasons, respectively. peak period also varied annually during the three seasons, from the second week of may in 1997 and 1998 to the last week of july in 1996. the seasonality of milkfish fry was believed to be influenced by the following: ebb period or tidal advection, freshwater inputs from adjacent riverine systems, onset of the rainy season, and direction of water current (bagarinao 1984). a plot of the tidal range and total landed catch during the 1996 season (fig. 3) suggests that the period of highest tidal advection generally coincids with the period of highest recorded landed catch of milkfish fry. while there were still amounts of fry caught in the latter part of the season, these did not contribute as much to the income of the fry gatherers becuase of the low buying price imposed by the concessionaire. 1200 1000 800 600 400 200 j f m a m j j a s o m d v ol um e of f ry c au gh t (1 x 1 00 0) fig. 2. total landed catch of milkfish fry (1996-1998) in balingasay, bolinao, pangasinan 1996 1997 1998 milkfish (chanos chanos) fry concession system 63 institutional arrangements and equitability considerations the concessionaire took full control of the operation and management of the concession. neither the fry gatherers nor the municipal government had the right to oppose any regulation imposed by the concessionaire. the concessionaire allowed the fry gatherers to use the fry ground on the condition that all catches were sold to the concessionaire. the concessionaire also had control over the price of the fry. catches were presented to the concessionaire who issued payments for the catch. the sharing arrangement imposed was that, from the total catch, 1/3 would be deducted as a concessionaire’s fee. an additional deduction of 5% of the total catch was imposed as mortality rate charge. the concessionaire then paid the fry gatherer the value of the net catch. the concessionaire, on the other hand, sold the milkfish fry to buyers outside bolinao at a relatively higher price. this system primarily benefited the concessionaire who got the “lion’s share” and who aimed for higher economic profits. furthermore, the system did not have a provision on management measures and since the concessionaire would always want to increase income, resource extraction was further intensified. the milkfish fry gatherers engaged in fry gathering and its relationship with the buying price during the 1996 season is presented in fig. 4. fry gatherers were abundant during the start of fry gathering period even if supply of milkfish fry was still low and progressively decreased towards the end of the season. conversely, fry gatherers were fewer even when the supply of milkfish fry was high, e.g., third week of july. such inverse relationship was affected by the buying price imposed by the concessionaire. buying price was usually set at p1.00/fry at the start of the season and decreased to p0.70 and p0.50/fry during the middle of the season until it stabilized at p0.40/fry towards the end of the season. according to the concessionaire, the selling price was usually p0.20/fry higher than the imposed buying price. however, in reality, the selling price of the concessionaire reached p0.50/fry (i.e., p1500.00/ 1000 fry), which is much higher than the buying price especially during march up to the second week of may. the source of income for the fry gatherers was derived only from daily landed catch paid for by the concessionaire while the concessionaire derived his/ her income from the mark up price (selling price minus buying price), the imposed 1/3 cut on landed catch, and 5% mortality rate. thus for every thousand fry, the fry gatherer received p633.00 while the concessionaire earned p1,741.00, broken down as p1,425.00 (derived from the selling price) and p316.00 (from the 1/3 cut on landed catch). the proposed alternative management scheme – permit system for a long time, fry gatherers have been clamoring for the elimination of the concession system of the municipal government. unfortunately, the request has not been granted because the municipal government has needed to generate income from the bangus fry resource derived from the concession fee paid in december of each year. the concession fee was used by the municipal government as payment for christmas bonuses of 1.2 1 0.8 0.6 0.4 0.2 j f m a m j j a s o n d t id al r an ge (m .) fig. 3. relationship of tidal range and total landed catch (log) during the 1996 season in balingasay, bolinao, pangasinan 0 m ilk fis h fr y ca tc h, lo g f ry g at he re r b uy in g pr ic e (p ) fig. 4. the number of fry gatherers and buying price during the 1996 fry gathering season 110 90 70 50 30 10 -10 j f m a m j j a s o n d 1.1 0.9 0.7 0.5 0.3 0.1 0.1 tide catch fry gatherer buying price salmo iii et al. 64 municipal employees. cognizant of the financial needs of the municipal government, the fry gatherers, through sammabal, proposed a scheme that would generate revenue equal to at least the concession fee. the proposed permit system (fig. 5) is an alternative management scheme based on individual permits intended to eliminate the concession system and promote sustainable harvest of milkfish fry. the proposed permit system involves the milkfish fry gatherers, accredited buyers, respective barangay councils, and the municipal government. the permit system differs from the present system in that only the concessionaire has the main role in the concession system (table 1). the income of the municipal government will be derived from the licenses and taxes paid for by the fry gatherers and buyers amounting to p330,000.00 (table 2). in the present system, the municipal government derives its income from the concession fee paid by the concessionaire. the barangay council, which does not have a role in the present system, will have its share through the fishing permits paid by the fry gatherers (p35-50/gear/year). the barangay council will serve as the monitoring body that will oversee the implementation of open/closed season and check the authenticity of recorded catch. although the municipal government may get a slightly lower income in the proposed system, if the income derived by the barangay councils (p50,000.00) will be considered as part of the municipal government’s income, then the total income that will be generated by the proposed permit system is higher than the current income derived from the present system. more importantly, the monopolistic system of the concessionaire will be diminished in the proposed permit system. authorized buyers will come from accredited fishers’ organizations who will manage small-scale buying stations where catch can be brought and sold. capitalization of a number of smaller buying stations will be more affordable to the local community. estimated aggregate gross income of the buyers was p1,815,274.93. among its expenses were accreditation, business permit, and taxes, thus, the estimated net income was computed at p371,667.26. meanwhile, the fry gatherers who were traditionally just involved in selling their catch to the concessionaire may have the opportunity to monitor the buying/selling price and look for buyers offering relatively higher buying prices. fry gatherers, even with the added expenses for licenses and permits, may have an increase in aggregate average income from p1,500.00 to p2,292.22 (range: p1,000p12,000.00) due to the abolition of the 1/3 cut on submitted catch (table 1). meanwhile, in order to help ensure the sustainable harvest of milkfish fry, the proposed system will include a provision on the closed season (october april 15). estimatormunicipal government fry gatherer r eg is tr at io n a cc re di ta tio n li ce ns e (p 50 /g at he re r brgy. council p 15-30/gatherer/year buyer b us in es s pe rm it (t ax : p 0 .5 /fr y) a cc re di ta tio n (p 50 00 /b uy er /y ea r tx: p0.05/fry fig. 5. the proposed permit system table 1. comparative analysis of benefits in the concession system and the proposed system stakeholder legal process/ requirement institution business relationship between stakeholder and or institution sector present system (concession) proposed system (permit) • municipal government • brgy. council • concessionaire • buyer • fry gatherer • p300,000 • none • p1.7 million • p300,000 average net income • p1,5000 average net income • p345,000 • p80,000 • none • p300,000 • average net income • p2292 • average net income options direct benefit indirect benefit • role of concessionaire eliminated • buying price not monopolize • equitable and sustainable harvest of milkfish fry • fry gatherer may look for buyers offering relatively higher price • milkfish fry grow-out as added value milkfish (chanos chanos) fry concession system 65 table 2. projected comparative income analysis between the concession system and the permit system summary and conclusion the milkfish fry concession system in bolinao, pangasinan has been implemented by the municipal government for a long time. an evaluation of the concession system showed that it threatens the sustainability of the milkfish fry supply. the concessionaire tends to exploit the resource to achieve high profits because of his concern that he might not be able to win the concession the following year. furthermore, the municipal government, after acquiring the concession fee from the concessionaire, does not exercise any control over the fry gathering operation and administration. thus, the present system lacks the assurance of fair buying price for the fry gatherers. in addition, the fry gatherers are always on the losing end due to the monopolistic pricing system and the sharing scheme imposed by the concessionaire. the proposed permit system promotes sustainable harvest and equitable sharing of milkfish fry. a closed season, from october to april 15, is included in the proposal to help ensure the successful return of a substantial number of milkfish fry in the ocean. this increases the probability of a greater number of fry becoming breeders (sabalo). equitable allocation of economic benefits among resource-users will also be realized with the abolition of the 1/3 cut on catch and assurance of the transparency of selling and buying prices. furthermore, the proposed system will also enjoin active participation of the concerned stakeholders (e.g., fry gatherers, barangay council) in implementing the closed season and validating the recorded catch. a. municipal government buyer’s permit=p5000/buyer * 5 buyers tax (p0.15/fry * 1.6 million fry license for fry gatherers (500 gatherers * p50/gatherer) permit to transport for buyers (p0.15/fry * at least 50% of 1.6 million fry) gross income expenses, i.e. estimator (0.5% of total catch) b. brgy. councils permit to gather (p15/year * 500 gatherers) expenses: none c. buyers (assumptions: no 1/3 cut on submitted catch and selling price is p0.4/fry higher than the buying price during the first month (march-may), p0.3/fry higher during the middle period (june-july) and constant at p0.1/fry higher towards the end of the season, e.g. september-october (selling price – buying price) gross expenses business permits (p5000/year * 5 buying stations) tax (p0.15/fry * 1.6 million fry) price paid for the fry gatherers total expenses net income (gross income – total expenses) d. fry gatherers paid buying price/gross expenses licenses (p35-50/year * 500 gatherers) permit to gather for brgy. council (p15/year * 500 gatherers) total expenses net income average income per fry gatherer (net income for 500 fry gatherer) income/expenses (p) 25,000.00 240,000.00 25,000.00 40,000.00 330,000.00 8,000.00 7,500.00 1,815,274.93 1,815,274.93 25,000.00 240,000.00 1,178,607.66 1,443,607.66 371,667.26 1,178,607.66 25,000.00 7,500.00 32,500.00 1,146,107.66 2,292.22 salmo iii et al. 66 references adams w, montalban hr, martin c. 1932. cultivation of bangos in the philippines. philipp j sci 47(1): 1-38. bagarinao tu. 1984. the natural life history of milkfish. seafdec asian aquaculture publication. southeast asian fisheries development center. tigbauan, ilo-ilo. 8(3): 3-6. garcia gvh. 1997. morphological abnormalities in hatchery-bred milkfish (chanos chanos forsskaal) fry and juveniles. aquaculture 152: 155-166. guerrero rd. 1981. introduction to fish culture in the philippines. philippines: philippine education co. inc. 70 p. herre aw, mendoza j. 1929. bangus culture in the philippine islands. philipp j sci 38: 451-509. pimentel aq. 1993. the local government code of 1991 –the key to national development. mandaluyong city: cacho publishing house, inc. r.a. 8550. the philippine fisheries code of 1998. rodriguez s. 1997. concession system in bolinao: implications to coastal management [dissertation]. quezon city, philippines: university of the philippines. smith ir, panayotou t. 1984. territorial use rights and economic efficiency: the case of philippine fishing concessions. rome: fao. villacorta lg. 1984. milkfish industry profile and action research and development program. pcamrd-dost primer no. 22. 02_enhancement morales and villagonzalo 6 enhancement of fe magnetic moments in fe/co (001) multilayers marienette b. morales*1 and cristine r. villagonzalo structure and dynamics group, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: mmorales@ateneo.edu abstract science diliman (july-december 2004) 16:2, 6–7 in order to investigate the electronic and magnetic properties of a bcc fe/co (001) multilayer, we have performed electronic structure calculations employing the total energy full-potential linear muffin tin orbital method. the magnetic moments of the layers are calculated. based on these results, the magnetization profiles and the microscopic origin of the enhancement of fe moments in the multilayers of the same co content but with different interface qualities are reported. large enhancement of magnetic moment is observed in the fe monolayer located at the interface, and an even greater increase is obtained for the multilayer with one monolayer of intermixing between fe and co layers. the co atoms were found to have similar magnetic moments in the bulk and at the interface. introduction thin films are well known to exhibit features that are not observed in bulk materials. a lot of work has been devoted to the study of bcc co thin films and fe/co multilayers primarily for their great importance in investigating magnetism of surfaces and interfaces in unique structures. in this present work, the effects of interface quality to the magnetic moment of the constituent atoms in the multilayer are investigated. we have chosen to study magnetism in a multilayer consisting of fe and co, both of which are independently highly ferromagnetic, and their positions in the bcc lattice. an advantage of the fe/co system is that it makes it possible to investigate co atoms in the bcc phase. numerical method the computational method used is the full-potential linear muffin tin orbital (fp-lmto) method based on the density-functional theory. this provides the basis set in which the wave functions are expanded by dividing the three-dimensional space into nonoverlapping muffin-tin (mt) spheres region and interstitial region. in each mt assigned to each atomic site, the density and potential are expanded using spherical harmonics, while outside the basis are expanded by means of fourier series. the main input parameters are only the type of atoms and the structure of the system of atoms, which are information that have long been established or may be patterned from experimental results. the advantage of this method is that no approximation is made on the shape of the density or potential. structural parameters the supercell or the smallest volume of the infinitely periodic multilayer system is composed of eight atoms *corresponding author 1ms. morales is also from the department of physics of the ateneo de manila university. enhancement of fe 7 which are stacked along the (001) axis. the atoms form four conventional bcc unit cells with eight layers. the unit cell is perfectly cubic so the in-plane and out-ofplane lattice constants are equal at the numerically verified value of 5.20 bohr. to simulate the experimentally observed imperfect interfaces in fabricated fe/co multilayers, the atoms in the supercell containing 25% co concentration are rearranged such that co atoms are intermixed within the fe layers. summary of results we have successfully verified theoretically the enhancement of the magnetic moment of fe in fe/co (001) multilayer in the ground state without any restrictive assumptions. for multilayers with the same content concentration, the location of atoms in the multilayers affects the magnetic moment. fe atoms that are sandwiched between two co atoms show an even larger increase in magnetic moment than when placed adjacent to another fe layer and co. for the fe monolayer, we have shown that this is due to the presence of a different kind of atom as its nearest neighbor. no significant change in co moment is observed even when the number of neighboring fe and co atoms changes. these information are useful for multilayer devices where a large magnetic moment response is needed. draft-dynamic (2).pmd dynamic programming optimization of multi-rate 33 dynamic programming optimization of multi-rate multicast video streaming services nestor michael c. tiglao1,3, jânio miguel e.f. monteiro1,2, antónio manuel r. c. grilo1, mário serafim s. nunes1, joão manuel f. xavier4 1instituto de engenharia de sistemas e computadores investigação e desenvolvimento, inov, instituto superior técnico, lisboa, portugal 2universidade do algarve, faro, portugal 3 electrical and electronics engineering institute, university of the philippines, diliman, quezon city, philippines 4instituto de sistemas e robótica, instituto superior técnico, lisboa, portugal *corresponding author: email: nestor@eee.upd.edu.ph abstract in large scale ip television (iptv) and mobile tv distributions, the video signal is typically encoded and transmitted using several quality streams, over ip multicast channels, to several groups of receivers, which are classified in terms of their reception rate. as the number of video streams is usually constrained by both the number of tv channels and the maximum capacity of the content distribution network, it is necessary to find the selection of video stream transmission rates that maximizes the overall user satisfaction. in order to efficiently solve this problem, this paper proposes the dynamic programming multi-rate optimization (dpmo) algorithm. the latter was comparatively evaluated considering several user distributions, featuring different access rate patterns. the experimental results reveal that dpmo is significantly more efficient than exhaustive search, while presenting slightly higher execution times than the non-optimal multi-rate step search (mss) algorithm. keywords: quality of experience (qoe), dynamic programming , quality of service (qos), internet protocol television (iptv), multi-rate introduction the possibility of ubiquitous internet access brought about by the wide dissemination of mobile technologies led to a greater heterogeneity of access networks and terminal equipments with different capabilities, which, coupled with the natural unpredictability of internet qos, constitutes a technological challenge for internetbased video distribution services. while current networking technologies and architecture have enabled a significant increase of the transmitted video quality, this can mainly benefit high capability terminals operating from high capacity access networks. however, in order to cover the entire market spectrum, the video distribution services must also satisfy users using lower capability terminals operating from lower capacity access networks (e.g. mobile handsets using gprs). in large scale ip television (iptv) and mobile tv distributions, the video signal is typically encoded and transmitted using several quality streams, over ip multicast channels, to several groups of receivers, which are classified in terms of their reception rate. as the number of video streams is usually constrained by both the number of tv channels and the maximum capacity of the content distribution network, the selection of the best transmission rate of each video stream should be dynamically adjusted trying to meet the maximum user level of satisfaction. for huge numbers of dynamically changing heterogeneous receivers and video streams, the exhaustive search for the optimal mix of stream quality levels is unbearable given the real-time adaptation requirements. science diliman (january-june 2010) 22:1, 33-42 tiglao, n.m., et al 34 regarding video quality metrics, while networking quality of service (qos) metrics usually provide good hints regarding the true quality experienced by the user, their relationship is nonlinear. that is the reason why user centric metrics have been used in television services for more than twenty years to evaluate video quality, based on subjective assessment metrics that are obtained using a panel of human evaluators in standard defined methods. these metrics measure the impairment caused by a diversity of factors on the human visual system (hvs) and constitute what is called quality of experience (qoe) metrics. when available, the latter constitute more reliable criteria for the optimization of the video streaming service. this paper proposes a qoe oriented multi-rate optimization algorithm designated the dynamic programming multi-rate optimization (dpmo) algorithm. as the name suggests, dpmo is based on dynamic programming concepts. it is able to find the set of video stream transmission rates that optimizes the average qoe of a set of users, featuring a computational complexity that is significantly lower than the exhaustive search. the proposed mechanism was comparatively evaluated considering several user distributions, featuring different access rate patterns. related works on multi-rate switching in the early video distribution systems over internet, video streaming was provided at a single rate. this scheme was suitable for users with connection speeds close to the average media rate, which implies that users with large access speeds were unable to exploit their transmission capabilities and users with slower connections could not view any content at all. a natural evolution of this single rate system is to offer content at individually encoded bitrates chosen in order to cover the available connection types (adsl, cable, wimax, wifi, etc.). however, this proposal does not optimize the quality of the streams sent to the different users due to the fact that the bottleneck capacity is not defined only by the access link but also by the core network and the streaming server, which can also limit the available bandwidth due to congestion or high load. multi-rate switching or stream replication (li & liu, 2003) is a dynamic extension of the individually encoded bitrates. it allows mid-stream switching between different rates according to the detected network conditions. several commercial products (real networks, 2002) (birney, 2000) use this scheme, as for instance surestream technology from real networks (conklin, et al., 2001). the innovation of this approach lies in the use of multiple representations of the original content (each one encoded at a different bitrate) optimized for different access network and load conditions. the result is a single file wherein all encoded streams are bundled. during the streaming session, the player monitors the bandwidth and the loss characteristics of the connection and instructs the server to switch to the stream that will provide the best perceptual quality. more recently, microsoft introduced smooth streaming (zambelli, 2009), an adaptive streaming technique where the video/audio source is encoded at multiple bitrates, generating multiple chunks of various sizes each with 2 to 4 seconds of video. because web servers usually deliver data as fast as network bandwidth allows, the client can easily estimate user bandwidth and decide to download larger or smaller chunks ahead of time. smooth streaming could be used for stored content or for live content, which allows to dynamically adapt the rates of the different streams in real-time according to the network and user conditions. in the olympic project (patrikakis, et al., 2003), a platform that was able to deal with multi-rate switching was developed. it consisted of servers, reflectors (proxy streaming servers) and transcoders. servers are responsible for streaming stored or live content and are considered as the point where any stream originates. users may request a particular stream directly from a server or the request may be submitted to a proxy streaming server. in the latter case, content is transmitted to the proxy node before being forwarded to the client. a proxy streaming server node may serve a large number of clients by replicating and subsequently forwarding packets received from the server, thus reducing the server’s workload. for example, assume that a large number of users request the same stream from a specific proxy streaming server. in this case, only one copy of the stream must science diliman (january-june 2010) 22:1, 33-42 dynamic programming optimization of multi-rate 35 reach the proxy streaming server. there the stream is replicated and transmitted to the various clients, improving scalability as the system’s capacity (in terms of users) can be increased by the deployment of additional proxy streaming servers. nunes, monteiro, & grilo (2009) proposed an algorithm called multi-rate step search (mss) to efficiently search the optimal set of streams bitrates that maximizes the qoe of a set of users (see section iii). mss fixes the lowest data rate to the value of the lower user access rate and then sequentially adds additional streams whose bitrates must always match available user access rates. for each added stream, an exhaustive search is performed so that its data rate will correspond to the argmax of the qoe when fixing the bitrates of the streams that are already on the system. after the new bitrate is found, all bitrates are sequentially adjusted until a local maximum is found. then the algorithm proceeds to add another stream. the process continues until the target number of streams is reached. although mss does not provide any guarantees of finding the optimal set of streams, it is very efficient from the point of view of execution time and its results are usually not very far from the optimal ones. yang, kim, & lam (2000) have proposed the use dynamic programming to determine the optimal partitioning of multi-rate multicast receivers. they have formulated the partitioning problem as an optimization problem to maximize the sum of receiver utilities subject to some loss tolerance constraints for a general class of utility functions. in particular, they considered two different receiver utility functions: one is based on interreceiver fairness (irf), which was first defined in (jiang, ammar, & zegura, 2000), and the other based on isolated receiver rates. in this study, however, we used a different utility function, one that is based on the quality of experience (qoe) metric as explained in the next section. qoe video multi-rate streaming optimization: problem statement the problem of rate adaptation as it is related to the olympic project platform (patrikakis, et al., 2003) may be formulated as follows: consider a multimedia stream s encoded at k different bitrates b i , i∈{1, ..., k}, resulting in k streams {s 1 , s 2 . .., s k }. a streaming server proxy node relays the streams to various clients, where the clients are categorized to k groups {g 1 , g 2 . .., g k }. according to the received stream (for example, a terminal receiving stream s i (encoded at bitrate b i ) belongs to group g i ). a group g i contains n i terminals t i,j , where j∈{1, ..., ni},. this configuration is illustrated in figure 1. the objective is to develop algorithms that are able to respond to varying network and terminal conditions by dynamically adapting a subset of the parameters k and b i , in order to achieve maximization of user perceived quality (qoe). the parameters that affect stream quality include the available bandwidth, network congestion, terminal cpu load and the number of served terminals. considering a fixed number of k streams, each stream encoded at a fixed bitrate b i , a specific case of the generally stated problem emerges. the goal is to find the optimal distribution of terminals to available streams whereby minimization of terminal-side packet loss and maximization of network utilization and perceived quality is achieved. it is worth noting that the optimization goal is twofold. firstly, we wish to obtain a specific (optimal) distribution of terminals as a function of their performance (specifically the sustained packet loss). secondly, we wish to design an algorithm for enforcing this distribution dynamically over time, as a response to fluctuating terminal performance. as a simple example of the aforementioned algorithm, consider a streaming server proxy node that relays 3 streams s 1 (128 kbps), s 2 (256 kbps) and s 3 (512 kbps). the streams are received by terminals t 1 t 2 , and t 3 respectively. at some point the system is informed (by means of a reporting mechanism presumably established between the terminals and the streaming server proxy node, e.g. rtcp reports) that terminal t 2 is sustaining a significant packet loss that is deemed as unacceptable on the basis of the defined policy. as a response, the streaming server proxy node ‘switches’ the 512 kbps transmitted to t 2 with the 128 kbps stream (represented by the big vertical arrow in figure 1). this results in a decrease of the packet loss experienced by the terminal that leads to a smoother terminal-side science diliman (january-june 2010) 22:1, 33-42 tiglao, n.m., et al 36 playback of the received stream as well as better network utilization. furthermore, consider an algorithm that examines the packet loss of each terminal in a group and determines the worst and best performing terminals in the group. then, the algorithm initiates a ‘switch’ of the stream received by the worst performing terminal with a stream encoded at a lower bitrate and another ‘switch’ of the stream received by the best performing terminal with a stream encoded at a higher bitrate. the qoe video streaming optimization aims to maximize the objective function that represents the sum of quality ( ) of each stream ( ) that exist at one instant: figure 1. terminal grouping according to the received stream bitrate. science diliman (january-june 2010) 22:1, 33-42 itu-t has standardized a parametric computational model, as recommendation g.1070 (itu-t, 2007), for evaluating qoe of video telephony. the model estimates the qoe of video-telephony services based on quality design/management parameters in terminals and networks. for h.264 the quality of a video stream without losses can be approximated by the expression ( in kbps): q t = 1.2 log 10 (1 + b i ) (2) notice that is subject to the constraint , b i ≤ aru, ∀ u∈gi , where ar u is the access rate that user u experiences. a straightforward approach to solve this problem is to enumerate all possible bitrates combinations and choose the one that maximizes q t . however, this approach is combinatorial in nature and the time complexity of the solution can grow exponentially with the number of variables. the mss algorithm (nunes, monteiro, & grilo, 2009) tries to tackle this problem in an efficiently in a heuristic way, but it offers no guarantees of optimality. (1) 1 k t i i i q q n = = ⋅∑ dynamic programming optimization of multi-rate 37 dynamic programming multi-rate optimization dynamic programming (dp) is an optimization technique for solving complex problems by breaking them into smaller sub-problems. it is especially useful in problems where one needs to make decisions one after another. in general, problems that can be solved through dynamic programming possess two characteristic ingredients: optimal substructure and overlapping sub-problems (cormen, et al., 2001). a problem has an optimal substructure if the optimal solution of the problem can be derived from the optimal solution of the sub-problems. an optimization problem is said to have overlapping sub-problems if the algorithm solves the same problem over and over again. the problem can be modeled as a system with an initial state and several possible final states. starting from the initial state, a decision has to be made to move to a next state where a certain reward or benefit is associated with that decision, until a final state is achieved. the total reward is the sum of the rewards accumulated along the way from the initial state to the final state. an optimal solution is one that maximizes the total reward. the application of dp to the optimal rate allocation problem is designated the dp multi-rate optimization (dpmo). each user experiencing an access rate ar j , ∈{1, ..., m} shall be assigned rate rj = bi, i∈{1, ..., k} . the dpmo algorithm analyses each user in turn, departing from its initial state at the lowest available access rate (ar 1 ) and then progressing to the highest access rate (ar m ). for users experiencing ar 1 , rate b 1 is introduced and this rate necessarily corresponds to ar 1 , i.e. r 1 = b 1 = ar 1 . for every other access rate ar j ,one of two possible options can be chosen: (1) to assign the previous rate b i again (i.e. r j = b i ), keeping the number of used streams for the moment; (2) to add rate b i + 1 = ar j to the set of used stream rates, making r j = b i + 1 . for example in figure 2, given m = 10 and k = 3. at state 4, the reward in choosing the first option is 1.2 log (1 + ar 1 ) . n 4 whereas at state 5, the reward is 1.2 log (1 + ar 5 ) . n 5 taking the second option, assuming that where n 4 and n 5 are the number of users that experience access rates ar 4 and ar 5 , respectively. although it was not clearly evident that this problem can be solved through dp, realizing that decisions on when to jump to a higher rates subject to a finite number of jumps gives the clue that dp can be used. dpmo can be best understood with an example. the dpmo implementation requires two state variables, k∈{1, ..., j 1} and l∈{0, 1, ..., k 1}, where k is the index of the rate allocated to users figure 2. dp optimization of multi-rate video streaming. science diliman (january-june 2010) 22:1, 33-42 tiglao, n.m., et al 38 table 1. decision table for j = 10. science diliman (january-june 2010) 22:1, 33-42 experiencing ar j and l is the number of available steps (stream rates). these two variables serve as indices for the decision tables computed by the algorithm. for each <k, l> pair, two items are stored: one is the optimal option taken and the other is the corresponding reward for that option, where option 0 means the previous rate is maintained and option 1 means that there is a jump to a new rate. the rewards consist of qoe values calculated with expression (2). moving to j = 9, the table is calculated in the same manner but for each <k, l> pair, the algorithm considers which option provides a higher accumulated qoe value (v2,0) considering the reward in j = 10, and stores the corresponding option and accumulated reward in the table. for example, for k = 2, l = 0, there is no other option but option 0, because no jumps in the rate are possible. the corresponding accumulated value is (v2,0) = 1.2 log (1 + ar 2 ) . n 9 + (v2,0), which goes into the respective table entry. for k = 2, l = 1, two options are possible: • option 01: (v2,0) = 1.2 log (1 + ar 2 ) . n 9 + (v2,1) • option 12: (v2,1) = 1.2 log (1 + ar 2 ) . n 9 + (v 9,0) the algorithm chooses the option that gives the higher (v2,1) and stores it in the respective entry in the table for j = 9. a similar procedure is carried out for the other <k, l> pairs. 9 9 1 0 9 1 0 9 1 0 dpmo solves the problem backwards (i.e., from the final state to the initial state), such that j ∈{m, m 1, ..., 1}. referring to the example in figure 2, dpmo calculates the table for j =10 first. for l = 0, the previous rate indicated by k is maintained while for l > 0, jumping to a higher rate will always provide a higher q t since ar 1 < ar 2 < ⋅⋅⋅ < ar m (see table 1). each <k, l> value in the decision table is designated as vk,l . note that column 10 is empty and is blackened in table 1. 1 0 9 the analysis will now jump to the last two steps. table 2 shows the decision table for j = 2. columns for k = 2,..., 10 are empty and are blackened. the dpmo algorithm terminates and arrives at the optimal solution when it reaches j = 1, where the optimal value is 1.2 table 2. decision table for j = 2 . log (1 + ar 1 ) . n 1 + (v1,2). the optimal rates are then determined by checking each decision table from j = 1 to j = 10 where option 1 is taken. to visualize the operation of dpmo, consider three peaks access rate distribution (described in section v). executing dpmo results in a decision graph with 62 states as shown in figure 3. dpmo starts computing 1 the value of l remains equal to 1 in v2,1 because no jump was made. 2 the value of l is decremented by 1 and becomes 0 in v2,1 meaning that further jumps will be possible. 10 10 1 2 3 … 9 10 0 0 0 0 … 0 1 1 1 1 … 1 2 1 1 1 … 1 1 2 … 9 10 0 0 … 1 1 … 2 1 … dynamic programming optimization of multi-rate 39 figure 3. decision graph for the three peaks access rate distribution. within each circle, three items are indicated: <l, k> pair, best decision and the accumulated q t value. science diliman (january-june 2010) 22:1, 33-42 the decision table of the final state, j = 62. then, it proceeds to compute the tables for the other states until it terminates at j = 1, obtaining the optimal q t . the optimal decision as well as the corresponding optimal bitrates b i , i ∈{1, 2, ..., k} can be obtained by going through the decision tables tracing from state j = 1 to state j = 62 where option 1 is chosen. take note that at j = 1, option 1 is chosen automatically. at <-,3> 1 9418.871 <1,0> 0 9198.043 <1,1> 0 9359.465 <1,2> 0 9391.232 <1,0> 0 9142.767 <1,1> 0 9304.189 < 1,2> 0 9335.955 <2,0> 0 9151.322 <2,1> 0 9307.882 <2,2> 0 9338.141 < 1,0> 0 9059.852 <1,1> 0 9221.273 <1,2> 0 9253.040 < 2,0> 0 9068.330 < 3,1> 0 9228.487 < 3,2> 0 9257.310 < 1,0> 0 22.1107 <1,1> 1 23.1997 < 40,0> 0 22.85003 < 60,1> 1 23.1997 <60,2> 1 23.1997 < 1,0> 0 0 <1,1> 1 0 < 40,0> 0 0 < 61,1> 1 0 <61,2> 1 0 j=1 j=2 j=3 j=4 j=61 j=62 tiglao, n.m., et al 40 science diliman (january-june 2010) 22:1, 33-42 each succeeding state, the optimal decision and the corresponding reward value is already solved by dpmo. in this example, the resulting optimal streams rates are 200 kbps, 219 kbps and 239 kbps. figure 4 plots the value of q t as dpmo progresses from state j = 62 where q t = 0 to initial state j = 1 where q t = 9418.871, the optimal value. results the dpmo algorithm was compared with the mss algorithm (nunes, monteiro, & grilo, 2009) and exhaustive search in terms of execution time and the obtained . the algorithms were implemented and tested in matlab (2009) and performance results were obtained considering a fixed number of streams, k = 3. the algorithm can be easily adapted to other values of k. we considered the five different distribution profiles (also used in (nunes, monteiro & grilo, 2009)) of user access rates ar j to analyze the performance of the algorithms under various computational scenarios ranging from easy to difficult as follows: • uniform: the integer access rate values (ar j ) are uniformly spaced within the interval [ar 1 , ar m ] . • three peaks: three wide triangular-shaped peaks spanning uniformly spaced access rates, with maxima figure 4. evolution of q t as calculated by dpmo. at 208 kbps (90 users), 222 kbps (70 users) and 245 kbps (≈128 users), minima at 200 kbps (10 users), 216 kbps (10 users), 229 kbps (0 users) and 260 kbps (8 users). • three clusters: three tiny triangular-shape pulses spanning uniformly spaced access rates, located in intervals [102 kbps, 140 kbps] (maximum at 108 kbps with 70 users), [251 kbps, 283 kbps] (maximum at 267 kbps with 85 users) and [501 kbps, 529 kbps] (maximum at 515 kbps with 75 users). • six clusters: three tiny triangular-shape pulses spanning uniformly spaced access rates, located in intervals [100 kbps, 165 kbps] (maximum at 130 kbps with 99 users), [175 kbps, 245 kbps] (two maxima at 205 kbps and 210 kbps with 99 users) and [250 kbps, 315 kbps] (maxima at 280 kbps and 285 kbps with 99 users), [325 kbps, 390 kbps] (maximum at 360 kbps with 99 users), [400 kbps, 465 kbps] (maximum at 435 kbps with 100 users) and [475 kbps, 530 kbps] (maximum at 410 kbps with 100 users). • random: randomly chosen 300 access rates from the interval [10 kbps, 1000000 kbps], with random total number of users, where each access rate is assigned to a maximum of 1000 users. all the three algorithms (i.e. exhaustive search, dpmo and mss) are executed using the same random scenarios. dynamic programming optimization of multi-rate 41 table 4. execution times and q t error table 3 provides a summary of the different access rate distribution profiles considered in this study. the execution times are listed in table 4 for all the considered user rate distributions. for each distribution except the random, the optimal q t is listed together with the set of rates that achieve it are shown. in addition, we also included the average difference and the standard deviation of the difference between the optimal calculated by dpmo and the calculated by mss. while the exhaustive and dpmo algorithms always find the optimal q t , mss sometimes fails to find the optimal solution in random distributions, since it can become stuck at local maxima. however, the error is very low and it consistently presents a lower execution science diliman (january-june 2010) 22:1, 33-42 table 3. access rate distribution profiles time than dpmo. this suggests that mss may still be useful in extremely demanding settings featuring huge numbers of heterogeneous users and target streams (k). both mss and dpmo are significantly more efficient than exhaustive search. conclusions this paper has proposed the dpmo, a dynamic programming based algorithm whose objective is to calculate the optimal set of multicast video data rates that maximizes the overall qoe for a set of users with heterogeneous access network rates. dpmo guarantees that the optimum solution is reached by employing dynamic programming concepts. nevertheless, experimental results have demonstrated that despite not providing any optimality distribution (kbps) (kbps) number of access rates ( ) total number of users max users in each total number of users number of runs uniform 250 440 20 20 1 20 1 three peaks 200 261 62 128 3338 1 three clusters 102 529 75 85 3060 1 six clusters 100 530 87 100 4494 1 random 10 1000000 300 n/a 1000 random 20 distribution optimal optimal (kbps), execution time (seconds) average difference between calculated and optimal (mss only) standard deviation of difference between calculated and optimal (mss only) exhaustive dpmo mss uniform 59.9 250, 310, 380 0.65 0.07 0.04 0 0 three peaks 9418.9 200, 219, 239 1.93 0.28 0.26 0 0 three clusters 8994.2 102, 252, 501 3.49 0.38 0.22 0 0 six clusters 12692.7 100, 345, 195 5.40 0.47 0.34 0 0 random n/a n/a 228.05 6.05 4.24 tiglao, n.m., et al 42 guarantees, the solutions presented by mss are optimal in most configurations or at least approach the optimum solution with a very low average error. the results show that both mss and dpmo are significantly more efficient than exhaustive search in terms of execution time, with mss being slightly but consistently more efficient than dpmo, suggesting that mss may still be useful in extremely demanding settings featuring huge numbers of heterogeneous users and target streams. while the present work targets at dynamic video multicast environments where users and access rates dynamically change over time, both dpmo and mss assume that the user configurations are static most of the time and that they must be executed anew each time the user configuration changes. future work shall focus on adapting the dpmo algorithm in order to remain efficient in highly dynamic user configurations. acknowledgment nestor tiglao would like to acknowledge the funding support of the department of science and technology – science education institute (dost-sei) and the university of the philippines engineering research and development for technology (up erdt). references birney, b. 2003. intelligent streaming, microsoft corp. (http://www.microsoft.com/windows/windowsmedia/howto/ articles/intstreaming.aspx) broadband forum. 2006. tr-126 triple-play services quality of experience (qoe) requirements. conklin, g. j., g.s. greenbaum, k.o. lillevald, a.f. lippman & y.a. reznik. 2001. video coding for streaming media delivery on the internet. ieee t. circ. syst. vid. 11(3):269281. cormen, t., c. leiserson, r. rivest, & c. stein. 2001. introduction to algorithms. 2nd edition. mit press. patrikakis, ch. z., y. despotopoulos, a.m. rompotis, n. minogiannis, a.l. lambiris & a.d. salis. 2003. an implementation of an overlay network architecture scheme for streaming media distribution. proc. 29th euromicro conf. 207-214. itu-t recommendation g.1010. 2007. opinion model for video-telephony applications, international telecommunication union. jiang, t., m. ammar, & e.w. zegura. 2000. on the use of destination set grouping to improve inter-receiver fairness for multicast abr sessions. proc. 19th annual joint conference of the ieee computer and communications societies (infocom 2000), 1:42-51. li, b & j. liu. 2003. multirate video multicast over the internet: an overview. ieee network, 17(1):2429. matlab, version 7.8.0.347 (r2009a), mathworks, february 2009. nunes, m., j, monteiro, & a. grilo. 2009. a quality of experience optimization for multi-rate internet tv services. proc. 8th intl. conf. on decision support for telecommunications and information society – dstis. realnetworks.2002. introduction to streaming media with realone player (http://service.real.com/help/library/guides/ realone/introguide/html/htmlfiles/title.htm). winkler, s. & p. mohandas. 2008. the evolution of video quality measurement: from psnr to hybrid metrics. ieee t. broadcast., 54(3):1-9. yang, y.r., m. s. kim, & s. s. lam. 2000. optimal partitioning of multicast receivers. proc. 8th intl. conf. network protocols (icnp’00) pp.129-140, 2000. zambelli, a. 2009. iis smooth streaming technical overview. microsoft corporation (http:// w w w . m i c r o s o f t . c o m / d o w n l o a d s / e n / details.aspx?displaylang=en&familyid=03d22583-3ed644da-8464-b1b4b5ca7520 ). science diliman (january-june 2010) 22:1, 33-42 occurrence and determination of haloacetic acids in metro manila drinking water irene b. rodriguez and maria pythias b. espino* 1institute of chemistry, university of the philippines, diliman 1101 quezon city *corresponding author: mbespino@up.edu.ph received: 26 february 2009; revised: 5 july 2010; accepted: 12 july 2010 abstract haloacetic acids are found in chlorinated water with high organic matter content. an analytical method based on a us epa method for measuring these compounds in water is described. the optimized method used diethyl ether as extraction solvent with sulphuric acid-methanol as esterification agent and subsequent detection by gas chromatography-electron capture detection. evaluation of this method showed that it was linear in the concentration range of 10 to 150 µg l -1 and the method detection limits were from 17 to 57 µg l-1. although the method demonstrated low recoveries (16 to 43%), it is useful in the quantitative determination of monochloroacetic acid as well as the qualitative determination of other haloacetic acids in water. drinking water samples taken from different areas in metro manila serviced by the local treatment plants were analysed using the method. monochloroacetic acid, monobromoacetic acid, and bromochloroacetic acid were detected in these samples. monochloroacetic acid was quantified and found in concentrations ranging from 19 to 157 µg l-1. in most of the water samples, the concentration of monochloroacetic acid exceeded the us epa maximum allowable total concentration of 60 µg l-1 for the five haloacetic acids (monochloro-, dichloro-, trichloro-, monobromo-, and dibromoacetic acids) in drinking water. this initial study established the occurrence of potentially harmful haloacetic acids in the local drinking water supplies. keywords: haloacetic acids; disinfection by-products; drinking water; chlorination introduction maintaining a safe and high quality drinking water is a common goal all over the world. drinking water sources can be surface water, such as rivers and lakes, or groundwater for those that have rich water tables. in most countries, the disinfection method of choice is chlorination due to the efficacy of this process against waterborne illnesses and the relative ease in maintenance and operation of the necessary equipment (nieuwenhuijsen et al., 2000; kaur et al., 2004). chlorination as a disinfection process was introduced in the 19th century when there were deaths due to typhoid, cholera, dysentery, and other diseases caused by waterborne bacteria (lee et al., 2001). chlorination of drinking water is effective in preventing deaths from these diseases because chlorine is a good primary and residual disinfectant. in the early 1970s however, the presence of disinfection by-products (dbps) in drinking water was reported (lee et al., 2001). trihalomethanes (thms) were detected in drinking water as a result of the reaction of chlorine with humic substances that are present in the source water (hozalski et al., 2001). this raised concerns because chloroform, one of the thms, is classified as a probable human carcinogen (nikolaou et al., 2004). the detection of thms in drinking water led to the development of more rigorous water treatment processes and also paved the way to the development of methods that are suitable for the science diliman 21(2):35-41 35 mailto:mbespino@up.edu.ph rodriguez, espino determination of these substances even at low concentrations. recent technological advances also provided more options for research on drinking water quality. thus, other dbps like haloacetonitriles (hans), haloacetic acids (haas) and their brominated analogues have been detected. the brominated analogues are formed when bromine is present in the raw source water (cowman & singer, 1996; hozalski et al., 2001; hua et al., 2006). of the different dbps, the haas are gaining interest from researchers. unlike the thms and hans, haas are not volatile. thus, measures like allowing the water to stand or boiling before consumption do not guarantee removal of the haas. moreover, the identified haas are regarded to be more toxic than the volatile dbps (takino et al., 2000). some studies have reported that haas are probable causes of cancers targeting the reproductive organs (nieuwenhuijsen et al., 2000). the haas that have been detected in relatively high concentrations are monochloroacetic acid (mcaa), dichloroacetic acid (dcaa), trichloroacetic acid (tcaa), monobromoacetic acid (mbaa) and dibromoacetic acid (dbaa). together, the total concentration of these haas is regulated as total for haa5 for the allowed maximum contaminant level. the us environmental protection agency (epa) regulates these compounds and allows a maximum value of 60 µg l-1 for total haa5 in drinking water (usepa dbp rule). other countries also monitor the levels of the total concentration of haa5 and four other minor haas known to occur in drinking water. together, these are termed haa9 that includes the haa5 as well as bromochloroacetic acid (bcaa), bromodichloroacetic acid (bdcaa), chlorodibromoacetic acid (cdbaa), and tribromoacetic acid (tbaa), (cowman & singer, 1996; hua et al., 2006). the european union monitors the level of thms in drinking water and is now considering regulating the haa contaminant levels. urbansky (2000) have discussed various methods that have been reported for the analysis of haas and noted that most of these methods use gas chromatography coupled with electron capture detection (gc-ecd) or mass spectrometry (gcms). the haas have to be derivatized to esters before gc analysis. the us epa standard method 552.2 is generally the method used for the determination of haas (hodgeson et al., 1990). this method employs an extraction step using methyl tert-butyl ether (mtbe) and esterification using diazomethane. the use of mtbe is now minimized, if not avoided, because this is also a contaminant of interest due to its possible toxicity. the use of diazomethane, on the other hand, has special requirements and has inherent hazards. modifications to the us epa method using other extraction solvents and derivatizing agents have been reported in several studies. one such modified procedure used acidic methanol to derivatize the haas and subsequent microextraction step (cancho & ventura, 2005). another method used in situ derivatization using dimethyl sulfate with solid phase microextraction and gc-ms for determination (sarrion et al., 2000). for our study, the us epa method 552.2 was modified by using diethyl ether as extraction solvent. the extracts were derivatized with a sulphuric acid-methanol mixture and the subsequent determination was performed using gc-ecd. the modified method was applied in the analysis of haas in drinking water samples taken from selected areas in metro manila. we present here an available and accessible method for haas analysis as well as a relevant finding on the occurrence of haas in the tap water samples in metro manila. this is the first report that reveals the presence of three haas (mcaa, mbaa, and bcaa) in the local drinking water supply. materials and methods sampling the drinking water samples were collected from a total of 31 sampling sites from different cities in metro manila serviced by the local water treatment plants. figure 1 shows the collection sites. these sites were the same sampling sites for the study on volatile dbps reported earlier (rodriguez et al., 2006). prior to sampling, 1.5 g of nh4cl (merck, germany) was placed in thoroughly washed and dried 1 l glass bottles. samples were collected directly from the tap into the glass bottles after allowing it to flow for 1 min, filled to the brim, and immediately refrigerated at 4°c prior to analysis. extraction and analysis were done between one day and fourteen days after sample collection. 36 science diliman occurrence and determination of haloacetic acids extraction the extraction procedure was a modification to the us epa standard method 552.2. extraction was carried out by adding 30 ml aliquot of the water samples to 40-ml teflon-faced vials (daigger, il, usa) containing 3 g of copper sulphate (jt baker, nj, usa) and 10 g of acidified sodium sulphate (merck, germany) prepared as reported earlier (rodriguez et al., 2006). the surrogate standard 2,3dibromopropionic acid (aldrich, wi, usa) was spiked (spike level equivalent to 600 µg l-1 in the final extract) and 2.5 ml of concentrated sulphuric acid (merck, germany) was added to adjust the ph of the samples to about 0.5. the ph should be low enough to ensure that the haas are in the acid form. diethyl ether (jt baker, nj, usa) was then added (3 ml for 30 ml sample) and the vials were then shaken mechanically for 1 h. after shaking, the samples were allowed to stand for 5 min and then 2 ml of the diethyl ether layer of the samples were transferred to 5 ml supelco conical vials with teflon face. derivatization the diethyl ether extracts were dried using a gentle stream of nitrogen gas to about 0.5 ml each. then, 0.5 ml of 10% sulfuric acid in methanol solution was added to methylate the haloacetic acids. the vials were then placed in a water bath kept at 70°c for 2 h. after heating, the extracts were cooled before adding 2 ml of diethyl ether and 1 ml of 10% sodium bicarbonate solution to neutralize the excess sulphuric acid. these were shaken mechanically for 5 min and allowed to stand. finally, 1 ml aliquots were transferred from the diethyl ether layer of the derivatized extracts to 2 ml vials (shimadzu, japan). to these extracts, an internal standard mix composed of fluorobenzene and 3-bromo-1chloropropane (supelco, bellefonte, usa) was added prior to gc-ecd analysis. gc-ecd determination and quantitation the instrument used for this study was a shimadzu gc8a equipped with a 63ni for electron capture detection. the carrier gas was nitrogen at 5 ml min-1 flow and the column used was zb 624 (cyanopropylphenylmethylpolysiloxane, 30m × 0.53mm id × 3.00µm ft) from phenomenex, torrance, usa. using a 10-μl syringe (hamilton, usa), 3 µl of the extracts were manually injected. the analyses were carried out in these conditions : split mode (1:10) injection; 120°c column temperature for isothermal separation; 160°c injector temperature; 160°c detector temperature. these isothermal parameters were the settings for optimum separation of the methylated haas. the haloacetic acids were quantified against calibration standard solutions prepared using the methylated standards spiked in diethyl ether. internal standard correction was performed to account for loses during the analysis. science diliman 37 figure 1.location of sampling sites in some cities in metro manila (numbers indicate the sampling sites listed in table 2). [metro manila map modified from http://mapsof.net/uploads/static-maps/metro_manila _political_map.png] rodriguez, espino results and discussion optimization of extraction & derivatization the haloacetic acids included in this study were mcaa, dcaa, mbaa, tcaa, bcaa, and dbaa. the method for the haloacetic acid analysis was optimized by first converting the standard haloacetic acid mix to the methylated form. briefly, the haas were spiked in ultrapure water, ensured to be in their acid forms by adding concentrated sulphuric acid to achieve a ph of 0.5. the haas were subsequently extracted using 3 ml of diethyl ether, the haas were derivatized to the methylated forms by adding 10% h2so4 in methanol, and then a second diethyl ether extraction was performed. the second extraction process may be carried out by neutralizing the excess h2so4 in methanol with nahco3 prior to addition of the extraction solvent. however, it was observed that splattering occurs during the addition of the bicarbonate solution which may result to loss of the analytes. a parallel analysis was carried out wherein the diethyl ether was added prior to neutralization of the excess acid. extra care was observed during the addition of the bicarbonate after diethyl ether was added because the neutralization of the excess acid resulted in the evolution of gas. the recoveries of all analytes were significantly increased when diethyl ether was added before neutralization of the excess acid with sodium bicarbonate. loses due to the splattering may be minimized by the presence of the solvent. adding diethyl ether at this point may have hindered loses because the haas are already in the methylated form thus interaction of the analytes with the solvent is already favored. for the haa analysis, an internal standard solution composed of fluorobenzene and 2-bromo-1chloropropane was used. however, only absolute recoveries against 2-bromo-1-chloropropane can be calculated because the peak for fluorobenzene coeluted with the diethyl ether peak. separation of the haas was excellent using the isothermal gc-ecd parameters, the retention times of the analytes are given in table 1. the run time of each analysis was 18 min which was the optimum gc run time due to the isothermal oven temperature. improving the run time, i.e., shortening of time, leads to closely positioned peaks which is less favorable. method performance using the optimized method, recoveries of haas spiked in ultrapure water at 10 to 150 µg l -1 concentrations were determined. recoveries were calculated using commercially available methylated haa standard solutions. our results show that in this concentration range, the response of all haas with increasing concentration was linear having correlation coefficients of 0.997 to 0.999. table 1 lists the absolute recoveries of haas at 50.0 µg l-1 spike level. tcaa was not recovered nor detected even at higher spike concentrations. for this particular haa, the optimized method is not applicable. the percent absolute recoveries of the five other haloacetic acids ranged from 16.4 to 42.9 at 50.0 µg l-1 concentration in water; the standard deviations ranged from 4.15 to 12.6. these recoveries are low compared to those reported using the epa standard method (comparison shown in table 1) where all haas have relatively good recoveries. however, it should be noted that for the epa method, the derivatization was done using diazomethane and the extraction by mtbe. diazomethane derivatization is excellent for mcaa and tcaa analysis because of the difficulty of methylating these compounds. diazomethane and mtbe were not tried in this study because handling of these chemicals requires special set-up and equipment. the esterification agent used in this study was used by xie (2001) in the analysis of haas where liquidliquid microextraction with acidic methanol derivatization was employed. xie reported recoveries that ranged from 81 to 144% but the determination of the analytes was done by gc-ms, suggesting that acidic methanol esterification with sensitive analytical determination could provide better detection. the same results were obtained by domino et al. (2004) who compared various methods for haa determination. in their study, they noted that a higher boiling solvent such as tertiaryamyl methyl ether can improve extraction efficiency of haas and that extraction efficiency can also be improved by increasing the amount of sodium sulphate added. barron & paull (2006) also studied 38 science diliman occurrence and determination of haloacetic acids table 1. percent recoveries and mdl of the modified method and the us epa method haas retention time a (min) % absolute recoveryb, modified method mdl, modified method (µg l-1 )c % absolute recovery, epa method 552.2d mdl, epa method 552.2, (µg l-1 )d mcaa 6.09 16.4 16.9 94.7 0.27 dcaa 7.95 25.9 22.8 84.7 0.24 mbaa 8.3 42.9 38 102 0.2 tcaa 8.3 nd nd 93 0.08 bcaa 11.96 42.3 50.5 86.9 0.25 dbaa 17.67 42.6 56.8 95.4 0.07 nd = not detected using the modified extraction and derivatization method a column used: cyanopropylphenylmethylpolysiloxane, 30 m × 0.53 mm id × 3.00 µm ft; n2 carrier gas at 5 ml min-1 flow rate b n = 3; calculated at 50 µg l-1 spike level c n = 7; at 50 µg l-1 spike level d reference : hodgeson, et al., 1990 haas and used microbore ion chromatography with suppressed conductivity coupled to electrospray ionization mass spectrometric detection. they reported recoveries ranging from 13 to 84% for the haas they studied. kou et al. (2004) who used supported liquid membrane microextraction with hplc-uv detection reported extraction efficiencies of 3.89 to 39.6% for the haas they analysed. another determination procedure was reported by liu et al. (2004) where hydrophilic anion-exchange column with steep gradient sodium hydroxide addition and inductively coupled plasma mass spectroscopy as detector were used. for their method, the recoveries of the haas are in the range of 92 and 104%. the methods above that reported relatively good recoveries use mass spectrometric detection which provides sensitivity that outperforms other detection methods. in the present study, the method detection limit (mdl) for each haloacetic acid was determined using the optimized method. the standard haas in the acid form were spiked in ultrapure water (concentration equal to 50.0 µg l-1 in the final extract), extracted, derivatized and analyzed by gcecd. seven replicates were performed and the mdl for each haa was calculated using the standard deviation of the measurements and the equation (adapted from the us epa method by williams & maillard, 1997): mdl=3.143×sd  for n=7 (1) where mdl is a statistical estimate of the detection limit that is equal to the standard deviation multiplied by the student’s t-value at 99% confidence level. mdl values calculated for the haas studied are relatively high (given in table 1) which means that if the actual concentration of a target haa in the water sample is below the mdl, the actual level will not be statistically quantified. mdl values obtained using the optimized method and the epa standard method are given in table 1. the difference in the mdl values is attributed to the different extraction and derivatization procedures used in the two methods. the optimized method presented in this study, thus, requires further modifications aimed at lowering the detection limits. analysis of drinking water samples the occurrence and levels of haas in actual drinking water samples were studied using the optimized method. although the other haas were detected in the samples, only mcaa was quantified and its concentrations are shown in table 2. the mcaa concentrations were calculated against external calibration solutions prepared by using commercially available methylated standards in diethyl ether. mcaa in the water samples ranged from 19 to 157 µg l-1. these values correspond to corrected values of 98 to 958 µg l-1 when the recovery of the method is taken into account. the corrected values indicate that the levels of mcaa alone exceeded the suggested maximum guideline value allowable for total haa5 concentration (60 µg l-1, us epa). mbaa and bcaa were detected in the samples but were found in levels below mdl values. science diliman 39 rodriguez, espino table 2. concentrations (values in parenthesis are standard deviations) of monochloroacetic acid in drinking water samples from various sampling sites in metro manila sampling location no. sampling sitea mcaa concentration, mg l-1 b actualc correctedd 1 upd a 24 (4) 144 (22) 2 upd b 20 (5) 121 (28) 3 upd c 25(5) 152 (31) 4 upd d 19 (4) 98 (28) 5 upd e nd nd 6 upd f 30 (4) 113 (25) 7 marikina city a 46 (4) 278 (27) 8 marikina city b 56 (4) 343 (21) 9 marikina city c 96 (4) 588 (26) 10 manila a 120 (7) 735 (44) 11 san juan a 74 (3) 451 (20) 12 san juan b 101 (10) 614 (61) 13 mandaluyong city a 154 (19) 941 (114) 14 mandaluyong city b 75 (11) 457 (68) 15 muntinlupa city a nd nd 16 muntinlupa city b 157 ( 12) 958 (71) 17 makati city a 128 (24) 778 (146) 18 makati city b 152 (5) 928 (28) 19 pasig city a 112 (12) 683 (71) 20 pasig city b 71 (15) 434 (91) 21 caloocan city a 81(13) 494 (80) 22 quezon city a 96 (24) 583 (150) 23 quezon city b 55 (22) 336 (133) 24 quezon city c 68 (6) 412 (35) 25 quezon city d 97 (1) 590 (9) 26 quezon city e 86 (14) 523 (82) 27 quezon city f 86 (10) 524 (61) 28 quezon city g 91 (11) 553 (66) 29 quezon city h 89 (6) 541 (39) 30 valenzuela city a 71 (3) 433 (16) 31 valenzuela city b 97 (12) 589 (74) nd= not detected a sampling sites a,b, etc. are different households from the same city; upd samples were collected in february 2003; other samples were collected in december 2002 to june 2003 b n=3 c actual = quantified concentrations using standard calibration solutions d corrected = concentrations corrected for recovery of mcaa at 50 µg l-1 these findings prompt a need to study the formation, reduction or possible removal of haas in the local water supplies. there is also a need to improve the extraction recoveries and detection limits of the method by considering other analytical techniques. the use of alternative extraction solvents and derivatization reagents can be explored. mtbe and diazomethane may still be the best extraction solvent and derivatization reagent, respectively. however, hazards precaution, correct handling, and proper waste disposal should be in place. other determination techniques such as the use of ion chromatography (ic), high pressure liquid chromatography (hplc) or gc with ms detection may prove useful in studying haas as contaminants in the drinking water supplies in metro manila. conclusions the modified method for analysing haloacetic acids in drinking water described in this study was evaluated in terms of linearity, recovery, and method detection limits. the use of diethyl ether as solvent for extraction and a sulphuric acid-methanol mixture as methylating agent were explored. the optimized method showed linear correlation for a wide concentration range but suffers from low recoveries and high detection limits. application of the optimized method to analyse actual water samples detected three haas (mcaa, mbaa, and bcaa) in the drinking water supplies in metro manila. the method was successful in the quantitative determination of mcaa where its concentrations in most of the water samples analysed were above the 60 µg l-1 maximum contaminant level recommended by the us epa for total haa5 in drinking water. this study offers an available method that can be used to quantify mcaa and qualitatively determine other haas present in the local drinking water supplies. modifications such as the use of ic-, hplcor gcms and the use of other extraction solvents and derivatization reagents are suggested to improve the performance of the method. an improved method may be used to study the formation and possible control of haas found to occur in the drinking water in metro manila where to date, these compounds are not routinely monitored for public safety. 40 science diliman occurrence and determination of haloacetic acids acknowledgement m.p.b.e. is grateful to the up diliman office of the vice-chancellor for research and development for the funding support (project no. 010111psn). references barron, l. & b. paull, 2006. simultaneous determination of trace oxyhalides and haloacetic acids using suppressed ion chromatography-electrospray mass spectrometry. talanta, 69, 621-630. cancho, b. & f. ventura, 2005. optimization of methods for the determination of dbps. global nest journal, 7, 72-94. cowman, g. a. & p.c. singer, 1996. effect of bromide ion on haloacetic acid speciation resulting from chlorination and chloramination of aquatic humic substances. environ. sci. technol., 30, 16-24. domino, m., b. pepich, d. munch & p. fair, 2004. optimizing the determination of haloacetic acids in drinking waters. j. chromatogr. a, 1035, 9-16. hodgeson, j. w., j. collins & r.e barth, 1990. epa method 552.2. determination of haloacetic acids and dalapon in drinking water by liquid-liquid extraction, derivatization and gas chromatography with electron capture detection revision 1.0. u.s. environmental protection agency, cincinnati, oh. hozalski, r. m ., l. zhang & w.a. arnold, 2001. reduction of haloacetic acids by feo: implications for treatment and fate. environ. sci. technol., 35, 2258-2263. hua, g., d.a. reckhow & j. kim, 2006. effect of bromide and iodide ions on the formation and speciation of disinfection byproducts during chlorination. environ. sci. technol., 40, 3050-3056. kaur, s., m. j. nieuwenhuijsen, h. ferrier & p. steer, 2004. exposure of pregnant women to tap water related activities. occup. environ. med., 61, 454-460. kou, d., x. wang & s. mitra, 2004. supported liquid membrane microextraction with high-performance liquid chromatography–uv detection for monitoring trace haloacetic acids in water. j. chromatogr. a, 1055, 63-69. lee, k. j., b.h. kim, j.e. hong, h.s. pyo, s. park & d.w. lee, 2001. a study on the distribution of chlorination byproducts (cbps) in treated water in korea. wat. res., 35 (12) 2861-2872. liu, y., s. mou & d. chen, 2004. determination of tracelevel haloacetic acids in drinking water by ion chromatography–inductively coupled plasma mass spectrometry. j. chromatogr. a, 1039, 89-95. nieuwenhuijsen, m.j., toledano, m.b., n.e eaton, j. fawell & p. elliot, 2000. chlorination disinfection byproducts in water and their association with adverse reproductive outcomes: a review. occup. environ. med., 57, 73-85. nikolaou, a.d., s. k. golfinopoulos, t.d. lekkas & g.b arhonditsis, 2004. factors affecting the formation of organic by-products during water chlorination – a benchscale study. water, air, soil pollut.,159, 357-371. rodriguez, i., t.a.o. quibuyen & m.p.b. espino, 2006. analysis of volatile disinfection by-products in drinking water. kimika, 22, 1-6. sarrion, m. n., f.j. santos & m.t. galceran, 2000. in situ derivatization solid-phase microextraction for the determination of haloacetic acids in water. anal. chem., 72, 4865-4873. takino, m., s. daishima & k. yamaguchi, 2000. determination of haloacetic acids in water by liquid chromatography-electrospray ionization-mass spectrometry using volatile ion-pairing reagents. analyst, 125, 1097-1102 . united states environmental protection agency (usepa) disinfection by-products (dbp) rule. http://www.epa.gov.safewater urbansky, e. t., 2000. techniques and method for the determination of haloacetic acids in potable water. j. environ. monit., 2, 285-291. williams, c. &v. maillard, environmental sampling and monitoring primer. method detection limits. http://ewr.cee.vt.edu/environmental/teach/smprimer/mdl/ mdl.html xie, y., 2001. analyzing haloacetic acids using gas chromatograph/mass spectrometry. wat. res., 35, 15991602. science diliman 41 02_de ungria de ungria et al. science diliman (january-june 2002) 14:1, 8-16 resolving questioned paternity issues using a philippine genetic database maria corazon a. de ungria*, kristina a. tabbada, frederick c. delfin, alma m. frani, michelle m.f. magno, gayvelline c. calacal, and saturnina c. halos dna analysis laboratory, natural sciences research institute university of the philippines, diliman, q.c. 1101 e-mail: mcadu@uplink.com.ph; telefax: (632) 9252965 the utility of the philippine genetic database consisting of seven short tandem repeat (str) markers for testing of ten questioned paternity cases was investigated. the markers used were humvwa, humth01, humcsf1po, humfolp23, d8s306, humfes/fps, and humf13a01. these markers had a combined power of paternity exclusion of 99.17%. due to the gravity of some cases handled in the laboratory, routine procedures must be assessed to determine the capacity of the analysis to exclude a non-father or predict paternity. clients showed a preference for only testing father and child to lower costs and reduce conflicts, particularly when the mother objects to the conduct of dna tests, or when she is deceased or cannot be located. the probability of paternity was calculated with and without the mother’s profile in each of the cases. in all instances, results were more informative when the mother’s dna profile was included. moreover, variations in the allelic distribution of five str markers among eight caucasian, one african-american, and two amerindian (argentina) populations resulted in significant differences in probability of paternity estimates compared to those calculated using the philippine database. based on the results of the present study, it is recommended that tests on alleged father-child samples be performed to screen for at least two mismatches. in the absence of these mismatches, further analysis that includes the mother’s dna profile is recommended. moreover, it is recommended that a philippine genetic database be used for dna-based paternity testing in the philippines. key words: short tandem repeat markers, philippine genetic database, inclusions, exclusions, paternity trios, motherless cases *corresponding author abstract introduction paternity testing has changed from its early days, when conventional serum-based testing, such as abo blood typing and protein polymorphism, was the norm, to the current dna-based analysis using restriction fragment length polymorphism (rflp) and/or the polymerase chain reaction (pcr). dna typing is based on the uniqueness of the genetic make-up of all individuals, except identical twins (jeffreys et al., 1985). it is widely used in criminal investigations, in establishing familial relationships between individuals in simple paternity disputes and immigration cases, and identification of mass disaster and war victims. dnabased systems offer a higher exclusion power than protein-based systems, thereby minimizing the chance 8 resolving questioned paternity issues of falsely including a non-father (markowicz et al., 1990), as well as a more accurate inclusion probability for the identification of true biological fathers (chakraborty & stivers, 1996). dna testing also allows greater flexibility in terms of the types of sample that can be submitted for testing. blood, hair, tissues, buccal swabs, and exhumed materials can be used as sources of dna (chakraborty & stivers, 1996). in addition, dna-based systems are unaffected by blood transfusion (huckenbeck & rand, 1994) which has been known to result in erroneous conclusions in cases tested using conventional protein-based methods. numerous countries have reported the establishment of population databases relevant for dna-based paternity testing. in keeping with recent developments in forensic dna technology, a philippine population database of the national capital region (ncr) consisting of short tandem repeat (str) markers was constructed (halos et al., 1999). the seven str markers, comprising the database, namely d8s306, humfolp23, humth01, humvwa, humcsf1p0, humfes/fps, and humf13a01, with a combined power of paternity exclusion of 99.17%, are used to evaluate the results of dna tests employing wellestablished statistical parameters. in routine dnabased paternity tests, samples from paternity trios – the mother, the child, and the alleged father (af) – are obtained. however, to make testing more affordable, it was suggested that tests be performed using only samples from af-child pairs, i.e., motherless cases. in countries such as the united states, canada, germany, australia, and the united kingdom, where forensic dna technology is established, paternity testing guidelines have been formulated to aid in i n t e r p r e t i n g d n a r e s u l t s a n d i n u s i n g d n a evidence in court. these include requirements for a minimum number of mismatched markers prior to the exclusion of an af as father of the child (paternity exclusions), or a minimum value of the probability of paternity (w) prior to the presumption of paternity (paternity inclusions). w provides a numerical estimate for the likelihood of paternity of an af compared to the probability of a random match of two unrelated individuals. in many genetic testing laboratories, mismatches in at least two str markers are required for paternity exclusions ( m e r t e n s et al., 1 9 9 7 ) . h o w e v e r, d u e t o t h e probabilistic nature of paternity inclusions, w will never equal 100% and the minimum legally accepted value of w will vary in different localities/countries. legally accepted minimum w values in the united states range from 95.0% in new york to 99.9% in louisiana (www.cga.state.gov). in the philippines, similar guidelines and laws are not yet in place. in this study, we report the use of the philippine database in resolving questioned paternity issues in ten cases submitted to our laboratory, the result of testing only af and child pairs (motherless cases) and the effect of using population databases other than the philippine database in evaluating probabilities of paternity. the results of the present study will be used in formulating specific guidelines for dna-based paternity determination in the philippines. materials and methods sources of samples ten questioned paternity cases submitted to our laboratory, consisting of five paternity exclusions and five paternity inclusions, were used in the present study. brief case descriptions are listed in table 1. motherless cases were simulated using only the dna profiles of the af and child for statistical analysis. dna extraction blood samples were collected from the af, the mother, and the child, blotted on fta™ cards (flinders technologies pty ltd., fitzco inc.), and processed following manufacturer’s instructions. pcr amplification for amplification at seven str loci, unlabeled primers (gibco-brl, life technologies, gaithersburg, md) and cy5-labeled fluorescent primers (genset oligos, singapore) were used. for each 25 µl reaction, two fta™ discs (2 mm in diameter) were placed in a 0.2 ml pcr tube and amplified as described earlier (halos et al., 1999). 9 de ungria et al. dna fragment analysis amplified products were separated by size using a high resolution reprogel™ (amersham pharmacia biotech, sweden) and the alf express™ unit (amersham pharmacia biotech) according to manufacturer ’s instructions. sizes of pcr products were compared with those of allelic ladders as previously reported (halos et al., 1999). statistical analysis the probability of paternity (w) of paternity trios and simulated motherless cases in instances of paternity inclusions were calculated using dnaview™ program (brenner, 1997). the w values of paternity trios and the corresponding motherless cases (designated as w-mother) were compared. calculations of probability of paternity using various population databases the w and w-mother of each of the paternity inclusion cases were calculated using published genotypic frequencies of 11 other populations compiled by the dna serology group at the university of duesseldorf, germany (http://www.uniduesseldorf.de/ www.medfak/serology/dna.html). the population databases included in the study were from the autochthonous basque region (garcia et al., 1998a; garcia et al., 1998b); brescia region of north italy (cerri et al., 1998); pomerania-kujawy region of poland (miscicka-sliwka et al., 1998); north portugal (gusmao et al., 1995; lurdes-pontes et al., 1998); northeast spain (crespillo et al., 1997); usa caucasoid and african american populations (smith, 1997); french caucasoid population of q u e b e c , c a n a d a ( b u s q u e e t a l . , 1 9 9 7 ) ; a n d caucasoid (buenos aires), mapuche (rio negro province), and wichi (salta province) populations of argentina (sala et al., 1998). these population databases were selected based on the availability of the five str loci humf13a01, humfes/fps, h u m v wa , h u m c s f 1 p o , a n d h u m t h 0 1 . p o p u l a t i o n d a t a b a s e s w i t h t h e d 8 s 3 0 6 a n d humfolp23 markers were not available so w and w-mother values were calculated using only the d n a p r o f i l e s a t f i v e s t r m a r k e r s o f e a c h population including the philippines. the 11 population databases were grouped as nonphil, and the modal w and w-mother values were compared with those of the philippine database. results paternity exclusion cases the absence of common alleles (also called an incidence of a mismatch) as shown in fig. 1, was detected between the af and the child when the mother’s dna profile was known (cases 1 to 5). the presence of at least two mismatches in each of these cases results in the exclusion of the af from being the biological father of the child (w=0%). on the other hand, fewer mismatches between af and child were detected without the mother’s genotype (table 2) due to the presence 10 table 1. description of questioned paternity cases 1 2 3 4 5 6 7 8 9 10 criminal criminal civil civil civil civil civil civil civil civil sexual assault/ accused imprisoned > 5 years/ child > 7 years sexual assault/ accused imprisoned > 1 year/ child > 2 years recognition of illegitimate child/ child > 15 years woman has two lovers, needs to know the real father of her child/ child < 5 years petition for illegitimate child (immigration)/ child > 3 years child support (annulled marriage)/ child > 5 years recognition of illegitimate child by the father’s family/ child < 1 month girlfriend is suspected of having another relationship/ child < 2 years recognition of illegitimate child by the father/ child > 4 years continued support for illegitimate child/ child > 12 years cases nature of case case descriptions resolving questioned paternity issues fig. 1. (a) paternity exclusion at str locus d8s306. the absence of any allele-sharing between af and child (mismatch) indicates non-paternity. in this situation, the mother’s dna profile is no longer needed to exclude the af as the biological father of the child. (b) paternity exclusion at str locus humfolp23/dhfrp2. this case illustrates the necessity of obtaining the mother’s profile in some cases. without the mother’s profile, it appears that the child and the af share allele 7. with the mother’s profile it becomes evident that since the child shares allele 7 with the mother, then the child does not share any allele with the af. the af is therefore excluded as being the biological father of the child. 4 5 1076 8 9 11 12 d8s306 allelic ladder 250 265 270 275 280 285 290255 260 295 98 6 8 10 11 4 5 1076 8 9 11 12 positive control k562 alleged father child d8s306 allelic ladder negative control a by chance of similar alleles in the af and the child’s mother. it is possible that a random match between the af and child alleles masks a paternity exclusion if the paternal alleles are not properly identified in the child’s dna profile. moreover, although a mismatch in a single str marker does not automatically exclude an af (case 3) the presence of a mismatch is more consistent with a paternity exclusion, than a paternity inclusion. in case 2 no mismatch was detected between the af and child in the absence of the mother’s dna profile since the af and the child’s mother possessed similar alleles (fig. 1b). the absence of a mismatch between af and child already suggests possible paternity. however, the low w-mother value (w-mother = 73.26%) indicates that t h e a f a n d t h e c h i l d s h a r e a l l e l e s w h i c h commonly occur in the philippine population, and dhfrp2 allelic ladder positive control k562 mother child alleged father dhfrp2 allelic ladder negative control b 1076 8 9 87 7 87 6 7 1076 8 9 145 150 155 160 165 170 175 11 3/7 0/7 1/7 2/7 2/7 4/7 2/7 3/7 3/7 4/7 1 2 3 4 5 table 2. number of mismatches between af and child in paternity exclusion cases using seven str markers case no. no. of mismatches between af and child with mother’s dna profile without mother’s dna profile de ungria et al. the weight of dna evidence to support paternity is considerably less compared to higher w-mother values. paternity inclusion cases in cases where no mismatches were detected with and without the mother’s dna profile (fig. 2), the probability of paternity estimates using seven str markers range from 96.48% to 99.98% and 79.71% to 99.49%, respectively (table 3). w values were higher than the corresponding w-mother values in all five cases that demonstrate the greater accuracy of dna test results when the maternal genotype is known. in cases 6 and 9 for example, w-mother was significantly lower than the corresponding w. probability of paternity values calculated using different population databases using 11 population databases, w and w-mother can be calculated for cases 6 to 10 (inclusion cases) in five str loci, namely, humvwa, humth01, humcsf1p0, humfes/fps, and humf13a01 (tables 4 and 5). some w and w-mother values derived from non-philippine databases were found 12 vwa allelic ladder positive control k562 mother child dhfrp2 allelic ladder negative control 1714 14 120 125 135 140 150 155 165 fig. 2. paternity inclusion at str locus vwa. in this case, the af is not excluded as the biological father of the child. the weight of the dna evidence is subsequently assessed by calculating probability of paternity (w). 181514 16 17 19 20 16 160145130 vwa allelic ladder 181514 16 17 19 20 1716 alleged father probability of paternity (%) w-mother table 3. probability of paternity (w) estimates in five paternity inclusion cases using seven str markers case no. w 6 7 8 9 10 79.71 99.49 99.34 83.97 98.10 96.48 99.98 99.98 98.70 98.60 table 4. comparison of probability of paternity (w) values calculated using 12 population databases and five str markers population databases+ case 6 case 7 case 8 case 9 case 10 probability of paternity w (%) philippines basque italy poland portugal spain us-cau us-afr canada argentina-c argentina-m argentina-w 81.97* 98.91 99.99 98.72 98.84 98.19 99.35 99.77 99.22 99.28 99.98 99.98 99.89 99.50 99.99 98.93 98.52 98.60 98.67 99.56 98.88 98.78 99.55 99.52 99.07 99.88 99.99 99.86 99.58 99.75 99.86 99.74 99.80 99.60 99.98 99.99 97.04 98.99 98.59 98.78 98.39 98.25 98.08 98.17 98.77 98.47 98.97 99.70 86.77* 97.25 97.53 95.76 97.50 96.80 96.88 99.13 98.26 97.55 99.84 99.73 + population databases include the philippine (ncr); autochthonous basque region; brescia region of north italy; pomerania-kujawy region of poland; north portugal; northeast spain; usa caucasoid and african american populations; french caucasoid population of quebec, canada; and caucasoid (buenos aires), mapuche (rio negro province), and wichi (salta province) populations of argentina; * values are less than the minimum legally accepted w (=95.0%). resolving questioned paternity issues to vary significantly from values calculated using the philippine database. for example, w values in cases 6 and 10, and w-mother values of cases 6, 8, 9, and 10 calculated using the philippine database are less than 95%. in contrast, w and w-mother values obtained using databases other than the philippine database, were significantly higher than the 95% minimum legal threshold value which in some localities, are sufficient proof of biological paternity. discussion the present study investigated the utility of seven str markers, namely, d8s306, humfolp23, humcsf1p0, humvwa, humth01, humfes/ fps, and humf13a01, in the philippine population database for resolving paternity disputes of paternity trio and motherless cases (table 1). most clients showed a preference for dna testing of father and child (motherless) to lower cost and reduce conflicts, particularly when the mother is unavailable or refuses to participate in dna testing. however, dna results of paternity exclusion and inclusion cases presented here show the decreased capacity of dna analysis to distinguish fathers from non-fathers when the mother’s dna profile is absent. paternity exclusions were definitive in all five cases (cases 1 to 5) when the mother’s dna profile was included in the analysis (table 2). to exclude a man as a possible father, many genetic testing laboratories (including ours) require two mismatches between af and child. false paternity exclusions due to a single mutation across one generation, e.g., from a man to his child have been reported (mertens et al., 1997). hence, this guideline was formulated to take into account the possibility of false paternity exclusion d u e t o a m u t a t i o n . t h e o c c u r r e n c e o f t w o simultaneous mutations in separate locations of the father’s dna in a single meiotic event is highly improbable, and the presence of mismatches in two str loci is considered sufficient to prove nonpaternity (w=0%). when cases 1-5 were analyzed as simulated motherless cases, two out of the five paternity exclusion cases presented here (cases 2 and 3) had inconclusive results. the absence of at least two mismatches between af and child in both cases is due to the presence of common alleles between the af and the child’s mother (fig. 1b). hence, the maternal alleles in both instances were erroneously identified as paternal alleles and the af in each case could not be excluded. generally, the number of excluding markers is lower in motherless cases (table 2). although a mismatch in a single str locus does not automatically exclude the af in case 3, this sole mismatch is more consistent with non-paternity (exclusion) than paternity (inclusion). to rule out the probability of a mutation, further testing, such as the use of additional str markers or testing the mother’s sample, must be conducted. in this instance, the initial result supporting non-paternity was confirmed when the maternal genotype was included in the analysis (table 2). case 2 qualifies as a paternity inclusion case since no mismatch was detected in all seven str loci without the mother’s dna profile, albeit the w-mother value (=73.26%) is low. the low w-mother value is due to shared alleles of the af and the child, which commonly occur in the philippine population. the result of the 13 72.20* 97.91 99.99 94.38* 94.28* 93.49* 96.87 98.11 96.57 94.45* 99.77 99.79 98.40 96.67 99.99 94.87* 91.41* 90.68* 93.65* 96.66 91.44* 91.01* 95.62 98.93 93.57* 99.44 99.99 99.12 98.63 99.31 99.27 99.22 99.14 98.35 99.93 99.99 81.81* 96.13 94.00* 94.45* 94.13* 91.52* 92.48* 90.74* 93.75* 92.95* 95.12 98.26 table 5. comparison of probability of paternity (motherless) w-mother values calculated using 12 population databases and five str markers philippines basque italy poland portugal spain us-cau us-afr canada argentina-c argentina-m argentina-w 87.42* 95.21 92.44* 91.97* 92.11* 92.56* 89.63* 93.58* 93.49* 89.81* 99.05 96.86 + as for table 4. population databases+ case 6 case 7 case 8 case 9 case 10 probability of paternity w-mother de ungria et al. dna tests for case 2 is particularly significant since the af was charged with sexually assaulting a mentallyretarded woman, resulting in the birth of a child. without the maternal genotype, the af would not have been excluded as a suspect and his conviction for a heinous crime was highly probable, although in this case, clearly erroneous. in the five paternity inclusion cases, the w values are greater than the minimum legally accepted value of 95.0% (table 3). however, the absence of the mother’s dna profile decreases the probability of paternity estimates (w-mother) that in some instances would significantly affect the result of dna tests. since without the maternal genotype, it is not possible to assign a paternal and maternal allele in the child’s genotype, the equation to derive w-mother assumes the paternal allele to be either one of the alleles found in the child (brenner, 1993). due to the additional uncertainty in the identity of the paternal and maternal alleles in the child’s genotype, w-mother values in cases 6 and 9 were less than the minimum legally accepted value of 95.0%. in these two cases, the issue of paternity was resolved only upon submission of the mother’s sample for further testing. however, it is worth noting that in the remaining six cases (excepting cases 2, 3, 6, and 9), dna tests on the af and child were sufficient to exclude a nonfather (cases 1, 4, and 5) or to predict probable paternity (cases 7, 8, and 10). for developing countries such as the philippines, where the cost of dna testing is prohibitive, initial routine analysis of af-child samples in at least s e v e n s t r m a r k e r s i s recommended. in the absence of the required two mismatches (probable paternity inclusions), or w-mother value > 99.0%, the mother ’s sample should be obtained for testing. because of the uncertainty introduced by the unfeasibility of assigning paternal and maternal alleles in motherless cases, setting the m i n i m u m w m o t h e r a t 9 9 . 0 % i s r e c o m m e n d e d . likewise, it is also ideal that minimum value of w should be set at 99.0% to reduce the risk of false matches. when w=95%, there exists a 5% probability that the matching profiles of the alleged father and child may be attributed to chance, for example case 2. a higher minimum w value (=99.0%) prior to the presumption of paternity requires testing to be conducted using additional str markers in some cases. work is underway in our laboratory to add more markers to the existing database in order to increase the discriminatory power of routine dna tests. the use of the appropriate population database to evaluate w and w-mother is also important. the philippines is a multicultural country, with over 100 ethno-linguistic groups, all of which belong to the austronesian family of language groups (hagelberg et al., 1999), a rich history of spanish, american, and japanese colonization and a large chinese population. due to their unique cultural background and colonial history, the profile of filipinos is likely to differ from that of other more well-studied races such as caucasians and blacks, and this may affect the results of dna tests. in two of the five cases presented here (cases 6 and 10), the use of nonphilippine databases resulted in w estimates that were significantly higher than those calculated using the philippine database (table 4). in these cases, which can be seen as false positives, use of non-philippine databases to evaluate the results of dna analysis leads to the recommendation that the alleged father is the biological father of the child. false positives can be attributed to variation in the distribution of alleles in different populations; since w values are dependent on allelic frequencies, high allele frequencies result in lower w values, while low allele frequencies give rise to higher w values. for example, allele 9 is the most common allele at locus humth01 in filipinos (frequency = 0.3933), whereas this allele is not common in other populations included in the present study (frequency = 0.0140 to 0.2100). sharing of alleles between alleged father and child that are common in filipinos, but relatively uncommon in other populations explains the significant variation in w values in cases 6 and 10. as a result of the decrease in the discriminatory power of dna tests without the mother ’s dna profile, variations in w-mother become more pronounced as a result of using different databases (table 5). in all five cases presented here, the alleged father may or may not be the father of the child depending on the population database used to calculate w-mother. these results support the use of the philippine genetic database as a reference database for dna-based 14 resolving questioned paternity issues paternity testing in the philippines, particularly in dna tests for motherless cases. conclusion the present study demonstrated the utility of a sevenstr philippine genetic database to resolve ten questioned paternity issues. results of the dna tests conducted on complete paternity trio cases either supported paternity (w values are greater than the minimum legally accepted value of 95.0%), or excluded the af as the biological father of the child due to the presence of at least two mismatches. however, dna results of exclusion and inclusion cases show the decreased capacity of dna analysis to distinguish fathers from non-fathers when the mother’s dna profile is absent. hence to reduce the overall cost, it is recommended that initial tests should be conducted o n a f c h i l d p a i r s t o s c r e e n f o r a t l e a s t t w o mismatches or w-mother > 99.0%. if the results remain inconclusive, further testing to include the mother’s sample should be conducted to increase the level of certainty of the dna tests. moreover, due to variations in the allelic frequencies observed in different populations, it is highly recommended that a philippine genetic database be used as the reference database for paternity determination in the philippines. there is no law regulating the use of dna-based paternity testing for paternity trio and motherless cases in the philippines. however, more and more cases have been filed in court requesting dna tests. clearly, current legislation for paternity determination needs to be amended to incorporate scientific advances in the field of dna-based paternity testing. recommendations such as those presented here will be used in the formulation of appropriate national legislation that reflect the current developments in dna-based paternity testing in the philippines. acknowledgments this work was funded by the natural sciences research institute, university of the philippines. the authors would also like to thank ms. liza p. faustino for technical assistance and ms. nota f. magno for editing the manuscript. references brenner, c., 1993. a note on paternity computation in cases lacking a mother. transfusion. 33(1):51-54. brenner, c.h., 1997. dnaview: a software package for forensic use. copyright 1989-1997. california, usa. busque, l., d. desmarais, s. provost, j.w. schumm, y. zhong, & r. chakraborty, 1997. analysis of allele distribution of six short tandem repeat loci in the french canadian population of quebec. j forensic sci. 42(6):11471153. cerri, n., a. decarii, f. zorzi, & f. de ferrari, 1998. a statistical analysis by means of linear models on italian strs population data. progr. forensic genet. 7:559-561. chakraborty, r. & d.n. stivers, 1996. paternity exclusion by dna markers: effects of paternal mutations. j. forensic sci. 41(4):671-677. crespillo, m., j.a. luque, r. fernandez, e. ramirez, p. garcia, & j.l. valverde, 1997. allele frequency distribution of 13 pcr-based systems in the population from north-east spain. int. j. legal med. 110:223-225. garcia, o., p. martin, b. budowle, j. uriarte, c. albarran, & a. alonso, 1998a. basque country autochthonous population data on 7 short tandem repeat loci. int. j. legal med. 111:162-164. garcia, o., p. martin, j. uriarte, c. albarran, & a. alonso, 1998b. allele frequency distribution of 7 str loci in the basque country autochthonous population. progr. forensic genet. 7:282-284. gusmao, l., m.j. prata, & a. amorim, 1995. the str system htpo: population and segregation data. int. j. legal med. 108:167-169. hagelberg, e., m. kayser, m. nagy, l. roewer, h. zimdahl, m. krawczak, p. lio, & w. schiefenhovel, 1999. molecular 15 de ungria et al. genetic evidence for the human settlement of the pacific: analysis of mitochondrial dna, y chromosome, and hla markers. phil. trans. r. soc. london. b 354:141-152. halos, s.c., j.c. chu, a.c.m. ferreon, & m.m. magno, 1999. philippine population database at nine microsatellite loci for forensic and paternity applications. forensic sci. int. 101:27-32. huckenbeck, w. & s. rand, 1994. serological findings and efficiency of dna profiling in transfused patients and their significance for identity and paternity tests. int. j. legal. med. 106:178-192. jeffreys, a.j., a.v. wilson, & s.l. thein, 1985. individualspecific ‘fingerprints’ of human dna. nature. 316:76-79. lurdes-pontes, m., m.f. pinheiro, e. huguet, m. gene, & j . p i n t o d a c o s t a , 1 9 9 8 . a u t o m a t e d t y p i n g o f 4 tetrameric str: a north portugal database. progr. forensic genet. 7:335-337. markowicz, k.r., l.a. tonelli, m.b. anderson, d.j. green, g.l. herrin, r.w. cotton, j.l. gottschall, & d.d. garner, 1990. use of deoxyribonucleic acid (dna) fingerprints for identity determination: comparison with traditional paternity testing methods -part ii. j. forensic sci. 35(6):1270-1276. mertens, g., n. mommers, h. heylen, m. gielis, a. muylle, & a. vandenberghe, 1997. allele frequencies of nine str systems in the flemish population and application in parentage testing. int. j. legal med. 110:177-180. miscicka-sliwka, c.j., t. grzybowski, & m. wozniak, 1998. population genetics of 14 strs: vwa, th01, tpox, csf1po, d5s818, d13s317, d7s820, d16s539, f13a01, fes/ fps, f13b, lpl, ds31358, and fga in the pomerania-kujawy region of poland. progr. forensic genet. 7:261-263. sala, a., g. penacino, & d. corach, 1998. comparison of allele frequencies of eight str loci from argentinean, amerindian, and european population. hum. biol. 70: 937947. smith, t.a., 1997. west virginia state police. http:// www.uniduesseldorf.de/www.medfak/serology/dna.html. 16 01_device nutraceuticals: dietary flavonoids 9 irene m. villaseñor institute of chemistry, university of the philippines, diliman, quezon city 1101 irene.villasenor@up.edu.ph nutraceuticals: dietary flavonoids flavonoids include many of the most common plant pigments. they are consumed as part of the human diet. flavonoids can be divided into several classes depending on their structure. they are responsible for much of the flavor and color of fruits and vegetables. the flavones and flavonols give yellow or orange colors, the anthocyanins red, violet or blue colors. the aurones are golden yellow pigments while flavanones and flavanonols are either colorless or only slightly yellow. this mini review highlights the sources of dietary flavonoids, their bioactivities, and their mechanisms of actions. dietary flavonoids provide both health (nutrition) and medical (pharmaceutical) benefits. dietary flavonoids are preventives or prophylactics rather than therapeutics. the flavonoids are just one of the many classes of nutraceuticals present in our diet. dietary flavonoids sources and uses common flavan-3-ols present in fresh fruits, vegetables, and nuts are catechin, catechin gallate, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, gallocatechin, and gallocatechin gallate (harnly et al., 2006). dietary flavonoids also include the anthocyanins cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin together with the flavanones hesperetin and naringenin, the flavones apigenin and luteolin, and the flavonols myricetin, kaempferol, and quercetin. quercetin is the most abundant flavonol. it is present in black and green teas, onions, apples, grapes, beans (somerset & johannot, 2008), broccoli (vasanthi et al., 2009), lettuce (ribas-agusti et al., 2011), berries (harnly et al., 2006), oregano (mueller et al., 2008), mangoes (wilkinson et al., 2008), and tomatoes (slimestad et al., 2008), among others. yellow and red onion bulbs contain 270-1187 and 415-1917mg flavonols mg/kg fresh weight, respectively, as well as anthocyanins (39240 mg/kg fresh weight) and dihydroflavonols (slimestad et al., (2007). berries are also rich in anthocyanins while black and green teas are also sources of flavan-3-ols (somerset & johannot, 2008). citrus fruits contain flavanones. coffee contains myricetin. the major flavone sources are parley, celery, and english spinach while the major anthocyanidin source is wine. tea, legumes, and wines contributed to 48% of daily intake of proanthocyanidins (wang et al., 2011). durian, pomelo, guava, and ripe banana are good sources of flavonoids (kongkachuichai et al., 2010). differences in cultivars and growing conditions may lead to differences in the relative amounts of dietary flavonoids. natural food-derived flavonoids may help reduce the incidence of atherosclerosis (terao, 2009), cancer (gibellini et al., 2011; mishra et al., 2011; clere et al., 2011; nishiumi et al., 2011; yao et al., 2011), cardiovascular diseases (bojic et al., 2011, hubbard et al., 2006), diabetes (zheng et al., 2011), thrombosis (phang et al., 2011), inflammation in arthritis, asthma, encephalomyelitis, and atherosclerosis (gonzalez et al., 2011), neurodegenerative diseases such as alzheimer’s and parkinson’s diseases (mandel et al., 2011; craggs & kalaria, 2011), obesity (birari et al., 2011; bell et al., 2011), hyperlipidemia (wang et al., 2011), nerve injury (jager & saaby, 2011), and hypertension (cassidy et al., 2011). in vitro studies and animal models apples contain high levels of flavonols, catechins, and oligomeric procyanidins. red apples are rich in anthocyanidins. apple extracts prevented skin, mammary and colon cancers in animal models (gerhauser, 2008). apple extracts also exhibited antiscience diliman (january-june 2011) 23:1, 9-16 villasenor, i.m. 10 inflammatory activity (lauren et al., 2009). red wine, which is rich in anthocyanins and flavonoids, inhibited the growth of human breast cancer cells (mcf-7) with relatively low cytotoxicity towards normal human mammary epithelial cells and a non-tumorigenic mcf10a cell line (hakimuddin et al., 2004). broccoli provided anti-oxidants, regulated enzymes and controlled apoptosis and cell cycle (vasanthi et al., 2009). flavored black teas, with higher levels of catechins, quercetin, and rutin, showed stronger anti-oxidant activity than fruit teas, which contained higher levels of naringin and hesperidin (pekal et al., 2011). bananas also showed antioxidant activity against rats fed normal as well as high fat diets (vijayakumar et al., 2008). the concentrations of peroxidation products were significantly decreased whereas the activities of catalase, superoxide dismutase, and glutathione were significantly enhanced. the antioxidant activity of strawberry against pc12 cells, a model system for neuronal differentiation, treated with hydrogen peroxide was higher than banana and orange. the reduction of oxidative stress-induced neurotoxicity and the higher neuroprotective activity of strawberry may be due to its higher anthocyanins content compared to banana and orange (heo& lee, 2005). fruits and vegetables rich in anthocyanins, such as strawberry, raspberry and red plum, were potent antioxidants, followed by those rich in flavanones, such as orange and grapefruit, and flavonols, present in onion, leek, spinach and green cabbage, while the hydroxycinnamate-rich apple, tomato, pear and peach consistently elicited lower antioxidant activities. the antioxidant activities (teac) in terms of 100 g fresh weight uncooked portion size were in the order: strawberry>> raspberry = red plum >> red cabbage >>>grapefruit = orange > spinach > broccoli > green grape e” onion > green cabbage > pea > apple > cauliflower> tomato e” peach=leek >banana e” lettuce (proteggente et al., 2002). quercetin inhibited the growth of several human cancer cell lines at different phases of the cell cycle by direct pro-apoptosis with almost total absence of damage for normal, non-transformed cells (gibellini et al., 2011). quercetin also inhibited the activation of peroxisome proliferator-activated receptor isoforms (ppars) alpha, beta and gamma (wilkinson et al., 2008). ppars are drug targets in metabolic syndrome, wherein obesity, hyperglycemia, hypertension, and hyper/dyslipidemia are all present (mueller et al., 2008). quercetin and myricetin blocked the genotoxic effects of some cooked-food mutagens (alldrick et al., 1986). the most potent anti-aggregatory activity was shown by 3,6dihydroxyflavone and syringetin (bojic et al., 2011). luteolin was more cytotoxic than apigenin against human chronic myelogenous erythroleukemia (k562) and bladder carcinoma (rt112), mainly due to induction of apoptosis (kilani-jaziri et al., 2011). apigenin was able to induce apoptosis in human chronic lymphocytic leukemia (eheb) cell line (hashemi et al., 2010). apigenin, epigallocatechin gallate, delphinidin and genistein appear to be beneficial compounds in various stages of carcinogenesis (clere et al., 2011). genistein, an isoflavone present in soy, prevents breast, prostate, and colon cancer (ullah et. al., 2011). epidemiological studies several population-based studies suggested that intake of dietary flavonoids is associated with health. in the netherlands, the average intake of dietary flavonoids is 23 mg/day. the important sources of dietary flavonoids were tea, onions, and apples (hertog et al., 1993). tea was also the main dietary source of flavonoids in the uk but this tea-drinking population has a high incidence of colorectal cancer (kyle et al. 2010). results of a population-based case-control study showed that non-tea quercetin reduced the incidence of colon but not rectal cancer. in the rotterdam study, tea drinkers showed a lower relative risk of incident myocardial infarction (geleijnse et al., 2002) while in the zutphen study, habitual intake of dietary flavonoids, with tea as major source, protected them against stroke (keli et al., 1996). in a double-blind randomized cross-over study, ingestion of onion soup inhibited collagen-stimulated platelet aggregation, which may lead to reduced risk of thrombosis and potential cardiovascular disease (hubbard et al., 2006). in a study of japanese women, their high intake of flavonoids and isoflavones, whose major sources are onions and tofu, respectively, led to science diliman (january-june 2011) 23:1, 9-16 nutraceuticals: dietary flavonoids 11 low plasma total cholesterol and plasma low density lipoprotein (ldl) cholesterol concentrations, which may explain the low incidence of coronary heart disease among japanese women (arai et al., 2000). populations with high intake of isoflavones through soy consumption have lower incidence of breast, prostate, and colon cancers (ullah et al., 2011). among the finnish population, intake of apples, the major source of quercetin, decreased the relative risk of thrombotic or embolic stroke but quercetin intake was not associated with the incidence of cardiovascular disease (knekt et al., 2000). a cross-sectional study of u.s. women showed that intake of flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and polymeric flavonoids led to modestly lower concentrations of inflammatory and endothelia dysfunction markers (landberg et al., 2011). prospective epidemiologic studies and several short term controlled randomised clinical trials have shown that consumption of some foods or beverages with high flavonoid content, especially flavanols and anthocyanins, lowered the incidence of coronary stroke mortality or prevalence of neurodegenerative diseases including alzheimer’s and parkinson’s diseases (stoclet & schini-kerth, 2011). samples tested were red wine, some grape juices, red fruits, tea, and cocoa. black tea and green tea catechins also protected the aging brain and reduce the incidence of dementia and neurodegenerative diseases (mandel et. al., 2011). results of a cross-sectional survey of participants, with known childhood intelligence quotients (iq), who carried out various neuropsychological tests at age 70 did not show a relationship between flavonoids intake and the prevention of cognitive decline. total fruit, citrus fruits, apple and tea intakes were initially found to be associated with better scores in a variety of cognitive tests, but the associations were no longer statistically significant after adjusting for confounding factors, including childhood iq (butchart et al., 2011). a four-year intervention study on adenoma recurrence of colorectal cancers showed that interleukin single nucleotide polymorphisms (il snps), in combination with a flavonol-rich diet or decreased serum il, lowered the risk of adenoma recurrence (bobe et al., 2011). in an observational osteoporosis study of peri-menopausal scottish women, their mean dietary flavonoid intake was 307 ±199 mg/d with catechins contributing the most to flavonoid intakes (55%). annual % change in bone mineral density was associated with intakes of procyanidins and catechins, and flavanones were negatively associated with bone resorption markers (hardcastle et al., 2010). in a prospective study, the habitual intake of flavonoids by participants was associated with hypertension. during 14 years of followup, it was established that anthocyanins, some flavone and flavan-3-ol compounds showed vasodilatory activities and may contribute to the prevention of hypertension (cassidy et al., 2011). mechanisms of action the anti-carcinogenic effects of dietary flavonoids may be due to their antagonistic effect on the aryl hydrocarbon receptor (ahr), and regulation of phase i and ii drug metabolizing enzymes and phase iii transporters. dietary flavonoids also modulated cell signal transduction pathways (nishiumi et al., 2011) related to cellular proliferation, apoptosis, and angiogenesis (yao et al., 2011) in various tumor cells and animal models (shanmugam et. al., 2011) as well as pro-inflammatory and oncogenic signals (fraga and oteiza, 2011). apigenin increased the cellular levels of cd26 on ht-29 and hrt-18 human colorectal cancer cells. cd26 is a multifunctional cell-surface protein that through its associated dipeptidyl peptidase (dppiv) and ecto-adenosine deaminase (eada) enzyme activities is able to suppress pathways involved in tumour metastasis (lefort&blay , 2011). genistein-induced apoptosis is inhibited by reactive oxygen species (ros) scavengers and by mobilization of endogenous copper, thus disabling a copper specific chelator from inhibiting apoptosis (ullah et al., 2011). some dietary flavonoids modified protein kinases mediated signal transmission inducing antioxidant and anti-inflammatory genes expression and inhibiting oxidant and inflammatory gene expression (stoclet & schini-kerth, 2011). molecular targets, such as cyclooxygenase and lipoxygenase, and cellular targets, such as macrophages, lymphocytes, epithelial cells, and science diliman (january-june 2011) 23:1, 9-16 villasenor, i.m. 12 endothelium, have been used in identifying the mechanism of anti-inflammatory activity. flavonoids, despite their structural differences, consistently inhibited nf-êb(nuclear factor kappa-light-chain-enhancer of activated b cells)signaling and down-regulated the expression of pro-inflammatory markers in myeloid cells (gonzalez et al., 2011).assessment of the antiinflammatory mechanism of action of flavonoids showed that citrus fruits were modestly associated with lower plasma il-18 concentrations while higher intake of grapefruit was significantly related with lower concentrations of plasma c-reactive protein (crp) and soluble tumor necrosis factor receptor-2 (stnf-r2) (landberg et al., 2011). in the central nervous system, several flavones bind to the benzodiazepine site on the gaba(a)-receptor resulting in sedation, anxiolytic or anti-convulsive effects. flavonoids also inhibited monoamine oxidase a or b and functioned as anti-depressants, which may improve the symptoms of parkinson’s disease (jager & saaby, 2011). the neuroprotective effects of green tea catechins may be due to their ability to modulate intracellular neuronal signaling and metabolism, cell survival/death genes, and mitochondrial function as well as their anti-oxidant activity (mandel et al., 2011). rats fed with a diet containing 0.5% quercetin showed a relative increase in the levels of lactate, low-density lipoprotein/very low-density lipoprotein (ldl/vldl), and serum low-density lipoprotein cholesterol (ldlc) and a relative decrease in glucose, high-density lipoprotein (hdl), and some amino acids (zhao et al., 2011). biochemical measurements confirmed that the serum level was increased significantly after quercetin treatment, indicating that quercetin can induce a remarkable change in cholesterol metabolism. flavonoids also acted on reactive oxygen species (ros) (yao et al., 2011).dietary flavonoids may directly scavenge or quench ros as well as inhibit oxidative enzymes that generate these ros (terao, 2009). antioxidants are capable of preventing ros-induced oxidative damage or oxidative stress, which contributes to the initiation and progression of several chronic diseases. prevention of the formation and reaction of ros by antioxidants decreased the risk of major diseases. for example, the oxidation of low density lipoprotein (ldl) cholesterol yields a product that damages the vascular system. thus, a lower intake of saturated fats to decrease the levels of ldl cholesterol, together with an adequate intake of antioxidants, is the optimal approach to lower heart disease risk (weisburger 1999). direct-acting carcinogens and their formation are inhibited by antioxidants. the growth, cell proliferation and development of abnormal preneoplastic and neoplastic cells are also prevented by antioxidants. even the ageing process is slowed down by antioxidants. metabolism of dietary flavonoids flavonoids are found in plants as glycosides. before oral absorption, flavonoids undergo deglycosylation either by lactase phloridzin hydrolase or cytosolic âglucosidase. the absorbed aglycone is then conjugated (jager & saaby, 2011) forming glucuronide, methyl, and sulphate derivatives, which may have different bioactivities than the parent flavonoid.in simulated gastric digestion, anthocyanin glycosides remained stable but their aglycones were significantly degraded. during subsequent pancreatic/intestinal digestion only pelargonidin-3-glucoside remained stable while cyanidin-3-glucoside, cyanidin, and pelagonidin aglycones were completely degraded. after microsomal metabolism, pelargonidin formed 4-hydroxybenzoic acid, which was metabolized to form two additional glucuronide conjugates, while cyanidin formed protocatechic acid, which was further metabolized to form three glucuronide conjugates (woodward, et al., 2011). methyl derivatives of quercetin, 3'-o-methyl-quercetin and 4'-o-methyl-quercetin, exhibited anti-inflammatory activity by blocking the expression of intercellular adhesion molecule-1 (icam-1) while quercetin-3-oglucuronide, quercetin-3'-o-sulfate, and phenolic acid metabolites of quercetin did not inhibit adhesion molecule expression. 4',4'’-di-o-methyl-epigallotechin3-o-gallate (egcg) inhibited icam-1 expression but egcg and 4'’-o-methyl-egcg had no effect (lotito et al., 2011). cooking reduced antioxidant activity and phenolic content (kuti and konuru, 2004). science diliman (january-june 2011) 23:1, 9-16 nutraceuticals: dietary flavonoids 13 references a.j. alldrick, j. flynn & i.r. rowland, 1986. effects of plantderived flavonoids and polyphenolic 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e-mail: jon@msi01.cs.upd.edu.ph the coastal development plan that was passed into law as the municipal fisheries ordinance of bolinao, pangasinan, is a concrete example of community participation in policy development. among the effective metalegal strategies used during the evolution of the plan into an ordinance were the lobbies staged by the federation of people’s organizations (kaisaka), the municipal fisheries and aquatic resources management council (mfarmc), and the municipal mayor. the capacity of various sectors to participate actively in the passage of the plan was enhanced through legal consultations and training provided by the marine fisheries resources management project, in partnership with tanggol kalikasan, the legal arm of haribon foundation. the experience underscores the need for development projects to include the metalegal training of community constituents as a requisite for the latter to actively participate in the formulation of policies and laws for coastal resources management. key words: metalegal training, coastal development planning, cbcrm introduction the coastal waters of bolinao are considered to be one of the most diverse areas in lingayen gulf. yet many resource users have threatened the integrity of this coastal area. to address this problem, a number of management approaches have been introduced to protect the resources from degradation and overexploitation. one of the approaches used is the institutionalization of people’s organizations or volunteer action groups to manage the resources. the institutionalization of these groups had been a long drawn process for both the communities and the project facilitators. from calling introductory meetings to participating in political processes and actually shaping local policy, the communities, the organizers, as well as the local government had gone through numerous challenges to forge a functional partnership. the objectives of this paper are to: (1) summarize the process undergone by the various sectors in bolinao to formulate a coastal development plan (cdp) and a municipal fisheries ordinance (mfo); (2) highlight the importance of metalegal training and legal intervention as part of the empowerment process of local communities, including local government units, in the sustainable management of marine fisheries resources; and (3) draw lessons in participatory planning so that may be replicated in other municipal planning exercises. * corresponding author asido & talaue-mcmanus 102 coastal development planning the coastal area, like the rest of the marine environment, is perceived as the common patrimony of a nation. as is the fate of many other public properties in the philippines, it is open access for the citizenry. this open access regime has subjected coastal resources to overexploitation to a level that renders them unable to support the needs of the people, and which eventually results in heightened conflicts between and among users. this situation exacerbates the poverty issue in most of the coastal barangays, as exemplified by the displacement of sustenance fishers by fishpen and fish cage operators, among others. often, the use of the commons is rationalized through government planning and articulated as policies. the traditional and usual planning process in the local government can be characterized as top down or table planning. municipal development planning, as stipulated in section 444 of the local government code (lgc) of 1991 is a function of the municipal mayor, with the support of the municipal planning and development coordinator (mpdc) and the municipal local government operations officers (mlgoo). the role of the municipal council is limited to legislation. although mandated by the lgc, people’s participation is being ignored in the planning process particularly by the elected officials. this approach in planning contradicts the essence of people empowerment. participatory coastal development planning is an innovation in development planning especially in the fields of local government administration and coastal resources management (crm). it is a deliberate social and organizational activity that involves the preparation of a set of decisions and strategies for action in the future, and which are directed towards achieving goals and/or options. the planning should have the following elements (alexander 1992): (1) clear formulation of the goals that planning seeks to achieve (the purposes and broad outcomes desired from management); (2) identification of the current situation, including an environmental analysis of both the internal organizational factors and external consideration (often termed as swot– strengths, weaknesses, opportunities and threats– analysis ); (3) determination of objectives clearly stating the desired outcomes (goal should be specific, time bound, and measurable); (4) analysis of the difference between the current and desired situations; (5) definition of strategies; (6) implementation of strategies; and (7) monitoring, evaluation, and review. project facilitation two projects facilitated the development and current implementation of the cdp. the community-based coastal resource management project (cb-crmp) played an important role in the management of coastal resources in bolinao. the cb-crmp began in 1993 as a collaborative action research project of the university of the philippines-marine science institute (up-msi), the university of the philippines-college of social work and community development (upcswcd), and the haribon foundation for the conservation of natural resources (haribon). the cb-crmp was supported by a grant from the international development research center (idrc). the cb-crmp aimed “to develop an integrated program of approaches, strategies and action plans through an interactive and interdisciplinary research process, by which the bolinao community could evolve into an active, consolidated and self-reliant community, collectively nurturing and equitably benefiting from the sustainable management of its coastal resources,” (1993 project workshop). one of the major outcomes of the cb-crmp was the cdp which was formulated through a participatory process. the marine fisheries and resources management project (mfrmp) succeeded the cb-crmp in 1997. since the commencement of its operations, the project has been involved in organizing, training, resource enhancement and management initiatives in various barangays all over the municipality. a main activity was the formalization of the passage of the bolinao cdp through continuous advocacy and technical support. more importantly, to realize the development plan implementation, the project had four major components: (1) capability building; (2) resource enhancement; (3) coastal zoning; and (4) harvest regulation. the planning process traditionally, crm at the municipal level in the philippines, as in bolinao, moves around raising revenues for the local government unit (lgu) through taxation participatory metalegal and legal processes 103 of resources and/or area use (kalagayan 1992). for instance, the municipality of bolinao made almost p4m from milkfish fry and siganid concessions and leases for fishpens and cages in the early part of 1997. while these seem profitable, the municipal fisherfolk do not actually benefit from these. there is an observed lack of clear management strategy, and of a holistic plan in managing coastal resources. meanwhile, the successful campaign against the proposed cement plant in bolinao, which polarized the community, showed that the people have chosen environmental protection over unregulated development. this realization brought about the idea of formulating cdp for bolinao. the development of the bolinao cdp was divided into three major stages: the preparatory stage, the legislation stage, and the implementation stage. even while the process was in the legislation stage, there were provisions of the cdp which were already being implemented. the preparatory stage took the most time. initial discussions about the cdp began as early as 1994, at the cb-crmp. it was largely a reaction to the then brewing cement plant issue. in 1996, an orientation on coastal zoning was conducted for the four existing fisherfolk organizations in the municipality. the result was a resource use map indicating their proposed management activities for bolinao. this was a product of their discussions of coastal management-related issues, problems, and concerns, and proposed management actions and options. parallel efforts to introduce coastal management planning were also directed towards lgu officials and employees. although the efforts were temporarily stalled by the cement plant controversy, lgu acceptance of the cdp was realized afterwards. continuous information campaign and capacity-building activities were undertaken to prepare the communities for the planning process; secondary data were gathered and analyzed, and resource and socioeconomic maps were prepared. in fact, the cdp could be considered as one instrument the communities and the lgu could claim ownership of. as a sign of unquestionable support for the effort, the municipal mayor issued eo no. 6, series of 1996, formally constituting a 21-member multi-sectoral committee, and giving it a p100,000 budget. the multisectoral technical working group (cdp-twg) was tasked to draft the provisions of the cdp in consultation with the sectors they represented. it was agreed that a combination of all possible approaches would be considered to ensure that the active participation of the community and the municipal government was engendered. the process was then conceptualized with the intention of formulating a plan that would regulate coastal resource use, resolve conflicting human activities, and develop a model planning process, using a participatory approach. through the cdp, the coastal waters were divided into four zones. the cdp also provided for regulatory mechanisms for municipal fisheries, including the granting of fishery privileges. it highlighted conservation, protection, and resource rehabilitation. it also called for the creation of a bolinao coastal development and management council (bcdmc), a multi-sectoral body that would coordinate the implementation of the plan. the finalized cdp was submitted to the municipal government for adoption. copies were also given to the regional offices of various agencies and departments, such as the national economic development authority (neda), the department of agriculture (da), the department of interior and local government (dilg), the department of environment and natural resources (denr), and the lingayen gulf coastal area management commission (lgcamc). on january 19, 1998, the sangguniang bayan of bolinao adopted sb resolution no.6, s. 1998, approving the plan. the approved plan was submitted to the provincial council for review. the provincial council would later approve the fishery ordinance resulting from the cdp. throughout 1998, the adopted cdp was redrafted into an ordinance. in february 1998, the philippines fisheries code was passed and set the mold for municipal fisheries ordinances in general. the code also provided for the creation of municipal fisheries and aquatic resources management councils (mfarmc), multi-sectoral groups that would assist local governments in addressing fisheries issues. in bolinao, the mfarmc was created in february 1999 and strengthened during the first half of that year. to transform the approved cdp into an ordinance, the municipal council held a series of public hearings from july to september 1999, with the assistance of the asido & talaue-mcmanus 104 mfarmc. the mfarmc prepared a report reflecting the results of the public hearings. major changes in the cdp included the removal of the bcdmc provision because it would overlap with the functions of mfarmc and the addition of a provision to establish a crm office to be led by a crm officer. the crm officer shall have the following functions, duties, and powers: (1) formulate measures to ensure the delivery of basic services and provision for adequate facilities relative to coastal and fishery resource management; (2) develop plans and strategies and implement the same, particularly those which have to do with coastal resource management and fisheries resources programs and projects; (3) extend technical assistance to and ensure equitable access to municipal coastal and fisheries resources and municipal fisherfolk in the context of sustainable management and use of coastal and fisheries management; (4) enforce laws, administrative orders, rules and regulations, and ordinances relating to coastal resources and fisheries management; and (5) coordinate with government agencies, non-governmental organizations and people’s organizations that seek to promote sustainable management of coastal and fisheries resources, among others. the objective of the crm office is to attend to the needs of the coastal and fisheries resources of the municipality. the municipal fishery technician would function as crm officer. the crm office became functional in february 2000. a report on the inputs derived from the public hearings was presented during a special session of the sangguniang bayan on december 10, 1999. on the same day, the cdp was enacted as “the bolinao coastal and fisheries resources management ordinance of 1999.” by january 2000 the cdp became a municipal ordinance, an official and legally binding plan for implementation. it is significant to point out that the mfrmp facilitated the execution of action plans which, in essence, were activities implementing the cdp since 1998. metalegal strategies in coastal development planning because a coastal development plan has to evolve into a legal instrument, the participation of various community sectors had to be enhanced through the use of metalegal strategies. metalegal strategies, or tactics, are creative actions employed by the basic sectors to further their particular interests in cases where the relief or remedy provided by law is too slow or non-existent. they are, therefore, not prescribed by law nor are they prohibited by it. metalegal strategies derive from the basic rights of the people to express themselves freely, by speech, press, and assembly, and to petition the government for redress of grievances. they are also anchored on the freedom to form associations, and, most of all, on the right to self-determination. before employing metalegal tactics, it is necessary to appraise the group involved about the situation and the nature of metalegal tactics in general. the issues and demands should be made clear to all who will participate in decision making. their reasons and purposes, and their plan of action have to be drawn, as well as the general conduct of implementation. in planning metalegal tactics, a group has to assess its strengths and weaknesses. it has to be certain of its resources, support, and security. proper timing and relevance are essential, and alternative plans should be made for all possible scenarios. it is important to use all contacts in the media to ensure additional pressure and favorable public opinion. the mfrmp sought the help of paralegals to facilitate metalegal training for the cb-crmp. the scarcity of lawyers who are engaged in advocacy for the basic sectors gives paralegals the task of performing many things traditionally undertaken only by lawyers, and also of educating people who need legal services so that they can be freed from dependence on lawyers. the composite of activities provided by paralegals is called developmental legal aid. this includes studying the legal system so that enlightened and critical action within the legal system can take place for the welfare of the majority. it engages in paralegal education and uses as primary tools creative tactics, including legal remedies using the people’s initiatives and available resources. kaisaka lobby as a metalegal strategy used by the bolinao community in the process of coastal development planning, the kaisaka lobby was focused on consultations, lobbying, and networking with the local government of bolinao, including the office of participatory metalegal and legal processes 105 the sangguniang bayan. consultations with neighboring barangays were regularly conducted. community meetings and workshops were held to help clarify local perceptions about the living coastal resources, the legal and social mechanisms that govern access to them, and the problems and possible solutions associated with their utilization. the active participation of stakeholders in the process deepened their sense of involvement and commitment to achieving prospective solutions to the problems they identified. the kaisaka federation was formed in june 1996 in recognition of the common concerns of four people’s organizations (po) to promote the development of the bolinao cdp. (mcmanus and others 1999). the resource use map originally drawn by kaisaka was refined into a broader plan that evolved into the cdp. kaisaka initiatives became a collaborative effort with the municipal government. the federation version of the cdp was subjected to a validation and refinement workshop, with the different sectors represented. at the multisectoral forum held in november 1996, the four pos which at the time were federated into kaisaka, presented their version of cdp. comments and suggestions were raised by the participants in the forum. cognizant of their effort and willingness to help, the four pos were given representation in the cpd-twg created by the mayor in december 1996. the kaisaka lobby led to positive results, including (1) networking between the pos and other institutions and coastal communities; (2) training of selected po members as local resource persons; and (3) facilitation of participatory crm planning between the local government and the concerned communities. lobby by the municipal fisheries and aquatic resource management council (mfarmc). recognizing the need for a municipal fisheries ordinance, the sangguniang bayan decided to turn the cdp, which it had adopted as a resolution, into an ordinance. on february 25, 1999, the sangguniang bayan called a public hearing on the passage of cdp as an ordinance. the hearing, however, failed to attract public attention. so, the sangguniang bayan decided to conduct additional hearings in santiago island and in the mainland. unfortunately, the public hearings still did not materialize. a parallel development during the first half of 1999 was the creation and strengthening of mfarmcs as provided by the philippine fisheries code passed on february 25, 1998. the mfarmc in bolinao played a critical role in the formulation of the mfo. with the legislation process stalled, the mfarmc sought to assist the sangguniang bayan in facilitating mobilization for the public hearings. the sangguniang bayan in turn asked the mfarmc for assistence in conducting public consultations in the villages. the mfarmc obliged and held public consultations in july, august, and september 1999. to facilitate the activities, the vice-mayor and the chair of the environment committee of the sangguninang bayan, dempsey abogado, and some members of the cdp-twg participated in public discussions. by the first week of october 1999, the mfarmc was ready to consolidate and deliberate on the results of the consultations. the appropriate amendments to the draft mfo were made. on october 25, 1999, the mfarmc submitted its report to the sangguniang bayan in the form of a revised cdp, now called a draft fishery ordinance. mayor’s lobby. december 1999 was spent on the refinement and eventual approval of the ordinance. however, it was observed that the original cdp-twg was not interested in lobbying for the speedy approval of the cdp ordinance proposed by the sangguniang bayan. instead, mayor jesus f. celeste and the mfarmc played the important role of lobbyists. the lobby by the communities for the immediate enactment of the ordinance resulted in obtaining mayor celeste’s full support for the immediate passage of the ordinance. he recognized that there would be major controversial issues in the trailblazing ordinance. however, he realized that the draft provision had been made consistent with the revised fisheries code of 1998 (co january 2000 report). through his intercession, several special sessions were held by the sangguniang bayan to facilitate the enactment of the proposed ordinance. these were held on november 10, 14 and 17, and december 10, 1999 so that the ordinance could be approved before the end of 1999 . the members of the sangguniang bayan approved the ordinance on december 15, 1999, even if some members of the council requested more time to review the draft. the asido & talaue-mcmanus 106 draft was signed into law on january 5, 2000, with the approval date reckoned to december 10, 1999. the support given by mayor celeste for the speedy passage of the cdp, could not be overemphasized. initiatives of the zonal action teams (zats). like the farmc, a zat is also a multi-sectoral body designed to advocate, initiate policy and program for resource development and management under a provision of cdp at the zonal level ( pinat et al, this volume). a zat could facilitate discussions on the effectiveness of cdp in its particular area or zone. four zones were delineated: zone i for ecotourism; zone ii for multiple use; zone iii for fishery management; and zone iv for trade and navigation. zats were formed to identify other issues in their respective zones which were not previously identified by the cdp-twg. zats were formed in zones i and iii and an ad hoc body was created in zone ii. since october 1998, the zat’s succeeded in mobilizing barangay councils, conducting barangay consultations to deepen people’s understanding of the cdp, and in soliciting recommendations as inputs to the sangguniang bayan. they used various approaches to mobilize their constituents. most of the time, the zats resorted to filing or disseminating written documents as an advocacy strategy. these documents include petitions, position papers, draft resolutions, and ordinances (asido, 1999). among the zats formed, that for zone i has been the most active, effective, and most focused. the zat was active until september 1999, when it decided to merge with kaisaka. this decision was reached while planning for the management of the milkfish fry concession for year 2000. the move aimed to consolidate a management body of the concession. zat members would join the kaisaka and participate as part of the federation. the kaisaka would then address the issues formerly discussed and addressed by the zat. legal interventions perhaps a factor that is pronounced in the planning and enactment of the mfo was the involvement of legal resource persons in the process. recognizing that cdp involves policy review and formulation, the two projects (cb-crmp and mfrmp) provided for the conduct of studies on the local fisheries policies of bolinao as early as 1994 ( ferrer et al. unpublished, rodriguez 1997). their findings underscore the inadequacy and lack of innovative approaches to the management of the coastal and fishery resources; hence, the need for an integrated planing process. these studies and the participatory approaches that were used by the project engendered community sectors to actively take part in the planning process. what was needed however was the capacity and skills to engage government and stakeholders in the legal aspects of policy formulation and adoption. it was in this context that metalegal strategies and paralegal philosophy advocated by tanggol kalikasan (tk) of the haribon foundation came into play. although a core program of haribon foundation, tk was not active in bolinao until the cement plant controversy. in this case, tk acted as counsel for the movement of bolinao concerned citizens, inc. (mbcci), a multi-sectoral organization in bolinao that led the opposition to the cement plant proposal from 1994 to 1996. although not directly related to coastal development planning, such engagement led to a deeper involvement of tk in bolinao issues, most especially in the cdp and mfo. it was through the involvement of tk that the concepts of paralegalism and metalegal strategies were introduced to the community. more appropriately, it was through this interaction that the community members were trained in doing metalegal work. this led to more activities that enhanced the skills and knowledge of the people on legal work, and in formulating and lobbying for the passage of cdp as an mfo. when the cdp was presented in a multi-sectoral meeting in march 1998, the cdp-twg, the technical staff of the municipal government, and the mfrmp decided to draft an enabling ordinance which should be in harmony with the provisions of the philippine fisheries code that was passed in february 25 1998. in may 1998, when the conceptualization process of drafting and legislation of the proposed ordinance on cdp was ongoing, members of the cdp-twg agreed participatory metalegal and legal processes 107 that the draft must be reviewed by a lawyer before it was submitted to the sangguniang bayan. the cdp as a plan could not be fully implemented, particularly the provision on prohibition and penalties, without an enabling ordinance. the initial draft was reviewed by lawyers and discussed with the working group before the end of june 1998. during the latter half of 1998, the cdp-twg prioritized on its agenda the legislation of the proposed ordinance, information campaigns, networking and lobbying with the different stakeholders. on august 4 and 5, 1998, the mfrmp and the local government of bolinao jointly sponsored an orientation cum consultation on the philippines fisheries code of 1998 (republic act 8550), at the up-msi bolinao marine laboratory. the orientation aimed to harmonized the provisions of the draft ordinance with the national fisheries law. the activity also aimed to identify legal and jurisdictional action points to be considered in the implementation of programs and policies, thereby enhancing multi-sectoral participation in resource management. around sixty participants attended the orientation. the first day of the orientation was intended for leaders of the different people’s organizations in bolinao; the second day, as an orientation for the members of the sangguniang bayan, barangay councils of coastal barangays, municipal development councils, teachers of the bolinao school of fisheries, and leaders of other municipal-based organizations in bolinao. atty. rodolfo quicho, director of tanggol kalikasanharibon foundation and atty. marilyn cepe, president of community empowerment and resources for development (cerd) facilitated the two-day orientation and consultation. atty. quicho discussed the details of the current state policies on fisheries, the role and power of local government under the new fisheries code, rights of the small fisherfolk, fishery privileges and how the new fisheries code was legislated and its differences from the previous code (presidential decree 704 ). atty. cepe discussed how to enhance fisherfolk participation through the formation of fisheries and aquatic resources management councils (farmc) at the barangay and municipal levels. the orientation was critical for the participatory formulation of the ordinance. controversial provisions and legislative processes were discussed and clarified, including major issues that the participants were interested in. the drafting of the enabling ordinance was made by an ad hoc team composed of mpdc, mlgoo, an aquaculture technician, one member from the cdptwg and the mfrmp project staff, the latter acting as secretariat. as the drafting progressed, working drafts were circulated to the kaisaka for discussion. the mfrmp coordinators and staff reviewed the working draft. the ad hoc team deliberated on the provisions of the ordinance and came up with the final draft on the last week of september 1998. on october 5, 1998, the final draft was submitted to the municipal mayor. on october 9, 1998, the mayor endorsed the final draft to the sangguniang bayan and asked the body to legislate the ordinance at the soonest possible time. from january until september 1999, the fishery ordinance went through a series of public consultations. those conducted during the third quarter were facilitated by the mfarmc. the ordinance was discussed one section at a time with the major stakeholders in attendance. the public consultations ended in september 1999. in september 1999, the mfarmc of bolinao asked for the assistance of the cb-crmp to help them formulate the implementing rules and regulations (irr) needed for the cdp. a one-day discussion with atty. quicho was held on september 6, 1999 to discuss how the irr would be formulated. during this time, discussions between the sangguniang bayan and the mfarmc continued. the discussions focused on the provisions for coastal aquaculture, (sizes of and the distances between fish pens and cages), capture fisheries (allowable gear, closed and open seasons), and waste management and pollution, as well as the designation of marine protected areas. the discussions were done on a weekly basis. beginning september, the results of the public hearings were reviewed by atty. quicho before these were presented to the sangguniang bayan for deliberation and approval. the sangguniang bayan signified its intention to pass the ordinance before the end of 1999. on september 22, 1999 the mfarmc again asked atty. quicho to give his legal opinion on the proposed cdp. atty. quicho discussed clearly his legal opinions asido & talaue-mcmanus 108 on some critical provisions found in the cdp, especially, those that might have implications on the implementation of the philippine fisheries code. the mfarmc deliberated on the provisions of the ordinance and came up with the final draft on the third week of october 1999. on november 10, 12, 17, and 24, 1999, the final draft of the ordinance was prioritized for discussion by the sangguniang bayan. in the final phase of the legislation, the mfarmc submitted the draft to atty. quicho who facilitated discussions of the draft ordinance with the sangguniang bayan. having a vast experience in coastal policy and development, atty quicho pointed out that if the sangguniang bayan passed the ordinance, it would be the springboard to overhaul coastal management in bolinao. highlights of the municipal fishery ordinance the mfo provides for the division of the municipal waters of bolinao into four priority zones: zone 1 for ecotourism; zone 2 for multiple use; zone 3 for fishery management; and zone 4 for trade and navigation. this prioritization of use does not preclude the conduct and management of other activities as appropriate within these priority zones. an important feature of the mfo is an article providing for the creation of the municipal crm office that would be headed by a crm officer. the bolinao mfo reiterates the provisions of the philippine constitution and the 1991 local government code on the mandate of the municipal government to conserve, protect, and sustainably manage its municipal waters and its coastal and fishery resources. generally, the mfo recognizes, promotes, and adheres to the precautionary principle in the conservation, management, and utilization of coastal and fishery resources. it recognizes and promotes the principle of participatory stewardship of coastal resources, particularly fishery resources. it espouses to ensure the sustainability of these resources by promoting public awareness on their uses within ecological limits through appropriate education and training. the mfo also recognizes and promotes the principles of community empowerment, equity, and social justice, by protecting the rights of the small fishers through the preferential use of the coastal and fishery resources and by recognizing the rights of their organizations. furthermore, the mfo strengthens the partnership between the municipal government, the barangays, the communities, and other stakeholders, in the management of municipal waters and the resources therein. lessons learned 1. metalegal strategies empowered community sectors to participate in the formulation of a legal instrument like the municipal fishery ordinance. 2. advice and assistance from legal resource groups (e.g., tanggol kalikasan-haribon foundation) were crucial in the metalegal training of sectors in bolinao. 3. integral to facilitation by development projects, such as the cb-crmp and mfrmp, was the provision for metalegal training by, and access to, a legal council in developing legal instruments for integrated coastal resource management. to date, the challenge is not yet over . with the mfo in place, it is important to ensure its proper implementation. this must be done by both the government and the people if the participatory process is to continue. it is thus important to further enhance the information campaigns to explain to the community the features of the mfo. the local government should be assisted in performing its mandate under the mfo. in the long run, it is also important to continue evaluating this legal instrument to validate the appropriateness of its provisions and to respond to the changing needs of the community. references asido wn. 1998. training paralegals as strategy in protected area management: the mount isarog national park experience. quezon city, philippines: tanggol kalikaasan-haribon foundation (unpublished). participatory metalegal and legal processes 109 asido wn. 1999. activity proposal on paralegal training for zonal action team member. marine fisheries and resources management project, marine science institute, university of the philippines, bolinao marine laboratory, bolinao, pangasinan, august 20-21, 1999 (unpublished). asido wn. 1999. community organizing monthly reports from september 1999 to february 2000. marine fishery resources management project. marine science institute, university of the philippines, diliman, quezon city, philippines (unpublished). kalagayan nv. 1990. institutional and legal framework. in: talaue-mcmanus l, chua thia-eng, editors. the coastal environmental profile of lingayen gulf, philippines. manila, philippines: international center for living aquatic resources management, on behalf of the association of southeast asian nations/united state coastal resources management project. leonen m. 1991. harnessing creativity: tentative notes towards progressive lawyering. excerpt from a paper presented during the second cordillera lawyer ’s consultation; 1991 july 20-22; baguio city. quezon city, philippines: legal rights and natural resources center-kasama sa kalikasan. p 14-15. (unpublished). mcmanus lt, yambao ac, salmo sg, aliño pm. 1999. preparatory planning for coastal development in bolinao, philipines: a powerful tool for conflict resolution (case study). diliman, quezon city, philippines: marine science institute, university of the philippines. 152 p. pinat j, turion r, asido wn, talaue-mcmanus l, . 2000. strategies in mobilizing coastal communities for community-based coastal resource management in bolinao, pangasinan. science diliman. (this issue) tanggol kalikasan. paralegal philosophy and metalegal strategies: training materials for paralegal development and legal aid. quezon city, philippines: tanggol kalikasan-haribon foundation. (unpublished). yambao ac, salmo sg, aliño pm. 1998. a preliminary assessment of the coastal development planning in the municipal waters of bolinao, pangasinan, philippines . cbcrmp terminal report. philippines: communitybased coastal resources management project. p 220-231. yambao ac, mcmanus lt. 1998. the proposed cement plant complex in bolinao, pangasinan: a case of ill-conceived development. cbcrmp terminal report. up marine science institute, university of the philippines. in: conference on integrated management of the coastal fringe; 1998 june; puerto princesa city, palawan, philippines. philippines: voluntary service overseas and palawan council for sustainable development. 201 p. quicho r. 1999. the law on fisheries and its implications on local governance. quezon city, philippines: tanggol kalikasan-haribon foundation. (unpublished). 37-48_de ungria w corrections 37 str analysis of bloodstained denims science diliman (january-june 2004) 16:1, 37-48 * corresponding author evaluation and in-house validation of five dna extraction methods for pcr-based str analysis of bloodstained denims henry b. perdigon, gayvelline c. calacal, kristine l. co seng, saturnina c. halos, and maria corazon a. de ungria* dna analysis laboratory, natural sciences research institute miranda hall, university of the philippines 1101 diliman, quezon city, philippines telefax: (632)925-2965, (632)920-5301 local 6060 e-mail: mcadu@uplink.com.ph; website: www.dnaforensic.org date received: 7 may 2003; date accepted: june 30, 2004 abstract one type of crime scene evidence commonly submitted for analysis is bloodstain on denim. however, chemicals (e.g., indigo) used to produce denim materials may co-purify with dna and hence, affect subsequent dna analysis. the present study compared five methods (e.g., standard organic, organic with hydrogen peroxide (h2o2), modified fta™, organic/chelex®-centricon®, and qiaamp® dna mini kit-based procedures) for the isolation of blood dna from denim. a short tandem repeat (str)-based analysis across two to nine str markers, namely, humvwa, humth01, d8s306, humfes/fps, humdhfrp2, humf13a01, humfga, humtpox, and humcsf1po, was used to evaluate successful amplification of blood dna extracted from light indigo, dark indigo, indigo-sulfur, pure indigo, sulfur-top, and sulfur-bottom denim materials. the results of the present study support the utility of organic/ chelex®-centricon® and qiaamp® kit procedures in extracting pcr-amplifiable dna from five different types of denim materials for str analysis. furthermore, a solid-based method using fta™ classic cards was modified to provide a simple, rapid, safe, and cost-effective procedure for extracting blood dna from light, dark indigo and pure indigo denim materials. however, dna eluted from bloodstained sulfurdyed denims (e.g., sulfur-top and sulfur-bottom) using fta™ procedure was not readily amplifiable. keywords: dna extraction, bloodstained denims, pcr, short tandem repeat, fta™ classic card, inhibitors introduction for successful dna analysis, sufficient dna template of good quality and purity must first be recovered from samples. however, selection of dna isolation procedures is affected by the type of sample and the matrix to which a sample adheres. biological fluids such as saliva, semen, sweat, and blood on denim are frequently submitted as evidentiary material that were recovered from crime scenes. however, chemical substances in denim are known to inhibit the activity of restriction enzymes (prinz & berghaus, 1990) and dna polymerases (larkin & harbison, 1999; jung et al., 1991; del rio et al., 1996) during different typing procedures. denim is a tight twill cloth made up of cotton, sometimes combined with polyester, where the characteristic blue color is determined by the level of 38 perdigon et al. indigo dye absorbed in the material after dyeing and washing (larkin & harbison, 1999). for example, material that was dyed with indigo followed by sulfur is known as sulfur-top denim. if the dyeing procedure is reversed, the material produced is classified as sulfur-bottom denim (alviar, personal communications, 2002). the resulting denim material is subsequently processed to make branded clothing (e.g., jeans). several extraction methods are available to isolate dna from bloodstained denim. these methods include the use of chelex® 100 (bio-rad laboratories, hercules, ca, usa) (walsh et al., 1991), chelex® in tandem with centricon® 100 (amicon, beverly, ma, usa) (jung et al., 1991), and qiaamp® spin columns (qiagen inc., ca, usa) (greenspoon et al., 1998). however, most materials (i.e., reagents, columns, filters) involved in these procedures are expensive, and many laboratories continue to use phenol and chloroform in conventional organic solvent-based extraction procedures. these procedures unfortunately introduce the problem of organic waste disposal. there is thus a need to develop an affordable, non-phenol based procedure for extracting dna from bloodstained denim. in the present study, a modified fta™ classic card procedure for extracting pcr-ready dna from bloodstained denim was validated and compared with known methods, e.g., organic extraction (kirby, 1992), organic method with hydrogen peroxide (merck, germany) (akane, 1996), organic/chelex®centricon® (jung et al., 1991), and the qiaamp® dna mini kit (qiagen inc., chatsworth, ca, usa) procedures. a pcr-based short tandem repeat (str) analysis using two to nine markers was used to evaluate successful amplification of blood dna extracted from six types of denims. materials and methods sources of samples denim samples were collected from two denim factories at different times. the first set of samples consisting of light indigo, dark indigo, and dark indigosulfur denims was obtained in 1999, while another set of samples consisting of pure indigo, sulfur-top, and sulfur-bottom denims was obtained in 2002. ten (10) bloodstains were prepared by depositing 100 µl of fresh blood from a male volunteer on each of the six types of denim material, and left at room temperature (27°c-32°c) away from direct sunlight for 8-16 days. bloodstained portions of each denim were cut into small pieces using sterile scissors and placed in sterile 1.5 ml microfuge tubes. dna extraction standard organic extraction samples were washed with 1x ssc, followed by brief vortexing and centrifugation at 14,000 rpm for 3 minutes. pellets were lysed using a cell lysis solution consisting of 375 µl of 0.2 m naoac (ph 5.2), 50 µl 10% sds, and 7.5 µl 20 mg/ml proteinase k and incubated for 18-24 hours at 56°c (kirby, 1992). cell lysate was purified using phenol:chloroform:isoamyl alcohol or pci (25:24:1). dna was subjected to ethanol precipitation as described in sambrook et al. (1989). the resulting pellet was resuspended in 40 µl te-4 buffer. organic/hydrogen peroxide (h 2 o 2 ) method samples were incubated in 2 ml freshly-prepared stain extraction (se) buffer (0.6% n-lauroylsarcosine; 10 mm tris-hcl; 50 mm edta, ph 7.4; 0.4 mg proteinase k) at 56°c for 18-24 hours. the denim was transferred to a new microfuge tube and 2 ml se buffer was added. a second incubation at 37°c for 2 hours was performed upon addition of 100 µl 30% h2o2 (merck, germany) (akane, 1996). dna was precipitated following standard alcohol precipitation procedure as described in sambrook et al. (1989). the resulting pellet was resuspended in 40 µl te-4 buffer. modified fta™ classic card method samples were completely immersed in te-4 buffer (300-400 µl) at 56°c for 18-24 hrs and the resulting 39 str analysis of bloodstained denims stain solution was deposited on one circular area of an fta™ classic card. the card was air-dried at room temperature for at least 2 hours, punched using a harris micropunch™ apparatus (life technologies, inc., gaithersburg, md, usa), and processed according to manufacturer’s instructions (whatman® bioscience, ma, usa). organic/chelex®-centricon® samples were washed with 1x ssc, followed by brief vortexing and centrifugation at 14,000 rpm for 3 minutes (jung et al., 1991). pellets were processed following a standard phenol-chloroform extraction procedure, but without the alcohol precipitation step. instead, the aqueous layer was purified with 5% chelex® 100 (bio-rad laboratories, ca, usa) and centricon® 100 columns (amicon, ma, usa) (applied biosystems, forster city, ca, usa). dna was first immobilized on a biodyne b® membrane (pall life sciences, mi, usa) using the s & s minifold® i slot-blot system (schleicher and schuell, nh, usa) and probed using a biotin-labeled primatespecific alpha satellite d17z1 probe. human dna was detected using a horse-radish peroxidase-streptavidin (hrp-sa) and chromogen 3,3’5,5’tetramethylbenzidine (tmb) following manufacturer’s instructions (applied biosystems). dna concentration was estimated by comparing band intensities with those of k562 dna (gibco brl, life technologies, inc., gaithersburg, md, usa) of known concentrations (fig. 1). to check for the presence of inhibitors which co-eluted with dna during extraction, several dilutions of dna extracts (1/10, 1/100, and 1/1000) in te-4 buffer were prepared and analyzed. fig. 1. quantification of extracted dna using the quantiblot® kit. shown in the blot are: k562 dna standards at various dilutions (slots 1a-2b), standard dna solution a (slot 2c), reagent blank (slot 2d), and extracted dna samples (slots 2e-6h). numbers above the blue bands denote dna concentrations in ng/µl. the letters “nvb” refer to no visible bands (unquantifiable samples). arrows point to samples that were positive for dna but failed to produce amplification bands after pcr. pointed in arrows are samples which were positive for human dna, e.g., (1) bloodstained dark indigo-sulfur denim extracted using standard organic procedure labeled as dis-[so] with estimated yield of 25 ng and (2) light indigo denim extracted using organic/h2o2 method labeled as li-[oh] (approx. 20 ng), but produced negative amplification (fig. 3b). according to manufacturer ’s instructions. dna was eluted using 100-200 µl of te-4 buffer. qiaamp® dna mini kit, dried blood spot protocol, qiagen 2001 samples were immersed in 360 µl of buffer atl and incubated at 85°c for 10 minutes. the mixture was lysed using proteinase k and buffer al following manufacturer’s instructions (qiaamp® dna mini kit handbook, qiagen, 2001). the lysed mixture was transferred to a qiaamp® spin column (in a 2-ml collection tube) to allow binding of dna to the spin column followed by several washing steps. dna was eluted using 150 µl buffer ae. dna quantification the amount of dna extracted was quantified using the quantiblot® human dna quantitation kit 40 perdigon et al. pcr amplification dna was amplified at loci humfes/fps, humf13a01, humvwa, humth01, humdhfrp2, d8s306 (ffvtdd), humcsf1po, humtpox, and humfga (ctf) (table 1) using 2.5 µl of liquid dna extract or one 2-mm fta™ extracted disc as template. pcr amplification reaction for each locus was composed of 1x pcr buffer (50 mm kcl, 20 mm tris-hcl), 1.5 mm mgcl2, forward (cy5-labeled) primer (genset oligos, singapore), reverse (unlabeled) primer (gibco brl) (ffvtdd: 0.72 µm, ctf: 0.504 µm), dntp mix (ffvtdd: 37.5 µm, ctf: 50 µm), 60 ng/µl bovine serum albumin (bsa), and 0.02 u/µl taq polymerase (gibco brl). pcr was carried out in a biometra uno ii thermocycler (biometra, germany) or geneamp 9700 thermoycler (applied biosystems) following thermocycling conditions: 96°c initial denaturation for 2 min, followed by 30 cycles of 94°c denaturation for 1 min, 56°c annealing for 1 min, 72°c extension for 1.5 min, and a 72°c final extension for 10 min, for the amplification of ffvtdd loci, while the following parameters: 96°c initial denaturation for 2 min, followed by 10 cycles of 94°c for 1 min, 64°c for 1 min, and 70°c for 1.5 min, and 20 cycles of 90° c for 1 min, 60° c for 1 min, and 20° c for 1.5 min were used for the amplification of ctf loci (halos et al., 1999; tabbada et al., 2002). k562 dna (gibco brl) and in-house dna standard nsdna 3967 were used as positive controls while sterile distilled deionized water was used as negative control. all amplification reactions were done in duplicate. in addition, in-house control nsdna 3967 and an equal amount of purified dna (organic-based extraction) were mixed and subsequently amplified to confirm the presence of inhibitors in solution. dna fragment analysis amplified products were resolved and detected in an automated fluorescence detection system using the alf express™ dna sequencer and allelinks™ version 1.01 software (amersham pharmacia biotech, uppsala, sweden) according to manufacturer ’s table 1. characteristics of short tandem repeat (str) markers used. locus name locus definition chromosome number size range (bp) repeat motif (isfh) references humvwa humfes/fps humf13a01 humth01 humcsf1po d8s306 humdhfrp2 humfga humtpox human von willebrand factor gene human c-fes/fps human coagulation factor xiii a subunit gene human tyrosine hydroxylase gene human c-fms proto oncogene for csf-1 receptor gene not identified human dihydrofolate reductase psi-2 pseudogene human alpha fibrinogen gene 3rd intron human thyroid peroxidase 12 15 6 11 5 8 6 4 2 126-170 206-238 179-235 146-189 291-323 249-298 157-173 160-314 106-138 [tctg][tcta] [attt] [gaaa] [tcat] [taga] [aaag] [aaac] [cttt] [gaat] kimpton et al., 1992 polymeropoulos et al., 1991a polymeropoulos et al., 1991b polymeropoulos et al., 1991c hammond et al., 1994 nelson et al., 1993 polymeropoulos et al., 1991d mills et al., 1992 anker et al., 1992 41 str analysis of bloodstained denims instructions. sizes of pcr products were compared and scored using in-house allelic ladders described previously (halos et al., 1999; tabbada et al., 2002). heterozygous alleles were assigned only when the smaller peak area was at least 60% of the larger peak area (clayton et al., 1998). results dna quantity dna was successfully extracted from six bloodstained denim samples after 8-16 days of storage at room temperature using five methods included in the present study. estimated dna yield varied between the four liquid-based extraction procedures with organic/ chelex®-centricon® method consistently resulting in the highest dna yield (table 2). up to 800-1000 ng of dna per 100 µl of bloodstain was recovered from bloodstained denims except for the dark indigo denim with yield of 100 ng dna per 100 µl of bloodstain using organic/chelex®centricon® procedure. similar yields of 320 ng/100 µl bloodstain were obtained from bloodstained denims extracted using standard organic and organic/h2o2 methods, except for the dark indigo-sulfur where yield varied between the two methods. on the other hand, low dna yields (75-300 ng/100 µl of bloodstain) were observed using the qiaamp® kit procedure. dna quality the success rates of pcr amplification of template dna from bloodstained light indigo, dark indigo, and dark indigo sulfur (phase i) using five dna extraction methods are summarized in table 3. liquid and bound dna prepared from the three bloodstained denim samples using the modified fta™, organic/chelex®centricon®, and qiaamp® kit procedures were successfully amplified in two str markers (100%). peak signals generated at two loci (humvwa and humth01) by the modified fta™-prepared dna were distinct and comparable with those obtained using organic/chelex®-centricon® and qiaamp® kit procedures. fig. 2 shows an electropherogram of dna from bloodstained light indigo denim amplified at humvwa locus. on the other hand, low success rates of amplification in two str markers of 33.3%-66.7% and 33.3% were observed in liquid dna samples prepared using the standard organic and organic/h2o2 procedures, respectively. the co-elution of inhibitors with dna extracted by standard organic and organic/h2o2 was confirmed when control nsdna 3967 failed to amplify when an equal volume of dna extracted via organic procedures was added to the solution (fig. 3a). subsequent dilution of the same dna preparation led to successful amplification (fig. 3b). further comparison between the modified fta™, organic/chelex®-centricon® and qiaamp® kit table 2. typical dna yields (in ng) from different types of bloodstained denim extracted using four procedures. denim average dna yield (ng) per 100 µµµµµl of bloodstain standard organic organic/ h2o2 organic/chelex® centricon® qiaamp® kit (qiagen, inc.) phase i light indigo dark indigo dark indigo-sulfur phase ii pure indigo sulfur-top sulfur-bottom 320 320 400 nd nd nd 320 320 60 nd nd nd 1000 100 800 800 1000 800 262.5 300 150 150 120 75 nd: not done procedures was performed by analyzing another set of bloodstained denim samples, e.g., pure indigo, sulfur-top, sulfur-bottom denims (phase ii). results are summarized in table 3. liquid dna prepared using organic/chelex®centricon® and qiaamp® kit procedures were successfully amplified in two of three samples (66.7%) in all three markers. on the other hand, lower success rates (33.3%-66.7%) were obtained in amplifying modified 42 perdigon et al. fig. 2. electropherogram of allele peak signals at humvwa resulting from amplification of dna from bloodstained light indigo prepared using five different extraction procedures. shown in figure are in-house generated allelic ladders (b & i), negative (a), and k562 dna (positive) controls (c) used in the pcr reaction and amplicons from dna extracted from bloodstained light indigo denim from a male volunteer using standard organic (d), organic with h2o2 (e), modified fta™ procedure (f), organic/chelex®-centricon® (g), and qiaamp™ dna mini kit (i) procedures. clear and typable allele peak signals produced as a result of successful dna amplification using modified fta™ procedure were comparable to those extracted via organic/chelex®-centricon® and qiaamp® dna mini kit procedures. absence of dna amplification is denoted by the absence of any detectable peak. x-axis represents the size range of alleles (in bp). the y-axis is a quantitative scale based on % peak area reflecting peak signal with the largest peak area given automatically a value of 100%, while other peaks within the same lane are given values that are a percentage of the largest peak (pharmacia biotech). fta®-prepared dna due to non-amplification of two (sulfur-top and sulfur-bottom) out of three samples in two str markers (humvwa and d8s306: 33.3%) and one (sulfur-bottom) out of three samples at humth01 marker (66.7%). dna eluted from bloodstained pure indigo denim using the modified fta™ procedure was successfully amplified across the three str markers with peak signals that were clear and distinct to be comparable with those obtained using organic/chelex®centricon® and qiaamp® kit procedures (fig. 4, panels d-f). in contrast, variable amplification results were obtained from modified fta™-prepared dna from bloodstained sulfur-top denim, while negative amplification results were evident from dna prepared from bloodstained sulfur-bottom denim using either of the three dna extraction procedures (fig. 4, panels g-l). to determine the overall success rate of amplification of dna extracted using the modified fta™ procedure in the remaining six str markers routinely used in our laboratory, dna from bloodstained pure indigo, sulfur-top, and sulfur-bottom denims were amplified in six additional str markers—humtpox, humdhfrp2, humfga, humf13a01, humfes/ fps, and humcsf1po. dna typing of bloodstained pure indigo denim yielded a full str profile across the six markers, while partial and null profiles were generated from bloodstained sulfur-top and sulfurbottom denims, respectively (table 4). discussion to find the most suitable procedure for isolating dna from bloodstained denims for forensic applications, five dna extraction procedures were evaluated and compared in terms of dna quantity and quality. sufficient dna was recovered from bloodstained 43 str analysis of bloodstained denims fig. 3a. electrophoregram of allele peak signals at humvwa resulting from amplification of dna eluted from bloodstained dark indigo-sulfur denim and light indigo denim using organicbased methods. shown in the figure are two distinct peaks of control nsdna 3967 resulting from positive dna amplification (panel c). adding an equal volume of dna extracts from bloodstained dark indigo-sulfur denim extracted using standard organic procedure (panel e) and light indigo denim extracted using organic/h2o2 method (panel g) in the pcr mix resulted in negative amplification which indicate the presence of inhibitors in organic extracts. fig. 3b. electrophoregram of allele peak signals at humvwa resulting from amplification of serial dilutions of dna eluted from bloodstained dark indigo-sulfur denim and light indigo denim using organic-based methods. shown in the figure are extracts (described in figs. 1 & 3a) that previously failed to amplify in the presence or absence of control dna (panels d & h) but successfully amplified upon serial dilutions with te-4 buffer 10 to 1000 fold (panels e-g & i-k). dilution significantly reduces the amount of inhibitors in the dna solution, hence, successful dna amplification was achieved. 44 perdigon et al. denim samples (60-1000 ng of human dna) per 100 µl of bloodstain using the five extraction methods. for the same amount of sample applied onto the denim, variation in the amounts of dna recovered were observed across procedures and samples indicating that differences exist among the five dna extraction procedures in terms of extraction efficiency which in turn is affected by the physical/chemical properties of each denim sample. hence, our results demonstrate the need to validate procedures for analyzing different forensic samples on various types of substrate. dna concentration and yield were compared across the four procedures except the modified fta™ procedure since quantity could not be empirically determined with bound dna on fta™ card. organic/ chelex®-centricon® produced the highest dna yield, followed by the standard organic and organic/h2o2 and qiaamp® kit procedures. theoretically, one 2-mm fta-extracted punch of blood sample with a typical white blood cell count contains 83-100 ng dna. in this study, the success rates of dna amplification obtained using the five methods were determined at selected str markers to evaluate the quality of extracted dna. the low success rates of amplification consistently obtained when amplifying dna extracted using standard and organic/h2o2 procedures during the phase i experiment indicate the presence of inhibitors in these extracts which was confirmed in three ways. first, quantiblot results revealed that some organic table 3. rate of successful amplification of dna from different types of bloodstained denim extracted using five procedures. where: phase i samples–light indigo, dark indigo and dark-indigo sulfur; phase ii samples–pure indigo, sulfur-top, sulfur-bottom; nd = not done; success rate is expressed as (nsuccess/nt) x 100, where nsuccess = number of times dna successfully amplified and nt = total number of amplifications made str marker rate of successful amplification across five extraction methods (%) standard organic organic/ h2o2 modified ftatm qiaamp® kit (qiagen, inc.) phase i humvwa humth01 phase ii humvwa humth01 d8s306 66.7 33.3 nd nd nd 33.3 33.3 nd nd nd 100 100 33.3 66.7 33.3 100 100 66.7 66.7 66.7 100 100 66.7 66.7 66.7 organic/chelex® centricon® dna solutions showed negative amplification although positive for human dna with concentrations sufficient for dna amplification, e.g., bloodstained dark indigo denim (figs. 1 & 3b). the yield of qiamp® extraction was lower than that obtained using organic methods and yet qiaamp® extracted dna consistently amplified, thus, indicating the high quality of dna in solution. in a second set of experiment, the presence of pcr inhibitors in organic extracts was further demonstrated when amplifications were subsequently achieved upon 1/10 to 1/1000 dilutions of organic extracts in te-4 buffer. diluting liquid dna template effectively reduced the amount of inhibitors per given volume, leading to successful pcr amplification (butler, 2001). in addition, amplification of control dna (nsdna 3967) was unsuccessful when an equal volume of organic extract was added in the pcr mixture. direct observation of blue-colored precipitate in the qiaamp® membrane during purification process also shows that indigo dye coextracts with dna in aqueous solution which may have inhibited amplification. the problem of inhibition was not encountered with the modified fta™, organic/chelex®-centricon®, and qiaamp® kit procedures. notably, these procedures are all membrane or filter-based methods which involve cell lysis and selective capture of dna on a membrane or filter, followed by dna purification and concentration steps. in contrast, organic methods relied on the cell lysis and release of dna in the liquid 45 str analysis of bloodstained denims fig. 4. electrophoregram of allele peak signals at humvwa resulting from amplification of dna eluted from bloodstained pure indigo, sulfur-top and sulfur-bottom denims using the modified fta™, organic/chelex®-centricon® and qiaamp® kit procedures. shown in the figure are allele peaks obtained from modified fta®-prepared dna that were distinct and comparable with those of organic/chelex®-centricon® and qiaamp® kit procedures in yielding pcr-amplifiable dna from bloodstained pure indigo denim (d-f) but not from bloodstained sulfur-top denim (g-i). the absence of amplification in bloodstained sulfurbottom denim using the three procedures is clearly shown in panels j-l. suspension without any substrate to bind to, followed by subsequent dna purification and concentration by serial addition of chemicals. hence, our data indicate that capturing dna with a substrate, e.g., column or filter, may be more efficient in purifying inhibitors from bloodstained denim than plain liquid extraction. aside from the problem of inhibition observed in organic-based methods, the disadvantages of using organic-based methods are: (1) the methods are timeconsuming; (2) they involve the use of hazardous and toxic chemicals (i.e., phenol and chloroform, which present the additional problem of organic waste disposal); and (3) they involve multiple tube transfer of liquids, which may increase the risk of error or contamination. thus, standard organic and organic h2o2 methods were excluded in the phase ii experiment where the modified fta™, organic/chelex®centricon®, and qiaamp® kit procedures were further evaluated using another set of bloodstained denims. the efficiency of dna extraction using qiaamp® spin columns is based on the optimal binding of high molecular weight dna to the silica-gel membrane inside the column as well as the salt and ph conditions in the lysate that ensure proteins and other contaminants which can inhibit pcr and other downstream enzymatic reactions, are not retained in the membrane (qiaamp® dna mini kit handbook). the use of chelex® 100 effectively removes pcr inhibitors in crude organic dna extracts by chelation of polyvalent metal ions such as iron cations (associated with hematin in blood) and magnesium ions (that activate dna degrading enzymes) with iminodiacetate ions of the styrene divinylbenzene co-polymers (walsh et al., 1991; jung et al., 1991; butler, 2001). subsequent processing of chelex-purified dna by passing through centricon® filter devices greatly improves dna quality by providing additional concentration and desalting of dna solution by ultrafiltration. however, both procedures require multiple liquid transfers and are very expensive. 46 perdigon et al. this paper presents a possible alternative procedure for handling bloodstained denims. a modified procedure involving fta™ cards for dna isolation described here was successful when bloodstained light indigo, dark indigo, and dark indigo-sulfur denims were analyzed. the effectivity of this method is that dna is isolated immediately upon contact of the sample on the dry solid matrix of the fta™ paper impregnated with chelators, denaturants, and free-radical traps which lyse cell membranes, denature proteins, and immobilize the dna in the fta™ matrix; thus, dna is protected against chemical and/or biological degradation (whatman® bioscience, ma, usa). differences in the success of dna amplification between the modified fta™, organic/chelex®-centricon®, and qiaamp® kit procedures were noted in the analysis of the second set of bloodstained denims particularly sulfur-top and sulfur-bottom denims. organic/chelex®-centricon® and qiaamp® kit procedures were successful in typing dna from bloodstained sulfur-top denim in three str markers (100%), which was not achieved with the modified fta™ procedure (33.3%). hence, modified fta™ procedure may be regarded as secondary or alternative to organic/chelex®-centricon and qiaamp® kit procedures, which both obtained the same results in all markers. the complete amplification of bloodstained pure indigo denim and the variable amplification across 9 str markers of modified fta™-prepared dna from bloodstained sulfur-top denim and sulfur-bottom denim using either modified fta™, organic/chelex®centricon®, or qiaamp® kit procedures support possible co-extraction of sulfur dye with the dna in solution and subsequently inhibit pcr reaction as reported in previous studies (de ungria et al., 1999). sulfur dye is produced by fusing organic chemicals with sulfur; hence, sulfur and/or any organic chemical components may likely to affect the activity of the taq polymerase. caution must therefore be taken in handling samples such as bloodstained sulfur-top and sulfur-bottom denims. nonetheless, the use of fta™ cards for isolating dna from bloodstained pure indigo denim as demonstrated here provided an attractive alternative to the more expensive organic/chelex®-centricon® and qiaamp® kit procedures. one extraction using the modified fta™ procedure costs less than half of the expenses in purchasing chelex® resin and centricon® columns, or qiaamp® kits. in addition, modified fta™ procedure is a good alternative to organic/ chelex®-centricon® and qiaamp® kit procedures since it is (1) simple and rapid, involving few extraction steps and no quantitation step as dna is immobilized in the paper; (2) health and environmentfriendly, not involving the use of highly toxic chemicals which also introduces the problem of waste disposal, e.g., phenol and chloroform; (3) not prone to er rors and contamination, requiring only one tube throughout extraction; and (4) cost-effective, requiring minimal storage space at room temperature. conclusion and recommendation the data presented in this study demonstrate that the success of dna extraction and subsequent analysis depends on the selection of an appropriate dna extraction procedure. table 4. rate of successful amplification of dna from different types of bloodstained denims extracted using the modified fta™ procedure. denim rate of successful amplification in six str markers (%) tpox (nt=2) dhfrp2 (nt=2) fga (nt=2) f13a01 (nt=2) fes/fps (nt=2) csf1po (nt=2) pure indigo sulfur-top sulfur-bottom 100 100 0 100 0 0 100 100 0 100 50 0 100 0 0 100 0 0 47 str analysis of bloodstained denims of the five extraction procedures evaluated, organic/ chelex®-centricon® or qiaamp® dna mini kit procedures proved to be the most suitable dna extraction procedures for pcr-based str analysis of bloodstained denims for forensic applications. alternatively, the modified fta™ procedure provides a simple, rapid, less expensive, and health and environment-friendly means for successful dna typing of bloodstained light indigo, dark indigo, dark indigosulfur, and pure indigo denims. future development of this method to effectively prevent sulfur dye in sulfur-containing denims from co-purifying with dna is recommended. hence, further work to optimize procedures that incorporate its use is currently underway. acknowledgments this project was supported by the philippine council for advanced science and technology research and development (pcastrd) of the department of science and technology (dost) and the european commission (ec). the authors are grateful to mr. frederick c. delfin for reference blood samples. the authors wish to acknowledge mr. eric alviar (litton mills inc.) for his generous contribution of denim samples. references akane, a., 1996. hydrogen peroxide decomposes the heme compound in forensic specimens and improves the efficiency of pcr. biotechniques. 21: 392-394. anker, r., t. steinbrueck, & h. donis-keller, 1992. tetranucleotide repeat polymorphism at the human thyroid peroxidase (htpo) locus. nucleic acids res. 1: 137. butler, j.m., 2001. forensic dna typing: biology and technology behind str markers. san diego, ca, usa, academic press: 30 pp. clayton, t.m., j.p. whitaker, r. sparkes, & p. gill, 1998. analysis and interpretation of mixed forensic stains using dna str profiling. forensic sci. int. 91: 55-70. de ungria, m.c.a., g.c. calacal, k.a. tabbada, & s.c. halos, 1999. extraction and pcr amplification of dna from human bloodstains on different textiles and philippine woods. poster presented at 18th international 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forensic sci. 47(6): 1397-1398. walsh, p.s., d.a. metzger, & r. higuchi, 1991. chelex as a medium for simple extraction of dna for pcr-based typing from forensic material. biotechniques. 10: 506-513. ocr document page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 53 morphological characters of papaya (carica papaya l.) for drought tolerance pablito m. magdalita* institute of crop science university of the philippines los baños institute of plant breeding university of the philippines los baños michael r. noel(+) edna a. aguilar institute of crop science university of the philippines los baños alangelico o. san pascual institute of plant breeding university of the philippines los baños abstract papaya plants (carica papaya l.) were assessed for tree and fruit morphological traits. during eight months of drought conditions, papaya plants showed signs of drought from march to may 2016. drought affected fruit development and fruit qualities. significant decrease in fruit weight, length, width, flesh thickness, and seed weight were observed in drought-affected papayas; however, total soluble solids (tss) of fruit flesh were significantly higher compared to the tss of fruits harvested during normal conditions. drought-tolerant papaya trees were selected based on morphological responses. drought-tolerant papaya trees were significantly taller and had thicker stems, wider crowns, and more functional green leaves compared to drought-affected trees. selected plants that thrived well under drought condition were identified for use in breeding. recovery of plants was evident in increase in fruit weight, length, and width. correlation of fruit characters of selected drought-tolerant papaya trees revealed that fruit weight was strongly positively correlated with fruit length, fruit width, peel weight, flesh thickness, and tss. keywords: carica papaya, drought, fruit characters, recovery, tree characters * corresponding author science diliman (july-december 2021) 33:2, 53-69 morphological characters of papaya for drought tolerance 54 introduction the papaya (carica papaya l., 2n=18), a monotypic species in the family caricaceae, is commonly grown for its melon-like fruits, which are available throughout the year. it is relatively easy to grow and produces fruits within a short period of time, making it a good cash crop. if grown under the right conditions, papaya can produce high yield per tree and on a per hectare basis. in 2020, a total of 163,299.41 metric tons were produced in the philippines. papaya ranked 10th in area planted and 4th in production volume among the ten leading fruit crops in the philippines (psa 2021). the top three papaya-producing regions were socsargen (64,113.16 metric tons), northern mindanao (38,030.41 metric tons), and davao region (10,619.2 metric tons) (psa 2021). about 92% of the total papaya production is consumed locally as food and the rest is for industrial uses. papaya suffers from several diseases and pests—the most widespread and destructive of which is the papaya ringspot virus (prsv). the prsv was first detected in silang, cavite in 1982 (opina 1986), but it spread rapidly in luzon and in other islands like marinduque, mindoro, panay, negros, bohol, and cebu, to name a few, because of the explosive nature of the disease (magdalita et al. 1989). in 2001, prsv was detected in a few areas in davao del sur and south cotabato in mindanao (herradura 2001) but did not spread because of vigilant sanitation and quarantine measures. in the philippines, prsv practically decimated commercial papaya cultivation in southern luzon, leading to significant declines in papaya production, which then caused substantial losses of income to farmers, a relative scarcity of the fruit in the market, and higher costs to consumers (magdalita et al. 2016). however, in hawaii, the transgenic “rainbow” variety is resistant to prsv. in the philippines, the moderately prsv-tolerant variety “sinta” has been used for planting by farmers in luzon since 1995. bacterial crown rot (bcr) causing 100% damage to papayas at any stage of growth is another limiting factor to production. breeding for tolerance to bcr via regrowth selections is underway at the institute of plant breeding, college of agriculture and food science, university of the philippines los baños (magdalita et al. 2016). aside from prsv and bcr, papaya production is also constrained by abiotic factors such as drought during summer months and beyond, waterlogging, and strong wind especially during typhoons. there are almost 20 typhoons that visit the country each year (pagasa 2016). tall papaya trees with heavy leaves and large canopies in general can be toppled by strong winds and also suffer from too much water brought by heavy rains, causing rotting of the roots. thin-stemmed papayas also p.m. magdalita et al. 55 cannot withstand limited water and can become weak, causing them to fall to the ground, particularly if loaded with fruits. the availability of water in the soil affects papaya physiology and productivity. it has been reported that papaya plants exhibit both stomatal and non-stomatal responses to soil water deficits, and the sources of these response signals are both hydraulic and non-hydraulic in nature (campostrini and glenn 2007). marler et al. (1994) proposed that it is highly unlikely that stomata of drought-stressed papaya plants closed due to hydraulic signals from leaf dehydration since leaf relative water content and pre-drawn xylem potential (ψ pd ) were unrelated to gravity (g) at mild and moderate soil water deficits. it was also proposed that other nonhydraulic plant signals are controlling stomatal behaviour. non-hydraulic signals such as abscisic acid and jasmonic acid (but not indole-3-acetic acid) differed in their accumulation patterns under stress. jasmonic acid initially increased and then decreased in leaves and roots. mahouachi et al. (2007) proposed abscisic acid as an accumulative, non-hydraulic hormonal signal that could be involved in the induction of several physiological responses in papaya under progressive water stress such as the reduction in gas exchange parameters and leaf abscission. they further suggested that delaying dehydration appears to be the adaptation that papaya uses in response to drought, even though osmotic adjustment was not demonstrated. however, mahouachi et al. (2006) found that osmotic adjustment is a contributing factor in drought adaptation in papaya varieties such as baixinho de santa amalia. marler et al. (1994) and torres-netto (2005) demonstrated that there is genetic variability in papaya cultivar response to soil water deficits, providing clues to the mechanisms of drought adaptation. however, these constraints can be overcome by developing and designing papaya line/s and hybrids with multiple disease resistance and tolerance to drought. this kind of variety has been sought by many papaya growers, especially those in the drought-prone areas of the country. beneficial architectural characteristics of papaya plants desired by papaya growers include the following: stout and sturdy trunk that holds more water and resists wind; dwarf habit that escapes wind; erect leaves and shorter petioles that drain rainwater quickly; bigger roots that anchor the tree better on the ground; and fast maturing so that it can escape typhoons and drought. like any agricultural crop, the papaya has a narrow range of adaptations to climatic elements for optimum and maximized productivity. characterization of papaya responses to natural conditions (in situ) is important to establish the genotype-byenvironment interactions based on anticipated fluctuations of climatic elements and edaphic factors, taking into account the temporal variability of the production morphological characters of papaya for drought tolerance 56 areas. in terms of drought, tolerant lines/varieties should be selected and developed despite the narrow range of adaptation because of the pressing issues of drought in this time of climate change. to develop such lines, documentation and comparison of responses under drought and non-drought conditions are vital for the development of lines with drought tolerance in terms of drought tolerance traits. this study aimed to: i) determine the association of different traits of droughttolerant and non-drought affected (pre-drought or normal) papaya trees; and ii) evaluate the morphological responses, such as tree and fruit characters, of droughttolerant and non-drought-tolerant (drought phase) papaya trees, drought affected and non-drought affected trees (pre-drought phase or normal condition), and the trees that recovered after the drought condition (recovery or post-drought phase). materials and methods ninety plants of c. papaya l. variety “sinta” were planted on december 23, 2014 in tranca, bay, laguna in a replicated trial. the plants were planted 3 m apart between rows and 2.5 m between plants. the plants were provided with the recommended cultural practices for fertilization, irrigation and pest control. for fertilization, basal application of 1:1 complete fertilizer and urea before the planting of seedlings and monthly application of 100g of the aforementioned mixture were implemented. watering during seedling stage whenever needed was observed. application of pesticides whenever needed was also implemented. the cultural management of papaya was based on the philippine recommends for papaya (pcaarrd 2006). during the normal condition, tree parameters such as plant height (m), stem diameter (cm), crown diameter (cm), and number of green leaves were gathered. the plant height was measured using a meterstick from the base to the top of the tree, while the stem diameter was measured using a vernier caliper. the crown diameter was measured at the largest canopy spread using a meterstick, while the green leaves were counted from the most mature leaves to the last fully expanded leaves. at first fruiting (normal condition), 10 physiologically mature fruits from each sample tree were harvested at peel color index 2 (pci 2), i.e., when a tinge of yellow appears on the apex of the fruit, and allowed to ripe completely on a ripening rack. at full ripe stage, fruit characters such as fruit weight (g), fruit length (mm), fruit width (mm), peel weight (g), peel thickness (mm), flesh thickness (mm), total soluble solids (tss, ºbrix), and seed weight (g) were assessed. the fruit weight, peel weight, and seed weight were determined using an automatic top-loading balance while fruit length, fruit width, peel thickness, and flesh thickness were measured using a vernier caliper. the tss was measured using a digital refractometer (milwaukee p.m. magdalita et al. 57 instruments, inc., 2950 business park drive, rocky mount, north carolina 27804, usa). juice extracts from each fruit were dropped into the refractometer and the digital reading of tss was recorded. during the eight months of drought throughout the dry season of 2016, the papaya trees were especially affected by drought in the months of march, april, and may, during which there were spikes in temperature with an average of 31–35°c and no rainfall observed. the data on temperature and other climatic data were retrieved from the national agrometeorological station based in los baños (figure 1). figure 1. actual and average daily high and low temperatures (oc) at tranca, bay, laguna from march to may 2016. the blue arrows represent spikes in temperature. data from the national agrometeorological station in los baños. morphological characters of papaya for drought tolerance 58 again, tree characters and fruit qualities mentioned above were assessed to compare the effect of drought with normal condition (figure 2). drought-subjected plants were planted in an identified drought-rainfed area in the central experiment station at the institute of plant breeding. during this set-up, no rainfall was recorded for three months; hence, the plants were identified to be under drought condition. plants that were irrigated based on recommended water management practices for papaya were identified as those under normal condition. in addition, droughttolerant trees that thrived well under drought condition and showed good vigor and fruiting ability were selected and identified as putative drought-tolerant trees (figure 3). figure 2. papaya plants in the papaya breeding block, b4, tranca, bay, laguna (a) before drought and (b) after drought. p.m. magdalita et al. 59 figure 3. fruits of drought-affected papaya trees and those that were grown under normal condition. after the drought condition, the trees were again evaluated to assess the ability of the drought-affected trees to recover from drought. the same tree and fruit characters mentioned above were evaluated. in addition, the association of tree and fruit characters of drought-tolerant and normal papayas was determined. for the statistical design and analysis, the experiment was laid out in randomized complete block design with three replications. twenty tree samples were used in each replication for the evaluation of tree characters. twenty ripe fruit samples for each sample tree in a replicate were used in the evaluation of fruit characters. all data gathered was subjected to one-way analysis of variance using the star statistical package (irri 2014). the significance of means was tested using student t-test at 0.05 level of significance. morphological characters of papaya for drought tolerance 60 results and discussion morphological characteristics of putative drought-tolerant and non-drought tolerant papayas were evaluated. results showed that drought-tolerant papayas were significantly taller, had thicker stem diameter and wider crown, and had more green leaves compared to drought-affected papayas (table 1). generally, tree height, stem diameter, crown diameter and number of green leaves were reduced in drought-affected trees (table 1). this reduction in tree characters of droughtaffected trees is probably due to reduced photosynthesis resulting in decreased growth. taiz and zeiger (2010) discussed that, as water content in the plant decreases, the cells tend to shrink and relax. this also causes a decline in cell volume, causing lower turgor pressure following the solute concentration in the cell. further, they stated that as there is water deficit, plasma membrane becomes thicker and then more compressed, making the area of the cell smaller. the changes in turgor pressure affect the expansion of different organs such as leaves and roots. these organs are sensitive to turgor changes, which in turn make then sensitive to drought (taiz and zeiger 2010) similar observations were recorded for wheat (triticum sativum l.) where water deficiency had an inhibitory effect on plant growth (pireivatlou et al. 2008). further, the authors observed that dry matter accumulation in the kernels was decreased by water deficit. the dry weight of vegetative organs was also decreased during the grain filling period under stress condition. table 1. selected tree morphological characters of selected drought-tolerant and droughtaffected papaya plants at fruiting stage in a drought-affected condition condition tree height (m) stem diameter (cm) crown diameter (m) no. of leaves drought-tolerant trees 3.2 a 9.2 a 6.2 a 25.4 a drought-affected trees 1.8 b 6.2 b 4.1 b 19.5 b reduction (%) 43.75 32.61 33.87 23.23 means with the same letter are not significantly different at 0.05 level. the results showed that drought significantly affected the fruit development and fruiting habit of papaya (table 2). generally, trees grown under drought condition produced fewer number of fruits than the trees grown under normal condition (figure 2). few (1–4) to no fruits developed under drought-affected conditions while 5–18 fruits were observed in papayas grown in normal condition. papaya trees grown in the field throughout the eight months of drought during the dry season of 2016 were highly affected especially during the months of march, april and may during which there were spikes of high temperatures (figure 1). p.m. magdalita et al. 61 table 2. fruit characters and fruit character % reduction of drought-affected papaya trees in comparison with drought-tolerant papaya trees and papaya trees grown in normal condition condition fruit weight (g) fruit length (mm) fruit width (mm) peel weight (g) peel thickness (mm) flesh thickness (mm) total soluble solids (ºbrix) seed weight (g) drought-affected vs. normal condition trees under normal condition 1,400.3 a 170.5 a 132.7 a 96.7 a 13.6 a 35.5 a 10.6 b 159.3 a droughtaffected trees 400.4 b 85.5 b 57.8 b 87.7 b 11.6 a 21.7 a 13.6 a 54.7 b reduction in fruit characters (%) 71.41 49.85 56.44 9.3 14.7 38.87 -28.30 65.66 drought-tolerant vs. drought-affected drought tolerant trees 800.5 a 143.4 a 111.5 a 90.5 a 13.4 a 22.5 a 14.4 a 55.6 a droughtaffected trees 400.4 b 85.5 b 57.8 b 87.7 b 11.6 a 21.7 a 13.6 a 54.7 a reduction in fruit characters (%) 49.98 40.37 48.16 3.09 13.43 3.55 5.56 1.62 means with the same letter are not significantly different at 0.05 level. it could be surmised that under drought condition, papaya flowers that develop tend to turn into fruit but they eventually aborted due to lack of water; hence, fruit development is hampered and yield is reduced. this can also be observed in the decreased number of flowers that set into fruits. in drought conditions, 0–10% of the papaya flowers set to become fruits while 70–80% of the flowers set to become fruits under normal condition. this finding is similar to a recent report that mungbean (vigna radiata l.) grown under drought condition also had reduced yield (adorada et al. 2019). in terms of fruit characters, the drought-tolerant papaya trees had significantly higher fruit weight, fruit length, fruit width, peel weight, peel thickness, flesh thickness and seed weight than the drought-affected papaya trees (table 2). in general, the abovementioned fruit characters were reduced among the droughtaffected trees, but not in the drought-tolerant trees. this is due to the limited fruit morphological characters of papaya for drought tolerance 62 development caused by lack of water, resulting in limited photosynthesis and translocation of photosynthates of the tree. according to zhang et al. (2019), this restricted photosynthesis is attributed to the decrease in the chloroplast’s carbonfixing enzyme activity and metabolic pathways’ changes. however, tss of fruits harvested under drought was significantly higher compared to tss of fruits (10.6±0.09) harvested under normal condition (table 2). this higher tss observed among fruits of drought-affected papaya trees is due to the higher accumulation of reducing sugars like fructose, which is likely more concentrated than those found in trees grown under normal condition. while the fruits borne by the drought-affected trees are smaller, their flesh is sweeter than those borne by trees grown under normal condition. the tss of fruits from drought-affected trees was 35.85% higher than the tss of fruits from trees grown under normal condition. similarly, in sugar apple (annona squamosal l.), sugar increased in fruits of drought-stressed trees compared to well-watered trees (kowitcharoen et al. 2018). furthermore, the present finding agreed with a previous report that mild drought improved the fruit quality relating to hexose sugar accumulation, flesh firmness and organic acid content (van de wal et al. 2017). the present result also corroborated previous findings that drought causes fructose levels in peach fruit to increase because heat causes higher rates of photosynthesis, increasing sugar levels in the fruit (alexandra 2012). in drought-stressed plants, the observed increased sugar level concentration was due to the plants’ adjustment to osmotic homeostasis (giné-bordonaba and terry 2016). the results showed that fruit development and fruit qualities of papaya are significantly affected when exposed to prolonged drought condition. for example, the fruits of drought-affected trees are generally small, while the fruits of hermaphrodite trees are deformed (figure 3). fruit deformation occurs in hermaphrodite trees where the stamens become carpelloidic and produce deeply ridged or irregular shaped fruits called “cat-faced”. this condition is most probably due to the high temperature and low soil moisture during drought (purseglove 1968). in addition, this result corresponded with a previous report that drought causes fruit deformation and fruit size reduction in raspberry (rubusidaeus) (morales et al. 2013). however, it is interesting to note that some trees were identified as putative drought-tolerant since they can withstand drought, develop a regular crown with green leaves, and still bear fruits (figure 4). p.m. magdalita et al. 63 figure 4. selected drought-tolerant papaya trees based on tree characters at first and second fruiting cycle. these trees are good selections as sources of drought-tolerant traits in breeding for drought tolerance in papaya. after further purification followed by screening, selection of drought-tolerant individual plants can be done to increase their breeding value. table 3. fruit characters of papaya trees grown during normal condition, drought, and recovery phase after a drought condition conditions fruit weight (g) fruit length (mm) fruit width (mm) peel weight (g) peel thickness (mm) flesh thickness (mm) total soluble solids (ºbrix) edible portion (%) seed weight (g) normal (pre-drought) 1,533.08 a 195 a 132.46 a 107.32 a 17.0 a 28.31 a 11.42 b 87.17 a 89.82 a drought 400.4 c 85.5 c 57.8 c 87.7 b 11.6 c 21.7 c 13.6 a 84.2 b 85.1 b recovery phase 1,395.5 b 175.3 b 125.5 b 88.65 b 15.5 b 25.9 b 10.2 b 84.2 b 85.1 b recovery from drought (%) 71.31 51.17 53.94 0.97 24.68 15.44 -33.33 3.86 35.72 means with the same letter are not significantly different at 0.05 level. the morphological responses in terms of fruit qualities of putative droughttolerant and drought-affected papayas and those that recovered after drought are presented in table 3. the recovery of the plants after drought was shown by an increase in value of the different characters such as fruit weight (71.31%), fruit length (51.71%) and fruit width (53.84%), but not tss. this is possible because tss, a relative measure of reducing sugars in papaya, is affected by water during the recovery phase when the trees are rehydrated, thus diluting its concentrations in morphological characters of papaya for drought tolerance 64 the fruit and resulting in the decrease in value. this indicates that the fruit traits at recovery stage returned to sub-normal conditions wherein they are similar to those observed during the normal condition or pre-drought stage. fruit characters observed during the recovery stage are within the range of the values observed during the normal condition or pre-drought stage. this result is in agreement with a previous finding in soybean (glycine max l.) that after rehydration the plant height and leaf area exhibited an increase in value (dong et al. 2019). however, since the trees were already older during the recovery stage, their fruit characters were no longer similar to those traits shown by the papaya trees during the normal condition or pre-drought condition when the trees were still young and very vigorous. concomitant to this result, it was found in maize (zea mays l.) that, after reuptake of water of a plant subjected to drought, most of the physiological parameters like leaf water content, water potential, osmotic potential, gas exchange, and chlorophyll content returned to normal levels (chen et al. 2015). in addition, after reuptake of water, plant growth, and photosynthesis became normal as shown by growth of new plant parts (xu et al. 2010). phenotypic correlation of different characters of drought-tolerant papaya trees showed weak to strong positive correlation (table 4). fruit weight has strong positive correlation with fruit length (0.81), fruit width (0.75), peel weight (0.91), table 4. phenotypic correlation among different characters of the selected drought-tolerant papayas ph sd cd ch lp gl fwt fl fw pw pt ft tss sw ph 1.00 sd 0.22 1.00 cd 0.34 0.43 1.00 ch 0.23 0.56 0.60 1.00 lp 0.25 0.46 0.62 0.90 1.00 gl 0.10 0.63 0.26 0.41 0.28 1.00 fwt -0.01 0.28 0.11 0.12 0.08 0.10 1.00 fl 0.11 0.33 0.15 0.09 0.05 0.27 0.81 1.00 fw 0.11 0.38 0.14 0.09 0.02 0.37 0.75 0.93 1.00 pw 0.02 0.23 0.14 0.11 0.09 0.07 0.90 0.83 0.78 1.00 pt -0.17 0.24 0.12 0.14 0.15 0.21 0.69 0.82 0.83 0.73 1.00 ft -0.03 0.40 0.09 0.12 0.12 0.26 0.78 0.88 0.91 0.80 0.89 1.00 tss 0.03 0.36 0.03 0.16 0.19 0.17 0.71 0.83 0.84 0.72 0.82 0.92 1.00 sw -0.02 0.12 0.01 0.01 -0.06 0.21 0.56 0.64 0.71 0.59 0.58 0.61 0.53 1.00 phplant height, sdstem diameter, cdcrown diameter, chcrown height, lplength of petiole, glno. of green leaves, fwtfruit weight, flfruit length, fwfruit width, pwpeel weight, ptpeel thickness, ftflesh thickness, tsstotal soluble solids, swseed weight p.m. magdalita et al. 65 flesh thickness (0.78), and tss (0.71), indicating that the fruit weight of droughttolerant papayas is strongly correlated with several traits. this finding corroborated a previous result that fruit weight of rambutan (nephellium lapaceum) is also strongly positively correlated with fruit length, fruit width, and seed weight (magdalita and valencia 2004). in addition, the present result coincided with an earlier report on jackfruit, another hardy and drought-tolerant species of fruit crop, indicating that fruit weight is strongly positively correlated with fruit length and fruit width (magdalita et al. 2011). fruit length is also strongly positively correlated with peel weight (0.83), peel thickness (0.82), and tss (0.83). the same has been observed between fruit width and peel weight (0.78), fruit width and peel thickness (0.83), fruit width and tss (0.84), and fruit width and seed weight (0.71). these results indicate that fruit length and fruit width are strongly correlated with several fruit morphological traits, including the sugar content of the flesh of the fruits of drought-tolerant papaya trees. a similar observation was made in avocado, a relatively known hardy fruit, that fruit length is correlated with flesh thickness, while fruit width has strong positive correlation with seed width (magdalita and valencia 2004). also in the present study, peel weight is correlated with flesh thickness (0.73), peel thickness (0.80) and tss (0.72). similarly, peel thickness has strong positive correlation with flesh thickness (0.89) and tss (0.82). furthermore, papayas with thick flesh could also have high tss, suggesting they could be sweet. however, very weak to no correlation existed between peel weight and plant height (0.02), and tss and plant height (0.03). also, there was very weak positive correlation between crown diameter and flesh thickness (0.09), crown diameter and tss (0.03), and crown diameter and seed weight (0.08). furthermore, the number of green leaves and peel weight had very low positive correlation (0.07). this finding suggests that these non-correlated characters are independent from each other and that they are unique characters of drought-tolerant papayas. the present result corroborated a previous finding in known drought-tolerant fruit crops like jackfruit where fruit weight had little correlation with fruitlet width, fruit weight, and percent of edible portion (magdalita et al. 2011). morphological characters of papaya for drought tolerance 66 summary and conclusion the drought-tolerant papayas were significantly taller and had thicker stem diameter, wider crown, and more green leaves compared to drought-affected papayas. drought significantly affected the fruit development and fruiting habit of papaya in that trees grown under drought condition produced fewer number of fruits than trees grown under normal condition. however, the tss of papayas under drought was significantly higher compared to tss of fruits under normal condition. selected drought-tolerant papaya trees had significantly higher fruit weight, fruit length, fruit width, peel weight, peel thickness, flesh thickness, and seed weight than the drought-affected papaya trees. however, the abovementioned fruit characters were reduced among the drought-affected trees. fruit development and fruit qualities of papaya were significantly affected when exposed to drought condition. the fruits of drought-affected trees were generally small, while the fruits of hermaphrodite trees were deformed. after drought, recovery of the papaya trees was shown by an increase in value of the different characters, such as fruit weight (71.31%), fruit length (51.71%), and fruit width (53.84%), but not tss. a strong positive correlation was detected among different fruit characters. fruit weight was strongly and positively correlated with fruit length (0.81), fruit width (0.75), peel weight (0.91), flesh thickness (0.78), and tss (0.71). overall, drought affected the growth and fruit development of papaya but the selected drought-tolerant trees had better stature, thicker stem diameter, wider crown, and more green leaves compared to drought-affected papayas. in addition, selected drought-tolerant papaya trees had heavier, longer, and wider fruits with thicker peel and flesh and heavier seeds than fruits from drought-affected papaya trees. the morphological responses to drought of papaya are important information for the selection of lines with drought tolerance. the selection based on the responses to drought can be used to identify putative lines that can be used to create hybrids and utilize heterosis for increased drought tolerance to produce drought-resilient papaya varieties in the future. acknowledgements this study was based on two projects namely: i) monitoring and detection of ecosystems changes for enhancing resilience and adaptation in the philippines (modecera) program–project 3: monitoring of the responses and productivity of industrial and fruit crops and development of intervention strategies to enhance crop production to climate change, funded by the department of science and p.m. magdalita et al. 67 technology-philippine council for agriculture 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[psa] philippine statistics authority. 2021. crop statistics; [accessed 2021 jun 19]. https:// openstat.psa.gov.ph/. purseglove jw. 1968. caricaceae. in: tropical crops dicotyledons i. new york: longman green and co. ltd. p. 45. torres-netto a, campostrini e, oliviera jg, smith reb. 2005. photosynthetic pigments, nitrogen, chlorophyll a fluorescence and spad502 readings in coffee leaves. scientia horticulturae. 104(2):199–209. taiz l, zeiger e. plant physiology. 5th ed. sunderland (uk): sinauer associates, inc. van de wal b, van mealebroel l, steppe k. 2017. application of drought and salt stress can improve tomato fruit quality without jeopardizing production. acta hortic. 1170:729–736. xu z, zhon g, shimizu h. 2010. plant responses to drought and re-watering. plant signaling and behavior. 5(6):649–654. zhang yj, li yh, gao h, wang l, kong ds, wu y, lu wk, tian jw, lu yl. 2019. effect of drought stress on leaf gas exchange, chlorophyll content, and dry matter allocation of phragmitesaustralis in the heihe river basin. philipp agric scientist. 102(2):141–148. ______ dr. pablito m. magdalita <pmmagdalita@up.edu.ph > is a professor and up scientist iii at the institute of crop science, and an affiliate researcher and plant breeder at the institute of plant breeding, college of agriculture and food science (cafs), university of the philippines los baños (uplb). he also teaches horticulture and plant breeding courses at uplb. his research is focused on fruit breeding and biotechnology. dr. edna a. aguilar is a professor at the institute of crop science, cafs uplb. she is currently teaching advanced crop and fruit physiology, and farming systems courses. her research endeavors are focused on farming systems and fruit physiology. mr. alangelico o. san pascual, m.sc. is a university research associate ii at the institute of plant breeding, cafs uplb. he is currently involved in plant breeding projects involving fruits and ornamentals. mr. michael r. noel, m.sc.(+) was a former university researcher at the institute of crop science, cafs uplb. his short stint at the institute was focused on understanding the effect of drought and other adverse conditions on fruit crop production. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 ocr document page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 ocr document 16_bahague 75 static behaviors science diliman (january-june 2003) 15:1, 75-79 static behaviors of confined time-arrival operators r.t. bahague jr.* and e.a. galapon theoretical physics group, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: rbahague@nip.upd.edu.ph abstract we show that the quantization of the classical time-of-arrival (toa) for arbitrary position x still leads to a class of self-adjoint toa-operator for a confined particle. the spectrum of the toa-operator is studied for different cases. introduction when does a given particle prepared in some initial quantum state arrive at a given spatial point? in standard quantum formalism, this raises the timeof-arrival (toa) at the level of quantum observable where the toa distribution is supposedly derivable from the spectral resolution of a self-adjoint toaoperator canonically conjugated to the driving hamiltonian. recently, galapon (2000) has shown that objections in constructing such toa operators, due to pauli’s theorem, do not hold within the single hilbert space formulation of quantum mechanics. also, researchers have evidently not been discouraged from seeking an expression for the toa distribution within a consistent theoretical framework (muga & leavens, 2000). we construct the toa-operator for arbitrary detector position as a generalization of the operator constructed for the detector position at x = 0 (galapon, 2002), which has shown that a class of self-adjoint and canonical toa operator can be constructed for a spatially confined particle in the interval [-l,+l]. by considering the symmetry properties of the constructed toa operator, theoretical predictions for the probability distributions were obtained and compared with numerical results. confined toa and toa operators the toa at x of a classical particle with position q, momentum p, and mass µ is given by (1) symmetrizing the classical expression (eq. (1)) for the toa at x gives (muga & leavens, 2000) (2) in which t, q, and p are the operator versions of t, q, and p, respectively. we attach the hilbert space h = l2[-l,l]. the position operator is unique and is given by the bounded multiplicative operator, q, whose domain is the entire hilbert space. we rename the momentum operator p ( )t q x p µ = − − ( ) ( )1 1 2 t q x p p q x µ − −⎡ ⎤= − − + −⎣ ⎦ * corresponding author 76 bahague jr. and galapon by with domain . the hamiltonian operator is whose domain is . we consider to cover the entire symmetry of the classical toa in the quantum domain. different values of γ correspond to different physics. we also rename t by tγ , such that eq. (2) becomes (3) the momentum and the hamiltonian operators commute and have a common set of eigenvectors, and both have pure point spectra. non-periodic boundary condition since q appears in first power and x is just a parameter in eq. (3), tγ is an operator if the inverse of the momentum operator pγ -1 exists. for this non-periodic case, zero is not an eigenvalue of pγ , thus, the inverse of pγ exists. pγ is unbounded and self-adjoint, thus pγ -1 is bounded, everywhere defined (by extension) and selfadjoint. then it follows that for every tγ is bounded, everywhere defined, and is a symmetric operator. thus, tγ is self-adjoint. in coordinate representation, eq. (3) assumes the form of a fredholm integral operator (galapon, 2000) (4) with the non-periodic kernel (5) 2 2 2 qhγ φµ = − ∂ h ( ) {d hγ φ= ∈ ( ) ( ) ( ) ( ) ( )}: '' , ' exp 2 'd p q h l i lγ φ φ γ φ∈ − = − ( ),γ π π∈ − ( ) ( )1 1 2 t q x p p q xγ γ γ µ − −⎡ ⎤= − − + −⎣ ⎦ ( ) ( )1 exp , 0, 1, 2,..., 2 q q i n n ll γφ γ π ⎛ ⎞= + = ± ±⎜ ⎟ ⎝ ⎠ ( ) ( ) ( ), ' ' ' l l t q t q q q dqγ γφ φ−= ∫ ( ) ( ), ' ' 2 4 sin t q q q q xγ µ γ = − + − h ( ) ( ) ( ) ( )( )exp ' exp 'i h q q i h q qγ γ− + − − in which h(q,q’) is the heaviside function. tγ is canonically conjugate to hγ in the canonical domain (6) periodic boundary condition for the periodic case, zero is an eigenvalue of the momentum operator, thus, the inverse of pγ doesn’t exist. but toa is a valid question only if the particle is in motion, otherwise it goes nowhere. we then expect that the non-periodic kernel eq. (5) has a finite part corresponding to the non-vanishing momentum components in the limit as the finite part is extracted by removing the divergent contribution of the vanishing momentum eigenvalue (galapon, 2002), such that the kernel becomes (7) and the canonical domain for the hamiltonian and toa-operator, t 0 (8) both kernels corresponding to the non-periodic and the periodic boundary conditions are symmetric and bounded, reaffirming the self-adjointness of the toa operators. also, they are compact and the canonical domains are closed, such that the pair (hγ,tγ) forms a canonical pair on this closed subspace of the hilbert space (galapon, 2002). confined time-of-arrival (toa) symmetries by symmetry consideration, we derive some properties of the confined toa-operator and infer relationships among the eigenfunctions. we particularly consider the behavior of the toa-operator on the actions of the parity operator, π and the time reversal operator, θ. ( ) ( ) }0, 0,1 .k kl l kφ φ− = = = 0.γ → ( ) ( ) ( ) ( )0 1 , ' ' 2 sgn ' ' 4 i t q q q q x q q q q l µ− ⎡ ⎤= − − − − −⎢ ⎥ ⎣ ⎦h { ( ) ( ) ( )0 0 : ' ' ' 0, l c l d q d h q q dqφ φ − = ∈ =∫ ( ) ( ) }0, 0,1k kl l kφ φ− = = = ( ) ( ) ( )}' exp 2 'l i lφ γ φ− = − ( ) ( ) ( ){ : ' ' 0,lc ld q d h q dqγ γφ φ−= ∈ =∫ p i q γ ∂ = − ∂ h ( ) { : ' ,d p h hγ φ φ= ∈ ∈ ( ), ,γ π π∈ − 77 static behaviors the actions of π and θ are a n d , respectively, where are vectors of the hilbert space (we particularly considered the initial state, ). non-periodic case the symmetries of the non-periodic toa-operators follow directly from the invariance of their kernels under the following operations (9) (10) (11) we denote t [γ,x] as the toa-operator at x for the case γ with the kernel in eq. (4) as t [γ,x](q,q’,x) and ϕ[γ,x] as the corresponding eigenfunctions. for every , it can be shown that [ ] [ ], , ,x xt tγ γϕ ϕ− θπ = −θπ and using eq. (9), the probability density relations in coordinate and momentum representations are (12) (13) we also find and using eq. (11) leads to (14) (15) also, it can be shown that, with the following probability distributions from eq. (10) (16) (17) if we let , where is the eigenvalue of the toa-operator, we found that (18) [ ] ( ) [ ] ( ), ,, ', , ',x xt q q x t q q xγ γ ∗ −= − − − − [ ] ( ) [ ] ( ), ,, ', , ',x xt q q x t q q xπ γ π γ ∗ − − −= − [ ] ( ) [ ] ( ), ,, ', , ',x xt q q x t q q xγ γ ∗ − −= − hϕ ∈ [ ] ( ) [ ] ( ) 2 2 , ,x x q qγ γϕ ϕ− = − [ ] ( ) [ ] ( ) 2 2 , ,x x k kγ γϕ ϕ− = [ ] [ ], ,x xt tγ γϕ ϕ− θ = −θ [ ] ( ) [ ] ( ) 2 2 , ,x x q qγ γϕ ϕ− = [ ] ( ) [ ] ( ) 2 2 , ,x x k kγ γϕ ϕ− = − [ ] [ ], ,x xt tπ γ π γϕ ϕ− −θ = −θ [ ] ( ) [ ] ( ) 2 2 , ,x x q qπ γ γϕ ϕ− = [ ] ( ) [ ] ( ) 2 2 , ,x x k kπ γ γϕ ϕ− = − [ ] [ ], ,x xt γ γϕ τ ϕ= [ ], xγτ [ ] ( ) [ ] ( ), ,x xt q qπ γ γϕ τ ϕ− = − (19) (20) periodic (γγγγγ = 0 and γγγγγ = πππππ/2) case we note the following symmetries of the periodic kernel (21) (22) for every , we note . it can be shown that [ ]0, xt has positive and negative eigenvalues of equal magnitudes, with corresponding eigenfunctions, and . using eq. (21), we get the following probability densities (23) (24) for opposite x, we have and by using eq. (22), we find (25) (26) spectrum of the time-of-arrival (toa) operator the solution to eq. (4) reduces to an eigenvalue problem (27) which is solved using the nystrom method for second order homogenous fredholm integral equation (delves & mohamed, 1985). we produce eigenfunctions and eigenvalues for the toa-operator, which were not done in current literatures (muga & leavens, 2000), although we will not emphasize on the numerical values of the simulation, but more on the behaviors of eigenfunctions and the spectrum. [ ] ( ) [ ] ( )0, 0,, ', , ',x xt q q x t q q x ∗= − [ ] ( ) [ ] ( )0, 0,, ', , ',x xt q q x t q q x ∗ −= − − − − hϕ ∈ [ ] [ ]0, 0,x xt tϕ ϕθ = −θ ϕ + ϕ − ( ) ( ) 2 2 q qϕ ϕ− += ( ) ( ) 2 2 k kϕ ϕ− += [ ] [ ]0, 0,x xt tϕ ϕ− θπ = −θπ [ ] ( ) [ ] ( ) 2 2 0, 0,x x q qϕ ϕ− = − [ ] ( ) [ ] ( ) 2 2 0, 0,x x k kϕ ϕ− = ( ) ( ) ( ), ' ' l l t q q q dq qγ γτ γ τφ τ φ− =∫ ( )≠ / 2γ π ( ) ( ), ,q t q tϕ ϕπ = − ( ) ( ), ,q t q tϕθ = − ( ) (, ,q t qϕ ϕ= )0 ( ), 0qϕ [ ] ( ) [ ] ( ), ,x xt q qγ γϕ τ ϕ− = − [ ] ( ) [ ] ( ), ,x xt q qγ γϕ τ ϕ− = − 78 bahague jr. and galapon for the non-periodic case, we are able to verify eqs. (12) to (17). in particular, in fig. 1, relations of the position probability conforms with eq. (12) and the eigenvalue of operators [ ], xt γ and [ ], xt γ − , which are opposite in sign but are of equal magnitude are consistent with eq. (20). the corresponding momentum probability for fig. 1 is shown in fig. 2. this is consistent with eq. (13). for the periodic boundary case, we particularly focused on the detector at x = 0. but we have also confirmed eqs. (25) and (26). this operator was constructed in galapon (2000). on figs. 3 and 4, the probability densities corresponding to the negative and positive eigenvalue are again overlapping for both the momentum and position. these are consistent with eqs. (23) and (24). fig. 3. probability density of the position corresponding to the eigenfunctions of the negative (φ-) and positive (φ+) eigenvalue at x = 0. the probability densities overlap and their eigenvalues differ in signs only. 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -1 -0.5 0 0.5 1 position p ro b a b il it y d e n s it y φ+(q) φ-(q) fig. 2. momentum probability distribution for detector positions x = 0.5 and x = -0.5, with γ = 3. the x-axis is the momentum value nπ, where n = 0, +1, ... the distributions overlap. momentum p ro b a b il it y d e n s it y 0.35 0.3 0.25 0.2 0.15 0.10 0.05 0 -50 0 50 x = 0.5 x = -0.5 fig. 4. the momentum probability density for periodic boundary condition at x = 0. the probabilities corresponding to the negative and positive eigenvalues overlap. 0.06 0.05 0.04 0.03 0.02 0.01 0 -200 -150 -100 -50 0 50 100 150 200 momentum p ro b a b il it y d e n s it y φ+(k) φ-(k) fig. 1. position probability distribution for detector positions x = 0.5 and x = -0.5; with γ = 3 and eigenvalues τ[3,0.5] = -4.3944 and τ[3,-0.5] = 4.3944. the distributions are mirror images of each other. 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -1 -0.5 0 0.5 1 x = 0.5 x = -0.5 position p ro b a b il it y d e n s it y 79 static behaviors conclusion we have shown that the quantization of the classical toa at arbitrary x for a spatially confined particle allows a construction of a quantum mechanical counterpart, a toa-operator, which is canonically conjugate to the free hamiltonian. the eigenvalues are supposed to be the outcome of a toa measurement. from symmetry considerations, we derived some properties of the toa for different cases. but a better insight on the toa operator properties is to be obtained by evolution of the given stationary states. references delves, l.m. & j.l. mohamed, 1985. computational methods for integral equations. cambridge university press. galapon, e.a., 2000. canonical pairs, spatially-confined motion, and the quantum time-of-arrival problem. quant-ph/ 0001062. galapon, e.a., 2002. pauli’s theorem and quantum canonical pairs: the consistency of a bounded self-adjoint time operator canonically conjugate to a hamiltonian with non-empty point spectrum. proc. roc. soc. 487: 451. muga, j.g. & c.r. leavens, 2000. arrival time in quantum mechanics. phys. rep. 338: 353-438. press, w.h., et al., 1992. numerical recipes in fortran77: the art of scientific computing. cambridge university press. 06_baclayon 27 time-of-flight measurement science diliman (january-june 2003) 15:1, 27-31 time-of-flight measurement of a 355-nm nd:yag laser-produced aluminum plasma m.f. baclayon*, c.a. alonzo, and w.o. garcia photonics research laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines email: marian@nip.upd.edu.ph abstract an aluminum target in air was irradiated by a 355-nm nd:yag laser with a pulse width of 10 ns and a repetition rate of 10 hz. the emission spectra of the laser-produced aluminum plasma were investigated with varying distances from the target surface. the results show the presence of a strong continuum very close to the target surface, but as the plasma evolve in space, the continuum gradually disappears and the emitted spectra are dominated by stronger line emissions. the observed plasma species are the neutral and singly ionized aluminum and their speeds were investigated using an optical time-of-flight measurement technique. results show that the speeds of the plasma species decreases gradually with distance from the target surface. comparison of the computed speeds of the plasma species shows that the singly ionized species have relatively greater kinetic energy than the neutral species. introduction laser ablation of solids with short-pulsed, high-intensity light source has led to complicated light matter interactions. some of the fundamental physical features of these interactions have not yet been fully understood, but nevertheless various practical applications have been developed in recent years, such as a cutting tool for metals, an elemental and chemical composition characterization technique (corsi et al., 2001), and a promising chemical deposition technique (richter, 1990). pulsed laser deposition of thin films has evolved into a well-recognized technique for a wide range of materials and in a variety of devices, especially in forming multicomponent films from stoichiometrically complex target source. among other factors that affect the quality of the deposited film, a key parameter is the kinetic energy of the vaporized atoms and ionized species. the physical characteristics of the ejected species play a crucial role in the microscopic mechanism of film growth (corsi et al., 2001; willmott & huber, 2000). the study of laser-produced plasma (lpp) is essential to understanding the light-matter interactions and the numerous applications it entails. in this paper, we report a study that aims to investigate the parameters and characteristics of the plasma produced during this highenergy laser ablation of solid materials. the plasma parameters that were investigated were the spatial evolution of the plasma emission spectra and the velocities of the different species produced specifically by the neutral and singly ionized aluminum. optical emission time-of-flight measurement is the method used in the determination of the speeds of the plasma species. this emission spectroscopy method was chosen because it is non-intrusive, fast, and* corresponding author 28 baclayon, alonzo, and garcia provides an effective and reliable determination between the produced neutral and ionized species (willmott & huber, 2000; wang et al., 1996). experimental procedure the experimental setup is shown schematically in fig. 1. the 355-nm beam generated by the third harmonic frequency of a q-switched nd:yag laser (spectra physics gcr-230-10) was focused with a 254-mm focal length quartz uv lens, l1, onto the surface of the rotating aluminum target. the laser was operated with a pulse width of 10 ns and a repetition rate of 10 hz. the plasma plume was collected by a uv lens, l2, and imaged by another uv lens, l3, to a magnification of 5x. the emission spectra of the plasma was measured using an optical fiber mounted on a 1d micrometer translational stage to a monochromator (spex 1000m) with 1600 grooves/mm grating installed and equipped with a photomultiplier tube (pmt) detector (hamamatsu r212). the slits of the monochromator were set at 200-mm widths for a 1.6 å resolution. the signal was rid of noise and further amplified by a digital lock-in amplifier (srs sr830), digitized, and finally analyzed by a personal computer. the optical time-of-flight measurements were done by connecting the pmt directly to a 500-mhz digitizing oscilloscope (tektronix tds 644b) triggered by the ttl signal synchronized with the q-switching of the laser while the monochromator was centered to the observed peaks of aluminum. the optical fiber detector was placed on the image plane and aligned with the centerline of the plume to detect only the plasma species traveling axially above the target surface along the centerline of the plasma. results and discussion when a short-pulsed laser strikes a solid surface, the rapid rise in temperature leads to intense evaporation of atoms and molecules from the solid. even at relatively low intensities near the threshold for ablation, it is observed that the ablated material is significantly ionized, and the ions in the plasma plume can reach energies ranging up to several hundred ev. at the end of the laser pulse, the ablated material exists as a thin layer of plasma on the target surface. initially, the expansion of the plasma plume is primarily driven by the plasma pressure gradients, but there may be additional contribution from coulomb repulsion between the ions. when the plume has propagated more than a hundred micrometers from the target surface, the major part of the initial thermal energy in the plasma is converted to the directed kinetic energy of the ions (willmott & huber, 2000). plasma emission the emission spectrum of the laser-produced aluminum plasma was recorded at different distances from the target surface. this was done by moving the optical fiber detector to increasing distances away from the target. fig. 2 shows the spatially resolved and time-integrated spectra of selected neutral and singly ionized aluminum peaks. an intense continuum, very close to the target, was observed as the early development of the plasma. this continuum emission was attributed to both the elastic collisions of electrons with ions and atoms (free-free brehmsstrahlung) pulsed nd:yag laser (1064 nm) mirror sync out l1 l2 l3 plasma plume rotating target image plane spectrometer computer a/d converter lock-in amplifier digital oscilloscope pmt fig. 1. experimental setup. optical fiber 29 time-of-flight measurement and the recombination radiation accompanying the electron-ion recombination (free-bound brehmsstrahlung) (willmott & huber, 2000; wolf, 1992). an example is the recombination process of the singly ionized species and an electron to form a neutral aluminum (wolf, 1992). as the plasma expands away from the target, it can be observed that the continuum gradually decreased and line emissions started to appear and become stronger than the continuum. at these distances, line emissions dominated the radiation process. this is manifested in fig. 2, starting at distances 0.4 mm to 0.6 mm away from the target surface. however, as the plasma expands further starting from 1 mm, the intensity of the line emissions gradually decreased. it is shown that among the observed lines, al (i) (4663 å) decreased rapidly at 0.8 mm and diminished totally at 1.0 mm. this can again be attributed to the singly ionized ion-electron recombination, thereby producing a neutral aluminum (griem, 1964). table 1 enumerates the observed line emissions of aluminum in the produced plasma. the theoretical values are the air wavelengths of aluminum (crc handbook of physics and chemistry, 2000) and the observed values are the average of the spatially resolved spectra. the deviation of the observed values from the theoretical ones can be attributed to the different line broadening and shifting mechanisms occurring in the plasma, such as the doppler, pressure, and stark broadening and shifting mechanisms (bekefi, 1976; griem, 1964). the ejection/expansion velocities of the radiating plasma species cause the doppler shift while collisions in the plasma causes the pressure broadening. stark broadening, which is the more prominent, is attributed to the interaction between the radiating species and the charged particles in the plasma. expansion velocities of the ejected species fig. 3 shows the graph of the delay time of the al (i) species relative to the laser pulse versus its probable location in the plasma. from this data, the speeds of the species were deduced. estimates of their ejection speeds were obtained by determining their delay time as they propagate a certain distance along the plasma. table 1. observed al (i) and al (ii) peaks. aluminum and their emission lines (å) observed plasma emission lines (å) al (i) al (i) al (i) al (i) al (ii) al (ii) 3082.15 3092.71 3944.00 3961.52 4663.05 6183.45 3081.4 + 4.2 3091.6 + 5.4 3943.1 + 3.3 3960.8 + 4.1 4664.3 + 8.1 6185.0 + 5.2 1.4 mm 1.2 mm 1.0 mm 0.8 mm 0.6 mm 0.4 mm 0.2 mm 0.0 mm fig. 2. spatially resolved plasma emission lines of neutral (a & b) and singly-ionized (c & d) aluminum for distances of 0 mm to 1.4 mm from the target. 3070 3110 3920 3990 4500 4800 6100 6250 (a) (b) (c) (d) 30 baclayon, alonzo, and garcia thus, for the 3082 å line with a distance between 1.2 to 1.4 mm from the target surface, we get an estimate of its ejection speed of 4.0 x 104 cm/s. figs. 4 and 5 show the computed speeds of the neutral and singly ionized aluminum species. it can be observed that the species acquired a very high ejection speed as it was exfoliated from the target surface and gradually decreased as it propagates along the plasma. this gradual slowing down of the plasma species is attributed to its collisions with the other plasma species, such as the electrons and the ions, and the presence of the surrounding air, which can cause an impediment to its expansion in space. it can also be observed that in comparison to the computed speeds of the selected plasma species, the singly ionized aluminum species have relatively higher speeds than the neutral aluminum species. the difference in their speeds can be attributed to the lesser mass of the singly ionized aluminum species due to the absence of an electron. thus, it can be inferred that the singly ionized aluminum species have greater kinetic energy than the neutral species. conclusion the spatially resolved spectra of the laser-produced plasma show that a strong continuum was generated close to the target surface, but as the plasma expands in space, stronger line emissions gradually appear. the “masking” of the line emissions near the target surface can be attributed to the many-body collisions, which occur as the plasma constituent species are ejected from the material surface. as the different species expand in space, collisions with other plasma species are reduced, and its further evolution in space is determined by its interaction with the surrounding air by elastic and inelastic collisions and recombination with air molecules and particles. the emission time-of-flight measurements fig. 3. location of the al (i) species and its delay time with respect to the laser pulse. location from target surface (mm) d e la y t im e f ro m l a s e r p u ls e ( s ) 6 3 5 2 4 1 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 3082 å 3092 å 3944 å 3961 å fig. 4. computed speeds of the neutral aluminum plasma species. distance from target surface (mm) s p e e d ( x 1 0 3 m /s ) 0 25 0.4 0.6 0.8 1.0 1.2 1.4 1.6 5 10 15 20 3082 å 3092 å 3944 å 3961 å fig. 5. computed speeds for the singly ionized aluminum plasma species. 4663 å 6185 å 0.4 0.6 0.8 1.0 1.2 1.4 1.6 distance from target surface (mm) 0 100 20 40 60 80 s p e e d ( x 1 0 3 m /s ) 0.2 31 time-of-flight measurement were used to deduce the speeds of the individual species produced in the plasma. it was observed that the singly ionized species have greater kinetic energies than the neutral species. continuing research is done on the investigations of the ejection speeds of the plasma species by observing as many species as possible to further verify our present results. the current research is a part of a continuing project that aims to investigate the pulsed laser deposition parameters and eventually, the deposition of high quality thin films. acknowledgments the authors gratefully acknowledge the assistance provided by mr. jonathan a. palero and ms. marilyn a. hui in the experiment. this research is funded by the philippine department of science and technology through the engineering and science education project and the philippine council for advanced science and technology research and development (pcastrd). references bekefi, g., 1976. principles of laser plasmas. new york, wiley. corsi, m., et al., 2001. a fast and accurate method for the determination of precious alloy caratage by laser-induced plasma spectroscopy. eur. phys. j. d. 13: 373-377. crc handbook of physics and chemistry, 2000. griem, h.r., 1964. plasma spectroscopy. new york, mcgraw-hill. knudtson, j.t., et al., 1987. the uv-visible spectroscopy of laser-produced aluminum plasmas. j. appl. phys. 61(10): 4771-4780. richter, a., 1990. thin solid films. 188: 275. wang, x.t. et al., 1996. optical spectroscopy of plasma produced by laser ablation of ti alloy in air. j. appl. phys. 80(3): 1783-1786. willmott, p.r. & j.r. huber, 2000. pulsed laser vaporization and deposition. rev. mod. phys. 71(1). wolf, p., 1992. the plasma properties of laser-ablated sio2. appl. phys. 72(4): 1280-1289. 15_two two-photon optical beam 61 two-photon optical beam-induced current microscopy of light-emitting diodes godofredo s. bautista jr.*, carlo mar y. blanca, and caesar a. saloma national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: gbautista@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 61–65 *corresponding author we demonstrate two-photon optical beam-induced current (2p-obic) microscopy of light-emitting diodes (leds). we utilized a ti:sapphire femtosecond laser source operating at 800 nm to derive the 2p-obic signal from a 605 nm band-gap led. the spatial confinement of free carrier generation only at the focus and the quadratic dependence of the 2p-obic signal on excitation power are the key principles in twophoton excitation. as a consequence, superior image quality evident in the 2p-obic images of leds are obtained. these features decrease the linear absorption and wide-angle scattering effects plaguing singlephoton optical beam-induced current (1p-obic) technique, thereby increasing the resolution of the imaging system in the axial and lateral directions. thus, the attainment of good axial discrimination in the led samples is obtained even without a confocal pinhole. in addition, 2p-obic images reveal local variations in free carrier densities which are not evident in the single-photon excitation. introduction when a laser beam with energy higher than the bandgap energy of the p-n junction irradiates semiconductors, generation of electron-hole pairs occurs and flow in the semiconductor to produce electrical current (takasu, 2001). measuring this current and mapping them on a display result to an optical beam-induced current (obic) image. this has become a major tool in the detection of defects in semiconductor devices (wilson & sheppard, 1984). however, a disadvantage of this technique is its inability to discriminate the localizations of the current distribution in the semiconductor since it is insensitive to focal positions (koyama et al., 1999). recently, researchers have devised a method to impart axial resolution to obic images from information derived from corresponding confocal reflectance images (daria et al., 2002; miranda & saloma, 2003; cemine et al., 2004). we approach this problem by introducing nonlinearity in the photon absorption process to generate free carriers which can be externally detected as current flow. obic via two-photon excitation (2p-obic) has been developed to visualize free carrier densities in the semiconductor (xu & denk, 1997). in 2p-obic, each electron-hole pair is generated via absorption of the energies of two photons. it takes advantage of the quadratic dependence of the generated photocurrent on the excitation power and the confined illumination only at the focus as shown in two-photon fluorescence imaging (denk et al., 1990). 2p-obic microscopy has also been utilized in backside imaging of flip chips, integrated circuits, and indium gallium nitride lightemitting diodes (xu & denk, 1999; kao et al., 1999; ramsay & reid, 2002). bautista, blanca, and saloma 62 here, we construct a two-photon optical beam-induced current microscope to image and characterize lightemitting diodes using a femtosecond laser source and a stage-scanning microscope. we compare free carrier generation, axial resolution, and image formation of 2p-obic with 1p-obic using a 543 nm hene laser. results unraveled the characteristics of these semiconductor devices previously unseen by the 1pobic technique. experiment the schematic diagram of the stage-scanning custombuilt microscope used in investigating 2p-obic is shown in fig. 1. the illumination source used is a mode-locked ti:sapphire femtosecond pulse laser at 800 nm (tsunami, spectra-physics) pumped by a frequency-doubled solid-state laser (millennia, spectraphysics). a neutral density filter (ndf) is used to attenuate the excitation source. the beam is reflected by a relay of mirrors (m1 and m2) and later expanded by a two-lens telescope composed of l1 (f = 25 mm) and l2 (f = 200 mm). the resulting collimated beam is then directed to an infinity-corrected objective lens (obj, olympus uplan fl, 20x, 0.5 na) overfilling its back aperture. the objective focuses the excitation beam on the lightemitting diode (led) sample mounted on the threeaxis (x,y,z) motorized stage (thorlabs, pt3-z6) driven by a motion-controller card (thorlabs, dcx-pci100). codes for stage scanning and image acquisition are all written in labview. the obic signals as a function of sample position are utilized to build the corresponding image of the sample at the focal plane. analog-to-digital (a/d) and digital-to-analog (d/a) conversion is accomplished with a 12-bit data-acquisition board (national instruments, 6024e). the led samples are commercially available and exhibit electroluminescence with a peak at 605 nm. throughout the scans, the average power which is measured before the objective lens is varied from 0.5 to 2.5 mw. in order to generate 1p-obic, we replaced the femtosecond laser with an 8 mw hene laser operating at 543 nm. the imaging optics and acquisition system were not changed. results shown in fig. 2 is the incident power dependence of the obic signals for the led sample. the quadratic dependence of the obic signal on the excitation power is expected for 2p-obic and the linear dependence for 1p-obic. we experimentally verified this for the 2p-obic process where the exponent of the excitation power is 1.9962. on the other hand, 1p-obic exhibited a linear behavior with a corresponding slope of 0.9095. because of the nonlinear behavior of 2p-obic, only regions near the focal volume generate current. this fig. 1. schematic diagram of the stage-scanning 2p-obic microscope used. pca/d d/a y x z 3-axis stage sampleobj l1 l2m 2 m 1 ndf mode-locked ti:sapphire laser 532 nm solid state laser fig. 2. power dependence of the generated obic signal. the slopes of the 2p-obic and 1p-obic responses are 1.9962 and 0.9095, respectively. laser input power (mw) 2p -o b ic s ig na l ( m v ) 1p -o b ic signal (v ) 1p-obic (0.9095) 2p-obic (1.9962) 42 35 28 21 14 7 0 0.5 1 1.5 2 2.5 1.6 1.4 1.2 1 0.8 0.6 0.4 two-photon optical beam 63 characteristic is clearly seen in fig. 3 where the photocurrent signal is plotted against the axial position obtained by moving the sample along the z axis. the largest measured 2p-obic signal is found at the focus located at z = 0 mm. a full-width-at-half-maximum (fwhm) value equal to 50 mm is measured for the 2p-obic axial profile. it is evident that as the sample is moved away from the focus or active region, the 2pobic signal declines considerably. this behavior is a result of the spatial confinement of excitation in a twophoton process (denk et al., 1990). absorption of the energies of two-photons is restricted only at the focus resulting in the generation of free carriers merely on that small region. electron-hole combination is not promoted in the other layers. asymmetry in the axial profile is attributed to spherical aberration caused by the index of refraction mismatch introduced by the plastic packaging material (xu & denk, 1999; kao et al., 1999; ramsay & reid, 2002). a different situation is observed if the 1p-obic is considered. as shown in fig. 3, 1p-obic is insensitive throughout the scanned axial positions. this is primarily due to the detection of the average photocurrents from all free carriers that are generated in the double-cone excitation along the optical axis. this results to 1pobic having very poor axial resolution and can only convey information in two dimensions (2d). in contrast, the nonlinear generation of 2p-obic endows it with an intrinsic optical sectioning capability enabling it to derive three-dimensional (3d) images even without confocal detection pinhole. to illustrate further the sensitivity of 2p-obic signals, the led sample is imaged at the focal position corresponding to the highest average photocurrent detected occurring at the active layer or z = 0 mm. figure 4 shows the image of the led affected by the presence of mode-locked illumination. during the period when the upper part of fig. 4 is scanned, twophoton excitation occurs. the energy of two-photons is enough to induce a free carrier generation resulting in a detectable photocurrent. midway through the scanning procedure, the modelocking mechanism of the femtosecond laser is turned off, converting the source to a continuous-wave laser. the quadratic requirement of two-photon generation can no longer be satisfied, resulting in the nondetection of the photocurrents at the observed sample positions. the energy of a single photon is therefore not enough to induce a photocurrent signal because it cannot transcend the led band gap. by comparing the images obtained using the 2p-obic and 1p-obic techniques, the advantage of two-photon excitation becomes more pronounced. shown in fig. 5 are the 2p-obic and 1p-obic images of the led separated by 25 mm along the z axis. average excitation powers at the sample were 1.5 and 0.06 mw for the twoand single-photon cases, respectively. the 2pobic procedure exhibits superior image quality compared with 1p-obic. since excitation is confined in the focal volume, free carriers are generated only at the focus. absorption and scattering effects in 1p-obic images are eliminated (xu & denk, 1999). two examples that will illustrate these effects are the fig. 3. focal dependence of the generated obic signal. throughout the scanned axial positions, 2p-obic changes while 1p-obic remains insensitive. the 2p-obic axial profile has a fwhm value of 50 µm. axial position (µµµµµm) o b ic s ig na l ( m v ) 1p-obic 2p-obic -80 -40 0 40 80 0 1.2 1.4 1.6 fig. 4. 2p-obic imaging of led with and without modelocked illuminations. image size is 625 x 625 µm. with mode-locked illumination without mode-locked illumination bautista, blanca, and saloma 64 differences in contrast of the electrodes and sharpness of the edges. in 2p-obic, the electrode appears dark and the edges appear sharp at all axial positions. however, in 1p-obic, a considerable amount of photocurrent is detected in the electrode regions and edges become enhanced due to wide-angle scattering, fig. 5. representative 2p-obic and 1p-obic images of the led sample at different focal positions. 2p-obic 1p-obic z = –75 µµµµµm z = –50 µµµµµm z = –25 µµµµµm z = 0 µµµµµm z = 25 µµµµµm z = 50 µµµµµm z = 75 µµµµµm which becomes more prominent as the sample is moved towards the objective lens. these scattered photons are energetic enough to induce 1p-obic and introduce spurious signals to the image. the longer wavelength used for 2p-obic suffer less scattering and do not corrupt the image because they do not have enough power to satisfy the quadratic excitation power requirement. the intrinsic optical sectioning capability of 2p-obic is clearly demonstrated in fig. 5. it can be seen from the figures that the led is optically sectioned by twophoton excitation attesting to the increase in axial resolution. on the contrary, 1p-obic images do not exhibit optical sectioning since the detected current is the average current across the whole excitation length. the most important effect of the nonlinear excitation of 2p-obic is the localized current topography on the led surface. the nonuniformity of the obic generation points to the inhomogeneity of the absorption process which can be influenced by the 3d distribution of free carriers on the volume of the active region. this can be clearly mapped out by 2p-obic but cannot be seen by 1p-obic, giving us an insight to semiconductor properties that are inaccessible using 1p-obic. conclusion in summary, optical beam-induced current imaging via two-photon excitation is demonstrated using lightemitting diodes. high-contrast images of leds exhibit a significant reduction of light scattering and absorption, thereby increasing the axial and spatial resolutions of the 2p-obic microscope. the nonlinearity of the excitation process enables it to image in 3d even in the absence of a confocal aperture. this revealed a localized absorption map of the semiconductor surface previously unseen by the 1pobic technique. acknowledgment we thank b. buenaobra for help in the development of the stage-scanning system. two-photon optical beam 65 references cemine, v.j., b. buenaobra, c.m. blanca, & c. saloma, 2004. high-contrast microscopy of semiconductor and metals sites in integrated circuits via optical feedback detection. opt. lett. 29: 2479-2481. daria, v., j. miranda, & c. saloma, 2002. high-contrast semiconductor sites via one-photon optical beam-induced current imaging and confocal reflectance microscopy. appl. opt. 41: 4157–4160. denk, w., j. strickler, & w. webb, 1990. two-photon laser scanning fluorescence microscopy. science. 248: 73–75. kao, f., m. huang, y. wang, m. lee, & c. sun, 1999. twophoton optical beam-induced current imaging of indium gallium nitride blue light emitting diodes. opt. lett. 24: 1407– 1409. koyama, t., k. sonoda, j. komori, & y. mashiko, 1999. detection of defects in metal interconnects by the nonbias optical-beam-induced current technique. j. appl. phys. 86: 5949–5956. miranda, j. & c. saloma, 2003. four-dimensional microscopy of defects in integrated circuits. appl. opt. 42: 6520–6524. ramsay, e. & d. reid, 2002. three-dimensional imaging of a silicon flip chip using the two-photon optical beam-induced current effect. app. phys. lett. 81: 7–9. takasu, s., 2001. application of obic/obirch/obhic: semiconductor failure analysis. jeol news. 36e(1): 60-63. wilson, t. & c.j.r. sheppard, 1984. theory and practice of scanning optical microscopy. academic press inc., new york. xu, c. & w. denk, 1997. two-photon optical beam-induced current imaging through the backside of integrated circuits. appl. phys. lett. 71: 2578–2580. xu, c. & w. denk, 1999. comparison of oneand two-photon optical beam-induced current imaging. j. appl. phys. 86: 2226–2231. 11_joson 51 kinetics of non-isothermal crystallization science diliman (january-june 2003) 15:1, 51-56 kinetics of non-isothermal crystallization of coconut-based cholesteryl ester: avrami and ozawa approaches j.f. joson*, l.t. davila, and z.b. domingo liquid cystals laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: joihren@nip.upd.edu.ph abstract kinetics of non-isothermal crystallization of coconut-based cholesteryl ester was performed by differential scanning calorimetry under various heating rates. different analysis methods were used to describe the process of non-isothermal crystallization. the results showed that the avrami equation could describe the system very well. however, the ozawa analysis failed. a probable reason is the difference in the crystallization kinetics at high and low relative crystallization. the phase transitions of the coconut-based cholesteryl ester were also observed through optical polarizing microscopy. introduction coco-based cholesteryl ester is a thermotropic liquid crystalline material synthesized from esters of fatty acids extracted from locally available coconut oil. it is waxy and powdery at room temperature (cureg, 2000). cholesteryl ester is also known to exhibit two mesophases between the solid state and isotropic liquid state, namely the smectic phase and the cholesteric phase. this polymorphism of cholesteryl ester causes three phase transitions: crystalline to smectic, smectic to cholesteric, and cholesteric to isotropic. unlike other liquid crystals, cholesteryl ester can exist as crystalline solid at room temperature. this will help in the investigation of crystalline to mesophase transitions, which is hard to achieve from most liquid crystals. the most common approach used to describe the overall isothermal crystallization kinetics is the avrami equation (spruiell & supaphol, 1999): (1) where x t is the relative degree of crystallinity, t is the duration of the crystallization process, n is the avrami exponent, and k is the crystallization rate constant. the avrami parameters n and k are constants typical of a given crystalline morphology and type of nucleation. the constant k in eq. (1) is given by: (2) where u is the rate of lateral growth, i is the rate of nucleation, and ξ is the average vertical extension of the crystals (van antwerpen, 1971). alternatively, eq. (1) can be written in its natural logarithmic form (3) using eq. (3), parameters k and n can be calculated from the least-square line fit of the double logarithmic plot of ln[-ln(1-x t )] against ln t for each heating or 2 3 k u i π ξ⎛ ⎞= ⎜ ⎟ ⎝ ⎠ * corresponding author 1 exp( )ntx kt− = − ( )ln ln 1 ln lntx k n t− − = +⎡ ⎤⎣ ⎦ 52 joson, davila, and domingo cooling rate, where k is the anti-logarithmic value of the y-intercept and n is the slope (ziru he et al., 1997). in the study of non-isothermal crystallization using differential scanning calorimetry (dsc), the energy released during the crystallization process is a function of temperature rather than time as in the case of isothermal crystallization (ziru he et al., 1997). therefore, the relative crystallinity, x t is given by: (4) where ∆h t is the enthalpy of crystallization released during a temperature change and, ∆h c is the overall enthalpy of crystallization, which is equal to the area enclosed by the crystallization peak in the dsc plot. in order to use eqs. (1) & (3) in non-isothermal conditions, the thermal lag of the sample and the dsc furnace is assumed to be minimal, such that (5) where t o and t are the onset and the arbitrary temperature, respectively; t is the crystallization time; and φ is the heating or cooling rate (ziru he et al., 1997). ozawa proposes another kinetic expression as an extension to avrami’s equation. assuming that the nonisothermal process may be composed of infinitesimally small isothermal crystallization steps, the following equation has been derived: (6) where k(t) (in °c/min) is the cooling/heating function, φ is the heating and cooling rate, and m is the ozawa exponent, which depends on the dimension of the crystal growth (ziru he et al., 1997). to analyze the non-isothermal crystallization data, the natural logarithmic form of eq. (6) is derived: (7) with different heating/cooling rates and plotting ln[-ln(1-x t )] against ln φ at a given temperature, a straight line should be obtained. k(t) and m are determined from the y-intercept and slope, respectively (yuxian an et al., 1998). understanding the kinetics of crystallization, or any phase transition, gives further insight on what appropriate conditions a phase transition would occur. this would include the temperature at which transition occurs, heating or cooling rates, the amount of energy needed for a transition to happen, the ordering of molecules, and other parameters. this research work aims to investigate the kinetics of the crystal to smectic phase transition of cholesteryl ester under non-isothermal conditions because it is usually under this condition that liquid crystals are used. differential scanning calorimetry will be used with various heating rates, supplemented by microscope observations. methodology the coconut-based cholesteryl ester used in this study was taken from the liquid crystals laboratory of the up diliman institute of chemistry. for calorimetric measurements, cell samples were prepared using solid crimp cells. cholesteryl ester, in its solid form, was weighed in the sample cells with a net weight of 2.40 ± 0.01 mg. this sample weight is enough for the sensitivity of the shimadzu dsc-50 equipment for thermal analysis. the crimped samples were heated using the dsc from 25°c to 100°c with various heating rates (1, 2, 3, 4, and 5°c/min). analysis was done using the thermal analysis program of dsc. for microscopic investigations, a liquid crystal cell was prepared using two glass plates. the sample was then introduced through a gap by capillary method. the textures of cholesteryl ester at different temperatures t t c h x h ∆ = ∆ ot tt φ − = ( ) 1 expt m k t x φ −⎡ ⎤ − = ⎢ ⎥ ⎣ ⎦ ( ) ( )ln ln 1 ln lntx k t m φ− − = −⎡ ⎤⎣ ⎦ 53 kinetics of non-isothermal crystallization were observed and photographed using the bh-2 olympus polarizing microscope equipped with crossed polarizers and camera attachment. control of temperature was made using the mettler toledo fp90 central processor connected to a hot stage that can be mounted on the microscope stage. results and discussion non-isothermal crystallization kinetics based on avrami equation the dsc thermograms of cholesteryl ester at various heating rates are presented in fig. 1. table 1 shows a summary of the non-isothermal data gathered from the dsc. the data presented in table 1 reveal that the exothermic peak temperature t p , which corresponds to the crystal to smectic phase transition, shifts to a higher temperature as the heating rate increases. moreover, the transition is faster at higher heating rates (table 1) as shown by the peak crystallization time t max , which is the amount of time elapsed from the onset of transition up to the peak temperature. this was taken using dsc ta analysis. the relative crystallinity at the peak temperature, x tp ranges from 52.25% to 60.06%. the relative crystallinity is determined by dividing a crystallization peak into small strips. each small strip corresponds to the amount of heat involved in the crystallization process from the onset of crystallization up to a certain temperature, thus obtaining the relative crystallinity as a function of temperature. the results are shown in fig. 2. all the plots of relative degree of crystallinity versus temperature have essentially the same pattern. at approximately 52.55% to 60.06% x t , the shift to higher temperature of the transition peak as the rate increases is evident. in order for these data to be analyzed using the avrami equation, the horizontal temperature scale must be transformed into the time domain using eq. (5). fig. 3 shows the relative crystallinity as a function of time. the plots indicate that the faster the heating rate, the shorter the time needed for the completion of the transition. the sigmoidal shape of the curves in fig. 3 suggests that the avrami analysis might be applicable to the non-isothermal data from the dsc. non-isothermal dsc scans of cholesteryl ester fig. 1. dsc thermograms of cholesteryl ester at various heating rates. 100 90 80 70 60 50 40 20 30 10 40 50 60 70 0 x tp temperature (oc) 1 c/min 2 c/min 3 c/min 4 c/min 5 c/min fig. 2. relative crystallinity as a function of temperature. table 1. parameters of cholesteryl ester during nonisothermal crystallization process. φφφφφ (oc/min) t p (k) t max (min) x tp (%) ∆∆∆∆∆h c (j/g) 1 2 3 4 5 327.94 328.09 328.46 329.49 329.63 13.15 8.55 5.83 4.54 3.86 59.94 54.19 55.27 52.55 60.06 -50.81 -49.82 -59.38 -64.08 -57.64 54 joson, davila, and domingo fig. 4 presents the avrami analysis of the data shown in fig. 3. it shows the plots of ln[-ln(1-x t )] versus ln t at various heating rates. the linearity maintained from the initial stages of transition until very high degree of conversion indicates that the avrami equation correctly describes the non-isothermal crystallization process of cholesteryl ester. from the slopes and intercepts of the lines in fig. 4, the avrami exponent n and rate constant k can be determined. moreover, the halftime for crystallization t 1/2 , which is analogous to t max has been taken directly from fig. 3. this is calculated as a function of parameters k and n. all the data are listed in table 2. values of rate parameters show that the crystallization rate k increases with the increase of heating rate. this might have been caused by the effect of superheating as heating rate is increased (yuxian an et al., 1998). these values of k, together with the values of t 1/2 -1, show that the rate of non-isothermal crystallization is proportional to the heating rate. the avrami exponent is found to be ~3.0, which suggests either a three-dimensional sporadic nucleation or a two-dimensional spontaneous nucleation. however, according to price and wendorff, the crystallization from a smectic mesophase is three-dimensional sporadic nucleation (ziru he et al., 1997). therefore, the first case is more probable. non-isothermal crystallization kinetics based on ozawa equation for comparison, ozawa’s model is used to deal with the data of non-isothermal crystallization. the plots at temperatures from 316 k to 336 k are shown in fig. 5. table 3 gives a summary of the slope and yintercept values of the plots. the changing slopes indicate that m is not constant with temperature. one probable reason is the assumption of the ozawa model. it must be noted that the x t values chosen at a given temperature include the values selected from the earliest stage of the transition at high heating rate and the values from the end state at lower rate (http:/ / w w w. s h o d o r. o r g / u n c h e m / a d v a n c e d / k i n / arrhenius.html). the kinetics of transition should be different at lowand high-relative crystallization. also, shoulder transitions occur near the onset of the heating curves for rates 1, 2, 3, and 4°c/min. these shoulder transitions may be due to difference in the molecular dynamics of the sample when undergoing phase transitions. thus, ozawa analysis cannot adequately describe the non-isothermal crystallization of cholesteryl ester. fig. 3. relative crystallinity as a function of time. time (min) 100 90 80 70 60 50 40 20 30 10 0 x tp 5 10 15 200 25 1 c/min 2 c/min 3 c/min 4 c/min 5 c/min fig. 4. avrami analysis of the data shown in fig. 3. -1.5 -0.5 0.5 1.5 2.5 3.5 3 1 -1 -3 -5 -7 -9 ln (time) ln [ln (1 -x t) ] 1 c/min 2 c/min 3 c/min 4 c/min 5 c/min table 2. parameters k and n for non-isothermal crystallization of cholesteryl ester. φφφφφ (oc/min) k n t1/2(min) = (ln2/k)1/n t 1/2 -1 (min)-1 1 2 3 4 5 5.67 x 10-4 2.27 x 10-3 1.69 x 10-2 2.21 x 10-2 5.12 x 10-2 2.99 3.09 2.95 3.05 2.97 10.76 6.33 3.52 3.08 2.40 0.0929 0.1579 0.2839 0.3247 0.4167 55 kinetics of non-isothermal crystallization fig. 6. cholesteryl ester in its (a) crystalline state, (b) smectic phase, (c) cholesteric phase, and (d) isotropic phase. increasing temperature (a) (b) (c) (d) 5.61 5.615 5.62 5.625 5.63 ln (rate) 3 2 1 0 -1 -2 -3ln [ln (1 -x t) ] -4 -5 -6 316 k 318 k 320 k 322 k 324 k 326 k 328 k 330 k 332 k 334 k 336 k fig. 5. ozawa analysis of the data shown in fig.3 at different temperatures. optical microscopy at room temperature, the cholesteryl ester is observed in its crystalline state (fig. 6a). upon heating, focal conic texture (fig. 6b) is observed before changing to the grandjean planar texture (fig. 6c). this change in texture corresponds to the smectic phase and cholesteric phase, respectively. further heating results in an isotropic phase (fig. 6d). conclusion non-isothermal crystallization of coconut-based cholesteryl ester shows rate-dependent characteristics. the ozawa analysis, when applied to this system, failed to provide a description of the non-isothermal crystallization. this failure is due to the assumption of this treatment. an alternative method based on the avrami analysis was used. the non-isothermal crystallization data could be suitable over a wide range of relative crystallinities by using only two adjustable parameters. this method enables the calculations of crystallization rate parameters, which increase with increased heating rates, and half-time of crystallization, which decreases with heating rate. observations using a polarizing microscope confirmed the crystal to smectic phase transition. the smectic phase was exhibited by the focal conic texture. acknowledgments the samples given by the liquid crystals laboratory up institute of chemistry and the facilities of the liquid crystals laboratory of up national institute of physics are deeply acknowledged. table 3. slopes and intercepts for the ozawa analysis. temperature (k) slope intercept 316 318 320 322 324 326 328 330 332 334 336 7.4012 0.4740 6.0744 6.1609 4.4005 8.8955 21.1370 39.2660 26.7010 36.3120 99.0850 -40.0370 -1.2953 -33.0340 -33.7850 -24.1760 -49.8170 -119.1500 -221.6700 -151.9200 -206.9200 -561.3500 references cureg, g., 2000. cholesteric liquid crystals formulations: coco-based cholesteryl ester and tm74a. university of the philippines diliman, quezon city. spruiell, j. e. & p. supaphol, 1999. kinetics of nonisothermal crystallization of syndiotactic polypropylene: avrami, ozawa, and kissinger approaches. in benedikt, g.m. & b.l. goodall (eds.) metallocene technology and modern catalytic 56 joson, davila, and domingo methods in commercial applications. knoxville, university of tennessee. 263-273. van antwerpen, f., 1971. kinetics of crystallization phenomenon of spherulites in poly(ethylene terephthalate). yuxian an et al., 1998. non-isothermal crystallization kinetics of poly(β-hydroxybutyrate). journal of polymer science: polymer physics. 36(b): 1305-1312. ziru he et al., 1997. a kinetics study of crystallization from discotic mesophases. liquid crystals. 23(3): 317-325. h t t p : / / w w w. s h o d o r. o r g / u n c h e m / a d v a n c e d / k i n / arrhenius.html inside front cover-19-2 editorial board editor-in-chief maricor n. soriano, phd. associate editors mario a. aurelio, phd. earth sciences jose maria p. balmaceda, phd. mathematics zubaida u. basiao, phd. biology joel joseph s. marciano, jr., phd. computer science, engineering marco nemesio e. montaño, phd. biochemistry irene m. villaseñor, phd. chemistry corazon d. villareal, phd. managing editor dercylis g. mararac editorial assistant editorial advisors rigoberto c. advincula, phd dept. of chemistry university of houston radvincula@uh.edu alfonso m. albano, phd. dept. of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu jose b. cruz, phd. dept. of electrical and computer engineering ohio state university cruz@ece.osu.edu. victor c. gavino, phd. dept. of nutrition university of montreal, canada victor.gavino@umontreal.ca science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p300.00/year (two issues), exclusive of postage us$ 25.00/year (two issues), exclusive of postage tel. no: (632) 981-85-00 loc. 4047 (632) 927-2567; 927-2309; 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences cover photo nylon shells, paphia undulata, collected by diving along the coast hinigaran, negros occidental (october 2007). photo courtesy of fenelyn m. nabuab from up visayas. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. kelvin s. rodolfo, phd. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu rudolf a. roemer, phd. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk luis g. sison, phd. electrical and electronics engineering institute university of the philippines, diliman luis.sison@up.edu.ph raul k. suarez, phd. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu 03_dipolar tongco and villagonzalo 8 dipolar interaction in a one-dimensional ising ring gina rose tongco* and cristine villagonzalo structure and dynamics group, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: gtongco@nip.upd.edu.ph abstract science diliman (july-december 2004) 16:2, 8–9 as synthesis of low-dimensional magnetic systems become possible, the need for theoretical understanding of their behavior arises. in this work, the authors explore a one-dimensional magnetic structure with the spins having dipolar interaction. the effects of a long-range interaction in a onedimensional (1d) magnetic ring is studied. this ring consists of n equally spaced spins i. the spin interaction is described by a modified ising model and the system’s total energy e is given by (1) where i = ±1 and r ij is the distance between spins i and j. the first sum is the exchange interaction restricted to nearest-neighbor pairs in the original ising model (yeomans, 1997). the second sum represents the dipolar interaction over all pairs of moments within the ring. the energy is measured in units of the exchange constant j. in this study we set j = 1.0. here g is the strength of dipolar interaction in units of j. we investigate the behavior of the ring as the dipolar strength g is varied for n = 10 spins. we employ the metropolis algorithm (gould & tobochnik, 1996) to obtain the equilibrium state of the system of spins. this procedure realizes the importance sampling which restricts the sampling space only to statistically feasible configurations. figure 1 shows the plot of the average magnetization (〈m〉) as a function of temperature (t). 〈m〉 is obtained by summing all the spins and dividing the result by the total number of spins. at low temperatures, the behavior of the average magnetization as a function of t is shown to depend on the strength of the dipolar interaction (g). ferromagnetic states occur in the ground state (t = 0) for a small value of g. on the other hand, *corresponding author 3 i j i j i j i j ij s s e j s s g r≠ ≠ = − +∑ ∑ fig. 1. behavior of the magnetization with temperature for different values of g. the temperature is given in energy units (k b t), where k b is the boltzmann constant. 0 10 20 30 40 0 0.2 0.4 0.6 0.8 1 50 〈〈〈〈 〈m 〉〉〉〉 〉 k b t dipolar interaction 9 antiferromagnetic states are favored when g is large even at a finite temperature. the magnetization saturates at high t. also, an effective lowering of the total energy is achieved when smaller values of g approach zero and when large values of g are increased further. similar effects are observed for rings having a larger number of spins. increasing the number of spins in a ring decreases the value of the transition point in g. our findings, which show the dependence of the ground state with the dipolar strength, are in contrast to results in higher dimensions, where dipolar interactions favor ferromagnetic alignment of spins (tang & sun, 2002). indeed, the state of a magnetic system is dictated by its dimension and spin interaction. acknowledgment ms. tongco would like to acknowledge the dost for the scholarship (2000–2005) support under ra 7687. references gould, h. & j. tobochnik, 1996. introduction to computer simulation methods: applications to physical systems. addison-wesley, new york. tang, b. & g. sun, 2002. striped and rectangular phases in multilayered ising systems with dipolar interaction. phys. rev. b. 65: 132418. yeomans, j.m., 1997. statistical mechanics of phase transitions. oxford university press, new york. 24_near near-ir spectral imaging 101 near-ir spectral imaging of semiconductor absorption sites in integrated circuits e. c. samson*, c. m. blanca, and c. saloma national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: esamson@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 101-105 *corresponding author we derive spectral maps of absorption sites in integrated circuits (ics) by varying the wavelength of the optical probe within the near-ir range. this method has allowed us to improve the contrast of the acquired images by revealing structures that have a different optical absorption from neighboring sites. a false color composite image from those acquired at different wavelengths is generated from which the response of each semiconductor structure can be deduced. with the aid of the spectral maps, nonuniform absorption was also observed in a semiconductor structure located near an electrical overstress defect. this method may prove important in failure analysis of ics by uncovering areas exhibiting anomalous absorption, which could improve localization of defective edifices in the semiconductor parts of the microchip. introduction optical beam-induced current (obic) imaging by laser scanning microscopy is becoming an important technique in analyzing defects and failures in integrated circuits (ics). this method was further enhanced when coupled with traditional confocal reflectance microscopy (takasu, 2001; daria et al., 2002; miranda & saloma, 2003; cemine et al., 2004). an obic image is a map generated from the current magnitudes that are induced in an ic when an optical probe is scanned across it. this current arises from an illuminated semiconductor material due to carriers that have absorbed photon energy from the probe. this occurs if the photon energy exceeds the band-gap energy of the semiconductor (takasu, 2001; beauchêne et al., 2003). defects in integrated circuits are identified in obic images as sites having unusually high or low intensity values (takasu, 2001). sites exhibiting high obic magnitudes can be due to impurities in the semiconductors. impurities have been shown to increase the optical absorption coefficient of silicon, the customary semiconductor used in ics (falk, 2000), making them potential sites of anomalous optical absorption; thus the unusual magnitudes of the induced current. analysis of these anomalous absorption sites may become problematic due to the poor contrast and lack of axial resolution of obic images. in order to attain a high-quality image, high intensities of light must be utilized as a probe. this presents yet another problem since imaging will be confined to sites near the surface of the ic due to this high-intensity requirement. thus, it is essential to develop a method that gives highcontrast obic images and, at the same time, capable of imaging deep inside an ic. it has been reported that the absorption coefficient of silicon increases exponentially with wavelength in the near-infrared region (jellison & modine, 1982). the local change in absorption behavior can be exploited to samson, blanca, and saloma 102 provide contrast to reveal differences in absorption among semiconductor sites and provide a more thorough analysis of obic images. probing deep inside the ic can also be performed since long wavelengths will be utilized. in this work, we demonstrate the possibility of generating spectral maps of different absorption sites in ics by utilizing several wavelengths in the nearinfrared region as the illuminant probe. from these spectral maps, the optical response and architecture of the ic will be determined. improvement in contrast in obic images is also shown. materials and methods the imaging method in this study utilizes a confocal microscope (fig. 1) that consists of a tunable continuous wave ti:sapphire laser (3900s, spectraphysics, mountain view, ca) pumped by a 5 w solidstate laser (millenia pro, spectra-physics). the beam exiting the laser is focused by a doublet lens l1 (f = 100 mm) onto a 20 mm pinhole p in order to ensure a diffraction-limited illumination. the collimator l2 (f = 150 mm) expands the beam, which is redirected by a beamsplitter bs to overfill the back aperture of l3. the objective lens l3 (na = 0.50, olympus, japan) focuses the beam on the integrated circuit (ic) sample mounted on a scanning stage. the reflected light is collected by a tube lens l4 (f = 80 mm), focused onto a 52 mm multimode optical fiber, and then directed to a photomultiplier tube (h5784, hamamatsu, japan). the average power entering the microscope is approximately 40 mw. data acquisition and control are managed by a computer-mounted digital-to-analog converter card (national instruments). this enables automatic control of the scanning stage (pt3-z6, thorlabs, new jersey) down to a 50 nm step size in the x, y, and z directions using a custom-coded software (labview 6i). acquisition of detector data and obic measurements from the sample are controlled by the same software. utilizing an illumination wavelength of 750 nm, twodimensional (x-y plane) confocal reflectance images (100 × 100 pixels) are acquired from the ic sample, covering an area of 200 × 200 mm2. obic images (100 × 100 pixels) of the sample are acquired at the axial position giving the highest reflectance signal (z = 0). to observe the spectral response of the current generated by the sample, the illumination wavelength is varied (from 720 to 850 nm) within the tunable range of the laser. results and discussion obic images were first obtained from a photodiode array sample. a confocal reflectance image of the specific site in the array is shown in fig. 2(a). the obic images acquired at different illumination wavelengths from the system are shown in fig. 2(b). as can be noted from the images, certain structures in the sample do not generate any induced current when longer wavelengths (e.g., 825 nm) are used, despite these wavelengths being above the band gap of the semiconductor present in the sample [gallium arsenide (gaas), 1240 nm] (kittel, 1996). when shorter wavelengths illuminate the sample (e.g., 750 nm), a greater number of semiconductor structures become apparent in the obic images generated by the optical system. this is more evident if a line scan is taken along the 750 nm image and compared with that of the image obtained using 825 nm [refer to fig. 2(c)]. we can fig. 1. the optical setup. the tunable ti:sapphire laser is pumped by a solid-state laser. the laser output is spatially filtered, collimated, and focused on the sample by an objective lens. the reflected signal is detected by a pmt. obic measurements are directly obtained by the data-acquisition card. laser pump desktop computer servo motor controller tunable cw ti:sapphire laser l1 p l2 d l4 bs l3 sample x y z i daq card near-ir spectral imaging 103 observe high obic measurements in the 750 nm image, while the longer wavelength scan contained only background signal. clearly, higher contrast can be obtained when the illumination wavelength used to probe the ic sample is varied. to visualize further the appearance of the ic structures for each change of wavelength, three images corresponding to different illumination wavelengths (750, 800, and 825 nm) were rendered in false color. the image taken at 750 nm was done in red, the 800 nm in green, and the 825 nm image was done in blue. the three images were superimposed and the resulting composite obic image is shown in fig. 3(a). this image reveals the spatially resolved spectral absorption of the various semiconductor components. it should be noted that the structures appearing in white in this composite image generate obic in all three wavelengths because all three color components are present. if structures that are more of a shade of a particular color are seen, it can be inferred that these parts appear only when the corresponding wavelength is used as illuminant. for example, the red structures would tend to absorb more at 750 nm than longer wavelengths. a histogram of the color components of a specific structure in the composite image can be generated [fig. 3(b)] to analyze easily on which wavelength a structure appears. since all three color components are fig. 2. (a) confocal and (b) obic images of a photodiode array. the confocal image was acquired at the focus of the system using 750 nm. the obic images shown are taken using different illumination wavelengths (720, 750, 775, 800, 825, 850 nm). (c) line scans of the images reveal structures that appear on certain wavelengths (750 nm) which are previously absent on others (825 nm). (a) confocal (b) obic x position p ix el in te ns it y 0 20 40 60 80 100 20 40 60 80 100 120 750 nm 820 nm (c) fig. 3. (a) composite image (area of 200x200 mm2) of the photodiode array generated from the obic images taken at 750, 800, and 825 nm. the obic images are masked each in red (750 nm), green (800 nm), and blue (825 nm). the composite image reveals the optical response of the semiconductor with varying wavelengths as shown when (b) a histogram of a particular region [bounded area in (a)] is generated. (b) pixel intensity n o. o f p ix el s 0 20 40 60 80 100 120 140 0 2 4 6 750 nm 800 nm 825 nm samson, blanca, and saloma 104 present in the structure, it suggests that this particular structure generates obic in all three wavelengths. but as indicated by its high intensity from the histogram, a greater amount of obic is generated when 750 nm is used as the optical probe. the semiconductor components of the photodiode were fabricated using gaas that has a band gap of 1240 nm, which could easily be bridged by the longest wavelength we have used (850 nm) and obic should have already occurred. the absorption contrast of the image could have been provided by the varying thicknesses of the semiconductor architecture. this would still have to be verified using scanning electron microscopy. finally, in order to observe how optical absorption is affected by the presence of defects, an ic sample having a site with electrical overstress (eos) was placed in the system. a confocal image of the defect (encircled region) and its surrounding structures is seen in fig. 4(a), and the obic images acquired using 750 and 800 nm as illuminants are shown in figs. 4(b) and 4(c), respectively. to perceive the difference in the generation of obic in the sample, the two images were rendered in false color [figs. 4(d) and 4(e)]. it can be noticed in the 750 nm image that there is a slight disruption in obic generation in the area next to the defect (indicated by arrow), though it is expected that obic would occur continuously in this area since it is not damaged physically by eos, as shown in the confocal image. this is also true for the image taken under 800 nm, though the disruption is to a lesser extent. hence, the proper choice of illumination wavelength can facilitate easier localization and identification of defective sites. another observation from the images is the nonuniform distribution of obic even for a single structure. a greater concentration of obic is located at the upper right corner of the image as compared with the region below it even though they are of the same material and structure. a possible explanation is that the optical absorption of the material is not uniform throughout the structure, having a deviation of 33% of the maximum obic signal. another thing that can be noted is when 800 nm is used, there is an increase in obic at fig. 4. (a) confocal and (b) obic images of an integrated circuit with an electrical overstress defect (encircled region). the obic images are taken using 750 and 800 nm. (c) false color images of the obic reveal nonuniform generation of obic near the defect area. arrow indicates a disruption of obic that could be due to the defect. confocal 750 µm 800 µm 255 0 50 µm obic the upper and center right portions of the image albeit the intensity remains the same at the lower regions. this may again be attributed to nonuniform absorption throughout the semiconductor. though it is still needed to be compared with a sample having the same structure without a defect, the above observations are enough to suggest that the architecture of the imaged semiconductor is not homogeneous. this inhomogeneity is peculiar since the semiconductor is of a single type and belongs to a single structure in the ic. whether this is brought about by the defect will be determined when the comparison to a nondefective sample is performed. near-ir spectral imaging 105 conclusion we have derived composite spectral absorption maps of semiconductor sites on integrated circuits. various components respond differently to different illumination wavelengths, enabling us to map out the topography of the absorption characteristics of the ic. it is possible to improve the analysis of semiconductor structures in an integrated circuit when the wavelength of the optical probe in obic imaging is made to vary. this method allows us to image structures that are not evident with longer wavelength illumination; thus, enhancing the contrast of the images obtained. furthermore, the method revealed how electrical overstress changes the absorption properties of neighboring semiconductor regions disrupting the absorption homogeneity of the semiconductor. this disturbance can only be detected at specific wavelengths and cannot be seen if multiwavelength spectral imaging is not employed. acknowledgments the authors acknowledge the funding provided by the office of the vice chancellor for research and development of the university of the philippines, diliman, and bernardino buenaobra for the acquisition software he developed. references beauchêne, t., d. lewis, f. beaudoin, v. pouget, p. perdu, p. fouillat, & y. danto, 2003. a physical approach on scobic investigation in vlsi. microelectron. reliab. 43: 173–177. cemine, v.j., b. buenaobra, c.m. blanca, & c. saloma, 2004. high-contrast microscopy of semiconductor and metal sites in integrated circuits via optical feedback detection. opt. lett. 29: 2479-2481. daria, v.r., j.j. miranda, & c. saloma, 2002. high-contrast images of semiconductor sites via one-photon optical beaminduced current imaging and confocal reflectance microscopy. appl. opt. 41: 4157–4161. falk, r.a., 2000. near ir absorption in heavily-doped silicon: an empirical approach. international symposium for testing and failure analysis. jellison jr., g.e. & f.a. modine, 1982. optical absorption of silicon between 1.6 and 4.7 ev at elevated temperatures. appl. phys. lett. 41: 180–182. kittel, c., 1996. introduction to solid state physics, 7th ed. wiley, new york: chap. 8. miranda, j.j. & c. saloma, 2003. four-dimensional microscopy of defects in integrated circuits. appl. opt. 42: 6520–6524. takasu, s., 2001. application of obic/obirch/obhic: semiconductor failure analysis. jeol news. 36e: 60–63. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 03_otadoy 11 influence of the transverse width science diliman (january-june 2003) 15:1, 11-16 the influence of the transverse width on the andreev bound states and self-consistent gap function in clean sns system r. e. s. otadoy1 and a. lodder2* 1university of san carlos, nasipit, talamban 6000 cebu city, philippines 2natuurkunde en sterrenkunde, vrije universiteit de boelelaan amsterdam, the netherlands email: alod@nat.vu.nl introduction in clean layered structure of normal metal and superconductor, andreev-bound states are formed in the normal metallic part through multiple andreev reflections (andreev, 1964 & 1967) of the electron and hole waves. in the andreev approximation (aa) (andreev, 1964 & 1967), the incident electron towards a normal metal-superconductor (ns) interface will be reflected as a hole. exact analysis, however, shows a small amplitude of a normally reflected electron (sipr & gyorffy, 1996). in most studies (larkin & yu, 1975), andreev-bound states are described using the quasiclassical description, which can be shown to be equivalent to aa (ashida et al., 1982). interestingly, andreev approximation works remarkably well (blaauboer et al., 1996). in this paper, we want to investigate the reliability of aa by varying the transverse dimensions (dimensions perpendicular to the flow of current) of a mesoscopic superconductor-normal metalsuperconductor (sns) sample (bagwell, 1999). in most systems considered so far (tanaka & tsukada, 1991), the transverse dimensions which the breakdown of the andreev approximation can hardly show up are considered infinite. theory throughout the paper, rydberg atomic units are used– the energy is in rydberg, the distance is in bohr (1 bohr ~ 0.5å), = 1, and the electronic mass is 1/2. the green’s function formalism is outlined extensively by koperdraad et al. (1995). it is an extension of the microscopic theory used by tanaka & tsukada (1991), in that the electron-hole scattering properties are treated exactly. the matrix green’s function satisfies the equation (1) in which the differential operator is closely related to the operator in the bogoliubov-de gennes (bdg) equations (2) apart from the replacement of e by iω n . the quantity ω n , which is equal to ω n = πnk b t, is called the matsubara frequency. for a system of fermions, n is an odd integer. possible inhomogeneities of the system are fully represented by the r dependence of the gap function. the spinor wave function ψ(r) describes quasiparticle excitations, and the energy e is measured with respect to the fermi energy µ. eq. (1) is derived using the finite h * corresponding author ( ) ( ) ( ) ( ) 2 2 , ', = ' 1 n n n i r g r r i r i r r ω µ ω ω µ δ ∗ ⎛ ⎞+ ∇ + −∆ ⎜ ⎟⎜ ⎟−∆ − ∇ −⎝ ⎠ − ( ) ( ) ( ) ( ) ( ) ( ) 2 2 r r e r r u r e v r µ µ∗ ⎛ ⎞−∇ − ∆ ψ = ψ⎜ ⎟⎜ ⎟∆ ∇ +⎝ ⎠ ⎛ ⎞ ≡ ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ 12 otadoy and lodder temperature green’s function formalism (abrikosov et al., 1963 & 1965) by manipulating the equations of motion instead of the often used diagrammatic analysis. the sns system we consider is shown in fig. 1. the gap function ∆ has a constant complex value in the superconducting part and zero in the normal part. this model of the gap ignores the proximity effect. as far as the transverse directions are concerned, the general solution of eq. (1) can be expressed as a fourier series expansion in sin y k y and sin z k z in which and . the functions sin yk y and sin zk z are in fact the transverse solutions of the bdg equations with the boundary conditions ψ(x,0,0) = ψ(x, l y ,l z ) = 0. thus, we have (3) in eq. (3), the summation extends over all allowed values of . the fourier coefficient becomes the green’s function of the quasiparticle motion along the x-direction, which can be seen by substituting eq. (1) with eq. (3): (4) where . eq. (4) demonstrates that is diagonal to and . in calculating the local density of states and the selfconsistent gap function, we will need the green’s function for diagonal spatial coordinates. as long as we keep x x'≠ , we can already take y = y’ and z = z’ in eq. (3). since we are only interested in the variations over the longitudinal direction x, we can average over the transverse dimensions. by that, eq. (3) simplifies to the series: (5) the solution of eq. (4) for a superconducting bar, that is when the system shown in fig. 1 is composed mainly of a superconducting material without the normal metal, is (6) where (7) (8) (9) (10) (11) (12) s s s s n n 0 x lz ly ( )x∆ fig. 1. the geometry of the sns system considered. y y y n k l π = z z z n k l π = ( ) ( )4, ', , ', , ' , , ' , sin sin ' 'sin sin ' ' n y y z z n y z y y z z g r r i g x x k k k k i l l k y k y k z k z ω ω= × ∑ , ' , , 'y y z zk k k k ( ), ', , ' , , ' ,y y z z ng x x k k k k iω 2 2 2 2 * 2 2 x x n f n f d i k dx d i k dx ω ω ⎛ ⎞ + + −∆⎜ ⎟ ⎜ ⎟ ⎜ ⎟ −∆ − −⎜ ⎟ ⎝ ⎠ , ' , ' ( , ', , ' , , ' , ) ( ') y y z zy y z z n k k k kg x x k k k k i x xω δ δ δ× = − 2 2 2 xf y z k k kµ= − − ( , ', , ' , , ' , )y y z z ng x x k k k k iω yk zk ( ) , 1 , ', ( , ', , , ) y z n y z n k ky z g x x i g x x k k i l l ω ω= ∑ 0 ( , ', , , ) ( ) ( ) ( ) ( ) s y z n s s s s s s g x x k k i d x x d x x σ σ σ σ σ σ σ σ ω ψ ψ ψ ψ − + < > + − > < = = ∑ ∑ % % / 2 / 2 s i s i k x s i s u e e u e σ σ φ σνσν σ φ ψ − − ⎛ ⎞ = ⎜ ⎟⎜ ⎟ ⎝ ⎠ ( )/ 2 / 2( ) si k xi is s sx u e u e e σσνσν σ φ σ φψ − −=% 1 4 s s s d k σ σ= − ω 22( )s ni iωω = − ∆ 22 su e e σ σ= + − ∆ 22 2 xs f k k eσ σ= + − ∆ 13 influence of the transverse width (13) φ is the phase of the complex constant ∆. the index σ refers to the type of particle (electronlike for σ = +1 and holelike for σ = -1), and the index n indicates the direction of propagation (ν = +1 to the right and ν = -1 to the left). the solution of eq. (4) for an ns system (single interface) is, (14) where (15) with µ = sgn(x–x’). for multiple interfaces, that is, for arbitrary number of layers (16) the quantities and are obtained by imposing the continuity of the green’s function and its derivative at the interfaces. the bound-state energy is determined by looking through the local density of states (ldos) using the formula (17) in which g 11 is the upper left matrix element of the multiple scattering green’s function (eq. (16)). at the bound-state energy, the ldos has infinite peak. to avoid this singularity, the parameter δ is introduced to broaden the peak so that it acquires a finite height. the peaks in the plot of the ldos against the quasiparticle energy must correspond to the andreev-bound state energies. in this formalism, the andreev approximation can easily be implemented. this approximation amounts to the replacement (18) if kσ vj occurs in the exponential and to if occurs as a factor. it is valid when e, |d|<< . in the present paper, we investigate its limitation by looking at configurations in which e, . the gap function can be determined self-consistently, using the formula (19) where ( , ', , , )y z nf x x k k iω is the upper right element of the matrix green’s function ' ' ( , ', , , )j j y z ng x x k k iν ν ω and v is the pairing interaction amplitude. in carrying out the calculation, we first substitute in ( , ', , , )y z nf x x k k iω the step-like gap profile shown in fig. 1. we can determine a new value of the gap by using eq. (19). this new value is again substituted in ( , ', , , )y z nf x x k k iω , and another new value is again obtained using eq. (19). the iteration is continued until the difference in the gap values between two successive iterations is negligibly small. results local density of states to investigate the reliability of the andreev approximation, we focus on the choice of the transverse dimensions which we choose to be y z tl l l= = . the transverse components of the solutions of the bdg equations are sin( )yk y sin( )zk z in which y y y n k l π = and zz z n k l π = . the different combinations of ( , )y zk k . 0 ' ' ' ' ' ' ' ' ' ' ' ( , ', , , ) ( , ', , , ) ( ) ( ') j j y z n j y z n j j j j j j g x x k k i g x x k k i d d x t x ν ν ν νν σ σ σν σσ νν σ ν ν ν ν ν ν ν σσ ω ω δ ψ ψ = + ∑ % 0 ','( , ', , , ) ( ) ( ')j y z n j j jg x x k k i d x x σ σµ σ µ ν ν ν ν σ ω ψ ψ= ∑ % ' ' 0 ' ' ' ' ' , ' ' ' ' ' ' ' ' ' ' ' ' ' ' ( , ', , , ) ( , ', , , )( ) ( ) ( ') j j y z n j j y z n jj j j j j j j j j g x x k k i g x x k k i d d x t x ν ν ν ν νν νν ν σ σ σµ σσ µµ σ µ ν ν ν ν ν ν σµσ µ ω ω δ δ δ δ ψ ψ − + = + + ∑ % ' ' ' 'j jt σσ νν ν ν ' ' ' 'j jt σσ µµ ν ν 11 0 , 1 ( , ) lim im ( , ', , , ) y z y z y z k k ldos x e l l g x x k k e i δ π δ → = − × +∑ 22 2x x j f f e k k k σ ν σ − ∆ → + xj f k kσν → 2 xf k 2 xf k∆ ≈ , , ( ) ( , ', , , ) n y z y z n k ky z v x f x x k k i l l ω ω β ∆ = − ∑ jk σ ν 2 2 2 xf y z k k kµ= − − 14 otadoy and lodder or ( , )y zn n are called modes whose allowed values are determined by . (20) when the transverse dimension is small, the second term in the right becomes large, and as a result of which, only a few modes will be allowed. if this term exceeds the chemical potential µ , xf k becomes imaginary, the wave-function is damped, and consequently, such mode cannot exist. for larger transverse dimensions, the second term is smaller whereupon more modes are allowed. most of our calculations will be done for small tl so that only few modes will exist. we will tune tl such that 2 xf k is of the same order of magnitude as the gap energy ∆, in which regime the andreev approximation (eq. (18)) is not valid, and call such tl value a critical width. figs. 2 and 3 show the results for a configuration in which ( , ) (2, 2)y zn n = is the highest allowed mode. the chemical potentials in the superconductor and in the normal metal, µ s and µ n , respectively, are assumed equal with magnitude 0.5. the longitudinal dimension l of the normal-metal part is 4,000 bohr and the gap ∆ is treated as real, with magnitude 0.0001 ry. the ldos in the normal-metal part at x = 1,000 bohr is plotted against e/∆. the peaks represent discrete energy states (blaauboer et al., 1996). we make the width curves, determined by the parameter δ in e + iδ , wide enough so that the fundamental features can be seen. the numbers finally, we want to make a comment on our choices of the transverse widths. it will come out in the next section that superconductivity is suppressed for transverse widths in the order of 20 bohr or less. this means that our choices of tl are not at all appropriate. we made those choices to illustrate with clarity the fundamental features of the andreev-bound states. if we choose a larger transverse width in which no suppression of superconductivity occurs, many states will appear and the picture would have been quite crowded. the fundamental features, however, remain unchanged. e/∆∆∆∆∆ l o c a l d e n s it y o f s ta te s 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.0 0.8 0.6 0.4 0.2 0.0 (2,2) (1,1) (2,1) (1,2) ○ ○ aa exact l t = 13 bohr x = 1000 bohr fig. 2. the ldos against e/∆ for an sns system in which l t = 13 bohr. 0.0 0.2 0.4 0.6 0.8 1.0 e/∆∆∆∆∆ 0.3 0.1 0.8 0.6 0.4 0.2 0.0 l o c a l d e n s it y o f s ta te s exact 0.7 0.5 ○ ○ aa (2,2) (1,2) (2,2) (2,2)(2,2) (2,2) (2,2) (2,2) (1,1) (2,1) fig. 3. the ldos against e/∆ for x = 1,000 bohr, l t = 12.5676 bohr, l = 4,000 bohr, and ∆ = 0.0001 ry. in parentheses denote the mode to which the energy belongs. in fig. 3, the transverse width is determined by the condition that 2 xf k = ∆ for the mode (2,2) in which one finds that tl = 12.5676 bohr; and in fig. 2 the transverse width is tl = 13 bohr, which is slightly larger than the critical width, but has the same allowed modes. in fig. 2, the exact results and the aa results coincide and only three states are found, one for each mode. for the critical width shown in fig. 3, the states for the first two modes are almost unchanged, but for the (2,2) mode many states are found. the peaks corresponding to the aa are split in the exact treatment. ( ) 2 2 2 2 0 xf y z t k n n l π µ ⎛ ⎞ = − + >⎜ ⎟ ⎝ ⎠ 15 influence of the transverse width self-consistent gap function we first present the results of the selfconsistent gap calculation for a bar-shaped superconductor. in about 80 iterations, the gap values stabilize. in fig. 4, we show the self-consistent gap values plotted against tl for a bar-shaped superconductor. it can be seen that there are oscillations of the gap whose amplitudes decrease as tl increases. these oscillations can be attributed to the discreteness of the transverse wave vectors. as tl increases, the transverse wave vector approaches the continuous regime, which can be gauged from the gap becoming closer to its bulk value obtained by integrating, instead of summing over, the transverse wave vectors. another interesting thing which can be seen in the figure is the suppression of superconductivity for narrower transverse widths. we notice that as the temperature increases, the onset of the suppression of the superconductivity occurs at higher values of tl . for the sns system, our initial gap profile is the step-like gap shown in fig. 1. by following the algorithm outlined in the theory, we obtain the results shown in fig. 5. in fig. 5a, the gap is depressed near the interface. this occurs because of the proximity of the superconductor to the normal metal. this phenomenon is known as the “proximity effect”. in fig. 5b, we show the pair amplitude or density of cooper pairs. it is evident that even in the normal metal, cooper pairs still exist. this is another manifestation of the proximity effect. references abrikosov, a.a., l.p. gorkov, & i.e. dzyaloshinski, 1963. methods of field theory in statistical physics. prentice hall. fig. 4. the self-consistent gap function against the transverse width, l t , for a bar-shaped superconductor at different temperatures. the number of iterations is 100. s e lf -c o n s is te n t g a p , ∆∆∆∆ ∆ ( l t) (r y ) transverse width, l t (bohr) 500 1000 1500 2000 2500 30000 1.4x10-5 1.2x10-5 1.0x10-5 8.0x10-6 6.0x10-6 4.0x10-6 2.0x10-6 0.0 bulk at t = 0.2 k t = 0.2 k t = 0.6 k t = 1.0 k 1.2x10-6 1.0x10-6 8.0x10-7 6.0x10-7 4.0x10-7 2.0x10-7 0.0 -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 s sn distance from the middle of the system, x (bohr) p a ir a m p li tu d e , ∆∆∆∆ ∆ ( x )/ v ( r y ) (b) s sn 1.2x10-5 1.0x10-5 8.0x10-6 6.0x10-6 4.0x10-6 2.0x10-6 0.0 -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 distance from the middle of the system, x (bohr) g a p f u n c ti o n , ∆∆∆∆ ∆ ( x ) (r y ) (a) fig. 5. (a) the gap function and (b) the pair amplitude of an sns system against the distance from the middle of the system chosen at x = 0. the interfaces are located at x = +2000. l t = 1000 bohr l t = 100 bohr l t = 99.8514 bohr l t = 1000 bohr l t = 100 bohr l t = 99.8514 bohr 16 otadoy and lodder abrikosov, a.a., l.p. gorkov, & i.e. dzyaloshinski, 1965. methods of field theory in statistical physics. pergamon press. andreev, a.f., 1964. the thermal conductivity of the intermediate state in superconductors. sov. phys. jetp. 19: 1228. andreev, a.f., 1967. macroscopic theory of spin waves. sov. phys. jetp. 24: 1019. ashida, m., s. aoyama, j. hara, & k. nagai, 1989. green’s function in proximity-contact superconducting-normal double layers. phys. rev. b40: 8673-8686. bagwell, p.f. (ed.), 1999. mesoscopic superconductivity. superlatt. microstr. 25: 5-6. blaauboer, m., r.t.w. koperdraad, a. lodder, & d. lenstra, 1996. size effects in the density of states in normal-metalsuperconductor and superconductor-normal-metalsuperconductor junctions. phys. rev. b. 54: 4283-4288. koperdraad, r.t.w., r.e.s. otadoy, m. blaauboer, & a. lodder, 2001. multiple scattering theory in clean superconducting layered structures. j. phys. condens. matter. 13: 38. koperdraad, r.t.w., 1995. the proximity effect in superconducting metallic multilayers. ph.d. thesis. amsterdam, vrije universiteit. larkin, a.i. & yu.v. ovchinninkov, 1975. nonlinear fluctuation phenomena in the transport properties of superconductors. sov. phys. jetp. 41: 960. sipr, o. & b.l. györffy, 1996. interpretation of bound states in inhomogeneous superconductors: the role of andreev reflection. j. phys. condens. matter. 8(2): 169-191. tanaka, y. & m. tsukada, 1991. phase-dependent energy spectrum of quasiparticles in a superconducting superlattice. phys. rev. b. 44: 7578-7584. inside front cover-23-2.pmd editorial board editor-in-chief marco nemesio e. montaño, ph.d. associate editors ma. patricia v. azanza, ph.d. food science & nutrition jose maria p. balmaceda, ph.d. mathematics zubaida u. basiao, ph.d. biology carlos primo c. david, ph.d. earth sciences joel joseph s. marciano, jr., ph.d. computer science, engineering giovanni a. tapang, ph.d. physics irene m. villaseñor, ph.d. chemistry violeda a. umali, ph.d. director, rduo corazon d. villareal, ph.d. managing editor dercylis g. mararac editorial assistant science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p300.00/year (two issues), exclusive of postage us$ 25.00/year (two issues), exclusive of postage tel. no: (632) 981-85-00 loc. 4048 (632) 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences editorial advisors rigoberto c. advincula, phd. dept. of chemistry university of houston radvincula@uh.edu alfonso m. albano, phd. dept. of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu jose b. cruz, phd. dept. of electrical and computer engineering ohio state university cruz@ece.osu.edu. victor c. gavino, phd. dept. of nutrition university of montreal, canada victor.gavino@umontreal.ca kelvin s. rodolfo, phd. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu rudolf a. roemer, phd. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk luis g. sison, phd. electrical and electronics engineering institute university of the philippines, diliman luis.sison@up.edu.ph raul k. suarez, phd. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu cover photo “samples of each morphotype of p. grandiflora (hook). (a.1.) double orange petal, (b.1.) single pink petal (c.1.), double pink petal (d.1.), double pink-white petal (e.1.), single yellow petal (a.2.), double orange sepal (b.2.), single pink sepal (c.2.), double pink sepal (d.2.), double pink-white sepal (e.2.),single yellow sepal (a.3.), double orange leaf (b.3.), single pink leaf (c.3.), double pink leaf (d.3.), double pink-white leaf (e.3.),single yellow leaf. figure was taken from cao et al. p. 41-53 of this issue.” contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. early development-campos.pmd the early development of the short-necked clam, paphia undulata 13science diliman (july-december 2010) 22:2,13-20 abstract the population of paphia undulata in negros occidental waters continues to decline due to unregulated harvesting because of the increasing demand for this resource. one potential effort to mitigate this problem is reseeding or stock enhancement of the natural population. for these efforts to be effective however, successful laboratory rearing and breeding of this species is a prerequisite. thus, this study was conducted to describe the embryonic and larval development of p. undulata to provide information on laboratory larval rearing. broodstock were collected during the natural spawning peak from hinigaran, negros occidental and were maintained in a static system with a mean salinity and temperature of 36 ppt. and 26.7 oc, respectively and were fed with isochrysis galbana, chaetoceros calcitrans, and tetraselmis tetrahele. fertilized eggs started to divide 30 minutes after fertilization. ciliated swimming blastulas were observed after 1.5 hours with an average shell length (sl) of 40 μm. these further developed into trocophore larvae (average sl 60μm) after 7 hours and into d-veliger (average sl 80 μm) after 12 hours. fully developed d-larvae (average sl 90 μm) were attained after 17 hours. larvae with completely developed umbo were observed on day 9 with an average sl of 140 μm. on day 13, larvae (average sl 220 μm) started to settle and metamorphose. spontaneous spawning was observed during the peak of reproductive activities of p. undulata showing that collection of broodstock must be conducted during the peak of reproductive season to ensure successful spawning and production of viable gametes under laboratory conditions. further experiments should be conducted to determine optimum conditions to improve survival rates of the larvae during settlement. the results corroborate other studies and help explain the derived information on the species’ population and reproductive biology. keywords: paphia undulata, embryonic and larval development, reproductive biology, laboratory breeding, negros occidental the early development of the short-necked clam, paphia undulata (born 1778) (mollusca, pelecypoda: veneridae) in the laboratory annabelle g.c. del norte-campos*, fenelyn m. nabuab, rocille q. palla & michael ray burlas marine biology laboratory, college of arts and sciences university of the philippines visayas, 5023 miag-ao, iloilo, philippines *corresponding author email: upvmarbio@yahoo.com del norte-campos, a.g. et al. 14 science diliman (july-december 2010) 22:2,13-20 introduction the short-necked clam paphia undulata (born, 1778) is one of the most economically important bivalves in the philippines as it is a cheap source of high quality protein. in west central visayas, philippines, it is widely distributed in the coastal muddy bottoms of hinigaran, binalbagan and himamaylan, negros occidental. clams collected by compressor diving in these areas are sold to local markets as well as exported (live or processed) to other countries like taiwan (villarta & del norte-campos, 2010). the population of this commercially important species has declined substantially because of unregulated harvesting caused by increasing demand. agasen et al. (1998) reported that the population in negros occidental was overexploited with an exploitation rate (e) of 0.5. after a period of thirteen years of unregulated fishing, overexploitation has worsened (e = 0.75) due most likely to the continued absence of management policies (del norte-campos & villarta, 2010). this overexploitation with the familiar symptoms of decrease in the sizes of the catch and high proportions of immature in the catch has clearly led to growth overfishing of the natural stock in the area (del nortecampos & villarta, in prep.). one way to mitigate this problem is by reseeding the overfished area or stock enhancement. reseeding/restocking has the potential to increase fishery yields and rebuild overexploited stocks and dampen fluctuations in catch due to variable recruitment (rothlisberg, 1999). on the other hand, stock enhancement is the introduction of artificially produced organisms which are released after rearing to a desired stage of development to support or augment the wild population of the species and to increase the eventual yield, or occasionally to create new fisheries (howarth 1991). knowledge on the larval biology of p. undulata is quite limited with the few existing publications either being sketchy and incomplete (salleh et al. 1987), or published in more obscure literature (pongthana, 1990). this knowledge is however a prerequisite to eventually promote successful reseeding or stock enhancement procedures. knowledge gained from its early growth will also further complement the information on its population biology and help understand the dynamics of the species’ recruitment in its natural habitats. this study was therefore conducted simply to describe the early development of p. undulata reared under laboratory conditions. due to logistical constraints, no replications were made. using the derived information, including the duration of larval stages, inferences on the timing of settlement of the species in its natural habitat were made. materials and methods clam collection and hatchery maintenance broodstock were collected through compressor diving from hinigaran, negros occidental, west central philippines (fig. 1) during the peak spawning season (august-november) (nabuab et al. pams 10) of the species in the area. they were transported without water, chilled (2-4 °c) in an ice chest to the aquaculture facility of the university of the philippines visayas where they were stocked in a tank without substrate with a mean salinity of 36 ppt, mean temperature of 26.7 oc and mean dissolved oxygen of 5.68 mg l-1 for acclimatization prior to spawning. ten liters each of isochrysis galbana, tetraselmis tetrahele and chaetoceros calcitrans were fed to the broodstock daily. microalgal starters were obtained from phycology laboratory of the southeast asian fisheries development center (seafdec/aqd) and were cultured using f-media. feces and algal debris were siphoned daily to ensure good water quality during the acclimatization period. monitoring of embryonic and larval development when natural spawning took place in the broodstock tank, fertilized eggs were collected using a 25 micron sieve and were incubated in plastic bins at a stocking density of 5 eggs ml-1. embryonic development was monitored every three hours until d-larvae were observed. thereafter, larval development monitoring and water change were done every other day. d-larvae were stocked in a flat bottom container with mild aeration at a density of 5 inds.ml-1 and fed with 5 x 103 cells ml-1 of chaetoceros calcitrans. when the dlarvae reached the umbone stage, a mixed diet of chaetoceros calcitrans and isochrysis galbana was given at a cell density of 5 x 104 cells ml-1. settled larvae were transferred in an upwelling system (pongthana 1990) and were fed with c. calcitrans, i. the early development of the short-necked clam, paphia undulata 15science diliman (july-december 2010) 22:2,13-20 galbana, and t. tetrahele at a cell density of 8.3 x 104, 3.3 x 104, and 3 x 103 cells ml-1, respectively (utting and spencer 1991). shell lengths of larvae were measured to the nearest 0.01 μm every other day. survival (%) and growth rates (μm day-1) were monitored. three months after spawning, the spats/ seed clams were transferred to 40 l plastic basins for further rearing. seed clams were fed with c. calcitrans, i. galbana, and t. tetrahele each at a concentration of 1.5 x 105 cells ml-1. results and discussion spontaneous spawning was observed among clams kept in the broodstock tank. such was also further observed in august, the peak of spawning activities in p. undulata (tuaycharoen 1984; pongthana 1990; and nabuab et al., in prep.). these show that successful spawning of the short-necked clams in the laboratory figure 1. site of collection of paphia undulata broodstock in negros occidental waters. is achieved during the peak of their reproductive activities in the wild. table 1 summarizes and shows the embryonic and larval development of p. undulata. during fertilization, a mass of spermatozoa surrounds the egg and formation of jelly coat was observed. cleavage was attained after 30 minutes and developed into swimming blastula (sl 40 μm) 1 hr and 34 minutes after fertilization. trocophore larvae with a sl of 60 μm were actively swimming after 7 hours and 13 minutes which further developed into d-veliger after 11 hours and 55 minutes of fertilization. fully developed d-veliger (straighthinged) which is also the feeding stage was attained after 17 hours and 12 minutes. straight-hinged veligers developed into the umbo stage with visible gut 9 days after fertilization having a mean length of 140 μm. thirteen days after fertilization, larvae (220 μm) with fully developed foot started to metamorphose and settle at the bottom. del norte-campos, a.g. et al. 16 science diliman (july-december 2010) 22:2,13-20 table 1. summary of the embryonic and larval development of the short-necked clam paphia undulata reared in the laboratory age stage ave.length (µm) description pictures 0 fertilization 40 swarms of spermatozoa surround the egg 30 mins cleavage 40 cells actively dividing & forming daughter cells 11 hrs 55 mins d-veliger 80 straight-hinged larvae; ciliated velum and gut; actively swimming 9 days umbone 140 two identical umbones appeared at the hinge 13 days metamorphosed larvae 220 disappearance of velum and cilia of the gut; foot fully developed and other adult organs (gills and siphon) start to develop s ip hon umbo fo o t 1 month fully metamorphosed larvae 1000 fully developed organs; development of shell pigmentation 3 months seed clam/ spat 6000 fully developed organs and shell the early development of the short-necked clam, paphia undulata 17science diliman (july-december 2010) 22:2,13-20 average increase in shell length (μm) of the two batches of larvae is plotted against time (weeks) in figure 2. over the first two weeks, the mean growth rates were 29.6 and 20.5 μm day-1 for batches 1 & 2 respectively. these values are within the range of that (25.1 μmday-1) estimated by pongthana (1990). early growth appears to continue with the same rate until about the 4th week, after which daily growth rates more than double (fig. 2). mean survival rate (%) from fertilized eggs to metamorphosis (day 13) was higher in batch 2 (2.8%) than batch 1 (1.7%) (figs.3a-b). although low, this higher survival rate for batch 2 would most likely be a reflection of slightly improved lab rearing techniques. in terms of days, the trend is the same for both batches whereby the survival drastically decreased in day 3, coinciding with the d-veliger stage, the stage when the larva starts to feed on its own (exogenous feeding), a time referred to in classic fisheries literature as the “critical period” (hjort 1914). further experimentation is obviously necessary to refine methods and thus achieve optimum survival rates. from a simultaneous study, the growth of the species was studied resulting in a growth curve shown in figure 4 (del norte-campos & villarta, 2010). using the von bertalanffy growth parameters, asymptotic shell length (sl � = 79 mm) & growth function (k=1.0 yr-1) derived from the study, the modal length of the first curve at approximately 30 mm in mid-april has an equivalent age of 6 months. projecting this backwards will therefore mean that these individuals were spawned in october the previous year, confirming the findings on the peak spawning reported by nabuab et al. (2010). based on the present results, these individuals would also have settled by the middle of october. these results therefore corroborate earlier studies specifically on its growth and settlement in the natural habitat. acknowledgments this study was funded by the dost/pcamrd project “studies on the biology and fishery of the short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) in negros occidental waters”. additional comments from w.l. campos and 2 anonymous reviewers substantially improved the manuscript. figure. 2. increase in average shell length (øm) against age in weeks of 2 batches of paphia undulata spawned at aug. 20, 2009, and nov. 4, 2009 and reared in the laboratory. del norte-campos, a.g. et al. 18 science diliman (july-december 2010) 22:2,13-20 figure 3a. survival rate (%) of the first batch of p. undulata larvae from fertilized eggs to metamorphosis. figure 3b. survival rate (%) of the second batch of p. undulata larvae from fertilized eggs to metamorphosis. the early development of the short-necked clam, paphia undulata 19science diliman (july-december 2010) 22:2,13-20 figure 4. growth curve of the short-necked clam paphia undulata derived using fisat. von bertalanffy growth parameters are sl ” = 79 mm and k = 1.0 yr-1 (from del norte-campos and villarta, in prep). references agasen, e.v., c.m. del mundo, & g.o. matias. 1998. assessment of paphia undulata in negros occidental/ guimaras strait waters. j. shellfish res. 17(5): 1613-1617. del norte-campos, a.g.c. & k.a. villarta. 2010. use of population parameters in examining changes in the status of short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) in coastal waters of southern negros occidental. science diliman 22(1):53-60. gallardo, w.g., de castro, ma.t.r., buensuceso, r.t., espegadera, c.c. and baylon, c.c. 1992. gonad development of placuna placenta linnaeus fed isochrysis galbana parke, tetraselmis tetrahele (g.s. west) butch, or their 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laboratory seed production of the carpet shell (paphia undulata). naga. iclarm. 10: 5. tuaycharoen, s. 1984. gonadal development and sex ratio of the short-necked clam (paphia undulata) (born). technical paper no. 35. brackishwater fisheries div. dept. of fisheries, bangkok. 31pp. utting, s. and b. spencer. 1991. the hatchery culture of bivalve mollusk larvae and juveniles. lab leaf 1. maff fish. res.lowesoft. 68:31pp. villarta, k. a. & a.g.c. del norte-campos. 2010. fishery of the short-necked clam paphia undulata born, 1778 in southern negros occidental, central philippines. science diliman 22(1):43-51. zhijiang, z., f. li, and c. ke. 1991. on the sex gonad development and reproductive cycle of clam paphia undulata. journal of fisheries of china, shuichan xuebao. 15(1): 1-8. abstract. 01_device growth of gold-assisted gallium arsenide nanowires 31 *corresponding author science diliman 20:1, 31-38 growth of gold-assisted gallium arsenide nanowires on silicon substrates via molecular beam epitaxy ramon m. delos santosa, jasher john a. ibañesa, joel g. fernandoa rafael b. jaculbiaa, jorge michael m. prestoa, michael j. defensora michelle b. somintacb, paul concepcionb arnel a. salvador, armando somintaca* anational institute of physics, university of the philippines, diliman, quezon city bquality and reliability department, intel technology philippines inc. date submitted: july 25, 2008; date accepted: october 16, 2008 abstract gallium arsenide nanowires were grown on silicon (100) substrates by what is called the vapor-liquidsolid (vls) growth mechanism using a molecular beam epitaxy (mbe) system. good quality nanowires with surface density of approximately 108 nanowires per square centimeter were produced by utilizing gold nanoparticles, with density of 1011 nanoparticles per square centimeter, as catalysts for nanowire growth. x-ray diffraction measurements, scanning electron microscopy, transmission electron microscopy and raman spectroscopy revealed that the nanowires are epitaxially grown on the silicon substrates, are oriented along the [111] direction and have cubic zincblende structure. introduction nanowires, or nanowhiskers, are wire-like nanocrystals with diameters of several tens of nanometers and with length:diameter aspect ratios of 10 or more (dubrovskii, et al., 2005). they are one-dimensional structures with unique growth mechanism and remarkable physical properties. one-dimensional systems such as nanowires and nanotubes are the smallest dimension structures that can be used for efficient transport of electrons and optical excitations. in some aspects semiconducting nanowires are complementary to carbon nanotubes, but in contrast to the latter they offer more flexibility with the choice of materials, which makes them more useful for various device architectures and functionalities. there is a growing interest in the synthesis and properties of semiconducting nanowires because established conventional semiconductor technologies such as junction formation, (cui, et al., 2000, gudiksen, et al., 2002). growth of heterostructures (cui, et al., 2000, gudiksen, et al. 2002) and doping can be potentially applicable to them. current trends in nanowire technology at present, semiconducting nanowires are seen to be the most versatile building blocks for electrical, optical, and (opto) electronic circuits at the nanoscale. the use of semiconducting nanowires in electrical circuits ranges from transistor arrays, (patolsky, et al., 2006) to single electron tunneling devices, (franceschi et al. 2003) and nonvolatile memory (duan, et al., 2002). they could also be used as electrical sensors (patolsky, et al., 2006) due to their large surface-to-volume ratios that lead to higher sensitivity to changes at their surfaces or surroundings. a “bottom-up” approach to circuit assembly using nanowire building blocks (huang, et al., 2006) can be useful for complementary optoelectrical functions. some opto-electrical nanodevices based on semiconducting nanowires include polarizationdependent photo-detectors (wang, et al., 2001), light emitting diodes (gudiksen, et al., 2002) and solar cells (law, et al., 2005). in addition, the nanowires can act as nanocavities for light resulting in opticallyor electrically-driven nanolasers (huang, et al., 2001, duan, et al., 2003), and subwavelength waveguiding somintac, et al 32 of light over long distances and through sharp bends (barrelet, et al., 2004). the compound semiconductor gallium arsenide (gaas) is one of the most likely candidates as the choice for nanowire material because of its intrinsic direct bandgap which gives rise to attractive optical and optoelectrical properties, and also due to the already existing technological platform for this material. particularly, epitaxial growth of gaas nanowires on silicon (si) substrates is of considerable importance because it can pave the way for the possible integration of highperformance iii-v semiconductor nanoscale devices with well-established si technology. this will likely happen since the growth of iii-v compound semiconductor nanowires, such as gaas nanowires, on si can solve many problems associated with the large difference in lattice constant and structure (martensson, et al., 2004). vapor–liquid–solid (vls) growth mechanism overview in general, the development of device-quality iii-v semiconducting nanowires based on vapor–liquid–solid (vls) reaction (martensson, et al., 2004, wagner & ellis, 1964, khorenko, et al., 2004) has the advantage over techniques such as photolithography, ion beam lithography, ion etching, and others because the lateral size of the nanoparticle metal-catalysts predefines the growth areas for the nanowires and controls the morphology of the as-grown nanostructures. in the vls growth mode, the metal nanoparticles are used to direct growth in a highly anisotropic or onedimensional manner. this mechanism consists of three main stages which are illustrated in figure 1a (wagner & ellis, 1964). first, a liquid eutectic alloy is produced from the nanoparticle and the growth elements. next, the eutectic system absorbs more semiconductor material until a supersaturation condition is reached. when the supersaturated alloy droplet coexists with the solid phase of the semiconductor, nucleation occurs. finally, a steady state is formed in which the semiconductor crystal grows at the solid/liquid interface. the precipitated semiconductor material grows as a wire because the growth area is limited by the area of the alloy droplet or the catalyst itself (wagner & ellis, 1964, khorenko, et al., 2004). in the vls mechanism, the nanowire diameter is determined by the diameter of the alloy particle that is formed from the metal nanoparticle and the growth elements; while the length is dependent on the growth rate, growth time and even on the lateral size of nanowires or au seed particles (dubrovskii, et al., 2005). examining the feasibility of the vls wire growth for a certain compound semiconductor-metal system requires the study of the associated pseudo-binary phase diagram as illustrated in figure 1b (duan & leiber, 2000). an isothermal line in the diagram will show the subsequent phases involved when a gold nanoparticle absorbs the semiconductor material at a constant temperature. the primary condition for vls wire growth is that the metal should form an alloy with the figure 1. (a) the stages of: i) alloying, ii) nucleation and iii) development of nanowire synthesis, according to the vls growth mechanism. (b) pseudobinary phase diagram of a semiconductor-gold system. science diliman 20:1, 31-38 growth of gold-assisted gallium arsenide nanowires 33 semiconductor at a temperature that also allows the semiconductor to exist in the solid phase. materials and methodology samples used in this study were grown using the riber 32p mbe facility at the national institute of physics, university of the philippines diliman. prior to the growth itself, the si (100) substrates were cleaned using the standard degreasing procedures and were subsequently immersed for 5 minutes in a diluted hydrofluoric acid to remove the native oxides. right after the oxide removal, they were loaded inside an electron beam evaporator to deposit gold (au) clusters (or islands) on their surfaces for various time intervals (i.e. 10 sec, 20 sec and 30sec), at a base pressure of 8.8 × 10-6 torr, using an emission current of 34 milliamperes. (the emission current of the electron beam dictates the rate at which the material is deposited on the substrates.) the gold-deposited si substrates were then transferred into the n 2 -ambient annealing tube furnace and were annealed at 540°c for 10 minutes to generate au nanoparticles of diverse sizes and densities from the au islands grown at different time intervals. finally, they were all mounted onto a molybdenum substrate holder and were degassed inside the mbe growth chamber for 5 minutes at a temperature of 585°c under arsenic flux. gaas nanowires on silicon substrates were grown at 580°c with an arsenic:gallium (as:ga) flux ratio of 15 and a fixed ga beam flux, which is required for the homoepitaxial gaas growth rate of 0.25µm/hr on a gaas (100) substrate. the background pressure was 10-9 torr during growth. the growth of gaas nanowires was ended by switching off the gallium supply while maintaining the arsenic supply until the substrate temperature cooled down to 400°c to stabilize the nanowires. for the investigation of the crystalline quality and structural properties of the mbe-grown gaas nanowires, scanning electron microscopy (sem), transmission electron microscopy (tem), raman spectroscopy and x-ray diffraction (xrd) analyses were performed. the samples for tem were prepared by scratching a surface in order to separate the nanowires from the substrate and transfer them onto carbon-film-coated cu grids. results and discussion electron microscopy characterization of gold nanoparticles in this research, gold nanoparticles of different sizes and densities were utilized as catalysts for the vls growth of nanowires. surface morphology of the generated au nanoparticles on silicon substrates was observed by acquiring sem micrographs at different magnifications. figures 2a up to 2c are images of au nanoparticles on si (100) substrates that were formed from the clusters deposited at different time intervals. generally, the nanoparticles from the clusters grown for 30sec have the largest lateral size (approximately ranging from 10-30 nm) of the three sets, followed by the nanoparticles from the clusters grown for 20sec the diameters of which range from about 10-20 nm, while those that came from the clusters grown for 10sec figure 2a-c sem micrographs of au nanoparticles on si (100) substrates generated from clusters deposited for: (a) 30 seconds; (b) 20 seconds; and (c) 10 seconds. all images were taken at a magnification of 200,000x. science diliman 20:1, 31-38 somintac, et al 34 are the smallest (roughly varying from 8-10 nm in diameter). the surface densities of figures 2a, 2b and 2c are 1.70 × 1011, 2.68 × 1011 and 3.38 × 1011 nanoparticles per square centimeter, respectively. the differences in lateral sizes and surface densities among the three sets may be due to the fact that the density of the islands formed during the early stages of growth saturates quickly, and that the islands begin to grow in size by adsorbing atoms from the incident vapor beam and receiving atoms from the neighboring islands which eventually disappear (maissel & francombe, 1973, bassett & wenter & pashley, 1959). annealing the gold-deposited si substrates at 540°c for 10 minutes improves the uniformity of the nanoparticles by enhancing the island agglomeration (harmand, et al., 2007, ihn, et al., 2006, chan, et al., 2003, hiruma, et al., 1995, vosen, 1977). electron microscopy characterization of gallium arsenide nanowires sem micrographs of gaas nanowires on si (100) substrate with gold nanoparticles generated from clusters that were deposited for 10 seconds are given in figures 3a and 3b. the images were acquired from one sample using three different magnifications and viewing angles. the gaas nanowires were grown at 580°c for 15 minutes using a as:ga flux ratio equal to 15 and a growth rate of 0.25 µm/hr. the sample has a surface density of approximately 9 × 108 gaas nanowires per square centimeter. this value was obtained by considering a 1 µm × 1 µm area from the top view sem image of the sample (figure 3a) and counting the nanowires that can be found within that area. most of the nanowires of the sample were observed to be either parallel or perpendicular to one another, and at an angle of inclination roughly equal to 35° from the surface of the si (100) substrate. this measured angle of inclination can also be calculated by looking at a cubic zincblende structure along the [111] axis (see figure 4a) (yu & carolona, 1996). in this point of view, the computed value of the angle between the (111) and (100) planes is 54.7µ, so that a nanowire that is assumed to be perpendicular to the (111) plane has an angle of inclination equal to 35.3 with respect to the (100) plane as shown in figure 4b. martensson et al. (2004) observed that on si (100) substrate, gap nanowires preferably grew in four equivalent <111> directions which make an angle of 35.3° with the substrate surface, distributed 90° apart azimuthally as illustrated in figure 5 (martensson, et al., 2004). this is similar to what we have observed on the nanowire sample in figure 3. martensson et al. further argued that for epitaxial growth all four directions can be expected since the four <111> directions are equivalent.14 epitaxial growth is characterized by an oriented layer by layer growth; this is illustrated by expressed parallelism of the deposited plane (and axis) and the plane (and axis) of the substrate (maissel & francombe, 1973). thus, our results affirm that the synthesized gaas nanowires on si (100) substrate were the <111> family in growth direction and they were epitaxially grown nanowires. a tem micrograph of the tip region of a single gaas nanowire from a sample with gold nanoparticles generated from clusters that were deposited for 10 seconds is shown in figure 6. this image was acquired to clearly resolve the lateral sizes of a representative gaas nanowire and the au nanoparticle on its tip which figure 3a-b. (a) top view (with 50,000x magnification) and (b) 52°-tilted view (with 200,000x magnification), sem images of gaas nanowires grown at 580°c on gold-deposited si (100) substrate. the inset in (b) is a cross-sectional image (with 45,000x magnification) of the same sample showing a nanowire inclined at approximately 35° from the substrate surface. science diliman 20:1, 31-38 growth of gold-assisted gallium arsenide nanowires 35 cannot be done by employing sem alone. the lateral size of the nanoparticle was measured to be approximately 12.6 nm and the average diameter of the nanowire to be 24.7 nm. that the nanowire was grown via the vls growth method can be inferred from the very presence of the gold nanoparticle at the tip of the gaas nanowire, since this is a characteristic feature of such a mechanism. the electron diffraction pattern from the tem suggests that the majority of this region of the nanowire has cubic (zincblende) structure. this type of crystal structure is also the characteristic feature of bulk gaas. figure 4a-b. (a) the crystal structure of a cubic zincblende. note that if this figure is looked at along the [111] axis, we can visualize the orientation of the nanowires with respect to the (100) plane. (b) diagram illustrating the angle 35.3°. figure 5. azimuthal projections of four nanowires grown along the four equivalent <111> directions on si (100) substrate. the inset shows that the nanowires form an angle of 35.3° with the si (100) surface. 24.7 nm 12.6 nm figure 6. tem image of the tip region of a single gaas nanowire. the image was taken at a magnification of 500,000x. the scale bar is 100 nm long. xrd analysis and raman spectroscopy characterizations of sample with gaas nanowires the gold-assisted gaas nanowires were also characterized by xrd analysis and raman spectroscopy at room temperature. the result of theta/ two theta xrd scan from a sample with gaas nanowires on si (100) is shown in figure 7 the normalized peaks are located at 2 = 66.219°, 69.118° science diliman 20:1, 31-38 somintac, et al 36 and 69.358°. the peak at 2 = 66.219° corresponds to the cubic zincblende gaas (004) plane as confirmed by the bragg’s law. those at 2 = 69.118° and 69.358° are initially the characteristic peaks of a bare si (100) substrate and are related to the (004) plane of the silicon material. only one gaas peak pertaining to the (004) plane was observed because the {004} family of planes on the nanowires were the ones directly exposed to the x-rays as illustrated in figure 8. a reference sample with very thin gaas layer on bare (without gold nanoparticles) si (100) substrate was also grown side by side with the nanowire sample to ensure similar growth conditions. the presence of a weak gaas peak on the scan of the sample with gaas nanowires, and its absence from that of the reference figure 7. x-ray diffraction 2 scan from a sample with gaas nanowires on si (100) substrate. figure 8. diagram illustrating the {004} family of planes on a nanowire grown on si (100) substrate. the angle between the wire and the substrate surface is 35.3°. sample with a thin layer of gaas proves that the gaas peak came from these nanostructures and that they exhibit good crystallinity. due to the nanometer-sized diameters and large surface-to-volume ratios of the nanowires, the gaas peak is expected to be broad. the core of a nanowire, which makes up the majority of the nanostructure, is expected to have true crystal lattice while those located near or at the surface have distorted lattice, and these contribute to the broadening of the peak. also, according to duan et al. (duan, et al., 2000), a thin amorphous coat at the nanowire exterior is formed when gaas nanowires are exposed to air; figure 9. raman spectra from (top) a sample with gaas nanowires on si (100) substrate; and (bottom) a gold-deposited si (100) substrate. for the former sample, the raman signals of the gaas lo and to phonons were observed at 292 cm-1 and 268 cm-1, respectively. science diliman 20:1, 31-38 growth of gold-assisted gallium arsenide nanowires 37 this amorphous coat causes the broadening of the gaas diffraction peak. normalized raman spectrum from a sample with gaas nanowires on si (100) substrate, together with a reference spectrum obtained from a gold-deposited si (100) substrate are shown in figure 9. the raman shift (in terms of wavenumbers) scan range was intentionally restricted to examine only the vibrational modes for gaas material and exclude those associated with si and even au. for the sample with gaas nanowires, the raman signals of the gaas longitudinal optical (lo) and transverse optical (to) phonons were observed at 292 cm-1 and 268 cm-1, respectively. for bulk gaas, the signal for lo phonon is at 290.2 cm-1, while the signal for to phonons is located at around 268.6 cm-1. moreover, the peak of lo phonon for bulk gaas has a higher intensity than that of the to phonon (mahan, et al., 2003). according to the results, and as seen on the graph, the gaas to phonon signal is much stronger than that related to the gaas lo phonon, as also previously observed by begum et al. (begum, et al., 2008).the full width at half maximum (fwhm) of the lo phonon signal is equal to eight wavenumbers, while the one associated with the to phonon signal is about seven wavenumbers. the existence of these two prominent raman signals associated with the gaas nanowires indicates the good crystal quality of the gaas material. at present, further investigations on the nature of the relative intensities of the raman peaks are still being carried out. summary of results good quality gaas nanowires epitaxially grown on si (100) substrates by using au-catalyzed vls growth with an mbe system were demonstrated. the sample with nanowires was found to have a surface density of approximately 9 × 108 gaas nanowires per square centimeter. the synthesized nanowires on si (100) substrate were epitaxially grown along the [111] direction and they have cubic (zincblende) crystal structure. tem imagery showed that the average diameter of a representative nanowire from the sample with au nanoparticles generated from clusters that were deposited for 10 seconds is 24.7 nm. from the results of the xrd measurements, the presence of a gaas peak at 2 = 66.219° for a sample with gaas nanowires and its absence for a sample with only a thin layer of gaas on bare si (100) proves that the gaas peak came from these nanostructures and that they exhibit good crystallinity. lastly, the raman signals of the gaas longitudinal optical (lo) phonon observed at 292cm-1 and gaas transverse optical (to) phonon at 268cm-1 from a sample with gaas nanowires indicate the good crystal quality of the gaas material. acknowledgements this research was funded by the ovcrd outright grant. the authors would like to express their gratitude to the intel technology philippines inc. 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properties. berlin, springer-verlag: pp 134-135. science diliman 20:1, 31-38 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 an annotated-art.9 an annotated checklist of macrobenthic algae 67science diliman (july december 2000) 12:2, 67-74 abstract introduction records of the marine algae and its taxonomic studies for the entire western coast of zamboanga city are scanty. the latest treatise that compiles the reported seaweeds from the philippines prior to 1987 is the work of silva et al. (1987) who catalogued 911 species, including those found in zamboanga waters. the earliest account of marine algae collected from this area is probably that of goerg von martens in 1868, during the preussiche expedition, followed by the british challenger expedition in 1874-1875 and the dutch siboga expedition in 1899. subsequent reports that include seaweeds from zamboanga were published by velasquez (1955), taylor (1966), cordero (1980), and trono et al. (1983). the most recent collections done an annotated checklist of macrobenthic algae of the western coast of zamboanga city (mindanao, philippines) o. tito*, c. sescon*, c. adalla*, j. asdani, e. m. basilio, a. climaco, a. dagalea, m. luna, y. uddin, and r. a. vega marine biology department zamboanga state college of marine sciences and technology seaweed collections were made at several sites along the western coast of zamboanga city from july to august 1999. ninety-four (94) taxa were identified. these represent the three (3) major algal divisions broken down into fifteen (15) orders, twenty-six (26) families and forty-four (44) genera. six species: botryocladia leptopoda (j. agardh) kylin, halimeda distorta (yamada) colinvaux, halimeda melanesica valet, kallymenia perforata j. agardh, sarconema filiforme (sonder) kylin and solieria robusta (greville) kylin, and the genus sarconema zanardini represent new records for zamboanga and the philippines. all specimens are deposited in the zamboanga state college of marine sciences and technology herbarium. keywords: checklist, seaweed species, new records, zamboanga by japanese and filipino scientists in 1988 resulted in a list of species published by umezaki in 1989. however, some of these reports documented in zamboanga do not mention the specific locality of the area except those made in santa cruz islands, sacol island, and the calarian areas. the main purpose of this study is to identify the different macrobenthic algae growing along the littoral zones of the western coast of zamboanga city. materials and methods seaweeds were collected from littoral areas (10 sites). stations 1 to 6 were located from the southeastern portion of zamboanga city. these areas were partially protected from strong waves and currents. stations 4 and 5 were situated near the river mouth and * corresponding authors tito et al. 68 characterized by heavy siltation resulting from run-off waters due to heavy rains. stations 7 to 10 were narrow reef flats facing the sulu-celebes sea, and were exposed to strong waves and winds. snorkeling and wading on the reef flats were done to collect the specimens. topographical and oceanographic data and other relevant information were recorded. collection and preservation procedures were adopted and modified from abbott et al. (1976), lewmanomont & ogawa (1995), and ganzon-fortes (1991). collected specimens were initially sorted out on the beach before being transported to the zscmst laboratory for identification and preservation. materials were pressed and dried, following the methods described by ganzon-fortes (1991), tito (1999), and by dr. max h. hommersand and dr. laurence m. liao (pers. comm.). some specimens were preserved in clear glass bottle with 5% formalin-seawater solution. microscopic examination of samples was done by sectioning the materials using freehand method. samples were identified on the basis of external and internal morphological features using reliable taxonomic keys like those of magruder & hunt (1979), calumpong & meñez (1997), & trono (1997). results and discussion a total of ninety-four (94) species of marine macrobenthic algae were collected and identified. these represent the three major algal divisions (rhodophyta, phaeophyta, and chlorophyta). these are broken down into 15 orders, 26 families, and 44 genera. of these, six species, namely, botryocladia leptopoda, kallymenia perforata, sarconema filiforme, and soliera robusta (greville) for rhodophyta, halimeda melanesica and halimeda distorta for chlorophyta, and one genus, sarconema zanardini (a red alga), represent new records for the area and the philippines. the list of all the taxa included in this study is presented below following the classification scheme of silva, et al. (1996): division rhodophyta number of orders: 8 number of families: 12 number of genera: 21 number of species: 42 order nemaliales family galaxauraceae actinotrichia decaisne actinotrichia fragilis (forsskaal) boergesen specimen examined: mb-154. galaxaura lamouroux galaxaura apiculata kjellman specimen examined: mb-044a. galaxaura arborea kjellman specimens examined: mb-044, mb-079, mb-221. galaxaura fasciculata kjellman specimen examined: mb-079a. tricleocarpa huisman & borowitzka tricleocarpa cylindrica (ellis & sol.) huisman & borowitzka specimens examined: mb-045, mb-078, mb-125, mb-148, mb-183, mb-217, mb-230, mb-238. tricleocarpa fragilis (linnaeus) huisman & townsend specimens examined: mb-046, mb-080. order gelidiales family gelidiaceae gelidium lamouroux gelidiella acerosa (forsskaal) feldmann & hamel specimen examined: mb-177. an annotated checklist of macrobenthic algae 69 order gracilariales family gracilariaceae gracilaria greville gracilaria arcuata zanardini specimen examined: mb073a. gracilaria debilis (forsskaal) boergesen specimen examined: mb-127, mb-149, mb-184, mb-213. gracilaria edulis (s.g. gmelin) silva specimens examined: mb-050, mb-071, mb-128, mb-144. gracilaria eucheumoides harvey specimens examined: mb-051, mb-073, mb-129. gracilaria salicornia (c. agardh) dawson specimens examined: mb-119, mb-153. gracilaria textorii (suringar) de toni specimen examined: mb-035. order cryptonemiales family halymeniaceae halymenia c. agardh halymenia dilatata zanardini specimen examined: mb-041, mb-081, mb-122, mb-176. halymenia durvillaei bory de saint -vincent specimens examined: mb-047, mb-077, mb-124, mb-187. halymenia maculata j. agardh specimens examined: mb-039, mb-083, mb-145, mb-214. family kallymeniaceae kallymenia j. agardh kallymenia perforata j. agardh specimens examined: mb-042, mb-175. order corallinales family corallinaceae amphiroa lamouroux amphiroa fragilissima (linnaeus) lamouroux specimens examined: mb-246. amphiroa rigida lamouroux specimens examined: mb-043, mb-115, mb-229. amphiroa foliacea lamouroux specimen examined: mb-252. order gigartinales family hypneaceae hypnea lamouroux hypnea boergesenii tanaka specimens examined: mb-123, mb-152. hypnea charoides sonder specimens examined: mb-049, mb-076, mb-205. hypnea cervicornis j. agardh specimens examined: mb-178, mb-215. hypnea divaricata (c. agardh) greville specimens examined: mb-048, mb-219. hypnea spinella (c. agardh) kuetzing specimens examined: mb-074, mb-126, mb-147, mb-186. hypnea pannosa j.agardh specimen examined: mb-123a. family solieriaceae kappaphycus doty kappaphycus alvarezii (doty) doty ex silva specimens examined: mb-047, mb-077, mb-124, mb-187. kappaphycus cottonii (weber van-bosse) doty ex silva specimen examined: mb-052. kappaphycus striatum (schmitz) doty ex silva specimen examined: mb-040. tito et al. 70 sarconema zanardini sarconema filiforme (sonder) kylin specimen examined: mb-072. solieria j. agardh solieria robusta (greville) kylin specimen examined: mb-037. order rhodymeniales family champiaceae champia desvaux champia parvula (c. agardh) harvey specimen examined: mb-038. family rhodymeniaceae botryocladia (j. agardh) kylin botryocladia leptopoda (j. agardh) kylin specimens examined: mb-036, mb-116, mb-181, mb-218. ceratodictyon zanardini ceratodictyon spongiosum zanardini specimen examined: mb-146. order ceramiales family dasyaceae dictyurus bory de saintvincent dictyurus occidentalis j. agardh specimen examined: mb-180. vanvoorstia harvey vanvoorstia spectabilis harvey specimen examined: mb-118. family rhodomelaceae acanthopora lamouroux acanthopora muscoides (linnaeus) bory de saint-vincent specimens examined: mb-1311, mb-085, mb-150. acanthopora spicifera (vahl) boergesen specimens examined: mb-082, mb-121, mb-151, mb-182. chondria c. agardh chondria armata (kuetzing) okamura specimen examined: mb-032. laurencia lamouroux laurencia cartilagenea yamada specimen examined: mb-034. laurencia papillosa (c. agardh) greville specimen examined: mb-034a. melanamansia r.e. norris melanamansia glomerata (c. agardh) norris specimen examined: mb-117. division phaeophyta number of orders: 3 number of families: 4 number of genera: 8 number of species: 15 order dictyotales family dictyotaceae dictyota lamouroux dictyota cervicornis kuetzing specimens examined: mb-024, mb-207. dictyota dichotoma (hudson) lamouroux specimens examined: mb-025, mb-107, mb-141, mb-174. an annotated checklist of macrobenthic algae 71 dictyota friabilis setchell specimen examined: mb-023. dictyota mertensii (martius) kuetzing specimen examined: mb-020. lobophora j. agardh lobophora variegata (lamouroux) womersley ex oliviera specimens examined: mb-022, mb-108, mb-143, mb-172. padina adanson padina australis hauck specimens examined: mb-027, mb-069, mb-113, mb-142. padina tetrastromatica segawa specimen examined: mb-066. spatoglossum kuetzing spatoglossum pacificum yendo specimens examined: mb-026, mb-070, mb-206. order scytosiphonales family scytosiphonaceae colpomenia (endlicher) derbes and solier colpomenia sinuosa (martens ex roth) derbes and solier specimen examined: mb-018. order fucales family sargassaceae sargassum c. agardh sargassum crassifolium j. agardh specimens examined: mb-030, mb-140. sargassum cristaefolium c. agardh specimens examined: mb-021, mb-109. sargassum polycystum c. agardh specimens examined: mb-018, mb-110. sargassum oligocystum montagne specimens examined: mb-028, mb-067, mb-112, mb-173. turbinaria lamouroux turbinaria ornata (turner) j. agardh specimens examined: mb-029, mb-111. family cystoseriaceae hormophysa kuetzing hormophysa cuneiformis (gmelin) p. silva specimen examined: mb-143a. division chlorophyta number of orders: 4 number of families: 10 number of genera: 15 number of species: 37 order ulvales family ulvaceae enteromorpha link enteromorpha intestinalis (linnaeus) van den hoek specimens examined: mb-016, mb-058, mb-136, mb-165. enteromorpha flexuosa (wulfen) j. agardh specimen examined: mb-233. ulva linnaeus ulva lactuca linnaeus specimens examined: mb-006, mb-139, mb-162, mb-201. ulva reticulata forsskaal specimens examined: mb-008, mb-089, mb-167, mb-193. tito et al. 72 order cladophorales family cladophoraceae chaetomorpha kuetzing chaetomorpha crassa (c. agardh) kuetzing specimens examined: mb-001, mb-086, mb-135, mb-166. chaetomorpha spiralis (okamura) specimen examined: mb-235. cladophora kuetzing cladophora vagabunda (linnaeus) venden specimens examined: mb-093, mb-129. family siphonocladaceae boergesenia j. feldman boergesenia forbesii (harvey) j. feldmann specimens examined: mb-005, mb-088, mb-155. boodlea g. murray and de toni boodlea composita (harvey) brand specimen examined: mb-094. dictyosphaeria decaisne ex endlicher dictyosphaeria cavernosa (forsskaal) boergesen specimen examined: mb-092. family valoniaceae valonia c. agardh valonia aegagropila c. agardh specimens examined: mb-055, mb-104, mb-131. order bryopsidales family caulerpaceae caulerpa lamouroux caulerpa cupressoides (vahl) c. agardh specimen examined: mb-091. caulerpa lentillifera j. agardh var. compacta trono and ang specimen examined: mb-091. caulerpa peltata lamouroux specimens examined: mb-106, mb-156, mb-194, mb-226. caulerpa racemosa (forsskaal) j. agardh specimens examined: mb-007, mb-065, mb-132, mb-169, mb-190. caulerpa racemosa (forsskaal) agardh var. turbinata (agardh) eubank specimens examined: mb-105, mb-149. caulerpa serrulata (forsskaal) j. agardh specimen examined: mb-232, mb-242. caulerpa sertularioides (s. gmelin) howe specimens examined: mb-015, mb-060, mb-100, mb-130, mb-171, mb-202, mb-243, mb-248. caulerpa taxifolia (vahl) j. agardh specimens examined: mb-222, mb-239. family codiaceae codium stackhouse codium bartlettii tseng & gilbert specimens examined: mb-061, mb-158, mb-204, mb-225, mb-240. codium divaricatum tseng & gilbert specimen examined: mb-053. codium edule p. c. silva specimens examined: mb-010, mb-224, mb-236. codium geppii o. schmidt specimens examined: mb-188, mb-250. an annotated checklist of macrobenthic algae 73 family halimedaceae halimeda lamouroux halimeda bikiniensis taylor specimen examined: mb-056. halimeda discoidea decaisne specimens examined: mb-017, mb-059, mb-101, mb-137, mb-195. halimeda distorta decaisne specimen examined: mb013. halimeda gracilis harvey ex j. agardh specimen examined: mb014. halimeda macroloba decaisne specimens examined: mb-002, mb-090, mb-170, mb-191, mb244. halimeda melanesica valet specimens examined: mb-009, mb-063, mb-096, mb-161, mb-198. halimeda micronesica yamada specimen examined: mb-012. halimeda tuna (ellis & solander) lamouroux specimens examined: mb-003, mb-097, mb-132, mb-164, mb-234. family udoteaceae chlorodesmis harvey & barkley chlorodesmis hildebrandtii a. gepp & e. gepp specimens examined: mb-160, mb-200, mb-227. chlorodesmis fastigiata (c. agardh) ducker specimen examined: mb-247. order dasycladales family dasycladaceae bornetella munierchalmas bornetella sphaerica (yamada) solmslaubach specimens examined: mb-004, mb-102, mb-157. neomeris lamouroux neomeris annulata dickie specimens examined: mb-195, mb-241. family polyphysaceae acetabularia lamouroux acetabularia dentata solmslaubach specimens examined: mb-054, mb-103. acetabularia major g. martens specimens examined: mb-159, mb-203, mb-245, mb-249. references abbott ia, hollenberg ga. 1976. marine algae of california. california: stanford university press. 827 p. calumpong hp, menez eg. 1997. field guide to the common mangroves, seagrasses and algae of the philippines. makati, philippines: bookmark inc. 197 p. cordero pa jr. 1980. taxonomy and distribution of philippine useful seaweeds. national research council of the philippines. 81: 1-78. ganzon-fortes et. 1991. field and laboratory techniques in the collection, preservation and preparation of marine benthic algae for herbarium and for identification. in: pagdilao jr cr, acedera mm, editors. training on seaweed research, proceedings of the seaweed research training and workshop for project leaders. (book series no. 11/1991). los baños, laguna: philippine council for aquatic and marine research and development. p 67-71. lewmanomont k, ogawa h. 1995. common seaweeds and seagrasses of thailand. thailand: faculty of fisheries kasersart university. 163 p. magruder wh, hunt jw. 1979. seaweeds of hawaii. a photographic identification guide. honolulu, hawaii: oriental publishing company. 116 p. silva pc, meñez eg, moe rl. 1987. catalogue of the benthic marine algae of the philippines. smithsonian. contr mar sci 27: 1-179. tito et al. 74 silva pc, basson pw, moe rl. 1996. catalogue of the benthic marine algae of the indian ocean. london: university of california press. 1259 p. taylor wr. 1966. records of asian and western pacific marine algae, particularly algae from indonesia and the philippines. pac sci 20:342-359. tito od. 1999. taxonomy and species diversity of marine macrobenthic algae of the santa cruz islands, zamboanga city, philippines [dissertation]. zamboanga city., philippines: zamboanga state college of marine sciences and technology. trono gc jr, azanza-corrales r, manuel d. 1983. the genus gracilaria (gigartinales, rhodophyta) in the philippines. kalikasan philipp j biol 12: 15-41. trono gc jr. 1997. field guide and atlas of the seaweed resources of the philippines. makati city: bookmark inc. 306 p. velasquez gt. 1955. the ecological distribution of the myxophycean algae of eastern palawan and sulu archipelago. natur appl sci bull 15:153-184. 03_olbinado synthesis of bulk superconducting magnesium diboride margie p. olbinado*, leandro jose d. guerra, and roland v. sarmago condensed matter physics laboratory national institute of physics, college of science university of the philippines, diliman, quezon city tel. no.: (632) 434-4260; fax no.: (632) 920-5474 e-mail: marge@nip.upd.edu.ph abstract bulk polycrystalline superconducting magnesium diboride, mgb2, samples were successfully prepared via a one-step sintering program at 750°c, in pure argon with a pressure of 1atm. both electrical resistivity and magnetic susceptibility measurements confirmed the superconductivity of the material at 39k, with a transition width of 5k. the polycrystalline nature, granular morphology, and composition of the sintered bulk material were confirmed using x-ray diffractometry (xrd), scanning electron microscopy (sem), and energy dispersive x-ray analysis (edx). key words: magnesium diboride, synthesis, superconductivity introduction the recent discovery of superconductivity in the intermetallic, binary compound magnesium diboride, mgb2, with a superconducting transition temperature of 39k, (nagamatsu et al., 2001) stimulated numerous investigations on the synthesis and characterization of this compound. mgb2 shares many features with both ‘conventional’ superconductors and high-tc cuprates, thus offering real progress in understanding superconductivity (hirsch, 2001). unlike the cuprates, mgb2 has lower anisotropy, larger coherence lengths, and transparency of the grain boundaries to current flow. these make mgb2 a promising candidate for higher operating temperature and higher speed devices (buzea & yamashita, 2001). moreover, its simple crystal structure and metallic nature prove easier hurdles for the development of commercial applications (rogado et al., 2001) than what is presented by the hightemperature cuprates. c u r r e n t p r o g r e s s o n t h e s y n t h e s i s o f b u l k superconducting samples of this material are both complex and involve the use of exotic materials. in most works, powders of mg and b are mixed, then wrapped in iron (dou et al., 2001), or tantalum foils (larbalestier et al., 2001; rogado et al., 2001), sealed in quartz ampoules (zhao et al., 2001; larbalestier et al., 2001), and then sintered at relatively high temperatures and pressures, ≈8001000°c at ≥150mpa, (indrakanti et al., 2001; frederick et al., 2001) in ambient ar/h2 (larbalestier et al., 2001) or mg vapor atmospheres (zhu et al., 2001). these preparations are multi-step processes. a single-step process in bulk synthesis is necessary for the successful growth of thin films of this compound. this may be the reason for the relatively low transition temperatures of ~24k-37k (patnaik et al., 2001) reported for grown mgb2 films so far. transition temperatures reported for bulk mgb2 are in the range of ~38.2k-39k (dou et al., 2001; larbalestier et al., 2001; indrakanti et al., 2001; frederick et al., 2001). 17science diliman (january-june 2002) 14:1, 17-20 *corresponding author this paper discusses the efforts by the condensed matter physics laboratory to prepare superconducting mgb2. results indicate that a one-step sintering process at a relatively low temperature of 750°c in an ar atmosphere is promising for the synthesis of good, high quality bulk superconducting samples of mgb2. methodology solid state reaction method initial attempts involved the solid state reaction of mgco3 and h3bo3. stoichiometric amounts of these precursors were weighed and mixed, and then calcined at 500°c for 24 hours. sequential sintering was performed on the calcined material at 900, 950, 980, and 1000°c for periods of 24 hours until a visible reaction of the precursors was observed. compaction and sintering in ar commercial mgb2 powder (alfa aesar, 99.9% pure) was utilized as starting material. two samples weighing 4 grams each were compacted into pellets under 10 tons for 15 minutes. electrical characterization was difficult because the pellets were granular and porous. these pellets were then sintered at 750°c and 780°c for 5 hours, respectively, in a pure ar atmosphere of 1 atm pressure. the sintered pellets were then characterized via resistivity and ac magnetic susceptibility measurements. x-ray diffraction was used to identify the resulting phases in the sintered pellet while scanning electron microscopy was used to investigate its microstructure. composition of the sintered sample was also determined using energy dispersive x-ray analysis. results and discussion solid state reaction the initial attempts via solid state reaction of carbonates were unsuccessful. the calcined material was white in color and remained white despite the sequential sintering at high temperatures. note that commercial mgb2 powder is black in color. also, the sintered product became noticeably harder at each sintering step. it was apparent that the proposed reaction did not take place. this may be due to the high volatility of mg atoms at high temperatures (shields et al., 2001). the volatility of mg likely results in the formation of mgo (whitish in appearance) and/or other derivatives of mgxby. also note that these sintering steps were done in air. sintering in an ambient atmosphere without o2 was thus concluded to be necessary in any synthesis of bulk samples of mgb2. compaction and sintering in ar the sintered mgb2 pellets were black in color with some grayish parts and were less granular and porous than the ones not yet sintered. fig. 1 shows the measured resistivity plots of the two sintered mgb2 pellets. a sharper transition was observed for the pellet sintered at 750°c (750c5h), compared to the incomplete transition for the pellet sintered at 780°c (780c5h). also, pellet 750c5h has a higher transition temperature, tc = 39.4k compared to the tc = 31.5k of pellet 780c5h. the transition width for pellet 750c5h was 5k indicating good sample quality. on the other hand, the broad transition width of pellet 780c5h indicates that other phases are present in the bulk material. therefore, pellet 750c5h is the better superconducting sample between the two. 18 olbinado, guerra, and sarmago fig. 1. resistivity measurement plots of the sintered mgb2 pellets. open circles are for pellet 750c5h and closed circles are for pellet 780c5h. 750c5h 780c5h temperature (k) 20 30 40 50 60 70 80 r es is ta nc e (o hm s) 0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 fig. 2 is a plot of the ac magnetic susceptibility of pellet 750c5h taken at a frequency of 800 hz. the onset of the diamagnetic transition in the figure is at fig. 2. ac magnetic susceptibility measurement plot of sintered mgb2 (750c5h) at 800hz. diamagnetic transition starts at 39k. s us ce pt ib ili ty (a .u .) -0.002 -0.004 -0.006 -0.008 -0.010 -0.12 temperature (k) 200 40 60 10080 3 9 k , c o n f i r m i n g t h e r e s u l t o f r e s i s t i v i t y measurements. in addition, the diamagnetic transition is noticeably sharp, consistent with the results of resistivity measurements. the x-ray diffraction scan of pellet 750c5h is shown in fig. 3. in the figure, the xrd pattern of the pellet is compared to the starting powder. from the figure, no significant difference between the sintered sample and the starting powder was observed. this implies that the sintered pellet is stoichiometrically mgb2. lastly, the morphology of the sintered pellets is shown in the sem micrographs depicted in figs. 4 and 5. fig. 4 shows the sem image of sample 750c5h. the synthesis of bulk superconducting magnesium 19 alfa aesar 750c5h (1 02 )(1 10 ) (0 02 ) (1 01 ) (1 00 ) fig. 3. x-ray diffractometry plot of sintered mgb2 pellet 750c5h (light line) compared to that of the starting powder (dark line). in te ns ity ( a. u. ) 2000 1500 1000 500 0 temperature (k) 255 45 65 750c5h alfa aesar (1 00 ) (1 01 ) (0 02 ) (1 10 ) (1 02 ) fig. 5. scanning electron micrograph of sintered mgb2 pellet at 8000x (780c5h). fig. 4. scanning electron micrograph of sintered mgb2 pellet at 8000x (750c5h). micrograph shows a relatively flat surface of compacted grains with small grain sizes. note also the apparent black color of the grains (the color of mgb2). fig. 5, on the other hand, shows the sem image of sample 780c5h. the micrograph this time shows a rougher surface with larger grains. also, the apparent color of the grains is generally whiter or lighter in color than those shown in fig. 4, which may be attributed to more mgo present in this sample. however, edx analysis showed the presence of mgo on both samples. thus, the apparent color in the sem micrographs is not a reliable measure of mgo content of the samples. therefore, the higher transition temperature of sample 750c5h is not due to the presence or absence of mgo in that particular sample. instead, it is more likely that the smaller grain size observed in sample 750c5h plays a more crucial role in its superconductivity. conclusions bulk superconducting samples of magnesium diboride have been prepared via a single sintering step at 750°c in a pure ar atmosphere. the sintered pellet exhibit a tc of 39.4k, confirmed by both resistivity and ac magnetic susceptibility measurements. a sharp transition width, approximately 5k, indicates good sample quality of the sintered pellet, which was later confirmed by x-ray diffractometry and scanning electron microscopy. references buzea, c. & t. yamashita, 2001. preprint. review of superconducting properties of mgb2. cond-mat/0108265. dou, s.x., x.l. wang, j. horvat, d. milliken, e.w. collings, & m.d. sumption, 2001. preprint. flux jumping and a bulkto-granular transition in the magnetization of a compacted and sintered mgb2 superconductor. cond-mat/0102320. frederick, n.a., s. li, m.b. maple, v.f. nesterenko, & s.s. indrakanti, 2001. preprint. improved superconducting properties of mgb2. cond–mat/0106518. hirsch, j. e., 2001. preprint. hole superconductivity in mgb2, cuprates, and other materials. cond-mat/0106310. indrakanti, s.s., v.f. nesterenko, m.b. maple, n.a. frederick, w.m. yuhasz, & s. li, 2001. preprint. hot isostatic pressing of bulk magnesium diboride: mechanical and superconducting properties. cond-mat/0105485. larbalestier, d.c., m.o. rikel, l.d. cooley, a.a. polyanskii, j.y. jiang, s. patnaik, x.y. cai, d.m. feldmann, a. gurevich, a.a. squitieri, m.t. naus, c. b. eom, e.e. hellstromn, r.j. cava, k.a. regan, n. rogado, m.a. hayward, t. he, j.s. slusky, p. khalifah, k. inumaru, & m. haas, 2001. strongly linked current flow in polycrystalline forms of the superconductor mgb2. nature. 410:186-189. nagamatsu, j., n. nakagawa, t. muranaka, y. zenitani, & j. akimitsu, 2001. superconductivity at 39k in mgb2. nature. 410:63-64. patnaik, s., l.d. cooley, a. gurevich, a.a. polyanskii, j. jiang, x.y. cai, a.a. squitieri, m.t. naus, m.k. lee, j.h. choi, l. belenky, s.d. bu, j. letteri, x. song, d.g. schlom, s. e. babcock, c. b. eom, e.e. hellstrom, & d. c. larbalestier, 2001. electronic anisotropy, magnetic field-temperature phase diagram and their dependence on resistivity in c-axis oriented mgb2 thin films. supercond.sci.technol. 14:1-5. rogado, n., m.a. hayward, k.a. regan, y. wang, n.p. ong, j.m. rowell, & r.j. cava, 2001. preprint. low-temperature fabrication of mgb2. cond-mat/0107534. shields, t.c., k. kawano, d. holdon, & j.s. abell, 2001. preprint. microstructure and superconducting properties of hot isostatically pressed mgb2. cond-mat/0107034. zhao, y.g., x.p. zhang, p.t. qiao, h.t. zhang, s.l. jia, b.s. cao, m.h. zhu, z.h. han, x.l. wang, & b.l. gu, 2001. preprint. influence of the starting composition on the structural and superconducting properties of mgb2 phase. cond-mat/0105053. zhu, y., l. wu, v. volkov, q. li, g. gu, a. r. moodenbaugh, m. malac, m. suenaga, & j. tranquada, 2001. preprint. microstructure and structural defects in mgb 2 superconductor. cond-mat/0105311. 20 olbinado, guerra, and sarmago 02_sarmiento 6 sarmiento et al. abstract electron traps in gaas grown by molecular beam epitaxy on on-axis (100) and off-axis substrates r. sarmiento1*, a. somintac2, l. guiao2, f. agra2, and a. salvador2 1university of san carlos 6000 cebu city, philippines 2condensed matter physics laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines email: raymund@nip.upd.edu.ph deep level transient spectroscopy (dlts) was used to characterize the electron traps present in the bulk gaas grown by molecular beam epitaxy (mbe) on on-axis (100) and off-axis (4° towards the (111) a direction) substrates. two electron traps were obtained for each sample having identical corresponding peak locations in the dlts spectra. the layer grown on the on-axis substrate has electron traps with activation energies of e c –0.454 ev and e c –0.643 ev and capture cross-sections of 1.205 x 10-14 cm2 and 3.88 x 10-15 cm2, respectively. the layer grown on the off-axis substrate has traps with activation energies of e c –0.454 ev and e c –0.723 ev and capture cross-sections of 2.060 x 10-14 cm2 and 4.40 x 10-14 cm2. the electron traps are possibly the m4 (or el3) and el2 (or eb4) traps commonly found in gaas layers. due to the high trap concentrations obtained and to the non-uniform trap concentration profile, as desorption may be considerable during growth. introduction molecular beam epitaxial (mbe) growth of gaas started in the 1970s and film characterizations such as x-ray diffraction, hall measurements, and various optical methods have since followed to determine the quality of the grown layers. another very useful technique for layer quality evaluation is deep level transient spectroscopy (dlts) (lang, 1974) first developed by lang et al. as defects in the crystal structure may form deep levels in the forbidden gap, dlts could be used to measure the activation energy and apparent capture cross-section of carrier traps. it could also determine the concentrations of the traps in the semiconductor. mbe-grown gaas was found to have nine different traps labeled m0 to m8 depending on the growth conditions used (farrow, 1995; blood & harris, 1984).traps m1, m3, and m4 occur reproducibly for ngaas grown under the (2x4) asrich conditions (xu et al., 1987; blood & harris, 1984; gombia et al., 1994). it has also been found that optical, electron transport, and morphological epilayer properties were highly improved when (100) gaas substrates misoriented by a few degrees toward the (111) a direction were used (blood & harris, 1984; radulescu et al., 1987). it is the intent of this work to compare traps found in the gaas layers grown on an on-axis (100) substrate and on an off-axis substrate. science diliman (january-june 2003) 15:1, 6-10 * corresponding author 7 electron traps in gaas theory a reverse bias varies the width of the space-charge region in a metal n-type semiconductor schottky contact so that its capacitance changes. if deep levels are present, traps can be depleted and filled with electrons of different amounts of reverse bias applied. the very small change in capacitance (transient) due to the filling and emission of electrons from the trap sites is detected. assuming that the traps are initially filled, its emission rate then is (schroder, 1990): (1) where t is the absolute temperature, k is the boltzmann’s constant, e c is the conduction band edge, e t is the trap level, and σ n is the apparent capture cross-section of the trap. the parameter γ n = (v th / t1/2)(n c /t3/2) where v th is the thermal velocity of electrons and n c is the effective density of states in the conduction band. the emission rate is the inverse of the time constant, τ e , of the capacitance transient which, in turn, is known to obey the relation (schroder, 1990): (2) where c 0 is the capacitance at the original reverse bias, n t (0) is the original number of electrons per unit volume occupying the trap level during the transient, n d is the doping concentration, and t is the time. hence, if the doping concentration is known, n t (0) can be obtained. for a sufficient filling pulse, this can be taken as the trap concentration n t . the activation energy e act = e c – e t , and capture crosssection σ n along with the trap concentration n t and temperature peak location in the dlts spectrum comprise the signature of the particular trap present in the semiconductor. experimental method one and a half micron (1.5 µm) gaas was grown by mbe on the on-axis and off-axis substrate simultaneously in order to have identical growth conditions. the films were doped with si to obtain doping concentrations of around 1017 cm-3. a typical growth temperature of around 630°c and beam fluxes of the as 4 and ga that would give a (2x4) surface reconstruction were utilized. schottky barrier diodes (sbds) were made out of the samples by deposition of 1 mm-diameter aluminum dot schottky contacts on the top layer and indium as backside ohmic contact. after obtaining the doping concentrations, dlts characterization was performed on the two samples using a dls-83d apparatus made by semilab, hungary. different excitation frequencies were used for temperature scans of 77 k to 400 k each. this makes the dlts signal (proportional to the change in capacitance and, therefore, to the emission rate) peak at different temperatures. an emission rate-temperature data pair could then be obtained. from eq. (1), necessary trap parameters e act and σ n could be calculated. the height of the dlts peak is related to n t from eq. (2). depth profiling of the deep level was obtained by choosing a frequency-temperature data pair, which belongs to the given deep level. by using two reverse bias pulses of different heights, the depth region could be selected. these two pulses would be changed step by step while the difference of the pulse heights was kept constant during the measurement. the dlts signal coming from the selected deep level would, thus, be plotted as a function of depth. results and discussion the dlts spectra for the samples grown on the onaxis substrate (l047-on) and on the off-axis substrate (l047-off) are shown in fig. 1. a peak is assumed to be due to one kind of trap. the emission ratetemperature data pair for each peak can be plotted in an ln(e n /t2 ) vs 1/t axis called an arrhenius plot. those of peaks p1 are shown in fig. 2 where least-square lines were fitted. from eq. (1), it can be shown that the slope of the line is e act /k and the y-intercept is just ln(γ n σ n ). table 1 shows the obtained trap parameters along with the possible identities of the traps based from the work of others. finally, fig. 3 shows the depth profiles for trap p1 for l047-on. that for l047-off is very similar. 2/ γ σ −⎡ ⎤= −⎢ ⎥ ⎣ ⎦ c t n n n e e e t exp kt (0) 1 exp 2 ⎡ ⎤⎛ ⎞ = − −⎢ ⎥⎜ ⎟ ⎝ ⎠⎣ ⎦ t o d e n t c c n τ 8 sarmiento et al. it can be seen in table 1 that there is not much difference between the trap parameters of p1 for l047-on and l047-off. due to the difficulty of resolving the arrhenius plots for p2, the e act and σ n , data shown in table 1 are obtained from dlts measurements of the substrates as the second deep level was observed to be present also in the substrates. the p2 data for l047-off are more accurate since there were 3 scans (not shown fig. 1. dlts spectra of (a) l047-on and (b) l047-off at frequencies: (i) 2500 hz; (ii) 250 hz; (iii) 25 hz; and (iv) 2.5 hz. a quiescent reverse bias/ filling pulse of -3v/-1v was used for all the measurements. excitation pulse widths were varied. 0 -20 -40 -60 -80 -100 d l s ( m u ) 100 150 200 250 350 400300 temperature (k) 100 0 -20 -40 -60 -80 d l s ( m u ) 150 200 250 400300 temperature (k) (a) (b) 350 -3 -4 -5 -6 -7 ln ( e /t 2 ) 3.4 3.6 3.8 4 4.4 4.64.2 1000/t (1/k) -8 -9 -3 -4 -5 -6 -7 ln ( e /t 2 ) -8 -9 3.6 3.8 4 4.44.2 1000/t (1/k) fig. 2. arrhenius plots for p1 in the dlts spectra for (a) l047-on and (b) l047-off at frequencies (i) 2500 hz; (ii) 250 hz; (iii) 25 hz; and (iv) 2.5 hz. (a) (b) 9 electron traps in gaas here) performed. due to the closeness of the peaks p1 and p2 for the onand off-axis samples, the corresponding traps were assumed to be of the same chemical origin. the first electron trap with a lower temperature peak corresponds to either the trap m4 (blood & harris, 1977) found in mbe-grown gaas or the trap el3 (martin et al., 1977) commonly found in lpe-grown samples. m4 was identified to be an impurity-related trap involving as vacancies (blood & harris, 1984). if this is the trap observed, then some mechanism must have been desorbing much of the as atoms during growth. this might explain the high rap concentrations obtained which were only an order of magnitude lower than the doping concentrations. the substrate temperature might not be accurate since the thermocouple is behind the molybdenum block substrate holder. this is supported by the non-uniform distribution of this trap as seen in fig. 3. table 1. trap parameters for the deep levels of l047-on and l047-off and their possible identities. sample trap label peak location (k)+ activation energy, e act (ev) capture crosssection, σσσσσ n (cm2) trap concentration, n t (cm-3) possible trap identity l047-on l047-off p1 p2 p1 p2 272.1 382.8 265.1 382.1 0.454 + 0.001 0.643 + 0.028 0.514 + 0.006 0.723 + 0.010 (1.205+0.035) x 10-14 (3.88+2.55) x 10-15 (2.060+0.560) x 10-14 (4.40+1.47) x 10-14 4.37 x 1016 1.70 x 1016 1.97 x 1017 5.15 x 1016 el3, m4 el2, eb4 el3, m4 el2, eb4 +peak temperature measured at a scan of 250 hz. the second electron trap observed is either the trap el2 (martin et al., 1977) or eb4. if it is el2, which is an as antisite-related (asga-x) trap, then this explains the earlier depletion of as. some of the as atoms that did not desorb from the grown layers might have replaced some of the ga atoms in the lattice. conclusions at the growth conditions used, there seems to be no difference in trap incorporation in gaas films grown on on-axis (100) and off-axis (tilted 4° towards the (111)a direction) substrates. two traps are present in the bulk samples possibly corresponding to m4 or el3 and el2 or eb4, respectively. as m4 0.0 0.5 1.0 1.5 2.0 0 0.5 1 1.5 2 n tr a p x e 1 7 depth (µµµµµm) xe-1 fig. 3. depth profile of electron trap p1 for l047-on. is an impurity-related trap involving as vacancies, the growth temperature might be higher than expected and as desorption are not uncommon. it is likely that a fraction of as atoms that produced the vacancies occupied ga sites in the lattice producing the el2 defect. acknowledgments we thank the philippine council for advanced science and technology research and development (pcastrd) of the department of science and technology (dost) for supporting this research. we also thank mr. gil nonato santos of de la salle university for the metal contacts of some of our samples. 10 sarmiento et al. references blood, p. & j.j. harris, 1984. deep states in gaas grown by molecular beam epitaxy. j. appl. phys. 56(4): 993-1007. farrow, r.f.c. (ed.), 1995. molecular beam epitaxy: applications to key materials. noyes publications. gombia, e., r. mosca, a. bosacchi, m. madella, & s. franchi, 1994. deep levels in gaas grown by atomic layer molecular beam epitaxy. appl. phys. lett. 65(22): 2848-2850. lang, d.v., 1974. deep-level transient spectroscopy: a new method to characterize traps in semiconductors. j. appl. phys. 45(7): 3023-3032. martin, g.m., a. mitonneau, & a. mircea, 1977. electron traps in bulk and epitaxial gaas crystals. electron. lett. 13: 191. radulescu, d.c., w.j. schaff, g.w. wicks, a.r. calawa, & l.f. eastman, 1987. influence of an intentional substrate misorientation on deep electron traps in algaas grown by molecular beam epitaxy. appl. phys. lett. 51(26): 2248-2250. schroder, d.k., 1990. semiconductor material and device characterization. john wiley & sons, inc. xu, h.d., t.g. andersson, & j.m. westin, 1987. relation between growth conditions and deep levels in gaas grown by molecular beam epitaxy. j. appl. phys. 62(5): 2136-2137. sd122-c3 economic modeling of residual generation 17science diliman (july december 2000) 12:2, 17-27 abstract economic modeling of residual generation for the lingayen gulf watershed douglas h. mcglone and herminia r. caringal marine science institute, college of science, university of the philippines velasquez st., u.p. campus, diliman, quezon city 1101 philippines tel. no. (632) 922-3921; telfax: (632) 924-7678 the philippines is one of four countries involved in the southeast asian core project of loicz (land ocean interactions in the coastal zone) which has among its general goals the determination of how changes in human activities affect the fluxes of materials between land, sea, and atmosphere through the coastal zone. the economic component of the philippine project addresses the question: “how does a change in economic activity affect coastal waters?” of particular concern is the introduction of anthropogenically-derived residuals (n, p, c, ss) into coastal waters. a regional input-output (io) model for region 1 of the philippines has been developed to estimate how projected changes in economic activity may affect residual flows into lingayen gulf. a residual coefficient matrix, derived from information obtained with a rapid assessment model (ra) of residual generation in the lingayen gulf watershed, has been incorporated into the io model. such a model allows for analysis of various economic scenarios for the region, with projections of residual generation as the output. the resulting changes in residual flows may then serve as inputs to biogeochemical models of lingayen gulf. from this process, the impact of various economic scenarios on the water quality of lingayen gulf may be ascertained. this paper discusses and compares the ra and io models of residual generation for the lingayen gulf watershed and provides examples of the scenario analysis process. keywords: economic modeling, lingayen gulf, rapid assessment watershed, residual generation introduction changes in land use, climate, sea level, and human activities alter the fluxes and retention of nutrients in the coastal zone. how these changes can be quantified or determined is a general goal of the loicz (landocean interactions in the coastal zone). this project has devised a methodology for creating simple biogeochemical budget models for coastal waters (gordon and others 1996), with the ten-year objective of obtaining enough of these budgets throughout the world to estimate the net flux of carbon from coastal waters to the deep ocean waters. a complicating factor in the biogeochemical modeling of residual fluxes in coastal waters is that a substantial portion of the residuals entering these waters are generated by economic activity within the relevant watershed. as economic activity changes, so will the amount of residuals entering the coastal waters. thus, mcglone & caringal 18 changing economic activity has the potential of altering the overall flux of carbon from coastal to deep ocean waters. this paper describes the basic economic submodel for the philippines. residual generation models how will a change in economic activity affect the flow of residuals (c, n, p, ss) into coastal waters? a methodology is needed that is generally applicable and available across a wide variety of sites. for many of these sites, data is scarce in a number of areas. with these factors in mind, we begin with rather simple models. for site-specific purposes, these models may be expanded as data allows. a regional economic activity model may be used to estimate the generation of residuals (james 1985). in its simplest form, residual discharges are given as: r = cx (1) where r = a matrix of residual discharges (residual type by economic activity) c = a matrix of residual discharge coefficients with ckj = the quantity of residual k per unit of sectoral activity j x = a diagonal matrix of sectoral activity levels. total discharges of each residual type are then given by: r = rs = cx (2) where r = a vector of residuals by type (summed across all activities) s = a summation vector. so, each element of the column vector r represents the sum of the corresponding row in the r matrix. that is, the total discharge of residual type k is the sum of each activity’s discharge of residual k. in the above formulation, x is simply an exogenous estimation of output for each activity in the region. the model may be expanded by allowing economic activity to be represented by a regional input-output model. in such a model, production (x), or supply, is equated to the sum of intermediate (inter-industry) demand (ax) and final demand (y): x = ax + y (3) with a = [aij] where aij is the leontif io technical coefficient and aij = zij/xj, where zij is the monetary value of the input flow (3a) from sector i to sector j manipulation of equation 3 yields x = (i – a)-1 y (4) where (i – a)-1 represents the leontif inverse matrix. substituting equation 4 into equation 2 gives r = c (i – a)-1 y (5) the total change in residual generation brought about by a change in one or more components of final demand are determined by ∆r = c (i – a)-1 ∆y (6) in equations 5 6, matrix r represents the amount of residuals generated during both direct activities and the indirect “support” activities. for example, if fish aquaculture is the direct activity being addressed, agricultural activity may be considered an indirect or support activity, since aquaculture feeds are often derived from agricultural output. thus, an increase in aquaculture may increase nitrogen loading into coastal waters not only from the application of feeds, but also by increased use of fertilizers in the agricultural sector. economic modeling of residual generation 19 equations 2 and 5 6 represent two alternative but related approaches to addressing the question of how economic activity affects the generation of residuals. equations 5-6 represent the input-output (io) modeling approach, which has often been employed by economists in the past 30 years to describe residual flows (forsund and strom 1976, mendoza 1994, orbeta and others 1996, miller and blair 1985). equation 2 represents the rapid assessment (ra) method utilized by who (economopoulos 1993), which may readily be incorporated into a geographical information systems (gis) modeling approach such as that discussed in turner et al. (1997). each approach has its strengths and weaknesses. a favorable aspect of the io approach is that it captures the interrelationships between sectors of the economy. a change in activity of one sector typically requires changes in activity in other sectors. these interrelations are not captured in the ra modeling approach, and thus, may lead to an underestimation of residual discharges. on the other hand, data constraints typically require a considerable degree of aggregation of economic sectors in regional io models. an ra/gis model allows for a considerable degree of disaggregation, and allowance for consideration of spatial relationships. these relationships may be of particular importance when taking account of environmental assimilation of residuals. input-output model a 12-sector (with the household sector endogenized) regional io model was created for region i of the philippines. this regional model was based on the 229sector 1994 national model, adjusted using the simple location quotient method of reduction (secretario 1999). highlights of the input-output model components this section gives a brief description of the development and the components of the regional io model. table 1 identifies the 11 production sectors and the household sector as used in the model. table 2, the 1994 regional 12 x 12 sector io table for region 1 of the philippines, (rows 1-12 and columns 1-12, collectively referred to as the production sector), shows the inter-industry flow of goods (including an endogenized household sector). such an inter-industry transactions table is derived from a larger set of income and production accounts for a region (secretario 1999). each element of the transactions table (the zij terms of equation 3a, expressed in monetary units) represents the sale (supply) of sector i’s outputs to sector j for use as inputs to j’s production process. thus, by reading row 1 it is seen that sector 1 supplies an amount of 2,399,734 to sector 1 (itself), 200 to sector 2, 20 to sector 3, etc. the columns show the amount each sector purchases (demands) from all other sectors. thus, sector 2 demands (or buys) an amount 200 from sector 1; 149,876 from itself; 0 from sector 3; etc. such demand of one production sector for the output of other producing sectors for use as inputs is termed intermediate demand, and is represented by the “ax” vector in equation 3. table 2 is an account of the amounts that each sector demands from other sectors in order to satisfy their own production processes. that is, the inter-industry transactions table (in particular, the columns) represents intermediate demands. the values for the ax vector are determined by the summation of each row sector. thus, the right-most column, labeled “total intermediate demand,” is the ax column vector of equation 3. the column sum of each production sector is termed “total intermediate inputs.” table 2 can be expanded in a variety of ways. first, there are inputs to the production process that must be paid for, other than those produced by other industries. the primary example of these value-added items is employee compensation. for the table 1. sectoral description (ilocos region i/o table) code description 1 2 3 4 5 6 7 8 9 10 agriculture fishery forestry mining and quarrying manufacturing i manufacturing ii electricity, gas and water waterworks and supply construction transportation, communication and storage mcglone & caringal 20 c o d e 1 2 3 4 5 6 7 8 9 10 11 12 ti d 1 2 3 4 5 6 7 8 9 10 11 12 ti i n h o a tp i ti 23 99 73 4 35 5 0 42 4 10 12 23 2 15 90 26 8 14 15 34 43 65 64 31 35 42 29 74 39 45 92 80 96 0 15 53 44 77 81 87 86 8 81 87 86 8 23 72 23 45 20 0 14 98 76 0 26 17 83 97 25 26 89 26 81 1 0 11 33 2 57 70 5 62 34 0 10 06 35 3 17 45 72 9 11 51 55 6 11 51 55 6 28 97 28 5 20 0 19 8 0 15 19 64 47 0 24 10 28 67 7 15 16 3 19 13 6 16 16 19 15 2 0 2 34 34 0 18 16 9 18 93 95 49 00 43 12 44 9 12 83 7 49 01 4 24 29 78 53 32 21 40 78 50 40 78 50 94 10 71 32 16 44 24 34 8 11 51 5 29 8 58 09 05 30 75 13 81 81 1 11 75 2 24 84 6 86 84 0 30 25 84 46 39 76 22 18 03 2 84 83 47 84 83 47 30 66 37 9 29 42 6 58 36 55 25 08 47 25 09 94 16 75 04 3 30 38 96 18 1 64 53 1 13 29 22 39 76 75 47 23 64 35 81 59 2 16 65 25 6 16 65 25 6 52 46 84 8 0 0 57 8 17 19 74 6 13 29 09 10 47 9 0 47 88 0 40 73 6 83 56 1 27 55 16 18 70 65 18 70 65 46 25 81 0 0 2 0 26 2 52 89 47 45 0 15 79 47 8 29 32 12 36 6 27 65 3 16 11 0 16 11 0 43 76 3 0 0 13 00 22 54 40 35 76 65 33 73 16 5 10 17 83 11 38 3 30 93 22 59 29 56 99 58 23 13 04 46 3 72 73 30 0 24 12 55 1 24 12 55 1 96 85 85 1 0 0 1 19 64 55 73 07 50 38 25 9 75 22 59 06 8 60 97 2 44 94 27 88 94 17 22 41 89 0 80 68 45 80 68 45 30 48 73 5 98 98 6 49 33 3 1 15 91 71 98 63 11 26 51 0 62 14 18 85 95 4 95 56 12 77 68 79 25 45 19 0 12 09 13 16 18 92 43 33 19 18 96 24 19 18 96 24 57 33 52 30 23 90 32 1 17 53 26 7 0 0 19 75 17 59 53 47 77 4 13 29 91 3 23 87 03 75 46 8 47 08 61 3 19 91 50 57 0 55 51 08 75 0 0 55 51 08 75 28 50 01 0 22 39 72 20 68 4 48 03 64 31 25 70 3 93 85 49 5 13 35 68 3 12 12 00 14 49 98 2 20 76 84 6 55 90 34 3 25 86 29 17 52 52 31 99 25 27 82 76 25 27 82 76 77 80 14 75 n ot e: t ii: to ta l i nt er m ed ia te in pu ts tp i: to ta l p rim ar y in pu ts tp i = n h o a ti = ti i + t p i n h o a : n on -h ou se ho ld o pe ra tin g a ct iv iti es ti : to ta l i np ut s ti d = to ta l i nt er m ed ia te d em an d ta bl e 2. s ec to r t ra ns ac tio n ta bl e, 1 99 4 (i lo co s r eg io n, in th ou sa nd p es os ) economic modeling of residual generation 21 purposes of this model, other categories are lumped together under operating surpluses. this collection of inputs is known as the payments, or value-added sector. a second point of expansion for table 2 is to include the final demand sectors (table 3). final demands are demands derived from sources outside the production sector of the region. final demand sectors include personal consumption expenditures of households (pce), government consumption expenditures (gce), business investment (gross fixed capital formation, gfcf), and net exports (e-m) to other regions (both domestic and international). an adjustment for changes in stock inventory (cs) is also included. final demands are summed up across rows to give the total final demand column vector (tfd in table 3), denoted as ‘y’ in equation 3. adding the intermediate and final demand column vectors gives the total output (to) column vector (the right-most column of table 3, for sectors 1-12), denoted as ‘x’ in equation 3. table 4 is the technical coefficient matrix, represented by the matrix ‘a’ in equation 3. to derive this matrix, each of the zij elements of table 2 is divided by the appropriate column sum xj, as shown in equation 3a. the column sums xj are represented in table 2 by the total input (ti) row. it should be noted that the column sum xj is the sum of all inputs – those of both – the production and payment sectors. the technical coefficient aij may be interpreted as the (currency unit’s) worth of sector i input per (currency unit)’s worth of output of sector j. the technical coefficients are viewed as representing a fixed relationship between a sector ’s outputs and its inputs. if technology changes, then the values for the technical coefficients will change. an alternative definition of the technical coefficient is that it indicates the portion of a column sector j’s input demand that is provided for by row sector i. thus, sector 1 (agriculture) provides 10% of it’s own input demand. the vector and matrix requirements of equation 3 are now provided for. to gain the form of equation 3, the leontif inverse matrix (i – a)-1 is created (table 5). the elements of the leontif inverse are known as output multipliers. each element indicates the value of the change of a row sector’s output due to a unit change in final demand for the column sector’s output. this may be seen by rearranging equation 4 to give dx / dy = (i – a)-1 (7) a low column sum reveals a weak sectoral interlinkage; otherwise, it shows a sector ’s strong dependence on the other sectors’ output to meet a unit increase in final demand for its output. the sector with the largest multiplier provides the largest total impact on the economy. note: pce: private consumption expenditures e-m: net export gce: government consumption expenditures tfd: total final demand gfcf: gross fixed capital formation to: total output cs: change in stocks code pce gce gfcf cs e m tfd to 1 2 3 4 5 6 7 8 9 10 11 12 tii nhoa tpi ti 2390321 1753267 0 0 19751759 5347774 1329913 238703 75468 4708613 19915057 0 55510875 0 0 55510875 0 0 0 0 0 0 0 0 0 0 6704644 0 6704644 0 0 6704644 1983520 0 0 0 0 1235028 0 0 5809729 229882 536392 0 9794551 0 0 9794551 177951 0 0 11790 166076 21615 0 0 0 0 0 0 377432 0 0 377432 16320543 920046 -1532 448917 -19977159 -10743064 -2203015 -316140 2350672 -3966606 -4078971 0 -21246309 0 0 -21246309 20872335 2673313 -1532 460707 -59324 -4138647 -873102 -77437 8235869 971889 23077122 0 51141193 0 0 51141193 23722345 2897285 19152 941071 3066379 5246848 462581 43763 9685851 3048735 28667465 25862917 103664392 25278276 25278276 table 3. 12 sector transaction table (ilocos region 1994, in thousand pesos) mcglone & caringal 22 1 2 3 4 5 6 7 8 9 10 11 12 to m c o d e d es cr ip ti o n 1 2 3 4 5 6 7 8 9 10 11 12 a gr ic ul tu re f is he ry f or es tr y m in in g an d qu ar ry in g m an uf ac tu rin g i m an uf ac tu rin g ii e ke ct ric ity , ga s & w at er w at er w or ks & s up pl y c on st ru ct io n t ra ns po ., co m m & st or ag e o th er s er vi ce s h ou se ho ld s ec to r ta bl e 4. 1 2 se ct or te ch ni ca l c oe ff ic ie nt ta bl e (i lo co s re gi on 1 99 4, in th ou sa nd p es os ) 1 2 3 4 5 6 7 8 9 10 11 12 0. 10 11 59 0. 00 00 15 0. 00 00 00 0. 00 00 18 0. 04 26 70 0. 06 70 37 0. 00 59 66 0. 00 01 84 0. 00 02 71 0. 01 49 32 0. 03 13 61 0. 39 12 33 0. 00 00 69 0. 05 17 30 0. 00 00 00 0. 00 00 09 0. 06 15 74 0. 08 72 16 0. 00 92 54 0. 00 00 00 0. 00 39 11 0. 01 99 17 0. 02 15 17 0. 34 73 43 0. 00 10 44 0. 00 00 00 0. 01 03 38 0. 00 00 00 0. 00 07 83 0. 10 25 48 0. 00 24 54 0. 00 00 00 0. 00 12 53 0. 05 36 76 0. 03 53 49 0. 79 17 19 0. 00 00 00 0. 00 00 02 0. 00 36 49 0. 00 00 00 0. 01 93 07 0. 20 12 55 0. 00 52 07 0. 00 00 46 0. 01 32 29 0. 01 36 41 0. 05 20 83 0. 25 81 93 0. 10 48 94 0. 00 79 40 0. 00 37 55 0. 00 00 97 0. 18 94 43 0. 10 02 85 0. 02 66 80 0. 00 38 33 0. 00 81 03 0. 02 83 20 0. 09 86 78 0. 15 13 11 0. 00 56 08 0. 00 00 11 0. 00 06 97 0. 04 78 09 0. 04 78 37 0. 31 92 47 0. 05 79 20 0. 00 00 34 0. 01 22 99 0. 02 53 34 0. 07 57 93 0. 09 00 28 0. 00 00 00 0. 00 00 00 0. 00 12 50 0. 00 37 16 0. 00 16 13 0. 28 73 20 0. 02 26 53 0. 00 00 00 0. 01 03 51 0. 00 00 00 0. 08 80 62 0. 18 06 41 0. 00 00 00 0. 00 00 00 0. 00 00 46 0. 00 00 00 0. 00 59 87 0. 12 08 56 0. 10 84 25 0. 00 00 00 0. 03 60 81 0. 01 09 22 0. 06 69 97 0. 28 25 67 0. 00 00 00 0. 00 00 00 0. 00 01 34 0. 02 32 75 0. 03 69 27 0. 34 82 57 0. 01 05 08 0. 00 11 75 0. 03 19 35 0. 06 12 19 0. 10 28 12 0. 13 46 77 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 06 0. 00 21 17 0. 23 96 90 0. 01 25 49 0. 00 24 67 0. 01 93 75 0. 01 99 99 0. 14 74 14 0. 29 17 33 0. 00 17 26 0. 00 08 60 0. 00 00 00 0. 00 00 28 0. 01 25 55 0. 01 96 48 0. 01 08 38 0. 00 14 99 0. 01 66 67 0. 01 35 50 0. 04 43 91 0. 21 08 88 0. 04 30 60 0. 03 15 84 0. 00 00 00 0. 00 00 00 0. 35 58 18 0. 09 63 37 0. 02 39 58 0. 00 43 00 0. 00 13 60 0. 08 48 23 0. 35 87 60 0. 00 00 00 c o d e d es cr ip ti o n a gr ic ul tu re f is he ry f or es tr y m in in g an d qu ar ry in g m an uf ac tu rin g i m an uf ac tu rin g ii e ke ct ric ity , ga s & w at er w at er w or ks & s up pl y c on st ru ct io n t ra ns po ., co m m & st or ag e o th er s er vi ce s h ou se ho ld s ec to r to ta l o ut pu t m ul tip lie r ta bl e 5. 1 2 x 1 2 le on tie f in ve rs e m at ri x (t ot al o ut pu t m ul tip lie r) 1 2 3 4 5 6 7 8 9 10 11 12 1. 19 76 79 0. 02 76 19 0. 00 19 74 0. 01 88 57 0. 40 92 52 0. 37 80 36 0. 06 55 46 0. 00 57 79 0. 02 00 56 0. 10 95 71 0. 40 70 30 0. 71 45 32 3. 35 6 0. 07 92 94 1. 07 91 75 0. 00 19 23 0. 01 93 69 0. 39 32 99 0. 38 75 41 0. 06 61 43 0. 00 50 77 0. 02 29 88 0. 10 59 49 0. 36 13 04 0. 63 03 72 3. 15 2 0. 13 80 01 0. 04 76 25 1. 01 34 39 0. 03 01 40 0. 61 29 23 0. 60 45 42 0. 09 89 84 0. 00 93 78 0. 03 38 02 0. 21 26 86 0. 67 41 61 1. 25 71 44 4. 73 3 0. 06 14 91 0. 01 94 89 0. 00 53 13 1. 02 54 10 0. 28 73 39 0. 51 01 88 0. 06 21 59 0. 00 41 06 0. 03 21 47 0. 08 77 20 0. 34 06 52 0. 50 33 44 2. 94 0 0. 20 35 82 0. 02 99 87 0. 00 62 10 0. 02 17 68 1. 50 50 37 0. 43 04 60 0. 08 81 48 0. 00 89 53 0. 02 99 76 0. 11 09 08 0. 41 89 86 0. 51 02 87 3. 36 4 0. 06 22 75 0. 01 52 93 0. 00 27 41 0. 08 31 58 0. 28 10 44 1. 71 17 26 0. 12 51 71 0. 00 35 70 0. 03 51 40 0. 09 46 90 0. 34 40 71 0. 37 84 82 3. 13 7 0. 05 23 99 0. 01 64 01 0. 00 28 31 0. 03 43 85 0. 23 63 37 0. 61 96 69 1. 08 28 61 0. 00 34 87 0. 03 00 99 0. 06 80 07 0. 35 02 71 0. 42 32 20 2. 92 0 0. 06 43 70 0. 02 12 93 0. 00 17 88 0. 02 39 38 0. 29 28 50 0. 45 90 28 0. 16 69 97 1. 00 44 05 0. 05 71 50 0. 09 02 96 0. 38 48 53 0. 55 49 91 3. 12 2 0. 06 30 39 0. 01 80 29 0. 00 21 87 0. 06 21 02 0. 30 59 50 0. 77 63 73 0. 08 47 34 0. 00 53 75 1. 05 80 57 0. 14 22 82 0. 41 30 80 0. 45 28 23 3. 38 4 0. 07 06 12 0. 02 30 74 0. 00 18 62 0. 03 07 18 0. 31 81 65 0. 61 37 80 0. 08 15 76 0. 00 73 60 0. 04 37 00 1. 10 90 46 0. 49 48 31 0. 59 90 46 3. 39 4 0. 04 33 42 0. 01 47 69 0. 00 09 48 0. 00 93 53 0. 19 33 08 0. 17 78 94 0. 04 03 90 0. 00 43 44 0. 02 76 70 0. 06 14 34 1. 23 48 74 0. 36 11 10 2. 17 0 0. 15 56 72 0. 05 51 82 0. 00 31 96 0. 02 41 53 0. 69 63 15 0. 48 03 46 0. 09 65 21 0. 01 05 31 0. 03 16 80 0. 17 49 73 0. 70 67 72 1. 46 22 15 3. 89 8 economic modeling of residual generation 23 residual coefficient matrix. the basic components of the io model are now provided for. the next step in modeling residual generation with the io model is to create the residual coefficient matrix ‘c’ of equation 1. this first required the quantification of residual generation in the study site, and then applying the information to equation 3a. a rapid assessment (ra) approach was used to estimate residual generation in the lingayen gulf watershed. the first step in building ra model is to identify the relevant economic sectors for each residual type. the level of activity for each sector is determined, and then matched with the appropriate residual discharge coefficient, as in equation 2. residual discharge coefficients may be obtained from a variety of general sources, notably the world health organization (economopoulos 1993), or from specific studies of particular sites (e.g. hinga and others 1991, valiela and others 1997). when possible, however, discharge coefficients were obtained from local studies. padilla et al. (1997) proved to be a valuable starting point in obtaining discharge coefficients that could be applied to the lingayen gulf watershed. with appropriate data, a spatial dimension may be attached to each activity sector to allow a better account for assimilation of residuals as they pass from their point of origin, through the natural environment, and into the coastal waters. some studies (e.g. valiela and others 1997, hinga and others 1991, jaworski and others 1992) have accounted for assimilation by simply attaching fixed assimilation coefficients to each step of a residual’s movement through the environment. in this case the coefficient represents assimilation for an ‘average’ distance to the coastal waters. assimilation estimates may be incorporated directly into the residual coefficient (as in this study) or placed in their own distinct matrix. a detailed description of the creation of the residual coefficients may be found in the swol philippine core research site 1998 annual report. the resulting residual coefficient matrix is given in table 6. the set of total residual generation entering coastal waters is given in table 7. while the estimates of residual generation from the rapid assessment exercise are at best ‘guesstimates’, the quality of the estimates may be ascertained to some degree by comparing the values obtained with the results from biogeochemical modeling. the results shown in table 8 indicate the ambient concentrations of n, p, c, and ss in the water column, and the percentage of the ambient concentration that may be attributed to economic activity. the numbers appear reasonable. analogous to the concept of the output multiplier is that of the residual multiplier. the residual multiplier matrix m is given as: m = c (i-a)-1 (8) the elements of m = [mkj] show the amount of residual k generated for a one unit change in final demand in sector j. these residual multipliers are provided in table 9. as an example, in order to service a one unit (in the tables presented, one unit is equivalent to p 1,000 which was equivalent to about us $40.) increase in agricultural final demand, approximately 0.00057 metric tons (or 0.57 kg) of nitrogen will be discharged into coastal waters. the residual coefficient matrix is created from a preexisting estimate of residual generation. this is then allocated among the sectors of the io model. thus, the estimates of residual generation for the given levels of economic activity represented by the io model are as good as (in fact the same) the estimates provided by the ra exercise. it is hoped that, despite the high level of aggregation in the io models, estimates of changes in residual flows brought about by changes in economic activity will be better than estimates given by ra model. scenario analysis with the completion of the residual coefficient matrix, and using the model of equations 5 6, we are now prepared to perform some scenario analyses. scenario analysis may take the form of projecting changes in final demand (∆y in equation 6). the result will be an estimate of the overall change in residual generation brought about by the change in economic activity necessary to service the change in final demand. two scenarios are presented for the 12sector model (table 10) for the purpose of mcglone & caringal 24 d e s c r ip ti o n n p s s c ta bl e 9. r es id ua l m ul tip lie r m at ri x fo r 1 2 se ct or i/ o m od el 1 2 3 4 5 6 7 8 9 10 11 12 0. 00 05 73 0. 00 03 65 0. 14 00 97 0. 00 08 16 0. 00 01 25 0. 00 01 13 0. 01 05 48 0. 00 07 30 0. 00 01 92 0. 00 01 81 0. 01 83 27 0. 00 14 11 0. 00 00 81 0. 00 00 74 0. 03 05 56 0. 00 05 74 0. 00 01 70 0. 00 01 09 0. 02 53 30 0. 00 08 53 0. 00 00 70 0. 00 00 60 0. 00 98 96 0. 00 04 46 0. 00 00 69 0. 00 00 63 0. 00 76 83 0. 00 04 82 0. 00 00 88 0. 00 00 81 0. 00 89 30 0. 00 06 28 0. 00 00 79 0. 00 00 69 0. 00 96 85 0. 00 05 27 0. 00 00 96 0. 00 00 88 0. 01 00 62 0. 00 06 78 0. 00 00 70 0. 00 00 58 0. 00 81 18 0. 00 04 09 0. 00 02 20 0. 00 02 09 0. 02 03 34 0. 00 16 38 d es cr ip ti o m n p s s c ta bl e 6. 1 2 x 1 2 re si du al c oe ff ic ie nt m at ri x 1 2 3 4 5 6 7 8 9 10 11 12 0. 00 04 13 0. 00 02 34 0. 11 57 70 0 0. 00 00 00 0. 00 00 21 0. 00 00 19 0. 00 00 23 0. 00 00 03 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 02 20 30 0. 00 00 00 0. 00 00 23 0. 00 00 00 0. 00 02 08 0. 00 02 23 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 00 0. 00 00 12 0. 00 00 05 0. 00 23 11 1 0. 00 00 00 0. 00 00 88 0. 00 01 14 5 0. 00 00 00 0. 00 10 13 9 ta bl e 8. e st im at io n of to ta l m at er ia l c on ce nt ra tio ns in l in ga ye n g ul f a nd th os e co nt ri bu te d by e co no m ic a ct iv iti es m at er ia l d in di p ts s a m b ie n t co n ce n tr at io n c o n ce n tr at io n d er iv ed fr o m e co n o m ic a ct iv it ie s (% c o n st ri b u ti o n ) 3 0. 81 1 µm ol /l 0. 12 1 µ m ol /l 2. 5± 4 .5 m g/ l 6. 3 m g/ l2 0. 33 • m ol /l ( 41 ) 0. 04 • m ol /l ( 33 ) 2 .6 m g/ l (3 710 0) e co no m ic a ct iv it y h o u se h o ld a ct iv it ie s d om es tic s ew ag e s ol id w as te d et er ge nt s u rb an r un of f n o n -p o in t a g ri cu ltu ra l r u n o ff c ro p fe rt ili za tio n c ro pl an d er os io n l iv es to ck c om m er ci al p ig ge ry p ou ltr y a q u ac u lt u re e ro si o n m in in g to ta l ta bl e 7. e ff lu en ts p ro du ce d by e co no m ic a ct iv iti es in l in ga ye n g ul f ( in m et ri c to ns p er y ea r) c ar bo n/ b o d 5 s s n it ro g en p h o sp h o ru s 56 ,2 84 19 ,5 36 36 ,7 48 7, 87 6 64 ,1 60 66 ,2 53 2, 74 3, 59 2 2, 74 3, 59 2 2, 19 4 49 3 66 20 ,7 32 2, 83 0, 64 3 4, 91 2 4, 46 7 44 5 35 4 9, 70 6 5, 09 7 4, 60 9 83 71 12 61 .7 4, 60 9 19 ,7 25 .7 6, 17 6 2, 68 0 35 0 3, 14 6 14 9 1, 55 3 1, 55 3 69 69 5 5 8, 00 2. 0 economic modeling of residual generation 25 demonstrating the workings of the model, and making a comparison with results from a simpler rapid assessment (ra) approach. 1. 53% growth in the net export of agriculture, translates into a 20% growth in final demand for agriculture. final demand for all other sectors is held constant. table 11 shows that total output changes in all sectors. 2. 20% across-the-board growth in total final demands. this is a clear indication of the interrelationships present in the economy. the resulting changes in residual generation is shown in table 12. the rapid assessment model would estimate lower increases in each residual (the increase in agricultural output necessary to meet the increased final demand, multiplied by the residual coefficients for agriculture). the ra model would predict a 10% increase in n, a 7.1% increase in p, a 15.1% increase in ss, and no change in c. thus, the rapid assessment model would seem to underestimate residual generation by 28 % for n, 36 % for p, 17.4% for ss, and would completely ignore any changes in c. the second scenario again shows how the rapid assessment model may underestimate residual generation (table 12). the ra method essentially estimates a change in residuals equal to the change in sectoral output (set equal to the change in final demand) multiplied by the sector’s share in residual generation (e.g., the 20% increase in final demand for agriculture would be equivalent to a 15.6% increase in agricultural output. this would be multiplied by the 64.4% share that agriculture has in the generation of code description 1 2 3 4 5 6 7 8 9 10 11 12 agriculture fishery forestry mining and quarrying manufacturing i manufacturing ii electricity, gas and water waterworks and supply construction transportation, communication and storage other services households table 10. changes in the total final demand (∆y) 12 sector regional model, 1994 (in thousand pesos) scenarios 1 2 3696402.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3696402.80 184009.20 306.40 92141.40 3962216.60 1897284.20 440603.00 63228.00 1632080.20 747344.80 632413.00 0.00 code scenarios 1 2initial x ∆∆∆∆∆ x ∆∆∆∆∆ % ∆∆∆∆∆ x ∆∆∆∆∆ % 1 2 3 4 5 6 7 8 9 10 11 12 table 11. incuded changes in total output (∆x) (in thousand pesos) 23722345 2897285 19152 941071 3066379 5246848 462581 43763 9685851 3048735 28667465 25862917 4427104.603 102089.450 7297.831 69704.380 1512760.299 1397374.316 242285.253 21363.099 74134.871 405017.947 1504548.147 2641199.414 18.66 3.52 38.10 7.41 49.33 26.63 52.38 48.82 0.77 13.28 5.25 10.21 5582417.598 514890.217 45178.466 558673.958 9090308.156 8609449.000 1559430.644 146986.036 2060646.255 2187334.982 5919144.825 7180584.346 23.53 17.77 235.89 59.37 296.45 164.09 337.12 335.87 21.27 71.75 20.65 27.76 n p ss c table 12. induced changes in residual generation, ∆r (in metric tons) 15189 12117 2833967 56976 2116.829 1349.224 517855.724 3015.140 13.90 11.10 18.30 4.75 3236.472 2170.397 674166.894 9306.558 21.30 17.90 23.90 16.30 # io model scenario 1 scenario 2initial r ∆∆∆∆∆ x ∆∆∆∆∆ % ∆∆∆∆∆ x ∆∆∆∆∆ % # rapid assessment method scenario 1 scenario 2initial r ∆∆∆∆∆ x ∆∆∆∆∆ % ∆∆∆∆∆ x ∆∆∆∆∆ % n p ss c 15189 12117 2833967 56976 1525 866 427933 0 10.0 7.1 15.1 0.0 1630 873 432253 883 10.7 7.2 15.3 1.5 mcglone & caringal 26 nitrogen, to give an estimated 10% increase in nitrogen generation). the ra method would underestimate nitrogen generation by 49%, phosphorus by 60%, suspended solids by 36%, and carbon by 90%. the above scenarios serve to demonstrate how the rapid assessment methodology represented by equations 1 and 2 may result in a significant underestimation of residual generation. the input-output model, by capturing intersectoral linkages, provides a more thorough assessment of the changes in activities that lead to residual generation. it should be noted that the economy of the study site is dominated by agriculture, with relatively little industrial development. in an economy with a more robust industrial sector, particularly in the agricultural product-based manufacturing 1 sector, the inter-linkages among residual-generating sectors would be stronger, and the relative value of the input-output model would be that much greater. one potential weakness of the specific io model is that, because of time and data constraints, there is no distinction in the transaction table between commodities produced within the region and those imported from other regions, whether domestic or international in origin. this provides no great obstacle in using the io model for estimating residual generation. one must simply assume that during conditions of changing demand, the mix of regionally and non-regionally sourced inputs does not change. this assumption is an extension of the typical io assumption that the technological input mix is constant (that is, the coefficients of the a matrix are constant). of course, over longer time horizons, these assumptions become less tenable. typically, however, governments attempt to overcome this criticism by updating their io tables every 5 or 10 years. use of the competitive type io model becomes problematic for simple or total output multiplier analysis. in such cases, it becomes more important to make the distinction between inputs produced within the regional economy and those imported from outside. while analyzing the changes in multipliers and in the technological coefficient matrix over time could theoretically be of use, the practical fact is that the methods of constructing io tables have changed over time, and typically, io tables over time are not compatible for purposes of comparison. for example, the period between 1979 and 1985 saw the introduction of the distinction between commodities and industries, thus allowing an improved method of allocating secondary output of industries. the negative result of this is that preand post-1979 io tables are no longer directly comparable. acknowledgments we wish to thank wotro, loicz, and sarcs for the fruitful collaboration and support over the last four years. the assistance and encouragement of renee van kessel, john pernetta, steven smith, paul bodreau, bradley opdyke, neil adger, jean-luc de kok, hartwig kremer, and criss crossland are gratefully acknowledged. concurrent grants from the philippine department of science and technology to the university of the philippines-marine science institute, have allowed the philippine team access to major equipment. the partnerships with the u.p. national institute of geological sciences and the environmental science program and with the de la salle universitydepartment of biology have been invaluable in realizing the goals of the project. the kind assistance and support of the project team members are greatly appreciated. references economopoulos ap. 1993. rapid assessment of sources of air, water, and land pollution. geneva, switzerland: world health organization. forsund fr, strom s. 1976. the generation of residual flows in norway: an input-output approach. j environ econ man 3: 129-141. hinga kr, keller aa, oviatt ca. 1991. atmospheric deposition and nitrogen inputs to coastal waters. ambio 20: 256-260. jaworski na, groffman pm, keller aa, prager jc. 1992. a watershed nitrogen and phosphorus balance: the upper potomac river basin. estuaries 15: 83-95. mendoza nf. 1994. input-output modeling. the philippine environmental and natural resources economic modeling of residual generation 27 accounting project-phase 2-technical appendices. pasig city, philippines. miller re,blair d. 1985. input-output analysis: foundations and extensions. engelewood cliffs, n. j.: prentice hall. orbeta em, cortez am, calara an. 1996. region xi policy simulation study: application of enra framework: regional study 4. pasig city, philippines: philippine environmental and natural resources accounting project: padilla j, castro l, morales a, naz c. 1997. evaluation of economy-environment interactions in the lingayen gulf basin: a partial area – based environmental accounting approach. philippines: denr and usaid. secretario ft. 1999. research project on the construction of input-output tables for the ilocos region. turner rk, adger n. 1997. towards integrated assessment in the coastal zone: a draft discussion document. valiela i, collins g, kremer j, lajtha k, seely b, brawley j, sham ch. 1997. nitrogen loading from coastal watersheds to receiving estuaries: new method and application. ecol applic 7(2): 358-380. sd-sample article s.l. yap and others 67 science diliman (july-december 2013) 25:2, 67-77 an account of the accessioned specimens in the jose vera santos memorial herbarium, university of the phil ippines dil iman sandra l. yap* institute of biology university of the philippines diliman sarah cristina w. estacio institute of biology university of the philippines diliman joanne rae c. pales center for integrative and development studies university of the philippines leonard l. co† institute of biology university of the philippines diliman abstract _______________ *corresponding author t h e u n i v e r s i t y o f t h e p h i l i p p i n e s h e r b a r i u m w a s e s t a b l i s h e d i n 1 9 0 8 a n d o r i g i n a l l y l o c a t e d i n e r m i t a , m a n i l a . t h e m a j o r i t y o f i t s p r e w a r collections were destroyed during world war ii, and no formal records of its specimens were preserved. since then, multiple efforts to restore and improve the herbarium have been proposed and implemented, most notably its move to the up diliman campus. in 1999, the herbarium was of f i c i a l l y r e n a m ed a s t h e j o s e ve r a s a n to s m e m o r i a l h e r b a r i u m a fte r the noted grass expert, who initiated rehabilitation work in the herbarium after the war. the herbarium is registered with the international code p u h i n t h e i n d e x h e r b a r i o r u m , a g l o b a l d i r e c t o r y o f p u b l i c h e r b a r i a managed by the new york botanical garden. to assess the accessioned ( u n i q u e l y n u m b e r e d a n d r e c o r d e d ) c o l l e c t i o n o f t h e h e r b a r i u m , a n e l e c t r o n i c d a t a b a s e o f i t s a c c e s s i o n s w a s c r e a t e d . t h e h e r b a r i u m c u r r e n t l y c o n t a i n s 1 4 , 6 4 8 a c c e s s i o n s , 1 2 , 6 8 1 ( 8 6 . 6 % ) o f w h i c h w e r e c o l l e c t e d i n t h e p h i l i p p i n e s . t h i s i s c o m p r i s e d o f 3 0 9 f a m i l i e s , 1 9 0 3 genera, and 4485 distinct species. thirty-nine type specimens form part of the collection, only one of which is a holotype. on the basis of major issn 0115-7809 print / issn 2012-0818 online specimens in the jose vera santos memorial herbarium 68 plant groups, angiosperms make up 71% of the collection. unsurprisingly, family poaceae has the largest number of specimens at 2,759 accessions. the earliest dated philippine specimen was collected by e.d. merrill in 1902, and roughly half of the total accessioned specimens were collected in the 1950s and 1970s. the two most prolif ic collectors were santos and leonardo l. co, with 2,320 and 2,147 specimens, respectively. luzon is the most well-represented island group with 2,752 specimens collected i n m e t r o m a n i l a a l o n e . at p r e s e n t , p u h cu r a to r j a m e s v. la f r a n k i e i s w o r k i n g o n t h e e x p a n s i o n o f t h e c o l l e c t i o n a n d u p g r a d i n g o f t h e herbarium to encourage future educational and research activities. keyword s: herbarium, botany, museum, collection, philippine flora introduction the university of the philippines herbarium was established in 1908, during a period dubbed as the “golden age of philippine botany” (pelser and others 2011). american botanist elmer d. merrill (1876-1956) led much of the floristic research conducted at the time, unsurpassed in asia until he left the country in the early 1920s (robbins 1958). the herbarium was originally located in ermita, manila then the main campus of the university of the philippines (up) system. given its proximity to the bureau of science herbarium (now the philippine national herbarium, or the pnh), the up herbarium was largely overlooked. merrill, who was head of the up botany department from 1912 to 1918, lamented the usage of the herbarium merely as an instructional tool; to him, herbarium work necessitated museum activities (asis 1975). the lack of funds given to the herbarium also greatly impeded its development. tragically, the herbarium and its collections were destroyed during the liberation of manila in 1945, during the second world war. most, if not all, specimen accessions prior to the war were not preserved. in 1946, after the war, the rehabilitation of the herbarium was under taken by d r. jose vera santos † (1908-1987). most of the initial specimen accessions came from santos’ personal collection. by 1949, the herbarium was housed in up diliman, at pavilion 4, in what was then known as the college of arts and sciences. much of the herbarium’s collections at the time were either stored in cabinets along the corridors of pavilion 4 or in faculty rooms and laboratories. it was only in 1978 that the herbarium’s collections were consolidated and a room dedicated to its purpose was secured. the bulk of the work in organizing and expanding the herbarium’s collections were done by leonardo l. co † (1953 -2010) and dr. s.l. yap and others 69 prescillano m. zamora† (1933-2010), then director of the institute of biology (ll co, unpublished notes). efforts to improvise and expand the herbarium were formalized in 1989 by zamora, with the main purpose of restoring damaged specimens and acquiring new specimens. the herbarium gained international recognition in 1990, when it was listed in the index herbariorum with the acronym puh. in 1999, it was off icially renamed as the jose vera santos memorial herbarium. (as a sidenote, there appears to be a discrepancy in the herbarium’s name that was registered in the index herbariorum (jose vera santos memorial herbarium) and the name used in off icial documents f iled at the institute of biology regarding the herbarium’s renaming (jose vera santos herbarium)). this study aimed to assess the accessioned collection of the jose vera santos memorial herbarium (alternatively referred to in this article by its acronym puh) by analyzing the scope of its collections in terms of taxonomic composition, geographic distribution, and chronological breadth. methods an electronic database of the herbarium accessions was created (note: this database will be made available to interested parties upon request.). logbooks containing specimen accessions were encoded into an ms excel f ile. entries for each accession include the following: family, genus, species epithet, subspecies or variety, collector, date of collection, and locality in which the specimens were collected. a review of the database was done to determine the taxonomic composition of the collections, the representation of collectors, the number of collections per decade, and the representation of each province in the herbarium. for the purpose of analysis, outdated nomenclature was corrected based on the apg iii system for angiosperms (apg iii 2009), and according to the online database on the plant list (www.theplantlist.org). results and discussion the jose vera santos memorial herbarium contains 14,648 accessions, 12,681 (86.6%) of which were collected in the philippines. the entire herbarium collection is comprised of 309 families, 1903 genera, and 4,485 distinct species. angiosperms make up the majority of the collections, comprising 71% of the total specimens (figure 1). bryophytes and pteridophytes make up 12% and 11% of the collections, respectively, while only one percent of the collections are gymnosperms. five specimens in the jose vera santos memorial herbarium 70 figure 1. representation of the four major plant groups in the jose vera santos memorial herbarium based on the number of collections. while fungi and algae collections continue to be housed in the herbarium, no accession numbers have been given for said specimens at this time. percent of the collections have either unclear taxonomic aff iliations (unresolved identif ication) or are lacking in data (specimens that have not been identif ied or those with genera or species that could not be found on any database). out of the 309 families, family poaceae has the largest number of specimens at 2,759, followed by fabaceae (690), and asteraceae (539) (table 1). the herbarium’s namesake, santos, specialized in grasses, and a great number of the specimens under poaceae were collected by him. the puh’s earliest dated specimen collected in the philippines is the grass echinochloa crus-galli (poaceae), collected by elmer d. merrill on april 26, 1902. as the herbarium and its contents were destroyed during the war, the origins of 392 pre-war specimens in the puh cannot be determined. the herbarium also table 1. the top ten famil ies based on the number of herbarium collections *dicranaceae is a family under the major plant group bryophytes, comprised of mosses, liverworts and hornworts. rank family number of collections 1 poaceae 2,759 2 fabaceae 690 3 asteraceae 539 4 cyperaceae 517 5 malvaceae 334 6 rubiaceae 326 7 euphorbiaceae 322 8 dicranaceae* 295 9 lamiaceae 277 10 moraceae 247 s.l. yap and others 71 houses specimens dating from as early as the 1860s collected from overseas or acquired from exchanges with other herbaria. the rise in the number of collections in the 1940s and 1950s (figure 2) was mostly due to the contributions of santos and his undergraduate students, during the post-war rehabilitation of the herbarium. from the 1970s onwards, most of the specimens added to the herbarium were collected by leonard co. a sizeable backlog of specimens has yet to be processed, mounted, and given accession numbers, and this may explain why very few collections have been added from the 2000s up to the present. jose vera santos has the most number of collections in the herbarium with 2,320 specimens collected (table 2), followed closely by leonard co (2,147). together, their table2. the top ten collectors based on the number of collections rank collectors number of collections 1 jose vera santos 2,320 2 leonardo l. co 2,147 3 r.s. francia 488 4 a.r. alvarez jr. 346 5 d.t. busemeyer, a. ipolito & j.f. barcelona 272 6 liborio e. ebalo 209 7 mary strong clemens 206 8 m.q. lagrimas 200 9 juan v. pancho 195 10 prescillano m. zamora 189 figure 2. the number of specimens in the jose vera santos memorial herbarium collected in the philippines from the 1900s to present. note that collections prior to 1940 were mostly destroyed during world war ii. specimens in the jose vera santos memorial herbarium 72 collections make up roughly 30.5% of the total number of collections in the herbarium. once his unprocessed specimens are added to the herbarium, co will likely overtake santos in the size of collections in the herbarium. based on the number of collections per province, luzon is the most well-represented island group. of the 10 provinces listed in table 3, seven are found in luzon. metro manila tops the list with 2,572 accessions recorded to have been collected from the area. laguna comes in second with 2,335 specimens, a majority of which are from mount makiling and the up los baños campus. in contrast, three provinces in mindanao are represented by only one specimen (agusan del sur, agusan del norte, and surigao del sur; see figure 3). table 3. the top ten provinces based on the number of collections rank province number of collections 1 metro manila 2,572 2 laguna 2,335 3 benguet 951 4 quezon 811 5 mountain province 653 6 palawan 528 7 rizal 430 8 oriental mindoro 305 9 basilan 279 10 zambales 264 there are currently 39 type specimens deposited in the puh; the majority (28) of these are isotypes (table 4). there is one published holotype and two additional holotypes collected by zamora that were not published before his death. the earliest type specimen in the herbarium is an isosyntype of aristida culionensis pilger ex perkins (mez and pilger 1904) collected by merrill in 1902, while the most recent is an isotype of vacciniumoscarlopezianium co (co and others 2002) collected by co in 1991 from the northern sierra madre natural park in san mariano, isabela. all herbaria will always be biased toward certain geographic regions or taxonomic groups, or both. these biases may be considered as both strengths and limitations of a herbarium. the flora of luzon is relatively well-represented in the up herbarium’s collections. most specimens in the herbarium are incidental collections, with the exception of groups that were of special interest to contributors, such as grasses (santos) and ferns (zamora and some of co’s early collections). the s.l. yap and others 73 figure 3. the number of collections per province in the jose vera santos memorial herbarium. specimens in the jose vera santos memorial herbarium 74 puh scientific name type category publ ication accession # 11016 acrosorusnudicarpus p.m. zamora & co isotype zamora and co 1980 9990 ancistrachneancylotricha isotype quisumbing and merrill 1928 (quis et merr.) st. blake 12297 andreaeanivalis var. baileyi holz. type holzinger 1924 (undef ined type specimen) 10059 aristidaculionensis pilger ex perkins isosyntype mez and pilger 1904 11017 aspleniummantalingahanum isotype zamora and co 1980 p.m. zamora & co 10111 centothecaphilippinensis isotype monod de froideville 1971 (merr.) monod de froideville 8780 cheilanthesdilimanensis p.m. zamora holotype zamora 1974 10163 cheilanthesdilimanensis p.m. zamora paratype zamora 1974 10164 cheilanthesdilimanensis p.m. zamora paratype zamora 1974 10112 digitariaphilippinensis henr. isotype henrard 1950 10072 dimeriaciliata merr. isotype merrill 1928 9988 echinochloastagnina (retz.) beaun isotype quisumbing and merrill 1928 12315 fissidensmanateensis grout type holzinger 1926 (undef ined type specimen) 12178 fissidenspringlei cardot isotype cardot 1909 10099 garnotiaacutigluma var. longiaristata isotype santos 1950 (santos) jansen 83 garnotiamindanaensis santos isotype santos 1950 6302 garnottialongiaristata var. isotype santos 1950 basilanensis santos 9690 grammitisalepidota m.g.price paratype price 1973 12317 grimmiamoxleyi r.s. williams type williams 1926 (undef ined type specimen) 5284 isachnelutaria santos isotype santos 1943 10136 miscanthusfloridulus isotype merrill 1910 (labill.) warb. ex k. schum. & lauterb. 9963 monostachyacentrolepidioides merr. isotype merrill and merritt 1910 1842 opuntiacharlestonensis clokey isotype clokey 1943 1843 opuntiamultigeniculata clokey isotype clokey 1943 14580 pandanusleonardocoi p.m. zamora holotype unpublished 14581 pandanuspricei p.m. zamora holotype unpublished 3274 potentillacryptocaulis clokey isotype clokey 1938 7060 premnaodorata blanco isoneotype munir 1984 10366 pronephriumbalabacensis isotype zamora and co 1980 p.m. zamora & co 12680 rottboelliaparadoxa de koning & sosef isotype veldkamp and others 1986 374 scheffleraheteroclada frodin isotype frodin 1986 309 scheffleraminutipetiolata frodin isotype frodin 1986 10071 schizostachyum lima (blanco) merr. isotype merrill 1916 10081 schizostachyumlumampao isotype merrill 1916 (blanco) merr. 11015 sphaerostephanoscartilagidens isotype zamora and co 1980 p.m. zamora & co 11918 taiwanobryumrobustum veloira isotype veloira del rosario 1959 11931 thamnobryumquisumbingii isotype iwatsuki and tan 1977 (veloira) z. iwats. & b.c. tan 11932 thamnobryumquisumbingii paratype iwatsuki and tan 1977 (veloira) z. iwats. & b.c. tan 12120 trichosteleumaequorum fl. ex dixon type dixon 1916 (undef ined type specimen) 10687 vacciniumoscarlopezianum co isotype co and others 2002 10863 xanthostemonfruticosus isotype wilson and co 1998 peter g. wilson & co table 4. a l ist of the type specimens in the jose vera santos memorial herbarium s.l. yap and others 75 herbarium also has an unoff icial policy of not collecting replicates, especially for common, ornamental, or agricultural species. another important feature of the herbarium is that it reflects, in part, the state of floristic research in the country, which appears to have stagnated in the past two decades based on accessioned specimens. a major overhaul of the herbarium is currently under way. under new management, led by curator james v. lafrankie, an estimated 26,000 specimens (baja-lapis and others 2004) are now being processed and accessioned, including some 6,000 specimens collected by co. the addition of these specimens will allow for further analyses of the herbarium’s collection, and consequently, of how much or how little of the philippine flora is represented in puh. acknowledgments the authors would like to thank the center for integrative and development studies and the institute of biology for funding the study, herbarium staff ramon bandong and edwin balbin for providing herbarium records, the team of student assistants who encoded logbook records, and dr. james v. lafrankie for reviewing a draft of the manuscript. references apg iii. 2009. an update of the angiosperm phylogeny group classif ication for the orders and families of flowering plants: apg iii. bot. j. linn.soc. 161: 105-121. asis cv. 1975. quisumbing and friend. nat. appl. sci. bull. 27(1-2): 1-73. baja-lapis ac, servaz-audije bn, linatoc ac. 2004. a compendium of extant botanical co l l e c t i o n s i n t h e p h i l i p p i n e s . la g u n a , p h i l i p p i n e s : a s e a n re g i o n a l ce n t r e f o r biodiversity conservation (arcbc). cardot j. 1909. revue bryologique 36: 69. clokey iw. 1938. notes on the flora of the charleston mountains, clark county, nevada. bull. s. calif. acad. sci. 37: 4. clokey iw. 1943. notes on the flora of the charleston mountains, clark county, nevada. madroño. 7: 67-76. co ll, madulid d, argent g. 2002. a new species of vaccinium (ericaceae) from the philippines edinb. j. bot. 59(03): 373-376. dixon, h.n. 1916. on a collection of bornean mosses made by the rev. c.h. binstead. bot. j. linn. soc. 43: 291-323. specimens in the jose vera santos memorial herbarium 76 frodin dg. 1986.studies in schefflera (araliaceae) ii.northern luzon (philippines) species of the heptapleurum group. proc. acad. nat. sci. philadelphia 138: 403-425. holzinger. 1926. musci acrocarpi boreali-americani et europai. fasc. 24:590. bryol. 29(5): 63-64. henrard m. 1950. monograph of the genus digitaria 563–565, f. f ig. 874 holzinger jm. 1924. and reaea nivalis baileyi n. var. bryol. 27(6): 90-92+xi. iwatsuki z, tan bc. 1977. new names for philippine mosses. misc. bryol. lichenol. 7: 152. merrill ed. 1910. new or noteworthy philippine plants, viii. philipp. j. sci. c. bot. 5(3): 170. merrill ed. 1914. new or noteworthy philippine plants, x. philipp.j.sci. c. bot. 9: 262. merrill ed. 1916. on the identity of blanco’s species of bambusa.am.j.bot. 3(2): 58-64. merrill ed, merritt lm. 1910. the flora of mount pulog. philipp. j. sci. 5: 287-401. mez c, pilger r. 1904. gramineae. in: perkins j, editor. fragmenta florae philippinae: contributions to the flora of the philippine islands, vol. 1. leipzig: gebrüder borntraeger. p 137-150. monod de froideville c. 1971. notes on malesian grasses iv. blumea 19(1): 60. munir j. 1984. a taxonomic revision of the genus premna l. (verbenaceae) in australia. j. adel.bot.gard. 7(1): 1-91. pelser pb, barcelona jf, nickrent dl, editors. 2011 onwards. co’s digital flora of the philippines. www.philippineplants.org price, mg. 1973. a new species of grammitis from mount banahaw, luzon. philipp. agric. 57(1-2): 34-36. quisumbing e, merrill ed. 1928. new philippine plants. philipp. j. sci. 37: 133-213. robbins wj. 1958. elmer drew merrill 1876-1956: a biographical memoir. washington, dc: national academy of sciences. santos jv. 1943. new grasses from the philippines and south india. j. wash. acad. sci. 33: 135-140. santos jv. 1950. a revision of the grass genus garnotia. nat. app. sci. bull. 10: 3-175. veldkamp jf, de koning r, sosef msm. 1986. generic delimitation of rottboellia and related genera (gramineae). blumea 31(2): 281-307. veloira del rosario ne. 1959. two new mosses from the philippines.the bryologist 62(2): 104-108. s.l. yap and others 77 wilson pg, co ll. 1998. xanthostemon fruticosus (myrtaceae), a new species from the philippines. sida 18: 283-286. zamora pm. 1974. a new species of cheilanthes. kalikasan 3: 193-195. zamora pm, co ll. 1980. new species of ferns (filicopsida) from palawan, philippines. nat. app. sci. bull. 32: 43-52. _______________ sandra l. yap, phd <sandralimyap@yahoo.com> is an assistant professor at the institute of biology, university of the philippines, diliman. she received her phd in ecology and evolutionary biology from the university of michigan, ann arbor, usa. sarah christina w. estacio is a university research associate i and ms biology student at the institute of biology, university of the philippines diliman. she graduated with a bs biology degree from the same institution. joanne rae c. pales is a licensed forester having completed a bs forestry degree from university of the philippines los baños. she was a research assistant for the flora of the philippines project funded by up-cids. leonardo l. co, phd, was the philippines’ premier botanist up to his untimely death in 2010. he taught at the institute of biology, university of the philippines diliman after many years of ‘botanizing’ all over the philippines while working for conservation international-philippines. 01_device bird diversity and abundance in the up diliman 1 *corresponding author science diliman 20:1, 1-10 spatial patterns of bird diversity and abundance in an urban tropical landscape: the university of the philippines (up) diliman campus benjamin vallejo jr.a*, alexander aloyab, perry ongc, annette taminod, jonathan villaspere ainstitute of environmental science and meteorology, college of science university of the philippines, diliman, quezon city bnatural science research institute, college of science university of the philippines, diliman, quezon city c institute of biology, college of science, university of the philippines diliman, quezon city dcollege of science, university of the philippines, baguio city edepartment of geography, college of social science and philosophy university of the philippines, diliman, quezon city e-mail: bmvallejo@up.edu.ph date submitted: february 3, 2006; date accepted: may 7, 2008 abstract an analytical biogeographic analysis of urban bird diversity and abundance was conducted in the university of the philippines diliman campus from february to april 2005. using the jokimäki urban bird census technique on four different land use subplots we observed 36 species of birds and 4036 cumulative counts individuals of these species. the open field area had the highest number of species (23) while the residential area had the lowest species number (14). the residential area has the highest bird counts and the college of science complex had the lowest number of counts. linear regression analysis of landscape features on bird abundances reveals that the number of trees and buildings are most significant predictors of abundances. spatial features and the trophic characteristics of the bird species are not significant factors to account for abundance. trees and buildings affect the distribution and abundance of urban exploiting species passer montanus and pycnonotus goiavier with the former favoring built spaces and the latter favoring trees. analysis of species area curves suggests that the bird community of the campus is not homogenized and that certain habitats allow uncommon species to persist. we recommend that in order to increase bird biodiversity existing green spaces must be preserved, fruit trees planted and in areas reserved for building development, pocket gardens and rooftop gardens be included in the landscape architectural design. key words: birds, biogeography, urban ecosystems, university of the philippines, metro manila vallejo, et al. 2 science diliman 20:1, 1-10 introduction urban environments are generally areas characterized by a growing human population, pollution, and conversion of natural habitat into ‘built-up’ areas. like any major asian metropolis where at least 10% of the national population resides, metro manila has about 12% of the entire population of the philippines (ohmachi 2002). metro manila’s population growth rate is 3% per annum largely brought upon by migration from the provinces. based on landscape statistics by the philippines national mapping and resource information (namria 2003), built-up areas in metro manila was estimated to be around 37.8% of the total land cover. since urban areas are characterized as centers of human habitation, philippine cities have not been considered as important areas for biodiversity conservation. many studies have shown that metropolitan areas still support significant levels of animal and plant biodiversity which includes the occurrence of rare species in wooded, green spaces (gyllin 1999; szacki 1999; ong et al. 1999). landscape developers and architects have long realized the importance of green spaces in mitigating microclimatological effects of overheating (ohmachi 2002) and in helping maintain humidity levels (szacki 1999) in built-up areas. green areas are generally locally fragmented and patchy in cities (clemants & moore 2003; gilber 1992; hobbs 1988) and this fragmentation may result in localized extinctions (hengeveld 1993). in a recent study in a university campus in brazil, habitat fragmentation resulted in a failure to sustain avian biodiversity (manhães & loures-ribeiro 2005) species richness decreased with increasing urbanization in vancouver, canada (melles et al 2003). however, bird biodiversity loss in metro manila has been not well documented with the exception of possible extinctions recorded at the university of the philippines diliman campus in quezon city. four forest dwelling birds (mostly raptors) were recorded in the 1960s but have never been recorded since, making them locally extinct from the diliman district ( ong et al. 1999). localized extinctions may be a result of species invasions by non-native species (kowarik 1995; blair 2004). urban bird communities are usually characterized by the dominance of a few species (beissinger & osborne 1982; marzluff 2001) and most of the species making up the communities are introduced. a low spatial variation of urban bird communities is expected and may probably result to more similar bird communities all over the world (jokimäki et al. 1996). urban birds are generally classified in three general categories: urban avoiders, suburban adaptable and urban exploiters (blair 1996; mckinney 2002). a growing number of amateur bird watchers and clubs (e.g. wild bird society of the philippines) in the last 10 years have recorded about 117 bird species in metro manila. some of the accounted species are listed as endangered (collar et al. 1999) and were mostly seen in wooded areas of universities, memorial parks, reclamation sites and in watersheds (e.g. la mesa watershed). the numerous observations by amateur and professional birdwatchers may support the idea of the conservation value of metro manila’s green spaces. nonetheless, there is not much scientific information on the distribution and habitat association (e.g. landscape ecological characteristics) of bird biodiversity. in this research we investigate the possible effect of landscape features on the patterns of diversity and abundance of bird communities in the 493 hectare university of the philippines (up) diliman campus. several researchers have recommended local scale investigations in studying bird-habitat relationships aimed at the development of effective management decisions for the conservation of birds in urban habitats (jokimäki & jokimäki 2003; clergeau et al. 2001). methods study area our study area is the university of the philippines diliman campus in quezon city (140 n, 1210 e), metro manila (fig. 1). the campus is located 15 kilometers from downtown manila. it is a fully functional community and a local government unit. the campus has faculty and student residential areas, academic use areas, parks and commercial areas. the campus bird diversity and abundance in the up diliman 3science diliman 20:1, 1-10 daytime population is 40,000 composing of students, staff, employees and informal settlers. we use the campus as a model for metro manila’s urbanization. geophysical studies by midorikawa and bautista (2002) categorized the area to be part of the ‘central plateau’ or guadalupe tuff region of metro manila with an elevation of at least 10 meters above sea level. rapid urban development began as early as 1947 when the campus was transferred from ermita district in downtown manila after the second world war. the climate type is tropical monsoonal (type i) with a pronounced dry season from november to april, and a pronounced wet season for the remainder of the year. the area is protected from the northeast monsoon, but open to the southwest monsoon and tropical cyclones. four survey sub-plots (20-25 hectares) were established within a 2 km × 2.5 km area. these plots were defined by existing university road networks and characterized by varying levels of land use and degree of infrastructure developments. plots were spatially delineated as up academic oval (ov), open space along university avenue (op), college of science complex (cs), and the up faculty and staff residential site (re). bird census technique we employed a bird survey designed for urban communities developed by finnish biogeographers jokimäki and jokimäki (2003). we initially established four sub-plots (20-25 has.) within the up diliman campus which was normally surveyed during early mornings (0530-0800 h) and before sunset (1630-1800 h) at a pace of 10 ha/20 min walk. we surveyed each plot six times during the dry and hot season from february to april 2005. we conducted the census by walking a random zigzag route pattern in wooded and open spaces while following road pavements in the residential site. careful bird counts were observed continuously through a fast-paced walk, taking note of bird movement across the route. this criterion is intended to avoid double or multiple counts of an individual. birds which merely flew high overhead from figure 1. the university of the philippines (up) diliman campus in northeast quezon city, metro manila. bird survey subplots are indicated. vallejo, et al. 4 science diliman 20:1, 1-10 the upper canopies or buildings were not included in the counts. observations on bird species richness and abundance including microhabitat associations were noted on standard data sheets. all corresponding statistical analyses were based on cumulative counts of species recorded for the entire survey. the jokimäki method is designed to estimate the abundances of mainly resident perching birds (passeriformes) that are usually associated with tree canopies and urban landscape features. the method cannot be used to estimate the abundance of birds with large foraging areas such as raptors and to detect extremely rare or cryptic species. while raptors have been observed by other researchers in campus, their sighting frequencies are rare and it is likely that these birds are migrants passing over the campus. ecological statistical analyses we used the non-parametric statistic spearman’s coefficient (r s ) to determine the degree of correlation between species occurrence and abundance in all plots. to analyze bird community diversity, we used shannonwiener’s (h’) and simpson’s (d) diversity indices. shannon-wiener index was generally used in ecological studies concerned with the number and abundance of rare species while simpson’s index for more abundant or common species (peet, 1974). hill’s modified ratio (e 5 ) was used to compute for bird community evenness, where a value approaching 0 indicates single species dominance. thus, e 5 = n 2 1/ n 1 – 1 (1) where: n 1 = eh’ (exponential function of shannon-wiener index) n 2 = 1 / d (reciprocal of simpson’s index) to measure the general trend in spatial species diversity among sub-plots, we obtained a species-area curve from nested plots. the derived curve describes the linear association of species richness with increasing sample size (rosenzweig 1995). if a non-linear association emerges, then a case of homogeneity of bird fauna might exist between plots (rosenzweig 1995). in order to estimate effects of landscape features on bird abundances, we log-transformed abundance data before using stepwise linear regression analysis with statistical package for the social sciences (spss version 13.0). log-transformations were done to stabilize variances and fulfill parametric statistical assumptions. ecologists have long accepted that biotic communities generally follow a lognormal distribution as species abundance values are affected by a range of complex processes and interactions in a community (waiten 1999). species abundance was also subjected to simple linear regression analysis with up-diliman landscape variables (ballad et al. 2002, unpublished data). data on species richness and occurrence were used to compute qualitative community similarities among sub-plots (sörensen index): c n = 2 jn /(an + bn) (2) where an is the total number of species recorded in site a, bn, the total number of species recorded at site b, while jn is the number of joint species occurrences for all sites. results species richness and abundance a total of 36 species were accounted in four sub-plots in up diliman (this includes 3 species accounted through opportunistic observations, see table 1). twenty or more species occurred in 3 sites: college of science, up oval, and the open field site. only 14 species were detected in the residential area. philippine endemics comprise 14% of the total species list including the lowland white-eye zosterops meyeni, which only occurs in the luzon faunal region (lfr). other endemics were pink-bellied imperial pigeon ducula poliocephala, philippine hanging-parrot loriculus philippensis, philippine coucal centropus viridis, and philippine pygmy-woodpecker dendrocopus maculatus. migrants make up 19% while introduced species 8%. three out of the 4 known introduced breeding bird species in the philippines were present in the study sites. bird diversity and abundance in the up diliman 5science diliman 20:1, 1-10 bird density a total of 4,056 cumulative counts of individual birds were recorded for 33 species (appendix 1). positive correlation exists between abundance of birds and the number of site occurrences (r s = 0.902, n = 33, p < 0.01). the residential site (although with only 14 species) appeared to have the highest absolute bird density with of 1,685 individuals (table 2). dominant species were eurasian tree-sparrow passer montanus, yellow-vented bulbul pycnonotus goiavier, brown shrike lanius cristatus, and pied fantail rhipidura javanica comprising >5% of the total number of occurring birds in all areas. it can be noted that p. montanus (x = 520.8, sd = 374.4, n = 4) and p. goiavier (x = 179.5, sd = 56.9, n = 4) composes majority of avian populations in all study plots. bird community diversity and evenness shannon-wiener’s index which provides a quantitative description of the intensity of species richness was highest at the op site (h’ = 1.8704) followed by cs (1.8536), ov (1.7951), and very least at re site (1.3196, table 3). simpson’s index was marginally highest at re (d = 0.4337) with the other 3 sites having a range value of 0.24 to 0.26. hill’s index of diversity evenness was highest at ov (e 5 = 0.6048), followed by cs (0.5472), op (0.5172) and re (0.4763). linear regression analysis a low r2 value of 0.175 suggests the effects of scale in the regression model. the significant f-statistic from the anova table (table 5) shows the effectiveness of the regression model in explaining abundance as affected by various landscape variables (i.e. no. of trees, % cover of buildings and green spaces, feeding guilds). coefficient values generated by the linear model indicates the number of buildings (á=0.014) and trees (á=0.004) as significant landscape predictors of bird abundance (table 6). other spatial attributes such as building and green-space cover appeared to be nonsignificant. food preferences of bird species as indicated by feeding-guild values were also not significant. (1) cs (2) ov (3) op (4) re p. montanus 43.9% p. montanus 36.9% p. montanus 46.7% p. montanus 63.7% p. goiavier 20.6% p. goiavier 30.7% p. goiavier 14.6% p. goiavier 12.1% l. cristatus 9.4% l. cristatus 7.9% l. cristatus 9.0% r. javanica 9.3% r. javanica 6.1% r. javanica 7.7% g. striata 8.5% g. sulphurea 5.6% l. schach 4.3% g. sulphurea 6.0% r. javanica 5.1% l. cristatus 3.2% h. tahitica 3.8% c. livia 1.9% g. sulphurea 3.9% h. tahitica 2.3% g. gallus 2.5% h. chloris 1.5% g. gallus 2.6% g. gallus 1.2% g. sulphurea 2.5% z. meyeni 1.5% h. tahitica 2.4% m. palustris 1.7% h. tahitica 1.4% l. schach 2.1% n= 1,685 individuals g. striata 1.3% l. schach 1.2% c. livia 1.6% s. chinensis 1.0% n = 635 individuals n= 810 individuals n = 926 individuals cs, college of science; ov, academic oval; op, open field site; re, residential site table 2 summary of dominant bird species expressed as percentage of the cumulative number of birds counted in surveys (n=6) of four sub-plots in up diliman philippines luzon this survey (up diliman) total cs ov op re total 572 391 36 22 20 23 14 residents 198 137 19 14 11 14 8 philippine endemic 176 100 4 2 3 1 1 lfrb endemic 25 25 1 0 1 0 0 migrants 162 154 7 3 3 4 2 introduced 4 4 3 3 2 3 2 aresidency data is taken from bird listings of kennedy et al. (2000) bluzon faunal region cs, college of science; ov, academic oval; op, open field site; re, residential site table 1 summary of species richness and residency status of birds detected from february to april 2005 in up dilimana. vallejo, et al. 6 community similarity average bird community similarity among all sites is 61.4% (sd = 5.97, n = 6) of which sites cs and op have the highest pairwise comparison with a similarity value of 71.1% (table 7). pair plots cs-re and ovop were found out to have more different species occurrences with only about 55% similarity. the degree of species heterogeneity among the study plots can also be evaluated in the species-area curve derived from nested plot data (fig. 2). the 0.94r2 indiates strong correlation of species richness with increased sampling area. it implies further that some species may have special preferences on certain areas over the others. this could be attributed to habitat type and landscape factors (jokimäki & jokimäki, 2003), food availability, or competition from other types of species. figure 2. species-area curve for bird nested data over six subplots in up diliman. adjusted std. error model r r square r square of the estimate 1 .934(a) .871 .813 .39781(b) a predictors: feeding guilds, number of trees, number of buildings, spatial area of green spaces, spatial area of buildings b dependent variable: log abundance table 4. summary statistics of the regression model. table 3. indices of bird community diversity and evenness, also shown are landscape data* in the 4 study plots in up diliman no. of no. of building road area green plot h’ simpson’s evenness buildings trees area (m2) (m2) spaces (m2) cs 1.8536 0.2535 0.5472 24 3818 36,647.90 5,430 254,853.44 ov 1.7951 0.2478 0.6048 3 3737 6,546.83 2,760 144,771.09 op 1.8704 0.2604 0.5172 13 1225 11,787.35 32,400 269,731.86 re 1.3196 0.4337 0.4763 93 1662 33,169.73 17,100 114,171.21 *data courtesy of geodetic engineering department, and training center for applied geodesy and photogrammetry, college of engineering, up-diliman 2002 h’ = shannon-wiener index of diversity table 5 anova table of linear regression analysis between log-transformed data on bird abundance with feeding guilds (biotic factor) and landscape variables (abiotic factors). sum of mean model squares df square f sig. 1 regression 23.602 10 2.360 14.914 .000(a) residual 3.481 22 .158 total 27.083 32 a predictors: feeding guilds, number of trees, number of buildings, spatial area of green spaces, spatial area of buildings b dependent variable: log abundance unstandardized standardized coefficients coefficients model b std. beta t sig. error 1 no. of buildings .005 .002 .500 2.670 .014 no. of trees .000 .000 .666 3.228 .004 building area -1.58e-005 .000 -.534 -1.326 .198 greenspace area 1.44e-006 .000 .391 1.295 .209 insectivores .110 .230 .049 .476 .639 frugivores -.178 .355 -.047 -.502 .621 omnivores .381 .204 .198 1.873 .074 nectarivores .291 .461 .055 .631 .534 seed eaters .553 .333 .146 1.659 .111 carnivores .106 .282 .038 .377 .710 a dependent variable: log abundance table 6. correlation coefficients of various landscape variables and foraging habits with bird abundance over spatial scales. science diliman 20:1, 1-10 y = 0.4352x + 0.6858 r 2 = 0.9424 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 1.2 1.4 1.6 1.8 2 2.2 log area (has) lo g # spe cies bird diversity and abundance in the up diliman 7 discussion this study detected about 9.2% of birds that can be found in luzon with 5 species endemic to the philippines. nineteen species of the birds recorded by ong et al. (1999) during october 1997 to january 1998 and june 1998 were detected in this survey. difference in species accounts may be due to the time of survey (this one was conducted late february to april 2005), the method used, or some previously recorded species may have been locally extinct, escapees or were accidentally recorded. nevertheless, results of this survey indicate persistence of a relatively diverse avifauna in up diliman campus since 1997. it is also a fact that no major habitat conversions were made in the study sites from year 1997 to the first half months of year 2005. the presence of a number of philippine endemic species d. maculatus, d. poliocephala, z. meyeni, l. philippensis, and c. viridis makes the campus an important urban bird area. this study indicates that although endemics were present, their population represents only a mere 0.59% of the total bird community. this figure may have been affected by successful colonization by other introduced or resident species as a result of native faunal extinction (blair 2004) or as a direct result of habitat conversion from its original state. it is unknown whether this small fragment of endemic birds found in the study sites were remnant population, re-introduced or ‘regular foragers’ from adjacent disturbed forest blocks. what is certain is the vulnerability of the endemic population even to slight habitat disturbance or modifications. our data on bird densities showed two species dominating bird population in all study plots, p. montanus making up 51.4% and p. goiavier 17.7% of the cumulative bird count. p. montanus is a communal seed-eater that was introduced in the philippines in the 1930s (ong et al. 1999) while p. goiavier is resident breeding an omnivore, present in all terrestrial habitat type except in mature and secondary forests. another species which has also been found abundant in all four plots is the pied fantail r. javanica (7.5%). the ability of these species to dominate bird populations in the area categorizes them as ‘urban exploiters’ (blair 1996; mckinney 2002). thus, these species may have directly benefited from earliest land conversion and development in up diliman and in all other areas in metro manila. the community diversity and evenness indices showed how various land-use schemes determine bird species richness. species evenness was highest at the ov site with a value of 0.6048 indicating higher community diversity wherein large numbers of species are all similarly abundant (table 3). this may be attributed to the continuous block of wooded areas in the park and minimal number of buildings (n = 3). the low shannonwiener (h’) and evenness value (e 5 ) in the re site is the function of low species richness and diversity. re had the most number of recorded individuals with 1,685 but composed only of 14 species. it should be noted that this plot is residential with the most number of infrastructures (number of single-family houses = 93). accordingly, bird community structure is primarily composed of species that are commensal with humans such as p. montanus (64%). our data indicates contrary result from the findings of jokimäki & jokimäki (2003) in urban centers in finland wherein bird diversity was observed to be higher in single-family house areas. this could be the case since their data indicated minimal ‘built-up’ spaces in residential areas that are located in sub-urban localities in developed countries (i.e. finland). however there are lesser areas of green spaces in highly urbanized cities like metro manila. this was the case in re site wherein it has the lowest green space area of approximately 114,000 m2 among other plots. our findings suggest the influence of landscapegradient variables in determining local abundances structure of birds in a tropical urban environment. this plots % similarity cs-ov 61.9% cs-op 71.1% cs-re 55.6% ov-op 55.8% ov-re 58.8% op-re 64.9% cs, college of science; ov, academic oval; op, open field site; re, residential site table 7 percent similarities (sorensen index) of paired plots based on qualitative data (species occurrences). science diliman 20:1, 1-10 vallejo, et al. 8 science diliman 20:1, 1-10 is a result that supports melles et al (2003). it appears in our analysis that abundance was determined by the number of spatial entities (i.e. number of buildings and trees) and not by spatial area (i.e. area in m2 of builtup and green spaces) or food preference (i.e. feeding guilds). this finding contrasts with that of lim and sodhi (2004) who found that increasing urbanization adversely impacted on insectivorous and carnivorous feeding guilds. in our study, insectivorous birds seem to have adapted to the urbanizing landscape. we did not observe carnivores that were probably extirpated in the 1960s as suggested by ong et al (1999). this finding supports the general idea that typical urban birds primarily respond to architectural features and secondarily to natural features of the environment (savard & falls 2001). bird species following this scheme were the most abundant especially for the highly adaptable and communal species, p. montanus. the more numerous the number of buildings, the more places these species find suitable foraging and nesting grounds. this phenomenon was further observed in our data as the distribution patterns on bird abundance were highly maximal towards ‘urban exploiter’ species like r. javanica, l. cristatus, zebra dove geopelia striata, and p. goiavier. apparently, landscape homogeneity and increased proportion of built-up spaces (e.g. re site) lowers bird community diversity and evenness. without much habitat modifications since the wildlife inventory in 1999, the up diliman campus still harbors a viable complex ecosystem for bird communities. despite pressures by urban sprawl inherent in highly urbanized areas (blair 2004), a considerable number of endemic species still persist. however, populations of these birds were very marginal compared with other highly urban-adaptable species. our results indicate that increased number of infrastructure and homogenizing landscape (e.g. residential site) greatly contributes to lower biodiversity patterns and domination of commensal, urban exploiting species. however, various land-use types in the campus appear to contribute to the heterogeneity of bird communities. this was evident the fact that there was moderate similarity in terms of birds species composition among sites. in order to preserve and promote diversity of bird species in the area, complex habitats should be sustained by preserving green spaces such as gardens and parks. recent research on tropical urban bird communities in brazil indicate that isolated forest patches within urban areas fail to sustain high bird biodiversity (manhães & loures-ribeiro 2005). a possible solution to mitigate biodiversity loss is to ensure that these patches be preserved as to provide refuges and resource areas for birds (melles et al 2003). artificial habitats may also mitigate biodiversity loss. thus we recommend that in areas reserved for building development, pocket gardens, rooftop gardens and parks must be integral to the landscape design. the planting of fruiting trees (not necessarily of economic value to humans) should also be encouraged. in singapore, low density housing developments with suitable gardens planted to fruiting trees favored the persistence of frugivores (lim & sodhi) endemics such as the lowland white-eye z. meyeni were only seen once during this survey in a flock composed of 10-12 individuals in the upper canopies of fruiting mango trees in the ov site. a number of urban bird researchers in developed countries recommend increasing biodiversity by attracting minority species in the community but are capable of utilizing resources available in urban landscapes (blair 1996; jokimäki & suhonen 1998). a good example would be the endemic pygmy woodpecker d. maculatus, an insectivore which is locally uncommon in up diliman. the bird’s population has the potential to increase the fact that it appears to mimic more common birds (e.g. eurasian tree sparrows) and it does not depend on fruiting or flowering trees for food. it however is a cavity nesting species that probably existed in greater abundances before rapid urbanization. the planting of suitable nesting trees may help in its population recovery. we suggest that further research be conducted on the inability of rainforest birds to persist in an urbanizing landscape. this is a finding noted by lim and sodhi (2004) that is likely applicable to the philippine situation. as most of the escapee pet birds we have observed in campus were of rainforest origin, the question of whether they can persist in an urban landscape has important conservation implications. anecdotal evidence from the wbcp suggests that at least one parrot species has established itself in metro manila. bird diversity and abundance in the up diliman 9science diliman 20:1, 1-10 acknowledgements this work is funded by university of the philippines nsri grant esm-05-2-01. we would like to thank m. lu and a. jensen of the wild bird club of the philippines and prof. a. de villa, cssp department of philosophy for helping in bird identification, the delaware museum of natural history for sending us species and identification catalogues and all volunteers who participated in the surveys. we are also very thankful to dr. r. m. gonzales, chair of the up department geodetic engineering for providing us remotely sensed data and gis databases of the up diliman campus. we also would like to thank the institutional support of academic and research units of the university of the philippines; the institute of environmental science and meteorology, the institute of biology, the marine science institute, department of geography and the natural science research institute. references beissinger, s. r. & osborne, d. r. 1982. effects of urbanization on avian community organization. condor 84: 75-83. blair, r. 1996. land use and avian species diversity along an urban gradient. ecological applications 6: 506-519. blair, r. 2004. the effects of urban sprawl on birds at multiple levels of biological organization. ecology and society 9(5): 2. available fromhttp://www.ecologyandsociety.org/vol9/ iss5/art2 (accessed january 2005) clemants, s. & moore, g. 2003. patterns of species richness in eight northeastern united states cities. urban habitats 1: 4-16. clergeau, p., jokimäki, j. & savard, j-p.l. 2001. are urban bird communities influenced by the diversity of adjacent landscapes? journal of applied ecology 38: 1122-1134. collar, n., mallari, n. a. d. & tabaranza, b. r. j. 1999. threatened birds of the philippines. bookmark, manila. gilber, o. 1992. the ecology of urban habitats. chapman and hall, london. gyllin, m. 1999. integrating biodiversity in urban planning. urban density and green structure. university of gotheburg. hengeveld, r. 1993. biogeographical ecology. journal of biogeography 21:341-351. hobbs, e. r. 1988. species richness of urban forest patches and implications for landscape diversity. landscape ecology 1: 141-152. jokimäki, j. & jokimäki, m-l. k. 2003. spatial similarity of urban bird communities: a multiscale approach. journal of biogeography 30: 1183-1193. jokimäki, j. & suhonen, j. 1998. distribution and habitat selection of wintering birds in urban environments. landscape and urban planning 39: 253-263. jokimäki, j., suhonen, j., inki, k., & jokinen, s. 1996. biogeographical comparison of winter bird assemblages in urban environments in finland. journal of biogeography 23: 379-386. kennedy, r.s., gonzales, p.c., dickinson, e.c., miranda, h.c. & fisher, t.h. 2000. a guide to the birds of the philippines. oxford university press inc., ny, usa. kowarik, i. 1995. on the role of alien species in urban flora and vegetation. pages 85-103 in p.k. pysek, m. prach, m. rejmanek, and p.m. wade, editors. plant invasions: general aspects and special problems. spb academic, amsterdam, the netherlands. lim, hc and sodhi, ns (2004) responses of avian guilds to urbanization in a tropical city. landscape and urban planning. 66:199-215. manhães, m.a. and loures-ribeiro, a. 2005. spatial distribution and diversity of bird community in an urban area of southeast brazil. brazilian archives of biology and technology. 48 (2): 285-294 vallejo, et al. 10 marzluff, j. m. 2001. worldwide urbanization and its effects on birds. in j. m. marzluff, r. bowman and r. donelly, editors. avian ecology and conservation in an urbanizing world. kluwer academic publishers, boston, ma, usa. melles, s., glenn, s., and martin, k. 2003. urban bird diversity and landscape complexity: species-environment associations along a multiscale habitat gradient. conservation ecology 7(1)5. [online] url http:// www.consecol.org/vol7/iss1/art5 mckinney, m. l. 2002. urbanization, biodiversity, and conservation. bioscience 52(10): 883-890. midorikawa, s. and bautista, b.c. 2002. expansion of damage potential area due to urban sprawling. in t. ohmachi & e. r. roman, editors. pp 301-307 metro manila: in search of a sustainable future. university of the philippines press, quezon city, philippines. national mapping and resource information (namria). 2003. namria, fort bonifacio, makati city, philippines. available fromhttp://www.namria.gov.ph/downloads/ lcstat.zip (accessed march 2005) ohmachi, t. 2002. ending the cycle of environmental deterioration. in t. ohmachi & e. r. roman, editors. pp 3-9 metro manila: in search of a sustainable future. university of the philippines press, quezon city, philippines ong, p., pedregosa, m., & de guia, m. 1999. wildlife inventory of the up diliman and ateneo de manila university campuses, diliman, quezon city, luzon, philippines. science diliman 11: 6-20. peet, r.k. 1974. the measurement of species diversity. annual review of ecology and systematics 5: 285-307. rosenzweig, m.l. 1995. species diversity in space and time. cambridge university press, uk. savard, j-p.l. & falls, b. 2001. survey techniques and habitat relationships of breeding birds in residential areas of toronto, canada. in j.m. marzluff, r. bowman and r. donelly, editors. avian ecology and conservation in an urbanizing world. kluwer academic publishers, boston, ma, usa. szacki, j. 1999. ecological corridors and greenspace in the citya polish perspective. urban density and green structure. university of gotheburg, sweden. statistical package for the social sciences, version 13.0 (spss, v. 13.0) 2005. spss inc. headquarters, 233 s. wacker drive, 11th floor, chicago, illinois 60606 waite, s. 1999. statistical ecology in practice: a guide to analysing environmental and ecological field data. prentice hall, uk. science diliman 20:1, 1-10 species cs ov op re acridotheres cristatellus 0 0 4 0 alcedo atthis 0 1 0 0 anthus novaeseelandiae 2 0 1 0 cacomantis variolosus 4 0 0 0 centropus viridis 2 2 0 0 chalcochaps indica 0 0 1 0 columba livia 3 15 15 5 dendrocopus maculatus 0 5 1 5 dicaeum sp. 0 1 0 0 ducula poliocephala 0 2 0 0 galirallus sp. 0 0 0 1 gallus gallus 16 0 24 20 geopelia striata 8 4 79 13 gerygone sulphurea 16 49 36 95 halcyon chloris 5 12 6 0 hirundo tahitica 24 11 22 38 ixobrychus cinnamomeus 0 0 2 0 lanius cristatus 60 64 83 53 lanius schach 27 10 19 0 loriculus philippensis 2 0 0 0 megalurus palustris 11 3 5 0 megalurus timoriensis 3 2 0 0 motacilla cinerea 1 0 2 0 muscicapa griseistica 0 3 0 0 nectarinia jugularis 0 4 0 6 passer montanus 279 299 432 1073 phylloscopus borealis 0 0 1 10 pycnonotus goiavier 131 249 135 203 rhipidura javanica 39 62 47 157 sterna/chlidonia sp. 0 0 1 0 streptopelia chinensis 2 0 9 6 turdus sp. 0 0 1 0 zosterops meyeni 0 12 0 0 cs, college of science; ov, academic oval; op, open field site; re, residential site appendix 1 abundance matrix of regularly occurring birds in 4 survey plots in up diliman (cs, college of science complex; ov, academic oval-park; op, open field site; re, university residential area) 01_amarra an assessment of the selenium status of iodine-deficient and non-iodine-deficient filipino children ma. sofia v. amarra* and demetria c. bongga college of home economics, u.p. diliman *2 c freedom lane interville 2 subd. culiat, q.c. 1128 tel. no. 926-7542 e-mail: amasof@edsamail.com.ph * corresponding author abstract the aim of this study is to examine and compare blood selenium levels in iodine-deficient and non-iodine deficient children. two groups of children were examined: one group with iodine deficiency (n = 31) and the other group with normal iodine status (n = 32). blood was extracted by venipuncture from children aged 6-10 years attending first grade in commonwealth elementary school in quezon city. whole blood selenium was examined by electrothermal atomic absorption spectrophotometry (aas). iodine status was determined by goiter palpation and urinary iodine excretion. mean selenium levels of deficient and non-deficient children were compared using t-test. using a cut-off value of 60 µg se/l whole blood, the proportion of children with normal and deficient iodine status who fell below this cut-off was compared using chi-square test. whole blood selenium values ranged from 17.6 to 133.6 µg/l. there were no significant differences in mean selenium levels between children with normal and deficient iodine status. children with normal iodine status had a mean blood selenium level of 55.87 ± 26.3 µg/l while children with deficient iodine status had a mean level of 58.76 ± 26.4 µg/l. sixty percent of children had blood selenium levels below the arbitrary cut-off of 60 µg/l with no significant difference between groups (p = 0.165), indicating that selenium deficiency is prevalent in this group of children regardless of iodine status. since selenium deficiency limits the response to iodine supplementation, further investigation is needed to determine whether the same situation exists in children from other areas. key words: selenium status, iodine deficiency, schoolchildren, nutritional status introduction iodine deficiency is presently a public health problem in the philippines. results of the fifth national nutrition survey (fnri, 1998) showed that the country has a mild iodine deficiency problem. iodine is an essential trace element required for the synthesis of thyroid hormones, which are involved in normal growth and development. one consequence of iodine deficiency is the development of goiter or enlargement of the thyroid gland. during pregnancy, iodine deficiency has deleterious effects on the fetus, the most severe of science diliman (january-june 2002) 14:1, 1-7 1 which is endemic cretinism. cretinism is characterized by mental retardation, squinting, deafness, primitive brain reflexes, severe growth stunting, and sexual immaturity (maberly, 1994; hetzel, 1986). at lower levels of deficiency, modest but detectable neurological changes occur, such as impaired learning capacity and performance in school or reduced capacity to handle formal tests of psychomotor function (stanbury, 1998). children and adults with endemic iodine-deficient goiter have been shown to have impaired mental function associated with reduced levels of circulating thyroid hormones. because of its impact on the nation’s intellectual productivity, the government has launched a nationwide salt iodization program in order to correct and prevent iodine deficiency. aside from iodine, other essential trace elements are also needed to improve iodine status. selenium is one trace element that is closely involved in iodine metabolism and thyroid function. there are two known roles of selenium. one is its role as a component of the enzyme glutathione peroxidase, which acts as the cell antioxidant system to protect cells from injury by free radicals. the other role of selenium is as component of iodothyronine deiodinases, enzymes which convert the thyroid hormone thyroxine (t4) to its biologically active form triiodothyronine or t3 (arthur et al., 1993). studies have shown that selenium deficiency affects thyroid hormone metabolism and the activity of thyroid hormone deiodinases. under normal circumstances, increased plasma t4 results in a reduction in plasma thyroid stimulating hormone (tsh) produced by the pituitary. during selenium deficiency, the pituitary is unable to recognize increased plasma tsh concentrations. rats given a seleniumdeficient diet exhibited hyperthyroxinemia (increased levels of plasma t4) accompanied by increased plasma tsh. selenium deficiency in rats also resulted in lower plasma t3 concentrations due to the inhibition of deiodinase enzyme activity. an increase in thyroidal t4 and t3 synthesis at the expense of a decrease in total thyroidal iodine has been observed during selenium deficiency even when there is an adequate supply of iodine in the diet. the result is a 15 to 20 percent decrease in the total concentration of thyroidal iodine, t4 and t3. since iodine deficiency also decreases thyroidal iodine, t4 and t3 concentrations, it is therefore aggravated by selenium deficiency (arthur et al., 1993). the response to iodine intervention is influenced by preexisting selenium status. it has been shown that goitrous children aged 6 to 12 years who were selenium-deficient had a less vigorous response to oral supplementation with iodized oil (zimmermann et al., 2000). percentage decrease in thyroid volume was lower among the more severely selenium deficient children than in those whose deficiency was less severe. aside from its role in thyroid activity, other functions of selenium are currently being studied. these include its role in the prevention of cancer and cardiovascular disease (shortt et al., 1997; ip, 1998). unlike iron and vitamin a, there are no current standard cut-off levels to define selenium deficiency or adequacy (gibson, 1990; who, 1996). studies use arbitrary cutoff points to define selenium deficiency on the basis of results obtained from enzyme function tests. no local study has so far examined the selenium status of any population group in this country. this study aims to determine blood selenium levels in iodine-deficient and non-iodine deficient schoolchildren and compare these values with those obtained in studies from other countries. materials and methods this study is part of a larger study that examined several factors (i.e., dietary, biochemical, health, and environmental factors) affecting iodine status in schoolchildren, and compared the psychomotor and cognitive functions of iodine-deficient and non-iodine deficient children. only the results for selenium analysis are reported here. subjects this portion of the study included 63 children aged 6 to 10 years (30 males and 33 females) from commonwealth elementary school in quezon city. all the children were in first grade. the children were distributed in three classroom shifts: 6:00-10:00 a.m.; 10:00 a.m.-2:00 p.m.; and 2:00-6:00 p.m. two classes from each time shift were randomly selected for inclusion in the study. the consent of the parents of the children participating in the study was obtained. a two-stage sampling procedure was used to select the study sample. the first stage was the initial screening of 290 children from the three classroom shifts for amarra and bongga 2 urinary iodine excretion and goiter palpation. the second stage was the selection of a purposive sample of 40 children classified as having adequate iodine status and 40 children with deficient iodine status. due to attrition during the study period, only a total of 77 children participated in the entire study. children with normal iodine status did not have goiter and had urinary iodine excretion of >100 µg/l. children with iodine deficiency had urinary iodine excretion below 90 µg/l and had either grade 1 or grade 0 goiter. parents of 63 children agreed to have their children’s blood samples taken. these samples were included in this study. thirty-two children with normal iodine status and 31 children with iodine deficiency were examined for blood selenium levels. determination of urinary iodine and enlargement of the thyroid gland spot casual urine samples were collected from the children during class hours. samples were labeled, stored in ice, and immediately brought to the biological research laboratory of the department of health for determination of urinary iodine. the method prescribed by who/ unicef/ iccidd (1994) was used in the analysis. urine was digested with chloric acid under mild conditions and iodine was determined by its catalytic role in the reduction of ceric ammonium sulfate in the presence of arsenious acid. duplicate tests were done on each sample. palpation of the thyroid gland was done by visually inspecting the size of the thyroid gland when the subject swallowed. goiter was classified as grade 0 (no visible goiter), grade 1 (palpable but not visible when the neck is in the normal position), and grade 2 (swelling in the neck is visible when neck is in the normal position) following the criteria set by who (1994). in order to avoid inter-observer variability, a single trained researcher conducted the examinations. analysis of whole blood selenium blood samples were collected from the children by venipuncture. approximately 2.5 ml blood was extracted and placed in heparin-lined test tubes. samples were diluted by adding 2.5 ml distilled water and 17.5 ml triton x-100 (0.1%). diluted samples were mixed with 22.5 ml palladium-triton x matrix modifier solution (sigma cat. no.p-4400) and analyzed by electrothermal atomic absorption spectrophotometry (shimadzu model 6501-s atomic absorption spectrometer with hga-600 graphite furnace). values were calculated using a standard curve following the standard addition method described by fidanza (1991). the coefficient of variation (cv) for every addition of the standard was ± 5.0%. analyses were done by the analytical services laboratory, institute of chemistry in u.p. diliman. data analysis data were analyzed for normality using kolmogorovsmirnov test. whole blood selenium values were normally distributed. mean selenium values for iodinedeficient and non-iodine deficient children were compared using student’s t-test. an arbitrary cut-off value of 60 µg se/l whole blood was used to define normal and low blood selenium levels (neve, 2000). using this cut-off, the proportion of iodine-deficient and non-iodine deficient children whose selenium levels fell within each category were analyzed using chi-square test. results whole blood selenium values ranged from 17.6 to 133.6 µg/l. the mean selenium level in the entire study sample was 57.3 ± 26.2 µg se/l whole blood. ashour et al. (1999) showed that mean plasma selenium level in children with kwashiorkor was 47 ± 10.4 µg/l, while in children with marasmus had a mean level of 56 ± 10.4 µg/l. a control group of healthy children had a mean plasma selenium level of 64 ± 1.8 µg/l. the results of the present study showed that the children from commonwealth elementary school have low selenium levels, similar to the values obtained for children with protein-energy malnutrition. a comparison of the mean values of selenium in iodinedeficient and non-iodine deficient children showed that there is no significant difference between the two groups (table 1). in selenium deficiency, there is a strict hierarchy of supply to specific tissues and also to different selenoenzymes within a tissue (arthur & beckett, 1999). studies in rats showed that with insufficient selenium intake, the deiodinase is preferentially supplied an assessment of the selenium status 3 with the element. this suggests that selenium requirement for the activity of deiodinase is lower than that for optimum glutathione peroxidase (gshpx) activity, and that gshpx activity is therefore a good indicator of selenium requirement (behne et al., 1992; levander & burk, 1996). normal values that are adequate for the expression of red blood cell glutathione peroxidase activity were shown to fall within the range of 60 to 220 µg se/l whole blood (neve, 2000). the present study examined the number of children who would fall below 60 µg se/l whole blood. table 2 shows that if this arbitrary cut-off level is used, majority of the children (60%) would be considered selenium-deficient with no significant difference between groups. aside from selenium deficiency, the condition of selenium toxicity has been reported in some population groups. since there are as yet no sensitive and specific indicators of dietary selenium overexposure, toxicological standards for selenium have been proposed on the basis of clinical signs of selenosis. these include loss of hair and nails, skin lesions, tooth decay, and abnormalities of the nervous system (levander & burk, 1996). the common clinical sign of selenosis, i.e., loss of hair and nails, was not observed in the present study sample. discussion the present study showed that whole blood selenium values in this group of schoolchildren ranged from 17.6 to 133.6 µg/l. the mean selenium level was 57.3 ± 26.2 µg se/l whole blood. children with normal iodine status had 55.87 ± 26.3 µg se/l while children with iodine deficiency had 58.76 ± 26.4 µg se/l whole blood, with no significant difference between groups. the study of van bakel et al. (2000) on children aged 9 to 13 years with phenylketonuria (pku) and hyperphenylalaninemia (hpa) who are fed seleniumfree diets during therapy showed plasma selenium levels of 41.08 ± 15.8 µg/l and 72.68 ± 16.59 µg/l for pku and hpa patients, respectively. a comparison group of healthy children had a mean plasma selenium level of 97 ± 11.05 µg/l. malnourished children examined by ashour et al. (1999) had mean plasma selenium 4 amarra and bongga levels ranging from 47 to 56 µg/l while a comparison group of healthy children had a mean plasma selenium level of 64 µg/l. zimmerman et al. (2000) used a cutoff value of 67 µg/l serum selenium to define selenium deficiency in schoolchildren. the values for selenium that were obtained in the present study (55.87 ± 26.3 µg/l and 58.76 ± 26.4 µg/l for children with normal and deficient iodine status, respectively) fall within the range of values obtained in studies done on children table 1. mean levels of whole blood selenium by children’s iodine status children with normal iodine status (n=32) children with iodine deficiency (n=31) mean ± s.d. mean ± s.d. t-value sig. whole blood selenium (µµµµµg/l) 55.87 26.3 58.76 26.4 -0.435 0.665 table 2. selenium deficiency by iodine status using an arbitrary cut-off level of 60 µg se/l whole blood.* *chi-square value = 1.932; df = 1; significance = 0.165 children with normal iodine status (n=32) children with iodine deficiency (n=31) whole blood selenium (µµµµµg/l) 10 22 32 31.3 68.7 100.0 15 16 31 48.4 51.6 100.0 > 60 µµµµµg se/l < 60 µµµµµg se/l total no. % no. % 25 38 63 39.7 60.3 100.0 total no. % with protein-energy malnutrition and those fed selenium-free diets. neve (2000) reported published experimental values for whole blood selenium, ranging from 60 to 220 µg/l, that were found adequate for the expression of red blood cell glutathione peroxidase activity. using the lower level of 60 µg se/l whole blood as arbitrary cutoff point to define adequacy, a majority of the children in the present study (i.e., 60.3%) were classified as selenium-deficient. the effects of selenium deficiency have been demonstrated in certain areas in china that were once afflicted with keshan disease. keshan disease is a selenium-responsive endemic cardiomyopathy that mainly affects children and women of child-bearing age (who, 1996). its main clinical features are acute or chronic episodes of heart disorder characterized by cardiogenic shock and/or congestive heart failure. very low selenium concentrations were pointed out as the fundamental cause of keshan disease (ge & young, 1993), although it is now known that keshan disease is due to a virus that causes heart damage – the coxsackie virus. once the disease is established, selenium is of little or no therapeutic value (who, 1996). levander & beck (1999) found that selenium-deficient mice suffered more extensive heart damage when infected with a coxsackie b4 virus than mice supplemented with selenium. selenium deficiency also increased the virulence of an already virulent strain of coxsackie b3 virus and allowed the conversion of a non-virulent strain to virulence. when the non-virulent strain was inoculated into mice deficient in selenium or vitamin e, the strain caused a moderate amount of heart damage, whereas the same strain given to mice fed nutritionally adequate diets caused no apparent damage. the benign virus that replicated in selenium-deficient mice was then inoculated into normal mice. a moderate amount of cardiac damage was observed in these mice, indicating that selenium-deficient mice had somehow altered the benign strain of the virus such that it was now able to cause heart damage even in a selenium-supplemented mouse. when the benign virus from the seleniumdeficient mice was sequenced, six nucleotide changes were found. these nucleotides correspond with those found in the genome of a virulent strain. further studies showed that selenium deficiency leads to mutations in the viral genome via reduced glutathione peroxidase activity (beck, 2000). the increased oxidative stress causes nucleotide changes that result in a normally avirulent virus changing into a virulent one. rna viruses, such as those responsible for influenza, hepatitis, polio, or aids have high mutation rates and lack proofreading capability. outbreaks of these diseases may actually be the result of infection by a virus whose pathogenicity has changed as a result of replicating in a nutritionally deficient host. beck (2000) proposed that the current paradigm of nutritional deficiency affecting the host immune system, which leads to increased susceptibility to infection, be changed to one in which nutritional deficiency affects both the host and the pathogen. aside from selenium deficiency, selenium toxicity has also been reported based on the clinical signs of excess selenium. in china, high-selenium areas with toxicity have reported whole blood selenium levels of 3200 µg/ l, while high selenium areas without toxicity have levels of 440 µg se/l whole blood (diplock, 1993). in venezuela, high selenium areas have whole blood levels of 813 µg/l while a moderate selenium area has 355 µg/l. the range of whole blood selenium values obtained in the present study (i.e., 17.6 to 133.6 µg/l) indicates that the local situation is more inclined towards selenium deficiency rather than toxicity. the study of ashour et al. (1999) on children with protein-energy malnutrition showed that plasma levels of selenium and other antioxidants decreased in parallel with glutathione peroxidase activity in the presence of malnutrition (i.e., marasmus and kwashiorkor). they concluded that malnourished children were susceptible to high oxidative stress. the two groups of iodine-deficient and non-iodine deficient children in the present study may not have differed in their selenium status since both groups were generally malnourished. the findings of ashour et al. (1999), indicating high oxidative stress in malnourished children, might be applicable to the present study since 70.6% of the children in the present sample were stunted (results not included in this report) which is a sign of chronic undernutrition (amarra, 2001). aside from having low calorie and nutrient intakes (results also not presented here), the low selenium levels in these children 5 an assessment of the selenium status increase the potential for viral mutation in the event of an infection (amarra, 2001). further investigations among children in other areas are needed to determine whether the same situation of low selenium levels also exists. summary the study has shown that filipino schoolchildren living in an urban poor area tend to have low blood selenium levels, concurrent with iodine deficiency. aside from inhibiting the response to iodine supplementation, recent studies have shown that selenium deficiency in the host may induce the mutation of viruses, causing non-virulent strains to become virulent. given the recent outbreaks of infectious diseases such as influenza among schoolchildren, there may be a need to further examine the selenium status of children in urban poor areas and determine whether selenium supplementation in addition to iodine is called for. acknowledgments this research project was made possible through funding support from the following institutions: office of the vice chancellor for research and development, (ovcrd) university of the philippines, diliman; national research council of the philippines (nrcp); and the university of the philippines college of home economics foundation (upchef) diliman. the authors also wish to acknowledge the following individuals: dr. federico b. cruz for providing feedback on the original manuscript; dr. jose s. solis for supervising the analytical method; dr. erniel barrios and dr. leticia p. ho for reviewing the final draft; perlita torres and estefania santos for technical support. references amarra, s.v., 2001. iodine status, psychomotor and cognitive functions of children in an urban poor community. doctoral dissertation. college of home economics, university of the philippines, diliman, quezon city. arthur, j.r., f. nicol, & g.j. beckett, 1993. selenium deficiency, thyroid hormone metabolism, and thyroid hormone deiodinases. am. j. clin. nut. suppl. 57:236s-9s. arthur, j.r. & g.j. beckett, 1999. thyroid function. brit. med. bul. 55(3):658-668. ashour, m.n., s.i. salem, h.m. el-gadban, n.m. elwan, & t.k. basu, 1999. antioxidant status in children with proteinenergy malnutrition (pem) living in cairo, egypt. euro. j. of clin. nut. 52:669-73. beck, m.a., 2000. nutritionally induced oxidative stress: effect on viral disease. am. j. clin. nut. suppl. 7:1676s-9s. behne, d., a. kyriakopoulos, h. gessner, b. walzog, & h. meinhold, 1992. type i iodothyronine deiodinase activity after high selenium intake, and relations between selenium and iodine metabolism in rats. j. of nut. 122:1542-1546. diplock, a.t., 1993. indexes of selenium status in human populations. am. j. clin. nut. suppl. 57:256s-8s. fidanza, f., 1991. nutritional status assessment – a manual for population studies. new york, chapman and hall. food and nutrition research institute, 1998. fifth national nutrition survey. taguig, metro manila. ge, k. & g. yang, 1993. the epidemiology of selenium deficiency in the etiological study of endemic diseases in china. am. j. clin. nut. 57:259s-63s. gibson, r., 1990. principles of nutritional assessment. oxford, oxford university press. hetzel, b.s., 1986. the concept of iodine deficiency disorders (idd) and their eradication. in: towards the eradication of endemic goiter, cretinism and iodine deficiency. j.t. dunn, e.a. pretell, c.h. daza, & f.e. viteri (eds.). paho/who. washington, d.c. ip, c., 1998. lessons from basic research in selenium and cancer prevention. j. of nut. 124:1845-1854. levander, o.a. & r.f. burk, 1996. selenium. in: present knowledge in nutrition. 7th ed. e. ziegler & l. filer jr. (eds.). washington, d.c., ilsi press. 6 amarra and bongga levander, o.a. & m.a. beck, 1999. selenium and viral virulence. brit. med. bul. 55(3):528-33. maberly, g.f., 1994. iodine deficiency disorders: contemporary scientific issues. j. of nut. 124:1473s-1478s. neve, j., 2000. new approaches to assess selenium status and requirement. nut. rev. 58(12): 363-8. stanbury, j.b., 1998. prevention of iodine deficiency. in: prevention of micronutrient deficiencies. tools for policymakers and public health workers. c.p. howson, e.t. kennedy, & a. horwitz (eds.). institute of medicine. washington d.c., national academy press. shortt, c.t., g.g. duthie, j.d. robertson, p.c. morrice, f. nicol, & j.r. arthur, 1997. selenium status of a group of scottish adults. european j. clin. nut. 61:400-404. van bakel, m., g. printzen, b. wermuth, & u.n. weismann, 2000. antioxidant and thyroid hormone status in seleniumdeficient phenylketonuric and hyperphenylalaninemic patients. am. j. clin. nut. 72:976-81. who/unicef/iccidd, 1994. indicators for assessing iodine deficiency and their control through salt iodization. who, 1996. trace elements in human nutrition and health. geneva. zimmermann, m.b., p. adou, t. torresani, c. zader, & r.f. hurrell, 2000. effect of oral iodized oil on thyroid size and thyroid hormone metabolism in children with concurrent selenium and iodine deficiency. european j. of clin. nut. 54: 209-213. 7 an assessment of the selenium status 12_localization localization and imaging 47 localization and imaging of integrated circuit defect using simple optical feedback detection vernon julius cemine*, bernardino buenaobra, carlo mar blanca, and caesar saloma national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: vernon@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 47-51 *corresponding author high-contrast microscopy of semiconductor and metal edifices in integrated circuits is demonstrated by combining laser-scanning confocal reflectance microscopy, one-photon optical-beam-induced current (1pobic) imaging, and optical feedback detection via a commercially available semiconductor laser that also serves as the excitation source. the confocal microscope has a compact in-line arrangement with no external photodetector. confocal and 1p-obic images are obtained simultaneously from the same focused beam that is scanned across the sample plane. image pairs are processed to generate exclusive highcontrast distributions of the semiconductor, metal, and dielectric sites in a gaas photodiode array sample. the method is then utilized to demonstrate defect localization and imaging in an integrated circuit. introduction a key concern in semiconductor characterization and device failure analysis is the accurate determination of the electrical current distributions in an integrated circuit (ic) that can divulge the locations of dielectric breakdowns, doping inhomogeneities, electrical overstresses, and inversion layers (wagner, 2003; ross & boit, 1999). semiconductors, metals, and dielectrics are the main ic material components and characterizing an ic defect requires the accurate identification of the defect material. optical beam-induced current (obic) imaging is often utilized to determine the two-dimensional (2d) current distribution in an ic sample (takasu, 2001). because only semiconductors produce obic signals, an exclusive image of the semiconductor sites in an ic sample may be generated by scanning a focused beam of wavelength λ ≤ hc /e b, where e b is the semiconductor band-gap energy, h is planck’s constant, and c is the speed of light in vacuum. a serious drawback with one-photon (1p) obic images is their lack of information concerning the depth distribution of the semiconductor sites in an ic sample. 1p-obic images look similar even if taken at different axial positions. confocal reflectance microscopy yields high-contrast images, but metal and semiconductor sites are difficult to distinguish from each other in these images because both materials exhibit relatively high reflectivities. it was previously shown that a pair of confocal and 1p-obic images could be produced simultaneously from the same focused beam of a modified confocal microscope with an argon-ion laser light source (daria et al., 2002). we have also developed an image processing procedure for quickly obtaining from the image pair exclusive high-contrast images of the semiconductor, metal, and dielectric sites in an ic sample. the technique was employed to identify cemine et al. 48 different kinds of ic defects and their three-dimensional (3d) structures (miranda & saloma, 2003). high-contrast imaging of the semiconductor sites in an ic could also be obtained by two-photon (2p) obic (xu & denk, 1997; ramsay et al., 2002). however, the high cost of an ultrafast excitation light source has prevented the 2p-obic technique from gaining acceptance as a standard failure analysis tool among ic manufacturers. acquisition and maintenance costs are primary concerns of the semiconductor industry in selecting a failure analysis tool for their test and assembly lines. the need of industry for more versatile but economical failure analysis tools will grow fast to cope with the increasing architectural sophistication of the next-generation ic devices with very high transistor densities. here, we report the localization and imaging of an ic defect with our new inexpensive microscopy technique that obtains high-contrast images of an ic sample (cemine et al., 2004). our new technique combines confocal microscopy, 1p-obic imaging, and optical feedback (of) detection with a semiconductor laser (sl). with post-detection processing, the technique permits the generation of exclusive 3d distributions of semiconductors, metals, and dielectric sites. we have replaced the bulky and inefficient gas laser with the more efficient, smaller, and cheaper sl to excite the ic sample. the sl is also used to detect the optical signals that are reflected from the sample. the microscope features a compact in-line arrangement with no external photodetector (fig. 1). the of sensitivity of sls has been previously utilized in confocal microscopy (rodrigo et al., 2002) and directionsensitive velocimetry (rodrigo et al., 2001). confocal reflectance and 1p-obic images we obtain the confocal reflectance image r(x, y, z) by scanning an ic sample relative to a stationary focused sl beam where r(x, y, z) = |or(x, y, z) ⊗ h 2(x, y, z)|2, or(x, y, z) is the true (object) reflection amplitude distribution and ⊗ represents a convolution operation. the 1p-obic image is given by c(x, y) = ∫ os(x, y, z) ⊗ |h(x, y, z)|2dz, and os(x, y, z) is the true distribution of semiconductor materials in the ic sample. the integration is taken from ±∞. unlike r(x, y, z), the lowcontrast image c(x, y) does contain any information about axial distribution of the semiconductor sites in an ic sample. the product s(x, y, z) = r(x, y, z)c(x, y) yields an exclusive image of the semiconductor sites at the axial location z (daria et al., 2002; miranda & saloma, 2003). similarly, m(x, y, z) = r(x, y, z)c’(x, y) yields an exclusive image of the metal sites at the axial plane z, where c’(x, y) = k – c(x, y) and the constant k represents the highest possible c(x, y) pixel value that is measured from the sample. experiment the experimental setup is shown in fig. 1. we use a commercially available laser diode (ld) (l = 833 nm, sharp lt015mf) that is operated at 25oc with a ld current/temperature controller (melles-grio,t model 06 dld 105). the power incident on the sample is 22.8 mw. the ld output power is monitored using the builtin photodiode (pd) in the ld package. the divergent and elliptical ld output beam is collimated with a collimating lens (cl) [melles-griot, model fig. 1. the combined confocal reflectance and obic microscope using a laser diode (ld) source with a built-in photodiode (pd). the collimating lens (cl) has a f = 14 mm. the objective lens (ol) has a na = 0.5 and a f = 1.7 mm. anamorphic prism (ap) is used to partially circularize the beam. confocalobic amplifier iris diaphragm ld controller ld package servo pd array sample ol ld pd ap cl localization and imaging 49 06glc003, numerical aperture (na) = 0.276, focal length (f) = 14.5 mm] and circularized by mounted anamorphic prisms (ap) (melles-griot, model 06gpa004, 3x magnification). residual asymmetry in the transverse intensity distribution of the collimated ld beam is removed by the iris diaphragm. an infinity-corrected objective lens (ol) (olympus uplan fl, nominal na = 0.5, working distance = 1.7 mm) focuses the ld beam onto the sample that is mounted on a closed-loop xyz servomotor stage (thorlabs, model pt3-z6) which is driven by a motioncontroller card (thorlabs, dcx-pci100). all stagescanning and image acquisition protocols are coded using labview 7.0 (national instruments). analogto-digital conversion of the pd and 1p-obic signals is accomplished with a 12-bit data-acquisition board (national instruments, model 6024e; conversion rate: 200 khz). results and discussion the axial responses for the confocal reflectance and obic microscopes are obtained at a particular site in the photodiode array sample. the responses are shown in fig. 2. confocal reflectance signal varies with axial position, while the obic signal is approximately constant as exhibited in fig. 2. this manifests the axial sectioning capability of the confocal reflectance microscope and the limitation of the obic microscope for depth discrimination. the axial sectioning capability of the confocal reflectance microscope is brought about by the rejection of the out-of-focus beams that are reflected back by the sample to the ld front facet, which serves as the confocal pinhole. on the other hand, the inability of the obic microscope to attain axial sectioning is caused by the fact that the obic signal is the sum of all the currents generated at all axial positions that are being irradiated with light of photon energy higher than the band gap of a semiconductor p-n junction. this total of the generated currents does not vary significantly as the focus of the beam is scanned along the different axial positions. despite the limitation of obic imaging to axial sectioning, this imaging technique is capable of discriminating semiconductor parts from metal parts (and vice versa) in an observation site. we can endow axial discrimination to obic imaging and thereby build up a 3d representation of exclusive semiconductor and metal sites by the numerical process enumerated in the methodology. the result is shown in fig. 3, where the 200 mm x 200 mm confocal, obic, semiconductor, and metal images of a particular site in a pd array sample at different axial positions, z = –4, 0, and, 4 mm, are exhibited. the semiconductor images are obtained from the multiplication of the confocal and obic images. metal images are obtained from the multiplication of the obic complement and the confocal images. axial sectioning is clearly exhibited by the high-contrast confocal images. notice the differing intensity and the parts that are being highlighted as the axial position changes. axial sectioning is not exhibited by the lowcontrast obic images, but notice the discrimination between the semiconductor part (high intensity) and metallic part (low intensity). the semiconductor part is the one that produces a high obic signal when irradiated and is thus the high-intensity portion of the obic image. the metal part, on the other hand, produces a low obic signal when irradiated and is thus the low-intensity part of the image. upon multiplying the confocal and obic images, high-contrast images of the semiconductor sites (highlighted part) fig. 2. axial responses for confocal reflectance and obic microscopes. axial sectioning is observed by the confocal reflectance microscope. the obic microscope, on the other hand, exhibits no dependence with the axial position. effective na for the ol here is 0.39. z position (µµµµµm) c on fo ca l ( v ) o b ic (v ) cemine et al. 50 are obtained. moreover, axial sectioning is observed on the resulting images as exhibited by the differing semiconductor intensity as the axial position changes. high-contrast images of the metal portions on the observation site are observed upon multiplying the obic complement and confocal images. just like the semiconductor sites, axial sectioning is also observed on the resulting images as exhibited by the differing metal intensity and intensity distribution as the axial position changes. a particular strength of this protocol is the ease with which the metal and semiconductor parts are mapped out, making it effective in exactly situating defective sites. figure 4 shows the 200 mm x 200 mm (a) confocal, (b) obic, (c) metal, and (d) semiconductor images of a site in an ic (z84c0008fec) with a verified electrical overstress (eos) defect (boxed). imaging is done at the focus (z = 0) using a 780 nm ld (sharp lt024md). the third column is a magnified shot (25 mm x 25 mm) of the (e) confocal and (f) obic images of the eos defect, that is, the portion pointed by the arrows on the second column. it is apparent in figs. 4(c) and 4(d) that the eos is situated on the metallic portion of the ic. zooming in on the eos site, however, reveals a small obic signal generation [see fig. 4(f)], a possible leakage or current bleedthrough across the metal and the underlying semiconductor layers. the leakage current ranges from 60–150 na, which is amplified using a current preamplifier (gw specimen current amplifier type 103b/31). investigation on the exact nature of the phenomenon is still currently being undertaken. nevertheless, it was shown here that an ic defect can be localized and discriminated from the normal portions through its abnormal obic signal generation. conclusion we have demonstrated an inexpensive technique for high-contrast imaging of semiconductor and metal sites in an ic sample. the of microscope which utilizes an fig. 4. 200 mm x 200 mm (a) confocal, (b) obic, (c) metal, and (d) semiconductor images of a portion of an ic (z84c0008fec) with eos (boxed part). 25 mm x 25 mm or zoomed (e) confocal and (f) obic images of the eos. (a) confocal (c) metal (e) defect-confocal (b) obic (d) semiconductor (f) defect-obic fig. 3. 200 mm x 200 mm confocal, obic, semiconductor, and metal images of a portion of the pd array sample at different axial positions. axial sectioning is observed by the high-contrast confocal images. semiconductor and metal discrimination is observed by the low-contrast obic images. high-contrast semiconductor images are obtained from confocal and obic multiplication. high-contrast metal images are obtained from obic complement and confocal image multiplication. effective na for the ol here is 0.39. z = –4 µµµµµm z = 0 z = 4 µµµµµm c on fo ca l o b ic s em ic o n d u ct o r m et al 0 255 localization and imaging 51 sl for both sample excitation and detection is compact and economical. sls are versatile light sources that can be acquired cheaply at various emission wavelengths. the sl output wavelength is easily tuned by varying the cavity temperature or injection current. high-contrast imaging is demonstrated by distinguishing the three-dimensional distributions of semiconductor, metal, and oxide sites in a photodiode array sample. despite the low incident power on the sample, the method was sensitive enough to detect current in the 60–150 na range leaking through an eos defect site. the simple and compact optical setup makes for a costeffective failure analysis tool that can open the road to affordable, table-top semiconductor characterization and defect analysis. references cemine, v.j., b. buenaobra, c.m. blanca, & c. saloma, 2004. high-contrast microscopy of semiconductor and metal sites in integrated circuits via optical feedback detection. opt. lett., 2004 29: 2479-2481. daria, v., j. miranda, & c. saloma, 2002. high-contrast images of semiconductor sites via one-photon beam-induced current imaging and confocal reflectance microscopy. appl. opt. 41: 4157–4161. miranda, j. & c. saloma, 2003. four-dimensional microscopy of defects in integrated circuits. appl. opt. 42: 6520–6524. ramsay, e., d. reid, & k. wilsher, 2002. three-dimensional imaging of a silicon flip chip using the two-photon optical beam-induced current effect. appl. phys. lett. 81: 7–9. rodrigo, p., m. lim, & c. saloma, 2002. direction-sensitive subwavelength displacement measurements at diffractionlimited resolution. opt. lett. 27: 25–27. rodrigo, p., m. lim, & c. saloma, 2001. optical feedback semiconductor laser michelson interferometer for displacement measurements with directional discrimination. appl. opt. 40: 506–513. ross, r. & c. boit (eds.), 1999. microelectronic failure analysis desk reference, 4th ed. asm international, materials park, oh. takasu, s., 2001. application of obic/obirch/obhic: semiconductor failure analysis. jeol news. 36e: 60–63. wagner, l., 2003. trends in failure analysis. microelectron. reliab. 43: 1369–1375. xu, c. & w. denk, 1997. comparison of oneand two-photon optical beam-induced current imaging. j. appl. phys. 86: 2226–2231. ecosystem-based approach to aquaculture management patrick white1* and maria lourdes san diego-mcglone2 1akvaplan-niva as, bp 411 crest cedex 26402, france telefax : +33475431901 *corresponding author email: patrick.white@akvaplan.niva.no 2the marine science institute university of the philippines diliman, quezon city, 1101 philippines date submitted: january 10, 2008; date accepted: november 21, 2008 abstract ecosystems have real thresholds and limits which, when exceeded, can affect major system restructuring. once thresholds and limits have been exceeded, changes can be irreversible. diversity is important to ecosystem functioning. the ecosystem approach is a strategy for the integrated management of land, water, and living resources that promotes conservation and sustainable use in an equitable way. the application of the ecosystem approach will help to reach a balance of the three main objectives: conservation, sustainable use, and a fair and equitable sharing of the benefits and use of the natural resources. aquaculture development needs to be within the carrying capacity of the water resource so that it is sustainable and does not greatly impact the environment. the determination of the carrying capacity needs to be science-based. the planning of development in ecosystems has been done for freshwater ecosystems within the pamb (protected area management board) framework, but in many cases this does not give the correct significance to the impact of aquaculture on the water resources in the ecosystem. it also needs to be extended to river basins and estuaries, brackishwater areas, and inland bays, and seas. the planning and management of aquaculture needs to be undertaken at the local government unit (lgu) level in a coordinated manner by all the lgus that have a part of the water resource. the co-management of aquaculture, in terms of monitoring of the environment, monitoring of production, and monitoring of licenses, needs to be funded out of license fees and noncompliance fines collected by the lgus. a number of these management activities need to be undertaken jointly (monitoring the environment) and others separately but in a coordinated manner (e.g., checking licenses and checking compliance). keywords: aquaculture, ecosystem, management introduction aquaculture has been developing rapidly over the last 25 years. it is now contributing a significant supply of quality food for humanity and is provider of employment and economic benefits to those engaged in this activity especially in asia. in the philippines, aquaculture production comprises approximately 50% of total fisheries production (bas, 2007). however, with increased development of aquaculture, there is the realization that living aquatic resources, although renewable and adaptable, are not infinite and need to be properly managed, if their contribution to the nutritional, economic and social well-being of the growing world’s population is to be sustained. the adoption in 1982 of the united nations convention on the law of the sea provided a new framework for the better management of marine resources. the new legal regime of the oceans gave coastal states rights and responsibilities for the management and use of fishery resources within the areas of their national jurisdiction, which embrace science diliman 1 white, p. & san diego-mcglone, m.l. some 90% of the world’s marine fisheries. in recent years, world aquaculture has become a dynamically developing sector of the food industry, and many countries have striven to take advantage of their new opportunities by encouraging the aquaculture development in response to growing international demand for fish and fishery products. it has become clear, however, that many aquatic resources used for aquaculture cannot sustain an often uncontrolled increase of exploitation. the ecosystem approach is a management principle. as such, it builds on the recognition that the nature of the natural world is integrated and a holistic approach to environmental management must be taken. the science to support the ecosystem approach to management must also be integrated and holistic. a core element of this science is ecology, with focus on the properties and dynamics of ecosystems (fenchel, 1987). many scientists and managers have recognized the need for an ecosystem approach for fisheries (likens, 1992), although it is only during the last 5-10 years that a broad awareness of the need for such an approach has grown in aquaculture. the increased awareness and formalization of the ecosystem approach have emerged as a result of international environmental agreements within the framework of the united nations. a fundamental description of the basis of an “ecosystem approach” was first formalized in the stockholm declaration in 1972 (turrell, 2004). the most authoritative account of the ecosystem approach is probably that found in decision v/6 from the meeting of the conference of the parties to the un convention on biological diversity in nairobi, kenya, in 2000. this decision has an annex with the description, principles and operational guidance for application of the ecosystem approach (www.biodiv.org/decisions/ default.asp). the ecosystem-based approach to aquaculture management ecological integrity is a state of the ecosystem in which ecological diversity and resilience are present, allowing the ecosystem to sustain itself and the inhabitants dependent on it. integrity of the ecosystem cannot be achieved, however, when irresponsible actions impair the beneficial uses of resources. scientific inquiry, public policy development, and co-management programs are essential for achieving and maintaining ecological integrity. an ecosystem approach entails an integrated, multiresource emphasis, and broad precautionary strategies that anticipate and prevent environmental damage. this approach respects and affirms the interconnectedness of ecological processes and requires people to understand and conduct themselves as an integrated part of the ecosystem rather than as an entity separate from it. the ecosystem approach is a strategy for the integrated management of land, water and living resources that promotes conservation and sustainable use in an equitable way. thus, the application of the ecosystem approach will help to reach a balance of the three main objectives: conservation, sustainable use, and fair and equitable sharing of the benefits arising out of the utilization of the natural resources. design of ecosystem-based management systems aquaculture and fisheries ecosystem-based management plans should be designed and implemented based on the principles, goals and policies below, which have been adapted for aquaculture from the ecosystems principles advisory panel (1999). principles • define the goals and constraints that characterize the desired state of aquaculture and fisheries and undesirable ecosystem changes. • the ability to predict ecosystem behaviour or impact is limited. • ecosystems have real thresholds and limits which, when exceeded, can affect major system restructuring. • once thresholds and limits have been exceeded, changes can be irreversible. 2 science diliman ecosystem-based approach to aquaculture management • diversity is important to ecosystem functioning. • components of ecosystems are linked (watershed, water bodies). • ecosystem boundaries are relatively open. • ecosystems change with time. • impacts can be cumulative. goals • maintain a healthy and sustainable ecosystem • provide profitable and sustainable livelihoods • production within safe carrying capacity constraint • there should be no irreversible changes in ecosystems (including the human dimension). policies • implement precautionary management measures that take account of aquatic species and industry interactions, and are adaptive to local environment and conditions. • allocate fishing rights and fishing quotas that are fair in a transparent process. • allocate aquaculture licenses in a fair and transparent process that favors the poor or landless. • plan sustainable production capacity on best available scientific knowledge. • develop management plans holistically. • learn from historical experiences. • monitor environment regularly and compare with previous years. • enforce quotas, remove unlicensed facilities. • promote participation, fairness, and equity in policy and management. • ensure participatory and transparent decision making. • protect the ecosystem for habitat and species of special concern or that are vulnerable. • provide management support, including scientific information, enforcement, and performance evaluation. • provide incentives for conservation and efficient use of living resources. ecosystem management plans should highlight: • a hierarchy of management entities, from an ecosystem scale (national government) to the local scale (local government and communities); • zoning of areas for fisheries and aquaculture, including marine and freshwater protected areas and other geographically defined management measures; and • specification and sizing of authorized fishing and aquaculture activities, with protocols required for licenses, authorizations and production quotas. ecosystem management plans need to be based on scientific information that is relevant, responsive, respected, and sound. a multifaceted approach is needed, including monitoring of aquaculture, fisheries and ecosystems, monitoring of aquaculture production and fisheries catch, and monitoring fisheries resources. scientific support should be to undertake the necessary research on which informed management decisions can be made. these include studies on carrying capacity and ecological modeling. scientists should have collaborative research with the aquaculture and fishing industry, with transparent quality assurance of scientific advice. principles underlying ecosystem-based aquaculture management there are certain underlying principles that must be considered in the development of ecosystem based management of aquaculture (http://www.glc.org/ ecochart/principles.html#rights#rights). these are as follows: science diliman 3 white, p. & san diego-mcglone, m.l. responsible to look after the ecosystem people have a right to live in an ecosystem that supports their health and well-being and provides a livelihood. the diverse communities of other organisms living in that ecosystem also have their own rights. people must accept responsibility to conduct themselves, individually and collectively, in ways that support a healthy ecosystem. responsible use of natural resources people have the right to use natural resources for economic purpose and enjoyment, commensurate with the responsibility to rehabilitate and maintain the integrity of the ecosystem. people must take responsibility to enhance and maintain the health of the ecosystem for the use, benefit and enjoyment of the current and future generations. people must adopt, pursue and promote principles and practices of sustainable use of the ecosystem resources. people must accept the responsibility to minimize or prevent activities that cause environmental harm. scientific research and support there should be a coordinated, multidisciplinary research agenda, as it is necessary to improve understanding of the scientific, social, and economic dimensions of the ecosystem. scientific, social, and economic data and information should form the basis for public policies, agreements, and management plans for the ecosystem. many aspects of the ecosystem and its dynamics are not well understood. a scientific program of basic and applied research is necessary to improve understanding of the ecosystem and the interactions between aquaculture, fisheries, and the environment. partnerships should be formed among public agencies, academic institutions, businesses, and citizen organizations to conduct and coordinate basic and applied research on the ecosystem. action plans should be prepared for emergency response to pollution events. applied research should be conducted on methods to mitigate impact on the environment. the research results should be made available and understandable to the public and useful to decision makers. sustainable communities in a sustainable society, there are fundamental and inextricable links between economic activity and the natural ecosystem. sustainable economic activity should meet the needs of the present generation without compromising the ability of future generations to meet their own needs, and respect the limits imposed by the capacity of the ecosystem to absorb the impact of human activities. adopting principles of sustainability at the community and local government unit (lgu) levels will promote long-term economic viability and continued improvements in environmental quality. living in harmony with the ecosystem ecosystem integrity and the economic well-being of human communities are interdependent; achieving and protecting ecosystem integrity is, therefore, an essential part of economic activity within the area. natural resources within the ecosystem may supply people with drinking water, support a recreation/tourism industry, provide habitat for thousands of plant and animal species, offer livelihood opportunities, and support an agricultural industry. to ensure that natural resources in the ecosystem continue to provide such benefits, economic strategies and activities must ensure that essential ecological processes are maintained, natural resources are used sustainably, biological diversity is conserved, and infrastructure investment is appropriately pursued. principles of sustainability must be incorporated into public and private sector plans and programs that reflect an appropriate balance between ecosystem protection and economic development. policies and programs that provide for the efficient and sustainable use of natural resources should be supported to revise or eliminate those that do not. they should also encourage the development of the aquaculture industry but avoid pollution and mitigate impact. there should be systematic data collection 4 science diliman ecosystem-based approach to aquaculture management monitoring and impact indicators to measure the environmental, social, and economic health of the ecosystem and well being to gauge progress in achieving a sustainable aquaculture and fisheries industry and society. management the ecosystem governance and management should emphasize partnership arrangements among government entities, the private sector, community organizations, and other interests. the interdependence of the economy and the environment amplifies the consequences of the individual and collective actions of all agencies, organizations, businesses, and individuals within the ecosystem. their mutual interests must be explicitly acknowledged and partnerships developed to pursue public and private sector actions that benefit the ecosystem. existing partnerships that integrate interests and management approaches in the ecosystem, such as protected area management boards (pambs) and other water management plans, should be supported and expanded. following the development of the ecosystem-based management plan, there should be full implementation of relevant government, provincial and municipal laws and programs, and the dedication of adequate resources to accomplish the stated goals. partnerships among basin interests should be formed to address commonly identified problems and to harmonize institutional relationships and authorities. ecosystem policies and programs should be based on the findings of sound scientific research. dissemination of plans and information timely, accurate, and accessible information should be provided to the public regarding all planned activities that may significantly affect the ecosystem. timely information enables the public to respond to current issues and opportunities in an appropriate time frame; accurate information enables the people to make informed decisions about their interests and concerns; and accessible information allows for all interested persons to obtain the desired information with relative ease. programs that reflect these qualities help promote informed public policy, efficient and effective implementation and strong partnerships among the different users of the resource. timely, accurate, and meaningful information should be gathered about the state of the ecosystem. monitoring programs and reports made on the progress in implementing programs consistent with the management plan and other relevant laws and agreements and be made available to the public and local communities. full and equal access should be made available to the public on public data, policies and related information concerning current and prospective conditions of the ecosystem, and the associated impact of proposed actions. formal and informal information links should be created to ensure ongoing and substantive dialogue on and dissemination of data and information relating to the ecosystem. decision-making responsible aquaculture and fisheries management planning requires many decisions about goals and objectives, a precautionary sustainable carrying capacity, sustainable fishing catches, a rights-based allocation of licenses method that is deemed to be fair, and avoidance of unacceptable changes in ecosystems. as noted above, many of these decisions are subjective. therefore, stakeholders need to have the opportunity to participate in the decision-making process and they need to be able to understand the basis for decisions. stakeholders include the fish and mollusc farmers, fishermen, environmentalists with concerns about the effects of aquaculture on ecosystems, and anyone who is interested in the distribution of benefits. there are also many different types of arrangements for stakeholders to participate in decision-making, science diliman 5 white, p. & san diego-mcglone, m.l. ranging from having input into the initial stages of decision-making where options are being formulated, to having the opportunity to comment before final decisions are made. responsible fisheries and aquaculture management requires as much participation in decision-making as is practical, recognizing that ultimately, a management authority (e.g., government officials or an association of stakeholders) must be charged with weighing the options and making a decision. ecosystems and fisheries resource populations cover large geographic areas. conservation measures and ecosystem protection need to be effective over the entire range of resource populations and the area of ecosystems. they are usually affected by the activities of many local communities. the management plan therefore needs to be holistic and implemented in a cooperative manner over the entire ecosystem by the lgus and local communities. the local authority is likely to be limited to making decisions about the implementation of higher-level decisions. one purpose of participatory and transparent decision-making is to gather the broadest possible support for decisions. however, there will usually be some participants in the process who could be unhappy with the outcome. responsible aquaculture and fisheries management requires that even those who dislike decisions are nevertheless bound by them. management support fisheries and aquaculture management depends on scientific information. it is ineffective unless there is compliance with management legislation and regulations (sustainable carrying capacity, conservation measures, allocation rules, and restrictions for ecosystem protection). fisheries and aquaculture management needs to evaluate its own performance in order to be responsible. sissenwine & mace (2001) identified that management needs to be: • relevant by providing the type of information that is needed in a form that fisheries managers can use, and that stakeholders can understand; • responsive by being timely; • respected (i.e., credible), which means that it must be perceived to be unbiased, and based on science conducted according to high scientific standards, including quality assurance; and • right, which requires an investment in research and appropriate data, in addition to high scientific standards and quality assurance. compliance with fisheries and aquaculture management rules requires either rules that the aquaculture and fisheries industry believes in, such that most of the industry willingly comply and they do not tolerate noncompliance by others in the industry; or it requires enforcement capability and severe enough penalties to force compliance. the former is preferable. performance evaluation is a valuable element of a fisheries and aquaculture management system because it is a way of learning from experience, so that management can be improved. fao (1999) discusses indicators of sustainability for fisheries. indicators for aquaculture will include fish growth rate, survival, and food conversion rate (fcr). such indicators can serve as the basis for performance evaluation. since ecosystems are defined geographically, an ecosystem approach to responsible governance of fisheries and aquaculture requires management institutions or arrangements that are defined geographically. accordingly, there should be freshwater and marine ecosystems management, covering coastal waters that account for most aquaculture production, as logical units for research and governance. however, the geographic areas used for management should be sufficiently large to encompass reasonably self-contained ecosystems throughout their range. ecosystem-based environmental management and relevance to the philippines agriculture and fisheries in the philippines directly account for about a fifth of the total economy and directly and indirectly (which considers the 6 science diliman ecosystem-based approach to aquaculture management backward and forward linkages, or the cluster universe) three fifths of the economy. more importantly, these directly employ about 10 million people, nearly 40% of the labor force. in 2007, the philippines produced 2,214,826.16 metric tonnes from aquaculture (bas, 2007).aquaculture production is still rising rapidly. aquaculture tends to develop in “hot spots”, initially with pen culture in laguna de bay, and, recently, with milkfish cage and pen culture in dagupan and bolinao in pangasinan, and tilapia cage culture in taal lake. this rapid increase in production has put pressure on the aquatic ecosystems and incidences of fish kills have been observed. in general, the management of fisheries and aquaculture should be undertaken with an ecosystem approach based on their scientifically calculated safe carrying capacity and implemented in a coordinated way by the concerned lgus, through appropriate regulations for lake management. regulations covering aquaculture management in the philippines, the planning, management, monitoring, and control of fisheries and aquaculture have been devolved from the national government to lgus. in the case of lakes, there are three different regulations, namely: (a) the local government code or ra 7160 of 1991 which provides for the empowerment of the lgus; (b) the national integrated protected areas system act or ra 7586 of 1992 which provides for the establishment and management of national integrated areas system defining its scope and coverage. the act includes the protected area management board (pamb) which is responsible for the general administration of the area; and (c) the fisheries code or ra 8550 of 1998 which aims for the rehabilitation of fisheries and other aquatic resources through enforcement of laws and regulations. agencies governing aquaculture development at the national level, the two principal agencies with coastal management responsibilities that apply to aquaculture are the department of environment and natural resources (denr) and the department of agriculture-bureau of fisheries and aquatic resources (da-bfar). these two agencies have retained authority over some land and water uses, management activities, and specific geographic areas. while national government has devolved significant authority to the local level, national government agencies have maintained significant institutional presence especially at the regional, provincial, and in the case of one agency, municipal level (lowry, et al., 2005). da-bfar and denr have offices and staff at regional (multiple provinces) and provincial levels. the denr has staff responsible for covering jurisdictional responsibilities in multiple municipalities. the department of interior and local government (dilg) is the primary national government agency responsible for overseeing, monitoring, and evaluating lgus and the devolution process. every municipality has one member of staff assigned from dilg. in spite of the broad representation of national government agency staff at provincial and municipal levels, coordination between national and local government is weak and major capacity gaps exist. while lgus are generally well versed in the provisions of the local government codes, they are less knowledgeable about special laws, such as the fisheries code, and environmental laws that are primarily under the jurisdiction of national government agencies. the primary implementing agency for the 1998 fisheries code is the dabfar; however, many of the provisions in the law relate specifically to lgus. implementing rules and regulations and administrative orders are issued describing specific implementation requirements. unfortunately, the overlap in responsibilities, laws, and regulations has led to confusion as to the roles of dabfar, denr, dilg, and the lgus in terms of planning, monitoring and control of aquaculture and fisheries, and has resulted in uncoordinated, science diliman 7 white, p. & san diego-mcglone, m.l. uneven, and, often, unsustainable development of aquaculture. there is, therefore, a need to resolve inter-agency roles and improve linkages between dilg, dabfar, and denr and encourage cooperation between the main agencies involved with aquaculture development, management and control. the inter-agency councils could be used for resolving conflict, overlap between agencies and to encourage cooperation between the agencies. there may be a need to draft joint bfar/ dilg administrative orders or ordinances for monitoring and control of aquaculture development. there are a number of initiatives to coordinate the planning and management of aquatic ecosystems among key national agencies. the protected area management boards of national protected areas are usually comprised of representatives of these different government bodies and private sector stakeholders. the joint memorandum order on the implementation of the fisheries code between denr and da-bfar is a start at the national level. the philippine fisheries code of 1998 provided for the creation of fisheries and aquatic management councils (farmcs) to act as consultative bodies of the lgus in determining priorities on fishing activities of municipal fishermen and aquaculture development. they also assist lgus in the preparation of the municipal fishery development plans, recommend fishery and aquaculture ordinances and assist in the enforcement of laws. the da-bfar, lgu and coastal resource management plan (crmp) have worked together in establishing and strengthening the capacity of farmcs to fulfill their role in coastal resource management. identification of ecosystems and aquaculture zones aquatic ecosystems have already been identified within the clean water act prepared by denr. aquaculture zones have been identified by the conservation international priority areas, and the fisheries resource management project (bfar). these two resources should be used for identifying the aquatic ecosystems that are of significant importance for aquaculture. the identified areas should then be checked to ascertain if these aquatic ecosystems have significant aquaculture production and ranked in terms of scale of resource use (low, intermediate, high). carrying capacity of ecosystems there is, presently, little known about the sustainable aquaculture carrying capacities of water bodies in the philippines. however, there is a need to base aquaculture development on the sustainable carrying capacity of the resource and to develop quality standards for aquaculture that can act as a measure of compliance. carrying capacity can be calculated by using box models, such as the environmental state variable (or vector) model concept (dowd, 2005). these models can estimate the “state” or category, such as trophic state (eutrophic), or if the nutrient concentration in the water will be above a set quality standard. models can represent some or all of these variables by dynamic equations. state variables for assimilative capacity models include: • concentrations of drivers such as nutrients; • environmental factors such as temperature; • environmental quality variables (eqvs) defined by the regulators, such as dissolved oxygen concentrations. some variables may belong to several categories. the simplest models average values of each state variable over a substantial homogeneous boxed volume that, ideally, corresponds to a defined water body. more universal models, such as european regional seas ecosystem model (ersem), deal with a large number of linked volumes that may represent whole sea areas (baretta, et al, 1995). models such as these, need to be tested and validated in different types of water body in the philippines so that carrying capacities of the identified ecosystems suitable for aquaculture can be calculated. in addition, there is a need to review the water quality standards in waters where aquaculture is carried out and maximum limits set 8 science diliman ecosystem-based approach to aquaculture management so that these areas are not overly impacted by aquaculture. coordinated planning and zoning of aquaculture there are two major aquaculture zoning plans developed, one by da-bfar (coastal resource management plan) and the other by denr (coastal development plans). in order for coordinated planning and zoning of aquaculture development, these two agencies need to work together to harmonize the plans. the criteria used for zoning aquaculture areas in these plans also need to be reviewed to ensure that they were based on relevant scientific criteria. coordinated co-management of ecosystems by surrounding lgus the fisheries ordinances issued by da-bfar are implemented by the coastal lgus. in many cases, the lgus surrounding an aquatic ecosystem act independently with varying levels of implementation of the ordinances and management of aquaculture. there is a need for a framework that encourages these lgus to work together in a coordinated manner. however, there is need for an ecosystem-wide development plan for aquaculture and a need to motivate lgus to plan and manage aquaculture in each ecosystem responsibly and sustainably. in protected freshwater ecosystems, there are the pambs that bring lgus in the catchment area together for unified planning and management of that aquatic ecosystem. in marine and brackishwater areas, there are integrated fisheries and aquatic management councils (ifarmcs) that have been created in areas such as bays, gulfs, lakes and rivers, and dams, which are bounded by two or more municipalities/cities. ifarmcs could potentially undertake the same role as pambs but this needs to be prioritised and implemented within the ifarmcs. conclusions an ecosystem-based co-management should be applied to aquaculture planning and development in the philippines following a number of steps. aquatic ecosystems and ecosystems with aquaculture or potential aquaculture should be identified using the clean water act as basis. this data should be entered in a gis database for easy data storage and analysis. an estimate of the safe and sustainable aquaculture carrying capacity of the identified ecosystems should be made based on best available science. within each ecosystem, an assessment should be made on the industries that have greatest impacts on the ecosystem. within each ecosystem, an integrated development plan should be made (taking aquaculture into consideration) and prioritize commercial activities in the aquatic ecosystem. a framework of planning and management should be developed at national, municipal, and local government levels using pambs and ifarmcs as the bases. develop a management plan that is within the capabilities and funding of lgus that will have to implement it. find ways to encourage lgus to implement the management plan and enforce aquaculture regulations. the national government must support the lgus by formulating enlightened policy and sensible regulations, as well as undertaking or assisting with the collection of baseline environmental data on the ecosystem, estimation of the safe and sustainable aquaculture carrying capacities, and monitoring the environmental impacts of aquaculture. references baretta, j.w., ebenhöh, w. & ruardij, p., 1995. the european regional seas ecosystem model, a complex marine ecosystem model. netherlands journal of sea research 33:233-246. bureau of agricultural statistics, 2007. http://countrystat.bas.gov.ph dowd, m, 2005. a bio-physical coastal ecosystem model for assessing environmental effects of marine bivalve aquaculture. ecological modelling 183(2-3):323-34. white, p. and san diego-mcglone, m.l. 50 ecosystem principles advisory panel, 1999. ecosystem-based fishery management. u.s. department of commerce. washington, dc, usa. pp54 . science diliman 9 white, p. & san diego-mcglone, m.l. fao, 1999. indicators for sustainable development of marine capture fisheries. fao, rome. pp 68 fenchel, t., 1987. ecology – potentials and limitations. in kinne, o. (ed.). excellence in ecology. book 1. international ecology institute, oldendorf/luhe. likens, g., 1992. the ecosystem approach: its use and abuse. in kinne, o. (ed.). excellence in ecology. book 3. international ecology institute, oldendorf/luhe. lowry k., white, a. & courtney, c., 2005. national and local agency roles in integrated coastal management in the philippines. ocean & coastal management 48:314– 335. republic act no. 7586, nipas law . http://eelink.net/~asilwildlife/republicact7586.html republic act 8550. http://www.da.gov.ph/fishcode/ ra8550b.html sissenwine, m.p. & mace, p.m., 2001. governance for responsible fisheries: an ecosystem approach paper to the fao reykjavik conference on responsible fisheries. turrell, w.r., 2004. the policy basis of the “ecosystem approach” to fisheries management. eurogoos publication no. 21, eurogoos office, smhi, 601 76 norrköping, sweden. 10 science diliman page 1 images image 1 page 2 images image 1 page 3 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 21_hybrid a hybrid lbfgs-de algorithm 89 a hybrid lbfgs-de algorithm for global optimization of the lennard-jones cluster problem ernesto padernal adorio department of mathematics, college of science, university of the phillipines, diliman, quezon city 1101 e-mail: adorio@math.upd.edu.ph; eadorio@yahoo.com bobby ondoy corpus department of computer science, collge of engineering, university of the philippines, diliman, quezon city 1101 e-mail: bcorpus jr@yahoo.com science diliman (july–december 2004) 16:2, 89 the lennard-jones cluster conformation problem is to determine a configuration of n atoms in threedimensional space where the sum of the nonlinear pairwise potential function is at a minimum. in this formula, ri,j is the distance between atoms i and j. this optimization problem is a severe test for global optimization algorithms due to its computational complexity: the number of local minima grows exponentially large as the number of atoms in the cluster is increased. as a specific test case, a better cluster configuration than the previously published putative minimum for the 38atom case was found in the mid-1990s. various algorithms have been tried for determining putative global minimum of lennard-jones clusters, for example, simulated annealing and genetic algorithm. there is a fast multistart two-step algorithm which can sometimes find the minimum potential energy clusters in seconds, but it works with a modified lennard-jones potential formula. in this paper we present a hybrid limited memory bfgs (l-bfgs) algorithm and a modified differential evolution (de) algorithm for determining the global minimum potential energy configurations of atom clusters using the unbiased or unmodified potential lennard-jones function. it performed with 100% reliability for clusters containing 2–50 atoms. the algorithm has excellent potential for solving other difficult global nonlinear optimization problems. the work of the two authors was supported by a research grant from the natural sciences research institute (nsri). ( ) 12 6 , 1 2 , , i j i j ij f x y z r r< = −∑ 04_hopping marco and villagonzalo 10 hopping in a molecular wire r. marco*, r. banzon, and c. villagonzalo structure and dynamics group, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: rmarcojr@nip.upd.edu.ph science diliman (july-december 2004 ) 16:2, 10–11 *corresponding author we utilize the hubbard model approach in analyzing the effect of the hopping parameter in the transport properties of a two-atom molecular device (kostyrko & bulka, 2003). the system consists of a molecule with two identical atoms connecting two metal electrodes. in this model, two important and realistic energy parameters are considered which affects the transport properties. the first is the kinetic energy required to “hop” from one atomic site to the next. we call this parameter the hopping term denoted by t, in arbitrary units of energy. the next is an on-site coulombic repulsion, u, between two electrons on the same atomic site. the molecular wire is represented by the hubbard hamiltonian . (1) we then used the nonequilibrium green’s function formalism (datta, 2000) to obtain the transport equations for the system. these transport equations are solved self-consistently. we utilized a root finding method to implement such calculations. our results for the current at t = 1.0 agree with that in the literature (kostyrko & bulka, 2003). the behavior of the current-voltage characteristics was shown to depend on the position of the molecular selfconsistent energy levels with respect to the fermi energy levels. for u > 0, the molecular levels are pinned between the fermi levels of the source and drain electrode for values of t ≤ 0.5 . the threshold voltage is fixed for these values of t ( fig. 1) and continuous to increase for values to t < 0.5. for u = 0, the molecular levels continue to decrease as t decreases. the threshold fig. 1. current-voltage characteristics for u = 4 for different values of t. 1.5 1 0.5 0 1 2 3 4 5 6 v (t/e) j ( e γγγγ γ /2 h ) 0 fig. 2. current-voltage characteristics for u = 0 for different values of t. 2 1 0 1 2 3 4 5 6 v (t/e) j ( e γγγγ γ /2 h ) 0 ( ) †, , , , , , 'mol i i j i j i i i j i h e t c c u n nσ σ σ δ ↑ ↓= + +∑ ∑ ∑ hopping in a molecular wire 11 voltage reaches its minimum at t = 0.5 (fig. 2) corresponding to the point where the antibonding energy levels at v = 0 are located between the fermi level of the source and drain. when both molecular levels are below the fermi level of the drain, transport does not occur. references kostyrko, t. & b. bulka, 2003. hubbard model approach for the transport properties of short molecular chains. phys. rev. b. 67: 205331. datta, s., 2000. nanoscale device modeling: the green’s function method. superlattices and microstructures. 28: 4. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 5 freshwater and land mollusk diversity patterns along dakil river at the university of the philippines laguna land grant (upllg), paete, laguna, luzon island, philippines kinsley meg g. perez* school of environmental science and management university of the philippines los baños julius a. parcon museum of natural history university of the philippines los baños emmanuel ryan c. de chavez animal biology division institute of biological sciences university of the philippines los baños abstract prior to this study, there had been no malacofaunal study in the university of the philippines laguna land grant (upllg). to address this, a diversity survey of its freshwater and land mollusks was conducted. a total of 25 quadrats (15 m2) on upstream and downstream stations along dakil river and its tributaries for freshwater mollusks and 12 quadrats (100 m2) for land snails were set randomly to correlate their diversity patterns with environmental variables. from 115 individuals of freshwater mollusks, seven species (six gastropods, one bivalve) belonging to six families (ampullariidae, corbiculidae, lymnaeidae, neritinidae, thiaridae, and viviparidae) were identified. on the other hand, seven species belonging to three families (ariophantidae, camaenidae, and chronidae) were identified among 28 land snail individuals. malacofaunal survey revealed that the area along dakil river has low diversity in both freshwater mollusk (h’=1.40) and land snail (h’=1.19). generalized linear mixed models (glmm) revealed river velocity was the most significant predictor for species richness of freshwater mollusks, and abundance was highly affected by temperature and inversely affected by canopy cover. furthermore, altitude was the most significant predictor for species richness of land snails and canopy cover for abundance. understanding the molluscan diversity could help determine the environment and ecological conditions of the watershed for its effective management and conservation.  keywords: diversity, freshwater mollusks, land snails, dakil river, species richness, abundance * corresponding author science diliman (july-december 2021) 33:2, 5-29 freshwater and land mollusk diversity patterns 6 introduction mollusks have a significant role in the ecosystem by being key prey items, active detritivores, and efficient calcium recyclers. mollusks such as gastropods and bivalves are also good habitat indicators because they can be found in areas with good habitat quality. however, one of the factors in the decline in their diversity and abundance are anthropogenic activities resulting in habitat destruction and modification. riparian forests along tropical streams are one of the important ecosystems for the targeted conservation of mollusks (clements et al. 2006); however, very few studies have examined mollusk communities in the forests along such waterways. in the philippines, flores and zafaralla (2012) found water quality deterioration of mananga river in cebu based on physicochemical analyses. this deterioration was validated by low diversity index. with the extensive destruction and disturbance of freshwater and tropical forest ecosystems, there is a need for studies on malacofaunal biodiversity to provide useful and realistic data for conservation and management (pérez-quintero 2007). one of the ideal sites for malacofaunal study is the watershed streams of the university of the philippines laguna land grant (upllg) in paete, laguna. it is a naturally grown secondary forest between pakil and paete. in 2001, upllg was included as an important biodiversity area (iba) as well as key conservation site in the philippines since many threatened and restricted-range birds among the luzon endemics have been recorded in the area (mallari et al. 2001; birdlife international, 2016). upllg serves as a habitat for various lowland rat species, civets, and fruit bats, and supports heavily hunted large mammals such as the philippine warty pig (sus philippensis) and long-tailed macaque (macaca fascicularis). the threatened endemic gray’s monitor lizard (varanus olivaceus) is also known to occur in the area (birdlife international 2016). prior to this study, there had been no study on the diversity of freshwater and land mollusks in the watershed streams of upllg. understanding the molluscan diversity could help determine the environment and ecological conditions within this watershed for its effective management and conservation. thus, this study aimed to 1) determine the diversity of freshwater and land mollusks in the watershed streams of dakil river in upllg; 2) correlate the species richness and abundance with selected environmental variables; and 3) quantify the effect of each environmental variable on molluscan species richness and abundance. k.m. g. perez et al. 7 materials and methods study site the university of the philippines laguna land grant (14o 23’ n, 121o 29’ e), formerly known as paete land reservation, is a 3,435.4 ha land area that has been owned by the university of the philippines since 1964. it is located across the municipalities of paete, pakil, and kalayaan in the province of laguna and partly in real, quezon province (figure 1). it is a mountainous area with an elevation of 300 to 400 masl and is considered a part of the sierra madre mountain range. historically, the area was exploited by slash-and-burn farming and logging that started in the 1920s and was severely logged in the 1960s, mostly on the outlying areas of balian and saray in pakil, laguna (gonzalez 1995). in the mid-1990s, there was still evidence of illegal logging in the area, specifically that trees with a diameter of less than 50 cm were being cut. at present, upllg has mostly disturbed secondary growth lowland dipterocarp forests that include certain areas of permanent agriculture figure 1. location of the study site in the university of the philippines laguna land grant, paete, laguna, philippines. (map source: google earth satellite image using qgis version 3.4 madeira). freshwater and land mollusk diversity patterns 8 (including rice paddies), kaingin, plantations, small settlements, and rural gardens (birdlife international 2016). it is not considered a protected area but it is managed and protected through the efforts of the laguna land grant management office of the university of the philippines los baños. upllg has a total land cover of 3,435.4 ha with four major rivers running through it, namely dakil, tibag, papatahan, and ginabihan, all of which drain towards lamon bay in quezon province. the dakil river has the largest watershed area, covering 2,989 ha with four tributaries draining to the main river. sampling protocol the sampling method for freshwater mollusks used in this study was adapted from the method of clements (2006). there were four tributaries present in the area, namely upstream 1 (up1), upstream 2 (up2), downstream 1 (dw1), and downstream 2 (dw2), and the main river (mr). both the tributaries and the main river were sampled. in each of these locations, five 3 x 5 meter (15 m2) quadrats were set at least 10 meters apart to prevent pseudoreplication. overall, a total of 25 quadrats were established for the whole study. physicochemical parameters were measured in each quadrat simultaneously with the freshwater mollusk sampling. water temperature, ph, and conductivity were measured in situ using a multiparameter water quality meter (oakton 35425-00, usa). at least three readings were measured at each sampling site. the average water current velocity was also measured using the floatation method (singh 2003; dobriyal et al. 2016) with five readings taken at each sampling site at equally spaced distances along the quadrat and then averaged. the substrate type was determined as gravel, pebbles, sand, and mud. moreover, 1 l of sediments per plot was collected for cascade sieving using 3 mm, 2 mm, and 1 mm steel meshes. samples were all fixed in 95% ethanol. the land snail sampling method used in this study was adapted from the method of de chavez and de lara (2011). a total of twelve 10 by 10 meter (100 m2) quadrats were randomly selected along the dakil river. the sampling sites were at least 10 meters apart to prevent pseudoreplication. the air temperature was measured by a digital thermometer (uni-t, model ut333, china). the number of trees and their diameter at 1.3 m from the base was determined using a tape measure. leaf litter depth was measured using a ruler placed vertically in the forest floor in the middle point and all the four corners of each quadrat. at least 1 l of topsoil was collected per quadrat. soil ph was also measured (milwaukee ph tester, usa). all geographic coordinates and elevation were determined using a garmin® gps 12 personal navigator® (garmin international, inc., usa). percent canopy cover was k.m. g. perez et al. 9 measured using a concave spherical densiometer (forest suppliers, inc., usa). direct hand-search of freshwater mollusks and land snails with a two-hour sampling effort was allotted in each quadrat (de chavez 2014). both freshwater mollusks and land snails were identified to the species level using published literature such as faustino (1930), bartsch (1932), springsteen and leobrera (1986), and abbott (1989). the sampling was done during the rainy season of october 2016. identified specimens were deposited as vouchers to the university of the philippines los baños museum of natural history. statistical analyses the individual count (relative abundance) and the total number of species per site (species richness) were determined for both freshwater mollusks and land snails. standard diversity indices (shannon-weiner and evenness) were also calculated using the software paleontological statistics (past) version 3.15 (hammer et al. 2001). in addition, an information-theoretic technique through model averaging (grueber et al. 2011) was conducted to determine the corresponding effects of environmental variables measured on the species richness and abundance of freshwater mollusks and land snails along dakil river. a generalized linear mixed-effect model (glmm) was implemented using the lme4 and mumin packages that were installed in r statistical software version 3.3.2. (r development core team, vienna, austria). in defining model parameters, the number of species and abundance were coded as response variables while environmental variables were identified as fixed factors. sampling sites were identified as the random variable. after running the codes, akaike’s information criterion corrected for small sample size (aic c ) appeared. this was used to assess model support, ranking each set using ∆aic c (burnham and anderson 2002). the most parsimonious models were selected based on the largest akaike information criterion corrected for small sample size by extracting the top five aic c . freshwater and land mollusk diversity patterns 10 results environmental variables water temperature throughout the stations was relatively uniform (24°c) while water ph was slightly acidic with a range of 5.08–6.37. however, this will vary across different seasons of the year. the sampling was conducted during the rainy season. fast water velocities were recorded in up2 station and dw2 station with a velocity of 6.54±0.16 m/s and 7.07±0.71 m/s, respectively. among all the stations, the dw2 and mr stations were found to have the deepest water with 30.68±6.32 cm and 49.0±0.58 cm, respectively. dw1 has the highest canopy cover among all the stations at 76% (table 1). table 1. summary of physicochemical parameters of the upstream and downstream stations of dakil river and its tributaries, up laguna land grant (upllg) environmental variables up1 up2 dw1 dw2 mr temperature (°c) 24.54 ±0.02 24.1 ±0.00 24.0 ±0.00 24.23 ±0.12 24.7 ±0.38 ph 5.08 ±0.28 6.37 ±0.10 6.09 ±0.17 6.20 ±0.30 6.37 ±0.38 conductivity (μs/cm) 22.39 ±0.12 25.14 ±0.12 29.7 ±0.43 32.17 ±0.86 23.47 ±0.68 velocity (m/s) 8.51 ±0.91 6.54 ±0.16 10.17 ±1.89 7.07 ± 0.71 10.87 ±0.81 water depth (cm) 18.2 ±0.36 14.9 ±1.16 14.30 ±1.74 30.68 ±6.32 49.0 ±0.58 canopy cover (%) 37.0 ±6.24 61.0 ±8.72 76.0 ±7.31 60.0 ±10.61 0 *substrate type c, s, p c, s, p c, s, m c, s, m m *m=mud, s= sand, p=pebble, c=cobble the sampling sites along the dakil river were characterized by altitudes between 252 to 387 masl with extensive canopy cover (50–70%) except for sampling site 1 (25%). air temperature ranged from 24 to 26°c. the soil was slightly acidic in nature with a ph range of 5.00 to 6.00. moreover, a few trees (<30) with relatively small diameters (<90 cm) were present in all the sampling sites (table 2). k.m. g. perez et al. 11 table 2. summary of the environmental variables measured in all 100 m2 sampling sites along dakil river in up laguna land grant (upllg) environmental variables 1 2 3 4 5 6 7 8 9 10 11 12 altitude (masl) 365 369 354 350 353 252 335 344 349 387 372 335 temperature (°c) 25 26 26 25 26 26 24 24 24 25 25 25 canopy cover (%) 25 50 50 50 50 75 75 50 50 50 50 75 number of trees 16 26 18 12 14 22 11 18 17 18 18 13 soil ph 6 5 5.67 ±0.33 6 6 5 5.33 ±0.33 5.33 ±0.33 5 5.33 ±0.33 5.33 ±0.33 5.33 ±0.33 leaf litter depth (cm) 5.18 ±1.12 6.64 ±1.32 3.48 ±0.87 4.74 ±0.88 8.40 ±0.73 6.90 ±0.60 5.32 ±1.05 7.66 ±1.19 1.62 ±0.45 4.04 ±1.37 4.58 ±1.43 2.82 ±1.16 tree diameter (cm) 36.40 ±4.11 31.70 ±3.12 44.40 ±4.34 43.30 ±5.11 33.60 ±4.56 36.10 ±3.96 53.70 ±9.31 48.20 ±10.35 27.80 ±6.41 38.20 ±6.73 42.60 ±7.11 32.60 ±6.57 freshwater and land malacofaunal diversity for freshwater mollusk diversity, a total of 115 individuals belonging to seven species (six gastropods and one bivalve) were recorded (figure 2). the prosobranch gastropod species belong to family thiaridae, which is represented by stenomelania macilenta (menke 1830) and melanoides tuberculata (müller 1774); family neritinidae represented by neritina sp.; family viviparidae represented by sinotaia angularis (müller 1774); and family ampullariidae represented by pomacea canaliculata (lamarck 1822). lymnaeidae represented by radix quadrasi (möllendorff 1898) was the only pulmonate observed at the site. corbicula fluminea (müller 1774) was the only bivalve representative. for land snail diversity, a total of 28 individuals belonging to seven species were recorded. these were macrochlamys sp. from family ariophantidae; calocochlea chrysocheila (sowerby i 1841), dryocochlias metaformis (férussac 1821) and helicostyla rufogaster (lesson 1832) from family camaenidae; and hemitrichiella setigera (sowerby i 1841), hemiglypta sp. and ryssota otaheitana (ferussac 1820) from family chronidae (figures 2 and 3). freshwater and land mollusk diversity patterns 12 a d g h k l i j b e c f figure 2. diversity of freshwater mollusks (a to g) and land snails (h to l) along dakil river, upllg: a. corbicula fluminea; b. radix quadrasi; c. melanoides tuberculata; d. sinotaia angularis; e. pomacea canaliculata; f. stenomelania macilenta; g. neritina sp.; h. calocochlea chrysocheila; i. dryocochlias metaformis; j. hemiglypta sp.; k. helicostyla rufogaster; and l. ryssota otaheitana. figure 3. live land snails found along dakil river in upllg: a. macrochlamys sp. found on a banana plant trunk; b. dryocochlias metaformis on the moist leaf litters of the forest floor; c. hemitrichiella setigera seen near the river; and d. ryssota otaheitana present near roots of trees. k.m. g. perez et al. 13 as for the overall malacofaunal diversity along the dakil river in upllg, 14 species of freshwater mollusks and land snails were recorded. freshwater mollusks were more abundant (115 individuals) than land snails (28 individuals), but both groups had similarly low diversity indices (h’) at 1.40 and 1.19, respectively. moreover, freshwater mollusks have an evenness value of 0.58 while land snails have 0.47 (table 3). table 3. summary of freshwater and land malacofaunal diversity in upllg parameters freshwater mollusks land snails total species richness 7 7 total number of family 6 3 total abundance 115 28 shannon-wiener (h’) 1.40 1.19 evenness (e) 0.58 0.47 the relationship among freshwater mollusks, sampling sites, and environmental variables is shown in the canonical correspondence analysis (cca) biplot (figure 4a). results showed that the closer the variables to the snail species, the stronger the dependence or association of that species to specific environmental variables. unrestricted monte carlo test for cca 1 and cca 2 was not significant at p = 0.48 and p = 0.35, respectively. the first two cca axes accounted for 51.70% and 27.69% variation in the taxon composition. the biplot shows that pomacea canaliculata and sinotaia angularis are related to depth and temperature. the bivalve, corbicula fluminea, was inversely correlated to fast current with slightly acidic water while melanoides tuberculata, radix quadrasi, and stenomelania macilenta were associated with low conductivity. neritina sp. was not related with any of the environmental variables measured since it was only found in a single site; therefore, it could not account for a direct relationship. on the other hand, the relationship among land snail species, sampling sites, and environmental variables along dakil river in upllg was shown using the cca biplot in figure 4b. the continuous environmental variables (altitude, temperature, canopy cover, soil ph, number of trees, diameter of tree trunks, and leaf litter depth) were represented as vectors while the nominal variable, sampling sites, was marked as black spheroids. land snail species were designated as blue spheroids. the dominant variables found were altitude, soil ph, and the number of trees. unrestricted monte carlo test for cca 1 and cca 2 was not significant at p = 0.14 and p = 0.72, respectively. the first two cca axes accounted for 49.35% and 29.20% variation in the taxon composition. it can also be seen in the cca biplot that freshwater and land mollusk diversity patterns 14 most of the land snail assemblage is in an area opposite to the direction of the altitude and soil ph. ryssota otaheitana was closer to altitude and soil ph compared to other snail species. among the seven species, macrochlamys sp. was not directly affected by the existing conditions in secondary forests since it was found only in a single site; thus, it is not directly related to the environmental variables measured. figure 4. canonical correspondence analysis (cca) showing the relationship of freshwater mollusks (a) and land snails (b) against selected environmental variables and sampling sites. a b k.m. g. perez et al. 15 after running the glmm in a global model, the standardized parameter estimates revealed that river velocity is the most significant predictor in determining the species richness (e = 0.1769, p<0.05) of freshwater snails along the dakil river in upllg (table 4a). in general, the estimated values showed a positive relationship between the response variables and the quadrats, i.e. more species and more numerous individuals will be expected in slow water velocity. in case of abundance, additional factors were influential such canopy cover (e = -0.0145, p<0.001) and temperature (e = 0.9621, p<0.001) (table 4a). moreover, since the parameter estimate values were relatively close to each other, model averaging was implemented to generate sub-models to explain more clearly the variables by considering the top two aic c . river velocity is revealed to be the most significant predictor (∆aic c = 0.0, waic c = 0.35) for species richness while null (∆aic c = 0.0, waic c = 0.39) or none of the mentioned environmental variables influences abundance. in addition, the standardized parameter estimates revealed that altitude is the most significant predictor in determining the species richness (e = -0.0106, p<0.10) of land snails along dakil river in upllg (table 4a). in general, the estimated values showed an inverse relationship between the response variable and altitude, i.e. more species and more numerous individuals will be expected in the lowest altitude. in the case of abundance, canopy cover was also influential (e= 0.0286, p < 0.10) (table 4a). moreover, since the parameter estimate values were relatively close to each other, model averaging was implemented to generate sub-models to explain more clearly the response variables by considering the top two aic c . for species richness, altitude (∆aic c = 0.00, waic c = 0.24) and null (∆aic c = 0.03, waic c = 0.24) were the most parsimonious model; for abundance (∆aic c = 0.00, waic c = 0.19), the most parsimonious model was canopy cover. null means none of the mentioned environmental variables influence species richness (table 4b). table 4a. generalized linear mixed models of each environmental variable on their corresponding effect on species richness and abundance of freshwater mollusks and land snails along dakil river in up laguna land grant, paete, laguna parameter estimateδ se p relative importance species richness interceptψ -0.2055 6.3683 0.9758 river velocityψ 0.1769 0.0800 0.0476* 0.60 water phψ -1.1401 0.8085 0.2026 0.22 temperatureψ 0.4690 0.5795 0.4558 0.12 interceptϕ 1.2833 4.0214 0.7681 freshwater and land mollusk diversity patterns 16 parameter estimateδ se p relative importance altitudeϕ -0.0106 0.0055 0.0954* 0.40 number of treesϕ 0.0646 0.0531 0.2934 0.13 canopy coverϕ 0.0111 0.0208 0.6337 0.09 abundance interceptψ -0.8982 8.3421 0.9149 canopy coverψ -0.0145 0.0038 0.00059*** 0.16 temperatureψ 0.9621 0.0038 < 2e-16*** 0.13 depthψ 0.0146 0.0179 0.4637 0.09 interceptϕ 1.7709 3.9034 0.6726 canopy coverϕ 0.0286 0.0148 0.0955*** 0.35 altitudeϕ -0.0097 0.0050 0.0894 0.33 soil phϕ -0.7715 0.5322 0.2084 0.19 ψfreshwater mollusks ϕland snails δeffect sizes have been standardized on two sd following gelman (2008) *(p < 0.05) ***(p < 0.001) table 4b. summary statistics of model averaging for species richness and abundance of freshwater mollusks and land snails. models are ranked based on akaike’s information criterion corrected for small size (aicc) component models k* aicc δaicc waicc species richness river velocityψ 5 55.84 0.00 0.35 nullψ 3 57.35 1.51 0.10 altitudeϕ 3 39.99 0.00 0.24 nullϕ 2 40.02 0.03 0.24 abundance nullψ 3 102.82 0.00 0.39 canopy coverψ 4 104.59 1.77 0.16 canopy coverϕ 3 47.52 0.00 0.19 altitudeϕ 3 47.70 0.19 0.17 ψfreshwater mollusks ϕland snails *k = number of parameters table 4a. generalized linear mixed models of each environmental variable on their corresponding effect on species richness and abundance of freshwater mollusks and land snails along dakil river in up laguna land grant, paete, laguna (con’t) k.m. g. perez et al. 17 discussion freshwater mollusks found in dakil river in up laguna land grant showed specific habitat preference. the community distribution might be affected by environmental factors operating at a more local scale instead of global factors operating on a larger scale such as latitude and temperature. in this study, freshwater mollusks were observed to be independent of the site but dependent on their specific microhabitat in the sites. the presence of diverse microhabitats in the heterogeneous environment may favor the existence of six species of gastropods and one species of bivalve, as it allows species to avoid competition (pointier 1993). in the study of liongson et al. (2005), temperature and rainfall vary with altitude, thus affecting the microhabitat of freshwater mollusks in mt. makiling. however, this study did not include altitude as an environmental variable for freshwater mollusks. the overall shannon-weiner in this study was relatively low (h’ = 1.40). according to magurran (2004), the diversity index in a natural ecosystem ranges from 1.5 to 3.5 where a value above 3.0 indicates a stable habitat while under 1.0 indicates a highly disturbed environment. moreover, flores and zafaralla (2012) stated that a diversity index lesser than 2.5 is relatively low, thus mollusk diversity in this study has a low diversity index. however, this low diversity index value of the sampling sites would be significant if the sampling was done for a longer period of time and seasonality, noting that in this present work the sampling was done for only three days. species richness varied significantly while the opposite was true for abundance against the river velocity, which selectively favored some species of freshwater snails. in the tributaries of dakil river, juvenile corbicula fluminea was frequently found. this could be attributed to its reproduction capabilities since this species is a simultaneous hermaphrodite, capable of crossing and self-fertilization (mcmahon 1982). in addition, a flexible feeding strategy may enhance the success of c. fluminea when phytoplankton resources are scarce. this species can deposit feed on organic matter on sediments (palmer 1999 as cited by freedman 2013). during deposit feeding, c. fluminea uses cilia on its foot to collect organic matter from the surface of sediments or interstitially while burrowing (reid et al. 1992 as cited by freedman 2013). for land snails, ryssota otaheitana is the most abundant and widespread species. because of its large size, which can reach up to 85 mm in shell diameter, this species can be easily seen during sampling (mostly at the base of buttresses) compared to other snails (de chavez and de lara 2011). it has discoidal and thick shells with a chestnut brown color in its spire and base (bartsch 1938 as cited by de chavez freshwater and land mollusk diversity patterns 18 and de lara 2011). its abundance is an indication that this species is the most ecologically established endemic. similar to the diversity of freshwater mollusks, the abundance of land snails along the dakil river in upllg is relatively low. moreover, in terms of diversity, the area has a shannon-weiner value of 1.19 while the evenness value is 0.47. there are several factors that cause this low diversity index. first is the acidic nature of the rainforest soil in the area with a range of 5 to 6. leaching of calcium, which is necessary for the egg and shell production of snails, is facilitated by active forest litter decomposition (schilthuizen and rutjes 2001; schilthuizen et al. 2003). second, all sites are exposed to anthropogenic activities such as illegal logging, slash-and-burn farming, and collection of timber and minor forest products for firewood and charcoal production (birdlife international 2016). resource extraction such as logging and wood harvesting is the top major threat to land snails. the demand for these resources results in modification or loss of the native forest habitat that eventually leads to the declining population of land snails (parent 2008; posa et al. 2008). snails are characterized by low dispersal rate and vagility, which make them vulnerable to anthropogenic disturbance (solem et al. 1981; strayer et al. 1986). loss of forest cover can lead to intense sun exposure, which land snails cannot tolerate. this causes desiccation, which leads to their mortality (asami 1993; douglas 2011; hodges and mckinney 2018). forest fragmentation caused by human-induced activities leads to less stable humidity and temperature microclimates in the tropical rainforest (tattersfield et al. 2001). waldén (1981) reported the decline of molluscan diversity because of forest canopy fragmentation that led to changes in sun and wind exposures, which directly affect snail survival. structural simplification resulting in the conversion of indigenous forests to plantations may also lead to microclimate changes, and thus affect litterdwelling species (de chavez 2008). changes in the distribution and abundance of individual land snail species in recent decades were documented as species have responded to land use pressures and climate change (hawkins et al. 1997; oke and chokor 2009; oke and chokor 2011). this phenomenon was also observed in this study. the low diversity and abundance of land snails may be an indicator that the molluscan communities in the area have not yet recovered from the intense anthropogenic pressures that had dramatic effects on the snail population. in a lotic environment, the abundance of freshwater mollusks is correlated with different environmental variables. in this study, the biplot correlated pomacea caniculata and sinotaia angularis to depth and temperature. most aspects of p. canaliculata biology are strongly influenced by temperature, on which its growth rates are highly dependent (estebenet and martin 2002). in temperate climates, seasonality imposes interesting periods during which growth ceases k.m. g. perez et al. 19 almost completely (estebenet and cazzaniga 1992). in small streams, they remain motionless under boulders or while entangled in submerged plants with their cephalopodium partially withdrawn, but quickly become active when taken out of the water (estebenet and martin 2002). this phenomenon was also observed in this study. pomacea and sinotaia occurred exclusively in low-depth marshes with plenty of floating, emergent, and submerged vegetation (estebenet and martin 2002). pomacea canaliculata are widely distributed throughout the tropical latitudes, occupying environments with slow flow or stagnant water (thiengo 1995 cited by giovanelli et al. 2005). p. canaliculata is among the 100 most invasive species in the world and is typically regarded as a pest in countries where it has been introduced— subsequently damaging crops and threatening native ecosystems (naylor 1996; cowie 2002; carlsson et al. 2004 as cited by tan et al. 2013). c. fluminea was inversely correlated to fast current with slightly acidic water. the abundance of c. fluminea generally increases with distance upriver and high sand content. clams utilized all parts of the habitat in these systems, including sand, gravel, mud associated with marshes, the main channel, and depths up to 12.8 m (freedman 2013). on the other hand, melanoides tuberculata, stenomelania macilenta, and radix quadrasi were associated with substrate types and river velocity. m. tuberculata thrives in shallow, slow running water, and on soft mud and sand substrates—where they feed on microalgae, detritus, epiphytic algae, and decaying plants—and even on deep zones of freshwater pools composed largely of rocks. in the study of giovanelli et al. (2005), m. tuberculata was strongly affected by water flow and rain distribution, which is the same in our study based on cca. prosobranchs prefer larger substrate as it provides a larger area for attachment, facilitates mobility, and is a source of algal assemblages and periphyton, the snails’ preferred diet (giovanelli et al. 2005; martinez and thome 2006). thiarids are known to exhibit parthenogenesis and ovoviviparity, and have evolved a cephalic brood pouch from which their young emerge. this has facilitated their dispersal (ben-ami and hodgson 2005). the parthenogenetic character, wherein most of the population are female, of the family thiaridae may have contributed to their success and abundance in the five study sites examined. the pulmonate radix quadrasi was positively correlated with different substrate types, i.e., mud, sand, pebble, and cobble. this species prefers slow-moving rivers with a muddy bottom. radix sp. can live on boulders or in low or low high-flow environments and can tolerate anoxic conditions, but it tends to prefer very lentic waters like lakes, bogs or slow rivers where there is a silt substrate (clarke 1981; jokinen 1992; sytsma et al. 2004). neritina sp. was not related to the selected environmental variable. it was found at very low abundance (one in a single site), therefore it could not account for a direct relationship between the abiotic factors and the bivalves. freshwater and land mollusk diversity patterns 20 the biplot of land snail species, sampling sites, and environmental variables along the dakil river in upllg showed that the dominant variables were altitude, soil ph, and the number of trees. it can also be seen in the cca biplot that most of the land snail assemblage is located in an area opposite to the direction of the altitude and soil ph vector. tattersfield et al. (2006) suggested that the diversity of mollusks is present at intermediate altitudes because of the faunal mixing of lowland and highland groups. it was observed that ryssota otaheitana is closer to altitude and soil ph compared to other snail species. macrochlamys sp. was not directly affected by the existing conditions in the secondary forests since it is considered an incidental collection. it was found on a banana plant trunk located between sampling sites. this is the limitation of cca; macrochlamys sp. was found only in a single site and thus was not directly related to the environmental variables mentioned in cca. all generated axes (p-value of axis 1 = 0.14; p-value of axis 2 = 0.72) were significant at p<0.81 using the monte carlo permutation test. in terms of increase in species richness and abundance at lower altitudes, it may be related to the combined effects of low rainfall conditions at low elevations and increasing effect of soil leaching at high elevations, both of which may limit mollusk diversity and abundance (de chavez 2008). land snail richness and abundance increase with increasing soil ph. abundance and diversity depend on the factors underlying ph such as calcium and not directly on ph levels (de chavez 2008). calcium-poor sites are those that experienced slashand-burn farming, which was evident in upllg in the 1920s until the 1990s when soil calcium was severely leached (gonzalez 1995). however, one limitation of this study is soil exchangeable calcium was not measured. microhabitats can no longer support the nutrient requirements of the re-colonizing land snails (de chavez 2008). this can potentially cause a lack of data on micro snails. shells are largely made of calcium carbonate and, although a protein-based periostracum is present, this protective layer is often eroded at the top whorls, thus initiating shell breakdown under acidic conditions. in addition, schilthuizen and rutjes (2001) mentioned that acidic soil ph in rainforests is facilitated by a high rate of forest litter decomposition that causes leaching of minerals such as calcium, which is important to snails for shell and egg production. in contrast with shells of living snails, dead and fossil shells were primarily affected by fragmentation, ornament loss, color loss, and carbonate coating. these taphonomic features fluctuated across time and space because of variable environmental conditions and/or time of exposure prior to shell burial (yanes 2012). k.m. g. perez et al. 21 the color loss and fragmentation were visible among the empty shells collected along dakil river in upllg, some of which had partly eroded periostracum while others were severely bioeroded. this can be attributed to slightly acidic to slightly basic soil ph present in sampling sites. moisture and low ph facilitate the rate of shell decomposition (reitz and wing 1999). on the other hand, alkaline soils rich in calcium delay this decomposition process (schilthuizen and rutjes 2001). being key prey to larger organisms or predators that crush and break their shells is another reason for the low diversity of land snails (pearce 2008; de chavez 2014). a terrestrial ecosystem is a hostile environment to land snails, which face a taxonomically wide range of predators (barker 2004). among the predators that feed on land snails is the threatened endemic gray’s monitor lizard (varanus olivaceus), which is also known to occur in upllg (birdlife international 2016). the diet of v. olivaceus (butaan) of polillo island and v. bitatawa of the sierra madre mountains of northern luzon consist almost entirely of fruits and snails (bennett 2014; law et al. 2018). they search for snails in decayed wood and on forest floors rather than in trees (bennett 2014). specifically, the most common snail included in the diet of butaan in polillo are helicostyla species (de chavez 2014). after the butaan eats the snails, shells are usually crushed and discarded on the ground (bennett 2014). glmm revealed the effect of single and combined environmental variables on species richness and abundance. dominant variables were determined. in a global model using glmm, the environmental variables affect the species richness and abundance of freshwater snails in the upllg watershed. it showed that species richness was highly affected by river velocity, while abundance was highly affected by temperature and inversely affected by canopy cover. canopy cover decreased in the main river both upstream and downstream while the temperature was constant at 24°c. current is the most significant characteristic of running water, and many stream animals are adapted to this. freshwater organisms in the streams such as mollusks are highly adapted to rapid waters due to their conchological characteristics (turreted, cylindrical, or rounded shape). on the other hand, some gastropods such as pomacea canaliculata and sinotaia angularis preferred slow or non-moving water and low canopy cover. this can be explained by the light availability in non-shading areas that cause the abundance of algae, which serves as food for these gastropods. however, shading and nutrient limitation in forest streams usually result in low-standing stocks of algae (dudgeon 2000), which are probably insufficient in terms of sustaining diverse molluscan communities. surface runoff and decaying vegetation in forest streams also produce low ph waters, which can retard snail growth by increasing calcium dissolution rates (johnson freshwater and land mollusk diversity patterns 22 1973; dussart 1976; hunter 1990). the frequency of c. fluminea in all sampling sites suggests that the bivalve species is adapted to both turbid and non-turbid water conditions with sandy stratum and slightly acidic water in dakil river, upllg. in the study of freedman (2013), c. fluminea abundance generally increases with distance upriver and high sand content. clams utilized all parts of the habitat in these systems, including sand, mud associated with marshes, and the main channel. c. fluminea was found in mud, gravel, and depths up to 12.8 m. prabhakar and roy (2008) noted that gastropods exhibited a preference for still or slow-moving waters and extended their limits to fast-running waters. on other hand, bivalves generally colonized non-turbid water with a sandy substratum. the occurrence of bivalves is indicative of non-polluted water and oxygen-rich habitat so they may be considered as bioindicators in inland waters (prabhakar and roy 2008). the availability of a suitable microhabitat may determine the stream contribution of the freshwater mollusk (bandel and riedel 1998). for land snails, after running the glmm in a global model, the standardized parameter estimates revealed that altitude is the most significant predictor in determining the species richness of land snails along the dakil river in upllg. in general, the estimated values showed an inverse relationship between the response variable and altitude, i.e. more species and more numerous individuals will be expected in the lowest altitude. in the case of abundance, canopy cover was also influential. moreover, since the parameter estimate values were relatively close to each other, model averaging was implemented to generate sub-models to explain more clearly the above response variables by considering the top two aic c. for species richness, altitude and null were the most parsimonious model; for abundance, it was canopy cover. null means none of the mentioned environmental variables influence species richness. canopy gaps are characterized by having decreased humidity due to elevated temperature compared to close-canopy forests. terrestrial gastropods are subjected to desiccation stress in these gaps. a study revealed that individuals of a common land snail in montane forest in puerto rico, caracolus caracolla (l.), moved from canopy gaps to the closed-canopy forest (bloch and stock 2014). both cca and glmm determine altitude as the major limiting factor for land snail diversity and abundance; however, these seem to be indirect relationships. k.m. g. perez et al. 23 conclusion corbicula fluminea is the most abundant freshwater mollusk while ryssota otaheitana is the most abundant land snail along the dakil river in upllg, paete, laguna. the diversity and abundance of both freshwater and land mollusks were low. moreover, mollusks along the dakil river have preferred microhabitats within the sites as supported by cca biplots. based on glmm, the species richness of freshwater mollusks was highly affected by river velocity while the abundance was highly affected by temperature and inversely affected by canopy cover. for land snails, altitude was the most significant predictor for species richness and canopy cover for abundance. existing microhabitats can no longer support the nutrient requirements of the re-colonizing land snails. historical habitat destruction and modification might have caused low diversity in the snail population as reflected by the present data. to further assess the malacofaunal diversity, it is recommended to conduct more sampling activities across upllg to generate more conclusive data on current molluscan populations. in addition, there should be an increase in the number of variables that might explain the diversity patterns for both freshwater and land mollusks. moreover, a seasonal variation in the study of the malacofauna should be included for further study. lastly, this study should also be replicated in various areas with similar historical environmental degradation episodes. acknowledgements we would like to thank dr. virgilio villancio, mr. pablo quilao, mr. alvin buquid, and the staff of the u.p. laguna land grant in paete, laguna for allowing us to conduct this study, and for their assistance during our fieldwork. we also thank mr. jonas llamas for his assistance during fieldwork. we would also like to thank the department of science and technology-accelerated science and technology human resource development program (dost-asthrdp) for the funding. freshwater and land mollusk diversity patterns 24 references abbott r. 1989. compendium of landshells: a color guide to more than 2,000 of the world’s terrestrial shells. melbourne, florida: american malacologists. asami t. 1993. interspecific differences in desiccation tolerance of juvenile land snails. funct. ecol. 7(5):571-577. doi:10.2307/2390133. bandel k, riedel f. 1998. ecological zonation of gastropods in the matutinao river (cebu, philippines), with focus on their life cycles. j. limnol. 34(2):171-191. doi:10.1051/ limn/1998017. barker gm. 2004. natural enemies of terrestrial molluscs. wallingford, oxfordshire (uk); 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[accessed 2019 nov 13]. http://geofaculty.uwyo.edu/neil/teaching/4880_files/hydraulicgeometry.pdf. solem a, climo fm, roscoe dj. 1981. sympatric species diversity of new zealand land snails. n. z. j. zool. 8(4):453-485. doi:10.1080/03014223.1981.10427971. springsteen f, leobrera f. 1986. shells of the philippines. manila philippines: carfel seashell museum. strayer d, glitzenstein js, jones cg, kolasa j, likens ge, mcdonnell mj, parker gg, pickett sta. 1986. longterm ecological studies: an illustrated account of their design, operation, and importance to ecology. occas. publ. inst. ecosyst. stud. 2:1-38. sytsma m, cordell j, chapman j, draheim r. 2004. lower columbia river aquatic nonindigenous species survey 2001-2004. center for lakes and reservoirs publications and presentations; [accessed 2021 nov 25]. https://pdxscholar.library.pdx.edu/centerforlakes_pub/23/. tan sk, lee yl, ng th. 2013 the status of the apple snail, pila scutata (gastropoda:ampullariidae) in singapore. nat. singap. 6:135-141 tattersfield p, seddon mb, lange cn. 2001. land snail faunas in indigenous rainforest and commercial forestry plantations in kakamega forest, western kenya. biodivers.  conserv.. 10(11):1809–1829. doi:10.1023/a:1013167726704. tattersfield p, seddon mb, ngereza c, rowson b. 2006. elevational variation in diversity and composition of land-snail faunas in a tanzanian forest. afr. j. ecol. 44(1):47–60. doi:10.1111/ j.1365-2028.2006.00612.x. waldén hw. 1981. communities and diversity of land molluscs in scandinavian woodlands. i. high diversity communities in taluses and boulder slopes in sw sweden. harxheim, germany: conchbooks; [accessed 2021 nov 25]. https://www.abebooks.com/communities-diversityland-molluscs-scandinavian-woodlands/18844672446/bd. yanes y. 2012. shell taphonomy and fidelity of living, dead, holocene, and pleistocene land snail assemblages. palaios. 27(3):127-136. k.m. g. perez et al. 29 ______ kinsley meg g. perez <kgperez2@up.edu.ph> was an instructor at laguna state polytechnic university-san pablo city campus (lspu-spcc) from 2013 to 2016 where she taught various math and science subjects. she is a 2018 m.s. environmental science graduate from the university of the philippines los baños (uplb) where she did research on land snails across habitat gradients as her thesis, which was a recipient of the academic achievement award (ranking 2nd among the  graduating class of the school of environmental science and management). she is a current board member of the malacological society of the philippines, and a member of unitas malacologica. she is currently a scholar of the department of science and technology-accelerated science and technology human resource development program (dost-asthrdp), and pursuing a ph.d. in environmental science and specializing in research on morphological and molecular biological techniques on freshwater snails.  julius a. parcon is a 2019 m.s. zoology graduate from uplb and a university researcher at the uplb museum of natural history (mnh), specializing in freshwater and terrestrial snails. prior to joining mnh, he has had experiences as an environmental management specialist, marine park ranger, and community organizer in two world heritage sites in palawan and in non-governmental organizations. he also specialized in marine biology, particularly corals and marine fishes. emmanuel ryan c. de chavez, ph.d. is an associate professor 5 at the animal biology division, institute of biological sciences, uplb where he has taught both undergraduate and graduate courses for 19 years. he is also an mnh curator for mollusks, the current president of the malacological society of the philippines, and a member of unitas malacologica. his research expertise is on tropical malacology, community ecology, evolutionary biology, and ecotoxicology. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 17_gargar 80 gargar and esguerra science diliman (january-june 2003) 15:1, 80-83 numerical investigation of non-homologous collapse of the one-dimensional gravitational gas kim gargar* and jose perico esguerra theoretical physics group, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: kim@nip.upd.edu.ph abstract in this paper, the one-dimensional gravitational gas is evolved numerically using an eventdriven code. two initial conditions are considered: (1) an initially uniform isolated system with no velocity dispersion and where the initial velocities are sine functions of the position, and (2) two “clusters” with initially constant phase-space densities in elliptical regions of phase space. introduction there have been attempts to study the time evolution of the one-dimensional gravitational gas both analytically (mineau et al., 1990; muriel & esguerra, 1996; muriel et al., 1998) and numerically (hohl & feix, 1967; severne et al., 1985). these attempts may be traced to two streams: the first, resulting from a long tradition of applying the kinetic theory to a gas of self-gravitating particles (chandrasekhar, 1942; landau & lifshitz, 1980; spitzer, 1987; lightman & shapiro, 1978; saslaw, 1985) and the second, arising from poincare’s unsuccessful attempt to solve the threebody problem, the emergence of computers, and the development of numerical analysis. the motivation for these is the problem of structure formation in the universe–a challenging problem of classical physics, which is of great interest today because of recent and still improving data on the cosmic microwave background. in its full glory, the problem of structure formation is a complicated nonlinear problem, which is difficult to treat both analytically and numerically. because of the above reasons, the interest in simplified models of structure formation persists to this day. one model, that of collapsing globular clusters, involves concentric spherical shells representing groups of stars with equal radial velocities (youngkins & miller, 2000). a variation of the model gives each shell constant angular momentum (barkov et al., 2002; klinko et al., 2001). a simpler model, that of self-gravitating parallel “infinite” sheets has been investigated for a longer time. it has been used in the past to model the behavior of halo stars (prendergast, 1954; camm, 1950; schilt, 1950). it is sometimes referred to as the self-gravitating one-dimensional system (sog) (youngkins & miller, 2000; wright & miller, 1984) or as the one-dimensional gravitational gas (1dgg) (hohl & feix, 1967; mineau et al., 1990). recently, a modification of the 1dgg, one that takes into account mass and energy loss due to the evaporation of stars, was introduced as a toy model of globular cluster (fanelli et al., 2001). in this paper, we study the non-homologous collapse of the 1dgg using numerical methods. * corresponding author 81 numerical investigation method consider a model of parallel self-gravitating “infinite” flat sheets, each with mass m i and allowed to pass through each other. the hamiltonian is (1) in the present study, every sheet has mass m = 1 = n, where n is the number of sheets in the system. to simplify further, we choose the scale such that 4πg is equal to one. in this system, the force acting on a sheet is constant as long as the sheet does not intersect (or “collide”) with any other sheet–this makes it easy to implement numerically.with the choice of units, the acceleration of a sheet is (2) where n r and n l are the number of sheets to the right and to the left of the sheet, respectively. if the sheets are ordered, the acceleration can be written as (3) where i, a positive integer, is the order of the sheet defined as follows: . (4) the evolution is then achieved using an event-driven algorithm described by fanelli et al. (2001). in this algorithm, intersection times between adjacent sheets are calculated, and the system is evolved up to the minimum intersection time giving a solution that is exact to machine precision. (a more sophisticated heap-based algorithm (noullez et al., 2001) which may give a faster running time is not used here.) the phasespace coordinates (x i and v i ) at a later time t is then recorded. two different configurations are analyzed: (1) an initially uniform isolated system with no velocity dispersion, and where the initial velocities are negative sine functions of the position (note that the choice of a negative sine dependence of initial velocity on the position is not essential–any other functional dependence of initial velocity on position, which assures that all sheets go towards the origin initially could have been chosen); and (2) two “clusters” with initially constant phase-space densities in elliptical regions of phase-space. results the results are shown in figs. 1 and 2. one may think of the results illustrated in both figures as metaphors: fig. 1 for the evolution of a collapsing galaxy, and fig. 2 for the evolution of two neighbor galaxies for different initial separation distances. ( ) 2 1 1, 2 2 n n i i j i j i i j ii p h g m m x x m π = = > = + −∑ ∑ ( )1 2 r la n n n = − ( )1 2 1 2 ia n i n = − + i ji j x x< → ≤ t = 0 t = 10 t = 20 t = 40 t = 50 t = 60 t =7 0 t = 80 t = 90 t = 100 t = 150 t = 200 t = 250 t = 300 t = 1000 fig. 1. evolution in phase-space of an initially uniform isolated system with no velocity dispersion and where the initial velocities are negative sine functions of the position. the sheets at t = 0, 40, 70, 100, and 250 are moving to the right while the sheets at t = 20, 60, 90, 200, and 1,000 are moving to the left–a collapse. 82 gargar and esguerra the following features in fig. 1 should be noted. at t = 0, the system is uniform and there is no velocity dispersion. at t = 10, the density at the origin is high. in the region near the origin are positions in which sheets moving with different velocities and in opposite directions can be found–this is called multistreaming. the region exhibiting multistreaming gradually expands. the formation of a high-density core region surrounded by a low-density halo region is apparent, beginning at t = 250. the following features in fig. 2 should be noted. in the time interval shown, the shape of the 300-sheet cluster in phase-space hardly changes, while that of the 100-sheet cluster changes a lot (e.g., at t = 7, the sheets from the 100-sheet cluster occupy a length that is about twice the length occupied at t = 0). it is tempting to conclude that the sheets from the 100-sheet cluster will eventually form a halo around the 300-sheet cluster, but the numerical simulations have not gone that far to make the conclusion definitive. t = 1 t = 4 t = 5 t = 6 t = 7 t = 8 t = 9 t = 10 t = 3 t = 2 t = 0 fig. 2. evolution of two clusters with initially constant phase densities in elliptical regions of phase-space. four initial conditions, which differ in the separation distance of the clusters, are evolved. the more massive cluster has 300 sheets while the other has 100. final remarks in this paper, the one-dimensional gravitational gas has been evolved numerically using an event-driven code for two initial conditions. features such as the onset of multistreaming and the formation of a core-halo structure have been exhibited. the event-driven code gives results that are exact to machine precision, but is rather slow compared to numerical integration, especially when the system being evolved has regions with high sheet density. however, one should not interpret this statement as an endorsement of faster routines employing straight numerical integration, such as the runge-kutta method–as a naive implementation yields nonphysical, non-energy conserving results fast. from here, one may pursue several research directions: (1) one may run the existing code on a faster computer; (2) implement a faster, more sophisticated (and still event driven) heap-based algorithm (noullez et al., 2001); (3) develop and optimize algorithms based on numerical integration using the existing event-driven code as a benchmark for accuracy; and, (4) 83 numerical investigation perform analytical work. analytical efforts dealing with essentially the same initial conditions are now under way (gargar, 2002). acknowledgment we acknowledge useful discussions with d. fanelli. references barkov, m.v., v.a. belinski, & g.s. bisnovatyi-kogan, 2002. model of ejection of matter from non-stationary dense stellar clusters and chaotic motion of gravitating shells. mnras. 334:338 camm, g.l., 1950. self-gravitating star systems. mnras. 110: 305. chandrasekhar, s., 1942. principles of stellar dynamics. chicago, univeristy of chicago press. gargar, k., 2002. numerical and analytical studies on model gravitating systems. ms thesis. university of the philippines diliman, quezon city. fanelli, d., m. merafina, & s. ruffo, 2001. one-dimensional toy model of globular clusters. phys. rev. e. 63: 066614. hohl, f. & m.r. feix, 1967. numerical experiments with a one-dimensional model for a self-gravitating star system. astrophys. j. 147: 1164-1180. klinko, p., b.n. miller, & i. prokhorenkov, 2001. rotationinduced phase transition in a spherical gravitating system. phys. rev. e. 63: 06613. landau, l.d. & a.e. lifschitz, 1980. physical kinetics. pergamon press. lightman, a.p. & s.l. shapiro, 1978. the dynamical evolution of globular clusters. rev. mod. phys. 50: 437-481. mineau, p., m. feix, & a. muriel, 1990. a perturbation method for high-virial gravitational systems in the collisionless regime. astron. astrophys. 233(2): 422-426. muriel, a. & p. esguerra, 1996. exact time evolution of the density of a classical many-body system: the open onedimensional gravitational gas. phys. rev. e. 54: 1433-1441. muriel, a., a. miciano-carino, & r. carino, 1998. structure formation in the one-dimensional gravitational gas. astron. astrophys. 334: 746. noullez, a., d. fanelli, & e. aurell, 2001. condmat/0101336. http://xxx.lanl.gov. prendergast, k.h., 1954. one dimensional self-gravitating star systems. astron. j. 59: 260-261. saslaw, w., 1985. gravitational physics of stellar and galactic systems. cambridge university press. schilt, j., 1950. the gravitational galactic force andthe density of interstellar matter. astrophys. j. 55: 97-110. severne, g., m. luwel, & p.j. rousseeuw, 1985. collisionless mixing in one-dimensional gravitational systems initially in a stationary waterbag configuration. astron. astrophys. 138: 365. spitzer, l., 1987. dynamical evolution of globular clusters. princeton university press. wright, h.l. & b. miller, 1984. gravity in one dimension: a dynamical and statistical study. phys. rev. a. 29: 14111418. youngkins, v.p. & b.n. miller, 2000. gravitational phase transitions in a one-dimensional spherical system. phys. rev. e. 62: 4583-4596. 12_flauta 57 thin film formation of gan science diliman (january-june 2003) 15:1, 57-60 thin film formation of gallium nitride using plasma-sputter deposition technique r. flauta*, t. kasuya, t. ohachi, and m. wada department of electronics, doshisha university kyotanabe, kyoto 610-0321 japan e-mail: eta1103@mail4.doshisha.ac.jp abstract the formation of gallium nitride (gan) thin film using plasma-sputter deposition technique has been confirmed. the gan film deposited on a glass substrate at an optimum plasma condition has shown x-ray diffraction (xrd) peaks at angles corresponding to that of (002) and (101) reflections of gan. the remaining material on the sputtering target exhibited xrd reflections corresponding to that of bulk gan powder. to improve the system’s base pressure, a new uhv compatible system is being developed to minimize the impurities in residual gases during deposition. the sputtering target configuration was altered to allow the monitoring of target temperature using a molybdenum (mo) holder, which is more stable against ga amalgam formation than stainless steel. introduction a number of studies have been made on gan thin film formation for its wide range of applications not only in the area of optoelectronics, but also in the production of high-power and high-speed electronic devices. the crystal quality of the gan thin films has been considerably improved by employing different growth techniques. among the popular methods are metalorganic chemical vapor deposition (mocvd) and molecular beam epitaxy (mbe) (sun et al., 2000; strite et al., 1992; nakamura et al., 1999; shen et al., 2001). high-quality gan had been fabricated with mocvd using trimethylgallium (tmg) and ammonia (nh 3 ) as the ga and n sources (nakamura et al., 1999). one of the earlier problems in mbe was the generation of sufficient nitrogen radical atoms to react with the ga source material (shen et al., 2001). the use of plasmaassisted mbe employing radio frequency (rf) or electron cyclotron resonance (ecr) microwave sources was recently revitalized to enhance the nitrogen reaction to that of ga and also to avoid the use of ammonia, which is highly toxic. however, the fundamental reaction mechanism for film formation is still unclear, and in particular, the role of excited nitrogen species responsible for the formation of good quality gan film has not yet been fully understood (morkoc, 1999). the n 2 plasma-sputter ga deposition can be an alternative technique to form gan thin film. in this paper, a deposition technique using a multi-cusp plasma sputter source is used to deposit gan thin film. basic principle the plasma parameters and the principle of depositing gan thin film were investigated and confirmed using a plasma chamber sealed with rubber o-rings, as shown in fig. 1.* corresponding author 58 flauta et al. the basic principle of the system is that the neutral ga is sputtered out of the negatively-biased target and the plasma is formed inside the chamber, surrounded by the magnetic cusp on the chamber wall, as illustrated in fig. 2. the formation of excited gallium and nitrogen molecules is through electron impact given in eq. (1) and eq. (3), respectively. the formation of excited atomic nitrogen is by dissociation process, as given in eq. (2). the reaction rate, given in eq. (4), is determined from the reaction cross-section, σ, and the electron velocity distribution function, f(ν e ). the plasma confinement magnetic field forbids ions to reach the substrate to avoid ion bombardment leading to film damage. the fluxes of the excited species of ga, n, and n 2 contribute to the formation of gan thin film on the substrate. plasma diagnostics were conducted to confirm these reaction mechanisms as reported in the previous paper (flauta et al., 2001). (1) (2) (3) (4) principle confirmation the system consisting of a plasma chamber (lower part) and a process chamber (upper part), both shown in fig. 1, was used to confirm the principle of plasma-sputter gan deposition. the plasma chamber (300-mm length, 210-mm diameter) has 10 rows of sm-co magnets, with four rows of sm-co magnets on each end plate arranged to produce a multi-line-cusp magnetic field. the plasma chamber houses the filament attached to the current feedthroughs inserted from the side of the chamber. langmuir probe, sputtering target to hold ga, and the quartz window for spectra analysis are also installed. the substrate holder is attached to the upper end of the process chamber away from the magnetic cusps. the electron emission from two 0.3-mm diameter, 9 cm long tungsten filaments sustains the plasma. both chambers 2 2n e n e ∗+ → + 2n e n n e ∗+ → + + ga e ga e∗+ → + ( ) ( ) ( ) 0e e e e e e k f dσ ν ν ν σ ν ν ν ∞ = = ∫ fig. 2. the basic principle of a multi-cusp plasma-sputtertype ion source system. neutral ga ga n+ n 2 + n 2 + substrate flux of excited species n2 + ga+n+ fig. 1. (a) photo and (b) schematic diagram of the multicusp plasma-sputter-type ion source. to tmp substrate holder substrate magnet w filament quartz window cusp magnetic field co sputtering target manipulator insulating glass cusp magnetic field ionization vacuum gauge magnet langmuir probe (b) (a) 59 thin film formation of gan are evacuated by a 160-l/s turbo molecular pump (tmp) coupled to a 300 l/min rotary pump at a base pressure of low 10–4 pa. gallium pellet of 99.9999% purity was used in the experiment. it was preheated and melted using an incandescent lamp and was spread evenly on the surface area of the sputtering target. the sputtering target was made of a circular stainless steel disk 30 mm in diameter and 3 mm thick. the disk had a 28-mm diameter and 1-mm deep hollow to hold the molten ga by gravity. the plasma characteristics of a multi-cusp plasma sputter-type ion source were optimized and resulted in a deposit of gan thin film. the electron density, n e , and electron temperature, t e , directly affected the film growth. at higher pressure, n e ion source has a length of 216 mm and a diameter of 102 mm and 12 rows of so-co magnets for the plasma confinement. the system utilizes copper gaskets to achieve better vacuum for cleaner deposition environment. sputtering target it is important that the ga is applied evenly on the surface area of the sputtering target. however, the formation of amalgam of the metallic ga on the stainless steel (002) 2θθθθθ angle in te n s it y ( a u ) (100) (101) (110) (103) (112) 20 30 40 50 60 70 80 0 20 40 60 80 fig. 3. xrd spectrograph of remaining gan material on the sputtering target after film deposition. increased, but t e decreased, thus reducing the rate of excitation of n as observed by optical emission spectroscopy. the gan film deposited at an optimum condition for the n 2 excitation was characterized using an x-ray powder diffractometer and showed a crystalline orientation corresponding to that of (002) and (101) reflections of gan (flauta et al., 2001). the remaining material left on the sputtering target was also examined using xrd and exhibited numerous peaks corresponding to that of bulk gan powder as shown in fig. 3. this is due to the exposure of the ga sputtering target to the n 2 plasma during the deposition process. the bulk gan powder has a dark grayish color while the gan film was transparent with a reddish yellow color as shown in fig. 4. further investigation ultra-high vacuum compatible system the background pressure of the system cannot be improved better than 3 x 10-4 pa. for this reason, an ultra-high vacuum system is being developed to improve its performance. using the uhv system, better base pressure can be achieved and impurities in residual gases can be minimized prior to film deposition. the ultrahigh vacuum compatible multi-cusp plasma-sputter type fig. 4. (a) photo of the bulk gan and (b) gan thin film on sio 2 substrate. (a) (b) 60 flauta et al. fig. 5. the sputtering target assembly of ultra-high vacuum-compatible multi-cusp plasma-sputter-type ion source. circular disk target was observed in the experiment. for this reason, the new sputtering target, 20 mm in diameter and 2 mm thick and made of mo was used as the target holder, since mo holds ga better. this disk has an inner diameter of 18 mm and a 1 mm deep hollow to hold the molten ga by gravity. this further minimizes the incorporation of other material aside from that of ga to be sputtered out during film deposition. the mo sputtering target is attached to a cu base that is supported by hollow stainless steel rods through which sheathed thermocouple pass through and touch the bottom part of the sputtering target, as shown in fig. 5. this allows the monitoring of the target temperature during plasma discharge and film deposition. ion source the filaments are placed below the sputtering target covered with glass insulator, exposing the metallic ga applied all over the surface area of the mo target to the plasma. this setup minimizes the exposure of the filaments and other supporting materials of the sputtering target to the plasma . spectroscopy analysis optical spectrometer will be installed to detect the excited nitrogen species in the plasma. the ga flux will be measured with a magnetic deflection type momentum analyzer during plasma discharge and film deposition. summary the formation of gan thin film was confirmed using plasma-sputter technique. an ultra-high vacuum (uhv)compatible multi-cusp plasma-sputter type ion source is now being assembled together with diagnostic equipment to study in which condition the plasma characteristics of the ion source becomes suitable to deposit polycrystalline gan thin film. acknowledgment this work was partly supported by the high-tech research project at the research center of advanced science and technology of doshisha university. references flauta, r., t. kasuya, t. ohachi, & m. wada, 2001. proc. 13th international conference on crystal growth. iccg13. 194. morkoc, h., 1999. nitride semiconductors and devices. berlin heidelberg, springer-verlag. nakamura, s., s. pearton, & g. fasol, 1999. the blue laser diode: a complete story. berlin heidelberg, springer-verlag. shen, x.q., t. ide, m. shimizu, & h. okumura, 2001. highquality ingan/gan multiple quantum wells grown on gapolarity gan by plasma assisted molecular beam epitaxy. j. appl. phys. 89: 5731-5733. strite, s. & h. morkoc, 1992. gan, aln, and inn: a review. j. vac. sci. technol. b. 10: 1237-1266. sun, x.l., h. yang, y.t. wang, l.x. zheng, d.p. xu, d.g. zhao, s.f. li, & z.g. wang, 2000. effect of buffer layer growth conditions on the secondary hexagonal phase content in cubic gan films on gaas (001) substrates. j. cryst. growth. 212(3-4): 397-401. thermocouple water cooling filament 1 insulating glass target holder (mo) cu hollow tube sus304 filament 2 25_gradient pobre and saloma 106 gradient and scattering forces on a kerr nanosphere r. f. pobre de la salle university, 2401 taft avenue, manila 1004 e-mail: pobrer@dlsu.edu.ph c. a. saloma national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: csaloma@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 106-109 a theoretical model that computes both for the gradient and scattering forces being exerted on a nonresonant nonlinear (electro-optic kerr effect) rigid nanosphere by a strongly focused continuous-wave laser beam is presented. the incident wavelength of the laser beam is assumed to be appreciably larger than the nanosphere radius a. optical forces arising from the aforesaid interaction can be derived by a twocomponent approach which determines individually the gradient force and scattering force. the behavior of the trapping (gradient) force is plotted against several experimental parameters, e.g., incident beam power, axial distance, sphere radius, wavelength, and refractive index difference between the surrounding liquid and the nanosphere. results have shown that the kerr effect on the nanosphere can produce a maximum of tenfold increase in the trapping force. introduction the great advantage of using a single beam is that it can be used as an optical tweezer to manipulate small particles under typical inverted microscopes. optical tweezing has found its way to life science applications particularly in cell manipulations and single-molecule diagnostics. bockelmann et al. (2002) have studied mechanical properties of dna by unzipping short hairpin loops in an optical trap. much effort has been made to explain the nature of optical forces and describe them quantitatively through sound theoretical models. we have studied the behavior of the (time-averaged) radiation that is exerted by a tightly focused cw gaussian beam on a nonlinear microsphere both in the geometrical regime (pobre & saloma, 1997) (α=2πa/ λ > 100) and in the mie regime (pobre & saloma, 2002) (α ≤ 100). but to our knowledge, this problem has not been addressed before for the case when the sphere radius a is appreciably smaller than the incident wavelength λ (α << l) or in the rayleigh regime. from an academic standpoint, the problem is worth pursuing for it involves finding significant behaviors that could accurately account for nonlinear effect, in particular kerr effect, on the optical trapping force. theoretical framework consider a nonresonant and nonmagnetic kerr nanosphere whose orientation with the incident gaussian beam can be shown in three-dimensional cartesian axes as illustrated in fig. 1. the nonlinear nanosphere is illuminated by a linearly polarized gaussian beam of the fundamental mode with beam radius wo at it’s beam waist position propagating from the negative z to the positive z direction whose refractive index given by n2 = n2 (0)+n2 (1)i(r), where n2 (0) gradient and scattering forces 107 is the linear component of the refractive index, n2 (1) is the nonlinear component, and i(r) is the intensity of the gaussian beam. the reference system of the nanosphere is in the center of the cartesian coordinate system, which is located at the beam waist center. when the beam is incident on the nanosphere, the nanosphere in the rayleigh scattering regime behaves like a particle that acts as a simple point electric dipole. the total radiation force, as shown in fig. 2, acting on the dipole at r1 for +q and r2 for –q can be expressed as . (1) since the particle is very small compared with the beam width, incident electromagnetic (em) fields can be treated almost constant within the distance ⏐r⏐. moreover, the distance ⏐r⏐ is much smaller than the incident wavelength, eq. (1) can be expanded in r using a power series. if we only consider limited terms in the first order of ⏐r⏐, we can obtain the following equations from eq.(1): , (2) where µ = qr is the electric dipole moment. but the third term is very small in comparison to the first and second terms, thus we can neglect the third term. the time-averaged radiation force due to the inhomogeneous force and lorentz force can be written as . (3) for a complex monochromatic gaussian beam, the em fields can be expressed as , (4) , while the complex-induced complex electric dipole and it’s time derivative can be written as , (5) . using vector quantities and eqs.(4) and (5), we can reexpress eq. (3) as . (6) the first dispersive term corresponds to the dipole force or gradient force, and the second dissipative term is the scattering force. if you consider the kerr nonlinearity n2 = n2 (0)+n2 (1)i(r), eq. (6) becomes fig. 1. orientation of the microsphere with the gaussian beam in the 3d cartesian axes. ( ) ( )1 2q qf f r f r−= + fig. 2. an electric dipole moment µ whose opposite charges (+q,-q) are separated by distance⏐r⏐. ( ) ( )( )ˆ expe r i r i tε φ ω= − 1ˆ ˆb e iω = − ∇× where i(r) ≡⎥e(r)⎪2 is the intensity of the gaussian beam at r of fig. 1. notice that the first component is dependent on the gradient of the intensity, while the second term is intensity dependent. the first term points f = (µ.∇) e (r,t) + µ ×b(r,t) + r,t) + r× (µ.∇)b(r,t) 〈 f 〉t = re [(µ * . ∇) ê + µ* × β2 1 ˆˆ ˆ . . pobre and saloma 108 to the direction of increasing spatial intensity, while the second term points to the direction of the intensity spread or scattering of the incident gaussian beam on the kerr nanosphere. results and discussion figure 3 shows how the optical trapping force could generate a pulling action or a pushing action on the kerr nanosphere. when gradient force or trapping force pulls a nanosphere either 〈fz〉 is negative at z > 0 or 〈fz〉 is positive at z < 0. for other cases the nanosphere is pushed away from the beam focus at z = 0. it is shown that kerr nonlinearity increases the magnitude of the trapping force for all pulling situations. quantitatively, a tenfold increase between the maximum trapping force of a kerr nanosphere and the maximum trapping force on a linear nanosphere is evident from the figure. unlike the previous plot, fig. 4 demonstrates how stiff the trapping system is for both linear and nonlinear nanospheres. as illustrated in fig. 4, the trapping stiffness for the kerr nanosphere is much larger than the linear nanosphere which indicates a deeper trapping mechanism. figures 3 and 4 are consistent trapping force profiles for both the geometrical ray computation (pobre & saloma, 1997) and the mie computation (pobre & saloma, 2002) but at varying orders of magnitude. however, unlike previous works (pobre & saloma, 1997; pobre & saloma, 2002), the result in fig. 5 shows a decrease in the trapping stiffness as the particle size is reduced from 10 to 2 nm. smaller particles both in the geometrical ray regime and mie regime result in an increase in the trapping force which points to larger trapping stiffness. the last figure is a valuable data to experimentalists who work in a tight budget under an optical trapping system. instead of using high numerical aperture (na) objective lens (an expensive optics), fig. 6 would show that it is feasible to trap a kerr nanosphere even at low na. even at high na (e.g., 1.2) for the linear nanosphere, a low na (e.g., 0.5) can match the amount of trapping force under the nonlinear counterpart given the same optical parameters. conclusion the optical trapping force consists of two governing components. these are the gradient force component fig. 3. axial force as a function of z in (µm). parameters: zo=0, r=0, p=100 mw, a=5 nm, na=1.2, λ =1.064 um, n1=1.33 , n2 (0)=1.4, and n2 (1)=1.8 x 10–11 m2/w. axial distance, z (µµµµµm) a xi al f or ce , 〈〈〈〈 〈f z 〉〉〉〉 〉 ( n ) -5 -3 -1 1 3 5 150e-15 100e-15 500e-16 00e+00 -500e-16 -100e-15 -150e-15 linear nonlinear fig. 4. stiffness of the trap (df/dz) as a function of z. same parameters as in fig. 3. axial distance, z (µµµµµm) tw ee ze r st if n es s, d f ( n /m ) -5 -3 -1 1 3 5 3.00e-9 1.00e-9 1.00e-9 -3.00e-9 -5.00e-9 -7.00e-9 linear nonlinear 2 nm 5 nm 7 nm 10 nm fig. 5. tweezer stiffness versus power at different particle sizes with the same parameters as before. optical power, p (mv) tw ee ze r st if n es s, d f ( n /m ) 10 30 50 70 90 0.00e+0 -4.00e-9 -8.00e-9 -1.20e-8 -1.60e-8 -2.00e-8 gradient and scattering forces 109 fig. 6. axial force as a function of na with the same optical parameters as before. na a xi al fo rc e, f z (n /m ) 0.40 0.60 0.80 1.00 1.20 1.00e-11 5.00e-12 0.00e+00 -5.00e-12 -1.00e-11 -1.50e-11 1.40 nonlinear linear (the term that depends on the gradient of the intensity) and the scattering force component. the mechanism of the gradient force stems from two particular mechanisms. the inhomogeneous field force serves only the dipole or gradient force, whereas the lorentz force contributes both the gradient and scattering forces. thus, the mechanism of optical trapping is an interplay between the inhomogeneous field force and some component of the lorentz force when the trapping particle is within the rayleigh regime. calculations have shown that both trapping force and trapping stiffness are enhanced with kerr nonlinearity. two significant result can be derived from the study. first is the dispersivelike behavior of the trapping force for a kerr nanosphere, which means that by proper tuning of the incident gaussian beam you can control the amount of trapping force for nanosized kerr particles. second and last interesting observation is it’s ability to trap even at low na of the objective lens for kerr nanospheres. the theoretical model could be extended for the case when it is dependent on the polarization of the incident gaussian beam. it could be added that size validity between the mie regime and rayleigh regime is still an unexplored area of investigation in the size range of 0.1–1 mm. acknowledgment mr. pobre would like to express his appreciation for the support accorded by urco of de la salle university-manila upon this project. references bockelmann, u., et al., 2002. unzipping dna with optical tweezers: high sequence sensitivity and force flips. biophys. j. 82: 1537-1553. pobre, r.f. & c.a. saloma, 1997. single gaussian beam interaction with a kerr microsphere: characteristics of the radiation force. appl. opt. 36: 20. pobre, r.f. & c.a. saloma, 2002. radiation force on a nonlinear microsphere by a tightly focused gaussian beam. appl. opt. 41: 20. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 07_almoro 32 almoro, cadatal, and daza science diliman (january-june 2003) 15:1, 32-36 measurement of three-dimensional deformations by phase-shifting digital holographic interferometry percival f. almoro*, marilou m. cadatal, and marlon rosendo h. daza national institute of physics, college of science university of the philippines diliman 1101 quezon city, philippines e-mail: val@nip.upd.edu.ph abstract out-of-plane deformations of a cantilever were measured using phase-shifting digital holographic interferometry (psdhi) and the fourier transform method (ftm). the cantilever was recorded in two different states, and holograms were stored electronically with a charge-coupled device (ccd) camera. when the holograms are superimposed and reconstructed jointly, a holographic interferogram results. the three-dimensional (3d) surface deformations were successfully visualized by applying ftm to holographic interferogram analysis. the minimum surface displacement measured was 0.317 µm. the processing time for the digital reconstruction and visualization of 3d deformation took about 1 minute. the technique was calibrated using michelson interferometry setup. introduction non-contact measurement of surface deformations is much demanded in areas of science and engineering. conventional holographic techniques coupled with automatic fringe analysis provide a sensitive and reliable method for deformation analysis (almoro & daza, 1998). traditionally, photographic films were used to record the holograms. photographic films have a very high resolution, but have the disadvantage of needing wet chemical processing. after recording a hologram on such films, a physical reconstruction of the recorded wave field by illumination of the film with a laser is required. added to this, the intensity distribution of the reconstructed object still needs to be imaged for phase evaluation and processing. the procedure is time consuming. in digital holography, holograms are recorded by a ccd camera and image reconstruction is performed by a computer (schnars & jûptner, 1994). to increase the fringe spacing and to obtain the desired image of high quality, phase shifting technique is used (yamaguchi & zhang, 1997). a more detailed discussion on the principles and experimental results on digital phase-shifting holography are presented in reference (almoro et al., 2001). digital holography has spawned into various imaging (zhang & yamaguchi, 1998) and optical measurement applications (yamaguchi et al., 2001; yamaguchi, 2001; pedrini et al., 1999; pedrini et al., 1998; schnars & jûptner, 1994). evidently, this technique is better suited to an industrial environment than photographic films. this study will demonstrate the sensitivity of psdhi in measuring microscopic (sub-micron) out-of-plane displacements. displacement calculations using psdhi and ftm will be compared to michelson interferometry (mi). this study will show that digital holographic interferometry, coupled with ftm, is a fast, sensitive, and reliable technique for measurement and* corresponding author 33 measurement of 3-d deformations visualization of 3d out-of-plane deformations of real objects. theoretical background: digital holographic interferometry the approach to obtain the interference phase distribution ∆φ(x,y) and displacement distribution d(x,y) in digital holographic interferometry is summarized in fig. 1 (kreis, 1996). in the same way that holography can be extended to double-exposure holographic interferometry, digital holography can be employed to perform digital holographic interferometry. two holograms with amplitude transmittances τ 1 (k,l) and τ 2 (k,l) corresponding to different states of the object are recorded and stored subsequently and then added point wisely. since the fresnel transform is essentially a fourier transform and, thus is linear, it reconstructs the sum of the two wave fields. the resulting intensity exhibits the cosine-shaped interference pattern called “holographic interferogram”. this interferogram may be evaluated by one of the various interference phase determination methods like the ftm and phase unwrapping techniques (kreis, 1986). the continuous phase distribution obtained is proportional to the out-ofplane displacement distribution when the illumination and observation directions are nearly anti-parallel. the displacement distribution is then given by (kreis, 1986) (1) methodology the experimental set-up is shown in fig. 2. the laser used was a 1 mw frequency stabilized helium-neon laser (ë = 632 nm). the split beams are expanded and collimated. one of the beams illuminates the test object in a direction that is almost anti-parallel with the line from object to ccd camera. this makes the setup sensitive only to out-of-plane (perpendicular to object surface) direction. the other beam, which is reflected back from the mirror 1 attached to the piezoelectric transducer (pzt1), forms the reference plane wave at normal incidence to the ccd. the pzt1 was utilized to phase-shift the reference beam. the diffracted light from the object is collected by lens l1 onto the ccd ( , ) ( , ) 4 x y d x y π λ ∆φ = fig. 1. digital holographic interferometry for measurement of out-of-plane displacements. hologram τ 1 (k,l) (initial state) hologram τ 2 (k,l) (final state) addition τ 2 (k,l) + τ 1 (k,l) (double-exposure) numerical fresnel transformation e(x,y,z) = f{τ 1 + τ 2 } intensity distribution i(x,y) = | e(x,y,z) |2 interference phase determination (ftm) i(x,y) ↔ ∆φ(x,y) phase unwrapping and displacement distributions ∆φ(x,y) ↔ d(x,y) pzt driver mirror 2 pzt1 mirror 1 laser computer pzt2 test object lens (l1) beam splitter 2 ccd beam splitter 1 mirror 3 collimating lenses fig. 2. experimental setup. 34 almoro, cadatal, and daza array. the distance from l1 to the ccd is beyond the image plane of the test object, and as far as possible to increase the fringe spacing. the optimum distance from object to ccd in this experiment is 1.3 m. as a rule of thumb on the beam ratio, the object and the reference beams must form visible interference fringes on the ccd plane as seen from the computer monitor. results and discussion the reconstructed object without any deformation is shown in fig. 4a. the image is free from holographic interferogram (note: the bright curved pattern on the center of the image is not a fringe pattern but a strong reflection of light on the metal surface. note also that the dark square image on the lower left region is the beam splitter that stood on the path of the object beam). fig. 4b to 4f are samples of the digitally reconstructed objects covered with holographic interferograms, obtained for a voltage difference of 10 v, 30 v, 35 v, 40 v, and 50 v, respectively. the number of fringes increased as the voltage difference is increased. notice also that the slope of the fringes become uniformly horizontal as the voltage is increased. this means that for increased voltage difference the cantilever undergoes uniform out-of-plane deformation, which is attributed to increased tension. an increase in the tension removes the slackening of the cantilever, thereby making the (b) (c) (d) (e) (f) (a) fig. 4. (a) reconstructed object without deformation. holographic interferograms obtained for a voltage difference of (b) 10 v, (c) 30 v, (d) 35 v, (e) 40 v, and (f) 50 v. fig. 3 shows the cantilever and the out-of-plane displacement actuated by the pzt2. the cantilever was an aluminum sheet (1.5 cm x 2.0 cm x 0.05 cm) clamped from the top. pzt2 was attached to the free end of the cantilever to effect an out-ofplane displacement (d). a 5 v step increase in the voltage difference was applied to pzt2. the ccd and a frame-grabber capture the interference pattern (between object and reference beams) and store it as the hologram. four frames (phaseshifted by π/2) are obtained for each deformation state to obtain the complex amplitude at the ccd plane using the fourbucket algorithm (yamaguchi & zhang, 1997). the holographic interferograms are evaluated using the ftm to yield a phase distribution wrapped in 2π. after phaseunwrapping, the displacement distribution is obtained using eq. (1). in comparing digital holographic interferometry with mi, the optical path difference in mi is calculated using (2) where n is the fringe shift as the voltage applied to the pzt2, while attached to the movable mirror in mi setup, is increased. 2 n d λ = fig. 3. cantilever and displacement, d. clamp cantilever pzt2 d z y 35 measurement of 3-d deformations surface deformation uniform. for still greater displacements, the fringes become more closely spaced and difficult to resolve. the processing time for the digital reconstruction of the images and visualization of 3d deformation by the ftm took about 1 minute. note that this is a vast improvement compared to wet chemical processing in conventional films, which take at least 3 minutes to develop and bleach, plus a few more minutes for optical reconstruction and image processing. the total processing time involving holographic films take at least 5 minutes (almoro & daza, 1998). since the intensity distributions are already in digital format, the 3d deformations can be visualized directly by applying the ftm to the holographic interferogram analysis. as a demonstration, ftm was applied to the rectangular region in fig. 4e, chosen because it is free from spurious artifacts. the phase distribution wrapped in 2π is shown in fig. 5. the filtering process in the table 1. calculated displacements using digital holography and michelson interferometry. voltage difference (v) displacement (µµµµµm) psdhi michelson 6.0 11.4 16.1 21.0 24.9 29.3 34.1 38.8 42.9 47.0 50.4 55.4 59.9 0.317 0.633 0.950 1.266 1.583 1.899 2.216 2.532 2.849 3.165 3.482 3.798 4.414 0.317 0.633 0.950 1.266 1.583 1.899 2.216 2.532 2.849 3.165 3.482 3.798 4.414 ftm removes the high frequency noise and yields the desired phase distribution. fig. 6 is the 3d plot obtained after unwrapping the phase distribution in fig. 5 and then applying eq. (1). from this plot it is clear that for a voltage difference of 40 v the out-of-plane displacement is 2.53 microns. table 1 lists the values of the out-of-plane displacements for different pzt voltages obtained using psdhi and mi. the displacements obtained are consistent for the two techniques. using the psdhi, the smallest displacement measured was 0.317 µm (l/2) and the largest was 4.414 µm. this shows that psdhi, coupled with ftm, is a sensitive, fast, and reliable technique in measuring 3d deformations of real objects. conclusions phase-shifting digital holographic interferometry was used to measure 3d out-of-plane deformations of a cantilever. the smallest displacement measured was 0.317 µm and the largest displacement was 4.414 µm. the digital reconstruction, instead of the wet chemical processing in conventional films, greatly reduced the processing time from 5 minutes to about 1 minute. the measured displacements of the cantilever, for a given voltage difference applied to the pzt, were consistent with those obtained using mi. therefore, phase-shifting fig. 5. phase distribution (wrapped in 2π) obtained after applying the ftm (fourier transform method) on the rectangular region in fig. 4 e. y x 3.14 1.57 0 -3.14 -1.57 fig. 6. 3d plot of the surface deformation for a voltage difference of 40 v (z-axis is in meter). 2.53 x 10-6 1.9 x 10-6 1.27 x 10-6 6.32 x 10-7 0 z x = 0.75 cm y = 2.0 cm 36 almoro, cadatal, and daza digital holographic interferometry can be used as a fast, sensitive, and reliable technique in measuring 3d deformations of real objects. acknowledgments the authors would like to thank the dost-pcastrd for the equipment grant. m. cadatal would like to thank dost-sei for the scholarship grant. references almoro, p., m. cadatal, & m.r. daza, 2001. reconstruction of 3d objects using phase-shifting digital holography. (presented at the spp national physics congress 2001). almoro, p. & m.r. daza, 1998. measurement of microscopic deformations using double-exposure holographic interferometry and the fourier transform method. science. diliman. 10(2): 55-60. kreis, t., 1996. holographic interferometry: principles and methods. berlin, akademie verlag. kreis, t., 1986. digital holographic interference phase measurement using fourier transform method. josa. a. 3: 847-855. pedrini, g., p. froning , h. tiziani, & m. gusev, 1999. pulsed digital holography for high-speed contouring that uses a twowavelength method. ao. 38(16): 3460-3467. pedrini, g., p. froning, h. fessler, & h. tiziani, 1998. in-line digital holographic interferometry. ao. 37(26): 62626269. schnars, u. & w. jûptner, 1994. digital recording and reconstruction of holograms in hologram interferometry and shearography. ao. 33: 4373-4377. schnars, u. & w. jûptner, 1994. direct recording of holograms by a ccd target and numerical reconstruction. ao. 33(2): 179-181. yamaguchi, i., 2001. interferometry by holography and speckle. proc. regional workshop on lasers and optoelectronics. puspiptek, indonesia. yamaguchi, i., j. kato, & s. ohta, 2001. surface shape measurement by phase-shifting digital holography. opt. rev. 8(2): 85-89. yamaguchi, i. & t. zhang, 1997. phase-shifting digital holography. opt. lett. 22(16): 1268. zhang, t. & i. yamaguchi, 1998. three-dimensional microscopy with phase-shifting digital holography. opt. lett. 23(15): 1221. 06_llaguno genetically modified organisms 73science diliman (january-june 2001) 13:1, 73-76 genetically modified organisms claro t. llaguno institute of chemistry, college of science university of the philippines, diliman, quezon city 1101 perspective recent reports have brought to public attention concerns about bt corn and genetically modified organisms (gmo) in general. the timing, it seems, is most appropriate considering two related developments early this year: the final approval of the cartagena protocol on biosafety in montreal on january 29, 2001, and the oecd edinburgh conference on gm food safety last february 28march 1, 2001. the protocol makes clear that gmos include all living modified organisms (lmo) defined as “any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology”. this includes seeds, live fish, and other organisms intentionally obtained for release to the environment. it would seem that the common understanding about gmos as referring to farm-totable products is perforce expanded to embrace genetically modified farm animals and aquatic resources. being a trade agreement, the montreal accord primarily deals with the safety issues related to the transboundary movement of lmos around the globe. the oecd conference on the other hand, called for an international body “to address all sides of the gm debate” in response to the public outcry, particularly in western europe, regarding the risks the new products pose to human health and the environment. some points of contention, which remain unresolved, include issues such as whether countries should be allowed to develop their own gm food based on their needs, and whether a global moratorium on gmos and mandatory labeling should be enforced worldwide. bt corn the u.s., canada, and france are the leading producers of gm crops. brazil, argentina, west africa, japan, australia, and new zealand have also adopted this form of agrobiotechnology. recently, japan announced its plans to double its investment in biotechnology while taiwan is engaging u.s. expertise to enable it to become the biotechnology center in this part of the world. genetically engineered corn or maize is one of at least thirty-six gm food products available in the world market. soybean, cotton, canola, potato, and squash are the other top modified crops in terms of land area planted to them. in 1998 the total area planted to gm crops is 27.8 million hectares, 23.3 million hectares or 84 percent of which are in the u.s. and canada. compared to the 1997 figures (11 and 9.4 million hectares, for u.s. and canada respectively), the increase in total land area is about 153 percent. gmos are derived from traditional organisms or crops using the techniques of recombinant dna technology. a genetic package, consisting of a gene which encodes the protein that produces the desired characteristics in the modified organism, a promoter and a marker gene, are introduced into the dna of the parent organism. the promoter regulates the amount and distribution of the trait-determining gene in the new organism while the marker provides a means to test for successful transformations or events. for agricultural products, the desirable characteristics or outcomes include, for example, resistance to either pest or herbicide or both, protection against plant viruses, and the introduction of llaguno 74 micronutrients in plants. quite controversial is the use of the technology to produce sterile seeds, also called terminator seeds, which compels farmers to purchase new seeds for every planting season. protein crystals produced by the bacterium bacillus thuringiensis ( bt ) had been used for more than thirtyfive years as the active component of biological pesticides, which are commercially available in various formulations: liquid concentrates, powders and readyto-use dust and granules. organic growers generally prefer these insecticides over their chemical counterparts since the bacteria are naturally occurring organisms commonly found in soils. the obsolescence of this agricultural practice, brought about by the development of resistance to the genetically engineered bt toxin by the target insects, is precisely one of the more important reasons cited against the use of the technology. the gene of interest in modified corn is obtained from the bacterium, thus, the name bt corn or bt maize. its distribution in the plant varies among the different corn hybrids. knockout (novartis seeds) and naturegard (mycogen seeds), for example, have these genes only in the green tissues and pollen; yieldgard (monsanto and northrup king/novartis seeds) has genes distributed throughout the entire plant. the bt protein is toxic primarily to corn borers. when the insect feeds on the plant, the protein becomes activated by the enzyme in the insect’s gut, eventually killing it within two to three days. the modified corn in effect acquires a built-in pesticide. in the u.s., the target pests are the european corn borers which are responsible for losses amounting to as much as one billion dollars annually. in the philippines, the losses brought about by asian corn borers are estimated to be about 20 to 30 percent of the annual potential yield. among the objections raised against the use of bt corn are (a) the possibility of allergic reactions in humans; (b) the development of resistance by the insects against the toxin; (c) bt toxin leakage through the root system to the soil, hence the possibility of gene transfer to other soil bacteria; and (d) outcrossing with other corn varieties. proponents of the technology enumerate as advantages the desirable characteristics and nutrient transfers mentioned above, and the enhanced ability to increase world food production. in the case of cereals, according to nobel laureate norman borlaug, the production should be raised by eighty percent over the 1990 average by the year 2025. management strategies to contain the development of resistance against the bt toxin are incorporated in integrated pest management systems. moreover, strict registration requirements and regulations ensure safety against possible harm to humans and the environment. gmo controversy in switzerland the national debate from 1996 to 1998 on the so-called “excesses” of recombinant dna technology led to a final vote on the gene protection initiative which sought to outlaw, if not severely curtail, some activities, gmos included, in biotechnology in the country. what became a difficult battle for scientists and advocates of the technology ended up in their favor, with two out of three voters expressing support for their cause. shortly after the swiss settled their gmo issue, public concern for food safety in the u. k. was brought to greater heights after a british scientist announced on television that rats fed with genetically modified potatoes suffered from stunted growth and a damaged immune system. his research data were promptly reviewed by various groups, including one panel, convened by the british royal society, which dismissed his findings as “flawed”. the journal lancet, which published his research results, was later criticized as “irresponsible” by the u.k. biotechnology and biological research council. the controversy continued with prince charles siding with the scientist who chose to retire after an indefinite suspension was served upon him, and after his laboratory was unceremoniously padlocked by the institute director. although not as intense as in the british situation, the american public appears to be entertaining some reasons for alarm regarding the risks to which gmos expose consumers and the environment. this was genetically modified organisms 75 prompted by a 1999 report in nature by an american entomologist about his studies on monarch butterflies. he reported that larvae fed with leaves dusted with bt corn pollen died within four days. the mortality rate was 44%. the scientists however noted that their data are laboratory results which need to be validated in field experiments. to date only two laboratory studies, the potato and monarch butterfly experiments, which bear upon the gmo controversy appear to have been undertaken by independent groups. this may be surprising considering that the first bt corn was approved for commercial distribution as early as 1996 by the u. s. environmental protection agency. government response to protests against gmos in other countries has taken various forms: heightened public information campaigns about the new product, ban or moratorium on importation, stricter regulations such as separate storage and labeling, and regional monitoring and cooperation. international organizations continue to address the issues surrounding the gmos, but in many instances representatives from developing countries end up not entirely pleased with the final versions of legally binding agreements, that is, when these are the expected outputs of international negotiations. new concepts dealing with modified organisms have also evolved. substantial equivalence, for example, refers to the determination of the safety of the new product for human consumption. the procedures appear to be quite cumbersome, but they seem to have gained international acceptance in spite of criticisms that the concept is “pseudo-scientific”. precautionary principle, a concept which traces its origin to international agreements oncerning the environment, has found its place in the cartagena protocol, the trade agreement referred to earlier. under this principle, a country may choose, as appropriate, not to trade in lmos even in the absence of “scientific certainty” that the organisms are harmful to human health or the environment. what constitutes “scientific certainty”, however, is far from clear as the bt corn issue in general santos city, south cotabato, is likely to demonstrate. the general santos city bt corn issue the actions taken by the community and the local government of general santos city are not any different from the public response to gmos in other parts of the world. the fact that the issue has been brought to the courts is not difficult to appreciate at a time when modified genetic resources are fast becoming the newly discovered items of international commerce. through their collective decision, the community asserts in concrete terms their fundamental rights to health and to a balanced and healthful ecology as provided for in the 1987 constitution ( art. ii, secs. 15 and 16 ). no less than the supreme court has underscored the primordial importance of these rights when it noted, in juan antonio v. fulgencio factoran ( g. r. no.101083, july 30, 1993), that their advancement “may even be said to predate all governments and constitutions. as a matter of fact, these basic rights need not even be written in the constitution for they are assumed to exist from the inception of mankind.” the resolution of the case now belongs to the courts. as it appears, the issues could be fairly complicated and may require the assistance of expert testimony. hopefully, the equivalent daubert standard (daubeli v. merrell dow pharmaceuticals, 509 u. s. 579 (1993)), not the frye test of general acceptance ( frye v. united states, 54 app. d. c. 46, 293 f. 1013 (1923) ), which daubert set aside after seventy years, will be made to apply in the instant case. in this manner, the rigorous requirements of scientific evidence and scholarship demanded by daubert will help pave the way for a reasoned settlement of the bt corn controversy. as in other courts confronted with scientific issues, the likely outcome of the litigation may depend on the trial judge’s attitude toward law on one hand and science on the other. those who are confident that the law can deal with situations where science fails to serve public interest can be one of two types: a full believer of science or entirely the opposite. in the former case, the risk is a decision based on blind faith; in the latter, a decision based on caution. among those who feel that current legislation is inadequate—an example, it seems, is dna profiling in sex assault cases—the judge, while llaguno 76 maintaining faith in science, can opt to be restrictive in allowing it to proceed with its course. another possibility refers to a situation where mistrust for science leads to skepticism on the part of the judge and, hence, inaction or a delay in judicial decision. in any case, the goal is the pursuit of balance among competing considerations. a true disaster is when science and technology is severely curtailed in a manner that is reminiscent of the luddites of 19th century england who professed that technology is harmful to society. remarks bt technology is still a relatively new field. it may very well be labeled as a first generation technology. as novel and useful organisms are discovered and new transformations are successfully carried out in the laboratory, second generation technology capable of producing “smart proteins” is a possibility in the near future. coupled with the new knowledge that will be available when the ongoing genome projects on major crops—rice, corn, and cassava presently—are completed three to five years from now, the implications for improved world food production and nutrition can be far-reaching indeed. biotechnology goes beyond its applications in agricultural products. it has gone into the realm of animal science producing improved livestock and important pharmaceuticals, such as insulin to treat diabetes, from animals modified through the introduction of human genes into the animal’s dna. xenotransplantation, the use of animal tissues or organs for transplantation to humans, is another area of application which is actively being pursued in various laboratories. a significant breakthrough was the cloning of a sheep in 1996 in scotland. dolly, the cloned offspring, came into being with the use of a procedure referred to as somatic nuclear cell transfer. alarmed that this development has made the cloning of humans a not too distant reality, governments have responded by restricting research on human embryos, as well as passed legislation which expressly prohibit human cloning. gene therapy, screening for genetic disorders like breast cancer, and reproductive technologies to address fertility problems are some of the more common applications of biomedicine to humans. human stem cell research appears to be both promising and controversial. laboratories are now looking into the possibility of “instructing” these pluripotent cells to develop into human tissues for xenotransplantation work, thereby overcoming acute rejections which presently limit animal to human transplantation efforts. in 2003, the human genome project is expected to be completed and, like the developments which followed the elucidation of the dna structure in 1953, this could lead to great advances never before imagined in medical research and practice. nobel laureates james d. watson, co-discoverer of the dna structure, and robert f. curl both said that the 21st century will be the era of the life sciences— genetics, biology and their related disciplines. the above discussion suggests why. like any other scientific discipline, biotechnology has its own package of benefits for society. like the others, it is also not immune from causing possible harm brought about by the negligent or irresponsible use of knowledge it acquires through research and development. indeed, the bt corn controversy is far more than interesting. it has become a national issue where public policy, political will, community concerns, scientific evidence, and judicial wisdom will all come into play. disclaimer articles published as perspectives or comments are the opinions of the authors, and do not, in any way, represent the views of the editorial board members or the publisher. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 ethnobotany-art.10 ethnobotany of solieria robusta 75science diliman (july december 2000) 12:2, 75-77 abstract introduction ethnobotanical data on the economic macrobenthic seaweeds in the philippines are few. among the coastal regions of the country, northwestern luzon or the ilocos region has a long history of seaweed utilization. the scarcity of agricultural crops brought about by the lack of arable lands has necessitated the tapping of diverse marine food sources. moreland (1980) listed over a dozen species of marine algae used as food, either fresh or cooked. these include the edible species of the red algal genus porphyra, a centuries-old favorite among the chinese and japanese. other folk uses of seaweeds in northern luzon include medicinal, agricultural (fertilizer and pesticide), and animal feed applications. in other parts of the philippines, the use of seaweeds is restricted to a very few species and mainly for industrial purposes. in central visayas and southwestern mindanao, the red algal genera kappaphycus and eucheuma are cultivated extensively for the international carrageenan industry and partly for the local fresh seafood market. likewise, two species of the green algal genus caulerpa, c. lentillifera j. agardh and c. racemosa (forsskaal) j. agardh, are ethnobotany of solieria robusta (gigartinales, rhodophyta) in zamboanga, philippines oliver d. tito and lawrence m. liao marine biodiversity program zamboanga state college of marine sciences and technology fort pilar, 7000 zamboanga city, philippines a market survey was made at the zamboanga city public market to determine the diversity of economic macrobenthic algae sold by the local population. interviews were conducted to obtain information on local names, uses, stock distribution, method of harvest, seasonality, and some aspects of marketing practices. keywords: ethnobotany, solieria robusta, seaweed utilization harvested for both domestic and foreign seaweed markets although on a rather limited scale. the zamboanga area in western mindanao is known for its variety of marine products such as fishes, sea cucumbers, molluscs, and crustaceans. recently, the kappaphycus and eucheuma seaweed farms have contributed significantly to the economic progress of the region. while the economics and technical aspects of the seaweed industry in the region have been documented by trono (1974, 1992), nothing is known on the ethnobotanical aspect of seaweed utilization in the area. an ethnobotanical study is made more interesting because the region is the home of a number of ethnic groups with different sociocultural and religious backgrounds and dietary practices. the following study was done to document the ethnobotanical aspect of seaweed utilization in the zamboanga area. materials and methods interviews with seaweed vendors in the zamboanga city public market were conducted to obtain information on the kinds of seaweeds harvested, sold, and consumed tito & liao 76 by the local population. information on their folk uses, local names, distribution seasonality, marketing practices and other ethnobotanical information were obtained. photographs were taken of the species sold in the market. results and discussion interviews revealed that the seaweed vendors in zamboanga city public market were predominantly members of the samal ethnolinguistic group who dwell in coastal areas. many of them have been in the fresh seaweed retail business for some years. three species of seaweeds were sold in the market: (1) caulerpa racemosa, which is represented by at least two varieties, c. racemosa var. laetevirens (montagne) weber-van bosse and c. racemosa var. turbinata (j. agardh) eubank; (2) kappaphycus alvarezii (doty) doty ex silva, represented by a highly proliferous green color mutant; and (3) solieria robusta (greville) kylin, with soft, gel-filled branches distinctly constricted at the bases. while the occurrence of caulerpa and kappaphycus have been documented in many parts of the country (trono and ganzon-fortes 1988), this is the first report on the genus solieria. solieria robusta occurs in tropical and subtropical waters, in the indo-pacific region from japan, fiji (south 1993), and australia (gabrielson and kraft 1984) to mauritius, madagascar, and south africa (norris 1988). in southeast asia, it has been reported from thailand (lewmanomont and ogawa, 1995), indonesia, singapore (teo and wee 1983), and southern china (tseng 1983). this is the first report of the species in the philippines. it has been found in the southern parts of the philippines, notably zamboanga and davao del sur (r. lucero, pers. comm.). it was only in zamboanga, sulu, and tawi-tawi where s. robusta was harvested and marketed in significant amounts. among the species marketed is solieria robusta (greville) kylin, a rhodophytan species never before reported from the philippines. the species has a long history of utilization among the samal and tausug ethnic groups who call it “tajuk bau’no.” this is harvested almost year-round, with variable seasonal supply which the natives attribute to rainfall variations. they are sold to retail vendors by volume, rather than by weight, in rectangular rattan baskets of approximately 15 kg per basket at php120 (us$1=php38). seaweeds are prepared as salads; these are washed in tap water and garnished with sliced tomatoes, onions, vinegar, and green mango slivers. the harvesting of s. robusta from wild populations in the waters around zamboanga is mainly carried out by tausug and samal fisherfolk who are mostly marginal earners with practically no resource conservation knowledge. fisheries authorities lack statistical data for the harvested s. robusta for these are mixed with the predominant carrageenophytes and gracilarioids which are similar in appearance. many of the natural seaweed beds containing s. robusta face the threat of habitat degradation owing to intensive kappaphycus seaweed cultivation and pollution discharges from coastal villages. unless conservation measures are implemented, the combined effects of these ecological and anthropogenic factors on s. robusta may result in its local extinction even before it can formally be documented, a scenario that is prevalent in other critical ecosystems such as tropical rain forests and coral reefs. further studies on the species are recommended to include: 1) at least more than a year-round study on its seasonality, phenology, and biomass yield; 2) study on its reproductive biology, recruitment, and growth rate; 3) determination of its optimal requirements for growth and development; 4) characterization of its phycocolloid content including seasonality of yield and quality; 5) development of management scheme for its natural stocks 6) the development of a suitable, economically feasible cultivation technology. data from these studies will aid in the development and formulation of an effective resource management scheme for the species and eventual development of its mariculture technology. ethnobotany of solieria robusta 77 references gabrielson pw, kraft gt. 1984. the marine algae of lord howe island (n.s.w.): the family solieriaceae (gigartinales, rhodophyta). brunonia 7: 217-251. lewmanomont k, ogawa h. 1995. common seagrasses and seaweeds of thailand. thailand: faculty of fisheries kanetsort university. 163 p. moreland ps. 1980. edible seaweeds of northern luzon, philippines: market prices, local taste preference, seaweed recipes, and other local uses. philipp j sci 108: 41-53. norris re. 1988. a review of natalian solieriaceae (gigartinales, rhodophyta), including the first south african records of solieria and meristotheca, and an investigation of erythroclonium corallinum. s afr j bot 54: 103-108. south gr. 1993. edible seaweeds of fiji: an ethnobotanical study. bot mar 36: 335-349. teo lw, wee yc. 1983. seaweeds of singapore. singapore: singapore university press. trono gc. 1974. eucheuma seaweed farming in the philippines. philipp agric 57: 327-334. trono gc. 1992. eucheuma and kappaphycus: taxonomy and cultivation. bull mar sci fish 12: 51-65. trono gc jr, ganzon-fortes et. 1988. philippine seaweeds. metro manila, philippines: national book store, inc.330 p. tseng ck. 1983. common seaweeds of china. beijing, china: science press. 316 p. 16_femtosecond gabayno, alonzo, and garcia 66 femtosecond pulse propagation in a highly nonlinear photonic crystal fiber j. f. gabayno1, c. a. alonzo2, and w. o. garcia3 national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: 1jgabayno@nip.upd.edu.ph; 2cca@nip.upd.edu.ph; 3wgarcia@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 66–69 femtosecond pulses are launched into a highly nonlinear photonic crystal fiber (pcf). the input and output spectra were measured using a monochromator and streak camera. the spectrum of the output from a 50 cm pcf pumped at 794 nm for different pump powers features asymmetric side lobes due to intrapulse raman scattering. similar measurements on a 100 cm pcf pumped at 795 nm highlight the appearance of blueshifted peaks as a result of energy transfer of solitons to dispersive waves. broadening in the spectrum is observed and attributed to raman-scattering-induced soliton self-frequency shift. spectrograms of both input and output pulses into a 50 cm pcf are captured using a streak camera. the spectrum reveals that individual modes observed on the spectrogram are actually a decomposition of the input pulse. introduction highly nonlinear photonic crystal fiber (pcf) is a new type of microstructure material that is now drawing the interest of researchers working in the areas of spectroscopy and telecommunication. just recently, application of these materials with ultrafast laser sources such as those in the femtosecond regime had productively set a new trend on supercontinuum generation (scg). ultimately, the coherent broadband produced through scg will find application such as a light source for telecommunication’s wdm systems. on the other hand, as far as the basic research is concerned pcfs are attractive media for studies related to understanding the complexities of nonlinear processes. currently, customizing the structure of pcfs to increase its nonlinear properties has become a standard procedure during fabrication. this liberty has not been previously available with conventional fibers. even the dispersion of pcfs can be designed so that it could exhibit zero-dispersion wavelengths (zdws) that are within the visible range. as a result, pcf could well be integrated into transmission systems which require less dispersion-induced pulse broadening. the structure of conventional fibers is primarily divided into a core with a higher refractive index than the surrounding cladding. an incident light into this fiber is effectively guided inside the core only if its angle of incidence satisfies the condition imposed by snell’s law. efficient coupling of the incident light strongly depends on the mismatch in the indices of refraction between core and cladding. to address this requirement, dopants are mixed with the core material during fabrication. the inclusion of dopants increases the refractive index difference on the core-cladding boundaries. in highly nonlinear pcfs, however, an array of air-silica cladding compensates for the high index difference with the core, which is made up of pure silica. the cladding, as a result of the air holes and silica matrix, has a very low effective index. effectively, incident light is confined inside the core due to total internal reflection (tir). femtosecond pulse propagation 67 these properties of solid-core pcfs, e.g., high index mismatch of core and cladding, and tailored core diameter and zdw permit several nonlinear processes during light propagation. worth mentioning are the stimulated raman scattering (srs), self-phase modulation (spm), and four-wave mixing (fwm), all of which are principal factors towards scg. in this paper, we present our results on the propagation of femtosecond pulses inside a solid-core pcf. we investigated the spectra of the input and output pulses with different input pump powers. the pump wavelength used is very close to the zdw of the pcf. the spectrum from 50 and 100 cm pcfs is measured using a spectrometer and a streak camera. experiment setup the pcf is pumped at 795 nm with a mode-locked ti: sapphire laser (tsunami) producing ~100 fs pulses extending over the near-infrared region from 705 to 985 nm and operating at 82 mhz repetition rate (fig. 1). a glan-laser prism polarizes the pulses from the femtosecond laser. it also provides an easy control over the amount of pump power launched into the pcf. to obtain a small beam spot at the focus of l1, about the size of the pcf core, a beam expander is integrated in the system to collimate and expand the beam diameter. an aspheric with 8 mm focal length and 0.50 na couples the laser into the pcf input face. at the fiber end is another aspheric which guides the output pulses towards the entrance slit of a monochromator/ streak camera. the pcf is a highly nonlinear fiber from blazephotonics. its core diameter of 2.3 mm is designed for zero-dispersion wavelength at 790±5 nm. the 50 and 100 cm pcfs have their ends cleaved clean to minimize reflection losses especially at the input face. the pcf was attached to an xyz stage, which offers a great deal of freedom over positioning the input end at the focus of l1. scanning electron microscopy (sem) photos of the core and cladding structures of a highly nonlinear pcf are shown in fig. 2. results and discussion the input and output spectra of femtosecond pulses in a 50 cm pcf is shown in fig. 3. it features a series of spectra arranged according to increasing input pump power po. we had observed that an incremental increase with po yields a spectrum containing several orders of side lobes. however, we concede that the increased complexity of the featured side lobes cannot be solely attributed to the increase in po. the corresponding change in the pump polarization when varying po was observed to affect the coupling efficiency on the fiber input. apparently, however, figs. 3(b)–3(d) highlight fig. 1. a diagram of the setup reveals a beam of femtosecond pulses emanating from a ti:sapphire laser. the beam is attenuated by a glan-laser polarizer and expanded to a diameter of 4 mm. two mirrors m1 and m2 steer the collimated beam towards lens l1; l1 and l2 couple the pulses in and out of the photonic crystal fiber (pcf). the pcf is attached to an xyzflexure stage for efficient and easy positioning. the spectral profiles of the output pulse were detected and captured using a monochromator and streak camera, respectively. ti:sapphire attenuator beam expander m1 m2 l1 l2 pcf xyz stage monochromator/ streak camera fig. 2. scanning electron micrograph (sem) photos of an (a) array of air-silica cladding and (b) pure silica core in a highly nonlinear pcf. (source: http:// www.blazephotonics.com) gabayno, alonzo, and garcia 68 that the peak wavelength of each side lobe is insensitive to the input power. although spectral details are added with increasing po, the unique locations of the side lobes are retained. some authors suggest that the characteristic asymmetries in the lobe are due primarily to intrapulse raman scattering, a crucial factor in the route towards scg. figure 4 above shows the output spectra of measurements made on a 100 cm pcf pumped at 795 nm. the spectrum of the input pump is embedded on each figure for the purpose of clarity. broadening of the spectrum is induced by raman scattering (rs). as po increases, considerable shifting of the pulse towards the infrared region is observed. this is a result of raman-induced soliton self-frequency shift. consequently, with increasing po energy is efficiently transferred towards shorter wavelengths. new peaks have started to emerge at the region of shorter wavelengths. for an input power of 350 mw, a distinctly intense pulse appers at 732 nm. this dispersive waves accumulate energy as higher-order solitons are generated and shift towards longer wavelengths. spectrograms of both input and output pulses from a 50 cm pcf are shown in figs. 5 and 6. the input pump is centered at 795 nm. fig. 3. input and output spectra measured using a mochromator on a 50 cm pcf for different pump powers: (a) input pump, λp = 794 nm, (b) po = 300 mw, (c) po = 328 mw, and (d) po = 335 mw. 7 4 0 7 8 0 8 2 0 8 6 0 w a v e le n g t h , n m p o = 3 0 0 m w 0 7 4 0 7 8 0 8 2 0 8 6 0 w a v e le n g t h , n m λ p = 7 9 4 n m p o = 3 2 8 m w 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 p o = 3 3 5 m w (a) (b) (c) (d) wavelength (nm) n or m al iz ed i nt en si ty 740 780 820 860 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 (a) (b) (c) (d) 780 820 860 − − − λp = 795nm ____po = 350mw − − − λp = 795nm ____po = 345mw (a) (b) 700 750 800 850 900 (a) (b) wavelength (nm) n or m al iz ed i nt en si ty 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 fig. 4. input and output spectrum measured on a 100 cm pcf pumped at 795 nm for different pump powers: (a) 345 and (b) 350 mw. in te ns it y (a .u .) 775 785 795 805 wavelength (nm) 0 0.5 1 g in te ns ity , a .u . 0 0 .1 25 0. 25 0 .5 0 .6 25 0. 75 1 time w av el en g th in te ns it y (a .u .) 0.75 0.625 0.5 0.25 0.125 0 fig. 5. (a) spectrogram of the input pulse captured by a streak camera. (b) spectral profile of the input pulse. (a) (b) femtosecond pulse propagation 69 the pump spectrum in fig. 5(b) assumes a broad gaussian envelope which extends from 775 to 810 nm. meanwhile, the spectrogram of the output pulses (fig. 6) features a number of modes exhibiting different spectral profiles. the spectrogram of the output pulse shows that each of these modes evolve quite independently of each other. featured in figs. 7(a)– 7(c) are three of the output modes. each mode has generally narrower spectrum as compared with the pump. integrating the spectrum of all measured modes will reveal that each individual mode is summarily a decomposition of the input pump. conclusion trains of ~100 fs pulses at 794 and 795 nm are launched into 50 and 100 cm solid-core pcfs. the output spectrum of the 50 cm pcf measured by a monochromator reveals asymmetric side lobes induced through intrapulse raman scattering. the measured spectrum from a 100 cm pcf pumped at 795 nm shows an emergence of a dispersive wave peaked at 732 nm as a result of raman-scattering-induced soliton selffrequency (ssf) shift. rs and ssf have also been observed to lead to spectral broadening of the pump. the spectrogram of the 50 cm pcf captured by a streak camera shows decomposition of the input pump into different modes. references agrawal, g.p., 1995. nonlinear fiber optics. 2nd ed. academic, new york. cristiani, i., r. tediosi, l. tartara, & v. degiorgio, 2004. dispersive wave generation by solitons in microstructured optical fibers. opt. express. 12: 124 – 135. gaeta, a., 2002. nonlinear propagation and continuum generation in microstructured optical fibers. opt. lett. 27: 924–926. genty, g., m. lehtonen, h. ludvigsen, j. broeng, & m. kaivola, 2002. spectral broadening of femtosecond pulses into continuum radiation in microstructured fibers. opt. express. 10: 1083–1098. genty, g., m. lehtonen, h. ludvigsen, & m. kaivola, 2004. enhanced bandwidth of supercontinuum generated in microstructured fibers. opt. express. 12: 3471–3480. knight, j.c., 2003. photonic crystal fibers.nature. 424: 847– 851. 75 785 795 805 7 5 78 5 79 5 8 0 5 (a) (b) (c) wavelength (nm) n om in al iz ed i nt en si ty 775 785 795 805 (a) (b) (c) fig. 7. (a)–(c) measured spectrum of the modes indicated by the arrows in fig. 6. time w av el en g th in te ns it y (a .u .)(a) 0.75 0.625 0.5 0.25 0.125 0 (b) (c) fig. 6. spectrogram of the output pulse reveals decomposition of the input into different modes. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 strategies-art.14 pinat et al. 110 science diliman (july december 2000) 12:2, 110-120 abstract strategies in mobilizing coastal communities for community-based coastal resource management in bolinao, pangasinan jacquelyn pinat*, renato a. turion, wenceslao n. asido jr., and liana talaue-mcmanus marine science institute, college of science university of the philippines, diliman, quezon city 1101 philippines tel. no.: (632) 922-3921; e-mail: jacquelynpinat@hotmail.com introduction the marine fisheries resources management project (mfrmp) has been in bolinao since 1997. since the commencement of its operations, the project has been involved in organizing, training, enhancing resource, and managing initiatives in various barangays all over the the marine fishery resources management project (mfrmp) hopes to facilitate partnership between the local government unit and the local communities in managing the coastal resources of bolinao. mobilization, both at the community and municipal levels, has been very important in promoting community-based strategies in coastal resources management. the community organization process in the municipality has gone through several levels; and different organizations have been formed. in empowering individuals and organizations, strategies tend to be varied and fluid depending on the need, the reason for mobilization, and the resources at hand. the bolinao experience showcases different strategies used in implementing the resource enhancement, coastal zoning, harvest regulation, and capability building components of the program. these have included the formation of people's organizations, the mobilization of zonal action teams for each of the four zones, the creation and orientation of fishery and aquatic resources management councils at the barangay and municipal levels, and the active collaboration with the local government unit. these strategies and approaches have provided the people and the communities a wealth of experience and lessons that provide helpful insights in undertaking different endeavors. the strategies employed in the mobilization activities have significantly contributed to the empowerment of communities and individuals who are the primary managers of their resources. keywords: community mobilization, participatory planning, integrated management, cbcrm, coastal resources management municipality. one main activity was the formulation and passage of the bolinao coastal development plan (cdp) through advocacy and technical support. the project has four major components namely; (a) capability building, (b) resource enhancement, (c) coastal zoning, and (d) harvest regulation (fig. 1). prior to the mfrmp, pioneering initiatives in organization and development work were undertaken by a tripartite partnership among the university of the * corresponding author strategies in mobilizing coastal communities 111 philippines-marine science institute’s (up-msi) community-based coastal resource management project (cb-crmp), the haribon foundation for the conservation of natural resources, and the university of the philippines college of social work and community development (up-cswcd) from 1993 to 1997. since then, these institutions have regularly shared experiences and strategies while focusing on specific issues. haribon is focusing on livelihood research in several barangays in bolinao, while upcswcd is concentrating its efforts in the municipality of anda, pangasinan. in 1996, four people's organizations (po) were formed in bolinao: sapa in arnedo, sammabal in balingasay, sammabi in binabalian, and sammaka in pilar. other people's organizations were formed later. at present, mfrmp has focused on strengthening and expanding these organizations, and assisting them in specific coastal resource management (crm) activities, such as the establishment of marine sanctuaries and the formulation of policies and management mechanisms at the municipal level as provided for in the bolinao cdp. the project also provided technical support and skills training for the different organizations. through the years, the organizations have expanded their membership to include more fisherfolk, as well as other locals in the municipality. member-organizations of the municipal federation, kaisaka, expanded to include more organizations from other barangays. in 1998 at the thick of the cdp formulation, the new philippine fisheries code, republic act 8550 (ra 8550), was passed by the national government. the law mandated the formation of the municipal fisheries and aquatic resources management councils (mfarmc) which would represent the fisherfolk before the municipal government with respect to policy formulation and implementation of different fishery laws and provisions stated in ra 8550. the bolinao cdp, which was started in 1997 through a series of consulations with the fisherfolk of bolinao, is a model of people-centered policy advocacy. the cdp, as a blueprint for municipal resource management, was formulated by a technical working group (cdp-twg) and subsequently validated and refined through successive community consultations and public hearings (talaue-mcmanus and others 1999). the plan was proposed and after two years of continued advocacy, the bolinao cdp was passed into law in december 1999. implementation of the provisions in the ordinance commenced in january 2000. the bolinao experience has been highlighted in many instances, notably because of its pioneering efforts in formulating the first participatory cdp, which serves as the framework for people-centered coastal resource management in the municipality. this paper highlights the different strategies employed in mobilizing the communities and assisting them in implementing the cdp to make the bolinao experience a model that may be replicated in other coastal municipalities. the cdp as a framework for coastal resource management to maximize the use of bolinao’s coastal resources, and to limit the possible undesirable environmental effects of this use, the coast of bolinao was divided into four zones, namely: zone i, the eco-tourism zone; zone ii, the multiple-use zone; zone iii, the fishery management zone; and zone iv, the trade and navigational zone (fig. 1). zone i encompasses the barangays of patar, ilog malino, estanza, tupa balingasay, and arnedo. most of the possible tourist destinations, such as white sand beaches, caves, and resorts are found in these barangays. it was for this reason that this area was allocated for eco-tourism use. zone ii is the area along the caquipotan channel, between mainland bolinao and the municipality of anda. all aquaculture activities such as pens and cages for the culture of milkfish and other finfish species are to be limited to this area, which included the barangays of pinat et al. 112 provisions were drawn up by a technical working group composed of some sb members, municipal officials, and representatives from the fisherfolk organizations, with the mfrmp providing technical assistance. structures and organizations as avenues for mobilization people's organizations at the village level the backbone of bolinao's struggle for crm has been the number of pos formed by the fisherfolk since 1989. many of these organizations were, and still are, at the forefront of advocacy and resource management, both at the local and municipal levels. the mfrmp has eight partner pos in bolinao. many of these fisherfolk organizations were formed during the previous tripartite partnership of haribon, the up cswcd, and the up-msi which started in 1993 (mcmanus, 1995). among those formed earlier were sapa in arnedo, sammabal in balingasay, sammaka in pilar, saudi in ilog malino, and sammabi in binabalian. after the tripartite partnership, the pos sammal in luna and sammacu in culang were formed. the activities of these organizations are discussed later. the kaisaka federation the kaisaka federation was formed in 1995, in line with the formulation of the cdp, by sapa sammabal, sammabi, and sammaka. the federation's role was important in the municipal fight against the then proposed bolinao coastal development plan. it spearheaded the lobby for a pro-people cdp as an alternative to the industry-focused development plan being proposed.the federation took an active role in the community consultation and public hearings on the different issues tackled in the cdp. cdp-twg the technical working group (cdp-twg) for the cdp was formed in august of 1996, after an orientation on the proposed cdp. during this orientation, memberorganizations of the kaisaka presented their own lambes, zaragoza, catungi, tara, culang, luna, luciente ii, pilar, salud, and lucero. zone iii is composed of the barangays in santiago island. the extensive reef system in the island is home to various species of fish and other marine life and is also the migration route of the siganids, bolinao's most dominant fish species. this was the reason why the area was designated as the fishery management zone. the barangays included in zone iii are lucero, binabalian, salud, pilar, victory, goyoden, and dewey. the seat of government in bolinao is in germinal. together with the adjacent barangays of luciente i and concordia, the area was designated for trade and navigation and called zone iv. fish and agricultural trading, all modes of transport, and various small to largescale businesses are found in the zone. in the last quarter of 1999, the sangguniang bayan made the final comprehensive deliberation on the provisions of the cdp before passing it in december. the implementing rules and guidelines for the different balingasay bolinao santiago island town of cabarruyan zone ii multiple use zon e i eco -to uris m zone iv trade & navigation zone ii fishery management 16.45 16.40 16.35 16.30 16.25 16.20 119.80 119.85 119.90 119.95 120.00 120.05 fig. 1. the zoning of the municipality of bolinao into four major use zones according to the coastal development plan strategies in mobilizing coastal communities 113 version and interpretation of the major use zones, as well as the different issues and problems in crm to an audience from various sectors. in addition, the group proposed a range of management steps which could be taken as provisions in the cdp to answer the stated issues. during the discussion, more ideas came up and other plans were drawn. after the discussion, the cdptwg was formed. the group was composed of representatives from different sectors including fisherfolk, private entities, the business sector, academe, and the local government unit (lgu). the lgu expressed support for the group and provided the budget for its operations and consultations with the different communities. together with kaisaka, the group held workshops and community consultations which helped clarify local perceptions of the living coastal resources, the legal and social mechanisms that govern access to them, and the problems and possible solutions associated with their utilization. the active participation of the stakeholders in the process deepened their sense of involvement and commitment to achieving the solutions to the problems they had identified. zonal action teams (zats) zonal action teams (zats) were formed in each zone to serve as core groups to provide support for pos and kakaisa. the zats would be focused on the issues in each of the zones. mobilization and management of resources will be immediate in the zone, and each zat will have the autonomy to manage the zone at the zonal level and may seek outside support when needed. the zats are composed of representatives from the different sectors affected by the issues in the zones, and they do not have an established structure nor a formal leader. the formation of the zats in the different zones of the municipal waters was a strategy to complement the resource management and conservation initiatives undertaken by the different people's organizations and the kaisaka. with the legislation of the cdp in bolinao, the zats specifically looked at the issues arising in the four zones. initially, the zats for zones i and iii and an ad hoc team for zat ii were set up during the last quarter of 1998. for its part, the zat i was particularly keen on addressing issues revolving around the bangus fry industry in zone i. with zone ii designated as the area for aquaculture, the focus of the ad hoc team were on fishpens and cages, as well as the different navigation routes. meanwhile, zat iii was envisioned as a group that would support the initiative for a marine protected area in the malilnep channel in santiago island, as well as address the siganid concession and reef fishery issues. since every zat was a multi-sectoral group, the membership varied depending on the zone. aside from fisherfolk, representatives from the private sector, the church, the academe, and the barangays, other members included those involved in the pertinent issues per zone. for instance, zat i members included fry gatherers and resort owners; zat ii members included fishpen and fish cage owners and caretakers . zat iii members included fishers using both passive and active gears. bfarmc executive order no. 240 (eo 240) provided for the formation of barangay fisheries and aquatic resources management councils (bfarmc) which would assist in the implementation of the law and recommend activities and action points to the barangay council. initially, 11 bfarmcs were formed in zones i, ii, and iii, with the help of the respective zats for each zone. the bfarmcs were important in championing the cause of crm through the cdp at the barangay levels. more bfarmcs were formed to increase the membership of the mfarmc, and to allow for the participation of the other barangays in the implemention of ra 8550. however, not all of these were actively participating in the mfarmc meetings. several of the bfarmcs have found specific issues in their own barangays, such as the bfarmc of germinal which was addressing the barangay’s waste problem. many of the bfarmcs were not yet active since they were newly formed and still lacked orientation and training. nineteen of the 23 coastal barangays formed their bfarmcs; but the rest of the barangays will be visited again when the mfarmc has finished with some of its more immediate activities. mfarmc the passage of ra 8550 called for the formation of the municipal fisheries and aquatic resources pinat et al. 114 management councils (mfarmc) to recommend and assist in the implementation of ra 8550 at the municipal level. the bolinao mfarmc was organized in late 1998. the council had its first meeting in early february 1999 and was officially launched on february 23, 1999. the members included representatives from the different sectors, the zats of the different zones, and from the different barangay fisheries and aquatic resources management councils (bfarmc) which were earlier organized in 11 barangays. several members were added from the lgu, including the municipal aquaculture technician, the municipal planning and development officer, representatives from the department of agriculture, and from the sangguniang bayan. the sangguniang bayan secretary, the up marine science institute, and the haribon acted as secretariat for the mfarmc. the mfarmc hoped to prepare plans to address the conservation, utilization, management, protection, and development of the coastal resources of the municipality. the mfarmc took the lead role in advocating the passage of the cdp ordinance. since its formation, the council has actively participated in public hearings, and has spearheaded some of the education and information campaigns at the local level. in support of the small and marginal fisherfolk, the mfarmc also addressed local issues which have been brought to attention of the municipal government. the mfarmc made its presence felt in the sangguniang bayan meetings as to support the reform of the milkfish fry concession system. other activities of the mfarmc were the preparation of the fisherfolk for registration, recommendation to the provincial board for the immediate approval of the cdp, training and orientation regarding mfarmc and bfarmc, socio-economic profiling of bolinao, alternative livelihood identification, information dissemination in coastal barangays, and the formation of implementing rules and guidelines for the cdp. the body also targeted the construction of the mfarmc office which was provided by the office of the mayor. community mobilization the process of organizing the institutions and structures discussed above varied depending on the issue at hand and the time available for organization. several realizations have been made in the course of advocating effective crm. the continuing development of the cdp process coupled with the government initiatives brought about by the passage of ra 8550, the value systems and the behavior of the communities, are factors that affected the organization and mobilization process. mobilization and action from the zonal and local levels zone i: eco-tourism zone • eco-tourism zone i • milkfish fry • marine protected area tourism the activities in zone i revolved around issues concerning eco-tourism, the marine protected area, and the milkfish fry concession. because long, white sand beaches abound in the zone, the area was designated for eco-tourism. in one year, the number of beach resorts and beach huts increased considerably. the increase of structures and the number of expected tourists raises issues such as the pollution of the coastal areas, the source of potable water and construction materials, the conversion of foreshore lands, and other threats to the carrying capacity of the area. to support the growing tourism industry, the bolinao hotel and restaurant owners association and the municipal tourism council were established. the barangay councils in the zone were supportive of the initiatives taken by these two groups, in line with the existing organizations, to maintain the quality of the coastal resources, while nurturing the budding eco-tourism industry. milkfish fry the balingasay river is believed to be the spawning ground for milkfish fry. the milkfish fry industry in bolinao supports many families during the fry season. strategies in mobilizing coastal communities 115 however, for a few years now, the fry industry has been run by a concession system. the concessionaire may be an individual, family, or group, and must have the capacity to raise the concessionaire's fee which is paid to the municipal government, as well as to buy the fry in bulk from the gatherers. this practice allows the concessionaire to dictate the buying price, to the detriment of the fry gatherers. the concessionaire profits well in the arrangement. the samahan ng maliliit na mangingisda ng balingasay (sammabal) proposed an alternative scheme for running the fry industry, but it has been put on hold by the sangguniang bayan of bolinao, and will be looked into for possible implementation in the next fry season. meanwhile, the municipal government has awarded the concession to the federation, provided it can pay the concession fee set for the year. the federation has been able to raise this amount fromm pledges and loans. at present, the management scheme, projected profits, and sharing schemes are being studied. mpa the first marine protected area in bolinao is also situated in barangay balingasay. the mpa is being managed by sammabal in cooperation with the barangay council and the barangay resource management council. sammabal has been responsible for monitoring activities while the council has helped in patrolling the area to prevent encroachment.3 strategies for mobilization in zone i involve the formation and reorganization of different groups (see fig.2 ). the fisherfolk organizations work on issues at the local level while zat i, at the zonal and municipal levels, with the kaisaka and mfarmc providing support. however, as activities and issues became well understood and evolved into zonal and municipal concerns, the zat i became a member of kaisaka to prevent duplication of tasks, to consolidate the strengths of both organization, and to speed up the coordination process. the kaisaka was strengthened by the merger with zat i because many of the latter’s members were municipal and baranggay officials. strategies like this proved to be wise and helpful in furthering the cause of the people. zone ii: multiple use zone • aquaculture zone ii • water quality • navigation • mariculture water quality and aquaculture water quality is the main issue in zone ii, which lies along caquipotan channel, and is used for aquaculture, fishing, and navigation. fishpens and cages abound in the zone. the area has supported the milkfish culture industry in bolinao. crm activities in the area include bi-monthly water quality monitoring and the quarterly report of data gathered. these activities are undertaken to help the fishpen and cage operators understand the impact of the industry on the environment. there are two partner fisherfolk organizations in the zone, but both are newly formed and are not directly involved in large scale aquaculture. this is why the monitoring of the water quality is done with some of the fishpen/cage operators. at present, there are nine operators who have cooperated in the activity. the monitoring is done by a team composed of students from the bolinao school of fisheries which provides technical expertise. this monitoring activity also serves to hone the technical skills of the students. the local government unit provides the boat and the fuel while kaisaka federation mfarmc sammabalbrmc sapasaudi people’s organization bfarmc zonal action team zonal action team fig. 2. organizational mobilization in zone 1 pinat et al. 116 the mfrmp provides assistance in skills training, discussions, and data analysis. results are reported to the people in the zone, and to the sangguniang bayan and the office of the mayor for action on recommendations or as reference for future legislation. this tripartite partnership is hoped to ensure the sustainability of the activity. after the mfrmp ends, the water quality monitoring may be continued by the students and the co-operators who will have learned skills in analysis as well. water quality monitoring can then become a local activity and it is hoped that other operators may become aware of its importance. the ad hoc committee for zat ii was formalized during a workshop where water quality data gathered from fishpens and cages in zone ii were shared and discussed with zone i members. some members of the zat ii have pledged to participate in the monitoring activities as co-operators and in informing other fishpen and cage owners of the activity. they will participate in information gathering and dissemination, and in supporting issues on navigation and abandoned nets and fishpens. mariculture initial experimental mariculture of siganids is being introduced in culang and luna. to date, only sammacu is active while samalu awaits siganid fry for stocking. organizational mobilization in zone ii include the groups shown in fig. 3. mobilization has been challenging because the barangays are far apart. also, the fisherfolk organizations are not active participants in the milkfish culture, and hence, are not keen on participating in water quality monitoring. also, the bfarmcs are not active and have yet to be visited. along the channel, issues vary in every barangay. in zaragoza for example, the primary issue is the threat posed by a salt farm and its proposed expansion. the bfarmcs and the organization will still have to be strengthened along with the other organizations. zone iii: fisheries management zone • siganid industry zone iii • reef fisheries • marine protected area • mangrove reforestation mpa zone iii is the area of santiago island where organizing and mobilization is very important because of its vast reef complex that supports the fishing industry of the municipality. some of the earliest organizing efforts were done in the zone. sammabi, the samahan ng mga mangingisda at mamamayan ng binabalian, was one of the first fisherfolk organizations formed in bolinao and was a founding member of kaisaka. with the protection and the preservation of the reef complex in mind, the organization worked for the declaration of a marine protected area (mpa) in the malilnep channel. an ad hoc team for zone iii was formed in september 1998, after a fisheries forum conducted in santiago island. the group was formed to provide support in the zone, especially in advocating the proposed mpa in the malilnep channel. initial meetings were set by the team to refine plans on the topics discussed in the forum, such as siganids, close and open season, gear use, and mpa. the group also received an orientation on mpas, but the discussion was later diverted to theconcerns mentioned above. the group formulated a statement of unity which was submitted to the municipal government. after meeting schedules were mfarmc kaisaka federation sammacusammal people’s organization bfarmc ad hoc zonal action team barangay councils fig. 3. organizational mobilization in zone ii strategies in mobilizing coastal communities 117 set for the last quarter of 1998, membership participation started to dwindle and many of the planned activities were not realized. sammabi also underwent organizational problems which limited its effectivity. at present, the zat iii has not been formed. the proposal for an mpa in malilnep channel is still waiting for support from groups in the island. to make up for the inactivity of the ad hoc team, several interest groups were approached to address specific issues. the siganids group was established in goyoden, and it has made a resolution on siganids. the barangay council of lucero has expressed interest in having an mpa in its barangay waters. to support this, environmental education, information dissemination, and community consultations are being held. the issue on the use of triplets has also cropped up and people are being consulted on how to address this in the island. the issuebased approach has been effective so far in realizing the initial plans of the zat. some members of the interest groups are also members of the ad hoc zat, but since it has not met for a while, these people are participating in the interest groups instead. siganid industry/reef fisheries there have been reports that the fishing industry, especially that of the siganids, has been experiencing setbacks compared to past years. as a result, the municipal government has suspended the awarding of rights to fish corrals; it will operate the fishery lots instead. as an alternative to the siganid industry, the project is now trying to develop a system for the grow out of siganids from fry to mature sizes. this initiative is being presented to the interest group for siganids. another fisherfolk organization, the samahan ng maliliit na mangingisda ng victory or smmv is also interested in undertaking the grow out of siganids. this newly formed organization is also planning to put up a four-hectare mpa in their barangay. mangrove reforestation the sammaka (samahan ng mga mangingisda at magsasaka in pilar) has been successful in maintaining a mangrove reforestation project which covers 12 hectares. it is also assisting the smmv to put up a similar mangrove reforestation project in victory. zone iii has provided much challenge in terms of islandwide organizing (see fig. 4). the barangays are far apart and the people have different interests. almost every barangay is interested in a different initiative. the planned sanctuary in the malilnep channel, for instance, did not progress because of lack of support at the local level. sammabi, being the closest barangay to the proposed site, was tapped to support the issue, but problems arose within the organization. also, the lack of support on the ground because of disinterest and lack of information has kept the malilnep channel open to the fishers who unknowingly continue to deplete the resources in the area. while another barangay may be interested in supporting the malilnep mpa, it may be geographically far from the area. instead of forcing the issue on the people and resurrecting some of the weakened organizations, interest groups were formed to support the issue. the interest groups are composed of persons who have expressed interest in discussing and taking initiatives regarding the issues. for instance, the siganid group is composed of fish corral operators from goyoden and some fisherfolk from pilar and victory. this strategy has allowed the participation of other barangays on other issues which did not concern them in the past. several bfarmcs have also been formed in the island to provide representation to the municipal farmc, as well as support in various issues. however, these bfarmcs have yet to be strengthened; not a single one has reportedly been active. barangay councils fig. 4. organizational mobilization in zone iii people’s organization sammacu bfarmcbfarmc sammal group interests kaisaka federationmfarmc pinat et al. 118 zone iv: trade and navigation • solid waste management zone iv • fisheries trade • navigation solid waste management there are no partner fisherfolk organizations in zone iv, and the proposed mobilization organization (fig. 5) was not realized. however, two bfarmcs have been formed. in addressing the primary issue of solid waste in the zone, the bfarmc in germinal was initially tapped. garbage collection in the town proper is done through the office of the mayor. however, past experiences showed that collection is not done on a regular basis and that the trucks are not able to reach foreshore areas where garbage is a huge problem and waste disposal is unhealthy. the bfarmc worked with the barangay council, but has since laid low for lack of support. the issue has been brought to the attention of the municipal administrator. because zone iv is the town proper, many of the activities are dealt with at the municipal level. since solid-waste is a municipal-wide problem, initial plans to address the issue were done through an ad hoc solid waste management council. the support of other stakeholders, such as business establishments, market associations, as well as transportation operators are needed. the municipal health officer chairs the committee. mobilization in the zone usually involves the sangguniang bayan, local government unit, and other municipal offices with the support of the other stakeholders and the bfarmcs at the local level. challenges amidst all the resource management initiatives, the prime challenge in bolinao has been how to sustain all efforts at all levels. in many instances, the initiatives are not continued after support from the mfrmp has ended. in many instances, the people become too dependent on funding and technical support and are not able to act without guidance. at the municipal level, the lgu has also come to depend on the technical assistance provided by the project. in the formulation of the cdp for instance, the write up and the sourcing of data are all left to the mfrmp staff. at times, even the correspondence and facilitation of the meetings are shouldered by the staff. activities in bolinao are often done to either corroborate or negate data gathered by the msi or the mfrmp. for instance, declining fish catch has spurred the mfrmp to propose experimental grow out of siganids. this activity aims to give enough time for siganid eggs to hatch and ensure stock on the next run. declaring an area for marine protection is another way to enhance fish stock in the area, while providing sanctuary for other marine life threatened by overfishing. these initiatives may be welcomed, but their long term impact on the environment and the lives of the people have yet to be seen. with the mfrmp on its last year, many of the results will not be seen before it ends and many support mechanisms may not be put in place in time. many of the activities have taken too much time. the cdp formulation has taken years, yet the ordinance still has to be signed, although, it has been "approved in principle." to speed things up, the staff have provided extra support and assistance beyond what is necessary. this is also true at the community level. if left alone, the people tend to become complacent, and without guidance, activities stretch and take longer routes even if these are not needed at all. bureaucracy has also been a factor, although this is expected in any community. because many of the activities of the mfrmp are rooted in scientific data, there is a need for an information and environmental education campaign each time an initiative is undertaken. yet there are still cases when lack of information is the cause of the delay or fig. 5. proposed organizational mobilization in zone iv barangay councils people’s organization sammacusammal kaisaka federation mfarmc strategies in mobilizing coastal communities 119 failure of an initiative. for example, fisherfolk tend to feel threatened whenever an mpa is proposed. their initial understanding is that the mpa will be guarded, thereby preventing them from gathering in the area. they do not immediately appreciate that the mpa is for their benefit. this is why the proposed mpas in arnedo, malilnep channel, and lucero have not succeeded. many people, including the lgu, have yet to appreciate and understand the advantage of having the msi in bolinao. the participatory monitoring of the mpa in balingasay, while still being done by some members of sammabal, still see it as a task given to them by the project; many have yet to find use for the data. the challenge of localizing information and maximizing the bolinao marine laboratory still lies ahead. sustaining mechanisms training and capability building to ensure that the people, organizations, and the local government units are equipped with the skills to continue the initiatives of the mfrmp, training and skills development work continues. also, the project provides equipment and reading materials, as well as a venue for activities. training, assessments and planning workshops for capability building and strengthening are continually being given. the lgu, key leaders and organizations have been identified and are being prepared them for the eventual phase out of the mfrmp. slowly, many activities are being relegated to the municipal government. mechanisms that ensure continued support for mfrmp initiatives from the lgu are being taken, such as the passage of the ordinance and the formulation of the implementing rules for the cdp, and moas that still require that they provide support. networking the organizations have been introduced to different organizations which may be of big help in terms of funding and technical assistance. the patrol boat for the mpa in balingasay was solicited from lgcamc and plan international. the mangrove reforestation projects in two barangays in santiago island are also supported by lgcamc and the denr. to ensure the representation of the people in policy and implementation, the mfarmc decided to form bfarmcs in all coastal barangays even though is not mandated by ra 8550. at present, the mfarmc is a large group of multi-sectoral representatives, the fisherfolk sector. the kaisaka has also taken it upon itself to participate in the mfarmc meetings, as well as sit in the other municipal activities for policy advocacy. the kaisaka has been recognized by the lgu as having a legal identity; in many cases these two have a good working relationship and coordination. advocacy advocacy is still a continuing activity in bolinao, even branching out to the youth sector. environment education is still done every time a new initiative is taken. the importance of taking care of the environment is still being instilled in the people both at the local and municipal levels. advocacy is done in many forms and the initiatives taken by the people are examples of concrete advocacy for the good of the environment. conclusion the people of bolinao grabbed the headlines a few years ago when they fought against the construction of a cement plant. many years have passed since, and the municipality faces another challenge–that of productivity and equitable access to coastal resources. the current threat is not an outside force, but one that people have created themselves. the deteriorating water quality along caquipotan channel, the declining siganid industry, among others, are examples of the threats to livelihood and environment facing them. this reality has been understood by many and as an answer to this, the cdp was formulated by the people. the bolinao experience has been hailed by many because of its pioneering effort in formulating a cdp. as the framework for local community resource management, the cdp does not end with its passage. the bigger challenge would be to ensure its pinat et al. 120 implementation and effectiveness in uplifting the lives of the people. many people’s organizations have been formed in bolinao and many initiatives have been undertaken to prepare the people for the implementation of, and as implementors of, the cdp, with bright hope for abundance and peace. references aguinaldo m, talaue-mcmanus l. 1999. facilitating the formation of an environmental advocacy program in bolinao: enhancing science and information use for community based management. unpublished. asido wn and others. 1999. the coastal development plan of bolinao pangasinan: a participatory process. unpublished. mcmanus j, and others. 1992. resource ecology of the bolinao coral reef system. iclarm. mcmanus l. 1995. community based coastal resources in bolinao, philippines: an evolving partnership among academe, ngos and local communities. coastal management in tropical asia. mcmanus l and others. 1999. participatory planning for coastal development in bolinao, philippines: a powerful tool for conflict resolution.conflicts and collaboration in natural resources management journal. 273 pp. menez l, and others. 1991. survey of the coral reef resources of western lingayen gulf, philippines. multi-sectoral committee for coastal development planning in bolinao. 1997. bolinao coastal development plan. rodriguez s, and others. 1997. the barangaen fishing concession in bolinao: an ethnographic study of a customary marine tenure system [dissertation]. quezon city, philippines: university of the philippines. salmo s iii. 1999. establishment and implementation of the balingasay marine protected area: a community -based approach. unpublished. salmo s iii. 1999. milkfish fry concession system in bolinao, pangasinan: implications to coastal resources management. unpublished. verceles l, talaue-mcmanus l. 1999. initiating a participatory monitoring feedback system: an important entry towards sustainable aquaculture in bolinao, northwestern philippines. unpublished. yambao a, salmo s iii. 1998. a preliminary assessment of coastal development planning in the municipal waters of bolinao, pangasinan. out of the shell 6 (2): 1-5. development of feature set, classification implementation and applications for vowel migration/modification in sung filipino (tagalog) texts and perceived intelligibility virginia b. bustos1, triah joyce g. dela cruz1, ramon maria g. acoymo2 and rowena cristina l. guevara 1* 1electrical and electronics engineering institute, college of engineering , university of the philippines diliman 1101 quezon city 2voice and music theater/dance department, college of music university of the philippines, diliman 1101 quezon city *corresponding author: gev@eee.upd.edu.ph received: 5 november 2009; revised: 18 february 2010; accepted: 24 june 2010 abstract with the emergence of research on real-time visual feedback to supplement vocal pedagogy, the utilization of technology in the world of music is now seen to accelerate skills learning and enhance cognitive development. the researchers of this project aim to further analyze vowel intelligibility and develop software applications intended to be used not only by professional singers but also by individuals who wish to improve their singing capability. data in the form of sung vowels and song pieces were obtained from 46 singers. a listening test was then conducted on these samples to obtain the ground truth for vowel classification based on human perception. simulation of the human auditory perception of sung filipino vowels was performed using formant frequencies and mel-frequency cepstral coefficients as feature vector inputs to a two-stage discriminant analysis classifier. the setup resulted in an over-all training set accuracy of 89.4% and an over-all test set accuracy of 90.9%. the accuracy of the classifier, measured in terms of the correspondence of vowel classifications obtained from the classifier with the results of the listening test, reached 92.3%. using information obtained from the classifier, offline and online/real-time software applications were developed. the main application features include the display of the spectral envelope and spectrogram, pitch and vibrato analysis and direct feedback on the classification of the sung vowel. these features were recommended by singers who were surveyed and were incorporated in the applications to aid singers to adjust formant locations, directly determine listener’s perception of sung vowels, perform modeling effectively and carry out vowel migration. keywords: filipino, vowel migration, intelligibility introduction one of the objectives of the singers of bel canto is the development of a vocal scale without interruption throughout its length. (appelman, 1986) this demands vowel modification in the upper notes to preserve the true vowel sound and to prevent notes from becoming disagreeable or harsh. the technique has become a means of transition to the upper voice for many centuries. the singer, science diliman 21(2):13-24 13 mailto:gev@eee.upd.edu.ph bustos, dela cruz, acoymo & guevara when modifying a vowel, is actually causing the vowel to migrate in the direction of another recognizable vowel. vowel migration causes the vowel to lose its integrity for the sake of enhancing musical aesthetics. thus, intelligibility, the degree or level to which the intended vowel of the singer is perceived correctly by the listeners, is diminished. for example, when the vowel /a/ sung by a singer in its unmigrated or unmodified form is correctly perceived as /a/ by all the listeners, the intelligibility is 100%. however, when the same vowel is migrated to another vowel, fewer listeners might perceive the vowel correctly. for example, when 52 out of 100 listeners correctly perceive the intended vowel, resulting intelligibility is 52%. a more quantitative and objective measure of these vowel modifications can be achieved through software applications that offer real-time visual feedback on vowel intelligibility, together with the analysis of pitch and frequency spectra. the assessment is helpful in determining the extent with which the acoustical demands can be met with minimal compromise in intelligibility. the classification of sung vowels is usually performed by listeners. research in vowel classification generally applies to spoken vowels through the use of automatic speech recognition (asr) systems. these systems can be implemented using numerous feature extraction algorithms and classification models as shown by different studies presented in table 1. table 1. studies in vowel classification for spoken vowels researcher/s data (number of speech samples) features used classification model achieved accuracy merkx and miles 17213 mfccs single layer feedforward ann 91.50% dumitri and gavat 145 mfccs 3-layer mlp 96.4% for male speakers 77.9%% for female speakers schmid and barnard 58268 formant features and mfccs mlp 73.40% the study made by merkx and miles (2005) utilized thirteen mel-frequency cepstral coefficients (mfccs) as feature vectors. mfccs are coefficients derived from a logarithmic scaling of audio frequencies using mel filterbanks. for pattern classification, the study used a feed-forward artificial neural network (ann) with 28 internal nodes. ann is an adaptive, often nonlinear, system that is trained to perform an input/output mapping using a given input and a target data. the classifier reached a recognition accuracy of 91.5% on a subset of 5 vowel phonemes. another study made by dumitru and gavat (2007) used twelve mfccs, a 3layer multilayer perceptron (mlp) classifier and 145 speech samples as input data. mlp is a feedforward ann that uses three or more layers of nodes with nonlinear activation functions. vowel recognition rates reached 96.4% for male speakers and 77.9% for female speakers. schmid and barnard (1997) tested the efficiencies of cepstral-based features, mfccs and formant features including formant trajectory, amplitude, bandwidth, pitch and segment duration in vowel classification. formants are resonating frequencies that show up as peaks in the sound spectrum. results showed that formant features alone reached an accuracy rate of 71.8%, while mfccs alone reached an accuracy rate of 71.6%. the combination of the two features proved optimal, reaching an accuracy rate of 73.4%. this project is part of a research track at the up digital signal processing laboratory. the preceding study (dimaculangan & felias, 2008) applied strategies used in asrs to the classification of sung vowels and determined the relationship between vowel migration in sung filipino text and perceived intelligibility from the perspective of the audience. several parameters including formants, spectral envelopes, vowel triangles and mfccs were investigated to identify the parameter that is most appropriate in simulating vowel perception. results showed that nine mfccs, extracted from each of the 281 sung unmigrated vowel samples, best discriminated the sung vowels. these features were used as inputs to a linear discriminant analysis (lda) classifier that proved optimal compared to an ann. lda computes a linear predictor from two sets of normally distributed data to allow for classification of new observations. the accuracies achieved were 68.1% for the training set and 52.9% for the test set, based on the correspondence of the vowel classification determined by the 14 science diliman development of feature set, classification implementation and application for vowel migration classifier and results of the intelligibility tests that were conducted. in the intelligibility tests the ground truth for vowel classification was based on the perception of 45 listeners. wilson et al. (2008) studied the effects of real-time visual feedback on teaching pitch accuracy in singing. they investigated whether the style of feedback affects the amount of learning achieved and whether the provision of concurrent visual feedback hampers the simultaneous performance of the singing task. through the implementation of a baseline-intervention-post test between-groups design, it was determined that real-time visual feedback to the learner promotes the acquisition of the neuromuscular skills underlying the task of singing the correct pitch. moreover, different styles of visual feedback did not produce differences in the amount and rate of learning. in this paper, the objective of the researchers is to develop software applications that provide an objective assessment of the intelligibility of sung vowels, based on the perception of listeners, through real-time visual feedback. furthermore, the applications should help singers readily assimilate feedback and improve their singing ability. the novelty of this project is the application of asr system techniques on sung vowels in unmigrated and migrated forms with emphasis on intelligibility based on the perception of listeners. methodology this section contains a description of the data, a discussion of the various extracted features, the pattern classification methods, the application development and testing of these applications. data audio recording of the data was held inside a whisperroom®, a sound isolation enclosure. the recording equipment includes tascam dvra1000 high definition audio master recorder, sennheiser ew100 g2 wireless microphone and behringer ultravoice xm8500 wired microphone. all audio data samples are recorded in stereo wav format with the following attributes: pcm signed 24bit, 44.1 khz sampling rate and 2116 kbps bit rate. video recordings were also done using a sony handycam sr220 to document the singers’ mouth shapes. data in the form of sung vowels and song pieces were obtained from 46 singers. the audio recording consists of two parts, vocalization and singing of folk songs. vocalization was divided into six sets: unmigrated, migrated to [a], migrated to [e], migrated to [i], migrated to [o] and migrated to [u]. each set comprised vocalizes for each of the five filipino vowels in increasing pitch, spanning the singer’s stable range of frequency. the second part of the recording involved singing of three folk songs: “si pilemon”, “lubi-lubi” and “neneng at nonoy”. each of the songs was sung unmigrated, migrated to [a], migrated to [e], migrated to [i], migrated to [o] and migrated to [u]. a survey regarding application design was conducted among 38 of the singers who participated in the project. singers were asked about software features that would be helpful in improving their singing ability and how they visualize these features to appear in the applications. information gathered in this stage served as the basis for the design of the applications that were developed. an intelligibility/listening test was done to establish the ground truth for the intelligibility of the recorded vocalizes. 100 listeners composed of 51 non-music major students and 49 students from the up college of music participated in this test. a total of 1350 sliced vowels were included in the test, utilizing the vowel preceding the last vowel note for singers who chose to vocalize within an octave, while the second to the last vowel note was used for singers whose vocalises exceeded an octave. data obtained from the intelligibility test served as the ground truth for vowel classification based on human perception. feature extraction before extracting the features from the data, signal pre-processing was done to remove the dc offset of the signal and normalize the average audio volume levels to -18 db. vowel detection was implemented by computing short-time energy and short-time zero crossing rate per signal frame then comparing these parameters to set thresholds. several features have been extracted from the science diliman 15 bustos, dela cruz, acoymo & guevara vowels and were used as inputs to different pattern classifiers. below is a summary of the extracted features from the vowels. • mel-frequency cepstral coefficients (mfcc) is a representation of cepstral coefficients, taken from the fourier transform of the decibel spectrum, wherein the analysis is done on a non-linear frequency scale known as the mel scale. the mfcc extraction algorithm used was developed by slaney (1998). the computation process is as follows. the signal is divided into short time windows, where the discrete fourier transform (dft) of each time window for the discrete-time signal x(n) with length n is computed using x k =∑ n= 0 n −1 w n x n exp− j2  kn/ n  (1) for k = 0, 1, . . . ,n − 1, where k corresponds to the frequency f(k) = kfs/n, fs is the sampling frequency in hertz and w(n) is a hamming window, given by w n=0.54−0.46 cos n/ n . (2) the magnitude spectrum |x(k)| is scaled in both frequency and magnitude. the frequency is scaled logarithmically using the mel filter bank h(k,m) using x ' m=ln∑ k =0 n −1 ∣x k∣⋅h k , m (3) for m = 1, 2, . . . ,m, where m is the number of filter banks. the mel filter bank is a collection of triangular filters defined by the center frequencies fc(m), as shown in eq. 4. h  k , m x={ 0 for f k f c m−1 f  k − f cm−1 f cm− f c m−1 for f c m−1≤ f  k  f c m f  k − f cm−1 f cm− f c m1 for f c m≤ f  k f c m1 0 for f  k≥ f c m1 } (4) the center frequencies of the filter banks are computed by approximating the mel scale using eq. 5. =2595 log10 f100 1. (5) a fixed frequency resolution in the mel scale is computed, corresponding to a logarithmic scaling of the repetition frequency, using eq. 6, where φmax is the highest frequency of the filter bank on the mel scale, φmin is the lowest frequency in mel scale. =max−min/m 1 (6) the center frequencies on the mel scale are given by eq. 7 for m = 1, 2, . . . ,m. the center frequencies in hertz can be obtained using eq. 8. cm=m⋅ (7) f cm=70010 c m 2595 −1 (8) the mfccs are obtained by computing the discrete cosine transform (dct) of x′(m) from eq. 3 using eq. 9 for l = 1, 2, . . . ,m, where c(l) is the lth mfcc. cl =∑ m=1 m x ' mcosl m m−1 (9) • f1+f2+mfcc is a combination of the first and second formants, f1 and f2, of the vowels and the extracted mfccs. formant frequencies characterize the acoustic structure of each vowel, enabling listeners to perceptually identify the vowel. the vowel formants were appended to the mfccs in two ways: on a formants per frame basis (f1f+f2f+mfcc) as shown in figure 1 and on a formants per vowel basis (f1v+f2v+mfcc) as shown in figure 2. the extraction of formant frequencies involves the determination of resonance peaks from the filter coefficients obtained through linear prediction coding (lpc) analysis of the signal. (makhoul, 16 science diliman figure 1. formants computed per frame appended to mfccs development of feature set, classification implementation and application for vowel migration 1972) once the prediction polynomial a(z), shown in eq. 10, has been calculated, the formant parameters are determined by solving for the roots of the equation a(z) = 0. a z=1a1 x −1a2 z −2...a p z − p (10) • mfcc+δmfccs is a combination of the mfccs and its derivatives calculated using a simple linear slope. • line spectral frequencies (lsf) or line spectral pairs is a representation of lpc coefficients that represents glottal activity. to determine the lsfs, the linear prediction polynomial shown in eq. 10 is decomposed into p(z) and q(z) as shown in eq. 11 where p(z) corresponds to the vocal tract with the glottis closed and q(z) with the glottis open. the roots of both polynomials represent the line spectral pairs. p  z= azz− p1 a z−1 q z= az−z− p1 a z−1 (11) • mfcc-rasta is the addition of a rasta (relative spectral) filter block after mfcc extraction. the rasta filter was approximated by a simple fourth order butterworth bandpass filter. (slaney, 1998) pattern classification the extracted features from the audio signals were then used as feature vector inputs to pattern classifiers. below is a summary of the pattern classification methods that were implemented. • discriminant analysis (da) is used to find a number of projection directions that are efficient in separating the features into classes. the process involves maximizing the ratio of between-class variance to within-class variance so that adequate class discrimination is obtained. the different types of da that were implemented were linear (lda), quadratic (qda), mahalanobis (mda), diagonal linear and diagonal quadratic. • feedforward backpropagation artificial neural network is a type of ann that is trained using input vectors and corresponding target output vectors until it can approximate a function and associate input vectors with specific output vectors. this is done by reducing calculated errors between the input and output data and consequently adjusting the weights of the network’s forward-connected layers. • classification tree (ct) is a type of machine learning algorithm used for non-parametric data classification. a classification tree is a structural mapping of binary decisions that lead to a decision about the class (interpretation) of an object. • support vector machines (svm) are decisionbased prediction algorithms which can classify data into two groups. the training data is mapped to a higher dimensional space and separated by a plane defining the two classes of data. input data are classified based on the side of the plane they fall on. cross validation a comparison of the performance of the classifiers was done to determine the optimal sung filipino vowel discriminator. the classifiers were compared using the training set, consisting of sung vowels from 80% of the singers, and the test set, consisting of sung vowels from the remaining 20%. singers belonging to each set were randomly chosen. the listening test vowels were also tested to determine the perception accuracy. development of applications in this project, two software applications have been developed using matlab, a command-line software development program. one software application runs offline and processes audio files while the other software application runs in real-time/online. screen display interface was developed for both software applications. the following features were incorporated in the applications based on the recommendation of the 38 singers who were surveyed. science diliman 17 figure 2. formants computed per vowel appended to mfccs bustos, dela cruz, acoymo & guevara a. pitch detection two algorithms, average magnitude difference function autocorrelation function (amdf-acf) and the correlogram model of pitch perception (slaney, 1998), were tested to develop an accurate pitch estimator. the amdf-acf algorithm extracts the pitch period of signals from the short-term autocorrelation of computed amdf values as shown in eq. 12. rk = ∑ n=0 n −k−1 xn x nk (12) the correlogram model of pitch perception uses the largest peak from a summarized autocorrelation plot, a plot of sample autocorrelations versus time lags, as the pitch estimate. b. calculation of vibrato parameters vibrato was represented by three parameters: intonation, rate and extent. the intonation curve was obtained by passing the instantaneous pitch frequency curve or pitch contour of a signal through a moving average filter. vibrato rate was estimated as the reciprocal of the maximum period of the pitch contour. moreover, vibrato extent was estimated as the mean amplitude difference between the pitch and intonation contours. testing of developed software applications for preliminary testing, dean ramon acoymo of the college of music assessed the performance and usefulness of the application’s features. further testing was implemented using vocalises of singers. vocalises were recorded while singers tested the online application. the recorded vocalises were then run on the offline application to match the results of both applications. singers were also asked to fill out a questionnaire assessing the performance and usefulness of the application. results and analysis optimal unmigrated vowel classifier in order to develop an effective vowel classifier, initial tests on unmigrated vowels were conducted. the samples were assumed to be perceived correctly by the listeners, thus accuracy of the tests depended on the intended vowel classification. several feature extraction and pattern classification methods were tested on the vowels to determine the optimal vowel classifier elements. the features that were extracted included: mfccs, f1f+f2f+mfcc, f1v+f2v+mfcc and mfcc+δmfccs. moreover, the implemented pattern classification methods were discriminant analysis, classification tree and feedforward backpropagation neural network. this phase was done in parallel with the listening test. table 2 shows the top 5 classifiers ranked based on training set and test set accuracies computed as the average accuracy per vowel. table 2. top 5 classifiers based on training set and test set accuracies number of coefficients features extracted pattern classification method training set accuracy test set accuracy 15 f1f+f2f+mfcc qda 85.90% 89.40% 21 f1f+f2f+mfcc qda 87.80% 86.50% 15 f1v+f2v+mfcc qda 86.40% 89.10% 21 f1v+f2v+mfcc qda 87.80% 88.60% 21 f1v+f2v+mfcc ct 100.00% 84.90% development of data set based on listener perception the listening test that was conducted resulted in 1041 consistently perceived vowels out of the 1350 sung vowels that were presented to the listeners. a consistently perceived vowel has the same classification for 60% or more of the listeners in the listening test. to increase the data used in the development of the classifier based on listener perception, k-means clustering based on formants was implemented on consistently perceived vowels. k-means clustering is a partitioning method that finds a partition in which objects within each cluster are as close to each other as possible, and as far from objects in other clusters as possible. the resulting clusters of the vowels were labeled with vowel classifications similar to the consistently perceived vowels from the listening test and included in the data set. unless stated otherwise, this is the developed data set cited in this paper. 18 science diliman development of feature set, classification implementation and application for vowel migration the number of vowels that are included in the training set and test set using the data set is shown in figures 3 and 4, respectively. figure 4. vowel distribution of test set. test on project 1 listening test data to determine the optimal classifier based on listener perception, the top 5 unmigrated vowel classifiers from table 2 were tested on the listening test data gathered from project 1 (dimaculangan & felias, 2008). project 1 data were recorded in a large hall. the classifiers were trained using data from 80% of the singers included in the developed data set based on listener perception. the results are summarized in table 3. the trained classifier with the highest accuracy is the 21 f1v+f2v+mfcc qda. classifier optimizations the trained classifier with the highest accuracy, 21 f1v+f2v+mfcc qda, was used to test the remaining data from 20% of the singers in the developed data set. the resulting accuracies, computed based on the correspondence of the classifier outputs to the labeled vowel classifications based on listener perception, are shown for each vowel in table 4. table 4. accuracies for each vowel using the developed data set vowel training setaccuracy test set accuracy perception accuracy /a/ 80.00% 91.60% 87.20% /e/ 91.40% 90.40% 94.70% /i/ 93.10% 96.90% 94.00% /o/ 81.60% 63.40% 84.30% /u/ 86.90% 89.50% 86.60% to address the speed constraints of the software applications, minimization of the training set size was employed. feature vectors from the fifteen middle frames of each training set vowels were taken to form a new training set. the minimization has reduced classification time from 1245.5ms to 131.5ms. furthermore, accuracies have improved as shown in table 5. however, using the new training set, the perception accuracies for the vowel /o/ have decreased due to misclassification of the vowel /a/. table 5. accuracies for the minimized training set vowel training setaccuracy test set accuracy perception accuracy /a/ 82.30% 92.10% 88.90% /e/ 91.90% 92.50% 94.70% /i/ 95.50% 98.00% 96.50% /o/ 80.60% 68.30% 81.90% /u/ 89.50% 90.40% 91.10% a second stage classifier was developed to address the confusion between the vowels /a/ and /o/, with the goal of increasing the accuracies for both vowels. the features that were extracted included lsf, formants, mfcc and mfcc-rasta. moreover, the pattern classification methods that were implemented were discriminant analysis, support vector machine and classification tree. comparing the results of testing the combination of features and pattern classification methods showed that the f1v+f2v+mfcc and lda combination had the highest accuracy in classifying the vowels. the second-stage classifier takes the first-stage classifier /a/ and /o/ outputs whenever the classification accuracy is below 50% for /a/ and below 80% for science diliman 19 figure 3. vowel distribution of training set. bustos, dela cruz, acoymo & guevara /o/, following computed optimal thresholds. the resulting accuracies for vowels /a/ and /o/ after adding the second stage classifier are shown in table 6, the perception accuracy has improved for both vowels. table 6. accuracies for vowels /a/ and /o/ after adding the second stage classifier vowel training set test set perception accuracy /a/ 84.40% 92.50% 89.20% /o/ 79.70% 70.30% 83.10% final classifier the final vowel classifier based on listener perception consists of a first-stage 21 f1v+f2v+mfcc qda classifier with a secondstage classifier 21 f1v+f2v+mfcc lda classifier for the vowels /a/ and /o/. the training set size was minimized by using only the 15 middle frames of the training set vowels. confusion matrices of the final classifier for the training set, test set and listening test vowels used to compute the perception accuracy are shown in tables 7, 8 and 9, respectively. the diagonal elements of the confusion matrix represent the correctly classified vowels. off-diagonal elements denote the confusion of one vowel for another vowel – each row of the confusion matrix sums up to 100%. the first row in table 7 is interpreted as follows: the vowel /a/ in the training set is classified as /a/ 84.4% of the time and classified as /e/ 2.4% of the time, as /i/ 0.3% of the time, as /o/ 11.8% of the time and as /u/ 1% of the time. it can be observed that there is confusion among the back and central vowels /a/, /o/ and /u/ which are mainly caused by the overlapping formant values (f1 and f2) of the three vowels. moreover, among all the vowels, /i/ is the most accurately classified vowel. table 7. confusion matrices for the training set training set /a/ /e/ /i/ /o/ /u/ /a/ 84.40% 2.40% 0.30% 11.80% 1.00% /e/ 2.80% 91.90% 4.70% 0.10% 0.50% /i/ 0.20% 3.60% 95.50% 0.00% 0.70% /o/ 14.40% 0.00% 0.00% 79.70% 5.90% /u/ 1.70% 0.70% 2.80% 5.40% 89.50% table 8. confusion matrices for the test set test set /a/ /e/ /i/ /o/ /u/ /a/ 92.50% 1.30% 0.00% 5.40% 0.80% /e/ 0.40% 92.50% 7.00% 0.00% 0.00% /i/ 0.50% 1.50% 98.00% 0.00% 0.00% /o/ 19.80% 1.00% 0.00% 70.30% 8.90% /u/ 4.40% 1.80% 1.80% 1.80% 90.40% table 9. confusion matrices for the listening test vowels listening test vowels /a/ /e/ /i/ /o/ /u/ /a/ 89.20% 2.00% 0.70% 8.10% 0.00% /e/ 1.90% 94.70% 3.00% 0.00% 0.40% /i/ 0.00% 2.80% 96.50% 0.00% 0.70% /o/ 10.80% 0.00% 0.00% 83.10% 6.00% /u/ 0.00% 0.90% 4.50% 3.60% 91.10% the final classifier was used in the developed offline application. the online application, on the other hand, used a further minimized classifier that utilizes only the five middle frames of the training set vowels to address the greater need for classification speed. overall accuracies for the offline application vowel classifier are 89.4% for the training set and 90.9% for the test set. overall accuracies for the online application vowel classifier are 89.4% for the training set and 89.7% for the test set. the overall perception accuracy for both classifiers is 92.3%. it was observed that the test set had a higher accuracy than the training set. the same result was observed after changing the singers included in both sets. the improvement over project 1 baseline accuracy (dimaculangan & felias, 2008) for vowel classification is 21.3% for the training set and 38.0% for the test set using the offline classifier. the improvement for the online classifier is 21.3% for the training set and 36.8% for the test set. the objective of improving the accuracy of the classifier was achieved. 20 science diliman development of feature set, classification implementation and application for vowel migration pitch estimation algorithms two algorithms, amdf-acf and the correlogram model of pitch perception, were tested to develop an accurate pitch estimator. sinusoids with fundamental frequencies ranging from 65 to 932 hz (c2 to a#5) were used as inputs to the pitch estimation algorithms. for the amdf-acf algorithm, large deviations, averaging 23.86 hz, of the pitch estimates from the test fundamental frequencies were observed especially in high frequencies. on the other hand, the correlogram model resulted in more accurate pitch estimates with smaller deviations, averaging 4.95 hz, from the fundamental frequencies occurring only at very high frequencies. from this result, the correlogram model of pitch perception was set as the pitch estimation algorithm for the software applications. vibrato rate and extent estimation the performance of the vibrato rate and extent estimation was tested using synthesized vowels with duration of 2 seconds and formant frequencies of 300 and 870 hz. the synthesized vowels had pitch ranging from 65 to 835 hz. vibrato extent was set to be 3% of the pitch value of the vowel and the vibrato rates ranged from 4 hz to 7 hz. the average deviation of the estimated vibrato rates from the set vibrato rates is 0.42 hz while average deviation of estimated vibrato extents from the values computed as 3% of the pitch frequencies, is 4.77 hz. inconsistencies in the estimation, especially at high frequencies, are attributed to deviations in the pitch estimate and ripple effects from the pitch interpolation and moving average filter. offline application the screen display of the offline application with the corresponding feature labels is shown in figure 5. the offline application takes transcribed audio files as input and displays the following features: spectral envelope, pitch contour, intonation contour, and estimates for pitch, vibrato rate and vibrato extent, formant frequencies, spectrogram and vowel classification. the vowel classification uses the 21 f1v+f2v+mfcc qda-lda classifier with 15 frames per training set vowel. options for vowel detection using short-time energy, transcription file reading and vowel synthesis are also included in the application. online/real-time application the screen display of the online application with the corresponding feature labels is shown in figure 6. the application continuously takes 250ms of audio input from a microphone. vowels are detected using short-time energy and zcr. the following features are included in the application and are displayed: spectral envelope, pitch contour, pitch estimate, formants, vibrato rate, vibrato extent, spectrogram and vowel classification. the vowel classification uses the 21 f1v+f2v+mfcc qda-lda classifier utilizing 5 frames per training set vowel. options for audio logging, audio exporting and accompaniment playback are also included in the application. the average processing time for each detected vowel was computed to be 193ms. testing of software applications testing with dean ramon acoymo of the up college of music was conducted to gauge the performance and usefulness of the features included in the applications. according to dean acoymo, the vowel classifications made by the classifier were consistent with human perception. the human ear usually has trouble discriminating among the vowels /a/, /o/ and /u/ due to the intersection of the formant values of these vowels. the display of the vowel classification and spectral envelope was advantageous in the assessment of the trade-off between vocal color and quality. moreover, the features are applicable in singing pedagogy wherein preserving both vocal color and quality is important. the display of the pitch and vibrato parameters, on the other hand, is especially useful for the singers who perform different styles of singing. science diliman 21 bustos, dela cruz, acoymo & guevara three other singers tested the software using vocalises. the correspondence between the intended vowels of the singers and the vowel classifications made by the online application was observed before and after the singer had a chance to use the software. the results showed an average increase of 7.0% in the correspondence between the intended vowels of the singers and the vowel classifications made by the online application. moreover, the singers gave positive feedback in the usefulness, ease of use, layout and performance of the online application. conclusion the developed vowel classifier based on listener perception utilizes the formant frequencies, f1 and f2, and mfccs as features. vowel classification is made by a first-stage qda classifier and a secondstage lda classifier for the vowels /a/ and /o/. the classifier was optimized for speeds applicable to the offline and online applications through the reduction of the training set size while preserving the integrity of the data. resulting overall accuracies for the classifier used in the offline application are 89.4% and 90.9% for the training set and test set, respectively. on the other hand, overall accuracies for the classifier used in the online application are 89.4% and 89.7% for the training set and test set, respectively. using the listening test vowels as inputs to the classifiers, the overall perception accuracy for both offline and online classifiers is 92.3%. aside from vowel classification assessing the intelligibility of sung vowels, additional features have been incorporated in the developed software applications. the added features are spectral envelope and spectrogram displays, pitch estimation and computation of vibrato parameters; these parameters were chosen based on the suggestions of the singers who were recorded. vowel classification and spectral envelope displays help singers assess 22 science diliman figure 5. graphical user interface of the offline application. development of feature set, classification implementation and application for vowel migration their vocal color and quality which are important elements in singing and given significant consideration in vowel pedagogy. moreover, the display of pitch, vibrato parameters and spectrogram will help singers improve their vocal tonalities and assess their adherence to the musical style that they are performing. other features such as voice recording and accompaniment playback were included in the applications to further enhance usage. in conclusion, the researchers have been able to develop a novel approach for assessing the intelligibility of sung vowels which performs with an accuracy exceeding 89% and effectively emulates the human auditory vowel perception. the implementation of the algorithm was based on the vowel classification made by 100 listeners. moreover, software applications were developed based on this algorithm. initial tests of these software applications show them to have potential use in vocal pedagogy and have been enhanced with features that prove to be beneficial to the intended users. recommendations the inclusion of a lip-shape detector and online video feedback of the user’s lips should be part of the next version of the software. it would be also interesting to study the pedagogical impact of the software on both students at the college of music and pop singers. it is expected that an increase in the database of recorded sung vowels and song pieces, as well as listeners in the listening test, will lead to a higher accuracy in the vowel classifier. acknowledgement this research project is funded by the office of the vice-chancellor for research and development open grant of the university of the philippines diliman and the office of the vice-president for science diliman 23 figure 6. graphical user interface of the online application. bustos, dela cruz, acoymo & guevara academic affairs emerging fields grant, and is part of interdisciplinary signal processing for pinoys (isip) program. references d.r. appelman. 1986. the science of vocal pedagogy (theory and application), indiana university press. j. dimaculangan, and r. felias. 2008.“vowel migration in sung filipino text and perceived intelligibility,” undergraduate student project, department of electrical and electronics engineering, university of the philippines, diliman. c. dumitru, and i. gavat. 2007. “vowel, digit and continuous speech recognition based on statistical, neural and hybrid modelling by using asrs_rl”, eurocon, the international conference on "computer as a tool, warsaw, poland, 856-863. p. merkx, and j. miles. 2005. automatic vowel classification in speech, duke project paper in math 196s, duke university, durham, nc, usa. p. schmid, and e. barnard. 1997. “explicit, n-best formant features for vowel classsification”, proc. intl. conf. on acoustics, speech, and signal processing, munich, germany, 991-994. m. slaney. 1998. auditory toolbox for matlab technical report, interval research technical report. p. wilson, k. lee, j. callaghan, and c. w. thorpe. 2008. “learning to sing in tune: does real-time visual feedback help?”. journal of interdisciplinary music studies 2(12):157-172. j. makhoul. 1972. “linear prediction: a tutorial review,” in proceedings of the ieee, pp. 1973–1986. 24 science diliman population biology-regalado.pmd population biology of tripneustes gratilla 41science diliman (july-december 2010) 22:2, 41-49 population biology of tripneustes gratilla (linnaeus) (echinodermata) in seagrass beds of southern guimaras, philippines joshua m. regalado*, wilfredo l. campos and august s. santillan oceanbio lab, division of biological sciences, college of arts and sciences university of the philippines, miagao, iloilotelefax: +63-33-3159271 telephone: +63-33-5007599 *corresponding author: joshud_regalado214@yahoo.com abstract the sea urchin tripneustes gratilla is a major grazer found in seagrass beds of southern guimaras, philippines. monthly length-frequency data from january 2008 to june 2009 were used to estimate the growth, recruitment pattern and mortality rate with the use of fisat ii. the estimated values for the von bertalanffy growth parameters l¥ (asymptotic length) and k (growth coefficient) were 114.2 mm and 1.08 respectively. monthly densities ranged from 0.06 to 0.58 per m² with a mean value of 0.26 per m². monthly biomass ranged from 4.1 to 49.5 grams per m² with a mean value of 21.15 grams per m². t. gratilla density and biomass were observed to be highest during the month of november 2008. the recruitment pattern showed a major broad peak and a minor peak separated by four months. total mortality (z) from the length-converted catch curve was computed to be 4.74 per year. keywords: sea urchin, growth, mortality, recruitment, tripneustes gratilla, guimaras introduction sea urchins are among the major grazers of seagrass coastal habitats. the presence of sea urchins in has been known to influence the community structure of marine plants and algae populations in different habitats. in some areas, overpopulation and overgrazing of sea urchins on macrophyte communities have been a major problem while in others, sea urchin stocks are collapsing due to overexploitation and improper management. population explosions of sea urchins in some areas have caused rich kelp beds into barren sandy areas creating a significant impact in the ecosystem and species composition of the area. because of its sedentary and lifestyle and shallow water habitat, sea urchins are vulnerable to overexploitation. sea urchin roe is considered a delicacy among coastal communities. in the philippines, tripneustes gratilla is the most targeted because its roe which commands a high price and is preferred over roes of diadema setosum and diadema savignyi. urchin roe are also used as feed for fattening lobsters. urchins can be easily harvested by hand picking them. divers gather urchins in subtidal areas with the use of rakes, spears or dredges. high demand for urchin roe have led to overexploitation in sea urchin stocks and led to under supplied markets (kessing and hall 1998 as cited by kelly 2002). cage culture of sea urchins and restocking activities the wild have been done in order to help maintain sea urchin stocks. tripneustes gratilla is the most abundant sea urchin found in the seagrass beds of southern guimaras. in the taklong island national marine reserve and neighboring areas, it occurs together with salmacis belli (belida et al 2003). this study examined the growth, recruitment, and mortality t. gratilla in southern guimaras. regalado, j.m., et al. 42 materials and methods monthly surveys were conducted from january 2008 to june 2009 in seven stations in southern guimaras. three of these sites namely nabinbinan (n 10.438 e 122.504) , san roque (n 10.427 e 122.505) , and kalaparan ( n 10.404e 122.507) are located in the taklong island national marine reserve and the other four stations santo nino(n 10.454 e 122.502), sikangkang beach (n 10.459 e 122.501), pulo ni ramon (n 10.465 e 122.496), tando (n 10.466 e 122.486) are located north of the reserve (fig. 1). in each station, two 50 x 2m belt transects were laid to delimit the area where the necessary data were recorded. the test diameter (from the tip of the interambulacral area to its opposite interambulacral area) of all tripneustes gratilla individuals occurring within the belt transect were measured using plastic vernier calipers. measurements were rounded off to the nearest mm. after measuring the test diameter, the sea urchins were placed outside the area covered by the transect to avoid being measured twice. other species of sea urchins, fishes and other invertebrates which were found within the delimited area were also noted. salinity and temperature were also recorded. length-frequency distributions using 5mm size classes were constructed for each month and were used to estimate growth, mortality and recruitment employing the elefan routine in the fisat ii software ( fao, 2000). size and weight measurements were conducted periodically to derive a length-weight relationship following the model w = alb and provide monthly sea urchin biomass. figure1. map showing the study area and seven sampling stations in southern guimaras. science diliman (july-december 2010) 22:2, 41-49 population biology of tripneustes gratilla 43 results density a total of 5631 sea urchin individuals were recorded from january 2008 to june 2009. a peak in, t. gratlla density was observed in the month of november 2008 and another peak in late january 2009. density was observed to be lowest in june 8, 2009. density ranged form 0.06 -0.58 ind./m2, with an average density of 0.26 ind./m2 (fig. 2). biomass length and weight of individuals used for the lengthweight analysis ranged from 44mm-82mm and 40g183g respectively. the length-weight relationship is shown in fig. 3. data from this model were used to compute biomass estimates for each month. biomass ranged from 4.8-49.5g / m2, with an average value of 21.15 g / m2. overall mean biomass across all stations was highest in november 2008 and showed a smaller peak in january 2009 (fig. 2). seasonal patterns in density and biomass were very similar except for the months of june 2008 and february 2009, where there were relatively large differences in their values. growth the best fitting growth curve showed the values 114.2 mm for l¥ and 1.08 for the growth constant k. the growth curve superimposed on the monthly length frequency distributions of t. gratila is shown in figure 4. the four (4) curves follow the same growth model but have different starting points, which in this study corresponded to sampling dates where the smallest test diameters were recorded. recruitment figure 5 shows the estimated recruitment pattern of t. gratilla based from the length-frequency data. it can be observed that recruitment occurs almost all throughout the year with a major and minor peak about four months apart. figure 2. monthly average density and biomass of t. gratilla in southern guimaras from january 2008 to june 2009. science diliman (july-december 2010) 22:2, 41-49 regalado, j.m., et al. 44 figure 3. size-frequency data of t. gratilla in southern guimaras with superimpoised von bertalanffy growth curve as estimated by fisat ii. figure 4. length-weight relationship data of t. gratilla in southern guimaras. science diliman (july-december 2010) 22:2, 41-49 population biology of tripneustes gratilla 45 figure 5. recruitment pattern of t. gratilla in southern guimaras showing the two recruitment pulses. figure 6. length converted catch curve for t. gratilla in southern guimaras. science diliman (july-december 2010) 22:2, 41-49 regalado, j.m., et al. 46 mortality total annual mortality (z = 4.74) from the lengthconverted catch curve is shown in figure 6. discussion the estimated values for the growth constant k and asymptotic length l for t. gratilla in southern guimaras were 1.08 yr-1 and 114.2mm respectively. t. gratilla in southern guimaras can reach a size of 60 mm in about eight months. a study by also conducted in southern guimaras beldia et. al (2001) showed tripneustes gratilla reach sizes of around 40-60mm in 10 months. southern guimaras grow as much as 40 mm in the first four months and tapers off as it grows reaching a size of 60mm within ten months. dafni (1992) estimated a high growth rate for tripneustes gratilla elatensis which grows to 60mm in about three months. different values for l¥ and k obtained by bacolod and dy (1986) and juinio-menez et al. (2008) are shown in table 1. differences in estimates of growth parameters show that conditions of a particular site affect the growth of rate and maximum sizes of tripneustes gratilla. seasonality and type of available food might be significant factors causing differences in growth rates and maximum size attained by sea urchins. sea urchins under shortage of food resorb skeletal material and shrink while an abundance of food is maximized by high allocation to gonadal and somatic growth (see note by andrew 1989). a number of studies have shown that type of food influences the growth rate of different species of sea urchin. species of strongylocentrotus (swan 1961), psammechinus miliaris (kelly 2002), tripneustes gratilla (juinio-menez 2008), and tripneustes gratilla elatensis (dafni 1998) showed variations in growth rate in relation to the type of food they ingest. the seagrass beds in southern guimaras are mainly dominated by thalassia and enhalus sp.. during the summer months from march to may, other macroalgae species such as sargassum sp. also become abundant. the estimated growth parameters of tripneustes gratilla in southern guimaras may reflect the seasonality in seagrass and algae in the area. tripneustes gratilla abundance was generally high during the southwest monsoon transitions and decreased towards the summer seasons. highest recorded densities during the study were recorded in october and november 2008. a gradual decrease followed until may 2009 (fig. 2). a combination of different factors such as physical exposure, recruitment variability, substratum type and disease could determine echinoid population abundance (andrew 1989). man-made structures like passive fishing gears and human activities like aquaculture and gleaning were also pointed out by beldia et al. (2003) to affect sea urchin densities. juvenile migration and movement from one place to another by adults might also affect the number of observable sea urchins in the area. a high annual mortality rate of 99.3% (z=4.74) was estimated using the length-converted catch curve. bacolod and ty (1986) estimated values of 91% table 1. comparison of estimated growth parameters for tripneustes gratilla from different locations in the philippines from various studies. science diliman (july-december 2010) 22:2, 41-49 location k l reference danahon bank, bohol 1.8 108 bocolod and dy, 1986 pisalayan, bolinao 1.7 8.2 menez et al., 2008 quezon island, bolinao 1.3 113 menez et al., 2008 southern guimaras 1.08 114.2 …   population biology of tripneustes gratilla 47 mortality for t. gratilla in danajon reef, central philippines. juinio-menez (2008) also report high natural mortality rates of 91-96% from different sites in northwestern luzon. even in the absence of a local fishery for sea urchins, the stock in southern guimaras still has a high annual mortality rate. this may be attributed to such factors like habitat conditions, predation and disease. heavy rains and bad weather have been observed to kill sea urchins (vaitilingon 2005). different species of starfish, wrass, wolf fish and decapods have been reported to be among predators of sea urchins (pearse 1987, bernstein 1981). on rare occasions during field surveys, the starfish protoreaster nodosus was observed to prey on sea urchins. starfish have also been reported to feed on juvenile sea urchins, which eventually would affect the visible population of the area (pearse 1987). further investigations on predator pressure may give more insight on how predation affects changes in sea urchin population. sea urchin survivorship in southern guimaras may be dependent on the changes in its habitat which is in this case the seagrass beds. storms and heavy waves can destroy or damage the seagrass which are the main diet of sea urchins. without a thick seagrass cover sea urchins particularly the juveniles may also be more exposed to predators. recruitment patterns based on length frequency data show recruitment occurring almost all throughout the year with two peaks separated by four to five months. beldia (2003) determined two major recruitment pulses in southern guimaras occur annually: a small peak in june and a major peak in november. the number of recruits in an area greatly depends on the availability of competent larvae in the water column that could settle in the appropriate substratum on the one hand, and the mortality of newly settled juveniles (cameron and schroeter 1980, tegner and dayton 1977) on the other. successful larval settlement in an area may also be influenced by biotic factors such as natural chemical settlement cues from different algae (swanson et al. 2004, swanson et al. 2006, williamson 2000) marine biofilms (naidenko 1996) and presence of conspecific adults (dworjanyn, and pirozzi 2007, see also reviews by pawlik 1992 and rodriguez 1993). the reproductive activity of t. gratilla has an extended breeding season lasting for several months, a resting period and another period with less intense spawning activity (formacion 1985 and tuason 1975). this pattern of spawning behavior agrees well with the derived recruitment pattern, having one major peak and a minor peak occurring about four months later. comparison of density and biomass estimates shows younger and lighter individuals to occur in june 2008 and february 2009. this may indicate high number of recruitment for this month (beldia et. al 2003). the occurrence of spawning activity of t. gratilla from march to july and another in december (formacion 1985) may be used as basis for the high number of recruits. t. gratilla densities in southern guimaras right after the mv solar 1 oilspill were found to be about half of what were previously occurred (beldia et al., 2003 and campos & regalado 2009). densities obtained by beldia et. al (2003) from 2000-2002 can be characterized by heavy recruitment, followed by a sharp decrease in population. numbers. such pattern of rare (once in ten years) episodic increase in urchin populations (strongylocentrotus purpuratus) has been observed by pearse (1987) in temperate waters. there are no such reports for urchins in the tropics. examining other factors such as seasonal changes in habitat and other species that may directly affect sea urchin population must also be conducted. only by this and by continuously monitoring long term changes in sea urchin population can we characterize the pattern of urchin fluctuations in southern guimaras. short term data may not be sufficient to give substantial information to the extent of variability and fluctuation of sea urchin density. long term monitoring of urchin populations may give a clearer picture on the fluctuations of densities and confirm if indeed the recorded high by beldia et al. (2003) and the low densities in months after the mv solar 1 oilspill is part of the natural fluctuation in urchin densities in southern guimaras or could be attributed to effects of the oilspill. in taklong island national marine reserve, the large difference in densities from the time of beldia et al ’s science diliman (july-december 2010) 22:2, 41-49 regalado, j.m., et al. 48 (2003) study to the present suggests that growth and mortality would also likely differ. because of their dominance in seagrass beds in the study area, it is important that variability in population dynamics of t. gratila between years and over longer periods are examined to better understand their role in seagrass bed trophodynamics. acknowledgements this study was funded by the oil spill response program of the university of the philippines visayas. the authors are thankful for the assistance rendered by the following: mr. joseph gajo of the up marine biological station in taklong island, mr. nelson gajo and the researchers of the oceanbio and marine bio laboratories. this paper was presented at the 10th pams national symposium held in davao city in october 2009. references andrew, n. l. 1989. contrasting ecological implications of food limitation in sea urchins and herbivorous gastropods. mar. ecol. prog. ser. 51: 189-193. andrew, n.l & j. h. choat. 1985. habitat related differences in the survivorship and growth of juvenile sea urchins. mar. ecol. prog. ser. 27: 155-161. bacolod, p. t. & d. t. dy. 1986. growth, recruitment pattern and mortality rate of the sea urchin tripneustes gratilla linnaeus, in a seaweed farm in danahon reef central philippines. the philipp. scient. 23: 1-14. beldia ii, p.d., n. p. evano, w.l. campos, a.s. santillan, j.b. bitoon-on & p.b. jalandoni. 2003. population parameter estimates of two seagrass associated sea urchins echinodermata in southern guimaras, philippines: preliminary findings. philipp. scient. 40:122-142. bernstein b. b. & k. h. mann. 1981. changes in the nearshore ecosystem of the atlantic coast of nova scotia, 1968-81. nafo sci. coun. studies. 5: 101-105. campos, w.l. & j.m. regalado. 2009. monitoring seagrassassociated echinoderm populations in the vicinity of taklong island national marine reserve: year 2. annual report. university of the philippines visayas, miagao, iloilo, 5p. cameron, r. a. & s.c. schroeter. 1980. sea urchin recruitment: effect of substrate selection on juvenile distribution. mar. ecol. prog. ser. 2: 243-247. dafni, j.1992. growth rate of the sea urchin tripneustes gratilla elatensis. isr. j. zool. 38: 25-33. dworjanyn, s.a. & i. pirozzi. 2007. induction of settlement in the sea urchin tripneustes gratilla by macroalgae, biofilms and conspecifics: a role for bacteria?. aqua-628002 (in press). fao. fao-iclarm fish stock assessment tool ii (fisat ii) fao, rome (2000). formacion, m. 1985. reproductive biology of the sea urchin trineustes gratilla linnaeus at taklong island and vicinity, nueva valencia, guimaras sub-province. final report. university of the philippines in the visayas, iloilo city, 11p. juinio-meñez m.a., h. g. p. bangi, & m.c. d. malay. 2008. effect of type of feed, stocking density and grow-out site on gonad index, growth and survivorship of cultured sea urchin (tripneustes gratilla). philipp. agric. scientist. 91(4): 439-449. juinio-meñez, m.a. h. g. bangi, m. c. malay and d. pastor. 2008. enhancing the recovery of depleted tripneustes gratilla stocks through grow-out culture and restocking. res. fish. sci. 6(1–3):35–43. kelly, m. s. 2002. survivorship and growth rates of hatcheryreared sea urchins. aquacult. int. 10: 309-316. naidenko, t. k. 1996. induction of metamorphosis of two species of sea urchin from sea of japan. mar. biol. 126: 685692. pawlik, j. r. 1992. chemical ecology of the settlement of benthic marine invertebrates. oceanogr. mar. biol. ann. rev. 30: 273-335. pearse, j. s. & a. h. hines. 1987. long-term population dynamics of sea urchin in a central california kelp forest: science diliman (july-december 2010) 22:2, 41-49 population biology of tripneustes gratilla 49 rare recruitment and rapid decline. mar. ecol. prog. ser. 39: 275-283. rodriguez, s. r., f. p. ojeda & n.c. inestrosa. 1993. settlement of benthic marine invertebrates. mar. ecol. prog. ser. 97: 193-207. swan, e.f. 1961. some observations on the growth rate of sea urchins in the genus strongylocentrotus. biol. bull. 120:420-427. swanson r. l., j. e. williamson, r. de nys, n. kumar, m. p. bucknall & p. steinberg .2004. induction of settlement of larvae of the sea urchin holopneustes purpurascens by histamine from a host alga. biol. bull. 206: 161–172. swanson r. l., j. e. williamson, r. de nys, n. kumar, m. j. hugget, j. k. green & p. steinberg. 2006. in situ quantification of a natural settlement cue and recruitment of the australian sea urchin holopneustes purpurascens. mar. ecol. prog. ser. 314: 1–14. tegner m. j. and p. k. dayton. 1977. sea urchin recruitment patterns and implications on commercial fishing. science. 196: 324-326. tuazon, a. y. & e. d. gomez. 1979. the reproductive biology of tripneustes gratilla linnaeus (echinoidea: echinodermata) with some notes on diadema setosum leske. proc. int. symp. mar. biogeogr. evol. s. hem. 2: 707-716. vaitilingon, d., r. rasolofonirina & m. jangoux. 2005. reproductive cycle of edible echinoderms from the southwestern indian ocean i. tripneustes gratilla l. (echinoidea, echinodermata). western indian ocean j. mar. sci. 4 (1): 47-60. williamson j.e., r. de nys, n. kumar, d.g. carson & p.d. steinberg. 2000. biol bull 198(3):332-45. science diliman (july-december 2010) 22:2, 41-49 nitrogen-art.7 nitrogen and phosphorus in coastal systems 51 introduction nutrient studies conducted in coastal systems often determine only the inorganic fraction of nitrogen (nh3, no3, no2) and phosphorus (po4). little attention has been given to the organic fraction, which, from previous works constitutes a large portion of the total dissolved n and p in the water column (jacinto 1992). science diliman (july december 2000) 12:2, 51-58 abstract nitrogen and phosphorus in coastal systems: focus on dissolved organic n and p daisy o. padayao* and maria lourdes san diego-mcglone marine science institute, college of science university of the philippines, diliman, quezon city 1101 philippines tel. no.: (632) 922-3944; fax: (632) 924-7678; e-mail: daisyp@upmsi.ph quantification of dissolved organic phosphorus (dop) and dissolved organic nitrogen (don) levels, and the relative importance of the organic fraction at various habitats (river, seagrass bed, mangrove area, coral reef, fishpen, and ocean) were the focus of this study. don concentrations ranged from 9.5 to 44.3 µm during the dry season and from 10.9 to 23.7 µm during the wet season. dop values ranged from 0.3 to 0.4 µm during the dry season and from 1.0 to 1.6 µm during the wet season. don was 70-90% of the dissolved fraction in the first five habitats for both dry and wet seasons. dop was approximately 15-35% during the dry season and 50-60% during the wet season. don was highest in the river and lowest in the coral reef area for both seasons. high dop concentrations were determined in the river and mangrove area in the dry season while lowest values were seen in the coral reef area. during the wet season, dop was highest in the coral reef area and lowest in the mangrove area. when compared with oceanic systems, dissolved inorganic nitrogen (din) and dissolved inorganic phosphorus (dip) have higher percentages (25-58% for din and 71-83% for dip) in the open ocean than in coastal areas (10-32% for din and 62-67% for dip). however, don and dop were the dominant forms in the coastal sites (42-75% don vs 7-32% din and 17-30% dop vs 35-67% dip). the smaller fraction of the organic forms of n and p in the open ocean may be indicative of the greater efficiency in nutrient recycling/regeneration in the open ocean than in the coastal area. n:p ratios in the five habitats ranged from 2 to14 with the highest ratio in the coral reef area. keywords: dissolved organic nitrogen (don), dissolved organic phosphorus (dop), dissolved inorganic nitrogen (din), dissolved inorganic phosphorus (dip), coastal habitats * corresponding author a comprehensive understanding of the nutrient cycle requires knowledge of the various forms and transformations undergone by nitrogen and phosphorus. the roles of the organic fractions of n and p in the nitrogen and phosphorus cycles have been well illustrated (parsons and others 1984b). the major constituents of total dissolved nitrogen (tdn) are the dissolved inorganic nitrogen (din) and the dissolved organic nitrogen (don). don can come from external inputs via runoffs or through the fixing of din. inorganic nitrogen sources include precipitation and the fixing of n2 gas from the atmosphere. this inorganic n padayao & san diego-mcglone 52 is transformed to particulate organic nitrogen when taken in by organisms, and through bacterial action during decomposition, it is converted into don. don can then be used directly by organisms as food source in the form of urea and amino acids, or it can be converted into the inorganic form via bacterial action (parsons and others 1984b). total dissolved phosphorus (tdp) is partitioned between dissolved inorganic phosphorus (dip) and dissolved organic phosphorus (dop) (orrett and karl 1987). it is apparent from the phosphorus cycle that the various forms of phosphorus are often readily exchangeable as metabolites. soluble inorganic phosphorus is taken up by organisms that release dissolved organic phosphorus as an excretion product. similar to don, dop can be directly assimilated by organisms or it can be converted back into its inorganic form through bacterial action (parsons and others 1984b). don and dop are poorly characterized compounds but are present at elevated concentrations (jackson and williams 1985). hence, it is important to quantify their potential contributions to the nutrient pool and better understand their roles in the n and p cycles. this study was undertaken to quantify levels of don and dop from various habitats (i.e., seagrass bed, fishpen, coral reef, mangrove, river, ocean) in order to assess their importance in different systems, and relate these fractions to the inorganic forms of n and p. materials and methods water samples were collected during the dry (may 36, 1999) and wet (august 25-26, 1999) seasons from 5 different habitats in bolinao, pangasinan: (1) river, (2) seagrass bed, (3) fishpen, (4) coral reef, and (5) mangrove area. three sites were sampled from each of the habitat (fig. 1). samples were taken at 1 m depth using a 5 l niskin sampler. samples for din, don, dip and dop determinations were filtered through whatman gfc filters, stored in high density polyethylene (hdpe) bottles and kept frozen until analysis in the laboratory. inorganic n and p were determined using methods described by parsons, et al. (1984a). tdn and tdp were analyzed after persulfate oxidation using the methods of koroleff (1983). don and dop were operationally defined as the difference between tdn and din, and tdp and dip, respectively. results and discussion nitrogen constituents fig. 2 shows the horizontal distribution of nitrogen constituents during the dry and wet seasons. the highest din concentrations which were measured in the river in both seasons could be due to external inputs, such as waste discharges. the high concentrations of din in the mangrove area could be due to the inputs of a small river system in this area. high concentrations of don g1 g2 g3 r1 r2 r3 f1 f2 f3 m3 c1 c2 c3 16.48 16.46 16.44 16.42 16.36 ` ` ` ` ` 8 • r ` 119.88 119.92119.94 119.96 119.98 120.00 119.02 fig. 1. map of stations in bolinao, pangasinan during the dry and wet seasons; sampling. (r1, r2, r3 = river stations; s1, s2, s3 = seagrass stations; f1, f2, f3 = fishpen stations; c1, c2, c3 = coral stations; m1, m2, m3 = mangrove stations) nitrogen and phosphorus in coastal systems 53 fig. 2. horizontal distribution of the nitrogen constituents during the dry and wet seasons were, likewise, observed in the river area during both seasons, albeit concentration gradients for don were less defined than din. levels of din and don in all habitats in both seasons are shown in fig. 3. din and don were consistently higher in the river area. din concentrations were lowest in the seagrass beds, while don was lowest in the coral reef area. din and don concentrations were higher during the wet season. table 1 summarizes the average concentrations of the n constituents from the five habitats. the ranges of din concentration were 3.05 to 4.44 µm and 1.23 to 14.00 10.00 8.00 6.00 2.00 0.00 12.00 4.00 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 5.80 5.20 4.60 4.00 3.40 2.80 2.20 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 55.00 45.00 35.00 25.00 15.00 5.00 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 din dry season don dry season din wet season 55.00 45.00 35.00 15.00 5.00 25.00 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 don wet season padayao & san diego-mcglone 54 80.00 60.00 40.00 20.00 0.00 r s f c m 20.00 15.00 10.00 5.00 0.00 dry season 80.00 60.00 40.00 20.00 0.00 r s f c m 20.00 15.00 10.00 5.00 0.00 wet season d o n d i n fig. 3. concentration of din and don in the five habitats (r-river; s-seagrass; f-fishpen; c-coral reef; m-mangrove) during the dry and wet season d i n d o n d i n d o n 9.71 µm during the dry and wet seasons, respectively. don values ranged from 9.52 to 44.29 µm during dry season and 10.86 to 23.73 µm during wet season. don concentrations were approximately 3-10 times higher than din during the dry season and 2 -15 times higher during the wet season. mean don concentrations in the coral reef area which were 9.52 µm and 10.86 µm during dry and wet seasons, respectively, were within the range of don concentrations for tropical coral reef areas, at 3.0-13.8 µm as reported by crossland (1983). don constituted about 70-90% of the total dissolved nitrogen in the five habitats in both seasons (table 2). as reported by jacinto (1992), about 90% of total dissolved nitrogen in the reef waters of bolinao, pangasinan is in the form of don. the percentage of don determined in the river sites (>70%) in both seasons agrees well with the results of stepanauskas and leonardson (1999) for organic n transported by river systems. table 1. mean concentrations of the n constituents in the five habitats during the dry (may) and wet (august) seasons. (values expressed in µm) *din is no3 + no2 + nh3 dry wet sampling sites river seagrass bed fishpen coral reef mangrove t d n 48.73 21.10 20.54 13.17 18.00 d i n* 4.44 3.18 3.05 3.65 3.74 d o n 44.29 17.92 17.48 9.52 14.26 t dn 31.33 19.60 22.84 12.73 33.44 d i n* 9.08 1.23 3.35 1.86 9.71 d o n 22.25 18.37 19.49 10.86 23.73 table 2. percentage composition of din and don in the five habitats during the dry and wet seasons dry wet sampling sites river seagrass bed fishpen coral reef mangrove % d i n 9.55 16.25 14.96 32.31 22.71 d o n 90.45 83.75 85.04 67.69 77.29 d i n 25.84 6.89 13.22 15.47 29.08 d o n 74.16 93.11 86.78 84.53 70.92 nitrogen and phosphorus in coastal systems 55 phosphorus constituents the horizontal distribution of the p constituents are shown in fig. 4. dip was highest in the river areas in both seasons and its concentration varied within the river system. this was not the observation in the other c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 2.10 0.00 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 1.80 1.50 1.20 0.90 0.60 0.30 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 2.10 0.00 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 1.80 1.50 1.20 0.90 0.60 0.30 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 1.05 0.00 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 0.90 0.75 0.60 0.45 0.30 0.15 c1 c2 c3 m1 m2 m3 f1 f2 f3 r1 r2 r3 g1 g2 g3 2.30 0.20 119.88 119.90 119.92 119.94 119.96 119.98 120.00 120.02 16.48 16.46 16.44 16.42 16.40 16.38 16.36 16.34 16.32 16.30 2.00 1.70 1.40 1.10 0.80 0.50 fig. 4. horizontal distribution of the phosphorous constituents during the dry and wet seasons dip dry season dop dry season dip wet season dop wet season habitats. higher dop was observed during the wet season. fig. 5 shows the dip and dop concentrations in the five habitats. highest dip was measured in the river for both seasons; lowest dip was measured in padayao & san diego-mcglone 56 3.00 2.25 1.50 0.75 0.00 r s f c m 4.00 3.00 2.00 1.00 0.00 dry season 3.00 2.25 1.50 0.75 0.00 r s f c m 4.00 3.00 2.00 1.00 0.00 wet season d o p d i p d i p d o p fig. 5. dip and dop concentrations in the 5 habitats sampled: r = river, s = seagrass, f = fishpen, c = coral reef, m = mangrove, during the dry and wet seasons d i p d o p the seagrass bed during the dry season, and in the coral reef area during the wet season. high dop values prevailed in the river and mangrove areas, while low values characterized the coral reef area during the dry season. during the wet season, dop was highest in the coral reef area and lowest in the mangrove area.during the wet season, higher dop concentrations were observed in all habitats. concentrations of the p constituents are shown in table 3. except for the river system which had the highest dip concentrations, dip and dop levels did not vary much within the 5 habitats during both seasons. dip ranged from 0.42 to 2.71 µm during dry season and 0.51 to 1.03 µm during wet season; dop ranged from 0.25 to 0.42 µm during dry season and 1.01 to 1.56 µm during wet season. average dop concentrations in the coral reef area (0.25 and 1.56 µm) obtained in this study were higher than that reported by crossland (1983) for tropical coral reef areas (<0.15 µm). dop constituted approximately 13-40% of the p constituents during the dry season and about 50-70% during the wet season. dip constituted the greater portion of total dissolved p during the dry season (at 60-87%), but not during the wet season (at 30-50%, table 4). n/p ratio table 5 shows the calculated n/p ratio in each habitat. these were compared with the known redfield ratio (n/p =16) which explains that major plant nutrients (i.e., no3, po4) change concentrations in seawater in a fixed ratio, that is, the same as the n and p stoichiometry of planktonic organisms (redfield 1934, 1958). the inorganic n/p ratios in all the habitats sampled were lower than the redfield ratio. the n/p value of 14, determined in the coral reef area, was highest among table 3. mean concentrations of the p constituents in the five habitats during the dry (may) and wet (august) seasons. (values expressed in µm) dry wet sampling sites river seagrass bed fishpen coral reef mangrove t d p 3.13 0.59 0.97 0.63 0.84 d i p (po4) 2.71 0.42 0.60 0.43 0.64 d o p 0.42 0.27 0.37 0.25 0.42 t dp 2.22 1.72 1.67 2.07 1.56 d i p (po4) 1.03 0.59 0.60 0.51 0.72 d o p 1.19 1.36 1.31 1.56 1.01 nitrogen and phosphorus in coastal systems 57 by hydrolysis at the alkaline ph of seawater or by phosphatases, which are hydrolytic enzymes present in many bacteria and on the surface of some phytoplankton, particularly those from environments low in inorganic phosphate. in contrast, the conversion of organic n to its inorganic form is more difficult since its fixation, reduction and oxidation back to nitrate requires, the exchange of energy (parsons and others 1984b). coastal area vs. open ocean table 6 compares the n and p constituents in the coastal area (bolinao, pangasinan) and in the open ocean (sulu sea). the percentages of don (42-75%) and dop (17-30%) in the open ocean were relatively lower compared to the coastal area. however, the difference was not significant because samples were collected in the upper 100 meters where production of organic matter occurred and regeneration or decomposition processes were not predominant. nonetheless, the lower percentage of the organic forms of n and p in the open ocean was indicative of the efficiency of the system for nutrient recycling. from general knowledge of geochemical cycling, there is greater efficiency for nutrient recycling in the open ocean due to absence of proximity to possible sources as compared to the coastal area which receives inputs from various sources (rivers, land, etc.). the inorganic fractions of n and p were the dominant forms in the open ocean, while the organic forms of n and p were the dominant forms in the coastal area. references crossland cj. 1983. dissolved nutrients in coral reef waters. in: barnes dj, editor. perspectives in coral reefs. australia: brian clouston publisher. p 56-68. jacinto gs. 1992. is dissolved organic nitrogen (don) more important than dissolved inorganic nitrogen (din) in tropical coastal waters [abstract]. in: chou lm, wilkinson cr, editors. third asean science and technology week conference proceedings, vol. 6, marine science: living coastal resources; 1992 sept 21-23; singapore. 219 p. table 5. n/p ratios in the five habitats during the dry and wet seasons table 4. percentage composition of dip and dop in the five habitats during dry and wet seasons sampling sites river seagrass bed fishpen coral reef mangrove d i p 86.76 62.46 62.13 67.42 65.59 d o p 13.24 37.54 37.87 32.58 34.51 d ip 44.93 31.33 34.46 33.48 50.59 d o p 55.07 68.67 65.54 66.52 49.41 dry wet sampling sites river seagrass bed fishpen coral reef mangrove din/dip 2.18 8.31 5.67 8.01 6.13 don/dop 347.82 127.58 43.61 31.20 48.27 din/dip 8.07 2.21 5.32 13.64 6.49 don/dop 23.83 16.53 15.59 22.65 49.45 table 6. percentage compositions of n and p constituents in the open ocean and coastal areas. * river data not included constituents open ocean (sulu sea) don din dop dip coastal area (bolinao) wet dry 42.49 74.89 25.11 57.71 16.66 29.39 70.71 83.34 70.92 93.11 6.89 29.08 49.13 68.67 31.33 50.59 67.69 90.45 9.55 32.3 34.41 37.87* 31.33 50.59 dry wet all habitats sampled. this value is greater than that reported for water flowing through the eniwetok atoll by webb et al. (1975). the n/p ratios calculated from the organic fractions were relatively high because of the high don, which was 2-15 times more than the dop. the disparity in the don and dop values could also be due to the greater ease of converting the organic form of p to its inorganic form than the conversion of n. organic p is readily hydrolysed to inorganic p, either padayao & san diego-mcglone 58 jackson ga, williams pm. 1985. importance of dissolved organic nitrogen and phosphorus to biological nutrient cycling. deep sea res 32(2): 223-235. koroleff f. 1983a. determination of total phosphorus by acid persulphate oxidation. in: grashoff k, ehrhardt m, kremling k, editors. methods of seawater analysis. 2nd ed. germany: verlag chemie. p 134-139. koroleff f. 1983b. total and organic nitrogen. in: grashoff k, ehrhardt m, kremling k, editors. methods of seawater analysis. 2nd ed. germany: verlag chemie. p 162-173. orrett k, karl dm. 1987. dissolved organic phosphorus production in surface seawaters. limnol oceanogr 32(2): 383-395. parsons tr, maita y, lalli cm. 1984a. a manual of chemical and biological methods for seawater analysis. oxford: pergamon press. p 3-25. parsons tr, takahashi m, hargrave b. 1984b. biological oceanographic processes. 3rd ed. oxford: pergamon press. p 164-166. redfield ac. 1934. on the proportions of organic derivations in sea water and their relation to the composition of plankton. in: james johnstone memorial volume. liverpool: liverpool university press. p 177-92. redfield ac. 1958. the biological control of chemical factors in the environment. amer sci 46: 205-221. stepanauskas r, leonardson l. 1999. bioavailability of wetland-derived don to freshwater and marine bacterioplankton. limnol oceanogr 44(6): 1477-1485. webb kl, dupaul wd, wiebe w, sottile w, johannes re. 1975. enewetak (eniwetok) atoll: aspects of the nitrogen cycle on a coral reef. limnol oceanogr 20: 198-210. ocr document a 01_metillo 1 abstract science diliman (july-december 2002) 14:2, 1-17 temporal patterns in the abundance and diet of acetes erythraeus nobili 1905 (crustacea, decapoda, sergestidae) in the nearshore waters of iligan bay, northern mindanao, philippines ephrime b. metillo department of biological sciences mindanao state university iligan institute of technology andres bonifacio ave., iligan city, philippines 9200 tel/fax: (063) 221-4056; (063) 221-4068 e-mail: ovcre-ebm@sulat.msuiit.edu.ph two adjacent nearshore estuaries in iligan bay (8on, 124oe), northern mindanao were sampled to investigate annual temporal patterns in the abundance and stomach contents of acetes erythraeus, and selected hydrometeorological factors. principal components analysis (pca) reveals temporal variations in a. erythraeus abundance and diet composition that appear to conform not only to the typical northeast and southwest monsoons, but also to three-month seasons (march-april-may [dry hot], june-july-august [wet hot], september-october-november [wet cold], december-january-february [dry cold]). rainfall and tide largely explain variations of abundance and stomach contents. peaks in a. erythraeus abundance are similar to those found in other tropical acetes species whose bimodal temporal distribution patterns coincide with the start of the southwest monsoon and the end of the northeast monsoon. a. erythraeus is a zooplanktivorous omnivore with copepods, ostracods, other crustaceans, and molluscan veligers as the predominant food. a total of twelve (12) diet categories were recorded, including, for the first time, dinoflagellates and tintinnids. a comparison of stomach contents of shrimps, caught before and after midnight, showed higher ingestion after midnight. diet composition of juvenile and adult shrimps, generally, is independent of sex and sampling location. although there is overlap in the diet between adults and juveniles, the latter ingest more small-sized food categories. key words: acetes erythraeus, abundance, diet analysis, temporal patterns introduction hutchinson (1953) defined pattern in ecological communities as the structure that results from the distributions of organisms in, or from, their interactions with their physical and biological environments. understanding the processes that generate these patterns is very important in ecology, and is the key to the development of principles for management (levin, 1992). the quest for pattern in tropical community ecology is as disputed as in temperate and sub-tropical ecological realms (krebs, 1985; deshmukh, 1986; pulman, 1994). there is disagreement on whether or not species components of a community do reveal any pattern at all. nevertheless, communities do show properties, such as species richness and diversity, food web and food chains, and stability and resilience, and that these change with time and space (steele, 1974). for instance, the maintenance of the structure and function in marine pelagic communities is often viewed 2 metillo within the context of biological (predation and competition) and/or physical control (mcgowan & walker, 1985; longhurst, 1985). the role of physical or biological factors on the spatiotemporal dynamics of tropical plankton remains less understood. although the effect of monsoons has been identified as a strong predictor of these dynamics (edra, 1975; longhurst & pauly, 1987; schalk, 1987), much still have to be studied particularly on the role of a micronekton that directly feeds upon phytoplankton and zooplankton. for example, the micronektonic sergestid shrimps of the genus acetes are quite common in many coastal areas of the tropics and sub-tropics, and they are known to be selective omnivores upon phytoplankton and zooplankton (xiao & greenwood, 1993; mcleay & alexander, 1998). acetes is caught from 0.5 cm to 150 m depth range, depending on geographic location. adult size ranges from 1.5 to 2.5 cm long. almost all species are naturally gregarious and exhibit spatio-temporal migration patterns. acetes is an extremely euryhaline micronekton which could tolerate freshwater to full strength seawater. it is eurythermal, based on its warm-temperate to tropical distribution. periodicity in feeding is evident in their intensive feeding at night. in the sub-tropical neritic waters of the south china sea, acetes shows positive selection for certain species of diatoms during summer. however, data from other biotopes and from laboratory experiments proved strong preference for animal-based food, mainly zooplankton. acetes is somehow the antarctic krill in their habitats because of its importance to many predators, such as squids, 151 species of fish (including whale shark), prawns, young crocodiles, and the many peoples of asia (xiao & greenwood, 1993; omori 1974, 1975, 1977). during certain times of the year, they become extremely abundant. the average annual catch of acetes from indo-asia during the period 1979 to 1989 exceeded 228,850 tons, 25% of the world’s annual total shrimp catch. in the philippines, an acetes fishery production of 17,260 tons was registered in 1989. artisanal fishers catch acetes spp. from iligan bay for bait and shrimp paste industry. acetes’ potential in the aquaculture industry (as fish food) remains to be explored. xiao & greenwood (1993) emphasize a huge information gap in the tropical waters where most acetes species are found. except for very little data on its fishery, no published data on the ecology of acetes in the philippine waters is available. the abundance and feeding patterns of the tropical micronektonic sergestid shrimp acetes erythraeus were investigated in an attempt to relate these patterns with temporal changes of selected physical and chemical factors in iligan bay. the abundance of a. erythraeus in two sampling sites for one year, and the relative importance of the different prey items in the diet of a. erythraeus from the two study sites between february 1999 to january 2000 were estimated. hydrometeorological factors (salinity, temperature, total suspended solids, rainfall, and tide) in the nearshore waters of iligan bay were studied in order to explain temporal changes in the abundance and the stomach contents of a. erythraeus. materials and methods sampling of a. erythraeus and seawater monthly sampling of a. erythraeus and various physical and chemical parameters was made monthly from october 1998 to january 2000 in two coastal sites (fig.1) of iligan bay. one of the sampling sites is located off tambacan, iligan city (8o 12’ 50” n; 124o 11’ 7” e), adjacent to the iligan city port. close to a reef flat, the second sampling site is off larapan, kauswagan (8 o 11’ 58” n; 124 o 6’ 20”e), and is located at about 20 km from the other site. both sampling sites are estuarine, with characteristic muddy-sand substratum and freshwater (riverine) input. fifteen-minute sub-surface collection of a. erythraeus was done once a month from 2000h to 0400h at depths of 20 to 45 m using a general oceanics (go) conical plankton net with 275 µm mesh and 0.3 m mouth diameter. sampling was done at nighttime, following results that zooplankton and micronektonic shrimps normally peak at night (mauchline, 1980; longhurst & pauly, 1987; flock & hopkins, 1992; takahashi & kawaguchi, 1998). from february 1999 to january 2000, a different conical net with a bigger mesh (1000 µm) and 1 m mouth diameter was used to prevent a. erythraeus net feeding. preliminary diet analysis of shrimps caught using the 3 abundance and diet of acetes erythraeus go net from october 1998 to january 1999 showed strong net feeding with a. erythraeus stomachs stuffed with calanoid copepod prey. the diet data reported in this study came from shrimps caught from february 1999 to january 2000. the use of the go net was continued, though, up to september 1999 mainly for the abundance estimate of a. erythraeus. horizontal tows using both nets were made parallel to the shore at a speed of 0.5 m/s. both nets were mounted in such a manner that no briddles obstructed the mouth in order to maximize capture efficiency. immediately after hauling of catch, a. erythraeus were fixed in 5% formaldehyde in millipore (pore size = 1 µm)-filtered seawater. a total of four samples were collected; two for shrimp abundance and another two for shrimp diet analysis. soon after shrimp sampling, replicate sub-surface water samples were collected for the determination of water temperature, salinity, and total suspended solids. subsurface water temperature was measured using a mercury thermometer, salinity using a titration-calibrated refractometer, and total suspended solids by gravimetric methods. total monthly rainfall data was obtained from a national power corporation (npc) weather station closest to the two sampling sites. tidal height values were obtained from published tidal predictions. fig. 1. map of iligan bay showing sampling sites a (tambacan) and b (larapan). inset is the map of the republic of the philippines with iligan bay enclosed in a square. 122o 123o 124o 125o 126o 117o 127o19o 12o 5o 9o 8o 7o iligan bay iliganab sorting of shrimps was done after samples were processed in a laboratory equipped with an exhaust fan in order to remove formaldehyde. after analysis, samples were stored in glass jars still using 5% formaldehyde as preservative. analysis of diet composition prior to the dissection of a. erythraeus, total body lengths of 35 sexed individuals were measured using a vernier caliper with an accuracy of 0.01 mm. sexing was based on the descriptions of xiao & greenwood (1993). the stomach of the shrimp was removed under a stemi 2000 zeiss stereomicroscope using a microdissecting needle (dumont, switzerland), slitopened dorsally, and spread apart. an opened gut was placed on a glass slide with a drop of glycerol and covered with a glass cover slip. gut contents were examined under the zeiss stereomicroscope and an olympus compound microscope at low and high magnifications, counting and identifying meticulously all ingested prey to the lowest taxonomic level possible. scoring of gut contents was on per individual shrimp basis. since micronektonic shrimps, like most crustaceans, macerate captured food prior to ingestion, the individual prey item of a. erythraeus was enumerated by weighted points method (williams, 1981; takahashi & kawaguchi, 1998), following the equation: percentage weighted points for the ith prey is equal to: where: aij = number of points of prey item in the foregut of the jth shrimp wj = weighting with the value dependent on the class (1,2,3,4,5) of stomach fullness of the jth shrimp n = number of shrimps examined for diet analysis s = number of prey categories n s a = σ . σ wjaij j=1 j=1 n σ wjaij x 100 j=1__________ a 4 chi square test was used to test the independence of gut contents from sex and sampling location. analysis of diet overlap between juveniles and adults the diet overlap between juveniles and adults within site was compared using the formula of schoener (1970): where ro is the overlap index expressed as percentage, and pxi and pyi are the relative importance (ratio of the points) of each food item i in the stomachs of predator species x and y. principal components analysis of stomach contents data the principal component analysis (pca) (ludwig & reynolds, 1988; grossman et al., 1991) was used to determine if sampling months could be grouped on the basis of the composition of a. erythraeus diet. pca was also used to explore possible groupings of the different categories of stomach contents of a. erythraeus. multiple partial regression was employed on the eigenvalues of the first principal component of each analysis and the different monthly hydrometeorological data to find out the degree to which these physico-chemical factors contribute to the variations in a. erythraeus stomach contents. results temporal variation in abundance a. erythraeus was present all throughout the year in both sites, but in some months numbers per haul were too few (fig. 2). very similar temporal patterns of abundance are seen in both sites that show major peaks occurring in october, february, and june. these peaks ro = 100 (1 σ | pxi – pyi | / 2) fig. 2. mean abundance (number of individuals per haul) of acetes erythraeus in two coastal areas of iligan bay. error bars = standard error. 5000 4000 3000 2000 1000 0 oct 1998 nov 1998 dec 1998 jan 1999 feb 1999 mar 1999 apr 1999 may 1999 jun 1999 jul 1999 aug 1999 sep 1999 tambacan site 5000 4000 3000 2000 1000 0 oct 1998 nov 1998 dec 1998 jan 1999 feb 1999 mar 1999 apr 1999 may 1999 jun 1999 jul 1999 aug 1999 sep 1999 6000 larapan site metillo 5 are attributed to the dominance of juvenile individuals in the sample. larapan showed relatively higher abundance during peak months than those in the tambacan site. temporal variability in the diet composition of adult a. erythraeus a total of 12 food categories were identified from the foregut contents of a. erythraeus (table 1). tambacan site except for the months of july, september, and january, intact and fragmented copepods form the bulk of the foregut contents of adult shrimps throughout the sampling period (table 2). it contributed 15% (september) to 55% (may). bivalve veliger fragments ranged from 0% to 36% and showed three peaks that occurred in march, may, and november. gastropod veligers also have three peaks (february, october, and december), with values ranging from 0% to 20%. ostracods peaked in august and january. other materials were highest in february and lowest in april. crustacean remains showed a range of 0% to 17%, with a peak occurring in january. chaetognath spines showed a range of 0% to 2.4% with the peak observed in november. pteropods and tintinnids peaked in september. although diatoms were ingested in september, relatively more dinoflagellates were ingested in july and october. echinoderm larvae were only observed in august. larapan site as in the other site, copepod fragments dominated the diet (table 3), with two major peaks occurring in june and january, and a contribution with a range of 17% to table 1. summary of food categories found in the foreguts of acetes erythraeus in iligan bay, northern mindanao, philippines. (codes used in the diet analysis and in pca graphs are in parenthesis). bacillariophycea (df) protoctista tintinnida (ti) dinoflagellida (di) crustacea copepoda (cpf) ostracoda (of) crustacean remains (cr) mollusca gastropoda (gf) lamellibranchia (bf) pteropoda (pf) chaetognatha (cs) echinodermata (el) others (ot) fragments of diatoms, primarily coscinodiscus spp. entire cells and thecae of favella spp. and tintinnopsis spp. entire cells and thecae of peridinium spp. and dinophysis spp. entire individuals, but predominantly fragments of calanoid copepods including antennae, peraepods, mouth parts, and prosome; occasional entire individuals and fragments of harpacticoid and cyclopoid copepods carapace, appendages, and other fragments of ostracods spines and appendages of other crustaceans, e.g., decapods intact and crushed individuals and fragments of gastropods veligers including spires, operculum, and soft mantle tissue intact and crushed individuals and fragments of bivalve veligers including the umbo, shells, and mantle tissues fragments of pteropod shells fragments of oral spines and body tissues entire bipinnaria and auricularia larvae amorphous materials and fine particles; foraminiferans (globigerina spp.), sponge spicules, and macrophyte fragments that are mixed with silt or fine particles abundance and diet of acetes erythraeus 6 80%. bivalve veliger fragments peaked in june and october, with nothing in august and january. gastropod veligers peaked in may and november. ostracods have one peak in august. other materials and/or fine particles range from 0% to 22%. although crustacean remains peaked in march and while chaetognaths have one peak in october, both prey types were not ingested in other months. tintinnids peaked in september, while pteropod fragments, in january, but both prey types were also not ingested in other months. as in the other site, dinoflagellates contributed higher to the diet than diatoms. temporal change in the diet composition of juvenile a. erythraeus tambacan site brownish green amorphous materials (fine particles) were the main item in the gut of juveniles. two peaks occurred in june and september (table 4). copepod fragments that peaked in june were also common in juvenile foreguts. its contribution ranges from 6% to 33%. bivalve veligers peaked in october, with nothing in september. gastropod veligers were also highest in table 2. proportion of stomachs containing food and the stomach contents of adult a. erythraeus from the tambacan site. the ratio under each sampling date represents the number of stomachs examined against the number of stomachs with food. 08 february 1999 (35:24) 26 march 1999 (35:33) 27 april 1999 (35:30) 30 may 1999 (35:33) 29 june 1999 (35:31) 30 july 1999 (35:34) 28 august 1999 (35:35) 29 september 1999 (35:33) 29 october 1999 (35:35) 29 november 1999 (35:25) 29 december 1999 (35:27) 10 january 2000 (35:35) date stomach contents (% contribution) cpf 34.1 29.9 54.9 43.0 38.6 21.0 35.0 15.0 40.0 33.2 26.0 31.2 12.0 25.0 18.0 36.1 34.0 27.0 21.0 14.0 19.4 20.8 14.7 0.0 21.0 16.0 20.0 13.6 0.8 7.8 4.4 14.0 20.4 16.3 23.0 0.0 1.6 0.2 1.0 1.3 1.2 29.0 32.0 31.0 12.0 21.0 19.4 32.0 0.0 4.7 3.5 0.0 3.1 1.9 0.0 0.0 1.1 0.0 0.0 17.0 0.0 0.0 0.9 0.0 0.2 0.4 0.5 2.4 0.0 6.0 4.9 3.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.5 0.0 0.0 0.0 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.6 0.0 1.6 0.2 0.2 0.2 0.0 dibf gf of cr cs pf tidf 0.0 0.0 0.0 0.0 0.0 0.0 1.4 0.0 0.0 0.0 0.0 0.0 30.0 23.0 1.7 5.8 20.7 8.8 5.8 16.0 5.1 2.2 11.3 15.0 otel metillo 7 october, and nothing for the months of may and september. the highest ostracod contribution to the diet was in july, with the only other record in august. crustacean fragments peaked in september and lowest in october. tintinnids peaked in august, but were only present from july to september. pteropod fragments were only seen in july. dinoflagellates were found from june to august and peaked in october while diatoms were only found in august. ingested phytoplankton is higher in juvenile shrimps than in adults caught in the same site. larapan site as in the other site, fine particles were the major ingested food item (table 5). it peaked in september, and became lowest in october. copepod fragments had its highest contribution in june and lowest was in july. peak values for bivalve veliger fragments were in may and october. gastropod veliger fragments were only found in may and july, ostracods in july, while crustacean fragments peaked in october. tintinnids peaked in may while pteropods were only observed in june. dinoflagellates were surprisingly very high in may, table 3. proportion of stomachs containing food and the stomach contents of adult a. erythraeus from the larapan site. the ratio under each sampling date represents the number of stomachs examined against the number of stomachs with food. 08 february 1999 (35:33) 26 march 1999 (35:35) 27 april 1999 (35:35) 30 may 1999 (35:35) 29 june 1999 (35:29) 30 july 1999 (35:35) 28 august 1999 (35:27) 29 september 1999 (35:26) 29 october 1999 (35:32) 29 november 1999 (35:34) 29 december 1999 (35:25) 10 january 2000 (35:34) date stomach contents (% contribution) cpf 24.6 57.1 39.0 36.4 69.8 17.0 19.0 43.0 18.0 18.0 23.0 79.9 12.0 1.9 22.0 24.3 11.3 9.9 0.0 16.0 29.0 27.0 24.0 0.0 23.0 9.5 18.0 23..2 0.0 10.0 2.4 12.0 3.9 28.0 26.0 0.5 5.3 3.0 8.0 3.2 0.9 37.0 79.0 6.8 29.4 20.2 26.4 5.0 0.0 8.2 3.0 5.5 0.0 2.1 0.0 0.0 6.7 1.0 0.0 0.1 0.0 0.1 0.8 0.0 0.0 0.9 0.0 0.0 8.7 0.0 0.5 0.0 0.0 0.0 0.1 0.0 1.4 0.0 0.0 0.0 0.0 0.0 0.0 1.9 0.0 1.1 3.0 0.3 0.0 0.1 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 1.5 0.4 0.0 0.9 0.0 14.0 0.0 0.2 0.0 0.0 dibf gf of cr cs pf tidf 0.0 0.0 0.0 0.0 0.0 4.2 0.0 0.0 0.0 0.0 0.0 0.0 22.0 19.0 11.0 4.2 17.9 16.0 0.0 8..2 4.0 1.1 0.0 14.0 otel abundance and diet of acetes erythraeus 8 table 5. proportion of stomachs containing food and the stomach contents of juvenile a. erythraeus from the larapan site. the ratio under each sampling date represents the number of stomachs examined against the number of stomachs with food. table 4. proportion of stomachs containing food and the stomach contents of juvenile a. erythraeus from the tambacan site. the ratio under each sampling date represents the number of stomachs examined against the number of stomachs with food. and present again in october. diatoms peaked in july, but became rare in may and september. dinoflagellates and diatoms are more abundant in the guts of juveniles than those in adults from the same site. august samples from this site were lost. differences in diet composition between location and sex adult shrimps taken in march, june, august, september, october, and january had stomach contents that are metillo 30 may 1999 (35:35) 29 june 1999 (35:29) 30 july 1999 (35:31) 29 september 1999 (35:32) 29 october 1999 (35:27) date stomach contents (% contribution) cpf 16.0 46.2 6.1 7.2 18.0 19.4 0.0 5.4 2.3 17.0 0.8 0.0 2.5 0.0 0.0 0.0 0.0 6.7 0.0 0.0 1.2 0.0 0.0 5.4 33.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 21.7 0.0 0.1 0.0 4.7 0.2 0.0 9.1 1.1 0.0 2.6 0.7 2.3 0.0 2.0 dibf gf of cr cs pf t idf 0.0 0.0 0.0 0.0 0.0 34.9 52.9 62.0 83.0 21.0 otel 30 may 1999 (35:35) 29 june 1999 (35:35) 30 july 1999 (35:29) 28 august 1999 (35:34) 29 september 1999 (35:24) 29 october 1999 (35:30) date stomach contents (% contribution) cpf 31.7 32.6 18.0 25.0 6.3 29.0 13.0 3.7 13.0 5.8 0.0 18.0 0.0 0.6 6.0 1.3 0.0 7.1 0.0 0.0 8.4 1.0 0.0 0.0 0.0 0.0 7.6 2.1 15.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.2 0.4 1.2 0.0 7.9 0.0 0.0 0.0 2.4 0.0 0.0 0.0 0.3 1.3 16.0 11.0 0.0 dibf gf of cr cs pf t idf 0.0 0.0 0.0 0.0 0.0 0.0 50.0 57.0 40.0 45.7 69.0 35.0 otel 9 not independent of sampling site (chi-square test, p < 0.05 for all). this is also true among juvenile shrimps collected in may, july, and september (chi-square test, p < 0.05 for all). adult shrimps from the tambacan site in august had stomach contents that are not independent of sex (chi-square test, p < 0.001). this is also shown in the larapan shrimps caught in november and december (chi-square test, p < 0.025). stomach contents are independent of sex among juvenile shrimps taken from the two sites. comparison of feeding intensity before and after midnight except for other materials (amorphous) and crustacean remains, all other diet categories observed in the stomachs of shrimps from the tambacan site showed higher feeding intensity after midnight (fig. 3). shrimps caught in the larapan site showed high feeding intensity after midnight, except those shown in copepod fragments and other (amorphous) dietary items (fig. 3). diet niche overlap between adults and juveniles the diet overlap between juvenile and adult individuals was computed from may to october 1999. in the tambacan site <50% feeding niche overlap was shown in august (39%) and september (24%), while in the other site this was seen in may (44%), july (40%), september (17%), and october (48%). the rest of the months for both sites showed overlap values >50%. ordination analysis of the diet composition of a. erythraeus from the tambacan site in general, the pci of the biplots (figs. 4-5) for gut contents reveals the grouping of dietary items on the basis of similar peak months and stomach contents abundance trends, i.e., those with positive loadings reflect dietary items that have higher % contribution. for adult shrimps in the tambacan site (fig. 4a.1), copepod fragments, bivalve veliger fragments, and dinoflagellates form group i with common peaks in may, june, and october. group ii members (gastropod veliger and crustacean fragments, echinoderm larvae, and others) peaked in december, january, and february. ostracod fragments and chaetognath spines form group iii with peaks in august and november. group iv is composed of diatoms, pteropod fragments, and tintinnids that peak in september. the monthly biplot (fig. 4b.1) shows three groups, namely: may-june-october; february-march-april; november-december, januaryaugust, and july-september. as in the diet of adults, pca of juvenile shrimps diet reveals groupings based on peaks of dietary items. dietary items were grouped into four (fig. 4a.2). having a common peak in october, group i is composed of gastropod and bivalve veliger fragments, dinoflagellates, and copepod fragments. group ii comprises ostracod and pteropod fragments with common peaks in july. group iii is composed of diatom fragments, tintinnids, and others. only crustacean remains with peak in september belong to group iv. monthly pca generated four groups, namely, october, july, may-june-august, and september (fig. 4b.2). abundance and diet of acetes erythraeus fig. 3. percentage contribution of stomach contents of adult a. erythraeus caught before and after midnight in the tambacan and larapan sites. 60 50 40 30 20 10 0 cf bf gf of ti cs pf di df el cr ot tambacan site 60 50 40 30 20 10 0 cf bf gf of ti cs pf di df el cr ot larapan site 8 pm 2 pm 9 pm 3 pm 10 ordination analysis of the diet composition a. erythraeus from the larapan site group i comprises chaetognath spines, gastropod and bivalve veliger fragments, crustacean and diatom fragments, and dinoflagellates (fig. 5a.1). these items share peaks in march-april and september-october. group ii is composed of ostracod fragments, tintinnids, and echinoderm larvae that show common peaks in julyaugust and september. copepod and pteropod fragments, and other materials comprise group iii that show peaks in january-february. three groupings (julyaugust; march-april-may and october-novemberdecember; and january-february, june and september) are formed in the monthly biplots with each group comprising months reflective of those for the dietary items biplot (fig. 5b.1). for juvenile shrimps, three groups are depicted in fig. 5a.2. group i has a peak in july, and comprises gastropod veliger, diatom, and ostracod fragments. group ii (copepod and pteropod fragments and others) peaked in september, while group iii constitute the remaining dietary items (crustacean, dinoflagellate and bivalve veliger fragments, and tintinnids) that have common peaks in may and october. the monthly biplot shows the same monthly groupings as those in the dietary items (fig. 5b.2). metillo fig. 4. bivariate plots of principal components i (x-axis) and ii (y-axis) for the variations in abundance of adult and juvenile a. eryhthraeus diet categories (a) and sampling months (b) in the tambacan site. pf of cf di bf gf df mu ti cr a.2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2-1.2 cr gf cf di mu ti df pf of bf feb may jun oct mar apr sep jul dec nov aug jan 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -1.2 -1.4 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 b.1 sep oct jan aug nov dec jul apr mar feb may jun df ti pf of cs cr el ot gf di cpf bf a.1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 pf ti cs df gf of bf cr ot el cpf di jun sep aug may oct jul1.4 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -1.2 -0.8 -0.4 0 0.4 0.8-1.6 -0.8 1.2 b.2 jul octaug sep mayjun 11 variations in hydrometeorological parameters salinity from the tambacan site ranged from 11% to 34‰ while that of the other site was 17% to 30‰ (fig. 6a). lowest records of salinity in the tambacan site was in september and october 1999, but salinities below 25‰ were recorded in november and december 1998, and from march to november 1999. below 25‰ in the larapan site was observed in the months of february, august, and september 1999. sub-surface water temperature in the two sites also varied with the tambacan site, showing a range of 26oc to 29oc, while that of the other site ranged from 27oc to 29oc (fig. 6b). the lowest water temperature at the tambacan site was recorded in the months of february and july 1999, while in the other site, it was in december 1999. average total suspended solids values ranged from 0.013 to 0.280 g in tambacan site, while those in the abundance and diet of acetes erythraeus jun sep aug jul oct may1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 1.2 -1 -0.6 -0.2 0.2 0.6 1 1.4 1.8 2.2 b.2 may oct jul jun sept aug el of df gf pf otcf ti cr di bf 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 a.2 bf di ti cr cf ot pf gf el of df di df cr bf gfti of mu pf cs el of 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 a.1 di gf cr of el pf ti cs bf df mu of oct novdec may apr mar sep febjun jan jul aug b.1 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -1.2 -1.2 -1 -0.8 -0.4 -0.2 0 0.2 0.4 0.6 0.8-0.6 jun jul mar aug dec nov oct may sept jan feb apr fig. 5. bivariate plots of principal components i (x-axis) and ii (y-axis) for the variations in abundance of adult and juvenile a. erythraeus diet categories (a) and sampling months (b) in the larapan site. 12 other site was 0.01 0.21 g (fig. 6c). total suspended solids values in both sites remained below 0.05 g from october 1998 to february 1999, but showed a drastic increase in march 1999 and remained >0.05 g. tidal heights from the two sites showed a very similar pattern (fig. 6d). high tides were observed in the months of september to december, average heights in february and from april to july, and low tides in january, march, and august. the highs and lows depict the dominant 24-h semi-diurnal pattern in iligan bay, with low tides observed from 0500h to 0700h and high tide at the approach of midnight to early dawn. the lowest low tides which occurred during the january and march 1999 sampling would explain the negative values. normally, a unimodal annual pattern occurs with low rainfall in the months of march and april and heavy rainfall normally occurring from june to february. old records show that from 1995 to 1997, low monthly rainfall would commence earlier on the last two weeks of february. an el niño that occurred in 1997 resulted in very low rainfall starting in january 1998 and placed the low rainfall months (march to april 1998) to unusually very low rainfall levels. the 1998 el niño was subsequently followed by a la niña which started june 1998 that extended up to the usually low rainfall months of march and april 1999, resulting in an unusually high rainfall values in these months (fig. 6e). no data were available for the months of october to december 1999. correlation between hydrometeorological parameters and the abundance of stomach contents of a. erythraeus multiple partial regression between the different hydrometeorological parameters and the first principal components (pci) of the abundance of stomach contents reveal that rainfall and tide best explain variations of the abundance and feeding patterns of a. erythraeus. rainfall explained 49.8% of the diet variations in the tambacan site, while tide explained 40.6% of stomach contents variations in the larapan site. discussion temporal pattern in abundance the pattern of abundance of a. erythraeus in iligan bay is similar to the other species of acetes (xiao & greenwood, 1993), and penaeid shrimps (vance et al., 1998) in having a marked seasonality. despite large sample variations, the pattern of abundance in a. erythraeus is similar in both sites. three peaks occurred in october, february, and july. although abundance could range from 0.02 – 2.5 individuals m-2 for both sites, this is a very rough estimate considering the extremely patchy distribution of most acetes (omori, 1974; xiao & greenwood, 1993). in bangladeshi waters, a. erythraeus has two peaks that coincide with the northeast and southwest monsoons, particularly in the months of february and august (zafar & alam, 1997). in this study, the peak in october 1998 is most probably an effect of the la niña that started two months earlier in 1998. the major peak in abundance occurring in drycold month (february) and the minor peak during a wethot month (june) differ from those shown by subtropical species that generally has a single peak in warmer months and the lowest density occurring in colder months (xiao & greenwood, 1993). the temporal pattern of a. erythraeus abundance in iligan bay is similar to those species found in the bay of bengal in that the two peaks in abundance occur at the start of southwest monsoonal rains (june-july) and at the end of the northeast monsoon (february) (deshmukh, 1993; zafar & alam, 1997). feeding ecology burkenroad (1945, as cited by xiao & greenwood, 1993) first reported that the diet of a single south american specimen of acetes comprised small crustaceans and thin-shelled mollusks. xiao & greenwood (1993) noted 11 food categories have so far been identified in the diet of acetes. these are diatoms, protozoans, chaetognaths or arrow worms, copepodan and branchiopodan crustaceans, molluscan larvae, small scales, amorphous materials, debris, sand grains, foraminiferans, and mud. an intensive and detailed study on diet composition of the sub-tropical a. chinensis revealed seasonality in feeding and food selection (xiao & greenwood, 1993). the diet of other metillo 13 fig. 6. temporal variation in salinity (a), temperature (b), total suspended solids (c), and tidal height (d) in the tambacan (open triangle) and larapan (solid triangle) sampling sites. both sampling sites share rainfall data (e). 30 28 26 24 45 35 25 15 5 ‰ oc 0.3 0.2 0.1 0 g 2 1.5 1 0.5 0 -0.5 m 500 300 100 98o 98n 98d 99j 99f 99m 99a 99m 99j 99j 99a 99s 99o 99n 99d 00j mm year/month a b c d e species (a. indicus and a. japonicus) is also mainly composed of animal prey items. le reste (1970) reported a sole diet of copepods in a. erythraeus, but this was based on a very few specimens (n=10) collected in ambaro bay, madagascar. the analysis of feeding habits among crustaceans based on stomach contents needs to be taken with reservation due to biases associated with this method. for instance, crustaceans usually macerate their food using their powerful mandibles, making identification of prey items abundance and diet of acetes erythraeus 14 very difficult (flock & hopkins, 1992). observations in the feeding mechanisms of a. sibogae australis indicated that the microand macrophagous opportunistic feeding of the species may underestimate the importance of large prey items and overestimate the contribution of smaller ones (mcleay & alexander, 1998). however, this study has shown that the high frequency of full stomachs with diverse types of identifiable prey may reduce the inherent bias of stomach content analysis. furthermore, the problem of overestimating the importance of a small quantity of one form that appear in many stomachs, as against other items with large amounts, but found only in fewer stomachs has been mitigated by using a combination of methods (williams, 1981; takahashi & kawaguchi, 1998) in estimating the relative contribution of dietary items. by far, this comprises the only report on the diet composition of a. erythraeus over several months in a tropical setting. twelve (12) categories of foregut contents were identified. like other species of acetes, a. erythraeus is a selective omnivore that tend to include more animal prey than plant in their diet (xiao & greenwood, 1993; mcleay & alexander, 1998). acetes’ high capture efficiency towards animal prey may be associated with its elaborate antennal sensilla which allow them to feed intensively at night (ball & cowan, 1977) and track amino acids from animal prey in highly turbid coastal waters (hamner & hamner, 1977). a. erythraeus ingested a fairly small amount of plant materials (diatoms and algal fragments), but this study reports for the first time relatively abundant dinoflagellates (peridinium spp. and dinophysis spp.) and tintinnids (favella spp. and tintinnopsis spp.) in the shrimp’s diet. a. erythraeus appears to feed intensely after midnight as also observed by takahashi & kawaguchi (1998) for benthopelagic mysids and in other species of acetes (xiao & greenwood, 1993). there is overlap in the diet composition between adults and juveniles, but more small-sized food categories in the stomachs of juveniles indicate diet differences due to size. size-selective feeding is also common among different life history stages of micronektonic mysids and euphausiids (mauchline, 1980). lower values in the index of niche overlap in certain months further support differences in prey size ingested. diet composition of juvenile and adult shrimps, in general, is independent of sex and sampling location, which is similar to some species of micronektonic mysids (metillo & ritz, 1993; takahashi & kawaguchi, 1998). instances where diet composition is not independent of sex and location may be explained by inherent variability of feeding state within a population. we cannot rule out possibilities of some individuals regurgitating food upon being captured in the net, non-feeding due to moulting and senescence. small-scale variations in prey concentration and availability may also explain differences in ingested food by males and females. principal components analysis (pca) indicates seasonality in the relative proportions of stomach contents, which appear to be similar to the seasonal peaks of certain planktonic prey items collected during the field sampling of this study (metillo, unpublished data). mcmanus et al. (1992) first reported that the neritic zooplankton in the waters of bolinao, northwestern philippines follow a three-month seasonal peaks which occur during march, april, may (dry-hot); june, july, august (wet-hot); september, october, november (wet-cold); and december, january, february (dry-cold). peaks of certain dietary items seem to coincide with the three-month and monsoonal seasonal patterns. for example, combinations of the dry-cold (january-february), wet-cold (september), and the southwest (may-june) and northeast (october) monsoons are evident. feeding in juvenile a. erythraeus appears to peak during wet-cold (september) and wet-hot (july) seasons. a. erythraeus, therefore, seems to show coupling of temporal peaks of certain stomach prey items with seasonal patterns of potential prey. like other sergestids and micronektonic crustaceans (e.g., mysids, krill), a. erythraeus also closely align with what is relatively abundant available prey (flock & hopkins, 1992; le reste, 1970; ball & cowan, 1970; atkinson et al., 1999; rudstam et al., 1989; takahashi & kawaguchi, 1998). similar diet composition of shrimps from the two sampling sites may be due to shared hydrometeorological dynamics and composition of potential prey. for instance, similar plankton assemblage may be transported by horizontal mixing of water masses in metillo 15 the two sites that may result in similar prey assemblages available to the shrimps. the differences in the relative proportion of prey items in the two sites may be attributed to site-specific physical processes. for example, multiple partial regression analysis indicates rainfall for the tambacan site and tide for the other site. these are two dominant hydro-meteorological features explaining temporal variability in the shrimp diet. acting singly or simultaneously, tide and rainfall probably have a strong influence on the availability of prey to a. erythraeus. for instance, in the neritic waters of the gulf of thailand, higher zooplankton biomass was observed during months with heavy monsoonal rain, high tides, and strong winddriven onshore currents (brinton, 1979, as cited by schalk, 1987). heavy rainfall produces runoff that elevates levels of dissolved phosphates and nitrates in the estuary. elevated levels of nutrients promote localized algal bloom that in turn increases zooplanktonic production (schalk, 1987). high tides and intense winds that drive surface currents towards the shore tend to restrict a narrow band of inshore water resulting in the aggregation of zooplankton (vance et al., 1998). a. erythraeus may exploit this aggregated and abundant supply of prey. conclusions to conclude, the temporal fluctuations in the population density and the diet composition of a. erythraeus appear to conform not only to typical monsoonal patterns but also to a three-month season in a year. during these periods certain hydrometeorological processes most probably influence the increased availability of planktonic prey to a. erythraeus. adult and juvenile a. erythraeus occupy a trophic niche belonging to a primarily zooplanktivorous omnivore. as an intermediate micronektonic zooplanktivore, a. erythraeus may form an important link between its fish predators and zooplankton, and may potentially compete for similar prey with other micronekton, e.g., fish larvae and post-larval penaeid shrimps that also feed intensely upon plankton. a. erythraeus may assume an important role in the structuring of pelagic communities in iligan bay. acknowledgments my deepest gratitude goes to the international foundation for science, sweden for the grant (#a/27101), and to the three anonymous reviewers whose comments immeasurably improved the manuscript into its more concise form. i also wish to thank mr. jullibert castillon for help in the field sampling, and dr. rachel d. sanchez-metillo for technical assistance. references atkinson, a., p. ward, a. hill, a.s. brierly, & g.c. cripps, 1999. krill-copepod interactions at south georgia, antarctica, ii. euphausia superba as a major control on copepod abundance. mar. ecol. prog. ser. 176: 63-79. ball, e.e. & a.n. cowan, 1977. ultrastructure of the antennal sensilla of acetes (crustacea, decapoda, natantia, sergestidae). philos. trans. r. soc. lond. (b. biol. sci.) 277: 429-476. deshmukh, i., 1986. ecology and tropical biology. london, blackwell scientific publications: 387pp. deshmukh, v.d., 1993. status of non-penaeid prawn fishery of india and stock assessment of acetes indicus milne edwards off maharashtra. indian j. fish. 40(1,2): 50-62. edra, r.b., 1975. on the plankton succession off changi point. philipp. j. fish. 13(1): 18-71. flock, m.e. & t.l. hopkins, 1992. species composition, vertical distribution, and food habits of the sergestid shrimp assemblage in the eastern gulf of mexico. j. crust. biol. 12(2): 210-223. grossman, g.d., d.m. nickerson, & m.c. freeman, 1991. principal component analyses of assemblage structure data: utility of tests based on eigenvalues. ecology. 72(1): 341347. hamner, p. & w.m. hamner, 1977. chemosensory tracking of scent trails by the planktonic shrimp acetes sibogae australis. science. 195: 886-888. abundance and diet of acetes erythraeus 16 hutchinson, g.e., 1953. the concept of pattern in ecology. proc. natl. acad. sci. usa. 105: 1-12. krebs, c.j., 1985. ecology: the experimental analysis of distribution and abundance. 3rd ed. new york, harper and row: 800 pp. le reste, l., 1970. biologie de acetes erythraeus (sergestidae) dans une baie du n.w. de madagascar (baie d’ambaro). cahiers o.r.s.t.o.m.: office de la recherche scientifique et technique outre-mer oceanographique. 8: 35-56. levin, s.a., 1992. the problem of pattern and scale in ecology. ecology. 73(6): 1943-1967. longhurst, a.r., 1985. relationship between diversity and the vertical structure of the upper ocean. deep-sea res. 32(12): 1535-1570. longhurst, a.r. & d. pauly, 1987. ecology of tropical oceans. london, academic press, inc.: 393 pp. ludwig, j.a. & j.f. reynolds, 1988. statistical ecology, a primer on methods and computing. canada, john wiley and sons, inc.: 337 pp. mauchline, j., 1980. the biology of mysids and euphausiids. adv. mar. biol. 18: 1-681. mcgowan, j.a. & p.w. walker, 1985. dominance and 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epiplanktonic shrimps of the genera lucifer and acetes (macrura, penaeidea, sergestidae). in proceedings of the symposium on warm water zooplankton (goa: special publication nio): 1-12. pulman, r.j., 1994. community ecology. london, chapman and hall: 177 pp. rudstam, l.g., k. danielsson, s. hansson, & s. johansson, 1989. diel vertical migration and feeding patterns of mysis mixta (crustacea, mysidacea) in the baltic sea. mar. biol. 101: 43-52. schalk, p.h., 1987. monsoon-related changes in zooplankton biomass in the eastern banda sea and aru basin. biol. oceanogr. 5: 1-12. schoener, t.w., 1970. nonsynchronous spatial overlap of lizards in patchy habitats. ecology. 51: 408-418. steele, j.h., 1974. the structure of marine ecosystems. harvard, harvard university press: 128 pp. takahashi, k. & k. kawaguchi, 1998. diet and feeding rhythm of the sand burrowing mysids archaeomysis kokuboi and a. japonica in otsuchi bay, northeastern japan. mar. ecol. prog. ser. 162: 191-199. vance, d.j., m.d.e. haywood, d.s. heales, r.a. kenyon, & n.r. loneragan, 1998. seasonal and annual variation in abundance of postlarval and juvenile banana prawns penaeus merguiensis and environmental variation in two estuaries in tropical northeastern australia: a six year study. mar. ecol. progr. ser. 163: 21-36. williams, m.j., 1981. methods for analysis of natural diet in portunid crabs (crustacea: decapoda: portunidae). j. exp. mar. biol. ecol. 52: 103-113. metillo 17 abundance and diet of acetes erythraeus xiao, y. & j.g. greenwood, 1993. the biology of acetes (crustacea: sergestidae). oceanogr. mar. biol. annu. rev. 31: 259-444. zafar, m. & m.d. alam, 1997. occurrence and abundance of acetes shrimps in the kutubdia channel of bangladesh coastal water. bangladesh j. fish. res. 1(2): 91-96. page 1 images image 1 page 2 images image 1 page 3 images image 1 sc i e n c e di l i m a n is published semi-annually (june and december) by the university of the philippines diliman through the office of 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issn online 2012-0818 international advisory board teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus university of illinois, usa university of california, irvine, usa the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheffield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. department of physics university of warwick, united kingdom raul k. suarez, ph.d. professor emeritus university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences graduate school university of tsukuba, japan 05_ndyag with corrections fr dr. saloma_1nov nd:yag laser-pumped hydrogen 37 abstract science diliman (january-june 2005) 17:1, 37-46 a nd:yag laser-pumped hydrogen raman shifter with capillary waveguide maria leilani y. torres*, marilou m. cadatal, and wilson o. garcia photonics research laboratory national institute of physics, college of science university of the philippines, diliman, 1101 quezon city telefax: (632)920-5474 e-mail: mltorres@nip.upd.edu.ph date received: january 12, 2005 ; date accepted: august 30, 2005 operation of a 355/532 nm nd:yag laser-pumped hydrogen raman shifter with capillary waveguide (cwg) is demonstrated. for both pump wavelengths, more laser lines are generated using the raman shifter with cwg compared to a conventional raman shifter. both 355 and 532 nm pumps showed a 60% decrease in threshold power for the generated first stokes (s1) laser line. the 355 nm-pumped raman shifter with cwg generated s1 at 2.1 mw pump power at a hydrogen pressure of 1.38 mpa. on the other hand, for the 532 nm pumped waveguide raman shifter at a hydrogen pressure of 1.72 mpa, the threshold power for s1 is at 8.3 mw. in addition, an improvement of the output powers is observed for the stokes and anti-stokes generated by raman shifter with cwg. keywords: waveguide, stimulated raman scattering introduction the hydrogen raman shifter is an efficient simple and economical method of simultaneously generating multiple laser lines. it has been used as a light source for two-photon two-color excitation studies (palero et al., 2002) and pulsed color digital holography (almoro et al., 2004). the raman shifter utilizes a nonlinear phenomenon known as stimulated raman scattering, which extends the tuning range of lasers into the vacuum ultraviolet (vuv) and to the far infrared (fir) (papayanis et al., 1998). of the possible raman medium, hydrogen is by far the most widely used due to its large raman shift and high raman transition probability leading to efficient shifting (bhagavantam, 1942). however, srs requires high pump intensity to reach its threshold (benabid et al., 2002). several techniques such as high-finess fabry perot resonator (benabid et al., 2002), multiple pass configurations and beam guidance with hollow dielectric waveguides (hdw) (rabinowitz et al., 1976), and hollow core photonic crystal fibers (benabid et al., 2002) have been utilized to achieve lower srs threshold intensity. classified as hdw (marcuse,1974), capillary waveguides (cwg) are inexpensive tools to confine the gas and provide beam guidance. with the cwg inside the raman shifter, an improvement of the conversion efficiency is achieved. developing a method to achieve low threshold intensity for srs provides an avenue for numerous applications *corresponding author torres, cadatal, and garcia 38 in nonlinear optics and technology such as spectroscopy, remote sensing, and atomic physics (benabid et al., 2002). low threshold intensity of srs can provide a light source suitable for image sectioning via multi-color multi-photon fluorescence microscopy, without damaging biological samples (palero et al., 2002). with low pump beam intensity requirement, laser pump sources with lower output intensity can also be utilized. a further advantage of achieving a low threshold is that the available pump power can be divided to drive a raman oscillator and amplifier. this configuration can result in a more efficient channeling of the pump power to generate the intended stokes radiation (berry et al., 1982). in this paper, we compare the performances of a conventional raman shifter and a raman shifter with cwg using the 532 and 355 nm output of a nd:yag laser as pump beam in terms of the number of raman components generated and threshold powers. similarly, we investigate the dependence of output power of the raman components with the hydrogen raman pressure and input power. stimulated raman scattering in capillary waveguide the basic theory behind srs is called the raman effect. an illustration of the frequency shifts involved in raman effect is shown in fig. 1. a pump photon interacts with a molecule in the ground state and excites it to a virtual state. when the molecule relaxes, it produces a photon of lower frequency compared to the pump photon. this shifted photon is called the stokes (s) photon. on the other hand, when a molecule initially in the first excited state absorbs a pump photon and is excited to a higher virtual state, a photon of higher frequency to the pump photon is emitted when it relaxes spontaneously. this photon of higher frequency is known as the anti-stokes (as) photon. srs occurs when a sufficiently intense light beam interacts with a raman medium to produce coherent radiation. furthermore, when the first stokes (s1) attains sufficient intensity, it may act as pump photon to produce higher order second stokes (s2) and third stokes (s3) lines as shown in fig. 2. this process is known as the srs cascade. another nonlinear phenomenon known as the four-wave raman mixing (fwrm) is also involved in the generations of the antistokes and higher-ordered stokes lines. fwrm is depleted at higher pressures due to an increase in wave vector mismatch (shoulepnikoff,1997). conventional raman shifters use a lens to focus the pump beam inside a gas cell. the nonlinear phenomena such as srs and fwrm occur in the focal region known as the interaction region where the threshold for srs to occur is achieved. this technique requires high input intensity for srs to occur (benabid et al., 2002). virtual state 1st excited state ground state stokes shifting virtual state 1st excited state ground state anti-stokes shifting fig. 1. an illustration of the frequency shifts due to the raman effect. nd:yag laser-pumped hydrogen 39 1997). in addition, a longer region of interaction provides longer region where phase matching between wave vectors can be satisfied. hence, the effect of fourwave raman mixing (fwrm) becomes prevalent. under steady-state conditions and for a focused singlemode pump laser, the stokes power will grow as exp(g) where the cumulative gain coefficient g is given by . (2) here pp is the laser pump power, λs and λp are the stokes and pump wavelengths, respectively, and b is the confocal parameter (perrone et al., 1997). according to eq. (2), higher pump laser wavelengths lead to higher threshold energies and lower stokes conversion efficiencies. methodology the schematic diagram of the experimental setup is shown in fig. 4. the laser pump is a q-switched neodymium-doped yttrium-aluminum garnet (nd:yag) laser (spectra physics gcr-230) operating at 10 hz repetition rate. the 1064 nm fundamental output is converted to its second (532 nm) and third (355 nm) harmonics using potassium dideuterium phosphate (kdp) crystals with pulse width of 5-6 ns. the mirrors m1 and m2 steers the beam into the setup. the pump beam passes through a diaphragm d and expanded using a telescope. a 1 mm aperture a approximates a plane wave and lens l1 (f = 290 mm) focuses the beam into a 50-cm long, 1 mm bore diameter silica glass (index of refraction ~1.5) capillary waveguide inside a 58-cm long raman cell. the raman cell consists of a pressurized stainless steel chamber made with both ends fitted with fused silica optical windows for laser beam input and output. after evacuating with a mechanical pump, it is filled with ultra high purity hydrogen (cryogenic rare gas, 99.9999% purity) with pressure up to 4.14 mpa. the cwg is placed on top of a sheet of stainless steel made to fit the walls of the raman cell. lens l2 collimates the beam into a pellin broca prism (pb) which separates the raman output into its components. the spectral characterization of the pump, stokes, anti-stokes, and with the use of a cwg inside the raman shifter, the pump beam is confined and propagated by multiple grazing reflections (verdeyen, 1981) which are equivalent to periodical focusing by a series of lenses (arnaud, 1976) as shown in fig. 3. this introduces foci along the hollow bore of the cwg. in effect, the length of interaction, z, between the pump and the medium increases. according to eq. (1), which gives the stokes intensity, is(z) (papayanis et al., 1998), the increase in z will decrease the srs threshold of the device and increase the conversion efficiency of the raman lines: . (1) here ip(0) is the pump beam intensity and gr is the steady state raman gain. the gr is proportional to the raman active gas pressure. moreover, gr is observed to saturate at high pressures (schoulepnikoff et al., ( ) ( ) ( )( )0 exp 0=s s p ri z i i g z virtual states v = 1 v = 0 (1) (2) (3) fig. 2. generation of (1) s1, (2) s2, and (3) s3 by srs cascade. fig. 3. the (b) propagation of light inside the capillary waveguide can be simulated by the focusing of light by (a) a series of lenses. regions of high intensity where srs threshold is achieved. l1 l2 l3 l4 (a) (b) l1 torres, cadatal, and garcia 40 rayleigh is executed using a computer-controlled monochromator (spex 1000m). the separated raman components are placed incident to an optical fiber bundle input connected to the grating monochromator. output powers are measured using a melles-griot broadband power meter. the cwg is removed from the raman shifter for the operation of the conventional raman shifter. results and discussion 355 nm pump beam the spectral profile of the generated raman components for a 355 nm pump beam is shown in fig. 5(a) for the raman shifter with cwg and fig.5(b) for the conventional raman shifter. maximum number of five (5) raman lines, three (3) stokes, and (2) antistokes lines with wavelengths ranging from 273.8 to 635.9 nm are generated using a raman shifter with cwg at h2 pressure = 1.38 mpa and input power (pin) = 26.5 mw. only two (2) raman components s1 and s2 with wavelengths 415.9 and 502.9 nm, respectively, are generated via a conventional raman shifter. the generation of the anti-stokes lines in fig. 5(a) shows the enhancement of the fwrm process in the presence of the cwg. the dependence of the output power with h2 pressure is shown in fig. 6 for the conventional raman shifter and for the raman shifter with cwg at pin = 26.5 mw. in the presence of a cwg, the rayleigh, otherwise known as the depleted pump rapidly decreases with increasing pressure due to its conversion to raman components. on other hand, s1 and s2 increase with increasing pressure then saturate at h2 pressure > 2.76 mpa. this is due to the saturation of the h2 raman gain at high pressures (schoulepnikoff et al., 1997). higher output power can be observed for the raman lines with cwg. s2 generated with cwg shows an improvement in its output powers with peak power of 1.3 mw at h2 pressure = 3.45 mpa compared to only 0.03 mw without cwg. interestingly, the output powers of s1 and the other raman lines are exceeded by the output powers of s2 generated with cwg at pressures 2.07-3.05 mpa. s2 is primarily generated from s1 by srs cascade. however, s1 is also utilized to generate anti-stokes lines by fwrm. because the output power of s1 is expended to generate not only higher ordered stokes but anti-stokes as well, s2 has a higher output power compared to s1. this is due to the longer interaction region provided by the cwg, which enhances fwrm and srs cascade. s1 is observed to have the same peak power of 1.5 mw at h2 pressure = 0.69 mpa for the raman shifter with cwg and h2 pressure = 3.79 mpa for the conventional raman shifter. this is in accordance with eq. (1), where at the same pump power with shorter z, a higher gr and hence a higher pressure must be attained. hydrogen raman shifter with capillary waveguidetelescopea d raman lines q-switched nd:yag laser (355/532 nm) l1 l2 m1 m2 pb fig. 4. experimental setup for the generation of raman lines, where m1 and m2–mirrors, a–aperture, d–diaphragm, l1–focusing lens, l2–collimating lens, and pb–pellin broca prism. nd:yag laser-pumped hydrogen 41 figure 7 shows the dependence of the output power with pump power for the conventional and the raman shifter with cwg operating at h2 pressure =1.38 mpa. a threshold power is observed for the raman lines. with increasing pin, more raman lines are generated. the threshold power of the raman components with and without cwg is summarized in table 1. at h2 pressure = 1.38 mpa, s1 is generated at pin = 2.1 mw for a raman shifter with cwg compared to the threshold power of s1 at pin = 5.2 mpa generated without the waveguide. with the aid of cwg, the threshold power for s1 is reduced by 60% at of h2 pressure = 1.38 mpa. 532 nm pump beam figure 8 shows the spectral profile of the generated raman lines for a 532 nm pump beam with [fig. 8(a)] fig. 5(a). spectral profile of the raman components generated from a raman shifter with cwg at 26.5 mw input power and 1.38 mpa hydrogen pressure. inset shows the actual location of the raman lines. intensity of raman lines is normalized with respect to the rayleigh. as2 as1 s3 r s2 s1 273.8 309.0 354.6 415.9 502.9 635.9 center wavelength (nm) 0.0 0.2 0.4 0.6 0.8 r el at iv e in te ns it y (a .u .) fig. 5(b). spectral profile of the raman components from a conventional raman shifter at 26.5 mw input power and 1.38 mpa hydrogen pressure. inset shows the actual location of the raman lines. intensity of raman lines is normalized with respect to the rayleigh. 0.0 0.2 0.4 0.6 0.8 r el at iv e in te ns it y (a .u .) center wavelength (nm) r s1 s2 354.6 415.9 502.9 torres, cadatal, and garcia 42 fig. 7. the output power of raman lines as a function of the 355 nm pin at h2 pressure of 1.38 mpa with and without a cwg. r el at iv e in te ns it y (a .u .) as2 as1 r s1 282.1 319.6 368.7 435.5 531.9 682.9 center wavelength (nm) 0.0 0.2 0.4 0.5 0.3 as3as4 0.1 fig. 6. the output power of the raman lines as a function of the hydrogen gas pressure at 355 nm pump power of pin = 26.4 mw with and without a capillary waveguide. r s1 s2 s3 as2 as1 pressure (mpa) pressure (mpa) o ut pu t po w er ( m w ) o ut pu t po w er ( m w ) 0.0 0.69 1.38 2.07 2.76 3.45 4.140.0 0.69 1.38 2.07 2.76 3.45 4.14 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5raman shifter with cwg conventional table 1. threshold power for the generated raman components with and without cwg at h2 pressure = 1.38 mpa for the 355 nm pump beam. without cwg only s1 and s2 are generated. with cwg without cwg threshold power (mw) 2.1 5.2 2.5 6.5 5.1 4.2 11.3 s1 s2 s3 as1 as2 nd:yag laser-pumped hydrogen 43 and without cwg [fig. 8(b)]. with a cwg, five (5) raman lines, s1 (682.9 nm), as1 (435.5 nm), as2 (368.7 nm), as3 (319.6 nm), and as4 (282.1 nm) are generated at h2 pressure = 1.72 mpa and pin= 46.5 mw. at the same h2 pressure and pin, the conventional raman shifter generated only two (2) raman lines, s1 and as1. with the longer interaction region provided by the cwg, phase-matching between interacting wave vectors is improved. hence, the enhancement of fwrm as manifested by the production of more antistokes lines for a raman shifter with cwg. the dependence of output power with h2 pressure is shown in fig. 9. with cwg, s1 reaches its peak output fig. 8(a). spectral profile of the raman components generated from a 532 nm pumped raman shifter with cwg at pin = 46.5 mw and h2 pressure =1.72 mpa. inset shows the actual location of the raman lines. intensity of the raman lines are normalized with respect to the rayleigh. r el at iv e in te ns it y (a .u .) 0.0 0.2 0.4 0.5 0.3 0.1 as2 as1 r s1 282.1 319.6 368.7 435.5 531.9 682.9 center wavelength (nm) as3as4 fig. 8(b). spectral profile of the raman components generated from a 532 nm pumped raman shifter without a cwg at pin = 46.5 mw and h2 pressure =1.72 mpa. inset shows the actual location of the raman laser lines. the intensities of the raman lines are normalized with respect to the rayleigh. r el at iv e in te ns it y (a .u .) 0.0 0.2 0.4 0.5 0.3 0.1 435.5 531.9 682.9 center wavelength (nm) s1 as1 r torres, cadatal, and garcia 44 0.0 0.2 0.4 0.6 0.8 r s1 s2 s3 as2 as1 o ut pu t po w er ( m w ) 0 5 10 15 20 25 pressure (mpa) 0.69 1.38 2.07 2.76 3.45 4.14 pressure (mpa) 0.69 1.38 2.07 2.76 3.45 4.14 o ut pu t po w er ( m w ) 0 5 10 15 20 25 a nti-s tokes output (m w ) raman shifter with cwg conventional fig. 9. the power of various raman lines as a function of the h2 pressure at 532 nm pump power of pin = 46.5 mw with and without a capillary waveguide. fig. 10. the power of various raman lines as a function of the 532 nm pump power at h2 p = 1.38 mpa with and without a capillary waveguide. r s1 s2 s3 as2 as1 input power (mw) 5 10 15 20 25 30 35 40 45 50 o ut pu t po w er ( m w ) 0 1 2 3 4 5 6 7 8 9 input power (mw) 5 10 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 8 9 o ut pu t po w er ( m w ) raman shifter with cwg conventional table 2. threshold power of the raman components generated from a 532 nm pumped h2 raman shifter with and without cwg at h2 pressure = 1.72 mpa. without cwg only s1 and as1 are generated. with cwg without cwg threshold power (mw) 8.3 21 9.5 36 19.1 46.3 46.5 s1 as1 as2 as3 as4 nd:yag laser-pumped hydrogen 45 power at h2 pressure =1.04 mpa and saturates at higher pressures due to the saturation of the h2 raman gain. the output power of the as1 generated from the raman shifter with cwg reaches its peak at h2 p = 1.38 mpa. at midrange h2 pressure (1.38-2.07 mpa), fwrm is most efficient. hence, higher output power is attained by the anti-stokes lines are at this pressure range. at h2 pressure > 2.07 mpa, the as lines are rapidly depleted since it is difficult to satisfy phase matching conditions at high pressures. higher output powers are achieved for the raman lines generated by the raman shifter with cwg. figure 10 shows plots of the output powers of the raman lines from the raman shifter with cwg and the conventional raman shifter as functions of pin. as with the 355 nm pump beam, there is a threshold power to generate srs. the threshold power of the raman components is summarized in table 2. however, because of lower cumulative gain g for longer pump wavelengths, higher threshold power can be observed using a 532 nm compared to a 355 nm pump beam. at h2 pressure = 1.72 mpa, the threshold power for s1 generated from a raman shifter with cwg is 8.1 mw. with longer interaction region, the threshold power of the device decreases. conclusion operation of a hydrogen raman shifter with capillary waveguide is demonstrated. for a 355 nm pump beam, s1 is generated at 2.1 mw at h2 pressure = 1.38 mpa while for the 532 nm-pump beam it is generated at 8.3 mw at h2 pressure = 1.72 mpa. with the presence of cwg for the 355 nm excitation wavelength, s2 is observed to generate higher power compared to s1 and other raman lines. more anti-stokes lines are observed for a 532-nm pumped raman shifter with cwg. for both pump wavelengths, a 60% reduction in threshold power is achieved. with a cwg, more raman lines are generated and effects of fwrm are enhanced. acknowledgments the authors gratefully acknowledge the instrumentation physics laboratory for lending their detector and the philippine department of science and technology (dost) through the engineering and science education project (esep) for the equipment grant. this project is also supported by university of the philippines and the philippine council for advance science and technology research and development (pcastrd). references almoro, p., m. cadatal, w. garcia, and c. saloma, 2004. pulsed full-color digital holography with a hydrogen raman shifter. appl. opt. 43: 2267-2271. arnaud, j., 1976. beam and fiber optics. bell tel. labs, inc. berry, a.j., d.c. hanna, and d.b. hearn, 1982. low threshold operation of a waveguide h2 raman laser. opt. commun. 43: 229-232. benabid, f., j.c. knight, g. antonopoulos, and p. russel, 2002. stimulated raman scattering in hydrogen-filled hollow-core photonic crystal fiber. science. 298: 399-402. bhagavantam, s., 1942. scattering of light and the raman effect. chemical publishing company, new york. garcia, w.o., 2002. temporal coherence control of a qswitched frequency tripled (355 nm) nd:yag pumped hydrogen raman shifter. ph.d. dissertation, university of the philippines. mannik, l. & s.k. brown, 1986. tunable infrared generation using third stokes output from a waveguide raman shifter. opt. commun. 57: 360-364. marcuse, d.,1974. theory of dielectric optical waveguides. bell tel. labs, inc. palero, j., w. garcia, and c. saloma, 2002. two-color (twophoton) excitation fluorescence with two confocal beams and a raman shifter. opt. commun. 211: 57-63. papayannis, a., g. tsikrikas, and a. serafetinides, 1998. generation of uv and vis laser light by stimulated raman scattering in h2, d2, and h2/he using a pulsed nd:yag laser at 355 nm. appl. phys. b. 67: 563-568. torres, cadatal, and garcia 46 perrone, m.r. et al., 1997. dependence of rotational and vibrational raman scattering on focusing geometry. ieee j. quantum electron. 33: 6. schoulepnikoff et al., 1997. experimental investigation of high-power single-pass raman shifters in the ultraviolet with nd:yag and krf lasers. appl. opt. 36: 5026-5043. verdeyen, j., 1981. laser electronics. prentice hall, inc., englewoods cliff, new jersey. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 sd-sample article abstracts of up diliman’s registered ips 86 patents and utility models • coatin® (titanium nitride thin film formation on henry j. ramos metal substrate by chemical vapor deposition in a magnetized sheet plasma source) • 1p-obic (method for generating high-contrast caesar a. saloma, jelda jayne c. images of semiconductor sites via one-photon miranda & vincent ricardo m. optical beam-induced current imaging and daria confocal reflectance microscopy) • 2-photon (two color (two photon) excitation with caesar a. saloma, jonathan a. focused excitation beams and a raman shifter) palero & wilson o. garcia • rte cooked rice (retrogradation-resistant ma. patricia v. azanza shelf-stable cooked rice) patent pending • moblocks® (time-sensitive assimilation device michael l. abundo, luther for use in tangible user interface (tui) devices) paul d. caranguian, carlos primo c. david, josef karlo s. diaz, luis g. sison & pablito tolentino jr. • oral vaccine (clay microencapsulation of anacleto m. argayosa, chelo s. non-infective pathogens for oral vaccine pascua, florentino c. sumera, development) john anthony d.l. yason & alpha rae m. espigar • bioactive coating for stents (bioactive coating jhalique jane r. fojas for intravascular drug eluting stents and implantable medical devices using carrageenanchitosanhyaluronan complex) abstracts of up dil iman's registered intellectual proper ties technology transfer is one of the major programs through which up diliman facilitates the dissemination of its research and creative work outputs. within the framework of the revised intellectual property rights (ipr) policy of the university of the philippines and the country’s technology transfer act, the university assists researchers and creative workers in the protection, licensing, patenting, copyrighting, commercialization and/or deployment of their works. tangible achievements along these lines are showcased through the registered intellectual properties of up diliman that are described in this section. the registered ips include: 87 abstracts of up diliman’s registered ips • algal bloom detection kit (a gamma-based lourdes j. cruz, elvira z. receptor binding assay for in-site monitoring sombrito & aileen l. de leon of paralytic shellf ish toxins) • amebiasis kit (rapid and accurate detection windell l. rivera, angeline kit for amebiasis patients through salivary iga) odelia c. concepcion & alexander edward dy for patent application • rte noodles (retrogradation-resistant ready-to-eat ma. patricia v. azanza, (rte) asian noodles) kristia may t. marte & maria lorina c. morales • motesartxp (motesartxp: wireless sensor networks ian christopher tolentino, for monitoring temperature and relative humidity in robin christian s. juson, art spaces) bernard james tan, marc caesar r. talampas, luis g. sison & maricor n. soriano trade secrets, trademarks, service marks, and copyrights • pyrodinium culture (development of rhodora v. azanza & lourdes semi-commercial scale (tank culture) of j. cruz pyrodinium for neosaxitoxin and saxitoxin production) • seamoy® (seaweed-based air freshener gel) marco nemesio e. montaño & banaag glorioso-lajera • up sablay (up in alibata characters and device) carmen g. diaz de ventanilla, antoinette b. hernandez, rogelio l. juliano jr., virginia d. monje, consuelo j. paz & abraham p. sakili industrial designs • lual – kiln firing as art and metaphor of birthing ma. rita badilla-gudiño • takeaway packaging system (greener fast food patricia t. mallare packaging in the philippines) abstracts of up diliman’s registered ips 88 coat in™: t in coating technology (t itanium nitride thin film formation on metal substrate by chemical vapor deposition in a magnetized sheet plasma source) henry j. ramos the invention relates to a titanium nitride f ilm chemical vapor deposition process on metal substrate using a magnetized sheet plasma source. interest in new coatings and surface treatment methods has been on the upsurge during the last decade of the 1990s, especially for titanium nitride (tin). a tin f ilm is a remarkably hard and wear-resistant coating on tools since it decreases the rate of abrasive wear during the cutting process as well as the chemical interaction between the tool and the work piece because of its chemical inertness. several techniques such as chemical vapor deposition, physical vapor deposition, ion plating, ion beam-assisted deposition, sputtering, and hybrid processes have been used to prepare tin f ilms. the f ilms produced by these techniques, however, often exhibit poor adhesion to the substrate, require high deposition temperature, need a relatively long duration of time for thin f ilm formation, and cover a limited substrate surface. the present invention surmounts these problems through the discovery of a deposition process where: a) there is no heating mechanism introduced, b) the synthesis of the f ilm is relatively short, and c) the synthesis can be done over a wide area of substrate surface, without sacrif icing the quality of the f ilm. although the invention demonstrates the capacity of synthesizing titanium nitride for small-sized samples, the wide area plasma could very well serve the coating of larger samples. (source: coatin® patent document) this invention was granted patents in the philippines (patent no. 1-2004-502138 issued on 5 november 2009), the united states (patent no. 7,438,955 issued on 21 october 2008), japan (patent no. 4481657 issued on 26 march 2010), europe (patent no. 1485516 issued on 24 august 2011), and malaysia (patent no. my148608-a issued on 15 may 2013). 89 abstracts of up diliman’s registered ips 1p-obic microscopic imaging for semiconductor sites (method for generating high-contrast images of semiconductor sites via one-photon optical beam-induced current imaging and confocal reflectance microscopy) caesar a. saloma jelda jayne c. miranda v incent ricardo m. daria this invention relates to a method of precisely determining the location of defects in an integrated circuit (ic). the invention, in one broad sense, is about the discovery that exclusive high-contrast images of semiconductor sites can be generated quickly and accurately from the 1p-obic image and the confocal reflectance image which are obtained via one and the same excitation beam that is focused on the ic sample. the process makes use of the fact that: (1) confocal images are optically-sectioned images while 1p-obic images are exclusive low-contrast images of semiconductor sites, and (2) both the confocal image and 1p-obic image are produced with an optical beam. (source: 1p-obic patent document) because the method can produce two images in a single scan, there is no need for separate imaging instruments, thus making 1p-obic microscopic imaging signif icantly less expensive than commercial failure detection facility. this invention was granted patents in the philippines (patent no. 1-2005-500056 issued on 23 november 2007) and the united states (patent no. 7,235,988 issued on 26 june 2007). 2-photon (two-color, two-photon excitation with focused excitation beams and a raman shifter) caesar a. saloma jonathan a. palero w ilson o. garcia this invention relates to a method for inducing highly localized light absorption in materials via two-color (two-photon) excitation (2ce). the invention, in one broad sense, is the discovery that two-color (two-photon) excitation with focused beam(s) abstracts of up diliman’s registered ips 90 may be achieved with a raman shifter. the process makes use of the fact that the raman shifter could act as the light source for all the excitation wavelengths (l 1 ,l 2 ) that are needed in two-color excitation. this work provides a promising f irst step towards the realization of a practical 2ce microscope. the raman shifter is a versatile excitation light source for 2ce. it is inexpensive and simpler to construct and operate than a dye laser which requires a cavity resonator, a spectrometer for spectral tuning, and a dye regulator assembly. with a raman shifter, the optimal conditions for spatial and temporal overlap between the two excitation pulses is achieved without great diff iculty unlike in set-ups where l 1 and l 2 are obtained from two different light sources. (source: 2ce patent document) this invention was granted patents in the philippines (patent no. 1-2005-500584 issued on 15 july 2008) and the united states (patent no. 8,227,256 issued on 24 july 2012). rte cooked rice (retrogradation-resistant shelf-stable cooked rice) ma. patricia v. azanza during calamities, people sometimes have to be evacuated to safety in evacuation centers. evacuees could stay for days in these centers. these places usually do not have adequate cooking equipment, and evacuees usually rely on ready-to-eat canned goods, biscuits, or “instant noodles” from disaster-relief agencies for their meals. in military f ield operations, filipino soldiers do not risk their lives by giving away their position to the enemies via the smell and smoke from cooking. as such, they also rely on ready-to-eat food. however, these foods are usually not nutritionally adequate for a whole day’s meal, let alone a single meal. this invention provides a novel solution to the aforementioned problems by producing cooked rice or kanin that is shelf-stable for several months without being subject to retrogradation or f irming, while still exhibiting the characteristics of newly-cooked rice even without the reheating step. the invention relates to pouched acid pasteurized cooked milled rice product that is resistant to retrogradation during storage at ambient conditions for three months. resistance to retrogradation of the treated product is attributed to the synergistic effects of the 91 abstracts of up diliman’s registered ips use of acidic carbohydrates, incorporation of cooking oil, and selection of rice cultivar that belongs to low apparent amylase content. this invention is registered as philippine utility model no. 1-2011-000008 issued on 3 october 2011. moblocks® (t ime-sensitive assimilation device for use in tangible user interface (tui) devices) michael l. abundo luther paul d. caranguian carlos primo c. david josef karlo s. diaz luis g. sison pabl ito tolentino jr. moblocks® is a fun, educational toy for mission-based games and creative play. it teaches direction and orientation, strategic planning and basic programming through a series of story missions and problem solving adventures. it aims to develop an embedded system for mobile robot (mobot) control-based on tangible user interface (tui). the system is composed of a mobot, wooden blocks, a tray and a play area. the blocks represent the instructions to the robot. the tray has slots for four blocks and sensors to detect the sequence of blocks. it commands the robot over a wireless link. moblocks® appeals to the senses. all parts of its playset are real and tangible objects. compared with manipulating elements on a computer screen, kids go through a direct and experiential learning. it also promotes critical thinking and problemsolving skills when kids sequentially program the robot. in remote control cars, kids can simply employ trial-and-error to drive the car to a target location. but in moblocks®, kids learn to strategize by thinking ahead and planning before making the robot move. a philippine patent application has been f iled for this invention (application no. 12009-000107; patent pending). moblocks® is a registered trademark with registration no. 4-2008-002702. abstracts of up diliman’s registered ips 92 oral vaccine (clay microencapsulation of non-infective pathogens for oral vaccine development) anacleto m. argayosa chelo s. pascua florentino c. sumera john anthony d.l. yason alpha rae m. espigar as with any type of farming, aquaculture’s viability and prof itability depend on the volume and quality of the yield. this requires, among other things, that the f ish be free from illnesses, in order to avoid f ish deaths that in turn will reduce harvest. some factors that cause diseases in f ish are poor water quality, high density (overcrowding), high water salinity, and the presence of predators such as snails and birds. there are available vaccines to prevent diseases, which are administered to the f ish either by injection, immersion, or oral administration. administration by injection is a tedious task because it requires a lot of time and effort, and may even cause stress to the f ish. on the other hand, immersion allows f ingerlings to be vaccinated at an early stage but it is still a more tedious process when compared to oral vaccination. however, the problem with current oral vaccines in the market is that they are known to be less effective than the other methods since the protection they provide is only short-term. the current invention involves a process that yields an oral a vaccine for f ishes that provides both long-term immunization for the f ish and ease of administration for the farmers. the process enhances the effectiveness of the oral vaccine through the clay microencapsulation of the antigen. the clay protects the antigen so that it is still intact when it reaches the f ish’s hind gut, where immunization takes place more effectively. the vaccine can be incorporated into f ish pellets to make administration easier. while the invention’s initial application is for f ish vaccine development, the inventors’ research also showed that it may be applicable for use in shrimps and even other animals. more impor tantly, the invention has the potential for use in human vaccine development. this invention won 1st place in the 2013 ambassador alfredo m. yao intellectual property awards organized by the philippine chamber of commerce and industry 93 abstracts of up diliman’s registered ips in collaboration with the intellectual property off ice of the philippines and the department of science and technology. a philippine patent application for this invention has been f iled on 30 august 2013 (application no. 1-2013-000256; patent pending). bioactive coating for stents (bioactive coating for intravascular drug eluting stents and implantable med ical devices using carrageenanchitosanhyaluronan complex) jhalique jane r. fojas the number of heart disease cases in the philippines is on the rise, and heart ailment is one of the leading causes of deaths in the country. while there are no data on angioplasty procedures in the country, the cost of a vascular stent, let alone a procedure, is no doubt high. although health insurances, both from the private and government sectors, are available, they might not be suff icient to cover the cost of procedures and treatments for heart disease. a more affordable stent will therefore offer f inancial relief to heart patients. the main purpose of the invention is to produce a bioactive coating for intravascular stainless steel stents and stainless steel implantable medical devices using a polymer complex of carrageenanchitosanhyaluronan. the bioactive coating synthesized with the layer-by-layer self-assembly method using carrageenan, chitosan, and hyaluronan is an improvement of the coating process for metal using bioactive polymers and existing stainless steel stent products. moreover, the biopolymer coating is capable for drug encapsulation and release. the f inal product is characterized as biocompatible and hemocompatible, antineoplastic, anti-inflammatory and capable of drug elution that can help in healing the damaged area. a philippine patent application has been f iled for this invention on 17 december 2 0 1 3 . abstracts of up diliman’s registered ips 94 algal bloom detection kit (a gamma-based receptor bind ing assay for in-site monitoring of paralytic shellf ish toxins) lourdes j. cruz elvira z. sombrito aileen l. de leon paralytic shellf ish poisoning (psp) is among the contamination problems that greatly affect the waters worldwide. hence, there is a high demand for a sensitive and specif ic method that can be easily used in f ield laboratories for the eff icient monitoring of paralytic shellf ish toxins. algal bloom detection kit is a novel gamma-based assay system that enhances the utilization of the radioactivity-based receptor binding assay (rba) technology by expanding its application for f ield/on-site monitoring of the paralytic shellf ish toxins, such as saxitoxin. detection by the assay is achieved through the use of more common, portable, and cheaper materials and counting equipment. regional and provincial agencies, especially in developing countries with no means to establish and maintain a central lab rba facility, will largely benef it from the technology, since the system can be promptly deployed to affected remote sites, with only a provisional laboratory as requirement. a philippine patent application has been f iled for this invention on 16 october 2 0 1 3 . amebiasis kit (rapid and accurate detection kit for amebiasis patients through salivary iga) w indell l. rivera angel ine odel ia c. concepcion alexander edward dy worldwide, deaths due to amebiasis may reach up to 100,000 annually. the infection is prevalent in areas where proper sanitation systems are lacking. in the philippines, prevalence rate is between 2% and 8%. current diagnostic tools for amebiasis all require stool samples, which are inconvenient to acquire and may even cause further 95 abstracts of up diliman’s registered ips contamination. one of these methods, stool microscopy or fecalysis, yields results within hours of submitting a sample but requires a no-meat diet of three to four days. other diagnostic methods have no dietary requirements and also provide results in a matter of hours; however, they are more expensive than stool microscopy. the present invention features an amebiasis kit that provides a more convenient and a cheaper tool to diagnose amebiasis infections. the kit only requires saliva samples rather than stool samples from patients. it is also self-contained and results can be visualized in about 3.5 hours. the kit has a sensitivity of 94.1% and specif icity of 97.6% with an overall diagnostic accuracy of 98% when compared with polymerase chain reaction. its cost is much cheaper than other e. histolytica detection kits currently available in the market. a philippine patent application has been f iled for this invention on 28 november 2008 (application no. 1-2008-000437; patent pending). rte noodles (retrogradation-resistant ready-to-eat (rte) asian noodles) ma. patricia v. azanza kristia may t. mar te maria lorina c. morales the invention relates to a process of producing pasteurized ready-to-eat (rte) cooked asian noodles comprising mung bean and rice-cornstarch noodles, which are resistant to retrogradation and shelf-stable during storage at ambient temperature for two months. moreover, they do not do not require further cooking, reheating, or hot water hydration. retrogradation control is attributed to the synergistic action of a mixture of humectants, oils, and acids added during the steeping and cooking of noodles. a philippine patent application will be f iled for this invention. abstracts of up diliman’s registered ips 96 motesar txp (motesar txp: w ireless sensor networks for monitoring temperature and relative humid ity in art spaces) ian christopher m. tolentino robin christian s. juson bernard james tan marc caesar r. talampas luis g. sison maricor n. soriano manual monitoring of an area’s microclimate is tedious, in that a person needs to periodically take readings at multiple points in an area at certain time intervals. an alternative would be to use dataloggers, which are devices equipped with sensors, memory, and a power source. they are usually deployed at certain points in an area, left for some time, and collected afterwards. though this approach is less tedious than manual monitoring, the readings it provides are not real-time. dataloggers typically have limited battery life, which also limits the amount of time that they can be deployed in an area. both manual and automatic monitoring using dataloggers can be expensive in terms of time and equipment cost. motesartxp allows a user to measure and monitor the microclimate of an area automatically, unobtrusively, and in real-time. since it employs power optimization schemes, the monitoring period is longer than that can be achieved by using dataloggers. motesartxp also allows the monitoring of more points within an area than that can be monitored by using dataloggers for the same price. a philippine patent application will be f iled for this invention. 97 abstracts of up diliman’s registered ips pyrodinium culture (development of semi-commercial scale (tank culture) of pyrodinium for neosaxitoxin and saxitoxin production) rhodora v. azanza lourdes j. cruz saxitoxin is a neurotoxin responsible for paralytic shellf ish poisoning (psp) that is prevalent during algal blooms or “red tides”. analysts require purif ied saxitoxin samples to calibrate test used to measure seawater toxin levels and shellf ish toxin concentrations. however, aside from its use in chemical analysis, saxitoxin is considered a schedule 1 substance by the chemical weapons convention. as such, export and import of the product is limited, and these trade restrictions are causing the decrease of available saxitoxin as calibration chemicals used for algal bloom monitoring and water safety analysis. in this invention, laboratory maintained strains of pyrodinium bahamense var. compressum are used to produce pyrodinium cells for semi-commercial scale production of neosaxitoxin and saxitoxin. the product is used strictly for chemical analysis and calibration of psp assays, as well as for saxitoxin research. pyrodinium culture is protected as a trade secret. seamoy™: seaweed-based air freshener marco nemesio e. montaño banaag glorioso-lajera many air fresheners contain substances that are harmful to the nervous system and respiratory tract. they can cause nausea, vomiting, dizziness, watery eyes, and chest pain. thus, healthier and natural alternatives to commercial air-fresheners are needed. seamoy™ , a seaweed-based air freshener, uses a minimal amount of chemicals and does not generate any hazardous waste. it makes use of whole seaweeds as base, instead of the extracted polysaccharide, thus reducing production costs. although the gel produced using the whole dried seaweed is not translucent in form, the gel strength remains the same as that of the carrageenan-based gel. abstracts of up diliman’s registered ips 98 the base allows a slow release of the essence, thus prolonging the air freshener’s shelf life. its residue is biodegradable, thereby making the product environmentfriendly. the addition of citric acid inhibits microbe growth. the freshener also has anti-clogging properties, which makes it appropriate for use in rooms with airconditioning units. finally, unlike the fresheners commercially available, seamoy™ can be divided into shapes and sizes for easier packing and utilization. seamoy™ is protected as a trade secret and is a registered trademark. up sablay (up in al ibata characters and device) carmen g. diaz de ventanilla antoinette b. hernandez rogel io l. jul iano jr. v irginia d. monje consuelo j. paz abraham p. sakili the up sablay is the off icial academic costume of the university of the philippines. the sablay uses the off icial colors of the university, maroon and green, as well as yellow gold, which stands for high standards of values and excellence. the colors, based on the pantone prosim color chart, are pantone 195 cvp (maroon), pantone 349 cvp (green), and pantone 138 cvp (yellow gold). the name of the university is represented by the indigenous letters o and m, originating from the indigenous alphabet called baybayin or katitikan, which are equivalent to the roman letters u and p, respectively. the curvilinear design called ukkil or ukit, which resembles a sprouting plant, signif ies life. the geometric designs (in zigzag and diamond patterns) are common design elements gracing the attires and functional objects of indigenous peoples from batanes to tawi-tawi. arranged continuously and rhythmically, these geometric designs highlight the diverse cultural communities in the philippines and the university’s pursuit of knowledge, cultural enrichment, and scientif ic advancement. the up sablay is a registered copyright since 2002. it is also a registered service mark with registration no. 4-2002-0010939 issued on 26 april 2006. 99 abstracts of up diliman’s registered ips lual – kiln firing as ar t and metaphor of birthing ma. rita badilla-gud iño having experienced f iring and birthing, the artist was convinced that a consonance exists between the two processes. in ceramic art, the ceramic pieces are the artworks. the process that leads to the production of these objects is conventionally considered as expedient to the form, but not the form itself. however, in light of the contemporary notions that process can also be formed, the artist was stimulated to pursue an assertion that a kind of kiln f iring can be created and framed as the art itself, because it is the embodiment of its metaphorical relation to birthing. kiln art is relatively new in the f ield of international ceramic arts. in the small local community of potters and ceramic artists, no one has pursued kiln art. this knowledge provided an artistic challenge and a heuristic opportunity for the artist. lual is made from clay, shaped into a sculptural kiln in the form of a bir thing woman. it was f ired inside kumot kiln, a removable kiln that was customized to transform lual from a clay sculpture to a ceramic kiln structure. while lual is being f ired, it is simultaneously f iring sibol, the sculptural clay babies with human and plant features into ceramic sculptures. thus, the f iring of lual utilizes a double kiln design concept. lual is a registered industrial design. takeaway packaging system (greener fast food packaging in the phil ippines) patricia ann t. mallare packaging plays an integral role in anything that we do. we interact with it every day. it protects the products that we buy. it ensures that these products will not be contaminated; likewise, it increases the products’ shelf-life. in turn, it protects us from harmful toxins that can get to us through the products we purchase. packaging therefore, in any shape or form, affects our lives directly and indirectly. like everything else around us, packaging needs to be linked with the ‘green’ lifestyle. this means that the environmental impact of the packaging system – from its abstracts of up diliman’s registered ips 100 production, which includes material selection and processing, to its disposal – needs to be considered. this project developed an eco-friendly takeaway packaging system for local fast food chains. the design features a new clamshell system and takeaway strap made from paper-based materials. a reusable strap made from recycled plastic is introduced to encourage customers to reuse it for another takeaway order. this new design addresses compliance to the ban on plastic and styrofoam packaging, and promotes a greener lifestyle among filipinos. the takeaway packaging system is a registered industrial design. 01_device polyclonal igg response to 23 kda antigen of e. histolytica 11 *corresponding author science diliman 20:1, 11-17 polyclonal igg response of balb/c mice to the 23 kda antigen of entamoeba histolytica herbert j. santos1, sheena marie b. maiquilla1, and windell l. rivera1,2* 1institute of biology, college of science, university of the philippines, diliman, quezon city 2molecular protozoology laboratory, natural sciences research institute, university of the philippines, diliman, quezon city telefax: (63-2) 920-5471 email: wlrivera@science.upd.edu.ph date submitted: january 22, 2008; date accepted: june 23, 2008 entamoeba histolytica is one of the most significant protozoan pathogens found in developing countries like the philippines. this intestinal parasite causes the disease amebiasis, which has a yearly average mortality of about 100,000 people worldwide. thus, it is essential to develop new diagnostic markers and possible treatment against this disease. the crude cell extract of e. histolytica, was used to induce polyclonal antibody response in mice. balb/c mice were given immunizations of the prepared crude e. histolytica antigens for a period of twelve weeks. indirect fluorescent antibody test showed the specificity of polyclonal igg in recognizing the cytosolic components of e. histolytica trophozoites. enzyme-linked immunosorbent assay was performed to determine the antibody titers in sera collected at various time intervals. antibody titers for the mouse serum taken 10 and 20 days after the third booster immunization were known to be 16,384 and 4,096 respectively. sds-page profile of the crude e. histolytica antigens revealed three bands with molecular weights of 23, 41, and 47 kda. western immunoblot results indicated that the polyclonal igg produced by mice targets the potentially novel 23 kda antigen from an axenic e. histolytica culture. key words: amebiasis, entamoeba histolytica, igg, immunoblot, indirect fluorescent antibody test, polyclonal antibodies abstract santos, h.j., et al. 12 science diliman 20:1, 11-17 glycoprotein galactose-inhibitable lectin (petri jr. et al., 1990a; ravdin et al., 1990). a 260-kda galactoseinhibitable adherence protein (giap) of e. histolytica is a mucosal antigen in naturally occurring invasive infection inhibited by monoclonal antibodies (kelsall et al., 1994), which could also be used to distinguish pathogenic from nonpathogenic amebae in culture (petri jr. et al., 1990b). an intermediate subunit (igl) of gal/ galnac (galactose n-acetyl-d-galactosamine) lectin, a 150-kda surface antigen has also been identified (cheng et al., 1998) and was well recognized in not only symptomatic but also asymptomatic patients with e. histolytica infection and that the carboxyl terminus of igl is a especially useful antigen for the serodiagnosis of amebiasis. it has been well known that there are a number of antigenic markers in e. histolytica that play crucial roles in its pathogenic effects. this study aimed to develop and partially characterize polyclonal antibodies in mice against a crude preparation of cellular antigens of e. histolytica. this could be a precedent for the development of new diagnostic markers and possible treatment against amebiasis. materials and methods animal care and handling four 7-week old balb/c mice were kept in separate cages in a well-ventilated animal house of the natural sciences research institute, university of the philippines, diliman, quezon city. the mice were provided with sterile water, food, and beddings. they were exposed to the normal daylight conditions. the cages were regularly cleaned during the immunization period. axenic cultivation of e. histolytica e. histolytica was cultured axenically using bi-s-33 medium (diamond et al., 1978). antibiotic solution containing 100 u/ml penicillin and 100 µg/ml streptomycin (1% of the volume of the medium) was also added. culture tubes were incubated at 35.5°c in slanted (5° from the horizontal) position. subculturing was done every 3-4 days. introduction amebiasis, an infection with the protozoan entamoeba histolytica, causes 100,000 deaths per annum, placing it second only to malaria based on mortality caused by protozoan parasites (who, 1997). e. histolytica is normally found in the large intestine, occasionally penetrating the intestinal mucosa and may disseminate into other organs such as the liver. initially, it was believed that the virulence of this parasite is variable due to the common observation that many people are apparently infected with it but never develop symptoms and spontaneously clear the infection. to answer this issue, emile brumpt in 1925 suggested the existence of two species of entamoeba – e. histolytica that causes the invasive disease and e. dispar which never causes disease. after which, years passed without any support to this view until the 1970s when studies began to arise giving way to the formal redescription of e. histolytica separating it from e. dispar in 1993 (diamond & clark, 1993). factors that trigger invasion are still not very clear. it was concluded in a study of mondal (mondal, et al., 2006) that ill-nutrition does not considerably contribute to e. histolytica-associated diarrhea. however, proteins associated with the virulence have already been identified prominent are the lectin that is responsible for the epithelial adherence of the parasite, pore-forming peptides lysing host cells, and secreted proteases degrading host tissues. clearly, the pathogenic mechanism of e. histolytica is a multifactorial phenomenon that occurs in 3 steps: adhesion, cytolytic and cytotoxic effect, and phagocytosis (horstmann et al., 1992). trophozoite surface antigens recognized by immune sera that have been identified may represent important components of the host-parasite interaction. the 29kda surface antigen (thiol-dependent peroxidase; eh29) of e. histolytica exhibits peroxidative and protective antioxidant activities (torian et al., 1990a, sen et al., 2007). several studies identified different surface antigens of different sizes and proposed these antigens as bases for differentiating isolates, whether pathogenic or nonpathogenic: 125-kda (edman et al., 1990), 30kda (blakely et al,. 1990), 96-kda (torian et al., 1990b), 170and 35-kda subunits of a surface polyclonal igg response to 23 kda antigen of e. histolytica 13science diliman 20:1, 11-17 cell counting trophozoites of e. histolytica were harvested during logarithmic phase of growth (3-4 days) and the cells were counted using hemocytometer. several culture tubes were pooled to have a total of 105 to 107cells/ml. preparation of cell lysates the collected trophozoites were centrifuged at 2,500 rpm for 3 min at 4°c. then, the medium was decanted. after which, the collected trophozoites were washed two times with 1x phosphate buffered saline (pbs). after washing, the trophozoites were resuspended in a solution containing 10mm tris-hcl (ph 7.5), 2mm phenylmethylsulfonyl fluoride (pmsf), and 1mm mgcl 2 . using a clean glass homogenizer, lysis of the cells was done (40 strokes) in ice-cold bath. the cell lysates were transferred into 1.5 ml microfuge tubes, centrifuged at 13, 200 rpm for 1.5 hr. finally, it was resuspended in 1x pbs prior to storage at -20°c. determination of protein concentration the concentrations of the cell lysates were measured using bradford reagent at an optical density (od) of 595 nm through a spectrophotometer (bradford, 1976). bovine serum albumin (bsa) was used as protein standard. several increasing concentrations of the bsa protein standard were measured through the spectrophotometer to generate a linear curve in relation to the absorbance readings. the crude antigen prepared was then quantified using the linear equation. immunizations blood samples from balb/c mice were collected from the tail vein. serum was separated from the whole blood by centrifugation (2,500 rpm for 10 min). then, it was stored in the freezer until use for antibody assay. prior to immunization, the mice were weighed individually. to prepare the antigen emulsion, complete freund’s adjuvant (cfa) was initially shaken to disperse the insoluble components. after which, the same proportion of antigen (212 µg/µl) in pbs to the cfa was added and both were mixed thoroughly until homogeneous by attaching two syringes to each other’s end and repeatedly forcing the mixture back and forth from one syringe to another. the cfa/antigen emulsion was injected into the mice intraperitoneally. after 1014 days, blood samples were again collected from the mice. serum was prepared as mentioned above and likewise stored in the freezer. indirect fluorescent antibody test (ifat) e. histolytica antigen slides were blocked with 1% bsa in pbs for 1 hr in a moist chamber at room temperature. afterwards, heat-inactivated serum samples in pbs at four-fold dilutions were added. the wells were washed 4x in 15 min using pbs. then, the anti-mouse igg conjugated with flourescein isothiocyanate (fitc) in 1% bsa (1:50) was applied to each well for 30 min at room temperature. the slides were then rinsed with pbs prior to viewing under a uv microscope (zeiss® axiovert). enzyme-linked immunosorbent assay (elisa) microtiter plates were coated with 10µg/ml of the e. histolytica antigen in 0.1 m carbonate buffer at 4°c overnight. the wells were washed with 0.05% tweenpbs (tpbs) thrice. then, blocking was done using 1% bsa in pbs at 37°c for 1 hr. subsequently, the plates were incubated with the serum in 4-fold dilutions at 37°c for 2 hr. washing using tpbs was again done thrice. alkaline phosphatase conjugated-anti-mouse igg in 1% bsa (1:1000) was then added at 37°c for 1 hr followed by washing with tpbs thrice. then, 1 mg/ml of the substrate, p-nitrophenylphosphate in 1m diethanolamine buffer (ph9.8) was coated at 37°c for 30 min. finally, the samples were read at 405 nm using an elisa plate reader (multiskan ex, thermo electron corp.). to test for cross-reactivity, the collected serum from mice immunized with e. histolytica antigen was reacted with blastocystis hominis antigen using elisa. western immunoblot prepared crude antigen was mixed with laemmli sample buffer and β-mercaptoethanol to make a 13 µg/µl loading sample into sodium dodecyl sulphatesantos, h.j., et al. 14 polyacrylamide gel electrophoresis (sds-page). proteins separated through the gel was transferred to a nitrocellulose membrane (nitrobind, osmonics inc.) then immunostained using serum (1:250) collected 1014 days after booster immunizations as primary antibody and horseradish-peroxidase-conjugated anti-mouse igg (1:1000) as secondary antibody. the reaction was visualized using diaminobenzine (dab) with 30% hydrogen peroxide. results and discussion approximately 0.8 µg/µl of the e. histolytica crude cellular antigen was injected into mice. accordingly, the antigen concentration was readjusted to the desired measurements for elisa (10 µg/ml), and western blot (13 µg/µl). ifat results showed fluorescent reactions of the mouse serum igg to the trophozoite antigen fixed on slides. the reaction was localized on the cytosolic component of the trophozoite (figure 1). immune response to intestinal and extraintestinal infection caused by e. histolytica was predominantly composed of igg class of immunoglobulin in sera. high titers had been reported even long after successful treatment (shetty et al. 1990). it was determined from the result of the elisa analysis that the titers for the sera collected 10 and 20 days after third boost were 1:16,384 and 1:4,096, respectively (figure 2). to determine cross-reactivity of the serum from the mice immunized with e. histolytica antigen, elisa was also done using blastocystis hominis antigen. b. hominis is a protozoan parasite normally found in the gastrointestinal tract and is commonly found in association with e. histolytica infections. as shown in figure 3, the serum antibody taken from the mice reacted with e. histolytica antigen coated on elisa plates at higher dilutions compared to b. hominis antigen. here we can conclude that the antibody produced is specific to e. histolytica and does not react with the protozoan, b. hominis. sds-page separated the crude antigen suspension into three distinct protein bands, with sizes 23, 41, and 47 kda. serum which was taken 20 days after the 3rd booster injection had a positive immunoblot on the 23 kda subunit of the crude e. histolytica cellular antigen (figure 4). the same serum contains highly specific antibodies against e. histolytica antigens since it did not react with any of the protein bands of candida albicans nor of pseudomonas aeruginosa from whole cells (data not shown). this protein is presumed to be a novel antigen since this is the first report of a 23 kda e. histolytica antigen. thus, it is warranted that further characterization of this antigenic protein reactive to the murine polyclonal igg be conducted. this is significant because this protein from an axenic culture may be another immunogenic marker which may be used for improving diagnosis and treatment of amebic infection. axenized strains of e. histolytica may lose their virulence to various degrees during adaptation to culture figure 1. a: phase contrast photomicrograph of e. histolytica trophozoites fixed on slides. b: immunofluorescence photomicrograph of the e. histolytica trophozoites reacted with the serum antibodies produced from mice. (colored images appear online.) science diliman 20:1, 11-17 b a b polyclonal igg response to 23 kda antigen of e. histolytica 15 figure 2. plot of the absorption at 405 nm versus reciprocal of dilution for mouse 4 sera taken before and after immunization (10 and 20 days after the third boost). without bacteria (blakely et al., 1990). however, this study was able to characterize a potentially novel antigen from axenic cultures of e. histolytica. the cellular antigen was found to be 23 kda by western immunoblot. immunoassays elisa and ifat were also utilized to determine the polyclonal igg serum antibody response of the balb/c mice to the e. histolytica crude antigen. it is suggested that further characterization of the igg produced be done by isotype analysis. acknowledgement we thank the natural sciences research institute (nsri) of the university of the philippines diliman for partially funding this research. references blakely, p., sargeaunt, p.g. and reed, s.l.1990. an immunogenic 30-kda surface antigen of pathogenic clinical isolates of entamoeba histolytica. j. infect. dis. 162: 949954. bradford, m. m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72:248-254. cheng, x.j., tsukamoto, h., kaneda, y., tachibana, h. 1998. identification of the 150-kda surface antigen of entamoeba histolytica as a galactoseand n-acetyl-d-galactosamineinhibitable lectin. parasitol. res. 84:632-639. science diliman 20:1, 11-17 santos, h.j., et al. 16 figure 3. test of cross-reactivity with b. hominis antigen. by elisa, od values were taken against increasing dilutions of the mouse serum. figure 4. western immunoblot analysis of the reactivity of polyclonal antibodies to trophozoites of e. histolytica. a: molecular weight marker; b: e. histolytica protein antigens (47, 41, and 23 kda); c and d: two lanes of serum igg antibody targeting the 23 kda antigen (arrows pointing to the 23kda e.histolytica specific antigen). diamond, l.s., and clark, g.c.1993. a redescription of entamoeba histolytica schaudinn, 1903 (emended walker, 1911) separating it from entamoeba dispar brumpt, 1925. j. euk. microbiol. 40:340-344. diamond, l.s., harlow, d.r. and cunnick, c.c. 1978. a new medium for the axenic cultivation of entamoeba histolytica and other entamoeba. trans. r. soc. trop. med. hyg. 72: 431-432. edman, u., meraz, m.a., rausser, s., agabian, n. and meza, i. 1990. characterization of an immune-dominant variable surface antigen from pathogenic and nonpathogenic entamoeba histolytica. j. exp. med. 172: 879-888. horstmann, r.d., leippe, m. and tannich, e. 1992. recent progress in the molecular biology of entamoeba histolytica. trop. med. parasitol. 43: 213-218. kelsall, b.l., jackson, t.g.h.f., gathiram, v., salig, s.b., vaithilingum, m., pearson, r.d. and ravdin, j.i. 1994. secretory immunoglobulin a antibodies to the galactoseinhibitable adherence protein in the saliva of patients with amebic liver disease. am. j. trop. med. hyg. 51: 454-459. science diliman 20:1, 11-17 a b c d 47 41 23kda 200 97 66 44 29 17polyclonal igg response to 23 kda antigen of e. histolytica 17 mondal, d., petri, w.a. jr., sack, r.b. , kirkpatrick, b.d. and haque, r. 2006. entamoeba histolytica-associated diarrheal illness is negatively associated with the growth of preschool children: evidence from a prospective study. trans. r. soc. trop. med. hyg. 100: 1032-1038. petri jr., w.a., snodgrass, t.l., jackson, t.f.h.g., gathiram, v. , simjee, a.e., chadee, k. and chapman, m.d. 1990a. monoclonal antibodies directed against the galactosebinding lectin of entamoeba histolytica enhance adherence. j. immunol. 144: 4803-4809. petri jr., w.a., jackson, t.f.h.g.., gathiram, v., kress, k., saffer, l.d., snodgrass, t.l., chapman, m.d. , keren, z. and mirelman, d. 1990b. pathogenic and nonpathogenic strains of entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin. infect. immun. 58: 1802-1806. ravdin, j.i., f.h.g. jackson, w.a. petri jr., c.e. murphy, b.l.p. ungar, v. gathiram, j. skilogiannis, and a.e. simjee, 1990. association of serum antibodies to adherence lectin with invasive amebiasis and asymptomatic infection with pathogenic entamoeba histolytica. j. infect. dis. 162: 768772. sen, a., chatterjee, n.s., akbar, m.a., nandi, n. and das, p. 2007. the 29-kilodalton thiol-dependent peroxidase of entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress. eukaryot. cell 6: 664-673. shetty, n., nagpal, s., subba rao, p.v., and schroder, h. 1990. detection of igg, iga, igm, and ige antibodies in invasive amoebiasis in endemic areas. scand. j. infect. dis. 22: 485-491. torian b.e., flores, b.m., stroeher, v.l., . hagen, f.s., stamm, w.e.. 1990a. cdna sequence analysis of a 29-kda cysteinerich surface antigen of pathogenic entamoeba histolytica. proc. natl. acad. sci. usa. 87: 6358–6362. torian, b.e.,reed, s.l. , flores, b.m., creely, c.m., coward, j.e., vial, k. and . stamm, w.e. 1990b. the 96-kilodalton antigen as an integral membrane protein in pathogenic entamoeba histolytica: potential differences in pathogenic and nonpathogenic isolates. infect. immun. 58: 753-760. world health organization, 1997. entamoeba taxonomy. bull. who. 75: 291-292. science diliman 20:1, 11-17 49-54_valencia w corrections 49 international scientific productivity science diliman (january-june 2004) 16:1, 49-54 international scientific productivity of selected universities in the philippines marshall n. valencia psychology department, de la salle university-manila 2401 taft avenue, 1004 metro manila, philippines tel. no.: (632)524-4611 local 142; fax no.: (632)526-5915 e-mail: valenciamn@dlsu.edu.ph, marshall@bhanvad.com date received: 29 january 2004; date accepted: june 30, 2004 abstract this paper presents a survey of the productivity, in terms of international scientific publications, of 465 phds in the science and engineering colleges of selected research universities in the philippines. it covers the period 1998-2002. publication counts are based on research articles listed in the science citation index of the institute for scientific information (isi). results indicate that the average productivity of the faculty surveyed is less than one (1) international publication in five (5) years. only one of the academic units included in the survey was able to achieve the world-class benchmark for research excellence, which is at least one international publication per faculty member per year. keywords: international scientific publications, scientific productivity, isi knowledge generation (research) is a primary mission, along with knowledge dissemination (teaching) in most reputable universities. the research function is vital to a university’s reputation and pursuit of academic excellence. in the philippines, there are more than 1,500 higher education institutions. however, almost all of these are mainly teaching institutions. only a handful could be truly considered as research universities. a bibliometric search of the international scientific publications using the institute for scientific information (isi) database indicates that in recent years, an overwhelming majority of international scientific publications from universities in the country came from only two institutions: the university of the philippines (up) and de la salle university (dlsu). the up has long been the reputed premiere research university in the country. however, compared with other universities abroad, even the top in the country dims in comparison as reflected in asiaweek’s survey of asia’s best universities (bacani, 1998, 1999, & 2000). in various studies using isi publication data as a measure of science and technology capability of nations, institutions, and individuals, the philippines as a country, and up as an institution lag way behind international standards. this paper presents a survey of the productivity, in terms of international scientific publications, in selected science and engineering departments of the two lead research universities of the philippines. in particular, it focuses on the productivity of science and engineering phd faculty members of up and dlsu for the period 1998-2002. the survey provides recent and comprehensive research productivity information at the individual and department level. it reflects local benchmark information and it could be useful for international benchmarking purposes. 50 valencia methodology the survey population the survey covered all full time phd faculty members (as of the school year 2001-2002) in the science and engineering departments of the university of the philippines (diliman and los baños campuses) and the de la salle university (manila campus). a total of 465 faculty members from the two universities were included. their academic rank distributions are as follows: 20% full professors, 42% associate professors, and 38% assistant professors. table 1 details the academic rank distributions by university. the list of up faculty members was obtained from the personnel database maintained by the human resource development office of up diliman and up los baños. the dlsu list came from the “research@dlsumanila, 2001-2002” of the university research coordination office of dlsu-manila. source of publication data publication data used in this survey came from the science citation index (sci), which is a database produced and maintained by the institute for scientific information (isi). the isi is a database publishing company that provides the most comprehensive coverage of the world’s most important and influential research conducted throughout the world (testa, 1997). it covers more than 16,000 international journals, books, and proceedings in the sciences, arts, and humanities. it includes more than 15,000,000 papers published since 1945 (garfield & welljams-dorof, 1992). inclusion of a journal in the isi database requires an extensive evaluation process. factors such as the journal’s publication standards, editorial board membership, internationality of authorship, and citation data are considered. measure of publication productivity publication productivity was measured at the individual and department level. productivity of individual faculty members was determined using the following ratio: in effect, the measure was the average number of individual isi publications per year for the 5-year period (1998-2002). a faculty member was given an equivalent of 1 publication count for each isi article where his or her name appeared, regardless of whether the individual concerned was a sole or co-author. the following ratio was used for determining the department level productivity: note that in computing the average productivity, the two ratios may yield slightly different values when a single publication has more than one author coming from the same department. the period covered was chosen to reflect the most recent isi data available during the time of data no. of isi indexed publications of individual faculty members for the period 1998-2002 5 years number of isi indexed publications (1998-2002) number of phd faculty members in the department 5÷ table 1. number of phd faculty members and academic rank distributions: up diliman campus, up los baños campus, and dlsu manila campus academic rank university professor associate professor assistant professor total up diliman up los baños dlsu manila total 57 (30%) 19 (9%) 18 (30%) 94 (20%) 65 (35%) 90 (41%) 39 (65%) 194 (42%) 65 (35%) 109 (50%) 3 (5%) 177 (38%) 187 218 60 465 51 international scientific productivity gathering. the beginning period, 1998, complements a previous research productivity survey done by lim and saloma (1998) which covered the period september 1988 to may 1998. database search procedure the number of isi publication counts for each faculty member was obtained by accessing the on-line version of isi’s web of science database, in particular the science citation index. an exhaustive search was done by retrieving all entries within 1998-2002 using the keyword “philippines” in the author’s affiliation search field. the search was restricted to cover only journal articles. from the entries generated, the names of the faculty members were manually searched and their publication counts per year were tallied. for the department level analysis, the publications identified for each individual faculty member were sorted to identify duplications due to multiple authors coming from the same department. results the overall individual average research productivity of the 465 faculty members was 0.20 for the period 1998-2002. this is equivalent to an average of 1 international publication per faculty member in 5 years. however, from a department level analysis, productivity is slightly lower with an average of 0.17 isi publication per faculty member. table 2 presents the detailed international publication productivity per area and per university. the basic science departments are the most productive in terms of international publications. in particular, departments in the college of science of up diliman produced the highest number of international publications per faculty member, per year at 0.32. this productivity level is equivalent to an average of slightly less than two international publications per faculty member for the period 1998-2002. the frequency distribution of individual productivity levels indicated that majority (62.4%) of the surveyed faculty members had no international publications at all during the period covered (table 3). only 4.3% were able to publish at least one isi publication per year, which is considered the “world c l a s s ” b e n c h m a r k f o r e x c e l l e n c e i n r e s e a r c h productivity. the most productive individual faculty member came from the national institute of physics of up diliman with an average of 7.8 isi publications per year or a total of 39 isi publications for the 5year period covered. tables 4, 5, and 6 detail the international publications productivity at the departmental level for the science, engineering, and agriculture colleges included in the survey. the best performing department was the marine science institute of up diliman with an average of 1.15 isi publications per year per faculty member. it is table 2. international publications productivity (1998-2002) of the university of the philippines (diliman and los baños campus) and de la salle university (manila) field / area up diliman up los baños dlsu overall basic science engineering statistics agriculture forestry veterinary medicine environmental science overall 137 42 5 3 187 0.32 0.05 0.04 0.20 0.25 no. of phds productivity no. of phds productivity no. of phds productivity no. of phds productivity 56 21 5 88 29 10 9 218 0.14 0.14 0.04 0.17 0.04 0.18 0.04 0.13 38 22 60 0.11 0.09 0.10 231 85 10 88 29 10 12 465 0.24 0.10 0.04 0.17 0.04 0.18 0.08 0.17 52 valencia perhaps the only academic department in the country that has reached the world class standard of at least one international publication per faculty member, per year. the national institute of physics of up diliman came next with a notable average of 0.62 isi publications per faculty member per year. except for the molecular biology units, all other academic units included in the survey averaged less than 0.40 publications per faculty member, per year or less than two international publications per faculty member for the entire 5-year period covered. possible sources of error some of the faculty members included in the survey may have been affiliated with a foreign institution in some years of the period covered. therefore, there may be cases of under-reporting individual productivity because the publication counts for this survey were based on isi entries containing the word “philippines” in the author’s affiliation field. there were also rare typographical errors in the isi entries that may have affected the publication counts. as an example, some of the entries retrieved had foreign university affiliations but the country indicated was philippines. in the same manner, it is also possible that a philippine institution entry had been erroneously encoded with a different country address. in this case, the entry would not have surfaced using the search procedure for this study. it should also be noted that some of the articles had multiple authors coming from different departments, colleges, or even universities. therefore, in these few cases, the articles were counted under two or more departments. discussion international scientific publications in the philippines are produced mostly by international organizations situated in the country such as the international rice research institute. a significant proportion also comes from academic institutions. table 4. international publication productivity (1998-2002) of the science departments of up (diliman and los baños) and dlsu (manila). dlsuup dilimanunit / department up los baños biology chemistry mathematics physics marine science geology molecular biology & biotechnology environmental science statistics overall 0.07 (n=20) 0.13 (n=30) 0.07 (n=27) 0.62 (n=24) 1.15 (n=13) 0.28 (n=18) 0.44 (n=5) 0.20 (n=3) 0.04 (n=5) 0.31 (n=145) 0.18 (n=25) 0.11 (n=18) 0.05 (n=11)* 0.50 (n=2) 0.04 (n=9) 0.04 (n=5) 0.12 (n=70) 0.15 (n=11) 0.13 (n=11) 0.10 (n=10) 0.03 (n=6) 0.11 (n=38) *the math and physics faculty are under one department. table 3. frequency distribution of international publication productivity (1998-2002) of science and engineering phds in up (diliman and los baños) and dlsu (manila). publication productivity frequency percentage 0.00 0.20 (1 publication in 5 years) 0.40 0.60 0.80 1.00 (5 publications in 5 years) 1.20 1.40 2.40 3.00 (15 publications in 5 years) 3.40 7.80 total 290 85 31 24 14 5 7 4 1 2 1 1 465 62.4 18.3 6.7 5.2 3.0 1.0 1.5 0.8 0.2 0.4 0.2 0.2 100.0 53 international scientific productivity table 5. international publication productivity (1998-2002) in the engineering departments of up (diliman and los baños) and dlsu (manila). dlsuup diliman unit / department up los baños chemical engineering civil engineering electronics & communications engineering industrial/manufacturing engineering mechanical engineering agricultural engineering geodetic engineering material engineering engineering sciences overall 0.05 (n=8) 0.04 (n=11) 0.04 (n=9) 0 (n=1) 0 (n=6) 0.20 (n=1) 0.10 (n=4) 0 (n=2) 0.04 (n=42) 0.16 (n=5) 0 (n=2) 0.16 (n=14) 0.14 (n=21) 0.12 (n=10) 0.10 (n=2) 0 (n=2) 0.07 (n=3) 0.08 (n=5) 0.09 (n=22) however, as pointed out earlier, the overwhelming majority of publications from academic institutions in the philippines came from the two research universities included in this survey. in general, the survey results indicated that even the top science and engineering academic departments of the country were performing way below satisfactory levels in terms of research productivity. in contrast with some of the reputable asian universities, indeed, we dim in comparison. the university of tokyo, as a whole, had an average of 2.10 international publications per faculty member based on the 1998 asiaweek survey of asia’s best universities (bacani, 1998). the australian national university, kyoto university, and the chinese university of hongkong had productivity levels ranging from 1.34 to 1.48. to be more specific with the benchmarks, consider the following figures 10 years ago from the national university of singapore: although the measure of publication productivity employed in this survey is very strict and limited, most academic scientists would argue that this has got to be the major indicator of research performance especially in the hard sciences and engineering. for the scientific community, publication is the culmination of a research process. as lacanilao (1999) puts it, proper publication means publishing results in a primary journal that is adequately peer-reviewed and accessible. adequate peer review ensures the correctness of methods and journal accessibility allows verification of results. it is in this regard that publications in journals that are indexed in the isi database are now used as standard indicators of science and technology performance of nations, institutions, or individuals. department no. of phds international publications no. per phd chemistry physics zoology mathematics botany total 36 31 23 40 24 154 182 113 36 42 14 389 5.08 3.65 1.57 1.07 0.67 2.53 (source: lacanilao, 1999) table 6. international publication productivity (1998-2002) in the college of agriculture of up los baños. unit / department agronomy dairy training & research institute entomology horticulture institute of animal science institute of food science & technology national crop protection center post harvest training & research center plant pathology soil science overall productivity 0.22 0 0.20 0.18 0.08 0.20 0.07 0.10 0.20 0.30 0.17 (n=10) (n=3) (n=11) (n=16) (n=16) (n=7) (n=6) (n=2) (n=9) (n=8) (n=88) 54 valencia there are substantial research activities on-going in the major research universities in the philippines. however, it seems that very few research projects find their way to being published internationally. to a large extent, lack of funding, inadequate research facilities, and heavy teaching loads are serious impediments to research productivity of academic scientists in the philippines. but perhaps beyond these limitations, the research culture in philippine universities is such that publication is not the targeted culmination of a research activity. as noted by lacanilao (1999), most research projects in the university of the philippines end up only as project reports, presentations at conferences, or published in “grey” literature. it can be observed that in recent years, the university of the philippines and de la salle university have been actively addressing issues of research productivity among their faculty members. administrations have been sending out the message clearly that they give a premium to research productivity of their faculty members. the two universities have been implementing more deliberate measures in improving research support systems and instituting rewards and incentive schemes. we could therefore most likely expect improving trends in their scientific productivity. but for now, the kind of research culture necessary for philippine universities to achieve world class standards is definitely not well entrenched yet even in the premiere research university of the country. it would still take a lot more of continuous organizational reorientation and realignment of systems and policies in order to foster the desired conditions. culture takes years or even generations to become fully imbibed in the system. references bacani, c., 1998. secrets of success. asiaweek. may 15: 3659. bacani, c., 1999. asia’s top universities. asiaweek. april 23: 46-65. bacani, c., 2000. time of ferment. asiaweek. june 30: 3849. garfield, e. & a. welljams-dorof, 1992. citation data: their use as quantitative indicators for science and technology evaluation and policy-making. science and public policy. 19(5): 321-327. lacanilao, f., 1999. science in the university of the philippines. diliman review. 47(2): 11-18. lim, m. & c. saloma, 1998. scientific publications by the faculty of the college of science, up diliman: september 1988 to may 1998. science diliman. 10(1): 1-6. testa, j., 1997. the isi database: the journal selection process. essay prepared by the editorial department, publisher relations, isi. �‰µ\nîuk�›âä 04_regis comparison of pollen abortiveness in four weed species treated with mercuric chloride emelina g. regis*a and daniel a. lagunzadb adirector, institute for environmental conservation and research (inecar) ateneo de naga university, naga city, philippines bassociate professor, institute of biology university of the philippines, diliman, quezon city, philippines a tel. no. (054) 473-84-47 loc. 2217; e-mail: egregis@sili.adnu.edu.ph btel. no. (02) 920-53-01 loc.6536; e-mail: dal@nib.upd.edu.ph abstract pollen grain abortiveness due to mercury exposure was investigated in four species of weeds, namely cleome rutidosperma mart., commelina diffusa burm.f., ludwigia micrantha (l.) hara, and stachytarpheta jamaicensis (l.) vahl. all four species tested showed mean pollen grain abortion rates significantly higher than those of their unexposed cohorts. pollen grain abortion was manifested by reduced size and staining deficiencies. scheffe’s test for variability indicated that higher mercury concentrations are required to effect changes in pollen grain abortiveness. the weed species tested can possibly be used as bioindicators of mercury pollution. because of the plant’s ability to absorb mercury, these species can also be considered as possible bioremediators. key words: pollen abortiveness, mercury, bioindicator, cleome rutidosperma, commelina diffusa, stachytarpheta jamaicensis, ludwigia micrantha introduction weeds are sturdy plants that are able to survive in a variety of adverse conditions such as abandoned patch, wasteland, roadside, footpaths, and trails. in the philippines, weeds abound in a variety of places both rural and urban, including agricultural and industrial areas, cemeteries, and waste dumps. they are known to possess growth rates higher than the crops with which they compete. even in mining areas contaminated with mercury, weeds have been found to thrive (regis, 1999). in the studies of murin (1995) and micieta & murin (1996), the weeds were included as indicators of genotoxicity of polluted environment because of their cosmopolitan nature and because their microspores respond to heavy metal pollution by aborting. of the forty one (41) species tested for genotoxicity, the researchers found thirty (30) that showed significant pollen grain abortion. polluted environments in the philippines include gold mining areas. when the area is favorable to acid mine drainage, heavy metals can come into solution (jackson & jackson, 1996). mercury, a heavy metal used during the amalgamation process in alluvial gold extraction, can be introduced in the mining areas (kim & kim, 1996; jackson & jackson, 1996). the process of recovering gold from the amalgam is by heating it in an open pan, causing mercury to vaporize. consequently, the areas within the vicinity of the gold mines become contaminated not only through water runoff and mine tailings but also through dry deposition. thus, there is a 21science diliman (january-june 2002) 14:1, 21-27 *corresponding author need to monitor this kind of environmental problem to safeguard the health of people and other living organisms. monitoring pollution may be done through pollen studies (murin, 1995; micieta & murin, 1996). for instance, high pollen abortive tendency above the normal level of 5% (murrin, 1995) can be used as indicator of mercury pollution of contaminated environments. the studies of murin (1995) and micieta & murin (1996) suggest that the abortive tendencies exhibited by the pollen grains may be due to genetic and physiological damages. these damages were manifested as (a) larger than normal size, (b) altered form, and (c) staining deficiency. chapman & mulcahy (1997) reported that pollen grain abortion is due, at least in part, to nondisjunctional meiotic events because of nucleocytoplasmic incompatibility in experimented hybrids. other researchers provided additional observation on pollen grain abortion. larson (1965) for instance observed cytoplasmic death that occured in aborting pollen grains. the cytoplasm became compartmentalized, and this compartmentalization may be due to a modified endoplasmic reticulum (er). this occurrence was also observed in injured maize root tip cells. the event was followed by organelle degeneration. in this study, the degree of pollen grain abortiveness as a result of mercury exposure was studied in four species of weeds, namely, cleome rutidosperma mart., commelina diffusa burm.f., ludwigia micrantha (l.) hara, and stachytarpheta jamaicensis (l.) vahl, to identify possible bioindicators of mercury pollution. possible bioindicators will be especially useful in remote areas where testing facilities and services are inaccessible and/or expensive. furthermore, results of this study can help identify promising phytoremediators for mercury pollution. materials and methods preliminary investigation a preliminary investigation of at least twenty four weeds growing in comparable sites in camarines norte and in naga city was conducted to be able to select the target weed species for the experiment. the two sites were initially compared as to their weed composition. based on the vegetational analysis conducted, sorensen’s coefficient of similarity (brower, zar, & von ende, 1990) was computed at 66.67% during the dry season and 70.69% during the rainy season. in the first site in labo, camarines norte, gold mining activities were mostly illicit and miners used mercury for amalgamation. chemical analyses of soil taken from this site showed an average of 0.747 mg/kg mercury content (regis, 1999). in the second site, a rural area in naga city, there was no known history of gold mining activities conducted and chemical analyses of soil recorded only an average of 0.047 mg/kg mercury content. of the twenty four weeds examined, four showed pollen grain abortion above the normal 5% abortiveness (murin, 1995). other characteristics deemed convenient for the selection of possible bioindicator species were: (a) anther can be easily distinguished in the flower, (b) pollen grains were large enough to be seen with ease under 100x magnification of the compound microscope, (c) are present in the two comparable sites chosen during the preliminary investigation, and (d) could be easily grown in hydrophonics set-up. hydrophonic set-up the experiment was conducted inside a greenhouse measuring 75 m2 located within the school campus of ateneo de naga university. plants were raised in liquid media using the hydrophonic method described by dushenkov et al. (1995) and hershey (1995). a modified hoagland solution was used in the preparation of the liquid media (hershey, 1995). for each species studied, sixty hydrophonics bottle were filled with 1 liter nutrient solution each. ten of these served as control – no hgcl 2 was added. appropriate amounts of hgcl 2 were added to the nutrient solution in the other bottles to make the following concentrations: 0.1, 0.3, 0.9, 2.7, and 8.1 ppm. ten bottles per species were used for each of these concentrations. full strength hoagland solution was used in growing c. rutidosperma and s. jamaicensis while half strength solution was used for l. micrantha and c. diffusa because these two latter species became vegetative and rarely flowered in full strength hoagland solution during the first trial. regis and lagunzad 22 two days prior to the preparation of the hydrophonics bottle, young plants of the species for study were collected from the rural area of naga city (second site mentioned in the preliminary investigation). these plants were given preliminary treatment before being transferred to purely liquid media. they were first planted in clean sand placed in make-shift clean plastic containers and watered with distilled water. after two days, the plants were transferred into the hydrophonics bottles. the hydrophonics bottles were arranged on six experimental tables. only one type of treatment was placed in each table. each bottle was covered with black plastic to prevent light from entering the lower part of the plant. the bottle was also fitted with a hose which was attached to an aerator. once the young plant was securely set in the bottle, another black plastic covered the set-up to the point where the stem was inserted through a hole in the bottle cap. in addition, each table was provided with a transparent plastic cover to reduce contamination from other sources, such as dust carried by strong winds and splashes from the rain. the cover was also provided with slit openings to allow interchange of air. every day, the aerators were switched on for 10 hours, from 7:00 am to 5:00 pm. for plants raised in full-strength nutrient solution, the various preparations (control and with pollutant) were continually added as soon as the level of solutions fell below the designated 1-liter level. for those raised in half-strength nutrient solution, the exposure to the pollutant was only done once a week and only the nutrient solution was added daily to each set-up when the solution inside the bottles fell below the designated 1-liter level. every two weeks, the solutions were replaced with new stock. as soon as the plants bore flowers, flower buds just before anthesis were collected (usually in the early morning or late afternoon). the buds were fixed in ethanol-acetic acid (3:1 v/v). after 24 hours, the solution was replaced with 70% ethanol as preservative, following the methods of murin (1995). after having collected enough flower buds (about 50 samples), the plants were removed from the hydrophonics bottles, washed, and air-dried. during times of high humidity or rainy days, drying of the samples was done inside a drying oven maintained at 40o-50oc for 12 hours or for several days until the tissues were dried crisp. the dried samples were then pulverized. two grams of each sample were brought to the natural science research institute, university of the philippines (up nsri) for chemical analysis of total mercury content using atomic absorption spectrometer or aas (bouchard, 1973). pollen grain analysis thirty flower buds were selected at random from the preserved samples. they were washed in water, placed on a glass slide, and with the aid of a stereomicroscope, one or two anthers, depending upon the species, were separated from the flower. then, the anther was treated with a drop of stain (1% aniline blue in lactophenol), cut, and crushed carefully using two fine-point needles. a minute drop o f h c l w a s a d d e d t o t h e p r e p a r a t i o n o f c . rutidosperma and l. micrantha in order to enhance the stain while h 2 so 4 was used for s. jamaicensis to remove the outer thin cover and enhance the stained protoplasm. no acid was added to c. diffusa because of the tendency of its pollen to burst. a coverslip was then placed over the glass slide and after a few hours (6 hours at the least), the pollen sample was examined under the microscope. the counting of normal and abortive pollen grains followed the method of micieta & murin (1996). this was done with the aid of a compound microscope, a mechanical counter, and a grid measuring 0.5 in. x 0.5 in. (0.25 sq. in). ten grids were used for both c. rutidosperma and c. diffusa while 20 grids were used for l. micrantha. a total count was made for s. jamaicensis because there are only two anthers per flower, and the number of pollen grains of this species is small. according to murin (1995), normal pollen abortiveness is 5%. above this value, the abortive tendency may be attributed to other factors s u c h a s m e r c u r y c o n t a m i n a t i o n , w h i c h w a s introduced in the experiment. results and discussion based on the results of the chemical analyses by up nsri, the total mercury content of all the comparison of pollen abortiveness 23 experimented plants (composite of leaves, stems, roots, flowers) treated with 0.3 ppm hgcl 2 were as follows: c. rutidosperma 7.6 ppm, l. micrantha 17 ppm, and stachytarpheta jamaicensis 18 ppm. for c. diffusa, mercury accumulated was 56.6 ppm at 0.9 ppm treatment. pollen grain abortion normal and aborted pollen grains are shown in figs. 1, 1a, 2, 2a, 3, 3a, and 4. the figures reveal that aborted pollen grains exhibit two types of pollen abnormalities – staining deficiencies and reduction in pollen sizes. staining deficiencies are manifested as: (a) protoplasm seemed absent due to the pollen’s inability to absorb stain, and (b) the protoplasm seemed undeveloped because only part of it is distinct. figs. 1a, 2a, and 3a present the structures of the aborted pollen under oil immersion objective (1000x). aborted pollen grains of s. jamaicensis are large, so that they are easily distinguishable even at low magnification (fig. 4). moreover, none exhibited larger sizes than normal although murin fig. 1. pollen grains of cleome rutidosperma mart. viewed at 100x magnification of the compound microscope. aborted pollen grains do not stain dark. normal (nrm) pollen grains measure 10m to 12m in diameter whereas aborted (abt) pollen measures 8m or less. fig. 1a (inset). normal (nrm) and aborted (abt) pollen grains of cleome rutidosperma mart. viewed under the oil immersion objective at 1000x magnification of the compound microscope. the thick exine is distinct. nrm abt abt exine nrm 10µ nrm abt exine10mmmmm abt nrm (1995) and micieta & murin (1996) have reported this type of abnormality in some species growing in areas contaminated with several kinds of heavy metals. the results of pollen grain analyses are presented in table 1. the mean average pollen abortion in all the four species shows abortive tendencies above the controls. anova analysis under the sas system suggests a significant difference between the control plants and those raised under various concentrations of hgcl 2 . scheffe’s test for variability also indicated a reduction in groupings involving concentrations that elicited pollen abortion, but the results did not differ much from their immediate neighbors. for instance, results obtained for commelina diffusa showed that there is no significant difference in pollen abortiveness at concentrations of 0.1 ppm, 0.3 ppm, and 0.9 ppm hgcl 2 (table 1). also, results obtained for the concentration of 2.7 ppm are not significantly different from 8.1 ppm. thus, instead of six groups being significantly different from each other (including the control), only 3 groupings were found to be significant: (a) control; (b) 0.1, 0.3, fig. 2. normal (nrm) and aborted (abt) pollen grains of commelina diffusa burm. f. viewed at 100x magnification of the compound microscope. size ranges of normal pollen is between 20µ to 30µ and aborted pollen, 15µ to 20µ long. fig. 2a (inset). normal (nrm) and aborted (abt) pollen grains of commelina diffusa burm. f. viewed under the oil immersion objective at 1000x magnification. the undeveloped protoplasm can be seen in the aborted pollen. n r m a b t nrm abt n r m a b t 5 µ e x i n e nrm abt exine 5mmmmm regis and lagunzad 24 and 0.9 ppm cluster; and (c) 2.7 and 8.1 ppm cluster. thus, it can be concluded that changes in pollen abortiveness require higher increases in the concentration of pollutants to elicit a response. for cleome rutidosperma, all the treatments were not significantly different from each other in terms of pollen abortiveness. only the control was significantly different from the rest of the treatments. it can be concluded, therefore, that any concentrations above the natural concentration of pollutants in plant tissues will elicit abortion of pollen grains. scheffe’s test for ludwigia micrantha on the other hand, revealed that results obtained for control plants are not significantly different from those treated with 0.1 ppm and 0.9 ppm. these were, however, significantly different from those obtained at 2.7 ppm and 8.1 ppm (table 1). for this species, pollen abortiveness is more distinct when fig. 3. normal (nrm) and aborted (abt) pollen grains of ludwigia micrantha (l.) hara viewed at 100x magnification of the compound microscope. the size of normal pollen is about 36µ while aborted ones measure 20µ in diameter. fig. 3a (inset). normal (nrm) and aborted (abt) pollen grains of ludwigia micrantha (l.) hara viewed under the oil immersion objective at 1000x magnification of the compound microscope. abtnrm abt nrm abt 17mmmmm fig. 4. normal (nrm) and aborted (abt) pollen grains of stachytarpheta jamaicensis (l.) vahl. viewed at 100x magnification of the compound microscope. the pollens were treated with small amount of acid to make the protoplasm distinct from the exine. n r m a b t e x i n e 5 0 µ nrm abt exine 50mmmmm table 1. mean % pollen grain abortion in flowers from plants under various treatments of hgcl 2 (in ppm) comparison of pollen abortiveness 25 cleome rutidosperma commelina diffusa ludwigia micrantha stachytarpheta jamaicensis hgcl 2 treatment (ppm)+ control+ 0.30.1 0.9 2.7 8.1species 2.47b 2.22d 3.51c 2.89c 9.74a 5.21c 9.83cb 11.73cb 8.54a 6.59bc * 17.61b 10.27a 6.83abc 9.89cb 19.29b 9.50a 9.57a 12.27b 21.53b 11.44a 9.30ab 11.84b 41.71a * results were not included due to observed microbial infection during the experiment. + scheffe’s test for variable: means with the same letter (shown as superscript) are not significantly different from each other; n = 30. the pollutant is at higher concentrations, which are 2.7 ppm and above. stachytarpheta jamaicensis exhibited 3 groups in the scheffe’s test for variable. group a at 8.1 ppm corresponds to the highest concentration used in the experiment. group b, which includes 0.3 ppm, 0.9 ppm, and 2.7 ppm, showed no significant differences in their variability. group c covered both the control and 0.1 ppm hgcl 2 . it can be concluded, therefore, that the critical level by which pollen abortiveness becomes significant is at 0.3 ppm and above. below this concentration, pollen abortiveness is not much different from that of the control. the absence of protoplasm in some aborted pollen grains of the experimented plants may be due to physiological damage (micieta & murin, 1996). it was also observed that aborted pollen grains have thicker exines. this occurrence was seen in c. rutidosperma (fig. 1a) and s. jamaicensis (fig. 4). this observation was similar with the study of rowley et al. (1997) wherein aborted pollen grains had thicker exines than the mature normal pollen grains. they concluded that the thicker wall represented early stages of development of the microspore, but its death prevented further development of the exine. bioindicators and bioremediators from among the four species studied, stachytarpheta jamaicensis can be considered as the best bioindicator of mercury pollution because its responses to the various treatments are clearly delineated in the scheffe’s test for variables. when determining only the presence of the pollutant, cleome rutidosperma may be used initially. the two other species are highly variable in their responses to contamination. however, they can also be used only to determine the presence of contamination in the absence of s. jamaicensis and c. rutidosperma. this study was also able to discover that the four species experimented have the ability to absorb mercury in their tissues. thus, they may be considered as potential bioremediators of mercury pollution in contaminated sites. conclusion based on the results presented above, the accumulation of mercury in plants at higher concentration above the critical level of 0.3 ppm for plant tissues (pfeiffer et al., 1988) resulted in pollen grain abortion. pollen abortiveness has also been confirmed by the results of statistical analyses. the variability between treatments, as well as within the samples examined per laboratory treatment showed significant differences between the control and contaminated materials. furthermore, pollen grain abortion was manifested by abnormalities in the structure of the pollen grains through staining deficiencies and reduction in sizes. these results suggest that the experimented species can be used as bioindicators of mercury pollution. likewise, the ability of the experimented plants to absorb mercury also imply that these species can be possible bioremediators of mercury-polluted areas. acknowledgments in achieving the objectives of this study, the authors wish to express gratitude to the following for their valuable assistance: dr. titos a. quibuyen of the institute of chemistry and dr. joselito p. duyanen of the national institute of geological sciences, both from the university of the philippines, diliman, quezon city; dr. marilou g. nicolas of the university of the philippines, manila; dr. gustav murin of comenius university, slovakia; the fund for assistance to private education (fape); and the late rev. raul j. bonoan, s.j., former president of the ateneo de naga university, naga city. references bouchard, a., 1973. determination of mercury after room temperature digestion by flameless atomic absorption. atomic absorption newsletter. 12(5):115-117. brower, z. & von ende, 1990. field and laboratory methods for general ecology. 3rd ed. dubuque, ia, wm. c. brown publishers: 167 pp. regis and lagunzad 26 chapman, m.j. & d.l. mulcahy, 1997. effect of genomeplastome interaction on meiosis and pollen development in oenothera species and hybrids. sexual plant reproduction. 10(5):288-292. dushenkov, v., p.b.a. nanda kumar, h. motto, & i. raskin, 1995. rhizofiltration: the use of plants to remove heavy metals from aqueous streams. environmental science & technology. 29(5):1235-1245. hershey, d.r., 1995. hydroponics in plant biology projects. new york, john wiley & sons, inc.: 107 pp. jackson, a.r.w. & j.m. jackson, 1996. the major extractive industry. in environmental science: the natural environment and human impact. harlow, essex, england, longman group ltd.: 236 pp. kim, k. & d. kim, 1996. heavy metal pollution in agricultural soils: measurements in the proximity of abandoned mine land sites (amls). j. environ. sci. health. a31(4):783-795. larson, d.a., 1965. fine-structural changes in the cytoplasm of germinating pollen. am. j. botany. 52(2):139-154. lee, h.s., b. karunanandaa, a. mccubbin, s. gilroy, & t. kao, 1996. prk1 a receptor-like kinase of petunia inflata is essential for postmeiotic development of pollen. plant j. 9(5): 613-624. micieta, k. & g. murin, 1996. microspore analysis for genotoxicity of a polluted environment. environ. and exp. bot. 36(1):21-27. murin, a., 1995. basic criteria for selection of plant bioindicators from the regional flora for monitoring of an environmental pollution. biologia. 50(1):37-40. moore, p.d., j.a. webb, & m.e. collinson, 1991. basis and applications. in pollen analysis. 2nd ed. blackwell scientific publication. pp. 1-9. pfeiffer, e.m., j. freytag, h.w. scharpenseel, g. meihlich, & v. vicente, 1988. trace elements and heavy metals in soil and plants of the southeast asian metropolis metro manila and of some rice cultivation provinces in luzon, philippines. hamburger bodenkundliche arbeiter: 129 pp. comparison of pollen abortiveness 27 regis, e.g., 1999. pollen grain abortion as indicator of mercury pollution near gold mining sites in camarines norte. a doctoral dissertation for the degree of ph.d. in environmental science from the college of science, university of the philippines, diliman, quezon city. rowley, j.r., j.j. skvarla, & w.f. chissoe, 1997. exine, onciform zone, and intine structure in ravenala and phenakospermum and early wall development in strelitzia and phenakospermum (strelitziaceae) based on aborted microspores. rev. of palaeobotany & palynology. 98(3-4): 293-301. 22_cesium villorente, noguera, and ramos 90 effect of cesium seeding on the production of h– ions in a magnetized sheet plasma source l. m. m. villorente*, v. r. noguera, and h. j. ramos plasma physics laboratory, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: lvillorente@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 90-94 *corresponding author the effect of the addition of cesium on the production of negative hydrogen ions (h–) in a magnetized sheet plasma source (spnis) is investigated. plasma parameters and h– yields were determined from langmuir probe and e x b probe measurements, respectively. significant increase on h– yield is observed with the addition of a controlled flux of neutral cs vapor. the maximum enhancement of 88 times compared with the uncesiated case is extracted at 5 cm away from the sheet plasma core at a discharge current of 0.5 a and initial gas filling pressure of 9 mtorr. the value is increased from 5.51 x 10–8 a/m2 for the uncesiated case to 4.86 x 10–6 a/m2 for the cesiated case. the largest negative hydrogen ion current density extracted is 0.0155 a/m2 at 2.5 a discharge current, 9 mtorr initial gas filling pressure, and 1 cm probe distance from the plasma center. here, enhancement is only 9.8 times compared with pure hydrogen discharge. the increase in h– current density is attributed mainly to the cooling effect of cs as evidenced by the considerable decrease in electron temperature especially at the periphery of the plasma. introduction the generation of negative hydrogen ions (h–) is being pursued due to its wide variety of significant applications. h– ion sources are crucial components of fusion plasma devices and particle accelerators, as well as of other plasma machines. numerous particle accelerators such as cyclotrons, synchrotrons, tandem accelerators, and proton storage rings require h– or d– ions extracted directly from negative ion sources (leung, 1991; dudnikov, 2002). h– ion sources are also used in diagnostics for fusion plasmas and semiconductor-etching-process plasmas, high-energy neutral beam heating, and medical applications (belchenko & grogoryev, 2002). one of the most important aspects of any ion source is its h– output. cesium (cs) vapor has been injected into various h– ion sources since the discovery of cesiation of surfaces at inp by dudnikov in 1971, resulting in dramatic increases of attainable h– currents (peters, 1998). extracted negative ion currents rose by as much as 370 times after cesium was added in certain surface production sources (dudnikov, 2001). various mechanisms to explain the enhancement of h– production by cs seeding have been proposed such as the decrease of the work function of the converter or plasma electrode surface due to cesium adsorption (okuyama & mori, 1989; bacal et al., 1998); reduction of electron temperature due to more collisions with the larger cs cross section; gettering or absorption of atomic hydrogen by cesium, resulting in reduction of h density; and formation of csh (fukumasa, 1994), among others. effect of cesium seeding 91 in general there are three distinct types of h– ion sources classified by how the ions are produced: surface production sources, volume production sources, and hybrid (surface + volume) production sources (sanchez & ramos, 1996). the magnetized sheet plasma negative ion source (abate & ramos, 2000; arciaga et al., 2003) is of the volume type. this is related to the geometry of the ion source. the vibrationally excited hydrogen molecules are produced at the sheet plasma core due to collisions with energetic electrons, then drift to the periphery and combine with cold plasma electrons via dissociative attachment to form h–. the presence of an anode at one end of the extraction chamber instead of a converter electrode lessens, if not actually hinders, the surface production process. consequently, positive ions are repelled and thus, obstruct its conversion to negative hydrogen ions by collision with the anode surface. moreover, the magnetic confinement of the source keeps the positive ions within the center of the sheet plasma. hence, conversion of positive hydrogen ions to h– due to collisions with the cs-covered chamber wall is unlikely to occur. h– being short-lived means that the production probability via collisions of neutral particles with the chamber wall is small and the probability of destruction as they enter the bulk plasma is great. specifically, the effect of cs on h– production in this source is investigated for the first time. different system parameters are investigated to determine the efficiency of cs in enhancing h– density. methodology the magnetized sheet plasma ion source has been described previously (abate & ramos, 2000; arciaga et al., 2003). a schematic diagram of apparatus and photo are shown in fig. 1. the plasma is generated inside the production chamber by thermionic emission of a single 0.5-mm-diameter, 13.0-cm-long hairpin-shaped tungsten wire using a 150 v dc, 150 a power supply (v4 in fig. 1). the filament potential ranged from 14 to 18v at a corresponding current range of 20–23 a. the tungsten filament is negatively biased with respect to the second intermediate electrode (limiter e2 in fig. 1). electrons and negative ions are accelerated into the extraction chamber by the secondary power supply v2 rated at 100 v dc and several amperes. v2 supplies the potential (between 60–70 v) at the production chamber at a current range of 3–4 a. the potential between the extraction chamber (from the limiter e1 to the anode) is supplied by the source v1 between 125 and 150 v, giving rise to a current in the extraction chamber ranging from 0.5 to 1.5 a. a switching sequence from s2 to s1 completes the circuit from the cathode to the anode. the conversion of the cylindrical plasma that is formed in the extraction chamber to a sheet plasma is done by using a pair of strong dipole magnets (~1.5 kg on the surface) with opposing fields and a pair of helmholtz coils producing a magnetic mirror field. this technique was successfully demonstrated by abate and ramos (2000). the sheet plasma form includes fast electrons within the core plasma (of several millimeters) accompanied by cold diffused plasma electrons at the periphery. negative hydrogen ion current can then be extracted from the sheet plasma configuration using the exb probe (arciaga et al., 2003). plasma parameters and h– yields are determined from langmuir probe and exb probe measurements at pressures of 5, 7, and 9 mtorr and at discharge currents of 0.5–2.5 a at 0.5 a increment for both the uncesiated and cesiated cases. results and discussion emission spectra the presence of cs in the hydrogen plasma is detected by an emission spectrometer. a typical spectroscopic fig. 1. schematic diagram of the spnis. a1 a2 helmholtz coils anode cs oven probe v1 s1 s1’ v2 s2 w filament v4 limiter e2 limiter e1 gas inlet s n s n sm-co magnets pump cathode villorente, noguera, and ramos 92 scan of the cesiated hydrogen plasma is shown in fig. 2. the balmer emission lines and the peaks associated with cesium are seen. plasma parameters a single langmuir probe of tungsten wire with an exposed tip of 0.5 mm diameter and 5 mm long determines the electron density and electron temperature of the source. shown in fig. 3 is the distribution of electron temperature te as a function of probe distance from the sheet plasma center at different initial gas filling pressures for 2.5 a discharge currents. plots in solid lines show the trend for the pure plasma, while that of the broken lines are for the cesiated h2 discharge. for the magnetized sheet plasma, te generally decreases from the sheet core to the periphery, that is, energetic electrons are present in the center and low-energy electrons are seen at the margins of the sheet plasma. high-energy electrons are responsible for raising the hydrogen molecules to highly rovibrationally excited states. on the other hand, the cold electrons at the periphery are necessary for the formation of negative hydrogen ions by dissociative attachment with the highly rovibrationally excited molecules. a comparison of the results in the cesiated and pure hydrogen plasma for all gas pressures and distances clearly indicates a general decrease in te for the cesiated case, although overlapping of values can still be seen especially for the 9 mtorr, 2.5 a plasma conditions. as much as 40% decrease in te is observed for the conditions of 2.5 a discharge current, 7 mtorr hydrogen pressure, and 4 cm probe distance from the plasma center. this lowering of the electron temperature is important since low-energy electrons play an important role in h– formation through the dissociative attachment process. furthermore, higher electron temperatures tend to reduce h– production because more energetic electrons mean more electron impact processes that neutralize the already produced negative hydrogen ions. the observed reduction in electron temperature is attributed to the much larger cross sections of excitation and ionization of cs by electron impact; hence, there are more inelastic excitation and ionization collisions of electrons with the presence of cs than with hydrogen alone (bacal et al., 2003). figure 3 also shows the behavior of electron temperature as a function of input gas pressure. the increase in pressure causes a corresponding decrease in te especially at the periphery. this trend is observed in both the uncesiated and cesiated hydrogen plasmas. the reduction of the effective mean free path between fig. 2. spectroscopic scan of cs-seeded plasma. wavelength (nm) a rb it ra ry c on st an t 400 450 500 550 600 650 700 fig. 3. spatial distribution of electron temperature at various pressures and discharge current of 2.5 a. probe distance (cm) e le ct ro n te m pe ra tu re , t e (e v ) 0 1 2 3 4 5 6 0 2 4 6 8 10 12 h, 5 mtorr, 2.5 a h, 7 mtorr, 2.5 a h, 9 mtorr, 2.5 a h+cs, 5 mtorr, 2.5 a h+cs, 7 mtorr, 2.5 a h+cs, 9 mtorr, 2.5 a effect of cesium seeding 93 particles within the bulk of the plasma is likely to be the cause of the decreasing trend of te for higher pressures. the mean free path is smaller on account of higher densities, and the frequent collisions contribute to the continuous loss of initial energies of the electrons. the spatial variation of electron density ne at various gas pressures is illustrated in fig. 4. the graphs in solid lines are for uncesiated hydrogen discharge, while those of the dotted lines are for the cesiated hydrogen plasma. the same spatial distribution as the electron temperature is seen in this plot. electron density generally decreases with probe distance from the sheet of the plasma. this condition arises from the source’s geometry, limiting the high-density energetic electrons at the center in order to minimize the interaction of “hot” electrons with the formed h–. few low-energy electrons drift towards the periphery. the addition of cs has not much effect on the electron density. there is no detected decrease in ne contrary to what others have observed. h– enhancement with the addition of cs vapor to pure h2 plasma the effect of the addition of cs on the production of h– is shown in fig. 5. upon addition of cs on the h2 discharge, a significant increase on h– yield is observed. the maximum enhancement of 88 times occurred at 5 cm away from the central plasma with discharge current of 0.5 a and initial gas filling pressure of 9 mtorr. the value is increased from 5.51 x 10–8 to 4.86 x 10–6 a/ m2. however, the largest h– yield is achieved at 1 cm relative to the plasma core, with 2.5 a discharge current and 9 mtorr initial gas filling pressure. here, enhancement is only 9.8 times compared with pure hydrogen discharge. the value obtained is 0.0155 a/ m2 from 0.00156 a/m2. this increase in the negative ion current density with the addition of cs is closely related to the drop of electron temperature at the periphery of the sheet plasma as presented in the section on plasma parameters. conclusions the effect of the addition of cs vapor on the h– production in a volume production source was investigated. the h– current density was compared for the pure and cesiated hydrogen plasmas. a maximum increase of 88 times corresponding to h– current density fig. 5. dependence of h– production with discharge current for varying gas pressures at 5 cm away from the core plasma. discharge current (a) lo g (h – cu rr en t de ns it y) [l og ( a /m 2 ) ] fig. 4. spatial distribution of electron density at various pressures and discharge current of 2.5 a. probe distance (cm) e le ct ro n de ns it y, n e (c m –3 ) 8.0e+10 7.0e+10 6.0e+10 5.0e+10 4.0e+10 3.0e+10 2.0e+10 1.0e+10 0.0e+10 0 1 2 3 4 5 6 h, 5 mtorr, 2.5 a h, 7 mtorr, 2.5 a h, 9 mtorr, 2.5 a h+cs, 5 mtorr, 2.5 a h+cs, 7 mtorr, 2.5 a h+cs, 9 mtorr, 2.5 a villorente, noguera, and ramos 94 of 4.86 x 10–6 a/m2 is achieved at 5 cm away from the central plasma with discharge current of 0.5 a and initial gas filling pressure of 9 mtorr. the increase in h– current density is attributed mainly to the cooling effect of cs as evidenced by the considerable decrease in electron temperature especially at the periphery of the plasma. however, the highest h– yield of 0.0155 a/m2 is obtained at 1 cm relative to the sheet plasma core, with 2.5 a discharge current and 9 mtorr initial gas filling pressure. here, enhancement is only 9.8 times compared with pure hydrogen discharge. references abate, y. & h.j. ramos, 2000. rev. sci. instrum. 71: 3689. arciaga, m.e., a.g. mendenilla, & h.j. ramos, 2003. rev. sci. instrum. 74: 951. bacal, m., f. el balghitin-sube, l. i. elizarov, & a. y. tontegode, 1998. rev. sci. instrum. 69: 932. bacal, m., et al., 2003. rev. sci. instrum. 74: 951. belchenko, yu. i. & e. v. grogoryev, 2002. rev. sci. instrum. 73: 939. dudnikov, v., 2001. 30 years of high intensity negative ion sources for accelerators. proceedings of the 2001 particle accelerator conference. 2087. dudnikov, v., 2002. rev. sci. instrum. 73: 992. fukumasa, o., t. tanebe, & h. nation, 1994. rev. sci. instrum. 65: 1213. leung, k.n., 1991. pac. 2076. okuyama, t. & y. mori, 1989. research report ippj-914. nagoya, japan. peters, j., 1998. review of negative hydrogen ion sources: high brightness/high current. proceedings of linac ’98. 1031. sanchez, j.k.c.d. & h.j. ramos, 1996. plasma sources sci. technol. 5: 416. inside front cover-19-2 editorial board editor-in-chief maricor n. soriano, ph.d. associate editors zubaida u. basiao, ph.d. biology joel joseph s. marciano, jr., ph.d. computer science, engineering nemesio e. montano, ph.d. biochemistry rosana b. tarriela, ph.d. earth sciences fernando p. siringan, ph.d. marine sciences cristine d. villagonzalo, dr. rer. nat. physics corazon d. villareal, ph.d. managing editor dercylis g. mararac editorial assistant science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p 300.00/year (two issues), inclusive of postage us $ 25.00/year (two issues), inclusive of postage tel. no: (632) 981-85-00 loc. 4047 (632) 927-2567; 927-2309; 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences cover photo scanning electron microscope image of gaas nanowires at 200,000x magnification grown on gold-deposited si (100) substrate. image courtesy of dr. armando somintac from the paper “growth of gold-assisted gallium arsenide nanowires on silicon substrates via molecular beam epitaxy” in this volume. editorial advisors kelvin s. rodolfo, ph.d. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu alfonso m. albano, ph.d. dept. of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu victor c. gavino, ph.d. dept. of nutrition university of montreal, canada victor.gavino@umontreal.ca raul k. suarez, ph.d. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 page 13 images image 1 anions-de vera.pmd anions analysis in ground and tap waters by sequential chemical 31science diliman (january-june 2011) 23:1, 31-41 anions analysis in ground and tap waters by sequential chemical and co 2 -suppressed ion chromatography glen andrew d. de vera* and ma. pythias b. espino institute of chemistry, college of science, university of the philippines, diliman 1101 quezon city tel. no.: (632) 920-54-30; fax no.: (632) 920-54-27 corresponding author: devera.glen@yahoo.com abstract an ion chromatographic method using conductivity detection with sequential chemical and co 2 suppression was optimized for the simultaneous determination of fluoride, chloride, bromide, nitrate, phosphate and sulfate in ground and tap water. the separation was done using an anion exchange column with an eluent of 3.2 mm na 2 co 3 and 3.2 mm nahco 3 mixture. the method was linear in the concentration range of 5 to 300 μg/l with correlation coefficients greater than 0.99 for the six inorganic anions. the method was also shown to be applicable in trace anions analysis as given by the low method detection limits (mdl). the mdl was 1μg/l for both fluoride and chloride. bromide, nitrate, phosphate and sulfate had mdls of 7 μg/l, 10 μg/l, 9 μg/l and 2 μg/l, respectively. good precision was obtained as shown in the relative standard deviation of 0.1 to 12% for peak area and 0.1 to 0.3% for retention time. the sensitivity of the method improved with the addition of co 2 suppressor to chemical suppression as shown in the lower background conductivity and detection limits. the recoveries of the anions spiked in water at 300 μg/l level ranged from 100 to 104%. the method was demonstrated to be sensitive, accurate and precise for trace analysis of the six anions and was applied in the anions analysis in ground and tap waters in malolos, bulacan. the water samples were found to contain high concentrations of chloride of up to 476 mg/l followed by sulfate (38 mg/l), bromide (1 mg/l), phosphate (0.4 mg/l), fluoride (0.2 mg/l) and nitrate (0.1 mg/l). keywords: ion chromatography, chemical suppression, co 2 suppression, anions de vera, gad and espino, mpb 32 introduction ion chromatography (ic) provides the advantage of being a simple, fast, small sample volume demanding, and fit for purpose technique for the routine analysis of anions in water samples (miskaki et al., 2007). much importance is given to monitoring water quality to ensure safe and potable water for public consumption. along with microorganisms, heavy metals and organic pollutants, inorganic anion contaminants are monitored. fluoride and nitrate anions are considered contaminants in drinking water by the us epa national primary drinking water regulations. high concentrations of fluoride when ingested can result to bone disease while nitrate can cause birth defects (us epa, 2011). although not considered harmful, chloride and sulfate should not exceed 250 mg/l for water to become potable according to the epa’s national secondary drinking water regulations (us epa, 2011). in addition, based on the philippine national standards for drinking water (pnsdw), fluoride and nitrate concentrations in water should not exceed 1 mg/l and 50 mg/l, respectively. chloride and sulfate levels have limits of 250 mg/l in accordance with the standards used by the us epa (doh, 2007). elevated levels of bromide in water were shown to increase the formation of disinfection by-products (dbps) such as trihalomethanes and haloacetic acids during chlorination (sun et al., 2009); while with ozonation, the formation of bromate was observed (richardson et al., 2007). these dbps were reported to cause increased risk of cancer (us epa, 2011). phosphate concentrations in groundwater are also monitored because they were found to be contributing to eutrophication in surface waters (holman et al., 2008). to comply with strict regulations in water standards, analytical methods need to constantly provide accurate and reliable results on the levels of contaminants in water such as inorganic anions. ion chromatography has been used extensively for this purpose employing different approaches and detection techniques. ion chromatography, in the early years, has been used for anions analysis with spectrophotometric uv detection. in those methods, prior formation of organic derivatives and ion-pairs were necessary before the samples are analyzed using uv detection (stefanovic et al., 2001). other detection techniques are amperometry, potentiometry and post-column reaction (buchberger, 2001). currently, the method used for anions analysis is by conductimetric detection because of its better sensitivity for ions. new techniques are also explored such as hyphenation of ic systems using atomic absorption, fluorescence or emission detection as well as mass spectrometric detection (haddad et al., 2008). the analytical performance of ic with conductimetric detection can be improved with suppression techniques although non-suppressed ic as reported by stefanovic et al. (2001) can achieve good separation using computer programs for optimization. in chemically suppressed ic, a cation exchanger is used to replace the metal cations accompanying the anions of interest with more conductive hydronium ions. this process also transforms highly conductive eluents like na 2 co 3 / nahco 3 to weakly conductive substances. this significantly improves the conductivity signal of the analyte (kleimann et al., 2007). further improvement in anions detection may be done using a co 2 suppressor after chemical suppression. the carbonic acid formed during chemical suppression is in equilibrium with co 2 and water. installing a co 2 suppressor removes co 2 from the eluent and shifts the equilibrium forward towards the formation of water (kleimann et al., 2007). as a result, the background conductivity is reduced to that of water which leads to lower detection limits. we report here a study that used an ion chromatographic-conductivity detection system with sequential chemical and co 2 suppression to analyze fluoride, chloride, bromide, nitrate, phosphate and sulfate in local ground and tap waters. the optimized method was applied to actual samples taken from groundwater and water from the distribution lines in malolos, bulacan. the results of this study not only established the levels of the anions in the water samples but also provided an accessible method for routine anions analysis in potable water that can be used by water providers and government monitoring agencies in the philippines. science diliman (january-june 2011) 23:1, 31-41 anions analysis in ground and tap waters by sequential chemical 33 employed sequential chemical and co 2 suppression. the same solution was also subjected to eluent compositions of 5.0 mm na 2 co 3 /nahco 3 and 3.2 mm na 2 co 3 /nahco 3 to determine the optimum mobile phase based on sensitivity, resolution and analysis time. studies on linearity, method detection limit, repeatability and accuracy of the method were performed using the optimum eluent composition and sequential suppression technique. anions analysis of ground and tap water samples ground and tap water samples were obtained from household deepwells and distribution lines in baranggay panasahan and baranggay tikay in malolos, bulacan. the sampling sites are shown in figure 1. the water samples were collected in pre-cleaned 330 ml mineral water bottles. during sampling, water was allowed to flow for 1 min before pouring into the water-rinsed bottles. turbid water samples were vacuum filtered through 0.45 μm nylon membrane filters (whatman, maidstone, england). using the optimized chromatographic parameters, the anions in ground and tap water samples were identified based on individual retention times which were evaluated using standard anions solutions. serial dilution of water samples was performed to quantify anions that were initially out of the established linear range. all analyses were done at room temperature. the concentrations of the anions in the water samples were calculated using linear regression and external standard calibration method. results and discussion optimization of ion chromatographic system common ion chromatographic systems using na 2 co 3 / nahco 3 eluent produce weakly conductive h 2 co 3 which exists in equilibrium with co 2 and h 2 o. the instrument’s sensitivity for the anions may be enhanced by reducing the concentration of co 2 generated through the use of a co 2 suppressor. in this way, the conductivity of water will be approached to serve as the chromatogram’s baseline. science diliman (january-june 2011) 23:1, 31-41 materials and methods instrumentation the chromatographic system used was a metrohm 881 compact ic pro (metrohm, switzerland) consisting of a sample and eluent degasser, ic high-pressure pump, six-port injection valve, column heater, separation column and conductivity detector. the separation column used was a metrohm anion column metrosep a supp 5-150, 150 mm x 4.0 mm, packed with polyvinyl alcohol with quaternary ammonium groups and was used with a 5 mm x 4 mm metrosep a sup 4/5 guard column. the chromatograph was equipped with a metrohm suppressor module for chemical suppression and a metrohm co 2 suppressor. samples and standards were injected into a 20 μl sample loop. the eluent flow was 0.80 ml/min with a total run time of 14 min. data acquisition was performed using a magic net 2.2 chromatography software. reagents standard solutions for calibration were prepared by dilution of the stock 10.0 mg/kg certified multianion standard solution (sigma-aldrich, switzerland) containing fluoride, chloride, bromide, nitrate, phosphate and sulfate. the concentrations of the calibration solutions ranged from 5 to 300 μg/l. all solutions were prepared with 16 to 18 megaohm-cm ultrapure water (barnstead, dubuque, iowa). the eluent composition was a mixture of 3.2 mm na 2 co 3 and 3.2 mm nahco 3 prepared by dissolving appropriate amounts of nahco 3 and na 2 co 3 (himedia, mumbai, india) in ultrapure water. the 100 mm h 2 so 4 regenerant solution used for the anion conductivity suppressor device was prepared from 18.0 m h 2 so 4 (mallinckrodt baker, inc., paris, ky, usa). optimization and validation of instrument parameters preliminary studies on suppression effects and eluent composition were performed using a 300 μg/l standard solution. chromatograms of the standard solution were obtained from the ic setup that used chemical suppression only, and from the modified system which de vera, gad and espino, mpb 34 the anions included in this study were fluoride, chloride, bromide, nitrate, phosphate and sulfate, six of the commonly analyzed inorganic anions in environmental monitoring. optimization of the ion chromatographic method that uses na 2 co 3 /nahco 3 was performed by first evaluating the effects on the sensitivity of measurement using chemical suppression alone, and using chemical and co 2 suppression in sequence. the effects of co 2 suppression illustrated in figure 2 show that the incorporation of co 2 suppressor after chemical suppression significantly improves detection of the six anions. co 2 suppression decreased the baseline conductivity to less than 1.6 μs/cm. in effect, the sensitivity increased as evident from sharper and enhanced anion peaks. additionally, the co 2 suppressor dramatically reduced the water dip in the beginning of the plot that may interfere with the first eluting anions. with the lowered background conductivity, anions separation and analysis time were optimized. good resolution with shorter analysis time was achieved by using the right eluent composition. an initial run with chemical suppression alone was done with an eluent of 3.2 mm na 2 co 3 / 1.0 mm nahco 3 . although complete separation was achieved, the analysis required 28 min to elute all the anions. with chemical plus co 2 suppression, the eluent composition was modified to 5 mm na 2 co 3 / 5 mm nahco 3 resulting in separation of the anions in 9 min (figure 3). however, bromide and nitrate peaks overlapped to form a broad peak at about 7 min. peak separation of these two anions was achieved when the eluent was changed to 3.2 mm na 2 co 3 / 3.2 mm nahco 3 . all the anions were completely separated in a total analysis time of 14 min. a chromatogram presenting the ion chromatographic science diliman (january-june 2011) 23:1, 31-41 figure 1. sampling sites: baranggays panasahan and tikay in malolos, bulacan (maps adapted from cpdo, 2008 and gonzales, 2005). anions analysis in ground and tap waters by sequential chemical 35 figure 4. chromatogram showing separation of the six anions using the optimized method. [conditions: metrosep a supp 5 150/4.0 column, 3.2 mm nahco 3 / 3.2 mm na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, 300 μg/l standard solution, sequential chemical and co 2 suppressor] figure 3. effects of eluent composition in anions separation and analysis time. [conditions: metrosep a supp 5 150/4.0 column, nahco 3 / na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, 300 μg/l standard solution, sequential chemical and co 2 suppressor] science diliman (january-june 2011) 23:1, 31-41 figure 2. chromatograms showing water dip reduction upon addition of co 2 suppressor to chemical suppressor. [conditions: metrosep a supp 5 150/4.0 column, 3.2 mm nahco 3 /3.2 mm na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, 300 μg/l standard solution] de vera, gad and espino, mpb 36 analysis of the six anions under the optimized conditions is shown in figure 4. performance of the optimized ion chromatographic method with the optimized method, calibration plots (n=3) covering the concentration range of 5 to 300 μg/l were constructed and are shown in figure 5. the linear relationship of peak area with concentration was assessed using linear regression. the results from the calibration data show that in this range, the response for all anions with increasing concentration was linear with near unity correlation coefficients of 0.9903 to 0.9998. moreover, the 10 μg/l standard solution that was within the calibration range was analyzed to confirm if the anion peaks for this concentration would have signal to noise ratio of greater than 3. all anions came out in the chromatogram with acceptable signal to noise ratios and were clearly detectable with good resolution. the standard solution of 10 μg/l anions concentration was analyzed eight consecutive times to determine the method’s detection limit for each ion. the detection limit, in accordance to epa method 300.1, was estimated from the standard deviation multiplied by the student’s t-value at 99% confidence level. the detection limit was calculated using the equation mdl = 2.998 × sd (for n=8) (1) where mdl is the method detection limit; 2.998 is the student’s t-value for n=8; and sd is the standard deviation of all the measurements. the calculated mdls were comparable to the mdls reported in us epa method 300.1. nitrate had the highest mdl of 10 μg/l followed by phosphate (9 μg/l), bromide (7 μg/l), sulfate (2 μg/l), and lastly, chloride and fluoride with the same mdl of 1 μg/l. the results signify that trace anions level can be quantified using the optimized method given that the anions concentrations are above the reported detection limits. the mdl for each anion from the optimized method and from the epa method 300.1 (hautman et al., 1997) are shown in table 1. except for nitrate, the mdls for all the anions obtained using the optimized method are lower than the values reported by the us epa method 300.1. the difference can be attributed to the different ic systems used for both methods. more importantly, a dionex ag9-hc / as9-hc column and a 10 μl sample loop were used in the us epa method 300.1. in order to verify precision, replicate measurements of 10 μg/l and 300 μg/l standard solutions were carried out using the optimized method. precision was assessed in terms of repeatability of the retention time of each anion, peak area, and concentration expressed as percent relative standard deviation (%rsd). except for phosphate in 10 μg/l solution, the anions’ retention times, calculated concentrations and areas remained stable as shown from the %rsd that are less than science diliman (january-june 2011) 23:1, 31-41 figure 5. calibration plots in the linear range of 5 to 300 μg/l for the quantification of the six anions (n=3 for each concentration). [conditions: metrosep a supp 5 150/ 4.0 column, 3.2 mm nahco 3 / 3.2 mm na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, sequential chemical and co 2 suppressor.]   anions analysis in ground and tap waters by sequential chemical 37 10%. these data are shown in table 2 while the reproducibility of the measurements is graphically illustrated in figure 6. the recoveries of the anions spiked in ultrapure water at 10 μg/l and 300 μg/l concentrations were determined and reported in table 2. the actual concentrations were calculated from the established calibration curves and compared to the expected concentrations. the calculated recoveries for all the anions in both low and high concentrations were above the 90% minimum for the us epa method 300.1 (hautman et al., 1997). acceptable recoveries of 94 to 154% for the 10 μg/l spiked anions and 100 to 104% for the 300 μg/l spiked anions were obtained. in recent years, priority anions in water systems are determined using ion chromatography evolving from non-suppressed ic to new technologies of suppression which include the method that was used in this study. studies on non-suppressed ic were done by stefanovic et al. (2001) using a phthalic acid based eluent. for all the six anions, the method detection limits ranged from 3 to 4 μg/l, the % recoveries were 98 to 103%, and the linear range was 500 to 50000 μg/l. miskaki et al. (2007) also proposed a method for the six anions in science diliman (january-june 2011) 23:1, 31-41 figure 6. stacked chromatograms showing reproducibility of anions measurement at 10 μg/l spike level using the optimized method. [conditions: metrosep a supp 5 150/4.0 column, 3.2 mm nahco 3 / 3.2 mm na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, sequential chemical and co 2 suppressor]   anion mdl of optimized method in this study, in µg/l a mdl of us epa method 300.1, in μg/l b fluoride 1 9 chloride 1 4 bromide 7 14 nitrate 10 8 phosphate 9 19 sulfate 2 19   table 1. comparison of detection limits for us epa method 300.1 and the optimized method a conditions: metrosep a supp 5 150/4.0 column (150 x 4 mm); 3.2 mm na 2 co 3 /3.2 mm nahco 3 ; conductivity detector after chemical and co 2 suppression; 0.80 ml/min; 10 μg/l standard solution in ultrapure water;20 μl injection volume; 14 min analysis time; n=8. b reference: usepa, ord, nerl, 1997; conditions: dionex ag9-hc/as9-h (250 x 2 mm); 9.0 mm na 2 co 3 ; suppressed conductivity detector, dionex cd20 with an asrs-i external source electrolytic mode at 100 ma; 0.40 ml/min; 20 μg/l fluoride, 20 μg/l chloride, 40 μg/l bromide, 10 μg/l nitrate, 40 μg/l phosphate, 40 μg/l sulfate standard in reagent water, 10 μl injection volume; 25 min analysis time; n=7. de vera, gad and espino, mpb 38 surface, ground and potable waters. their method utilized chemical suppression alone with 0.10 n h 2 so 4 and 1.3 mm na 2 co 3 / 2.0 mm nahco 3 eluent. the detection limits ranged from 2.0 to 27.0 µg/l and the %rsd for concentration were from 0.80 to 4.60%. douglas et al. (2002) demonstrated the use of co 2 suppression technology with the na 2 co 3 /nahco 3 eluent with detection limits of 0.3 to 2.8 µg/l. a similar suppression technique using a metrosep a supp 5 – 100 column but a different eluent composition of 1 mm nahco 3 /3.2 mm na 2 co 3 was employed by kleimann et al. (2007). the method had a good performance as evident in the low detection limits of 0.7 – 1.9 µg/l. overall, the optimized method in our study is acceptable and comparable to those reported in literature. it was determined to be suitable for the analysis of fluoride, chloride, bromide, nitrate, phosphate and sulfate in water at a concentration range of 5 to 300 µg/l. analysis of anions in ground and tap waters the optimized method was applied in the simultaneous analysis of the six anions in groundwater and tap water in malolos, bulacan. the concentrations of fluoride, chloride, bromide, nitrate, phosphate and sulfate in the water samples were determined simultaneously. the concentrations are summarized in table 3. the water samples were found to contain high concentrations of chloride of up to 476 mg/l followed by sulfate (38 mg/ l), bromide (1.07 mg/l), phosphate (0.42 mg/l), fluoride (0.20 mg/l) and nitrate (0.09 mg/l). the samples were taken from malolos city in bulacan whose main water supplies come from either the local water district or from household artificial pumps. one set of samples was taken from baranggay panasahan which is in the vicinity of seawater connecting to manila bay. another set was taken from baranggay tikay which is the entrance point of malolos from metro manila. both sampling sites are located in build up areas that consume approximately 220,000 to 320, 000 cu. m. and 4,000 to 13,000 cu. m. of water daily for household and commercial uses, respectively (cpdo, 2008). during the analysis, serial dilution of water samples was performed to fit the areas of some ions within the linear range of the method. thus, there is a drawback when the same linear range for all ions is used to simultaneously determine the anion levels in samples having extremely low and extremely high anion concentrations. this is particularly true for chloride and sulfate in the actual water samples. quantifying sulfate required 1:50 dilution while chloride needed two 1:50 dilutions to fit in the linear range of the quantification method. diluting sample solutions several times caused some of the anions to be below the detection limits. hence, for these types of water samples, a further work would be to establish a wider linear range which is realistically up to 10 mg/l only; this is limited by the concentration of the commercially available mixed standard solution. another approach, albeit timeconsuming, would be to work with different sets of calibration solutions after doing preliminary analyses to estimate the anions level in the water samples. science diliman (january-june 2011) 23:1, 31-41 conditions: metrosep a supp 5 150/4.0 column, 3.2 mm nahco 3 / 3.2 mm na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, sequential chemical and co 2 suppressor, a n=8 at 10 μg/l anions concentration table 2. repeatability and accuracy of the optimized method in low (10 μg/l) and high (300 μg/l) spike levels fluoride 3.73 + 0.00 0.08 1.16 1.16 116 0.12 0.12 102 chloride 5.31 + 0.01 0.10 1.35 1.59 122 0.21 0.22 102 bromide 7.92 + 0.01 0.09 3.39 4.86 94 0.19 0.20 100 nitrate 9.07 + 0.01 0.08 4.86 4.38 96 0.55 0.55 100 phosphate 10.16 + 0.03 0.34 11.79 7.86 154 5.03 4.95 104 sulfate 12.33 + 0.04 0.29 1.71 2.60 102 0.11 0.11 101 10 μg/l (n=5) spike levelret. time %rsda anion ret. time (min)a 300 μg/l (n=3) spike level peak area %rsd conc. %rsd %rec. peak area %rsd conc. %rsd %rec. anions analysis in ground and tap waters by sequential chemical 39 significantly, the results here show that chloride ions, at the time of sampling, exceeded the maximum contaminant level (mcl) of the pnsdw for chloride which is 250 mg/l (doh, 2007). panasahan ground water has higher chloride levels than its tap water treated with chlorine disinfectant. this possibly indicates that saltwater intrusion is prevalent in the area. in tikay, higher chloride levels were observed for tap water. chloride levels from this sample may be dictated primarily by the dose of disinfectant added to the water supply. in addition, the presence of chloride and bromide in the water samples implies the possible formation of halogenated dbps such as trihalomethanes and haloacetic acids in the water systems. the sampling sites were in areas far from agricultural lands which may be a probable reason why nitrate and phosphate levels in the water samples were found to be low. an interesting further study would be to compare phosphate and nitrate concentrations around bulacan particularly in areas where synthetic fertilizers are used in farming; this is to determine their effects on water quality. sulfate in the water samples was below the mcl. sulfate in groundwater may be caused by the presence of sulfate minerals in the soil. however, sulfate can also be found in water as a result of discharges from agricultural and manufacturing industries present in malolos. hence, to completely see an overall picture of anions contamination as a result of the different industrial activities in bulacan, more samples should be analyzed using the optimized ion chromatographic method. in malolos city, drinking water quality is assessed by analyzing the turbidity, color, ph, total dissolved solids, levels of iron, manganese, arsenic, cadmium, lead, and inorganic anions such as nitrate, sulfate and chloride in the water supply (cmwd, 2011). the levels of each water quality parameter are compared to the limits set by the pnsdw and are measured using the standard methods for the examination of water and wastewater (apha, awwa, wef, 2006). none of the tests currently employed in malolos use ion chromatography in monitoring inorganic anions. nitrate is measured by colorimetric detection after cadmium reduction; sulfate is by turbidimetry; and chloride is by argentometry (cmwd, 2011). thus, this study offers an available alternative method in the form of the optimized ion chromatographic method to evaluate levels of nitrate, sulfate and chloride as well as anions not routinely analyzed in water supplies in malolos such as bromide, fluoride and phosphate. this method requires simple sample preparation, involves fast analysis time and more importantly, offers a procedure for the simultaneous determination of anions. other asian countries evaluate levels of inorganic anions in water to correlate their concentrations with principal contaminant sources which include industrial wastes, municipal landfills, agricultural chemicals, leaks science diliman (january-june 2011) 23:1, 31-41 asampled in february 13, 2011 and analyzed within six days; bn=1; cn=2; d1:10 dilution; e1:50 dilution; fdiluted twice using 1:50 dilution; g1:50 to 1:25 dilution; hevaluated using 5 to 500 μg/l calibration curve with equation: y=0.0929x + 0.0008, r2=0.9983; ireference: the philippine national standards for drinking water, 2007. conditions: metrosep a supp 5 150/4.0 column, 3.2 mm nahco 3 / 3.2 mm na 2 co 3 eluent, 0.8 ml/min flow rate, 20 μl injection volume, sequential chemical and co 2 suppressor; concentrations were corrected using 300 μg/l spike level recovery measurements; n.d. = not detected table 3. anion concentration in ground and tap waters a fluoride 0.08c 0.06c 0.20c 0.13c 1.0 chloride 190.73b,f 337.35b,f 476.26b,f 184.66b,g 250.0 bromide 0.49c,h 0.51b,e 1.07b,e 0.39b,d nitrate n.d. 0.08c 0.01c 0.09c 50 phosphate 0.42 c,d 0.14 c 0.01c 0.20b,e sulfate 4.74b,e 4.74 b,e 38.04b,e 22.51b,e 250.0 anion concentration, in mg/l panasahan tap panasahan ground tikay tap tikay ground mcl i de vera, gad and espino, mpb 40 from petroleum pipelines and storage tanks, animal wastes, saltwater intrusion, and irrigation return flow (park et al., 2002). with the knowledge of the origins of contamination, stricter regulations may be implemented to limit industrial and human waste generating activities. in korea, inorganic anions were investigated using chemically suppressed single column ic with conductimetric detection and the anions contamination was not severe with respect to their standards (park et al., 2002). in contrast, problems associated with high fluoride concentrations as evident in the high cases of fluorosis disease occur in china (wang et al., 2007), india (gautum, r. et al., 2011) and sri lanka (dissanayake c. b., 1996). in indonesia, ic was applied to assess their water quality affected directly by environmental problems such as increasing pollutants of various types, acid rain, and hazardous wastes (amin et al., 2008). hence, investigations like these in the philippines are essential to draw attention to the use of good water quality monitoring procedures such as the ion chromatographic method optimized in this study. the results of our work provide relevant information on the levels of fluoride, chloride, bromide, nitrate, phosphate and sulfate in ground and tap waters in malolos, bulacan. our findings suggest the need to routinely analyze water samples for these anions, particularly chloride that was found in relatively high levels in order to ensure public safety. more importantly, the optimized method described here can be used as an available method for assessing anions contamination in potable water not only in malolos city but also in water supply systems in other parts of the country. a planned research application of this method is on spatial and temporal variations of the six anions in environmental waters. another is its application, specifically for bromide and chloride analysis, in tandem determination of halogenated dbps in tap waters. finally, a future work on this sequential chemical and co 2 suppressed ion chromatographic method would be its application in other priority anion contaminants including bromate, chlorite, chlorate, perchlorate, nitrite and chromate. conclusions an ion chromatographic method with sequential chemical and co 2 suppression was optimized and validated in this study. the optimized method used to analyze fluoride, chloride, bromide, nitrate, phosphate and sulfate in ground and tap waters was evaluated to be linear in the concentration range of the analysis, sensitive and specific to these anions, highly reproducible, accurate and of comparable detection limits as in the us epa method 300.1. in addition, this ion chromatographic method required simple sample preparation. the method was successfully applied in measuring the anions even in the low µg/l levels in ground and tap water. acknowledgement we thank dynalab corporation for allowing us to use their metrohm 881 compact ic pro unit and for providing the analytical standard solution used in this study. we gratefully acknowledge ms. sheila mappala, ms. lynneth bagsic and ms. nadine arejola for their invaluable assistance. references apha, awwa, wef, 2006. standard methods for the examination of water and wastewater. www.standardmethods.org. amin, m., lim l.w., & takeuchi, t., 2008. recent environmental problems in indonesia and the application of advanced ion chromatography to water quality monitoring. discussion paper series vol. 2007-11. buchberger, w.w., 2001. detection techniques in ion chromatography of inorganic anions. trends anal. chem., 20: 296-303. city of malolos water district (cmwd), 2011. city planning and development office (cpdo), city government of malolos, 2008. city of malolos ecological profile. science diliman (january-june 2011) 23:1, 31-41 anions analysis in ground and tap waters by sequential chemical 41 department of health (doh), 2007. philippine national standards for drinking water. www.lwua.gov.ph. dissanayake, c.b., 1996. water quality and dental health in the dry zone of sri lanka. geological society, london, special publications, 113:131-140. douglas, d.r., saari-nordhaus, r., despres, p., & anderson, j.m., 2002. new suppressor technology improves trace level anion analysis with carbonate–hydrogencarbonate mobile phases. j. chromatogr., a, 956: 47–51. gautum, r., bhardwaj, n., & saini, y., 2011. study of fluoride content in groundwater of nawa tehsil in nagaur, rajasthan. j. environ. biol., 32 (1) 85-89. gonzales, m., 2005. map of bulacan showing the location of malolos. http: // en. wikipedia. org / wiki / file: ph _ locator _bulacan_malolos.png. haddad, p.r., nesterenko, p.n., & buchberger, w.w., 2008. recent developments and emerging directions in ion chromatography. j. chromatogr., a., 1184: 456-473. hautman, d.p., & munch, d.j., 1997. united states environmental protection agency method 300.1. determination of inorganic anions in drinking water by ion chromatography. holman, i.p., whelan, m.j., howden, n.j.k., bellamy, p.h., willby, n.j., rivas-casado, m., & mcconvey, p., 2008. phosphorus in groundwater-an overlooked contributor to eutrophication? hydrol. processes, 22: 5121-5127. kleimann, j., schäfer, h., & viehweger, k.h., 2007. sequential suppression for conductivity detection in ion chromatography. http://www.metrohm.com/applications/ic/ ic-reprints.html. science diliman (january-june 2011) 23:1, 31-41 miskaki, p., lytras, e., kousouris, l., & tzoumerkas, p., 2007. data quality in water analysis: validation of ion chromatographic method for the determination of routine ions in potable water. desalination 213: 182–188. park, h.-m., kim, y.-m.., lee, d.w., lee, s.w., & lee, k.b., 2002. ion chromatographic determination of inorganic anions in environmental samples of korea. anal. sci., 18: 343-346. richardson, s.d., plewa, m., wagner, e.d., schoeny, r., & demarini, d.m., 2007. occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection byproducts in drinking water: a review and roadmap for research. mutat. res., 636: 178–242. stefanovic, s.c., bolanca, t., & curkovic, l., 2001. simultaneous determination of six inorganic anions in drinking water by non-suppressed ion chromatography. j. chromatogr., a, 918: 325–334. sun, y.x., wu, q.y., hu, h.y., & tian, j., 2009. effect of bromide on the formation of disinfection by-products during wastewater chlorination. water res., 4: 2391-2398. us environmental protection agency (us epa), 2011. drinking water contaminants. http://water.epa.gov/drink/ contaminants/index.cfm wang s-x, wang z-h, cheng x-t, li j, sang z-p, zhang, x.-d., han, l.-l., qiao, x.-y., wu, z.-m.,wang, & z.q., 2007. arsenic and fluoride exposure in drinking water: children’s iq and growth in shanyin county, shanxi province, china. environ. health perspect., 115(4): doi:10.1289/ehp.9270. 18_cruz 84 cruz and villarin science diliman (january-june 2003) 15:1, 84-87 an analysis of the precipitable water vapor observed over the pimo gps station f.t. cruza* and j.t. villarinb adepartment of physics, ateneo de manila university loyola heights, quezon city, philippines bmanila observatory, ateneo de manila university campus loyola heights, quezon city, philippines e-mail: aastinus@zuma.ps.admu.edu.ph abstract remote sensing of the atmosphere using global positioning systems has been made possible with the derivation of the precipitable water vapor (pwv) from the tropospheric wet delay experienced by the signal propagation. although limited by the missing observational data from the receiver, it is observed that the pwv obtained from this data gives reasonable values when considered for the cases of wet and dry seasons and when analyzed with a measurable meteorological variable, such as the amount of rainfall. a continual update of the record for pwv is highly recommended for further studies on the behavior of the atmospheric water vapor and its contribution to the changing climate. introduction water vapor plays a significant role in the physical and chemical processes of the atmosphere. it is also considered as one of the major contributors to the greenhouse effect. analyses of the water vapor would contribute greatly to studies, such as weather forecasting and climate change. global positioning systems (gps) have been used in the remote sensing of the atmosphere. due to their good spatial and temporal coverage, the gps’s complement radiosondes and groundand space-based water vapor radiometers which are either limited in cold, dry regions or in the presence of clouds and rain (bevis et al., 1992). gps signals experience delays as they propagate through the atmosphere. in the troposphere, the delays are classified as hydrostatic delay or as wet delay. the wet delay due to the atmospheric water vapor is more variable and harder to remove. however, it is from these delays that the precipitable water vapor can be estimated (bevis et al., 1992). derivation of the precipitable water vapor from the zenith wet delay the precipitable water vapor (pwv) is related to the zenith wet delay (zwd) by a factor of π, (1) where both the pwv and zwd are in units of length (m). the dimensionless constant of proportionality, π is given by: (2) pwv zwd= π × 6 '3 2 10 v m k r k t ρ π = ⎡ ⎤⎛ ⎞ +⎢ ⎥⎜ ⎟ ⎝ ⎠⎣ ⎦ * corresponding author 85 analysis of the precipitable water vapor where ρ is the density of water, r v is the specific gas constant for water vapor, t m is a weighted mean atmospheric temperature and k 3 and k’ 3 are the refractivity constants (bevis et al., 1994) the mean atmospheric temperature is related to the surface temperature, t s , (3) where t s can be obtained from any meteorological station (bevis et al., 1994). since the values of ρ, r v are well-defined, the uncertainty of π is derived from the uncertainties of t m and the refractivity constants. if the term k 3 /t m is large enough, the contribution of k’ 2 can be neglected and thus, the error in π can be approximated to 2% (bevis et al., 1994). methodology the pimo station is one of the national aeronautics and space administration, jet propulsion laboratory (nasa jpl) gps ground-based receivers in the philippines. it is located at a latitude of 14°35’ n and a longitude of 121° e, within the grounds of the manila observatory at the ateneo de manila university, quezon city. the gps receiver records the observation data every 300 seconds in gps time. these are then sent to the regional data centers, to the global data centers, and then to the data analysis centers for processing. the observation files in the receiver independent exchange (rinex) format can be obtained from the crustal dynamics data information service (cddis) at http://cddisa.gsfc.nasa.gov. these files would be submitted to jpl’s autogipsy for the processing for tropospheric delay. the zwd and pwv are derived using the utility programs written in c language by reyes, which was later modified for this study (reyes & mcnamara, 2000). temperature profiles are recorded every hour by the advanced meteorological station, located within the grounds of the manila observatory. it is very important to consider the time indicated in the records. observation files from the gps receivers are in gps time, which differs from the coordinated universal time (utc) by a few leap seconds. temperature profiles, on the other hand are recorded in the local time, which is 8 hours ahead of the utc. for consistency in this study, the utc is chosen as the standard time. in the process of obtaining the observation files, it is noted that there were days with no available data, as well as days with incomplete data. therefore, in these cases, the zwd and pwv were set to zero, and consequently, not included in the results. results and discussion the philippine climate is described mainly by its wet and dry seasons. in this study, two months were selected for each season based on the relative completeness of the data for that month. the dynamic change of the amount of pwv is observed in figs. 1 and 2. the amount of pwv for the months of august and september 2000 (fig. 1) varies from a minimum value of 0.134 m to a maximum value of 0.167 m. on the other hand, for the months of december 2000 and january 2001 (fig. 2), it varies from a minimum of 0.122 m to a maximum of 0.164 m. it is observed that the pwv lowered in general during the dry season. 70.2 0.72m st t= + 0.17 0.16 0.15 0.14 0.13 0.12 7/31/00 0:00 8/20/00 0:00 9/9/00 0:00 9/29/00 0:00 time (utc) p w v ( m ) fig. 1. plot of the precipitable water vapor (pwv) for august 2000 to september 2000 (wet season). 86 cruz and villarin a comparison of the pwv with a measurable meteorological variable would give some insight into its behavior or variation. the pwv is described as the height of the equivalent column of liquid water directly above the receiver. thus, the variation of the amount of pwv can be analyzed with rainfall data. to illustrate, the case of december 31, 2000 is selected since there is a noticeable dip in the pwv at this time (fig. 2). the hourly averages of the pwv are derived (fig. 3) since the rainfall data are recorded every hour (fig. 4). in this case, after a slight variation during the early hours, the pwv continually decreased at 1200 utc. it 0.17 0.16 0.15 0.14 0.13 0.12 11/30/00 0:00 12/20/00 0:00 1/9/01 0:00 1/29/01 0:00 p w v ( m ) time (utc) fig. 2. plot of the precipitable water vapor (pwv) for december 2000 to january 2001 (dry season). 0.145 0.14 0.135 0.13 p w v ( m ) 0:00 4:48 time (utc) 9:36 14:24 19:12 0:00 fig. 3. plot of the hourly average precipitable water vapor for december 31, 2000. 0:00 7:12 14:24 21:36 time (utc) 5 4 3 2 1 0 r a in fa ll a m o u n t (m m ) fig. 4. plot of the rainfall amount for december 31, 2000 where the peak occurs at 14:00 utc. is at this time when there was a sudden occurrence of rain, which eventually lessened towards the end of the day. therefore, it is reasonable to infer that when it rains, there is an associated decrease in the pwv. it should be noted that one of the considerations for this was the relatively short distance between the gps receiver and the meteorological station since the pwv is defined directly over the gps receiver. the archive for the observation data for the pimo station started only in april 1999. thus, it is recommended that there should be a continuous update of the record and the analyses on pwv for studies such as weather forecasting and climate variability. acknowledgments the authors are grateful for the help and support offered by the staff of the jet propulsion laboratory (jpl) and crustal dynamics data information service (cddis) of the national aeronautics and space administration (nasa); the environmental management bureau of the department of environment and natural resources of the philippines; and conexor for the advanced meteorological station data; michael reyes, gemma narisma, armelle remedio, luisito agustin, and joanne dy. 87 analysis of the precipitable water vapor references bevis, m., s. businger, t. herring, c. rocken, r. anthes, & r. ware, 1992. gps meteorology: remote sensing of atmospheric water vapor using the global positioning system. j. geophys. res. 97(d14): 15787-15801. bevis, m., s. businger, s. chiswell, t. herring, r. anthes, c. rocken, & r. ware, 1994. gps meteorology: mapping zenith wet delays onto precipitable water. j. appl. met. 33(3): 379-386. reyes, m. & d. mcnamara, 2000. observing zenith wet delays and precipitable water vapor over jpl gps stations in the philippines. manila observatory scientific report. 4: 1-12. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 inside back cover-24-1.pmd reviewers for this issue volume 24 no. 1 ian jasper agulo, phd university of the philippines baguio marvin a. albao, phd university of the philippines los baños porfirio m. aliño, phd marine science institute college of science university of the philippines diliman evangeline c. amor, phd institute of chemistry college of science university of the philippines diliman alberto v. amorsolo, jr., phd department of mining, metallurgical and materials engineering college of engineering university of the philippines diliman susan d. arco, phd institute of chemistry college of science university of the philippines diliman wilfredo l. campos, phd university of the philippines visayas lilibeth c. coo, phd institute of chemistry college of science university of the philippines diliman luis maria b. garcia, dfsc institute of biology college of science university of the philippines diliman arnold v. hallare, dr rer nat department of biology cas, university of the philippines manila hilton y. lam, mha, phd department of clinical epidemiology college of medicine university of the philippines manila jonas p. quilang, phd institute of biology college of science university of the philippines diliman leni l. quirit, phd natural sciences research institute university of the philippines diliman jose s. solis, phd institute of chemistry college of science university of the philippines diliman florentino c. sumera, phd institute of chemistry college of science university of the philippines diliman ocr document ocr document page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 differentiating ac and dc field effects on the magnetic susceptibility of bulk yba2cu3o7-δ jessica pauline c. afalla* and roland v. sarmago national institute of physics , university of the philippines, diliman 1101 quezon city *corresponding author: jcafalla@up.edu.ph received: 6 january 2010; revised: 29 may 2010; accepted: 28 july 2010 abstract a low field ac magnetic susceptibility has been measured for a superconducting bulk yba 2cu3o7-δ sample with an ac excitation field superimposed with a dc field. the effects on the susceptibility due to either type of field have been interpreted without any assumption regarding the presence of vortices in the material. from the in-phase susceptibility data, saturation values show that increasing the ac field strength causes a decrease in shielding ability and a persistence of intergranular losses to lower temperatures. the intergranular loss peaks in the out of phase susceptibility data show shifting to lower temperature in accordance with the in-phase data. increasing the dc field strength does not cause the saturation values to decrease, but rather, saturation values remain at the same level for the in-phase susceptibility data, showing consistency in the sample’s shielding ability. however, increasing dc field strength increases the peak height for the intergranular loss peaks, but the peak does not shift to lower temperatures, thus greater energy is expended to shield the dc excitation, but without causing losses to persist to lower temperatures. keywords: 74.25.ha magnetic properties, 74.62.-c transition temperature variations, 74.72.bk y-based cuprates introduction ac magnetic susceptibility is a widely used technique in characterizing electrical and magnetic properties of superconductors. the technique has been used to study frequency or field dependence of high temperature superconductor samples to allow for improvement in the application properties of these materials and the study of the physics such as investigating irreversibility lines (deak, 1994), critical current densities (bertman and strongin, 1966), granularity (couach and khouder, 1991) and the pseudogap (wang et.al., 2005). the principle of ac susceptibility measurements is given by the sample’s response to an external magnetic field. the magnetization m(ωt) of a material under a time dependent (sinusoidal) field, is given by: h  t =h dch ac re [exp i t ] (1) which can be expressed in a fourier expansion: m t =o h dch ac∑ n=1 ∞ re [n exp i n t ] (2) where χ0hdc is the dc magnetization brought about by the superimposed dc magnetic field hdc while the second magnetization term is the time-varying component (yamamoto, et.al., 1992). in order to interpret susceptibility data, several frameworks are used (couach and khouder, 1991) such as eddy current-based models, bcs theory-based interpretation and critical state models (t. ishida and r.b.goldfarb,1990, enomoto and okada, 1996). critical state models are most common in interpreting susceptibility for type-ii science diliman 21(2):25-34 25 mailto:jcafalla@up.edu.ph afalla, sarmago superconductor, although there is no delineation between the meissner (vortex-free) and the mixed (superconducting state with vortices forming normal cores) states (poole, farach and ceswick, 1995), despite requiring low values for the applied magnetic fields. in critical state models, the dependence of the critical current density and the field needs to be established. in a previous study (sarmago and singidas, 2004), low field ac susceptibility, without dc field, in the absence of vortices, has been explained using an eddy-current loss model. the model is an alternative means of interpreting data without having to create a field and critical current density relationship for the system. the model has been found to be applicable for several superconducting samples such as yba2cu3o7-δ (sarmago and singidas, 2004) and mgb2 (sarmago and olbinado, 2004). in this present study, we have introduced the material ybco to a superimposed dc field. we present susceptibility measurements using the parameters of the same range (ishida and goldfarb, 1996, jeffries, et al., 1989, shinde, et al. 1990) as that used in numerous studies that have utilized critical state models in data interpretation. these studies along with others (chen, 1991, qin et al. 1999, xenikos and lemberger 1989) have shown that the magnetic response of superconducting material to a dc magnetic field is different from its response to an ac field. in most of these works, critical state models have been employed in interpreting results. the addition of small amplitude dc fields to this work is aimed at identifying difference in behavior of susceptibility with either type of field and whether the additional dc field can drive the superconductor out of the vortex-free state. however, results show that the field strengths used are yet insufficient to drive the material out of the vortex-free state, thus the electrodynamic model by singidas and sarmago has been utilized in order to interpret magnetic susceptibility data obtained in this study. ybco is a ceramic superconductor and when fabricated in bulk, is treated as a network of grains connected by weak links (muller, et al. 1987). due to this granular nature of the sample, magnetic susceptibility often shows distinct features, first being the intrinsic or intragranular response, whereby the field acts on the individual grains of the sample and second when the grains achieve coherence, the sample acts as a singular body, thus the effects on the sample are referred to as intergranular (y.yang, et al. 1992). typical coupling measurements have been presented by goldfarb, et al. (1991) identifying intragranular and intergranular responses in ac susceptibility measurements. intragranular response coincides with the onset of the critical temperature, and when phase-coherence (rose-innes and rhoderick, 1978) is achieved, the much larger intergranular response becomes evident. when phase coherence is achieved, it is reflected in the in-phase susceptibility (χ’) as a second transition in the in-phase (aksu, et al. 2003). the out of phase susceptibility (χ”) may show two loss peaks. a smaller peak appears coinciding with tc, and is this loss peak is attributed to intragranular losses, meanwhile, the larger peak appearing at lower temperature corresponds to the intergranular losses. depending on the strength of grain coupling, the peaks may be distinctly separated or may overlap. methodology the behavior of bulk superconducting yb2cu3o7-δ (ybco) was investigated using a hartshorn-type mutual inductance bridge (hartshorn, 1925), shown in figure 1. a mutual inductance bridge is composed of identical and coaxial pickup coils which are oppositely wound, enclosed by a primary coil. the primary coil supplies the applied field and a corresponding emf is induced in both the two pickup coils. placing a magnetic sample in one of the pick-up coils introduces an imbalance in the emf, which can be detected by a lock-in amplifier. the emf responses are proportional to the magnetization and susceptibility of the sample. an additional coil was added in order to superimpose a dc field such that the total excitation field experienced by the sample is an ac field with an offset: b=bdc+baccosωt. the dc coil is coaxial with the mib such that bdc is parallel to bac. the range of fields have been limited such that b dc (2.04mt, 1.56mt, 0.72mt, 0.24mt and 0mt) and bac (0.512mt, 0.409mt,0.307mt and 0.205mt) are around the same order of magnitude as other published results. susceptibility measurements are obtained via a lock-in amplifier that automatically decomposes the in-phase and out of phase components of the susceptibility χ=χ’+iχ”, χ’ being 26 science diliman differentiating ac and dc field effects on the magnetic susceptibility the in phase and χ” being the out of phase component. the excitation frequency for all measurements is 3200hz. two sets of magnetic susceptibility data were gathered. the first set contains magnetic susceptibility where bdc is held fixed and bac is varied (0.512, mt, 0.409mt, 0.307mt and 0.205). the second set is a measurement of magnetic susceptibility responses for when bac is fixed and bdc is then varied (2.04mt, 1.56mt, 0.72mt, 0.24mt and 0mt). these two sets will distinguish the response of the material to either type of field. the ybco sample was fabricated through standard solid-state reaction method for bulk sample fabrication, where powders of y2o3, bao, cuo are ground and pressed into pellets.. the resulting pellets are sintered twice at 900c and annealed in flowing oxygen. a 1mm × 1mm × 10mm bar was obtained from the produced ybco pellet. results and discussion figure 2 shows the susceptibility of ybco without a dc field (bdc=0). the measurements were obtained still with the dc coil attached, but turned off. the in-phase susceptibility (χ’) is characterized by a single transition, which broadens as bac is increased. the corresponding out-of-phase susceptibility (χ”) shows a single peak reflecting intergranular losses. the absence of the intragranular loss peak indicates strong coupling in the sample, thus the in phase transition can immediately be associated to intergranular shielding. the saturation values of χ’ (low temperature) decrease in magnitude as the field is increased, such that the shielding ability of the sample decreases as field strength increases. the corresponding intergranular loss peak broadens, decreases in magnitude and shifts to lower temperature as the field is increased. fixed dc field, varying ac to illustrate the effects due only to the ac field, we gathered a set of data where in the dc field is held constant, while the ac field is varied for each value of the dc field. the data is shown in figures 3-6. figure 3 shows the susceptibility data for upon the addition of a dc field (bdc=0.24 mt). the χ’ science diliman 27 figure 1. hartshorn-type mutual inductance bridge. an alternating current creates an alternating magnetic field through the primary coil, which induces emf across the two secondary pick-up coils a and b. figure 2. in phase susceptibility χ’ and out of phase susceptibility χ” measured at 3200hz. dc field is turned off and the ac field amplitude is varied from 0.512mt to 0.204mt. afalla, sarmago behaves similar to the case where no dc field is applied. the transition temperature is ~81k. the single transition broadens as bac is increased. and χ” has a single loss peak which broadens, decreases in magnitude and shifts to lower temperature as the ac field is increased. figure 4 shows the susceptibility when bdc is 0.72 mt, the in phase susceptibility (χ’) is still seen to broaden as the ac field is increased. however at this dc field, the in phase response begins to show two transitions. immediately after tc (~83k), a short temperature range of ~5-8k appears, before a second (intergranular) transition occurs. the first transition is associated with the shielding of individual grains (intragranular), and is observed to broaden slightly with increasing ac field strength. the second transition is brought about by the coupling of grains, enabling the sample to act as a single grain (intergranular). the out of phase susceptibility still displays a single loss peak which broadens, decreases in magnitude and shifts to lower temperature with increasing field. figure 5 shows the susceptibility data when b dc is 1.56mt, the in phase susceptibility shows two transitions. the first transition, occurring at tc (~84k), is seen to broaden with increasing field, such that the second transition occurs at a lower temperature when the ac field is greater. the second (intergranular) transition is also seen to broaden with increasing field, as seen in the inset. the out of phase behavior is still consistent as before. figure 6 shows the susceptibility data at the highest bdc of 2.04mt, the slopes are at their broadest in the data set. as the ac field is increased both transitions in the in-phase susceptibility broaden, and is most apparent for 0.512mt, the highest ac field. while the out of phase susceptibility again follows the trend as before of decreasing in 28 science diliman figure 3. ac susceptibility measurements at 3200hz. the dc field strength is fixed at 0.24 mt while bac is varied. figure 4. ac susceptibility measurements at 3200hz. the dc field strength is fixed at 0.72 mt while bac is varied. differentiating ac and dc field effects on the magnetic susceptibility magnitude, broadening of the peak and shifting to lower temperature as the field is increased. the second transition appears 6.5k after tc, and slightly broadens with increasing field strength as shown in the inset. for all fixed values of the dc field, it has been consistently observed that increasing the ac field strength causes the intergranular transition to broaden and the saturation values to become less negative, as seen from the in-phase susceptibility. the intergranular loss peak on the other hand, decreases in magnitude, broadens and shifts to lower temperature as the bac is increased, as seen from the out-of-phase susceptibility curves. fixed ac field, varying dc field in the second data set, we focus on the behavior due to the dc field. in the same respect, the ac field is held fixed while the dc field is varied (0, 0.24mt, 0.72mt, 1.56mt and 2.04mt) for every fixed ac field value. figure 7 shows the susceptibility data at bac=0.204mt. the in phase susceptibility (χ’) shows two transitions for those at higher dc fields, the first transition (tc ~85k) confined in a very short temperature range. as the dc field is increased, only slight broadening is observed. the corresponding out of phase (χ”) shows a single loss peak. as the dc field is increased, hardly any shifting of peaks to lower temperatures nor changes in behavior can be seen. figure 8 shows susceptibility data for when bac is 0.307mt. χ’ shows two transitions for those at higher dc fields. both the first (tc~84k) and second transitions broaden with increasing applied field. while the corresponding χ” has a single loss science diliman 29 figure 5. ac susceptibility measurements at 3200hz. the dc field strength is fixed at 1.56 mt while bac is varied. the inset for χ’ shows a closer inspection of the transition that appears at tc. the first transition width broadens. figure 6. ac susceptibility measurements at 3200hz. the dc field strength is fixed at 2.04 mt while b ac is varied. the inset shows a closer look at the first transition appearing near tc. the first transition width is approximately 6.5k . afalla, sarmago peak, showing slight broadening, but no decrease in magnitude and no shifting to lower temperature as the dc field is increased. figure 9 shows the susceptibility data at bac=0.409mt. the slope of the first transition (χ’, t¬c~84k) shows broadening as the dc field is increased, such that at higher dc fields, the second transition occurs at lower temperature. the second transition also broadens with increasing dc field. the intergranular loss peak (χ”) on the other hand, displays slight broadening, slight decrease in magnitude but barely any shifting to lower temperature as the dc field is increased. figure 10 shows the susceptibility data at bac=0.512mt. the in phase susceptibility now shows two transitions for all values of the dc field. the first (tc~84k) and second transitions are seen to broaden significantly with increasing dc field. the out of phase response on the other hand shows an increase in magnitude, but no broadening and no shifting to lower temperature as the dc field is increased. differentiating the effects of bac from bdc on the behavior of magnetic susceptibility the behavior of the magnetic susceptibility data with increasing bac and bdc is summarized in table1. from the first data set (fixed dc, varying ac), we can identify the behavior of susceptibility data due to increasing the ac field. the behavior of the susceptibility when the dc field is increased can also be identified from the second data set (fixed 30 science diliman figure 7. ac susceptibility measurements at 3200hz. the ac field strength is fixed at 0.2047mt while bdc is varied. the inset for χ’ shows two transitions for all measurements with the dc field is turned on. figure 8. ac susceptibility measurements at 3200hz. the ac field strength is fixed at 0.307 mt while bdc is varied. the χ‘ curves at higher dc field shows slight signs of developing an intragranular transition. differentiating ac and dc field effects on the magnetic susceptibility ac, varying dc). when increasing the ac field strength, the intergranular transition (in-phase) broadens and becomes less negative. only when the fixed dc field is applied does the intragranular transition appear. the intragranular transition is seen to be independent of the ac field strength. on the other hand, increasing the ac field strength causes the intergranular loss peak (out-of-phase) to decrease in height, broaden and shift to lower temperature. this behavior is consistent with the observed behavior of the intergranular loss peak in other studies (ishida and goldfarb, 1990, sarmago and singidas, 2004, shinde, et al., 1990). from this observation, it appears that the phase coherence between order parameters of individual grains is affected by the amplitude of the applied magnetic field. when the dc field is varied, the susceptibility data behaves differently. as dc field strength is increased, the intergranular transition (in-phase) does not broaden significantly and the saturation values do not become less negative. increasing the dc field strength causes an increase in height and broadening, but no shifting to lower temperature of the intergranular loss peak (out-of-phase). the lack of sensitivity of the intergranular loss peak to the dc field has also been observed in other studies (couach and khoder, 1991) the decrease in saturation values in the in-phase susceptibility reflects the material’s decreased ability in expelling the applied magnetic field. increasing the ac field strength reduces the material’s ability to shield its interior. increasing the dc field strength however does not cause the same effect. essentially superimposing a dc field science diliman 31 figure 9. ac susceptibility measurements at 3200hz. the ac field strength is fixed at 0.409 mt while bdc is varied. the inset for χ’ shows two transitions for susceptibility measurements obtained for bdc>=1.56mt figure 10. ac susceptibility measurements at 3200hz. the ac field strength is fixed at 0.512 mt while bdc is varied. the inset for χ’ shows two transitions for measurements done at bdc>=0.72mt. afalla, sarmago table 1. susceptibility behavior with increasing dc and ac fields in phase (χ’) out of phase (χ”) increasing ac field strength (constant dc field) -intergranular transition broadens -saturation values decrease -peak height decreases -fwhm increases -peak moves to lower temperature increasing dc field strength (constant ac field) -intragranular transition widens in temperature -intergranular transition broadens (slightly) -saturation values are constant -peak height increases -fwhm slightly increases -movement of peak to lower temperature less significant introduces an offset in the applied field and the sample is still able to shield its interior by the same amount despite the range of dc field strength values applied. the corresponding out-of-phase reflects the material’s losses incurred during the transition from normal to superconducting. increasing the ac field strength causes the intergranular losses to persist into lower temperatures. the material loses energy to expel the field even at the lower temperatures. other studies (couach and khoder, 1991) have postulated that as bac increases, shielding breaks down when the induced currents needed to shield out bac exceeds the critical currents of the intergranular weak links. the field thus penetrates more of the volume of the sample. the currents induced in the grains contribute to the dissipation. eddy currents travel through the surface of the material, shielding the interior. when eddy currents traverse through the normal surface material, it crosses grain boundaries when it achieves sufficient phase coherence. the weak links (provided by the grain boundaries) act as resistance encountered by the eddy current. increased resistance translates to increased heating in the material (quirion, et al. 2004). increasing the dc field strength on the other hand, increases the intergranular loss peak. although the energy loss increases, the intergranular loss peak does not persist to lower temperature. this and the saturation values that have no dependence on dc field strength, show that the material utilizes more energy to expel the dc field, but is able to shield its interior by the same amount. however, using the range of fields used in this study, the effects caused by the ac and dc fields have shown no concrete delineation between the meissner and mixed states. an eddy current model may be best suited to interpret the data presented. the behavior of the intergranular transitions and loss peaks are gradual. previous studies (singidas and sarmago, 2004, torralba and sarmago, 2004 olbinado and sarmago, 2004) have utilized an eddy-current based model for ybco, without a superimposed dc field. the eddy current model previously proposed (sarmago and singidas, 2004) has been successful in explaining the behavior of the susceptibility in the absence of vortices. the addition of the dc field in this experiment has not deviated from the predictions of the model. this suggests that for this range of fields, it is not necessary that vortices be assumed present and that an electromagnetic framework is sufficient in explaining susceptibility behavior. conclusion ac suceptibility data gathered for a ybco sample shows that when the dc excitation field is fixed, increasing the ac field strength causes the in phase susceptibility saturation values to decrease. the inphase susceptibility reflects the sample’s ability to shield its interior from the excitation field, at larger fields, greater area of the sample is penetrated. meanwhile, the out of phase susceptibility reveals that the intergranular loss peak broadens, decreases in peak height and shifts to lower temperature. the out of phase susceptibility reflects the sample’s losses incurred in shielding its interior. increasing the ac field strength causes losses to persist to lower temperature. however, when the dc field strength is varied and the ac field strength is fixed, susceptibility data behaves differently. the in-phase susceptibility shows constant saturation values, indicating that the sample is able to shield the same amount of material from the total excitation field, despite increasing the 32 science diliman differentiating ac and dc field effects on the magnetic susceptibility dc field strength. the out of phase susceptibility data show that the intergranular loss peak increases in peak height, maintains its width and shows no shifting to lower temperatures. this indicates that energy needed to shield the sample by the same amount increases. the increase in field strength does not cause losses to persist to lower temperature. it is recommended that an electromagnetic (eddy current based) framework be utilized in order to gain more insight in the mechanism by which ac field strength effects differ from dc field strength effects. references k.a. muller, m. takashige and j.g. bednorz. 1987. “flux trapping and superconductive glass state in la2cuo4-y:ba”, phys. rev. lett. 58(11): 1143-1146. m. polichetti, m.g. adesso, and s. pace. 2003. “third harmonicsof the ac magnetic susceptibility: a method for the study of flux dynamics in high temperature superconductors,” eur. phys. j b 36, p.27-36. c. poole, h. farach and r. creswick. 1995. superconductivity, chapter 10, academic press, inc, san diego. a.c. rose-innes and e.h. rhoderick. 1978. introduction to superconductivity 2nd ed. pergamon pess. t. ishida and r.b.goldfarb. 1990. “fundamental and harmonic susceptibilities of yb2cu3o7-δ”, phys. rev. b, 41(13):8937-8948. y. enomoto and k. okada. 1996. “the ac magnetic response in type-ii superconductors”, j. phys.: condens. matter 8(33):l445-l453. r.v. sarmago and b.g. singidas. 2004. “low field ac susceptibility of ybco: the frequency and field dependence of intraand intergrain coupling losses in the absence of vortices”. supercond. sci. technol. 17(9):s578-s582. k. yamamoto, h. mazaki, h. yasuoka, s. katsuyama, and k. kosuge. 1992. “harmonic susceptibilities of a sintered oxide superconductor”. phys. rev. b. 46(2):1122-1129. m.j. qin and x.x.yao. 1996. “ac susceptibility of high temperature superconductors”. phys rev b. 54(10):75367544. y. wang, l. li, m. j. naughton, g. d. gu, s. uchida, and n. p. ong. 2005.“field-enhanced diamagnetism in the pseudogap state of the cuprate bi2sr2cacu2o8 superconductor in an intense magnetic field” phys. rev. lett. 95 247002. c. d. jeffries, q. h. lam, y. kim, c.m. kim, a. kettl, m.p. klein. 1989. “nonlinear electrodynamics in the granular superconductor yba2cu3o7-δ: experiments and interpretation” phys. rev. b. 39(16):11526-11537. s.l. shinde, j. morrill, d. goland, d.a. chance and t. mcguire. 1990. “ac susceptibility and grain-boundary pinning strengths in yba2cu3o7-δ and yba2cu2.985ag0.015o7-δ phys. rev. b 41 13. j.s. shilling, j. diederichs, s. klotz, and r. sieburger. 1991. “ac susceptibility studies of superconducting properties under high hydrostatic pressure”. magnetic susceptibility of superconductors and other spin systems. plenum press, new york, p107. e. aksu, a. gencer, h. yilmaz, s. neriz. 2003. “fundamental and high-order harmonic susceptibilities of a bscco (2223) bulk superconductor” physica c 391(1): 67-74. y. yang, c.beduz, z. yi and r.g. scurlock. 1992. “ac susceptibility of high-tc superconductors hysteresis and intergranular critical state” physica c 201(3-4): 325-326. m. couach and a.f. khoder. 1991. “ac susceptibility responses of superconductors: cryogenic aspects, investigation of inhomogeneous systems and of the equilibrium mixed state”. magnetic susceptibility of superconductors and other spin systems. plenum press, new york, p25. a.f. khoder and m. couach. 1991. “early theories of x’ and x” of superconductors: the controversial aspects”. magnetic susceptibility of superconductors and other spin systems. plenum press, new york, p107. j. deak, m. mcelfresh, j. clem, z. hao, m. konczykowski, r. muenchausen, s. foltyn, and r. dye. 1994. “irreversibility line in ybco thin films: correlation of transport and magnetic behavior” phys rev b 49(9):6270-6279. science diliman 33 afalla, sarmago bertman and m.strongin. 1966. “ac susceptibiilty transition in type ii superconductors and surface critical currents” phys rev. 147(1):266-272. d. quirion, f. lefloch, m sanquer. 2002. “transport and heating effect in proximity superconducting structures” physica e 12(1-4): 934-937. rv sarmago and mvs torralba. 2004. “harmonic response of coupled and uncoupled ybco” supercond. sci. technol. 17(12):1381–1388. rv sarmago and mp olbinado. 2005. “ac loss intrinsic to mgb2 at low magnetic fields” supercond. sci. technol. 18(3):307-310. 34 science diliman sd-sample article m.p.b. espino and j.p. mendoza 51 science diliman (july-december 2013) 25:2, 51-66 determination of monochloroacetic acid in swimming pool water by ion chromatography-conductivity detection maria pythias b. espino* institute of chemistry university of the philippines diliman jamie p. mendoza natural science research institute university of the philippines diliman abstract in this study, an analytical method involving ion chromatography with c o n d u c t i v i t y d e t e c t i o n w a s d e v e l o p e d a n d o p t i m i z e d f o r t h e d e te r m i n a t i o n of m o n o c h l o r o a ce t i c a c i d i n s w i m m i n g p o o l w a te r. t h e ion chromatographic method has a detection limit of 0.02 mg l -1 and linear range of 0.05 to 1.0 mg l-1 with correlation coeff icient of 0.9992. t h e m e t h o d i s r e p r o d u c i b l e w i t h p e r c e n t r s d o f 0 . 0 5 2 % ( n = 1 0 ) . t h e r e c o v e r y o f m o n o c h l o r o a c e t i c a c i d s p i k e d i n d i f f e r e n t w a t e r t y p e s (bottled, tap and swimming pool water) ranged from 28 to 122%. in dilute solutions, chloride and bromide were simultaneously analyzed along with m o n o c h l o r o a c e t i c a c i d u s i n g t h e o p t i m i z e d m e t h o d . c h l o r i d e a n d bromide have detection limits of 0.01 to 0.05 mg l-1, respectively. the usefulness of the ion chromatographic method was demonstrated in the analysis of monochloroacetic acid in swimming pool water samples. in such highly-chlorinated samples, an ag/h cartridge was used prior to the ion chromatographic determination so as to minimize the signal due to chloride ion. monochloroacetic acid was detected in concentrations between 0.020 and 0.093 mg l-1 in three of the six swimming pool water samples studied. the presence of monochloroacetic acid in the swimming p o o l w a t e r s a m p l e s s u g g e s t s t h e p o s s i b l e o c c u r r e n c e o f o t h e r disinfection by-products in these waters. keywords: monochloroacetic acid, chloride, bromide, water, ion chromatography _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online determination of monochloroacetic acid in swimming pool water 52 introduction water is impor tant in sustaining life. to safeguard human and ecological health, contaminants must be absent or kept at the minimum possible levels. because water is used for human consumption, the highest quality of water should thus be maintained. it is common to disinfect water by ozonation or chlorination to prevent the spread of disease. however, disinfection by-products are unintentionally produced during water treatment. when bromide-containing water is treated using ozone, the potentially carcinogenic bromate may be formed (von gunten and hoigne 1994, who 2008). chlorination of water containing naturally-occurring organic matter, on the other hand, results in the formation of a variety of disinfection byproducts (liang and singer 2003, richardson and others 2007). chlorine species such as hocl and oclreact with humic and fulvic substances in water to form the regulated disinfection by-products including haloacetic acids and trihalomethanes. haloacetic acids are more soluble in water and are reported to be as potentially harmful as the commonly-analyzed trihalomethanes (nieuwenhuijsen and others 2000, richardson and others 2007, plewa and others 2010, pals and others 2011). the us epa regulates f ive haloacetic acids in drinking water, which are collectively known as haa5. monochloro-, dichloro-, trichloro-, monobromoand dibromoacetic acids constitute the haa5 and these have a total allowable limit of 60 ug l-1 in drinking water (us epa 2009). the 2007 philippine national standards for drinking water (pnsdw) drawn from the world health organization (who) register of standard values for disinfection by-products include three haloacetic acids namely: monochloroacetic acid at 0.02 mg l-1, dichloroacetic acid at 0.05 mg l-1, and trichloroacetic acid at 0.2 mg l-1 (pnsdw 2007, who 2008). of these compounds, only dichloroacetic acid is categorized as group b or possibly carcinogenic to humans by the international agency for research on cancer (who iarc 2004). nevertheless, monochloroacetic acid and trichloroacetic acid have been reported to exhibit cytotoxicity, genotoxicity, mutagenicity and teratogenicity in animal studies (liang and singer 2003, plewa and others 2010, pals and others 2011). water is important not only for drinking but also for bathing and cleaning. likewise, it is essential in recreational activities such as swimming, diving or water aerobics. swimming in pools, for example, provides exercise, relaxation, therapy and wellness to man. for hygiene and health protection, swimming pool water is usually disinfected. it has been shown that microorganisms can thrive in swimming pools and cause outbreak of disease (friedman and others 1999, leoni and others 1999). chlorination is the disinfection method of choice for swimming pools because of the residual effects of chlorine. in some countries like germany, it is recommended to maintain chlorine levels of 0.3 to 0.6 mg l-1 or higher to safeguard the wellbeing m.p.b. espino and j.p. mendoza 53 of the swimmers (uhl and hartmann 2005). chlorinated swimming pool water has more organic matter than chlorinated drinking water because of continuous inputs from the swimmers. as a consequence, disinfection by-products including haloacetic acids are formed in swimming pools. likely, recirculation or reuse of this water may result in disinfection by-products accumulation. lee and others (2010) demonstrated that haloacetic acids represent over 60% of the disinfection byproducts found in swimming pool waters in korea which were treated with chlorine, ozone-chlorine, or electrochemically-generated mixed oxidants. in their chlorinated water samples, haloacetic acids were measured at 14.1 to 636 ug l-1 concentrations. catto and others in 2012 reported haloacetic acids in water from two swimming pools in canada with concentrations of less than the limit of detection (<lod) to 201.0 ug l-1. in swiss swimming pool waters, the haloacetic acids ranged from 0.9 to 240 ug l-1 where the concentrations of monochloroacetic acid alone were 11117 ug l-1 (berg and others 2000). haloacetic acids in water samples from swimming pools in portugal were detected in concentrations between 0.1 and 72.9 ug l-1 (sa and others 2000). the monochloroacetic acid in these samples ranged from 0.6 to 13.2 ug l-1. haloacetic acids in drinking water are commonly analyzed using us epa method 552.2. this method involves solvent extraction with methyl tert-butyl ether (mtbe), methylation with 10% sulphuric acid in methanol, and determination by gas chromatography-electron capture detection or gc-ecd. studies on the analysis of haloacetic acids in swimming pool water are also carried out using us epa method 552.2 (lee and others 2010, catto and others 2012). berg and others (2000) used diazomethane to derivatize the haloacetic acids before determination by gas chromatography-mass spectrometry or gc-ms. sa and others (2012), on the other hand, used dimethyl sulphate for derivatization and headspace solid phase microextraction followed by gc-ecd. electrophoresis has also been demonstrated to be an alternative method for haloacetic acids analysis particularly in drinking water. haloacetic acids may be extracted by solid-phase extraction (spe) and determined by capillary electrophoresis with diode array detection (martinez and others 1998), capillary electrophoresis with contactless conductivity detection (kuban and others 2012) or microchip capillary electrophoresis with capacitatively coupled contactless conductivity detection (ding and rogers 2010). capillary zone electrophoresis with direct uv detection and contactless conductivity detection w a s u s ed by lo p ezav i l a a n d o t h e r s ( 2 0 0 3 ) . ca r r e r o a n d ru s l i n g ( 1 9 9 9 ) demonstrated the use of high pressure liquid chromatography and an electrochemical detector coated with a f ilm of naf ion and dido-decyldimethylammonium bromide in haloacetic acids determination. ion chromatography or ic is another technique for haloacetic acids analysis. direct injection ion chromatography and mass determination of monochloroacetic acid in swimming pool water 54 spectrometry (ms) were used to determine trace levels of haloacetic acids in drinking water (matthew and others 2009). solid-phase extraction followed by ion-pair liquid chromatography with electrospray ionization-mass spectrometry was also used to study haloacetic acids in different water samples including tap, river and swimming pool waters (loos and barcelo 2001, prieto-blanco and others 2012). sun and gu (2007) used ion chromatography with suppressed conductivity to determine haloacetic acids in chlorinated hospital effluents. monochloro-, monobromoand bromochloroacetic acids were reported to be detected in metro manila drinking water samples (rodriguez and espino 2010). only monochloroacetic acid was quantif ied and it was found to be in 19-157 ug l-1 concentrations. the analytical method was a modif ied us epa method 552.2 in which diethyl ether was used as extraction solvent instead of mtbe, resulting in low recoveries. this preliminary study demonstrated that haloacetic acids may be formed in the water supplies in metro manila. it was thus deemed that haloacetic acids may also occur in recreational waters such as in swimming pools where expectedly the water source would come from the main distribution lines of the drinking water supply. the aim of this present study was to develop and optimize an ion chromatographicconductivity detection method for monochloroacetic acid in swimming pool water. if detected in the water samples, monochloroacetic acid may serve as a marker for the occurrence of disinfection by-products including the other priority haloacetic acids. experimental method chemicals and solvents chloride and bromide analytical standards, both labeled ic standard in 100 ml of 1 0 0 0 µ g m l 1 , w e r e p u r c h a s e d f r o m f l u k a ( b u c h s , s w i t z e r l a n d ) . t h e monochloroacetic acid analytical standard (99.9% chloroacetic acid, 1000 mg/ ampule) was purchased from sigma-aldrich (st. louis, mo, usa). the seven calibration solutions of these standards were in the range of 0.05 to 1 mg l-1. all solutions were prepared in absolute® distilled water (asia brewery inc. , philippines). the mobile phase was a mixture consisting of 3.2 mm na 2 co 3 and 1.0 mm nahco 3 prepared by dissolving appropriate amounts of nahco 3 and na 2 co 3 (j.t. baker, philipsburg, nj, usa) in distilled water. the 100 mm h 2 so 4 regenerant solution used in the anion conductivity suppressor device was prepared from 18 m h 2 so 4 solution (labscan, bangkok, thailand). m.p.b. espino and j.p. mendoza 55 ordinary tap water, swimming pool water and bottled water (summit® and evian® bottled drinking water purchased from a local supermarket) were used in spiking and recovery experiments. sample collection and preparation surface water samples were taken from six quezon city swimming pools in august 19-28, 2012. these pools are rectangular in shape and small to medium in size, typical of swimming pool facilities in metro manila. these were generally f illed with chlorinated water from the tap. the water samples were collected in clean 500-ml pet bottled water containers; headspace was avoided during collection. the containers were then sealed, covered on the outside with paper, and brought immediately to the laboratory for analysis. the samples were f iltered through 125 mm whatman grade no. 40 f ilters (whatman, nj, usa). for chloride and bromide determination, the samples were diluted 20 to 150 times with distilled water to appropriate concentrations such that the analytes can be measured against the calibration solutions. before injection into the ion chromatograph, the samples were f iltered using 0.45 um nylon syringe f ilters (whatman, nj, usa). for monochloroacetic acid determination, undiluted swimming pool water samples were passed through 2.5 cc dionex ii onguard ag/h cartridges (thermo scientif ic, waltham, ma, usa). prior to use, the cartridges were conditioned with 15 ml distilled water. eight milliliters of the samples were passed through the cartridges. the f irst 6 ml fractions were discarded, while the last 2 ml fractions were used for monochloroacetic acid analysis. the swimming pool water samples were also measured for ph using a milwaukee ph 600 pocket-sized ph meter (milwaukee, nc, usa) as well as ultraviolet absorbance at 254 nm using a uv mini 1240 uv-vis spectrophotometer (shimadzu, kyoto, japan). instrumentation a m e t r o h m 8 8 1 c o m p a c t i c p r o ( m e t r o h m a g , h e r i s a u , s w i t z e r l a n d ) i o n chromatographic system was used. the ion chromatograph was equipped with a sample and eluent degasser, chemical suppressor device, high-pressure pump, sixport injection valve, column heater, and conductivity detector. the ions were separated on an anion column (metrohm metrosep a supp 5-150) with dimensions of 150 mm x 4.0 mm and a stationary phase material containing polyvinyl alcohol plus quaternary ammonium groups. the guard column used was a metrohm 5 mm x 4 mm metrosep a supp 4/5. samples and standard solutions were introduced into a f ixed 20-ul loop using a 10-ml syringe. the 3.2 mm na 2 co 3 -1.0 mm nahco 3 determination of monochloroacetic acid in swimming pool water 56 mobile phase had a constant flow rate of 0.50 ml min-1 which resulted in a total run time of 13 min. monochloroacetic acid, chloride and bromide elute at 6.3, 7.1 and 10.5 min, respectively. data acquisition and processing were performed using magic net 2.2 chromatography software. analysis and quantitation twenty microliters of the calibration solutions and water samples were injected into the ion chromatograph. blank solutions (distilled water) were injected at the start of the analysis and after each injection of relatively high concentrations of the analytes to flush the system as well as avoid baseline drift. no peaks were observed at the retention times of the analytes in the blank solutions. the target analytes were separated on the anion analytical column using a 3.2 mm na 2 co 3 -1.0 mm nahco 3 mobile phase at 25 oc. all measurements were done in three replicates. more replicate measurements were done for validation, specif ically for recovery and detection limit determinations. monochloroacetic acid, chloride and bromide in the water samples were quantif ied by external calibration and linear regression techniques. data analyses including computations of detection limits, recoveries, concentrations and standard deviations were performed using excel microsoft office 2 0 0 7. results and discussion ion chromatographic determination of monochloroacetic acid, chloride and bromide the analytical determination of monochloroacetic acid in water was performed on an anion column and initially using different mobile phases of various proportions of na 2 co 3 and nahco 3 . at the f inal conditions of 3.2 mm na 2 co 3 -1.0 mm nahco 3 mobile phase, 0.5 ml min-1 flow and 25oc, monochloroacetic acid eluted at 6.3 min. monochloroacetic acid is detectable in water solutions containing low concentrations of chloride and bromide. the simultaneous determination of the three analytes is shown in figure 1 where monochloroacetic acid, chloride and bromide were separately eluted on the anion column at the optimum conditions. the linear range for the determination of these analytes was from 0.05 to 1.0 mg l-1 with correlation coeff icients, r2, of 0.9986-0.9993 (figure 2). the instrument detection limits (idl) were 0.01, 0.02 and 0.05 mg l-1 for chloride, monochloroactic acid and bromide, respectively (table 1). the repeatability of the determination method was good with percent rsd of 0.052-0.27% for these analytes. the 0.02 mg l -1 detection limit for monochloroacetic acid using this ion chromatographic method is comparable to that of the gc-ecd method developed m.p.b. espino and j.p. mendoza 57 figure 1. ion chromatograms of 0.6 mg l-1 monochloroacetic acid (mcaa), chloride and bromide spiked in [a] bottled water and [b] tap water. conditions: 3.2 mm na 2 co 3 /1.0 mm nahco 3 mobile phase, 0.5 ml min-1 flow rate, 20 ul injection volume. y = 0 .0 4 7 9 x 0 .0 0 1 4 r 2 = 0 . 9 9 9 2 y = 0 .2 6 3 1 x + 0 .0 0 0 5 r 2 = 0 . 9 9 8 6 y = 0 .0 9 0 1 x + 0 .0 0 7 3 r 2 = 0 . 9 9 9 3 0 0 .0 5 0 .1 0 .1 5 0 .2 0 .2 5 0 .3 0 0 . 2 0 .4 0 .6 0 .8 1 1 . 2 c o n ce n tr a ti o n , m g l -1 a re a, ( µ s /c m )x m in m ca a c hlo ride bro m ide figure 2. calibration plots of 0.05 to 1 mg l-1 monochloroacetic acid (mcaa), chloride and bromide solutions (n=3 for each concentration). determination of monochloroacetic acid in swimming pool water 58 and optimized by rodriguez and espino (2010). using gc-ecd, monochloroacetic acid that was derivatized with acidif ied methanol had a quantitation limit of 0.0169 mg l -1. however, this method had low recoveries mainly due to the sample preparation steps involved, including microextraction, derivatization and preconcentration. table 2 summarizes the recoveries of monochloroacetic acid spiked in two brands of commercial bottled water, tap water and swimming pool water and analysed by ion chromatography-conductivity detection. at spike levels of 0.1 to 0.6 mg l-1, the recoveries were between 43 and 122%. in the gc-ecd method, the recovery of monochloroacetic acid spiked in ultrapure water was only 16.4%. the detection limit for monochloroacetic acid in this present study is also comparable to the detection limits of other methods reported in literature. sun a n d g u ( 2 0 0 7 ) s t u d i e d h a l o a c e t i c a c i d s i n h o s p i t a l e f f l u e n t s u s i n g i o n chromatography and reported a monochloroacetic acid detection limit of 0.01235 mg l-1. hplc with electrochemical detection was also used for monochloroacetic acid determination with a relatively high detection limit of 10 mg l-1 (carrero and rusling 1999). various electrophoresis methods were also reported in which the detection limits were 2.1-2.7 mg l-1 (ding and rogers 2010), 0.044 mg l-1 (kuban and others 2012), and 0.005 mg l-1 (martinez and calull 1998). matthew and others (2009) studied haloacetic acids in drinking water using ion chromatography-mass s p e c t r o m e t r y w i t h a s u p e r i o r d e t e c t i o n l i m i t o f 2 . 1 5 8 x 1 0 5 m g l 1 f o r monochloroacetic acid. it should be noted that the recoveries of these methods varied widely as the sample preparation involved different extraction and pretreatment techniques using a variety of sorbent materials. monochloroacetic acid 6.3 0.05-1.0 0.9992 0.052 0.02 (0.03) (15) chloride 7.1 0.05-1.0 0.9986 0.27 0.01 (0.05) (8) bromide 10.5 0.05-1.0 0.9993 0.11 0.05 (0.1) (8) analyte retention time, min (sd), n=3 linear range, mg l-1 correlation coeff icient, r2 repeatabilitya of peak area, (µs/cm) x min (% rsd) n=10 idlb mg l-1 a interday analyses of a 1 mg l-1 standard solution for 6 days b measured using a 0.05 mg l-1 standard solution; idl= sd x 3.143 (n=7; student’s t-value at 99% confidence level) table 1. analytical performance of the optimized ion chromatographic method m.p.b. espino and j.p. mendoza 59 for chloride and bromide determination in this study, the detection limits were a little higher than those of the sequential chemical and co 2 -suppressed ion chromatographic method reported by de vera and espino in 2011. with chemical and co 2 suppression, the background conductivity was reduced, which resulted in lower detection limits for chloride and bromide at 0.001 and 0.007 mg l -1, respectively. this tandem suppression technique was used solely for the analysis of inorganic anions namely fluoride, chloride, bromide, nitrate, phosphate and sulphate. the recoveries of these anions in ultrapure water ranged from 94 to 154%. in this present study wherein the ion chromatographic method involved only chemical suppression for detection, the recoveries of chloride and bromide in bottled, tap and swimming pool waters ranged between 28 and 121%. this wider range of recoveries may be attributed to the different water matrices that were used. overall, the optimized ion chromatography with conductivity detection method developed in this study has low detection limits, relatively low linear concentration range for trace analysis, good reproducibility, and acceptable recoveries. bottled water: a low (0.1 mg l-1) 100 (17) 73( 4) 43 (10) high (0.4 mg l-1) 117 (15) 82( 4) 67 ( 7) b low (0.2 mg l-1) 70 (17) 71 ( 2) 51 (10) high (0.4 mg l-1) 65 (16) 90 ( 8) 72 (14) tap water low (0.2 mg l-1) 82 (14) 121 (14 ) 72 ( 6) high (0.6 mg l-1) 76 ( 2) 79 ( 1) 86 ( 2) swimming pool water* low (0.2 mg l-1) 122 (36) 102 (15) 28 ( 4) high (0.6 mg l-1) 43 ( 0) 95 ( 1) 65 ( 2) a= summit®; b= evian®; *swimming pool water sample that does not contain monochloroacetic acid was used spiked water % recoveries (sd), n=3 monochloroacetic acid chloride bromide table 2 percent recoveries in d ifferent water matrices determination of monochloroacetic acid in swimming pool water 60 analysis of monochloroacetic acid in swimming pool water the applicability of the ion chromatography-conductivity detection method was tested in the analysis of monochloroacetic acid in swimming pool water samples. i n w a t e r c o n t a i n i n g h i g h c o n c e n t r a t i o n s o f c h l o r i d e , t h e d e t e c t i o n o f monochloroacetic acid was found to be affected by the large chloride ion signal. thus, the analysis of monochloroacetic acid and chloride in highly chlorinated water samples requires special sample preparation. dilution was necessary particularly for chloride determination because the concentration was anticipated to be above the highest concentration in the established linear range of the optimized method. monochloroacetic acid was separately analysed after chloride was removed from the sample or after its concentration was minimized. an ag/h cartridge was used for the sample preparation in the analysis of monochloroacetic acid in chlorinated swimming pool water. this cartridge consists of layers of ag and h resins that retain chloride, bromide, iodide and carbonate ions. figure 3 compares the ion chromatograms of a swimming pool water sample that was not passed through an ag/h cartridge and the same water sample that was passed though an ag/h cartridge. the use of an ag/h cartridge minimized the signal due to chloride allowing the detection and quantitation of monochloroacetic acid. figure 3. ion chromatograms of [a] a swimming pool water sample and [b] the same swimming pool water sample after passing through an onguard ag/h cartridge. conditions: 3.2 mm na 2 co 3 /1.0 mm nahco 3 mobile phase, 0.5 ml min-1 flow rate, 20 ul injection volume. m.p.b. espino and j.p. mendoza 61 in a spiking experiment of 0.6 mg l-1 monochloroacetic acid and chloride in distilled water using ag/h cartridges, the retention time of monochloroacetic acid on the anion column was invariable at 6.3 min and the recovery was good at 122% (n=6, sd 13). monochloroacetic acid, chloride and bromide concentrations in six swimming pool water samples were determined using the optimized ion chromatographyconductivity detection method. because the ag/h cartridge removes the inorganic anions, chloride and bromide were determined separately without pre-treatment using ag/h cartridges. monochloroacetic acid was detected in the swimming pool water samples with an average ph of 7.1 and high chloride concentrations. monochloroacetic acid was found in three of the six samples with concentrations r a n g i n g f r o m 0 . 0 2 0 to 0 . 0 9 3 m g l -1 ( ta b l e 3 ) . t h e c h l o r i d e a n d b r o m i d e concentrations in all samples were 24-104 mg l-1 and 0.16-7.52 mg l-1, respectively. these water samples registered uv absorbances at 254 nm ranging from 0.002 to 0.060 a.u. , suggesting the presence of dissolved organic compounds which may serve as precursors of monochloroacetic acid. bromide found in the water samples also indicate the possible occurrence of brominated haloacetic acids such as monobromoand dibromoacetic acids belonging to the f ive regulated haloacetic acids. and because monochloroacetic acid was detected in the swimming pool table 3. concentrations of monochloroacetic acid, chloride and bromide in swimming pool water samples a n=3 b measured using water samples diluted 20, 50, 80, 100 or 150x depending on the response which should be within the range of the calibration solutions a 8/19/12 6.8 0.002 0.020 104 5.98 (0) (0) (0) b 8/22/12 6.9 0.010 not detected 66 2.04 (0.69) (0.97) c 8/22/12 6.5 0.002 not detected 24 1.86 (0.34) (0.58) d 8/22/12 7.1 0.012 0.083(0) 67 7.52 (0.96) (1.89) e 8/28/12 7.8 0.014 0.093(0) 53 0.80 (0.50) (0.47) f 8/28/12 7.6 0.060 not detected 24 0.16 (0.13) (0) water sample code sampl ing date ph absorbance at 254 nm mean concentration in mg l-1 (%rsd)a monochloroacetic acid chlorideb bromideb determination of monochloroacetic acid in swimming pool water 62 water samples, it could be expected that other disinfection by-products may also be present. the concentrations of monochloroacetic acid found in these swimming pool samples are within the 0.011-0.117 mg l-1 range of monochloroacetic acid concentrations in swiss swimming pools reported by berg and others (2000). however, the concentrations are relatively high compared to the 0.0006-0.0132 mg l-1 monocloroacetic acid concentrations in swimming pools in portugal (sa and others 2012). to the best of our knowledge, this is the f irst study on the occurrence of the disinfection by-product monochloroacetic acid in the swimming pools in the philippines. although disinfection by-products such as trihalomethanes and haloacetic acids have been studied in the local drinking water supplies (rodriguez and others 2006, rodriguez and others 2010), these compounds have not been studied yet in the local swimming pool waters. ion chromatographyag/h cartridge 0.02 mg l-1 0.05-1.0 122 this study conductivity mg l-1 (0.2 mg l-1 detection spike level in swimming pool water and 0.6 mg l-1 in distilled water) gc-ms mtbe extraction; 2 ng l-1 0-0.005 96 berg and diazomethane mg l-1 (100-430 others derivatization ng l-1 spike 2000 level in nanopure water) gc-ecd mtbe extraction; n o t n o t n o t lee and 10% sulfuric acid reported reported reported others in methanol 2010 derivatization gc-ecd mtbe extraction; 0.1-1.6 n o t n o t catto and 10% sulfuric acid µg l-1 reported reported others in methanol 2012 derivatization gc-ecd mtbe extraction; 1.3 µg l-1 n o t n o t simard 10% sulfuric acid reported reported and others in methanol 2013 derivatization ultraperformance membrane-protected 0.07 µg l-1 1-150 91.6-95.1 nsubuga liquid chromatographymicro-solid phase µg l-1 (10 µg l-1 and diode array detection extraction spike level in basheer swimming 2013 pool water) table 4. comparison of analytical methods for monochloroacetic acid in swimming pool water method performance analytical technique sample preparation lod linearity % recoveries reference m.p.b. espino and j.p. mendoza 63 table 4 lists analytical methods used in the determination of monochloroacetic acid in swimming pool water. the main advantage of the ion chromatographic method used in this present study is that it eliminates the use of harmful derivatization reagents such as diazomethane and extraction solvents such as mtbe. solvent extraction and gc-ecd or ms are the common techniques in monochloroacetic acid analysis and when combined with sample preconcentration steps, very low detection limits can be achieved such as those reported by catto and others (2012), and berg and others (2000). although the detection limit of monochloroacetic acid using the ion chromatographic method is relatively high, this method is less costly and easier to use than the gc-based methods. the results of this initial study prompt the need to examine further the occurrence of disinfection by-products in swimming pool waters in order to protect the public from exposure to these compounds. extensive data are required as basis for setting up measures in minimizing the levels of disinfection by-products in public swimming pools. the analytical method involving ion chromatography with conductivity detection developed in this study provides a simple method for use in determining monochloroacetic acid in swimming pool water. in addition, this method has potential application in the determination of monochloroacetic acid in chlorinated drinking water. the method may be used by laboratories of water providers and regulatory agencies in the philippines. if used in drinking water analysis, a pre-concentration step may be necessary because drinking water contains less organic matter than swimming pool water; thus, the monochloroacetic acid concentration may be lower. the 2007 philippine national standards for drinking water stipulates that monochloroacetic acid in our drinking water should not exceed 0.02 mg l-1. the ion chromatographic method presented in this study has a detection limit of 0.02 mg l-1. this is thus an available method for detecting monochloroacetic acid in concentrations above the guideline value for drinking water. this method can still be modif ied and improved if used in the determination of very low concentrations of monochloroacetic acid (<0.02 mg l-1) especially in relatively clean water such as drinking water. the detection limit can be lowered with the use of spe and preconcentration steps and more sensitive detection techniques such as mass spectrometry in ion chromatography. nonetheless, this method in its present form can still be used in compliance monitoring of monochloroacetic acid in the local water supplies. determination of monochloroacetic acid in swimming pool water 64 conclusions ion chromatography with conductivity detection is applicable for the determination of monochloroacetic acid in swimming pool water. this method has low detection limit at 0.02 mg l-1. it is reproducible and does not require harmful reagents such as diazomethane and mtbe extraction solvent. low concentration chloride and bromide may be analyzed together with monochloroacetic acid in water using the optimized ion chromatographic method. in high chloride content water samples like swimming pool water, an ag/h car tridge is necessary for monochloroacetic acid determination. the detection of monochloroacetic acid in the water of some swimming pools studied suggest the need to further investigate the occurrence of monochloroacetic acid as well as the other disinfection by-products in swimming pools in order to protect public health. acknowledgments m.p.b. espino thanks the up diliman natural science research institute for the research grant through project no. che-12-2-07. dr. nathaniel diola and engineer mabel globio are gratefully 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z o n a t i o n o f b r o m i d e containing waters: interaction of ozone and hydroxyl radical reactions. environ. sci. technol. 28(7): 1234-1242. world health organization 2008. guidelines for drinking water quality, third edition i n co r p o r a t i n g t h e f i r s t a n d s eco n d a g e n d a , vo l u m e 1 reco m m e n d a t i o n s . g e n eva. available from: http: //www.who.int/water_sanitation_health/dwq/gdwq3rev/en/ world health organization international agency for research 2004. iarc monographs on the evaluation of carcinogenic risks to humans. volume 84, some drinking water d i s i n f e c t a n t s a n d c o n t a m i n a n t s , i n c l u d i n g a r s e n i c . a v a i l a b l e f r o m : h t t p : / / monographs.iarc.fr/eng/monographs/vol84/index.php _______________ maria pythias b. espino, phd <mbespino@upd.edu.ph> is an associate professor at the institute of chemistry, university of the philippines diliman. jamie p. mendoza was a university research associate at the natural science research institute and is currently a graduate student at the college of science, university of the philippines diliman. ocr document 01_device negotiating stakeholder agreements for conservation 47science diliman (january-june 2007) 19:1, 47-63 *corresponding author negotiating stakeholder agreements for conservation: the case of tubbataha reefs, philippines edgardo tongson vice-president programmes, wwf-philippines 4f, jbd plaza, mindanao ave., quezon city 1105 tel.+632-920-7923/7926 fax: +632-426-3927 etongson@wwf.org.ph raoul cola consultant, wwf-philippines many conservation projects fail because local stakeholders share a disproportionate burden of the cost arising from a no-take zone compared to benefits accruing to global and national stakeholders and more powerful groups. conflicts arising from the establishment of marine protected areas are usually caused by outside interests colliding with local interests and priorities. ensuring fishers who will lose access to fishing grounds will be able to negotiate and obtain benefits in return for their losses is key to gaining their support to no-take provisions of a marine protected area. the case of establishing an offshore marine protected area in the tubbataha reefs, in the center of the sulu sea, philippines, offers a practitioners' perspective in reconciling competing interests based on the sharing of costs and benefits that all stakeholders consider satisfactory and equitable. user fees from diver groups and grant payments from outside donors that supported local livelihoods and park operations offer lessons in cost and benefit sharing. the experience highlights the importance of generating stakeholders' agreements based on cost and benefit-sharing as a platform for conservation actions. keywords: marine protected areas, stakeholders, tubbataha, coral reefs, marine biodiversity, conservation abstract tongson, edgardo 48 introduction stakeholders are individuals, groups or institutions sharing common or conflicting concerns, values or interests in a park and who depend on it for some given reason, whether economic, political, social, ecological or even aesthetic. the stakeholder community is a site of both solidarity and conflict, shifting alliances and power structures. people have various interests based on their status and sources of income, and logically, people will look after their own interests. at the onset of any conservation project, the overlapping and differing stakeholder interests will have to be negotiated to arrive at an arrangement beneficial to all stakeholders. negotiation is oftentimes embedded in the participatory processes and used with other terms such as social dialogue, consensusbuilding, mediated agreements, conflict resolution and the like. most people would agree that the sustainable use and management of natural resources requires collaboration between different stakeholders. however, collaboration does not develop by people merely agreeing to it. there are barriers to consensus -building among multiple stakeholders that need to be overcome. there are also considerable differences in interests and power amongst those using or dealing with natural resources. power is not neutral within any given community. there will always be those who possess more influence, higher authority, more knowledge or material wealth, which affect their social, economic and political power. these dynamics strongly influence public decisions including the establishment of conservation areas. these aspects of natural resource management are often weakly dealt by government departments, local institutions and development agencies that are used to centralized top-down approaches to conservation. in some cases, inappropriately designed development projects impede the creation of social capital and the mobilization of internal capital both vital ingredients to developing and sustaining institutions for managing common pool resources. social capital is measured by the quality of social relations, degree of cooperation and trust and willingness to sacrifice one's own selfinterest for the collective good. internal or local capital comprises the skills, financial or in-kind resources that the community can mobilize collectively to achieve a common good. the role of social capital as an important pre-condition to promoting sustainable use of common pool resources has been extensively discussed in the commons literature (ostrom 1990, wade 1994, baland & platteau 1996, agrawal 2002). the experience of integrating conservation and development in the last two decades show that there are few win-win solutions and that there are winners and losers (brandon & wells 1992, larsen et al 1998, mcshane & wells 2004). the lessons point to the role of agreements and win-win platforms as a necessary part of the design, planning and implementation of conservation projects. trade-offs exists between different interests and priorities, most often sharply divided between economic development, social welfare and environmental goals. a large part of the literature is beginning to detail the tradeoffs between sections of society and biodiversity conservation, often suggesting the rich people benefiting from conservation while the poor bear the brunt of the costs (wells 1992, ghimire & pimbert 1997, cartwright 1991, hulme & infield 2001, adams & infield, 2001). lately, conservation agents with their huge financial backing and political connections, have been criticized for imposing their preservationist agendas to the detriment of the majority of poorer, fragmented and more powerless groups particularly indigenous peoples who have less access and voice in decisions affecting their lives (chapin 2004). conservation areas established under these conditions face an uncertain future as displaced user groups resent and may even undermine the continued operation of the conservation area. to avoid conflict, local communities' economic losses should be compensated for in the form of cash payments, goods or services (brandon & wells 1992, abbot & thomas, 2001). ensuring fishers who will lose access to fishing grounds will be able to negotiate and obtain benefits in return for their losses is key to gaining their support to no-take provisions of a marine protected area. the history of conservation projects offers important lessons to the achievement of negotiated solutions as a negotiating stakeholder agreements for conservation 49 platform for a conservation strategy. the conservation literature views negotiation as an essential part of the participatory process leading to a common vision of a conservation area. it is the part of a process in forging win-win outcomes as a platform for developing a shared vision (allen at al 2000; borrini-feyerband 2000, babbit et al 1994). in protected areas settings, the conservation literature cautions on the likely existence of unequal power relationships among stakeholders and potential pitfalls of limiting participation to the rural elite who tend to capture most of the benefits (allen at al 2000, borrinifeyerabend et al 2004). they caution against disenfranchising the poor and marginal groups in the participatory process and encourage practitioners to be more inclusive in their methodologies. in their attempt to be more inclusive, development practitioners developed parallel streams of consultations to reach out to inaccessible groups particularly women, indigenous peoples and other "invisible" groups. in these parallel meetings, the collective aspirations, strategies, expected outcomes are processed and distilled through facilitated exercises. the group elects a representative to articulate their interests and negotiate in their behalf in the bigger multi-stakeholder settings. this way, the plurality of interests and stakes are considered in the visioning and planning exercise. negotiations based on more pluralistic platforms tend to be more credible and respected by stakeholders (borrini-feyerabend 2000). the consultation process applied in tubbataha national marine park (trnmp) takes cue from the need to include the fishers and stakeholders of cagayancillo in the park planning process. this attempt departs from the centralized approaches of national government agencies in establishing protected areas without the participation of affected communities. oftentimes, parks established this way were met with resistance and rejection, as in the case of the mangyan and tagabanua tribes when they rejected attempts in establishing parks in mindoro and coron island, palawan, respectively (nipap 2003). this paper describes the process, results and lessons generated from establishing the tubbataha reefs national marine park (trnmp), an offshore marine protected area in the sulu sea, philippines. 1.1 site description the philippines, together with malaysia and the indonesia, form part of the sulu-sulwesi ecoregion that encompass the "coral triangle" which is known as the global epicenter for coral diversity. the cagayan ridge in the sulu sea is home to extensive coral reefs, underwater sea mounts and a high biodiversity of species and habitat types. located at the center of the sulu sea (n 8°50'677" e 119°55'734"), the tubbataha reefs national marine park (trnmp) is the largest coral atoll in the philippines and the country's only national marine park. the park covers an area of 33,200 hectares and is located about 160 kilometers southeast of puerto princesa city and 80 kilometers southwest of cagayancillo municipality. (fig.1). biologists, oceanographers, ornithologists, fishery and coral experts as well as fishing operators, environmental ngos, scuba divers, dive boat operators and government agencies have taken interest in tubbataha's rich marine life and abundant fishery. lately, the discovery of commercial quantities of natural gas has fuelled a gold rush among energy producers to stake their claims in untapped offshore gas fields in the sulu sea off the coast of eastern palawan. puerto princesa cagayancillo tubbataha reefs sulu sea pacific ocean south china sea fig. 1. map of tubbataha reef natural marine park tongson, edgardo 50 the trnmp reef complex consists of two coral atolls separated by a channel eight kilometers wide. the larger north reef is about 16 kilometers long and 4.5 kilometers wide. the south reef is about 5 kilometers long and 3 kilometers wide. since 1982, biologists, oceanographers and geologists have been fascinated by the manner of reef formation and the high diversity in terms of species numbers and habitat types (alcala 1993, dollar & alcala 1993, dollar1999). the complex harbors 6 species of sharks, 79 algae species and 7 species of seagrass (white & arquiza, 1999). the south islet has a lighthouse and serves as nesting ground for 2 species of turtles and 5 out of 12 species of seabirds (manamtam, 1996). for divers, the more interesting places are the fringing reefs, walls and drop-offs that provide home to 396 species of coral and 463 species of fish (wwf 2004). the united nations education, science and cultural office (unesco) declared tubbataha reefs a world heritage site in 1993. the tubbataha reef is believed to provide the sulu seas and eastern coastline of palawan with fish and invertebrate larvae (alcala, 1993). biologists believe the water current disperses these larvae and enriches the coral reefs and fishing grounds around the sulu sea. studies show the beneficial effects of mpas to surrounding fishing grounds (alcala 1999, alcala & russ 1990, ballantine, 1994, 1995, 1997, 1998; bohnsack, 1994; pdt, 1990; russ and alcala, 1989, 1996). 1.2 legal basis on 11 august 1988, president corazon aquino signed presidential proclamation 306, creating the tubbataha reef national marine park (trnmp), making it the first marine protected area (mpa) in the country. this proclamation also transferred tubbataha's management jurisdiction from the municipal government of cagayancillo to the national government through the denr. by the 1990s, the unique and outstanding natural characteristics of tubbataha have earned enough renown to command international attention. the united nations educational, scientific and cultural organization (unesco) declared it a world heritage site on 11 december 1993. it is the only marine world heritage site in southeast asia. on 19 november 1999 tubbataha was inscribed in the list of wetlands of international importance, also known as the ramsar list. 1.3 issues and threats since the early 1980s, tubbataha has been a prime destination for scuba divers from the philippines and other countries. at the same time, tubbataha has been the prime fishing ground for commercial fishing vessels plying the sulu sea and for fishers from cagayancillo municipality that has jurisdiction over the area. a commercial seaweed farm operated within the reef before it was aborted due to opposition from environmental groups. in 1989, the near pristine condition of the reefs deteriorated due to illegal fishing, including use of explosives, indiscriminate dropping of anchors and unscrupulous collection of wildlife. fishers from china and taiwan encroach into the sulu sea to catch turtles, live fish and sharks using destructive gears. the living coral cover on the outer reefs had decreased by 24 percent (white et al, 2003). in 1992, the condition of the reefs improved but the el niño event in 1998 coincided with the hottest year in recorded history and the worldwide increase in sea surface temperatures affected the reefs. tubbataha lost more than 20% living hard coral since 1998 due to warm water bleaching (white et al, 2003). 1.4 management history a timeline of management events up to the present follows: 1988 park declared by presidential decree 1989 seaweed farm established but stopped in 1990. first draft of park management plan based on limited information 1990 sporadic patrols started to stop illegal and destructive fishing 1991 illegal seaweed farm removed from the park 1992 research expeditions by silliman university to collect baseline data on the coral reef 1993 park management plan re-drafted; illegal activities increase negotiating stakeholder agreements for conservation 51 1993 unesco world heritage status declared 1995 japan international cooperation agency (jica), marine parks center of japan, department of environment and natural resources (denr) and haribon foundation conduct marine and bird studies; presidential task force set up to implement management plan and provide funds; philippine navy assigned to guard park 1996 management plan refined with support from jica, denr, palawan council for sustainable development (pcsd), world wide fund for nature (wwf) and stakeholders in palawan and cagayancillo 1998 protected area management board (pamb) formed; management plans endorsed in a workshop with all stakeholders with support from pcsd, denr, wwf, sulu fund; coral bleaching event kills more than 20% of living coral cover 1999 pamb becomes operational with a park manager appointed and supported by wwf; global environment facility (gef) approves 5-year funding through united nations development program (undp) and wwf to implement park management plan; park ranger station constructed 2000 management plan fully endorsed by the pamb for implementation and user fees based on willingness-to-pay study of wwf established; tubbataha management office (tmo) organized. coastal resources management project (crmp) and the sulu fund jointly implement reef monitoring; pamb & wwf implements ecosystem research & monitoring plan 2001 lgu of cagayancillo establishes five marine protected areas (mpa). wwf launches credit and livelihood program in cagayancillo municipality. 2005 end of gef-undp funded wwf project. participatory stakeholder evaluation conducted. 1.5 stakeholders the stakeholders of tubbataha, as well as in many other protected areas, reflect heterogeneity across stakeholder groups and changing power relationships. table 1 presents the stakeholder organizations, mandates, interests and scale of operation as they relate to the trnmp. 1.5.1 national government agencies the national government agencies comprises the dept of environment and natural resources (denr) and the office of the president. the denr is mandated to manage the protected areas system of the country under the national integrated protected areas act. the former president fidel v. ramos, being an avid diver himself, has brought the powers of his office to trouble shoot the park. the creation of the task force in 1995 by the office of the president was in line with the purpose of establishing tubbataha as a national park. 1.5.2 local government units the local government units of cagayancillo comprising the municipal and barangay governments have a stake in the park as it used to be under their political jurisdiction. the interest of the local government units lies not only in the preservation of the park's resources but also in the generation of funds to benefit the lgu, and to finance social services particularly to its constituency whose livelihood is affected by the cessation of access to park resources. the municipal and barangay government officials of cagayancilllo wanted to regain their management control over the park. their main reason was their exclusion from any benefits derived from the park which was their traditional fishing grounds. the barangay officials wanted not just management control but also extraction rights. these rights were not just limited to fisheries but also to shells, birds and turtle eggs and the establishment of seaweed farm within the park. one reason behind the "aggressive" position of the barangay leadership was that nobody had explained to them and to their constituency the reason behind the sudden ban on extraction activities in tubbataha reef. the mayor, then a member of the executive committee of tubbataha reef national marine parks task force was understandably interested in the municipality's share in the proceeds from tourism in tubbataha reefs. given these explicit aspirations, fixing the user fee tongson, edgardo 52 stakeholder mandate interests level government office of the president execute laws through preservation of resources national national agencies department of national defense (dnd) defense and security enforcement of existing national regulations department of environment and conservation of preservation of resources national natural resources (denr) natural resources department of tourism (dot) tourism promotion preservation of resources national department of budget allocation of financial resources preservation of resources national management (dbm) local government units palawan council for sustainable implementation of ra 7611 preservation of resources; provincial development (pcsd) jurisdiction under ra 7611 provincial government of palawan governance generation of funds for provincial park management and other activities; jurisdiction under ra 7611 municipal government of cagayancillo governance access to park’s resources, local generation of funds for local governance, alternative livelihood barangay government of cagayancillo governance access to park’s resources local and generation of funds for local governance private sector diving tour operators live-aboard dive business preservation of resources local and safe park access cagayancillo fisherfolks fishing access to park’s resources local palawan fishermen fishing access to park’s resources local outside fishermen fishing access to park’s resources local ngos wwf biodiversity preservation of the resource global conservation while addressing concerns of communities in cagayancillo saguda conservation preservation of the resources local research institutions research; applied research preservation of the resources national/local marine science institution (msi), silliman university, western palawan university system and benefit-sharing arrangements were definitely considered key to resolving the issue of management control. while the vice-mayor also lobbied for municipal control of tubbataha reef, he admitted their lack of knowledge and skills in environmental management and their difficulty in stopping the use of destructive fishing methods. he was considering alternative livelihood projects for the residents of cagayancillo as part of the solution to the issue. it appeared that the poverty level in the municipal and barangay level was such that the leadership cannot overemphasize their need to derive economic benefits from the project. surprisingly, the community members were not as adamant as their leadership to regain management control over tubbataha reef. they demanded more intensely to have persons of authority explain to them the reason for banning extractive activities in the park. they also wanted clarification on their participation in park management particularly in areas of decisionmaking and rule enforcement and their share from proceeds derived from the beneficial use of the park. it was only in brgy. nusa that they raised the need to table 1. tubbataha reef stakeholders, mandates, interests and level of involvement in tubbataha negotiating stakeholder agreements for conservation 53 revive their fishing rights in the park. likewise, it was only in brgy. magsaysay that the ban on the slaughter of sea turtles for meat was raised. the generally open position of the rest of the communities and their eagerness to participate augured well for the existing management arrangement. 1.5.3 pcsd between the divergence of interests of the national agencies and those of the municipal and barangay government units, was the provincial government in the person of the governor, who chairs the palawan council for sustainable development (pcsd). the pcsd is mandated to implement the environmental critical areas network of the strategic environmental plan of palawan passed under a congressional act (ra 7611). the pcsd had a stake in exercising its devolved powers under ra 7611 over tubbataha and to manage it under their own terms. 1.5.4 fishing operators the fishing operators in tubbataha catch a range of species from coral associated species such as napoleon wrasses, groupers, lobsters, sharks (for their fins) and sea cucumbers to pelagics and small pelagics. the dominant fishing gears used are long lines, hook and lines, purse seines and ring nets. there are occasional reports of destructive and illegal fishing including the use of dynamite and cyanide. chinese poachers have been reported to enter tubbataha to catch turtles and live fish. the resources extracted by these fishing methods were not limited to fisheries. sea turtle and eggs of sea birds, giant clams and collector's shells were also included (white & palaganas, 1991). the problem of resource extraction in tubbataha reefs is a recent phenomenon resulting from a confluence of factors: resource depletion of other traditional fishing grounds, commercialization of fishing production, modernization & improvement of fishing and sea transport technology and increasing unavailability of farmlands in out-migration areas in the visayas. the lack of effective enforcement allows illegal, unregulated and unreported fishing to prosper in tubbataha. 1.5.5 dive tour operators simultaneous with intensified fishing in the tubbataha reefs was the development of the diving industry in the philippines. the reef hosted the first diving expedition in 1982 and from then on became one of the world's top diving destinations. it is estimated that during march to may of 2006, there are about 80 boattrips and about 1,500 guest-visits to tubbataha's diving spots. total spending is estimated at us$ 1.9 million per year excluding foreign travel. the presence of dive boats discourages illegal fishing activities during the tourist season. members of the diving industry were the first to raise their voices against the fishing operation in the area. because of the prohibitive cost of diving to tubbataha, these divers are mostly rich and influential people. some of them have cross-memberships with ngos, research institutions and government agencies. 1.5.6 ngos the environmental ngos (wwf-philippines, sulu fund, palawan ngo network inc. & saguda) in palawan have always supported the preservation of the tubbataha reef, as evidenced by their involvement in a number of conservation activities benefiting the reef. their involvement has mainly been in information, education and communication campaigns, research and monitoring, community organizing in cagayancillo, policy advocacy, installation of mooring buoys and participation in policymaking bodies. but the remoteness of the tubbataha reefs (the reason for its relatively pristine condition) and their limited resources constrain their participation in on-site activities. later, the environmental legal assistance center (elac) provided the legal services to help the park on its enforcement work. 1.5.7 research institutions research institutions were occasionally present in tubbataha reef. their presence was dictated by research interest and fund availability. silliman university conducted earlier researches in tubbataha in the 1990's and published their findings in 1993 and tongson, edgardo 54 1999 (alcala 1993, dollar & alcala 1993, dollar1999). the ccef and earthwatch volunteer group undertook survey expeditions to monitor reef health in tubbataha reefs in the early 2000's and published their findings. the western palawan university and university of the philippines visayas conducted various studies in 2004. these research institutions wanted to preserve tubbataha reefs as a living laboratory for their scientific and educational use. methods and materials this section describes the process used to ensure participation of the more marginalized stakeholders, reconcile the different interests of stakeholders and generate the agreements that serve as basis in managing and sustaining the park. the process initiated in 1998 involves five steps: (1) conduct of stakeholders' analysis, (2) community workshops with cagayancillo' s small-scale fishers to prepare for their participation in the stakeholders' meeting, (3) small meetings between small-scale fishers and their local government officials to consolidate their positions for the stakeholders meetings, (4) small meetings between personnel of environmental ngos and government agencies to consolidate their positions for the stakeholders meetings; (5) conduct of the stakeholders' meeting where the agreements together with vision, issues and action plan were generated and (6) monitoring and evaluation. stakeholders analysis the stakeholders' analysis was conducted to identify the stakeholders, characterize their interests and determine their capability and potential contribution to manage the park. knowing their capability was important because they would prepare and implement the management plan and the capability of a stakeholder would define its role in the implementation. through the stakeholders' analysis and the extent of their involvement in preparatory meetings and small group workshops, the management measures to be implemented could also be designed to be within their capacity. figure 2 depicts the flow of activities completing the full cycle of project management beginning from an analysis of stakeholder interests to the formulation, implementation and monitoring of the management plan. the cycle culminated with the presentation of the project outcomes, participatory evaluation, generation of lessons and recommendations from a stakeholder workshop in 2005. results 3.1 conduct of stakeholders' analysis the stakeholders were characterized as to mandate, interest, level of involvement in tubbataha (table 1). the detailed explanation of each stakeholder group is presented in the preceding section. the stakeholders in tubbataha reefs vary with respect to their interests and to degree of power they hold. we classified stakeholder groups by their interests, namely: pro-preservation, pro-extraction, fund generation, alternative livelihoods and national security . the following agencies comprise the "preservationist" category: office of the president, department of national defense (dnd), department of environment and natural resources (denr), department of tourism (dot), department of budget and management (dbm), palawan council for sustainable development (pcsd), silliman university, marine stakeholder analysis preparatory meetings negotiated agreements plan formulation baseline information & trends implementation cagayancillo workshops stakeholder validation monitoring & evaluation fig. 2. stakeholder process in the formulation and implementation of the management plan negotiating stakeholder agreements for conservation 55 science institute, saguda ngo, spcp, dive operations and divers, world wide fund for nature philippines. the "pro-extraction" group consists of the municipal and barangay local government units of cagayancillo, cagayancillo fisherfolks, palawan fishers and outside fishers. a large sub-group of fishing operators are usually in the area for 6-7 months in a year coming not only from cagayancillo and palawan but from luzon and visayas and even from china and taiwan. local residents of cagayancillo, known as cagayanen, enjoyed the privilege of fishing on the coral reefs and in the offshore waters by virtue of their residence. outsiders were required to get a fishing permit from the municipal mayor's office in the town center. the coverage of the fishing permit included the tubbataha reefs. the cagayanen are no longer considered a stakeholder by themselves, i.e., separate from the islands' fishing operators or from the constituency represented by its local government. this is because migration from the visayas and the resulting acculturation and intermarriage has made it difficult to differentiate the cagayanen from the new settlers who are engaged in fishing. the municipal lgu, wwf and other ngos constituted another group that promoted alternative livelihoods for affected fishers. those expecting cash benefits from park user fees are the provincial government and cagancillo lgus. the dnd through the philippine navy has a continuous presence in the park. the area is important for safeguarding national security during the terrorist threats emanating from the southern philippines. the diving tour and fishing operators are seasonally physically present although the latter's activities are heavily curtailed by the presence of the philippine navy. in the case of tubbataha, the stakeholders' interests follow the typical conservation-development divide. interests are sharply contrasted between those pushing for more preservationist approaches such as banning fishing within the park and those claiming their rights to extract resources in the park. these interests also differ across scales. interests of the provincial, national and global stakeholders collide with those directly dependent on the park for livelihoods and government allotments. not only are interests sharply divided, power imbalances exist across stakeholders and also within a stakeholder group. for example, the category of "preservationist" stakeholders has large political and financial backing. this category consists of the dive boat operators, international conservation ngos, donor agencies, provincial and municipal officials and national government agencies. this block lobbied to declare the entire 33,000 park as a no-take zone for fishing. only recreational diving and research were proposed as allowable activities. within the "extractive" group, power differs as the commercial fishing operators have more money and flexibility to fish elsewhere within the sulu sea. they are also more vocal during meetings and are able to articulate their concerns. in contrast, the smaller fishers from cagayancillo are fragmented, marginalized and have little voice to influence policies that may affect their condition. they fish in tubbataha for livelihood and capture sea turtles for ritual food. in the middle are the local government units and the some ngo members who may be open to limited and regulated use of the reef's area fisheries subject to limits with respect to its sustainable yield and carrying capacity. although this option was discussed, the cost of monitoring sustainable catches and the viability of launching fishing expeditions to tubbataha given prescribed limits to catches rendered this option unfeasible. thus, the conflict is sharper between the "extractive" and "preservationist" groups. the conflict seems to be skewed in favor of the "preservationist" block since the position had powerful backing from wealthier and politically connected stakeholders. those losing out in the park are the cagayanen and outside fishing operators. in contrast to cagayancillo fishers, outside fishing operators can fish elsewhere given the no-take policy. for cagayancillo residents, the conflict remains unresolved until the issue of livelihood is addressed. cagayancillo residents consider tubbataha reef as part of their traditional food range and felt entitled to its tongson, edgardo 56 use. this means that if other stakeholders are directly and indirectly benefiting from tubbataha reef, they should pay for their use. ideally, the share of the local residents from the proceeds must be enough to replace their foregone benefits. although they proposed a 7% sharing scheme from user fees, the basis of this proposal is unknown outside the cagayancillo community. 3.2 community workshops and small meetings it is recognized that negotiations based on stakeholders' interests alone cannot start given the poor quality of relationships between government agencies, ngos and fishers of tubbataha. for example, relations between the palawan-based ngos, phil. navy and and local cagayancillo fishers were strained over past enforcement actions in tubbataha. their relationship must improve for agreements to come out from the process. recognizing the relational problems and disparities in power, workshops and meetings were held before the final multi-stakeholder planning workshop. the community workshops were done to draw out the common interest of cagayancillo fishers in tubbataha reef, identify areas of compromise and improve their skills in expressing their interest and negotiating with other groups. a palawan-based ngo, known to be a local advocate for fishers' rights, facilitated the focus group discussions in five island barangays in cagayancillo. results from the cagayancillo workshop show that majority of its fishers wanted a share derived from user fees, followed by management control, participation in enforcement, fishing and extraction rights and access to alternative livelihoods. one barangay wanted to revive fishing rights to tubbataha reefs. the municipal officials were briefed on the result of the workshops and a common position was agreed upon which the officials agreed to present in the stakeholder meeting. 3.3 conduct of stakeholders workshop preparations based on a stakeholders list, a list of stakeholders who would attend the workshop was made. wwf sent a letter to each listed stakeholder requesting for an interview. a set of guide questions was prepared for the interview. the guide questions asked for the stakeholders' interests, current use of the reefs resources, potential contribution to its management and role in managing it. the interview did not only draw out information from the stakeholders. the process also enabled the stakeholders to reflect on the current state of tubbataha, weigh its options for the future and prepare their positions for the stakeholders' workshop. within two weeks before the workshop, the small meetings between wwf and other stakeholders further enhanced ground working activities. the cagayancillo workshops were part of this process. the stakeholders include other ngo, provincial government, denr, philippine navy, dive shop operators and scientists who have done work in the reef. the meetings validated the positions of each stakeholder, hew their differences to a common view and consolidate a common position for the reef's closure to extractive activities. within a week before the workshop, wwf and the facilitating ngo, palawan network of ngos inc. (pnni), called on the stakeholders to ensure their attendance to the meeting. validation of findings and resolving conflicts the stakeholder validation workshop began with two presentations: one is the physical and biological characteristics of tubbataha and another on its history and results of the stakeholders' analysis and preparatory consultations. the stakeholders validated the information contained in the presentation afterward. the differing positions of the pro-extraction and preservationist groups were presented and validated. the cagayancillo group presented their request to be granted fishing access to tubbataha in one of zones designated for the purpose. but the tubbataha park management was concerned with cost of conducting fishery studies, estimating sustainable yields and monitoring allowable catches for each fisher that entered tubbataha. this imposed a heavy burden on park management. besides, with catch limits, the potential returns from the yield may not be enough to cover for the cost of traveling all the way to tubbataha. in response, the cagayancillo lgu proposed instead negotiating stakeholder agreements for conservation 57 for technical assistance in coastal resources management and enforcement against illegal fishing. the lgu also asked for technical and credit support for seaweed production (where most fishers are engaged in one way or another). the lgu also asked for a 7% share from future user fees since the idea of charging entry was presented during the preparatory meetings. the bigger group agreed to have cagayancillo lgu to take on a greater role in management and enforcement in tubbataha through their membership in the tpamb but the group avoided making financial commitments. at that time, wwf was accessing grant support for park infrastructure and cagayancillo livelihoods from the global environment facility through the united nations development program. in return for this future support, wwf asked the bigger group to support a user fee system that will be charged among divers and boat operators for entry to the park. the group acceded to this proposal and the benefit sharing with cagayancillo. the purpose of the presentations was to draw out the stakeholders from their view of tubbataha as confined by their current interest and enable them to see it in a broader picture including its diversity of users. the validation enabled the stakeholders to share similar understanding of the condition of tubbataha by providing new information and rectifying misinformation. this facilitated consensus to be arrived on the proposals, counter-proposals and their refinements. the facilitator ensured that consensus was reached for each decision point reached. no decision was reached using voting as the means to resolve the issue. after the stakeholders agreed on the access and compensation issue, they put together their vision of tubbataha reef. they subsequently worked as small groups and identified the issues that must be addressed to attain the vision. the small group output is consolidated into plenary output. the consolidation includes categorizing the issues. the categories include enforcement, research, alternative livelihood and funds among others. based on the validated issues, the participants again worked in small groups and formulated the actions that will be done on each category of issue. the actions are presented in the plenary session. the break-though agreements on the actions were negotiated during the plenary session. the agreements were reached through consensus and the facilitation process used prevented a potential chaos from erupting. when the set of actions is finalized, the stakeholders proceeded to match the doers of the actions. a framework plan was derived from the workshop output. a management plan for the trnmp was completed based on the agreements reached among the stakeholders in the multi-stakeholder workshop in 1998 and subsequently endorsed by the tpamb. the plan incorporated both conservation and development aspects to reflect these tradeoffs. wwf helped find donors to finance the plan and execute a project to implement agreed actions. a proposal was designed to set up park management systems, provide livelihoods and help establish coastal management in cagayancillo. 3.4 stakeholder agreements a summary of the stakeholder agreements resulting from the workshop were as follows: cagayancillo fishers to respect no-take zone commercial & palawan fishers to respect no-take zone divers and dive operators to pay user fees 7% share from user fees allocated for cagayancillo livelihood and crm support for cagayancillo pcsd to draft bill and authorize pilot collection of user fees pamb to establish tubbataha management office phil navy & coast guard to establish and staff ranger station the response from the commercial operators to the notake policy was unexpected. one would expect opposition from the bigger fishing boat operators but this did not materialize when they realized tubbataha's ecological importance in the sulu sea. the theory posited by alcala (1993) that tubbataha is a larval source for palawan fishery and would need protection tongson, edgardo 58 efforts if fishers were to benefit from spillover to adjacent fishing grounds caused a turnaround among the commercial fishers. the realization on the importance of tubbataha to the sulu sea and to their fishery operations was enough for them to give up their access rights to the park. discussion 4.1 outcomes five years after with funding from gef through the undp and cofinancing from packard foundation and wwf-us, a 5-year project implemented activities under seven components biological research and monitoring, information and education, enforcement, sustainable financing, visitor management and coastal management and livelihood development for cagayancillo municipality. the resources leveraged internal resources from other stakeholders. the results and outcomes are summarized below. after 5 years, tubbataha reefs is now operated by the tubbataha management office which is staffed with eight rangers providing year-round enforcement and monitoring support using modern equipment including radar technology. the philippine navy and coast guard provide the manpower and the bi-monthly relieving trips to the ranger station. since 1999 when enforcement campaign was launched, there were fewer reported illegal fishing violations coming from cagayancilo and palawan-based fishing boats. most infractions to the park came from ships who failed to notify the rangers about their passage through the park while on their way to fish aggregating devices outside the park boundaries. in 2001-2003, taiwanese and chinese vessels were found poaching in the park and were jailed in puerto princesa. also, some dive boat operators were suspended for various violations. visitor arrivals and user fee collections are on the uptrend since user fees were started in 2000. park collections have reached p18 million since 1998 with 2006 collections approximating 80% of the core costs to run the park. approximately 6 million pesos is needed annually to run the park, about half of which are provided through in-kind support by the philippine navy and coast guard. while it is acknowledged that no single group or institution can provide all the required resources to run the park, a multi-stakeholder management model allows for cost and benefit-sharing which was not possible under centralized command-and-control models. few parks in the philippines can boast of full park staffing and a mechanism for sustainable financing through user fees. compared to other parks, the trnmp is well funded with financing sources ranging from user fee systems and grants from foreign donors. returns from investments in protection showed improvements in biological health. reef health, fish biomass and densities have improved or have stabilized. live coral cover stabilized at 40% from 1999-2003 before reaching 50% in 2004 (wwf 2004). for 2004, commercial fish biomass are at its highest at 60 mt/sq-km (wwf 2004) and is twice that reported by alcala (2001) for a healthy reef at 30 mt/sq-km. total biomass and density averaged 166 mt/sq-km and 60 individuals/100 sq-m respectively, the highest since 1998 (wwf 2004). visitor satisfaction is high with most boat operators claiming more sightings of mega fauna species (i.e. manta rays, turtles, whalesharks, etc.). the cagayancillo municipality is a model in "bootstrap" development. illegal fishing has been contained due to large part to strong leadership supported by an active citizenry. on their own initiative, local officials established five mpas as part of their coastal resources management program. live coral cover and fish biomass in these mpa are at their highest levels (wwf 2004). perceived fish catches outside the mpas by fishers during the focus group discussion reportedly increased from 10 kg/day to 15-20 kg/day for the period 1999-2004 (todd & nunez 2004). in 2004, the lgu of cagayancillo finally collected on their 7% annual share accumulating to php 435,000. the money was used to build a section of a farm-tomarket road which improved access of farm products. a micro-credit program provides the needed financial negotiating stakeholder agreements for conservation 59 services that support a broad number of families and their livelihoods. the micro-credit facility released php 680,000 in loans to 175 borrowers for their livelihood, education and health needs and enjoys a healthy repayment rate of 90%. the participatory evaluation in 2005 was attended by cagayancillo lgu officials led by the mayor, and fisherfolk representatives. the delegation affirmed the positive outcomes of the project. interestingly, the cagayanen participants in the participatory evaluation did not raise the issue of reviving their fishing rights indicator 2000 2004 (nso) (subade) households who owned lots 82% 86% households who owned houses 85% 95% houses with gi roof 58% 72% households using kerosene for lighting 65% 50% households using lpg as cooking fuel 11% 10% houses with water-sealed toilets 46% 56% houses with television 5% 6% houses with refrigerator 5% 7% table 2. data on selected living standard indicators in cagayancillo: 2000 and 2004 to tubbataha. it seems the recovery of fish stocks in cagayancillo due to enforcement of fishing laws are providing benefits on its own. the level of biodiversity conservation attained in trnmp has not sacrificed socio-economic development and living standards. they are in fact the twin fruits of integrated conservation and development. to measure changes in living standard, selected data from nso census in 2000 and wwf-sponsored study in 2004 (subade 2004) are compared.the living standard shows positive change in the eight indicators used. lot ownership increases from 82 percent in 2000 to 86 percent in 2004 (table 2). the difference is 4 percent. the increase is even greater in house ownership jumping from 85 percent in 2000 to 95 percent in 2004. the difference is 10 percent. the quality of houses has improved in even larger scale as seen in the type of roof. in 2000, only 58 of the houses have galvanized iron (gi). this percentage rises to 72 percent or 14 percent increase from the 2000. the utilities that the households enjoy have also improved for many. the users of kerosene lamp for lighting are reduced from 65 percent in 2000 to 50 percent 2004. it means that 15 percent more households benefit from electricity in 2004 than in 2000. the percentage of liquefied petroleum gas (lpg) users as cooking fuel is about the same (11% versus 10%). but toilet ownership dramatically rises from 46 to 56 percent. the ownership of appliance has not changed much. households with television is about the same (5% versus 6%) but slight increase in noted in refrigerator (5% versus 7%).. although no quantitative data exists, the fishing benefit derived by cagyancillo from tubbataha seems to have been offset by the higher fish catch in the fishing grounds around their islands. the small fishers using hook and line, spear and net gears are the main beneficiaries. sources of income have diversified partly through the rising membership in livelihood cooperative. the loan that the members obtained serves as capital for general merchandise (sari-sari) stores, poultry, livestock, seaweed production and fishing. an important component of the management plan which was not fulfilled was the enactment of the protected area bill for tubbataha by congress. as part of the nipas process, the pcsd drafted a protected area bill for tubbataha and filed the bill with the office of cong. mitra who became the bill sponsor. the bill incorporates innovative elements to avoid problems experienced by other protected areas such as devolving chairmanship of the pamb from denr to the provincial governor; localizing the collection, management and accounting of protected area funds and creating a special account for user fee collections, fines and donations. without this bill, the institutional framework and legal mandate of the tpamb and tmo, to effect arrests at sea, are not secured by law. finally, the participatory evaluation conducted with the stakeholders in 2005 validated these positive results. the workshop concluded that: (1) agreements reached in 1998 were complied with, (2) current bio-physical tongson, edgardo 60 condition is closer to the vision through the evident improvements in coral cover, fish biomass, higher occurrences of mega-fauna species, and (4) general improvement in the socio-economic condition of cagayancillo residents (although attribution to the project needs to be ascertained). the evaluation completes one cycle as the lessons and recommendations from the workshop are carried into the next cycle of planning and implementation. rarely have parks benefited from completing an entire project life cycle. 4.2 balancing costs and benefits of conservation central to the issue of compliance by stakeholders to these agreements was the incentive systems created resulting from the distribution of costs and benefits, rights and responsibilities among stakeholders. higher levels of responsibility (costs) on the part of one stakeholder without parallel increase in incentives have proven unworkable in practice. on the other hand, private operators benefiting from resource rents from public goods are associated with low burdens of responsibility (or costs). in the case of tubbataha, one camp comprising the "preservationists" are those who directly benefited from establishing the no-take zone, this group consists of divers, dive operators, researchers, environmental ngos and national government agencies. on the opposite camp was the "pro-extraction" group such as the fishers and lgu officials from cagayancillo or those who depend on tubbataha resources for their livelihoods. with the imposition of a park and a no-take zone policy, this group will directly bear the cost by giving up their access rights and political jurisdiction to tubbataha. although it can be argued that the cagayancillo and the commercial fishers were to indirectly benefit from the park through a "spillover" effect, their costs were short-term and immediate. in principle, by having the beneficiaries "pay" for the costs borne by the fishers and service providers, each will have a positive "return" from establishing a notake zone for tubbataha. the objective is to create residual incentives by netting out benefits and costs arising from conservation actions. to create the financing needed to "pay" the fishers and services provided, , a user fee system for divers based on their "willingness to pay" was introduced in 2000 (tongson & dygico 2004). the user fees contributed to financing the cost of park management, and fulfill the 7% share expected by the cagayancillo lgu. 4.3 win-win arrangement as platform for stakeholder compliance the success of the trnmp can be attributed to the high degree of compliance by stakeholders to the agreements reached in 1999 and corresponding actions by the stakeholders themselves. compliance is the fruit of win-win arrangements designed from the beginning. except for infractions by a few dive boat operators at the early part of the user fee system, majority of the boats followed the park rules including mandatory anchoring on mooring buoys, ban on visitors to set foot on bird island and registration upon entry to the park. the fishers from cagayancillo became busy tending to their seaweed farms. some island barangays set up marine sanctuaries. the lgu allocated a budget to seed 50% of a livelihood fund, provided manpower complement to manage the credit program and passed ordinances setting up five barangay-managed mpas. the project introduced new technologies in seaweed culture to prevent diseases ("ice-ice"), shortening the harvesting cycle, organizing the farmers into cooperatives and marketing support. the coast guard and navy fulfilled staffing and provisioning commitments despite all year round including voyages across bad sea conditions during the off-season. on the part of the pcsd, they supported drafting of a draft bill for tubbataha and provided secretariat support to every tpamb meeting. other ngos provided legal and technical support to the park staff for their litigation and reef monitoring needs. the stakeholder understanding of the reason for park set-up, banning of extractive activities and need for negotiating stakeholder agreements for conservation 61 cooperation is rooted on the collective vision of restoring tubbataha to its original state for the longterm benefit of stakeholders including the fishers of cagayancillo. these stakeholders collectively experienced the rise and fall of tubbataha since the early 90's. this shared experience enhanced social capital that is benefiting the park. the case affirms essence of social capital as a condition to develop trust and mutually supportive relationships and as pre-condition in achieving self-enforcing agreements. according to game theory, trust is developed through repetitive games among parties. these games play out in every stakeholder meeting where issues and decisions are deliberated and accounted for. it also affirms stakeholder management as an operational paradigm ensuring ownership and sustainability of actions. 4.4 creating a sea change the bigger challenge the successes of tubbataha are still dwarfed by the wide spread destruction of marine resources in the sulu sea. the disparity in economic yield between these areas surrounding tubbataha and the park itself attracts poachers and violators to fish in tubbataha especially during the off-season. basterra and jesse beazely reefs, for example, used to be highly productive reef systems but are not part of the trnmp park boundaries. reports indicate wide spread poaching and destruction in these reefs which is indicative of the fate of tubbataha had interventions not materialized in the park. there is need to embed these parks and mpas in broader integrated coastal management programmes to manage "push and pull factors" to ensure sustainable development of the entire area. this means zoning a larger area for commercial fishing, municipal fishing, strict protection, oil exploration and development, tourism and seasonal closures (in the case of spawning aggreagations). sea change needs to happen at a larger scale. as the range of stakeholders concerned with natural resource management broadens, the complexity of the interlinkages between them increases. in such contexts, the potential for conflicts is high and tools are needed to examine and address these relationships. these tools will help inform the allocation of costs and benefits and elicit workable solutions from affected stakeholders. more work needs to be done in developing capacities of park managers throughout our park system in using participatory tools in designing win-win arrangements and resolving conflicts through facilitation. as shown by the tubbataha case, a win-win arrangement in turn, can transform "battlefield" natural resource use into shared assets capable of meeting divergent needs and aspirations. the greater challenge is bringing the lessons and tools from these small successes to create the sea change that is needed. acknowledgements the authors wish to thank ms. marivel dygico, wwf tubbataha project manager; ms. angelique songco, tubbataha park superintendent, the palawan provincial government, the municipal government and fisherfolks of cagayancillo; and to the management of wwf-philippines for giving us the time and support in completing this manuscript. references: abbot, j. & thomas, d., 2001. understanding the links between conservation and development in the bamenda highlands, cameroon. world development. 29(7):11151136. adams, w. and infield, m., 2001. park outreach and 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tubbataha, natural history, resource use, conservation of tubbataha reefs, palawan, philippines (rev. ed.). manila: bookmark. white a & palaganas v., 1991. philippines tubbataha reef national park: status, management issues, and proposed plan. environ. conserv. 18:148-157, 136. white a, ledesma m, ovenden m., 2003. tubbataha reefs national marine park, palawan. in coral reef information network of the philippines (philreefs), alino p (ed). university of the philippines marine science institute, quezon city. pp 144-151. wwf (world wide fund for nature)., 2004. sabater m. & ledesma m. eds. project monitoring report. world wide fund for nature philippines (wwf). quezon city (unpublished). page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 17_broadband maraña, gabayno, and garcia 70 broadband continuum generation in single-mode optical fiber b. d. maraña*, j. f. gabayno, and w. o. garcia photonics research laboratory, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: bmarana@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 70-73 *corresponding author we report broadband continuum generation in a single-mode step-index optical fiber pumped by the second harmonic (532 nm) of a nd:yag laser. the continuum started to appear together with the firstorder stokes at 545 nm with an input power of 11 mw. the appearance of the second-order stokes is distinctly observed at 557.5 nm for 96 mw input power. the generated continuum spans 123.12 thz (550–712 nm). asymmetric cross-phase modulation-induced spectral sidebands were also observed. introduction frequency conversion and broad supercontinuum generation (scg) techniques are presently developed for a variety of applications where laser sources of several emission wavelengths play a significant role. in optical fibers, nonlinear and dispersion processes permit frequency shifting and generation of supercontinuum. the most studied nonlinear effects are stimulated raman scattering (srs) for multiple wavelength generation and together with dispersion, self-phase modulation (spm), and cross-phase modulation (xpm), these processes act together to generate a continuum. srs in an active media has found applications such as a tunable light source for imaging, spectroscopy, and ultrashort pulse generation (palero et al., 2002). in optical fibers, conversion of a single wavelength into several frequencies opens up new possibilities especially as highly tunable light source (ilev et al., 1996; gavrilovic et al., 1998) for wavelength-divisionmultiplexed (wdm) systems (arya, 1997), signal amplification (desurvire, 1994), and as a new tool for beam cleanup and combining (russel et al., 2002). on the other hand, supercontinua find applications in optical metrology, spectroscopy, optical coherence tomography, and optical communications (zhao et al., 2003; mussot et al., 2003; lu & knox, 2004). when a pump wave of energy greater than or equal to the threshold is coupled into an optical fiber, nonlinear effects can occur. srs in optical fibers results from inelastic scattering of high-energy photons by the molecules of the ramanactive media. the high-energy photons excite silica molecules from lowerto higher-energy levels which generate frequency-shifted photons during its relaxation to lower-energy states. this shift in frequency occurs in units of the vibrational frequencies of silica. the shifting to lower and higher frequencies is called stokes and anti-stokes shifting, respectively. multiple raman lines are generated by srs cascade and four-wave raman mixing (fwrm). spm and xpm are nonlinear refractions due to the intensity dependence of the refractive index. spm refers broadband continuum generation 71 to the self-induced time-dependent phase shift experienced by the field as it propagates along the fiber. time dependence of the phase shift results in the creation of new frequencies. xpm is similar to spm except that shifting is induced by copropagating fields of different wavelengths. xpm induces an asymmetrically broadened spectrum (agrawal, 1995). broadband continuum generation can be enhanced by using highly nonlinear fibers. this can be done by increasing the optical density of the fiber, which includes a tapering method (lu & knox, 2004) and launching high-power lasers. supercontinuum has already been observed in birefringent (zhao et al., 2003), dispersion-shifted (mussot et al., 2003), and photonic crystal fibers. in this paper, we report the generation of broadband continuum of the 532 nm laser pulse in a 51 m stepindex optical fiber. pump power dependence of the spatial extent of the continuum is presented as well as the observed xpm-induced modulational instability of the rayleigh scattered line. experimental setup figure 1 shows a schematic of the experimental setup employed. the excitation source is the linearly polarized second-harmonic (532 nm) output of a qswitched nd:yag laser (spectra physics gcr-230) operating with a pulse width of ~7 ns and 10 hz repetition rate. an iris (d) is introduced for spatial filtering of the laser beam. light is collimated using two lenses, l1 and l2, of focal lengths 250 and 11 mm, respectively. the input is focused into a 51-mlong optical fiber (of). light is coupled onto the fiber using an aspheric lens (l3), which has a numerical aperture (na) of 0.25 and a focal length of 8 mm. the medium is a commercially available step-index single-mode optical fiber (newport model f-sv). it is designed to operate at 633 nm with the single-mode cutoff condition at 550±50 nm. the fiber has a mode field diameter of 4.3 mm and na of 0.14. the outer cladding diameter of 125 mm is exposed by manual stripping and cleaving. the quality of the cut end faces are examined using a microscope. the output light of the fiber is fed directly into the monochromator (spex 1000m) of which the slit width is set at 200 mm. the monochromator is connected to a photomultiplier tube (pmt) (hamamatsu r-1328u5) and the signal is digitized through a lock-in amplifier (srs-sr830). input power measurement was done using the molectron detector and power meter. results and discussion figure 2 shows the initial spectral evolution of the 532 nm pump. at the lowest input power of 11 mw, the first-order stoke (s1) was observed at 545 nm corresponding to a shift of 448.37 cm–1. this line is observed to broaden from a width of 4 nm at 11 mw to 5.5 nm at 96 mw pump. a continuum starting at 550 nm spanning 11.64 thz was generated together with the first raman line. as the pump power is increased (fig. 3), the continuum broadens extending up to the red region at 712 nm spanning a 123.12 thz frequency range. table 1 summarizes the frequencies spanned by the scg with increasing power. following the spectral evolution up to 96 mw input, the second-order stokes (s2) is clearly seen at 557.5 nm with a spectral shift of 859.77 cm–1. this line with a full width at half maximum (fwhm) of 5 nm has a lower intensity compared with s1 as a result of energy transfer during multiple scattering. the s1 line increases in intensity up to 50 mw and experiences a noticeable decrease at 80 mw and a considerable gain at 96 mw. d q -s w it c h e d n d: ya g l1 l2 l3 of m p m t spex monochromator lock-in amplifier fig. 1. schematic diagram of the experimental setup. maraña, gabayno, and garcia 72 a decrease in the intensity of s1 can be due to the transfer of energy to the continuum and the generation of s2. no anti-stokes orders are generated even at this intense energy of 96 mw. the absence of blueshifted lines can be attributed to the fact that the phasematching condition, a requirement of four-wave mixing, is difficult to satisfy in silica fibers (agrawal, 1995). an average decrease in the energy of the continuum was observed for the maximum input power employed probably because of the transfer of energy to longer wavelengths. a continuum was also observed by krylov et al. (1998) after the first stokes was generated in srs by a femtosecond laser in hydrogen gas and has attributed the continuum to white-light generation by spm. another interesting feature is the presence of spectral side lobes with a frequency separation of 4.77 thz (4.5 nm) near the rayleigh line (figs. 2 and 3). the presence of these sidebands results from the spectral manifestation of modulational instability induced by xpm. a thorough discussion on xpm and xpminduced modulational instability is given by agrawal (1987 & 1995). this observation suggests the possibility that xpm can sustain solitons in the normal dispersion regime (agrawal, 1987). the intensities of the first (529.5 nm) and second (534 nm) sidelobes reached their maximum at 35 and 50 mw, respectively. their energies begin to drop at 50 and 80 mw, correspondingly. no change in the relative positions of the sidelobes was observed for different pump powers. conclusion broadband continuum generation of a pulsed nd:yag laser operating at 532 nm has been observed in a 51m-long step-index single-mode optical fiber. the continuum supports a 123.12 thz frequency range extending from 550 to 712 nm for an average pump power of 96 mw. the first raman line (s1) occurs at the lowest input of 11 mw with a spectral shift of 448 cm–1 at 545 nm. the second-order stokes is clearly defined at the fig. 2. spectrum on the fiber output featuring the evolution of the 532 nm laser pulse towards the first-order stokes s1 and generation of spectral sidebands with increasing power. wavelength (nm) n or m al iz ed i nt en si ty ( a. u. ) 528 536 544 552 560 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 s1 pump 7 mw 11 mw wavelength (nm) in te ns it y (n ot s ca le d) 520 560 600 640 680 720 0 1 2 3 4 5 6 7 8 pump 7 mw 11 mw 23 mw 35 mw 50 mw 80 mw 96 mw fig. 3. output spectra of the 51-m-long single-mode fiber for different pump powers. table 1. frequency spanned by the broadband continuum generated for different input power. ave. pump power (mw) spanned frequency (thz) 7 11 23 35 50 80 96 — 11.64 17.28 69.26 80.34 101.01 123.12 broadband continuum generation 73 maximum pump where the longest continuum occurs. it has a shift of 859.77 cm–1 at 557 nm. spectral sidelobes induced by xpm was also observed. acknowledgment we acknowledge the support provided by intel philippines. references agrawal, g.p., 1987. modulation instability induced by crossphase modulation. phys. rev. lett. 59: 880–883. agrawal, g.p., 1995. nonlinear fiber optics, 2nd ed. academic, san diego: chap. 1. arya, v., 1997. analysis, design, and performance evaluation of optical fiber spectrum-sliced wdm systems. dissertation submitted to virginia polytechnic institute, virginia. desurvire, e., 1994. erbium-doped fiber amplifiers. wiley , canada. gavrilovic, p., et al., 1998. raman-effect-based modulator for high-power fiber lasers. appl. phys. lett. 72: 401–403. ilev, i., et al., 1996. a widely tunable (0.54–1.01 mm) doublepass fiber raman laser. appl. phys. lett. 69: 1846–1848. krylov, v., et al., 1998. femtosecond stimulated raman scattering in pressurized gases in the ultraviolet and visible spectral ranges. j. opt. soc. am. b. 15: 2910-2916. lu, f. & w. knox, 2004. generation of broadband continuum with high spectral coherence in tapered single-mode optical fibers. opt. exp. 12: 347–353. mussot, a., et al., 2003. generation of broadband singlemode continuum in a conventional dispersion-shifted fiber by use of a subnanosecond microchip laser. opt. lett. 28: 1820–1822. palero, j., w. garcia, & c. saloma, 2002. two-color (twophoton) excitation fluorescence with two confocal beams and a raman shifter. opt. comm. 211: 65-71. russel, t., et al., 2002. stimulated raman scattering in multimode fibers and its application to beam cleanup and combining. j. nonlinear opt. phys. mat. 11: 303–316. zhao, y., et al., 2003. characteristics of supercontinuum generation in birefringent optical fibers. proc. symp. ieee/ leos benelux chap. 217–220. 2editor's note.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online this issue of science diliman features four main articles and two short communications. the f irst research article offers a solution to a basic problem in color symmetry— the classif ication of symmetrically colored symmetrical patterns. the paper presented a method of determining the non-trivial colorings of perfect and transitive 2-uniform tilings, and looked into the equivalence of the obtain colorings. the second article studied surface migration kinetics of chemical additives in vulcanized natural rubber compounds, as a function of ingredient loading. the third article is a contribution from the up biology invertebrate museum, accounting accessioned collections and reporting the availability of these collections to the community of researchers studying invertebrates. these collections were assessed in terms of taxonomic scope, geographical range, and chronological breadth. the fourth article is a discussion on a widespread parasite, toxoplasma gondii; specif ically, its seroprevalence in stray and domestic cats in metro manila, along with risk factors associated with its seropositivity. the two short communications tackle the geochemistry of a mining site, and a nematode parasite infection in a snail species from mindanao.  the science diliman community would also like to congratulate four distinguished scientists, academicians gavino c. trono, angel c. alcala, ramon c. barba, and edgardo d. gomez, for being conferred the rank and title of national scientist by president benigno s. aquino iii by virtue of malacañang proclamation nos. 737, 782, 783, and 843 on 12 august 2014 at the malacañang palace, in recognition of their outstanding works and contributions to science and technology in the country. academician trono was recognized for his extensive studies on the culture of seaweed species, which benef ited numerous people among the coastal populations. he identif ied and described 25 new species of marine benthic algae and successfully implemented 45 research projects, which resulted in the publication of 142 scientif ic papers. he established the largest phycological herbarium in the country— the g.t. velasquez herbarium in the marine science institute of the university of the philippines, which houses more than 70,000 herbarium specimens of the seaweed flora. 2 academician alcala was recognized for his seminal research on the systematics, ecology, and conservation of vertebrates, particularly in herpetology, by providing valuable basic knowledge on the country’s rich biodiversity and ecology. he served as pioneer scientist and advocate in the protection of coral reefs leading to a national policy and program that established the no-take marine reserves, a specific type of marine protected area (mpa) set aside by the government where no extractive activity is allowed. academician barba was recognized for his distinguished achievements in the f ield of plant physiology, specif ically on the induction of flowering of mango and on micropropagation of important crop species that have earned him national and international accolades. his pioneering work on the induction of flowering and fruiting of mango resulted in the change from the seasonal supply of fresh fruits to all year round availability of abundant fresh mangoes. academician gomez was recognized for his sterling contributions in invertebrate biology and ecology, giant clam culture and restoration, and coral reef assessment and conservation. he steered the world’s f irst national-scale assessment of damage to coral reefs that led to worldwide conservation initiatives, such as the global reefs and risk analysis, global coral reef monitoring network, and the international coral reef action. he shepherded the development of the marine science institute (msi) at the university of the philippines (up), where he served as the founding director. it is important to note that ns trono, ns barba, and ns gomez are up professors. ns alcala had research collaborations with up marine science institute and was a consultant to the institute in the late 1980s. we in the science diliman are very proud of the new national scientists. more power to them. marco nemesio e. montaño, phd editor-in-chief 10_marcos 45 low-level color and texture science diliman (january-june 2003) 15:1, 45-50 low-level color and texture feature extraction of coral reef components ma. sheila angeli marcos*, maricor soriano, and caesar saloma instrumentation physics laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: sheila@nip.upd.edu.ph abstract the purpose of this study is to develop a computer-based classifier that automates coral reef assessment from digitized underwater video. we extract low-level color and texture features from coral images to serve as input to a high-level classifier. low-level features for color were labeled blue, green, yellow/ brown/orange, and gray/white, which are described by the normalized chromaticity histograms of these major colors. the color matching capability of these features was determined through a technique called “histogram backprojection”. the low-level texture feature marks a region as coarse or fine depending on the gray-level variance of the region. introduction coral reefs are considered as one of the richest ecosystems on earth and an essential source of livelihood for many people. marine scientists assess the condition of coral reefs from population estimates of biotics and abiotics in the reef area (alcala & gomez, 1982). in coral reef assessment, marine scientists classify videos or images of transects of corals into six main categories or “benthos”: (1) abiotic (rock, rubble, sand); (2) live coral; (3) dead coral (and dead coral with algae); (4) algae; (5) soft coral; and (6) other fauna. some popular methods used for reef assessment are line intercept transect (litr) and in-situ mapping (ismp). both methods employ a diver to assess or film, in-situ, the coral reef, which is lined with transects, a kind of tape measure utilized by marine scientists to record the length or area of the reef. although filming the benthic organisms in a reef area reduces diving time, video analysis still requires identification of items in each frame by expert individuals (uychiaoco et al., 1992; carleton & done, 1995). a software known as pointcount’99 (http://www.cofc.edu/~coral/pc99/ pcppintro.htm) has been developed to aid in the assessment, but user-intervention is still required. most of these techniques are often labor-intensive, requiring experience and a trained eye. there are works on remote sensing or spectral imaging of seafloors using airborne multispectral cameras (bierwirth et al., 1993; carper et al., 1990), but close-up inspection of the coral reefs are still needed to correlate spectral signatures with actual reef conditions. hence, the purpose of this study is to computer-automate the coral reef assessment from digitized underwater video, thus making the analysis less tedious. it is also worth noting that this is, to the best of our knowledge, the first attempt to automate coral reef assessment through a computer. in computer vision, “color” is a point property in the digitized image. pattern recognition techniques using color often operate on the color distribution alone, ignoring the spatial, black and white (tonal) property of* corresponding author 46 marcos, soriano, and saloma regions in the image which defines the texture. incidentally, color and texture are some indicators used by marine scientists to identify components in a reef. hence, this study aims to show the feasibility of using color and texture as features for classifying coral reef components. one approach in pattern recognition is classification by minimum distance between model and test image features, such as color, texture, and color-texture (huang et al., 1997; kondepudy & healey, 1994; ojala & pietikainen, 1999). in marcos et al. (2001), we attempted to classify coral reef components directly from color and texture feature vectors. recognition rates were low, which could mean that such a direct approach to classification is not suitable for corals. we attempt another approach to image classification based on the presence or absence of features. for example, abiotics such as rocks would normally be gray and craggy. dead corals are bright white, while live corals are mostly colorful and regularly textured. note that a combination of features such as “irregular+gray” would most likely point to the existence of rocks, while a region which is “regular+colorful” would most likely mean the region has a living coral. if we have a binaryvalued feature such as “regularity”, which has a value of 1 when the region is regularly textured, and 0 otherwise; and if we have another feature such as “colorfulness”, which is 0 when the region is gray or white, and 1 if otherwise, then we can preclassify an image region with binary numbers. for example, rock will be 00 (regularity-colorfulness) and coral will be 11. if we have f binary-valued features, then the total number of combinations is 2f. in this paper, we test two features, color and coarseness-fineness, in preclassifying image regions in a video frame of a coral reef. methodology image data fifty frames from a digitized video of australia’s great barrier reef (© australian institute of marine science) were used for testing. the video (frame rate = 25 frames/second) was taken from a shallow depth, with sunlight as the illumination source. distance between camera and corals were kept to 30 cm. still frames were digitized to an image size of 640 x 480 pixels. low-level color feature extraction humans perceive color in an object through the nature of light reflected by that object. the characteristics generally used to distinguish one color from another are brightness, hue, and saturation (gonzales & woods, 1992). in computer vision, color represents a property of a point picture element or a “pixel” in a digitized image. for a given colored pixel, hue and saturation represent the chromaticity, while brightness, the intensity of the pixel. the purpose of color spaces or color models is to represent how color is to be perceived according to some standard. some color spaces are modeled on how the human eye perceives color. other color spaces are used as hardware-oriented models, such as the rgb (red, green, blue) for color monitors. in this study, we transformed image rgb into the normalized chromaticity coordinates (ncc) or normalized rg (gonzales & woods, 1992). the normalized rg coordinates are computed from rgb space by the following expressions: i = r + g + b; r = r/i; g = g/i (1) where i is the brightness value and r and g are the chromaticity values. note that b is no longer unique because of the relationship r + g + b = 1. this color space has three advantages: (1) it is easy to compute; (2) it separates brightness and chromaticity information (intrinsic color); and (3) the colors created in combining two colors in different brightness proportion appear in the line joining the two chromaticities of the two colors in rg-space. the rg chromaticity histogram of an image will then be a 3d plot where the xand y-coordinates are the chromaticity values (r and g) while the zcoordinate is the frequency of occurrence in the image. most live corals in the video are either blue, yellowish, brown, or green, while dead corals have a bleached or white color. rocks, on the other hand, have grayish color, or sometimes greenish due to algae growth. 47 low-level color and texture heuristically, it was determined that four groups of colors, namely blue, green, yellow/orange/brown, and gray/white, are dominant in the coral reef images. since gray is merely an incidence of white, gray and white are grouped into one major color. for each major color, an average rg chromaticity histogram was obtained from manually cropped image patches which visually possess the major color. these major colors are the low-level color features utilized in this study. histogram backprojection the dominance of each group of color at each region of an image was tested using histogram backprojection (swain & ballard, 1991) (fig. 1). the chromaticity histogram of a target image is first obtained before the test image is transformed into chromaticity space. the chromaticity values of each pixel in the test image are traced onto the target chromaticity histogram, and the corresponding frequency value is assigned to the same pixel in the test image (backprojection). this process produces a bright region in the backprojected image where the image matches the colors in the histogram with high frequencies. in this paper, we aim to match regions in the coral images exhibiting the four major colors (blue, green, yellow/brown/orange, and gray/ white). dead corals and sand have the same chromaticity and differ only in brightness. to distinguish between the two, intensity information was included in the color matching. thus, whenever a region is labeled as gray, we test further if its intensity is beyond a certain threshold. low-level texture feature extraction in computer vision, “texture” is the spatial or black and white or tonal property of an image (haralick, 1979). an image is composed of texture primitives, which is a region in an image with little variation in tone. texture is exhibited by an image when there is a large variation in the texture primitives. fine textures have higher spatial frequencies and its texture primitives are small, while coarse textures have larger primitives and lower spatial frequencies. most texture paradigms utilize methods that extract the spatial frequency information of an image, for example, fourier transform. some analyze the spatial distribution of the gray level of each region in an image and derive statistical information from the distribution. in this study, gray-level variance was utilized to characterize the texture of coral reef components. a gray-level image of the frame to be analyzed is obtained using i in eq. (1). the gray-level values may be scaled from 0 to 1 or 0 to 255. a region of the image has fine texture if the gray-level variance relative to the average gray value of that region is small, and has coarse texture if the variance is large. rocks, live corals, algae, and dead corals usually have coarse texture, while sand and certain live corals perceptually have fine textures. these characteristics of texture, or low-level features, aid marine scientists in the classification of corals. thus, coarse and fine will be the low-level texture features to be used in this study. fig. 1. illustration of the histogram backprojection technique. the chromaticity values of a pixel are traced into the color histogram of a query image and its frequency value is assigned to that pixel. image frequency chromacity 2 5 2 model histogram chromacity 1 histogram backprojected image 48 marcos, soriano, and saloma results and discussion color matching results although colors are illumination dependent, the light source for the video used in this study is constant all throughout the film (sunlight). therefore, no color shifting due to light source change is observed in the images. the average major color histograms were used as model histograms. on the upper left side of fig. 2a is a massive blue-colored coral; on the upper right, a dead coral. histogram backprojection was applied to fig. 2a using the average blue histogram, resulting in fig. 2b; in fig. 2c, the gray/white histogram was used. the results for both show that the blue and white parts registered as bright areas. for effectively matching bleached or pale white regions in the image, we incorporated the intensity information into the histogram backprojection of the test image. we emphasize that from this technique alone, the dead coral population of a reef can be determined. we have implemented histogram backprojection in video. an example showing the labeling of blue corals and dead corals may be downloaded from http:// www.nip.upd.edu.ph/ipl/members/jing/corals. texture labeling the mean of the gray-level variances of each low-level texture was computed using 10 cut-out image samples per low-level class (i.e., images exhibiting coarse and fine textures). it was found that the mean of the variances for coarse texture regions fall in the range of 0.01 to 0.1, while that for fine texture regions have an order of magnitude of 0.001. the results imply that these low-level texture features are dissimilar or nonoverlapping. we further tested the low-level feature by getting the variance of each n x n block in an image. if the variance of the image is within the range of 0 to 0.01, this area is classified as “of fine texture”, and the center of this block is marked by a green letter “f”. if the variance of the n x n block is within 0.01 to 1, this area is “of coarse texture”, and we mark a red letter “c” on the center of the block. this approach was tested for different window sizes and it was found that effective texture characterization is achieved using window sizes in the range of 40 x 40 to 70 x 70 pixels. fig. 3 shows the result of the block processing using a 60 x 60 block size. perceptually, we can see that fine and coarse textures in the image are effectively recognized by merely using the gray-level variance of the image. coarseness or fineness of an image region can depend on illumination direction. in our case, the videos are illuminated by diffused sunlight (corals were shallow); thus, image coarseness does not change. (c) (g) (a) (b) (d) (f) (h) (e) fig. 2. test images (a, c, e, and g) and their corresponding histogram backprojected images (b, d, f, and h) using the four image chromaticity histograms: averaged green rg histogram (a & b); averaged yellow/ brown/orange rg histogram (c & d); averaged blue rg histogram (e & f); and averaged gray/white histogram (g & h). 49 low-level color and texture conclusion in this work, we introduced an approach to coral reef image labeling utilizing low-level features for both color and texture. the low-level color features using four major colors (blue, green, yellow/brown/orange, gray/white) can effectively label colored reef images with histogram backprojection. the low-level texture features (coarse and fine texture as described by graylevel variances) can effectively label perceptually coarse and fine texture regions in the images. having obtained the low-level features, the next step would be a higher-level of classification. recommendations these low-level features can serve as weights, or “onoff” inputs to the classifier, such that certain combinations of these low-level features can describe a certain benthic category. if color and texture are not enough as lowlevel inputs, then a third feature, shape, may be included. if there are f features, each having only two states, then the number of possible combination of feature states is 2f. for example, three binary features (f = 3) would be able to label up to eight classes. in our case, we have six benthic categories; therefore, we only need three dual-state features for combinatorial classification. fig. 3. a frame classified into coarse (c) and fine (f) blocks. each block is 60 x 60 pixels. the whole image is 640 x 480 pixels. acknowledgment this project was granted financial support by the office of the vice-chancellor for research and development, university of the philippines diliman, grant no. 00013.2 nsep. references alcala, a.c. & e.d. gomez, 1982. coral growth rates on a reef flat in sumilon island, central philippines. kalikasan: philippine journal of biology. 11: 179-184. bierwirth, p.n., t.j. lee, & r.v. burne, 1993. shallow seafloor reflectance and water depth derived by unmixing multispectral imagery. photogrammetric engineering & remote sensing. 59(3): 331-338. carleton, j.h. & t.j. done, 1995. qualitative video sampling of coral reef benthos: large-scale application. coral reefs. 14: 35-46. carper, m.w.j., t.m. lillesand, & r.w. kiefer, 1990. the use of intensity-hue-saturation transformations for merging spot panchromatic and multispectral image data. photogrammetric engineering and remote sensing. 56(4): 459-467. gonzales, r. & r. woods, 1992. digital image processing. usa, addison-wesley publishing company. haralick, r.m., 1979. statistical and structural approaches to texture. proc. ieee. 67: 786-804. huang, j., s.r. kumar, m. mitra, w.j. zhu, & r. zabih, 1997. image indexing using color correlograms. ieee conference on computer vision and pattern recognition. 762-768. kondepudy, r. & g. healey, 1994. use of invariants for recognition of three-dimensional color textures. journal of the optical society of america. 11: 3037-3049. marcos, s., m. soriano, m. quibilan, p. alino, & c. saloma, 2001. image classification of coral reef components from underwater video. proc. oceans 2001. mts/ieee. honolulu, hawaii. november 5-8, 2001. 50 marcos, soriano, and saloma ojala, t. & m. pietikainen, 1999. unsupervised texture segmentation using feature distributions. pattern recognition. 32: 477-486. swain, m. & d. ballard, 1991. color indexing. international journal of computer vision. 7(1): 11-32. uychiaoco, a.j., p.m. alino, & m.p. atrigenio, 1992. video and other monitoring techniques for coral reef communities. 3rd asean science and technology week conference proceedings. marine science: living coastal resources. 6. http://www.cofc.edu/~coral/pc99/pcppintro.htm page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 manuscript submision form_sd-new-p57.pmd author certifications: i certify that this manuscript contains new content not previously published or submitted elsewhere for simultaneous consideration. i warrant that the article will not be submitted to another publication/publisher while under consideration for publication in science diliman. i further certify that all the other authors, if any, have agreed to the submission of this manuscript to the journal. ____________________________________________________ _________________ printed name and signature date return this form to: up rduo-ovcrd attn: science diliman lg/f phivolcs bldg., c. p. garcia ave., up diliman 1101, quezon city, philippines tel. nos. (632) 981-85-00 loc. 4048; (632) 436-8720 fax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph; rduo.ovcrd2012@gmail.com title: _______________________________________________________________________________ author/s: ___________________________________________________________________________ corresponding author: ______________________________________________________________ address: _________________________________________________________________________ telephone: __________________ fax: _________________ e-mail: ________________________ please list down the names of at least three individuals who may be appropriate reviewers of your manuscript. these suggestions will be used at the editorial board’s discretion. name affiliation tel.no./fax no./e-mail ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ as an aid to the indexer, please list down a maximum of six keywords that describe the content of your manuscript: ____________________________________________________________________________________ ____________________________________________________________________________________ manuscript submission form rduo form 3b science diliman research dissemination and utilization office up office of the vice chancellor for research and development a model to estimate aquaculture carrying capacity in three areas of the philippines tarzan legović1*, rune palerud2, guttorm christensen2 patrick white2, and regie regpala3 1centre for marine research, r. boškoviæ institute, p.o. box 180, bijenièka 54, hr-10002 zagreb, croatia tel +385 1 4680230; fax +385 1 4680242 * corresponding author; email legovic@rudjer.irb.hr 2akvaplan-niva as, tromsø polar environmental centre, n-9296 tromsø, norway 3national integrated fisheries technology development centre-bureau of fisheries and aquatic resources (bfar-niftdc), bonuan binloc, 2400 dagupan city, philippines date submitted: august 21, 2007; date accepted: august 21, 2008 abstract a model was developed to estimate the production carrying capacity of water bodies based on nutrient inputs from aquaculture and other sources, flushing rates, and the risk of algal blooms for three different areas of the philippines – bolinao (marine site), dagupan (brackishwater site) and taal lake (freshwater site). the results suggest that aquaculture production in the taal lake was greater than the sustainable carrying capacity. aquaculture structures in bolinao were close to carrying capacity during average tidal exchange but greater than the carrying capacity during low tidal exchange and no winds. aquaculture production in the dagupan estuary has not overcome its carrying capacity even during low flow. however, during very low flow and no tidal flushing, carrying capacity has been overcome. introduction modeling carrying capacity environmental carrying capacity for fish aquaculture is defined as the maximum number of fish of a given species that may be safely grown in the considered water body. the maximum number is limited by a variety of factors. certainly, if the maximum number exists for a single aquaculture occupying a given area, then the available area for fish cultures induces the upper limit. however, this limit may be much higher than the carrying capacity. computation of carrying capacity must be based on the condition which limits the stock maximally. in other words, it must be based on the limiting condition. a well known factor which could limit the maximum number more than the available area is the oxygen content in water. dissolved oxygen is used by fish and its content must not fall below a certain limit. during a normal sunny day, fish in high density is one of the major oxygen users. however, not all days are sunny. during several overcast days phytoplankton in high concentration is more intensive user of oxygen and hence one must ensure that phytoplankton is not able to reach very high concentration. otherwise, within a few days, phytoplankton will decrease oxygen content to a value which will dramatically increase fish mortality. since fish in aquacultures emits waste to the water body, and this waste contains nutrients used by phytoplankton, increasing the fish stock will cause unacceptably high phytoplankton concentration in water. hence this will limit the science diliman 31 legović, t., et al. standing stock of fish that we may have in the water body. consequently, our strategy to compute carrying capacity is composed of three steps: 1) consider characteristics of a water body. how quickly is the water exchanged with neighboring water where fish is not grown and hence has higher oxygen content? what are concentration and locations of other nutrient sources that enter considered water body? since fish is usually grown close to the coast, impact of land based sources may be considerable and may cause smaller carrying capacity for fish grown in aquacultures. 2) consider how the phytoplankton is grown and the concentration it is able to reach with given external sources. this will give us the remaining concentration of phytoplankton that we may reach by increasing aquaculture size. 3) increase (or decrease) fish stock until critical phytoplankton concentration is reached. so obtained fish stock will define the carrying capacity of the water body for a given species of fish. figure 1 depicts the process graphically. modeling methods a model of dependence of phytoplankton growth on nutrient let us consider a well mixed water body such as an upper layer of a lake or a coastal sea. denote the nutrient concentration by s and the nutrient concentration in phytoplankton concentration by x. nutrient concentration in phytoplankton is proportional to phytoplankton concentration. inflow of nutrient is: inflow of water, q, times concentration in the inflow, i. the equivalent rate of change in the concentration is q*i/v = d*i, where v is the volume of the water body. d is the flushing rate because water inflow is assumed to be equal to water outflow. then, the loss of concentration from a water body is d*s. concentration of nutrient in water is also lost by phytoplankton uptake: u*x. phytoplankton is lost from a water body due to outflow of water. this loss is equal to d*x. finally, the equations of the model are: ds /dt=d  i −s −ux (1) dx /dt=u−d x (2) where u = vmaxs /(h+s) = specific uptake rate = specific growth rate. the term u is known as the michaelis-menten-monod uptake. the parameter, vmax, is a fixed maximum growth rate = maximum uptake rate, while parameter h is the half-saturation constant. terms ds/dt and dx/dt denote rates of change of nutrient concentration in water, s, and the nutrient concentration in phytoplankton, x, respectively. let us investigate steady states of this model i.e. possible states when t →∞. steady states are solutions of ds/dt = 0 and dx/dt = 0. with this requirement, equations (1) and (2) become a system of two algebraic equations: d i−s *= v max s * x *  hs* (3)  v max s * hs * − d x *=0 (4) where (*) denote steady states. steady state (s*=0, x*=0) is called the total extinction state and it does not exist. indeed if we insert these values into (3) and (4) we see that equation (3) cannot be satisfied. for equation (3) to be satisfied: d*i = 0, which is impossible given that d > 0 and i >0. steady state (s*=i , x*=0) exists and it is called the phytoplankton extinction steady state. in this state the phytoplankton has been washed out from the reactor. for this state to be stable, flushing rate d must be greater than the maximum possible specific division rate of phytoplankton which is vmaxi/(h +i). 32 science diliman a model to estimate aquaculture carrying capacity finally, assuming s*≠0 and x* ≠ 0, from equation (4) we easily find: s *= dh v max−d (5) hence, s* can be positive only if v max d (6) from (3) and (4) it also follows that: i = x *s * (7) in other words, the sum of nutrient concentration in water and in the phytoplankton is equal to the inflowing concentration. by substituting (5) into (7) it follows: x *=i − dh v max− d (8) for x*> 0 the condition i > d h/(vmax-d) must hold. this gives a more stringent condition on vmax: v max d1h / i  (9) namely, it is not enough that vmax > d which ensures that s* > 0, but also s* < i and hence the condition (9). that is, even if the condition (6) is satisfied but the condition (9) is not, as t → ∞ the system starting with s(t=0) = so > 0 and x(t=0) = xo>0 will end up in (s*=i, x*=0 i.e. the extinction of phytoplankton. this will occur because wash out of phytoplankton dx will eventually overcome the growth vmaxsx/(h+s). hence, the condition (9) must be satisfied if we want that the non-extinction steady state (i.e. the state in which x persists) be stable (in fact a stable node). since we cannot control the maximum uptake rate, vmax, because this parameter is a property of existing phytoplankton in the lake, the condition (9) has to be rewritten in terms of d: d v max 1h / i  (10) hence, we have a conclusion: by controlling flushing rate we can control the concentration of phytoplankton in the lake. alternatively, since (9) and (10) means: i  dh v max−d . (11) we also conclude: by controlling the inflow of nutrients in the lake we can control phytoplankton in the lake. in figure 2 we display dynamics of two systems, each starting with its own initial condition. the first system starts with a large nutrient concentration so=200 and a low phytoplankton concentration, xo=50. wee see a phytoplankton bloom and then a tendency to a lower steady state x*. in the second system which starts with a low nutrient concentration, so=20, and a very low concentration of phytoplankton, xo=10, a transition to steady state is without phytoplankton bloom. science diliman 33 figure 1. the process of determining carrying capacity for fish aquaculture based on arriving at the critical phytoplankton concentration legović, t., et al. both systems tend to the same steady state since they are characterized with identical flushing rate, inflow of nutrient and phytoplankton characteristics vmax and h. consequences for the carrying capacity of a water body for fish farms the preceding section contains interesting information concerning carrying capacity of a water body for fish aquacultures. fish aquacultures emit nutrients into the lake and this is seen as an increase in the inflow d i. since the inflow of water to the water body does not change, and hence d is the same, an increase in fish cultures is seen as an increase in average nutrient concentration, i, that enters the reactor. we see from the equation (5) that as a consequence of an increase in i, the steady state of nutrient concentration, s*, in the reactor does not change. all the benefit of increasing nutrient inflow due to the increase in i goes into the increase of phytoplankton concentration. as equation (7) shows, the increase in steady state of phytoplankton concentration is linearly related to the increase in nutrient inflow. in other words, if the nutrient inflow doubles, the phytoplankton concentration will double. since we know that there exists a phytoplankton concentration which will induce fish kill due to excessive consumption of dissolved oxygen during night and an overcast day, by limiting phytoplankton concentration we limit the standing stock of fish which are the source of nutrient inflow. in case that rivers, other land based sources, and atmospheric input, bring so much nutrient that the critical steady state phytoplankton concentration has been reached already, the carrying capacity of the water body for the standing stock of fish is zero. of course, this conclusion may change if these other sources decrease. estimation of the carrying capacity from equation (7) when fish aquacultures do not exist, we have: i o= x o *s * (12) in the above expression, io is derived from other nutrient sources that drain into the water body and results into the background concentration of phytoplankton xo*. let us add a contribution from fish aquacultures: ia. then equation (12) changes into:  i oi a= x o * x a *s * (13) but we know that a critically high concentration of xc*, call it xc* = xo* + xa* will induce a critically 34 science diliman figure 2. dynamics of two systems. the first system starts with concentrations of nutrient so=200 and phytoplankton xo=50. the second system starts with so=20 and phytoplankton xo=10. parameters for both systems are i=100, d=0.04, vmax = 0.1, h=20. 0 20 40 60 80 100 0 50 100 150 200 s w ith so=200 x w ith xo=50 s w ith so=20 x w ith xo=10 time (a.u.) c on ce nt ra tio n a model to estimate aquaculture carrying capacity low dissolved oxygen. this will be achieved for a value called the carrying capacity for aquacultures. carrying capacity of aquacultures translates into a critical increase in nutrient concentration. denote this value by ic. now using (13), we have: i c= x c *s *− i o (14) if xc* is greater than xo we have ic > 0. note one more property of equation (14): since the phytoplankton keeps the nutrient concentration, s* , constant regardless of the increase in i, it is likely that xc* >>s*, so to a good approximation: i c= x c *−i o . (15) application issues the problem of the limiting nutrient in the above, we assumed that there exists a nutrient which limits the production of phytoplankton. we know that phytoplankton needs many nutrients to grow. all those nutrients which exist in higher concentration than the one upon which the production depends, are not of our concern. if we would use any of them in equation (15), we would get a smaller carrying capacity of fish aquacultures. hence, to benefit from the above results, we must use the limiting nutrient. it is only for this nutrient that all of the above results are relevant. so it is obvious that somehow we must know the limiting nutrient in advance. most oceanographers approach this problem as follows: we know that there exists a redfield ratio in phytoplankton. this ratio is an optimum ratio of nutrients that phytoplankton needs for growth. hence, if one has the excess of one nutrient over the other in water than the redfield ratio dictates in phytoplankton, the other is the limiting nutrient. legovic and cruzado (1997) have shown that this line of reasoning is misleading. they concluded that the redfield ratio of nutrients in phytoplankton does not translate into the redfield ratio in water as one to one relationship. the exception is only in the environment where the growth of phytoplankton is negligible (ultra oligotrophic waters). but, in a water body in which we drive phytoplankton to a high concentration, phytoplankton growth is not negligible. in other words, we are on the opposite side of the mentioned exception. to know which nutrient is limiting at a given time, the correct approach is to do separate experiments for each potentially limiting nutrient. the experiment is to increase one nutrient while keeping the others the same as they occur in the water body, and see if phytoplankton grows faster. the procedure needs to be repeated with all candidates for a limiting nutrient. the candidates are: reactive nitrogen, reactive phosphorus and reactive silica. from a number of experiments of the above kind it is known that for lakes and brackish waters the most likely limiting nutrient is phosphorus. hence for these kinds of environments we are advised to take phosphorus as the limiting nutrient. similar experiments for the seas have resulted into nitrogen being limiting for the atlantic, while phosphorus is slightly more limiting for the mediterranean. according to: dufour and berland (1999) and dufour et al., (1999), south pacific waters are nitrogen limited. however, these results are derived from very oligotrophic sub-tropical tuamotu archipelago and are probably not valid for bolinao. based on short-term responses of coral reef microphytobenthic communities to inorganic nutrient loading dizon and yap (1999) found that n and p are limiting when added together while neither n nor p seems to be limiting when added alone. in the area of a dominant impact of aquacultures, the limiting nutrient will be determined by the ratio in which aquacultures emanate nutrients. if we look at the distribution of n and p in fish feed: 73 kg n/ton and p=14 kg n/ton we have the ratio n/p = 5.21. if we were to feed phytoplankton with fish feed, p would be given in excess of n, since the ideal ratio in phytoplankton is n/p = 7 (by weight), and hence science diliman 35 legović, t., et al. n would be limiting. however, fish farms emanate 68 % of n and 28% of p from fish feed into the water column through excretion and soluble feces (lupatsch and kissil, 1998). this makes the ratio in emission: n/p = 13.13 by weight and hence, a fish farm induces p limitation of phytoplankton. finally, one more caution. it is possible that bioassay studies show phosphorus limitation at one time instant and nitrogen limitation at another instant, when at both instances the same ratio of nutrients has been emitted to water. the phytoplankton species composition is dynamic in time due to existing seasonal succession of phytoplankton species and hence for the same nutrient ratio in emission one species is limited by nitrogen while another may be limited by phosphorus or even silica. this latter is due to the fact that different phytoplankton species require different optimum n/p ratio. hence, bioassay results are a function of phytoplankton composition and dominance for a given time instant. critical phytoplankton concentration the critical phytoplankton concentration is one of the key parameters in equations (14) and (15) which determine the carrying capacity of aquacultures in a studied area. hence, our next problem is to determine the highest phytoplankton concentration which guarantees that oxygen concentration will not drop below the healthy level for fish. sowles (2005) gives the critical mean phytoplankton concentration as 4 μg chl-a/l. this concentration would bring dissolved oxygen concentration at the lowest value of 6 mg/l. perhaps this is acceptable critical dissolved oxygen content for salmon aquacultures in the gulf of maine, usa, but many agree that this dissolved oxygen value is too high as a critical value for freshwater tilapia species or trophic fish cultures. masser (1988) writes: “in general, warmwater species such as catfish and tilapia need a dissolved oxygen concentration of 4 mg/l do (or ppm) or greater to maintain good health and feed conversion. healthy warmwater fish can tolerate 1 mg/l do for short periods of time but will die if exposure is prolonged. prolonged exposure to 1.5 mg/l do causes tissue damage, and any prolonged exposure to low dissolved oxygen levels will stop growth and increase the incidence of secondary diseases, apparently by reducing fish ability to resist infection.” according to u.s. environmental protection agency (usepa) the maximum allowable total p should be 0.17 mg/l while the maximum allowable phytoplankton related chl-a should be 10 μg/l. if we assume that p in water is found almost exclusively in phytoplankton, then by using a relationship between total phosphorus (tp) and chl-a we find the upper value of chl-a that corresponds to tp found in water. according to dillon and rigler (1974): log 10μg chl-a/l=−1.134 1.5383 log10 μg tp / l. (16) the expression gave excellent correlation of r = 0.975 between the log10 (chl-a) and log10 (tp) for canadian lakes. usepa total p of 0.17 mg/l would mean 198 μg chl-a/l. this tells us that there is a gross mismatch between the standard for total p and chl-a. as it concerns us, the relationship for canadian lakes may not hold for tropic lakes. from 534 florida lakes, the following relationship has been found by researchers at the florida lakewatch (2000): log10 μg chl-a / l = – 0.369 1.053 log10 μg tp / l (17) given tp of 0.17 mg/l we get 95 μg chl-a/l. it is instructive to keep in mind (florida lakewatch, 2000): “in florida, when chlorophyll concentrations reach a level over 40 μg/l, some scientists will call it an algae or algal bloom.” “when algal biomass exceeds 100μg/l (measured as chlorophyll concentrations), there is an increased probability of a fish kill. fish kills, however, typically only occur after three 36 science diliman a model to estimate aquaculture carrying capacity or four cloudy days. during this time, algae consume oxygen rather than produce it because they don’t have sunlight available to help them photosynthesize more oxygen. this can lead to oxygen depletion. without oxygen, aquatic organisms, including fish, die. chlorophyll concentrations below 100 μg/l generally do not adversely affect fish and wildlife, but dead fish and wildlife can occasionally be found.” hence the above value of 0.17 mg p/l and corresponding 100 μg chl-a/l we may take as the indication that carrying capacity has been reached. let us also mention that the department of environment and natural resources has set the standard for total p as < 0.4 mg p/l. however, when compared to usepa and equation (17), the upper limit of 0.4 mg p/l is obviously an overestimate because it would result into unacceptably high phytoplankton concentration. this is an indication that the standard of < 0.4 mg p/l should be changed into < 0.17 mg p/l. background concentration of nutrient in the inflow, io the background concentration of nutrient, io, means an average value of nutrient concentration in the inflow from other sources. since there are two seasons, dry and wet: should we take the average value of the limiting nutrient for the dry season or the average value for the wet season? to resolve this dilemma we have to consider time scales of processes responsible for the phytoplankton dynamics and choose the most critical one. dry season in this season the phytoplankton dynamics will be driven by a small value of flushing rate d, small nutrient inflow in terms of d*i, but a high value of i (more concentrated nutrient in small streams that enter the water body). from the equations (5) and (8) we see that if phytoplankton has enough time to come close to steady state, the steady state would be higher than in case d is higher and i lower. since dry season is long enough for phytoplankton to grow to whatever value limits its growth, it follows that it is important to measure nutrient concentration in the inflow, because this concentration will determine carrying capacity of aquacultures. wet season wet season is characterized by much higher precipitation. direct precipitation contains small concentration of limiting nutrient. the effect on phytoplankton dynamics is basically determined by high flushing and this decreases existing concentration of phytoplankton and presents a weak basis for further phytoplankton growth. however, wet season is not characterized by a continuous higher precipitation but by a series of storms, sometimes violent ones. although the dilution phenomenon are more prominent, storms are followed by an increased erosion and flushing of agricultural fields which are rich in nutrients. during such storms up to 80 % of nutrients are flushed into the recipient water body, estuary, coastal bay or a lake. the first significant storm after the dry season is the one which brings most nutrients into the recipient water body. a representative total concentration made up of averaging across a series of streams and diffuse inflows is difficult to measure. however, the total inflow of water is usually available since it is equal to the outflow. a single outflow such as in the taal lake is not difficult to measure. the average concentration in such a case would be: i ave= q 1∗i 1q2∗i 2...qn∗i n q 1q2...qn (18) where qi and ii are the inflow of water through the ith stream and ii is the nutrient concentration in the ith stream, where the number of streams is i=1,…, n. given the fact that the inflow of water through the streams and the concentration of nutrient in each stream are highly variable in time, the problem of precisely estimating the average nutrient concentration is very difficult and time consuming process. the process is further complicated by the existence of diffuse inflows from agricultural lands, forests and meadows. the above shows that the precise determination of io will be difficult to obtain directly, and yet the carrying capacity depends on this determination. science diliman 37 legović, t., et al. the alternative is to resort to indirect methods. indirect methods would involve measurements of the nutrient concentration in the water body and possibly extract io from such measurements. indeed, equation (13) holds some potential to succeed by assuming much smaller measurement cost. when one makes measurements of the limiting nutrient in the water body, aquacultures are already there, hence io is masked by emission from aquacultures. now, if we would know the emission of aquacultures and the corresponding nutrient in the phytoplankton, then by using the above expression and neglecting s*, the determination of io would be straightforward. emission of nutrients by fish cultures in order to apply the equation (15) with all units being in mass nutrient per volume, for example, μg(nutrient)/l, we need to convert production of fish into the emission of nutrient into the environment. the fish stock in aquacultures is not constant but varies during the year. suppose, we are interested in the maximum stock, say fm (tons). this stock of fish emits fn mass of nutrient in a time interval, for example in kg (nutrient)/day): f n=a f m (19) the parameter a specifies how many kg of nutrient are emitted per one ton of standing stock of fish. from the value of fn, the equivalent concentration addition in the water body may be calculated from: i c= f n dv = f n inflow or outflow of water into or from lake (20) now, by imposing the ic value we may calculate back the value of fm i.e. the carrying capacity of the water body in terms of tons of fish. modeling carrying capacity in taal lake volume v of the receiving body of water in taal is v = 2.43×109 m3. this volume is derived from the surface area of the taal lake, which is 2.43×108 m2 and the depth of the upper 10m. average yearly rainfall is 1882.9 mm and average evaporation is 116.75 mm, so the net water layer entering the lake is 1766.15 mm over the watershed and the lake. in addition to the lake, the watershed is 4.2×108 m2. hence the yearly inflow is 1.2×109 m3. thus d = (inflow=outflow)/v = 0.494/year. therefore the lake needs two years to renew one volume of water to the depth of ten meters. the net inflow of aquacultures is: fn = 2.13×103 kg (phosphorus)/day = 730 t/y. this value may be compared to 816 t/y reported by vista et al., 2006. hence, the contribution of fish cultures to the nutrient concentration in phytoplankton is: i a = 730×103kg p / y 0.494/ y∗2.43×109 m3  i a = 730×103 kg p / y 1.2×109 m3 / y =608mg p / m3 i a = 608 g p / l (21) according to the florida lakewatch relationship in equation (17) the value of total phosphorus translates to 365 μg chl-a/l. from the above calculation it would appear that the carrying capacity of the taal lake has been overcome due to fish cultures alone by a factor of 3.7. let us now assume that nitrogen is limiting. input of nitrogen to the water column from aquacultures is 26 259.56 kg/day. i a= 9.6×106 kg n / y 1.2×109 m3 / y =8 g n / m3 =8000  g n / l (22) now using the lakewater relationship between total nitrogen (tn) and chl-a: log 10chl-a /l =−2.421.206 log 10 g tn / l (23) we obtain 193 μg chl-a/l. we conclude that if nitrogen were limiting, and other sources of nitrogen are negligible, the carrying capacity for aquacultures in the taal lake would be 38 science diliman a model to estimate aquaculture carrying capacity overcome by a factor of 2. finally, with n/p of 13.13 (by weight) simultaneous limitation of n and p can not be ruled out. in this case the following relationship would hold (florida lakewatch 2000): chl-a=0.628 tp−2.402 . (24) for the taal lake, with tn = 8000 μgn/l, tp = 609 μg p/l the resulting chlorophyll-a would then be 380 μgchl-a/l. modeling carrying capacity dagupan estuary the volume of the receiving body of water is v = 0.3×109 m3. the volume is derived from the surface area of the estuary, which is 68×106 m2 and the depth of 4.4 m. the depth is obtained from detailed bathymetric measurements obtained in the project. average inflow of the river feeding the estuary during the most critical period (end of the dry season) is 38.4 m3/s = 3.3×106 m3/day. this is based on the current measurements within the project. the average current at the center of the measurement station is 10.5 cm/s with a current distribution factor of 0.5, river width of 140 m, the depth of 8.7 m and a cross-sectional shape factor of 0.6. the flushing rate is d = (inflow=outflow)/v = 0.011 (1/day). the estuary needs 91 days to renew one volume of water. net inflow of aquacultures including resuspension is fn = 146 kg (phosphorus)/day. hence, the contribution of fish cultures to the nutrient concentration in phytoplankton is: i a = 146kg p /day  0.011l / day ∗0.3×109 m3 =44.2mg p /m3= g p / l  (25) using equation (17) the value of total phosphorus translates to 23.1 μg chl-a/l. from the above calculation it would appear that the carrying capacity of the dagupan estuary has not been overcome. however, the above contribution to chl-a value has been derived only from limiting nutrient inflow from fish cultures. two other nutrient inflows have not been considered: a) from people, their agriculture and animal farming activities. this input is very important and it probably contributes as much as the existing fish cultures. b) nutrient concentration in the river water feeding the estuary. depending on the activities upstream the estuary, this input could contribute one quarter to one half of fish cultures. if a) and b) were also taken into account, it would still turn out that the natural carrying capacity of the dagupan estuary under consideration has not yet been reached. the above calculation is based on the river inflow of 38 m3/s. we know that the river inflow feeding the estuary is not constant but varies greatly. during the dry season, the river inflow may be smaller. when the river inflow is half the one that has been measured, the contribution of fish cultures together with other existing inflows, overcome the natural carrying capacity. during this time of the year, fish kills would be imminent. according to the existing information, this has already happened at the end of the dry season, which is characterized by the lowest river inflow. modeling carrying capacity in bolinao bay surface area of 28.88×106 m2 with an average depth of 4.8 m leads to the volume v= 138.6×106 m3. residence time of particles at bolinao, varies from several days to over 25 days, so it would be reasonable to use 20 days. excretion of phosphorus from aquacultures amounts to 339 kg/day, a contribution from soluble feces is 143 kg/day and resuspension from the bottom is science diliman 39 legović, t., et al. estimated as 94 kg/day. together, this amounts to 576 kg/day. i a = 576kg p / day 0.051/ day∗138.8×106 m3 = 83 g p / l (26) from equation (17), we obtain the corresponding contribution to the phytoplankton concentration of 44 μg chl-a/l inclusion of external sources the above contribution to phytoplankton concentration is on top of all external sources of nutrients: a) land based sources, b) concentration of nutrients entering from lingayen bay and c) atmospheric fall out. if external sources were negligible or even if they are of the same order of magnitude as the contribution from fish cultures, which is unlikely, it would appear that the maximum concentration of 100 μg chl-a/l has not been reached and hence the carrying capacity of the bay has not been overcome. flushing of the bay the above calculation is based on a particle residence time of 20 days which is computed from an average including neap and spring tides. we know that the marine through flow is not constant but varies greatly. during the neap tide, flushing is much slower. if the water through flow is half the one that has been assumed, the contribution of fish cultures alone would get close to natural carrying capacity. then the inclusion of all external nutrient sources would well overcome the carrying capacity. according to the existing information, this has already happened at the neap tide, which is characterized by the lowest through flow. modeling conclusions the calculations suggest that aquacultures in the taal lake have overcome the carrying capacity. aquacultures in bolinao bay are close to carrying capacity during average tidal exchange. this means that during low tidal exchange and no wind, carrying capacity has been overcome. aquacultures in dagupan section of the estuary have not overcome carrying capacity even during low flow. however during very low flow and no tidal flushing carrying, capacity has been overcome. references dillon p.j. & rigler f.h., 1974. the phosphoruschlorophyll relationship in lakes, limnol. oceanogr, 19: 767-773. dizon r.m. & yap h.t., 1999. short-term responses of coral reef microphytobenthic communities to inorganic nutrient loading, limnol. oceanogr., 44(5):1259–1267. dufour p. & berland b., 1999. nutrient control of phytoplanktonic biomass in atoll lagoons and pacific ocean waters: studies with factorial enrichment bioassays. j. exp. mar. biol. ecol., 234:147-166. dufour p., charpy, l., bonne, s. & garcia, n., 1999. phytoplankton nutrient control in the oligotrophic south pacific sub tropical (tuamotu archipelago). marine ecology progress series, 179:285-290. florida lakewatch, beginner's guide to water management – nutrients information circular, no. 102 2000. university of florida. legovic t. & cruzado a., 1997. a model of phytoplankton growth on multiple nutrients. ecological modelling, 99:19-31. lupatsch i. & kissil g.w., 1998. predicting aquaculture waste from gilthead seabream (sparus aurata) culture using a nutritional approach. aquat. living resources, 11: 265-268. masser m., 1997. cage culture: site selection and water quality, oklahoma cooperative extension service, srac161, http://srac.tamu.edu/ sowles j.w., 2005. assessing nitrogen carrying capacity for blue hill bay , maine : a management case study. in hargrave b., ed., environmental effects of marine finfish aquaculture, springer, pp 359-380. 40 science diliman call for papers-revised.pmd call for papers for more information about editorial requirements, please contact: the editor-in-chief science diliman c/o research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg., c.p. garcia ave. up diliman, quezon city 1101 tel. nos.: (632) 981-8500 voip 4048; 436-87-20 telfax: 927-2568 e-mail: rduo.ovcrd@up.edu.ph; rduo.ovcrd2012@gmail.com website: http://www.ovcrd.upd.edu.ph the office of the vice-chancellor for research and development university of the philippines diliman is accepting papers for publication in science diliman science diliman is an internationally refereed semi-annual journal of pure and applied sciences. results of inter-disciplinary research projects may also be submitted for publication. papers submitted will undergo peer review before final approval for publication. editorial board marco nemesio e. montaño, phd editor-in-chief associate editors patricia v. azanza, phd food science and nutrition jose maria p. balmaceda, phd mathematics zubaida u. basiao, phd biology carlos primo c. david, phd earth sciences joel joseph s. marciano, jr., phd computer science, engineering giovanni a. tapang, phd physics irene m. villaseñor, phd chemistry ocr document ocr document ocr document inside front cover-19-2 editorial board editor-in-chief maricor n. soriano, ph.d. associate editors mario a. aurelio, ph.d. earth 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in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p300.00/year (two issues), exclusive of postage us$ 25.00/year (two issues), exclusive of postage tel. no: (632) 981-85-00 loc. 4047 (632) 927-2567; 927-2309; 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences cover art the illustration shows a multimedia server distributing content to different clients grouped according to how fast they receive the content (tiglao et al. pp. 33-42, this issue.) cover art courtesy of engr. nestor michael tiglao of the electrical and electronics engineering institute, university of the philippines, diliman. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. kelvin s. rodolfo, phd. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu rudolf a. roemer, phd. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk luis g. sison, phd. electrical and electronics engineering institute university of the philippines, diliman luis.sison@up.edu.ph raul k. suarez, phd. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu 01_device device fabrication 1 device fabrication of 60 µm resonant cavity light-emitting diode j. j. c. reyes1*, w. bisquerra1, r. v. sarmago1, and a. a. salvador2 1materials science and engineering program, college of engineering, university of the philippines, diliman, quezon city 1101 2condensed matter physics laboratory, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: jreyes@nip.upd.edu.ph abstract science diliman (july-december 2004) 16:2, 1–5 an array of 60-mm-diameter resonant cavity light-emitting diodes suited for coupling with fiber optic were fabricated using standard device fabrication technique. i-v characterization was used to determine the viability of the device fabricating process. under forward bias, the turn-on voltage of the devices is 1.95–2.45 v with a series resistance of 17–14 kw. under reverse bias, the devices showed a breakdown voltage of 35 v. introduction laser diode and light-emitting diodes (leds) are effective light sources (koudelka & woodall, 2002) for optical communication system (dumitrescu et al., 2002). for this purpose, vcsel/rce geometry structure (dumitrescu et al., 2002) is better preferred over conventional laser and led structure for its superior optical properties, which include higher spectral purity (dumitrescu et al., 2002; schubert et al., 1992), better directionality and coupling efficiency (dumitrescu et al., 2002; gökkavas et al., 2001; corbett, 1993), high brightness, increased efficiency, and ease of fabricating two-dimensional arrays of devices (dumitrescu et al., 2002). for many applications, like low-cost short-haul communication system (dumitrescu et al., 2002; schnitzer et al., 1993), resonant cavity leds (rcleds) emitting in the same red wavelength range offer a suitable alternative over vcsels (dumitrescu et al., 2002; schnitzer et al., 1993) because of its thresholdless operation, better thermal behavior, and robustness. while the geometry structure allows easy coupling with optic fibers, it is also desirable to have an active window size that is comparable to the fiber optics, which has typical core diameters of 50 and 62.5 mm (gökkavas et al., 2001). the condensed matter physics laboratory of the national institute of physics, up diliman, is making an active participation in the development of optoelectronic devices that can be used for optoelectronics applications. in the past years, we were able to grow gaas-based heterostructure semiconductor layers and fabricate them into devices. these devices include vertical cavity surface-emitting lasers (vcsels) (manasan et al., 2004; samson et al., 2003; estacio et al., 2003; agra, 2003), edge-emitting lasers (manasan et al., 2003), light-emitting diodes (leds) (agra et al., 2004), and photodetectors (somintac et al., 2001). the vcsels and leds that the cmpl had fabricated, 250 mm device diameter, have relatively large active window compared with the size of the optical fiber. in *corresponding author sarmago, reyes, and bisquerra 2 this work, we designed a fabrication process for 60mm-diameter rcleds that can be easily coupled with the typical fiber optic size. the challenge in the new design is to provide the device with a large enough p contact dimension (fig. 2) for ease in interconnects. the thickness of the ring p contact is approximately 10 mm, which is relatively small for wire bonding. the solution is to add a top metal pad, which is made feasible by the addition of a polyimide overlay. experimental method sample description the sample used, a vcsel/rce p-i-n layer, was grown on n-type (001) gaas substrate (somintac et al., 2001) using a riber 32-p molecular beam epitaxy (mbe). figure 1 shows the schematic diagram of the layer structure grown. the sequence consists of a gaas (1.0 mm) buffer layer followed by 28 pairs of silicondoped alas (710 å) and algaas (623 å) layers, three pairs of algaas (60 å) and gaas (95 å) quantum wells (qw), then 24 pairs of beryllium-doped alas (710 å) and algaas (623 å) layers, and finally, a beryllium-doped gaas (100 å) cap layer. device design figure 2 is the device structure design for a 60 mm device. in addition to the conventional device structure, polyimide is added as an overlay to make the deposition of a top pad contact possible. device fabrication before the sample was fabricated into the 60 mm device, the indium on the backside was removed using an indium removal solution (10% ammonium hydroxide). the sample was then degreased using the standard degreasing procedure, which includes 2–5 min soak in tce, acetone, and methanol baths with ultrasonic agitation. the sample was rinsed under free-flowing de-ionized (di) water, then finally blown dry with highpurity nitrogen gas. to fabricate the designed 60 mm device structure, the sample underwent the standard device fabrication technique. this includes photolithography using the karl suss mj3 mask aligner system, etching, and metallization processes. the summary of the fabrication process is shown in fig. 3. figure 3(a) is the prefabricated mbe sample. the ring contact was defined under uv exposure of the negative photoresist (ma-n 425 b ) [figs. 3(b) and 3(c)], spin coated over the sample, using patterned mask. after developing, the top contact, auzn/ti/au, was deposited using an in-house-built metallization setup. photoresist was stripped off using acetone and the metal was alloyed [fig. 3(d)] using a tube furnace with high-purity nitrogen purge. the 60 mm mesa was created by, initially, pattern definition of positive photoresist (map 275) under uv exposure [figs. 3(e) and 3(f)] and then etching [fig. 3(g)] of the patterned sample using a h 2 so 4 :h 2 o 2 :h 2 o (1:8:80) etchant solution. the pattern for the bottom contact was defined using uv exposure [figs. 3(h) and 3(i)]. afterwards, the bottom contact, auge/ni/au, was deposited, the photoresist was lifted off, and then the metal was alloyed [fig. 3(j)]. to create the top metal pad, a polyimide overlay was added. an overlay pattern was defined using uv exposure [figs. 3(k) and 3(l)] of a durimide 7005 1234567890123456789012 1234567890123456789012 1234567890123456789012 1234567890123456789012 123456789012345678901234567890121234567890 123456789012345678901234567890121234567890 123456789012345678901234567890121234567890 123456789012345678901234567890121234567890 123456789012345678901234567890121234567890 123456789012345678901234567890121234567890 123456789012345678901234567890121234567890 gaas (be) cap be-doped algaas/alas dbr algaas/gaas qw si-doped algaas/alas dbr gaas buffer r-type (001) gaas substrate fig. 1. schematic diagram of vcsel/rce layer structure. 65 µm 40 µm 20 µm 40 µm fig. 2. schematic diagram of the 60 µm device structure showing dimensions. (a) top view of the metal pad. (b) side view of the device structure. (a) (b) device fabrication 3 negative polyimide spin coated over the sample. an opening on the polyimide overlay was created next to the top and bottom contacts (fig. 2). the top metal pad was deposited by, first, pattern definition using the photolithography process [figs. 3(m) and 3(n)], then metal deposition, and finally, removal of the photoresist. the final device structure is shown in figs. 2(b) and 3(o). device characterization current-voltage (i-v) measurement, which gives information such as turn-on voltage and sample resistance, was used to check the viability of the 60 mm device fabricated. the characterization setup used is shown in fig. 4. the sample was mounted on a needle-probe xyz translation stage and the currentvoltage (i-v) curves were taken using a tektronix i-v curve tracer. results and discussion figure 5 is the sem micrograph of a 60 mm device in fig. 3(j), showing a clearly defined layer profile of the sample. in fig. 6 is the sem micrograph of a 60 mm device with a polyimide overlay, showing an opening next to the top and bottom contacts. notice that the layer profile is completely covered with polyimide, which therefore prevents short circuit in the device. the micrograph of a complete 60 mm device fabricated is shown in fig. 7. fig. 3. schematic diagram for fabrication of the 60 µm device. (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (o) curve tracer fig. 4. schematic diagram of i-v characterization setup. fig. 5. sem micrograph of the 60 mm device structure (4.96k x magnification). n contact p contact fig. 6. isometric view of polyimide overlay (1.81k x magnification). p contact polyimide n contact sarmago, reyes, and bisquerra 4 the i-v characteristic of a 60 mm rcled device is shown in fig. 8. the curve shows an exponential behavior, which agrees with the diode equation, i = i o exp[(ev/k b t)–1] (where i o is the leakage current in fig. 7. isometric view of the 60 µm device with polyimide overlay and top metal pad (1.5k x magnification). n contact top metal pad contact polyimide fig. 8. i-v characteristic of the 60 mm rcled (200 ma x 5 v). fig. 9. i-v characteristic of the 250 mm rcled (200 ma x 5 v). reverse bias, e is the electronic charge, v is the bias voltage, k b is the boltzman constant, and t is the absolute temperature). under forward bias, the i-v characteristic shows a turn-on voltage at 1.95 v. at high forward bias, the current increase differs from exponential and shows linear i-v characteristics. this area exhibits a relatively low differential conductivity (di/dv) indicating a high device series resistance, around 6 kw. under reverse bias, the sample shows a breakdown voltage of 35 v. figure 9 is the i-v characteristic of a 250 mm rcled device. the sample shows a turn-on voltage of 1.3 v and a series resistance of around 6 kw. both devices of different dimensions have different turn-on voltages. this could be possible since the 60 mm device underwent numerous steps during device fabrication. a previous step may leave a residue on the surface of the device prior to metallization, which could be a possible cause of the device’s high resistance. both devices show a high series resistance, which was initially attributed to the high resistance of dbr layers, but a non-dbr layer also shows a high series resistance of 4–8 kw. high resistance can then be attributed to alloying of the metal contact, since the alloying parameters were not optimized. conclusion the sem micrograph and the i-v curve demonstrated the viability the cmpl to fabricate small devices such as 60 mm rcleds. this represents a substantial improvement in our device fabrication capabilities. further improvements in alloying for metal contact are needed to lower the device’s high series resistance. references agra, f.a., 2003. thermal oxidation of alas and the possibility of confinement in an optical device. university of the philippines, diliman. agra, f., e. estacio, a. somintac, and a.a. salvador, 2004. fabrication and characterization of an oxide confined front surface light-emitting diode. 13th asemep national technical symposium. device fabrication 5 corbett, b., l. considine, s. walsh, and w.m. kelly, 1993. resonant cavity light-emitting diode and detector using epitaxial lift-off. ieee photonics tech. lett. 5(9): pp. dumitrescu, m., o. tampere et al. 2002. rc-leds for plastic optical fiber applications. semiconductor international, april 2002. estacio, e., c. alonzo, a. samson, a. garcia, a. somintac, and a. salvador, (submitted). time response characteristics of an oxide-confined gaas/algaas resonant cavityenhanced photodetector. appl. phys. lett. estacio, e., g. manasan, a. somintac, a. podpod, a.j. samson, f. agra, and a.a. salvador, 2003. characteristics of an oxide-confined gaas/algaas vcsel emitting at 842 nm. proceedings of the 21st spp physics congress. gökkavas, m., o. dosunmu, m.s. ünlü, g. ulu, r.p. mirin, d.h. christensen, and e. özbay, 2001. high-speed highefficiency large-area resonant cavity enhanced p-i-n photodiodes for multimode fiber communications. ieee photonics tech. lett. 13(12): pp. koudelka, robert d. & j.m. woodall, 2002. light-emitting device with increased modulation bandwidth, yale university. manasan, g., e. estacio, and a.a. salvador, 2003. investigation of the quantum efficiency of an edge-emitting ingaas laser. proceedings of the 21st spp physics congress. manasan, g.g., a.j. samson, a.t. garcia, and a.a. salvador, 2004. device fabrication of gaas-based lasers. 14th asemep national technical symposium. samson, a.j., f. agra, g. manasan, e. estacio, a. podpod, a. somintac, and a.a. salvador, 2003. current confinement in an optical device using alas oxide. proceedings of the 21st spp physics congress. schnitzer, i., e. yablonovich et al., 1993. 30% external quantum efficiency from surface-textured, thin-film leds. appl. phys. lett. 63(16): pp. schubert, e.f., y.h. wang et al., 1992. resonant cavity lightemitting diode. appl. phys. lett. 60 (8): pp. somintac, a.s., e. estacio, m.f. bailon, and a.a. salvador, 2001. growth of gaas-based vcsel/rce structures for optoelectronic applications via molecular beam epitaxy. proceedings of the 19th spp philippine congress. sd122-c1 enhancement effect of sea urchin 1science diliman (july december 2000) 12:2, 1-9 abstract enhancement effect of sea urchin grow-out cages in lucero, bolinao, pangasinan maria celia defrance malay*, helen grace p. bangi, and marie antonette r. juinio-meñez marine science institute, college of science, university of the philippines diliman, quezon city 1101 philippines tel. no. (632) 922-3959; e-mail: machel@upmsi.ph a preliminary study was conducted on the environmental impact of sea urchin (tripneustes gratilla linnaeas) grow-out culture in lucero, bolinao, pangasinan. it was hypothesized that the feces generated by the caged urchins (~6,000 individuals at any one time) might cause localised sediment organic enrichment and subsequent shifts in benthic faunal communities. results from preliminary surveys conducted in april and august of 1999 indicated minimal impact of sea urchin grow-out culture on the local reef flat community. some enhancement of faunal abundance and sediment organic matter content in the cage area were noted; however, the impact was limited to a radius of 5-25 meters from the grow-out cages. the enhancement effects appeared to be seasonal occurrences that were dependent on local currents and degree of wave exposure. epiphyte biomass, total suspended solids, sediment grain size, and relative water movement seemed largely unaffected by sea urchin grow-out culture. however, more frequent and thorough samplings are needed to validate these initial results. the presence of localised enrichment in sediment organic content and epibenthic faunal density suggest the possibility of converting the sea urchin grow-out area into polyculture systems that would make more efficient use of the food resources available while minimizing potential anthropogenic impacts on the environment. keywords: sea urchin, aquaculture, environmental impact, enhancement effect, organic enrichment introduction numerous studies have documented the impact of aquaculture activities on the marine environment (reviewed in buschmann and others 1996). for the most part, research has been centered on the effects of fish farms and mussel farms on the sediment and water column. nutrients and waste emanating from fish farms may cause localised changes in the physical and chemical characteristics of the sediment, elevating organic matter content, nitrogen and phosphorous levels, and sediment metabolism and sulfate reduction (findlay and others 1995, holmer and kristensen 1992, karakassis and others 1998). these changes in physicochemical parameters may in turn cause shifts in benthic faunal assemblages and increase epibenthic faunal densities (findlay and others 1995, karakassis and others 1999). while mussel farming generally has less environmental impact than fish farming, biodeposition from mussel lines may also result in local increases in sediment nutrient and organic matter content, consequently changing the structure of benthic communities (castel and others 1989, grant and others 1995, hatcher and others 1994). high nutrient levels in the water column can also cause eutrophication and *corresponding author defrance malay et al. 2 increase algal epiphyte biomass. large epiphyte loads may have detrimental effects on the productivity of submerged macrophytes through shading and reduced nutrient availability (duarte 1995, frankovich and fourqurean 1997, wear and others 1999). the grow-out culture of the commercially-important sea urchin tripneustes gratilla is an emerging technology currently being refined and promoted in bolinao, pangasinan by the university of the philippines marine science institute (up-msi). the up-msi’s sea urchin research project aims to aid in the recovery of natural spawning populations of t. gratilla that were severely depleted in the early 1990s because of uncontrolled harvesting of fishery stocks (reviewed in juinio-meñez and others 1998). while grow-out culture in sea pens is deemed a very promising fishery management strategy (juinio-meñez and others 1998), the potential impact of small-scale sea urchin cage culture on the local environment has yet to be studied. t. gratilla are herbivores that consume approximately 20% of their body weight in plant matter per day (bangi, unpubl. data), and produce correspondingly large amounts of feces on a daily basis. their feces have been shown to contain large amounts of ammonium that enhance the nitrogen levels of the water column (koike and others 1987) and to contribute to the biodeposition of organic matter in the sediment. the culture pens in the main pilot grow-out site in lucero, bolinao occupy an area of 64 m2 and typically hold up to 6,000 sea urchins at any one time. hence, cage grow-out in this site may have a significant impact on the local reef flat community. however, it is postulated that the overall effect of small-scale sea urchin culture is minimal compared to finfish culture because only natural, not artificial algal feeds (e.g., the brown seaweed sargassum sp.) are used. this paper presents the initial findings of a study on the environmental impact of a small-scale sea urchin grow-out culture on a shallow seagrass reef flat. to quantify the impact, the following parameters were assessed: epibenthic faunal abundances, epiphyte biomass, sediment grain size, total sediment organic matter, total suspended solids, and relative water movement with respect to distance from the grow-out cages. methodology study area and sampling station the study was conducted on a seagrass bed along a reef flat in bo. lucero, bolinao, pangasinan, where sea urchin pens were set up three years ago (fig. 1). four 50-m transects radiating from the grow-out cages (two transects are parallel to the shore, and the other two are perpendicular to the shore) were established. in each transect, permanent sampling stations were set up at 0 (right beside the cages), 5, 25, and 50 meters from the cages. samplings of all parameters were done in triplicate, in april and august 1999 (the 50-m stations were not sampled in august, except for the total suspended solids, as detailed below). benthic macrofauna using a 0.25 m2 quadrat, the benthic macrofauna were collected, sorted into epiphytic (“leaf”) or epibenthic (“sediment”) component, stored in plastic bags, preserved in 10% buffered formalin, identified to genus bolinao silaqui lucero santiago island bml sea urchin cages 0m 5m 25m 50m lucero fig. 1. diagram showing experimental sampling design. inset: location map of study site in lucero, bolinao, pangasinan enhancement effect of sea urchin 3 level (when possible), and counted. their faunal density data were expressed in number of animals per m2. two-way anova (p<0.05) was used to determine significance of results. epiflora (seagrass epiphytes) shoots of the dominant, seagrass enhalus acoroides, were collected from all sampling stations for epiphyte biomass studies. these were preserved in 10% buffered formalin upon reaching the laboratory. the epiphyte were carefully scraped off from every excised centimeter of the seagrass blade. their biomass were measured after drying in the oven to constant weight at 60°c. two-way anova (p<0.05) was used to test the significance of variation in epiphyte dry weights with respect to the distance from the grow-out site and the transect surveyed. sediment analyses sediment samples were obtained using cylindrical corers and were immediately placed on ice and stored at 20ºc upon reaching the laboratory. the upper layer (0-1 cm) was analyzed separately from the lower layer (15 cm). to determine sediment grain sizes, dried sediments were wet-sieved and graded using the wentworth scale (buchanan 1984). total sediment organic matter (tom) was calculated as the difference between the weight of oven dried sediment and its ash (buchanan 1984). two-way anova (p<0.05) was used to analyse results. the dependent variable was the percent organic matter content of the sediment while the independent variables were the transect sampled and the distance from the cages. scatter plots were also constructed relating tom and abundance of total fauna, gastropods, and the major taxa (strombus sp., columbella sp., and hermit crabs). the % tom was assumed to be the independent variable while faunal density, the dependent variable. total suspended solids one-liter samples of surface seawater were obtained from extreme sides of the transect (0 m and 50 m). these were filtered and the particulate material obtained was oven-dried to constant weight. water movement the degree of water movement in all sampling stations was determined using clod cards (doty 1971). results benthic macrofauna total epibenthic fauna was higher in august (105±1.63 to 213±6.15 individuals m-2) than in april (57±11.63 to 71±9.68 individuals m-2) when the small (~1 mm shell length) gastropods columbella sp.were dominant. crustaceans (mainly hermit crabs) constituted the second most abundant group, followed by the holothurians (figs. 2 and 3). in april, total epibenthic faunal density, both leafand sediment-associated epibenthic fauna, were not significantly affected by distance from the grow-out cages (fig. 4a). however, values differed significantly between transects (p<0.05). the interaction of distance and transect was not significant. likewise, total gastropod density was not significantly affected by distance (fig. 4b). transect was a significant factor for leaf-associated but not for the sediment-associated. the interaction of gastropod density, transect and distance was not significant. in august, total faunal density was significantly affected by distance from the cages for both leafand sedimentassociated fauna (fig.5a).neither transect, nor the interaction of transect and distance significantly affected total faunal density (p<0.05). the density of leafassociated fauna was significantly lower at 25 m than at 5 m and beside the grow-out area (tukey’s hsd test, p<0.05). the density of leaf-associated gastropods was significantly affected by distance (fig.5b), but not by transect nor the interaction of distance and transect (p<0.05). gastropod densities at 5 m from the cages were significantly different from densities at 25 m defrance malay et al. 4 figs. 2a & b. average composition and abundance (± s.e.) of epifauna collected from (a) seagrass leaf blades and (b) sediment substrate, april 1999 0 0 0 0 0 0 0 0 0 5 25 50 gastropods crustaceans other taxa 0 0 0 0 0 0 0 0 0 5 25 50 gastropods crustaceans other taxa 140 120 100 80 60 40 20 0 140 120 100 80 60 40 20 0 502550 502550 a bu nd an ce m -2 a bu nd an ce m -2 (a) (b) distance from cages (m) distance from cages (m) figs. 3a & b. average composition and abundance (± s.e.) of epifauna collected from (a) seagrass leaf blades and (b) sediment substrate, august 1999 80 60 40 20 0 140 120 100 80 60 40 20 0 2550 502550 a bu nd an ce m -2 a bu nd an ce m -2 (a) (b) distance from cages (m) distance from cages (m) 0 0 0 0 0 0 0 0 gastropods crustaceans other taxa 140 120 100 gastropods crustaceans other taxa (tukey’s hsd test, p<0.05). analysis of variance of densities of the dominant leaf-associated gastropod, columbella sp., showed that densities of collumbelids were not significantly affected by distance but by the transect. the interaction of distance and transect was not significant. on the other hand, the density of sediment-associated gastropods was affected significantly (p<0.05) by transect, but not by distance nor the interaction of distance and transect. densities of the dominant sediment-associated gastropods, strombus sp., were significantly (p<0.05) affected by distance from the cages, transect, and the interaction of distance and transect. strombus sp. densities were significantly higher beside the cages than at distances of 5 meters and 25 meters from the cages (tukey’s hsd test, p<0.05) (fig. 5c). sediment analyses fine sand grains predominated in the sediment across all sampling stations for both april and august. average organic matter levels were higher in august (6.98 ± enhancement effect of sea urchin 5 0.78%) than in april (5.78 ± 0.35%). during both months, average tom was consistently higher in the top layer of the sediment (8.68±1.74% in april, 6.07± 0.40% in august) than in the lower sediment layer (6.89±0.35% in april, 5.49± 0.26% in august). moreover, total organic matter in the lower sediment layer was not significantly affected by the distance and transect, nor by the interaction of distance and transect. in april, tom of the top sediment layer was significantly (p<0.05) affected by distance from the cages, but not by transect or the interaction of figs. 4a & b. mean abundance (± s.e.) of (a) all faunal taxa and (b) all gastropod taxa, april 1999 0 502550 502550 a bu nd an ce m -2 a bu nd an ce m -2 (a) (b) distance from cages (m) distance from cages (m) 0 5 0 5 0 5 0 leaf fauna sediment fauna 150 125 100 75 50 25 150 125 100 75 50 25 leaf fauna sediment fauna distance from 150 cages (m) 25 20 15 10 5 0 2550 a bu nd an ce m -2 (c) leaf fauna sediment fauna distance from cages (m) 150 125 100 75 50 25 0 2550 a bu nd an ce m -2 (a) distance from cages (m) leaf fauna sediment fauna 150 125 100 75 50 25 0 2550 a bu nd an ce m -2 (b) distance from cages (m) leaf fauna sediment fauna figs. 5a, b, & c. mean abundance (± s.e.) of (a) all fauna taxa (b) all gastropod taxa, and (c) strombus sp, august 1999 defrance malay et al. 6 distance and transect. tukey’s hsd test (p<0.05) showed that tom levels were significantly higher beside the cages than 50 meters from the cages (fig. 6a). however, in august, organic matter levels of the upper sediment layer were not significantly different with respect to distance, transect, or the interaction of distance and transect (fig. 6b). scatter plots of tom in the upper sediment layer vs. abundance of epibenthic fauna are shown in figs. 7 ad. in april 1999, total faunal density varied inversely with the percent total organic matter of the top sediment layer (r2=69.3%, fig. 7a). in august, however, tom levels did not show a good fit with total faunal densities (r2=26.6%, fig. 7b). likewise, little variation in the densities of total gastropods (fig. 7c-d), strombus sp., columbella sp., and hermit crabs was explained by percent tom on either sampling date. seagrass epiphyte biomass average epiphyte biomass did not vary in the two sampling periods (3.00mg ±0.55 se in april; 3.08mg ±0.31 se in august). there was no significant difference in epiphyte biomass with respect to distance, transect, or the interaction of distance and transect for either sampling date (figs. 8a & b). total suspended solids (tss) and water movement amount of tss was higher at 50 m from the cages than beside the cages. the tss levels were higher in april (12.89±1.66 g se) than in august (7.94g ±0.74 se). average clod card dissolution rates were higher in april (5.10g/h±0.04 se) than in august (2.86g/h±0.05 se), indicating higher water movement in april than in august: very little spatial variation in water movement was observed, however, with consistently lower values recorded beside the cages. discussion data on faunal densities and sediment total organic matter indicated the presence of enrichment in the vicinity of the sea urchin grow-out cages. the 20 15 10 5 0 0 5 25 50 % t o m distance from cages (m) 0-1 layer 1-5 cm layer figs. 6a & b. average percent total organic matter (± s.e.) in the sediment, in (a) april and (b) august 1999 epibenthic faunal density, notably, of the edible gastropod strombus sp., was significantly higher around the cage area in august while no faunal enhancement was observed in april. on the other hand, tom in the upper sediment layer was significantly elevated in the vicinity of the sea urchin cages in april, but not in august. the results suggest that the enhancement effect may be temporally variable due to seasonality or changes in tidal regimes. the enrichment effect in tom and faunal abundances was limited to a radius of about 5-25 meters from the cages, suggesting the minimal effect of the culture activity. the often significant differences in faunal abundances between the transects, however, did not pinpoint specific transects as being consistently different (a) distance from cages (m) 20 15 10 5 0 0 5 25 50 % t o m 0-1 layer 1-5 cm layer (b) enhancement effect of sea urchin 7 april 1999 august 1999 figs. 7a, b, c, & d. scatter plots of %tom vs. total faunal abundance (a & b) and total gastropod abundance (c & d). open squares are individual data points, filled circles represent mean values 0 ga st ro po d ab un da nc e m -2 10 20 30 40 50 60 70 80 0 5 10 15 20 25 30 ga st ro po dl a bu nd an ce m -2 0 20 40 60 80 100 120 140 160 r2 = 0.2367 180 200 0 5 10 15 % tom % tom r2 = 0.0614 2 120 100 80 60 40 20 0 0 5 10 15 20 25 30 to ta l f au na l a bu nd an ce m -2 % tom 300 fa un al a bu nd an ce m -2 250 200 150 100 50 0 0 5 10 15 % tom r2 = 0.6934 r2 = 0.2662 (a) (b) (c) (d) from the others. these results indicate heterogeneous distribution of organic matter and fauna in the seagrass bed surveyed. in addition, organic material generated by the urchins may be distributed assymetrically around the cages because of prevailing local currents. lucero has relatively strong water currents and wave exposure (agawin and others 1996), hence it was possible for cage grow-out in this area to have less potential ecological impact than in other, more protected, sites in bolinao. buschmann et al. (1996) similarly noted that between-site differences in current strength is an important factor determining the degree of impact of aquaculture activities in coastal areas. high tom levels did not cause an increase in epibenthic fauna, suggesting that sea urchin detritus may not have been palatable for the taxa surveyed, or that food supply was not limited in the area during the survey period. scatter plots showed that in april, increased %tom in the sediment actually depressed total faunal densities. it was possible that elevated tom rendered the substrate unsuitable for epibenthic fauna. however, in defrance malay et al. 8 0.008 0.006 0.004 0.002 0 0 5 25 50 g dr ie d ep ip hy te s pe r cm s ea gr as s le af 0.008 0.006 0.004 0.002 0.000 0 5 25 g dr ie d ep ip hy te s pe r cm s ea gr as s le af distance from cages (m) distance from cages (m) (a) (b) figs. 8a & b. average epiphyte dry (± s.e.) per 1-cm length of enhalus acoroides leaf blade, in (a) april and (b) august 1999 august, total faunal abundance varied less with tom. also, during both sampling periods, no significant trend was observed between tom and densities of gastropods, strombus sp., columbella sp., and hermit crabs. it appeared that any inhibitory effect that tom may have on the epibenthic fauna was temporally variable and limited in scale. unlike finfish and mussel cultivation, sea urchin growout culture appeared to have no effect on parameters such as epiphyte biomass and total suspended solids as was previously reported (see review of buschmann and others 1996). the nutrient and organic input from the culture activity may not have been of sufficient magnitude as to have a significant impact on these factors. evidence of the presence of organic enrichment in the cage area has important implications for future directions in sea urchin grow-out research. excess organic matter generated by the cages may be utilized in a more efficient manner by setting up a polyculture system within the culture area. the feasibility of culturing edible holothurians and gastropods that can utilize sea urchin detritus as food alongside the sea urchins should be explored. aside from increasing profitability, such polyculture system would likely have the least impact on the environment because of rapid and efficient recycling of wastes within the system. future studies should survey more parameters, (i.e., faunal biomass, infaunal and microbial community structure, dissolved nutrients in the water column, dissolved oxygen, sediment metabolism, and sulfate reduction), that may be more sensitive to localized enhancement effects. more surveys should also be undertaken to provide a more complete picture of the impact of the sea urchin cages across different times of the year. most importantly, future studies should include an unimpacted “control” site alongside the “experimental” sea urchin grow-out site. acknowledgments we are grateful to hildie marie nacorda for her guidance and indispensable advice in planning and executing this study and to tirso catbagan sr. for assistance in the field. funding was provided by the bureau of agricultural research department of agriculture. references agawin nsr, duarte cm, fortes md. 1996. nutrient limitation of philippine seagrasses (cape bolinao, nw philippines): in situ experimental evidence. mar ecol prog ser 138: 233-243. enhancement effect of sea urchin 9 buchanan jb. 1984. sediment analysis. in: holme na, mcintyre ad, editors. methods for the study of marine benthos. 2nd ed. oxford: blackwell scientific publications. p 41-65. buschmann ah, lopez da, medina a. 1996. a review of the environmental effects and alternative production strategies of marine aquaculture in chile. aquacul eng 15: 397-421. castel j, labourg pj, escaravage v, auby i, garcia me. 1989. influence of seagrass beds and oyster parks on the abundance and biomass patterns of meioand macrobenthos in tidal flats. estua coast shelf sci 28: 71-85. doty ms. 1971. measurement of water movement in reference to benthic algal growth. bot mar 14: 32-35. duarte cm. 1995. submerged aquatic vegetation in relation to different nutrient regimes. ophelia 41: 87-112. findlay rh, watling l, mayer lm.1995. environmental impact of salmon net-pen culture on marine benthic communities in maine: a case study. estuaries 18: 145-79. frankovich ta, fourqurean jw. 1997. seagrass epiphyte loads along a nutrient availability gradient, florida bay, usa. mar ecol prog ser 159: 37-50. grant j, hatcher a, scott db, pocklington p, schafer ct, winters, gv. 1995. a multidisciplinary approach to evaluating impacts of shellfish aquaculture on benthic communities. estuaries 18: 124-144. hatcher a, grant j, schofield b. 1994. effects of suspended mussel culture (mytilus spp.) on sedimentation, benthic respiration and sediment nutrient dynamics in a coastal bay. mar ecol prog ser 115: 219235. holmer m, kristensen e. 1992. impact of marine fish cage farming on metabolism and sulfate reduction of underlying sediments. mar ecol prog ser 80: 191-201. juinio-meñez ma, macawaris nnd, bangi hgp. 1998. community-based sea urchin (tripneustes gratilla) grow-out culture as a resource management tool. in: jamieson gs, campbell a, editors. proceedings of the north pacific symposium on invertebrate stock assessment and management. canada: can spec publ fish aquat sci 125: 393-399. karakassis i, hatziyanni e, tsapakis m, plaiti w. 1999. benthic recovery following cessation of fish farming: a series of successes and catastrophes. mar ecol prog ser 184: 205-218. karakassis i, tsapakis m, hatziyanni e. 1998. seasonal variability in sediment profiles beneath fish farm cages in the mediterranean. mar ecol prog ser 162: 243252. koike i, mukai h, nojima s. 1987. the role of the sea urchin, tripneustes gratilla (linnæus) in decomposition and nutrient cycling in a tropical seagrass bed. ecol res 2: 19-29. wear dj, sullivan mj, moore ad, millie df. 1999. effects of water-column enrichment on the production dynamics of three seagrass species and their epiphytic algae. mar ecol prog ser 179: 201-213. 14_fernandez 66 fernandez and ramos science diliman (january-june 2003) 15:1, 66-70 optimized extraction of h– by three-electrode faraday cup system in magnetized sheet plasma ion source m.s. fernandeza and h.j. ramosb* auniversity of san carlos 6000 cebu city, philippines bplasma physics laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: bplasma@nip.upd.edu.ph abstract a locally designed rectangular parallelepiped, three-electrode faraday cup system has been developed. its design incorporates the capability of simultaneous extraction and deposition of the h– ions on substrates. the device functions to attain prescribed selectivity conditions of extracted ions, with controlled energies, for deposition or adsorption. it has been proven to detect the ions at filter bias voltage of 13.61 v with a current density of 5.3 a/m2 that is relatively higher than reported (abate & ramos, 2000). introduction up to now, there is still a question as to how the deposited hydrogen can be responsible for the changes that it causes on some substrates or films. there are still some lapses in the detailed explanation on why estimated range of values of hydrogen densities deposited in the crystal can lead to the modification of the structure from amorphous to polycrystalline (abelson, 1993), to trivalent rare-earth elements forming hydrides, especially yttrium and lanthanum hydrides, and can even cause the element to exhibit metal-semiconductor transitions (vajda, 1995). the continuous absorption of hydrogen also caused some optical changes in the grown films from shiny mirror to a yellow, transparent window (huiberts et al., 1996). on other elements, it has been known that the effect of the addition of hydrogen during the processing on the plasma properties and the growing film serve to dramatically improve the electrical and electronic properties of the deposited film of amorphous and crystalline silicon (abelson, 1993). silicon hydrides (sih x ) have been studied carefully as they continue to be prominent materials in microelectronic device manufacturing. they are suited to numerous applications, including photovoltaics and thin film transistors (tfts) (srinivasan et al., 1997). the most common method of preparation of hydrogenated silicon (a-si:h) and polycrystalline silicon films is done by the plasma enhanced chemical vapor deposition (pecvd) from discharges composed of sih 4 or mixtures of sih 4 diluted in argon and/or h 2 (marra et al., 1998). in this study, ion deposition is employed. unlike most of the various researches illustrating ion-surface interaction that utilized positive ions, negative hydrogen ions shall instead be used in this study. the h– ions (hydrides) will be deposited on the silicon substrate to form the silicon hydrides by ion-surface interaction phenomenon (williams & poate, 1984). the objective of this work is to explore the feasibility of a* corresponding author 67 optimized extraction of h– locally designed device for the extraction of the ions to be deposited. the h– ions are extracted, accelerated, and transported from pre-mixed 10% ar/h 2 sheet plasma and are deposited into a silicon substrate. in this paper, we show some initial data of the extraction, acceleration, and transport towards a detector that will subsequently be replaced with a si substrate. the transport is done using a developed multi-electrode faraday cup system. theory it is necessary to suppress electrons that are extracted with the anions because collisions are inevitable. the h– ion-electron collision can lead to the destruction of h– ions, which are commonly called “electronic collisional impact detachment”. the incident electron deionizes a hydride by the emission of the orbital electron. this process is briefly expressed by the reaction (1) to minimize this, the plasma electrode bias potential is set at a value slightly greater than the floating potential at a point from the sheet plasma. the floating potential at a certain point from the sheet plasma is determined by the langmuir probe measurements as shown in fig. 3. it is at that value of the bias potential of the probe tip in which net current is zero. hence, adjustments must be made to increase the plasma electrode bias potential to a value slightly greater than the floating potential of the probe tip until a low current arises, implying an attraction of anions and electrons. a plasma electrode bias potential is chosen to produce a relatively high detected current. the position of the plasma electrode from the sheet plasma is chosen with the significantly reduced electron density. the reduction of electron density with the increase in the distance between the probe tip and the core sheet plasma is determined and shown in fig. 4. in the event the ions are extracted through the plasma electrode apertures into the first region bounded by the plasma electrode and the extraction electrode, they appreciably interact with each other by coulombic repulsion. this repulsion causes the monodirectionally-drifted ions to diverge while moving along the path towards the detector. hence, they spread radially. for weak electric field intensity, this radial component of the ionic velocities obviously can strongly reduce the ion flux. to avoid this radial ionic spreading, the electric field between electrodes to the diffusion distance ratio, e/l, needs to be of such value as to offset the space charge effect. this criterion can be taken impliedly from the poisson’s equation, (2) in terms of electric field e, the above equation can be written as (3) where the following replacements ρ = ne; ion charge density n = density of the hion e = charge of the hion are used. the criterion can be expressed simply as follows: (4) the ion density within the working distance l can be determined. in fact it has been reported (sanchez & ramos, 1994) that an h– ion density n was found in the chamber used here at an optimum condition of approximately 1.24 x 1010 cm-3. this low ion density would guarantee a space-charge effect-free transport of extracted ions across the electrodes. ion transport across the electrodes in the device is therefore possible. experimental method the magnetized sheet plasma negative ion source (spnis) is the facility used here. the schematic diagram of this machine is shown in fig. 1. langmuir probe measurements are done to determine the corresponding values of the floating potentials at various transverse distances of the probe tip from the sheet plasma. at the same time, the measurement provides information on how electron density varies o x xe h h 2e+ → + 2 4v πρ∇ = − 4e neπ∇ =g 0 4 e n elπ = 68 fernandez and ramos with these distances. the results of this measurement serve to set values from which the plasma electrode parameters are based. the device is installed in such a manner that the magnetic fields of the helmholtz coils of the facility are perpendicular to electric fields between the electrodes of the device while the flat surface of the plasma electrode is parallel to the surface of the sheet plasma. the bias potentials of the electrodes as well as their separation have been adjusted to meet the criterion set by eq. (4). to offset the magnetic force on an extracted ion by the helmholtz coils, a pair of parallel conducting plates, one with varying bias potentials while the other fixed on the ground, are installed. the pair of plates spans the separation of the electrodes so as to have the electric field of the plates cause the offset. with the device properly installed, the plasma electrode fixed at the predetermined bias potential and positioned relative to the core’s sheet plasma in the extraction chamber, the extraction experiment is conducted at a relatively low gas-filling pressure and discharge current. the hions are detected using the faraday cup as the detector that is attached to the computer. results and discussion the ignition produces the plasma at a discharge current of 0.5 a, with a gas-filling pressure of 0.005 torr. the langmuir probe measurements are conducted primarily to determine corresponding values, the floating potential at various positions of the probe, and more importantly, to determine the electron density at every chosen position. with the probe positioned at 3 cm from the sheet plasma, an i-v characteristic curve with a floating potential value of 2.71 v is produced. this floating potential value then serves as the basis for the plasma electrode bias potential. the choice of the position relative to the sheet plasma and the bias potential of the plasma electrode are decided from the graph, as shown in figs. 3 and 4. fig. 1. the schematic diagram for the magnetized sheet plasma negative ion source facility. the device (fig. 2) installed in the extraction chamber is drawn (encircled). pump limiters e 1 e 2 anode extraction chamber mass spectrometer s 1v 1 am v 2 s 2 am v 3 am s 3 v 4 samarium-co magnets helmholtz coils production chamber hot assembly fig. 2. the three-electrode faraday cup system. shown here is the (1) plasma electrode with an array of nine circular apertures, (2) extraction electrode, and (3) acceleration electrode. 3 2 1 v fc v a v f2 v e v f1 v p electrometer 12 10 8 6 4 2 -2 -30 -20 -10 10 20 30 voltage (v) c u rr e n t (a ) fig. 3. the characteristic i-v curve of the 10% ar/h 2 sheet plasma showing the floating potential (encircled). the value is found to be 2.71 v. 69 optimized extraction of h– the plasma electrode is fixed at a bias potential value of ~3.1 v, where a high detected current is produced while the extraction electrode, which is the next electrode (fig. 2), is biased at 57 v. similar to the plasma electrode, the extraction electrode has a rectangular array of 3 x 3 apertures, each with a diameter of 1.5 mm. just 1 mm behind the aperture on the center of the electrode is the 9-mm disk faraday cup, the detector, which is also biased using a 68-v battery. the bias potential of one of the plates that are 1.49 cm apart, is scanned from -30 to 30 v. with the device attached to the computer, automatic data acquisition routines store the ionic signals. a peak is detected at deflecting plate voltage of 13.61 v. this would mean that the magnetic force causing the ions to deflect away from the detector is offsetted by the electric force ee, corresponding to this voltage, of the fields between the plates, (5) the potential difference (57 v – 3.1 v) across the plasma and the extraction electrodes provides the energy for the ion transport, and is calculated at 53.9 v while the separation of the electrodes is set at 3.0 cm: (6) ee evb= ( )21 53.9 volts 2 mv e= using the values of the potential difference across the plasma and extraction electrodes, and the distance between them, eq. (4) is clearly satisfied with the righthand side of the inequality estimated at ~1020 cm-3. hence, we have the peak shown in fig. 5, indicating an unobstructed extracted ion transport. equating eq. (5) and eq. (6), an expression for the mass of the ion can be derived. with the measured b = 57.6 gauss of the helmholtz coils, m = 1.16 x 10-27 kg. the error is attributed to instrumental error of the langmuir probe measurements known to be about 30%. the current density is easily calculated as follows, (7) references abate, y. & h.j. ramos, 2000. optimization and enhancement of hions in a magnetized sheet plasma. rev. sci. instrum. 71(10): 3694. fig. 4. using the characteristic curve (fig.1), the variation between the electron density and the position of the probe relative to the sheet plasma can be derived. the result of the derivation is illustrated. 16 14 12 10 8 6 4 2 0 1 2 3 4 distance (cm) e le c tr o n d e n s it y ( 1 0 + 1 0 , c m -3 ) fig. 5. the value of the deflecting plates’ voltage offsets the effect of the helmholtz magnetic field on the extracted h– ions. the coordinates of the peak are (13.61 volts, 11.97 µa). -30 -20 -10 10 20 30 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 voltage (v) c u rr e n t (1 0 -5 a ) ( ) 2 2 11.97 a 5.3 a/m 1.5 mm i j a µ = = = 70 fernandez and ramos abelson, j.r., 1993. plasma deposition of hydrogenated amorphous silicon: studies of the growth surface. appl. phys. 56(a): 493-512. huiberts, j.n., r. griessen, j.h. rector, r.j. wijngaarden, j.p. dekker, d.j. de groot, & n.j. koeman, 1996. yttrium and lanthanum hydride films with switchable optical properties. nature. 380(6571): 231. marra, d.c., e.a. edelberg, r.l. naone, & e.s. aydil, 1998. silicon hydride composition of plasma-deposited hydrogenated amorphous and nanocrystalline silicon films and surfaces. j. vac. sci. technol. a. 16: 3199-3210. sanchez, j.k.c.d. & h.j. ramos, 1994. characteristics of a magnetized sheet plasma produced by a hot cathode. abstracts of the 6th asia pacific conference. brisbane, australia (july 4-8, 1994). 459. srinivasan, e., d.a. lloyd, & g.n. parsons, 1997. dominant monohydride bonding in hydrogenated amorphous silicon thin films formed by plasma enhanced chemical vapor deposition at room temperature. j. vac. sci. technol. a. 15: 77-84. vajda, p., 1995. in geschneider, k.a. & l. eyring (eds.) handbook on the physics and chemistry of rare earths. elsevier, amsterdam. 20: 207-291. williams, j.s. & j.m. poate, 1984. ion implantation and beam processing. science diliman (july-december 1999) 11: 2,15-17 abstract !\erworc/s: superconductors, resistivity, vortices, flux flow resistivity, magnetoresistance introduction as expedient as high critical temperature superconductors (htsc) are, their properties in the presence of an external magnetic field should inexorably be studied since these materials would necessarily be exposed to these fields in technological use. an applied magnetic field penetrates a superconductor in the mixed state. the pendration occurs in the form of tubes, called vortices, which serve to confine the flux. the interaction between vortices is repulsive so that they arrange themselves as far away from each other, as in a flux lattice in clean samples or as vortex glass in the presence of randomly distributed pinning centers. the pinning centers may be point defects, grain clarina de la cruz', leandro guerra, roland sarmago, and arnel salvador condensed matter physics laboratory, national institute of physics l;niversity of the philippines diliman. quezon city 1101 philippines email: c1ar«(l)nip.upd.edu.ph; fax: +632-920-5474 boundary or intra/intergranular non-superconducting regions. on the other hand, transport currents and thermal fluctuations can both induce flux motion, due to the lorentz force, and produce dissipation. this can involve, for example, the release and transportation of vortices to other pinning centers, and the pinning and depinning of flux bundles. flux flow can be viewed as a rate of change of flux which, by faraday's law, produces a voltage drop in the superconductor along the direction of the transport current. the resistance associated with this motion provides a mechanism for heat dissipation (poole et ai., 1995). in this study, the contribution of these vortices were investigated through resistivity measurements in varying magnetic fields. this contribution was quantitatively determined as the flux flow resitivity. 15 the behavior of high-temperature superconductors in the presence of an external magnetic field is of particular interest in light of its technological application and commercialization. in this paper, we perfonned resistivity measurements on bulk superconducting pellets of in the presence of external magnetic fields below 0.5t. the broadening of the transition region below tc in the resistivity plots, was attributed to the residual resistance imparted by flux flow in the sample. from 1-v measurements at 50 k at fields below 0.6t, the contribution of vortices was quantitatively measured as a flux flow resistivity which range from 0.1231 to 1.700 (m( -mm) for applied magnetic fields from 0.04t to 0.6t. the increase in the flux flow resistivity with increasing applied field was due to the increase in the number of vortices moving in steady state motion brought about by the interaction of the vortices with the transport current. fig. 2 shows that the critical current decreases with increasing magnetic fields. this is because, the external magnetic field destroys the superconductivity in the sample resulting in the lowering of the amount of supercurrent that the sample can carry. methodology after the sample was cooled down to -10k, using a closed-cycle helium cryostat, resistivity measurements were done using a current of soma in different magnetic fields below 0.5t. the contribution of the vortices when the resistivity measurements were done with an external magnetic field was determined through i-v measurements with fields below 0.6t. resul 1's and discussion fig. i. the resulting resistivity plots at varying magnetic fields below 0.5 t. inset shows a typical resistivity plot at 0 t 16 this residual resistance termed as flux flow resistance was quantitatively determined from: in equation (i), ie is the critical current which was determined through i-v measurements done at 50 k for varying magnetic fields. where 0" is the sample cross-section and dl is the separation of the voltage contacts. de la cruz et al. fig. 2. magnetic field dependence of the transport critical current ofa bulk bi,sr,cacup8+& sample determined at 50 k fig. 3 shows that the flux flow resistivity increases with respect to the magnitude of the applied magnetic field. this increase in the resistance indicates that the vortices are interacting with the transport current (poole et ai., 1995; bishop, 1993). if the lorentz force on the vortices due to the transport current is large enough to unpin the samples were bulk superconducting pellets of which were prepared via the solid state reaction method. the pellets were cut into 3 .ox8.0x 1.0 mm rods. the electrical contacts used were gold wires attached by pressed indium solder. the transport measurements were done using the in-line contact configuration to minimize errors from leads and contact resistances (ison, 1999). from the resistivity measurements performed at varying fields below 0.5t (fig. 1), a broadening of the transition region below the critical temperature was observed. this is attributed to the residual resistance imparted by flux flow in the sample. also, it can be seen that the plots coincide in the high temperature region above te since there are no more defined vortices in the sample when it is in its normal state (bishop et ai., 1993). the flux flow resistance, , was denved as the slope of the linear part of the corresponding i-v curves. knowing the dimensions of the sample, the corresponding flux flow resistivity , was calculated usmg: the vortices so that they move from one pinning center to the next, then the resistance effectively increases. on the other hand, the granular nature of the sample would establish that the vortices would remain pinned due to the random orientation of the supercooducting planes. therefore, the increase in the resistance implies that there is an increase in the number of vortices in the liquid phase, present in the sample. the increase in the flux flow resistivity corresponds to an increase in the amolmt of field that was able to penetrate the sample. since vortices are quantized, an increase in the internal field would be due to the addition of vortices moving in steady-state motion. the dependence of the critical current and the flux flow resistivity on the externally applied magnetic field, as detennined in this investigation, conformed with previous studies (poole et ai., 1995; ison, 1993; yeshurun, 1996). 17 page 1 images image 1 image 2 image 3 titles science diliman (july-december 1999) 11: 2,15-17 abstract !\erworc/s: superconductors, resistivity, vortices, flux flow resistivity, magnetoresistance introduction 15 page 2 images image 1 image 2 image 3 image 4 image 5 image 6 image 7 image 8 titles methodology resul 1's and discussion 16 de la cruz et al. the samples were bulk superconducting pellets of page 3 images image 1 image 2 image 3 image 4 image 5 image 6 image 7 image 8 titles 17 establishment-art.15 establishment and implementation of the balingasay mpa 121science diliman (july december 2000) 12:2, 121-128 abstract establishment and implementation of the balingasay marine protected area: a community-based approach severino g. salmo iii, renato a. turion, marie antonette r. juinio-meñez, and porfirio m. aliño marine science institute, college of science university of the philippines, diliman, quezon city 1101 philippines tel. no.: (632) 922-3921; (632) 920-5301 to 99 loc. 7428; telfax: (632) 924-7678; e-mail: jon@upmsi.ph a community-based approach in the establishment and implementation of a marine protected area (mpa) in balingasay, bolinao, pangasinan is presented. the factors necessary to facilitate the successful establishment and implementation of a community-managed mpa include heightening of environmental awareness, community mobilization, and legal/institutional and financial assistance. a heightened environmental awareness encouraged the community to undertake resource management action. the formation of a people’s organization, sammabal (samahan ng mga mangingisda at mamamayan ng balingasay), was crucial in assessing environmental problems (e.g., overfishing) and identifying the establishment of an mpa as a management tool to address the problem. sammabal was also instrumental in eliciting community support for the issuance of a municipal ordinance in the establishment of the mpa. subsequently, the organization initiated the patrolling of the mpa. institutions involved in the communitybased management of the mpa also included the multi-sectoral council (brmc – balingasay resource management council) and representatives from the barangay council and the municipal government. this institutional arrangement has proven to be very resilient, indicating a high probability of sustaining its successes despite some obstacles and shortcomings. clear delineation of the role and functions of the institutions and the stakeholders was essential in advancing the initiative. this case study will draw on the lessons from the experience of a four-year community-managed mpa. keywords: community-based approach, environmental awareness, management tool, marine protected area (mpa), community mobilization, inter-institutional interaction, cbcrm introduction the balingasay-mpa (marine protected area) is located in bolinao, pangasinan along western lingayen gulf, northwestern luzon (fig. 1). this has two components: a marine sanctuary covering approximately 14.77 hectares and a marine reserve of approximately 200.86 hectares. in the marine sanctuary, all kinds of fishing access are prohibited while in the marine reserve area, fishing is allowed but regulated (e.g., use of nondestructive/passive fishing gears, regulation of of fishing net mesh size). the establishment of an mpa was conceptualized in october 1995, after the formation of sammabal (samahan ng mga mangingisda at mamamayan ng balingasay) in september 1995. the establishment of the mpa was conceived by sammabal in an attempt to improve the poor condition of their coastal resource, which was salmo iii et al. 122 endorsement of the mfarmc, modified the ordinance accordingly, and re-submitted it to the provincial council in may 1999. at present, the ordinance is still in the office of the provincial council pending its formal legislation. regardless of the lack of formal legislation, sammabal initiated the patrolling of the mpa in january 1998. since then, the community, through sammabal and with some assistance from the barangay council, has implemented the mpa. this paper presents the important features of the mpa as a case study, and draws lessons on the establishment and implementation of a community-managed mpa. methodology presentation and analysis of this case study is divided into two phases: establishment and implementation. the establishment phase covers preparatory activities involving environmental education, planning, legislation, and organizational development. the implementation phase covers training of the people’s organizations for bio-physical monitoring and sourcing of external support and assistance. all information used in this case study were gathered from 1995 to 1999 mainly from barangay balingasay through sammabal, balingasay resource management council (brmc) and the barangay council. pertinent legal documents, (e.g., resolution, ordinance) were gathered from the municipal government of bolinao while other technical information were obtained from the community-based coastal resources management project (cbcrmp)and the marine fisheries resources management project (mfrmp). the cbcrmp, with funding from the international development research center of canada (idrc), was implemented from 1995 to 1997 by the university of the philippines marine science institute (up-msi), the university of the philippines college of social work and community development (up-cswcd), and the haribon foundation, an environmental nongovernmental organization (ngo). the mfrmp, on the other hand, was implemented by up-msi from 1998 to present with funding from the royal netherlands embassy. 16.44 16.42 16.40 16.38 16.36 16.34 16.32 119.79 119.81 119.83 119.85 119.87 119.89 fig. 1. map of the balingasay-mpa (inset: map of lingayen gulf) ilog malino patar tupa estanza balingasay arnedo lingayen gulf bolinao characterized by low fish abundance, and deteriorating coral cover (dizon and miranda 1996). after attending a series of environmental education training and conducting barangay consultations, sammabal, together with the barangay council, submitted a resolution to the municipal council of bolinao in august 1997 seeking the establishment of the mpa. through the subsequent lobbying of sammabal, the municipal council adopted the resolution in october 1997, after a number of public consultations. as provided for by law, the municipal ordinance was submitted to the provincial council of the pangasinan provincial government for review and ratification in may 1998. in november 1998, the provincial council recommended that the municipal council revise the ordinance to include the municipal government’s role in the mpa management. furthermore, the ordinance required the endorsement of the municipal fisheries and aquatic resources management council (mfarmc), as provided for by r.a. 8550. the municipal council, after getting the establishment and implementation of the balingasay mpa 123 results and discussion establishment phase the establishment of a community-managed mpa required much preparation. this started with the formation of the people’s organization (po) sammabal, in september 1995. this organization served as the core institution moving for the establishment of the mpa. sammabal has about 100 members mostly fishers with some fisher-farmers, barangay officials, and local professionals. the organizational structure of sammabal includes the executive committee as the operational executing body, the board of directors (bod) as the legislative body, and the general assembly as the ultimate decisionmaking body (fig. 2). the executive committee constituted different sub-committees, each with its specific roles and functions. for instance, the environment committee was responsible for the conceptualization and implementation of the environmental management programs while the livelihood committee took care of the incomegenerating projects of the po. the decision-making process of the po started when a committee proposed a particular project which would then be endorsed by the executive committee to the bod for approval. the bod, after evaluating the proposal, would in turn endorse it to the general assembly for final decision. the proposal for the establishment of the mpa went through this process. prior to the establishment process of the mpa, sammabal undertook a series of organizational strengthening activities from october 1995 to february 1996. these were the creation of a vision, mission, and goal; formulation of a constitution and by-laws; leadership training; paralegal training; and enhancement of networking and advocacy skills. such activities enabled the sammabal to be a vigilant advocate of coastal resources management. the establishment of an mpa was the priority activity of sammabal. concrete action was catalyzed by the heightened environmental awareness of its members brought about by a series of formal and non-formal environmental education and training activities facilitated by the cbcrmp. the training programs comprised three modules: (1) basic environmental education; (2) advanced environmental education; and (3) mpa management. the first two modules were attended by sammabal members and representatives from the barangay council, while the third module, was attended by the members of the environment committee of sammabal. the first module covered general general assembly board of directors (bod) executive committee environmental committee education & training committee fund-raising committee livelihood committee membership committee fig. 2. organizational structure of sammabal salmo iii et al. 124 ecological concepts, i.e., components of the marine ecosystems and interconnectivity, and was held on february 24, 1996. an important highlight during this session was the identification of environmental problems. different resource management options to address the identified environmental problems (e.g. uychiaoco and others 1998), were discussed in an advanced environmental education session held on march 9, 1996. among the potential options, the establishment of an mpa was chosen by sammabal as the most appropriate management tool that could be employed to address the depletion of fishery resources (russ 1996). the last environmental education session was held on april 1996, focusing on the details of mpa establishment, implementation, and management. to further help sammabal appreciate mpa management, the cbcrmp sponsored an exposure trip to a community-managed mpa in san salvador island, masinloc, zambales on june 1996. this activity gave the sammabal leaders first hand knowledge about the impressive environmental status which could be achieved by establishing an mpa. the testimonies given by members of the samahang pang-kalikasan ng san salvador (spss) during an experience-sharing session (madamba-nuñez 1998) further inspired them to establish their own mpa. the identification of the proposed mpa site was based on the resource/resource-use map of the area prepared by sammabal. this map featured the spatial and temporal distributions of different marine resources, together with the areal extent of different fishing gear operating in the area. initially, three sites were considered by sammabal. preliminary cursory surveys narrowed the proposed sites to just one as it was the only site that had relatively good coral cover. the proposed mpa sites were evaluated by the cbcrmp by conducting bio-physical surveys using manta tow, fish visual census, and benthic life form monitoring. results of the survey indicated that the proposed site had an average of 10% live coral cover and fish density of 161 fish individuals per 500 m2. other socioeconomic data, i.e., average fish catch, resource seasonality map, were gleaned from the participatory action research (par) conducted by the cbcrmp in barangay balingasay from january to july 1995. the par was conducted by the cbcrmp, during the prepo formation stage to gather socioeconomic and biophysical data. the activity facilitated the involvement of the community in identifying environmental problems and mobilizing them as a partner for coastal resources management. the information gathered were then presented to the environment committee and general assembly of sammabal. despite the mediocre status of the proposed site, sammabal was still willing to establish the mpa because of their belief that the area could be improved if it is properly managed. the exact extent and location of the mpa was selected and agreed upon by the general assembly of sammabal in august 1996. ideally, the harmonization of ecological (e.g., habitat and resource status), socioeconomic (e.g., dependency of the fishers in the site), and practical (e.g., relative distance of the site for patrolling) considerations should be used in selecting the most appropriate site for an mpa (kelleher and kenchington 1991). however, in actual practice, socioeconomic considerations, most especially social acceptability, prevail in deciding the actual size and location of the mpa. legislation after almost a year of conceptualization, training, and planning, sammabal, with the assistance of the barangay council, conducted information campaigns in the entire barangay through one-on-one talks and distribution of leaflets in september 1996. after the information campaign, a series of community consultations and public hearings were launched by members of the po to broaden mass advocacy in july 1997. in all the consultations, the majority favored the establishment of the mpa. after sammabal explained the purpose of the mpa, how it would be managed, and the benefits which could be acquired from it, most residents agreed, including some fishers who were initially hesitant. with a strong mass-base support, sammabal submitted a resolution to the barangay council. the barangay council in turn supported the initiative of sammabal. a joint resolution between the barangay council and sammabal seeking the establishment of mpa was submitted to the municipal council for legislation in august 1997. prior to the establishment and implementation of the balingasay mpa 125 sammabal brmc brgy. council implementing body multi-sectoral monitoring body fig. 3. inter-institutional arrangement in the management of the balingasay-mpa submission of the joint resolution, sammabal also held consultation in three other neighboring areas – barangays tupa, estanza, and ilog malino – to further strengthen community support for their initiative. in august 1997,the respective councils of these barangays issued a supporting endorsement for the establishment of the mpa to the municipal council. the municipal council responded by sponsoring public hearing in september 1997. the public hearing was attended by fishers from barangays balingasay, estanza, ilog malino, and patar. because of the favorable response from the participants, the municipal council adopted and approved the resolution in december 1997. with this development, the mpa was immediately inaugurated. all expenses incurred in the said occasion were paid for by the office of the mayor and the lingayen gulf coastal area management commission (lgcamc). however, in the philippines’ legal context, enforcement of a new law requires the issuance of a municipal ordinance, which, in this case, took nearly a year (municipal ordinance no.2 s1998, september 1998). however, even before the issuance of the municipal ordinance, sammabal initiated the patrolling of the mpa, albeit quite irregular because of limited logistical supplies. in addition, the passage of r.a. 8550 (the new philippine fisheries code) in september 1998, required an endorsement from the provincial council prior to the implementation of a municipal ordinance. the provincial council managed to sponsor an environment committee hearing in november 1998 which was attended by sammabal and some officials from the municipal council. the recommendation of the committee was to return the ordinance to the municipal council of bolinao for refinement so that all of its provisions will be in consonance with r.a. 8550, i.e., clearly defining the role of the municipal government in mpa implementation, and securing endorsement from the municipal fisheries and aquatic resources management council (mfarmc). the municipal council, upon completing all the requirements, resubmitted the ordinance to the provincial council in may 1999. re-submission of the ordinance could not be done sooner because there was no mfarmc yet. the municipal government initiated the formation of the mfarmc only in january 1999. unfortunately, to this date the mpa ordinance has not yet been included in the agenda of the provincial council meetings. institutional arrangement clearly, sammabal as an environmental organization, had the most significant role in the establishment of the mpa, from conceptualization to legislation and implementation (1995-present). consistent with reports from other community-managed mpas (white and savina 1987), the crucial activities that enabled sammabal to become a local crm institution were the training programs attended by its members. these programs heightened their environmental and legal awareness, and improved their leadership and organizational management skills. furthermore, sammabal also networked with concerned government and non-government institutions at the municipal, provincial, regional, and national levels. sammabal and the barangay council were the institutions that were most active in pursuing the establishment and implementation of the mpa. however, the municipal ordinance stipulated multisectoral tasking in the implementation of the mpa. based on the ordinance, sammabal would serve as the implementing body that would directly oversee the patrolling and bio-physical monitoring of the area. meanwhile, the multi-sectoral council balingasay resource management council (brmc) would serve as the overall coordinating body responsible for administrative activities such as sourcing of funds and providing updates to the municipal government on the status of the mpa. furthermore, the brmc was also responsible for overseeing sammabal. fig. 3 salmo iii et al. 126 provide the community with the skills to monitor the changes and improvements occurring in the mpa. external assistance considering all the difficulties encountered, sammabal and the brmc could not have been successful without external assistance. the efforts of the barangay council and the municipal government, as well as the assistance provided by lgcamc and mfrmp contributed to the success of the balingasaympa. the barangay council was working with sammabal since the conceptualization phase. the municipal government provided some financial assistance for the operations, e.g., buoys, markers, gas and oil for patrolling. the other hand, the lgcamc provided a grant for a motorized boat to be used for patrolling. other logistical supplies such as binoculars, flash lights, etc. were provided by the mfrmp. they also facilitated training sessions and programs for institutional strengthening, and provided seed stock for reseeding activity (e.g., sea urchin). nonetheless mechanisms to make the mpa selfsustaining has been a major consideration. interim partnerships with the private sector have been explored for the establishment of an environmental fund to support the cost of maintening the mpa until the mpa can generate sufficient income (e.g., visitor’s fee). environmental impacts despite difficulties involving logistical constraints and inter-institutional dynamics, the balingasay-mpa gained some successes in improving the environmental status of the mpa. as shown in table 1, bottom coral cover increased from 10% in july 1996 to 27% in june 1999. meanwhile, fish abundance also increased from 161 to 326 fish individuals per 500 m2, or an estimated 51% increase. although the increase in fish abundance was relatively low compared to what was reported for the presents the inter-institutional interaction arrangement in the implementation of the mpa. the interaction between sammabal and brmc brought about leadership conflicts at some instances. fortunately the barangay council effectively served as a mediator between the two institutions. intervention from the barangay council facilitated the sustenance of mpa implementation despite the occasional conflicts. implementation phase recognizing the importance of environmental awareness, sammabal, brmc, and the barangay council in balingasay and neighboring areas undertook information campaigns through the distribution of leaflets and posters. while patrolling was initiated by sammabal in january 1998, its conduct was irregular because of logistical constraints, e.g., insufficiency of patrol boats, binoculars, etc. such constraints resulted in instances of encroachment into the mpa which diminished the objective of restoring natural stock. while there were some apprehensions, some of the apprehended encroachers claimed ignorance about the municipal ordinance declaring the mpa. despite some of these difficulties, the mpa was still enforced because of the motivation and persistence of sammabal members who, most of the time, contributed their personal resources (e.g., boat, time) so that patrolling could be conducted. they knew that visibility of enforcement was essential. institutional strengthening after the organizational development phase, follow-up training programs were facilitated by the mfrmp to improve the capability of sammabal and brmc to implement the mpa. a training session on mpa management was held in february 1999 to equip the two institutions with the knowledge and skills needed to manage the mpa efficiently. this resulted in the preparation of an administrative, financial, and operational plan. meanwhile, deputation of fish wardens was conducted in may 1999 to teach them, and bantaydagat members in the barangay, the legal procedure for arresting encroachers in the mpa. likewise, training on participatory bio-physical monitoring using manta tow, fish visual census, and landed catch monitoring (de la cruz and uychiaoco 1998) were conducted to table 1. result of bio-physical monitoring in balingasay-mpa parameter july 1996 % increasejune 1999 % coral cover fish abundance (fish individual per 500 m2) 10 161 27 326 62.96 50.61 establishment and implementation of the balingasay mpa 127 the support of various external groups encouraged sammabal to implement the mpa. the balingasaympa also benefited from sufficient scientific/technical support from the mfrmp (e.g., providing seed stocks, sponsoring training for bio-physical monitoring). the scientific/technical support helped the po to systematically manage the mpa. despite obstacles and weaknesses, the balingasay-mpa had some positive impacts. the immediate indicator were the increases in fish density and percent coral cover. if sustained in the longer term, increased fish productivity can improve the livelihood of the local coastal communities. more importantly, the process of establishing and implementing a community-managed mpa has empowered local institutions to become more responsible stewards of fishery resources. acknowledgments the authors wish to acknowledge the participation and cooperation of the people of barangay balingasay, especially the members of sammabal and the officials of the barangay council, who shared their comments and thoughts in documenting this case study. thanks is due to the municipal government of bolinao for providing the necessary legal documents. likewise, we want to acknowledge the international development research center of canada (idrc) and the royal netherlands embassy for providing financial support to the coastal management activities related to the cbcrmp and mfrmp in bolinao, pangasinan. references de la cruz mt, uychiaoco a. 1998. monitoring the effectiveness of marine sanctuaries. in: participatory methods in community-based coastal resources management. 3 vols. silang, cavite, philippines: international institute of rural reconstruction. dizon jac, miranda gc. 1996. the coastal resource management experience in santiago island. in: ferrer em, polotan-dela cruz l, agoncillo-domingo m, editors. seeds of hope. quezon city, philippines: college of social work and community development, university of the philippines. p 116-129. community-managed mpas in apo and pamilacan islands (i.e., 173% and 89% increase per 750 sq m in two years, respectively) (white 1988), such improvement was significant enough to demonstrate the regenerating potential of the mpa to the fishers. this improvement was perceived by the local fishers who reported that fish abundance was increasing in and adjacent to the mpa. other parameters that were monitored by sammabal were landed catch and fishing area to gear mapping. however the results of these two parameters have not yet been evaluated. there was an increase in the density of sea urchins from practically zero in december 1997 to 0.11-2.14 individuals per square meter (average test diameter: 1.4-8.0 cm) in october 1999. reseeding of cultured sea urchins was conducted at the mpa in december 1997. however the sea urchins monitored in october 1999 were mostly new recruits. this is significant because natural recruitment of sea urchins has not been recorded in bolinao since the collapse of the sea urchin fishery in 1992 (juinio-meñez and others 1998). conclusion an mpa that utilized a community-based approach was able to draw mass-based support from the local community through multi-sectoral participation facilitated by a po (sammabal) and other external support. this multi-sectoral participation was necessary and critical to the development and maintenance of the mpa, as in the case of the sumilon island reserve (russ and others 1994). however, too many sectors involved in mpa management (barangay to municipal level), resulted in a long legislation process. despite the length of this process, the establishment of the balingasay-mpa was relatively systematic and wellplanned from conceptualization to planning, implementation, and monitoring. in mpa implementation, inter-institutional interaction among local institutions was necessary to provide complementary actions and a wide support base. at the same time, the participation of various local institutions created leadership conflicts, such as in the case of sammabal and brmc. such conflict resulted in delays in conducting some of the necessary management activities. salmo iii et al. 128 juinio-meñez mar, macarawaris nnd, bangi hgp. 1998. community-based sea urchin (tripneustes gratilla) grow-out culture as a resource management tool. in: north pacific symposium on invertebrate stock assessment. can spec publ fish aquat sci 125: 393399. kelleher g, kenchington r. 1991. guidelines for establishing marine protected areas. a marine conservation and development report. gland, switzerland: international union for the conservation of nature. 79 p. madamba-nuñez. 1998. study tours. in: participatory methods in community-based coastal resources management. 3 vols. silang, cavite, philippines: international institute of rural reconstruction. p 96-106. municipality of bolinao, pangasinan, 1998. municipal resolution no. 32 s1998. an ordinance to declare and promulgate the municipal marine rehabilitation and replenishment area along barangay balingasay, providing the implementing rules and regulations thereto. municipality of bolinao, pangasinan, 1998. municipal ordinance no. 2 s1998. adopting the implementing rules and regulations and providing penalty clause thereto, for effective and efficient operation and management of the municipal marine rehabilitation and replenishment area at barangay balingasay, bolinao, pangasinan. russ gr. 1996. fisheries management: what chance on coral reefs? naga: the iclarm quarterly. russ gr and alcala ac. 1994. sumilon island reserve: 20 years of hopes and frustrations. naga: the iclarm quarterly. uychiaoco a, de la cruz mt, salmo iii sg. 1998. conservation and rehabilitation strategies. in: participatory methods in community-based coastal resources management. 3 vols. silang, cavite, philippines: international institute of rural reconstruction. p 55-62. white at. 1988. the effect of community-managed marine reserves in the philippines on their associated coral reef fish populations. asian fish sci 2: 27-41. white at, savina gc. 1987. community-based marine reserves, a philippine first. in: magoon ot, converse h, miner d, tobin lt, clark d, domurat g, editors. coastal zone ’87. vol.2. new york: american society of civil engineers. p 2022-2036. guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors . mandatory actions: ✔ if changes are made, choose a new title for the paper. ✔ use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality. ✔ reference your conference paper in the appropriate locations. ✔ include a footnote in the submitted manuscript stating, e.g., "an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings." ✔ indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed. ✔ provide the original conference paper (upload a pdf file during the submission process). ✔ if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions: ✔ expand the background section and include additional references. ✔ include novel scientific content and expanded descriptions of procedures. ✔ provide data that was not published at the conference. ✔ revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission from the editors of ieee sensors journal) guidelines for expanding conference papers for submission to science diliman mandatory actions: recommended actions: (adapted with permission from the editors of ieee sensors journal) page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 sedimentation rate in fringing reefs of honda bay, puerto princesa city, palawan, philippines with reference to coral reef condition joel g. becira western philippines university-puerto princesa campus, puerto princesa city, palawan, e-mail: j_becira@yahoo.com received: 5 january 2007; revised: 23 september 2009; accepted: 23 september 2009 abstract the study was conducted to determine and compare the rate of sedimentation in two fringing reefs in honda bay during two seasons, to discuss sedimentation rate in relation to selected environmental parameters, to determine the coral cover in the two stations, to compare it with data from 2000 and to explain eventual changes in coral cover in relation to the selected environmental parameters, especially sedimentation. the study was conducted from march 1, 2003 to november 30, 2003. two stations, bush and meara islands, in honda bay were established. each station was mounted with three sets of 3-replicated sediment traps that were collected one week after installation. each station was further assessed in terms of coral cover. the average sedimentation rates in honda bay during dry and wet season were 3.50 and 10.00 mg/cm2/day, respectively. the sedimentation rates in the two islands did not show significant difference at 5% level of significance. benthic cover of both bush and meara was higher in 2003 than in 2000. the fact that coral cover is able to recover despite sedimentation seems to indicate that sedimentation has not yet reached critical levels in honda bay. keywords: coral reefs, sedimentation, honda bay, philippines introduction sediment is a particularly troublesome pollutant in coral reefs. it clouds seawater, which reduces photosynthesis of zooxanthellae that live in a mutualistic relationship with many species of corals. in water clouded by sediment, the amount of food available to corals is reduced. particularly, heavy sedimentation can bury a reef, choking the life out of it. according to el-swaify (2000), movement of sediment to low lying and shoreline areas after erosive storms – which often takes only a few hours or even minutes – can induce serious impairment to habitat quality. as a result, deposited sediments in the coastal area may create valuable deltas and islands which may lead to the change of the morphological structure of the coastal ecosystem (personal observation). of the 27,000 km2 of philippine reefs, 60% lies on the shelf surrounding the large western island of palawan (white, 2001). honda bay is one of the important fishing grounds and tourist destinations in palawan. destructive fishing methods, overexploitation and devastating land-based activities that cause erosion and siltation resulted in a decrease of fish catch per unit effort from 36.5 kg in 1985 to 8.4 kg in 1989 (sandalo, 1994) and to 5 kg in 1996 (iclarm, 1996). live cover in coral reefs is currently estimated to be 36.5% (gonzales, in press). the protection of honda bay’s marine resources has been the concern of the department of environment and natural resources (denr), the fisheries resource management project (frmp) science diliman 21(1):7-13 7 mailto:j_becira@yahoo.com becira and some non-governmental organizations (e.g., environmental legal assistance center) particularly in the barangays of san jose, santa lourdes, tagburos, babuyan and manalo (gonzales, 2000). indicators of the success of their efforts, such as a decrease in fishing pressure, an increase in fish stocks and the rehabilitation of coral cover, have been observed, though not sufficiently monitored. frmp started a monitoring program of coastal ecosystems and fishes in 2000 (frmp, 2001) which was not continued. thereafter, palla (2003) conducted the only other recent study; he analyzed the population dynamics of demersal fishes in puerto princesa and honda bay. despite the claim of some authors that one of the causes of coral cover reduction in palawan is siltation and erosion (cruz et al., 1988 as cited by frmp, 2001), studies related to erosion, siltation and sedimentation and its potential impact on the coastal ecosystems have no concrete evidence. in fact, the present study is the first that ever looked into sedimentation in honda bay. to describe the status of sedimentation in honda bay, this study was conducted with the following objectives: 1) to determine and compare the rate of sedimentation between bush and meara island in honda bay during the two seasons, 2) to discuss sedimentation rate in relation to selected environmental parameters, 3) to determine the coral cover in the two stations and to compare it with data collected in 2000, 4) to explain eventual changes in coral cover in relation to the selected environmental parameters, especially sedimentation. materials and methods locale of the study honda bay is one of the bays of puerto princesa city, which is a major fishing ground of both artisanal and commercial fishers (fig. 1). it is a land indentation embayment with an area of approximately 280 km2 located northeast of mainland palawan between 9o50’ to 10o00’ n latitude and 118o44’ to 119o00’ e longitude. it is composed of several islands and islets and has a coastline of 100 km. the islands are small, ranging from 0.0125 to 0.45 km2 (fig. 1). honda bay is surrounded by 18 coastal barangays. six major rivers, namely bacungan, tandayak, babuyan, tanabag, langugan and tapul and numerous small tributaries drain into the bay (fig. 1). during rainy season, these rivers carry high amounts of sediments into the bay (personal observation). two reef sites were selected near the islands of bush and meara (fig. 1). bush island is located near bgy. sta. cruz and is 1.70 km away from the shoreline of mainland palawan, lying at 118o47.268’ e longitude and 9o55.272’ n latitude. meara is approximately 3.00 km from the nearest shore and 4.40 km from bush island. it is located at 118o46.410’ e longitude and 9o53.304’ n latitude. at each of these islands, a sampling station was established along a demarcated coral transect, which was assessed in terms of coral cover by frmp in 2000. the two stations were assessed in terms of sedimentation rate, water temperature, salinity, depth, ph and total dissolved solids. the stations were further assessed in terms of coral cover. sampling of physical and chemical parameters was conducted thrice during the dry season (april, may and june 2003) and thrice during rainy months (august, september and november 2003). sampling was conducted within one day. the coral cover at the reef stations was assessed on may 31, 2003 when the visibility was high and the sea was calm. three sets of sediment traps (length: 11.50 cm; diameter: 5.00 cm) were installed in each station with three pseudo-replicates / sub-samples per set (hurlbert 1984) (i.e., a set was composed of three individual traps) on the same day. the traps were installed along a 100-m transect line, 1 m away from the frmp-markers. the three replicates were mounted close (0.30 m) to each other. during the samplings in april, may and june, sediment traps were left for one month at the sites. however, during the remaining samplings (i.e., august, september and november), traps were only left for 24 hours at the sampling sites. direct comparison on the result will probably not be possible due to the time difference in the deployment of traps. traps were sealed with cellophane before removing them from the rod to prevent loss of material while bringing the sample to the surface. in the laboratory, the content of the traps was filtered to 8 science diliman sedimentation rate in fringing reefs of honda bay separate the sediment from seawater. samples were dried overnight in an oven at 60oc. dried samples were weighed to the nearest milligram. sediment traps were thoroughly cleaned of encrusting organisms before re-use. water temperature, salinity, depth, ph and total dissolved solids were measured during the collection of sediment traps. parameters were determined at the subsurface of the place where sediment traps were installed. triplicate readings were taken using the following measuring devices: standard mercury thermometer, ph meter (corning – checkmate tm ii) and conductivity meter (corning – checkmate tm ii) for salinity and total dissolved solids. the line intercept transect (lit) method described by english et al. (1997) which was utilized by the frmp team in 2000 was used in assessing benthic lifeforms. in each station, a 100 m transect line was laid along the reef crest at 6-7 m depth. to ensure that transects are laid exactly in the same position previously used by the frmp team, their concrete markers – which were found at 5-m intervals – were used. the following lifeform categories and codes modified after unep (1993) and english et al. (1997) were used in this study: hard coral (hc), soft coral (sc), sponge (sp), others (ot), dead corals with algae (dca), sand (s), rock (rck) and rubble (r). for the three replicate sets, the mean (± sd) amount of sediment was calculated. the mean sedimentation rate was determined in mg/cm2/day. for comparative reasons, the overall mean sedimentation rate in honda bay was converted into g/m2/day. analysis of variance (anova) was used science diliman 9 figure 1. map showing palawan (lower right), the study area and the location of the sampling sites (+) in honday bay, puerto princesa city. becira to determine eventual differences in sedimentation rate between the two stations, and between the data gathered during the dry (1st three sets of data) and rainy season (2nd three sets of data) at 5% level of significance. mean physico-chemical parameters were correlated with sedimentation. line intercept transect (lit) data was used to analyze the percentage of the different lifeforms. results were then summarized into two major categories: biotic and abiotic. results sedimentation rate the monthly sedimentation rate in bush island ranged from 2.00 to 21.00 mg/cm2/day while in meara island it ranged from 2.00 to 16.00 mg/cm2/day (fig. 2). the overall mean sedimentation rate was 7.10 and 6.50 mg/cm2/day for bush and meara islands, respectively. between the sites, sedimentation was higher (7.00 mg/cm2/day) in bush island than in meara island (6.00 mg/cm2/day) (tab. 1). differences in sedimentation rate between sampling events (6 events) and sampling seasons (wet & dry) in bush island were not significant. on the other hand, significant differences were detected in meara island between sampling events and also between sampling seasons. between the stations in bush and meara islands, no significant differences were established in both sampling events and sampling seasons. total dissolved solids were high in the two island stations (bush island, 24.88 ± 0.31 mg/l; meara island, 24.98 ± 0.29 mg/l). the correlation of tds with the sedimentation rate in bush island was low (r = 0.37) and with that of meara island was negatively high (r = -0.80). salinity readings were relatively stable in the two islands, i.e., meara island (32.71 ± 0.41 ppt) and bush island (32.61 ± 0.41 ppt). it was moderately correlated with the sedimentation rates in the two islands (r=0.50). the values of the correlation coefficient (r) in both islands were not significantly different. mean temperatures at bush and meara islands were 30.19oc and 29.43oc, respectively. the mean ph readings in the two stations were slightly basic. the ph reading was almost similar in both islands with 8.36 in meara and 8.44 in bush island (tab. 2). the correlation coefficients (r) between ph and the sedimentation rates of both islands, bush and meara, were relatively high (i.e., r=0.90 for meara and r=0.99 for bush). table 1. mean sedimentation (mg/cm2/day) during dry and wet season in the different stations in honda bay, puerto princesa city. season station bush meara dry 3.00 4.00 wet 11.00 9.00 mean 7.00 6.50 coral cover the hard coral cover had the largest contribution to the benthic category. in bush island, hard corals contributed 45.83% and in meara island 43.20% to the benthic community (tab. 3). in both islands soft coral cover was 3.80%. the abiotic components contributed 35.52% and 48.30% to the total benthic lifeforms of the two islands wherein dead corals covered with algae and rubble contributed most for both islands (tab. 3). discussion sedimentation rate average sedimentation in honda bay was higher (6.80 mg/cm2/day) compared to dona paula bay, west coast of india (bhaskar et al., 2000) which ranged from 0.11 to 1.34 mg/cm2/day (pvc pipe used had a diameter of 20.00 cm) with an average of 10 science diliman figure 2. mean sedimentation rate at the two reef stations during the six (6) sampling events. sedimentation rate in fringing reefs of honda bay table 2. mean of the physico-chemical parameters considered in bush and meara islands in honda bay, puerto princesa city. physico-chemical parameters sampling season average bush island meara island bush island meara island dry wet dry wet temperature (oc) 30.57 29.80 29.83 29.03 30.19 29.43 salinity (ppt) 32.43 32.80 32.83 32.60 32.61 32.71 ph 8.14 8.75 8.14 8.58 8.44 8.36 total dissolved solid (mg/l) 24.75 25.00 25.05 24.90 24.88 24.98 0.54 mg/cm2/day. however, direct comparison may not be advisable due to differences in trap designs, exposure periods and hydrological aspects of the different marine environments. a study conducted by aliño (1983) in three areas (matab-ang, bato and looc) in toledo city, cebu using 5 cm  20 cm pvc pipe revealed an average sedimentation of 16.20, 10.10 and 33.50 mg/cm2/day, respectively. compared with these values the two island stations in honda bay had relatively low values. in singapore, increasing coastal development has been claimed to cause increasing levels of sedimentation. studies of chan (1980 as cited by lane, 1991) revealed a sedimentation rate of 3.00-6.00 mg/cm2/day in 1979. later studies found sedimentation to be at 5.45 mg/cm2/day (lane, 1991; low and chow, 1994). their data is similar to those gathered from honda bay. sediments collected in both islands were composed of sand, shell fragments and organic matter. table 3. benthic lifeform cover (%) in bush and meara islands in 2000 (frmp,2001) and 2003 (this study). benthic categories bush meara 2000 2003 2000 2003 hc 40.65 45.83 25.65 43.20 sc 2.90 3.80 0.20 3.82 sp 4.05 0.00 6.45 0.00 ot 0.30 17.84 0.85 4.68 dc 0.00 0.00 0.40 0.00 dca 5.80 12.72 3.70 33.00 s 0.00 6.50 0.00 7.50 r 17.60 13.30 9.15 7.80 rck 17.00 0.00 24.45 0.00 si 0.00 0.00 1.00 0.00 hc=hard coral, sc=soft coral, sp=sponge, ot=others, dc=recently dead corals, dca=dead corals with algae, s=sand, r=rubble, rck=rock, si=silt total dissolved solids (tds) are minerals (salt) in the water. they are high in saltwater in line with salinity. sedimentation brought in from watersheds with high mineral content could eventually increase tds. however, total dissolved solid values during dry and wet season at both islands were almost the same, which shows that freshwater does not influence the water quality of both islands as evidenced by its high and relatively stable salinity. salinity had no significant changes between dry and wet season at both islands under study. ph in both islands was higher during wet season. although a lot of basic ions are deposited in the coastal environment during the onset of the rainy days, saltwater has the capacity to normalize ph. the ph measurements in both islands are typical for the characteristics of the stations, wherein the stations are well buffered with ph above 8. a ph of 8.1 to 8.3 is typical for seawater (lalli & parson 1997). coral cover a study conducted by frmp in 2000 using the same method revealed that hard coral cover in bush and meara islands was 40.65% and 25.65%, respectively. with regards to soft coral cover, it was 2.90% in bush island and 0.20% in meara (tab. 3). in both island sites, hard corals and sponges constituted the largest biotic components. likewise, rock, sand and rubble contributed to almost 50% of the abiotic components in both islands. live coral cover went down after el niño in 1997 and 1998. in 2000, coral cover in honda bay was probably not yet back to its previous state, though there was recovery in 2003. from 2000 to 2003, the biotic component had increased in both sites (fig. 3).the fact that they are able to recover despite sedimentation seems to indicate that sedimentation has not yet reached critical levels. live coral cover in honda bay is higher compared to coral cover in science diliman 11 becira nearby provinces. in romblon, live coral cover was 17.92% and in mindoro, it was 19.94% (spcpasti, 1999). earlier assessment of honda bay, particularly the islands of fondeado, arrecife, snake, pandan and canyon showed that the live coral cover ranged from 19.38 to 50.63% (spcpasti, 1999). assessment of live coral cover in palawan in 1981 using transect/quadrat method had a value of 12-40.80% (gomez, 1981). in port barton, san vicente, palawan, live coral cover had an average value of 27.17% (philreefs, 2003) which is lower compared to the live coral cover in 1981. it was only in panglao island (45.18%) and bais bay (50.26%) where the live coral cover was almost similar with live coral cover in honda bay (aliño et al., 2002). the philippines has an average coral cover of 25 to 49.90% (white and trinidad, 1998). other factors that may favor the growth of corals in honda bay are the temperature, salinity and ph. the two island stations, bush and meara, had a mean temperature of 30.19oc and 29.43oc, respectively. these values are within the minimum and the maximum temperature requirement for corals to exist (18-35oc) (sale, 1991; tait and dipper, 1998; fabricus and alderslade, 2001). the salinity measured at the two stations seems to be quite stable and the difference between dry and wet seasons is negligible (tab. 2). this indicates that no freshwater reaches the island stations from the nearby river systems. the values measured are typical for marine waters of this geographical region. corals are favorably grown in areas with a salinity of 32-38 ppt with an average of 35 ppt. (lalli and parsons, 1997). ph, like any other water quality parameters, can also affect the growth of corals. a value of 8.1-8.3 is normal for seawater because it is always well buffered. ph in bush island ranged from 8 to 9.69 while in meara island, it ranged from 8.11 to 8.99. mean ph in bush island was 8.44 while in meara island it was 8.36. the fact that coral cover is able to recover despite sedimentation seems to indicate that sedimentation has not yet reached critical levels in honda bay. acknowledgement the author wishes to extend his sincere gratitude and appreciation to all individuals, who in one way or another, contributed towards the accomplishment of this study: his wife and kids, dr. sabine schoppe, dr. filipina sotto, dr. benjamin gonzales, bs fisheries and aquatic biology students and the wpu administration. references aliño, p.m., miclat, e.f.b., nañola, c.n., quiaoit, h.a.r., campos, r.t. (eds.) 2002. atlas of philippines coral reefs. goodwill trading co., inc. (goodwill bookstore). quezon city, philippines. 264 pp. aliño, p.f., 1983. the effects of mine tailings on the structure of coral communities in toledo, cebu. ms thesis in marine biology, university of the philippines. 105 pp. bhaskar, d.v., cardozo, e, giriyan, a, garg, a., bhosle, n.b. 2000. sedimentation of particulate matter in the dona paula bay, west coast of india during november to may 1995 to 1997. estuaries. 23(5): 722 734. reef information network of the philippines (philreefs), 2003. philippine coral reefs through time: workshop proceedings. second of the atlas of philippine coral reefs series. coral reefs information network of the philippines, university of the philippines-marine science institute. quezon city, philippines and marine park center, tokyo, japan. 197 pp. el-swaify, s.a., 2000. operative processes for sedimentbased watershed degradation in small, tropical volcanic island ecosystem. pages 35-49 in: r. lal (ed.), integrated 12 science diliman figure 3. relative contribution of biotic and abiotic components in the reefs of bush and meara islands in years 2000 and 2003. sedimentation rate in fringing reefs of honda bay watershed management in the global ecosystem. soil and water conservation society. crc press. usa. pp. 395. english, s., wilkinson, c., baker, v. 1997. survey manual for tropical marine resources. 2nd edition. australian institute of marine science. townsville, australia. 390 pp. fabricus, k., alderslade, p. 2001. soft corals and sea fans. a comprehensive guide to the tropical shallowwater genera of the central-west pacific, the indian ocean and the red sea. australian institute of marine science, queensland, australia. 264 pp. gomez, e.d., alcala, a.c., san diego, a.c. 1981. status of philippine coral reefs. prodeeding of the 4th international coral reef symposium, manila. 1:275-282. marine science center, university of the philippines, diliman, quezon city, philippines gonzales, b.j. 2000. puerto princesa city integrated coastal resource management network. a concept paper. frmp-puerto princesa city. 9 pp. gonzales, b.j. in press. puerto princesa bay and honda bay, palawan: an ecological profile. in: bfar turbulence, 15 pp. hurlbert, s. h. 1984. pseudoreplication and design of ecological field experiments. ecological monographs 54: 187-211. ecological society of america frmp, 2001. resource and social assessment of honda bay and puerto princesa bay: a terminal report, philippines denr, 635 pp. iclarm (international center for living aquatic resources management). 1996. resource and ecological assessment of honda bay, palawan, philippines, iclarm, makati city, philippines lalli, m.c., parsons, t.r. 1997. biological oceanography. 2nd edition. butterworth heinemann. woburn, great britain, 314 pp. lane, d.j.w. 1991. growth of scleractinian corals on sediment –stressed reefs in singapore. in: alcala, a.c. (ed.) proceedings of the regional symposium in living resources in coastal areas. university of the philippines, manila, pp. 97-106 low, j.k.y., chow, l.m . 1994a. sedimentation rates in singapore waters. in: sudara, s., wilkinson, c.r. & l.m. chow (eds.). proceedings 3rd asean-australia symposium on living coastal resources. vol.: 2 research papers. chulalongkorn university, bangkok, thailand, may 1994. palla, h.p., 2003. population dynamics of demersal fishes in honda bay, palawan, philippines. ms thesis in international studies in aquatic tropical ecology. university of bremen, federal republic of germany, 65 pp sale, p.f. 1991. the ecology of fishes on coral reefs. academic press. san diego, new york, 754 pp. sandalo, r.m. 1994. community-based coastal resources management: the palawan experience, p. 165-181. in pomeroy, r.s. (ed.). community management and common property of coastal fisheries in asia and the pacific: concepts, methods and experiences. iclarm conf. proc. 45, 189 pp. spalding, m.d., ravilious, c., green, e.p. 2001. world atlas of coral reefs. university of california press, usa, 424 pp. spcp-asti 1999. a report on the rapid resource assessment in some coastal areas of north and west sulu sea. department of environment and natural resources – dost-philippine council for aquatic and marine research and development, 404 pp. tait, r.v., dipper, f.a. 1998. elements of marine ecology. butterworth-heinemann. woburn, 462 pp. unep, 1993. monitoring coral reefs for global change: reference methods for marine pollution studies no. 61, 72 pp. white, a.t. 2001. philippine coral reefs. a natural history guide, 2nd edition. bookmark, inc., hong kong, 276 pp. white, a.t., trinidad, a.c. 1998. the values of philippines coastal resources: why protection and management are critical. coastal resource management project. department of environment and natural resources. united states agency for international development. cebu city, philippines, 96 pp. science diliman 13 13_wall ponce, lazarte, and ramos 52 effect of wall material on h– production in a plasma sputter-type ion source y. d. m. ponce*, j. r. s. lazarte, and h. j. ramos plasma physics laboratory, national institute of physics, university of the philippines, diliman, quezon city 1001 e-mail: dponce@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 52–56 *corresponding author the effect of wall material on negative hydrogen ion (h–) production was investigated in a multicusp plasma sputter-type ion source (pstis). steady-state cesium-seeded hydrogen plasma was generated by a tungsten filament, while h– was produced through surface production using a molybdenum sputter target. plasma parameters and h– yields were determined from langmuir probe and faraday cup measurements, respectively. at an input hydrogen pressure of 1.2 mtorr and optimum plasma discharge parameters vd = –90 v and id = –2.25 a, the plasma parameters ne was highest and t–e was lowest as determined from langmuir probe measurements. at these conditions, aluminum generates the highest ion current density of 0.01697 ma/cm2, which is 64% more than the 0.01085 ma/cm2 that stainless steel produces. the yield of copper, meanwhile, falls between the two materials at 0.01164 ma/cm2. the beam is maximum at vt = –125 v. focusing is achieved at vl = –70 v for stainless steel, vt = –60 v for aluminum, and vt = –50 v for copper. the results demonstrate that proper selection of wall material can greatly enhance the h– production of the pstis. introduction negative ion beams are extensively used in several fields including nuclear fusion and high-energy physics (nishiura et al., 1998). such beams are generally produced with three types of sources: volume, surface, and hybrid sources. the plasma sputter-type ion source (pstis) in the plasma physics laboratory designed by ramos (1995) for thin-film formation via ion beam deposition is distinctly of the surface conversion type due to the presence of a mo converter electrode, which, when biased negatively with respect to the chamber wall (anode), causes the ions to self-extract towards the beam diagnostic chamber for beam analysis. aside from the surface production mechanism occurring in the converter surface, h– can also be generated from the plasma bulk by a dissociative attachment process and along the walls as described in eqs. (1) and (2), respectively: , (1a) , (1b) , (2) where h2 (ν’’=0) = hydrogen molecule in ground state, h2*(ν’’) = highly rovibrationally excited h2 molecule, e–fast = fast or high-energy electron, and e–cold = cold or low-energy electron. for practical applications like thin-film deposition, the ion beam must have high negative ion current output. effect of wall material 53 however, in a previous study conducted on the same device, the extracted h– current (~0.097 na) was very minimal (ranay, 2002). several optimization and enhancement procedures have been performed on the extracted beam including argon-mixing (ubarro, 2003), beam focusing (yambot, 1999), and cs seeding, which so far has proven to be the most effective with the highest h– yield of 33.1 na (yambot, 2003). another possible technique that has considerable effect on the production rate of ions is the modification of the wall material of the ion source by installing various metal liners. leung et al. (1985) investigated the effect of different metal liners on the chamber wall of a magnetically filtered multicusp source. for the specific device used, the differences in current yield were attributed to the amount of secondary electrons emitted from the wall surfaces. the presence of secondary electrons of energies >> 8 ev reduced the h– yield considerably. the relatively low yield of stainless steel indicates that the walls of the all-stainless-steel ion source of the pstis pose an inherent limitation to the production of h– beams. hence in the study, the effect of varying the wall material of the ion source on the h– yield was carried out to resolve this limitation. experiment the facility utilized in this experiment, the pstis, is shown schematically fig. 1. hydrogen plasma is produced within the ion source chamber made up of an all-stainless-steel cylinder measuring 10.8 cm in diameter and 14 cm in length, with an approximate volume of one liter. eight columns of sm–co magnets surround the cylinder, while the side flanges each have six columns forming a multicusp magnetic configuration for plasma confinement, reducing the loss of primary electrons and thereby increasing the plasma density. h are produced through surface production using a mo converter (concave disc with 10 cm focal length, 1.5 cm thickness, and 2.4 cm diameter), while thermionic emission from a tungsten filament (0.05 cm diameter and 9 cm length) provides the primary electrons. al and cu metal liners with thickness of 0.28 mm were used as substitutes to the purely stainless steel ion source chamber to be able to study the effect of varying the wall material in the production of negative ions. plasma characteristics are obtained by the langmuir probe, while the h– current is measured by a faraday cup. the effect of altering the wall material on plasma parameters was investigated in a pure and a cesiatedhydrogen discharge at 1.2 mtorr input hydrogen pressure in a vacuum system with a base pressure of 10–6 torr. results and discussion prior to h– beam extraction, plasma characteristics were determined to establish the optimum plasma conditions for the production of h–. the conditions of the plasma influence h– production and destruction. langmuir probe measurements yield the pertinent parameters: electron temperature te and electron density ne from the i-v curve of the plasma. te and ne are indicators of the rate of formation, destruction, and extraction of h– ions in a surface production multicusp source (uramoto, 1985). higher ne and lower te are positive conditions for the formation of negative ions. te describes the energy of the electrons present in the plasma. low te coupled with high ne point to the presence of more slow or cold electrons, necessary for the formation of h– from an excited molecular hydrogen h2(ν”) by dissociative electron attachment (wengrow et al., 1998). electrons of high energies, called “hot” electrons, tend to destroy the already formed h– within the bulk of the plasma.fig. 1. schematic diagram of the pstis. gas inlet converter electrode diagnostic chamber multicusp ion source c on ve rt er po w er s up pl y discharge power supply filament power supply sm-co magnets langmuir probe faraday cup einzel lens le ns p ow er su pp ly pump ponce, lazarte, and ramos 54 shown in figs. 2 and 3 are the te and ne at varying discharge current id. from fig. 2, the electron temperature generally decreases with increasing discharge current. such behavior is attributed to the increased electron-ion and electron-neutral interactions at higher id due to shorter mean free paths of the particles, offsetting the initial energies of the electrons. ne, on the other hand, exhibits a converse behavior; that is, ne increases with id as illustrated in fig. 3. this trend agrees with expected results since the number of electrons per unit time is increased with increasing id. addition of cs vapor further reduced te and increased ne. hence, it is expected that the ion yield from the cesiated plasma would be much higher than that of the uncesiated case. the increase in the ion yield can be accounted from the following underlying mechanisms (fukumasa et al., 1996): electron cooling (i.e., lower te), production of h2(ν”) due to reaction between cs atoms and h3 +, h– surface production caused by h atoms and positive hydrogen ions and lowering of the work function of the surface material. similar behaviors of te and ne are observed for the three materials, both for the cesiated and uncesiated cases. the value of ne peaks at id = –2.25 a and at the same time, te has a low value at this condition. thus, the discharge condition most suitable for beam characterization is at id = –2.25 a and vd = –90 v. it is also notable from the plots that among the three wall materials, the plasma with highest ne and lowest te is produced with the al liner, most defined upon addition of cs, followed by the cu liner, and the stainless steel obtained the lowest. this behavior is due to the dependence of ne on the work function of the metals used. lower work function means easy removal of electrons from the boundary of the metal, thereby increasing the presence of more low-energy electrons in the plasma upon bombardment of primary electrons into the chamber walls. results for the uncesiated case, on the other hand, are inconclusive due to overlapping values obtained. hence only the cesiated case is considered in the characterization of the h– beam. upon determination of the optimum ne and te, optimization of the target potential vt was carried out. results show that the h– current values produced by the three wall materials increase with vt, consistently peak at –125 v, and then gradually decrease thereafter as depicted in fig. 4. the target potential determines the energy of the ions leaving the surface of the mo target. thus, increasing vt is expected to increase the energies of the h – ions repelled towards the diagnostic chamber, leading to an increased number of h– with enough energy to reach the detector, hence, the greater h– current with increasing vt. the subsequent diminishing values of h– current density at vt greater (more negative) than –125 v may seem anomalous. however, this may be accounted for by the secondary electron emission at the collector of the faraday cup upon impact of the negative ions. the faraday cup is made of brass, which is 60%–80% copper. copper begins to emit secondary electrons when hit by particles of energies approaching 200 ev (weast, 1986). hence, at higher target potentials these electrons may reduce the detected current. at fig. 2. comparison of te and id for stainless steel, al, and cu metal liners in pure and cs-seeded h plasma at vt = 0. t e (e v ) id (a) fig. 3. comparison of te and id in pure and cs-seeded h plasma at vt = 0. id (a) n e ( x1 01 0 c m 3 ) effect of wall material 55 optimum vt = –125 v, the extracted h – current densities are 0.0106, 0.00592, and 0.00730 ma/cm2 for al, cu, and stainless steel (ss), respectively. the high current density values detected for the cesiated case, even without focusing of the beam, are three orders of magnitude higher than the previously optimized uncesiated results (ranay, 2002). even when compared with the previous results obtained with cesium-seeded hydrogen plasma (yambot, 2003), the difference is approximately two orders of magnitude higher. this is attributed to the difference in the operating temperature of the cesium oven (180°c compared with 300°c in previous experimental runs). overcoverage of cesium on the surface of the mo converter is known to cause a diminishing of h– yield. for the present case, the high h– current suggests that no destructive effect occured in the production of ions due to excessive amount of cesium (wengrow et al., 1998). at optimum target voltage, vt = –125 v, the input voltage to the einzel lens is varied from 0 v to –150 v. figure 5 clearly describes the effect of vl on the detected current. the current density generally increases until the optimal value of vl is achieved. focusing of the beam with the different wall materials is achieved at almost similar values of vl: –70 v for ss, –60 v for al, and –50 for cu. at optimized vt and vl, the values of h– current density for al, cu, and ss are 0.01697, 0.01164, and 0.01085 ma/cm2, respectively. for the case of aluminum, a 63% increase of h– current density at optimum vl is obtained compared with the extracted unfocused beam, clearly showing the focusing effect of the einzel lens. the same enhancement in h– yield is observed for copper (51%) and stainless steel (67%) at their corresponding optimum vl. the presented results from the plots above clearly indicate that the wall material of the ion source plays an important role in the production of h– ions. under the same discharge conditions, the performance of the different wall materials in terms of h– yield differs significantly. from the plots, aluminum consistently generates the highest ion current, indicating that among the three it is the best metal liner for optimum beam extraction. stainless steel, on the other hand, yields the lowest amount of ions, 64% lower than aluminum at optimum beam extraction parameters. this is consistent with the expected results based on the plasma parameter investigation since aluminum has the highest ne and lowest te values. the first leads to a more efficient use of rovibrationally excited molecules, resulting in more negative ion formation (bacal et al., 2002), and the second leads to less reduction of h– within the plasma due to less impact processes with energetic or “hot” electrons that can contribute to neutralization of produced negative ions (ramos et al., 1989). the higher ion yield produced by aluminum is attributed to its lower work function as compared with stainless steel and copper. aluminum’s work function f is only 4.28 ev, while cu has f = 4.65 ev. stainless steel is approximately 65% iron (f = 4.7 ev), 20% chromium (f = 4.5 ev), and 10% nickel (f = 5.15 ev). the work function of a material is the energy needed to remove an electron from the fermi level in a metal to a point at infinite distance away outside the surface. the lower fig. 4. optimization of the mo target potential. the h– current density peaks at vt = –125 v. target voltage, vt (–v) h – cu rr en t de ns it y (m a /c m ) fig. 5. optimization of the focusing effect of the einzel lens. h – cu rr en t de ns it y (m a /c m ) lens potential, vl (–v) ponce, lazarte, and ramos 56 the work function of the material is, the easier it would be to eject the electrons from the wall material into the bulk of the plasma. bombardment by neutrals and ions along anode cusps would facilitate the removal of electrons. within the plasma, the ejected electrons from the wall materials would contribute greatly to the formation of more h– through the surface production process. additional reactions that generate h– within the bulk of the plasma as described by the dissociative electron attachment process [eqs. (1a) & (1b)] require the presence of low-energy electrons. the wall material of lower work function can contribute more electrons necessary for the aforementioned processes, yielding more h–. thus, aside from the surface production process occurring at the mo converter, additional h– ions are produced at the walls and within the plasma. comparing the values of work functions of the metal liners and using the argument presented above, it is logical that the wall material that produces the most h– is the aluminum liner as more electrons are easier to remove for electron dissociative electron attachment necessary for h– production than from copper and stainless steel. the greater current obtained from the experiments agree well with this argument. similarly, the almost identical values of the work functions of copper and stainless steel explain the comparable ion yield detected for the wall materials. stainless steel, with the highest work function, yielded the least as expected. this also justifies why the yields of cu and stainless steel overlap at certain plasma and beam conditions. conclusion enhancement in the production of h– may be achieved with proper selection of the wall material in the operation of the pstis. at optimum plasma parameters, results show that at 1.2 mtorr, aluminum generates the highest yield of 0.01697 ma/cm2, which is 64% more than the 0.01085 ma/cm2 that stainless steel produces. the yield of copper, meanwhile, falls between the two materials at 0.011644 ma/cm2. the discrepancy in the metals’ work functions accounts for the differences in the h– yield among the wall materials. aluminum having the lowest work function among the three yields the highest current. references bacal, m., et al., 2002. rev. sci. instrum. 73: 903. fukumasa, o., et al., 1996. jpn. j. appl. phys. 35: l1528–31. leung, k.n., k. w. ehlers, & r. v. pyle, 1985. appl. phys. lett. 47: 227. nishiura, m., m. sasao, & m. wada, rev. sci. instrum. 69: 974. ramos, h.j., et al., 1989. ippj-914 research report, nagoya, japan. ramos, h.j., 1995. development of negative ion sources for thin film formation. proceedings of the 15th annual conference of the japan society of fusion and plasma science research, osaka. ranay, j.p., 2002. investigation of plasma parameters for negative hydrogen ion extraction in a sputter-type negative ion source, b.s. thesis. national institute of physics, college of science, university of the philippines, diliman. ubarro, a.d., 2003. effect of converter potential and ar– h2 mixing in the production of negative ions in a multicusp source, b.s. thesis. national institute of physics, college of science, university of the philippines, diliman. uramoto, j., 1985. ippj research report, nagoya, japan. weast, r.c. (ed.), 1986. crc handbook of chemistry and physics, 67th ed. crc, boca raton, fl. wengrow, a.b., et al., 1998. development of a high duty factor, surface conversion h– ion source for the lansce facility. ieee. yambot, m.l., 1999. negative ion beam focusing study in a pstnis, b.s. thesis. national institute of physics, college of science, university of the philippines, diliman. yambot, m.l., 2003. effect of cesium seeding on h– ion production in a plasma sputter-type ion source, m.s. thesis. national institute of physics, college of science, university of the philippines, diliman. 01_device taganna and rivera 24 *corresponding author science diliman 20:1, 24-30 epigallocatechin gallate from camellia sinensis l. (kuntze) is a potential quorum sensing inhibitor in chromobacterium violaceum joemar c. taganna1 and windell l. rivera1,2* 1institute of biology, college of science, university of the philippines, diliman, quezon city 2molecular protozoology laboratory, natural sciences research institute, university of the philippines, diliman, quezon city telefax: (63-2) 920-5471 email: wlrivera@science.upd.edu.ph date submitted: january 22, 2008; date accepted: may 6, 2008 abstract the problem on the widespread occurrence of antibiotic resistant strains of bacteria calls for novel methods of control of bacterial activity. one of the new viable alternatives to antibiotics is the use of substances that inhibit quorum sensing (qs) – a bacterial communication system that has been known to regulate the expression of virulence genes during infection. in this study, epigallocatechin gallate (egcg) from green tea, camellia sinensis l. (kuntze) was tested for its ability to inhibit qs in a test organism, chromobacterium violaceum. this microorganism produces a violet-colored substance, violacein, through qs. this study aimed to detect inhibition of qs-regulated violacein production in c. violaceum by egcg and to determine the dynamics of qs inhibition relative to the concentration of egcg. the effect of increasing concentration of egcg on both violacein production and cell density of treated and untreated c. violaceum was determined in a 96-well-microplate format and read at 570nm and 620nm for violacein production and growth, respectively. the results show that addition of egcg increased the growth of the organism while there is concentration-dependent decrease in the qs-controlled production of violacein. this study thus establishes that egcg is a potential qs inhibitor and can be further studied and developed for its use as an anti-pathogenic but non-toxic drug. keywords: antibiotic resistance, anti-pathogenic, camellia sinensis l. (kuntze), chromobacterium violaceum, epigallocatechin gallate, quorum sensing inhibition egcg is a potential quorum sensing inhibitor 25science diliman 20:1, 24-30 introduction quorum sensing (qs) is the production and reception of diffusible signal molecules or autoinducers by bacteria to regulate the expression of phenotypes that are dependent on population density (shauder & bassler, 2001). after the first mechanism of this communication system in vibrio fischeri was elucidated (nealson et al., 1970; eberhard et al., 1981), other studies found the same system at work in many other groups of bacteria – mainly differing in the nature and structure of the diffusible signals and on the phenotypes being regulated (de kievit & iglewski, 2000; schauder & bassler, 2001). some of the phenotypes regulated by qs are antibiotic production in erwinia carotovora (bainton et al., 1992); expression of virulence factor in pseudomonas aeruginosa (gambello & iglewski, 1991); swimming and swarming motility in yersinia enterocolitica (atkinson et al., 2006); and biofilm formation in a number bacterial species including p. aeruginosa (davies et al., 1998; favre-bonte et al., 2003), vibrio cholerae (hammer & bassler, 2003) and serratia marcescens (labbate et al., 2004; rice et al., 2005). in the quest for qs inhibitors or antagonists, studies have found that many eukaryotes, particularly plants, and even bacteria themselves produce anti-qs substances (gonzalez & keshavan, 2006). the first one to be studied was the australian red alga (dilesia pulchra), which was found to produce furanones that inhibit the qs system of a marine bacterium, serratia liquefaciens (givskov et al., 1996). several species of higher plants, including pea seedlings, secrete a series of unidentified signals that are capable of interfering with the qs of reporter strains (teplitski et al., 2000). the work of keshavan and colleagues in 2005 found that l-canavanine, secreted by the legume alfalfa (medicago sativa) interferes with the qs of sinorhizobium meliloti, a nitrogen-fixing bacterium that invade its roots. in vivo studies have documented the anti-pathogenic effects of qs inhibitors. synthetic furanones were found to enhance bacterial clearance of p. aeruginosa from lung infection in mice (wu et al., 2004). qs inhibitory extract from garlic renders p. aeruginosa susceptible to tobramycin in lungs of mice (bjarnsholt et al., 2005). disruption of ai-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic artemia from the pathogenic isolates in in vivo challenge tests (defoirdt et al., 2006). qs inhibitor rip injected to rats prevented methicillin-resistant staphylococcus aureus infections (balaban et al., 2007). these studies establish that the use of qs inhibitors is a viable alternative to the use of antibiotics and that there is a need to search for other natural qs inhibitors from plants or other sources. in this regard, we checked for the possibility that extracts from green tea, camellia sinensis l. (kuntze) can be a source of such qs inhibitory substances. the main component of green tea extracts, epigallocatechin gallate (egcg) has been previously shown to inhibit biofilm formation in staphylococcus spp. (blanco et al., 2005), that it inhibits penicillinase leading to restoration of antibiotic activity of penicillin (zhao et al., 2002) and that it possesses direct bactericidal activity on certain species of bacteria (toda et al., 1989, 1991). the objective of the present study was to evaluate the ability of egcg to inhibit quorum sensing using a test organism, chromobacterium violaceum. specifically, it aimed to detect inhibition of qs-dependent violacein production in c. violaceum by egcg and to determine the dynamics of qs inhibition by egcg in relation to its concentration. materials and methods bacterial cultures and egcg the pure culture of wild type c. violaceum jcm1249 was obtained from japan collection of microorganisms (saitama, japan) while the non-pigmented mutant strain, c. violaceum cv026 (nctc13278) was acquired from the national collection of type cultures (london, u.k.). these were maintained and cultured in luria-bertani (lb) broth and lb agar. the egcg used was teavigo® green tea extract powder (dsm nutritional products, philippines) with a claimed 94 to 99% egcg content derived from camellia sinensis l. (kuntze). taganna and rivera 26 science diliman 20:1, 24-30 tube-based qualitative assessment of qs inhibition by egcg a stock solution of 10,000 µg ml-1 egcg was prepared. from this stock solution, dilutions using nutrient (nb) as diluent were prepared. three lanes composed of seven sterile test tubes at varying concentrations were set. a single lane contained tubes with 5 ml of 1000, 500, 250, 125, 62.5, 31.25 and 0 µg ml-1 of egcg in nutrient broth. two lanes were inoculated with 100 µl of c. violaceum culture with an od 660 of 1.6. the remaining lane was used as a negative control. the whole set-up was then incubated at 37oc for 48 hours. inhibition of quorum sensing or of growth was seen as a reduction in violet pigmentation of the treated broth cultures. quantitative qs inhibition and toxicity assays in c. violaceum the effect of egcg on the qs-controlled production of violacein was determined using the wild type pigment-producing strain of c. violaceum while the potential toxic effects was monitored using nonpigmented c. violaceum strain to avoid inaccuracy due to light scattering of violacein at 620 nm. this protocol was a modified version of that described by martinelli et al. (2004). a two-fold serial dilution of the sample was prepared in 8-well lanes of a 96-well microtiter plate (1000, 500, 250, 125, 62.5, 31.25, 15.625 and 0 ug/ml) at 150 ul each using lb broth as diluent. fifty microliters per well of an overnight culture of the wild type c. violaceum in lb broth was added to three lanes. the same amount of an overnight culture of cv026 in lb was added to the next three lanes. another three lanes contained serially diluted egcg at the indicated concentrations dissolved in lb but without any culture added to measure the absorbance of egcg in lb alone for normalization purposes. the plate was then incubated at 37°c for 24 hours; after which, it was read at 570 nm (for violacein) and 620 nm (for cell density) using a microplate spectrophotometer (tecan, spectra iii, austria). data analyses the data was analyzed using anova followed by dmrt when the data set satisfy the assumptions of parametric tests; while friedman test followed by conover’s post hoc test was used for the data sets that are non-parametric. statistical analyses were done using spss 15 and statsdirect 2.6.5. results and discussion the test organism, c. violaceum, produces a violetcolored substance, violacein, which has been identified to have antibiotic properties. mcclean et al. (1997) have shown that the production of this antibiotic is regulated by qs following the luxi-luxr circuit. this became the basis for the development of a simple procedure of screening for qs inhibitors using the decrease in violet pigmentation of c. violaceum as indicator of qs inhibition (mclean et al., 2004). but since any reduction in violacein production could also be a result of reduction in bacterial cell density, another criterion has to be met in order to consider the violacein-inhibiting substance as a qs inhibitor. methods should be done to check the growth or cell density of the test organism. if the violacein-inhibiting substance causes a corresponding decrease in cell density, then it is not to be considered as a qs inhibitor. an effective and pharmacologically important qs inhibition should be able to considerably reduce the expression or production of a qs-regulated phenotype without significantly affecting the population density of the treated bacteria. the results for the violacein inhibition assay based on violacein’s absorbance at 570 nm revealed that its production is negatively affected by increasing concentration of egcg in the medium (figures 1 & 2). anova and duncan’s post hoc test for this data set have shown that violacein production has started to significantly decrease at egcg concentration of 250 µg ml-1 and that pigment production is significantly decreasing from this point when egcg concentration is further increased. it is quite interesting that at concentration lower than 125 µg ml-1, violacein production is enhanced, which correlates with an increase in cell density of the non-pigmented c. violaceum at this range of concentration. this could be due to the presence of other components (e.g. impurities in the teavigo® preparation of egcg) that may have been utilized by c. violaceum as a carbon or nitrogen source. this may also have been elicited egcg is a potential quorum sensing inhibitor 27 by egcg itself, which the test organism may have construed to be a chemical offensive from a possible competitor microbial population. the data on effects of egcg on growth (see figure 3) have shown that there was a general increase in c. violaceum cv026 cell density in the presence of increasing concentration of egcg. friedman test and conover’s post hoc test have confirmed that from the lowest (15.625 µg ml-1) to the highest egcg concentration (1000 µg ml-1), the growth of the test organism has gone significantly higher than that of the untreated. the slower growth of the test organism at concentrations between 15.625 and 62.5 µg ml-1 then a sudden increase in growth from 125 to 250 µg ml-1 can be thought to be a result of interactions between various components in the teavigo® preparation, which may contain components other than egcg. otherwise, this could be due to changes in binding preference of egcg to different targets at different levels or concentrations. it may, for instance, bind or react to certain metabolites at a particular range of concentration that may adversely affect the growth of the organism but may preferentially bind other targets at higher concentrations. however, what is worth noting is that growth is generally enhanced by egcg at a figure 2. graph showing the relationship between epigallocatechin gallate (egcg) concentration and the amount of violacein produced by treated c. violaceum jcm1249. figure 1. photograph showing the increasing production of violacein by c. violaceum at decreasing concentrations of egcg. (colored image appears online.) science diliman 20:1, 24-30 taganna and rivera 28 concentration range when violacein production is significantly reduced starting at around 250 µg ml-1. while a previous work by huber et al. (2003) already found that egcg antagonizes qs in two recombinant reporter strains – escherichia coli mt102 (psb403) and pseudomonas putida (pkr-c12), the present work proceeded to test the anti-qs activity of this compound on another test organism, c. violaceum, that normally expresses an easily observable and quantifiable qscontrolled phenotype. furthermore, the teavigo® powder is a food grade egcg preparation, which has less purity than the analytical grade egcg used by the previous report; and yet, it is still qs inhibitory at a higher concentration but within the working formulations of many teavigo®-supplemented food products. therefore, the findings of the present work strongly indicate that egcg derived from c. sinensis l. inhibits qs in c. violaceum, which strengthens a previous report that it can be a potential qs inhibitor in other gram-negative bacteria and can be further studied and developed for its use as an anti-pathogenic but nontoxic drug. other plants, especially those that are used in traditional medicine, can be screened for qs inhibitory compounds. screening, isolation and purification of these types of compounds from philippine medicinal plants are currently in progress, of which this study is a preliminary work for assay optimization. acknowledgements we thank the dsm nutritional products, philippines and tubu food manufacturing corporation for providing a few grams of teavigo® for this study; and ms. catherine espinosa for her assistance in the statistical analyses. we are also grateful for the financial support of the u.p. institute of biology and the philippine council for advanced science and technology research and development (pcastrd). references atkinson, s., c.y. chang, r.s. sockett, m. camara, & p. williams, 2006. quorum sensing in yersinia enterocolitica controls swimming and swarming motility. j. bacteriol. 188:1451-1461. bainton, n.j., b.w. bycroft, s.r. chhabra, p. stead, l. gledhill, p. hill, c.e.d. rees, m.k. winson, g.p.c. 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ison*, e.s. estacio, m.f. bailon, and a.a. salvador condensed matter physics laboratory, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines e-mail: cison@nip.upd.edu.ph abstract room temperature photocurrent spectroscopy is performed on an mbe-grown gaas/algaas mqw p-i-n device. an observed shift to longer wavelengths is seen with increasing reverse bias voltages. this behavior is explained through a mechanism called the quantum-confined stark effect. applied electric fields are estimated using second-order correction for infinite quantum wells. the estimated built-in electric field is 20 kv/cm corresponding to a 9-mev shift from the flatband energy transition. an observed shift to shorter wavelengths is seen under an optically applied field for both biased and unbiased conditions. introduction gaas-based devices have recently found widespread use in modern telecommunications. being a direct band-gap semiconductor, gaas boasts of efficient and fast carrier recombination rates (sze, 1969). the incorporation of an undoped multiple quantum well (mqw) region between the conventional p-n junction has allowed for the further improvement of the absorption and transit time of carriers. in addition, the absorption of these p-i-n devices peaks abruptly at the energy levels of the mqw. thus, the region of operation of a p-i-n device may be specified by appropriately choosing the well width of the active mqw region (singh, 1993). this property of quantum well-based devices provides many important and useful benefits and effects for optoelectronic applications. the application of an electric field perpendicular to the plane of the qw influences its properties. among the prominent effects observed is the quantumconfined stark effect (qcse). at flat band condition (zero-field), a qw is simply treated as a quantum mechanical “particle-in-a-box”, wherein the electron and hole have symmetric sinusoidal wavefunctions from which the energy of inter-subband transitions for the excitons (e-h pairs) can be easily obtained. with electric field, band bending occurs, forming a tiltedquantum well, which results in the lowering of energy band transitions, i.e., the electron subband energy level drops and the hole subband energy level rises. also the excitons are “polarized” since the wavefunctions of the electrons and holes are pulled towards opposite directions, as illustrated in fig. 1. as an effect, the band edge absorption is quadratically reduced, broadened, and shifted to lower energy (loehr, 1996; klingshirn, 1997.). photocurrent (pc) spectra are considered to imitate the photoabsorption spectral lineshape, when the photogenerated carriers escape the mqw active region and cross the heterojunction. this involves photoabsorption* corresponding author 89 observation of the quantum-confined stark effect and transport of photo-carriers across the junction yielding the photocurrent (kawasaki et al., 1999). as direct absorption experiments in devices require intricate substrate-etching sample preparations, pc spectroscopy offers the best alternative in studying the band structure of an mqw active region incorporated in a p-i-n structure. this band-gap tailoring capability, together with the exploitation of the qcse, has led to novel devices, such as optically bistable self-electrooptic effect devices (seed)(miller et al., 1986; lentine & miller, 1993). the authors present results on the observation of the qcse in the mqw active region of an mbegrown p-i-n diode. room temperature photocurrent spectroscopy was performed with reverse bias voltage and an optically applied forward bias. experimental method the sample used was grown on an n-type gaas (100) substrate by molecular beam epitaxy. the designed quantum structure consists of 3 periods of 90 å gaas quantum wells and 100 å algaas barriers. this undoped quantum structure is confined by undoped 100 å al 0.3 ga 0.7 as layers. these layers contained in the intrinsic region are sandwiched by nand p-al 0.3 ga 0.7 as cladding layers to form a p-i-n structure. ohmic indium contacts were soldered on a 1.5 mm2 piece of the mbegrown p-i-n structure. fig. 2 shows the current-voltage characteristics of the device. the breakdown voltage is estimated to be 2.0 v. room temperature pc measurements were done using the same experimental setup utilized by ison et al. (2000). the spectra were probed by light from a 100 w tungsten-halogen lamp dispersed by a spex 500m monochromator. the probe beam is mechanically chopped at 200 hz and focused into the sample by suitable optics. the photo-induced current was then fed to an sr510 lock-in amplifier using the chopping frequency as reference. system control and data acquisitions were done by computer. pc spectra were acquired for no reverse bias, 0.5 v, and 1 v reverse bias. in addition, an optically applied forward bias (using an ar+ laser) was employed to counteract band bending due to qcse. results and discussion the flat band energy transitions for the excitons were measured via photoluminescence (pl) of a different multiple quantum well sample. the pl peaks provide the excitonic transitions (1hh-1c and 1lh-1c) for a 90 å gaas/algaas quantum well. these peaks were verified using the effective mass approximation method e = 0 e 0 e g e g ’ 1c 1hh 1c’ 1hh’ (a) (b) fig. 1. the band structure of a quantum well (a) without and (b) with an applied electric field perpendicular to the wells. the interband transition energy decreases as greater field strength is applied. 0.0011 0.0009 0.0007 0.0005 0.0003 0.0001 -0.0001 -0.0003 -0.0005 -6 -5 -4 -3 -2 -1 0 1 2 3 voltage (v) c u rr e n t (a ) fig. 2. current-voltage characteristics of the p-i-n structure. the estimated breakdown voltage is 2.0 v. 90 ison et al. (chang, 1996). the energy position for the first light hole transition (1lh-1c) occurs at 8430 å (1.471 ev) while the first heavy-hole transition (1hh-1c) occurs at 8480 å (1.462 ev). comparing the flat band energy locations of the allowed transitions (pl peaks) with the features observed in the zero-bias pc spectrum in fig. 3, the excitonic energy levels are shifted toward longer wavelengths (red-shift). for the pc spectra, the energy position for the 1lh-1c transition occurs at 8423 å (1.470 ev) while the 1hh-1c transition occurs at 8531 å (1.453 ev). this red shift corresponds to a calculated 20 kv/cm built-in field. the shifting of the excitonic transitions toward lower energies is an evidence of qcse. eq. (1) gives the energy level of a zero-field infinite quantum well. (1) using a second order correction for the infinite quantum well, bastard et al. obtained the relationship of the field (f) with the change in the energy level, as given by eq. (2), (2) where f is the electric field strength, m* is the effective mass, and l is the width of the quantum well. the other features at lower wavelengths correspond to fabry-perot oscillations resulting from the abrupt interface between the mqw and the cladding region (lacap et al., 2000). these equally spaced modes are generally unaffected by the application of an electric field. to investigate further the effects of electric fields on the qw in a p-i-n structure, pc measurements were done at different reverse bias voltages. fig. 4 shows the pc spectra under an applied voltage of -0.5 v and -1.0 v. under reverse bias, the additional electric field causes the tilting of the quantum wells to increase. the observed energy shifts due to band tilting were used to estimate the electric field strengths in the i region. these shifts were taken relative to the flatband condition. table 1 shows the excitonic transitions with their corresponding applied electric field strengths. the first order approximation of the electric field strength uses 2 2 2 2 ; 1, 2, 3,... 2 n n e n m l π ∗= = h 15 2 2 2 33 1 3 2 2 e fl e f e m∗ ⎛ ⎞⎛ ⎞∆ = − + ⎜ ⎟⎜ ⎟ ⎝ ⎠ ⎝ ⎠ h 5100 6600 7100 7600 8100 8600 9100 wavelength (angstrom) p h o to c u rr e n t (a rb it ra ry u n it s ) pl spectrum 1lh-1c 1hh-1c ∆e fig. 3. pl spectra showing the 1hh-1c and 1lh-1c excitonic spectra under flat band conditions and the pc spectra at zero bias. 8100 8200 8300 8400 8500 8600 8700 wavelength (angstrom) p h o to c u rr e n t (a rb it ra ry u n it s ) 0 v 0.5 v fig. 4. pc spectra at zero bias, 0.5 v, and 1 v reverse bias showing shifts to higher wavelengths with increasing reverse bias voltages. the dotted spectrum corresponds to 1 v reverse bias. 91 observation of the quantum-confined stark effect parallel-plate capacitor calculations. the second order correction terms for the infinite quantum well assumption were also taken into account, but did not introduce any significant deviation from the first order approximation. as expected, the increase in the applied field results in longer transition wavelengths (lower energies). it is also apparent in fig. 4 that the intensity of the pc spectra increases for biased samples. this can be explained by the distortion of the electron and hole wavefunctions, wherein an applied field decreases the exciton binding energy, causing an increase in the tunneling efficiency (haug, 1988). as a further note, the “smoothing” of the pc spectra at 1.0 v reversed bias compared to the 0.5 v spectra is a direct consequence of the relaxation of the allowed transitions (band edge) and filling up of the forbidden transitions (lower wavelengths). the electric field essentially does not increase the amount of absorption (miller et al., 1985). the sample was also subjected to an optically applied field, effectively forward biasing the p-i-n junction. this was done by illuminating the sample with an ar+ laser (488 nm). fig. 5 shows the pc spectra under an optically applied field. the estimated incident photon flux is in the order of 1,015 photons/second with an input power of 2 mw. the number of electronhole pairs was determined from the estimated fields; each is in the order of 1,010/cm2. the comparatively lower number of calculated electron-hole pairs is attributed to many factors, among which is the difference in tunneling probabilities of the algaas barriers and the gaas wells, gaas being slightly p-type (unintentionally doped), and due to reflection of the incident photons, by the thick n-type and p-type layers. an observed blue shift to shorter wavelengths is seen for both no bias and reverse biased conditions, with no significant increase in pc intensity. the blue shift in the no bias condition closely approximates the flatband transition seen in pl spectra in fig. 2. illumination of the sample under 0.5 v reverse bias causes the shifted excitonic peaks to revert back to the excitonic peak positions of the unbiased state because the field created by the laser counteracts the field caused by the applied bias. summary of results room temperature pc spectroscopy was performed on an mbe-grown gaas/algaas mqw p-i-n device. an observed shift to longer wavelengths is seen with increasing reverse bias voltages characteristic of the quantum-confined stark effect. applied electric fields were estimated using second-order correction for 0 v with laser 0.5 v with laser 8100 8200 8300 8400 8500 8600 8700 wavelength (angstrom) p h o to c u rr e n t (a rb it ra ry u n it s ) fig. 5. pc spectra under an optically applied field for zero bias and 0.5 v reverse bias. dotted lines correspond to pc spectra without illumination. table 1. excitonic transition energies with estimated electric field calculated from observed energy shifts. field (kv/cm) 1st order 2nd order transition energies (ev) 1hh-1c 1lh-1c energy shift relative to flatband (mev) 1hh-1c 1lh-1cflatband unbiased 0.5 v reverse bias 1.0 v reverse bias 0.0 20.0 31.1 37.8 0.00 20.03 31.15 37.85 1.462 1.453 1.448 1.445 1.471 1.470 1.468 1.465 9 14 17 1 3 6 92 ison et al. infinite quantum wells. table 2 gives a summary of the calculated electric field strengths and transition energy shifts under applied electric and optical fields. the estimated built-in electric field is 20 kv/cm corresponding to a 9-mev shift from the flatband transition. an observed blue shift is seen under an applied optical field for both biased and unbiased conditions, reverting the tilted band edge to a nearly “flatband” condition. acknowledgment the authors would like to thank dost-esep for their continued support in this research. references chang, y., 1996. band structures of iii-v quantum wells and superlattices. in bhattacharya, p. (ed.) properties of iiiv quantum wells and superlattices. inspec. 35-41. haug, h., 1988. optical nonlinearities and instabilities in semiconductors. new york, academic press. ison, c., e. estacio, j. laniog, & a. salvador, 2000. timeresolved photocurrent spectroscopy of an lpe-grown gaas/ algaas heterojunction device. proc. 18th spp physics congress. 85-87. kawasaki, k., et.al., 1999. interplay of excitonic radiative recombination and ionization in photocurrent spectra of thick barrier gaas/alas multiple quantum wells. jpn. j. appl. phys. 38: 2552-2554. klingshirn, c.f., 1997. semiconductor optics. berlin heiddelberg, springer-verlag. 259-263. lacap, n., e. estacio, a. podpod, & a. salvador, 2000. photocurrent spectroscopy of a resonant cavity enhanced photodetector. proc. 18th spp physics congress. 76-78. lentine, a.l. & d.a.b. miller, 1993. evolution of the seed technology: bistable logic gates to optoelectronic smart pixels. ieee. j. quant. electr. 29(2): 655-669. loehr, j.p., 1996. effects of electric fields in quantum wells and superattices. in bhattacharya, p. (ed.) properties of iii-v quantum wells and superlattices. inspec. 71-73. miller, d.a.b., j.s. weiner, & d.s. chemla, 1986. electricfield dependence of linear optical properties in quantum well structures: waveguide, electroabsorption, and sum rules. ieee. j. quant. electr. qe-22(9): 1816-1830. miller, d.a.b., d.s. chemla, t.c. damen, a.c. gosard, w. wiegmann, t.h. wood, & c.a burrus, 1985. electric field dependence of optical absorption near the band-gap of quantum well structures. phys. rev. b. 32(2): 1043. singh, j., 1993. physics of semiconductors and their heterostructures. sze, s.m., 1969. physics of semiconductor devices. john wiley and sons. 57. table 2. summary of transition energies as a function of applied electric and optical fields. estimated field (kv/cm) transition energies (ev) 1hh-1c 1lh-1c 1hh-1c 1lh-1c flatband unbiased 0.5 v reverse bias 1.0 v reverse bias 0.0 20.0 31.1 37.8 1.462 1.453 1.448 1.445 1.471 1.470 1.468 1.465 without applied optical field with applied optical field --1.460 1.453 ----1.482 1.472 --comparison-acabaldo.pmd a comparison of zooplankton assemblages 51science diliman (july-december 2010) 22:2, 51-60 introduction the sulu sea is one of the major regions that contribute to high fisheries production in the philippines. it is a deep marine basin with sills shallower than 200 m, restricting deep water circulation that may lead to anoxic conditions (linsley et al., 1985). the water is fresher and more oxygenated in the northern part of the sulu sea because deep waters are replenished only through mindoro strait, where a deep (420m) channel connects the sulu sea to the south china sea (linsley et al., 1985; quadfasel et al., 1990; jones, 2002). this type of marine habitat exhibits a characteristic faunal composition reflecting its distinctive warm waters in the mesopelagic and deeper layers. the extent of a comparison of zooplankton assemblages in a coastal upwelling and offshore station in east sulu sea cristy s. acabado*, wilfredo l. campos, mary mar p. noblezada and dm g. estremadura oceanbio laboratory, division of biological sciences, college of arts and sciences, university of the philippines visayas, 5023 miagao, iloilo *corresponding author email: cristy.acabado@yahoo.com,telefax: +63-33-3159271 abstract partially enclosed marine basins often display characteristic pelagic faunal compositions reflecting their distinctive hydrographic conditions. seasonal upwelling along the northern coast of the zamboanga peninsula in east sulu sea results to the high fisheries production in the area likely by enhancing local plankton abundance. zooplankton in this area were investigated during the joint philex oceanographic cruise on board the r/v melville in december 2007. data on the abundance and vertical distribution of major groups of zooplankton were analyzed and compared between two sets of mocness samples collected from a coastal upwelling station and the other from an offshore station. eighty four (84) zooplankton taxa belonging to 20 major groups were identified in the coastal station, while ninety five (95) taxa under 28 major groups were found in the offshore station. the total zooplankton density was much higher in the coastal station (98.9 ind./m3) than in the offshore station (47.5 ind./m3). copepods dominated in both stations, comprising up to 70% of the total zooplankton. no distinct variation in the abundance, composition and distribution of zooplankton with depth was observed except in 150 – 200 m depth stratum in the coastal station wherein a dramatic increase in the abundance of copepods, ostracods and chaetognaths was noted. in the offshore station, the crustaceans also showed a drastic increase in abundance in the 50 – 100 m depths. these appear to be related to the thermocline in both stations, where typically higher upper layer primary productivity can support dense communities of zooplankton. keywords: upwelling, thermocline, zooplankton, sulu sea decrease of zooplankton with depth in this semienclosed basin is greater than in the open oceans. this observation suggests that the vertical distribution of zooplankton could be set by the local and unique processes that differ from those in typical open oceans (nishikawa et al., 2007). the environment of the sulu sea is strongly influenced by the monsoons (beaufort et al., 2003). upwelling develops in the leeward side of major islands (mindoro, panay, negros and zamboanga peninsula) along the eastern border of the sulu sea during the northeast monsoon (miki et al., 2008). such processes provide an opportunity to examine the response of zooplankton to increased primary productivity. acabado, c.s. et al. 52 the information on the pelagic fauna in the sulu sea is limited to primary production and chlorophyll distribution (san diego-mcglone et al., 1999; jones, 2002; gomez et al., 2005; miki et al., 2008). the few studies on zooplankton focused on fish larvae (campos and estremadura, 2003), chaetognaths (johnson et al., 2006) and mesozooplankton (nishikawa et al., 2007), but were based on samples collected in oceanic areas of the sulu sea. this study compares vertical patterns in the abundance and distribution of zooplankton in a coastal upwelling and offshore station in eastern sulu sea off the coast of the zamboanga peninsula. materials and methods plankton were collected in the sulu sea during the philippine strait dynamics experiment (philex) cruise on board the r/v melville from 20 november to 17 december 2007. a total of ten (10) stations along a transect extending from the coast of zamboanga peninsula (n8o11.47’, e122o38.16’) to about 206 km offshore were surveyed. a 10-net mocness (multiple opening-closing net and environmental sensing system), with net dimensions of 1m x 1.25m and bags with 335 µm mesh size (wiebe et al., 1976), was deployed in two of the stations, namely station 37 along figure 1. location of stations along the zamboanga peninsula transect. legend: – mocness stations compared in this study; o – mocness excluded in this study; + other stations where nutrient and chlorophyll data presented in this study were collected. science diliman (july-december 2010) 22:2, 51-60 • table 1. depth layers where zooplankton were sampled in the sulu sea. net no. depth strata net no. depth strata 1 0 – 400m 6 200 – 150m 2 400 – 350m 7 150 – 100m 3 350 – 300m 8 100 – 50m 4 300 250m 9 50 0m 5 250 200m 10 0m a comparison of zooplankton assemblages 53 the coast (11.5 km out) and station 33 about 72 km from the coast. samples were collected from ten (10) different depth strata at 50m depth intervals from the surface down to a maximum depth of 400 m (table 1). aside from the first net, which was open from the surface to the deepest (400 m) point sampled, all other nets were fished for approximately 15 minutes at their designated strata. at each station, temperature and salinity were measured with a ctd profiler attached to the mocness. station 37 is a potential upwelling area located along the coast of zamboanga peninsula and station 33 is an oligotrophic area located about 72 km from the coast (figure 1). unfortunately, nets 7 to 10 assigned to the depth strata from 150 m to 0m in the coastal station (37) malfunctioned. upon retrieval of the mocness, a double oblique tow (dot) was done using a 60 cm diameter ringnet fitted with a 335 um mesh net to cover the layer from 100 m to the surface. this dot sample was used in place of the missed mocness samples. all samples were fixed in 10% seawater-formalin solution and taken back to the lab for processing, sorting and identification. subsampling was done using a plankton splitter. aliquots ranged from 1/32 to ¼ of the original sample. zooplankton were first identified into major taxonomic groups; then calanoid copepods, the most abundant group, were further identified to family level, to allow a more detailed examination of their vertical distribution. densities were computed as number of individuals per m3. to examine the decrease in abundance with depth, linear regression analysis was done to the log 10 -transformed abundance data. cluster analysis was done on relative abundance (%) data using the bray-curtis index of dissimilarity and flexible (ß = -0.25) sorting strategy to group species and stations. the comm program was used to do the cluster analysis (piepenburg and piatkowski, 1992). data for chlorophyll a and nutrients were provided by the chemical oceanography component of the cruise, which analyzed samples collected along most of the stations along the transect shown earlier, aside from 2 – 3 stations wherein the ctd rosette water sampler malfunctioned. figure 2. vertical profile of temperature in stations 33 and 37. a) 0 – 400m, and b) 0 – 100m. a b science diliman (july-december 2010) 22:2, 51-60 acabado, c.s. et al. 54 results and discussion hydrography the depth and shape of the thermocline is similar in the two stations, where temperature decreased rapidly from 50 – 200 m (figure 2a). temperature decreased more gradually below 200 m and seemed to be nearly constant at 10 oc in deeper layers. this agrees with the vertical profiles reported by quadfasel et al. (1990) and jones (2002) for the sulu sea. figure 2b shows that for the same depth, water in station 37 is slightly cooler down to 75 m (25.5 – 28.0 oc) than water in the same layer in the offshore station (26.5 – 28.3 oc). this is an indication of a weak upwelling near the coast, where vertical mixing results to a decrease in surface temperature by entraining cold water from deeper layers (wang et al., 2006; miki et al., 2008). this pattern changed at depths below 75 m where the deeper water was slightly warmer closer to the coast. overall, the concentration of chlorophyll a was lower in the 200 – 400 m depth layer compared to the layer above it (figure 3). the spatial variation in chlorophyll a detected in the 0 – 200 m layer showed a gradual decrease in concentration from the coast toward the central part of the basin. below 200 m, the concentration of chlorophyll remained the same throughout the transect and was lower than concentrations nearer the surface. the difference in chlorophyll concentrations between surface water and the layer below the thermocline showed a parallel trend across the whole transect, except for the slight decline before the offshore end of the transect. the relatively higher concentration of chlorophyll a near the coast is also indicative of a potential upwelling. nitrate-nitrite concentrations at given depths also showed higher values closer to the coast (jacinto, pers comm.). this is also consistent with the upwelling area near the coast, although not indicative of a strong upwelling. zooplankton abundance the combination of environmental conditions and phytoplankton concentration are some of the main factors controlling the spatial and vertical distribution of the pelagic fauna in the sulu sea. zooplankton concentrations over the water column from 0 – 400 m in station 37 ranged from 15.7 – 373.7 ind./m3, and showed high values up to 200 m (average = 254.3 ind./ m3), before dropping to an average of 21.3 ind./m3 in deeper water. this planktocline corresponds closely to the highly stratified water column. linear regression did not show a significant decrease in zooplankton abundance with increasing depth in the coastal station figure 3. spatial distribution of chlorophyll a above and below the thermocline (200m) in stations along the sulu sea transect. arrows indicate location of stations 37 (11.5 km) and 33 (72 km) from the coast. science diliman (july-december 2010) 22:2, 51-60 a comparison of zooplankton assemblages 55 (p > 0.05; r2 = 0.50). this is due more to the lack of data points for depths above the thermocline (fig. 4), resulting from the malfunctioning of the mocness, rather than the absence of a trend. the latter of course is reflected by high abundances in near-surface waters and low abundances beneath the thermocline. in both coastal and offshore stations, densities (mean values = 21.3 and 14.3 ind./m3, respectively) in layers below the thermocline (200 – 400 m) were from 6 – 12 times less than those at the surface (254.3 and 80.6 ind./m3, respectively; 0 – 200 m). this is typical of most waters since primary productivity and the resulting food availability is higher in the photic zone. it is only in the offshore station (33) that the decrease is significant (p < 0.05, r2 = 0.70). hence, even with ill-fitting regressions, the decrease in abundance from upper to deeper layers is clear in both stations. furthermore, the change in the coastal station is somewhat steeper as shown by the fitted lines. zooplankton abundance in station 33 ranged from 8.2 – 147.7 ind./m3, with highest values in the 0 – 100 m depth layer. the average density for the upper layer in this station (80.6 ind./m3) is only about 1/3 the average density in the upper layer in the coastal station (table 2). higher average above-thermocline zooplankton density along the coast, as well as deeper occurrences of higher densities (offshore: 0 – 100 m; coast: 0 – 200 m; table 2) and larger vertical density differences in the coastal station are not inconsistent with upwelling conditions in this area. taxonomic composition and vertical distribution there were no differences in the ratio of holoplankton and meroplankton although there were generally more organisms recorded in the coastal station (table 3). since local production of meroplankton by benthic adults is generally higher along the coast (archambault et al., 1998), the lack of a difference in holoplankton – meroplankton ratio might suggest that horizontal mixing extends from the coast to the offshore station. however, there are substantial differences in the composition of the meroplankton between the 2 stations. meroplankton in station 33 were more diverse and included different kinds of worms like platyhelminthes, annelids and sipunculids (table 3). because of the depth in the offshore station, it is possible that the diverse assemblage of meroplankton in this station is the result of mixing with waters other than from the zamboanga coast. it therefore seems that there is little horizontal mixing between coastal waters and those 70 km offshore. this is consistent with the slight differences in chlorophyll a concentrations (figure 3) mentioned above and the differences in the vertical distribution of zooplankton abundance (table 2). among the major groups of zooplankton, copepods were the most abundant, contributing 69.9% on average (69.3 – 70.6%) to the total zooplankton abundance (table 3) with little difference between the two stations. science diliman (july-december 2010) 22:2, 51-60 table 2. total abundance of zooplankton for every depth layer sampled from 0 – 400 m. highest densities for each station are highlighted. stn 33(off) stn 37(coast) depth density % total density % total (m) (ind/m 3 ) (ind/m 3 ) 0-50 147.7 38.9 134.9 22.7 50-100 130.9 34.5 100-150 27.5 7.2 150-200 16.1 4.3 373.7 62.9 200-250 8.2 2.2 15.7 2.6 250-300 24.1 6.3 25 4.2 300-350 13.8 3.6 25.2 4.2 350-400 11.2 2.9 19.3 3.2 total (0 400m) 379.5 100 593.7 100 *% refers to the entire water column sampled acabado, c.s. et al. 56 science diliman (july-december 2010) 22:2, 51-60 other zooplankton taxa contributed less than 9% to the overall zooplankton. ostracods ranked second to copepods in the offshore and coastal stations, contributing 5.7% and 8.7%, respectively. other crustaceans were of minor importance and the remaining taxa had relative abundances of < 5%. this result where copepoda is the dominant taxa is similar to those reported for other marginal tropical seas and coastal areas (vinogradov, 1970). the variability in the vertical distribution of numbers of copepods is subject to almost the same factors as those affecting the changes in the total amount of zooplankton since they make up the majority of the plankton at all depths. copepods were observed to have higher densities in the upper 200 m where other biological factors, such as light intensity and food availability, are most favorable (figure 5). protozoans, chaetognaths, ostracods and mollusks were also recorded mainly in the upper 200 m in both stations, especially in the 150 – 200 m layer near the coast. the vertical pattern of distribution of chaetognaths was similar to the profile reported by johnson et al. (2006), where they are concentrated only in the upper 200 m. mollusks, with the high contribution of gastropods, were also distributed within the thermocline, but with considerable abundance that extends to deeper layers in the coast (figure 5). this may be due to the local reproduction of adult mollusks near the coast (archambault et al., 1998). the oceanographic conditions along the coast may have been generally favorable for upwelling to occur. physico-chemical information was consistent with a weak upwelling, and may explain the elevated densities in the 150 – 200 m layer in the coastal station. cluster analysis showed three clusters of samples (figure 6) grouped on the basis of similarities in zooplankton species assemblages: the deeper waters from 200 – 400 m in the coast, the deeper layer (200 – 400m) in the offshore station, and the thermocline layer up to the surface (0 – 200m) in both stations. the deeper coastal layer was characterized mainly by relatively high abundance of the ubiquitous groups of zooplankton, like the calanidae and heterorhabdidae families of calanoids, oncaeid cyclopoids, ostracods and the pelagic mollusk, hydromyles. calanids and oncaeids are among the more abundant copepod families and they are found in the epipelagic down to the mesopelagic layer, while heterorhabdids are more commonly found in deeper water (boxshall and halsey, 2004). ostracods in tropical waters are abundant in the mesopelagic layer especially in marginal seas and coastal areas of the ocean (vinogradov, 1968). in this study, these zooplankton groups were also present in the thermocline layer but in lower densities. this suggests that these taxa are those that can tolerate the environmental conditions of deeper water in the coastal station. table 3. total abundance (0 – 400 m) of major groups of zooplankton and their relative compositions to the total abundance in the 2 stations in the sulu sea. stn 33 stn37 major groups density % density % (ind./m 3 ) (ind./m 3 ) holoplankton 42.9 92.4 90.5 91.5 copepoda 32.45 70.00 68.52 69.25 ostracoda 2.62 5.66 8.65 8.74 protozoa 2.24 4.83 4.43 4.48 chaetognath 2.05 4.43 4.25 4.30 mysidacea 1.19 2.56 1.35 1.37 urochordata 0.94 2.02 0.22 0.22 euphausiacea 0.69 1.50 2.93 2.96 cnidaria 0.65 1.40 0.18 0.18 hydroida 0.02 0.04 meroplankton 3.5 7.6 8.4 8.5 mollusca 1.17 2.53 4.90 4.95 amphipoda 0.69 1.49 0.69 0.70 decapoda 0.39 0.85 1.56 1.58 fish 0.35 0.75 0.61 0.62 cladocera 0.23 0.50 0.13 0.13 polychaete 0.21 0.46 0.39 0.40 echinodermata 0.13 0.28 0.05 0.05 cirripedia 0.06 0.12 0.01 0.01 isopoda 0.01 0.02 0.02 0.02 tanaidacea 0.001 0.00 0.04 0.04 bryozoa 0.25 0.54 sipunculida 0.001 0.01 platyhelminthes 0.001 0.01 brachiopoda 0.001 0.01 nematoda 0.001 0.001 46.4 100 98.9 100 a comparison of zooplankton assemblages 57 figure 4. linear regressions of log 10 –transformed density data in stations 33 and 37, where x = depth (independent) and y = density (dependent) in the equation. note: open circles and broken line for station 37; filled circles and solid line for station 33. meroplankton groups such as gastropods, pagurids, echinoderms, bivalves and cirripedes, on the other hand were more abundant in the thermocline layer. low to moderate frequencies of these meroplankton characterize the more productive surface layer especially with their high abundance in the 0 – 50 m layer offshore (table 2). a larger group of zooplankton (including scolecitrichidae, corycaeidae, forams and mysids) was recorded in both the thermocline and the deep offshore clusters, but in somewhat higher abundances in the former. conclusions the abundance values presented in this study are lower compared to other published data for the sulu sea or for other internal water basins in the philippines (table table 4. zooplankton densities reported for the internal waters of the philippines. note: dot – double oblique tow; moc – mocness; ht – horizontal tow; vt – vertical tow. 4). nishikawa et al. (2007) reported much higher densities (20 – 800 ind./m3) for zooplankton in the central portion of the sulu sea, but showed peaks in the upper (< 200 m) layer similar to what was observed in the offshore and coastal stations in this study. a mocness of the same dimensions and manufacturer was used by nishikawa et al. (2007). seasonal differences might also be discounted because nishikawa’s work was also done in november. since it is not related to gear or to season, the difference might be related to the location of the stations surveyed in this study. high ambient nutrient concentrations are not always observed in upwelling areas (primavera et al. 2002). upwelling generally leads to high nutrient concentrations in the surface, but high nutrient concentrations are consumed by high phytoplankton abundance. chlorophyll information (figure 3) showed higher concentrations along the coast and lower away from the coast. if there were upwelling, phytoplankton abundance could have been high and nutrients would have been used up quickly, resulting in relatively low concentrations. under these circumstances, high zooplankton concentrations would be expected. however, if planktivores such as sardines, are also numerous in the area, predation rates may also be high such that ambient zooplankton concentrations are much reduced due to fast cropping rates. hence, low zooplankton abundance alone need not indicate low productivity. put in another way, high zooplankton abundance need not always be the case in productive areas. thus, low abundance in this transect suggests that conditions in the study area are different from other locations in the sulu sea. science diliman (july-december 2010) 22:2, 51-60 zooplankton reference gear type density (ind./m 3 ) central philippines campos et al. (2002) vt (200um) 3012 tawi-tawi campos (2004) ht (335um) 244 surigao campos (2004) ht (335um) 477 calamianes campos (2004) ht (335um) 364 sulu sea nishikawa et al. (2007) moc (335um) 10 – 800 sulu sea this study dot (335) 8 – 373 acabado, c.s. et al. 58 science diliman (july-december 2010) 22:2, 51-60 figure 5. vertical distribution of abundances of dominant zooplankton near the coast and offshore. figure 6. clusters of samples showing similar assemblages of major zooplankton taxa. samples form three groups: offshore deep, upper layer in both stations and the coastal deep. a comparison of zooplankton assemblages 59 station 37 is located in a known upwelling area. satellite images of sea surface color and temperature (pullen et al., 2008) during the transect survey period, however, did not show elevated surface chlorophyll concentrations in this vicinity. on the other hand, the distribution of surface chlorophyll a concentrations along the transect showing somewhat higher values close to the coast is indicative of at least weak upwelling. high zooplankton concentration extending to deeper strata, along with a more abundant, but less diverse meroplankton assemblage closer to the coast are indicative of different processes influencing the above-thermocline layers along the coast. these results are not inconsistent with weak upwelling in the area. the results of the study suggest that vertical mixing is more extensive close to the coast (consistent with upwelling area) and that this does not extend far (~70 km) from the coast, at least under conditions similar to what occurred during the present survey. there seems to be little (horizontal) mixing between the northwest coast of zamboanga peninsula and offshore waters, although this may differ under strong upwelling conditions. oceanographic conditions near the coast may be conducive for upwelling that becomes intensified when all conditions are favorable. there is still a need to examine temporal variability (e.g., within seasons) in the intensity of upwelling and the different conditions leading to such. acknowledgments the authors are grateful to dr. cesar villanoy and dr. gil jacinto of the physical oceanography and chemical oceanography teams during the philex oceanographic cruise for providing the data and information on chlorophyll a and nutrient concentrations. we also thank the chief scientist, dr. pierre flament (university of hawaii), and all the participants in the cruise, as well as the crew of the r/v melville. the philex joint cruise was funded by the u.s office of naval research. travel funds for wlc, mmp and dme to join the cruise were provided by a research grant from conservation international inc., philippines for an investigation of the distribution and dispersal of fish larvae in the sulu sea. science diliman (july-december 2010) 22:2, 51-60 references archambault, p., j.c. roff, e. bourget, b. bang, and g.r. ingram. 1998. nearshore abundance of zooplankton in relation to shoreline configuration and mechanisms involved. journal of plankton research 20: 671 – 690. beaufort, l., t. degaridel-thoron, b. linsley, d. oppo, n. buchet. 2003. biomass burning and oceanic primary production estimates in the sulu sea area over the last 380 kyr and the east asian monsoon dynamics. marine geology 201: 53 – 65. boxshall, g. and s. halsey. 2004. an introduction to copepod diversity. the ray society. campos, w.l. & d.g. estremadura. 2003. a comparison of fish larval assemblages in the sulu sea and south china sea. in: proceeding of the first joint seminar on coastal oceanography, chulalongkorn university, bangkok: 68 – 74. chu, p.c., q. liu, y. jia, c. fan. 2002. evidence of a barrier layer in the sulu and celebes seas. american meterological society: 3299 – 3309. gomez, f., k. furuya, s. takeda. 2005. distribution of the cyanobacterium richellia intracellularis as an epiphyte of the diatom chaetoceros compressus in the western pacific ocean. journal of plankton research. 27 (4): 323 – 330. johnson, t.b., j. nishikawa, m. terazaki. 2006. community structure and vertical distribution of chaetognaths in the celebes and the sulu seas. coastal marine science 30: 360 – 372. jones, i.s.f. 2002. primary production in the sulu sea. proceedings of the indian academy of sciences. (earth and planetary science) 111: 209 – 213. linsley, b.k., r.c. thunnel, c. morgan, d.f. williams. 1985. oxygen minimum expansion in the sulu sea, western equatorial pacific, during the last glacial low stand of sea level. marine micropaleontology 9: 395 – 418. miki, m., n. ramaiah, s. takeda, k. furuya. 2008. phytoplankton dynamics associated with the monsoon in acabado, c.s. et al. 60 the sulu sea as revealed by pigment signature. journal of oceanography 64: 663 – 673. nishikawa, j., h. matsuura, l.v. castillo, w.l. campos, s. nishida. 2007. biomass, vertical distribution and community structure of mesozooplankton in the sulu sea and its adjacent waters. deep-sea research ii 54: 114 – 130. piepenburg, d. and u. piatkowski. 1992. a program for computer-based analyses of ecological field data. cabios 8 (6): 587 – 590. primavera, k., m.l. san diego-mcglone and g. jacinto. 2002. chemical hydrography and primary production on the bicol shelf and the pacific seaboard (east of the philippines). upv j. nat. sci. 7 (1-2): 32 – 41. pullen, j., j. doyle, p. may, c. chavanne, p. flament, r. arnone. 2008. monsoon surges trigger oceanic eddy formation and propagation in the lee of the philippine islands. geophys. res. lett. 35, l07604. doi:10.1029/ 2007gl033109. science diliman (july-december 2010) 22:2, 51-60 quadfasel, d., h. kudrass, a. frische. 1990. deep-water renewal by turbidity currents in the sulu sea. nature 348: 320 – 322. san diego-mcglone, m.l., g.s. jacinto, v.c. dupra, i.s. narcise, d.o. padayao, i.b. velasquez. 1999. a comparison of nutrient characteristics and primary productivity in the sulu sea and south china sea. acta oceanographica taiwanica 37: 219 – 229. vinogradov, m.e. 1970. vertical distribution of the oceanic zooplankton. israel program for scientific translations ltd. pp. 186 – 187. wang, j., qi, y., jones i. 2006. an analysis of the characteristics of chlorophyll in the sulu sea. journal of marine systems 59 (1-2): 111 – 119. wiebe, p.h., k.h. burt, s.h. boyd, a.w. morton. 1976. multiple opening-closing net and environmental sensing system for sampling zooplankton. journal of marine research 34: 313 – 326. ocr document tulungan: a consensus-independent reputation system 17science diliman (july-december 2011) 23:2, 17-39 tulungan: a consensus-independent reputation system for collaborative web filtering systems alexis v. pantola*1, susan pancho-festin2, florante salvador1 1de la salle university, 2401 taft avenue, manila, philippines telephone: +632-5264247 2university of the philippines, diliman, quezon city, metro manila, philippines *corresponding author: pong.pantola@delasalle.ph abstract web filtering systems allow or prohibit access to websites based on categories (e.g., pornography, violence, sports, etc.). categorization of websites can be done automatically or manually. automatic categorization is prone to underand over-blocking. on the other hand, manual approach is typically performed by a limited number of people making it not scalable. collaborative web filtering systems, a variation of manual categorization, allow anyone to categorize websites in order to determine which domain these sites belong (e.g., pornography, violence, sports, etc.). this attempts to solve the scalability issue of the typical manual method. the approach offered by collaborative web filtering relies heavily on the contribution of users in order to make the system scalable and less prone to errors. however, its success is greatly dependent on user cooperation. to promote cooperation, reputation system can be used in web filtering. a previous study called rater-rating promotes cooperation and explores the use of a user-driven reputation system that measures both the contributor and rater reputation of users of a collaborative web system. however, rater-rating is consensus dependent. if the number of malicious users are more than their good counterparts, the reputation system can be defeated. in other words, the system can mistakenly give malicious users a high reputation value. this study discusses a reputation system called tulungan that is consensus-independent. it can detect the presence of malicious users even if the number of their good counterparts are fewer. a simulation result that compares the effectiveness of tulungan relative to rater-rating is presented in this paper. the simulation shows that tulungan is still effective even with 25% good users while rater-rating requires at least 50% good users to be effective. keywords: reputation system web filtering pantola, a.v., et al 18 science diliman (july-december 2011) 23:2, 17-39 introduction collaborative web systems such as wikipedia (wikipedia, the free encyclopedia [online] , 2001) and untangle (untangle multi-functional firewall software open source content filter and spam filter [online] , n.d.) allow anyone to contribute information. such set-up allows these systems to grow since contribution can come from different parts of the world (ebner & zechner, 2006). however, open collaboration also is prone to issues such as low credibility and inaccuracy of contents. these can be caused by malicious contributors who vandalize contents in such systems (chesney, 2006). in some cases, “lazy” contributors, as opposed to malicious ones, may haphazardly post entries without verifying their correctness. there are also deviant contributors, whose intentions are good, however, their view deviates from other users such that their contributions are generally considered as incorrect. because of such problems, reputation systems are developed to measure the credibility of contents and/ or contributors. a reputation system is composed of several interacting modules that address problems such as existence of bad contributors and contributions. to solve such problems, algorithms on distinguishing good and bad contributors and contributions are developed and implemented in these modules. these algorithms utilize information gathered from transactions in a web system to assess contributors and contributions (kennes & schiff, 2003). transactions can be in the form of purchase or sale in e-c ommerce sites or cont ribution in collaborative systems. this paper presents a reputation system called tulungan that can detect the presence of malicious users even if the number of their good counterpart are fewer. a simulation result that compares the effectiveness of tulungan relative to rater-rating is presented in this paper. aside from this, the simulation also determines the minimum percentage of good users needed in order to ensure that the system can differentiate good from malicious users. review of related work reputation systems can be classified into two: (a) content-driven and (b) user-driven. 2.1 content-driven reputation system content-driven reputation system relies on the content to determine the accuracy of information. as an example, wikitrust (adler & alfaro, 2007; adler, chatterjee, et al., 2008; adler, alfaro, pye, & raman, 2008) relies on the contents of wikipedia pages to determine their accuracy. the stability of a content is equated to its credibility. it assumes that the less frequent an entry of a wikipedia page is changed, the more credible it is since reviewers find it as already accurate and does not require any correction. however, such an assumption may lead to a wrong conclusion since a “non-edit” to an entry does not necessarily imply that the said entry is correct. there are cases when authors are “lazy” in reviewing and editing an entire article and focus only in entries that interest them thereby leaving other entries unedited. for example, a biography entry in wikipedia is proven inaccurate even if it is not edited for 132 days (cross, 2006). if the credibility of this entry is measured using wikitrust, it is possible that it will be incorrectly labelled as accurate. 2.2 user-driven reputation system user-driven reputation system relies on user rating to measure the accuracy of contents. for instance, users of reddit can provide contents and these are assessed by other users by rating them as good or junk (reddit.com: help [online] , n.d.). it solves the issue of its content-driven counterpart by not relying on the frequency of edits to determine if submitted contents are accurate. its accuracy is measured based on the rating of users. in order to make user-driven reputation system successful, cooperation among raters is needed. without cooperation, raters may not provide any feedback (steiner, 2003) or if ever they provide one, tulungan: a consensus-independent reputation system science diliman (july-december 2011) 23:2, 17-39 19 it is biased or negatively influenced by external factors. in other cases, the users may even manipulate the system to improve their own reputation or degrade the reputation of others. encouraging raters to provide honest rating can be a challenge as seen in websites that utilize such system. in ebay, some raters refuse to give negative ratings for fear of retaliation. such incorrect rating has negative effects to users of collaborative sites (yao, fang, dineen, & yao, 2009). there are even cases when users resort to self-rpomotion by resorting to sybil attack (douceur, 2002). sellers in ebay can create alter egos or phantom user accounts and use these to give a positive rating to their original account (zittrain, 2008; some ebay users abuse auction site’s feedback system, professor finds [online], 2007). aside from self-promotion, users may resort to other form of attacks such as whitewashing, slandering, orchestrated, and denial of service (hoffman, zage, & nita-rotaru, 2009). there are reputation systems that encourage cooperation1. however, they are designed to work in mobile ad-hoc networks (manets) (ietf manet working group. mobi le ad hoc networks (manet). workinggroup charter [online], n.d.) and not in collaborative web systems. a manet is a collection of mobile devices or nodes that communicate with each other through wireless technology. since wireless communication has a finite and limited range, nodes in a manet need to rely on each other to forward and route messages. several routing algorithms such as dynamic source routing (johnson, maltz, & broch, 2001) and ad-hoc on demand distance vector routing (perkins & royer, 1999) allow mobile nodes to determine the path a message should pass through in order for it to be routed from one node to another. these routing algorithms assume that nodes are cooperative and are expected to forward messages in behalf of other nodes (trivedi et al., 2009). however, due to resource constraints such as battery life, nodes tend to be selfish and refuse to forward messages in order to limit battery consumption. there are also cases where nodes are programmed to be malicious and cause problems in a manet. due to selfish and malicious nodes, several reputation systems were introduced to mitigate the effects of such nodes (marti, giuli, lai, & baker, 2000; buchegger & le boudec, 2002a, 2002b; michiardi & molva, 2002; bansal & baker, 2003; hu & burmester, 2006). in a system proposed by (marti et al., 2000), misbehaving nodes are mitigated through the use of two components, a watchdog and a pathrater. the watchdog is used to identify misbehaving nodes. this is accomplished by checking which nodes do not forward packets. once misbehaving nodes are determined, the pathrater uses this information to know which nodes are more reliable in forwarding packets. the pathrater prevents messages from not being forwarded by avoiding a path that includes misbehaving nodes. however, such system may ironically encourage misbehavior of nodes since a misbehaving node will be off-loaded from forwarding messages. as a result, its battery life can be conserved. a modification of this system, called confidant, was presented in (buchegger & le boudec, 2002a) and its result shown in (buchegger & le boudec, 2002b). confidant implements a concept called neighbor watch. in this scheme, neighboring nodes monitor and detect malicious activities. this increases the chance of all legitimate nodes detecting bad behavior since neighbor watch allows multiple nodes to report malicious activities. aside from this, it punishes bad behavior by not providing services to misbehaving nodes. this solves the issue presented by (marti et al., 2000). two systems core (michiardi & molva, 2002) and ocean (bansal & baker, 2003) provide a similar scheme presented by (marti et al., 2000; buchegger & le boudec, 2002a). in addition, they addressed problems concerning reporting false misbehavior which can result to denial of service. core accomplishes this by ignoring negative reports and focusing only on positive ones. on the other hand, 1in rep u t at i on syst ems, en cou ra gi n g c oop era t ion c a n b e accomplished by making misbehavior unattractive or by providing incentive for good behavior (buchegger & le boudec, 2002b). pantola, a.v., et al 20 science diliman (july-december 2011) 23:2, 17-39 second-hand information regardless if the report is negative or positive. another reputation system called lars (hu & burmester, 2006) addresses the same concern solved by core and ocean. however, lars does not ignore negative reports. reports on misbehavior are still processed in this scheme as long as they are reported by a significant number of nodes.this reduces the chance that a well-behaved node will be denied service. several concepts applied in manet reputation systems can be applied in collaborative web systems (pantola, pancho-festin, & salvador, 2010). users of collaborative web systems can be given an additional task of reviewing and rating the work of contributors in order to improve the accuracy of the contributions. however, such extra work may not be attractive to “lazy” users who tend not to review the contribution of others. systems applied to mobile nodes to encourage message passing can also be applied to users to encourage them to review and rate. collaborative web systems may require users to review the work of others. however, there is no assurance that they will do it properly. the concept of watchdog in the system of (marti et al., 2000) can be applied in collaborative systems in order to detect raters who “misbehave” or those who do not review and rate properly. once misbehaving users are detected, there should be a corresponding penalty similar to confidant. one way to implement this is to compute not only the reputation of users based on the quality of their contribution but also consider their reputation when rating the contribution of others. bad raters can be penalized by not requiring other users to review their work. an unreviewed work should be given a low rating and thus the reputation of the author of a substandard work is also negatively affected. however, caution must be exercised when implementing such approach since other users of such collaborative web systems may be ultima tely affected if many contributions remain unreviewed. the implementation of the reputation system must also consider denial of service. the rating made by a good rater may be manipulated by misbehaving ones in order to bring down the reputation of the former. core, ocean, and lars can be adopted to address such concern. as mentioned above, concepts behind these reputation systems for manets can be adopted by collaborative web systems such as community-based web filtering. web filtering is the evaluation of web resources in relation to a set of parameters. such evaluation can be used to regulate access to web contents (bertino, ferrari, & perego, 2010) such as pornography, violence, and racism. (bertino, ferrari, perego, & zarri, 2005) presented an integrated approach to rating and filtering web content. it combines existing approaches (i.e. list-based and metadata-based) with content labelling. this mitigates problems of previous systems that enforce a restrictive and often ineffective filtering. such ineffectiveness results to under and/ or over-blocking of web access (e.g., pornographic and gynecology sites are considered as having similar category). however, these list-based and metadatabased filtering approaches have deficiencies and are inadequate (noll & meinel, 2006). these methods do not scale with the rapid growth of the internet. manually updating a list or metadata cannot catch up with the increasing number of new websites. in 2005, there were approximately 63 million active websites, with an average increase of 1.2 million sites per month (noll & meinel, 2005). a survey conducted by netcraft (netcraft internet research, antiphishing and pci security services [online], n.d.) in november, 2010 saw an increase on the number from 63 to 100 million active websites. there are algorithms available that can potentially automate the process of list-based and metadatabased approaches. however, such algorithms face particular challenges. aside from difficulty in extracting information from web documents containing different types of data (e.g., images, videos, java applets, or flash animation), these algorithms depend heavily on the quantity and quality of training input tulungan: a consensus-independent reputation system 21science diliman (july-december 2011) 23:2, 17-39 (noll & meinel, 2006). with the current number of active websites, the training phase would either be inadequate or take too long. due to these challenges, social or collaborative web filtering becomes a practical alternative. this approach empowers the end users (i.e., actual recipients of web content) to categorize websites (noll & meinel, 2006). this is a form of crowdsourcing that utilizes the internet (following the crowd [online], 2008). crowdsourcing (howe, 2006), is the act of taking a job traditionally performed by a designated agent (usually an employee) and outsourcing it to an undefined, generally large group of people in the form of an open call. this large group can be the community of internet users that can collaborate in performing web filtering. taggybear (noll & meinel, 2008) is an example of a scalable collaborative web filter. it allows end users to collaborately categorize web pages and mark them as objectionable (e.g. pornographic content, violence, racism) or dangerous (e.g. phishing, malware). it uses several types of information like user and community2. the user information provides the category of a particular website based on the perception of a user. an aggregate of the categories made by several users is known as “community information”. as an example, a website with a community information of porn:13:68 means that 13 out of 68 users categorized the website as a pornographic site. other established websites such as opendns (opendns solutions business/enterprise web content filtering [online] , 2008) and untangle (untangle multi-functional firewall software open source content filter and spam filter [online], n.d.) apply a similar concept of categorizing other websites through the help of end users. opendns started as a dns service in 2006 and eventually widened its function by including collaborative web filtering. registered users can categorize websites (e.g., gambling, video sharing, social networking). other users can vote on the accuracy of the category made (broersma, 2008). unlike taggbear, the web filtering service offered by opendns is cloud-based and is meant for enterprise deployment (a radically simpler approach to web content filtering and security [white paper], n.d.). untangle also offers web filtering service. using its web filter submission tool, registered users can contribute by categorizing different web urls (untangle webfilter technical specifications [online], n.d.). however, unlike taggybear and opendns, contributions made by the users are verified and approved by the technical support of untangle (untangle web filter submission tool [online], n.d.) that act as community or content managers. these managers are responsible for sustaining and nurturing the contributions (gray, 2010). this makes its web filtering less scalable since the verification process is done by a relatively fewer number of people. the three collaborative web filtering systems enable registered users to categorize websites. however, taggybear relies on the consensus of the majority to determine the accuracy of a category. on the other hand, opendns depends on a voting mechanism to measure the accuracy of categories. this presents a new problem since the accuracy of the voting mechanism itself is not measured. untangle solves the accuracy issue by relying on its technical support. but as mentioned, such approach is not scalable. to solve issues regarding the accuracy of categories, (ravikumar, mcafee, & tomkins, 2009) use the same strategy offered by opendns in their web filtering system. however, they proposed to solve the deficiency of opendns (i.e., accuracy of the votes) by having a system that rates the raters. reputation systems in manets discussed earlier can be used as a basis in developing such a strategy. websites categorized by registered users are treated as contribution and votes made by others are treated as 2the term used in taggybear is actually types of rating and not types of information. however, in order not to confuse with the rating that this study is focusing on, the word information is used instead. pantola, a.v., et al 22 science diliman (july-december 2011) 23:2, 17-39 ratings. by adopting reputation systems in manet, the contributors as well as the raters are encouraged to cooperate. rater-rating, a reputation system for a collaborative web-based system, at tempts to encourage cooperation by adopting concepts in manet (pantola et al., 2010). it explores the use of a user-driven reputation system that measures both the contributor and rater reputation of users of a collaborative web system. however, rater-rating is consensus dependent. the effectiveness of the system relies heavily/primarily on the opinion of the majority. if the number of malicious users are more than their good counterparts, the rep utation system can be manipulated. in other words, the system can mistakenly give malicious users a high reputation value relative to the reputation of good users. the tulungan algorithm this section discusses the algorithm tulungan. the discussion is divided into four sections: overview, notation, tuple definition, and actual algorithm. 3.1 overview the algorithm considers several entities such as urls, categories, users, contributions, and ratings. a url, w represents a webpage or website (e.g., www.nba.com). each url has a set of categories. a category a specifies a category that a url can have such as pornographic site, violent site, search engine, sports site, etc. a category for a particular url has three possible levels: positive (+) negative (-), and unknown (0). as an example, if the url www.nba.com has a category of sports site with a level α of positive, it means that www.nba.com is categorized as a sports site. in the same way, if it has a category of pornographic with a level of negative, it means that www.nba.com is not a pornographic site. there are two types of urls used in the algorithm: control and unverified. the control urls are those urls whose category levels are known already (e.g. α is positive or negative). unverified urls have category levels that are unknown (e.g., α is unknown). a user u provides contribution c and rating r in the algorithm. the contribution provided by a user is in the form of a url, category, and answer. the answer represents the level of the category that the user perceives that the url possesses. as an example, if a user contributes the following: www.nba.com (url), violent site (category), and negative (answer), it means that the user perceives www.nba.com as a non-violent site. the rating provided by a user is also in the form of a url, category, and answer. however, unlike a contribution, the answer in a rating signifies if a rater agrees or disagrees with the contribution. as an example, if a user rates the following: www.nba.com (url), violent site (category), and negative (answer), it means that the user disagrees that www.nba.com is a non-violent site. ratings performed by users are grouped into three urls. two of the urls are controls while the third one is unverified. the purpose of such grouping is discussed in the succeeding sections. 3.2 notation the different entities (e.g., user and contribution) used in the algorithm are tuples. a tuple is denoted by an italicized small letter (e.g., u and c). a set of tuples is denoted by a bold capital letter (e.g., u and c). an element of a tuple is referred by specifying the symbol of the tuple, followed by a dot (.), and followed by the symbol of the element. as an example, to refer to the contributor reputation (ρc )of the user u, the following notation is used: u.ρc. unlike elements which are preceded by the symbol of the tuple, constants are not preceded by any symbol. as an example ρcmax, which is the maximum user contributor reputation, is denoted without the symbol u. tulungan: a consensus-independent reputation system 23science diliman (july-december 2011) 23:2, 17-39 functions are denoted by characters followed by parameters enclosed in parentheses (e.g., cs(s, α, ti, tf ) and r(u, s, α, ti, tf )). two types of functions are defined in this paper: functions that return a scalar value and functions that return a set. functions that return a scalar value such as cs(s, α, ti, tf) use italicized small letters. functions that return a set use a bold capital letter for its name such as r(u, s, α, ti, tf)). aside from this, the name of the function determines the superset of the set returned by the function. as an example, r(u, s, α, ti, tf )). is a function that returns a set that is a subset of r. 3.3 tuple definition the tuples used in this section are summarized in table 1. the value of the contributor reputation penalty multiplier should satisfy the condition below. (2) this discourages contributors to randomly provide contributions since a randomly made contribution has a 50% chance of being correct. table 2. user-related constants ρcpm < ρcrm 1 name symbol user u url category s contribution c potential rater p rating group g rating r table 1. tuple definition a user u is a tuple defined as u =<ρc, ρr, ρo> (1) where ρc contributor reputation ρr rater reputation ρo overall reputation similarly, the value of the rater reputation lazy penalty multiplier ρrlpm should satisfy the following condition. (3) this makes sure that even if only one of the three urls (refer to the discussion of the rating group tuple) that needs to be rated is incorrect, its corresponding penalty is more than the effect of a single reward. this will discourage malicious raters to intentionally make the rating of two urls correct and the third one incorrect. in addition, the rater reputation incorrect penalty multiplier should be smaller than the rater reputation lazy penalty multiplier (ρripm < ρrlpm) in order to encourage raters to rate. a url category s is a 5-tuple defined as s = < w, a, jc,jr ,α > (4) symbol description ρcrm user contributor reputation reward multiplier ρc p m user contributor reputation penalty multiplier ρci initial user contributor reputation ρcmax maximum user contributor reputation ρcmin minimum user contributor reputation ρrrm user rater reputation reward multiplier ρrlpm user rater reputation lazy penalty multiplier ρripm user rater reputation incorrect penalty multiplier ρri initial user rater reputation ρrmax maximum user rater reputation ρrmin minimum user rater reputation (ρcpm < ρcrm 1 )  table 2 summarizes the user-related constants used in this document. pantola, a.v., et al 24 science diliman (july-december 2011) 23:2, 17-39 where w url a category jc contribution reputation jr rating reputation α level table 3 summarizes the url category-related constants used in this document. table 3. url category-related constants a contribution c is a 4-tuple defined as c =< u, s, α t > (5) where u user who provides the contribution s url category α answer (i.e., +1 means s.w is categorized as s.a, -1 otherwise) t time the contribution was made a potential rater p is a 6-tuple defined as p =< u, wca, wcb, wx , ti, tf > (6) u user who can provide a rating wca 1st url used as control wcb 2nd url used as control wx url with unverified categories ti time user is requested to rate tf expiration time of request a rating group g is a 5-tuple defined as g =< u, wca, wcb, wx , t > (7) symbol description jcat url category contribution reputation absolute threshold jrct url category rating reputation credibility threshold jrdt url category rating reputation difference threshold where u user who provides the rating wca 1st url used as control wcb 2nd url used as control wx url with unverified categories t time rating was made the group rating accuracy threshold â is the only rating group-related constant used in the algorithm. a rating r is a triple defined as r =< g, s, α > (8) where g rating group s url category α answer 3.4 algorithm algorithm 1 enumerates the steps involved in the tulungan reputation system. algorithm 1 tulungan algorithm 1: initialize the contribution reputation j c, rating reputation j r , and level α of all url categories s in s. 2: initialize the contributor reputation ρc and rater reputation ρr of user u and add it in u. 3: allow all users u to add contribution c in c. 4: determine potential raters p and add them in p. 5: allow all users u that are potential raters to add rating group g in g and rating r in r. 6: update the rating reputation j r of all url categories s in s. 7: update the contribution reputation j c of all url categories s in s. 8: update the level α of all url categories s in s. 9: update the contributor reputation ρc of all users u in u. 10: update the rater reputation ρr of all users u in u. 11: update the overall reputation ρo of all users u in u. tulungan: a consensus-independent reputation system 25science diliman (july-december 2011) 23:2, 17-39 1. initialize the contribution reputation j c, rating reputation j r, and level α of all url categories s in s. since the url categories s are not yet determined (i.e., α is unknown), the contribution reputation jc and rating reputation jr of all url categories s in s are initialized to 0. aside from this, the url category level α is also initialized to 0 to denote that the level for the url category is unknown. (9) (10) (11) 2. initialize the contributor reputation ρc and rater reputation ρr of user u and add it in u. unlike the contribution reputation and rating reputation of the url category, the contributor reputation ρc and rater reputation ρr of a new user u are initialized to ρci and ρri u.ρc = ρci (12) u.ρr = ρri (13) once the reputation of user u is initialized, it is added to u. this step is performed everytime a new user registers in the reputation system. 3. allow all users u to add contribution c in c. all users are allowed to contribute a contribution c regardless of his/her contributor and rater reputation. however, the elements c. u and c.s of the contribution c should have values that satisfy the condition c(u, s) = ø. this ensures that a particular user u will only contribute a single contribution that involves a particular url category s. this prevents users from contributing the same contribution. c(u, s) = {c|c.u = u c.s = s} (14) si.α = 0 si w 4. determine potential raters p and add them in p. it is essential that the contributions that will be rated are prioritized such that those contributions with a higher probability of being correct are assigned with enough raters. to have enough raters for contributions involving a particular url wx and time frame ti to tf , the condition below must be satisfied. (15) (16) in order to determine which contributions have higher probability of being correct, the contribution reputation of each contribution can be computed (please refer to step 7 on computing the contribution reputation). however, since computing the contribution reputati on requires that the contributions are rated already, computations that involve information on ratings are ignored. specifically, equations in step 7 that refers to the rating reputation update condition (refer to eq. 18) assumes that z(s) results to a value of true. 5. allow all users u that are potential raters to add rating group g in g and rating r in r. take note that the previous step determines the contributions that a particular user should rate. this prevents a user from choosing a particular contribution to rate. this decreases the chance that a particular user will intentionally and incorrectly rate a particular contribution (e.g., giving a +1 to a wrong contribution just because it is contributed by a friend). aside from this, for each rating group, categories of three urls are rated (i.e., two control urls wca and wcb, and an unknown url wx ). the two control urls, as the name implies, are used only as controls. the url categories of these controls are actually known by the system. since the url categories are already known, the reputation si.jc = 0 si s si.jr = 0 si s p(wx, ti, tf ) = {p|p.wx = wx p.ti = ti’ p.tf = tf } 2αrct  (pi.u.ρr )2 where pi p(wx, ti, tf ) i pantola, a.v., et al 26 science diliman (july-december 2011) 23:2, 17-39 table 4. rating reputation function description the conditions are explained as follows: (i) rjw (s, ti, tf) > jrct this ensures that the rating reputation of the winning rating rαw (s, ti, tf) is credible enough to be considered since it reaches the url category rating reputation credibility threshold jrct. (ii) rjw (s, ti, tf ) rjl (s, ti, tf ) > jrdt this ensures that difference between the rating reputation of the winning rating and the losing system can determine already if the ratings made by a user to the control urls are correct. this aids the reputation system in gauging if a user is performing the rating seriously. if the ratings of the controls are wrong, then the reputation system will assume also that the user’s rating for the unknown url is also wrong. this approach is based from recaptcha, where in users are asked to type the two words (i.e., an unverified word and a control word) that are presented to them (recaptcha stop spam, read books[online] , n.d.). similar to recaptcha, the three urls are presented to the user for rating in a way that the user has no idea which are the controls and whichis the unknown. this prevents malicious raters from making the rating of the two controls correct while intentionally making the rating of the unknown url wrong. 6. update the rating reputation j r of all url categories s in s. a summary of the functions that will be used in computing the rating reputation is presented in table 4. the rating reputation j r of the url category s is computed as follows: rjw (s, ti, tf ) if z(s) s. jr otherwise (17) where ti initial time to consider tf final time to consider the function rjw (s, ti, tf ) computes the winning rating reputation. users provide a set of ratings r on a particular url category s. the set of ratings is divided into two groups: those that are positive ratings (i.e., r.α = +1) and those that are negative ratings (i.e., r.α = “1). the rating reputation of both groups of ratings are computed. the higher (and therefore the winning) rating reputation is returned by the function r j w (s, ti, tf ). s. jr = take note that the function rαw (s, ti, tf ) serves as the value of the rating reputation s. jr only if the conditions in z(s) are satisfied otherwise the current value of s.jr is retained. the function z(s) is defined as z(s) = (rjw (s, ti, tf ) > jrct) (rjw (s, ti, tf ) rjl(s, ti, tf ) > jrdt) (18) (rvw (s, ti, tf ) = +1) name description z(s) rating reputation update condition r α w (s, ti, tf ) winning ratingreputation rα (s, ti, tf ) losing rating reputation rcw (s, ti, tf ) winning rating count rcl (s, ti, tf ) losing rating count rcp(wx , ti, tf ) positive rating count rcn (s, ti, tf ) negative rating count rc(s, α, ti, tf ) rating count v(g) url controls correct condition ra(g, w) rating accuracy rvw (s, ti, tf ) winning rating vote rsw (s, ti, tf ) winning rater reputation summation rsl (s, ti, tf ) losing rater reputation summation rsp(s, ti, tf ) positive rater reputation summation rsn (s, ti, tf ) negative rater reputation summation rs(s, α, ti, tf ) rater reputation summation pantola, a.v., et al 28 (27) science diliman (july-december 2011) 23:2, 17-39 the rating count function simply counts the number of elements returned by the ur (s, α, ti, tf) function. the ur (s, α, ti, tf) function returns a set of users that rated the url category s with an level α. this function is defined as follows: ur (s, α, ti, tf ) = {u|r(u, s, α, ti, tf )  ø} (26) the function r(u, s, α, ti, tf) is utilized by the function above. this function returns all ratings rated by user u, and rated the url category s with an answer α. this function is defined below. r(u, s, α, ti, tf ) = {r|r.g.u = u r.s = s r.α = α r.g.t [ti ..tf ) v(r.g)} the r(u, s, α, ti, tf ) function ensures that only ratings that correctly answered the control urls (i.e., r.g.wca and r.g.wcb) are considered. this is checked by the function v(g) shown below. v(g) = ra(g, g.wca) > β ra(g, g.wcb) > β the function above is based on the rating accuracy ra(g, w) of the two control urls: g.wca and g.wcb. the rating accuracy for both controls are compared to the group rating accuracy threshold β. the rating accuracy ra(g, w) function is indicated below. (28) the function considers the number of correct ratings performed on a particular url w in a rating group g. this can be accomplished by getting the number of elements of the set below. aside from this, it also considers all the ratings (i.e., correct and incorrect) on the same url w in the same rating group g. similarly, this is accomplished by getting the number of elements of the set below. the winning rating vote rvw (s, ti, tf ) determines which group of rating (i.e., positive or negative) has the higher rater reputation summation. the function is defined below. the winning rater reputation rsw (s, ti, tf) returns the rater reputation summation of the winning rating vote. the function is shown below. (31) rall(g, w) = {r|r.g = g r.s.w = w r.s.α 0} rvw (s, ti, tf ) = +1 if rsp (s, ti, tf) > rsn (s, ti, tf) -1 if rsn (s, ti, tf ) > rsp(s, ti, tf) 0 otherwise (32) (33) the losing rater reputation rsl(s, ti, tf ) provides the opposite result of the winning rater reputation. the function is defined as follows: the functions rsp (s, ti, tf) and rsn(s, ti, tf), which are the positive and negative rater reputation summation functions, respectively, use the rater reputation summation function rs(s, α, ti, tf) to compute the summation of the rater reputation of raters that provide positive and negative rating. these functions are defined below. rsp(s, ti, tf ) = rs(s, +1, ti, tf) (35) rsn(s, ti, tf ) = rs(s, -1, ti, tf) (36) (29)ra(g, w) = |rcorrect(g,w)| |rall (g,w)| 0 if |rall(g, w)| > 0 otherwise (30) rcorrect(g, w) = {r|r.g = g r.s.w = w r.s.α  0 r.s.α = r.α} rsw (s, ti, tf) = rsp(s, ti, tf) if rsp (s, ti, tf) > rsn (s, ti, tf) rsn(s, ti, tf) if rsn (s, ti, tf) > rsp (s, ti, tf) 0 otherwise rsl (s, ti, tf) = rsp(s, ti, tf) if rsp (s, ti, tf) < rsn (s, ti, tf) rsn(s, ti, tf) if rsn (s, ti, tf) < rsp (s, ti, tf) 0 otherwise (34) tulungan: a consensus-independent reputation system 29 the rater reputation summation rs(s, α, ti, tf) is defined below. rs(s, α, ti, tf) = (ui.ρr )2 where ui ur (s, α, ti, tf) (37) instead of adding only the rater reputation of the users (ui.ρr), their square ((ui.ρr)2) are added. this makes the effect of low (i.e., reputation less than 1) reputation less significant and in the same way increases the effect of high reputation (i.e., reputation greater than 1). 7. update the contribution reputation jc of all url categories s in s. a summary of the functions that will be used in computing the contribution reputation is presented in table 5. the contribution reputation jc of the url category s is equal to the winning contribution reputation cjw (s, ti, tf). however, two conditions must be satisfied before the winning contribution reputation is used:  i z(s) and the current contribution reputation of the url category s.jc should be less than the url category contribution reputation absolute threshold jcat . s.jc = cjw (s, ti, tf) if s.jc < jcat z(s) s.jc otherwise (38) the winning contribution reputation cjw (s, ti, tf) is defined as follows: where (39) cjw (s, ti, tf) = [log(x)]x y if x > 0 0 otherwise { x = ccw (s, ti, tf) y = csw (s, ti, tf) the equation above uses another function csw(s,ti, tf), which is the winning contributor reputation summation. however, using this as the only basis is not sufficient. a single malicious contributor that intentionally increase its contributor reputation to eventually cause problems in the future can manipulate the value of c j w(s, ti, tf), if csw(s, ti, tf) is the only basis. to ensure that no single user can manipulate the result, the winning contribution reputation function also considers the winning contribution count ccw(s, ti, tf). similar to its rating counterpart, the winning contribution count is used as a parameter in a logarithmic function to ensure two things. first, if the count is less than 10 (e.g., only one contributor), then the effect of the contribution is very small. as mentioned earlier, this prevents few contributors from manipulating the result. second, even if there are many new malicious users (e.g., 20), the count is not enough to provide a significant effect. this makes it harder for malicious users to plan a sybil attack since they still need to increase their contributor reputation before they have a significant effect. the winning contribution count ccw(s, ti, tf) and losing contribution count csl(s, ti, tf) are computed by comparing the positive and the negative contributor reputation summation. the contributor count of the higher contributor reputation summation is returned by the winning contribution count function while the lower one is returned by the losing contribution count function. the two functions are shown below. ccw (s, ti, tf) = ccp(s, ti, tf) if ccp (s, ti, tf ) > ccn(s, ti, tf) ccn (s, ti, tf) if ccn (s, ti, tf) > ccp (s, ti, tf) 0 otherwise (40) ccl (s, ti, tf) = ccp(s, ti, tf) if ccp (s, ti, tf ) < ccn(s, ti, tf) ccn (s, ti, tf) if ccn (s, ti, tf) < ccp (s, ti, tf) 0 otherwise (41) science diliman (july-december 2011) 23:2, 17-39 name description cjw (s, ti, tf) winning contribution reputation ccw (s, ti, tf ) winning contribution count ccl (s, ti, tf ) losing contribution count ccp (s, ti, tf) positive contribution count ccn (s, ti, tf) negative contribution count cc(s, α, ti, tf) contribution count cvw (s, ti, tf) winning contribution vote csw (s, ti, tf) winning contributor reputation summation csl (s, ti, tf) losing contributor reputation summation csp (s, ti, tf) positive contributor reputation summation csn (s, ti, tf) negative contributor reputation summation cs(s, α, ti, tf) contributor reputation summation table 5. contribution reputation function description tulungan: a consensus-independent reputation system 31 i to update the contributor reputation, contributions made by user u are considered. in addition, only contributions whose url category s is classified (i.e., c.s.α  0) are considered. if ci.α matches c.s.α, then the contribution is considered correct and the user is rewarded by increasing its contributor reputation by multiplying it with the user contributor reputation reward multiplier ρcrm. otherwise, it is penalized with the user contributor reputation penalty multiplier ρcpm. to ensure that the contributor reputation is within the range, it is compared with the maximum user contributor reputation ρcmax and the minimum user contributor reputation ρcmin as shown below. u.ρc = max(ρcmin, min(ρcmax, u.ρc)) (55) to get the set of contributions made by a particular user u, the following function is used: c(u, ti, tf) = {c|c.u = u c.t [ti ..tf)} (56) 10.update the rater reputation ρr of all users u in u. the rater reputation ρr of user u is computed as follows: u.ρr = u.ρr rρ(pi) where pi p(u, ti, tf) (57) u.ρr = max(ρrmin, min(ρrmax, u.ρr )) (58) to update the rater reputation, the potential ratings that should be made by user u are considered. these potential ratings may be derived from the subset of p and is defined in the following function: p(u, ti, tf) = {p|p.u = u p.ti [ti ..tf ) p.tf [ti ..tf ) each potential rating is examined if the user did in fact perform the rating. if the potential rating is not found in g, then it means the user failed to rate. the user is penalized by multiplying its current rater reputation with the user rater reputation lazy penalty multiplier ρrlpm. the penalty multiplier is cubed ((ρrlpm)3) since in a single potential rating, three urls need to be rated (i.e., wca, wcb, and wx). if the user successfully rated, its rating is examined. this process is summarized in the two function below. (59) m(g, p) = (gi.wca = p.wca) (gi.wcb = p.wcb) (gi.wx = p.wx) (g.u = p.u) (61) the function rm(g) is used to examine the rating. the three urls involved in the rating are examined separately. science diliman (july-december 2011) 23:2, 17-39 cvw (s, ti, tf) = +1 if csp (s, ti, tf) > csn (s, ti, tf) -1 if csn (s, ti, tf) > csp(s, ti, tf) 0 otherwise (53) 9. update the contributor reputation ρc of all users u in u. the contributor reputation ρc of user u is computed as follows: 1 otherwise ρcrm if ci .α = c.s.α c.s.α = 0 ρcrm if ci .α = c.s.α c.s.α = 0 u.ρc = u.ρc i (54) similar to the contributor reputation, to ensure that the rater reputation is within the range, it is compared with the maximum user rater reputation ρrmax and the minimum user rater reputation ρrmin as shown below. rm(gi) if {g|g g m(g, p)} = ø where gi gm(gi, p) rρ(p) = (ρrlpm)3 otherwise  rm(g) = rmc(g, g.wca)rmc(g, g.wcb)rmx(ri ) i where ri r(g, g.wx) (62) where ci c(u, ti , tf) pantola, a.v., et al 32 evaluation of the reputation system 4.1 evaluation set-up the system is evaluated using a simulation. the different types of users are modelled based on the behaviour they are expected to have. the behaviour of a user is dictated by the type of contributor and rater he or she is. a contributor or a rater can either be good or bad (i.e., lazy, deviant, and malicious). the behaviour of the different types of contributors and raters are discussed below. a good contributor provides correct categorization of urls most of the time. in the simulation, there is only 1 in 5000 chances a good contributor provides a wrong contribution. a lazy contributor randomly chooses whether a url falls under a particular category. as an example, a url that has pornographic content has a 50% chance of being categorized as pornographic. a deviant contributor provides incorrect categorization of urls most of the time. it has a 1 in 5000 chances of providing a correct contribution. a malicious contributor has the same behaviour as the deviant contributor. the behaviour of the different types of raters are modelled similarly to their contributor counterpart. however, instead of providing contribution, the raters rates the contributions made by the contributors. only the malicious rater is modelled differently from its contributor counterpart. since the rating process requires rating the url categories of three urls (i.e., two control urls wca and wcb, and an unknown url wx ), a malicious rater needs to rate correctly the two control urls and intentionally make an incorrect rating for the unknown. giving a correct rating for the control urls is essential, otherwise, the reputation system can detect that the rater is giving ρrrm if ra(g, w) β ρripm otherwise rmc(g, w) =  (63) similar to rmc(g, w), the function rmx(r) examines the unverified url. however, since it is unverified, it is not appropriate to rely on the rating accuracy function ra(g, w). the set of ratings that is related to rating group g and the unknown url wx must be considered. this can be derived using the function below. r(g, w) = {r|r.g = g  r.s.w = w} (64) for each rating r in r(g, g.wx), a user is rewarded or penalized based on the rvw (r.s, ti , tf), which is the winning rating vote. this is defined in the function below. ρrrm if rvw .(r.s, ti , tf ) = r. α rvw (r.s, ti, tf ) = 0 ρripm otherwise rmx(r) = (65) 11.update the overall reputation ρo of all users u in u. tulungan ensures that if a user fails to be good in at least one of its roles as a contributor and rater, its overall reputation will be affected. this is accomplished by getting the product of the contributor and rater reputations of a user. as an example, if a user is good as a contributor and has a high contributor rating, but is delinquent in being a rater and thus gets a low rater rating, the user’s overall reputation is also low. the equation in deriving the overall reputation of a user is shown below. u.ρo =  u.ρc u.ρr science diliman (july-december 2011) 23:2, 17-39 the function rmc(g, w) is used to examine the two control urls. it simply checks if the rating accuracy ra(g, w) meets the group rating accuracy threshold β. if it is greater than or equal the threshold, the user is rewarded by multiplying its rater reputation with the user rater reputation reward multiplier ρrrm. otherwise, it is multiplied with the user rater reputation incorrect penalty multiplier ρripm. (66) tulungan: a consensus-independent reputation system 33 a wrong rating and may be doing something malicious. however, since the malicious rater has no idea which of the three urls are the controls and the unknown, it needs to make a guess. there is 1 in 3 chances that a malicious rater will correctly guess which is the unknown url. therefore, it also has 1 in 3 chances of being successful in giving a wrong rating for the unknown url while remaining undetected by the reputation system. to keep track of the user type (e.g., a good contributor and rater), the simulation uses two fields, contributor_type and rater_type, for each user. this is essential to determine the contribution and rating behaviour that a user will perform in the simulation. however, these fields are not used in the actual computation of the contributor, rater, and overall reputation of each user. the simulation does not only cover the tulungan reputation system but as well as rater-rating in order to compare the two systems in terms of differentiating good and bad users based on their reputation calculation. the simulation is divided into four (4) parts as illustrated in table 6. for each part, the simulation is executed with a varying percentage of good users relative to their bad counterpart. it starts with 5% good users and 95% bad (in the case of the 3rd and 4th part of the simulation, only the malicious users are used among the bad users). it is executed again with10% good and 90% bad users. this is repeated with an increment of 5% in the number of good users until the percentage of good users reach 95%. for each execution, the simulation is composed of 500 users and runs for 366 simulated days (i.e., january 1 to december 31). everyday, each user in the simulation provides one contribution. on the 1st day of each month, the potential raters are determined type of users involved rater-rating tulungan good vs. bad part 1 part 2 good vs. malicious part 3 part 4 table 6: simulation experiments by the reputation system. on the 2nd day of each month, each user provides a rating based on the potential rater list generated on the 1st day. on the 28th day of each month, the contributor reputation (ρc) and rater reputation (ρr) of all the users are determined. at the end of the 366th day, the average of the contributor reputation, rater reputation, and overall reputation of all the good users is computed. the same thing is done with the reputation of each type of bad user. to get the average reputation for each user type, the fields contributor_type and rater_type are used. take note that fields are used in getting the averge reputation and not in the computation of individual reputation of the users. the maximum user contributor reputation (ρcmax) and rater reputation (ρrmax) are both set to 10.0. similarly, the minimum user contributor reputation (ρcmin) and rater reputation (ρrmin) are both set to 0.001. the initial user contributor reputation (ρci) and rater reputation (ρri) are both set to 0.5. 4.2 results and analysis 4.2.1 good vs. bad users (parts 1 and 2) figures 1a, 1c, and 1e are the results of good versus bad users using raterrating reputation system (part 1 results). this can be compared with figures 1b, 1d, and 1f, which are the results of good versus bad users using tujunga reputation system (part 2 results). the contributor reputation and rater reputation of good users in part 1 are dependent on their number. the more good users there are, the higher their reputation compared to the bad users. before the simulation result was available, it was expected that 50% of good users is needed to overcome the reputation of the bad users when rater-rating is used. however, as seen in figures 1a and 1c, only 40% of good users is needed in order to give the former a higher reputation relative to their deviant and malicious counterpart. this relatively good performance of rater-rating can be attributed to the presence of lazy users. since the lazy users randomly choose the category of urls and randomly rate contributions, there may be cases that the contribution and rating done by lazy users science diliman (july-december 2011) 23:2, 17-39 pantola, a.v., et al 34 their number. the more good users there are, the higher their reputation compared to their malicious counterpart. it can be noted in figures 2a and 2c, both the contributor reputation and rater reputation of the good users are lower than the malicious users when the population of good users is less than 50%. this means that the rater-rating reputation system is effective only when the good users are more than the malicious users. in part 4, a significant improvement in the contributor and rater reputation can be seen when the tujunga reputation system is used. as seen in figures 2b and 2d, even if there is only 1 good user for every 3 malicious users (or 25% good users), the tujunga reputation system is still effective in giving the good users a higher reputation than their malicious counterpart. similar to parts 1 and 2, there are cases where the rater reputation of good users in rater-rating and tujunga decreases to a certain level (e.g., when there are 85% good users in rater-rating and 50% and 65% in tulungan). this can be attributed to the composition of potential raters and the small possibility of good raters in making incorrect rating. in terms of overall reputation, when there are more malicious users compared to good users, the minimum average overall reputation given by raterrating to malicious users is 3.7 and it goes as high as 6.2 (refer to figure 2e). tulungan, on the other hand, limits the overall reputation of malicious users to less than 1 even if there are only 25% good users. this can be seen in figure 2f. support the contributions and rating made by good users. when it comes to tulungan, the contributor reputation of good users are already higher than their bad counterpart when there are only 25% good users (refer to figure 1b). in addition, even if there are only 5% good users, the rater reputation of good users are already higher than the lazy, deviant, and malicious users. this can be seen in figure 1d. there are cases where the rater reputation of good users in rater-rating and tulungan decreases to a certain level (e.g., when there are 70% and 85% good users). this can be attributed to the composition of potential raters (e.g., a set of good raters are grouped with some bad users to rate a particular unknown url). aside from this, good raters have a 1 in 5000 chance of making incorrect rating which may contributed to the decrease in rater reputation. in terms of overall reputation, when there is at least 60% bad users, the minimum average overall reputation given by rater-rating to deviant and malicious users is 1.5 and it goes as high as 4 (refer to figure 1e). tulungan, on the other hand, limits the overall reputation to less than 2.5 even if there are only 25% good users. this can be seen in figure 1f. although rater-rating allows good users to outperform the deviant and malicious users even if there are less than 50% good users (i.e., only 40% good users are needed), this can be attributed to the presence of lazy users, as explained earlier. in order to confirm this, the good users are compared against only their malicious counterpart in parts 3 and 4 of the simulation. 4.2.2 good vs. malicious users (parts 3 and 4) figures 2a, 2c, and 2e are the results of good versus malicious users using rater-rating reputation system (part 3 results). this can be compared with figures 2b, 2d, and 2f, which are the results of good versus malicious users using tulungan reputation system (part 4 results). as expected, the contributor reputation and rater reputation of good users in part 3 are dependent on science diliman (july-december 2011) 23:2, 17-39 conclusion and future work the simulation shows that tulungan is capable of distinguishing good users from their bad counterpart even if majority of the population of users are bad. when good users are pitted against malicious users, tulungan requires only 25% good users to be effective. this is a 100% improvement relative to rater-rating that requires 50% of the population to be good. tulungan: a consensus-independent reputation system 35science diliman (july-december 2011) 23:2, 17-39 in terms of overall reputation, tulungan limits the reputation of bad users (i.e., lazy, deviant, and malicious) to 2.5. this is 37.5% less than raterrating where the overall reputation of bad users can go as high as 4. in addition, when the good users are compared against only to their malicious counterpart, the discrepancy of tulungan and rater-rating is even more evident. tulungan limits the overall reputation of malicious users to 83.88% less than rater-rating. in tulungan, malicious users have a maximum average of overall reputation of less than 1, regardless of their percentage in the total user population. on the other hand, rater-rating allows malicious users to get an overall reputation of 6.2. tulungan can be used to effectively categorize the urls of websites without the under and overblocking issues of the automatic approach as well as the scalability issue of the traditional manual method. more importantly, the system is still effective even if the number of good users involved in the categorization is fewer than their bad counterpart. although tulungan is consensus-independent, it may be possible to manipulate this reputation system if the bad users collude. an improvement in tulungan can be made in order to address scenarios when bad users create multiple accounts (e.g., phantom accounts). such situation allows these multiple accounts to have a coordinated attack towards tulungan, and as a result, these multiple accounts may be given a high reputation value by the reputation system. references adler, b. t., & alfaro, l. de. 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(2010). rating the raters: a reputation system for wiki-like domains. in proceedings of the 3rd international conference on security of information and networks (pp. 71–80). new york, ny, usa: acm. available from http://doi.acm.org/10.1145/1854099.1854116 perkins, c. e., & royer, e. m. (1999, february). ad-hoc on demand distance vector routing. in second ieee workshop on mobile computingsystems and applications (p. 99-100). a radically simpler approach to web content filtering and security [white paper]. (n.d.). available from http:// www.opendns.com/support/whitepapers/? ravikumar, s., mcafee, r. p., & tomkins, a. (2009, april). community-based web filtering. available from http://www.freepatentsonline.com/20090112974.pdf recaptcha stop spam, read books[online]. (n.d.). available from http://www.google.com/recaptcha/ reddit.com: help [online]. (n.d.). available from http:// www.reddit.com/help/faq some ebay users abuse auction site’s feedback system, professor finds [online]. (2007, january). availab le fr om http://www.p hyso rg.com/ news87832472.html (haas school of business, uc berkeley) pantola, a.v., et al 38 science diliman (july-december 2011) 23:2, 17-39 figure 1: good versus bad users: rater-rating reputation system results (left figures: a, c, e) and tulungan reputation system results (right figures: b, d, f) tulungan: a consensus-independent reputation system 39science diliman (july-december 2011) 23:2, 17-39 figure 2: good versus malicious users: rater-rating reputation system results (left figures: a, c, e) and tulungan reputation system results (right figures: b, d, f) science diliman cover vol 23, no. 2 slide 1 inside front cover-23-2 fish kill-pp1-3 guidelines for expanding conference papers p4 tetrabromobispheno-pp5-16 tulungan-p17-final (2)-for authors review how to register as author pp 40 intraspecific-pp41-53 info for authors_revised-pp54-55 call for papers-revisedpp56 manuscript submision form_sd-new-p57 inside back cover-23-2 participatory monitoring-art.11 verceles et al. 78 science diliman (july december 2000) 12:2, 78-87 abstract participatory monitoring and feedback system: an important entry towards sustainable aquaculture in bolinao, northern philippines lailany f. verceles*, liana talaue-mcmanus, and porfirio m. aliño marine science institute, college of science, university of the philippines diliman, quezon city 1101 philippines email: lany@msi01.cs.upd.edu.ph the aquaculture industry in caquiputan channel contributed p2.3m to municipal revenues in 1998. however, the uncontrolled construction of fish pens and fish cages have contributed to the deterioration of the water quality in the caquiputan channel. despite monitoring of parameters (e.g. do, salinity, and temperature), low production was implicated because of limited dissolved oxygen supply. a participatory monitoring of fish pens and fish cages was facilitated to pave the way for sustainable aquaculture. a system for monitoring and disseminating information on water quality, production and zonation of pens and cages, has been pursued to assess the situation and identify mechanisms to regulate aquaculture activities. the feedback system adopted has raised and facilitated environmental awareness, issue identification, and implementation, of solutions to some major issues. furthermore, the results have resulted in policy reforms, as embodied in the provisions on aquaculture in the municipal fisheries ordinance. keywords: participatory monitoring, fishpens and fishcages, caquiputan channel introduction aquaculture in bolinao , northern philippines, started in the early 1970's through brackish-water fishponds. the semi-intensive form of milkfish culture was adopted. supplemental food in the form of bread crumbs and commercial feeds were given to the fish when natural food (lab-lab) in the pond was exhausted. to maximize income, shrimps and other fishes entering the pond during inflow of seawater were grown out together with the milkfish. this type of aquaculture accelerated in bolinao in the 1980's and many mangrove areas were converted to fishponds. in 1995, the culture of milkfish on brackish-water fishponds was expanded to coastal waters through the use of fishpens and fish cages. since then, fishpens and fish cages have proliferated in the caquiputan channel, reaching tambac bay and parts of victory and dewey. to regulate the coastal aquaculture of finfishes in bolinao, the municipal council passed on october 5, 1995 ordinance number 1-series of 1995 regulating the establishment, erection, and/or construction of fishpens and fish cages in the municipal waters of bolinao, pangasinan and the granting of permits for their operation. despite the passage of the ordinance, fishpens and fish cages have continued to be illegally constructed and operated. many fishpens have exceeded the maximum* corresponding author participatory monitoring and feedback system 79 pen area at which water flow and quality in the channel could be maintained. because of the shortcomings of the local government unit (lgu) in implementing the ordinance, several issues have been raised regarding the proliferation of fishpens and fish cages, and their ecological and socioeconomic impacts on the different sectors. among these issues are: (1) reduction of traditional fishing grounds; (2) unregulated construction and expansion of fishpens and fish cages; (3) constriction of navigational routes; (4) conflicts in the allocation of water space and; (5) deterioration of water quality. in 1997, 703 permits (around 88 hectares) were given to individuals to operate fishpens and fish cages (municipality of bolinao,1997), excluding expansions. the number of permits granted decreased to 476 units in 1998, but ocular inspection in 1999 showed a total of 797units (around 165 hectares), excluding those inaccessible by boat (fig. 1). this trend showed that the number of permitees exceeded the 544 units (130 hectares) estimated as the carrying capacity of the channel and proposed in the coastal development plan (cdp) (fig. 2). with the high stocking density and excessive feeding, the water quality of the channel has been degraded, as indicated by fish kills and phytoplankton blooms. because of low fish growth, the number of pens and cages decreased by 43% from 1997 to 1999 (table 1). this paper documents how a mechanism for participatory monitoring of aquaculture was established in bolinao. results obtained on the first monitoring year in 1999 are presented. the monitoring scheme is evaluated for its sustainability and impact in making finfish grow-out a viable source of income. materials and methods setting up a mechanism for participatory aquaculture monitoring beginning in 1996, the number of coastal aquaculture facilities in caquiputan channel has been increasing. the channel has been identified as a multiple-use fig. 1. location of fishpens and fish cages (as of february 1999) 119.90 119.95 120.00 legend: _ fish cages _ fish pens santiago island bolinao luciente ii luna culang anda 16.30 16.35 16.40 pen cage total ocular 1997 permit 1997 permit 1998 ocular 1999 operational nonoperational no data no data 1,076 no data no data 703 362 114 476 277 147 424 283 90 373 table 1. status of milkfish aquaculture in bolinao (1997-1999) fig. 2. proposed areas for fishpens and fish cages in the cdp scale 1:50,000 fishpens fishcages 119.30 119.40 119.50 119.60 119.70 119.80 119.90 16.34 16.37 16.39 16.42 16.44 lambes zaragosa catungi tara culang luna luciente ii luciente i lu ce ro sa lud pi la r ii-6=9 ii-8=44 ii-5=154 ii-4= 92 ii-3=108 ii-1=55 ii-2=38 ii-7=44 16.24 16.27 16.29 16.32 verceles et al. 80 table 2. location and designs of the sampling pens and cages c--cage: p--pen: *-24 hour sampling station c1 c1 p1* p2 p3* p4 p5 location depth area( s q m ) n16 23.189 e119 54.961 n16 23.304 e119 55.425 n16 22.488 e119 56.810 n16 20.745 e119 55.646 n16 19.438 e119 55.693 n16 19.515 e119 55.808 n16.31578 e119.91873 13 m 18 m 5 m 3 m 3 m 3 m 2 m 225 324 3,000 4,800 3,200 2,800 4,800 25 60 250 25 400 500 3 station distance f r o m shore (m) water residence t i m e (unobstructed flow) 0-4 days 0-4 days 16-18 days 10-12 days 10-12 days 10-12 days 14-16 days zone (zone ii) in the coastal development planning exercise that was conducted from 1996-1999. the marine fisheries resources management project (mfrmp) initiated a series of meetings in zone ii to begin the implementation of the plan, particularly the regulation of coastal aquaculture. a major step was to begin the monitoring of caquiputan channel and to engender the participation of the community in this activity. villages belonging to zone ii include luciente 1 and 2, luna, culang, tara, zaragoza, lambes, catungi, part of pilar, part of salud, and part of lucero. discussed below are the major activities carried out in creating the monitoring team and setting up their operation. a “seminar on the sustainable management of milkfish aquaculture” was held on 5 november 1998 it was attended by 65 participants, including fishpen and fish cage owners, students of the bolinao school of fisheries, municipal officials, barangay officials, and marginal fishers of zone ii. it aimed to: (a) orient the participants on methods and practices of sustainable coastal aquaculture; (b) identify issues/problems and their solutions in zone ii and; (c) form a multi-sectoral monitoring team. on 28 november 1998, the team members –(students from bolinao school of fisheries, fisahpen/cage owners/operators, fishers, members of barangay councils, and the municipal aquaculture technician) – underwent training on the use of water quality monitoring instruments, including a dissolved oxygen (do) meter, refractometer, secchi disk, and thermometer. the group scheduled their first zone visit and water sampling (three fishpens) on 2 december 1998 and implemented regular monitoring since. monitoring proper site selection. the team started monitoring water quality in two sites (one fish cage in lucero and one fishpen in luna). the number of monitoring sites has increased through informal information campaigns with other pen/cage owners and caretakers. during the first half of 1999, the team established two cages (one each in lucero and salud) and five pens (one each in pilar, luciente 2, and culang; two in luna) as sampling sites. fig. 3 shows the locations of the sampling sites, and table 2 lists information about them. c1 and c2 were about 1 to 2 m apart. p1 is found beside other pens while p2 is located adjacent to a navigational route. p3 has a small fingerling enclosure attached to it and is located at the center of many pens (at about 4m distance). p4 is situated at the outermost row of fishpens in luna. p5 is just 3 m away from the shore, and is the most shallow, especially at low tide. it is also the most turbid. (fig. 3 and table 2). fig. 3. sampling sites 119.88 119.90 119.94 119.96 120.00 120.02119.92 119.98 16.24 16.27 16.29 16.32 16.34 16.37 16.39 16.42 16.44 c1 c2 p1 p2 p3 p4 p5 participatory monitoring and feedback system 81 protocols. the water quality parameters of dissolved oxygen, ph, salinity, temperature, and transparency were monitored monthly using do meter, ph paper, refractometer, and sechi disk, respectively. in p1 and p2, temperature, dissolved oxygen, and salinity were measured every 4 hours over 24 hours. for milkfish production, the frequency and amount of feeding, stocking density, growth, and harvest yields were recorded once a month in p2, p3, and p4, to evaluate production levels. census of aquaculture structures was conducted twice a year, while the number of licenses and permits issued by the municipality was assessed yearly using municipal records. monitoring of water quality was conducted between 9:30am to 3:00pm. the monitoring scheme is shown in fig. 4. information campaigns the monitoring results were discussed quarterly with stakeholders from the barangays of tara, luna, luciente 2, and culang. the municipal aquaculture technician, the office of the mayor, and the bolinao school of fisheries were given copies of the report of the results of monitoring. the data would serve as a basis for aquaculture policy reforms that would need to be made by the government of bolinao. the feedback system adopted is illustrated in fig. 5. results and discussion biophysical factors temperature and salinity changes over a monitoring period of 12 months showed seasonal trends (fig. 6 and table 3). during the dry months (january to may; november december), temperature ranged from 27°c, recorded in c2 in february, to 33°c, observed in p5 in may. during this period, salinity changed in p3 from 28 ppt in february to 40 ppt in may. with the onset of rains in june, temperature was at or above 31°c in mid-june, dipping to below 29°c in late september, and then increasing to nearly 34°c in p3 in mid-october. during the humid and rainy period, salinity decreased in all stations, with p5 in culang experiencing the most fresh salinity values among all 7 stations at 7 ppt in late september. over an annual period, the temperature changes were from a two-degree difference in p2 to as much as sixdegree change in c1. fish cages located at 13 to 18 m depth had a 10-15 ppt change in salinity. the pens in shallow waters experienced a change ranging from 16 to 28 ppt over a year. fishpen monitoring team -technical students (bsf) -pen/cage operators -fishers -lgu (brgy./mat) production (monthy) -stocking density -growth of the stock -volume & frequency of feeding -volume & price of harvest water quality (2x a month) -dissolved oxygen -temperature -salinity -ph -turbidity inventory of structures (2x a year) -size and distance -location -permit & license semi-annual analyses & feedback quarterly analyses & feedback fig. 4. the flowchart of the monitoring scheme verceles et al. 82 sites in shallow waters had an annual minimum of less than 4.0 mg/l for their annual minimum, except in the case of p4. p1 and p3 sites, were monitored during the wet season (fig. 7 & table 4). over a 24-hour cycle, temperature ranged from 27 to 31°c, and salinity changed from 15 to 24 ppt. dissolved oxygen fluctuated from 4 to 7 mg/l in p1 and from 2.9 to 4.5 mg/l in p3. boyd and lichtkoppler (1979) gave optional levels of dissolved oxygen (about 5 mg/l), temperature (25-32°c), and salinity (<32 ppt) for milkfish growth in tropical waters. given that milkfish is euryhaline and can tolerate brackish conditions, salinity is probably the least limiting factor for its growth. provided that salinity does not exceed 36 ppt, during which feeding is reduced, growth is not hampered (bautista and others 1994). dissolved oxygen and temperature, because of its effect on gas solubility, are both critical determinants of growth rates. dissolved oxygen is replenished via photosynthesis and diffusion; the latter enhanced by fast flow and turbulent regimes. in a 24-hour period, the dissolved oxygen is miimized during the night hours when respiration dominates, until photosynthesis produces oxygen by mid-morning. the strong northeast wind, which blows during the cool months from december to early march, creates sufficient turbulence to also enhance solubility of oxygen; hence, the high dissolved oxygen levels during the dry and cool months. low dissolved oxygen values dominate during the warm and humid rainy months. although runoff from land fig. 5. flowchart on the feedback system information quarterly feedback with: brgy. council (zone ii) zonal action team 2 fishers pen/cage operators feedback every after monitoring with the operator on site with needed recommendations quarterly feedback with municipality of bolinao and the mun. aquaculture technician bolinao school of fisheries billboard primers • affect pricing changes • update perceptions • response options • information dissemination • management response implications on permits and operation practices good practice evaluation environmental credits • guide in policy reforms table 3. annual range of water quality parameters station (depth m) temp. (c) salinity (ppt) do (mg/l) secchi disk depth (m) c1 (13) c2 (18) p1 * (5) p2 (3) p3 * (3) p4 (3) p5 (2) 28-34 27-32 28-33 30-32 30-34 29-33 28-33 21-36 28-38 21-38 12-40 14-35 20-36 7-35 4.2-7 5-6.8 1.5-7.6 2.5-7.8 3.5-6.9 4.3-7.8 3.8-8.5 2-3 1.5-3 1.5-4.5 .5-1.9 1-2 1.5-2 .8-1 c cage; ppen; do dissolved oxygen; * 24 hour sampling station fluctuations in dissolved oxygen indicated the interactions between water residence time in the study sites, the influence of monsoon winds, and the prevailing temperature. during the northeast monsoons that prevailed during the dry months (november to march), oxygen values across stations were tighter and ranged from over 5 mg/l to 7 mg/l. with the onset of the doldrums (april to may) and the rains brought about by the southwest monsoon, dissolved oxygen, in general, decreased in value in all stations from june to late november. it increased beginning in december with the shift to the northeast winds. over an annual cycle, fish cages in deep waters had dissolved oxygen concentrations greater than 4.0 mg/l. all the fishpen table 4. daily range of water parameters station date of sampling temp (c) salinity do p1 p3 sept. 21-22, 99 sept. 25-26, 200 27-30 27-31 17-21 15-24 4-7 2.9-4.5 participatory monitoring and feedback system 83 brings nutrients, siltation limits oxygen production by reducing photosynthesis.in addition, warm temperature decreases gas solubility. however, temperature enhances growth rates provided that food is provided. thus, in terms of overall effect on biomass production, harvest was higher for the august to november period at 14 tons, compared to 11 tons for the same stocking density in the january-april culture cycle in p3. this result needs further validation. the effects of the aquaculture facilities on water flow and exchange, as well as those of stocking and feeding practices would need to be considered in determining overall production. in the case of p3 for example, extremely low dissolved oxygen levels were obtained in november. the fish were given feeds at the rate of 680 kg/day with a stocking density of 14 fingerlings per square meter in a 3,200 m2 pen. the excess feeds used up oxygen for decomposition and caused it to reach sub-optimal concentrations. in general, chiu et al. (1986) found that milkfish stops feeding when dissolved oxygen is <1.0 mg/l, but feeds before sunrise and continues after sunset provided dissolved oxygen is maintained at >3.0 mg/l. to assess better growth differences, additional pen sites with variable biophysical conditions would be established and monitored, in collaboration with indepth technical studies looking at water column and sediment parameters as influenced by aquaculture. production from milkfish grow-out two-month old fingerlings (about 3-5 centimeters) sold at p2.40 to p2.50/fingerling were stocked at a stocking density of 10-16 and 110 123 per square meter in pens and cages, respectively. fingerlings come from suppliers in anda, bani, bolinao, or bulacan. other operators who have fishponds buy milkfish fry for php 0.35 to php 0.70/fry from fry catchers or php 1.00 to php 1.25/fry from milkfish fry dealers, and grow these out in a nursery pond or small net enclosures within the pond. mortality occurs during transport especially when the fry come from distant sources. cage number 2 (c2) experienced 15% mortality rate of milkfish fry from bulacan during stocking while only 2% mortality occurred for stocks coming from nearby sources like santiago island, anda, and bani. higher mortality rates were observed during stocking when fry were not properly acclimatized. the fingerlings could not quickly adapt to the water conditions in the cage which were different from those in the seed stock container. where mortalities were caused by parasites (e.g., pen number 4 experienced 0.33% mortality due to blood spots in d is so lv ed o xy ge n 0 1 2 3 4 5 6 7 8 9 l l v v 26 27 28 29 30 31 32 33 34 35 25 te m pe ra tu re 5 10 15 20 25 30 35 40 45 0 s al in ity 21 -j an 28 -j an 11 -f eb 23 -a pr il 28 -a pr il 15 m ay 28 m ay 11 -j un 29 -j un 17 -j ul 24 -j ul 21 -s ep 29 -s ep 15 -o ct 15 -n ov 26 -n ov 27 -d ec lucero (c1) salud (c2) pilar (p2) luc.2 (p2) luna (p3) luna (4) culang (p5) fig. 6. dissolved oxygen (do), salinity and temperature fluctuation verceles et al. 84 month. at the other extreme are cases where feeds could not be given because of budgetary limitations. marketable weight for one milkfish was usually about 0.35 to 0.75 kg/fish (wet weight). selective harvesting took place in fishpens and fish cages where a small portion of the stock was left in the structure for the following harvest. instances of unexpected harvesting also occurred in cases of typhoons and strong winds. operators in culang revealed that harvesting in 1995-1996 required only 3 months for fingerlings to reach marketable size (about 2-5 pieces/kilo). in 1999, grow-out cycle took 4 to 5 months. some operators reported that they needed 6 months of grow-out period in order to produce a viable harvest. they were not able to reach the expected output of three culture cycles in a year. in addition to poor water quality, other reasons for low production include miscounting of fry during trading, poaching and food limitations, and calamities. operators preferred the culture cycle where harvest took place during christmas, fiestas, and holy week (december, march, and april) when the demand for milkfish was high; the increased demand resulted in the high price of milkfish. a systematic way of harvesting was done by encircling the stock with a net, driving them to the center for scooping. after harvest, the milkfish were landed at catubig, picocobuan, and guiguiwanen for sorting (according to size) and packing (with ice in a tub). benefits from fishpen and fish cage culture an average net income for each pen was ph p 174,000 per cropping (table 5). market destination for harvests were usually dagupan and metro manila. milkfish aquaculture benefited not only the owners of fishpens and fish cages, but also the people of bolinao and other municipalities. they were hired (and paid as contract laborers) as caretakers, fishpen/cage fixers, harvesters, classifiers, packers, and laborers during transport. likewise, the municipality earns from annual fees. in site: p1 on september 21-22, 1999 d is so lv ed o xy ge n 25 20 15 10 5 0 4 pm 8 pm 12 mn 4 am 8 am 11 am 30.5 30 29.5 29 28.5 28 27.5 27 26.5 26 25.5 s al in ity /t em pe ra tu re d is so lv ed o xy ge n 25 20 15 10 5 0 4 pm 8 pm 12 mn 4 am 8 am 11 am 31.5 31 30.5 30 29.5 29 28.5 28 27.5 27 26.5 s al in ity /t em pe ra tu re site: p3 on september 25-26, 1999 do salinity temperature fig. 7. 24 hours of dissolved oxygen fluctuation gills of fish in the third month of the culture period), blue water and vetracin antibiotics were administered to the stock. p4 and p5 were given commercial feeds (vitarich, tateh, ram, or welgrow) three times a day (morning, noon, and afternoon) at .01kg/fingerling/day during the first three to four months. starter mash and crumble were offered on the first month, grower feeds in the second and third months; and finisher feeds in the fourth month. two weeks before the harvest, noodles or bread crumbs were given to the stock as food supplement at 10:00 am and 3:00 pm. in certain instances such as in c2, as much as 10 sacks of feeds per day (or 250 kg/ day) were given to 65,000 fingerlings on the third month. this amounted to 300 sacks or 7,500 kilograms per participatory monitoring and feedback system 85 1998, it earned an estimated amount of ph p 2.3 m from these fees (table 6). evaluation of participatory monitoring impact of monitoring. the essence of motivating the participation of the community is to allow its members to direct their resources in solving problems collectively. their participation is important not only for their personal interests on the resource but also in consideration of the interests of other sectors. a participatory process adopted in the monitoring (and also in the formulation of the cdp) played a major role in raising the awareness among the major stakeholders in zone ii. an awareness on the need for sustainable aquaculture practices was initiated, especially on the part of the fishpen cooperators. sustaining monitoring. the best reward received by the monitoring team was the recognition by the municipality of the activities of the team. the data shared with the municipality helped to facilitate deliberations on the cdp ordinance. this recognition was manifested when the municipal mayor approved the team's request for a monitoring boat and gasoline. a major concern now is how to sustain monitoring efforts. given the voluntary and ad hoc nature of the monitoring team, it was expected that levels of participation would vary. for the bsf students, their participation waned when they had to start their onthe-job training. among operators, only a few had time for post monitoring work. technical constraints were also expected. the participatory monitoring team lacked the technical expertise in data analysis so that the assistance of the mfrmp was vital during the quarterly information campaigns. the maintenance of water quality, size, and distance and zonation of pens and cages are among the critical provisions in the cdp that should be monitored regularly. as the lead officer in the cdp, the role of the coastal resource management (crm) officer is crucial in its implementation. however, the crm officer would need assistance from the major stakeholders. the ad hoc zonal action team for zone ii that was formed to facilitate the information campaigns and the implementation of cdp would particularly be a major partner. there is, therefore a need to mobilize the whole zone ii so that provisions on coastal aquaculture could be properly and successfully implemented. implementing the municipal fishery ordinance provisions. the implementation of the previous ordinance on milkfish pen and cage regulation (especially on the issuance of permits) since its approval on october 9, 1995 has not been effective. the bolinao coastal development plan, now called the municipal fishery ordinance, provides clear guidelines on how to regulate coastal aquaculture, the most controversial part of the ordinance. since its submission to the municipal council on october 1998 until its enactment in december 1999, several consultations and public hearings were conducted by the municipal fisheries and aquatic resources management council and the municipal council regarding pen (5 yr.) pen (3 yr.) cage (5 yr.) cage (3 yr.) total stocking density (fingerlings/ sqm) grow-out period mortality rate harvest (tons) pen area (sqm) 7 9 7 9 13 dec ‘98-mar ‘99 jan-april ‘99 may-july ‘99 aug-nov ‘99 june-oct ‘99 table 5. average production per cropping source: mfrmp data, december 1998 to october 1999 pen no. p1 p2 p3 income/ pen (php) 25 % 16 % 12 % 14 % 14 % 10 10.8 8.1 13.8 12 3,000 3.200 2,800 170,000 150,000 70,000 230,000 202,133 annual fee (in php) number of structures wharfage f e e filing fee (in php) 6,000 4,000 3,000 2,000 p1,936,000 table 6. estimated revenues of the lgu from milkfish aquaculture in bolinao (1998) source: municipality of bolinao, 1996. revenue ordinance no. 01 s-1996, mfrmp, 1998 116 246 28 86 476 p2.5 per tub of during harvest p1,190,00 php 2.3m 1,000 1,000 500 500 p419,000 verceles et al. 86 its provisions. recommendations on aquaculture include the following: 1) revision in sizes from 2,400 to 1,200-4000 square meters in pens and from 225 to 144-324 square meters for cages. 2) construction of fish cages in clusters (10 units per cluster), with, a distance of 100 meters between clusters. 3) change in the maximum number of allowed pens and cages from 1 unit to 5 units per permitee. provisions on the issuance of permits, maintenance of water quality, and monitoring of fish production which are broadly stated should likewise be addressed in the implementing rules and regulations. summary enjoining the community's participation is an effective strategy to any coastal resource management initiative. other initiatives like the coastal development planning, a participatory aquaculture monitoring mechanism was set up through information dissemination. a group of pen/cage owners, barangay council members, technical students, fishers, and representatives from the municipality comprised the monitoring body. the monitoring of the parameters dissolved oxygen, salinity, and temperature, showed that low production of milkfish was caused by limited dissolved oxygen supply. regulation is needed to ensure optimal production of pens and cages which would need to be located in optimal environmental conditions where dissolved oxygen remains high for fish growth. the monitoring scheme allowed the formulation of policies to regulate milkfish grow-out. sustaining monitoring efforts remains to be seen, but the municipal fishery ordinance provisions at least ensure that the industry is regulated by law. municipal coastal development plans would be operational if coastal aquaculture is squarely addressed. although often contentious because of intense conflicts, the bolinao experience indicates that advocacy and biophysical participatory monitoring of water quality, production, and incomes are vital in ensuring sustainable coastal aquacutlure. references boyd c, lichtkoppler f. 1979. research and development of international center for aquaculture, agricultural experiment station. series (22): 3-10. bautista mn and others. monitoring water quality. feeds and feeding of milkfish, nile tilapia, asian sea bass, and tiger shrimp. tigbauan, iloilo, philippines, seafdec aquaculture department: p 83. chiu yn, makahilig p, sastrillo mas. 1986. factors affecting the feeding rhythm of milkfish (chanos chanos forsskaal). in: maclean jl, dizon lb, hosillos lv, editors. the first asian fish forum. manila, philippines: asian fisheries society. p 547-550. municipality of bolinao, list of fishpen/cage operators, 1997. municipality of bolinao, list of fishpen/cage operators, 1998. municipality of bolinao,the proposed bolinao coastal development plan (cdp) of bolinao, 1997. municipality of bolinao,1995. ordinance no.1 s-1995, regulating the establishment, erection or construction of fish corral, such as fishpens and fish cages in the municipal waters of bolinao, pangasinan and granting permits for their operations prescribing payments and penalties thereof. municipality of bolinao, 1995. resolution no.27 s-1995, to regulate the establishment, erection and/or construction of fish corrals in the municipal waters of bolinao, pangasinan pursuant to republic act no. 7160. municipality of bolinao, 1996. resolution no. 61 s-1996, to enact a revenue ordinance on wharfage. municipality of bolinao, 1996. revenue ordinance no. 01 s-1996, an ordinance to impose and collect wharfage participatory monitoring and feedback system 87 on agricultural and marine products in all municipal operated and maintained wharves and deep-sea fishing landing sites within the municipality of bolinao, pangasinan. info for authors-revised2012.pmd 57 information for authors science 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manuscript templates in word, open office and latex are now available online at www.sciencediliman.edu.ph. authors may opt to submit their typeset manuscripts as an email attachment to rduo.ovcrd@up.edu.ph and rduo.ovcrd2012@gmail.com. manuscripts may also be submitted in hard copy (3 copies) to: the editor-in-chief science diliman office of the vice-chancellor for research and development lgf phivolcs bldg., c.p. garcia ave. university of the philippines diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. ocr document ocr document ocr document 08_juanico 37 topologically evolving networks science diliman (january-june 2003) 15:1, 37-40 onset of small-world behavior in topologically evolving networks d.e. juanico*, c.p. monterola, and c.a. saloma national institute of physics, college of science university of the philippines diliman 1101 quezon city, philippines e-mail: earl@nip.upd.edu.ph abstract we evolve topology of a network of n fully-coupled nodes that interact according to repulsion-attraction dynamics within a confining wall. the dynamics portrays each node’s tendency to keep distance from its competitors while maintaining a lighter tendency to resist relative isolation. each node is characterized by two parameters: an intrinsic mobility µ and a preferred neighboring distance ρ. onset of clustering is found to occur at a critical variance in mobility, σµ 2 = 1, and in preferred neighboring distance, σµ 2 = 10. this result implies that small-world behavior manifested in clustering can be triggered by the diversity of node population. introduction the current interest in networks is part of a broader movement towards research on complex systems. a network of interacting individuals is considered to be a complex system because the collective behavior of the entire network is not deducible from the properties of the individual. this is called “emergent behavior”. many of the systems in the real world, such as neural networks, social networks, and even the world-wide web, that are yet to be completely understood can be considered to be complex networks (strogatz, 2001). interactions between nodes that constitute a network is difficult to determine exactly. however, there are subtle manifestations of this interaction that can be used to motivate a simple dynamical model of the network. nodes are always located at some finite, nonzero distance from each other. nodes do not overlap because each node should minimize competition among its neighbors for resources, and the maximum distance is bounded because it needs to interact considerably such that it prevents itself from becoming isolated. in the frame of one node, all other nodes are viewed both as competitors and allies. these qualities can be modelled in terms of shortdistance repulsion and long-distance attraction adopted from the interaction between molecular species defined by the lennard-jones potential in the field of molecular dynamics. furthermore, the population of nodes is inherently diverse, such that any two nodes take on different states. in recent studies of complex networks (amaral et al., 2000), the nodes are characterized by some preferential attachment, which result in sparse connections. as a new perspective, we set aside this property and instead consider a fully-connected network wherein all the nodes interact with one another. the reason is that we believe that the diversity of the nodes has something to do with the emergence of self-organized behavior even if the connections are static.* corresponding author 38 juanico, monterola, and saloma model we consider a system composed of n agents represented by nodes in a network. the entire network evolves within a bounded region in space, considering limited resources. the state of agent k is represented by φ k = {µ k , ρ k , ϑ k }, wherein k is a measure of the node’s mobility, ρ k is the node’s preferred neighboring distance, and ϑ k determines its sensitivity. the response of the node (k) is defined by its displacement velocity υ k , a function of its separation distance r = r kj = r jk from another node at state φ j . it is defined to be (1) the value for µ k and ρ k is normally different for each particle. the distribution of the value is gaussian centered at 〈µ〉 = 0.125 and 〈ρ〉 = 10, with variance denoted by σµ 2 and σµ 2 , respectively. the value of ϑ k is set to unity for all particles. eq. (1) is positive if r < ρ k , such that node k recedes from node j, and negative if r > ρ k , such that node k approaches toward node j. the network evolves topologically in the following way: at each time step t i (1) a connection is randomly selected for update, and (2) node k is displaced by ∆r k = υ kj ∆t due to its interaction with node j according to eq. (1), wherein ∆t is a fixed time step. the total displacement of a node due to all other nodes is given as (2) wherein ∆r kj is the contribution of one particle to the total displacement. the goal of the evolution is to minimize an energy function e. this energy is defined to be (3) which follows from the assumption that a stable configuration of the network is attained if the nodes settle down at their respective positions. results initially, the network is distributed uniformly within a confining wall (fig. 1). the side of the wall is set to have a length l = 5 〈ρ〉. the network is allowed, not forced, to evolve until a stability criterion has been reached, as given by (4) let us denote the variance in the states to be (5) in fig. 1b, the cumulative distribution is expected to be smooth, corresponding to a random distribution of the k kj j k r r ≠ ∆ = ∆∑ 2 2 n n k kj k j j k e r r ≠ ⎛ ⎞ = ∆ = ∆⎜ ⎟ ⎝ ⎠ ∑ ∑ ∑ 610 e e −∆ ≤ { }2 2 2, ,µ ρ ϑδφ σ σ σ= ( ) ( )( )tanh .= − −k k k kr rυ µ ϑ ρ fig. 1. (a) initial distribution of the nodes. the length of one side of the confining wall is l = 50; (b) cumulative distribution of internode separation. (a) (b) 39 topologically evolving networks nodes within the bounded region. if all the nodes have the same mobility and preference, the resulting topology does not exhibit clustering as in fig.2. instead, the cumulative distribution (fig. 2b) has a linear region, which implies that there is approximately a uniform distribution of internode distances. consequently, the stable configuration appears as a nearly perfect circle (fig. 2a). if the nodes have different preferences but of similar mobility, the stable configuration has only one cluster at the center of the distribution, as shown in fig. 3. the cluster consists of nodes that settle with more neighbors. the nodes at the periphery tend to prefer isolation. clustering is seen to produce three “plateaus” in the cumulative distribution, as shown in fig. 4b. the first plateau corresponds to the distribution of nodes, with a mean separation 〈r〉 ≈ 2 in each of the six clusters formed (5 arms, 1 center). the second plateau corresponds to 〈r〉 ≈ 9 between adjacent clusters. the third plateau corresponds to 〈r〉 ≈ 16.5 between opposite clusters. discussion small-world behavior was first suggested by watts and strogatz (watts & strogatz, 1998) to explain the collective dynamics of complex networks fig. 2. parameters: δφ = {0,0,0}. (a) stable configuration with final energy, e = 7.03 x 10-7; (b) cumulative distribution of internode separation of the final configuration. (a) (b) fig. 3. parameters: δφ = {0, 10, 0}. (a) stable configuration with final energy, e = 8.13 x 10-7; (b) cumulative distribution of internode separation of the final configuration. (a) (b) characterized by fluid connectivity between nodes. small-world networks are so called because of the surprisingly small average path lengths between nodes in the presence of a large degree of clustering. it has been shown that real complex systems such as the network of movie-actor collaborations, the neuronal network of the worm c. elegans, the world-wide web, and the network of citations of scientific papers behave as small-world networks (amaral et al., 2000). in this paper, we show that diversity in the node population can trigger the onset of small-world clustering 40 juanico, monterola, and saloma behavior. adding dynamics in the connectivity between nodes (i.e., taking the possibility of growth of new links and death of old ones) has been shown (strogatz, 2001; amaral et al., 2000; watts & strogatz, 1998; bornholdt & rohlf, 2000; eguiluz & zimmerman, 2000) to bring about the emergence of clustering or herding behavior in communication and information networks. however, we have shown that this is not necessarily the only reason that can explain the small-world behavior because a network with static connections may exhibit small-world behavior. to summarize, we have shown that variance in nodespecific properties allows us to construct a topology consistent with small-world distributions. in the future, more subtle properties of real-world complex systems may surface out by considering the interplay between node diversity and link dynamics in evolving topology of complex networks. acknowledgment this work is partially supported by the commission on higher education (ched). references amaral et al., 2000. classes of small-world networks. proc. natl. acad. sci. usa. 97: 11149-11152. bornholdt, s. & t. rohlf, 2000. topological evolution of dynamical networks: global criticality from local dynamics. phys. rev. lett. 84: 6114-6117. eguiluz, v.m. & m.g. zimmerman, 2000. transmission of information and herd behavior: an application to financial markets. phys. rev. lett. 85: 5659-662. strogatz, s.h., 2001. exploring complex networks. nature. 410: 268-276. watts, d.j. & s.h. strogatz, 1998. collective dynamics of small-world networks. nature. 393: 440-442. fig. 4. parameters: δφ = {10, 1, 0}. (a) stable configuration with final energy, e = 5.22 x 10-3; (b) cumulative distribution of internode separation of the final configuration. (a) (b) 29-36_rivero w corrections 29 subcellular localization of cadmium science diliman (january-june 2004) 16:1, 29-36 * corresponding author subcellular localization of cadmium in chlorella vulgaris beijerinck strain bt-09 p.b. lintongan1,2, f.a. cariño1,3, and g.c. rivero*1,2 1natural sciences research institute, 2institute of biology and 3institute of chemistry college of science, university of the philippines 1101 diliman, quezon city, philippines e-mail: gilda.rivero@up.edu.ph tel. no.: (632)920-5301 local 4266; fax no.: (632)928-2888 date received: 10 july 2003; date accepted: 16 april 2004 abstract growth response curves of chlorella vulgaris beijerinck strain bt-09 to sublethal concentrations of cadmium were evaluated. the growth responses of this microalgal isolate was determined through analysis of chlorophyll a levels. cadmium was effectively taken up by the cells as determined by flame atomic absorption spectrophotometry (f-aas). subcellular fractionation was undertaken to locate sites that accumulate cadmium. keywords: chlorella vulgaris, cadmium uptake, subcellular localization introduction the heavy metal, cadmium, is found in the environment as inorganic cations or as complexed species. cadmium serves no known biological roles and is a powerful toxicant even when present at low concentrations (babich & stotzky, 1981). as an environmental contaminant, cadmium ions may act on a number of cellular and biochemical processes essential to growth and reproduction of microalgae. among the deleterious effects of cadmium on photosynthetic organisms are inefficient photosynthesis, unproductive nucleic acid, protein and lipid biosynthesis, ineffective nitrogen fixation, decreased uptake of inorganic nutrients, and eventual death (boyle, 1984). cadmium enters the aquatic environment through various anthropogenic activities, which include mining, smelting, and electroplating. this heavy metal is then released into the waterways where it is readily assimilated and concentrated by microorganisms in the primary trophic levels. cadmium is incorporated and biomagnified through the food chain. as essential components of aquatic ecosystems, microalgae represent a major proportion of primary producers in the food chain. these organisms are known to bioaccumulate heavy metals, and as such play important roles in the redistribution and bioconcentration of heavy metals. as survivors in a metal contaminated environment, microalgae have evolved a number of metabolismdependent and -independent processes for the uptake and accumulation of heavy metals. these processes include exclusion, chelation, binding to cell surfaces, and compartmentalization of the metal ions (cobbett, 2000). a general response mechanism for heavy metal detoxification is the expression of stress biomolecules, e.g., metallothioneins and phytochelatins. 30 lintongan, cariño, and rivero chlorella vulgaris, as well as many species of microalgae, have been observed to bioaccumulate significant amounts of cadmium ions from the medium. carr et al. (1998) and matsunaga et al. (1999) observed the ability of c. vulgaris to withstand deleterious concentrations of cadmium. the capacity of c. vulgaris for taking up cadmium ions makes it a suitable organism for remediation of aqueous surface environments. the cells can be used either as live or inanimate bioscrubbers. the removal of harmful heavy metals from effluents and wastewaters provides an additional or alternative process of metal recovery for environmental protection or economic reasons. the actual biochemical mechanism responsible for the tolerance of c. vulgaris to cadmium has not been fully elucidated. furthermore, the distribution of cadmium ions inside the cells has not been thoroughly investigated. this study reports on the cadmiumbinding ability of chlorella vulgaris beijerinck strain bt-09 in culture and on the subcellular localization of cadmium. this report comprises the first part of the investigation into the physiological mechanism behind the ability of c. vulgaris strain bt-09 to tolerate cadmium in its growth medium. materials and methods microalgal isolate, culture conditions, and treatment chlorella vulgaris beijerinck strain bt-09 cells were collected in bataan and the cultures acclimatized in bg-11 (ph 7.4) at 28 oc ± 2, under constant illumination (60 me m-2 s-1). the composition of the basal medium (in mm) was as follows: macronutrients, viz., 17.65 nano3, 0.18 k2hpo4, 0.3 mgso4, 0.25 cacl2, 0.19 na2co3, 0.0003 edta, 0.029 citric acid, 0.03 ferric ammonium citrate; micronutrients, viz., 0.46 b, 0.17 co, 0.32 cu, 9.2 mn, 1.6 mo, and 0.77 zn. all glassware were acid washed. variable concentrations of cadmium were added to the basal medium to come up with the following concentrations: 0.05 ppm, 0.5 ppm, and 5.0 ppm cdcl2. these were each inoculated with c. vulgaris strain bt-09 at its mid-log growth phase. cultures without cadmium served as control. growth analysis the growth response was assessed spectrophotometrically using chlorophyll a (chl a) content, monitored at various time intervals for 30 days adopting the modified combined methods of mackinney (1941), butterwick et al. (1982), and sartory & grobelaar (1984). cells from the cultures in test tubes were harvested by centrifugation (13,000 x g, 4oc, 5 min). the cell pellet was recovered, rinsed with fresh bg-11 medium plus nitrogen, taken up in 5 ml of 2:1 methanol-petroleum ether, sonicated (20 khz, 2 min), and subsequently incubated in a water bath (50oc, 4 min). the mixture was then cooled and passed through a coarse filter paper, with the filtrate read at 663 nm. the generation time (g) and specific growth rate (µ) of the cadmium-exposed, as well as unexposed, c. vulgaris cultures were calculated using the formulae shown below (brock,1979), where a0 and a1 are the absorbances at the initial time (t0) and the final time (t1), respectively. cadmium binding capacity of chlorella vulgaris cells cells grown in medium with or without cadmium were harvested by centrifugation (10,000 x g , 4oc, 10 min). after which, they were subjected to a series of water and 0.1 m edta washes. bg-11 medium augmented with cadmium served as positive control, while bg-11 medium without cadmium served as the negative control. the harvested cell pellet was rinsed thrice with ice cold ultrapure water. the recovered cell pellet was then subjected to triple washings with room temp ultrapure water and recovered each time by high speed centrifugation (10,000 x g, 5 min). the supernatants from each centrifugation step were pooled and set aside for aas analysis. each of the resulting cell pellet was washed thrice with 0.1 m edta solution, the edta 1 0 1 0 ln lna a t t µ − = − ln 2 g µ = 31 subcellular localization of cadmium washes were likewise pooled and set aside for aas analysis. the final pellet was recovered, weighed, and set aside also for aas analysis. subcellular localization of cadmium microsomes and cytosols cells exposed to 0.5 ppm cadmium for 2 days were harvested by centrifugation (10,000 x g, 4oc, 10 min). the resulting cell pellet was resuspended in ice cold ultrapure water and the resulting mixture was sonicated (20 khz, 6 min). the resulting suspension was centrifuged (1,000 x g, 4oc, 7 min) and the resulting pellet (mostly cell walls and cell debris) was washed twice with ice cold ultrapure water and submitted for aas analysis. one ml aliquot of the supernatant resulting from the first centrifugation was submitted for aas analysis, with the remaining supernatant further centrifuged (15,000 x g, 4oc, 15 min). the pellet (containing intact mitochondria and chloroplast) was washed twice with room temperature ultrapure water and submitted for gf-aas analysis. likewise, 1 ml aliquot of the supernatant resulting from 15,000 x gcentrifugation was submitted for gf-aas. the remaining supernatant was finally centrifuged (100,000 x g, 4oc, 30 min). the resulting pellet (representing microsomal fraction) and supernatant were also submitted for gf-aas. plasma membrane to determine the amount of cadmium bound to cell membranes, membranes were isolated following the procedure described by hodges & mills (1988). harvested cadmium-exposed cells were disrupted in 5 ml cold grinding medium [0.25 m sucrose, 3 mm edta, 2.5 mm dtt, and 25 mm tris-mes (ph 7.2)]. the suspension was centrifuged (13,000 x g, 4oc, 5 min). the pellet (presumably containing chloroplast, nuclei, and mitochondria) was collected and set aside for further fractionation, while the supernatant was centrifuged (80,000 x g, 4oc, 1 h) and the resulting supernatant was submitted for aas analysis. the recovered pellet was resuspended in 4 ml of 0.25 m sucrose and 5 mm potassium phosphate buffer (ph 7.8). the suspension was layered onto a sucrose step gradient (i.e., 20%, 30%, 34%, and 45%) containing 1 mm mgso4, 2.5 mm dtt, and 1 mm tris-mes (ph 7.2). the resulting mixture was centrifuged (95,000 x g, 4oc, 2 h). the plasma membrane and microsomes (concentrated at the 34%-45% interface) were pipetted out and submitted for gf-aas analysis. chloroplast and nuclei to confirm the presence of cadmium in the chloroplast and nuclei, the fraction obtained from the 13,000 x g centrifugation was further fractionated following the procedure described by orozco et al. (1988). the pellet was resuspended in 4 ml (shemna) buffer (330 mm sorbitol, 50 mm hepes-koh (ph 8.0), 2 mm edta, 1 mm mgcl2, and 5 mm naoac. the resulting mixture was layered onto a 40%-85% percoll step gradient and was centrifuged (6,650 x g, 4oc, 6 min), concentrating broken chloroplasts and intact mitochondria at the 0%40% interface. the interface was pipetted out and set aside for subsequent isolation of the mitochondria. the intact chloroplasts, which were concentrated at the 40%-85% interface, were similarly taken out. the remaining white pellets presumably contain the nuclei. the fractions containing the intact chloroplasts and nuclei were submitted for gf-aas analysis. mitochondria cadmium in the mitochondria was likewise determined following the method of hanson et al. (1988). the fraction containing the crude mitochondria and broken chloroplasts was resuspended in 2 ml mtesb buffer [0.3 m mannitol, 50 mm tris-hcl (ph 8.0), 3 mm edta, 0.1% bsa, 20 mm 2-mercaptoethanol, and 10 mm mgcl2]. the resulting suspension was mixed with 10 ml of sucrose-buffered solution (sbs) [0.3 m sucrose, 50 mm tris-hcl (ph 8.0), 20 mm edta, and 0.1% bsa]. the mixture was centrifuged (15,000 x g, 4oc, 15 min) and the recovered pellet was resuspended in 4 ml sbs specified above. this suspension was then layered onto a sucrose step gradient [1.6 m, 1.2 m, and 0.6 m sucrose, with 50 mm tris-hcl, 20 mm 32 lintongan, cariño, and rivero edta, and 0.1 % bsa, all at ph 8]. this layered mixture was centrifuged (15,000 x g at 4oc for 1 h) and the resulting cream colored bands at the interfaces were collected for gf-aas analysis. results and discussion growth analysis the chlorophyll a levels of the control, 0.05, 0.5 and 5 ppm cdcl2-treated cultures increased as the cultures aged (fig. 1). based on chl a levels, the log phase of the treated and untreated cultures occurred from days 1-18, followed by the stationary phase at day 19. five ppm cadmium adversely affected the normal growth of c. vulgaris strain bt-09 cultures. cultures grown at 5 ppm cdcl2 showed reduced chl a content, decreased specific growth rate and increased generation time (table 1). other studies have also reported that cadmium indeed decreases the growth rates of several species of microalgae. payne & price (1999) observed that chlamydomonas reinhardtii cells were sensitive to cadmium, showing an inhibition for growth at 3 x 10-8 m (0.005 ppm). other microalgae, including our c. vulgaris strain bt-09, are resilient to cadmium, not showing any marked growth inhibition when the cadmium concentration is below 5 ppm. in another study, a decrease in the number of anacystis nidulans cells was noted when the cells were exposed to 5 ppm cdcl2 (lee et al., 1992). exposure of cells to lower cadmium concentrations (i.e., 0.05 and 0.5 ppm) did not significantly affect chl a levels even when the respective chl a content for these cultures were lower than that of the control. on the otherhand, the chl a levels of the cells were 54% lower in cultures exposed to 5.0 ppm cdcl2. lee et al. (1992) also observed a decrease in the chl a content of anacystis nidulans when cells were grown in 5 ppm cdcl2. such a decrease was attributed to the disruption of thylakoid membranes by cadmium ions, resulting to the degradation of pigments. in a study conducted by masojidek et al. (2000), a decrease in the total chlorophyll content of algal cells was observed when cells were grown in culture media containing either cd, cu, hg, ni, zn, or pb. the total chlorophyll deterioration was attributed to the abstraction and replacement of mg2+ from chlorophyll molecules by the heavy metal atoms, leading to a change in the 10 1 0.1 0.01 0.001 20 6 10 14 19 23 27 30 a bs or ba nc e (6 63 n m ) time (days) 0 ppm 0.05 ppm 0.5 ppm 5 ppm fig. 1. chlorophyll a profile of c. vulgaris strain bt-09 exposed to varying levels of cdcl2 for 30 days. table 1. effects of cadmium on growth rate and generation time of c. vulgaris strain bt-09. cadmium concentration (ppm) cell density specific growth rate (µµµµµ/day) generation time (g/day) % inhibition chorophyll a profile specific growth rate (µµµµµ/day) generation time (g/day) % inhibition control 0.05 0.5 5.0 0.5270 0.5274 0.4999 0.3923 1.3152 1.3142 1.3865 1.7668 -----0 5.14 25.56 0.6562 0.6901 0.6678 0.4122 1.056 1.004 1.037 1.682 -----0 0 37.18 33 subcellular localization of cadmium functional characteristic of the specific chlorophyll molecules. uptake and subcellular localization of cadmium approximately 64% of the original cadmium concentration in the culture medium were associated with the c. vulgaris cells, as evidenced by the presence of the cadmium ions in their cytosol and cell wall fractions (table 2). the cell wall of c. vulgaris strain bt-09 played a major role in metal uptake as was demonstrated by the 5-fold higher cadmium content in the cell wall as compared to that in the cytosol. the same observation was noted by mapoy et al. (2001) in their study on chlorella vulgaris strain zs-22. they reported that cadmium associated with the cell walls of c. vulgaris strain zs-22 was 21 times higher than the cadmium associated with the cytoplasm. our result does not agree with that of carr et al. (1998) who found that more cadmium ions were found in the cytosol than in the cell wall when cells were treated with 11 ppm cd. our result, however, agrees with the observation of sandau et al. (1996) on the metal binding capacity of chlorella vulgaris and spirulina platenesis. both live and dead cells demonstrated their capacities to bind metal ions. the dead cells of c. vulgaris and s. platenesis were observed to adsorb heavy metal cations much more quickly and more effectively than the live cells (sandau et al., 1996). in our study, no measure was taken to separate the dead and live cells in the harvested cultures. thus, it is possible that the cell walls are derived from both live and dead cells. the cell wall of dead cells could have contributed to the number of binding sites of our cellular fractions. a substantial amount of cadmium ions was associated with edta washes. the cadmium ions that were stripped off by the edta washings correspond to the cadmium which was more intimately bound to the cell wall. these cadmium ions were probably held by electrostatic attractions to divalent functional groups (e.g., sulfates and sulfites) of polysaccharide components of the cell wall. the cadmium ions which were not dissociated by the edta chelating agent represented the metal ions very tightly bound to the cell walls. these cadmium ions are bound irreversibly, perhaps by covalent bonding, to the cell wall matrix. cadmium content was found to be unevenly distributed among the five subcellular fractions of c. vulgaris strain bt-09 (fig. 2). the cytosolic fraction contained the highest amount of cadmium. this amount, 198 µg/ l cd, represents 66% of the total cadmium that had either passively or actively entered the interior of cells. it must be noted that our cell fractionation methods do not always preserve the structural integrity of subcellular organelles. and also, there was no attempt to check the purity of the cytosolic fractions. thus, our cytosolic fraction can be a composite of soluble proteins from mitochondria, chloroplast, and/or vacuoles that have been broken down by our cell fractionation procedures. table 2. uptake of cadmium in c. vulgaris strain bt-091 cells. fraction µµµµµg/l cadmium associated % cadmium associated total cadmium associated with cell wall loosely associated (dissociated with water washes) tightly bound (dissociated with 100 mm edta washes) non-dissociable (residual cadmium in cell wall) cadmium associated with cytoplasm total cadmium recovered from cell fraction total cadmium used for treatment 217 6.86 172.00 38.00 45.10 262.10 52.61 1.67 41.72 9.22 10.95 63.58 372.5 µg 1one l of late log phase cells were grown for 2 days in bg-11 medium with 0.5 ppm cdcl2. 34 lintongan, cariño, and rivero in higher plants, the cytosol is generally the site for important metabolic enzyme activities and is the prime cellular compartment where metal-binding complexes are located (clemens, 2001). the study of carr et al. (1998) indicates that this situation can also be true for c. vulgaris, where they reported that when cells were treated with 11 ppm cd, most of the cadmium were associated with the cytosolic fraction rather than with the cell wall fraction. part of their results revealed that cadmium was associated with a low molecular weight polypeptide, presumed to be the metal-binding phytochelatin. the cytosolic enzymes of tolerant and non-tolerant plants are generally metal-sensitive (ernst et al., 1992; clemens, 2001). this situation may also be true for microalgae. the presence of cadmium ions in the cytosolic fraction of healthy cells could activate biochemical mechanisms that can alleviate cadmium toxicity. removal of excess metal ions from the cytosol can be achieved by efflux or by compartmentalization. a substantial proportion of cadmium (44 µg) was also detected in the chloroplast of c. vulgaris strain bt-09 cells. in higher plants, apart from the cytosol, chloroplasts contain glutathione. klapheck et al. (1987 & 1988) observed the localization and distribution of glutathione synthetase, glutathione and homoglutathione in plastids, and cytoplasm of pisum sativum and phaseolus coccineus leaves. glutathione serves to keep plastids in reduced state apart from its protective role against heavy metal toxicity by complexing with the metals. glutathione-metal binding has been observed in algae although this was not localized in the chloroplast (gekeler et al., 1988). it is possible that in our case, cadmium may be complexed with glutathione. the amount of cadmium detected in the mitochondrial fraction of c. vulgaris strain bt-09 was low (1.53 µg). nevertheless, our results are in agreement with the data reported by silverberg (1976) who studied the effect of three levels of cadmium (0.03, 0.05, and 0.1 ppm cdcl2) on the mitochondria of chlorella pyrenoidosa. he observed cd-containing granular inclusions attached to the cristae, detecting these inclusions by energy dispersive x-ray. still smaller amounts of cadmium (0.91 µg) were also detected in the plasma membrane fraction of our strain. since one of the functions of plasma membrane is transport, numerous carrier and channel proteins are embedded in the membrane. the cadmium ions detected in the plasma membrane fraction are probably entrapped metals in transit. one of the protein families important for heavy metal transport is the metalinorganic transport system which is found ubiquitously in eukaryotic organisms (nies, 1999). among the principal structural components of membranes are the phospholipids. it is also possible that the cadmium ions were bound to the phosphate and other charged group of the phospholipids. our strain accumulated more cadmium through its cell wall or cell wall-associated biomolecules as compared to its cytosolic fraction. the observed subcellular distribution of cadmium would indicate the presence of more than one mechanism to alleviate heavy metal stress. matsunaga et al. (1999) screened 191 strains of microalgae for potential organisms that could be used for on-site removal of heavy metals from the marine environment. of the 191 strains studied, 24 strains showed high capacity for cadmium uptake. chlorella sp. nkg16014 exhibited the highest cadmium uptake when exposed to 50 mm cd, taking up 65% of the metal from the medium and was deemed most promising for sequestering cadmium from the marine environment. when exposed to 91.6 mm, c. vulgaris 250 200 150 100 50 0 nickel chloroplast mitochondria plasma membrane cytosol subcellular fractions c ad m iu m ( µµµµ µg /l ) fig. 2. subcellular distribution of cd in c. vulgaris strain bt-09 exposed to 0.5 ppm cdcl2 for 2 days. 35 subcellular localization of cadmium strain bt-09 cells were able to take up 63.58%. thus, c. vulgaris strain bt-09 can be likewise classified as a potential bioscrubber, either as whole cells or by using their non-living cell walls. acknowledgments the authors thank the natural sciences research institute for the grant, and the institute of biology, the institute of chemistry and the marine science institute, of the college of science, university of the philippines, diliman for the use of equipment in their laboratories. references babich, h. & g. stotzky, 1981. effects of cadmium ions on the biota: influence of environmental factors. adv. appl. microbiol. 23: 55-177. boyle, t.p., 1984. the effect of environmental contaminants on aquatic algae. in whitton, b.a. (ed.) algae as ecological indicators. san diego, california, usa, academic press inc.: 237-256. brock, t.d., 1979. biology of microorganisms. microbiology. 3rd ed. englewood cliffs, new jersey, prentice hall, inc. 07632: 30-35. butterwick, c., s.i. heaney, & j.f. talling, 1982. a comparison of eight methods for estimating the biomass and growth of planktonic algae. br. phycol. j. 17: 69-79. carr, h.p., f.a. cariño, m.s. yang, & m.h. wong, 1998. characterization of cadmium binding capacity of chlorella vulgaris. bull. environ. contam. toxicol. 60: 433-440. clemens, s., 2001. molecular mechanisms of plant metal tolerance and homeostasis. planta. 212: 475-486. cobbett, c.s., 2000. phytochelatins and their roles in heavy metal detoxification. plant physiol. 123: 825-832. ernst, w.h.o., j.a.c. verkleij, & h. schat, 1992. metal tolerance in plants. acta bot. neerl. 41(3): 229-248. gekeler, w., e. grill, e.l. winnacker, & m.h. zenk, 1988. algae sequester heavy metal via synthesis of phytochelatin complexes. arch. microbiol. 150: 197-202. hanson, m.r., m.l. boeshore, p.e. mcclean, m.a. o’connell, & a.t. nivison, 1988. the isolation of mitochondria and mitochondrial dna. methods for plant molecular biology. 118: 257-273. hodges, t.k. & d. mills, 1988. isolation of the plasma membrane. methods for plant molecular biology. 118: 41-54. klapheck, s., c. latus, & l. bergmann, 1987. localization of glutathione synthetase and distribution of glutathione in leaf cells of pisum sativum l. j. plant physiol. 131: 123131. klapheck, s., h. zopes, h.g. levels, & l. bergmann, 1988. properties and localization of the homnoglutathione synthetase from phaseolus coccineus leaves. physiol. plant. 74: 733-739. lee, h.l., b. lustigman, v. schwinge, i-yu chiu, & s. hsu, 1992. effect of mercury and cadmium on the growth of anacystis nidulans. bull. environ. contam. toxicol. 49: 272278. mackinney, g., 1941. absorption of light by chlorophyll solutions. j. biol. chem. 140: 315-322. mapoy, r.d., c.a.m. untalan, s.l. vergara, & k.a.s. villegas, 2000. isolation and characterization of cadmiumbinding polypeptide from philippine strains: chlorella sp. (zam-22, cdo-13, and cav-25) and anabaena sp. (btg-01). bs biology thesis. institute of biology, university of the philippines, diliman, quezon city, philippines. 25-30. masojidek, j., j. maly, s. alessandrelli, m. koblizek, m. rizzuto, b. geiken, j. kopecky, j. komenda, o. prasil, & m.t. giardi, 2000. effect of heavy metals on the structure and function of photosystem ii: potential and prospects for use as bioindicator. proceedings of the 2nd workshop on chemical sensors and biosensors. 249-256. matsunaga, t., h. takeyama, t. nakao, & a. yamazawa, 1999. screening of marine microalgae for bioremediation of cadmium-polluted seawater. j. biotech. 70: 33-38. 36 lintongan, cariño, and rivero nies, d.h., 1999. microbial heavy-metal resistance. appl. microbiol. biotechnol. 51: 730-750. payne. c.d. & n.m. price, 1999. effects of cadmium toxicity on growth and elemental composition of marine phytoplankton. j. phycol. 35: 293-302. orozco jr., e.m., j. e. mullet, l. bowdoin, & n. chua, 1988. in vitro transcription of chloroplast protein genes. methods for plant molecular biology. 118: 125-146. sandau, e., p. sandau, o. pulz, & m. zimmermann, 1996. heavy metal sorption of marine algae and algal by-products. acta. biotechnol. 16(2-3): 103-119. sartory, d.p. & j.u. grobbelaar, 1984. extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis. hydrobiologia. 114: 177-187. silverberg, b.a., 1976. cadmium-induced ultrastructural changes in mitochondria of freshwater green algae. phycologia. 15(2): 155-159. inside front cover-21-2.pmd editorial board editor-in-chief maricor n. soriano, ph.d. associate editors mario a. aurelio, ph.d. earth sciences jose maria p. balmaceda, ph.d. mathematics zubaida u. basiao, ph.d. biology joel joseph s. marciano, jr., ph.d. computer science, engineering nemesio e. montaño, ph.d. biochemistry irene m. villaseñor, ph.d. chemistry corazon d. villareal, ph.d. managing editor dercylis g. mararac editorial assistant editorial advisors kelvin s. rodolfo, ph.d. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu alfonso m. albano, ph.d. dept. of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu victor c. gavino, ph.d. dept. of nutrition university of montreal, canada victor.gavino@umontreal.ca rudolf a. roemer, ph.d. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk raul k. suarez, ph.d. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu luis g. sison, ph.d. electrical and electronics engineering institute university of the philippines, diliman luis.sison@up.edu.ph science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p300.00/year (two issues), exclusive of postage us$ 25.00/year (two issues), exclusive of postage tel. no: (632) 981-85-00 loc. 4047 (632) 927-2567; 927-2309; 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences cover photo the cover shows four species of ferns thriving near the copper-rich soils of the lepanto mine in benguet. they are found to absorb the most copper in their roots and thus may be used for phytoremediation (claveria et al. page 1-12 this issue). photo courtesy of dr. rene claveria from the department of environmental science, ateneo de manila. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. 18_svd oblefias, soriano, and saloma 74 svd vs pca: comparison of performance in an imaging spectrometer wilma r. oblefias1, maricor n. soriano2, and caesar a. saloma3 national institute of physics, university of the philippines diliman, quezon city 1101 e-mail: 1woblefias@nip.upd.edu.ph; 2msoriano@nip.upd.edu.ph; 3csaloma@nip.upd.edu.ph abstract science diliman (july–december 2004) 16:2, 74–78 the calculation of basis spectra from a spectral library is an important prerequisite of any compact imaging spectrometer. in this paper, we compare the basis spectra computed by singular-value decomposition (svd) and principal component analysis (pca) in terms of estimation performance with respect to resolution, presence of noise, intensity variation, and quantization error. results show that svd is robust in intensity variation while pca is not. however, pca performs better with signals of low signal-to-noise ratio. no significant difference is seen between svd and pca in terms of resolution and quantization error. introduction a common method to measure image spectrum is to use a monochrome charge-coupled device (ccd) camera with narrowband interference filters. for example, to measure the spectrum from 400 to 700 nm at 1 nm resolution requires 301 filters and the same number of images. each color or grayscale value of the image corresponds to the intensity of the band pass of the filter used. singular-value decomposition (svd) and principal component analysis (pca) (hair et al., 1998) are used to compress multivariate data such as spectra. weighted spectral estimation can be obtained by linear superposition of the basis spectra computed using these techniques. we have designed, implemented, and characterized an imaging spectrometer microscope for measuring fluorescent and bioluminescent spectra (oblefias, 2004). an imaging spectrometer microscope, as its name suggests, is a microscope that not only delivers the magnified image of a slide specimen but gives its spectrum at each image point as well. this paper presents the estimation performance of our device using the basis spectra calculated from svd and pca. the two methods are compared by measuring the fidelity of estimation with respect to spectral resolution, noise level, intensity variation, and quantization. methods svd is a statistical method that looks for the component space where the data are most efficiently represented. if the data to be analyzed are spectra, each wavelength l of the spectrum will be considered as one component or axis. thus, a spectral library with spectrum from 400 to 700 nm at resolution dl equal to 1 nm will have 301 axes. each spectrum will be considered as data point. figure 1 shows an example of data set with two components (x1 and x2). svd vs pca 75 in svd the optimum component space is found by rotating the coordinate axes with respect to the origin. the first basis is the axis about which the data are most widely spread, or where the data have the largest variance. the second basis is the second axis, perpendicular to the first, which has the next highest data variance, and so on. pca, on the other hand, is a variation of svd. its main difference with svd is that the origin is translated to the mean. then the first basis is the axis about which the data are most widely spread. succeeding bases are calculated in the same way as that of the svd. when the set of basis spectra ei(l) are obtained, the estimated spectrum cest(l; x, y) of the reference spectrum c(l; x, y) can be expressed as a weighted linear superposition of the first few significant ei(l): , (1a) , (1b) where 〈c(l)〉 is the mean of the spectral library {ck(l)} and an is the coefficient of the nth e(l). the summation is taken from n = 1 to n = n, where n is the number of ei(l) that is utilized for estimation. “a” in the equation number refers to svd, while “b” refers to pca. relating the estimated spectrum to the output of the camera, we obtain , (2a) , (2b) where q and qmean are m-column vectors containing the channel output of a color camera and average color of the spectral library, respectively. t is an m×n transformation matrix that maps the expansion coefficients in a to the image space colors q (soriano et al., 2002; saloma et al., 2004). in spectral imaging, the image output channels {qm(x, y)}, the spectral library {ck(l)}, and the basis spectra ei(l)’s are known and the immediate task is to determine the component values of a. after a is known, one proceeds via eq. (1) to solve for the corresponding spectral estimation cest(l; x, y) which describes the optical spectrum at location (x, y) of the image of the spatially extended fluorescent sample. for a colored image, the values for the different qm(x, y)’s for every pixel location (x, y) of the twodimensional image are taken from the r, g, and b channel outputs (m = 3) of the camera. the unknown coefficients {an(x, y)} are determined via , (3a) , (3b) where t–1 is the inverse of t. the inversion matrix t–1 is defined only if t is a square matrix because the size of t is equal to m×n, i.e., t–1 exists only if n = m. to increase the number m of color channels, the sample is image-captured with a lightly colored transmission filter placed before the camera. with the insertion of a filter, the fluorescent sample is imaged under three more independent channels (m = 6), in addition to the original three (for r, g, b) provided by the three ccd camera in the absence of a filter (imai et al., 2000). accuracy of estimation was measured using fidelity f given by (4) q ta= fig. 1. diagram of svd for two-dimensional data. first basis x2 original axis second basis x1 original axis oblefias, soriano, and saloma 76 where 〈·〉 is the average value and c is the theoretical spectrum. fidelity describes the general similarity between theoretical and estimated spectra. perfect estimation occurs when f = 1. results and discussions bases spectra were computed using pca and svd from 423 spectra of fluorescence emissions. a gaussian emission spectrum , (5) where lo is the peak wavelength and s is the variance related to the width of the spectrum, was used to determine the narrowest spectrum that can be estimated. it was found that 15 basis spectra can be used to estimate a spectrum whose s is 14 nm and fidelity is approximately equal to unity. this is equivalent to a full width at half maximum (fwhm) of 33 nm. variance of less than 14 nm, a narrower spectrum, does not give estimation merit of unity even when the number of basis spectra are equal to 15. using s = 14 nm, two gaussian curves were superimposed with different lo. the minimum peak separation that can be resolved both by svd and pca is 28 nm. two peaks are said to be resolved if the ratio of the intensity at the midpoint to that at the maxima is 0.811 in accordance with the rayleigh criterion. the smaller peak separation of the two gaussian curves is estimated as a unimodal spectrum. more than 15 basis spectra, equivalent to more than five colored images, are needed to estimate an emission spectrum that has a fwhm of less than 33 nm. the same number of basis spectra can also resolve two peaks that are separated by less than 28 nm. in this study, however, the above result is already sufficient to estimate the emission of bioluminescent and naturally fluorescing samples since they are not usually narrow. two emission peaks also rarely occur (herring, 1993). figure 2 shows the minimum fwhm and peak separation of a bimodal spectrum that can be resolved as the number of basis spectra increases using svd and pca. bimodal spectrum cannot be resolved with less than five bases no matter how large the fwhm is. using the best fit curve, a fwhm of 1 nm and a peak separation of 2 nm can be resolved by using 301 basis spectra. however, this is not practical for our purpose because fluorescing bioluminescent samples do not have narrow spectra. experimentally, the increase or decrease in the intensity of emission of the sample cannot be fully controlled. the greater the intensity of the excitation light, the greater the intensity of the emission. the sample may also undergo photobleaching after long exposure to the excitation source that decreases the emission intensity. ideally, the estimated emission spectrum should not change if the intensity of the excitation is varied since emission spectrum is independent of the intensity of the excitation source. figure 3 shows the effect in average fidelity computed from 423 spectra in changing the intensity of the sample emission. svd is robust against changes in intensity. number of basis spectra p ea k se pa ra ti on (n m ) (b) y = 645.37x–1.0603 r2 = 0.98 number of basis spectra fw h m y = –43.919 ln(x) + 151.02 r2 = 0.93 (a) fig. 2. resolution with increasing number of basis spectra. (a) minimum fwhm that can be estimated and (b) minimum peak separation that can be resolved. svd vs pca 77 estimation merit is not affected by either an increase or a decrease of intensity. pca, however, is intensity dependent. increase in intensity has little effect as the number of basis spectra is increased. at the seventh basis spectra, estimation is the same as if there is no change in intensity. to remove the dependency of pca with intensity, at least 12 basis spectra must be used. this is equivalent to using at least four images. noise becomes more apparent with signals of low intensity. increase in noise results in low snr. figure 4 shows the effect of additive white gaussian noise at estimation merit. the snr was varied and calculated as , (6) where co is the energy of the theoretical signal and cd is the difference between the energy of the theoretical signal and the energy of the actual signal. the unit of the snr is decibel (db). at low snr, estimation merit is greatly affected. increasing the number of basis spectra when snr is equal to 20 db does not improve the estimation. as snr increases, the difference between the estimation merit with noise and that without noise decreases. at high snr, even if noise is present, its effect is negligible when the number of basis spectra is increased. when snr is gradually increased, noise is negligible in svd when snr is 42 db, while in pca, noise is negligible when snr is 40 db. thus, at low snr pca performs better than svd. quantization error is the result of the finite number of bits in the digitizer that converts the voltage output of the camera into grayvalue levels. figure 5 shows the fidelity using 8 bits for each camera channel. improvement in spectral estimation can be seen from one to seven basis spectra. spectral estimation degrades after the seventh basis spectra. this means that for such number of bits, using up to three images only is advisable. fig. 3. fidelity with intensity. light and dark lines are the result with intensity reduced to one fourth and intensity increased by four, respectively. (a) svd and (b) pca. continuous light line in b is for no variation in intensity. standard deviation is not shown. f number of basis spectra f number of basis spectra (a) (b) number of basis spectra f number of basis spectra f fig. 4. fidelity with the addition of white gaussian noise. (a) snr = 20 db, and (b) snr = 40 db. dark and light lines are the result using svd and pca, respectively. continuous dark line is for svd without noise and continuous light line is for pca. standard deviation is not shown. (a) (b) oblefias, soriano, and saloma 78 number of basis spectra f id el it y 1 0 -1 -2 -3 3 6 9 15 fig. 5. fidelity with 8 bits digitizer. the dark line is for svd without noise and the light line is for pca. standard deviation is not shown. it was observed, however, that using more than three images can be used to get better estimation for samples having a fwhm of greater than 56 nm. fidelity of narrower spectra follows the curve of fig. 5. as the number of bits increases, the graph of fidelity with the number of basis spectra approaches the value of fig. 3. significant improvement is observed up to 16 bits. using more than 16 bits does not give further improvement. comparison of performance of svd and pca when quantization is present shows no significant difference. summary singular-value decomposition (svd) and principal component analysis (pca) were used to calculate the basis spectra. using five colored images (15 basis spectra), both svd and pca can accurately estimate a unimodal spectrum whose minimum fwhm is 33 nm and a bimodal spectrum with peak separation of 28 nm. when intensity variation is considered, svd has a greater advantage than pca. thus, in an actual experiment provided that the sample has enough snr, svd is recommended since the intensity of emission cannot be controlled due to photobleaching and intensity variation of the excitation source. the threshold snr to have good estimation merit is 42 db for svd, while it is 40 db for pca. a snr that is lower than the threshold fails to estimate the spectrum correctly even when the number of basis spectra is increased. thus, pca should be used for low snr as long as photobleaching and intensity variation of the sample can be ignored. the digitizer should have at least 8 bits for samples with fwhm greater than 56 nm. increasing the number of bits shows improvement in spectral estimation. however, using more than 16 bits does not give further information. acknowledgment this work received financial support from the office of the vice chancellor for research and development (ovcrd) of the university of the philippines diliman. references hair jr., j., r. anderson, r. tatham, & w. black, 1998. multivariate data analysis, 5th ed. prentice-hall, engelwood cliffs, new jersey. herring, p., 1993. the spectral characteristics of luminous marine organisms. proc. r. soc. london. b220: 183–217. imai, f., r. berns, & d. tzeng, 2000. a comparative analysis of spectral reflectance estimated in various spaces using a trichromatic camera system. j. imaging sci. technol. 44: 280–287. oblefias, w., 2004. spectral imaging of fluorescent and bioluminescent samples. m.s. thesis, university of the philippines, diliman. saloma, c., w. oblefias, & m. soriano, 2004. spectral microscopy of live luminescent samples. in nanophotonics: integrating photochemistry, optics, and nano/bio materials studies. elsevier: chap. 30. soriano, m., w. oblefias, & c. saloma, 2002. fluorescence spectrum recovery from image color and non-negativity constraint. opt. express. 10: 1458–1464. sdinside front cove-jan.-june2015.pmd january-june 2015 • vol. 27 no. 1 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief irene m. villaseñor, ph.d. associate editors jose maria p. balmaceda, ph.d. louis angelo m. danao, ph.d. carlos primo c. david, ph.d. alonzo a. gabriel, ph.d. marco nemesio e. montaño, ph.d. jonas p. quilang, ph.d. rene n. rollon, ph.d. arnel a . salvador, ph.d. terence p. tumolva, ph.d. managing editor gonzalo a. campoamor ii, ph.d. editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor arvin a. mangohig on the cover: a coral reef in oriental mindoro, philippines. many reef areas throughout the philippines have become dominated by small-bodied species and individuals due to pressures such as over-f ishing and habitat degradation. description of the image by jonathan anticamara lab group. teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university cleveland, ohio, usa kenneth buckle, ph.d. food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, jr., ph.d. department of electrical and computer engineering ohio state university university of california, irvine university of illinois, urbana, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield sheff ield, united kingdom jeanmiare e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. centre for scientif ic computing and department of physics university of warwick united kingdom raul k. suarez, ph.d. department of ecology, evolution and marine biology university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. 05_tubal effect of the diurnal variation of the convective boundary layer height over metro manila on pollutant concentration genelita b. tubal1*, mariano estoque2, john holdsworth3, and jose villarin, sj3 1environmental science program, college of science university of the philippines, diliman, 1101 quezon city, philippines; 2department of meteorology and oceanography university of the philippines, diliman, 1101 quezon city, philippines; 3ateneo de manila university (admu), loyola heights, quezon city air pollutants are dispersed throughout a very thin layer of the atmosphere called the boundary layer (bl), and concentrations would be influenced by the thickness of the bl. a monostatic, biaxial, verticallypointing mie scattering 532 nm nd:yag lidar was used to observe the development of the daytime bl over metro manila in may 1999. the data profiles were background-subtracted, energy-normalized, and rangecorrected; 20,000 profiles (30-32 minute period files) were arranged in arrays in time sequential order. a matlab program with color enhancement capability was developed to display the range-time indicator (rti) image to visualize the bl. the convective bl height developed with a general pattern; it increased gradually in the early morning, rapidly from mid-morning until noontime, then slowly reaching its maximum height in the early afternoon. (the maximum height reached by the bl from 1– 4 may 1999 was 1635 m). bl height is maintained or lowered very slowly from mid-afternoon until sunset. the bl grew higher when the surface temperature and solar radiation received was greater. fair-weather active clouds inhibited the growth of the bl. when the relative humidity was higher, the base of the fairweather cloud field was lower; therefore, the mean bl height was also lower. prolonged sea breeze modified the convective bl by creating a much lower bl than when there was no sea breeze. around 75% of the total suspended particulates (tsp) in metro manila comes from traffic emissions. traffic volume over most part of metro manila including the main thoroughfares near the lidar site, peaks at around 09:00 local standard time (lst) and between 17:00 – 18:00 lst, although traffic volume is lower than at 09:00 lst. the traffic volume reduces to 80% from its morning peak at around noontime. the morning peak of the pollution concentration occurred earlier than that of the traffic. this could be due to the fact that the bl before 09:00 lst was much lower than after it. the pollution concentration on may 1 and 2 was reduced to less than 50% from its morning peak, a reduction much less than expected based on the traffic volume. this could be ascribed to the much higher bl around noontime. the may 2 pollution profile did not have a peak corresponding to the afternoon traffic volume peak because at that time the mean bl height was still very high. the may 3 and 4 pollution profiles were different from the two previous days, with values much greater around noontime. pollution during those times was concentrated in a much lower layer due to the sea breeze effect. key words: lidar, boundary layer, sea breeze, air pollution abstract science diliman (january-june 2002) 14:1, 28-3728 introduction air pollutants are dispersed and transported throughout a very thin layer of the atmosphere called the boundary layer (bl), a generally turbulent region whose characteristics are directly influenced by the ground (stull, 1988). turbulence in the bl is primarily convectively driven during calm sunny days and mechanically driven at night or even during windy overcast days (panofsky, 1985; benkley & schulman, 1979). the boundary layer driven by convection, hence called a convective boundary layer (cbl), grows from sunrise and stays until around sunset. the nocturnal or stable boundary layer (sbl) grows from around sunset through the night until around 08:00 lst (local standard time) the next day when it is eroded by the young cbl. see fig. 1. pollutants emitted at the surface are carried aloft by convective thermals due to turbulence in the cbl (ferrare et al., 1991). above the cbl is an inversion or stable layer that prevents the further movement of thermals upward. momentum causes a thermal to overshoot the inversion base, reaching the warmer region called the free atmosphere (fa). the negatively buoyant thermal decelerates and eventually sinks back any pollutant and moisture within it (stull, 1988). thus, the stable layer acts as a barrier to the transfer of pollutants from the cbl to the fa (beyrich & gryning, 1998). fa air is therefore much cleaner than the aerosol laden cbl. during the overshoot into the inversion, parcels of warm, usually drier fa air are entrained into the cbl, contributing to the increase of the cbl thickness. this region is called the entrainment zone (ez), (fig. 2). modification of the cbl by the much lower marine down into the cbl, which is mostly intact, trapping a lidar, working in the same principle as radar, detects the suspended air particulates and determines clearly the extent of the cbl from a distinct drop in the return lidar signal strength at the interface between the cbl and the fa. sometimes convective or fair-weather clouds, can be found at the top portion of thermals. the bases of the clouds of neighboring thermals are usually at different levels because of the variability of moisture contents between thermals. the top of the cbl in a field of cloud-topped thermals is a little higher than the lowest cloud base. during prolonged sea breeze, pollutants are trapped within a much thinner bl. this is caused by the fig. 1. the boundary layer in high-pressure regions over land consists of three major parts: a very turbulent convective boundary layer, a less turbulent residual layer, and a stable (nocturnal) boundary layer (stull, 1988). midnight sunrise noon 2000 m 1000 m 0 m sunset midnight local time fig. 2. the entrainment zone and the mean cbl height zi (stull, 1988). range a lt it ud e x local cbl top h2 zi ho convective boundary layer z 29 effect of the diurnal variation metro manila is under an air shed where pollutant concentrations are regularly over the standard values set by the world health organization (who) and the philippine guidelines. ill effects of air pollutants on human health make the study of air pollutant transport and dispersion indispensable. one of the basic parameters that need to be observed is the diurnal variation of the bl height. no observation of the bl over metro manila (or over any part of the philippines) has been recorded previously. this paper presents the results of the cbl observation on 1 4 may 1999 over metro manila using a lidar and meteorological instruments. methodology data acquisition the mie scattering lidar system used in this study was situated on top (15 m agl) of the climate studies building of the manila observatory (mo), ateneo de manila university, quezon city. the mo lidar system was, at that time, monostatic, vertically pointing with biaxial configuration (fig. 3). the axes of the laser beam and the receiver, a 28 cm-diameter schmidt cassegraine telescope, were separated by 30 cm. the laser source was an nd-yag laser that provided 532 nm light pulses at a repetition rate of 20 hz. the laser pulses were sent vertically to the atmosphere via dielectric mirrors after being expanded three times and collimated. the backscattered signal was received by the telescope and directed to a photomultiplier tube (pmt) via a 3-mm diameter iris, a collimating lens, a 10% transmitting neutral density filter, and 532 nm bandpass filter all positioned along the telescope optic axis. the electrical output of the pmt was amplified 15 times by a high bandwidth operational amplifier before it was fed into a compuscope cs1012 oscilloscope pc card. this card digitized the signal waveforms every laser pulse at a sampling rate of 20 mhz (single channel). this means that data points in one lidar data profile are 7.5 m apart. the data received was averaged to 1second ensemble. the energy per pulse was measured by an analog module laser energy monitor and recorded to a pc through the dt300 data acquisition board. a labview control and data-handling program allowed the automated data acquisition from around 04:30 lst to 19:00 lst daily. a real time display of every averaged lidar profile saved unto the computer’s hard disk was displayed on the monitor. with this, the alignment of the lidar system was closely monitored. bl advected from offshore to onshore by changes in the roughness or temperature over land. fig. 3. schematic diagram of the manila observatory lidar system. fig. 4. lidar site is represented by the big dot inside metro manila. (map courtesy of dr. e. ramos of the philippine institute of volcanology and seismology (phivolcs).) tubal et al. 30 the lidar site is situated near large bodies of water (fig. 4). around 14 km on its western side is manila bay, and around 14 km on its southeastern side is the laguna de bay. metro manila is characterized by nearly flat terrain. to characterize the meteorology of the area, surface meteorological measurements were obtained from four meteorological stations. three were situated near the lidar site – (a) the environmental management bureau (emb) air quality monitoring van, located 50 m ne of the lidar site, which measured solar radiation, surface temperature, relative humidity, and wind speed; (b) the university of the philippines – national center for transport studies (up ncts) horiba air quality monitoring van, 50 m nw of the mo lidar, which measured surface wind velocity; (c) the philippine atmospheric, geophysical, and astronomical services administration (pagasa) monitoring van, situated 600 m nw of the lidar site, which measured surface temperature and wind velocity. the fourth one, (d) the pagasa monitoring site at the science garden, situated 4 km ne of the lidar site, recorded wind velocity and cloud cover. theodolite balloons were released every 1.5 hours from 6:00 lst until 18:00 lst 600 m nw of the lidar site to determine the vertical wind velocity profile. at this same site, two to three radiosondes were launched daily to determine the vertical profiles of temperature and humidity. cloud cover was recorded every 15 minutes at the lidar site. the surface temperature used in the analysis of the cbl growth was the average of the temperature measurements obtained by the emb and pagasa monitoring vans. there were very slight variations between the two readings. the wind speed was the average of the wind speeds from the emb and upncts vans. although the two monitoring vans were very near each other, their wind speed measurements had slight variations. lidar data processing and analysis the lidar data processing, done after the intensive lidar observation period, took several steps. ten successive files of 100 profiles each were concatenated to have 1000 profiles in a bigger file with filename bearing the date and the nth set of 1000 profiles. a matlab program was developed to automatically correct each lidar profile and arrange them in an array. the correction done were subtraction of background signal, energy normalization to correct variation of laser output between laser shots, and range correction for the 1/r2 fall-off of laser energy. the background signal was taken to be the average value of the last 40 data points. at the range from where the data was taken (above 4.5 km), particulate matter was considered absent. two 1000-profile arrays were further joined to have a bigger array of 2000 profiles. this corresponds to a sampling time of about 32 minutes. taking the average wind speed during the observation period of about 3 ms-1, assuming that the convective thermals were advected across the laser beam path with this speed, then a sampling time of 32 min corresponds to sampling the cbl along a horizontal distance of about 5.7 km, a long enough range to obtain a good average of the cbl height. a matlab program was used to display the time sequence of the 30-minute data (2000 data profiles) in an image display called the range-time indicator (rti) map/image (piironen & eloranta, 1995) as shown in fig. 5. the range of intensity was chosen such that the corresponding color scale could display the contrast of the cbl, fa, and the clouds. in fig. 5, the dark region from 375 m and below corresponds to the height below the full overlap (fol) of the lidar where it could not detect any scatterers. the lighter regions above the fol region correspond to the bl and clouds. the white regions above 1.5 km in figs. 5c and 5d were cloud patches. note that figs. 5a and 5b are rtis of the same file only, the color enhancement in fig. 5b was chosen in such a way as to delineate the cloud tops (much lighter portions) from the cbl. figs. 5c and 5d are also rtis of the same file. note that in fig. 5d the cloud tops of the thermals (much lighter portions) are made more visible than in fig. 5c. the convective clouds in fig. 4b were forced clouds, in which the top portion of the cloud patches could still be seen, while the convective clouds in fig. 4d were active clouds. their top portions were no longer detectable and could be much higher than ez. the dark region above the convective clouds and bl was the fa that was devoid of scatterers. the normalized covariance of each 30-minute or so array was calculated and superimposed on the rti map in order to have an immediate comparison between effect of the diurnal variation 31 the manual and the automatic methods of determining the mean cbl height. the mean cbl height or the base of the inversion layer was estimated using 3 methods, depending on the amount and type of convective cloud cover. if there were no clouds along the lidar beam’s path, either above or at the cbl top, the automatic and manual methods described by hooper & eloranta (1986), and piironen & eloranta (1995) were adopted. in the automatic method, the lowest-altitude local maximum peak of the covariance profile for all data points taken over the 2000 or so lidar profiles was the mean cbl height (fig. 6c). with the manual or visual method, the height where there was 50% cbl air above and 50% fa air below was taken as the mean cbl top. the horizontal line in fig. 6c shows this level. this 5050 level was the definition of deardorff (1983) of the mean cbl top adopted by boers & eloranta (1986), ferrare et al. (1991), piironen & eloranta (1995). note that the 50-50 level and the peak of the covariance profile are at the same altitude. if clouds were present along the beam’s path as in fig. 6a, the covariance profile had its maximum at the cloud level, usually making the covariance below it very small. in this case, only the data points below the cloud signals were considered in the recalculation of the covariance. if there is a significant peak in the recalculated covariance profile, that level was taken as the height of the mean cbl as shown in figs. 6a fig. 5. (a) processed data for 08:51-09:51 lst on may 1, 1999; (b) the same period as (a) but delineated clouds (red areas) by adjusting the color enhancement scale; (c) similar to (a) for 08:47-09:16 lst on may 3, 1999; (d) similar to (b) for the same period as (c). may 1, 1999 normalized covariance 0 2 3 4 3.0 1.5 0 847 901 a lt it ud e (k m ) r el at iv e in te ns ity 14 x104 10 6 1 local time: 0847-0916(c) may 1, 1999 normalized covariance 0 2 3 4 3.0 1.5 0 847 901 a lt it ud e (k m ) r el at iv e in te ns ity 20 x104 12 8 1 4 16 local time: 0847-0916(d) may 1, 1999 normalized covariance 0 0.1 0.2 0.3 3.0 1.5 0 851 907 a lt it ud e (k m ) r el at iv e in te ns ity 16 x104 12 8 4 local time: 0851-0921(b) may 1, 1999 normalized covariance 0 0.1 0.2 0.3 a lt it ud e (k m ) 3.0 1.5 0 10 8 6 4 r el at iv e in te ns ity 851 907 x104 (a) local time: 0851-0921 and 6b. the lowest maximum peak of the covariance profile was taken to be the mean cbl top (horizontal line in fig. 6b). the bottom-up approach (or choosing the lowest significant peak of the covariance profile) was used to eliminate errors due to strong aerosol variability caused by aerosol layers above the cbl (piironen & eloranta, 1995) as in fig. 6b. the second covariance peak in fig. 6b pointed by the vertical arrow was due to the variability in the aerosols above the cbl and was not caused by the advection of the domeshaped tops of the thermals. it is clear from the figure 32 tubal et al. fig. 6. (a) in the presence of clouds, normalized covariance (ncov) profile at cbl becomes insignificant; (b) cloud signals eliminated and ncov recalculated to reveal ncov profile at cbl; (c) ncov profile peaks at cbl mean top if there is no cloud signal; (d) ncov profile peaks at cloud signal, cbl mean top is at 50-50 level below the ncov peak; (e) cbl mean top evaluated manually and automatically. normalized covariance a lti tu de ( m ) 0 .04.02 1125 375 906 922 9 3 5 7 x104 r el at iv e in te ns ity may 2, 1999: (9:06-9:36)(e) normalized covariance a lti tu de ( m ) 0 .04.02 0 1125 375 1706 1721 7 1 3 5 x104 r el at iv e in te ns ity may 4, 1999: (17:06-17:41)(b) normalized covariance a lti tu de ( m ) 0 21 0 3000 1500 1706 1721 7 1 3 5 x104 r el at iv e in te ns ity may 4, 1999: (17:06-17:41)(a) normalized covariance a lti tu de ( m ) 0 .04.02 0 1125 375 848 904 7 1 3 5 x104 r el at iv e in te ns ity may 4, 1999: (8:48-9:19)(c) normalized covariance a lti tu de ( m ) 0 .4.2 0 1125 375 927 943 7 1 3 5 x104 r el at iv e in te ns ity 9 may 4, 1999: (9:27-9:58)(d) 33 effect of the diurnal variation that the thermal tops are way below the second covariance peak. in cases where the covariance profile had multiple peaks (fig. 6e), the cbl mean top was evaluated manually by visual inspection of the 50-50 level (horizontal line in fig. 6e) and the result compared with the covariance profile. the covariance peak nearest the 50-50 level was considered as the mean cbl top. the automatic method could not be used when at least one thermal was cloud-topped since the covariance profile had its maximum at the cloud level, which was more often different from the 50-50 level as shown in fig. 6d. if the convective cloud cover was forced cloud and cover was less than 10%, the manual method was used. the 50-50 level in fig. 6d is shown by the horizontal line. if the convective cloud cover was more than 10%, the relationship of the inversion base (or the mean cbl top) and the lifting condensation level (lcl) zone near the base of the convective cloud as defined by stull (1988) was used. here, the mean cbl height is around 6% higher than the base of the lcl zone as shown in fig. 7 (stull, 1988). the lcl zone was determined from the base of the clouds in the convective cloud field. because of the subjectivity in evaluating the mean cbl height using the manual and the lcl zone-based methods, rti images were reevaluated for the mean cbl height at least four times, with succeeding evaluations separated by around one month. results from one-time evaluation were not very different (up to around 30 m difference) from the previous estimate of the mean cbl height. in cases where both the manual and the automatic methods could be employed to evaluate the mean cbl height, the two methods were both used and the results compared. it was found that the mean cbl height resulting from the two methods agreed very well (fig. 8). results and discussions the mean cbl height for the four observation days, 1-4 may 1999 is presented in fig. 9. there were no measurements at least one hour around noontime. with the sun directly overhead, the photomultiplier tube (pmt) was saturated and no useful data could be taken. the solid lines in figs. 9a-d denote the mean cbl height for the day. the dashed line in fig. 9a represents the average height of the part of the cbl without cloud top. this shows that the presence of clouds affects the development of the cbl, which in this case made the mean cbl height lower. the height of the convective cloud, on the other hand is affected by relative humidity. the higher the relative humidity, the lower is the base of the convective cloud, and hence, the lower the mean cbl height. the dashed lines in figs. 9c and d represent the height of the mixed layer due to sea breeze (fig. 9) fig. 7. relative frequency of occurence of various lcl and mean cbl height zi in a convective cloud field (stull, 1988). probability density a lt it ud e z entrainment zone median zi median lcl lcl zone 34 tubal et al. fig. 8. scatter plot for the manual and automatic methods of determining the mean cbl height. scatter plot for mean cbl height 800400 1200 1600 1600 800 400 1200 legend may 2 (15:44-16:55; 16:1616:47; 16:48-17:19; 17:2017:51) may 4 (16:02-16:31; 16:3417:03; 17:06-17:14) mean cbl height (m): automatic method m ea n c b l h ei gh t ( m ): m an ua l m et ho d and the solid lines, the mean height of the modified cbl. it can be observed from the different graphs shown in fig. 9 that the cbl growth, though different each day, has similarities. in general, the cbl growth can be divided into four stages: (a) slow cbl growth, which was evident from sunrise until around 08:00 lst; (b) rapid cbl growth recorded from around 08:00 lst to mid-morning; (c) cbl of nearly constant height which occurred between mid-morning and mid-afternoon; (d) decay of turbulence, which happened around midafternoon until sundown. here, either the mean cbl height remained the same or decreased a little. in events of prolonged sea breeze a lower mixed layer developed which remained low until sundown. there may be spatial variations of the cbl heights, but differences may not be very significant if the land cover is more or less uniform over the area as in metro (valeroso et al., 1992; teodoro, 1998). traffic volume over most part of metro manila especially in main thoroughfares near the lidar site, are seen to peak at around 09:00 lst and a smaller peak between 17:0018:00 lst, corresponding to the morning and afternoon peak hours. the traffic volume reduces by 20% at around noontime. the morning peak of the pollution concentration (fig. 10) occured earlier than that of the traffic. this could be due to the fact that the cbl before 09:00 lst was much lower than after it. pollution was concentrated in a much lower cbl. the reduction of the pollution concentration on may 1 & 2 was more than twice its morning peak, much higher than expected based on the traffic volume. this could be ascribed to the much higher cbl around noontime. the may 2 pollution profile did not have a peak corresponding to the manila. also, it should be remembered that the air sampled over the lidar site was advected from several kilometers from the lidar site – an equivalent of 3840 m for a wind speed of 2 ms–1 and 7680 m for a wind speed of 4 ms–1 within a 32-min period, the period over which the mean cbl height was estimated. hence, the derived cbl height over the lidar site can be taken more or less to be the average cbl height over most part of metro manila particularly the determination of the mean cbl height is a crucial element in understanding the intensity and transport of pollution in the metropolis. around 75% of the air pollution in metro manila come from traffic emissions the loyola heights and diliman areas. 35 effect of the diurnal variation a lti tu de ( m ) 7 9 11 mean cbl height (may 1, 1999) local standard time 13 15 17 1500 750 0 a lti tu de ( m ) 7 9 11 mean cbl height (may 3, 1999) local standard time 13 15 17 1500 750 0 a lti tu de ( m ) 7 9 11 mean cbl height (may 2, 1999) local standard time 13 15 17 1500 750 0 a lti tu de ( m ) 7 9 11 mean cbl height (may 4, 1999) local standard time 13 15 17 1500 750 0 fig. 9. time history of the mean cbl height per day for 1-4 may 1999. (a) (b) (c) (d) afternoon peak hour because at that time the mean cbl height was still very high. the may 3 and 4 pollution profiles were different from the two previous days, with values much greater around noontime. pollution during those times was concentrated in a much lower mixed layer generated by the sea breeze. figure 11 compares the effect of the much lower mixed layer due to sea breeze on pollution concentration. on may 1 (fig. 11a), the pollution concentration dropped very significantly at around noontime as a result of the reduction of pollution emission (decreased traffic volume) and the high cbl around that time. on may 3 (fig. 11b), although there was a reduction in pollution emission, the pollution concentration was much higher at around noontime. this was because of the very low mixed layer due to sea breeze. the very high peak of pollution concentration at 1600 lst on may 3 corresponds to a very low mixed layer that developed at that time. the pollution concentration peak on may 3 occurred earlier than on may 4 (fig. 10) since the sea breeze was felt at the lidar site much earlier on that day than on may 4. the disproportionate high pollution concentration in the events of sea breeze could also be due partly to the advection of a much polluted air to the lidar site by the sea breeze. acknowledgments this research study was made possible through the assistance of the department of science and technology (dost); ateneo de manila university (admu)–manila observatory (mo); office of the vice chancellor for research and development (ovcrd), university of the philippines, diliman; philippine atmospheric, geophysical, and astronomical services administration (pagasa); national center for transportation studies (ncts); department of environment and natural resources–environmental management bureau; university of the philippines national institute of science and mathematics education development (up nismed); the valuable training on lidar data processing and analysis given by local standard time carbon monoxide concentration (may 1 4, 1999) c on ce nt ra tio n (p pm ) 0.8 1.2 0.4 10 15 20 0 5 m1 m2 m3 m4 0 5 fig. 10. diurnal variation of co concentration on 1-4 may 1999 measured at the lidar site by the up-ncts air quality monitoring van. fig. 11. pollution concentration profile superimposed on the mean bl height for may 1 and 3, 1999. local standard time may 1, 1999 b l h ei gh t ( m ) c on ce nt ra tio n (p pm ) 0 0.6 1.2 5 9 13 17 750 1500 36 tubal et al. (a) local standard time may 3, 1999 b l h ei gh t ( m ) c on ce nt ra tio n (p pm ) 0 0.6 1.2 5 9 13 17 750 1500 (b) 37 effect of the diurnal variation dr. edwin eloranta of the university of wisconsin madison to the primary author; initiation of dr. minella alarcon of the existing mo lidar; and the admu lidar team, especially anne, aaron, and c. enaje for their help in the lidar operations. references benkley, c.w., & l.l. schulman, 1979. estimating hourly mixing depths from historical meteorological data. j. appl. meteor. 18:772-780. beyrich, f., & s. gryning, 1998. estimation of the entrainment zone depth in a shallow convective boundary layer from sodar data. j. appl. meteor. 37:255-268. boers, r., & e.w. eloranta, 1986. lidar measurements of the atmospheric entrainment zone and the potential temperature jump across the top of the mixed. bound.-layer meteor. 34: 357-375. ferrare, r.a., j.l. schols, e.w. eloranta, & r.l. coulter, 1991. lidar observations of banded convection during blx83. j. appl. meteor. 30:312-326. hooper, w.p., & e.w. eloranta, 1986. lidar measurements of wind in the planetary boundary layer: the method, accuracy and results from joint measurements with radiosonde and kytoon. j. climate appl. meteor. 25:990-1000. panofsky, h.a., 1985. the planetary boundary layer. adv. geophys. 28b:359-385. piironen, a.k., & e.w. eloranta, 1995. convective boundary layer mean depths and cloud geometrical properties obtained from volume imaging lidar data. j. geophys. res. 100:25 56925 576. stull, r.b., 1988. an introduction to boundary layer metereology. kluwer academic publishers: 647pp. page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 full gauge-art.2 full gauge-parameter-independent 29science diliman (january june 2000) 12:1, 29-34 abstract introduction full gauge-parameter-independent higgs-boson self-energy bernd a. kniehl1 and caesar p. palisoc2 1 ii. institut für theoretische physik, universität hamburg luruper chaussee 149, 22761 hamburg, germany email: bernd.kniehl@desy.de 2 national institute of physics, university of the philippines diliman, quezon city 1101, philippines email: cpalisoc@nip.upd.edu.ph we calculate the self-energy of the standard-model higgs boson by means of the pinch technique. we work in the general rξ gauge and show that the final result is independent of the gauge parameters. this result is useful in order to demonstrate that the threshold singularities encountered in the description of higgs-boson production and decay in the on-shell renormalization scheme only arise at physical thresholds and are independent of the chosen gauge. keywords: sm higgs-boson self-energy, pinch technique, s-matrix pt framework, gauge invariance, quantum corrections, perturbation theory the standard model (sm) of glashow, salam and weinberg is a non-abelian gauge field theory of the electroweak and strong interactions. it involves a yangmills sector in its classical lagrangian, which on the whole is invariant under a local su(2) and u(1) gauge transformation. this gauge invariance property is compromised when one wishes to canonically quantize the theory. in order to achieve this, one needs to introduce gauge-fixing terms in the lagrangian, which break the underlying gauge symmetry and introduce unphysical degrees of freedom. in order to compensate for this, one needs to add terms involving anticommuting faddeev-popov ghost fields into the lagrangian. both these requirements for the quantization complicate matters concerning practical calculations, for example, of radiative corrections involving quantum-loop diagrams, in the vectorand scalar-boson portion of the theory (papavassiliou & pilaftsis, 1998). as for the higgs-boson, its full oneloop self-energy, when calculated from the assumption that the higgs-boson is an asymptotic state of the scattering (s) matrix, while gauge independent on-shell, turns out to depend on the gauge parameter ξ off-shell. this property, among others, is by no means an obstacle in conventional perturbation theory, which predicts meaningful observables independent of the gauge-fixing procedure. however, this is not the case where the conventional perturbation theory breaks down, like in the strongly coupled theory of quantum chromodynamics (qcd) and in the vicinity of resonances in a weakly coupled theory (papavassiliou & pilaftsis, 1996) such as the electroweak sector of the standard model. the search for a self-consistent scheme for constructing off-shell green’s functions resulted in a formalism based on what is called the pinch technique (pt). kniehl & palisoc 30 as has been observed in refs. (kniehl, 1991; 1992; 1994), the one-loop corrected decay width of the sm higgs-boson as calculated in the conventional on-shell renormalization scheme exhibits singularities if the mass relations mh = 2mw or mh = 2mz happen to be satisfied. these threshold singularities arise from the wavefunction renormalization of the higgs-boson, which is given in terms of the derivative of the self-energy of the latter. one important application of the pt is to verify that these threshold singularities only occur at desirable properties like being resummable and process independent and complying with the unitarity of the s matrix (papavassiliou & pilaftsis, 1995; 1996; 1998). conventional higgs-boson self-energy in rξ gauge the feynman diagrams contributing to the conventional self-energy of the sm higgs-boson are depicted in fig. 1. in rξ gauge, we findphysical thresholds and are independent of the chosen gauge. this will be exhibited in a forthcoming paper (kniehl et al., in preparation), which will also explain how the threshold singularities are removed by the consequent application of the pole definitions of mass and width. in this paper, we derive the full higgs-boson self-energy in the pt framework. we work in the general renormalizable rξ gauge (fujikawa et al., 1972) and explicitly show that the result obtained is independent of the gauge parameters. our result generalizes a previous analysis (papavassiliou & pilaftsis, 1998). where g is related to the fermi constant by g = gf/ (2π√2), s is the higgs-boson virtuality, mh and mw are the masses of the higgs and w bosons, respectively, ξw is the gauge parameter associated with the w boson, the functions a0 and b0 are the so-called scalar oneand two-point functions (kniehl, 1994), respectively, and the term (w → z) signifies the contribution involving the z boson, which is obtained from the one involving the w boson by replacing mw 2 and ξw with mz 2 and ξz, respectively. in the ’t hooft-feynman gauge, with ξw = ξz = 1, eq. (1) agrees with the corresponding result given in eqs. (b.2) and (b.3) of ref. (kniehl, 1991). pinch technique the pt is an algorithm that renders one-loop gaugeboson self-energies gauge independent. it has rich applications in qcd (cornwall & papavassiliou, 1989) and in the electroweak sector of the sm (degrassi & sirlin, 1992; papavassiliou, 1990; papavassiliou & sirlin, 1994). at the one-loop level, the technique unravels self-energy contributions from vertex and box diagrams that are otherwise excluded in the conventional manner of computing the self-energy. by themselves, these pt self-energy contributions are gauge dependent. when combined with the conventional self-energy, these contributions exactly cancel the gauge-dependent terms of the former rendering the combined self-energy gauge independent. the resulting pt self-energy satisfies ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ,,,2 2 2 ,, 8 9 4 3 2 1 ,, 4 1 ,,3 2 2 1 3 2 22 0 22 0 2 22 0 42 0 2 22 0 42 22 0 42 2 2 0 22 0 2 ⎭ ⎬ ⎫ ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −−− ++ →+−− ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ +−+ ⎩ ⎨ ⎧ −−⎟ ⎠ ⎞ ⎜ ⎝ ⎛ +=π ∑ ffff f ff hhhhh wwwwh wwww wwhwwhh mmsbm s mamn mmsbmmam zwmmsbms mmsbmsm s mamsmam sg s ξξ ξ π (1) full gauge-parameter-independent 31 figure 1. feynman diagrams pertinent to the conventional self-energy of the sm higgs boson in rξ gauge. kniehl & palisoc 32 full pt contribution in rξ gauge in calculating the full pt contribution to the higgs-boson self-energy in rξ gauge, we use the s-matrix pt framework elaborated in ref. (degrassi & sirlin, 1992). the scattering process considered is a four-fermion process with the higgs-boson as intermediate state. in the formulation of ref. (degrassi & sirlin, 1992), the relevant amplitudes reflecting the gauge-boson and external-fermion interactions are described in terms of matrix elements of fourier transforms of time-ordered products of current operators. through successive current contractions with the longitudinal four-momenta found in the propagators of the massive vector bosons, ward identities are triggered, after which the relevant pinch contribution is identified upon application of appropriate equal-time commutators of currents. the relevant feynman diagrams are depicted in fig. 2. setting aside the details, the full pt contribution is found to have the following form, discussion of the results some comments on eqs. (1) and (2) are in order. as expected (papavassiliou & pilaftsis, 1998), their sum, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ,,,2 2 2 ,, 8 9 4 3 2 1 ,,32 4 3 2 22 0 22 0 2 22 0 42 0 2 22 0 422 2 2 2 0 2 2 ⎭ ⎬ ⎫ ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −−− ++ →+⎥ ⎦ ⎤ ⎢ ⎣ ⎡ +−⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ ++ ⎩ ⎨ ⎧ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ += ∆π+π=π ∑ ffff f ff hhhhh wwwww h h ww h hhhh pt hh mmsbm s mamn mmsbmmam zwmmsbmsmm m m mam mg sss π ( ) ( ) ( ) ( )[ ] ( ) ( ) ( ) ( ) ( ) ( ) . 2 1 ,, 4 1 ,, 4 1 2 1 22 0 42 22 0 222 2 0 2 0 2 ⎭ ⎬ ⎫ →+−+ ⎥⎦ ⎤ ⎢⎣ ⎡ ++−− ⎩ ⎨ ⎧ −−=∆π zwmmsbms mmsbmmsms mamams g s wwwwh wwwhh wwwhhh ξξ ξ π(2) (3) which represents the full pt self-energy of the higgsboson, is manifestly independent of ξw and ξz for all values of s. in the special case of s = mh 2, πhh(s) is separately gauge independent, while ∆πhh(s) vanishes. in fact, this ensures that the total decay width of the higgs-boson, which is proportional to the absorptive part of its self-energy, is gauge independent and does not receive additional contribution due to the application of the pt. in the ’t hooft-feynman gauge, our full pt result agrees with that reported in ref. (papavassiliou & pilaftsis, 1998). for gauges other than the ’t hooft-feynman gauge, our result only differs from the one in ref. (papavassiliou & pilaftsis, 1998) with respect to the first term in eq. (2). however, consistency between the two results remains and has not been compromised, since the authors of ref. (papavassiliou & pilaftsis, 1998) have explicitly omitted contributions from tadpole and seagull graphs, which are included in our consideration and are the ones responsible for the presence of the first term in eq. (2). on the other hand, their significance must not be underestimated, since they render the dispersive part of the higgs-boson selfenergy gauge independent as well. full gauge-parameter-independent 33 figure 2. feynman diagrams pertinent to the pinch parts of the self-energy of the sm higgs boson kniehl & palisoc 34 acknowledgments the work of b.a.k. was supported in part by the bundesministerium für bildung und forschung under contract no. 05 ht9gua 3, and by the european commission through the research training network quantum chromodynamics and the deep structure of elementary particles under contract no. erbfmrxct980194. the work of c.p.p. was supported by the german academic exchange service (daad) through grant no. a/97/00746. references cornwall, j. m. & j. papavassiliou, 1989. gauge invariant three gluon vertex in qcd. phys. rev. d40: 3474-3485. degrassi, g. & a. sirlin, 1992. gauge-invariant selfenergies and vertex parts of the standard model in the pinch technique framework. phys. rev. d46: 31043116. fujikawa, k., b. w. lee, & a. sanda, 1972. generalized renormalizable gauge formulation of spontaneously broken gauge theories. phys. rev. d6: 2923-2943. kniehl, b. a., 1991. radiative corrections for η → zz in the standard model. nucl. phys. b352: 1-26. _______, 1991. radiative corrections for h→w+w (δ) in the standard model. nucl. phys. b357: 439-466. _______, 1992. radiative corrections for h→f f (δ) in the standard model. nucl. phys. b356: 3-28. _______, 1994. higgs phenomenology at one loop in the standard model. phys. rep. 240: 211-300. kniehl, b. a., c. p. palisoc, & a. sirlin. july 2000. hep-ph/0007002. papavassiliou, j., 1990. gauge invariant proper selfenergies and vertices in gauge theories with broken symmetry. phys. rev. d41: 3179-3191. papavassiliou, j. & a. pilaftsis, 1995. gauge invariance and unstable particles. phys. rev. lett. 75: 3060-3063. _______, 1996. a gauge-independent approach to resonant transition amplitudes. phys. rev. d53: 21282149. _______, 1996. gauge-invariant resummation formalism for two-point correlation function. phys. rev. d54: 5315-5335. _______, 1998. gauge-and renormalization-groupinvariant formulation of the higgs-boson resonance. phys. rev. d58: 053002-1-053002-26. papavassiliou, j. & a. sirlin, 1994. renormalizable w self-energy in the unitary gauge via the pinch technique. phys. rev. d50: 5951-5957. − page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 page 8 images image 1 page 9 images image 1 page 10 images image 1 page 11 images image 1 page 12 images image 1 page 13 images image 1 page 1 images image 1 page 2 images image 1 page 3 images image 1 page 4 images image 1 page 5 images image 1 page 6 images image 1 page 7 images image 1 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(ed.) the molecular structure of membranes. berlin heidelberg, new york, springer: pp. 53 responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 8.0 figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each figure or table on a separate page. at the lower hand corner, the name of the author and the figure number should be indicated. illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2” x 11” size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructed photographic prints (not photocopies), trimmed at right angles and in the final size desired by the author. ocr document 04_herrera 51 synthesis of pb-doped bi-2223 * corresponding author abstract science diliman (july-december 2002) 14:2, 51-58 synthesis of pb-doped bi-2223 from pb-doped bi-2212, ca 2 cuo 3 , and cuo above the glass transition temperature of bi-2212 marvin u. herrera1* and roland v. sarmago2 1materials science and engineering program, college of science university of the philippines, diliman 2condensed matter physics laboratory national institute of physics, college of science university of the philippines, diliman tel no.: 434-4260; fax no. 920-5474 email: 1mherrera@nip.upd.edu.ph synthesis of pb-doped bi-2223 from pb-doped bi-2212 (pb=0.3), ca 2 cuo 3 , and cuo was done by sintering at the glass-phase temperature of pb-doped bi-2212. the sample sintered at 850°c possesses nearly 100% pb-doped bi-2223, as revealed from the xrd pattern and magnetic susceptibility data. the presence of holes, terrace-like features, and magma-like flow features in the sem micrographs of the sample strongly support a glass-state pb-doped bi-2223 formation. key words: pb-doped bi-2223, glass transition temperature introduction bi-2223 has the highest critical temperature (110k) among the superconducting phases of the bi-sr-cacu-o system, but it is the most difficult to synthesize (khaled et al., 1996; murayama et al., 1988; shimojima et al., 1989). the difficulty of preparing bi-2223 lies in the complexity of its structure and its narrow phase stability. considerable effort has been made in order to synthesize single-phase bi-2223 (zhu et al., 1999). methods of increasing the volume fraction of bi-2223 includes long sintering time (yu et al., 1996; hadano et al., 1988), reduced oxygen partial pressure (oka et al., 1989; hadano et al., 1988), control of composition (endo et al., 1989; hadano et al., 1988), and pb-doping (hadano et al., 1988). although bi-2223 in pure form is quite difficult to achieve, substitution of lead enhances its formation, and it is the most common way to increase the volume fraction of bi-2223. the role of lead in the formation of bi-2223, however, is still obscure. studies on bi-2223 revealed that: (1) bi-2212 precedes the formation of bi-2223 (holesinger et al., 1996); and (2) the presence of a liquid phase (ono, 1988) and the occurrence of partial melting (hadano et al., 1988; oka et al., 1989) enhances its formation. intercalation models take into account that bi-2223 comes from bi-2212 via insertion of ca-cu-o units. one variation of this model states that cuo 2 /ca bilayers are inserted into the cuo 2 / ca/cuo 2 blocks of bi-2212 to form bi-2223 (garnier et al., 2000). another states that insertion of cacuo 2 slab into bi-2212 results in the formation of bi-2223 (zhu et al., 1999). nucleation and growth models are about the involvement of a liquid phase in bi-2223 52 herrera and sarmago formation. dissolution precipitation, low-level mobile liquid droplet, partial molten-phase precipitation, and twodimensional nucleation growth describe the role of the liquid in bi-2223 formation (garnier et al., 2000). since it was observed that bi-2212 precedes bi-2223 formation, using bi-2212 as the starting material would offer easier reaction since it would readily transform to bi-2223, as opposed to using carbonate and oxides as precursors. the observed fluid-like movement that is associated with increased bi-2223 formation could be bi-2212 in its glassy phase. we use the term “glassy phase” to describe the state in which a substance retains some degree of crystallinity but possesses fluid-like mobility. glass state occurs above the glass transition temperature and below the melting point. we expect that a substance is more mobile in glassy phase, which gives them the ability for faster reaction, than in solid phase. the melting point of bi-2212 (for certain pbdoped level ) is below the melting point of bi-2223, so it is possible to synthesize bi-2223 at the glassy state of bi-2212. differential thermal analysis (dta) curves of bi-2212 is presented in the works of liu et al. (1988). other substances which precedes bi-2223 formation, such as those belonging to the cao-cuo system, have melting points higher than that of the bi-2223 system. the phase diagram of the cao-cuo system is presented in the works of risold et al. (1995). the possibility of having a glassy state in bi-2212 lies in its structural anisotropy. the a,b plane of the superconducting phases of the bi-sr-ca-cu-o system (bi-2212 and bi-2223) are weakly bonded between the neighboring bio layers (majewski, 2000). the weaker bonding of a,b planes are dissociated at a lower temperature (glass transition temperature) than the bonding at other directions (that dissociates at the melting point). when the bonding between the bio layer are dissociated, the a,b plane layers can move more freely. there is still crystallinity within a layer since the bonding between the b,c planes and a,c planes are still present. the concept previously stated indicates that to facilitate the formation of bi-2223, it is necessary to sinter the sample at a temperature just below the melting point where the glassy state is expected and has optimum effect. for pb-doped bi-2223 (pb=0.3), the candidate sintering temperature for a fast-forming bi-2223 would be at 850°c. this temperature corresponds to the position of the pre-peak of bi-2212 endothermic curve (liu et al., 1998), which also corresponds to melting. in this paper, pb-doped bi-2223 was synthesized from pb-doped bi-2212 via partial melting in the glass state. by avoiding decomposition, results indicate that bi-2223 formation becomes faster and more homogeneous. magnetic susceptibility measurements suggest that conversion of bi-2212 phase to the bi-2223 is almost 100% complete. methodology stoichiometric amounts of oxide/carbonate precursor powder were used to prepare pb-doped bi-2212 (pb=0.3) and ca 2 cuo 3 via solid state reaction. these were used as precursors for the formation of pb-doped bi-2223. these were mixed with cuo to achieve a stoichiometric amount of bi-2223. the mixture was then sintered at 830°c, 840°c, and 850°c for 50 to 150 hours. the presence of bi-2223 was detected using x-ray diffraction (xrd) and was verified using magnetic susceptibility. confirmation using susceptibility measurements was preferred over resistivity since the former does not rely on granular connectivity to manifest a signal. the samples were observed using a scanning electron microscope (sem). results and discussion our success in producing bi-2223 from bi-2212 (pb=0.3), ca 2 cuo 3 , and cuo is verified by the magnetic susceptibility data (fig. 1) and the xrd patterns (fig. 2). from the magnetic susceptibility measurements (fig. 1b), the sample sintered at 850°c shows an almost single phase bi-2223, with a critical temperature of 103k. the sample sintered at 830°c shows small amounts of bi-2223 (fig. 2a). the sample is composed of unreacted bi-2212. increasing the sintering time up to 150 hours does not show any significant increase in bi2223 volume fraction. fig. 2b shows the xrd pattern of the sample sintered at 840°c, which shows 53 synthesis of pb-doped bi-2223 fig. 1. magnetic susceptibility of samples sintered for 50 hrs at (a) 840°c and (b) 850°c. -2.5 -3.5 -4.5 -5.5 -6.5 -7.5 0 20 40 60 80 100 120 temperature (ok) s u s c e p ti b il it y ( a rb . u n it s ) a -8.5 0 20 40 60 80 100 120 temperature (ok) -2.5 -3.5 -4.5 -5.5 -6.5 s u s c e p ti b il it y ( a rb . u n it s ) b proportionate amounts of bi-2212 and bi-2223 (the volume fraction of bi-2223 is approximately 40%). large bi-2223 volume fraction is exhibited by the sample sintered at 850°c (fig. 2c). practically, most bi-2212 is reacted to form bi-2223. the volume fraction at 830°c shows very slow reaction rate. in fact, bi-2223 is known to have a sluggish phase formation (zhu et al., 1999). sintering at 850°c greatly improved the volume fraction of bi-2223. the temperature 850°c is at the pre-peak of bi2212 endothermic curve (liu et al., 1998), which2qqqqq r e la ti v e i n te n s it y 0 0.5 1 20 25 30 35 2qqqqq 2 2 1 2 2 2 1 2 2 2 1 2 2 2 1 2 2 2 1 2 2 2 2 3 2 2 1 2 2 2 2 3 2 2 1 2 c a 2 c uo 3 a 0 0.5 1 r e la ti v e i n te n s it y 20 25 30 0 0.5 1 c 2 2 2 3 2 2 2 3 2 2 1 2 2 2 2 3 2 2 2 3 2 2 2 3 2 2 2 3 c a 2 c uo 3 35 2 2 2 3 fig. 2. xrd pattern of samples sintered for 50 hrs at (a) 830°c, (b) 840°c, and (c) 850°c. 20 25 30 35 2qqqqq 2 2 1 2 2 2 2 3 2 2 1 2 2 2 2 3 2 2 1 2 2 2 1 2 , 2 2 2 3 2 2 1 2 2 2 2 3 2 2 1 2 , 2 2 2 3 2 2 2 3 2 2 1 2 , 2 2 2 3 c a 2 c uo 3 b 54 herrera and sarmago corresponds to melting. in this temperature, bi-2212 is expected to be at its glassy phase, which possesses higher mobility than the bi-2212 in the solid phase. the corresponding sem images of the three samples are presented in fig. 3. the image in fig. 3a is the sample sintered at 830°c, showing unreacted precursors. the grains are smaller in comparison with bi-2223 grains in the sample sintered at 850°c (fig. 3c). the grain size of the samples sintered at 850°c is the largest since it is sintered at a higher temperature. owing to the anisotropy of the superconducting phases of bi-sr-ca-cu-o (bi-2212 and bi-2223), the rate of crystallization about the a,b planes is much faster (1000x) than in the c-direction (majewski, 2000). due to the slow crystallization rate, the c-direction would have the thinnest part in the grain. for comparison with fig. 3. sem images of samples sintered for 50 hrs at (a) 830°c, (b) 840°c, and (c) 850°c. 2 mmmmmm 2 mmmmmm 2 mmmmmm fig. 4. (a) crystallographic axis of bi-sr-ca-cu-o grain; (b) sem image of grains sintered at 830°c; and (c) sem image of grains sintered at 840°c. b a c a b a c 1mm acc. v. spot magn det wd exp 6.00kv 3.0 20000x se 10.0 415 a c b a b c b 5mm acc. v. spot magn det wd exp 6.00kv 3.0 4000x se 15.0 429 a-b plane c 55 synthesis of pb-doped bi-2223 sem images, the crystallographic direction of bi-srca-cu-o grains is presented in fig. 4a. the aand bdirections could be treated isotropically. they can be labeled arbitrarily in any direction in the plane perpendicular to c-direction. the relative proportions of both bi-2212 and bi-2223 phases from samples sintered at 840°c suggest that it would be possible to observe the transformation of bi2212 (frozen in the sample) to bi-2223. since the reaction at 840°c is slower than the reaction at 850°c (because the later temperature is higher), it is possible to see remnants of bi-2212 movements. these movements are no longer apparent in samples sintered at 850°c due to the rapid reaction rate. by comparing the sem images of the samples sintered at 830°c and 840°c (fig. 4b and fig. 4c, respectively), we could see the difference between the grains of mostly unreacted sample (sample sintered at 830°c) and those of the sample which reacted (sample sintered at 840°c). the grains of samples sintered at 830°c are unfused and stacked with other grains, while the grains of the samples sintered at 840°c are fused with other grains. these observations indicate that at 840°c the sample becomes very soft and attain fluid-like movement as compared with samples at 830°c, which are solid. this agrees with the idea that the presence of fluid enhances bi-2223 formation (khaled et al., 1996; ono, 1988). 5 mm acc.v spot magn det wd exp 6.00kv 3.0 4000x se 19.5 429 terrace-like feature hole magma-like flow fig. 5. sem image of sample sintered at 840°c showing terrace-like feature, magma-like flow, and holes. further examination of the sample sintered at 840°c reveals the presence of terrace-like structures, fluidlike movements, and holes (figs. 5, 6, and 7). two important characteristics of these features include: (1) movement between planes that are manifested by the displacement of one layer with respect to other layers; and (2) there exists a degree of order within a plane as exemplified by the layered orientation of the plane. these are the features of a substance in the glassy state. fig. 6 shows the layered structure where one layer is displaced with respect to other layers, resembling a terrace. fig. 7 shows the presence of a magma-like flow feature (fig. 7a) and holes (figs. 7b, 7c, and 7d). holes also have a structure similar to a terrace. fig. 8 shows the possible mechanism, which happens to the bi-2212 grains. fig. 8a shows the initial condition of bi-2212. the bonding in all directions is still intact. when bi-2212 is brought above the glass transition temperature (fig. 8b), the bonding between the bi-o layers (a-b planes) is dissociated. when this happens, the a-b planes can move freely with respect to each other. since the bonding in the other directions is still intact, a degree of order still exists within a plane. the fast formation of bi-2223 from the glassy phase of bi-2212 could explain the relationship between the existence of fluid-like movement and large bi-2223 volume fraction. the increase in the rate of bi-2223 formation is a result of the higher mobility of bi-2212, due to the freely moving a-b planes of bi-2212 above the glass transition temperature. this allows bi-2212 to move faster and react with other reactants to form bi-2223. this shows the importance of fluid-like movement in bi-2223 formation. the above concept offers a possible explanation regarding the relationship between the increase in bi2223 volume fraction and the increase in pb-doping. likewise, it could also explain the existence of an optimum value of pb-doping in bi-2223. the melting point of bi-2212 decreases with an increase in pbdoping. dta of bi-2212 with different pb-doping levels is presented in the works of liu et al. (1998). for a sample sintered at a particular sintering temperature (below the melting point of undoped bi-2212), increasing the amount of pb-doping entails the glass state to be nearer the sintering temperature, which results in faster reaction. optimum pb-doping exists when the melting 56 herrera and sarmago temperature of pb-doped bi-2212 is just below the sintering temperature. further increase in the amount of pb will result in the melting of the sample. summary and conclusion pb-doped bi-2223 (pb=0.3) from pb-doped bi-2212 (pb=0.3), ca 2 cuo 3 , and cuo, at the glassy phase of bi-2212, was successfully prepared. the bi-2212 phase dominates the samples sintered at 830oc. at 840°c, bi-2212 and bi-2223 phases coexist at almost equal proportions. at 850°c, bi-2223 dominates such that the presence of bi-2212 is no longer discernible from the susceptibility measurements. sem investigation reveals the presence of frozen, fluid-like movement in the material, which is attributed to the formation of a glassy phase of the sample sintered at 840°c and above. this glassy phase is responsible for the enhancement of bi-2223 formation because bi-2212 can move freely to react with ca 2 cuo 3 and cuo. references endo, u., s. koyama, & t. kawai, 1989. composition dependence on the superconducting properties of bi-pbsr-ca-cu-o. jpn. j. appl. phys. 28(2): l190-l192. garnier, v., i. monet-laffez, & g. desgardin, 2000. kinetics study of the bi-2223 grain-growth thickness. physica. c. 349: 103-112. hadano, t., k. aota , s. ikeda, k. nakamura, & k. ogawa, 1988. growth of the 2223 phase in leaded bi-sr-ca-cu-o system. jpn. j. appl. phys. l2055-l2058. holesinger, t.g., k.v. salazar, d.s. phillips, b.l. sargent, j.k. bremser, j.f. bingert, j.o. willis, & d.e. peterson, 1996. a two-powder process for bi-2223 precursors. j. mater. res. 11(1): 28-38. fig 6. sem image of sample sintered at 840°c showing terrace-like feature. 5 mmmmmm 2 mmmmmm 5 mmmmmm 2 mmmmmm 57 synthesis of pb-doped bi-2223 khaled, j., t. komatsu, & k. matusita, 1996. a new model for the formation of high-tc phase in superconductive (bi,pb) 2 sr 2 ca 2 cu 3 o x glass-ceramics. j. mater. sci. mater. elect. 7: 261-266. liu, h., l. liu, y. zhang, & z. jin, 1998. effects of pb and ca on the melting point of the 2212 phase in the (bi,pb)srcacuo system. j. mat. sci. let. 17: 665-667. majewski, p., 2000. material aspects of the high-temperature superconductors in bi 2 o 3 -sro-cao-cuo system. j. mat. res. 15(4): 854-870. murayama, n., m. awano, e. sudo, & y. torii, 1988. cation contents and superconducting properties of the high-tc phase of bi-pb-sr-ca-cu-o ceramics. jpn. j. appl. phys. 27(12): l2280-l2282. oka, y., n. yamamoto, h. kitaguchi, k. oda, & j. takada, 1989. crystallization behavior and partially melted states in bi-sr-ca-cu-o. jap. j. appl. phys. 28(2): l213-l216. b hole c a c aa fig 8. (a) bi-2212 grains below glass transition temperature; and (b) movement of bi-2212 above the glass transition temperature. 2 mmmmmm 5 mmmmmm 5 mmmmmm 2 mmmmmm fig 7. sem image of sample sintered at 840°c showing magma-like flow feature (a) and holes (b-d). 58 herrera and sarmago ono, a., 1988. crystallization of 107k superconducting phase and partial melting in the bi-(pb)-sr-ca-cu-o system. jap. j. appl. phys. 27(12): l2276-l2279. risold, d., b. hallstedt, & l. gauckler, 1995. thermodynamics assessment of ca-cu-o system. j. am. ceram. soc. 78(10): 2655-2661. shimojima, h., k. tsukamoto, & c. yamagishi, 1989. preparation of the high-tc superconductive bi-pb-sr-ca-cu-o film pyrolysis of organic acid salts. jpn. j. appl. phys. 28(2): l226-l228. yu, s., y. okuda, & e. takayama-muromachi, 1996. critical current densities and irreversibility fields in pb-doped and pb-free bi 2 sr 2 ca 2 cu 3 o y superconductors. jpn. j. appl. phys. 35: 2619-2623. zhu, w., c. kuo, & p. nicholson, 1999. diffusion calculations for the 80-kto-110k bi(pb)srcacuo superconducting phase formation. j. mat. res. 14(11): 4143-4147. guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions: ✔ if changes are made, choose a new title for the paper. ✔ use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality. ✔ reference your conference paper in the appropriate locations. ✔ include a footnote in the submitted manuscript stating, e.g., "an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings." ✔ indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed. ✔ provide the original conference paper (upload a pdf file during the submission process). ✔ if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions: ✔ expand the background section and include additional references. ✔ include novel scientific content and expanded descriptions of procedures. ✔ provide data that was not published at the conference. ✔ revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission from the editors of ieee sensors journal) ocr document ocr document optimizing-matias.pmd optimizing the utility of allium cepa l. var. aggregatum (sibuyas tagalog) 43science diliman (january-june 2011) 23:1, 43-51 optimizing the utility of allium cepa l. var. aggregatum (sibuyas tagalog) for the allium test by elucidating its mitotic periodicity and rhythmicity under varying light conditions ambrocio melvin a. matias* and ian kendrich c. fontanilla institute of biology, college of science, university of the philippines, diliman, quezon city telefax: (63-2) 920-5471 *corresponding author: email: kelvin4225@yahoo.com abstract the occurrence of pattern of mitotic activity has long been studied in different plants; in the onion allium cepa, determination of its mitotic activity has led to its utilization in the allium test for cytotoxicity and mutagenicity of test substances. in this study, the pattern of mitotic activity of a. cepa var. aggregatum and the effect of light exposure on mitotic activity were determined to test the utility of a. cepa l. var. aggregatum as an alternative to the common onion, a. cepa, for the allium test. bulblets of a. cepa var. aggregatum were allowed to root for three days in tap water under three different set-ups: constant light exposure set-up (light), constant dark set-up (dark) and 12 hours light-12 hours dark set-up (light-dark). the root tips from the bulblets were then excised and subjected to microscopic observation for the mitotic index (mi) every hour after the third day. the results showed no significant difference observed across the three set-ups. however, mi for the dark and light set-ups were periodic, showing peaks or maxima of mi falling between 11 am and 12 pm, whereas that of light-dark set-up was rhythmic, having an hourly fluctuation, but also showed maximum between 11 am and 12 pm. it is recommended that a. cepa var. aggregatum root tips be excised between 11 am and 12 pm for the allium test. keywords: allium cepa var. aggregatum, periodicity, rhythmicity, mitotic index, light, allium test matias, ama and fontanilla, ikc 44 introduction the allium test is widely used as an environmental monitoring tool due to its ease of use and low cost as well as high correlation of its results with animal models (evandri et al. 2000; fijesko 1979; quilang et al. 2008; rathore et al. 2006; vidakoviæ et al. 1993; yi & meng 2003). the test took its origin from levan (1938), who studied the effects of colchicine on mitosis using allium cepa. since then, a. cepa has been used as a test organism for cytotoxicity and mutagenicity for environmental monitoring, which involved exposing the roots of allium in a test substance, cutting the root and observing the root cells under the microscope for mitotic activity and chromosomal aberrations (fijesko 1985). in the allium test, root tips should be cut during optimal mitotic activity, which is measured in terms of the mitotic index or mi (the ratio of observed dividing and non-dividing cells). jong (1997) suggested that cutting should be done during midday (12:00-1:00 pm) based on previous studies that determined the optimal mitotic activity of a. cepa. however, studies determining optimal mitotic activity show inconsistent results (lewis 1901; kellicot 1904; solomon & trent 1941). lewis (1901), using a 4-hour interval set up, showed mitotic maxima at 12 pm and 12 am, demonstrating periodicity, which is characterized by the occurrence of one or two peaks of high mitotic activity. however, winter (1929) argued that lewis’ results were uniform rather than showing periodicity as shown by the comparison of the percentage of dividing cells at different times. in a similar work by kellicot (1904) where the onions were planted in soil and a 2-hour interval was employed, maxima were achieved at 11 am and 1 pm. later on, solomon & trent (1941) observed mitotic activity under varying light conditions at one-hour intervals. the results showed rhythmicity, or the occurrence of hourly fluctuations in mitotic activity, under normal light condition. however, periodicity was evident in continuous light and continuous dark set-ups. effect of light on mitosis is demonstrated in other systems (yeoman & davidson 1971; nemota & furuya 1985). in chattonella antiqua (red tide flagellate), light can induce cell division, but it can also inhibit division by exposing cells that divide under continuous light (nemota & furuya 1985). in callus cultures, light tends to have inhibitory effect on cell division (yeoman & davidson 1971; fraser et al. 1967). this decreased cell division, resulting to decreased callus formation, might be due to reduction of some substance by light (yeoman & davidson 1971). it is currently suggested that cell division is affected by circadian clock since cell cycle genes, i.e. cyclins and cyclin-dependent-kinase (cdk), are under circadian control (moulager et al. 2007). thus, alteration of exposure to light might change patterns of mitotic activity. recent works involving the allium test include those of evandri et al. (2000), which showed that water in polyethylene bottles could induce cytogenetic aberration; and quilang et al. (2008), which demonstrated the effects of polychlorinated biphenyls (pcb’s) on allium root cells. both studies made use of the common onion variety of a. cepa, though other studies also employed other varieties of a. cepa and even other species. for instance, yi & meng (2003) observed increased anaphase aberration and micronuclei as well as the presence of pycnotic cells in root tips of a. sativum (garlic). in another modified allium test, vidakovic (1993) used a. cepa var. aggregatum (a. ascalonicum in the literature) as test organism for waste drilling fluid, which promoted cytotoxicity, particularly inhibition of mitotic activity, in the onion roots. allium cepa var. aggregatum, also known as the bunching onion or locally as “sibuyas tagalog,” has since been synonymized with a. ascalonicum (rabinovitch & currah 2002). its utility by vidakovic (1993) could attest to its suitability, perhaps even more so than the common onion variety of a. cepa since a. cepa var. aggregatum were found to grow faster and in greater numbers than those of the common onions based on authors’ personal observations. since different species and varieties are being employed for the allium test, there is a need to ascertain the period of optimal mitotic activity for each type of allium other than the common onion variety in order to optimize the time to cut the root tips. this study therefore aimed to investigate the periodicity and rhythmicity of mitotic activity in a. cepa var. aggregatum root tips. furthermore, since mitotic science diliman (january-june 2011) 23:1, 43-51 optimizing the utility of allium cepa l. var. aggregatum (sibuyas tagalog) 45 activity is hypothesized to be affected by light, this study also investigated the effects of varying durations of light exposure to the periodicity of mitotic activity in a. cepa var. aggregatum, by measuring the mitotic index (mi). other factors that could affect periodicity of mitotic activity such as hormone activity and temperature were not considered in this study. materials and methods allium cepa var. aggregatum were purchased from the local market in san jose, nueva ecija. scaly leaves were removed until the bulb leaves were exposed. for each bulb, only one bulblet was used. three set-ups were prepared similar to solomon & trent (1941) with modification of light source: 24 hour light exposure (light), 12-hour-light-exposure then 12-hour-dark exposure (light-dark) and no light exposure (dark). for each set-up, 18 bulblets were prepared as in quilang et al. (2008) in which each was placed on top of a 100 ml glass bottle filled with tap water to expose the bottom end of the bulblet to water. the light set up was exposed to continuous light, the light-dark set-up was alternately exposed to light and placed in a dark locker every 12 hours (i.e. 7 am and 7 pm, respectively), and the dark set-up was placed in a dark locker during the entire duration of the experiment. the light and light-dark set-ups were exposed to light by placing them in front of fluorescent bulbs; light intensity ranged from 70 to 171.5 fcandle and was measured using an extech ea31 easy viewtm light meter. only 18 bulblets were prepared for each set up due to the limited space on the laboratory bench that allowed uniform exposure of the set-ups to the light apparatus. the roots were allowed to grow for three days. on the 4th day, roots were excised every hour for 24 hours starting at 7 pm until 6 pm the next day. for every hour, 3 bulblets from each set-up were selected for root excision. around 3-5 roots were cut from each bulblet using a pair of dissecting scissors. since there were only 18 bulblets for each set up, each bulblet was sampled for roots for a total of four times to complete the hourly sampling over a 24 hour period. all 18 bulblets for each set up were randomly arranged prior to root excision. the roots were placed into 1.5-ml microfuge tubes containing farmer’s solution (1 part acetic acid, 3 parts ethanol) for fixation, after which they were stored at 40 c prior to use. the root tips were prepared for examination through the squash method following el-shahaby et al. (2003) with some modifications. around 1-3 mm of the root tip was cut and placed on a glass slide, then exposed to 1 n hcl for 1-2 minutes, after which the hcl was blotted off using tissue paper. the root tip was then macerated using the cover slip. aceto-orcein was added onto the macerated tissue; after 5-8 minutes, the slide was passed through a flame to heat-fix the stain on the tissue. a cover slip was placed on top of the tissue, and the excess aceto-orcein was blotted off using tissue paper. the slide was subsequently viewed under a 40x objective. for each root tip sample, 1000 cells were scored, taking note at which stage the cell was in: interphase, prophase, metaphase, anaphase or telophase. scoring of cells for all the slides began in the upper left side of the slide and proceeded through a convention of moving the slides from left to right until 1000 cells were counted. the mitotic index (mi) of each root tip sample for every hour (or the number of mitotic cells over 1000) was determined. for the determination of periodicity and rhytmicity, the mi for each hour was plotted. oneway analysis of variance (anova) was performed for every hour, comparing the mi of each set-up. for each hour, there were 3 bulblets, and root tips were excised from these bulblets. each root tip cut from a bublet for every hour was considered as a replicate, yielding three replicates for each hour. to determine whether there was difference in the mi obtained from different treatments (light set-up, dark set-up and light-dark set-up), one-way analysis of variance (anova) was employed using excel 2007. prior to anova, data was transformed using arc sin (zar 1999). two trials were done for this experiment. results for trial 1, the mi obtained from the dark set-up across different time intervals were very similar with those of the light-set-up, with the curves of the two set-ups showing a similar pattern (figure 1). in the light setscience diliman (january-june 2011) 23:1, 43-51 matias, ama and fontanilla, ikc 46 up, the maxima mi were observed at 1am, 12 pm and 8 pm, with the highest mi value obtained at 12 pm. in the dark set-up, 11 am had the highest mi followed by 12 am and 8 pm. in both set ups, three maxima mi were observed: (1) 12 am to 1 am; (2) 11 am to 12 pm; and (3) 8 pm; highest maxima was obtained at 11 am to 12 pm. in the light-dark setup, the maxima mi were at 12 am, 6 am and 12 pm; two of these maxima were within the range of the maxima mi in the other two set-ups. unlike the other two set-ups, however, the mi of light-dark set-up appeared to be fluctuating at an hourly interval characterized by an increase in one hour followed by a decrease in the succeeding hour. furthermore, the mi’s of light-dark set-up were generally lower than in the other set-ups. in trial 2, the light set-up had maxima mi at 12 am, 12 pm, and 10 pm with highest maxima at 12 pm; dark set-up at 12 am, 12 pm and 11 pm with highest at 12 am; the light-dark at 12 am, 12 pm and 7 pm, with highest at 12 pm. the light and dark set-ups followed a similar pattern in trial 1 in terms of the presence of maxima mi which were distinctly higher than the other points. in comparison to these two, the light-dark set-up had a fluctuating pattern from 12 am to 12 pm, but continually decreased at 1 pm to 5 pm and increased at 7 pm then followed a fluctuating pattern (figure 2). based on the plots, trial 1 and trial 2 had almost similar results in terms of the patterns. however, unlike in trial 1, the mi of light-dark setup in trial 2 was relatively high making it similar with the other two set-ups at 11 am to 1 pm; hence the three plots seemed to overlap at this time interval. in comparing the mi for the three set-ups, a significant difference based on anova was obtained in trial 1, though this was only observed at two time periods, 1 science diliman (january-june 2011) 23:1, 43-51 figure 1. plot of mitotic index for each hour of the day obtained from the first trial showing periodicity for dark and light while rhythmicity for light-dark set-up. error bar corresponds to standard error. optimizing the utility of allium cepa l. var. aggregatum (sibuyas tagalog) 47 am and 9 pm, (p= 0.063 and p= 0.036, respectively) (table 1). no significant difference was obtained from trial 2 based on anova (table 2). discussion in both trials, the maxima fell in the range of 11 am to 12 pm and 11 pm to 1 am, which is in agreement with previous works on the common onion variety of a. cepa by kellicot (1904) and lewis (1901). these results agree with those of solomon and trent (1941); however, the maxima for the continuous light differed slightly, which was observed at 7 am, 11 am, 3 pm and 10 pm. the results for the dark set-up were similar with those of solomon & trent (1941) except that the frequency of mitosis in the dark-set-up in this study was lower than that obtained from the common onion. the light-dark set-up appeared to have lower mi than the other two set-ups, particularly in trial 1. in trial 2, the light-dark set-up appeared to have a similar mi value with the other two set-ups. this result, however, differed with that of solomon and trent (1941) in which the day-night set-up (light-dark) had the highest mi of all the three set-ups. in all set-ups, the frequency of mitosis was lower than that observed for the common onion variety by solomon & trent (1941). the intensity of light used in the experiment was 70171.5 fcandle for both trials. this intensity is low compared to the presumed intensity of light from the sun (720 – 11000 fcandle), which was used by solomon & trent (1941) for their light-dark set-up. the disparity in terms of the mi for this study and that of solomon & trent (1941) could be attributed to the different light intensities. by definition of periodicity by solomon & trent (1941), which is the occurrence of waves or peaks for the science diliman (january-june 2011) 23:1, 43-51 figure 2. plot of the mitotic index for each hour of the day obtained from the second trial showing periodicity for dark and light while rhythmicity for light-dark set-up. error bar corresponds to standard error. matias, ama and fontanilla, ikc 48 mitotic activity at different times of the day, the mi for a. cepa var. aggregatum is periodic in both dark and light set-ups. the maxima for both were distinct and higher than the other points. on the other hand, the mi of the light-dark set-up appeared to be rhythmic, defined by the occurrence of hourly fluctuations in mitosis. in this study, we tested how light could cause these variations in mitosis. using anova, it was found that there was no significant difference between each treatment for each hour in trial 2 (table 2). furthermore, the significant difference observed in trial 1 did not justify the significance of light in defining the pattern for the mitotic activity, particularly as the difference occurred only in two time periods, at 1 am and at 9 pm (table 1). the dark-set up and light-set up had almost similar pattern (periodic pattern). this suggested that alteration of condition (i.e. from light to dark) affects mitotic activity, thus showing different    trial 1     dark set‐up  light set‐up  light‐dark set‐up  time  mean mi     sd  mean mi     sd  mean mi     sd  12 am  0.0270  ± 0.0010  0.0210  ± 0.0000  0.0247  ± 0.0042  1 am  0.0197  ± 0.0080  0.0240  ± 0.0036  0.0117  ± 0.0006*  2 am  0.0190  ± 0.0035  0.0140  ± 0.0046  0.0177  ± 0.0023  3 am  0.0173  ± 0.0029  0.0167  ± 0.0040  0.0170  ± 0.0046  4 am  0.0163  ± 0.0021  0.0143  ± 0.0081  0.0107  ± 0.0015  5 am  0.0150  ± 0.0044  0.0130  ± 0.0030  0.0113  ± 0.0059  6 am  0.0153  ± 0.0025  0.0127  ± 0.0042  0.0200  ± 0.0046  7 am  0.0180  ± 0.0060  0.0120  ± 0.0056  0.0137  ± 0.0015  8 am  0.0153  ± 0.0124  0.0167  ± 0.0074  0.0137  ± 0.0029  9 am  0.0153  ± 0.0085  0.0137  ± 0.0065  0.0140  ± 0.0046  10 am  0.0173  ± 0.0081  0.0173  ± 0.0067  0.0170  ± 0.0053  11 am   0.0333  ± 0.0160  0.0173  ± 0.0042  0.0150  ± 0.0046  12 pm  0.0197  ± 0.0035  0.0310  ± 0.0130  0.0230  ± 0.0072  1 pm  0.0160  ± 0.0035  0.0217  ± 0.0084  0.0140  ± 0.0035  2 pm  0.0133  ± 0.0072  0.0157  ± 0.0040  0.0080  ± 0.0044  3 pm  0.0157  ± 0.0060  0.0120  ± 0.0087  0.0183  ± 0.0060  4 pm  0.0120  ± 0.0010  0.0167  ± 0.0055  0.0090  ± 0.0030  5 pm  0.0163  ± 0.0042  0.0107  ± 0.0107  0.0167  ± 0.0102  6 pm  0.0173  ± 0.0136  0.0160  ± 0.0061  0.0140  ± 0.0062  7 pm  0.0187  ± 0.0029  0.0183  ± 0.0059  0.0110  ± 0.0035  8 pm  0.0250  ± 0.0078  0.0257  ± 0.0134  0.0150  ± 0.0044  9 pm  0.0177  ± 0.0025  0.0230  ± 0.0035  0.0133  ± 0.0040*  10 pm  0.0217  ± 0.0076  0.0157  ± 0.0031  0.0173  ± 0.0006  11 pm  0.0167  ± 0.0064  0.0143  ± 0.0035  0.0160  ± 0.0035   * corresponds to set-up in which there is significant difference (á=0.05) table 1 the average mitotic index for each hour and set-up obtained from trial 1 starting from 12 am to 11 pm. anova was used to determine the difference between each set-up for each hour. values expressed as mean±sd (standard deviation) science diliman (january-june 2011) 23:1, 43-51 optimizing the utility of allium cepa l. var. aggregatum (sibuyas tagalog) 49 patterns. similarly, the effect of light could have been due to the previous light/dark regime in which cells were exposed; exposure to light after cell division could therefore have prevented further divisions (nemota & furuya 1985). in addition to light, cell division is also regulated by cyclin and cyclin-dependent-kinase (cdk) proteins, which increases activity of cdk and release the transcription factor for genes of dna replications, respectively. moulager et al. (2007) suggested that cyclins and cdk’s are under circadian control. these proteins, in turn, are induced by phytohormones, particularly cytokinin. cytokinin is said to regulate cell cycle at both the g1/s phase and g2/m phase progressions (zhang et al. 2004). cytokinin concentrations also vary across different times of exposure to light (nova’kova et al. 2005). the role of cylins, cdk’s and cytokinins in the rate of cell division should therefore not be ruled out.    trial 2     dark set‐up  light set‐up  light‐dark set‐up  time  mean mi     sd  mean mi     sd  mean mi     sd  12 am  0.0267  ± 0.0021  0.0203  ± 0.0081  0.0210  ± 0.0087  1 am  0.0150  ± 0.0035  0.0137  ± 0.0046  0.0143  ± 0.0045  2 am  0.0127  ± 0.0021  0.0137  ± 0.0025  0.0190  ± 0.0062  3 am  0.0147  ± 0.0083  0.0127  ± 0.0031  0.0107  ± 0.0055  4 am  0.0167  ± 0.0029  0.0157  ± 0.0116  0.0130  ± 0.0053  5 am  0.0163  ± 0.0057  0.0050  ± 0.0044  0.0087  ± 0.0042  6 am  0.0123  ± 0.0040  0.0137  ± 0.0035  0.0190  ± 0.0090  7 am  0.0120  ± 0.0072  0.0113  ± 0.0055  0.0147  ± 0.0015  8 am  0.0077  ± 0.0038  0.0140  ± 0.0052  0.0207  ± 0.0083  9 am  0.0083  ± 0.0029  0.0117  ± 0.0081  0.0127  ± 0.0064  10 am  0.0147  ± 0.0050  0.0107  ± 0.0059  0.0210  ± 0.0010  11 am   0.0107  ± 0.0055  0.0163  ± 0.0081  0.0110  ± 0.0069  12 pm  0.0230  ± 0.0030  0.0243  ± 0.0074  0.0233  ± 0.0091  1 pm  0.0133  ± 0.0093  0.0133  ± 0.0025  0.0170  ± 0.0082  2 pm  0.0077  ± 0.0032  0.0100  ± 0.0046  0.0150  ± 0.0046  3 pm  0.0150  ± 0.0026  0.0080  ± 0.0066  0.0117  ± 0.0012  4 pm  0.0150  ± 0.0035  0.0070  ± 0.0026  0.0087  ± 0.0035  5 pm  0.0127  ± 0.0051  0.0133  ± 0.0103  0.0080  ± 0.0044  6 pm  0.0113  ± 0.0023  0.0087  ± 0.0015  0.0117  ± 0.0032  7 pm  0.0130  ± 0.0026  0.0180  ± 0.0026  0.0210  ± 0.0104  8 pm  0.0110  ± 0.0035  0.0170  ± 0.0026  0.0147  ± 0.0049  9 pm  0.0143  ± 0.0015  0.0170  ± 0.0046  0.0177  ± 0.0065  10 pm  0.0127  ± 0.0015  0.0183  ± 0.0040  0.0177  ± 0.0015  11 pm  0.0220  ± 0.0085  0.0090  ± 0.0060  0.0167  ± 0.0025   no significant difference in all groups (á=0.05) table 2 the average mitotic index for each hour and set-up obtained from trial 2 starting from 12 am to 11 pm. anova was used to determine the difference between each set-up for each hour. values expressed as mean±sd (standard deviation) science diliman (january-june 2011) 23:1, 43-51 matias, ama and fontanilla, ikc 50 conclusion even though it was not shown that light affects the pattern of mitotic activity, the periodicity and rhythmicity of mitosis was still evident in the experiment. in this study, the duration of exposure to light at 70-171.5 fcandle illumination did not affect the mitotic activity in the root tips of a. cepa var. aggregatum. however, a pattern of mitotic activity was still seen, and these were periodic for the light and dark set-ups and rhythmic for the light-dark set-up. the mechanism causing these patterns is still not clear but is being suggested to be controlled by the cyclin and cdk proteins as well as the plant hormone cytokinin. these observations have practical applications for the allium test, particularly in the usage of a. cepa. var. aggregatum, a close relative of a. cepa, as model organism. root excision is suggested to be done around 11 am – 1pm where mi is highest; this time period also falls within the range given by jong (1997) for root excision of a. cepa. acknowledgements the authors wish to thank the institute of biology, particularly the genetics research laboratory, for the use of equipment and chemicals, and ms. erika alvero, dr. janet puzon and dr. lilian ungson for their helpful comments on the study. references el-shahaby, o.a., h.m. abdel migid, m.i. soliman and i.a. mashaly. 2003. genotoxicity screening of industrial wastewater using the allium cepa chromosome aberration assay. pakistan j. biol. sci. 6(1): 23-28. evandri m. g., p. tucci, and p. bolle. 2000. toxicological evaluation of commercial mineral water bottled in polyethylene terephthalate: a cytogenetic approach with allium cepa. food addit. contam. 17(12): 1037-1045. fijesko g. 1985. the allium test as a standard in environmental monitoring. hereditas 91: 169-178. fijesko g.1979. mercury and selenium in a modified allium test. hereditas 91: 169-178. fraser r., u. loening and m. yeoman. 1967. effect of light on cell division in tissue culture. nature 215:873. jong k. 1997. laboratory manual of plant cytological techniques. edinburgh, royal botanic garden: 97 pp. kellicott, w. e. 1904. the daily periodicity of cell division and of elongation in the root of allium. bull. torr. bot. club. 31: 529-550. levan a. 1938. the effect of colchicine on root mitoses in allium. hereditas 24: 471-486. lewis, a. c. 1901. contributions to the knowledge of the physiology of karyokinesis. bot. gaz. 32:423-425. moulager m., a. monnier, b. jesson, r. bouvet, j. mosser, c. schwartz, l. garnier, f. corellou and f. bouget. 2007. lightdependent regulation of cell division in ostreococcus: evidence for a major transcriptional input. plant physiol. 144:1360-1369. nemota y. and m. furuya. 1985. inductive and inhibitory effects of light on cell division in chattonella antiqua. plant cell physiol 26(4):669-674. nova’kova, m., v. motyka, p. dobrevi, j. malbecki, a. gaudinova’ and r. vankova. 2005. diurnal variation of cytokinin, auxin and abscissic acid levels in tobacco leaves. j. exp. bot. 56(421): 2877-2883. quilang j., m de guzman, m. h. de hitta-catalan, r. rubio, s. jacinto, e. santiago, and e. cao. 2008. effects of polychlorinated biphenyls (pcbs) on root meristem cells of common onion (allium cepa l.) phil. j. sci. 137(2): 141-151. rabinowitch h.d. and r. kamensetsky. 2002. shallot (allium cepa, aggregatum group). in rabinowitch, h.d., and l. currah (eds). allium crop sciences: recent advances. uk, cabi publishing: 515 pp. science diliman (january-june 2011) 23:1, 43-51 optimizing the utility of allium cepa l. var. aggregatum (sibuyas tagalog) 51 rathore h.s., s. bi, a. sharma, and m. makwana. 2006. prevention of aluminium chloride-induced mitodepression with myrobalan (fruit of terminalia chebula, retz, combretaceae) in allium cepa model. ethnobot. leaflets 10: 272-279. solomon d. and j. trent. 1941. preliminary report on periodicity and rhythmicity of mitotic phases of root tips under varying light conditions. trans. kans. acad. sci. 44: 202-207. vidakoviæ •., d. papeš and m. tomiæ. 1993. toxicity in waste drilling fluids in modified allium test. water air soil poll. 69: 413423. winter j. 1929. some observations on the rate of mitosis in root tip meristems of gladiolus. trans. am. microsc. soc. 48(3): 276 -291. yeoman m. and a. davidson. 1971. effect of light on cell division in developing callus cultures. ann bot 35(5):10851100. yi, h. and z. heng. 2003. genotoxicity of hydrated sulfur dioxide on root tips of allium sativum and vicia faba. mut. res. 537: 109-114. zar j. 1999. biostatistical analysis 4th ed. new jersey, prentice hall: 663 pp. zhang k., l. diederich, and p. john. 2004. the cytokinin requirement for cell division in cultured nicotiana plumbaginifolia cells can be satisfied by yeas cdc25 protein tyrosine phosphate. implications for mechanisims of cytokinin response and plant development. plant physiol. 137:308-316. science diliman (january-june 2011) 23:1, 43-51 2editor's note-jan-june2016.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online in this june 2016 issue of science diliman, we feature three research articles, one short communication, and a review. the three research articles addressed problems related to coffee, natural rubber, and the natural environment, whereas the contents of the review and short communication are pertinent to our seaweed industry. with the commercial importance of coffee in mind, authors santos, besa, victoria, and cao used simple sequence repeat (ssr) markers to correctly identify the varieties arabica, robusta, and liberica. the correct identification of coffee varieties increases their market value. the research of lamorenalim and rosales determined the potential risks brought about by inhalable or airborne particulate matter at places within the vicinity of a landf ill facility. the paper by pajarito, berba, par to, and yabut detailed the blooming behavior of paraff in wax in natural rubber composites as a function of three types of zeolite treatment. the research group of santiañez, suan-flandez, and trono, jr. described the occurrence and detrimental effects of white rot disease and epiphyte infection on cultured red seaweed. we also celebrate the retirement from the university of the philippines of our immediate past editor-in-chief, academician marco nemesio e. montaño, with a review, albeit incomplete, of his research on post-harvest technology. most of his work was done in conjunction with the research on seaweed production of national scientist gavino c. trono, jr. and co-authors. irene m. v illaseñor, ph.d. editor-in-chief 2editor's note-jan.-june2015.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online the june 2015 issue of science diliman features four articles on diverse topics. authors anticamara, go, ongsyping, valdecañas, and madrid used underwater visual census (uvc) belt transect sampling methods to systematically monitor reef f ish diversity to be able to implement science-based reef management throughout the philippines. the beautiful cover was provided by the authors. toralba, quiming, and palacpac validated the use of chemical f ingerprinting to ensure the safety of herbal medicines through correct authentication and identif ication. the authors were able to quantify the amount of quercetin present in sambong collected from nueva ecija, cotabato, and leyte. authors magdalita and saludes recommended the proper choice of crops to mitigate the adverse effects of climate change that may impact on the agricultural sector and on our food security. the crop selections can adapt to marginal and drought-prone areas as well as in flooded areas. authors gueco, merino, and paras studied all the possible inertia of the hermitian h when the jordan canonical form of b contains only type (i) or one block of type (ii). we requested the authors to summarize their scientif ic research for non-specialist readers to make the papers accessible to a wider audience. we aim to inspire budding scientists, among them junior and senior high school students, to appreciate the impact of doing scientif ic research to the different sectors in the philippines. i highlighted above the practical relevance and new theoretical perspectives of the f ive manuscripts. the layman’s abstracts are found after the technical abstracts. we are also inviting readers for a further discussion of the scientif ic merits of the papers by writing letters to the editor. irene m. v illaseñor, ph.d. editor-in-chief inside front cover-20-2.pmd editorial board editor-in-chief maricor n. soriano, ph.d. associate editors zubaida u. basiao, ph.d. biology joel joseph s. marciano, jr., ph.d. computer science, engineering nemesio e. montano, ph.d. biochemistry rosana b. tarriela, ph.d. earth sciences fernando p. siringan, ph.d. marine sciences cristine d. villagonzalo, dr. rer. nat. physics corazon d. villareal, ph.d. managing editor dercylis g. mararac editorial assistant alfie r. pelicano copy editor science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p 300.00/year (two issues), 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roemer, ph.d. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk raul k. suarez, ph.d. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu 8guidelines.pmd 68 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a 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(adapted with permission f rom the editors of ieee sensors journal) 3eclarin.pmd r.p. felix and l.a . eclarin 1 science diliman (july-december 2014) 26:2, 1-20 transitive perfect colorings of 2-uniform til ings rene p. fel ix university of the philippines diliman lawrence a. eclarin* mariano marcos state university _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract in this work, a method to determine the nontrivial colorings of perfect a n d t r a n s i t i v e 2 u n i f o r m t i l i n g s i s p r e s e n t e d . t h i s m e t h o d h a s b e e n applied to determine all nontrivial transitive perfect colorings of 2-uniform tilings that use the least number of colors. in addition, the equivalence of the colorings obtained was also ascertained. keywords: perfect colorings, 2-uniform tilings, equivalent colorings introduction numerous tilings of the plane by regular polygons have long been known, such as the regular tilings 36, 44, and 63 as well as the 8 semi-regular tilings 3.122, 4.6.12, 4.82, 3.4.6.4, 3.6.3.6, 34.6, 32.4.3.4, and 33.42, as illustrated by grünbaum and shepard (1987). these tilings are also known as archimedean tilings. given any pair of vertices of the tiling, the archimedean tilings would exhibit symmetry (translation, rotation, reflection, or glide reflection) that sends one vertex to the other. that is, the vertices of an archimedean tiling form one transitivity class under the action of the symmetry group of the tiling. for this reason, the arrangement of polygons about a vertex is the same for every vertex of an archimedean tiling. for example, 3.4.6.4 means that a vertex is surrounded in cyclic order by a triangle (3-gon), a square (4-gon), a hexagon (6-gon) and a square. on the other hand, 63 is just 6.6.6, meaning a vertex is surrounded by three hexagons. lesser known are the 2-uniform tilings, which are edge to edge tilings by regular polygons and where vertices of the tiling form two transitivity classes. these transitive perfect colorings of 2-uniform t ilings 2 tilings have 20 types, as shown in figure 1. the enumeration of these tilings is attributed to krötenheerdt (1969). each of the 2-uniform tilings is described through the vertex types of the two transitivity classes. for example, (32.4.3.4; 3.4.6.4) describes a 2-uniform tiling where the vertices are of types 32.4.3.4, and 3.4.6.4. figure 1. the twenty 2-uniform tilings in the euclidean plane. r.p. felix and l.a . eclarin 3 in this study, the colorings of 2-uniform tilings, which fall under the theory of color s y m m e t r y, we r e co n s i d e r ed . t h e b a s i c p r o b l e m i n co l o r s y m m e t r y i s t h e classif ication of symmetrically colored symmetrical patterns. the work of schwarzenberger (1984) provides a compendium of results on color symmetry, spanning decades of works. senechal (1988) discussed results of interest and posed some problems on color symmetry. the paper of rapanut (1988) provided useful results on subgroups of the seventeen plane crystallographic groups. in his paper, roth (1993) determined that the minimum number n of colors that suff ice to color any multipattern with an associated symmetry group is 2 < n < 25. more recent works on coloring symmetrical patterns in the case of hyperbolic plane patterns have been done by de las peñas, felix, and laigo (2006). frettlöh (2008) listed possible values for perfect k-colorings of some hyperbolic regular and laves tilings. felix and loquias (2008) worked on semiperfect colorings. precise perfect colorings were studied by santos and felix (2011). a study on transitive perfect colorings on semi-regular tilings was done by gentuya (2013). preliminaries let x be a set of objects in the plane and g the symmetry group of x. a coloring of x (using n colors ) is a surjective or onto function from x to . the coloring results in a partition of x where two elements x and y in x are assigned the same color ci if and only if they are elements of the same set pi . we may therefore treat a coloring as a partition p of x. if for every , we say that the partition p is g-invariant and that the associated coloring is perfect. we also say that each induces a permutation of the colors . a special class of perfect colorings of x is the class of transitive perfect colorings of x . a perfect coloring of x is transitive if g acts transitively on the set of colors , i.e. , if ci and cj are any two colors in there is an element in g that sends ci to cj . thus, not only is each symmetry in g associated with a unique permutation of the colors of the colored set, but given any two colors, the pattern formed by elements of one color is congruent to the pattern formed by elements of the other color. hence, the colored pattern may be thought of as a disjoint union of colored subpatterns that are congruent to each other. 1, 2,…,   1, 2,…,  1, 2,…,      ∈   ∈   1, 2,…,   1, 2,…,   1, 2,…,   transitive perfect colorings of 2-uniform t ilings 4 consider two colorings of the same set x and the corresponding colored patterns arising from the two colorings. the two colorings are said to be equivalent if one of the colored patterns may be obtained from the other colored pattern by (1) a bijection from the set of colors used in the f irst coloring to the set of colors used in the second coloring, (2) a symmetry in the symmetry group g of x, or (3) a combination of (1) and (2). this def inition of equivalence is adapted from roth (1982). the concepts are illustrated using the colored patterns in figure 2. in figure 2(a), x = {1, 2, 3, 4, 5, 6, 7, 8} is a set of eight points with symmetry group g = <a, b>= {e, a, a2, a3, b, ab, a 2b, a3b} d 4 where a is a 90o-counterclockwise rotation about the center of the conf iguration and b is a mirror reflection about the horizontal line passing through the center of a. figure 2(b) exhibits a transitive perfect coloring of x. the coloring corresponds to the partition p = {{1, 2}, {3, 4}, {5, 6}, {7, 8}}, where the points in {1, 2}, {3, 4}, {5, 6}, and {7, 8} are colored red (r), blue (b), green (g), and yellow (y), respectively. the 90o rotation a results in the permutation (rygb), whereas the reflection b results in the permutation (by). given any two colors in the set { r, y, g, b}, there is a symmetry in g that sends one color to the other color. the coloring of x in figure 2(c) is not perfect. the only elements of g that induce a permutation of the colors are e, a 2, b, and a2b. figure 2(d) and 〈 , 〉 ≅ 4  ′  〈 , 〉 ≅ 4  figure 2. the set x consisting of eight distinct points with symmetry group showing (a) the mirror elements of g, (b) a transitive and perfect coloring, (c) a non-transitive and non-perfect coloring, (d)–(e) equivalent colorings, and (f ) the set of eight points with symmetry group with a perfect but non-transitive coloring. ≅  r.p. felix and l.a . eclarin 5 figure 2(e) exhibit equivalent colorings of x. using the bijection redblue and green  yellow and then applying the 90o rotation a we obtain the colored pattern in figure 2(e) from the colored pattern in figure 2(d). in figure 2(f ), we illustrate a coloring of a set x’ which is perfect but not transitive. the symmetry group of x’ is also g = <a, b> d 4 . let x denote the upper right hand corner point and the point immediately below as shown in the f igure. the g-orbit of x refers to the set and consists of the images of x under the elements of g. the set of corner points of x’ is the g-orbit of x whereas the g-orbit of y is the remaining set of points in x’. the rotation a induces the permutations (rb) (gy) and the reflection b induces the permutation (gy). hence, the coloring is perfect. the coloring is not transitive because there is no symmetry in g that will send the color yellow to blue. coloring framework in this study, nontrivial transitive perfect colorings of 2-uniform tilings were considered. the approach of felix (2011) where a coloring of a set is treated as a partition of the set was used. we made use of the theorem described below. in the theorem, denotes the set and is called the stabilizer in g of x. theorem. let x be a set and let g be a group acting transitively on x . 1 . if is a coloring of x for which g permutes the colors, then for e v e r y , t h e r e e x i s t s s u c h t h a t a n d t h e c o l o r i n g is described by the partition 2. let and such that and . then is a coloring of x with n colors for which g permutes the colors. (see evidente, 2012 for the proof ). remark: the above theorem determines all perfect colorings of x on the assumption that g acts transitively on the set x. the partition p above corresponds to a coloring of x that is perfect and transitive.   1  ∈       : ∈ .  ∈       ∶ ∞  : ∈   ≅  ∈ :   : ∈   transitive perfect colorings of 2-uniform t ilings 6 based on the theorem, a procedure for arriving at nontrivial transitive perfect colorings of a 2-uniform tiling where the number of colors used is minimal is described. method for determining transitive perfect colorings of a 2-uniform tiling using the least number of colors 1. given a 2-uniform tiling, let g denote the symmetry group of the tiling. this group g is a plane crystallographic group. 2. the set x of tiles of the tiling is partitioned into a f inite number of g-orbits x i , i = 1, 2, ..., m. 3. for each g-orbit , x i , i = 1, 2, ..., m, obtain a g-orbit representative . 4. obtain , i = 1, 2, ..., m. this group is a finite group, which is cyclic or dihedral . 5. look for a proper subgroup of g of least index such that for each i = 1, 2, ..., m, a conjugate of is contained in . assume there is a subgroup that was obtained in 5. 6. obtain the -orbits of tiles of the tiling. 7. for each i = 1, 2, ..., m, choose a tile such that or equivalently . 8. form the set and denote by the set ; i.e. , is the union of the -orbits i = 1, 2, ..., m. 9. let be a complete set of left coset representatives of in g. 10. the partition describes a nontrivial transitive perfect coloring of the tiling using n colors . the coloring is given by the assignment , i = 1, 2, ..., n; i.e. , the tiles in are all colored (assigned the color) . ∈     ≅   ≅         ∈       1, 2,…,     1 ∪ 2 ∪ …  …∪       ,  1, 2,…,     , 1,2,…,    1, 2,…,   ⟶          r.p. felix and l.a . eclarin 7 for the basic ideas involved in formulating the procedure, some explanations are provided below. first, g-orbits x i was considered to be independently colored. based on the theorem, the coloring or partition where and was used. note that if , there exists such that and thus ; i.e. , and are conjugate. the number of colors corresponding to the partition is given by . since using the least number of colors in the coloring is preferred, the same set of colors was used in coloring the g -orbits , and thus we take for i = 1, 2, ..., m for some where . if instead of , was used to represent the g-orbit x i then should be obtained. however, if for some . is required. all of the transitive perfect colorings of 2-uniform tilings were looked into, with the least number of colors (which were f inite) and in all cases, a subgroup was found. otherwise, if no proper subgroup of g was found, then the option will be that = g, and the coloring will be trivial. the procedure used to arrive at the results for four of the twenty 2-uniform tilings, namely, the tilings (3 2.4.3.4; 3.4.6.4), (3 3.42; 32.4.3.4) 1 , (3 3.4 2; 32.4.3.4) 2 , and (36; 32.4.3.4) is illustrated as follows. the 2-uniform t il ing (32.4.3.4; 3.4.6.4) consider the 2-uniform tiling (32.4.3.4; 3.4.6.4) given in figure 1. if we let g denote the symmetry group of the tiling, then , where are two linearly independent translations, a is a six-fold rotation centered at a hexagonal tile, and r is a reflection with symmetry axis passing through the center of a. these are shown in figure 3 together with g-orbits of tiles where tiles of the tiling belonging to the same g-orbit have the same color. there are four g-orbits of tiles of the tiling: x 1 , the set of hexagons; x 2 , the set of squares; x 3 , the set of triangles whose sides are sides of squares, and; x 4 , the set of triangles that share one side with another triangle. without loss of generality, tiles and can be chosen, as given in figure 3. the stabilizer of the hexagonal tile 1 in x 1 , generated by the six-fold rotation a and the reflection r, is isomorphic to d 6 . for the square tile 2 in x 2 , the stabilizer is isomorphic to d 1 generated by the reflection r. the triangular : ∈   ∈     , ∈   ∈     1     : ∈   :   1, 2,…,   1, 2,…,   ,  1,2,…,         ,  1,2,…,   ∈   ∈     1    ∈           〈 , , , 〉 ≅ 6   ,   1 ∈ 1, 2 ∈ 2, 3 ∈ 3,  4 ∈ 4  transitive perfect colorings of 2-uniform t ilings 8 tile 3 in x 3 has a stabilizer generated by a three-fold rotation about its center and the reflection r. the subgroup is isomorphic to d 3 . the stabilizer of the triangular tile 4 is generated by a reflection r and is isomorphic to d 1 . · ·  figure 3. the (32.4.3.4; 3.4.6.4) tiling showing the generators and the distinct g-orbits with the tiles , and .1 ∈ 1, 2 ∈ 2, 3 ∈ 3  4 ∈ 4  to f ind the subgroup of smallest index in g that yields a nontrivial transitive perfect coloring of the (32.4.3.4; 3.4.6.4) tiling, a plane crystallographic group h that contains subgroups of type d 6 , d 3 , and d 1 must be identif ied. among the subgroups of g isomorphic to h is the subgroup , which is needed in this instance. the results of rapanut (1988) indicate that . moreover, the subgroups of g of type are of index n 2 or 3n 2, where n is a natural number. this gives the possible indices 1, 3, 4, 9, and so on. since the least possible index n is being determined such that the coloring is nontrivial, the subgroup of index 3 is f irst considered. figure 4 shows a unit cell corresponding to the subgroup . for simplicity, a unit cell of the tilings will be looked into. schattschneider (1978) can be referred to for the unit cells corresponding to the 17 plane crystallographic groups and the symbols used to denote centers of rotations. an inspection of the unit cell shows that no 3-fold rotation of stabilizes a triangle in x 3 hence another low index subgroup must be considered. if we let , a subgroup of index 4 in g, each g-orbit splits into 2 or more -orbits. as shown in figure 5, there are two -orbits of hexagons, three -orbits of squares, two -orbits of triangles in and three -orbits of triangles in x 4 .     ≅ 6   6   0 〈 2 1, 1 2, , 〉   0  0  〈 2, 2, , 〉            r.p. felix and l.a . eclarin 9 figure 4. the (3 2.4.3.4; 3.4.6.4) tiling with the stabilizers in shown in a unit cell (shaded region). 0  figure 5. the -orbits of the tiles of the tiling in (a) x 1 , (b) x 2 , (c) x 3 , and (d) x 4 .  next, the set t = {t 1 , t 2 , t 3 , t 4 } is formed, where and . any blue hexagonal tile in x 1 can be chosen as t 1 , any grey or green square tile in x 2 can be chosen as t 2 , any purple triangular tile in x 3 can be chosen as t 3 , and any orange or blue triangular tile in x 4 can be chosen as t 4 . note that in the -orbits of tiles of the tiling in each x i , . form the partition, assigning distinct colors to each results in a transitive perfect coloring of the tiling using only four colors. considering all possible combinations of tiles for t results in exactly four inequivalent transitive perfect 4-colorings of the (3 2.4.3.4; 3.4.6.4) tiling, as given in figure 6(a)–(d). ∈           , , , .  transitive perfect colorings of 2-uniform t ilings 10 figure 6. the four possible partitions for the (32.4.3.4; 3.4.6.4) tiling and their corresponding inequivalent transitive perfect 4-colorings. 〈 , , r.p. felix and l.a . eclarin 11 〈 , , , 〉 ≅ 4   in each of the 4-colorings generated, color permutations are given in table 1 (where the colors red, green, blue, and yellow are denoted by r, g, b, and y, respectively). table 1. the color permutations correspond ing to generators of g generator color permutation u (rg) (by) v (rb (gy) a (byg) r (bg) the 2-uniform t il ings and (33.42.32.4.3.4) 1 and (33.42.32.4.3.4) 2 consider the 2-uniform tiling (33.42.32.4.3.4) 1 with symmetry group , where u and v are two linearly independent translations, a is a 4-fold rotation and r is a reflection with symmetry axis not passing the center of rotation a, as shown in figure 7. there are four g-orbits of tiles of the tiling: (1) the squares that share no side with other squares, (2) the squares that share one side with another square, (3) the triangles that share exactly one side with a square, and (4) the triangles that share two sides with squares. the stabilizers in g for a tile in each g-orbit are isomorphic to c 4 , d 1 , d 1 , and c 1 , respectively. the proper subgroup of g that contains them must be isomorphic to . the subgroup of least possible index n 2 where n is a natural number is , as generated by gap and . 〈 , , , 〉 ≅ 4     4     〈 3, 3, , 〉  ∶ 9  figure 7. the (33.42;32.4.3.4) 1 tiling with the generators u, v, a, and r. transitive perfect colorings of 2-uniform t ilings 12 form where for i = 1, 2, 3, 4. observe that in figure 8(a) any yellow square tile can be chosen as t 1 , in (b) t 2 can be chosen from any of the square tiles of colors red, pink, or peach, in (c) t 3 can be chosen from any of the triangular tiles of the tiling of colors orange, purple, or green, and in (d) t 4 can be any of the nine colored triangular tiles of the tiling. from all the possible choices of tiles for inequivalent nontrivial transitive perfect colorings of the (33.42.32.4.3.4) 1 tiling is obtained. it may be checked that these colorings are inequivalent. one such transitive perfect coloring of the (33.42.32.4.3.4) 1 tiling using nine distinct colors is shown in figure 9. the 2-uniform tiling (33.42.32.4.3.4) 2 has the same vertex types 33.42 and 32.4.3.4 but its symmetry group , where u and v are two linearly independent translations, p is a glide reflection, and q is a glide reflection with glide axis perpendicular to the glide axis of p, as shown in figure 10.  1, 2, 3, 4   〈 , , , 〉 ≅   1 ⋅ 3 ⋅ 3 ⋅ 9 81 1, 2, 3, 4 ,  figure 8. the -orbits of square and triangular tiles t i with .    r.p. felix and l.a . eclarin 13 figure 9. a transitive perfect 9-coloring of the (33.42; 32.4.3.4) 1 tiling. there are three g-orbits of tiles of the tiling: x1 consisting of squares, x2 consisting of triangles that share two sides with squares, x3 and consisting of the triangles not included in x2. the stabilizers of the tiles of the tiling in each g-orbit are all isomorphic to c1. this is contained in any subgroup of g. thus, we only need subgroups of least possible index greater than 1. using gap, three subgroups of index 2 are obtained, and these are given in table 2. figure 10. the (33.42; 32.4.3.4) 2 tiling with its generators and tiles belonging to the three g-orbits. transitive perfect colorings of 2-uniform t ilings 14 〈 , 2〉 〈 , 2〉          ∪ ,  ,     ,   ,   ,     (a)  1, (b)  2, (c)  3 where  〈 , 2〉  if , each g-orbit splits into two -orbits, as shown in figure 11. form t = {t 1 , t 2 , t 3 }, where t i is in x i for each i = 1, 2, 3 and . in figure 11(a), t 1 can be any yellow square tile or grey square tile. in (b), t 2 can be any orange tile or purple tile, and in (c), t 3 can be any pink tile or blue tile. these give eight possible combinations for the set t. the subgroup is of index 2 in g and we have where h is the 2-fold rotation in g whose center is shown in figure 11. if a set of f i xed tiles for and the half-turn h in g are considered, the partition could be obtained. assigning the color red to and the color green to results in a transitive perfect 2-coloring of the (33.42; 3 2.4.3.4) 2 tiling. nevertheless, it should be noted that 〈 2, 1 , 1 1〉  subgroup index symmetry group 2 pg 2 pg 2 p2 〈 , 2〉  〈 2, 〉  table 2. subgroups of of index 2〈 , , , 〉    , , 2 , .  that is, the symmetry maps the partition to the partition . hence, the coloring described by is equivalent to the coloring described by . ∈   ,   figure 11. -orbits of tiles of the tiling in and a half-turn h in g.   1, 2, 3   r.p. felix and l.a . eclarin 15   〈 , 2〉  in turn, this reduces the possible number of nontrivial transitive perfect colorings to four instead of eight, as presented in figures 12(a)-(h). the coloring in (a) is equivalent to (b), (c) is equivalent to (d), (e) is equivalent to (f ), and (g) is equivalent to (h). figure 12. the eight transitive perfect 2-colorings of (33.42; 32.4.3.4) 2 when . transitive perfect colorings of 2-uniform t ilings 16 similarly, when we let , each g-orbit of tiles of the tiling splits into 2 -orbits and results into four inequivalent transitive perfect 2-colorings, as seen in figure 13. if we let , each g-orbit of tiles of the tiling also splits into two -orbits. the resulting 2-colorings are shown in figure 14. in all, there are 12 inequivalent transitive perfect 2-colorings of the (33.42; 32.4.3.4) 2 tiling. 〈 2, 〉 〈 2, 〉  〈 2, 1 , 1 1〉 〈 2, 1 , 1 1〉    figure 14. the four inequivalent transitive perfect 2-colorings of (33.42; 32.4.3.4) 2 when .〈 2, 1 , 1 1〉  figure 13. the four inequivalent transitive perfect 2-colorings of (33.42; 32.4.3.4) 2 when .〈 2, 〉    r.p. felix and l.a . eclarin 17 the 2-uniform t il ing (36; 32.4.3.4) using the method discussed, f ive inequivalent transitive perfect 25-colorings of (36; 32.4.3.4) were obtained. the least number of colors that can be used to color the tiling is 25. one such coloring is shown in figure 15 where tiles of the same number are assigned the same color. figure 16 indicates the remaining four transitive perfect 25-colorings of (36; 32.4.3.4). applying the method for finding nontrivial transitive perfect colorings to all 2-uniform tilings, results were obtained, as summarized in table 3. the patterns for all of the inequivalent colorings in each of the twenty 2-uniform tilings were illustrated. the results show that if n is the least number of colors needed in coloring a 2-uniform tiling in such a way that it is nontrivial, transitive, and perfect then . this r e s u l t i s ex p ected b a s ed o n t h e w o r k of ro t h ( 1 9 9 3 ) . applying the method to 3-uniform tilings, i.e. , tilings by regular polygons where the vertices of the tiling form three transitivity classes, is also of interest. additional insights may be acquired from looking at transitive perfect colorings of 3-uniform tilings. the complete list of drawings for the 61 3-uniform tilings are found in chavey (1989). 2 25  figure 15. a transitive perfect 25-coloring of the (3 6; 32.4.3.4) tiling. transitive perfect colorings of 2-uniform t ilings 18 figure 16. the other four motifs of a 25-coloring of the (36; 32.4.3.4) tiling. table 3. the 2-uniform til ings with their correspond ing symmetry groups and the least number of colors needed for generating transitive perfect colorings (36; 34.6) 1 p6 4 64 (36; 34.6) 2 p6m 3 1 (36; 33.42) 1 cmm 2 8 (36; 33.42) 2 pmm 2 4 (36; 32.4.3.4) p6m 25 5 (36; 32.4.12) p6m 3 1 (36; 32.62) p6m 2 2 (34.6; 32.62) cmm 2 2 (33.42; 32.4.3.4) 1 p4g 9 81 (33.42; 32.4.3.4) 2 pgg 2 12 (33.42; 3.4.6.4) p6m 4 4 (33.42; 44) 1 cmm 2 2 (33.42; 44) 2 cmm 2 4 (33.4.3.4; 3.4.6.4) p6m 4 4 (32.62; 3.6.3.6) pmm 2 2 (3.4.3.12; 3.122 ) p4m 9 3 (3.42.6; 3.4.6.4) p6m 25 25 (3.42.6; 3.6.3.6) 1 pmm 3 3 (3.42.6; 3.6.3.6) 2 cmm 2 2 (3.4.6.4; 4.6.12) p6m 25 4 2-uniform til ings symmetry group least number n of colors number of inequivalent n-colorings r.p. felix and l.a . eclarin 19 acknowledgments we wish to thank the commission on higher education for the f inancial support provided in the preparation of this research through the off ice of the vice-chancellor for research and development of the university of the philippines diliman. references chavey d. 1989. tilings by regular polygons–ii: a catalog of tilings. computers math. applic. 17(1-3): 147-165. conway j. , burgiel h. , goodman-strauss c. 2008. the symmetries of things. wellesley, ma: a.k. peters, ltd. de las peñas m. , felix r. , laigo g. 2006. colorings of hyperbolic plane crystallographic patterns. z. kristallogr. 221: 665-672. evidente i. 2012. colorings of regular tilings with a singular center (unpublished doctoral dissertation). university of the philippines, diliman. felix r. color symmetry. available from http://www.crystallography.fr/mathcryst/pdf/ manila/felix.pdf. accessed on 3 november 2011. f e l i x r . , lo q u i a s m . 2 0 0 8 . e n u m e r a t i n g a n d i d e n t i f y i n g s e m i p e r fec t co l o r i n g s of symmetrical patterns. z. kristallogr. 223(8): 483-491. frettlöh d. 2008. counting perfect colourings of plane regular tilings. z. kristallogr. 223: 773-776. the gap group. 2014. gap – groups, algorithms, and programming ( version 4.7.4) [software]. available from http: //www.gap-system.org. gentuya j. 2013. transitive perfect colorings of semi-regular tilings (unpublished master’s thesis). university of the philippines, diliman. grunbaüm b. , shephard g.c. 1977. perfect colorings of transitive tilings and patterns in the plane. discrete math. 20: 235-247. grunbaüm b. , shephard g.c. 1987. t ilings and patterns. new york: w. h. freeman and company. k r ö t e n h e e r d t o . 1 9 6 9 . d i e h o m o g e n e m o s a i k e n t e r o r d n u n g i n d e r e u k l i d i s c h e n ebene. i, wiss. z. mar tin-luther-univ. halle-wittenberg. math.-natur. reihe 18: 273-290. rapanut t. 1988. subgroups, conjugate subgroups, and -color groups of the seventeen plane crystallographic groups (unpublished doctoral dissertation). university of the philippines, diliman. roth r. 1982. color symmetry and group theory. discrete math. 38: 273-296. transitive perfect colorings of 2-uniform t ilings 20 roth r. 1993. perfect colorings of multipatterns in the plane. discrete math. 122: 269-286. santos r. , felix r. 2011. perfect precise colorings of plane regular tilings. z. kristallogr. 226: 726-730. schattschneider d. 1978. the plane symmetry groups: their recognition and notation. amer. math. monthly 85(6): 439-450. schwarzenberger r. 1984. colour symmetry. bull. london math. soc. 16: 209-240. senechal m. 1988. color symmetry. comput. math. appl. 16(5-8): 545-553. _____________ lawrence a. eclarin <olens16@gmail.com> is an assistant professor at the department of mathematics, mariano marcos state university. rene p. fel ix is a professor at the institute of mathematics, university of the philippines diliman. he is a member of the commission on mathematical and theoretical crystallography, international union of crystalography, 2008 2014. 2editor's note-july-dec.2016.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online irene m. v illaseñor, ph.d. editor-in-chief welcome again to science diliman. four ar ticles and a short communication are featured in this december 2016 issue. the topics discussed are a mixture of physical and natural sciences. rather than the usual measurement of physicochemical parameters in the assessment of water quality of freshwater streams, deborde, hernandez and magbanua used benthic macroinvertebrates as bioindicators. the paper concludes that macroinvertebrates can be successfully used to differentiate water quality of the streams. the manuscripts by the research groups of mathematicians merino, kunwor, and walls; aala, jose and roque; and physicists paraan and laurente are quite challenging and only experts in the respective f ields can fully appreciate their conclusions. let me try to go over the main points. the manuscript of aala, jose and roque gave a unique solution to a parabolic partial differential equation whose application is on the heat transfer in a perforated cell. the research group of merino, kunwor, and walls discussed the application of mobius transformation into a sub-group of vahlen matrices, and in the process, provided results on eigenvalues with entries in clifford algebra. paraan and laurente discussed the work they did on the quantum ising model. a preliminary study by chichioco-hernandez and isah showed the potential cholesterol lowering activity of plant extracts.the activity was measured by the inhibition of β-hydroxy-βmethylglutaryl-coenzyme a reductase (hmg-coa reductase). layman’s abstracts for the non-specialists are also included. 01_device subade and subade 18 socio-economic conditions and perceptions on the conservation of tubbataha reefs and vicinity: a households survey in cagayancillo, palawan rodelio f. subade*1 and ana liza a. subade2 1associate professor in economics division of social sciences, college of arts and sciences, university of the philippines in the visayas, miag-ao, iloilo 5023a 2ph.d. candidate and wwf-usa russel e. train fellow school of environmental science and management university of the philippines los baños, los baños, laguna date submitted: march 2, 2006; date accepted: october 12, 2006 abstract this paper presents the results of a socio-economic monitoring survey of 110 sampled respondents/ households across the 12 barangays of cagayancillo, using a survey instrument through personal interviews. findings show that on the average, the respondent was 48 years old, with 7 years of formal education, has lived in cagayancillo for 31 years and belonged to a family/household with 6 members. cagayanens had shifted to farming as the main source of their livelihood and income, while fishing was only second. based on their income data, cagayanens are living below poverty threshold level of income. poverty incidence ranged from 67-79% of the cagayanen households. the income data for 1999 and 2004 when compared, plus the respondents' perception that present aquatic resources are not in good condition, somehow allude to the possibility that the cagayanens may just easily fall on the vicious cycle of poverty and environmental degradation. the good news can be the increasing involvement of people in coastal resource management and conservation. by including the people in the main cagayancillo islands as beneficiaries of and participants in the conservation efforts and projects, wwf-philippines/ kkp has perhaps initiated a good momentum for a democratized and more sustainable stakeholders management of coastal resources in cagayancillo. in order to sustain this momentum for continuous people's participation in coastal resource management and conservation, and eventually the alleviation of poverty in cagayancillo, concerned policy makers and other entities need to consider some options, one of which is the continuation of conservation efforts started by wwf-philippines for another 2-3 years. key words: cagayancillo, tubbataha reefs, socio-economic, perceptions, conservation, wwf philippines science diliman (july-december 2006) 18:2, 18-33 *corresponding author socio-economic conditions and perceptions on the conservation of tubbataha reefs 19 introduction cagayancillo (or cagayancillo islands) is an archipelagic town of the province of palawan, consisting of 31 islands and islets. it lies approximately 320 kilometers east of puerto princesa and about 98 kilometers southwest of panay island. it has 12 barangays and a population of 6,348 people or 947 households (2000 census of the national census and statistics coordinating board). the islands of this municipality are part of an extensive coral atoll system, hence they are of coralline origin (alcala, 1993 as cited by arquiza and white, 1994). as the nearest town and closest human settlement to the tubbataha reefs national marine park (trnmp) and unesco world heritage site, cagayancillo covers the said reefs under its political jurisdiction. as such, any conservation measure to protect and preserve the trnmp will inevitably include and consider the role of and impacts on cagayanen people. considering that cagayancillo residents are major stakeholders in the conservation of trnmp, the geffunded tubbataha conservation project, which was implemented by wwf-philippines (or the kabang kalikasan ng pilipinas, i.e. kkp) included among others, sustainable resource management and livelihood. this component aimed at:(a) increased understanding of the resource use and socio-economic factors leading to resource depletion in trnmp, cagayancillo and cavili; (b) implementation of community-based resource management, including management of local reserves; (c) and setting up of community-based livelihood projects that are linked to conservation management (wwf-philippines, 1999). as part of the end-of-project activities, wwfphilippines funded this study in order to provide updated information on the socio-economic conditions of households in cagayancillo, palawan. specifically, this study aims: 1. to assess the current socio-economic conditions in cagayancillo. 2. to determine changes in the over-all socioeconomic conditions in cagayancillo, comparing the present conditions with those prior to the wwfgef-undp conservation project; and 3. to assess people's perception of conservation efforts in cagayancillo, particularly during the wwf-gef-undp five-year conservation project. conservation efforts in cagayancillo the emergence of coastal resource management in cagayancillo can be traced from the evolution of multi-sector arrangements for the conservation of tubbataha reefs which is part of the municipality (figure 1). it took the initiative of one person to ring the alarm bells for tubbataha, and subsequently the various effort of stakeholders non-government organizations and government agencies, for these reefs to become a national marine park. eventually, these initiatives become the launch pad for coastal resource management in the whole municipality. arquiza and white (1994) discussed the evolution of multi-sector social response to the decline of marine environmental quality at tubbataha reefs mainly due to illegal fishing. in 1987, a native of palawan, ernesto sta. cruz, who was working with task force pawikan wrote a request addressed to vice governor ventura to declare tubbataha as a marine park. in response, the palawan provincial legislative board passed resolution no. 244 on september 7, 1987, requesting the department of environment and natural resources (denr) to declare tubbataha as a marine park in order to protect its fisheries and aquatic resources. responding to this request, president aquino issued proclamation 306, which declared tubbataha as the first national marine park. what followed was a protracted battle on the establishment of shemberg's (a private company based in cebu) seaweed farm in tubbataha, the endorsement for which was marked with controversy and dirty politics. eventually, the joint efforts of various stakeholders, initiated by sta. cruz whistle blow, led subade and subade 20 to shemberg's abandonment of the plan, the politicians backing off from the amendment to proclamation 306 which was to allow the seaweed farm in tubbataha, and the ejection of the seaweed farmers at the park per order by the denr. in the late 1980s, conservationists, scuba divers, dive boat owners and manila-based scientists, organized the tubbataha foundation. this foundation worked for the conservation and protection of tubbataha, and was also instrumental in the discontinuation of the seaweed farm. the foundation was appointed eviction team leader. in 1990, denr signed a memorandum of agreement with the foundation for the protection of tubbataha. from 1991-1993 the foundation received funds for conservation efforts through the debt-fornature swap. however, the foundation was perceived as lacking in networking with other groups or stakeholders. recognizing the rich biodiversity in tubbataha and its global value/importance, the unesco declared it as a world heritage site on december 11, 1993. on july 20, 1995, presidential fidel ramos issued memorandum circular no. 128 which created the presidential task force for tubbataha jointly headed by the denr secretary and the chairman of the palawan council for sustainable development (pcsd). on november 7, 1996, the co-chairmanship of the task force was designated to the denr secretary, secretary of national defense and the governor of palawan, representing pcsd. in this year the management plan for tubbataha was drafted and refined through the efforts of coastal resource management project (crmp), japan international cooperation agency (jica), denr, pcsd, wwfphilippines and the stakeholders of palawan and cagayancillo. in june 1998, the tubbataha protected area management board (tpamb) was created through the memorandum of agreement between governor salvador socrates and denr secretary victor ramos. in october 13, 1999 the trnmp management plan was approved by the tpamb, and in november 26, 1999, the pcsd also approved the plan. it is in this year that trnmp was enlisted in the ramsar list of wetlands of international importance, figure 1. cagayancillo islands with tubbataha reefs. socio-economic conditions and perceptions on the conservation of tubbataha reefs 21 and also when the gef-supported conservation project administered by wwf-philippines started. it must be pointed out that most of the efforts for coastal/marine resource management have been focused on the conservation of tubbataha, and less on the other cagayancillo islands. though the tubbataha foundation started some work on setting up cooperative in cawili island in 1990, not so much were undertaken by any party for coastal resource management in the archipelagic municipality. it could have been ideal to do so, but perhaps society's focus at that time was glued more on the urgency to save tubbataha. as part of the 5-year gef grant through wwf-philippines, community organization/ development work was undertaken in cagayancillo itself, in order to increase awareness and seek the municipality's long-term partnership in conserving tubbataha and other marine resources of cagayancillo. methods a perusal of available secondary materials previous study of wwf-philippines, municipal documents and other research studies, was done. for primary sources of data, a household survey was undertaken using a stratified random sampling of cagayancillo households during the month of may 2004. sample size was computed at 95, given a 95% level of confidence. to maximize interviewers fieldwork, a total of 102 households were surveyed, proportionately allocated across the 12 barangays of cagayancillo. initially, due to big waves and difficulty of reaching the islands by boat, the two sitios, cavili and calusa were not included in the sampling. however, due to better weather, a field survey was undertaken in the these island sitios of bgy. magsaysay. eight (8) respondents were then sampled, thereby increasing total sample to 110. eleven field interviewers from cagayancillo were trained on the use of the survey instrument, and were supervised on field while conducting personal interviews of respondents who were usually household heads or the spouse. the survey instrument comprised the following sections: (1) eight demographic questions; (2) a household composition matrix; (3) six questions on economic activities; (4) nine questions on economic conditions; (5) a appliance composition matrix; (6) two matrixes consisting of a six-point likert-type scale were used to evaluate residents perception on changes in over-all economic conditions (7) 13 questions on residents' perception on conservation programs; (8) a martrix on respondent's involvemen in conservation programs; (9) a problemsolution matrix in the management of coastal resources. to complement with survey data, key informant interviews were also conducted with the town mayor, municipal agricultural officer, some barangay captains, an old age resident who have lived in the village through the years and seaweed farmers. these were conducted to obtain background information and history of the study site and conservation programs implemented in tubbataha and cagayancillo. a focus group discussion with a group of fishermen was also undertaken to evaluate the status of coastal resources and to determine problems in the management of these resources. results and discussions socio-economic characteristics of respondents results of the study showed that on the average, respondents interviewed were 47.6 years old. the biggest age group or cohort was the age group of 4049 years old. seventy six percent of the respondents were male while 24% were female. average number of years of education was 7.45 years (table 1). almost all (97.3%) of the cagayanens were catholic while only 2.7% were baptists. main language spoken was the native cagayanen, while many people can also speak and understand tagalog, cuyonin, kinaray-a, ilonggo and cebuano. the cagayanen household had an average number of 6 members. only 46.4% (or 51 of 110) of the respondents had lived there since birth. a significant 23.4% or 26 people had lived in the town for only 10 years or less, signifying that there have been many people who have moved into cagayancillo for the past decade. socio-economic conditions of respondents most household respondents owned their house and lot. of the total respondents, 95% owned their house, while 86% owned their lots where their house stood. subade and subade 22 majority (72%) of the used galvanized iron (g.i.) sheets for their house roofing, while 17% used nipa. for their house walling, 33% used sawali (bamboo slits), 22.7% used mixture of cement and wood, 18% used lawanit and wood and 17.3% used mainly cement. the above information may imply that cagayanen households had limited capability in house construction expenditures. eighty-eight percent of households (97 of 110) had their own toilets. of these 97 households, 62 had water sealed toilets, 17 used open pit, and 14 used closed pit toilets. for water source, 60% were totally dependent on installed water tanks which gather/ store rain water, 19.1% relied mainly on open wells, and combination of the two. the main fuel used for cooking was wood-based (wood or charcoal), which was cited by 69% of the sampled respondents. seventeen percent (or 19 of 110) used a combination of lpg, and wood (& charcoal), while 10% used only lpg. about 3.6% used kerosene gas. sources varied, but lighting were mainly sourced from kerosene gas (50% of households). only few households depend on electricity (23%). this is attributed to the limited and insufficient electric supply, which lasts only for the daily period of 6:00 pm to 11:00pm among poblacion barangays. generator, both owned and others'/neighbors constitute 10% as lighting source. in terms of ownership of household appliance, majority (or 82%) owned at least one appliance, 23.6% owned two appliances, and only 12% owned three appliances. this indicates the simple or very modest living conditions of cagayanen households. the most common appliance owned was the radio (65.6%). economic activities and livelihood income sources and livelihood occupations/jobs. the distribution of jobs/ occupation of cagayanens showed variability and less dependence on fishing as source of livelihood. compared to the results of the 1999 socio-economic survey conducted by wwfphilippines that showed fishing and seaweed farming as the major sources of livelihood. it is interesting to note that the leading occupation of the respondents was not fishing, but farming as evidenced by 27% who cited this as their first occupation (table 2). this was followed by fishing, (24.5%). other jobs were along table 1. socio-economic characteristics of respondents characteristics minimum maximum mean std. dev. years of education (n=109) 1 14 7.45 2.83 household size 1 14 6.13 2.50 age 25 78 47.58 13.72 years of residence in cagayancillo 1 73 31.27 20.99 age distribution: frequency percent below 30 12 10.9% 30-39 23 20.9% 40-49 28 25.45% 50-59 24 21.8% 60-69 15 13.64% 70-above 8 7.27% sex frequency percent male 84 76.36% female 26 23.64% total 110 100% socio-economic conditions and perceptions on the conservation of tubbataha reefs 23 construction and home-based livelihood. there were very few professionals. very few cited seaweed farming as their occupation/source of income, and this may be attributed to the so-called "ice-ice" disease which has infested farmed seaweed for the past few years. the few former seaweed farmers admitted abandoning it as a source of livelihood due to the said seaweed disease, which resulted to severe losses. to upgrade/ supplement household income, 45.5% (or 50 respondents) of the respondents had a concurrent second job/occupation. fishing had become a second occupation (36%) followed by farming (26%). others were into home-based livelihood, and seaweed farming (2%). if the different main occupations/ livelihood sources of all working family members are considered and tabulated, farming remained the widest employer, attributed to 63 family members, compared to 53 for fishing, across the 110 households of respondents, furthermore, farmers would outnumber fishers, 89 vs. 81, respectively, if we tabulate all main and concurrent second (and third) livelihood sources/ occupations of family members. per capita and family income. based on the information on family members occupations/ sources of income, household or family income was computed. the average family (household) income for cagayanens was p3418.48 per month or p41021.760 per year. however, this figure is good for the average household size of 6.13 members for cagayancillo, as found in the same survey. on a per capita (per person) basis, cagayanens earned p557.66 per month or p6691.97 per year. translating such amount to a national standard of a five-member family/ household, monthly household income would have been p2788.32, while annual household income would be p33459.84. the poverty threshold level of income for rural palawan in year 2002, based on the national census and statistics board, was a monthly income of p894.08 per capita per person or p4470.42 per month per family/ household of five members. hence, a rural person of palawan should then earn p10729 to be able to live at the poverty threshold, or p53645.00/year. it is evident that the 2004 level of income of cagayanens is way below the 2002 poverty threshold level of income, and much less for 2004 since prices have gone up in 2004 compared to 2002 (note: available poverty threshold level of income at nscb is only for 2002). based on the results of the study on the total family income earned by cagayanens, the poverty incidence can range between 67% to 79% of the population. perceptions on changes in the over-all socioeconomic conditions to determine perceived changes in over-all economic conditions, respondents were asked to compare quality of life in 1999 with that of the present 2004 (table 3). table 2. respondent's occupation/ job type of job/occupation frequency as frequency as 1st job 2nd job 1 fisherman 27 18 (24.5) (16.4) 2 seaweed farmer 2 4 (1.8) (3.6) 3 farmer (crops)/farming 30 13 (27.27) (11.8) 4 teacher 3 --(2.7) 5 gov't employee 1 --(0.9) 6 construction workers 1 --(0.9) 7 carpenters 6 3 (5.5) (2.7) 8 home-based livelihood 2 4 (poultry, livestock) (1.8) (3.6) 9 home-based livelihood 4 3 (banig making) (3.6) (2.7) 12 housewife/housekeeper 11 --(10) 15 businessman 3 1 (2.7) (0.9) 17 brgy. officia/sk 10 --(9.1) 18 day care worker/ 2 3 brgy health worker (1.8) (2.7) 20 retiree/pensioner 1 --(0.9) 21 laborer/maid/janitor/ 3 1 security/utility worker (2.7) (0.9) 0 no job/occupation/ none 4 60 (3.64) (54.5) total 110 110 figures in parentheses are percentage of column totals subade and subade 24 most of them (45.5%) mentioned that the economic condition did not change. only 25.5% believed that 2004 quality of life is better than that of 1999, the rest believed other wise that 2004 was worse than 1999. in terms of household income, 49.1% believed that their income decreased in 2004 compared to that of 1999 (table 4). around 24% on the other hand, said that although their income increased this year, they could only buy less goods, due to increasing prices. similar to conservation programs in belize (alexander, 2000), residents perceived that their living conditions hardly improved with the establishment of the community baboon sanctuary. though many residents felt that neither their households nor themselves were benefiting, majority of them did not want the sanctuary abolished and strongly supported maintaining its status. moreover, several questions on perceived changes in socio-economic conditions since 1999 were asked (table 5). on the number of students studying in elementary and high school, 73% of the respondents thought they were increasing. only 8% believed this indicator was decreasing, while 10% thought there were table 3. comparing quality of life since 1999 to present frequency percent better 28 25.5 worse 28 25.5 the same 50 45.5 can't tell 2 1.8 no answer 2 1.8 total 110 100.0 table 4. comparing income since 1999 to present frequency percent higher, but can buy less goods 26 23.6 higher, and can buy more goods 7 6.4 lower 54 49.1 can't tell 12 10.9 no answer 3 2.7 the same 8 7.3 total 110 100.0 table 5. perceptions on changes in socio-economic conditions compare present higher/ lower/ no changes can't tell no conditions to that of 1999 increasing decreasing answers number of students studying in elementary and 80 9 11 10 -high school (72.7) (8.2) (10.0) (9.1) number of students in elementary and high school 21 55 15 17 1 jwho dropped out (19.1) (50.0) (13.6) (15.5) (0.9) numbver of families who can eat 3 times a day & has 49 10 42 7 2 enough food to eat (44.5) (9.1) (38.18) (6.4) (1.8) number of families who 58 20 17 13 1 moved out to other places (n=109) (52.7) (18.2) (15.5) (11.8) (0.9) number of people in cagayancillo/ 89 7 8 5 1 population (80.9) (6.4) (7.3) (4.5) (0.9) number of transient fishers in cagayancillo from other places 12 48 14 21 13 (n=108) (10.9) (43.6) (12.7) (19.1) (11.8) number of migrants who settled 9 39 29 16 14 in cagayancillo (n=107) (8.2) (35.5) (26.36) (14.5) (12.7) level of health sources 33 8 61 6 2 (30.0) (7.3) (55.5) (5.5) (1.8) frequency/number of crimes 6 29 32 18 20 (5.5) (26.4) (29.1) (16.4) (18.2) amount of fish catch (n=108) 15 54 24 9 6 (13.6) (49.1) (21.8) (8.2) (5.5) socio-economic conditions and perceptions on the conservation of tubbataha reefs 25 no changes. on the other hand, 50% of the respondents also thought that the number of drop-out have decreased, while 19% thought they were increasing, and 13.6% said there was no change. on another positive note, 44.5% believed that more families can eat three times a day and have enough food were as compared to that of 1999. although 38% perceived that there is no change. as regards people's mobility/ out-migration, 53% of the respondents thought that in 2004 higher number of people moved to other places compared with 1999. moreover, despite such movements, 81% of the respondents believed that the municipality's population has increased. corollary to this, 36% believed that the number of migrants who decided to settle in cagayancillo in 2004 has decreased, while 26% said there was no change on this indicator. considering increased population, it is important that health services increased in its level of provision to the community. however, 56% believed that there is no change, or that the level of services remains the same compared to 1999, while 30% thought that level of health services increased for the same comparative periods. as to peace and order, 29% believed that there is no change in the peaceful atmosphere of the town, while 26% even believed that there are even less crimes nowadays than before. only 6% believed that crimes have increased. perhaps due to conservation/ enforcement efforts, 44% of the respondents believed that the transient fishers who come to cagayancillo nowadays have decreased compared to 1999. however, a big 19% could not really tell if it was so, while 12% had no answer. for fish catch, 49% believed that it decreased in 2004 compared to 1999, 22% believed there is no change, while 14% thought it increased in 2004. perception on the condition of aquatic resources with regards to the present condition of aquatic resources, about 46.4% of the respondents believed that these resources are not in good condition and only 30.0.% say otherwise. on the other hand, more respondents observed that aquatic resources in 1994 and in 1998 were in good condition (figure 2). figure 2. perceived condition of aquatic resources through time the primary reason for the deteriorating condition of aquatic resources is the effect of illegal/destructive fishing (17.2%). it was reported in one study that one blast of dynamite fishing could damage about 7.5 square meters of coral reef area (mcmanus et. al., 1992). considering that corals have very slow growth rate which ranges from 2-20 cm. per year depending on the life form (wells, 1984), it would indeed be difficult for the corals to recover for a span of 10 years, and much more difficult for just 5 years (1999-2004). in the study of alcala and gomez (1979) as cited in uychiaoco et. al. (1995) it was reported that recovery of adult coral communities from blast fishing would take about 38 years, while yap et. al. (1990) reported a slow rate of 1-3% coral cover per year in his study. thus, despite the five-year conservation program in tubbataha and cagayancillo, and the recent fisheries law enforcement in cagayancillo, the negative effects of destructive fishing are still evident, while the tangible effects of conservation still need to be felt by the cagayanens. other reasons for the deterioration were implementation of conservation programs were not implemented throughout the municipality (1.8%), due to bad weather or climate (1.8%), increasing number of fishers (1.8%), the aquatic resource is not taken cared of (0.9%) and the increasing number of population (0.9%). on the other hand, the major reasons of those who perceived why the aquatic resources are in good subade and subade 26 condition at present, are: the disappearance of illegal fishing (10%), existence of coast patrols and guards (7.3%), lessening of illegal activities (5.5%), and establishment of programs of local government unit (3.6%) and of kabang kalikasan ng pilipinas (3.6%). perception on and awareness of conservation programs half (50%) of the respondents were members of various organizations. only five respondents were officers and members at the same time. they were either chairman or vice-chairman of the organization. among the different organizations mentioned, fisherfolk (26.4%), a fishermen's organization, was the most cited organization that the respondent was involved with. even some of those who were no longer engaged in fishing were still members of the fisherfolk organization. other organizations were barangay based, religious based and people's organizations. respondents (30%) involvement in the different organizations were within the period of 2002-2003, which is within the period when conservation programs of kkp-wwf were on its implementation. for those who were not yet members, majority (81%) was willing to be a member of an organization. only a few respondents (12.7%) named organizations existing before kkp-wwf implemented programs in the area. the aims of these organizations are shown in table 6. when asked of who started conservation programs in tubbataha and cagayancillo, the respondents (47.3%) cited kkp-wwf. this was followed by government agency (e.g. denr or da) at 18.2%. with regards to those persons or institutions that significantly contributed in the management and improvement of marine resources, 67.27% of the respondents cited kkp-wwf. the local government unit and government agency (e.g. da and denr) were also mentioned with 14.54% and 8.18% respectively. the contributions of the abovementioned institutions are shown in tables 7 and 8. others mentioned are pnp/tanod, mayor joel carceler, fisherfolk, bantay dagat, bfarmc and individuals who in one way or another helped in various activities or programs. the major contribution of kkp-wwf is in the management and protection of marine resources. other major contributions are provision of training and seminars and assistance in addressing illegal fishing. these were also mandates of lgu and government agencies. it could be observed that the management and improvement of marine resources covers wider tasks and needs support of different individuals and institutions for its implementation. while many of the respondents (45.5%) were not aware of any conservation project/program to protect marine resources of cagayancillo and tubbataha, it could be noted that 40% were aware of these programs. some of these programs mentioned were the kkp-wwf table 6. organizations with its aims before wwf conservation programs name of organization aim frequency percent fisherfolk 1. for the good of fishermen and seaweed farmers/growers 2 16.67 2. as guards of the sea.3. for livelihood program 1 8.33 3. for livelihood program 1 8.33 samaka none marine reserve 1. as guards of the sea. 1 8.33 bfarmc 1. for the good of fishermen and seaweed farmers/growers 1 8.33 2. to help people. 1 8.33 3. for livelihood programs 4 33.33 cooperative 1. for the good of fishermen and seaweed farmers/growers 1 8.33 total 12 100.00 socio-economic conditions and perceptions on the conservation of tubbataha reefs 27 conservation project (32.56%), coastal resource management e.g. establishment of marine reserves (20.93%), programs of the fisherfolks (16.28%), creation of bfarmc (9.3%), and creation of bantay dagat (4.65%). the activities cited by the respondents involved in the different programs are shown in table 9. among these activities, only 29.1% of the respondents participated in these projects and programs. the level of participation of the residents is quite higher than in other areas in the country. uychiaoco and aliño (1995) estimated that only a small proportion of the population (less than 10% of fishers in a given area) participate in a community-based coastal resource management programs in the philippines. among those who participate, their contributions to these programs are shown in table 10. with regards to whether the conservation programs benefited the cagayancillo community, majority of the respondents who were aware of or involved in these programs (93.18%) answered yes. a number of them (61.70%) also believed that the mechanisms on how these conservation programs were implemented were not contradictory to their indigenous culture and tradition. table 11 shows how the conservation programs benefited the cagayancillo community. the perceived benefits from conservation programs were interrelated, e.g. the lessening of illegal fishing activities (17.%%) could be due to the presence of table 7. contributions of kkp-wwf in the management and improvement of marine resources. contributions frequency percent spearheaded the conservation program 2 2.90 helped so many and without them all the corals are destroyed 2 2.90 provided projects 2 2.90 provided seaweeds 1 1.45 helped in prohibiting illegal fishing 9 13.04 manage & protect marine resources 33 47.83 patrol 1 1.45 established reserve areas 2 2.90 helped in the right way of fishing 3 4.35 provided training & seminar to obtain information 14 20.29 total 69 100 table 8. contributions of lgu in the management and improvement of marine resources. contributions frequency percent helped in prohibiting illegal fishing 8 57.14 manage and protect marine resources 1 7.14 helped in the right way of fishing 1 7.14 provided training and seminar to obtain information 3 21.43 patrol 1 7.14 total 14 100 table 10. residents' contribution to conservation program. activities involved frequency percent as part of the enforcement of fishery laws through bantay dagat and patrolling 10 38.46 informing the community to avoid illegal activities 3 11.54 prohibiting of illegal fishing 2 7.69 mangrove planting 2 7.69 seaweeds cultivating 1 3.85 reporting illegal activities 2 7.69 participation in what ever activities 2 7.69 assisting in monitoring of coastal resources 1 3.85 paying due fees e.g. fishing license 1 3.85 attending trainings and seminars 2 7.69 total 26 100.0 table 9. projects/programs to protect marine resources of cagayancillo and tubbataha activities frequency percent 1. enforcement of fisheries laws 6 25 2. information dissemination of management and protection of marine resources 3 12.50 3. implementation of reserve areas per barangay 3 12.50 4. prohibition of illegal fishing methods 3 12.50 5. patrol of coastal waters 3 12.50 6. planting of seaweeds or mangroves 2 8.33 7. implementation of protected areas to enhance fish growth. 2 8.33 8. alternative livelihood. 1 4.17 9. prohibition of dumping of waste in the coastal waters 1 4.17 total 24 100.00 subade and subade 28 bantay dagat and patrols (7.5%). this in turn could have resulted to increased fish growth (10%) and thus could be one of the people sources of income (10%). another benefit is the increase in people awareness and information regarding marine resources.this benefit could be due to the information and education campaign activities of the wwf. there were only two reasons cited by those who perceived that the community did not benefit from conservation programs. first, programs were not realized and second people were not able to benefit from the programs of kkp-wwf. it would be politically naïve to conclude that all forms of participation are always beneficial to the participants. in most cases, it would take some time for the community to enjoy the benefits of conservation programs considering that it would take years for the natural resources to recover. nevertheless, the community's contribution in the programs through the provision of their peoples' labor is worth noting. the enlistment of members of the community, often on a voluntary basis, is a good form of participation. later, some may act as catalysts for other community development effort. most (98%) of those involved in conservation programs agreed that these should be continued for a number of reasons (table 12). perceived problems in the management of coastal resources the use of illegal fishing methods seems to be a major problem in cagayancillo. this is further exacerbated by the lack of patrol in area and intrusion of outsiders using illegal fishing. solutions given were to increase patrol and to report and arrest illegal fishers. according to fiscal julius conception, the problem with illegal fishing is the lack of witnesses to testify against those illegal fishers. in most cases, the illegal fishers were acquitted due to lack of witnesses (table 13). to address problems on illegal fishing in tubbataha, the tubbataha management office had encouraged its support groups and stakeholders to help in the enforcement of laws. in particular, the navy and the coast guard have been mobilized to enforce park rules in tubbataha, in addition to the tmo's hired park rangers and logistical inputs. in addition, the local government of cagayancillo and the provincial government have deputized the kilusan sagip kalikasan (ksk) to help in the enforcement of local table 11. perceived benefits of the cagayancillo community from conservation program benefits frequency percent alternative livelihood 1 2.5 informal education 3 7.5 illegal activities were lessen 7 17.5 able to catch fish 1 2.5 increased fish growth 4 10 presence of bantay dagat and patrols 3 7.5 conservation of the marine resources 6 15 implementation of reserve areas 1 2.5 people source of income 4 10 kkp gives fry and fingerlings/capital to the community 2 5 increase knowledge/information about the conservation of marine resource 3 7.5 helping the poor 3 7.5 the mangroves planted became the spawning grounds of fishes 1 2.5 gives satisfaction to the people 1 2.5 total 40 100.0 table 12. reasons why conservation programs should be continued reasons frequency percent for the good and development of the community in cagayancillo. 22 50.00 the program is now operating/ has started. 1 2.27 to encourage growth of fishes and the marine resources 4 9.09 to control illegal fishing 4 9.09 so that marine resources and spawning grounds will not be destroyed 3 6.82 to give the people of cagayancillo enough income 3 6.82 provided seaweeds for cultivation and materials needed. 3 6.82 to be able to help the poor like us 1 2.27 to inform and spread the programs of kkp to develop our locality 2 4.55 the intentions are good 1 2.27 total 44 100.0 socio-economic conditions and perceptions on the conservation of tubbataha reefs 29 and national fishery laws in coordination with the local government and other agencies. another major problem is the lack of fund or financial support for fishery and other livelihood in the area. funding is quite difficult to obtain considering that cagayancillo is a fifth class municipality with no sufficient income to support government's projects. the islands are also isolated from mainland palawan thus there are difficulty in the transportation of goods and services. poor soil condition and lack of irrigated lands make it also difficult for rice production in the area. in addition, despite being a pristine area, only upper level tourist can afford to travel and dive in cagayancillo particularly in tubbataha. changes in the over-all socio-economic conditions over the span of 5 years from 1999, i.e. when the wwf-philippines tubbataha conservation project started until 2004, the last year of the project, some changes on the socio-economic conditions in cagayancillo could be stated. table 13. problems with solutions in the management of coastal resources problems frequency solutions frequency lack of patrol 8 police should patrol always 3 increase patrol 2 poverty 3 go fishing 1 use of illegal fishing 17 avoid use of illegal fishing 12 increase patrol 4 report and arrest illegal fishers 7 give due punishment to illegal fishers 4 to stop outsiders in using illegal fishing 3 prohibit intrusion of outsiders to fish 3 lack of access to tubbataha 1 to make a resolution to allow fishing in tubbataha 1 the sea is deteriorating 2 intrusion of outsiders 5 authorities should make inspection 2 prohibit intrusion of outsiders to fish 3 death of sea weeds 5 ask help from the government 3 no means of transportation 1 provide vehicle 1 lack of funds for fishery, seaweeds and others 11 help, assist, provide funds for livelihood/jobs 3 look for funding for seaweeds farming 1 lack of fish net 2 provide credit 1 lack of nylon 1 lack of boat 2 to work hard in order to earnhelp, assist, provide funds for livelihood/jobs 11 loss of octopus 1 insufficient/lack of products e.g. fish. guard the fish, seaweeds and other sea weeds etc. 11 marine resources 2 lack of patrol boats 6 ask support from the ngo/government for a vehicle to patrol 5 lack of weapons for patrol guards 1 request weapons from the national government 1 lack of radio communication in patrolling 1 provide radio communication 1 lack of information 3 information education through training and seminar 3 lack of source of income/livelihood 2 to pray for the development of seaweed project 1 help, assist, provide funds for livelihood 1 lack of fishing tools and equipment 2 lack of transportation vehicle 1 provide rice that could be bought in cagayancillo 1 lack of sufficient fish to catch 1 avoid destroying corals 1 destroyed coral reefs 1 avoid destroying corals 1 lack of information regarding kkp 1 information education through training and seminar 1 subade and subade 30 1. more movement/ migration of people. the 1999 survey of wwf-philippines showed that only 17% have resided in the municipality only for the last 10 years (decade). this study found that such constituent has grown to 23.4%, perhaps as a product of people's in-migration from mainland palawan, and other provinces in the visayas. 2. slight increase in high school educational attainment. a small improvement in the educational attainment could be noted. from a 17%, those who completed high school was about 24% based in this survey for 2004. assuming that the high school education was of good quality, cagayancillo now have more trainable people for other livelihood activities other than fishing. improved literacy of these families will have a higher chance of gainful employment, thus veering away from high dependence on fishing. 3. decreased/lower income level. at current prices, the 2004 level of family income is p3418.48, which is lower than the 1999 level of p3812 pesos. computing the real value of peso, the family income in 2004 is only p2784.792 in 1999. considering constant prices, or converting it to actual purchasing power, the 2004 level of income is then much lower than that of 1999. this is an indication of a "worse off " condition or lower level of welfare for the cagayanens. this may imply that the effects or impacts of illegal/ destructive fishing that has occurred in cagayancillo have lingered. it would take longer for coral reefs to recover and restore the productivity of fishing grounds. moreover, the disease which struck seaweeds for the past few years diluted a supposed-to-be very good source of income and livelihood for many cagayanens. no wonder why more residents have resorted to farming as their main source of income/livelihood. 4. change in income sources composition/ livelihood. as found by this survey, and compared to the 1999 survey by wwf-philippines, the cagayanens have shifted towards farming as main source of income and livelihood, relegating fishing only as second source of livelihood. this is understandable since households must ensure that staple food (rice, cassava and corn) has to be made available given that fishing is not a stable source of income. in several occasions, respondents interviewed mentioned they cultivate cassava and corn which can provide them food / staple for about one to two months. seaweed farming is also no longer a major source of livelihood because of the seaweed disease that resulted to severe losses. the occurrences of illegal fishing have had also severe impact on the population of cagayancillo, and on the conservation of tubbataha reefs. in the study of subade and subade (2004) shows that 76% of the surveyed residents in cagayancillo cited illegal fishing has negative impact on people's economic conditions and livelihood. they believed that the destructive methods of fishing would eventually result to no catch, and hence no income, which would results to poor living conditions, suffering and hunger. indeed, illegal/ destructive fishing is not sustainable in the short run, it may give abundant catch or lots of money to the fishers, however, it would eventually destroy the marine environment, which is the source of livelihood of people. in addition,. the declaration of tubbataha reefs as no-take zone reduced fishing as the primary source of income of cagayanens. the shift to farming as another source of livelihood may indicate their coping mechanism to ensure more stable supply of food. 5. some indication of ageing population. comparing the 2004 with 1999 data, population structure shows ageing of cagayanen population, whereby the senior citizens now compose 21% of the population, compared to 18% in 1999. the 50-59 age cohort this year has reached 22%% compared to 17% in 1999. this will imply higher need for health services for these people, particularly the senior citizens. 6. higher level of people's involvement in governance system. people's organizations and people's involvement in several activities seem to show greater participation of cagayanens in governance. though this data were not gathered in 1999 study, 73% (35 of 48) of those who mentioned the years/ period of their involvement indicated that it was only in the past twothree years that they have been involved in organizations. people's involvement in organizations, particularly fisher folks and other conservation socio-economic conditions and perceptions on the conservation of tubbataha reefs 31 organizations are effective ways of hastening better governance towards sustainable resource management. the increasing level of participation of the coastal residents can be a good take off point and must be sustained. summary and conclusions as an end-of-project activity of the wwf-philippines tubbataha conservation project, the present socioeconomic monitoring survey was conducted. a total of 110 respondents were randomly selected, proportionately distributed across the 12 barangays of cagayancillo. field interviews were undertaken by 11 interviewers, spot-checked and supervised by three field supervisors. on the average, the respondent was 48 years old, with 7 years of formal education, has lived in cagayancillo for 31 years and belonged to a family/household with 6 members. almost all respondents owned their lots and homes, more than 50%of which were made of g.i sheets for roof and sawali, wood, wood/lawanit and or pawid/nipa for walling. at least 60% admitted they relied solely on rain-fed water tanks for water source. electricity could barely meet a quarter of the respondents' lighting needs. as to livelihood and income sources, cagayanens had shifted to farming as the main source of their livelihood and income, while fishing was only second. household income per month was p 3418.48 for a 6-member family/household, or p 2788.32 for the standard 5member household/family. these shows below poverty threshold level of income for cagayanens, since rural palawan had a poverty threshold income benchmark of p4470.42 way back in 2002. poverty incidence ranged form 67-79%of the cagayanen households, an indication of further threat to environmental resources in cagayancillo. cagayanens can/may just easily fall on the vicious cycle of poverty and environmental degradation, i.e. due to poverty, people would be pushed to further overextract from environmental resources and may even use destructive methods to do so, further depriving themselves of sustainable sources of livelihood, and even worsening their poverty conditions. the income data for 1999 and 2004 when compared, plus the people's perception that present aquatic resources are not in good condition, somehow allude to the possibility that the cycle is happening. some changes in socio-economic conditions may however show that cagayanens are able to cope or are coping with the difficulties they are confronting. there is perception that many of the cagayanens have moved to other places in search for better opportunities, a sign of less stress to the environmental resources. however, more people have moved into cagayancillo in the past decade, which may be good if these people are wellskilled and would utilize resources in more sustainable manner. secondly, more people were able to complete their high school education, an indication of better trainability of the populace, perhaps for other alternative livelihood outside fishing. despite the decreased income level for cagayanen households (for the period 1999 to 2004), they were able to cope, such that, the perception is that there are more families/people who could eat 3 times a day and have enough food nowadays. the shift to farming as main source of livelihood may indicate their coping mechanisms to ensure more stable supply of staples corn and cassava, since rice had to be imported yet at higher price from mainland palawan and iloilo. the good news can be the increasing involvement of people in coastal resource management and conservation. perhaps more people have now realized the importance of protecting and sustainably managing coastal resources, which was their main source of livelihood. by including the people in the main cagayancillo islands as beneficiaries of and participants in the conservation efforts and projects, wwf-philippines/kkp has perhaps initiated a good momentum for a democratized and more sustainable stakeholders management of marine/coastal resources in cagayancillo (which also includes tubbataha reefs). the momentum need to be sustained to ensure genuine success. subade and subade 32 recommendations to sustain the momentum for continuous people's participation in coastal resource management and conservation, and eventually the alleviation of poverty in cagayancillo, concerned policy makers and other entities need to consider the following options: 1. formal and non-formal education and training in sustainable agriculture and farming for selected qualified cagayanens need to be provided as soon as possible. soil analysis by experts will provide best advise on the well-suited crops for different areas of cagayancillo. moreover, due to the unsustainability of chemical and fertilizer dependent farming/crop system, organic farming need to be explored as an option for higher yield. 2. scholarships for selected qualified high school graduates from cagayancillo need to be provided in order to increase income possibilities for these households, particularly the poorest of the poor households. various sources can be tapped – municipal budget can request for yearly appropriation; palawan congressmen; first gentleman arroyo will likely donate if given the proper communication and information; an ngo-sponsored program may be designed to ensure success of this scholarship. 3. in order to address the problem on illegal fishing, increase the bantay dagat or law enforcement capabilities of cagayancillo lgu. the establishment of a bantay dagay is a laudable move. however, with only two boats running at 7-8 knots, patrolling the vast municipal waters of the 31-islands and islets municipality would be a gargantuan task. additional patrol boats, and even faster boats will be needed to increase law enforcement and further minimize illegal fishing activities. moreover, incentives on those who help arrest or find illegal fishers must be continued. in fact, these (cash) incentives must be expedited as soon as the arrest has been done, verified and documented. 4. community-based ecotourism mainly based on marine and coastal resources and environment will be an option with very bright prospects for cagayancillo. details of this is discussed in subade and subade (2004). this will properly complement environmental conservation with increasing household and municipal incomes. 5. the conservation efforts started by wwfphilippines, should be continued for another 2-3 years. these conservation activities include among others, conservation management (includes financing, policy making), conservation awareness through information education campaign (iec), community development, and research and monitoring. gef or other funding sources may be tapped, and should seriously consider providing additional budgetary support for the fruitful work started by wwf-philippines. as an experienced environmental ngo, and since it has already established good rapport and networking in cagayancillo, wwf-philippines is in the best position to continue and sustain the conservation efforts in the next 2-3 years. as found by this study, it seems that the positive effects and fruits of conservation work still need to be felt and realized by many of the households. conservation work and community development efforts take many years, not just five years, to take root and bear fruits. continuity and sustainability of the efforts need to be ensured. wwf-philippines need to seriously consider this matter. 6. partnership and alliances with other government and non-government institutions need to be explored. they may provide needed manpower, financial support and scientific inputs to the coastal resource management in cagayancillo. 7. socio-economic monitoring study and survey can be done every two-three years for database development. given the data recording can be put in place in the respective barangays the needed time and resources for data collection and field interviews can be lessened later on. indicators of success/needs based from future monitoring studies will provide proper signals on what further interventions still need to be undertaken. these information and data are usually needed crucial inputs for good proposals for funding projects for the localities. socio-economic conditions and perceptions on the conservation of tubbataha reefs 33 acknowledgments this study was funded by the kabang kalikasan ng pilipinas inc. (wwf-philippines) and by unesco (united nations educational scientific and cultural organization) contract no. 4500014225 through the tubbataha protected area management board. the authors would like to acknowledge the support of ms. marivel dygico, team leader of the wwfphilippinestubbataha conservation project, and ms. angelique sonco, tubbataha park manager and head of the tubbataha management office, and the field assistance of ms. zoe latumbo of wwf-philippines. assistance in data encoding and analysis was ably provided by ms. dianne hope tormon. in addition, the authors also thank the travel support of conservation international-philippines through the philippine association of marine science foundation inc., and of the university of the philippines in the visayas, to enable the authors in presenting the paper and participate in the 8th national symposium in marine science held in palawan state university, puerto princesa city on october 20-22, 2005. references arquiza, y. and a. white. 1994. tales from tubbataha: natural history, resource use, and conservation of the tubbataha reefs, palawan, philippines. mandaluyong city, philippines: rainee trading and publishing inc. cesar, h.j. 2000. coral reefs: their functions, threats and economic value. in cesar, h. 2000. collected essays on the economics of coral reefs. boras, sweden: cordio and sida. mcmanus. j.w., nañola jr. c.l., reyes jr. r.b. & kesner, k.n. 1992. resource ecology of the bolinao coral reef system. international center for living aquatic resources management, manil, philippines. subade, r.f. and a.l.a. subade. 2004. socio-economic impacts of illegal fishing on the conservation of tubbataha reefs national marine park and unesco world heritage site, cagayancillo, palawan. final report of research project commissioned by the tubataha protected area management board under unesco contract number 4500014225 and the wwf-philippines. uychiaoco a.j., aliño p.m., miclat e.f.b. & campos r.t. 1995. coral reef science and management in the philippines. in philippines coral reef information network (philreefs). uychiaoco a.j. & aliño p.m. 1995. coastal management in the philippines. in philippine coral reef information network (philreefs). wells, s.m. 1984. what's happening to coral reefs? iclarm newsletter. metro manila. philippines. wwf philippines. 2000. tubbataha reef national marine park and world heritage site: management objectives, strategies and actions, p. 15. white a.t., ledesma m.c. & ovenden m. 2003. tubbataha reefs national marine park, palawan. in philippine coral reefs through time: workshop proceedings. second of the atlas of the philippine coral reefs series. coral reef information network of the philippines (philreefs), university of the philippines marine science institute, quezon city, philippines and the marine parks center, tokyo, japan. wwf philippines 2000. conservation of the tubbataha reef national marine park and world heritage site. a project proposal submitted to the undp-gef. sd inside back cover-ched.pmd s c i e n c e d i l i m a n : a p h i l i p p i n e j o u r n a l o f p u r e a n d a p p l i e d s c i e n c e s i s a c c r e d i t e d b y t h e commission on higher education ( c h e d ) t h r o u g h i t s j o u r n a l accreditation service project, as category a-2 journal from 2014 to 2016. 6penuliar.pmd c. b. t. garcia and others 49 science diliman (july-december 2014) 26:2, 49-60 seroprevalence and risk factors associated with seropositivity to toxoplasma gond ii among stray and domestic cats (fel is silvestris catus) in metro manila christel bohn t. garcia ma. jill ian p. talavera gil m. penul iar* university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract toxoplasma gond ii is a protozoan parasite that causes toxoplasmosis. it is widespread in the environment and infects a variety of warm-blooded animals, causing miscarriages and birth problems. previous studies in the philippines have determined the seropositivity of t. gondii in humans. h ow ev e r, t h e s e r o p r e v a l e n ce o f t h e p a r a s i t e a m o n g h o u s e h o l d p e t s , par t i c u l a r l y i t s fe l i n e d e f i n i t i ve h o s t , r e m a i n s i n s u f f i c i e n t . t h i s s t u d y aimed to: (1) determine the seroprevalence of t. gond ii antibodies among domestic and stray cats in the philippines; and, (2) to analyze the risk factors associated with seropositivity. blood samples from 59 domestic and stray cats were collected and tested for t. gond ii seropositivity using a c o m m e r c i a l l y a v a i l a b l e i n d i r e c t e l i s a k i t , w h i l e p e t o w n e r s a n d handlers were given questionnaires about their cats. thirteen or 22.03% o f t h e c a t s w e r e s e r o p o s i t i v e to t. g o n d i i , a n d r i s k f a c t o r a n a l y s i s revealed a signif icant difference betw een domestic and stray cats with regard to diet (p = 0.026, or = 8.333, c = 0.299) and domestication (p = 0.039, or = 5.000, c = 0.276). cats fed with table food tested 31.43% seropositive compared to the 4.35% of those fed with cat food, whereas 33.33% of the stray cats were seropositive compare d to 7.69% for domestic cats. o d d s r a t i o t e s t s h o w e d t h a t t h e r i s k f a c t o r s s t u d i e d w e r e a s s o c i a t e d with higher likelihood of t. gond ii seropositivity. these results implicate diet and environment in the transmission dynamics of t. gond ii among cats. keywords: toxoplasma gond ii, seroprevalence, risk factor analysis, indirect elisa φ  φ  seroprevalence and risk factors associated with seropositivity 50 introduction toxoplasma gondii is an intracellular parasite that infects a wide range of warmblooded animals, including cats, dogs, and humans. it is present worldwide and is of medical and veterinary importance due to its ability to cause miscarriages and birth defects in intermediate hosts (garcia and others 2012). infection by this zoonotic parasite causes toxoplasmosis, occurring through the ingestion of cysts in undercooked and raw meat or the accidental ingestion of oocysts from the environment (duan and others 2012). t. gondii only undergoes sexual reproduction and gametogenesis within its def initive host, the members of family felidae; this serves as the only method for the production of oocysts (webster 2007). oocysts are environmentally stable and are shed in the cat’s feces. these remain infectious for approximately two years, causing widespread contamination and providing a source of infection to humans and other intermediate hosts (yan and others 2012). previous studies on t. gondii in the philippines have covered toxoplasmosis in rats, cats, pigs, and humans (advincula and others 2010). an earlier study on the seroprevalence of t. gondii among filipinos in the philippines revealed an overall seropositivity of approximately 11.1% in the metro manila area, which is a relatively low seropositivity compared to rural areas like leyte (30.1%) and mindoro (61.2%) (kawashima 2000). domestic cats (felis silvestris catus) are household pets that are exposed to similar environments as humans but are less cautious of the cleanliness of their immediate environment. this increases their exposure to t. gondii and should provide a more accurate estimate of the prevalence of the parasite in the environment. as the parasite’s def initive host, cats are crucial for the parasite to reach maturity and complete its life cycle. consequently, t. gondii is able to manipulate the behavior of its intermediate host to enhance transmission to the def initive host (webster 2007). domestic cats, therefore, represent a major source of contamination and infection for humans and other potential hosts. t. gond ii infection can be detected through several diagnostic methods. one of these is the indirect enzyme-linked immunosorbent assay (elisa), a serological test that is one of the most widely used methods for the diagnosis of toxoplasmosis. another technique is the polymerase chain reaction (pcr), a highly specif ic and c. b. t. garcia and others 51 sensitive molecular test that enables detection of the parasite dna (castillo-morales and others 2012). this method, however, requires the use of costly reagents. this study aimed to (1) determine the seroprevalence of t. gondii antibodies among domestic and stray cats in the philippines using indirect elisa and (2) to analyze the risk factors associated with seropositivity. findings from this study will be signif icant in monitoring and controlling the possible infection of intermediate hosts, particularly humans, because of their interaction with cats. materials and methods consent forms an introductory letter indicating the purpose of the study and the assistance needed had been sent to pet owners, shelter administrators, and veterinarians from different sampling sites in metro manila for approval. a consent form explaining the purpose of the study and rights regarding participation had also been provided. questionnaire prior to blood extraction, information regarding the age, sex, breed, diet, location, presence of other cats, contact with the outdoor environment (for domestic cats), health condition, medical history, litter information, and domestication was obtained through questionnaires f illed out by the owners, shelter administrators, and veterinarians. sampl ing sites and blood collection blood samples from domestic cats were collected from the makati dog and cat hospital, riverside village, and companion animal veterinary clinic, whereas samples from stray cats were collected from the philippine society for the prevention of cruelty to animals and the marikina city vet off ice. one milliliter of whole blood sample was collected by licensed veterinarians via venipuncture of the cephalic veins (mccurnin and bassert 2002). proper animal restraint was accomplished before venipuncture. the elbow of the cats was kept extended and pressure was applied such that the vein remains f illed with blood. after the venipuncture, f irm pressure was applied to the puncture site for 60 s to prevent hematoma formation (mccurnin and bassert 2002). fifty-nine cat blood samples were successfully obtained. the blood was stored in red-topped vacutainer tubes seroprevalence and risk factors associated with seropositivity 52 (contains no anticoagulant), labeled, and left in a slanting position for 10 min at room temperature (rt ) to facilitate clotting. the tubes were then kept inside a styrofoam box with ice (stevens and others 2007) to keep the temperature low. the blood samples were then transported to the medical microbiology laboratory in up diliman, where they were either stored in the refrigerator or immediately processed. serum preparation the vacutainer tubes were taken out of storage and left undisturbed at rt for 10 min, to aid in clotting (www.invitrogen.com). three hundred microliters of serum was then taken from the tubes, and transferred into sterile 1.5 ml microcentrifuge tubes. the serum was centrifuged at 1000 x g for 10 min, and immediately transferred to a sterile 1.5 ml microcentrifuge tube in aliquots of 250 μl. the tubes were stored in the freezer (www.invitrogen.com) until a suff icient number of samples were obtained. serological assay igg antibodies against t. gondii in the serum samples were detected through indirect elisa using a commercially available kit (id screen® toxoplasmosis indirect multispecies from id.vet innovative diagnostics, montpellier, france) following the manufacturer’s instructions. samples were prepared in a 96-well microplate coated with p30 antigen of t. gond ii. to each well, 90 μl dilution buffer 2 was added, followed by 10 μl of the negative control in wells a1 and b1, and 10 μl of the positive control in wells c1 and d1. serum samples were thawed and 10 μl were dispensed into the remaining wells. microplates were then incubated for 45 min at rt. the wells were washed thrice with 300 μl wash solution, then 100 μl of conjugate was added, followed by incubation for 30 min at rt. the wells were washed thrice with 300 μl wash solution, then 100 μl substrate solution was added, followed by incubation in the dark for 15 min at rt. the reaction was stopped by adding 100 μl stop solution. the optical density (od) of the samples and controls were measured at 450 nm and recorded using a microplate reader. this assay was performed twice. interpretation and val idation of results the od values were tested for validity using the readings of the positive and negative controls. the test was considered valid if the mean od values of the c. b. t. garcia and others 53 (1)s/p = od sample –od nc od pc –od nc × 100 positive control was greater than 0.350 (od pc >0.350), and if the ratio of the od values of the positive and negative controls was greater than 3.5 (od pc /od nc >3.5). the sample/positive (s/p) percentage was computed for each sample using equation 1. the results for each sample were labeled as either negative (s/p < 40%), doubtful (40% < s/p < 50%), or positive (s/p > 50%). analysis of data statistical analysis of the prevalence of t. gondii across potential risk factors for infection was performed using chi-square test in spss software (release 20.0 standard version, spss inc. , armonk, new york). risk factors that were analyzed included sex, diet, domestication (domestic versus stray), and location (from veterinary clinics versus from animal shelters). p values less than 0.05 were considered to be statistically signif icant (duan and others 2012). otherwise, the null hypothesis was accepted, and the risk factor in question was concluded to play no role in t. gondii infection. phi and cramer’s v values were also reported using cramer’s phi test to determine the strength of association between t. gondii infection and the risk factors. phi values from 0–0.30 indicated absence of relationship to weak relationship, 0.31–0.70 implied a moderate relationship, and 0.71–1.0 suggested a strong relationship (release 20.0 standard version, spss inc. , armonk, new york). odds ratios (ors) were also noted to compare the relative odds of the occurrence of t. gondii infection across the risk factors involved. odds ratio equal to 1 indicated that the risk factor does not affect the likelihood of infection, ors greater than 1 indicated that the risk factor is associated with higher likelihood of infection, and ors less than 1 indicated that the risk factor is associated with lower likelihood of infection (szumilas 2010). waste disposal potentially infectious materials were decontaminated using an autoclave prior to disposal (cdc 2009). seroprevalence and risk factors associated with seropositivity 54 results val id ity of the assay two separate assays were performed and both were deemed valid as each set met the conditions for validity. the ratios of the od values of the positive and negative controls for the f irst and second batches were 3.51 and 3.53, respectively. seroprevalence out of the 59 blood samples collected, 22.03% were seropositive to t. gondii and 72.88% were negative for infection. three samples exhibited a doubtful result. the seroprevalence of t. gondii in cats from marikina, manila, and makati were 27.78%, 40%, and 14.29%, respectively (table 1). table 1. seroprevalence of t. gond ii in cats (fel is catus) based on geographic location in the phil ippines makati city 2 10 2 14 14.29 manila city 6 9 0 15 40 marikina city 5 12 1 18 27.78 pasig city 0 2 0 2 antipolo city 0 10 0 10 total 13 43 3 59 prevalence of t. gond ii(%) positive negative doubtful total table 2. seroprevalence of t. gond ii infection in cats (fel is catus) across each risk factor total 13 43 3 59 stray 11 21 1 33 59 data domestic 2 22 2 26 completed in clinics 2 20 2 24 57 2 from in shelters 11 21 1 33 private homes male 6 15 2 23 56 3 without female 7 25 1 33 information table food 11 23 1 35 58 1 without cat food 1 20 2 23 information positive negative doubtful total sum details c. b. t. garcia and others 55 table 3. summary of statistical analysis for potential risk factors associated with t. gond ii infection * or=1 means that the risk factor does not affect the odds of infection, or<1 means that the risk factor is associated with lower odds of infection, and or>1 means that the risk factor is associated with higher odds of infection. * * p-values<0.05 are considered statistically significant. * * * phi values from 0–0.30 indicate absence of relationship to weak relationship, 0.31–0.70 with a moderate relationship, and 0.71–1.0 with a strong relationship. risk factor odds ratio(or) * p-value** phi value( c)*** sex 1.733 0.403 0.115 diet 8.333 0.026 0.299 domestication 5.000 0.039 0.276 location 5.000 0.054 0.262 φ  the seroprevalence across risk factors are summarized in table 2. each risk factor was observed to indicate at least one cat to be positive. those shown to be seropositive to t. gondii were 33.33% (11/33) in stray cats, 7.69% (2/26) in domestic cats, 8.33% (2/24) in cats kept in clinics, 33.33% (11/33) in cats from shelters, 26.09% (6/23) in male cats, 21.21% (7/33) in female cats, 31.43% (11/ 35) in cats fed with table food, and 4.35% (1/23) in cats fed with cat food. risk factor analysis a statistically signif icant association with seropositivity was found with domestication and diet (p<0.05). the proportion of seropositive cats was found to be higher in stray cats and in cats fed with table food. cramer’s phi, however, indicated that the correlation is not strong, but the or test suggested that all risk factors are associated with higher odds of t. gondii infection (or>1, table 3). discussion cats are considered reservoirs of zoonotic diseases such as toxoplasmosis. these animals that live in close contact with humans increase the risk of transfer of potential infections, especially to immuno-compromised individuals (grøndalen and others 2004). whether domestic or stray, cats are exposed to the same environment as humans and may be used as sentinels that reflect the spread of t. gondii in the environment. a high seropositivity in cats would indicate a high risk of infection in the community. the seropositivity obtained in this study was 22.03% (13/59). this rate of t. gondii infection is rather low relative to those reported in other countries, which can be as high as 80% of tested cats (alvarado-esquivel and others 2007). seroprevalence and risk factors associated with seropositivity 56 a third of the stray cats (33.33%) were seropositive compared to 7.69% in domestic cats. this difference in seropositivity was based on statistical tests (p<0.05). studies conducted in other countries similarly reveal that stray cats generally have higher seropositivity rates and are more prone to infection (meireles and others 2004, miró and others 2004, raeghi and others 2011). the differences in the lifestyles of stray cats and domestic cats affect their daily encounters with the potential parasite, which subsequently affect their health. domestic cats are kept indoors, and are often personally carried or kept in cages when brought outside the house. they are generally exposed to clean, controlled environments free of t. gondii contamination. they may, however, get infected by the ingestion of oocysts that have been left by other cats, or the ingestion of cysts in raw or uncooked contaminated meat. stray cats, on the other hand, are exposed to environments that are not maintained to the same degree of sanitation compared to the inside of a house. the outside environment contains numerous possible sources of contamination, such as raw or uncooked food scraps from domestic garbage, and droppings from intermediate hosts like mice, birds, and reptiles, which may contain cysts. interaction with other free-roaming stray cats also hastens the spread of infections. this is supported by the ors for domestication, which suggested the risk factor is likely to increase the odds of seropostivity to t. gond ii in cats (or=5.000, table 3). a difference in seropositivity values was also seen in cats according to their diets. a higher percentage of positive result was seen in cats fed with table food or leftovers (31.43%), relative to cats fed with cat food (4.35%). the odds ratio test also indicated that diet has the highest or value (8.333), corresponding to the increased chances of infection in cats fed with table food (table 3). these results are similar to those obtained in studies conducted in brazil and mexico by lucas and others (1999), and alvarado-esquivel and others (2007), respectively. table food, when fed to cats, is often mixed with uncooked or undercooked meat and by-products that are easily contaminated. cat food, on the other hand, is factory-processed and is less exposed to the environment prior to feeding, as it is kept in containers or packages. companies that manufacture animal food undergo regular monitoring procedures to ensure the safety of their products. protocols in these companies involve the application high temperatures (>100 °c) in the production of commercial cat food. this ensures the elimination of viable tissue cysts, as t. gondii becomes nonviable at temperatures above 66 °c (meireles and others 2004). in addition, cats fed with cat food are most likely domestic, which as previously mentioned, are less prone to infection. c. b. t. garcia and others 57 a larger percentage of males (26.09%) were seropositive to t. gondii compared to females (21.21%). the difference, however, was not statistically signif icant (p>0.05). this was also seen in studies conducted by alvarado-esquivel and others (2007), hooshyar and others (2007), and wu and others (2011) in mexico, iran, and china, respectively. this may be attributed to the territoriality and roaming behaviors associated with male cats. these allow them to cover a wider range of areas, which increases their chances of coming into contact with oocysts in contaminated meat and environments, and with other cats, particularly females (reyes and others 2013). a study by maruyama and others (1998) suggested that male cats may have increased chances of infection through aggressive encounters during estrus period. the or test, however, confirmed that sex is a risk factor involved with higher likelihood of t. gondii infection (or=1.733, table 3). seropositivity was also higher in cats being cared for in animal shelters (33.33%) compared to those kept in veterinary clinics (8.33%). or test conf irmed that location as a risk factor can possibly increase the likelihood of t. gondii infection (or=5.000). generally, the cats from animal shelters were usually stray cats, whereas cats from veterinary clinics were usually domestic cats. in this study, the same holds true. the main difference was the sample size, with the risk factor for location having fewer samples due to incomplete data supplied by the participants. as it is, the higher seropositivity in cats from animal shelters may be explained by the same reasons justifying the higher seropositivity among stray cats. in conclusion, the seroprevalence of t. gondii among cats in metro manila is relatively low compared to other countries, which may indicate that the parasite is not as widespread in the areas covered by the study. results from the risk factor analysis also emphasize the role of diet and environment in the transmission dynamics of t. gondii among cats. acknowledgments the results of this paper were presented as poster during the 43rd annual convention and scientif ic meeting of the philippine society for microbiology, cebu city, 2014. we thank juvy ann c. palma for technical assistance. this work was supported by the off ice of the vice chancellor for research & development of the university of the philippines diliman, through the ph.d. incentive awards (project no. 121218 phdia). seroprevalence and risk factors associated with seropositivity 58 references advincula j.k.d.c. , iewida s.y.p. , salibay c.c. 2010. serologic detection of toxoplasma gondii infection in stray and household cats and its hematologic evaluation. scientia medica (porto alegre) 20: 76-82. alvarado-esquivel c. , liesenfeld o. , herrera-flores r.g. , sanchez-ramirez b.e. , gonzalezherrera a. , matrinez-garcia s.a. , dubey j.p. 2007. seroprevalence of toxoplasma gond ii antibodies in cats from durango city, mexico. j. parasitol 93(5): 1214-1216. castillo-morales v.j. , v iana k.y.a , ed. guzmán-marín s. , jiménez-coello m.j. , seguracorrea j.c. , aguilar-caballero a.j. , ortega-pacheco a. 2012. prevalence and risk factors of toxoplasma gond ii infection in domesticated cats from the tropics of mexico using serological and molecular tests. interdisciplinary perspectives on infectious diseases 2012. center for disease control and prevention (cdc), 2009. biosafety in microbiological and 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kawabata m. , barzaga n. , matsuda h. , konishi e. 2000. prevalence of antibodies to toxoplasma gondii among urban and rural residents in the philippines. southeast asian j trop med public health 4: 742-6. l i f e te c h n o l o g i e s t m p r o t o c o l s . i n v i v o r n a i p r o t o c o l s . av a i l a b l e f r o m h t t p : / / www.invitrogen.com/. accessed 30 april 2013. lucas s.r.r. , hagiwara m.k. , loureiro vd.s. , ikesaki j.y.h. , birgel e.h. 1999. toxoplasma gond ii infection in brazilian outpatient cats. rev. inst. med. trop. s. paulo 41(4): 221-224. c. b. t. garcia and others 59 maruyama s. , hiraga s. , yokoyama e. , naoi m. , tsuruoka y. , ogura y. , tamura k. , namba, s. , kameyama y. , nakamura s. , katsube y. 1998. seroprevalence o f bartonella henselae and toxoplasma gondii infections among pet cats in kanagawa and saitama prefectures. journal of veterinary medical science 60(9): 997-1000. mccurnin d.m. , basser t j.m. 2002. diagnostic sampling and treatment techniques. in: clinical textbook for veterinary technicians, 5th edition. philadelphia, usa: elsevier inc. available from http: //www.catnmore.com/animals/pdfs/venipuncturehow to.pdf. accessed 30 april 2013. meireles l.r. , galist eo a .j. j r. , pompeu e. , andrade h.f. j r. 2004. toxoplasma gond i i spreading in an urban area evaluated by seroprevalence in free-living cats and dogs. tropical medicine and international health 9(8): 876-881. miró g. , montoya a. , jiménez s. , frisuelos c. , mateo m. , fuentes i. 2004. prevalence of antibodies to toxoplasma gond ii and intestinal parasites in stray, farm and household cats in spain. veterinary parasitology 126: 249-255. raeghi s. , sedeghi s. , sedeghi s. 2011. prevalence of toxoplasma gond ii antibodies in cats in urmia, northwest of iran. the journal of animal & plant sciences 21(2): 132134. rey e s m . f . , g u ev a r a v. g . , s a n ro q u e d. g . d. g . , f l o r e s m . l . s . , l a s t i c a e . a . 2 0 1 3 . seroprevalence o f toxoplasma gond ii antibodies in domestic shor t-haired cats ( fel is ca t u s ) in a wildlife facility in manila. philippine journal of ve terinary and animal sciences 39(1): 99-106. s t ev e n s v. l . , pa te l a .v. , f e i g e l s o n h . s . , ro d r i q u e z c . , t h u s m . j . , ca l l e e . e . 2 0 0 7. c r y o p r e s e r v a t i o n o f w h o l e b l o o d s a m p l e s c o l l e c t e d i n t h e f i e l d f o r a l a r g e epidemiologic study. cancer epidemiol biomarkers prev 16: 2160-2163. szumilas m. 2010. explaining odds ratios. j can acad child adolesc psychiatry 19(3): 227-229. webster j.p. 2007. the effect of toxoplasma gond ii on animal behaviour: playing cat and mouse. schizophrenia bulletin 33: 752-6. wu s.m. , zhu x.q. , zhou d.h. , fu b.q. , chen j. , yang j.f. , song h.q. , went y.b. , ye d.h. 2011. seroprevalence of toxoplasma gond ii infection in household and stray cats in lanzhou, northwest china. parasites & vectors 4: 214. yan c. , fu l.l. , yue c.l. , tang r.x. , liu y.s. , lv l. , shi n. , zeng p. , zhang p. , wang d.h. , zhou d.h. , zhu x.q. , zheng k.y. 2012. stray dogs as indicators of toxoplasma gond ii distribut ed in the environment: the f irst repor t across an urban-rural gradient in china. parasites & vectors 5: 5. seroprevalence and risk factors associated with seropositivity 60 _______________ christel bohn t. garcia has a bs biology degree from the institute of biology, up diliman, and is an active member of the up association of biology majors and up pre-medical society. ma. jill ian p. talavera is a fourth year bs biology student of the institute of biology, up diliman. she is currently conducting her undergraduate thesis on microbiology under the medical microbiology laboratory and plans to take up graduate studies on microbiology as well. gil m. penul iar <gil.penuliar@gmail.com> is an assistant professor of the institute of biology, up diliman. he has a doctorate degree in medical science from gunma university graduate school medicine, and a master’s degree in microbiology from the up diliman. his f ield of specialization is systems microbiology. ocr document ocr document ocr document laboratory experiments-catane.pmd laboratory experiments on steady state seepage-induced landslides 17science diliman (january-june 2011) 23:1, 17-30 laboratory experiments on steady state seepage-induced landslides using slope models and sensors sandra g. catane1, mark albert h. zarco2, cathleen joyce n. cordero1, roy albert n. kaimo1, ricarido m. saturay, jr.1 1engineering geology laboratory, national institute of geological sciences university of the philippines, diliman, quezon city 1101 2geotechnical engineering group, institute of civil engineering university of the philippines, diliman, quezon city 1101 a thorough understanding of the failure initiation process is crucial in the development of physicallybased early warning system for landslides and slope failures. laboratory-scale slope models were constructed and subjected to instability through simulated groundwater infiltration. this is done by progressively increasing the water level in the upslope tank and allowing water to infiltrate laterally towards the toe of the slope. physical changes in the slope models were recorded by tilt sensors and video cameras. when the model slope was destabilized, the chronology of events occurred in the following sequence: (1) bulging at the toe, (2) seepage at the toe, (3) initial failure of soil mass, (4) piping, (5) retrogressive failure, (6) formation of tension cracks and (7) major failure of soil mass. tension cracks, piping and eventual failure are manifestations of differential settlements due to variations in void ratio. finite element analysis indicates that instability and subsequent failures in the model slope were induced primarily by high hydraulic gradients in the toe area. seepage, initial deformation and subsequent failures were manifested in the toe area prior to failure, providing a maximum of 36 min lead time. similar lead times are expected in slopes of the same material as shown in many case studies of dam failure. the potential of having a longer lead time is high for natural slopes made of materials with higher shear strength thus evacuation is possible. the tilt sensors were able to detect the initial changes before visual changes manifested, indicating the importance of instrumental monitoring. keywords: seepage-induced landslides, landslide initiation, wireless sensors, early warning system, philippines abstract catane, et al 18 introduction landslides caused serious human and property losses to vulnerable communities and have contributed longterm environmental degradation. refinement of methods and technologies in landslide hazard assessment and risk reduction has been the focus of research in the past decade. most of these, however, are applicable to small landslides and only affluent communities are capable of installing rather expensive monitoring systems and maintaining engineering mitigating structures. in the case of massive and potentially disastrous landslides, there is no effective yet economically viable structural mitigation measure. in the end, the only options are to permanently relocate threatened communities or provide a reliable early warning system. an early warning system which is cost effective yet efficient is desired in order to make it affordable to marginalized communities, which are the most susceptible to landslides. existing early warning system for landslides are mostly based on instrumental monitoring of rainfall and/or distressed slopes (mittal et al., 2008; simeoni and mongiovi, 2007; tommasi et al., 2006). parameters used in early warning systems are commonly rainfall threshold and ground deformation. in particular, rainfall thresholds have been widely used in community-based warning systems (e.g., larsen and simon, 1992; guzzetti et al., 2007) but only limited areas have established rainfall thresholds worldwide. moreover, landslide initiation does not solely depends on rainfall volume and intensity but also on other factors such as antecedent precipitation, fluctuations of groundwater level, geologic and topographic conditions (tohari et al., 2007). ground deformation that precedes failure has also been investigated in recent studies (sakai, 2001; araiba, 2006). hong and adler (2007) attempted to develop a real-time global satellite-based landslide prediction system based on two essential data sets, landslide susceptibility mapping and rainfall data analysis. potential improvement to early warning systems can be drawn from the results of laboratory experiments. the effect of changes in parameters such as moisture content (orense et al., 2004; tohari et al., 2007), porewater pressure (kuriakose et al., 2008), matric suction (huat et al., 2005; gofar et al., 2008) and deformation (sakai, 2001; araiba, 2006) that leads to failure were investigated. orense et al. (2004) and tohari et al. (2007) found that failure of experimental slopes is preceded by an increase in moisture content. we conducted nine laboratory experiments in a landslide box to investigate the changes in various parameters that initiate failure such as moisture content and deformation using sensors. this paper summarizes the results of the experiments such as the physical changes in the model slope and response of tilt sensors with rising water level and correlation of parameters with visual observations. a two-dimensional modeling to simulate the landslide process was also performed based on parameters used in the experiment. the result of the study is useful in identifying the most sensitive factors in an attempt to forecast landslides. early warning system in the philippines the philippines has been identified as one of the landslide hotspots in the world (kjestad, 2007) due to its steep topography, wet climate and active tectonic setting. among the 162 landslide-prone countries, the philippines ranks 4th in terms of risk of human exposure to landslide and 6th in terms of potential economic loss (united nations international strategy for disaster reduction secretariat, 2009). disastrous landslides in the philippines happened more frequently since 1999. these include: (1) 1999 cherry hills landslide in antipolo, (2) 2003 panaon island-surigao flooding landslides, (3) 2004 aurora-quezon flooding landslides, (4) 2006 guinsaugon landslide in southern leyte, (5) 2006 mayon lahars in albay (catane, et al., 2008). about 3,000 loss of lives and billions of pesos of property damage were incurred by these events. all the above mentioned landslides were preceded by heavy rainfall but no prior monitoring and early warning system were in place. the high casualty and enormous property damage highlight the need for an early warning system, especially in high-risk areas. soon after the panaon disaster in 2003, the philippine government launched the national geohazards mapping program. landslide susceptibility maps of 1:50,000 scale were produced for the entire country. more recently, a science diliman (january-june 2011) 23:1, 17-30 laboratory experiments on steady state seepage-induced landslides 19 multi-agency program called hazards mapping for effective community-based disaster risk management (ready) was implemented at the local level (ndcc, 2008). the program has been conducted in 27 selected and high risk philippine provinces. one of the components is community-based disaster risk mitigation using community-based early warning system (cbews). meanwhile, various non-government organizations (ngos) such as manila observatory (mo), center for environmental concerns (cec), care philippines and center for disaster preparedness (cdp), have organized local communities affected by landslides (e.g., auroraquezon, guinsaugon and legaspi). early warning system is an integral part of their programs. current early warning systems in the philippines are underdeveloped since values for rainfall threshold is based on limited instrumental and historical data, or global values rather than site-specific values. moreover, since the relation between rainfall and landslide occurrence is empirically determined and site-specific, threshold values established for an area could not always be applied to other areas. thus, the relation of rainfall to landslide initiation needs further clarification. an effective early warning system requires full understanding of landslide triggering process prior to failure. a research program, disaster risk management using sensors, networks and computing: early warning system for landslides, slope failures and debris flows (drms), was conceived to investigate landslide initiation on model slopes using wireless sensor networks (wsn). this research program aims to develop an alternative means for monitoring slopes that is both cost effective yet efficient. it is a collaborative research between the college of engineering and college of science of the university of the philippines, diliman. methods a landslide box was used to reproduce seepage-induced failure where slope geometry and soil properties were kept constant in all the experiments except for experiment 1 where loose soil was used. sensor columns were inserted in the upper slope and in the toe. initial condition on water level was established after which water column was raised at an approximate rate of 10 cm/10 min to 10 cm/15 min until the water level reached 115 cm. given the initial conditions and observations, a numerical model simulation was performed to check the consistency of the results. soil sample the soil used to construct the model slopes in the experiments is porac sand obtained from porac river, pampanga province. porac sand is a lahar deposit derived from pinatubo volcano. it is composed of plagioclase, quartz and ferromagnesian minerals. to improve the homogeneity of the sample, it was sieved using a 1 mm wire mesh to remove greater than 1 mm coarse fraction. the specific gravity of the soil is 2.67. the maximum and minimum void ratios are 0.918 and 0.699, respectively (orense et al., 2006). loose soils used in experiment 1 represent behavior for high void ratio whereas dense soils used in experiments 2 through 9 represent behavior for low void ratio. landslide box the landslide box, shown in fig. 1a, is 240 cm long, 90 cm wide and 120 cm high. its walls, except for one, are made of steel plates. a transparent plexi glass was used in one of the side walls to visually observe changes in the model slope. the box is divided into three sections: the upslope water tank, the downslope water tank and the main chamber (fig. 1a). the water tanks are 30 cm x 90 cm while the main chamber is 180 cm x 90 cm. separating the said chambers are perforated steel walls covered with wire mesh. the walls allow water to pass through without flushing out the sand grains. a plastic hose was used to fill in the upstream water tank. faucets are installed at the bottom of the water tanks to control water outflow. the plexiglass and steel side panels were lubricated using petrolatinum jelly to minimize the friction between the panel and the soil. this resulted in a condition in which shear stresses were assumed to be negligible. the side panels were considered to be rigid such that science diliman (january-june 2011) 23:1, 17-30 catane, et al 20 no deformation in the direction perpendicular to the wall resulting in negligible out-of-plane strains. also, the impermeable side panels prevented flow perpendicular to the wall which made the seepage two dimensional. the perforated panels at the upslope and downslope were assumed to be completely pervious such that the hydraulic head at these boundaries were assumed equal to that in the tanks. these boundaries were also assumed to be sufficiently rough and rigid so as to prevent displacements both in the vertical and horizontal directions. (a) (b) science diliman (january-june 2011) 23:1, 17-30 monitoring sensors sensor columns containing several nodes for measuring tilt are installed at the upper slope and at the toe of the model slope. the nodes with a sampling interval of 1 sec measure acceleration and transmit collected data to a central repository computer. the data is converted to tilt angle and analyzed for ground deformation. the details of the sensor column development were described in de dios et al. (2010). laboratory experiments on steady state seepage-induced landslides 21 visual monitoring two video cameras were set-up to monitor the changes in the model slope such as development and movement of wetting front and deformation. camera 1 is positioned to monitor the cross-section and camera 2 is placed in front of the slope face. camera 2 provides a more detailed view of the saturation process, movement of the soil mass and failure sequence. this visual monitoring set-up facilitated correlation of tilt sensor readings and visual observations. initial conditions except for experiment 1, where the soil was loosely placed, the model slopes were constructed in the main chamber and were manually compacted in lifts of 10 cm thickness. the first three layers of soil were spread uniformly on the surface of the chamber to form the base of the slope. the final dimension of the slope was 90 cm high and 120 cm wide with a slope angle of 37°. after constructing the model slope, the upslope tank was filled gradually with water up to the 40 cm mark. water was allowed to seep through the soil and percolate laterally towards the downslope tank. initial condition was set at 40 cm and 20 cm water level at the upslope and downslope tanks, respectively. in the series of experiments, it took 3 to 7 hours to establish the initial conditions. science diliman (january-june 2011) 23:1, 17-30 figure1. schematic diagram of the landslide box used in the experiments. (a) section view and (b) plan view. (c) laboratory set-up showing the landslide box, sensors and data acquisition system. (c) the experiment proceeded by increasing water inflow in the upslope tank while keeping the 20 cm mark in the downslope tank. to initiate failure by seepage, the water level in the upstream tank was increased at a rate of 10 cm/10 min from l=40 cm to l=80 cm then 10 cm/15 mins from l= 80 cm until the head of the slope collapses. this condition is steady-state seepage. in the real world, this set-up closely resembles a steadystate flow of groundwater in (1) natural slopes, and (2) accumulation of water behind natural landslide debris dams and earth dams used in containing mine tailings. the experiment is completed when no further failure is observed. finite element modeling a two-dimensional elastoplastic finite element modeling of the landslide box was performed using rocscience phase 2 software to determine which factors significantly affect the failure of the slope. the soil mass was assumed homogenous and isotropic. it has a permeability of k=1x10-3 cm/sec, a young’s modulus of e=15 mpa, and a poisson’s ratio of v=0.3 based on laboratory tests performed on the same material type and void ratio (zarco et al., 1999). based on direct shear strength tests performed on reconstituted samples, the mohr coulomb parameters, effective friction angle ( ’=34°) and effective cohesion (c’=0.001 mpa) for a sample with a saturated unit weight of 18 φ’  catane, et al 22 kn/m3 were used for the analyses. the mesh used consisted of 175 6-node interpolated triangular elements. this mesh provides the optimum solution as further refinement of the mesh will not significantly change the results. the problem was analyzed as plane strain problem in which the resistance between the soil and the walls of the landslide box was considered negligible. details regarding the implications of this assumption in comparison with experimental results are given in the section under discussion. the loading sequence consisted of computing the insitu state of stress from gravity loads, then increasing the height of the water in the upslope water tank in seven stages to a height of h 0 = 0.3 m, 0.6 m, 0.75 m, 0.9 m, 0.95 m, 1.0 m, and 1.07 m. results we conducted a total of 9 experiments (experiment 1 to 9) using similar conditions. appropriate initial conditions for the slope model were tested while tilt, pore-water pressure and soil moisture sensors (de dios et al., 2010) were tested and calibrated in the first 6 experiments. the subsequent experiments, 7, 8 and 9 produced consistent and reasonable results. only visual monitoring and tilt measurements were used in the last three experiments because the other sensors still need calibration and refinement. experiment 7 is described in detail in this paper. experiments 8 and 9 produced similar results, which demonstrate the repeatability of the experiment. although details of each experiment were unique, the results and sequence of events were consistent in the last three experiments. table 1 summarizes water levels, significant events and sensor reading changes. development of the saturation zone in order to establish the initial conditions, water was allowed to seep through the perforated steel wall and migrate laterally toward the downslope tank, producing a saturation zone. the profile of the wetting front is asymmetric as shown in fig. 2. a capillary zone has developed adjacent to the upslope tank which resulted to the higher level of the saturation zone than the water science diliman (january-june 2011) 23:1, 17-30 level in the upslope tank. when the initial conditions were attained, the experiment was set to commence at t 0 =0. when the water level was increased, the saturation zone rose and expanded laterally. seepage occurred at the toe when the water level in the upslope water tank reached 70 cm at t 1 =30 min. the first movement of the soil mass was observed after 44 min (t 2 ) when almost half of the slope face was saturated. the water level at this time was 84 cm. piping at the sand/wall interface began at t 3 =53 min at 90 cm water level (fig. 3). at t 4 =94 min, water from the upslope tank flooded the main chamber due to retrogressive failure of the slope. slope deformation movement of the soil mass was preceded by the development of bulges or pressure ridges at the toe area at t 1d =21 min and water level of 61 cm (fig. 4). minor failures of the slope in the form of surface erosion occurred at t 2d =44 min and water level of 84 cm. these movements marked the onset of retrogressive failure. meanwhile, a sand slurry was formed at the base of the slope when seepage water was absorbed in the failed soil mass. tension cracks started to form in the unsaturated portions of the slope face. the first tension crack was observed at t 3d =71 min and a water level of 99 cm. a series of failure occurred along the cracks. the largest tension crack appeared near the head of the slope (fig 5). the failure of the soil mass along this crack happened at t 4d =81 min. the experiment was terminated at t 5d =94 min when the head of the slope finally collapsed and water surged from the upslope tank. correlation with sensors the nodes in the sensor column installed at the toe area show changes in tilt up to 0.9 deg (fig. 6). movements were detected 14 to 22 min prior to the laboratory experiments on steady state seepage-induced landslides 23 table 1. correlation of water levels, significant events and sensor reading changes time (min) upslope tank water level (l) (cm) visual observation: saturation zone visual observation: deformation toe sensor reading upslope sensor reading 0 40 t0: establishment of initial condition t0: establishment of initial condition t0: establishment of initial condition t0: establishment of initial condition 8 48 tn3.1: initial change in tilt in toe sensor node 3 21 61 -----t1d: bulging at the toe ---------- 22 62 ---------- tn3.2: change in tilt in toe sensor node 3 ----- 30 70 t1 : formation of seepage area at the toe --------------- 31 71 ---------- tn1: change in tilt in toe sensor node 1 ----- 40 80 ---------- tn2: change in tilt in toe sensor node 2 ----- 44 84 t2: initial movement of soil mass t2d: initial movement of soil mass ---------- 53 90 t3: piping --------------- 55 90 --------------- tn4: change in tilt in upslope sensor node 4 60 93 --------------- tn5:change in tilt in upslope sensor node 5 62 94 --------------- tn6: change in tilt in upslope sensor node 6 63 95 --------------- tn1:change in tilt in toe sensor node 1 64 96 --------------- tn3=tn7: change in tilt in upslope sensor nodes 2 and 7 65 96 --------------- tn3: change in tilt in upslope sensor node 3 71 99 ----- t3d: first tension crack appears ---------- 81 110 ----- t4d: major failure of soil mass ---------- 93 113 t4: water from the upslope tank flooded the main chamber t5d: head of the slope collapsed ----------   science diliman (january-june 2011) 23:1, 17-30 catane, et al 24 figure 2. profile of the asymmetric wetting front at 80 cm water level. figure 3. piping at the toe of the slope model (view from camera 2). figure 4. bulging at the toe (view from camera 2). figure 5. plan view of the largest tension crack just above the upslope sensor column. (view from camera 2). figure 6. toe sensor readings. all nodes have positive tilt (node tilts away from the slope). science diliman (january-june 2011) 23:1, 17-30 laboratory experiments on steady state seepage-induced landslides 25 visually observed seepage at the toe and 9 to 13 min before bulging manifested. in experiment 7, the sensors detected the initial tilt 36 minutes before the first failure (t 2d ) and 72 minutes before the major failure (t 4d ) of the slope. the upslope sensors did not detect any of the earlier movements in the toe area. however, changes were recorded when movements progressed to the upper portion of the slope model but prior to the main failure (fig. 7). finite element modeling results of the analyses for each stage are illustrated in fig 8. as shown in the figures, progressive saturation of the slope results in a vertical downward displacement of the top of the slope, followed by lateral bulging at the toe of the slope beginning at h 0 = 0.75 m (fig. 8d). the bulging continued to increase as the head in the upstream water tank was increased until failure occurred at h 0 = 1.07 m (fig. 8h). the bulging continued to increase as the head in the upstream water tank was increased until failure occurs at h 0 = 1.07 m (fig. 8h). also noticeable was the development of very high hydraulic gradients in a localized zone at toe area of the slope which indicates the presence of large seepage forces locally occurring in this area. during numerical experiment, the hydraulic gradient in the toe zone has increased from 0.7 when initial bulging occurs in the toe zone at h 0 = 0.7 m to a maximum value of 0.96 when failure occurs at h 0 = 1.07 m. these values are within the critical hydraulic gradient required to cause erosion and piping which is estimated to be within range of 0.87 to 1.06 based on the given range of void ratios of the material. figure 9 illustrates the predicted conditions within the slope during failure at h 0 = 1.07 m. the finite element mesh together with the enforced boundary seepage and deformation boundary conditions is shown in fig. 9a. figure 9b shows the corresponding deformed mesh and displacement vectors at the initiation of failure, while fig. 9c is a contour plot showing the variation of the shear strain within the slope during failure. figures 9b and 9c indicate a rotational type failure for the slope. figure 9d is a contour plot of the pore pressure distribution within the slope. comparing this figure with corresponding fig 8h, failure in the slope occurs primarily due to high hydraulic gradients that resulted in large seepage forces, rather than the decrease of shear strength resulting from high pore pressures. in experiments 2 through 9 where the slope model was constructed by placing and compacting material in layers as previously described in the methods, no significant vertical settlements or slumping was observed with increasing water level. slumping at the figure 7. upslope sensor readings from t 1d to t 4d . positive tilt values=node tilts away from the slope; negative tilt values=node tilts towards the slope. science diliman (january-june 2011) 23:1, 17-30 catane, et al 26 total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 (a) (b) (c) (d) total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 (e) (f) figure 8. contour plot showing the change in hydraulic gradient distribution within test embankment with increase in upstream head. (a) initial gravity load, (b) h 0 =0.3 m, (c) h 0 =0.6 m,(d) h 0 =0.75 m, (e) h 0 =0.9 m, (f) h 0 =0.95 m, (g) h 0 =1.0 m, (h) h 0 =1.07 m. (g) (h) total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 total hydraulic gradient 0.00e+000 9.00e-002 1.80e-001 2.70e-001 3.60e-001 4.50e-001 5.40e-001 6.30e-001 7.20e-001 8.10e-001 9.00e-001 9.90e-001 1.08e+000 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 science diliman (january-june 2011) 23:1, 17-30 laboratory experiments on steady state seepage-induced landslides 27 toe was noticed only when the critical hydraulic gradient was reached. furthermore, the failure surface was observed to be shallow and elongated in shape. in experiment 1, where the material was loosely placed during construction of the slope model, significant vertical settlements at the crest together with slumping of the slope were observed as the water level increased. the failure mechanism was observed to be deep seated with a circular-shaped failure surface. these observations are consistent with that predicted by the numerical simulation. it is noteworthy that deformations during the increase of water level are predominantly inelastic, i.e. these deformations are permanent. the differences in the observed movements and the failure mechanism described above can be attributed to the dilative behavior of dense or stiff soils upon shearing (roscoe et al., 1958). during shearing at failure, this dilative tendency results in resistance figure 9. results of finite element analysis of landslide box experiment summarizing conditions at failure. (a) finite element mesh, (b) deformed mesh and displacements vectors at failure, (c) contour plot of shear strain profile at failure, (d) contour plot of pore pressures at failure. between the soil and the landslide box. this resistance increases with depth within the slope model. at the surface where the soil is not confined, the resistance along the sides of the landslide box is less. this phenomenon can be simulated in the numerical model by including side resistance as upward vertical body force proportional to the vertical stress at a given point. for numerical simulations of experiments 7 through 9, imposing a body force corresponding to 80% of the vertical stress results in similar displacements and failure mechanism with that obtained experimentally. discussions the slow infiltration of water into the soil mass has led to the initiation of various phases of slope failure. the formation of bulges or pressure ridges, seepage and surface erosion at the toe area were all related to the development of very high hydraulic gradients, which indicates the presence of large seepage forces locally occurring in this area. 0 0 .2 0 .4 0 .6 0 .8 1 1 .2 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 (a) critical srf: 1 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 (b) critical srf: 1 maximum shear strain 0.00e+000 7.00e-004 1.40e-003 2.10e-003 2.80e-003 3.50e-003 4.20e-003 4.90e-003 5.60e-003 6.30e-003 7.00e-003 7.70e-003 8.40e-003 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 (c) critical srf: 1 pore pressure mpa -0.00 -0.00 -0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0 0 .2 5 0 .5 0 .7 5 1 1 .2 5 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 (d) science diliman (january-june 2011) 23:1, 17-30 catane, et al 28 this condition was first captured by the toe sensor node 3 at t=30 min and water level of 70 cm (fig. 6). the above result justifies the importance of installing sensors at the toe of slopes since they are most sensitive to changes. finite element modeling predicted the very high hydraulic gradients (fig. 8) rather than the reduction of shear strength resulting from high pore pressures as the main mechanism for failure. high hydraulic gradients are physically manifested by pressure ridges, seepage and surface erosion. the modeling confirms that the appropriate site for installation of monitoring instrument and sensors is the toe area to capture the changes in hydraulic gradients. initial failure of the toe caused the removal of support and increased the slope angle and consequently resulted to retrogressive failure. the change in tilt in upslope sensor nodes 1 to 7 are all related to retrogressive failure (fig. 7). tension crack is an extension fracture caused by tensile stress. it reduces the overall stability of a slope by decreasing the shear strength. even before the tension cracks manifested, all nodes in the upslope sensor column have detected change in tilt (fig. 7). in natural slopes, abrupt variations in void ratio naturally occur. the experiments highlight the differences in settlement patterns and failure mechanisms due to these variations. the key implication to this is that large differential settlements can occur during saturation which then can cause fracturing, giving rise to conditions ideal for piping, erosion and eventual slope failure. lead times in large scale embankments are expected to be similar to those in the laboratory model. based on documented failures of large-scale earth dams, the time between initiation of piping and embankment failure is generally in a matter of a few hours. for example, in the case of the teton dam (idaho, usa) and baldwin hills dam (california, usa) failure, the lag time was 4-4.5 hrs and 2-3 hrs, respectively (randle et al., 2000). laboratory experiments indicate that instrumental and visual observations of deformation are useful indicators of impending failure apart from changes in saturation, which was visually observed. in many landslides, precursors such as appearance of seepage, deformation at the base of the slope and the formation of tension cracks were observed prior to failure. similar features were noted in the experiments even if the conditions in natural slopes are more complex due to variability of earth materials and seepage conditions. although the lead time for physical changes is rather short in the experiments, the potential of having a longer lead time is high for natural slopes made of materials with higher shear strength. conclusion the toe area manifests the most significant visual and instrumental measurement changes. the physical changes observed such as pressure ridges, seepage and surface erosions are indications of high hydraulic gradients as demonstrated by the finite element modeling. differences in void ratio give rise to differential settlements in the slope that manifest as fractures, piping and eventual failure. a maximum of 36 min lead time prior to failure is possible for a slope of the same soil properties, geometry and initial conditions. similar lead times have been observed to occur in large scale embankments. the initial result of this study indicates that instrumental and visual observations of deformation are useful indicators of impending failure apart from changes in saturation and pore-water pressure. indeed, initial changes in the model slope were detected by the sensors highlighting their importance in monitoring and prediction. instrumental monitoring of the upper part of the slope is not as significant as those observed in the toe since the deformation features appeared only after failure has started. however, monitoring the upper slope is still recommended because changes occurred prior to the main failure, which is the most disastrous phase in the evolution of a landslide. science diliman (january-june 2011) 23:1, 17-30 laboratory experiments on steady state seepage-induced landslides 29 acknowledgement this paper is a result of a continuing research program funded by the department of science and technology and the university of the philippines, diliman. it leverages on synergistic collaboration between scientists and engineers of the college of science and college of engineering of the university of the philippines, diliman. we thank our colleagues and students for their invaluable inputs and support: dr. joel joseph marciano, marc caesar talampas, earl anthony mendoza, rosanno jc de dios, jason enriquez, francis victorino, franz libao, al john lexter lozano, andrew sandoval, krestabelle futalan, jane kristine teves and edna patricia mendoza. references araiba, k., 2006. study on the method for detecting and monitoring of pre-failure deformation in slope. disaster mitigation of debris flows, slope failures and landslides 2: 581-589 catane, s.g., zarco, m.a.h. and saturay, r.m., 2008. landslide-risk reduction strategies and practices in the philippines: in: the proceedings of the first world landslide forum, tokyo, japan. de dios, r.j., enriquez, j., victorino, f.g., mendoza, e.a.v., talampas, m.c. and marciano, j.j.jr. 2010. a tilt, soil moisture, and pore water pressure sensor system for slope monitoring applications. science diliman 21: 15-27. gofar, n., lee, m.l. and kassim, a., 2008. response of suction distribution to rainfall infiltration in soil slope. electronic journal of geotechnical engineering 13: 1-13. guzzetti, f., perucacci, s., rossi, m. and stark, c. p., 2008. the rainfall intensity–duration control of shallow landslides and debris flows: an update. landslides 5: 3-17. hong, y., adler, r. and huffman, g., 2006. evaluation of the potential of nasa multi-satellite precipitation analysis in global landslide hazard assassment. geophysical research letters 33: l22402, doi:10.1029/2006gl028010. huat, b.b.k., ali, f.hj. and mariappan, s., 2005. a study on suction-rainfall response of a cut slope in unsaturated residual soil using a field rain 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h., parayno, f. c., tuibeo, l. l. s., papa, j. r. p. jr., and de leon, g. r. 1999. geotechnical properties of redeposited lahar derived from the 1991 mt. pinatubo eruption. terminal report to pcierd-dost. technical report no., up/brs-1-1-99. science diliman (january-june 2011) 23:1, 17-30 manuscript submision form_vol.22-2.pmd author certifications: i certify that this manuscript contains new content not previously published or submitted elsewhere for simultaneous consideration. i warrant that the article will not be submitted to another publication/publisher while under consideration for publication in science diliman. i further certify that all the other authors, if any, have agreed to the submission of this manuscript to the journal. ____________________________________________________ _________________ printed name and signature date return this form to: up rduo-ovcrd attn: science diliman lg/f phivolcs bldg., c. p. garcia ave., up diliman 1101, quezon city, philippines tel. nos. 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____________________________________________________________________________________ as an aid to the indexer, please list down a maximum of six keywords that describe the content of your manuscript: ____________________________________________________________________________________ ____________________________________________________________________________________ manuscript submission form rduo form 3b science diliman research dissemination and utilization office up office of the vice chancellor for research and development total mercury in three fish species sold in a metro manila public market: monitoring and health risk assessment criselda r. africaa, artemio e. pascuala and evangeline c. santiagoa* anatural sciences research institute, university of the philippines, diliman, quezon city 1101, philippines, telephone: +6329207731 telefax: +6329286868 *corresponding author: ecs@nsri.upd.edu.ph received: 23 january 2007; revised: 28 april 2009; accepted: 28 april 2009 abstract the total mercury concentrations in bangus or milkfish (chanos chanos forskal), tilapia (oreochromis niloticus) and galunggong or round scad (decapterus spp.) purchased from a local market in metro manila from 5 august to 20 october 2004 were determined by cold vapor atomic absorption spectrophotometry. the ranges of total mercury concentrations observed from about 30 composite test samples for each fish species were 0.0060 to 0.015 mg kg-1 (wet weight) for bangus, 0.0041 to 0.017 mg kg-1 (wet weight) for tilapia and 0.014 to 0.05 mg kg-1 (wet weight) for galunggong. risk assessment for neurological effects associated with the consumption of the fish species with the highest concentration of mercury (0.05 mg kg-1 for galunggong) was done. the calculated daily dose of total mercury of 0.06 µg d-1 kg-1 body weight indicates that consumption of any one or any combination of bangus, tilapia, and galunggong sold in nepa-q-mart from august 5 to october 20 in 2004 does not entail risk of adverse neurological effects. keywords: milkfish, cold vapor atomic absorption spectrophotometry, round scad, health risk assessment, mercury monitoring, tilapia introduction among the heavy metals that cause adverse health effects in humans, mercury is one of the most prevalent in the environment. degassing of the earth’s crust is the most important natural source of mercury in the environment; however, human activities result in increasing considerably the presence of mercury in the environment. burning fossil fuels in incinerators and power plants, mining operations, lead smelting, and pulp and paper processing are some of the activities that release mercury into the air, soil and water. when mercury from the natural and man-made sources finds its way into the water, it can be transformed into its most toxic form, organic methyl mercury, through the action of anaerobic bacteria in the water system. methyl mercury accumulates in fish and the extent of mercury accumulation in fish depends on the level of mercury in the water and on the place of the fish in the food web (sloof et al., 1995). consumption of methyl mercury-contaminated fish by man poses risks especially to children and childbearing women who are the most vulnerable (cox et al., 1989, sloof et al., 1995, fao/who 2003). the adverse health effects of high level mercury contamination that include cardiovascular effects, severe nervous system damage and death have been documented (atsdr 1992, goyer 1996). health risk through fish consumption can be science diliman 21(1):1-6 1 mailto:ecs@nsri.upd.edu.ph africa, pascual & santiago evaluated by measuring the rate of mercury intake based on the mercury content of the fish (seg 1971, who-ipcs 1991). to minimize the health risk of mercury, government regulatory agencies have issued health advisories on the kinds of fish that consumers should avoid and have provided regulatory limits on mercury in fish. the maximum allowed/recommended levels of methyl mercury in fish are 0.5 mg kg-1 in the united states, european union, korea, thailand, philippines, and the world health organization/food and agriculture organization (who/fao), and 0.3 mg kg-1 methyl mercury in japan, china and the united kingdom (unep 2003). other countries have set maximum levels for total mercury in fish at 0.4 mg kg-1 in japan and 0.2 mg kg-1 in australia (unep 2003). the philippines is rich in mineral resources and the exploitation of these mineral resources by big and small scale mining operations in the country is expected to increase the level of mercury in the aquatic environment. the philippines, being an archipelago, is blessed with long coastal waters as sources of fish, making fish a major source of protein for most filipinos. in a survey done in 1994-96 (unep 2003), the country ranked third among the biggest consumers of fish in asia (75 g d1 per person) after japan (107 g d-1 per person) and korea (74 94 g d-1 per person). in view of the potential enhancement of mercury contamination of the aquatic system by mining operations and industrial wastes and the importance of fish in the filipino diet, it is important to investigate if the fish that filipinos consume will not pose adverse health effects due to mercury. the concentrations of mercury in some commercial fish species from albay gulf (santiago and africa, 2008), manila bay (prudente et al., 1997) and laguna lake (cuvin-aralar, 1990) have been reported. this study, however, is the first investigation of the mercury levels of widely consumed fish species sold in a public market in the greater manila area. the health risk associated with the consumption of these fishes based on standard estimation of health risk is also reported. materials and methods sampling design samples of bangus (c. chanos f.) 19-32 cms, tilapia (o. nilotica) 16-24 cms, and galunggong (decapterus spp.) 10-30 cms, were purchased in nepa-q-mart, quezon city in eight batches from 5 august to 20 october 2004. the three species of fish were selected because of the abundance of supply and relatively cheaper price that make them the most affordable among the fishes sold in the market. for each sampling batch, eight fish stalls were chosen at random. depending on the size of the fish, representative samples (1-5 fishes) from each fish stall were taken at random on each sampling period. for each species, all eight representative fish samples from eight fish stalls were cleaned, cut into pieces, and combined before homogenization. test procedures preparation of samples. the procedure for preparation of fresh fish samples before analysis is the aoac standard method 937.07 (hollingworth et al., 1990). briefly, the fresh fish samples were cleaned, scaled and eviscerated, and cut up according to size. large fishes (≥ 20 cm) were cut into several cross-sectional slices approximately 2.5 cm thick and had their bones removed. for small fishes (≤15 cm) and intermediate-sized fishes, heads, scales, tails, fins, guts, and inedible bones were removed and discarded. all body flesh from head to tail was taken. the fish flesh was homogenized in a waring blender and subsamples were prepared by quartering technique. the samples were kept in the freezer when analysis could not be done immediately. before weighing, the sample was thawed to room temperature and rehomogenized. a portion of the subsample (5.0000 ± 0.0001 g) was weighed in a 250 ml erlenmeyer flask for mercury analysis. digestion of sample and analysis of total mercury. the procedures for digestion and analysis of the fish sample for mercury were adopted with some modifications from a published method (bouchard 1973). concentrated nitric acid (5 ml) was added to the sample and the flask was covered with polyethylene film to allow digestion of the sample overnight. five percent chromic acid (10 ml) was added and the digestion was allowed to continue for at least 30 minutes. ultrapure water (15 ml) was added after 2 science diliman total mercury in three fish species sold in mm public market digestion was completed. hydroxylamine crystals (4 g) were added prior to instrumental analysis. total mercury was analyzed by cold vapor (flameless) atomic absorption spectrophotometry using thermo jarrell ash video 11e equipped with a hamamatsu mercury hollow cathode lamp operated at 3 ma and 1.0 nm spectral bandwidth. absorption of light was measured at 253.6 nm. the digested sample was transferred quantitatively into a reaction flask which was attached to an aeration apparatus (see figure 1). tributylphosphate (8 drops) was added to the sample to minimize foaming. ten percent stannous chloride solution (10 ml) was immediately added and reaction was allowed to proceed. the absorbance of mercury that was volatilized and carried by air into the absorption cell was measured. the calibration curve was constructed from absorbance data of mercury standards against concentration of standards (0, 0.02, 0.05, 0.10, 0.20, 0.50 and 1.0 µg) using linear regression (microsoft excel program). the mercury standards were prepared from mercury standard solution (titrisol brand) from merck, usa. the mercury concentration of the samples is expressed in mg kg-1 units based on the wet weight of the sample. method validation. the method was validated using spiked samples with 0.02 µg (low level) and 0.2 µg (high level) mercury in fish and with reference material dorm-2 (nrc·cnrc dogfish muscle certified reference material for trace metals). the method detection limit (mdl) is 0.003 mg kg-1 total mercury (n=8) calculated based on 5 g of sample. the analysis of dorm-2 showed a bias of + 0.02 mg kg-1 and a precision of 3.8 % (n=10). the mean recovery and precision of the 0.2 µg hg spike samples (n=20) are 137 % and 5 % rsd, respectively. the uncertainty for the measurements was calculated from the uncertainty due to random effects. a summary of the validation and quality control data is presented in table 1. quality control. duplicate samples of reagent blank and method control sample (sample spiked with 0.4 µg hg) were included in the analysis of each batch of samples. the absorbance obtained from the reagent blank was subtracted from the absorbance obtained for the sample. mean recovery and precision of the method control sample (n=8) are 104 % and 13 % rsd, respectively. the fish samples were analyzed in three or four replicates; the mean concentration is reported for the sample from each batch. the concentration of total mercury obtained in the sample was not corrected for recovery. health risk assessment. the allowed concentration of mercury in fish is calculated from the daily reference dose (rfd ) and the daily consumption of fish. the rfd for mercury is the daily dose that is considered safe or the dose that does not entail an appreciable risk of adverse effects of mercury (usepa 2001). the rfd is based on the benchmark dose, obtained from the lower 95% confidence limit for a 5% effect in a linear model of the doseresponse curve; the response is usually a neurological endpoint (usepa 2001). the usepa calculated an rfd of 0.1 µg kg-1 body weight d-1 for mercury based on the risk to the adult woman, the population sector which is most vulnerable to the adverse effects of mercury (usepa 2001). health risk is estimated by comparing the daily dose of mercury from consumption of fish with the reference dose rfd. consumption of mercurycontaminated fish will not entail neurological effects if the daily dose of mercury will not exceed the rfd of 0.1 µg kg-1 body weight d-1 (usepa 2001). the daily dose or estimated daily intake (edi) can be calculated using equation 1 (kotsonis et al., 2001): edi = hg concentration in fish (g/g) × daily consumption of fish (g d -1 per person) / weight of person (kg) (1) science diliman 3 figure 1.setup for mercury determination by flameless atomic absorption spectrophotometry africa, pascual & santiago table 1. data on validation of method and quality control for analysis of total hg in fish test material n mean experimental concentration* standard deviation mean theoretical concentration mdl † mean % recovery‡ rsd (%)¶ unspiked tilapia sample 12 0.0087 0.0003 0.001 3 0.02mg hg spiked on tilapia 8 0.01349 0.00009 0.0039 123 7 0.2mg hg spiked on tilapia 5 0.0622 0.003 0.039 137 5 unspiked bangus 3 0.015 0.001 7 0.4mg hg spiked on bangus 8 0.097 0.013 0.079 104 13 mean experimental concentration certified concentration dorm-2(nrc-cnrc) reference material 10 4.66 0.18 4.64 100 3 * all concentrations are expressed as mg kg-1total hg, wet weight † method detection limit is calculated as 3standard deviation ‡ mean %recovery is calculated as the (difference of the mean experimental concentrations of spiked and unspiked samples divided by the mean theoretical concentration)  100; mean concentration is the average concentration of the number of samples (n) analyzed ¶ rsd (%) is relative standard deviation – calculated as (standard deviation / mean experimental concentration) 100 results and discussion measurement results for mercury in bangus, tilapia, and galunggong table 2 shows the results of analysis for total mercury in all the samples collected. total mercury was found in the range of 0.006 to 0.015 mg kg-1 (wet weight) for bangus, 0.0041 to 0.017 mg kg-1 (wet weight) for tilapia and 0.014 to 0.05 mg kg-1 (wet weight) for galunggong. the average mean of the means of total mercury concentration are 0.010 mg kg-1 for bangus, 0.009 mg kg-1 for tilapia, and 0.032mg kg-1 for galunggong. however, test of significance using two-tailed tests related to means showed evidence (table 2) that some random batches among the samples collected for bangus and tilapia species are not drawn from the population having the measured population average total mercury concentration. hence, the total mercury concentration for these species is reported as a range of concentration. the average total hg and expanded uncertainty for galunggong is reported as 0.03±0.01 mg kg-1 since all the samples have been shown statistically to come from the measured population average. galunggong, which is caught in marine waters, showed the highest contamination with mercury, followed by bangus, which is grown in fish cultures in brackish or estuarine waters. tilapia, which is grown in fresh water fish cultures, showed the least contamination. in laguna lake where both tilapia and bangus are grown in aquaculture, the concentrations of mercury were found to be higher in tilapia than in bangus; with the highest concentrations of 0.1 mg/kg dry weight and 0.057 mg/kg dry weight respectively (cuvin-aralar, 1990). since the fish species investigated are all non-predators, the result suggests that the marine water where the galunggong were caught is more polluted with mercury than the aquatic environments where bangus and tilapia were raised. fishes, belonging to decapterus spp., including galunggong, are near shore pelagic fishes that feed mostly on zooplanktons such as hyperiid amphipods and crab megalops (mc naughton, b., 2008). unlike in the big pelagic fishes which prey on other fishes, the main pathway of accumulation of mercury in galunggong may not be through the food chain. this observation agrees with the result of an assessment of mercury levels in commonly-consumed marine fishes in malaysia (hajeb, p. et al., 2009) where the mercury concentration found in scad (0.04 µg/g dry weight) was much lower compared to the concentrations in short=bodied mackerel (0.45 µg/g dry weight) and long-tailed tuna (0.5 µg/g dry weight). it is most likely that the mercury found in galunggong is the result of the exposure of the fish to the marine waters. it is expected that the marine waters would have more methyl mercury in the water column than in freshwater because the sea is a bigger sink for mercury than rivers and lakes. in addition, the water column is deeper and the presence of anaerobic bacteria is greater in marine waters than in the estuarine and fresh waters. 4 science diliman total mercury in three fish species sold in mm public market table 2. result of three-month monitoring of total hg in fishes sampled from a metro manila market bangus (chanos chanos forskal) sampling batch date of sampling mean batch concentration n standard deviation repeatibility rsd (%) z 1 5-aug-04 0.0152 3 0.001 7.4 7.4 2 24-aug-04 0.0095 4 0.002 19 -0.7 3 6-sep-04 0.0116 3 0.0008 6.9 2.3 4 14-sep-04 0.0104 4 0.001 9.5 0.57 5 22-sep-04 0.0089 4 0.002 16 -1.6 6 29-sep-04 0.0096 4 0.0006 6.6 -0.57 7 13-oct-04 0.0060 4 0.0007 12 -5.7 8 20-oct-04 0.0103 4 0.0009 9.1 0.42 range of hg concentration 0.0041-0.017 average of means 0.009 standard deviationreproducibility 0.004 tilapia (oreochromis nilotica) sampling batch date of sampling mean batch concentration n standard deviation repeatibility rsd (%) z 1 5-aug-04 0.0066 3 0.00069 10 -1.7 2 24-aug-04 0.011 4 0.00066 6 1.4 3 6-sep-04 0.0088 3 0.0004 4.1 -0.14 4 14-sep-04 0.0044 4 0.0004 10 -3.2 5 22-sep-04 0.0166 4 0.0018 11 5.4 6 29-sep-04 0.0093 4 0.0004 4.6 0.21 7 13-oct-04 0.0041 4 0.0004 10 -3.5 8 20-oct-04 0.0091 4 0.0004 4.5 0.07 range of hg concentration 0.0041-0.017 average of means 0.009 standard deviationreproducibility 0.004 galunggong (decapterus spp) sampling batch date of sampling mean batch concentration n standard deviation repeatibility rsd (%) z 1 5-aug-04 0.014 3 0.0013 9.4 -1.1 2 24-aug-04 0.0426 4 0.0022 5.2 0.90 3 6-sep-04 0.0167 4 0.0025 15 -0.95 4 14-sep-04 0.0184 3 0.0026 14 -0.83 5 22-sep-04 0.0357 3 0.0054 15 0.41 6 29-sep-04 0.0463 4 0.0023 5 1.16 7 13-oct-04 0.0367 4 0.0022 6 0..47 8 20-oct-04 0.0503 4 0.0027 5.3 0.74 range of hg concentration 0.014-0.050 concentration of average of means 0.03 standard deviation of the average of means 0.014 pooled standard deviation for repeatability 0.00231 combined standard deviation for random effects 0.014468 combined standard uncertainty for random effects 0.005 u, expanded uncertainty 0.01 *all concentrations are expressed as mg kg-1 total hg, wet weight †z value from two tailed test of means, calculated as [ave of means – batch mean /std of the average of means /8] where std is standard deviation; a z value of more than ± 2.58 indicates evidence at 95% ci that the batch does not belong to the population with total mercury concentration equal to the average of means. ‡combined standard uncertainty for random effects is combined uncertainty due to repeatability and reproducibility, calculated as (combined std due to random effects) 2/8 where combined std due to random effects is calculated as std reproducibility 2 pooled stdrepeatability 2 ¶u is calculated as 2 standard uncertainty for random effects science diliman 5 africa, pascual & santiago health risk assessment the per capita fish consumption for the filipino adult was reported as 75 g d-1 in 1998 (unep 2003) and 69 g d-1 in 2003 (fnri 2003a). the published average weight for an adult filipino woman is 54 kg and for an adult filipino male, 60 kg (fnri 2003b). the health risk assessment was calculated for the adult filipino woman to give bias to the sector of the population most vulnerable to the effects of mercury. based on the 2003 data on fish consumption and average weight of a filipino adult woman, a daily dose of total mercury of 0.06 µg kg-1 body weight d-1 was estimated for the consumption of fish with the maximum total mercury contamination (0.05 µg g-1). the calculated daily dose or the estimated daily exposure due to consumption of fish is less than the rfd = 0.1 µg kg-1 body weight.d-1. to exceed the rfd , the same fish consumption rate would require a maximum concentration of 0.08 mg kg-1 of fish. the risk assessment indicates that the consumption of any one or any combination of bangus, tilapia, and galunggong bought from nepa-q-mart within august 5 to october 20, 2004 will not entail risk of adverse neurological effects for an average adult filipino consumer. acknowledgment the authors acknowledge the support of the natural sciences research institute for this research project. references [atsdr] agency for toxic substances and diseases registry. 1992. mercury washington dc, us department of health services bouchard a. 1973. determination of mercury after room temperature digestion by flameless atomic absorption. atomic absorption newsletter 12(5):115-117. cox, c., clarkson, t.w., marsh, d.o. 1989. doseresponse analysis of infants pre-natally exposed to methylmercury – an application of a single computed model to single strand hair analysis. environment research. (49):318-332. cuvin-aralar, l. 1990. mercury levels in the sediment, water and selected finfishes of laguna lake, the philippines. aquaculture 84(3):277-288 [fao/who] food and agriculture organization/ world health organization. 2003. jecfa/61/sc summary and conclusions of the sixty-first meeting of the joint fao/who expert committee on food additives (jefca), rome. [fnri] food and nutrition research institute. 2003. food consumption survey component. in 6th national nutrition survey, fnri, department of science and technology. [fnri] food and nutrition research institute. 2003. anthropometric survey component. in 6th national nutrition survey, fnri, department of science and technology. goyer r a. 1996. toxic effects of metals. in: klaasen c.d ed . casarett and doull’s toxicology: the basic science of poisons, 6th ed, mc graw hill, 834-837. hajeb. p., jinap, s., ismail, a., fatimah, a.b., jamilah, b., abdul rahim, m. 2009. assessment of mercury levels in commonly-consumed marine fishes in malaysia. food control 20(1):79-84. hollingworth, t., wekell, m. 1990. fish and other marine products. in: helrich k, editor. official methods of analysis of the association of official analytical chemists, 15th ed. p. 864. kotsonis, f., burdock, g.a., flamm, w.g. 2001. food toxicology. in: klaasen cd, editor. casarett and doull’s toxicology: the basic science of poisons, 6th ed, mc graw hill, p.1058. mcnaughton, b.d. 2008. a biological and social examination of opelu (decapterus spp) fisheries in west hawaii, hawaii island. ms. thesis. conservation biology and environmental science program, university of hawaii at hilo, hawaii. prudente, m., kim, e.y., tanabe, s., tatsukawa, r.. 1997. metal levels in some commercial fish species from manila bay, the philippines. marine pollution bulletin, 34(8):671-674. santiago, e.c., africa, c.r. 2008. trace metal concentrations in the aquatic environment of albay gulf in the philippines after a reported mine tailings spill. baseline/ marine pollution bulletin, 56:1650-1667. [seg] swedish expert group. 1971. methyl mercury in fish, a toxicological–epidemiological evaluation of risk. nord-hyg. tidskr 4 (supp) 19-34. slooff, w., van beelen, p., annema, j.a., janus, a. 1995. integrated criteria document: mercury. rivm no. 601014. [unep] united nations environment program. 2003. global mercury assessment report. retrieved march 23, 2006 from http://www.chem.unep.ch/mercury/report/ chapter4.htm#4.4 6 science diliman 01_device kazanidis, p.a. 18 *corresponding author science diliman 20:1, 18-23 some important definitions definition 1. let ω be any non-empty set. let r be a partition of ω × ω. (ω,r) is a coherent configuration if the following axioms are satisfied: (a) the set {(α, α) | α ∈ ω} is a union of some elements of r. this set is called the diagonal. (b) for each class c ∈ r, the transpose c* which is defined as {(β, α) | (α, β) ∈ c} is a class in r. (c) for all c i ,c j and c k in r and for any (α, β) ∈ c k , there is a non-negative number pk ij such that the number of γ satisfying (α, γ) ∈ c i ∧ (γ, β) ∈ c j (1) is equal to pk ij . this number is independent of the choice of (α, β) ∈ c k . if g is a permutation group on ω and r is the set of orbitals then, (ω, r) is called the coherent configuration associated with g. the group g is said fission and fusion schemes and the action of the diagonal group d(t, n) priscila alejandro kazanidis* institute of mathematics, college of science university of the philippines, diliman, quezon city email: cristyale35@yahoo.com date submitted: march 15, 2006; date accepted: july 9, 2008 abstract this paper is a closer look at associate classes of an association scheme. the orbitals of a subgroup d(t, n) of the full automorphism group of the association schemes served as building blocks for the fusion schemes. keywords: association scheme, fusion and fission schemes, orbital, intersection number, permutation, group action to be generously transitive on ω if given any (α, β) ∈ ω × ω, there is an element g ∈ g such that (α, β)g = (β, α) (2) replace item (b) by (b’) for each class c, c*= c. with (b’), the coherent configuration is called association scheme. if item (a) is replaced by (a’) the diagonal is one class in r, then, (ω, r) with r as a set of orbitals is called an association scheme associated with the transitive permutation group g. a group g with association scheme (ω, r) which is generously transitive is necessarily transitive on the elements of ω. some relations between association schemes are defined here. definition 2. consider the following association schemes. a1 = (ω, r 1 ) ∧ a 2 = (ω,r 2 ) (3) fission and fusion schemes and the action of the diagonal group d(t; n) 19science diliman 20:1, 18-23 the association scheme a 1 is finer than a 2 if for all c k ∈r 1 , there exists c l ∈ r 2 such that c k ⊂ c l . also, a 2 is said to be coarser than a 1 . the association scheme a 1 is fission scheme while a 2 is called a fusion scheme. the diagonal group diagonal groups were defined by cameron (cameron, 1999) and the following are their definition in terms of generators (alejandro, et al., 2003). a diagonal group d(t, n) where t is a group and n a positive integer is a permutation group acting on a non-empty set ω given by ω = {[x 1 , x 2 , . . . , x n ]|x i ∈ t} (4) the following are the generators of d(t, n): (a) the group tn acting by right translation, that is the permutations [x 1 , x 2 , . . . , x n ] [x 1 t 1 , x 2 t 2 , . . . , x n t n ] (5) for t 1 , t 2 , . . . , t n ∈ t; (b) the automorphism group of t, acting coordinatewise, that is, [x 1 , x 2 ,..., x n ] [x 1 α, x 2 α, ... , x n α] (6) for α ∈ aut (t); (c) the symmetric group s n , acting by permuting the coordinates, that is, [x 1 , x 2 ,..., x n ] � [x1 , x2 , ..., xn ] (7) where π ∈ s n ; (d) the permutation τ acting as follows: [x 1 , x 2 ,..., x n ] � [x1 −1, x 1 −1x 2 , ..., x 1 −1x n ]. (8) another way of looking at diagonal groups is by their action on the right cosets of a point-stabilizer. define a group g as follows: g = tn+1(out(t) × sn+1) (9) consider the subgroup h = aut(t) × sn+1 (10) the inner automorphisms are identified with the diagonal subgroup {(t, t, ..., t)⏐t ∈ t} (11) of tn+1. this is how diagonal groups are described in the o’nan-scott theorem. each right coset of h in g has a unique coset representative (1, x 1 , ... , x n ) ∈ tn+1. this representative is denoted [x 1 , ... , x n ]. now, let us observe the action of g on the coset representatives: [x 1 , ... , x n ](1, t 1 , ..., t n ) = h(1, x 1 , ... , x n ) (1, t 1 , ..., t n ) = h(1, x 1 t,1 ..., x n t n ) = [x 1 t 1 , ..., x n t n ]. (12) thus we obtain the permutations in (a). a conjugation by an element t ∈ t is uniquely a product of an element of tn+1 with first coordinate 1 and the diagonal element (t, ..., t) as seen below: [x 1 , ..., x n ](t, ..., t) = h(1, x 1 , ..., x n )(t, ..., t) = h(t, x 1 t, ..., x n t) = h(1, t-1x 1 t, ..., t-1x n t) = [t-1x 1 t, ..., t-1x n t] (13) hence, inn(t) is a subset of tn+1. the remaining automorphisms in (b) are exactly the outer automorphisms of t. observe the following action of τ: [x 1 , ..., x n ]τ = h(1, x 1 , ..., x n )τ = h(x 1 ,1, x 2 , ..., x n ) = h(1, x 1 -1, x 2 , ..., x 1 -1x n ) = [x 1 -1, x 1 -1x 2 , ..., x 1 -1x n ] (14) the symmetric group s n+1 is generated by the symmetric group s n in (c) and the transposition τ = (1, 2) in (d). diagonal groups d(t, n) where n ≥ 8 and t abelian are generously transitive permutation groups. all orbitals in orbl(ω, d(t, n)) are symmetric and this gives a commutative association scheme. some association schemes result from fission schemes. for the case of orbl(ω, d(t, n)), a fission scheme is formed by looking → → π π π kazanidis, p.a. 20 science diliman 20:1, 18-23 at a proper subgroup k of d(t, n). a special set of generators is used to construct the subgroup k. by taking union of elements of orbl(ω, k), the association scheme from orbl(ω, d(t, n)), is obtained. the character tables corresponding to the basis algebra of orbl(ω, k) and orbl(ω, d(t, n)) will be investigated by making each entry a function of t and n. the orbitals let t be a permutation group acting on itself by right translation. let autt be the automorphism group of t acting on the elements of t naturally. the semi-direct product t2 × outt acts on t the following way: x((t1,t2),γ) = (t 1 -1 xt 2 )γ (15) for all x ∈ t and for all (t 1 , t 2 )γ ∈ t2 × autt. the orbitals of t2 acting on t is the set of orbits resulting from the induced action of t2 on t × t. the orbitals of outt in t is defined similarly. the two actions are shown below: (x 1 , x 2 )(t1,t2) = (t 1 -1 x 1 t 2 , t 1 -1 x 2 . t 2 ) (x 1 , x 2 )γ = (xγ 1 , xγ 2 ) (16) the discussion below shows that the orbitals of t2 × outt can be computed using the above orbitals. the following is a detailed discussion of an important part of the proposition. let (α 1 , β 1 ) be any element of (ω × ω). suppose that for some γ ∈ autt, (α 1 , β 1 )γ = (α, β). also, for some (t 1 , t 2 ) ∈ t × t (α, β)(t1, t2 ) = (α2, β2). then, (α2, β2) is in a t 2-orbital containing an image of (α1, β1) under an automorphism γ. proposition 1. let t be a group and outt its outer automorphism group. then, an orbital o of t2 × outt containing the element (α1, β1) in ω × ω in the action previously defined is the union of all t2orbitals and all autt-orbitals containing images of (α1, β1). proof. let o be an orbital of t2 × autt containing (α1, β1). suppose that (α2, β2) is an element of o satisfying the following: (a) for ι ∈ autt (α1, β1) ((t1, t2),ι) = (t 1 -1 α1t2, t1 -1 β 1 t 2 ) = (α2, β2) (17) (b) for t ∈ t (α1, β1) ((1,1),γ) = (α1 γ, β1 γ) = (α2, β2) (18) (c) for some non-identity elements t 1 , t 2 ∈ t and also non-identity auto-morphism γ ∈ autt (α1, β1) ((t1,t2),γ) = ((t 1 -1 α1t2) γ, (t 1 -1 β1t2) γ) = (α2, β2) (19) in case (a) (α2, β2) is trivially in the t 2-orbital containing (α1, β1). in case (b) (α2, β2) is again trivially in the autt-orbital containing (α1, β1). for case (c), let α : = α1 γ, β : = β1 γ, t 1 = t 1 γ and t 2 : = t 2 γ. then (α2, β2) = (t1 -1 αt 2 , t 1 -1βt) (20) this proves that (α2, β2) is in the t 2-orbital containing (α, β) which in turn is the image of (α1, β1) under γ. the three cases cover all possible images of (α1, β1) under the induced action of the semi-direct product t2 × autt. therefore, ( ∪ ot ) ∪ ( ∪ oautt ) ⊂ o (21) where the union is taken over all orbitals containing images of (α1, β1). � as a remark, to get the orbital o containing (α1, β1) under the semi-direct product t2 × autt, all autt-orbitals containing all elements of the t2-orbital must be listed. additionally, all t2-orbitals containing all elements of the autt-orbital containing (α1, β1) must also be listed. this result was used in a gap program (the gap group, gapgroups, algorithms and programming, version 4, http://www.gap-system.org) implemented to compute fission and fusion schemes. the set of orbitals as the finest association scheme the fusion schemes will be constructed based on the fact that the finest association scheme preserved by γ γ fission and fusion schemes and the action of the diagonal group d(t; n) 21science diliman 20:1, 18-23 tn+1 × outt is its set of orbitals resulting from its action on t. recall that an automorphism of an association scheme is a permutation of the set of points ω which preserves associate class c i for each i = 0, 1, ..., m where m is the number of associate classes. the set of all orbitals o forms association scheme as discussed here. consider the orbitals o i , o j and o k which are not necessarily distinct. let (α, β) ∈ o k . it can be shown that the intersection number pk ij is independent of the choice of (α, β). denote by g the group tn+1 × outt . suppose that pk ij is the non-negative number which gives the cardinality of the set {γ⏐(α, γ) ∈ o i ∧ (γ, β) ∈ οj} (22) let (δ,λ) ∈ o k . it can be shown that (δ,λ) has the same intersection number. let g ∈ g such that (δ,λ) = (α, β)g. then {γg⏐(αg, γg) ∈ o i ∧ (γ g, β) ∈ ο j } = {γg⏐(δ, γg) ∈ o i ∧ (γ g, λ) ∈ ο j }. since g is a permutation of t, the cardinality of this set is constant given by pk ij . this means that the intersection number is independent of the choice of (α, β). the set of orbitals o is the finest association scheme preserved by the the permutation group g. the following is an explanation. suppose that there is a finer association scheme preserved by g. let õ i be an associate class of a finer one which is properly contained in orbital o i . for some g ∈ g, õ i g ≠ õi because the group g is transitive on the elements of its orbitals. therefore, õ i is not preserved by g. this contradicts our assumption that the finer association scheme is preserved by the group g. the associate classes of fusion schemes are union of orbitals of tn+1 × outt acting on the elements of t. the group tn+1 × outt preserves these associate classes because it preserves its orbitals. hence, it is a subgroup of the full automorphism group of the fusion scheme. intersection numbers and incidence matrices the last axiom of the definition of association scheme deals with intersection numbers. this section discusses intersection numbers and its relation to the product of incidence matrices. the incidence matrix a i corresponding to the associate class c i will be constructed by using the elements of ω as the row and column indices. the matrix a i is defined as follows: 1 if (α, β) ∈ c i a i (α, β) = 0 otherwise (23) if a i , a j and a k are respectively the incidence matrices of associate classes c i , c j and c k , then the product a i ⋅ aj can be written as a i ⋅ a j = p0 ij ⋅ a 0 +p1 ij ⋅ a 1 + ⋅⋅⋅ +pm ij ⋅ a m = σ k=mpk ij a k (24) where m is the number of associate classes. the (α, β)-entry of the product a i ⋅ a j is non-zero if and only if there is at least one γ such that (α, γ)-entry of a i and (γ, β)-entry of a j are both 1. this is equivalent in saying that there is at least one γ such that (α, γ) ∈ c i and (γ, β) ∈ c j . the intersection number pk ij gives the number of such γ if and only if (α, β) ∈ c k . hence, the intersection number pk ij is the coefficient of a k in the linear expansion of a i ⋅ a j . fusion schemes from groups isomorphic to z p proposition 2. the semi-direct product t2 × autt is sharply 2-transitive on ω where t ≅ zp for some prime number p. proof. let ρ be a generator of the group z p . consider the following arbitrary pairs of distinct elements of z p × z p . (ρa, ρb) ^ (ρc, ρd) (25) k=1 kazanidis, p.a. 22 where a, b, c, d ∈ {0, 1, ... , p 1}. since we would like to prove sharp 2-transitivity, we shall consider ordered pairs whose first component is distinct from the second component. hence, the following conditions are satisfied. a ≡ b mod p and c ≡ d mod p (26) consequently, a b ≡ 0 mod p and c d ≡ 0 mod p (27) we shall find an element ((t 1 , t 2 ), γ) ∈ t2 × autt such that (ρa, ρb)((t1, t2),γ) = (ρc, ρd) (28) there is an element (ρv1, ρv2) ∈ t × t such that (ρa, ρb)((ρv1, ρv2 ),ι) = (ρ-v1+a+v2 , ρ0) = (ρw, ρ0) (29) from above, we deduce the following equation: b ≡ (v 1 v 2 ) mod p (30) the element w of z p satisfy the following: w ≡ (a b) mod p (31) there is an ordered pair (d 1 ,d 2 ) where d 1 ,d 2 ∈{0, 1,⋅⋅⋅,p1} such that d ≡ (-d 1 + d 2 ) mod p (32) now, let x ∈ {0, 1, ⋅⋅⋅ , p 1} such that x ≡ (c d) mod p (33) then, using the assumptions above, we have w ≡ 0 mod p and x ≡ 0 mod p (34) it is known that the full automorphism group autt of t is transitive on its non-identity elements. hence, there exists γ ∈ autt such that (ρw)γ = ρx. thus, (ρa, ρb)((ρv1, ρv2 ),ι), ((ρ0, ρ0),γ) = (ρx, ρ0) (35) applying ((ρd1, ρd2),ι), we get (ρx, ρ0)((ρd1, ρd2 ),ι) = (ρx-d1+d2, ρ-d1+d2) = (ρc, ρd) (36) therefore, the element ((t 1 , t 2 ),γ) will be the product of the three elements of t2 × autt given below: ((ρ(v1, ρv2),ι)((ρ0, ρ0), γ)((ρ-d1+d2, ρ-d1+d2),ι) (37) hence, the semi-direct product t2 × autt is sharply 2transitive on t. � corollary 1. if t ≅ zp where p is a prime number, then the semi-direct product t2 × autt is generously transitive in its action on t. proof. take the two ordered pairs of distinct elements in the proposition above to be (ρa, ρb) and (ρb, ρa).� these results explain why there is only one fusion scheme for the case when the group t is isomorphic to the cyclic group z p of prime order. such fusion schemes are the set of orbitals {c 0 , c 1 } where c 0 is the diagonal orbital and c 1 is its complement in ω × ω. it can also be observed that for all the t considered, the orbitals are all symmetric. this implies that the semidirect product t2 × autt for these groups t are generously transitive in its action on the elements of t. the following is a discussion of commutativity of incidence matrices of these orbitals. let a i and a j be arbitrary incidence matrices. recall the following equation a i ⋅ a j = p0 ij ⋅ a 0 +p1 ij ⋅ a 1 + ⋅⋅⋅ +pm ij ⋅ a m where m is the number of associate classes. the (α, β)entry of the product a i a j depends on the associate class where (α, β) belongs. it is pk ij if (α, β) ∈ c k . since c k is symmetric and pk ij is independent of the choice of (α, β) by axiom (c) of the definition, the (β, α)-entry of the product is also pk ij . this means that the product of incidence matrices a i and a j for i, j ∈{0, 1, ⋅⋅⋅ , m} is symmetric. this implies that the algebra of science diliman 20:1, 18-23 ∼ ∼ ∼ σ pkijak= k=m k=1 (38) • fission and fusion schemes and the action of the diagonal group d(t; n) 23 matrices over the set of reals ℜ generated by a 0 , a 1 , ⋅⋅⋅ , a m is a commutative algebra. the diagonal group and the fusion schemes the diagonal group d(t, n) contains tn+1 × outt as a proper subgroup for n ≥ 1. if an association scheme is preserved by d(t, n), then it is also preserved by every subgroup of d(t, n). as a consequence, the fusion schemes preserved by tn+1 × outt are candidate association schemes preserved by d(t, n). a complete list of all possible association schemes preserved by d(t, n) can be derived from the fusion schemes preserved by tn+1 × outt . this claim is shown in the following result: corollary 2. all association schemes preserved by d(t, n) have associate classes which are union of orbitals of tn+1 × outt proof. let c be an associate class in an association scheme preserved by d(t, n). suppose that c is not a union of orbitals of tn+1 × outt . then there exists an orbital oi of tn+1 × outt and o ic ⊂ oi such that o ic = c ∩ o i . consider x ∈ o ic and y ∈ o i o ic . there exists g ∈ tn+1 × outt such that xg = y and hence cg ≠ c. this is a contradiction because c is an associate class for d(t, n). � association schemes preserved by d(t, n) are coarser as compared with the association schemes preserved by tn+1 × outt . for the case when n = 1 and t ≅ zp for some prime number p, only the trivial association scheme is preserved by d(t, n). the generous transitivity of t2 × outt on ω implies that of the diagonal group. discussion the automorphism groups of finitely generated groups can further be studied to characterize the resulting orbitals in the action of t2 × outt. a diagonal group d(t, n) is isomorphic to (tn+1 × outt ) o s n+1 . the generators are as enumerated in the introductory section of diagonal groups. the gap algorithm may further be implemented for those cases when n ≥ 2. acknowledgements this research was funded by the natural science research institute, university of the philippines, diliman. references alejandro, p.p., bailey, r.a. and cameron, p.j., 2003. permutation groups and association schemes. discrete mathematics 266, 47-67. cameron, p.j. 1999. permutation groups. cambridge university press. dixon, j.d. and mortimer, b. 1996. permutation groups. springer-verlag, new york. science diliman 20:1, 18-23 04_jirkovsky 17 turbulence in planar and circular pipe science diliman (january-june 2003) 15:1, 17-21 onset of turbulence in planar and circular pipe l. jirkovsky* and l. bo-ot theory group, national institute of physics college of science, university of the philippines diliman 1101 quezon city, philippines email: jir@nip.upd.edu.ph abstract a third-order hydrodynamic equation with a molecular structure parameter, obtained through a projection and perturbation formalism from the liouville equation is applied to circular and planar poisseuille-hagen flow. it is shown that there is no principal difference in the resulting parabolic velocity profiles as long as the flows remain laminar. however, a difference is noted in the onset of turbulence in consistency with observations, showing larger stability of the parabolic velocity profile in circular pipe. introduction the origin of turbulence and laminar-turbulent transition are among the most important unresolved problems of fluid dynamics (fasel & saric, 1999). the traditional method in standard hydrodynamics is to solve navierstokes equation for stationary velocity profiles, which are parabolic for poisseuille-hagen flow in laminar regime in good agreement with experiments. however, any agreement breaks down at the onset of turbulence as velocity profiles flatten and become non-stationary (landau & lifshic, 1988; fox & germano, 1961). an explanation for the flattened velocity profiles was given by prandtl and von karman (fox & germano, 1961) using the transverse component of the fluctuation of the velocity. the faster molecules of the central region of the pipe show up in the boundary layer mix with slower molecules, and the velocity profile becomes roughly uniform except boundary layers. for the description of turbulent flows, many renormalized perturbation theories all based on reynolds equation (mccomb, 1992) were developed in the past. the reynolds equation, which aside from the mean velocity considers its arbitrary time fluctuations, like navier-stokes equation, ignores the structure of the molecules. limitations of the reynolds equation in the laminar-turbulent transition and its difficulty in explaining the origin of turbulence suggest study of the phenomenon directly from the liouville equation. from the liouville equation we derive third order hydrodynamic equation containing two control parameters related to the internal structure and geometry of the molecule (muriel & dresden, 1995). for the incompressible flows, we then obtain corresponding flow equations for planar and circular pipe. the flow equations with just one control parameter related to the internal structure of the molecule are solved numerically for the velocity profiles for each system separately to see the differences in the onset of turbulence and the change from the parabolic velocity profile into the flattened one. the control parameter is interpreted as the strength of inelastic interactions among the molecules of the fluid. the inelasticity of the collisions is easily explained if one adopts a hypothesis of quantum origin of turbulence (muriel & dresden, 1995). there are dissipative effects due to the excitations of internal degrees of freeedom of the molecules, inducing the occurence of inelastic interactions and irregular motion. the quantum kinetic model of turbulence (muriel & dresden, 1995) is based* corresponding author 18 jirkovsky and bo-ot on the well-known landau idea (landau & lifshic, 1988), which associates turbulence with deterministic chaos. hydrodynamic equations following the projection formalism of zwanzig, dresden, and muriel (zwansig, 1961; muriel & dresden, 1969) and the perturbation procedure outlined in jirkovsky & muriel (1994), the kinetic equations correct to the appropriate order k = 0,1,2 are derived from the liouville equation for n-particle distribution function: (1) with liouville operator , where and define a projector , where ω is the volume of the system. the complementary projector is 1–p. applying both projectors to the liouville equation, we reduce it to an exact equivalent of the first equation in the bbgky hierarchy for the one particle distribution function (2) we then take a very simple approach: formally expand the distribution function in orders of λ and the propagator in taylor series, and then substitute into eq. (2). we pick the terms to the appropriate order k. ( ) ( ) n nfi lf t ∂ = ∂ o il l lλ= + i rio p l i m = − ∑ ∇ v uv 1 ( ) 2 j j jr jj p pi j j l i v ≠ = ∇ ∇ − ∇∑ uv uv 21 1 ... ... nnp dr dr−= ω ∫ ∫ v v ( ) 2 (1 ) (0) ( ) ( ) n o i t i i o f il f i pl g p f t pl g t s l f s ds λ λ ∂ = − − − ∂ − −∫ ( ) 0 λ ∞ = = ∑ k k n f f (1 )−= i p ltg e zero order (k=0) zero order kinetic equation for the one-particle distribution function f(0) is the boltzmann equation, with bbgky-like elastic collision term (3) in the absence of an external field, reducible to the standard hydrodynamic equations. first order (k=1) we obtain a hydrodynamic equation with one correction term , known as the reynolds equation. corresponding fourier transformed form is used in almost all theories of turbulence. the turbulence is considered merely as a flow phenomenon and a mathematical problem, molecular structure being unimportant or unnecessary. although some theories, such as the kolmogorov theory (mccomb, 1992), correctly predict the power spectrum, and fully developed turbulence is considered as well understood, the question of laminar-turbulent transition and origin of turbulence, on the other hand, is still open. second order (k=2) the second order kinetic equation is reduced to the equation for the mean velocity of the fluid using a renormalization attributed to mccomb (mccomb, 1992) (4) where p is the mean pressure, ρ is the density, and ν is the kinematic viscosity. the control parameter is 2 ( ) ( ) ω = ∇ ω∫ v b v d , with ω as ∂ < >j uiuj t uv u 2 2 2 0 0 ( ) = 1 1 ( ) ( ) ρ ρυ ρ ⎛ ⎞∂ + ∇ + ∇ − ∇⎜ ⎟∂⎝ ⎠ − − ∇ − − ∇∫ ∫ v v v v v v v uvv vt t u u u p u t b t s ds b t s u ds m m (0) (0) (0) ⎛ ⎞∂ ∂ + ∇ = ⎜ ⎟∂ ∂⎝ ⎠ v v coll f p f f t m t 19 turbulence in planar and circular pipe the volume and v, the interaction potential. we have b=0 for elastic collisions. this suggests the interpretation of b > 0 as a measure of the strength of inelastic interactions. the parameter b manifests its presence in the turbulent regime and an application such as the planar poisseuille-hagen flow (jirkovsky & bo-ot, 1999) is used to illustrate the effect of b. numerical simulation has shown time development of parabolic velocity profiles into nearly uniform velocity profiles indicative of the onset of instability. an adhoc assumption was utilized, specifically, the control parameter was increased linearly with the mean velocity. also, there was a short time limitation in the validity of the second order equations. for large times, the velocities changed signs. third order (k=3) to obtain the third order kinetic equation, we pick terms containing λ3 in eq. (2) using the appropriate expansions for the one-particle distribution function and propagator g. the third order kinetic equation can be written as: (5) using explicit forms of the liouville operators, multiplying eq. (5) by one component of momentum and integrating over velocity space, it may be reduced to momentum transport equation of the third order: (6) (3) (3) 3 2 2 0 2 ( 0) 3 (1) 2 2 (1) 1 { ( ) [( 6 ) ( ) ( )] 1 [ ( ) ( ) ] ( )} 2 ∂ = − − − ∂ + + + − − − − ∫ t o i o i o i o i i o i o i o i o i f il f pl i t s l l t l l l l l l l f s l l f s i t s l l t s l l f s ds 2 2 2 0 3 2 3 2 2 0 ( ) 1 [( ) ( ) ] 4 ( ) [5 ] ρ υρ ρ ρ ⎡ ⎤∂ + ∇ + ∇ − ∇ =⎢ ⎥∂⎣ ⎦ − − ∇ + − ∇ − − ∇ + ∇ ∫ ∫ uv uv uv uvv v v uvv v v v vv t t u u u p u t b t s t s u ds m t s c b u ds m where the vector parameter 3 ( ) ( ) ω = ∇ ω∫ vv c v d is related to the geometry of the molecule and is interpreted as a measure of the assymetry of the molecule. we used the renormalization of the density, pressure, and velocity to their true values attributed to mccomb (mccomb, 1992). eq. (6) is the hydrodynamic equation with correction terms in integral form correct to the third order. it contains the second order equation and new terms with vector parameter v c and scalar parameter b. it is interesting to note that the geometry of the molecule plays no role in the flow of an incompressible fluid, although there is another term with control parameter b. planar and circular poisseuille-hagen flow if one uses the incompressibility condition, eq. (6) may be reduced to non-linear equation for the poisseuillehagen flow, with configuration where ( ( , ), 0, 0)= uv u u z t for the fluid enclosed between two infinite parallel planar boundaries at fixed distance l: (7) utilizing a similar procedure for the flow in infinite circular pipe of radius r, with configuration where (0, 0, ( , ))= uv u u r t in cylindrical polar cordinates we have: (8) 5 6 3 5 4 2 2 2 3 2 2 3 2 4!2 3 0 ρ υ υ ∂ ∂ ∂ − − ∂ ∂ ∂ ∂ ⎛ ⎞∂ ∂ ∂ − + =⎜ ⎟∂ ∂ ∂⎝ ⎠ u u b u t t z m t z b u u u u m z z z 5 6 5 2 2 5 2 4 4 3 2 2 2 22 2 2 2 3 2 2 3 2 2 3 2 4! 0 ρ υ υ ∂ ∂ ∂ − − ∂ ∂ ∂ ∂ ∂ ⎛ ⎞∂ ∂ − +⎜ ⎟∂ ∂ ∂ ∂⎝ ⎠ ⎡ ⎤∂ ∂ ∂⎛ ⎞−⎢ ⎥⎜ ⎟∂ ∂ ∂⎝ ⎠⎢ ⎥+ = ⎢ ⎥∂ ∂ ∂ + − −⎢ ⎥ ∂ ∂ ∂⎣ ⎦ u u u r r r t r t r t b u u r r m r t r t u u u r r b r r r m u u u r u u ru r r r we use non-slip boundary conditions and static fluid as initial conditions to get numeric solutions of standard navier-stokes equations, which are subsequently used as initial guess in simulation of third order hydrodynamic equations for the flow in planar and circular pipe (figs. 3 & 4). fixed parameters for the numeric simulation are as l = 1, r = 0.5, ρ = 1, ν = 0.1, and m = 1. since constant pressure gradients drive the motion of the flows, they are adjusted in standard navier-stokes equations to give equal maximum velocity and reynolds number of the flow in planar and circular pipe. the reynolds number is max υ =n u l r with characteristic length l = 0.5l = r. fig. 2. bottom to top, circular pipe flow: (a) b = 0.00000005, t = 1.10; (b) b = 0.0000001, t = 1.10. -0.4 -0.2 0 0.2 0.4 120 100 80 60 40 20 0 (a) -0.4 -0.2 0 0.2 0.4 (b) 120 100 80 60 40 20 0 140 0 0.2 0.4 0.6 0.8 1 120 100 80 60 40 20 0 fig.3. bottom to top, circular pipe flow: b = 0, t = 1.10. fig.1. bottom to top, planar flow: (a) b = 0.00000005, t = 1.10; (b) b = 0.0000001, t = 1.10. 200 150 100 50 0 0.1 0.2 0.3 0.4 0.5 0.6 0.90.7 0.8 (a) 300 250 200 150 100 50 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 (b) jirkovsky and bo-ot 20 the numeric results (figs. 1a & 1b) for planar poisseuille-hagen flow show the onset of instability and a flattened velocity profile for control parameter b = 0.00000005. on the other hand, velocity profiles in circular pipe with the same control parameter (figs. 2a & 2b) are still parabolic and the flow is laminar, though quasi-stationary. higher control parameter is needed to induce laminar-turbulent transition. this is consistent with the observation (landau & lifshic, 1988) of critical reynolds numbers for the circular pipe, rc = 1800, and for the planar flow, rc = 1000. results for the third order theory represent improvement over second order in the time validity and shape of the profiles. also, velocities do not change signs for large times. references fasel, h.f. & w.s. saric (eds.), 1999. laminar-turbulent transition. iutam symposium. sedona, arizona. fox, g. & f. germano, 1961. fluid mechanics. princeton, van nostrand co., inc. jirkovsky, l. & a. muriel, 1994. derivation of the reynolds equation from a microscopic picture using projection techniques. phys. lett. a: 191. jirkovsky, l. & l. bo-ot, 1999. proc. 14th international symposium. prague. plasma chem. 1. 120 100 80 60 40 20 0 -0.4 -0.2 0 0.2 0.4 fig. 4. bottom to top, planar flow: b = 0, t = 1.10. turbulence in planar and circular pipe 21 landau, l.d. & a.e. lifschitz, 1988. theoretical physics, vol. 6: hydrodynamics. nauka, moscow. mccomb, w.d., 1992. the physics of fluid turbulence. oxford, clarendon press. muriel, a. & m. dresden, 1969. projection techniques in nonequilibrium statistical mechanics. physica. 43. muriel, a. & m. dresden, 1995. a microscopic theory of turbulence. physica. 81. zwanzig, r., 1961. lectures in theoretical physics. new york, wiley/interscience. 3. 7david.pmd j.p.t. domingo and c.p.c. david 61 science diliman (july-december 2014) 26:2, 61-71 geochemical characterization of copper tail ings after legume revegetation justine perry t. domingo* carlos primo c. david university of the philippines diliman issn 0115-7809 print / issn 2012-0818 online abstract k n o w l e d g e o n t h e g e o c h e m i s t r y o f m i n e t a i l i n g s i s i m p o r t a n t i n understanding the challenges in establishing vegetation cover on tailings d u m p s a n d m i n e d o u t a r e a s . i n t h i s s t u d y, t h e m i n e r a l o g y a n d t r a c e e l e m e n t c o m p o s i t i o n o f co p p e r t a i l i n g s w e r e e x a m i n e d . tw o l e g u m e species, calopogonium mucunoides and centrosema molle, were utilized to i n v e s t i g a t e t h e p o s s i b l e e f f e c t s o f t h e s e p l a n t s i n t h e g e o c h e m i c a l d e v e l o p m e n t o f m i n e t a i l i n g s i n t o s o i l l i k e m a t e r i a l . t h e i n i t i a l mineralogical and chemical analysis of the tailings samples indicated poor conditions for plant growth—minimal levels of major nutrients and organic matter as well as elevated copper concentrations. despite these conditions, the two legume species exhibited good growth rates. both legumes have likewise signif icantly reduced heavy metal concentrations in the tailings, indicating the possibility of metal hyperaccumulation in the plant tissue. the mineral composition has been retained even after revegetation; nevertheless, breakdown of primary minerals and subsequent formation of clay minerals were detected. these results provide insights on the transformation of toxic materials into habitable substrates for sustained plant growth. keywords: tailings characterization, mineralogy, heavy metals, revegetation introduction mine tailings have been identif ied as both a hazardous waste and an economically potential resource—a metal-rich material that can generate acid mine drainage and leachates but could also be an easily extractable low grade ore (garcia-meza and others 2004). as by-products of ore separation, tailings usually consist of f inesand to silt-size particles of quartz, aluminosilicates, carbonates, oxides, and sulf ides (quispe and others 2013). this waste material naturally undergoes _______________ *corresponding author geochemical charact erization of copper tailings 62 weathering due to exposure to air, water, ambient temperatures, and vegetation. subsequently, the minerals present in mine tailings will determine the physical and geochemical stability of the material under different weathering conditions, and consequently, the nature and concentration of toxic, acid generating/neutralizing chemical species that may be released (raudsepp 2011). for example, sulf idic minerals in tailings can weather within months or years and could lead to the formation of acid mine drainage (robbed and robison 1995). tailings also frequently contain high concentrations of heavy metals, which contribute to plant growth def iciency in tailings dumps and mined out areas (asensio and others 2013). revegetating these tailings dumps is usually considered to stabilize the substrate (martinez-ruiz and fernandez-santos 2005) therefore reducing the risk of polluting the surrounding environment. it can also improve soil quality with the increase in levels of organic matter and nutrients as well as in biological activity (arienzo and others 2004). consequently, selecting the appropriate plant species is another signif icant consideration to achieve successful revegetation. indigenous plants are usually selected because these plants are often better able to grow and proliferate under the environmental extremes of the area, compared with other plants originating in other environments (brown and amacher 1997). consideration should also be given to plants that have the ability to improve soil characteristics, such as leguminous species that can enhance organic matter and nutrient concentration (parotta 1992, domingo and david 2014). in addition, leguminous species exhibit rapid growth and high adaptation capacities in degraded lands (maiti and maiti 2014), making them viable candidates in rehabilitation programs. the purpose of this paper is to present data on the mineralogy and trace element composition of copper tailings before and after a short-term revegetation using legumes. in addition, this study will also examine the potential effect of the selected plants in the geochemical tailings development. the results of this study would promote a better understanding on the challenges associated with revegetating mine tailings and the contribution of plants in its development. methodology site description and sampl ing the sampling site is located in philex padcal mine (figure 1). it is situated 15 km southeast of baguio city, benguet province, with an elevation of approximately 1500 m above sea level. the area experiences an average annual rainfall of 4500 mm, with maximum rainfall occurring on the months of may to october. j.p.t. domingo and c.p.c. david 63 the primary mineral deposit in the area, called sto. tomas ii deposit, is classif ied as a porphyry copper type. the mine started its underground block cave operations in 1958, and has since produced copper concentrates comprising copper, gold, and silver. it has three tailings storage facilities (tsfs), two of which have been decommissioned. samples were collected at three random points on the upper 30 cm layer of tsf no. 3, and were homogenized prior to the pot experiment. soil samples were also collected from a hill adjacent to the tsf. plant species leguminous species have been previously identif ied to naturally occur on the decommissioned tailings ponds of philex padcal mine, particularly calopogonium mucunoides desv. (rillorta and lagunzad 2004). seeds of this species, along with another legume, centrosema molle mart. ex benth were obtained from the bureau of animal industry, department of agriculture, philippines. the seeds were germinated on garden soil for 10 d prior to the pot experiment. figure 1. location map of philex padcal mine, benguet province, philippines.   baguio city philex padcal mine metro manila geochemical charact erization of copper tailings 64 experimental design the control group has two setups—the f irst consisted of 1 kg of pure mine tailings only to serve as baseline for the tailings parameters, while the other setup has 1 kg of soil and 10 legume seedlings. meanwhile, the experimental setup is comprised of 1 kg of tailings and 10 legume seedlings. different setups were prepared for each legume species and for different collection periods, all in triplicates. the pots were watered daily with approximately 200 ml of tap water and placed in a fenced 5 m x 5 m outdoor area in a randomized block design. plant growth was monitored throughout the experiment. after four months, the plants were carefully removed before collecting the tailings material on each pot and dish. the samples were subjected to various analyses to examine the changes in the soil properties. geochemical characterization mineralogy entire tailings materials were removed from the pots after the experiment. samples were dried at room temperature to avoid possible phase transformation when heated inside an oven (essene and peacor 1995). approximately 50 g of each sample was pulverized into f ine powder with texture < 10 μm, using quartz mortar and pestle, to optimize the diffraction pattern from crystal lattice in the sample. minerals present in the samples were scanned using an x-ray diffractometer (shimadzu maxima xrd-7000) from the national institute of geological sciences, university of the philippines. the samples were further subjected to glycolation process to determine the type of clay present in the sample (i.e. , non-expanding or expanding). peak identif ication was performed by manual search using materials data, inc. (mdi) mineral and auto-search method with pdf-2, both of which are supplied by the international centre for diffraction data. trace metal s each sample was f iltered through a 63 μm sieve to obtain the clay fraction, and was oven dried at 60°c overnight. digestion of samples was carried out in teflon digestion tubes using concentrated hf and hno 3 , in a 2:1 volume ratio, with 1.5 ml mixed acid for every 0.01 g of the sample. river sediment (gbw08301) certif ied reference material was used, having the following certif ied values: as 56 μg g-1, ba 375 μg g-1, cd 2.45 μg g-1, co 16.5 μg g-1, cr 90 μg g-1, cu 53 μg g-1, mn 975 μg g-1, hg 0.22 μg g-1, pb 79 μg g-1, se 0.39 μg g-1, fe 3.94%, and indicative values for be, ni, v, zn (measurement standards laboratory of new zealand 2009). the vessels j.p.t. domingo and c.p.c. david 65 ·  were placed on a hot plate at 80°c overnight. addition of hno 3 was repeatedly done upon drying of the samples to ensure complete digestion. digests were transferred into 15 ml centrifuge tubes before being f iltered through a 0.45 ìm sieve onto 50 ml centrifuge tubes. samples were diluted to 50 ml using ultrapure water, from which 5 ml aliquots were collected and used to scan for the following heavy metals: chromium, nickel, copper, zinc, arsenic, cadmium, and lead, using the inductively coupled plasma–mass spectrophotometer (agilent 7500cx) from the national institute of geological sciences, university of the philippines, quezon city, philippines. results and discussion tail ings characterization analysis of the general characteristics of the tailings samples indicate neutral to slightly basic ph and poor soil nutrients (table 1). total nitrogen in the sample was minimal at 0.01%. phosphorus was not detected initially in the samples, i.e. , less than 0.1 mg kg-1, whereas the potassium content of the sample was 78.0 mg kg-1. the organic matter content of the tailings material was likewise very low at 0.2%. meanwhile, the initial concentration of the heavy metals examined in the tailings were below the effects range median sediment quality guidelines set by the national oceanic and atmospheric administration and the australian and new zealand environment and conservation council, with the exception of copper (domingo and david 2014). these heavy metals are considered to be the most common trace elements of mine tailings (allan 1995). with these conditions, the tailings present unsuitable conditions for plant development, lacking the essential nutrients while containing heavy metal concentrations that could hinder biological processes in the substrate (liu and others 2012). plant growth one of the primary considerations in rehabilitating degraded areas such as tailings dumps and mined out lands is the proper selection of plant species. indigenous plants, or plants that naturally grow in the area, are recommended because they are already adapted to the environmental conditions and because they minimize the risk of introduced species being invasive to the native ones. in the experiment, a discrepancy in the plant heights was observed between the soil and tailings setups for c. molle (figure 2). on the other hand, no signif icant geochemical charact erization of copper tailings 66 difference exists among the c. mucunoides setups. this could be explained by the fact that c. mucunoides is naturally occurring in the tailings ponds (rillorta and lagunzad 2004). hence, the plant has already adapted to the poor tailings status and will likely exhibit better growth compared with an introduced species such as c. molle. this also emphasizes the advantage of using native or indigenous plants in rehabilitation schemes. table 1. general physico-chemical characteristics of pure mine tail ings used the experiment tailings control (t = 0 months) ph 7.63±0.13 total nitrogen % 0.01±0.00 phosphorus (mg kg-1) not detected potassium (mg kg-1) 78.0±0.0 organic matter % 0.19±0.03 chromium (ppm) 166.1±17.5 nickel (ppm) 23.0±2.4 copper (ppm) 3355±608 zinc (ppm) 269±27 arsenic (ppm) 2.87±0.40 cadmium (ppm) 0.69±0.04 lead (ppm) 15.5±0.6 values indicate mean ± se; n = 3. figure 2. plant heights in the soil and tailings setups; values indicate mean ± se. j.p.t. domingo and c.p.c. david 67 trace metals one-way analysis of variance (anova) was utilized to determine whether the heavy metals had been substantially reduced in legume-planted tailings after the experiment . results of the anova indicate signif icant differences in the concentrations of copper, arsenic, and cadmium (p < 0.05) (domingo and david, 2014). in particular, the c. molle setup had undergone 14% as, 15% cd, and 10% cu reduction; while an even greater decrease in the heavy metals was observed in the c. mucunoides setup, having 52% as, 38% cd, and 78% cu removed from the tailings (figure 3). the results suggest that the selected plants could hyperaccumulate the metals in their tissues, particularly c. mucunoides . the reduction of these heavy metals will promote the proliferation of nitrogen-f ixing bacteria, nitrogen mineralization, and decomposition of organic matter (liu and others 2012), and thus could help in the soil development of mine tailings. mineralogy the results of the x-ray diffraction (xrd) analysis for the tailings indicate that the material is composed of quartz, plagioclase-feldspars (albite, anorthite), amphiboles (actinolite), muscovite, and clay minerals (figure 4a). after the experiment, the legume-planted tailings exhibited similar mineralogical composition, with anorthoclase and tremolite detected in addition to the previous minerals (figures 4b and 4c). the detected minerals are consistent with the dioritic/andesitic parent rock in the philex area, and belong to the most abundant gangue silicate and oxide minerals in porphyry copper deposits, as well as biotite, magnetite, anhydrite, and epidote (berger and others 2008). furthermore, these minerals are also included in the most common alteration minerals in silicate-rich rocks, which may indicate that a percentage of these minerals are products of weathering of the original material. other minerals associated with the detected minerals, e.g. , sulf ides and magnetite, are most likely present in the tailings but in amounts below the detection limit of the xrd apparatus. moreover, the peaks of the more crystalline minerals (i.e. , quartz, plagioclase) in the sample could have masked the peaks of the other minerals. still, evidence of smectite clay minerals was observed in the samples that have been planted with legumes, indicating hastened breakdown of primary minerals due to the plants. a longer experimental period is recommended to observe signif icant effects in the mineralogy of mine tailings. experimental work on the clay type and accurate quantif ication of the clay content would further help in understanding the contribution of these plants in the improvement of tailings mineralogy. geochemical charact erization of copper tailings 68 · ·  figure 3. reduction of as, cd, and cu concentrations in mine tailings before and after the experiment. values indicate mean ± se; n = 3; in parts per million. j.p.t. domingo and c.p.c. david 69 figure 4. mineralogical composition of (a) copper-gold tailings; (b) c. molle-planted tailings; (c) c. mucunoides-planted tailings. geochemical charact erization of copper tailings 70 conclusions and recommendations the mineralogical and chemical analysis of the mine tailings showed minimal levels of major plant nutrients and organic matter, and high copper concentrations. between the two plants used in the study, the legume species c. mucunoides has exhibited better growth in tailings than c. molle. the presence of these legumes signif icantly reduced heavy metal concentrations in the tailings, indicating the possibility of metal hyperaccumulation. lastly, evidence of clay mineral formation indicates the hastened breakdown of the material into more soil-like substrates that are dominated by clays and organic matter. acknowledgments the authors would like to thank the off ice of the chancellor of the university of the philippines diliman, through the off ice of the vice-chancellor for research and development, for funding support through the outright research grants; philex mining corporation for providing tailings material; and to the xrd and icp-ms laboratories in up nigs for the analysis. contributions from pamela tolentino, joan de vera, carmela tupaz, cherisse ferrer, russel ong, and dr. carlo arcilla have been very helpful in the conduct of the study and are greatly appreciated. references arienzo m. , adamo p. , cozzolino v. 2004. the potential of lolium perenne for revegetation of contaminated soil from a metallurgical site. science of the total environment 319: 13-25. asensio v. , covelo e.f. , kandeler e. 2013. soil management of copper mine tailing soils–sludge amendment and tree vegetation could improve biological soil quality. science of the total environment 456-457: 82-90. berger b.r. , ayuso r.a . , wynn j.c. , seal r.r. 2008. preliminary model of porphyry copper deposits: u.s. geological survey open-file repor t 2008-1321: 8-9. brown r.w. , amacher m.c. 1997. selecting plant species for ecological restoration: a p e r s p e c t i v e f o r l a n d m a n a g e r s . re v e g e t a t i o n w i t h n a t i v e s p e c i e s , p p . 1 1 6 i n : proceedings, 1997 society for ecological restoration annual meeting. u.s.d.a . forest service rocky mountain research station. proceedings rmrs-p8. domingo j.p.t. , david c.p.c. 2014. soil amelioration pot ential of le gumes for mine tailings. philippine journal of science 143 (1): 1-8. essene e.j. , peacor d.r. 1995. clay mineral thermometry: a critical perspective. clays and clay minerals 43: 540-553. j.p.t. domingo and c.p.c. david 71 garcia-meza j.v. , ramos e. , carrillo-chavez a. , duran-de-bazua c. 2004. mineralogical and chemical characterization of historical mine tailings from the valenciana mine, g u a n a j u a t o , m e x i c o : e n v i r o n m e n t a l i m p l i c a t i o n s . b u l l e t i n o f e n v i r o n m e n t a l contamination and toxicology. 72: 170-177. maiti d. , maiti s.k. 2014. ecorestoration of waste dump by the establishment of grasslegume cover. international journal of scientif ic and technology research 3 (3): 37-41. parrotta j.a. 1992. the role of plantation forests in rehabilitating degraded tropical ecosystems. agriculture, ecosystems and environment 41: 115–133. price w.a . 2009. prediction manual f or drainage chemistry from sulphidic geologic materials. mend repor t 1.20.1. quispe d. , perez-lopez r. , acero p. , ayora c. , nieto j.m. 2013. the role of mineralogy on element mobility in two sulf ide mine tailings from the iberian pyrite belt (sw spain). chemical geology 345: 119-129. raudsepp m. 2011. rietveld quantitative phase analysis of mineralogical materials. pa p e r p r e s e n t e d a t t h e d e n v e r xra y co n f e r e n ce : 6 0 t h a n n u a l c o n f e r e n ce o n applications of x-ray analysis, crowne plaza hotel, colorado springs, colorado, u.s.a . robbed g.a . , robison j.d.f. 1995. acid drainage from mines. geographical journal 161: 47-54. _____________ justine perry t. domingo <jptdomingo@yahoo.com> is a research associate in the environment monitoring laboratory in up diliman national intitute of geological sciences. with a background in biology, mr. domingo is aiming to develop an effective way for revegetation of mine tailings and mined out areas based on an alternative plant colonization strategy. he has recently completed his master’s degree in geology and is currently handling different geo-environmental studies. carlos primo c. david, phd <cp.david@yahoo.com>, <cpdavid@nigs.upd.edu.ph> is a licensed geologist and professor of geology and environmental science in up diliman. a project leader of the department of science and technology’s project noah, dr. david pioneers short term rainfall forecasting in the country and climate change-related research on water resources. he concurrently serves as dost’s spokesperson for climate change and a resident resource person for disaster preparedness for gma news and public affairs. dr. david is a member of the panel of experts (poe) of the climate change commission and was the 2013 awardee of the oscar m. lopez professorial chair for climate change research. inside front cover-19-2 editorial board editor-in-chief maricor n. soriano, ph.d. associate editors zubaida u. basiao, ph.d. biology joel joseph s. marciano, jr., ph.d. computer science, engineering nemesio e. montano, ph.d. biochemistry rosana b. tarriela, ph.d. earth sciences fernando p. siringan, ph.d. marine sciences cristine d. villagonzalo, dr. rer. nat. physics corazon d. villareal, ph.d. managing editor dercylis g. mararac editorial assistant hildie m. nacorda copy editor science diliman (issn 0115-7809) is published bi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university of the philippines diliman. address all communications to the editor in chief, science diliman, research dissemination and utilization office, office of the vice chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p 300.00/year (two issues), inclusive of postage us $ 25.00/year (two issues), inclusive of postage tel. no: (632) 981-85-00 loc. 4047 (632) 927-2567; 927-2309; 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences cover photo aerial shot taken in 2001 of fish cages in guiguiwanen channel, bolinao, pangasinan several months before the february 2002 massive fish kill in the area. photo courtesy of dr. gil jacinto of the marine science institute, university of the philippines, diliman. editorial advisors kelvin s. rodolfo, ph.d. dept. of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu alfonso m. albano, ph.d. dept. of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu victor c. gavino, ph.d. dept. of nutrition university of montreal, canada victor.gavino@umontreal.ca rudolf a. roemer, ph.d. centre for scientific computing and dept. of physics university of warwick r.roemer@warwick.ac.uk raul k. suarez, ph.d. dept. of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu sd-sample article b. edullantes and r. galapate 25 science diliman (january-june 2014) 26:1 25-40 embryotoxicity of copper and zinc in tropical sea urchin tripneustes gratilla brisneve edullantes* ritchel ita p. galapate university of the philippines cebu _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract the study determined the individual toxicity of copper (cu) and zinc (zn) i n s e a u r c h i n t r i p n e u s t e s g r a t i l l a . b i o a s s a y u s i n g i n h i b i t i o n s o n fertilization, early cleavage, mid cleavage, late cleavage and blastulation as endpoints involved exposure of viable gametes to cu and zn for 0.5, 3 , 6 , 9 a n d 1 2 h , r e s p ec t i ve l y. i n h i b i t i o n s i n c r e a s ed s i g n i f i c a n t l y w i t h concentration of cu and zn. probit analysis estimated ec 50 values for cu and zn, respectively, at 32 and 67 µg·l-1 on fer tilization; 31 and 93 µg·l-1 on early cleavage; 43 and 61 µg·l -1 on mid cleavage; 42 and 42 µg·l -1 o n late cleavage; and 20 and 44 µg·l-1 on blastulation. results showed that t ox i c i t y o f cu i s s i g n i f i c a n t l y h i g h e r ( p < 0 . 0 5 ) t h a n t h a t o f zn i n a l l d e v e l o p m e n t a l s t a g e s , e x c e p t i n l a t e c l e a v a g e . a l s o , t h e i n h i b i t i o n s e l i c i t e d b y c u s h o w e d s e n s i t i v i t y t o l i f e s t a g e s . t h i s s t u d y p r o v i d e d evidence on heavy metal species-sensitive, concentration-dependent and stage-specif ic inhibitions on embryonic development in t. gratilla to cu and zn. keywords: embryotoxicity, sea urchin development , individual toxicity, h e a v y m e t a l s introduction waste disposal from mines and industries discharges complex mixtures of pollutants to coastal areas. these anthropogenic activities expose aquatic wildlife to various heavy metals such as copper and zinc (us epa 2007), which at elevated levels often subject aquatic organisms to heavy metal poisoning (eisler 1998). sea urchins dwell in marine environment, and they respond readily to heavy metal pollution, making them an ideal bioindicator of ecosystem health (kobayashi and okamura 2 0 0 4 ) . embryotoxicity of copper and zinc in tropical sea urchin 26 known to act as teratogen, heavy metals cause developmental delay, malformations and mortalities among exposed aquatic organisms (eisler 1998). studies have found that different heavy metals cause developmental anomalies among sea urchins (kobayashi and okamura 2004), and elevated concentrations of cu and zn inhibit the development of echinoid species (phillips and others 2003, kobayashi and okamura 2004, kobayashi and okamura 2005). although there have been several studies on the effects of toxic heavy metals on marine organisms (see for example king and riddle 2001, phillips and others 2003, kobayashi and okamura 2005), further research is needed to have a better understanding of the embryotoxic effects of heavy metals. moreover, endpoints of sea urchin bioassay are limited to spermiotoxicity, inhibitions of fertilization, and malformations. few studies have been undertaken to investigate the inhibitory effect of cu and zn on early stages such as cleavage and blastulation, which are critical stages in sea urchin development. to help address these research gaps, the present study was undertaken. this study may be regarded as an initial attempt to evaluate the inhibitory effects of cu and zn on the early life stages of the tropical sea urchin, tripneustes gratilla, in the philippines. using bioassay testing, this study aimed to: (a) determine the percentage of inhibitions on fertilization, early cleavage, mid cleavage, late cleavage, and blastulation; and (b) compare the inhibitions across heavy metal species, concentration, and developmental stages. materials and methods preparation of test solutions five nominal concentrations of cu and zn (0, 25, 50, 100 and 150 µg·l-1 each) were used to examine the toxicity of heavy metal on sea urchin. the test solutions were prepared by adding copper sulfate and zinc sulfate into f iltered natural seawater. the temperature (28.10 ± 1.84°c), salinity (30.67 ± 0.58 µg·l-1), and ph (6.99 ± 0.6) of the test solutions were maintained. collection of sea urchin gametes forty-two adult sea urchins t. gratilla, 6.60 ± 0.47 cm in diameter, were collected from marigodon, lapu-lapu city. each organism was isolated in a plastic container f illed with seawater to ensure that none of the sea urchins would induce others to spawn. they were transported to the laboratory immediately after sampling. b. edullantes and r. galapate 27 procedures for gamete collection were adapted from the us epa (1995) protocols, with a few modif ications. each sea urchin was inverted over a 100 ml beaker fully f illed with f iltered natural seawater. the gonadal openings on the aboral side were immersed in the seawater. about 1 ml of 0.5 m kcl was injected through the tough leathery peristomial membrane into the perivisceral cavity of each sea urchin. this resulted in the contraction of the smooth muscles of the gonad and induced spawning of the specimen. injections were repeated after 2-5 minutes to induce heavier spawning. the sex of the sea urchin was determined. t. gratilla males ejected cream-colored semen while females released yellow eggs. a drop of the gametes from each sea urchin was examined under the microscope to conf irm its sex. each spawning sea urchin male was transferred into a petri dish in oral side up position and was allowed to shed into the dish. a drop of the dry sperm (semen) was examined under the microscope to observe the motility of the sperm. the sea urchin males with high sperm motility were used in the test to ensure sperm viability. the viable sperm cells were pooled into a 100 ml beaker, which was covered with paraf ilm to prevent exposure of semen to air that may reduce the viability of the sperm by altering the surrounding ph. sperm stock was stored at 5°c. female sea urchins were left to shed eggs into the 100-ml beakers f illed with f iltered natural seawater. a small sample of the eggs from each female was examined under the microscope to determine the presence of mature eggs. mature eggs were characterized as having a) small nucleus found near the periphery of the cell membrane and b) large amount of cytoplasm. mature eggs were pooled into a 1 l beaker. the eggs were suspended in 600 ml f iltered seawater, and allowed to settle for 15 minutes. about 500 ml of the overlying water was siphoned off and the volume was brought back to 600 ml with f iltered natural seawater. the eggs were resuspended and allowed to settle for 15 minutes. after siphoning off the overlying 500 ml, the eggs were f inally resuspended in 600 ml f iltered natural seawater. egg suspension was stored at 12°c. the gametes were used in the toxicity assay after 2 h following the collection. gametes were exposed to different treatments of cu and zn (25, 50, 100 and 150 µg·l^-1 each). same batch of gametes were exposed to 0 µg·l^-1 cu and µg·l^-1 zn, which serve as control. embryotoxicity of copper and zinc in tropical sea urchin 28 toxicity assay the exposure experiments were adapted from the protocol used by kobayashi and okamura (2004, 2005), with a few modif ications. inhibitions on fertilization, early cleavage, mid cleavage, late cleavage and blastulation were the endpoints. exposure experiment for every endpoint was conducted separately in a plastic container with 10 ml of test solution. one drop of dry sperm stock and 1 ml of the egg suspension were added into the container. the gametes that were exposed to different treatments were of the same batch. incubation temperature (28 ± 2 °c), salinity (30 ± 1 µg·l-1), and ph (7 ± 0.5) were maintained throughout the exposure experiment. fertilization, early cleavage, mid cleavage, late cleavage, and blastulation were arrested by adding 1 ml of 10% formaldehyde after 0.5, 3, 6, 9 and 12 h exposure to test solutions, respectively. exposure experiments were triplicated. a drop of the treatment solution was mounted on a slide. about four mounts were prepared for each treatment. each mount was observed under the compound microscope in a single f ield of vision at 100x magnif ication. one hundred eggs and/or embryos were randomly selected and their development stage, as described in table 1, was identif ied. inhibitions on fertilization, early cleavage, mid cleavage, late cleavage, and blastulation were determined. table 1. distinguishing features of early developmental stages in sea urchin stages description unfertilized egg mature eggs without fertilization cone or envelope fertilized egg mature eggs with fertilization cone or envelope early cleavage 2and 4-cell stage embryos mid cleavage 8and 16-cell stage embryos late cleavage 32and 64-cell stage embryos blastulation embryos with a sphere of cells surrounding a cavity data analyses the toxicity responses were reported as percent inhibitions on fertilization (if), early cleavage (iec), mid cleavage (imc), late cleavage (ilc), and blastulation (ib) using the following formulas: = 100 eq. 1 b. edullantes and r. galapate 29 = + + + 100 = + + + + 100 eq. 4 eq. 5 where u is the number of unfertilized eggs, f is the number of fertilized eggs, ec is the number 2and 4-cell stage embryos, mc is the number 8and 16-cell stage embryos, lc is the number of 32and 64-cell stage embryos, and n is the total number of eggs and/or embryos evaluated. the toxicity responses were f itted against the concentration through a probit model to estimate the concentration at which 50% inhibition is observed (ec 50 ). kruskal wallis anova was used to compare the toxicity responses among treatments. all statistical analyses were done using the ibm sppss statistics version 20 software, at 95% conf idence interval. results the effects of varying concentrations of cu and zn on the different developmental stages of t. gratilla are shown in figures 1 to 5. both cu and zn elicited logarithmic concentration-dependent inhibitions on t. gratilla fertilization, early cleavage, mid cleavage, late cleavage and blastulation (figures 1 to 5, respectively). comparison between ec 50 of cu and zn across different embryonic stages is shown in figure 6. inhibitions on fer til ization (if) if increased with increasing cu and zn concentration at 0.5 h exposure (figure 1). all treatments of cu and zn elicited a signif icantly higher if than in control (17 ± 7%). if at 25 µg·l-1 cu increased threefold from the control (62 ± 3%). increasing the cu concentration to 50 and 100 µg·l-1 elicited 70 ± 6% and 75 ± 3% if, respectively, which were four times higher than in control. at 150 µg·l-1, a f ivefold increase in if was observed (87 ± 3%). in zinc treatment, if doubled at 25 µg·l-1 zn (39 ± 3%). it increased threefold at 50 and 100 µg·l-1 zn, eliciting 56 ± 3% and 61 ± 2% if, respectively. it was signif icantly high at 150 µg·l-1 zn (73 ± 5%), which was four = + 100 = + + 100 eq. 2 eq. 3 embryotoxicity of copper and zinc in tropical sea urchin 30 times higher than in control. the ec 50 of cu (32 ± 11 µg·l-1) was signif icantly lower than zn (67 ± 3 µg·l-1) (figure 6), suggesting that cu is twice as toxic as zn in eliciting inhibitions on fertilization. inhibitions on early cleavage (iec) concentration-dependent inhibitions on early cleavage of t. gratilla were also observed at 3 h exposure period to increasing cu and zn concentration (figure 2). iec in all cu treatments were signif icantly higher than in control (15 ± 5%). at 25 µg·l-1 cu, iec increased threefold to 50 ± 6%. increasing the concentration to 50 µg·l-1 cu elicited 68 ± 7% iec, which was four times higher than in control. iec increased six times from the control at more elevated concentration (>90%). iec at 25 and 50 µg·l-1 zn showed no signif icant difference from the control. at higher zn concentrations, iec increased more than threefold, with values signif icantly higher than those in control. ec 5 0 of cu and zn on iec were 31 ± 3 µg·l 1 and 93 ± 43 µg·l -1, respectively (figure 6). there was a signif icant difference between the ec 50 of cu and zn in eliciting inhibitions on early cleavage, indicating that cu is three times more toxic than zn. figure 1. inhibitions on fertilization to varying concentrations of cu (black circles) and zn (white circles). b. edullantes and r. galapate 31 figure 2. inhibitions on early cleavage to varying concentrations of cu (black circles) and zn (white circles). figure 3. inhibitions on mid cleavage to varying concentrations of cu (black circles) and zn (white circles). embryotoxicity of copper and zinc in tropical sea urchin 32 inhibitions on mid cleavage (imc) the inhibitions on mid cleavage were evaluated at 6 h exposure to varying concentrations of cu and zn (figure 3). similar to the previous observations, imc increased with increasing concentration of cu and zn. imc in both treatments did not vary signif icantly from the control (23 ± 13%) at 25 µg·l-1, but showed a signif icant difference from the control at elevated concentrations. in cu treatment, imc doubled at 50 µg·l-1 (59 ± 7%). increasing the cu concentration to 100 µg·l-1 elicited 75± 5%, which was threefold higher than in control. imc increased four times at 150 µg·l-1 (93 ± 9%). there was a twofold increase in imc at 50 and 100 µg·l-1 zn (51 ± 3% and 63 ± 5%, respectively). at 150 µg·l-1 zn, imc had increased to 81 ± 6%, which was three times higher than in control. the ec 50 of cu (43 ± 11 µg·l-1) was signif icantly lower than zn (61 ± 4 µg·l-1) (figure 6), which suggests that cu is more toxic than zn in inhibiting mid cleavage. inhibitions on late cleavage (ilc) inhibitions on t. gratilla late cleavage were evaluated at 9 h exposure to cu and zn (figure 4). trends similar to those for the previous developmental stages were observed between ilc and concentration of cu and zn. all treatments showed a figure 4. inhibitions on late cleavage to varying concentrations of cu (black circles) and zn (white circles). b. edullantes and r. galapate 33 signif icant difference from the control (17 ± 7 %). ilc at 25 µg·l-1 cu increased twofold from the control (39 ± 5%). increasing the cu concentration to 50 µg·l-1 caused 52 ± 10% ilc, which was three times higher than in control. at higher concentration, cu elicited a f ivefold increase in ilc (>90%). in zinc treatment, ilc doubled at 25 µg·l-1 zn (40 ± 7%). it increased threefold at 50 µg·l-1 zn, producing 57 ± 6% inhibitions. ilc at 100 µg·l-1 cu quadrupled from the control (81 ± 2%). it was signif icantly high at 150 µg·l-1 zn (98 ± 2%), which was f ive times higher than in control. cu and zn elicited ec 50 at 42 ± 4% and 42 ± 9%, respectively (figure 6). no signif icant difference was observed between ec 50 of cu and zn, which indicate that cu is as toxic as zn. this is a different trend from that obtained in the previous observations. inhibitions on blastulation (ib) the percent inhibitions on blastulation were determined at 12 h exposure to varying concentrations of cu and zn (figure 5). similar trends of inhibitions were observed in the blastulation, which was found to increase with increases in the concentration of cu and zn. ib in all cu treatments varied signif icantly from the control (17 ± 6%). at 25 µg·l-1 cu, ib was quadrupled (70 ± 6%). at elevated cu concentration, ib was f ive times higher compared to control (>82%). ib at low concentration of zn showed figure 5. inhibitions on blastulation to varying concentrations of cu (black circles) and zn (white circles). embryotoxicity of copper and zinc in tropical sea urchin 34 no signif icant difference from control. concentration at 100 µg·l-1 zn elicited an ib of 73 ± 4%, which was four times higher than in control. increasing the concentration to 150µg·l-1 zn increased the ib to f ive times (86 ± 17%). the ec 50 of cu and zn were 20 ± 7 µg·l -1 and 44 ± 6 µg·l-1, respectively (figure 6). based on this ec 50 values, cu appeared to be twice as toxic as zn in inhibiting blastulation. discussion industrial and agricultural wastes discharge heavy metals such as cu and zn that pollute coastal areas and endanger aquatic organisms, including the sea urchins. elevated concentrations of these heavy metals may cause adverse effects on the growth, survival and reproduction of the echinoid species (thongra-ar 1997, kobayashi and okamura 2004). findings of this study showed that elevated concentrations of cu and zn lead to signif icantly higher inhibitions on fertilization, early cleavage, mid cleavage, late cleavage and blastulation in t. gratilla. the signif icant variation of percent inhibitions between control and treatments manifests toxicity of cu and zn in early developmental stages. previous studies reported similar f indings on embryotoxicity of heavy metal when elevated from their natural figure 6. comparison of ec 50 values of cu and zn in eliciting inhibitions of different stages of embryonic development in sea urchin t. gratilla. lower-case letters indicate significant difference between inhibitory effects of cu and zn for a particular embryonic stage. black and white circles indicate signif icant difference between the inhibitions of two stages for a certain heavy metal. b. edullantes and r. galapate 35 concentrations in seawater (us epa 1987, nakamura and others 1989, king and riddle 2001). the observed inhibitions on fertilization can be attributed to the spermiotoxic effects of cu and zn. motility and fertilizing capacity of t. gratilla spermatozoa are reduced by these toxicants (thongra-ar 1997), hence lowering the fertilization success. results also revealed inhibitions on cleavage and blastula stages, which are clear mitotoxic responses of cu and zn. exposure to high levels retards the division of cells, thus delaying the formation of blastula (kobayashi and okamura 2004). copper may elicit inhibitions on the early life stages in sea urchin by (1) respiratory acidosis (bielmyera and others 2005) or (2) disruption of ionic balances by alteration of atpases (li and others 1996) and carbonic anhydrase (zimmer and others 2012). zinc, on the other hand, possibly causes embryotoxic effects by: (1) inhibition of glucose-6-phosphate dehydrogenase that transforms carbohydrate via the pentose phosphate pathway (durkina and evtushenko 1991), (2) inhibition of the synthesis of ribosomal rna (pirrone and others 1970), (3) restriction of the development of endoderm as well as mesenchyme derivatives causing abnormalities to developing embryos (timourian 1968), and (4) intervention with the action of cortical granulederived protease that inhibit the formation of the fertilization membrane in sea urchin eggs (nakamura and others 1989). the results of this study provide evidence of concentration-dependent inhibitions caused by cu and zn on the embryonic development of sea urchin (figures 1 to 5). generally, inhibitions on developmental stages in sea urchin followed a logarithmic pattern when plotted against the concentration of cu and zn. inhibitions increased exponentially at low concentration but slowed at elevated concentration. although the effects of other stressors (e.g. particulate materials) on the inhibitions could not be completely excluded, these were minimized in the experiment. in fact, samples of natural seawater were collected in a pristine area and were f iltered to remove particulate materials. hence, any variation between the nominal and actual would have been kept at minimum; the same would be true for the observed concentration-response relationships. one limitation of this study is that inhibitions in the control group (>15%) were higher than the value (<10%) ideal for toxicity testing. as such, it could be argued that sources of stress other than cu and zn could have contributed to the embryotoxicity of copper and zinc in tropical sea urchin 36 concentration-response relationship observed. also, levels of cu and zn were not determined in the test solutions, consequently casting some questions regarding the accuracy of the ec 50 values. corollary to this, some inhibitions in cu treatments showed higher than 50% for all concentrations (e.g. , figures 1, 2 and 5). a possible explanation for this is that the ec 50 might be overestimated due to lack of information on the inhibitions at concentrations lower than the tested levels. it must be pointed out, however, that although range f inding tests were not conducted before the def initive tests, the exposure experiments were designed to determine inhibitions at concentrations within the range of the ec 50 values specif ied in the literature (see for example kobayashi 1990, king and riddle 2001, phillips and others 2003). hence, any differences in the threshold values between experiments with or without range f inding test would be insignificant. there would be a negligible difference in ec 50 values between def initive tests conducted with range f inding test and def initive tests conducted without range f inding test since the concentration used in the def initive tests were at concentrations within the range in the literature. as observed, cu was toxic within the range of 20 to 43 µg·l -1. ec 50 of cu. comparatively, this is below (king and riddle 2001, phillips and others 2003), within (heslinga 1976, pagano and others 1986) and above (kobayashi 1985, ramachandran and others 1997) the observed threshold range reported in other studies. on the other hand, ec 50 of zn from the past studies is below (kobayashi 1990), within (phillips and others 2003) and above (bay and others 1993, thongra-ar 1997, king and riddle 2001) the observed range of ec 50 of zn (42 93 µg·l -1). findings showed the sensitivity of sea urchin bioassay to the heavy metal species (figure 6). generally, ec 50 of cu was signif icantly lower than that of zn in all developmental stages, except in late cleavage. this comparison of cu and zn toxicity tests on developmental stages in t. gratilla suggests that cu is more toxic than zn. the potential toxicity of cu to t. gratilla was found to be 2–4 times greater than zn. previous studies also observed the same trend (thongra-ar 1997, kobayashi and okamura 2004 and 2005). responses of embryonic development to toxicants are stage specif ic (pagano and others 1986, dinnel and others 1987, bay and others 1993). this can be seen in the f indings regarding the inhibitions caused by cu in the different developmental b. edullantes and r. galapate 37 stages of t. gratilla (figure 6). ec 50 of cu in blastulation was signif icantly higher compared to the threshold value in mid and late cleavage, suggesting that cu is more toxic during blastulation than during the earlier developmental stages. kobayashi (1980) reported that cu is more disruptive in the later embryonic stages than in the earlier stages. in contrast to the f indings for cu, inhibitions elicited by zn did not vary signif icantly across developmental stages, which indicate that the toxicity of zn is not stage-dependent. accumulative trend in the inhibitions from early to late embryonic development was not observed in the present study. that is, it appears that the toxicity endpoints are independent of each other. the inhibitions in early development do not seem to influence the inhibitions in the latter stages. one possible explanation is that heavy metal exposure experiments were not carried out continuously. in continuous bioassay testing, it is expected that inhibitions from fertilization to blastula will show remarkable differences. conclusion the study examined the inhibitory effect of cu and zn on fertilization, early cleavage, mid cleavage, late cleavage and blastulation of t. gratilla. the inhibitions exhibited logarithmic concentration dependence where it increases exponentially at low concentration, but more slowly at elevated concentrations of heavy metals. the f indings conf irmed the sensitivity of sea urchin bioassay to heavy metal pollution, with cu eliciting greater toxicity than zn in the early developmental stages of t. gratilla. also, the study revealed the sensitivity of the assay to the developmental stages, although only cu showed stage-specif ic inhibitions. generally, the study provided a clear evidence of the dependence of heavy metal toxicity on heavy metal species, their concentration and the developmental stage they inhibit. the f indings may contribute to the improvement of the bioassay, particularly the use of the sea urchin t. gratilla in the assessment of toxicity of harmful anthropogenic substances. acknowledgments the authors are grateful to the reviewers and the editor whose critical review helped improve this manuscript. we thank the faculty members of the biology program in up cebu for their helpful comments and suggestions. embryotoxicity of copper and zinc in tropical sea urchin 38 references b a y s , b u r g e s s r , n a c c i d . 1 9 9 3 . s t a t u s a n d a p p l i c a t i o n s o f e c h i n o i d ( p h y l u m e c h i n o d e r m a t a ) tox i c i t y te s t m e t h o d s . i n : la n d i s w, h u g h e s j s , lew i s m a , ed i to r s . environmental 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[us epa] united state environmental protection agency. 1995. short-term methods for estimating the chronic toxicity of effluents and receiving waters to west coast marine and estuarine organisms. epa/600/r-95-136. us epa . 1987. ambient water quality criteria for zinc. epa-440/5-87-003. us epa. 2007. aquatic life ambient freshwater quality criteria – copper. epa-822-r-07001. zimmer am, barcarolli if, wood cm, bianchini a. 2012. waterborne copper exposure inhibits ammonia excretion and branchial carbonic anhydrase activity in euryhaline guppies acclimated to both fresh water and seawater. aquatic toxicology 122-123: 172-180. embryotoxicity of copper and zinc in tropical sea urchin 40 _______________ brisneve edullantes <brisneve713@hotmail.com> is currently an assistant professor of biology of the university of the philippines cebu. his research interests are aquatic ecology, water quality assessment and environmental toxicology. his research outputs have been presented in international and local scientif ic conferences. he received his bachelor’s degree in biology from university of the philippines cebu. he earned his master’s degree in environmental engineering from mokpo national maritime university, south korea. ritchel ita p. galapate is currently an associate professor of environmental science of the university of the philippines cebu. her research interests are water quality assessment, water pollution, water treatment, and environmental toxicology. she has published in international and local refereed journals of which the international publications have received at least 84 citations. her research outputs have likewise been presented in international and local conferences of professional and scientif ic organizations. she received her master’s and doctoral degrees in engineering (major in environmental science) from hiroshima university, japan. 11info for authors.pmd 79 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the off ice of the vicechancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); 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(adapted with permission f rom the editors of ieee sensors journal) 6santianez-white rot disease.pmd white rot disease and epiphytism on halymenia d urvillei 54 science diliman (january-june 2016) 28:1, 54-60 white rot disease and epiphytism on halymenia durvillei bory de saint-vincent (halymeniaceae, rhodophyta) in culture w ilfred john e. santiañez* university of the philippines diliman hokkaido university, japan hera j. suan-flandez university of the philippines diliman gavino c. trono jr. university of the philippines diliman keywords: e p i p h y t e , neosiphonia, red algae, seaweed culture, seaweed d i s e a s e _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online among the targeted seaweed species for resource development in the philippines is the red seaweed halymenia durvillei bory de saint-vincent, which is commonly known as “red sea lettuce”. locally, it is known as lablabig, gayong-gayong, gargarnatis, gamet, aragantiilek, and guraman (trono and ganzon-fortes 1988). aside from being used as food in many countries (lewmanomont and kawaguchi 2002), including the philippines, h. durvillei is also a source of the red pigment r-phycoerythrin, as well a s a l a m b d a c a r r a g e e n a n l i k e p o l y s a c c h a r i d e ( f e n o r a d o s o a e t a l . 2 0 0 9 ) . phycoerythrin is an important food and cosmetic colorant, a therapeutic agent owing to its immunomodulating and anti-cancer activity, and a fluorescent agent, among others (bermejo román et al. 2002; spolaore 2006). the current market value for 1 mg of r-phycoerythrin from porphyra tenera “nori” is us$ 1,030 (sg$ 1,400; sigma-aldrich online catalog, 2016a) lambda-carrageenan is a type of carrageenan (an integral part of red algal cell wall), but it only occurs naturally to a few species. as with other types of carrageenan, lambda-carrageenan is widely used in the food industry as an emulsif ier (fenoradosoa et al. 2009), but it can also act as an allergy suppressant against cer tain food products (tsuji et al. 2003). currently, 5 g of lambda-carrageenan is priced at us$ 114.79 (sg$ 156; sigma-aldrich online catalog, 2016b). w.j.e. santiañez et al. 55 in view of the high market value of h. durvillei, dr. gavino c. trono jr. of the marine science institute, university of the philippines (upmsi), in collaboration with the then philippine council for aquatic and marine research and development (pcamrd) of the depar tment of science and technology (dost ), developed a culture technology for the sustainable mass production of h. durvillei. a booklet entitled ‘a primer on the land-based culture of halymenia durvillaei bory de saint vincent (rhodophyta)’ (trono 2010) was published as a culmination of the research and development process. however, in a recent work to ref ine the technology on vegetative culture as well as in developing the spore to sea-out-planting mariculture of h. durvillei, we encountered two challenges that were detrimental to the alga’s growth and health: white rot disease and epiphytism. diseases and epiphytes are perennial challenges to the culture of seaweeds (e.g. , eucheuma denticulatum and kappaphycus alvarezii), which often lead to signif icant losses in biomass and prof it for the farmer. we describe herein the nature and progression of the disease, as well as the identity of the epiphytic organism. we also offer possible explanation for the infestation of epiphytes on h. durvillei. figure 1. white rot disease infected sea-out-planted carpospores-cultured halymenia durvillei showing discolored and decaying portions of each thallus (arrows). note that the circular infection often affects the basal portion of the thallus. white rot disease and epiphytism on halymenia d urvillei 56 we described the occurrence and nature of the disease based on the observations we made on the open sea-out-planted carpospores-cultured h. durvillei thalli grown at the bolinao marine laboratory (bml) of upmsi in guiguiwanen, bolinao, pangasinan. for the land-based (tank) culture of h. durvillei using vegetative propagules, we used the methods described by trono (2008), while the methods utilized on the spore culture to sea-out-planting portion of the project are detailed in trono (2014). we determined the identity of the epiphyte based solely on the morphological characters of ethanol-preserved samples. epiphytes were stained with aniline blue and mounted on corn syrup with phenol and were observed under the light microscope. white rot disease on open sea-outplanted carpospores-cultured h. d urvillei the white rot disease appears as a semi-circular spot of discoloration on the surface of the thallus. the initial site of infection enlarges while becoming progressively lighter, and its color turns from bright orange to light yellow until it becomes translucent as pigments appear to disintegrate. the affected part of the thallus becomes increasingly soft, with the white to translucent area being the most delicate and often decomposing (figure 1, arrows). large and soft infected portions of the thallus are the parts which facilitate the breakage and the consequent loss of the distal portion of the thallus. we noted that, in most cases, the infected parts are found near the base of the thallus. thus, during rough seas or in the presence of strong waves or currents, the majority of the thalli is broken and washed away, considerably reducing the amount of biomass. the characteristic symptoms of the disease in h. durvillei were similar to the white rot disease reported in porphyra, where vibrio (as beneckea) was indicated as among the possible pathogens (tsukidate 1977). the discoloration, decay of affected tissues, and consequent loss of biomass in h. durvillei due to breakage were also reminiscent of the ice-ice disease common among the carrageenan-producing eucheumatoids, kappaphycus and eucheuma (trono 1974). ice-ice disease in eucheumatoids, in addition to unfavorable environmental conditions, is also associated with pathogenic bacteria (uyenco et al. 1981), particularly vibrio sp. p11 and cytophaga sp. p25 (largo et al. 1995; largo 2002). we were not able to conduct further studies on what environmental factors induced the advent of the disease, but we hypothesize that, as in the case of other seaweed diseases, pathogenic microorganisms may also be involved in the white rot disease in h. durvillei. w.j.e. santiañez et al. 57 identity of the epiphyte and potential triggers of epiphytism on tank-cultured vegetative h. durvillei the thalli of the epiphytes are brownish-red, mostly erect but sometimes prostrate, and generally less than 3 cm (figure 2a). the epiphyte’s main axes are attached to h. durvillei by rhizoids that are cut-off from pericentral cells (figure 2b). branching is pseudodichotomous, with each branch abruptly tapering at the apices (figure 2f ). branch segment has four ecor ticate pericentral cells. trichoblasts are few, rudimentary, and limited at the apices. these are quickly shed, leaving spirally arranged scar cells (figure 2e, arrows). the species is dioecious. female g a m e t o p h y t e s p r o d u c e o v o i d m a t u r e c y s t o c a r p s ( f i g u r e 2 c ) , w h i l e m a l e gametophytes produce spermatia on conical spermatangial branches (immature) that develop as a trichoblast fork (figure 2d). tetrasporophytes produce figure 2. details of the alga, neosiphonia apiculata, found epiphytic on tank-cultured halymenia durvillei. (a) dried herbarium specimen; (b) prostrate portion of the main axis showing rhizoids cut off from the pericentral cells (arrow); (c) female plant showing ovoid mature cystocarp; (d) male plant bearing immature spermatangial branch (sp) issuing from trichoblast (tr); (e) portion of the branch showing spirally arranged scar cells in every segment (arrows); (f) distal branch portion showing spiral arrangement of tetraspores and the abrupt apical apices (a, scale bar = 1cm; b,c, scale bar = 50μm; d-f, scale bar = 100μm). white rot disease and epiphytism on halymenia d urvillei 58 tetrasporangia in slightly spiral series, associated with lateral cover cells (figure 2f ). despite being relatively larger, the characters of the epiphyte agree with the original descriptions of the polysiphonous alga described by hollenberg (1968), currently known as neosiphonia apiculata (hollenberg) masuda et kogame (ceramiales, rhodophyceae) (tani et al. 2003). the polysiphonous alga, n. apiculata appears to be a common epiphyte affecting cultured seaweeds (e.g. , eucheumatoids in calatagan, batangas and green island, palawan (wje santiañez, personal observation)) but may pose as a threat to seaweed farmers, such as the case of the heavy epiphytism in calaguas is. , camarines norte (hurtado et al. 2006; vairappan et al. 2008). heavy epiphytism by n. apiculata has negatively affected the development of h. durvillei, resulting in stunted growth, forcing us to prematurely discontinue our ongoing study. moreover, epiphytism became apparent in march 2013. the occurrence of heavy growth of epiphytes on cultured h. durvillei was preceded by algal blooms in the area that are often associated with high nutrient fluxes. during the said period, temperature (29-30°c) and salinity (34 ppt) levels in culture tanks were within optimum levels, but high water nutrient concentration was observed (ammonia: 2.17-3.87 μm; nitrate: 0.89-6.69 μm; phosphate: 2.13-2.54 μm). we believe that high nutrient concentrations coupled with warm water temperature and high incident light during the summer months encouraged the growth and proliferation of n . apiculata. in eucheumoid culture in the philippines, incidence of epiphytism by the alga was also high during summer (santiañez and trono, unpublished data). acknowledgments this study was funded by the philippines’ department of agriculture – bureau of agricultural research (da-bar). the authors wish to acknowledge mr. jerry arboleda and mr. ronaldo de guzman for their assistance in the conduct of laboratory and f ield work, ms. marilyn dayao for the administrative help, and mr. christian ace guerta for mounting some of the specimens used in this study. this is the up marine science institute contribution no. 449. w.j.e. santiañez et al. 59 references bermejo román r, alvárez-pez jm, acién fernández fg, molina grima e. 2002. recovery of pure b-phycoerythrin from the microalga porphyridium cruentum. j biotech. 93(1):7385. fenoradosoa ta , laroche c, wadouachi a, dulong v, pictan l, andriamadio p, michaud p. 2 0 0 9 . h i g h l y s u l p h a t e d g a l a c t a n f r o m h a l y m e n i a d u r v i l l a e i ( h a l y m e n i a l e s , rhodophyta), a red seaweed of madagascar marine coasts. int j bio macromol. 45:140145. hollenberg gj. 1968. an account of the species of polysiphonia of the central and western tropical pacif ic ocean. pac sci. 22:56-98. hurtado aq, critchley at, trespoey a , lhonneur gb. 2006. occurrence of polysiphonia epiphytes in kappaphycus farms at calaguas is. , camarines nor te philippines. j appl phycol. 18:301-306. largo db, fukami k, nishijima t. 1995. occasional pathogenic bacteria promoting “ice-ice” disease in the carrageenan-producing red algae kappaphycus alvarezii and eucheuma denticulatum (solieriaceae, gigartinales, rhodophyta). j appl phycol. 7:545554. largo db. 2002. recent developments in seaweed diseases. in: hur tado aq, guanzon ng jr. , de castro-mallare tr, luhan mrj, editors. proceedings of the national seaweed p l a n n i n g w o r k s h o p h e l d o n a u g u s t 2 3 , 2 0 0 1 , s e a f d e c a q u a c u l t u r e d e p a r t m e n t , tigbauan, iloilo. iloilo: seafdec aquaculture department. p. 35-42. le w m a n o m o n t k , k a w a g u c h i s . 2 0 0 2 . f o l i o s e h a l y m e n i a ( h a l y m e n i a c e a e , cryptonemiales, rhodophyta) from thailand. in: abott ia , mcdermid k, editors. taxonomy of economic seaweeds with reference to some pacif ic species volume iii. california: sea grant college program. p. 267-277. sigma-aldrich online catalog 2016a. r-phycoerythrin from porphyra tenera “nori”. accessed 13 june 2016 at http://www.sigmaaldrich.com/catalog/product/sigma/p8912? l a n g = e n & r e g i o n = p h . sigma-aldrich online catalog 2016b. -carrageenan. accessed 13 june 2016 at http: // www.sigmaaldrich.com/catalog/product/sigma/22049?lang=en&region=ph. spolaore p, joannis-cassan c, duran e, isamber t a . 2006. commercial applications of algae. j biosci bioeng. 101(2):87-96. tani m, yamagishi y, masuda m, kogame k, kawaguchi s, phang sm. 2003. taxonomic notes on marine algae from malaysia. ix. four species of rhodophyceae, with the description of chond ria decidua sp. nov. bot mar. 46:24-35. trono gc jr. 1974. eucheuma farming in the philippines. quezon city: natural science research center. λ  white rot disease and epiphytism on halymenia d urvillei 60 trono gc jr. 2010. a primer on the land-based culture of halymenia d urvillaei bory de saint-vincent (rhodophyta). quezon city: marine science institute, university of the philippines diliman. trono gc jr. 2014. mariculture of the red alga halymenia d urvillei bory de saint-v incent: tec h n i q u e s f r o m s p o r e s to s e a o u t p l a n t i n g . q u ezo n c i t y : b u r e a u of a g r i c u l t u r a l research, depar tment of agriculture. 10 p. trono gc jr. , ganzonfor tes et. 1988 philippine seaweeds. technology resource center. 400 p. tsuji rf, hoshino k, noro y, tsuji nm, kurokawa t, masuda t, akira s, nowak b. 2003. suppression of allergic reaction by lambda-carrageenan: toll like receptor–dependent and independent modulation of immunity. clin exp allergy. 33(2):249-58. tsukidate j. 1977. microbiological studies of porphyra plants – v. on the relation between bacteria and porphyra diseases. bul nansei reg fish res lab. 10:101-112. uyengco fr, saniel ls, jacinto gj. 1981. the “ice-ice” problem in seaweed farming. in: trono gc jr. and ganzon-for tes, editors. repor t on the training course on gracilaria algae. manila: food and agriculture organization of the united nations. vairappan cs, chung cs, hur tado aq, soya fe, lhonneur gb, critchley a . 2008. distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms. j appl phycol. 20:477-83. _____________ wilfred john e. santiañez <santianez@mail.sci.hokudai.ac.jp> was formerly a research associate at the marine science institute of the university of the philippines (up-msi), diliman, quezon city, where he worked on seaweed diversity, ecology, and culture. he has a master’s degree in environment and natural resource management from the university of the philippines open university and is now pursuing his phd in natural history sciences at hokkaido university in sapporo, japan. his current research is focused on the diversity and molecular phylogeny of the brown algal genus hydroclathrus. hera j. suan-flandez holds a master’s degree in aquaculture from the university of ghent, belgium and has worked as a senior research assistant at the up-msi. she has conducted ecological as well as spore and vegetative culture studies on the red alga halymenia durvillei, which were essential to the ref ining of the culture technology for this economically important species. gavino c. trono jr. is a national scientist at the national academy of science and technology (nast )-philippines and professor emeritus of marine science at upmsi. considered as 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(subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total 3abstracts.pmd layman’s abstracts 1 science diliman (january-june 2016) 28:1, 1-4 layman’s abstracts simple sequence repeat analysis of selected nsic-registered coffee varieties in the phil ippines daisy may c. santos, carla francesca f. besa, angelo joshua a. v ictoria, and ernelea p. cao coffee is an important commercial crop worldwide. c. arabica and c. canephora, commonly known as arabica and robusta coffee, respectively, comprise most of the global coffee production. the latter is inferior in terms of taste and aroma but is more resistant to coffee diseases, such as leaf rust, berry borer, and wilt disease. another coffee species, c. liberica, commonly known as liberica coffee, is also cultivated in the philippines because of its strong taste and flavor. the philippines has been trying to revive the coffee industry by producing specialty coffee with nsic-registered varieties. the differences in the cup quality of the varieties are the main factors that determine market value. therefore, there is a pressing need for the correct identif ication and isolation of pure coffee beans. local farms usually misidentify and mix coffee beans of different varieties, depreciating their value. this study used simple sequence repeats (ssrs) to distinguish philippine nsic-registered coffee varieties. ssrs are tandem repeats in dna sequences, with each repeat consisting of about two to six nucleotides. ssrs differ in terms of the number and kinds of repeats. the markers used in this study were able to separate the arabica, robusta, and liberica coffee from each other. issn 0115-7809 print / issn 2012-0818 online layman’s abstracts 2 chemical characterization and behavior of respirable fractions of indoor dusts colleged near a landf ill facil ity rheo b. lamorena-lim and colleen marciel f. rosales f r a c t i o n a t e d a i r b o r n e p a r t i c u l a t e m a t t e r ( t o t a l s u s p e n d e d p a r t i c u l a t e , coarse, and f ine) were separately collected from a junkshop, school, and money changer shop situated near a landf ill facility. par ticulate matter samples were extracted and chemically analyzed for water-soluble metals, as well as organic constituents. in general, lead and cadmium were found to be abundant in the total suspended particulate fraction (10-100 μm), while copper was abundant in pm 2.5 (<2.5 μm). in addition, manganese, arsenic, strontium, cadmium, and lead were detected to be signif icant in the pm 10 fraction (2.5-10 μm) compared to the pm 2.5 fraction. the metal a n d p h t h a l a t e c o n c e n t r a t i o n s w e r e u s e d i n a g e o c h e m i c a l m o d e l i n g software for speciation characterization under different relative humidity conditions. several solution complexes of the metals were predicted to form from the simulation runs. results of the study indicate the potential f o r m a t i o n o f i n o r g a n i c a n d o r g a n i c s p e c i e s o n i n h a l a b l e p a r t i c u l a t e surfaces under different relative humidity conditions. effect of zeol ite treatment on the blooming behavior of paraffin wax in natural rubber composites bryan b. pajarito, nico v. berba, jadreign keisheen c. par to, and raechel anne v. yabut soluble additives of natural rubber are known to bloom and form solid p r e c i p i t a t e s i n t h e p r o d u c t s u r f a c e . w h i l e s o m e a p p l i c a t i o n s f i n d blooming to be benef icial, the presence of bloom in products is usually visually offensive and unattractive. this work studied how three different chemical treatments of natural zeolite f iller, namely acid activation, ion exc h a n g e w i t h a te r t i a r y a m i n e s a l t , a n d o r g a n i c m o d i f i c a t i o n w i t h a non-ionic surfactant, affect the blooming of a model compound (paraff in layman’s abstracts 3 wax) in vulcanized natural rubber. specif ically, we want to know: (1) how paraff in wax blooms with time in rubber; (2) what will happen to wax blooming in natural rubber if raw and treated zeolite f illers are added; and (3) how zeolite treatments affect blooming. we found out that: (1) bloom amount varies linearly with the square root of time; (2) raw, acid-activated, and ion-exchanged zeolite f illers reduce bloom, while surfactant-treated f illers increase bloom in rubber; and (3) ion exchange and surfactant treatments enhance the speed and initial amount of bloom in natural r u b b e r co m p o s i te s . a m o n g t h e f i l l e r s , r a w a n d a c i d a c t i v a ted zeo l i t es g r e a t l y r e d u c e t h e a m o u n t of w a x b l o o m i n g o u t o f n a t u r a l r u b b e r. meanwhile, surfactant-treated zeolites increase the amount of bloom. white rot disease and epiphytism on halmenia durvillei bory de saint-v incent (halymeniaceae, rhodophyta) in culture w ilfred john e. santiañez, hera j. suan-flandez, and gavino c. trono jr. the current drive for research and development on philippine seaweed resources is directed towards developing culture technologies that expand the current seaweed industry, which is largely based on eucheuma and kappaphycus (locally known as gusô), by tapping on the large majority of seaweeds that remain underdeveloped. the focus of our current research is the red alga halymenia durvillei, which produces high-priced chemicals, such as carrageenan (a gelling agent) and the pigment r-phycoerythrin. we highlight herein the challenges we faced during the development and ref inement of the vegetative and spore culture technology of h. d urvillei, particularly while tackling white rot disease and heavy infestation of epiphytes. to our knowledge, this is the f irst repor t of both white rot disease and heavy infestation of cultured h. d urvillei. we identif ied the epiphyte as neosiphonia apiculata, a common epiphyte infesting other seaweeds in culture. we also describe the nature of the white rot disease, which is characterized by a distinct discoloration and disintegration of the affected portion. both white rot disease and epiphytes have negative e f f e c t s o n t h e g r o w t h a n d d e v e l o p m e n t o f h . d u r v i l l e i , r e s u l t i n g i n signif icant losses in biomass and potential prof it . layman’s abstracts 4 marine macroalgae: a review irene m. v illaseñor t h i s r e v i e w h i g h l i g h t s t h e r o l e o f s c i e n c e a n d t e c h n o l o g y i n t h e deve l o p m e n t , s u s t a i n a b i l i t y, a n d co m m e r c i a l v i a b i l i t y of t h e s e a weed i n d u s t r y i n t h e p h i l i p p i n e s . i t c o v e r s t h e s u b s t a n t i a l c o n t r i b u t i o n o f academician marco nemesio e. montaño and his co-authors on the posth a r v e s t s i d e o f s e a w e e d p r o d u c t i o n . t h i s r e v i e w i s l i m i t e d t o t h e r e s e a r c h d o n e o n t h e o p t i m i z a t i o n o f e x t r a c t i o n m e t h o d s t o e n h a n c e t h e q u a n t i t y a n d q u a l i t y o f h y d r o c o l l o i d s , s p e c i f i c a l l y a g a r a n d c a r r a g e e n a n , f r o m r e d s e a w e e d s ; a n d t h e c h a r a c t e r i z a t i o n o f t h e physicochemical properties of the hydrocolloid extracts. gaps requiring the conduct of sound research and development initiatives still exist in the seaweed industry. keyword s : seaweeds, hydrocolloids, agar, carrageenan, postharvest use of popparameters-delnorte-villarta.pmd use of population parameters in examining changes in the status of short-necked clam 53 use of population parameters in examining changes in the status of the short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) in coastal waters of southern negros occidental* annabelle del norte-campos* and karen a. villarta marine biology lab, college of arts & sciences university of the philippines visayas, miagao, iloilo *correspondng author: email: upvmarbio@yahoo.com abstract growth, recruitment, mortality and exploitation rate of the short-necked clam paphia undulata (born, 1778) were studied in southern negros occidental waters between august 2007 and july 2008 from length-frequency data derived from catches of divers, to be able to compare with earlier data derived and analyzed 13 years ago by agasen et al. (1998). both sets of data were analyzed using the fisat software (gayanilo & pauly, 1997). the asymptotic shell length (s∞ = 79 mm) derived from the present data proved to be smaller compared to the earlier data (sl∞ = 81.5 mm) due to the lack of bigger sizes in the present samples. the growth constant (k = 1.0 yr-1) was however comparable indicating it to be a more species-characteristic parameter. two recruitment pulses for each study were derived and were found to be correlated with the spawning pattern in the species. the value of natural mortality (m = 1.57 yr-1) derived from bivalve literature, is deemed more appropriate compared to the earlier estimate (m = 2.89 yr-1) based on pauly’s (1980) empirical equation developed for fish. total mortality (z) values for both studies were comparable, but a higher level of fishing mortality (f = 4.61 yr-1) was estimated for the present data set, thus resulting likewise in a higher exploitation rate (e = 0.75). these, together with fishery information from an accompanying paper (villarta & del norte-campos, 2010), not only validate the earlier findings of overexploitation due to lack of management, but reveal a worsening condition of the stock, most likely as a result of growth overfishing. overexploitation can only be mitigated by imposing stringent restrictions in terms of the minimum size for exploitation (45 mm shell length) and closed seasons during the spawning peak (august-november). keywords: paphia undulata, population parameters, negros occidental science diliman (january-june 2010) 22:1, 53-60 *this paper is part of the proceedings of the 10th national symposium on marine science held in davao city in october 2009. del norte-campos a.g. c. and k. villarta 54 science diliman (january-june 2010) 22:1, 53-60 introduction the short-necked clam paphia undulata (born, 1778) (figure 1), locally known in western visayas as “nylon shell”, is a popular shellfish resource harvested mainly for food. aside from negros occidental (agasen et al. 1998), it is likewise found in bolinao, pangasinan, where it is locally called “kabloy”, and in sorsogon bay in bicol region, where it is called “badoy” (soliman et al., 1999). it is reputed to have a “boom and bust” fishery (pastor & juinio-meñez 2003), just like its related species p. textile off bantayan island, cebu (dacles 1998). agasen et al. (1998) in their study thirteen years ago reported the species to be overexploited in coastal waters of negros occidental. the suitability of size-based, single-species assessment for bivalves has already been demonstrated in a number of papers in the country, such as those on scallops (del norte, 1988; gabral-llana 1988; soliman & dioneda 2004; del norte-campos & villarta, 2008) or even clams (del norte-campos 2004). due to the commercial importance of the p. undulata, as well as its polyculture potential, there is a need to re-assess its status considering that there are still no known management policies governing its exploitation. comparing the status of the stocks at different times is important as it provides insights on the impact of unregulated exploitation on the resource. the objectives of this study were to (a) examine the population biology of the species, by estimating the parameters of its figure 1. the short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) growth, recruitment, mortality, and exploitation rate, and (b) compare the results with the those of a study on the same species in the same fishing ground conducted thirteen years ago (agasen et al., 1998). the results are then used in the context of other information from parallel investigations on the fishery (villarta & del norte-campos, 2010) and reproductive biology (nabuab et al., 2010) as the scientific basis for the formulation of management guidelines for the rational exploitation of short-necked clam stocks in coastal waters of negros occidental, central philippines. materials and methods the study was conducted in the waters of hinigaran, binalbagan, and himamaylan, negros occidental, central west visayas (figure 2). length-frequency data from catches of compressor divers covering the period august 2007 to july 2008 were analyzed. shell lengths (sl) (i.e. shell anterior to posterior) of clams were measured to the nearest 0.05 mm using a vernier caliper. sl’s were grouped in size classes of 5 mm and analyzed using the fisat software (gayanilo & pauly, 1997). the growth parameters, asymptotic shell length (sl∞ in mm) and growth coefficient (k, yr-1) were derived using the elefan i routine. the growth function (φ’) of pauly & munro (1984) was computed using the following formula: φ’ = log k + 2logl∞ (1) recruitment, and total mortality (z, yr-1), were derived with the use of the elefan ii routine. the estimate of natural mortality (m) was derived by averaging m/ k values from bivalve literature and multiplying it with the k value derived from this study. fishing mortality (f, yr-1) was computed by subtracting m from z, total mortality being equivalent to: z = f + m (2) exploitation rate was then computed with the formula: e = f/z (3) use of population parameters in examining changes in the status of short-necked clam 55science diliman (january-june 2010) 22:1, 53-60 results and discussion population parameters (growth, recruitment, mortality) are useful bases in comparing the status of exploited resources as they provide valuable information on how exploitation affects the population (pauly 1984). the sl∞ of 79 mm (figure 3) derived from this study is smaller than the 81.5 mm) obtained by agasen et al. figure 2. location of the study area in southern negros occidental (1998), which can be attributed to the smaller sizes of the present data (figure 4). a t-test performed on random samples taken from the original data sets to compare mean sizes of clams from current (sl = 44.8 mm) and previous (sl = 55.9 mm) studies showed that the difference in sizes (i.e. a reduction of almost 24%) is significant (t = 6.38, df = 39, p<0.001). therefore, whereas clams > 75 mm sl were still caught in 199697 during the study of agasen et al. (1998), sizes from figure 3. von bertalanffy growth curve of the short-necked clam paphia undulata derived using elefan i in the fisat software (sl ” = 79 mm, k = 1.0 yr-1, rn = 0.561) del norte-campos a.g. c. and k. villarta 56 science diliman (january-june 2010) 22:1, 53-60 current catches did not exceed 65 mm (villarta & del norte-campos, 2010). the value of k in the present study (1.0 yr-1) differed only slightly from the estimate of agasen et al. (1998) (1.2 yr-1), thereby resulting in close estimates of φ’ (3.791 and 3.901, respectively). this shows that the difference in sl∞ values stems from the difference in maximum sizes in the two sets of samples, and that the growth function (φ’) can truly be used to characterize not only similar species (pauly & munro 1984), but also related species as in the case of scallops (del norte 1988). nabuab et al. (2010) reported that the minimum size at sexual maturity is 42.6 mm for males and 44. 8 mm for females. thus, in addition to being smaller clams, the bulk of current catches are also immature (sl< 45mm) (figure 4) indicating that growth and perhaps even recruitment overfishing (gulland 1971) set in sometime during the past 13 years. two recruitment pulses were derived (figure 5) just as in agasen et al. (1998). whereas reproduction was not examined in the earlier study, these pulses indicate spawning peaks which were observed between august to november (nabuab et al., 2010). these pulses not only coincide with the spawning peak, but are also related to the monsoons. spawning and subsequent recruitment are believed to be timed during a “survival window” during the monsoons. this pattern is similar to those observed in many tropical species (pauly & navaluna, 1983), including many bivalve species in the philippines like scallops (del norte 1988; gabral-llana 1988) and ark shells (ledesma-fernandez & del norte-campos 2004). bivalve literature data used in computing natural mortality (m) are shown in table 1. based on these, m was estimated to be 1.57 yr-1. this estimate is deemed more proper than the estimate of agasen et al. (1998) (2.89 yr-1) which was based on pauly’s (1980) empirical equation. the latter was based purely on information from fish, and may thus be inappropriate for invertebrates, including bivalves. natural mortality, just like k, are speciesor taxon-specific parameters reflecting the niche of the species and/or the taxon in m in i mu m si ze a t se x u a l m a tu r it y 0 5 1 0 1 5 2 0 2 5 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 s h e ll len g th (m m) f re q u en c y (% ) e a rlie r d a ta (1 9 9 6 -9 7 ) p re s e n t d a t a (2 0 0 7 -0 8 ) figure 4. comparison of sizes (shell length in mm) of the short necked clam paphia undulata caught 13 years ago based on agasen et al. (1998) and current sizes in southern negros occidental with the range of sizes at sexual maturity for males (42.3 mm) and females (44.8 mm) superimposed. use of population parameters in examining changes in the status of short-necked clam 57science diliman (january-june 2010) 22:1, 53-60 question. as such, their values do not deviate widely among related species and/or within taxonomic groups. averaged m/k values taken from bivalve literature are therefore biologically suitable to use as basis for computing m for the species in the present study. mortality and exploitation estimates are shown in table 2. from these, comparable values of total mortality, z (6.18 vs. 6.22 yr-1) were derived but a higher fishing mortality (f = 4.61 yr-1) was computed, thereby resulting also in a higher value of exploitation rate (e = 0.75). these results not only confirm the previous finding that the negros stock is overfished (agasen et al., 1998), but even further emphasize that the situation has become far more serious at the present. details of overfishing are presented in another paper (villarta & del nortecampos, 2010). the present results therefore further highlight the urgent need to manage this resource. this problem can only be mitigated by imposing strict management guidelines that should include (1) fishing only sizes that are sexually mature, i.e. >45 mm (pongthana 1990; nabuab et al., 2010), and (2) imposing closed seasons during the spawning season (augustfigure 5. recruitment pattern of paphia undulata showing two recruitment pulses november). these guidelines, albeit unpopular due to their initial short-term impact on fisher livelihoods, can restore the stock to a sustainable status if properly implemented, thereby assuring a more stable income for the fishers. acknowledgments this study was funded by the dost/pcamrd project “studies on the biology and fishery of the short-necked clam paphia undulata born, 1778 (mollusca, pelecypopda: veneridae) in negros occidental waters”. field and lab assistance were provided by f. nabuab and r. palla. w.l. campos and two other reviewers provided suggestions to improve the manuscript. references agasen, e.v., c.m. del mundo, & g.o. matias. 1998. assessment of paphia undulata in negros occidental/ guimaras strait waters. j. shellfish res. 17(5): 1613-1617. del norte-campos a.g. c. and k. villarta 58 science diliman (january-june 2010) 22:1, 53-60 table 1. natural mortality (m, yr-1) and growth coefficient (k, yr-1) literature estimates for different bivalve species used to compute the m value for the short-necked clam, paphia undulata in the present study. *given values obtained from different methods were averaged table 2. mortality and exploitation rate estimates for the southern negros occidental stocks of the short-necked clam paphia undulata, derived from the present study and those from agasen et al. (1998). *derived from averaged bivalve literature ** inappropriately derived from fish stocks (pauly, 1980) species common name (fao, 1998) m (yr-1) k (yr-1) m/k literature amusium pleuronectes asian moon scallop 1.29 0.92 1.4 del norte (1988) a. pleuronectes “ 1.3 0.94 1.38 gabral-llana (1998) a. pleuronectes “ 1.49 0.90 1.66 del norte-campos & villarta (2008) tridacna maxima elongate giant clam 0.21 0.074 2.84 munro & heslinga (1982) t. maxima “ 0.15 0.132 1.14 mckoy (1980) tapes philippinarum japanese carpet shell 0.2005 0.9125 0.22 yap (1977) chlamys funebris 0.506* 0.457 1.11 soliman & dioneda (2004) c. senatoria nobilis senatorial scallop 0.506* 0.457 1.11 “ decatopecten striatus 0.525* 0.507 1.04 “ gari elongata elongate sunset clam 2.95 1.0 2.95 del norte-campos (2004) paphia undulata short-necked clam 2.89 1.2 2.41 agasen et al. (1998) 1.57 mean m/k paphia undulata short-necked clam 1.57 1.0 this study total mortality (z, yr-1) fishing mortality (f, yr-1) natural mortality (m, yr-1) exploitation rate (e) source 6.18 4.61 1.57* 0.75 this study 6.22 3.33 2.89** 0.54 agasen et al. (1998) use of population parameters in examining changes in the status of short-necked clam 59science diliman (january-june 2010) 22:1, 53-60 dacles, t.p.u. 1998. the population dynamics of the nailon clam paphia textiles (gmelin, 1791) and notes on its communal clam fishery off bantayan island, cebu. ms thesis. u. san carlos, cebu city. del norte, a.g.c. 1988. some aspects of the growth, recruitment, mortality and reproduction of the asian moon scallop, amusium pleuronectes (linné) in the lingayen gulf, philippines. ophelia. 29(2): 153-168. del norte-campos, a.g.c. 2004. some aspects of the population biology of the elongate sunset clam gari elongata (lamarck 1818) (mollusca, pelecypoda: psammobiidae) from the banate bay area, west central philippines. asian fish. sci. 17: 299-312. del norte-campos, a.g.c. & k.a. villarta. 2008. population biology of the asian moon scallop amusium pleuronectes linné (bivalvia: pectinidae) from pilar and capiz bays, northern panay, philippines. upv j. nat. sci. 13(2): 1-10. fao species identification guide for fishery purposes. 1998. carpenter, k.e. & v.h. niem (eds.). the living marine resources of the western central pacific. vol. 1. seaweeds, corals, bivalves and gastropods. rome, fao: 1-686. gabral-llana, e. 1988. growth, mortality and recruitment of the asian moon scallop (amusium pleuronectes) in the visayan sea, philippines. in: contributions to trop. fish. bio: papers by participants of fao/danida follow-up training courses. venema, s., j. möller-christensen & d. pauly (eds.). fao fish. rpt.389, rome. gayanilo, f. c. & d. pauly (eds.). 1997. fao-iclarm stock assessment tools. (fisat). reference manual. fao computerized information series (fisheries). no. 8. rome, fao: 262 p. gulland, j.a. 1971. the fish resources of the oceans. fao/ fishing news books, ltd. surrey, england. ledesma-fernandez, l.n. & a.g.c. del norte-campos. 2004. reproductive cycle of the ark shell scapharca inaequivalvis (brugiere, 1789) (mollusca, pelecypoda: arcidae) in banate bay, west central philippines. upv j. nat. sci. 9(1): 111-123. mckoy, j.l. 1980. biology, exploitation and management of giant clams (tridacnidae) in the kingdom of tonga. fish. bull. 1: 5-61. munro, j.l. & g.a. heslinga. 1982. prospects for the commercial exploitation of giant clams (bivalvia: tridacnidae). paper pres. 35th mtg. gulf & carrib. fish. inst. nassau, bahamas, nov. nabuab, f.m., l. ledesma-fernandez & a.g.c. del nortecampos. 2010. reproductive biology of the short-necked clam paphia undulata (born, 1778) from negros occidental waters. science diliman (in press). pastor, d.s. & m.a. juinio-meñez. 2003. boom and bust kabloy fishery-the bolinao experience. philippine scientist. 40: 88-100. pauly, d. 1980. on the interrelationships between natural mortality, growth parameters and mean environmental temperature in 175 fish stocks. j. cons. int. explor. mer. 39: 175-192. pauly, d. 1984. fish population dynamics in tropical waters: a manual for use with programmable calculators. iclarm studs. & revs. 8: 325 p. pauly d. & j.l. munro 1984. once more on the comparison of growth in fish and invertebrates. fishbyte. 2(1): 21. pauly & navaluna. 1983. monsoon-induced seasonality in the recruitment of philippine fishes: pp. 823-833. in: g. sharp & j. csirke (eds.) proceedings of the expert consultation to examine changes in abundance and composition of neritic fish stocks, san jose, costa rica, 18-29 april 1983. fao fish. tech. rep. 231. vol. 3. pongthana, n. 1990. breeding and rearing of short-necked clam (paphia undulata). thai. mar. fish. res. bull. 1: 6973. soliman, v.s. & r.r. dioneda sr. 2004. quick stock assessment of the commercial scallops (bivalvia: pectinidae) in asid gulf, masbate. upv j. nat. sci. 9(1): 165-176. del norte-campos a.g. c. and k. villarta 60 science diliman (january-june 2010) 22:1, 53-60 soliman, v.s., r.r. dioneda, s.g. borbe, & l.m alcantara. 1999. assessment and management of short-necked clam paphia undulata (bivalvia: veneridae) and blue crab portunus pelagicus linnaeus (crustacea: portunidae) in sorsogon bay, philippines. bu r. & devt. j. 12: 1-7. villarta, k.a. & del norte-campos. 2010. fishery of the shortnecked clam paphia undulata in southern negros occidental waters. science diliman 21(1):pp-pp. yap, w. g. 1977. population biology of the japanese littleneck clam tapes philippinarum in kaneohe bay, oahu, hawaiian islands. pac. sci. 31(3): 223-244. the water-art.3 the water and sediment quality of chanos chanos 35science diliman (january june 2000) 12:1, 35-44 abstract the water and sediment quality of chanos chanos monoculture and chanos chanos gracilariopsis bailinae biculture in pond lota b. alcantara aquatic science and technology institute state polytechnic college of palawan (spcp-asti), sta. monica p.o. box 93, puerto princesa city 5300, phone: (048)434-3908, fax: (048)433-4367 email: spcpasti@pal-onl.com a short-term study on the physical-chemical parameters in chanos chanos monoculture and its biculture with gracilariopsis bailinae indicated that the biculture might be advantageous for the growth of milkfish. dissolved oxygen of the biculture and monoculture was not significantly different early in the morning. oxygen produced by g. bailinae from late afternoon until evening was probably compensated by larger c. chanos that consumed more oxygen in the biculture. the afternoon do of the biculture, however, was higher than that of the monoculture. there was no difference in ph readings between the monoculture and the biculture. water temperature ranged from 23 39oc, and salinity ranged from 14 42% for both monoculture and biculture. the presence of g. bailinae did not affect water ph, temperature, and salinity of the biculture pond. during the culture period, phosphate in the water of the biculture decreased while it increased in the monoculture. the change in nitrate was insignificant for both monoculture and biculture. the ammonium decrease in the biculture was higher than that in the monoculture while the rise in phosphorus in the sediment was higher in the monoculture than in the biculture. the increase in nitrate and ammonia was higher in the monoculture sediments than in biculture sediments, but the difference was insignificant. some of the phosphate and ammonium lost in the biculture pond may be attributed to the phosphorus and the nitrogen utilized by the red seaweed or stored in its tissues. chanos chanos grew better in biculture with g. bailinae as the effect of more favorable water and sediment quality in the pond during the culture period. furthermore, the nutrients present in the pond water and sediment were probably utilized by g. bailinae for their growth or stored in their tissues. keywords: chanos chanos, gracilariopsis bailinae, water quality, sediment quality, monoculture, biculture. alcantara 36 introduction milkfish (chanos chanos forsskål) culture using ponds, pens and cages is popular in the philippines. many aquaculturists today engage in high-density stocking to increase production. such systems require supplemental feeding and may be stressful to the environment (bagarinao, 1996). the limited area of the pond is prone to accumulation of certain nutrients from animal excreta and uneaten feeds both of which may become toxic to the reared organisms or lead to proliferation of pests or diseases. in the long run such conditions may contribute to the decline in the yield of the culture organisms as well as to the yield of other organisms in other bodies of water in the environs into which the pond drains. it is hypothesized that the metabolic wastes and excess feed of c. chanos are used as source of nutrients for the photosynthetic processes of the agarophyte, gracilariopsis bailinae zhang et xia. previous studies have noted that gracilaria spp. absorb ammonia produced in shrimp ponds (dela cruz, 1995). furthermore, gracilaria spp. photosynthesize, therefore, produce oxygen that in effect accelerates oxidation of organic matter and utilizes ammonia. as a result, ammonia in the water will be reduced and dissolved oxygen will increase, leading to favorable conditions for the growth of c. chanos. the biculture of seaweed and fish has not been adopted commercially in the philippines. supplemental income from the seaweed produced and reduced environmental impact are some of the advantages of this biculture technique (hurtado-ponce, 1993; buschmann, 1996). nowadays, environmental concerns are given high consideration in any technology development. this is important in aquaculture especially because the medium is in continuity with other bodies of water that can easily disperse substances such as nutrients and pesticides. this study examines the effect of the biculture of g. bailinae with c. chanos on the water and sediment characteristics of the pond. it also presents the differences in the water quality and sediment quality of the pond for the culture of c. chanos alone, and the biculture of c. chanos and g. bailinae. materials and methods the study was conducted in the fishponds of the silliman university marine laboratory (suml), dumaguete city from february 3 to march 17, 1998. there were two treatments in the study: (1) the c. chanos monoculture, and (2) the c. chanos-g. bailinae biculture. each treatment was replicated six times in 12m2 earthen ponds, which were further subdivided by netting material into three compartments. one compartment for all the replicates was measured, without replacement of experimental organisms, at two weeks interval. the first samples were referred to as series 1; the second series 2; and the third, series 3. net production rates and growth rates of c. chanos were determined from the weights measured in every series. water exchange between pond and incoming water occurred every other day during high tides. vegetative cuttings of g. bailinae were broadcasted in the biculture treatment at 1,000 kg ha-1 one day ahead of c. chanos stocking. the stocking density of milkfish was 5,000ha-1. the milkfish were given supplemental food (tateh bangus feeds, manila, philippines) three times daily at 4.0 5.0% of the initial body weight, and adjusted accordingly every other week. temperature, ph and salinity were monitored daily at 1000-100h and 1500-1600h using an ordinary mercury thermometer, a portable ph meter orion research model sa 250 (orion research incorporated, usa), and a hand-held temperature compensated reichert refractometer (japan), respectively. dissolved oxygen was determined every seventh day of the week at 0600-0700h and 1500-1600h using winkler’s method. water samples collected from each pond were filtered and stored in a freezer until the laboratory determination. sediment samples were collected using an improvised pipe borer then air dried, powdered and sieved for laboratory analysis. dissolved phosphate (po4), nitrate (no3), ammonium (nh4) in the water, and phosphorus (p), nitrate (no3) and ammonia (nh3) in the sediment were determined at the start and at the end of the study. chemical analysis followed the methods in grasshoff et al. (1983). pond sediment particle size profile was determined using the methods prescribed by dartnall & jones (1986) and the water and sediment quality of chanos chanos 37 english et al. (1994). soil ph was determined every two weeks. data were subjected to analysis of variance (anova). results water ph the minimum and maximum morning ph in the pond compartments were recorded at 6.8 and 9.0, respectively. the initial morning ph ranged from 7.8 to 8.0. during the culture period, the weekly morning ph of the biculture treatment fluctuated from 8.1 to 8.3 while in the monoculture ph ranged from 8.2 to 8.3. the range of the afternoon ph was from 6.2 to 9.3. the initial afternoon ph was 8.1. in the biculture, ph varied from 7.9 to 8.3, and in the monoculture, the range was 7.9 to 8.4 (fig. 1). analysis of variance indicated no significant difference between the two treatments in both morning and afternoon ph readings. dissolved oxygen the morning do ranged from 0.1 to 6.6mg l-1. highest morning do was recorded before the stocking of experimental organisms (4.5 to 5.1mg l-1). for six weeks of culture, the do of the biculture treatment fluctuated from 0.4 to 1.3mg l-1, and for the monoculture treatment do ranged from 0.6 to 1.3mg l-1. the afternoon do ranged from 4.4 to 19.3mg l-1. initial afternoon do in the pond ranged from 10.6 to 11.9mg l-1. during the culture period, the do of the biculture fluctuated from 9.6 to 12.5mg l-1 while in the monoculture do ranged from 6.4 to 7.2mg l-1 (fig 2). no significant difference (p>0.05) was determined between the two treatments in the morning do but significant difference (p<0.05) existed in the afternoon do using anova. phosphate chemical analysis of the water showed that the amount of po4 in the pond water decreased from 2.9 to 2.2µmol l-1 in the biculture treatment. in contrast, the monoculture treatment was found to increase from 2.9 to 4.0µmol l-1 (fig. 3). analysis of variance showed that the difference between the two treatments was significant (p<0.05). nitrate the amount of no3 decreased from 1.9 to 1.3µmol l -1 in the biculture. on the contrary, in the monoculture treatment, a small increase of 0.1µmol l-1 was seen (fig. 4). analysis of variance revealed that the two treatments were not significantly different (p>0.05). ammonium the amount of nh4 lost in the biculture treatment was 1.7µmol l-1, from 1.9 to 0.2µmol l-1 . on the other hand, in the monoculture, there was a decrease of 0.7µmol l1 in nh4 from 1.9 to 1.2µmol l -1 (fig. 5). the amount of nh4 lost in the biculture treatment was significantly higher (p<0.05) than that in the monoculture treatment using anova. temperature morning pond water temperature ranged from 25 to 35oc while in the afternoon, it fluctuated from 22 to 39oc. minimum-maximum temperature readings showed that the lowest water temperature was 22 to 29oc, and the highest was 32 to 39oc. there was no difference in water temperature between the monoculture and biculture ponds (fig. 6). salinity the range of morning pond salinity was from 14 to 42% and the afternoon salinity ranged from 14 to 44%. generally, afternoon salinity (30.2%) was higher than morning salinity (29.8%). the salinity of the monoculture and biculture ponds were similar (fig 7). alcantara 38 figure 2. comparison of the average dissolved oxygen between the ponds of c. chanos monoculture and c. chanos g. bailinae biculture taken in the morning and afternoon from initial to six weeks of culture from february 3 to march 17, 1998 (mono = monoculture, bi = biculture, am = morning, pm = afternoon). initial week 1 week 2 week 3 week 4 week 5 week 6 am-mono am-bi pm-mono pm-bi culture period d is so lv ed o xy ge n [m g l-1 ] sediment particle size the grain size analysis using the wentworth grade scale showed that the sediment was sandy mud. the principal grain component was fine to very fine sand, 62-250µm (45.3%), followed by very coarse to coarse sand, 500-200µm (22.3%), medium sand, 250-500µm (21.7%), and silt, 3.9-62µm (10.6%). sediment ph the sediment ph ranged from 6.8 to 9.0. initially the ph was 6.9. in the first series, the ph in the biculture was 8.9 and in the monoculture, it was 8.8. on the second and third series, both treatments had a ph of 8.3 and 8.5, respectively (fig. 8). phosphorus the amount of p in the sediment increased during the culture period from 301.4 to 312.1mol l-1 or an increase of 10.7mol l-1 in the biculture. the monoculture increased by 45.5mol l-1 from 271.6 to 346.9mol l-1 in p (fig. 9). analysis of variance showed that the increase in the amount of total p in the monoculture was significantly higher (p<0.05) than the biculture. nitrate there was an increase in the amount of no3 in the sediment at the end of the study. the amount increased by 7.7mol l-1 from 26.2 to 33.9mol l-1 (fig. 10). analysis of variance revealed a significant (p<0.05) increase of no3 in the sediment; however, the two figure 1. comparison of the average ph between the ponds of c. chanos monoculture and c. chanos g. bailinae biculture taken in the morning and afternoon from initial to six weeks of culture from february 3 to march 17, 1998 (mono = monoculture, bi = biculture, am = morning, pm = afternoon). 8.6 8.4 8.2 8 7.8 7.6 7.4 initial week 1 week 2 week 3 week 4 week 5 week 6 am-mono am-bi pm-mono pm-bi culture period w at er p h 14 12 10 6 4 2 0 8 figure 3. comparison of the average phospate between the pond water of c. chanos monoculture and c. chanos g. bailanae biculture taken before and after the culture period from february 3 to march 17, 1998. 4.5 4 3.5 2.5 2 1.5 0.5 culture period p ho sp ha te [ µm ol l -1 ] 3 initial final 1 0 monoculture biculture the water and sediment quality of chanos chanos 39 figure 4. comparison of the average nitrate between the pond water of c.chanos monoculture and c. chanos g. bailinae biculture taken before and after the culture period from february 3 to march 17, 1998. 2.5 2.0 1.5 1.0 culture period n itr at e [µ m ol l -1 ] initial final 0 monoculture biculture 0.5 figure 5. comparison of the average ammonium between the pond water of c. chanos and c. chanos g. bailinae biculture taken before and after the culture period from february 3 to march 17, 1998. 2 1.8 1.6 1.2 1 0.8 0.2 culture period a m m on iu m [ µm ol l -1 ] 1.4 initial final 0.6 0 monoculture biculture 0.4 figure 6. comparison of the average water temperature between the ponds of c.chanos monoculture and c. chanos g. bailinae biculture taken in the morning and afternoon from initial to six weeks of culture from february 3 to march 17, 1998 (mono = monoculture, bi = biculture, am = morning, pm = afternoon). week 1 week 2 week 3 week 4 week 5 week 6 am-mono am-bi pm-mono pm-bi culture period te m pe ar at ur e [° c ] 40 35 30 20 15 10 0 25 5 treatments have no significant difference (p>0.05). ammonia the biculture treatment had higher nh3 in the sediment at the start than at the end of the study. the difference was 3.7mol l-1 nh3 from 33.8 to 30.1mol l-1. in the monoculture, the increase was 1.0mol l-1 (fig. 11). however, no significant difference (p>0.05) was shown by the two treatments. growth of c. chanos two-way anova showed that the weight increment of c. chanos in the biculture treatment was significantly (p<0.05) greater than in the monoculture and that significant (p <0.05) differences exist between series. weight increments of c. chanos were observed to increase with longer culture period (table 1 & 2). there was no significant difference (p>0.05) between the weight gained by milkfish in both monoculture and biculture treatments during the first two weeks of culture. after one month of culture until the end of the culture period, the milkfish cultured with the agarophyte had higher weight increment compared with those in monoculture. in both the daily and the monthly net production rate estimates, anova of logarithmically transformed data showed that the biculture treatment was significantly (p<0.05) higher than the monoculture. net production rate after two weeks of culture did not alcantara 40 figure 7. comparison of the average salinity between the ponds of c.chanos monoculture and c. chanos g. bailinae biculture taken in the morning and afternoon from initial to six weeks of culture from february 3 to march 17, 1998 (mono = monoculture, bi = biculture, am = morning, pm = afternoon). week 1 week 2 week 3 week 4 week 5 week 6 am-mono am-bi pm-mono pm-bi culture period s al in ity [ ‰ ] 40 35 30 20 15 10 0 25 5 figure 8. comparison of the average ph between the pond sediment of c. chanos monoculture and c. chanos g. bailinae biculture taken once every two weeks period from february 3 to march 17, 1998. week 1 week 2 week 4 week 6 culture period 10 9 8 5 4 3 0 6 1 monoculture biculture 7 2 vary significantly (p>0.05) from those cultured for four weeks or six weeks in both the monoculture and biculture. the net production rate in the biculture decreased with time. highest net production rate was obtained during the first two weeks while lowest was obtained in the sixth week (table 2). both the biculture and monoculture treatments obtained the fastest growth rate during the first two weeks of culture. the growth rates of the biculture were significantly (p<0.05) higher than those of the monoculture. moreover, there were significant differences (p<0.05) between the culture time series. comparison of means using duncan’s multiple range test (dmrt) revealed that growth rates in the biculture increased with time. however, the monoculture mean growth rates in all series were statistically similar. discussion growth performance of chanos chanos was higher in the biculture than in the monoculture. several physical and chemical parameters most likely contributed to this. the dissolved oxygen content probably influenced the growth performance of c. chanos. the early morning do of the two treatments did not differ, but the afternoon do in the biculture was usually higher than that in the monoculture. afternoon do may represent most of the oxygen produced by photosynthesizing algae, including the cultured g. bailinae, that started to build-up from midfigure 9. comparison of the average phosphorus between the pond sediment of c. chanos monoculture and c. chanos g. bailinae biculture taken once every two weeks period from february 3 to march 17, 1998. 360 350 340 320 310 300 280 culture period p ho sp ha te [ m ol l1 ] 330 initial final 290 270 monoculture biculture the water and sediment quality of chanos chanos 41 figure 10. comparison of the average nitrate between the pond sediment of c. chanos monoculture and c. chanos g. bailinae biculture taken before and after the culture period from february 3 to march 17, 1998. 50 45 40 30 20 15 5 culture period n itr at e [m ol l -1 ] 35 initial final 10 0 monoculture biculture 25 figure 11. comparison of the average ammonia between the pond sediment of c. chanos monoculture and c. chanos g. bailinae biculture taken before and after the culture period from february 3 to march 17, 1998. 36 35 34 32 31 30 28 culture period a m m on ia [ m ol l1 ] 33 initial final 29 27 monoculture biculture basis of comparison biculture monoculture overall weight increment weight increment with longer culture period daily net production rate monthly net production rate overall growth rate growth rate with longer culture period ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↑ ↑ ↑ table 1. comparison of the performance of chanos chanos in monoculture and biculture with gracilariopsis bailinae based on analysis of variance from february 3 to march 17, 1998 (↑ = higher, ↓ = lower). morning till late afternoon. with higher do and additional food source from the epiphytic organisms of the agarophyte, the milkfish in the biculture treatment benefited and probably converted to higher weight. higher do in the biculture was probably caused by the photosynthetic activity of g. bailinae (fig. 2). lower do in the pond early in the morning may have been caused by several respiring organisms present in the sediment and water. dissolved oxygen greatly affects growth and production of milkfish through its direct effect on feed consumption and metabolism, and indirect effect on environmental parameters (dela vega, 1996). bagarinao (1996) reported that fish grew favorably when do was maintained at 5 or >5mg l-1 but grew slowly at do <5mg l-1. in this study, however, all readings are still within the range of 5.0mg l-1 but the biculture were much higher than those of the monoculture. further study is needed to elucidate this matter. carbon dioxide and ions in equilibrium with photosynthesizing organisms largely influence changes in water ph. thus, ph is low at dawn and high in the afternoon (dela vega, 1996). water ph (9 to 10) in ponds recorded by haglund & pedersen (1993) was higher than reported in this study (7.5 to 8.5). nevertheless, the results here were very near to what santelices & doty (1989) suggested (ph 8.1) for pond culture of gracilaria spp. nelson et al. (1980) found that gracilaria edulis and gracilaria arcuata have the capability of removing ammonia from seawater. likewise, gracilaria tenuistipitata alcantara 42 treatment/ series initial weight (g) final weight (g) weight increment* (g) net production* rate (g m-2 day-1) net production* rate (kg ha-2 mo-1) growth rate (% day-1) biculture, series 1 biculture, series 2 biculture, series 3 monoculture, series 1 monoculture, series 2 monoculture, series 3 11.0 ± 1.2 12.7 ± 1.7 15.8 ± 1.7 14.1 ± 1.0 16.4 ± 1.4 17.6 ± 1.8 27.7 ± 2.3 46.8 ± 4.1 68.8 ± 5.4 23.1 ± 1.8 38.1 ± 2.4 56.3 ± 5.5 16.7 ± 2.0dc 34.1 ± 3.5b 53.1 ± 4.7a 9.0 ± 1.2d 21.7 ± 1.9c 38.8 ± 4.2b 0.9 ± 0.1a 0.6 ± 0.1a 0.6 ± 0.1a 0.3 ± 0.04b 0.39 ± 0.03b 0.46 ± 0.05b 262.9 ± 90.3a 182.8 ± 18.7a 174.8 ± 18.4a 95.8 ± 13.0b 116.2 ± 10.3b 138.5 ± 14.8b 6.2 ± 0.6a 4.8 ± 0.5a 3.3 ± 0.2c 3.3 ± 0.4c 2.89 ± 0.2c 2.61 ± 0.2b * means having common letter superscript are not significantly different at p = 0.05. table 2. mean (±se) initial weight, final weight, weight increment, net production rate, and growth rate of chanos chanos in monoculture and biculture ponds from february 3 to march 17, 1998 (n = 12). was found to remove no3 -, nh4 + , and po4 3(haglund & pedersen, 1993). results of the study show that the removal of po4 and nh4 in the biculture of c. chanos and g. bailinae was significantly higher than in the monoculture (figs. 4 & 5). the phosphorus from the sediment is a good source of phosphorus for phytoplankton and algal growth. ammonia, ammonium, nitrite and nitrate are inorganic forms of nitrogen used by photosynthetic organisms. the range of p and n for average to high fish production has been reported by dela vega (1996) to be 30 to >60 mol l-1 and 250 750 mol l-1, respectively. the fish production referred to here relates to natural food production. the monoculture of milkfish increased the quantity of total p in the pond soil. the amount of nitrogen in the form of nitrate and ammonia in this study is lower than the optimum range given above. the nutrients in the sediment are probably accumulated through time from fish excreta and other inputs. the maximum growth rates of c. chanos in the biculture of this study are higher (6.2% day-1) than those obtained in 500m2 ponds stocked at 7,000ha-1 (2.1% day-1) (sumagaysay & borlongan, 1995). the culture system, area, and stocking density are different. biculture with g. bailinae probably improved the growth performance of c. chanos. milkfish cultured with the red alga obtained higher mean growth rate (3.19% day-1) than monoculture (1.57% day-1) did. the biculture of fish and macroalga has synergistic interaction, and the culture medium is favorable to both organisms. it has been documented that feeding the cultured fish increases production and profits. however, nutrients and organic matter from excess feeds and feces can cause rapid deterioration of water quality (feed dev’t. section, 1994). cruz (1996a) claimed that the direction of the milkfish aquaculture in the philippines is towards intensification. it should be considered, though, that highdensity culture systems have ecological limits (bagarinao, 1996). in line with this, it is relevant to look for an alternative milkfish culture system that will be economically and ecologically viable for a longer period. the simultaneous culture of milkfish and the agarophyte may be an alternative method to minimize, if not prevent pollution in the pond and the coastal waters. the water and sediment quality of chanos chanos 43 environmental conditions in the culture pond are significant because these may have some effect on the physiology of fish, its appetite and growth (chiba, 1986). for example, it has been noted that the ideal feeding temperature for milkfish is at 28-34oc (cruz, 1996b). chiu et al. (1986) observed that peak feeding of c. chanos occurs during the day and stop feeding when do is below 1.5mg l-1. conclusions dissolved oxygen in the monoculture of c. chanos was significantly lower than in the biculture of c. chanos and g. bailinae during afternoon. the biculture significantly lost higher phosphate and ammonia from the culture medium compared to the c. chanos monoculture. on the other hand, the monoculture of c. chanos accumulated a higher amount of total phosphorus in the pond sediment than the biculture treatment. in general, c. chanos grew better in biculture with g. bailinae. recommendations results on the biculture of g. bailinae and c. chanos look promising. pilot testing of this farming system is, therefore, recommended using longer culture periods and larger ponds. management measures to minimize proliferation of opportunistic green algae and epiphytes should be developed to produce good quality g. bailinae. the recommended procedure is to grow the milkfish for two months before stocking the red alga and there should be a regular monitoring of water quality for management purposes. acknowledgments the author is very grateful to suml, dumaguete city, especially to the director, dr. nida p. calumpong for the use of experimental area and laboratory facilities. the study was partly funded by uplb-fi-usaid cdr project no. 96-009 through dr. mila martinez-goss and the pcamrd ms thesis grant. thanks to dr. sabine schoppe for reviewing the draft. references bagarinao, t.u., 1996. ecological limits of high-density milkfish culture. paper presented during the conferenceexhibit, technicon2: technical considerations for the management and operation of high-density milkfish culture system. 24-26 october 1996, diliman, quezon city. buschmann, a.h., 1996. an introduction to integrated farming and the use of seaweed as biofilters. hydrobiologia. 326/327: 59-60. chiba, k., 1986. the cycle of nitrogen, carbon and phosphorus in an eel culture pond. in: mclean, j.l., b. dizon, and l.v. hosillos (eds.) the first asian fisheries forum. asian fisheries society, manila, philippines: 31 pp. chiu, y.n., p. macahilig & m.a.s. sastrillo, 1986. factors affecting feeding rhythm of milkfish (chanos chanos forsskal). in: mclean, j.l., b. dizon, and l.v. hosillos (eds.) the first asian fisheries forum. asian fisheries society, manila, philippines: 547 pp. cruz, p.s., 1996a. overview of high-density milkfish culture. paper presented during the conference-exhibit, technicon2: technical considerations for the management and operation of high-density milkfish culture system. 24-26 october 1996, diliman, quezon city. cruz, p.s., 1996b. milkfish feeding management and economics. paper presented during the conferenceexhibit, technicon2: technical considerations for the management and operation of high-density milkfish culture system. 24-26 october 1996, diliman, quezon city. dartnall a.j. & m. jones, 1986. a manual of survey methods living resources in coastal areas. asean-australian cooperative program on marine science handbook. australian institute of marine science, townsville: 167 pp. alcantara 44 dela cruz, c.r., 1995. brackishwater integrated farming systems in southeast asia. in: bagarinao, t.u. and e.e.c. flores (eds.) towards sustainable aquaculture in southeast asia and japan. proceedings of the seminar-workshop on aquaculture development in southeast asia. iloilo city, philippines: 26-28 july 1994. aqd/seafdec, iloilo, philippines: 32 pp. dela vega, a.m., 1996. soil and water quality. paper presented during the conference-exhibit, technicon2: technical considerations for the management and operation of high-density milkfish culture system. 24-26 october 1996, diliman, quezon city. english, s., c. wilkinson, & v. baker, (eds.), 1994. survey manual for tropical marine resources. australian institute of marine science, townsville: 368 pp. feed development section, 1994. feeds and feeding of milkfish, nile tilapia, asian sea bass and tiger shrimp. seafdec/aqd, tigbauan, iloilo, philippines: 97 pp. grasshoff, k., m. ehrhardt, & k. kremling (eds.), 1983. methods of seawater analysis. second revised edition. verlag chemie gmbh, d. weinhein: 61 pp. haglund, k. & m. pedersen, 1993. outdoor pond cultivation of the subtropical marine red alga gracilaria tenuistipitata in brackishwater in sweden. growth, nutrient uptake, co-cultivation with rainbow trout and epiphyte control. j. appl. phycol. 5: 271-284. hurtado-ponce, a.q., 1993. growth rate of gracilariopsis heteroclada (zhang et xia) zhang et xia (rhodophyta) in floating cages as influenced by lates calcarifer bloch. in: calumpong, h.p. and e.g. meñez (eds.) proceedings of the 2 nd rp-usa phycology symposium/workshop: supplement. 6-18 january 1992, cebu city & dumaguete city, philippines: 13 pp. nelson, s., r. tsutsui, & b. best, 1980. a preliminary evaluation of the mariculture potential of gracilaria (rhodophyta) in micronesia: growth and ammonium uptake. in: abbott, i., m. foster, and l. eklund, (eds.) pacific seaweed aquaculture. proceedings of the symposium on useful seaweed, march 1980 at pacific grove. california sea grant college program, institute of marine resources, university of california, la jolla, california: 72 pp. santelices, b. & m.s. doty, 1989. a review of gracilaria farming. aquaculture. 78: 95-133. sumagaysay, n.s. & i.g. borlongan, 1995. growth and production of milkfish (chanos chanos) in brackishwater ponds: effects of dietary protein and feeding levels. aquaculture. 132: 273-283. sd inside back cover-ched.pmd science diliman: a philippine journal of pure and applied sciences is accredited by the commission on higher education (ched) through its journal accreditation service project, as category a-2 journal from 2014 to 2016 and is available via www.doaj. org and www.ebsco.com. science diliman has been selected for coverage in the emerging sources citation index of thomson reuters and is available via www.doaj.org and www.ebsco.com 4balela-nanoparticles.pmd formation of highly antimicrobial copper nanoparticles 10 science diliman (july-december 2015) 27:2, 10-20 formation of highly antimicrobial copper nanopar ticles by electroless deposition in water mary donnabelle l. balela* university of the philippines diliman kathy lois s. amores university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract metallic copper (cu)nanoparticles (cunps)with mean diametersranging f r o m 3 7 n m t o 4 4 n m w e r e s y n t h e s i z e d b y e l e c t r o l e s s d e p o s i t i o n (chemical reduction)in an aqueous solution at 353 k. cupric oxide (cuo) powder, which has low solubility in water, was used as the cu(ii) precursor. gelatin and hydrazine (n 2 h 4 ) were employed as the protective agent and reductant , respectively. small spherical cu nanopar ticles having mean diameter of 37 nm were formed using 2.25 wt% gelatin. in the absence o f g e l a t i n , l a r g e c u n a n o p a r t i c l e s o f 3 7 7 n m i n m e a n d i a m e t e r w e r e p r o d u c e d . b o t h c u p r o u s o x i d e ( c u 2 o ) a n d m e t a l l i c c u p e a k s w e r e identif ied from the x-ray diffraction pattern of the samples. the results suggest that gelatin hinders the growth of cu nanoparticles in solution and protects the nanoparticles from oxidation. interestingly, the as-prepared cu nanoparticles exhibit strong antimicrobial activity against escherichia coli and staphylococcus aureus. keywords: c o p p e r n a n o p a r t i c l e s , e l e c t r o l e s s d e p o s i t i o n , h y d r a z i n e antimicrobial layman’s abstract spherical copper (cu) nanoparticles with average diameter in the range of 37-44 nm were formed by simple chemical reduction in water at 80°c. g e l a t i n w a s u s e d t o p r o t e c t t h e c u n a n o p a r t i c l e s f r o m o x i d a t i o n a n d prevent their agglomeration in solution. in fact, larger cu nanoparticles of about 377 nm in average diameter were produced when gelatin was absent in the solution. in addition, oxides of cu (cu 2 o) were observed in the x-ray diffraction pattern of the same sample. m.d.l. balela and k.l.s. amores 11 introduction metal nanoparticles are widely gaining recognition due to their unique properties, which result to diverse applications in the f ields of microelectronics and biotechnology(usman et al. 2012). in par ticular, silver (ag)and copper (cu) nanoparticles are drawing attention due to their excellent thermal, electrical, catalytic, and optical properties. for example, ag and cu nanoparticles have been shown to exhibit superior bactericidal effect because of their large surface areas, which allow them to closely interact with bacteria. thus, these nanomaterials could be potential antimicrobial agents for antibiotic-resistant microorganisms, which are alarmingly becoming more widespread (ruparelia et al. 2008; zhang and yang 2013; chatterjee et al. 2014). the antimicrobial properties of ag nanoparticles have been extensively studied over the years. many hypotheses that attempt to elucidate the mechanism of its antimicrobial activity have been proposed. ag nanoparticles can penetrate the cell membrane and can result to the leakage of intracellular substances, ultimately leading to cell death (ruparelia et al. 2008). on the other hand, recent studies have shown that cu nanoparticles could also be a promising antimicrobial agent, and hence, a cheaper alternative to ag (bogdanovic et al. 2014; chatterjee et al. 2014; shankar and rhim 2014). cu has the ability to act as an electron donor or acceptor depending on its oxidation state. cu can easily change between oxidation states because of its high electrochemical potential, allowing it to interact freely with bacterial proteins (konieczny and rdzawski 2012). for instance, the oxidation of cu+ generates hydroxyl radicals in fenton’s reaction as in equation (1). the generated hydroxyl radicals consequently breakdown bacterial proteins and dna (hajipour et al. 2012). cu+ + h 2 o 2 → cu2+ + oh+ oh(1) rispoli et al. (2010) investigated the antimicrobial activity of cu nanoparticles against escherichia coli (e. coli) and found out that the toxicity of cu nanoparticles against e. coli is dependent on temperature, aeration rate, ph, and the concentration of cu nanoparticles. moreover, a separate study has shown that the antimicrobial activity of cu nanoparticles is more effective against bacteria than fungi (ramyadevi et al. 2012). cu nanoparticles have also been incorporated in natural f ibers and tested for their bactericidal effect against e. coli and staphylococcus aureus (s. aureus) (chowdhury et al. 2013). the cu nanoparticles immobilized in natural f ibers exhibited 7% antifungal activity and greater bactericidal effect against e. coli compared with s. aureus. formation of highly antimicrobial copper nanoparticles 12 cu nanoparticles can be synthesized by numerous methods, such as the polyol process (zhang et al. 2014), sonochemical method (dhas et al. 1998), nanosphere lithography (chan et al. 2007), thermal reduction(habibi and kamrani 2010), laser ablation (sadrolhosseini et al. 2013), hydrothermal synthesis (giannousi et al. 2014), and electroless deposition (chemical reduction) ( yagi et al. 2008; yagi et al. 2009; tan et al. 2014). though the fabrication of cu nanoparticles can also be carried out in gas or solid phases, the liquid phase via the “liquid-phase reduction” is the most preferred due to its simplicity ( yagi et al. 2008; yagi et al. 2009; magdassi et al. 2010; tan et al. 2014). synthesis of cu nanopar ticles in liquid does not require a vacuum environment and can be done at low temperatures, making the process more cost-effective(umer et al. 2012). additionally, the morphology of cu nanoparticles can be easily controlled in solution by varying parameters, such as temperature, ph, and concentrations of the reacting species (usman et al. 2012). though many studies have reported the preparation of cu nanoparticles in solution, it remains a challenge, particularly in water, due to the high propensity of cu nanopar ticles for oxidation (yagi et al. 2008; yagi et al. 2009; tan et al. 2014). in this study, oxidation-stable cu nanopar ticles were prepared by electroless deposition (chemical reduction) in an aqueous solution using gelatin as protective agent. the effect of gelatin concentration on the particle size of cu nanoparticles was investigated. the use of gelatin as protective agent has been previously reported (chatterjee et al. 2012; zhang and yang 2013). by contrast to the use of cu(ii) salts in the said studies, the present work used cupric oxide (cuo) as the cu(ii) precursor. cuo has low solubility in aqueous solution, particularly at high ph, favoring the formation of small and uniform cu nanoparticles.the oxidation of cu nanopar ticles in solution was monitored by uv-v is spectroscopy, while the antimicrobial activity of cu nanoparticles against e. coli and s. aureus was determined by the agar disk diffusion method. materials and methods materials reagent grade cupric oxide powder (cuo, himedia labs), 98 wt% hydrazine solution in water (n 2 h 4 , sigma aldrich inc.), sodium hydroxide pellets (naoh, rsi labscan), and gelatin granules (nacalai tesque inc.) were utilized as received. deionized water was used throughout the synthesis. m.d.l. balela and k.l.s. amores 13 electroless deposition of cu nanoparticles cuo suspension solution was prepared by dispersing 4.87 g of cuo powder in 42 ml deionized water by sonication. n2 h 4 solution was prepared by dissolving 3.06 g of n 2 h 4 in 42 ml of deionized water. afterwards, 18 g of 0% to 15% gelatin solution in water was added to both the cuo suspension and then 2 h 4 solution. the ph values of the two solutions were adjusted to 12 at room temperature using 1.5 m naoh aqueous solution. nitrogen (n 2 ) gas was introduced into the solution 30 mins prior to the reaction to eliminate dissolved oxygen. the n 2 h 4 solution was then slowly added to the cuo suspension while stirring at 500 rpm. the total solution was allowed to react for 2 h at 353 k under continuous nitrogen gas purging. the as-prepared cunps were collected by centrifugation and washed by deionized water several times. characterization the size and morphology of the cu nano particles were observed using a scanning electron microscope with an acceleration voltage of 10 kv (sem, hitachi sem s3700n). image analyses of 300 nanoparticles from several sem images were used to determine the mean particle size. the structure of the cu nanoparticles was investigated in an x-ray diffractometer using a voltage of 40 kv and a current of 30 ma (xrd, maxima_x xrd-700). the ultraviolet-visible (uv-vis) absorption spectra of the cu nanoparticles were obtained using a uv-vis spectrometer (shimadzu uv1700) within the wavelength range of 400 nm to 800 nm. antimicrobial activity gram-positive bacteria s. aureus (mtcc-3160) and gram-negative bacteria e. coli (mtcc-2642) were suspended in 0.1% peptone water and inoculated on 3-mm thick nutrient agar plates. a total of 200 μl of the cu nanoparticle solutions were added on three equidistant wells with diameters of 10 mm on the nutrient agar plates. antibiotic discs were used on the center of the nutrient agar plates as the positive control. nutrient agar plates were then incubated at 308 k for 24 h. the average diameters of the clearing zones were measured, and the antimicrobial index (ai) was calculated using the formula below: (2)ai diameter of clearing zone diameter of well diameter of well   formation of highly antimicrobial copper nanoparticles 14 results and discussion electroless deposition (chemical reduction) of copper nanoparticles figure 1 shows the sem images of cu nanoparticles formed by electroless deposition (chemical reduction) in an aqueous solution using increasing concentrations of gelatin. in the absence of gelatin, large cu particles with mean diameter of 377 nm were formed in the solution. the particles were irregularly shaped and agglomerated as seen in figure 1a, possibly due to the lack of steric hindrance by gelatin. on the other hand, addition of 0.75 wt% gelatin to the reaction suspension yielded spherical cu nanoparticles with mean diameter of 44 nm. figure 1. sem images of cu nanoparticles prepared by electroless deposition (chemical reduction) at 353 k in aqueous solution using (a) 0 wt%, (b) 0.75 wt%, (c) 1.50 wt%, and (d) 2.25 wt% gelatin as protective agent. m.d.l. balela and k.l.s. amores 15 when the amount of gelatin was increased to 1.50 wt% and 2.25 wt%, smaller cu spherical nanoparticles with mean diameters of 37 nm to 38 nm were generated. however, the smaller cu nanoparticles were surrounded by an organic layer, probably gelatin, as seen in figure 1c. the presence of the organic layer suggests that the washing process was insuff icient to remove the excess gelatin around the cu nanoparticles. except for the sample prepared without gelatin, the spherical cu nanoparticles were uniform in size with standard deviations less than 10% of the mean particle size. it is possible that gelatin effectively restricts the growth of cu nanoparticles in solution. as a result, uniform and minute cu nanoparticles were formed. adherence of gelatin on the surface of cu nanoparticles also increases the space steric hindrance among the nanoparticles, effectively inhibiting agglomeration (magdassi et al. 2010; zhang and yang 2013; tan et al. 2014). figure 2 shows the corresponding xrd patterns of cu nanoparticles formed with increasing amount of gelatin. for the samples with gelatin, peaks at 43.44°, 50.62°, and 74.20° were attributed to 111, 200, and 220 peaks of face-centered (fcc) cu, respectively. the presence of these peaks indicates that only metallic cu was formed in the solution. the cu peaks also appear broadened compared to the sample figure 2. xrd patterns of cu nanoparticles synthesized by electroless deposition (chemical reduction) at 353 k using (a) 0 wt%, (b) 0.75 wt%, (c) 1.50 wt%, and (d) 2.25 wt% gelatin as protective agent. formation of highly antimicrobial copper nanoparticles 16 without gelatin, suggesting smaller crystallite size. such observationis in agreement with the apparent diameter determined from the sem images in figure 1. in the absence of gelatin, peaks of cu 2 o at 2q = 64.22° and 77.42°, which are reflections of the 220 and 222 planes, respectively, were indexed together with peaks of fcc cu as shown in figure 2a. oxidation of cu nanoparticles possibly occurred in the absence of gelatin, explaining the cu 2 o peaks (tan et al. 2014). figure 3 shows the uv-vis spectra of cu nanoparticles generated using 2.25 wt% gelatin after 0 to 60 days of storage in aqueous solution. this particular sample was selected for the oxidation study due to its small particle size of 37 nm in mean diameter. the small particle diameter suggests large surface-to-volume ratio and a high tendency for oxidation. the as-prepared cu nanoparticles exhibit an absorption peak at about 584 nm, a value well within the range of the cu absorption peak (573 nm to 600 nm). interestingly, there were no signif icant changes in the cu absorption peak after 14 to 60 days of storage. no other absorption peak besides that of cu was observed after 60 days, suggesting that neither agglomeration nor oxidation occurred for the cu nanoparticles in the solution. such result is signif icant for its future applicationas an antimicrobial agent. figure 3. uv-v is absorption spectra of cu nanoparticles prepared by electroless deposition (chemical reduction) with 2.25 wt% gelatin after (a) 0,(b) 14, and (c) 60day(s) of storage. m.d.l. balela and k.l.s. amores 17 antimicrobial activity of cu nanoparticles figure 4 shows the actual images of the nutrient agar plates containing e. coli and s. aureus inoculates incubated with200 μl of theas-prepared cu nanoparticles with mean diameters of 38 nm to 377 nm. compared to the samples with larger particle diameters, cu nanoparticles with mean diameter of 38 nm exhibit higher average inhibition zone at 55 mm and an antimicrobial index of at least 4.5. such performance figure 4. clearing zones of 200 μl cu nanoparticles with mean particle sizes of (a-b) 377 nm, (c-d) 44 nm, and (e-f ) 38 nm against gram (-) e. coli and gram (+) s. aureus. formation of highly antimicrobial copper nanoparticles 18 can be attributed to the large specific surface area of these cu nanoparticles. hydroxyl radicalsare generated from the interaction of cu with the bacteria, leading to its high antimicrobial activity by oxidative damage to the bacteria (ruparelia et al. 2008; chatterjee et al. 2014). though the 377-nm cu nanoparticles have lower antimicrobial properties than the smaller ones, they exhibit antimicrobial properties comparable to chloramphenicol, which was used as the positive control. the antimicrobial properties of the as-prepared cu nanoparticles against e. coli and s. aureus are summarized in table 1. table 1. comparison of the antimicrobial activities of the synthesized cu nanoparticles against gram (-) e. col i and gram (+) s. aureus 38 55 4.5 gram (-) 44 53 4.3 escherichia coli 377 38.3 3.2 chloramphenicol* 25 4.0 38 >55 >4.5 gram (+) 44 53.3 4.3 staphylococcus aureus 377 42.3 3.2 chloramphenicol* 25 3.2 antimicrobial index (ai) test organism cu nanoparticles diameter (nm) average inhibition zone (mm) conclusion spherical cu nanoparticles with mean diameters of 37 nm to 44 nm were synthesized via electroless deposition (chemical reduction) using cuo suspension in water. the presence of gelatin as protective agent provides excellent oxidation and agglomeration protection for the cu nanoparticles. the particle size of cu nanoparticles was controlled to some extent by changing the concentration of the gelatin in the solution. smaller cu nanoparticles were generally formed with higher gelatin concentration. this observation can be attributed to the steric hindrance provided by the gelatin, which thereby inhibits particle growth. oxidation studies exhibited that minute cu nanoparticles of 37 nm in mean diameter were stable against oxidation even after 60 days of storage. the cu nanoparticles also exhibited high microbial activity against gram (-) e. coli and gram (+) s. aureus with clearing zones rangingfrom 38 nm to 55 mm in diameter. further study is needed, in order to fully elucidate the antimicrobial property of the as-prepared cu nanoparticles. *6-mm disc contains 30 μg chloramphenicol m.d.l. balela and k.l.s. amores 19 acknowledgments this study was supported by the university of the philippines under the off ice of the v ice-chancellor for research and development (phd incentive award), the department of science and technology philippine council for industry, energy and emerging technology research and development (dost-pcieerd), and the college of engineering professorial chair award (uratex). references bogdanovic u, lazic l, 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o u s c u o s u s p e n s i o n . j o u r n a l of e l e c t r o c h e m i c a l s o c i e t y. 155(6):474–479. zhang d, yang h. 2013. gelatin-stabilized copper nanopar ticles: synthesis, morphology and their surface-enhanced raman scattering properties. physica b. 415:44-48. z h a n g y, z h u p, l i g , z h a o t, f u x , s u n r , wo n g c p. 2 0 1 4 . f a c i l e p r e p a r a t i o n o f monodisperse, impurity-free, and antioxidation copper nanoparticles on a large scale for application in conductive ink. acs applied materials and interfaces. 6(1):560-567. _____________ mary donnabelle l. balela, ph.d. <mdlbalela@gmail.com> is an associate professor from the department of mining, metallurgical and materials engineering, university of the philippines diliman. she is the head of the sustainable electronic materials research group. she obtained her ph.d. in materials science and engineering from kyoto university in 2011. kathy lois s. amores is a f ifth year b.s. materials engineering student of the department of mining, metallurgical and materials engineering. she worked on the synthesis of cu nanoparticles during her summer apprenticeship in the sustainable electronic materials laboratory. 7short comm.pmd a manifestation of climate change? 78 science diliman (july-december 2013) 25:2, 78-85 a manifestation of cl imate change? a look at typhoon yolanda in relation to the historical tropical cyclone archive carlos primo c. david bernard alan b. racoma jonathan gonzales and mark vincent clutario environment monitoring laboratory national institute of geological sciences university of the philippines diliman abstract the ibtracs world database of tropical cyclone(tc) tracks was analysed to determine potential historical trends in tc characteristics for the west pacif ic basin. trends are then related to the characteristics of typhoon yolanda to see if this individual event constitutes as a data outlier or is par t of a trend that can be related to climate change. in terms of tc frequency, it is deduced that there is a decreasing pattern in tropical cyclone formation starting in 1970. it is also noted that while there is no trend observed in the annual mean maximum wind speed, a decrease in the number of high wind speed tcs is measured for the months of n ove m b e r a n d d ece m b e r. t h e l o c a t i o n of tc fo r m a t i o n h a s a l s o b ee n changing towards a higher latitude but closer to the philippines in terms o f l o n g i t u d e . l a s t l y, t y p h o o n s m a k i n g l a n d f a l l i n t h e v i s a y a s a n d mindanao region have also become slightly more frequent in the last decade. except for the last f inding, the 2013 typhoon season does not f it in these general trends. this year may be the star t of a new trend or shift in tc characteristics (which we will only know after a few more years) but is most likely part of the inherent annual variability of typhoon characteristics. yolanda goes against perceived trends but its occurrence signif ies that there is still much to learn about tropical cyclones and the impending impacts of climate change in general. introduction on 8 november 2013, super typhoon yolanda (international name: haiyan) made landfall in guiuan, eastern samar. it was, by many accounts, the most powerful tropical cyclone (tc) that made landfall ever recorded in history. according to the issn 0115-7809 print / issn 2012-0818 online c.p. david and others 79 philippine atmospheric, geophysical and astronomical services administration (pagasa), the recorded maximum 10-minute sustained winds of yolanda were 230 kilometers per hour (kph) with gustiness reaching 250kph shortly after landfall. the joint typhoon warning center (jtwc) calculated 1-minute maximum sustained windspeed of 315kph with gustiness reaching 380kph. the national disaster risk reduction and management council (ndrrmc) report dated 16 december 2013 stated that 6,069 individuals perished while 1,779 are still missing because of super typhoon yolanda. more than 5,000 of the casualties came from the province of leyte and this was mainly due to the storm surge that affected its coastline. the estimated total worth of damages is pegged at php35.5 billion. one question that often arises during discussions in the aftermath of the disaster is whether this extreme event can be considered to be the “new normal” and therefore attributable to climate change. among meteorologists, the consensus is that the warming of ocean waters due to climate change will theoretically influence cyclogenesis, but the high level of uncertainty in results precludes any def initive cause-effect relationship (wmo 2006). the intergovernmental panel on climate change (ipcc) report conceded that the resolution of coupled ocean and atmospheric modelling is still too coarse to completely resolve climate change-related changes to tropical cyclone characteristics (ipcc 2007). still, based on various modelling studies, the ipcc report projected a decrease in mid-latitude storms globally per year but an increase in average wind intensity. this statement was slightly revised in its fifth assessment repor t (ipcc 2013) wherein it said that current datasets indicate no signif icant observed trends in global tropical cyclone frequency over the past century. conflicting results were provided by mcdonald and others (2005): they reported an almost insignif icant decrease in the number of typhoons (6% decrease) but an increase in wind intensity. still, emoriet and others (2005) projected that both the number and intensity of cyclones in the northern pacif ic basin will decrease but related precipitation will increase. one of the more recent works on tropical cyclone modelling was done by knutson and others (2010), who projected that globally, the number of tropical cyclones will decrease by 6-34%, but the number of very intense cyclones will increase by about 20% by 2100. the same paper suggested a poleward shift in tropical cyclone formation. considering the apparent uncer tainty of tc frequency and intensity trends from global climate models, the other technique that can be used in f iguring out tropical cyclone trends is to analyze historical archives while looking at a single event, such as yolanda, in relation to the typhoon database. this work aims to contribute to this form of analysis by looking at not only annual frequency and intensity trends but a manifestation of climate change? 80 also other typhoon metrics by dissecting typhoon characteristics on a month-bymonth scale. methodology the us national oceanic and atmospheric administration (noaa) maintains a database that archives all recorded tropical cyclones by various weather agencies. the international best track archive for climate stewardship (ibtracs) boasts of more than 300,000 tropical cyclone-related entries, one-third of which are western pacif ic cyclones (knapp and others 2010). the dataset covers the years 18842012, and includes tc tracks (6-hourly), calculated wind speed and barometric pressure, among other information. the ibtracs has been endorsed by the world meteorological organization as an off icial archiving program for tropical cyclone information. the present analysis primarily uses the ibtracs data; however, the 2013 tc data from jtwc are included, whenever applicable, for completeness. the ibtracs data is parsed using the python programming language and microsoft excel, and plotted using esri’s arcgis. in many of the interpreted data, a 10-year moving average is employed to reduce annual variability and highlight the longer term changes in the parameters measured. statistical analysis is performed to conf irm any possible trends from the dataset. results and discussion tropical cyclone frequency figure 1 shows tropical cyclone formation in the west pacif ic basin on an annual basis. evident in this plot is the increase in tcs recorded from the start of the dataset until around the 1970s wherein the highest total number of typhoons recorded was 61 in 1971; the 10-yr moving average in 1971 was 47.7 typhoons per year. the apparent 300% increase over the 86-year period is partly due to incomplete tropical cyclone reporting in the early years; reports were based on data dependent on the density of shipping vessels reporting such weather disturbances (knuttson and others 2010). higher ship density started in the 1960s and satellite-based reporting only became operational in 1966. starting in 1970, a decreasing trend spanning 43 years is recorded in the 10-year moving average. the current 10-year average stands at 28.4 typhoons per year. typhoon yolanda was the 34th of 35 tropical cyclones that formed in the west pacif ic basin in 2013. with seven more typhoons than the 10-year average, 2013 c.p. david and others 81 figure 1. number of tropical cyclones recorded annually in the west pacif ic basin. the black line is the 10-yr moving average. has the highest recorded number of tcs in the last 12 years and constitutes a 3 -year increasing trend starting in 2010. tropical cyclone intensity tropical cyclone intensity is measured via the maximum wind speed each tc system has attained during its lifetime. the ibtracs database has a record of historical wind speeds between the years 1977 and 2012. the measure of wind speed is based on the 10-minute maximum sustained winds, which is similar to pagasa’s signal system but different from the storm category system used in the united states. the latter is based on a 1-minute maximum sustained winds measurement. figure 2 shows the annual maximum wind data obtained from the ibtracs dataset. average annual maximum wind speeds do not show any def inite trend; if at all, there is a slight decrease in mean typhoon intensity in the 25th to 75th percentile of annual typhoons since 2007. typhoon yolanda’s 230kph matches 2010’s typhoon juan (international name: megi) wind speed. the 2013 maximum wind speed average falls within historical range despite recording f ive typhoons that exceeded 185kph maximum wind speeds (two made landfall in the philippines: odette and yolanda). a manifestation of climate change? 82 the ibtracs dataset is further analysed to determine monthly trends for typhoon intensity. table 1 shows the average number of signif icant typhoons (>150kph) that formed in the west pacif ic basin for each month per decade. august and september record the most number of signif icant typhoons and the number is increasing throughout the decades. signif icant typhoons in november and december show a decreasing trend. figure 2.maximum wind speed data. the dashed line shows the range of wind speeds per year, the box denotes the range of typhoons falling within the 25 th to 75th percentile (interquartile range) and the horizontal line inside the box represents the mean of the annual maximum wind speeds. table 1. average frequency of significant typhoons per decade (>150kph) c.p. david and others 83 location of formation the location of tropical cyclone formation will indirectly have a bearing on whether or not typhoons will make landfall in the philippines. with a general west-northwest typhoon track, the higher the formation latitude and further east longitude, the lower the chance of the typhoon passing by our country. the philippines is located at latitude 5pn to 20pn and longitude 117pe to 127pe. this latitude range is roughly the same range as the western pacif ic typhoon formation. figure 3a shows a def inite shift in annual mean latitude of formation from about 17pn to as low as 12pn in the mid-1990s. this is coupled with an increase in annual mean formation longitude (farther east from the philippines) from 125pe to 145pe (figure 3b). since then, however, a shift back to higher latitudes but closer longitude of formation is recorded in the last 18 years. this means that on average, typhoons have been more recently forming nearer the philippines but at a higher latitude equivalent to metro manila. this is despite the fact that signif icant typhoons within the recent past have originated from very near the equator. typhoon sendong (international name: washi) became a tropical depression at 6p north; typhoon pablo (international name: bopha), at 5p north; and typhoon yolanda, at 7pn of the equator. number and location of landfall figure 4a shows that annually a range of 8-76% of tcs that form in the west pacif ic basin make landfall in the philippines. the historical average is 30.3% with the highest recorded percentage happening in 1991 and culminating in the highest 10yr average of 37.7% in 1995. however, since then, this percentage has gone down to 28%, with 2013 recording only 31.4% of the tcs making landfall in the country. to determine whether typhoon tracks are changing through time, the number of tcs figures 3a and 3b. mean latitude and longitude of typhoon formation per year. a manifestation of climate change? 84 making landfall in northern luzon, southern luzon-bicol, and visayas-mindanao are plotted (figures 4b-d). northern luzon still accounts for most tcs making landfall (20.2% of all tcs formed), followed by v isayas-mindanao (9.8%) and southern luzon-bicol (8.4%). however, noticeable in these plots is the slight decrease in tcs entering luzon and bicol and increase in the percent of tcs entering v isayasmindanao, which is up by 0.8% to 10.6%. the peak occurred in the 1990s when 1215% of tcs passed by visayas-mindanao. the 2013 typhoon season recorded 7 of 11 tropical cyclones making landfall in visayas or mindanao. this also constitutes 20% of all tcs that formed in the west pacif ic basin, double the 10.6% average for the region. figures 4a-d. percent of tropical cyclones formed that made landfall in the philippines. the dark line shows the 10-year moving average for the dataset. conclusions there are evident tropical cyclone trends as shown by the analyses of the ibtracs database. these include: the decreasing number of tcs forming in the west pacif ic basin, the increase in latitude (and decrease in longitude) of mean tc formation, the decrease in the number of signif icant storms in november and december, and the increase in tcs entering visayas and mindanao. further analyses of the ibtracs c.p. david and others 85 database are already underway, including the separation of apparent linear trends relatable to climate change with possible inter annual cyclical occurrences such as the el nino southern oscillation. acknowledgment this study is funded through a research project of the philippine council for industry, energy and emerging technology research and development (pcieerd-dost) and a professorial chair award from the oscar m. lopez (oml) center for climate change. references e m o r i s , h a s e g a w a a , s u z u k i t, d a i r a k u k . 2 0 0 5 . va l i d a t i o n , p a r a m e t e r i z a t i o n d e p e n d e n c e , a n d f u t u r e p r o j e c t i o n o f d a i l y p r e c i p i t a t i o n s i m u l a t e d w i t h a h i g h r e s o l u t i o n a t m o s p h e r i c g c m . g e o p h y s . re s . le t t . 3 2 l 0 6 7 0 8 . d o i : 1 0 . 1 0 2 9 / 2004gl022306. [ipcc] intergovernmental panel on climate change. 2007. climate change 2007: the physical science basis. contribution of working group i to the fourth assessment repor t of the intergovernmental panel on climate change. solomon s, qin d, manning m, chen z, marquis m, averyt kb, tignor m, miller hl, editors. cambridge, uk and new york, usa: cambridge university press. [ipcc] intergovernmental panel on climate change. 2013. climate change 2013: the physical science basis. contribution of working group i to the fifth assessment repor t of the intergovernmental panel on climate change. stocker tf, qin d, plattner gk, tignor m, allen sk, boschung j, nauels a , xia y, bex v, midgley pm, editors. cambridge, uk and new york, usa: cambridge university press. in press. knapp kr, kruk mc, levinson dh, diamond hj, neumann cj. 2010. the international best track archive for climate stewardship (ibtracs): unifying tropical cyclone best track data. bull. amer. meteor. soc. 91: 363-376. knutson tr, mcbride jl, chan j, emmanuel k, holland g, landsea c, held i, kossin jp, srivastava ak, sugi m. 2010. tropical cyclones and climate change. nature geosci. 3: 157-163. doi 10.1038/ngeo779. [wmo] world meteorological organization. 2006. wmo international workshop on tropical cyclones statement on tropical cyclones and climate change. available from: http: //www.wmo.int/pages/prog/arep/tmrp/documents/iwtc_statement .pdf subscription form method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address _______________________________ _____________________________________________________ telephone no. _______________________________ ___________________________________________________ fax no. ________________________________________ please send accomplished subscription form to the rduo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city (02) 436-8720 fax (02) 927-2568  research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. 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lower hand corner, the name of the author and the f igure number should be indicated. 1 6 . illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2" x 11" size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: wellconstructed photo-graphic prints (not photocopies), trimmed at right angles and in the f inal size desired by the author. 1 7. when possible, all organisms must be identif ied by the scientif ic binomen. 1 8 . mathematical equations should be clearly presented so that they can be interpreted properly. 1 9 . obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 2 0 . all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc. , especially those adopted by the council of science editors (cse) scientif ic style and format, 7 th edition, 2006. 22. less common abbreviations may be printed as footnotes. 23. authors may opt to submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor-in-chief science diliman off ice of the vice-chancellor for research and development university of the philippines lower ground floor, phivolcs bldg. , c.p. garcia avenue up campus, diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. 2editor's note-july-dec.2017.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online irene m. v illaseñor, ph.d. editor-in-chief welcome again to science diliman! in this second issue of science diliman for 2017, we present four articles and one short communication with rather diverse topics. the layman’s abstracts will make the technical papers more comprehensible to non-specialists. in the f irst ar ticle, engineers tanguilig and danao studied the aerodynamic performance of a cut-out hollow pipe blade prof ile in small horizontal axis wind turbines, which have the potential to replace conventional blades with aerofoil prof iles. initial results show poor aerodynamic characteristics, and improvements thereof will be investigated again. according to the website of the department of health, schistosomiasis, caused by the schistosoma japonicum, is still endemic in 12 regions, affecting about 12 million filipinos. the intermediate host of this parasitic fluke is a snail, which is the object of morphometric and genetic studies by a group of doctors and biologists, with chua as principal author. preliminary results show the presence of four haplotypes of the snail. the paper by cabarrubias et al. shifts the topic from animals to plants, specif ically hibiscus. the group of agricultural scientists characterized 57 hibiscus hybrid progenies to select hybrids with unique morphological traits, such as the color and size of flowers, among others. another research on plants, specif ically herbal products, was conducted by a group of chemists. the quality and safety of herbal products are very impor tant, which make the research of de vera et al. on trace metal analysis timely. results show that most herbal products they tested contained trace metals that were below the maximum limits in herbal products. another paper on medicinal plants by singson and hernandez measured the ability of plants to inhibit the activity of angiotensin-converting enzyme, which is one mechanism to lower blood pressure. allow me to thank the authors and reviewers for their contributions in making science diliman a ched journal incubation grant recipient for 2017-2019, on top of being selected for coverage in the web of science emerging sources citation index. 9guidelines.pmd 85 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) 05_vicente 59 harmful algal bloom * corresponding author abstract science diliman (july-december 2002) 14:2, 59-65 harmful algal bloom in iligan bay, southern philippines helen j. vicente1*, ruth d. gaid1, henry e. dejarme1, elnor c. roa1, and rhodora v. azanza2 1mindanao state university at naawan, naawan, misamis oriental, 9023 philippines email: helena@eudoramail.com 2the marine science institute, college of science university of the philippines, diliman, quezon city we report the first occurrence of harmful algal bloom (hab) caused by a non-toxic dinoflagellate, cochlodinium sp. in philippine waters, particularly, in kalangahan pt.-manticao pt., iligan bay on march 13-18, 2002. two patches of cochlodinium sp. bloom, associated with fish kills in kalangahan pt.-manticao pt., iligan bay, caused localized water discoloration from the usual ocean blue to rusty brown or reddish brown to blackish. the first patch, located near fish-aggregating device (fad) areas, spanned 2 km wide, while the second patch, located near a fish corral, spanned 500 m wide. these patches occupied the water column from surface to 5 m depth, but a thick mat formed at 0.5 m to surface. patches decreased as the bloom began to decline. the observed dead demersal and pelagic fishes coincided with highest bloom density of 3.1 x 104 to 3.8 x 104 cells ml-1 of cochlodinium. dissected gills and stomach contents of fishes killed in hab-affected areas did not reveal any indication of clogging of gills by cochlodinium sp. fishes covered by the “shading effect” of cochlodinium bloom may have suffered anoxia or asphyxation due to oxygen depletion. no poisoning of people who consumed the dead fishes was reported. laboratory analyses revealed lower do values, 2.4 to 0.5 mg l-1 from 2400 to 0600 hr; 14n:1p ratio; air-water temperature ranged from 28-29 oc; ph 7.89-8.29; and salinity, 33-35o/oo. favella sp., a tintinnid grazer of dinoflagellate was developing in the area at the termination of the cochlodinium bloom on march 18. keywords: habs, discoloration, cochlodinium sp., fish kill, favella sp. introduction harmful algal blooms (habs) or “red tides” as known traditionally, refers to localized natural phenomena, which occur in polluted, warm sea, river, lake, or lagoon. discoloration of the water could be observed as red, reddish brown, rusty brown or chocolate, maroon, blue, and yellowish brown (taylor, 1987; smayda, 1997). this water discoloration happens when hab-causing organisms such as dinoflagellates, diatoms, or cyanobacteria reach a bloom density, i.e., cellular abundance of the causative species, that exceeds 103 cells ml-1 (sournia, 1995). based on the reported habs in other parts of the world, the bloom density ranges from 9 x 103 to 2.50 x 108 cells l-1 (or 9 to 250 x 103 cells ml-1) (holmes et al., 1967; tracey, 1988; sotto & young, 1995; bajarias & relox, 1996; wiadnyana et al., 1996). in certain areas in the asia pacific, most habs have been reported to occur due to eutrophication, favorable wind and weather conditions, 60 vicente et al. and the advent of enso (el niño southern oscillation) (maclean, 1989). yet the hab or “red tide” phenomenon is not completely understood. further research studies are still needed to understand the dynamics of habs (azanza & taylor, 2001). habs have negative impacts on health, economy, and ecology (maclean, 1989; corrales & gomez, 1990; smayda, 1997; azanza & taylor, 2001). in the asia-pacific region, four negative impacts of habs affecting fishery resources were recorded in 72 incidents in a span of 60 years. these are fish kills (53%), paralytic shellfish poisoning (42%), shrimp kills (4%), and ciguatera fish poisoning (1%) (corrales & maclean, 1995). mindanao state university (msu) naawan officials received a report on the occurrence of water discoloration in lugait and manticao municipalities in the province of misamis oriental from mr. aldin gabe, manager, semaña hatchery, on march 13-14, 2002. this was followed by a report of fish kill in some coastal towns of the province on march 14, 2002. acting on these reports, msu naawan immediately organized a research task force to investigate the phenomenon. methodology the monitoring of the alleged fish kill and water discoloration started with field interviews of ten (10) fishers, living along the coastal barangays of iligan bay, initially in kalangahan, biga, paitan, punta silum, poblacion manticao, and maputi (fig. 1). similar interviews of five (5) fishers were also conducted in tubajon and molugan, in adjacent barangays in the western side of macajalar bay, misamis oriental. the interviewees’ ages ranged from 29 to 56 years old, all male, and all engaged in fishing activities only. the collection of water samples was focused in lugait-punta silum-manticao coastal area, where the reddish-brown to blackish water covered a relatively large area (approximately 8 km span according to fishermen’s estimation on march 13, 2002). water was sampled using a nansen water sampler at 0, 1, 2, 3, 4, and 5 m depth in duplicate. additional surface water was sampled using 1l and 500 ml plastic bottles. water samples from nansen water sampler were placed in 1l and 500 ml plastic bottles to determine: (1) phytoplankton population density, by microscopic counting with a modified sedgwick-rafter counting chamber; (2) ph, by a pam 210 (copenhagen) ph meter; (3) salinity, by an atago s/mill (japan) refractometer; (4) nutrient analysis, by standard methods: cadmium reduction method for no 3 -n and stannous chloride method for po 4 -p (franson, 1995); and (5) air-water temperature readings, by a thermometer. do was likewise, measured using a wtw (germany) oximeter. gills and stomach of dead fishes were dissected and analyzed to determine the presence or absence of cochlodinium sp. on the corresponding organ tissues, which may indicate clogging of gills and filtering of cochlodinium sp. by fishes. results and discussion there was an observed localized discoloration of seawater in iligan bay, particularly in kalangahan pt. and manticao pt. on march 13-14, 2002. the discolored water formed two patches: one patch was observed bukidnon lanao del norte iligan bay marawi city cagayan de oro city macajalar bay misamis oriental 1 2 3 4 key map fig. 1. map of iligan bay showing harmful algal bloom sites (circular blackened areas along neritic and oceanic zones): (1) patch in fad area; (2) patch near fish corral area in lugait-manticao pts.; (3) patch in tubajon pt.; (4) patch in molugan pt. 61 harmful algal bloom about 2 km from the shoreline of manticao pt., where fads were located, and the other was observed 500 m from the shoreline near a fish corral (fig.1). the first patch spanned about 2 km wide while the other one was about 100 m wide. patches decreased as the bloom began to decline (table 1). the causative organisms of the bloom was found to be a dinoflagellate, cochlodinium sp. (fig. 2). this species is characterized by a large displacement of the median girdle and a spiral sulcus, with apical and antapical loops (kofoid & swezy, 1921; schiller, 1937; wood, 1968) (fig. 3). this cochlodinium species contracts date density (cells ml-1) location from shoreline (km) span/remarks (km) impact march 13 14 15 16 17 18 5 10 31,000 38,000 2,500 7,200 800 4,800 0 0 6* 4* 3** 2** 8* 2* 0.5** 0.1** fish kill table 1. cochlodinium sp. population densities during a harmful algal bloom (hab) occurrence in iligan bay. *fisherman’s estimation **msu-naawan estimation and extends its apical and antapical pellicular loops while swimming and feeding. the body is subovate to ellipsoidal, with a length of 36-54 µm. cochlodinium sp. hab transformed the color, from the usual ocean blue to rusty brown or reddish brown to blackish, of some patches in iligan bay. these patches were observed to occupy the water column from the surface to 5 m depth, but a thick mat formed at 0.5 m to surface. the thick 0.5 m to surface matting consequently formed “shading effect” on the pelagic and benthic fishes below it. in this ecological scenario, there could be a competition for do demand by fishes during the cochlodinium hab, and the dead fishes in localized hab span could have suffered anoxia due to oxygen depletion. oxygen depletion means that do value is below the asean marine water quality criteria value for the protection of aquatic life which is 4.0 mg l-1. denr (1990) critical do values ranged from 5.0 mg l-1. values below this range becomes unsuitable for most aquatic life. likewise, due to vertical migration of cochlodinium sp. at night till dawn to a depth of approximately 10 fathoms (16.62 m), as observed by fishermen spearfishing in tubajon pt., macajalar bay, the dinoflagellate could have been a competitor to do consumption of fishes in the area. fishermen recounted that corral fishes went out of their burrows and were weak and sluggish. however, no fish kills were observed in tubajon, unlike in the coastal areas of lugait, punta silum, manticao, and maputi, where dead demersal and pelagic fishes (table 2) were collected in the shoreline on march 14, 2002. dead fishes in these coastal areas were collected by fishermen and eaten broiled or raw. there was no reported poisoning of people who consumed the fish. fig. 2. (a) cochlodinium sp. (lm, enlarged from 100x = 220x; scale: 100x); (b) line drawing of the cell; (c) very motile cell arrows indicate cell furrows; (d) cell starting to feed; and (e) cell fully immobile and extended while feeding; (b) to (e) cells documented with a microscopemounted video camera. b c d e a 62 vicente et al. the observed fish kill in iligan bay coincided with highest bloom density of 3.1 x 104 to 3.8 x 104 cells ml-1 of cochlodinium sp. (table 1) on march 14, which is higher than the reported bloom density of 2,000 x 104 cells-l (or 2.0 x 104 cells ml-1) (holmes et al., 1967). fish kills are one of the consequences of habs (maclean, 1989; corrales & maclean, 1995). a similar incident of cochlodinium sp. bloom was observed in australia in 1989 during which mass wild fish kills was reported by sarjeant (1989). fishes covered by the “shading effect” of the cochlodinium bloom may have suffered anoxia or asphyxation (respiratory failure) due to oxygen depletion. cochlodinium was not reported as a toxic species (taylor, 1987; matsuoka & fukuyo, 2002). cochlodinium is a harmful, but not toxic, dinoflagellate. cochlodinium sp. is different from other dinoflagellates like gambierdiscus toxicus, which cause tropical fish contamination and inflect ciguatera fish poisoning to humans. those who consume the fish suffer from gastrointestinal (diarrhea, abdominal pains, nausea, and vomiting) or neurological symptoms (numbness and tingling of hands and feet; cold objects feel hot to the touch; difficulty in balance; low heart rate and blood pressure; rashes; and death, in extreme cases) (sarjeant, 1989; lewis & holmes, 1993). it is also different from the hab-causing organism, prorocentrum minimum, associated with the bolinao fish kills (fuertes, 2002). prorocentrum minimum was reported to cause diarrhetic shellfish poisoning in north sea-netherlands (kat, 1979). the cochlodinium-caused fish kills in iligan bay did not manifest any of the symptoms specified for humans consuming the dead fishes. based on interviews of fishermen in the mapalad, paitan, lugait, kalangahan, and manticao areas, residents ate the dead fishes on march 14, 2002 (table 2). none of them felt any of the symptoms of poisoning. in short, there was no deleterious health impact on humans. dissected gills and stomach contents of frozen fishes killed in the hab affected areas did not reveal any indication of clogging of gills by cochlodinium sp. however, mechanical damage leading to death of larvae of the american oyster, crassostrea virginica shellfish, which resulted from collisions (bumping) with cochlodinium heterolobatum bloom, was reported by ho & zubkoff (1979). the nutrient analyses revealed a mean of 2.678 (nitrate) and 0.190 mg l -1 (phosphate) (fig. 3), with nitrate:phosphate ratio of 14:1 comparable to the 16:1 (n:p) ratios of the pyrodinium bloom in zambales (corrales et al., 1995). these data indicate that the cochlodinium sp. bloom used up more nitrates in the discolorized water patches. air-water temperature fig. 3. nitrate-n and phosphate-p concentrations at 5 m to surface collected at 0900 hr at a bloom patch located 2 km from the shoreline. nitrate-n concentration (mg/l) 6 5 4 3 2 1 0 1 2 3 4 5 6 7 phosphate-p concentration (mg/l) d e p th ( m ) nitrate depth (m) phosphate legend: fig. 4. do monitoring of water samples from 1600 to 0600 hr at a bloom patch located at 2 km from the shoreline. 10 8 6 4 2 0 time d o c o n c e n tr a ti o n (m g /l ) 4: 00 p m 7: 00 p m 10 :0 0 p m 12 :0 0 p m 4: 00 a m 2: 00 p m 6: 00 a m 63 harmful algal bloom ranged from 28-29 oc; ph, 7.89-8.29; and salinity, 3335 o/oo. do measurement registered lower values, 2.4 to 0.5 mg l-1 (fig. 4), from 2400 to 0600 h. at the termination of the cochlodinium bloom on march 17, population of favella sp., a tintinnid grazer of dinoflagellate, was developing in the area. cochlodinium sp. was not reported to form cyst or hypnozygotes (sarjeant, 1974; taylor, 1987; matsuoka & fukuyo, 2000); thus, seed population for the recurrence of the species depends on its vegetative stage. so far, this paper is the first documented outbreak of cochlodinium sp. in philippine waters. recommendation and action plans 1. conduct of information drive on habs in mindanao; 2. establishment of fish kill “hotline” – zonal centers/ bfar; 3. immediate formation of fish-kill task force and fishkill contingency plan at the regional level; 4. adequate funding of monitoring and surveillance activities and establishment of facilities for environmental monitoring at the regional level; and, 5. review of policies on the illegal use of chemicals (which may add to the nutrient build-up in northern mindanao waters) in fishing. acknowledgments the authors wish to thank engr. joey semaña and mr. aldin gabe, who reported the occurrence and provided the hab samples for march 13 and 14 and the pump boat and operator during the collection of the samples on march 15, 2002. the authors also acknowledge the assistance of the chancellor of msu naawan, dr. marcelino i. tumanda jr.; dean warnita h. destajo; and jenis amarga for the support extended throughout the monitoring period. special thanks to mr. rey l. table 2. respiratory organs that could be affected due to clogging by filtering cochlodinium sp.*** municipalities/ local name of resources scientific name respiratory organs paitan & lugait lapulapu, pugapu molmol mamsa danggit tabogok (small octopus) kugita (big octopus) kalangahan bolinao uburan barungoy swasid maputi ba-at epinephelus sp. scarus sp. caranx sp. siganus sp. stolephorus sp. muraenesox sp. cyoselurus sp. hemiramphus sp. holothuria gills gills gills gills high oxygen requirement provided by one pair of gills without cilia; water exchange by radial muscles in mantle wall and expel water forcibly through a funnel. gills gills gills gills respiratory tree (analogous “gill rakers” in fishes) for respiration and excretion. gas exchange in skin and tube feet. ***based on interviews. likewise, the fishermen in the municipalities of tubajon and molugan observed weak fishes. dead fishes were not observed by fishermen in macajalar bay on march 11,13, and 16 when harmful algal bloom (hab) occurred in these areas. 64 vicente et al. roa for the video and photomicrograph of the specimens. references azanza, r.v. & f.j.r. taylor, 2001. are pyrodinium blooms in the southeast asian region recurring and spreading? a view at the end of the millennium. ambio. 30(6): 356-364. bajarias, f.f.a. & j.r. relox, 1996. hydrological and climatological parameters associated with the pyrodinium blooms in manila bay, philippines. in yasumoto, t., y. oshima, & y. fukuyo (eds.). harmful and toxic algal blooms. paris, ioc-unesco: 49-52. corrales, r.a. & e.d. gomez, 1990. red tide outbreaks and their management in the philippines. in granelli, e., et al., (eds.) toxic marine phytoplankton. new york, elsevier: 453458. corrales, r.a. & j.l. maclean, 1995. impacts of harmful algae on sea-farming in the asia-pacific areas. j. appl. phycol. 7: 151-162. corrales, r.a., m. reyes, & m. martin, 1995. notes on the encystment and excystment of pyrodinium bahamense var. compressum in vitro. in lassus, p., et al., (eds.) harmful marine algal blooms. paris, lavoisier science publ.: 573-578. denr administrative order no. 34 series, 1990. revised water usage and classification of water quality criteria amending section nos. 68 & 69. chapter iii of the 1978 npcc rules and regulations, manila, philippines: 15 pp. franson, m.a.h., a.d. eaton, l.s. clesceri, & a.e. greenberg (eds.), 1995. standard methods for the examination of water and wastewater. american public health association, american water works association and environmental federation. fuertes, y., 2002. up agency proposes suspension of bolinao fish pen operations. philippine daily inquirer, february 7, 2002: a10. ho, m.s. & p.l. zubkoff, 1979. the effects of cochlodinium heterolobatum bloom on the survival and calcium uptake by larvae of the american oyster, crasostrea virginica. in taylor, d.l. & h.h. seliger (eds.) toxic dinoflagellate blooms. north holland, elsevier: 409-412. holmes, r.w., p.m. williams, & r.w. eppley, 1967. red water in la jolla bay, 1964-1966. limnol. oceanogr. 12: 503-512. kat, m., 1979. the occurrence of prorocentrum species and coincidental gastrointestinal illness of mussel consumers. in taylor, d.l. & h.h. seliger (eds.) toxic dinoflagellate blooms. north holland, elsevier: 215-220. kofoid, c.a. & o. swezy, 1921. the free-living unarmored dinoflagellata. mem. univ. calif. 5: 1-562. lewis, r.j. & m.j. holmes, 1993. origin and transfer of toxins involved in ciguatera. comp. biochem. physiol. 106c(3): 615628. maclean, j.l., 1989. indo-pacific red tides, 1985-1988. mar. pollut. bull. 20: 304-310. matsuoka, k. & y. fukuyo, 2000. technical guide for modern dinoflagellate cyst study. westpac-hab/wespac ioc. sarjeant, w.a.s., 1974. fossil and living dinoflagellates. academic press, london: 182 pp. sarjeant, w.a.s., 1989. what are algae? micropaleontology. 35(2): 1-9. schiller, j., 1937. dinoflagellata (peridineae) in monographischer behandlung. acad. verlages, leipzig: 1590. smayda, t.j., 1997. harmful algal blooms: their ecophysiology and general relevance to phytoplankton blooms in the sea. limnol. oceanogr. 42(5): 1137-1153. sournia, a., 1995. red-tide and toxic marine phytoplankton of the world ocean: an inquiry into biodiversity. in lassus, p., et al., (eds.) harmful marine algal blooms. technique et documentation-lavoisier, intercept ltd: 103-112. sotto, f.b. & j.g. young, 1995. pyrodinium bahamense var. compressum cells in the visayan sea, philippines: transport by water currents. in lassus, p. et al., (eds.) harmful marine algal blooms. technique et documentation-lavoisier, intercept ltd: 243-247. 65 harmful algal bloom taylor, f.j.r., 1987. the biology of dinoflagellates. botanical monographs. london, blackwell scientific publications. tracey, g., 1988. feeding reduction, reproduction failure, and mortality of mytilus edulis during the 1985 “brown tide” in naragansett bay, rhode island. mar. ecol. prog. ser. 50: 7381. wiadnyana, n.n., t. sidabutar, k. matsuoka, t. oshi, m. kodama, & y. fukuyo, 1996. note on the occurrence of pyrodinium bahamense in eastern indonesian waters. in yasumoto, t., y. oshima, & y. fukuyo (eds.) harmful and toxic algal blooms. intergovernmental oceanographic commission of unesco: 53-56. wood, e.j., 1968. dinoflagellates of the caribbean sea and adjacent areas. florida, university of miami press: 399 pp. sd inside back cover-ched.pmd 1 s c i e n c e d i l i m a n : a p h i l i p p i n e j o u r n a l o f p u r e a n d a p p l i e d s c i e n c e s i s a c c r e d i t e d b y t h e commission on higher education ( c h e d ) t h r o u g h i t s j o u r n a l accreditation service project, as category a-2 journal from 2014 to 2016 and is available via www.doaj. org and www.ebsco.com. 3layman'sabstracts.pmd layman’s abstracts 1 science diliman (july-december 2016) 28:2, 1-3 layman’s abstracts benthic macroinver tebrate community as an ind icator of stream health: the effects of land use on stream benthic macroinver tebrates danielle dominique d. deborde, maria brenda m. hernandez and francis s. magbanua issn 0115-7809 print / issn 2012-0818 online biological monitoring of stream health in the tropical countries still uses standard water chemistry methods, which require expensive measuring tools. in this study, the use of benthic macroinver tebrates as biological i n d i c a t o r s o f f r e s h w a t e r s t r e a m s w a s c a r r i e d o u t . t h e s t u d y a l s o a t t e m p t e d t o d e t e r m i n e t h e a p p l i c a b i l i t y o f d i f f e r e n t b i o t i c i n d i c e s (numerical scores for stream health evaluation) in characterizing sites across land use type. benthic macroinvertebrate samples were obtained from nine streams in silago, southern leyte and were identif ied up to f a m i l y l eve l . ave r a g e to l e r a n ce s co r e p e r taxo n ( at s p t ) w a s t h e o n l y b i o t i c i n d e x t o s u c c e s s f u l l y d i f f e r e n t i a t e t h e w a t e r q u a l i t y o f n i n e s t r e a m s b a s e d o n l a n d u s e , w h i c h r a n g e d f r o m m o d e r a t e l y p o o r t o excellent. forested sites achieved the lowest atspt score, whereas mixed f o r e s ted a g r i c u l t u r a l s i t e s h a d t h e h i g h e s t at s pt s co r e s . t h e r e s u l t s establish that benthic macroinvertebrates can be used as biomonitoring tool in evaluating the ecological integrity of waterways in the country. layman’s abstracts 2 detailed calculation of the average work done in a ground state quench of the quantum ising model salvador t. laurente jr. and francis n.c. paraan work is done on the ising model of a quantum magnet when an external m a g n e t i c f i e l d i s c h a n g e d i n s t a n t a n e o u s l y. ve r t i c a l t a n g e n t s a n d inflection points appear in graphs of the work done versus the initial f ield. these singular features arise from an underlying zero-temperature p h a s e t r a n s i t i o n b e t w e e n a f e r r o m a g n e t i c o r d e r e d p h a s e a n d a p a r a m a g n e t i c d i s o r d e r e d p h a s e . le t a n i n t e g e r n ≥ 2 b e g i v e n . le t r n = r n ∪ { ∞ } b e t h e ex t e n d e d euclidean space. a möbius transformation is a function from rn onto rn t h a t t r a n s f o r m s s p h e r e s a n d h y p e r p l a n e s t o e i t h e r s p h e r e s o r hyperplanes. of particular interest are the möbius transformations that map the unit sphere sn-1 onto a hyperplane that is orthogonal to a given u n i t vec to r. t h e r e s t r i c t i o n of s u c h a m ö b i u s t r a n s fo r m a t i o n to s n -1 i s called a stereographic projection. let ∏ : s n-1 → r n-1 be a stereographic p r o j e c t i o n . i t i s k n o w n ( d u e t o l a r s a h l f o r s ) t h a t a n y m ö b i u s t r a n s f o r m a t i o n m a y b e e x p r e s s e d a s a p s e u d o l i n e a r f r a c t i o n a l t r a n s f o r m a t i o n d e f i n ed by a 2 b y 2 va h l e n m a t r i x w i t h e n t r i e s i n a clifford algebra. conjugating by ∏ , we embed the real orthogonal group o(n) into a projective group of vahlen matrices. we also provide results on eigenvalues of 2-by-2 matrices with entries in a clifford algebra. the real orthogonal group and vahlen matrices bimal kunwor, dennis i. merino, edgar n. reyes and gary a. walls         layman’s abstracts 3 potential cholesterol-lowering activity of selected plant extracts raff i r. isah and christine l. chichioco-hernandez β-hydroxy-β-methylglutaryl-coenzyme a reductase (hmg-coa reductase) catalyzes the nadph-dependent reduction of hmg-coa to mevalonate. this reaction is the rate-limiting step in cholesterol synthesis. inhibition of hmg-coa reductase helps in the reduction of the high levels of plasma cholesterol, which is a primary risk factor in coronary artery diseases, s u c h a s a t h e r o s c l e r o s i s . t h i s s t u d y e x a m i n e d t h e h m g c o a r e d u c t a s e inhibitory activities of f ive philippine plants, namely pouteria campechiana (kunth) baehni, barringtonia asiatica (l.), vitex parviflora a . juss. , antidesma bunius (l.) spreng. , and diospyros blancoi a .dc. the hmg-coa reductase inhibitory activities of the plants were determined spectrophotometrically at 340 nm, utilizing the oxidation of nadph and with hmg-coa as the substrate. pouteria campechiana, a. bunius, and d. blancoi exhibited percent inhibition values of more than 70% among the f ive methanol extracts. the last two plants were further partitioned, and their respective hexane, ethyl acetate, and aqueous extracts displayed more than 80% inhibition. keywords: β-hydroxy-β-methylglutaryl-coenzyme a reductase, medicinal plants, plant extracts p a r a b o l i c p a r t i a l d i f f e r e n t i a l e q u a t i o n s ( p d e s ) a r e w i d e l y k n o w n f o r modeling physical phenomena. they have many applications, especially i n t h e a r e a s o f s c i e n ce a n d e n g i n e e r i n g . i n t h i s p a p e r, w e s t u d y a parabolic pde which models heat transfer in a perforated cell. in our system, the thermal conductivity represented by a matrix is dependent on the temperature f ield and the temperature itself. we impose a cer tain co n d i t i o n fo r t h e h e a t f l u x o n t h e b o u n d a r y, a s we l l a s s o m e i n i t i a l c o n d i t i o n . w e s h o w t h e e x i s t e n c e o f a s o l u t i o n t o o u r p r o b l e m i n a n a p p r o p r i a t e f u n c t i o n a l s p a c e . w e a l s o p r o v e t h a t t h e s o l u t i o n w e obtained is unique. on the solvabil ity of a quasil inear parabol ic problem with neumann boundary cond ition mylen l. aala, ed itha c. jose and marian p. roque 10call for papers-new.pmd social science diliman, vol. 9, number 1, january-june 2013 humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) vargas museum & angelo joshua a. victoria call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development the scientific basis of marine fish farm regulation kenneth d. black1* and christopher j. cromey2 1scottish association for marine science dunstaffnage marine laboratory oban, argyll pa37 1aq, scotland, uk *corresponding author, email: kenny.black@sams.ac.uk 1tel. +44 1631 559259, fax +44 1631 559001, 2tel. +44 1631 559266 date submitted: august 21, 2007; date accepted: august 21, 2008 abstract as aquaculture expands, regulation to prevent environmental damage is an essential requirement for sustainability. in this paper, we discuss three aspects of aquaculture regulation pertaining to 1) protection of other resource users, 2) protection of ecosystem structure (conservation), and 3) protection of ecosystem function (recycling). some of the approaches taken to regulation of aquaculture in several countries are presented, emphasizing the need for these to be based firmly in a good scientific understanding of the ecosystem and the processes by which it interacts with aquaculture. introduction marine fin-fish aquaculture continues to expand, although expansion has to some extent, plateaued in several of the developed countries, where modern intensive aquaculture was pioneered. for example, in scotland, production increased steadily reaching a peak in 2003, but has since been variable (fig. 1). there are several reasons for this, including disease and market conditions, but one reason has been constraints placed on farmers by planners and regulators. planners must ensure that aquaculture developments meet aesthetic, social and economic criteria, and that there is harmonization between new developments and local infrastructure capacity or other resource use e.g., tourism. planners and regulators have duties to ensure that developments do not adversely affect the environment. the objectives of regulation can be separated into three areas: 1. protection of legitimate users of the environment, such as tourists or fishermen, such that resources are fairly distributed; 2. protection of the environment for its biological structure including protection of important/rare habitats and species; and 3. protection of ecosystem functions such as the recycling of nutrients and the maintenance of oxygen levels. the first of these is the subject of the evolving "discipline" of integrated coastal zone management (iczm) which has eight broad principles (defra, 2006). it is worth presenting these here in full: a. a broad holistic approach the objective of a holistic approach is to forego piecemeal management and decision making in favor of a more strategic approach, which looks at the 'bigger picture', including cumulative causes and science diliman 11 black, k. & cromey, c. effects. this means considering the conservation value of natural systems alongside the human activities which take place on land and coastal waters. taking a holistic approach will also involve looking at the problems and issues on the coast in the widest possible context, including looking at the marine and terrestrial components of the coastal zone and considering how different issues conflict or interact together. b. taking a long-term perspective successful coastal management must consider the needs of present and future generations. therefore, administrative structures and policies required to manage the environmental, social, and economic impacts now, must also be adaptable to take account of, and acknowledge, uncertainties in the future. c. adaptive management the coastline has been subject to constant physical and economic changes over the years, and the management of such a dynamic environment requires measures which are able to adapt and evolve accordingly. successful management should reflect this principle by working towards solutions which can be monitored effectively. d. specific solutions and flexible measures coastal management measures for each stretch of coast must reflect and accommodate the many variations in the topography, biodiversity, and local decision-making structures. integrated management should therefore be rooted in a thorough understanding of the specific characteristics of an area, i.e., its local specificity. e . working with natural processes the natural processes of coastal systems are continual, so it becomes necessary in some instances to adopt a different approach that works with natural processes rather than against them. by recognising the physical impacts and the limits imposed by natural processes, decisions regarding the human impact on the coastal zone are made in a more responsible manner and are more likely to respond to environmental change. f. participatory planning in the past, stakeholders may not have had sufficient opportunity to contribute towards the development and implementation of coastal management measures or programmes. participatory planning incorporates the views of all of the relevant stakeholders (including maritime interests, recreational users, and fishing communities) into the planning process. it can also help to promote a real sense of shared responsibility and coastal stewardship by reducing conflict as real issues, information, and activities which affect the coast can be aired more openly. g. support and involvement of all relevant administrative bodies administrative policies, programmes and plans (land use, spatial, energy, tourism and regional development, for example) set the context for the management of coastal areas and their natural and historical resources. addressing the problems faced by coastal zones will therefore require the support and involvement of all relevant administrative bodies at all levels of government to ensure cooperation, coordination and that commons goals are achieved. it is therefore essential to engage key bodies from the start so that decisions are consistent and firmly based on local circumstances. h. use of a combination of instruments 12 science diliman figure 1. scottish annual salmon production, in tons (frs, 2006). 0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000 180,000 1 98 6 1 9 87 1 98 8 1 98 9 1 99 0 1 99 1 1 99 2 1 99 3 1 99 4 19 9 5 1 99 6 19 9 7 19 98 1 99 9 20 00 20 01 20 0 2 20 03 2 0 04 20 05 2 0 06 the scientific basis for marine fish farm regulation managing the different activities which take place on the coast requires the use of a number of different policies, laws, and voluntary agreements. while each of these approaches is important, achieving the right combination is key to resolving conflicts, as these instruments should work together to achieve coherent objectives for the planning and sustainable management of coastal areas. the second objective, the protection of ecosystem structure, may be intimately linked to the third, protecting ecosystem function, especially where the structure of habitats have dominant functional roles. for example, mangroves have been shown to have key functional roles in flood protection, nutrient recycling and as nursery areas (holmer, 2003; primavera, 1998; primavera, 2005). however, habitats may be deemed worthy of protection when their precise contribution to ecosystem function is unknown but they are considered to be rare or have rare species assemblages, e.g., cold water corals and moves to protect them from trawling damage (roberts et al., 2006). interactions between aquaculture and sensitive habitats or species can be minimized by establishing aquaculture zones in areas with less sensitive/important/rare habitats, or by designations that more closely regulate developments with respect to their interactions with particular features of concern. in europe, special areas of conservation (sacs) have been established under the habitats directive (92/43/eec) for the protection of specific habitats. as this is an important piece of legislation, with wide ranging impact, it is worth exploring this further. the uk joint nature conservation council gives the following background on its website (www.jncc.gov.uk). "in 1992 the european community adopted council directive 92/43/eec on the conservation of natural habitats and of wild fauna and flora (ec habitats directive). this is the means by which the community meets its obligations as a signatory of the convention on the conservation of european wildlife and natural habitats (bern convention). the provisions of the directive require member states to introduce a range of measures including the protection of species listed in the annexes; to undertake surveillance of habitats and species and produce a report every six years on the implementation of the directive. the 189 habitats listed in annex i of the directive and the 788 species listed in annex ii, are to be protected by means of a network of sites. each member state is required to prepare and propose a national list of sites for evaluation in order to form a european network of sites of community importance (scis). once adopted, these are designated by member states as special areas of conservation (sacs), and along with special protection areas (spas) classified under the ec birds directive, form a network of protected areas known as natura 2000. the directive was amended in 1997 by a technical adaptation directive. the annexes were further amended by the environment chapter of the treaty of accession 2003. the habitats directive introduces for the first time for protected areas, the precautionary principle; that is that projects can only be permitted having ascertained no adverse effect on the integrity of the site. projects may still be permitted if there are no alternatives, and there are imperative reasons of overriding public interest. in such cases compensation measures will be necessary to ensure the overall integrity of network of sites." in loch creran on the west of scotland, for example, an sac has been implemented to protect the unique reefs of the tube building polychaete serpula vermicularis and reef forming horse mussels modiolus modiolus. several management principles are invoked in marine sacs (www.argyllmarinesac.org): • management should enable the natural habitat types and the species habitats concerned to be maintained or, where appropriate, restored at a favorable conservation status. • steps must be taken to avoid deterioration or disturbance of the habitats and species for which the site has been designated. • activities, plans or projects likely to have a significant effect upon the features of the site must be subject to an appropriate assessment. a development that would have an adverse effect on the conservation interests of the site should only be permitted science diliman 13 black, k. & cromey, c. if there is no alternative solution and there are imperative reasons of overriding public interest, including those of a social or economic nature. • monitoring must be undertaken at each site to monitor the condition of the conservation features and to assess the effectiveness of management measures. • management of the site must take into account the economic, social, cultural and recreational needs of the local people. loch creran was one of the original sites in scotland developed for fish farming with continuing major aquaculture activity both from shellfish and salmon farming. however, any proposed change to the aquaculture activity must be assessed against these principles. in practice, the operator has found it possible to continue to farm in loch creran, despite the sac, by being able to show that any proposed changes will have little impact on the conservation feature. in general, the science of interactions between sensitive habitats and aquaculture is not well developed, and it is only where catastrophic impacts occur on relatively visible habitats, such as mangroves or seagrasses (holmer et al., 2003; orth et al., 2006), that research effort is focused. thus for many habitats, neither the precise functional significance nor the sensitivity to aquaculture is known with any certainty and regulation is more problematic. in addition to loss of habitats, reduction in genetic diversity can also be regarded as having a structural component that may or may not also have a functional aspect. for example, the release of genes, disease organisms and parasites for salmon culture and their effects on wild populations have received considerable attention. wild atlantic salmon maintain their high degree of population structure by homing to natal rivers for breeding. the resulting genetic diversity has been shown to be important in terms of fitness and can be severely eroded by interbreeding with escaped cultured salmon or from intentional translocations of fish with maladapted genes (mcginnity et al., 2003). modeling work has shown that, under high rates of intrusion of cultured fish, significant changes to populations will occur that may not recover even if the intrusion rate is reduced (hindar et al., 2006). for marine species, such as atlantic cod and european sea bass, the consequences of genetic interactions on populations are not well established. the potential interactions between parasitic sea lice on wild and cultured populations have also received considerable attention and publicity. salmon host ectoparasitic parasitic sea lice lepeophtheirus salmonis and caligis elongatus which feed on the skin and underlying tissue. in salmon culture these can increase rapidly to high abundance if left untreated nd infestations can result in severe legions and mortality. sea lice larvae are adept at find hosts and increasing infestations on juvenile atlantic salmon and sea trout salmo trutta were blamed on infection pressure from salmon farms. this in turn was linked to reduced survival and population declines of these salmonids. while causal links are extremely difficult to prove (mcvicar, 1997), there seems little doubt that sea lice from farmed salmon can (murray & gillibrand, 2006) and do (bjorn et al., 2001) infect wild salmonids cause mortality, particularly to sea trout. different approaches have been taken to regulate escapes and parasites in different countries. in scotland, the government now require statutory declaration of escapes including the approximate number and size of fish, the location and date of escape (scottish statutory instrument 2002 no. 193, the registration of fish farming and shellfish farming businesses amendment (scotland) order 2002). in the planning process, developments of farms near important salmon rivers is discouraged, and farmers are obliged to show how they have taken steps to minimize escapes and improve containment. in scotland, there is no statutory limit at present for the average lice infestation of salmon that is permitted before treatment with antiparasitic medicines, although there are several voluntary agreements in place between the fish farming sector and the fisheries sector (area management agreements). this is in contrast to norway, where when statutory lice limits are exceeded, treatment is compulsory (heuch & mo, 2001). the situation is likely to change in scotland as new legislation, aimed at ensuring farmers keep lice levels low, is enacted (aquaculture and freshwater fisheries bill). this will require fish farmers to record levels of parasites and to ensure that burdens are kept low, but the precise mechanism for achieving this has yet to be announced. 14 science diliman the scientific basis for marine fish farm regulation the state of knowledge of the structural effects of fish farming and their sound regulation is rather weak. in contrast, the third objective of regulation, to protect ecosystem function from the consequences of aquaculture, is often better understood and quantified. we now briefly review the current state of knowledge for the main environmental outputs from aquaculture, focusing on marine fish farming, and for each of these we comment on issues related to regulation. in general, the environmental interactions posed by intensive marine shellfish culture are fewer than for fin fish culture owing to the fact that shellfish are net extractive of nutrients. however, they do concentrate organic material and deposit wastes as faeces and pseudofaeces, causing enrichment of the local benthos (chamberlain et al., 2001) and, if cultured in sufficiently high density in some areas, can clear the water to such an extent that they reduce productivity (smaal et al., 2001). regulation on shellfish farming is, however, less well developed than for marine finfish farming, owing to its lower perceived environmental risk. wastes from fish farming are usually considered in 3 overlapping categories: science diliman 15 figure 2. a budget for the flow of nutrients from oceanic wild caught fish to the coastal environment for a harvest of 1kg of farmed salmon assuming no substitution with vegetable protein or oil and a ratio of fish feed o product of 1.2:1 (black, 2001). wild fish 17% protein 7-10% oil 75% water fish feed 40% protein 30% oil 9% water 1200g: n 96g p 18g c 660g particulate wastes n 22g p 9.5g c 185g soluble wastes n 46g p 4.9g c 323g harvest fish 1kg n 26g p 3.2g c 139g mortalities & escapes n 1.9g p 0.4g c 13g 2.8kg for protein plus 0.8-2.3kg extra for oil black, k. & cromey, c. 1. soluble wastes, being the products of fish excretion and including reactive nitrogen species such as ammonia; 2. solid wastes, being mainly uneaten feed material and faeces; and 3. chemical wastes, being medicines such as antibiotics and antiparasitics, disinfectants, and antifoulants such as tin or copper compounds formulated into coatings or paints. soluble wastes soluble nutrients from aquaculture may constitute a risk of eutrophication. the eu definition of eutrophication is: "the enrichment of water by nutrients especially compounds of nitrogen and phosphorus, causing an accelerated growth of algae and higher forms of plant life to produce an undesirable disturbance to the balance of organisms and the quality of the water concerned". the undesirable consequences of eutrophication include (black et al., 2002): • increased abundance of micro-algae, perhaps sufficient to discolor the sea and be recognized as a bloom or "red tide"; • foaming of seawater; • killing of free-living or farmed fish, or seabed animals • poisoning of shellfish; • changes in marine food chains; and • removal of oxygen from deep water and sediments as a consequence of the sinking and decay of blooming algae. a nutrient budget for atlantic salmon shows that the majority of the nutrients that are input to the system are subsequently lost either directly as soluble wastes or through losses from solid particulate wastes (fig. 2). one method of determining the risk of elevated nutrient concentrations is called the equilibrium concentration enhancement (ece) model. the ece model is a box model of dissolved ammoniacal nitrogen arising from farmed fish occurring within an enclosed body of water of known dimensions that is being exchanged at a steady rate (gillibrand & turrell, 1997). model input data include the flushing time and volume of the system, rate of excretion of ammonia by fish, and annual production. using this model, systems can be ranked in terms of the potential contribution of aquaculture to raise nutrient levels and assigned a nutrient enhancement index (nui). in scotland, this index was proposed in a government document called the locational guidelines for fish farming (gillibrand et al., 2002). table 1 shows the predicted ece for scottish sea loch systems, their nui and the distribution of lochs by nui. ece(μmol/l) no. of scottish sea lochs nutrient enhancement index >10.0 5 (4.5%) 5 3.0-10.0 15 (13.5%) 4 1.0-3.0 23 (20.7%) 3 0.3-1.0 22 (19.8%) 2 <0.3 46 (41.4%) 1 total 111 table 1. ece values for nitrogen for scottish sea lochs and classification using the nutrient enhancement index (gillibrand et al., 2002). for an ece of 0 (i.e. where no emission from aquaculture exist) a value of 0 is assigned. more complex models exist, where the biological response from nutrient additions is predicted in terms of phytoplankton biomass or chlorophyll. one such model, currently being adapted for an aquaculture (laurent et al., 2006) was initially developed for predictions of the eutrophication potential of nutrient discharges from sewerage marine outfalls. this model, originally called the csst model, has been described by tett et al. (2003). such models are capable of being applied at a range of scales and in non-enclosed water bodies, provided appropriate boundary conditions are known, can be measured, or can be predicted from larger scale models. absolute standards for what constitutes eutrophication are not available. what constitutes a harmful disturbance to the ecosystem will vary according to the normal seasonal cycle which may be very different in temperate, subtropical and tropical regions. thus to assess the acceptable level of phytoplankton biomass will require some knowledge of annual nutrient cycles and the primary production response for different 16 science diliman the scientific basis for marine fish farm regulation environments and such measurements have often not been made. an alternative is to look for a deleterious ecosystem response such as the hypoxia caused by enhanced carbon inputs to deeper waters and sediments. hypoxia may constitute a threat not only to the ecosystem but to the farmed fish themselves. solid wastes the major solid wastes from fish farming are from uneaten feed and faecal material. the quantity of both these components will vary by species, food conversion ratio, food digestibility, and the skill of the operator in matching feed availability to demand. benthic macrofaunal communities in sediments receiving normal detrital inputs derived from planktonic production in the overlying water column are species rich, have a relatively low total abundance/species richness ratio, and include a wide range of higher taxa, body sizes, and functional types, i.e. they are highly diverse communities (pearson, 1992). the total productivity of the system is dependent on the availability of food organic matter, and its quality. animals have evolved to maximize the utilization of the available resource by virtue of a wide range of feeding modes and some species can vary their mode of feeding depending on environmental factors. benthic types include filter feeders that gather detrital material from the water column above the sediment, surface deposit feeders that feed on material deposited on the sediment surface, sub-surface deposit feeders that consume buried organic material by burrowing, and carnivores that prey on other macrofauna. microbes degrade organic material and are themselves consumed by macrofauna, mediating the transfer of nutrients up the food chain. a variety of terminal electron acceptors are used by different bacterial communities in marine sediments. the oxygen concentration at any point in the sediment is dependent on the rate of its uptake, either to fuel aerobic metabolism, or to re-oxidize reduced products released from deeper in the sediment. when the oxygen demand caused by input of organic matter exceeds the oxygen diffusion rate from overlying waters, sediments become anoxic and anaerobic processes dominate. as sediments become more reducing with increasing distance from the water column interface, a range of microbiological processes become successively dominant in the order: • aerobic respiration, ammonium oxidation (to nitrite) and nitrite oxidation (to nitrate). these aerobic nitrifying processes are inhibited by sulphide and are, therefore, of limited importance in sediments beneath marine fish farms; • denitrification (producing dinitrogen from nitrate); • nitrate reduction (producing ammonium from nitrate) and manganese reduction; • iron reduction; • sulphate reduction (producing hydrogen sulphide); • and lastly, under the most reducing conditions, methanogenesis (producing methane). to some extent, these processes may overlap spatially. oxygen in sediment porewaters is rapidly depleted and sulphides are generated by sulphate reduction, which is the dominant anaerobic process in coastal sediments (holmer & kristensen, 1992). these effects on sediment biogeochemical processes have profound consequences for the seafloor fauna that becomes dominated by a few small, opportunistic species, often at very high abundances, and confined to the upper few centimetres of the sediment (brooks & mahnken, 2003a; brooks et al., 2003a; brooks et al., 2003b; hargrave et al., 1997; heilskov & holmer, 2001; holmer et al., 2005; karakassis et al., 1999; pearson & black, 2001; pearson & rosenberg, 1978; weston, 1990). away from the farm, as organic material flux and oxygen demand decreases, animal communities return to background conditions typified by high species diversity and functionality (gowen & bradbury, 1987; nickell et al., 2003; pereira et al., 2004). the redox potential (eh) profile measured the sediment column to a depth of 10-15 cm gives a useful guide to the relative degree of carbon enrichment in the sediments (pearson & stanley, 1979). positive eh values are indicative of aerobic science diliman 17 black, k. & cromey, c. conditions whereas negative values are associated with anaerobic microbial processes. under normal rates of detrital carbon input to sediments, the redox discontinuity level (rdl), i.e., the point at which anaerobic processes become predominant, lies some centimetres below the surface. as carbon inputs increase, the rdl approaches ever closer to the surface as the bod (biological oxygen demand) within the sediments increases. eventually, under very high detrital inputs, the rdl coincides with the sediment/water interface, where, under low flow conditions, it might even rise into the water column. it is important to emphasize that highly organically enriched sediments can occur naturally from large marine or terrestrial inputs of detritus. this may be transient and localized or long-lived and wide scale. hypoxia/anoxia in sediments and overlying water occurs when the supply of new oxygenated water is poor as may be the case, for example, in deep silted fjordic systems. in such systems, benthic communities are modified and specialist opportunist animals may dominate. the process of organic particulate material impacting the seabed and causing benthic effects is amenable to modeling (cromey & black, 2005; cromey et al., 2002a; cromey et al., 2002b; silvert & cromey, 2001; silvert & sowles, 1996). a widely used model is that of cromey et al. (2002a) depomod. the outputs of this model are in terms of accumulated carbon per unit area of sea bed per unit time the word accumulation is used here as resuspension processes are accommodated in the model. the output is visualized as a contour plot of carbon deposition on a spatial grid (an example is shown in fig. 3). in scotland, as in several other countries, the regulator (scottish environment protection agency, sepa) is required to manage the impacts of fish farming to avoid unacceptable damage of the sea bed and its fauna. sepa have gone a little further than most regulators in giving some examples of where they think the boundary between acceptable and unacceptable seabed conditions lies. they have established sediment quality criteria (sqc, table 2) as indicators of when they will take action in order to reduce impacts, e.g., by reducing the maximum allowable biomass or by entirely revoking the discharge consent. the sqc are not the only criteria used sepa will accept and consider all the available evidence but as many benthic indicators co-vary, they do offer a meaningful insight into what sepa considers to be unacceptable benthic conditions. discharge consents have monitoring conditions specified in detail: both their level (i.e., the number of stations, types of measurement and analysis) and their frequency are matched to the perceived risk of the farm. for example, a small farm over hard sediment with strong currents will be monitored less intensively than a large farm over a soft substrate with weak currents. this process is given in great detail, together with its underlying philosophy and science, in the regularly-updated fish farm manual that can be downloaded from the sepa website (www.sepa.org.uk). the sqc (or action levels, table 2) are levels at which sepa may take action against the farmer, i.e., reduce or remove the consent to discharge. implicit within the approach are: (a) that the farmer is required to monitor the sediments around the farm to measure compliance or otherwise, and (b) the concept of the allowable zone of effects (aze). 18 science diliman figure 3. example of a depomod particle tracking model output. cages are represented by squares; the contours show the accumulation of organic matter on the seabed beneath and around the cages. depomod site l a3 b3 cages sediment traps key group 3 model predictions of solids deposition from a cage group m position of traps a1 and b1 a2 is positioned between a1 and a3 b2 is positioned between b1 and b3 0 50 100 150 200 250 300 350 400 450 500 easting (m) 0 50 100 150 200 250 300 350 400 450 n or th in g (m ) a3b3m 25 500 2500 5000 15000 g solids m bed yr -2 -1 the scientific basis for marine fish farm regulation determinand action level within allowable zone of effects action level outside allowable zone of effects number of taxa less than 2 polychaete taxa present (replicates bulked) must be at least 50% of reference station value number of taxa two or more replicates with no taxa present aundance organic enrichment polychaetes present in abnormally low densities organic enrichment polychaetes must not exceed 200% of reference station value shannon-weiner diversity n/a must be at least 60% of reference station value inafunal trophic index (iti) n/a must be at least 50% of reference station value beggiatoa n/a mats present feed pellets accumulations of pellets pellets present teflubenzuron 10.0 mg kg-1 (dry wt)/5cm core 2.0 μg kg-1 (dry wt)/5cm core copper* probable effects 270 mg kg-1 dry sediment possible effects 108 mg kg-1 dry sediment 34 mg kg-1 dry sediment zinc* probable effects 410 mg kg-1 dry sediment possible effects 270 mg kg-1 dry sediment 150 mg kg-1 dry sediment fruu sulphide 4800 mg kg-1 (dry wt) 3200 mg kg-1 (dry wt) organic carbon 9.00% redox potential values lower than -150mv (as a depth average profile or values lower than -125mv (in surface sediments 0-3 cm) loss on ignition 27.00% *a detailed description of the derivation of these action levels may be obtained from sepa on request table 2. sediment quality criteria (sepa fish farm manual, annex a). the aze represents an area around the farm where some deterioration is expected and permitted. thus for several determinands, two sqcs are proposed: one within the aze and one at any point outside the aze. the sqc inside the aze is less demanding than that outside the aze. the sqc approach thus constrains the level of ecological change while the aze limits the spatial extent of major changes. chemical wastes several classes of chemicals are used in fish farming. three of the most important are antibiotics, antiparasitics and antifoulants. since the advent of effective fish vaccines for several important bacterial diseases, the use of antibiotics in salmon culture has declined since the early 1990s (alderman, 2002), but less is known about the use of antibiotics in other species, particularly those in the developing world (gräslund & bengtsson, 2001; holmström et al., 2003; tacon et al., 1995). the main concerns relating to antibiotic use relate to the possibility of the development of bacterial resistance that can be transferred to human pathogens reducing the efficacy of antibiotics in human medicine (cabello, 2004; 2006). prophylactic use of antibiotics is a particular concern, as is adequate testing of aquaculture products for antibacterial residues. where residues persist, low levels of antibiotic intake can stimulate resistance in human pathogens but another concern is that some of the antibiotics used in aquaculture may not be approved for human medicine and carry a health risk. for example, nitrofurans (e.g., furazolidone) are a group of antimicrobials which possess either carcinogenic or mutagenic properties whose use is banned in many countries but still permitted in some. in general, limits have not been set for the concentrations of antibiotics permitted in the environment. of the antiparasitics, the best studied are those that science diliman 19 black, k. & cromey, c. treat infestations of sea lice on salmonids. these are generally highly toxic substances where the use is tightly regulated. in scotland, for example, ecological quality criteria have been set both for both sediments and the water column (www.sepa.org.uk) and access to these medicines is strictly controlled. medicines are only available to farmers after a multi-step regulatory process of authorization that includes testing for efficacy, food safety, and environmental effects. the most infamous antifoulant product, now banned, is tributyltin (tbt). tbt disrupts the endocrine system in invertebrates leading to imposex (miller et al., 1999) and has now been replaced by products with a high concentration of copper. copper, however, is also a potent toxin, hence it's utility as an antifoulant, and quality criteria have been set for both the water column and sediments. a recent study at a fish farm in scotland found copper at higher concentrations than the sediment quality criteria (dean et al., 2007) although its toxicity in anoxic sediments may be limited by its precipitation as the sulphide (brooks & mahnken, 2003b). conclusions the regulation of aquaculture in developed countries has developed considerably over the past decade. this has been driven by the need to improve the scientific basis for management of this very high economic value sector. regulators must base their decisions of good science in order to protect the environment but at the same time allowing development with its economic benefits. this has particularly been the case for the major salmon growing countries, although chile perhaps has still to catch up in a regulatory sense with its rapidly expanding industry. in the developing world, where aquaculture products are primarily for export, market pressures are increasingly brought to bear to ensure food safety, for example concerning residues, and there is also a growing awareness of environmental issues, particularly relating to habitat destruction related to shrimp farming. in the philippines, regulation of the very large number of small scale fish farms where the market is local represents a significant regulatory challenge, but the potential environmental costs make rational regulation essential for the future of this industry. in this paper we have outlined some of the approaches being taken by aquaculture regulators in other countries. it is highly likely that some of these approaches will be relevant and adaptable to the philippine industry. acknowledgements the authors are grateful for support from the eu fp6 ssa philminaq: mitigating impact from aquaculture in the philippines and from the eu fp6 strep ecasa: ecosystem approach for sustainable aquaculture (www.ecasa.org.uk). references alderman, d.j., 2002. trends in therapy and prophylaxis 1991-2001. bulletin of the european association of fish pathologists 22: 117-125. bjorn, p.a., finstad b. & kristoffersen, r., 2001. salmon lice infection of wild sea trout and arctic char in marine and freshwaters: the effects of salmon farms. aquaculture research 32: 947-962. black, k.d., 2001. environmental, economic and social impacts of mariculture. in steele, j.s. thorpe, & k. turekian (eds.) encyclopedia of ocean sciences, academic press, london, pp. 1578-1584. black, k.d., cook, e.j., jones, k.j., kelly, m.s., leakey, r.j., nickell, t.d., sayer, m.d.j., tett, p., & willis, k.j., 2002. review and synthesis of the environmental impacts of aquaculture. scottish executive central research unit, edinburgh. brooks, k.m. & mahnken, c.v.w., 2003a. interactions of atlantic salmon in the pacific northwest environment ii. organic wastes. fisheries research 62: 255-293. brooks, k.m. & mahnken, c.v.w., 2003b. interactions of atlantic salmon in the pacific northwest environment iii. accumulation of zinc and copper. fisheries research 62: 295-305. brooks, k.m., stierns, a.r. & mahnken, c.v.w., 2003a. chemical and biological 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(ed.) environmental effects of marine finfish aquaculture, the handbook of environmental chemistry, volume 5: water pollution, springer verlag, pp. 181-206. holmström, k., gräslund, s., wahlström, a., poungshompoo, s., bengtsson, b.e., & kautsky, n., science diliman 21 black, k. & cromey, c. 2003. antibiotic use in shrimp farming & implications for environmental impacts and human health. international journal of food science and technology 38: 255-266. karakassis, i., hatziyanni, e., tsapakis, m. & plaiti, w., 1999. benthic recovery following cessation of fish farming: a series of successes and catastrophes. marine ecology progress series 184: 205-218. laurent, c., tett, p., fernandes, t., gilpin, l. & jones, k., 2006. a dynamic cstt model for the effects of added nutrients in loch creran, a shallow fjord. journal of marine systems 61: 149-164. mcginnity, p., prodöhl, p., ferguson, k., hynes, r., o'maoiléidigh, n., baker, n., cotter, d., o'hea, b., cooke, d., rogan, g., taggart, j. & cross, t., 2003. fitness reduction and potential extinction of wild populations of atlantic salmon, salmo salar, as a result of interactions with escaped farm salmon. proceedings of the royal society of london series b-biological sciences 270: 2443-2450. mcvicar, a.h., 1997. disease and parasite implications of the coexistence of wild and cultured atlantic salmon populations. ices journal of marine science 54: 10931103. miller, k.l., fernandes, t.f. & read, p.a., 1999. the recovery of populations of dogwhelks suffering from imposex in the firth of forth 1987-1997/98. environmental pollution 106: 183-192. murray, a.g. & gillibrand, p.a., 2006. modelling salmon lice dispersal in loch torridon, scotland. marine pollution bulletin 53: 128-135. nickell, l.a., black, k.d., hughes, d.j., overnell, j., brand, t., nickell, t.d., breuer, e. & harvey, s.m., 2003. bioturbation, sediment fluxes and benthic community structure around a salmon cage farm in loch creran, scotland. journal of experimental marine biology and ecology 285: 221-233. orth, r.j., carruthers, t.j.b., dennison, w.c., duarte, c.m., fourqurean, j.w., heck, k.l., hughes, a.r., kendrick, g.a., kenworthy, w.j., olyarnik, s., short, f.t., waycott, m. & williams, s.l., 2006. a global crisis for seagrass ecosystems. bioscience 56: 987-996. pearson, t.h., 1992. the benthos of soft sublittoral habitats. proceedings of the royal society of edinburgh section biological sciences 100: 113-122. pearson, t.h. & black, k.d., 2001. the environmental impact of marine fish cage culture. in environmental impacts of aquaculture (ed. k. d. black), pp. 1-31. sheffield academic press, sheffield. pearson, t.h. & rosenberg, r., 1978. macrobenthic succession in relation to organic enrichment and pollution of the marine environment. oceanography and marine biology annual reviews 16: 229-311. pearson, t.h. & stanley, s.o., 1979. comparative measurement of redox potential of marine sediments as a rapid means of assessing the effects of organic pollution. marine biology 53: 371-379. pereira, p.m.f., black, k.d., mclusky, d.s. & nickell, t.d., 2004. recovery of sediments after cessation of marine fish farm production. aquaculture 235: 315-330. primavera, j.h., 1998. mangroves as nurseries: shrimp populations in mangrove and non-mangrove habitats. estuarine coastal and shelf science 46: 457-464. primavera, j.h., 2005. mangroves, fishponds, and the quest for sustainability. science 310: 57-59. roberts, j.m., wheeler, a.j. & freiwald, a. 2006. reefs of the deep: the biology and geology of cold-water coral ecosystems. science 312: 543-547. silvert, w. & cromey, c.j., 2001. modelling impacts. in black, k.d. (ed.), environmental impacts of aquaculture, sheffield academic press, sheffield, pp. 154-181. silvert, w. & sowles, j.w., 1996. modeling environmental impacts of marine finfish aquaculture. journal of applied ichthyology 12: 75-81. smaal, a., van stralen, m. & schuiling, e., 2001. the interaction between shellfish culture and ecosystem processes. canadian journal of fisheries and aquatic sciences 58: 991-1002. tacon, a. g. j., phillips, m.j. & barg, u.c., 1995. aquaculture feeds and the environment the asian experience. water science and technology 31: 41-59. tett, p., gilpin, l., svendsen, h., erlandsson, c.p., larsson, u., kratzer, s., fouilland, e., janzen, c., lee, j.y., grenz, c., newton, a., ferreira, j.g., fernandes, t. & scory, s., 2003. eutrophication and some european waters of restricted exchange. continental shelf research 23: 1635-1671. weston, d.p., 1990. quantitative examination of macrobenthic community changes along an organic enrichment gradient. marine ecology progress series 61: 233-244. 22 science diliman draft-optimization (2).pmd optimization of anodized aluminum oxide 19science diliman (january-june 2010) 22:1, 19-25 introduction anodic aluminum oxide (aao) is one of the widely used templates in patterning nanoparticles. this is due to its relative ease of fabrication, mechanical strength, thermal stability, structural uniformity and tunability of the pore morphology (kim, kim & cho 2006; naitabdi, ono, & cuenya 2006; masuda & fukuda, 1995; masuda, hasegwa & ono, 1997). these properties prove useful, for example, in the synthesis of nanoparticle catalytic synthesis nanowires. in this case, the nanowire properties are influenced by the nanoparticle dimensions which, in turn, are patterned by the aao template (gudiksen, wang & lieber, 2001; gudiksen & lieber, 2000). aao is a naturally-occurring, self-ordered porous structure. the geometry of the anodic porous alumina may be described as a honeycomb structure of columnar abstract anodic aluminum oxide (aao) films were produced by anodization of sputtered aluminum thin films on silicon substrates. the effects of anodization voltage and aqueous oxalic acid solution on the pore diameter and interpore distance were studied. parameters were sequentially varied to optimize the pore uniformity. pore morphology was most uniform at 40v anodization voltage and 0.3m solution concentration. average pore diameter and interpore distance for these parameters are 26.14nm ± 13% and 74.62 ± 8%, respectively. pore diameter uniformity was further improved by etching with phosphoric acid solution. the aao films were also successfully used to pattern gold nanoparticle catalysts for the synthesis of semiconductor nanowires. keywords: anodic films, electrolysis, nanoparticles optimization of anodized aluminum oxide pore morphology for gaas nanowire growth regine a. loberternos, oliver d. semblante, rogelio g. dizon, claude r. ceniza, jasher john a. ibanes*, arnel a. salvador, armando s. somintac condensed matter physics lab national institute of physics university of the philippines, diliman 1101, quezon city *corresponding author: email: jaibanes1@up.edu.ph, mobile: +639393585473 pores. these pores feature a high depth-to-diameter ratio as shown in figure 1. the pore dimensions can be tuned via the anodization parameters (masuda & fukuda 1995; masuda, hasegwa & ono, 1997). wet etching can also be used to alter the pore dimensions and improve uniformity(chen & zhang, 2005; masuda et al. 1997). figure 1. schematic of an idealized honeycomb aao structure. loberternos r., et al. 20 science diliman (january-june 2010) 22:1,19-25 in this work, aao thin films were synthesized from aluminum (al) thin films on silicon (si) substrates. anodization parameters were optimized to achieve the pore diameter and interpore distance uniformity. effect of pore etching on the pore dimensions was also explored. in addition, the resulting aao films were used to pattern gold (au) nanoparticles which were subsequently used to synthesize iii-v semiconductor nanowires. methodology pre-cleaned si (111) substrates were deposited with al thin films (~300nm) via rf magnetron sputtering. anodization followed in an oxalic acid solution. anodization parameters were based on the work of masuda and yamada (1997). the anodization voltage, solution concentration, and solution temperature were varied sequentially to optimize the aao pore uniformity. initially, the anodization voltage was varied (30v, 40v and 50v) while keeping the oxalic acid concentration constant at 0.3m. the resulting optimal anodization voltage was then carried over in the optimization of the other parameters. the oxalic acid concentrations used were 0.2m, 0.3m, and 0.4m and the solution temperatures used were 5°c, 15°c, and 25°c. pore widening was subsequently done on the aao thin films using 5% phosphoric acid solution at room temperature. an anodization parameter was said to be optimized when the pore diameter and inter-pore distance standard deviations were minimized. pore dimensions were measured using a phillips field emission scanning electron microscope (fe-sem). pore diameter was determined by measuring 50 randomly chosen pores while inter-pore spacing was evaluated by measuring 50 pairs of randomly selected pores. in addition, the resulting aao thin films were used to pattern au nanoparticles which were used to grow iii-v nanowires. gold of 90å film thickness was deposited on the aao thin films via e-beam evaporation. the aao thin film was then removed using a phosphoric and chromic acid solution leaving behind the au nanoparticles. the resulting au nanoparticles were then used to synthesize iii-v nanowires via molecular beam epitaxy (mbe). details of the au nanoparticle and nanowire synthesis were reported elsewhere (loberternos et al., 2008; bailonsomintac et al, 2010). results and discussions a fe-sem micrograph of the aao film on si is shown in figure 2. variation of the pore dimensions with the anodization parameters is shown in figures 3-5. the y-error bars represent the standard deviation. the variation of the pore diameter and interpore distance with anodization voltage are shown in figure 3. the oxalic acid concentration is kept at 0.3m. the average pore diameter linearly increased with anodization voltage. this confirms that the pore diameter can be controlled by the anodization voltage. similarly, the interpore spacing increased with the anodization voltage. note that the pore diameter extrapolates to zero since anodization cannot occur when there is no anodization voltage. no significant difference was seen in the pore diameter uniformity for varying anodization voltages. however, it can be seen that the interpore distance uniformity was best for an anodization voltage of 40v. pore diameter and interpore distance for this setting were 26.14nm ± 13% and 74.62nm ± 8%, respectively. the variation of the pore diameter and interpore distance with oxalic acid concentration are shown in figure 4. the optimized anodization voltage of 40v was used. a strong linear relationship can be found between the pore figure 2. fe-sem micrograph of anodized aluminum oxide (aao) on silicon (si) substrate. optimization of anodized aluminum oxide 21science diliman (january-june 2010) 22:1, 19-25 figure 3. variation of the aao pore dimensions with the anodization voltage. the error bars represent the standard deviation. figure 4. variation of the aao pore dimensions with the oxalic acid concentration. the error bars represent the standard deviation. loberternos r., et al. 22 science diliman (january-june 2010) 22:1,19-25 figure 5. variation of the aao pore dimensions with the solution temperature. the error bars represent the standard deviation. diameter and solution concentration. note that the pore diameter can be extrapolated to zero since the anodization voltage is relatively low. the pore diameter and interpore distance was most uniform for a solution concentration of 0.3m. the variation of the pore diameter and interpore distance with solution temperature are shown in figure 5. the optimized anodization voltage (40v) and solution concentration (0.3m) were used. it was observed that the pore diameter and interpore distance were independent of the solution temperature. however, the interpore distance standard deviation was minimum at 5% for a solution temperature of 5°c. pore morphology was most uniform for an anodization voltage of 40v, oxalic acid solution concentration of 0.3m, and solution temperature of 5°c. the resulting pore diameter and interpore distance for these parameters were 26nm and 75nm, respectively. pore diameter can be further increased by wet etching with 5% by volume phosphoric acid solution. figure 6 shows the pore diameter variation with etching time. the pore diameter has a linear relation with the etching time; this allows for further control of the pore diameter. furthermore, it can be observed that the pore diameters become more uniform with etching time. this is manifested in the decreasing trend of the pore diameter standard deviation with increasing etching time. in addition, the resulting aao pores were used to pattern au nanoparticles which were then used in the catalytic synthesis of gaas-based nanowires. figure 7 shows the deposition of the au nanoparticles and the resulting nanowires. details of the nanoparticle and nanowire growth were reported elsewhere (loberternos et al., 2008; bailon-somintac et al. 2010). summary anodic aluminum oxide (aao) was synthesized from aluminum thin films on silicon substrate via anodization with oxalic acid. pore diameter and inter-pore spacing of aao were found to be strongly controlled by the anodization voltage and concentration of the oxalic acid solution. optimum parameters for uniformity of pore morphology were also determined. etching using phosphoric acid further improved the pore uniformity. furthermore, the obtained aao-templates were used optimization of anodized aluminum oxide 23science diliman (january-june 2010) 22:1, 19-25 figure 6. (a) variation of the aao pore diameter with the etching time. the error bars represent the standard deviation. fe-sem micrographs of (b) as anodized sample and (c) after 20 min etching. c b loberternos r., et al. 24 figure 7. fe-sem micrographs of the resulting aao films for iii-v nanowire catalytic growth. (a) au deposited on si substrate with aao. (b) au nanoparticles after removal (etching) of aao. (c) iii-v nanowires grown via mbe using the au nanoparticle catalyst. science diliman (january-june 2010) 22:1,19-25 optimization of anodized aluminum oxide 25science diliman (january-june 2010) 22:1,19-25 as a template for au nanoparticles and iii-v nanowire synthesis. acknowledgement this work is supported in part by grants from the department of science and technology – philippine council for advanced science and technology research and development (dost-pcastrd) and the university of the philippines diliman – office of the vice chancellor for research and development (upd-ovcrd). references bailon-somintac, m.f., j.j. ibanez, r.b. jaculbia, r.a. loberternos, m.j. defensor, a.a. salvador and a.s. somintac, 2011. low temperature photoluminescence and raman phonon modes of au-catalyzed mbe-grown gaasalgaas core-shell nanowires grown on a pre-patterned si (111) substrate. j. crys. growth. 314:268-273. chen, z. and h. zhang, 2005. mechanisms for formation of a one-dimensional horizontal anodic aluminum oxide nanopore array on a si substrate. j. electrochem. soc. 152(12):d227-d231. gudiksen, m. s. and c.m. lieber, 2000. diameter-selective synthesis of semiconductor nanowires. j. am. chem. soc. 122: 8801–8802. gudiksen, m.s., j. wang, and c.m. lieber, 2001. synthetic control of the diameter and length of single crystal semiconductor nanowires. j. phys. chem. b 105: 4062-4064. kim, k., m. kim, and s.m. cho, 2006. pulse electrodeposition of palladium nanowire arrays using aao template. mater. chem. phys. 96: 278-282. loberternos, r., o. semblante, r. dizon, d. taguba, o. valdez, j. bugne, a. salvador and a. somintac, 2008. metal nanoparticles deposition on silicon substrate using ultrathin anodic aluminum oxide (aao) template. proc. from 26th spp physics congress, baguio city, october 2008. masuda, h. and k. fukuda, 1995. ordered metal nanohole arrays made by a two-step replication of honeycomb structures of anodic alumina. science. 268(5216): 1466–1468. masuda, h., f. hasegwa, and s. ono, 1997. self-ordering of cell arrangement of anodic porous alumina formed in sulfuric acid solution. j. electrochem. soc. 144(5): l127–l130. masuda, h., h. yamada, m. satoh and h. asoh, 1997. highly ordered nanochannel-array architecture in anodic alumina. appl. phys. lett.71(19):2770-2772. naitabdi, a., l.k. ono and b. roldan cuenya, 2006. local investigation of the electronic properties of size-selected au nanoparticles by scanning tunneling spectroscopy. appl. phys. lett. 89: 043101. 8ahernandez-singson-short com-supplementary.pmd angiotensin-converting enzyme inhibitory action of selected plants 102 science diliman (july-december 2017) 29:2, 102-105 angiotensin-converting enzyme inhibitory action of selected plants supplementary material dionisio bong b. singson and christine l. chichioco-hernandez* university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online bixa orellana (bixaceae), commonly known as annatto, is locally referred to as atsuete. the methanol extracts from the leaves and seeds of annatto exhibited antibacterial activity (medina-flores et al. 2016). the methanol leaf extract of annatto showed neuropharmacological, anticonvulsant, analgesic, and antidiarrhoeal activities in mice (shilpi et al. 2006). the bark extract rendered protective anti-oxidant activity in acetaminophen-induced hepatic damage in rats (bell et al. 2012). artocarpus heterophyllus (moraceae),commonly known as jack fruit, is locally referred to as langka. the polysaccharide isolated from its pulp has a strong anti-oxidant activity (zhu et al. 2017). the ethanol extract from its stem bark inhibited the action of a-amylase and a-glucosidase, an activity with potential anti-diabetes applications (ajiboye et al. 2016). preclinical studies have shown that jackfruit possesses several bioactivities including anti-inflammatory, antibacterial, anticariogenic, antifungal, antineoplastic, and wound healing effects (baliga et al. 2011).new phenolic compounds have also been isolated from its leaves (wang et al. 2017). morus alba (moraceae), commonly known as white mulberry, is locally referred to as moras. flavonoids and cinnamic acids were identif ied from the ethanol extracts obtained from its leaves. the same extract showed a high degree of activity against inflammation but was found to be toxic in mice (de oliveira et al. 2016). its root bark has shown therapeutic potential against diabetes-induced depression in mice (ye et al. 2017).compounds isolated from its root bark exhibited potential antiobesity activity by inhibiting the action of pancreatic lipase (ha et al. 2016). new glycoside and flavones were also isolated from its stem bark (ali and ali 2016). d.b. b. singson and c.l. chichioco-hernandez 103 nymphaea pubescens or water lily is part of the nymphaeacea family. its aqueous extracts exhibited anti-inflammatory and hepatoprotective effects in rats (debnath et al. 2013). syzygium samarangense (myrtaceae), commonly known as water apple, is locally referred to as makopa. the pulp and seeds contain cytotoxic chalcones and antioxidant glycosides (simirgiotis et al. 2008). vescalagin isolated from its fruit exhibited therapeutic value against diabetes through its anti-hypertriglyceridemic and anti-hyperglycemic effects (shen and chang 2013). aurentiacin from the said plant displayed anti-inflammatory effects in lipopolysaccharide (lps)-stimulated mouse macrophages (kim et al. 2012). extraction the leaves of b. orellana, a. heterophyllus, m. alba, n. pubescens and s. samarangense were collected within the university of the philippines diliman campus and submitted to the dr. jose vera santos herbarium, institute of biology, up diliman for verif ication. the accession number of b. orellana is 3649, a . heterophyllus is 9431, m. alba is 14626, n. pubescens is 3498, and s. samarangense is 14258. the leaves were washed and air dried. the dried leaves were macerated using a blender and soaked in methanol. the solution was f iltered to obtain the crude organic extract. the crude methanol extract was partitioned between distilled water and hexane in a 1:2 ratio, followed by the partitioning of the remaining aqueous layer with ethyl acetate using the same ratio. the hexane and ethyl acetate partitions were then concentrated in vacuo. phytochemical screening the phytochemical screening protocol was modif ied from the works of harborne (1984), edeoga et al. (2005), and onwukaeme et al. (2007). the qualitative test for the presence of saponins, flavonoids, tannins, cardiac glycosides, phenolic compounds, alkaloids, and terpenoids were performed on 20 mg of the methanol leaf extracts dissolved in 200 μl dmso. angiotensin converting enzyme inhibitory assay the method for the determination of the ace inhibition of the plant extracts was modif ied from the procedure of jimsheena and gowda (2009). a stock solution was angiotensin-converting enzyme inhibitory action of selected plants 104 prepared using one mg of the sample dissolved in 20 μl methanol. the mixture was diluted to 0.5 ml using the 0.05m sodium borate buffer. thirty f ive μl of the stock solution was mixed with an equal volume of the sodium borate buffer in a microplate. a positive control solution was made using 35μl of captopril as the ace inhibitor. a blank solution containing twice the volume of the buffer solution was also prepared. the reaction was initiated by the addition of 10 μl of ace in each solution. the plate was shaken for 15 seconds and incubated at 37oc for 10 minutes. twenty μl of the hippuryl-histidyl-leucine (hhl) substrate was added to the solutions. the plate was shaken for an additional 15 seconds and incubated at 37oc for 30 minutes. the reaction was stopped by the addition of 50 μl hydrochloric acid. one hundred μl pyridine and 50 μl benzene sulfonyl chloride (bsc) were added to the solutions to produce a change in color. the absorbance of each solution was measured at 410nm. all measurements were performed in triplicates. using the absorbance measurements of the solutions, the extent of inhibition was calculated using the following formula: (1) where b is the absorbance of control (buffer was added instead of the test sample), c the absorbance of the reaction blank, and a isthe absorbance in the presence of the sample. references ajiboye bo, ojo oa, adeyonu o, imiere o, olayide i, fadaka a, oyinloye be.2016. inhibitory effect on key enzymes relevant to acute type-2 diabetes and antioxidative activity of ethanolic extract of artocarpus heterophyllus stem bark. journal of acute disease.5(1):423-429. ali a, ali m. 2016. isolation and structure elucidation of a new linoleiyl glycoside and f l a v o n e s f r o m t h e s t e m b a r k o f m o r u s a l b a l . f u t u r e j o u r n a l o f p h a r m a c e u t i c a l sciences.2(2):82-86. baliga ms, shivashankara ar, haniadka r, dsouza j, bhat hp. 2011.phytochemistry, nutritional and pharmacological properties of artocarpus heterophyllus lam (jackfruit):a review. food research int ernational. 44(7):1800-1811. bell gas, shamna r, sangeetha b, sasikumar jm. 2012. in vivo antioxidant activity of bark extract of bixa orellana l. against acetaminophen-induced oxidative stress. asian pacif ic journal of tropical biomedicine. 2(2):s700-s705. debnath s, ghosh s, hazra b. 2013.inhibitory effect of nymphaea pubescens wild. flower extract on carrageenan-induced inflammation and ccl 4 -induced hepatotoxicity in rats. food and chemical toxicology. 59:485-491. ace inhibitory activity % – – 100,  d.b. b. singson and c.l. chichioco-hernandez 105 de oliveira am, do nascimento fd, ferreira mra, de moura df, dos santos souza tg, da silva gc, da silva ramos eh, paiva pmg, de medeiros pl, da silva tg, lira soares la, chagas ca , de souza ia, napoleao th. 2016. evaluation of acute toxicity, genotoxicity and inhibitory effect on acute inflammation of an ethanol extract of morus alba l. (moraceae) in mice. journal of ethnopharmacology. 194:162-168. edeoga ho, okwu de, mbaebie bo. 2005. phytochemical constituents of some nigerian medicinal plants. african journal of biotechnology. 4(7):685-688. ha mt, tran mh, ah kj, jo kj, kim j, kim wd, cheon wj, woo mh, ryu sh, min bs. 2016. potential pancreatic lipase inhibitory activity of phenolic constituents from the root bark of morus alba l. bioorganic & medicinal chemistry letters. 26(12):2788-2794. harborne j. 1984. phytochemical methods, a guide to modern techniques of plant analysis. 2nd ed. london: chapman and hall. j i m s h e e n a v k , g o w d a l r . 2 0 0 9 . c o l o r i m e t i c , h i g h t h r o u g h p u t a s s a y f o r s c r e e n i n g angiotensin 1-conver ting enzyme inhibitors. analytical chemistry. 15(81):9388-9394. kim yj, kim hc, ko h, amor ec, lee jw, yang ho. 2012. inhibitory effects of aurentiacin from syzygium samarangense on lipopolysaccharide-induced inflammatory response in mouse macrophages. food and chemical toxicology.50(3-4):1027-1035. m e d i n a f l o r e s d , u l l o a u r i z a r g , c a m e r e c o l a r o s s i r , c a b a l l e r o g a r c i a s , m a y t a tovallino f, del valle-mendoza j. 2016. antibacterial activity of bixa orellana l. (achiote) a g a i n s t s t r e p t o cocc u s m u t a n s and s t r e p t o cocc u s s a n g u i n i s . a s i a n pa c i f i c j o u r n a l of tropical biomedicine. 6(5):400-403. o n w u k a e m e d n , i k u e g b v we h a t b , a s o n y e cc . 2 0 0 7. e v a l u a t i o n o f p h y to c h e m i c a l constituents, antibacterial activities and effect of exudate of pycanthus angolensis we l d w a r b ( m y r i s t i c a c e a e ) o n c o r n e a l u l c e r s i n r a b b i t s . tr o p i c a l j o u r n a l o f pharmaceutical research. 6(2):725-730. shen sc, chang wc.2013. hypotriglyceridemic and hypoglycemic effects of vescalagin from pink wax apple [syzygium samarangense (blume) merrill and perry cv pink] in high-fructose diet-induced diabetic rats.food chemistry. 136(2):858-863. shilpi ja , tauf ig-ur-rahman m, uddin sj, alam ms, sadhu sk, seidel v. 2006. preliminary pharmacological screening of bixa orellana l. leaves. journal of ethnopharmacology. 108(2):264-271. simirgiotis mj, adachi s, to s, yang h, reyner tson ka, basile mj, gil rr, weinstein ib, kennelly ej.2008. cytotoxic chalcones and antioxidants from the fruits of syzygium samarangense (wax jambu). food chemistry. 107(2):813-819. wang xl, di xx, shen t, wang sq, wang xn. 2017. new phenolic compounds from the leaves of artocarpus heterophyllus. chinese chemical letters. 28(1):37-40. ye m, ke y, liu b, yuan y, wang f, bu s, zhang y. 2017. root bark of morus alba ameliorates the depressive-like behaviors in diabetic rats. neuroscience letters. 637:136-141. zhu k, zhang y, nie s, xu f, he s, gong d, wu g, tan l.2017. physicochemical proper ties and in vitro antioxidant activities of polysaccharide from artocarpus heterophyllus lam. pulp. carbohydrate polymers. 155:354-361. 12journal policy.pmd 96 journal pol icy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the off ice of the vice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsif ication is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 97 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research area concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “notes to contributors” stipulates that “all ar ticles must have a high degree of scholarship,” that “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a cer tif ication from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right… .” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as def ined above and; b.) is specif ic and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the bases for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specif ic advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the 98 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientif ic misconduct policy. in attendance were: d r. co r a zo n d. v i l l a r e a l , r d u o d i r ec to r, p r e s i d i n g ; d r h e n r y j . ra m o s , p m rg o director and professor, nip; atty. vyva v ictoria aguirre, ovcrd legal consultant; e d i to r s i n c h i e f d r. m a r i co r s o r i a n o (science dil iman) a n d d r. m a r i a m a n g a h a s (s o c i a l s c i e n c e d i l i m a n ) . m s . n a n i e d o m i n g o a n d m s . d e r c y m a r a r a c , e d i t o r i a l assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. 99 references council of science editors. “ white paper on promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ec to r s o n s e p te m b e r 3 , 2 0 0 6 .” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct .php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http: //www.kzoo.edu/ ajp/submit.html the use of-art.12 aguinaldo & talaue-mcmanus 88 science diliman (july december 2000) 12:2, 88-100 abstract the use of science in environmental advocacy for coastal resource management maria maida s. aguinaldo and liana talaue-mcmanus marine science institute, college of science university of the philippines, diliman, quezon city 1101 philippines tel. no.: (632) 922-3921; telfax.: (632) 924-7678; e-mail: maguinaldo@upmsi.ph environmental advocacy in bolinao has played an important role in the prevention, remediation, and rehabilitation of potential and felt impacts of the various activities in the coastal zone. most initiatives have been spurred by the sharing of knowledge and information in mobilizing community advocates. facilitating action in four key areas–development planning, coastal aquaculture, concession systems, and tourism– involved the provision of venues for information transfer. these included the conduct of orientations and consultations, sharing of results of research project undertaken, lobbying, and use of primers, newsletters, and theater. mechanisms for sustaining these actions and upholding the coastal resource management (crm) principles (sustainable, equitable, empowering) long after projects have been phased out were initiated through the establishment of a coastal resource management center, and the institutionalization activities through existing institutions, such as the local government, academic institutions, and peoples’ organizations. maximizing knowledge and information, popularizing information, and sharing this with members of the community and getting them to use it, as well as enjoining them to act, are the challenges that must be faced. environmental advocacy, as a tool for empowering different community sectors in evolving a consensus for crm has become an integral feature of development work in bolinao. keywords: environmental advocacy, information transfer, coastal zone management, cbcrm, resource management introduction community-based coastal resource management (cbcrm) projects have been undertaken in bolinao, pangasinan since the late 1980’s. initiatives and activities spanning ten years have gained a number of victories for both bolinao’s coastal resources and its people. various programs have been conducted with various sectors of the municipality. significant success in coastal resource management (crm) have been facilitated by the popularization of science and its dissemination. the identification of habitats was used as a basis for zoning the municipal waters. data on water quality and on fish production were used as basis for policy reforms in regulating the aquaculture industry. in addition, levels of catch and income derived from fishery resources have helped policy makers formulate possible reforms on the existing concession systems. the bolinao coastal development plan (cdp) was based on community practices and perceptions and on scientific research and technical advice from the marine science institute (up-msi) of the university of the philippines in diliman. the use of science in environmental advocacy 89 cbcrm action plans begin when stakeholders evolve into advocates, with the knowledge and information they receive through environmental advocacy. an advocate is defined as one “who defends, maintains, publicly recommends, or raises his voice in behalf of a proposal or tenet,” (oxford english dictionary 1989). thus, a cbcrm-oriented advocacy activity encourages community members to publicly take a stand as a group, a prerequisite to implementing action as a collective steward. this paper discusses key advocacy activities which were implemented in bolinao for the period 1998-1999 and facilitated by the marine fishery resources management project (mfrmp) based at the msi. this period was crucial in that substantive cbcrm action plans integral to the municipal coastal development plan were to be carried out. an advocacy program was formulated to develop the broad-based mass support needed to effectively implement the cdp. this support was also critical in rationalizing what was an unregulated coastal aquaculture and in reconfiguring the traditional concession systems that have dominated the rabbitfish and milkfish fry fisheries since the postwar years. the proposal to open the local tourism industry to multi-national ventures was a nascent issue that required the formation of a local group that could engage in forward planning. the nature of each advocacy activity, and its strengths and weaknesses, are discussed and evaluated in order to identify replicable strategies that promote advocacy toward sustained collective action. in addition, the extent to which scientific information was used and popularized in each activity are underscored. conceptual framework environmental advocacy as practiced in bolinao has evolved into an empirical framework (fig.1) which may not be much different from what is being practiced in other areas (o’malley 1999). issues and problems in the community are either perceived and expressed by the locals (e.g., declining catch, use conflicts) or are identified through scientific research (e.g., impact of gear use, causes and impact of water quality deterioration). when issues have been identified, information and data are gathered and shared with target sectors through different activities that provide a venue through which stakeholders can publicly declare their stand and make a call for collective action. environmental advocacy has three main objectives: (1) the generation of awareness; (2) the enhancement of knowledge; and 3) the mobilization of community sectors (people’s organizations, local government units both at the municipal and barangay levels, academic institutions, communities) to participate in the public declaration of cbcrm as a means to sustainable development. different activities were used to meet these objectives and were designed and chosen based on their relevance and potential to realize the targeted action program (table 1). science identified community identified and/or collective action/ program fig. 1. conceptual framework for the advocacy program of the marine fishery resources management project (mfrmp) issue/ problem information/ data gathering sharing information w/ target sector information/ data gathering aguinaldo & talaue-mcmanus 90 target action major issues information & data needs target sectors advocacy activities 1. implement the coastal development plan (cdp) 2. rationalize coastal aquaculture 3. modify concession systems for milkfish fry and siganid fisheries 4. (regulations) for the local tourism industry > lack of popular support > lack of understanding by municipal legislative and executive branches > lack of political will to implement the cdp > deteriorating water quality > unregulated proliferation of fishpens and cages > reduced culture production > blocked navigation routes and reduced fishing grounds > inequitable resources access > overexploitation > low returns for marginal fishers > unregulated tourism > pollution > entry of external players > destruction of resources > resource use conflicts > anticipated entry of socio-cultural problems (i.e., prostitution, drugs, etc.) > popularize the aim and substance of the plan > popularize the legal provisions and their relationship with regional plans and national laws > impacts of coastal aquaculture on water quality and fisheries > sustainability of coastal aquaculture > mitigation measures > experience of other coastal communities in aquaculture > status of and impact on the fisheries > status of and impact on income generation > options to improve status of access and resources > potential impacts on resources and livelihood > options for regulation > all sectors > pen and cage operators > local government > marginal captures fisheries > local fisheries school > marginal fishers > local government > concessionairies and fish dealers > resort owners > local government > federation of fisherfolk org., transportation sector, health sector > cdp forum > primers and leaflets > sectoral orientation > school-based municipal newsletter on the environment > community theater > crm center > lobbying > aquaculture forum > zonal orientation > primers > lobbying > sharing of results of participatory monitoring activities to target sectors > school-based municipal newsletter > formation of monitoring team > fisheries fora > sharing of results of participatory monitoring activities to target sectors > formation of a zonal action team for zone 1 > lobbying > multi-sectoral orientation > primers and leaflets > formation of municipal tourism council and bolinao hotel, resort, and restaurant owners association > community theater table 1. advocacy program for community-based coastal resources management in bolinao, pangasinan advocacy program the following activities sought to enhance awareness and enjoin public participation in four key areas: development planning, coastal aquaculture, concession systems, and tourism. they were conducted from 1998 up to the first quarter of 2000 within mfrmp in the municipality of bolinao, pangasinan. engender participation in the implementation of the coastal development plan the bolinao cdp, which started from the initiatives of a federation of local fisherfolk organizations, went through a two-year process of community consultations and validation. on january 19, 1998, through the collaboration of the local government unit, the federation, and the different sectors, the cdp of bolinao was adopted by the municipal council. its implementation hinged on its passage as an ordinance by both the municipal and provincial councils. a multi-sectoral technical working group (cdp-twg) started drafting the implementing rules and regulations of the municipal fisheries ordinance immediately after its enactment in december 1999. between its adoption as a plan and its passage the use of science in environmental advocacy 91 as an ordinance, the municipal council required that the document be validated again in all the coastal villages. it was during this period that advocacy for the immediate implementation of the cdp needed to be intensified. it was also then that those most involved in the plan formulation realized how unfamiliar most of the community sectors, including the municipal council, were with the plan. pooling together information that needed to be disseminated to all sectors, as well as planning for advocacy activities, followed. the local government sponsored a cdp forum for the village councils and sectoral representatives in march 1998. the forum aimed to inform the sectors of the salient features of the plan, as well as to provide a venue to clarify controversial provisions. the forum also took advantage of the representation of the participants to lobby for its immediate implementation. prior to the forum, villagelevel meetings were conducted to present the cdp and to get initial recommendations on how the plan would best be implemented. the pre-forum meetings were also a mechanism to ensure the attendance and active participation of all the sectors in the forum itself. it also helped the facilitators get a sense of the concerns and controversial issues that needed to be discussed in the forum. information on the fishery status, technical information on coastal resources, as well as a working knowledge on national laws and fishery rules and regulations were helpful in facilitating the discussions. experts on the field of environmental law were invited to help ensure that the cdp conformed to national laws and appropriate legal processes. the forum proved to be an effective strategy because it started off another phase in the process of cdp implementation–the drafting of the ordinance and its implementing rules and regulations. after the forum, a cdp primer was prepared in tagalog (the cdp was originally drafted in english), a language understood by a majority of the citizens. the primer contained the major provisions of the cdp, the issues in the zones which the cdp hoped to address, and pertinent national fisheries laws. copies of the primer were disseminated to the coastal villages, peoples’ organizations, local government units, fisheries schools, and government agencies. village-level cdp orientations were continually conducted after the forum to ensure that the citizens were well-informed. the cdp was set to be implemented as soon as the ordinance and its implementing rules and regulations were approved. cdp advocacy also utilized existing programs and structures such as student bodies and school papers. in the second quarter of 1999, a cbcrm orientation and a popular education workshop were conducted by the mfrmp for 50 youth representatives from different schools in bolinao. it aimed to orient the youth on environmental and popular education. it also hoped to provide a venue for the formation of an environmental advocacy group for bolinao. a follow-up training on popular education, which focused on theater, journalism, public speaking, and production of information materials, was conducted in june 1999 to help the participants hone their skills to enable them to undertake environmental advocacy initiatives. an advocacy group was formed in june 1999 with the objective of advancing environmental awareness through the use of their skills in theater and journalism. although the group was supposed to include students from different schools, only the students from the bolinao school of fisheries (bsf) comprised the advisory group. instead of a representation of different schools, the formation of a youth advocacy group was taken up only by the bsf. establishing linkages with school officials to formalize the organization and to seek institutional support was explored. what followed was the forging of the memorandum of agreement in december 1999 which included the recognition of, and support for, the youth advocacy group, and the promotion of collaboration in other cbcrm-related activities, such as participatory aquaculture monitoring, community activities on environmental protection, review and enforcement of environmental and fishery laws, and operations of the crm center. the school also agreed to devote one of three issues of their school paper, which they have been able to produce yearly, to environmental advocacy. linking with the fisheries school for specific cbcrm-related activities, such as advocacy, was deemed strategic in engendering the active participation of more cbcrm advocates. institutionalizing programs such as this increases the likelihood that the activities would be sustained beyond the lifespan of the mfrmp. aguinaldo & talaue-mcmanus 92 in april 2000, specific provisions of the cdp which were already being implemented, such as aquaculture monitoring and the establishment of a marine protected area, were featured in a school newsletter which was circulated in the municipality. from the last quarter of 1999 to april 2000, other crm issues were featured in a play by the youth advocacy group. the group performed in the village plazas of patar, estanza, liwaliwa, and in the church grounds in the town center. the group also performed in bsf school programs and in a youth camp sponsored by plan international for youth representatives of region 1. cbcrm activities in bolinao, which have cdp for their framework, were sought to be institutionalized through the establishment of a crm center in december 1999. the center aimed to aid in sustaining these activities in bolinao by providing a structure, a system, and a cbcrm program which would facilitate the flow of information, activities, and support from the various groups existing in bolinao. the crm center was preceded by efforts to establish an ecology center. the operation of the ecology center, which was put up in march 1999, entailed coordinating with the local government, soliciting support from the local schools and from different local and national organizations, and setting up a program to start off its operations. its functional use was never maximized because of lack of financial and institutional support and a non-conducive environment. in december 1999, construction of the crm center began just after a memorandum of agreement was signed by the municipal government, through the mayor, and the mfrm project. the creation of the crm center and a position for a crm officer are provided for in the municipal fisheries ordinance (article 15, bolinao coastal and fisheries resource management ordinance of 1999). operations of the crm office began in february 2000. rationalized coastal aquaculture. milkfish aquaculture in bolinao started in 1995 (verceles and mcmanus, this issue). it reached its peak in 1997, with over 3000 units in operation (lgcamc reports); it dwindled to about 1000 units spread over 88 hectares of municipal waters in 1998 (bolinao municipal records 1998). among the issues that surfaced were deteriorating water quality, reduced culture production, fishkills, and use conflicts between the aquaculture operators and the marginal fishers and boat owners; conflicts which are the results of unregulated proliferation of fishpens and cages blocking navigational routes, thereby reducing fishing grounds. all these issues that were experienced and expressed by the locals clearly needed to be addressed. to share information on the impact of coastal aquaculture on water quality, fisheries, and culture production, measures to mitigate their effects and help sustain the industry-stimulated initiatives to address issues attendant to coastal aquaculture, an orientation on sustainable aquaculture was conducted in november 1998. it was attended by fishpen and cage operators, local government units, marginal fishers, and representatives from the local fisheries school . it aimed to orient the participants on sustainable and environmentfriendly aquaculture practices, to share technical studies on the milkfish culture industry in bolinao, and to provide a venue to encourage cooperation and participation in the management of the caquiputan channel. the orientation resulted in the realization of a need to regulate and monitor the industry. a follow-up training on monitoring was held soon after the orientation. the training was participated in by pen and cage owners and operators and students from the fisheries school. resource persons from the bureau of fisheries and aquatic resources (bfar) and the marine science institute (msi) were invited to train the participants in basic water quality monitoring and sustainable milkfish culture. the training facilitated the creation of a sectoral monitoring body for the aquaculture zone. a monitoring scheme was drafted and regular monitoring of water quality, production, and zoning of the area was conducted starting december 1998. results of the monitoring activities were shared quarterly with the villages, the fisheries school, and the local government unit. these were used by the local government unit (lgu) as bases for drafting policies on aquaculture. the results, as well as the technical studies done by experts at the msi were often referred to in zoning and regulating aquaculture structures in the area (cdp appendix b 1998). the results also provided guidance to aquaculture operators in addressing problems in their culture operations. the sharing sessions served as venue for the village councils and residents to generate awareness on the status of the industry and milkfish culture practices. it also aimed to the use of science in environmental advocacy 93 encourage participation of the community sectors in the management of their zone, as well as in other crmrelated initiatives. as a result of the sharing sessions, some participants also requested assistance in the monitoring of the growing grouper industry in the area. in the second half of 1999, primers were produced and disseminated to the coastal villages in the zone, as well as to the policy makers. at present, sustainable aquaculture advocates are being encouraged to participate more actively through the creation of a zonal body that would oversee the management of the aquaculture zone. modify concession systems for milkfish fry and siganid fisheries. the milkfish fry and the siganid resources, which are major fishery resources in bolinao, are managed through a concession system. the concession is a system through which user rights are conceded by a management body to an applicant. in bolinao, the highest bidder gains exclusive rights over fishery resources for a year. the concessionaire, having paid a considerable sum to the municipal government for exclusive rights to the area for a year, implements certain management prerogatives in the use of the fishery resource (rodriguez 1997). this practice has elicited issues of resource accessibility and sustainability, as well as social equity. the need to modify the existing concession systems for both the milkfish fry and the siganid fisheries was borne out of a realization that there were inequities in access and income derived from these resources. furthermore, catches were declining. advocacy activities included fora and orientations on fisheries monitoring. sharing information on yearly catch based on monitoring studies, impact of unsustainable use, and on the biology and life cycle of the fish coupled with their indigenous knowledge, gave the stakeholders an understanding of how the milkfish fry and the siganid resources may be managed for sustainable use. reef fisheries. to facilitate action on issues on the reef fisheries, including the siganid concession system, a reef fisheries forum was conducted in september 1998. representatives from the different fisheries sectors and local government units participated in discussions on the status of the reef fisheries, the siganid fishery and the concession system, and resource management strategies for the zone. results of technical studies on the reef resources (i.e., seaweeds, sea urchins, siganids) and of fisheries monitoring activities conducted by the project were shared. the monitoring results included a resource-use map, seasonality of the resource, and volume and catch composition. the information helped in planning for management interventions in the zone. among the identified management interventions were participation in fishery monitoring activities, establishment of marine protected areas, and mangrove reforestation, as well as implemention fishery regulations as stipulated in the cdp. a core group was initially formed to pursue the planned actions. because many issues beset a multi-gear reef fishery, the task of forming a zonal action team proved daunting. coordination between sectors was difficult. this problem was coupled with a lack of follow-up activities on the part of the facilitators. interest groups have now been formed. different views on approaches concerning the siganid concession system caused temporary inaction. at present, an experimental culture of siganids is being undertaken by the mfrmp with a local partner to find new practices that would hopefully lead to changes in the concession system and ultimately, in the sustainable use of the resource. milkfish fisheries. a milkfish fry forum was held in november 1998 to facilitate information sharing among milkfish fry gatherers on fry management and to promote the concept of sustainable management of the resource. lectures on the status of the resource, its biology, life history, and seasonality, as well as trends on milkfish fry catch and gear-use and a review of the concession system, from bidding to trading, were substantial inputs to the discussions that followed. the participants focused on concerns regarding the inequity of the concession system. this stimulated lobbying for reforms to the municipal government in the existing system at the level of the municipal government. a follow-up forum in march 1999 provided a venue for the fisher group to draft plans for the legislation of policy reforms in the concession system. the forum also resulted in the drafting of an alternative system that proposed the changing of the concession system to a “permit system”. the permit system would mean the removal of the concessionaire and the use of licenses and permits for the milkfish fry gatherers. the aguinaldo & talaue-mcmanus 94 alternative system hoped to provide the gatherers more opportunity to increase income through the removal of fees and other shares the concessionaire usually imposes. despite the municipal council’s refusal to implement the proposed system in the coming year, changes in the concession system that were also included in the cdp were made. transparency in buying and selling prices, monitoring of the fishery resource, and closed and open seasons were among the provisions that were to be implemented in the coming year. in november 1999, representatives from the village councils and the people’s organizations from the villages of patar, ilog malino, balingasay, arnedo, pilar, (zone i under the cdp) sought an audience before both the legislative and executive branches of the local government requesting that the concession for the following year be awarded to a fisherfolk organization. as a result of their lobbying efforts and in conformity with sec. 17 of the fisheries code of 1998 (grant of fishery privileges in municipal waters) and sec. 24 of the bolinao coastal and fisheries resource management ordinance of 1999, the concession for the year 2000 was awarded to a federation of people’s organizations, the kaisaka. provide regulations for the local tourism industry. the municipality of bolinao has been a favorite destination of a moderate number of tourists. the number of resorts has increased from 23 in 1998 to about 40 in 1999. multi-million dollar proposals to develop and operate a tourism estate and retirement village have been submitted to the municipality. in anticipation of the eventual boom of the tourism industry, provisions for its regulation, though in broad terms, have been included in the cdp of bolinao; the municipality has been divided in its opinions regarding tourism. some see it as a welcome infusion of capital and livelihood opportunities; others are anxious of the possible effects on their resources (i.e., drinking water, corals and beaches, landscape) and their way of life (i.e., congestion, pollution, increase in incidences of drug use, prostitution, crime). advocacy initiatives would prove to be difficult given that no sector had any strong feeling regarding tourism. no issues were pressing and views were varied. even among the mfrmp staff, there were differing stands. in february 1999, a resource person from the asian institute of tourism of the university of the philippines (up ait) was invited by the mfrmp to help orient the staff on tourism and ecotourism concepts and principles, issues on and impact of the industry, and the limits of ecotourism development. the orientation also hoped to give the staff a unified vision for the development of tourism in bolinao. in may 1999, a multi-sectoral forum on tourism was planned with the municipal planning and development coordinator (mpdc). it aimed to bring out issues (felt and anticipated), identify stakeholders and their roles in the tourism industry and level off on where the industry was at and what their vision for it was. unfortunately, the forum was repeatedly postponed by the mpdc. it seemed to be a low priority compared to other pressing concerns (i.e., conflicts arising from the use of a new fishing gear, reentry of a cement plant project, formation of barangay fishery and aquatic resource management councils, public hearings for the municipal ordinance, and drafting of a land-use plan) which the mpdc also had to facilitate. to stimulate discussions among stakeholders and to get a sense of stakeholder perceptions about tourism, questionnaires were fielded in april 1999 to the different sectors involved: resort owners, the transportation sector, media, lgus in the tourism zone, academic institutions, and people’s organizations. the results were to become part of the multi-sectoral consultation on tourism. in july 1999, a tourism class from the upait visited bolinao to assist in drafting a micro-tourism plan for bolinao. the class presented their micro-plans to the mfrmp and the mpdc in october 1999. a multi-sectoral orientation was held in september 1999. it aimed to share information on the potential environmental, sociocultural and economic impact and control measures; and to surface issues on the existing tourism industry. a resource person from the up-ait discussed the different aspects of the tourism industry. a speaker from the department of environment and natural resources (denr) briefed the group on denr policies on tourism, underscored the importance of the environmental impact assessment (eia) system, and clarified regulations regarding the construction and development of tourism structures. in the last quarter of 1999, primers were disseminated to give the stakeholders and the general public the use of science in environmental advocacy 95 information on the industry, so that they could make more informed decisions and influence policies on local tourism. information on social considerations, development and sanitation standards and guidelines, and on laws governing tourism concerns was given to major stakeholders through orientations and seminars from october 1999 to march 2000. a youth advocacy theater group conducted a play on the entry of development projects in an area from december 1999 to april 2000 in four villages to surface these concerns. the lgu, through the office of the municipal planning and development coordinator, facilitated the creation of the bolinao municipal tourism council and the bolinao hotel, resort, and restaurant owners association (bhrroa). the municipal tourism council was first convened in july 1999. its primary objective was to oversee the sustainable development of the tourism industry. this included recommending policies, reviewing, evaluating, and monitoring tourism projects, and promoting a sustainable tourism plan for the municipality. to aid the council, orientations and lecture-meetings with resource speakers from the department of health (doh), the department of tourism (dot), and the protected area management board (pamb) have been held to access as much information and technical advise as possible. the target of the municipal tourism council was to formulate a draft tourism plan by december 2000. before then, a series of planning sessions and consultations would be conducted as soon as an executive order tasking the council to draft the plan is given. the bhrroa was formed in august 1998, and since then has been working on organizational strengthening, and on small projects such as the putting up of information billboards. they, too, have been recipients of information since september 1999. the bhrroa members are currently working on meeting the respective standards and guidelines of the dot and doh for tourism facilities. this followed orientations given by the two departments from october to march 2000 to the sector. the continued assistance by the dot and the doh is the result of the efforts of the bhrroa to regularly coordinate with the departments. the possible-ill effects of unregulated tourism are not yet apparent to the residents of bolinao, while its positive contributions are evident. information and advocacy on appropriate tourism are needed for the effective management of the industry. institutionalizing initiatives to sustain cbcrm advocacy for various issues in the management and use of coastal resources in bolinao, it was deemed strategic to institutionalize major activities. the establishment of a crm center, which was provided for in the municipal fisheries ordinance, resulted from collaborations between the local government, the federation of peoples’ organizations, and the mfrmp. the crm center aimed: (1) to coordinate the crm actions undertaken by various groups and advocates; (2) provide a venue for the provision of information and technical support needed to strengthen existing groups; (3) enhance the knowledge and skills of these groups; (4) enjoin the participation of more advocates. utilizing existing systems, such as a school paper, and widening its theme (environmental advocacy vs. school concerns) and reach (municipal-wide vs. school level) were also explored. enjoining student organizations to participate in environmental advocacy and encouraging school officials to support the initiatives were pursued. these were done by enhancing their knowledge and skills and capitalizing on their interests. collaborations with the bolinao school of fisheries (bsf) in activities, such as environmental theater, newsletter production, and aquaculture monitoring were formalized through a memorandum of agreement. the participation of peoples’ organizations in traditional systems, such as the milkfish fry concession, was a significant step in realizing changes in the traditional practices which exacerbate unsustainable and inequitable use of resources. lobbying and information dissemination efforts aided in influencing policies and in gaining advocates and supporters. significant crm activities, such as the monitoring of fishery resources, were legally provided for in the municipal fishery ordinance and were institutionalized in peoples’ organizations and sectoral groups. the monitoring activities have enhanced the capability and knowledge of groups in the multiple-use zone have resulted in a better understanding of their resource base. aguinaldo & talaue-mcmanus 96 the federation of peoples’ organizations, kaisaka, five active people’s organizations, the municipal fishery and aquatic resources management council (mfarmc), and village-level farmcs that evolved from being resource users to cbcrm advocates and that have been implementing and advocating cbcrm actions for the last six years will ensure that the use of bolinao’s coastal resources adhere to the cbcrm principles of sustainability and equitability. the role of science and information in cbcrm advocacy the use of science and information through advocacy initiatives has contributed significantly to the management of bolinao’s coastal resources. institutions such as the msi and the bsf have been valuable sources of information and technical support that have provided the scientific bases for policy, resolution of issues and conflicting interests, and community action. actions such as the zoning of municipal waters, regulating fishery activities (closed season, number, type), and the establishment of mpas have relied on scientific advise. the resolution of the cement plant issue in bolinao in 1996 used scientific data. accessing different agencies such as the bureau of fisheries and aquatic resources (bfar), the department of environment and natural resources (denr), the protected areas management bureau (pamb), the department of tourism (dot), and other groups (peoples’ organizations, non-government organizations, etc.) for information and technical advice has contributed to the success of advocacy initiatives. science and information as preventive measures for tourism. science and information have served as an eye-opener to an otherwise uninformed community. issues or impacts of community practices such as unregulated tourism that the community may not have been aware of have been put forward by the sharing of information. information dissemination facilitated the action of the community on such issues even before the negative impacts could be observed. in bolinao, the effects of tourism have yet to be felt and perceived by the whole community. the formation of the municipal tourism council and the bhrroa, and the formulation of plans and regulations for the local tourism industry, started from the dissemination of information on the possible negative consequences of tourism activities and the establishment of tourism facilities on the environment, the community, and on the economy. the information disseminated to the stakeholders was prepared by the facilitators using various materials (e.g., libosada c 1997, urquico c 1998, national and regional tourism master plans-department of tourism 1992-2010, economic and social commission for asia and the pacific 1992, whelan 1991). the choice of information used was based on what the facilitators deemed appropriate, given the status of the industry in bolinao, existing policies, practices and perceptions, and the potential impacts based on the experiences in other areas. consultations concerning the information were held with community members, the project staff, as well as external consultants. information sources were the dot, the up-ait, non-governmental organizations with community-based sustainable tourism projects (i.e., asset, inc., haribon foundation), the doh, and secondary literature gathered from libraries and from the network of cbcrm practitioners. resource persons from the ait, denr, pcmard, and dot as well as the experiences of other communities in tourism helped give a more realistic perspective to tourism development. science and information for remediation of unregulated aquaculture. in seeking solutions to current issues, science found its role in enhancing the knowledge and capability of stakeholders to enable them to understand the nature of the issue and options to mitigate it. in the case of the aquaculture industry, the community did not need to be warned of the potential damage because they were already experiencing the negative consequences of unregulated aquaculture. what they needed was a means to remedy their situation. information gathered from secondary literature and from technical studies on the impacts of congested aquaculture structures in an area, unregulated aquaculture practices in feeding and stocking, the need for monitoring water quality and production were highlighted in an orientation held in november 1999. stakeholders were trained to use water quality monitoring instruments. they were taught to monitor their production and to take account of their stocking densities and consumption of feeds. these data were later analyzed for use in policy formulation. aside from the advocacy facilitators, resource persons from the the use of science in environmental advocacy 97 bfar, up-msi, and aquaculture department of the southeast asian fisheries development center (seafdec) helped in the transfer of knowledge and skills in aquaculture. valuable sources of information that were used in the advocacy activities came from site specific studies (e.g., aliño and villanoy 1997, meñez and others 1991, verceles and talaue-mcmanus this volume). other sources included materials from bfar, fao, and seafdec, and secondary literature on topics such as water quality, feeds and chemicals, environmental impacts, trade and marketing, social equity, and advances in aquaculture. as in tourism advocacy, information used in the form of primers, newsletters, orientations, and sharing sessions for aquaculture advocacy were processed and packaged by the facilitators. science and information for rehabilitation of declining capture fisheries. science has been looked upon as a source of alternative strategies for traditional practices that have caused significant negative impacts on the resource base and on cultural structures. information on the fishery resources of bolinao, status of habitats and the biology and life cycles of the fisheries, interaction of ecosystems, and impacts of activities on the resources were bases for management interventions in the cdp. the need for and importance of establishing marine protected areas was facilitated by advocacy. the identification of areas for protection depended on the expertise of scientists from the msi. results of fisheries monitoring have influenced policies on the management of the reef resources. solutions of issues in the concession systems, dwindling catch, destruction of habitats, and pollution were sought through technical research and scientific advice. impacts and issues, such as dwindling fishery, destruction of habitats, and inequitable resource use, that arose from traditional practices may take some time before rehabilitative solutions take significant effect. identifying alternative strategies for the management of the concession systems entailed gathering information and conducting advocacy activities. significant sources of information for the capture fisheries were mostly site-specific studies (e.g. talaue-mcmanus and others 1992, estepa 1997, ochavillo 1998, rodriguez, 1997) and results of the mfrmp’s fishery monitoring activities (1997present). all the site-specific information was simplified and presented in fora, meetings, and sharing sessions with the affected fisherfolk sectors and representatives of the local government. as mentioned above, these sessions provided venues for the participants to identify strategies and alternative options to address both the inequitable access to the resources and the dwindling fisheries. advocacy activities that followed were limited to lobbying the municipal council and the lgu to influence changes in policies in the use and management of the capture fisheries. challenges environmental advocacy in bolinao has not been without challenges (table 2). the project, having access to technical information and the means to share this with the larger community, was responsible for facilitating the advocacy process toward constructively addressing negative impacts of unregulated use of coastal resources. in identifying issues and problems, there is the challenge of non-issue perception if the issue or problem is not perceived by the community. this is usually the case when common practices or economic activities are the sources of negative impacts (e.g., decline in siganid fish catch, concession system, deteriorating water quality). an issue must be accepted by the community as an issue before a sector or community can act on that issue. it must prove to be pressing enough for the community members to put in their time and resources. otherwise, the initiatives to act on the issues would eventually lose momentum. mobilizing stakeholders to establish regulations for the tourism industry entailed almost a year of advocating and coordinating with the local government, bhrroa, the transportation sector, health sector, local media, academic institutions, and table 2. challenges faced in mobilizing community advocates > not anticipated > non-issue identifying an issue/ problem gathering information sharing information participatory action > choosing appropriate information > unpopular forms > naccessible sources > inability to identify target sectors > heterogeneity of views within a sector > non-use of information > biased use of information aguinaldo & talaue-mcmanus 98 religious groups. having engaged even a fraction of all these sectors to participate in the drafting of a plan, in practicing information they have received, and in advocating a precautionary approach in the development of tourism in the area is proof of that the challenge has been meet. in gathering information, the challenge to the facilitator lies in choosing the information to use, popularizing the data, and finding the most appropriate method and venue to transfer the information. data and information must be related to the present conditions of a community for it to want to act on the issue. in addition, the information must be simple enough to understand and use. there are times when what science deems important (e.g., research objectives) is different compared to what the community perceives. science has a natural bias for ecological concerns. communities are almost always concerned about livelihood and basic survival. it usually takes a compromise between these two concerns to evolve an effective management strategy. information may be very powerful if it is clear and simple and connected to the realities a community is faced with. finding the most appropriate venue to transfer valuable information is a balance between available resources, time, and scope of target sectors. the most often used strategy was the conduct of orientations and meetings. while orientations and meeting gave opportunities for deepening the discussions and information exchange, these required financial resources that were most often provided by the mfrmp, and at times by the local government. the sessions were at times too long for participants to stay for the duration of the activities because of economic and familial responsibilities. venues that required less mobilization of finances and were less time-consuming would allow a wider reach of information. in sharing information, given constraints on funding, time, and manpower, identifying target sectors has been a challenge. should an issue be shared by the whole community or should one take heed of opinions of other sectors not directly affected but nevertheless part of the community? it was expected that not everyone would be equally receptive to available information when this would require them to rethink their practices. it may also take time for the community to accept and act on the information that has been disseminated, sometimes taking years or however long it would take for negative impacts to be observed. identification of recipients and potential advocates was crucial to the sustainability or even the progress of an action on an issue. issues on reef fisheries affected various sectors in the island. identified interventions for the management of the reef (e.g., regulating fish corrals and implementing a closed season for the siganid fishery) required consultations with the different resource users (e.g., gleaners, fish corral owners and operators, hook and line, net, fishtrap, and other marginal fishers), the village councils, and the community members. advocacy initiatives involved the conduct of orientations and validation sessions that took a long time and did not amount to any real action that would address the issue. sometimes it was because the participants to the meetings had neither a direct stake on the issue nor the means to implement the actions. there were also times when the sessions were participated in by representatives of various sectors who had various interests, causing the discussion to veer from issue to issue. focusing on one issue would mean the exclusion of the other sectors. advocacy strategies shifted from village-level consultations to focused sectoral discussions. though the reach may not be as wide, the focused sectoral discussions have resulted in a number of initiatives that were being taken on by small partner groups. at present, there are five groups initiating action on mangrove reforestation, marine protected area establishment, experimental siganid culture, and lobbying for regulations on the use of the municipal waters. after the information had been shared, the next challenge was how the community would use the information. groups or individuals who facilitated the flow of information have taken a social responsibility to maximize and exhaust means to make the community or the affected groups act on the information with equal responsibility. if the community would use the information, would they use the information properly, or would they just use it for their own motives? a common perception would be that the community had only one stand on issues and that a community consultation would validate this. in reality, the demands or opinions of all sectors of the community when given a voice and the chance to participate reflected different the use of science in environmental advocacy 99 views on issues. the greatest challenge was resolving these differences and arriving at a consensus to pursue appropriate action. an example was the advocacy for the establishment of a marine protected area (mpa) in malilinep channel. efforts toward this end have taken more than five years of information dissemination and consultations. the proposed mpa site has been transferred and its size reduced and enlarged a number of times to accommodate all affected users. until now, the mpa has yet to be established. effective strategies in facilitating the flow of information lgus, peoples’ organizations, and cbcrm practitioners from other areas have considered bolinao as a valuable and rich source of replicable cbcrm strategies in coastal development planning, resource enhancement, capability building, networking, and issue advocacy. ten years of cbcrm work in bolinao has given both the community and cbcrm facilitators and stakeholders the opportunity to try out a suite of strategies that would be most effective in sustainably and equitably managing the municipality’s coastal resources. the advocacy efforts of the different groups in the area for the past three years have had its share of both unsuitable and effective strategies. strategies which have been effective in the use of science to mobilize cbcrm actions have been cognizant of the following: 1. issues should be both community-related and scientifically grounded. if an issue is identified by the community, utilize science to help the community understand its nature. if an issue is identified by science, show the community how it relates to them and their resources. 2. popularize scientific information, but be transparent about the state of the art of scientific knowledge. use the precautionary principle when scientific evidence is equivocal, and act with preference for the protection of the environment and marginal users. 3. among the most appropriate methods of dissemination are: (1) written materials for wider reach and longer duration of use; (2) community theater for the novelty of audio-visual appeal from fellow community members; and (3) sectoral orientations and meetings for deepening knowledge and understanding of issues. 4. target sectors should include those who are negatively affected and those who can do something constructive about the issues (i.e. peoples’ organizations and lgus). also, include sectors along the chain of resource production (aquaculture) or distribution (capture fisheries) who directly use the coastal resources. 5. action must represent a process of consensus, to sustain the action and to minimize conflict. 6. institutionalize initiatives when possible (i.e., crm office) to ensure that initiatives are sustained. 7. work within existing structures and programs in different sectors of the community instead of creating new ones. sometimes existing structures may need to be reconfigured, but this will be less resource-intensive (time, material, manpower) compared to creating new ones. it also ensures sustainability while creating a venue for crm. the capability of the mfrmp and other groups to make information accessible to the communities and the local government, and to train stakeholders in looking after their own resources through monitoring, have more often than not helped provide solutions. bridging the communities with agencies, groups and organizations, experts who have the knowledge, technical know-how, and experience has spelled the difference between talking about solutions and actually doing them. references aliño p, villanoy c. 1997. technical considerations for the evaluation of the sustainable management of fishpens and fishcages in bolinao and anda, pangasinan. in the coastal development plan of bolinao. estepa b. 1997. assessment of siganid concession as baseline information for community-based coastal resources management. (unpublished). aguinaldo & talaue-mcmanus 100 libosada c. 1997. ecotourism in the philippines. makati city: bookmark, inc. 203 p. mcmanus j, nañola c, reyes r, kesner k. 1992. resource ecology of bolinao. manila, philippines: international center for living aquatic resources management (iclarm). 117 p. o’ malley s. 1999. environmental education and iec: information, education and communication of environmental messages in palawan. in: communitybased strategies in natural resources management. philippines: volunteer service organization. rodriguez s. 1997. the barangen fishing concession in bolinao: an ethnographic study of a customary marine tenure system [dissertation]: quezon city, philippines: university of the philippines. talaue-mcmanus l, yambao a, salmo s, aliño p, 1999. participatory planning for coastal development in bolinao, philippines: a powerful tool for conflict resolution. in: buckles d, editor. conflict and collaboration in natural resource management. idrc/ worldbank. p 149-157. urquico c, editor. 1998. community-based sustainable tourism. quezon city, philippines: asset, inc. 92 p. verceles l, talaue-mcmanus l, alino pm. 1999. participatory monitoring and feedback system: an important entry towards sustainable aquaculture in bolinao, northwestern philippines. science diliman (this issue). whelan t. 1991. nature tourism. washington d.c.: island press. 223 p. the bolinao coastal and fisheries resources management ordinance of 1999. 2000. northern journ 4: 38. bolinao municipal records, 1998. coastal development plan of bolinao, 1997. (unpublished) fisheries code of the philippines, 1998. lingayen gulf coastal area management commission (lgcamc) reports. 1997. (unpublished) lingayen gulf coastal area management commission (lgcamc) resolution no. 12 s. 1996. lingayen, philippines. 13info for authors.pmd 113 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the off ice of the vicechancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles and short communications may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out. the letter to the editor usually contains these: that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1" margins on all sides. each page of the manuscript must include continuous line numbers in the margin. all pages should be numbered consecutively on the upper right hand corner of the page. information for authors 114 7. page 1 should contain the article title, author(s), aff iliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. the author must also submit a layman’s abstract of not more than 200 words. 9. the paper should be organized as follows: abstract and layman’s abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, f igures, tables, and f igure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be def ined at f irst mention. 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the f irst author’s name plus “et al.” need to be given. references in the text should follow the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 115 examples: articles from journals: print ( section 29.3.7.1 p. 518-527) format: author(s). date. article title. journal title. volume(issue):location. example: smar t n, fang zy, marwick th. 2003. a practical guide to exercise raining for heart failure patients. j card fail. 9(1):49-58. ar ticles from journals: onl ine (section 29.3.7.13 p. 557-558) format: author(s) of article. date of publication. title of article. title of journal (edition) [medium designator]. 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dissertation and thesis [content designator]. place of publication: publisher. extent. notes. example: lutz m. 1989. 1903: american nervousness and the economy of cultural change [dissertation]. stanford (ca): stanford university. 118 group/corporate author (section 29.2.1.2.5 p. 494) format: [abbreviation of group] name of group (country). date. title. place of publication: publisher. notes. example: [iom] institute of medicine (us). 1975. legalized abortion and the public health; report of a study by a committee of the institute of medicine. washington (dc): national academy of sciences. other internet materials (section 29.3.7.13 p. 556-564) homepage format: title of homepage [medium designator]. date of publication. edition. place of publication: publisher; [date updated; date cited]. notes. example: apsnet: plant pathology online [internet]. c1994-2005. st paul (mn): american phytopathological association; [cited 2005 jun 20]. available from:http://www.apsnet.org/. for more detailed examples please refer to the cse manual 7th edition. 12. the list of references at the end of the paper should include only works mentioned in the text and should be arranged alphabetically by the name of the author. 13. responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. 14. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 15. figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each f igure or table on a separate page. at the lower hand corner, the name of the author and the f igure number should be indicated. 119 16. illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2" x 11" size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructedphoto-graphic prints (not photocopies), trimmed at right angles and in the f inal size desired by the author. 17. when possible, all organisms must be identif ied by the scientif ic binomen. 18. mathematical equations should be clearly presented so that they can be interpreted properly. 19. obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 20. all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc. , especially those adopted by the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 22. less common abbreviations may be printed as footnotes. 23. short communications must be guided by the following points: • short communications are reports of limited data or important findings that warrant publication before the completion of the study; • short communications are reports of signif icant new data arising from problems with narrow, well def ined limits before broader studies are completed; and results have not been published in print elsewhere, e x c e p t a s p a r t i a l c o m m u n i c a t i o n s o r p o s t e r s i n c o n f e r e n c e proceedings; 120 • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; • abstracts will be required on submission but not to be part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages ) in length with simple layout, a maximum of two tables and two f igures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors may opt to submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor in chief science diliman off ice of the vice-chancellor for research and development university of the philippines lower ground floor, phivolcs bldg. , c.p. garcia avenue up campus, diliman, quezon city 1101, philippines camera-ready illustrations must accompany the manuscript. 11ethics.pmd 71 journal pol icy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the off ice of the v ice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 72 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research area concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “notes to contributors” stipulates that “all ar ticles must have a high degree of scholarship,” that “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a cer tif ication from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right… .” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as def ined above and; b.) is specif ic and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the bases for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specif ic advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the 73 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientif ic misconduct policy. in attendance were: d r. co r a zo n d. v i l l a r e a l , r d u o d i r ec to r, p r e s i d i n g ; d r h e n r y j . ra m o s , p m rg o director and professor, nip; atty. vy va v ictoria aguirre, ovcrd legal consultant; e d i to r s i n c h i e f d r. m a r i co r s o r i a n o (science dil iman) a n d d r. m a r i a m a n g a h a s (s o c i a l s c i e n c e d i l i m a n ) . m s . n a n i e d o m i n g o a n d m s . d e r c y m a r a r a c , e d i t o r i a l assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. 74 references council of science editors. “ white paper on promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ec to r s o n s e p te m b e r 3 , 2 0 0 6 .” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ ajp/submit.html 3layman'sabstracts.pmd layman’s abstracts 1 science diliman (july-december 2017) 29:2, 1-3 layman’s abstracts a hybrid cfd-bem analysis of the aerodynamic performance of a cut-out hollow pipe horizontal axis w ind turbine blade neil richard p. tanguil ig and louis angelo m. danao issn 0115-7809 print / issn 2012-0818 online this study investigated the performance of a cut-out hollow-pipe blade prof ile in small horizontal axis wind turbines (hawt). although this type of blade was expected to have losses in eff iciency, such blade prof ile can be easily manufactured locally, and could potentially have a lower c o s t c o m p a r e d t o c o n v e n t i o n a l b l a d e s w i t h a e r o f o i l p r o f i l e s . computational fluid dynamics (cfd) was used to derive the aerodynamic characteristics of the cut-out hollow-pipe sections. the blade element momentum (bem) method was then used to investigate the performance of the hawt’s rotor. cfd results show that cut-out hollow-pipe sections have poor aerodynamic characteristics due to their simple geometry and crude design. bem results demonstrate that rotor with cut-out hollowpipe blades can still work but only at low tip speed ratios. other relevant a n a l y s e s s h o w t h a t t h e p e r f o r m a n c e c a n b e i m p r o v e d b y a l t e r i n g t h e pitch of the blades and by adding additional blades to the rotor. genetic comparison of oncomelania hupensis quad rasi (möllendorf, 1895) (gastropoda: pomatiopsidae), the intermed iate host of schistosoma japonicum in the phil ippines, based on 16s ribosomal rna sequence james christopher c. chua, ian kim b. tabios, pebbles grayle p. tamayo, lyd ia r. leonardo, ian kendrich c. fontanilla, raffy c. fornillos, emmanuel ryan c. de chavez, takeshi agatsuma, mihoko kikuchi, naoko kato-hayashi and yuichi chigusa schistosoma japonicum is a parasitic fluke that makes use of humans and other vertebrates as hosts. its infective stage is the water-borne cercaria that burrows through skin. one of its intermediate hosts that release crecariae is the freshwater snail oncomelania hupensis. there are many layman’s abstracts 2 morphological characterization, evaluation and selection of hibiscus (hibiscus rosasinensis l) hybrids elena may n. cabarrubias, pabl ito m. magdal ita, antonio g. lalusin and norma g. med ina fifty-seven hibiscus siblings derived from the cross-breeding of different parents were assessed for various characteristics to select plants with g o o d f l owe r co l o r a n d fo r m . o u t of 5 7, 1 4 h y b r i d s we r e s e l ec ted. t h e c h a r a c t e r i s t i c s o f t h e s i b l i n g s w e r e s u b j e c t e d t o p r i n c i p a l c o m p o n e n t analysis (pca) and cluster analysis. pca revealed three major pcs with e i g e n v a l u e > 1 c o n t r i b u t i n g 7 8 % o f t h e t o t a l a c c u m u l a t e d d i f f e r e n c e s among the siblings. for instance, in pc-i, the contribution of flower size, leaf size, and style length to variation in flower traits was the highest. u s i n g t h e t h r e e m a j o r p c s , t h e 5 7 s i b l i n g s w e r e g r o u p e d i n t o f i v e different clusters. cluster-i had 16 yellow-orange and purple members. cluster-ii had 15 white and red-purple members. cluster-iii comprised f ive yellow members. cluster-iv was composed of 13 yellow and yellowo r a n g e m e m b e r s . c l u s t e r v h a d 8 m e m b e r s w i t h r e d a n d r e d p u r p l e flowers. the mean size of the flowers of all siblings was 130.21 mm. pca revealed that the siblings with large flowers and longer petioles tend to have wider leaves, which agrees with the dendrogram groupings o f t h e 5 7 h i b i s c u s h y b r i d s . c o r r e l a t i o n b a s e d o n p c a r e v e a l e d a s u b s p e c i e s o f o . h u p e n s i s i n a s i a ; i n t h e p h i l i p p i n e s , t h e s u b s p e c i e s p r e s e n t i s o . h u p e n s i s q u a d r a s i . t h e g e n e t i c d i v e r s i t y o f t h e s n a i l intermediate host may also reflect the genetic diversity of the parasite, which in turn, may have implications on the susceptibility of a snail to the parasite as both may co-evolve with each other. this study aimed to assess the genetic diversity of o. h. quadrasi isolates based on the 16s r r n a g e n e f r o m n i n e p r o v i n c e s k n o w n t o h a v e s . j a p o n i c u m i n t h e philippines, namely cagayan valley, bohol, negros occidental, leyte, davao, davao del sur, mindoro oriental, samar, and sorsogon. o. h. hupensis and o. h. nosophora subspecies were also collected from china and japan, respectively. the results reveal that the philippine o. h. quadrasi had four distinct sequences, but these were not correlated with their geographical location. furthermore, the philippine isolates formed a distinct group from the other subspecies, conf irming its taxonomic designation. layman’s abstracts 3 determination of cr, cd, sn and pb in selected herbal products available in phil ippine markets joan s. de vera, leni b. quirit and irene b. rodriguez m a n y f i l i p i n o s r e l y o n h e r b a l p r o d u c t s d e s p i t e t h e l a c k o f s t u d i e s validating their safety. one concern in the use of herbal supplements is m e t a l c o n t a m i n a t i o n , w h i c h m a y b e i n t r o d u c e d f r o m t h e e n v i r o n m e n t w h e r e t h e p l a n t s a r e g r o w n , a s w e l l a s d u r i n g m a n u f a c t u r i n g . w h e n ingested, metals, such as lead and cadmium, can accumulate in the body and cause various health problems like liver and kidney damage. some forms of tin and chromium are also known to be toxic to humans. in light of this, we conducted a study to analyze the amount of these trace metals in selected herbal products with various plant ingredients. most of the products tested had measurable trace metal concentrations, which w e r e b e l o w t h e s u g g e s t e d m a x i m u m l i m i t s f o r c a d m i u m a n d l e a d i n h e r b a l p r o d u c t s . h o w e v e r, o n e p r o d u c t d e r i v e d f r o m m a n g o s t e e n exceeded the limit for cadmium. the variability of metal concentrations i n h e r b a l p r o d u c t s u n d e r l i n e s t h e f a c t t h a t p l a n t s a r e s u s c e p t i b l e t o contamination, and quality control during processing must be improved t o m i n i m i z e p o s s i b i l i t y o f c o n t a m i n a t i o n . t h e r e s u l t s o f t h i s s t u d y suggest that vigilant monitoring of herbal products is imperative to avoid exposure to trace metal contaminants. angiotensin-converting enzyme inhibitory action of selected plants dionisio bong b. singson and christine l. chichioco-hernandez hypertension is a leading cause of morbidity in the philippines affecting 2 0 % o f f i l i p i n o a d u l t s . u n t r e a t e d h y p e r t e n s i o n m a y l e a d t o s t r o k e , blindness, heart attack, and kidney and heart failure. one way to lower blood pressure is through the use of angiotensin-converting enzyme (ace) inhibitors like captopril. howeve r, the use of such drugs is associated with unwanted side effects. plants are good sources of potential effective anti-hypertensive compounds with minimal side effects. signif icant positive association between flower size and leaf size, and between petiole length and leaf size. sdinside front cover-july-dec.2016.pmd july-december 2016 • vol. 28 no. 2 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief irene m. villaseñor, ph.d. associate editors jose maria p. balmaceda, ph.d. louis angelo m. danao, ph.d. carlos primo c. david, ph.d. alonzo a. gabriel, ph.d. gil s. jacinto, ph.d. jonas p. quilang, ph.d. arnel a . salvador, ph.d. terence p. tumolva, d.eng. managing editor gonzalo a. campoamor ii, ph.d. editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor sarah mae u. penir on the cover: a p r i s t i n e f o r e s t e d s t r e a m i n s i l a g o , southern leyte, philippines. photo taken by alyssa fontanilla of the institute of biology, university of the philippines diliman. teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university cleveland, ohio, usa kenneth buckle, ph.d. food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, jr., ph.d. department of electrical and computer engineering ohio state university university of california, irvine university of illinois, urbana, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield sheff ield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. centre for scientif ic computing and department of physics university of warwick united kingdom raul k. suarez, ph.d. department of ecology, evolution and marine biology university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. sdinside front cove-july-december 2014.pmd july-december 2014 • vol. 26 no. 2 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june a n d d e c e m b e r ) b y t h e u n i v e r s i t y o f t h e philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief marco nemesio e. montaño, phd associate editors jose maria p. balmaceda, phd carlos primo c. david, phd joel joseph s. marciano jr. , phd jonas p. quilang, phd arnel a . salvador, phd irene m. villaseñor, phd managing editor gonzalo a. campoamor ii, phd editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor patricia s. san jose on the cover: a 2-uniform tiling of the plane by squares and equilateral triangles wherein a set of tiles of the same color form the same pattern as other sets of colored tiles. moreover, the symmetry group of the tiling induces a permutation of the colors. the coloring is a transitive and perfect coloring using 9 distinct colors. description of the image by lawrence a. eclarin of mariano marcos state university rigoberto c. advincula, phd department of chemistry university of houston alfonso m. albano, phd department of physics bryn mawr college, bryn mawr, pennsylvania kenneth buckel, phd food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, phd department of electrical and computer engineering ohio state university flor crisanta f. galvez, phd quality assurance and technical manager kerry ingredients and flavours (americas region) 7989-82nd st. , delta, vc v4g 1l7, canada victor c. gavino, phd department of nutrition university of montreal, canada kelvin s. rodolfo, phd department of earth and environmental sciences university of illinois, chicago, illinois rudolf a. roemer, phd centre for scientif ic computing and department of physics university of warwick luis g. sison, phd electrical and electronics engineering institute university of the philippines diliman raul k. suarez, phd department of ecology, evolution and marine biology university of california, sta. barbara contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. 10call for papers-new.pmd 86 humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) analyn salvador-amores, myles capareda & fenelyn nabuab call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 2editor's note-july-dec.2015.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online dear colleagues, welcome to the december 2015 issue of science diliman. let me f irst thank the authors for sharing basic knowledge as well as applications of researches in their f ields of expertise. i also thank the science diliman team for completing the publication of this issue. this issue presents four diverse researches covering quaternion matrices , synthesis of copper nanoparticles, barcoding of catf ish, and optimized extraction of mango kernel butter. quaternions are also used in electromechanics, quantum mechanics, and 3d animation to calculate movement and rotation. i viewed a real-world application of quaternions in computer graphics with the open source program nasa worldwind at http://worldwind.arc.nasa.gov/java/. the research paper of ralph john dela cruz gave a mathematical description of quaternion matrices. metallic copper nanoparticles were successfully synthesized and characterized by mary donnabelle l. balela and kathy lois s. amores. the synthesized copper nanoparticles also exhibited antimicrobial activity. nanoparticles or nanopowders or nanoclusters or nanocrystals are submicroscopic with dimensions up to 100 nanometers. nanoparticles have varied applications in medicine (e.g. , in drug delivery systems), in manufacturing (e.g. , as industrial catalysts) and materials (e.g. , as n a n o co m p o s i t e s ) , i n e n e r g y ( e . g . , a s b a t t e r i e s ) a n d e l e c t r o n i c s ( e . g . , a s supercapacitors), and in the environment (e.g. , in remediation). different species of catf ish may look very similar in terms of their morphological features to the untrained eye. dna barcoding, which uses a very short genetic sequence from a part of the genome, was utilized by the research group of brian s. santos to distinguish the different catf ish species in the philippines. the gene region used by the research group is the 648 base-pair region in the mitochondrial cytochrome c oxidase 1 gene “coi”. santos et al. concluded that c. batrachus and c. macrocephalus are native to the philippines while c. gariepinus is an introduced species. 2 irene m. v illaseñor, ph.d. editor-in-chief the mango seed, which is a major waste of mango, is a potential source of oil. mango oil may be used as edible oil, as extenders in manufacturing soap and cosmetics, and it may also provide nutritional benef its. mango kernel butter is well-characterized in terms of its chemical composition with oil content of 8 to 10%. it has a similar chemical composition to shea and cocoa butter. its major constituents are oleic acid and stearic acid. the research group of edgardo v. casas optimized the parameters used in the microwave pretreatment of mango kernels, which increased the yield of mechanically extracted mango kernel butter. microwave is a non-ionizing electromagnetic radiation. please read the complete articles including the layman’s abstracts to better understand the researches. cadmium and lead determination-rodriguez.pmd cadmium and lead determination by icpms 1science diliman (january-june 2012) 24:1, 1-11 cadmium and lead determination by icpms: method optimization and application in carabao milk samples riza a. magbitang1 and irene b. rodriguez1,2* 1natural sciences research institute, college of science university of the philippines diliman, quezon city, 1101 philippines 2institute of chemistry, college of science university of the philippines diliman, quezon city, 1101 philippines *corresponding author: tel/fax. +632 9205427; email: ibrodriguez@upd.edu.ph abstract a method utilizing inductively coupled plasma mass spectrometry (icpms) as the element-selective detector with microwave-assisted nitric acid digestion as the sample pre-treatment technique was developed for the simultaneous determination of cadmium (cd) and lead (pb) in milk samples. the estimated detection limits were 0.09µg kg-1 and 0.33µg kg-1 for cd and pb, respectively. the method was linear in the concentration range 0.01 to 500µg kg-1with correlation coefficients of 0.999 for both analytes.the method was validated using certified reference material bcr 150 and the determined values for cd and pb were 18.24 ± 0.18 µg kg-1 and 807.57 ± 7.07µg kg-1, respectively. further validation using another certified reference material, nist 1643e, resulted in determined concentrations of 6.48 ± 0.10 µg l-1 for cd and 21.96 ± 0.87 µg l-1 for pb. these determined values agree well with the certified values in the reference materials.the method was applied to processed and raw carabao milk samples collected in nueva ecija, philippines.the cd levels determined in the samples were in the range 0.11 ± 0.07 to 5.17 ± 0.13 µg kg-1 for the processed milk samples, and 0.11 ± 0.07 to 0.45 ± 0.09 µg kg-1 for the raw milk samples. the concentrations of pb were in the range 0.49 ± 0.21 to 5.82 ± 0.17 µg kg-1 for the processed milk samples, and 0.72 ± 0.18 to 6.79 ± 0.20 µg kg-1 for the raw milk samples. keywords: trace metal, carabao milk, icpms, microwave digestion, food contaminant rodriguez, i.b. and magbitang, r.a. 2 science diliman (january-june 2012) 24:1, 1-11 introduction the occurrence of heavy metals like cadmium (cd) and lead (pb) in the environment has been the subject of a wide range of published work from analytical methods for their determination to understanding the mechanism of action leading to their toxicities (lidsky & schneider, 2003; bertin & averdeck, 2006; patrick, 2006; patra et al., 2011). both cd and pb are ubiquitous in nature and the concentrations are highly dependent on anthropological activities. pb was popularly used during the early days in large scale manufacture of tableware and pipes for water supply (garcia-lestün et al., 2010). the use of pb in pipes, which was prevalent until the 19th century, eventually declined due to incidence of lead poisoning (tong, 2000; järup, 2003). but the use of pb as anti-knock agent and as pigment in paints has persisted until recently (garcialeston et al., 2010). cd is commonly used in industries such as electroplating, pigments, synthetic chemicals, ceramics, metallurgical and photographic products, electronic and other industries (sadegh safarzadeh et al., 2007). these widespread uses are the most common sources of these contaminants in the environment. cd and pb are of interest due to health concerns associated with these metals. the effects of cd have been linked to higher risks of cardiovascular disease mortality in males, increased bone fragility, children’s nephrotoxicity, and decreased visual ability. pb is considered to affect the brain heavily, especially during children’s developmental stage, which can result in a change in cognitive function, social behavior and slow learning ability. in adults, exposure to pb may cause kidney problems and neurodegeneration (genuis et al., 2010). these effects have pushed authorities to set limits for these contaminants. the joint fao/who food standard programme codex committee on contaminants in foods (cccf) released a new list of maximum levels (mls) for contaminants and toxins in food last march 2011. for cd, the committee established a provisional tolerable monthly intake (ptmi) of 25 µg kg-1 body weight. however, no ml was established for cd in milk. in the case of pb, the committee estimated that the previously established provisional tolerable weekly intake (ptwi) of 25µg kg-1 body weight was associated with a decrease of at least 3 iq points in children and an increase in systolic blood pressure of approximately 3 mmhg (0.4 kpa) in adults. these consequences were considered important effects when viewed on a population level and the committee therefore withdrew the ptwi as it could no longer be considered health protective. nevertheless, they retained the ml of 0.020 mg kg-1pb in milk. carabaos (water buffalos) in the philippines were mainly raised to augment manpower in farming until early 1990s when the use was diverted towards meat and milk production. buffalos contribute 72 million tons of milk and 3 million tons of meat annually to world food, much of it in areas that are prone to nutritional imbalances (singh & barwal, 2010). in 2010, the total carabao milk production in the philippines reached about 5.8 million liters (bas, 2011). the nutritional content of milk makes it important as a food source in the filipino diet either as raw milk or processed as cheese, yogurt, butter, cream and native filipino sweets. the increasing utilization of carabao milk for food production in the philippines intensifies the need for a method suitable for the determination of relevant heavy metals. in this work, we present the use of microwaveassisted acid digestion for the complete mineralization of milk samples prior to the simultaneous detection of cd and pb using icpms as the element-selective detector. this simple and reliable method was aimed for use in routine monitoring of these environmental pollutants in milk samples. in addition, we report for the first time the analysis of these elements in carabao milk, both raw (obtained directly from the farm) and processed (commercially available) milk samples in the philippines. methodology chemicals and reagents all chemicals and reagents used in this work were of analytical reagent grade unless otherwise specified. nitric acid was obtained from merck (darmstadt, germany), hydrochloric acid was obtained from mallinckrodt chemicals (thailand), and hydrogen peroxide was from sigma-aldrich (steinheim, germany). single-element standards of cd, pb and rhenium (re) with concentrations equivalent to 10,000 cadmium and lead determination by icpms 3science diliman (january-june 2012) 24:1, 1-11 ± 30 µg ml-1 and indium (in) with concentration equal to 1,000 ± 3 µg ml-1were purchased from cpi international (santa rosa, ca, usa). a certified reference material, bcr 150 (spiked skim milk powder), was obtained from the european commission, joint research center, institute for reference materials and measurements (ec-jrc-irmm, geel, belgium). another reference material, nist 1643e (trace elements in water) was obtained from the national institute of science and technology (gaithersburg, md, usa) and was used for validation. all dilutions and solution preparations were done using ultrapure water prepared using a barnstead system (18.2 mωcm resistivity, thermo fisher scientific, selangor darul ehsan, malaysia). instrumentation a multiwave 3000 microwave digestion system (anton paar, graz, austria) fitted with a 16-position rotor for high digestion performance was used for the complete mineralization of the samples. an agilent 7500cx icpms (agilent technologies, germany) equipped with a micromist glass concentric nebulizer and an integrated autosampler (i-as with type a vials, 89 x 6 ml capacity) was used as the element-selective detector. the monitored masses were m/z 111 for cd and m/z 208 for pb. the analysis was performed using standard mode and the optimum conditions typically used for the analyses, based on a previous study (torres et al., 2010), are as follows: rf power = 1550 w, carrier gas flow rate = 0.85 lmin-1, make-up gas flow rate = 0.25 l min-1, nebulizer pump operated at 0.01 rps, sampling depth at 8.0 mm and the spray chamber was kept at 2.0°c. samples and sample preparation raw milk samples and processed milk samples were obtained from different towns and cities in nueva ecija, philippines. nueva ecija is the largest province in central luzon covering an area of 5, 284 km2 where majority of its land is utilized for agricultural purposes. as of january 1, 2011, the total carabao population in the province is 51,442 (bas, 2011). nueva ecija gathers more milk from carabao than any other place in the philippines and is the major supplier of carabao milk in metro manila (pcc, 2011). the location, age, breed and type of diet of the animal sources for the raw milk samples are summarized in table 1, along with the details about the processed (commercially available) milk samples. all samples were stored in polyethylene containers and kept at 4.0°c during transport to the laboratory. triplicate aliquots (0.500 ml each) of the milk samples were subjected to the optimized mineralization procedure prior to icpms analysis. results and discussion optimization and validation of method the certified reference material bcr 150 was used for the method optimization and validation studies. this material is available in powder form but it is the only available certified reference material which is closely matched in terms of matrix to the target samples for the application of the method. in the method optimization, parameters were varied such as amount of sample, maximum power, and different acid and acid mixtures used in digestion.the use of microwave digestion for sample pre-treatment has been reported by dobrzañski et al. (2005) and ataro et al. (2008). these previous studies used different microwave digestion systems compared to the one available to us, thus we found it necessary to optimize the conditions. initially, different mineralization agents were added: nitric acid only, acid mixtures (nitric acid and hydrochloric acid), as well as mixtures of acid and oxidizing agent (nitric acid: hydrogen peroxide) at different proportions (2:1 and 3:1, v:v) while other parameters(amount of sample: 0.2 g, digestion period: 45 min, and maximum power of digestion: 600 w) were held constant. figure 1a shows the percent recovery of cd and pb achieved with the different acid and acid mixtures used in the mineralization process. after subjecting to anova, it was apparent that the results obtained do not have significant differences (at α <0.05), thus the subsequent optimization step was focused on the use of lowest amount of acid (2 ml nitric acid) added to 6.0 ml of water. this choice of mineralization agent will ensure that possible contamination during the sample preparation is minimized due to addition of only one rodriguez, i.b. and magbitang, r.a. 4 science diliman (january-june 2012) 24:1, 1-11 table 1. milk samples obtained from the different cities and municipalities of nueva ecija with product description (for the processed milk samples) and information about the animal source (for the raw milk samples) processed milk samples (commercially available) sample place of origin product description cmca1 talavera, nueva ecija pasteurized milk with coffee flavor cmca2 talavera, nueva ecija pasteurized milk with buko-pandanflavor cmca3 talavera, nueva ecija pasturized milk with chocolate flavor cmca4 talavera, nueva ecija pasteurized milk raw milk samples sample place of origin age diet breed (years) cmc1 palayan city, nueva ecija 5 grass only philippine carabao cmc2 cabanatuan city, nueva ecija 7 grass only philippine carabao cmc3 cabanatuan city, nueva ecija 10 grass only philippine carabao cmc4 palayan city, nueva ecija 7 grass only philippine carabao cmq1 quezon, nueva ecija 4 grass and rice hay bulgarian murrah buffalo cmq2 quezon, nueva ecija 7 grass and rice hay bulgarian murrah buffalo cmq3 quezon, nueva ecija 5 grassand rice hay philippine carabao cmq4 quezon, nueva ecija 7 grassand rice hay bulgarian murrah buffalo cmq5 quezon, nueva ecija 6 grass and rice hay bulgarian murrah buffalo cmq6 quezon, nueva ecija 6 grass and rice hay mestiza (hybrid carabao) cmq7 quezon, nueva ecija 6 grass and rice hay mestiza (hybrid carabao) cmq8 quezon, nueva ecija 7 grass and rice hay mestiza (hybrid carabao) cmsd1 sto. domingo, nueva ecija 12 grass and rice hay bulgarian murrah buffalo cmsd2 sto. domingo, nueva ecija 11 grass and rice hay bulgarian murrah buffalo cmsd3 sto. domingo, nueva ecija 4.5 concentrate bulgarian murrah buffalo cmsd4 sto. domingo, nueva ecija 5 concentrate bulgarian murrah buffalo cmsd5 sto. domingo, nueva ecija 11 concentrate bulgarian murrah buffalo cmsd6 sto. domingo, nueva ecija 10 concentrate bulgarian murrah buffalo cmsd7 sto. domingo, nueva ecija 12 concentrate bulgarian murrah buffalo cmsl1 san leonardo, nueva ecija 15 grass only philippine carabao cmsl2 san leonardo, nueva ecija 10 grass only philippine carabao cmsl3 san leonardo, nueva ecija 8 grass only philippine carabao cmsl4 san leonardo, nueva ecija 5 grass and rice hay bulgarian murrah buffalo cmsl5 san leonardo, nueva ecija 10 grass only philippine carabao cmsl6 san leonardo, nueva ecija 12 grass only philippine carabao cadmium and lead determination by icpms 5science diliman (january-june 2012) 24:1, 1-11 reagent. also, the use of only one mineralization agent is more economical if the method is intended for use in routine monitoring. the maximum power used in the digestion was then varied (from 600 w to 800 w, and then to 1000 w) while keeping all other parameters constant. the power used in the microwave system linearly affects the temperature inside the digestion vessels. higher digestion power (1000 w) used ensures that the temperature inside the vessels reaches the range between 180°c to 200°c, which favors mineralization of the organic constituents in the samples. the digestion program employed was as follows: the system was ramped to 600 w for 5 min, kept at this condition for 5 min, ramped to 1000 w for 5 min and held at this condition for another 20 min, before allowing the system to cool. this digestion program made certain that the temperature in the system reached only a maximum value of 200°c. this maximum power was chosen to provide a safe distance from the set value of 210°c, which is the maximum tolerable temperature of the microwave digestion unit. when the set value is reached, the instrument will force the microwave system to instantaneously abort the program. the apparent increase in the percent recoveries of both cd and pb with increasing maximum power used can be clearly seen in figure 1b. it was then decided to use 1000 w for the succeeding optimization steps. to further optimize the method, the amount of sample was increased from 0.2 g to 0.5 g, while maintaining figure 1. percent recoveries of cd and pb as a function of different digestion parameters (acid used in the digestion, maximum power and amount of sample): (1a) effect of the use of different acid, acid mixtures, and mixture of acid and oxidizing agent on the percent recovery of the analytes in the reference material (amount of sample: 0.2 g; power: 600 w; digestion period: 45 min); (1b) effect of the maximum power used in digestion and (1c) effect of the amount of sample digested rodriguez, i.b. and magbitang, r.a. 6 other parameters at their respective values (period of digestion: 45 min; maximum power: 1000 w; and acid used in digestion: 2 ml nitric acid). the suggested minimum amount of the reference material that should be used for validation studies is 0.1 g. the data shown in figure 1c reflect that the recovery of both cd and pb slightly improved when higher amount of sample was used. this was expected due to the use of more of the homogenized sample. although there may be insufficient mineralization with larger amount of sample used, the results suggest that the digestion parameters were still suitable with the higher amount of sample. with this, it was deemed better to use higher amount of sample for better results. for subsequent analyses, the optimized parameters were as follows: 0.5 g of sample was digested with 2 ml nitric acid and 6.0 ml of water at 1000 w for a period of 45 min. after digestion, the digests were transferred to 15 ml polyethylene containers and the volume was adjusted to 10 ml using ultrapure water. these pretreatment procedures take about 2 hr (for triplicate runs of 5 samples/crms) from the weighing step until the digests are ready for icpms analysis. the digests were then subjected to icpms analysis using the optimized parameters in the agilent 7500 icpms system (torres et al., 2010). internal standards composed of in (m/z= 115) and re (m/z= 185) were incorporated to the sample via peristaltic pump. internal standards were added to compensate for any instrument drift and possible signal suppression or enhancement from matrix effects (vanhaecke et al., 1992). the choice of internal standards is based on several requirements: that it should be near the m/z ratio of the target analytes, it should have comparable ionization energy and it should not be widely available in nature (park & song, 2005). for our purpose, we selected the internal standards in for cd and re for pb. figure 2. evaluation of the linearity of the calibration standard solutions. (2a) shows the linearity of the calibrations standard solutions; (2b) and (2c) show results of the residual analyses for cd and pb standard solutions, respectively. science diliman (january-june 2012) 24:1, 1-11 cadmium and lead determination by icpms 7 table 2. determined concentration of cd and pb in the milk samples (expressed as mean concentration, n = 3) science diliman (january-june 2012) 24:1, 1-11 s a m p l e cadmium lead concentration (ì gkg-1) s d concentration (ì gkg-1) s d processed milk samples (commercially available) cmca1 < mdl 5.82 0.17 cmca2 < mdl 3.48 0.12 cmca3 5.17 0.13 2.74 0.17 cmca4 0.11 0.07 0.49 0.21 raw milk samples cmc1 < mdl < mdl cmc2 0.20 0.05 < mdl cmc3 < mdl 1.17 0.12 cmc4 0.31 0.07 1.09 0.05 cmq1 0.12 0.08 2.12 0.31 cmq2 0.22 0.08 2.98 0.21 cmq3 0.18 0.08 0.78 0.17 cmq4 0.11 0.08 2.77 0.20 cmq5 0.12 0.07 0.72 0.18 cmq6 0.17 0.07 1.08 0.08 cmq7 0.11 0.09 3.99 0.15 cmq8 0.45 0.09 < mdl cmsd1 0.20 0.06 2.75 0.11 cmsd2 < mdl < mdl cmsd3 0.14 0.06 < mdl cmsd4 0.16 0.05 < mdl cmsd5 0.20 0.07 < mdl cmsd6 0.12 0.07 1.29 0.07 cmsd7 0.11 0.07 6.79 0.20 cmsl1 0.23 0.08 6.10 0.23 cmsl2 0.29 0.08 2.89 0.16 cmsl3 0.26 0.08 2.63 0.16 cmsl4 0.33 0.07 4.93 0.16 cmsl5 0.25 0.08 2.97 0.14 cmsl6 0.15 0.07 1.89 0.14 *mdl – method detection limit rodriguez, i.b. and magbitang, r.a. 8 for the method parameters, we assessed linearity, reproducibility and the method detection limit. the correlation coefficients calculated were 0.999 for both cd and pb within the range of 0.01 to 500 µg kg-1of the calibration solutions as shown in figure 2a. further analysis of residuals reflected no visible trend with the determined concentration against the known concentration of the calibration standard solutions indicating that the change in counts monitored is linear with the change in concentration (shown in figures 2b and 2c). the instrument drift was monitored by analyzing repeatedly the calibration standard containing 1.0 µgkg-1 of both cd and pb throughout the icpms analysis sequence. the repeated determination of one standard will give an indication on the instrument performance and day-to-day variability of the measurements. the calculated rsd for the drift standards, on different icpms analysis days, were less than 2% for both cd and pb (n = 7) indicating that the instrument performance was reliable throughout the measurements. the estimated method detection limits were 0.09 µgkg-1 and 0.33 µgkg-1 for science diliman (january-june 2012) 24:1, 1-11 table 3. comparison of reported concentrations of cd and pb concentrations in milk samples by other researchers location concentration milk method reference cd pb source c r o a t i a 0.037 ± 0.007 mg kg -1 0.27 ± 0.06 mg kg -1 cow (raw) e a a s pavlovic et al. ( 2 0 0 4 ) e g y p t 0.007 mg kg -1 0.327 mg kg -1 cow (raw) i c p a e s elsayed et al. (2011) t u r k e y 0.257 ì g l-1 6.83 ì g l -1 cow (raw) gfaas ay and karayunlu ( 2 0 0 8 ) 0.162 ì g g-1 0.109 ì g g-1 sheep (raw) i c p a e s coni et al. (1996) 0.128 ì g g-1 0.059 ì g g-1 i t a l y 0.183 ì g g-1 0.078 ì g g-1 0.181 ì g g-1 0.094 ì g g-1 0.161 ì g g-1 0.050 ì g g-1 goat (raw) 0.130 ì g g-1 0.049 ì g g-1 p a k i s t a n 0.076 ± 0.014mg l -1 18.870 ± 0.062mg l -1 cattle (raw) a a s javed et al. (2009) 0.084 ± 0.003 mg l -1 42.687 ± 0.051 mg l-1 goat (raw) i n d i a 0.23 ± 0.02 ì g m l-1 0.85 ± 0.11 ìg ml -1 cow (raw) a a s patra et al. (2008) s p a i n 4.88 mgl -1 14.82 mg l -1 cow (raw) e a a s rodríguez et al. 4.30 mgl -1 10.25 mg l -1 cow (processed) e a a s ( 1 9 9 9 ) 7.81 mg l -1 11.86 mg l -1 goat (raw) e a a s l i t h u a n i a 0 . 3 7 ì g k g -1 0 . 4 7 ì g kg -1 cow (raw) i c p s f m s valiukenaite et al. ( 2 0 0 6 ) 0 . 1 8 ì g k g -1 0 . 5 4 ì g k g -1 nueva ecija, 0.11 ± 0.07 to 5.17 ± 0.13 ì g kg -1 0.49 ± 0.21 to 5.82 ± 0.17 ì g kg -1 carabao (processed) i c p m s this study p h i l i p p i n e s 0.11 ± 0.07 to 0.45 ± 0.09 ì g kg -1 0.72 ± 0.18 to 6.79 ± 0.20 ì g kg -1 carabao (raw) i c p m s this study cadmium and lead determination by icpms 9science diliman (january-june 2012) 24:1, 1-11 cd and pb, respectively. these were calculated by getting the standard deviation of the measurements of seven replicates of a solution containing 1 µg kg-1of both cd and pb, then subsequently multiplying it by the student’s t-value (n = 7; student’s t-value = 3.143; ripp, 1996). after assessment of the method performance, the optimized conditions were applied to triplicate aliquots (0.5 g of bcr 150 weighed to the nearest 0.1 mg) of the reference material and the determined values were 18.24 ±0.18 µgkg-1 and 807.57 ± 7.07µg kg-1 for cd and pb, respectively. these determined values correlate well with the certified values (cd: 21.8 ± 1.4 µg kg-1 and pb: 1000 ± 40.0 µg kg-1) in the reference material. these recovery values account for 83.7% and 80.7% in the reference material of cd and pb, respectively. the values fall within the limits of acceptable recovery percentages (80-110%) as a function of the analyte concentration, as suggested by taverniers et al. (2004). since the reference material available to us is in the solid form, we also analyzed another certified reference material nist 1643e (triplicate samples of 0.500 ml) and the determined concentrations were 6.48 ± 0.10 µg l-1 for cd and 21.96 ± 0.87 µg l-1 for pb, which were in good agreement with the certified values in the material 6.568 ± 0.073µg l-1 and 19.63 ± 0.21µg l-1, for cd and pb, respectively. after these validation experiments, we subjected actual samples to the pretreatment procedures prior to the actual analysis by icpms. analysis of actual samples the samples analyzed in this study were carabao milk samples from the different cities and municipalities in the province of nueva ecija, which is the top producer of carabao milk in the philippines. the determined cd and pb levels for all samples are summarized in table 2. all the determined pb levels were below the suggested maximum level of the joint fao/who food standards programme for milk, which is 0.020 mg kg-1. the concentrations of pb were in the range 0.49 ± 0.21 to 5.82 ± 0.17µg kg-1for the processed milk samples, and 0.72 ± 0.18 to 6.79 ± 0.20 µg kg-1 for the raw milk samples. although there is no maximum limit established for cd in milk, it can be observed that the levels of cd in the samples were detectable. the cd levels determined were in the range 0.11 ± 0.07 to 5.17 ± 0.13 µg kg-1for the processed milk samples, and 0.11 ± 0.07 to 0.45 ± 0.09 µg kg-1 for the raw milk samples. the detection of these metal contaminants in carabao milk being supplied for human consumption highlights the need for monitoring of these metals in this foodstuff. the values reported in this study were generally lower compared to the values reported by other researchers from other countries. table 3 shows the results obtained in the analysis of cd and pb in milk samples from different countries as well as the animal source and method used in the analysis. the differences in the reported values with what was determined in this study may be due to the animal source in the other studies, which is cow, and in this study, which is carabao. there are studies showing that the concentrations of heavy metals in cow’s milk are lower as compared to buffalo’s or even goat’s milk (enb et al., 2009). other factors that may have influenced the differences in the reported values are the analytical techniques used for the determination and the actual grazing areas for the animals used as sources of the milk samples. aside from these factors, the heavy metal concentrations may have been affected by conditions such as age, type of diet and breed of the carabao. although we have noted the information about the carabaos used as milk sources in this work, the correlation that we got is only indicative of possible effect of these factors on the metal load of the milk samples. for further studies, a wider scope with more sampling periods may be conducted since the results show that the optimized method offers a simple way of determining these contaminants in this type of sample. this is the first time that the analysis of cd and pb in carabao milk in the philippines was performed. thus, this information can be useful for further toxicological studies and to estimate the average intake of cd and pb due to consumption of carabao milk. for future work, it might be worthwhile to conduct a comparative study of the metal content of the different milk samples (cow, carabao, goat, formula and breast milk) in the philippine setting. processing of the milk samples might also have effects on the metal concentrations. it has been reported that the processing was related to the observed increase of metal concentrations in the rodriguez, i.b. and magbitang, r.a. 10 science diliman (january-june 2012) 24:1, 1-11 processed milk and milk products (enb et al., 2009). also for further work, it might be sensible to carry out more detailed correlation studies which will look at the animal source, the raw milk and the processed milk. analysis of more samples may also give more conclusive results. conclusion a method suitable for the simultaneous determination of cd and pb in milk samples was optimized and validated. the method is based on the use of microwave-assisted acid digestion as the sample pretreatment step prior to detection using icpms. the optimized method showed excellent linearity and low estimated detection limits, which are desirable for trace analysis of these contaminants in foodstuffs. the method was validated using certified reference materials, bcr 150 (spiked-skim milk powder) and nist 1643e (trace elements in water), and the determined cd and pb values agree well with the certified values in the reference materials. the application of the method to milk samples collected from different cities and municipalities in nueva ecija in the philippines showed that low concentrations of cd and pb were detected in these samples. the determined amounts were below the maximum limits set by the authorities but the presence of these contaminants in this important nutrient source necessitates for routine monitoring of these metals in these samples. the method presented is a simple, rapid and repeatable method which can be used for the routine monitoring of both cd and pb in milk samples. acknowledgement the authors would like to thank the natural sciences research institute – up diliman for the financial support. references ataro, a., r.i. mccrindle, b.m. botha, c.m.e. mccrindle, & p.p. ndibewu, 2008. quantification of trace elements in raw cow’s milk by inductively coupled plasma mass spectrometry (icp-ms). food chem, 111: 243-248. ay, ü. & s. karayünlü, 2008. modification in direct analysis method: metal levels in raw milk at the region of izmit by graphite furnace atomic absorption spectrophotometer. int j food sci tech, 43:326–329. bencko, v., 1995. use of human hair as a biomarker in exposure to pollutants in occupational settings. toxicology, 101:29-39. bertin, g. 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int, 36:623-636. garland, m., j.s. morris, b.a. rosner, m.j. stampfer, v.l. spate, c.j. baskett, w.c. willett & d.j. hunter, 1993. toenail trace element as biomarkers: reproducibility over a 6-year period. cancer epidembiomar, 2:493-497. genuis, s.j., d. birkholz, i. rodushkin & s. beesoon, 2010. blood, urine and sweat (bus) study; monitoring and elimination of bioaccumulated toxic elements. arch environ contamtoxicol, doi: 10.1007/s00244-010-9611-5. cadmium and lead determination by icpms 11science diliman (january-june 2012) 24:1, 1-11 järup, l., 2003. hazards of heavy metal contamination. brit med bull,68: 167-182. javed, i., i. jan, f. muhammad, zia-ur-rahman, zargham m. khan, b. aslam & j.i. sultan, 2009. heavy metal residues in the milk of cattle and goats during winter season. bull environ contam toxicol, 82:616-620. joint fao/who food standards programme. codex committee on contaminants in foods (cccf) fifth session, march 2011.codex alimentarius commission. lidsky, t.i. & j.s. schneider, 2003. lead neurotoxicity in children: basic mechanisms and clinical correlates. brain, 126:5-19. park, c. j. & h. song, 2005. determination of arsenic in biological samples by inductively coupled plasma mass spectrometry with selenium as an internal standard. j anal at spectrom, 20: 436-440. patra, r.c., a.k. rautray & d. swarup, 2011. oxidative stress in lead and cadmium toxicity and its amelioration. vet med int, doi: 10.4061/2011/457327 patra, r.c., d. swarup, p. kumar, d. nandi, r. naresh & s.l. ali, 2008. milk trace elements in lactating cows environmentally exposed to higher level of lead and cadmium around different industrial units. sci total environ, 40:36-43. patrick, l., 2006. lead toxicity part ii: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. altern med rev, 11:114-127. pavlovic, i., m. sikiric, j.l. havranek, n. plavljanic & n. brajenovic, 2004. lead and cadmium levels in raw cow’s milk from an industrialized croatian region determined by electrothermal atomic absorption spectrometry. czech j anim sci, 49:164–168. ripp, j., 1996. analytical detection limit guidance & laboratory guide for determining method detection limits. wisconsin department of natural resources, laboratory certification program. rodríguez, e.m., e. uretra & c. romero, 1999. concentrations of cadmium and lead in different types of milk. z lebensm unters forsch a, 208:162-168. sadegh safarzadeh, m., m.s. bafghi, d. moradkhani & m. ojaghiilkhchi, 2007. a review on hydrometallurgical extraction and recovery of cadmium from various resources. miner eng, 20:211-220. singh, c.v. & r.s. barwal, 2010. buffalo breeding research and improvement strategies in india. 9th world buffalo congress, p. 1024. taverniers, i., m. de loose & e. van bockstaele, 2004. trends in quality in the analytical laboratory. ii. analytical method validation and quality assurance. trends anal chem, 23:535-552. tong, s., y.e. von schirnding & t. prapamontol, 2000. enviromental lead exposure: a public health problem of global dimensions. b world health organ, 78: 1068-1077. torres, j. r. p., m.k.g. banaag & i.b. rodriguez, 2010. a rapid method for simultaneous determination of arsenic, cadmium and lead in drinking water by inductively coupled plasma mass spectrometry. science diliman, 22: 1-8. valiukenaite, r., m. stankeviciene, h. stankevicius & k.a. skibniewska, 2006. lead and cadmium levels in raw cow’s milk in lithuania determined by inductively coupled plasma sector field mass spectrometry. pol j food nutr sci, 56:243-246. vanhaecke, f., h. vanhoe, r. dams & c. vandecasteele, 1992. the use of internal standard in icp-ms. talanta, 39:737-42. sd-sample article 80 1. science diliman is a journal of pure and applied sciences. considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out; that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright 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1994) and the journal of american chemical society. 2 2 . less common abbreviations may be printed as footnotes. 23. authors may opt to submit their typeset manuscripts (word f ile) as an email attachment to <rduo.ovcrd@upd.edu.ph>, <science.diliman@journals.upd.edu.ph> and <rduo.ovcrd2012@gmail.com>. manuscripts may also be submitted in hard copy (3 copies) to: the editor-in-chief science diliman off ice of the vice-chancellor for research and development lgf phivolcs bldg. , c.p. garcia avenue university of the philippines diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. preliminary study-art.4 preliminary study on the agar quality 45science diliman (january june 2000) 12:1, 45-47 abstract introduction preliminary study on the agar quality of laboratory-generated carposporelings of gracilariopsis bailinae zhang et xia grown in the field (a short communication) susan f. rabanal marine science institute, university of the philippines diliman, quezon city 1101 philippines *cagayan state university, 3515 maura, aparri, cagayan the agar quality (gel strength, gelling and melting temperature) of laboratory-generated carposporelings of gracilariopsis bailinae grown in the field for six weeks off amunitan, gonzaga, cagayan was investigated. cut sporelings grown at 1.0 m depth showed good quality agar (492 gm cm-2 gel strength, 43oc gelling temperature, 84oc melting temperature). this constitutes the first report on the agar quality of this species from this area. keywords: gracilariopsis, agar, agarophyte *present address gracilariopsis bailinae zhang et xia grows abundantly in panay island (de castro et al., 1991). growth rate studies using vegetative cuttings have been done (hurtado-ponce, 1990) and a good quality agar has been extracted (hurtado-ponce & umezaki, 1988; luhan, 1992; pondevida & hurtado-ponce, 1996). the importance of agar in food preparations and in other industries is well established. the agar quality and yield however, differ according to species, locality, environmental conditions, time of harvest and extraction time or process (hoyle, 1975; thomas & krishnamurthy, 1976; oza, 1978; bird et al., 1981; guerin & bird 1987; daugherty & bird 1988; bird, 1988; hurtado-ponce 1992; pondevida & hurtado-ponce, 1996; chirapart et al., 1997). it was also found to differ depending upon the reproductive states of the species (whyte et al., 1981). doty and santos (1975) reported that from among the gracilaria species they have studied in the philippines (g. arcuata, g. salicornia and g. eucheumoides), g. arcuata seemed to be the best source of agar for bacteriological use. recent studies by hurtado-ponce (1992) observed that g. bailinae is also good for bacteriological use. this study describes for the first time the agar quality of laboratory-generated carposporelings of the species which were outplanted off amunitan, gonzaga, cagayan at 1.0 m depth. this is part of a research aimed at the development of mariculture techniques for the species (rabanal et al., 1997). materials and methods agar extraction and analysis the sporelings of g. bailinae with cut apices cultured 1.0 m below the lowest tide level for six weeks in rabanal 46 february to march 1996 off amunitan, gonzaga, cagayan (122 o 02’ e, 18 o 19’ n) was harvested and analyzed for agar quality. the sporelings were cleaned of foreign materials and washed thoroughly with tap water and air dried and then dried at 60oc. agar extraction was done using the modified procedure of hurtado-ponce and umezaki (1988). only 3.125 g dried sample was utilized because of limited raw material and therefore ratio and proportion was employed. the dried material was treated with 5% naoh solution for 1 h at 90oc and then washed with tap water. the specimens were soaked in 0.5% acetic acid for 1 h and extraction was done by boiling with distilled water for 1 hour. two replicates were used. gel strength was measured using marine colloids gel tester (model gt-1). the plunger had a diameter of 1 cm and a descent rate of 2.5 mm sec-1. the gelling and melting temperatures were determined from a 1.5% agar solution (whyte & englar 1980). the agar solution was poured in three test tubes provided with clinical thermometer and allowed to gel. glass beads (3 mm in diameter) were dropped one after the other and the temperature was noted when the bead failed to drop through the agar. this then was recorded as the gelling temperature. melting temperature was also determined using the same test tube previously used in gelling temperature. the samples were heated in a water bath slowly and the temperature was noted as the beads dropped to the bottom. analysis of these properties were done at the seaweed laboratory at the marine science institute. results and discussions cut sporelings generated from the laboratory and grown at 1.0 m depth showed good quality agar (492 gm cm-2 gel strength, 43oc gelling temperature, 84oc melting temperature). pondevida and hurtado-ponce (1996) reported that the highest gel strength shown by gracilariopsis bailinae collected from the field was 784 g cm-2 . although the gel strenght of this species generated from the laboratory is lower, its gel strength is higher than those of gracilaria manilaensis and gracilaria changii. the gelling and melting temperature recorded conform with the values obtained for the same species collected in the field with 39.0 + 0.5 – 45.7 + 0.5 gelling temperature and 83.7 + 0.5 – 91.0 + 0.5 melting temperature (pondevida & hurtadoponce, 1996). the results show that the agar quality seemed unaffected by cutting despite the mechanical damage or stress inflicted to the plants. the cutting induced greater activity and hence, greater regeneration which could have produced greater molecular-sized agar polymer (bird, 1988) thereby giving high gel strength and lower gelling temperature. in addition, the presence of young tissues could have also contributed to the high gel strength. pondevida and hurtado-ponce (1996) noted that older tissues have higher concentration of stable sulfate groups which cannot be removed by naoh, hence, affecting the quality of agar. the temperature and salinity readings were done at weekly intervals from 10:00 am to 3:00 pm for six weeks. during the harvest period, the temperature and salinity in the field was 26.9oc and 32.5% respectively. these results agree with the findings of luhan (1992) where higher gel strength was observed when temperature was lowest and salinity was higher. high gel strength was also observed in gracilaria gracilis when water temperature was between 23 to 25oc (rebello et al., 1996). in contrast, bird (1988) found higher gel strength at higher temperature (32oc) but similar higher salinity (33%) was demonstrated in gracilaria sp-16. while the data are very limited, it is apparent that sporelings of gracilariopsis bailinae are also a potential source of agar. hurtado-ponce (1992) demonstrated that agar quality could be improved. she also mentioned that hard and brittle gels are best for bacteriological purposes while soft, elastic gels are best for food preparations. cutting the apices of the sporelings could be an important factor to consider for farming but its effect on agar production and agar quality needs further research. preliminary study on the agar quality 47 references bird, k.t., 1988. agar production and quality from gracilaria sp. strain g-16: effects of environmental factors. bot. mar. 31: 33-39. bird, k.t., m.d. hanisak & j. ryther, 1981. chemical quality and production of agars extracted from gracilaria tikvahiae grown in different nitrogen enrichment conditions. bot. mar. 24: 441-444. chirapart, a. et al., 1997. effects of partial acid hydrolysis on physical and chemical properties of agar from a newly reported japanese agarophyte (gracilariopsis lemaneiformis). j. appl. phycol. 9: 73-76. daugherty, b.k. & k.t. bird, 1988. salinity and temperature effects on agar production from gracilaria verrucosa strain g-16. aquaculture. 74: 1-9. de castro, t.r., n.g. guanzon jr., & m.r.j. luhan, 1991. assessment of natural stocks of gracilaria in panay island, philippines. bot. mar. 34: 383-391. doty, m.s. & g.a. santos, 1975. gracilaria for the manufacture of agar. fish res. j. philipp. 3(2): 2934. guerin, j.m. & k.t. bird, 1987. effects of aeration period on the productivity and agar quality of gracilaria sp. aquaculture. 64: 15-110. hoyle, m.d., 1975. the literature pertinent to the red algal genus gracilaria in hawaii. marine agronomy sea grant program. hawaii tech. report. 3: 34 pp. hurtado-ponce, a.q., 1990. vertical rope cultivation of gracilaria (rhodophyta) using vegetative fragments. bot. mar. 33: 477-481. _______________, 1992a. rheological properties of agar from gracilariopsis heteroclada (zhang et xia) zhang et xia (gracilariales, rhodophyta) treated with powdered commercial lime and aqueous alkaline solution. bot. mar. 35: 365-369. _______________, 1992b. influence of extraction time on the rheological properties of agar from some gracilaria species from the philippines. bot. mar. 35: 441-445. hurtado-ponce, a.q. & i. umezaki, 1988. physical properties of agar gel from gracilaria (rhodophyta) of the philippines. bot. mar. 31: 171-174. luhan, m.r.j., 1992. agar yield and gel strength of gracilaria heteroclada collected from iloilo, central philippines. bot. mar. 35: 169-172. oza, r.m., 1978. studies on indian gracilaria. iv. seasonal variation in agar and gel strength of gracilaria corticata j. ag. occurring on the coast of verval. bot. mar. 21: 165-67. pondevida, h.b. & a.q. hurtado-ponce, 1996. assessment of some agarophytes from the coastal areas of iloilo, philippines. ii. seasonal variations in the agar of gracilaria changii, g. manilaensis and gracilariopsis bailinae (gracilariales, rhodophyta). bot. mar. 39: 117-122. rabanal, s.f., r. azanza, & a. hurtado-ponce, 1997. laboratory manipulation of gracilariopsis bailinae zhang et xia (gracilariales, rhodophyta). bot. mar. 40: 547-556. rebello, j. et al., 1996. growth rates and agar quality of gracilariagracilis (stackhouse) steentoft from namibia, southern africa. bot. mar. 39: 273-279. thomas, p.c. & v. krishnamurthy, 1976. agar from cultured gracilaria edulis (gmel.) silva. bot mar. 19: 115-117. whyte, j.n.c. & j. r. englar, 1980. chemical composition and quality of agar in the morphotypes of gracilaria from british columbia. bot. mar. 23: 277283. whyte, j.n.c. et al., 1981. seasonal variation in the biomass, quantity and quality of agar from the reproductive and vegetative stages of gracilaria (verrucosa). bot. mar. 24: 493-501. 10ethics.pmd 105 journal pol icy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the off ice of the v ice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 106 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research area concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “notes to contributors” stipulates that “all ar ticles must have a high degree of scholarship,” that “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a cer tif ication from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right… .” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as def ined above and; b.) is specif ic and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the bases for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specif ic advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the 107 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science dilimanto formulate a scientif ic misconduct policy. in attendance were: dr. corazon d. villareal, rduo director, presiding; dr henry j. ramos, pmrgo director and professor , nip; atty. vyva victoria aguirre, ovcrd legal consultant; editorsin-chief dr. maricor soriano (science diliman) and dr. maria mangahas (social science diliman). ms. nanie domingo and ms. dercy mararac, editorial assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct , united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. 108 references council of science editors. “ white paper on promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ecto r s o n s e p tember 3, 2006.” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ ajp/submit.html 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile 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phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city (02) 436-8720 fax (02) 927-2568  research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. (subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total dichlorophen and-waseem ahmad.pmd dichlorophen and dichlorovos mediated genotoxic 13science diliman (january-june 2012) 24:1, 13-22 abstract sibhghatulla shaikh, nazia nazam, mohammad iqbal lone and waseem ahmad* gene tox laboratory, division of genetics, department of zoology aligarh muslim university, aligarh, 202002, up, india *corresponding author: tel: +91-571-2700920(ext-3443) fax: +91-571-2720920 email: wafgenomics@gmail.com dichlorophen and dichlorovos mediated genotoxic and cytotoxic assessment on root meristem cells of allium cepa plants are direct recipients of agro – toxics and therefore important materials for assessing environmental chemicals for genotoxicity. the meristematic mitotic cell of allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. onion root tips were grown on moistened filter paper in petri dish at room temperature. germinated root tips were then exposed to three concentrations of each pesticide for 24 h. about 1 – 2 mm length of root tip was cut, fixed in cornoy’s fixative, hydrolyzed in warm 1 n hcl, stained with acetocarmine and squashed on glass slide. about 3000 cells were scored and classified into interphase and normal or aberrant division stage. cytotoxicity was determined by comparing the mitotic index (mi) of treated cells with that of the negative control. the mi of cells treated with dichlorophen and dichlorovos at one or more concentration was half or less than that of control are said to be cytotoxic. genotoxicity was measured by comparing the number of cells/1000 in aberrant division stages at each dose with the negative control using mann – whitney u test. both dichlorophen and dichlorovos are genotoxic at higher concentrations i.e. 0.001%, 0.002% and 0.028%, 0.056% inducing chromosome fragment, chromosome lagging and bridges, stick chromosome and multipolar anaphase. keywords: allium cepa, cytotoxicity, genotoxicity, mitotic index, pesticides, root tip cells 14 sibhghatulla, s. et al. science diliman (january-june 2012) 24:1, 13-22 introduction pesticides have been used in modern agriculture to greatly improve the yield through inhibition of disease causing organisms and by acting against pest in the fields and during storage of agricultural products (taylor et al., 1997; mackenzie et al., 1998). pesticides form an important group of environmental pollutants and genotoxic effects of several chemical groups of pesticides have been shown by in vivo and in vitro experiments (bolognesi, 2003; abdollahi et al., 2004; kaushik & kaushik, 2007). however, genotoxicity data for few pesticides exist (gandhi et al., 1995), while reports on majority of them are lacking. among pesticides, organophosphates and organochlorines are constantly a matter of worry because of their wide use. both group of chemicals bear the potentiality to cause genotoxicity and carcinogenicity (kaushik & kaushik, 2007). however, in addition to intended effects of pesticides, they are sometimes found to affect non – target organisms, including humans (chantelli-forti et al., 1993; chaudhuri et al., 1999). the mutagenic and carcinogenic action of herbicides, insecticides and fungicides on experimental animal is well known and several studies have shown that chronic exposure to low levels of pesticides can cause mutation and/or carcinogenicity (international agency for research on cancer, 1990; international agency for research on cancer, 1991; karabay & gunnehir, 2005; bull et al., 2006). organophosphorus (op) pesticides, which are widely used in agriculture as insecticides, leave residues to varying extent in agricultural produce such as vegetables and fruits (iram et al., 2009). op compounds exert acute toxic effect that are mainly due to suppression of neuronal acetylcholinesterase activity (sachanaa et al., 2003). the widespread uses of op insecticides indicate the extensive availability and potential for accidental and intentional human exposure (el-behissy et al., 2001). dichlorovos (2, 2 – dichlorovinyl dimethyl phosphate – ddvp) is an op compound used to control household, public health and stored product insects. it is effective against mushroom flies, aphids, spider, mites, caterpillars, thrips and white flies in greenhouse, outdoor fruits and vegetable crop (lotti, 2001). organochlorine pesticides are endocrine disrupting chemicals, meaning they have subtle toxic effect on the body’s hormonal system (lemaire et al., 2004). endocrine disrupting chemicals often mimic the body’s hormones, disrupting normal functions and contributing to adverse health effects. dichlorophen (2, 2 – methylene bis 5 – chlorophenol – dddm) is an organochlorine compound that is incompatible with strong oxidizing agents and can be slowly oxidized in air. it is hazardous in case of eye contact or ingestion, but less severe in case of skin contact or inhalation (kintz et al., 1997). no studies have been carried out, to our knowledge, on the cytotoxic and genotoxic effects of dichlorophen and dichlorovos on onion root tips, despite the fact that it is commonly used. the use of plant test systems for the evaluation of the mutagenic potential of pesticides is particularly important, since they largely enter the human food chain. materials and methods onion (allium cepa, 2n = 16) bulbs equal in size 1.5 – 2.0 cm diameter were chosen from a population of locally available commercial variety, nasik red (n53). dichlorovos cas no. 62 – 73 – 7 and dichlorophen cas no. 97 – 23 – 4 are products of sigma. ethanol (merck) is of analytical grade. glacial acetic acid cas no. 64 – 19 – 7 and hydrochloric acid are products of fisher scientific. methyl methane – sulfonate (mms, 99%) cas no. 66 – 27 – 3, a product of super religare laboratories limited (formerly srl ranbaxy ltd.), was used as positive control. acetocarmine cas no. 64 – 19 – 7 is a product of loba cheme. dichlorophen and dichlorovos mediated genotoxic 15science diliman (january-june 2012) 24:1, 13-22 determination of lc50 and dose selection fifty (50) onion seeds were spread on filter paper moistened with different concentrations of pesticide in a petri dish and left to germinate at room temperature for about three days. the number of seeds which produced radicle were recorded at the end of three days and compared to the number of seeds that germinated in the concurrent water treated control to derive the percentage germinating at each concentration. the lc50 for both pesticides was determined from the curve of percentage of root length that germinated against dose. as per above procedure the lc50 for dichlorophen and dichlorovos was determined to be 0.004 % and 0.224 %, respectively. on the basis of determined lc50, three random concentrations in increasing order within the lc50 were selected for both pesticides. the three concentrations taken in this study do not correlate with the concentration used in the field, as the concentrations used in the field vary from place to place. genotoxicity assay the method used was similar to the method of asita & matebesi (2010). a. cepa (onion) seeds were germinated in petri dishes containing pesticide – soaked filter paper (test), water – soaked filter paper (negative control) and on filter paper soaked in aqueous solution of 1% methyl methanesulfonate (positive control). in this study, a discontinuous treatment protocol was used. a. cepa seeds were first soaked in distilled water until the radicles reached a length of about 1 – 3 cm. germinated seeds were transferred to petri plates containing chemicals at different concentration in which they were left for 24 h at room temperature. at the end of the 24 h exposure, some seeds were collected at random and assessed. root harvest and slide preparation root tips 1 – 3 cm long were cut and placed in a watch glass and fixed in acetic alcohol (ethanol: glacial acetic acid in 3:1 ratio) for 12 h at room temperature. after this the root tips were hydrolyzed in 1 n hcl at 60 oc for 10 minutes and stained with acetocarmine for 20 minutes, then squashed on glass slide under 45% acetic acid to determine the mitotic index and the presence of chromosomal aberrations. scoring of slides the slides were viewed under the light microscope (olympus ch 20 i) using the 100x objective lens with oil immersion. a total of 3000 cells were scored on each slide. the cells were recorded as normal or aberrant in the different stages of the cell cycle namely: interphase, prophase, metaphase, anaphase or telophase. all cells with aberrations were counted and the most representative ones for each abnormality were photographed using an olympus u – pmtv microscope mounted with optical zoom camera. data analysis cytotoxic determination mitotic index method was used for determination of cytotoxicity, which was similar to the method of asita & matebesi (2010). the mitotic index (mi) was calculated as the number of cells containing visible chromosomes (i.e. cells in the division stages) divided by the total number of cells scored. the mitotic indices of the treated cells at each dose were compared with that of the negative control group. any dose of a test substance was said to be cytotoxic if the mitotic index of treated cells was half or less, compared to the mitotic index of the concurrent water treated cells. genotoxicity determination dividing cells with any of the under listed abnormalities were recorded, namely; c-mitosis (no spindle fibres), stick chromosomes, chromosome bridges, lagging chromosomes or chromosome breaks. the number of aberrant cells /1000 cells in each of the four division stages for pesticides treated cells were compared with the numbers in the aberrant division stages for the water treated (negative control) cells by the mann – whitney u test using the spss 10.0 for windows statistical package. the calculated u value for each comparison (pesticide and negative control) was obtained. if the calculated u value was less than the critical value from the table at the appropriate 16 sibhghatulla, s. et al. science diliman (january-june 2012) 24:1, 13-22 degrees of freedom (in our own case, n 1 = 4 and n 2 = 4) at the 0.05 probability, then a statistically significant difference existed between the medians and the pesticide was adjudged to be genotoxic at the dose of the pesticide. results cytotoxicity of pesticides table 1 presents the cytotoxicity of pesticides on onion root tips. cells treated with dichlorophen and dichlorovos had reduced mitotic indices compared with the cell treated with water, which indicate inhibition of cell division by these pesticides. dichlorophen and dichlorovos are therefore said be cytotoxic at one or more doses. however, the decrease of the mi was not dose dependent. significant levels of inhibition were apparent at lower concentrations of both pesticides, which means that they are cytotoxic at lower concentration. in addition to lower concentration dichlorovos was also cytotoxic at higher concentration. the positive control chemical, methyl – methane sulfonate at 0.2% concentration in water did not inhibit mitotic cell division of the onion root tip cells. genotoxicity of pesticides table 2 presents the genotoxic effect of pesticides on onion root tips. significant differences were detected in the frequency of aberrant division stages in dichlorophen and dichlorovos treated cells when compared with water (negative control) treated group, at two higher doses (p < 0.05), while at lower dose of both compounds the genotoxic effect was not observed. in comparison with the positive control (mms) used in the present investigation, a known genotoxic compound. table 3 presents the types of aberration observed in the cells treated with dichlorovos and dichlorophen. the different types of mitotic abnormalities due to genotoxic effects of the pesticides and the normal anaphase in negative control on a. cepa cells observed on glass slides are presented in plate 1 (a – i) table 1. cytotoxicity of pesticides on onion root tip cells ddm = dichlorophen; ddvp = dichlorovos; mms = methyl methane sulfonate; se = standard error, conc = concentration; mi = mitotic index;* =cytotoxic (mi control: mi test > 2) treatment conc mi mean±se mi control/mi test (%) water 100 0.158 39.55±9.08 1.00 mms 0.2 0.244 61.03±1.64 0.65 dddm 0.0005 0.020 4.97±0.80 7.9* 0.001 0.145 36.14±5.89 1.09 0.002 0.172 43.07±1.50 0.92 ddvp 0.014 0.050 12.47±3.39 3.16* 0.028 0.136 34.08±1.02 1.61 0.056 0.056 14.04±3.21 2.82* dichlorophen and dichlorovos mediated genotoxic 17science diliman (january-june 2012) 24:1, 13-22 discussion organophosphorus and organochlorine pesticides are widely used by farmers in india and in other countries because of their high efficiency towards the target organisms (levine, 1991; mineau, 1991). extensive use of these pesticides in crop protection and for household purposes has resulted in their widespread distribution in the environment (mineau, 1991). while they contribute greatly to the animal and human prevention of vectors of diseases, their use also creates many problems because of their toxicity to non– target organisms, persistence and combined effects with other agro – biochemicals and environmental factors (levine, 1991; mineau, 1991; u.s. environmental protection agency, 1999). the allium cepa assay is an efficient test for chemical screening and in situ monitoring for genotoxicity of many pesticides. results have shown that these compounds can induce chromosomal aberration in root meristem of a. cepa (feretti et al., 2007). pesticides residues can be present in fruit and vegetables and represent a risk for human health. table 2. mutagenic potencies of the pesticides on onion root tip cell treat men t conc (%) number of cell s in different divisi on stages/1000 cel ls scored m – wu value (calculated) prop metap anap te lop total n ab n n ab n n ab n n abn n abn water 100 58.42 0.00 51.25 0.00 28.17 0.00 2 0. 39 0.00 1000.0 0.00 20 m ms 0.2 105.11 6.01 41.77 7.65 37.42 5.03 3 1. 27 0.90 971.45 28.55 0** dddm 0.0005 1.25 2.66 4.12 0.87 4 .6 6 1.00 2.33 0.00 992.48 7.52 6 0.001 23.62 1.35 23.96 4.39 40.16 5.40 3 5. 10 1.01 978.28 21.72 0** 0.002 32.64 1.87 26.05 4.77 43.12 5.80 3 8. 75 1.12 976.28 23.72 0** ddvp 0.014 19.24 5.66 9.64 3.64 4 .3 1 4.99 1.32 4.65 984.63 15.37 10 0.028 104.37 5.96 11.61 1.03 9 .8 0 0.51 7.25 0.06 996.70 3.30 0** 0.056 23.55 1.35 11.22 2.06 8 .2 0 1.10 6.80 0.17 993.58 6.42 0** dddm = dichlorophen; ddvp = dichlorovos; n = normal cells; abn = abnormal cells; conc = concentration; m – wu = mann – whitney u; prop = prophase; metap = metaphase; anap = anaphase; telop = telophase. ** = mutagenic (u < 0; p < 0.05, mann – whitney u test) table 3: types of mutation induced by pesticides pesticides mutation type cb ma cm cb’ sc lc ddvp + + + + + dddm + + + ddvp = dichl orovos; dddm = dichlorophen; c b = chromosome break; ma = m ultipol ar anaphase; c m = c – mit osis; c b’ = chrom osome b rid ge; sc = stick chromosom e; lc = lag chrom osome table 3. types of mutation induced by pesticides 18 sibhghatulla, s. et al. science diliman (january-june 2012) 24:1, 13-22 the findings of the present study indicate that ddvp and dddm can induce cytotoxic and genotoxic effect on the meristematic cells of allium cepa. the mi inhibition and induction of chromosomal aberration in plant cells by several pesticides have been reported earlier by different researchers (chauhan et al., 1999; sudhakar et al., 2001). the observations of the present study are in accord with the earlier report, wherein a clear indication of the mitoclastic and clastogenic actions are seen, which are evident from the lowering of the mi and manifestation of spindle abnormalities (chauhan et al., 1999; sudhakar et al., 2001). the lower chemical concentration stimulates the rate of cell division. mitotic activity reduction could be due to the inhibition of dna synthesis (schneiderman et al., 1971; sudhakar et al., 2001) or due to a block in the g 2 – phase of the cell cycle, thus preventing the cell from entering mitosis (van’t hof, 1968). the mitotic activity suppression is often used to assess cytotoxicity (smaka-kincl et al., 1996). it has been reported by many investigators that a depression of the mitotic index is the result of treatment with pesticides (amer & farah, 1974; panda & sahu, 1985; asita & makhalemele, 2008). the insecticides showed more effectiveness in the s – phase in comparison to the g 1 and g 2 phases of cell cycle (srivastava et al., 2008). several chromosomal aberrations (cas) like stickiness, chromosomal break, chromosomal bridges, laggard, c-mitotic effect and multipolar anaphase have been formed. c – mitosis was observed in root tips treated with ddvp. the occurrence of c – mitosis in a. cepa indicates that spindle formation was adversely affected (el-ghamery et al., 2000). chromosome break was one of the most frequent chromosome aberrations induced by ddvp. the induction of chromosome breaks by pesticides indicates the clastogenic potential of the test compounds (chauhan & gupta, 2005). chromosome break induced by ddvp indicates that ddvp has more clastogenic activity compared to dddm. chemicals that induce chromosome breakage are known as clastogens and their action on chromosome is generally regarded to involve an action on dna (grant, 1978; chauhan & sundararaman, 1990 ). breaks and unequal distribution were noticed in diverse materials as a result of treatment with various chemicals (aly et al., 2002; borah & talukdar, 2002; gomürgen, 2005). chromosome bridges were noticed in both ddvp and dddm treated onion root tips, which were probably formed by breakage and fusion of chromosomes and chromatids. gomürgen (2005) reported that potassium metabisulphite and potassium nitrate caused anaphase bridges in a. cepa. according to gomürgen (2005), chromosome bridges may be due to the stickiness of chromosome and subsequent failure of free anaphase separation or may be attributed to unequal translocation or inversion of chromosome segments. laggards were observed after the treatment with both dddm and ddvp, which are due to the failure of the chromosome to move to either of the poles. according to permjit & grover (1985), the lagging chromosomes can be attributed to the delayed terminalization, stickiness of chromosome ends, or because of the failure of chromosomal movement. gomürgen (2005) reported that potassium sulphite and potassium nitrate, which are food preservatives, caused laggard chromosomes in a. cepa. chromosome stickiness was another frequent chromosomal abnormality induced by dddm in meristematic cells of a.cepa. this stickiness is presumably due to the intermingling of chromatin fibers, which lead to subchromatid connection between chromosomes (mcgill et al.,1974; klasterska et al., 1976). according to saxena et al. (2005), subchromatid connections were observed in plants cells exposed to pyrethrin insecticides. stickiness can also be explained as physical adhesion of the proteins of the chromosome (patil & bhat, 1992). stickiness is accepted as an indicator of toxicity, which results in cell death (elghamery et al., 2000). anonymous (1980), reported that plant test systems among known test system are more sensitive for determining these effects of pesticides. dichlorophen and dichlorovos mediated genotoxic 19 plate 1. allium cepa root tip cells showing normal anaphase (a) in negative control group; mitotic abnormalities induced by pesticide treated group showing – multipolar anaphase (b – c); chromosome bridge (d – e); chromosome break (f); lag chromosome (g); stick chromosome (h); and c – mitotic effect (i) rank & nielsen (1994) shown that allium test in some way is more sensitive than both the microscreen assay and the ames test. it can even detect some carcinogenic substances that are not detected in the ames test. the allium test has proved to be a reliable test for monitoring cyto – and genotoxicity of the different chemical substances. for in situ monitoring, meristematic cells of allium and vicia are very efficient cytogenetic materials for the detection of mutagenicity of the environmental chemicals (ma et al., 1995). the study has further demonstrated the usefulness of the a.cepa chromosome assay in assessing the genotoxicity and environmental chemicals as mixtures or pure products. science diliman (january-june 2012) 24:1, 13-22 20 sibhghatulla, s. et al. references abdollahi, m., a. ranjbar, s. shadnia, s. nikfar & a. rezaiee, 2004. pesticides and oxidative stress: a review. med. sci. monit, 10: 144-147. aly, f.a.e., s.m. donya & k.m. aly, 2002. protective effects of the folic acid and vitamin b12 against chromosome damage induced by manganese sulfate in cultured mouse spleen cells. cytologia, 67: 221-228. amer, s.m. & o.r. farah, 1974. cytological effects of pesticides. vi. effect of the insecticide “roger” on the mitosis of vicia faba and gossypium barbadense. cytologia, 39: 507-514. anonymous, 1980. current status of bioassays in genetic toxicology. gene tox, 1-69. asita, a.o. & l.p. matebesi, 2010. genotoxicity of hormoban and seven other pesticides to onion root tip meristematic cells. afr. j. biotechnol, 9(27): 4225-4232. asita, a.o. & r. makhalemele, 2008. genotoxicity of chlorpyrifos, alphathrin, efekto virikop and springbok to onion root tip cells. afr. j. biotechnol, 7(23): 4244-4250. bolognesi, c., 2003. genotoxicity of pesticides: a review of human biomonitoring studies. mutat. res, 543: 251-272. borah, s.p. & j. talukdar, 2002. studies on the cytotoxic effects of extract of castor seed (ricinus communis l.). cytologia, 67: 235-243. bull, s., k. fletcher, a.r. boobis & j.m. battershill, 2006. evidence for genotoxicity of pesticides in pesticide applicators: a review. mutagenesis, 21: 93-103. chaudhuri, k., s. selvaraj & a.k. pal, 1999. studies on the genotoxicity of endosulfan in bacterial systems. mutat. res, 439: 63-67. chantelli-forti, g., m. paolini & p. hrelia, 1993. multiple end point procedure to evaluate risk from pesticides. environ. health perspect 101: 15-20. chauhan, l.k.s., p.n. saxena & s.k. gupta, 1999. effects of deltamethrin on the ultrstructure of root meristem cells of allium cepa, pestic. biochem. physiol 64: 181-189. chauhan, l.k.s. & s.k. gupta, 2005. combined cytogenetic and ultra structural effects of substituted urea herbicides and synthetic pyrethroids insecticide on root meristem cells of allium cepa. pestic. biochem. physiol, 82: 27-35. chauhan, l.k.s. & v. sundararaman, 1990. effects of substituted ureas on plant cells. i. cytological effects of isopruturon on the root meristem cells of a. cepa. cytologia, 55: 91–98. el-behissy, e.y., r.d. king, m.m. ahmed & a.m. youssef, 2001. fate of postharvest-applied dichlorvos in stored and processed dates. j agr food chem, 49: 1239-1245. el-ghamery, a.a., a.i. el-nahas & m.m. mansour, 2000. the action of atrazine herbicide as an indicator of cell division on chromosomes and nucleic acid content in root meristems of allium cepa and vicia faba. cytologia, 65: 277-287. feretti, d., i. zerbini, c. zani, e. ceretti, m. moretti & s. monarca, 2007. allium cepa chromosome abberation and micronucleus tests applied to study genotoxicity of extracts from pesticide-treated vegetables and grapes. food addit. contam, 24 (6): 561-572. gandhi, g., j.b. chowdhury, p.k. sareen &v.p.s. dhillon,1995. genotoxic effects of deltamethrin in the mouse bone marrow micronucleus assay. mutat. res, 346: 203-206. science diliman (january-june 2012) 24:1, 13-22 acknowledgements the authors especially want to mention the financial help provided by the up-cst, lucknow to sibhghatulla; 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jon stewart h. dy ian kendrich c. fontanilla institute of biology university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract achatina fulica is a ubiquitous land snail commonly found throughout the philippines. as a generalist feeder and being able to survive in a wide range of habitat types and conditions, the snail can easily establish itself in a new area after introduction. it also acts as host to a variety of parasites, including nematodes, which may accidentally infect humans. in this study, a. fulica individuals from 13 areas in the philippines were sampled and analyzed for nematode infection rate and load. of the 393 i n d i v i d u a l s s a m p l e d , 8 0 ( 2 0 % ) w e r e f o u n d t o b e i n f e c t e d , w i t h 5 0 4 9 nematodes isolated. the infection rates and parasite load were highly v a r i a b l e . o v e r a l l , t h e p a r a s i t e l o a d r a n g e s f r o m 1 t o 8 6 7 p e r s n a i l . representative nematodes from a . fulica from plaridel (n=8) and davao c i t y ( n = 2 6 ) i n m i n d a n a o w e r e s u b j e c t e d t o d n a e x t r a c t i o n , p c r a m p l i f i c a t i o n , a n d s e q u e n c i n g o f t h e s s u r r n a g e n e , w h i c h i s t h e universal barcode for nematodes. sequences successfully matched with the dog lungworm oslerus osleri for the plaridel nematodes and the rat l u n g w o r m a n g i o s t r o n g y l u s c a n t o n e n s i s f o r t h e d a v a o c i t y n e m a t o d e s , r e s p e c t i v e l y. t h e l a t t e r i s k n o w n t o i n f e c t h u m a n s a n d c a n c a u s e eo s i n o p h i l i c m e n i n g o e n ce p h a l i t i s . t h i s s t u d y p r e s e n t s t h e f i r s t r e p o r t of a. cantonensis in a. fulica from mindanao and raises a public health concern. keywords: achatina fulica, nematode, philippines, ssu rrna , oslerus osleri, angiostrongylus cantonensis d.m.a. constantino-santos and others 73 introduction the giant african land snail, achatina fulica (family achatinidae), is a ubiquitous snail with characteristic reddish brown markings on the shell (jarrett 1931, raut and barker 2002) and has the ability to adapt to a wide range of environmental conditions (cobbinah and others 2008). its habitat and diet include a wide variety of plant species (jarrett 1931, raut and barker 2002). a. fulica originated from eastern coastal africa but has spread throughout asia and the pacif ic, including the philippines, during world war ii. a . fulica was introduced to these regions by the japanese to be used as an alternative food source (alicata 1966, latonio 1971). a. fulica is capable of carrying large numbers of parasitic nematodes that may infect humans (kliks and palumbo 1992). the snail is a known intermediate host of the rat lungworm angiostrongylus cantonensis (tsai and others 2004, cobbinah and others 2008, zhang and others 2009, fontanilla and wade 2012, constantinosantos and others 2014). a. cantonensis infection causes eosinophilic meningitis in humans, and outbreaks of the disease have been associated with exposure to infected a . f u l i ca (tsai and others 2004). humans may become infected by a. cantonensis via consumption or contact with an infected snail. consumption of a. fulica is quite common due to its high protein, iron, and calcium content ( ts a i a n d others 2003, cobbinah and others 2008). infection by contact with a . fulica was documented in taiwan, especially among children playing with the snail (tsai and others 2004). apart from a. cantonensis, a. fulica has also been shown to harbor rhabditis sp. (seehabutr 2005), the cat lungworm aelurostrongylus abstrusus (de andrade-porto and others 2012), and the dog lungworm ancylostoma caninum (constantino-santos and others 2014). numerous studies in the philippines have targeted gastropods, including a. fulica, to determine the presence of a specif ic nematode, a . cantonensis, but these were limited to luzon (salazar and cabrera 1969, westerlund and chamberlain 1969, latonio 1971, fontanilla and wade 2008). a recent study by constantino-santos and others (2014) demonstrated the presence of two medically important nematodes, a . cantonensis and a . caninum, and 12 other unidentif ied nematodes in a. fulica populations found in metropolitan manila. they used the ssu rrna gene to identify the nematodes or determine their closest match. in this study, a survey of the nematode parasite load and infection rate in a. fulica populations was conducted in 13 different parts of the philippines. furthermore, nematodes in the mindanao populations, which were surveyed for the f irst time, were identif ied through dna sequencing of the ssu rrna gene. philippine survey of nematode parasite infection 76 ·  results and discussion all areas sampled were found to have infected snails except for batac, ilocos norte. infection rates for infected populations, however, varied, ranging from 3% to 39%. even the parasite load of infected snails exhibited high variability, ranging from 1 to 867 nematodes per snail. davao city, in particular, had the highest infection rate and total number of parasites (table 1). the variation in parasite load could be a function of the relative age of the snail. sithithaworn and others (1991) found a positive correlation between the age of the snail based on its shell size and the parasite load; they noted a mean parasite load of 5478 per infected snail in the oldest snail group (>6.60 cm shell length). on the other hand, the variation in rate of infection across the different a. fulica populations may be due to the patchy distribution of both the parasite and snail host. for instance, bisseru (1971) observed high variation of infection rates in a. fulica even within a small geographic area in west malaysia, with two populations exhibiting no infection. the prevalence of the parasite is subject to the availability of the hosts, both def initive and intermediate, for the parasite to complete its life cycle. eight nematode samples from plaridel were successfully subjected to direct sequencing of the 5’ end of the ssu rrna gene and gave higher than 99.5% identity with nematoda sp. fontanilla (genbank ef514918) and oslerus osleri (genbank ay295812) based on blast results. the blast results for the plaridel nematodes bacoor, cavite 30 3 (10%) 53 (2-49) baguio city, benguet 30 4 (13%) 82 (1-68) batac, ilocos norte 30 0 (0%) 0 (0) biasong, cebu 30 4 (13%) 10 (1-5) legazpi, albay 30 8 (27%) 792 (1-743) boac, marinduque 30 11 (37%) 184 (2-160) butuan city, agusan del norte 30 5 (17%) 1478 (5-867) davao city, davao 30 11 (37%) 2300 (1-791) matanao, davao del sur 30 2 (7%) 8 (2-6) plaridel, misamis occidental 33 13 (39%) 74 (1-27) tagbilaran city, bohol 30 10 (33%) 43 (1-18) taytay, rizal 30 8 (24%) 24 (1-7) tres de avril, cebu 30 1 (3%) 1 (1) total 393 80 (20%) 5049 (0-867) table 1. infection rates and parasite load of a. ful ica specimens from 13 sites in the philippines site sample size number of infected snails (percentage) total number of nematodes (parasite load range) d.m.a. constantino-santos and others 77 are summarized in table 2. following the 99.5% threshold value proposed by floyd and others (2002) in identifying nematodes using the ssu rrna gene, these nematodes are therefore in the same molecular operational taxonomic unit (motu) as oslerus osleri. o. osleri is previously classif ied as filaroides osleri. it is a metastrongyle parasite with worldwide distribution and is a widely occurring tracheal parasite capable of causing respiratory disease in domestic and wild canids such as dogs. it is transmitted by direct contact and oral ingestion of the f irst-stage larva (foreyt and foreyt 1981, outerbridge and taylor 1998). the life cycle of this nematode does not involve an intermediate host (outerbridge and taylor 1998). mucociliary apparatus carries the eggs and the infective f irststage larva from the tracheal bifurcation to the oropharynx, where they are either swallowed and shed in the feces or shed in the saliva. the primary sources of the parasite are the asymptomatic dogs (outerbridge and taylor 1998). following infection, the f irst-stage larvae penetrate the mucosa of the gastrointestinal tract. it then travels to the right side of the heart via lymphatics or the hepatic venous circulation where the larva migrates to the lungs via pulmonary arteries (yao and others 2011). development from the f irst-stage larva to adult occurs in the respiratory tract where tracheobronchial nodules are formed, thus completing the life cycle (clayton and lindsay 1979). maternal grooming is assumed to be the major transmission route in domestic dogs; for free-ranging canids, the regurgitative feeding of the young by parents appeared to be the major means of infection (clayton and lindsay 1979, bowman 2009). the presence of o. osleri in a. fulica in plaridel, misamis occidental could be a result of accidental infection as the snail is not the def initive or f inal host of the parasite and may have only been shed via the feces by an infected dog. a previous study by constantino-santos and others (2014) also detected the presence of worms most similar to o. osleri (99.1% identity) and o. rostratus (99.5%–99.7% identity) in a. fulica populations in metro manila, philippines. nevertheless, detection of o. osleri in plaridel indicated that a. fulica could serve as another route of infection for humans, especially to those who come in contact with these snails. twenty six nematode samples from davao city had the 5’ end of their ssu rrna gene successfully sequenced and identif ied as angiostrongylus cantonensis (genbank gq181114) based on blast results and floyd’s 99.5% threshold value, with f ive of them having 100% identity with a. cantonensis, as shown in table 2. philippine survey of nematode parasite infection 78 plaridel, misamis occidental 15-2a ef514918 nematoda sp. fontanilla d17-d1 446/446(100%) 0/446(0%) ay295812 oslerus osleri 444/446(99.6%) 0/446(0%) 15-2b ef514918 nematoda sp. fontanilla d17-d1 406/406(100%) 0/406(0%) ay295812 oslerus osleri 405/406(99.8%) 0/406(0%) 15-3a ef514918 nematoda sp. fontanilla d17-d1 423/423(100%) 0/423(0%) ay295812 oslerus osleri 421/423(99.5%) 0/423(0%) 15-3b ef514918 nematoda sp. fontanilla d17-d1 416/416(100%) 0/416(0%) ay295812 oslerus osleri 414/416(99.5%) 0/416(0%) 15-5a ef514918 nematoda sp. fontanilla d17-d1 387/388(99.7%) 0/388(0%) ay295812 oslerus osleri 386/388(99.5%) 0/388(0%) 15-5b ef514918 nematoda sp. fontanilla d17-d1 420/420(100%) 0/420(0%) ay295812 oslerus osleri 418/420(99.5%) 0/420(0%) 15-13 ef514918 nematoda sp. fontanilla d17-d1 422/422(100%) 0/422(0%) ay295812 oslerus osleri 420/422(99.5%) 0/422(0%) 22-4 ef514918 nematoda sp. fontanilla d17-d1 420/421(99.8%) 0/421(0%) ay295812 oslerus osleri 418/421(99.3%) 0/421(0%) davao city, davao dv1 gq181114 angiostrongylus cantonensis 428/429(99.8%) 0/429(0%) dv3 gq181114 angiostrongylus cantonensis 439/439(100%) 0/439(0%) dv4 gq181114 angiostrongylus cantonensis 439/439(100%) 0/439(0%) dv5 gq181114 angiostrongylus cantonensis 428/429(99.8%) 0/429(0%) dv6 gq181114 angiostrongylus cantonensis 438/439(99.8%) 0/439(0%) dv7 gq181114 angiostrongylus cantonensis 434/436(99.5%) 0/436(0%) dv3a jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3b jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3c jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3d jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3e jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3f jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3g jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3h jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3i jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3j jn663725 angiostrongylus cantonensis 460/460(100%) 0/460(0%) dv3k jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3l jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3n jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3o jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3q jn663725 angiostrongylus cantonensis 460/460(100%) 0/460(0%) dv3s jn663725 angiostrongylus cantonensis 460/460(100%) 0/460(0%) dv3t jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3x jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3y jn663725 angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) dv3aa jn663725 angiostrongylus cantonensis 468/469(99.8%) 1/469(0.2%) table 2. identity of nematodes from achatina ful ica in plaridel, misamis occidental and davao city, davao based on blast specimen code accession number best match % similarity gaps d.m.a. constantino-santos and others 79 this study demonstrates that a. fulica in davao facilitates the spread of a. cantonensis. the absence of a. cantonensis in plaridel, on the other hand, is not unexpected. in a study by bisseru (1971), 27 sites in west malaya were surveyed for a. cantonensis from a. fulica, and two of these sites did not even yield any parasite. for instance, padang besar in perlis had 0% infection rate from 100 snails sampled, whereas alor star, kedah, which was 65 km away, yielded 28.5% infection rate from 70 snails. the snail intermediate hosts, the rat def initive hosts, or even the parasites, have a patchy distribution themselves; most likely, only those snails found in areas with rats that harbored the parasite were the ones infected (fontanilla and wade 2012). cases of a. cantonensis infection in rodents and molluscs have been documented in the philippines; however, most of these were found in several provinces in luzon (salazar and cabrera 1969, westerlund and chamberlain 1969, garcia 1979, antolin and others 2006) and one area in the visayas (guerrero and guerrero 1972). this is the f irst reported case of a. cantonensis in mindanao in either the snail intermediate host or the rat def initive host. the presence of a . cantonensis in mindanao is not surprising considering the distribution of rats and a. fulica all over the philippines. this information, however, is signif icant from a public health perspective. these nematodes cause eosinophilic meningitis or meningoencephalitis (wan and weng 2004). they are now generally recognized as the causative agent of human eosinophilic meningitis, also called angiostrongyliasis (panha 1988), which is characterized by inflammation of the meninges in the human brain and the presence of higher levels of eosinophils in the cerebral spinal fluid (senanayake and others 2 0 0 3 ) . a three-year study done by latonio (1971) focused on four cases of eosinophilic meningoencephalitis and two cases of myeloencephalitis symptom-complex from patients in the philippines. he reasoned that infection might not be due to direct consumption, since a. fulica is not normally consumed by filipinos. rather, it is possibly due to the prevalence of a. fulica among edible plants, which could have been contaminated by the snail with a. cantonensis, either through their feces or mucus, before they were consumed by humans (wallace and rosen 1969, marquardt and others 2000). alternatively, handling infected snails, particularly by children who play with them, could have been another possible route of infection (wan and weng, 2004). occurrence of a. cantonensis in canton, china was f irst described by chen (1935) in rats. rodents are considered its def initive host (e.g. , rattus rattus, r. norvegicus) whereas intermediate hosts include snails (e.g. , a c h a t i n a f u l i ca , p o m a ce a philippine survey of nematode parasite infection 80 canaliculata) and slugs (e.g. , imerinia plebeia, leavicaulis alte) (bartschi and others 2003). the basic life cyle of a . cantonensis involves a def initive mammalian host and an intermediate molluscan host. the adult worms live in the pulmonary arteries of their def initive hosts, where the females also lay their eggs. these hatch into f irststage larvae, which are then transmitted to the rats’ feces via the trachea and gastrointestinal tract. these larvae enter their intermediate hosts, such as molluscs, through ingestion of the excrement, wherein they turn into third-stage larvae after two successive molts (lee and yen 2005). an infected mollusc can carry a highly variable number of second– and third–stage juveniles depending on the degree of infection (caldeira and others 2007). these third-stage larvae infect the rat host through consumption of the intermediate host. the larvae migrate to the central nervous system where further development occurs until they reach adulthood. from there, they then return to the pulmonary arteries where they undergo sexual maturation (qvarnstrom 2007). humans, upon ingestion of these molluscs in their raw forms, become accidental hosts, manifesting the infection as eosinophilic meningitis (senanayake and others 2003). the third-stage larvae, similar to what occurs in rodents, migrate to the central nervous system, which consists of the brain and spinal cord tissues. however, these larvae often remain in the central nervous system of human hosts; nevertheless, rare cases exist wherein they continue migration to the lungs. their presence in the brain and spinal cord causes tissue damage and subsequent inflammation (qvarnstrom 2007). to prevent human infection, the most effective method is to educate people to maintain good sanitation in food preparation areas, not to eat raw or undercooked snails, and to avoid eating raw vegetables that may harbor inconspicuous or juvenile snails or slugs in regions where a . cantonensis is present (yang and others 2013). as davao a. fulica snails carry the said parasite, it is important to heighten the awareness of people in the area of the possibility of infection through consuming raw vegetable crops associated with the snails or playing or handling these snails. moreover, control and monitoring of various intermediate hosts, in this case a . fulica, and def initive hosts in areas of epidemiological relevance should be undertaken to lessen the risk to humans. acknowledgements this study was funded through a phd incentive grant (project number: 101017) awarded to dr. ian kendrich c. fontanilla by the university of the philippines diliman d.m.a. constantino-santos and others 81 off ice of the v ice-chancellor for research and development . the authors acknowledge the logistical support provided the 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o b s e r v a t i o n s o n a n g i o s t r o n g y l u s cantonensis in the philippines. acta medica philippina 6: 3-11. yang t. , wu z. , lun z. 2013. the apple snail pomacea canaliculata, a novel vector of the rat lungworm, angiostrongylus cantonensis: its introduction, spread, and control in china. hawai‘i journal of medicine & public health 72: 23-25. yao c. , o’toole d. , driscoll m. , mcfarland w. , fox j. , cornish t. , jolley w. 2011. filaroides o sle r i (osle r u s o sle r i) : t w o c a s e r e p o r t s a n d a r e v i e w o f c a n i d i n f e c t i o n s i n n o r t h america. veterninary parasitology 179(1-3): 123-9. zhang l.h. , chen j.l. , dong w.r. 2009. analysis of a survey of the infection of achatina f u l i c a w i t h a n g i o s t r o n g y l u s c a n t o n e n s i s i n f o s h a n , g u a n g d o n g p r o v i n c e . a c t a parasitologica et medica entomologica sinica 16: 244-246. _____________ daisy may a. constantino-santos <daisymay_constantino@yahoo.com> is a phd student at the institute of biology, university of the philippines (up), diliman and a university research associate at the natural sciences research institute, up diliman. she obtained her bsc biology at up baguio and her msc biology (genetics) at up diliman. she specializes in molecular genetics. brian s. santos <bryzeel13@yahoo.com> is an instructor and a phd student at the institute of biology, up diliman. he obtained his bsc biology and msc biology (genetics) at the institute of biology, up diliman. he specializes in molecular population genetics and morphometrics. johanne myrrh e. soriano is currently a medical student at the far eastern university nicanor reyes medical foundation. she obtained her bsc biology at up diliman. jon stewar t h. dy is currently a medical student at the st. luke’s college of medicine. he obtained his bsc biology at up diliman. ian kendrich c. fontanilla <ianfontanilla@hotmail.com> is an assistant professor and head of the dna barcoding laboratory, institute of biology, university of the philippines diliman. he received his phd in genetics from the university of nottingham, united kingdom. he specializes in molecular genetics and molecular phylogenetics. sd inside back cover-ched.pmd science diliman: a philippine journal of pure and applied sciences is accredited by the commission on higher education (ched) through its journal incentive program, as journal incubation category. science diliman has been selected for coverage in the emerging sources citation index of thomson reuters. science diliman is listed in asean citation index and is available via www.doaj.org and www.ebsco.com reproductive biology-nabuab.pmd reproductive biology of the short-necked clam, paphia undulata 31science diliman (july-december 2010) 22:2, 31-40 reproductive biology of the short-necked clam, paphia undulata (born 1778) from southern negros occidental, central philippines fenelyn m. nabuab1, leah ledesma-fernandez 2 and annabelle del norte-campos 1* 1marine biology laboratory, cas, university of the philippines visayas, miagao, iloilo 2west negros university, bacolod city *corresponding author: upvmarbio@yahoo.com abstract the short-necked clam, paphia undulata, occurs in coastal muddy bottoms of hinigaran, binalbagan, and himamaylan, negros occidental where it has an important contribution to fisheries. however, stocks in these areas are dwindling and in order to formulate sound management strategies, information on the reproductive biology of this species is a prerequisite. also, knowledge on the reproduction of this species is a prelude to its laboratory breeding and rearing. reproductive biology of the short-necked clam from the three areas was studied over an annual cycle. short-necked clams were collected monthly from catches of compressor divers. five gonad stages were described based on histological examinations: developing, mature, partially spawned, redeveloping, and spent. immature or indeterminate gonads were also noted. although occasional presence of hermaphrodite gonads was observed this species is still functionally dioecious with 1.00m: 1.02f sex ratio. sexual maturity is attained at 42.6 mm for males and 44.8 mm for females. short-necked clams have continuous breeding season. however, peak of spawning activities is on the months of august to november which coincide with the monsoon transition months probably due to the decrease in salinity and nutrient influx during these months. organisms smaller than 45mm must not be collected and collection must be regulated during peaks of spawning activities to leave a portion of reproductively mature individuals to ensure sustainability of fisheries. however, in terms of laboratory breeding and rearing of this species, it is fundamental to consider the spawning peaks and size at sexual maturity to ensure successful spawning in the laboratory. hence, broodstock to be used in the experiments must be sexually mature (>45mm) and collected during peaks of spawning activities. key words: paphia undulata, reproductive biology, gonad stages, dioecious, size at sexual maturity, continuous breeding season, sustainability of stocks nabuab, f.m. et al. 32 science diliman (july-december 2010) 22:2, 31-40 introduction paphia undulata (born), or the short necked-clam (fig 1.), occurs in muddy substrate (4-16m depth) along the coasts of hinigaran, binalbagan, and himamaylan, negros occidental in central philippines. because of their value to both local and export markets, there is indiscriminate harvesting of this resource. in fact, shortnecked clams have already been determined as overexploited in these areas (agasen et al. 1998), a situation which has worsened even more today (del norte – campos and villarta 2010). overexploitation will ultimately lead to biological and economic maturity. in thailand (tuaycharoen 1984; http: www.fisheries.go.th/mf-umdec/subject3.htm) and in china (zhijiang et al 1991), the reproductive biology of p. undulata has already been documented and the results of the present study are compared with these reports. materials and methods a maximum of sixty (60) short-necked clams were collected monthly from august 2007 to july 2008 from catches of compressor divers in hinigaran, binalbagan, and himamaylan, negros occidental (fig. 2). clams were packed in ice for transport to the laboratory where specimens were weighed, measured, and dissected. shell length and shell width were measured to the nearest 0.05mm using a vernier caliper. total (tw), viscera (vw), and gonad (gw) weights were figure 1. the short-necked clam, p. undulata, locally known as “ nylon shell”. overfishing, especially so since the high demand for this resource can only be supplied by harvesting from natural stocks. information on the reproductive biology of this species is a prerequisite for the formulation of management initiatives to develop sustainable exploitation of the resource in these areas. this is imperative since one potential cause of mismanagement is a poor understanding of the biology of the species. furthermore, knowledge on the reproduction of this species is a prelude to its laboratory breeding and rearing. this study was conducted to describe the reproductive biology of the short-necked clam, specifically to describe gonad stage development, determine spawning periodicity, sex ratios and minimum size at sexual figure 2. site of collection p. undulata in coastal waters of negros occidental, central philippines. hinigaran binalbagan himamaylan reproductive biology of the short-necked clam, paphia undulata 33science diliman (july-december 2010) 22:2, 31-40 measured to the nearest 0.01mg using a digital electronic balance. the gonadosomatic index (gsi %) of each individual was computed using the formula gsi = vw/gw *100 (sastry 1979) and plotted against time. after measurements, clams were dissected and the gonads were fixed in bouin’s solution. gonads were then processed using standard histological procedures (humason 1972). sex was determined by examining clams under a compound microscope. monthly and overall sex ratios were computed and their skewness tested ( 2test). gonad stages were described and classified into the following: developing, mature, partially spawned, redeveloping, and spent based on the predominant morphological appearance of the gametes (tuaycharoen 1984; rose et al. 1990; ledesmafernandez and del norte-campos 2004; kang et al. 2007). monthly frequencies of gonad stages were then plotted against time. size at sexual maturity is defined as the smallest shell length staged with mature gonads. results and discussion sex ratio, gonad morphology, and size at sexual maturity a total of 692 short-necked clams were collected. of these 11.9% (82) were sexually indeterminate, 0.4 % (3) were hermaphrodites, 43.4% (300) were males and 44.4% (307) were females (table 1). male to female ratio was 1.00: 1.02 and based on chi-square test, males and females are equally represented in the population. this means that the population is still in equilibrium, wherein inbreeding and competition for mates does not occur (downing et al. 1989). this also shows that even if a portion of the potential spawning stock is left in the population, there will still be fertilization which eventually will lead to recruitment. eventually this recruitment will translate to recovery of the stocks provided that sustainable exploitation of the resource is practiced in the area. short-necked clams are functionally dioecious wherein male and female sexes are separate, with very low incidences of hermaphroditism. however, this species does not exhibit sexual dimorphism. both male and female gonads are cream to pale orange in color regardless of sex, stage, χ table 1. sex ratios of the short-necked clam of p. undulata from monthly samples (august 2007 – july 2008) collected from hinigaran, binalbagan, and himamaylan negros occidental (m: male, f: female, h: hermaphrodite, in: indeterminate). months male female h in total(n) sex ratio m:f aug ‘07 23 36 1 60 1:1.57 sept 24 36 60 1:1.50 oct 26 31 1 1 59 1:1.19 nov 13 20 33 1:1.53 dec 31 25 4 60 1:1.81 jan ‘08 29 30 1 60 1:1.03 feb 31 29 60 1:0.94 mar 37 22 1 60 1:0.60 apr 14 14 32 60 1:1.00 may 17 16 27 60 1:0.94 jun 21 22 17 60 1:1.05 jul 34 26 60 1:0.76 total (n) 300 306 3 82 692 1:1.02 % 43.35 44.36 0.43 11.85 nabuab, f.m. et al. 34 science diliman (july-december 2010) 22:2, 31-40 and size. thus, it is not possible to determine the sex and characterize the gonad development of this species through external morphological examination. shell lengths of the collected clams ranged from 20.7mm to 71.9mm. the smallest shell length staged with mature gonads was 42.6mm for males and 44.8mm for females. since this species does not exhibit sexual dimorphism, a nominal size of sexual maturity for both sexes is set at 45mm. setting this as the minimum harvestable size ensures that at least a portion of the population is allowed to spawn and contribute to recruitment, enhancing the sustainability of the resource. in thailand, the average size at sexual maturity is reported to be greater than 40mm (tuaycharoen et al. 1984; pongthana 1990), similar to the results in this study. gonad stages histological analysis of the gonads showed four determinate stages for male (figs. 3a-d) and five for female (figs. 4a-e). table 2 summarizes the description of the gonad stages. reproductive stages and spawning periodicity p. undulata has a protracted or continuous breeding season as supported by the co-occurrence of different reproductive stages in all the monthly samples of the population (figs. 5-6). the presence of mature gonads in almost all months and the infrequent occurrence of spent stages are consistent with this species having a prolonged reproductive cycle. however, variation in the intensity of the reproductive activities results in peaks or periodicity in the reproductive phases. this pattern figure 3a-d. gonad stages of male short-necked clam, p. undulata a. developing, b. mature, c. partially spawned, d. redeveloping (magnification (100x). reproductive biology of the short-necked clam, paphia undulata 35science diliman (july-december 2010) 22:2, 31-40 table 2. gonad stages of p. undulata collected from hinigaran, binalbagan, and himamaylan, negros occidental on august 2007 to july 2008.   stage male female 1. immature/ sexually indeterminate no definite gonad region or follicle. no gamete proliferation. sex cannot be determined. 2.developing rounded to expanded tubules with spermatogonia and spermatocytes. few spermatozoa proliferate at the center. oogonia at different stages of development. oocytes are attached to the edges but starting to fill the follicles. 3.ripe tubules are elongated assuming rosette formation and filled with spermatozoa. follicles are filled with mature oocytes with prominent nucleus and nucleolus. 4. partially spawned tubules show streaky appearance of streaming sperms. spermatozoa still occupy the lumina but have numerous gaps. spacious acini, remaining mature oocytes at some acini. gaps on the follicle walls. 5.redeveloping connective tissue present. some tubules are empty but generally surrounded by residual spermatozoa. connective tissues present. oocytes at different stages are present. some follicles are empty. 6.spent empty follicles. may contain residual oocytes at the connective tissues. nabuab, f.m. et al. 36 of reproduction is exhibited by both male and female clams. at the start of the study (august 2007), a high frequency of partially spawned females was observed and this remained high until november. this was followed by prolonged gametogenesis from december to july, wherein the dominant stages were developing and mature. on the other hand, male short-necked clams showed a more protracted breeding pattern as shown by the dominance of developing and mature gametes in almost all months, and by the absence of spent stages. the latter is attributed to the smaller size of sperm relative to egg cells, resulting to a lower energetic cost of reproduction typical in males. this is believed to be a strategy to ensure availability of mature sperm whenever females spawn, thus allowing synchronicity between male and female reproductive cycles. since short-necked clams are broadcast spawners, synchronicity of reproductive cycles between the sexes is necessary to ensure successful fertilization. science diliman (july-december 2010) 22:2, 31-40 fig. 4a-e. gonad stages of female short-necked clam, p. undulata a. developing, b. mature, c. partially spawned, d. redeveloping, e. spent (magnification (100x). reproductive biology of the short-necked clam, paphia undulata 37science diliman (july-december 2010) 22:2, 31-40 protracted breeding seasons with periodicity over an annual cycle is commonly exhibited by tropical bivalve species such as scapharca inequivalvis (ledesmafernandez and del norte-campos 2004), anodontia edentula (samentar et al. 2004), and gari elongata (nabuab and del norte-campos 2006). this is because seasonal patterns of gonad development result from responses to within-year changes in environmental factors (sastry 1979; mackie 1984), although seasonal variation in the tropics may be minimal. high spawning activities of male and female short-necked clams coincides during the months of august to november (transition months to the northeast monsoon). a stronger signal is observed during these months likely due to lower salinities related to the influx of riverine water (palla et al. in prep.). influx of riverine water, furthermore, increases terriginous inflow of organic matter which enhances nutrients in the water. nutrient influx-induced spawning has also been observed in s. inequivalvis (ledesma-fernandez and del nortecampos 2004) and g. elongata (nabuab and del norte-campos 2006) from banate bay in eastern panay, about 60km north of the study area. the months of august to november are considered as the peak in spawning for both male and female clams. subsequent recruitment occurred in the following months of january to march (del norte-campos and villarta 2010). gonadosomatic indices (gsi) for male (8.53-14.15 %) and female (9.59-13.63%) short-necked clams showed minimal variation over an annual cycle (figs. 7-8), which is consistent with extended gametogenesis in this species. however, above average gsi values were observed during the northeast monsoon months coinciding with the peak of reproductive activities. other studies show that p. undulata has two spawning peaks: april – may and august – november in trat province, thailand (tuaycharoen 1984) and in may and october in china (zhijiang et al 1991). these studies show that the species spawns during preand postmonsoon months, possibly with varying degrees in peak activity. however, the spawning season in the present study is more similar to the spawning peak (august to october) observed in the population of short-necked clams in samut sakhon province, thailand (jindalikit, undated). this illustrates that reproductive activity (gametogenesis and spawning) of p. undulata varies from one area to another, possibly in response to the local environmental conditions. summary and conclusions p. undulata is a functionally dioecious bivalve that exhibits no sexual dimorphism. however, occasional incidence of hermaphroditism was observed. sex ratio is 1.00m: 1.02f, which shows that the male and female portions of the population are equally represented. it attains sexual maturity at 42.6mm in males and 44.8mm in females. the effective size at sexual maturity can be taken as 45mm. five gonad stages were described in the short-necked clams: developing, mature, partially spawned, redeveloping, and spent. based on the monthly frequency distribution of the gonad stages and the gsi, p. undulata has a protracted breeding season with high spawning activities in the transition months to the northeast monsoon (august to november) probably due to high nutrient influx during these months. spawning of male and female clams also coincides during these months, showing synchronicity between male and female gonad development. the reproductive cycle of p. undulata varies in different locations probably due to the differences in the local conditions. in terms of laboratory breeding and rearing of this species, it is fundamental to consider the spawning peaks and size at sexual maturity to ensure successful spawning in the laboratory. thus, broodstock to be used in the experiments must be sexually mature (>45mm) and collected during peak spawning months (august to november). regarding sustainable exploitation of this resource, it is imperative to leave a portion of the spawning stock in the population to allow continuous breeding and recruitment. thus, gathering of individuals smaller than 45mm must be prohibited and collection must be regulated during the months of peak spawning activity (august to november). acknowledgments this study was funded by a grant from the philippine council of aquatic and marine research and development (pcamrd) of the dost. field assistance was provided by the mije and laurel families of brgy. tagda, hinigaran, negros occidental. nabuab, f.m. et al. 38 figure 5. frequency distribution of gonad stages in female (n=307) p. undulata collected from august 2007 to july 2008 in hinigaran, binalbagan, and himamaylan, negros occidental. figure 6. frequency distribution of gonad stages in male (n = 300) p. undulata collected from august 2007 to july 2008 in hinigaran, binalbagan, and himamaylan, negros occidental. science diliman (july-december 2010) 22:2, 31-40 reproductive biology of the short-necked clam, paphia undulata 39science diliman (july-december 2010) 22:2, 31-40 figure 7. the gonadosomatic index (gsi) of female p. undulata collected from hinigaran, binalbagan, and himamaylan, negros occidental from august 2007 to july 2008. figure 8. the gonadosomatic index (gsi) of female p. undulata collected from hinigaran, binalbagan, and himamaylan, negros occidental from august 2007 to july 2008. nabuab, f.m. et al. 40 science diliman (july-december 2010) 22:2, 31-40 references agasen, e.v., c.m. del mundo, & g.o. matias. 1998. assessment of paphia undulata in negros occidental/ guimaras strait waters. j. shellfish res. 17(5): 1613-1617 del norte-campos, a.n.c. and k.a. villarta. 2010. “use of population parameters in examining changes in the status of the short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) in coastal watersof southern negros occidental. sci. dil. 22(1):53-60. downing, j.a., j.p. amyot, m. perusse and y. rochon. 1989. visceral sex, hermaphroditism, and protandry in a population of the freshwater bivalve elliptio complanata. j. n. am. benthol. soc. 8(1): 92-99. jindalikit, j. undated. reproductive biology of shortnecked clam paphia undulate (born,1778) in mahachai bay, samut sakhon province. abstract from upper gulf marine fisheries research and development center (http: www.fisheries.go.th/mf-umdec/subject3.htm). humason, g.l. 1972. animal tissue techniques. w.h. freeman and co., san francisco: 725 p. kang, d., hyun, c., limpanont, y., and k. choi. 2007. annual gametogenesis of the chinese anapella clam coecella chinensis (deshayes 1855) at en upper intertidal sandy beach on the east coast of jeju, korea. j. shellfish res. 26(2): 433-441. ledesma-fernandez, l.n. & a.g.c. del norte-campos. 2004. reproductive cycle of the ark shell scapharca inaequivalvis (brugiere, 1789) (mollusca, pelecypoda: arcidae) in banate bay, west central philippines. upv j. nat. sci. 9(1): 111-123. mackie, g.l. 1984. bivalves. in: “the mollusca”. (eds a.s. tompa, n.h. verdonk and j.a.m. van den biggelaar.) vol. 7, pp. 351-418. academic press: new york. nabuab, f.m. and a.g.c. del norte-campos. 2006. “some aspects of the reproduction in the elongate sunset clam, gari elongata (lamarck 1818) from banate bay area, west central philippines. sci. dil. 18(2): 34-46. palla, r.q., w.l. campos, and a.n. campos. sled trawl survey of megabenthic assemblages in heavily-fished coastal waters of western negros island, central philippines. pams 10, davao, phils. pongthana, n. 1990. breeding and rearing of short-necked clam (paphia undulata). thai. mar. fish. res. bull. 1: 6973. rose, r.a., dybdahl, r.e., and s. harders. 1990. reproductive cycle of the western australian silverlip pearl oyster, pinctada maxima (jameson)(mollusca: pteriidae). j. shellfish res. 9(2): 261-272. samentar, l., m. formacion, and j. geduspan. 2004. reproductive biology of the mud-dwelling clam, imbaw (anodontia edentula) in guimaras province, central philippines. upv j. of nat. sci. 9:217-228. sastry, a.n. 1979. pelecypoda (excluding ostreidae). v. mollusks: pelecypoda and lesser classes. acad. press, n.y. tuaycharoen, s. 1984. gonadal development and sex ratio of the short-necked clam (paphia undulata) (born). technical paper no. 35. brackishwater fisheries div. dept. of fisheries, bangkok. 31pp. zhijiang, z., f. li, and c. ke. 1991. on the sex gonad development and reproductive cycle of clam paphia undulata. abstract from journal of fisheries of china, shuichan xuebao. 15(1): 1-8. 30 isolation and antimicrobial activity of fructophilic lactic acid bacteria from flowers in the university of the philippines diliman, quezon city gil m. penuliar* renz joseph r. artezuela institute of biology university of the philippines diliman abstract fructophilic lactic acid bacteria (flab) is a recently discovered group of lactic acid bacteria that prefers fructose as carbohydrate source. the isolation of flab from fructose-rich niches like flowers, in particular, and the gut of insect pollinators suggests that it may be used as probiotics. the objective of this study was to determine if flab can be isolated from flowers at the institute of biology, university of the philippines diliman, and to screen them for antimicrobial activity against bacteria that are commonly associated with intestinal diseases. a total of 20 different isolates were obtained from 14 species of flowers. all isolates were identified as lab, but only 17 out of 18 isolates were osmotolerant in 30% fructose, and only 8 out of 15 isolates had higher absorbance in fructose yeast peptone broth, which are characteristics of presumptive flab. seven isolates exhibited inhibitory activity in at least three test bacteria in the primary screening and only four isolates had inhibitory activity in at least two test bacteria, particularly against enterococcus faecalis and campylobacter jejuni, in the secondary screening. dna sequencing and phylogenetic analysis identified isolates mfps 4.1 and mfru 7.2 as weissella spp. the in vitro antimicrobial activities of these isolates can be studied further for possible applications in food and medicine, and their low sequence similarities suggest that the isolates might be novel weissella species. keywords: flab, antimicrobial activity, weissella spp. * corresponding author science diliman (july-december 2021) 33:2, 30-52 g. m. penuliar and r.j. r. artezuela 31 introduction fructophilic lactic acid bacteria (flab) is a unique group of lactic acid bacteria that preferentially utilizes fructose as their main carbohydrate source under anaerobic conditions. in addition, key characteristics of flab include a need for electron acceptors, such as pyruvate and oxygen, for effective glucose utilization, and poor carbohydrate fermentation capabilities (endo et al. 2009). studies have shown that flab can be isolated from fructose-rich environments such as flowers, fruits, fermented products made from fruits, and animal by-products such as honey (endo and okada 2008; endo et al. 2011; syed yaacob et al. 2018). some species of flab, like lactobacillus apinorum, lactobacillus kunkeei, and fructobacillus fructosus, have been isolated from the guts of honeybees and are reported to exhibit antibacterial activity against bacteria that cause diseases in honeybees (endo and salminen 2013; olofsson et al. 2014; maeno et al. 2017). development on possible probiotic application of flab has advanced in recent years with the discovery of kunkecin a, a bacteriocin produced by apilactobacillus kunkeei ff30-6 isolated from honeybees, which had effectively inhibited melissococcus plutonius, which causes european foulbrood in honeybee larvae (zendo et al. 2020). it is possible that other members of flab isolated from other sources with observed antimicrobial activity can be used as probiotics for other animals. probiotics are live microorganisms that confer health benefits when consumed in adequate amounts, such as restoration of normal gut microflora, prevention of colonization of pathogenic bacteria, prevention of viral infections, and promotion of oral health care by preventing or treating oral diseases (reid 2002; kang et al. 2019). the mechanism of action of probiotics includes the production of anti-adhering molecules to prevent the attachment of pathogenic bacteria to the epithelial lining of the gut; alteration of gut ph through the production of lactic acid and acetic acid; and modulation of the host’s immune response by enhancing the expression of genes involved in the integrity of the intestinal barrier (bermudez-brito et al. 2012; collins et al. 2019). currently, there are no published studies related to flab in the philippines. fructobacillus tropaeoli, the most recent species of the genus fructobacillus, was isolated from the nasturtium flower, tropaeolum majus (endo et al. 2011). recent studies had also shown that other members of flab such as l. kunkeei and f. durionis were isolated from various fruit and flowers (ruiz rodriguez et al. 2019; sakandar et al. 2019). it would be interesting to study the flab population present in the diverse flora found in the philippines, especially the endemic floral species. isolation and antimicrobial activity of fructophilic lactic acid bacteria 32 the objectives of this study were (1) to isolate presumptive flab from flowers in the institute of biology, university of the philippines diliman, (2) to characterize and screen the isolates for in vitro antimicrobial activity, and (3) to identify the isolates through dna sequencing. the study aims to contribute information on flab by detecting their presence in local flowers and by screening for isolates for antimicrobial activity against selected bacteria associated with intestinal diseases. materials and methods sample collection fourteen flower species were aseptically collected from the institute of biology, university of the philippines diliman from 30 january 2019 to 8 february 2019. the samples were identified up to the genus and species level by the jose vera santos memorial herbarium and were placed in sterile plastic bags before briefly storing in a refrigerator at 4°c prior to processing. samples were dissected inside a biosafety cabinet, using sterile scalpel and forceps, to collect 1 g of floral nectaries (stamen and pistil), which were macerated inside a sterile 50 ml conical tube. isolation and purification of isolates enrichment, isolation, and purification methods were adapted and modified based on the study of endo et al. (2009). samples were enriched separately in 10 ml of fructose yeast peptone broth (fypb) (himedia laboratories, mumbai, india), de man, rogosa and sharpe broth (mrsb) (himedia laboratories, mumbai, india), and sobremisana, artezuela and penuliar broth (sapb), supplemented with 0.02% sodium azide and 0.01% nystatin as antifungal agent (acme laboratories, dhaka, bangladesh), and were incubated at room temperature (28°c) for 24 h. a loopful of enrichment culture was streaked on fructose yeast peptone agar (fypa), de man, rogosa and sharpe agar (mrsa), mrsa supplemented with 1% fructose, and sobremisana, artezuela and penuliar agar (sapa) plates, each supplemented with 0.5% caco 3 . the plates were incubated at room temperature (30°c) for 24– 48 h, and isolated colonies with distinct zones of clearing were purified on their respective isolation media using the 3-quadrant streak method. purification plates were incubated at room temperature (30°c) for 24–48 h. gram-staining gram-staining was performed based on a method adapted from the american society for microbiology (smith and hussey 2005). briefly, one loopful of a 24-h old bacterial culture was smeared onto a glass slide with one drop of distilled water g. m. penuliar and r.j. r. artezuela 33 and allowed to air dry. the smear was heat fixed by passing the glass slide over a flame three times. the slide was flooded with crystal violet for 30 s, rinsed with distilled water, then flooded with gram’s iodine for 1 min. the slide was then rinsed with 95% ethanol drop-wise until the runoffs were clear. the slide was flooded with safranin for 30 s and was rinsed with distilled water. the slide was then blotted dry and viewed using a microscope with 100х magnification. staphylococcus aureus and escherichia coli were used as positive and negative controls, respectively. catalase test approximately 3 ml of 3% hydrogen peroxide was dispensed in each slant of 24-h old cultures. the presence of effervescence indicated that the isolate was catalasepositive. s. aureus and streptococcus mutans were used as positive and negative controls, respectively. polymerase chain reaction confirmation using lab-specific primers the dna of the isolates were extracted using the g-spintm genomic dna extraction kit (intron biotechnology), following the protocol for gram-positive bacteria. a preliminary pcr amplification of the isolates was performed to confirm their identity as lab. each 10 μl reaction mixture contained 5 μl gotaq® master mix (promega, wisconsin, usa) (with 50 units/ml of taq dna polymerase in a proprietary reaction buffer [ph 8.5], 400 μm each of datp, dgtp, dctp and dttp, and 3 mm of mgcl 2 ), 2.4 μl nuclease-free water, 1 μl dna template, and 0.8 μl each of forward (l15f, 5’ gctcaggaygaacgcygg 3’) and reverse (l687r, 5’ caccgctacacatgradttc 3’) primers (hou et al. 2018). pcr was performed in a mycyclertm thermal cycler system (bio-rad, california, usa), with the following cycling conditions: initial denaturation at 95°c for 2 min, 35 cycles of denaturation at 95°c for 1 min, annealing at 60°c for 1 min, extension at 72°c for 1 min, and final extension at 72°c for 5 min. the pcr products were electrophoresed in a 1% agarose gel with 0.5 μg/ml ethidium bromide at 100 v for 24 min. the amplicons were visualized using a uv transilluminator. fructose osmotolerance test the method used to determine the fructose tolerance of the isolates was adapted and modified from the work of endo (endo et al. 2009). approximately 1.5 x 108 cfu/ml of 24-h old cultures were prepared in 0.9% saline, and 500 μl of each suspension was inoculated into scintillation vials containing 5 ml of fypb with 30% (w/v) fructose. the vials were incubated at 37°c for 24 h under aerobic conditions on an orbital shaker (120 rpm) and observed for growth. isolation and antimicrobial activity of fructophilic lactic acid bacteria 34 growth comparison in fyp broth and gyp broth growth of the isolates was compared using fypb and glucose, yeast and peptone broth (gypb) under microaerophilic conditions. gypb has the same components as fypb except fructose is replaced with glucose. approximately 1.5 x 108 cfu/ml of 24-h old cultures were prepared in 0.9% saline and 500 μl of each suspension was inoculated into separate scintillation vials containing 5 ml of fypb and gypb. the vials were incubated at 37°c for 24 h under microaerophilic conditions. an aliquot of 200 μl of each culture was transferred into a 96-well plate, and the absorbance of the cultures was measured by spectrophotometry (bmg labtech, offenburg, germany) using 600 nm wavelength in triplicate and using sterile media as blank. isolates with higher absorbance readings in fypb compared to gypb were considered flab. agar plug diffusion assay the agar plug diffusion assay was used as primary screening method for antimicrobial activity against the following test bacteria: acinetobacter baumannii mml esab1701, campylobacter jejuni atcc 33560, enterococcus faecalis biotech 10348, e. coli biotech 1634, klebsiella pneumoniae biotech 1754, proteus mirabilis mml espm1701, pseudomonas aeruginosa biotech 1335, and salmonella typhimurium biotech 1756. approximately 1.5 x 108 cfu/ml of 24-h old cultures of the isolates were prepared in 0.9% saline and were lawned on their respective agar medium. the plates were incubated at 37 °c for 24–48 h until the lawns were confluent. similarly, about 1.5 x 108 cfu/ml of 24-h old cultures of the test bacteria were prepared in 0.9% saline and were lawned on mueller-hinton agar (mha) plates, except for c. jejuni, which was prepared using mha with 5% lysed horse blood. agar plugs 4.5 mm in diameter were made from the flab lawns and were aseptically transferred onto lawns of the test bacteria. the plates were incubated at 37°c, and 42°c for c. jejuni, for 24 h. the assay was performed in duplicates. the diameters of zone of inhibition (zoi) were measured using a caliper and were classified as weak (diameter ≤ 3 mm), moderate (diameter ≤ 6 mm) or strong inhibition (diameter > 6 mm) (pan et al. 2009). isolates that exhibited moderate to strong inhibition were selected for secondary screening. agar well diffusion assay the agar well diffusion assay was used to screen the cell free supernatant (cfs) of the isolates for antimicrobial activity. approximately 1.5 x 108 cfu/ml of 24-h old cultures were prepared in 0.9% saline and 1 ml of each suspension was inoculated g. m. penuliar and r.j. r. artezuela 35 in a test tube containing 10 ml of their respective broth medium and incubated at 37°c for 24 h. an aliquot of 1 ml from each culture was transferred into separate 1.5 ml microcentrifuge tubes and centrifuged at 12,000 rpm for 1 min. the supernatants were collected and transferred into new 1.5 ml microcentrifuge tubes. about 1.5 x 108 cfu/ml of 24-h old test bacteria cultures were prepared in 0.9% saline and lawned on mha plates using cotton applicators. agar wells with a diameter of 6 mm were made on the lawns of test bacteria, and 50 μl of cfs was added into the wells. the cfs was allowed to dry and the plates were incubated at 37°c, and 42°c for c. jejuni, for 24 h. the assay was performed in duplicates. the zoi of the cfs were measured using a caliper and were classified as previously mentioned. identification of isolates by dna sequence analysis the dna of isolates were extracted as previously described, and uniplex pcr amplification of the 16s rrna gene was performed for dna sequencing. each 30 μl reaction mixture contained 15 μl gotaq® master mix (promega, wisconsin, usa) (with 50 units/ml of taq dna polymerase in a proprietary reaction buffer [ph 8.5], 400 μm each of datp, dgtp, dctp and dttp, and 3 mm of mgcl 2 ), 7.2 μl nuclease-free water, 3 μl dna template, and 2.4 μl each of forward (27f, 5’ agagtttgatcctggctcag 3’) and reverse (1492r, 5’ tacggytaccttgttacgactt 3’) primers (lane 1991). pcr was performed in a mycyclertm thermal cycler system (bio-rad, california, usa) with the following cycling conditions: initial denaturation at 95°c for 2 min, 35 cycles of denaturation at 94°c for 1 min, annealing at 56°c for 1 min, extension at 74°c for 1 min, and final extension at 74°c for 5 min. agarose gel electrophoresis was performed as previously described. purified pcr products were sent to macrogen (10f world meridian center, 60-24 gasan-dong, geumcheon-gu, seoul 153-781, south korea) for dna sequencing. the consensus sequences were determined from the forward and reverse sequences using the bioedit sequence alignment editor 7.2.6.1 (hall 1999), and were used to identify the isolates using the basic local alignment search tool (blast) in the national center for biotechnology information (ncbi) nucleotide database. the sequences were then deposited in genbank. phylogenetic analysis a phylogenetic tree was constructed to determine the relationship of the isolates to type strains of flab and lab in genbank. full formats of 16s rrna gene sequences of 43 type strains were downloaded from the nucleotide database of ncbi, including isolation and antimicrobial activity of fructophilic lactic acid bacteria 36 the sequence of lactococcus lactis, which was used as outgroup. multiple alignments of the sequences were performed using mega x and clustal_w algorithm (kumar et al. 2018). the kimura two-parameter model with gamma distribution was used in calculating the distance matrices for the aligned sequences (kimura 1980). the neighbor-joining (nj) method (saitou and nei 1987) was used to construct the phylogenetic tree, and a bootstrap of 1000 replicates was used to estimate the robustness of each branch (felsenstein 1985). statistical analysis significant differences in the growth of the isolates in fypb and gypb were determined using student’s t-test (α=0.05) in microsoft® excel® for microsoft 365 mso. results isolation of presumptive flab flowers from 14 plant species were collected in the institute of biology, university of the philippines diliman. the plants were identified up to the genus and species level with the assistance of the staff from the jose vera santos memorial herbarium at the institute of biology. the plant species were as follows: acalypha indica, begonia sp., calotropis gigantea, clerodendrum intermedium, clerodendrum quadriloculare, cosmos sulphureus, hibiscus rosa-sinensis, impatiens sp., plumbago sp., pseuderanthemum sp., rosa sp., ruellia sp., tabernaemontana pandacaqui, and thunbergia grandiflora. enrichment and plating of the samples in four isolation media produced colonies of different morphologies ranging from raised and convex elevations, entire and undulate margins, and smooth and glistening appearance. presumptive flab isolates were isolated in all samples except in t. grandiflora. twenty-eight colonies with distinct morphologies and clearing zones were selected for purification and characterization (table 1). g. m. penuliar and r.j. r. artezuela 37 table 1: number of distinct colonies observed in each flower species specimen media fypa mrsa mrsa + 1% fructose sapa acalypha indica 0 1 1 0 begonia sp. 0 1 1 1 calotropis gigantea 0 0 0 1 clerodendrum intermedium 0 0 1 1 clerodendrum quadriloculare 1 1 1 0 cosmos sulphureus 1 1 1 0 hibiscus rosa-sinensis 0 1 1 1 impatiens sp. 1 0 0 0 plumbago sp. 1 0 0 1 pseuderanthemum sp. 1 1 2 0 rosa sp. 0 0 0 0 ruellia sp. 1 1 1 0 tabernaemontana pandacaqui 0 1 0 0 thunbergia grandiflora 0 0 0 0 subtotal 6 8 9 5 total 28 all 28 isolates were gram-positive, but only 20 isolates were screened for catalase activity due to loss of viability in eight isolates. eighteen isolates were catalasenegative, while two isolates exhibited catalase activity. all 20 isolates were confirmed as lab using lab-specific primers (table 2, figure 1). seventeen isolates were fructose osmotolerant, while eight isolates (facq 9, faim 21, maps 4, mabe 25, mafps 4.1, mfru 7.2, mafai 8, and mfhr 16) exhibited higher absorbance in fypb compared to gypb and were selected for in vitro antimicrobial activity screening. isolation and antimicrobial activity of fructophilic lactic acid bacteria 38 table 2: characterization of the isolates based on flower origin, catalase test, amplification of lab-specific primers, fructose osmotolerance and growth in fypb and gypb isolate flower source catalase test pcr with lab-specific primers fructose osmotolerance absorbance (600 nm) fypb gypb p-value facs 2 c. sulphureus negative positive negative facq 9 c. quadriloculare negative positive positive 0.649 0.505 0.074 faim 21 impatiens sp. negative positive positive 0.310 0.237 0.007 fapl 23 plumbago sp. negative positive macs 1 c. sulphureus negative positive positive 0.530 0.767 0.303 maps 4 pseuderanthenum sp. negative positive positive 0.553 0.446 0.059 maru 7.1 ruellia sp. negative positive positive maai 8 a. indica negative positive positive macq 10 c. quadriloculare negative positive positive 0.548 0.856 0.166 matp 17 t. pandacaqui negative positive positive 0.138 0.169 0.048 mabe 25 begonia sp. negative positive positive 0.354 0.286 0.355 mfcs 1 c. sulphureus negative positive positive 0.693 1.123 0.163 mfps 4.1 pseuderanthenum sp. negative positive positive 0.734 0.517 0.043 mfps 4.2 pseuderanthenum sp. negative positive positive 0.830 0.857 0.471 mfru 7.2 ruellia sp. negative positive positive 0.838 0.483 0.001 mfai 8 a. indica negative positive positive 0.494 0.356 0.008 mfhr 16 h. rosa-sinensis negative positive positive 0.523 0.404 0.011 mfbe 25 begonia sp. positive positive positive 0.034 0.113 0.051 sabe 30 begonia sp. negative positive positive 0.018 0.020 0.378 sahr 35 h. rosa-sinensis positive positive figure 1: representative polymerase chain reaction (pcr) results using lab-specific primers through agarose gel electrophoresis (age). expected amplicon size was around 750 base pairs. g. m. penuliar and r.j. r. artezuela 39 in vitro antimicrobial activity of the isolates seven isolates exhibited medium to strong in vitro antimicrobial activity in at least three test bacteria in the primary screening (figure 2 and figure 3). the largest zoi was observed in isolate mfps 4.1 against e. coli (9.7 mm) and salmonella (8.925 mm), although a few colonies were seen in the inhibition zone. the largest complete zoi was observed in isolate maps 4 against c. jejuni (8.3 mm), and in isolate mfru 7.2 against e. faecalis (7.4 mm). inhibition of all test bacteria was observed in isolates mfps 4.1, mfru 7.2, mfai 8, and mfhr 16. figure 2: representative result of growth inhibition of isolate mfps 4.1 against test bacterium e. faecalis. measurements were done using a digital caliper and expressed in millimeter. agar plug size was measured at 4.5 mm. figure 3: results of the primary screening of the isolates against four test bacteria. the grey dotted line represents the 3 mm diameter zone of inhibition (zoi) threshold for isolates with moderate inhibition and the black dotted line represents the 6 mm diameter threshold for isolates with strong inhibition. isolates that exhibited partial inhibition are marked with an asterisk (*). isolates that did not exhibit inhibition are not included. error bars represent standard errors. isolation and antimicrobial activity of fructophilic lactic acid bacteria 40 only four isolates had inhibitory activity in at least two test bacteria in the secondary screening (figure 4). the largest inhibition was observed against c. jejuni by cfs from isolates mfps 4.1 (6.7 mm) and maps 4 (6.2 mm), although the cfs from isolate mfru 7.2 also gave moderate to strong inhibition against c. jejuni (4.4 mm) and e. faecalis (3.5 mm). figure 4: results of the secondary screening of the isolates against test bacteria. the grey dotted line represents the 3 mm diameter zone of inhibition (zoi) threshold for isolates with moderate inhibition and the black dotted line represents the 6 mm diameter threshold for isolates with strong inhibition. isolates that exhibited partial inhibition are marked with asterisk (*). isolates that did not exhibit inhibition are not included. error bars represent standard errors. identification of the isolates only two isolates were selected for molecular identification based on their in vitro antimicrobial activity. dna sequencing and blast identified isolates mfps 4.1 and mfru 7.2 as weissella cibaria, although the sequence similarity with the top hit was less than 97%, 95.6% for mfps 4.1 and 92.2% for mfru 7.2. the two isolates formed a sub-cluster with w. cibaria and w. confusa with 95% and 100% bootstrap support (figure 5). g. m. penuliar and r.j. r. artezuela 41 fi gu re 5 : p hy lo ge ne ti c po si ti on o f th e is ol at es m fp s 4. 1 an d m fr u 7 .2 . t he n ei gh bo rjo in in g tr ee b as ed o n th e 16 s rr n a ge ne s of t he i so la te s an d ty pe st ra in s of f la b a nd l a b c on fi rm s th e id en ti fi ca ti on o f th e is ol at es a s m em be rs o f th e ge nu s w ei ss el la . isolation and antimicrobial activity of fructophilic lactic acid bacteria 42 discussion the institute of biology, university of the philippines diliman was chosen as the sampling site for the flower collection as it is deemed a suitable starting point because it is one of the few remaining green spaces in metro manila and also because it houses several native and endemic flowering plant species (cabel 2019; rodriguez 2019). flowers possess nectaries that can produce nectars that are composed of varying concentration of sugars, most commonly a combination of sucrose, glucose and fructose, making flowers a fructose-rich niche (chalcoff et al. 2006). fructose-rich niches are probable sources of flab since fructose is the preferred carbohydrate source of flab over glucose in the absence of external electron acceptors (endo and okada 2008). among the four different culture media used, both mrsa and mrsa with 1% fructose medium had more isolates observed than both fypa and sapa medium (table 1). a possible explanation might be that formulated media like mrsa would be a better growth media than media created by mixing separate components like fypa and sapa. a similar study yielded the same results wherein mrsa with fructose medium was able to isolate both lab and flab (ruiz rodriguez et al. 2019) while fypa was able to isolate strictly flab species (endo et al. 2009). isolation of flab from fructose-rich niches requires enrichment with a medium that contains 1% 2% (w/v) fructose or, alternatively, conventional medium for lab isolation, such as mrsa, with fructose supplementation (endo and salminen 2013; olofsson et al. 2014; endo 2019). optimum temperatures for isolation range from 30–35°c, depending on the sample used, to simulate the environment where the sample was taken, while the optimum ph for growth is ph 6.5, which was achieved by placing the isolation setup at room temperature (30°c) during the enrichment step (endo and okada 2008; endo et al. 2011; syed yaacob et al. 2018; olofsson et al. 2014). the addition of sodium azide to the enrichment medium inhibits the growth of gram-negative bacteria, while the addition of nystatin inhibits the growth of yeasts (snyder and herman 1940; barney et al. 2003). two out of 14 flower species have the greatest number of distinct isolates. these are c. quadriloculare and pseuderanthemun sp., which have four distinct isolates each. both flower species have deep corolla tubes. a study conducted in temperate forests in south america stated that flowers with deep corolla tubes have low viscosity nectars due to low evaporation rates compared to more open flowers (chalcoff et al. 2006). this might explain the number of distinct isolates obtained from the two flower species and agrees with the absence of isolates from the g. m. penuliar and r.j. r. artezuela 43 t. grandiflora, which are relatively open flowers with short corolla tubes, although further studies are needed to establish the correlation. all 28 isolates were gram-positive and 18 out of 20 isolates tested were catalasenegative, which are typical characteristics of flab (endo and okada 2008; endo et al. 2012). similar results can be found in a study conducted by endo et al. (2009) wherein isolates identified as fructobacillus sp. were catalase-negative while other lab isolated exhibited catalase-like activity. members of flab are expected to have higher absorbance values when cultured in fypb compared to gypb under microaerophilic conditions, as observed in this study and particularly seen in eight isolates (facq 9, faim 21, maps 4, mabe 25, mafps 4.1, mfru 7.2, mafai 8, and mfhr 16). however, two of the isolates (maps 4 and mabe 25) did not have significant difference when analyzed using t-test (p>0.05). another study, however, reported that all their presumptive flab isolates were isolated using fypb and fypa, and had better growth in fypb than gypb (endo et al. 2010). in contrast, the isolates in the current study were isolated using mrsa, mrsa with fructose, and fypa. members of flab are xerotolerant and can survive in an environment with low water activity (endo and okada 2008). increasing the fructose content to 30% (w/v) in fypb results in an environment with high osmotic pressure and low water activity, which is a condition conducive for flab selection (endo 2012; endo 2019). all eight isolates still proceeded to the next step based on the higher absorbance observed in fypb compared to gypb and to test more isolates for antimicrobial activity. although not currently recognized as flab, our isolates that were identified as weissella spp. were obtained using standard methods for isolating flab. weissella is a genus of gram-positive, catalase-negative, and non-spore forming lab, with some species reported as osmotolerant. weissella spp. have been isolated from fermented food, vegetation, and silage. many of its species are heterofermentative lab that utilize a wide variety of carbohydrate sources, including glucose and fructose, due to their numerous phosphotransferase systems (fusco et al. 2015; assamoi et al. 2016; säde and björkroth 2019). antimicrobial activity of flab isolates seven test bacteria that cause several gut related diseases, such as a variety of infections, diarrhea, campylobacteriosis, and salmonellosis, were chosen for the study. several studies also included some of the test bacteria in determining the antimicrobial activity of lab isolated from various sources particularly against e. coli and salmonella sp. (gao et al. 2019; sakandar et al. 2019; lakra et al. 2020). isolation and antimicrobial activity of fructophilic lactic acid bacteria 44 numerous members of lab have been used as probiotics in recent years, particularly w. cibaria, which had been studied for its possible growth inhibitory activity against different pathogens. both isolates mfps 4.1 and mfru 7.2, identified as w. cibaria, exhibited strong antagonistic activity against c. jejuni, which causes campylobacteriosis. w. cibaria jw15 was shown to have inhibitory effects on the growth of c. jejuni in the study conducted by yu et al. (2019). the same study also showed that w. cibaria jw15 inhibited the growth of e. coli and salmonella typhimurium, which was also observed on isolates mfps 4.1 and mfru 7.2, respectively. based on the study, w. cibaria jw15 was shown to have an increased acetic acid production compared to other lab, which explains its ability to inhibit the growth of several pathogens. another study also observed that w. cibaria was able to inhibit the growth of e. coli (lakra et al. 2020). in the study, it was concluded that acid production in the supernatant of w. cibaria inhibited the growth of e. coli, and loss of inhibitory activity was observed when the ph was neutralized. to date, studies on specific inhibition of w. cibaria against e. faecalis are scarce but other members of lab, such as lactococcus sp., were able to inhibit the growth of said test pathogen (gonzález et al. 2007). similar published studies on the antimicrobial activity of flab from the philippines are also scarce, indicating that the current study might be the first one. several studies also reported the production of other antimicrobial metabolites by weissella sp. the bacteriocin weissellicin 110 from w. cibaria inhibits growth of other gram-positive bacteria, while weissellin a from weissella paramesenteroides has been reported to inhibit food spoilage bacteria and listeria (srionnual et al. 2007; papagianni and papamichael 2011). production of hydrogen peroxide by w. cibaria was found to inhibit the growth of fusobacterium nucleatum (kang et al. 2006). this was supported by another study that reported that w. cibaria cmu, an oral care probiotic, inhibits the growth of common cariogenic bacteria such as f. nucleatum and porphyromonas gingivalis. the same strain had also effectively inhibited the biofilm formation of streptococcus mutans (jang et al. 2016). as mentioned in the previous studies, isolates mfps 4.1 and mfru 7.2, which were identified as w. cibaria, can have potential use in the food industry as food additive to prevent food spoilage and in the medical field as a probiotic. the growth inhibition exhibited by the isolates against the test bacteria in the primary screening were either reduced or absent in the secondary screening. several studies have shown that the presence of stress in a microbial environment plays an important role in the production of antimicrobial substances. cell-tocell contact of the isolates with the test bacteria in the primary screening may g. m. penuliar and r.j. r. artezuela 45 be interpreted as stress in the form of competitive exclusion, thus the production of antimicrobial substance. however, the same stress was not present in the preparation of cfs since the isolates were grown in optimized culturing condition in broth media, therefore production of antimicrobial substance may have been reduced or limited. assuming that the inhibitory substances produced by the isolates were bacteriocin or bacteriolysins, a possible explanation might be that the inhibitory substances produced were stress-induced. it has been reported that l. kunkeei produces bacteriolysin in the presence of microbial stressors, such as peptidoglycan, lipopolysaccharides, and lipoteichoic acid (butler et al. 2013). this is in accordance with another study where the gene promoter for the production of bacteriocin, bactofencin a from l. salivarius, observed an increase in activity when cells were subjected to microbial stress such as exposure to bile salts, gastric fluids, and target microbiota (guinane et al. 2015). addition of sodium acetate in weissella cibaria culture has been reported to produce an increased amount of h 2 o 2, which have inhibitory activity against f. nucleatum (kang et al. 2006; bendo et al. 2009). another possible explanation for the lack of antimicrobial activity of the cfs is that the conditions, like temperature, ph, amount of carbohydrate source, and oxygen requirements, for the optimized production of either bacteriocin, bacteriolysin or organic acids were not met when the isolates were grown in broth cultures. cfs used in the assay were collected from cultures that were grown in optimized conditions for growth rather than for optimized bacteriocin, bacteriolysin or organic acid production. several studies, however, used similar culture conditions for optimized bacterial growth and cfs preparation for antimicrobial assays (pan et al. 2009; endo and salminen 2013; syed yaacob et al. 2018). a study on the production of bacteriocin-like inhibitory substance (blis) produced by pediococcus acidilactici, for example, had different growth conditions when grown for the optimized blis production compared to conditions optimized for cell growth. in the study, 37°c was used as the optimal temperature and the culture was grown for 24 h for the growth of p. acidilactici, while 28.5°c was used as optimal temperature and the culture was grown for 28 h for optimized blis production (md sidek et al. 2018). another study on carnobacterium divergens reported different culturing conditions for optimized growth and for optimized bacteriocin production. in the study, optimized condition for growth of c. divergens were 15 °c, ph 6.5–8, and the amount of carbohydrate source was 2% (w/v) while the optimized condition for its bacteriocin activity were 15°c, ph 6.5, and the amount of carbohydrate source ranged from 0.2% 2% (brilletviel et al. 2016). isolation and antimicrobial activity of fructophilic lactic acid bacteria 46 phylogenetic tree analysis the phylogenetic tree has 93% bootstrap support for the isolates clustering with the genus weissella, and 95% and 100% bootstrap support for the isolates forming a sub-cluster with w. cibaria and w. confusa, respectively, which is consistent with the blast identification of the isolates based on their 16s rrna gene sequences. the high support for the phylogenetic positions of the isolates confirms the identity of the isolates as belonging to genus weissella. with regard to the species identification of the isolates, it has been reported that the recommended range of 16s rrna gene sequence similarity for species differentiation is 98.9% 99.0% (stackerbrandt and ebers 2006). it is therefore possible that isolates mfps 4.1 and mfru 7.2 are new species under the genus weissella, or new species that are closely related to w. cibaria. however, this needs to be confirmed by further studies on the molecular and physiological level, which should include an extensive study of their cell morphology using scanning electron microscope (sem), assessment of their carbohydrate fermentation capabilities and antimicrobial susceptibility, and additional phylogenetic analyses using multiple housekeeping genes to definitively determine the taxonomic positions of the isolates. conclusions this work describes the first attempt to isolate flab from flowers in the philippines. the results show that eight presumptive flab isolates were isolated and two weissella spp. were identified from flowers from the university of the philippines diliman based on the antimicrobial activity. isolates mfps 4.1 and mfru 7.2 displayed flab features such as fructose osmotolerance and preferential growth in fypb, and exhibited in vitro antimicrobial activity against several enteric pathogens, particularly c. jejuni and e. faecalis, which can be studied further for possible applications in both the food industry and the medical field, such as as probiotics. data availability the sequences of the isolates described in the study have been deposited in genbank with the following accession numbers: mw836947.1, mw836946.1, mw836945.1, mw836944.1, and ol629482. g. m. penuliar and r.j. r. artezuela 47 conflicts of interest the authors declare that there is no conflict of interest regarding the publication of this paper. acknowledgements the authors would like to acknowledge gilbert s. sobremisana, ma. socorro edden p. subejano and jann eldy l. daquioag. this work was supported by the up diliman research load credit/creative work load credit (rlc/cwlc) and the up diliman office of the vice-chancellor for research and development, with project code ib-pfgmp-2020-01. isolation and antimicrobial activity of fructophilic lactic acid bacteria 48 references assamoi aa, krabi er, ehon af, n’guessan ga, niamké ls, thonart p. 2016. isolation and screening of weissella strains for their potential use as starter during attiéké production. biotechnol agron soc environ [internet]. 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[cited 2021 nov 18]; 11. doi:10.3389/fmicb.2020.571903. ______ renz joseph r. artezuela is a graduate of the b.s. in biology program of the institute of biology. he was the first undergraduate student of the medical microbiology laboratory to work on fructophilic lactic acid bacteria.. gil m. penuliar <gmpenuliar@up.edu.ph> is an associate professor of the institute of biology. he has a ph.d. in medical science and is the current principal investigator of the medical microbiology laboratory. ion chromatographic method with post-column fuchsin reaction for measurement of bromate in chlorinated water *homer c. genuino, maria pythias b. espino institute of chemistry , university of the philippines, diliman, quezon city 1101 *corresponding author: homer.genuino@uconn.edu received: 15 july 2009; revised: 10 february 2010; accepted: 10 february 2010 abstract an ion chromatographic method that employs a post-column reaction with fuchsin and spectrophotometric detection was optimized for measuring bromate (bro3-) in water. bro3is converted to br2 by sodium metabisulfite and then reacted with acidic fuchsin to form a red-colored product that strongly absorbs at 530 nm. the reaction of bro3and fuchsin reagent is optimum at ph 3.5 and 65 oc. the method has a limit of quantitation of 4.5 µg l-1 and is linear up to 150 µg l-1 bro3-. recoveries from spiked samples were high ranging from 95 to 102 % using external standard calibration and 87 to 103 % using standard addition method. intra-batch and inter-batch reproducibility studies of the method resulted to rsd values ranging from 0.62 to 2.01 % and percent relative error of 0.12 to 2.94 % for bro3concentrations of 10 µg l-1 and 50 µg l-1. this method is free of interferences from common inorganic anions at levels typically found in chlorinated tap drinking water without preconcentration. the optimized method can be applied to trace analysis of bromate in chlorinated tap drinking water samples. keywords: bromate, fuchsin, chlorinated water, post-column reaction, ion chromatography introduction bromate (bro3-) may be present in various water types, including those intended for human consumption, either as a major disinfection byproduct of the ozonation of water containing naturally occurring bromide ions or as a contaminant of hypochlorite disinfection (fawell & walker, 2006; haag & hoigné, 1983; legube, et. al., 2004; von gunten & hoigné, 1994; weinberg, et. al., 2003). once generated and found in water, bro3does not easily degrade. toxicological studies of bro3in rats have provided evidence of its possible carcinogenicity (fuji, et. al., 1984; kurokawa, et. al., 1990). acute exposure of rodents to bro3has been shown to cause neuropathological disorders and induce tumors of the kidney, peritoneum and thyroid (de borba, et. al., 2005; kurokawa, et. al., 1990). the lifetime cancer risk determined for bro3in drinking water for humans was 2×10−5 per µg l-1 assuming a 2-l daily water consumption (de borba, et. al., 2005; fawell & walker, 2006). lifetime risks of 10-4, 10-5 and 10-6 were theoretically associated with exposures to bro3concentrations of 5, 0.5 and 0.05 µg l-1, respectively. the availability of analytical methods to monitor and determine bro3in drinking water at sub-µg l-1 levels is thus important. a maximum admissible concentration (mac) of 10 µg l-1 in drinking water is recommended by the us environmental protection agency (us epa), the european commission (ec) and the world health organization (who) (de borba, et. al., 2005; fawell & walker, 2006; guinamant & ingrant, science diliman 21(1):29-36 29 mailto:homer.genuino@uconn.edu genuino, espino 2000; uraisin, et. al., 2006). this guideline was defined primarily on the basis of the detection capabilities of existing ion chromatographic methodologies. the philippine national standards for drinking water of 2007 has a maximum guideline level of 10 µg l-1 bro3in drinking water based on the recent risk assessment of the who. the proposed detection limit of less than 2.5 µg l-1 bro3by the ec has called for the development of more sensitive analytical methods and alternative techniques (ingrant & guinamant, 2002). a number of methods have been developed and adapted to meet the objectives of setting quality standards for bro3in water. official methods for bro3 determination include ion chromatography with conductivity detection. recent studies have shown that the sensitivity of these analytical methods may be improved by coupling the separation using ion chromatography with a specific post-column reaction (delcomyn, et. al., 2001; uraisin, et. al., 2006). three different post-column reaction techniques in ion chromatography have been compared. post-column reactions with ki(nh4)6mo7o24, nabr-nano2 and o-dianisidine showed low detection limits ranging from 0.17 to 0.24 µg l-1 for bro3in water (hautman, et. al., 2001; salhi & von gunten, 1999). performance evaluation of the us epa method 317.0, which employs both suppressed conductivity and spectrophotometric detection after post-column reaction with o-dianiside, demonstrated specificity and sensitivity for bro3with a detection limit of 0.042 µg l-1 (wagner, et. al., 2001). two similar studies based on the hyphenation of an ion chromatographic system and post-column fuchsin reaction with visible absorbance detection showed the ph and temperature dependence of the bro3--fuchsin reaction (archilli, et. al., 1999; valsecchi, et. al., 1999). using a standard carbonate-bicarbonate mobile phase, a linear range of 0.1 to 100 µg l-1 and a detection limit of 0.1 µg l-1 bro3 were reported by archilli, et. al. (1999). valsecchi, et. al. (1999), on the other hand, used a tetraborate mobile phase and obtained a linear range of 0.5 to 10 µg l-1 and detection limit of 0.4 µg l-1 bro3-. their methods were successfully used in quantifying trace levels of bro3in actual drinking water samples. this study aimed to optimize an ion chromatographic method involving post-column fuchsin reaction followed by spectrophotometric detection for trace bro3in chlorinated water as well as provide an alternative analytical technique useful in the strict compliance of water quality standards in the philippines. materials and methods reagents and standard solutions all chemicals used were analytical reagent grade. the standards and blank solutions were prepared using ultrapure water at resistance of 18.0 mω (nanopure® barnstead, usa). bromate standard solutions were made by dilution of a 1000 µg l-1 stock solution of potassium bromate (merck, germany). the fuchsin stock solution (fss) was prepared by dissolving 100 mg basic fuchsin (c19h18n3cl, beijing chemical works, china) in 100 ml of ultrapure water. stored at 4 oc in a glass amber bottle, this solution is stable for several months. the color developing solution which acts as post-column reagent was prepared by acidifying 10 ml of fss with 0.5 ml of 12 m hcl (merck, germany) followed by the addition of 350 mg of sodium metabisulfite (mallinckrodt, usa). the solution was made up to 100 ml with ultrapure water in a glass flask and was left to stand overnight for complete decoloration. the mobile phase solutions were prepared from 2.5 mm phthalic acid (merck, germany) and 2.4 mm tris(hydroxymethyl)aminoethane (merck, germany) solutions buffered at ph 3.5. these solutions were filtered through 47 mm×0.2 μm cellulose nitrate membrane filters (whatman, england) using a filtering apparatus attached to a vacuum source and degassed for several minutes by sonication (branson model 8510 ultrasonic cleaner, usa) prior to use. for the interference studies, inorganic anions (hco3-, cl-, so42-, br-, f-, no2-, no3-, po43and clo3-) of different concentrations (1, 5, 50 and 100 mg l-1) were prepared by dissolving appropriate amounts of the anions in their potassium and sodium salts. ion chromatographic analysis a perkin elmer lambda 40 uv-vis spectrophotometer was used for visible spectrum scanning. the shimadzu hic-6a ion 30 science diliman ion chromatographic method with post-column fuchsin reaction chromatograph system used in this study is composed of the following modules: lp-6a delivery pump unit (lc-6a liquid pump with a high-sensitivity noise filter), spd-6av uv-vis spectrophotometric detector, cto-6as column oven, scl-6b system controller, lc-9a liquid pump used for post-column reagent, sil-6a auto sample injector, and c-r4a integrator and printer/plotter for data processing. three stainless steel columns (shimadzu, japan) were used in the chromatographic system: shim-pack ic-pc1 precolumn, shim-pack ic-ga1 guard column and shim-pack ic-a1 analytical column. the injected samples passed through the preheater before entering the guard column and analytical column inside an oven set at 65 oc. the 4.6 mm (i.d.) × 10 cm (length) × 12.5 μm (particle size) analytical column was packed with an anion exchange resin on a polymethacrylate support incorporating a quaternary ammonium base which is a strong anionexchange functional group. the ions that elute from the column mix with the fuchsin post-column reagent in the mixing tee. bro3and fuchsin react completely in the 204-cm reaction coil to form the red-colored product that is directed to the uv-vis detector. measurement of bro3concentration was carried out using external standard calibration and standard addition methods. results and discussion bromate, upon reaction with fuchsin reagent, is detected in the visible region (espino & cimatu, 2003; romele & achilli, 1998). in this study, a maximum absorbance was obtained at 530 nm when a 10 µg l-1 bro3and acidified fuchsin solution was scanned in the visible range of 400 to 635 nm. the 530 nm wavelength also gave a maximum peak response for the same bro3--fuchsin solution when determined by ion chromatography using different wavelength settings from 520 to 535 nm. the 530 nm wavelength was then used in the subsequent ion chromatographic analyses. previous studies revealed that the reaction of bro3 with fuchsin reagent occurs in the acidic range (achilli & romele, 1999; romele & achilli, 1998; valsecchi, et. al., 1999). in this study, the optimum ph for the bro3--fuchsin reaction was investigated by varying the ph of the mobile phase from 2.0 to 7.0. figure 1 shows that a maximum peak response is obtained when the ph of the mobile phase was maintained at 3.5. similar to ph, temperature also affects the bro3-fuchsin reaction. previous ion chromatographic studies found that the bro3peak response increases as the temperature is raised from ambient to an optimum reaction temperature, e.g., 65 or 80 oc (achilli & romele, 1999; valsecchi, et. al., 1999). in the present study, the optimum temperature is 65 oc as shown in figure 2. a blank measurement experiment was performed to validate the use of ultrapure water as solvent in preparing calibration or sample solutions. no peak response was observed in the chromatographic measurements of seven replicate blank solutions consisting only of ultrapure water and fuchsin reagent. this confirms that ultrapure water does not contain bro3-. in addition, separate chromatographic measurements were performed on two sets of fortified solutions. the first set of seven replicate solutions were prepared using ultrapure science diliman 31 figure 2. average peak areas (n=3) and retention times (n=3) for 10 µg l-1 bro3at varying oven temperatures in oc. figure 1. average peak areas (n=3) for 10 µg l-1 bro3-at varying ph of the mobile phase. genuino, espino water spiked with 10 µg l-1 bro3-; the second set of seven solutions consisted of bromate-free drinking water spiked with 10 µg l-1 bro3-. the average peak areas and % rsd of the two sets of data are in good agreement as shown in table 1. this further validates the use of ultrapure water in procedural blanks and sample analysis using ion chromatography with post-column fuchsin reaction. table 1. comparison of the average peak areas and percent relative standard deviations of spiked blanks using ultrapure water and bromate-free drinking water as solvents. spiked blanks average peak areaa (n=7) sd rsd,% blank using ultrapure water +10 μg l-1 bro338212 708 1.85 blank using bromate-free bottled waterb +10 μg l-1 bro338010 729 1.92 aseven replicates with three measurements per replicate. bbottled distilled drinking water (brand:absolute). inorganic anion interferences in the ion chromatographic determination of bro3were also investigated. figure 3 shows the effect of adding common inorganic anions (hco3-, cl-, no3-, so42-, br-, f-, no2-, po43-, and clo3-) in four different concentrations to 10 µg l-1 bro3solutions prepared in ultrapure water. a paired t-test was performed to determine whether or not the inorganic anions were interfering at different concentrations. the mean peak area differences of solutions of 10 µg l-1 bro3 in ultrapure water were not significantly greater than zero before and after the same solutions were spiked with the inorganic anions at four different concentrations. student’s t-test gave test statistic t values for inorganic anions (hco3= -0.0392, cl= -3.07, no3= 0.556, so42= 1.09, br= 1.55, f= -3.93, no2= -2.32, po43= 1.00, and clo3= 1.14) which were lower than the critical t value of 3.18 at 95 % confidence interval from the t-distribution table. these statistical tests proved that the inorganic anions do not interfere in the determination of bro3-. further, low rsd values ranging from 0.24 to 2.5 % were obtained for the 10 µg l-1 bro3solutions spiked with different concentrations of these anions. it was also observed that in the absence of bro3-, the red-colored product that absorbs at 530 nm was not formed in solutions spiked with the anions. hence, these anions do not react with the fuchsin reagent which appeared to be bro3--specific. using the optimum conditions, eleven bro3 standard solutions ranging from 2 to 1500 µg l-1 were prepared to get the range of concentration from the estimated lowest detectable concentration to a concentration where a departure from linearity will be observed. a linear response was obtained and the limits of the linear dynamic range are presented in figure 4. based on the results of the analysis in wide concentration range (2 to 1500 µg l-1), low-bromate (2 to 150 µg l-1) and high-bromate (150 to 2000 µg l-1) calibration solutions were then measured and compared. table 2 summarizes the regression and residual analyses for low and high-bromate concentration calibration curves. linear (first-order polynomial) and quadratic (second-order polynomial) least square (ls) regression models 32 science diliman figure 3. comparison of inorganic anions [(a): hco3-, cl-, no3-; (b): so42-, br-, f-; (c): no2-, po43-, clo3-] at different concentrations spiked in ultrapure water containing 10 µg l-1 bro3and ultrapure water containing 10 µg l-1 bro3only. ion chromatographic method with post-column fuchsin reaction were used to assess the degree of goodness of fit of the experimental calibration data. the correlation coefficients and residual values were also derived using these models. the linear and quadratic least squares are polynomials described by a set of coefficients in the following equations: y=a oa1 x (linear, rdf=2) (1) y=a oa1 xa 2 x 2 (quadratic, rdf=3) (2) where y is the peak area, a0, a1 and a2 are the coefficients of the polynomial, x is the concentration in µg l-1, and rdf is the required degrees of freedom. the results of the regression and residual analysis showed good agreement between the two ls regression models. this means that the values of x from the linear equation 1 and quadratic equation 2 will almost be the same. in this study, the simpler linear ls regression was used to calculate bro3 concentration. table 2. comparison between low-bro3and high-bro3 calibration data in terms of regression and residual analyses. parameters low-bromate concentration range 2 to 150 μg l-1 (n=3) high-bromate concentration range 150 to 2000 μg l-1 (n=3) linear least square quadratic least square linear least square quadratic least square regression equation y=10628 + 2724x y=7198 +2985x – 1.80 x2 y=45267 0+ 257.9x y=396704 + 258x – 0.0855x2 correlation coefficient, r 0.9994 0.9997 0.8402 0.9704 residuals (absorbance units) -7.5152 460.991 -483.49 -6824.1 4575.93 8630.05 -6351.8 2907.06 2629.89 514.680 -7897.1 -549.0 3958.0 -1563.4 -56522 -8339.4 29972 30753 33831 4694.8 -34389 -25343 15432.8 18273.4 1247.76 -2798.91 -14132.2 7319.78 the average % rsd (n=3) of the peak areas in the low-bromate concentration curve was 1.47 %, while in high-bromate concentration it was 0.97 %. as expected, the precision improved in high-bromate concentration due to less deviation in replicate analysis that is almost always associated with measurements in higher concentrations. nevertheless, both values are within the required ± 15 % level. regression analyses given in table 2 were used to assess the linearity and uncertainty in low and high-bromate concentration calibration curves. the low-bromate concentration calibration curve has correlation coefficient nearer to unity and residual values that are more random, making it more linear than the high-bromate concentration calibration curve. figure 5 displays a graphical view of a seven-point regression plot of the low-bromate concentration range showing 95 % confidence interval levels about the regression. this lowbromate concentration range was used in quantifying bro3in this study. science diliman 33 figure 5. a linear calibration graph for 2 to 150 µg l-1 bro3solutions (n=3) using the optimized ion chromatographic method. [solid line represents linear regression and dotted lines show 95 % confidence interval levels about the regression.] figure 4. graphical representation of the peak area response of 2 to 1500 µg l-1 bro3(n=3) in ultrapure water showing linear dynamic range. genuino, espino table 3. intra-batch and inter-batch reproducibility studies for precision and accuracy of the ion chromatographic method. average concentration intra-batch reproducibility inter-batch reproducibility concentrationa measured concentrationb (sd)c rsd,% rel. error, % concentrationa measured concentrationb (sd)d rsd,% rel. error, % 10.00 9.988(0.19) 1.93 0.12 10.00 9.900(0.13) 2.01 1.00 50.00 51.47(0.42) 0.81 2.94 50.00 50.88(1.16) 0.62 1.76 a concentration of prepared bro3solution in µg l-1. b measured by ion chromatography and calculated using external standard calibration method. c mean for seven replicates with three measurements per replicate in one day. d mean for seven replicates with three measurements per replicate in five days. the limit of quantitation of the optimized method was calculated using the following equations: s loq=sblank 10 sblank (3) x loq= s loq−s blank /m (4) where sloq is the detectable signal, sblank is the mean signal of the blank, sblank is the standard deviation in the blank signal, xloq is the limit of quantitation, and m is the slope of the regression line (loconto, 2006). baseline absorbance readings in milliabsorbance (mabs) units of a blank solution flowing through the ion chromatographic system were recorded for 20 min in 30 s intervals. the average values of the absolute difference between two successive absorbance readings were taken. this process was repeated for six more days. an average value of 0.142 mabs (sblank) and a standard deviation of 0.0727 (sblank) were derived from the resulting seven sets of data. these values were used to solve for sloq in equation 3. finally, an xloq of 4.5 µg l-1 bro3was obtained using equation 4 and m equal to 0.162. this limit of quantitation is lower than the detection limit of 50 µg l-1 previously reported by espino & cimatu (2003), making the optimized method more sensitive.intra-batch and inter-batch (long-term precision) reproducibility studies were performed within one day and five consecutive days, respectively, to determine the accuracy and precision of the optimized method. these were done by carrying out seven replicate analyses of 10.00 and 50.00 µg l-1 bro3solutions. table 3 presents the calculated rsd and relative error values. overall, the optimized method showed good repeatability with rsd values within the required ± 15 % for each concentration level. the optimized method is accurate with the relative error of 0.12 to 2.94 %. percent recoveries of bro3spiked in tap drinking water samples in three different concentrations (low, 5.0 µg l-1; medium, 50.0 µg l-1; and high, 100.0 µg l-1) were determined to test the reliability and accuracy of the method using an actual sample matrix. table 4 gives the % recoveries using external standard calibration and standard addition quantitation methods. using the external standard calibration method, % recoveries were generally acceptable based on the recommended 80 to 120 % as reported by lesnik (1992). low % rsd values were found across the three bro3concentrations. notably, better recoveries were obtained for medium and high concentrations when using standard addition method. table 4. percent recovery data for low, medium and high bromate concentrations in tap drinking watera. quantification method %recovery (rsd)b concentration low 50 μg l-1 medium 50.0 μg l-1 high 100.0 μg l-1 (using peak area) external standard calibration 99 (1.84) 101 (0.89) 102 (0.77) standard addition 89 (1.07) 101 (1.37) 99 (1.08) (using peak height) external standard calibration 97(1.54) 95(0.86) 96(1.40) standard addition 87(1.13) 98(1.60) 103(0.77) atap water sample taken from the faculty lounge of the institute of chemistry, university of the philippines. bmean for seven replicates with three measurements per replicate. the ion chromatographic method described in this study is reproducible, accurate, sensitive and suitable for the analysis of bro3in chlorinated tap drinking water at trace levels. the method can be used for monitoring bro3in chlorinated water in the treatment plants as well as in the distribution 34 science diliman ion chromatographic method with post-column fuchsin reaction lines. this is an available method that can be used to ensure strict compliance of the 10 µg l-1 bro3 stipulated in the philippine national standards for drinking water. acknowledgements the authors gratefully acknowledge the research and development division of the environmental management bureau (rdd-emb) for the use of their ion chromatograph and laboratory facilities. the commission on higher education (ched) provided a master’s thesis grant to h. c. genuino to conduct this study. references achilli, m. and romele, l., 1999, ion chromatographic determination of bromated in drinking water by postcolumn reaction with fuchsin, j. chromatogr. a., 847: 271-277. de borba, b. m., rohrer, j. s., pohl, c. a. and saini, c., 2005, a. determination of trace concentrations of bromate in municipal and bottled drinking waters using a hydroxide-selective column with ion chromatography, j. chromatogr. a., 1085 (1): 23-32. delcomyn, c. a., weinberg, h. s. and singer, p. c., 2001, use of ion chromatography with post-column reaction for the measurement of tribromide to evaluate bromate levels in drinking water,. j. chromatogr. a., 920: 213-219. espino, m. p. and cimatu, k., 2003, bromate levels in metro manila drinking water, kimika, 19 (1): 35-39. fawell, j. and walker, m., 2006, approaches to determining regulatory values for carcinogens with particular reference to bromate, toxicology, 221 (2-3): 149-153. fujii, m., oikawa, k., saito, h., fukuhara, c., onosaka, s. and tanaka, t., 1984, metabolism of potassium bromate in rats i. in vivo studies, chemosphere, 13 (11): 1207–1212. guinamant j. and ingrant, v., 2000, laboratory and field methods for determination of bromate in drinking water, eur report, en., 19601: 178. haag, w.r. and hoigné, j., 1983, ozonation of bromidecontaining waters: kinetics of formation of hypobromous acid and bromate, environ. sci. technol. 17 (5): 261-267. hautman, d. p., munch, d. j., frebis, c., wagner, h. p. and pepich, b. v., 2001, review of the methods of the us environmental protection agency for bromate determination and validation of method 317.0 for disinfection by-product anions and low-level bromated, j. chromatogr. a., 920 (1-2): 221-229. ingrant, v. and guinamant, j., 2002, determination of bromate in drinking water: development of lab and field methods, trends anal. chem., 21: 356-365. kurokawa, y., mackawa, a. and takahasi, m., 1990, toxicity and carcinogenicity of potassium bromated—a new renal carcinogen, environ. health perspect., 87, 309-335. legube b., parinet, b., geliner, k., berne, f. and croue, j., 2004, modeling of bromate formation by ozonation of surface waters in drinking water treatment, water res., 38 (8): 2185-2195. lesnik, b., 1992. guidance for methods development and methods validation for the rcra program. office of solid waste, us environmental protection agency, us epa, cincinnatti, oh, usa. 1-32. loconto, p., 2006. trace environmental quantitative analysis, principles, techniques and applications, 2nd ed., crc press, taylor & francis, inc., or, usa. 82-83. romele, l. and achilli, m., 1998, spectrophotometric determination of low levels of bromated in drinking water after reaction with fuchsin, analyst, 123 (2): 291-294. salhi, e. and von gunten, u., 1999, simultaneous determination of bromide, bromate and nitrite in low μg l−1 levels by ion chromatography without sample pretreatment, water res., 33 (15): 3239-3244. uraisin, k., takayanagi, t., nacapricha, d. and motomizu, s., 2006, novel oxidation reaction of prochlorperazine with bromate in the presence of synergistic activators and its application to trace determination by flow injection/spectrophotometric method, anal. chim. acta., 580 (1): 68-74. valsecchi, s., isernia, a., polesello, s. and cavalli, s., 1999, ion chromatography determination of trace level bromate by large volume injection with conductivity and spectrophotometric detection after post column derivatisation, j. chromatogr. a., 864 (2): 263-270. science diliman 35 genuino, espino von gunten, u. and hoigné, j., 1994, bromate formation during ozonation of bromide-containing waters: interaction of ozone and hydroxyl radical reactions, environ. sci. technol., 28: 1234-1242. wagner, h. p., pepich, b. v., hautman, d. p. and munch, d. j., 2000, performance evaluation of a method for the determination of bromate in drinking water by ion chromatography (epa method 317.0) and validation of epa method 324.0, j. chromatogr. a., 884 (1-2): 201210. weinberg, h. s., delcomyn, c. a., and unnam v., 2003, bromate in chlorinated drinking waters: occurrence and implications for future regulation, environ. sci. technol., 37: 3104-3110. 36 science diliman 22_mamolo 97 teaching physics modeling science diliman (january-june 2003) 15:1, 97-99 teaching physics modeling using computer interfaced video: the case of paper baskets c. mamolo1*, n. capistrano, and e. van den berg2 science and math education institute university of san carlos cebu city, philippines e-mail: 1cbmamolo@yahoo.com; 2edberg@cnms.net modeling in physics in typical high school and bs physics courses, students study the results of physics rather than the process of doing physics. even most laboratory work are verification of known results of physics, rather than doing physics with yet unknown results. students are rarely exposed to a core activity of physicists: modeling. the availability of computer-video interfacing and analysis software, such as videopoint and coach, has greatly expanded the possibilities of doing real modeling in physics education at the high school and college levels. in this paper, we present and work out a simple modeling problem on the free fall of paper baskets. our purpose is to illustrate the possibilities of modeling in the classroom. the problem when leaves of trees or sheets of paper fall, they fall in a very irregular way. the paper zigzags slowly to the ground in an unpredictable way. however, if we fold the edges of the paper and make it into a little “basket”, the fall is much more regular. the paper basket moves slowly, but nearly straight to the ground. this is not free fall; air resistance plays a big role. how can we describe this motion mathematically? is it possible to make a model so that we will be able to predict what influence different factors have, such as the mass of the paper, the area of the bottom of the basket, etc.? when observing the basket falling, we notice that almost immediately a constant velocity is reached: the terminal velocity (v). different baskets have different terminal velocities. factors which influence the terminal velocity might be the mass (m) of the basket (greater m, greater v) or the area (a) of the basket (greater a, smaller v). in high school level physics courses, we may use aerodynamics to get a proper model. in lower level courses, we can do some smart trial and error. let us try this: (1) * corresponding author this is a simple formula that mathematizes our idea that a greater mass should result in a greater terminal velocity and that a greater area should result in a smaller terminal velocity. the k is just a constant that we can try to find experimentally. also, perhaps it should not be m, but m2 or m1/2. similarly, we might have to take a function of a rather than a straight, or if we know something about aerodynamics, we might already want to write v2 rather than v. our next task is to try to test the model in an experiment. then we can use the results to improve our formula and get a better fit with data. please note that so far we have not used any physics beyond high school level. at this stage we could still introduce a force equation: our task is now to design an experiment to test the model represented by eq. (1). m k a ν = ⋅ dragf mg f= −∑ 98 mamolo, capistrano, and van den berg the computer-video interface we can capture our own video of a falling paper basket or we can right away analyze a video which had been captured and edited earlier. videos are recorded by way of a video capturing device like the vidcap. the videos are processed using a video editing software such as corel draw. the essential series of frames are saved. the edited videos are then converted to a position-time graph by marking the center of mass of the paper basket at every frame. fig. 1 shows an example of a graph recorded with the coach software. with the same software, a velocity-time graph is derived from the position-time graph as shown in fig. 2. the slope at any point of each of these graphs can be determined, and therefore, the terminal velocity. the variables from the graphs, we determine the terminal velocity of the falling paper basket (varying bottom area and mass). then, we can commence the investigation of the correctness of the model (center for microcomputer applications, 1999). with the mass of the paper basket held constant while varying the cross sectional area of the paper basket, we obtain a graph of v versus 1/a as shown in fig. 3. the curve formed by the data points seems parabolic. taking the square root of a (fig. 4), the result looks linear. from this we can then revise eq. (1) to: (2) to investigate the influence of mass on terminal velocity, we keep the cross sectional area constant and vary the mass by adding whole pieces of paper of the same kind into the basket. the result looks linear (fig. 5). based on these experiments, our best model is represented by eq. (2). we could experiment further and try some other variations of the basic formula. we could be more sophisticated in deciding which graphs provide the best p o s it io n time (s) 0.0 -0.4 -0.8 -1.2 -1.6 0.1 0.2 0.3 0.4 0.5 0.6 0.7 fig. 1. position-time graph recorded with the coach software. m k a ν = data points linear (data points) 5 4 3 2 1 0 0 0.1 0.2 0.3 t e rm in a l v e lo c it y 1/a fig. 3. graph of v versus 1/a. fig. 2. velocity-time graph derived by the coach software from the position-time graph shown in fig. 1. 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.5 -0.1 -1.5 -2.0 -2.5 time (s) v e lo c it y ( m /s ) 99 teaching physics modeling fit, for example, we could use least squares techniques. but remember that this modeling exercise is intended for students in introductory physics courses. we think this semi-quantitative way of working with data provides an attractive experience in modeling. sources of error in this modeling exercise are the following: (1) some inherent instability in the motion of the paper basket; even in basket shape, it still wobbles a bit; (2) the clicking on the marked point could be off by a few millimeters, and in addition, there could be a slight parallax error; (3) within the series, no frame should be dropped in order to have a reliable time base; and (4) differentiation of the position-time graph in order to obtain a velocity-time graph introduces some instability. in spite of these inaccuracies, it is possible to obtain and verify a reasonable model for students in introductory physics courses. conclusion in physics, we try to describe phenomena. once we have a good description, we can use that to predict or even control phenomena. we try to describe phenomena as precisely as possible using mathematics to create our models. with a creative use of theory we try to make a model and then we conduct experiments to test the model and to improve it. that is what we did in this experiment. the purpose of doing it was to illustrate the way we work in physics. this experiment shows that modeling in physics can also be done with the simple physics available to high school and lower level college physics. reference cma centre of microcomputer applications, 1999. coach system workbook. amstel institute, universiteit van amsterdam. fig. 4. graph of v versus a . 5 4 3 2 1 0 data points linear (data points) 0 0.1 0.2 0.3 0.4 0.5 0.6 t e rm in a l v e lo c it y a fig. 5. graph of v versus m. data points linear (data points) 0 -1 -2 -3 -4 0 2 4 6 8 mass t e rm in a l v e lo c it y 4pajarito.pmd b. pajarito and others 21 science diliman (july-december 2014) 26:2, 21-39 effect of ingredient loading on surface migration kinetics of additives in vulcanized natural rubber compounds bryan b. pajarito* christia angel ine de torres marienne maningding university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract surface migration kinetics of chemical additives in vulcanized natural rubber compounds were studied as function of ingredient loading. rubber sheets were compounded according to a 212-8 fractional factorial design of experiment, where ingredients were treated as factors varied at two levels of loading. amount of migrated additives in surface of rubber sheets was monitored through time at ambient conditions. the maximum amount and estimated rate of additive migration were determined from weight loss kinetic curves. a t t e n u a t e d t o t a l r e f l e c t i o n – f o u r i e r t r a n s fo r m i n f r a r e d ( at rf t i r ) spectroscopy and optical microscopy were used to characterize the chemical s t r u c t u r e a n d s u r f a c e m o r p h o l o g y o f s h e e t s p e c i m e n s d u r i n g a d d i t i v e migration. anova results showed that increased loading of reclaimed rubber, caco 3 , and paraff in wax signif icantly decreased the maximum amount of additive migration; by contrast, increased loading of used oil, asphalt, and mercaptobenzothiazole disulphide (mbts) increased the maximum amount. increased loading of sulfur, diphenylguanidine (dpg), and paraff in wax signif icantly decreased the additive migration rate; increased loading of used oil, asphalt, and stearic acid elicited an opposite effect. comparison of atrftir spectra of migrated and cleaned rubber surfaces showed signif icant variation in intensity of specif ic absorbance bands that are also present in infrared spectra of migrating chemicals. paraff in wax, used oil, stearic acid, mbts, asphalt, and zinc stearate were identif ied to bloom and bleed in the rubber sheets. optical micrographs of migrated rubber surfaces revealed formation of white precipitates due to blooming and of semi-transparent wet patches due to bleeding. keyword s: rubber, migration, blooming, bleeding, vulcanizate, ingredient loading effect of ingredient loading on surface migration kinetics 22 introduction rubber compounds are mixtures made of a matrix based on one or more types of raw gum elastomer and dispersed amounts of different chemical ingredients. these ingredients include vulcanizing agents, activators, accelerators, fillers, antidegradants, processing aids, and other additives designed to modify and convert the raw gum elastomer matrix into a useful material. during compounding, selected ingredients are added to the rubber matrix at specif ic loadings to achieve good processing characteristics, yield the best balance of physical properties, lower the price of formulation, and even limit the hazard of the desired compound and rubber endproduct to health, safety, and environment (dick 2009). however, when high loadings of ingredients are mixed to the rubber matrix, some of the chemical additives migrate from the bulk matrix to the surface of the rubber because of oversaturation and limited solubility. in most cases, the migrated additives accumulate at the surface and form solid precipitates, resulting in a bloom. according to literature, a large number of compounding ingredients are known to migrate and bloom in rubber. these include sulfur (venable and greene 1922, auerbach and gehman 1954, wake and others 1983, jurkowski and jurkowska 1998, ciesielski 1999, mark and others 2005, bart 2006, dick 2009, basak and others 2010, dick 2014); activators such as zinc oxide (wake and others 1983, saeed and others 2011, saeed and others 2012a and 2012b, dick 2014) and stearic acid (sugiura and others 1996, bielinski and others 2005, dick 2009); antidegradants such as wax (nah and thomas 1980, wake and others 1983, choi 1999a and 1999b, ciesielski 1999, mark and others 2005, bart 2006, dick 2009, torregrossa-coque and others 2011a and 2011b, dick 2014), paraphenylenediamines (wake and others 1983, choi 1997, 1998, 1999a, and 1999b, parra and others 2000, mark and others 2005, bart 2006, dick 2009, saeed and others 2011, saeed and others 2012a and 2012b, dick 2014), and butylated hydroxytoluene (keen and others 1992, choi 1997 and 1998); and various accelerators such as dithiocarbamates (zinc dimethyl-, diethyl-, and dibutylsalts), thiazoles (mercaptobenzothiazole and its disulf ide), sulfenamides (tert-butyl-benzothiazole sulfenamide), and thiurams (tetramethylthiuram monoand disulf ide) (wake and others 1983, bart 2006, dick 2009, saeed and others 2011, torregrossa-coque and others 2011a and 2011b, saeed and others 2012a and 2012b, dick 2014). the high loading of process oils and plasticizers in the rubber matrix also results in migration and surface bleeding, leading to a wet and oily bloom (wake and others 1983, sugiura and others 1996, bart 2006, dick 2009 and 2014). antidegradants are designed to deliberately migrate to the rubber surface and create a passive barrier against ozone attack (wake and others 1983, choi 1997, 1998, and 1999, ciesielski 1999, dick 2009); nevertheless, the undesirable b. pajarito and others 23 deposition of other migrated additives at the surface can cause several problems. the appearance of a bloom at the rubber surface is visually offensive and can cause rejection and return of the rubber product. it destroys building tack and interferes with the adhesion of rubber to other material surfaces (wake and others 1983, ciesielski 1999, dick 2009 and 2014). human contact to bloom can also cause skin irritation (bart 2006). blooming can also facilitate the initiation of cracks at the reagglomeration sites and reduce the fatigue life of rubber because of the reagglomeration of additives at the surface and in bulk (saeed and others 2011). the migration of chemical additives in rubber is related to diffusion kinetics, and is dependent on several factors such as additive-rubber compatibility, molecular size of migrating additive, physical and chemical interactions between additive and rubber molecules, conf iguration of rubber chains and voids, and others (bart 2006). loading a rubber matrix with a soluble ingredient beyond its solubility limit is already established to cause the excess amount to simply migrate and bloom or bleed at the surface. however, the effect of insoluble and/or non-migrating compounding ingredients to the blooming and bleeding behavior of soluble additives in rubber has scarcely been investigated. sugiura and others (1996) reported that loading ethylene-propylene diene monomer (epdm) rubber with sepiolite, a clay mineral and adsorbent, prevents blooming and bleeding of paraff in process oil, stearic acid, and curing product zinc stearate at the rubber surface. choi (1998) showed that increased loading of silica f iller in natural rubber vulcanizates reduce the migration rates of butylated hydroxytoluene, paraphenylenediamines, and wax. talc, a semi-reinforcing f iller, was also reported to reduce iridescent bloom (dick 2014). the effect of loading other insoluble and/or non-migrating compounding ingredients such as reclaimed rubber, calcium carbonate, kaolin clay, and asphalt in the blooming and bleeding of oversaturated additives in rubber is not yet investigated. this work studied surface migration kinetics of chemical additives in vulcanized natural rubber compounds as a function of ingredient loading. a fractional factorial design was used during the formulation of rubber compounds, where ingredients were assigned as factors and were varied at two different loadings. in experimental design, reclaimed rubber, calcium carbonate, kaolin clay, and asphalt were treated as unconfounded factors, whereas the remaining ingredients were assigned as factors confounded with twoor more factor interactions. the amount of migrated additives at the surface of rubber sheet specimens was monitored through time at ambient conditions. mean effects and factor ranking were calculated and performed from the results. anova was used to determine the compounding ingredients that have a signif icant effect on blooming and bleeding behavior. attenuated total reflection– effect of ingredient loading on surface migration kinetics 24 fourier transform infrared (atr-ftir) spectroscopy and optical microscopy were used to identify the migrating additives and to characterize the surface morphology of rubber sheet specimens during blooming and bleeding, respectively. methodology table 1 shows the basic and the selected upper and lower loadings of compounding ingredients used in the formulation of rubber sheet specimens. ingredient loading was expressed as parts per hundred (phr) parts of standard philippine rubber (spr 20). reclaimed rubber is vulcanized scrap rubber recovered from grinded tire threads and is chemically treated. it is also commonly used as an economical f iller for natural rubber. asphalt is an inexpensive tackif ier. calcium carbonate and kaolin clay are non-black f illers for cost reduction and hardness improvement. as seen in table 1, sulfur was used as the vulcanizing agent, zinc oxide and stearic acid as the activators, paraff in wax as an antidegradant, petroleum oil as a processing aid, and mercaptobenzothiazole disulf ide (mbts), mercaptobenzothiazole (mbt ), and diphenylguanidine (dpg) as the accelerator system. each ingredient was assigned as a factor x i varied at two levels of loading (upper and lower) relative from their respective basic loading. to evaluate the effect of ingredient loading on the migration kinetics of additives at the surface of rubber sheets, eighteen natural rubber compounds were prepared at different ingredient loading combinations. the formulations are given in table 2. ingred ient load ing, phr reclaimed rubber, x 1 caco 3 , x 2 kaolin clay, x 3 asphalt, x 4 zno, x 5 stearic acid, x 6 basic 33 400 35 3.0 6 1.5 variation interval 15 100 15 1.5 3 1.0 upper 48 500 50 4.5 9 2.5 lower 18 300 20 1.5 3 0.5 basic 2.5 95 2.5 1.5 1.5 1.5 variation interval 1.5 25 1.5 1 1 1 upper 4 120 4 2.5 2.5 2.5 lower 1 70 1 0.5 0.5 0.5 ingred ient load ing, phr paraffin wax, x 7 used oil, x 8 sulfur, x 9 mbts, x 10 mbt, x 11 dpg, x 12 table 1. basic, upper, and lower loadings of compounding ingred ients in rubber formulations b. pajarito and others 25 the ingredient loading combinations for compounds 1–16, as shown in table 2, were derived from a 212-8 fractional factorial design of experiment (lazic 2004). the design matrix was generated from the fracfactgen and fracfact functions of matlab 6.5 using 12 main factor terms, with k = 4 for 2k runs under design resolution 3. the total number of runs or formulations (2 k = 16) of the generated design matrix corresponds to 1/256 fraction of a 212 full factorial experiment. the f irst four factor ingredients (reclaimed rubber, caco 3 , kaolin clay, and asphalt) were unconfounded, whereas the effects of the remaining factor ingredients were the coded value of the i-th factor ingredient x i in the table is calculated by the equation (upper or lower loading – basic loading) (variation interval)-1. a value of x i = +1 corresponds to the upper loading of i-th factor ingredient; x i = -1 for the lower loading, and x i = 0 for the basic loading (lazic 2004). for example, rubber compound 1 in table 2 was formulated using 18 parts reclaimed rubber, 300 parts caco 3 , 20 parts kaolin clay, 1.5 parts asphalt, 9 parts zno, 0.5 parts stearic acid, 1 part paraff in wax, 120 parts used oil, 1 part sulfur, 2.5 parts mbts, 2.5 parts mbt, and 0.5 part dpg per 100 parts of natural rubber. compound number factor ingred ients x 1 x 2 x 3 x 4 x 5 x 6 x 7 x 8 x 9 x 10 x 11 x 12 -1 -1 -1 -1 +1 -1 -1 +1 -1 +1 +1 -1 -1 -1 -1 +1 -1 +1 +1 -1 +1 -1 -1 -1 -1 -1 +1 -1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 +1 +1 +1 -1 -1 +1 +1 -1 -1 +1 -1 +1 -1 -1 -1 +1 -1 +1 +1 -1 +1 +1 -1 +1 -1 +1 +1 -1 +1 -1 -1 +1 -1 +1 -1 +1 +1 -1 +1 -1 +1 -1 +1 -1 +1 -1 -1 +1 +1 +1 -1 +1 -1 +1 -1 +1 -1 -1 +1 -1 -1 -1 -1 -1 +1 +1 +1 +1 -1 +1 +1 -1 -1 +1 +1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 -1 +1 +1 -1 -1 +1 +1 -1 -1 +1 -1 +1 +1 -1 -1 +1 +1 -1 -1 +1 -1 +1 +1 -1 -1 +1 +1 +1 +1 -1 -1 -1 -1 +1 +1 -1 +1 -1 -1 -1 -1 +1 +1 +1 -1 +1 +1 +1 -1 -1 -1 -1 -1 -1 -1 -1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 table 2. formulations of rubber compounds ·  effect of ingredient loading on surface migration kinetics 26 confounded with factor interactions. the confounding factor interactions are given by the following generating ratios: x 5 = x 1 x 2 x 3 x 4 (1) x 6 = x 2 x 3 x 4 (2) x 7 = x 1 x 3 x 4 (3) x 8 = x 3 x 4 (4) x 9 = x 1 x 2 x 4 (5) x 10 = x 2 x 4 (6) x 11 = x 1 x 4 (7) x 12 = x 1 x 2 x 3 . (8) thus, if e and e’ are the true and estimated factor effects to an experimental response, respectively, the estimated effect of reclaimed rubber e’ x1 is equal to e x1 because it is unconfounded; on the other hand, the estimated effect of paraff in wax e’ x7 is equal to e x7 + e x1x3x4 , where e x1x3x4 is the effect due to three-way factor interaction of reclaimed rubber, kaolin clay, and asphalt. compounds 17 (all ingredients are at low loading) and 18 (all ingredients are at basic loading) were also formulated for comparison. rubber compounds listed in table 2 were mixed in a heated two roll mill. all ingredients were added on the f irst batch of mixing, except sulfur and the accelerator system. the resulting master batch was aged for 24 hrs at ambient conditions before roll milling with sulfur and accelerators. compounds were then cured using a heated compression molding press at 160 °c for 20 mins. the vulcanized rubber sheets have f inal dimensions of 300 mm × 300 mm × 3 mm. the preparation of ingredients, compounding, and vulcanization of rubber sheets were performed by rhodeco rubber processing services, inc. rubber sheet specimens of 50 mm × 50 mm × 3 mm size were cut from the vulcanized sheets. the migration experiments were performed in open air at ambient conditions for 32 d. sheet specimens were clipped and left hanging in a metal rack to allow unhindered migration of additives to the exposed surface. the amount of additives that migrated to the surface of the rubber sheet specimens were determined by periodically removing the additives that have bloomed and bled to the surface, and measuring the weight loss (nah and thomas 1980). the removal was done by means of a single pass of adhesive tape (scotch tape). percent weight loss due to additive migration m t in rubber sheet specimens was calculated by: (1)mt = ×100 w i –w t w i b. pajarito and others 27 where w i is the initial weight of the rubber sheet specimen and w t is the weight of the rubber sheet specimen after application of adhesive tape at time t. using equation 1, the total amount of additives that migrated to the surface of specimens were recorded as a function of time. three specimens from each compound were used to report the average weight loss. other rubber sheet specimens were left unmonitored for 32 d, and their migrated surfaces were analyzed using atr-ftir spectroscopy and optical microscopy. afterwards, the migrated surfaces were cleaned with adhesive tape, and the resulting infrared spectra were recorded. for comparison and possible identif ication of migrating additives, atr-ftir spectra of compounding ingredients were also measured. results and discussion figures 1–3 illustrate the amount of additive migration in terms of cumulative weight loss in rubber sheet specimens as a function of time. all compounds exhibit an increasing amount of migrating additives to the rubber surface through observation time at ambient conditions. moreover, the migration kinetic curves have non-zero weight loss value at t = 0, indicating the presence of migrated additives at the specimen surface before the migration experiments were started. in terms of maximum amount of additive migration, compounds 1, 8, and 4 (0.59%, 0.57%, and 0.48%, respectively) exhibited the highest amount, whereas compounds 14, 18, and 17 (0.13%, 0.14%, and 0.15%, respectively) exhibited the lowest amount, after 32 d figure 1. migration kinetic curves of additives in rubber compounds 1–6. effect of ingredient loading on surface migration kinetics 28 of migration. aside from determining the maximum amount of additive migration, the migration rate was also measured from the initial slope of the kinetic curves. the migration rate was calculated as the slope of the regressed line using data points of the kinetic curves at t = 0, 4, and 8 d. from this procedure, compounds 8, 1, and 10 (2.80 × 10-2% d-1, 1.38 × 10-2% d-1 and 1.30 × 10-2% d-1, respectively) were found to have the highest migration rate, whereas compounds 17, 14, and 18 (3.9 × 1 0 -3% d-1, 4.0 × 10-3% d-1, 4.3 × 10 -3% d-1, respectively) have the lowest rate. figures 4 and 5 illustrate the effect of ingredient loading on the maximum amount figure 2. migration kinetic curves of additives in rubber compounds 7–12 figure 3. migration kinetic curves of additives in rubber compounds 13–18 · ·  ·  ·  ·  ·  b. pajarito and others 29 highylow highyhigh and the rate of additive migration on surfaces of rubber sheet specimens, respectively. the bars indicate the mean values of maximum amount and rate of additive migration at low and high ingredient loadings. the mean values were calculated using table 2. for example, let be the maximum amount of additive migration of j-th rubber compound. following the coded values of factor ingredients in table 2, the mean value of maximum amount of migration at low loading of reclaimed rubber (x 1 ) was computed as: (2) likewise, the mean value at high loading of reclaimed rubber was: (3) figure 4. effect of ingredient loading on maximum amount of additive migration. figure 5. effect of ingredient loading on surface migration rate of additives. yj    9 1787654321 l ow yyyyyyyyy y ++++++++ =   8 161 514131 21 1109 hi g h yyyyyyyy y +++++++ = effect of ingredient loading on surface migration kinetics 30 the mean values at low and high loading of i-th compounding ingredient were calculated following the distribution of coded values of x i (-1 for low, +1 for high loading) in table 2. in figure 4, the maximum amount of additive migration in rubber sheet specimens was observed to increase when the following compounding ingredients were increased in loading in the formulations: kaolin clay, asphalt, zinc oxide, stearic acid, used oil, and mbts. by contrast, increased loading of reclaimed rubber, caco 3 , paraff in wax, sulfur, mbt, and dpg in the rubber formulations resulted to decrease in maximum amount of additive migration in rubber sheet specimens. in figure 5, the migration rate of additives to the rubber surface was found to be enhanced when loading of kaolin clay, asphalt, stearic acid, sed oil, and mbts were increased in the formulations. however, high loading of reclaimed rubber, caco 3 , zno, paraff in wax, sulfur, mbt, and dpg in the formulations was observed to decrease the migration rate of additives. using the mean values at low and high ingredient loadings, as seen in figures 4 and 5, the estimated effect of factor ingredients on maximum amount and rate of surface migration of additives in rubber sheet specimens were quantif ied, and were used to rank the ingredients according to the magnitude of estimated effect. these are shown in figures 6 and 7. the bars in figures 6 and 7 demonstrate the effect e’ of each compounding ingredient on maximum amount and rate of migration, expressed as: (4) figure 6. ranking of compounding ingredients in terms of their effect on maximum amount of additive migration.   100' lo w lowh igh � � = y yy e ×  _ b. pajarito and others 31 as shown in figure 6, used oil, asphalt, and mbts have the highest effect in increasing the maximum amount of additive migration (59.6%, 19.5%, and 18.4%, respectively) among the compounding ingredients. on the other hand, reclaimed rubber, caco 3 , and paraff in wax have the largest effect in decreasing the maximum amount of additive migration (-33.9%, -26.7%, and -15.8%, respectively). anova using matlab’s anovan function reveals that the effect of these compounding ingredients was statistically signif icant at a 95% conf idence level. likewise, in figure 7, ingredients such as used oil, asphalt, and stearic acid have the greatest effect in enhancing the rate of additive migration (73.3%, 43.3%, and 34.4%, respectively), whereas sulfur, dpg, and paraffin wax have the highest effect in lowering the migration rate (-30.4%, -26.5%, and -25.6%, respectively). the observed effects of these ingredients on migration rate were also found to be statistically signif icant at a 95% conf idence level. in terms of increased additive migration, high levels of processing oils, stearic acid, and mbts in rubber recipes are known to cause blooming and bleeding (sugiura and others 1996, bielinski and others 2005, bart 2006, dick 2009 and 2014). asphalt, commonly used as building material in roof ing and paving applications, is a natural organic end-product subject to chemical oxidation through reactions with atmospheric oxygen (petersen 2009). increased loading of asphalt in rubber compounds may have caused a pseudo bloom (wake and others 1983), as observed from black specks found in adhesive tapes used during the late stages of migration experiments. with respect to the effect of the other signif icant ingredients, the decrease in migration rate due to high loading of sulfur and dpg can be attributed to increased cured modulus and ultimate crosslink density (dick 2014), resulting in slow diffusion figure 7. ranking of compounding ingredients in terms of their effect on surface migration rate of additives. effect of ingredient loading on surface migration kinetics 32 of additives from bulk to the surface of the rubber sheet. the observed decrease in additive migration due to high loading of wax can be explained by the interface between the wax bloom and the surface of the rubber vulcanizate. choi (1999) found the surface migration of paraphenylenediamines in natural rubber vulcanizates compounded with wax to be slower and lower than those in vulcanizates without wax. a discontinuous concentration gradient of migrating additives was observed at the bloom-vulcanizate interface, where higher concentration of additives is found at the interface than at the bulk regions of the rubber and wax bloom. the wax bloom impeded migration and caused accumulation of migrating additives at the bloom-vulcanizate interface. meanwhile, the increased loading of caco 3 reduced migration through an increased diffusion path length of migrating additives from bulk to the surface of rubber sheet. lastly, high loading of reclaimed rubber in the formulation extended the rubber matrix greatly and contributed to the absorption and retention of migrating additives. figures 8 and 9 show the atr-ftir spectra of the rubber sheet specimens after 32 d of additive migration at ambient conditions. high absorption bands were found at wave numbers 500 cm-1–1600 cm-1 and 2800 cm-1–3000 cm-1; nevertheless, not all compounds exhibited signif icant peaks at the latter wave number range (see atr-ftir spectra of compound 4 in figure 8; compounds 11 and 14–18 in figure 9). migrated surfaces of rubber sheet specimens were then cleaned several times with adhesive tape to ensure removal of migrated additives. for each compound, the atr-ftir spectrum of the cleaned surface was measured and superimposed to the figure 8. atr-ftir spectra of migrated rubber surfaces for compounds 1–9. b. pajarito and others 33 figure 9. atr-ftir spectra of migrated rubber surfaces for compounds 10–18. infrared spectrum of the migrated surface. the superimposed spectra were manually compared to indicate changes in position and intensity of absorption bands. figures 10 and 11 illustrate this procedure for compounds 1 and 15, respectively. in figure 10, decrease in band intensity at wave numbers 2916 cm-1, 2848 cm-1, and 1535 cm-1 were observed after the removal of bloom and bleed in the rubber surface. moreover, absorption bands at 1455 cm-1 and 1397 cm-1 disappeared after cleaning. in some specimens, such as compound 15 in figure 11, new absorption bands with high intensity emerged at 2916 cm-1 and 2848 cm-1, accompanied by disappearance of the band at 1257 cm-1 after removal of migrated additives. the atr-ftir spectra of compounding ingredients shown in figures 12 and 13 were also used as reference to identify the migrated additives. based on the superimposed atr-ftir spectra of cleaned and migrated rubber surfaces, as well as the measured spectra of compounding ingredients, the following absorption bands were found to be characteristic of the migrated additives in rubber compounds (sugiura and others 1996): 2954 cm-1, 2916 cm-1, and 2848 cm-1 for antisymmetric and symmetric ch stretching of wax, used oil, and stearic acid; 1535 cm-1 and 1397 cm-1 for asymmetric and symmetric -coostretching of zinc stearate, the reaction product of zno and stearic acid; 1455 cm-1 for antisymmetric ch deformation of wax, used oil, and stearic acid; 1257 cm-1 for co stretch of stearic acid; and 724 cm-1 for -ch 2 rocking and wagging of wax, used oil, and stearic acid. absorption bands found in asphalt (1095 cm-1 and 1025 cm-1) and mbts (755 cm-1) also exhibited changes in band intensity after cleaning of migrated rubber surfaces. effect of ingredient loading on surface migration kinetics 34 figure 10. atr-ftir spectra of cleaned and migrated rubber surfaces of compound 1. figure 11. atr-ftir spectra of cleaned and migrated rubber surfaces of compound 15. b. pajarito and others 35 figure 12. atr-ftir spectra of sample migrated and cleaned rubber specimen, raw natural rubber, and compounding ingredients 1-5. figure 13. atr-ftir spectra of sample migrated and cleaned rubber specimen, raw natural rubber, and compounding ingredients 6-12. effect of ingredient loading on surface migration kinetics 36 figures 14–16 illustrate the actual additive blooming and bleeding in the rubber sheet specimens due to surface migration of additives. in rubber compounds with low band intensity at 2800 cm-1–3000 cm-1, small (white spots in figure 14) and large (figure 15) precipitates of migrated additives bloomed at the rubber surface. in figure 16, used oil was shown bleeding from rubber sheet specimens of compound 12, forming semi-transparent wet patches at the rubber surface. additive bleeding was mostly observed on rubber compounds with high absorption bands at 2800 cm -1–3000 cm -1. figure 14. additive blooming in compound 14 at ambient conditions. figure 15. additive blooming in compound 16 at ambient conditions. b. pajarito and others 37 figure 16. additive bleeding in compound 12 at ambient conditions. conclusions loading of ingredients in formulations of natural rubber compounds has a signif icant effect on the surface migration kinetics of chemical additives at ambient conditions. in the studied formulation, increased loading of soluble ingredients such as used oil, stearic acid, and mbts promoted blooming and bleeding in rubber sheets. high loading of asphalt in the formulation resulted in pseudo bloom due to surface degradation. in reducing overall migration of additives at the rubber surface, increased loading of other soluble ingredients such as sulfur and dpg was found to be effective. wax, although a migrating additive, is observed to hinder the migration of other additives at increased loading. non-soluble and non-migrating ingredients such as caco 3 lowered additive migration by increasing the diffusion path length within the rubber matrix. high loading of reclaimed rubber decreased blooming and bleeding by absorbing and retaining the migrating additives in the bulk. acknowledgments the authors acknowledge the off ice of the chancellor of the university of the philippines diliman, through the off ice of the vice-chancellor for research and development, for funding support through the ph.d. incentive awards (project no. 131309 phdia). the authors also express their gratitude to rhodora dela cruzmedalla of rhodeco rubber processing services, inc. for compounding and vulcanization of rubber sheets. effect of ingredient loading on surface migration kinetics 38 references auerbach i. , gehman s.d. 1954. tracer method for sulfur solubility and diffusivity in rubber. anal chem. 26(4): 685-690. b a r t j . c . 2 0 0 6 . p o l y m e r a d d i t i v e a n a l y t i c s : i n d u s t r i a l p r a c t i c e a n d c a s e s t u d i e s . universita degli studi di firenze, 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diliman. 5rambutan-magdalita.pmd influence of changing rainfall patterns on the yield of rambutan 64 science diliman (january-june 2015) 27:1, 64-90 influence of changing rainfall patterns on the y ield of rambutan (nephel ium lappaceum l.) and selection of genotypes in known drought-tolerant fruit species for cl imate change adaptation pabl ito m. magdalita* university of the philippines los baños ronaldo b. saludes university of the philippines los baños _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract in fruit crop production, rainfall, water stress, temperature, and wind are key variables for success, and the present changes in rainfall patterns c o u l d a f f e c t t h e f l o w e r i n g a n d y i e l d o f t h e r a m b u t a n ( n e p h e l i u m lappaceum l). other fruit species like macopa (syzygium samarangense), s i n i g u e l a s ( s p o n d i a s p u r p u r e a ) , a n d n a t i v e s a n t o l o r c o t t o n f r u i t (sandoricum koetjape) remain productive despite extreme climatic changes. t h i s s t u d y a s s e s s e d t h e i n f l u e n c e o f r a i n f a l l o n r a m b u t a n y i e l d a n d evaluated and selected tree genotypes of known drought-tolerant fruit s p e c i e s . r a m b u t a n y i e l d i n a s e l e c t e d f a r m i n c a l a u a n , l a g u n a , philippines, dropped remarkably from 152.2 kg/tree in 2008 to 8.6 kg/tree in 2009. this reduction could be attributed to the high rainfall in april 2 0 0 9 a t 3 3 4 . 4 m m , a n d p o s s i b l y o t h e r e n v i r o n m e n t a l f a c t o r s l i k e t e m p e r a t u r e , r e l a t i v e h u m i d i t y, s o l a r r a d i a t i o n , a n d s t r o n g w i n d . f u r t h e r m o r e , w e t m o n t h s i n 2 0 0 9 a l s o i n h i b i t e d t h e f l o w e r i n g o f rambutan. however, a low yield obtained in 2010 at 45.5 kg/tree could be partly attributed to the very low rainfall in may 2010 at only 9.1 mm. o n t h e o t h e r h a n d , i n r e l a t i o n t o c h a n g i n g c l i m a t e , s e l e c t i o n o f t r e e genotypes for use as varieties in known droughtand flood-tolerant fruit species based on important fruit qualities like sweetness, juiciness, and high edible por tion was done. among 103 macopa genotypes, mc-13, 43, and 91 were selected and the best (i.e. , mc-13) had sweet (7.15 °brix) and crispy fruits weighing 49.44 g, creamy white (rhcc 155 a), and had high edible por tion (ep, 93.22%). among 114 siniguelas genotypes, sg-41, pm magdalita and rb saludes 65 42 and 105 were selected and the best selection (i.e. , sg-41), had sweet (12.50 °brix) and juicy fruit weighing 20.42 g, ruby red (rhcc 59 a), and had high ep (83.27%). among 101 native santol genotypes, sn-47, 59, and 74 were selected and the best selection (i.e. , sn-59) had relatively sweet (5.56 °brix) and juicy fruits weighing 51.96 g, maize yellow (rhcc 21 b), a n d h a d h i g h e p ( 8 2 . 2 0 % ) . t h e s e s e l e c t i o n s a r e r e c o m m e n d e d f o r p l a n t i n g i n m a r g i n a l a n d d r o u g h t p r o n e a r e a s f o r c l i m a t e c h a n g e adaptation. in addition, they can fare better in flooded areas in the face of climate change since they are very hardy, and have woody and strong r o o t s t h a t c a n r e s i s t s t r o n g w i n d a n d i n c r e a s i n g a m o u n t o f r a i n f a l l brought about by climate change. k e y w o r d s : c l i m a t e c h a n g e , r a i n f a l l , m a c o p a , n a t i v e s a n t o l , r a m b u t a n , s i n i g u e l a s layman’s abstract in fruit production, the rainfall, water stress, temperature, and wind are impor tant variables for success. however, the changing rainfall patterns c o u l d a f f e c t t h e f l o w e r i n g a n d y i e l d o f r a m b u t a n . o t h e r f r u i t s p e c i e s l i k e m a c o p a , s i n i g u e l a s , a n d n a t i v e s a n t o l r e m a i n p r o d u c t i v e d e s p i t e extreme climatic changes. in relation to this, the influence of rainfall on rambutan yield and evaluation of genotypes of known drought-tolerant fruit species were conducted in this study. the rambutan yield in a farm in calauan, laguna, philippines, decreased remarkably from 152.2 kg/tree in 2008 to 8.6 kg/tree in 2009. this could be due the high rainfall in april 2 0 0 9 a t 3 3 4 . 4 m m , a n d p o s s i b l y o t h e r e n v i r o n m e n t a l f a c t o r s l i k e t e m p e r a t u r e , r e l a t i v e h u m i d i t y, s o l a r r a d i a t i o n , a n d s t r o n g w i n d . i n addition, the low yield in 2010 at 45.5 kg/tree could be due to the very low rainfall in may 2010 at only 9.1 mm. in relation to climate change, selection of genotypes for future use as varieties in droughtand floodt o l e r a n t f r u i t s p e c i e s b a s e d o n s w e e t n e s s , j u i c i n e s s , a n d h i g h e d i b l e portion was done. among 103 macopa genotypes evaluated, three were selected ( m c1 3 , mc4 3 , a n d m c9 1 ) a n d t h e b e s t m c1 3 , i s s wee t a n d crispy, weighs 49.44 g, creamy white and had high edible por tion (ep). among 114 siniguelas genotypes selected, sg-41, sg-42 and sg-105 were selected, and the best selection sg-41, is sweet , juicy, weighs 20.42 g, ruby red and had high ep. in addition, among 101 native santol genotypes evaluation, sn-47, sn-59, and sn-74 were selected and the best selection sn-59 is relatively sweet , juicy, weighs 51.96 g, maize yellow, and had high ep. these selections can be planted in marginal and drought-prone areas for climate change adaptation. also they can fare better in flooded influence of changing rainfall patterns on the yield of rambutan 66 introduction climate change causes increases in the temperature of the earth, carbon dioxide concentration in the atmosphere, destruction of the ozone layer, and more intense hydrologic events causing flooding during typhoons (lansigan 2009 ). this causes global warming that brings sea level rise, changes in hydrologic regimes, and more frequent and more intense extreme events like el niño and la niña. these adverse conditions brought about by climate change have pervasive effects on crop productivity and yield, poses a serious threat to the agricultural sector and consequently a threat to the country's food security (da-bar 2011). in fruit crops production, the onset of rain, duration of the rainy period, water stress following the vegetative stage (e.g. , flowering), occurrence of extreme events like strong wind, intense rainfall, and high temperature are the key variables for determining success. some fruit species like rambutan, avocado, and sugar apple are affected by climate change because their flowering behavior changes and fruit yield decreases signif icantly either during el niño or la niña (magdalita 2012). the rambutan (nephelium lappaceum l.) is a tropical fruit species that thrives best in humid and hot regions with rainfall that is evenly distributed throughout the year. it has fruited successfully in areas with pronounced dry season if irrigated during the reproductive period. it is unable to withstand cold temperature, and its growth is thus restricted to areas with an elevation below 700 m. the moisture content of the soil should always be maintained at a high level so that the best places to grow rambutan are those with evenly distributed rainfall or where the dry season is short (coronel 1998). it also grows well from sea level to medium altitudes in places with a long rainy season (coronel 2011). however, with the occurrence of changing rainfall patterns in places where the rambutan is welladapted like in calauan, laguna, it has been observed that there were corresponding changes in the flowering and fruiting behavior. hence, one of the objectives of this study is to f ind out if rainfall has an influence on flowering of rambutan in calauan. this study used a government-owned orchard found in the area, wherein the researchers have an access to conduct the study and the only orchard with a complete data on rambutan yield for the last ten years, needed in studying climate change, hence, the reason for using only one experimental area using purposive sampling. a r e a s b ec a u s e t h ey a r e ve r y h a r d y, a n d h a ve wo o d y a n d s t r o n g r o o t s t h a t c a n r e s i s t s t r o n g w i n d a n d i n c r e a s i n g a m o u n t o f r a i n f a l l i n t h e face of climate change. pm magdalita and rb saludes 67 before selections of these known drought-tolerant fruit species can be made available, they f irst need to be evaluated for fruit qualities. the native santol, sandoricum koetjape (burm. f. merr.), is widely distributed in the philippines and is esteemed by many people as a fresh fruit. it is a potential money earner for the country because it can be manufactured commercially into preserve, candies, chutney, jam, and jelly (coronel 1998). at this time of drought and sometimes flooding, native santol can be tapped for planting since it is very hardy, vigorous, and has a very strong root system that can thrive successfully in dry and humid areas. two varieties exist: the native santol and the big-fruited “bangkok” santol. because of so much interest in the “bangkok” santol, the native santol has been neglected. however, efforts must be focused on the selection of native santol because of its very hardy nature that can stand either drought or flooded conditions aside from being a fresh fruit having sweet aril and pulp. macopa, syzygium samarangense (blume) merr. & perr. , a very prolif ic tree and a popular symbol of “good luck,” is being grown in many frontyards of filipino homes. it is another attractive summer fresh fruit because of its crunchy and striking red, maroon, pink, white, or green color. the astringent bark is made into a mouthwash, the dried powder leaves are applied to cracked tongue, and the root is used as a diuretic. it grows well from sea level to 750 m elevation with well distributed rainfall, although it also grows successfully in both dry and humid areas (coronel 1998). this makes the macopa another potential income earner despite extreme changes in weather conditions. nevertheless, selection/s that are sweet, juicy, and small-seeded are still limited. the siniguelas, spondias purpurea l., a very hardy and prolif ic tree is one of the most popular seasonal fruit during summer in the philippines because of its attractive color, delicious taste, and wholesome and excellent flavor. the ripe fruit has a ready market, while the mature green fruit is made into pickles and used as the souring ingredient for cooking “sinigang.” it grows equally well in both dry and wet places, but better quality fruits are apparently produced in places with long dry season. siniguelas grows successfully in dry areas that are otherwise not suited to many fruits (coronel 1998), thus making it another potential income generator. while the siniguelas is clonally propagated, selections of bud mutant with big fruits, sweet and small-seeded are wanted. while the effect of rainfall on rambutan flowering is important to fruit production, similarly, the evaluation of known drought-tolerant fruit species like macopa, siniguelas, and native santol needs to be done since they are also important for fruit production in the face of climate change. particularly, specif ic genotypes in influence of changing rainfall patterns on the yield of rambutan 68 each species with desirable fruit qualities are also of paramount importance in the future production of these lesser known fruits because they are drought-resistant, hence selections of these species were also identif ied in this study. they can be used and traded as alternative fruits during drought periods. hence, this study was conducted to assess the influence of changing rainfall patterns on the yield of rambutan in calauan, laguna, and evaluate, select, and identify genotypes with good fruit qualities in known drought-tolerant fruit species including macopa, siniguelas, and native santol. materials and methods the annual yield of rambutan for a 12-year period was obtained from a selected orchard located in lamot ii, calauan, laguna, philippines. the site is a governmentowned orchard, fenced, and planted to several species of fruit crops including rambutan, lanzones, caimito, pomelo, banana, macopa, and other fruit crops. a propagation nursery is also located in the site. the area is flat, rainfed, and has clay loam soil. the orchard is presently being managed by the nursery chief, mario tenorio. the total rambutan yield was taken from 2001 to 2012 fruiting trees growing in a portion of a 15-ha orchard planted to different fruit crops. the number of rambutan trees growing in the orchard on a year basis from 2001 to 2012, respectively is as follows: 300, 310, 353, 266, 310, 296, 164, 134, 134, 154, 242, and 143. in addition, the annual rainfall accumulation from 2001 to 2012 was o b t a i n e d f r o m t h e u n i v e r s i t y o f t h e p h i l i p p i n e s lo s b a ñ o s n a t i o n a l agrometeorological station in los baños, laguna. since this is the only weather station available in the area, it was used and assumed that there are small differences in the climatic pattern between the two municipalities (pagasa 2012). calauan is very close to los baños, laguna, which is only about 10 km away via the national highway going to san pablo city. the rambutan yield data from 2001 to 2012 were used for correlation analysis with the rainfall data using pearson r correlation (gomez and gomez 1984). in addition, drought-tolerant fruit crop species including macopa, siniguelas, and native santol, were identif ied based on literature (coronel 1998). while excessive moisture from high rainfall during la niña is a known effect of climate change, the other scenario being experienced is the occurrence of very low soil moisture during el niño. because of this, an investigation on the utilization of drought-tolerant species becomes a truly important component study. more than 100 trees of each species growing in different places primarily in laguna, batangas, and cavite were selected and used as sources of ripe fruits for evaluation of selected fruit characters. pm magdalita and rb saludes 69 selection of trees and collection of fruits for macopa, siniguelas, and native santol was conducted from march to june 2012. the places and owners of the tree/s where the fruit samples of the different trees of macopa, siniguelas and native santol were collected were indicated in tables 1, 2, and 3. mc-01 & mc-02 sta. maria, talisay, batangas bobet luna mc-03 to mc-06 caloocan, talisay, batangas lucita ramos, cynthia carandang & archie trinidad mc-07 ambulong, talisay, batangas gigit panganiban mc-08 to mc-10 santor, tanauan city, batangas pastor landicho & ethel vivas mc-11 darasa, tanauan city, batangas rose pecho mc-12 luta sur, malvar, batangas evelyn viaje ledesma mc-13 inisluban, lipa city, batangas aristides lindog mc-14 & mc-15 tangway, lipa city tangway elem. school mc-16 san felix, sto. tomas, batangas orlando maloles mc-17 talahiban, sta. cruz, bay, laguna dominador hernandez mc-18 masaya, bay, laguna cleotilde caldo mc-19 to mc-22 lamot ii, calauan, laguna sta. cruz nursery c/o mr. tenorio mc-23 orchard, college of agriculture, uplb college, laguna mc-24 bpi, san andres bukid, manila bpi, manila mc-25 imus, cavite dr. emer s. borromeo mc-26 ambulong, talisay, batangas adel rapadas mc-27 to mc-29 santor, tanauan city, batangas fely macadilo & ricky cariño mc-30 balayhangin, calauan, laguna guillermo del valle mc-31 to mc-33 lamot ii, calauan, laguna sta. cruz nursery c/o mr. tenorio & mc-35 mc-34 calamba city, laguna calamba city council (beside national road in licheria) mc-36 balayhangin, calauan, laguna corazon melba borton mc-37 & mc-38 tanauan city, batangas norma perez & lilibeth dayta mc-39 talaga, tanauan city, batangas dominga robles mc-40 & mc-41 sto. cristo, san jose, batangas sto. cristo brgy. council (t#1&3) mc-42 darasa, tanauan city, batangas dolor magsino mc-43 to mc-46 sto. cristo, san jose, batangas clara mendoza, conchita atienza, pablo mendoza & cleofe mendoza mc-47 maraouy, lipa city, batangas aristides lindog mc-48 351 vallejo st. , sta. rosa, laguna mercy alvarez mc-49 to mc-53 ipb fruit & ornamentals section csc & ipb, uplb, ca mc-54 social garden, uplb uplb mc-55 raymundo subdivision, laila perez los baños, laguna tree genotypes location owner of the tree table 1. different macopa genotypes or trees where fruit samples were obtained, the location, and the owner of the tree influence of changing rainfall patterns on the yield of rambutan 70 tree genotypes location owner of the tree table 1. different macopa genotypes or trees where fruit samples were obtained, the location, and the owner of the tree (cont’d.) mc-56 & mc-57 falklands, csc & ipb (t#1&2) uplb mc-58 & mc-59 magnetic hill, los baños, laguna danilo ubaldo (non-seeded variety) mc-60 to mc-62 san antonio, pila, laguna gil garcia, jose lat & carmelita hernandez mc-63 timugan, los baños, laguna mrs. umali mc-64 masiit, calauan, laguna mercy dreje mc-65 to mc-67 imok, calauan, laguna alberto peñaranda & rosalinda alcantara mc-68 balayhangin, calauan, laguna aurora san gabriel mc-69 pansol, calamba city, laguna johnny amparo mc-70 pulo, indang, cavite delma guevarra mc-71 & mc-72 bagong pook, san jose, batangas lina harina & yoyong harina mc-73 banay-banay ii, san jose, batangas aquilino lizardo mc-74 pulo, indang, cavite analie r. mateo mc-75 sungay east, tagaytay city loraine cumabig mc-76 & mc-77 carasuchi, indang, cavite felix feraer & ester rosela mc-78 mendez, cavite asuncion encarnacion mc-79 & mc-80 san vicente, san pablo city amada ibarra & glicerio perinog mc-81 & mc-83 san mateo, san pablo city cristina reyes & dennis amante mc-82 san antonio, quezon averina vicedo mc-84 san francisso, san pablo city hick cayton mc-85 santa maria, san pablo city maita fajardo mc-86 to mc-88 mahanadyong, taysan, batangas amada ibarra (t#1,3&4) mc-89 bagong kalsada, calamba city, rodolfo jimenez laguna mc-90 to mc-93 magnetic hill, los baños, laguna emma leonzon (t#1-4) mc-94 maahas, los baños, laguna miguel lauang mc-95 & mc-96 san juan, san pablo city billy matandig & concepcion federizo cortez mc-97 & mc-98 padre garcia, batangas (t#1&2) paolo sison mc-99 sto. domingo, bay, laguna mr. ordobesa mc-100 csc & ipb compound uplb mc-101 & mc-102 san isidro, batangas city efren forto mc-103 san pablo nayon, sto. tomas, celia mansit batangas pm magdalita and rb saludes 71 sg-01 to sg-09 bagong pook, san jose, rodel de la peña & lina harina batangas (t#14, 5, 10, 9, 15, 7, 1, 11 & 8) sg-10 sitio paligawan, taysan, batangas lydia lontoc sg-11 & sg-12 paligawan, taysan, batangas eleuterio ramirez (t#1 & 2) sg-13 taysan, batangas nicanor tolentino sg-14 sitio paligawan, taysan, batangas eleuterio ramirez (t#3) sg-15 to sg-17, sto. niño, taysan, batangas ciriaco, lontoc & lydia lontoc sn-21, sg-26 (t#1, 2, 3, 4, 5, 6) & sg-31 sg-18 & sg-19 taysan, batangas (t#1 & 2) art cayap sg-20 & sg-25 pansol, padre garcia, batangas ernesto padilla sg-22, sg 28, mahanadyong, taysan, batangas manny ona, buddy cariño sg-32 & sg-33 & nicanor tolentino sg-23 paligawan, taysan, batangas (t#4) eleuterio ramirez sg-24 national highway, san antonio, municipality of san antonio quezon sg-27 paligawan, taysan, batangas (t#5) eleuterio ramirez sg-29 mataas na lupa, taysan, batangas anadeto evangelo sg-30 namuco rosario, batangas lorna mendoza sg-34 mataas na lugar, taysan, batangas violeta untalan sg-35, sg-36, bacao, taysan, batangas benjamin africa & martin atcheco sg-41 to sg 45 (t#6, 7, 8, 55, 69, 70, 71 & 95) sg-37 & sg-38 malapad na parang, lobo, batangas gabriel biacaro & marcelino manalo sg-39 & sg-40 pinya, taysan, batangas adelaida magtibay & alejandra plata sg-46, sg-48 & lobo ii, batangas (t# 9, 10 & 14) apolinario catipon, benjamin sg-49 africa & gabriel binuro sg-47 bacao, taysan, batangas (t# 9) benjamin africa sg-50 & sg-51 pinatubo, taysan, batangas adelaida magtibay sg-52 & sg-53 sto. niño, taysan, batangas cristina macatangay (t# 69 & 70) sg-54 to sg-87 highway, taysan, batangas remedios azoilo, martin ateneco, (t#13, 16, 19, 51, 66, 25, 20, 22, 24, roger azoilo, rodrigo azoilo & 27, 30, 31, 32, 33, 34, 36, 39, 40, apolonio catipon 41, 26, 43, 46, 48, 52, 54, 56, 58, 59, 60, 63, 64, 65, 68, 69) sg-88 to sg-106 seaside, lobo, batangas reynaldo hernandez, remedios (t# 12, 16, 17, 18, 23, 28, 29, 42, 44, azoilo, martin atcheco & renato 47, 49, 50, 53, 57, 67, 71, 75, 91) catipon sg-107 to sg-114 san miguel, lobo, batangas gina adoyo & apolonio (t# 81, 84, 85, 88, 89, 91, 98) macatangay tree genotypes location owner of the tree table 2. the d ifferent siniguelas genotypes or trees where fruit samples were obtained, their location, and the owner of the tree influence of changing rainfall patterns on the yield of rambutan 72 tree genotypes location owner of the tree table 3. the d ifferent native santol genotypes or trees where fruit samples were obtained, their location, and the owner of the tree sn-01 barangay uno, tanauan, batangas angelito javier sn-02 & sn-03 santor, tanauan, batangas (t# 1&2) myrna bathan sn-04 & sn-05 janopol occidental, tanauan, karen flores batangas (t# 1&2) sn-06 & sn-07 de leon's nursery, talisay, batangas asuncion carandang (t# 1&2) sn-08 to sn-11 janopol, tanauan, batangas (t#1-4) johsua landicho & mauro landicho sn-12 to sn-16 bitin, bay, laguna (t# 1-5) eric bautista sn-17 makban geothermal, bay, laguna cesar alcantara sn-18 to sn-25 puypuy, bay, laguna (t# 1-9) romel punzalan & alex vermi sn-26 to sn-33 riverside, puypuy, bay, laguna raymundo salvador & barak (t#1-8) virgilio sn-34 to sn-39 roadside, puypuy, bay, laguna fe batino, efren pura, jehova (t# 1-6) witnesses church & sof ia paril sn-40 furniture shop, puypuy, bay, laguna jerry peñaranda sn-41 & sn-42 calauan, laguna (t# 1&2) councilor gulay sn-43 to sn-50 san juan, kalayaan, laguna (t# 1-8) gregorio macanilo, pingot lagumloay, may gonzales & wenceslao macalintal sn-51 to sn-55 pagsanjan, laguna (t# 1-5) iglesia ni kristo church, teresita nadal, weny macalintal & joseph quizon sn-56 gatid, sta. cruz, laguna susan maro sn-57 & sn-58 kalayaan, laguna (t# 2&3) ariel wenceslao sn-59 to sn-76 labuin, sta. cruz, laguna (t#1-18) lotlot banoknok, iglesia ni kristo church & irene baltazar sn-77 to sn-84, uplb compound, college, laguna uplb sn-88 & sn-89, (t# 1-17) sn-91, sn-93, sn-95 to sn-99 sn-85 to sn-87, jamboree, los baños, laguna boy scouts of the philippines sn-90, sn-92, (t# 1, 3, 5-10) sn-94, sn-96, sn-101 sn-95 upco (t# 1) uplb sn-100 inclineville, upco uplb pm magdalita and rb saludes 73 twenty to thirty fruit samples were gathered from each tree and taken to the laboratory for analysis. in all three fruit species, fruit weight, fruit length, fruit width, total soluble solids, and percent edible portion were evaluated. fruit weight (g) was determined using a triple beam balance, while fruit length (cm) and width (cm) were measured using a vernier caliper. a hand-held refractometer was used to measure the total soluble solids (tss, ºbrix), while the edible portion (%) was determined by dividing the edible portion by the total fruit weight and the quotient was multiplied by 100. the colors of the skin and flesh were characterized based on the colour chart of the royal horticultural society (rhs) of london (rhs 2007). based on the evaluation of fruit qualities, the best three genotypes were selected and identif ied. the selection of the best genotypes of macopa, siniguelas, and native santol was based on the standard or criteria focusing on fruit qualities and found on the “guidelines for evaluation, selection, and registration of new fruit crop varieties” developed by the fruit crops technical working group (fctwg) of the national seed industry council (fctwg-nsic 2009). in selecting the three best macopa genotypes, the criteria set by the fctwg-nsic (2009) must be satsif ied. according to these criteria, the whole fruit is large (> 50 g); the shape is widely obovoid; the skin or peel color at maturity is either pink, maroon, or red; and the texture is smooth and shiny. the flesh color is creamy white; texture is crispy, smooth, and f irm; flesh aroma is mild; flavor is sweet to sub-acid (> 15°brix) and juicy; while the seed is small (< 1 g) and the fruit is either seedless or has one seed only. in the case of the native santol, the following selection criteria were used: the fruit is either large (>100 g), medium (80-100 g), or small (<80 g); the fruit shape is spheroid; the skin color is yellow to yellow orange; and the skin texture is smooth. pulp is either thick (> 6 mm) or intermediate (4-6 mm); texture is f ine and smooth and pulp flavor is sub-acid; the flavor of the aril is sub-acid to sweet (>18°brix); color is cottony white, juicy, thick (> 5 mm); and has none to scanty f ibers. the seed is small (<2.5 g) and the percent edible portion is high (> 40%). on the other hand, in selecting the best three genotypes of siniguelas, the following criteria were used: the whole fruit is big (> 15 g), the shape is oblong, the skin or peel color at maturity is dark brown, and the texture is smooth and shiny. in addition, the flesh color is yellow orange, texture is smooth and f irm, no f ibers, flavor is sweet to sub-acid (> 10ºbrix) and juicy, while the seed is small (< 3 g). the mean, range standard deviation, and shannon-weaver diversity index (h') were taken for all fruit characters evaluated. the shannon-weaver diversity index was influence of changing rainfall patterns on the yield of rambutan 74 used to estimate phenotypic diversity and guide to selecting the best genotypes. it was computed for the different characters using the method of tolbert and others (1979) given below. where pi = frequency of each descriptor state, n = the number of states per descriptor. the formula gives h' within 0-1.0 and the value nearest to 1.0 is most diverse. for the shannon-weaver diversity index, a value of 0.80-1.0 is arbitrarily considered high to indicate a wide variability, while a value of 0.20-0.79 is considered low, indicating narrow variability. results and discussion effect of rainfall on rambutan in calauan, laguna rambutan thrives well in tropical and humid regions with high rainfall over a fairly long season. the dry season should not last much over three months. it can live from sea-level to 500 m or even up to 600 m. it is also very sensitive to water stress, but it cannot withstand poor drainage condition. in southeast asia, rambutan flowers normally appear shortly after the dry season when the climate is highly influenced by monsoon. in the philippines, rambutan flowering occurs from late march to early may and the fruits mature from july to october or occasionally to november (morton 1987). a dry month is def ined as one with a total rainfall of less than 50 mm (pagasa 2010). as shown in figure 1, the climate of los baños and of nearby municipalities have two distinct seasons similar to a type i climate (i.e. , dry from january to april and wet during the rest of the year) (pagasa 2010). majority of rambutan production in the philippines are found in areas with distinct wet and dry seasons (type i and type iii) including laguna. it has also fruited successfully in areas with a pronounced dry season (coronel 1998). figure 2 shows the annual rambutan yield from 2001 to 2012 in calauan and the amount of rainfall for april 2001 to 2012. the yield of rambutan remarkably dropped from 152.2 kg/tree in 2008 to 8.6 kg/tree in 2009. analysis of monthly rainfall data from 2001 to 2012 revealed that the flowering of rambutan could have been affected by the abnormally high rainfall at 334.4 mm observed for the h’ = ∑pi(log2pi)/log2n pm magdalita and rb saludes 75 month of april 2009 due to the la niña phenomenon. furthermore, other environmental factors including temperature, relative humidity, solar radiation, and wind contributed to the decrease in rambutan yield. in this month, a moderate negative correlation (r=-0.64) between rambutan yield with rainfall was detected (table 5). this negative correlation suggests that while there is an increase in the amount of rainfall, there is a corresponding decrease in the amount of yield of rambutan. the month of april is usually considered a dry month with a normal rainfall of 41.6 mm from 1971 to 2000 (figure 2). however, in this case, the reverse happened where there is high rainfall in april (figure 2). in addition, the yield also decreased from 152 kg/tree in 2008 to 9 kg/tree in 2009 (table 5). in relation to this, there is a moderate negative correlation (r=-0.51) between yield and amount of rainfall (table 5). this negative moderate correlation also partially implies that while the amount of rainfall is increased, the yield of rambutan decreased. this is shown in the data where in march 2009 the amount of rainfall increased to 127.3 mm (table 4) when, in fact, march should be a dry month instead, which is a typical effect of climate change. in addition, other weather factors like temperature, relative humidity, and solar radiation may also have affected flowering of rambutan. corollary to this, it has been reported that excessive rainfall before the expected flowering of rambutan is detrimental since it promotes vegetative growth rather than flowering (tindall and others 1994). figure 1. normal rainfall (mm) from 1971 to 2000 and monthly rainfall in 2009 at the university of the philippines los baños national agromet station, college, laguna, philippines. normal rainfall is the average of the rainfall values over a thirty-year period and rainfall may very often be either well-above or well-below the seasonal average or normal (williams 2008). each bar corresponds for the month from 1971 to 2000 and for 2009. influence of changing rainfall patterns on the yield of rambutan 76 figure 2. amount of rainfall (mm) for april 2001 to 2012 and the annual rambutan yield in calauan, laguna, philippines. each bar represents the amount of rainfall for april of each year. jan feb mar apr may jun jul aug sep oct nov dec 2001 7.1* 132.9** 43.6* 57.9** 191.0** 243.6** 252.7** 278.8** 127.5** 168.6** 261.0** 187.4** 2002 16.4* 11.8* 16.1* 6.8* 131.6** 88.6** 621.6** 214.7** 166.0** 188.9** 173.8** 80.9** 2003 7.8* 3.2* 14.5* 19.9* 350.4** 131.1** 356.0** 180.1** 189.5** 87.4** 226.2** 27.2* 2004 19.0* 51.2** 4.3* 11* 128.9** 249.0** 409.2** 236.5** 114.4** 152.1** 342.6** 60.1** 2005 16.6* 17.4* 45.0* 65.5** 61.20** 79.5** 110.8** 192.3** 298.3** 242.4** 118.6** 397.0** 2006 140.5** 39.6* 54.6 0.7* 143.8** 243.7** 285.9** 208.8** 637.1** 80.6** 198.5** 265.3** 2007 38.1* 15.0* 47.7* 6.4* 69.5** 75.4** 228.3** 469.1** 161.0** 226.6** 451.1** 199.2** 2008 164.5** 62.5** 4.7* 54.1** 211.8** 319.3** 165.9** 238.8** 149.6** 174.9** 162.1** 184.0** 2009 136.6** 56.4** 127.3** 334.4** 233.2** 299.7** 377.0** 178.1** 502.9** 389.4** 171.2** 23.4* 2010 15.3* 3.0* 26.2* 56.2** 9.1* 171.2** 762.5** 211.3** 124.3** 368.7** 159.8** 170.0** 2011 49.7* 6.1* 67.9** 11.4* 143.2** 476.1** 302.6** 294.4** 242.4** 275.6** 290.6** 254.1** 2012 81.6** 86.5** 116.4** 40.0* 178.5** 72.3** 464.4** 486.1** 136.5** 337.2** 76.7** 340.0** table 4. amount of monthly rainfall (mm) from 2001 to 2012 showing the critical amount during dry (*) and wet (**) months prior to and at the start of flowering of rambutan * dry month ** wet month pm magdalita and rb saludes 77 furthermore, wet months were observed during 2009 and this further inhibited the flowering of rambutan trees (table 4). according to kawabata and others (2007), excessive rainfall during the expected flowering season is detrimental to rambutan flowering and enhances vegetative growth. instead, in order for rambutan to flower, it should be naturally induced by water stress for 2 to 4 weeks period with a temperature greater than 22°c (nakasone and paull 1998; salakpetch 2005; tindall and others 1994). however, for 2009, there was a high amount of rainfall for march (127.3 mm) and april (334.4 mm) which coincided with the flowering season of rambutan. this could be one of the factors that induced vegetative growth of the trees instead of being reproductive. in addition, a lower yield of rambutan was also observed in 2010 at 45.5 kg/tree (figure 2). this could be partly explained by the abnormally reduced rainfall at 9.1 mm for the month of may 2010 (table 4). the month of may is considered a wet month with a normal rainfall of 159.7 mm from 1971 to 2000 (figure 3). however, an extended water stress period in march and may (table 4) could have inhibited the flower initiation and flower bud growth, hence a low yield. in addition, high solar radiation, strong wind, and high humidity are contributory factors to this low rambutan yield. in rice farming, for example, it has been reported that increases in table 5. the yield of rambutan in calauan, laguna from 2001 to 2012 and the monthly rainfall from january to december (2001-2012). the correlation of yield and rainfall is also shown. jan feb mar apr may jun jul aug sept oct nov dec 2001 91 7.1 132.9 43.6 57.5 191 243.6 252.7 278.8 127.5 168.6 261 187.4 2002 82 16.4 11.8 16.1 6.8 131.6 88.6 621.6 214.7 166 188.9 173.8 80.9 2003 65 7.8 3.2 14.5 19.9 350.4 131.1 356 180.1 189.5 87.4 226.2 27.2 2004 100 19 51.2 4.3 11 128.9 249 409.2 236.5 114.4 152.1 342.6 60.1 2005 75 16.6 17.4 45 65.5 61.2 79.5 110.8 192.3 298.3 242.4 118.6 397 2006 92 140.5 39.6 54.6 0.7 143.8 243.7 285.9 208.8 637.1 80.6 198.5 265.3 2007 102 38.1 15 47.7 6.4 63.5 75.4 228.3 469.1 161 226.8 451.1 199.2 2008 152 164.5 62.5 4.7 54.1 211.8 319.3 165.9 238.8 149.6 174.9 162.1 184 2009 9 136.6 56.4 127.3 334.4 233.2 299.7 377 178.1 502.9 389.4 171.2 23.4 2010 45 15.3 3 26.2 56.2 9.1 171.2 762.5 211.3 124.3 368.7 159.8 170 2011 74 49.7 6.1 67.9 11.4 143.2 476.1 302.6 294.4 242.4 275.6 290.6 254.1 2012 94 81.6 86.5 116.4 40 178.5 72.3 464.4 486.1 136.5 337.2 76.7 68.1 mean 57.8 40.5 47.4 55.3 153.9 204.1 361.4 265.8 237.5 224.4 219.3 159.7 r 0.18 0.26 -0.51 -0.64 -0.02 -0.01 -0.41 0.35 -0.37 -0.54 0.18 0.24 r= pearson r value or correlation coefficient 0-0.3 (0 to -0.30) = little correlation 0.31-0.5 (-0.31 to -0.5) = low correlation 0.51-0.7 (-0.51 to -0.7) = moderate correlation 0.91-1.0 (-0.91 to -1.0) = very high correlation 0.71-0.9 (-0.71 to -0.9) = high correlation monthly rainfall (mm)year yield (kg/ tree) influence of changing rainfall patterns on the yield of rambutan 78 figure 3. amount of rainfall (mm) for may 2001 to 2012 and the annual rambutan yield in calauan, laguna, philippines minimum temperature causing water stress in los baños and other areas in the philippines led to a reduced rice yield (ipcc 2007; lansigan 2009). selection of genotypes of known-drought tolerant macopa, siniguelas, and native santol macopa the mean, range, standard deviation, and shannon-weaver diversity index of different fruit characters such as fruit weight, fruit length, fruit width, total soluble solids, flesh thickness, seed number, seed weight, and edible portion for 103 macopa genotypes evaluated are shown in table 6. a wide variability was observed as indicated by the shannon-weaver diversity index for fruit weight (h'=0.87), total soluble solids (h'=0.86), and flesh thickness (h'=0.87). the same degree of variability was obtained for fruit weight and fruit length in rambutan (magdalita and valencia 2004). the wide variability in the present study suggests that a mass selection strategy for desirable tree genotypes of macopa could be done effectively in the natural population. the differences in phenotypic characteristics of the different genotypes evaluated are shown in figure 4. the fruit weight of 103 pm magdalita and rb saludes 79 macopa genotypes ranged from 6.70 to 78.15 g with a mean of 28.25 g, while the total soluble solids ranged from 0 to 13.66 °brix with a mean of 5.27 °brix. flesh thickness ranged from 0.10 to 1.89 cm with a mean of 1.01 cm, while fruit length ranged from 2.01 to 9.05 cm with a mean of 3.77 cm. in contrast, the variability is narrow as indicated by the shannon-weaver diversity index for fruit width (h'=0.29), seed weight (h'=0.69), seed number (h'=0.79), and percentage of edible portion (h'=0.67). this narrow variability is expected because of the distinct varietal characteristics of each individual macopa trees evaluated. figure 4. the differences in phenotypic characteristics of different macopa genotypes evaluated for fruit qualities table 6. the mean, range, standard deviation, and shannon-weaver d iversity index (h') of selected fruit characters in 103 tree genotypes of macopa fruit characters mean range sd h'* fruit weight (g) 28.25 6.70-78.15 11.95 0.87 fruit length (cm) 3.77 2.01-9.05 0.80 0.80 fruit width (cm) 4.60 2.25-43.01 3.94 0.29 total soluble solids (obrix) 5.27 0.00-13.66 3.03 0.86 flesh thickness (cm) 1.01 0.10-1.89 0.44 0.87 seed number 0.59 0.00-3.05 0.50 0.79 seed weight (g) 1.62 0.00-13.53 2.11 0.69 edible portion (%) 92.87 55.96-100.00 9.08 0.67 obrix degree brix sd standard deviation h' shannon-weaver diversity index *nearest to 1.0 means most diverse influence of changing rainfall patterns on the yield of rambutan 80 while fruit crops are generally eaten fresh once they are ripe, the most important general criterion for selecting the best three genotypes in each fruit species is primarily centered on the most desirable qualitative traits like sweet taste and flavor of the flesh, flesh color, texture of the flesh, and edible portion or the fleshly part. these traits were given higher weights during the selection process and scored accordingly based on the criteria set by the fctwg-nsic (2009) because fruits are eaten fresh so taste is the primary consideration rather than using analyzed quantitative data which are of secondary importance. in the case of macopa, based on the shannon-weaver diversity index, and fruit qualities particularly flavor of the flesh, crunchiness and texture of the flesh, skin color, and edible portion, three genotypes of macopa out of 103 trees evaluated were selected, namely: mc-13, mc-43 and mc-95 (table 7). mc-13 is sweet (7.15°brix) and crispy, creamy white (rhcc 155 a) with fruits weighing 49.44 g, and has high edible portion (93.22%) (figure 5-a). mc-43 is a currant red (rhcc 46 a) selection with big fruits (42.46 g), crispy and sweet (6.83°brix), and has very high edible portion (94.32%) (figure 5-b). mc-91 is a pea green (rhcc 149 b) selection with medium-size fruits (32.14 g), crispy and sweet (7.69%), and has high edible portion (92.41%) (figure 5-c). the tree of mc-13, mc-45, and mc-91 is hardy; a very prolif ic and regular bearer; and can stand both drought and flooding, and those places with less water and erratic rainfall. they can be also used for agro-reforestation in marginal areas. siniguelas table 8 shows the mean, range, standard deviation, and shannon-weaver diversity index of different fruit characters such as fruit weight, fruit length, fruit width, flesh thickness, total soluble solids, individual seed weight, seed length, seed width, seed thickness and edible portion of 114 siniguelas genotypes evaluated. the shannon-weaver diversity index indicated that a wide variability existed for fruit weight (h'=0.85), fruit length (h'=0.85), fruit width (h'=0.90), flesh thickness (h'=0.88), total soluble solids (h'=0.90), individual seed weight (h'=0.87) and percent edible portion (h'=0.88) (table 8). this wide variability is not expected for siniguelas since it is an asexually propagated crop, usually by stem cuttings, hence, uniformity is expected. however, this wide variability could be due to somatic mutations that occurred within the mother plant, and when stem cuttings were taken and grown into another tree, the variation was expressed. in sweetsop, a similarly high degree of variability was observed for fruit weight, fruit length, fruit width, flesh thickness, total soluble solids and percent edible portion (magdalita and valencia 2004). since, there is a wide variability observed for important traits like fruit weight, total soluble solids, and edible portion in the present study, this pm magdalita and rb saludes 81 suggests that on-site selection, a strategy similar to mass selection process for desirable tree genotypes of siniguelas can be done in the natural population to identify superior siniguelas genotypes. the phenotypic characteristics of the different siniguelas genotypes evaluated are shown in figure 6. the fruit weight of 114 siniguelas ranged from 11.03 to 30.88 g with a mean of 16.88 g, while the total soluble solids ranged from 6.55 to 17.40°brix with a mean of 11.56°brix (table 8). flesh thickness ranged from 0.38 to 0.89 cm with a mean of 0.61 cm, while the individual seed weight ranged from 2.50 to 3.77 g with a mean of 3.38 g. the fruit length ranged from 2.50 to 3.77 cm with a mean of 3.26 cm, while the fruit width ranged from 2.42 to 3.26 cm with a mean of 2.82 cm. in contrast, the shannon-weaver diversity index indicated a narrow variability for seed length (h'=0.30), seed width (h'=0.72), and seed thickness (h'=0.66). this narrow variability indicates that the seed characteristics of siniguelas are distinct to this species. siniguelas is another fresh fruit that is popularly eaten during summer because of its sweet flavor, its shiny skin, and fleshy part. in selecting the three best genotypes of siniguelas, the qualitative traits that were given more emphasis in selection and provided with more weight were flavor or taste, skin color, texture, and shiny skin and fleshy part. among the 114 different siniguelas trees evaluated based on diversity index and fruit qualities like flavor of the flesh, skin color and texture, shininess of the skin, and edible part, three genotypes of siniguelas, namely sg-41, sg-42, and sg-105, were identif ied as promising genotypes. sg-41 is sweet table 7. the average fruit qual ities of selected tree genotypes of macopa, siniguelas, and native santol no. of genotypes evaluated fruit species selected genotypes fruit weight (g) fruit length (cm) fruit width (cm) total soluble sol ids (0brix) ed ible portion (%) trait for cl imate change adaptation macopa 103 mc-13 49.44 4.25 5.41 7.15 93.22 droughtmc-43 42.46 3.69 5.33 6.83 94.32 tolerant mc91 32.14 4.57 4.38 7.69 92.41 siniguelas 114 sg-41 20.42 3.30 2.87 12.50 83.27 droughtsg-42 19.67 3.58 3.11 12.75 81.73 tolerant sg-105 16.05 3.26 2.79 16.79 82.13 native 101 sn-47 105.43 5.18 5.84 4.76 86.44 droughtsantol sn-59 58.27 3.91 4.99 5.56 84.99 tolerant sn-74 51.96 4.00 4.69 4.17 82.20 influence of changing rainfall patterns on the yield of rambutan 82 figure 5. the selected genotypes of macopa (mc-13) [a], (mc-43) [b], (mc-91) [c]; siniguelas (sg-41) [d], (sg-42) [e], (sg-105) [f]; and native santol (sn-47) [g], (sn59) [h], (sn-74) [i] pm magdalita and rb saludes 83 fruit weight (g) 16.88 11.03-30.88 2.99 0.85 fruit length (cm) 3.26 2.50-3.77 0.18 0.85 fruit width (cm) 2.82 2.42-3.26 0.17 0.90 flesh thickness (cm) 0.61 0.38-0.89 0.09 0.88 total soluble solids (obrix) 11.56 6.55-17.40 2.39 0.90 individual seed weight (g) 3.38 1.56-4.98 0.70 0.87 seed length (cm) 2.30 1.62-10.00 0.80 0.30 seed width (cm) 1.60 1.43-2.57 0.12 0.72 seed thickness (cm) 1.47 1.31-2.35 0.12 0.66 edible portion (%) 78.77 61.86-91.58 5.96 0.88 (12.50°brix), ruby red (rhcc 59 a), and has shiny and smooth skin, and the fruit weighs an average of 20.42 g, and has high edible portion of 83.27% (figure 9-d). sg42 is also ruby red (rhcc 59 a) with a shiny and smooth skin, has an average weight of 19.67 g, has sweet flesh with a tss of 12.75°brix and has high edible portion of 81.73%. sg-105 has grenadine red (rhcc n34 a) fruits with shiny and smooth skin weighing 16.05 g that are very sweet with 16.79°brix and has high edible portion of 82.13%. the tree of sg-41, sg-42, and sg-105 is very strong, hardy, prolif ic, regular bearer, and can withstand drought. this suggests that these siniguelas selections are ideal for growing in drought-prone areas and those places with less water and erratic rainfall. they can be also used for planting in marginal areas and for agro-reforestation in mined and less fertile areas. figure 6. the differences in phenotypic characteristics of different siniguelas genotypes evaluated table 8. the mean, range, standard deviation, and shannon-weaver d iversity index (h') of some fruit characters evaluated in 114 genotypes of siniguelas fruit characters mean range sd h’* obrix – degree brix sd – standard deviation h’ shannon-weaver diversity index *nearest to 1.0 means most diverse influence of changing rainfall patterns on the yield of rambutan 84 figure 7. the differences in phenotypic characteristics of some native santol genotypes evaluated native santol the mean, range, standard deviation, and shannon-weaver diversity index of different fruit characters such as fruit weight, fruit length, fruit width, pulp thickness, aril thickness, total number of seeds, total seed weight, skin weight, seed weight, seed length, seed width, seed thickness, total soluble solids, and percent edible portion of 101 santol genotypes evaluated are shown in table 9. a wide variability indicated by the shannon-weaver diversity index existed for fruit weight (h'=0.87), fruit length (h' = 0.89), fruit width (h=0.89), aril thickness (0.90), total seed weight (h' = 0.89), skin weight (h'=0.87), seed width (h=0.87), seed thickness (h'=0.85), total soluble solids (h'=0.85), and percent edible portion (h'=0.85) (table 8). similarly, a high degree of variability was observed for fruit weight, seed weight, total soluble solids, and percent edible portion in rambutan (magdalita and valencia 2004), papaya (magdalita and others 1984), and cashew (ramadas and thatham 1982). this wide variability observed for important traits in the present study also suggests that desirable tree genotypes of native santol could be selected effectively in the natural population. the fruit phenotypic characteristics of the different genotypes evaluated are shown in figure 7. fruit weight of 101 native santol pm magdalita and rb saludes 85 ranged from 49.18 to 151.49 g with a mean of 86.48 g, while total soluble solids ranged from 1.11 to 5.56°brix with a mean of 2.28°brix. fruit length ranged from 3.80 to 6.0 cm with a mean of 4.72 cm while fruit width ranged from 4.60 to 6.89 cm with a mean of 5.56 cm. aril thickness ranged from 0.18 to 0.66 cm with a mean of 0.40 cm, while the total seed weight ranged from 4.40 to 15.20 cm. the weight of the skin ranged from 11.73 to 48.37 g with a mean of 24.16 g. seed width ranged from 2.29 to 1.71 cm with a mean of 1.48 cm, while seed thickness ranged from 0.96 to 1.60 cm with a mean of 1.16 cm. the total soluble solids, a relative measure of sweetness ranged from 1.11 to 5.56 with a mean of 2.28°brix, while the percent edible portion ranged from 26.81 to 86.44% with a mean of 58.56%. contrastingly, a narrow variability as indicated by the shannon-weaver diversity index was observed for pulp thickness (h'=0.73), total number of seeds (h'=0.73), and seed length (h'=0.75) (table 9). this narrow variability for pulp thickness is unique for native santol since many native santol genotypes have relatively thin pulp. the narrow variability for total number of seeds and seed length are distinct traits to native santol, as an indicator trait of many native santol genotypes. in selecting the best three native santol genotypes, the traits that were given emphasis in selection included pulp and aril flavor, fleshy part, and pulp thickness that were all scored accordingly based on the criteria of fctwg-nsic (2009). among the 101 different native santol trees evaluated, based on the shannonweaver diversity index, and fruit qualities particularly flavor of the pulp and aril, three genotypes of native santol namely: sn-47, sn-59, and sn-74 were identif ied as promising genotypes. sn-47 is sweet (4.76°brix), has lemon yellow (rhcc 13 a) fruit with an average weight of 105.43 g, and an edible portion of 86.44%. on the other hand, sn-59 has maize yellow (rhcc 21 b) fruit that weighs 58.77 g, with tss of 5.56 °brix and percent edible portion of 84.99%, while sn-74 has butter cup yellow (rhcc 168 b) fruits that weigh 51.96 g, with tss of 4.17°brix and percent edible portion of 82.20%. the tree of sn-47, sn-59, and sn-74 is a very prolif ic, deep rooted, regular bearer, very hardy, and can withstand drought. this suggests that these santol selections can be grown in droughtand flood-prone areas, and those areas with limiting water and erratic rainfall. like any other hardy trees, native santol can be also used as border trees in windy areas and typhoonprone areas to protect any crop susceptible to wind damage. in general, the selections of macopa, siniguelas, and native santol can be used for agro-reforestation of marginal areas and denuded forests since they are all hardy, have strong root systems, and are fast growers. they can be also planted in places that are frequently visited by typhoons. in addition, the selections of native santol influence of changing rainfall patterns on the yield of rambutan 86 can be also grown in flood-prone areas since they are also tolerant to excessive water. like other trees, these selections, besides being used as sources of food, also have environmental benef its for mitigating the ill effects of climate change. for instance, they can improve air quality, prevent soil erosion, improve soil quality, increase biodiversity, and provide aesthetic value to the natural landscape (putz and pinard 1993). in addition, these tree selections can be the best bet for storing carbon at very little cost and high return (dixon and others 1994). table 9. the mean, range, standard deviation, and shannon-weaver d iversity index (h') of some fruit characters evaluated in 101 genotypes of siniguelas fruit weight (g) 86.48 49.18-151.49 21.41 0.87 fruit length (cm) 4.72 3.80-6.04 0.47 0.89 fruit width (cm) 5.56 4.60-6.89 0.50 0.89 pulp thickness (cm) 0.32 0.11-0.93 0.15 0.73 aril thickness (cm) 0.40 0.18-0.66 0.10 0.90 total number of seeds 4.09 2.90-10.17 0.77 0.73 total seed weight (g) 10.87 4.40-16.20 2.20 0.89 skin weight (g) 24.16 11.73-48.37 7.60 0.87 seed length (cm) 2.61 2.19-4.60 0.28 0.75 seed width (cm) 1.48 1.29-1.71 0.08 0.87 seed thickness (cm) 1.16 0.96-1.60 0.09 0.85 total soluble solids 2.28 1.11-5.56 0.87 0.85 (obrix) edible portion (%) 58.56 26.81-86.44 8.10 0.85 obrix-degree brix sd standard deviation h' shannon-weaver diversity index *nearest to 1.0 means most diverse fruit characters mean range sd h’* conclusion and recommendation the fruit yield of rambutan trees growing in a selected farm in calauan, laguna, philippines, remarkably dropped from 152.2 kg/tree in 2008 to 8.6 kg/tree in 2009. this decline in yield could be traced back from the abnormal flowering of rambutan. this could be due to the effect of the abnormally high rainfall observed for the month of april 2009 at 334.4 mm, based on the analysis of monthly data on rainfall from 2001 to 2012. other environmental factors like temperature, solar radiation, humidity, and wind contributed to this yield decline. the month of april is usually considered a dry month with a normal rainfall of 41.6 mm that was observed from 1971 to 2000. in addition, no dry months were observed during 2009 which promoted the vegetative growth of rambutan instead of flowering. also, a lower yield was recorded at 45.5 kg/tree in 2010. this can be explained by an abnormally pm magdalita and rb saludes 87 reduced rainfall at only 9.1 mm for may 2010 which could be inadequate for the flowering of rambutan, hence the yield decreased. usually, the month of may is considered a wet month with a normal rainfall at 159.7 mm based on rainfall data from 1971 to 2000. preliminary evaluation and selection among 103 macopa genotypes resulted in selection of three most promising selections, namely: mc-13, mc-43, and mc-91. however, a follow-up study is needed to ascertain the performance of these selections in the face of climate change. mc-13 is sweet (7.15 °brix) and crispy, creamy white (rhcc 155 a) and has fruit weighing an average of 49.44 g, with high edible portion (93.22%) while mc-43 is a currant red (rhcc 46 a) selection with fruits weighing 42.46 g, with very high edible portion (94.32%). mc-91 is a pea green (rhcc 59 a) selection with medium fruits (32.14 g), crispy and sweet (7.69 °brix) and has high edible portion (92.41%). among 114 siniguelas genotypes evaluated, three genotypes namely: sg-41, sg-42 and sg-105 were selected. sg-41 has sweet (12.50 °brix) and juicy fruits weighing an average of 20.42 g, ruby red (rhcc 59 a), and has high edible portion (83.27%) while sg-42 has fruits weighing an average of 19.67 g, ruby red (rhcc 59 a), juicy and sweet (12.75 °brix) with high edible portion (81.73%). sg-105 has medium fruits (16.05 g) that are grenadine red (rhcc n34 a), very sweet (16.79 °brix) and has high edible portion (82.13%). among 101 native santol genotypes evaluated, sn-47, sn-59 and sn-74 were selected. sn-47 has sweet (4.76b °brix) and juicy fruits weighing an average of 105.43 g, lemon yellow (rhcc 13 a), and has high edible portion (86.44%) while sn-59 has medium (58.27 g) fruits that are maize yellow (rhcc 21 b), juicy and sweet (5.56 °brix) fruits with high edible portion (84.99%). sn-74 has also medium fruits (51.96 g) that are butter cup yellow (rhcc 16 b), sweet (4.17 °brix) with high edible portion (82.20%). the selections of macopa including mc-13, mc-43, and mc-91; siniguelas such as sg-41, sg-42, and sg-105; and native santol like sn-47, sn-59, and sn-74 are being recommended for planting in marginal, droughtand flood-prone areas around the country. it is further recommended that grafted materials should be used for planting to maintain the true-to-type nature of the selections. acknowledgments the project entitled, “selection/breeding known drought-resistant, pest/disease resistant, and flood-tolerant species for climate change adaptation” on which this article is based is being funded by the department of agriculture bureau of agricultural research (da-bar). the authors also acknowledge mr. mario tenorio, influence of changing rainfall patterns on the yield of rambutan 88 chief of the sta. cruz nursery, calauan, laguna, for providing the yield data on rambutan, the farmer-cooperators in batangas, laguna, and cavite who provided the fruit samples for evaluation, mr. marcelino t. gregorio, mr. jessie v. silverio, mr. albert dichoso, and ms. maria fe h. cayaban for the various assistance extended to the authors, and the anonymous reviewers for the useful comments and constructive criticism. references coronel re. 1998. promising fruits of the philippines. laguna, philippines:uplbcollege of agriculture. coronel re. 2011. important and underutilized edible fruits of the philippines. diliman, q u e z o n c i t y, p h i l i p p i n e s : u p l b f o u n d a t i o n i n c . 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[internet]. fao plant production and protection paper 121. rome, italy; [cited 2013 mar 10]. available from: http: //www.booksgoogle.com.ph/books?ishn=925 103250. tolber t dm, qualset co, jain sk, craddock jc. 1979. a diversity analysis of a world collection of barley. crop sci. 19(17):789-94. influence of changing rainfall patterns on the yield of rambutan 90 williams j. 2008. understanding weather normals. national disaster and management council drought indices, usa today p 1 [internet]. [cited 2013 mar 10]. available from: http: //www.usatoday30usatoday.com/weather/whermals.htm. _____________ pabl ito m. magdal ita <pabsmagdalita@gmail.com> is professor i and up scientist i at the crop science cluster and institute of plant breeding, college of agriculture, uplb. he obtained his ph.d. at the university of queensland (uq), brisbane, australia and currently teaches agronomy, crop science and biotechnology courses, and has been conducting breeding researches on fruit and ornamental crops for 33 years. he published or co-published 28 isi articles, 25 of which received the international publication award, 22 non-isi, two book chapters and presented or co-presented 44 papers and 40 posters in national and international conferences. ronaldo b. saludes is an associate professor ii at the institute of agricultural engineering, college of engineering and agro-industrial technology. he obtained his ph.d. at tokyo university of agriculture and technology (tuat), japan. he has been teaching agrometeorology, geometeorology, and waste management for 17 years. he published six peer-reviewed articles, three of which in isi-indexed journals. 2editor's note-jan-june2017.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online irene m. v illaseñor, ph.d. editor-in-chief welcome to the f irst issue of science diliman for 2017! let me introduce the research articles in this issue. the journal cover features the work done by authors pascual and guevara on the development of automated reading tutors (art) in filipino language to improve reading literacy. the paper presents the positive effects of art on the oral reading fluency and comprehension of a small group of children during pilot testing. another paper by mathematicians monterde and paras characterized “square matrices which can be expressed as a sum of strictly k-zero matrices”. in their paper on quantum communication, physicists dumigpe and galapon concluded that they have achieved “nearly perfect to perfect state transfer at an earlier time as the spin quantum number of the channel goes higher.” another group of physicists mentored by salvador and somintac controlled the geometry of the nanowires produced using metal-assisted chemical etching by changing the concentrations of etchants and etching times. a group of marine scientists supervised by montaño determined the presence of quorum sensing inhibitors with potential anti-biof ilm/biofouling applications in philippine seaweeds and surface-associated microorganisms. layman’s abstracts are also included for the better appreciation of the results of the research included in this issue. 9guidelines.pmd 106 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) ssd-sample article s c i e n c e d i l i m a n : a p h i l i p p i n e j o u r n a l o f p u r e a n d a p p l i e d sciences has been declared by the commission on higher education (ched) as a category a-2 journal for 2014 to 2016 through its journal accreditation service project. 3santos-coffee varieties.pmd d.m. c. santos et al. 5 science diliman (january-june 2016) 28:1, 5-16 simple sequence repeat analysis of selected nsic-registered coffee varieties in the phil ippines daisy may c. santos* university of the philippines diliman carla francesca f. besa university of the philippines diliman angelo joshua a. v ictoria university of the philippines diliman ernelea p. cao university of the philippines diliman abstract c o f f e e (coffea s p . ) i s a n i m p o r t a n t c o m m e r c i a l c r o p w o r l d w i d e . t h r e e species of coffee are used as beverage, namely coffea arabica, c. canephora, and c. liberica. coffea arabica l. is the most cultivated among the three coffee species due to its taste quality, rich aroma, and low caffeine content. despite its inferior taste and aroma, c. canephora pierre ex a . froehner, w h i c h h a s t h e h i g h e s t c a f f e i n e c o n t e n t , i s t h e s e c o n d m o s t w i d e l y cultivated because of its resistance to coffee diseases. on the other hand, c. l iberica w.bull ex hierncomes is characterized by its very strong taste and flavor. the philippines used to be a leading expor ter of coffee until coffee rust destroyed the farms in batangas, home of the famous kapeng barako. the country has been attempting to revive the coffee industry by focusing on the production of specialty coffee with registered varieties on the national seed industry council (nsic). correct identif ication and i s o l a t i o n o f p u r e c o f f e e b e a n s a r e t h e m a i n f a c t o r s t h a t d e t e r m i n e cof fee’s m a r ke t v a l u e . lo c a l f a r m s u s u a l l y m i s i d e n t i f y a n d m i x cof fee beans of different varieties, leading to the depreciation of their value. this study used simple sequence repeat (ssr) markers to evaluate and distinguish philippine nsic-registered coffee species and varieties. the _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online s i m p l e s eq u e n ce re p e a t a n a l y s i s of s e l ec ted n s i cr eg i s tered coffee 6 n e i g h b o r j o i n i n g t r e e g e n e r a t e d u s i n g pa u p s h o w ed h i g h bootstrap support, separating c. arabica, c. canephora, and c. liberica from each other. among the twenty primer pairs used, seven were able to distinguish c. arabica, nine for c. liberica, and one for c. canephora. keyword s: coffea, nsic-registered varieties, ssr introduction coffee is an economically important crop in the global market. it belongs to the caffeine-containing subgenus coffea from the family rubiaceae, which comprises over a hundred species originating from the african region (charrier and berthaud 1985). among the coffee species used for commercial consumption, c. arabica l. is the most cultivated, accounting for 70% of the global coffee production. it is the only allotetraploid in the genus and is self-pollinating. this species also has the highest market value (tornincasa et al. 2010) because of its low caffeine content, excellent taste, and aroma (vidal et al. 2010; vieira et al. 2010). the species c. canephora pierre ex a. froehner is second to c. arabica in terms of production, contributing the remaining 30% of global coffee production. it has certain advantages in terms of production due its high-yielding properties and tolerance to diseases. however, its taste, which is characterized as woody bitter and of high caffeine content, is inferior to c. arabica (reyes 2010). most studies report that only c. arabica and c. canephora are cultivated for commercial consumption. the philippines is one of the few countries that commercially produce, in addition to c. arabica and c. canephora, varieties of the species c. liberica w. bull ex hierncomes. the liberica variety, c. liberica var. liberica, was an economically important commodity during the 1930s. locally known as the kapeng barako, it is distinguished for its strong, woody, and bitter taste, acidic aftertaste, and pungent aroma. apart from its strong taste, this variety also possesses desirable reproductive characteristics in terms of fruit clusters, bean size (the largest among the four varieties), and low caffeine content (n’diaye et al. 2005). coffea liberica var. dewevrei, commonly known as excelsa coffee, has a woody taste, and sweet, fruity aroma (reyes 2010). the identity and purity of the coffee produce determine its market value. owing to the economic importance of coffee, it is of interest to assess its genetic diversity d.m. c. santos et al. 7 and to come up with markers that will identify and distinguish species, as well as varieties within a species. since morphological methods are sometimes not reliable in differentiating coffee species and varieties, molecular techniques are being used and developed to address this concern. the cbol (consortium for the barcoding of life) plant working group has recommended two universal plant barcodes for species identif ication, namely the matk and rbcl genes (janzen 2009). these two genes have been used in verifying the identities of the coffee species in the farms located in cavite, philippines. the said genes were able to distinguish among the species c. arabica, c. canephora, and c. liberica. however, the varieties c. liberica var. liberica and c. liberica var. dewevrei were not successfully differentiated and clustered together in a single clade (cao et al. 2014). the matk and rbcl markers could discriminate between species but not varieties within species. microsatellite or simple sequence repeat (ssr) markers are short, tandem repeats present in the coding and non-coding portions of the genome (wang et al. 2009). ssrs require only a small amount of dna for polymerase chain reaction (pcr)-based screening and can reveal multiple alleles at a single locus. automated allele detection and sizing are also readily available (schlotterer et al. 2000). the abundance and highly polymorphic property of ssrs make it a good marker for plant genetic studies, identif ication of cultivars, and evaluation of varieties with a narrow genetic base (vieira et al. 2010; wang et al. 2009). ssrs have been used in varietal identif ication and the evaluation of genetic diversity in c. arabica varieties (vieira et al. 2010). in 2012, low genetic diversity was observed in the c. arabica populations in the nicaraguan regions due to their narrow genetic base, but signif icant differentiation was found among the varieties (geleta et al. 2012). both c. arabica and c. canephora have also been shown to have narrow diversity using ssr markers (anthony et al. 2001; anthony et al. 2002; lashermes et al. 1999). in other studies, c. arabica dna f ingerprinting using ssr markers has also been developed as a method to test against c. canephora, in order to ensure the authenticity of the coffee products sold in the market (tornincasa et al. 2010). ssrs have also been used to evaluate leaf miner resistance in arabica coffee (pereira et al. 2011). the diversity of the c. canephora gene pool was also assessed using ssrs (prakash et al. 2005). since coffee variety misidentif ication and coffee bean sample impurity are major factors that affect the income of small-scale farmers, this study aims to identify potential molecular markers with different ssr primers for variety identif ication s i m p l e s eq u e n ce re p e a t a n a l y s i s of s e l ec ted n s i cr eg i s tered coffee 8 using nsic-registered varieties as standards. the nsic under the department of agriculture, bureau of plant industry was established in 1992 under republic act 7308. this off ice functions to approve and register crop varieties. currently, there are 22 registered coffee varieties across the country (nsic 2012). materials and methods plant material and dna extraction nsic-registered coffee samples were collected from benguet, cavite, and bukidnon (table 1). two plants from each available variety were collected. around 100 mg of young leaves were obtained from each plant for dna extraction. genomic dna was extracted using qiagen dneasy plant mini kit (qiagen, valencia, ca, usa) according to the manufacturer’s instructions. automated quantification of the amount and purity of the extracted dna was performed using nanodrop. on average, about 100 ng per μl of dna was extracted for each specimen. polymerase chain reaction and electrophoresis twenty ssr primers reported in published literature (table 2) were used for amplification in each specimen. the concentration of the pcr components for a 14 μl reaction were as follows: 3.44 μl qiagen master mix, 1.2 μl q buffer, 0.5 μl 25 mm mgcl2, 0.24 μl 10 μm primers, 7.38 μl dnase/rnase-free water, and 1.0 μl 20 ng dna. the following pcr conditions were used: initial denaturation at 94ºc for 10 min; 35 cycles of denaturation at 94ºc for 30 s, annealing at 50ºc for 30 s, and extension species identity variety source coffea arabica red bourbon bureau of plant industry, baguio city, benguet coffea arabica yellow bourbon bureau of plant industry, baguio city, benguet coffea arabica caturra bureau of plant industry, baguio city, benguet coffea canephora ivory coast 2 cavite state university, indang, cavite coffea canephora ivory coast 7 cavite state university, indang, cavite coffea canephora ivory coast 8 cavite state university, indang, cavite coffea canephora s247 cavite state university, indang, cavite coffea liberica bs1 (for registry) cavite state university, indang, cavite coffea canephora frt23 nestle philippines, inc., malaybalay, bukidnon coffea canephora frt 65 nestle philippines, inc., malaybalay, bukidnon table 1. nsic varieties used in this study d.m. c. santos et al. 9 at 72ºc for 1 min; and f inal extension at 72ºc for 7 min (teressa et al. 2010). the pcr products were run in 2% agarose gels for conf irmation. for better resolution of the bands, the pcr products were run in 10% native polyacrylamide gels. both 100 bp (kapa) and 25 bp (bioline) dna ladders were used as molecular weight markers. primer name sequence repeats reference ssrr209 f 5’cgggggtaaaaagattgtaa3’ ga (16) teressa et al. 2010 ssrr209 r 5’ttggtgggaggggagta3’ ssrr268 f 5’gtatcccacaatgaaatcac3’ ga (19) teressa et al. 2010 ssrr268 r 5’agtagaattttcaacatataag3’ ssr124577 f 5’gatggcttttctccgttatcc3’ aag (6) teressa et al. 2010 ssr124577 r 5’ggattcgactgctggatgat3’ ssr122850 f 5’tccagtttgatcagcaacca3’ (agag)3 teressa et al. 2010 ssr122850 r 5’ccatcttggggatagagcaa3’ ssr124195 f 5’atccccatcagaagacctca3’ (agc)6 teressa et al. 2010 ssr124195 r 5’cctccaccgcctgtttatta3’ ssr123557 f 5’atctcctcgttcttccccat3’ ctct (4) teressa et al. 2010 ssr123557 r 5’gcttgtagcaggcaggaaac3’ ssrcma008 f 5’cattctggtcctgatgctct3’ (ct)14. .(tg)10 teressa et al. 2010 ssrcma008 r 5’tcattcacttattaacgtccatc3’ m-24 f 5’ggctcgagatatctgtttag3’ not specified bigirimana et al. 2013 m-24 r 5’tttaatgggcatagggtcc3’ sat235 f 5’tcgttctgtcattaaatcgtcaa3’ not specified bigirimana et al. 2013 sat235 r 5’gcaaaatcatgaaaatagttggtg3’ sat172 f 5’acgcaggtggtagaagaatg3’ not specified bigirimana et al. 2013 sat172 r 5’tcaaagcagtagtagcggatg3’ sat227 f 5’tgcttggtatcctcacattca3’ not specified bigirimana et al. 2013 sat227 r 5’atccaatggagtgtgttgct3’ sat229 f 5’ttctaagttgttaaacgagacgctta3’ not specified bigirimana et al. 2013 sat229 r 5’ttcctccatgcccatattg3’ sat254 f 5’atgttcttcgcttcgctaac3’ not specified bigirimana et al. 2013 sat254 r 5’aagtgtgggagtgtctgcat3’ ssrcma059 f 5’gatggacaggagttgatggt3’ (ct9)(ca)8 teressa et al. 2010 ssrcma059 r 5’ttttaacactcattttgccaat3’ ssrcma198 f 5’agcaactccagtcctcaggt3’ (tg)9(ag)18 teressa et al. 2010 ssrcma198 r 5’tggaagcccgcatatagttt3’ ssrca068 f 5’atgttgttggaggcattttc3’ (agg)7//(gaa)4 missio et al. 2011 ssrca068 r 5’aggagcagttgttgttttcc3’ ssrca087 f 5’tcactctcgcagacacactac3’ (tc)22 missio et al. 2011 ssrca087 r 5’gcagagatgatcacaagtcc3’ ssrca094 f 5’gtgtcctagggaagggtaag3’ (tc)4(ttct)3// missio et al. 2011 (tttcct)3 (ttct)5 ssrca094 r 5’gagtgctaggagagggagag3’ ssrca091 f 5’cgtctcgtatcacgctctc3’ (gt)8(ga)10 missio et al. 2011 ssrca091 r 5’tgttcctcgttcctctctct3’ sat207 f 5’aagccgtttcaagcc3’ pereira et al. 2011 sat207 r 5’caatctctttccgatgctct3’ table 2. primer sequences used for ssr analysis s i m p l e s eq u e n ce re p e a t a n a l y s i s of s e l ec ted n s i cr eg i s tered coffee 10 data analysis the pcr products were evaluated by scoring the presence (1) or absence (0) of clear and unambiguous bands. a neighbor-joining tree with 1,000 bootstrap replicates was constructed using paup version 4.0b10 for microsoft windows 95/ nt and viewed using treeexplorer 2.12 by koichiro tamura 1997-1999. pairwise genetic distances were also calculated using paup. results and discussion a total of 236 unique bands were identif ied from the 20 ssr markers. based on the neighbor-joining tree generated, the c. arabica, c. canephora, and c. liberica species were differentiated into separate clades (figure 1). of the 20 ssr markers, seven primer pairs distinguished c. arabica, nine for c. liberica, and one for c. canephora (table 3). this shows that the ssr markers can be used in delineating species despite figure 1. neighbor-joining tree of 20 nsic-registered coffee varieties generated from banding prof iles from 20 microsatellite markers. branch lengths are drawn to scale and represent uncorrected p-distances. bootstrap supports of 1000 replicates are shown. d.m. c. santos et al. 11 pairwise comparisons p average between species (n=108) 0.382 average between varieties of the same species (n=72) 0.285 average between varieties of c. arabica (n=12) 0.060 average between varieties of c. canephora (n=72) 0.330 average within varieties (n=10) 0.034 red bourbon (n=1) 0.017 yellow bourbon (n=1) 0.008 caturra (n=1) 0.009 yellow bourbon and caturra combined (n=4) 0.024 ivory coast2 (n=1) 0.083 ivory coast7 (n=1) 0.068 ivory coast8 (n=1) 0.072 s247 (n=1) 0.000 frt23 (n=1) 0.021 frt65 (n=1) 0.004 bs1 (n=1) 0.057 table 4. generic d istances in coffee species and varieties. n, number of pairwise comparison; p, uncorrected d istance table 3. species d istinguished by each primer pair primer pair diagnosable species ssrr209 ssrr268 ssr124577 c. arabica ssr122850 c. liberica ssr124195 c. arabica ssr123557 c. arabica ssrcma008 c. arabica, c. liberica, c. canephora m-24 c. liberica sat235 sat172 sat227 c. liberica sat229 c. liberica sat254 c. liberica ssrcma059 c. arabica, c. liberica ssrcma198 c. arabica ssrca068 c. arabica ssrca087 ssrca094 ssrca091 c. liberica sat207 c. liberica s i m p l e s eq u e n ce re p e a t a n a l y s i s of s e l ec ted n s i cr eg i s tered coffee 12 very high polymorphisms. in par ticular, the ssrcma008 primer pair was able to differentiate the three species. teressa et al. (2010) used this primer pair to compare varieties of c. arabica. this is the f irst study that demonstrates its utility for species diagnosis. a 100% bootstrap support was observed for c. arabica and c. liberica species, whereas the support for c. canephora was only at 50%. the low bootstrap support for c. canephora is likely due to the large genetic distance between the ivory coast and s247 varieties from cavite, and the frt varieties from bukidnon. the average genetic distance among varieties of c. canephora (p = 0.330) was comparable to the distances among species (p = 0.382; table 4). the ssr markers were also able to differentiate among the varieties. bootstrap supports of 100% were observed for the red bourbon, ivory coast 2, ivory coast 7, s247, frt23, frt65, and bs1 varieties. a bootstrap support of 97% was observed for ivory coast 8. bootstrap supports of 95% and 88% were observed for yellow bourbon and caturra varieties, respectively. among the c. arabica varieties, the red bourbon variety can be distinguished from the others using the ssr124577 (figure 2) primer pair. the allele number for this primer pair was higher in this study (n=8) compared to that of teressa et al. (2010), indicating higher diversity among the c. arabica varieties in the philippines. the red bourbon variety was shown to be distinct: a 150-bp band from ssr124577, and 150-bp and 350-bp bands from sat229 primer pairs can distinguish the red bourbon from the other c. arabica varieties. the yellow bourbon and caturra varieties clustered together with 93% bootstrap support. although the bootstrap support for each of these clades is moderately high, the values obtained were lower compared to the support for the clades of the other varieties (figure 1). the average pairwise figure 2. banding patterns observed for the different coffea varieties using the ssr124577 marker. d.m. c. santos et al. 13 genetic distance within these two varieties combined is small (p = 0.024) and is even lower than the average genetic distance within single varieties (p = 0.033; table 4). these two varieties were distinguished by the ssrca087 primer pair. apart from this, they share the same banding prof ile based on the other markers. moreover, the 140-bp, 1,000-bp, and 1,200-bp bands from ssr124577 (figure 2) were found to be unique to yellow bourbon and caturra. these varieties are commonly considered to be identical, but were registered as distinct varieties (prof. valentino macanes, pers. comm.). results in this study show partial support for this claim, but the current dataset is insuff icient to generate conclusions, considering that the two varieties did form distinct clades. the yellow bourbon and caturra varieties were observed to have leaves that are similar in shape and size, but caturra had shorter internodes. according to the nsic registry (nsic 2012), they also differ in berry color, but this was not observed in this study because there were no berries during the time of sampling. the sat235 is linked to disease resistance against coffee berry disease (gichimu et al. 2014; gichuru et al. 2008). it is not clear from these papers, however, what fragment size correspond to the marker for the disease. based on the nsic registry (nsic 2012), only ic8 has a record of moderate resistance against coffee berry disease. the bands exhibited by ic8 for the sat235 primer pair is shared by other varieties of c. canephora, except for the frt varieties from bukidnon. no entries are available for other varieties. among the c. canephora varieties, the frt varieties developed by nestle philippines, inc. in bukidnon highly diverged from the ivory coast and s247 varieties from cavite—a phenomenon manifesting even in terms of morphology. frt varieties take a much longer time to flower and fruit, but produce greater yield and more berries per leaf node. these results show the potential of ssr markers for use in varietal identif ication of coffee. the f indings also indicate the possible application of ssr markers in other existing cultivars available in the country. proper identif ication is important to ensure homogeneity and increase marketability. moreover, the application of ssrs could later be extended for marker-assisted selection of important traits, such as disease resistance, aroma, and yield. marker-assisted selection would provide a bottom-up evolutionary approach in genetic improvement, which is more acceptable to society compared to the top-down approach of genetic engineering. acknowledgment s i m p l e s eq u e n ce re p e a t a n a l y s i s of s e l ec ted n s i cr eg i s tered coffee 14 the authors would like to thank the following: dost – pcaarrd, up ovcrd, up natural sciences research institute, and up institute of biology for the funding and support of this project; bureau of plant industry in baguio city, benguet, cavite state university in indang, cavite, and nestle philippines, inc. in malaybalay, bukidnon for providing the nsic-registered coffee varieties; and professor valentino macanes of the benguet state university and his staff for providing resources and information. references anthony f, ber trand b, quiros o, wilches a, lashermes p, berthaud j, charrier a . 2001. genetic diversity of wild coffee (coffea arabica l.) using molecular markers. euphytica. 118(1):53-65. anthony f, combes mc, astorga c, ber trand b, graziosi g, lashermes p. 2002. the origin of cultivated coffea arabica l. varieties revealed by aflp and ssr markers. theoretical and applied genetics. 104(5):894-900. bigirimana j, njoroge k, muthomi jw, gahakwa d, phiri na , gichuru ek, walyaro dj. 2 0 1 3 . g e n e t i c d i v e r s i t y a m o n g d i s e a s e r e s i s t a n t c o f f e e v a r i e t i e s a n d c u l t i v a r s i n rwanda based on rapd and ssr markers. journal of renewable agriculture. 1(6):106112. cao ep, constantino-santos dm, ramos lap, santos bs, quilang jp, mojica rm. 2014. m o l e c u l a r a n d m o r p h o l o g i c a l d i f f e r e n t i a t i o n a m o n g c o f f e a ( r u b i a c e a e ) v a r i e t i e s grown in the farms of cavite province, philippines. philippine science letters. 7(2):387397. charrier a, ber thaud j. 1985. coffee: botany, biochemistry and production of beans and beverage. in: clifford mn, wislon kc, editors. london: croom helm. p. 13-47. geleta m, herrera i, monzon a , bryngelsson t. 2012. genetic diversity of arabica coffee (coffea arabica l.) in nicaragua estimated by simple sequence repeat (ssr) markers. the scientif ic world journal. 1-11. g i c h i m u b m , g i c h u r u e k , m a m a t i g e , n ye n d e a b . 2 0 1 4 . o cc u r r e n ce of c k1 g e n e conferring resistance to coffee berry disease in c. arabica v. ruiru 11 and its parental genotypes. journal of agricultural and crop research. 2(3):51-81. gichuru ek, agwanda co, combes mc, mutitu ew, ngugi eck, ber trand b, lashermes p. 2008. identif ication of molecular markers linked to a gene conferring resistance to cof fee b e r r y d i s e a s e ( co l l e t o t r i c h u m ka h a w a e ) i n cof fe a a r a b i c a . p l a n t pa t h o l o g y. 57:1117-1124. janzen dh. 2009. a dna barcode for land plants. pnas. 106(31):12794-12797. lashermes p, combes mc, rober t j, trouslot r, d’hont a , anthony f, charrier a . 1999. molecular characterization and origin of the coffea arabica l. genome. molecular and general genetics. 261(2):25266. d.m. c. santos et al. 15 n’diaye a , poncet v, louarn j, hamon s, noirot m. 2005. genetic differentiation between co f f e a l i b e r i ca v a r. l i b e r i c a a n d c . l i b e r i ca v a r. d e wev r e i a n d co m p a r i s o n w i t h c . canephora. plant systematics and evolution. 253:95-104. [ n s i c ] n a t i o n a l s e ed i n d u s t r y co u n c i l . [ i n te r n e t ] . 2 0 1 2 . quezo n c i t y. [c i ted 2 0 1 5 december 1]. available from http: //www.nseedcouncil.bpinsicpvpo.com.ph/. pereira gs, padilha l, pinho ev, teixeira rd, carvalho ch, maluf mp, carvalho bl. 2011. microsatellite markers in analysis of resistance to coffee leaf miner in arabica coffee. pesquisa agropecuária brasileira. 46(12):1650-6. prakash ns, combes m, dusser t s, naveen s, lashermes p. 2005. analysis of genetic diversity in indian robusta coffee genepool (coffea canephora) in comparison with a representative core collection using ssrs and aflps. genetic resources and crop evolution. 52:333-343. reyes b. [internet]. 2012. coffee origins. las pinas: bote central, inc.; [cited 2016]. available from http: //www.botecentral.net/coffee-origins. schlotterer c. 2000. evolutionary dynamics of microsatellite dna. chromosoma. 109: 365-371. teressa a , crouzillat d, petiard v, brouhan p. 2010. genetic diversity of arabica coffee (coffea arabica l.) collections. ethiopian journal of applied sciences and technology. 1(1):63-79. to r n i n c a s a p, f u r l a n m , p a l l a v i c i n i a , g r a z i o s i g . co f f e e s p e c i e s a n d v a r i e t a l identif ication. in: morrison da, editor. tools for identifying biodiversity: progress and problems. 2010. p. 307-313. vieira esn, vonpinho evd, carvalho mgg, esselink dg, vosman b. 2010. development of microsatellite markers for identifying brazilian coffea arabica varieties. genetics and molecular biology. 33(3):507-514. wang ml, barkley, na , jenkins tm. 2009. microsatellite markers in plants and insects. par t i: applications of biotechnology. genes, genomes and genomics. 3(1):1-14. _____________ daisy may c. santos <daisymay_constantino@yahoo.com> is currently working as a science research specialist and as a lecturer at the institute of biology, university of the philippines diliman. she f inished her bachelor of science degree in biology from the university of the philippines baguio and her master of science degree in biology (major in genetics) from the university of the philippines diliman. her research interests include plant genetics and molecular biology. s i m p l e s eq u e n ce re p e a t a n a l y s i s of s e l ec ted n s i cr eg i s tered coffee 16 carla francesca besa is a graduate of bs agricultural biotechnology at the university of the philippines los baños. she is also a member of the alpha chi chapter of phi sigma society, and is currently working as a science research s p e c i a l i s t u n d e r t h e c o f f e e g e n o m i c s p r o j e c t a t t h e p l a n t g e n e t i c s a n d cyanobacterial biotechnology laboratory of the university of the philippines diliman. angelo joshua v ictoria is a graduate student at the institute of biology where he also works as a researcher. his research interests include molecular genetics and dabbling with bioinformatics. he also has a keen interest in foreign languages and learns german and swedish during his time out of the lab. dr. ernelea p. cao is currently professor 12 and director of the institute of biology, college of science, university of the philippines diliman. she was former deputy executive director of the philippine genome center and was also the former director of the natural sciences research institute, college of science, university of the philippines diliman. she f inished her bachelor of science degree in biology from the university of the philippines los baños (major in genetics) and her m.s. and ph.d. in biology at the university of the philippines diliman (major in genetics). she conducted her dissertation under a sandwich program at the department of biology, university of north carolina in chapel hill, north carolina, u.s.a . her research interests include plant genetics and cyanobacterial biotechnology. 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address _______________________________ 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(subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total 7geometric study-tingzon.pmd geometric study on silicon nanowires 66 science diliman (january-june 2017) 29:1, 66-86 geometric study on sil icon nanowires fabricated via silver-assisted electroless etching neil irvin f. cabello university of the philippines diliman eloise p. anguluan university of the philippines diliman joseph christopher r. ragasa university of the philippines diliman phil ippe mar tin b. t ingzon* university of the philippines diliman kerr a. cervantes university of the philippines diliman arvin jay s. escolano university of the philippines diliman arnel a. salvador university of the philippines diliman armando s. somintac university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract c o n t r o l l i n g t h e g e o m e t r y o f s i l i c o n n a n o w i r e s ( s i n w s ) h a s b e e n o f paramount necessity for the viability of mass-producing nanostructured devices. the length, radius, and crystallinity of sinws grown via onestep and two-step electroless chemical etching of p-type si(100) in this s t u d y w e r e c o n t r o l l e d b y v a r y i n g t h e c o n c e n t r a t i o n s o f e t c h a n t s a n d etching times. scanning electron microscope images conf irmed that the length of the nanowires varied directly with increasing concentrations of hf and agno 3 for the one-step etching process, and hf, h 2 o 2 , a n d agno 3 for the two-step etching process. diameters cannot be controlled via the electroless etching methods in the one-step process, but can be manipulated in the two-step process to some extent. x-ray diffractometry n.i.f. cabello et al. 67 analysis exhibited that the sinw 's peak broadening can be attributed to the slight degradation of crystallinity of sinws compared to bulk silicon. f r o m t h e r a m a n s p e c t r a , s i n w s , r e g a r d l e s s o f t h e i r g e o m e t r i c parameters, make excellent thermo-insulators due to the one-directional movement of phonons. the slight shift in peaks can be attributed to laser heating. finally, photoluminescence analysis of sinws demonstrated that the length of the sinws varied the ratio of the surface defects of both the one-step and two-step processes, but not the intensity. ke y w o r d s : n a n ow i r e s ( 8 1 . 0 7. g f ) , s i l i co n n a n ow i r e s ( 7 8 . 6 7. u h ) , s i l v e r nanopar ticles (78.67.bf ) introduction studies on the nanostructure formation on the most essential and economical semiconductor available, silicon (si), have gained a new driving force with the advent of low-cost chemical preparation of nanostructures and the approaching end of moore's law. primary among these nanostructures are silicon nanowires (sinws), believed to be promising building blocks for the next-generation devices in the f ield of optoelectronics, nanoelectronics, sensing, energy storage, and harvesting. sinws have a huge array of advantageous optical and optoelectronic properties. these include low thermal conductivity (curtin et al. 2012), wide broadband optical absorption (tsaklakos 2007), high carrier mobility (liu et al. 2013), quantum conf inement effects, and phononic properties (bandaru and pichanusakorn 2010). these properties are all dependent on the geometry and crystallinity of the fabricated sinws. different methods have been developed in the past years for the fabrication of sinws. some commonly used techniques in the fabrication of sinws are the vaporliquid-solid (vls) method (gunawan and guha 2009), and the chemical-assisted beam etching. however, these methods are time-consuming and make use of expensive equipment to produce the required high-vacuum environment at high temperatures for the fabrication of vertically-oriented sinws. a novel technique used in the fabrication of sinws is silver-assisted electroless etching (ee), which has gained signif icant interest in the recent years (schmidt et al. 2009), as it offers a low-cost method of fabricating vertically-aligned sinws using a simple table-top set-up. the process utilizes silver (ag) which oxidizes the geometric study on silicon nanowires 68 si substrate and is subsequently dissolved by an etchant solution (peng et al. 2006). this simultaneous redox reaction and etching in the areas in contact with the noble metal forms voids in the substrate, leaving the unetched areas as sinws (peng et al. 2002). the etching process used in this study can be categorized into two types: one-step process and two-step process. one-step etching involves the immersion of the si substrate in an etchant solution composed of hydrofluoric acid (hf) and agno 3 (figure 1, left). the ag+ ions in the solution are reduced and deposited as ag nanoparticles (agnps) near the defective sites on top of the si substrate are reduced by the more electronegative si (lin 2010) (figure 1, right). this reduction is coupled with the oxidation of the si beneath the agnp and is subsequently dissolved by hf (peng et al. 2006). the reaction is described by the following: ag++e–→ ag (s) (1) si (s) +2h 2 o (1) sio 2 +4h++4e– (2) furthermore, the etching of the newly formed silicon oxide layer in the one-step process is described by the following reaction: sio 2 +6hf →h 2 sif 6 +2h 2 o (3) figure 1. etching mechanism in lateral view (left) and top view (right). n.i.f. cabello et al. 69 for the one-step process, the net chemical reaction using equations (1), (2), and (3) yield: 4ag+ si (s) +6f –®[sif 6 ] 2+4ag (s) (4) meanwhile, the two-step etching process involves an additional immersion of the si substrate in hf-hydrogen peroxide (h 2 o 2 ) etchant solution after a prior immersion in hf-agno 3 solution (figure 1, right). the f irst step deposits agnps onto the surface of the si. the second step oxidizes the deposited agnps into ag+, which are then reduced to ag (s) by injecting holes in the valence band of si. the oxidized si is then dissolved by hf in the etchant, which causes the etching of the substrate. the following reaction governs the two-step process: si+2h 2 o 2 +6f–+4h +®[sif 6 ]2+4h 2 o (5) the study aims to fabricate vertically-oriented sinws and to attain control of the geometry and crystallinity of these nanostructures by varying the etching p a r a m e t e r s . t h e e f f e c t o f e a c h p a r a m e t e r w a s o b s e r v e d u s i n g d i f f e r e n t characterization tools. the dimensions of the nws were examined and measured using a scanning electron microscope (sem). x-ray diffractometry (xrd) was used to determine any changes in the quality of the crystal structures in the si substrate. raman spectroscopy was performed to determine whether the etched sinws retained their crystallinity. photoluminescence (pl) spectroscopy was used to determine the possible radiative transitions of the sinws. methodology a monocrystalline p-type si (100) wafer with a resistivity of 0.3-300ω•cm and thickness of 500 μm was used as a substrate. the wafer was cleaned using standard degreasing procedures. it was then immersed in a solution of 48% hf and di water (1:9 volumetric ratio) to remove the oxides formed at the surface. the surface was cleaved into 1 cm × 1 cm samples, upon which the nws were etched. for the one-step standard etching, ag deposition and etching was achieved through the immersion of the substrate in a solution of 5.0 m hf and 0.02 m agno 3 for 60 minutes. each substrate was rinsed with di water after etching. the ag dendrites formed at the surface of the sample after immersion (representative image in the supplementary material) were carefully removed using tweezers, and the remaining ag particles on the samples were chemically removed using a solution of nh 4 oh and h 2 o 2 at a 3:1 volumetric ratio. geometric study on silicon nanowires 70 for two-step standard etching, ag deposition was performed through the immersion of the samples in an etchant solution of 5.0 m hf and 0.02 m agno 3 for 2 minutes. the samples were then rinsed with di water to remove loose ag particles and excess ions from the etchant solution. to etch the nws, the samples were immersed in a solution of 5.0 m hf and 0.3 m h 2 o 2 for 40 minutes. the samples were again washed with di water after the etching. the deposited ag particles were removed in the samples via immersion in a solution of nh 4 oh and h 2 o 2 with a 3:1 volumetric ratio. for the one-step etching process, agno 3 concentration was varied from 0.01 to 0.04 m, hf concentration from 2.5 to 7.5 m, and the etch time between 120 and 180 minutes. for the two-step etching ag deposition, the deposition time was varied from 1 to 3 minutes, hf concentration from 2.5 to 7.5 m, h 2 o 2 concentration from 0.1 to 0.5 m, and the etch time to 120 and 180 minutes. the concentrations of the chemicals, ag deposition time, and etch time are summarized in tables 1 and 2. only one parameter was changed from the standard procedure per sample, in order to determine the individual effects of the parameters on the sinw structure. table 1. the mod ified parameters (highl ighted) in the one-step etching process. the first row of parameters is the standard used by peng et al. (2002) 5.0 0.02 60 2.5 0.02 60 7.5 0.02 60 5.0 0.01 60 5.0 0.04 60 5.0 0.02 120 5.0 0.02 180 hf concentration (m) agno 3 concentration (m) etch t ime (minutes) 120 5.0 0.3 40 60 5.0 0.3 40 180 5.0 0.3 40 120 2.5 0.3 40 120 7.5 0.3 40 120 5.0 0.1 40 120 5.0 0.5 40 120 5.0 0.3 20 120 5.0 0.3 60 ag deposition t ime (seconds) hf concentration (m) h 2 o 2 concentration (m) etch t ime (minutes) table 2. the mod ified parameters (highl ighted) in the two-step etching process. the first row of parameters is the standard used by peng et al. (2002) n.i.f. cabello et al. 71 sinw images were captured using a philips xl-30 feg sem. measurements from the images were obtained using sem software's built-in measurement application, along with an external application tsview. the xrd pattern was obtained using a bede d3 high resolution x-ray diffractometer. the wavelength of the incident x-ray from a cu kα source was 1.541 å, and the scan resolution was 0.002º. raman spectroscopy was performed with an excitation provided by a 60-mw, 514.5-nm ar+ laser, and the scan resolution was 1.2 cm-1. the scattered light was detected by a horiba jobin-yvon ihr 550 imaging spectrometer with a charge-coupled device (ccd) detector. pl was executed with an excitation provided by a 488-nm ar+ laser, and the detection apparatus was a spex 500m equipped with a photomultiplier tube. results and discussion one-step etching dependence on hf concentration the sem image in figure 2 shows that the increase in hf concentration was directly proportional with the length of the sinws. following the implementation of the nernst equation on reactions (1), (2), (3) by lin et al. (2010), the potential of the one-step etching process is governed by: (6) from the equation (6), an increase in concentration of the hf directly affects the amount of free f– ions. thus, we should see an increase in the electric f ield produced by the etching process. f– theoretically has the highest contribution to the electric f ield process. figure 2. sem micrographs showing increasing lengths of sinws (8, 13, and 19 ìm) as hf concentration was increased (2.5, 5.0, and 7.5 m) (left to right). [ag+]4[f-]6 δe= δe0 – 0.059 [sif 6 2–]log 4 geometric study on silicon nanowires 72 the increase in length of the nanowires consequently results to the agglomeration at the tips of the nanowires. this agglomeration results from van der waals forces (li et al. 2010) and capillary forces due to water (choi et al. 2010). by lengthening the sinws, the elastic deformation force (f ed ), which counters the bending forces from the van der waals and its capillary effect, becomes weak (togonal et al. 2014). the weakening of the elastic deformation is given by the following equation: (7), where e is the young's modulus, l is the length of the nanowires, and r is the radius of the nanowires. the hf concentration has no signif icant effect to the diameter or to the nanowire density as shown in table 3. dependence on agno 3 concentration from equation (6), the ag+ contribution is only raised to 4, but the increment in the agno 3 molarity increased by 2.0× (from 0.02 to 0.04 m), while the hf molarity increased by 1.5× (from 5 to 7.5 m). hence, changing the agno 3 concentration has a larger contribution to the increase in nanowire length. the sem image in figure 3 shows that an increase in agno 3 concentration also enhanced the length of the sinws. this observation can be attributed to the increase in the hole sources being injected to the silicon substrate. from the coupled equations (1) and (2), more ag+ extract electrons from the silicon surface, which eventually becomes ag(s) and simultaneously oxidizes silicon. the increase in ag+ ions does not only cause an increase in the amount of ag deposited at the surface of (3πer4) 4l3 f e d = figure 3. sem micrographs showing increasing lengths of sinws (10, 13, and 21 μm) as agno 3 concentration was increased (0.01, 0.02, and 0.04 m) (left to right). n.i.f. cabello et al. 73 the silicon but also the surface area available for etching. consequently, this caused an increase in the nanowire density (table 3). dependence on etch time the sem image in figure 4 shows that the increase in the etch time also caused an increase in the length of the sinws. the increase in etch time allows the reactions to proceed, and as a result, the agnps burrow more deeply into the substrate. the increase in the length of the nanowire results to the agglomeration at their tips. the top-view sem in figure 5 shows the formation of distinctive clusters of nws as the sinw length increased. there is an observed increase in the nanowire diameter for the 180-minute etch. figure 4. sem micrographs showing increasing lengths of sinws (13, 31, and 37 ìm) as etch time was increased (60, 120, and 180 minutes) (left to right). . table 3. summary of the measurements and characterizations of the sinw samples produced with varying parameters in the one-step etching process 69.112 0.012 520.0 5.0 0.02 60 12.8 ± 0.3 170 ± 122 44 69.112 0.012 517.4 2.5 0.02 60 7.9 ± 0.3 99 ± 39 131 69.112 0.024 517.8 7.5 0.02 60 19.3 ± 1.0 122 ± 62 86 69.112 0.027 518.0 5.0 0.01 60 9.8 ± 0.3 137 ± 98 68 69.112 0.010 517.5 5.0 0.04 60 21.4 ± 1.0 120 ± 48 88 69.112 0.008 516.4 5.0 0.02 120 30.8 ± 0.9 157 ± 104 52 69.112 0.030 515.6 5.0 0.02 180 37.2 ± 2.5 172 ± 88 43 69.112 0.046 515.5 hf conc. (m) agno 3 conc. (m) etch t ime (minutes) nw length ( μμμμμm ) nw diameter (nm) nw count density (no./μμμμμm2) xrd peak (0) xrd fwhm raman peak (cm-1) geometric study on silicon nanowires 74 figure 5. top-view sem micrographs of the samples as etch time was increased (60, 120, and 180 minutes). the degree of agglomeration or the clustering at the tips of the nws increased as the sinw length increased. the xrd plot from figure 6 reveals that, as the etch time was increased, the fullwidth at half-maximum (fwhm) also exhibited a slightly increasing trend. the samples demonstrated a somewhat lower crystal quality, as can be observed from their broadened xrd peaks. moreover, compared to the fwhm of bulk si, the samples displayed degradation in the crystal quality after etching sinws to the samples, which can be a consequence of non-uniform surface stress encountered by the nanowire (song et al. 2011). the raman spectroscopy plot shown in figure 7 conf irms that the sinws are indeed crystalline. the sinw raman peaks are downshifted relative to the bulk raman peaks, but this is not a result of phonon conf inement because the maximum diameter for nanowires to exhibit quantum conf inement is at 20 nm (richter et al. 1981; figure 6. xrd peaks of one-step electroless etching sinws as etch time was increased (60, 120, and 180 minutes). n.i.f. cabello et al. 75 campbell and fauchet 1986). instead, this can be ascribed to laser heating as a consequence of subjecting the sinws to a high-power laser in the raman set-up, a phenomenon encountered by scheel et al. (2008). the dissipation of heat in sinws is eff icient only along one direction, which makes sinws excellent thermoinsulators. the down-shifting is different for each sample due to the non-linear relationship between thermal conductivity and sinw length (wang et al. 2012). f i g u r e 7. r a m a n p e a k s fo r o n e s te p e l ec t r o l e s s e tc h i n g s i n ws a s e tc h t i m e w a s increased (60, 120, and 180 minutes). a downshift from the bulk si peak (solid line) is observed for all sinw samples. two-step etching dependence on the ag deposition time for the two-step process, the potential for the etching process from the nernst equation (lin et al. 2010) becomes: thus, theoretically, ag deposition should be independent of the etching process. at low deposition times, little agglomeration occurred at the tip of the nws (figure 8). meanwhile, the mean diameter of nws decreased as deposition time increased (figure 8). this can be attributed to the increase in the area covered by the deposited δe= δe0– 0.059 4 [sif 6 2–] [ h 2 o 2 ] 2[ h +] 4[f –] 6 log (8) geometric study on silicon nanowires 76 ag as the deposition time increased. because the unetched area decreased, then the diameters of the nws formed in the process also decreased. there was a slight increase in the etch depth as the deposition time increased (figure 9). longer deposition time yielded increased area covered by ag deposition on the surface of si, resulting to better electron capture from the electrolyte. as a consequence, the sinw density increased while the aspect ratio did not exhibit a clear trend. figure 9. sem micrographs showing independence of the length of sinws (16, 19, and 16 ìm) on increasing ag deposition time (60, 120, and 180 seconds) (left to right). dependence on hf concentration from the nernst equation (8), hf has the highest contributing factor, as demonstrated by the 29-μm length of sinws etched. the etch depth increased as the concentration of hf increased (figure 10). meanwhile, the mean diameter (table 3) of sinws decreased with increasing hf concentration. such trend is attributed to the faster anisotropic etching along the direction of ag particles, as well at the sidewalls of sinws, due to the large amount of hf available to etch the oxidized si. thus, increasing the hf concentration resulted to a great increase in the density and aspect ratio of sinws. figure 8. top-view sem micrographs of the samples showing the agglomeration of sinw tips as ag deposition time was increased (60, 120, and 180 seconds). n.i.f. cabello et al. 77 dependence on h 2 o 2 concentration at low h 2 o 2 concentrations, agnps catalyze with just the small amount of h 2 o 2 , thereby exhausting the h 2 o 2 supply. with increasing h 2 o 2 supply, the process of agno 3 is hastened and greater etch depth is observed. however, at higher concentrations (0.5 m h 2 o 2 ), the length of sinws decreased. as described by li et al. (2014), an increase in h 2 o 2 concentration leads to horizontal and vertical etching on the silicon nanowire (figure 11). dependence on etch time similar to the one-step etch, the increase in the etch time also caused an increase in the length of the sinws (figure 12). the degree of agglomeration of the nanowires increased as the time of etching increased, similar to what is observed in the onestep etching (figure 13). no change was observed in the diameter or the density of the nanowires as the etch time increased (table 4). figure 10. sem micrographs showing increasing lengths of sinws (7, 19, and 29 ìm) as hf concentration was increased (2.5, 5.0, and 7.5 m) (left to right). figure 11. sem micrographs showing varying sinw lengths (2, 19, and 16 ìm) as h 2 o 2 concentration was increased (0.01, 0.02, and 0.04 m) (left to right). geometric study on silicon nanowires 78 from the xrd data summarized in table 4, no clear trend was observed in the crystal quality of the samples. the samples were crystalline; however, they possessed lower crystal quality compared to bulk si, as suggested by their broadened peaks in figure 14. the decrease in crystal quality can be attributed to the surface roughness of the sinws brought about by the etching step in the twostep process. there are two salient features in the raman spectrum for all two-step etched sinws, a broad peak and a sharp peak. the sharp peak is attributed to the crystalline si (c-si), and the broad peak is imputed to the contributions of lower energy phonon modes due to the porous sidewalls. table 3 shows both sharp and broad peaks. figure 12. sem micrographs showing increasing lengths of sinws (11, 19, and 26μm) as etch time was increased (20, 40, and 60 minutes) (left to right). 69.112 0.012 520.0 120 5.0 0.3 40 19.3 ± 0.5 193 ±108 34 69.112 0.033 514.0 489.9 60 5.0 0.3 40 15.7 ± 0.4 210 ±135 28 69.112 0.035 513.3 496.7 180 5.0 0.3 40 16.4 ± 0.3 166 ± 92 46 69.112 0.045 516.1 504.0 120 2.5 0.3 40 7.3 ± 0.4 164 ± 90 47 69.112 0.045 517.8 502.3 120 7.5 0.3 40 28.9 ± 0.9 154 ± 73 53 69.112 0.038 515.7 500.3 120 5.0 0.1 40 2.3 ± 0.7 80 ± 57 198 69.112 0.036 519.2 ——— 120 5.0 0.5 40 15.7 ± 0.4 189 ± 99 36 69.112 0.056 512.9 488.7 120 5.0 0.3 20 11.0 ± 0.5 172 ±106 43 69.112 0.056 519.0 505.5 120 5.0 0.3 60 26.0 ± 0.8 197 ± 90 33 69.112 0.049 513.5 496.2 ag deposition time (seconds) hf conc. (m) table 4. summary of the measurements and characterizations of the sinw samples produced with varying parameters in the two-step etching process h 2 o2 conc. (m) etch time (mins.) nw length ( μμμμμm ) nw diameter (nm) nw couynt density (no./ (μμμμμm 2) xrd peak (o) xrd fwhm raman peak 1 (sharp) and peak 2 (broad) (cm-1) n.i.f. cabello et al. 79 figure 13. top-view sem micrographs of the samples as etch time was increased (20, 40, and 60 minutes). the degree of agglomeration or the clustering at the tips of the sinws increased as the sinw length increased. figure 14. xrd peaks of two-step electroless etching sinws as etch time was increased (20, 40, and 60 minutes). there was no apparent shift from the peak of bulk si (solid line). no trend was observed on the fwhm. figure 15. raman peaks for the bulk si and the two-step electroless etching sinws as etch time was increased (20, 40, and 60 minutes). broad and sharp peaks were observed in the sinw plots. geometric study on silicon nanowires 80 photoluminescence (pl) spectra of sinws analyses based solely on intensities of the pl peaks reveal insuff icient information on the possible radiative transitions of the sinws. in reality, these pl peaks are just a sum of several gaussian peaks from unique defects (muldera et al. 2013; ghosh et al. 2014). to gain more information from the pl peaks for both the one-step and the two-step electroless etching, each peak was deconvolved to four gaussian peaks centering at approximately 590 nm, 663 nm, 759.9 nm, and 850 nm. the 590-nm peak can be attributed to the defects from si-si dimer, which is commonly known as the host dioxide matrix (hdm) by kamenev and nassiopoulou (2010). this peak is intrinsic to silicon nanowires. as observed by najar et al. (2012), the 663-nm peak comes from the oxygen defect of si. the two pl peaks which approximately lie at 760 nm and 850 nm can be assigned to two unique oxide surface defects (lin 2010). the ratios of the area under the curve of these four gaussian peaks were obtained with respect to the area under the f itted plot. the areas under the curve of the f itted plot were obtained using simpson's rule. figure 16 and 17 show the deconvolution of the pl peaks and their ratios in percent using a peak fit program. peaks 3 and 4 are the oxide surface defects which come from the oxide dangling defects in the oxide layer of the silicon (lin et al. 2010). figure 16. pl intensity of increasing lengths of one-step electroless etching sinws (0.5, 0.75, and 1.0 μm), with each peak deconvolved into four gaussian peaks. n.i.f. cabello et al. 81 figure 17. pl intensity of increasing lengths of one-step electroless etching sinws (0.5, 0.75, and 1.0 m), with each peak deconvolved into four gaussian peaks. the area under the curve ratios for one-step and two-step etching processes are listed in tables 5 and 6, respectively. as the hdm (590 nm) defect increased, the defect from the si-o bond (663 nm) decreased, suggesting an inverse relationship between the two defects. this inverse relationship between the two defects was observed in both one-step and two-step processes with increasing nanowire length. for the two other oxide surface defects (759 nm and 850 nm), the values were too small (<5% of the actual intensity) to show any trend. pl due to quantum conf inement is also negligible due to the large diameters of the sinws formed. table 5. area under the curve pl values in % for the one-step electroless etching process 0.5 μm 37.55 % 58.48 % 4.20 % 0.23% 0.75 μm 35.59 % 60.88 % 3.43 % 0.1 % 1.0 μm 35.35 % 63.86 % 0.1 % 0.71 % sinw length (μm) 590.5 nm 663 nm 760 nm 590.5 nm geometric study on silicon nanowires 82 0.5 μm 34.87 % 62.43 % 0.92 % 1.78% 0.75 μm 38.08 % 56.74 % 4.03 % 1.14 % 1.0 μm 38.12 % 56.07 % 5.06 % 0.75 % sinw length (μm) 593 nm 663 nm 760 nm 850 nm table 6. area under the curve pl values in % for the two-step electroless etching process conclusion as the parametric values were increased, the wire lengths also increased, with etch time being the most signif icant factor, followed by agno 3 and hf concentration. t h e u n i fo r m i t y o f t h e n a n ow i r e d i a m e te r c a n b e m o d i f i ed by t h e ag n o 3 concentration. increasing the h 2 o 2 concentration of the second etchant caused a decrease in the nanowire diameter. from the xrd analysis of the one-step etching, the crystallinity of the sinws was slightly compromised with increasing parametric values. however, for the two-step etching, no clear trend was observed. the broadening and downshifting of the sinw 's raman spectra relative to the bulk si can be attributed to laser heating and the low thermal conductivity of the sinws. the photoluminescence of the sinws were obtained as well, relating the area under the curve plots of the deconvolved gaussian peaks to their corresponding defects. an inverse relationship was observed for the host dioxide matrix defect and the si-o bond defect. acknowledgments the authors would like to acknowledge support from the department of science and technology grants-in-aid program, the university of the philippines diliman off ice of the vice-chancellor for research and development, and the national research council of the philippines. n.i.f. cabello et al. 83 references bandaru p, pichanusakorn p. 2010. an outline of the synthesis and properties of silicon nanowires. semiconductor science and technology. 25(2):024003. campbell i, fauchet p. 1986. the effects of microcrystal size and shape on the one phonon raman spectra of crystalline semiconductors. solid state communications. 58(10):39-741. choi c, yoon y, hong d, jin s. 2010. strongly superhydrophobic silicon nanowires by supercritical co2 drying. electronic materials letters. 6(2):59-64. curtin b, fang e, bowers j. 2012. highly ordered vertical silicon nanowire array composite thin f ilms for thermoelectric devices. journal of electronic materials. 41(5):887-894. gunawan o, guha s. 2009. characteristics of vapor-liquid-solid grown silicon nanowire solar cells. solar energy materials and solar cells. 95(1):1388-1393. g h o s h r , g i r i p k , i m a k i t a k , f u j i i m . 2 0 1 4 . o r i g i n o f v i s i b l e a n d n e a r i n f r a r e d photoluminescence from chemically etched si nanowires decorated with arbitrarily shaped si nanocrystals. nanotechnology. 25(4):045703. kamenev bv, nassiopoulou ag. 2001. self-trapped excitons in silicon nanocrystals with sizes below 1.5 nm in si/sio2 multilayers. journal of applied physics. 90(11):57355740. li c, fang g, sheng s, chen z, wang j, ma s, zhao x. 2005. raman spectroscopy and f ield electron emission proper ties of aligned silicon nanowire arrays. physica e: lowdimensional systems and nanostructures. 30(1):169-173. li s, ma w, zhou y, chen x, xiao y, ma m, zhu w, wei f. 2014. fabrication of porous silicon nanowires by mace method in hf/h 2 o 2 /agno 3 system at room temperature. nanoscale research letters. 9(1):196. lin l, guo s, sun x, feng j, wang y. 2010. synthesis and photoluminescence proper ties of porous silicon nanowire arrays. nanoscale research letters. 5(11):1822-1828. liu k, qu s, zhang x , tan f, wang z. 2013. improved photovoltaic performance of s i l i c o n n a n o w i r e / o r g a n i c h y b r i d s o l a r c e l l s b y i n c o r p o r a t i n g s i l v e r n a n o p a r t i c l e s . nanoscale research letters. 8(1):88. muldera j, cabello n, ragasa jc, mabilangan a, balgos mh, jaculbia r, somintac a, estacio e, salvador a. 2013. photocarrier transpor t and carrier recombination eff iciency i n v e r t i c a l l y a l i g n e d s i n a n o w i r e a r r a y s s y n t h e s i z e d v i a m e t a l a s s i s t e d c h e m i c a l etching. applied physics express. 6(8):082101. geometric study on silicon nanowires 84 najar a, dalaver ah, hedhili mn, ng tk, ooi bs, slimane ab, sougrat r. 2012. effect of hydrofluoric acid concentration on the evolution of photoluminescence characteristics i n p o r o u s s i l i c o n n a n o w i r e s p r e p a r e d b y a g a s s i s t e d e l e c t r o l e s s e t c h i n g m e t h o d . journal of applied physics. 112(3):033502. pan h, chen w, lim sh, poh ck, wu x, feng y, ji w, lin j. 2005. photoluminescence and o p t i c a l l i m i t i n g p r o p e r t i e s o f s i l i c o n n a n o w i r e s . j o u r n a l o f n a n o s c i e n c e a n d nanotechnology. 5(5):733-737. peng k, yan y, gao s, zhu j. 2002. synthesis of large-area silicon nanowire arrays via self-assembling nanoelectrochemistry. advanced materials. 14(16):1164-1167. peng kq, hu jj, yan yj, wu y, fang h, xu y, lee s, zhu j. 2006. fabrication of singlec r y s t a l l i n e s i l i c o n n a n o w i r e s b y s c r a t c h i n g a s i l i c o n s 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r o c a s p. 2 0 1 4 . e f f e c t o f w e t t a b i l i t y o n t h e a g g l o m e r a t i o n o f s i l i c o n n a n o w i r e a r r a y s f a b r i c a t e d b y m e t a l a s s i s t e d c h e m i c a l etching. langmuir. 30(34):10290-10298. tsakalakos l, balch j, fronheiser j, shih my, leboeuf sf, pietrzykowski m, codella pj, korevaar ba, sulima o, rand j, davuluru a. 2007. strong broadband optical absorption in silicon nanowire f ilms. journal of nanophotonics. 1(1):013552. wang y, schmidt v, senz s, gösele u. 2006. epitaxial growth of silicon nanowires using an aluminum catalyst. nature nanotechnology. 1(3):186-189. wang m, shan x, and yang n. 2012. understanding length dependences of effective thermal conductivity of nanowires. physics letters a . 376(46):3514-3517. n.i.f. cabello et al. 85 _____________ neil irvin f. cabello graduated b.s. applied physics (instrumentation) and m.s. physics from the university of the philippines diliman. he is currently a ph.d. physics student at the university of the philippines diliman and a research fellow in the chef-pcari project “3v-recon.” his research interests include silicon nanowires and porous silicon (psi) application, photoluminescence and thz applications of these nanostructures, and vertical cavity surface emitting lasers (vcsels). eloise p. anguluan graduated b.s. applied physics (instrumentation) and m.s. physics from the university of the philippines diliman. she is currently a researcher in gwangju institute of science and technology, south korea. her research interests include electro-optical characterization of psi and sinws, enhanced raman spectroscopy, and biophotonics. joseph christopher r. ragasa graduated b.s. physics from the university of the philippines diliman. he is currently employed in hgst company. his research interests include sinws fabrication, harvest, and application for optoelectronic devices. phil ippe martin b. t ingzon <ptingzon@gmail.com> graduated b.s. applied physics (instrumentation) and m.s. materials science and engineering from the university of the philippines diliman. he is currently a ph.d. materials science and engineering student at the university of the philippines and a research fellow in the chefpcari project “3v-recon”. his research interests include silicon nanowires applications, gaas-based solar cells, and vertical cavity surface emitting lasers (vcsels). kerr a. cervantes is currently an undergraduate student taking up b.s. applied physics (materials physics) at the university of the philippines diliman. his research interests include sinws fabrication and application. arvin jay s. escolano graduated b.s. applied physics (materials physics) and is currently taking up m.s. in materials science and engineering at the university of the philippines diliman. he is currently a dost scholar under the dost-asthrd program. his research interests include sinws fabrication and application, and sno 2 / fe 2 o 3 pyroelectric f ilms. geometric study on silicon nanowires 86 dr. arnel a. salvador graduated b.s. physics from the university of the philippines diliman and ph.d. in physics from university of illinois. he is currently a professor at the national institute of physics and the chairperson of the physics division of the national research council of the philippines. his research specialization include mbe (molecular beam epitaxy) grown nanostructures with their corresponding properties and applications such as vcsels. dr. armando s. somintac graduated b.s. physics in the university of the philippines baguio as his undergraduate course, took his ms in materials science and engineering from the university of the philippines diliman and f inished his ph.d. in physics from the same university. he is currently a professor at the national institute of physics and the director of the project management and resource generation off ice of the off ice of the vice-chancellor for research and development. his research 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ieee computer society. p. 307313. unpubl ished (section 29.3.7.15 p. 566-568) format: authors(s). date of conference. title of paper. paper presented at: title of conference. number and name of the conference; place of the conference. example: antani s, long lr, thomas gr, lee dj. 2003. anatomical shape representation in spine x-ray images. paper presented at: viip 2003: proceedings of the 3rd iasted international conference on visualization, imaging and image processing; benalmadena, spain. technical repor ts (section 29.3.7.4 p. 537) format: author(s). date. title of report. edition. place of publication: publisher. extent. report no.: notes. example: feller ba. 1981. health characteristics of persons with chronic activity limitation, united states, 1979. hyattsville (md): national center for health statistics (us). report no.: vhs-ser-10/137. available from: ntis, springf ield, va; pb88-228622. dissertations and theses (section 29.3.7.5 p. 539-541) format: authors(s). date. title of dissertation and thesis [content designator]. place of publication: publisher. extent. notes. example: lutz m. 1989. 1903: american nervousness and the economy of cultural change [dissertation]. stanford (ca): stanford university. 105 group/corporate author (section 29.2.1.2.5 p. 494) format: [abbreviation of group] name of group (country). date. title. place of publication: publisher. notes. example: [iom] institute of medicine (us). 1975. legalized abortion and the public health; report of a study by a committee of the institute of medicine. washington (dc): national academy of sciences. other internet materials (section 29.3.7.13 p. 556-564) homepage format: title of homepage [medium designator]. date of publication. edition. place of publication: publisher; [date updated; date cited]. notes. example: apsnet: plant pathology online [internet]. c1994-2005. st paul (mn): american phytopathological association; [cited 2005 jun 20]. available from:http://www.apsnet.org/. for more detailed examples please refer to the cse manual 7th edition. 12. the list of references at the end of the paper should include only works mentioned in the text and should be arranged alphabetically by the name of the author. 13. responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. 14. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 15. figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each f igure or table on a separate page. at the lower hand corner, the name of the author and the f igure number should be indicated. 106 16. illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2" x 11" size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructedphoto-graphic prints (not photocopies), trimmed at right angles and in the f inal size desired by the author. 17. when possible, all organisms must be identif ied by the scientif ic binomen. 18. mathematical equations should be clearly presented so that they can be interpreted properly. 19. obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 20. all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc. , especially those adopted by the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 22. less common abbreviations may be printed as footnotes. 23. short communications must be guided by the following points: • short communications are reports of limited data or important findings that warrant publication before the completion of the study; • short communications are reports of signif icant new data arising from problems with narrow, well def ined limits before broader studies are completed; and results have not been published in print elsewhere, e x c e p t a s p a r t i a l c o m m u n i c a t i o n s o r p o s t e r s i n c o n f e r e n c e proceedings; 107 • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; • abstracts will be required on submission but not to be part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages ) in length with simple layout, a maximum of two tables and two f igures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors may opt to submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor in chief science diliman off ice of the vice-chancellor for research and development university of the philippines lower ground floor, phivolcs bldg. , c.p. garcia avenue up campus, diliman, quezon city 1101, philippines camera-ready illustrations must accompany the manuscript. sdinside front cove-july-dec.2015.pmd july-december 2015 • vol. 27 no. 2 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief irene m. villaseñor, ph.d. associate editors jose maria p. balmaceda, ph.d. louis angelo m. danao, ph.d. carlos primo c. david, ph.d. alonzo a. gabriel, ph.d. marco nemesio e. montaño, ph.d. jonas p. quilang, ph.d. rene n. rollon, ph.d. arnel a . salvador, ph.d. terence p. tumolva, ph.d. managing editor gonzalo a. campoamor ii, ph.d. editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor sarah penir on the cover: the cover image is an artist's rendition of the equations used in one of the main articles of this science diliman issue. image by aina ysabel b. ramolete, university of the philippines diliman. teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university cleveland, ohio, usa kenneth buckle, ph.d. food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, jr., ph.d. department of electrical and computer engineering ohio state university university of california, irvine university of illinois, urbana, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield sheff ield, united kingdom jeanmiare e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. centre for scientif ic computing and department of physics university of warwick united kingdom raul k. suarez, ph.d. department of ecology, evolution and marine biology university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. 12info for authors.pmd 75 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the off ice of the vicechancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles and short communications may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out. the letter to the editor usually contains these: that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1" margins on all sides. each page of the manuscriptmust include continuous line numbers in the margin. all pages should be numbered consecutively on the upper right hand corner of the page. information for authors 76 7. page 1 should contain the article title, author(s), aff iliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. the author must also submit a layman’s abstract of not more than 200 words. 9. the paper should be organized as follows: abstract and layman’s abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, f igures, tables, and f igure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be def ined at f irst mention. 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the f irst author’s name plus “et al.” need to be given. references in the text should follow the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 77 examples: articles from journals: print ( section 29.3.7.1 p. 518-527) format: author(s). date. article title. journal title. volume(issue):location. example: smart n, fang zy, marwick th. 2003. a practical guide to exercise raining for heart failure patients. j card fail. 9(1):49-58. ar ticles from journals: onl ine (section 29.3.7.13 p. 557-558) format: author(s) of article. date of publication. title of article. title of journal (edition) [medium designator]. [date updated; date cited]; volume(issue):location. notes. example: savage e, ramsay m, white j, beard s, lawson h, hunjan r, brown d. 2005. mumps outbreaks across england and wales in 2004: observational study. bmj [internet]. [cited 2005 may 31]; 330(7500):11191120. available from: http://www.bmj.bmjjournals.com/cgi/repr ting/ 330/7500/1119 doi:10.1136/bmj.330.7500.1119. articles from newspapers: print (section 29.3.7.8 p. 543) format: author(s). date. title of article. title of newspaper (edition). section: beginning page of article (column no.). example: weiss r. 2003 apr 11. study shows problems in cloning people: researchers f ind replicating primates will be harder than other mammals. washington post (home ed.). sect. a:12 (col. 1). books: print (section 29.3.7.2 p. 527-534) format: author(s). date. title. edition. place of publication: publisher. extent. notes. example: schott j, priest j. 2002. leading antenatal classes: a practical guide. 2nd ed. boston (ma): books for midwives. 78 books: onl ine (section 29.3.7.13 p. 556-564) format: author(s). date of publication. title of book [medium designator]. edition. place of publication: publisher; [date updated; date cited]. notes. example: griff iths ajf, miller jh, suzuki dt, loweontin rc, gelbart wm. c2000. introduction to genetic analysis [internet]. 7th ed. new york (ny): w. h. freeman & co.; [cited 2005 may 31]. available from: http:// www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.view. .showtoc&rid=iga. toc. book chapter (section 29.3.7.2.10 p. 533) w ith ed itors format: author(s). title of article. in: editors. title of book. edition. place of publication: publisher; date of publication. notes. example: anderson rj, schrier rw. acute renal failure. in: braunwald e, isselbacher kj, petersdorf rd, editors. harrison’s principles of internal medicine. 15th ed. new york (ny); mcgraw-hill; c2001. p. 1149-1155. w ithout ed itors format: author(s). title of article. in: title of book. edition. place of publication: publisher; date of publication. notes. example: hazeltine wa. aids. in: the encyclopedia americana. international ed. danbury (ct): grolier incorporated; 1990. p. 365-366. conference proceed ings/papers (section 29.3.7.3 p. 534) publ ished without author(s) format: editor(s). date. title of book. number and name of conference; date of conference; place of conference. place of publication: publisher. extent. notes. example: callaos n, margenstern m, zhang j, castillo o, doberkat ee, editors. c2003. sci 2003. proceedingsof the 7th world multiconference on systemics, cybernetics and informatics; orlando, fl. orlando (fl): international institute of informatics and systematics. 79 publ ished with author(s) format: author(s) of paper. date. title of paper. in: editors. title of book. number and name of conference; date of conference; place of conference. place ofpublication: publisher. location. notes. example: lee dj, bates d, dromey c, xu x, antani s. c2003. an imaging system correlating lip shapes with tongue contact patterns for speech pathology research. in: krol m, mitra s, lee dj, editors. cms 2003. proceedings of the 16thieee symposium on computer-based medical systems; new york. los alamitos (ca): ieee computer society. p. 307313. unpubl ished (section 29.3.7.15 p. 566-568) format: authors(s). date of conference. title of paper. paper presented at: title of conference. number and name of the conference; place of the conference. example: antani s, long lr, thomas gr, lee dj. 2003. anatomical shape representation in spine x-ray images. paper presented at: viip 2003: proceedings of the 3rdiasted international conference on visualization, imaging and image processing; benalmadena, spain. technical repor ts (section 29.3.7.4 p. 537) format: author(s). date. title of report. edition. place of publication: publisher. extent. report no.: notes. example: feller ba. 1981. health characteristics of persons with chronic activity limitation, united states, 1979. hyattsville (md): national center for health statistics (us). report no.: vhs-ser-10/137. available from: ntis, springf ield, va; pb88-228622. dissertations and theses (section 29.3.7.5 p. 539-541) format: authors(s). date. title of dissertation and thesis [content designator]. place of publication: publisher. extent. notes. example: lutz m. 1989. 1903: american nervousness and the economy of cultural change [dissertation]. stanford (ca): stanford university. 80 group/corporate author (section 29.2.1.2.5 p. 494) format: [abbreviation of group] name of group (country). date. title. place of publication. publisher. notes. example: [iom] institute of medicine (us). 1975. legalized abor tion and the public health; report of a study by a committee of the institute of medicine. washington (dc): national academy of sciences. other internet materials (section 29.3.7.13 p. 556-564) homepage format: title of homepage [medium designator]. date of publication. edition. place of publication:publisher; [date updated; date cited]. notes. example: apsnet: plant pathology online [internet]. c1994-2005. st paul (mn): american phytopathologicalassociation; [cited 2005 jun 20]. available from:http://www.apsnet.org/. for more detailed examples please refer to the cse manual 7th edition. 12. the list of references at the end of the paper should include only works mentioned in the text and should be arranged alphabetically by the name of the author. 13. responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. 14. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 15. figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each f igure or table on a separate page. at the lower hand corner, the name of the author and the f igure number should be indicated. 81 16. illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2" x 11" size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructed photo-graphic prints (not photocopies), trimmed at right angles and in the f inal size desired by the author. 17. when possible, all organisms must be identif ied by the scientif ic binomen. 18. mathematical equations should be clearly presented so that they can be interpreted properly. 19. obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 20. all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc. , especially those adopted by the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 22. less common abbreviations may be printed as footnotes. 23. short communications must be guided by the following points: • short communications are reports of limited data or important findings that warrant publication before the completion of the study; • short communications are reports of signif icant new data arising from problems with narrow, well def ined limits before broader studies are completed; and results have not been published in print elsewhere, e x c e p t a s p a r t i a l c o m m u n i c a t i o n s o r p o s t e r s i n c o n f e r e n c e proceedings; 82 • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; • abstracts will be required on submission but not to be part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages ) in length with simple layout, a maximum of two tables and two f igures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors may opt to submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor-in-chief science diliman off ice of the vice-chancellor for research and development university of the philippines lower ground floor, phivolcs bldg. , c.p. garcia avenue up campus, diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. 13info for authors.pmd 89 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the off ice of the vicechancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles and short communications may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out. the letter to the editor usually contains these: that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1" margins on all sides. each page of the manuscript must include continuous line numbers in the margin. all pages should be numbered consecutively on the upper right hand corner of the page. information for authors 90 7. page 1 should contain the article title, author(s), aff iliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. the author must also submit a layman’s abstract of not more than 200 words. 9. the paper should be organized as follows: abstract and layman’s abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, f igures, tables, and f igure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be def ined at f irst mention. 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the f irst author’s name plus “et al.” need to be given. references in the text should follow the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 91 examples: articles from journals: print ( section 29.3.7.1 p. 518-527) format: author(s). date. article title. journal title. volume(issue):location. example: smar t n, fang zy, marwick th. 2003. a practical guide to exercise raining for heart failure patients. j card fail. 9(1):49-58. ar ticles from journals: onl ine (section 29.3.7.13 p. 557-558) format: author(s) of article. date of publication. title of article. title of journal (edition) [medium designator]. [date updated; date cited]; volume(issue):location. notes. example: savage e, ramsay m, white j, beard s, lawson h, hunjan r, brown d. 2005. mumps outbreaks across england and wales in 2004: observational study. bmj [internet]. [cited 2005 may 31]; 330(7500):11191120. available from: http://www.bmj.bmjjournals.com/cgi/reprting/330/ 7500/1119 doi:10.1136/bmj.330.7500.1119. articles from newspapers: print (section 29.3.7.8 p. 543) format: author(s). date. title of article. title of newspaper (edition). section:beginning page of article (column no.). example: weiss r. 2003 apr 11. study shows problems in cloning people: researchers f ind replicating primates will be harder than other mammals. washington post (home ed.). sect. a:12 (col. 1). books: print (section 29.3.7.2 p. 527-534) format: author(s). date. title. edition. place of publication: publisher. extent. notes. example: schott j, priest j. 2002. leading antenatal classes: a practical guide. 2nd ed. boston (ma): books for midwives. 92 books: onl ine (section 29.3.7.13 p. 556-564) format: author(s). date of publication. title of book [medium designator]. edition. place of publication: publisher; [date updated; date cited]. notes. example: griff iths ajf, miller jh, suzuki dt, loweontin rc, gelbart wm. c2000. introduction to genetic analysis [internet]. 7th ed. new york (ny): w. h. freeman & co.; [cited 2005 may 31]. available from: http:// www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.view. .showtoc&rid=iga. toc. book chapter (section 29.3.7.2.10 p. 533) w ith editors format: author(s). title of article. in: editors. title of book. edition. place of publication: publisher; date of publication. notes. example: anderson rj, schrier rw. acute renal failure. in: braunwald e, isselbacher kj, petersdorf rd, editors. harrison’s principles of internal medicine. 15th ed. new york (ny); mcgraw-hill; c2001. p. 1149-1155. w ithout editors format: author(s). title of article. in: title of book. edition. place of publication: publisher; date of publication. notes. example: hazeltine wa. aids. in: the encyclopedia americana. international ed. danbury (ct): grolier incorporated; 1990. p. 365-366. conference proceed ings/papers (section 29.3.7.3 p. 534) publ ished without author(s) format: editor(s). date. title of book. number and name of conference; date of conference; place of conference. place of publication: publisher. extent. notes. example: callaos n, margenstern m, zhang j, castillo o, doberkat ee, editors. c2003. sci 2003. proceedingsof the 7th world multiconference on systemics, cybernetics and informatics; orlando, fl. orlando (fl): international institute of informatics and systematics. 93 publ ished with author(s) format: author(s) of paper. date. title of paper. in: editor(s). t itle of book. number and name of conference; date of conference; place of conference. place ofpublication: publisher. location. notes. example: lee dj, bates d, dromey c, xu x, antani s. c2003. an imaging system correlating lip shapes with tongue contact patterns for speech pathology research. in: krol m, mitra s, lee dj, editors. cms 2003. proceedings of the 16th ieee symposium on computer-based medical systems; new york. los alamitos (ca): ieee computer society. p. 307313. unpubl ished (section 29.3.7.15 p. 566-568) format: authors(s). date of conference. title of paper. paper presented at: title of conference. number and name of the conference; place of the conference. example: antani s, long lr, thomas gr, lee dj. 2003. anatomical shape representation in spine x-ray images. paper presented at: viip 2003: proceedings of the 3rd iasted international conference on visualization, imaging and image processing; benalmadena, spain. technical repor ts (section 29.3.7.4 p. 537) format: author(s). date. title of report. edition. place of publication: publisher. extent. report no.: notes. example: feller ba. 1981. health characteristics of persons with chronic activity limitation, united states, 1979. hyattsville (md): national center for health statistics (us). report no.: vhs-ser-10/137. available from: ntis, springf ield, va; pb88-228622. dissertations and theses (section 29.3.7.5 p. 539-541) format: authors(s). date. title of dissertation and thesis [content designator]. place of publication: publisher. extent. notes. example: lutz m. 1989. 1903: american nervousness and the economy of cultural change [dissertation]. stanford (ca): stanford university. 94 group/corporate author (section 29.2.1.2.5 p. 494) format: [abbreviation of group] name of group (country). date. title. place of publication: publisher. notes. example: [iom] institute of medicine (us). 1975. legalized abortion and the public health; report of a study by a committee of the institute of medicine. washington (dc): national academy of sciences. other internet materials (section 29.3.7.13 p. 556-564) homepage format: title of homepage [medium designator]. date of publication. edition. place of publication: publisher; [date updated; date cited]. notes. example: apsnet: plant pathology online [internet]. c1994-2005. st paul (mn): american phytopathological association; [cited 2005 jun 20]. available from:http://www.apsnet.org/. for more detailed examples please refer to the cse manual 7th edition. 12. the list of references at the end of the paper should include only works mentioned in the text and should be arranged alphabetically by the name of the author. 13. responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. 14. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 15. figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each f igure or table on a separate page. at the lower hand corner, the name of the author and the f igure number should be indicated. 95 16. illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2" x 11" size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructedphoto-graphic prints (not photocopies), trimmed at right angles and in the f inal size desired by the author. 17. when possible, all organisms must be identif ied by the scientif ic binomen. 18. mathematical equations should be clearly presented so that they can be interpreted properly. 19. obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 20. all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc. , especially those adopted by the council of science editors (cse) 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promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ec to r s o n s e p te m b e r 3 , 2 0 0 6 .” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ ajp/submit.html 8guidelines.pmd 102 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a 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(adapted with permission f rom the editors of ieee sensors journal) 10call for papers-new.pmd social science diliman, vol. 9, number 1, january-june 2013 humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) vargas museum & ronald m. pascual call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 03_nable deformation quantization and representations of the real rotation group 41 deformation quantization and representations of the real rotation group job a. nable department of mathematics, college of science university of the philippines, diliman, quezon city job@math. upd.edu. ph abstract in this paper we present concrete computations of representations of the real rotation group so(3,r) arising from deformation quantization of its coadjoint orbit ωξξξξξ ≅ s 2. in particular, we construct the quantization mapping l, ls  i fs : c∞(ωξξξξξ) [[λ]] ⎯→ c∞(ωξξξξξ) [[λ]]. . where l is a is a lie algebra representation of g = so(3,r) g s | → ls ∈ εnd (c ∞(m) [[λ]]). we will prove that the left-regular representation t of so(3,r) is just t = exp ( l̂ ), where l̂ is unitarily equivalent to l. so instead of the usual hilbert space of representation, h, we need to have an associative algebra aaaaa = (c∞(m) [[λ]], λ) which gives a deformation of the commutative algebra and lie algebra structures of c∞(m). aaaaa is called a deformation quantization of the symplectic manifold m. the requirement that l be a lie algebra representation is that the equation i fs i ft − i ft i fs = { i fs , i ft} = i f[ s, t ], (s,t ∈ g ) should be satisfied. this means that the coadjoint action of the real rotation group on its orbits should be strictly homogeneous, and furthermore, that the star-product λ is a covariant star-product, i.e., the lie subalgebra h .. = span { fs : s ∈ g } of c ∞(m) is an h-relative quantization with respect to λ. an appropriate fourier transform intertwines ls and a differential operator l̂ s. it is proved that l̂ s equivalent to the action of rotation given by the element exp s of the rotation group g = so(3,r), that is, l̂ s is the differential of the left regular representation. exponentiation of the representation l̂ s gives the corresponding representations of g. key words: deformation quantization, orbit method, coadjoint orbit, symplectic manifold, representation theory, rotation group ∈ science diliman (january-june 2001) 13:1, 41-53 nable 42 introduction quantization is a process by which quantum systems are assigned to classical mechanical systems. to illustrate, n free particles are classically described by 2n coordinates (pl ,..., pn , q 1, ..., qn) where the pj’s are momenta and the qi’s are the position coordinates. h. weyl (1931) gave the following prescription for the quantization of this system: qi → q̂ i = multiplication by qi, pj → p̂ j = -i ∂ , h ∂q j where the operators on the right hand side act on hilbert space l2(rn). the theory of quantum mechanics requires that the correspondence principle holds: [ p̂ j , q̂ ] = i h { pj, q i }=δj i i , where { , } is the poisson bracket on r2n. in general, quantization means the assignment of hilbert space operators to functions on phase space. recall that the hamiltonian formulation of classical mechanics has for its framework a symplectic manifold (m,ω). the classical observables are smooth real functions on m, denoted by c∞(m), and the evolution of observables satisfies the differential equation d ft = {h, ft } dt here h ∈ c∞(m) is the hamiltonian (e.g., energy) of the system. on the other hand, heisenberg’s formulation of quantum mechanics considers a hilbert space h, the quantum observables being self-adjoint operators on h. the time evolution of observables satisfy dat ___ = -i [h, at ] = h ° at at° h, dt hwhere the hamiltonian h is a self-adjoint operator on h. a natural definition for quantization is that it is a linear mapping q : c∞(m) → {self-adjoint operators on h,} such that (q1) q(1) = idh , (q2) q({f, g}) = -i [ q ( f ), q (g)]. hthe requirement (q2) means that the lie algebra structure of functions on phase space under poisson bracket goes over to the lie algebra structure of operators under commutators. moreover (q2) limits the class of functions that can be “quantized,” that is, there is no correspondence q defined on all of the smooth functions on m when irreducibility requirements are imposed. the fact that it is not possible that all elements of c∞(m) may be made to correspond to self-adjoint operators and still satisfy heisenberg’s correspondence principle has been known for quite a while as groenewold-van hove theorem (abraham & marsden, 1978; groenewold, 1946; van hove, 1951). in the example given above, the quantizable functions are those which belong to some symbol class. mathematical theories addressing the”irreducibility” problem are the geometric quantization theory of kostant & souriau (kostant, 1970; auslander & kostant, 1971; kirillov, 1962; kirillov, 1976), berezin’s quantization (berezin, 1975, berezin 1974) and deformation quantization. the idea of quantization by deformation of structures of the algebra of classical observables was proposed by bayen, flato, fronsdal, lichnerowicz, and sternheimer in the mid 1970’s (bayen et al., 1977; 1978). it consists of replacing operators on hilbert space as the quantum observables by formal power series in some variable λ, and interpreting the correspondence principle as an equality only up to second order in λ. indeed, bayen and others suggested that: “quantum mechanics be interpreted as a deformation of the algebra of observables, and not as a radical change in the nature of observables.” as was mentioned above, there is no quantization of r2n, i.e. , there is no linear mapping f→ q (f ) satisfying deformation quantization and representations of the real rotation group 43 (ql) and (q2). one does not even have a quantization of the polynomial algebra on r2n as symmetric operators in hilbert space for which qi and pj are represented irreducibly (groenewold-van hove theorem). quite recently an analogue of this theorem was found for the sphere s2 (gotay et al., 1996) the following list apparently points to some general obstruction phenomena to quantization, that is, the examples satisfy a groenewold-van hove type theorem: 1. nilpotent basic algebras on connected symplectic manifolds; 2. t*s1 (the cylinder r × s1 ), with basic algebra e(2) which is the lie algebra of the solvable group e(2) (euclidean group); 3. basic lie algebras on compact symplectic manifolds, in particular, s2; 4. finite-dimensional quantizations of basic lie algebras on noncompact symplectic manifolds, in particular, r2n with lie algebra h (2n) (heisenberg lie algebra). in gotay (1995); gotay & grundling (1997a, 1997b); gotay & grabowski (1999); gotay, grabowski, & grundling (1997); gotay, grundling, & hurst (1995) and gotay, grundling, & tuynman (1998) the full details and the precise meaning of basic algebra are discussed. the conjecture of the existence of a general obstruction to quantization is false, however, as gotay and co-workers showed. they demonstrated that there is no obstruction to quantization of the torus t2. furthermore, as item 3 in the case of the lie algebra e(2) indicates, a corresponding obstruction should appear for basic solvable lie algebras, in analogy with item 2. this is not true since there is an infinitedimensional quantization of t*r+ ≅ r × r+ with affine basic algebra aff (r). the paper by gotay, grundling, & tuynman (1998) discusses at length the circumstances under which obstructions to quantization appear and why no such obstruction occur for the examples just mentioned. the aim of this paper is to present concrete computations of the representations of so(3,r), in particular the left-regular one, coming from deformation quantization of s2, which appear as a (semisimple) coadjoint orbit. in particular, we will show that the quantization mapping ifs :c∞(m)[[i]]→c∞(m)[[i]] is in fact the differential of the left-regular representation of so(3,r) in s2. to perform these computations, one needs to look for an appropriate coordinate system on the sphere that reflects certain properties of the lie group action on it. the polar coordinate system on s2 is a natural candidate, but it does not reflect these properties of the group action. the coordinate system arising from geodesics on s2 satisfies the requisite properties, this time on the universal covering, but the ensuing integrability of the lie algebra representation presents a problem and requires extra care in computing. thus, the application of deformation quantization to the representation theory of concrete lie groups is dependent on the proper choice of coordinate systems on coadjoint orbits. the quantization we obtain is actually a prequantization (quantization sans the irreducibility requirement), and this is consistent with the groenewold-van hove result for s2. the final step of finding the irreducible representations from among those constructed does not come from deformation quantization but from geometric quantization or the theory of induced representations. the computations performed here, it is hoped, should hint to further methods on how to treat the general case of compact semisimple lie groups, in, particular so(n, r), and also the discrete series of the noncompact semisimple classical groups. it is difficult to say the same thing for the continuous series of the noncompact groups since they require a considerable modification of the orbit method, for example, the representation space of the principal continuous series of sl(2, r) is not a coadjoint orbit in the usual sense. ideally, each of the more important considerations in representation theory (namely, characters, intertwining operators, induction, restriction, and others), or some suitable modification thereof, should be defined in terms of deformation quantization. this has been done for the case of connected, simply connected nilpotent groups and exponential groups but is not yet complete for solvable groups (arnal 1984; arnal & cortet 1990a; 1990b; 1985; arnal et al., 1983) and semisimple groups. the framework with which we shall work within is the so-called method of orbits nable 44 they allow one to see clearly what is going on. we remark further that the results in these papers are consistent with the works of gotay and coworkers. for example, there is a complete description of the list of infinite-dimensional unitary irreducible representations of aff(r) (do ngoc diep & nguyen viet hai, 1999a) in terms of deformation quantization of the coadjoint orbit r×r+, although the authors seem to have forgotten about the series of one-dimensional irreducible representations. the paper by arnal & cortet (1990b) gives a description of unitary, not necessarily irreducible, representations of e(2) in terms of the deformation quantization of the cylinder t* s l. deformation quantization definition 1 let (m,ω) be a symplectic manifold. by deformation quantization of (m,ω) we mean an associative algebra structure on the space c∞(m) [[λ]] of formal power series, in the variable λ with coefficients in c∞(m), with respect to some product λ. λ is called a star-product and we shall write λ-product. the λ-product has the form a λ b = (σ as λ s) λ (σ bt λ t ) = σ ck λ k (1) where (dq1) the coefficients ck = ck (a,b) depends not only on a and b but also on the partial derivatives ∂i α a, ∂ j β b where i + j + |α | + | β | ≤ k, (dq2) c0 = a0 b0 , (dq3) c1(a,b)-c1(b,a)=λ{a,b}, for a, b ∈ c ∞(m). (dq1) means that the λ-product is local. this is also equivalent to the condition that the ck are given by differential operators. (dq2) means that the λ-product is a deformation of the commutative pointwise product of functions in c∞(m). lastly, by defining the lie bracket [a, b ] λ = 1 (a λ b b λ a), 2λ (dq3) means that the λ generates a deformation of the poisson bracket { , } on c∞(m). introduced by a. a. kirillov in 1962. this is the framework employed in the articles by arnal (1978), arnal & cortet, (1985, 1990a, 1990b), and arnal et al., (1990). in the orbit method the basic object is a coadjoint orbit of a lie group g in the dual space ggggg* of its lie algebrag g g g g = lie(g) . the application of deformation theory to the theory of lie group representations has been pursued since the mid-1970s (fronsdal, 1989; bayen et al., 1977) although only a few results have been obtained. it was only in the early to mid-1980s that a better understanding of the application came to light. what is needed is an appropriate notion of group invariance of the starproduct. in bayen et al., (1977) the notion of geometrical invariance was introduced. however, it finds only limited application since almost all important examples of symplectic manifolds with lie group action do not have this invariance property. a correct notion of invariance, called covariance, is investigated by arnal et al., (1983). the properties of covariance is compared with those of other invariance concepts, and its applicability to representation theory of nilpotent lie groups commenced. arnal (1984) gave the first concrete application of deformation quantization to representation theory. the lie groups considered consisted of the class of connected, simply connected nilpotent lie groups and works within the framework of the orbit method of kirillov (1962). further investigations into this same class of lie groups followed (arnal & cortet, 1990a), where other familiar considerations in representation theory, such as fourier transform and group c*-algebras (gelfand-naimark-segal construction), are defined in terms of or found connections with deformation quantization. arnal & cortet (1985) and arnal et al. (1995) continued this program to exponential and type i solvable lie groups, respectively. in short articles by do ngoc diep & nguyen viet hai (1999a; 1999b), nguyen viet hai (2000a; 2000b), and arnal & cortet (1990b), the techniques that appear in the papers mentioned previously are applied to concrete lie groups, namely the motion group e(2), the real and complex affine groups aff(r) and aff(c), and the diamond groups introduced by do ngop diep (1999). we note, for example, that aff(r) is solvable but not type i. indeed these papers attempt to apply deformation quantization to other classes of lie groups. moreover deformation quantization and representations of the real rotation group 45 a more general definition of deformation quantization may be given for poisson manifolds m, manifolds with a contravariant skew-symmetric 2-tensor. the main difference with symplectic manifolds is that the bilinear mapping induced on c∞(m) may be degenerate. we shall, however, consider only symplectic manifolds in this paper . example the basic example here is that of the symplectic manifold (r2n, ω = σdpi /\ dqi), which has for its deformation quantization the algebra (c∞(r2n) [[ih/2]], m) where, u m v = exp ( ih ω−1 ∂r u ∂r v ) 2 r ∂r u ∂rv=σ ih  1 ωi1 j1...ωir jr ________ __________ 2 r! ∂xi1 ... ∂xir ∂ xj1 ... ∂ xjr = u·v + terms of higher order in ih, where u, v ∈ c∞(r2n), (x1,..., x 2n) = ( p 1,...,pn, q1,...,qn), and (ωij)=0 in. the lie bracket between u and v is -in 0 [u, v ] m = 1 sinh ( ih ω -1 ∂ru ∂rv ) 2i 2 = {u,v} + terms of higher order in (ih)2, showing the deformation of the poisson bracket. the symbol m is read as moyal star-product (moyal, 1949). it is intimately connected with the weyl quantization of r2n. for functions a on r2n belonging to symbol class (fedosov, 1993) there corresponds the operator a = a acting on the schwartz class s (rn) ˆ (au)(q)=∫ exp( i p(q-q’ )) a (q+q’, p)u(q’)dq’dp r2n h 2 this is actually the formula which gives the correspondence given in the introduction, i.e., qi |→ q ̂ i p j |→ p ̂ j. the moyal -product enters into the formula for the composition of operators â ° b̂ = a m b where a and b are symbols. the moyal -product will be very important for us in applications to the representation theory of lie groups. we introduce next the notion of covariance of a starproduct. definition 2 let (m,ω) be a symplectic manifold. a subalgebra h of c∞(m) is called an h-relative quantization with respect to the star-product λ if [u, v] λ  1 (u λ v v λ u) = {u, v}, ∀ u, v ∈ h. 2λ definition 3 let the lie group g act on the symplectic manifold m in a strictly homogeneous manner. the star-product λ on c∞(m) is said to be a covariant star-product if [ λ fs, λ ft ] λ = λ{ fs , ft} for all generating functions fs, ft , s, t, ∈ g = lie (g). the point of these definitions is that the operator of left λ-multiplication ls  λ fs : c ∞(m) [[λ]] → c∞(m) [[λ]], ls (u) = λ fs λ u, u ∈ c ∞(m) [[λ]] is a lie algebra representation of g. it remains to find some equivalent expression (e.g., differential operators), if possible, for ease of computations and, more importantly, to find invariant subspaces. exponentiation gives unitary representations of the corresponding lie group of g. deformation quantization (of poisson, symplectic, kahler manifolds, etc.) itself, i.e., without considering the representation theory of symmetry groups of the manifolds concerned, is of course of separate interest. nable 46 renewed interest in deformation quantization came along because of the works of drinfeld on quantum groups (which are deformation of lie groups), and because of b. fedosov’s solution to the problem of deformation quantization of symplectic manifolds, which employed only geometric concepts, and did not make any cohomological constructions. the corresponding and considerably more difficult deformation quantization problem for poisson manifolds was solved fairly recently by m. kontsevich (1997). fedosov’s book (1993) deals with the formulation of an index theory using deformation quantization. the works of karabegov (1996) and reshetikhin & takhtajan (1999) and others, employed similar methods as those discovered by fedosov to solve the corresponding deformation quantization of kahler manifolds and other special classes of complex manifolds. on the other hand m. rieffel (1994) gives stricter criteria for deformation quantization which takes into account c*-algebra structures on manifolds, in particular heisenberg manifolds. hamiltonian mechanics and coadjoint orbits of so (3,r) throughout the rest of this paper we let g = so (3, r), g = so(3,r) and g* = so(3,r)*. for semisimple lie subgroups of the full linear group gl(n,r), the coadjoint representation takes the following form: ad (g)s = gsg-1, g ∈ g, s ∈ g there is a nondegenerate bilinear form on g, namely, < s,t >= trace (s.t). because of this and the fact that g is semisimple, we have that g* ≅ g. (this is not true, say, for nilpotent lie groups). therefore the coadjoint representation k : g → aut (g*) is given by k(g)ξ = gξg-1, ξ ∈ g*, and the determination of coadjoint orbits is equivalent to describing conjugacy classes. choose and fix ξ ∈ g*. we may look at gξ as the group of rotations of 3-space fixing the axis determined by the vector ξ ∈ g* ≅ g ≅ r3. this means gξ ≅ so(2,r) and hence g/gξ ≅ so(3,r) ≅ so(2,r) ≅ ωξ. it is well-known that so(n+1, r)/ so(n, r) ≅ sn, the unit n-sphere. thus ωξ≅ s 2. now, coadjoint orbits are symplectic manifolds (kirillov, 1962), in which case hamiltonian mechanics may be performed on them. if there is a lie group action on a symplectic manifold m by symplectomorphisms, that is, for each g ∈ g g*ω = ω, ∀ g ∈ g (pullback of the action) we call m a homogeneous symplectic manifold and there is a structure of a g-module on c∞(m) defined by (g.u)(ξ) = u (g -1.ξ) where g ∈ g, ξ ∈ m, u ∈ c∞(m). derivation gives a g-module structure on c∞(m) and this is given by the following: to each s ∈ g = lie (g) of the lie algebra of g there corresponds a hamiltonian vector field ηs ∈ vect (m) given by (ηsu)(ξ) = d u (exp(-ts).ξ)⏐t = 0 dt the module structure is now given by (s.u)(ξ) = (ηsu)(ξ). if the ηs are strictly hamiltonian and the generating functions fs and ft satisfy f[s,t] = { fs , ft } we call m a strictly homogeneous symplectic manifold. the generating function fs is defined as the solution of the differential equation i(ηs )ω = -d fs . the following short exact sequence of lie algebras reflects the strictly hamiltonian character of the action of g on m. 0 → r → c∞(m,r) → g → 0 (2) deformation quantization and representations of the real rotation group 47 although the mapping s |→ ηs is a lie algebra representation of g, it does not give us a quantization since ηs sends all constants to zero. instead we work on the space of generating functions fs and perform a deformation quantization of this space. a very important class of strictly homogeneous symplectic manifolds are coadjoint orbits of a connected lie group g in the dual space g*. in fact, we know the following from kostant: theorem 1 (kostant) any symplectic manifold m with a hamiltonian g-action, where g is a connected lie group, is locally isomorphic to a coadjoint orbit of g or a real central extension g~ of g. this theorem implies that the coadjoint orbits are all the classical systems that we need. quantization of the sphere a criteria in choosing the appropriate coordinates in (ωξ, ωξ) is that it must reflect the strictly g-homogeneous action of the group g on ωξ, i.e., { fs , ft} = f[s, t] , for generating functions fs , ft ∈ c ∞(m). another criteria is the relation of relative quantization or the covariance of star-product [λ fs ,λ ft ] λ  1 (λ fs λ ft − λ ft, λ fs) =λ{fs , ft}. 2λ with respect to the polar coordinate system τ : r 2(p,q)|→ (sin p sin q, sin p cos q, cos p) ∈ s 2 ≅ ωξ, the moyal star-product is not a covariant star-product. this means that the lie algebra of generating functions g~ does not have a relative quantization with respect to this star-product. however, this coordinate system is interesting since it provides a deformation quantization of the algebra c∞(s 2) arising from the moyal -product on r2. since the polar coordinate system is global, -products of functions hold globally (up to linear transformations). a parametrization which suits the requirement of covariance will now be explained. this time, however, the action of g on ωξ is not strictly g-homogeneous, in which case, we use theorem 1 which states that it is the real central extension g~ of g which acts on the simply connected covering ω ~ of ωξ. in a strict g-homogeneous manner. fix the functional ξ=x* ∈ g* = so(3,r)*. the coordinate system that we need is the one coming from the exponential mapping exp : tx* (ωx*) → ωx* , where tx*(ωx*) is the tangent space to ωx* at x*. since tx*(ω x*) ≅ tx* (s 2) ≅ r2, exp will give a local diffeomorphism ϕ :r2→ωx* . this local diffeomorphism is given ϕ (p,q) := exp(py*+qz*) = (cos1)x* + (sin1)(py*+qz*) we modify this mapping and use the simpler ϕ (p,q) :=z* + py* + qz* replacing cos 1 and sin 1 with 1. the effect is that the symplectic form ω appearing below will not have the factor sin21. the symplectic form on ωx* is now given by ω (ηs ⊗ηt) = < x * , [s,t] >. the hamiltonian function f s associated to s = α1x + β1y + γ1z ∈ g is given by fs (p,q) = < ξ, s>= < x *+py* +qz*, α1 x+β1y + γ1z > = α1 + β1p + γ1q the hamiltonian vector field ηs is given by ηs = β1 ∂ − γ1 ∂ . ∂q ∂p indeed, ηs( f ) = ∂ fs ∂ f − ∂ fs ∂ f , ∂p ∂q ∂q ∂p ___ __ ___ __ the right-hand side being the poisson bracket in c∞ (r2 ). nable 48 write ω = ϕ*ωx* and let t = α2x + β2y + γ2z be another element of g. then ft = α2 + β2 p + γ2 q and ηt = β2 ∂ − γ2 ∂ . we have ∂q ∂p <ω, ηs ⊗ηt > = < ω, (β1γ2 − β2γ1) ∂ ⊗ ∂ + ...> ∂p ∂q = β1γ2 − β2γ1 = < x *, (β1γ2 − β2γ1) x + ...> = < x *, [s, t] > = <ωx* , ηs ⊗ηt >. therefore, we conclude that ω = ϕ∗ωx* = dpdq. before proceeding, let us first summarize our discussion. theorem 2 1. the exponential mapping exp: tx* (ωx*) → ωx* gives rise to a local diffeomorphism (in fact, a symplectomorphism) ϕ : r2 → ωx* given by ϕ (p,q) = x* + py* +qz*. 2. under the mapping ϕ in 1., the hamiltonian function fs associated to a vector s = αx +βy + γz∈g has the form fs (p,q) = α + β p + γ q; the hamiltonian vector field ηs ∈ vect{ ωx*) has the form ηs = β ∂ − γ ∂ ; ∂q ∂p and the kirillov symplectic form ω is ω = ϕ∗ωx* = dp  dq. the operator lllll̂s the action of the real rotation group g on ωx* is only hamiltonian (or homogeneous) and not strictly homogeneous on account of the relation { fs , ft} = f[s, t] c (s,t) where c( . , .) is some 2-cocycle. the form c disappears when we consider, instead of g, the lie algebra g~ = {s ~ = (s, t ) : s ∈ g , t ∈ r} ≅ g ⊗ r with lie bracket [(s, t1),(t,t2)] = ([s,t], c (s,t)). the lie algebra g~ may be obtained in another way. it is simply the lie algebra under poisson bracket which is generated by generating functions fs, s ∈ g. indeed from the exact sequence (2) of lie algebras c∞(m) ≅ g ⊗ r ≅ g~.. what this direct product means is the following. the solution of the differential equation df = −i(ηs)ω is unique only up to a constant addend, that is, fs and fs + const solve the differential equation. thus, for a vector s ∈ g, fs and fs~ differ only by a constant. although fs ft and fs~ ft~ differ by a constant, [ fs , ft] = [fs~ , ft~ ] . therefore the covariance property of the moyal star-product still reads the same, where, upon choosing λ= i [ ifs~ , ift~ ] = i{ fs~ , ft~ } = if[s~ t~ ] . this means that the operator ls : c∞(m) [[i]] ⎯→ c∞(m) [[i]], m = r2 ≅ ωx* , , , , , ls := ifs is a representation of the lie algebra g. to be more precise, the operator should be ifs~ and s |→ ls = ifs~ is a representation of the lie algebra g~ = su (2) (coming from the lie group g~ = su(2) which is the universal covering group of g=so(3,r)). however, the appearance of a constant addend merely goes into the constant multiplier appearing in the differential operator expression for ls (to be obtained as follows). deformation quantization and representations of the real rotation group 49 theorem 3 let s=αx+βy+γz ∈ g and let f ∈ c∞0 (r2) be a smooth function with compact support. then we have l̂ s ( f )  f1°ls°f1 -1 ( f ) = β ( 1 ∂ − ∂ )( f ) + iγ (q − x )· f + iα· f . 2 ∂q ∂x 2 here, f1( f )(x,q) = 1 ∫e-ipx f (p,q) dp √2π r is the fourier transform with respect to the first variable. the inverse fourier transform is f1 -1 ( f )(p,q) = 1 ∫ eipx f (x,q) dx √ 2π r proof of theorem 3 l̂ s  f  = f1°ls°f1 -1 f  = f1 i fs °ls°f1 -1  f  = if1 fs ·f1-1  f  + σ 1  1 )r p r  fs ,f1-1  f  r ≥1 r! 2i = if1fs·f1-1 f + 1 p1( fs ,f1-1 f  2i = if1α+β p+γ q·f1-1 f  + 1 βf1 -1 ∂f −γ ∂ f1 -1f  2i ∂q ∂p __ __ __ = if1 α·f1-1  f  + iβf1-1  ∂f  +γ f1 -1 q·f  ∂x + β f1 -1 ∂f  − iγ f1 -1 x·f  2i ∂q 2i __ __ __ __ = iα · f − β ∂f + iγ q· f + β ∂f − i γ x·f ∂ x 2 ∂q 2 ______ ____ _____ ____ = β 1 ∂ − ∂ · f + iγ q − x · f + iα · f . 2 ∂q ∂x 2 therefore, for s= α x +β y + γ z ∈ g, l̂ s = β  1 ∂ − ∂ + iγ q x  + iα , 2 ∂q ∂x 2 which takes the form l̂ s = β ∂ + iγs + α 3 ∂s upon changing to new variables s = q− x/2, t = q + x/2. the (generating) function fs = α +β p+γ q is called the symbol of the differential operator l̂ s . if, instead of ξ =x*, we chose ξ =y* (resp. ξ =z*), then elements of ωy* (resp. ωz*) have the form ξ p,q = px * + y * + qz *, (resp. ξ =px* + qy* + z*). of course, ωx* , ωy* ,ωz* are one and the same orbit since the definition of the coadjoint representation (k(g)ξ = gξg-l) and of an orbit ωξ imply that the elements x *, y *, z * of g * are conjugate to each other by rotations of the sphere. we note also that if, say, ξ= px * + y * + qz * ∈ ωy* and s = αx + βy + γz ∈ g then fs p,q = αp + β + γq, l̂ s = α ∂ + i γs + β . ∂s in this connection we need to make sure that for a given element s ∈ g, l̂ s is a unique operator, independent of change of charts. for a linear symplectomorphism f, say, from ωy* to ωx* , f  ϕx ° ϕy : ωy* → r 2 → ωx* , there corresponds a unitary operator u (fedosov, 1993), such that l̂ s = u ° l̂ (f,s) ° u-1 where the operator on the left hand side is with respect to the chart ωy*. and the operator in the middle of the right hand side is l̂ s, with respect to the chart ωx*. nable 50 furthermore, the local operator l̂ s has a unique global extension to all of ωξ ≅ s 2 since s 2 is simply connected (monodromy theorem (kirrilov, 1976)). the conclusion is that there is a unique operator, l̂ s up to a unitary operator u. finally, we make the remark that the operator l̂ s , aside from allowing for the fairly simple computations below, exhibits the invariant subspace c∞(ωx*) ⊕ ic ∞(ωx*) ⊂ c∞(ωx*)[[i]]. in effect the fourier transform takes care of convergence issues regarding the star-product. we next show that the mapping g  exp(ts)| → exp(tl̂ s) ∈aut (l2(s2)) is just the left (or right) regular representation t of the rotation group g acting on the hilbert space l 2(s2). restricting to the (2l +l)-dimensional (l = 0,1, ...) subspace of harmonic functions on the sphere gives the complete list of unitary irreducible representations of g. to do this, we first set s = y to obtain the operator exp(tl̂ y). then we shift charts and, in turns, let s = x and s = z to get the operators exp(sl̂ x) and exp(rl̂ z). since the group elements exp x, exp y, exp z generate g, the operators give the unitary irreducible representations upon restricting to the space of harmonic functions inside l2(s2). let now s = y so that, ∞ (ty)n cos t 0 sin t exp (ty) = σ _____ =( 0 1 0 ) n=0 n! -sin t 0 cos t 1 0 0 similarly, we have exp (sx) =( 0 cos s sin s) 0 -sin s cos s cos r sin r 0 and exp (rz) =(-sin r cos r 0 ) 0 0 1 . then t(exp(ty)) f (xi, x2, x3) = f (xi cos t + x3sin t, x2, x1sin t + x3cos t). this is just the action of rotation (by an angle t) in 3-space preserving the y-axis. consider a function f ∈ c ∞(ωx*). we look at the restriction of f = f (xi, x2, x3), to the great circle x1 2 + x3 2 = 1 in s2, in which case we may write it as f = f(eis) (eis = x1 + ix3). consequently, t(exp(ty)) f(eis) = f(ei(t+s)). now l̂ s = l̂ y= 1• ∂ + i • 0 • s = ∂ , so that if we put ∂s ∂s w = ei(t+s), we have ∂ t(exp(ty)) f (w) = ∂ f (exp ty • eis) ∂t ∂t = ∂ f (ei(t+s)) ∂t = ∂w ∂ f (w) ∂t ∂w = ieiseit ∂f _ ∂w = ∂ f (ei(t+s)) ∂s = l̂ y t (exp (ty )) f (e is). because t (exp (ty)) f (w)⏐t=0 = f(w), the unique solution to the cauchy problem { ∂ u(t,w) = l̂ y u (t ,w) u(0,w) = id ∂t is u(t,w) = exp (tl̂ y ) f (w) thus exp (tl̂ y ) f (w) = t (exp (ty )) f (w) = f (exp(ty )·w). deformation quantization and representations of the real rotation group 51 similarly exp(sl̂ x) f (w) = f (exp (sx)·w), exp(rl̂ z) f (w)=f (exp (rz)·w). theorem 4 the operators exp(sl̂ x), exp(tl̂ y), exp(rl̂z), where x, y, z are basis elements for the lie algebra so(3,r) provide the irreducible unitary representations of s0(3,r) by restricting them to the space of harmonic functions on the sphere. these representations are exactly the representations t = t ωx* associated to the orbit ω x* ≅ s 2, in accordance with the method of orbits. more precisely, to each positive integer l , tωx* is the (2l +1)-dimensional irreducible unitary representation on the space l2(s2) of spherical functions of order l, that is, the square integrable functions on the sphere satisfying _________ ______ ______ __________ _________ 1 ∂ (sin θ ∂ f )+ 1 ∂ 2 f + l (l+1) f = 0 (4) sinθ ∂θ ∂θ sin2θ ∂2ϕ we call this the space of harmonic functions on the sphere. acknowledgments i would like to express my deep gratitude to professor do ngoc diep, of the hanoi institute of mathematics, for his help in introducing me to the kind of mathematics presented here and who greatly influenced me in the kinds of 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pp. van hove, l., 1951. sur certaines representations unitaire d’un groupe infini des transformations. proc. roy. acad. sci. belg. 26: 1-102. vey,. j., 1975. deformation du crochet de poisson sur une variete symplectique. comment math helv. 50: 421-454. weyl, h., 1931. group theory and quantum mechanics. new york, dover: 383 p. subscription form method of payment (please check one)  pay cash at the ovcrd (see address above)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address 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his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. 4-bernardo-low-complexity.pmd n.i.m. bernardo and f.a . de leon 5 science diliman (january-june 2018) 30:1, 5-23 low-complexity physical layer security scheme for heterogeneous cellular networks based on coordinated precoding design and artif icial noise generation neil irwin m. bernardo* electrical and electronics engineering institute university of the philippines franz de leon electrical and electronics engineering institute university of the philippines abstract the undertaking for higher capacity and seamless wireless connectivity i n n e x t g e n e r a t i o n m o b i l e n e t w o r k s w h i l e m a i n t a i n i n g a n e n e r g y eff icient transmission requires a fundamental redesign of the existing cellular architecture. heterogeneous network (hetnet) deployment is a p r o m i s i n g a r c h i t e c t u r a l f r a m e w o r k f o r m e e t i n g t h e s e d e s i g n g o a l s . however, an increase in cellular capacity and device connectivity would also result in an increase of sensitive data and classif ied information being exchanged over the network, thus making security another critical aspect in cellular network design. in this study, a convex optimization model was formulated that minimizes the total power consumption of t h e n e t w o r k w h i l e s a t i s f y i n g c e r t a i n l e v e l o f p e r u s e r d a t a r a t e r eq u i r e m e n t a n d i n fo r m a t i o n s ec r ecy a t t h e p h y s i c a l l a ye r. f r o m t h i s model, a low-complexity physical layer security scheme was developed that exploits coordinated precoding design, ar tif icial noise generation, a n d a s u b o p t i m a l s l e e p m o d e s t r a t e g y i n h e t n e t s . s i m u l a t i o n r e s u l t s s h o w t h a t j o i n t o p t i m i z a t i o n o f c o o r d i n a t e d p r e c o d i n g s c h e m e a n d a r t i f i c i a l n o i s e g e n e r a t i o n i s a n e f f e c t i v e a p p r o a c h f o r i n c r e a s i n g cellular capacity while simultaneously lowering the transmit power of the base stations and risk of eavesdropping attacks. incorporating sleep m o d e m e c h a n i s m i n p h y s i c a l l a y e r s e c u r i t y t r a n s m i s s i o n s c h e m e o f hetnets also reduced the total power consumption while maintaining a _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online low-complexity physical layer security scheme 6 s e c u r e d a n d r e l i a b l e c o m m u n i c a t i o n d u r i n g l o w t r a f f i c p e r i o d s . f u r t h e r m o r e , o u r p r o p o s e d p h y s i c a l l a y e r s e c u r i t y s c h e m e e x h i b i t e d signif icant reduction in computational complexity, but at the expense of slight performance degradation in terms of energy eff iciency. keywords: physical layer security, heterogeneous networks, small cells, 5g introduction the rapid growth of the number of mobile devices and data traff ic created a demand for cellular technologies that can provide extremely high data throughput and seamless wireless coverage. one approach to meet this demand is through the dense deployment of multiple low-power small cell access nodes (scas) in order to complement the performance of high-power base stations (bs), thus forming a heterogeneous network (hetnet) (nigam et al. 2014). scas can be deployed on hot spots with high throughput demand to offload the traff ic from the macro-cell bs or on coverage holes to ensure uninterrupted connectivity. this results in large cellular capacity improvement. several studies have also shown that, with proper coordinated transmission and interference mitigation schemes, heterogeneous network deployment is an energy-eff icient alternative to traditional macro-cell-only cellular architecture in meeting the data rate requirement of future wireless applications (bjornson et al. 2013; tang et al. 2015; nguyen et al. 2016; vu et al. 2016). due to these advantages, several regulatory bodies, as well as some major players in te l e co m m u n i c a t i o n i n d u s t r y, h a ve a c k n ow l e d g ed h e t e r o g e n e o u s n e t w o r k deployment as an enabler for f ifth generation mobile networks or 5g (international telecommunication union 2014; samsung electronics co. 2015). aside from improvements in energy and spectral eff iciency, cellular network security has also become a signif icant concern in mobile networks. the increase in data traff ic and number of connected device had increased the risk of data theft and eavesdropping as more and more conf idential data are being exchanged over the network (yang et al. 2015). furthermore, the growth of internet-of-things (iot) would also require a suff icient security scheme that is both computationallyeff icient and energy-eff icient for low-power iot devices (trappe 2015). security solutions for wireless devices are traditionally implemented at the application layer in the form of cryptography. however, the performance of these cryptographic protocols is highly dependent on the assumption of limited computational power of the eavesdropper, thus introducing potential vulnerabilities to network’s data conf identiality (mukherjee et al. 2014). n.i.m. bernardo and f.a . de leon 7 an alternative approach to encryption strategies is to implement security at the physical layer (phy) of the network. phy security can provide a quantif iable and i n f o r m a t i o n t h e o r e t i c s e c u r i t y t o t h e n e t w o r k b y e x p l o i t i n g t h e r a n d o m characteristics of wireless channel. hetnets are able to provide reliable security at the physical layer using coordinated precoding design (lv et al. 2015; bernardo and de leon 2016), resource allocation strategy (shiqi et al. 2016), and artif icial noise (an) generation (deng et al. 2015; wang et al. 2016) in fending off eavesdropping attacks. however, most studies on phy security performance of hetnets assume that scas are always in active mode. in an energy eff iciency perspective, time periods with low user density and low traff ic would leave multiple scas idle, thus resulting in wasted energy. in practice, scas have sleep mode mechanism to adapt with high spatiotemporal variations in user density and mobile data traff ic (hoydis et al. 2011). furthermore, phy security approaches presented in the work of lv et al. (2015) and bernardo and de leon (2016) have very high computational complexity, making them infeasible to implement in multi-tier cellular networks with dense layer of scas. as such, there is a need to implement computationallyeff icient phy security solutions for hetnets that consider sleep mode capabilities of scas. this study presents a low-complexity phy security scheme for heterogeneous cellular networks. we formulate an optimization model that solves for the optimal precoding vectors and an signals which minimize the total power consumption of a heterogeneous network, while satisfying the quality-of-service (qos) requirement of every user, the transmit power limitations of macro-cell bs and scas, and a certain degree of secrecy against eavesdropping attacks. moreover, an algorithm that incorporates the sleep mode capability of scas in precoding and an an design to further reduce the total power consumption is proposed. to the best of our knowledge, no prior study has been done with regards to the development of fast and low-power phy security techniques for hetnets that consider their sleep mode capability. system model and algorithm formulation system model for heterogeneous networks we consider a single cell downlink scenario of a two-tier heterogeneous cellular network serving k authorized single-antenna user equipment (ue) (figure 1). the macro-cell bs and scas are connected via a high capacity backhaul network which enables joint spatial soft-cell resource allocation (parkvall et al. 2011; bjornson et al. 2013). the backhaul network facilitates the information exchange and low-complexity physical layer security scheme 8 interference coordination of the network. furthermore, the bs and scas allow spatial multi-flow transmission so that a ue can be served by multiple bs. in spatial multi-flow transmission, the bs and scas convey the same information symbol for kth ue, denoted by s k , but independently apply precoding on the information symbol before transmission (holma and toskala 2012). in addition, the macro-cell bs also transmits an an signal to degrade eavesdropper reception. an signals do not carry any user information and are only emitted for the sole purpose of disrupting the eavesdroppers. the transmitted signals of the macro-cell bs and scas are given by: where w k,0 ∈cnbsx1 and w k,0 ∈cnscx1 are the precoding vectors of the bs and jth sca, respectively. n bs denotes the number of antennas at the macro-cell bs and n sc denotes the number of antennas at each scas. v 0 ∈cnbsx1is the an vector transmitted by the macro-cell bs. k eve single-antenna eavesdropping terminals are placed within the macro-cell and try to listen to the transmit signals x 0 and x j . furthermore, the eavesdroppers are assumed to be colluding; that is, an eavesdropper can share its observation of x 0 and x j with other eavesdroppers. collusion of eavesdroppers was modeled as a single eavesdropper with multiple antennas located at different locations in the cell k k k,0 k, j k 1 k 1 s and s      0 k,0 0 j k, jx w v x w   (1) figure 1. illustration of a generic heterogeneous network: a macro-cell base station with nbs antennas and multiple small-cell access points communicate with k single-antenna ues uniformly distributed over the macro-cell area. low-complexity physical layer security scheme 10 p static is the static power consumption which models the total power dissipation due to rf circuitry of the macro-cell bs and m scas. p cir , j and p idle , j denote the perantenna circuit power consumption and non-transmission power consumption of the jth bs, respectively. the dynamic power consumption, denoted as p dynamic , accounts for the aggregated emitted power of the macro-cell bs and m scas. the parameter m][0,j1,/ρ1 j  models the power amplif ier eff iciency at jth transmitter. higher value of jρ means lower power amplif ier eff iciency, thus resulting in higher transmit power. since the amplitude of signal transmission at each antenna element is controlled by the precoding vectors, the dynamic power consumption is proportional to the power allocated to each precoding vectors. furthermore, the model for the dynamic power consumption presented in this work also accounts for the power allocated by the macro-cell bs in generating the an signals. to limit the maximum allowable transmission power of the macro-cell bs and scas, transmit power constraints should be imposed on the optimization model. these transmission power limits can be represented using the following inequality constraints:   m s t a t i c c ir , j s c i d le , j j 0 p p n p    (4) m k 2 2 dynam ic j 0 j 0 k 1 p ρ ρ      k, j 0w v (5) subcarrier bandwidth of 15 khz (similar to what is currently used in 4g cellular systems). furthermore, it was also assumed that the separation between antenna elements is wide enough, such that users and eavesdroppers experience independent and identically distributed rayleigh fading. formulation of the optimization model as a starting point in the formulation of the optimization model, the objective function presented in our previous work (bernardo and de leon 2016) was modif ied. the objective is to optimize the power consumption of the network, while satisfying the qos constraints and phy security requirement of each ue. the objective function in the model is the total power consumption, denoted as p total = p dynamic + p static , where k k 1 0 1, ... ,j,l scq j , l n       hk, j j,l k, jw q w (6) n.i.m. bernardo and f.a . de leon 11 where (6) denotes the transmit power limit for each scas and (7) denotes the transmit power limit for the macro-cell bs. the terms q j , l and q 0 , l denote the transmit power limit imposed on the lth antenna element at the jth sca and macro-cell bs, respectively. the main difference between the (6) and (7) is the inclusion of the an signal term in the inequality. q j,1 ∈cnsc× nsc and q 0,1 ∈cnbs × nbs are weighting matrices for the constraints. since per-antenna transmit power limits are desired, the weighting matrices are only non-zero at the lth diagonal element and zero elsewhere. in addition to transmit power constraints, data rate requirement of each user should be def ined. as such, quality-of-service constraints are included in the optimization model which specif ies a minimum target for the information rate (in bits/s/hz). these constraints can be def ined as follows: where γ k is the minimum information rate that the kth ue must satisfy; and sinr k is the signal-to-interference and noise ratio at the kth ue, and can be expressed as: m 2 j 0 k k m 2 2 2 k i k j 0 s i n r σ               h k , j k , j h h k , j i , j k , 0 0 h w h w h v (9) denotes the noise power at the kth ue. information symbols not intended to the kth ue, as well as non-information bearing an signals, are treated as interference. the inequality constraint for the qos is based from the well-known shannon’s capacity formula (shannon 1948) which relates the theoretical limit for information rate to the received signal quality. this qos constraint formulation has also been adopted in several research works (bjornson et al. 2013; lv et al. 2015; bernardo and de leon 2016; nguyen et al. 2016).     k 0 k 1 1,... ,,l bsq l n       h hk,0 0 0,l k,0 0w v q w v (7) σ k 2 2 k klo g (1 s in r ) , k {0 , 1 , ..., }k    (8) low-complexity physical layer security scheme 12 finally, a set of phy security constraints that limits the signal quality at the eavesdroppers should be incorporated to the optimization model. this set of constraints can be expressed as: using the total power consumption in (4) and (5) as the objective function and inequalities (6), (7), (8), (10) as constraints, the optimization model can be formulated as: δ k is the maximum allowable signal-to-noise ratio (snr) that the eavesdropper can detect from the downlink transmission of the kth ue. snr eve , k describes the combined signal quality of the signals intended to the kth ue at the colluding eavesdroppers’ side. snr eve , k excludes the interference terms from signals intended to other ue, and is only degraded by the transmitted an signal and eavesdropper noise power . this was implemented to assume the worst-case scenario, wherein the eavesdropper can decouple the information for multiple ue. phy security constraint in (10) is derived from the secrecy capacity formula given as: 2 k 2 eve, klo g (1 s in r ) lo g (1 s n r )sc     (11) which follows the work of lv et al. (2015). secrecy capacity is the highest information rate at which the transmitter and the intended receiver can communicate while the eavesdroppers receive an arbitrarily small amount of information. equation (11) also relates the secrecy capacity of the communication channel to the received signal quality of the eavesdroppers. reduction of the signal quality at the eavesdroppers would increase the secrecy capacity. by setting a target secrecy capacity c s and minimum qos target γ k , the lower bound value for δ k can be obtained using the following expression: 22 ^ (log (1 ) ) 1k k sc     (12) eve, k m 2 j 0 eve, k 2 2 eve s n r k s n r σ k w h ere        heve, j k, j h ev e, 0 0 h w h v (10) σ eve2     dyna m ic sta tic k , j 2 k k k k 1 k 0 0 k 1 eve , k m in . p p s . t . lo g (1 s in r ) k , , , s n r k j,l ,l k q j 0 ,l q l                     0 k, jv , w h k, j j,l k , j h h k,0 0 ,l k,0 0 w q w w v q w v (13) n.i.m. bernardo and f.a . de leon 13 the above optimization model is not a convex optimization problem due to the qos constraints and phy security constraints. as such, the model must be reformulated in order to be computationally tractable. semi-def inite relaxation trick, similar to what was used in the work of bjornson et al. (2016), was applied to the model. by letting , , a n d i g n o r i n g t h e requirement that w k, j and v 0 should be rank-1 matrices, the original optimization model is transformed to:        k j 0 static k, j j 0 k 1 k 2 k j 0 i 1k k k 1 k 0 k 1 2 eve m in . ρ tr( ) ρ tr( ) p 1 s . t . 1 σ , k tr , ,1 j m tr σ tr t m m j,l ,l q l q , l                                       k, j k, j 0 w h h k, j k, j i, j k, j k,0 0 k,0 j,l k, j 0 ,l 0, j 0 h eve,0 0 eve,0 w v h w w h h v h q w q w v h v h  j 0 k r , k m                k, jhev e, j ev e, j w h h (14) where . the removal of rank constraint implies that multiple transmitters can serve a ue, which is justif ied since spatial multi-flow transmission is allowed in our system model. furthermore, the constraints were transformed into linear matrix inequalities (lmi) which inherently have a convex structure. thus, the resulting optimization model is a convex semi-def inite program (sdp) and the global optimum solution can be solved numerically using convex solvers. design of low computational complexity algorithm for phy security the solution obtained from the derived optimization model in the previous section is our benchmark for the achievable performance of heterogeneous network. the solution to the benchmark model can be calculated in polynomial time. however, the problem becomes infeasible to implement in real-time if n bs and n sc have large values. thus, it is necessary to develop an alternative optimization model with complexity independent on n bs and n sc . this is achieved by assuming that the precoding vectors and an vector can be expressed as: 2 1kk     w k, j ∈sn = w k, j w k, j+ h v 0 ∈ sn = v 0 v 0+ h ,k jpk, j k, jw u 0 1 p p p p r   v fand (15) low-complexity physical layer security scheme 14 2 2 2 , , , , ,k i j eve k j eve pg g d   h h h k, j i, j ev e, j k, j eve,0 ph u h u h f (18) 2 ) the jth sca sends the scalar values g k,i,j and g evek,j to the macro-cell bs. the macro-cell bs solves the convex optimization problem in (19) to obtain the optimal power allocation strategy, denoted by p k, j and r p . where p k,j is the power allocated to w k,j , and u k,j is the unit vector that specif ies the direction of the w k,j . the expression for the regularized zero-forcing (rzf) precoding presented in the work of bjornson et al. (2013) is used to compute u k,j . the rzf precoding expression is given as: 1 k 2 i 1 i , 1 k 2 i 1 i , 1 σ 1 σ k j l k j l k q k q                       h i, j i, j k k, j h i, j i, j k h h i h u h h i h (16) v 0 is expressed as a linear combination of the p column vectors f 0, … , f p . the unit an vector components f p are chosen such that ,p  h 0 p h f 0 where k i 1   h0 i , 0 i , 0h h h (17) i.e. the an vector components should lie on the null space of h 0 . this an generation scheme is known as the null space method (goel and negi 2005; zhou and mckay 2010). p denotes the nullity of matrix h 0 . r p is the power allocated to f p . to ensure that the nullity is nonzero, n bs is set to be greater than k. since the direction of the precoding vectors and an vector components are known, the optimization model can be formulated as a power allocation strategy problem. the proposed low-complexity algorithm is implemented as follows: 1) each transmitter j=0 to m computes the following quantities in parallel: * * n.i.m. bernardo and f.a . de leon 15 (19) 3 ) the optimal values *, jkp that satisfy (19) are sent to the jth sca. the precoding vectors and an vector can now be computed by the macro-cell bs and scas using equation (15). the optimization model presented in (19) is a linear problem (lp) which can be eff iciently solved by convex solvers. moreover, the unknown quantities are realvalued variables instead of complex-valued semi-def inite matrices. the interference term caused by the an signal to the ue is removed since an signals are orthogonal to all ue channel vectors. the use of chebyshev norm (   ) for the transmit power constraints provides an upper bound value on the per-antenna transmit power. this was done to remove the dependency of the optimization model on the total number of antenna elements. modif ications to incorporate sleep mode capabil ities in proposed algorithm sleep mode mechanism of hetnets is essential in order to reduce power consumption at low traff ic periods. to include the sleep mode capability of scas in the optimization model given in (19), boolean variables that determine the state of the jth small cell node, denoted as λ j , were incorporated in the optimization model. a value of ‘1’ indicates that the small cell is in active mode and a value of ‘0’ indicates that the small cell is in sleep mode. with this, the optimization model can be stated as follows: low-complexity physical layer security scheme 16 (20) where p static,0 and p static, j are the static power consumption of the macro-cell bs and jth sca, respectively. p static, j is the power consumption of the jth sca when it is in sleep mode. the last term in the objective function indicates that an sca can only be either in sleep mode or in active mode, and that its static power consumption will depend on its state. the introduction of the boolean variable λ j makes the problem intractable due to loss of convexity. lp relaxation can be applied in order to obtain an approximate solution to the problem. this is done by replacing the boolean variables by continuous variables (i.e. 0 ≤ λ j ≤ 1  j ). replacing the boolean constraints in (20) provides a convex structure to the model. the resulting optimization model can be used to determine an approximate value of λ j that lies within the interval [0, 1]. decision on whether the jth sca should be in sleep state or active state can be determined by comparing the approximate value of λ j with some threshold τ. λ j assumes a value of ‘1’ if the approximate value is greater than τ and ‘0’ if otherwise. once a decision on the states of scas has been made after solving optimization model (20), the proposed low-complexity algorithm in (19) can be used to calculate the precoding and an vectors, wherein scas in sleep mode are ignored in the power allocation strategy. although the proposed phy security algorithm which incorporates sleep mode mechanism of hetnets has a suboptimal power allocation strategy due to constraint relaxations, it is shown in the next section that it outperforms the achievable performance of hetnets without sleep mode mechanism when qos requirement is low. results and discussion the total power consumption of the proposed low-complexity algorithm is analyzed for varying per-user qos requirement. the optimal solution of the model derived n.i.m. bernardo and f.a . de leon 17 in (14) serves as a baseline for performance assessment. the hetnet consists of one macro-cell bs placed at the center and 18 scas strategically deployed as shown in figure 2. the hetnet serves 10 ue, while 10 eavesdroppers uniformly distributed within the macro-cell coverage area try to intercept the data intended for each ue. the positions of ue and eavesdroppers are taken from a uniform distribution with lower and upper bound distances from the macro-cell bs of 35m and 500m, respectively. furthermore, a constraint was added that the minimum distance between a mobile terminal and an sca is 5m. these distance constraints are necessary to ensure the validity of the channel model parameters in table 1. the value of δ k is f ixed at 20.1-1 = 0.0717 for all users. hardware parameters used in the simulation are listed in table 2, which mainly follow those used in the works of nguyen et al. (2016) and tang et al. (2015). figure 2. illustration of a single-cell downlink scenario. bs and scas are f ixed, while ue and eavesdroppers are uniformly distributed within the cell. table 2. list of hardware parameters used in simulation number of antennas 16 4 max transmit power p 0,max = 43 dbm p j,max = 30 dbm q 0,l = p 0,max /n bs q j,l = p j,max /n sc per-antenna circuit power 189 mw 5.6 mw power amplif ier eff iciency 38.8% 5.2% non-transmission power 30 dbm 20 dbm sleep mode power 5 dbm macro-cell small-cell low-complexity physical layer security scheme 18 average total power consumption per 15 khz subcarrier for different values of peruser qos target is depicted in figure 3. the optimization models presented in the previous section were implemented using a matlab-based modeling system for convex optimization called cvx (2010). the simulation results show that progression in average total power consumption is observed as per-user qos target is increased. although the proposed scheme enables fast precoding and an vector design, deviation from the baseline performance is observed. the discrepancy in performance can be attributed to the heuristic structure of rzf precoding scheme, as discussed in the work of bjornson et al. (2014). in the derivation of the rzf precoding structure, an assumption that the lagrange multipliers for each user are of equal value was made, in order to reduce the complexity of the problem. however, this assumption causes a slight degradation in performance– a necessary trade-off to achieve practical implementation. rzf precoding is discussed in detail in the work of bjornson et al. (2014). nevertheless, the performance gap between the proposed algorithm and the optimal solution is small, with less than 1 db (or 25%) difference in power consumption of the whole network at γ k = 3.0 bits/s/hz. figure 3. average total power consumption of proposed low-complexity algorithm, low-complexity algorithm with sleep mode, and optimal solution for different qos target. n.i.m. bernardo and f.a . de leon 19 the performance of the proposed algorithm allowing sleep mode in scas was also investigated. the total power consumption of the proposed algorithm allowing sleep mode is lower compared to the optimal performance of hetnets without sleep mode mechanism at γ k < 2.5 bits/s/hz. this shows that incorporating sleep mode capability to scas can reduce the power consumption at low traff ic periods, while still maintaining a reliable and secure downlink transmission. at high peruser qos requirement, the power consumption of the proposed algorithm allowing sleep mode approaches that of the proposed algorithm without sleep mode mechanism. the impact of eavesdropper presence on the generated solution of the proposed algorithms was also analyzed and is depicted in figure 4. parameters used for this simulation experiment are similar to those used in figure 3, but the per-user qos targets γ k are f ixed at 2.0 bits/s/hz and k eve is varied instead of γ k . the sudden jump on the average total power consumption in figure 4 was caused by the an signal. figure 4. average total power consumption of proposed low-complexity algorithm and low-complexity algorithm with sleep mode for varying eavesdropper count. low-complexity physical layer security scheme 20 since the purpose of the an signal is to degrade the performance of the eavesdroppers, the an signal will only have a non-zero power allocation if the eavesdroppers are present. in addition, the average total power consumption has a steadily increasing trend as the number of eavesdroppers grows. this result is not entirely unexpected due to the assumption that the eavesdroppers are colluding. placement of additional eavesdroppers at random locations exploits receive spatial diversity which improves the quality of eavesdropped signals. as such, a more secured transmission strategy is required to degrade their signal reception. this is achieved by increasing the an signal power or increasing the power allocation in rzf precoding. the average runtimes of solving the benchmark performance and the proposed low-complexity schemes for different number of scas are listed in table 3. the hardware used in conducting the simulation is an intel core i7-4770 with 3.40 ghz processing speed and 4 gb ram. this hardware runs a windows 7 64-bit operating system with matlab 2015a installed. runtime measurements were acquired using the built-in timeit function of matlab. measured runtime in solving the benchmark performance exponentially increases as more scas are added to the simulation setup. this is expected since the matrix dimensions of the unknown semidef inite matrices w k,j scale up drastically with the addition of more scas. meanwhile, runtime performances of the proposed phy security schemes were not signif icantly affected by the addition of scas. however, the runtime of the proposed phy security scheme doubled when sleep mode was considered. this is because the algorithm needs to solve two linear optimization problem: f irst is to determine whether the scas are in the active state or in sleep state using (20), and then solve the precoding vectors of all active scas using (19). table 3. list of average runtimes in solving the original optimization model and the proposed low-complexity physical layer security schemes 3 scas 2.534 0.165 0.337 6 scas 5.258 0.175 0.358 9 scas 10.227 0.187 0.382 12 scas 20.4917 0.198 0.404 15 scas 37.415 0.2083 0.425 18 scas 66.338 0.217 0.442 number of sca runtime of benchmark performance (in seconds) runtime of proposed scheme (no sleep mode) (in seconds) runtime of proposed scheme (no sleep mode) (in seconds) n.i.m. bernardo and f.a . de leon 21 conclusion in this study, an optimization model which solves for the optimal values of the precoding vectors and an an vector that minimizes the total power consumption, while satisfying certain level of data rate requirement and secrecy performance at the phy layer, was developed. using the formulated optimization model, a lowcomplexity algorithm which determines the optimal power allocation strategy for the precomputed an vector and precoding vectors was designed. the performance of the proposed algorithm was analyzed by comparing it with the derived benchmark performance. despite the decrease in computational complexity, deviation from the achievable performance was observed. however, the small increase in power c o n s u m p t i o n i s a n a c c e p t a b l e t r a d e o f f f o r t h e f e a s i b i l i t y o f r e a l t i m e implementation. the algorithm was also extended to take into account the sleep mode capability of scas. allowing scas to sleep during idle/low traff ic periods could lessen the power consumption without sacrif icing secrecy of communication— even outperforming the optimal solution for hetnets without sleep mode at low data rate requirement. increase in eavesdropper also resulted in an increase in total power consumption. finally, we note that, although perfect knowledge of ue and eavesdropper channel state information (csi) was assumed in this study, modif ication of the proposed algorithm to be robust against imperfect csi knowledge is considered for future work. acknowledgements the authors would like to acknowledge the up wireless communications engineering laboratory for their support to the study. references bernardo ni, de leon f. 2016. on the trade-off between physical layer security and energy eff iciency of massive mimo with small cells. in: international conference on ad v a n ced tec h n o l o g i e s fo r co m m u n i c a t 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for small-cell networks. ieee transactions on communications. 64:790-804. wa n g h m , z h e n g tx , yu a n j , tow s l ey d, le e m h . 2 0 1 6 . p h y s i c a l l a ye r s e c u r i t y i n h e te r o g e n eo u s ce l l u l a r n e t wo r k s . i e e e tr a n s a c t i o n s o n co m m u n i c a t i o n s . 6 4 : 1 2 0 4 1219. yang n, wang l, geraci g, elkashlan m, yuan j, di renzo m. 2015. safeguarding 5g wireless communication networks using physical layer security. ieee communications magazine. 53:20-27. zhou x, mckay mr. 2010. secure transmission with ar tif icial noise over fading channels: a c h i ev a b l e r a t e a n d o p t i m a l p o w e r a l l o c a t i o n . i e e e tr a n s a c t i o n s o n ve h i c u l a r technology. 59:3831-3842. _____________ neil irwin bernardo <neil.bernardo@eee.upd.edu.ph> is an assistant professor of the electrical and electronics engineering institute, university of the philippines diliman. he received both his m.s. degree in electrical engineering and b.s. degree in electronics and communications engineering from the same institute. his research interests include communication signal processing, software-def ined radios, and mimo. franz de leon <franz.de.leon@eee.upd.edu.ph> received both his b.s. degree in electronics and communications engineering in 2003 and m.s. degree in electrical engineering in 2005 from the university of the philippines diliman. he received his ph.d. degree in electrical and electronic engineering in 2014 from the university of southampton, united kingdom. he is an assistant professor in the electrical and electronics engineering institute, up diliman, whose interests include applications of digital signal processing in audio and communications. 12info for authors.pmd 88 1. science diliman is a journal of pure and applied sciences. considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out; that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without 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editors style manual (6th ed. , 1994) and the journal of american chemical society. 2 2 . less common abbreviations may be printed as footnotes. 23. authors may opt to submit their typeset manuscripts (word f ile) as an email attachment to <rduo.ovcrd@upd.edu.ph>, <science.diliman@journals.upd.edu.ph> and <rduo.ovcrd2012@gmail.com>. manuscripts may also be submitted in hard copy (3 copies) to: the editor-in-chief science diliman off ice of the vice-chancellor for research and development lgf phivolcs bldg. , c.p. garcia avenue university of the philippines diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. 01_device meñez, villanoy and david 10 *corresponding author science diliman (july-december 2006) 18:2, 10-17 introduction water masses are identified by their characteristic combination of properties (pickard and emery, 1990). from temperature and salinity alone, four different water masses in the upper 1000m (fig. 1) are found around the philippine archipelago (udarbe-walker et al, 2003; amedo et al., 2002; qu et al., 1998; bingham and lukas, 1995; fine et al., 1994). at the surface, warm and fresh water corresponds to the tropical surface water (tsw) that forms at the surface of the inter tropical convergence zone. a subsurface layer with maximum salinity is equated with the core of the north pacific tropical water (nptw) and below this the north pacific intermediate water (npiw) is recognized by its minimum salinity value. the cold deeper layer with increasing salinity towards the bottom movement of water across passages connecting philippine inland sea basins lambert anthony b. meñez*, cesar l. villanoy and laura t. david marine science institute, university of the philippines, diliman, qc 1101 contact no.: 632/922-3921; mobile: 0919/319-5974 email: bebetmenez@yahoo.com date submitted: march 2, 2006; date accepted: november 6, 2006 abstract advection of pacific water to the inland seas is through a number of straits bordering the archipelago. movement of water was demonstrated by temperature-salinity diagrams plotted for a number of stations situated along the various passages. as water from the pacific flowed through the straits its characteristic t-s profile was modified as it mixed with waters of different properties. this was best seen along the san bernardino-verde island transect where strong surface flow during the ne monsoon resulted in separation of profiles at the surface indicating dilution as water moved away from the source. for deeper water, the erosion of the subsurface salinity minimum and maximum representing the core of the intermediate waters showed transport. these waters were restricted by shallow sill along the eastern coast of the country and limited to a depth of 441m by the sill across the mindoro strait. keywords: temperature, salinity, water mass, inland sea, t-s profile, water transport 33.2 33.6 34 34.4 34.8 35.2 35.6 salinity (psu) 0 10 20 30 40 t e m p e ra tu re (º c ) tsw nptw npiw aaiw figure 1. temperature salinity scatter plot of water east of the philippines. dashed boxes indicate property ranges of the different water mass present in the region. these are the tropical surface water (tsw), north pacific tropical water (nptw), north pacific intermediate water (npiw) and the antarctic intermediate water(aaiw). movement of water across passages 11 is identified with the antarctic intermediate water (aaiw) that moves into the region from the south. pacific water moves into the inner basins through various passages surrounding the archipelago and flow was inferred from the spatial distribution of properties described in the previous sections. temperature-salinity (t-s) diagrams are commonly used in oceanography to trace movement of ocean water (tolmazin, 1985). as water moves farther away from its source (in this study the western pacific) extreme salinity values characterizing the core of the water mass decreases. transport of water between basins was inferred from the transition of water property distributions from one basin to its neighboring basin. based on t-s curves plotted for stations on transects sampled along passages, flow direction and seasonal variation was determined. methods the temperature-salinity (t-s) diagram introduced by helland-hansen in 1916 (sverdrup et al. 1970) was used to identify water masses present in the area and traced its movement from the pacific to the inland basins. waterways linking the open ocean to the inner seas were identified. these were the san bernardino and surigao straits along the eastern coast of the philippines, sibutu passage on the southeastern end of the sulu archipelago, balabac strait off the southern end of palawan and the mindoro strait on the northwest. data was extracted from the word ocean database 2001 (conkright et al 2002) produced by the national oceanographic data center (nodc-noaa). water temperature and salinity for stations along the various passages were extracted. information sampled from single cruises (done within the same year or consecutive months) was preferred. however, in its absence, data from other cruises were used. the data was sorted to season (monsoon) to show variation brought about by prevailing weather patterns. temperature and salinity were plotted against each other and plots for a particular water way was combined to indicate behavior of the curves with respect to neighboring stations. results water masses exhibit temperature and salinity characteristics that can identify them as they move in and out of the basins. profiles for different stations along passages between basins are shown in figures 2 to 6 with data taken during the sw and ne monsoons designated as b and c, respectively. little deviation is observed between the seasons except for the surface layer that has relatively higher temperature and slightly lower salinity during the sw monsoon months. temperature-salinity profiles across the eastern straits that allow pacific water access to the archipelago are illustrated in figures 2 and 3 for the san bernardino and surigao straits respectively. for san bernardino, data were collected all the way to the south china sea through the ticao pass, burias strait, the northern edge of the sibuyan sea and the verde island passage. along this transect four groups representing stations with similar t-s diagrams were noted. the easternmost stations (fig. 2a stn 8-11) displayed patterns characteristic of pacific water with relatively high salinity, a subsurface salinity maximum located at 100200m and a salinity minimum at 400-500m. at the other end of the transect, stations 1 and 2 showed profiles similar to that of the south china sea with salinity extrema (lower than the pacific) found between 150 and 200m and a salinity minimum at 500m. along the waterways linking the san bernardino to the verde island passage, the remaining stations (3 to 7) had a range of surface salinity values that decreased with distance from the san bernardino strait. temperaturesalinity plots for these stations were near vertical with a wider range of values observed during the ne monsoon (fig. 2b). as with san bernardino, the eastern stations (fig. 3 stn. 1-2) across the surigao strait showed distinct pacific water characteristics. salinity maximum and minimum values were observed at 100-150m and 300500m respectively. west of these, stations located along the shallow surigao shelf (sw monsoon stn 3-5, no stations for the ne monsoon) can be differentiated from those within the bohol sea (stn 5-8). unlike san bernardino, where no distinct subsurface salinity maximum and minimum values were observed, high salinity was present in the shallow stations at 50 to meñez, villanoy and david 12 100m decreasing to a minimum value at 75 to 125m. beyond this minimum, salinity gradually increased towards the bottom. across the bohol sea no distinct maxima was seen. instead, salinity increased gradually before dropping monotonically towards the bottom at around 200m. aside from the usual shift in surface temperature and salinity brought about by the change in season, a significant variation in pattern between sw and ne monsoons can be seen with lower salinity extremes recorded during the ne monsoon. from the eastern straits pacific water eventually replenishes the surface waters of the sulu sea via the bohol sea, guimaras and tablas straits. access to the sulu sea however is not limited to these waterways since three other passages allow movement of pacific figure 2. temperature-salinity diagrams for stations along the passage from the south china sea to the pacific running through the verde is. and san bernardino strait. sw monsoon station 1 extracted from cruise wod98_49003566 (1941); 2,4 wod98_26000000 (1929); 3,5,6,7 31000586 (1949); 8 wod98_31008482 (1974); 9 wod98_49001338; 10 wod98_49001316 (1965); 11 wod98_13009650 (1966). ne monsoon stations 1 to 8 from cruise wod98_31000586 (1949); station 9 wod98_49003393 (1934). 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 1 1 9 1 2 0 1 2 1 1 2 2 1 2 3 1 2 4 1 2 5 1 2 6 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 2 3 4 5 6 7 8 9 1 1 1 2 3 4 5 6 7 8 9 te m pe ra tu re (o c ) te m pe ra tu re (o c ) a. sw and ne monsoon stations along the verde is. – san bernardino transect. ? sw monsoon ? ne monsoon --s. china sea (1,2) — inner seas (3-7) … pacific ocean (8-11) --s. china sea (1) — inner seas (2-7) … pacific ocean (8,9) b. sw monsoon c. ne monsoon • figure 3. temperature-salinity diagrams for stations along the passage from the pacific ocean to the bohol sea running through the leyte gulf and surigao strait. sw monsoon stations 1,2 extracted from cruise wod98_66001300 (1968); 3 to 5 cruise wod98_31008482 (1974); 6 to 8 cruise wod98_31000586 (1949). ne monsoon stations 1,2 from cruise wod98_66001301 (1969); 3,5 cruise wod98_32008478 (1974) and station 4 from cruise wod98_31000586 sampled in 1949. 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 3 2 3 3 3 4 3 5 s a lin ity (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 1 2 2 1 2 3 1 2 4 1 2 5 1 2 6 1 2 7 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 ? sw monsoon ? ne monsoon b. sw monsoon c. ne monsoon a. sw and ne monsoon stations along the surigao strait – bohol sea transect. … pacific ocean (1,2) --surigao strait (3,4,5) — bohol sea (6,7,8) … pacific ocean (1,2) — bohol sea (3,4,5) • te m pe ra tu re (o c ) te m pe ra tu re (o c ) movement of water across passages 13 water into the basin. pacific water feeds the basin by way of the sulawesi sea through the sibutu strait on the sw tip of the sulu archipelago, from the south china sea via the mindoro strait on the north and the balabac strait off the sw end of balabac is., palawan. figure 4 illustrates the t-s curves for transects made across the sibutu strait. influence of pacific water can be seen from the profiles of the southern stations (fig. 4b stn. 6-8 and 5c stn. 4) with salinity maximum at 100 to150m and minimum salinity at 300 to 400m. stations 1 to 3 located in the deeper sections of the sulu sea show a gradual increase in salinity with depth while stations 4 and 6 (sw monsoon) on the sibutu shelf although having the same pattern as the former exhibited lower salinity values. the northern access to the sulu sea and consequently other inland basins is through the mindoro strait. the outer stations (fig. 5b stn.1-2 and 6c stn. 1) show relatively low surface salinity characteristic of south 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 3 2 3 3 3 4 3 5 s a lin ity (p s u ) 0 5 10 15 20 25 30 35 t e m p e r a t u r e ( º c ) 1 1 6 1 1 7 1 1 8 1 1 9 1 2 0 1 2 1 1 2 2 1 2 3 1 2 4 3 4 5 6 7 8 9 1 0 1 1 1 2 3 4 5 6 7 8 1 2 3 4 b. sw monsoon c. ne monsoon a. sw and ne monsoon stations along the sibutu strait. — sulu sea (1,2,3) --n sibutu pass. (4,5) … sulawesi sea (6,7,8) — sulu sea (1,2,3) … sulawesi sea (4) ? sw monsoon ? ne monsoon • te m pe ra tu re (o c ) te m pe ra tu re (o c ) figure 4. temperature-salinity diagrams for stations along the sibutu passage that links the sulawesi (south) to the sulu sea (north). sw monsoon data extracted from cruise wod98_64000198 sampled in 1929. ne monsoon stations from cruise wod98_31000586 sampled 1947. • figure 5. temperature-salinity diagrams for stations along the mindoro strait linking the south china sea to the sulu sea and other inland basins. ne monsoon stations and sw monsoon stations 3 to 7 extracted from cruise wod98_31000586. sw monsoon stations 1 and 2 samples 1941. 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 1 1 8 1 1 9 1 2 0 1 2 1 1 2 2 1 2 3 1 2 4 1 2 5 1 2 6 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 2 4 5 6 7 1 3 4 5 6 7 a. sw and ne monsoon stations along the mindoro strait. b. sw monsoon c. ne monsoon ? sw monsoon ? ne monsoon --s. china sea (1,2) — mindoro strait (3-7) --s. china sea (1) … outer strait (2,3) — mindoro strait (4-7) • te m pe ra tu re (o c ) te m pe ra tu re (o c ) meñez, villanoy and david 14 china sea water with maximum and minimum salinity values at 150 to 200m and 400m, respectively. the other stations have maximum salinity at 75 to 100m that dropped almost monotonically to the bottom. the ne monsoon profiles however show a third group of stations (stn. 2-3) with higher surface salinity than the more southern stations indicating a possible alternate source of water. although the mindoro strait is the primary link of water to the inland seas, the verde island passage may have significant input to the surface layer particularly during periods of strong westward flow. the balabac strait sw of palawan is a relatively shallow passage between the sulu sea and the southern section of the south china sea. western stations (fig. 6b stn 1-3 and 6c stn 1-2) show t-s plots typical of the south china sea with low salinity compared to the pacific and extreme salinity of 34.48 to 34.52 psu at 200m and minimum value of 34.43 to 34.45 psu at figure 6. temperature-salinity diagrams for stations along the balabac strait that links the south china sea to the sulu sea. data extracted from cruise wod98_31000586. sw monsoon stations. 1, 2 6 sampled in 1949; 3, 4, 5, 8 sampled 1948. ne monsoon stations 1 and 2 sampled 1949; stations 3 to 7 sampled 1948. figure 7. inferred net flow of water across the various passages linking the pacific. sulawesi and the south china seas to the inland basins. 114 116 118 120 122 124 126 128 6 8 10 12 14 16 18 20 114 116 118 120 122 124 126 128 6 8 10 12 14 16 18 20 a. surface water b. deep water below 300m 3 2 3 3 3 4 3 5 s a lin ity (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 3 2 3 3 3 4 3 5 s a lin it y (p s u ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 t e m p e r a t u r e ( º c ) 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 9 1 2 0 1 2 1 1 2 2 4 5 6 7 8 9 1 0 1 1 1 2 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 ? sw monsoon ? ne monsoon a. sw and ne monsoon stations along the balabac strait. b. sw monsoon c. ne monsoon --s. china sea (1,2,3) … e of strait (4,5) — sulu sea (6,7) --s. china sea (1,2) … e of strait (3,4) — sulu sea (5,6,7) • te m pe ra tu re (o c ) te m pe ra tu re (o c ) movement of water across passages 15 400m. east of balabac, t-s patterns mimic distribution for the sulu sea with a near vertical drop from a salinity maximum at 50 to 75m. ne monsoon profiles illustrate decreasing salinity from the central basin of the sulu sea towards the balabac shelf with the vertical profile at station 3 indicating a well mixed column from surface to bottom. discussion the shallow sills along the eastern coast of the country hinder the flow of the intermediate water masses entering the archipelago. the salinity minima and cold deeper layer characteristic of these water masses are therefore absent from the t-s plots across the san bernardino and surigao straits (fig. 2 and 3). only the upper layers are present showing decreasing salinity away from the source (pacific). decrease in the salinity gradient is more pronounced during the ne monsoon since the flow from the pacific is strongest this time of the year (wyrtki, 1961). this is depicted in figure 2c where a gradient of decreasing surface salinity is observed farther away from the pacific. in contrast during the sw monsoon (fig. 2b) the surface salinity gradient is weaker within the inner passages indicating reduced flow from the pacific. amedo et al., (2002) observed similar anomalies in the course of their analysis of water characteristics taken from the ticao pass and adjacent water ways leading away from the surigao and san bernardino straits. intense mixing likewise accounts for the homogeneous nature of the water column as shown by the vertical to near vertical behavior of plots west of the straits. although unable to flow through the eastern straits, the npiw is reported to influence the deep water of the sulu sea (quadfasal et al., 1990). this water mass feed the inner basins through the mindoro strait from the south china sea by way of the luzon strait. its subsurface salinity minima can be seen in plots for the outer station of the verde is.-san bernardino (fig. 2), mindoro (fig. 5) and balabac (fig.6) transects and can be traced further into the mindoro strait at depths of 400 to 500m. as with the eastern straits, shallow sills across the two other passages hinder the flow of deep water to the sulu sea. by tracing the erosion of core properties of water masses, it is possible to infer direction of the general flow through the strait (wust (1935) as described in sverdrup, et al., 1970). the high salinity values of the inner stations along the mindoro strait (fig. 5b and c) at shallower depths relative to the outer stations indicate movement of water from the sulu sea to the south china sea. in contrast, below 125m a reverse flow into the sulu basin can be inferred by the change in salinities relative to each other. the change in direction between the south china sea and the mindoro strait is due to different forces acting on the water column. at the surface movement of water towards the south china sea is due to wind stress induced by the monsoons and the prevailing easterlies. below the surface, the horizontal pressure gradient force due to the difference in density between the south china sea and the sulu sea causes a shift towards the mindoro strait. relatively low density of the sulu water is the result of turbulent mixing from sill over flow and other lesser-understood processes that might be occurring with the basin. this behavior is present in both sw and ne monsoons although plots for the sw monsoon aside from being warmer, show a more vertical curve which is associated with intense mixing brought about by the sw winds. the ne monsoon stations 2 and 3 off mindoro likewise display higher salinity compared to the inner stations (fig. 5c). this is most likely the result of mixing of sulu sea water with water flowing out from the verde island passage. relatively higher salinity is expected since water from the passage would have traveled a more direct route from the pacific that has higher salt content. the wide variation in salinity at the surface is due to periodical changes at the frictional layer and is normally disregarded in the analysis (tolmazin, 1985). next to the mindoro strait, the sibutu passage has the deepest sill allowing entry of deep water into the sulu basin. it connects the sulu sea with the sulawesi sea that is fed from the pacific by the mindanao current (qu et al., 1998). as with the mindoro strait, direction of flow is inferred by the concentration of core properties. the inference however should be made with caution since the inner stations are also influenced by waters flowing into the basin from other sources (e.g. balabac, mindoro, bohol, etc.). the decrease in salinity from outer to inner station for the column of water down to about the sill depth (275m) shows a general flow towards the sulu sea during the sw monsoon. meñez, villanoy and david 16 conversely, the ne monsoon profile shows a two directional flow with the upper layer to about 125m flowing out of the sulu basin and the deeper layer moving into the basin. metzger and hurlburt (1996) suggest that transport through the sibutu passage is associated with the shift in the bifurcation latitude of the north equatorial current. the northward migration of the bifurcation latitude towards the end of the sw monsoon (autumn) intensifies transport of the mindanao current bringing pacific water towards the sulawesi sea. transport is reduced in spring (end of ne monsoon) when the bifurcation latitude moves southward (qiu and lukas, 1998). the core of the npiw can be seen at 400-500m in the sulawesi sea stations but it is not clear from the charts whether the npiw is able to move into the sulu basin. the large difference in t-s characteristic between the sulawesi and the rest of the sibutu transect stations suggests a limited exchange between the sulu and sulawesi seas. this could be due to the location of the sibutu strait relative to the eastward flow south of the sulu archipelago which transports water from the pacific to the sulawesi. summary and conclusion the movement of water through the various passages linking the open seas with the inland basins was demonstrated by the series of t-s profiles plotted for samples along these waterways. as water from the pacific flowed through the straits its characteristic t-s profile was modified as it mixed with waters having different properties. where transport was strong, the gradients between the t-s curves were wider indicating dilution farther away from the source. this was most obvious for the surface layer of the san bernardino strait during the ne monsoon (fig. 2c). reduced flow during the sw monsoon on the other hand showed a narrower spread of the profiles (fig. 2b). advection of the nptw and npiw from the pacific to the other basins was suggested from the erosion of the subsurface salinity maximum and minimum that indicates the core of these water masses. restricted by narrow sills across the eastern straits, the nptw rejuvenates the inland basins through the mindoro strait by way of the south china sea (fig. 5). the 441m sill across the mindoro strait however, prevents lateral flow of the npiw from feeding the inland basin. its characteristic salinity minimum is not evident in the t-s profiles of the inner seas. the net flow of surface and deep water below 300m across the various passages linking the pacific, sulawesi and the south china seas to the inland basins is illustrated in figures 7a and b respectively. acknowledgments this study is part of the first author's msc research that looked at how pacific deep water influences the hydrographic character of philippine inland seas. presentation of the paper at the 8th national symposium for marine science was made possible by a travel grant provided by conservation international. references amedo, c.l.a., c.l. villanoy & m.j. udarbe-walker. 2002. composition and transport of pacific water masses along the philippine pacific seaboard. upv journal of natural sciences, 7(1-2): 90-102. bingham, f. & r. lukas. 1995. the southward intrusion of north pacific intermediate water along the mindanao coast. journal of physical oceanography, 24(1): 141-153. conkright, m.e., s. levitus, t.p. boyer, t. o'brien, c. stephens, d. johnson, l. stathoplos, o. baranova, j. antonov, r. gelfeld, j. burney, j. rochester & c. forgy. 2002. world ocean database 2001. ocean climate laboratory, national oceanographic data center, national oceanographic and atmospheric administration, silver spring, md usa. fine, r.a., r. lukas, f. bingham, m. warner & r.h. gammon. 1994. the western equatorial pacific. a water mass crossroads. journal geophysical research 99(c12): 25063-25080. metzger, e.j. & h.e. hurlburt. 1996. coupled dynamics of the south china sea, the sulu sea and pacific ocean. journal of geophysical research 101: 12331-12352. movement of water across passages 17 pickard, g.l. & w.j. emery. 1990. descriptive physical oceanography, 5th edition. pergamon press, ny usa. 320p. qiu, b. & r. lukas. 1998. seasonal and interannual variability of the north equatorial current, the mindanao current, and the kuroshio along the pacific western boundary. j. geophysical res. 101(c5): 12,315-12,330. qu, t., h. mitsudera & t. yamagata. 1998. on the western boundary currents in the philippine sea. journal of geophysical research 103: 7537-7548. quadfasel, d., h. kudrass & a. frische. 1990. deep water renewal by turbidity currents in the sulu sea. nature 348(6299):320-322 sverdrup, h.u., m.w. johnson & r.h. fleming. 1970. the oceans. their physics, chemistry and general biology. prentice-hall, englewood cliffs, nj usa. 1087. tolmazin, d. 1985. elements of dynamic oceanography. allen and unwin, ma usa. 181p. udarbe-walker, m.j., m.m. magno, s.a. moh & c.l. villanoy. 2003. inter-basin comparison of mean hydrographic characteristics around the philippines. upv journal of natural sciences, 7(1-2): 53-65. wyrtki, k. 1961. physical oceanography of the southeast asian waters. naga report 2, scripps institute of oceanography, university of california, san diego, la jolla, ca, 195p. 3layman'sabstracts.pmd layman’s abstracts 1 science diliman (january-june 2017) 29:1, 1-3 layman’s abstracts experiments and pilot study evaluating the performance of read ing miscue detector and automated read ing tutor for fil ipino: a children’s speech technology for improving literacy ronald m. pascual and rowena cristina l. guevara issn 0115-7809 print / issn 2012-0818 online thi s wo rk i s abo u t the performance evaluation of a children’s speech technology which is known in the literature as automated reading tutors (art ). the art is a computer-assisted learning system that provides oral r e a d i n g f l u e n cy i n s t r u c t i o n , i n o r d e r to i m p r ove t h e l e a r n e r ’s r e a d i n g literacy. however, the design of an art system is language-specif ic, thus requiring the authors to specif ically develop a system for the filipino language. in the f irst par t of the paper, the authors present the results of the experiments that evaluate the performance of the core technology of the art called the reading miscue detector (rmd) which automatically spots reading errors. in the second part of the paper, the authors present the design and results of the pilot study that evaluates the effectiveness of the art in improving the learner’s oral reading fluency. experimental results show that the rmd’s performance is at par with those from stateo f t h e a r t r m d s r e p o r t e d i n l i t e r a t u r e . t h e r e s u l t s o f t h e p i l o t s t u d y d e m o n s t r a te s t h a t t h e s t u d e n t s w h o u s ed t h e a rt fo r a m o n t h h a ve g e n e r a l l y i m p r o v e d i n t h e i r o r a l r e a d i n g f l u e n c y a n d c o m p r e h e n s i o n by more than four times compared to the period when they were not using the art yet . a square matrix is said to be strictly k-zero if a k = 0 for some positive i n t e g e r k a n d a m ≠ 0 f o r a l l p o s i t i v e i n t e g e r s m w i t h m < k . w e characterize square matrices which can be expressed as a sum of strictly k -zero matrices. on the sum of strictly k-zero matrices little hermie b. monterde and agnes t. paras k layman’s abstracts 2 effect of spin dimensional ity in the fidel ity of spin chain ar t graeson b. dumigpe and eric a. galapon we consider a linear chain of three spins (two qubits at the ends with o n e a r b i t r a r y s p i n b e t w e e n t h e m ) , w h e r e i n w e t r a n s f e r a n u n k n o w n q u a n t u m s t a te f r o m o n e e n d to a n o t h e r. we l o o k a t t h e e f fec t of t h e spin quantum number of the arbitrary spin in the probability that the state transfer is successful. we also study the phenomenon in which we open the system to an environment/noise. geometric study on sil icon nanowires fabricated via silver-assisted electroless etching neil irvin f. cabello, eloise p. anguluan, joseph christopher r. ragasa, phil ippe mar tin b. t ingzon, kerr a. cervantes, arvin jay s. escolano, arnel salvador and armando s. somintac n a n o s t r u c t u r e d d e v i c e s a r e h i g h l y p r o m i s i n g d u e t o t h e i r n o v e l applications. nanowires (nws) are among the possible building blocks o f f u t u r e n a n o s t r u c t u r e d d e v i ce s . to m a s s p r o d u c e t h e s e n ws , t h e process to synthesize them should be simple, convenient, and economical. in this work, we study the geometry of the nanowires produced using a process called metal-assisted chemical etching. it involves immersing a crystalline silicon wafer (si) in several solutions, which can either be a one-step or a two-step process. the one-step process involves immersion in a solution containing hydrofluoric acid (hf) and silver nitrate, while t h e t w o s t e p p r o c e s s i n v o l v e s a n a d d i t i o n a l i m m e r s i o n i n t o a n o t h e r s o l u t i o n c o n t a i n i n g h f a n d h y d r o g e n p e r o x i d e . a s s u c h , t h i s p r o c e s s m e e t s t h e a f o r e m e n t i o n e d r e q u i r e m e n t s f o r t h e m a s s p r o d u c t i o n o f sinws, with the added benef it of si being nontoxic. the effect of varying the synthesis parameters on the lengths, diameters, crystallinities, and n w d e n s i t i e s o f t h e p r o d u c e d s i n w s w e r e e x a m i n e d . t h e o p t i c a l p r o p e r t i e s o f t h e s i n w s w e r e c h e c k e d a n d v e r i f i e d f o r o p t i c a l o r optoelectronic applications. the differences between the one-step and two-step processes were also noted. sinws produced from the one-step process may be more suited to cer tain applications over the other. layman’s abstracts 3 q u o r u m s e n s i n g ( q s ) i s a p r o c e s s o f b a c t e r i a l c o m m u n i c a t i o n w h i c h s e n s e s s i g n a l m o l e c u l e s t h a t i n d u c e t h e f o r m a t i o n o f m a t r i c e s ( o r biof ilms) that facilitate growth and contribute to virulence. the disruption o f b i o f i l m f o r m a t i o n m a y p r o v i d e a n a l t e r n a t i v e p a t h w a y f o r t h e p r e v e n t i o n o f d i s e a s e s a n d a d v e r s e e n v i r o n m e n t a l p r o b l e m s o f t e n attri bu te d to bi o f i l ms and biofouling. in this study, we determine the presence of qs inhibitors in philippine seaweeds and surface-associated microorganisms. a screening method utilizing a bacterial strain whose purple-colored pigmentation is inhibited in the presence of qs inhibitors was used. agar well diffusion and liquid broth assays were conducted to test for the presence of qs inhibition in crude seaweed extracts, as w e l l a s s u r f a c e a s s o c i a t e d m i c r o o r g a n i s m s . t h e d e c r e a s e i n p i g m e n t a t i o n i n t h e b a c t e r i a l s t r a i n w i t h a r e l a t i v e l y c o n s t a n t c e l l density indicates that qs is disrupted without cell death. over 7% of the seaweed fragments and 5% of microbial isolates tested positive in the preliminary screening, while almost 50% of the crude extracts were positive for qs inhibition. these f indings provide relevant information for fur ther isolation, purif ication, and characterization of qs inhibitory c o m p o u n d s f r o m p h i l i p p i n e m a r i n e s e a w e e d s a n d s u r f a c e a s s o c i a t e d microorganisms with potential anti-biof ilm/biofouling applications. high-throughput screening for quorum sensing-inhibitory compounds from selected phil ippine marine algae and surface-associated marine microorganisms for potential anti-biofilm/biofoul ing appl ications aira sacha nadine s. ferrer, aljon francis koji p. elegado, mel iton r. chiong iii, laude karina g. alcober, dang marviluz l. espita, marco nemesio e. montaño 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing 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(subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total growth of anatase-p33-.pmd growth of anatase titanium dioxide nanotubes via anodization 33 ed adrian dilla*, renato daclan, michael j. defensor, celestino andrew m. borja, arnel a. salvador and armando s. somintac condensed matter physics laboratory, national institute of physics university of the philippines, diliman 1101, quezon city *corresponding author: edilla@nip.upd.edu.ph science diliman (january-june 2012) 24:1, 33-42 abstract growth of anatase titanium dioxide nanotubes via anodization in this work, titanium dioxide nanotubes were grown via anodization of sputtered titanium thin films using different anodization parameters in order to formulate a method of producing long anatase titanium dioxide nanotubes intended for solar cell applications. the morphological features of the nanotubes grown via anodization were explored using a philips xl30 field emission scanning electron microscope. furthermore, the grown nanotubes were also subjected to x-ray diffraction and raman spectroscopy in order to investigate the effect of the predominant crystal orientation of the parent titanium thin film on the crystal phase of the nanotubes. after optimizing the anodization parameters, nanotubes with anatase tio 2 crystal phase and tube length more than 2 microns was produced from parent titanium thin films with predominant ti(010) crystal orientation and using ammonium fluoride in ethylene glycol as an electrolyte with a working voltage equal to 60v during 1-hour anodization runs. keywords: nanotechnology, titanium dioxide, nanotubes, anodization 34 dilla, e.a. et al. science diliman (january-june 2012) 24:1, 33-42 introduction titanium dioxide (tio 2 ) is a useful material known for its unique properties such as its high refractive index, self-cleaning effect and photocatalytic properties (zhao et al., 2005; jaroenworaluck et al., 2007). titanium dioxide is often utilized as a component in biocompatible materials for bone implants (kaneco et al., 2007; li et al., 2009), gas sensors (mor et al., 2006; li et al., 2009), electronics (kaneco et al., 2007), optical coatings (kaneco et al., 2007) and photovoltaic cells (mohapatra et al., 2007; su & zhou, 2009). titanium dioxide commonly exists either in rutile or anatase crystal phase. these crystal phases possess high band gaps, 3ev for rutile and 3.2ev for anatase (zhao et al., 2005; mor et al., 2006; mohapatra et al., 2007). the crystal phase of titanium dioxide often affects the applications where it can be used. anatase tio 2 is often used in photocatalysis, dye-sensitized solar cells and photolysis while rutile tio 2 is commonly used in industrial pigments and coatings (mor et al., 2006). titanium dioxide is often fabricated as thin films or coatings. in order to fully utilize the properties of tio 2 , it must be formed into structures that will enable it to be used efficiently for specific applications. production of nanostructures based on tio 2 is one of the ways to improve and utilize the properties of this material. with recent advances in materials engineering and nanotechnology, titanium dioxide nanostructures such as nanotubes, nanowires and nanorods which rectify the properties of titanium dioxide are now being used as replacements for thin films (huang & choi, 2007). among these nanostructures, tio 2 nanotubes are widely investigated due to their enhanced photocatalytic and sensing properties (mor et al., 2006). titanium dioxide nanotubes are oriented nanostructures with tunable lengths and pore diameters (paulose et al., 2006). these nanostructures are now commonly used as components in dye-sensitized solar cells and gas sensors (li et al., 2009). tio 2 nanotubes can be fabricated using different techniques, such as template or lithography method, hydrothermal method, sol-gel process and anodization (kaneco et al., 2007; li et al., 2009). among these techniques, anodization is the most efficient and cost effective method of fabricating selfordered and oriented nanotubes since the completion of the whole anodization process only requires relatively cheap and accessible materials and a short duration of time unlike other techniques in fabricating of titanium dioxide nanotubes (jaroenworaluck et al., 2007; macak et al., 2007). anodization is an electrochemical method of growing an oxide layer on top of a metallic layer. this method is also used to fabricate oxide nanostructures from valve metals other than titanium, such as aluminum, tantalum and niobium (li et al., 2009). in this work, a way of producing anatase titanium dioxide nanotubes, intended for solar cell applications, with tube lengths greater than 2 microns was explored by investigating the effects of the parameters such as the crystal orientation of the parent titanium film, the type of electrolyte, anodization duration and voltage on the morphology and crystal phase of tio 2 nanotubes. methodology glass and n-type silicon(100) were used as substrates for nanotube samples grown via anodization. the substrates were cut and subjected to a degreasing process using trichloroethylene, acetone, methanol and deionized water in order to remove unwanted grease and contaminants on the substrates. titanium thin film deposition was carried out after substrate preparation. deposition was conducted for 30 minutes and 1 hour inside a chamber with a working pressure equal to 8x10-3mbar and a working power equal to 150 watts to produce titanium layers with thickness approximately equal to 1µm and 2µm for samples anodized in hydrofluoric acid and ammonium fluoride in ethylene glycol electrolyte, respectively. in order to investigate the effects of the parent titanium thin films on the grown nanostructures, titanium thin films were deposited via rf magnetron sputtering on the substrates using two different commercial titanium sources from chemiston co. (source 1) and advent research materials (source 2). after deposition, the substrates deposited with a titanium film were cut into 0.5cm by 1cm samples. the samples were then mounted on the anodization setup growth of anatase titanium dioxide nanotubes via anodization 35science diliman (january-june 2012) 24:1, 33-42 presented in figure 1. the current of the system for each anodization run was monitored and recorded using a circuit connected to a computer with a labview interface. initial anodization runs were conducted in order to determine the metal source from which anatase titanium dioxide nanotubes will be produced. the crystal phase of the grown nanotubes was determined through raman spectroscopy. after the initial runs, thin films from the metal source which produced anatase titanium dioxide nanotubes were subjected to anodization using different parameters. two types of electrolytes were used during anodization, namely: hydrofluoric acid (hf) and ammonium fluoride (nh 4 f) in ethylene glycol (eg). in the anodization setup, lead and silver was used as the cathode for hf and nh 4 f in eg electrolyte, respectively, with the titanium thin film samples serving as the anode. each anodization run was carried out using a constant voltage. for hf electrolytes, voltages equal to 7v, 10v, 15v and 18v were used while voltages equal to 60v, 80v, and 100v were used for nh 4 f in eg electrolyte. anodization was also conducted at different durations. for hf electrolytes, anodization was done for 3, 4, 5, and 7 minutes while anodization was conducted for 5, 15, 30 and 60 minutes for nh 4 f in eg electrolyte, using a selected constant voltage. after anodization, the samples were washed with deionized water and dried with n 2 gas. different characterization techniques were implemented in order to investigate the effects of the parameters on the morphology and crystallinity of the grown nanotubes. a philips xl30 field emission scanning electron microscope was used to observe the morphology of the nanostructures grown. the crystal structure of the parent titanium film was investigated through x-ray diffraction using a bede d3 system. since the as-grown nanotubes were generally amorphous, the crystal phase of the grown samples was investigated using raman spectroscopy instead of x-ray diffraction. results and discussion crystal phase of the titanium dioxide nanotubes controlling the crystal phase of the grown titanium dioxide nanotubes is essential since the applications to which these nanostructures will be used are dependent on their crystal phase. in order to control the crystal phase of the grown nanotubes, the effects of the quality of the parent titanium source was investigated. the xray diffraction spectra of the titanium thin films are shown in figure 2. it can be observed in figure 2 that two crystal orientations namely, ti(010) and ti(002), indicated by the peaks at 35° and 38° are predominant for the titanium samples deposited using two different sources. for source 1, the intensity of the peak at 35° for ti(010) is greater than the intensity of the peak for ti(002) while the opposite is observed for source 2. this implies that the predominant crystal orientation of figure 1. setup diagram and actual anodization setup 36 dilla, e.a. et al. science diliman (january-june 2012) 24:1, 33-42 the titanium samples from sources 1 and 2 are different from each other. ti(010) is predominant for source 1 (chemiston co.) while ti(002) is predominant for source 2 (advent research materials). figure 3 presents the raman spectra of the titanium dioxide nanotubes formed from the titanium samples deposited from two titanium sources. it can be observed that the nanotubes formed from different parent titanium sources exhibited different sets of peaks. for tio 2 nanotubes grown from titanium from source 1, characteristic peaks of anatase titanium dioxide at 388, 502 and 635cm-1 were observed while rutile tio 2 peaks at 443 and 606cm-1 were observed for tio 2 nanotubes grown from the titanium from source 2. it can be observed in figures 2 and 3 that the crystal phase of the fabricated titanium dioxide array can be surmised from the dominant crystal orientation of the parent titanium thin film. when the intensity of the peak ascribing ti(010) is greater than the peak for ti(002), the resulting titanium dioxide produced during anodization is in anatase tio 2 crystal phase. on the other hand, when ti(002) is predominant on the parent titanium film, the nanotubes produced during anodization is in the rutile tio 2 phase. optimization of anodization parameters the effects of the parameters used during anodization such as voltage and anodization time are presented in this section. the discussion of these effects is divided into two subsections according to the electrolyte used during anodization, namely: a) hydrofluoric acid and b) ammonium fluoride in ethylene glycol electrolyte. a. hydrofluoric acid as an electrolyte titanium dioxide nanotubes are often grown via anodization using fluorine-based aqueous solutions. among fluorine-based aqueous solutions, hydrofluoric acid (hf) is most commonly used as electrolyte in growing titanium dioxide nanotubes via anodization due to its availability and high etching rate. this section discusses the growth of titanium dioxide nanotubes via anodization in aqueous hf electrolyte. figure 4 shows the representative current density behaviour during anodization of samples grown using hf electrolyte. the behaviour of the current density of the system agrees with the observations made by tang et al. (2008) and macak et al. (2007). the periods in between each maximum or minimum in the graph, indicated by the letters a to c, correspond to the stages figure 3. raman spectra of tio 2 nanotubes grown from the titanium thin films sputtered from sources (1) and (2). the letters a and r denote the anatase and rutile phase, respectively, of the titanium dioxide. figure 2. x-ray diffraction spectra of thin films from sources (1) and (2) growth of anatase titanium dioxide nanotubes via anodization 37 figure 4. current density of the anodization system during anodization science diliman (january-june 2012) 24:1, 33-42 of nanotube growth (macak et al., 2007). according to macak et al., the growth of the nanotubes is mainly governed by two processes, namely: oxide formation and oxide dissolution (macak et al., 2007). these processes are described by the chemical equations: oxide formation: ti + 2h 2 o→tio 2 + 4h+ + 4eoxide dissolution: tio 2 + 6f→ [tif 6 ]2the sudden current drop from t=0 to the first minimum point at the first period, a, indicates the rapid formation of a barrier oxide layer on top of the titanium film layer. the drop in the current density is due to the heightened resistance of the outer layer of the titanium sample caused by the formation of the barrier oxide layer. after the period a, a slight increase in the current is observed which signifies the second stage of the nanotube growth. the slight increase in current density in period b is attributed to the localized oxide dissolution which leads to the formation of pores on selected sites in the barrier oxide layer. the third stage, period c, starts after the initial growth of pores on the oxide layer. a stable current density is observed in this stage as a result of the equal rate of oxide formation and dissolution. the initial pores begin to widen and grow uniformly as the current present in the system is distributed evenly among the growing pores. the pores also deepen as the fluoride ions start to dissolve the barrier oxide layer and react with the titanium ions to form the oxide nanostructures (macak et al., 2007; li et al., 2009). figure 5 presents the sem micrograph of the samples grown at different anodization voltages. nanotubes with different diameters, from 50nm to 130nm, were produced based on the voltage used during anodization. from the images in figure 5, it can be observed that the diameter and length of the nanotubes increase as the anodization voltage used is increased. this agrees with the results from the work of macak et al. (2007) and mor et al. (2006). this increase in tube diameter and length can be attributed to effect of the distribution of the electrical current, supplied to the system, on the growing nanostructures during anodization. the variation of the tube diameter with respect to the anodization voltage used is presented in figure 6. figure 5. fe-sem micrographs of the samples grown using a) 10v, b) 15v, and c) 18v 38 dilla, e.a. et al. science diliman (january-june 2012) 24:1, 33-42 figure 7 presents the sem micrographs of the titanium dioxide nanotubes grown after different anodization durations. it can be observed that the nanotubes become more defined as the duration of anodization is increased. at t=180 seconds, the majority of the nanostructures are covered with a white precipitate, ti(oh) 4 , which results from the reaction of ti4+ and ohions on the oxide/electrolyte interface. at longer durations of anodization, the precipitate layer recedes mainly due figure 6. variation of the nanotube diameter with the anodization voltage to the formation [tif 6 ]2complex instead of ti(oh) 4 in the electrolyte (macak et al., 2007). this is shown in figure 5c, where no precipitate layer can be observed at the top of the nanotube layer. another view of the nanotube layer, for selected anodization durations, is also presented in figure 7. the thickness of the nanotube layer decreased from 242nm to 184nm when the duration of anodization was increased from 180 seconds to 300 seconds. the decrease in the thickness of the nanotubes may be attributed to the faster rate of oxide dissolution compared to the rate of oxide formation when the anodization time exceeds 180 seconds since the stage of uniform nanotube growth, where the rate of dissolution and oxide formation is equal, ends shortly after 180 seconds for this system as illustrated in figure 4 (tang et al., 2008). figure 8 presents the representative raman spectra of the samples grown in hf electrolyte. characteristic peaks of anatase titanium dioxide at 388, 502 and 635cm-1are observed in the raman spectra.this signifies that anatase titanium dioxide is predominant in the nanotubes formed during anodization. figure 7. fe-sem micrographs of the samples anodized for a) 180, b) 240 and c) 300 seconds. inset shows the crosssection of the nanotube layer. growth of anatase titanium dioxide nanotubes via anodization 39science diliman (january-june 2012) 24:1, 33-42 b. ammonium fluoride in ethylene glycol as an electrolyte the photocatalytic property of titanium dioxide nanotubes improves with the increase in the length of the nanotubes. since the tube length of the nanotubes formed via anodization using hf electrolyte is limited, other electrolytes can be used to produce nanotubes with greater tube lengths. in this study, ammonium fluoride in ethylene glycol (eg) electrolyte was used in order to grow longer titanium dioxide nanotubes. the behaviour of the current density during anodization in nh 4 f in ethylene glycol(eg) electrolyte is shown in figure 9. it can be observed that the current density of the system during anodization in nh 4 f in eg exhibits a curve similar to the current density of the system when hf was used. it can be noted, however, that the changes in the current density of the system, which indicates the stages of nanotube formation, are set apart by longer time intervals compared to the behaviour of the current density in the case of the hf electrolyte. the mechanisms of the growth of titanium nanotubes in nh 4 f in eg electrolyte and hf electrolyte are similar, although differences in the morphologies of the grown nanostructures may arise due to the differences in the chemical properties of the electrolytes. according to prakasam et. al., the steep drop in the current density figure 8. raman spectra of the as-grown tio 2 nanotube array. the symbol a denotes the anatase phase of the titanium dioxide nanotubes. observed in the first few seconds of anodization, period a, is due to the initial formation of an insulating oxide layer (prakasam et al., 2007). due to the insulating oxide layer, electronic conduction decreases and ionic conduction through the oxide layer increases. electronic conduction, which is predominant mechanism at the first few seconds of anodization, is replaced by ionic conduction once the entire surface of the sample is covered by the insulating oxide layer (prakasam et al., 2007). the stable current density following the sudden drop in current density of the system in period b signifies the initial formation of porous nanostructures on the oxide layer. in this stage, pores start to form on the oxide layer and continually grow until the electric field present in the system is uniformly distributed to each pore. finally, the gradual drop of the current density followed by a region with stable current density, period c, signifies the formation of uniform and self-organized nanotubes which are formed as the result of the oxide formation, ti4+ migration and oxide dissolution on the bottom of the initial pores (macak et al., 2007). the images presented in figure 10 shows the sem micrograph of the samples grown in nh 4 f in eg electrolyte using different anodization potentials. it can be observed that the morphology of the nanostructures varies with respect to the anodization potential used. high anodization voltages lead to wider tube diameters as seen in figures 10a-10c. it was also observed that figure 9. current density of the anodization system during anodization 40 dilla, e.a. et al. science diliman (january-june 2012) 24:1, 33-42 the thickness of the nanotube layer is almost the same for samples grown using 60v and 80v (2.3 microns) while a thinner nanotube layer (1.8 microns) is produced for nanotubes produced using 100v. the thinner layer produced after a 1-hour anodization run under a voltage equal to 100v is attributed the breaking of the nanotube/ oxide layer at anodization potentials greater than 60v. this is attributed to the weakening of the ti-o bond due to polarization at high applied electric field (tang et al., 2008). due to the tendency of the nanotubes produced using anodization potentials higher than 60v to breaking and peeling off from the glass substrate, the use of voltages higher than 60v may not be suitable for the production of titanium dioxide nanotubes from deposited thin films. the variation of the tube diameter with the anodization voltage is presented in figure 11. the graph in figure 11 exhibits a similar trend as to the case of the hf electrolyte shown in figure 6. this result also agrees with the results of the work of macak et al. (2007). figure 10. fe-sem micrographs of the samples grown using a) 60v, b) 80v, and c) 100v figure 12 shows the sem micrograph of the samples grown at different durations of anodization using a constant voltage in nh 4 f in eg electrolyte. titanium dioxide nanotubes with diameters ranging from 5080nm can be observed in the sem images shown in figure 12. from the presented images, it can be observed that the nanostructures are already welldeveloped after 5 minutes of anodization. no noticeable changes were observed in the tube diameter and the spacing of the nanotubes when the duration of anodization was increased from 5 minutes to 60 minutes. the length of the nanotubes, however, increased from 690nm to 2.3µm when the duration of anodization was prolonged from 5 minutes to 60 minutes contrary to the result obtained in the case of the hf electrolytes. this may be due to the rapid growth rate of tio 2 figure 11. variation of the nanotube diameter with the anodization voltage growth of anatase titanium dioxide nanotubes via anodization 41science diliman (january-june 2012) 24:1, 33-42 nanotubes and more stable balance between oxide formation and dissolution in nh 4 f in eg electrolytes compared to aqueous hf electrolytes (prakasam et al., 2007). the representative raman spectra of the sample grown using nh 4 f in eg electrolyte is shown in figure 13. it can be observed that the peaks present in the raman spectra are characteristic peaks of anatase titanium dioxide which suggests that the titanium dioxide nanotubes grown during anodization is in the anatase phase. summary anatase titanium dioxide nanotubes, with diameters ranging from 30nm to 110nm and tube length greater than 2 microns, were successfully grown using anodization of sputtered titanium thin films. anodization parameters, such as crystal orientation of the parent titanium thin film, anodization time, electrolyte and anodization voltage, were varied in order to explore ways of producing anatase titanium dioxide nanotubes with tube lengths exceeding 2 microns. based on the data acquired from x-ray diffraction and raman spectroscopy, it was observed that the crystal orientation of the parent titanium thin film dictates the crystal phase of the titanium dioxide nanotubes formed during anodization. titanium thin films with predominant ti(010) peak result in anatase tio 2 nanotubes while thin films with predominant ti(002) peak result in rutile tio 2 nanotubes . from the images acquired using the fe-sem, it was observed that the diameter of the nanotubes depend on the anodization voltage. long nanotubes with lengths greater than 2 microns can be grown by using ammonium fluoride in ethylene glycol electrolyte instead of the common aqueous hf electrolyte and by conducting the anodization process for durations equal or more than 1 hour. figure 12. fe-sem micrographs of the samples anodized for a) 5, b) 15, c) 30 and 60 minutes. inset shows the crosssection of the nanotube layer. figure 13. raman spectra of the as-grown tio 2 nanotube array. the symbol a denotes the anatase phase of the titanium dioxide nanotubes. 42 dilla, e.a. et al. science diliman (january-june 2012) 24:1, 33-42 acknowledgement this work is supported in part by grants from the university of the philippines-office of the vicechancellor for research and development (updovcrd). references huang, x.-j. & y.-k., 2007. chemical sensors based on nanostructured materials. sensors and actuators b: chemical, 122(2), 659-671. doi:10.1016/j.snb.2006.06.022 jaroenworaluck, a., d. regonini, c.r. bowen, r. stevens & d. allsopp, 2007. macro, micro and nanostructure of tio2 anodised films prepared in a fluorine-containing electrolyte. journal of materials science, 42, 6729-6734. doi:10.1007/ s10853-006-1474-9 kaneco, s., y. chen, p. westerhoff & j.c. crittenden, 2007. fabrication of uniform size titanium oxide nanotubes: impact of current density and solution conditions. scripta materialia, 56(5), 373-376. doi:10.1016/j.scriptamat. 2006.11.001 li, y., x. yu & q. yang, 2009. fabrication of tio2 nanotube thin films and their gas sensing properties. journal of sensors, 1-19. doi:10.1155/2009/402174 macak, j. m., h. tsuchiya, a. ghicov, k. yasuda, r. hahn, s. bauer & p. schmuki, 2007. tio2 nanotubes: self-organized electrochemical formation, properties and applications. materials science, 11, 3-18. doi:10.1016/j.cossms. 2007.08.004 mohapatra, s. k., m. misra, v.k. mahajan & k.s. raja, 2007. design of a highly efficient photoelectrolytic cell for hydrogen generation by water splitting: application of tio2x c x nanotubes as a photoanode and pt/tio2 nanotubes as a cathode. the journal of physical chemistry c, 111(24), 8677–8685. acs publications. retrieved from http:// pubs.acs.org/doi/abs/10.1021/jp071906v mor, g. k., o.k. varghese, m. paulose, k. shankar & c.a. grimes, 2006. a review on highly ordered, vertically oriented tio2 nanotube arrays: fabrication, material properties, and solar 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zhu, 2008. preparation of tio 2 nanotube on glass by anodization of ti films at room temperature. transactions of nonferrous metals society of china, 19(1), 192-198. the nonferrous metals society of china. doi:10.1016/s10036326(08)60251-4 zhao, j., x. wang, r. chen & l. li, 2005. fabrication of titanium oxide nanotube arrays by anodic oxidation. solid state communications, 134(10), 705-710. doi:10.1016/ j.ssc.2005.02.028 5santos.pmd b.s. santos et al. 21 science diliman (july-december 2015) 27:2, 21-40 status assessment of clarias species in the phil ippines: insights from dna barcodes brian s. santos* university of the philippines diliman francis peter c. vesagas university of the philippines diliman marc t imothy c. tan university of the philippines diliman joycelyn c. jumawan caraga state university jonas p. quilang university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract catf ishes of the genus clarias are important food f ishes in aquaculture. in the philippines, six species are documented but only three, namely c. batrachus, c. macrocephalus, and c. gariepinus, are found in the market today. of these, c . macrocephalus is both native and near threatened. in this study, the cytochrome c oxidase i (coi) gene was amplif ied for 20 agusan marsh, agusan d e l s u r s p ec i m e n s p r ov i s i o n a l l y i d e n t i f i ed a s c . m a c r o ce p h a l u s. t h e s e specimens have a different morphology compared to other c. macrocephalus specimens previously obtained elsewhere. the coi sequences all matched the philippine coi sequences of c. macrocephalus, thus conf irming its identity. reanalysis of barcode sequences was also conducted to resolve the conflicting claims regarding the status of some clarias species. a total of 179 coi sequences from clarias species present in genbank were included in the analyses. the average intraspecif ic and interspecif ic kimura-2-parameter distances were 2.99% and 13.26%, respectively. there was very little sequence diversity observed in the philippine samples of c. macrocephalus. philippine samples of c. batrachus and c. macrocephalus formed distinct clades, while status assessment of clarias species in the philippines 22 philippine c. gariepinus specimens clustered with those of other countries, s u p p o r t i n g t h e c l a i m t h a t t h e f o r m e r t w o s p e c i e s a r e n a t i v e a n d t h e l a t te r w a s i n t r o d u ced to t h e co u n t r y. t h e s t a t u s of t h e o t h e r c l a r i a s species in the philippines is also discussed. keyword s: catf ish, lariidae, coi, dna barcoding layman’s abstract ca t f i s h e s of t h e g e n u s c l a r i a s a r e a b u n d a n t a n d a r e i m p o r t a n t fo o d f ishes in the philippine market. six clarias species have been repor ted in the country in the past. today, however, only c. batrachus, c. gariepinus, and c. macrocephalus are found in the market. the other species have not been seen in recent years. of these species, c. macrocephalus is of primary importance because it is both native to the philippines and near threatened. as such it is important to identify them from their natural populations. dna barcoding is a technique that utilizes a single gene to identify an organism to the species level. this was performed in this study by amplifying and sequencing the cytochrome c oxidase subunit i gene (coi) of 20 specimens of c. macrocephalus from agusan marsh to co n f irm their identity. these specimens appear different compared to other c. macrocephalus specimens from other locations. the coi sequences f r o m a g u s a n m a r s h w e r e h i g h l y s i m i l a r t o t h e s e q u e n c e s o f o t h e r philippine c. macrocephalus, thus conf irming their identity. as for the other clarias species, there are conflicting claims regarding their status. to r e s o l v e t h i s , a l l a v a i l a b l e b a r c o d e s e q u e n c e s o f c l a r i a s s p e c i e s w e r e analyzed. a total of 179 coi sequences from the genbank database were included. philippine samples of c. batrachus and c. macrocephalus formed distinct groupings, while philippine c. gariepinus specimens grouped with those of other countries. this supports the claim that c. batrachus and c. macrocephalus are native to the philippines while c. gariepinus is only introduced to the country. the presence of the other clarias species in the philippines is questionable. b.s. santos et al. 23 introduction clarias species are abundant and are important food f ishes in the philippine market. six clarias species have been reported in the country. of these, only c. batrachus, c. gariepinus, and c. macrocephalus are commonly found in the market today. the others, c. nieuhofii, c. meladerma, and c. fuscus, have not been reported in recent years. the status of these clarias species is quite controversial due to conflicting reports from old checklists, recent surveys, as well as current f indings. the status of the philippine native catf ish, c. macrocephalus, is of primary interest. while c. batrachus and c. gariepinus are abundantly used in f ish farming, c. macrocephalus is listed as near threatened by the international union for conservation of nature (iucn). natural populations of c. macrocephalus have been declining all over southeast asia (vidthayanon and allen 2013). in the philippines, c. macrocephalus was previously described by conlu (1986) as widely distributed. however, the abundance of this species was greatly reduced in the years that followed due to habitat loss, poor water quality, and the presence of larger-sized competitors, c. batrachus and c. gariepinus (vidthayanon and allen 2013). the presence of c. macrocephalus in the philippines was also reported by fowler (1941) and herre (1953). fishbase also indicates that c. macrocephalus is native to the philippines (froese and pauly, 2014). ‘native’ is def ined as naturally occurring in the country as opposed to those that are introduced. bycontrast, teugels et al. (1999) claimed that c. macrocephalus is a species introduced to the philippines for aquaculture, falsely citing conlu (1986), who stated that c. macrocephalus is endemic, or is found exclusive, to the philippines. this, however, is also false since c. macrocephalus is also nativeto cambodia, laos, thailand, and vietnam (froese and pauly 2014). in recent sampling activities, c. macrocephalus specimens from agusan marsh, agusan del sur were found to be much larger than others. the standard lengths of agusan marsh specimens ranged from 22.7–34.8 cm with an average of 29.4 cm, whereas those of cagayan specimens ranged from 11.2–20.2 cm with an average of 16.3 cm. conlu (1986) indicated that the size range of c. macrocephalusis 20–30cm; however, it was not clear whether the length indicated was the total length or the standard length. the average size was not described as well. the agusan marsh and cagayan specimens were larger and smaller, respectively, than conlu’s estimates. these specimens were still assigned to c. macrocephalus due to the characteristic obtuse and rounded shape of their supraoccipital process. nonetheless, it is still important status assessment of clarias species in the philippines 24 to verify whether the large-size and small-size morphotypes are merely variants of the same species or whether they are genetically divergent lineages. the mitochondrial dna (mtdna) cytochrome c oxidase subunit i (coi) gene has been extensively used in the past decade as a dna barcode for species identif ication and delineation (hebert et al. 2004). the coi gene is useful for fast, simple, robust, and precise species identif ication of f ish species (costa and carvalho 2007). it has been applied for the detection of cryptic species, possible market mislabeling, and taxa requiring taxonomic re-evaluation (smith et al. 2008; ward et al. 2008; smith et al. 2011). to date, 179 clarias coi sequences are available in genbank (table 1). some of the sequences have been published in various papers, with some having conflicting claims regarding the status of clarias species in different countries. for example, wong et al. (2011) observed low sequence identities between thai specimens of c. batrachus and those from genbank. although it was not stated, the genbank sequences were most likely from india since these were the only ones available at that time. in 2012, bhattacharjee et al. (2012) barcoded catf ishes, including c. batrachus, from india. they did not include the thai specimens from the study of clarias angolensis congo hm880232 international barcode of life, direct submission clarias batrachus india fj459456-59 lakra et al. 2011 india gq466399-403 barman et al. (unpublished) india jn628880,924 bhattacharjee et al. 2012 india kf214293-96; khedkar et al. 2014 jq667517-18 india jq699205-208 aneesha et al. (unpublished) india jx946369 singh et al. (unpublished) india kf742432 subedi et al. (unpublished) india kj720696 premdass et al. (unpublished) thailand jf292297-309 wong et al. 2011 philippines hq654701 aquilino et al. 2011 philippines hq682679-81 aquino et al. 2011 philippines kc789523-27 yambot et al. (unpublished) philippines kf604645-56 quilang and yu 2015 vietnam ef609334 ward and holmes 2007 clarias camerunensis nigeria hm882808 nwani et al. 2011 clarias dussumieri india hm579862 simi et al. (unpublished) india jq699209-13 aneesha et al. (unpublished) clarias fuscus china jn020071 wang et al. (unpublished) china kf011504-05 xiao and peng (unpublished) species country accession reference number table 1. list of clarias coi sequences obtained from genbank b.s. santos et al. 25 species country accession reference number table 1. list of clarias coi sequences obtained from genbank (cont’d.) clarias gabonensis congo hm880231,33 international barcode of life direct submission nigeria jf510511, nwani et al. 2011 hm882815-16,29 32-37 clarias gariepinus brazil gu701825-29 pereira et al. 2013 indonesia hm345933-34 muchlisin et al. 2013 nigeria hm882809-14,17, nwani et al. 2011 20-21,23-31 thailand jf292310-320 wong et al. 2011 turkey jq623925; keskin and atar 2013 kc500413-32 india jq699199-203 aneesha et al. (unpublished) india kf742418 chaulagain et al. (unpublished) india jx260853 kalyankar et al. (unpublished) philippines kf604657-61 quilang and yu 2015 ethiopia kf929769 bentley and wiley (unpublished) clarias jaensis nigeria hm882818-19 nwani et al. 2011 clarias macrocephalus philippines kf604662-66 quilang and yu 2015 thailand jf292321-37 wong et al. 2011 clarias nieuhofii malaysia jf280833-34 othman et al. (unpublished) clarias teijsmanni malaysia jn646093 sade and biun 2012 wong et al. (2011), but they cited the c. batrachus accession of aquilino et al. (2011) from taal lake, philippines as a possible misidentif ication. quilang and yu (2015) addressed this issue by including philippine c. batrachus sequences from laguna de bay (aquino et al. 2011) and from their own specimens obtainedfrom cagayan and three lakes from camarines sur. they observed that all c. batrachus specimens clustered within their country of origin. philippine specimens are closer to those from thailand with an average distance of 2.6%, whereas the average distance of philippine and thai specimens with those from india were 11.3% and 12%, respectively. these results were taken as supporting proof to the status of c. batrachus as native to the philippines (herre 1924; herre 1926) even though it was widely believed to have been introduced later (vallejo 1985; juliano et al. 1989; asap 1996; table 2). in subsequent years, more coi sequences of clarias became available in genbank, enabling the representation of various countries (table 1). as such, reanalyzing the barcodes would provide more information that could possibly resolve the conflicting claims. in this study, the coi gene was utilized to ascer tain the identity of the agusan marsh specimens. this was also used to assess the status of other clarias species in the philippines. status assessment of clarias species in the philippines 26 materials and methods a total of 20 specimens of c. macrocephalus were collected from agusan marsh, agusan del sur (8.21 n; 125.96 e) from a local contact. these were brought to the institute of biology, molecular population genetics laboratory for processing and identif ication. specimens were initially identif ied based on the morphology of the supraoccipital process (conlu 1986; teugels et al. 1999). the weight and length measurements were obtained. each specimen was photographed on the dorsal and left side views using a nikon d90 slr camera. a piece of white muscle tissue was excised from the right body side of each specimen. the tissue was placed in a 2-ml microfuge tube containing absolute ethanol and stored in the freezer until further use. approximately 20 mg of the muscle tissue from each specimen was used for dna extraction using promegawizard® genomic dna purif ication kit (madison, wi) following the manufacturer’s protocol. the following primers from ward et al. ( 2 0 0 5 ) we r e u s ed fo r t h e a m p l i f i c a t i o n of a p p r ox i m a te l y 6 5 5 b p of t h e mitochondrial cytochrome c oxidase i (coi) gene: fishf1: 5’-tcaaccaaccacaaagacat tggcac-3’ fishr2: 5’-acttcagggtgaccgaagaatcagaa-3’ species country status reference clarias batrachus philippines native herre 1924 thailand native welcomme and vidthayanon 2003 india misidentif ication ng and kottelat 2008 vietnam misidentif ication kottelat 1998 clarias gariepinus philippines introduced juliano et al. 1989 india introduced shaji et al. 2000 thailand introduced vidthayanon 2003 brazil introduced vitule et al. 2006 indonesia introduced cambray 2005 turkey native solak et al. 2001 nigeria native olaosebikan and raji 1998. ethiopia native rocha 2008 clarias macrocephalus philippines native conlu 1986 thailand native vidthayanon et al. 1997 clarias fuscus philippines native herre 1953 philippines misidentif ication ng 1999 china native sudarto et al. 2004 table 2. status of clarias species in countries where the coi sequences were obtained b.s. santos et al. 27 pcr amplif ication was performedin 50-μl volumes. the pcr mix consisted of 1.0 μl of dntp (0.05 μm), 2.5 μl of each primer (0.1 μm), 5.0 μl of 1x pcr buffer, 0.5 μl of (1.25 u) taq polymerase (roche taq dntpack), 34.5 μl of ultrapure water, and 4.0 ml of dna template. the pcr conditions were as follows (ward et al. 2005): initiation for 2 min at 95oc, followed by 35 cycles of denaturation for 0.5 min at 94oc, primer annealing for 0.5 min at 54 oc, and primer extension for 1 min at 72oc. a f inal extension step at 72oc for 10 min completed the reaction. the pcr products were visualized on 1% agarose gels with ethidium bromide. bands with approximate size of 650 bp were excised from the gel. the excised gels were then purif ied with qiaquick® gel extraction kit (qiagen, valencia, ca) following the manufacturer’s protocol. the purif ied dna products were sent to 1st base in selangor, malaysia for bidirectional sequencing. the consensus sequence of each specimen was assembled using the staden package v4.10 (staden et al. 2000). these were aligned and analyzed using mega version 6 software (tamura et al. 2013). the 20 c. macrocephalus coi sequence were submitted to genbank and barcode of life data systems (bold). additional sequences were downloaded from genbank (table 1). all clarias coi sequences from bold have corresponding genbank accessions. as such, only genbank accessions were noted. pairwise genetic distances within species and between species were calculated using the kimura 2-parameter (k2p) model of nucleotide substitution (kimura 1980). a neighbor-joining (nj) tree was constructed at 1000 bootstrap replicates (saitou and nei 1987) using the k2p model. results identity of c. macrocephal us specimens out of the 20 c. macrocephalus specimens from agusan marsh, two distinct haplotypes were observed: one haplotype, represented by 19 sequences, matched c. macrocephalus from cagayan (accession numbers kf604663-65) with 100% identity based on blastn results; and the other haplotype matched the same sequences above but with 99.7% identity. k2p d istances within and be tween species a total of 199 coi sequences representing 11 species were analyzed. the average withinand between-species k2p distances were 2.99% and 13.26%, respectively (table 3). average within-species k2p distance greater than 2% was observed for status assessment of clarias species in the philippines 28 c. batrachus (6.98%) and c. fuscus (5.60%; table 4). some intraspecif ic k2p distances were very high, if only a priori species assignments were considered, such as those for c. batrachus and c. fuscus sequences. to facilitate subsequent analyses, highly diverging conspecif ic sequences were given a posteriori assignments based on their phylogeny (figure 1). for c. batrachus, the sequences were grouped according to country, namely philippines, thailand, and india. indian c. batrachus sequences still exhibited very high within-group distances and were further subdivided into the following: india-1, which forms a distinct clade; india-2, which clustered with c. macrocephalus sequences; and india-3, which clustered more closely with philippine and thai sequences of c. batrachus. for c. fuscus, the three available sequences were subdivided into the following: c. fuscus -1, which corresponds to an accession from yunnan province; and c. fuscus2, which represents two accessions from the guangxi region. sequences of c. macrocephalus were also subdivided into philippine and thai samples for comparison. the subgroups described above were treated as separate species for calculations of average withinand between-species genetic distances (tables 4 and 5). the average k2p distances within subgroups were lower compared to the whole. species del ineation of the 11 species, three, namely c. angolensis, c. camerunensis, and c. teijsmanni, were represented by only one sequence. the average k2p distances between these three species and others range from 5.2% to 16.1% (table 5). five others, namely c. dussumieri, c. gabonensis, c. gariepinus, c. jaensis, and c. nieuhofii, form distinct clades with average k2p distances within species ranging from 0.00% to 0.98% (table 4) and a minimum average distance of 5.2% with a non-conspecif ic (table 5). the clades representing these f ive species all had 99% bootstrap support (figure 1). the remaining three species, namely c. batrachus, c. macrocephalus, and c. fuscus, did not form distinct clades and were not completely delineated. six c. batrachus sequences from india (india-2) and one c. batrachus sequence from vietnam clustered with c. macrocephalus (figure 1). one c. fuscus sequence (china-1) grouped with philippine sequences of c. batrachus, while the other two (china-2) formed a distinct clade (figure 1). table 3. summary of percent k2p genetic d istances within and between species within species 5063 0.00 2.99 14.24 0.06 between species 14638 0.00 13.26 17.14 0.02 k2p genetic distance (%) standard error n minimum average maximum b.s. santos et al. 29 table 4. average intraspecific percent k2p genetic d istances species k2p genetic distance (%) clarias angolensis (n=1) clarias batrachus – all (n=59) 6.98 clarias batrachus – philippines (n=21) 0.09 clarias batrachus – thailand (n=13) 0.12 clarias batrachus – india all (n=24) 6.98 clarias batrachus – india 1 (n=17) 0.99 clarias batrachus – india 2 (n=6) 0.58 clarias batrachus – india 3 (n=1) clarias batrachus – vietnam (n=1) clarias camerunensis (n=1) clarias dussumieri (n=6) 0.31 clarias fuscus – all (n=3) 5.60 clarias fuscus – 1 (n=1) clarias fuscus – 2 (n=2) 0.31 clarias gabonensis (n=12) 0.64 clarias gariepinus (n=70) 0.98 clarias jaensis (n=2) 0.00 clarias macrocephalus – all (n=42) 0.50 clarias macrocephalus – philippines (n=25) 0.04 clarias macrocephalus – thailand (n=17) 0.60 clarias nieuhofii (n=2) 0.00 clarias teijsmanni (n=1) add itional phil ippine sequences of c. batrachus the coi sequences of f ive c. batrachus specimens from pantabangan dam, nueva ecija obtained by yambot et al. (unpublished) were included this study. all f ive sequences clustered with the other philippine c. batrachus specimens. the average within group distance for philippine c. batrachus specimens is 0.09% (table 4). the c. batrachus sequences from thailand were closest to the philippine group. the average distance between the two groups is 2.4% (table 5). the average within group distance for thai c. batrachus specimens is 0.12% (table 4). discussion the 100% sequence identity between c. macrocephalus specimens from agusan marsh and those of cagayan conf irms that the two morphotypes belong to the same species. moreover, the philippine specimens of c. macrocephalus continue to form a group distinct from thai specimens (figure 1). the increase in size among specimens from agusan marsh may have been an effect of protecting the area as a status assessment of clarias species in the philippines 30 t ab le 5 . p ai rw is e a ve ra g e p e rc e n t k 2 p g e n e ti c d is ta n ce s b e tw ee n s p ec ie s b.s. santos et al. 31 wildlife sanctuary. despite the geographic separation and overall habitat difference between cagayan and agusan, the lack of genetic diversity is still a cause of concern. although coi is not a standard tool for detecting genetic diversity, it still exhibited a relatively high degree of conspecif ic divergence in other catf ish taxa (wong et al. 2011; bhattacharjee et al. 2012; quilang and yu 2014). while c. macrocephalus specimens from the philippines are genetically diverged from thailand based on coi sequences, very little diversity was observed within the philippine samples. figure 1.neighbor-joining tree of 199 coi sequences of clarias species computed using the k2p model of nucleotide substitution. branch lengths are drawn to scale and represent average k2p distances. numbers in parentheses following the taxon names represent the number of sequences included in the clade. accession numbers of sequences from genbank are shown. bootstrap supports of 1000 replicates are shown. taxa indicated in red text indicate possible misidentif ications. status assessment of clarias species in the philippines 32 only three haplotypes were observed from 25 sequences (π = 0.036) in philippine samples compared to that of thailand where ten haplotypes are present from 17 sequences (π = 0.600). it is thus important to further assess the genetic diversity of c. macrocephalus in these areas. this is especially true since c. macrocephalus is now very seldom caught in areas where they were previously found. tan et al. (unpublished) noted the absence of c. macrocephalus among the catchments in several major water bodies in the country despite repeated sampling. these areas include laguna de bay, taal lake, and the provinces of quezon, camarines sur, albay, samar, leyte, nueva ecija, and iloilo. the addition of c. batrachus coi sequences from pantabangan, nueva ecija, increased the number of philippine c. batrachus samples. this group now represents seven localities all over luzon, which adds support to the distinct genetic lineage formed by c. batrachus from the philippines. philippine and thai specimens of c. batrachus may actually represent different species due to the >10-fold higher genetic distance between them compared to the average distance within groups (hebert et al. 2004). sequences of c. batrachus from india were even more diverged. one group of sequences formed a distinct clade (india-1) with 99% bootstrap support. the average distances of this clade with philippine and thai sequences were 10.4% and 11.3%, respectively. the second group (india-2), together with the coi sequence from vietnam, clustered with c. macrocephalus sequences with 99% bootstrap support. two of the sequences from india were identical with some c. macrocephalus specimens from thailand, while the others, including the sequence from vietnam, were closer to the c. macrocephalus specimens from the philippines (figure 1). one c. batrachus sequence from india (india-3) did not cluster with either of the f irst two indian subgroups, but was closer to philippine and thai sequences with average distances of 1.5% and 3.6%, respectively. ng and kottelat (2008) observed that c. batrachus from northeastern india had the same head shape and pectoral spine serration as c. magur and should be designated to the latter species. eight c. batrachus sequences from india have map coordinates data from genbank. two of the india-1 sequences had map coordinates (jn628880, 924), and both were from northeast india. four out of the six india-2 sequences (fj459456-59) were from central india, while the other two india-2 sequences (jq667517-18) were from west india. based on these data, the india-1 clade in this study may actually be the c. magur described by ng and kottelat (2008) from northeast india. this subgroup formed a distinct clade with 99% bootstrap support (figure 1). the average genetic distances between india-1 specimens and other species or subgroups ranged from 7.5% to 13.5%. none of the india-2 specimens b.s. santos et al. 33 were obtained from northeast india, and their average distance from india-1 sequences was 13.0%. india-2 specimens clustered with c. microcephalus, which has not yet been reported in india (figure1; table 2). it is possible that the lack of reports on c. macrocephalus is due to the widespread mislabeling of catf ish species as c. batrachus, a s i n t h e c a s e of o t h e r f i s h s p e c i e s ( s m i t h e t a l . 2 0 0 8 ; m a r a l i t e t a l . 2 0 1 3 ) . t h e c . batrachus sequence from vietnam was likewise grouped with c. macrocephalus. kottelat (1998) claimed that the c. batrachus found in v ietnam were also misidentif ied (table 2). kottelat (2001) listed c. fuscus as the only clariidae species in vietnam, citing pellegrin (1907) who identif ied the species as c. macrocephalus. although c. fuscus is native to vietnam, it is possible that pellegrin (1907) also examined specimens correctly identif ied as c. macrocephalus. one sequence from india (india-3) grouped more closely to c. batrachus from the philippines and thailand than to india-1 and india-2 sequences. this observation suggests that actual c. batrachusis present in india, apart from those reported as misidentif ications. the 1.5%and 3.6%-distance of this sequence from the philippine and thai samples indicate geographic divergence. ng and kottelat (2008) also suggested three provisional species making up what is widely recognized as c. batrachus. they designated a neotype of c. batrachus from java and species from mainland southeast asia, including thailand, as c. aff. batrachus “indochina”. the species from the rest of sundaic southeast asia were assigned as c. aff. batrachus “sundaland.” ng and kottelat (2008) were unable to analyze philippine samples. however, they were able to analyze specimens from insular malaysia and borneo and assigned them to c. aff. batrachus “sundaland.” based on the geographic proximity of the philippines to borneo, the philippine samples may be more similar to c. aff. batrachus “sundaland.” the calculated average distance supports the distinction of philippine samples from those of thailand. of the three c. fuscus specimens from china, two formed a distinct clade with 99% bootstrap support (figure 1), while one sequence grouped more closely with c. batrachus from the philippines. the average distance between the two subgroups is 8.3%. although herre (1953) lists c. fuscus as native to the philippines, ng (1999) and sudarto et al. (2004) claimed that this is most likely a case of misidentif ication, since c. fuscus is a known chinese taxon. because the diverging sequence showed close similarity with philippine samples, it is possible that the accession from the yunnan province is actually a c. batrachus specimen introduced status assessment of clarias species in the philippines 34 from sundaic southeast asia. importation of c. batrachus to china has been documented by brummett (2008). among the clarias species included in this study, the african catf ish c. gariepinus has the most sequences available in genbank. a total of 70 coi sequences of the species from eight countries were used (table 1). among these, c. gariepinus is native to nigeria, ethiopia, and turkey, and is exoticto the rest. all 70 sequences clustered together with 99% bootstrap support and the average within-species distance is 0.98%. no case of misidentif ication was observed in this study. there is also no case of misidentif ication documented in fishbase (froese and pauly 2014). the large-scale importation of c. gariepinus to many countries, including the philippines, is due to its superior size (juliano et al. 1989; vitule et al. 2006). in contrast to c. gariepinus, the distribution of c. macrocephalus is limited to southeast asia with little reports of importation. it is inferior in size and is threatened by exotic larger-sized competitors (vidthayanon and allen 2013). apart from the 20 generated in this study and the f ive of quilang and yu (2015), only 17 coi sequences were available from genbank for this species. sequences from the philippines formed a clade distinct from the thai sequences. the average withingroup distances for the philippine and thai sequences were 0.04% and 0.60%, respectively (table 4), while the average distance between the two subgroups was 0.8%. these values show that the two subgroups belong to the same species. the c. macrocephalus sequences from thailand show greater diversity compared to c. macrocephalus from the philippines. as discussed earlier, it is impor tant to assess the genetic diversity of this threatened species for management and conservation purposes. the other clarias species, namely c. angolensis, c. camerunensis, c. dussumieri, c. gabonensis, c. jaensis, c. nieuhofii, and c. teijsmanni, were also delineated based on coi sequences. the average within-species distances ranged from 0.0% to 0.64% (table 4), while the average between-species distances ranged from 5.2% to 13.9% (table 5). thus, dna barcoding can be used for the identif ication of the above mentioned species, as well as c. gariepinus and c. macrocephalus as previously discussed. further taxonomic review is necessary for c. batrachus and c. fuscus. status of phil ippine clarias species six clarias species have been reported in the philippines (table 6): f ive of these were reported as native species (herre1953), while one is introduced (juliano et al. b.s. santos et al. 35 1989). as was dicussed earlier, the status of c. batrachus, c. macrocephalus, and c. gariepinus were conf irmed in recent collections. the three other species, namely c. nieuhofii, c. fuscus, and c. meladerma, were not present in recent collections in various freshwaters of luzon (quilang and yu 2015), thus raising questions regarding the past reports. fishbase (froese and pauly 2014) lists these species as native to the philippines citing herre (1953), but these were not listed by conlu (1986). herre (1953) reported the presence of c. nieuhofii, under the synonyms c. nieuhofii and c. gilli, from luzon and mindanao. although there are no recent reports of this species in luzon, its presence in mindanao freshwaters is yet to be verif ied. sudarto et al. (2004) were able to examine a type specimen of this species from mindanao. the morphological features of this type specimen were characteristic of one belonging to the species c. nieuhofii. this f inding conf irms that the early reports of this species in the country are correct, but whether or not this species is still present in the country today remains to be verif ied. herre (1953) reported the presence of c. fuscus in the country, citing fowler (1941). ng (1999), on the other hand, suggested that the c. fuscus identif ied by fowler (1941) might actually be a previous report current status c. batrachus native (herre 1924; verif ied native; currently herre 1953) present (quilang and yu 2015; this study) c. macrocephalus native (herre 1953) verif ied native; currently present (quilang and yu 2015; this study) c. fuscus native (fowler 1941; questionable herre 1953) probable introduction (cui and zhao 2013) probable misidentif ication (ng 1999) not listed (conlu 1986) c. meladerma native (herre 1953) questionable listed as present in the country (rainboth 1996) not listed (conlu 1986; ng 2013) c. nieuhofii native (herre 1924; verif ied native; current presence not herre 1953; fowler 1941) verif ied (sudarto et al. 2004) not listed (conlu 1986) c. gariepinus introduced verif ied introduced; currently present (juliano et al. 1989) (quilang and yu 2015; this study) table 6. list of reported clarias species in the philippines and their current status status assessment of clarias species in the philippines 36 case of misidentif ication. interestingly, iucn lists c. fuscus as an introduced species in the country (cui and zhao 2012). this report is also questionable because cui and zhao (2012) cited no reference for the philippines, but they did so for the introduction of c. fuscus to hawaii and japan. with no other information available, the presence of c. fuscus in the philippines could not be conf irmed. herre (1953) also noted previous reports of c. meladerma in the philippines, particularly in laguna de bay. he indicated that the actual presence of the species in the country is “doubtful.” no c. meladerma specimen was observed in the catf ish samples obtained from laguna de bay in recent years (quilang and yu 2015). rainboth (1996) included the philippines in the geographic distribution of c. meladerma, but did not further specify the actual location nor cite any previous reports. iucn, on the other hand, did not list c. meladerma as present in the philippines (ng 2013). as such, the presence of c. meladerma in the philippines could not be conf irmed. conclusions based on the coi sequences generated from this and past studies, it is clear that the philippine specimens of c. batrachus and c. macrocephalus form distinct lineages, thus conf irming their native status, despite reports of introductions of the former. the philippine sample of c. gariepinus clustered with c. gariepinus from other countries, where the species was also introduced and somewhat divergent from native populations. with the questionable status of c. fuscus and c. meladerma, it is best to limit the list of native clarias species to three, namely c. batracus, c. macrocephalus, and c. nieuhofii, until concrete evidence of the presence of the former two species is reported. the use of highly polymorphic markers like mitochondrial control region and microsatellite dna is recommended to further assess the seemingly low genetic diversity of c. macrocephalus, which is currently listed as threatened. likewise, the current presence of c. nieuhoffi in the philippines still has to be conf irmed, and if so, the genetic diversity of this will have to be assessed. the coi barcodes of philippine and thai samples of c. batrachus should also be compared with those from java and the rest of southeast asia in order to conf irm their species identities. acknowledgments this project was funded by the off ice of the chancellor of the university of the philippines diliman, in collaboration with the off ice of the vice-chancellor for research and development (ovcrd) through the ovcrd outright research grant b.s. santos et al. 37 (project no. 131320 pnse) awarded to j. p. quilang. we would like to thank shiny cathlynne yu, john carlos del pozo, kevin ferrer, and justin bryan maranan for technical assistance. references aquilino sv, tango jm, fontanilla ik, pagulayan rc, basiao zu, ong ps, quilang jp. 2011. dna barcoding of the icrocephalus of taal lake, philippines. molecol res. 11:612-619. 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[internet]. clarias macrocephalus. the iucn red list of t h r e a te n e d s p ec i e s . ve r s i o n 2 0 1 4 . 2 . [ c i t ed 2 0 1 4 s e p t e m b e r 1 6 ] . av a i l a b l e f r o m : www.iucnredlist .org. v itule jrs, umbria sc, aranha jmr. 2006. introduction of the african catf ish clarias gariepinus (burchell, 1822) into southern brazil. biol invas. 8:677-681. ward rd, holmes bh. 2007. an analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase i (cox1) in f ishes. molecol notes. 7:899-907. w a r d r d , h o l m e s b h , w h i t e w t, l a s t p r . 2 0 0 8 . d n a b a r c o d i n g a u s t r a l a s i a n chondrichtyans: results and potential uses in ward rd, zemlak ts, innes bh, last pr, heber t p. 2005. dna barcoding australia’s f ish species. phil. trans. r. soc. b. 360:1847– 1857. welcomme rl, vidthayanon c. 2003. the impacts of introductions and stocking of exotic species in the mekong basin and policies for their control. cambodia: mekong river commission. 65 p. wong ll, peatman e, lu j, kucuktas h, he s, zhou c, na-nakorn u, liu z. 2011. dna barcoding of catf ish: species authentication and phylogenetic assessment . plos one. 6:e17812. yambot av, juanico cst, alcantara sg, fernandez mc. unpublished. dna barcoding of commercially-impor tant f ish species in pantabangan dam, nueva ecija philippines. status assessment of clarias species in the philippines 40 _____________ brian s. santos <bryzee13@yahoo.com> is an instructor at the institute of biology, up diliman. he obtained his ms in biology degree from the same institute in 2011 and is currently enrolled in a phd program. francis peter c. vesagas is an ms student at the institute of biology, up diliman. he obtained his bs in biology degree from the same institute in 2012. marc t imothy c. tan obtained his bs in biology degree from the institute of biology, up diliman in 2014. he is currently enrolled at the ateneo school of medicine and public health. joycelyn c. jumawan, ph.d. is an associate professor at the biology department, caraga state university. she obtained her ph.d. in biology degree from the institute of biology, up diliman in 2012. jonas p. quilang, ph.d. is an associate professor at the institute of biology, up diliman. he obtained his ph.d. in biology degree from the same institute in 2008. 6narsico-fucoidan.pmd j.t. narsico et al. 45 science diliman (january-june 2018) 30:1, 45-59 fucoidan content in phil ippine brown seaweeds joemark t. narsico hokkaido university joyce a. nieva alfred wegener institute helmholtz centre for polar and marine research alper james g. alcaraz university of saskatchewan, canada elad io g.m. anino v university of the philippines manila norchel corcia f. gomez marco nemesio e. montaño* marine science institute university of the philippines diliman abstract this study aims to determine which brown macroalgae in the philippines has the highest content of par tially purif ied fucoidan. percent fucoidan content of brown seaweeds sargassum spp. , pad ina sp. , hyd roclathrus sp. , turbinaria ornata j. agardh, hormophyza cuneiformis pc silva, and dictyota d ichotoma lamouroux were determined in f ifty sites across 14 provinces in northern luzon (cagayan, ilocos), west luzon (pangasinan), the eastern seaboard of luzon (quezon province, camarines, sorsogon), central and eastern visayas (bohol, cebu, negros oriental, negros occidental), and n o r t h e r n m i n d a n a o ( c a m i g u i n , l a n a o d e l n o r t e , m i s a m i s o r i e n t a l , m i s a m i s o c c i d e n t a l ) . c r u d e a n d s e m i p u r e f u c o i d a n w e r e e x t r a c t e d t h r o u g h a c i d h y d r o l y s i s a n d e t h a n o l p r e c i p i t a t i o n u s i n g 5 0 g r a m s o f dried and milled seaweed biomass. extracts were verif ied using infrared spectroscopy with fucoidan from fucus vesiculosus as standard. sargassum spp. is the most widely distributed source of fucoidan found in all sites. t. ornata was found in only 11 sites. both have signif icantly higher percent _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online fucoidan content in philippine brown seaweeds 46 c o n t e n t ( p ≥ 0 . 0 5 ) o f f u c o i d a n t h a n o t h e r s a m p l e d s e a w e e d s . h i g h e r percent content of semi-purif ied fucoidan were observed in d. d ichotoma from bohol (1.53%), h. cuneiformis from cebu (2.17%), hydroclathrus sp. f r o m p a n g a s i n a n ( 2 . 2 3 % ) , p a d i n a s p . f r o m q u e z o n p r o v i n c e ( 3 . 6 9 % ) , sargassum spp. from camiguin (4.30%), and t. ornata from cagayan (7.03%). keywords: brown seaweeds, distribution, fucoidan, fucoidan yield introduction the philippines is known for its diverse marine flora, particularly seaweeds. the country contains 966 taxa of macroalgae, with 893 species in 82 families. brown macroalgae (order ochrophyta, phaeophyceae) are represented in 171 taxa with 153 species in 10 families (ang et al. 2013). brown algae contain fucoxanthin, a xanthophyll pigment responsible for its distinguishing brown color. their cell walls are composed of cellulose, alginic acid, and other algal polysaccharides (trono 1 9 9 7 ) . fucoidan is a sulfated polysaccharide commonly found in brown seaweeds, as well as in marine invertebrates (mak et al. 2013). fucoidan is absent in green algae (chlorophyceae) and red algae (rhodophyceae) (berteau and molloy 2003). fucoidans are structurally diverse macromolecules with a backbone of a (1,3)and (1,4)linked α(1,4)-bonded α-l-fucopyranose residues. the polysaccharide may be arranged in stretches of alternating α(1,3)and α(1,4)-bonded l-fucopyranose residues or through (13)-α-fucan (ale et al. 2011). fucoidans have been extensively studied due to their biological activity which includes anti-inflammatory, antioxidant, anticoagulant, antitumor, antiangiogenic, antithrombotic, antiviral, and immunomodulatory properties. it is widely studied because it comes from different inexpensive sources, and has a potential for drug development or as a functional food resource (li et al. 2008). despite the diverse potential applications of fucoidans, information on the fucoidan content of seaweeds in the philippines remain limited. this information is necessary to guide manufacturers and researchers on which species and sites should be utilized for the maximization of the extraction of the algal polysaccharide. in this study, we determined the fucoidan content of brown seaweeds collected from different j.t. narsico et al. 47 figure 1. percent fucoidan content (semi-purif ied) of dictyota collected in pangasinan, sorsogon, negros occidental and bohol. provinces all over the philippines through acid hydrolysis extraction and fractional ethanol precipitation. we further compared which among the species has the highest content of fucoidan. materials and methods sample collection gratuitous permits were issued by the different regional off ices of the bureau of fisheries and aquatic resources (bfar). fresh seaweed thalli were collected from the intertidal zones across f ifty sites in fourteen provinces in the philippines (figure 1, table 1): northern luzon (cagayan, ilocos); west luzon (pangasinan); the eastern seaboard of luzon island (quezon, camarines, sorsogon); central and eastern visayas islands (bohol, cebu, negros oriental, negros occidental); and northern mindanao island (camiguin, lanao del norte, misamis oriental, misamis occidental). one-time sampling was conducted in all the sites. not all species were found in fucoidan content in philippine brown seaweeds 48 sargassum patar, bolinao, pangasinan 17-19 may 2010 16°19’59.11"n 119°47’13.95"e patar, bolinao, pangasinan 17-19 may 2010 16°19’59.11"n 119°47’13.95"e trensiera, bolinao, pangasinan 17-19 may 2010 16°26' 24.84"n 119°56' 45.87"e lucero, bolinao, pangasinan 17-19 may 2010 16°24’10.50"n 119°54’22.91"e villa manzano norte, alabat, quezon 25-30 nov 2011 14°1’43.17"n 122°5’17.56"e sabang, alabat, quezon 25-30 nov 2011 14°3’28.52"n 122°9’33.78"e gonzaga, cagayan 25-29 july 2010 18°17’16.30"n 121°59’24.89"e sta. ana, cagayan 25-29 july 2010 18°28’49.06"n 122°8’26.09"e sta. ana, cagayan 25-29 july 2010 18°28’49.06"n 122°8’26.09"e blue lagoon, ilocos norte 25-29 july 2010 18°37’22.49"n 120°51’34.16"e burgos, ilocos norte 25-29 july 2010 16°2’44.93"n 119°45’13.97"e burgos, ilocos norte 25-29 july 2010 16°2’44.93"n 119°45’13.97"e burgos, ilocos norte 25-29 july 2010 16°2’44.93"n 119°45’13.97"e lioes, ilocos norte 25-29 july 2010 18°0' 59.68"n 120°29' 11.37"e pangil, ilocos norte 25-29 july 2010 18°0’24.19"n 120°29’20.19"e bgy nailon, bogo city, cebu 10-16 aug 2010 11°2’52.02"n 124°2’28.96"e bgy nailon, bogo city, cebu 10-16 aug 2010 11°2’52.02"n 124°2’28.96"e alcoy, cebu 10-16 aug 2010 9°40’25.97"n 123°30’18.65"e alcoy, cebu 10-16 aug 2010 9°40’25.97"n 123°30’18.65"e maribago, mactan, cebu 10-16 aug 2010 10°17’8.17"n 124°0’26.30"e catmon, cebu 10-16 aug 2010 10°43’20.72"n 124°1’4.35"e dalaguete, cebu 10-16 aug 2010 9°45’54.64"n 123°32’10.47"e dalaguete, cebu 10-16 aug 2010 9°45’54.64"n 123°32’10.47"e bgy. paypay, daanbantayan, cebu 10-16 aug 2010 11°13’20.67"n 123°58’56.78"e ubojan east, garcia-hernandez, bohol 10-16 aug 2010 9°36’32.40"n 124°18’7.14"e larapan, jagna, bohol 10-16 aug 2010 9°38’55.90"n 124°22’7.31"e pamilacan island, baclayon, bohol 10-16 aug 2010 9°29’25.84"n 123°54’59.53"e pamilacan island, baclayon, bohol 10-16 aug 2010 9°29’25.84"n 123°54’59.53"e sitio basdio, loon, bohol 10-16 aug 2010 9°48' 2.22"n 123°47' 20.61"e punta-cruz, maribojoc, bohol 10-16 aug 2010 9°43’56.69"n 123°47’54.99"e sitio daorong, bgy. danao, panglao, bohol 10-16 aug 2010 9°32’41.70"n 123°46’1.16"e bgy. lawis, panggangan island, calape, bohol 10-16 aug 2010 9°54’13.80"n 123°50’27.84"e bgy. lawis, panggangan island, calape, bohol 10-16 aug 2010 9°53' 40.86" n 123°50' 29.37"e pungtod island, panglao, bohol 10-16 aug 2010 9°40’41.37"n 123°51’0.69"e sitio hoyohoy, bgy. tawala, panglao, bohol 10-16 aug 2010 9°33’23.51"n 123°48’32.91"e sto. domingo, (bicol) 8-13 nov 2010 13°23’42.40"n 123°11’29.20"e sto. domingo, (bicol) 8-13 nov 2010 13°23’42.40"n 123°11’29.20"e pasacao, (bicol) 8-13 nov 2010 13°30’30.59"n 123°0’27.70"e bulusan, sorsogon 8-13 nov 2010 12°44’52.69"n 124°8’33.59"e bulusan, sorsogon 8-13 nov 2010 12°44’52.69"n 124°8’33.59"e sangay, sorsogon 8-13 nov 2010 13°36’22.02"n 123°32’51.26"e matnog, sorsogon 8-13 nov 2010 12°35’59.45"n 124°6’24.24"e pinagtigasan, sorsogon 8-13 nov 2010 14°9’56.41"n 122°58’34.42"e poblacion, oroqueta city, misamis occidental 18-24 oct 2010 8°28’55.05"n 123°49’37.89"e poblacion, oroqueta city, misamis occidental 18-24 oct 2010 8°28’55.05"n 123°49’37.89"e talisayan, poblacion, misamis oriental 18-24 oct 2010 9°0’53.98"n 124°52’15.25"e cantaan, guinsilaban, camiguin 18-24 oct 2010 9°5’36.28"n 124°47’29.84"e tagcatong, carmen, misamis oriental 18-24 oct 2010 9°5’36.28"n 124°47’29.84"e tagcatong, carmen, misamis oriental 18-24 oct 2010 8°31’8.56"n 124°37’48.51"e gingoog, misamis oriental 18-24 oct 2010 8°49’57.16"n 125°5’49.66"e kauswagan, lanao del norte 18-24 oct 2010 8°12’3.60"n 124°4’57.57"e liangan, maigo, misamis oriental 18-24 oct 2010 8°9’36.72"n 123°56’20.24"e bgy. roque, mantigue island, camiguin 18-24 oct 2010 9°10’14.48"n 124°49’26.41"e san jose, negros occidental 17-20 sept 2010 9°25’10.65"n 123°14’36.85"e sipalay city, negros occidental 17-20 sept 2010 9°44’47.75"n 122°23’50.14"e dumaguete, negros oriental 17-20 sept 2010 9°18’17.53"n 123°18’40.40"e genera site date latitude longitude sample collection table 1. sampl ing sites and gps coord inates j.t. narsico et al. 49 genera site date latitude longitude sample collection table 1. sampl ing sites and gps coord inates (cont.) hinobaan 2, (negros) 17-20 sept 2010 9°32’23.05"n 122°30’56.19"e san juan, (negros) 17-20 sept 2010 10°36’32.86"n 122°54’36.25"e bais city, (negros) 17-20 sept 2010 9°34’44.33"n 123°10’19.55"e hinobaan 1, (negros) 17-20 sept 2010 9°35’57.65"n 122°27’50.81"e sta. catalina, (negros) 17-20 sept 2010 9°19’40.42"n 122°51’54.28"e lazi, (negros) 17-20 sept 2010 9°19’21.00"n 123°19’33.01"e maria 2, (negros) 17-20 sept 2010 10°43’52.81"n 122°56’2.08"e turbinaria trensiera, bolinao, pangasinan 17-19 may 2010 16°26' 24.84"n 119°56' 45.87"e villa manzano norte, alabat, quezon 25-30 nov 2011 14°1’43.17"n 122°5’17.56"e gonzaga, cagayan 25-29 july 2010 18°17’16.30"n 121°59’24.89"e blue lagoon, ilocos norte 25-29 july 2010 18°37’22.49"n 120°51’34.16"e maribago, mactan, cebu 10-16 aug 2010 10°17’8.17"n 124°0’26.30"e punta-cruz, maribojoc, bohol 10-16 aug 2010 9°44' 4.20" n 123°47' 26.04" e sitio hoyohoy, bgy. tawala, panglao, bohol 10-16 aug 2010 9°32' 57.91"n 123°46' 52.84"e sangay, sorsogon 8-13 nov 2010 13°36’22.02"n 123°32’51.26"e talisayan, poblacion, misamis oriental 18-24 oct 2010 9°0’53.98"n 124°52’15.25"e bgy. balite, sagay, camiguin 18-24 oct 2010 9°6’14.35"n 124°42’48.10"e bgy. roque, mantigue island, camiguin 18-24 oct 2010 9°10' 16.99"n 124°49' 22.38"e sipalay city, negros occidental 17-20 sept 2010 9°44’47.75"n 122°23’50.14"e hinobaan 2, (negros) 17-20 sept 2010 9°32’23.05"n 122°30’56.19"e san juan, (negros) 17-20 sept 2010 10°36’32.86"n 122°54’36.25"e padina patar, bolinao, pangasinan 17-19 may 2010 16°19’59.11"n 119°47’13.95"e perez, alabat, quezon 25-30 nov 2011 14°10’48.33"n 121°55’27.49"e gonzaga, cagayan 25-29 july 2010 18°17’16.30"n 121°59’24.89"e sta. ana, cagayan 25-29 july 2010 18°28’49.06"n 122°8’26.09"e burgos, ilocos norte 25-29 july 2010 16° 2’44.93"n 119°45’13.97"e alcoy, cebu 10-16 aug 2010 9°40’25.97"n 123°30’18.65"e maribago, mactan, cebu 10-16 aug 2010 10°17’8.17"n 124°0’26.30"e bgy. paypay, daanbantayan, cebu 10-16 aug 2010 11°13’20.67"n 123°58’56.78"e pamilacan island, baclayon, bohol 10-16 aug 2010 9°29’25.84"n 123°54’59.53"e sitio basdio, loon, bohol 10-16 aug 2010 9°47’51.87"n 123°47’1.22"e sto. domingo, (bicol) 8-13 nov 2010 13°23’42.40"n 123°11’29.20"e bulusan, sorsogon 8-13 nov 2010 12°44’52.69"n 124°8’33.59"e sipalay city, negros occidental 17-20 sept 2010 9°44’47.75"n 122°23’50.14"e dumaguete, negros oriental 17-20 sept 2010 9°18’17.53"n 123°18’40.40"e caliling, (negros) 17-20 sept 2010 9°59’32.31"n 122°28’14.94"e hinobaan 1, (negros) 17-20 sept 2010 9°35’57.65"n 122°27’50.81"e hormophyza patar, bolinao, pangasinan 17-19 may 2010 16°19’59.11"n 119°47’13.95"e trensiera, bolinao, pangasinan 17-19 may 2010 16°26' 24.84"n 119°56'45.87"e lucero, bolinao, pangasinan 17-19 may 2010 16°24’10.50"n 119°54’22.91"e maribago, mactan, cebu 10-16 aug 2010 10°17’8.17"n 124°0’26.30"e dalaguete, cebu 10-16 aug 2010 9°45’54.64"n 123°32’10.47"e sto. domingo, (bicol) 8-13 nov 2010 13°23’42.40"n 123°11’29.20"e bulusan, sorsogon 8-13 nov 2010 12°44’52.69"n 124° 8’33.59"e talisayan, poblacion, misamis oriental 18-24 oct 2010 9°0’53.98"n 124°52’15.25"e hinobaan 2, (negros) 17-20 sept 2010 9°32’23.05"n 122°30’56.19"e san juan, (negros) 17-20 sept 2010 10°36’32.86"n 122°54’36.25"e hydroclathrus patar, bolinao, pangasinan 17-19 may 2010 16°19’59.11"n 119°47’13.95"e patar, bolinao, pangasinan 17-19 may 2010 16°19’59.11"n 119°47’13.95"e dictyota lucero, bolinao, pangasinan 17-19 may 2010 16°24’10.50"n 119°54’22.91"e ubojan east, garcia-hernandez, bohol 10-16 aug 2010 9°36’32.40"n 124°18’7.14"e pamilacan island, baclayon, bohol 10-16 aug 2010 9°29’25.84"n 123°54’59.53"e bulusan, sorsogon 8-13 nov 2010 12°44’52.69"n 124°8’33.59"e hinobaan 2, (negros) 17-20 sept 2010 9°32’23.05"n 122°30’56.19"e san juan, (negros) 17-20 sept 2010 10°36’32.86"n 122°54’36.25"e maria, (negros) 17-20 sept 2010 10°20’58.91"n 122°50’54.02"e fucoidan content in philippine brown seaweeds 50 the same locations. the type of seaweeds collected varied among sampling sites. samples were washed with distilled water to remove salt and epiphytes, air dried, and milled for extraction. voucher specimens were dried, identif ied, labeled, and kept at the gt velasquez phycological herbarium. study collection sites were limited by geopolitical and accessibility considerations. extraction of fucoidan milled samples were used for extraction following the methods presented by ale et al. (2011) with slight modif ications. fifty grams of dried and milled seaweed thalli were briefly acid-treated using dilute hydrochloric acid, heated, allowed to cool, and centrifuged. residues were discarded afterwards. the solution was neutralized to ph 7.0 using sodium hydroxide pellets, forming brown precipitate of crude fucoidan. partial purif ication was performed by fractional precipitation using 30% and 60% ethanol, in order to remove alginate contamination and to precipitate semi-pure fucoidan. semi-pure fucoidan extracts were subjected to infrared spectroscopy to verify signature peaks of functional groups of fucoidan from fucus vesiculosus. using ft-ir spectrophotometer, dry fucoidan was mounted to the attenuated total reflectance (atr) accessory sample holder and scanned from 400 cm -1 to 4000 cm-1. fucoidan from f. vesiculosus (sigma aldrich: f5631) was used as standard for structure elucidation. data analyses percent fucoidan content of the sampled seaweeds from different sites were pooled within respective provinces. percent fucoidan content was calculated using the weight of the semi-purif ied fucoidan (in grams) divided by the dried and milled biomass (50 g) of seaweeds. the resulting value was then multiplied by 100. the average value of the percent fucoidan content from its corresponding seaweed sources were plotted on the philippine map through qgis, an open-source geographic information software. there were differences in the occurrence of seaweeds per sampling site. statistical analyses on the percent content were conducted via oneway anova and tukey’s multiple comparison test. results the diversity and occurrence of brown seaweeds varied signif icantly among sampling sites. samples were grouped and pooled up to genus level. of the major j.t. narsico et al. 51 groups, sargassum was consistently present and collected in all of the 14 provinces, turbinaria in 11 provinces, padina in 10 provinces, hormophysa in six provinces, dictyota in four provinces, and hydroclathrus in only one province (figures 1-6). crude fucoidan content varied from 10.23% to 24.55% (sargassum spp. , turbinaria ornata), whereas partially-purif ied fucoidan yield were at 1.89% to 7.03% (padina sp. , t. ornata) based on dry weight. sargassum samples from camiguin had the highest fucoidan content (4.3%) among the provinces while samples from pangasinan had the lowest content (1.89%). turbinaria from the northern philippine provinces of cagayan and ilocos had the highest content (7.03% and 6.85%, respectively), figure 2. percent fucoidan content (semi-purif ied) of the brown seaweed hormophysa collected in six provinces in the philippines. figure 3. percent fucoidan content (semi-purif ied) of hydroclathrus. hydroclathrus was only observed in pangasinan. fucoidan content in philippine brown seaweeds 52 figure 4. percent fucoidan content (semi-purif ied) of padina collected in 10 provinces in the philippines. figure 5. percent fucoidan content (semi-purif ied) of sargassum. samples were collected in 14 provinces in the philippines. highest fucoidan yield was observed in camiguin. j.t. narsico et al. 53 while those obtained in cebu had the lowest fucoidan content at 0.74%. padina from the quezon province had the highest content at 3.69%, while the negros occidental samples had the lowest content at 1.3%. hormophysa from cebu had the highest content at 2.17%, while the misamis oriental samples obtained the lowest content at 0.99%. dictyota from bohol had the highest content at 1.53%, while the pangasinan samples had the lowest content at 0.19%. hydroclathrus from pangasinan averaged at 2.23% fucoidan content. fucoidan percent content among the genus were also compared. there was no signif icant difference between the contents of sargassum, and hormophysa, hyd roclathrus, padina, and turbinaria (p ≤ 0.05). however, the percent content of sargassum compared to dictyota was signif icantly higher (p ≥ 0.05). percent content from turbinaria was signif icantly higher compared to dictyota, hormophysa, and padina (p ≤ 0.05). there was no signif icant difference between the yields of sargassum and turbinaria (p ≤ 0.05). additionally, there was no signif icant difference between the fucoidan contents of padina and hormophysa, hydroclathrus and dictyota, and hormophysa against hydroclathrus and dictyota (figure 7). figure 6. percent fucoidan content (semi-purif ied) of turbinaria collected in 11 provinces in the philippines. samples from cagayan obtained the highest fucoidan yield. fucoidan content in philippine brown seaweeds 54 representative data on the semi-purif ied fucoidan from sargassum, turbinaria, and padina showed peaks similar to the standard fucoidan from f. vesiculosus (figure 8). there were broad bands at 3321–3415 cm-1 and small peaks at 2941-2945 cm-1, indicating signature vibrations of oh groups and ch of pyranoid rings, and c6 of fucose and galactose, respectively (kim et al. 2010). a peak at 1732 cm-1 indicates the o-acetyl group (chandia and matsuhiro 2008; kim et al. 2010; synytsya et al. 2010). sulfate stretch was observed at 1241–1242 cm-1, which are peaks unique to ester sulfates. finally, centered peaks were observed between 830–842cm-1, corresponding to c-o-s with sulfate at equatorial and/or axial positions (bilan et al. 2004; kim et al. 2010). figure 8. ir spectra of semi-purif ied fucoidan from different brown seaweed species and f. vesiculosus. figure 7. percent fucoidan content of brown seaweeds in the philippines. comparison of the fucoidan yield of samples gathered from different provinces in the country from 2010-2011. signif icant differences between the following comparisons were observed at 95% conf idence interval: sargassum and dictyota, turbinaria and padina, turbinaria and hormophysa, and turbinaria and dictyota. j.t. narsico et al. 55 discussion in this study, we sampled six genera of brown macroalgae from more than 50 sites within 14 provinces in the philippines. some seaweeds were at minimal distribution, if not absent, in sampling sites; hence, it was not feasible to extract suff icient fucoidan for analyses. all samples were not collected in the same sites at the same time during the year. seasonal differences and varying distribution patterns could account for the absence or presence of certain species in the sampling sites. for instance, dictyota is known to be widely distributed in the luzon and visayas regions (trono 1997), but it was not found during cer tain collection periods in sampling sites in mindanao. on the other hand, sargassum was collected in all provinces because it is widely distributed and grows during wet and dry seasons. trono (1997) detailed the distribution and seasonal variation of brown seaweeds in the philippines. thus, the differences in their distribution affect the comparison of fucoidan content among the provinces. as a consequence of this irregularity, we pooled the data collected in each genus or species per provinces. this gives us an estimate of how much content can be obtained in seaweeds from representative sites per province. at present, there is no standardized purif ication procedure for fucoidans. classical methods of extracting fucoidan involve a multi-step aqueous extraction using an acid which is usually hydrochloric acid (ale et al. 2012). fucoidan extracted using hcl is similar to the fucoidan supplied by sigma-aldrich. it is important to note that the characteristics of fucoidan are dependent on the extraction technique. different extraction methods and purification treatments of fucoidans have resulted to varied compositional results and structural suggestions for fucoidan and other polysaccharides (ale and meyer 2013). among the seaweeds sampled, dictyota had the lowest fucoidan content while sargassum and turbinaria had the highest. it is expected that dictyota will have the lowest fucoidan content because of its fleshy and soft fronds. fucoidan yield and monosaccharide composition are also affected by plant age or maturity (skriptsova et al. 2010). aside from differences in fucoidan content, seaweeds are also reported to exhibit a relatively large variation in composition and structural properties, even those belonging to the same order or family (ale et al. 2011), resulting to an array of varied intensities of bioactivities. the amount and composition of algal metabolites are influenced by complex exogenous factors and endogenous biological and biochemical processes. mature and reproductive stages of the macrophyte reportedly produce signif icant amounts of fucoidan compared to young thalli (zvyagintseva et al. 2003; skriptsova et al. 2010). fucoidan content of reproductive fucoidan content in philippine brown seaweeds 56 ҁ  tissues of f ive macroalgal species were 1.3-1.5 times higher compared to their sterile counterparts. fucoidan generally accumulates in the reproductive structures of brown seaweeds, whose reproduction cycle also affect the changes in fucoidan monosaccharide composition (skriptsova et al. 2012). geographical location, seaweed species, and seasonal variations may also influence the differences in polysaccharide composition and their chemical structure. in a study conducted by sinurat et al. (2016), sargassum polycystum from three different sampling sites in indonesia exhibited differences in their fucoidan and ash contents. the maturation cycle of the seaweed also influences the changes in fucoidan content. this cycle is primarily influenced by the changes in season. in temperate countries, the increase in water temperature influences the growth and maturity of the seaweeds. this was observed in the brown seaweed undaria pinnatifida collected from september-october 2011 in three different mussel farms in new zealand, wherein seaweed samples exhibited low fucoidan content on july, an increase in the content on september, and a drastic drop on october 2011. aside from the changes in the fucoidan content, uronic, sulphate, fucose, and protein contents of the seaweeds were also affected by the month of harvest. the study also suggested that there were variations in the crude fucoidan content and composition between the two different sampling sites (mak et al. 2013). sargassum and turbinaria are potential sources of fucoidan because of their greater fucoidan content and wide distribution in the philippines. for future studies, it is recommended that the relationships between the fucoidan content of the seaweeds, and the seasonal variations and the reproductive cycles should be investigated. it is also recommended to f ine scale the sampling sites, and to focus on locations where there is a high chance of collecting the same type of species in certain months. for example, the preliminary results of this study suggest that the provinces of camiguin, negros oriental, and pangasinan are possible locations for establishing multiple collection sites for sargassum. for turbinaria, possible sampling sites can be established in cagayan, ilocos norte, and quezon province. by establishing multiple sites in these provinces, researchers and investors can be assisted in deciding which areas should be prioritized for collection and which months the seaweeds should be collected. it is recommended to further study the life cycle and physiology of these fucoidan-yielding species. it is also important to develop culture techniques for local brown seaweeds to prevent the overharvesting of these species in the wild and to create a steady supply of brown seaweeds in the j.t. narsico et al. 57 market. for instance, culture techniques for brown seaweeds undaria and laminaria were already developed (tseng and fei 1986). possible investors and farmers may culture the brown macroalgal species in the sites identif ied. acknowledgements this study was funded by the bureau of agricultural research of the department of agriculture, republic of the philippines. we thank ms. hannah faith dormido for making the maps presented in this study. this is contribution no. 452 of the marine science institute, university of the philippines. references ale mt, meyer as. 2013. fucoidans from brown seaweeds: an update on structures, extraction techniques and use of enzymes as tools for structural elucidation. royal chemistry society advances. 3:8131-8141. ale mt, mikkelsen jd, meyer as. 2011. important determinants for fucoidan bioactivity: a critical review of structure-function relations and extraction methods for fucosecontaining sulphated polysaccharides from brown seaweeds. marine drugs. 9(10):21062130. ale mt, mikkelsen jd, meyer as. 2012. designed optimization of a single-step extraction of fucose-containing sulfated polysaccharides from sargassum sp. journal of applied phycology. 24(4):715-723 ang po, leung sm, choi mm. 2013. a verif ication of repor ts of marine algal species from the philippines. philippine journal of sciences. 142:5-49 berteau o, mulloy b. 2003. sulfated fucans, fresh perspectives: structures, functions, b i o a c t i v i t i e s , a n d b i o l o g i c a l p r o p e r t i e s o f s u l p h a t e d f u c a n s a n d a n o v e r v i e w o f enzymes active toward the class of polysaccharides. glycobiology. 13(6):29r-40r. bilan mi, grachev aa, ustuzhanina ne, shashkov as, nifantiev ne, usov ai. 2004. a highly regular fraction of a fucoidan from the brown seaweed fucus distichus l. carbohydrate research. 339:511–517. chandia n, matsuhiro b. 2008. characterization of a fucoidan from lessonia vadosa (phaephyta) and its anticoagulant and elicitor properties. international journal of biological macromolecules. 42:235-240. k i m e j , park s y, lee j y, pa r k j h y. 2 0 1 0 . f u co i d a n p r e s e n t i n b r ow n a l g a e i n d u ces apoptosis of human colon cancer cells. bmc gastroentorology. 10:96. fucoidan content in philippine brown seaweeds 58 li b, lu f, wei x, zhao r. 2008. fucoidan: structure and bioactivity. molecules. 13:16711695. mak w, hamid n, liu t, lu j, white wl. 2013. fucoidan from new zealand undaria pinnatifida: monthly variations and determination of antioxidant activities. carbohydrate polymers. 95(1):606-614. sinurat e, saepudin e, peranginangin r, hudiyono s. 2016. characterization of fucoidan e x t r a c t e d f r o m s a r g a s s u m p o l y c y s t u m d i f f e r e n t h a b i t a t s . i n t e r n a t i o n a l j o u r n a l o f chemical, environmental and biological sciences. 4(1):96-99. skriptsova av, shevchenko nm, zvyagintseva tn, imbs ti. 2010. monthly changes in the c o n t e n t a n d m o n o s a c c h a r i d e c o m p o s i t i o n o f f u c o i d a n f r o m u n d a r i a p i n n a t i f i d a (laminariales, phaeophyta). journal of applied phycology. 22:79–86. skriptsova av, shevchenko nm, tarbeeva dv, zvyagintseva tn. 2012. comparative study o f p o l y s a cc h a r i d e s f r o m r e p r o d u c t i ve a n d s te r i l e t i s s u e s of f ive b r ow n s e a weed s . marine biotechnology. 14:304-311. synytsya a , kim w, kim s, pohl r, synytsya a, kvasnicka f, park y. 2010. structure and antitumour activity of fucoidan isolated from sporophyll of korean brown seaweed undaria pinnatifida. carbohydrate polymers. 81:41-48. trono gc. 1997. field guide and atlas of the seaweed resources of the philippines. makati: bookmark. tseng ck, fei xg. macroalgal commercialization in the orient. in: ragan ma , bird cj, e d i t o r s . pr o ce e d i n g s o f t h e tw e l f t h i n t e r n a t i o n a l s e a w ee d s y m p o s i u m . tw e l f t h i n t e r n a t i o n a l s e a w e e d s y m p o s i u m ; j u l y 2 7a u g u s t 1 , 1 9 8 6 ; s a o pa o l o , b r a z i l . netherlands: springer. 1986. pp 167-172. zvyagintseva tn, shevchenko nm, chizhov ao, krupnova tn, sundukova ev, isakov vv. 2003. water-soluble polysaccharides of some far eastern brown seaweeds. distribution, structure, and their dependence on the developmental conditions. journal of experimental marine biology and ecology. 294(1):1-13. _____________ joemark t. narsico is a graduate of the marine science institute under the marine biology program. he is currently a ph.d. student at hokkaido university in japan. his dissertation focuses on the biochemical studies of polysaccharide lyases from a marine bacterium. his research interests include microbial biotechnology and algal polysaccharides. joyce a. nieva is a graduate of the marine science institute under the marine biotechnology program. she is currently pursuing ecological chemistry for her ph.d. in alfred wegener institute in bremerhaven, germany. j.t. narsico et al. 59 alper james alcaraz is currently a ph.d. research fellow on molecular and environmental toxicology at the university of saskatchewan, canada. he was a research associate at the marine science institute working on inland macroalgal farming techniques and seaweed polysaccharide chemistry. he also lead an ecotoxicology research project that was used in the development of offshore effluent standards, partnering with government and private sectors. he graduated with a bachelor’s degree in chemistry and an m.s. in environmental science in up where he also worked with leading scientists in the marine sciences. elad io g.m. anino v was one of the research assistants in dr. montaño’s team, working on algal polysaccharides from brown and red seaweeds during his time in the seaweed chemistry laboratory of the marine science institute. he is currently a fourth year medical student in the university of the philippines college of medicine. his research interests include biomedical applications of marine natural products and drug delivery systems. norchel corcia gomez is a graduate student in the marine science institute under the program marine biotechnology. as a research assistant in the seaweed chemistry lab, she has worked with algal polysaccharides from brown and red seaweeds under the supervision of dr. montaño. her research interests include marine natural products, and marine pollution. she plans to study about microplastics in the marine environment for her ms thesis. marco nemesio e. montano, ph.d. is a retired professor from the marine science institute. he is a member of the national academy of science and technology of the philippines. he was awarded by the integrated chemists of the philippines as the most outstanding chemist award for the year 2012 for his dedication and professional excellence in supporting the seaweed industry in the philippines. he has published numerous papers and spearheaded projects on various research topics on algal polysaccharides, marine pollution and marine natural products. 10subscription form.pmd method of payment (please check one) £ pay cash at the ovcrd (see address above) £ pay in check (please make check payable to the university of the philippines diliman-ovcrd) £ money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address _______________________________ _____________________________________________________ telephone no. _______________________________ ___________________________________________________ fax no. ________________________________________ please send accomplished subscription form to the rduo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city ((02) 436-8720 fax (02) 927-2568 * research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. (subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650) £ humanities diliman £ science diliman £ social science diliman grand total sd inside back cover-ched.pmd science diliman: a philippine journal of pure and applied sciences is accredited by the commission on higher education (ched) through its journal accreditation service project, as category a-2 journal from 2014 to 2016 and is available via www.doaj. org and www.ebsco.com. science diliman has been selected for coverage in the emerging sources citation index of thomson reuters and is available via www.doaj.org and www.ebsco.com 8montano-high throughput-supplementary.pmd a.s.n.s. ferrer et al 93 science diliman (january-june 2017) 29:1, 93-97 high-throughput screening for quorum sensing-inhibitory compounds from selected phil ippine marine algae and surface-associated marine microorganisms for potential anti-biofilm/biofoul ing appl ications supplementary material aira sacha nad ine s. ferrer, aljon francis koji p. elegado, mel iton r. chiong iii, laude karina g. alcober, dang marviluz l. espita and marco nemesio e. montaño university of the philippines diliman field collection of samples and in-situ detection of quorum sensing inhibition seaweed samples were obtained from 36 sites around luzon, visayas, and mindanao (table 1). necessary permits were obtained from relevant government units in coordination with the regional off ices of the bureau of fisheries and aquatic resources (bfar). since a greater variety of seaweed species is observed during specif ic months, sampling visits accounted for the seasonality of seaweeds. voucher specimens were submitted to the gt velasquez herbarium at the up marine science institute for proper identification and future reference. issn 0115-7809 print / issn 2012-0818 online table 1. field collection sites site gps coord inates/municipal ity luzon poblacion dos, calatagan, batangas 13°49’47.0"n 120°37’02.9"e san antonio, zambales 14°54’50.6"n 120°00’29.7"e minanga weste, buguey, cagayan 18°16’5.69"n 121°52’1.77"e minanga weste, buguey, cagayan 18°16’9.82"n 121°51’39.12"e gonzaga, cagayan 18°22’48.73"n 122° 5’57.62"e santa ana, cagayan 18°22’48.62"n 122° 5’57.97"e taggat sur, claveria, cagayan 18°36’44.00"n 121° 2’55.29"e sitio banwa, balaoi, pagudpud, ilocos norte 18°35’38.46"n 120°53’5.99"e casa teresita, pagudpud, ilocos norte 18°36’29.84"n 120°46’42.55"e paayas, burgos, ilocos norte 18°29’56.31"n 120°34’11.04"e currimao, ilocos sur 17°59’30.47"n 120°29’46.39"e sinait, ilocos sur 17°51’53.33"n 120°26’25.30"e santa maria, ilocos sur 17°22’4.12"n 120°27’2.28"e balaoan, la union 16°48’12.89"n 120°19’42.38"e rosario, ilocos sur 16°13’0.44"n 120°24’45.66"e bolinao, pangasinan 16°22’40.7"n 119°54’42.0"e high-throughput screening for quorum sensing-inhibitory compounds 94 table 1. field collection sites (cont’n.) visayas marine sanctuary, luyang, carmen, cebu 10°35’53.88"n 124° 1’45.30"e punta engaño, lapu-lapu, cebu 10°19’51.81"n 124° 2’40.01"e moalboal beach resort, saavedra, moalboal, cebu 9°59’7.76"n 123°23’5.31"e saavedra fish sanctuary, moalboal, cebu 9°59’51.36"n 123°22’46.79"e bcd’s pace, tan-awan, oslob, cebu 9°27’46.80"n 123°22’45.91"e calaguyan sur, loon, bohol 9°50’32.13"n 123°47’42.03"e tambulian shoal, tubigon, bohol 10° 3’20.16"n 123°56’34.17"e ubay island, tubigon, bohol 10° 1’21.26"n 123°58’0.57"e poblacion, bien unido, bohol 10° 8’10.16"n 124°22’51.06"e larapan, jagna, bohol 9°39’10.39"n 124°22’51.31"e doljo, panglao, bohol 9°35’10.27"n 123°43’27.94"e mindanao tibungco, davao city, davao del sur 7°11’07.4"n 125°39’17.2"e bunawan, davao city, davao del sur 7°14’28.4"n 125°39’10.1"e passig islet, digos, davao del sur 6°47’10.1"n 125°23’42.2"e sarangani island, davao occidental 5°25’30.5"n 125°27’44.1"e moncado poblacion, samal island, davao del norte 7°07’50.2"n 125°40’55.0"e miranda poblacion, samal island, davao del norte 7°08’19.1"n 125°40’59.8"e tambo, samal island, davao del norte 7°08’57.9"n 125°41’07.7"e kawas beach, alabel, saranggani 6°04’00.2"n 125°16’37.2"e bula beach, general santos city, saranggani 6°05’58.7"n 125°11’55.7"e site gps coord inates/municipal ity immediately after the f ield collection, preliminary qualitative high-throughput screening was performed with the seaweed fragments and microbial isolates using the method described by mclean et al. (2004). this determines whether qs inhibitory compounds are present on the seaweed surface and eff iciently tests all surfaceassociated microorganisms. the bacterial sensor chromobacterium violaceum (cv 12472), which characteristically produces a water-insoluble purple pigment called violacein through the qs network, was used as an indicator strain. quorum sensing inhibition activity would appear as zones around seaweed fragments or microbial isolates, indicating cv 12472 growths devoid of purple coloration. seaweed fragments were cut into smaller size fragments (ca. 1 cm) aseptically and surface-sterilized. the fragments were subsequently washed in sterile seawater and placed on luria broth-agar (lba). surface-associated microbial isolates were streaked on lba and were incubated for 24 hours. soft lba inoculated with an overnight culture of cv 12472 was then poured over the plates with the seaweed fragments and isolates. the plates were observed after 24 hours of incubation. results of the in-situ assays are depicted in figure 1. colorless zones around the seaweed fragment or microbial isolate indicate the presence of potential qs inhibitory compounds. a.s.n.s. ferrer et al 95 crude extraction and agar well diffusion a total of 51 crude methanol extracts from seaweed were obtained, and percent yields based on dry weight are presented in figure 2. in order to test for qs inhibitory compounds in seaweed metabolites, all crude extracts were screened for qs inhibitory activity through agar well diffusion assay, similar to the method used by rasmussen et al. (2005), but with cv12472 as reporter strain. wells were bored on layers of lba and soft lba inoculated with an overnight culture of cv12472, in order to accommodate 50 ì l of extract. plates were incubated for 24 hours at room temperature and observed. the absence of the purple pigmentation in areas surrounding the wells signif ies the inhibition of quorum sensing. figure 3 depicts the inhibition of violacein production in cv12472 agar plates by qsi-positive extracts. figure 3 shows the presence of zones of inhibition for the crude extract, as well as the hexane and ethyl acetate partitions. quorum sensing inhibition assay in broth cultures the extract, together with an overnight culture of c. violaceum cv12472 in luria broth, was incubated at room temperature for 24 hours without agitation. violacein production and cell density were quantif ied by measuring the optical density (i.e. absorbance) at 585 nm and 600 nm, respectively. as illustrated in figure 4, the crude extract of h. edulis has a lower absorbance reading for violacein compared to the negative control (methanol). figure 1. results of the in-situ assay. (a) seaweed fragments of chaetomorpha crassa exhibiting qs inhibitory activity (with replicates labelled as 1, 2, 3); (b) noninhibitory seaweed fragments of padina sp.; (c) microbial isolate showing the decolorization of chromobacteria violaceum; and (d) a non-inhibitory microbial isolate. high-throughput screening for quorum sensing-inhibitory compounds 96 f ig u re 2 . p e rc e n t y ie ld s ( b a s e d o n d ry w e ig h t) o f c ru d e e x tr a c ts o f s e a w e e d s p e c ie s f ro m l u z o n ( b lu e ), v is a y a s ( o ra n g e ), a n d m in d a n a o ( g re e n ). halymenia durvellei chaetomorpha crassa gracilaria sp. halimeda opuntia galaxaura oblongata padina sp. cheilosporum sagittatum halimeda macroloba halimeda opuntia padina sp. hormophysa cuneiformis chaetomorpha crassa mastophora rosea turbinaria ornata gelidiella acerosa gracilaria edulis gracilaria edulis kappaphycus contonii hydropuntia edulis gracilaria sp. laurencia sp. halimeda macroloba mastophora rosea ulva reticulata galaxaura oblongata galaxaura fasciculata turbinaria ornata halimeda opuntia amphiroa foliacea laurencia cartilaginea padina sp. galaxaura oblongata galaxaura apiculata ulva reticulata padina sp. halimeda macroloba turbinaria conoides galaxaura fasciculata halimeda opuntia halimeda macroloba turbinaria ornata mastophora rosea ulva reticulata gracilaria sp. gracilaria salicornia caulerpa racemosa halimeda sp. laurencia tronoi galaxaura fasciculata ulva lactuca bornetella sp. 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 a.s.n.s. ferrer et al 97 figure 4. liquid assay of crude h. edulis extract (100 mg/ml), methanol, and cinnamaldehyde (75mm). methanol and cinnamaldehyde served as the negative and positive controls, respectively. data presented as mean ± sd (n=6). figure 3. agar well plate diffusion assay for the (a) crude extract, (b) hexane partition, (c) ethyl acetate partition, and (d) aqueous partition of hydropuntia edulis. on each plate, the negative control (methanol, hexane, ethyl acetate, and water, repectively) is located on the lower right, while the positive control (75 mm cinnamaldehyde) was placed on the lower left. references mclean rj, pierson ls, fuqua c. 2004. a simple screening protocol for the identif ication of quorum signal antagonists. journal of microbiological methods. 58:351–360. rasmussen tb, bjarnsholt t, skindersoe me, hentzer m, kristoffersen p, kote m, nielsen j, eberl l, givskov m. 2005. screening for quorum-sensing inhibitors (qsi) by use of a novel genetic system, the qsi selector. journal of bacteriology. 187(5):1799–1814. 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address _______________________________ _____________________________________________________ telephone no. _______________________________ ___________________________________________________ fax no. ________________________________________ please send accomplished subscription form to the rduo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city (02) 436-8720 fax (02) 927-2568  research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. (subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total non-carcinogenic health risk-molina.pmd non-carcinogenic health risks of heavy metal in mudfish 23 victorio b. molina associate professor and chair department of environmental and occupational health college of public health, university of the philippines manila 625 pedro gil street, ermita manila 1000 philippines tel. (632) 524-7102; fax (632) 523-7745; email: vbmolina@post.upm.edu.ph science diliman (january-june 2012) 24:1, 23-32 abstract non-carcinogenic health risks of heavy metal in mudfish from laguna lake this paper examines the potential risks to human health associated with exposure to heavy metal that have bioaccumulated in mudfish (ophicephalus striatus) from laguna lake. fish samples were collected in eight sampling stations in three major areas of the lake during the dry and wet seasons. dry season samples were collected from may to june 2010 and wet season samples, from september to november 2010. coordinates of sampling site locations were recorded using global positioning system (gps) and plotted in geographic information system (gis) digital maps. heavy metal analyses for cadmium (cd), lead (pb), mercury (hg), arsenic (as), and chromium (cr) were conducted using atomic absorption spectrophotometer (aas) and a mercury analyzer (mercur-duo). estimates of health risks associated with mudfish consumption were summarized according to non-carcinogenic effects. non-carcinogenic hazard quotient (nhq) values of five heavy metal showed that lead is the most urgent pollutant of concern in terms of adverse health effects from risks associated with mudfish consumption from all sampling locations in the lake. from the point of view of human health protection and disease prevention, mudfish from laguna lake is not fit for long-term human consumption primarily due to lead and mercury contamination. keywords: bioaccumulation, health risk assessment, heavy metal, laguna lake, mudfish 24 molina, v.b. science diliman (january-june 2012) 24:1, 24-32 introduction heavy metals from natural sources and anthropogenic activities are continually released into aquatic environment, causing serious threat due to their toxicity, bioaccumulation, long persistence and bio-magnification in the food chain. fish is considered as one of the most significant indicators in freshwater ecosystems for estimating extent of trace metals pollution (yousafzai et al., 2010). with growing urbanization and industrialization, there has been a rapid increase in domestic and industrial wastewater which has intensified environmental pollution in different environmental compartments. the major sources of contamination in surface waters can be traced to industrial discharges, domestic waste disposal and application of agrochemicals on farmlands, among others. pollutants like heavy metal, after entering into aquatic environment, accumulate in tissues and organs of aquatic organisms (akan et al., 2009). these contaminants entering the aquatic ecosystem may not directly damage the organisms but they can be deposited into aquatic organisms through the effects of bioaccumulation, biomagnifications and food chain process and eventually threaten the health of humans through fish consumption (lakshmanan et al., 2009). biomagnification of trace elements in living organisms describes the processes and pathways of these potential pollutants from one trophic level to another. increasing concentration through the food chain causes higher retention time of toxic substances than that of the other normal food components (sreedhara nayaka et al., 2009). fish being situated at high trophic level of the food web may accumulate large amounts of heavy metal from the water and often in concentrations several times higher than in the ambient water (yousafzai et al., 2010). some of the metals found in the fish might be essential as they play important role in biological system of the fish as well as in human beings. however, some of them may also be toxic and might cause serious damage in human health even in trace amount at a certain limit or threshold (hosseinkhezri et al., 2011). in contrast, fish has been known for its reputation as the established health food for most of the world’s population, particularly in developing countries, as compared to meat, poultry and eggs. the protein content in fish averages from 15 to 20 percent; hence fish provides comparatively cheap and readily available protein sources in complement with long chains of n-3 fatty acids, amino acids, vitamins and minerals that further contribute to healthy nutritional options for a balance dietary intake (nor hasyimah et al., 2011). health risk assessment of heavy metal bioaccumulation in fish therefore is highly important to establish scientific basis for understanding risks versus benefits of fish consumption. the philippine millennium ecosystem assessment subglobal assessment for laguna lake emphasized that the laguna lake basin is a classic model of a multiple resource with multiple users. its capacity to provide various ecosystem services to various users is continuously being challenged mainly by anthropogenic factors. deforestation of its watersheds in favor of other uses such as agriculture, industry, and human settlements is expected to cause an imbalance in the lake hydraulic processes. lake water quality has deteriorated through the years due to various point sources of pollution from industry, agriculture, and domestic sources. detection of traces of heavy metal like copper, cadmium, chromium, and lead in the water and sediment is a major concern for human health. (ecosystems and people: the philippine millennium ecosystem assessment, sub-global assessment, lasco, espaldon & tapia, 2005. the main objective of the study is to assess the risks to human health associated with the exposure to heavy metal bioaccumulation of mudfish (ophicephalus striatus) from laguna lake. materials and methods sampling zones and sites laguna lake being the largest inland body of water in the philippines with surface area of approximately 900 square kilometers was arbitrarily divided into five sampling zones: namely, northern west bay, central west bay, central bay, south bay, and east bay. mudfish samples were collected from each of the five designated sampling zones in the lake. there were two non-carcinogenic health risks of heavy metal in mudfish 25science diliman (january-june 2012) 24:1, 24-32 sampling sites each for northern west bay, central west bay, and central bay; and one sampling site each for south bay, and east bay; for a total of eight sampling sites. the summary of sampling zones and sites is shown in table 1. the coordinates of the sampling locations of the eight stations in the different zones were recorded using a global positioning system (gps) instrument and plotted in geographic information system (gis) digital maps. the locations and coordinates of the sampling sites are shown in table 2. this facilitated re-sampling activities and ensured that subsequent samples for the wet season were collected in the area as that of the dry season samples. a gis map of laguna de bay showing the sampling sites is shown in figure 1. sampling frequency there were two batches of mudfish (ophicephalus striatus) samples collected using fish net. the first batch of fish samples was collected in may to june 2010 to represent the dry season conditions in the study area. the second batch was collected during the months of september to november 2010 to represent wet season conditions. samples were collected for both seasons to determine potential variations in health risk. sampling zones name number of sampling site/s 1 nort hern west bay 2 2 central west bay 2 3 central bay 2 4 south bay 1 5 east bay 1 total 8 table 1. sampling zones and sites fish sampling site location coordinates 1a (binangonan) northern west bay n 14 o 28’ 57.8’’ e 121 o 09’ 22.6’’ 1b (taguig) northern west bay n 14 o 27’ 50.6’’ e 121 o 05’ 19.3’’ 2a (talim island) cent ral west bay n 14 o 22’ 34.1’’ e 121 o 12’ 03.6’’ 2b (sta rosa) cent ral west bay n 14 o 22’ 43.4’’ e 121 o 04’ 30.1’’ 3a (jal a-jala) central bay n 14 o 22’ 43.9’’ e 121 o 19’ 25.5’’ 3b (cardona) central bay n 14 o 28’ 13.5’’ e 121 o 13’ 19.4’’ 4 (calamba) south bay n 14 o 11’ 41.4’’ e 121 o 11’ 43.5’’ 5 (paki l) east bay n 14 o 22’ 12.9’’ e 121 o 25’ 28.8’’ table 2. sampling site locations and coordinates 26 molina, v.b. science diliman (january-june 2012) 24:1, 24-32 figure 1. location of sampling sites (gis map) heavy metals included in the study the heavy metals included in the study were cadmium (cd), lead (pb), mercury (hg), arsenic (as), and chromium (cr). these non-essential metals from the point of view of human health are also known to have the ability to bioaccumulate through the food chain. sample packaging and preservation fish samples were individually wrapped in a waterproof plastic sampling bag. the edible portions of the fish samples were processed on-site to avoid puncturing of the packaging material by fish spines. individual fish samples were sealed in three layers of plastic bags. each sample was provided with identification tag and sample code. after packaging, samples were kept in an ice chest (with ice) and immediately brought to the industrial technology development institute, department of science and technology laboratory. laboratory procedures and analysis samples submitted to the laboratory were stored in freezer until all the samples had been collected to ensure uniform sample preparation. prior to analyses, samples were thawed then osterized for homogeneity. replicates were prepared and all quality control parameters were conducted to ensure integrity of the analyses. cadmium, chromium and lead were analyzed using the aas (atomic absorption spectrometer). the sample solutions were aspirated into a flame and atomized. non-carcinogenic health risks of heavy metal in mudfish 27science diliman (january-june 2012) 24:1, 24-32 arsenic analysis involves the generation of arsine gas by reacting the arsenic in the sample with sodium borohydride. reaction takes place in a hydride generation assembly that is attached to an aas system. mercury was analyzed using the mercur-duo mercury analyzer, a single-beam instrument with a mercury lowpressure lamp as a light source for the excitation of mercury atoms, and a photomultiplier to record the fluorescent or absorption radiation. results and discussion the results on the heavy metal concentrations in the edible portions of collected mudfish are divided into two: (1) heavy metal levels in mudfish for dry season, and (2) heavy metal levels in mudfish for wet season. heavy metal levels in mudfish for dry season table 3 shows the concentrations of heavy metals (cd, cr, pb, hg and as) in mudfish from eight sampling stations during the dry season. cadmium (cd) concentration ranged from 0.03742 mg/kg in sampling station 3a to 0.0695 mg/kg in station 2b. chromium (cr) ranged from 0.02243 mg/kg in station 5 to 0.42589 mg/kg in station 3a. lead (pb) ranged from 0.58037 mg/kg in station 2a to 2.80447 mg/kg in station 4. mercury (hg) ranged from 0.00314 mg/kg in station 4 to 0.17685 mg/kg in station 3b. arsenic (as) ranged from 0.00003 mg/kg in station 3b to 0.36976 mg/kg in station 1a. figure 2 shows the spatial distribution of heavy metal concentrations in mudfish during the dry season. heavy metal levels in mudfish for wet season table 4 shows the concentrations of heavy metals (cd, cr, pb, hg and as) in mudfish from eight sampling stations during the wet season. cadmium (cd) concentration ranged from 0.00241 mg/kg in sampling station 5 to 0.30122 mg/kg in station 1b. chromium (cr) ranged from 0.00148 mg/kg in station 3b to 0.29289 mg/kg in station 1b. lead (pb) ranged from 0.007 mg/kg in station 2b to 4.41776 mg /kg in station 1a. mercury (hg) ranged from 0.01192 mg/kg in station 2b to 0.07668 mg/kg in station 1a. arsenic (as) ranged from 0.001 mg/kg in stations 2a and 3b, to 0.04696 mg/kg in station 1b. figure 3 shows the spatial distribution of heavy metal concentrations in mudfish during the wet season. analysis of laboratory data for the dry and wet seasons showed that the onset of the rainy season can either increase or decrease the heavy metal concentrations in mudfish depending on where the fish was located in the lake. the positive effect of the rainy season could be due to the dilution of rainwater run-off which was apparent in the south bay, central bay and east bay. on the other hand, the negative effect of the rainy season could be due to the “flushing-effect” from tributaries and run-off from adjoining areas with significant sources of heavy metal in the environment. sampling si te (dry season) heavy metal concentration mg/ kg ds cd cr pb hg as 1a 0.04243 0.0 4833 1.09690 0.03574 0.36976 1b 0.06087 0.0 8773 0.65377 0.17506 0.03194 2a 0.03947 0.0 6029 0.58037 0.09953 0.15524 2b 0.06950 0.1 1788 0.85857 0.03432 0.22737 3a 0.03742 0.4 2589 1.11713 0.10578 0.08388 3b 0.04859 0.0 3203 0.74580 0.17685 0.00003 4 0.03782 0.0 3707 2.80447 0.00314 0.11371 5 0.06180 0.0 2243 1.15327 0.01630 0.21853 table 3. heavy metal concentration, mudfish (mg/kg), dry season 28 molina, v.b. science diliman (january-june 2012) 24:1, 24-32 this was observed in the west bay where lead was highest during the wet season. estimate of potential human exposure to heavy metal in mudfish (ophicephalus striatus) the basic equation for calculating systemic toxicity (non-carcinogenic hazard) is: non-carcinogenic hazard quotient (nhq) = cdi/rfd figure 2. heavy metal concentrations, mudfish (mg/kg), dry season where: cdi = chronic daily intake for the toxicantexpressed in mg/kg-day rfd = chronic (oral) reference dose for the toxicant expressed in mg/kg-day chronic oral rfd is defined as an estimate (with uncertainty spanning perhaps an order of magnitude or greater) of a daily oral exposure level for the human population, including sensitive subpopulations, that is table 4. heavy metal concentration, mudfish (mg/kg), wet season sampling site (wet season) heavy metal concentration mg/kg ws cd cr pb h g a s 1a 0.22665 0.06178 4.41776 0.07668 0.03807 1b 0.30122 0.29289 4.10531 0.03514 0.04696 2a 0.01017 0.00314 0.47623 0.02420 0.00100 2b 0.00245 0.00469 0.00700 0.01192 0.00718 3a 0.00386 0.00532 0.01104 0.05341 0.00100 3b 0.01161 0.00148 0.10115 0.03584 0.00100 4 0.00278 0.00311 0.05372 0.03560 0.02558 5 0.00241 0.00301 0.02392 0.01241 0.02755 non-carcinogenic health risks of heavy metal in mudfish 29science diliman (january-june 2012) 24:1, 24-32 likely to be without an appreciable risk of deleterious effects during a lifetime. chronic oral rfds are specifically developed to be protective for long-term exposure to a compound. as a guideline, chronic oral rfds generally should be used to evaluate the potential non-carcinogenic effects associated with exposure periods greater than 7 years (approximately 10 percent of a human lifetime). chronic oral reference doses are expressed in units of mg/kg-day. the rfd values of heavy metal in this study were adopted from usepa. for arsenic, rfd=0.0003 mg/kg-day, chromium, rfd=0.003 mg/kg-day, mercury, 0.0001mg/kg-day, cadmium, rfd=0.001 mg/kg-day, and lead, rfd=0.0000001 mg/kg-day. non-carcinogenic fish ingestion equation: cdi cdi = c x ef x ed x irf x (kg/1000g) (365 days/year) x lt x bw where: cdi = chronic daily intake for the toxicant expressed in mg/kg-day c = concentration of heavy metal in fish (mg/kg) bw = body weight (for filipino adult ~ 65kg) ed = exposure duration (30 years) ef = exposure frequency (350 days per year) irf = ingestion rate fish (fish consumption) = 102.74 g/ day (fao). this is the estimated average daily per capita consumption of fish in the philippines from the fao fisheries and aquatic department. lt = lifetime (average), 30 years for non-carcinogenic health effects the non-carcinogenic hazard quotient (nhq) is one of the measures of non-carcinogenic health effects of exposure to chemical contaminants. it is the ratio of an exposure level by a contaminant to a reference dose or value selected for the health risk assessment of a particular substance or chemical. if the exposure level is higher than the toxicity value, then there is the potential for risk to the receptor. computed nhq value of greater than 1.0 indicates that the exposure to a single chemical or substance will likely result in adverse health effects. the potential health effects are dependent on the type of chemical or substance of concern. nhq values of 1.0 or below indicate that daily oral exposure level for the human population, including sensitive subpopulations, is likely to be without an appreciable risk of deleterious effects during a lifetime. computed values of chronic daily intake (cdi) and non-carcinogenic hazard quotient (nhq) of cadmium, chromium, lead, mercury and arsenic in mudfish for all sampling stations during the dry season are summarized in table 5. nhq values for cadmium, chromium, and arsenic are less than 1.0 (unit less value) in all sampling stations, showing that the daily oral exposure level for the human population, including sensitive subpopulations, is likely to be without an appreciable risk of deleterious effects during a lifetime. however, nhq values for mercury have values ranging from 1.36 to 2.41 indicating that long-term mudfish consumption would likely result in adverse health effects. nhq values of lead in all sampling stations are way above 1.0 (ranging from 8,796 in sampling station 2a to 42,506 in station 4), indicating high risk for adverse human health effects associated with long-term mudfish consumption. for the wet season, computed values of noncarcinogenic hazard quotient (nhq) of cadmium, chromium, lead, mercury and arsenic in mudfish for all sampling stations are summarized in table 6. as with the dry season findings, the nhq values for cadmium, chromium, and arsenic were less than 1.0 in all sampling stations, showing that the daily oral exposure level for the human population, including sensitive subpopulations, is likely to be without an appreciable risk of deleterious effects during a lifetime. in sampling station 1a nhq value of mercury is slightly greater than 1.0 (nhq=1.046), which indicates that the risk value is at the borderline with probable risk for adverse human health effects associated with long-term mudfish consumption. the nhq values for lead in all sampling stations were way above 1.0 (ranging from 106 in sampling station 2b to 66,958 in station 1a), indicating high risk for adverse human health effects associated with long-term mudfish consumption. conclusion and recommendations results of the study showed that arsenic, cadmium, and chromium do not pose significant non-carcinogenic health effects associated with the consumption of mudfish from laguna de bay. however, 30 molina, v.b. science diliman (january-june 2012) 24:1, 24-32 summary of nhq values for dry season sampling station c d cr pb hg as 1a 0.0643 0.0146 16625 0.4870 0.1864 1b 0.0923 0.0266 9909 2.3880 0.0161 2a 0.0598 0.0183 8796 1.3577 0.0784 2b 0.1053 0.0357 13013 0.4682 0.1149 3a 0.0567 0.1291 16932 1.4429 0.0424 3b 0.0736 0.0097 11304 2.4124 0.0000 4 0.0573 0.0112 42506 0.0428 0.0574 5 0.0937 0.0068 17480 0.2223 0.1104 table 5. summary of nhq values for dry season concentrations of mercury and lead showed elevated levels that are likely to cause adverse health effects on fish long-term consumers. this study therefore concludes that from the point of view of human health protection and disease prevention, long-term human consumption of mudfish from laguna de bay is not safe due to elevated levels of mercury and lead that were found to be above the safe nhq values. in light of the above findings, the following recommendations are presented to help policy makers and stakeholders in decision-making as well as in crafting lake management policies and mitigating measures: 1. urgent measures should be done by concerned authorities to protect health of communities consuming mudfish from the lake especially the children. the immediate goal should be to minimize exposure by minimizing the amount of fish intake and the frequency of consumption. 2. regular monitoring of heavy metal in fishes should be done at least twice a year (wet and dry seasons) by concerned government agencies at all levels. 3. regular health advisories regarding quantitative health risks associated with fish consumption should be issued by the laguna lake development authority or the regional office of the department of health. 4. local government units, especially the lakeshore communities, should be involved in the heavy metal monitoring in fish and in developing and disseminating advisories and other health-related information to the communities and other stakeholders. 5. inventory and assessment of potential sources of heavy metal in the lake (e g., industrial sources) most especially for lead and mercury, should be undertaken. non-carcinogenic health risks of heavy metal in mudfish 31science diliman (january-june 2012) 24:1, 24-32 summary of nhq v alues for wet season sampling station cd cr pb hg as 1a 0.3435 0.0187 66958 1.0460 0 .0192 1b 0.4565 0.0888 62222 0.4793 0 .0237 2a 0.0154 0.0010 7218 0.3301 0 .0005 2b 0.0037 0.0014 106 0.1626 0 .0036 3a 0.0059 0.0016 167 0.7286 0 .0005 3b 0.0176 0.0004 1533 0.4889 0 .0005 4 0.0042 0.0009 814 0.4856 0 .0129 5 0.0037 0.0009 363 0.1693 0 .0139 table 6. summary of nhq values for wet season given back to our heavenly father, our god almighty, for giving me strength, spiritual gift of perseverance, wisdom and discernment, to god be the glory! references akan, j.c., f.i. abdulrahman, o.a. sodipo &p.i. akandu, 2009. bioaccumulation of some heavy metals of six fresh water fishes caught from lake chad in doron buhari, maiduguri, borno state, nigeria. journal of applied sciences and sanitation. 4(2):103-114. annalee, y., t. kjellstrom, t. dekok &t. gidotti, 1998. basic environmental health. office of global and integrated environmental health. world health organization, geneva. arshad, j., j. muhammad & a. sajid, 2007. nickel bioaccumulation in the bodies of catla catla, labeo rohita and cirrhina mrigala during 96-hr lc50 exposures. international journal of agriculture & biology. 9 (1): 139142. barwick, m. & w. maher, 2003. biotransference and biomagnification of selenium, copper, cadmium, zinc, arsenic and lead in a temperate seagrass ecosystem from lake macquarie estuary, nsw, australia. marine environmental research. 56: 471–502. 6. more stringent regulation of effluents from industries around the lake should be enforced. 7. there should be regular monitoring of heavy metal in major rivers and tributaries draining into the lake. acknowledgements i wish to extend my gratitude to dr. maria victoria o. espaldon, dr. enrique p. pacardo, dr. maxima e. flavier, dr. carmelita m. rebancos, dr. lynn panganiban and ms. lennie borja for their support and guidance. my thanks to the hardworking staff of lake management division, mr. dong estoy, mr. jess futalan, mr. noely sumadia, mr. val ablaza and mr. melvin martinez. my special thanks to the fishermen in lakeshore communities for their assistance during the fish sampling activities. my heartfelt gratitude to the industrial technology development institute, department of science and technology laboratory for their patience in analyzing voluminous fish samples. thanks also to the department of environmental and occupational health, college of public health up manila, the philippine council for health research and development dost, and the research institute for humanity and nature, kyoto, japan for the financial assistance. above all, highest honor and gratitude is 32 molina, v.b. cadmium exposure and human health. http:// www.cadmium.org/env_exp.html. accessed november 23, 2009. campbell, l.m., j.s. balirwa, d.g. dixon & r.e. hecky, 2004. biomagnification of mercury in fish from thruston bay, napoleon gulf, lake victoria (east africa). african journal of aquatic science. 29 (1): 91–96. castro-gonzalez, m.i. & m. mendez-armenta, 2008. heavy metals: implications associated to fish consumption. environmental toxicology and pharmacology. 26 (2008): 263–271. developing species-specific fish consumption advice. environmental health perspectives, 117 (2): 267-275. ecosystems and people: the philippine millennium ecosystem assessment (ma) sub-global assessment, 2005. edited by lasco, r.d., m.v.o. espaldon & m.a. tapia. environmental forestry program college of forestry and natural resources university of the philippines los baños, department of environment and natural resources (denr), and laguna lake development authority (llda). extension toxicology network : pesticide information project of cooperative extension offices of cornell university, oregon state university, the university of idaho, and the university of california at davis and the institute for environmental toxicology, michigan state university. fact sheet: health effects of lead (usepa). http:// www.epa.gov/dclead/epa_lead_health_effects_final208 _12.pdf. accessed november 19, 2009. hammerschmidt, c.r. & w.f. fitzgerald, 2006. bioaccumulation and trophic transfer of methylmercury in long island sound. archives of environmental contamination and toxicology. 51: 416-424. hosseinkhezri, p. & j. tashkhourian, 2011. determination of heavy metals in acanthopagrus latus (yellowfin seabream) from the bushehr seaport (coastal of persian gulf), iran. international food research journal. 18: 791-794. lakshmanan, r., k. kesavan, p. vijayanan, v. rajaram & s. rajagopal, 2009. heavy metals accumulation in five commercially important fishes of parangipettai, southeast coast of india. advance journal of food science and technology. 1(1): 63-65. mercury and the environment. http://www.ec.gc.ca/ mercury/eh/en/eh-hc.cfm. accessed october 27, 2009. nor hasyimah, a.k., v. james noik, y.y. teh, c.y. lee & h.c. pearline ng, 2011. assessment of cadmium (cd) and lead (pb) levels in commercial marine fish organs between wet markets and supermarkets in klang valley, malaysia. international food research journal. 18: 795-802. obasohan, e.e. & o.i. eguavoen, 2008. seasonal variations of bioaccumulation of heavy metals in a freshwater fish (erpetoichthys calabaricus) from ogba river, benin city, nigeria. african journal of general agriculture. 4 (3): 153163. silva, e.i.l. & a. shimizu, 2004. concentrations of trace metals in the flesh of nine fish species found in a hydropower reservoir in sri lanka. asian fisheries science. 17: 377-384. sreedhara nayaka, b.m, s. ramakrishna, jayaprakash & m.r. delvi, 2009. impact of heavy metals on water, fish (cyprinus carpio) and toxic substances and health: lead. http://www.atsdr.cdc.gov/tfacts13 .html. accessed november 27, 2009. yousafzai, a.m., d.p. chivers, a.r. abdur rehman khan, i. ahmad & m. muhammad siraj, 2010. comparison of heavy metals burden in two freshwater fishes wallago attu and labeo dyocheilus with regard to their feeding habits in natural ecosystem. pakistan journal of zoology. 42(5): 537544. science diliman (january-june 2012) 24:1, 24-32 01-1 notes for contributors v4n1&2 january-june 2013 • volume 25 no. 1 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june a n d d e c e m b e r ) b y t h e u n i v e r s i t y o f t h e philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief marco nemesio e. montaño, phd associate editors ma. patricia v. azanza, phd jose maria p. balmaceda, phd carlos primo c. david, phd joel joseph s. marciano jr. , phd jonas p. quilang, phd arnel a . salvador, phd irene m. villaseñor, phd managing editor violeda a. umali, phd editorial assistants epifania m. domingo dercylis g. mararac on the cover: variety of fruits displayed at the roma’s fruits stand, up shopping center, diliman. photo taken by marvic a. pastrana of the ovcrd, up diliman. rigoberto c. advincula, phd department of chemistry university of houston alfonso m. albano, phd department of physics bryn mawr college, bryn mawr, pennsylvania kenneth buckel, phd food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, phd department of electrical and computer engineering ohio state university flor crisanta f. galvez, phd quality assurance and technical manager kerry ingredients and flavours (americas region) 7989-82nd st. , delta, vc v4g 1l7, canada victor c. gavino, phd department of nutrition university of montreal, canada kelvin s. rodolfo, phd department of earth and environmental sciences university of illinois, chicago, illinois rudolf a. roemer, phd centre for scientif ic computing and department of physics university of warwick luis g. sison, phd electrical and electronics engineering institute university of the philippines diliman raul k. suarez, phd department of ecology, evolution and marine biology university of california, sta. barbara the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. 4benthic macroinvertebrate-deborde.pmd d.d.d. deborde et al. 5 science diliman (july-december 2016) 28:2, 5-26 benthic macroinver tebrate community as an ind icator of stream health: the effects of land use on stream benthic macroinver tebrates danielle dominique d. deborde* university of the philippines diliman maria brenda m. hernandez university of the philippines diliman francis s. magbanua university of the philippines diliman abstract biomonitoring of stream health in the tropics still emphasize on the use of standard water chemistry methods (physicochemical variables), which require expensive and elaborate measuring apparatus. in this study, the reliability of benthic macroinvertebrates as bioindicators of freshwater s t r e a m s w a s c a r r i e d o u t . t h e s t u d y a l s o a t t e m p t e d t o d e t e r m i n e t h e d i s c r i m i n a t i n g p o w e r o f v a r i o u s b i o t i c i n d i c e s i n c h a r a c t e r i z i n g s i t e s across land use. benthic macroinvertebrate samples were obtained from n i n e s t r e a m s i n s i l a g o, s o u t h e r n ley te a n d w e r e i d e n t i f ied to f a m i l y l e v e l . o n e w a y a n a l y s i s o f v a r i a n c e w a s p e r f o r m e d o n v a r i o u s b i o t i c indices to assess the water quality of streams based on land use. average to l e r a n c e s c o r e p e r ta xo n ( at s p t ) w a s t h e o n l y b i o t i c i n d e x t h a t differentiated the nine streams based on land use (p <0.001). forested s i t e s a c h i e v e d t h e l o w e s t at s p t s c o r e , w h e r e a s m i x e d f o r e s t e d a g r i c u l t u r a l s i t e s h a d t h e h i g h e s t at s p t s c o r e s . p h y s i c o c h e m i c a l variables (e.g. , stream width, conductivity, total dissolved solids, water temperature) and biological metrics (e.g. , simpson’s diversity index, total macroinvertebrate density) used in the study supported this assessment. t h e r e s u l t s s h o w t h a t b e n t h i c m a c r o i n v e r t e b r a t e s c a n b e u s e d a s p o t e n t i a l b i o m o n i t o r i n g t o o l t o e v a l u a t e t h e e c o l o g i c a l i n t e g r i t y o f _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online benthic macroinvertebrate community 6 waterways in the country. long-term data sets will be generated from f u t u r e s a m p l i n g e f f o r t s f o r t h e d e v e l o p m e n t o f t h e p h i l i p p i n e b i o t i c index. ke y w o r d s : ave r a g e to l e r a n ce s co r e p e r ta xo n ( at s pt ), b i o t i c i n d i ce s , stream monitoring, physicochemical, philippines introduction habitat degradation due to rapid population growth and economic development intensif ies global decline in biodiversity and ecological functionality of freshwater ecosystems. because of the increase in human land use pressure, the following threats imperil stream habitats (karr 1991; brisbois et al. 2008; miserendino et al. 2011; mcgoff et al. 2013): deforestation, land conversion, contaminant pollution, alteration of stream channels, and excessive nutrient input. such disturbances have led to the disruption of ecological integrity because of the resulting decrease in primary production (henley et al. 2000), altered trophic structure (gregory et al. 1991), modif ied channel dynamics (walsh et al. 2001), and reduced bank stability (findlay et al. 2001). several assessment and monitoring strategies have been implemented to assess the biological quality of freshwater habitats and to sustain human and ecological demands for fresh waters. for example, traditional stream assessments are generally performed using water chemistry, wherein physicochemical parameters, namely dissolved oxygen (do), temperature, conductivity, total dissolved solids, water hardness, and water flow rate are recorded and analyzed in situ (dinka et al. 2004; halstead et al. 2014). however, this method was deemed ineff icient in providing thorough habitat evaluation due to underlying constraints (scrimgeour and wicklum 1996; heatherly et al. 2007). this then paved the way for the emergence of new approaches (i.e. , biological monitoring or biomonitoring) in making comprehensive analysis of the overall condition of freshwater ecosystems. biomonitoring utilizes a wide array of organisms as biological indicators (or simply bioindicators) to determine the overall status of stream habitats. diatoms are used because of their ubiquity, short generation time, broad range of tolerance against contaminants, ease of use, and well-documented taxonomy (kireta et al. 2012; mendes et al. 2012). fishes are also used due to their well-known community s t r u c t u r e a n d r e c r e a t i o n a l v a l u e ( c a r ey a n d m a t h e r 2 0 0 8 ; re s h 2 0 0 8 ) . macroinvertebrates, which are indispensable components of aquatic ecosystems, d.d.d. deborde et al. 7 are widely used indicator species in freshwater biomonitoring because of a set of distinct advantages they offer (reece and richardson 2000; barbosa et al. 2001; clements et al. 2002; bae et al. 2005): their ubiquity and sedentary nature makes them good representatives for spatial analyses of pollutants; their relatively longer life cycles compared to other freshwater organisms can elucidate temporal changes; their constant exposure to varying water quality conditions allows them to accumulate toxins from the sediments they live in and feed on; and their welldescribed taxonomy aids in the ease of identif ication and evaluation of collected samples. several studies have considered the use of abundance and species richness among macroinvertebrates to detect environmental responses because of their variable sensitivity towards multiple disturbances (davis 2003; ferreira et al. 2011; friberg et al. 2011). moreover, this set of organisms does not experience rapid blooms and death in response to nutrient inputs compared to algae. they also do not possess great mobility similar to that of f ishes, preventing them to escape pollution by moving towards unaffected tributaries (morse et al. 2007). unlike in temperate regions, benthic macroinvertebrates are underutilized, poorly established, and rarely applied to tropical freshwater assessments. this happens due to a great deal of challenges occurring among tropical streams (clews et al. 2014; feio et al. 2015), such as the paucity of information on the taxonomy of faunal groups, low eff iciency of biotic indices, differences in community structure, variation in functional processes, and seasonal variation. however, there is an increasing interest in studying tropical streams using benthic macroinvertebrates. the municipality of silago, southern leyte (10º 31’45" n, 125º 9’56" e, total land area = 21,505 ha) provides an excellent study site for macroinvertebrate assemblages. the study attempts to determine the validity of extensively used biotic indices (e.g. , hilsenhoff’s family biotic index, biological monitoring working party (bmwp), average score per taxon (aspt ), singscore, and average tolerance score per taxon) in providing preliminary assessment of silago’s current stream condition across land use. the study also tested the eff iciency of several physicochemical parameters (e.g. , water temperature, conductivity, total dissolved solids) and biological metrics (e.g. , simpson’s diversity index, total macroinvertebrate density) in describing the ecological integrity of the selected streams. since there are no published studies on benthic macroinvertebrates in silago, baseline data from the results will be useful for the development of the philippine biotic index for freshwater streams. benthic macroinvertebrate community 8 materials and methods physicochemical parameters previously collected data on various physicochemical parameters were used in this study to assess the water chemistry of the nine selected streams in silago, southern leyte. using a multiparameter water quality meter, the following variables were measured: (i) dissolved oxygen, (ii) ph, (iii) temperature, (iv) conductivity, and (v) total dissolved solids (tds). in addition, wetted width, water depth, and water velocity were recorded. benthic macroinvertebrates this study used the macroinvertebrate samples previously collected from selected streams in silago, southern leyte in june and july 2014, which were deposited at the aquatic biology research laboratory of the institute of biology, university of the philippines diliman. nine streams were surveyed, with each stream having six macroinvertebrate sample collections per location: upstream, midstream, and downstream. these samples were collected using a surber sampler, stored in 50 ml centrifuge tubes containing 95% ethanol, and were brought to the laboratory for identification. using a fluorescent illuminated magnif ier, relatively large benthic macroinvertebrates were initially sorted based on morphology. a stereomicroscope was then used to group relatively small individuals. all morphologically-similar organisms were immediately placed in properly labeled 15-ml centrifuge tubes containing 95% ethanol. after sorting, the taxonomic family level of the macroinvertebrates were identif ied using the keys of dudgeon (1999), yong and yule (2004), and the mekong river commission (2006). finally, all identif ied samples were transferred into individual 20-ml scintillation vials, with each vial containing only one family per sampling site. all vials were properly labelled with the name of the site, the date of collection, and the respective taxonomic family. using the macroinvertebrate data, the following biological metrics were calculated: (i) total invertebrate density, (ii) taxon richness, (iii) richness of ephemeroptera, plecoptera and trichoptera (ept) insect orders, and (iv) simpson’s index of diversity. moreover, widely accepted biological scoring systems were calculated to determine d.d.d. deborde et al. 9 the current condition of the streams in silago southern leyte: (i) hilsenhoff’s family biotic index (hbi), a biotic index for assessing organic and nutrient pollution using tolerance values of arthropod families (hilsenhoff 1988); (ii) biological monitoring working party (bmwp), a standardized score system based on tolerance scores of macroinvertebrate families to organic pollution (mustow 2002); (iii) average score per taxa (aspt), a biotic index which measures river status using the calculated bmwp score divided by number of taxa (mustow 2002); (iv) stream invertebrate grade number – average level version 2 (signal 2), a biotic index for australian river macroinvertebrates (chessman 1995, 2003); (v) singscore, a newly developed biotic index for measuring the health of singapore’s streams using benthic macroinvertebrates (blakely et al. 2014); and (vi) average tolerance score per taxon (atspt), a biotic index for evaluating stream health integrity using site disturbance scores and benthic macroinvertebrate abundance (chessman and giap 2 0 1 0 ) . data analysis data were log10(x) or log10(x + 1) transformed to improve normality and homoscedasticity after exploratory data analysis (quinn and keough 2002), where necessary. one-way anova was performed to determine signif icant difference across land use for the various physicochemical, benthic macroinvertebrate metrics, and biotic indices (magbanua et al. 2010; narangarvuu et al. 2014; aguiar et al. 2015). if land use had a signif icant effect, pairwise comparisons with tukey’s hsd (or games-howell, in cases of persisting heteroscedasticity) post hoc tests were conducted. results and discussion physicochemical variables across land use all variables, except stream depth and do, showed signif icant differences across land use (p<0.05 in all cases; table 1). forested land use had the lowest mean values for all parameters other than ph (figure 1). water physicochemistry, particularly water temperature, conductivity, tds, ph, water velocity, and stream width, showed signif icant results in discriminating selected streams across land use. benthic macroinvertebrate community 10 data analysis revealed that forested areas had the lowest water temperature as opposed to the other land uses. this supports the prediction that forested sites are abundant in diverse sets of trees and vegetation, contributing to the canopy cover which provides shade (studinski et al. 2012). on the other hand, both agricultural and mixed areas achieved a relatively warmer temperature due to the decrease in the surrounding riparian zone. moreover, as reflected in its narrow stream width, forested sites had stable banks, which is indicative of the rich vegetation that holds the soil intact and reduces the effects of erosion. however, the case was different among agricultural and mixed sites, which generated higher measurements for their respective stream width due to poor bank stability caused by farming practices and other land development occurring in the area. the high water conductivities within agricultural and mixed areas suggest excess nutrient inputs in these particular sites. this is expected due to the presence of farming activities, which contribute to increased fertilizer and pesticide loading via terrestrial runoff (al-shami et al. 2011; piggott et al. 2012). forested sites, in turn, had low measurements for both conductivity and tds, indicating minimal anthropogenic activity. table 1. mean (± standard error) stream physicochemical parameter values of selected streams in silago, southern leyte across d ifferent land uses f = forested; a = agricultural; m = mixed land use parameter forested agricultural mixed p-value ranking stream 4.67 (0.42) 8.66 (1.48) 17.87 (1.49) <0.001 f<a<m width stream 0.13 (0.01) 0.16 (0.01) 0.17 (0.03) 0.341 depth water 0.31 (0.09) 0.42 (0.09) 0.54 (0.20) 0.003 f<a<m velocity temperature 23.60 (0.09) 25.24 (0.35) 26.59 (0.18) <0.001 f<a<m conductivity 6105.09 12216.31 12965.23 <0.001 f<a<m (912.07) (1201.71) (231.08) tds 4113.73 7931.57 8173.66 <0.001 f<a<m (611.34) (795.90) (139.15) ph 7.79 (0.19) 7.17 (0.34) 9.62 (0.74) 0.012 a<f<m do 5.09 (0.35) 5.39 (0.61) 5.54 (0.52) 0.930 d.d.d. deborde et al. 11 figure 1. mean (± standard error) stream physicochemical parameter values of selected streams in silago, southern leyte across different land uses. benthic macroinvertebrate community 12 biological response variables among streams two of the biological metrics used, namely total benthic macroinvertebrate density (p=0.001) and simpson’s diversity index (p=0.030), markedly differed across land use (figure 2). apart from physicochemical values, diversity indices are also extensively used in assessing the health of freshwater streams. diversity indices measure the degree of diversity of benthic macroinvertebrates in a particular site and provide an evaluation of its condition (lenat 1988; death and winterbourn 1995; linke et al. 1999). in fact, there is a wide selection of diversity indices that can be used to determine water quality (carter et al. 2009); however, only the simpson’s diversity index was selected for this study. simpson’s diversity index does not only give the number of different taxa present across sites, but it also measures the evenness of the distribution of individuals amongst taxa (bailey et al. 1 9 9 8 ) . forested sites had the greatest number of taxa, whereas agricultural sites exhibited the least number of taxa. this alone immediately suggests that the water quality among forested sites is indeed excellent as opposed to the other land uses. however, care should be taken on solely using diversity indices because of the possibility of obtaining a false assessment (wilsey et al. 2005; heino et al. 2008). accurate stream health assessments could be constructed from considering the type and abundance of the present taxa (e.g. , pollution-tolerant, pollution-sensitive). for example, macroinvertebrates belonging to the ephemeroptera-plecopteratrichoptera (ept ) orders are highly sensitive to organic pollution (henriques-deoliveira et al. 2007; narangarvuu et al. 2014; zaiha et al. 2015), which makes them good water quality indicators. pollution tolerant species, on the other hand, are insensitive to various environmental stressors, allowing them to thrive even in heavily degraded habitats (guimaraes et al. 2009; frizzera and alves 2012; rosa et al. 2014). the taxa compositions across the three land uses were different (figure 3). agricultural and mixed land uses exhibited a greater number of ept insect orders as opposed to those of forested sites. this observed pattern was most likely due to increased input of organic nutrients from farmlands, which could have possibly given the macroinvertebrates additional opportunity to increase in number (niyogi et al. 2007; wagenhoff et al. 2011). interestingly, the preponderance of baetidae and hydropsychidae among agricultural and mixed areas could also be attributed to the introduction of nutrients from the surrounding land uses. several studies have shown that both taxa are commonly abundant in mildly polluted, nutrient enriched areas (czerniawska-kusza 2005; ratia et al. 2012; xu et al. 2014). d.d.d. deborde et al. 13 figure 2. mean (± standard error) biological metric values and indices of selected streams in silago, southern leyte across different land uses. benthic macroinvertebrate community 14 figure 3. benthic macroinvertebrate density of the f ive most dominant taxa across different land uses. white = pollution-sensitive taxa; gray = moderately pollutiontolerant taxa; black = pollution tolerant taxa. macroinvertebrate classif ication followed chang et al. (2014). d.d.d. deborde et al. 15 hydropsychidae larvae (trichoptera) are reported to be sedentary f ilter feeders that commonly inhabit fast flowing rivers (andersen and klubnes 1983). due to their mode of feeding, this taxon greatly prefers streams with high concentration of suspended organic matter, which may originate from various sources (e.g. , f ish farm effluents, waste treatment plants) (guilpart et al. 2012). the observed high density of hydropschidae in agricultural sites was consistent with other studies that showed increase in hydropsychidae density in areas influenced by farm-related activities (strand and merritt 1997; kyriakeas and watzin 2006; dahal et al. 2007). baetidae larvae (ephemeroptera) are regarded as tolerant species against organic pollution (zamora-munoz and alba-tercedor 1996; buss et al. 2002), which explains their occurrence in sites experiencing intermediate levels of degradation. however, the results failed to support the f indings of t imm et al. (2001) and buluta et al. table 2. mean (± standard error) biological metric values and ind ices of selected streams in silago, southern leyte across d ifferent land uses. ept = ephemeroptera-trichoptera-plecoptera insect orders; hbi = hilsenhoff’s family biotic index; bmwp = biological monitoring working party; aspt = average score per taxa; signal 2 = stream invertebrate grade number – average level version 2; singscore = singapore’s macroinvertebrate biotic index score; atspt = average tolerance score per taxon. f = forested; a = agricultural; m = mixed land use parameter forested agricultural mixed p-value ranking taxon richness 19.83 (1.54) 16.33 (1.68) 18.17 (0.70) 0.117 invertebrate 1054.11 1894.11 3239.44 <0.001 f<a<m density (165.06) (374.49) (492.78) simpson’s 10.52 (0.86) 6.53 (0.81) 7.79 (0.41) 0.002 a<m<f diversity ept richness 8.56 (0.64) 8.00 (0.72) 9.56 (0.34) 0.116 hbi 3.35 (0.12) 3.19 (0.10) 3.34 (0.07) 0.462 bmwp 80.78 (6.03) 73.89 (7.06) 85.94 (3.35) 0.157 aspt 6.34 (0.08) 6.16 (0.20) 6.40 (0.06) 0.287 signal 2 5.04 (0.09) 5.15 (0.11) 5.06 (0.04) 0.672 singscore 131.61 (1.45) 131.33 (3.01) 134.17 (1.71) 0.567 atspt 25.65 (0.63) 29.74 (0.45) 32.39 (0.36) <0.001 f<a<m benthic macroinvertebrate community 16 (2010) that consider baetidae as an excellent indicator of pristine freshwater habitats. this then implies that the stream health among agricultural and mixed sites is considered moderately poor as reflected by the gathered macroinvertebrate data. chironomidae, on the other hand, was observed in all types of land uses, which was expected, on account of the ability of this specif ic taxon to thrive in all habitat types: both in highly polluted and minimally disturbed habitats (de haas et al. 2005; loayza-muro et al. 2012). these taxa are described as pollution tolerant species due to their capability to withstand a wide range of environmental conditions (i.e. , temperature, ph, dissolved oxygen). it is also impor tant to note that chironomidae tend to occur in higher densities when oxygen levels are low and organic pollution is high (buss et al. 2002; bacey and spurlock 2007). however, the direct or indirect effects of nutrient enrichment to chironomidae density could not be assessed as no manipulative test was performed in our study (miracle et al. 2006; wagenhoff et al. 2012). despite the low number of ept orders, only forested areas supported a signif icant number of taxa belonging to plecoptera (i.e. , perlidae), which has long been regarded as the most pollution intolerant of the aquatic insect orders. this general claim is supported by various studies concerning the evolution of this particular taxa in the cold mountain streams, where oxygen stress was scarce (zwick 2000; chang et al. 2014). based on this observation, it is reasonable to conclude that the water quality among forested sites is in good condition, especially since these areas were minimally disturbed by anthropogenic activities. traditional measures, such as total taxa richness and total ept richness, were considered helpful in providing stream health evaluation according to several studies (lammert and allan 1999; roy et al. 2003; moya et al. 2011). however, the results obtained from the data analysis show that the values for both parameters were not signif icant across sites, suggesting that the two are not reliable tools for biomonitoring. stream health assessment using biotic scoring systems average tolerance score per taxa (atspt ) signif icantly differed across land use (p<0.001). in contrast, hbi, signal 2, singscore, bmwp, and aspt did not show any signif icant changes across land use (p>0.05 in all cases; table 2). d.d.d. deborde et al. 17 modern stream monitoring and habitat assessments are being conducted using a relatively new technique that uses different biotic indices (armitage et al. 1983; hilsenhoff 1988; chessman 1995; mustow 2002; blakely et al. 2014). the method is an example of a numerical estimation, wherein specif ic taxa are given corresponding tolerance scores depending on their sensitivity towards organic pollution. a f inal score that indicates the current state of the freshwater system is then obtained. originally, it was developed for monitoring temperate freshwater system, but it is now being used in tropical countries, including in southeast asia. in this study, only the average tolarance score per taxa (atspt ) of chessman and giap (2010) generated highly signif icant values across sites. the remaining f ive biotic indices, namely bmwp, aspt, hbi, signal 2, and singscore, failed to discriminate the three land uses in terms of stream health conditions, as evidenced by their corresponding p-values. based on the results from singscore, all sites within different land uses achieved excellent water quality, since it has been suggested that singscore values (>120) indicate optimal stream conditions (blakely et al. 2014). similarly, the data obtained from hbi exhibited the same pattern in line with the proposed values (0.00 – 3.75) for excellent water conditions (hilsenhoff 1988). on another note, it is interesting to mention that, despite the presence of farming activities and human impairment among agricultural and mixed sites, their corresponding water qualities remain excellent. this observation could be due to the lack of large-scale industries (factories and manufacturing plants) in the municipality of silago, southern leyte, which explains why the ongoing anthropogenic activities are not suff icient to heavily impact the waterways. furthermore, this indicates that the discriminatory powers of singscore and hbi were not sensitive enough to be used for freshwater habitat assessment. signal 2, bmwp, and aspt failed to discriminate the streams across the three types of land use, proving to be consistent with the works of wyzga et al. (2013) and mohmad et al. (2015). this was because the development of these three biotic indices only accounted for organic pollution, which could potentially underestimate/ overestimate the extent of disturbance occurring among impacted sites. it should also be emphasized that the response of benthic macroinvertebrates to different stressors (i.e. , organic enrichment, heavy metal contamination) varies across taxa and is greatly influenced by its geographical setting (chutter 1972). benthic macroinvertebrate community 18 in contrast, atspt characterized the water quality of the streams across the three land uses, which ranged from moderately poor to excellent. sites within mixed areas were observed to have the highest atspt scores, implying their ability to support a great number of pollution tolerant taxa. these moderately poor quality reference sites augment the occurrence of high-surrounding impervious surfaces within these areas, ultimately leading to increased sediment deposition as observed in other studies (allan 2004; walsh et al. 2005; mantyka-pringle et al. 2014). in turn, forested sites possessed excellent water quality, as evidenced by their low atspt scores. from these f indings, atspt is a potential bioindicator of water quality that can be used in the philippines. accordingly, several key points about this biotic index should be re-assessed and re-evaluated. first, atspt is advantageous over the other biotic scoring systems due to the fact that all of the identif ied taxonomic families across sites were provided with respective tolerance values, which were obtained from the calculated site disturbance score (sds) from the time of sampling (chessman and giap 2010). this essentially removes the idea of excluding all identif ied taxa not having preassigned tolerance values, as employed by other biotic indices. second, the habitat assessment performed by assigning values of 1 to 3 (1 = best possible condition; 3 = worst possible condition) for the computation of sds remains subjective, bringing about changes depending on the person performing the f ield sampling. finally, the tolerance value for each taxa remains dependent to the condition of its immediate habitat at the time of collection. conclusions the results show that benthic macroinvertebrates can be used as a bioassessment tool, as it was able to successfully evaluate and determine the conditions of the stream ecosystems under varying land use in silago, southern leyte. out of the six biotic indices tested, atspt shows potential in distinguishing polluted sites from unpolluted ones. this result was also supported by the data reflected in simpson’s diversity index, benthic macroinvertebrate composition, and the physicochemical variables. the atspt approach is considered advantageous over the widely used physicochemical method for stream bioassessment and biomonitoring, as atspt provides a rapid and cost-effective stream health evaluation without requiring expensive sets of elaborate equipment for data collection. finally, the f indings indicate that a long-term data set generated from future sampling efforts will signif icantly contribute in the protection, conservation, and restoration of the country’s freshwater through the development of the philippine biotic index. d.d.d. deborde et al. 19 acknowledgements this project was funded by the off ice of the chancellor of the university of the philippines diliman, in collaboration with the off ice of the vice chancellor for research and development (ovcrd), through ovcrd open grant (project no. 151503 og) awarded to f.s. magbanua. special thanks to alyssa fontanilla, irvin rondolo, and prana renee pambid for their help in the f ield. we are grateful to the three anonymous reviewers whose suggestions improved the manuscript. references aguiar acf, gücker b, brauns m, hille s, boëchat ig. 2015. benthic inver tebrate density, biomass, and instantaneous secondary production along a f ifth-order human-impacted tropical river. 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associate at the institute of biology, university of the philippines diliman. he obtained his bsc biology from up diliman. maria brenda m. hernandez <embeemh@gmail.com> is an instructor at the institute of biology, university of the philippines diliman. she is a phd candidate at the department of biology, university of waterloo, ontario, canada. she specializes in limnology and benthic communities (freshwater algae and macroinvertebrates). francis s. magbanua <fsmagbanua@gmail.com> is an assistant professor and head of the aquatic biology research laboratory, institute of biology, university of the philippines diliman. he received his phd in zoology from the university of otago, dunedin, new zealand. he specializes in freshwater ecology and biomonitoring using f ish and benthic macroinvertebrates. 7guidelines.pmd 98 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) 7de vera-quirit-rodriguez.pmd determination of cr, cd, sn and pb in selected herbal products 82 science diliman (july-december 2017) 29:2, 82-96 determination of cr, cd, sn, and pb in selected herbal products available in phil ippine markets joan s. de vera university of the philippines diliman leni b. quirit university of the philippines diliman irene b. rodriguez* university of the philippines diliman academia sinica, taiwan abstract the growing popularity of herbal products in the philippines makes it i m p e r a t i v e t o m o n i t o r a n d e n s u r e s a f e t y o f c o n s u m e r s f r o m m e t a l contaminants. in this study, trace concentrations of cr, cd, sn, and pb in h e r b a l p r o d u c t s w e r e s i m u l t a n e o u s l y m e a s u r e d u s i n g a m i c r o w a v e a s s i s t e d d i g e s t i o n a s s a m p l e p r e t r e a t m e n t a n d i n d u c t i v e l y c o u p l e d plasma mass spectrometry (icp-ms) for elemental detection. using the o p t i m i z e d m e t h o d , r e c o v e r i e s o f e r m c d 2 8 1 , t h e p r i m a r y ce r t i f i e d reference material (crm) used, were found to be between 80-89%, and the method detection limits (mdl) for cr, cd, sn, and pb were 0.15, 0.07, 0 . 3 , a n d 0 . 1 4 μg / l , r e s p e c t i v e l y. t h e l i n e a r r a n g e s f o r c r a n d o t h e r elements (cd, sn, and pb) were 0.01-500 and 0.01-50 μg/l, respectively. all correlation coeff icients were 0.9999 or better. most of the products t e s t e d h a d m e a s u r a b l e t r a c e m e t a l c o n c e n t r a t i o n s , w h i c h w e r e b e l o w the suggested maximum limits in herbal products. however, one product d e r i v e d f r o m m a n g o s t e e n e x c e e d e d t h e l i m i t f o r c d ( 0 . 4 2 m g / k g ) . subsequent analysis of metal content in tea infusions showed that only a small fraction of metals may leach out, suggesting that consumption o f t e a i n f u s i o n s p o s e l e s s e r r i s k s . t h e o r d e r o f a b u n d a n c e o f m e t a l s f o u n d i n h e r b a l p r o d u c t s w a s c r > p b > c d > s n . t h e v a r i a b i l i t y o f m e t a l concentrations in herbal products underlines the fact that many plant ingredients are susceptible to contamination, and quality control during p r o c e s s i n g m u s t b e i m p r o v e d t o m i n i m i z e t h e p o s s i b i l i t y o f _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online determination of cr, cd, sn and pb in selected herbal products 84 accumulate in this organ (hutton 1987; satarug and moore 2004; navas-acien et al. 2009). pb also accumulates and affects the bones and liver (hutton 1987). in addition, persistent low-level exposure to pb can cause neurotoxic effects to children because of the permeability of their blood brain barrier to pb (jarup 2003). no guideline values have been set for cr and sn despite the known serious health conditions associated with these two metals. some forms of cr and sn, such as cr (vi) and organotin compounds, are considered toxic for humans (kimbrough 1976; kotaœ and stasicka 2000; yu et al. 2008). in consideration of the known risks of these metals, they should be monitored in herbal products, especially because these products are sold as supplements and are regularly consumed for a long period of time. this work is aimed towards the development and optimization of a method suitable for the determination of metal concentrations in herbal products commonly sold and consumed in the philippines. an analytical method was optimized and validated using the certif ied reference materials (crm) and spiked samples prior to the application of the method to actual samples. the data from this study may serve as a background on the extent of metal contamination in herbal products in the country since publications regarding metal levels are scant. materials and methods the following crm were used for the optimization and validation of the sample preparation and analytical method: erm-cd281 (rye grass obtained from institute for reference materials and measurements, belgium), bcr 150 (skim milk powder from institute for reference materials and measurements, belgium), dorm-3 (f ish protein from national research council, canada), and nist 1643e (trace elements in water, national institute of standards and technology, usa). erm-cd281 was the primary crm used because it has the closest matrix as the samples. bcr 150 and dorm-3 were analyzed to check the applicability of the method to more complex sample matrices. nist 1643e was used as an additional crm for tea infusions because it was the available reference material that has the closest matrix to liquid samples. for further validation, the applicability of the sample pre-treatment scheme for liquid samples was evaluated following the standard addition method on one of the herbal liquid samples (noni plant juice), which was spiked with known analyte concentrations between 0.67 μg/l and 13.33 μg/l. a total of 33 commonly available herbal products were purchased from local drug stores and supermarkets in quezon city, philippines between september 2011 and j.s. de vera, et al. 85 november 2011. all products analyzed were manufactured in the philippines and made from plant ingredients that are endemic to the country except for the noni juice sample, which was included in the study to serve as the matrix for standard addition validation. the products were chosen according to their availability and whether consumers can easily acquire them through over-the-counter purchases and without prescription. the plant ingredients, formulations, and purported health benef its of the herbal products included in the study are summarized in table 1. of the samples analyzed, four plants, namely lagundi, sambong, amplaya and garlic, are listed in the circular of the department of health (1995) as safe, effective and scientif ically-validated herbal medicinal plants. malunggay, ginger, mangosteen, coconut and papaya plants are listed as medicinal plants with folkloric basis, and thereby require fur ther scientif ic validation (department of health 1995). other plants that were not mentioned in the list are also believed to have medicinal values based on folkloric evidences. herbal products made from lagundi were already approved as drugs to cure cough, while other lagundi-derived products are marketed as dietary supplements. plant ingred ient (scientific name) formulations common med icinal benefits ampalaya (momordica charantia) capsule, tea anti-diabetes banaba (lagerstroemia speciosa) capsule, tea good remedy for diabetes, high blood pressure and kidney problems coconut (cocos nucifera) oil f ights viral, bacterial, fungal and protozoan infections; aids in digestion and nutrient absorption garlic (allium sativa) capsule anti-cholesterol ginger (zingiber officinale) tea relief for gaseous distention, cough, arthritis and wounds lagundi (vitex negundo) capsule, syrup, tea anti-cough, asthma relief malunggay (moringa oleifera) capsule, coffee relief for arthritis, scabies, wounds and constipation mangosteen (garcinia mangostana) capsule, tea relief for diarrhea and stomach pain; contains anti-oxidant narra (pterocarpus indicus) capsule immune system support noni (morinda citrifolia) syrup anti-oxidant; supports immune system; has tumor-f ighting properties sambong (blumea balsamifera) capsule, tea diuretic spirulina (arthrospira) capsule antioxidant, anti-inflammatory and detoxif ier. turmeric (curcuma longa) capsule relief for digestive disorders papaya (carica papaya) syrup relief for constipation and wounds table 1. plant ingred ients, formulations and common med icinal benefits of the herbal products included in this study determination of cr, cd, sn and pb in selected herbal products 86 for the solid samples, tea capsules/tea bags/coffee sachets were pooled and mixed to achieve sample homogeneity. from the pooled sample, triplicate samples (0.2500 g weighed to the nearest 0.0001 g) were obtained and digested using a multiwave 3000 microwave system (anton paar, austria). for samples in liquid form, triplicate aliquots (1 ml) were obtained for digestion. the digestion mixture is composed of 2 ml concentrated superspure hno 3 , 1 ml concentrated superspure hcl, and 5 ml ultrapure water. the digestion program for the complete mineralization of samples was as follows: digestion was initially carried out at 250 w for 1 minute; then ramped to 400 w for 5 minutes; 600 w for 5 minutes; and, 800 w for 20 minutes. the digestion system was allowed to cool before the digested samples were transferred to polyethylene containers, bulked to 15 ml with ultrapure water, and subsequently f iltered through 0.45 μm nylon f ilters prior to elemental analysis. the concentrations of metals in tea infusions were also measured. for every brand of tea, three infusions prepared from different tea bags obtained from the same batch were analyzed. tea infusion was made by adding 40 ml boiled (~100 oc) ultrapure water to a tea bag. the infusion was kept for 3 minutes, which is the commonly suggested length of infusion when preparing tea drinks. the infusion was f iltered through a 0.45 μm nylon f ilter, mixed with 0.5 ml concentrated hno 3 , and bulked to 50 ml with ultrapure water. because of the formation of precipitates upon acidif ication, the infusions were f iltered again with new nylon f ilters prior to elemental analysis. simultaneous analyses of cr, cd, sn and pb in acid-digested sample solutions and tea infusions were performed using an agilent 7500cx icp-ms (agilent, germany) equipped with a micromist glass concentric nebulizer and an integrated autosampler (i-as with type a vials, 89 x 6 ml capacity). calibration standards ranging between 0.01 μg/l and 500 μg/l were prepared from stock standard solutions of cr, cd, sn and pb (10000 μg/ml, cpi technology, usa). for cr, the linear range was established from 0.01 μg/l to 500 μg/l. a wider range of concentration was necessary since erm cd281 and the samples contain relatively higher amount of cr. the linearity for cd, sn and pb were set at a much narrower range of concentration (0.01 μg/l 50 μg/l). the correlation coeff icients (r2) for all the calibration curves were 0.9999 or better. all solutions analyzed were spiked on-line with internal standard mixture containing ge (1 mg/l), in (10 mg/l), and re (10 mg/l) for signal drift correction. in addition, intraand inter-day variabilities of the method were assessed by repeated measurements of a solution containing a f inal concentration equivalent to 1 ppb of each analyte and erm cd281-digested solutions, respectively. the detection limit was estimated by analyzing seven individually prepared solutions containing 1 μg/l of each analyte in 1% hno 3 , which were subjected to digestion and subsequent j.s. de vera, et al. 87 icpms analysis. the detection limit was only estimated for aqueous digests and not for solid samples because of the diff iculty of ensuring homogeneity of spiked standards in solid samples. the standard deviations of the seven measurements multiplied with the student’s t-value (i.e. , student’s t-value for n = 7 at 99% conf idence level is 3.143) represents the estimated detection limit described in epa document 40 cfr part 136. results and discussion a prerequisite to trace metal analysis in actual samples is the existence of an analytical method that is optimized for the specif ic measurement and duly validated to be suitable for the deemed purpose. prior to the analysis of herbal products, the reliability of the method was established by analyzing crm and spiked liquid herbal product. the results from the analysis of the different crm are summarized in table 2. results from the analysis of the primary crm, erm-cd281 (n = 15), revealed that the recoveries for cr, cd, sn and pb were at least 80%. results from other solid r e f e r e n c e m a t e r i a l s d o r m 3 a n d b c r 1 5 0 s h o w e d t h a t m e a s u r e d v a l u e s approximate well the cer tif ied values of the analytes, indicating that the mineralization procedure and subsequent elemental determination were robust for the determination of more complex sample matrices. this is particularly advantageous for the samples in this study which were of complex nature. results of the analysis of nist 1643e revealed that the optimized method was suitable for the analysis of cr, cd and pb as reflected by the agreement between certif ied and measured values. to further validate the suitability of the method for liquid samples, as tea infusions may have more complex matrix than water, we also carried out the standard addition table 2. val idation of d igestion method and icpms detection in crm (mean ± sd, n = 15 for erm-cd281, n =3 for other crm) ermcertif ied value 24.8 ± 1.3 0.120 ± 0.072 0.062 ± 0.011 1.67 ± 0.11 cd281 measured value 19.8 ± 0.5 0.101 ± 0.003 0.067 ± 0.003 1.46 ± 0.03 dorm-3 certif ied value 1.89 ± 0.17 0.290 ± 0.020 0.066 ± 0.012 0.39 ± 0.05 measured value 2.08 ± 0.10 0.27 ± 0.01 0.08 ± 0.03 0.33 ± 0.01 bcr 150 certif ied value not certif ied 0.0218 ± 0.014 not certif ied 1.00 ± 0.04 measured value 0.018 ± 0.001 0.76 ± 0.01 nist certif ied value 20.4 ± 0.24 6.568 ± 0.073 not certif ied 19.63 ± 0.21 measured value 21.4 ± 0.4 6.7 ± 0.1 20.2 ± 0.7 certified reference material cr mg/kg cd mg/kg sn mg/kg pb mg/kg certified reference material crμg/l cdμg/l snμg/l pbμg/l determination of cr, cd, sn and pb in selected herbal products 88 method (table 3). one of the liquid samples, noni juice, was spiked with mixtures containing different concentrations of the target analytes. except for cr at the lowest spiked concentration, most of the analytes at different concentrations resulted in acceptable recoveries within the range of 90% to 120%. the high standard deviation observed in the values obtained for cr at the lowest spiked concentration may have been due to the diff iculty in completely mineralizing cr after it has equilibrated with the noni juice. evaluation of the method detection limits (mdl) following the suggested protocol by the us environmental protection agency resulted in the following values: 0.15, 0.07, 0.3, and 0.14 μg/l for cr, cd, sn and pb, respectively. these values were low enough to allow trace determination of the analytes should they be present as contaminants in the target samples. digested samples and tea infusions with metal concentrations below mdl were reported as <mdl. 0.67 163 ± 20 109 ± 1 116 ± 8 120 ± 20 3.33 95 ± 4 100 ± 1 111 ± 3 100 ± 1 6.67 99 ± 4 100 ± 1 112 ± 1 103 ± 3 13.33 109 ± 3 97 ± 2 111 ± 2 93 ± 1 33.33 99 ± 3 92 ± 1 105 ± 1 91 ± 1 concentrations of the spiked standards, μg/l % recovery (n = 3) cr cd sn pb table 3. val idation of d igestion method and icpms detection by standard add ition. various concentrations of cr, cd, sn and pb standards were spiked in noni juice the precision of the method was further assessed by measuring intra-batch and inter-day repeatability. intra-batch repeatability was determined by monitoring the response of a 1.0 μg/l standard solution. during an entire icp-ms run, the same solution was read at regular intervals to check if the instrument gave stable signals. from the response of the instrument, %rsd was calculated for every icp-ms run. the calculated %rsd obtained for cr, cd, sn and pb during the entire course of the study (6 different icp-ms runs were performed) were within 5% and were generally within 2% for each analysis day. inter-batch repeatability was evaluated by analyzing erm cd281 samples in triplicates for f ive different days. the concentrations measured for cr, cd, sn and pb are shown in table 4. the method was suitable for the determination of the target elements whose measured concentrations were comparable to certif ied values. overall, the results obtained on different days of analyses suggest that the optimized method is reliable and robust enough to be used over time. j.s. de vera, et al. 89 the mean concentrations of cr, cd, sn and pb from triplicate measurements of solid and liquid herbal supplement and products are summarized in tables 5 and 6, respectively. generally, the order of abundance of metals found in herbal products was cr > pb > cd > sn. for cr, four samples obtained concentrations that were below the mdl. in solid samples, including tea leaves, coffee powder, and capsules, cr was determined to be within the range of 0.14 to 2.6 mg/kg. in liquid samples, including juice and oils, the values ranged from 50 to 1100 μg/l. we observed that the values obtained for cr are relatively higher than what solidum et al. (2012) measured in philippine herbal products, wherein only 3 out of 10 products have measurable cr concentrations that range between 0.0084 and 0.0578 mg/kg. the cr concentrations measured in this study are comparable to values (0.34–2.12 mg/kg in herbs) reported in a study performed in turkey (baº gel and erdemo lu 2006). however, compared to the results obtained in studies done in pakistan (11.4–66.6 mg/kg) and poland (0.3–63.1 mg/kg), the samples in the present study have much lower concentrations of cr (leœniewicz et al. 2006; mahmood et al. 2013). the detection of cr in most samples warrants further studies that focus on speciation studies that attempt to f ind out whether cr is present in these products as cr (vi), which poses a higher health risk compared to cr (iii). pb, another ubiquitous contaminant in the environment, was determined in 30 samples and was below the detection limit in three samples. the concentrations were within the range 0.02–1.2 mg/kg and 5–51 μg/l in solid and liquid products, respectively. although none was found to exceed the 10 mg/kg guideline value, the determined values suggest that some products are susceptible to pb contamination at signif icant concentrations. pb is known to be bio-accumulated, and since most of these products are taken regularly, presence of pb at trace 24.8 24.9 19.4 22.8 19.6 19.8 (1.3) (0.9) (0.6) (0.3) (0.2) (0.5) 0.12 0.098 0.086 0.099 0.096 0.101 (0.007) (0.008) (0.003) (0.001) (0.002) (0.003) 0.062 0.071 0.055 0.04 0.049 0.067 (0.011) (0.004) (0.009) (0.01) (0.008) (0.003) 1.67 1.38 1.18 1.42 1.24 1.46 (0.11) (0.01) (0.05) (0.02) (0.04) (0.03) cr cd sn pb measured concentration (mg/kg) and standard deviation of measurements (sd) day 3 day 4 day 5day 1 day 2 certified values element table 4. inter-batch repeatabil ity study using erm cd281 measured during five d ifferent analyses days (n = 3 per day of analysis) ğ  determination of cr, cd, sn and pb in selected herbal products 90 concentrations may lead to health risks. in the measurements made by solidum (2014) for pb, medicinal plants and herbal tea ingredients in the philippines were in the range of 0.006–0.873 mg/kg, which is comparable to this study. compared to herbal products from other countries, the levels of pb measured in the philippines (e.g. , this study and solidum 2014) were lower. for instance, some samples exceeded the 10 mg/kg limit in studies performed in pakistan (0.5–41.01 mg/kg) by mahmood et al. (2013) and in malaysia (2.34–13.2 mg/kg) by ang (2008). the coffee in this study was also found to have lower pb concentration (0.02 mg/kg) than that of the study made by kapur and west (1974) in england (0.45–1.65 mg/ kg). a1 ampalaya capsule 2.6 ± 0.2 0.127 ± 0.002 < mdl 0.33 ± 0.02 a2 ampalaya capsule 0.98 ± 0.04 0.059 ± 0.001 < mdl 0.6 ± 0.1 a3 ampalaya tea leaves 0.8 ± 0.2 0.052 ± 0.001 0.059 ± 0.003 0.58 ± 0.03 a4 ampalaya tea leaves 0.25 ± 0.01 0.013 ± 0.001 0.010 ± 0.003 0.13 ± 0.02 b1 banaba capsule 0.9 ± 0.1 0.187 ± 0.004 < mdl 1.0 ± 0.3 b2 banaba tea leaves 0.8 ± 0.1 0.223 ± 0.008 0.054 ± 0.004 0.19 ± 0.01 b3 banaba tea leaves 0.7 ± 0.1 0.187 ± 0.003 0.027 ± 0.003 0.19 ± 0.02 g1 garlic capsule 0.43 ± 0.05 0.074 ± 0.004 < mdl 0.2 ± 0.1 r1 ginger tea leaves 4.2 ± 0.2 0.057 ± 0.001 0.013 ± 0.004 0.11 ± 0.02 l1 lagundi capsule 1.1 ± 0.3 0.011 ± 0.001 < mdl 0.5 ± 0.1 l2 lagundi capsule 0.6 ± 0.1 0.11 ± 0.01 < mdl 0.20 ± 0.03 l3 lagundi tea leaves 0.58 ± 0.04 0.010 ± 0.002 0.035 ± 0.001 0.19 ± 0.01 l4 lagundi tea leaves 0.6 ± 0.1 0.014 ± 0.002 0.028 ± 0.001 0.15 ± 0.01 m1 malunggay capsule 0.5 ± 0.1 0.03 ± 0.01 0.04 ± 0.01 0.30 ± 0.03 m2 malunggay capsule < mdl 0.025 ± 0.001 0.07 ± 0.01 0.71 ± 0.01 m3 malunggay coffee 0.16 ± 0.04 < mdl 0.003 ± 0.001 0.02 ± 0.01 m4 malunggay tea leaves 0.8 ± 0.1 0.007 ± 0.001 0.02 ± 0.01 0.12 ± 0.04 m5 malunggay tea leaves 0.14 ± 0.02 0.012 ± 0.001 0.029 ± 0.003 0.15 ± 0.02 t1 mangosteen capsule 0.21 ± 0.03 0.083 ± 0.001 <mdl 0.07 ± 0.03 t2 mangosteen capsule < mdl 0.29 ± 0.01 0.02 ± 0.01 0.10 ± 0.01 t3 mangosteen tea leaves 0.4 ± 0.1 0.43 ± 0.02 0.02 ± 0.01 0.09 ± 0.01 n1 narra capsule < mdl 0.010 ± 0.001 0.036 ± 0.002 0.44 ± 0.02 s1 sambong capsule 2.8 ± 0.1 0.216 ± 0.002 0.20 ± 0.02 1.2 ± 0.2 s2 sambong tea leaves 0.23 ± 0.04 0.22 ± 0.01 0.012 ± 0.002 0.3 ± 0.1 s3 sambong tea leaves 0.89 ± 0.01 0.18 ± 0.01 0.03 ± 0.01 0.3 ± 0.1 p1 spirulina capsule 0.6 ± 0.1 0.018 ± 0.001 0.013 ± 0.001 < mdl the detection limits for cr, cd, sn, and pb were 0.15, 0.07, 0.3, and 0.14 μg/l, respectively. the solutions of digested samples with concentrations below the detection limits were reported as <mdl. tea samples from which infusions were prepared from are indicated. the trace metals in the infusions are summarized in table 7. sample id herbal ingred ient form cr cd sn pb mg/kg ± standard deviation (n = 3) table 5. concentrations of cr, cd, sn and pb in solid herbal products. values represent mean ± sd of tripl icate samples determination of cr, cd, sn and pb in selected herbal products 92 of different organotin compounds. in this study, sn was found to be the least abundant, and was only quantif ied in 21 samples whose concentrations ranged between 0.003–0.20 mg/kg and 11–36 μg/l in solid and liquid products, respectively. the highest concentration of sn (0.20 mg/kg) was measured in a sambong capsule. the results from the present study were much lower compared to the results observed by ª ahan et al. (2007), wherein the concentrations of sn in olives were in the range of 14.40–53.62 mg/kg. the results from the analysis of tea leaves showed that most tea samples have measurable concentrations of cr, cd, sn and pb (table 5). however, these are not directly ingested like capsules and syrups, hence we deemed it necessary to analyze tea infusions. the amount of metals measured in tea infusions were normalized to the weight of the tea leaf powder used (table 7). only small fractions of metals leached out to the tea infusions. the metal concentrations measured in tea infusions were in the range of 0.006 –0.40 mg/kg for cr, cd and pb. sn, which was the least abundant among the four metals, was not detected at all in any of the infusions, while cr and pb were measured in almost all samples. cd was only measured in f ive products, which include the mangosteen tea leaves that exceeded the limit. the mean concentration of cd in the infusion of mangosteen tea leaves that table 7. amount of cr, cd and pb that leached out in tea infusions (mg) normal ized to the weight of tea leaves (kg) values represent the average concentration (± sd) or the range of metal concentrations determined in tripl icate tea infusion preparations a3 bitter gourd 0.04 ± 0.02 < mdl 0.03 ± 0.01 a4 bitter gourd 0.012 ± 0.005 < mdl 0.01 ± 0.01 b2 banaba 0.05 ± 0.02 < mdl to 0.004 0.02 ± 0.02 b3 banaba 0.06 ± 0.01 < mdl to 0.003 0.02 ± 0.02 r1 ginger 0.40 ± 0.03 < mdl 0.01 ± 0.01 l3 lagundi 0.09 ± 0.05 < mdl 0.03 ± 0.01 l4 lagundi 0.28 ± 0.06 < mdl 0.03 ± 0.01 m4 malunggay 0.30 ± 0.04 < mdl 0.01 ± 0.01 m5 malunggay 0.06 ± 0.02 < mdl 0.02 ± 0.02 t3 mangosteen < mdl to 0.006 0.005 ± 0.001 0.01 ± 0.01 s2 sambong 0.08 ± 0.04 < mdl to 0.01 0.03 ± 0.03 s3 sambong 0.110 ± 0.003 0.0034 ± 0.0003 0.01 ± 0.01 sn was below mdl for all samples. the method detection limits for cr, cd, sn and pb were 0.15, 0.07, 0.3, and 0.14 μg/l, respectively. the solutions of digested samples with concentrations below the detection limits were reported as <mdl. sample id herbal ingred ient mg/kg ± sd cr cd pb j.s. de vera, et al. 93 exceeded the limit was 0.005 mg/kg, which is about 80-fold lower than the cd found in the actual tea leaves. from the results of triplicate analyses, it can be observed that some of the infused samples have varied concentrations of the metals (high sd especially for pb). although the temperature of the water used and the length of infusions were constant, minor variations during the actual preparation were inevitable. the actual level of metals in each tea bag may vary as well. during preparation, the colors of the resulting infusions were observed to be not similar even when the same product was being compared. these factors were considered as possible reasons as to why the results of the replicates vary in some samples. although the extent of metal contamination is minimal, with only one product exceeding the limit, vigilant monitoring is still crucial, since bioaccumulation from prolonged exposure is highly possible. moreover, the existence of at least one product that exceeds the prescribed limit suggests that, despite the presence of regulatory bodies, there could still be products in the market that do not adhere to the guideline values. the high variability of metal concentrations of herbal products from the same plant ingredients underlines the fact that all plants are susceptible to contamination, and factors, such as the environment at which the plants were grown and the level of quality control employed during processing, may have influenced the variations observed. as for the analysis of tea infusions, the results show that the elemental impurities present in the tea leaves may not be completely transferred to infusions. since we only sampled a small fraction of the herbal products available in the market, it is encouraged that more herbal products be tested for heavy metal contamination. finally, the presence of cr in almost all samples and sn in some samples suggests that herbal products may possibly contain their harmful forms (e.g. , cr (vi) and organotin). it is therefore recommended to carry out speciation in order to determine the toxic forms of cr and sn, such as cr (vi), trimethyltin, and triethyltin. the data provided in this study may be used as preliminary information for future speciation analysis of cr and sn. acknowledgments the authors would like to thank the natural sciences research institute university of the philippines diliman for funding the study, and the environment monitoring laboratory of up nigs headed by dr. cp david for the use of icp-ms. determination of cr, cd, sn and pb in selected herbal products 96 _____________ joan s. de vera <jsdevera.43@gmail.com> earned her bs and ms chemistry degrees from the ic-upd. this work comprised her ms thesis. js de vera is currently a ph.d. candidate at the department of earth sciences of the university of toronto, canada. leni l. quirit <lquirit@yahoo.com> is a professor at the ic-upd with specializations in analytical chemistry, radiochemistry, and nuclear chemistry. ll quirit earned her ph.d. chemistry from the ateneo de manila university. irene b. rodriguez <hanrodriguez@yahoo.com> is currently a postdoctoral fellow at the academia sinica in taiwan. ib rodriguez earned her doctoral degree in chemistry from the karl-franzens university of graz in austria. filtration-manalo.pmd filtration and respiration rates of the short-necked clam 21science diliman (july-december 2010) 22:2, 21-29 filtration and respiration rates of the short-necked clam paphia undulata (born, 1778) (mollusca, pelecypoda: veneridae) under laboratory conditions laureen morillo-manalo* and annabelle del norte-campos marine biology laboratory, college of arts and sciences university of the philippines visayas miagao, iloilo *corresponding authore email: luvkosea@yahoo.com abstract the filtration and respiration rates of various size classes (35-39.99, 40-44.99,45-49.99, 50-54.99 and 5559.99 mm) of the short-necked clam paphia undulata were measured in the laboratory. the effects of three light regimes (0 lux, 172.22 lux and 645.83 lux), three microalgal species (isochrysis galbana, tetraselmis tetrahele and chaetoceros calcitrans) and four microalgal concentrations (10, 25, 50 and 100 x 104 cells ml-1) on filtration rates were investigated. mean filtration rate was highest (0.57 ± 0.04 lh-1ind.-1) under total darkness. this can be attributed to the natural environment of this species which is characterized by silty substrate and low visibility. filtration was also highest in the microalga isochrysis galbana (0.67 ± 0.05). rates initially increased from low to moderate microalgal concentrations (25 x 104 cells ml-1) and decreased at higher concentrations. filtration generally decreased with increase in clam size. light intensity, microalgal species and microalgal concentration showed significant effects on filtration. respiration of fed clams was higher (0.138 ± 0.026 ml o 2 h-1ind.-1) than unfed clams (0.053± 0.025 ml o 2 h-1 nd.-1) and increased with clam size. key words: paphia undulata, filtration rate, respiration rate, microalgae manalo, l. and campos, a.n. 22 introduction the short-necked clam paphia undulata (born, 1778) (fig. 1) is a commercially important bivalve species in the philippines and neighboring countries such as thailand and malaysia. the meat is consumed not only locally but a large quantity goes to the export market (agasen et al. 1998; pongthana 1990). it is locally known as “nylon shell” in western visayas. agasen et al. (1998) reported that the species in negros occidental is overexploited. del norte-campos & villarta (2010) showed the worsened state of its exploitation not only based on the higher exploitation rate (e= 0.75) compared to that reported by agasen et al. (1998) (e= 0.54) 13 years ago, but more importantly the decrease in the sizes of the catch and increase in proportion of immature clams in the catch. p. undulata has a moderately inflated and transversely elongate shell. its outer surface is smooth and glossy, with fine, slightly oblique undulating grooves (fao 1998). it is usually found in intertidal and sublittoral areas with muddy substrates at a depth of about 30 meters (fao 1998). despite the number of scientific studies on this commercially important clam in the philippines, there is no published information on its physiology. knowledge of the feeding habits gained from observations of filtration rates and respiration rates in relation to body size is important in understanding the nutritional biology of this filter feeding bivalve. the objectives of this study were to 1) determine the light condition, microalgal species and microalgal cell concentration at which filtration rate is highest and 2) compare the respiration rates (r) for unfed and fed clams in relation to body size of the short-necked clam paphia undulata. materials and methods sample collection and laboratory experiments paphia undulata clams were collected from the catches of hired compressor divers in negros occidental. the clams were brought to the upv institute of aquaculture hatchery and biology laboratory in miagao, iloilo. the clams were grouped according to 5 mm size classes: 35-39.99, 40-44.99, 45-49.99, 50-54.99 and 55-59.99 mm to determine the effect of size on filtration and respiration rates. mean tissue dry weights (g) for each size class were determined. clams were acclimated in aerated uvfsw (ultraviolet treated 1µm-filtered seawater) at 37-38 ppt inside an air-conditioned laboratory at 26-27 ºc for 3-7 days before the start of the experiments. the salinity and temperature conditions used were similar to the clam’s natural environment. a. filtration rates 1. light intensity experiment three light intensity treatments were used to determine the light condition at which filtration rate is highest: a) 0 lux (total darkness) by using 8l experimental basin covered with black plastic bag; b) 172.22 lux (ambient laboratory room lighting); c) 645.83 lux (using 20 watts fluorescent illumination placed two feet from the containers). a light meter was used to measure the light intensity for each treatment. the clams were selected at random from the pool of acclimated clams grouped according to the five size classes. for each size class, one set up is composed of three clams and three set ups were used for all experiments. each set up was placed in separate experimental basins and fed with a mixed microalgal diet in equal proportions of chaetoceros calcitrans, isochrysis galbana and tetraselmis tetrahele with a final concentration of 3 x 105 cells ml-1. this concentration corresponds to the levels usually found in aquaculture set ups (duerr et al. 1998). the mixed microalgal diet was added with ultraviolet filtered seawater to obtain the desiredfigure 1. the short-necked clam paphia undulata. science diliman (july-december 2010) 22:2, 21-29 filtration and respiration rates of the short-necked clam 23 concentration and volume of the suspension by using the formula: v 1 x c 1 = v 2 x c 2 (1) where v 1 is the volume of the undiluted microalgal culture in l; c 1 is the concentration of the undiluted microalgal culture in cells ml-1; v 2 is the desired volume of the final microalgal suspension in l and c 2 is the desired concentration of the final microalgal suspension in cells ml-1. for each light treatment, control suspensions without clams were made under similar conditions to monitor the reproduction of the microalgae during the experiment. all set ups were aerated to enable proper mixing of the microalgae and to keep them in suspension. the volume of water that each bivalve filtered (f, lh-1) was determined from the experimental basins with a volume of 8 liters. a 5 ml sample was collected from the center of the experimental basins using a 5 ml syringe every 30 minutes for a period of 3 hours. the samples were stored in plastic canisters and fixed with 2 drops of lugol’s solution. density of microalgal population in the samples was measured by direct microalgal cell count using a haemacytometer and a microscope. three replicate counts were made for each sample using one mm2 block with 16 squares. the filtration rate was determined using the formula used in del norte-campos (2004): fr = v x r (2) where r is the rate constant (h-1) or the negative of the slope obtained from regressing ln c or cell concentration (cells ml-1) against time (h) and v (= 8l) is the set up volume with the diluted microalgal suspension (l) and the clams. filtration rate was expressed in l h-1 ind-1. the light intensity with the highest mean filtration rate was chosen for the succeeding experiments. two-way anova with replication was used to examine the effects of size and light intensity on filtration rates. 2. microalgal preference experiment after the light intensity experiment, microalgal feeding experiment was done. unialgal cultures of c. calcitrans (cell diameter, c.d. = 5.4 ± 0.26 µm), i. galbana (c.d. =3.8 ± 0.45 µm) and t. tetrahele (c.d. = 10.2 ± 0.70 µm) with a concentration of 1.75 x 105 cells ml-1 each were used as food. this concentration was set after a series of unialgal feeding trials. these species were chosen because of their nutritive value and they are also considered as excellent food for many bivalves and crustaceans when fed alone or in combination (kurosawa 1994; fao 1996; 2004). feeding was carried out under the preferred light resulting from the light intensity experiment. procedures and analysis of samples were the same as in the preceding experiment using a different set of clams for each microalgal species set up. water sampling was done at 45-minute intervals for a period of 4 hours and 30 minutes. the microalgal species with the highest mean filtration rate was chosen for the succeeding experiments. two-way anova with replication was used to determine if there is a significant difference in filtration rates between sizes and microalgal species. 3. microalgal concentration experiment after the microalgal feeding experiment, four concentrations of the preferred microalgal species were tested: 10, 25, 50 and 100 x 104 cells ml-1 under the preferred light resulting from the light intensity experiment. three set ups were made for each size class. water sampling was done every thirty minutes for a period of 3 hours. procedure and analysis of samples were the same as the preceding experiment using a different set of clams. the microalgal concentration with the highest mean filtration rate was chosen for the succeeding experiment. to show the relationship between filtration rate and size in terms of dry weight, filtration rates (l h-1ind.-1) were plotted against dry weights (g) of the organisms. two-way anova with replication was used to determine if there is a significant difference in filtration rates between sizes and microalgal concentrations. b. respiration rates respiration rates were measured for both unfed (ration = 0; starved for 24 h) and fed clams at 26-27 ºc and 37-38 ppt. the light condition, microalgal species and microalgal concentration used were based from the results of the preceding filtration rate experiments. fed clams were given isochrysis galbana at 25 x 104 cells science diliman (july-december 2010) 22:2, 21-29 manalo, l. and campos, a.n. 24 ml-1 under 0 lux for at least 15 minutes prior to the respiration measurements. for each of the 5 size classes, three set ups were made. the respirometer consisted of an erlenmeyer flask filled with uvfsw to the brim (1.150 l total volume) with the mouth of the flask covered tightly with a rubber sheath. the ysi do meter was first calibrated based on the procedure provided by the manufacturer. preliminary runs with varying intervals and duration were conducted. a 20min acclimation period following clausen and riisgard (1996) and zhang et al. (2004) was allowed after which oxygen concentration was measured every 3 mins until the oxygen concentration approached 70% of the initial reading. oxygen concentration was expressed in ml o 2 l-1. a control set up with no clams was first made to test the stability of the chamber or to ensure that there was no leak. respiration rate (ml o 2 h-1 ind.-1) was determined as the negative of the slope of ln o 2 concentration versus time (h). to show the relationship between respiration rate and size in terms of dry weight, respiration rates (ml o 2 h-1 ind.-1) were plotted against dry weights (g) of the organisms. one-way anova with replication was used to determine if there is a significant difference in respiration rates between sizes. results a. filtration rates 1. light intensity experiment it was observed that the clams opened their valves and extended their siphons when the microalgal suspension was given during the experiment. the filtration rate was highest under 0 lux (total darkness) with a mean value of 0.57 ± 0.04 lh-1ind.-1 smaller clams had higher filtration rates than bigger ones (fig. 2).two-way anova with replication showed that there was a significant difference in filtration rates between sizes (f = 59.41, p < 0.001) and light intensities (f = 734.73, p < 0.001). interaction between light intensity and size was not significant (f = 0.3102, p = 0.9561) which suggests that these factors are independent. 2. microalgal preference experiment the highest mean filtration rate of 0.67 ± 0.05 lh-1ind.1 was observed in the microalga isochrysis galbana. filtration rate also decreased with increasing size (fig. 3) two-way anova with replication showed that there was a significant difference in filtration rates between sizes (f = 16.46, p <0.001) and microalgal species (f = 4.70, p < 0.001). interaction between microalgal species and clam size was not significant (f = 0.1845, p = 0.9913) which also suggests that the two factors are independent. figure 2. filtration rates (l h-1 ind-1) of various size classes of p. undulata under different light intensities (n = 45). figure 3. filtration rates (l h-1 ind-1) of various size classes of p. undulata at different microalgal species under 0 lux (n = 45). 3. microalgal concentration experiment the filtration rates of p. undulata increased from low to moderate concentrations (100,000 to 250,000 cells ml-1). a decrease in filtration rate was observed at 500,000 cells ml-1, decreasing further at 1,000,000 cells ml-1. when the plastic bag cover was opened after the set up, the clams were observed to have narrow valve gape, their mantle edges were almost invisible, the exhalant siphon was narrow and there was a production of pseudofeces. more pseudofeces were observed at 500,000 and 1,000,000 cells ml-1. the mean filtration science diliman (july-december 2010) 22:2, 21-29 filtration and respiration rates of the short-necked clam 25 rate values for the different microalgal concentrations ranged from 0.23 ± 0.06 to 0.99 ± 0.07 lh-1ind.-1. filtration rates initially increased from low to moderate concentrations and decreased at higher concentrations (fig. 4). of these concentrations, the highest mean filtration rate of 0.99 ± 0.07 lh-1ind.-1 was observed under 25 x 104 cells ml-1 thus considered as the optimum concentration of i. galbana for feeding. filtration rates of p. undulata plotted against dry weights under the preferred light intensity, microalgal species and microalgal concentration were observed to decrease with an increase in size with a slope of 0.314 (r2 = 0.692) (fig. 5). two-way anova with replication showed that there was a significant difference in filtration rates between sizes (f = 55.41, p < 0.001) and microalgal concentrations (f = 1794.23, p < 0.001). the interaction between these factors was not significant (f = 0.2538, p = 0.9931) which means that they are also independent. b. respiration rates respiration rates of unfed clams were observed to increase with size, i. e. from 0.028 ± 0.002 ml o 2 h1ind.-1 for 35-39.99 mm individuals to 0.089 ± 0.010 ml o 2 h-1 ind.-1 for 55-59.99 mm (fig. 6). respiration rates for fed individuals also increased with size. the respiration rate values obtained ranged from 0.099 ± 0.006 ml o 2 h-1ind.-1 for 35-39.99 mm individuals to 0.166 ± 0.003 ml o 2 h-1ind.-1 for 55-59.99 mm (fig. 7). mean respiration rate for all size classes was observed to be higher (0.138 ± 0.026 ml o 2 h-1ind.-1) for fed clams than for unfed clams (0.053 ± 0.025 ml o 2 h-1ind.-1). respiration rates for fed clams plotted against dry weights (g) were observed to increase with size with a slope of 0.113 (r2 = 0.825) (fig. 8). one-way anova showed that there was a significant difference in respiration rates between sizes for both unfed (f = 31.99, p < 0.001) and fed clams (f = 22.18, p < 0.001). figure 4. filtration rates (l h-1 ind-1) of various size classes of p. undulata at different microalgal concentrations (cells ml-1) of isochrysis galbana (n = 60). figure 5. filtration rates (lh-1ind.-1) of p. undulata fed with isochrysis galbana at 25 x 104 cells ml-1 under 0 lux versus dry weights (g) of 35-39.99, 40-44.99, 45-49.99, 50-54.99 and 55-59.99 mm size classes. figure 6. respiration rates (ml o 2 h-1 ind.-1) of unfed p. undulata versus size classes (mm) (n = 15). figure 7. respiration rates (ml o 2 h-1 ind.-1) of unfed p. undulata versus size classes (mm) (n = 15) science diliman (july-december 2010) 22:2, 21-29 manalo, l. and campos, a.n. 26 discussion jørgensen (1990) reported that a relaxed and unrestrained bivalve fully opens its valves and extends its siphons. these conditions result to an efficient food uptake and maximum filtration rates. the highest filtration rate was observed under 0 lux (total darkness) treatment for all size classes used. paphia undulata is a mud burrower adapted to dark conditions thus it may be sensitive to direct light. similar results were observed by corda & del norte-campos (1998) for the angelwing clam pholas orientalis which is also a mud burrower. the high filtration rate of p. undulata under dark condition can be attributed to its natural environment which is characterized by a silty substrate and low visibility. the difference in the filtration rates between microalgal species was significant. although i. galbana may not be in the clam’s natural habitat, the highest filtration rate (0.67 lh-1ind.-1) of p. undulata to this microalga can be due to its smaller size (3.8 ± 0.45 µm, the smallest among the three microalgal species) which makes it much easier to ingest and faster to digest than c. calcitrans (5.4 ± 0.26 µm) and t. tetrahele (10.2 ± 0.70 µm). the results also suggest that algal size and quality (not availability) are the determining factors in food selection if the clam is given a variety of choices such as the three species used in the experiment. in addition, i. galbana has a higher nutritive value specifically its carbohydrate and lipid content which makes it an excellent food for many bivalves and crustaceans (fao 1996; taylor et al. 1997; brown et al. 1999; phatarpekar et al. 2000; fao 2004). in bivalves, particle size and quality play a major role in filtration. those that are too large are not utilized as food so they are rejected and those that are too small may pass through the filter (dame 1996). the feeding mechanism of bivalves is said to be a process of actively sorting particles of different sizes prior to ingesting them (jørgensen 1996). the result of this study agrees with that of epifanio & ewart (1977) wherein the oyster crassostrea virginica had higher filtration rates for the smaller algae isochrysis and thalassiosira than the larger croomonas and carteria. this was also observed in the mussel limnoperna fortunei which filter faster when fed with the smaller microalga scenedesmus sp. than schizochytrium sp. which is twice its size (pestana et al. 2009). the filtration rates of p. undulata initially increased from low to moderate concentrations and decreased at higher concentrations. a decrease in filtration rates at high concentrations were also observed in the surf clam paphies donacina (marsden 1999), the yellow clam paphia malabarica, the mussel perna viridis, the oysters crassostrea madrasensis (rajesh et al. 2001) and crassostrea gigas (gerdes 1983), the clam tapes decussatus (khalil 1996) and the mussel mytilus chilensis (navarro & winter 1982). low algal concentrations stimulate valve opening and filtration activity thus filtration rate is increased whereas high algal concentrations cause an overloading of the alimentary canal which in turn results to valve closure thereby reducing filtration rate (riisgard 1988; jørgensen 1990). in all the microalgal concentration set ups [in this present work], the clams were fully open and siphons extended during the experiment. it was also observed that filtration rate was reduced and more pseudofaeces were produced at higher microalgal concentrations of 500,000 and 1,000,000 cells ml-1. these results showed that there is an inverse relationship between filtration rate and pseudofaeces production. production of pseudofaeces or undigested food particles in mucus form implies that bivalves maximize energy gain during feeding by rejecting excess particles filtered by the gills (instead of utilizing more energy to metabolize excess particles). it also allows bivalves to regulate ingestion rate and prevent saturation of the alimentary system. foster-smith (1975) also figure 8. respiration rates (ml o 2 h-1ind.-1) of fed p. undulata versus dry weights (g) of 35-39.99, 40-44.99, 45-49.99, 5054.99 and 55-59.99 mm size classes. science diliman (july-december 2010) 22:2, 21-29 filtration and respiration rates of the short-necked clam 27 reported a decrease in filtration rates and increase in pseudofaeces production with increasing concentrations of the diatom phaeodactylum in the mussel mytilus edulis, the cockle cerastoderma edule and the clam venerupis pullastra. results of this study showed that filtration rates of p. undulata decreased with an increase in size in terms of dry weight. this implies that there is a higher energetic cost on filtration rate for larger clams than smaller ones. this is also supported by the results on the respiration rate experiment in this study which showed an increase in respiration rate with size. the higher filtration rates of smaller sized p. undulata can support its faster growth rate with k = 1.0-1.2 yr-1 (agasen et al 1998; del norte-campos & villarta, 2010) compared to subtropical bivalve species such as the pearl oyster pinctada maxima with k = 0.720.79 yr-1 ( hart & joll 2006). the observed higher growth rates of p. undulata before they reach sexual maturity (agasen et al. 1998) around 40-50 mm confirms the need for smaller individuals to filter faster in order to support the energetic demands of growth and development. filtration rates of the hard clam meretrix lusoria also decrease with size (chien & hsu 2006). in contrast, the filtration rates of paphia malabarica, perna viridis and crassostrea madrasensis (rajesh et al. 2001), crassostrea gigas (ren et al. 2000, gerdes 1983), pinctada margaritifera , pinctada maxima (yukihira et al. 1998), chlamys farreri (kuang et al. 1997),tapes decussatus (khalil 1996) and mytilus chilensis (navarro & winter 1982) increase with size. filtration rate in general is said to increase with increasing body size (in w) and this is represented by an allometric equation f = awb (bayne et al. 1976; winter 1978). the respiration rate of p. undulata was lower (0.053 ± 0.025 ml o 2 h-1ind.-1) when starved and increased (0.138 ± 0.026 ml o 2 h-1ind.-1) with feeding. similar results were obtained in the study of del norte-campos (2004) for the sunset clam gari elongata. when a bivalve is starved, the rate of oxygen consumption declines to a standard rate or steady state due to a decreased filtration and ventilation activity. when a bivalve is fed, the oxygen consumption is in the active rate which is marked by an increased uptake of oxygen and ventilation activity (navarro & winter 1982). feeding results to an increase in respiration rate two to three times compared to the standard rate of an organism at rest (jørgensen 1990). there was an increase in the respiration rate of p. undulata by 0.085 ml o 2 h-1ind-1 with feeding. the respiration rates increased with size and this observation was also reported in the pearl oysters pinctada margaritifera and pinctada maxima (yukihira et al. 1998) and the mussel mytilus chilensis (navarro & winter 1982). oxygen consumption increases with bivalve size according to the power function r = awb (bayne et al. 1976; winter 1978; dame 1996). based on the results of the study, filtration rate of p. undulata was highest under 0 lux (total darkness) treatment, microalga isochrysis galbana and 25 x 104 cells ml-1 concentration of i. galbana under laboratory conditions. filtration rates of p. undulata also decreased with an increase in clam size. light intensity, microalgal species and microalgal concentration have significant effect on filtration rate. respiration rate of fed clams was higher than unfed ones. respiration rate also increased with an increase in clam size. acknowledgements the authors would like to thank dost/pcamrd for funding, dr. s.s. mingoa-licuanan and dr. w. l. campos for suggestions, f. nabuab, r. palla and m. burlas for lab and field assistance. this paper was presented at the 10th pams national symposium held in davao city in october 2009. references agasen, e. v., c. m. del mundo, g. o. matias. 1998. assessment of paphia undulata in negros occidental/ guimaras strait waters. j. shellfish res. 17(5): 1613-1617. bayne, b. l., r. j. thompson & j. widdows. 1976. marine mussels: their ecology and physiology. (ed. b. l. bayne). great britain, cambridge univ. press. p. 25-57. brown, m. r., m. mular, i. miller, c. farmer & c. trenerry. 1999. the vitamin content of microalgae used in aquaculture. j. appl. phyco. 11: 247-255. science diliman (july-december 2010) 22:2, 21-29 manalo, l. and campos, a.n. 28 chien, y. h. & w. h. hsu. 2006. effects of diets, their concentrations and clam size on filtration rate of hard clams meretrix lusoria. j. shellfish res. 25 (1): 1522. clausen, i. b. & h. u. riisgard. 1996. growth, filtration and respiration in the mussel mytilus edulis : no evidence for physiological regulation of the filter-pump to nutritional needs. mar. ecol. prog. ser. 141: 37-45. corda, d. r. & a. g. c. del norte-campos. 1998. filtration rate of the angelwing clam, pholas orientalis (gmelin, 1970). upv j. nat. sci. 3: 26-36. dame, r. f. 1996. ecology of marine bivalves: an ecosystem approach. boca raton, florida, crc press. 254 pp. del norte-campos, a.g.c. & k.m. villarta. 2010. comparison of the status of the southern negros occidental stocks of the short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) using population parameters.science diliman 22(2):51-60. del norte-campos, a. g. c. 2004. filtration and respiration rates of the elongate sunset clam gari elongata lamarck 1818 under natural light conditions. j. shellfish res. 23 (1): 107-111. duerr, e. o., a. molnar & v. sato. 1998. cultured microalgae as aquaculture feeds. j. mar. biotech. 7: 65-70. epifanio, c. & j. ewart. 1977. maximum ration of four algal diets for the oyster crassostrea virginica gmelin. aquacult. 11: 13-29. fao. 2004. lovatelli, a. 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(adapted with permission f rom the editors of ieee sensors journal) 5pedales.pmd an account of the accessioned collections of the up biology 40 science diliman (july-december 2014) 26:2, 40-48 an account of the accessioned collections of the up biology invertebrate museum ronniel d.c. pedales* gizelle a. batomalaque university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract the university of the philippines (up) biology invertebrate museum has r e c e n t l y c o m p l e t e d t h e c u r a t i o n o f i t s a c c e s s i o n e d c o l l e c t i o n s o f invertebrates. this paper reports on the availability of the said collections to the community of researchers studying invertebrates. the accessioned collections were assessed in terms of their taxonomic scope, geographical range, and chronological breadth. a total of 4,238 accessioned specimens are in the museum, which is composed of 1,108 non-insectan arthropods, 1 , 1 4 9 c n i d a r i a n s , 1 7 8 e c h i n o d e r m s , a n d 1 , 8 0 3 m o l l u s k s . t h e i n s e c t specimens, all of which do not have any accession numbers, are yet to be curated. a total of 1,185 species belonging to 621 genera are found i n t h e co l l ec t i o n s . t h e m u s e u m ’s s a m p l i n g a c t i v i t i e s we r e g r e a te s t i n t h e w e s t e r n p a r t o f t h e p h i l i p p i n e s , s p e c i f i c a l l y i n pu e r t o g a l e r a , o r i e n t a l m i n d o r o . m u c h o f t h e e a s t e r n r e g i o n s i n t h e p h i l i p p i n e s a r e yet to be sampled, par ticularly the terrestrial habitats. prolif ic museum contributors include francisco nemenzo, sr. (709 specimen lots), neon rosell (327 specimen lots), and fernando dayrit (233 specimen lots). at p r e s e n t , p l a n s f o r c o l l e c t i o n ex p a n s i o n i s u n d e r w a y, t o e n c o u r a g e collaborative research with other natural history museums. keyword s: inver tebrate zoology, museum, philippine inver tebrate fauna, collection r.d. pedales and g.a. batomalaque 41 introduction museums are institutions that function to support research in various disciplines (winston 2007) and are regarded as massive information resources (graham and others 2004). winston (2007) outlined ten key points regarding the signif icance of museum collections: 1) they support research in numerous disciplines; 2) they are non-renewable resources; 3) they are cost-effective; 4) they have an important function in human medicine, public health, and security; 5) they can be used to monitor climate change; 6) they can be used to demonstrate biological differences and/or changes in genetic diversity; 7) they can be used for research on the history of a discipline; 8) they educate new generations of students; 9) they have aesthetic importance; and 10) they are the foundation for taxonomic research and the study of biodiversity. invertebrates make up almost 96% of known metazoan species (brusca and brusca 2003 c. in winston 2007), and museum collections (both living and fossil) provide a valuable resource in studying their diversity and evolutionary relationships (winston 2007). in the university of the philippines diliman, the up biology invertebrate museum has served as a repository for several well-known invertebrate collections. among these are the hermatypic corals of francisco nemenzo sr. (the father of philippine coral taxonomy) that date back to 1935, and the cirriped type specimens of neon rosell from the musorstom campaign in the 1980s (rosell 1981, rosell 1989, rosell 1991). however, the museum has not been prolif ic as a resource in taxonomic research since then. thus, this study aimed to accomplish the following: 1) report the extant accessioned collections, 2) determine the chronological breadth of the museum’s activity in terms of taxonomic research, 3) determine the scope— in terms of taxa and geographic range—of invertebrate research done by the museum, and 4) identify the gaps in invertebrate research and the possible future directions for the museum. methodology due to the neglect of the museum for decades, the initial step was to restore the specimens’ conditions and storage. collections processed included non-insectan arthropods, soft and hard corals, echinoderms, and marine and terrestrial mollusks. an electronic database, in ms excel (microsoft off ice excel 2013) format, was created by encoding records from the card catalogues and logbooks. the entries included the 1) unique specimen code, 2) species binomen, 3) collection data (collector, collection date, and locality), and 4) other pertinent details about the an account of the accessioned collections of the up biology 42 organism (e.g. , type designation). however, not all specimens have collection details. the scientif ic names were updated using publication records and online databases, such as the world register of marine species (worms editorial board 2014) and encyclopedia of life (eol) (encyclopedia of life 2014). the database was then used to determine the number of specimens under each phylum, the chronological breadth, and the geographical range of invertebrate sampling. qgis 2.2.0-valmera (quantum gis development team 2014) was used to illustrate the locality data from the specimens. results and discussion the up biology invertebrate museum holds a total of 4,238 accessioned specimens, comprising 1,108 (26%) non-insectan arthropods, 1,149 (27%) cnidarians, 178 (4%) echinoderms, and 1,803 (43%) mollusks (figure 1). the entire collection comprises 226 families, 621 genera, and 1,185distinct species. among these, 2,440 specimens have collector data, 2,427 specimens have locality data, and 1,773 have collection date entries. the earliest recorded specimen in the museum is a bivalve mollusk pteria peasei (pteriidae) collected in 1909 by an unknown contributor. the collections started to figure 1. percentage of specimens per phylum; verif ied specimens distributed in four phyla: non-insectan arthropoda (1,110 specimens), cnidaria (1,148 specimens), echinodermata (181 specimens), and mollusca (1,809 specimens). mollusca 4 3 % anthropoda 2 6 % cnidaria 2 7 % echinodermata 4 % percentage of specimens per phylum r.d. pedales and g.a. batomalaque 43 grow after the war during the 1940s, with expeditions led by p. de mesa, who specialized on marine mollusks, and m. delgado, who specialized on cnidarians. table 1 lists the primary contributors to the museum’s collection. table 1. primary contributors to the up biology invertebrate museum collector verified specimens in collection fidel nemenzo 709 neon c. rosell 327 fernando dayrit 233 f. abad santos 210 neon rosell staff 113 biology class* 96 banzon, militante, and zarco 80 e.s. muego and r.g. visconde 68 dela cruz 48 p. de mesa 47 *collection of students as a requirement in ecology and/or taxonomy classes the peak of museum activity was observed in the 1950s through the efforts of nemenzo, sr. , who spearheaded the collection and description of philippine hermatypic corals. his collection comprises 57% of all coral specimens, 65 species of which he has described (note: type designations of these specimens are yet to be verif ied). this particular collection, comprising 335 species under 48 families, is of great importance to philippine coral taxonomy (nemenzo 1986, gomez and others, 1994). notable contributors to this collection were banzon, militante, zarco, estampador, dela cruz, and zambo. nemenzo and his colleagues’ work flourished until the late 1980s. echinoderms have been included in the collection only during the 1960s through the efforts of the up zoological society, d.z. llamas, e.s. muego, and r.g. visconde, who collected sea feathers (crinoidea) and sea stars (asteroidea). during this period, n.c. rosell also started building the arthropod collection, particularly the barnacles (cirripedia). rosell’s research on philippine barnacles proliferated from the 1970s to the 1980s. his participation in the musorstom 1, 2, and 3 expeditions resulted in the description of 14 new species (rosell 1981, rosell 1989, rosell 1991). nines species described by rosell are housed in the museum (note: type designations of the specimens are yet to be verif ied). another notable contributor is f. dayrit, whose donation comprised 13% of all the mollusk specimens in the collection. however, only locality data were aff ixed to his collection. an account of the accessioned collections of the up biology 44 figure 2 shows the chronological breadth of museum activity based on the number of specimens for each taxonomic group, added to the collection per decade from 1909–2007. peaks from the 1940s to the 1970s are attributed to the expeditions described above. however, the taxa representation was observed to be very limited. this verif ies alberch’s (1993) observation that museum collections grow indiscriminately, which is mainly dependent on the curator’s interest or preference. alberch (1993) further states that natural history collections have not properly been appreciated, and consequently unfunded, because of the lack of standard datakeeping procedures and “failure to identify new potential users”. in 1995, president ramos signed an executive order no. 247 (e.o. 247 issued may 18, 1995), which requires the securing of permits when collecting biological specimens for scientif ic, commercial, and other purposes. this may have dampened the collection activities. furthermore, the institute stopped offering the field biology course (biology 161) in the mid-90s, and only resumed in 2007. the taxonomy and ecology courses, although having systematic sampling activities, did not assign catalogue numbers, thus the specimens have not been included in the database (i.k.c. fontanilla, personal communication, september 5, 2014). this has been the case for all the insects, worms (both terrestrial and aquatic) and most freshwater invertebrates in the collection (note: these uncatalogued specimens are in the process of being assigned unique specimen codes). another reason for the lack of growth in the museum’s collections beginning from the 1990s was the absence of a designated curator in the institute. figure 2. collection of the up biology inver tebrate museum from 1904–2007. r.d. pedales and g.a. batomalaque 45 using the locality data from 2,427 (55.6%) specimens, maps were generated to show the geographical range of invertebrate sampling by the museum (figure 3) the data were sorted according to phylum. sampling of all taxa was greatest in puerto galera, oriental mindoro, with 732 specimens. the southern luzon region was the most represented, having 919 specimens. an apparent bias towards aquatic— mostly marine—invertebrates (4,129 specimens) also persists, as opposed to terrestrial invertebrates (109 specimens). in general, the eastern region of the philippines appears to be poorly sampled. samples from the northern and central luzon, and central and eastern mindanao, figure 3. gis map representing sampling sites of museum researchers throughout the philippine islands. points represent locality of museum specimen with biogeographical data. an account of the accessioned collections of the up biology 46 are also evidently lacking. the island of palawan, which is known to have a high biodiversity (phelps and others 2010) is represented only by 166 specimens in the collection. natural history museums must “change their mode of operation and public image” to contribute in addressing the biodiversity crisis (alberch 1993). the current holdings of the up biology invertebrate museum may be reflective of the work done by up diliman in the philippines. the museum must, at this stage, create networks with other natural history institutions. the zoological collection of the philippine national museum boasts a collection of 292,628 invertebrate specimens, amounting to 86% of its total holdings (the national museum zoological collection, n.d.). the university of santo tomas natural history collection is also of great importance, as their collections date as early as 1682 (natural history collection, n.d.). the center for research and documentation of phlippine biota is the up los baños museum of natural history (uplb-mnh), which includes 700 shells on display, and the largest entomological (insect) collection of 200,000 specimens (uplb museum of natural history, n.d.). the uplb-mnh is also the leader in museum research in the philippines; it has had research projects since 1985, expeditions from 2006–2013, and has its own refereed journal, the uplb museum publications in natural history (uplb museum of natural history, n.d.). finding ways to do collaborative work with these institutions to complement them in their undertaking is essential for the museum. under-represented taxa must be given attention in terms of sampling and consequent processing. these include the terrestrial arthropods (excluding insects), worms (from different habitats), sponges, and invertebrate chordates (e.g. , tunicates/ ascidians). the lack of experts in these specif ic groups does not warrant them to be excluded in the collection, as communications can be done with experts from other museums. furthermore, the museum must not simply focus on building its collection. rather, museum-based research must also be conducted, as the present collections are important tools in comparative biology (alberch 1993), conservation, and genomics (austin and melville 2006). acknowledgments the authors would like to thank the off ice of the vice chancellor for research and development, particularly the source of solutions (sos) grant for funding the study, and the numerous volunteers, summer interns, graduate, and undergraduate assistants who helped this project come into fruition. r.d. pedales and g.a. batomalaque 47 references alberch p. 1993. museums, collections and biodiversity inventories. trends in ecology & evolution, 8(10): 372-375. austin j.j. , melville j. 2006. incorporating historical museum specimens into molecular systematic and conservation genetics research. molecular ecology notes, 6(4): 10891092. encyclopedia of life. available from http://www.eol.org. accessed 08 may 2014. gomez e.d. , alino p.m. , yap h.t. , licuanan w.y. 1994. a review of the status of philippine reefs. marine pollution bulletin 29(1): 62-68. graham c.h. , ferrier s. , huettman f. , moritz c. , peterson a .t. 2004. new developments i n m u s e u m b a s ed i n f o r m a t i c s a n d a p p l i c a t i o n s i n b i o d i ve r s i t y a n a l y s i s . tr e n d s i n ecology & evolution 19(9): 497-503. microsoft . 2003. microsoft excel [computer software]. redmond, washington: microsoft . natural history collection (n.d.). university of sto. tomas museum. available from http://ustmuseum.ust.edu.ph/collections/collection.aspx?id=txal6jkvaexrpnf2qme9tj% 2buhxxv6tvptqurlnlxqre%3d. accessed 17 may2014. nemenzo f. 1986. guide to philippine flora and fauna. vol. 5. corals. natural resources management centre and university of the philippines, philippines. phelps j. , guerrero m.c. , dalabajan d. a . , young b. , webb e.l. 2010. what makes a ‘redd’country?. global environmental change 20(2): 322-332. quantum gis development team. 2014. quantum gis geographic information system. o p e n s o u r c e g e o s p a t i a l f o u n d a t i o n p r o j e c t . a v a i l a b l e f r o m h t t p : / / q g i s . o s g e o . o r g . accessed 20 may 2014. ro s e l l n . c . 1 9 8 1 . cr u s t a ce a : c i r r i p ed i a . ré s u l t a t s d e s c a m p a g n e s m u s o r s to m i philippines (18–28 mars 1976). éditions de l’off ice de la récherche scientif ique et tec h n i q u e o u t r e m e r a vec l e co n co u r s d u m u s é u m n a t i o n a l d . h i s to i r e n a t u r e l l e collection mémoires orstom (91): 277-307. rosell n.c. 1989. thoracic cirripeds from the musorstom 2 expedition. in: forest , j. (ed.) resultats des campagnes musorstom, volume 5. mémoires du muséum national d’histoire naturelle (a) (144): 9-35. rosell n.c. (1991). crustacea cirripedia thoracica: musorstom 3 philippine collection. in: crosnier, a . (ed.) resultats des campagnes musorstom, volume 9. mémoires du muséum national d’histoire naturelle (a) (152): 9-61. t h e n a t i o n a l m u s e u m zo o l o g i c a l co l l e c t i o n ( n . d . ) . t h e n a t i o n a l m u s e u m of t h e philippines. available from http: //www.nationalmuseum.gov.ph/nationalmuseumbeta/ collections/zoology/zoology.html. accessed 17 may 2014. an account of the accessioned collections of the up biology 48 uplb museum of natural history (n.d.). available from http://mnh.uplb.edu.ph/. accessed 17 may 2014. winston j.e. 2007. archives of a small planet: the signif icance of museum collections and museum-based research in inver tebrate taxonomy. zootaxa 1668: 47-54. worms editorial board, 2014. world register of marine species. available from http:// www.marinespecies.org at vliz. accessed 08 may 2014. _____________ ronniel d.c. pedales <ronnipedals@gmail.com> is a volunteer for the up biology invertebrate museum since 2012 and has worked there as a student assistant during his undergraduate studies. he currently works as a molecular biologist for a cancer research laboratory. he will pursue an ms in the institute of biology early next year with research focusing on forensic entomology and the ecology of philippine flies. gizelle a. batomalaque is an instructor at the institute of biology, university of the philippines diliman. she received her master's degree from the same institution, and is currently pursuing a ph.d. degree in environmental science at drexel university, philadelphia, usa. her research focuses on molluscan systematics, particularly philippine land snails. 4lim-chemical characterization.pmd r.b. lamorena-lim and c.m.f. rosales 17 science diliman (january-june 2016) 28:1, 17-33 chemical characterization and behavior of respirable fractions of indoor dusts collected near a landf ill facil ity rheo b. lamorena-lim* university of the philippines diliman colleen marciel f. rosales university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract t h e s t u d y a i m s t o d e t e r m i n e t h e i n o r g a n i c a n d o r g a n i c p h a s e s i n airborne par ticulate matter (pm) collected near a landf ill facility. the establishments within the vicinity of the landf ill considered in the study w e r e a j u n k s h o p , a s c h o o l , a n d a m o n e y c h a n g e r s h o p . f r o m t h e elemental analysis using inductively-coupled plasma mass spectrometry ( i c p m s ) , l e a d a n d c a d m i u m w e r e d i s c o v e r e d t o b e m o r e a b u n d a n t i n the total suspended particulate (tsp) fraction, whereas copper was more abundant in the smaller pm 2.5 . manganese, arsenic, strontium, cadmium, a n d l e a d w e r e m o r e a b u n d a n t i n t h e p m 10 f r a c t i o n t h a n i n p m 2.5 . t h e results of the chemical characterization were compiled and evaluated in a geochemical modelling code (phreeqc) to determine the potential speciation of these chemical constituents. solution complexes of as, pb, cd and phthalates, and metal species, such as h 2 aso3, cd 2 oh3+, pb(oh)3-, were predicted to form by the phreeqc simulation runs once the endmember components interact with water. the results contribute to the b a c k g r o u n d i n f o r m a t i o n o n t h e p o t e n t i a l i m p a c t s f r o m e x p o s u r e t o airborne pm at workplaces around landf ill facilities. moreover, the data g a t h e r e d p r o v i d e a b a s e l i n e f o r t h e c h e m i c a l c h a r a c t e r i z a t i o n a n d behavior of chemical constituents of pm possibly present in this specif ic type of environment. keyword s: airborne par ticulate matter, landf ill facility, indoor air quality, solution complexes, phreeqc speciation, respirable fractions chemical characterization and behavior of respirable fractions 18 introduction air pollution is almost always considered ambient. however, humans are estimated to spend 70-90% of their time indoors, thus they tend to be more exposed to pollutants present indoors (raunemaa et al. 1989). the world health organization reports that 2 million premature deaths per year are attributed to exposure to indoor air pollution. indoor air pollution also accounts for up to 4.0% of the burden of disease for low-income countries (world health organization [date unknown]). pollutants may come from combustion sources indoors (e.g. cook stoves), building materials and furnishings, household consumer products, appliances, or outdoor sources (lamuth 2008). in the philippines, a number of studies regarding ambient air quality have been performed in the past (bautista et al. 2014; pabroa et al. 2011). however, local studies on the characterization of indoor air aerosols and their airborne behavior are still very limited. published data regarding the exposure of filipino workers to indoor particulate matter (pm), particularly in the inhalable and respirable mass fractions, are sparse. one of the most abundant pollutants indoors is pm. pm is a mixture of small particles and liquid droplets. the small particles in pm may be made up of several components, such as acids, organic chemicals, dust particles, soil, or metals (us epa [date unknown]). pm is generally classif ied based on its size, sampling methodology, and cut-off point of the sampler used for its collection. for example, pm with a diameter of 10 μm is called pm 10 , whereas pm with a diameter of 2.5 μm is called pm 2.5 . pm, especially the smaller size fractions, is a health concern because of its capability to enter the respiratory system and cause health issues, such as chronic obstructive pulmonary diseases and reduced lung function for children and adults (w.h.o. regional off ice for europe 2003). furthermore, toxic metals in pm have been proven to cause acute inflammatory responses. water-soluble metals, on the other hand, have been observed to affect the cardiopulmonary system (costa and dreher 1997). hence, it is of importance to be able to characterize and identify the toxic metals present in pm. some sites of concern with respect to indoor air pollution are plastic/electronic recycling/dismantling centers near landf ill facilities. these facilities collect and sort recyclable wastes as prof itable businesses that flourish as an unorganized sector. due to unsupervised and uncontrolled practices, improper handling techniques may be prevalent in such facilities. these improper handling techniques can bring about serious health risks not only to workers in such facilities but also to local residents. most of these facilities situated in metro manila lack proper r.b. lamorena-lim and c.m.f. rosales 19 equipment to control or diminish pollutant emissions. hence, the characterization of pollutants in such complex microenvironments is a crucial f irst step in preparing a comprehensive methodology for def ining acceptable indoor air quality in occupational settings in metro manila. the flourishing community near payatas dumpsite, which is the main controlled and organized waste disposal site of quezon city, is an appropriate place to initiate the study. the study aims to determine the inorganic and organic phases in airborne pm collected near a landf ill facility. the results of the chemical characterization were compiled and evaluated in a geochemical modelling code to determine the potential speciation of these chemical constituents. several establishments within the vicinity of the landf ill site were considered in the study. the indoor areas of a junk shop, a school, and money changer shop were selected as the sampling sites. the study will provide data on the prof iles of total suspended particulates, elemental species, and organic phases in airborne pm. materials and methods prel iminary stud ies to assess the presence of toxic metals in indoor air, preliminary analyses of ordinary house dust using inductively coupled plasma mass spectrometry (icp-ms) and scanning electron microscopy/energy dispersive x-ray fluorescence (sem/edx) w e r e p e r f o r m e d . s a m p l e s w e r e c o l l e c t e d f r o m t a b l e t o p s u r f a c e s a n d a i r conditioning systems in different residential and commercial establishments, digested using hot acid (hno 3 /h 2 o 2 system), and then subjected to icp-ms (calepa 2007). the remaining undigested samples were subjected to sem/edx. indoor airborne pm sampl ing for the landf ill samples, pm was collected on teflon f ilters using the airmetrics minivol sampler with a flow rate of 5 l/min. three fractions were separately collected (pm 2.5 , pm 10 , and tsp) due to the limitation of the sampler (i.e. only one fraction can be collected at a time). at least three trials per fraction and location were performed. no tsp fraction was collected from the money changer shop. the chemical characterization and behavior of respirable fractions 20 sampling was conducted for only three hours due to personnel security, from 10 am to 1 pm, and 1 pm to 4 pm. sampling was performed in three sites, namely a junk shop, a school, and a money changer shop. the sites were chosen based on their 1) distances from the landf ill and 2) sampler and personnel security. field and instrument f ilter blanks were also obtained prior to each sampling. the f ilters were weighed using a sartorius me5-f microbalance with a minimum resolution of 0.001 mg. mass concentrations are reported in μg/m3, which was calculated using equation (1). after weighing, the f ilters were halved for inorganic and organic phase characterization. conc in μg/m3 = (1) where volume of air sampled, m3 = flow rate of minivol length of sampling conversion factor = 0.9m 3 sample analysis half of the teflon f ilter was set aside for icp-ms analysis, whereas the other half was further halved for other characterization techniques. one-fourth of the teflon f ilter was subjected to 1h-nmr characterization. the other one-fourth was stored for a morphological analysis using sem-edx (data will not be shown). microwaveassisted acid digestion using ethos closed-vessel microwave digester and 18.5% hno 3 (4% f inal acid concentration after diluting to 50 ml and upon introduction to icp-ms), following the method of kulkarni et al. (2007), was employed as the sample preparation technique prior to elemental analysis using icp-ms. recovery studies by means of spiking 0.1, 0.2, 0.25, 0.5, 0.75, and 1 ml of 10 ppm multielemental standard into different teflon f ilters and subjecting them to the same digestion method were performed as a check for the extraction method. seven solutions containing 1 ppb of the multi-elemental standard were prepared and then subjected to icp-ms analysis for the determination of the method detection limit. a 500-mhz agilent nmr spectrometer was used for the 1h-nmr analysis. pm samples were dissolved in deuterated chloroform (cdcl 3 ) to obtain the spectra for the nonpolar compounds, while deuterated water (d 2 o) was used to obtain the spectra for the polar compounds. however, due to the limitation of the nmr spectrometer, no quantif ication was performed for the organic components. 5 l min 3 hr 60 min hr 0.001 m3 1 l weight of sample in μg volume of air sampled in filter, m3 r.b. lamorena-lim and c.m.f. rosales 21 geochemical modell ing to model the possible speciation and reactions between water and toxic metals in pm, as well as the influence of organic phases on metal speciation, phreeqc (phredox-equilibrium, written in c language), a program developed by the united states geological survey (usgs), was used. phreeqc is designed to perform a wide variety of aqueous geochemical equations and is based on equilibrium chemistry of aqueous solutions interacting with minerals, gases, solid solutions, exchangers and sorption surfaces, and one-dimensional transport (parkhurst and appelo 2013). model simulations performed in the study considered the formation of solution complexes and precipitation of solid phases. measured parameters, such as ph, temperature, % humidity, and relative stoichiometry, were set as variables in different sets of reaction runs. all speciation calculations used the dissolved elemental concentrations (i.e. chemical analyses previously performed). metals were indicated in their mineral phases and solution species form. iso-phthalate containing aromatic and short aliphatic constituents was used as the representative for organic phases. the llnl.dat phreeqc thermodynamic database supplied with the software was used for the analyses. the f irst data block for the input statement describes a solution of water containing the elements mn, as, sr, cd, and pb. the temperature was also specif ied. mole fractions of the elements, which were based on the total metal concentration measured with icp-ms, were also included. the second data block describes the removal of water from the solution, in order to achieve the humidity conditions as measured. the last data block in the run simulates the reaction of an organic component, isophthalate, to the solution under the specif ied humidity condition. the concentration of isophthalate added was based on literature values since no quantif ication of the organic species was performed. results and discussion prel iminary stud ies preliminary results are shown in figure 1. among the elements found in the samples, the crustal/naturally found elements are na, mg, al, ca (figure 1a), and ba (figure 1b). meanwhile, the non-crustal elements, which are likely associated with anthropogenic activities, are cr, mn, ni, cu, zn, sr, and pb (figure 1b). ca was chemical characterization and behavior of respirable fractions 22 the most abundant among the crustal elements. for the non-crustal elements, zn concentration was the highest for all locations, followed by mn, cu, ba, and pb. ni and cr were present at low concentrations. figure 1. total concentration of metals present in airborne pm. (a) metals associated with crustal fraction and (b) trace metals. t01, t02, t04 – residential-singledetached houses; t08, t09 – laboratory; t10 – restaurant; t11, t21– residentialcondominium; t17 – dental clinic; t19 – residential. r.b. lamorena-lim and c.m.f. rosales 23 no source apportionment was performed, thus the exact sources of the elements could be determined. however, high concentrations of zn, in addition to the natural amounts present in the crust, are associated with the wear and tear of vulcanized vehicle tires and the corrosion of galvanic automobile parts (wahab et al. 2012). such could also the source of the zn for the sampling sites since they were located within close range of some roads (commonwealth road and katipunan road). similarly, cu and mn may originate from both natural and anthropogenic sources. soil contains natural amounts of mn and cu, but anthropogenic sources, such as industrial processes (battery and electronics manufacturing, steel productions, welding and motor vehicle exhaust), that may be present nearby are also possible sources of these elements (datta et al. 2012; midander, 2006). in addition, cu may also result from brake wear. pb may be part of the road dust as an element adsorbed and continuously resuspended in the upper layers of the soil. anthropogenic sources of pb, such as leaded gasoline, lead-based paint, and lead-arsenate pesticides, have long been eliminated from the industry; however, the presence of pb in the environment may still be attributed to these sources due to the persistence of pb. other anthropogenic sources of pb may also include the manufacture of lead-containing products, combustion of coal and oil, and waste incineration (atsdr 2014). ni, on the other hand, may also come from both natural and anthropogenic sources. the natural sources of ni, which include the weathering of rocks and soils from volcanic eruptions, are unlikely reservoirs in the urban setting. anthropogenic sources of ni, such as industrial processes (combustion, incineration, metallurgical operations, ni production, chemicals and catalyst manufacturing) may be more probable in the urban setting. in addition, mobile sources, such as engine wear and tear and impurities, and engine oil and fuel additives, may also be possible sources of ni in an urban setting (galbreath et al. 2003). possible sources of cr may include stainless steel, paint pigments, and wood preservatives (galarpe and parilla 2014). moreover, cr is involved in many industrial processes, such as chromium plating and cement manufacture. it is also used as an additive to anti-corrosion coatings on vehicles (mcsheehy 2008). this preliminary study serves to measure the concentrations of metals in areas far from a landf ill facility. preliminary measurements show that metal concentration is signif icant even in locations distal from the landf ill area. based on these f indings, analysis of the airborne pm at the landf ill area is deemed important and necessary. chemical characterization and behavior of respirable fractions 24 description of the main sampl ing sites payatas dumpsite, a 22-hectare open pit, is located in quezon city, philippines. it is the largest and oldest solid waste dumpsite in metro manila. many material recovery facilities (junk shops) are also operational along the payatas road. a large community of families thrives near the dumpsite, thus facilities, such as schools, small stores, and small commercial establishments, are present within the vicinity of the site. the three chosen sampling sites are as follows: a junk shop, a school (around 1.6 km from the landf ill), and a money changer shop (near commonwealth road, a main highway in quezon city). these sampling sites are situated along a busy road (i.e. which garbage trucks use to enter the landf ill facility). furnishing and f itting conditions, such as air-condition settings, room and ceiling-height sizes, and room content, were variable. variation in the organic and inorganic components of pm table 1 summarizes the results obtained from the sampling, while figure 2 shows a comparison of the weight distribution per pm fraction per location. from the data, it can be observed that the tsp fraction in the junk shop has the highest mass concentration, while the lowest mean for the pm 2.5 fraction was obtained from the elementary school. moreover, the values obtained from the junk shop display a generally higher trend than the elementary school and money changer, possibly because it is not a fully enclosed (indoor) location. thus, pm from outside sources (e.g. road dust, fugitive dusts) may have also contributed to the higher mass concentration. the mass concentration is directly proportional to size, because larger particles are heavier and have higher masses. numerical values obtained from the study, as shown in table 1, are larger compared to literature values for indoor air in a classroom in munich during winter time (19.8 μg/m3 pm 2.5 and 91.5 μg/m 3 pm 10 ) (fromme et al. 2007). moreover, the measured parameters are higher than those measured in a typical residential environment in athens, where the mean 24-hour indoor pm 10 concentration is 35 μg/m3 during the warm period and 31.8 μg/m 3 during the cold period (diapouli et al. 2011). furthermore, since no guideline values have been set for indoor air pm, 2005 ambient air guidelines set by the world health organization were used to compare the values. the guideline values are 10 μg/m3 (annual mean) and 25 μg/m3 (24-hour mean) for pm 2.5 , and 20 μg/m3 (annual mean) and 50 μg/m 3 (24-hour mean) for pm 10 . it should be of immediate concern that the values obtained from the landf ill r.b. lamorena-lim and c.m.f. rosales 25 site are well above the guideline values. however, it is recommended that a longer sampling time be carried out, in order to be able to fully quantify the pm mass concentration for the selected sampling locations. table 1. three-hour averages for pm 2.5 and pm 10 in the three locations, namely the junk shop, elementary school, and the money changer shop. n mean min max pm2.5 (μg/m 3, 3-hour average, junk shop) 3 70.19 57.78 76.67 pm2.5 (μg/m 3 , 3-hour average, elem. school) 4 45.00 33.89 56.11 pm2.5 (μg/m 3, 3-hour average, money changer) 4 77.08 60.00 102.22 pm10 (μg/m 3, 3-hour average, junk shop) 3 129.44 114.44 143.33 pm10 (μg/m 3, 3-hour average, elem. school) 4 126.25 98.89 176.11 pm10 (μg/m 3, 3-hour average, money changer) 4 105.69 78.33 142.22 tsp (μg/m3, 3-hour average, junk shop) 4 209.72 187.78 247.22 tsp (μg/m3, 3-hour average, elem. school) 4 110.00 33.89 176.11 pm 2.5 (ju nk sh op ) pm 2.5 (e lem . s ch oo l) pm 2.5 (m on ey ch an ge r) pm 10 (j un ks ho p) pm 10 (e lem . s ch oo l) ts p ( ju nk sh op ) ts p ( el em . s ch oo l) figure 2. mass concentration of pm fraction per location. pm 10 (m on ey ch an ge r) chemical characterization and behavior of respirable fractions 26 (3)mdl = t x s where t = student’s t-value at 99% conf idence level (t 7 =3.14) s = standard deviation of the seven replicates % recovery = (c s -c)/s x 100 where c s = measured concentration of spiked sample c = measured concentration of unspiked sample (background concentration) s = theoretical concentration of the spiked sample (2) method val idation (closed-vessel microwave-assisted acid d igestion) to assess the accuracy of the method, recovery studies were performed. percent recovery was calculated according to the following formula (us epa 1994): upon the analysis of the spiked samples using icp-ms, eight out of the 13 elements (manganese, cobalt, nickel, copper, arsenic, strontium, cadmium, and lead) analyzed have acceptable % recovery values (i.e. between 70-130%) for all the concentrations of the spike. to determine the method detection limit (mdl), equation (3) was used, and mdl values calculated for the elements were observed to have acceptable recoveries (us epa 1994). inorganic and organic characterization of airborne pm elemental analysis of the pm fractions allowed the determination of the dominant toxic metals on each fraction. lead and cadmium were more abundant on the tsp fraction, whereas copper was more abundant on the smaller pm 2.5 . on the other hand, manganese, arsenic, strontium, cadmium, and lead were also present on pm 10 and were more abundant on this fraction than on pm 2.5 . figures 3-5 show the distribution of toxic metals for different size fractions of pm. 1h-nmr analysis indicates the presence of organic constituents, such as hydrocarbons, from airborne pm. spectra with similar peaks at 0.8-1.6 ppm and 7.0-7.5 ppm were obtained for all fractions and locations, signifying similar organic compositions for r.b. lamorena-lim and c.m.f. rosales 27 figure 3. metal concentrations in tsp fractions collected from sampling sites. (a) junk shop and (b) elementary school. no tsp fractions were collected from the money changer shop. a b airborne pm present near landf ill areas. peaks found at 0.8-1.6 ppm indicate the presence of aliphatic groups for all the samples. an additional peak at around 4.7 ppm for the tsp fraction of the junk shop, which was also present in the pm 10 fraction from the elementary school, may indicate the presence of some alcohols. aromatic groups were also detected, as represented by peaks at 7.2-7.3 ppm (overlapping with the solvent peak, therefore not conclusive) (figures 6a and 6b). chemical characterization and behavior of respirable fractions 28 a b c figure 4. metal concentrations in pm 10 fractions collected from sampling sites. (a) junk shop, (b) elementary school, and (c) money changer shop. r.b. lamorena-lim and c.m.f. rosales 29 figure 5. metal concentrations in pm 2.5 fractions collected from sampling sites. (a) junk shop, (b) elementary school, and (c) money changer shop. a b c chemical characterization and behavior of respirable fractions 30 potential speciation of inorganic and organic constituents in pm simulation results show that solution complexes of h 2 aso3-, haso 3 2-, aso 3 3-, h 3 aso 3 , cd(oh) 2 , cdoh +, cd(oh)3-, cd2+, cd 2 oh3+, cd(oh) 4 2-, pb(oh) 4 2-, pb(oh) 3-, pb 3 (oh) 4 2+, pb(oh) 2 , sr2+, and sroh+ could potentially form when elemental components interact with the surrounding water vapor. moreover, phases, such as cd(oh) 2 , cd (s) , litharge, massicot, monteponite, pb(oh) 2 , pb 2 o(oh) 2 , pb (s) , and pbo:0.3h 2 o, could possibly be precipitated after equilibration. signif icant formation of isophthalate and protonated pb-isophthalate were also identif ied by the simulation runs. on the other hand, cd-isophthalate species were found at lower concentrations. a b figure 6. small peaks detected for aliphatic and aromatic groups in all pm fractions collected. (a) tsp and (b) pm 2.5 fractions. both fractions were collected from the junk shop. r.b. lamorena-lim and c.m.f. rosales 31 conclusions and recommendations the results contribute to the discussion on local issues with indoor air quality in the workplace by providing background information on the potential impacts from exposure to airborne pm around landf ill facilities. the gathered background information provides a baseline data on the chemical characterization and behavior of chemical constituents of pm possibly present in this specif ic type of environment. this places importance on occupational health in workplaces where filipino workers (especially women and children) are exposed to environmental agents on respiratory health. signif icant levels of metals were observed from all tsp and respirable fractions. presence of such metals may be attributed to activities involving the production of such metals. however, since no topographical or meteorological parameters were taken in to account, attribution to anthropogenic sources cannot be made with certainty. organic phases with aromatic and aliphatic characters were also detected in all airborne fractions. an additional peak for the tsp fraction may also be correlated to the presence of some alcohols. the data gathered from this study will be used for further modeling studies on the speciation of the chemical constituents of pm. the results will also be utilized in developing methods to quantitatively determine the species formed during the simulations. likewise, the information collected from the characterization of the respirable pm regarding indoor or workplace air quality is important in the assessment of the exposure of the workers in such occupational settings and the determination of its influence on nearby regions. acknowledgments the author acknowledges the off ice of the chancellor of the university of the philippines diliman, through the off ice of the vice chancellor for research and development, for funding support through the outright research grants. references [ atsdr] agency for toxic substances and diseases registry [internet]. 2014. lead: p o t e n t i a l f o r h u m a n x p o s u r e . 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[date unknown]. us: epa; [cited 2013 september 06]. available from: http: //www.epa.gov/airquality/par ticlepollution/. us epa . 1994. method 200.8: determination of trace elements in waters and wastes b y i n d u c t i v e l y co u p l e d p l a s m a m a s s s p e c t r o m e t r y. c i n c i n n a t i : u s e pa : o f f i ce o f research and development . wahab naa, darus fm, isa n, sumari sm, hanaf i nfmh. 2012. heavy metal concentration of settled surface dust in residential building. malaysian journal of analytical sciences. 16(1):18–23. [who] world health organization regional off ice for europe. 2003. health aspects of air pollution with par ticulate matter, ozone and nitrogen dioxide. germany: report on a who working group. world health organization q & as: air quality and health [internet]. [date unknown]. [cited 2015 may 19]. available from: http://www.who.int/entity/phe/air_quality_q&a.pdf? u a = 1 . _____________ rheo b. lamorena-lim is an associate professor of institute of chemistry, university of the philippines-diliman. she earned her ph.d. degree in environmental engineering in korea advanced institute of science and technology (kaist) in daejeon, south korea in 2011. her scope of research interest includes carbon dioxide sequestration in geological formations, atmospheric and indoor air chemistry, soil chemistry, soil and waste remediation. colleen marciel f. rosales is an ms chemistry student at the institute of chemistry, university of the philippines diliman. she f inished her undergraduate degree in chemistry, and was an instructor in the same institute for four years. her research interests include indoor air quality, air quality management, and environmental analytical chemistry. sd-sample article l.v. aragones and others 1 science diliman (july-december 2013) 25:2, 1-33 issn 0115-7809 print / issn 2012-0818 online dolphin watching in the southern tañon strait protected seascape, phil ippines: issues and challenges lemnuel v. aragones* institute of environmental science and meteorology natural sciences research institute university of the philippines diliman liana talaue-mcmanus rosentiel school of marine and atmospheric science university of miami mary anne a. roque-borigas institute of environmental science and meteorology natural sciences research institute university of the philippines diliman apple kristine s. amor institute of environmental science and meteorology natural sciences research institute university of the philippines diliman edward o.keith† nova southeastern university, usa abstract dolphin watching is a growing economic activity in the southern tañon strait protected seascape (tsps), the philippines, an area that is also h e a v i l y e x p l o i t e d b y f i s h e r i e s . i n o r d e r t o ex a m i n e t h e i s s u e s a n d challenges in this growing industry, we monitored relevant information regarding cetacean watching, conducted focus group discussions (fgds) and educational seminar-workshops for various local stakeholders from 2004 to 2006, and followed these up from 2008-2012. from 9 may to 16 a u g u s t 2 0 0 4 , w e c o n d u c t e d s t r u c t u r e d i n t e r v i e w s t o d e t e r m i n e t h e perceptions of cetacean-watching tourists (cwts) and assess the level of l o c a l k n ow l ed g e of f i s h e r s a n d n o n f i s h e r s ( n f s ) r eg a r d i n g m a r i n e mammals and environmental management in this area. ninety f ive (95) cwts, 100 local f ishers, and 64 nfs were interviewed. sixty seven percent _______________ *corresponding author dolphin watching in the southern tañon strait protected seascape 2 ( n = 6 4 ) o f t h e c w ts b e l i e v e d t h a t t h e o v e r a l l q u a l i t y o f t o u r s w a s i m p r e s s i v e p r i m a r i l y b e c a u s e t h e y w e r e a b l e t o w a t c h , a t r e a s o n a b l e c o s t s , l a r g e g r o u p s o f d o l p h i n s i n c l o s e p r o x i m i t y a n d i n a n a l m o s t pristine environment . the majority of cwts (~91%) felt that there is a n eed to d eve l o p a ‘ s p ec i a l m a n a g e m e n t p l a n ’ ( s m p ) fo r t h e s o u t h e r n tsps focusing on cetaceans and their habitats. the increasing number o f d o l p h i n w a t c h i n g b o a t s , h e a v y e x p l o i t a t i o n o f f i s h i n g g r o u n d , m i s p e r c e p t i o n o f l o c a l f i s h e r s t h a t ce t a ce a n s a r e co m p e t i t o r s w i t h f i s h e r i e s , a n d l a c k o f a s m p o r a m a n a g e m e n t p l a n p e r s e fo r t s p s w a r r a n t e d t h e f a c i l i t a t i o n o f a p a r t i c i p a t o r y m a n a g e m e n t p r o c e s s t o protect the cetaceans and their habitats. this study has shown that even with only preliminary results, survey interviews of key stakeholders in c o m b i n a t i o n w i t h f g d s a n d s e m i n a r w o r k s h o p c o u l d b e c r i t i c a l i n facilitating a participatory management process. in the case of the tsps, t h i s p a r t i c i p a to r y a p p r o a c h l ed to t h e f o r m a t i o n o f t h e ta ñ o n s t r a i t association of dolphin and whale watching operators, inc. (tasadowwi), and eventual development of cetacean watching protocols for the area. all of these highlight the importance of following a participatory process, e m p ow e r i n g s t a ke h o l d e r s , a n d m o n i to r i n g r e l ev a n t i n fo r m a t i o n ( e . g . , numbers of cetacean watching tourists, cetacean watching boats and its impacts, f isheries, dolphin behaviour and abundance) to ensure the longterm sustainability of dolphin watching and f isheries in southern tsps a r e a . keywords: d o l p h i n w a t c h i n g , c e t a c e a n w a t c h i n g , d o l p h i n s , w h a l e s , p e r c e p t i o n , p a r t i c i p a t o r y m a n a g e m e n t p r o c e s s , f i s h e r i e s , tañon strait , philippines introduction the philippine archipelago is noted for the diversity of its marine organisms, particularly its coral reefs. however, its resident marine mammals, particularly cetaceans, are still an undervalued resource in the country. to date, there are 29 (28 marine and 1 estuarine) aquatic mammal species conf irmed to inhabit philippine territorial waters (aragones 2008, 2013; aragones and others 2010). this list includes 27 cetaceans, the dugong (dugong dugon), a sirenian, and the asian smallclawed otter (amblonyx cinereus). “cetacean watching” is used in this paper as a collective term to refer to both whale watching and dolphin watching, since a growing number of people use these terms interchangeably. globally, cetacean watching is a multi-billion dollar (usd) ecotourism industry (goodwin 1996; hoyt 2002; corkeron 2004). in the central l.v. aragones and others 3 visayas, it is a growing economic activity (abrenica and calumpong 2002). there are three areas in the philippines where cetacean watching is currently offered: tañon strait, bohol sea (abrenica and calumpong 2002), and waters off puerto princesa, palawan. cetacean watching in the bohol sea is seasonal (primarily during april to july) and limited to pamilacan and balicasag islands and adjacent coastal towns (i.e. , panglao and baclayon). tañon strait, specif ically in the southern section, is available year-round, but understandably contingent on weather conditions (aragones and keith 2004) (figure 1). in 1995, cetacean watching in the tañon strait protected seascape (tsps) was initiated by the government of bais city, negros oriental. cetacean watching boats usually embark from bais city, located about 45 km north of dumaguete city, the provincial capital of negros oriental (figure 1). to date, there have been very limited studies in the philippines that examined the perception of cetacean-watching tourists (cwts) (rosales 2003) or the level of local knowledge possessed by f ishers and non-f ishers (nfs) regarding marine mammals, f isheries and their management, particularly for a protected seascape established for cetaceans. understanding the different stakeholders’ perceptions and knowledge is critical to the development of both conservation and management plans for this protected seascape. crucial issues must be addressed, such as the growing perception among locals that dolphins and whales are a nuisance because they compete with f ishers for f ish and other marine resources (see read 2005, for review of depredation by dolphins). moreover, direct interactions between marine mammals and f isheries have been reported as one of the major threats to these animals worldwide (read 2005). also, dolphin and whale watching activities have been shown to have negative impacts on the animals’ behaviors (lusseau 2006; arcangeli and crosti 2009; roque 2011). furthermore, a clear set of implementable protocols for cetacean watching in the host areas has been wanting for some time now. for the study site, the southern section of tsps, the basic principles of stakeholder analysis, i.e. , the process of identifying important individuals or groups that may be affected by any proposed activities (after grimble and chan 1995), were applied to recognize and enlist support from the key stakeholders. structured interviews using questionnaires, and consensus building through focus group discussions and seminar-workshops, were conducted as part of a larger, ongoing study on the ecology of cetaceans in the southern section of tsps. this paper presents the results of these interviews, as well as the challenges and issues in the area, and efforts toward a participatory management process that may serve as the basis for the development of policies that would improve and regulate cetacean watching tourism. dolphin watching in the southern tañon strait protected seascape 4 figure 1. map showing location of tañon strait, project site (southern tañon strait) and other relevant details mentioned in the text. inset shows relative location of the north bais bay (n), south bais bay (s), ‘sandbar’, capiñahan port (cap p), and cannibol port (can p). l.v. aragones and others 5 materials and methods study site tañon strait is predominantly a narrow but deep channel located between the islands of negros and cebu (figure 1). the strait is about 200 km long and connects the visayan sea to the bohol sea and is bordered by about 40 municipalities and cities (figure 1). based on f isheries data presented by green and others (2004), tañon strait can be considered as one of the top 10 major f ishing grounds in the philippines. the southern section of tañon strait has been classif ied as very heavily exploited f ishing grounds with >70 f ishers per km (green and others 2003). the high productivity of the strait makes it a favorable cetacean habitat. fourteen of the 29 marine mammals found in the philippines have been recorded in this area (aragones 2008, 2013; aragones and others unpublished data) (see appendix d for species listing). also, tañon strait has the highest density of dwarf sperm whales (kogia sima) among the surveyed waters in the philippines (dolar and others 2006). the diversity and abundance of cetaceans are the main reasons why tañon strait was declared as a protected seascape through a philippine presidential order issued in 1998. interview surveys specif ic sets of written interview questionnaires (aragones and others 1997) were used to assess: (1) the perception or views of cetacean watchers in the southern tsps, and (2) the level of knowledge of local f ishers and (3) nfs regarding marine mammals, fisheries and per tinent environmental management. interviews with cetacean watching tourists (cwts) were conducted from 9 may to 16 august 2004, in bais city, negros oriental. interviews with the local f ishers and nfs were conducted in bais city, negros oriental and in ginatilan, malabujoc, and alegria, all of which are par t of the island province of cebu. interviews with local f ishers and nfs were conducted from 9 may 9 to 15 june 2004, in bais city while those in cebu were done on 20 may 2004. the local cetacean watching tour guides assisted in administering all of these surveys. all interviewed tourists took only licensed and authorized tour boats (i.e. , motorized boat or ‘banca’ = mba) embarking from the capiñahan wharf, bais city (southern bais bay, figure 1). two tour boats were operated by the bais city government (mba dolphin i & mba dolphin ii), while a private group operated the third boat (mba dolly). at least one tour guide was aboard each cetacean watching boat. the total number of individuals per group or total number of family members was recorded to determine the average total dolphin watching in the southern tañon strait protected seascape 6 number of cwts per boat. only the head of a family or group leader for each group of tourists was asked to answer a questionnaire. interviews and fgds with local f ishers and nfs were administered using the local language (cebuano). there were two sets of interview questionnaires. the f irst was for the cwts and included questions that evaluated the respondents’ dolphin watching experience. this used a four-point rating system (where 1 is not aware, 2 is poor, 3 is fair and 4 is good) patterned after the likert scale (likert 1932) to assess the cwts’ dolphin watching experience and their perception of the condition of local marine environment, among others. the second questionnaire was for local f ishers and nfs; it focused on these two sectors’ level of knowledge regarding f isheries and cetaceans in the southern tañon strait area. all questionnaires included questions that asked demographic details to facilitate comparison across various demographic prof iles (for a copy of the questionnaires, please email the corresponding author). the questionnaire for nfs included similar topics covered for the f ishers. the difference is that while the questionnaire for f ishers had questions on f ishing, that for nfs asked about their overall awareness of the marine environment. for example, instead of asking nfs about their length of f ishing experience, their length of residence in the locality was asked in order to have some measure of their familiarity with the area. similarly, instead of asking types of f ish they catch, the kinds of f ish they usually buy or eat were asked to gauge the nfs’ knowledge of the local f isheries. as for their encounters with local marine mammals, nfs were asked if they observed them during their boat trips. the survey responses were encoded in separate spreadsheets according to group. if the respondent neglected to answer a particular question, a column for “no answer” was added to account for these in the data analysis. the results were presented as frequency distributions expressed as percentages of total responses, noting that the sample sizes varied across questions. in relevant cases, one-way anova was performed using spss (version 15.0, 2008) to test whether the total number of respondents varied signif icantly. dolphin watching in tañon strait we examined the dolphin watching operation by gathering relevant information including boat types used and their costs, as well as the number of boats that operated through time. to determine the cetacean watching tourists’ arrivals, both l.v. aragones and others 7 local and foreign, and the frequency of boat trips, data were collected from the 2007 to 2012 philippine coast guard manifesto of bais city. data on cetacean watching tourists’ arrivals prior to 2007 were unfortunately unavailable. the number of tourists were gathered and segregated into local and foreign. the number of boat trips and boat trips per cetacean watching operator were determined. environmental awareness and participatory management to enhance environmental awareness about cetaceans, several focus group discussions (fgds) and environmental education seminar-workshops were conducted after the interviews. identif ication of the important stakeholders was conducted following grimble and chan (1995). the f irst seminar-workshop on “protocols for dolphin and whale watching” was held in bais city on 21 august 2004 as requested by local government off icials, since boat operators and crew indicated in the initial fgds the absence of any cetacean watching protocols. a series of seminar-workshops were conducted the following year (1 and 14-15 june, and 12 october 2005) in bais city for a larger audience that included local off icials, f ishers, and groups interested in cetacean watching (including resort owners) surrounding southern tsps. the workshop held on 12 october 2005 discussed the development of management planning skills for surrounding cities and municipalities. also, a seminar-workshop on ‘tour guiding’ for current and aspiring tour guides for dolphin watching was held in 17-18 august 2006. results eight fgds (n=8) and eight (n=8) seminars or workshops were conducted in the area to enhance the understanding of the major stakeholders on the need to protect and conserve not only the habitats of the dolphins and whales but also the entire system. a total of 95 heads of families or groups representing the cwts in bais city accomplished the interview questionnaires. the average family or group size was 13, and ranged from 2 to 20 people (e.g. , parents plus their children, an extended family or group of friends). a total of 79 f ishers and 36 nfs from bais city, and 21 f ishers and 28 nfs from cebu were interviewed during the study period (table 1). the sample sizes were uneven as it was diff icult to gather local f ishers, particularly in cebu, because they were either out f ishing or resting; for nfs, it was observed that there was not much interest among them to accomplish the questionnaires. dolphin watching in the southern tañon strait protected seascape 8 socio-demographic profile of local fishers, non-fishers, and cetacean watchers the age class distribution of the cwts, local f ishers and nfs respondents is summarized in table 1. among cwts, 36% were 18-30 yrs old, with 23% each from the 31-40 and 41-50 yrs old groups. the oldest age class (>60 years old) had the least representatives in all of the three groups of interviewees. fishers interviewed in bais city were either subsistence f ishers or those employed mainly as crew for the purse seine f ishing fleets based in capiñahan, bais city (figure 2). in cebu, 15 f ishers (71%) in malabujoc and six f ishers (29%) in ginatilan were interviewed. as for the nfs, 23 were from malabujoc consisting of 21 residents (75%) and two (7%) from the nearby town of alegria, and f ive (18%) from ginatilan. for simplicity, these groups will be referred to here as f ishers and nfs from cebu. all but one of the f ishers interviewed were male; the lone female f isher was from bais city (figure 2). most of the f ishers had at least some primary (elementary) and secondary (high school) education. most of those from bais city (82%, n=65) and cebu (90%, n=19) claimed that f ishing was their primary occupation (table 2). a considerable proportion of the f ishers interviewed in bais city (41%, n=32) and cebu (52%, n=11) have been engaged in f ishing for more than 20 yrs (figure 2). there was an almost even distribution between female and male respondents among the nfs interviewed (figure 2). the nfs mainly had secondary to tertiary (college) education. their primary jobs varied (figure 2). some were employees, cetacean watching 36 23 23 16 2 tourists (34) (22) (22) (15) ( 2) (95) 24 24 20 23 9 bais city (19) (19) (16) (18) ( 7) (79) 19 38 19 24 cebu ( 4) ( 8) ( 4) ( 5) -(21) 56 22 8 8 6 bais city (29) ( 8) ( 3) ( 3) ( 2) (36) 21 21 21 26 11 cebu ( 6) ( 6) ( 6) ( 7) ( 3) (28) local fishers local non-fishers age bracket (yrs old) sector total (n)>6051-6041-5031-4018-30 table 1. percentage d istribution of respondents accord ing to age* *note: figures in parentheses represent raw frequencies l.v. aragones and others 9 f ig u re 2 . t h e so cio -d em o g rap h ic p ro file o f fish ers an d n o n -fish ers in terview ed fro m 9 m ay to 1 5 ju n e 1 5 2 0 0 4 sh o w in g h o m eto w n , g en d er, ed u catio n al attain m en t, p rim ary jo b , an d n u m b er o f years o f ex p erien ce o r resid en ce in th e area. t h o se fro m m alab u jo c an d a leg ria w e re lu m p ed to g e th e r, re p re se n tin g c eb u . s e e tex t fo r m o re d e tails. dolphin watching in the southern tañon strait protected seascape 10 while others were students (25% in bais city only) and vendors (29% in cebu only). a number of local medical doctors, nurses, priests and businessmen were interviewed, and they were accounted for in the category ‘other’. majority of the non-f ishers have been residing in their localities for more than 20 years (figure 2). most of the cwts came from the province of negros oriental (39%) (table 2), but the adjacent provinces of cebu (16%), negros occidental (11%) were also represented in the sample. the rest are divided among those coming from other countries (16%), the national capital region (8%), and other parts of the country (10%). most of the tourists interviewed were married (table 2), and 39% of them were single (39%). most of the cwts belonged to the highest income bracket (>500,000 philippine pesos/ php per annum) (table 2); however, 11% belonged to the lowest income bracket (<50,000 php per annum) and 14% of interviewees were either unwilling or unable to provide information about their income. in terms of the number of times the tourists have gone cetacean watching, most were f irst timers (75%) (table 2). the respondents’ main source of information on cetacean watching varied, although their friends or relatives were the most cited (50%) sources (table 2). most of the cwts believed that the overall condition of tañon strait was still good (50%) to fair (42%) (figure 2). similarly, most of them believed that there was good (44%) to fair (40%) levels of protection given to marine mammals in the strait (figure 3). however, their perceptions about tañon strait differed signif icantly from their perceptions about the overall condition of marine habitats in the philippines (t = 3.760, df = 3, p = 0.033). specif ically about half (~47%) of the respondents believed that the overall condition of marine habitats in the philippines was fair (figure 3), and only about 28% rated the overall condition of marine habitats to be good. in the same vein, almost half of the respondents (44%) believed that there was only a fair level of protection afforded to marine mammals in the whole of the philippines, and only 22% perceived that it was at a good level; 20% replied that they were ‘unaware’ of the overall condition of marine habitats in the country (figure 3). in terms of their cetacean watching experience, many of the cwts believed that the current level of dolphin-watching tours was good (58%) to fair (41%) (figure 3). also, almost all of the respondents (96%) were willing to do cetacean watching again in the near future (figure 4). nonetheless, the tourists believed that the tours could still be improved. a list of the major components of cetacean watching that the respondents enjoyed most and those that needed improvement is shown in table 3. the most cited enjoyable component was seeing the wild dolphins or toothed whales (28%), followed by swimming and sunbathing in the ‘sandbar’ (21%), l.v. aragones and others 11 ta bl e 2 . pe rc en ta ge d is tr ib ut io n of c et ac ea n w at ch er -r es po nd en ts a cc or d in g to s el ec te d de m og ra ph ic v ar ia bl es , n um be r of t im es t he y ha ve go ne c et ac ea n w at ch in g, a nd s ou rc es o f in fo rm at io n ab ou t ce ta ce an w at ch in g* dolphin watching in the southern tañon strait protected seascape 12 figure 4. the need for user fees, special management plan for tañon strait and the willingness to cetacean watch again in the near future according to cetacean watching tourists interviewed from may 11 to august 19, 2004 (n=95). figure 3. perception of cetacean watching tourists interviewed from may 11 to august 19, 2004 in the capiñahan wharf, bais city, negros oriental, philippines (n=95) regarding some relevant issues on marine mammals (marmams), marine ecosystem and dolphin watching. 0 1 0 2 0 30 40 50 60 70 8 0 90 10 0 ove rall condition of tañ on strait o verall con dition o f marine ha bitats in the philipp ines current level of prote ction af forde d marmams in ta ño n s trait curren t le ve l of prot ectio n a ffo rd ed ma rmams in whole p hilipp ines curren t le vel o f d olphin wat ching to urs p er c en ta g e r es p on d e n ts ( % ) go od fair po or unawa re 0 10 20 30 40 50 60 70 80 90 100 need f or "user fee s" nee d f or sp ecial mgt plan fo r tañon st rait? willin g to dolphin wat ch a gain in t he near future? p er ce n ta g e r es po n d e nt s (% ) (% ) y es no don 't know no answer l.v. aragones and others 13 table 3. list of the components of cetacean watching tours that the cetacean watchers enjoyed the most and those that they recommended for improvement and enjoying nature (18%). the provision of shower rooms and/or toilets on the wharf for cwts’ use was the most cited component (29%) that could improve this ecotourism activity. however, 37% of the cetacean watchers interviewed did not provide information about their cetacean watching experience. almost all of the cwts (91%) believed that there was a need to develop a ‘special management plan’ (smp) for tsps, whose primary purpose would be to protect the local cetaceans and their habitats (figure 4). eighty percent (80%) were willing to pay additional money or user fees for the protection and conservation of marine mammals in the area (figure 4). the respondents believed that the user fees should be managed or administered either by the local government unit (50%) or a special unit (40%). the amount visitors were willing to pay for user fees varied signif icantly (t = 4.398, df = 5, p = 0.007) ranging from <10 php (~0.20 usd) (31%) to >50 php (~1 usd) (25%). knowledge of local fishers and non-fishers about marine mammals and cetacean watching only 50% of the f ishers interviewed in bais city were familiar with marine mammals, i.e. able to describe these animals, in comparison to those in cebu where all of the interviewees (100%) showed good knowledge of marine mammals (figure 5). similarly, the nfs in cebu were more familiar with marine mammals (~80%) than their counterparts in bais city (~50%) (figure 5). in bais city, a majority (~60%) seeing wild whales/dolphins 55 28 shower rooms & toilets 40 29 swim/snorkelling and sunbathing 41 21 coordination with regards to 10 7 at ‘sandbar’ booking of boats seeing nature itself (apart from 36 18 more information regarding 10 7 dolphin) dolphins, whales, marine habitats and marine conservation in general courteous service by staff 17 8 protection of dolphins 7 6 fun and great experience 18 9 snorkelling gear (rental?) 6 4 safety f irst and well maintained 10 5 boats (e.g. more space) 4 3 boats other 8 4 other 10 7 no answer 15 7 no answer 51 37 total = 200 total = 138 component most enjoyed n % component most need ing n % improvement dolphin watching in the southern tañon strait protected seascape 14 of f ishers familiar with marine mammals claimed that the summer season is the best time to see cetaceans (figure 6). almost half of the f ishers from cebu believed they could sight cetaceans year-round (figure 6). however, 38% of these f ishers also suggested that summer is the best time to go cetacean watching. it should be noted that the entire negros island is more of an agricultural area as evidenced by its extensive sugarcane farms while cebu is famous for its f ishing industry as manifested in its extensive seafood products. around seven out of 10 f ishers from both bais city (68%) and cebu (72%) perceived that the relative abundance of cetaceans in their area has been increasing in the last f ive years (figure 7). on the other hand, a considerable proportion of the nfs in both areas believed that there have been no changes in the cetaceans’ relative abundance (figure 7). only 28% of the f ishers in bais city claimed some form of f ishery-cetacean interactions (e.g. dolphins eating f ish caught by f ishers) in comparison to 57% from cebu (figure 8). the f ishers who acknowledged f isherycetacean interactions (i.e. dolphins either eating their catch or dolphins being caught in their gear) were mainly gill netters, and hook and liners. the f ishers interviewed in bais city employed a variety of f ishing gears, with the biggest numbers using figure 5.familiarity with marine mammals of local fishers (bais city, n= 79; cebu, n=21) and non-fishers (bais city, n=36; cebu, n=28). 0 10 20 30 40 50 60 70 80 90 100 fishers-bais city fishers-cebu non-fishers bais city non-fishers ceb u p e rc e n ta g e r e s p o n d e n ts ( % ) (% ) yes no not sure l.v. aragones and others 15 figure 7. perception of local fishers (bais city, n=53; cebu, n=21) and non-fishers (bais city, n=17; cebu, n=22) regarding the trend in relative abundance of cetaceans in the area. figure 6. season when cetaceans were most frequently sighted by fishers (bais city, n=53; cebu, n=21) and non fishers (bais city, n=17; cebu n=22). see text for complete explanation. 0 10 20 30 40 50 60 70 80 90 100 am ihan (ne) habagat (sw ) sum mer whole year other p e rc e n ta g e r e s p o n d e n s t (% ) f ishers-bais city f ishers-cebu n on-fis hers bais city n on-fis hers cebu 0 10 20 30 40 50 60 70 80 90 100 amihan (ne) habagat (sw ) sum mer whole year other p e rc e n ta g e r e s p o n d e n s t (% ) fishers-bais city fishers-cebu n on-fishers bais city n on-fishers cebu dolphin watching in the southern tañon strait protected seascape 16 gill nets (39%) and beach/boat seine (22%) (table 4). the f ishers in cebu employed mostly gill nets (41%) and hook and line (45%). interestingly, all f ishers and nfs interviewed agreed that marine mammals should be protected and conserved. dolphin watching boats in southern tañon strait – types and costs the boats used for dolphin watching are basically modif ied (i.e. , with elevated floor to allow better viewing) pumpboats or canoes (locals call them ‘motorized banca’) with outriggers. initially, there were two big (~30 ft long) boats (mba dolphin i and dolly): each could accommodate as many as 20 people (plus crew) and charged 3,000 php (~58 usd) per trip. later, the smaller (~20 ft long) boat mba dolphin ii was added; it could accommodate as many as 15 people, and charged 2,500 php (~48 usd) per trip. these prices are not cheap by philippine standards (1 usd = 52 php in 2004). however, this price has remained nominally the same as when dolphin watching started in 1995. the most recent price increase occurred only during the summer of 2009. the big boats now charge 4,000 php (~80 usd at 1 usd =47 in 2009) per trip, while the smaller boats charge 3,500 php (~70 usd). cetacean watching tourist arrivals and boat trips from 2007 to 2012 during the period 2007 to 2012, the number of cetacean watching tourists peaked in 2007 and 2010, as shown in figure 8. in 2007, there were a total of 7,497 tourists; of these, 86% were local tourists and only 14% were foreigners. on its fig. 8. perception of local fishers (bais city, n=53; cebu, n=21) and non-fishers (bais city, n=17; cebu, n=22) regarding cetacean-fishery interactions in the area. 0 10 20 30 40 50 60 70 80 90 100 f ishers -bais cit y fis hers-c ebu non-f ishers b ais c ity non-f ishe rs c ebu p e rc e n ta g e r e s p o n d e n ts ( % ) ye s no not s ure l.v. aragones and others 17 table 4. percentage of fishers from bais city and cebu who reported cetacean-fishery interactions, by type of fishing gear used* purse seine 14 13 (14) (2) gill net 39 40 41 34 (38) (6) (12) (4) driftnet 1 7 7 (1) (1) (2) spear f ishing 7 7 8 (7) (2) (1) cage/f ish trap 3 13 (3) (2) fish corral 3 (3) beach/boat seine 22 (21) hook and line 11 27 45 58 (11) (4) (13) (7) % of total (n=98) fishing gear bais city cebu % acknowledged cetacean-fishery interactions (n=15) % of total (n=29) % acknowledged cetacean-fishery interactions (n=12) *note: (1) figures in parentheses represent raw frequencies; (2) multiple response: some f ishers used more than one type of f ishing gear figure 9. cetacean watching tourists’ arrivals in bais city from 2007 to 2012. dolphin watching in the southern tañon strait protected seascape 18 second peak in 2010, the number of tourists totaled 8,415. after these peak years, however, tourists arrivals decreased by 56% and 37% in 2008 and 2011, respectively. tourist turn-out in 2008 was the lowest during the period 2007 to 2012, with 3,293 visitors registered (figure 9). in each year, on the average, 90% of the visitors were locals while 10% were foreigners. not surprisingly, the number of boat trips per year during the period 2007 to 2012 showed similar trends as those for tourist arrivals (see figures 9 & 10). boat trips were most frequent in 2007 and 2010, with 613 and 742 trips, respectively. the number of boat trips declined by 61% from 2007 to 2008 and by 42% from 2010 to 2011. the lowest frequency of yearly boat trips for cetacean watching was in 2008, which also registered the lowest number of tourists. however, the number of cetacean watching boats operating increased from nine in 2007 to 14 in 2010 despite the relatively low tourist turnout and boat trips in 2008 (figure 10). in terms of the monthly boat trip data, boat trips were frequent during the months of march to july and peaked during the summer months of april and may, as shown in figure 11. in the latter part of the year, boat trips increased during the months of september and october during the inter-monsoon season. figure 10. cetacean watching boat trips from 2007 to 2012 (note: figures in parentheses represent the number of boats operating per year) l.v. aragones and others 19 figure11. monthly cetacean watching boat trips from 2007 to 2012 initiatives on environmental awareness and participatory management the main stakeholders that the study identif ied for the initiatives on environmental awareness and participatory management included individuals involved in cetacean watching operations (boat owners, boat captains, crew, tour guides and local officials from bais city and manjuyod), local f ishers (f ishing within the area) and local government off icials. the workshops, meant to serve as a forum to discuss regulation of cetacean watching and protection of cetaceans, were all held upon the request of local stakeholder groups. these workshops eventually led to the formation (in october 2005) of the tañon strait association for dolphin and whale watching incorporated (tasadowwi). a total of 38 members signed up to become the charter members for tasadowwi. they were mainly dolphin-watching tour boat operators and employees (both from negros and cebu), employees of bais city government (e.g. , bais city tourism off ice and mayor’s off ice), and other concerned locals. aff iliate organizations such as the bais city government (city tourism off ice), university of the philippines, university of miami, and nova southeastern university were also acknowledged as institutional members since personnel of these organizations were involved in the formation of tasadowwi or are active members. thus, the tasadowwi is composed dolphin watching in the southern tañon strait protected seascape 20 of local and non-local stakeholders who share similar interests in cetacean watching, with a common goal to protect cetaceans and to develop and promote sustainable ecotourism in tañon strait. discussion this study showed that information from monitoring the cetacean watching ecotourism and key stakeholders (cwts, f ishers and nfs) in tsps through interview surveys and fgds, even if just preliminary, are critical in facilitating participatory management and building consensus about cetacean conservation and habitat protection. the survey results were used as the bases for consequent activities including workshops, which resulted in this case to the establishment of the tasadowwi and development of cetacean watching protocols. building consensus is often challenging and our experience in negros and cebu gave us f irst-hand experience about this. fortunately, using scientif ic methods (e.g. interviews and monitoring) guided us in the facilitation of a participatory process towards consensus building. from the interviews, a preliminary picture of the perceptions of cwts and the level of local knowledge of f ishers and nfs, who are the key stakeholders in the tsps, has emerged. in general, younger people (< 30 years old) from negros oriental and those whose annual income was >500,000 php (~10,000 $us) were the primary clients of dolphin watching in the southern tañon strait. it should be noted that the average per capita income in the philippines in 2006 was only 1,175 usd (adb, 2007), thus majority of those who availed of cetacean watching were above average income earners. further, it is not surprising to know that the bulk of non-local clients were from the nearby provinces of cebu (16%) and negros occidental (11%). these two provinces plus the home province of negros oriental comprised 66% of the client base of the cetacean watching industry. most tourists from adjacent provinces had to travel for at least four hours to get to bais city. those from cebu have to cross the strait via ferry and then travel by land either from the ports in dumaguete or tampi (san jose), while those from negros occidental traveled solely by land (figure 1). furthermore, bais city is not easily accessible to those from metro manila as they have to fly to dumaguete city and then travel farther north to get to bais city. the annual average percentage (10%) of foreign tourists recorded by the study, although small, was surprising considering the minimal advertising campaigns on cetacean watching locally and nationally (table 2), and expectedly much less internationally. however, since its inception in 1995, dolphin watching in bais city has been featured occasionally in various national tv programs in the philippines . l.v. aragones and others 21 as for the local f ishers interviewed, they were predominantly male, belonging to a wide range of age brackets, had some secondary education, mostly experienced (>20 yrs) f ishers, and knowledgeable of cetaceans (table 1 & figure 2). meanwhile, the nfs interviewed represented both gender groups, had tertiary-level education, held various white-collared occupations, have been residents of their respective localities for a considerable time (>20 yrs), and had some awareness of local cetaceans. the major differences in the perceptions of f ishers and nfs regarding cetaceans (e.g. f ishers believed that cetaceans are increasing in the area while nfs perceived otherwise) may be explained by the disparity in their experiences with local marine resources. however, aragones and others (unpublished) show that the population has actually been slightly declining since 2007. that the f ishers and nfs agree on the need to conserve and protect these cetaceans is interesting, and may be attributed to the growing cetacean watching industry in the area and the threats this may pose to their f ishing grounds (further discussed below). cetacean watching in southern tañon strait dolphin/whale watching in southern tañon strait is conducted in a straightforward manner and appears to be quite fulf illing for its participants. it appears to be still in its infancy stage, as the local boat design is still used as opposed to modern boats (without outriggers). it is also evident that cetacean watching has yet to reach a stable and sustainable stage, based on the fluctuating number of tourist arrivals and boat trips from 2007 to 2012. it is interesting to note that the mandated discounts for locals (bais city residents) have made a difference in allowing the lower income residents to also go cetacean watching. in our study, all respondents who belonged to the lowest income bracket came from bais city). the bais city boats (mba dolphins i and ii) were the most preferred boats by the cwts. in fact, the smaller mba dolphin ii boat was the most popular choice of the tourist-respondents. cetacean watching is def initely becoming a major economic activity not only for bais city, but for the whole region. in 2006, dolphin watching in bais city generated a gross income of ~1.5 million php (a. serrano jr. , bais city tourism off icer, personal communication). in contrast, in 1999-2000, bais city’s whale watching gross earnings was only ~0.50 million php (abrenica and calumpong 2002). it is therefore not surprising that the adjacent localities are trying to emulate the bais city model for cetacean watching. in anticipation of the proliferation of cetacean watching, the local government has been cautioned that there should be studies examining the impacts of dolphin watching boats on dolphins, and setting maximum limits on the number of tourists and boat trips that could be permitted on a daily basis so that these do not interfere dolphin watching in the southern tañon strait protected seascape 22 with cetacean habits (i.e. , carrying capacity studies) before any additional licenses for dolphin watching boat operators are issued. in april 2005, another tour boat (mba alfer) was licensed to operate in bais city, making it the fourth boat that holds a license for offering cetacean watching tours in the area. this is the second boat of the owners operating mba dolly. according to bais city tourism records (2005) the number of tourists increased by 12% from 2003 to 2004. this trend, along with the current lack of regulation of dolphin watching boats, has resulted in an alarming increase in the number of dolphin watching boats in southern tañon strait. as of 2009, another 10 to 12 boats were operating in bais city through another por t – cannibol (northern bais bay, see figure 1 inset). in the adjacent island of cebu (from liloan, malabujoc to badian, figure 1), a total of f ive to 10 boats are operating seasonally – that is, during the summer season, the peak period for cetacean watching in the area. in 2009, several adjacent municipalities namely ginatilan, alegria, and aloguinsan, all from the cebu side, have shown interest in offering cetacean watching tours. this increase in the number of operators may lead to a decline in the quality of the cetacean watching experience of the tourists. as such, the growing interest in, and popularity of, cetacean watching warrants the need for more studies and monitoring of the area. the tourists’ impressive experience in southern tañon strait another f inding of our study that merits highlighting is the tourists’ perception that the quality of cetacean watching package offered by the three legally permitted and appropriately trained tour boats embarking from bais city ranged from good (58%) to fair (41%). this appreciation or show of approval was evident in the willingness of cwts to cetacean watch again (figure 4) as quite a few have already done (see table 2). what appears to be most appealing to the tourists is the opportunity to watch the marine mammals in an almost pristine environment. it is also a memorable experience to watch large groups of dolphins ranging from 100 to 300 individuals. further, it is almost rare to see a large area (~150 h) of almost white sand, still undeveloped. this place is popularly referred to as ‘sandbar’ or ‘white sand’ (figure 1) and is found between bais city and manjuyod. this area is most likely the savior of the dolphins from tourists. instead of overstaying with the dolphins, the tourists go sunbathing, swimming, and taking their lunch in this area. impl ications of results for the management process for protecting cetaceans there is consensus among conservationists that protecting areas and habitats critical to the life cycle of organisms is fundamental to sound species management (e.g. , shipley 2004). however, setting up a marine protected area for cetaceans is often l.v. aragones and others 23 problematic because of their extensive migration routes that greatly exceed what most resource managers would be willing to allocate, much less manage as an mpa (hoyt 2005). this is one of the challenges in the tsps area and the reason why we believe it is imperative to develop a participatory management plan for this body of water so as to not only protect the ecological integrity of the system but also sustain the local sustenance f isheries and cetacean watching in the long term. unfortunately, with the increasing number of boats, the logistics required to ensure maximum compliance with dolphin watching protocols would become increasingly limited. the interviews revealed that among the cwts, there was a clear consensus that the local cetaceans should be protected. this is an overwhelming show of support for conservation of these animals and their habitat. this was the opposite of the usual response that the local f ishers and nfs gave during the workshop discussions, who believed that cetaceans are pests to f ishing. this is obviously a misperception as dolar (1999) has established that the most dominant cetacean species in the area, spinner dolphins, predominantly eat small mesopelagic f ishes and squids, which f ishing gears can neither reach nor target. despite their misperceptions, however, the f ishers and nfs from both cebu and bais were unanimous on the need to protect and conserve cetaceans. as for the cwts, they perceived a need to develop a special management plan (smp) for tsps. as mentioned, a series of seminars and workshops on the ecology and conservation of cetaceans in tsps and on the proposed protocols or guidelines for cetacean watching were conducted. these seminarworkshops were borne out of the overwhelming requests from the locals, and were instrumental in organizing the cetacean watching operators, including selected local resort owners, to form the tasadowwi. the main mission of the tasadowwi is to provide and promote ecologically-friendly dolphin watching services, and to elevate the standards of this ecotourism activity. the consensus built by the locals through these seminars, workshops and organization (tasadowwi) are manifestations of the participatory management process. in contrast, the protected area management board (pamb) for the tañon strait protected seascape, which has been mandated to manage the strait (as per philippine republic act 7586, the national integrated protected areas system act of 1992) by developing a management plan (mp) involving its stakeholders, is perceived to have failed its mandate. the organization is perceived to be composed of dubious members, and is alleged to be nontransparent because it issues prescriptive memoranda without appropriate consultation with the concerned stakeholders. the respondent cwts were willing to pay additional money for user fees, on top of the tour fees, in order to augment the funding for the development and implementation of the smp for the tsps. however, the amount they are willing to pay as user fees was too small (<10 php) dolphin watching in the southern tañon strait protected seascape 24 (~0.20 usd). the body they identif ied to be most appropriate for administering the funds is either the local government unit or a special agency that will be established by and through the smp. recommendations for improving the cetacean watching experience as stated above, the cwts were generally very pleased with their cetacean watching experience. however, there are still areas for improvement to make the cetacean watching tours even more memorable and satisfying for cwts. as the respondentcwts have articulated, there should be shower rooms that tourists could use after swimming in the waters near the sandbar. steps should be taken to make the tour booking process more eff icient and the content of the tours more informative/ educational. both of these concerns are already being addressed by the cetacean watching ecotourism industry stakeholders. for instance, a workshop for tour guides was conducted in the summer of 2006, to provide the guides with information about the basic biology and ecology of the cetaceans found in the area and other related facts about the city of bais, the province of negros oriental, and tañon strait. in addition, the newly formed tasadowwi will spearhead the implementation of a centralized process for booking reservations, which is expected to minimize conflicting trip schedules. the current tour service is def initely very simple and very much wanting (table 3) in comparison to those offered in developed countries. however, these shortcomings are easily outweighed by the near-pristine condition of the environment and the memorable experience of being in close proximity to wild dolphins and sometimes toothed whales (table 3), the major draw for repeat watchers (table 2). the need to protect and manage the tañon strait protected seascape tañon strait is considered as one of the most productive waters in the visayan region (yambao and others 2001; green and others 2003, 2004). it is one of the most unique aquatic systems in the country as 14 of the 28 cetaceans recorded in the philippines have been sighted in the area through the years (aragones and others 2003; aragones and keith 2004; aragones 2008; aragones and others unpublished data). the area also features many natural assets such as coral reefs (e.g., piscador island, moalboal, and mantalip reef, a single barrier reef) and mangrove forests (300 hectares, talabong mangrove reserve in bais city, yambao and others 2001). the marine ecosystem of the whole philippines is primarily threatened by overexploitation, pollution and coastal development (gomez and others 1981; white and cruz-trinidad 1998; alcala 2001; christie and others 2003). l.v. aragones and others 25 this study has shown that in general the cwts believed that the overall condition of tañon strait is better than that of the marine ecosystem for the whole philippines (figure 3). however, some data show that there was a signif icant decline in the population of spinner dolphins in the southern tañon strait area in 2005-2006, most likely due to activities related to the oil exploration in 2004 (aragones and others unpublished data). as noted above, the spinner dolphin is the main species that is watched in this area (see appendix d). under the current set up provided in the philippine wildlife conservation and protection act of 2001 (philippine republic act 9147), the protection of marine mammals is a responsibility shared between the bureau of fisheries and aquatic resources (bfar) and the protected areas and wildlife bureau (pawb). the bfar, which is under the department of agriculture, takes care of cetaceans; the pawb, which is under the department of environment and natural resources, handles the dugong. as provided for in ra 9147, the f ines and penalties for killing, maltreating, harming, and illegal trading of wildlife, including marine mammals, will range from from 50,000 to 1m php. however, monitoring of the implementation of the act has been weak. for instance, neither the negros oriental nor the cebu portions of tañon strait area are regularly patrolled. most patrolling is limited to the respective municipal territorial waters (ranges from 3 to 14 km from shoreline in tañon strait). further, there has been a considerable incidence of strandings of cetaceans in this area: specif ically, 7 stranding incidences in 6 years; 4 of the 7 incidences involved spinner dolphins (aragones and others 2010). furthermore, with the signif icant increase in the number of dolphin watching boats and the absence of clear policies and regulatory measures for cetacean watching tour operators, we predict that the quality of dolphin watching tours will decline. for one, an increase in the number of visitors is bound to cause some damage to the currently still pristine environment of the tsps. moreover, with more boats, competition among operators will increase, and they would likely try to outdo one another, which in turn would lead to the harassment of dolphins (i.e. , analogous to malthusian f isheries). a recent study by roque (2011), which examined the impacts of cetacean watching boats on the behavior of spinner dolphins in the southern tañon strait area, showed signif icant negative effects of the former on the latter. simultaneous with the aims of developing an appropriate management plan for the cetacean watching ecotourism industry and of making this industry sustainable, one should bear in mind that other activities in the area such as f isheries and other forms of livelihood and ecotourism should be harmonized. all of these economic activities should be carried out at a sustainable level. to ensure this, participatory management process should be facilitated, involving local f ishers, residents, tourists dolphin watching in the southern tañon strait protected seascape 26 and investors, to guarantee equitable resource use (after thiele and others 2005). in general, it is perceived that tourism is benef icial to local and developing communities. however, thiele and others (2005), in their assessment of the integrated coastal management sustainability in the central visayas, found that it is not entirely true. their analysis revealed that there was an inverse relationship with the quality of life and the presence of tourism, and that there seemed to be a higher level of community tension between local f ishers and proponents of tourism. as noted earlier, the southern tsps area is a f ishing ground with very high f ishing efforts (green and others 2003). in addition, an increasing number of illegal f ishing has also been recorded in the tsps (green and others 2004). it is our hope that the tourists’ impressive cetacean watching experience in the tsps would be maintained, alongside the development of other similar sustainable ecotourism activities in this area so that the ecological integrity of the entire tsps would be valued and be sustained by the locals themselves. acknowledgments funding for the study was provided by the nova southeastern university president’s grant, seaworld and busch gardens conservation fund, natural sciences and research institute, and the university of the philippines research grant (through the off ice of the vice president for academic affairs). access to dolphin watching boats from 2004 to 2005 was provided by the city government of bais. we would like to thank the previous mayors of bais city hon. hector ‘tata’ villanueva and hon. karen villanueva, and the bais city tourism off ice, especially cerilo mantua, ricardo ‘tariric’ reynado, marissa diaz, carmen bermejo, lorelie barnido, harold infante, sharon arapoc, cindy amas, joana burgos, antonio serrano jr. and the rest of the boat crew of mba dolphins i and ii for their help, support and cooperation. special thanks also to the amor family in sibulan for their unwavering support, especially to doc avelex amor, and all the other volunteers who helped collect data. many thanks also to kathleen dudzinski and two anonymous reviewers for their suggestions for improving the manuscript. this paper is up-iesm contribution number 33 and is in memory of one of our co-authors, dr. edward o. keith of nsu, who passed away last sept 14, 2012. he will be missed as he had supported this project and admired the southern tsps since day one. references a b r e n i c a b , ca l u m p o n g h p. 2 0 0 2 . w h a l e w a t c h i n g i n b a i s c i t y, n e g r o s o r i e n t a l , philippines: an ecotourism enterprise. university of the philippines-visayas journal of natural sciences 7: 158-168. l.v. aragones and others 27 [adb] asian development bank. 2007. philippines: critical development constraints. publication stock 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marine protected areas for whales, dolphins and porpoises: a world handbook for cetacean habitat conservation. sterling, va: earthscan. likert r. 1932. a technique for the measurement of attitudes. archives of psychology 140: 1-55. lusseau d. 2006. the short-term behavioral reactions of bottlenose to interactions with boats in doubtful sound, new zealand. marine mammal science 22(4): 802-818. re a d a j . 2 0 0 5 . b yc a tc h a n d d e p r ed a t i o n . i n : rey n o l d s j e , pe r r i n w f , reev e s r r , m o n t g o m e r y s , r a g e n t j , e d i t o r s . m a r i n e m a m m a l r e s e a r c h : c o n s e r v a t i o n b e y o n d crisis. baltimore, md: johns hopkins university press. p 5-17. roque ma . 2011. the s ho r tte rm i mpact of cetacean watching on the behavior of spinner dolphins (stenella longirostris) in southern tañon strait [ms thesis]. quezon city: university of the philippines diliman. 120 p. ro s a l e s r p. 2 0 0 3 . a s u r vey to e s t i m a t e t h e r ec r e a t i o n a l v a l u e o f s e l ec ted m pa s : moalboal-cebu, siquijor and pamilacan island-bohol. cebu city, philippines: coastal conservation education foundation, inc. l.v. aragones and others 29 shipley jb, editor. 2004. aquatic protected areas as f isheries management tools: design, use, and evaluation of these fully protected areas. bethesda, md: american fisheries society. spss. 2008. spss for windows. release 15.0. chicago, il: spss inc. thiele mt, pollnac rb, christie p. 2005. relationships between coastal tourism and icm sustainability in the central visayas region of the philippines. ocean and coastal management 48: 378-392. yambao a, white at, ablong w, alcala m. 2001. coastal environmental prof ile of negros oriental, philippines. cebu city, philippines: coastal resource management project. appendix a. recommendations for the protection and conservation of the cetaceans and their habitats in the tañon strait protected seascape (tsps) based on the results of this study, the following are our recommendations: • the pamb for tsps should reorganize and invite to the discussion table the real concerned stakeholders (e.g. , mayors from negros and cebu bordering tañon strait, tasadowwi, associations of resort owners and f isherfolk organizations, local ngos, researchers working in the area). • all concerned stakeholders (as mentioned above) should map out through a participatory management process where the key activities (e.g., ecotourism, fishing) are conducted and where important habitats are found (e.g., cetacean habitats, coral reef and mangrove reserves) and establish multiple use zones and core critical areas in the tsps. • all concerned local government units (lgus) (e.g. , bais city, manjuyod, bindoy) should plan and develop facilities (e.g. , road access, ports, souvenir shops, comfort rooms, etc.) within the focal areas to ensure the quality and safety of dolphin watching tours, as well as improve and broaden entrepreneurial opportunities among the other sectors of the communities (e.g. , f ishers, resort owners). • all concerned lgus (e.g. bais city, manjuyod) should continue the training and certif ication of dolphin boat operators from boat captains to tour guides (e.g., cetacean watching protocols) and even make this mandatory to ensure the professionalization of the cetacean watching industry. these lgus should continue the environmental awareness through seminars and workshops (e.g., integrated coastal management, fisheries and marine mammal interactions). • all concerned lgus who operate dolphin watching boats (e.g. bais city, manjuyod, malabujoc) with the assistance of the tsps pamb should develop, institutionalize and implement appropriate regulations for the dolphin watching in the southern tañon strait protected seascape 30 boat operators (e.g., minimum boat sizes, boat captain license, life craft and f ishing gear license) through participatory management process. some of these trainings and patrolling could be funded by ‘conservation fees’. • the local and national governments should never allow a foreign company to conduct seismic surveys (2004) and exploratory drilling (2007) in this protected area without an appropriate environmental impact assessment (eia) and proper consultations with key stakeholders. appendix b. cetacean watching protocols in southern tañon strait 1. slow down when approaching cetaceans (dolphins and whales) 2. approach cetaceans or group of cetaceans from sides and never from front nor behind the group (see also appendix c). 3. the allowed number of boats per group size of dolphins are the following: a. if group is < 10 animals, only 1 boat b. if group is ~11-50 animals, max. 2 boats c. if group is > 50 animals, max. 3 boats d. when several boats are sharing groups, never crowd the dolphins 4. active approaches by boats (whether motorized or not) should follow minimum distance and should stop (“low gear”; do not idle engine) and wait when they reach such distance or zones. 5. give the dolphins and whales the choice to interact. if a group of animals chooses to interact, it will approach and often remain with the boat and maybe “bow ride”. 6. do not harass the dolphins and whales by chasing them or driving through their herd. if you are actively motoring to stay with the animals at a certain distance, this means that the animals are moving away and are choosing not to interact. 7. when leaving the group, make sure that there is enough distance (> 200 m) before speeding up or revving up the engine. 8. when watching dolphins and whales, adopt the following zones (see appendix c for an illustration of these zones): a. caution zone – the designated or predetermined distance and area designed to reduce possible adverse effects on animals while allowing reasonable viewing: i. 300 meters for whales ii. 150 meters for dolphins l.v. aragones and others 31 b. no vessel zone – 50 meters from the animals (measured from outer section of herds) c. calf exclusion zone – 100 meters away d. no approach zone – within 45 degrees of the animals’ direction e. no follow zone – within 45 degrees of the animals’ tail section 9. do not feed the dolphins and whales. 10. do not touch these animals. 11. do not swim with these animals. 12. hire only accredited dolphin and whale watching boats. 13. all commercial dolphin and whale watching boats should employ trained boat captain, crew and tour guides. appendix c. diagram of the cetacean watching protocol dolphin watching in the southern tañon strait protected seascape 32 _______________ lemnuel v. aragones, phd <lemaragones@gmail.com> is currently an associate professor at the institute of environmental science and meteorology (iesm) in the university of the philippines diliman. he has been conducting research and conservation programs on cetaceans and dugongs throughout the philippines since 1990, and had been particularly interested in the southern tañon strait area since 1997. liana talaue-mcmanus is currently the project manager for a global environment facility project entitled “transboundary waters assessment programme” with the un environment programme as main executing agency. she leads the socioeconomic assessment of large marine ecosystems and the impacts of sea level rise on coastal populations for the same project. append ix d. list of cetacean species recorded in tañon strait from 1994 to 2012; all data from aragones and others (unpubl ished data) except 1994 and 2003 (see reference below) l.v. aragones and others 33 mary anne a. roque-borigas is a former university research associate for nsri from 2008-2011 for the dolphin ecology and conservation in southern tañon strait project, and an assistant professorial lecturer at the de la salle university (taft avenue) from 2012 to 2013. she earned her master of science on environmental science from iesm (re impacts of dolphin watching on spinner dolphin behavior) in 2 0 1 2 . apple kristine s. amor is currently a university research associate for the iesm. she also worked on the dolphin ecology in southern tañon strait through an nsri funded project from 2011-2013. she is currently doing her graduate studies on environmental science at iesm. edward o keith† was an associate professor at the nova southeastern university, florida, usa. he was instrumental in getting the initial funding to do this research in 2004 to 2006. he loved studying the dolphins and whales off tañon strait as well as interacting with the local people at bais city, negros oriental. 9guidelines.pmd 96 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) 9guidelines.pmd 99 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) 8montano-high throughput-shortened.pmd a.s.n.s. ferrer et al. 87 science diliman (january-june 2017) 29:1, 87-92 high-throughput screening for quorum sensing-inhibitory compounds from selected phil ippine marine algae and surface-associated marine microorganisms for potential anti-biofilm/biofoul ing appl ications aira sacha nadine s. ferrer university of the philippines diliman aljon francis koji p. elegado university of the philippines diliman mel iton r. chiong iii university of the philippines diliman laude karina g. alcober university of the philippines diliman dang marviluz l. espita university of the philippines diliman marco nemesio e. montaño* university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online one of the main global problems that cause significant losses in mariculture, medical, and industrial f ields is biof ilm formation and biofouling (bixler and bhushan 2012). biof ilms, which are composed of thick layers of cells embedded in an intricate exopolysaccharide matrix, are often the preferred mode of growth for most bacteria. biof ilms manifest as the slime often found attached to surfaces in aquatic or marine environments, or even in medical polymers such as catheters and implants. biof ilm growth provides a strategic niche for planktonic bacteria to thrive in environments prone to mechanical and chemical disruptions. in the complex domain of the extracellular carbohydrate matrix of biof ilms, bacterial aggregates become much less susceptible to treatment with the most diverse chemical biocides and antibiotics than the planktonic cells (joint et al. 2007). it has been shown that bacteria and biof ilms are able to act together concertedly through the metabolism of compounds formed within the intact community of bacterial biof ilm cells. these compounds were then later associated with quorum sensing (qs) (rasmussen and high-throughput screening for quorum sensing-inhibitory compounds 88 givskov 2006). the formation of biof ilms, which lead to biofouling or the development of complex biological communities on surfaces, such as ship hulls and aquaculture substrates, results in massive material and economic loss (lehaitre et al. 2008). in different ecological environments, nature has given advantage to organisms that are able to diminish or eradicate detrimental biofouling. quorum sensing is def ined as a community genetic regulation mechanism that controls microbiological functions of medical, agricultural, and industrial importance in response to population density (zhang and dong 2004). concomitant to an increase in bacterial population, small, freely diffusible signal molecules excreted by the organisms will accumulate. a critical threshold concentration is necessary to initiate a response in the whole population and to activate target genes essential in the synchronization of gene expression and functional coordination among bacteria (swift et al. 2001; zhang and dong 2004). studies show that some human and plant pathogens, such as pseudomonas aeruginosa and erwinia carotovora, regulate virulence through quorum signaling. quorum sensing has been suggested as an ideal target for treatment of both gram-negative and gram-positive bacterial infections often associated with the formation of biof ilms (kim et al. 2007). interception of this communication pathway thus implies a potential for qs inhibition to prevent diseases and adverse environmental problems, such as biof ilm growth and biofouling. qs-inhibitory molecules that were initially identif ied include triclosan, furanone, 3-oxo-c12-(2aminocyclohexanone), furanylhydrazides and macrolides, 2-o-glycerolalpha-d-galactopyranoside (floridoside), betoncine, isethionic acid, and several other molecules (zhang and dong 2004). the red alga delisea pulchra was shown to produce a class of halogenated furanones that accelerate the turnover of the n-acyl homoserine lactone (ahl)-responsive regulatory protein of the luxr family and inhibit bacterial quorum sensing and biof ilm formation (manef ield et al. 2002). on the other hand, the green alga ulva lactuca was demonstrated to rely on the epiphytic bacterium pseudoalteromonas tunicata to inhibit ahl-dependent transcriptional control by means of synthesizing pigmented substances (egan et al. 2002). there are numerous studies worldwide reporting the bioactivity of marine-sourced organisms, and these provide impetus to utilize the rich biodiversity of the relatively untapped seaweed resources of the philippines. this work utilized a high-throughput screening method for quorum sensing-inhibitory molecules from selected philippine marine algae and associated marine microorganisms. this will subsequently aid in the systematic fast identif ication, extraction, and upscale a.s.n.s. ferrer et al. 89 production of the bioactive molecules for applications in the prevention of biof ilm formation and biofouling, especially in mariculture systems. seaweed samples were obtained from 36 sites around luzon, visayas, and mindanao. out of 86 seaweed fragments collected from the respective sampling sites, six yielded positive results for qsi. of the 179 microbial isolates that were assayed, nine exhibited qs inhibitory activity. a total of 51 crude methanol extracts from seaweed were obtained. a total of 25 out of 51 crude seaweed extracts tested positive for qs inhibitory activity (table 1). solvent fractions for the crude methanol extracts of hydropuntia edulis, halymenia durvillei, chaetomorpha crassa, and halimeda macroloba were also tested using agar well diffusion assay (table 2). the putative qs inhibitor(s) in h. edulis possesses non-polar to partially non-polar characteristics, similar to that of h . d u r v i l l e i . comparison of the size of the pigment inhibition in h. edulis shows more pronounced qsi activity in the hexane partition compared to the ethyl acetate partition. the putative qs inhibitors in both c. crassa and h. macroloba are partially polar. seaweed site collection period halymenia durvillei bolinao, pangasinan october 2015 chaetomorpha crassa balaoan, la union june 2014 gracilaria sp. buguey, cagayan june 2014 halimeda opuntia santa ana, cagayan june 2014 tricleocarpa fragilis (=galaxaura oblongata) santa ana, cagayan june 2014 padina sp. santa ana, cagayan june 2014 jania sagittata (=cheilosporum sagittatum) santa ana, cagayan june 2014 halimeda macroloba gonzaga, cagayan june 2014 halimeda opuntia gonzaga, cagayan june 2014 hormohysa cuneiformis gonzaga, cagayan june 2014 kappaphycus cottonii calatagan, batangas april 2015 hydropuntia edulis calatagan, batangas october 2015 mastophora rosea lapu-lapu, cebu september 2014 ulva reticulata lapu-lapu, cebu september 2014 turbinaria ornata moalboal, cebu september 2014 amphiroa foliacea moalboal, cebu september 2014 chondrophycus cartilagineus (=laurencia cartilaginea) oslob, cebu september 2014 padina sp. oslob, cebu september 2014 padina sp. panglao, bohol september 2014 halimeda macroloba panglao, bohol september 2014 galaxaura divaricata (=galaxaura fasciculata) panglao, bohol september 2014 halimeda macroloba loon, bohol september 2014 mastophora rosea loon, bohol september 2014 ulva reticulata jagna, bohol september 2014 chondrophycus tronoi (=laurencia tronoi) sarangani island, davao occidental february 2016 table 1. seaweed extract positive for qsi high-throughput screening for quorum sensing-inhibitory compounds 90 the method described by chaudhari et al. (2014) and choo et al. (2006) quantif ies changes in violacein production and cell density according to the addition of seaweed and microbial extracts. the results obtained using this method provide more def initive data for qs inhibitory activity. inhibition of purple pigmentation with constant cell density indicated qsi. on the other hand, a decrease in optical density with diminished pigmentation is attributed to cell death, which often implies antibacterial activity and not qsi. the crude extract of h. edulis has a lower absorbance reading for violacein compared to the negative control (methanol). there is a 75.6% decrease in the violacein absorbance in the crude h. edulis extract compared to the negative control. the cell density remained relatively constant, with a decrease of only 14.5%, similar to the results obtained using the positive control cinnamaldehyde. the decrease in qsregulated violacein production without manifestations of bacterial death conf irms the qs-inhibitory activity of the crude h. ed ulis extract. a tukey post-hoc test conf irmed that , while the difference in violacein between methanol and crude h. edulis extract is signif icant (p= 5.801e-09), the difference in cell density is not (p=.011). the in-situ method provides a rapid and systematic means of screening for qs inhibition exhibited by seaweeds and associated marine microorganisms in the f ield. the agar well diffusion assay, although quick and convenient, has several limiting factors in terms of miscibility, volume, and molecule size of the possible qs inhibitors. on the other hand, the broth assay provides more quantitative information on the relative absorbance range of potential bioactive compounds. this further facilitates the isolation and characterization process, and validates the results obtained in the agar well diffusion assay. overall, the three assays described provide high throughput methods to maximize the screening of available qs inhibitory compounds from marine seaweeds and associated microorganisms. chaetomorpha crassa + + hydropuntia edulis + + + halimeda macroloba + + halymenia durvillei + + + seaweed species crude extract partition hexane ethyl acetate aqueous table 2. qsi activity of seaweed partitions a.s.n.s. ferrer et al. 91 supplementary material e x p e r i m e n t a l d e t a i l s r e l a t i n g t o t h i s p a p e r a r e a v a i l a b l e o n l i n e : h t t p : / / www.journals.upd.edu.ph/index.php/sciencediliman/article/view/5626/5046 acknowledgments this research was funded by the philippine department of agriculture – bureau of agricultural research (da-bar). the authors would like to acknowledge the staff of the seaweed chemistry laboratory for their assistance, the seaweed taxonomy and ecology laboratory headed by dr. edna ganzon-fortes for identifying the seaweed specimens, and dr. t ilman harder of the university of new south wales for generously providing the indicator strain used in this study. references b i x l e r g d a n d b h u s h a n b . 2 0 1 2 . b i o f o u l i n g : le s s o n s f r o m n a t u r e . p h i l o s o p h i c a l transactions of the royal society. 370:2381-2417. chaudhari v, gosai h, raval s, kothari v. 2014. effect of cer tain natural products and organic solvents on quorum sensing in chromobacterium violaceum. asian pacif ic journal tropical biomedicine. 4:s890-s897. choo jh, rukayadi y, hwang jk. 2006. inhibition of bacterial quorum sensing by vanilla extract . letters in applied microbiology. 42:637–641. egan s, james s, kjelleberg s. 2002. identif ication and characterization of a putative regulator controlling the expression of fouling inhibitors in pseudoalteromonas tunicate. applied & environmental microbiology. 68:372-378. joint i, tait k, wheeler g. 2007. cross-kingdom signalling: exploitation of bacterial quorum sensing molecules by the green seaweed ulva. philosophical transactions of the royal society of london b: biological sciences. 362:1223-1233. k i m j , k i m y, s e o y, pa r k s . 2 0 0 7. q u o r u m s e n s i n g i n h i b i t o r s f r o m t h e r e d a l g a ahnfeltiopsis flabelliformis.biotechnology and bioprocess engineering. 12:308-311. le h a i t r e m , d e l a u n ey l, co m p e r e c . b i ofo u l i n g a n d u n d e r w a te r m e a s u r e m e n t s . i n : babin m, roesler cs, cullen jj, editors. real-time coastal observing systems for marine ecosystem dynamics and harmful algal blooms: theory, instrumentation and modelling. oceanographic methodology series. paris: unesco publishing; 2008. p. 463-493. manef ield m, rasmussen tb, henzter m, andersen jb, steinberg p, kjelleberg s, givskov high-throughput screening for quorum sensing-inhibitory compounds 92 mclean rj, pierson ls, fuqua c. 2004. a simple screening protocol for the identif ication of quorum signal antagonists. journal of microbiological methods. 58:351–360. rasmussen tb, bjarnsholt t, skindersoe me, hentzer m, kristoffersen p, kote m, nielsen j, eberl l, givskov m. 2005. screening for quorum-sensing inhibitors (qsi) by use of a novel genetic system, the qsi selector. journal of bacteriology. 187(5):1799–1814. rasmussen tb, givskov m. 2006. quorum sensing inhibitors: a bargain of effects. microbiology. 152:895-904. swift s, downie ja , whithead n, barnard aml, salmond gpc, williams p. 2001. quorum sensing population density dependent determination of bacterial physiology. advances in microbial physiology. 45:199270. z h a n g l h a n d d o n g y h . 2 0 0 4 . q u o r u m s e n s i n g a n d s i g n a l i n t e r f e r e n c e : d i v e r s e implications. molecular microbiology. 53:1 563-1571. _____________ aira sacha ferrer <aira.sacha@gmail.com> obtained her undergraduate degree from the institute of chemistry, university of the philippines diliman and is currently a research associate, as well as a graduate student, at the institute of environmental science and meteorology, university of the philippines diliman. her work now centers on marine and aquatic pollution studies and health risk assessment. aljon francis koji elegado is a graduate student in the marine science institute, university of philippines. his research interests include marine microbiology, organic geochemistry and metagenomics. mel iton r. chiong iii is a graduate student of materials science and engineering in the college of science, university of the philippines diliman. he received his bachelor of science in chemistry degree from the institute of chemistry, university of the philippines diliman in 2015. he is currently pursuing his m.s. in materials science and engineering degree under the structure and dynamics laboratory, national institute of physics, university of the philippines diliman. his research i n te r e s t s i n c l u d e s o l i d s t a te c h e m i s t r y, b i o m a te r i a l s , n a n o m a te r i a l s , a n d computational chemistry. marco nemesio e. montaño <coke.montano2@gmail.com> is a retired professor at the marine science institute, university of the philippines diliman. he has devoted his career working on seaweeds since 1977 and is known for his extensive work on philippine marine algal chemistry research. he obtained his ph.d. in biological chemistry from griff ith university in australia where he worked on isolation and characterization of secondary metabolites from marine organisms. 12journal policy.pmd 85 journal pol icy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the off ice of the v ice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 86 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research area concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “notes to contributors” stipulates that “all ar ticles must have a high degree of scholarship,” that “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a cer tif ication from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right… .” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as def ined above and; b.) is specif ic and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the bases for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specif ic advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the 87 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientif ic misconduct policy. in attendance were: d r. co r a zo n d. v i l l a r e a l , r d u o d i r ec to r, p r e s i d i n g ; d r h e n r y j . ra m o s , p m rg o director and professor, nip; atty. vy va v ictoria aguirre, ovcrd legal consultant; e d i to r s i n c h i e f d r. m a r i co r s o r i a n o (science dil iman) a n d d r. m a r i a m a n g a h a s (s o c i a l s c i e n c e d i l i m a n ) . m s . n a n i e d o m i n g o a n d m s . d e r c y m a r a r a c , e d i t o r i a l assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. 88 references council of science editors. “ white paper on promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ec to r s o n s e p te m b e r 3 , 2 0 0 6 .” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ ajp/submit.html inside front cover-24-2.pmd editorial board editor-in-chief marco nemesio e. montaño, phd associate editors ma. patricia v. azanza, phd food science & nutrition jose maria p. balmaceda, phd mathematics zubaida u. basiao, phd biology carlos primo c. david, phd earth sciences joel joseph s. marciano, jr., phd engineering giovanni a. tapang, phd physics irene m. villaseñor, phd chemistry managing editor violeda a. umali, phd editorial assistant dercylis g. mararac science diliman (issn 0115-7809) is published bi-annually by the university of the philippines diliman through the office of the vicechancellor for research and development (ovcrd). address all communications to the editor-in-chief, science diliman, research dissemination and utilization office, office of the vice-chancellor for research and development, lower ground floor, phivolcs bldg., c. p. garcia ave., university of the philippines, diliman, quezon city 1101 philippines. subscription rates: p650.00/year (two issues), inclusive of postage us$50.00/year (two issues), inclusive of postage tel. no: (632) 981-85-00 loc. 4048 (632) 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph rduo.ovcrd2012@gmail.com website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences editorial advisors rigoberto c. advincula, phd department of chemistry university of houston radvincula@uh.edu alfonso m. albano, phd department of physics bryn mawr college, bryn mawr, pennsylvania aalbano@brynmawr.edu kenneth buckel, phd food science and technology group school of chemical sciences and engineering the university of new south wales, sydney, australia k.buckle@unsw.edu.au jose b. cruz, phd department of electrical and computer engineering ohio state university cruz@ece.osu.edu. flor crisanta f. galvez, phd quality assurance & technical manager kerry ingredients and flavours (americas region) 7989-82nd st., delta, vc v4g 1l7, canada ffgalvez1@yahoo.com victor c. gavino, phd department of nutrition university of montreal, canada victor.gavino@umontreal.ca kelvin s. rodolfo, phd department of earth and environmental sciences university of illinois, chicago, illinois krodolfo@uic.edu rudolf a. roemer, phd centre for scientific computing and department of physics university of warwick r.roemer@warwick.ac.uk luis g. sison, phd electrical and electronics engineering institute university of the philippines diliman luis.sison@up.edu.ph raul k. suarez, phd department of ecology, evolution and marine biology university of california, sta. barbara suarez@lifesci.ucsb.edu the hippopus porcellanus in tubbataha reefs national park. note the fringing tentacles on the inhalant siphon, top photo and the shell that is globose in shape and semicircular in outline with smooth and regularly shaped dorsal margin, bottom photo (see dolorosa, this issue). photo courtesy of r.g. dolorosa. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and re-publication in any other journal or compilation is likewise prohibited except as provided in the copyright agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. cover photo sd-sample article 1 from the editor on the aspect of scientif ic productivity, it is delightful to know that up diliman is the leading contributor to the thompson reuters’ web of science among the autonomous campuses of the university of the philippines, during the period of 2008 to 2013 (www.webofknowledge.com). the data illustrate how the up diliman campus has improved in terms of research. we also would like to congratulate humanities diliman for being listed in the scopus coverage. with the mutualistic synergy between the parties involved, we hope that science diliman would also be included in the said database soon, as well as in the web of science. considering the number of faculty and students in the colleges that offer science and technology graduate courses, a greater number of scientif ic papers should be expected to be published in both local and international categories. nevertheless, the diliman campus’ feat in scientific productivity leadership among the up campuses is something to be distinguished. we proudly present this issue of science diliman, which has been reformatted in sync with other current journals of the ovcrd-up diliman. to celebrate the university’s continued endeavor for scientif ic productivity, we highlight articles traversing both life and physical sciences. one article features the use of advanced instrumentation to quantitatively measure levels of lead and cadmium in commercial beverages and condiments. another article discusses the fabrication of porous silicon for use in photonic applications. the third article reports on a survey and taxonomy of an ecologically and economically important brown seaweed. the last article updates readers on the status and mariculture of abalone f ishery in pangasinan. this set of articles emphasizes the impact of research in our university on macro to micro systems. we commend the authors and reviewers of the articles for their invaluable contributions to this journal. marco nemesio e. montaño, phd editor-in-chief issn 0115-7809 print/issn 2012-0818 online 12journal policy.pmd 109 journal pol icy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the off ice of the v ice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 110 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research area concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “notes to contributors” stipulates that “all ar ticles must have a high degree of scholarship,” that “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a cer tif ication from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right… .” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as def ined above and; b.) is specif ic and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the bases for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specif ic advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the 111 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientif ic misconduct policy. in attendance were: d r. co r a zo n d. v i l l a r e a l , r d u o d i r ec to r, p r e s i d i n g ; d r h e n r y j . ra m o s , p m rg o director and professor, nip; atty. vy va v ictoria aguirre, ovcrd legal consultant; e d i to r s i n c h i e f d r. m a r i co r s o r i a n o (science dil iman) a n d d r. m a r i a m a n g a h a s (s o c i a l s c i e n c e d i l i m a n ) . m s . n a n i e d o m i n g o a n d m s . d e r c y m a r a r a c , e d i t o r i a l assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. 112 references council of science editors. “ white paper on promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ec to r s o n s e p te m b e r 3 , 2 0 0 6 .” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ ajp/submit.html sdinside front cover-july-dec.2017.pmd july-december 2017 • vol. 29 no. 2 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief irene m. villaseñor, ph.d. university of the philippines associate editors jose maria p. balmaceda, ph.d. university of the philippines louis angelo m. danao, ph.d. university of the philippines carlos primo c. david, ph.d. university of the philippines christian n. della, ph.d. university of glasgow singapore alonzo a. gabriel, ph.d. university of the philippines arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines dennis i. merino, ph.d. southeastern louisiana university jonas p. quilang, ph.d. university of the philippines 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science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) vargas museum, jonathan anticamara lab group & mary jocelyn l. tarnate call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 01_device will mangrove reforestation provide net benefits 21 will mangrove reforestation provide net benefits: a case in sibunag, guimaras1 cheryl joy j. fernandez2, rodelio f. subade3 and paul erwen t. parreño4 1paper presented n the 8th national symposium in marine science held in palawan state university, puerto princesa city on october 20-22, 2005. 2instructor in economics, university of the philippines in the visayas division of social sciences. 3associate professor in economics, university of the philippines in the visayas division of social sciences 4research assistant, environmental economics program for southeast asia (eepsea) date received: march 2, 2006; date accepted: july 7, 2006 abstract science diliman (july-december 2005) 17:2, 21-38 *corresponding author in response to the threats in mangrove resources such as massive fishpond conversion, industrialization, and increased human settlements in coastal areas, the province of guimaras answered these threats by widespread mangrove reforestation projects in its coastal communities. these projects were found out to be beneficial, as depicted on large gap on the mangroves overall benefits and the costs of implementation of the mangrove reforestation project. results of the study show that the present total benefit of mangrove per hectare with sustainable harvesting in the first year is lesser than the costs. however after the first year, the net benefits are positive. however, in compliance with republic act 7161 (r.a. 7161) that banned the cutting/using of all mangrove species, cost-benefit analysis of mangrove reforestation without harvesting was also computed. the net benefits exceed the costs from the start of the year up to the 20th year. both the scenarios include the mean wtp equivalent to php 142.75, which is the amount people are willing to give for the conservation of mangroves. the net present values (net benefits) of mangrove reforestation were found positive for both scenarios: with sustainable harvesting and without harvesting. key words: mangroves, mangrove reforestation, total economic value, contingent valuation method, willingness to pay, cost-benefit analysis introduction mangroves is a community of intertidal plants including all species of trees, shrubs, vines and herbs found on coast, swamps, or border of swamps (melana, et al., 1998). in the past, mangrove areas were regarded as wastelands that should be reclaimed for better economic purposes. through the years, however, science has revealed that mangrove ecosystem is not a wasteland but rather an area with high natural productivity in terms of plant growth and all associated organisms. the diversity of the mangrove ecosystem can be seen through the abundance of species of flora and fauna. about a quarter of the 18 million hectares of mangroves are found in southeast asia. the philippines with its 18,000 kilometer-shoreline, has a mangrove area of about 500,000 hectares in at the early 1990s but has shrunk to 117,700 in 1993. in guimaras island, the total number of mangrove cover has declined to 395.6 hectares in year 1990s. there is no record of the province's previous number of hectares but a map of the bureau of coast and geodetic survey (bcgs) in 1956 noted extensive mangrove areas in the southern region of the province (babaran and ingles, 1997). fernandez, subade & parreño 22 there has been a continuing decline of mangrove areas not only in the world but also in the philippines. even in the province of guimaras, substantial decrease of mangrove cover is noted. main reasons for this decline are the following: clearing of mangrove areas for fishpond and other aquaculture purposes, industrialization, and increase of population. people go for their short-term benefits by exploiting mangroves i.e. cutting for timber use, and not for the trees' long term benefits. in response to the threat to mangroves, conservation must be considered. one way to conserve and preserve the mangrove ecosystem is through reforestation. in planting mangrove seedlings/propagules, it could restore the ecosystem, gaining benefits from an increase in fish catch to coastal protection. the main problem was people do not realize or are not aware that mangroves can provide huge net benefits. considering the efforts and costs involved, sustaining mangrove reforestation is not an easy task. the coastal community as project implementers can just weaken their commitment considering that benefits from mangrove reforestation will be reaped after several years yet. this paper examined the costs and benefits of mangrove reforestation projects in three coastal barangays in sibunag, guimaras, namely, bubog, sabang, and sebaste. cost-benefit analysis (cba) of mangroves resources was computed in two scenarios: with sustainable harvesting and without harvesting. costs and benefits of mangrove reforestation the mangrove ecosystem is an open-access resource. according to field (1996), open access resource is a resource or facility that is open to uncontrolled access by individuals who wish to use the resource. it is important, therefore to know the values of these resources. the total economic value (tev) of the natural resource composes of the use and non-use values (figure 1). white and trinidad (1998) defined use values as one that measures the consumptive value (direct use values) of tangible natural resources as well as nonconsumptive (indirect use values) ecological and recreational uses of natural resources. use value can be classified as direct use value ("goods") and indirect use value ("services"). the former can be outputs or services that can be consumed directly while the latter can be functional benefits enjoyed directly. on the nonuse value side, there are three classifications: (i) option, (ii) bequest and (iii) existence or preservation values. the first one refers to the future direct and indirect use of the natural resource. the second one pertains to how much the present generation values the use and nonfigure 1. total economic value of natural resources source: white and trinidad (1998) t o t a l e c o n o m i c v a l u e u s e v a l u e n o n u s e v a lu e d ir e c t u se v a l u e (g o o d s) i n d ire c t u s e v a l u e (g o o d s) o u tp u ts/ se r v ic e s th a t c a n b e c o n s u m e d d ir e c tl y f u n c tio n a l b e n e fits e n jo y e d in d ire c tl y o p tio n b e q u e s t e x is te n c e o r v a l u e v a l u e p r e s e rv a tio n v a l u e f u tu r e d ir e c t a n d in d ir e c t u se v a l u e o f l iv in g u s e a n d n o n u se v a l u e s to o ff sp r in g v a l u e fr o m k n o w l e d g e o f c o n tin u e d e x is te n c e o f p re s e rv a tio n will mangrove reforestation provide net benefits 23 use values of the resource for their offspring. lastly, the third classification is the value from knowledge of continued existence of preservation. since non-use values are intangible, this posits difficulties to measure the true (or total economic) value of a natural resource. thus, some valuation techniques have evolved to measure and capture non-use values of natural resources. in this study, contingent valuation method (cvm) was used to assess the willingness to pay (wtp) for conservation of mangroves. alternatively, wtp provides a measure of the conservation value or benefit for the natural resource concerned, mangroves in the case of this study. a contingent evaluation study, according to boyle (2001), requires very careful design and data analysis. on the cost side of the analysis, social costs can be measured by the opportunity costs of using resources in certain ways, and the costs of price changes. the opportunity cost of using resources in a particular way is the highest-valued alternative use to which they might otherwise have been put. costs are incurred by all sorts of individuals, firms, agencies, industries, and groups. such costs are the capital costs of initial construction (initial implementation of the reforestation project), and the annual operating and maintenance costs that will extend over the life of the project. the source of data on costs of this type is normally from engineering or scientific authorities that can specify in detail the inputs needed for various phases of the projects. according to white and trinidad (1998), cost-benefit analysis compares the present value of all benefits (environmental, financial and social) with all costs associated with achieving a proposed outcome. it can give valuable insights into the economic efficiency of management and regulatory actions. the more benefits exceed the costs; the better off the society in economic terms as a result of the activity. the study focused on the costs and benefits of mangrove reforestation in two scenarios: with sustainable harvesting and without harvesting. two values were determined, the costs (c) and benefits (b). all the costs incurred for the implementation of the mangrove reforestation were broken down for each of the barangay studied. the total cost in general form is, tc = tfc + tvc oc, where tc = total cost tfc = total fixed cost tvc = total variable cost oc = total opportunity cost table 1: items of costs of mangrove reforestation in sibunag, guimaras costs variable representation definition labor for planting x1 physical strength exerted in the planting of mangrove propagules labor for maintenance x2 physical strength exerted in putting up bamboos poles; cleaning mangroves trees from “lumot”; putting up nets and other works under maintenance tree planting snacks x3 snacks during the plantingperiod seedling/propagules x4 mangrove seedling nylon x5 a synthetic material used in the nets rope to be used as latid x6 a string used to tie up the nets nets for fencing x7 a piece of fabric used in confining mangrove areas bamboo pole x8 pole made of bamboo used as a support for young mangrove trees bamboo post x9 post made of bamboo used in supporting the net straw x10 a piece made of plastic used to tie up mangrove seedling to the pole land x11 solid part of the earth surface, pertaining to mangrove areas fernandez, subade & parreño 24 the total cost (tc) in the equation is the total cost of the mangrove reforestation projects in sibunag, guimaras. the total fixed cost (tfc) on the other hand is part of the budget that stays the same regardless of whether the output (mangrove trees) increases or not. total variable cost (tvc) is part that varies as one produce more or less. total opportunity cost (oc) includes the foregone benefits one incurred in participating in the mangrove reforestation. it was assumed that oc is zero, to simplify our analysis. but must be pointed out that opportunity cost method i.e. foregone benefits, is used in quantifying tfc and tvc. specifically the model is, ∑ = = 11 1i ixc ; i = integer table 1 shows the costs incurred in reforesting mangroves with their representation: on the benefit side of analysis, specifically the model is, ∑ = = 12 1i iyb , where i=integer table 2 shows the benefits incurred in reforesting mangroves with their corresponding representation. net present value (npv) will be used to determine the viability of the project. the general formula for npv is: ( ) ( )∑= + − = n t t tt r cb npv 1 1 , wherein b=benefits of mangrove reforestation table 2: benefits of mangrove reforestation in barangay bubog, sabang and sebaste with their corresponding representation benefits variable representation definition fuel y1 something that is burned to provide power or heat i.e. branches of mangroves medicine any part of mangroves trees as treatments for · cough y2 cough, stomachache and body pains (usually · stomachache the leaves and the bark) · body pains household items · christmas tree y3 upper portion of the mangrove tree cut usually during ber months mangrove roots for aquarium trade y4 the roots of the mangrove tree use as a decoration in aquarium control of shoreline and riverbank erosion y5 the capacity of the mangrove tree to hold soil carbon sequestration y6 a chemical process of binding oin i.e carbon baluk y7 usually in the form of the roots, used as a cork dye y8 red coloring from the bark of the mangrove tree agriculture · fodder for pigs y9 use as a medicine and food for animals construction (furniture) · sala set · cabinet · dining table y10 mangrove trees as an input in building · single bed something · table fishing poles y11 refers to the braches used in catching fishes wtp y12 willingness to pay for conservation of mangroves will mangrove reforestation provide net benefits 25 c=costs of mangrove reforestation t= number of years r= rate of interest n= duration of the reforestation project on the other hand, benefit-cost ratio will also be used, assuming the formula: ( ) ( )∑ ∑ = = + + = n t t t n t t t r c r b bcr 1 1 1 1 , wherein b=benefits of mangrove reforestation c=costs of mangrove reforestation t= number of years r= rate of interest n= duration of the reforestation project the willingness to pay (wtp), denoted as y16 in the benefit table is dependent o other variables, assuming a formula of ( )921 ,..., zzzfwtp = , wherein; z1= age z2= sex z3= civil status z4= scale of knowledge on mangroves z5= occupation z6= wtp amount z7= mode of payment z8= educational attainment z9= household income the z variables above show the factors that could affect the willingness to pay of respondents. the age is length of time (expressed in years) that the respondent has lived. sex refers to the male and female duality of biology and reproduction of the respondent. civil status is presented as whether the respondent was "single" or "married", a variable illustrating respondent's marital status. the scale for knowledge is a variable which measure the level of awareness and information of respondents regarding mangrove ecosystem. occupation pertains to the job, profession, work, career, livelihood, living, employmentof the respondent. the wtp amount in the equation would be the bid prices cited on the questionnaires. the bid prices are php 10, 50, 100, 200, 500. these prices were based on the pretest in barangay baguingin, tigbauan, iloilo. the test for the prices was an open-ended question. the top five prices assumed the bid prices in the actual survey. in the questionnaire, there are two modes of payment or payment vehicles. one is through surcharges in electric bill and the other one is through an addition charge on cedula. these are the ways to collect the charges for mangrove reforestation. hanemann's formula was also used to get the mean wtp, assuming the formula: ( )⎥ ⎦ ⎤ ⎢ ⎣ ⎡ +⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ = σ+ iiaemeanwtp δβ β 01ln 1 1 where: 1β = coefficient of wtp amount 0a = coefficient of the constant e = natural logarithm iβ = coefficient of the independent variables iδ = mean of the independent variables however, there are situations when hanemann's mean wtp can be overestimated. this can happen when the percentages of no responses are not consistently increasing as bid price increases (hanemann, 1984). to deal with this haab and mcconnel (2002) devised the turnbull mean wtp as a conservative lower bound mean wtp estimate. in this situation given the present data set, turnbull wtp offers a better estimate. to get the turbull mean wtp, the formula that was used was: 1* +σ= jj ftmeanwtp ; where j j j t n f = , or the ratio of number of no responses and the number offered in the specific bid jn = number of no responses jt = number offered in the specific bid jt = bid prices fernandez, subade & parreño 26 jf * = turbull estimate of j j t n to get the social mean wtp, the formula that was used was: social mean wtp= (percentage of the respondent who are willing to pay) x (total households of the barangay) x (mean wtp) methodology the duration of the study was from june 2004 to february 2005. actual gathering of data was conducted from october 2004 to january 2005. purposive sampling was used in choosing the sites. these were three barangays in sibunag, guimaras, namely; brgy. bubog, sabang, and sebaste, where mangrove reforestation projects were being conducted . eight barangays were selected from two different municipalities in guimaras (jordan and buenavista) to determine people's willingness to pay. the selection of these barangay to determine the variable wtp was due to logistics, e.g. time and money. benefit-transfer method was then used to transfer the wtp of jordan and buenavista to that of sibunag's. table 3: respondent distribution of the study barangay total number of male female total households 1. hoskyn 423 16* 24 40 *(40%) (60%) ** (20%) 2. sinapsapan 293 16 12 28 57.14% (42.86%) (14%) 3. lawi 280 15 12 27 (55.56%) (44.44%) (13.5%) 4. buluangan 149 6 8 14 (42.86%) (57.14%) (7%) 5. santo rosario 502 28 20 48 (58.33%) (41.67%) (24%) 6. rizal 206 7 13 20 (35%) (65%) (10%) 7. umilig 121 8 4 12 (66.67%) (33.33%) (6%) 8. san miguel 102 7 4 11 (63.64%) (36.36%) (5.5%) total 2076 103 97 200.00 (51.5%) (48.5%) note : the figures in columns three, four and five with parenthesis are percentage of raw total. legend: * means that 40% are males in barangay (percentages by rows) ** means that percentage of the total number of respondents (n=200) table 3 shows the distribution of respondents in the eight barangays where the wtp survey was conducted. the first four barangays in the table were from the municipality of jordan and the last four were from the municipality of buenavista. sibunag is a two and a half -hour puj ride to buenavista and an hour puj ride to jordan.it should be noted however, that due to logistics reason, the wtp survey was done in buenavista and jordan, not from sibunag. the authors then used benefit transfer analysis to transfer the benefits to the municipality of sibunag. the contingent valuation method survey instrument was divided into six sections. the first section was background framing and information. this contains an overview of what the survey is all about. the second section was nine questions on knowledge of marine environment. it contains specific areas such as politics, economics, and environment, which the respondents gets to choose the one they are most familiar and will mangrove reforestation provide net benefits 27 concern with. the third section was a 10-point likert scale knowledge on study site information. this section contains the rating that the respondent would rate to themselves as to the level of knowledge that he/she has regarding mangrove conservation. the fourth section was an information box on background information about mangrove reforestation. the conservation efforts of the province as well as the reasons for the continuing decline of mangrove areas were highlighted on this section. the fifth section comprised the background information on trust fund and wtp questions. this section illustrates the hypothetical scenario. it is n this section where the conservation plan for mangrove reforestation was presented. it includes the willingness to conserve question of whether to contribute for the mangrove conservation or not. the last section was on socioeconomic background of the respondent. it contains information about income, age, civil status and other socio-economic variables. regression analysis in the cvm logit model was used to derive mean wtp. other tools such as correlation, frequency and percent distribution, and average cost were also used in the interpretation of data. basically, the study monetized the costs and benefits of the mangrove reforestation by averaging cost data across three barangays for the two scenarios: with sustainable harvesting and without harvesting. the costs of mangrove reforestation were quantified using key informants. two mangrove experts were interviewed to determine the sustainability use of mangroves species. this information was important in the cba analysis. in quantifying the benefits through opportunity costs, prices in the prevailing market were used. in the pricing of the shoreline protection benefit, data were gathered through interviews with the respondents who experienced the effect of coastal erosion thereby incurring costs which were quantified through the costs of repairing the damage. carbon sequestration value was adopted from the study of guanzon and lagera table 4: frequency of the wtp reply wtp amount wtp reply total yes no 10 33 7 40 50 29 11 40 100 30 10 40 200 28 12 40 500 28 12 40 (unpublished, 2006). the value was revised for the only species present in the area which were rhizophora and avicennia. this is a non-use value of mangroves. results and discussion contingent valuation method (cvm) the contingent valuation method (cvm) was used to elicit the non-use value of conservation of mangroves depicted on their willingness to pay (wtp). the wtp is expresses in monetary terms. table 4 shows the frequency of the willingness to pay reply of the people in the eight barangays. the highest number of people willing to pay was php 10 while the lowest was php 200 and php 500. the law of demand tells that as the wtp amount increases the people will less likely pay for the conservation of it, holding other factors constant. however on the table, as the price of the bid goes higher, the table 5: coefficient and mean of the different independent variables variables coefficient t statistics mean 1. constant -0.99264800 -0.989 2. age -0.00433351 -0.318 45.64 3. sex -0.09752380 -0.290 0.4850 4. knowledge scale 0.14250700 2.252 7.1750 5. mode 0.18794500 0.553 0.51 6. educational years 0.03705550 0.529 7.9450 7. total household 0.31388100 1.724 2.3650 income 8. civil status 0.431706 0.780 0.87 9 wtp amount -0.00116414 -1.240 172.0 willingness to pay declines but, at php100, it goes up and decline again. this trend was corrected by using the turnbull formula in solving for the mean wtp as shown in the succeeding paragraphs. fernandez, subade & parreño 28 mean wtp the mean wtp is value or price for conservation of mangroves. table 5 shows the coefficient and mean of the different independent variables used to get the mean wtp. using the hanemann's (1984) formula the mean wtp of the study was php 1605. this estimate is an overestimation since it is even greater than the maximum bid of 500. a more conservative estimate as formulated by haab and mcconnel (2002) provides a lower bound wtp which they called turnbull wtp. the details are presented in their work. for this study table 6 presents how the turnbull wtp is completed. turnbull wtp = 1* +σ jj ft = 0 (0.175) + 10 (0.0875) + 50 (0.0375) + 100 (0) + 200 (0.7) table 7: benefits of mangroves as cited by the respondents benefits (goods and services) of mangroves as cited respondents · flora and fauna (shells), alimango (crabs), shrimps, babuy-baboy, palu-palo (fingerlings), iras, pala, dawat (small crabs), suso, tipsay, lusaw, bangi-bangi, sisi and samaral or gusaw · households uses christmas trees, tables, chairs, dye, firewood, corks, decoration and walls for houses, driftwood for orchids, boats as souvenir, and landay (small boats) etc. · medicine stomachache, cough, body pains · bird sanctuary kalansiyang and kalaksahan · others increase fish catch of the fishermen, coastal protection society acquires if conservation or reforestation is done. since this conservation is non-market value, cvm provides the estimate for this non-use value of mangroves. benefits the study also conducted interview to the residents on benefits they acquire from mangroves. table 7 shows the benefits of mangroves as cited by the respondents. the flora and the fauna that can be obtained from mangroves as cited by the respondents are the following: tuway (shells), alimango (crabs), shrimps, babuy-baboy, palu-palo (fingerlings), iras, pala, dawat (small crabs), suso, tipsay, lusaw, bangi-bangi, sisi and samaral or gusaw. the birds like kalansiyang and kalaksahan can also be seen in mangroves area. other important benefits were discussed in the succeeding paragraphs namely, as christmas trees; as dye; as a shade; as an aid to vinegar production; as a protector of big waves; as medicine; and as a source of income. one of the benefits from mangroves is that a part of it can be made into a christmas tree. moreover the sap of the bark can also be used as a dye which is mahogany brown or red in color is from the bark of the tree. mangroves can also serve as a shade. it also acts as a protection from big waves and can prevent soil erosion and flood in the coastal communities. the "balok", which when fermented becomes vinegar, can also be table 6: computation of turnbull wtp for conservation of mangroves bid number of unestimated turnbull price (tj) no’s nj tj f*j f*j 10 7 40 0.175 0.175 0.75 50 11 40 0.275 0.2625 0.0875 100 10 40 0.25 pooled pooled back back 200 12 40 0.3 0.3 0.0375 500 12 40 0.3 0.3 0 500+ 1 0.7 jt jn jf = = 0 + 0.875 + 1.875 + 0 + 140 = 142.75 php 143 the mean, therefore is equal to 143. social mean wtp social mean willingness to pay method was used to determine the willingness to pay of the sampled barangay. since there are surveyed barangay eight barangays, there are also eight social mean wtps. in order to get the overall social mean wtp for all the barangays, the same formula was used, thus the value php 219682. this social mean wtp represents the conservation value for mangroves, or the benefit which will mangrove reforestation provide net benefits 29 extracted from mangrove trees. firewood, corks, decoration and walls for houses, driftwood for orchids, boats as souvenir, and landay (small boats) are some of the benefits mangroves can offer. another benefit that a mangrove can provide is medicine. it can be use as a remedy for cough, stomach ache, and body pains. the roots and leaves of mangroves were used to alleviate cough and stomach ache of an individual as cited by the respondents. on the other hand, remedy for body pains can be obtain from the barks of a mangrove. moreover, mangrove area also acts as a bird sanctuary. furthermore, mangrove can also increase the income of fishermen through their fish catch. mangrove areas can attract many fishes especially fingerlings since they serve as nursery ground for a variety of marine organisms. mangroves also act as providers of coastal protection, especially from big waves and soil erosion. during the last december 2004 tsunami tragedy over indian ocean and andaman sea, sumatra suffered fewer casualty and destruction due to protection provided by mangroves. since monetary values of mangroves are in question here, table 8 shows the benefits of mangroves. it provides different benefits from mangroves as well as the corresponding value in philippine peso (php). it must noted however, that the estimation is based on sustainability. this means that the third column (quantity consumed per year) assumed that this amount will not kill the mangrove trees. hence, there is no total benefit derived from this listing. the following paragraphs will explain the value estimation. through the documents provided by penro (2004), it was found out that the average survival rate of mangroves in the three barangays was 51.67%. it was table 8: benefits of mangroves per hectare per year (in php) benefit price (brand) quantity consumed total benefit (households)/ year fuel p15/bundle‘ 1 bundle per tree 34440 medicine · stomachache p12/capsule (imodium) twice a month 288 · cough 8.50 php(tuseran forte) twice a month 204 · body pains 13.70 php (25 ml of efficascent oil) everyday 5000.5 household items · christmas trees p200/tree once a year 229600 mangroves roots for aquarium trade p75/qty once a year 172200 control of shoreline and riverbank erosion p2475/year throughout the year 2475 carbon sequestration p4664.93/ha/year throughout the year 4275.18 baluk p10/liter 1 liter per tree 22960 dye p3.50/pack 1 pack per tree 8036 agriculture · fodder for pig p25/kilo 1 kilo per tree 57400 construction (furniture) · sala set p20000 (5 trees) once in 10 years 20000 · cabinet p6000 (3 trees) once in 10 years 6000 · dining-table p8000 (2 trees) once in 10 years 8000 · single bed p2500 (2 trees) once in 10 years 2500 · table p2500 (2 trees) once in 10 years 2500 fishing poles p50/piece 1 piece per tree 114800 wtp p142.75/hh once a year 296349 · total benefits will vary each year, refer to tables 16-17 · 2296 mangrove trees per hectare out of 4445 tress in a hectare · note: 51.67% survival rate which is the average of 55% (bubog), 40% (sabang), and 60% (sebaste) fernandez, subade & parreño 30 found out that an average of 4445 trees of mangroves was planted in one hectare. it was estimated that about 2296 of mangrove trees survive in a year. this is one of the major assumptions of the estimation. as shown on table, mangroves could be a source of fuel or firewood. it was monetized by an indirect opportunity cost approach. the php 15 is the regular cost of a bundle of firewood in the market in the year 2005. this could also be the value of the firewood benefit of mangroves. its total benefit in a year per hectare was php 34440, which was obtained by multiplying php 15 by 2296 trees. another benefit from mangroves is medicine. mangroves can provide remedy for cough, stomach ache, and body pains. these services were also monetized by an indirect opportunity cost approach. the php 12, php 9, php 14 value of medicine for stomachache, cough, and body pain were obtained by assuming that these values are equal to the costs of one capsule of imodium for stomach ache, one capsule of tuseran forte for cough and 25 ml of efficascent oil for body pains in the market. the prices were taken from a convenient store. the respondents also cited that at least twice a month an individual can experienced stomachache and cough. however, they are prone to body pains everyday since most of the respondents were drivers. using indirect opportunity cost approach, total price for medicine from mangroves is php 5493. the value of medicine for stomachache was computed by multiplying php 12 with 2 (assuming that individuals can get a stomachache at least twice a month) and 12 (months in a year). the value of medicine for cough has the same computation while that for stomachache is only php 9. the value of medicine for body pains was computed by multiplying 365, which is the number of days in a year, and php 14 which is the price of medicine for body pain. the table also shows that mangroves can control the shoreline and river banks erosion. the value for this benefit from mangrove was quantified using opportunity cost method. it was assumed that the opportunity cost of a person for one hour's work is equal to php 23. this was derived from the minimum 10-hour wage of php 180. two respondents cited their restoration costs from erosion. one respondent spent a total of php 2590 per hectare of mangroves. this cost includes total labor cost of 900, for ten hectares of land and equipment cost of 2500. this equipment cost includes 5 shovels that cost 500 each. another respondent cited a cost of php 2360 per hectare of mangrove. this cost includes total labor cost of 360, for 2 persons who worked 10 hours. materials used were bamboo that cost 50 per bamboo, garnering 2000 for the materials used. getting their average, the total value for mangrove control of shoreline and restoration was equal to, php 2475 per hectare. carbon sequestration value at php 4275 was adopted from the study of guanzon and lagera (unpublished, 2006). the value was revised for the only species present in the area which were rhizophora and avicennia. this is a non-use value of mangroves. another benefit from mangroves is that its barks can be made into christmas tree and its roots can be made as an aquarium decoration. indirect opportunity cost approach was used to determine the monetary value of these two benefits. the price of one quantity of christmas tree and aquarium decoration is php 200 and php 75, respectively. the table also shows that half of the 2296 trees in a hectare were utilized for christmas trees and other half for mangrove roots for aquarium trade. to compute for the total benefits of the household use of mangroves, particularly in making a christmas tree, the value of one christmas tree which is php 200 was multiplied to half of the number of mangrove trees in a hectare which is 1148. its total benefit is php 459200. on the other hand, the benefit for mangrove roots for aquarium trade was php 344400 which was from the product of php 75 which is the value of one mangrove root for aquarium trade, 2 from an assumption that this item will be derived twice in a year and 1148 which is half of the number of mangrove trees in a hectare. the "baluk", which when fermented can turn into vinegar, is another benefit from mangroves. again, using indirect opportunity cost approach, the monetary value of "baluk" was computed. the market price for "tuba" which is extracted from coconut trees is php 10 per liter. assuming that the value of tuba is equal to baluk, then 1 liter of baluk is also equal to php 10 per liter. to compute for its total benefit which was php will mangrove reforestation provide net benefits 31 22960, price per liter of baluk was multiplied to the number of mangrove trees in a hectare. the table also shows that dye can also be derived from mangrove trees. the monetary value attach to it is from the price of 1 pack of dye in a market that costs php 4. the indirect opportunity cost approach was again used in order to attach an equal value for dye from mangroves and dye in the market. the total benefit derived from dye was php 80 which was from the product of 4 and 2296 mangrove trees in a hectare. mangroves can also be used for fodder for pigs. in the market, the price of one kilogram of "lintok" which is used as feeds for pigs costs php 25. indirect opportunity cost approach was used to attach monetary value on the fodder for pigs. therefore the value attach on fodder for pigs was php 25. to compute for the total benefit of agricultural benefit of mangroves which was php 57400, monetary value of fodder for pigs was multiplied to the total number of trees in a hectare. mangroves can also provide fishing poles. the monetary value attach on the fishing poles was obtained table 9: the preliminary planting cost of mangrove reforestation in barangay sabang, sibunag (7 ha.) item agency involved unit unit cost econ life dep cost total cost bamboo penro 50 pcs. 40 ¼ year 500 2500 poles barangay counterpart 100 pcs. 20 ¼ year 5000 7000 save 187.5 40 ¼ year 1875 9375 bamboo save 562.5 pcs. 15 ¼ year 2109.38 10546.88 posts penro 150 pcs. 15 ¼ year 562.5 2812.5 seedlings/ penro 4445 1 4445 propagules pesco-dev 10000 1 10000 save 16000 1 16000 barangay counterpart 20 rolls 35 1/12 year 58.33 758.33 nylon penro 2 legs 200 ¼ year 100 500 pesco-dev 6 rolls 90 ¼ year 135 675 save 8 legs 200 ¼ year 400 2000 fish net penro 3 bundles 2300 ½ year 3450 10350 save 9 bundles 2300 ½ year 10350 31050 labor for barangay planting counterpart 40 pax 180 7200 nets for municipal 400 m 30 ½ year 6000 18000 fencing counterpart billboards barangay 1 300 5 60 360 counterpart tree barangay 700 planting counterpart snacks straws barangay 20 rolls 35 1/12 year 58.33 758.33 counterpart save 19 35 1/12 year 55 720.39 penro 5 rolls 35 1/12 year 14.58 189.58 total 135941.01 fernandez, subade & parreño 32 with the use of indirect opportunity cost approach. the price of a piece of bamboo is php 50 per piece. this was used to attach the monetary value of fishing poles from mangrove trees. to compute for the total benefit of mangrove trees used as fishing poles, the value attach to the fishing pole from mangroves was multiplied from the number of mangrove trees in a hectare. the total benefit from mangrove trees as fishing poles was php 114800. the benefits in question here are not complete. it does not include other mangrove benefits such as increase in fish catch; bequest value; and other benefits because of time constraints. costs the costs of mangrove reforestation in three barangays were divided into two: preliminary and maintenance cost. the preliminary cost is the cost incurred at the start of the mangrove reforestation project. this usually includes the planting costs. on the other hand the maintenance cost includes all the cost incurred in maintaining the reforested area. table 9 showed the total preliminary cost of mangrove reforestation in barangay sabang. the agencies involved in the reforestation in this barangay were the following: fisherfolk association, penro, save, barangay council and local government unit (lgumunicipal). it was assumed that the labor for planting was php 22.5 per hour of work. since, cost of labor for planting was not shouldered by any agency, the labor cost was quantified using opportunity cost method. this assumption was also used in the two remaining barangays. table 10 shows the maintenance cost of mangrove reforestation in the barangay. again, the same assumption was considered for labor cost. this cost was used throughout the years of implementation of the project. the total cost here was the yearly cost of mangrove refo in barangay sabang. the total table 11: the preliminary planting cost of mangrove reforestation in barangay bubog, sibunag (2.5 ha.) item agency unit econ total involved unit cost life dep cost cost bamboo poles penro 125 pcs. 40 ¼ year 1250 6250 bamboo posts penro 375 pcs. 15 ¼ year 1406.25 7031.25 seedlings/propagules penro 11113 1 11113 straw penro 12.5 rolls 35 1/12 year 36.44 473.94 nylon penro 5 legs 200 ¼ year 250 1250 fish net penro 7.5 bundles 2300 ½ year 8625 25875 billboard municipal counterpart 1 300 5 60 360 stick barangay counterpart 4 30 ¼ year 30 150 labor for planting barangay counterpart 28 180 5040 total 57543.19 table 10: the maintenance cost of mangrove reforestation in barangay sabang, sibunag (7 ha.) item agency unit unit total involved cost cost seedlings for replanting penro 445 1 445 labor for monitoring barangay counterpart 24 22.5/day 197100 total 197545 will mangrove reforestation provide net benefits 33 table 12: the maintenance cost of mangrove reforestation in barangay bubog, sibunag (2.5 ha.) item agency unit unit total involved cost cost labor for monitoring barangay counterpart 28 22.5/day 229950 seedlings for replanting penro 445 1 445 total 230395 reforested mangrove area was 7 hectares. in the computation of the depreciation cost, it was assumed that the salvage value or the replace cost was equal to zero. this would break down the equation of the depreciation to total cost over the economic life of the equipment/material. this assumption was also used in the entire cost tables presented on this research. table 11 shows the preliminary costs of putting up mangrove reforestation project in brgy. bubog. similar to brgy. sabang, the same labor cost was used. opportunity cost method was also used, as well as the zero-salvage cost. the agencies involved in the table 14: the maintenance cost of mangrove reforestation in barangay sebaste, sibunag (5 ha.) item agency unit unit total involved cost cost labor for monitoring barangay counterpart 18 22.5/day 147825 seedlings for replanting penro 668 1 668 total 148493 reforestation project in the area were almost the same with that of sabang except for the fisherfolk association, which was replaced by local fisherfolk association. the save, an ngo, which actively participated in the mangrove reforestation in sabang and sebaste, did not participate on this project. table 12 shows the maintenance costs of mangrove reforestation in barangay bubog, sibunag, guimaras. it includes labor costs for monitoring and seedlings for replanting. the total maintenance cost was 230395 php. item agency unit econ total involved unit cost life dep cost cost seedlings/propagules save 16000 1 16000 penro 6668 1 6668 bamboo poles barangay counterpart 75 pcs. 20 ¼ year 5000 7000 save 187.5 40 ¼ year 1875 9375 penro 75 pcs. 40 ¼ year 750 3750 straws barangay counterpart 20 rolls 35 1/12 year 58.33 758.33 save 19 35 1/12 year 55 720.39 penro 7.5 rolls 35 1/12 year 21.87 284.37 billboards barangay counterpart 1 300 5 60 360 bamboo posts barangay counterpart 150 pcs. 15 ¼ year 562.5 2812.5 save 562.5 pcs. 15 ¼ year 2109.38 10546.88 penro 225 pcs. 15 ¼ year 843.75 4218.75 nylon save 8 legs 200 ¼ year 400 2000 penro 3 legs 200 ¼ year 150 750 fish net save 9 bundles 2300 ½ year 10350 31050 penro 4.5 bundles 2300 ½ year 5175 15525 labor for planting barangay counterpart 18 3240 total 99534.22 table 13: the preliminary planting cost of mangrove reforestation in barangay sebaste, sibunag (5 ha.) fernandez, subade & parreño 34 table 13 and 14 showed the details of preliminary and maintenance cost of mangrove reforestation in sebaste. the total reforested area of this barangay is 5 hectares. the computation was also done with the use of similar assumptions mentioned earlier. table 15, summarizes the costs of mangrove reforestation projects in the three barangays. on the average the preliminary cost would approximately at php 69514 and the maintenance cost at php 192144. it noted that most of the cost incurred is on labor. this means that initiating and maintaining a mangrove reforestation project is labor-intensive. the labor costs were calculated using opportunity cost method. this implies that the 'payment" for laborers in the area are actually their foregone benefits and not the actual payment they received. these laborers were members of organization of fisher folks who believe that participating in the project would give them more fishes to catch. cost-benefit analysis the cost-benefit analysis is tool to weigh down the benefits and the foregone benefits of a particular project, i.e. mangrove reforestation. the authors wanted to know if the net benefits will be positive in two different situations. one situation is the with sustainable use of mangrove trees. in here, people are using the resource but without damaging the ecological balance or depriving others from consuming it in the future. scenario 1: with sustainable harvesting table 16 shows the comparison of the costs and the benefits of mangroves on the span of 20 years. it was compounded at different interest levels: 5, 8, 10, 12, 15 and 20 %. using the turbull estimate the benefit through the social mean wtp was php 219298. this is the willingness to pay of all the households for the mangrove conservation. on this scenario, aside from the non-use value (wtp) there are also used values in the benefit equation. all the benefits listed in table 7 are depicted on this scenario. but it must be noted however that they are add added up in every year. that's why the benefits in table 16 vary from year to year. the variation is due to that fact that one cannot acquire the same benefits to the same trees all year round. the fuel benefit of mangrove can be ripped in 10 years time and onward. remember that the value for the tree is equivalent one bundle of firewood and the wood will be harvested once a year. the medicinal value of mangrove trees is further expressed into three uses. the first cure is for stomachache, since leaves are used, the values can be acquired in 10 years time and onwards. this is also true in the case of the treatment for cough. the last cure would be for body pain, which will be valued after 10 years, for the bark uses. the christmas tree benefit can be ripped after 10 years, and it will is assumed that only half of all the mangrove trees will be used. the christmas tree is actually the top part of the tree, thus, not killing it at all. in the case for aquarium trade, it is cost barangay barangay barangay average sabang bubog sebaste (7 ha.) (2.5 ha.) (5ha.) preliminary cost php 135941.01 57542.69 115059.20 69514.47 maintenance cost 197545.00 230395.00 148493.00 192144.3 total cost 233486.01 287937.69 263552.20 261658.8 table 15: summary of cost incurred in mangrove reforestation in sibunag, guimaras (in php) will mangrove reforestation provide net benefits 35 also assumed that half of the trees will be used and the value can be ripped off after 9 years. according to mangrove experts, in order for a mangrove tree to fully participate in control of a riverbanks or coast, it should be a grown tree. that is why the value for shoreline control is used after 9 years. since after 10 years, a mangrove tree has grown, balok and dye benefits can be added up. the fodder for pigs (actually associated its acapacity to treat pig's parasites) will be ripped after 5 years. in the case of carbon sequestration, from year 0 to 2, there are no values for it. in the 3rd to 5th year, only 25% of the value is assumed to be present; 50% in the 6th to 8 years and 75% in the 9th to 20th years. on the cost side of analysis, it was assumed that the cost for the land was at php 500. this is based on the fishpond lease agreement (fla) fee set by the philippine government. there is a 100 php increment for the next three years, and became fixed at 500 php in the fifth year onwards. on the first year of the project, the cost is higher, than the second year onwards. this was for the reason that the cost in the first year includes cost for the first planting of project. this is also the cost of putting a reforestation project per hectare of mangrove. it can be seen that the npvs in zero year is table 16: cost and benefit analysis of mangrove reforestation in sibunag, guimaras (with sustainable harvesting) year total total net discounted net benefit benefit cost benefit 5% 8% 10% 12% 15% 20% 0 219298 268909 -49611 -41674 -41674 -41674 -41674 17771 17308 1 219298 199394 19904 -18956 18430 18095 17771 14711 13954 2 219298 200844 18454 16738 15821 15251 14711 23134 21370 3 234796 202294 32502 28076 25801 24419 23134 19734 17754 4 234796 203744 31052 25546 22824 21209 19734 15974 13996 5 234796 206644 28152 24330 21133 17480 15974 445282 379976 6 1085551 206644 878907 655854 553861 496120 445282 397573 330414 7 1085551 206644 878907 624623 512834 451018 397573 354976 287316 8 1085551 206644 878907 594879 474846 410017 354976 322531 254246 9 1101049 206644 894405 576541 447425 379315 322531 2293928 1761090 10 7331236 206644 7124592 4373881 3300065 2746839 2293928 2164780 1618586 11 7736941 206644 7530297 4402809 3229615 2639323 2164780 1932840 1407466 12 7736941 206644 7530297 4193151 2990384 2399385 1932840 1725750 1223884 13 7736941 206644 7530297 3993477 2768875 2181259 1725750 1540848 1064247 14 7736941 206644 7530297 3803312 2563773 1982963 1540848 1375757 925432 15 7736941 206644 7530297 3622202 2373864 1802693 1375757 1228355 804723 16 7736941 206644 7530297 3449716 2198022 1638812 1228355 1096745 699760 17 7736941 206644 7530297 3285444 2035205 1489829 1096745 979237 608487 18 7736941 206644 7530297 3128994 1884449 1354390 979237 874318 529119 19 7736941 206644 7530297 2979994 1744861 1231264 874318 784866 462593 20 7777691 206644 7571047 2853448 1624355 1125388 784866 462593 197484 negative in 5%, 8%, 10% and 12%. but after the 1st year, the npvs are all positive in all levels of interest rates. scenario 2: without harvesting in the analysis of cba, it was assumed that the person will use mangrove resources (bark, leaves, etc.) at sustainable level for their consumption. this would reflect mangroves direct use/benefit to the society. in compliance with r.a. 7161 of 1990, which banned all cutting of all mangrove species, cba analysis for without mangrove harvesting was computed. this implies that utilization is no longer permitted. however, it was found out that this ra does not explicitly specify that reforested mangrove species could not be harvested. if this is the case, there is an unclear policy/ regulation for this utilization. if planted mangrove species can be cut down, then the previous cba will hold true. moreover, there are other policies that allow the cutting of mangrove species. memorandum circular no. 5, series of 1990, prescribed guidelines on the cutting of mangrove species within approved fla areas. there fernandez, subade & parreño 36 table 17: cost and benefit analysis of mangrove reforestation in sibunag, guimaras (without harvesting) year total total net discounted net benefit benefit cost benefit 5% 8% 10% 12% 15% 20% 0 219298 76764 142534 -41674 -41674 -41674 -41674 -41674 -41674 1 219298 199394 19904 -18956 18430 18095 17771 17308 16587 2 219298 200844 18454 16738 15821 15251 14711 13954 12815 3 234796 202294 32502 28076 25801 24419 23134 21370 18809 4 234796 203744 31052 25546 22824 21209 19734 17754 14975 5 234796 206644 28152 24330 21133 17480 15974 13996 11313 6 250293 206644 43649 32572 27506 24639 22114 18871 14618 7 250293 206644 43649 31021 25469 22399 19745 16409 12182 8 250293 206644 43649 29543 23582 20363 17629 14269 10151 9 265791 206644 59147 38126 29588 25084 21329 16813 11463 10 265791 206644 59147 36311 27396 22804 19044 14620 9553 11 265791 206644 59147 34582 25367 20731 17003 12713 7960 12 265791 206644 59147 32935 23488 18846 15181 11055 6634 13 265791 206644 59147 31367 21748 17133 13555 9613 5528 14 265791 206644 59147 29873 20137 15575 12103 8359 4607 15 265791 206644 59147 28451 18645 14159 10806 7269 3839 16 265791 206644 59147 27096 17264 12872 9648 6321 3199 17 265791 206644 59147 25805 15985 11702 8614 5496 2666 18 265791 206644 59147 24577 14801 10638 7691 4779 2222 19 265791 206644 59147 23406 13705 9671 6867 4156 1851 20 265791 206644 59147 22292 12690 8792 6132 3614 1543 is also this dao no. 2000-29, series of 2000 which prescribed guidelines regulating the harvesting and utilization of forest products within cbfm (community-based forest management) areas. however, according to the philippine national committee, following the hierarchy of policy, republic act is above other laws and therefore, cannot be amended by a mere administrative order. nevertheless, for the sake of comparison, cba for mangrove without harvesting is presented in table 17. this is in accordance to the policies that banned the cutting and using of all mangrove species. only the non-use values comprised the benefits under the cba without harvesting. these were the following: shoreline protection, carbon sequestration, and social willingness to pay. this implies that there would be no benefits on mangrove direct uses i.e. for medicine, construction, aquarium, etc. similar to the first scenario (with harvesting), benefits derived from carbon sequestration and shoreline protection will only be reaped starting ten years onwards when mangrove trees have already grown. the benefits for the first nine years will largely depend on the social mean wtp of the community. based on these tables, it was found out that over 20years time, the benefit will also outweigh the cost, at different interest rates. however, the net benefit from without harvesting is lower than the values obtained from allowing harvesting. this is may be due to the fact that the benefits calculated were only non-use table 18: summary of net present values (npv) at different rates of interests for the two scenarios interest npv npv (w/o benefit benefit rates (with harvesting) cost ratio cost ratio harvesting) (with (without harvesting) (harvesting) 5% 42614057 523690 16 1.27 8% 28806441 421382 13 1.27 10% 22425067 351859 12 1.27 12% 17609110 298785 11 1.28 15% 12441720 238739 9 1.28 20% 7242423 172514 7 1.29 will mangrove reforestation provide net benefits 37 values of mangrove. this is the case if r.a 7161 is strictly implemented. however, there were other policies such as dao no. 15 and m. c. no 5, where cutting/using these mangrove resources are allowed --with corresponding guidelines. on the other hand, dao 15 do not allow cutting of mangrove trees within existing fishpond lease agreement (fla) unless permit were obtained from the department of environment and natural resources. the trees cut in fla areas through a permit shall be turned over to the denr for disposition through public bidding. fla holders are given the right to compete the highest bidder, in which case the bid is automatically awarded to him. in the case of commercial plantations, mangrove plantation developers shall be allowed to cut the planted trees found within their respective plantations through clear cutting by strips system, whether such action is intended for personal or commercial purposes. provided they secure a permit from the immediate office of the denr. if sustainable harvesting can be allowed, higher net benefits can be reaped from mangroves (table 18). the benefit-cost ratios is also greater than one in all levels of interest rates, whether on the first or second scenarios. this implies that the benefits really exceed the costs. summary, conclusion and recommendations summary this study examined the benefits and costs of mangrove reforestation in three selected barangay of sibunag, guimaras. key informants were interviewed to determine the different costs and benefits involved in mangrove reforestation. before the proper survey was conducted a pretest survey was done, this is to determine the bid prices to be used in the survey proper. survey of 200 respondents were undertaken to determine the conservation value of mangroves. the survey of 200 respondents involved personal interviews using contingent valuation method to determine people's wtp reply. these 200 respondents are the usual size personal interview cvm survey. cvm respondents were comprised of 51.5% males and 48.5% female. it was noted that as the wtp amount increases the people will less likely pay for the conservation fee. the mean wtp of the study was at php 142.75, while the social wtp was php 219298. in the cost side of analysis, the costs were divided into the cost of preliminary planting and the maintenance cost, for the entire 14.5 hectares of mangrove reforested area. the average cost of preliminary work in mangrove reforestation was at php 69514. on the other hand, the aveage maintenance cost approximately at php 261259. this means with this amount, any agency or community can start up a mangrove reforestation project. comparing the costs and the benefits, it was found out that at different interest rate levels, the npv was still positive until the next 20-years for the two scenarios: with sustainable harvesting and without harvesting. this means that the benefits outweigh the cost. whether the mangrove trees were utilized or not, still, the npvs were positive at all interest rates. therefore, the mangrove reforestation is beneficial, either in accordance to ra 7161 or not. conclusion in conclusion, the mangrove reforestation projects in sibunag, guimaras were successful. the benefits of the project outweigh its costs. this implies that the project should be continued and expanded. there were indeed, many benefits that could be reaped in the mangrove ecosystem; however that these benefits would entail time i.e. twenty years or so. some people do not realize this. oftentimes, they want immediate benefits from any natural resources like mangroves. as shown in the cvm survey, some people are also aware of the non-use benefits of mangroves. the social mean wtp equal to php 219298. cba was also conducted for mangrove reforestation without harvesting. this is in accordance to the republic act 7161 that banned the cutting/using all mangrove species. the net present value was also positive indicating that the mangrove reforestation is also beneficial in this scenario. recommendations a wider scope of cvm survey involving more respondents can update and maybe enlarge the nonuse values of mangroves during reforestation. benefit fernandez, subade & parreño 38 transfer of non-use values could only be used if mangrove species were similar in the area where cvm was conducted thus; more studies are needed to determine the economic value of non-use benefits of mangroves. other benefits of mangroves such a bequest value, increase fish catch and other ecosystem uses should also be included in further studies such as this. this can make the values of mangroves even higher than what is concluded here. acknowledgements the authors hereby acknowledge the contributions of people who provided valuable insights for this paper, in particular, dr. jurgenne primavera and dr. rex sadaba. the travel grant of u.p. in the visayas made it possible for the second author to present this paper in the 8th national symposium of the philippine association in marine science. ms. ana liza a. subade's technical editing greatly improved the first draft of this paper. references babaran, r. and j. ingles. 1997. philippine coastal marine habitats at risk: a case study of guimaras island. university of the philippines press. boyle, k.j. 2003. contingent valuation in practice. in p.a. champ, k.j. boyle and t.c. brown (eds). a primer on nonmarket valuation. the economics of non-market goods and resources. batemann, i.j. series editor. kluwer academic publishers. dordrecht/london/boston. dao no. 15-20 regulation governing the utilization, development and management of mangrove resources. dao 2000-29 guidelines regulating the harvesting and utilization of forest products within community based fores management area, dept. of env. & natural resources. field, colin d. (editor), 1996. "restoration of mangrove ecosystem". international society for mangroves ecosystems, okinawa, japan. guanzon, t. and j. lagera. cost-benefit analysis of sagay marine reserve. unpublished. university of the philippines in the visayas, division of social sciences, economics 199.2. 2006. haab t. and k. mcconnel. 2002. valuing environmental and natural resources: the econometrics of non-markets valuation. mpg books ltd., bodmir, cornwall. hanemann, w.m. 1984. welfare evaluations in contingent valuation experiments with discrete responses. american journal of agricultural economics 66:332-41. melana, d. m. et al. 2000. mangrove management handbook. manila: coastal resource management project of the department of environment and natural resources. melana, dioscoro m. et. al. "mangrove management and development in the philippines". retrieved from: http:// w w w . o n e o c e a n . o r g / d o w n l o a d / 2 0 0 0 0 4 2 7 / mangrove_management_phils.pdf philippine national committee. initial analysis on mangrove and mangrove-related policy issuance. available on line: primavera, j.h. 1999. mangroves of southeast asia. mangrove-friendly aquaculture. "project proposal of barangay sabang, sibunag, guimaras for mangrove plantation." denr: provincial office, guimaras.2001. ra 7161 tax laws incorporated in the revised forestry code. republic of the philippines. white a. and trinidad. 1998. the values of philippine coastal resources. 8call for papers-new.pmd 99 humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) analyn salvador-amores, myles capareda & fenelyn nabuab call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 4-yolanda-anticamara.pmd j.a . anticamara and b.c.a . tan 5 science diliman (july-december 2018) 30:2, 5-31 survival and growth of re-attached storm-generated coral fragments post super-typhoon haiyan (a.k.a. yolanda) jonathan a. anticamara* institute of biology natural science research institute university of the philippines diliman barron cedric a. tan institute of biology university of the philippines diliman abstract coral reefs in eastern samar, philippines were badly damaged by super typhoon haiyan, which left many reefs in a fragmented state – with many b r a n c h i n g c o r a l s a n d o t h e r c o r a l f o r m s s c a t t e r e d i n l o o s e p i e c e s . a s part of the efforts to address this problem, we tested the re-attachment of 43 species of coral fragments to sturdy natural substrates in three r e e f s i t e s i n e a s t e r n s a m a r ( c a n u s o d a n d m o n b o n i n l a w a a n , a n d panaloytoyon in quinapondan). the results revealed that 88% of re-attached coral fragments survived (45% showed positive growth, and 43% survived with partial tissue mortality). those that showed positive growth exhibited high growth rates. we also found that fragments of some coral species are more fast-growing (e.g. , cyphastrea decad ia, echinopora pacificus, and millepora tenella) than others (e.g. , porites lobata o r pectinia paeonia ). overall, our results suggest that if local government units (lgus) invest in the re-attachment of fragmented corals (e.g. , reefs damaged by super typhoons or by various human activities such as f ishing), then coral reef degradation in the philippines would have a better chance of recovering. keywords: coastal management , conservation, leyte gulf, reef restoration, super typhoon _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online survival and growth of a re-attached storm-generated coral fragments 6 introduction philippine coral reefs have been experiencing degradation since the 1980s – caused mainly by exploitative activities, such as f ishing, including destructive f ishing (e.g. , dynamite and cyanide f ishing) (gomez et al. 1994; white and vogt 2000). in addition, human activities, such as deforestation of upland and coastal areas, land conversion and industrialization, urbanization, and over-fertilization of farmlands, have added more sediments, nutrients, and pollutant run-offs onto philippines coastal areas, where most reefs can be found. recent accounts of the status of philippine reef benthos using meta-analysis techniques have demonstrated that philippine reefs remain in degraded conditions (magdaong et al. 2014). moreover, climate-related disturbances, such as ocean warming and acidif ication (mcleod et al. 2010; pandolf i et al. 2011), and super typhoons (knutson et al. 2010; anticamara and go 2016), have added more stresses to many degraded philippine reefs. thus, there is a need to test options for actively recovering extensive degraded reef areas in the philippines, in order to meet the increasing demands for reef ecosystem services, such as food f ish, tourism areas, and livelihoods, from the rapidly growing filipino population. over the last three decades, there has been a growing global interest in testing active restoration methods for coral reefs (yap and molina 2003; abelson 2006; rinkevich 2014). the existing coral restoration techniques can be generally categorized into three types: (1) direct transplantation of fragments (garrison and ward 2008; boch and morse 2012); (2) coral transplantation using nursery and coral gardening techniques (shaish et al. 2008; lohr et al. 2015); and (3) growing coral nubbins from coral spawn for later transplantation (shaf ir et al. 2003; guest et al. 2014; leal et al. 2016). a less popular coral restoration technique involves the use of electrodes to augment coral larval settlement in a chosen area (van treeck and schuhmacher 1999; benedetti et al. 2011). among the existing coral transplantation methods, the most promising one for the philippines is direct fragment transplantation, simply because of the cheaper cost and the abundance of coral fragments in many philippine reef areas, especially those that are subjected to frequent storms or human activities, such as unregulated diving (anticamara et al. 2015; go et al. 2015). in recent years, some of the philippine coral reefs, especially those facing the western pacif ic ocean (wpo), have been subjected to super typhoons, resulting to the eradication of some shallow reefs and the extensive damage to many reefs j.a . anticamara and b.c.a . tan 7 (anticamara and go 2016). to date, there is no published literature on any attempts to recover degraded reefs facing the wpo following the impacts of a super typhoon using available coral restoration techniques. in fact, we have noted that, in most coastal and reef areas that experienced devastation by super typhoons, most of the responses from the international donor agencies or local government units (lgus), national government agencies (ngas), and other aid institutions (ais) were the provision of more f ishing boats, engines, and f ishing gears in the affected areas (anticamara and go 2016). this is counterintuitive, considering that the devastation of corals due to typhoons have obvious negative effects on the productivity (f isheries) of impacted reefs based on established trophic interactions and the loss of habitats for many f ishes (lassig 1983; dollar and tribble 1993, tan et al. 2017). thus, there is a great need to test and demonstrate the potential of currently available coral restoration techniques in typhoon-prone areas of the philippines to help alleviate the negative consequences of storms on the reef productivity of the said areas. in the philippines, coral restoration techniques have been tested and have demonstrated promising results, such as relatively high coral growth rates and survival post transplantation (shaish et al. 2010; dela cruz et al. 2014; cabaitan et al. 2015). however, to date, coral restoration in the philippines has been mostly conducted in limited reef sites (mainly bolinao in northwest luzon) using few sets of coral species (dizon et al. 2008; shaish et al. 2010; gomez et al. 2014; cabaitan et al. 2015) (appendix table 1). thus, there is still a need to expand current studies and tests on coral restoration in many sites in the philippines for many coral species. the main goal of this research is to quantify the growth rates and survival of 43 species of re-attached storm-generated coral fragments in three reef sites of eastern samar– an area that was heavily damaged by super-typhoon haiyan in 2013. here, we used direct transplantation of coral fragments collected from the same reef site where the fragments were re-attached. we believe that this preliminary study (of similar duration to most published literature on coral restoration, appendix table 1) is necessary for providing insights on the possibility of recovering stormimpacted reefs in the philippines, which have been mostly abandoned and left to further unregulated exploitation and degradation. survival and growth of a re-attached storm-generated coral fragments 8 materials and methods study sites for re-attaching coral fragments we re-attached storm-generated coral fragments in three reef sites (i.e. , can-usod, monbon, and panaloytoyon) in eastern samar (figure 1). the reef sites of eastern samar were generally in a degraded state due to (a) over-exploitation, including destructive f ishing, such as the use of dynamite (anticamara et al. 2015; go et al. 2015), and (b) frequent impacts of strong storms– among which, the strongest was super-typhoon haiyan in 2013 (anticamara and go 2016). however, the three sites selected for the re-attachment of storm-generated fragments were within the regulated and well-enforced marine reserves (mrs) in eastern samar (figure 1), to ensure that blast f ishing, which is still practiced in eastern samar (personal observation) will not damage the re-attached coral fragments. the selected three study sites are very important in eastern samar as these are the last remaining protected reefs in the area, supporting the f isheries demands of the largely f isheries-dependent communities living in nearby coasts (anticamara and go 2016). figure 1. (a) philippine map; (b) southern coast of eastern visayas showing location of the three coral fragment re-attachment sites: can-usod (red circle; coordinates: lat11.133323, long125.284957), monbon (yellow circle; coordinates: lat11.125751, long125.302617), panaloytoyon (green circle; lat11.127674, long125.543586); (c) can-usod with 11 attachment sites; (d) panaloytoyon with nine attachment sites; and (e) monbon with five attachment sites. j.a . anticamara and b.c.a . tan 9 coral fragment attachment protocol within each reef site, we selected 5-11 square attachment areas, each measuring 10x10 m2, with their borders marked by a rope. within each attachment square, coral fragments were attached to sturdy substrates, such as dead massive or submassive corals, rocks, or to a pvc pipe, using concrete nails for hard substrate and cable ties. the choice substrate type was mainly based on what was found in the attachment square, and we did not test the effects of substrate types for this particular study. in addition, within each attachment square, up to 30 re-attached coral fragments were haphazardly selected and tagged using numbered security seals. all re-attached coral fragments were collected from coral fragments found within the study reef site, and only coral fragments that were alive and free of algae or bleaching marks were re-attached. within each attachment square, we reattached over 30 fragments, but did not count or track those that were not tagged due to limitations in terms of time and monitoring resources (i.e. , limited funding). monitoring growth rates and survival of attached coral fragments after re-attachment, the tagged coral fragments were visited every quarter and were monitored from july 2015 to june 2016. at the start of the study and during each visit, all tagged coral fragments were photographed with a ruler in the frame beside the re-attached coral fragment to provide a scale for growth measurements later in the lab. data processing and analyses photographs of the tagged corals were processed using the software coral point count with excel extensions cpce v4.1 (kohler and gill 2006) to compute for the area of each coral colony fragment. there have been reports of many different ways of measuring coral growth, such as linear extension, surface area, and ecological volume (shaish et al. 2008; gomez et al. 2011; guest et al. 2011; rinkevich 2014; cabaitan et al. 2015). we deemed it best to monitor the coral growth in terms of area (in cm2) so as to account for the differences in coral growth forms (i.e. , branching, foliose, or submassive). in addition, the mean monthly growth rate per re-attached coral species were also expressed as percentage of original colony area during initial re-attachment (appendix table 2). all tagged and re-attached coral fragments across the three reef sites were later identif ied at the species level and according to the life form using online resources (http://coral.aims.gov.au/) and publications (veron 1986; veron and hodgson 1989; veron et al. 1996). all survival and growth of a re-attached storm-generated coral fragments 10 analyses presented in this manuscript are descriptive in nature. our main purpose is to demonstrate the preliminary results of re-attaching storm-generated coral fragments, which were mostly left to die in eastern samar, since there has not been much effort in reef assessment or recovery in the area post super-typhoon haiyan. we hope the preliminary results presented here, albeit descriptive, will provide some insights on the possibility of actively recovering degraded reefs in eastern samar. we also presented calculations of the incurred costs of re-attaching coral fragments in eastern samar, to help guide lgus, ngas (e.g. , bfar, denr, dswd), and ais interested in investing in reef recovery in eastern samar. literature review and data compilation we compiled published peer-reviewed l i terature to extract the following information: (1) types of coral restoration (i.e. , direct re-attachment of fragments, coral gardening using nursery, or growing coral nubbins from larva); (2) the re-attached species; (3) the survival per species; (4) the growth rate per species (standardized as mean monthly growth rates (%) for comparison and discussion purposes); and (5) the incurred cost per re-attached fragment. the compiled information were utilized for discussion purposes (appendix table 1). results general f indings a total of 651 tagged coral fragments (belonging to 43 species and 15 families) were re-attached across the three study sites (appendix table 2). majority of the storm-generated fragments that were re-attached belong to the poritiidae family, since they comprise most of the live fragments that were left after super-typhoon haiyan impacted the reefs of eastern samar (figure 2). of the 651 tagged coral fragments, 320 were re-attached in can-usod, 75 in monbon, and 256 in panaloytoyon (figure 3). after a year of quarterly monitoring, 295 (45%) of the tagged and re-attached coral fragments showed positive growth, 282 (43%) stayed alive but with partial tissue mor tality, and 75 (11%) were lost (either dead or detached, and no longer found during the monitoring period). j.a . anticamara and b.c.a . tan 11 figure 2. barplot showing the mean (±se) monthly growth rates by coral species. the number above the bar shows the number of re-attached fragments for each coral species. numbers in bold are species with ≥10 re-attached fragments/colonies. green bars represent species with ≥3 re-attached fragments. figure 3. bar plot showing the mean (±se) monthly growth rates (%) of coral species in the three study sites. the number above the bar shows the number of re-attached fragments for each coral species. numbers in bold are species with ≥10 re-attached fragments/colonies. green bars represent species with ≥3 re-attached fragments. survival and growth of a re-attached storm-generated coral fragments 12 growth rates of re-attached coral fragments by species for those tagged and re-attached fragments that showed positive growth, the calculated mean (± standard error se) monthly growth rates by species (those with ≥3 colonies) ranged from 7-24%, but with high variability within and across species (figure 2a). the tagged and re-attached coral fragments exhibited mean monthly growth rates by species (those with ≥3 colonies) of about 1-5 cm2, albeit with high variability within and across species (figure 2b). among those corals with ≥10 tagged and re-attached fragments, the following showed mean monthly growth rates ranging from 2-3 cm2 (or 10-20%): echinopora horrida; millepora tenella; pavona cactus; porites attenuata; porites cyclindrica; and porites deformis (figure 2a-b; appendix figure 1a-c) growth rates of re-attached coral fragments by species and site the tagged and re-attached coral species with ≥3 fragments showed variable growth rates across and within species, but also exhibited mean monthly growth rates ranging from 5-25% in all the three study sites (figure 3a-c). it was diff icult to compare mean monthly growth rates across species and across sites because of the differences in numbers of tagged and re-attached fragments across sites by species (figure 3a-c). however, those species with ≥10 tagged and re-attached fragments across the three study sites demonstrated that coral fragments can achieve high growth rates within a month of at least 3% and up to 25% (figure 3a-c). growth rates of re-attached coral fragments by coral l ife form five coral life forms were represented by the tagged and re-attached coral fragments across the three sites in eastern samar (figure 4a-e). most of the tagged and re-attached coral fragments were of branching forms (figure 4a-e). it was diff icult to compare the growth rates of re-attached coral fragments by life form across the study sites due to the unequal and variable number of re-attached fragments per site. however, those tagged and re-attached coral fragments with ≥10 fragments showed mean monthly growth rates of at least 3% and up to 25% (figure 4a-e). j.a . anticamara and b.c.a . tan 13 figure 4. barplot showing the mean (±se) monthly growth rates (%) by coral life forms. the number above the bar shows the number of re-attached fragments for each coral species. numbers in bold are species with ≥10 re-attached fragments/ colonies. green bars represent species with ≥3 re-attached fragments. incurred cost of coral fragment re-attachment in eastern samar the total cost of re-attaching 300 coral fragments was 544 usd (us dollar), based on 50 php (philippine peso) per 1 usd conversion rate, which is equivalent to 1.8 usd per fragment (table 1). majority of the incurred cost went to logistics (e.g. , boat, land transportation, scuba rentals, and accommodation), while the cost of materials (e.g. , cable ties, concrete nails) were minimal (table 1). discussion overall, the preliminary results of this study suggest that re-attaching stormgenerated coral fragments may aid in the recovery of degrading reefs in eastern samar, as indicated by the high survival and growth rates of re-attached fragments after a year of observation. in addition, estimated growth rates by species indicated that some coral species fragments may grow relatively faster than other species survival and growth of a re-attached storm-generated coral fragments 14 by a factor of 2-3 times, although variability in growth rates was also observed within species. moreover, the incurred cost of directly re-attaching coral fragments is relatively cheaper compared to other methods that require additional costs of nursery maintenance or growing coral nubbins from coral spawns up to sizeable coral colonies for transplantation. a detailed discussion of the key f indings in relation to published literature is presented below. on survival of re-attached coral fragments this study shows that re-attached coral fragments mostly survived (88%) after a year, although some of the colonies showed partial mortality (43%). this survival rate is comparable to those observed in other places based on published literature data (appendix table 1), although we noticed that many of the studies did not account or report partial mortalities of surviving re-attached coral fragments. our table 1. the cost of re-attaching storm-generated coral fragments in eastern samar per day based on three scuba divers doing three d ives in a day. each d iver was able to re-attach 100 coral fragments per day item unit cost (usd) remarks boat 1 60 honorarium-local aids 2 20 local aids help collect fragments honorarium-divers 3 75 honorarium for 3 divers re-attaching the coral fragments underwater accommodation 1 60 scuba tanks 9 45 rental costs of 3 tanks per diver; 3 divers diving 3 times per day scuba gears set 3 150 rental costs of 3 sets of scuba gear rental per diver per day for 3 divers land transportation 1 80 transportation used to carry gear and personnel from the base to the location of the boat and back food 6 24 food for all the crew involved in coral re-attachment activities cable ties 300 20 concrete nails/pcv 300 10 total cost/day 544 j.a . anticamara and b.c.a . tan 15 results show that, if coral fragments (produced by disturbances such as typhoons, anchoring damage, or diver impacts) were regularly re-attached, then most of the reefs can retain live cover. we noticed that, during the monitoring of reefs impacted by super-typhoon haiyan, most coral fragments that were left un-attached died after sometime from the abrasive impacts of other rubble (produced by supertyphoon haiyan) when the tides and currents changed and moved. leaving unattended coral fragments produced by large-scale (typhoons) or frequent disturbances (f isher or diver impacts) have largely contributed to the decrease in live coral cover of philippine reefs. thus, investments on regular re-attachment of coral fragments per lgu (i.e. , municipalities and cities with coral reefs) should be promoted to help alleviate the decline of live coral cover in most reefs of the country. however, our results also show that re-attached coral fragments may experience partial death and some mortalities, suggesting that the re-attachment of coral fragments as conservation or recovery strategy should not be a one-shot activity, but rather a regular part of monitoring and management of both protected and exploited reefs of lgus. other studies based on reviews of published literature on coral restoration also highlighted the importance of regular monitoring of re-attached coral fragments and perhaps regular re-attachment of coral fragments within managed reef sites, in order to help reefs cope with the ever expanding and intensifying impacts of human-induced disturbances on reefs (appendix table 1). on growth of re-attached coral fragments based on our one-year observation of re-attached coral fragments in eastern samar, we found that most of the coral species that we re-attached show considerably high mean monthly growth rates, comparable to those reported in published literature (figure 2a-b, appendix table 1, and appendix figure 1a-c). most of the coral species with ≥3 re-attached colonies showed mean monthly growth rates ranging from 7-24% (1-5 cm2) (figure 2a-b). we noticed during our observations of the re-attached coral fragments that if the goal of the coral recovery was to increase the volume of coral habitable areas, then re-attaching branching species from the genera acropora, hydnopora, millepora, and porites can greatly help achieve such goal (appendix figure 1a-c). however, if the goal is to stabilize reef areas that are covered with great amount of rubble, as in the case of eastern samar reefs after super-typhoon haiyan’s impacts, then re-attaching encrusting corals from the genera echinopora, montipora, and pachyseris is benef icial in achieving such objective (appendix figure 1a-c). however, we would like to emphasize that the long-term survival and growth of a re-attached storm-generated coral fragments 16 goal of any coral recovery project and management should be to establish diversity of coral life forms in an area, ideally resembling the natural coral assemblages found in the area. thus, re-attaching coral fragments from the same vicinity should be encouraged and enforced as a protocol. nonetheless, the concerns about changes in coral disturbances related to climate change should also be considered in coral recovery activities. our observations based on the assessment of super-typhoon haiyan’s impacts in eastern samar indicate that branching coral species found in shallow reef flats and exposed to typhoons’ path will be wiped-out in the event of a typhoon impact (anticamara and go 2016). therefore, if typhoons become a frequent event impacting what used to be a branching coral reef area, then re-attachment of typhoon-resistant species (e.g. , from encrusting genera) should be considered, in order to maintain live coral cover in such a reef and to maintain its habitat and productivity, as opposed to leaving it dead. this is perhaps one climate change adaptation strategy that should be explored in philippine reef recovery and management. cost of re-attaching coral fragments the costs we incurred in re-attaching corals in eastern samar were modest, especially if we consider only the cost of the materials (i.e. , if lgus already have suff icient logistics, scuba, and personnel to suppor t such activities). we f ind it diff icult to compare our costs with those coral restoration and recovery projects in published literature because (1) most of them did not report costs, and (2) when the costs were reported, they usually did not present all the categories of coral recovery costs that we considered (i.e. , materials, logistics, scuba, personnel, and technology costs) (appendix table 1). nonetheless, we think that the costs of re-attaching coral fragments is small, and thus, should be affordable to most lgus if initial investments in big item costs, such as scuba equipment, boat, logistics, and personnel support, are already in place. the cost of materials such as cable ties, concrete nails, or pvc pipes is considerably minimal. also, the cost of re-attaching fragments should be viewed in light of all the ecosystem services and benef its provided by every km2 of reefs to the coastal communities of each lgu, from food, livelihood, recreation, and protection (de groot 2013). however, to date, in most of the lgus that we surveyed throughout the philippines and in eastern samar, there was no allocation for reef management cost, especially j.a . anticamara and b.c.a . tan 17 recovery cost in case of damage. in fact, many of the reefs that we surveyed were not assessed after super-typhoon haiyan due to lack of assessment funding allocation. this is most ironic since majority of coastal communities living near reefs in the philippines or eastern samar are heavily dependent on reefs and coastal resources for food, livelihood, and income, but there has never been any funding for regular assessment or recovery of damaged reefs. allocating a budget for reef and resource management should be tackled when improving coastal management of many reef and coastal resource-dependent lgus. interestingly, even in reefs that generate a lot of money from tourism (e.g. , batangas, hundred islands, etc.), we noticed the lack of allotment for regular reef assessment and recovery, and the huge allocation for enforcement of mpas, patrols, or fur ther infrastructure development (e.g. , building more sheds and accommodations). thus, funds for reef or coastal assessment, monitoring, and recovery are always lacking in most philippine lgus. the revision and implementation of funding allocation should be considered for each lgu for the maintenance of reef productivity, in light of increasing humaninduced disturbances and exploitation of reefs and coastal resources. conclusions and recommendations this study demonstrates that many species of coral fragments produced by disturbances (in this case, a super-typhoon) can be successfully re-attached, highlighting its potential for the recovery of degraded and impacted reefs. the results also show that different species perform variably, but in general, the branching species can produce a rapid increase in volume of available reef habitat, while the encrusting species can help stabilize rubble-covered reefs. moreover, results from this study demonstrate that reef recovery via re-attachment of coral fragments need to be executed as part of the regular program or strategies of lgus to assess, monitor, and recover degraded and exploited reefs, rather than as a onetime activity, in order to ensure continuous provisions of ecosystem benef its that coastal communities derive from reefs and coastal resources. re-attachment of coral fragments from various human-induced disturbances or large-scale impacts such as typhoons should be given consideration and funding allocation in every lgu that derive huge benef its from reefs to sustain their communities. long-term studies on coral re-attachment using multiple local species in representative sites of the philippines should be implemented to help improve the techniques and scientif ic understanding of this strategy of recovering degraded reefs of the philippines or degraded reefs in general. survival and growth of a re-attached storm-generated coral fragments 18 acknowledgements field work expenses were mainly supported by the foundation for the philippine environment and the university of the philippines-natural sciences research institute (up-nsri) research project bio-14-2-05. additional support came from the up ovcrd (off ice of the vice-chancellor for research and development) open grant project no. 141406og and the off ice of the vice president for academic affairs (ovpaa) creative writing grant, for the salaries of research assistants who helped work on this project. we would also like to thank pio angelo c. ballesteros, mark angelo b. pontica, and marlon p. gutierrez for their help during the f ield work for this study. finally, we would like to thank professor margarita de la cruz and professor filemon romero for their help in this research work. references a b e l s o n a . 2 0 0 6 . ar t i f i c i a l r ee f s v s co r a l t r a n s p l a n t a t i o n a s r e s to r a t i o n to o l s fo r m i t i g a t i n g co r a l r e e f d e te r i o r a t i o n : b e n e f i t s , co n ce r n s , a n d p r o p o s ed g u i d e l i n e s . bulletin of marine science. 78(1):151-159. ammar msa, amin em, gundacker d, mueller weg. 2000. one rational strategy for restoration of coral reefs: application of molecular biological tools to select sites for rehabilitation by asexual recruits. marine pollution bulletin. 40(7):618-627. a n t i c a m a r a j a , g o kt b , o n g s y 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evolutionary relationships of scleractinian corals. coral reefs. 15(1):1-9. white at, vogt hp. 2000. philippine coral reefs under threat: lesson learned after 25 years of community-based reef conservation. marine pollution bulletin. 40(6):537550. yap ht, alino pm, gomez e d. 1992. trends in growth and mor tality of three co r a l species (anthozoa: scleractinia), including effects of transplantation. marine ecology progress series. 83(1):91-101. yap ht, molina ra . 2003. comparison of coral growth and survival under enclosed, semi-natural conditions and in the f ield. marine pollution bulletin. 46(7):858-864. j.a . anticamara and b.c.a . tan 23 _____________ jonathan a. anticamara <jonathan.anticamara@gmail.com> is an associate professor at the institute of biology, university of the philippines diliman, quezon city, philippines. he is also a researcher at the natural science research institute, miranda hall, p. velasquez st, diliman quezon city, philippines. he is currently a coordinating lead author for the intergovernmental platform for biodiversity and ecosystem services (ipbes) asia-pacif ic regional assessment. barron cedric a. tan <tanbarroncedric@gmail.com> is a lecturer at the institute of biology, university of the philippines-diliman, quezon city, philippines. he is also a research associate in jonathan anticamara ja lab group. he is currently pursuing his graduate degree in canada. survival and growth of a re-attached storm-generated coral fragments 24 appendix figure 1 photos showing the growth selected re-attached coral colonies in (a) can-usod, (b) monbon and (c) panaloytoyon, eastern samar within one year. j.a . anticamara and b.c.a . tan 25 appendix figure 1 (cont’n.) photos showing the growth selected re-attached coral colonies in (a) can-usod, (b) monbon and (c) panaloytoyon, eastern samar within one year. survival and growth of a re-attached storm-generated coral fragments 26 appendix figure 1 (cont’n.) photos showing the growth selected re-attached coral colonies in (a) can-usod, (b) monbon and (c) panaloytoyon, eastern samar within one year. j.a . anticamara and b.c.a . tan 27 1 (cabaitan et al. philippines bolinao 12 direct 4 13,32,75,86 15 7-95 2015) transplant 2 (dela cruz et al. philippines bolinao 12 gardening 2 9,17 2-6 8-20 2015) 3 (griffin et al. 2015) us virgin 4 direct 1 45 35 98 islands transplant 4 (griffin et al. 2015) puerto rico tallaboa 48 direct 1 45 2 transplant 5 (lohr et al. 2015) cayman cayman 3 gardening 1 45 islands islands 6 (dela cruz et al. philippines bolinao 19 direct 2 55,63 47-67 68-69 0.37a,b 2014) transplant 7 (gomez et al. 2014) philippines bolinao 20 direct 1 32 91 80-98 transplant 8 (ng and chou 2014) singapore singapore 5 gardening 2 26,28 21-50 64-97 9 (romatzki 2014) indonesia north 9 electric field 2 63,66 1-6 47-100 sulawesi 10 (tortolero-langarica mexico bahía de 12 direct 3 88,89,90 10-11 75-99 et al. 2014) banderas transplant 11 (ngai et al. 2013) vietnam co to 12 direct 10 68,70,71, 0-1 0-100 11 archipelago transplant 72, 73,85, 87,93, 97, 99 12 (boch and morse palau palau 12 direct 1 47 64 47-81 12 2012) transplant 13 (garrison and ward us virgin 60 direct 1 59 4 9 13 2012) islands transplant 14 (griffin et al. 2012) puerto rico caribbean 12 gardening 1 45 99 96 14 15 (bongiorni et al. 2011) singapore singapore 12 nubbins 11 13,54,56, 0-8 34 from spawn 64,67,74, 81,89,93, 95,96 16 (guest et al. 2011) philippines bolinao 12 direct 1 81 7 10-70 transplant 17 (nakamura et al. japan okinotori 14 nubbins 1 5 37 60 9.46a, 2011) shima from spawn b,c,d 18 (borell et al. 2010) indonesia north 5 electric 2 63,66 7-21 65-100 sulawesi field ref # author and year country duration (months) site restoration type no. of species species code mean monthly % growth rate % survival cost per coral fragment or colony (usd) appendix table 1 list of coral restoration publications, their location, duration, restoration type, number of species, coral growth, coral survival, and cost per colony restored or re-attached survival and growth of a re-attached storm-generated coral fragments 28 ref # author and year country duration (months) site restoration type no. of species species code mean monthly % growth rate % survival cost per coral fragment or colony (usd) appendix table 1 list of coral restoration publications, their location, duration, restoration type, number of species, coral growth, coral survival, and cost per colony restored or re-attached (cont’n.) 19 (ferse 2010) indonesia north 24 gardening 4 57,66,76,90 5-33 0.08a sulawesi 20 (mbije et al. 2010) tanzania zanzibar 9 gardening 5 32,51,52, 26-59 56-100 0.11 58,90 21 (shafir and rinkevich israel eilat 60 gardening 3 60,89,98 36-59 50-95 2010) 22 (shaish et al. 2010) philippines bolinao 24 gardening 8 9,17,40,51, 5-20 20-100 74,80,81,89 23 (garrison and ward us virgin 60 direct 3 45,59,94 25 21.00a,b,c 2008) islands transplant 24 (shaish et al. 2008) philippines bolinao 4 gardening 7 9,17,40,51, 6-22 30-98 0.24a 80,81,89 25 (raymundo et al. philippines negros 10 recruitment 64 2007) oriental 26 (forsman et al. 2006) us hawaii 10 gardening 2 35,91 48-100 27 (shafir et al. 2006) israel eilat 5 gardening 3 50,60,64 47-56 60-90 28 (soong and chen 2003) taiwan henchun 4 gardening 1 63 17 29 (bowden-kerby 2001) puerto rico la parguera 12 direct 2 45,61 14-18 reef system transplant 30 (bruckner and puerto rico mona island 24 direct 1 59 14 69 673.13a, bruckner 2001) transplant b,c,d,e 31 (ammar et al. 2000) egypt red sea 6-12 direct 6 52,53,65, 8-93 transplant 69,89,98 32 (nagelkerken et al. curacao caribbean 4 direct 1 78 1 39 2000) transplant 33 (rinkevich 2000) israel red sea 12 gardening 1 98 0-5 50-89 34 (clark 1998) maldives high energy 28 direct 9 35,39,46, 50-80 reef flat transplant 48,53,54, 90,92,93 35 (clark and edwards maldives galu falhu 8 direct 6 39,46,53, 62-77 1994) transplant 54,90,93 36 (yap et al. 1992) philippines cangaluyan 18 direct 3 54,86,89 0-10 0-100 island transplant 37 (harriot and fisk great britain 5-7 direct 2 32,89 75 1988)f transplant j.a . anticamara and b.c.a . tan 29 38 (plucer-rosario and guam direct 4 27,49,77,83 0-100 randall 1987) f transplant 39 (auberson 1982) f philippines 12 direct 6 12,18,44,62, 44-100 transplant 79,82 40 (birkeland et al. guam 2-10 nubbins from 1 32 0-47 1979) f spawn 41 (maragos 1974) f hawaii direct 2 84,91 6-71 transplant ref # author and year country duration (months) site restoration type no. of species species code mean monthly % growth rate % survival cost per coral fragment or colony (usd) appendix table 1 list of coral restoration publications, their location, duration, restoration type, number of species, coral growth, coral survival, and cost per colony restored or re-attached (cont’n.) items included in the estimated cost per fragments: a materials (e.g., cable ties, nails, pvc, etc). b logistic cost: transportation, accommodation c scuba cost: gear rental, oxygen tank d technology cost (e.g., spawning corals and growing larvae) e other unspecified costs f taken from (harriot and fisk 1988) review paper, but cannot trace or access the original source of data. survival and growth of a re-attached storm-generated coral fragments 30 1 acropora aculeus acroporidae 2 acropora cerealis acroporidae 52.6 (1) 2.4 3 acropora latistella acroporidae 23.6 (1) 40.6 4 acropora parilis acroporidae 28.7 ± 12 (2) 17.8 ± 1.3 5 acropora tenuis acroporidae 18.4 (1) 24.7 37.3 (1) -12.6 6 cyphastrea decad ia faviidae 37.2 ± 2 (4) 15.8 ± 7.1 7 cyphastrea microphthalma faviidae 8 echinopora horrida faviidae 36.1 ± 4 (14) 9.3 ± 3.2 9 echinopora lamellosa faviidae 10.2 ± 1 (2) 21.9 ± 11.0 15.1 ± 4.8 (3) +6.8 10 echinopora mammiformis faviidae 27.2 ± 5 (2) 1.9 ± 0.5 1 1 echinopora pacificus faviidae 17.9 ± 4 (7) 16.3 ± 3.3 1 2 heliopora coerulea helioporidae 49.0 ± 7 (5) 8.7 ± 2.6 1 3 hyd nophora rigida merulinidae 34.1 ± 7 (3) 10.5 ± 4.9 14 lithophylon undulatum fungiidae 27.9 (1) 13.9 15 lobophyllia hemprichii mussidae 1 6 merulina ampliata merulinidae 16.4 ± 2 (3) 10.1 ± 5.9 17 merulina scabricula merulinidae 8.9 (1) 19.7 4.7 ± 1.4 (3) +15.0 18 millepora platyphylla milleporidae 1 9 millepora tenella milleporidae 19.9 ± 6 (16) 23.0 ± 13.0 2 0 montipora grisea acroporidae 15.9 ± 6 (3) 13.4 ± 5.4 21 montipora hirsuta acroporidae 11.4 (1) 62.6 22 montipora tuberculosa acroporidae 7.0 ± 1 (2) 37.1 ± 17.8 23 mycedium lacera pectiniidae 24 oxypora glabra pectiniidae 6.5 (1) 1.0 25 pachyseris foliosa agariciidae 15.1 (1) 26.5 2 6 pachyseris speciosa agariciidae 16.7 ± 3 (9) 24.1 ± 4.6 21.1 (1) +3.0 2 7 pavona cactus agariciidae 29.3 ± 6 (12) 7.9 ± 1.6 0.4 (1) +7.5 28 pectinia paenioa pectiniidae 29.2 ± 8 (2) 2.5 ± 1.4 50.3 (1) -47.8 2 9 porites annae poritiidae 27.9 ± 9 (5) 11.6 ± 3.2 3 0 porites attenuata poritiidae 31.3 ± 5 (20) 11.8 ± 5.1 31 porites cocosensis poritiidae 17.8 ± 1 (2) 16.0 ± 15.3 3 2 porites cyl ind rica poritiidae 26.1 ± 1 (137) 9.4 ± 1.1 55.1 ± 22.3 (3) -45.7 3 3 porites deformis poritiidae 20.2 ± 5 (15) 12.8 ± 4.0 34 porites latistella poritiidae 35 porites lobata poritiidae 22.7 ± 1 (2) 3.1 ± 1.4 0.2 (1) +2.9 36 porites monticulosa poritiidae 3 7 porites napopora poritiidae 26.6 ± 5 (8) 9.8 ± 4.0 38 porites negrosensis poritiidae 13.8 ± 4 (2) 6.8 ± 0.5 39 porites nigrescens poritiidae 14.2 (1) 1.6 4 0 porites rus poritiidae 17.3 ± 4 (3) 7.3 ± 3.5 15.6 ± 1.5 (2) -8.3 41 porites tuberculosa poritiidae 34.0 ± 0 (2) 1.2 ± 1.1 42 psammocora contigua siderastidae 43 turbinaria irregularis dendrophyliidae 44 acropora brueggemani acroporidae 45 acropora cervicornis acroporidae 30.7 ± 18.3 (5) 46 acropora cyatherea acroporidae 47 acropora digitifera acroporidae 64.2 (1) 48 acropora divaricata acroporidae 49 acropora echinata acroporidae 1.2 (1) 50 acropora eurystoma acroporidae 47.2 (1) species code species family mean initial coral size in cm2 ± se (n) e. samar mean monthly % growth rate ± se literature mean monthly % growth rate ± se (n) differences appendix table 2 list of coral species re-attached (1) in eastern samar (species code 1-43) and (2) in other areas as reported in literature (species code 44-99). also presented are the mean monthly growth rates (%) (1) observed in samar, (2) reported in literature, and (3) the difference between samar and literature growth rates whenever available. highlighted in bold are species with ≥3 re-attached colonies. j.a . anticamara and b.c.a . tan 31 species code species family mean initial coral size in cm2 ± se (n) e. samar mean monthly % growth rate ± se literature mean monthly % growth rate ± se (n) differences appendix table 2 list of coral species re-attached (1) in eastern samar (species code 1-43) and (2) in other areas as reported in literature (species code 44-99). also presented are the mean monthly growth rates (%) (1) observed in samar, (2) reported in literature, and (3) the difference between samar and literature growth rates whenever available. highlighted in bold are species with ≥3 re-attached colonies. (cont’n.) 51 acropora formosa acroporidae 21.2 ± 15.0 (2) 52 acropora hemprichii acroporidae 30.6 (1) 53 acropora humilis acroporidae 54 acropora hyacinthus acroporidae 3.8 ± 1.2 (2) 55 acropora intermedia acroporidae 46.7 (1) 56 acropora millepora acroporidae 2.72 (1) 57 acropora muricata acroporidae 58 acropora nasuta acroporidae 26.0 (1) 59 acropora palmata acroporidae 8.8 ± 4.9 (2) 60 acropora pharaonis acroporidae 45.9 ± 9.9 (2) 61 acropora prolifera acroporidae 14.2 (1) 62 acropora prominens acroporidae 63 acropora pulchra acroporidae 23.1 ± 14.9 (4) 64 acropora valida acroporidae 26.6 ± 23.5 (2) 65 acropora verweyi acroporidae 66 acropora yongei acroporidae 13.6 ± 7.9 (2) 67 diploastrea heliopora faviidae 0.02 (1) 68 echinophyllia aspera pectiniidae 1.2 (1) 69 favia stelligera mussidae 70 galaxea fascicularis oculinidae 1.1 (1) 71 goniastrea favulus faviidae 0.6 (1) 72 goniopora columna poritiidae 1.1 (1) 73 goniopora lobata poritiidae 0.01 (1) 74 heliopora coerulea helioporidae 3.0 (1) 75 hydnophora rigida merulinidae 76 isopora brueggemani acroporidae 77 leptoseris gardineri agariciidae 1.1 (1) 78 madracis mirabilis astrocoeniidae 0.9 (1) 79 millepora dichotoma milleporidae 80 montipora aequituberculata acroporidae 2.3 (1) 81 montipora digitata acroporidae 9.4 ± 1.8 (3) 82 montipora prolifera acroporidae 83 montipora pulcherrina acroporidae 0.7 (1) 84 montipora verrucosa acroporidae 85 pavona decussata agariciidae 0.6 (1) 86 pavona frondifera agariciidae 10.0 (1) 87 plesiatrea versipora faviidae 0.5 (1) 88 pocillopora capitata pocilloporidae 10.2 (1) 89 pocillopora damicornis pocilloporidae 20.0 ± 10.2 (5) 90 pocillopora verrucosa pocilloporidae 20.3 ± 9.6 (2) 91 porites compressa poritiidae 0.2 (1) 92 porites lichen poritiidae 93 porites lutea poritiidae 0.2 ± 0.2 (2) 94 porites porites poritiidae 95 porites sillimaniana poritiidae 4.1 (1) 96 psammocora digitata siderastreidae 0.01 (1) 97 pseudosiderastrea tayami siderastreidae 0.01 (1) 98 stylophora pistillata pocilloporidae 21.5 ± 19.4 (2) 99 turbinaria peltata dendrophyliidae 0.5 (1) sdinside front cover-jan.-june2019.pmd january-june 2019 • vol. 31 no. 1 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief jonas p. quilang, ph.d. university of the philippines associate editors jose maria p. balmaceda, ph.d. university of the philippines louis angelo m. danao, ph.d. university of the philippines carlos primo c. david, ph.d. university of the philippines christian n. della, ph.d. university of glasgow singapore alonzo a. gabriel, ph.d. university of the philippines arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines dennis i. merino, ph.d. southeastern louisiana university arnel a. salvador, ph.d. university of the philippines terence p. tumolva, d.eng. university of the philippines irene m. villaseñor, ph.d. university of the philippines managing editor gonzalo a. campoamor ii, ph.d. university of the philippines editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor sarah mae u. penir on the cover: teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus university of illinois, usa university of california, irvine, usa the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. department of physics university of warwick, united kingdom raul k. suarez, ph.d. professor emeritus university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences graduate school university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. ten most abudant macroinvertebrates in up diliman waterways. from top left to bottom right: chironomous sp. (non-biting midge). oligochaeta, cricotopus sp. (non-biting midge), psychoda sp. (drain fly), lymnaea sp. (air breathing freshwater snail), brachydentera sp. (shore fly), brechmorhoga sp. (club skimmer), helobdella sp. (freshwater jawless leech), labiobaetis sp. (tiny blue-winged olives), and thienamannimyia sp. (non-biting midge). background: study site beside institute of mathematics. photo taken by angelo joshua c. luciano of the institute of biology, university of the philippines diliman. 10call for papers-new.pmd social science diliman, vol. 9, number 1, january-june 2013 humanities diliman, science diliman and social science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> journal cover images courtesy of (l-r) vargas museum & magdalita et al. call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development sd inside back cover-ched-jan.-june2018.pmd science diliman: a philippine journal of pure and applied sciences is an awardee of the journal incubation grant through the journal incubation program of the commission on higher education (ched). science diliman has been selected for coverage in the emerging sources citation index of clarivate analytics (formerly thomson reuters intellectual property and science business). science diliman is listed in asean citation index and is available via www.doaj.org and www.ebsco.com 10call for papers-new.pmd editor’s note humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) vargas museum & angelo joshua a. victoria call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development notes on common-dolorosa.pmd common macrobenthic reef invertebrates of tubbataha 1science diliman (july-december 2012) 24:2, 1-11 notes on common macrobenthic reef invertebrates of tubbataha reefs natural park, philippines roger g . dolorosa* and jean beth s. jontila college of fisheries and maritime technology western philippines university, puerto princesa city 5300, philippines *corresponding author: tel. no. (+63) 048-4343908/fax. (+63) 048-4334367; email: rogerdolorosa@yahoo.com abstract macrobenthic reef invertebrates are important reef health indicators and fishery resources but are not very well documented in tubbataha reefs natural park. to provide notes on the species composition and the abundance and size of commonly encountered macrobenthic reef invertebrates, belt transects survey in intertidal, shallow, and deep subtidal reef habitats were conducted. in total, 18 species were recorded, six of which were echinoderms and 12 were mollusks, which include the rare giant clam hippopus porcellanus. only the giant clam tridacna crocea and the top shell trochus niloticus occurred in all seven permanent monitoring sites but the two species varied in densities across depths. there was also an outbreak of crown-of-thorns (cots) sea stars in some sites. the large variation in the density of each species across sites and depths suggests niche differences, overharvesting, or their recovery from having been overly exploited. separate monitoring areas for each commercially important species are suggested to determine how their populations respond to poaching and their implications on the park’s long term management. key words: macrobenthic invertebrates, marine protected area, tubbataha reefs natural park dolorosa, r.g. and jontila, j.b.s. 2 science diliman (july-december 2012) 24:2, 1-11 introduction situated within the coral triangle, the tubbataha reefs natural park (trnp) harbors some of the richest marine ecosystems in the world (white and cruztrinidad 1998, arquiza and white 1999, white and vogt 2000). covering an area of 96,828 ha, trnp is the country’s largest marine protected area (mpa) and the sole, pure marine world heritage site in southeast asia (songco and jack 2009). the north and south atolls and jessie beazley reef that comprise it feature spectacular reef formations teeming with marine life (dygico 2006). its strategic location in the middle of the sulu sea makes it an important source of fish and coral larvae that enrich the fisheries of eastern coast of palawan (see white and vogt 2000). but like many other reefs, trnp has always been a target of illegal fishermen before and even after its declaration as a national park in 1988 (tmo unpublished data, arquiza and white 1999, dygico 2006, dolorosa and others 2010, jontila and others 2011). such is a challenge to the management in preserving the globally significant biological diversity and ecological processes of trnp. to efficiently manage the park, monitoring efforts were conducted as early as the 1990s, but it was only in 1997 that monitoring and evaluation were undertaken more systematically. seven permanent monitoring sites for fish and corals at 10 m depths were established then and in 2002, replicates at depths of 5 m were added in the same sites (ledesma and others 2008). between 1997 and 2005, studies on seabirds, seagrass, planktons, large predators including sharks and cetaceans, and focal benthic mollusks species were conducted sporadically. a survey of focal benthic mollusks in 2005 followed the transect lines for coral monitoring, which was at 5 m and 10 m depths, with one additional transect in the intertidal area (dolorosa and schoppe 2005). with the incidence of illegal collection of the reef gastropod topshell trochus niloticus or ‘trochus’ in 2006, the management immediately facilitated the gathering of baseline data. seven permanent monitoring stations in the shallow subtidal areas (~1 m deep during low tide) were established and marked with cement blocks (see dolorosa and others 2010). during the second trochus monitoring in 2008, efforts were exerted at noting other macrobenthic invertebrates occurring within the trochus habitat in terms of species figure 1. tubbataha reefs natural park map showing its location in the philippines (upper right) and the permanent monitoring sites (modified after tubbataha management office brochure) composition, abundance and size. information on such is necessary to aid the management in safeguarding the species against illegal collectors. knowing where these species abound would help the management maximize their resources by focusing more on “hot spot” areas that need greater protection. methods the study was conducted in the south and north atolls of trnp (8°43’-8°57’ n latitude and 119°48’-120°3’ e longitude). the park is located amidst sulu sea, some 150 km southeast of puerto princesa city, palawan and 130 km south of cagayancillo, the municipality where the park belongs (figure 1). between may 27 and june 3, 2008, the seven permanent monitoring sites established in 2006 (see dolorosa and others 2010) at the shallow subtidal (~1.0 m deep at low tide) areas were revisited (figure 1) and replicates in intertidal (exposed at low tide) areas and at ~5 m deep reef slope were added. the selected areas were generally composed of rock substrates that favor the abundance of trochus. belt common macrobenthic reef invertebrates of tubbataha 3science diliman (july-december 2012) 24:2, 1-11 transects (100 m x 2 m) positioned parallel to the shoreline to record the abundance of echinoderms and macrobenthic mollusks were surveyed with the use of scuba gears. in total, 21 transects were surveyed covering 4,200 m2. the sizes of reef gastropods were measured either with a plastic ruler glued on a slate board or with calipers. the relaxed lengths of sea cucumbers were measured with a tape measure. noteworthy macrobenthic reef invertebrates encountered outside but along the transect lines were noted. field identification of mollusks were based on the works of poutiers (1998a, 1998b) and springsteen and leobrera (1986), while for echinoderms, the works of conand (1998) and schoppe (2000) were used. results species composition in total, 18 species were recorded, six of which were echinoderms and the rests were mollusks. the echinoderms were composed of crown-of-thorns (cots) sea star, acanthaster planci, and five species of sea cucumbers. the 12 macrobenthic mollusks were composed of eight gastropods and four bivalves, all of which were giant clams. all, except the three sea cucumber species (bohadscia argus, stichopus chloronotus, and thelenota ananas) and three mollusk species (bursa bubo, chiragra chiragra, and tridacna squamosa), occurred within the belt transects (table 1). abundance and size there was a large variation in the abundance of species across sites (table 2) and depths (table 3). the cots were only noted in deep reef slopes of sites 1 and 4 (tables 2 and 3). no size measurement was taken for the cots but their approximate diameter ranged between 20 and 40 cm. of the five sea cucumber species noted during the survey, only holothuria atra and pearsonothuria graeffei occurred within the shallow and deep subtidal family species echinodermata (asteroidea) acanthasteridae acanthaster planci echinodermata (holothuroidea) holothuriidae bohadscia argus* holothuria atra pearsonothuria graeffei stichopodidae stichopus chloronotus* thelenota ananas* mollusca (gastropoda) bursidae bursa bubo* conidae conus lividus cypraeacidae cypraea tigris strombidae chiragra chiragra* strombus luhuanus trochidae tectus pyramis trochus niloticus turbinidae turbo chrysostomus mollusca (bivalvia) tridacnidae hipoppus porcellanus tridacna crocea t. maxima t. squamosa* *noted outside the transect table 1. species of echinoderms and mollusks encountered during the 2008 survey dolorosa, r.g. and jontila, j.b.s. 4 science diliman (july-december 2012) 24:2, 1-11 habitats at relatively too low densities (tables 2 and 3). the two largest sea cucumbers found were thelenota ananas, each measuring 60 cm in length. the mean sizes of other species ranged between 24 and 33 cm (fig. 2, table 4). among the four giant clam species, only tridacna crocea consistently occurred in seven sites (table 2) with declining abundance as depth of habitat increased (table 3). for other tridacnid species, abundance was very low (tables 2 and 3). the average (± sd) shell length of t. crocea was 7.19±4.32 cm, while the hippopus porcellanus measured 28.85±6.91 cm, and the tridacna maxima was 13.12±5.64 cm (figure 3). among the reef gastropods, only trochus niloticus occured in all seven sampling sites, mainly concentrating in the shallow subtidal habitats (~1.0 m deep during low tide) of sites 1 and 2 (tables 2 and 3). the not-socommon trochid species tectus pyramis appeared to favor deeper habitats. there was a high number of strombus luhuanus in the intertidal area of site 6 (table figure 2. mean (+sd) body length of sea cucumbers encountered at trnp in 2008 figure 3. mean (+sd) shell sizes of bivalves and gastropods encountered during the survey at the trnp in 2008. all sizes were shell lengths except for maximum basal diameter for t. niloticus and t. pyramis. common macrobenthic reef invertebrates of tubbataha 5science diliman (july-december 2012) 24:2, 1-11 2). in terms of average size (±sd), t. niloticus measured (8.2±1.6 cm) while t. pyramis was about 4.8±1.1 cm in diameter. the sizes of other mollusks species are shown in figure 3. discussion the limited number of species encountered in this study could be site-related. transects were established in rocky areas suitable for the trochid species, but possibly not for others. the surveyed sites were only at the seaward reef flat and slope and did not include other important habitats within the lagoon. sea cucumbers generally favor sandy-muddy grounds with rubble (conand 1998, schoppe 2000) instead of seaward rocky habitats, which are preferred by the trochus (nash 1993). some species are nocturnal (kerr and others 2006) making them difficult to spot during the day. there are over 170 sea cucumber species in the philippines (see olavides and others 2010), thus further surveys on other potential habitats within the park may yield additional species. all five sea cucumber species recorded in trnp are harvested and sold as bêche-de-mer with varying demands (conand 1998, akamine 2002, olavides and others 2010). among these five species, pearsonothuria graeffei is of pharmacological importance because it contains substances with anticancer properties (zhao and others 2011). however, the indiscriminate harvesting of sea cucumbers has caused their populations to decline, forcing fishermen to venture to deeper and offshore reefs (anderson and others 2011), which may include marine protected areas (choo 2008). to date, all the seven giant clam species in the philippines have been recorded in the trnp. species of giant clams noted during the survey include: tridacna crocea, tridacna maxima, tridacna squamosa, and hippopus porcellanus. in 2009, tridacna derasa and empty shells of the tridacna gigas were noted (dolorosa unpublished data) and the presence of the hippopus hippopus has been previously reported (yamaguchi 1996, dolorosa and schoppe 2005). however, it can be that the finding of the h. hippopus was a misidentification of h. porcellanus (fig. 4), although the possibility that these two species can coexist cannot be disregarded. the densities of commonly encountered macrobenthic reef invertebrates in the trnp appear to vary among sampling sites and depths. considerable numbers of the a. planci in the deep subtidal (~5 m) reef slope of sites 1 and 4, with densities at 28 and 37 ind/200 m2, can be noted. these species can be seen aggregating, figure 4. the hippopus porcellanus in tubbataha reefs natural park. note the fringing tentacles on the inhalant siphon (left) and the shell that is globose in shape and semicircular in outline with smooth and regularly shaped dorsal margin (right). dolorosa, r.g. and jontila, j.b.s. 6 science diliman (july-december 2012) 24:2, 1-11 often on top of each other, while actively feeding during the day. as early as 2007, a. planci had been commonly sighted in some reefs within the trnp and in many reef areas in the mainland palawan (pers. obs.). in 2008, researchers collected more than 3,000 individual cots in the northwestern part of the north atoll, which measures about 2 h (ledesma and others 2008). in the great barrier reef, outbreaks of cots have been thought to be a natural phenomenon but have also been linked with the overexploitation of their predators and the influx of nutrients (harriott and others 2003). in trnp, the reported cots predators, such as the giant reef gastropods charonia tritonis and cassis cornuta, were not noted during the survey, but in january 2010, two c. tritonis on the reef slope and six c. cornuta partly buried in exposed sand during low tides (dolorosa unpublished data) were noted, suggesting a variation in habitats between the two species. the densities of sea cucumbers in the trnp are quite low compared with some reported densities in the pacific (see conand 1998), but their sizes appear larger or comparable with the maximum and common sizes (table 4) as reported by conand (1998) and kerr and others (2006). shiell (2004) and eriksson (2012) report that adult and juvenile sea cucumbers have separate distinct habitats; this survey was only conducted at the seaward reefs, which could be one of the reasons for having low densities and limited but large-sized samples. the density of t. crocea, which declined with depth, could also be substrate and site related. in an inner reef surrounding the ranger station of the trnp, high numbers of t. crocea aggregated in coral heads, exposed at low tide (pers. obs.), make this area a potential monitoring site for the said species. hammer and jones (1976) show that the density of the t. crocea on top and underside of coral boulders varies with distance from the shore, writing that the highest density occurs at habitats 140 m (24.1 ind m-2) from shore compared with those found at 90 m (7.6 ind m-2) and 160 m (14.4 ind m-2) from shore. in the trnp, the density of the giant clam t. crocea is higher than reported densities from other parts of the country and in many areas in the pacific (see othman and others 2010). based on the previous studies conducted in the park, it appears that the population of t. crocea has declined considerably from 440 ind/200 m2 in 1993 to 200 ind/200 m2 in 1995, down to 27 ind/200 m2 in 2005 (see dolorosa and schoppe 2005). these figures provide a snapshot of information on the general status of the species in the trnp. however, sites covered by each survey differ and results cannot be compared directly and would be insufficient to say that population of t. crocea has indeed declined over the years. to have a reliable data trend, there should be a representative number of sites and regular monitoring over the years. data should be site-specific rather than generalized, for it is important to take into account the inherent differences of each site. the density of tridacna maxima in some sites at the trnp is much higher than the reported densities from other parts of the country (othman and others 2010), but comparable with the densities in fished (0.6 ind/ 200 m2) and unfished (0.8 ind/200 m2) reefs in the maldives (basker 1991). the t. maxima, however, can reach an extremely high density of 88.3 million clams in 11.46 km-2 (gilbert and others 2006), equivalent to 1,541 ind/200 m-2. in the reao atoll, the density can be as high as 244 ind m-2 or 48,800 ind/200 m-2 (salvat 1971 in gilbert and others 2006). conversely, the hippopus porcellanus appears to be very rare in the philippines as its density is only reported in el nido, palawan at 0.0008 ind/200 m2 (see othman and others 2010). however, the high number of h. porcellanus in site 6 indicates a recovering population. it may also suggest that it is not a current target species compared to trochus whose population declined sharply in site 6 due to poaching (dolorosa and others 2010, jontila and others 2011). the mean sizes of tridacnid species recorded in the trnp, which were much smaller than their common sizes as reported by poutiers (1998a), also suggest that most individuals have yet to reach their maximum sizes. the locally threatened reef gastropod t. niloticus (da 2001) in the trnp had its mean density in 2006 (about 6,000 ind ha-1) (dolorosa and others 2010) reduced to 1,714 ind ha-1 or 34 ind/200 m2 in only two years of exploitation (jontila and others 2011). results of this common macrobenthic reef invertebrates of tubbataha 7science diliman (july-december 2012) 24:2, 1-11 study further show that only sites close to the ranger station (sites 1 and 2) contained considerable populations (table 2). despite this, the population of trochus in the trnp is exceptionally higher than at any other sites in the country (see dolorosa and others 2010) and even in some pacific islands (smith and others 2002, lasi 2010). conversely, the mean size of trochus (82 mm) is smaller than in the cartier reef (smith and others 2002), in tongavera, cook islands (chambers 2007), and in aitutaki (ponia and others 1997). the other trochid species tectus pyramis appear to prefer deeper areas although few individuals occur in the intertidal and shallow subtidal sites. its average density (2 ind/200 m2) and size (4.8±1.1 cm) are comparable with the previous report (2.38 ind/200 m2; 4.7±0.5 cm) of dolorosa and others (2010), suggesting the absence of exploitation. unlike the trochus, the shells of t. pyramis have low commmercial value (gillespie 1997) and their inclusion in the confiscated trochus collected by poachers from the trnp in 2007 (dolorosa and others 2010) is rather accidental and because the poaching happened at night. the other invertebrates in the trnp are sparsely distributed with no distinct pattern, oftentimes noted in intertidal and shallow subtidal sites. it is only strombus luhuanus that has a considerable number recorded in the intertidal areas of site 6 (70 ind/200 m2) and fewer in the intertidal and deep areas of site 5. the species seems not at all exploited because of its small size or may have quickly recovered given the similarities in mean sizes between the recent (fig. 3) and previous studies. yamaguchi (1996) reports an average size of 5.03 cm, while a common size of 5 cm is reported by poutiers (1998b). aside from the possible effects of harvesting, the low numbers of macrobenthic reef invertebrates might be related to slow recovery. overfishing of sea cucumbers (uthicke and conand 2005) and giant clams (gomez and mingoa-licuanan 2006, othman and others 2010) are common problems in the philippines and in most parts of their habitat range. once overfished, these organisms are difficult to revive because of their large size, slow growth, mode of reproduction (broadcast spawner), low fecundity, and late maturity (bruckner 2006, anderson and others 2011). it is important to note that prior to having been declared as a national park in 1988, the tubbataha reefs had already been overexploited (arquiza and white 1999) and possibly, many slow growing reef species (e.g. t. gigas) are still in the process of recovering. it is estimated that after harvesting closure, decades are required for sea cucumber populations to recover; recovery for other large macro invertebrates require a much longer time (see anderson and others 2011). to fully understand the status of sea cucumbers, giant clams, and other important reef invertebrates like crustaceans, there is a need to establish separate survey sites for each species for regular monitoring. assessment is suggested in areas dominated by sand, rubble, and sea grass at the seaward and leeward parts of the south and north atolls. different survey methods (e.g. traps) may be needed to study the crustacean resources in the trnp. the trnp is among the few mpas in the country where protection and management really work. over two decades of protection has made the fish population steadily increase and coral cover recovery relatively stable (dygico 2006, ledesma and others 2008). fish population may be overwhelming but other resources, such as macrobenthic invertebrates, may already be dwindling as with the t. niloticus. the large scale extraction of reef invertebrates (arquiza and white 1999) particularly giant clams (ticke 2002, benaventevillena and pido 2004) in the early 2000s could have seriously affected their populations, especially those of the t. squamosa and the t. gigas. all giant clam species are listed in appendix ii of cites, suggesting that “these species are not necessarily threatened with extinction but that may become so unless trade so closely controlled” (cites 2013). however, separate populations of species may have different conservation needs as some species may have become extinct in other areas (iucn 2012). as the trnp serves as a seed source for the heavily depleted reefs in mainland palawan and the surrounding reef areas within the sulu sea (see white and vogt 2000), the protection of the remaining populations of these reef invertebrates and other marine organisms in trnp is deemed important. dolorosa, r.g. and jontila, j.b.s. 8 science diliman (july-december 2012) 24:2, 1-11 table 2. mean (+sd) abundance (ind/200 m2) of echinoderms and mollusks per sampling site species sites 1 2 3 4 5 6 7 echinoderms a. plancii 9.3±16.7 12.3±21.4 h. atra 1.0±1.0 1.0±1.7 p. graeffei 0.3±0.6 mollusks t. crocea 12.7±11.0 4.7±4.6 4.3±5.8 27.0±37.3 26.3±30.8 10.7±7.1 10.7±6.0 t. maxima 1.3±2.3 h. porcellanus 3.7±5.5 0.3±0.6 t. niloticus 29.3±37.8 34.3±56.0 6.3±11.0 6.0±7.2 1.3±1.5 0.7±1.2 10.0±17.3 t. pyramis 3.3±5.8 2.3±4.0 c. tigris 0.3±0.6 c. lividus 18.0±31.2 t. chrysostomus 0.3±0.6 s. luhuanus 3.7±6.4 2.0±1.7 27.3±45.6 echinoderms a. plancii 9.3±16.1 3.1±9.9 h. atra 0.1±0.4 0.7±1.2 0.3±0.8 p. graeffei 0.1±0.4 0.1±0.2 mollusks t. crocea 26.6±27.9 10.9±5.0 3.9±3.8 13.8±18.4 t. maxima 0.6±1.5 0.2±0.9 h. porcellanus 1.4±3.8 0.1±0.4 0.1±0.4 0.6±2.2 t. niloticus 2.6±6.0 34.1±37.2 1.0±1.5 12.6±25.9 t. pyramis 2.4±4.2 0.8±2.6 c. tigris 0.1±0.4 0.1±0.2 c. lividus 7.7±20.4 2.6±11.8 t. chrysostomus 0.1±0.4 0.1±0.2 s. luhuanus 13.0±30.2 0.7±1.2 0.4±1.1 4.7±17.4 shallow subtidal (~1m during low tide) deep subtidal (~5 m)species intertidal overall table 3. mean (+sd) density (ind/200 m2) of reef invertebrates encountered during the 2008 survey at intertidal, shallow, and deep subtidal reef areas s. chloronotus (n=23) 35 (18) 39 (25) t. ananas (n= 2) 80 (45) 60 (60) b. argus (n=15) 60 (36) at least 50 37 (30) h. atra (n=28) 45 (20) about 40 52 (33) p. graeffei (n= 5) 45 (35) about 30 35 (24) maximum (common) lengths (cm) in the indo-pacific region (conand 1998) maximum size (cm) in the central philippines (kerr and others 2006) maximum (mean) lengths (cm) in this studyspecies table 4. maximum and mean lengths of sea cucumbers from trnp and those from other studies common macrobenthic reef invertebrates of tubbataha 9science diliman (july-december 2012) 24:2, 1-11 acknowledgments this study was an offshoot of the 2008 trochus survey at the trnp, which was made possible through the support of world wide fund for nature philippines (wwf-phils), the tubbataha project, and the tubbataha management office (tmo). the authors likewise acknowledge the members of the 2008 research team, the trnp rangers and the crew of minerva for their assistance during the survey and ms. allaine baaco for her comments on the early draft of the manuscript. references akamine j. 2002. trepang exploitation in the philippines: updated information. spc beche-de-mer inf. bull. 17: 17-21. anderson sc, flemming jm, watson r, lotze hk. 2011. serial exploitation of global sea cucumber fisheries. fish fish. 12: 317-339. arquiza yd, white at. 1999. tales from tubbataha. makati city, metro manila, philippines: the bookmark, inc. 190 p. basker jr. 1991. giant clams in the maldives – a stock assessment and study of their potential for culture. bay of bengal programme, reef fish research and resources survey: food and agriculture 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giant clams in the philippines. fisheries research 80: 46-52. hammer wm, jones ms. 1976. distribution, burrowing and growth rates of the clam tridacna crocea on interior reef flats – formation structures of resembling micro atolls. oecologia 24: 207-27. harriot v, goggin l, sweatman h. 2003. crown-of-thorns starfish on the great barrier reef queensland, australia. australia: crc reef research centre ltd. 6 p. iucn (international union for conservation of nature). 2012. iucn red list of threatened species, version 2012.1 [cited 2012 september 21]. available from www.iucnredlist.org jontila jbs, dolorosa rg, gonzales jb. 2011. trochus niloticus: threatened yet commercially exploited. paper presented at the international conference on biodiversity and climate change; 2011 february 1-3; manila, philippines. kerr am, netchy k, gawel am. 2006. survey of the shallowwater sea cucumbers of the central philippines: a report to the municipalities of negros oriental, cebu and bohol local bantay dagat groups. coastal conservation and education foundation, inc. and siliman university-angelo king center for research and environmental management, university of guam marine laboratory. 56 p. lasi f. 2010. trochus production in solomon islands from 1953 to 2006. spc trochus inf. bull. 15: 24-27. ledesma mc, jontila jbs, dygico mp, conales s, songco am, dolorosa rg, calderon v. 2008. tubbataha reefs natural park: research and monitoring report 2008. 65 p. nash wj. 1993. trochus. in: wright a, hill l, editors. nearshore marine resources of the south pacific: information for fisheries development and management. institute of pacific studies, suva and international centre for ocean development, canada. p 451-96. olavides r, edullantes c, juinio-meñez m. 2010. assessment of the sea cucumber resource and fishery in the bolinaoanda reef system. science diliman 22: 1-12. othman as, goh ghs, todd pa. 2010. the distribution and status of giant clams (family tridacnidae) – a short review. raffles bull. zool. 58(1): 103-11. ponia b, terekia o, taime t. 1997. study of trochus introduced to penrhyn, cook islands: 10 years later. spc trochus inf. bull. 5: 18-24. poutiers jm. 1998a. bivalves (acephala, lamellibranchia, pelecypoda). in: carpenter ke, niem vh, editors. the living marine resources of the western central pacific, volume 1: seaweeds, corals, bivalves and gastropods. rome: fao. p 123-362. poutiers jm. 1998b. gastropods. in: carpenter ke, niem vh, editors. the living marine resources of the western central pacific, volume 1: seaweeds, corals, bivalves and gastropods. rome: fao. p 364-686. schoppe s. 2000. echinoderms of the philippines: a guide to common shallow water sea stars, brittle stars, sea urchins, sea cucumbers and feather stars. visca-gtz program on applied tropical ecology, visayas state college of agriculture. 114 p. shiell g. 2004. field observation of juvenile sea cucumbers. spc beche-de-mer inf. bull. 20: 6-11. smith l, rees m, heyward a, colquhoun 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lessons learned after 25 years of community-based reef conservation. mar. pollut. bull. 40: 537-50. yamaguchi m. 1996. shallow water molluscan assemblages of the tubbataha reef, republic of the philippines. in: department of environment and natural resources (denr) and marine parks center of japan (mpcj). the report of the project for resources survey and conservation of tubbataha reefs national marine park. 200 p. zhao q, xue y, wang j-f, li h, long t-t, li z, wang y-m, dong p, xue c-h. 2011. in vitro and in vivo anti-tumour activities of echinoside a and ds-echinoside a from pearsonothuria graeffei. j. sci. food agric. 92(4): 965-74. 01-1 notes for contributors v4n1&2 january-june 2014 • vol. 26 no. 1 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june a n d d e c e m b e r ) b y t h e u n i v e r s i t y o f t h e philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief marco nemesio e. montaño, phd associate editors jose maria p. balmaceda, phd carlos primo c. david, phd joel joseph s. marciano jr. , phd jonas p. quilang, phd arnel a . salvador, phd irene m. villaseñor, phd managing editor violeda a. umali, phd editorial assistants narita e.c. de las alas dercylis g. mararac on the cover: rush hour traffic along north edsa, quezon city. photo taken by marvic a. pastrana of the ovcrd, up diliman. rigoberto c. advincula, phd department of chemistry university of houston alfonso m. albano, phd department of physics bryn mawr college, bryn mawr, pennsylvania kenneth buckel, phd food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, phd department of electrical and computer engineering ohio state university flor crisanta f. galvez, phd quality assurance and technical manager kerry ingredients and flavours (americas region) 7989-82nd st. , delta, vc v4g 1l7, canada victor c. gavino, phd department of nutrition university of montreal, canada kelvin s. rodolfo, phd department of earth and environmental sciences university of illinois, chicago, illinois rudolf a. roemer, phd centre for scientif ic computing and department of physics university of warwick luis g. sison, phd electrical and electronics engineering institute university of the philippines diliman raul k. suarez, phd department of ecology, evolution and marine biology university of california, sta. barbara contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. sd-sample article c.o. talaue and c.g. tapia 1 science diliman (january-june 2014) 26:1 1-24 solving a multi-objective transportation problem in a group decision-making setting cherryl o. talaue* institute of mathematics university of the philippines diliman cesar g. tapia institute of mathematics university of the philippines diliman _______________ *corresponding author abstract i n s o l v i n g t r a n s p o r t a t i o n p r o b l e m s , r e c e n t d e v e l o p m e n t s h a v e s e e n interest in including several kinds of attribute (other than the classical cost and prof it attributes) that may even be incommensurate with one a n o t h e r. t h e r e ex i s t s eve r a l a p p r o a c h e s i n s o l v i n g a t r a n s p o r t a t i o n p r o b l e m w i t h m u l t i p l e a t t r i b u t e s / o b j e c t i v e s . s o m e o f t h e a p p r o a c h e s allow a decision maker to input his/her preferences with respect to the multiple number of objectives that need to be concurrently optimized in a co m p r o m i s e d w a y. t h e l i te r a t u r e , h o w eve r, s e e m s t o l a c k s o l u t i o n techniques that would deal with a real life decision-making situation wherein a group of decision makers is involved but would probably have d i f f e r e n t ( e v e n c o n f l i c t i n g ) p r e f e r e n c e s i n s o l v i n g a t r a n s p o r t a t i o n problem with multiple objectives. in this research, we propose to utilize a fuzzy programming formulation and binary search technique (adopted from tapia and murtagh 1992) as a methodology to solve multi-objective t r a n s p o r t a t i o n p r o b l e m a s a g r o u p d e c i s i o n m a k i n g c o n c e r n . f u z z y programming allows the decision makers to vary at any given iteration t h e i r f u z z y a s p i r a t i o n l e v e l s i n t e r m s o f p r e f e r e n c e c r i t e r i a a n d underachievement tolerance values. since conflict in aspiration levels usually results in an infeasible situation, binary search is applied until a feasible and acceptable compromise solution is achieved. the main o b j e c t i ve o f t h i s p a p e r i s to p r o p o s e a v a l i d a n d n e w m e t h o d o l o g y. inasmuch as comparing existing methodologies is not our primary aim, we believe this is another major and serious research undertaking. keywords: transportation problem, group decision-making, fuzzy programming issn 0115-7809 print / issn 2012-0818 online solving a multi-objective transpor tation problem 2 introduction the classic transportation problem is a special class of linear program that deals with transporting a commodity from sources (e.g. , factories) to destinations (e.g. , warehouses) through a network of arcs (e.g. , highways). each source has a given supply, each destination has a given demand, and each arc that connects the sourcedestination pair has a given transportation cost (or prof it) per unit of shipment. the objective is to determine the amount of commodity to be transported from each source to each destination so that the total transportation cost (or prof it) is minimized (or maximized) while satisfying the supply and demand constraints. however, in real-life applications, for each possible transportation, other than cost (or prof it), several kinds of attribute (e.g. , average delivery time of commodities, reliability of transportation, product deterioration, etc.) are also important to consider. usually, these attributes are incommensurate with one another. moreover, in a corporate setting where several decision makers actively participate in problem solving and each one can pick his/her own shipment goal different from the others, the group of decision makers may initially express a set of conflicting or incoherent goals. hence, a number of researches have considered the transportation problem with multiple objectives and offered different solution approaches. abd el-wahed and lee (2006) have classif ied the solution approaches to the multiple objective transportation problem (motp) into four categories: (1) interactive approaches, (2) non-interactive approaches, (3) goal programming approaches, and (4) fuzzy programming approaches. ringuest and rinks (1987) and climaco and others (1993) have developed two interactive approaches that determine the satisfactory solution while maintaining the special structure of the transportation problem. the decision maker controls the search direction during the solution procedure through his/her own preferences. therefore, the main advantage of these approaches is that the eff icient solution satisf ies the preferences of the decision maker. the shortcomings of these methods are on a) the convergence of the solution procedure, which is dependent on the decision maker’s rationale and consistency in expressing his/her preferences, and b) the diff iculty in enumerating the set of eff icient solutions in large-scale problems. some of the non-interactive methods found in the literature are in aneja and nair (1979), diaz (1976 and 1979), isermann (1979), and kasana and kumar (2000). these authors have proposed different approaches to generate the set of eff icient solutions from where a decision maker chooses his/her preferred solution. one diff iculty with these techniques is that the solution process may take a long time in scanning the feasible region for the eff icient solutions. another is the diff iculty c.o. talaue and c.g. tapia 3 of the decision maker in making trade-offs among the eff icient solutions due to his/her inexperience and/or the incomplete information about the decision environment. lee and moore (1973) and aenaida and kwak (1994) have used goal programming to solve the motp. with goal programming, a decision maker can obtain a satisfying solution and likewise analyze his/her aspiration levels. as mentioned in tamiz and others (1998) and romero (1991), goal programming also has some disadvantages. the naive setting of the weights in the formulation of goal programming models can lead to wrong results. likewise, the goal programming formulation changes the well-known mathematical structure of the multi-objective transportation problem. problems could also come up during the determination of the aspiration levels. fuzzy programming is recommended as a tool to solve the motp where there is incomplete preference information provided by the decision maker. abd el-wahed (2001) and li and lai (2000) have used fuzzy programming to obtain a compromise solution to the motp. other research works utilizing fuzzy programming are described in chanas and others (1984), bit and others (1992, 1993a, 1993b), chanas and kuchta (1998), ehrgott and verma (2001), challam (1994), abd el-wahed and abo-sinna (2001), and lai and hwang (1996). fuzzy programming has likewise some shortcomings. abd el-wahed (2001) has shown that using fuzzy programming in solving motp changes the standard form of a transportation problem. li and lai (2000) have proven that the min-operator does not guarantee a solution. abd elwahed and lee (2006) have combined categories 1, 3 and 4 (see paragraph 2 of this section for the categories) of the solution approaches. some other solution approaches not mentioned in abd el-wahed and lee (2006) are found in the works of mistuo and others (1999), who used a spanning tree-based genetic algorithm for solving the motp; hong-wei and others (2009), who applied the thinking of lamarckina evolution based on fuzzy-genetic algorithm and proposed the lam-genetic algorithm technique; and amirteimoori (2010), who utilized the concept of data envelopment analysis to compute the eff iciency measure in his proposed approach. however, in our literature search, we have not found a study wherein group decisionmaking scenario is considered in motp. it is a given fact that group decision-making is a common feature in today’s corporate world. hence, we feel the need to consider a decision-making situation involving a number of decision makers who are all concerned with determining a compromise solution to the motp. solving a multi-objective transpor tation problem 4 a number of papers have proposed to solve multi-objective problems under a group decision-making environment using fuzzy programming techniques. one of these is the work of xiong and others (2013), which deals with group decisionmaking for multi-objective problem where the preferences of the decision makers are expressed by fuzzy reference points using a multi-objective evolutionary approach. a decision support model to consider consensus measure and robustness measure for fuzzy group decision-making for multi-objective problems has also been presented. wang and others (2012) have proposed to solve the multi-criteria group decision-making by using intuitionistic interval information aggregation operators. based on the intuitionistic interval, a method that applies the knowledge level of the experts to the group decision-making problem is developed. wu and others (2007) have presented a method that integrates fuzzy multi-objective linear programming with fuzzy group decision-making techniques. based on the method, a fuzzy multiple objective group decision support system is developed. xu and chen (2007) have proposed an interactive method that can be used in multi-objective group decision-making scenario where the information about attribute weights is partly known. the weights of the decision makers as well as the attribute values are expressed in triangular fuzzy numbers. the method constructs the normalized expected decision matrices by using two simple formulas and aggregates these normalized expected decision matrices into a complex decision matrix. the decision makers are then asked to provide their preference gradually in the course of interactions and by solving linear programming models, the method diminishes the given set gradually and f inds the most preferred alternative. zhang and lu (2003) have proposed an integrated fuzzy group decision-making method. this method allows group members to express fuzzy preferences for alternatives and individual judgments for solution selection criteria. the method likewise allows for the weighting of group members. the technique then aggregates these elements into a compromise group decision that is the most acceptable for the group as a whole. herrera and others (1995) have utilized the linguistic ordered weighted averaging (lowa) operator to solve group decision-making problems from individual linguistic preference relations. in most of the proposed approaches that use fuzzy techniques, the decision makers are asked to input certain preferences. infeasibility is likely to occur if the preferences are highly conflicting. asking the decision makers to input a new set of preferences usually becomes a waste of time. in this paper, we propose to obtain a satisfying solution to the motp under a group decision-making scenario that would reasonably be acceptable to all the decision makers by utilizing interactive group decision-making using fuzzy programming with preference criteria developed by tapia and murtagh (1992). c.o. talaue and c.g. tapia 5 ( = 1, 2, … , ) ( ) = 1 ( ), 2 ( ), … , ( ) min ( ) subject to = , = 1, … , (1)= = , = 1, … , (2)=1 ≥ 0 , ( ) the multi-objective transportation problem in the classic transportation problem, a commodity is to be transported from several sources to several destinations in such a way that the total transportation c o s t i s m i n i m u m . s u p p o s e t h e r e a r e m s o u r c e s s i ( i = 1 , 2 , . . . , m ) a n d n destinations d j (j = 1, 2, ..., n). each source s i has available supply a i and each destination d j has demand b j . c ij is the cost of transporting a unit of the commodity from s i to d j . we let x ij as the number of commodity to be transported from s i to d j . in real life, most transportation problems are not single objective.the mathematical model of motp can be stated as follows : where f is a vector of k objective functions and ( ) = ==1 . minf (x) f(x) the superscript on is used to identify the number of objective functions . a solution to this multi-objective problem is called a nondominated solution and we def ine it as follows: (1) (2) solving a multi-objective transpor tation problem 6 definition 1 a feasible vector (s is the feasible region) yields a nondominated solution, of the motp if, and only if, there is no other vector such that: but a number of techniques have been proposed in the literature to solve the motp. as far as we know, none of the published approaches have considered a group decisionmaking situation involving several decision makers with different and usually conflicting preferences due to differences in their choice of nondominated solutions. in this paper, we wish to obtain a nondominated solution that would satisfy all the decision makers despite their incoherent preferences. we use fuzzy programming with preference criteria technique as proposed in tapia and murtagh (1992). fuzzy programming enables the decision makers to vary at any given iteration their fuzzy aspiration levels in terms of input information, known as preference criteria and underachievement tolerance values. should there be conflict in preferences, which usually results in infeasibility, a binary search method will be employed until a feasible, eff icient and acceptable solution is obtained. these concepts will be expounded in the next section. group decision-making in solving the motp involving a number of, say l decision makers, this research applies the interactive group decision-making technique utilizing fuzzy programming with preference criteria by tapia and murtagh (1992). notions of preference criteria and percentages of achievement (concepts borrowed from tapia and murtagh 1989) and underachievement tolerance values are considered important to quantify for mathematical modeling purposes. these are used to define the membership function of a set of nondominated solutions. the value of the membership function of an element in a fuzzy set would indicate the degrees of satisfaction of the decision makers, which are expressed in terms of some aspiration levels. a solution with a high membership value signif ies that the solution is preferred compared to one with a low membership value. the proposed solution technique utilizes iteratively (a) fuzzy programming with preference criteria to obtain nondominated solutions to the motp, and (b) binary search algorithm to explore progressively nearer to a commonly desired solution. the binary search is used to arrive at a compromise ( ) 0 ∈ 0 ≤=1=1 =1=1 0 <=1=1 =1=1 , = 1,2, … , . c.o. talaue and c.g. tapia 7 ( ) 1,2, … , , ( ∗ ) 1,2, … , , . ( ) = ( ∗ ) = ( ) (3) ( ) = = max1≤ ≤ ( ∗ ) (4) (4) ( ) = ( ∗ ) (5) signifies the closeness of the k-th objective’s computed value at , to the true minimum, , over the range of values between and . ( ∗ ) ( ∗ ) 1,2, … , , 1,2, … , , ∗ = 1, … , ( ) = ( ) = 1 − ( ) − ( ∗ )− ( ∗ ) ∗ 100% ( ) , ( ) ( ) = solution at every iteration stage. the interaction of the group of l decision makers takes the form of (1) initially indicating their preference information and then (2) signifying their acceptance (or non-acceptance) of the results after each binary search for a feasible solution. in the course of the decision process, the binary search algorithm is expected to reduce the degrees of the interpersonal conflicts among l decision makers. the best acceptable compromise solution is the one that can be associated with the least possible degree of conflict. the following def initions of some concepts borrowed from tapia and murtagh (1989) are needed for the formulation of the fuzzy programming model and the binary search algorithm. definition 2 the optimal functional value of the k-th objective function, of the motp denoted by is given by subject to (1) and (2) definition 3 the worst functional value of the k-th objective function, of the motp denoted by is given by where are the k optimal decision support vectors given in (3). definition 4 the preference criterion for the k-th objective function, of the motp, denoted by , quantifies the l-th decision maker’s desire to attain the optimal functional value, , over the range of values between this optimal value and the worst value, is expressed as a value between 0 and 100. the higher the value, the higher the preference of the l-th decision maker to achieve the optimal value of the k-th objective function. definition 5 the percentage of achievement of the k-th objective function, of the motp with respect to the solution vector x, denoted by or is given by max solving a multi-objective transpor tation problem 8 ∈ ( ) ∈ definition 6 the nonnegative underachievement size that the l-th decision maker is willing to accept for the k-th objective function of the motp is denoted by . definition 7 for each objective function, , the selection criteria, , which the l-th decision maker, is going to use in searching for the best compromise solution should satisfy the following requirement: the best compromise solution should satisfy the requirement that the k selection criteria are satisf ied simultaneously for all the l decision makers. these selection criteria can be regarded as expressions of each of the l decision makers’ aspiration level for the k-th objective function. we want to search for a best compromise solution, x, in which the percentage of achievement, , of the k-th objective function is better or at least equal to the l-th decision maker’s aspiration level expressed in terms of the preference criterion, , and the underachievement tolerance value, . fuzzy programming to solve motp with multiple decision makers preliminary to fuzzy programming, the l-th decision maker, among a group of l decision makers, has to generate a f inite number of nondominated solutions to the motp satisfying his/her implicit preference structure (see tapia and murtagh 1989, 1991). we denote a nondominated solution as and let u l be the index set of alternative solutions, , favored by the l-th decision maker. we can let such nondominated solutions favored by the l-th decision maker constitute the following set: we also form the set = 1, … , = 1, … , (6) ( ) ( ) = ( ), ∈ ℱ = =1 which is the union of all the sets of favored nondominated solutions to the motp of the l decision makers. from this set, the l decision makers have a task of choosing the best compromise solution. ( ): 0 ≤ −∈ ≤ ≤ 100 for = 1, … , and = 1, … , ( ) c.o. talaue and c.g. tapia 9 ( ) ( ) using the selection criteria in (6), we now def ine the membership function, , for ; and , of a favored nondominated solution in f as follows: = 1, … , ; = 1, … , = 1, … , ( ) = ( ) − +∈100 − + ∈ 0 ≤ −∈ ≤ ( ) ≤ 1000 ( ) < − ∈ each solution can be considered to be a member of a fuzzy set whose degree of membership can be calculated using (7), with values ranging from 0 to 1. a solution with a membership value near 1 is taken to be a strongly desirable solution, while a membership value near 0 means a weakly desirable solution. we can def ine the following fuzzy sets: = ( ( ), ( )): ( ) ∈ ℱ = 1, … , ; = 1, … , where is the set of nondominated solutions of the motp and is the membership function given in (7). these fuzzy sets are also called fuzzy objectives. one of the ways of obtaining the intersection of these fuzzy objectives is by taking the minimum membership value among all the fuzzy objectives that can make up a non-empty fuzzy set . the fuzzy set can be expressed as follows: = ( ( ), min, ( )) ∶ ( ) ∈ ℱ the fuzzy set contains the solutions common to all the fuzzy objectives, and their membership values are determined by the membership function that gives the smallest membership value. the best compromise solution is then the nondominated solution in having the largest membership value, which means that it corresponds to the optimal solution of the following maximin program: max min, ( ) (8) (8) the above discussion considers a discrete set of alternatives among the number of selected nondominated solutions. the maximin program (8) can be extended to one of determining the best compromise solution among all nondominated solutions of the motp by considering the following mathematical program based on the model in tapia and murtagh ℱ ( ) (7) solving a multi-objective transpor tation problem 10 ( ∗ ) ∈ (1992), which incorporates this fuzzy algorithm into a single-objective mathematical model. we propose the fuzzy model that allows the search for a nondominated solution vector, x, which is found not only in the union of sets of favored solutions but also from among the feasible space of the nondominated solutions. (fp-motp): max z subject to: satisfies (1) and (2) of motp ( ) − + ∈100 − + ∈ ≥ ∀ , 0 ≤ −∈ ≤ ( ) ≤ 100 ∀ , ( ) ( ) = 1 − ( ) − ( ∗ )− ( ∗ ) ∗ 100 (9) (10) (11) where is as given in (3) and is as given in (4). according to a proposition in tapia (1992), fp-motp can be used to generate solutions that correspond to nondominated solutions of motp. in cases where the solution is unsatisfactory (e.g. , the value of z is very close to zero), or in the event of infeasibility, or in case the resulting feasible solution is not acceptable to the decision makers, fpmotp can be used interactively and iteratively as needed in such a way that the values of the input parameters, including the decision makers' preference criteria, , and the underachievement tolerance values, , can be modif ied by the decision makers according to their individual preference structures. infeasibility is also expected to occur should the l decision makers have highly conflicting preferences. this situation arises when one of the decision makers holds a very high aspiration for a particular objective function while the rest of the decision makers, on the contrary, hold a very low aspiration level for it. c.o. talaue and c.g. tapia 11 ∈ we apply the interactive procedure proposed in tapia and murtagh (1992), which makes use of a binary search technique to determine a combination of input parameters, and , that can give a feasible solution to the fp-motp. it is necessary to modify fp-motp in order to f ind a compromise nondominated solution that will be acceptable to all the decision makers. it is possible to reduce constraints of type (9) and of type (10) from in fp-motp by considering the most stringent constraint falling under each type. the revised fuzzy program has the following form: (rfp1) subject to: satisfies (1) and (2) of motp ( ) − (1)100 − (1) ≥ ∀ (12) 0 ≤ (1) ≤ ( ) ≤ 100 ∀ (13) (12) (13) (1) = max −∈ (14) constraints (12) and (13) represent the most stringent constraints among those of types (9) and (10), respectively, as we consider here the maximum among the aspiration levels of all decision makers for each of the objective function. if rfp1 has a feasible solution, then this must be the best compromise solution as it satisf ies all the decision makers' preferences. however, rfp1 has constraints that require the most rigorous degree of satisfaction. infeasibility therefore is likely to occur. if rfp1 does not provide a feasible solution, then the decision makers should make a compromise by way of ignoring some constraints that require a certain amount of rigor. in this case, it is necessary to solve rfp2. rfp2 is exactly the same as rfp1 except that (1) in (12) and (13) is changed to (2) where (2) = min −∈ (15) this means that rfp2 is the least rigorous model to solve for the given decision problem. hence, if rfp2 does not provide a feasible solution, then a compromise solution acceptable to all the decision makers is impossible to achieve. the proposed where pk (x) is given in (11) and l l solving a multi-objective transpor tation problem 12 ( ) ( ) algorithm is therefore not carried out for the initial set of preference criteria, , and underachievement tolerance values . the decision makers can then be required to input another set of parameters associated with aspiration levels of lower priority ranking. in case rfp2 has a feasible solution, then it should be possible to determine some revised models rfpn, n = 3, 4, . . . , n having the following formulation: (rfpn) ∈ subject to: x satisfies (1) and (2) of motp − ( )100 − ( ) ≥ ∀ (16) 0 ≤ ( ) ≤ 100 ∀ (16) (17) where is as given in (11) and for (where n is a predetermined number of iterations) can be determined by using a binary search technique applied to the set of l expressed preference criteria and underachievement tolerance values. binary search algorithm constraints (16) and (17) can be characterized as having an intermediate degree of rigor to satisfy between the most rigorous requirements, which have most recently led to an infeasible solution, and the least rigorous requirements, which have most recently led to a feasible solution. to accomplish this, a binary search technique proves to be useful in determining the next value of needed in the revised models rfpn as follows: for n = 3, 4, . . . , n ( ) = 3, 4, . . . , ( ) = 12 ( + ) (18) where inf i is the most recent value of for which rfpninf has an infeasible solution, and fes i is the most recent value of for which a feasible solution exists for rfpnfes. ( ) ( ) c.o. talaue and c.g. tapia 13 max| ( ) − ( − 1)| ≤ this interactive solution procedure for rfpn may be terminated under any one of the following circumstances: • the prescribed maximum number of iterations n is reached, in which case the most recent feasible solution may be acceptable to all the decision makers as their best compromise solution. • the decision makers have accepted the feasible solution of the n-th iteration, where n < n. • the binary search algorithm has reached an iteration stage at which a prescribed degree of convergence, say , has been reached, i.e. , if, however, the binary search algorithm has already reached an iteration stage at which a prescribed degree of convergence has been reached but the decision makers are still not satisf ied with the solution, then we ask them to input a new set of preference criteria and underachievement tolerance values, and repeat the binary search until a compromise solution will have been reached. we are expecting all of the decision makers to be consistently in a compromising posture in any decisionmaking activity. numerical example to illustrate our fuzzy motp technique for multiple decision makers, we have developed computer codes using the advanced integrated multidimensional modeling software (aimms 2012). the free aimms academic software can solve up to 5000 variables and constraints. for models with almost unlimited number of variables and constraints for commercial or industrial use, the aimms pro is recommended. the use of parameter inputs necessary for the interactive solution of our proposed multi-objective transportation methodology in a group decisionmaking setting is very convenient. complexity analysis is not our real concern and the problems of dominated momp or non-pareto-eff icient solutions is non-existent here inasmuch as we are dealing with a purely linear mathematical model, which in a known real-world setting may not hopefully become excessively so large that existing software such aimms pro would not be able to handle it. we use published data in amir teimoori (2010), as follows: an automobile manufacturer has assembly plants located in eight towns: a, b, c, d, e, f, g and h. the cars are assembled and sent to major markets in three towns: i, j, and k. the solving a multi-objective transpor tation problem 14 1 , 2 , 3 , ( ) 1 1 2 3 2 manufacturer considers one input (shipping cost) and two outputs (the value of shipment and prof it). hence, the objective functions to be optimized are the total shipping cost, total value of shipment and total cost, which we denote as and respectively. the appropriate input-output, , availabilities (a i ), and demands (b j ) are listed in table 1. these are the parameter values that we input in the computer codes that we have written in aimms. a (531, 3500, 500) (431, 380, 600) (395, 3950, 400) 10 b (394, 2850, 600) (418, 2395, 700) (512, 2590, 485) 13 c (405, 310, 800) (512, 409, 1000) (412, 390, 1100) 11 d (355, 290, 705) (493, 385, 617) (570, 419, 518) 7 e (299, 415, 585) (398, 512, 490) (315, 255, 380) 9 f (319, 512, 488) (464, 215, 305) (435, 355, 512) 9 g (619, 612, 619) (490, 510, 505) (354, 550, 490) 4 h (456, 299, 601) (394, 512, 432) (439, 499, 519) 6 dj 30 25 14 i/j i j k si table 1. the data for example. each ordered triple represents the shipping cost (k = 1), value of shipment (k = 2), profit (k = 3) of each unit of shipment from source i to destination j (i.e. ) = (531, 3500, 500), , to guide the decision makers in their input preferences (i.e. , preference criteria and underachievement tolerance values), we perform single optimization computations using our computer codes written in aimms, one for each function of the motp subject to the same constraints of the said model. the nondominated solution obtained by solely minimizing gives 25924 as the best value of while the worst value of is 29243 obtained from solely minimizing . the nondominated solution from solely maximizing gives 98234 as its best value. the worst value of is 53093 obtained from solely minimizing . lastly, the nondominated solution as a result of solely maximizing gives 47794 as the best value. the worst value of is 40952 obtained from solely maximizing . the nondominated solutions obtained are given as row-vectors in table 2. 1 2 3 3 min f 1 25924 68750 44044 max f 2 29243 98234 40952 max f 3 29343 53093 47794 f 1 f 2 f 3 table 2. the nondominated solutions from the single objective optimizations 2 c.o. talaue and c.g. tapia 15 , , = , = , = ) table 3. solution vectors, obtained from the single objective optimizations and the correspond ing percentages of achievement, of each objective function (i.e., for min = , … , = , , , i\j i j k min f 1 pa k a 4 1 5 pa 1 100.00% b 13 pa 2 34.69% c 1 3 7 pa 3 45.19% d 7 e 9 f 9 g 2 2 h 6 max f 2 pa k a 4 4 2 pa 1 0.00% b 13 pa 2 100.00% c 11 pa 3 0.00% d 7 e 9 f 9 g 2 2 h 1 5 max f 3 pa k a 3 4 3 pa 1 27.96% b 3 10 pa 2 0.00% c 1 10 pa 3 100.00% d 6 1 e 7 2 f 9 g 3 1 h 2 1 the decision vectors, x ij i = a, ..., h, j = i, j, k, and the corresponding percentages of achievement (computed using (5) of def inition (5)), are given in table 3. each l-th decision maker can use these values as a guide to determine his/her preference criterion, which is his/her aspiration level to achieve the optimal value of an objective function (see def inition 4) and his/her underachievement tolerance value which he/she is willing to accept for the k-th objective function (see def inition 6). the percentage of achievement, , of the k-th objective function corresponding to a nondominated solution serves as a guide to a decision maker as to the solving a multi-objective transpor tation problem 16 1 1 , = , . . . , h, = i, j, k , acceptability to him/her of a solution as this signifies the closeness of the objective's value at a decision vector to the true optimum over the range of values of an objective function. hence, also serves as a guide for each decision maker in assigning the inputs and for fuzzy programming. should it happen that all the decision makers are willing to accept any of the nondominated solutions out of the three single-objective optimization results, then the problem is already solved. for example, if all decision makers would choose a nondominated solution which has the highest average of percentages of achievement of the three objective functions, then all of them have the same choice, which is the nondominated solution optimizing the function, (see table 3). however, in real life, this scenario is rather unlikely to happen as each decision maker does not normally place full preference on a particular objective but rather holds partial preferences on all the objective functions. suppose there are three decision makers (l = 3). for discussion purposes, let us suppose that the decision makers have individually engaged in interactive and iterative solution processes to solve the motp (see tapia and murtagh 1989, 1991). each decision maker is assumed to have tried solving the motp a number of times and used this as a learning process to come up with his/her own preference criteria and underachievement tolerance values, which are given in table 4. these are also used as input parameters in our aimms computer codes. for example, decision maker 1 chooses a preference criterion for to be 70, because he wants to obtain a nondominated solution vector, x, for which the value of evaluated at x is 70% close to its best value of 25924 measured from its worst value of 29243. additionally, he/she chooses an underachievement tolerance value of 5 for the f irst objective function. the same explanation holds for all the other input values in table 4. using these preference structures and applying the interactive and iterative solution approach for a single decision maker proposed in tapia and murtagh (1991), we perform three multi-objective optimizations using our aimms computer codes and obtain three decision vectors, , which are given in table 4. each nondominated solution's percentage of achievement, also computed using (5), is likewise given in table 4. realistically, not all the decision makers will favor the same nondominated solution out of the three multi-objective optimization results because different decision makers are more likely to have different or inconsistent preference structures relative to the multiple objective functions (see, for example, table 4). a compromise has then to be reached and hence we now propose to apply the binary search technique. ∈ 1, 11 c.o. talaue and c.g. tapia 17 table 4. different preference structures, i.e., preference criteria and underachievement tolerance values chosen by decision makers l = 1, 2, 3; solution vectors obtained after using these preference structures in the method proposed in tapia and murtagh (1991); and the correspond ing percentages of achievement, of each objective function, k = 1, 2, 3 = , … , = , , , , 1 1 70 5 a 6 4 pa 1 70.96% 2 70 5 b 13 pa 2 70.90% 3 50 5 c 5 6 pa 3 50.01% d 7 e 9 f 9 g 4 h 6 2 1 70 5 a 10 pa 1 70.56% 2 45 5 b 13 pa 2 46.66% 3 70 10 c 11 pa 3 70.24% d 3 4 e 1 8 f 9 g 4 h 6 3 1 60 3 a 10 pa 1 60.44% 2 50 4 b 13 pa 2 50.02% 3 60 5 c 2 9 pa 3 60.06% d 7 e 9 f 4 g 4 h 6 l (decision maker) k i/j i j k pak∈ the binary search starts with the preference structures of the three decision makers g i ve n i n ta b l e 4 f r o m w h e r e t h e v a l u e s of t h e i r p r e f e r e n ce c r i te r i a a n d underachievement tolerance values are used as input parameters into our fuzzy model for group decision-making. the proposed algorithm, as implemented in the aimms environment applied to these input parameters, gives the results (presented in table 5) after eight (i.e. , n = 1, 2, . . . ,8) iterations assuming the decision makers have agreed to do this maximum number of iterations. each nondominated solution's solving a multi-objective transpor tation problem 18 ( ) 1(1) = max 70 − 5, 70 − 5, 60 − 3 = 65; 70 − 10, 60 − 5 = 60 percentage of achievement, , is likewise given in table 5. the detailed description of the inputs in each iteration step is given below. = , , … , a 5 2 3 pa 1 = 68.44% a 4 2 4 pa 1 = 67.68% b 13 pa 2 = 62.00% b 13 pa 2 = 62.56% c 1 10 pa 3 = 61.30% c 2 9 pa 3 = 61.47% d 7 d 7 e 9 e 9 f 9 f 9 g 3 1 g 3 1 h 6 h 1 5 p2(1)=65p1(1)=65 p3(1)=60 n = 3 pa k p2(2)=40p1(2)=57 p3(2)=45 n = 4 pak p 2 (3) =52.5 p 1 (3)=61 p 3 (3) =52.5n = 5 p2(4) =58.7 p1(4)=63 p3(4) =56.3n = 6 pa k p2(5) =61.9 p1(5)=64 p3(5) =58.1n = 7 p 2 (6) = 63.4 p1(6) =64.5 p3(6) =59.1n = 8 p 1 (7) =64.7 p 3 (7) =59.5 p 2 (7) =64.2 p 1 (8) =64.6 p 3 (8) =59.3 p 2 (8) =63.8 pa k n = 1 n = 2 i / j i j k i / j i j k a a b b c c d d e inf. e inf. f f g g h h a 3 2 5 pa 1 = 66.92% a 2 2 6 pa 1 = 66.16% b 13 pa 2 = 63.13% b 13 pa 2 = 63.70% c 3 8 pa 3 = 61.02% c 4 7 pa 3 = 60.57% d 7 d 7 e 9 e 9 f 9 f 9 g 3 1 g 3 1 h 2 4 h 1 5 pa k a a 2 3 5 pa 1 = 70.55% b b 1 12 pa 2 = 57.23% c c 3 8 pa 3 = 61.02% d d 7 e inf. e 9 f f 9 g g 3 1 h h 2 4 table 5. solution vectors, obtained after iterations of the binary search model rfpn and the correspond ing percentages of achievement, pak , of each objective function. = , … , = , , , c.o. talaue and c.g. tapia 19 1(1) = max 70 − 5, 70 − 5, 60 − 3 = 65; 2(1) = max 70 − 5, 45 − 5, 50 − 4 = 6570 − 10, 60 − 5 = 60 2(1) = max 70 − 5, 45 − 5, 50 − 4 = 65; and 3(1) = max 50 − 5, (1) (2) (1) (2) 3(1) 3(3) (4) 1(4) 2(4) (7) 1(7) 2(7) for the f irst iteration, the fuzzy model rfp1 needs as input parameters, , for k = 1,2,3. these input parameters are computed using (14) as follows: using the input values in table 4, . t h i s m e a n s that all the decision makers' maximum preferences for all the objectives are utilized. rfp1 gives an infeasible solution. table 5 capsulizes the result of this iteration together with the results of succeeding iterations. for the next iteration, we use (15) to compute for the values of for k = 1, 2, 3 (i.e. , . these are the least s t r i n g e n t preferences of the decision makers. since rfp2 turns out to be feasible, we can proceed to further iterations in order to search for a more stringent set of preferences that could result possibly in another feasible solution. in the third iteration, for k = 1, 2, 3 is taken to be the average of and (see (18), i.e. , average {65, 57} = 61, = average {65, 40}= 52.5, and = average {60; 45} = 52.5). by taking the average here, there is an intermediate degree of rigor that satisf ies both the most stringent preferences and the least stringent preferences of the decision makers. rfp3 turns out to be feasible. the solution obtained from rpf3 can still be improved by proceeding to the fourth iteration. noting that iteration 3 is the last iteration that gives a feasible solution while iteration 1 is the last iteration that gives an infeasible solution, we then use in rfp4: for k = 1, 2, 3 the average of and (i.e. , = average {65, 61} = 63; = average {65, 52.5} = 58.7; and = average {60, 52.5} = 56.3). rfp4 gives us a feasible solution. we can fur ther improve the solution given in rfp4 by proceeding to the next two iterations by solving rfp5 and rfp6, which both give feasible solutions. in an attempt to further improve the solution given in rfp6, we proceed to the seventh iteration and solve rfp7. in rfp7, for k = 1, 2, 3 is the average of and (i.e. , = average {65, 64.5} = 64.7; = average {65, 63.4} = 64.2; and = average {60, 59.1} =59.5). for k = 1, 2, 3 are computed in this manner since iteration 6 is the last iteration that gives a feasible solution, while iteration 1 is the last iteration that gives an infeasible solution. rfp7, however, turns out to be infeasible. 50 − 5, 70 − 10, 60 − 5 = 60 , 1(2) = min 70 − 5, 70 − 5, 60 − 3 = 57; 2(2) = min 70 − 5, 45 − 5, 50 − 4 = 40 = 40; and 3(2) = min 50 − 5, 70 − 10, 60 − 5 = 45) (3) 3(2) (1) (3) 3(4) (1) (6) 3(7) (7) solving a multi-objective transpor tation problem 20 (8) (7) 1(8) 3(8) 2(8) = 13, = 4, = 7, noting that rfp6 is the last iteration that gives a feasible solution while rfp7 is the last iteration that gives an infeasible solution, we perform the last (i.e. , predetermined) maximum number of iterations (that is n=8, i.e. solve rfp8) where for i = 1,2,3 is the average of and (i.e. , = average {64.5, 64.7} = 64.6; = average {63.4, 64.2} = 63.8; and = average {59.1, 59.5} = 59.3). rfp8 likewise turns out to be infeasible. hence, the best compromise solution is given by rfp6: . in this group decision exercise, the last feasible result can be considered as the best compromise solution because it corresponds to the most stringent combination of preferences that the binary search methodology attempts to identify. conclusion many real applications of the transportation problem consider arcs with various attributes (input/output). as a result, a number of researches have been done to solve the motp. however, literature lacks methodologies on solving the motp that involves a group of decision makers who may have varying and conflicting preferences relative to the attributes of the arcs. this paper presents a model that makes it possible to solve the motp in a group decision-making setting. the proposed motp model (fp-motp) requires the decision makers to input their fuzzy aspiration levels in the form of preference criteria and underachievement tolerance values, which signify the relative importance of each attribute of the arcs to each decision maker. to illustrate our model and methodology, we use published data in amirteimoori (2010) wherein each arc in the transportation problem involves one input and two output attributes. we demonstrate a scenario with three (3) hypothetical decision makers wherein they are required to input their fuzzy preferences signifying differently the importance of every attribute to them individually. in summary, we describe the methodology being proposed in this paper for solving a multi-objective transportation problem (motp) in a group decision-making setting as follows: step 1. each function (arc attribute) in the multiple objective transportation problem is separately optimized subject to the same set of transportation constraints. the results yield a decision support matrix that describes to the multiple decision makers the worst and the best values that could possibly be expected for each objective function (see def initions (3) and (4)). (6) = 2, = 2, = 6, = 13, = 4,= 7, = 9, = 9, = 3, = 1, = 1, = 5 c.o. talaue and c.g. tapia 21 step 2. each decision maker is allowed individually to engage in several interactive and iterative fuzzy programming attempts (see fp-motp) wherein, as in a learning process, he/she can find a preference structure consisting of preference criteria and underachievement tolerance values (see definitions (5) and (6), respectively) that he/she actually favors most, depending on the corresponding percentages of achievement of the objective functions (see def inition (5)). inevitably, it is possible that not all decision makers will have one and the same preference structure. therefore, there is a need for the decision makers to come together and look for a compromise. step 3. the binary search methodology (see section (5)) is performed. it is an impartial tool that can provide a compromise nondominated solution to the motp. ultimately, a best compromise nondominated solution is assured because it corresponds to the combination of preference criteria for all the objectives that are as high as possible relative to the initial various preference structures chosen by all the decision makers. we f inally note that in any decision-making activity involving multiple decision makers, it is to be expected that to arrive at the ultimate solution, every decision maker should be ready to compromise. acknowledgment this study received funding from the up diliman off ice of the vice-chancellor for research and development through the outright research grants. references abd el-wahed wf. 2001. a multi-objective transportation problem under fuzziness. fuzzy sets and systems 117: 27-33. abd el-wahed wf, abo-sinna ma. 2001. a hybrid fuzzy-goal programming approach to multiple objective decision-making problems. fuzzy sets and systems 119: 71-85. abd el-wahed wf, lee s. 2006. interactive fuzzy goal programming for multi-objective transportation problems.the international journal of management 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a . 1 9 9 4 . f u z z y g o a l p r o g r a m m i n g ( f g p ) a p p r o a c h t o a s t o c h a s t i c transportation problem under budgetary constraints. fuzzy sets and systems 66(3): 29-39. chanas s, kolodziejckzy w, machaj aa . 1984. a fuzzy approach to the transportation problem. fuzzy sets and systems 13: 211-221. chanas s, kuchta d. 1998. fuzzy integer transportation problem. fuzzy sets and systems 98: 18-29. climaco jn, antunes ch, alves mj. 1993. interactive decision support for multi-objective transpor tation problems. european journal of operational research 65: 58-67. diaz a. 1976. solving multi-objective problems. ekonomicko-matematicky obzor 14: 267-274. diaz a . 1979. finding a complete description of all eff icient solutions to a multiobjective transportation problem. ekonomicko-matematicky obzor 15: 62-73. ehrgott m, verma ra . 2001. note on solving multi-criteria transpor tation-location problems by fuzzy programming. asia-pacif ic operational research 18: 149-164. herrera f, herrera-v iedma e, verdegay j. 1995. direct approach processes in group decision making using linguistic owa operators. fuzzy sets and systems 79: 175-190. hong-wei z, hong-ping s, jian-qiang, l. 2009. multi-objective transportation optimization based on lam-ga. in: proceedings of the ieee international conference on intelligent computing and intelligent systems (icis 2009); 2009 november 20-22; shanghai, china. ieee. p 214-217. isermann h. 1979. the enumeration of all eff icient solutions for a linear multi-objective transpor tation problem. naval research logistics quar terly 26: 123-139. kasana hs, kumar kd. 2000. an eff icient algorithm for multi-objective transpor tation problems. asia-pacif ic operational research 17: 27-40. c.o. talaue and c.g. tapia 23 l a i y, h w a n g c . 1 9 9 6 . f u z z y m u l t i p l e o b j ec t i v e d ec i s i o n s m a k i n g : m e t h o d s a n d applications. berlin: springer. le e s a n g m , m o o r e l j . 1 9 9 3 . o p t i m i z i n g t r a n s p o r t a t i o n p r o b l e m s w i t h m u l t i p l e objectives. aiee transactions 5: 33-38. li l, lai kk. 2000. a fuzzy approach to the multi-objective transportation problem. computers & operational research 27: 43-57. mistuo g, yinzhen l, kenichi i. 1999. solving multi-objective transportation problem by spanning tree-based genetic algorithm. ieice transactions on fundamentals 12: 28022810. paragon decision technology. 2013. advanced integrated multidimensional modeling software (aimms). ri n g u e s t j l , ri n k s d b . 1 9 8 7. i n t e r a c t i ve s o l u t i o n s f o r t h e l i n e a r m u l t i o b j e c t i ve transpor tation problems. european journal of operational research 32: 96-106. romero c. 1991. handbook of critical issues in goal programming. oxford, new york: pergamon press. tamiz m, jones df, romero c. 1998. goal programming for decision-making: an overview of the current state-of-the-ar t . european journal of operational research 111: 569581. tapia c, mur tagh b. 1989. the use of preference criteria in interactive multi-objective mathematical programming. asia-pacif ic journal of operations research 6: 131-147. tapia c, mur tagh b. 1991. interactive fuzzy programming with preference criteria in multi-objective decision-making. computers and operations research 3: 307-316. tapia c, murtagh b. 1992. interactive group decision-making using fuzzy programming with preference criteria. fuzzy sets and systems 45: 13-23. wang j, han z, zhang h. 2012. multi-criteria group decision-making method based on intuitionistic interval fuzzy information. group decis negot (september 2012) doi 10.1007/s10726-012-9316-4. wu f, lu j, zhang g, ruan d. 2007. the development of a fuzzy multi-objective group decision-making support system. in: proceedings of the ieee international conference on fuzzy systems (fuzz-ieee 2007); 2007 july 23-26; imperial college, london, uk. ieee. p 670-675. xiong j, tan x, yang k, chen y. 2013. fuzzy group decision-making for multi-objective problems: tradeoff between consensus and robustness. journal of applied mathematics (2013), article id 657978, http://dx.doi.org/10.1155/2013/657978. xu z, chen j. 2007. an interactive method for fuzzy multiple attribute group decision making. information sciences 1: 248-263. solving a multi-objective transpor tation problem 24 zhang g, lu j. 2004. a group decisionmaking method with fuzzy weights for decision makers, fuzzy preferences for alternatives and fuzzy judgments for selection criteria, i n : p r o c e e d i n g s o f t h e i n t e r n a t i o n a l c o n f e r e n c e o n f u z z y i n f o r m a t i o n p r o c e s s i n g theories and applications, beijing, china, march 2003, volume 2, tsinghua university press, china, p 655-661. _______________ cherryl o. talaue, phd <cherryl@math.upd.edu.ph> is an assistant professor at the institute of mathematics, university of the philippines diliman. cesar g. tapia, phd is a retired professor of the institute of mathematics, university of the philippines diliman. editorial.pmd editorial i in this issue, we feature articles on applied mathematics, ecotoxicology, material science and engineering, and biodiversity. the diversity of data and information reflects the dynamism of the science and technology fields in the university. it is noteworthy that two of the articles featured in this issue are very relevant to current events in the country. the article on the “optimized drying parameters of water hyacinths” describes the utilization of the aquatic plant that may clog the waterways. the article on the “notes on common macro benthic reef invertebrates of tubbataha reef natural park” reveals the probable damage brought about by the uss guardian and, recently, a chinese fishing vessel that ran aground in the world heritage site. all the articles in this issue show that developing countries like the philippines practice science and technology that are significant to human endeavors. again, this is a manifestation of the nationalism of our scientists and technologists. more power to them! marco nemesio e. montaño, phd editor-in-chief sdinside front cover-jan-june2016.pmd january-june 2017 • vol. 29 no. 1 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief irene m. villaseñor, ph.d. university of the philippines diliman associate editors jose maria p. balmaceda, ph.d. university of the philippines diliman louis angelo m. danao, ph.d. university of the philippines diliman carlos primo c. david, ph.d. university of the philippines diliman christian n. della, ph.d. university of glasgow singapore alonzo a. gabriel, ph.d. university of the philippines diliman arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines diliman dennis i. merino, ph.d. southeastern louisiana university jonas p. quilang, ph.d. university of the philippines diliman arnel a. salvador, ph.d. university of the philippines diliman terence p. tumolva, d.eng. university of the philippines diliman managing editor gonzalo a. campoamor ii, ph.d. university of the philippines diliman editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor sarah mae u. penir on the cover: teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university cleveland, ohio, usa kenneth buckle, ph.d. food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, jr., ph.d. department of electrical and computer engineering ohio state university university of california, irvine university of illinois, urbana, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield sheff ield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. centre for scientif ic computing and department of physics university of warwick united kingdom raul k. suarez, ph.d. department of ecology, evolution and marine biology university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e., for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. the figure presents an overview and basic components of an automated reading tutor (art) for filipino. the art is a computer-assisted learning system that employs children’s speech signal processing technology, and oral reading fluency (orf) instruction for improving literacy. figure was taken from pascual & guevara of this issue. sd-sample article a.d.a. aniñon and others 41 science diliman (january-june 2014) 26:1 41-52 spectrophotometric determination of losartan potassium in tablets _______________ *corresponding author abstract i n t h e q u a l i t y c o n t r o l o f p h a r m a c e u t i c a l p r o d u c t s , i t i s o f u t m o s t i m p o r t a n c e t h a t v a l i d a t e d a n a l y t i c a l m e t h o d s a r e u s e d t o e n s u r e t h e cred i b i l i t y of t h e r e s u l t s g e n e r a ted . at t h e t i m e of t h e s t u d y, of f i c i a l monographs from the united states pharmacopeia and national formulary (usp-nf) for the quantif ication of losar tan potassium in tablets were unavailable, denoting the need for a validated analytical procedure for the analysis of the drug. the study adapted direct and f irst-derivative uv spectrophotometry methods proposed by bonf ilio and others (2010) for the assay of losar tan potassium in losar tan 50 mg. capsules, then m o d i f i ed a n d v a l i d a ted t h e s a i d p r o ced u r e s fo r t h e a s s a y of lo s a r t a n p o t a s s i u m i n lo s a r t a n 1 0 0 m g . t a b l e t s f o l l o w i n g t h e i n t e r n a t i o n a l conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (ich) guidelines on method validation for accuracy, precision, specif icity, linearity, limit of detection, and limit o f q u a n t i t a t i o n . re s u l t s d e m o n s t r a t e d t h a t a l l t h e p e r f o r m a n c e characteristics of both methods were highly satisfactory and conf irmed the possible application of the methods in routine analysis of losar tan potassium tablets. keywords: lo s a r t a n po t a s s i u m , u v s p ec t r o p h o t o m e t r i c d e te r m i n a t i o n , assay validation, direct and f irst-derivative spectra, hypertension issn 0115-7809 print / issn 2012-0818 online arianne diane a. aniñon richard simon r. binos karen mae m. brizuela marlyn c. corpuz w ill ison john e. de luna regine phill ine s. del rosario jesus john c. dimalala t imothy joseph p. dueñas isaac ireneo b. linatoc juan paolo d. recto melanie v. sal inas west kristian d. paraiso* university of the philippines manila spectrophotometric determination of losar tan 42 spectrophotometric determination of losartan potassium in tablets hypertension is a condition characterized by persistently elevated arterial blood pressure (wells and others 2009). it is considered as one of the most signif icant risk factors in the development of heart disease (wells and others 2009), which has been identif ied as the leading cause of mortality in the philippines (world health organization 2010). angiotensin ii receptor blockers (arb) are among the primary agents used as f irst line treatment for hypertension with compelling indications. arbs potently and selectively inhibit angiotensin ii generally by competitive binding to the at 1 receptor. losartan, an example of arb, is approved for stroke prophylaxis and is well tolerated in patients with heart failure (jackson 2006). it is available in two dosage strengths (50 mg.and 100 mg.-tablet), in different tablet preparations (core and f ilm-coated), and in combination with hydrochlorothiazide (wai fun and others 2008). although available literature has described several analytical methods for the assay of losar tan at the time of the study, no pharmacopeia has yet described a monograph for losartan drug products. the absence of a universal procedure deprives the public of an assurance on safety, quality and eff icacy; thus, validated analytical procedures are needed to quantify losartan potassium found in various drug preparations. two methods are presented in this paper by the researchers for the analysis of losartan drug products. this study aimed to validate the conditions and modify, if necessary, the direct and f irst-derivative uv spectrophotometry methods to quantify losartan potassium in 100 mg. tablets with an appropriate level of conf idence. this study is of significance to the pharmaceutical industry for the routine analysis of losartan potassium in tablet dosage form. the study also contributes to safeguarding the public against the presence of low-quality losartan drug products in the market. lastly, the study provides new and additional knowledge which can be used for future method development for the quantif ication of losartan in tablet dosage forms. the paper of bonf ilio and others (2010) described an analytical procedure for the quantitative analysis of losar tan in the capsule but not in the tablet form. by determining the validity of their analytical procedure as it applies to the quantitative analysis of losartan in tablets, and applying the international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (ich) validation parameters, the robustness of the methodology developed by bonf ilio and others (2010) were fur ther tested. a.d.a. aniñon and others 43 the study evaluated the following performance characteristics of the two methods: specif icity, accuracy, precision, linearity, range, limit of detection (lod), and limit of quantitation (loq). due to limitations in resources, the study only used the available spectrophotometer at the college of pharmacy, university of the philippines manila and a single brand of losartan potassium 100 mg. tablet (cozaar®) preparation. the study did not include simultaneous determination of possible degradation products or impurities. methods sample losartan potassium working standard with a purity of 99.8%, lot 2008-0111-06, and expiration date of october 2011 was used as reference standard and was generously provided by the institute of pharmaceutical sciences, national institutes of health, university of the philippines manila. losartan potassium (cozaar®) tablets, labeled 100 mg. losartan potassium, were obtained from a local distributor and were used as samples for the experiment. the tablets were described to contain excipients such as microcrystalline cellulose, lactose hydrous, pregelatinized starch, magnesium stearate, hydroxypropyl cellulose, hypromellose and titanium dioxide (merck and co. 2010). starch, magnesium stearate, microcrystalline cellulose, lactose, hydroxyethycellulose, hydroxymethylpropylcellulose and titanium dioxide used in the preparation of the simulated standard of excipients (sse) were of analytical grade and were provided by the department of industrial pharmacy, college of pharmacy, university of the philippines manila. locally manufactured distilled water was used as solvent in both standard and sample solutions. instrument a double-beam genesys 10s uv – visible spectrophotometer unit, with a visionlite™ se software and 1.8 nm bandwidth, was used for the spectrophotometric determination. sartorius ag analytical balance was used to weigh all reagents. all solutions were placed in a 1 cm. quartz cuvette during measurement. procedure the direct and f irst-derivative spectrophotometric procedures developed and validated by bonf ilio and others (2010) for the assay of losartan potassium in losartan 50 mg. capsules were validated in the study for the assay of losartan potassium in losartan 100 mg. tablets. twenty tablets were accurately weighed and powdered. one hundred milligrams (100 mg.) of losartan potassium was spectrophotometric determination of losar tan 44 weighed and placed on a 100 ml-volumetric flask to which 40 ml of distilled water was added. the solution was sonicated and the flask was f illed to volume. the absorbance of the standard solution (1 mg/ml) was measured from 200-300nm for both zero and f irst-order derivative spectrophotometry to obtain the . aliquots of the standard solution equivalent to 0.1-2 mg. losartan potassium were transferred into 10 ml volumetric flasks. the analytical curve was produced by plotting drug concentration versus the absorbance obtained, for both zeroorder and f irst-order derivative spectrophotometry. the calibration curve was plotted after measuring the concentration and recording the regression equation (youssef and taha 2007). the method validation was performed following ich specif ications for specif icity, accuracy, precision, linearity, range, limit of detection, and limit of quantitation. specificity specif icity was determined by comparing the sample solution to a simulated sample of excipients (sse). the sample solution (1 mg/ml) was spiked with a sse concentration of 25 ug/ml. using the handbook of pharmaceutical excipients as reference (rowe and others 2006), the percentages of the inactive components of the losartan tablet were approximated (appendix 1). the sse mixture, with an amount equal to the weight of one tablet, was mixed with 100 mg. losartan standard and dissolved in distilled water. solutions were subsequently prepared following the procedure of bonf ilio and others (2010) to obtain a f inal concentration of 5 mg/l and 10 mg/l for the direct and f irst-derivative spectra, respectively, using distilled water as diluent. accuracy accuracy was determined by recovery of known amounts of losar tan potassium standard added to the sample solution. sample solutions of 5.0 mg/l and 10 mg/l for the two spectra were mixed with adequate losartan standard solutions using serial dilutions to obtain a f inal concentration of 4.0, 5.0 and 6.0 mg/l for the direct spectrophotometry and of 8.0, 10, and 12 mg/l for the f irst-derivative spectrophotometry. all measurements were done in triplicates. precision precision was evaluated in terms of repeatability and intermediate precision. for repeatability, six standard solutions of the same concentration were measured twice λmax λmax a.d.a. aniñon and others 45 = 3 sd α (1) = 10 sd α (2) on the same day. for intermediate precision, six standard solutions of the same concentration were measured on a different day by a different analyst.the concentrations used were 5.0 mg/l and 10 mg/l solutions for the direct and f irstderivative spectra, respectively. linearity and range linearity and range were evaluated by using losartan sample solutions of 0, 4.0, 5.0, 6.0 and 7.0 mg/l for the direct derivative spectra, and 6.0, 8.0, 10, 12, 14 mg/l for the f irst-derivative spectra to produce an analytical curve. limits of detection and limits of quantitation the limits of detection (lod) and quantitation (loq) were calculated using equations (1) and (2): (1) (2) where sd is the standard deviation of the 10 blank readings (distilled water and mobile phase) and is the calibration curve slope obtained in the linearity. data analysis all data were entered and analyzed using microsoft® off ice excel® 2007. results and discussion validation of the assay procedure developed by bonf ilio and others (2010) started with the determination of the wavelength at which absorbance for the direct and f irst-derivative spectrophotometry was to be measured. the wavelength of maximum absorbance of the zero-order spectrum (appendix 2) obtained using 5.0 mg/l as the 100% nominal concentration was found to be at 205.4 nm. on the other hand, the f irst-derivative spectrum, calculated and graphed according to the absorbances measured by the instrument, showed an intense negative peak at 234 nm (appendix 3). at this wavelength, the absorbances of the losartan potassium standard solution of 4.0, 8.0, 12, 16, and 20 mg/l showed absorbance values of 0.201, 0.393, 0.564, 0.762, and 0.951, respectively. for the subsequent analyses, 10 mg/l was set at the 100% level of the analytical curve in the f irstderivative spectrophotometric method so that the analytical signal corresponds to the direct spectrophotometric method. based on the results, the wavelengths determined for both methods closely corresponded the f indings of bonf ilio and α (λmax) spectrophotometric determination of losar tan 46 others (2010) of 205 nm for the direct spectrophotometry and 234 nm for the f irst-derivative spectrophotometry. thus, subsequent absorbance for the direct spectrophotometry was done at 205.4 nm while absorbance was set at 229 and 239 nm to obtain the data for the f irst-derivative spectrophotometry. following the ich guidelines on analytical method validation, the adapted procedure from bonf ilio and others (2010) was validated for the assay of losartan potassium in losartan 100-mg tablet preparation. to reiterate, the performance characteristics tested were specif icity, accuracy, precision (repeatability and intermediate precision), linearity, range, loq, and lod. results are shown in table 1 together with the set acceptance criteria. specificity* no significant no significant passed no significant passed difference difference difference between between between spiked and spiked and spiked and unspiked unspiked unspiked absorbance absorbance absorbance curves curves curves accuracy 95-105% conc % passed conc % passed recovery per recovery# recovery concentration level* 80% 99.2 80% 104.3 (90-110%***) 100% 98.8 100% 98.5 120% 96.9 120% 101.1 precision** rsd<5% passed passed overall 2.21% repeatab. 3.28% intermed. 3.17% 3.77% precision linearity r2> 0.99**** 1.00 passed 1.00 passed p-value of y 0.66 passed 0.54 passed intercept >0.05***** range* 80-120% % conc passed % conc passed nominal nominal (mg/l) nominal (mg/l) concentration conc conc 80 4.00 80 8.00 100 5.00 100 10.00 120 6.00 120 12.00 lod* variable 0.03 mg/l passed 0.61 mg/l passed loq* variable 0.10 mg/l passed 1.86 mg/l passed * adapted from ich 2006 * * adapted from brazil, as cited in bonfilio and others 2010 * * * adapted from brazilian pharmacopeia **** adapted from bryan 2009 * * * * * adapted from chan and others 2004 #%recovery formula: % = (actual concentration/theoretical concentration) x 100 parameter acceptance criteria direct spectrophotometry result remark first-derivative spectrophotometry result remark table 1. summary of performance characteristics of direct and first-derivative spectrophotometry a.d.a. aniñon and others 47 in both methods, none of the excipients displayed any absorbance upon being spiked to a standard solution of known concentration. the excipients also did not interfere with the measurement of the losartan potassium contained in the solution as shown in the zero-order spectrum (appendix 4) and f irst-order spectrum (appendix 5) of the unspiked and spiked standard solutions. these f indings indicate that absorbance at 205.4 nm for the direct spectrophotometry, and at 229 and 239 nm for the f irstderivative spectrophotometry, is specif ic for losartan potassium. both methods exhibited good accuracy with a mean recovery range of 96.9-104.3 for the direct spectrophotometric method and 98.5-101.1 for the f irst-derivative method. being specif ic and accurate, both direct and f irst-derivative methods are therefore suitable for the determination of losartan potassium in tablets. the results of the precision study conformed to the acceptance criteria, with the overall repeatability value of 2.2120% and 3.2837%, for the direct and f irstderivative spectrophotometry, respectively, and intermediate precision of 3.1740% and 3.7659%, respectively, for the two methods. the methods were therefore expected to be insensitive to small changes in conditions (i.e. , sample preparation, weighing, dilution, time, operator). linearity was evaluated by linear regression. the coeff icient of determination (r2) values obtained from direct spectrophotometric method over the range of 4.0-6.0 mg/l was 1.00 and the equation produced was a=0.0949c-0.0068. for the f irstderivative spectrophotometric method, the r2 value obtained over the range of 8.0-12.0 mg/l was 1.00 and the calibration equation was da/dw = -0.0018c0.0002. the method was found to be linear, with the absorbance response being directly proportional to the concentration of losartan. using the formula for lod and loq, the limits of quantitation and detection were calculated to be 0.10 and 0.03 mg/l, respectively, for direct spectrophotometry whereas limits of quantitation and detection for f irst-derivative spectrophotometry were calculated to be 1.86 and 0.61 mg/l, respectively. the results indicate that the analyses were performed beyond the quantitation limit. in summary, all performance characteristics were found to be highly satisfactory. these results conf irmed the suitability of both methods for the assay of losartan potassium in losartan 100 mg. tablet preparation. no modifications to the procedure developed by bonf ilio and others (2010) were necessary to conform to the set acceptance criteria. robustness and inter-laboratory studies are recommended to provide further evidence for the applicability of both methods for routine analysis of losartan potassium in various losartan tablet preparations. spectrophotometric determination of losar tan 48 appendices appendix 1. composition of the simulated sample of excipients table 2. simulated sample of excipients* * composition based on rowe rc, sheskey pj, quinn me 2009 ingredients amount starch 0.6000 g magnesium stearate 0.1500 g microcrystalline cellulose 0.6000 g lactose 1.3500 g hydroethylcellulose 0.1922 g hydroxymethylpropylcellulose 0.0600 g titanium dioxide 0.0481 g total 3.0003 g appendix 2. losartan potassium zero-order absorption spectrum figure 1. losartan potassium zero-order absorption spectrum at 5 mg/l using distilled water as solvent. a.d.a. aniñon and others 49 appendix 3. losar tan potassium f irst-order absorption spectrum figure 2. losartan potassium f irst-order absorption spectrum at 10 mg/l using distilled water as solvent. appendix 4. comparison of the zero-order spectrum of unspiked and spiked solutions figure 3. zero-order absorption spectra of the unspiked and spiked losartan potassium aqueous solution at 5 mg/l (n=1). spectrophotometric determination of losar tan 50 acknowledgment the authors would like to thank the department of industrial pharmacy, college of pharmacy and the institute of pharmaceutical sciences, national institutes of health, university of the philippines manila for providing some of the reagents needed for the experiments. references b o n f i l i o r , f a vo r e t t o l b , pe r e i r a g r , d e ca s s i a r , azev ed o p, d e a r a u j o m . 2 0 1 0 . c o m p a r a t i v e s t u d y o f a n a l y t i c a l m e t h o d s b y d i r e c t a n d f i r s t d e r i v a t i v e u v spectrophotometry for evaluation of losar tan potassium in capsules. brazilian journal of pharmaceutical sciences 46(1): 147-155. bryan pd. 2009. what acceptance criteria and specif ications should be used for nonclinical dose formulation analysis (ncfda)? available from: http: //mediaserver.aaps pharmaceutica.com/meetings/09am/slides/11.12.09_thu/406%20ab/0700/peter%20 bryan.pdf. chan cc, lam h, lee yc, zhang x. 2004. analytical method validation and instrument performance verif ication. new jersey: john wiley & sons, inc. appendix 5. comparison of the f irst-order spectrum of unspiked and spiked solutions figure 4. first-order absorption spectra of the spiked and unspiked losartan potassium aqueous solution at 10 mg/l (n=1). a.d.a. aniñon and others 51 ermer j, miller, jh, editors. 2005. method validation in pharmaceutical analysis. germany: wiley-vch. ( i c h ) i n te r n a t i o n a l co n f e r e n ce o n h a r m o n i z a t i o n . 2 0 0 6 . va l i d a t i o n of a n a l y t i c a l p r o ced u r e s : tex t a n d m e t h o d o l o g y q 2 ( r 1 ) . av a i l a b l e f r o m : h t t p : / / p r i v a te . i c h . o r g / m ed i a s e r ve r. j s e r ? @ _ i d = 4 1 7 & @ _ m o d e = g l b . jackson ek, brunton ll, lazo js, parker kl. 2006. renin and angiotensin. in: goodman and gilman’s the pharmacological basis of therapeutics, 11thed. new york: mcgraw-hill companies, inc. p 810, 813-814. (mims) medical information management system. 2014. concise prescribing information of cozaar. available from: http://www.mims.com/philippines/drug/info/cozaar/. merck & co. , inc. 2010. cozaar: prescribing information, june 2010 ed. nj: merck & co. , inc. merck & co. , inc. 2010.the merck index, 13th ed. [cd-rom]. nj: cambridge soft. rowe rc, sheskey pj, owen sc, american pharmacists association. 2006. handbook of pharmaceutical excipients. london: pharmaceutical press. schmauser b. 2008. pharmaceutical development with focus on paediatric formulations. a v a i l a b l e f r o m : h t t p : / / a p p s . w h o . i n t / p r e q u a l / t r a i n i n g r e s o u r c e s / p q _ p r e s / w o r k s h o p _ india-aprilmay08/day_3/analy_meth_dev.ppt . wells, bg, dipiro, jt, schwinghammer, tl, dipiro, cv. 2009. pharmacotherapy handbook, 7th ed. new york: mcgraw-hill companies, inc. p 111. (who) the world health organization. 2010. who western pacif ic region – philippines – health situation and trends. available from: http://www.wpro.who.int/countries/2010/ phl/health_situation.htm. youssef ny, taha ea . 2007. development and validation of spectrophotometric, tlc and h p lc m e t h o d s fo r t h e d e te r m i n a t i o n of l a m o t r i g i n e i n p r e s e n c e of i t s i m p u r i t y. chemistry and pharmacy bulletin 55(4): 541-545. _______________ arianne diane a. aniñon is currently working as a quality assurance and regulatory pharmacist at a local distributing company. she previously worked as a research assistant for the technical team that prepared the country situational analysis on antimicrobial resistance of the philippines and as an associate to several pharmacy practice research studies. she is also currently taking her master's degree program in health policy studies at the university of the philippines manila alongside her regulatory work. richard simon r. binos is currently working at the product research and standards development division, center for drug regulation and research, food and drug spectrophotometric determination of losar tan 52 administration of the department of health, philippines. he is tasked with the formulation of technical guidelines and requirements for the registration of drug products, licensing and monitoring of establishments, and other related regulations. karen mae m. brizuela is currently a student at the university of manitoba in winnipeg city, canada under the chemistry program – specif ically geared towards its application towards pharmacological studies. she was a previous instructor at the department of pharmaceutical chemistry at the college of pharmacy, university of the philippines manila. she is also currently working on her pharmacist license registration in manitoba and is interning at a local clinical retail pharmacy. w ill ison john e. de luna is currently working at the licensing and registration division, center for drug regulation and research, food and drug administration of the department of health, philippines. he is assigned in the evaluation and processing of applications of pharmaceutical products, particularly prescription and over-the-counter drugs, herbal medicines and traditionally-used herbal products. juan paolo d. recto is a registered pharmacist who currently works in the quality assurance team of a multinational company with a corporate site in the philippines. he provides end-to-end quality oversight and ensures that local contract manufacturers, laboratories, and distributors comply with local and international quality and regulatory requirements. on top of routine operations, he also assists in cgmp audits and inspections. melanie v. sal inas is currently working on the completion of her master’s degree in pharmacology at the college of medicine, university of the philippines manila. she is particularly interested in investigating the mechanisms of action of herbal medicines. she was formerly an instructor at the department of pharmaceutical chemistry of the college of pharmacy, university of the philippines manila where she had been conducting research on pharmaceutical analysis. west kristian d. paraiso <wkdparaiso@post.upm.edu.ph> is currently a research student at the faculty of pharmaceutical sciences, hokkaido university in sapporo city, japan. he was an assistant professor at the depar tment of pharmaceutical chemistry, college of pharmacy, university of the philippines manila where he taught and performed research supervision in pharmaceutical assay validation. marlyn c. corpuz, regine phill ine s. del rosario, jesus john c. dimalala, t imothy joseph p. dueñas, and isaac ireneo b. linatoc were former up pharmacy students and were part of the class that performed this project. 01_device longakit, sotto and kelly 52 the shallow water marine sponges (porifera) of cebu, philippines ma. belinda a. longakit*1, filipina b. sotto2 and michelle kelly3 1extension services office, cebu state college of science and technology, cebu city, philippines, blongakit@yahoo.com; 2marine biology section, university of san carlos, cebu city, philippines; 3national centre for aquatic biodiversity and biosecurity, national institute of water and atmospheric (niwa) research, ltd., auckland, new zealand abstract science diliman (july-december 2005) 17:2, 52-74 thirty-three (33) species of marine sponge were identified in this study. four were identified as possibly new to science; a short description of these species is given here. in addition, one species has potential for bath sponge culture. percent similarity of species is low between stations suggesting a highly diverse sponge assemblage around the island. clustering of the stations appears to be related to distance between stations. keywords: sponges, cebu, percentage similarity, number of species *corresponding author introduction the coastline of the island of cebu has wide shallow water areas and reef flats. while many studies have been conducted on the island, few studies on sponges have been reported or published. so far, there are only the works of ruelo (1964), esmero (1978) and bakus and nishiyama (2000) that reported on the sponges in the collection of the university of san carlos, sponge fauna on artificial substrates in cebu harbor and the three species of toxic sponges, respectively. no comprehensive study on the sponge fauna of cebu has been completed, nor for the entire philippine regions, although many probate collections are known. ecologically, sponges are important components of coral reefs since their biomass and ecological tolerance frequently exceed that of the reef-building corals (ruetzler, 1978). they have unique symbiotic relationships with cyanobacteria or blue-green algae (hooper, 2000), with their own kind and with other marine organisms. they are also effective filters, filtering up to four to five times their own volume every minute (allen, 2000). they are capable of bioeroding as well as consolidating reef structures (hooper, 2000). economically, the growing preference for natural products has reinforced the market position of sponges (josupeit, 1990) as good sources of bath sponges for the cosmetic industry. some sponges (i.e. aplysina fulva and mycale microsigmatosa) showed potential to prevent marine biofouling (periera et al., 2002). the shallow water marine sponges (porifera) 53 sponges have become the focus of many medical and biochemical studies due to the presence of novel compounds and bioactive secondary metabolites which are hoped to inhibit cancerous growths and other diseases. there are about 7,000 recognized species worldwide however, it is believed that there are at least 15,000 living species (hooper, 2000). the indo-malay archipelago and south china sea have approximately 1,200 described species with the philippines having less than 500 species documented pers. com. caberoy. this region is thought to harbor high diversity of sponges estimated to range from 4,000 to 6,000 species. the many types of habitats (i.e. coral reefs, mangrove, muddy, sandy and rubble) in this region support such diverse fauna. this study aims to identify the sponges found in the shallow waters of cebu island, philippines focusing on the demosponge fauna of the intertidal and shallow subtidal (0-18 m). sponges that are possibly new to science will be described preliminarily awaiting more detailed study and specimens to finally allocate a new species name. materials and methods six sampling stations were established around cebu (fig. 1). in establishing the sampling stations, it was considered that all the bodies of water surrounding the island were represented. table 1 shows the six (6) sampling stations and the bodies of water that the stations represent. the station at san francisco (station 2) included a separate intertidal area (zone 1) due to observed abundance of sponges in one particular area. this is located about 2 km from the sampling area of the three deeper zones. the station inside a marine protected area of badian (station 4) was also included as a reference station for sponge distribution for mpas fig. 1. map of cebu showing the six sampling stations of the study. longakit, sotto and kelly 54 and at the same time as a comparison to the station outside an mpa (station 5). sponge specimens were collected from the six sampling stations from april to may 2003. collection of sponges for taxonomy was done together with the samplings for the distribution study thus the depth specification of the latter was used. four depth zones were considered for the six stations: depth zone 1 (0-2m); depth zone 2 (3-9m); depth zone 3 (10-12m) and depth table 1. the six (6) sampling stations with their respective location. station location position remarks no. ºn latitude ºe longitude 1 daanbantayan 11º13'25.8" 124º03'23.5" the station represents the shallow waters of the visayan sea and camotes sea 2 san francisco 10º41'23.2" 124º22'31.9" bound by camotes sea 3 marigondon 10º16'10.2" 123º59'30.1" located at hilutungan channel 4 badian (inside mpa) 9051'43" 123023'55" bound by tanon strait 5 badian (outside mpa) 09º53'38.8" 123º22'51.8" bound by tanon strait 6 argao 09º50'02.7" 123º34'08.0" bound by bohol strait zone 4 (13-18m). a 50-m transect line was laid parallel to the shore at every depth zone. quadrat sampling was then carried-out at 5-m interval using a 1-m2 quadrat. all the samples collected were coded (for later identification) and recorded. sponges were collected by scuba diving and snorkeling at a distance ranging from 80 m to 1,000 m from the shore. species richness, which is the total number of sponge species, is determined for each station and zone. jaccard's index of similarity and dissimilarity (bakus, 1990) was calculated to process clustering of stations using statistica, 2000. the formula used to compute this index is given below: jaccard cj = j/(a+b-j) where: j = number of species found in both stations; a = the number of species in station a; b = the number of species in station b figure 2 shows the schematic diagram of the laboratory process that was used to identify the sponges. spicule forms, sizes and their architecture together with some morphological characters (i.e. color, shape, texture, surface, sizes of pores and others) were used in the identification. only part of the sponge collection (those identified to the species level and the four sponges preliminarily identified as new species) is presented in this work. comparisons of biological data gathered will be presented in another paper.fig. 2. schematic diagram of the laboratory process in the preparation of permanent slides used in the microscopic analysis of sponges collected in the different stations of cebu, from april to may 2003. laboratory process spicule preparation section preparation cutting cutting bleaching dehydration washing clearing drying waxing mounting cutting clearing mounting the shallow water marine sponges (porifera) 55 results and discussion a total of thirty-three (33) species belonging to 29 genera, 22 families and 11 orders were identified in this study. sixteen (16) species are new to the philippines and four (4) species are possibly new to science, only a short description is given here, as the primary purpose of the paper is to provide an overall description of the fauna of cebu island. these will be described formally in a later publication. similarly, previous studies of sponges in the philippines reported several new species. wilson (1925) discovered 37 new sponge species of the 90 species he identified from the collection of the albatross expedition to the philippines in 1907-10. in 1935, de laubenfels also reported 2 new species out of the eight (8) species identified. in 1989, lèvi and lèvi reported 16 new species out of the 68 identified sponges of the south china sea with the majority collected in manila. the percentage distribution (by order) of sponges of cebu island, philippines (fig. 3) showed that the haplosclerids had the highest percentage composition at 28% followed by halichondrids and dictyoceratids at 18%. twenty-five percent (25%) of the haplosclerids were found in san francisco and marigondon, thirtythree percent (33%) of the halichondrids were recorded outside the marine protected area in badian, cebu and forty-five percent (45%) of the dictyoceratids were found in san francisco. the dominance of the haplosclerid sponges of cebu is comparable to what is reported for ilocos region (caberoy, 1997) which is 24% and the sponges of west central pacific (de laubenfels, 1954) which is 22%. this is lower compared to what was reported for the motupore island, papua new guinea sponge fauna (kelly-borges and bergquist, 1988) which is 40% (10 species out of 25 species). genus haliclona contains the most number of species within the order haplosclerida (4 haliclona species out of the 9 haplosclerid species). it has always been assumed that the observed habitat specialization of adult individuals results from selective mortality following unselective settlement. the combination of swimming or crawling behavior, duration of free life and phototactic response displayed by larvae such as haliclona sp., combined with unspecific requirements as to settlement surface, works to minimize the dispersion of the larvae into unsuitable habitats (bergquist, 1978), thus ensuring higher survival compared to the other genus. astrophorida astrophoridaastrophorida 3% ‘lithistid’ sponges 3% hadromerida 3% poecilosclerida 12%halichondrida 18% haplosclerida 28% dictyoceratida 18% dendroceratida 3% verongida 3% homosclerophorida 6% spirophorida 3% longakit, sotto and kelly 56 a synoptic list of the sponge species found in cebu, philippines is presented below followed by the description of each species. synoptic list of the sponge species found in six (6) stations around cebu island, philippines phylum porifera grant, 1836 class demospongiae sollas, 1885 subclass homoscleromorpha bergquist, 1978 order homosclerophorida dendy, 1905 family plakinidae schulze, 1880 genus corticium schmidt, 1862 corticium sp. nov. genus plakortis schulze, 1880 plakortis lita de laubenfels, 1954 subclass tetractinomorpha lèvi, 1953 order spirophorida bergquist and hogg, 1969 family tetillidae sollas, 1886 genus paratetilla dendy, 1905 paratetilla bacca (selenka, 1867) order astrophorida sollas, 1888 family ancorinidae schmidt, 1870 genus rhabdastrella thiele, 1903 rhabdastrella sp. nov. order hadromerida topsent, 1894 family clionaidae d'orbigny, 1851 genus spheciospongia marshall, 1892 spheciospongia vagabunda (ridley, 1884) 'lithistid' demospongiae family theonellidae lendenfeld, 1903 genus siliquariaspongia hoshino, 1981 siliquariaspongia cf. mirabilis (de laubenfels,1954) order poecilosclerida topsent, 1928 suborder microcionina hadju, van soest and hooper, 1994 family microcionidae carter, 1875 subfamily microcioninae carter, 1875 genus clathria schmidt, 1862 subgenus thalysias duchassaing and michelotti, 1864 clathria (thalysias) reinwardti vosmaer, 1880 family raspailiidae hentschel, 1923 subfamily echinodictyinae hooper and van soest, 2002 genus echinodictyum ridley,1881 echinodictyum cf. conulosum kieschnick, 1900 suborder myxillina hadju, van soest and hooper, 1994 family iotrochotidae dendy, 1922 genus iotrochota ridley, 1884 iotrochota baculifera ridley, 1884 suborder mycalina hadju, van soest and hooper, 1994 family desmacellidae ridley and dendy, 1886 genus biemna gray, 1867 biemna fortis (topsent, 1897) order halichondrida gray, 1867 family axinellidae carter, 1875 genus axinella schmidt, 1862 axinella carteri (dendy, 1889) genus phakellia bowerbank, 1862 phakellia cavernosa (dendy, 1922) family desmoxyidae hallman, 1917 genus higginsia higgin, 1877 higginsia cf. mixta (hentschel, 1912) family dictyonellidae van soest, diaz and pomponi, 1990 genus liosina thiele, 1899 liosina paradoxa thiele, 1899 genus stylissa hallmann, 1914 stylissa massa (carter, 1889) family halichondriidae gray, 1867 genus axinyssa lendenfeld, 1897 axinyssa cf. topsenti lendenfeld, 1897 order haplosclerida topsent, 1928 suborder haplosclerina topsent, 1928 family callyspongiidae de laubenfels, 1936 genus callyspongia duchassaing and michelotti, 1864 subgenus callyspongia duchassaing and michelotti, 1864 the shallow water marine sponges (porifera) 57 callyspongia (callyspongia) aerizusa desqueyroux-faundez, 1984 callyspongia (callyspongia) muricina (lamarck, 1813) family chalinidae gray, 1867 genus dendroxea griessinger, 1971 dendroxea sp. nov. genus haliclona grant, 1836 haliclona amboinensis (lèvi, 1961) haliclona cymiformis (esper, 1794) haliclona poseidon (de laubenfels, 1954) haliclona sp. nov. family niphatidae van soest, 1980 genus cribrochalina schmidt, 1870 cribrochalina olemda de laubenfels, 1954 suborder petrosina boury-esnault and van beveren, 1982 family petrosiidae van soest, 1980 genus xestospongia de laubenfels, 1932 xestospongia testudinaria (wilson, 1925) order dictyoceratida minchin, 1900 family thorectidae bergquist, 1978 subfamily thorectinae bergquist, 1978 genus hyrtios duchassaing and michelotti, 1864 hyrtios erecta (keller, 1889) genus luffariella thiele, 1899 luffariella cf. variabilis polejaeff, 1884 genus dactylospongia bergquist, 1965 dactylospongia cf. elegans thiele, 1899 subfamily phyllospongiinae bergquist, sorokin and karuso, 1999 genus carteriospongia hyatt, 1877 carteriospongia flabellifera (bowerbank, 1877) family spongiidae gray, 1867 genus spongia linnaeus, 1759 spongia zimocca sensu de laubenfels, 1954 family dysideidae gray, 1867 genus dysidea johnston, 1842 dysidea cf. arenaria bergquist, 1965 order dendroceratida minchin, 1900 family dictyodendrillidae bergquist, 1980 genus igernella topsent, 1905 igernella mirabilis lèvi, 1961 order verongida bergquist, 1978 family pseudoceratinidae carter, 1885 genus pseudoceratina carter, 1885 pseudoceratina verrucosa bergquist, 1995 description of the sponges of cebu island, philippines phylum porifera grant, 1836 class demospongiae sollas, 1885 subclass homoscleromorpha bergquist,1978 order homosclerophorida dendy, 1905 family plakinidae schulze, 1880 genus corticium schmidt, 1862 corticium sp. nov. (plate 1, fig. 1) description: encrusting sponge with round edges (38 mm at the longest portion, 28 mm wide and 10 mm thick); surface is smooth but granular to the touch; texture is firm, cartilaginous and difficult to tear. external color in life and in alcohol is shiny jet black, interior is grayish to light brown. mesoscleres are calthrops, irregular non-lophose (non-branching) in one size category (range: 38.4-76.8 µm; mean: 60.5 µm); and candelabrum, with three basal equally ramified actines and the fourth actine ramifies basally in 4-10 longer and thinner microspined rays (range: 24 36 µm; mean: 0.5 µm). the ectosome is well-defined, the choanosome is composed of spicules scattered between choanocyte chambers; candelabra are more concentrated at the surface and edges of canals. found encrusting on a rock at 9 m. remarks: corticium sp. nov. is closely related to c. candelabrum schmidt, 1862 in terms of the size of spicules but differ in color, the former is pale yellow or longakit, sotto and kelly 58 brown in life while the latter is black on the external and grayish on the internal. further specimens are required before a final species allocation can be made. distribution: philippines: cebu badian (present study) genus plakortis schulze, 1880 plakortis lita de laubenfels, 1954 (plate 1, fig. 8) description: thickly encrusting sponge (51 mm long, 25 mm wide and 20 mm thick); surface is smooth with contractile oscules that are hard to detect when the sponge is taken out of the water; texture is soft and compressible, fleshy and easy to tear. in life, the sponge is reddishbrown that is a little brighter and darker at the ectosome than the endosome. in alcohol, its color is brown with the ectosome darker than the endosome. mesoscleres are diods, with straight and sinuous rays (range: 99-120 x 2.9-3.5 µm; mean: 108 x 3.2 µm); triods are occasionally present. spicules are densely packed all throughout, without differential location of spicules. encrusting on empty bivalve shells and coral rubbles at depths 5-17 m. remarks: bakus and nishiyama, 1999 reported this sponge as one of the toxic sponges found in cebu. this is quite a common sponge in the island, present in three of the six stations. distribution: west-central pacific (de laubenfels, 1954); south korea (sim, 1985); vanuatu (niwa collection); indonesia (niwa collection); fiji (niwa collection); philippines: cebu mactan island (bakus and nishiyama, 1999); san francisco, daanbantayan and maribago (present study) subclass tetractinomorpha lèvi, 1953 order spirophorida bergquist and hogg, 1969 family tetillidae sollas, 1886 genus paratetilla dendy, 1905 paratetilla bacca (selenka, 1867) (plate 1, fig. 9) description: globular sponge (fragment: 110 mm at longest portion and 62 mm wide) with numerous porocalices 7-12 mm in diameter; surface is uniformly hispid caused by protruding spicules; texture is firm and slightly compressible. color of live specimen is bright yellow for the endosome and greenish to brown at the ectosome caused by either the epiphytic algae or the accumulated sand, mud or detritus trapped at the protruding spicules. in alcohol, the color of the endosome is light brown and the ectosome is dark brown. megascleres are protriaenes (only few were observed), with three straight clads and long, straight shaft anatriaenes, with sharply curved clads and long and thick shaft (range: 4,925-5,801 x 4.8-7.2 µm; mean: 5,363 x 6 µm), orthotriaenes, with shaft shorter than the clads resembling calthrops (range of shaft: 40-119 x 12-19.2 µm; mean, 83 x 16 µm; range of clad: 99-218 x 9.6-19.2 µm; mean: 155 x 15.5 µm), oxeas, huge and very long (range: 1,725 4,473 x 19.8-50 µm; mean: 2,841 x 28.4 µm); microscleres are sigmas, finely spined (range: 14-17 x 1 µm; mean: 16 x 1 µm). radial arrangement is very evident with bundles of oxea radiating from a central focus; a specialized dermal layer of modified triaenes, resembling calthrops is present. found at a depth of 18 m among coral rubbles. distribution: indo-west pacific: samoa, mayanmar, ne australia, sri lanka (van soest and hooper, 2002); mauritius (niwa collection); indonesia (van soest and hooper, 2002; niwa collection); maldives (niwa collection); philippines: la union, ilocos sur, ilocos norte (caberoy, 1997); cebu argao (present study) order astrophorida sollas, 1888 family ancorinidae schmidt, 1870 genus rhabdastrella thiele, 1903 rhabdastrella sp.nov. (plate 1, fig. 2) description: encrusting sponge (130 mm, 63 mm wide and 20-30 mm thick); surface is microscopically hispid with micropores evident at the portion in contact with haliclona amboinensis (lèvi, 1961), oscula (25 mm in diameter) are located at portions free of any attachments; texture is fleshy, slightly compressible, rubbery and difficult to tear. out of water and in preservative, the color is grayish black. spicules are oxeas (range: 239-873 x 7.1-28.6 µm; mean: 660.3 x 14.3 µm), spherasters (range: 16.8-40.8 µm; mean: 31.9 µm), spheroxyasters (range: 16.8-38.4 µm; mean: 27.7 µm), and oxyasters (range: 43.2-60 µm; mean: 53.3 µm). oxeas are radially arranged forming bundles that run perpendicular to the surface while the euasters are randomly scattered at the innermost zone, spherasters the shallow water marine sponges (porifera) 59 and spheroxyasters are mostly found at the cortex. found at 10-16 m in a coral reef area. remarks: rhabdastrella sp. nov. is found to be in close association (always appearing as the underside) with haliclona amboinensis (lèvi, 1961). this is closely related to rhabdastrella disctincta (thiele, 1900) from indonesia however there was no mention of any close association with another sponge. further specimens are required before a final species allocation can be made. distribution: indonesia (niwa collection); phillipines cebu: mactan island (niwa collection); badian (present study) order hadromerida topsent, 1894 family clionaidae d'orbigny, 1851 genus spheciospongia marshall,1892 spheciospongia vagabunda (ridley, 1884) (plate 1, fig. 10) description: irregular in shape (105 mm long, 60 mm wide and 15-30 mm thick); surface is hispid and has steep-sided conical projections (8-12 mm high and 5-9 mm wide), grooves filled with calcitic materials, ostia are not visible while the oscula located at the apex of the conules could not be easily seen out of water; texture is hard, corky, not readily compressible and difficult to tear. its color in life and in preservative, is brown with the top of conules darker than the other parts of the sponge due to heavy concentration of pigments. megascleres are tylostyles of two size categories, with terminal or sub-terminal heads slightly curve at the anterior half and pointed sharply (i. range: 429-600 x 7.1-14.2 µm; mean: 522.6 x 9.7 µm, ii. range: 143-329 x 2.8-8.1 µm; mean: 222 x 5.0 µm); microscleres are finely spined spirasters (range: 10.313.7 µm; mean : 11.3 µm). spicules are tightly packed and confused, crisscrossing each other with some protruding to the surface. found at 6 m depth in an area with coral rubbles and sandy substrate. remarks: as described by kelly-borges and bergquist (1988), the sponge specimen collected form cebu is a juvenile. distribution: indonesia (van soest, 1989); indowest pacific, fiji islands (tendal, 1969); palau (bergquist, 1965); papua new guinea (kelly-borges and bergquist, 1988); philippines: cebu mindoro (de laubenfels, 1935); la union, ilocos norte, ilocos sur (caberoy, 1997); san francisco (present study) 'lithistid' demospongiae family theonellidae lendenfeld, 1903 genus siliquariaspongia hoshino, 1981 siliquariaspongia cf. mirabilis (de laubenfels, 1954) description: irregularly encrusting (105 mm long and 43 mm wide) with short tubular projections (7-12 mm high, 10-11 mm wide) distributed 14-20 mm apart; surface is wrinkly and uneven but the tubes are smooth, ostia are not visible but the oscula are terminal located at each tube (4 mm in diameter); texture is spongy, crumbly and easy to tear. in life, ectosome is reddish brown and endosome is yellowish brown; in alcohol, the color is orange brown. spicules are strongyles, long and smooth (range: 393.6-556.8 x 4.8-10.8 µm; mean: 442.6 x 7.8 µm), desmas are non-articulated tetraclone (range: 268.8-374.4 µm; mean: 306 µm), microrhabds, are straight to slightly curved and spiny (range: 7.2-12 x 1.2 µm; mean: 10.6 x 1.2 µm). ectosome seems to be devoid of desmas or may be present sparsely but it has high concentration of rhabds; in areas with conules, ascending tracts of strongyles (55-82 µmnin diameter) are present terminating at its crest; desmas are present in great number at the choanosome or at the area below the conules, arranged in random. attached to a reef at 10 m. remarks: siliquariaspongia cf mirabilis (de laubenfels, 1954) is somewhat related to placinolopha mirabilis however, it is not a true member of the homosclerophorid genus due to the presence of non-articulated desmas and a skeleton highly reminiscent of the lithistid genus. the sponge differs from siliquariaspongia japonica hoshino, 1981 in the absence of discotriaenes. distribution: palau (de laubenfels, 1954; niwa collection); papua new guinea (niwa collection); indonesia (niwa collection); philippines: davao (niwa collection); panglao, bohol (niwa collection); longakit, sotto and kelly 60 sulu sea, north tubbataha reef (niwa collection); cebu mactan island (niwa collection); marigondon (present study) order poecilosclerida topsent, 1928 suborder microcionina hadju, van soest and hooper, 1994 family microcionidae carter, 1875 subfamily microcioninae carter, 1875 genus clathria schmidt, 1862 subgenus thalysias duchassaing and michelotti, 1864 clathria (thalysias) reinwardti vosmaer, 1880 description: massive, elongate and ramose (ramose: 87 mm long, 50 mm wide and 5-10 mm thick; elongate: 132 mm long and 10 mm wide) with primary branch giving rise to cylindrical fingers growing or rising just above the ground clinging into branching corals, some have as many as 5 branches growing at different directions forming a mass of branching network attached to the substrate through several points while others have only single elongate branch. surface is rough and hispid due to protruding spicules with oscula (1-2 mm in diameter) irregularly dispersed throughout the 'body'; texture is semi-elastic and difficult to tear. in life, ectosome is bright orange and endosome is brick brown; in alcohol, color is light orange. megascleres are styles with three size categories: principal style, smooth and slightly curved at the anterior third (range: 210-263 x 10-11.3 µm; mean: 243 x 10.4 µm), accessory style, generally straight with faintly microspined bases (i. range: 88-168 x 2.5-3.8 µm; mean: 115 x 2.9 µm, ii. range: 125-228 x 6.3-10 µm; mean: 174 x 7.5 µm), and acanthostyles, heavily spined towards the distal end (range: 53-70 x 4.3-5.5 µm; mean: 63 x 5 µm); microscleres are palmate isochela (range: 10-14 µm; mean: 11 µm), toxas (range: 55-129 x 2.3-4.6 µm; mean: 93 x 2.8 µm). ectosomal skeleton is madeup of a thin layer of smaller microspined styles that form discrete brushes erect on surface in a continuous palisade; choanosomal skeleton is irregularly reticulate with spongin fibers fully cored by principal styles forming oval, triangular or rectangular meshes with dense echinating acanthostyles at the surface. found attached to some dead coral (5-11m) at an area with sandy substrate and patches of corals. distribution: australia (bergquist et al. 1971; hooper, 1996); caroline islands (hooper, 1996); vietnam (hooper, 1996); indonesia (van soest, 1989; hooper, 1996; niwa collection); motupore island, papua new guinea (kelly-borges and bergquist, 1988; hooper, 1996); solomon island (bergquist et al., 1971); zanzibar (niwa collection); philippines: bohol (niwa collection); negros oriental (hooper, 1996); cebu daanbantayan, marigondon and san francisco (present study) family raspailiidae hentschel, 1923 subfamily echinodictyinae hooper and van soest, 2002 genus echinodictyum ridley, 1881 echinodictyum cf. conulosum kieschnick, 1900 description: anastomosing small branches forming an irregularly round to oval mass (105 mm long and 54 mm wide); surface is rugged with many projecting branches (4-7 mm long) and numerous interstitial holes covered with a thin membranous sheath that easily disintegrates upon preservation; texture is stiff, firm and brittle. in life and in preservative, the color is jet black with purple tinge due to dense deposit of pigment granules. megascleres are oxeas, straight to slightly curved (range: 78-243 x 4.2-14.3 µm; mean: 364 x 8.8 µm), acanthostyles are straight and tapering and with blunt ends (range: 86-157 x 4.2-7.1 µm; mean: 127 x 6.1 µm); microscleres are absent. the ectosome is membranous with protruding tips of extra-axial styles while the choanosome is differentiated into primary ascending fibers and secondary transverse connecting tracts, fully cored with oxeas and echinated by acanthostyles; pigment granules are embedded in the membrane. occurs at depths 15-18 m, in a coral reef area. remarks: pigment granules are only found in shallow water specimen (hooper, 1991). distribution: australia (hooper, 1991); philippines: cebu marigondon suborder myxillina hadju, van soest and hooper, 1994 family iotrochotidae dendy, 1922 genus iotrochota ridley, 1884 iotrochota baculifera ridley, 1884 the shallow water marine sponges (porifera) 61 description: irregularly thick encrusting sponge (fragment: 200 mm long, 87 mm wide and 20-30 mm thick), accumulates a lot of foreign materials into its 'body' which emits a purplish mucus that stains the hand when handled; surface is uneven and rough with no visible pores; texture is firm and barely compressible. color is purplish-black in life and in preserved state. megascleres are styles, smooth and slightly curved at the anterior portion (range: 153.6-172.8 x 4.8-6 µm; mean : 163.9 x 6 µm), strongyles are straight and thin (range: 204-249.6 x 3.6 µm; 12 mean : 225.3 x 3.6 µm); microscleres are birotula (range: 12-14.4 µm; mean : 13.7 µm). the skeleton is fibrous with irregular reticulate tracts of curved styles; strongyles are randomly arranged at the dermal membrane. found at 0-2 m in an area with muddy substrate. distribution: palau (de laubenfels, 1954; bergquist, 1965); papua new guinea (kelly-borges and bergquist, 1988); india (dendy, 1922); aru island (hentschel, 1912); philippines: cebu san francisco (present study) suborder mycalina hadju, van soest and hooper, 1994 family desmacellidae ridley and dendy,1886 genus biemna gray, 1867 biemna fortis (topsent, 1897) description: massive sponge (150 mm long and 90 mm in diameter), with chimney-like projections, base is buried in the substrate sometimes with only the tubular projections visible at the surface; the sponge is rugged and hispidous, ostia are not visible while the oscula (3-8 mm) are terminally located at each projection; texture is woody and cork-like. in live specimen and in preserved state, the portion buried to the ground is yellowish-green to yellowish-brown while the top of the projection is dark green to gray; variations in color is due to accumulated debris. megascleres are styles, smooth and slightly curved upwards (range: 9291,283 x 16.2-36.5 µm; mean : 1,121 x 28.6 µm); microscleres are sigmas, robust with pointed ends (range: 71-93 x 3.1-5.3 µm; mean : 85 µm x 4.3 µm). ectosomal skeleton is a mass of tangentially arranged spicules; choanosome occasionally contains fiber tracts but is mostly composed of abundant felted spicules interspersed with numerous sigmas. found at 9-10 m in two habitats (coral reef and sandy substrate with coral patches). remarks: thrives well in areas with high siltation. distribution: papua new guinea (kelly-borges and bergquist, 1988); straight of malacca, dead sea (hentschel, 1912); indonesia (van soest, 1989); palau and ponapé (de laubenfels, 1954); philippines: cebu san francisco and badian (present study) order halichondrida gray, 1867 family axinellidae carter, 1875 genus axinella schmidt, 1862 axinella carteri (dendy, 1889) description: flabellate sponge (94 mm long, 42 mm wide and 12 mm thick) with relatively thick buttressed lamellae having irregular margin, attached to the substrate by a small basal stalk; surface is hispid and rugged with ridges (5 mm high) and conules all throughout, only one osculum (1 mm in diameter) is found; texture is rubbery, compressible and easy to tear. the color is bright orange-brown in life and pale orangebrown in alcohol. megascleres are styles, relatively long, either slender or robust and slightly curved (range: 347 504 x 4.8-16.8 µm; mean: 448.5 x 9.8 µm); microscleres are absent. the ectosome is membranous with sparsely 13 protruding extra-axial spicules; choanosome is composed of multispicular bundles fully cored with styles running longitudinally through the lamellae interconnected by vaguely plumose, ascending paucispicular extra-axial tracts or individual spicules; fiber reticulation formed is relatively close-meshed. found at 15-18 m attached to a coral stone. distribution: indonesia (van soest, 1989; niwa collection); papua new guinea and the great barrier reef (hooper and lévi, 1993); new caledonia (hooper and lévi, 1993; laboute et al., 1995); red sea, saudi arabia (niwa collection); zanzibar (niwa collection); philippines: cebu badian (present study) genus phakellia bowerbank, 1862 phakellia cavernosa (dendy, 1922) description: rounded and clathrate-cavernous (90 mm long and 50 mm wide) consisting of intertwined longakit, sotto and kelly 62 taberculae forming small branches (2 mm in diameter) with blunt tips uniformly protruding to the outside forming rounded cavities between which is stretched a thin dermal membrane; surface of the branch is smooth and even while the entire mass is perforated; texture of the whole mass is compressible but not the individual branch, the membrane is very soft. its color is orange, darker in life than in alcohol. megascleres are strongyles, long and sinuous (range: 282-943 x 1.7-11.3 µm; mean: 603 x 5.7 µm), styles, straight to sinuous (range: 243-500 x 5.3-17.7 µm; mean : 332 x 9.2 µm), and anisoxeas (range: 239-521 x 4.2-9.9 µm; mean : 348 x 7.6 µm); microscleres are absent. individual branch is partially cored with dense spicules terminating to the surface; some are arranged perpendicular to the spicule tracts fully enclosed within the spongin fiber. found at 17 m in an area with sandy substrate and coral patches. distribution. indonesia (van soest, 1989); indian ocean (dendy, 1922); philippines: cebusan francisco (present study) family desmoxyidae hallmann, 1917 genus higginsia higgin, 1877 higginsia cf. mixta (hentschel, 1912) description: thickly encrusting (fragment: 82 mm long, 50 mm wide and 10 mm thick); surface is rough with broken ridges (thin and tapering 3-10 mm high), the underside is smoother with no ridges but with holes, ostia are not visible but the oscula (2-4 mm in diameter) are irregularly distributed; the sponge is stiff, compact and resilient. the color is dark orange in life and light brown in alcohol. spicules are oxeas with two size categories: dominant stout oxeas and thinner centrangulate oxeas ( i. range: 1,015-1,143 x 25.7-42.9 µm; mean: 1,078 x 30.2 µm: ii. range: 757-1,115 x 5.315.7 µm; mean: 889 x 9.7 µm), styles are very long and sinuous (range: 1,802-2,574 x 9.6-16.8 µm; mean: 2,117 x 14.2 µm), acanthoxeas are finely spined and centrangulate (range: 81-191 x 3.5-6 µm; mean : 154 x 4.6 µm). skeleton is a regular arrangement of ascending tracts formed by long and stout oxeas concentrated towards the center of vertical processes; spongin is present along spicule tracts but no actual spongin encased in fibers occur; acanthoxeas are present all over but mostly concentrated at the ectosomal area; long styles and thin oxeas protrude to the surface. found at 5-6 m deep in a coral reef area. distribution: palau (bergquist, 1965); philippines: cebu badian (present study) family dictyonellidae van soest, diaz and pomponi, 1990 genus liosina thiele, 1899 liosina paradoxa thiele, 1899 description: massive encrusting sponge (fragment 174 mm long and 72 mm in diameter); surface is conulose with raised and irregularly distributed oscules (5 mm in diameter); texture is spongy and slightly compressible. in life, the sponge is whitish while pale brown in alcohol. megascleres are oxeas (range: 287921 x 2.4-14.4 µm; mean: 468 x 7.1 µm), and strongyles (range: 337-970 x 3.6-12 µm; mean: 571.7 x 7.9 µm); microscleres are absent. spicule tracts are weakly developed and are widely separated by tangentially distributed small group of megascleres; pigment granules are distributed sparsely on the choanosome and dense at the surface and canal lining. habitat. found at 5-11 m deep in a coral reef area. distribution: indonesia (van soest, 1989); mauritius (niwa collection); new caledonia (laboute et al., 1998); solomon islands (bergquist et al., 1971); vanuatu (niwa collection); zanzibar (niwa collection); philippines: cebu-pescador island (niwa collection); buyong, mactan island (niwa collection); badian (present study); argao (present study). genus stylissa hallmann, 1914 stylissa massa (carter, 1889) plate 1, fig. 5 description: massive, erect, lamellate or globular (fragment: 100 mm long, 46 mm wide and 15 mm thick) attached to the ground through a narrow portion (10 mm diameter) or may grow laterally at the ground; surface is rugged and microhispid, ostia (1-2 mm in diameter) and oscula (3-4 mm in diameter) are plenty and randomly scattered; texture is very soft, compressible, firm and soggy. color is bright yellow in life and dull yellow in alcohol; it turns orange when exposed. megascleres are styles, straight to slightly curved (range: 443 572 x 7.1 19.5 µm; mean: 493 x the shallow water marine sponges (porifera) 63 13.5 µm); microsleres are absent. spicules are arranged in a loosely plumoreticulate structure, each tract ends with projecting spicules to the surface; other areas are devoid of spicules. found at 1 m deep among soft corals in a coral reef. distribution: palau, papua new guinea (kellyshanks and bergquist, 1988); philippines -zamboanga, batangas, davao del norte, mindoro occidental, marinduque, quezon, la union, ilocos sur, ilocos norte (caberoy, 1981); cebu, badian (present study) family halichondriidae gray, 1867 genus axinyssa lendenfeld, 1897 axinyssa cf. topsenti lendenfeld, 1897 description: massive (140 mm long and 84 mm wide), attached to the substrate by a narrow pedunclelike structure; surface is hispid and with many irregular depressions formed by raised portions at the surface, many of these are ostia but some are superficial pores without distinct channels to the interior, oscula (3-6 mm in diameter) are few and somewhat raised; the sponge is compressible but firm and easy to tear. its color is reddish brown or purplish in life, brownish in alcohol. megascleres are oxeas, straight and smooth (range: 364.8-710.4 x 3.6-14.4 µm; mean: 502.7 x 9.2 µm); microscleres are absent. the ectosome is a thick organic skeleton with sparsely scattered spicules; choanosome is made up of spicules scattered in confusion with regular tracts separated at regular intervals, giving rise to the raised portions at the surface. found at 17 m deep in a coral reef area. distribution: central atlantic (diaz et al., 1991); philippines: cebu marigondon (present study) order haplosclerida topsent, 1928 suborder haplosclerina topsent, 1928 family callyspongiidae de laubenfels, 1936 genus callyspongia duchassaing and michelotti, 1864 subgenus callyspongia duchassaing and michelotti, 1864 callyspongia (callyspongia) aerizusa desqueyroux-faundez, 1984 description: tubular and erect (178 mm long, 17.5 mm wide and walls at 2.5 mm thick), form clusters attached to the substrate by a common base. internal surface of tubes is smooth with plenty of small pores while the external surface is laden with tapering and distally directed spine-like projections (3-10 mm high and 2-5 mm wide), ostia are not visible but the oscula1(5 mm in diameter) are terminally located at each tube. texture is soft, spongy, compressible and easy to tear; the color is blue-green to green in life and fawn in alcohol. megascleres are oxeas, small and thin, straight to slightly curved (range: 79.2-96 x 2.4 µm; mean: 87.8 x 2.4 µm); microscleres are absent. ectosomal and choanosomal skeleton is ladder-like with fully cored primary fibers (28 µm in diameter) branching out to secondary fibers (7-10 µm in diameter) and unispicular tertiary fibers; meshes formed have oval or round shapes (69-183 µm wide), spongin is always present, fully or partially cored with spicules; primary fiber makes-up the skeletal support of the spine-like projections. found attached to a reef at 10 m. distribution: great barrier reef, australia (fromont, 1993); indonesia (niwa collection); new caledonia (laboute et al., 1998); tanzania (niwa collection); papua new guinea (niwa collection); palau (niwa collection); philippines: cebu badian (present study) callyspongia (callyspongia) muricina (lamarck, 1813) description: thin and long solid tubes with spinelike projections (210 mm long and 10 mm in diameter); surface is micropunctipore with spinelike projections (4-7 mm high) distributed at 3-5 mm apart; ostia are not visible but the oscula (2.5-5 mm in diameter) are distributed 6-10 mm apart at the surface of the sponge; texture is soft, compressible and a little difficult to tear; its color is greenish brown in life, light brown in alcohol. megascleres are oxeas, small and thin (range: 56-83 x 1-3 µm; mean: 77 x 2 µm); microscleres are absent. ectosomal skeleton is a tangential reticulation of sparsely cored primary and secondary fibers; choanosomal skeleton is a reticulation of fully cored primary fibers (34-59 µm in diameter), partially cored secondary fibers (14 µm in diameter) and unispicular tertiary fibers (7 µm in diameter) forming round to oval meshes, 55-247 µm wide; primary fibers support the spine-like projection. found at a depth of 13 m attached to a coral stone in an area with sandy substrate and coral patches. longakit, sotto and kelly 64 distribution: great barrier reef (fromont, 1993); philippines: cebu daanbantayan (present study) family chalinidae gray, 1867 genus dendroxea griessinger, 1971 dendroxea sp. nov. (plate 1, fig. 3) description: thinly encrusting (fully encrusting a coral fragment 64 mm long and 10 mm in diameter); surface is velvety and hispid; texture is soft and compressible. in life, color is olive to dark green while in preservative it is greenish brown. megascleres are oxeas, small and almost uniform, straight to slightly curved (range: 91.2-103.2 x 2.4-4.8 µm; mean: 96 x 4 µm); microscleres are absent. reticulate base gives rise to multispicular, plumose, branching spicular tracts that thin out to the surface; primary tracts (7.2-14 µm in diameter) are partially to fully cored with spicules; secondary tracts (4.8 µm in diameter) are partially cored with 2 or more spicules. found encrusting in coral fragments at coral reefs. remarks: morphologically this is different from the lone species of dendroxea, dendroxea lenis (topsent), 1892, which has smooth, even surface and grayish color. further specimens are required before a final species allocation can be made. distribution: philippines: cebu argao, daanbantayan, marigondon, san francisco (present study) genus haliclona grant, 1836 haliclona amboinensis (lèvi, 1961) (plate 1, fig. 11) description: encrusting sponge (140 mm long, 63 mm wide and 20-30 mm wide) spreading like a thick mat above rhabdastrella sp. nov.; surface is rough to the touch with no visible ostia, oscula (2-4 mm in diameter) are slightly raised and are irregularly distributed at the upper side of the sponge; texture is brittle, crumbly and easy to tear. in life, color is light blue and fawn in alcohol. megascleres are oxeas, curved at center, occasionally straight (range: 168-288 x 2.419.6 µm; mean: 238.9 x 10 µm); microscleres are sigmas, small and c-shaped (range: 12-14.4 µm; mean: 14.2 µm). choanosomal skeleton is confused isotropic to sub-isotropic reticulation of spicules; ectosome is an extension of the choanosomal skeleton forming a single layer of spicules parallel to the surface with occasional erect spicules extending beyond the parallel layer; sigmas occur throughout the membrane and around internal pores. found at 10 m deep in a coral reef. distribution: moluccas (kelly-borges and bergquist, 1988); great barrier reef, australia (fromont, 1993); philippines: cebu badian (present study). haliclona cymiformis (esper, 1794) (plate 1, fig. 12) description: thinly encrusting that completely surrounds the red algae ceratodictyon spongiosum; in general, it appears erect with bifurcate branches interconnected to form large spreading mass (140 mm long, 120 mm wide and 20-30 mm high) with branches (up to 2-8 mm in diameter and 8-11 mm high); surface is even and unornamented, porous and microscopically hispid with oscules (1-2 mm in diameter) that are irregularly scattered; texture is firm, incompressible, tough and a bit difficult to tear. in life, its color is green to greenish-brown; the one with the greenish color is observed to be robust with full tips while that of the greenishbrown coloration looks like it is being grazed on by other organisms with the tips broken; in alcohol, the color is fawn. megascleres are oxeas, slim and slightly curved at the center (range: 132-165.6 x 2.44.8 µm; mean: 146.2 x 3.6 µm); microscleres are sigmas (range: 16.8-21.6 µm; mean: 19.7 µm). a spongin-fiber reticulation is observed between anastomosing networks of thalli; surface skeleton has isodictyal reticulation. the sponge is relatively abundant in an intertidal area with muddy substrate, attached to a hard substrate by a narrow portion at the base that is sometimes burrowed in the mud. distribution: indonesia (van soest, 1989); papua new guinea (laboute et al., 1998); great barrier reef, australia (laboute et al., 1998); philippines ilocos sur, ilocos norte, la union (caberoy, 1997); cebu san francisco (present study) the shallow water marine sponges (porifera) 65 haliclona poseidon (de laubenfels, 1954) description: tubular to flabellate forms with very thin walls (long and thin tubes: height 115-172 mm and width 10 mm; wall thickness 1.5 mm; oscular opening 6-8 mm in diameter; short and stout tubes: height 115 mm and width 72 mm; flabellate form fragment: 254 mm long and 185 mm wide), some tubular forms have long and slender tubes that connect with each other at the base with narrow openings while others have shorter and larger tubes with wide openings, the tubes branchout at some point, usually at the middle. surface is generally smooth and micropunctipore but the stouter forms have some folds, oscula are sometimes visible (1-2 mm in diameter) but ostia are not; texture is very soft, compressible and easy to tear. color is highly variable; in water, it is faint blue-grey to light violet; out of the water, it turns to greenish brown, pink, dark rose, lavender or purple; in alcohol, it is light brown to cream. megascleres are oxeas, straight and thin (range: 67112.8 x 2.4-4.8 µm; mean : 85.6 x 3.8 µm); microscleres are absent. spicular arrangement is isotropic reticulation of unispicular fiber tracts devoid of spongin forming triangular and polygonal meshes (45-80 x 60-70 µm); ascending tracts of fibers (20 µm in diameter) containing small amount of spongin enclosing one or more spicules are present, arranged 233-644 µm apart; pigments are scattered between spicule tracts. found at 12-17 m deep, attached to a branching coral. distribution: indonesia (niwa collection); palau (de laubenfels, 1954); tanzania (niwa collection); philippines-camiguin (niwa collection); cebu: marigondon (present study) haliclona sp. nov. (plate 1, fig. 4) description: encrusting sponge with variable thickness and shape (fragment: 50 mm long, 42 mm wide and 10-20 mm thick); surface is slightly conulose (conules are 2 mm high) and micropunctipore, ostia are not visible but oscula are common (1-2 mm in diameter); texture is very soft, crumbly and compressible. its color is orange in life and light brown or cream in alcohol. megascleres are oxeas, almost uniform, straight to slightly curved, pointed, sometimes strongylote (range: 110-156 x 2.5-5 µm; mean: 131.8 x 3.88 µm); microscleres are absent. skeleton is isotropic reticulation that is unispicular all throughout with spicules not enclosed by spongin; meshes formed are irregular in sizes with some spicules protruding to the surface; organic content (brownish pigment) is scattered all throughout. found at 15-17 m encrusting in coral stones, the area is dominantly sandy with coral patches. remarks: the sponge shows high degree of plasticity; the specimens collected range from a thin encrusting sponge, encrusting with low tubular protrusions to encrusting forms with pronounced tubules; could be easily distinguished by the brightness of its color. further specimens are required before a final species allocation can be made. distribution. philippines cebu: san francisco and marigondon family niphatidae van soest, 1980 genus cribrochalina schmidt, 1870 cribrochalina olemda de laubenfels, 1954 description: fan shaped, thicker at the point of attachment than at the edges (32 mm long, 46 mm wide and 3-5 mm at its thickest portion); surface is rugged with protruding fiber tracts forming subdermal cavities while the underside is smoother, ostia are not visible but the oscula (5 mm in diameter) are distributed 8-15 mm apart; texture is soft, compressible and difficult to tear. color is brown with bluish tint when out of water while in preservative, it is light brown. megascleres are oxeas (range: 120-139.2 x 2.4-4.8 µm; mean: 126.2 x 3.6 µm); microscleres are absent. the skeleton is fibro-reticulate with cored spongin fibers (12-25 µm in diameter) forming irregular meshes; ascending fibers terminate at the protrusions at the surface of the sponge. found at 5 m in a coral reef area. distribution: palau (bergquist, 1965; de laubenfels, 1954); philippines: cebu badian (present study) suborder petrosina boury-esnault and van beveren, 1982 family petrosiidae van soest, 1980 genus xestospongia de laubenfels, 1932 xestospongia testudinaria (wilson, 1925) longakit, sotto and kelly 66 description: encrusting (fragment: 46 mm long, 22 mm wide and 15 mm thick) with ridges (7-15 mm high and 1-2 mm thick) arranged laterally forming deep canals; surface is micropunctipore, hispid and conulose (conules are 1 mm high and 3 mm wide); ostia and oscula are not visible; texture is hard, stiff and crumbly. in life, its color is light brown, darker at the endosome than the ectosome; in alcohol, it is fawn. megascleres are oxeas, slightly bent with blunt to pointed ends (range: 328.8-465.8 x 13.7-17.8 µm; mean: 387.7 x 14.8 µm); microsleres are absent. ectosomal and choanosomal skeleton are isotropic reticulation of spicules forming thick tracts (151-178 µm wide) with meshes (233-699 µm wide); many spicules are scattered in confusion obscuring the skeletal network; spongin is absent. attach to rubbles in an area with sandy substrate and patches of corals at a depth of 17 m. remarks: the specimen collected is a young sponge that has not fully attained its characteristic barrel shape. distribution: philippines (lévi and lévi, 1989; ruelo, 1964, wilson, 1925): cebu-san francisco (present study) order dictyoceratida minchin, 1900 family thorectidae bergquist, 1978 subfamily thorectinae bergquist, 1978 genus hyrtios duchassaing and michelotti, 1864 hyrtios erecta (keller, 1889) (plate 1, fig. 7) description: elongate to massive (105 mm long, 60 mm wide and 15-20 mm thick); surface is conulose (conules are 2 mm high and 3 mm wide); sponge is compressible and a bit difficult to tear. in life, the color of the ectosome ranges from brown to black while the endosome is light to dark brown; little change in the color is observed in alcohol. siliceous spicules are absent. skeleton is made up of primary (terminates at the conules) and secondary fibers, fully cored with detritus; the surface is darkly pigmented. found at 913 m deep in areas with patches of hard and soft corals. distribution: palau (de laubenfels, 1954; bergquist, 1965); philippines: cebu-daanbantayan, marigondon and san francisco (present study) genus luffariella thiele, 1899 luffariella cf. variabilis polejaeff, 1884 description: massive forming a shallow caliculate form (fragment: 130 mm wide, 80 mm and 5-10 mm thick); surface has many depressions formed by four protruding tracts (4-8 mm in diameter and 9 mm deep) distributed close to each other and connected by a membrane; the sponge is moderately firm and less compressible. out of water, the sponge is yellowishbrown to reddish-brown, in alcohol the external color is blackish brown while the internal is brown. siliceous spicules are absent. skeleton in irregular formed by branching primary (cored with foreign debris and almost fasciculate near the surface) and uncored secondary and tertiary spongin fibers. found at 10-18 m deep in a coral reef. distribution: philippines: cebu argao, badian (present study) genus dactylospongia bergquist, 1965 dactylospongia cf. elegans (thiele, 1889) description: repent sponge (fragment: 210 mm long, 38 mm wide and 5-10 mm thick) with irregular branches; the upper surface is rugged and irregularly conulose (with conules, 1-4 mm high) while the bottom is smoother, ostia are not visible but the oscula (2-6 mm in diameter) are irregularly distributed along the depressions; texture is rubbery, not readily compressible and difficult to tear. the color is reddish-brown, both out of water and in preservative. siliceous spicules are absent. skeleton is a reticulation of yellowish-brown spongin fibers made-up of primary fibers (27-41 µm in diameter), secondary fibers (10-14 µm in diameter) and fine tertiary fibers (3 µm in diameter), the resulting pattern is a beautiful and neat intricately woven fibers forming round to oval meshes (41-315 µm wide). attached to a coral stone at 11 m deep in an area with sandy substrate and coral patches. distribution: phillippines: cebu san francisco (present study) subfamily phyllospongiinae bergquist, sorokin and karuso, 1999 genus carteriospongia hyatt, 1877 carteriospongia flabellifera (bowerbank, 1877) description: foliose (fragment: 130 mm wide, 56 mm long and 2 mm thick) with a single and short the shallow water marine sponges (porifera) 67 attachment stalk; surface displays a characteristic pattern of low regularly aligned ridges and hispidous; texture is firm, flexible and granular. in life, color is beige; out of water and in preservative, it is brown. siliceous spicules are absent. irregular reticulation of primary and secondary spongin fibers cored with foreign materials; long, thin and vermiform tertiary fibers intertwined along the columns to form complex fiber tresses. found at 15-18 m deep in a coral reef. distribution. philippines: cebu marigondon, argao (present study) family spongiidae gray, 1867 genus spongia linnaeus, 1759 spongia zimocca sensu de laubenfels, 1954 (plate 1, fig. 6) description: massive (fragment: 70 mm high and 82 mm wide); surface is rugged with projections (7-10 mm high) found at the upper surface bearing the oscules (4-8 mm in diameter); texture is soft and spongy. in life and in preservative, ectosome is black and endosome is orange to rusty red with brownish tinge. siliceous spicules are absent. the skeleton is a fibroreticulate arrangement of fibers (20 µm in diameter) forming irregular sizes of polygonal meshes (about 100-133 µm across); ectosome of the sponge is thin and darkly pigmented. the specimen was found on some hard substrate in an intertidal area with generally muddy substrate. remarks: this is one of the sponges traded as bath sponge, often referred to as "yellow" sponge because the macerated fibers exhibit a somewhat yellowish or almost orange color (de laubenfels, 1954). distribution: eastern ponapè and palau (de laubenfels, 1954), philippines (wilson, 1925; ruelo, 1964): cebu san francisco (present study) family dysideidae gray, 1867 genus dysidea johnston, 1842 dysidea cf. arenaria bergquist, 1965 description: irregular, roughly conulose (fragment: 65 mm long, 16 mm wide and 2 mm thick); surface is conulose, with conules measuring 1-5 mm high and 2050 mm apart (bergquist, 1965) separated by deep pits; texture is rubbery and less compressible. its color in life is light brown while grayish white in alcohol. fibers are not differentiated into primary and secondary fibers (93.3-133.4 µm in diameter); arranged in a reticulate pattern forming irregular meshes; all fibers are fully cored with detritus. found at depth zone 15-18 m in an area with sandy substrate and coral patches. distribution: indonesia (van soest, 1989); palau (bergquist, 1965); new caledonia (laboute et al., 1998); philippines: cebu-san francisco (present study) order dendroceratida minchin, 1900 family dictyodendrillidae bergquist, 1980 genus igernella topsent, 1905 igernella mirabilis lèvi, 1961 description: massive (fragment: 154 mm long, 88 mm wide and 15-38 mm thick), accumulates a lot of shells and small stones in the body; round depressions (5-7 mm in diameter) are irregularly distributed at the surface with pores not visible on the outside but when sliced, a lot of them can be seen; texture is very soft but difficult to tear. the external color is dark brown in life and become lighter in alcohol while the internal is light brown in life and in alcohol. siliceous spicules are absent, replaced instead by spiculoids, which are yellowish-brown fibers taking the form of triactines and diactines; skeletal arrangement is reticulate forming regular to slightly irregular meshes. found at 9-12 m in a coral reef area. distribution: philippines: cebu marigondon (present study) order verongida bergquist, 1978 family pseudoceratinidae carter, 1885 genus pseudoceratina carter, 1885 pseudoceratina verrucosa bergquist, 1995 description: massive and repent (fragment: 64 mm long, 32 mm wide and 30 mm thick) with thick branches; surface is generally uneven, verrucose containing small rounded projections (1-2 mm high); texture is hard, incompressible and difficult to tear. its color is yellowish-brown with greenish patches when out of water while in preservative it is deep purple longakit, sotto and kelly 68 almost black. dendritic arrangement of irregular fibers composed of pith elements; a large portion of the deeper region of the choanosome is devoid of skeleton; bark is absent and the ectosome contains a layer of collagen. found at depths of 15-18 m in a steep reef front with abundant corals. distribution: new caledonia (bergquist, 1995); philippines: cebu marigondon (present study) of the thirty-three (33) species identified in this study, four (4) have medical and economic potentials (plate 1, figs. 5-8). the sponge stylissa massa (carter, 1889) contains eight (8) known alkaloids in which two showed significant enzyme inhibitory activity and inhibited the growth of human tumor lovo cells (tasdemir et al., 2002). hyrtios erecta (keller, 1889), one of the most common sponges in the island, has some associated bacteria (alpha proteobacteria spei-7) which are potential sources of bioactive metabolites (rodriguez et al., 2005). plakortis lita de laubenfels, 1954 has been reported to release allelochemicals toxic to hard corals (bakus and nishiyama, 1999; 2000). furthermore, spongia zimocca sensu de laubenfels, 1954 is considered as one of those traded as bath sponges commercially referred to as the "yellow sponge" because of the color of its fibers (de laubenfels, 1954). sponges can enter into complex epizoic relationships, growing over, upon or even inside one another without hampering their pumping and filtering activities on which they depend (bergquist, 1978). four (4) species of sponges in this study (plate 1, figs. 9-12) were found growing in symbiotic relationship with another sponge or other marine organisms. haliclona cymiformis (esper, 1794) had been known in association with red algae (ceratodictyon spongiosum) thinly but fully encrusting the algae (fromont, 1993). paratetilla bacca (selenka, 1867) have greenish tinge caused by cyanobacteria, symploca sp. (caberoy, 1997). sponge to sponge association is exhibited by haliclona amboinensis (lèvi, 1961) and rhabdastrella sp. nov. in all the specimens collected for this study. haliclona amboinensis (lèvi, 1961) grow on top of rhabdastrella sp. nov. with the latter attached to the substrate. fromont (1993) reported another sponge, niphates nitida fromont, 1993 in association with these two sponges however, this association is not well established in this study. kelly-borges and bergquist (1988) reported another sponge in association with h. amboinensis (lèvi, 1961), psammaplysilla purpurea carter, 1880 but such association was not observed in this study. percentage similarity figure 4 shows the species richness of the six stations. the station in san francisco (station 2) recorded the highest number of species (15 species) while daanbantayan (station 1) recorded the lowest (5 species). the highest number of species recorded at san francisco station is due to the presence of several sponge species in the intertidal zone, which is unusual for this zone. the study of diaz et al. (1985) revealed that sponges are generally absent in the intertidal zone of open reef habitats. sponges are not the major occupants in this zone, frequently occurring in pools, shades, under boulders, in crevices and on top of organisms (bergquist, 1978). light and wave action may play an important role affecting sponge distribution in this zone. wave stress may limit the colonization and growth of sponges by generating substrate instability, high turbidity and turbulence (diaz et al., 1985). unlike the other intertidal zones, a dike that runs fig. 4. species richness of the six sampling stations of cebu, philippines. a, argao; bi, badian in-mpa; bo, badian outmpa; d, daanbantayan; m. marigondon; s, san francisco. the shallow water marine sponges (porifera) 69 perpendicular to the shore is protecting the intertidal area of san francisco making it suitable for sponge growth and survival. species richness increases with depth as observed in this study (fig. 5). the highest number, 22 species was recorded at depth zone 4 (13-18 m) while the lowest number, 6 species, was at depth zone 1 (0-2 m). schmahl (1985) noted similar observations in his study of the four southern florida coral reefs. distribution of sponges along the depth gradient is indicative of their ecological tolerance in which species widely distributed are more tolerant than those restricted at certain depths (alvarez et al., 1985). ecological factors that vary with fig.5. species richness of the four depth zones of cebu, philippines, 1. 0-2m; 2, 3-9m; 3, 10-12m; 4, 13-18m. depth appear to be responsible for the observed distributions. one such factor is turbulence or physical disturbance due to wave action, which decreases substantially with depth (schmahl, 1985). species richness inside and outside the marine protected areas has almost the same values (6 and 7 species, respectively), which could mean that these marine organisms are not being harvested or utilized in the area. table 2 shows the species occurrence at the six stations of cebu. not a single sponge species was present in all of the six sampling stations. only three (3) species or nine percent (9%) occurred in four of the six stations, two of which are new to science, dendroxea sp. nov. and haliclona sp. nov. the other most common species is hyrtios erecta (keller, 1889). three (3) species or nine percent (9%) were present in three stations and these include plakortis lita de laubenfels, 1954; clathria (thalysias) reinwardti vosmaer, 1880; and liosina paradoxa thiele, 1899. six (6) species or eighteen percent (18%) were present in two stations and twenty-one (21) species or sixty-seven percent (67%) of the thirty-three (33) known sponges were recorded only once. this trend is lower than the one recorded by raymundo and harper (1995) in their study of the sponges in central visayas. in their study, only fifty-three percent (53%) of the 85 sponges collected were present at one site, twenty-four percent (24%) were found in more than one site and thirty-three percent (33%) were found in more than five stations. a fairly large number of their genera has widespread distribution in central visayas. however, it was not mentioned from which bodies of water the sponges were collected. the cluster results using jaccard's index of similarity and dissimilarity is shown in fig. 6. two major groupings were formed as if distance between stations is the major criteria. the first cluster is composed of the closest stations, inside and outside the marine protected area of badian, cebu (stations 4 and 5) situated at the shallow waters of tanon strait at the western portion of the island. the second cluster is composed of stations in daanbantayan, san francisco, fig. 6. cluster analysis of the different sampling stations of cebu, philippines using percentage similarity and weighted pair-group average. a, argao; bi, badian in-mpa; bo, badian out-mpa; d, daanbantayan; m, marigondon; s, san francisco. longakit, sotto and kelly 70 species a bi bo d m s 1. corticium sp. nov. + 2. plakortis lita de laubenfels, 1954 + + + 3. paratetilla bacca (selenka, 1867) + 4. rhabdastrella sp. nov. + 5. spheciospongia vagabunda (ridley, 1884) + 6. siliquariaspongia cf. mirabilis (de laubenfels, 1954) + 7. clathria (thalysias) reinwardti vosmaer, 1880 + + + 8. echinodictyum cf. conulosum kieschnick, 1900 + 9. iotrochota baculifera ridley, 1884 + 10. biemna fortis (topsent, 1897) + + 11. axinella carteri (dendy, 1889) + 12. phakellia cavernosa (dendy, 1922) + 13. higginsia cf. mixta (hentschel, 1912) + + 14. liosina paradoxa thiele, 1899 + + + 15. stylissa massa (carter, 1889) + 16. axynissa cf. topsenti lendenfeld, 1897 + 17. callyspongia (callyspongia) aerizusa desqueyroux-faundez, 1984 + 18. callyspongia (callyspongia) muricina (lamarck, 1813) + 19. dendroxea sp. nov. + + + + 20. haliclona cf. amboinensis (lèvi, 1961) + 21. haliclona cymiformis (esper, 1794) + 22. haliclona poseidon (de laubenfels, 1954) + 23. haliclona sp. nov. + + + + 24. cribrochalina olemda de laubenfels, 1954 + + 25. xestospongia testudinaria (wilson, 1925) + 26. hyrtios erecta(keller, 1889) + + + + 27. luffariella cf. variabilis polejaeff, 1884 + + 28. dactylospongia cf. elegans (thiele, 1889) + 29. carteriospongia flabellifera (bowerbank, 1877) + + 30. spongia zimocca sensu de laubenfels, 1954 + 31. dysidea cf. arenaria bergquist, 1965 + 32. igernella mirabilis lèvi, 1961 + 33. pseudoceratina verrucosa bergquist, 1995 + + table 2. occurrence of sponges at the six (6) sampling stations of cebu island, philippines (a, argao; bi, badian inside mpa, bo, badian outside mpa; d, daanbantayan; m. marigondon; s, san francisco). marigondon and argao located at the eastern part of the island. other closely related stations are stations 1 and 2 (daanbantayan and san francisco). though the distance between marigondon and san francisco is relatively shorter than san francisco and daanbantayan, the sampling site being located at the back of the island may have favored the flow towards daanbantayan than towards marigondon. the station in daanbantayan is still within the northern tip of camotes sea where san francisco is situated. marigondon and argao are closer to each other and are found in the same cluster with san francisco and daanbantayan. fig. 7. cluster analysis of the four depth zones of cebu, philippines using percentage similarity and weighted pairgroup average, depth zone 1, 0-2m; depth zone 2, 3-9m; depth zone 3, 10-12m; depth zone 4, 13-18m. the shallow water marine sponges (porifera) 71 plate 1. figs. 1-12. habit of sponges. figs. 1-4, sponges preliminarily identified as new species, figs. 5-8, those with economic, medical and ecological potentials; figs. 9-12, those with association to its kind or with other marine organisms. figure 6. spongia zimocca sensu de laubenfels, 1954 figure 1. corticium sp. nov. figure 2. rhabdastrella sp. nov. figure 3. dendroxea sp. nov. figure 4. haliclona sp. nov. figure 5. stylissa massa (carter, 1889), in situ. figure 7. hyrtios erecta (keller, 1889) figure 8. plakotis lita de laubenfels, 1954 figure 9. paratetilla bacca (selenka, 1867) figure 10. spheciospongia vagabunda (ridley, 1884) figure 11. haliclona, cf. amboinensis (levi, 1961) figure 12. haliclona cymiformis (esper, 1794) longakit, sotto and kelly 72 for depth zones (fig. 7), the two shallowest zones (zones 1 and 2) are clustered together while the two deepest zones are not. these two, however have high linkage distance than depth zones 1 and 2. conclusion the shallow marine areas of cebu are inhabited by a wide variety of sponge species. the low percentages of similarity among stations clearly showed that the bodies of water surrounding the island support different species of sponges. clustering of these areas occurred between nearest neighboring waters. the discovery of four possible new species shows that there are probably more undescribed species in philippine waters and that more taxonomic research is needed. the discovery of the species of bath sponges, spongia zimocca sensu de laubenfels, 1954, proves promising for aquaculture. protecting the coral reef from anthropogenic factors does not have big impact on sponge richness as shown by the sampling results of stations inside and outside a marine protected area. recommendations this study showed that there are still a lot of sponges yet to be discovered in philippine waters, hence the need to conduct similar study in other areas of the country, possibly using the advancements in molecular biology. since only few sponge species were found common among the different bodies of water surrounding cebu, it is recommended that studies related to current system of these waters be conducted to explain this pattern of distribution. spongia zimocca sensu de laubenfels, 1954, one of the commercial sponges, has potential for mariculture in the shallow waters of cebu. thus studies on growth, survival and culture methods of this sponge and other species with importance to economy, ecology and medicine should be conducted for these may provide livelihood opportunities for marginal fishermen. acknowledgments the first author is grateful to the professional association of diving instructors (padi) foundation for the financial grants (padi grant reference # 209 of year 2003 and padi grant reference #12 of year 2004); 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(eds.), new perspectives in sponge biology. 3rd int. con. biol. sponges: 316-319. tasdemir, d., r. mallon, m. greenstein, l.r. feldberg, s.c. kim, k. collins, d. wojciechowicz, g.c. mangalindan, g.p. concepcion, m.k. harper, c.m. ireland. 2002. aldisine alkaloids from the philippine sponge stylissa massa are potent inhibitors of mitogenactivated protein kinase kinase1 (mek-1). j. med. chem. 4:529-32. tendal, o.s. 1969. demospongiae from the fiji islands. vidensk. meddr. dansk. naturh. foren. 132:31-44. van soest, r.w.m. 1989. the indonesian sponge fauna: a status report. netherlands j. sea res. 23(2):223-230. wilson, h.v. 1925. silicious and horny sponges collected by the us fisheries steamer “albatross” during the philippine expedition, 1907-10. smithsonian institution, us national museum bull 100(2)4:273-530. 6detailed calculation-paraan.pmd average work done in a ground state quench 54 science diliman (july-december 2016) 28:2, 54-64 detailed calculation of the average work done in a ground state quench of the quantum ising model salvador t. laurente jr. university of the philippines diliman francis n.c. paraan* university of the philippines diliman abstract a detailed derivation of the exact expression for the average work done in a transverse f ield quench of the quantum ising model ground state is presented. in the thermodynamic limit, it is proved that the average work done generally has inflection points at the critical f ield for the pre-quench quantum phase transition. the f irst divergent f ield derivative i s c a l c u l a t e d a n d i s s h o w n t o d i v e r g e l o g a r i t h m i c a l l y. we a l s o demonstrate that the average work done is equal to the product of the transverse magnetization of the pre-quench ground state and the change in the magnetic f ield. k e y w o r d s : q u a n t u m q u e n c h , q u a n t u m i s i n g m o d e l , q u a n t u m p h a s e transition _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online introduction an initially prepared quantum state evolves unitarily after a rapid change in the hamiltonian in a quantum quench. the system is isolated from heat and particle baths, and its response time is much longer than the time scale of the hamiltonian change, so that no adiabatic approximation can be made. quenches like these generally lead to excited (non-stationary) states, and the resulting non-equilibrium dynamics has been the subject of several investigations on quantum many-body physics in the absence of decohering environmental effects (polkovnikov et al. 2011; eisert et al. 2015). preparing suff iciently isolated states under controllable s.t. laurente jr. and f.n.c. paraan 55 conditions is a technical challenge but experimental progress in the last two decades has allowed quench dynamics to be observed in, for example, ultracold gases in optical lattices (greiner et al. 2002; cheneau et al. 2012). because these isolated quantum systems evolve in time without exchanging matter and heat with a reservoir, conventional statistical mechanical notions, such as temperature, entropy, and equilibriation, are not directly applicable to them. thus, new theoretical frameworks have been developed to investigate quantum quenches and the novel phenomena that are associated with them. some recent new discoveries include the emergence of persistent fluctuations despite the absence of energy and matter exchanges with a bath (rossini et al. 2009; häppölä et al. 2012; cosme and fialko 2014), universal scaling behavior in the vicinity of quantum critical points (silva 2008; jacobson et al. 2011; gambassi and silva 2012), and the generation of quantum entanglement entropy (fagotti and calabrese 2008; cardy 2011; daley et al. 2012; schachenmayer et al. 2013; alba and heidrich-meisner 2014; alkurtass et al. 2014; torlai et al. 2014). in this manuscript we consider the case of a zero temperature instantaneous quench of the transverse magnetic f ield h in a quantum ising model. the hamiltonian is (1), where is the a-pauli operator on site j. there are n spins in the chain and periodic boundary conditions are imposed. the system is thermally isolated and prepared in the ground state of the pre-quench hamiltonian h 0 = h(h 0 ) with f ield h 0 . at time t = 0 + the f ield is suddenly changed to h 1 , and the system subsequently evolves according to the post-quench hamiltonian h 1 = h(h 1 ). throughout this work, the subscript index will refer to pre-quench (post-quench) quantities. the quantity of interest in this study is the average work done (or quantum work) in these quenches under the formalism developed by talkner et al. (2007, 2016). this quantum work w is def ined as the difference between a projective measurement of the system energy at some time t after the quench and the initial ground state energy at time t < 0 . that is, if is the state vector of the system at time t, the work done is (2). ) 1 2 σ σ 1 σ 1 | 0)〉 0 1) | )〉 )| 1 | ) 0)| 0| 0) average work done in a ground state quench 56 ) 1 2 0) 1 0 0) (5). for a given measurement of quantum work in this quench protocol, one gets the result w m = e 1 (m) – e 0 , where e 0 is the pre-quench ground state energy and e 1 (m) is the m th energy eigenvalue of the post-quench hamiltonian. this result occurs with total probability so that the quantum work distribution p(w) is equal to (3). taking the fourier transform of this distribution gives the characteristic function (4), whose logarithm in g(u) is the generating function for the cumulants of p(w). from the known properties of this cumulant generating function, the average work done is 〈w〉 = _ilim u→0∂ ln g(u)/∂u and the variance in the work done is ∑ 2 =_lim u→0 ∂2 ln g(u)/∂u2. these statistical measures of the work done in a quenched quantum ising model have been investigated at zero initial temperature (silva 2008), nonzero temperatures (dorner et al. 2012), and the more general anisotropic xy model (bayocboc jr. and paraan 2015). these studies have shown that signatures of the quantum phase transition of the model, such as the vanishing excitation gap, can be revealed through an analysis of the work statistics. in this paper, we focus on providing a detailed derivation of the exact solution for the average work done along the quantum ising line to complement the previous work of silva (2008) and bayocboc jr. and paraan (2015). in particular, we will prove that the average work done is not an analytic function of the pre-quench f ield h 0 when the excitation gap closes at the quantum critical point. the characteristic function g(u) is given as an expectation value (4) with respect to the pre-quench ground state . it is convenient to calculate this quantum average in the basis of single-particle fermionic jordan-wigner fock states. the result is (silva 2008; dorner et al. 2012): ) 0) 1 0 0) |0) | 1 )| 0) | 2 | 0)〉 ) δε cos2 δ 1 ) sin2 δ 1 0 s.t. laurente jr. and f.n.c. paraan 57 in the previous equation, is the difference between the ground state energies of the post-quench and pre-quench hamiltonians. additionally, the angle is the difference between the post-quench and pre-quench bogolyubov angles. these bogolyubov angles satisfy finally, the energy spectrum of elementary excitations is (7), where for odd n or for even n. the effect of n being odd or even is negligible in the thermodynamic limit . the excitation gap closes at the critical f ield value which signals a quantum phase transition between a ferromagnetic (⏐h i ⏐< 1) and paramagnetic (⏐h i ⏐>1) ground state. the average work done can be calculated from the f irst derivative of ln g, which yields we now take the thermodynamic limit , and f ind that the average work per spin becomes the def inite integral ∆n = θ1(qn) − θ0(qn) ∆e = e1 − e0 ) cos )2 sin2 qn 2πn/n qn 2π n ½)/n → ∞ ±1, 〈 〉 1 2 0 ) 1 ) cos 2δ 1 0 → ∞ 〈 〉 ≡ lim →∞ 〈 〉⁄ 〈 〉 0 1 4 0 cos 0 cos )2 sin2 2 0 (9). the average work per spin is singular when the pre-quench hamiltonian is quantum critical (h 0 = ±1). in the following section we prove that 〈w〉 is indeed non-analytic (not inf initely differentiable) at these quantum critical points. tan 2 ) sin cos (8). (6). average work done in a ground state quench 58 average work done the symmetry and periodicity of the trigonometric functions allow us to put the integral (9) in the form (10). after factoring and rearranging terms, the resulting integrals are recognized as the complete elliptic integrals (11), (12), with modulus we f ind thatκ. (13), where also, from the known connection formulas for the complete elliptic integrals (gradshteyn and ryzhik 2007; olver et al. 2010) (14), (15), (16), we can simplify our result to (17). a similar result for this compact formula (17) has been reported without derivation by bayocboc jr. and paraan (2015). 〈 〉 0 1 0 1 2 sin2 0 1)2 4 0 sin2 /2 0 . ) 1 2 sin2 ) 1/2 /2 0 ) 1 2 sin2 )1/2 /2 0 〈 〉 0 1 2 0 | 0 1| 0 1 0 1 ) ) 2 4 0 0 1) 2⁄ 1. ) 1 0) 0), ) 1 1 0 2 0) 1 0 2 ) 0) , 1) 1 ) 1 2) ) 〈 〉 sgn 0 0 1) 0 1) s.t. laurente jr. and f.n.c. paraan 59 singularities at criticality the exact expression (17) for reveals that the average work per spin is not analytic at (⏐h 0 ⏐= 1), since ε(κ) is not inf initely differentiable at⏐κ⏐= 1. as seen in graphs of 〈w〉 at different pre-quench and post-quench f ields (figure 1), quantum criticality is manifested as a sudden change of curvature of the average work done at the pre-quench critical f ields h 0 = ±1. in fact, we now prove that these points are actually inflection points by explicitly calculating the needed derivatives. 〈 〉 figure 1. the average work done per spin 〈w〉 for different post-quench f ields h 1 generally have inflection points at the pre-quench critical f ields h 0 = ±1 . the f irst derivative of with respect to h 0 is exactly (18). this derivative is graphed in figure 2 for several f ield quenches and we verify that it generally diverges at the critical points . the only exceptions are when the post-quench f ield is also at the same critical value as the post-quench f ield . we now investigate the divergence at critical h 0 using the asymptotic formulas for the elliptic integrals at unit modulus (olver et al. 2010). the leading term of the asymptotic expansion of the exact derivative (18) about the critical f ield h 0 = 1 is (19). 〈 〉 〈 〉 0 sgn 0 0 1 0 1 ) 0 1) 0 1 ) 0 ±1 0 1 ±1 〈 〉 0 1 1 1) 2 ln 0 1 8 , 0 1 average work done in a ground state quench 60 this result shows that the leading divergence in the derivative ∂〈w〉/∂h 0 is logarithmic in⏐h 0 –1⏐. this divergence vanishes when h 0 = h 1 = 1. similarly, an expansion about h 0 = –1 yields these approximations about h 0 = ±1 are compared with the exact results in figure 3 for the case when the post-quench hamiltonian is also critical (h 1 = ±1). the logarithmic divergence is seen to be captured by the approximate expansions about h 0 = ±1 (dashed lines), and we f ind that the derivative remains continuous when h 0 = h 1 = ±1: (21). to f inally prove that the average work per spin has inflection points at critical prequench f ields, we need to verify that the second derivative changes sign at h 0 = ±1, as depicted in figure 4. let us def ine the complement . the second derivative becomes (22). figure 2. the derivative ∂〈w〉/∂h 0 diverges at the pre-quench critical f ields h 0 = ±1, except when h 1 = h 0 . (20). 2〈 〉/ 0 2 2 〈 〉 0 2 sgn 0 0 2 0 ′ 2 0 3 0 2 1 2 1) 0 1) 0 ′ 2 0 1) 0 1 ) 0 ′ 2 ≡ 1 0 2 〈 〉 0 1 1 1) 2 ln 0 1 8 , 0 1 〈 〉 0 0 1 ±1 ± 1 s.t. laurente jr. and f.n.c. paraan 61 figure 3. the derivative (solid line) diverges logarithmically at the critical f ields except at h 0 = h 1 = ±1. the approximate expansions about h 0 = ±1 are also shown (dashed lines). 〈 〉/ 0 figure 4. the second derivative changes sign at the pre-quench critical f ields h 0 = ±1, except when h 1 = h 0 . in the special case when both hamiltonians are at the same critical f ield h 0 = h 1 = ±1 (black arrows), the second derivative of the average work per spin diverges logarithmically at h 0 = ±1. 2〈 〉/ 0 2 again, asymptotic expansions about h 0 give the following leading order divergences when h 0 ≠ h 1 ≠1: (23). 2〈 〉 0 2 ~ 1 2 1 ∓ 1 0 ∓ 1 , 0 ±1) average work done in a ground state quench 62 since this derivative changes sign at h 0 = ±1, we conf irm that the average work per spin indeed has inflection points when the pre-quench f ield is at its critical value and h 1 ≠ h 0 . this time, in the special case that the post-quench field is critical h 1 = ±1, the leading divergence of the second derivative at h 0 = h 1 is logarithmic: (24). discussion and conclusion we have calculated the average work done in a zero temperature f ield quench of the quantum ising model. in the thermodynamic limit, we presented an exact expression for the average work per spin in terms of complete elliptic integrals. we have shown that the average work per spin is not inf initely differentiable at the pre-quench f ields h 0 = ±1, where a quantum phase transition occurs. when the post-quench f ield h 1 ≠ ±1, the f irst derivative d o e s n o t e x i s t a n d diverges logarithmically at h 0 = ±1. in this case, the non-analyticity at the critical point takes the form of an inflection point. when the post-quench hamiltonian is also critical, the non-analyticity at h 0 = h 1 = ±1 is weakened. that is, the f irst derivative at h 0 = h 1 exists and the logarithmic divergence f irst appears in the second derivative . it is interesting to note that complete elliptic integrals are encountered frequently in the study of the quantum ising model. in fact, the derived expression for the average work done (10) is similar to that for the transverse magnetization of the quantum ising model (niemeijer 1967). some exact solutions on a chain of spins 1/2. physica 36:377-419). that is, if is the average magnetization of the ground state of the ising model with f ield h 0 : then . thus, the logarithmic divergence (19) of the f ield derivative at the pre-quench quantum critical point also mirrors the logarithmic divergence of the zero-temperature magnetic susceptibility of an unquenched ising model at the critical f ield h = ±1, where the excitation gap vanishes and the quantum phase transition occurs. 〈 〉 〈 〉/ 0 →0 ⁄ 2〈 〉 0 2 1 ±1 ~ 5 2 1 ln 0 ∓ 1 8 , 0 ±1) 〈 〉/ 0 2〈 〉/ 0 2 lim →∞ 1 0)| | 0) 1 (25), 〈 〉 0 1) 2⁄ 〈 〉/ 0 s.t. laurente jr. and f.n.c. paraan 63 acknowledgements the authors are supported by the university of the philippines diliman off ice of the chancellor and the off ice of the vice-chancellor for research and development through project no. 141420 phdia. note some of the results in this paper were f irst reported in the research poster pb-48 at the 33rd physics congress of the samahang pisika ng pilipinas and appeared in its conference handbook. references alba v, heidrich-meisner f. 2014. entanglement spreading after a geometric quench in quantum spin chains. physical review b. 90:075144. a l k u r t a s s b , b a n c h i l , b o s e s . 2 0 1 4 . o p t i m a l q u e n c h f o r d i s t a n c e i n d e p e n d e n t entanglement and maximal block entropy. physical review a . 90:042304. bayocboc jr fa, paraan fnc. 2015. exact work statistics of quantum quenches in the anisotropic xy model. physical review e. 92:032142. cardy j . 2011. measuring entanglement using quantum 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salvador t. laurente, jr. completed the bs physics program of the national institute of physics (nip), university of the philippines diliman last june 2016. f r a n c i s n . c . p a r a a n < f p a r a a n @ n i p . u p d . e d u . p h > i s a n a s s i s t a n t p r o f e s s o r a t t h e n a t i o n a l institute of physics, university of the philippines diliman. he is the program coordinator of the nip structure and dynamics research program (www.nip.upd.edu.ph/sand). 6casas-mango kernels.pmd ev casas et al. 41 science diliman (july-december 2015) 27:2, 41-75 optimizing microwave-assisted crude butter extraction from carabao mango (mangifera indica) kernels edgardo v. casas* university of the philippines los baños von jansen g. comed ia university of the philippines los baños arni g. gilbuena university of the philippines los baños ateneo de manila university kevin f. yaptenco university of the philippines los baños _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract c a r a b a o m a n g o e s a r e a m o n g t h e h i g h l y p r o d u c e d f r u i t c r o p s i n t h e philippines. the processing and consumption of carabao mangoes leave a signif icant amount of waste seeds. mango kernel butter extracted from waste seed kernels is a potential additive to cosmetic products or as a c o c o a b u t t e r s u b s t i t u t e . t h i s s t u d y d e t e r m i n e d t h e p r e t r e a t m e n t conditions that produce optimum yield prior to the mechanical extraction of t h e c r u d e b u t t e r. m o r eo ve r, t h i s s t u d y p r ov i d ed a g e n e r a l s e n s o r y evaluation of the f inished product. microwave power (160, 500, and 850 w), microwave exposure time (2.0, 3.5, and 5.0 min), and size levels (1.5, 3.0, a n d 4 . 5 m m ) w e r e t e s t e d f o r t h e i r e f f e c t s o n t h e y i e l d o f t h e m e c h a n i c a l l y e x t r a c t e d c r u d e b u t t e r i n w e t b a s i s p e r c e n t a g e . t h e optimization procedures resulted to optimum pretreatment conditions of 160 w, 4.25 min, and 1.5 mm. size level was the most signif icant factor in the crude butter yield. sensory evaluation of the crude butter extracted at optimum pretreatment conditions through acceptance test by a test panel resulted to below neutral scores in visual appearance and odor, optimizing microwave-assisted crude butter extraction 42 and above neutral score in texture, indicating the potential of mango butter as a good substitute to cocoa butter in cosmetic products. k e y w o r d s : m i c r o w a v e a s s i s t e d c r u d e b u t t e r e x t r a c t i o n f r o m c a r a b a o mango kernels, optimization layman’s abstract this paper discusses the microwave-assisted mechanical screw extraction of mango butter from waste carabao mango seed kernels resulting from t h e p r o ce s s i n g a n d co n s u m p t i o n of c a r a b a o m a n g o e s . m o r e ov e r, t h e p h y s i c a l p r o p e r t i e s o f t h e e x t r a c t e d b u t t e r, a s w e l l a s i t s s e n s o r y properties as evaluated by the sensory taste panel, were also examined. t h e s t u d y e x p l o r e d t h e e f f e c t s o f t h e f o l l o w i n g o n t h e m i c r o w a v e assisted screw pressing of mango seed kernels: microwave power (160, 500, and 850 w), exposure time (2.0, 3.5, and 5.0 min), and size levels (1.5, 3.0, and 4.5 mm). the optimum extraction conditions were identif ied b y t h e r s m m e t h o d o l o g y a s 1 6 0 w, 4 . 2 5 m i n , a n d 1 . 5 m m . s e n s o r y evaluation of the mango butter determined that it has a high overall acceptability as a substitute to cocoa butter in pharmaceutical, cosmetic, and skin care products. introduction the distinct proportions of different fatty acids and the presence of their minor components characterize the physical and chemical properties of oils (institute of shortening and edible oils 2006). vegetable oils are plant-derived triglycerides used for various purposes, including food, but not limited to fuels, cosmetics, lubrication, medicinal, and other industrial applications. almost 80% of worldwide vegetable oil consumption is for food and related purposes, while the remaining 20% is used in the industrial and biodiesel sector with recently increasing usage due to energy security concerns (rosillo-calle et al. 2009). mulimani et al. (2012) sought out the potential of deriving signif icant quantities of biodiesel from vegetable oils, such as using transesterif ication processes on plants that can be produced by smallholders and in rural areas, such as cotton, mahua seed oil, and neem seed oil. techniques in extraction are widely adapted for the isolation of bioactive compounds and vegetable oils from raw plant materials (azadmard-damirchi et al. 2011). the ev casas et al. 43 nature of the applied technique primarily determines the quality (not the inherent) of the resulting yield, and hence, discrepancies among different extraction methods and conditions can exist. cold pressing, which subjects the plant material to pressure, is the simplest and cheapest method to release crude oil bound within the matrices of the plant material. in terms of quality, cold pressing is best in preserving the natural properties of the extracted oil. hot pressing involves the introduction of heat and pressure to ease the flow of extracted oil. most of the time, this method also requires heat treatment and size reduction to increase its eff iciency. residual meal from pressing can serve as source of animal feed and other food components, or can be subjected to solvent extraction, which involves the immersion of the treated or non-treated raw material to a heated solvent, to further dissolve the oil. the resulting solution can be freed from the solvent via distillation to obtain the crude oil. in terms of oil yield, cold pressing is the least eff icient, followed by hot pressing, and solvent extraction. although solvent extraction is superior in production levels, it poses several disadvantages, including plant security issues, high operation costs, emission of volatile and harmful solvents, quality degradation, and the relative complexity of the process. however, the method is practical to use on plant materials with oil contents of less than 20%, such as soybeans (azadmard-damirchi et al. 2011). mango is a perennial crop native to south asia, primarily in india and pakistan, and cultivated in tropical to subtropical areas. mangifera indica, or widely known as indian mango, is the most cultivated mango (unctad 2003). the characteristic features of the fruit include its oval shape, smooth and leathery rind, fleshy to f ibrous pulp, and its stony seed. at least 60 known varieties of mangoes with fruit colors ranging from red to yellow can be identif ied (nzikou et al. 2010). the crop is mainly produced for the consumption of its fleshy pulp. other products derived from the pulp include mango puree, mango juice, dried mangoes, concentrates, frozen mangoes, mango glaze, edible parts, mango in brine, and mango preserves (boi 2011). the philippines produces the world’s sweetest and less f ibrous varieties of mangoes—an example of which is the carabao mango from the provinces of cebu and guimaras. the pico variety also offers the sour-sweet flavor comparable to the carabao when ripe. while being more f ibrous and smaller than the carabao, pico mangoes are still widely known because of their availability and cheaper price in the market. the high demand for mangoes consequently promotes the increase in production through efforts in developing technologies and maximization of land and resource optimizing microwave-assisted crude butter extraction 44 usage. although a seasonal crop, several means and techniques are available for application, in order to produce the fruit in any season and to continuously sustain the demand for the fruit at lesser price inconsistencies all year round. the annual production capacity of the philippines is approximately one million metric tons in average over the last few years. moreover, mango is the third top agricultural commodity exported by the country, next to coconut and banana (bas 2010). since the major use of mango is the consumption of the sour-sweet pulp being eaten raw or for different kinds of food preparation and processes, the seed and the skin, which comprise 30% to 50% of the weight depending on the variety, are typically treated as waste and immediately disposed without further utilization (philippine mango seedling farm corp. [date unknown]). the vast amount of wastes generated from the fruit after consumption can be estimated to provide a potential and sustainable raw material for further processing (kaphueakngam et al. 2009). in recent years, efforts have been made to f ind ways to utilize these wastes with acceptable economic and commercial feasibility. peels and seeds can become sources of oils and other organic compounds for food, cosmetic, and alternative medicinal applications (ashough and gadallah 2011). other research f inds ways to use these by-products as sources of biodiesel for internal combustion engines. the fuel properties of mango kernels were analyzed in the study by agbede et al. (2010). the low iodine value of the mango kernel oil limits its application as nondrying oil for lubrication purposes. however, the observed fuel properties still possess the potential for biofuel production, and further research on the improvement of these properties can make them into suitable biofuels. the mango kernel is rich in carbohydrates, fats, proteins, and minerals (anand and maini 1997; kittiphoom 2011). the kernel can also be used for extraction of edible oils and as a supplement to wheat flour (kittiphoom 2011; tandon and kalra 1989). ashoush and gadallah (2011) reported that the potential antioxidant properties and high phenolic content of mango kernels and peel powders can help improve the nutritional quality and antioxidant properties of foods, such as biscuits. among these promising applications, mango kernel oil has been bringing commercial benef its, especially in india, where mangoes are among the widely produced fruits. the oil from the kernel of the mango seed is typically light yellow in color with semi-solid texture and thick consistency at room temperature. it is also known as mango butter because it exhibits nearly solid consistency at room temperature and melts in contact with a warmer surface, such as the human skin. it is a good alternative natural ingredient or additive in various body and skin care products, ev casas et al. 45 such as lotions, sun protection creams, moisturizer, and soaps (pioneer enterprise [date unknown]). interests on natural oils, such as from seeds, for cosmetic application are steadily increasing. in mango-based products like soaps and lotions, 3% to 12% of mango kernel oil is added by the manufacturers (boi 2011). in some countries like india, mango kernel oil is sometimes used as a substitute for shea and cocoa butter. only a few countries can cultivate cocoa on a commercial scale, that consequently leads to an unstable supply of cocoa butter, the primary ingredient in making chocolates. cocoa butter is one of the most expensive vegetable oils, prompting many industries to seek out for alternative vegetable fats. cocoa butter equivalents or cbes are compounds that can be substituted to cocoa butter because of their almost similar fatty acid composition, causing minimal softening or hardening effects on the product where they are incorporated. only six vegetable fats can be substituted to cocoa butter (eu regulations 2003), and mango kernel oil is one of them, as it contains mango seed almond fat (maf) that is high in stearic acid content. when mango kernel oil is blended with palm oil, their resulting mixture contains palmitic, stearic, and oleic acids, similar to that of the cocoa butter. the blend of 80% kernel oil—20% palm oil has a melting behavior and slip melting point that is closest to that exhibited by cocoa butter. the mango kernel butter itself can also be used as edible oil (kaphueakngam et al. 2009). mango kernel butter reduces skin cell degeneration, protects the skin against ultraviolet radiation from the sun, and restores skin elasticity. the stearic acid content of mango kernel butter makes it suitable as a preservative in different cosmetic products, and the high unsaponif iable matter content (4.58%) guarantees the use of such oils in the cosmetic industry. it is also being developed for medicinal applications and for food items, such as confectionery and blended oils (nzikou et al. 2009). the kernel is about 75% of the seed weight, which is about 50% of the waste that can be derived from an average mango fruit. mango seed kernels contain about 10% to 13% oil, depending on the variety and the climatic conditions where it is grown. there are two intertwined extraction methods for mango kernel butter extraction: mechanical and solvent extraction. mechanical methods, such as hydraulic pressing and screw pressing, are generally cheaper in terms of acquisition and operation costs than solvent extraction. however, the screw pressing procedure generally produces smaller oil yields compared to solvent extraction upon use of the same amount of raw material. some processing plants in india employ both methods, wherein the by-product cake from the mechanical extraction process is further subjected to solvent extraction (mahale and goswami-giri 2011). the industry studies by boi (2011) presented a brief overview of the potential of establishing optimizing microwave-assisted crude butter extraction 46 processing units, that do not exist at the present, for mango kernel oil production in the philippines. the abundance of discarded seeds from processed and raw fresh mangoes is a potential cheap supply for a processing plant. patterned after the processing schemes in india, the process can be manual, or semi-mechanized and mechanized with imported designs and fabricated units, such as driers, decorticators, mills, solvent extraction unit, f inishing section equipment, and packaging units. with existing laws and government policies, as well as continuing research, the industry can be established feasibly for continuous operation in the country. up to date, there are 28 potential suppliers of mango seed kernels from different regions in the country, and 36 potential local manufacturers of mango kernel oil for cosmetic applications, including several multinational companies, such as procter and gamble, unilever, and sanof i-aventis. oil extraction technology for mango kernels is still under development in the country. however, several international companies involved in the extraction, manufacture, and distribution of mango seed oil, such as the american entities jeen international corporation and protamine chemicals inc. , already identif ied markets for mango oil in the philippines. microwave (mw) pretreatment of the raw material for oil extraction, especially pulps and seeds, recently became a suitable pretreatment process for increasing the yield of the extracted oil. moreno et al. (2003) revealed that mw pretreatment in avocados undergoing solvent extraction resulted to an increase in oil extraction eff iciency from 54% in soxhlet-hexane extraction to 97% in mw-assisted soxhlethexane extraction. uquiche et al. (2008) conducted mw pretreatment that signif icantly modif ied the microstructure of the substrate tissue, and helped in the increase of mass transfer of oils and other lipids of chilean hazelnuts which will undergo mechanical extraction. additionally, mw treatment further ruptured the cell wall of the treated sample, making cell permeability and oil movement throughout the cell walls more eff icient. they also claim the mw pretreatment brought positive effects on the oil quality by providing higher stability to oxidative deterioration. their data also showed that the time of exposure to microwave radiation has a more profound and signif icant effect (p<0.05) on the extraction yield compared to the potency of microwave power. similarly, momeny et al. (2012) studied the signif icant effects of mw pretreatment on the oil yield of an unidentif ied variety of mango kernels. the use of 300-w mw power and 180-sec exposure time resulted to an increase in oil yield to as much as 8.9% wet basis, in contrast to the 5.65% oil yield of the untreated sample, which was f inely ground prior to extraction to increase the surface area in contact with the solvent. after treatment at 450 w for 180 sec, the sample was burnt, indicating that microwave heating of f inely ground samples may exceed tolerance limit for extraction and analysis. ev casas et al. 47 using the conventional methods of solvent extraction in mango kernels, the boi (2011) projected a percentage of annual prof it of investment of about 27% if an oil extraction and processing facility for mango kernels will be built in the philippines. the introduction of an optimal mw pretreatment to the local supply of mango kernels should theoretically increase the yield returned per investment and consequently increase the prof it of the investor. today, no study that focuses on the optimization of mw pretreatment for crude butter extraction from the mango kernels of the philippines’ locally produced mangoes and conducts a sensory analysis on crude butter from carabao mango kernel exists. physical and chemical properties of mango kernel butter and its appl ications fresh mango butter is a thick and solid fat with the following properties: pale yellow in color at room temperature (mahale and goswami-giri 2011), slight fatty odor and a bland taste, thick consistency at room temperature but readily melts at temperature of around 31 to 39 oc (pioneer enterprise 2006), and specif ic gravity of around 0.885 to 0.925, making it less dense than water. kittiphoom (2011) cited that mango kernel oil has stearic (27%-38%) and palmitic (6.5%-10%) acids as its primary saturated fatty acid constituents, and oleic (42.6-48.3%) and linoleic acids (2.4-5.3%) as its major unsaturated fatty acids. such fatty acid proportions make them suitable as cocoa butter substitute and for applications in other industries without further neutralization of the oil. in addition, mango kernel oil is more stable than other vegetable oils that are also rich in unsaturated fatty acids. the applications of mango kernel oil can extend into incorporation with widely produced vegetable oils, stearin manufacturing, and the confectionery and soap industries. mechanical extraction and the microwave pretreatment process mechanical extraction is accomplished by exerting compressive force or pressure on the raw material to release the crude oil. this method is widely employed on plant materials, such as coconut and other vegetables, which provide edible oil. there are three major steps in mechanical screw extraction method, namely pretreatment, pressing, and oil clarif ication. pretreatment, which involves the application of heat and/or size reduction, is usually applied to increase the yield. in screw pressing, the pretreated raw material is fed to the hopper of the screw extruder and then to the screw conveyor where it is pressed. the freshly pressed optimizing microwave-assisted crude butter extraction 48 oil contains several other components, such as water, f ibers, dust, and other impurities from the raw material, which contribute to the unclear appearance of the oil. clarif ication, or the process of letting the oil stand for some time to form a purer oil layer, can remove these impurities. moreover, the oil can be cooked to remove the moisture and separate the oil. however, since the process extracts oil only through applied shear pressure (and temperature for an expeller with a heater), the recovery of the raw material from the matrix and the characteristics of its natural perforations, primarily the material cell wall, are severely limited. to address this problem, one possible and practical approach is the adoption of microwave pretreatment or microwave-assisted extraction to improve the extracting capability of an oil extraction method by making perforations larger (mandal et al. 2007). in contrast to solvent extraction methods executed alone, microwave or mw pretreatment prior to solvent extraction can bring several advantages in an oil extraction venture, such as improved yield and quality, direct extraction capability, lower energy consumption, faster processing time, and reduced solvent levels (azadmard-damirchi et al. 2011). microwaves are non-ionizing electromagnetic waves with frequency between 300 mhz to 300 ghz, which are between the x-ray and infrared rays in the electromagnetic spectrum. microwaves can be created from electric and magnetic f ields oscillating at perpendicular f ields. in mw pretreatment, heating occurs in a localized manner, resulting to minimal loss of heat to the surroundings. in studies involving microwave-assisted soxhlet apparatus, extraction time can be as low as thirty minutes compared to the conventional solvent extraction system accomplished in long hours. the practical application of microwave energy in pretreatment lies in the frequency of 2450 mhz, the common frequency found in domestic microwave ovens. the use of frequencies lesser or greater than 2450 mhz will result in the non-heating of the treated material. any plant material, such as seeds, can contain minute amounts of moisture in their interstices at atmospheric pressures. the microwave heats and evaporates the locked moisture from the plant material, giving immense pressure to the cell walls that bind the moisture, eventually causing their swelling and bursting. this allows the oil to move freely out of the matrices of the plant material, making it easier for the surrounding solvent during extraction to dissolve it. there are several factors that signif icantly affect the performance of microwaves for oil extraction, including microwave irradiation power, exposure time, nature and quantity feed material, matrix characteristics of the material to be extracted, and the temperature of the treatment ev casas et al. 49 (letellier and budzinski 1999; zuloaga et al. 1999; huie 2002; wang and weller 2006; mandal et al. 2007). other effects of microwave pretreatment aside from the oil extraction yield, mw pretreatment also affects the major and minor components, the physicochemical properties, and the overall quality of the oil produced. oleic and linoleic acids present in mango kernel oil are more dramatically affected by microwave pretreatment than palmitic and stearic acid. antioxidative phenolic content and tocopherol levels signif icantly increased after microwave pretreatment prior to extraction, primarily due to the damage and rupture of the plant cell membrane. in the mw pretreatment of sunflower oil, an increase in the exposure time caused the decrease in the refractive index, unsaponif iable matter, and the iodine values; and the increase in the saponif ication value and the free fatty acid content. increased heating times, temperature, and power settings can result in a higher degree of oil deterioration. however, microwave pretreatment of olive, sunflower, and rapeseed oils led to increased stability because of the increase in released antioxidants, such as tocopherols (yoshida et al. 1995; veldsink et al. 1999; anjum et al. 2006; azadmard-damirchi et al. 2011). optimization by response surface regression response surface regression is an analysis used to f it a polynomial regression model with cross product terms of variables, which may be raised up to the third power, and to calculate the minimum or maximum value of the surface (ncss 2014). many industrial experiments that seek out optimization solutions from a given set of factor variable employ response surface methodology. if a factor is measured at three or more values as determined by the experiment, a quadratic response surface can be estimated with the use of least squares regression. if the surface generated has a hill or valley-like shape, an optimal value can be determined and predicted. however, if the shape becomes more complicated or if the offered optimum solution is far from the range of experimentation, the shape of the surface can still be used for an analysis to create recommendations as to how new experiments should be performed (sas institute 2014). statistical software packages like statistica 10, sas 8.0, anddesign expert 7.0 can analyze and process optimization multiple response processes by response surface methodology via desirability functions. desirability functions approach optimizing microwave-assisted crude butter extraction 50 results by obtaining an optimum set of quantitative values from multiple responses, by assigning a score to a set of responses, and by selecting the factor setting that shows the relatively maximum or optimal score. it is one of the widely used methods for determining a set of conditions from tested and actual values from experimentation expected to yield a desired maximum output (nist/sematech 2012). desirability functions for each response can produce a desirability prof ile. the overall desirability can be obtained by determining the geometric mean of the desirability for each response. this is not only limited to maximization, but it can also f ind the set of responses that can achieve a minimum response score. statistical computer softwares can be used to compute for desirability functions and can provide a desirability prof ile for optimization studies (sas institute 2014). crude butter fat yield (y 1 ) is expressed as the following by response surface regression (rsm): y 1 = f (x 1, x 2, x 3 ). the true relationship between y 1 and x 1, x 2 , x 3 can be complicated, and in most cases, unknown. however, second-degree quadratic polynomial equations can represent them in the range of interest (annadurai and sheeja 1998). sensory evaluation and acceptance test ift (1975) def ined sensory evaluation as “a scientif ic discipline used to evoke, measure, analyze, and interpret reactions to those characteristics of food and materials as they are perceived by the senses of sight, smell, taste, touch, and hearing”. since food properties relevant to consumer perception studies, such as flavor, odor, and texture, are hardly quantif ied by product marketing, the use of the senses of a group of actual people with minimal bias as much as possible is applied to quantify properties in a predetermined scaling. the three broad methods of sensory evaluation include descriptive, discrimination, and acceptance. descriptive test (cernohous [date unknown]) is the sensory method that objectively identif ies attributes and qualities of a food or a product using human subjects (panelists) specif ically trained for this purpose. discrimination test is designed to measure the likelihood that two products are perceptibly different. acceptance test or affective test measures actual user or end user response in terms of likeliness or acceptability (gengler [date unknown]). acceptance tests, which are commonly used for new products for release in the market, do not require trained or professional panelists like in the other two methods, but necessitate the participation of larger number of people that represents the consumer body. the 9-point hedonic scaling quantif ies human responses. other factors that can affect ev casas et al. 51 the outcome of sensory evaluation include health, interest, availability and communication skill of the panelists, and the subjective nature or bias responses of the panelists (gengler [date unknown]). significance of the study the philippines contributes an average of one million tons (mt) to the 35-mt annual worldwide production (fao 2009). local production of fresh mangoes is tallied at 825423 metric tons, with 20114 metric ton of exports including processed forms and 4,200 tons of oil extracts (bas 2010). estimates in india reveal that 30000 tons of oil extracts come from their total annual harvest of 7 mt (boi 2011). the f igures in actual yield of mango kernel butter by solvent extraction change across the varieties, sizes, maturity, pretreatments, and environmental growth conditions of the mangoes. according to existing literature, the pure oil content of mango oil from the kernel is about 815% dry basis. objectives of the study this study explored the microwave-assisted mechanical screw extraction and optimized crude butter extraction from microwave pretreated carabao mango kernels. specif ically, this study: (1) evaluated the effects of size reduction, microwave power, and exposure time parameters for crude butter extraction during microwave-assisted mechanical screw extraction; (2) performed sensory evaluation through acceptance tests on mango kernel butter extracted at optimal conditions; and (3) determined the optimal pretreatment conditions prior to microwave-assisted mechanical screw extraction. materials and method materials and equipment the materials and equipment used in the study consisted of the following: mango kernels from ripe carabao variety, microwave-safe bowls, kitchen grater (diameters of 1.5 mm, 3.0 mm, and 4.5 mm)5, promac™ microwave oven (model svc-005038), scissors, plastic bags, electronic balance, funnel, large flat pan, domestic stove, small-scale motor-powered expeller, glass rod, evaporating dish, and a pair of pliers. optimizing microwave-assisted crude butter extraction 52 experimental design the experiment used three independent variables, namely microwave power in watts (w), size in millimeters (mm), and microwave exposure time in minutes (min). a three-level, three factorial box and behnken experimental design was formulated using the essential regression software. the design is an independent quadratic design, which does not contain embedded factorial or fractional factorial design. in addition, treatment combinations are at the midpoints of edges of the process space and at the center (nist/sematech 2012). this study used 15 basic experimental runs depicted in table 1. fresh mango seed samples weighing 10 grams were used in each run. the microwave power settings used were 160, 500, and 850 w; size levels were 1.5, 3.0, and 4.5 mm; and the exposure time settings were 2.0, 3.5, and 5.0 min. table 1 shows the matrix of the experimental runs. table 2 shows the combinations of the independent and dependent parameters for the pretreatment conditions of mango kernel butter extraction. preparation and dry weight determination of samples ripe mangoes of the same sizes and batch were purchased from the angono public market, philippines. the fruits were manually depulped to extract the stones. the fresh stones were immediately washed and decorticated using a pair of pliers to determine the suitability of the kernel inside for processing. as shown in figure 1, the collected kernels were then washed and stored in the vegetable compartment of a domestic refrigerator for preservation prior to pretreatment. the dry weights were determined using 10 grams of fresh sample in a pre-weighed tin container placed in an air oven for 12 hours. the weight of the tin can and the dried sample microwave power level (w) 160 500 850 oil yield, wet basis microwave exposure time (min) 2 3.5 5 and dry basis size reduction level (mm) 1.5 3 4.5 independent parameter symbol code dependent parameter -1 0 1 table 1. independent and dependent parameters of the study ev casas et al. 53 was measured, and from this, the weight of the tin can alone was deducted to determine the actual dry weight of the sample that will be used for the dry basis computation of the crude butter yield. moisture content of the fresh mango kernel sample was computed using the equation 100%, where mc is the actual moisture content (%), w 1 is the initial weight of the fresh sample (10 g), and w 2 is the f inal weight of the oven-dried sample (g). figure 1. hot water bath set-up for the evaporation of moisture. % 1 2 1 1 160 2 3 2 850 2 3 3 160 5 3 4 850 5 3 5 160 3.5 1.5 6 850 3.5 1.5 7 160 3.5 4.5 8 850 3.5 4.5 9 500 2 1.5 10 500 5 1.5 11 500 2 4.5 12 500 5 4.5 13 500 3.5 3 14 500 3.5 3 15 500 3.5 3 run # x 1, microwave power (w) x 2, microwave exposure t ime (mins) x 3, size reduction level (mm) y 1 crude butter yield %6 table 2. matrix of independent and dependent parameters 6 based on wet basis optimizing microwave-assisted crude butter extraction 54 microwave pretreatment and crude butter extraction the mango kernels were reduced in sizes using kitchen graters of different sizes as stated in the experimental design. approximately ten grams of the reduced fresh sample was placed in a bowl, in such a way that the fresh sample beneath is equally distributed to the floor of the bowl. a total of 30 ml of boiling water was poured in the bowl prior to the microwave pretreatment to properly distribute the effects of the microwave treatment during different exposure times and to eliminate the error of heat distribution that will only be used in boiling the water. hot water is also a safe solvent for the better dissolution of crude butter from the kernel, especially when combined with mechanical extraction. crude butter was then placed inside the domestic microwave promac™ model svc-005038 with a f ive-power level adjustable knob which provide specif ic power outputs convertible to watts. the mixture was cooked inside the microwave for a specif ic exposure time. the cooked mixture was then expelled from its solid constituents in the small-scale expeller. the water-butter mixture from each experimental run was placed in plastic bags as temporary containers for the mixture, in order to minimize the losses in transferring prior to evaporating the water from the mixture. evaporation of water the water-butter mixture from the plastic container was transferred into a reweighed evaporating dish. a large flat pan was f illed with ample amount of distilled water and placed onto a kitchen stove. the evaporating dish was then placed in the center of the pan as in a hot water bath as shown in figure 1. this process of moisture evaporation is much safer than direct heating and prevents excessive physical and chemical damage to the crude butter. crude butter contains high amounts of carbohydrates that are very susceptible to damage by high temperature. the water in the pan was consistently replaced until all free-flowing moisture inside the evaporating dish disappeared. the weighing of the evaporating dish with the dried butter inside followed. the dry weights of the crude butter used in subsequent computations were calculated as follows: dbw = wd 2 — wd 1 , where dbw is the crude butter dry weight (g), wd 2 is the weight of the evaporating dish and butter after the hot water bath (g), and wd 1 is the weight of the evaporating dish (g). ev casas et al. 55 crude butter y ield using the oven-dry weight of the fresh sample, the crude butter yield in dry basis was computed using * 100%, where cby db is the crude butter yield in dry basis (%), dbw is the dry weight of the crude butter (g), and w 2 is the f inal weight of the oven-dried sample (g). on the other hand, using the fresh sample weight prior to pretreatment, the crude butter yield in wet basis was computed using (5), where cby wb is the crude butter yield in wet basis (%), dbw is dry weight of the crude butter (g), and w 1 is the initial weight of the fresh sample (g). statistical analysis results from the experiment were analyzed using the sas 9.0 software to determine the effects of the independent parameters to the crude butter yield in wet basis. contour and surface plots were also generated from the same program. optimization analysis was performed using the statistica 10 software under polynomial regression to determine the pretreatment conditions that will theoretically result to an optimum yield. a desirability prof ile was also generated using the analysis. trial runs using the determined optimum pretreatment conditions from the polynomial regression analysis were also conducted. the predicted and actual values were compared and analyzed to determine if there exist signif icant differences between the predicted and actual values by duncan multiple range test (dmrt ) at 95% level of signif icance. sensory evaluation of the crude butter extracted the crude butter sample in the optimal pretreatment condition derived from the statistical analysis underwent sensory evaluation through acceptance test. ten men and ten women college students aged 17 to 24 years participated in the tests. to reduce the subjective responses of the participants, the test was performed in areas with adequate lighting and free from other odors that can be present in the area. the criteria for the sensory analysis were overall visual appearance, odor, and texture. they were also oriented that the sample will be used as a cosmetic additive. the 9-point hedonic scale was used for the acceptance test of the mango butter by   2   1  ∗ 100% optimizing microwave-assisted crude butter extraction 56 the sensory panel (table 3). dmrt analysis of the results from the evaluation determined the signif icant difference in the mean scores among panelists. the crude butter was evaluated according to its visual appearance, odor, and texture when applied to the palm of the hand. results and discussion effects of the independent parameters on crude butter y ield table 4 illustrates the crude mango butter extracted from mango kernels at different experimental runs as influenced by selected independent parameters at various levels. the extracted crude butter ranged from 1.2% wb to 26.4% wb or 2.26% d to 48.85% d , which is way below the yields of 60% to 90% when using the soxhlet-hexane extraction method. crude mango kernel butter is a combination of 1 160 2 3 0.65 6.5 12.03 2 850 2 3 1.5218 15.3 28.18 3 160 5 3 0.811 8.1 15.02 4 850 5 3 0.765 7.7 14.17 5 160 3.5 1.5 2.638 26.4 48.85 6 850 3.5 1.5 2.072 20.1 38.37 7 160 3.5 4.5 1.1369 11.4 21.05 8 850 3.5 4.5 0.41 4.1 7.59 9 500 2 1.5 1.2782 12.8 23.67 10 500 5 1.5 2.275 22.8 42.13 11 500 2 4.5 0.122 1.2 2.26 12 500 5 4.5 0.359 3.6 6.65 13 500 3.5 3 0.512 5.1 9.48 14 500 3.5 3 0.591 5.9 10.94 15 500 3.5 3 0.481 4.8 8.91 trial dependent parameters final crude butter dry weight (grams) extracted crude butter (% wet basis) extracted crude butter (@ dry basis) power level (w), x1 exposure t ime (min), x2 size reduction level (mm), x3 table 4. crude butter yield (%) from the experimental runs table 3. 9-point hedonic scale (gengler [date unknown]) dislike disl ike disl ike disl ike neither like like like like extremely very moderately slightly like nor slightly moderately very extremely much dislike much ev casas et al. 57 oil, proteins, and carbohydrates, such as starch. the effectiveness of the extraction relies on the suitable combinations of the pretreatment procedure and the mechanical extraction. mechanical extraction processes alone are inefficient because of the inherent stickiness or the sap-like texture of the fresh butter. the fresh butter adheres to the solid cake or to the inside walls of the screw conveyor in a screw-press expeller. application of direct heat to the expeller will usually burn the rich carbohydrate content of the kernel and will result in a stickier sap-like substance in the cake with no yield at all of low quality butter. the butter extracted from the mango kernel is readily biodegradable. if left in average room temperature, it may spoil in the course of a week. therefore, the storage of extracted butter in refrigerated spaces is highly advisable to prolong its shelf life in its fresh form. contrary to the reports in literature about the density of the butter, the extracted butter from the experimental runs had densities of over 1 g/cc. this is primarily due to the rich carbohydrate and protein content of the crude butter, which had not gone any ref ining process after its extraction. figure 2 shows the crude butter yield in both wet and dry basesin percent. as seen in the graph, the trends of the wet and dry bases do not differ, because of the same oven dry weight used in the computation of the dry basis per run. run 5 employing the parameters 160 w, 3.5-min exposure time, and 1.5-mm size reduction level produced the highest percent yield among the trials. run 5 was followed by run 10 at 500 w, 5-min exposure time, and 1.5-mm size reduction level. the third highest in oil yield is run 6 at figure 2. crude butter yield (%) prof ile from mango kernels in wet and dry basis by run. optimizing microwave-assisted crude butter extraction 58 850 w, 3.5-min exposure time, and 1.5-mm size reduction level. smaller particle sizes of the fresh sample prior to microwave pretreatment increased the butter yields. size reduction of the sample increased the yield by incrementing the surface area affected by the applied pretreatment process. the increased surface area also helped the mechanical extraction by easing the flow of the water-butter mixture in the interstices. the lowest yield occurred at run 11 at 500 w, 2-min exposure time, and 4.5-mm size reduction level. such result could be attributed to the small surface area in contact with the hot water and the microwave power. since the fresh sample is in hot water during the microwave exposure, only the outermost area of the sample is affected by the heating process, which removes the crude butter from its solid constituents with the help of water acting as a solvent. the solubility of butter to water is signif icantly affected by temperature: the warmer the water, the more soluble the butter will be. figure 6 shows the settling of the butter in cool temperatures of around 4oc. increased microwave power can also bring additional effects to the physical and chemical properties of the crude butter. table 5 shows the brief sensory evaluation of the trials as affected by microwave power. after the pretreatment, the mango butter, which was exposed to high microwave power, released strong characteristic odor, as opposed to the odorless description of mango butter in the literature and product catalogs for mango butter. this odor can bring undesirable properties to the f inished product especially when used in cosmetic applications normally applied near olfactory organs. high microwave power in combination with long exposure times can also result in the darkening of the crude butter from pale yellow to dark yellow. the increased surface area of the samples also makes them more susceptible to the changes brought by increased microwave power. figures 3 to 5 show the contour plots of the effects of the combination of two independent parameters on the crude butter yield. table 5. color and smell evaluation* of trials based on microwave power level 160 almost odorless, with slight yellowing of the crude butter. 500 with a characteristic odor with slight yellowing of the crude butter. 850 strong characteristic odors with darkening of the crude butter. microwave level (watts) sensory evaluation ev casas et al. 59 figure 3. contour (a) and surface (b) plots of crude butter yield (%) by microwave power (w) and exposure time (min). optimizing microwave-assisted crude butter extraction 60 figure 4. contour (a) and surface (b) plots of crude butter yield (%) by microwave power (w) and size reduction level (mm). ev casas et al. 61 figure 5. contour (a) and surface (b) plots of crude butter yield (%) by size reduction level (mm) and exposure time (min). optimizing microwave-assisted crude butter extraction 62 analysis of variance (anova) of the independent parameters affecting crude butter y ield the rsregression procedure of sas 8.0 performed the anova of the independent parameters to determine their signif icance to the crude butter yield from mango seeds. table 6 shows the results of the anova tests on the independent parameters. the most signif icant parameter is the size of the sample, followed by the microwave power at 95% conf idence interval. the size of the sample also reflects the degree in which microwave power can penetrate the sample, as well as the degree of contact in the hot water acting as the solvent. microwave power has a direct effect on the temperature of pretreatment, wherein higher temperatures ease the flow of the crude butter from its solid constituents. microwave exposure time has the least signif icance among the parameters. this might have been caused by the soaking of the sample in hot water prior to the actual microwave pretreatment that caused initial dissolution of the crude butter to the water. table 7 shows the signif icance of the response surface regression models for the crude butter yield in wet basis, as well as the r2 value, and coeff icient of variation. settled crude butter figure 6. settling of butter-water mixture prior to water bath. crude butter solidif ied and settled at the bottom of the mixture at low temperature. ev casas et al. 63 the r2 value of this regression model is 0.88, whereas the coeff icient of variation is 42.6365. these values explain the degree of the precision and reliability of the experiment. the high variation among the data can be attributed to the relatively small sample size used in the experiment, which is signif icantly susceptible to losses incurred. it is therefore recommended to use a larger sample size for further studies, in order to equalize the effects of microwave power and exposure time to larger sample sizes. optimization through polynomial regression via desirabil ity functions figures 4 to 6 show the plot of the predicted desirability values based on the minimum and maximum values of the study’s independent parameters. from this data, the predicted values of crude butter yield in wet basis per derived factor table 6. anova of the independent parameters on the response variable (yield % wet basis) at 95% confidence level microwave 4 1.108216 0.277054 1.40 0.3537ns* power exposure time 4 0.429478 0.107370 0.54 0.7125ns* sample size 4 6.157077 1.539269 7.80 0.0224** parameter df sum of squares mean square f value pr > f * not significant ** highly significant at 95% level of confidence; significantly affects crude butter yield based on dmrt at 95% conf idence interval table 7. anova showing the significance of the response surface regression model for the crude butter yield in wet basis. linear 3 4.944845ns* quadratic 3 1.941378ns cross product 3 0.363926** total model 9 7.250149ns lack of fit 3 0.980424ns pure error 2 0.006467 total error 5 0.986891 r2 0.8802 coeff icient of variation 42.6365 source df sum of squares * not significant ** signif icant at 90% confidence level; optimizing microwave-assisted crude butter extraction 64 level were determined. as analyzed using the general linear model polynomial regression in the statistica 10 software, factor levels from the respective independent parameters with the highest desirability values were selected and considered as the optimal pretreatment conditions for crude butter extraction (table 8). as highlighted, the optimal conditions are as follows: 160-wmicrowave power, 4.25-minute exposure time, and size reduction level of 1.5 mm diameter grater size. as expected from the initial results, the f inest sizes of the fresh samples were determined as optimal and the predicted values indicated a decrease in the increase rate in percent yield as the size of the sample increases (figure 7). the trend for the effect of microwave power shows high desirability values in both minimum and maximum values. the trend for time shows relatively little changes in the predicted yields along its range. equation (6) shows the polynomial equation generated from the analysis, with crude butter yield in wet basis as the response variable. y 1 = 48.4450 – 0.0424x 1 + 2.5037x 2 2 – 0.2815x 2 2 19.7278x 3 + 2.4296x 3 2 where: y 1 = crude butter yield in percent wet basis (%) x 1 = microwave power (w) x 2 = exposure time (min) x 3 = size reduction level (mm) table 8. regression coefficients for the optimization of crude butter yield in % wet basis intercept 48.4450 power -0.0424 power2 0.0000 time 2.5037 time2 -0.2815 size -19.7278 size2 2.4296 effect coefficient ev casas et al. 65 figure 7. desirability plot for the predicted values and desirability values of the dependent parameters for the extraction of crude butter. optimizing microwave-assisted crude butter extraction 66 verif ication of the optimal pretreatment cond ition the optimum pretreatment conditions for the crude butter extraction determined by the polynomial regression analysis shown in table 9 were tested by replicating the experiment in these conditions for three trials. the results of the verif ication test results are shown in table 10. table 9. pred icted responses at each level of each factor while hold ing all other factors constant at their current settings power 160.0000 24.11667 0.950520 power 332.5000 20.20885 0.822293 power 505.0000 18.68958 0.772442 power 677.5000 19.55885 0.800965 power 850.0000 22.81667 0.907864 time 2.000000 22.44167 0.895559 time 2.750000 23.31667 0.924270 time 3.500000 23.87500 0.942591 time 4.250000 24.11667 0.950520 time 5.000000 24.04167 0.948059 size 1.500000 24.11667 0.950520 size 2.250000 16.15417 0.689247 size 3.000000 10.92500 0.517663 size 3.750000 8.42917 0.435768 size 4.500000 8.66667 0.443561 factor-level predicted-y ield desirabil ity-value observed values % 23.54a11 21.96c11 22.15b* 22.55b* predicted value % 24.11667a percent difference % 6.5 trial 1 trial 2 trial 3 average table 10. comparison of the pred icted and actual values using the predetermined optimal cond itions * values in a row followed by the same letters are not signif icantly different from each other using dmrt at 95% level ev casas et al. 67 all verif ication values at optimum conditions were lower than the predicted values at optimum conditions, thereby requiring changes in the chosen independent parameters tested. this suggests that other combinations of factors may be more appropriate in producing optimum yields from microwave-assisted screw pressing of mango butter from seed kernels. lower values of factor levels may be examined in the future to lower the cost of production. lowering the power, exposure time, and particle size will reduce the total production costs. although hexane extraction by soxhlet method produces higher yields, the method is not recommended, in terms of the costs, product safety, and environmental considerations. problems on health among workers will rise and the setup of a hexane waste treatment will be necessary if hexane extraction is employed in extracting crude butter from mango seeds. the average percent difference between the actual and observed values is 6.5%. this error is primarily attributed to the observed losses incurred in the mechanical extraction of the butter, as well as in the transportation of the water-butter mixture from one container to another during laboratory analysis. the butter extracted from the verif ication runs had a yellowish white color with no signif icant odor. it is also vulnerable to spoilage when stored in average room conditions in less than a week. acceptance test of the crude butter in optimum pretreatment cond ition twenty untrained analysts evaluated the acceptance test of the crude butter processed from the optimal pretreatment condition using the 9-point hedonic scale. table 11 shows the results of the experiment. figure 8 depicts the radar graph of the sensory properties of the crude mango butter as graded by the sensory panel members. converting the score from the hedonic scale into numerical values, the signif icance between the mean values of the three sensory criteria were tested using the dmrt at 95% conf idence interval on sas 9.0. results from the analysis show signif icant differences between the scores per criterion as perceived by the individual evaluators. the sensory characteristics of crude mango butter undergoing a consumer level acceptance test is generally subjective in nature and based on the individual preferences of each evaluator (figure 8). however, comparison of the mean values between the three criteria shows that the mean texture score (above neutral) is signif icantly different from both visual appearance and odor mean scores (below neutral). based on the hedonic scaling, two of the important criteria, namely optimizing microwave-assisted crude butter extraction 68 visual appearance and odor, for the evaluation of a potential cosmetic product obtaineda below neutral average perception. visual scores ranged from 4 to 12, odor ranged from 3 to 20, and texture scores ranged from 2 to 19. the values indicate that odor and texture are the primary considerations of the panelists in expressing the olfactory properties of the mango butter. the observations suggest that further processing or ref ining of crude mango butter kernel is recommended to improve these sensory properties if it is intended to be marketed as a fresh product. 1 dislike slightly neutral like slightly 2 dislike slightly dislike slightly like slightly 3 like slightly dislike slightly like slightly 4 like slightly neutral like moderately 5 like slightly neutral like slightly 6 like moderately dislike moderately dislike slightly 7 dislike slightly dislike slightly dislike slightly 8 dislike slightly neutral like slightly 9 dislike slightly neutral neutral 10 like slightly neutral like slightly 11 dislike slightly dislike slightly like slightly 12 like slightly neutral like moderately 13 like slightly like slightly like moderately 14 dislike slightly dislike slightly like slightly 15 dislike slightly dislike slightly neutral 16 dislike slightly neutral neutral 17 dislike moderately dislike slightly like slightly 18 dislike slightly like slightly like slightly 19 neutral like slightly like moderately 20 dislike moderately neutral like slightly participant # visual appearance a odor b texture c table 11. acceptance test results of the crude butter in the optimal pretreatment cond ition as determined by the test panel ists. ev casas et al. 69 figure 8. radar graph of the criteria scores per category summary and conclusion the optimal pretreatment conditions for crude mango butter microwave-assisted extraction was studied using a box and behnken design of experiments with the following serving as the independent parameters of the experimental design: microwave power in w (160, 500, and 850), microwave exposure time in min (2.0, 3.5, and 5.0), and size reduction level through grating of the fresh sample in mm (1.5, 3, and 4.5). the pretreatment comprised a microwave set-up with varying power levels, different sizes of kitchen graters, and a mechanical extraction unit for the actual extraction process. results show that varying levels of microwave power affect the physical and chemical characteristics of the crude butter as observed in the differences in color and the odor it emitted. the anova of the data identif ied sample size and microwave power as the signif icant factors in the crude butter extraction and calculated an r2 value of 0.88. the variation in the data can be attributed to the small sample size used in the experiment, which is susceptible to signif icant losses along the execution of the trials. the optimum extraction conditions determined by polynomial regression via desirability analysis were 160 w, 4.25-min exposure time and 1.5-mm size reduction level. the predicted and average actual yield values had a 6.5% difference based on the verif ication runs and had no signif icant difference based on the dmrt at 95% conf idence interval. evaluation through acceptability test of the crude mango butter processed with the optimum pretreatment conditions was conducted using the dmrt. the scores for all criteria indicated signif icant differences. the mean score for texture was signif icantly different and below than the visual appearance and odor mean scores, indicating the acceptability and feasibility of mango butter for inclusion in cosmetic products. optimizing microwave-assisted crude butter extraction 70 recommendations in view of the obtained results, future studies must consider the following: (1) increasing the sample sizes to 1 kg achieve lesser variations among the response variables, (2) consider the use of other local mango varieties and feasibility of extracting butter from the their waste seed kernel, (3) performing descriptive sensory evaluation on the crude mango butter and other derived products using trained panelists, and (4) performing an extensive physical and chemical analysis on the extracted butter from carabao mangoes to further understand the effects brought by microwave pretreatment on the crude mango butter and the other relevant properties if it is to be used as a cosmetic ingredient or additive, or as a cocoa butter substitute. acknowledgments the authors gratefully acknowledge many people who contributed to the success of this study. sincere appreciation goes to mr. comedia for accepting the topic as his thesis problem; dr. k. f. yaptenco, chairman of abprod, for allowing mr. comedia to work outside off ice hours in the laboratory; dr. e. k. peralta and dr. a. g. gilbuena, the thesis committee members, for the valuable suggestions for the improvement of the thesis manuscript and for approving the same in fulf illment of the requirements for graduation; mr. art m. macandili for the assistance during data gathering in the laboratory; engr. mimi pardua and ms. abbie dacer for the company during data gathering at nights; and mr. kevin g. comedia for the assistance in hauling the raw materials from angono, rizal public market to the abprod laboratory. we deeply acknowledge the suggestions, support, and time allotted by the members of the guidance committee: dr. a .g. gilbuena and dr. e.k. peralta. moreover, the assistance of the whole division (abprod), especially art m. macandili for the unconditional help during the initial experiments, and to ms. abbie dacer for the sincere assistance during registration, form submissions, and coordination of the presentation of this study during thesis defense are greatly appreciated. jocelyn comedia, who continuously supported mr. comedia throughout the bs courses. the whole comedia and gatulayao families for the moral support and everything they extended; gabriel for lending his laptop unit for the presentation of both outline and thesis defenses. christian, sheena, camille, michael, eunice, ton, allen, and ian for serving as family during the f ive-year study in up los baños of mr. comedia. the uplb engineering society and uplb agham youth for molding mr. comedia. ev casas et al. 71 ignatius, paul, lorenzo, horace, fritz, and jericho for all the theories, games, fun, and wor thwhile moments extended to the 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[cited 2013 march 1]. available from: http://unctad.org/en/docs/ditccom20032_en.pdf. uquiche e, jeréz m, ortíz j. 2008. effect of pretreatment with microwaves on mechanical extraction yield and quality of vegetable oil from chilean hazelnuts (gevuina avellana mol). innovative food science & emerging technologies. 9(4):495-500. ve l d s i n k j h , g oy e r j a , o l s e n j l , s a m w t. 1 9 9 9 . g l a c i a l r e f u g i a a n d r eco l o n i z a t i o n pathways in the brown seaweed fucus serratus. molecular biology. 16:3606-3616. wang y, weller j. 2006. recent advances in extraction of nutraceuticals from plants. trends in food science and technology. 17(6):300-312. yoshida y, shimamura k, sugino n, nakamura y, ogino k, kato h. 19995. changes in l i p i d p e r o x i d e a n d a n t i o x i d a n t e n z y m e a c t i v i t i e s i n c o r p o r a l u t e a d u r i n g p s e u d o pregnancy in rats. j reprod fer til. 105(2):253-257. optimizing microwave-assisted crude butter extraction 74 z u l o a g a d g , m o r r i s j a , j o r d a n c , b r e e d l o v e s m . 1 9 9 9 . m i c e w i t h t h e t e s t i c u l a r feminization mutation demonstrate a role for androgen receptors in the regulation of anxiety-related behaviors and the hypothalamic-pituary-adrenal axis. horm behav. 54(5):758-766. _____________ prof. edgardo v. casas <evcasas2@up.edu.ph.com> was born in tiaong, quezon, philippines in 1951. he received his bsc in agricultural engineering in 1975 from the university of the philippines los baños. he went to the university of new south wales for master of applied science in food engineering in 1986, and enrolled in the ph.d. in food science program in 1992. he immediately worked as a research assistant after graduation and as atraining off icer in the former nfac grain postharvest engineering project of the then department of agricultural processing technology, institute of agricultural engineering. he began teaching at the same department in 1976, for the courses fundamentals of crop processing, materials handling equipment, basic rheology, and operations research and optimization to undergraduate and graduate students. he became a member of the graduate faculty in 2013. mr. von jansen g. comed ia <von_jansen@yahoo.com> was born on may 30, 1991 in binangonan, rizal by parents victor and jocelyn comedia. he has three younger siblings, namely jan kevin, mar jovic, and elenee may. he f inished his elementary education in the angono elementary school in angono, rizal (2003) and his secondary education as a part-time scholar at the angono private high school (2007). after failing the entrance examination at both up diliman and los baños campuses for a b.s. computer science or biology program in 2007, he decided to be apply as a candidate for a degree in agricultural engineering program in the college of engineering and agro-industrial engineering in uplb. he is interested in agricultural engineering as a profession and the challenges posed to prospective engineers in the demands for a much better sustainability in agricultural and food production. arni g. gilbuena, ph.d. <amigambe@gmail.com> is currently teaching full-time at the school of science and engineering, ateneo de manila university in quezon city. she earned her bsc agricultural engineering degree at the university of the philippines los baños in 2001. she briefly taught at the department of engineering science and at the agricultural and bio-process division of the institute of agricultural engineering in uplb. she holds a master’s degree in biotechnology and in energy engineering from osaka university and up diliman, respectively. she obtained her ph.d. in advanced science and biotechnology from osaka university. ev casas et al. 75 kevin f. yaptenco, ph.d. is presently the chair of the agricultural and bio-process division; a graduate of bsc in agricultural engineering, university of the philippines los baños in 1989. he obtained his ms in agricultural engineering from the university of illinois at urbana-champaign in 1993 and his ph.d. in bio-industry from the tokyo university of agriculture in 2000. he worked as a research engineer at the postharvest horticulture training & research center of uplb until 2008. he currently teaches full time at the institute of agricultural engineering of uplb, handling classes on crop processing, postharvest engineering, and process control theory for both undergraduate and graduate students of the iae. 04_mamauag mamauag, penolio, and aliño 54 science diliman (january-june 2001) 13:1, 54-65 abstract preliminary data on the patterns of occurrence of juvenile groupers in the philippines was examined. survey questionnaires were randomly distributed to respondents (i.e., fishers and traders of juvenile groupers). patterns of presence/absence of the juveniles were examined and recruitment and spawning patterns were inferred from survey results. results showed that patterns of recruitment of juvenile groupers in the philippines varied greatly with each area/region. the large variability in the recruitment patterns precluded a “general pattern”. upon closer examination, however, some emergent signals were noted, albeit not very strong. spatio-temporal patterns for recruitment of juvenile groupers seemed to be influenced by change in seasons (summer and wet) and monsoons (northeasterlies and southwesterlies). inferred spawning patterns likewise varied with area/region and an inter-specific variation in spawning behaviour may be possible. although results are preliminary due to lack of intensive data verification, a more rigorous type of sampling protocol is worth pursuing in the future. the results of this preliminary examination provided insights on the recruitment of the commercially important epinepheline serranids in the philippines. key words: groupers, lapu-lapu, fish recruitment, spawning patterns deriving recruitment and spawning patterns from a survey of juvenile grouper (pisces: serranidae) occurrences in the philippines samuel mamauag1*, lutgarda penolio2, & porfirio aliño1 1marine science institute, university of the philippines diliman, quezon city 1101 2bureau of fisheries and aquatic resources department of agriculture, quezon ave., quezon city *present address of corresponding author: international marinelife alliance 2268 sylvina bldg., tramo st., pasay city; tel. no.: (632) 831-2805 to 06, e-mail: smamauag@imamarinelife.org introduction fish resources are currently being fully-exploited on a global scale (hilborn & walters, 1992; alverson & larkin, 1992). increasing evidence of over-exploitation of fish stocks has been documented (e.g., munro & williams, 1985; russ, 1991; hilborn & walters, 1992; alverson & larkin, 1992) and management schemes are often either lacking or ineffective (longhurst & pauly, 1987). biological information such as stock size, age, growth, mortality, and recruitment provide the building blocks of population dynamics models which are essential to the development of sound management schemes for these exploited resources. * corresponding author deriving recruitment and spawning patterns 55 the family serranidae (subfamily epinephelinae), commonly known as groupers (heemstra & randall, 1993), is an important group among the marine fish species that are highly exploited. local names for groupers include lapu-lapu, pugapo, and suno, among others (rau & rau, 1980; schroeder, 1980) groupers are generally long-lived, slow growing species with low rates of natural mortality (ralston, 1987). these characteristics make them highly vulnerable to overfishing groupers are generally a high-priced food fish in most parts of the tropics and it has been reported that these are often heavily exploited (ralston, 1987). the coastal fisheries of the tropical seas which are often characterized by large artisanal and subsistence fisheries of the developing countries, recorded more than 97,000 tons of groupers captured worldwide in 1990 (heemstra & randall, 1993). coral reef fish have complex life cycles (roughgarden et al., 1988). almost all coral reef fish have two distinct life history stages occurring in two different environments (i.e., a sedentary reef-associated phase and a more mobile pelagic phase) (doherty, 1991; leis, 1991). it has been observed that recruitment (transition from the larval phase to a settled existence closely associated with the coral reef structure) may considerably influence the abundance of fish stock (doherty, 1987 & 1991; doherty & fowler, 1994). large variations in the abundance of recruits with time are likely to have a strong “forcing function” on the population dynamics of fish stocks on coral reefs (williams, 1980; victor, 1983 & 1986; doherty, 1987; doherty & fowler, 1994). local changes in the abundance and demography of a few reef fish families have been shown to be driven by historical patterns of recruitment (interacting with density-independent mortalities) (sale & ferrell, 1988). information on recruitment patterns will greatly assist management strategies for these important fish resources (doherty, 1987; roberts, 1996). theoretical models in fisheries, such as stock-recruitment relationships developed by ricker (1954) and beverton & holt (1957) have been utilized to estimate fish stock sizes which are dependent on the replenishment of populations through larval recruitment (roberts, 1996). difficulty in the identification of the juveniles and the scarcity of the newly settled larvae in the field complicate recruitment studies in groupers (leis, 1987). in the subfamily epinephelinae, available information deal mostly on their larval biology. in general, the distribution of grouper larvae has been found to be similar to that of the adults’ distribution over continental shelves (powles, 1977; young et al., 1986). doherty et al. (1994) elucidated well the dynamics of fish recruitment through field surveys of spatio-temporal settlement of plectropomus larvae on the northern reefs of the great barrier reef in australia. in the philippines, there is a paucity of published literature on larval biology, much less spatiotemporal variations of recruited juveniles, of groupers. the aim of this study was to carry out a cursory examination of the recruitment patterns of the commercially important epinepheline serranids in the philippines. specific objectives were the following: (a) to determine species of epinepheline serranids that are observed or captured as juveniles; (b) to determine temporal and spatial patterns of recruitment of juveniles of epinepheline serranids based on survey questionnaires; and (c) to obtain information on the spawning seasonality of groupers inferred from the timing of occurrences of settled juveniles this study was an initial assessment of data from a survey of local fishers and traders of juvenile groupers in selected regions in the philippines. no actual sampling of reef fish recruits was carried out. it should also be noted that the assessment was restricted to presence or absence data from the questionnaires because upon review of the data, the investigators observed that there were unreliable estimates of absolute abundance of settled groupers. thus, all abundance data from surveys were not included. mamauag, penolio, and aliño 56 methodology an extensive survey in 1990-1991 to determine the seasonal occurrence and relative abundance of juvenile groupers in the philippines was carried out by the research staff of the bureau of fisheries and aquatic resources (bfar) under the helm of l. penolio survey sheets (annex a) were distributed to regional offices of bfar nationwide. support staff handed out these questionnaires in various fishing localities in each region (fig. 1) where artisanal fishery has been known the questionnaires were handed out randomly to respondents, mainly fishers, and were then retrieved more questionnaires were distributed in areas where fisher population was big, like the areas adjacent to manila bay, while fewer questionnaires were given in areas with smaller fisher population, like those within fig. 1. map of the philippines showing the regional divisions with important fishing localities region x. a total of 200 questionnaires were distributed. the survey focused mainly on the seasonality of occurrences of juvenile groupers, but it also asked for estimates of catch rates of fingerlings and the types of artisanal fishery gears used although these were not analyzed due to the need for proper verification or validation of the abundance estimates the distribution of questionnaires was biased by prevailing weather conditions. approximately 50% of the questionnaires were returned and analyzed. a similar but less extensive survey was carried out in the period august 1993-february 1994 by s. mamauag. survey questionnaires (annex b; modified from a format of the lobster project of the marine science institute under dr. annette juinio-meñez) were distributed to several areas in the philippines, viz., pangasinan area, palawan area, samar area, cebu area, bohol area and misamis occidental-lanao del norte area (fig. 1). randomly chosen fishers and traders in juvenile groupers from each region were given questionnaires around 28 questionnaires were variably distributed to all areas depending on the population of fishers (table 1). the questionnaires initially asked the respondents to provide a list of species of juvenile groupers captured with an adequate description of each species. some respondents were shown photographs of grouper species in color to verify species identification a total of 17 questionnaires were returned and analyzed. additional sources of information on the presence/ absence of juvenile groupers (mostly taken from the traders’ activities) in a few areas in the philippines were table 1. number of questionnaires distributed in a separate survey for selected areas and number returned for analysis i ii iii luzon iv iv iv iv pa law an mindanao v v iv viii viii vi vi vii x ix ix xi sulu moro gulf davao g ulf mis am is o ccid ent al ilig an bay lan ao del no rte bohol guiuan leyte gulf ce bu sulu sea south china sea pacific ocean sam ar sam ar sea visayas cam otes sea sibuyan sea ilo ilo panay gu im ara s s tra ittaytay bay calamian group ragay gulf babuyan channel calauag bicol lamon bay quezon manila bay pangasinan bolinao lingayen gulf area pangasinan palawan samar cebu/bohol misamis total # distributed 6 8 8 3 3 28 # returned 4 5 5 2 1 17 deriving recruitment and spawning patterns 57 utilized to help verify the reliability of the results. for example, results of the resource ecological assessment (rea) funded by the philippine department of agriculture, which aimed to evaluate the grouper fry industry in calauag, quezon, in 1991 were used data from the southeast asian fisheries development center’s (seafdec) grouper research on the survey of finfish fry in iloilo (fig. 1) were also used. results juvenile groupers based on the survey data compiled, only a few species of groupers in their juvenile forms were observed (table 2). comparison with previous but unpublished data on species of groupers occurring in some areas in the philippines showed a disparity in the species composition (table 2). temporal and spatial recruitment patterns of juvenile serranids in the philippines based on the survey results, the peak occurrence of juvenile groupers within the year was highly variable across several regions (fig. 2). the occurrence of juveniles peaked in 12 of 16 areas (i.e., regions), generally around the summer months, although this pattern was not particularly strong (fig. 2) relatively fewer records were noted during the rainy season and some overlapped through summer-rainy months yearround occurrence of juveniles was also observed but peak months fell on the summer months (april-june), as in the case of juveniles found off the coast west of samar. year-round occurrence was specifically noted in davao gulf (fig. 2). although not very consistent, recruitment may follow a monsoonal pattern. peak in abundance occurred generally during the northeast monsoon (octobermarch) in areas like babuyan channel (cagayan valley: region ii), lamon bay (quezon: region iv), south of sibuyan sea (northern panay: region vi), and iligan bay (lanao del norte: region x) (fig. 2). during the southwest monsoon (april-september), the abundance of juveniles peaked in areas such as lingayen gulf table 2. juvenile groupers observed in the survey and list of grouper species reported in previous studies present study (=juveniles) cromileptes altiveles epinephelus fuscoguttatus e. coioides e. guttatus e. macrospilos e. merra e. ongus e. fasciatus e. sexfasciatus cephalopholis cyanostigma cephalopholis urodeta cephalopholis boenack plectropomus leopardus p. laevis variola louti aragones & mamauag (1992, unpublished) (=adults) aethaloperca rogaa anyperodon leucogrammicus cephalopholis argus c. boenack c. cyanostigma c. microprion c. miniata c. sexmaculata c. sonerrati c. urodeta c. altiveles epinephelus areolatus e. fasciatus e. fuscoguttatus e. haxagonatus e. macrospilos e. malabaricus e. merra e. ongus e. quoyanus e. sexfasciatus e. coioides p. laevis p. leopardus p. oligacanthus p. maculatus p. areolatus variola albimarginata v. louti pagdilao et al. (1992, unpublished) (=adults) a. leucogrammicus c. argus c. boenack c. urodeta c. altivelis e. areolatus e. fasciatus e. fuscoguttatus e. macrospilos e. merra e. microdon e. ryncholepis e. ongus e. coioides e. sexfasciatus p. leopardus p. melanoleucus p. oligacanthus p. truncatus (pangasinan: region i), off the western coast of luzon including manila bay (region iii), ragay gulf (camarines provinces: region v), and off the western coast of samar (region viii) (fig. 2). spawning patterns seasonality of spawning was inferred mainly from related patterns of recruitment in the survey, as spawning seasonality was not addressed directly in the survey similar to recruitment pattern, the inferred mamauag, penolio, and aliño 58 1), e. tauvina (=e. coioides) has been the main target species in the commercial fishery, and its juveniles became popular among the traders (silapan, pers. comm.). in iloilo (fig. 1), the seafdec’s finfish fry project staff reported that although juvenile groupers were scarce, e. suillus and e. sexfasciatus were collected during their sampling activities. both species were present in the fishery, but no data on species composition in the fishery was available for comparison variation in the species composition may be due in part to the different levels of intensity of sampling for each study identification of species depended largely on the traditional knowledge of fishers and traders which resulted in the absence of some species of groupers known to occur in the philippines (e.g., schroeder, 1980; rau & rau, 1980; aragones & mamauag, 1992; pagdilao et al., 1992; randall et al., 1990; heemstra & randall, 1993). this problem was exacerbated further fig. 3. inferred spawning patterns of groupers in the philippinesfig. 2. peak occurrences of juvenile groupers in the philippines seasonality of spawning of groupers in the philippines was highly variable (fig. 3) spawning patterns varied among the various areas/regions in the philippines some areas had annual periodicity while others showed biannual patterns. discussion juvenile groupers in the philippines few species of juvenile groupers were more frequently noted than the others in the survey. many respondents from bolinao, pangasinan, recounted that juveniles of e. merra, e. fasciatus, and e. summana were the most commonly observed. in an earlier separate cursory visual assessment made by mamauag in 1989, the three aforementioned species comprised the bulk of the caged juvenile groupers. in the cebu-bohol area (fig. summer months rainy months northeast monsoon southwest monsoon jun sept may fe bja n mar apr ju ly d ec au g nov oct summer months rainy months northeast monsoon southwest monsoon jun sept may fe bja n mar apr ju ly d ec au g nov oct iv iii ii i iv iv iv v v vi vi viii ix ix x xi vii viii deriving recruitment and spawning patterns 59 by the difficulty in identifying juveniles. also, except for some juvenile grouper species that were commonly caught (e.g., e. merra in bolinao), it has been the general consensus among the fishers and traders that juveniles of groupers were generally scarce. this is true for the epinepheline serranids in general. a review of the early life history of tropical groupers revealed the relative rarity of their larvae over continental shelves compared to other equally important coral reef fish species (e.g., snappers) (leis, 1987) juvenile groupers are generally cryptic, thus, reducing their ease in detection in the benthic environment (sale, 1991). although it remains to be established, the observed dominance of some species of juvenile groupers is key to prediction of established adult populations. the predominance of juveniles of e. merra, e. fasciatus, and e. summana, observed in fish ponds in bolinao, pangasinan during the 1989 cursory survey correlates well with the observed dominant species in the local fishery. aragones & mamauag (1992, unpublished data) found e. merra, e. fasciatus, and e. summana to be among the most abundant groupers in bolinao (table 3, column 2) based on a two-year fishery catch data carried out to provide biological information and some aspects of the fishery of bolinao groupers in guiuan, samar, the presence of young p. leopardus (about 10 cm tl) in relatively large quantities in several fish cages was noted by mamauag during a cursory survey made in 1993. pagdilao et al. (1992) reported that p. leopardus was the most abundant among the groupers (table 3) in catch records at the fish landing sites in guiuan collected during the period 1985-1986. these reports seem to imply that the presence of juveniles in an area can be roughly indicative of the presence of adults in the same area, assuming that other important factors (e.g., postrecruitment processes [see jones, 1991]) do not significantly alter abundance and composition of species. however, due to the limited number of juvenile grouper species observed and the absence of abundance estimates, as compared to the magnitude of the size of the adult populations recorded from the fishery, further conclusions regarding their potential relationship require further investigation. the findings of this study are promising and heuristic, as these results can be utilized as proxy estimator for recruitment processes (e.g., victor, 1983 & 1986; doherty, 1987). alternatively, the occurrence and distribution of adults in their habitats (i.e., fishing locality) provide keys in lieu of the problem in taxonomy, rarity, and absence of juveniles in the study there were similarities in the spatial occurrence of dominant species of juvenile groupers in the present study and conspecific adults in previous studies. the presence of the adults may give clues to the availability of potential juvenile recruits although recruitment is highly variable in time and space (sale, 1981; victor, 1986; doherty, 1987 & 1991; doherty & fowler, 1994) and pre-settlement fish larvae go through a hazardous pelagic phase (leis, 1991) before these settle into preferred areas (see victor, 1991), groupers, nonetheless, show similar adult and larvae distributions even over large continental shelves (powles, 1977; young et al., 1986; leis, 1987). although the data gathered in this study are preliminary, it is likely that patterns of adult distribution reflect the presence and/ or absence of juveniles. spatio-temporal recruitment patterns the relatively large spatio-temporal variability patterns in juvenile peak occurrences has placed a considerable constraint in providing a discernible, general pattern when preliminary data were re-examined closely, however, a seasonal trend (summer and wet) was noted juveniles were generally observed in relatively high numbers during summer months (when seawater temperatures are assumed to be relatively higher) in most regions. the observed peak of juveniles in the study during the summer is consistent with a number of work elsewhere. robertson (1991) noted that the growth and survival of juvenile fish have become stabilized after spawning output of the adults seemed to track the seasonal change of the suitability of the benthic environment due to changes in temperature, among others. an annual seawater temperature cycle was observed to regulate the onset and duration of the breeding season for the blackspot sergeant abudefduf sordidus in hawaii (stanton, 1985) and for the tropical damselfish pomacentrus in the great barrier reef, australia (doherty, 1983). in the coral trout plectropomus leopardus, large densities of pre-settling mamauag, penolio, and aliño 60 larvae were captured by light traps deployed in northern reefs off cairns in the gbr, australia, mainly during the summer (november), for two weeks, particularly around the new moon (doherty et al. 1994). variation in ambient seawater temperature has been recognized to be influenced by the general water circulation pattern (e.g., wyrtki, 1961; pickard & emery, 1982). the philippines has an average annual variation of sea surface temperature of less than 2oc (morgan & valencia, 1983). villanoy et al. (in prep.) reported seasonality in the sea surface temperature (sst) measurements in the south china sea, including the sulu sea and part of the celebes sea. during the southwest monsoon, temperature difference was almost negligible at 1oc (29-30oc) while in the northeast monsoon, sst ranged from 26oc to 29oc. however, information to further elucidate the effects of physicochemical factors on fish recruitment in the philippines is lacking. in addition, although data are few, some spatio-temporal peak occurrences of juveniles in this study may also suggest a monsoonal pattern. most of the regions observed to demonstrate peak occurrences during the northeast monsoon are found generally along the eastern coasts of the archipelago or on the windward side of any land mass (e.g., iligan bay) while regions with peak occurrences during the southwest monsoon are located on the windward side of the prevailing winds apparently, local water circulation patterns which are virtually wind-driven (see morgan & valencia, 1983) influence the distribution of the juveniles. during the northeast monsoon (ne; october-march), water circulation pattern (fig. 4) [with current speed ranging from 12 cm/sec to >100 cm/sec (wyrtki, 1971)] may possibly transport pelagic larvae of groupers to suitable areas onshelf for settlement along some of the areas found on the eastern side of the philippine shoreline (e.g., lamon bay) and potentially drive larvae to oceanic conditions on the western side of the archipelago; hence, the low abundance of juveniles on the leeward side during this time of the year (e.g., manila bay). conversely, prevailing water currents during the southwest monsoon (sw; june-september) (fig. 4) may bring the larvae onshore for settlement (eventually increasing the abundance of juveniles) along the western side (e.g., lingayen gulf) and, concomitantly, larvae on the eastern side may be dispersed further away from their natal reefs. however, the sw monsoon, with current speed of up to 25 cm/sec (wyrtki, 1971), has little effect on the general circulation pattern on the leeward side. this may reflect the presence of juvenile groupers albeit in very low numbers in areas like guiuan at this time of the year. a small percentage of respondents in the area recounted that juveniles can be observed throughout the year and peak around the summer months. aside from the temperature rise during fig. 4. water current patterns for the southwest monsoon (june; top) and for the northeast monsoon (october, bottom) in the philippines (after morgan and valencia 1984) deriving recruitment and spawning patterns 61 summer, predominant current speeds are generally slower with the shift of the monsoons (april-may). on this premise, it is not very surprising to expect that in at least some of the central islands in the philippines (visayas), recruitment may appear to occur during both monsoons. this would reflect recruits arriving from two different upstream source areas in any one year. results suggest that both monsoons influence the pattern of distribution of the juveniles, temporally and spatially aliño et al. (1992), using an ordination analysis correlated with localized environmental parameters, pinpointed the northeast (ne)-southwest (sw) monsoons as responsible for the observed groupings of coral reef fish species doherty et al. (1994) explained that the “pulse” in the supply of plectropomus larvae downstream to arlington and green reefs in cairns, australia, was correlated with the northerly winds albeit the absence of a mechanism to determine the influence of water circulation. dight et al. (1988) showed that patterns of passive juvenile recruitment and the maintenance of the cross-shelf species distribution of fish within the central great barrier reef, australia, were explained by models of larval dispersal primarily due to hydrodynamic processes in the area. however, evidence of the active role of larvae in settlement (in leis, 1991) provided additional mechanisms to the patchy distribution of reef fishes. sweatman (1983) showed that larvae of some coral reef fish actively choose settlement sites which were not habited by adult pomacentrid dascyllus. although the recruitment patterns presented above were derived solely from the survey, the results were promising and should dictate a more refined protocol to verify the spatio-temporal variations of juvenile groupers in the philippines. distribution, abundance, and biomass of juvenile groupers at the species level may be obtained by visual counts using transects (english et al., 1997) or by sampling using light-traps (e.g., doherty et al., 1994). future studies should sample both monsoons and select sites at both shorelines (western and eastern) which emerged in the present study as showing signals of peak juvenile occurrence. spawning patterns the variability in the recruitment patterns presented earlier may be reflective of the variability in the spawning patterns of groupers in the philippines (as the latter was inferred mainly from the former). it has been consistently observed that there is a close linkage between spawning and recruitment for coral reef fishes in general. robertson et al. (1988) reported the close linkage between spawning activity and settlement based on contemporaneous measurements of clutch size and recruitment of damselfish in panama. an australian damselfish, pomacentrus displayed matching periodicity of both spawning and settlement cycles (doherty, 1991). although there was no close comparison between both cycles, it generally suggested that production processes may have controlled the timing of settlement. for plectropomus, doherty et al. (1994) observed that the synchrony between spawning effort of the adults and the pattern of larval replenishment was due to a regional entrainment of spawning probably triggered by thermal and lunar cues. seasonality of spawning for the philippine groupers has been deduced to be bi-annual as noted in the banded grouper epinephelus sexfasciatus (pauly & ingles, 1982) recruitment pattern for the banded grouper was derived from the same overall shape of the “spawning pattern”. while some areas in this study were observed to have groupers with restricted spawning seasonalities (mainly based on the assumption that spawning periodicity and seasonality and occurrence of juveniles [settlement episodes] are closely matched), others displayed variation exemplified by biannual seasonality (e.g., region iv) or extended periods of spawning (e.g., region xi) which led to overlapping of seasonalities (fig. 3) this suggests that some groupers in the philippines may have spawned more than once in a year as in the case of the banded grouper e. sexfasciatus (pauly and ingles, 1982). however, these results (biannual seasonality and extended periods) are in contrast to the previously known seasonality of spawning common in groupers. all groupers for which there was evidence spawned during a restricted period (generally between early spring and summer in low latitudes [e.g., australia] and somewhat later in the year in higher latitudes [e.g., the caribbean]) (see reviews by shapiro, 1987; williams & russ, 1991). on the other hand, spawning records show that some grouper species are known to spawn over 6-8 months (nagelkerken, 1979; loubens, 1980; thompson & munro, 1983). mamauag, penolio, and aliño 62 moreover, some artifacts in the local spawning patterns of the present study (fig. 3) may have been introduced in part due to the manner the regions are subdivided, which was based on political classification rather than natural biogeographic criterion. this is shown in the case of region iv which covers a large area ranging from palawan in the western side influenced by the south china sea to quezon province in the eastern side facing the pacific ocean (fig. 1). the two provinces located at opposite shorelines showed two varying inferred spawning patterns which resulted in the display of a biannual seasonality for region iv. finally, the year round spawning period for region xi was rather unreliable. this result may be indicative of the poor sampling scheme of the survey. an interspecific difference in the behavior of groupers with regard to their spawning activity may explain the observed variability in the spawning episodes. this is difficult to discern, however, since this study depended mainly on the fisher’s traditional knowledge which may constrain species level identification although spawning patterns of groupers were presented for several areas in the philippines, these were preliminary and far from providing conclusive results a need to verify these results therefore exists verification studies include determination of gonado-somatic index (gsi), gonad histology analysis of samples, and actual study of spawning behavior since most species of groupers studied previously displayed spawning aggregations (e.g., shapiro, 1987; samoilys, 1997). conclusion recruitment patterns of groupers in the philippines which were inferred from the peak occurrences of juveniles derived from a survey among fishers and traders varied greatly with area/region, masking an overall, general trend. few, less discernible observations were noted, however. the spatiotemporal recruitment patterns of the juvenile groupers appeared to be related to changes in seasons (e.g., temperature) and monsoons as these seemed to affect the distribution of the juveniles. concomitantly, spawning patterns appeared highly variable. inferred patterns varied with most groupers spawning during a restricted period once or twice a year while others may presumably have extended spawning period. although preliminary in nature, the results of this study not only give a broad snapshot of the juvenile grouper occurrence, but also some hypotheses which may stimulate future local studies with refined question-specific sampling programs. in addition, the results are nonetheless promising since the study is the first attempt to investigate the spatiotemporal dynamics of recruitment for the commercially important groupers in the philippines. there is also a need to determine local oceanographic processes in the different regions to help explain the variation in spatiotemporal recruitment. retention of significant numbers of larvae throughout their entire pelagic phase is affected by the topography and hydrodynamics (e.g., flushing times) in areas like bays or lagoons (see hamner and wolanski, 1988). behaviour may also play a major role in larval distribution, aside from the more obvious cause of passive drift with mainstream currents, as well as spawning activities of adults of different grouper species. acknowledgments we thank vincent hilomen whose comments were valuable to the manuscript. al licuanan and ma. catalina ranola also read the manuscript and made some contributions. the technical staff of the former lapu-lapu research of bfar is also highly acknowledged. references aliño, p.m., a.j. uychiaoco, n.a. bermas, & e.d. gomez, 1992. assemblage structure of coral reef fish: multi-scale correlations with environmental variables in: chou, l.m. & c.r. wilkinson, editors. third asean science and technology week conference proceedings; 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science foundation. young, p.c., j.m. leis, & h.f. hausfield, 1986. seasonal and spatial distribution of fish larvae in waters over the north west continental shelf of western australia. mar ecol prog ser. 31:209-222. 7daquioag-staphylococcus aureus.pmd staphylococcus aureus and methicillin-resistant s. aureus (mrsa) carriage 60 science diliman (january-june 2018) 30:1, 60-73 staphylococcus aureus and methicill in-resistant s. aureus (mrsa) carriage in publ ic computer service providers and util ity jeepneys in up dil iman jann eldy l. daquioag ricardo bened ict c. almirol mary grace b. ayala ma. socorro edden p. subejano gil m. penul iar* instititute of biology college of science university of the philippines diliman abstract s t a p h y l o c o c c u s a u r e u s i s a g r a m p o s i t i v e b a c t e r i u m t h a t c a u s e s m i n o r s k i n i n f e c t i o n s t o l i f e t h r e a t e n i n g d i s e a s e s . i t i s t r a n s m i t t e d t h r o u g h direct contact with fomites, such as computer peripherals and handrails. tr e a t m e n t of s . a u r e u s i n fec t i o n s i s g e n e r a l l y s t r a i g h t fo r w a r d , b u t i s complicated by drug-resistant strains, particularly methicillin-resistant s. aureus (mrsa). the university of the philippines diliman (up diliman) h a s h u n d r e d s o f c o m p u t e r s e r v i c e p r o v i d e r s ( c s p s ) a n d p u b l i c u t i l i t y j ee p n ey s ( p u j s ) r eg u l a r l y u s ed by f a c u l t y, s t u d e n t s , s t a f f, a n d v i s i to r s . w h i l e n o o u t b r e a k s o f s . a u r e u s a n d m r s a h a v e b e e n r e p o r t e d , t h e possibility of infection with this pathogen through csps and pujs is very likely. the objectives of this study are to determine the carriage rates of s. aureus and mrsa in csps, computer peripherals, and handrails of pujs inside up diliman, and to identify the risk factors associated with s. aureus and mrsa contamination. a total of 162 computer peripherals from 27 csps and 196 puj handrails were swabbed. s. aureus isolates w e r e i d e n t i f i e d u s i n g c o l o n y m o r p h o l o g y, b i o c h e m i c a l t e s t s , a n d amplif ication of the nuc gene, whereas mrsa isolates were identif ied using the cefoxitin challenge and amplif ication of the meca gene. s. aureus w a s i d e n t i f ied i n 9 2 . 6 % of c s ps , 3 6 . 4 % of co m p u te r p e r i p h e r a l s , a n d _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online j.e.l. daquioag, et al. 61 7.1% of pujs, while mrsa carriage was 3.1% in csps and 2% in pujs. no signif icant associations between s. aureus/mrsa and the assessed risk factors were observed (p > 0.05). results indicate that while s. aureus prevalence is relatively high, mrsa carriage is low in csps and pujs in up diliman. keywords: staphylococcus aureus, mrsa, computer peripherals, handrails introduction staphylococcus aureus is a gram-positive, coccus-shaped bacterium belonging to the phylum firmicutes and the family staphylococcaceae. it is a facultative anaerobe which can ferment mannitol and produce enzymes, such as catalase, coagulase, and deoxyribonuclease (plata et al. 2009; kateete et al. 2010). it asymptomatically colonizes the skin and nose in humans, and is able to survive in fomites like plastic and steel surfaces (chiller et al. 2001; kusumaningrum et al. 2003). s. aureus is the causative agent of several diseases, ranging from mild skin infections like impetigo and folliculitis, to toxin-mediated conditions like food poisoning and toxic shock syndrome, and severe infections like staphylococcal bacteremia and endocarditis (lowy 1998; chiller et al. 2001). staphylococcal infections are common and can generally be treated without complications. however, methicillin-resistant strains of s. aureus (mrsa), mediated by the meca gene, are recently becoming more frequent in the community. in the philippines, 30% of community-acquired s. aureus (casa) and 38% of hospital-acquired s. aureus (hasa) infections are caused by mrsa (song et al. 2011). according to the antimicrobial resistance surveillance of the philippines (arsp), the prevalence of mrsa in the country is 31%. dicloxacillin remains as the antibiotic of choice for methicillin-susceptible s. aureus (mssa) infections, whereas mrsa infections are treated with vancomycin or teicoplanin (rayner and munckhof 2005). transmission of s. aureus commonly occurs through hand contact with fomites like computer peripherals and handrails contaminated by s. aureus (alkhezali and taha 2013). according to yahoo-nielsen (2009), 20% of filipinos in urban areas access the internet using public computer service providers (csps). the land transportation franchising and regulatory board listed 210,840 public utility jeepneys (pujs) nationwide in 2012, with 58,000 operating in metro manila (ronda 2012). csps and pujs are the dominant access points and mode of transportation, respectively, staphylococcus aureus and methicillin-resistant s. aureus (mrsa) carriage 62 for users from lower socio-economic classes. despite this, there is very little published data on the prevalence of s. aureus and mrsa in csps and pujs in the country. the objectives of this study are the following: to determine the prevalence of s. aureus and mrsa in computer peripherals in csps and handrails of pujs in the university of the philippines diliman (up diliman), and to analyze the risk factors associated with carriage. findings from this study may be used to influence university policies regarding the regular sanitation in csps and pujs to prevent or reduce further contamination. materials and methods sample size the number of csps in up diliman was obtained from two sources: the business permit licensing off ice of the quezon city hall for department of trade and industry-accredited internet cafés, and the website www.mainlib.upd.edu.ph (university library diliman 2010) for libraries aff iliated with up diliman. the number of pujs was obtained from the up diliman police through the off ice of the vice-chancellor for community affairs. only csps with at least three computers units, signed the consent forms, and allowed unannounced sampling dates were included in the study. the pujs per route were randomly sampled. the sample size was determined from each population with a 95% confidence level and a conf idence interval of 10 (creative research systems 2014). a total of 162 computer peripherals from 27 csps and 196 puj handrails were sampled. consent and survey forms an introductory letter and consent form explaining the purpose of the study and rights regarding participation were provided to the participants. head librarians and owners of internet cafés were given survey forms for the assessment of the following risk factors: years in service, service hours, comfort room availability, number of computer units, number of clients per day, usual gender of clients, duration of computer use, consumption of food and drink, frequency of cleaning the facility, frequency of cleaning the peripherals, and availability of hand sanitizers. j.e.l. daquioag, et al. 63 sample collection three computers were selected from each csps: the computers nearest to the door, fur thest to the door, and in the middle of the facility. sample collection in csps was performed on weekdays between 1:00 pm and 4:00 pm. sterile cotton swabs dipped into 0.9% sterile saline were swabbed on the entire surface of keyboards and mice. the number of pujs sampled per route is indicated in table 1. sampling of pujs was performed every thursday from 2:30 pm to 5:30 pm. for each handrail, a 10-cm length was swabbed with 10 sweeps of consistent length. the plate number of each puj was recorded to prevent duplication. the location and distribution of the csps are shown in figure 1. the cotton swabs were placed into 15-ml tubes of mannitol salt broth (msb) and delivered to the medical microbiology laboratory of the institute of biology, up diliman within 4 hours of collection for incubation at 37°c for 18-24 hours. ikot 2.9% (1/35) nd katipunan 11.6% (5/43) 2.3% (1/43) pantranco 6.4% (3/47) 4.3% (2/47) philcoa 3.3% (1/30) nd sm north edsa nd (0/28) nd toki 30.8% (4/13) 7.7% (1/13) total 7.1% (14/196) 2% (4/196) route s. aureus prevalence mrsa prevalence table 1. prevalence of s. aureus and mrsa in pujs figure 1. location and distribution of csps that participated in the study. red dots indicate csps where mrsa isolates were obtained. staphylococcus aureus and methicillin-resistant s. aureus (mrsa) carriage 64 isolation samples positive for mannitol fermentation were streaked on mannitol salt agar (msa) plates and incubated at 37°c for 18-24 hours. medium-sized yellow colonies with smooth surfaces were purif ied in msa, and the resulting isolated colonies were subcultured on nutrient agar (na) slants for maintenance. s. aureus identification gram staining, koh test, catalase test, coagulase test, and dnase test were used to identify s. aureus. identif ication was conf irmed through the pcr amplif ication of the nuc gene. genomic dna was extracted using the microwave lysis method (ahmed et al. 2014). the cell pellets were briefly washed and resuspended in 100 μl te buffer. fifty (50) μl of 10% sds was added to the mixture for incubation at 65°c for 30 minutes. the lysates were centrifuged at 10,000 x g for 10 minutes. supernatants were discarded and the cell pellets were heated three times for 1 minute at the high setting of a microwave oven (3d, model no. wp-70b17-65, input: 230v ~60 hz 1200 w, output: 700 w 2450 mhz). the pellets were dissolved in 200 μl te buffer. an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to extract the dna, followed by overnight absolute ethanol precipitation at -20°c. dna was recovered by centrifugation at 10,000 x g for 10 minutes, air-dried, and resuspended in 30 μl te buffer. dna yield and purity were determined using the nanodroptm 2000c spectrophotometer (thermo scientif ic, u s a ) . pcr was performed as previously reported with slight modif ications (brakstad et al. 1992) using the forward primer sa-01f 5’-gcgattgatggtgatacggtt-3’ and the reverse primer sa-02r 5’-agccaagcct tgacgaactaaagc-3’. each 25 μl reaction mixture was composed of 5 μl dna (48 to 50 ng), 3.5 μl sterile water, 2.0 μl of each primer (0.8 μm), and 12.5 μl 2x gotaq® master mix (promega, usa). pcr amplif ications were performed in a mycycler™ thermal cycler (bio-rad, usa) using the following conditions: 94oc for a 2-minute initial denaturation; 37 cycles of 94 oc for 1 minute, 42oc for 30 seconds, 72oc for 1 minute; and 72oc for a 7minute f inal extension. s. aureus biotech 1350 and s. epidermidis biotech 10098 were used as controls. pcr products were electrophoresed in 1.3% agarose gel p r e s t a i n e d w i t h 0 . 5 μg / m l e t h i d i u m b r o m i d e u s i n g t h e p o w e r p a c b a s i c electrophoresis system (bio-rad, usa) at 100 v for 22 minutes. the gel was viewed using a white/2uv transilluminator (thermo scientif ic, usa). j.e.l. daquioag, et al. 65 pcr amplif ication of the 16s rdna was performed as internal control, in order to rule out false-negative results (amit-romach et al. 2004). amplif ication of the 16s rdna was performed using the forward primer unibac-f 5’-cgtgccagccgcggtaatacg-3’ and the reverse primer unibac-r 5’-gggttgcgctcgttgcgggact taacccaacat3’ under the following conditions: 94oc for 3-minute initial denaturation; 37 cycles of 94 oc for 30 seconds, 60oc for 1 minute, 68oc for 2 minutes; and 68oc for a 7minute f inal extension. pcr products were electrophoresed and visualized as previously described. mrsa identification s. aureus isolates were challenged with 30 μg cefoxitin using the kirby-bauer disk diffusion assay as described in the clinical and laboratory standards institute (2014). isolates were identif ied as mrsa if the zone of inhibition was less than or equal to 21 millimeters. mrsa identif ication was conf irmed by pcr amplif ication of the meca gene. pcr was performed as described above using the forward primer m ec a 1 f 5 'a a a atcg atg g taaag g t tg g c3 ' a n d t h e r eve r s e p r i m e r m e c a 2 r 5 'agt tctg cagtaccggat t tg c-3'. pcr was performed using the following conditions: 94 oc for a 4-minute initial denaturation; 30 cycles of 94 oc for 30 seconds, 53oc for 30 seconds, 72oc for 1 minute; and 72 oc for a 7-minute f inal extension (murakami et al. 1991). mrsa biotech 10378 and s. aureus biotech 1350 were used as controls. pcr products were electrophoresed and visualized as previously described. statistical analysis the prevalence of s. aureus and mrsa was determined using the data from samples that yielded positive results. the association of potential risk factors in s. aureus and mrsa contamination was performed using the chi-square test in the ibm spss software (ibm corporation, 2013). p-values less than 0.05 were considered to be statistically signif icant. otherwise, the null hypothesis was accepted, and the risk factor in question was concluded to play no role in contamination. waste disposal all materials that were contaminated with s. aureus, mrsa, and reference microorganisms were decontaminated using an autoclave prior to disposal (cdc 2 0 0 9 ) . staphylococcus aureus and methicillin-resistant s. aureus (mrsa) carriage 66 results prevalence of s. aureus and mrsa among csps a total of 162 samples (81 keyboards and 81 mice) were collected from 27 csps, which were artif icially categorized into four quadrants based on their locations (figure 1). the prevalence of s. aureus among csps was 92.6% (table 2). s. aureus was detected in all csps located in quadrants ii and iii, while only 85.7% of the csps in quadrants i and iv were contaminated. the prevalence of s. aureus among keyboards was 40.7%, with more than half (56%) of the keyboards in quadrant ii contaminated with s. aureus. keyboards of the csps in quadrant iv were the least contaminated (24%). the prevalence of s. aureus among mice was 32.1%, which is lower compared to the keyboards. mice of the csps in quadrant ii were the least contaminated. the difference in contamination between quadrants was not statistically signif icant (p > 0.05). mrsa had a prevalence of 3.1% and was isolated from one keyboard in quadrant i, 2 keyboards in quadrant iv, and one keyboard and one mouse in quadrant iii. i 85.7% (6/7) 38.1% (8/21) 33.3% (7/21) 35.7% (15/42) nd ii 100% (6/6) 55.6% (10/18) 27.8% (5/18) 41.7% (15/36) 2.8% (1/36) iii 100% (7/7) 47.6% (10/21) 38.1% (8/21) 42.9% (18/42) 4.8% (2/42) iv 85.7% (6/7) 23.8% (5/21) 29.6% (6/21) 26.2% (11/42) 4.8% (2/42) total 92.6% (25/27) 40.7% (33/81) 32.1% (26/81) 36.4% (59/162) 3.1% (5/162) quadrant s. aureus prevalence in csps s. aureus prevalence in keyboards s. aureus prevalence in mice total mrsa prevalence table 2. prevalence of s. aureus and mrsa in csps prevalence of s. aureus and mrsa among pujs samples were collected from a total of 196 puj handrails designated in 6 different routes (table 1). the prevalence of s. aureus among pujs was 7.1%. s. aureus was detected in all routes except for pujs traveling to sm north edsa. pujs traveling to katipunan had the highest prevalence of 11.6%. the difference in contamination between routes was not statistically signif icant (p > 0.05). mrsa had a prevalence of 2% and was isolated from pujs traveling along the katipunan, pantranco, and toki routes. j.e.l. daquioag, et al. 67 risk factor analysis none of the risk factors assessed in this study was found to have a signif icant effect on the contamination of s. aureus and mrsa on computer peripherals and handrails (table 3). discussion the university of the philippines diliman has 59 csps and 324 pujs, which cater to faculty, students, administrative staff, and visitors. computer peripherals, such as keyboards and mice, and puj handrails can serve as fomites for the transmission of pathogenic bacteria like s. aureus. in the absence of clear sanitation guidelines and regular cleaning of computer peripherals and handrails, contaminated keyboards and mice in csps and handrails in pujs pose a risk to the health of computer users and commuters, respectively. the inclusion and exclusion criteria of the study specif ied that only csps with at least three computer units may participate, reducing the number of qualif ied csps in calculating our sample size. some internet cafés also refused to sign the consent forms on grounds of possible bad publicity despite a clause on the conf identiality of the study. in order to have a representation of up diliman, the effective sampling sizes were 21 libraries and 5 internet cafés. in this study, 22 libraries and 5 internet cafés were sampled. table 3. statistical analysis of risk factors assessed in the study years in service 0.270 2 0.874 service hours 0.909 2 0.635 comfort room availability 0.173 1 0.678 number of computer units 2.70 2 0.259 number of clients per day 0.513 3 0.163 usual gender of clients 0.376 1 0.540 duration of use of computer 1.392 4 0.846 food consumption in facility 0.376 1 0.540 drinking inside the facility 0.003 1 0.957 frequency of cleaning the facility 0.756 3 0.860 frequency of cleaning the peripherals 1.121 4 0.891 availability of hand sanitizers 0.270 1 0.603 risk factor assessed x2 df p value staphylococcus aureus and methicillin-resistant s. aureus (mrsa) carriage 68 among the 27 csps, 25 were positive for s. aureus contamination, resulting to a prevalence of 92.6%. no signif icant difference in the prevalence was observed among quadrants (p > 0.05) because most of the csps had at least one computer peripheral contaminated with s. aureus. out of the 162 peripherals sampled, 36.4% were positive for s. aureus contamination. the high prevalence observed among the csps is likely due to the lack of disinfection policies before and after use of the computers. library computers are high-traff ic computer units with high-contact surfaces. given the large number of students accessing these computers on a daily basis, contamination rates must be intuitively high. the lack of routine disinfection, coupled with the absence of hand sanitizers near the computer terminals, likely contribute to the high prevalence observed. s. aureus, including mrsa, has been reported to be a persistent pathogen because it can survive for months on dry surfaces. if no regular surface disinfection is performed, these dry surfaces can be a source of transmission (kramer et al. 2006). s. aureus and other pathogenic microorganisms have also been demonstrated to persist on non-porous surfaces, such as keyboards and mice, even in the absence of enrichment. unwashed moist or sweaty hands and a room temperature that favor the growth of s. aureus can also be factors in the high prevalence observed. s. aureus can survive in a salt environment, and sweat is a hospitable environment for the carriage and transfer of the bacterium onto various surfaces (kahanov et al. 2015). the prevalence of s. aureus was higher in keyboards (40.7%) compared to mice (32.1%), although the difference was not statistically signif icant (p > 0.05). the total surface area of a keyboard is larger than that of a mouse, allowing for the colonization by a greater number of microorganisms. keyboards also have spaces between keys where dirt and food particles can accumulate. moreover, both hands are in contact with the keyboard. the prevalence of s. aureus in csps observed in this study is lower compared to a similar study conducted in kogi state university in nigeria, where s. aureus was isolated from all csps (enemuor et al. 2012). it should be noted, however, that in their study, only 30 samples were collected from f ive sampling sites. by contrast, the prevalence of s. aureus among keyboards and mice in this study is higher compared to a study in al-mustansiriya university in iraq, where s. aureus had a prevalence of 18.6% among computer peripherals (alkhezali and taha 2013). the difference is likely due to the larger number of samples and sampling sites used in this study (162 versus 50 samples). understandably, a study conducted in ebonyi state university in nigeria observed a higher prevalence of 42.6% after sampling 250 keyboards and mice in three campuses (chukwudi et al. 2013), because it only j.e.l. daquioag, et al. 69 included internet cafés, where food and drinks are generally allowed, unlike in school libraries. the prevalence of s. aureus in pujs in up diliman is surprisingly low at 7.1%, considering the heavy traff ic of commuters pujs encounter during school days. however, previous studies on public transportation have reported prevalence values ranging from 8% in london (otter and french 2009) to 68% in the united states (lutz et al. 2014), or to even the absence of s. aureus (yeh et al. 2011). the prevalence of s. aureus in pujs is dependent on the nature of the fomite surveyed for the study. the handrails of pujs are made of smooth steel. the lack of rough surface limits the amount of dirt or organic material that s. aureus may use for attachment or nutrient, unless the turnover of passengers using the handrails is high. pujs along the toki route had the highest contamination of s. aureus at 30.8%, while pujs along the sm north edsa route had no contamination of s. aureus. the differences in the prevalence of s. aureus across puj routes is multifactorial and may be due to the following: passenger prof ile, personal hygiene of the passengers, bacterial contamination from paper bills and coins used as payment or change, and eating and drinking inside the pujs. different numbers and prof iles of passengers (students versus non-students) per puj were observed during sample collection. in this study, methicillin resistance was detected through the cefoxitin disk diffusion test and the pcr amplif ication of the meca gene. the cefoxitin disk test was used as a surrogate test for oxacillin and methicillin test because cefoxitin can better detect heteroresistant strains or strains that carry the resistance gene but express different levels of resistance (cdc 2015). furthermore, cefoxitin can better induce the meca gene and produce more reproducible and accurate results than oxacillin and methicillin. based on the results, the prevalence of mrsa in up diliman csps and pujs were low at 3.1% and 2%, respectively. different studies have varied reports on the prevalence of mrsa isolated from public transportation. stepanoviæ (2008) reported the presence of methicillin-resistant coagulase-negative staphylococci in the handrails of public buses in belgrade, serbia, but no mrsa was detected. a study in portugal reported a prevalence of 36.2% for mrsa in public buses ( et al. 2013). based on our search in published literature, no points of comparison could be found for mrsa colonization in csps and pujs in universities in other countries, but it would seem that mrsa prevalence is low in csps and pujs in up diliman. however, the isolation of mrsa from these places indicates a potential risk for the transmission of these bacteria in an out-hospital environment. concei ãoҁ  staphylococcus aureus and methicillin-resistant s. aureus (mrsa) carriage 70 based on the risk factor analysis, no correlation was observed between any of the risk factors considered and the contamination of csps and pujs by s. aureus and mrsa (table 3). previous studies showed similar results (kassem et al. 2007; oguzkaya et al. 2015). such observation could be due to the ubiquitous nature of s. aureus, its easy mode of transmission by hand contact, and its status as a normal microflora of the body, allowing s. aureus contamination of fomites to be prevalent and unnoticed. understanding the spread of infectious diseases involves gaining insight into its complex spatial diffusion through a network of people. individuals in a given population participate in various activities that may either be mobile or stationary. mobile activities include commuting through the pujs, while stationary activities take place at f ixed locations such as csps. tracking disease transmission not only involves the individual members of the population but also the physical environment, where these activities are carried out. the epidemiologic model of infectious disease propagation in the work by perez and dragicevic (2009) revealed that dynamic spatial interactions within a population lead to high numbers of exposed individuals who carried out stationary activities after moving between places within their environment. it was found that individuals at risk were concentrated in locations like universities. the f indings presented in the work support the signif icance of public areas, such as pujs and csps, in the transmission of microorganisms to commuters and computer users. in conclusion, this study documents the prevalence of s. aureus and mrsa in csps and pujs in up diliman, and emphasizes the potential of computer peripherals and handrails as environmental vehicles for the transmission of potentially pathogenic bacteria within the university. the isolation of mrsa, in particular, calls for a need to increase public awareness among computer users and commuters to disinfect hands after being in csps and pujs. acknowledgements the results of this study were presented in a poster paper during the 45th annual convention and scientif ic meeting of the philippine society for microbiology, inc. in vigan city, 2016. this study was supported by the off ice of the vice-chancellor for research and development of the university of the philippines diliman through the ph.d. incentive award (project no. 151505 phdia). j.e.l. daquioag, et al. 71 references ahmed ob, asghar ah, elhassan mm. 2014. comparison of three dna extraction methods for polymerase chain reaction (pcr) analysis 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[cited 2014 nov 20]. available from http://www.philstar.com/metro/ 2012/07/23/830576/too-many-vehicles-slowing-down-traff ic-group-says. song jh, hsueh pr, chung dr, ko ks, kang ci, peck kr, yeom js, kim sw, chang hh, kim ys, jung si, son js, so tm, lalitha mk, yang y, huang sg, wang h, lu q, carlos cc, perera ja , chiu ch, liu jw, chongthaleong a, thamlikitkul v, van ph,on behalf of the ansorp study group. 2011. spread of methicillin-resistant staphylococcus aureus between the co m m u n i t y a n d t h e h o s p i t a l s i n a s i a n c o u n t r i e s : a n a n s o r p s t u d y. j o u r n a l o f antimicrobial chemotherapy. 66(5):1061-1069. 2008. public transport as a r e s e r vo i r o f m e t h i c i l l i n r e s i s t a n t s t a p h y l o co cc i . le t t e r s i n a p p l i e d m i c r o b i o l o g y. 47(4):339-341. university library diliman. 2010. the university library, university of the philippines diliman. [cited 2016 may 15]. available from http: //www.mainlib.upd.edu.ph. yahoo-nielsen. 2009. yahoo-nielsen net index highlights. [cited 2016 may 15]. available from http://www.scribd.com yeh pj, simon dm, millar ja , alexander hf, franklin d. 2011. a diversity of antibioticresistant staphylococcus spp. in a public transportation system. osong public health and research perspectives. 2(3):202-209. _____________ gil m. penul iar <gil.penuliar@upd.edu.ph> is an assistant professor of the institute of biology. he has a ph.d. in medical science and is a specialist microbiology certif ied by the philippine academy of microbiology. jann eldy l. daquioag is a graduate student of the institute of biology. he is enrolled in the ms microbiology program and is aff iliated with the medical microbiology laboratory. his current research interests include bioprospecting for natural products against staphylococcus aureus. ricardo bened ict c. almirol is a graduate of the b.s. biology program of the institute of biology. he was the f irst undergraduate student of the medical microbiology laboratory to work on staphylococcus aureus. mary grace b. ayala is a graduate of the m.s. microbiology program of the institute of biology. she spearheaded the work on staphylococcus aureus in the medical microbiology laboratory. ma. socorro edden p. subejano is a graduate student of the institute of biology. she is enrolled in the m.s. microbiology program and is currently a university research associate. stepanović s, ćircović i, djukić d, vuković d, švabić-vlahović m.   journal subscription form.pmd journal subscription form using local ecological-p43-.pmd 43 using local ecological knowledge and environmental education emmanuel c. capinpin, jr. pangasinan state university, binmaley campus, 2417 binmaley, pangasinan telefax: (075) 540-1558 corresponding author e-mail: manny_capinpin@yahoo.com science diliman (january-june 2012) 24:1, 43-55 abstract using local ecological knowledge and environmental education in resource management of abalone in carot, anda, pangasinan the objectives of the present study were to (1) determine the local ecological knowledge (lek) of abalone gatherers through interviews and mentoring, and assess the correspondence between scientific information and lek, so that areas where local knowledge may be most useful in resource management could be identified, and (2) to empower selected gatherers/farmers with knowledge and technical skills through environmental education to help develop or build their capacity to become sustainable resource managers. the lek of abalone fishers was determined using three complementary approaches – group interview, individual interview, and mentoring sessions. local fishers possess a wealth of knowledge about the interactions of species gained through many years of observations, and this knowledge may be useful in guiding biologists in ecological restoration or management regimes. additionally, the fishers’ lek, validated by modern scientific ecological findings, could be a source of important and effective ideas in resource management. the knowledge of the abalone gatherers about important abalone fishing grounds should help in pinpointing critical areas that need to be managed. abalone mariculture in cages should be set up in these areas to routinely create dense breeding populations which can help in enhancing recovery and in providing fishers with a source of additional income. the continued enforcement of marine protected areas and the periodic release or reseeding of abalone in sanctuaries could also be considered viable resource management options. other recommendations for resource management based on gathered local knowledge and lessons learned from the environmental education (ee) seminars are also presented. keywords: abalone, local ecological knowledge, environmental education, resource management 44 capinpin, e.c., jr. science diliman (january-june 2012) 24:1, 43-55 introduction the coastal ecosystems of the philippines are some of the most productive and biologically diverse in the world. however, these coastal ecosystems are under severe stress from the combined impacts of human overexploitation, physical disturbance, pollution, sedimentation, and general neglect. unregulated harvesting of commercially important fish and invertebrates is rampant and the lack of resource management is a problem. one example is the abalone resource of anda, pangasinan, which has been degraded by activities related to rapidly expanding human populations such as overharvesting and destructive fishing (e.g. destroying rocks which are known habitats of cryptic abalone, cyanide fishing, etc.). one possible option for resource management is to educate local fishers to develop a corps of local resource managers who would promote sustainable fishing practices and resource conservation in carot, anda, pangasinan. in developing countries, many sites are poorly studied because of remoteness; in such areas, local or indigenous peoples may be the only source of biological information (johannes, 1982; poizat & baran, 1997; aswani & hamilton, 2004). through years of observation, local people acquire a wealth of knowledge about the interactions of species, and this knowledge may be useful in guiding biologists in formulating ecological restoration or management schemes (drew, 2005; gilchrist et al., 2005). moreover, soliciting local knowledge could also help identify areas of concern for communities and resource users, making conservation and management more locally relevant. hence, in this study, the objectives were to (1) determine the local ecological knowledge (lek) of abalone gatherers through interviews and mentoring, and assess the correspondence between scientific information and lek, so that areas where local knowledge may be most useful in resource management could be identified, and (2) empower six selected gatherers/farmers with knowledge and technical skills through environmental education, to develop their capacity to become sustainable resource managers. materials and methods compilation of local ecological knowledge (lek) data on the fishers’lek were collected using three complementary methods – group interview, individual interview, and mentoring sessions during the environmental education (ee) sessions. in all cases, data were recorded digitally and in a notebook, and maps and charts were used where applicable. data gathering was conducted in january to september 2011. the group and individual interviews began by outlining the objectives and procedures of the interview; this was done to secure the fishers’ cooperation. additionally, the benefits of the study for the community were explained. the researcher also took care to develop trust and mutually respectful relationships with the cooperators and enter into a dialogue on terms set by them, who are the holders of local knowledge (drew, 2005). focused group discussion/group interview with abalone gatherers the group interviews were conducted through informal meetings or dialogues with groups of local informants that included abalone fishers, elders, and the youth from the local community. this method helps in determining if there exists a consensus among the informants and allows both informants and scientists to better understand one another’s perspectives (fraser et al., 2006). to facilitate data gathering, an interview guide was developed. a half-day group interview was conducted in barangay carot in coordination with the office of the municipal agriculturist and the concerned barangay captain. selected community members who were able to provide information on particular subjects based on their local knowledge, skills, and experience were invited to participate in the activity. it was important to include older community residents since they could provide information about biology over longer periods of ~40 years. older fishermen are 45 using local ecological knowledge and environmental education science diliman (january-june 2012) 24:1, 43-55 familiar with environmental changes that younger residents might not have observed. for instance, a reduction in abalone sizes in the past decades might have been observed by people with longer fishing experiences but not by those who have been fishing for only a few years. individual interview with identified cooperators for the individual interview, the same interview guide used in the focused group discussion was employed. the advantage of the one-on-one interview is that it allows for in-depth clarification and follow-up or probing questions. as a form of validation, the informants’ lek were compared with available scientific information. further clarification and validation were also sought during the ee sessions and meetings with the informants. mentoring sessions a third and final method of gathering information about lek was undertaken during the process of ee sessions and meetings. meetings, one-on-one or group, were done regularly to discuss results and share experiences and reflections. selected environmental education (ee) programs/strategies experiential activity: abalone mariculture this activity was done following the farmer field school (ffs) concept and was designed to be experiential, participatory, hands-on work. six abalone gatherers participated in this activity. the activity addressed both management and economic needs, and was envisioned to contribute to increasing abalone populations while heightening the fishers’ecological awareness and knowledge of the life history of abalone and its culture. at the same time, abalone mariculture was viewed as a potential means to provide supplemental livelihood to the fishers. there are existing abalone mariculture protocols available (capinpin et al., 1999). ee seminars three seminar modules (biology and culture of abalone, basic marine ecology, and resource management options) were provided to the cooperators and other interested members of the community. seminars were conducted and the contents of the modules were translated into the vernacular using simple terms. activity-based modules were also provided during the ee seminars, such as identification of seaweeds, drawings, motivational exercises, games, and reflections to help the participants gain a better understanding of the topics. field trips the cooperators went on field trips to an abalone hatchery at the bureau of fisheries and aquatic resources (bfar rmatdec) in alaminos city and a sea cucumber hatchery at the up marine science institute in bolinao. this activity was intended to supplement the ee seminars, so that the cooperators will be able to better appreciate the issues discussed in the seminars. film/video showings documentary film showings on abalone and its culture and on marine protected areas (mpas) particularly in region 1 (localized examples) were shown to enhance the knowledge and interest of the participants on resource management options. results and discussion determination of local ecological knowledge knowledge on sites/locations of abalone habitats many research projects are being conducted in remote, infrequently visited areas. local people can aid researchers by furnishing information about species presence and distribution (poizat & baran, 1997), juvenile habitats (aswani & hamilton, 2004) or spawning aggregations (johannes, 1982). the local cooperators in this study have knowledge of the sites where abalone can be found (table 1 and 46 capinpin, e.c., jr. figure 1). they also have peculiar names for some islands/islets not seen on the map. all of the names used for the location of abalone fishing grounds date back to the past except for one, cory island, which got its name from former president corazon aquino.there are also places where abalone can be found but with no special name such as “sa tapat ng imbo”. however, the cooperators cannot pinpoint the location of recentlysettled abalone post-larvae or the places where very small abalone (<1 cm) can be found. this was not surprising because of the cryptic coloration and habits of small abalone. most of the cooperators in carot collect abalone at “mitumpa” or “mindanao” because they are near their residence and can be easily reached using only bamboo rafts or balsa. the fishers also have favorite areas for collection because these are where they can find more abalone. in nature, all adult abalone grounds are also the settlement places for abalone larvae. this is because science diliman (january-june 2012) 24:1, 43-55 table 1. abalone collection sites known to gatherers abalone collection site local meaning other meaning in filipino mitumpa the sound and movement of “nagsasalubong ang alon” merging strong waves remind one of the handclap (mitumpa in the vernacular) mindanao the extensive use of dynamite “kasi yung mga naghas caused accidents which, in didinamita napuputulan ng kamay” many instances, have led to the amputation of their victims’ arms saloy putot there is a sudden drop-off in the “putol yung lalim niya dahil sa water’s depth biglang pagbabaw” pangrapogan this area is exposed during low “pag-ihawan” tide, allowing fishermen to cook there mâdol the water in the area is too shallow, “pag sumisisid parang giving a sense of bumping into the nauumpog sila” sea floor when one dives into it imondayon the bottom topography is shaped “korteng duyan” like a moon purod alaki “alaki” means wide “malapad na batuhan” purod daikling “daikling” means small “maliit na batuhan” tapat ng imbo front of imbo 47 using local ecological knowledge and environmental education abalone do not migrate far from their natal reef; they also have short planktonic phase. it means that the areas where adult abalone can be found also are the settlement areas of larvae. according to the cooperators, they can collect more abalone during spring tides (“kapag malaki ang kati”). this is especially true for gatherers at night because spring tides occur during new and full moon periods, which are the known spawning periods of abalone. hence, during this time, most mature abalone are out of the crevices. knowledge of spawning patterns because many indigenous peoples view their environment in a holistic fashion, they have a sense of the linkages among various ecological processes, multiple species, and abiotic factors that influence species biology (nabhan, 2000; vogt et al., 2002). for instance, in this study, the knowledge of the spawning patterns of the abalone by local people closely matched the one formalized by researchers. according to the fishers, they were able to observe the abalone’s spawning every full moon and new moon periods. the informants have noted that during spawning, the abalone release a cloud of smoke (“nagpapausok sila”) with their cephalic tentacles extended. this happens beginning 11-12 pm up to 2 am. in an earlier study, abalone were observed to spawn year round and the spawning events coincided with the new and full moon for recently captured h. asinina held in tanks (capinpin & hosoya, 1995). this lunar periodicity in spawning lasted for two months. spawning still continued every two weeks thereafter but no longer coincided with the lunar cycle. in h. asinina, the time interval between successive spawning of hatcheryreared abalone provided with optimal rearing conditions and adequate algal food was 13-15 days (capinpin et al., 1998). similar results were observed in australia and malaysia (d. nair, university sains malaysia, personal communication). during the spawning season in australia, recently-captured abalone held in aquaria released gametes for two nights every two weeks during the new and full moons (counihan et al., 2001). the researchers observed that the time of spawning of either sex is highly correlated with evening high tides; males spawned about 19 min prior to high tide and females spawned 11 min after high tide. synchronous spawning patterns persisted for six weeks; after this period, spawning continued but were irregular and asynchronous. in the bolinao hatchery, h. asinina spawning begins at 10 pm and lasts up to 2 am. males usually spawn first, their cephalic tentacles extended in order to detect spawns from other abalone. milt from male abalone usually stimulates other nearby male abalone to spawn. females spawn after the males, and this ensures synchronous fertilization upon the release of eggs from female abalone (capinpin & hosoya, 1995). the fishers’ knowledge about abalone’s habitat and spawning behavior could help generate future management plans. for instance, in the present study, science diliman (january-june 2012) 24:1, 43-55 table 2. recommendations for abalone resource management based on gathered local knowledge local ecological knowledge resource management knowledge of spawning pattern 1. imposition of catch quotas for night gatherers 2. imposition of closed/temporarily-closed season knowledge of abalone habitat 1. setting up of abalone mariculture to help protect breeders and supply offspring to these areas 2. setting up of abalone sanctuary 48 capinpin, e.c., jr. science diliman (january-june 2012) 24:1, 43-55 the knowledge of the abalone gatherers on important abalone fishing grounds will help in pinpointing critical areas that need to be managed. perhaps, abalone cage culture should be set up in these areas to help in protecting adult breeders and help in supplying offspring to these areas. the continued enforcement of marine protected areas and reseeding of abalone in sanctuaries can also be considered viable resource management options. table 2 presents the recommendations for abalone management to the lgu based on collected information from local informants during interviews and mentoring sessions. an example of protected areas management that had its origin in traditional knowledge can be found in gladden spit in belize. gladden spit is an area of the mesoamerican barrier reef located off the coast of belize, which has long been known by fishers as a spawning aggregation site for mutton snappers (lutjanus analis). local fishers have known about this area since at least the 1920s (heyman et al., 2001), but the aggregations remained unreported in the scientific literature until 2001. additional examples of spawning sites that have gained protected-area status through the interaction of traditional fishers and researchers can be found in palau (johannes et al., 1999), the solomon islands (aswani & hamilton, 2004), and glover’s reef in belize (sala et al., 2001). there is growing recognition that ecological knowledge can and should play an important role in wildlife conservation, particularly in remote areas where standard scientific approaches may be impractical or difficult to apply (gilchrist et al., 2005; fraser et al., 2006). lek can be a useful companion to standard scientific approaches provided that it has undergone rigorous testing prior to its incorporation into management plans (gilchrist et al., 2005; rist et al., 2010). lek helps to engage local stakeholders as part of a team addressing a shared conservation concern, an approach that generally yields more productive results than scientific studies alone. environmental education (ee) modules/ strategies/approaches the ee seminar modules can serve as a venue for the cooperators to enhance their ecological awareness. in one of the ee seminars on resource management options, the researcher discussed the different management options specifically for abalone, one of which is mariculture. it was reiterated to the participants that mariculture activity can be used to address both management and economic needs. this strategy can be a potential source of supplemental livelihood for the community. at the same time, it was envisioned to contribute in enhancing depleted abalone stocks while improving the people’s ecological knowledge. residents should be taught that they should harvest only the sexually mature individuals at the right time, and that they should leave a few abalone to reseed the area for future replenishment. ee seminar on abalone biology and culture the ee session on the biology of abalone and its culture was held on march 23, 2011 at the barangay hall of carot. to capture the audience’s interest, the session started with a presentation on the different abalone species of the world (haliotis rufescens, h. discus hannai, h. iris, etc.). the advantages of abalone culture in the philippines, as compared to temperate countries, such as fast growth rates, availability of suitable sites and seaweeds, were noted. highlighted in the discussion was the biology of abalone, with emphasis on its life cycle. this gave the cooperators an understanding and appreciation of the development of abalone from eggs to sexually mature individuals. the cooperators also realized the significance of allowing abalone to spawn before they are harvested to ensure the replenishment of remaining natural stocks. it was pointed out to the participants that it was important to put together sexually mature individuals during spawning (e.g. mariculture cages) to ensure fertilization success which, among abalone, happens outside the bodies of the parents (external fertilization). additionally, posters and a film (local example aired on kapuso mo, jessica soho) on abalone culture and its life cycle were also shown to give participants a better appreciation of the experiential abalone mariculture project that they carried out. ee seminar on basic marine ecology the basic marine ecology seminar was held on may 9, 2011. the seminar took place at the house of mang 49 using local ecological knowledge and environmental education science diliman (january-june 2012) 24:1, 43-55 junior, one of the cooperators who joined the abalone mariculture activity. prior to the seminar proper, a video presentation on abalone hatchery practices was shown and a motivational exercise was given to the participants. highlighted in this seminar were discussions on the concepts of ecosystems and basic ecology terms such as food chain, habitat, niche, photosynthesis, producers, consumers, etc. a brief discussion on the important features of the marine environment, the different types of marine organisms, and the different marine ecosystems such as coral reefs, mangroves, seagrass, and estuaries also was provided. the discussions were linked to the seven environmental principles, especially when giving examples of human impacts on the environment. field trip in the morning of may 27, 2011, the group went on a field trip to bolinao marine laboratory of the university of the philippines marine science institute (up-msi). the first activity undertaken was an orientation on the projects of the research staff of the invertebrates laboratory. highlighted in the orientation was the community-based project on sea urchin culture and reseeding of protected areas, which resulted in the recovery of the sea urchin population in bolinao and nearby areas. the orientation was followed by a brief presentation on the progress of the research staff’s current research on the culture and resource management of tropical sea cucumbers. the venue also allowed for an exchange of ideas between the research staff and the group of cooperators. after the lecture-orientation, the group toured the algal room and also viewed the exhibit about the other studies completed and currently being done by up-msi on seaweeds, giant clams, corals, etc. the cooperators expressed appreciation for the type of work of the research staff, which involved simulating the work of nature (i.e. artificial propagation of seeds), as well as strengthening resource management. the participants themselves witnessed the recovery of sea urchin population in their coastal area in recent years and they now understood clearly the rationale of culturing sea urchin in cages to serve as reproductive reserves to supply offspring to different areas and reseeding some in protected areas. they appreciated this new knowledge which can be applied to abalone and other marine resources in their area. the group made a side trip to the bolinao museum on the way to the bureau of fisheries and aquatic resources (bfar) abalone hatchery in alaminos city. the cooperators enjoyed viewing the natural sciences section, which contains display of different types of rocks, a fossilized bill fish from the pleistocene period, preserved organisms such as birds and aquatic organisms, and dioramas depicting different aquatic ecosystems of bolinao such as coral reefs, mangroves, etc. there was also an anthropological exhibit showing the evolution of early tools of bolinao townspeople. after lunch, the group proceeded to bfar alaminos to visit the abalone hatchery. during this visit, they learned and appreciated the life history of the abalone and the critical life history stages that are vulnerable to unsustainable fishing practices. after the tour, the group had a brief meeting with the hatchery technician to discuss their concerns and observations. it was also an opportunity for them to ask assistance for such concerns as the possible supply of healthy abalone juveniles and technical help in the future. ee seminar on resource management on june 7, the last segment of the ee seminar series on resource management options was held. prior to the seminar proper, the cooperators viewed a video showing titled “on these grounds”. this film about the benefits of marine protected areas was produced by bfar region 1 and featured examples from pangasinan. the film features the different environmental advocates of various mpas in pangasinan such as barangay captains, students and teachers, i.e. people who are familiar to the cooperators. hence, they found the film showing interesting and personally relevant. the film showing was followed by a game called “fishing game/open access” (deguit et al., 2004). in this game, cut-out pictures of different marine 50 capinpin, e.c., jr. science diliman (january-june 2012) 24:1, 43-55 organisms were posted on different areas of the room, with some pictures hidden (behind curtains, under chairs) and some visible. the participants were asked to fish for the organisms. the person who fished the most number of organisms won the game and was given a prize. as expected, the participants scrambled to catch as many fish as they could. it was pointed out to them that if the training area was physically a coastal habitat, it would now be destroyed due to the frenetic fishing activity. it was explained to them that this game showed how open access fishing causes depletion of resources. as such, this activity illustrated what is now happening in the country, and the urgent need for coastal resource management. a good example of resource management for abalone done in tawi-tawi by researchers at mindanao state university was shared with the participants. the project involved translocation of adult abalone in a sanctuary. after only 10 months the researchers already observed a multiple increase in recruits (r. tajil, personal communication). the ensuing discussion revolved around fishery regulations and environmental interventions (juiniomeñez et al., 2000). fishery regulations include closed and open seasons/areas, catch and size quotas, and prohibition of illegal fishing methods. environmental interventions include habitat enhancement/rehabilitation, mariculture/sea ranching, reseeding/transplantation, marine protected areas, and pollution regulation. it was noted that the mariculture of abalone in sea cages undertaken by the participants was actually one resource management option. during this seminar, there was a lot of discussion on what resource management options the participants would like to take. one of these is the communitybased culture of abalone. there was a fruitful exchange of ideas. there was also a discussion regarding what table 3. recommendations for abalone resource management based on lessons learned from the ee activities ee activities resource management biology of abalone/resource management 1. only abalone that meet the minimum size limit of 5 cm should be harvested 2. ban on destructive fishing practices (i.e. destroying rocks to gather cryptic abalone) 3. stricter enforcement of existing mpas (i.e. carot mpa which is actually an abalone fishing ground) 4. conduct ee activities in other areas with the help of corps of enlightened abalone fishers in this study as resource persons 5. impose closed/temporarily-closed season experiential mariculture 1. setting aside a portion of harvest to reseed mpas or abalone sanctuary 2. harvest only sexually mature abalone (i.e. 5 cm) 3. consider setting up a network of marciulture cages in different areas to serve as reproductive reserves, or lobby for limited exclusive use rights for a mariculture area 4. continue mariculture and involve other community members in this activity 51 using local ecological knowledge and environmental education science diliman (january-june 2012) 24:1, 43-55 the participants could do to help raise the ecological awareness of the rest of the community. during the discussions, they realized the importance and the potential of the mariculture activity to enhance the recovery of abalone resources and they would like to continue the activity even after the current research was completed. they also realized the potential of saving some of their harvest for reseeding a nearby marine sanctuary. essential to building local capability for coastal resource management is raising awareness of other members of the community. this can be done through information dissemination about the activities being done by the group related to resources management, such as the abalone mariculture. this can take the form of informal conversations with fishers and between fishers, which has proved effective in convincing the other members of the community of the ecological and economic relevance of the resource management related activities of the group. other mechanisms include regular group meetings, ee seminars and field trip, all of which could be attended by other members of the community. the confidence generated by these formal and informal activities can convince more local residents to set up their own grow-out cages and practice sustainable fishing activities. table 3 presents a summary of the recommendations for resource management of abalone based on lessons learned from the ee activities. final assessment meeting on september 20, 2011, the group had their final assessment meeting to evaluate the abalone mariculture activity that they did (reported elsewhere) and other activities. the meeting was presided by the researcher as well as the group leader. it was attended by the municipal agriculturist, ms. elizabeth t. tomas, who also represented the municipal mayor aldrin cerdan. the municipal agriculturist is the one in charge of updating and spearheading the various coastal resources management plans and activities of the town. hence, the group decided that it would be proper and beneficial to be able to voice out their plans and concerns regarding their coastal resources to ms. tomas. discussed during the meeting were the various ee activities taken, lessons learned from the abalone mariculture activity, and recommendations to further improve the mariculture activity as a supplemental livelihood. the meeting also became a venue for the cooperators to discuss their experiences and opinions regarding the various ee activities. ms. tomas, who has more than 20 years of experience in coastal resources management, reiterated and reminded the attendees that it is urgent to conserve not just the abalone resources but all other important marine resources for use of future generations. she also emphasized that mariculture activity using small wild juveniles should be continued as well as other sustainable activities like regulating the harvest of abalone and other marine resources. the municipality, through the efforts of the office of the municipal agriculturist, also plans to incorporate the fishers’ recommendations into their coastal resource management plan, which will feature, among others, setting a legal size limit of 5 cm for abalone, prohibition of illegal fishing activities that threaten abalone habitat such as cyanide and dynamite fishing, and stricter enforcement of the network of mpas in anda when it is time to update their crm plan. need for sustainable resource management the abalone fishery of anda provides a significant source of employment and income to the coastal community. from a socio-economic perspective, the long-term sustainability of abalone fisheries is of great importance to coastal communities. unfortunately, abalone stocks have been overfished in many areas as a result of ever-increasing market demand, uncontrolled exploitation and/or inadequate fisheries management. unless the direct users and the policy makers are educated about the status and proper management of their resources, a cascading event of overexploitation of different species may eventuate. for instance, overfishing is the main problem contributing to the depletion of sea cucumber resources. except for japan, other asian countries are generally lacking in management measures to conserve and sustain their sea cucumber fisheries. the two most important 52 capinpin, e.c., jr. science diliman (january-june 2012) 24:1, 43-55 producing countries, indonesia and the philippines, do not have management plans specific to sea cucumber conservation (bruckner et al., 2003). it is not surprising that there are also no management measures for conserving abalone resources that exist in the philippines. in asia, japan has the longest history of managing sea cucumber resources. mitsukuri (1903 as cited by choo, 2008) noted that for hundreds of years, the people of oki island used to put up loose stone piles in the shallow seas to provide adult a. japonicus a place to aestivate, and metamorphosing larvae and juveniles to aggregate. japan has enforced regulations setting aside certain localities as breeding reserves where stone piles have been constructed, and in these places, sea cucumber fishing is strictly prohibited. in anda, enhancing abalone habitat by placing stones and boulders in the sea may not be that urgent. what is more important is the strict enforcement of mpas that are already identified. anda has five mpas situated in carot (13.3 ha), cabungan (18 ha), caniogan (9.8 ha), magsaysay (14.8 ha) and panacalan (48.59 ha). the mpas in carot and cabungan were the earliest established (1998) while panacalan is the most recent (2003) (salmo et al., 2005). in the broad context, mpas are areas managed to enhance conservation of marine resources. mpas are believed to serve as a management tool by supplementing fished stocks in surrounding areas (sale et al., 2005). mpas may be particularly useful alternative for abalone management because effective spawning and fertilization seems to require high densities of spawners, which may not occur in most of the “open” fishing grounds (bell et al., 2008). when it comes to sustainability, one of the very important things to consider is for the coastal fishers to reduce pressure on their marine resources by identifying other means of livelihood that are also sea-based. for this reason, the option of mariculture of abalone should be explored to address both resource management and the development of livelihood. as a resource enhancement activity, mariculture guarantees that abalones of high economic value are allowed to grow to sexual maturity before they are harvested. mariculture also ensures that some reproductive individuals are left for reseeding. as supplemental livelihood activity, the cultured abalone can help supplement the food and income of the communities. this resource conservation strategy had worked well with sea urchins in bolinao as a result of the intervention of the up-msi, as discussed earlier. presently, the sea urchin industry in bolinao has been revived and is thriving very well. the most important actions for fisheries managers are sociological in nature, indicating that management of abalone stocks must embrace social science more strongly than in the past. this can be achieved with greater involvement of stakeholders and capacity building of local-level institutions. agencies with modest capacity for developing management plans and enforcement should at least apply a minimum set of the most important and simple regulatory measures and actions in implementing management. hence, it is important for lgus to have a management plan featuring at least the minimum regulations (i.e. regulations imposed on fishers) and actions (by the manager). regulations may include a minimum legal size limit (5 cm for abalone), ban on destruction of rocks, marine reserves, licensing, monitoring, and reporting along the market chain. actions should include the conduct of fishery-independent stock surveys, of fishery-dependent surveys of catch and effort, of socioeconomic surveys, and education programs for various stakeholders. there should be a size limit or a minimum length or weight of abalone that can be legally fished or traded. two principal purposes of size limits in fisheries are to protect juveniles and to allow recently matured adults one or more seasons to spawn before they can be fished (purcell et al., 2009). minimum size limits must therefore have some biological basis, corresponding to the size at which individuals first become mature plus some additional buffer so that they have time to contribute to spawning. a recommended conservative approach is to add some centimeters to the minimum size at maturity which, for h. asinina, is 3.5 cm (capinpin et al., 1998). 53 using local ecological knowledge and environmental education by setting the size limit at 5 cm, individual mature abalone could have significantly contributed to replenishment of larvae in surrounding areas for several months and aided in the recovery of depleted abalone populations before they are harvested. purcell et al. (2009) also recommend that fishery managers prepare simple plastic rulers, with graduations to give to fishers, which they can use to verify sizes of animals in the water. the objective of enhancing the recovery of natural abalone populations through community-based figure 1. map showing the location-specific knowledge of the cooperators regarding known productive abalone grounds in the island municipality of anda in the province of pangasinan mariculture and through selective harvesting (i.e. the harvest, collection, and sale of abalone that have reached the agreed legal size limit of 5 cm), will largely depend on the success of the expansion of the activity to other areas, and hence will depend on the institutionalization of a system that will grant limited exclusive use rights for fishermen to set up sea cages in their areas and/or granting subsidy in the form of hatchery seed stock which the farmers could use in this activity. science diliman (january-june 2012) 24:1, 43-55 54 capinpin, e.c., jr. there are still many gaps in our current knowledge of abalone biology and fisheries. nonetheless, it should be stressed that uncertainty should not prevent the development of operational management strategies that aim to maintain or rebuild the productive capacity of abalone fisheries and to safeguard the long-term social and economic benefits to local communities. the lek and the lessons gained from the ee activities can be used as inputs in formulating management plans for abalone resource management by the lgu in anda, pangasinan, which should be incorporated into the larger, more encompassing coastal resource management plan. acknowledgement the author wishes to thank ms. elizabeth tomas, the municipal agriculturist, and the honorable municipal mayor aldrin cerdan, for allowing the researcher to conduct this study in anda, pangasinan; for providing all the necessary documents, maps, unpublished reports, etc.; and for their whole-hearted support during the entire conduct of the study. the author also acknowledges the helpful comments and suggestions of dr. ruth guzman of the rizal technological university before and during the conduct of the study, dr. phares parayno, dr. angelina galang, dr. donna paz reyes and dr. maria lourdes baybay of miriam college for advice, and prof. ceferino toledo of psu for reviewing this manuscript. references aswani, s. & r. j. hamilton, 2004. integrating indigenous ecological knowledge and customary sea tenure with marine and social science for conservation of bumphead parrotfish (bolbometopon muricatum) in the roviana lagoon, solomon islands. environmental conservation, 31:69–83. bell, j.d., s.w. purcell & w.j. nash, 2008. restoring smallscale fisheries for tropical sea cucumbers. ocean and coastal management, 51: 589-593. bruckner, a.w., k.a. johnson & j.d. field, 2003. conservation strategies for sea cucumbers: can a cites appendix ii listing promote sustainable international trade? spc beche-de-mer 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heron reef, australia. marine ecology progress series, 213: 193-202. deguit, e.t., r.p. smith, w.p. jatulan & a.t. white, 2004. participatory coastal resource assessment training guide, coastal resource management project of the department of environment and natural resources, cebu city, philippines. 134 p. drew, j.a., 2005. use of traditional ecological knowledge in marine conservation. conservation biology, 19(4):1286-1293. fraser, d.j., t. coon, m.r. prince, r. dion & l. bernatchez, 2006. integrating traditional and evolutionary knowledge in biodiversity conservation: a population level case study. ecology and society, 11(2):4-23. gilchrist, g., m. mallory, & f. merkel, 2005. can local ecological knowledge contribute to wildlife management? case studies of migratory birds. ecology and society, 10(1):20-31. heyman, w. d., r. t. graham, b. kjerfve, & r. e. johannes, 2001. whale sharks, rhinocodon typus aggregate to feed on fish spawn in belize. marine ecology progress series, 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pastor, 2008b. enhancing the recovery of depleted tripnuestes gratilla stocks through grow-out culture and restocking. reviews in fisheries science, 16:35-43. juinio-menez, m.a., s.g. salmo iii, e. tamayo, n. estepa, h.g. bangi, & p.m. alino, 2000. bugsay community environmental education: experiences from bolinao, northern philippines. community-based coastal resources management program, marine science institute, university of the philippines: 126 pp. nabhan, g. p., 2000. interspecific relationships affecting endangered species recognized by o’odham and comcaac cultures. ecological applications, 10:1288–1295. poizat, g. & e. baran, 1997. fishermen’s knowledge as background information in tropical fish ecology: a quantitative comparison with fish sampling results. environmental biology of fishes, 50:435–449. purcell, s.w., h. gossuin, & n.s. agudo, 2009. status and management of the sea cucumber fishery of la grande terre, new caledonia. worldfish center studies and reviews. the 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philippines (philreefs) and the marine science institute, university of the philippines, diliman, quezon city: 248 pp. vogt, k. a., k. h. beard, s. hammann, j. o’hara palmiotto, d. j. vogt, f. n. scatena, & b. p. hecht, 2002. indigenous knowledge informing management of tropical forests: the link between rhythms in plant secondary chemistry and lunar cycles. ambio, 31:485–490. science diliman (january-june 2012) 24:1, 43-55 9subscription form.pmd 100 method of payment (please check one)  pay cash at the ovcrd (see address above)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _______________________________________________________________________________________ contact person (for institutional subscribers) ___________________________________________________________________________ mailing address ___________________________________________ email address _________________________ ___________________________________________ telephone no. _________________________ ____________________________________________ fax no. __________________________________ please send accomplished subscription form to the rduo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city (02) 436-8720 fax (02) 927-2568  upjournal.ovcrd@gmail.com journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. (subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total university of the philippines diliman office of the vice chancellor for research and development call for papers humanities diliman, science diliman, and social science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> journal cover images courtesy of (l-r) vargas museum & biodiversity research laboratory (agno, pangasinan) 2editor's note-jan.-june2019.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online in this june 2019 issue of science diliman, we feature four research articles and one short communication. one article is on the use of benthic macroinvertebrate assemblages to assess stream condition in an urban landscape. two articles have relevance to dna forensics. another article discusses the isolation of fungal endophytes from leaves of medicinal plants. the short communication is on phytoplankton grown from ballast waters. magbanua et al. assessed the overall condition of the waterways in the university of the philippines (up) diliman campus based on benthic macroinvertebrate assemblages. they sampled 19 stream reaches across three different land use categories. their results revealed poor to severe stream conditions in all sampling sites. thus, they suggest restoration efforts in streams and waterways, as well as improvement of the wastewater treatment facility in the campus. sales et al. compared the utility of an organic procedure and a silica-based method for extracting dna from cigarette butt samples. they also tested the effect of storage time and conditions, and cigarette type on dna yield and allele recovery. they recommend the use of organic procedure for samples collected outdoors and stored for a long period of time, while the silica-based method is recommended for samples collected indoors and stored for a short period of time. guerrero et al. compared the occurrence, frequency, and isolation rates of fungal endophytes from the leaves of the 10 most frequently used medicinal plants in albay across three different locations. they obtained 120 fungal isolates belonging to 17 species. they found no signif icant difference in terms of the number of isolates and unique species across the sampling sites. they recommend further sampling and testing of the biological properties of these fungal endophytes. soliven et al. compared dna yield and quality from urine samples stored at room temperature, 4°c, and -20°c for 2 months and 9 months. they found that dna extracted from urine samples stored at cooler temperatures were amplif ied better, especially at 2 months of storage. their study is important as dna testing of urine samples can be used to resolve allegation of sample switching and laboratory misconduct. 2 austero et al. identif ied bloom-forming and potentially harmful diatom and dinoflagellate species in ballast waters from international vessels berthing at two ports in the country. their results showed that these harmful phytoplankton can be transported via shipping, which may lead to bioinvasion in the local aquatic environment. lastly, on behalf of the editorial board of science diliman, i would like to thank dr. irene m. villaseñor for her three years of service as editor-in-chief. under her able leadership, science diliman was included in the asean citation index and emerging sources citation index, and was a recipient of the journal incentive program awarded by the commission on higher education (ched). science diliman also deeply mourns the passing on march 2, 2019 of the up diliman college of science dean, dr. perry s. ong. he was a up scientist and a former director of the institute of biology. he was at the forefront of biodiversity research and conservation in the philippines. jonas p. quilang, ph.d. editor-in-chief 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address _______________________________ _____________________________________________________ telephone no. _______________________________ ___________________________________________________ fax no. ________________________________________ please send accomplished subscription form to the rduo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city (02) 436-8720 fax (02) 927-2568  research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. (subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total sd-sample article 1 from the editor marco nemesio e. montaño, phd editor-in-chief issn 0115-7809 print/issn 2012-0818 online similar to the previous issues, we have been exerting efforts to produce the issue on time. in this issue we feature two main articles and two communications. the vehicular traff ic in metro manila prompted researchers to use a programming formulation with binary technique in solving the multi-objective transportation problem. the second article is on the toxic effects of copper and zinc on the embryo of the tropical urchin tripneustes gratilla. it should be noted that the roe of the test organism is eaten by the local f isherfolks and is hence economically important. the importance of quality control and assurance in drug formulations are shown in the communication on the spectophotometric determination of losartan potassium in tablets. the other communication is a contribution to the literature on philippine biodiversity. a note on the biogeographical distribution of the informal group basommatophora, a highly ubiquitous group of mollusks, is presented. ******** it is noteworthy to mention that through presidential proclamation no 737, s. 2014 issued on 13 march 2014, dr. gavino c. trono jr. , professor emeritus of the marine science institute, college of science, up diliman, was raised to the rank and title of national scientist. prof. trono is recognized for his work on seaweed taxonomy and culture in the philippines. with this development, up diliman now has three national scientists who are still active. finally, we are very proud and happy to announce that through commission on higher education resolution no. 200-2014, science diliman has been placed in the a-2 category, effective this year, until 2016. journals under category a-2 have the highest accreditation among those not yet indexed in the web of knowledge (isi), scopus and other international citation databases. science diliman shall also receive a publication award of php 200,000.00 per year from ched during the period of accreditation. with this development, all three journals of ovcrd are now category a journals. we are thankful to the staff, led by the managing director dr. violeda umali, for the efforts to enable the journals to reach this category. 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email address _______________________________ _____________________________________________________ telephone no. _______________________________ ___________________________________________________ fax no. ________________________________________ please send accomplished subscription form to the rduo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. research dissemination and utilization office office of the vice-chancellor for research and development lower ground floor, phivolcs bldg. , c.p. garcia ave. , up diliman 1101 quezon city (02) 436-8720 fax (02) 927-2568  research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd), as follows: humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php650.00. (subscription price is subject to change without prior notice.) i/we would l ike to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650)  humanities diliman  science diliman  social science diliman grand total 01_olivera 1 larger forms in lophiotoma science diliman (january-june 2004) 16:1, 1-28 larger forms in lophiotoma: four new species described in the philippines and three from elsewhere in the indo-pacific baldomero m. olivera department of biology university of utah 257 south 1400 east salt lake city, utah 84112-0840 email: olivera@biology.utah.edu date received: june 16, 2003; date accepted: january 14, 2004 abstract a group of venomous turriform gastropods in the subfamily turrinae, genus lophiotoma, has been investigated. previously, forms in this group were identified as either lophiotoma unedo or lophiotoma indica. our analysis has led to the description of four new species from the philippines (l. bisaya, l. friedrichbonhoefferi, l. panglaoensis, and l. tayabasensis) and one each from australia (l. capricornica), south africa/mozambique (l. dickkilburni), and madagascar (l. madagascarensis). a new subspecies, l. indica queenslandica, is also described. in addition, 11 distinctive forms related to these taxa that may or may not deserve separate taxonomic status are defined; these need further evaluation. it is hypothesized that the forms of lophiotoma discussed in this report are closely related to a particular subset of gemmula, the g. kieneri/g. interpolata group. keywords: toxoglossate mollusc, venomous snail, turridae, lophiotoma, shell morphology, conacea introduction this paper is part of a series that has the long-term goal of defining and evaluating distinctive forms of indo-pacific turrinae. the first paper dealt with philippine forms of turris (olivera, 1999). in this and the second paper of this series (olivera, 2002), we have initiated a definition of the larger lophiotoma. the focus of the present paper are lophiotoma related to (and generally identified as) lophiotoma indica and lophiotoma unedo. we introduce the group through a broad review of the relevant taxonomy. the last comprehensive treatment of indo-pacific turrinae carried out by powell (1964 & 1966) used several shell-based morphological criteria to define genera or subgenera. these included the position of the sinus, the presence or absence of gemmules, protoconch characters, and the length of the siphonal canal. both kilburn (1983) and bouchet (1990) have suggested that protoconch morphology, while useful for species differentiation, should only be one of the many morphological parameters considered in proposing new genera. thus, the subgenus lophioturris proposed by powell for forms with blunt paucispiral protoconchs (including l. indica) will not be recognized here, in consonance with the suggestions of kilburn and bouchet. in the second paper of this series, it was suggested that the length of the siphonal canal was a morphological character that could change more rapidly than previously thought. some forms with long canals (presently assigned to lophiotoma) seemed more 2 olivera closely related to species with short siphonal canals (traditionally assigned to xenuroturris) than to other species conventionally included in lophiotoma (the example considered was “xenuroturris” cingulifera and lophiotoma albina, which except for the siphonal canal appear to be closely related). thus, we suggest that turrid genera or subgenera based on the length of the siphonal canal alone, without other distinguishing criteria, should also be avoided. finally, the presence or absence of gemmules may not be as straightforward a character as powell indicated; he argued that in the context of the fossil record, the presence or absence of gemmules on the sinus cord was a key indicator of the evolutionary history of the group. some of the species that we discuss in this manuscript were assigned by powell to the genus gemmula (in the subgenus unedogemmula). kilburn suggested a closer relationship between unedogemmula and the lophiotoma (s.s.) than powell’s monograph indicated, and felt that “phenetic resemblances between lophiotoma and unedogemmula are certainly greater than between gemmula and unedogemmula” (kilburn, 1983). his suggestion that “unedogemmula” spp. should be included in lophiotoma rather than gemmula has been adopted by taylor et al. (1993) and higo et al. (1999), and will be adopted here. in this series, as a response to the problem of generic assignments, we provisionally use only three major genera in the turrinae for forms that are broadly distributed over the indo-pacific: turris, gemmula and lophiotoma. some other groups in the turrinae, such as fusiturris, are small with a narrow geographic distribution; the other large genus in the turrinae, polystira, is a western atlantic/eastern pacific group. thus, lophiotoma is provisionally treated as conceptually broader than in powell’s treatise, encompassing not only all species he assigned to lophiotoma (s.s.), but all of the species assigned to lophioturris, xenuroturris, and unedogemmula as well. this is only a provisional, if convenient, solution: we recognize that the species we include in lophiotoma will ultimately be grouped into different infraspecific taxa. however, because the present divisions probably do not accurately represent the true evolutionary relationships between the subgroups, using lophiotoma broadly defined seems the best interim solution. consequently, all of the forms discussed in this paper are treated as lophiotoma (s.l.); in the literature, the two most commonly used specific names, often referred to as lophioturris indica and gemmula (unedogemmula) unedo, are now included in lophiotoma. the species related to lophiotoma indica and lophiotoma unedo comprise a confusing series of larger forms that bring to mind hedley’s remark that the turrids “are considered by those who meddle with them to be more perplexing than any other molluscan family” (hedley, 1922). we have several reasons for examining these species groups at this time. the immediate reason is somewhat pedestrian: our laboratories are engaged in an analysis of the venoms of toxoglossate molluscs, with an initial focus on conus. as we initiated work on venoms of the larger turrinae, we began to recognize the problematic taxonomy of even supposedly well-known forms. the imminent characterization of venoms provided an urgent need for defining distinct forms, and if possible, assigning appropriate names. every large lophiotoma is labeled as either l. indica or l. unedo in most collections. any larger shells that are relatively narrow and/or reticulated with darker markings are generally identified as lophiotoma indica, and those larger forms that are broader and lighter in color as lophiotoma unedo. these names have been applied to a confusing set of diverse forms. in this work, all distinct morphological forms conventionally assigned to these two taxa are evaluated, and new names for forms that are sufficiently distinctive to merit subspecific or specific status are proposed. however, for many forms, there is insufficient information to decide whether the form is a new species or subspecies, or an unusual variant of another species. in most cases, the material examined is collected from deeper water, with relatively few examples available. the decision as to which distinctive forms are separate species or subspecies is somewhat subjective at this time. in this manuscript, we have generally taken an approach in which only very distinctive forms that are well defined are given new specific or subspecific names. in describing new species and subspecies, we have deliberately taken a narrow set of specimens as the 3 larger forms in lophiotoma type material; it is possible that even forms that only differ subtly and are quite similar are, in fact, separate species. it seems wise to limit the type material to the narrowest possible variation and geographic range since the possibility that there will prove to be three or even ten times as many species as are being recognized below from the material examined cannot be eliminated. we also intend this initial evaluation, based on shell morphology, to facilitate the assessment of relationships between the various forms using a molecular approach. since the required live-collected material is generally not available for molecular studies, a systematic morphological evaluation of conchological characters should help focus collection efforts of the appropriate specimens for analysis. we anticipate that the molecular work will include the identification of genes encoding venom components; such genes have been useful in assessing relationships between species in conus (olivera, 2002). the prospect of systematic collection efforts of live material by the paris museum in the near future may provide an unparalleled opportunity to collect the live specimens needed for the requisite molecular analysis. we first discuss those species that are morphologically most distant from gemmula, namely the l. indica-like forms, followed by an analysis of the l. unedo-like forms. overview of lophiotoma indica-like forms lophiotoma indica is the largest shallow-water form in the genus, but specimens assigned to this species are morphologically heterogeneous. this heterogeneity may arise in part because l. indica is quite variable, but in addition, several different species have been lumped under this taxon. the description of kilburn (1983) on the general problematic taxonomy of the turridae particularly applies to this group: “intense regional speciation has produced a high percentage of often poorly studied endemics”. the l. indica species group comprises a confusing series of seemingly interrelated forms; the treatment that follows is a conservative evaluation of the available material. as more material is collected, and a more thorough characterization of the various forms becomes available, it seems probable that more species and subspecies will be recognized. the various forms that are often labeled in collections and in publications as l. indica can be divided into two groups on the basis of protoconch morphology: all shallow-water forms appear to have a blunt paucispiral protoconch. however, there are deep-water forms which may or may not be taxonomically distinct that also have a blunt paucispiral protoconch. in addition, several forms conventionally assigned to this species have a polygyrate protoconch, and can therefore be distinguished from other l. indica-like morphs based on this character. all of the latter are collected off-shore, and although they have been previously assigned to l. indica, the difference in protoconch morphology provides a firm basis for separating these from l. indica and other related forms with paucispiral protoconchs. in the treatment that follows, all forms with blunt paucispiral protoconchs are included in group i. among the forms assigned to l. indica (and sometimes misidentified as lophiotoma unedo) that have blunt paucispiral protoconchs, we propose a new subspecies; in addition, four distinctive forms in the l. indica complex of uncertain taxonomic status are defined. in group ii, we include the l. indica-like forms with polygyrate protoconchs; four new species are proposed and several distinctive forms of uncertain taxonomic status are described. group i species: lophiotoma indica-like forms with blunt paucispiral protoconchs even when all forms with polygyrate protoconchs are excluded from l. indica, this complex still encompasses a bewildering variety of forms as illustrated in figs. 1 and 2. within the l. indica complex, we recognize two subspecies as is discussed below. in the eastern indian ocean, indo-pacific arc (new guinea to the philippines), and japan, the “typical form” lophiotoma indica indica (röding, 1798) is found. we propose a new subspecies, lophiotoma indica queenslandica, for specimens collected in the southeastern edge of the range from queensland, australia to fiji. four additional forms in the l. indica complex are described which require further evaluation. 4 olivera lophiotoma indica indica (röding, 1798) description (adapted from powell, 1964) adult shells up to 100 mm in height, elongate-fusiform with a tall spire, and a long straight anterior canal. whorls, about 14 with a blunt, smooth paucispiral protoconch (1 to 1 1/2 whorls) with a half whorl of strong axial ridges. a strong, narrowly-rounded, smooth peripheral carina is located below medium whorl height. at the subsutural fold, there is a strong, smooth spiral cord at its lower extremity, with a weaker cord and several threads above it. between 1 to 3 smooth primary spiral cords are present between the periphery fig. 1a. lophiotoma indica indica from the philippines, variation series. a1 is the melanistic variety from murcielagos bay, misamis occidental, northern mindanao island, philippines. a7 is an albinistic example (form bulowi) trawled off negros island, philippines. a4 is from cuyo island, palawan, collected by divers in shallow water. a2, a3, a5, and a6 were trawled in deeper water from various philippine localities. fig. 1b. lophiotoma indica queenslandica. the holotype is shown in b1 and b2. b3, b4, and b5 are paratypes 1, 6, and 7, respectively, all live-collected specimens trawled off queensland, australia. b6 and b7 are dead-collected specimens from the swain reefs, paratypes 2 and 3, respectively (appendix). 1 2 3 4 5 6 7 1 2 3 4 5 6 7 5 larger forms in lophiotoma and the lower suture, with 18 to 20 primary spinal cords in the body whorl from the periphery to the end of the anterior canal. the entire surface is crowded with smooth spiral threads of varying strength. the color pattern is profusely spotted, with dark round maculations on a white, grayish-white or brownishwhite background. the maculations are confined to the primary spiral cords, but diffuse vertically in many specimens, presenting a sinuous eggshell pattern that varies in strength, giving the shell pattern a generally marbled effect. the peripheral maculations are always the strongest. the tip of the anterior canal often has a brownish tint, with the brownish cast extending over the entire canal in some specimens. fig. 2a. distinct forms within the lophiotoma indica complex. a1 and a2 are l. indica indica, typical forms. a3 and a4 are l.c.f. indica, variant/form 1, from south india (“arabian sea form”). a5 and a6 are l.c.f. indica, variant/form 2, from exmouth gulf, western australia. (bottom row) a7 and a8 are l.c.f. indica, variant/ form 3, thin shallow-water form from broome, western australia. a9 and a10 are l.c.f. indica, variant/form 4, from cagayan province, northeast luzon, philippines (los angeles county museum #75648). a11 (holotype) and a12 (paratype 6) are l. indica queenslandica, trawled off the queensland coast. figs. 2b, 2c, and 2d. comparison of protoconchs. fig. 2b. lophiotoma indica queenslandica (swain reefs, australia). fig. 2c. lophiotoma bisaya (aligway, philippines). fig. 2d. lophiotoma friedrichbonhoefferi (aligway, philippines). photos, 25x using a dp-10 olympus digital camera on an olympus szx9 s t e r e o m i c r o s c o p e . photomicrographs by nancy kurtzeborn. 1 2 3 4 5 6 7 8 9 10 11 12 6 olivera the recently collected specimens of the “typical form” of l. indica shown in fig.1 are from the philippines, north to japan and south to queensland. however, this form occurs in the indian ocean, since the type is reported to be from tranquebar, india (r. kilburn, personal communication). even within the typical form, considerable variation in color, pattern, and shape is observed—some of this variation appears to be a function of depth. in the central philippines, l. indica has been trawled by fishermen at depths of over 50 m; the deeper-water material tends to be narrower and often lighter in color (fig. 1). at intermediate depths, the typical form is less narrow, and shallowwater morphs from the philippines have broader, more robust shells. a range of diverse forms, likely all variants of l. indica indica, are shown in fig. 1a. atypical forms, probably variations of lophiotoma indica indica melanistic form in northern mindanao and the visayas islands, philippines, a slender melanistic form has been collected; one locality verified by trawlers is murcielagos bay, off misamis occidental, northcentral mindanao (fig. 1a, specimen 1). albinistic form (“form bulowi”) several morphs in the l. indica complex have a tendency to become albinistic. albinistic specimens from the philippines, which we include in l. indica indica, tend to have a broader canal than other forms of l. indica (fig. 1a, specimen 7). lophiotoma indica queenslandica, new subspecies most specimens have been collected from queensland, australia offshore that appear distinctive enough from any other form in the complex to justify at least subspecific separation. the unusual depth at which specimens are collected and the known geographic range also separate this form from l. indica indica. future work may justify separation into different species. description l. indica queenslandica has a blunt paucispiral protoconch (fig. 2b), typical of the l. indica complex, and 12-13 teleoconch whorls. each whorl has highly characteristic sculpture and markings between the suture and the periphery. there are two rows that are broadly maculated in brown: one set of maculations is centered around the periphery, which comes to a sharp apex in most specimens; the second row of maculations is subsutural and is centered around a cord that is usually well displaced from the suture. otherwise, the sculpture consists of very fine spiral threads (both the peripheral apex and the cord that is maculated in the subsutural region are generally lighter in color than the background except for the broad brownish maculations). the body whorl has a grayish-brown background below the periphery, with a transition to a brownish base and siphonal canal. there are about 5-6 spiral cords within the area of the grayish-brown background and a larger number in the siphonal canal. the body whorl is quite rounded, particularly in the suite of specimens from the swain reefs, giving the entire shell a generally more bulbous, less pagoda-form appearance than other forms in the l. indica complex. in the region from the suture to the periphery, only a subsutural cord and the periphery are maculated; the multiple cords in the region between the suture and the periphery are not maculated. some specimens tentatively assigned to l. indica queenslandica have strong axial markings. type material the holotype and some paratypes are shown in fig. 1b. the holotype will be deposited in the muséum national d’historie naturelle (mnhn), paris, france. paratypes will be deposited at the national museum of natural history (usnm), washington, d.c.; the academy of natural sciences (ansp), philadelphia, 7 larger forms in lophiotoma pa; the field museum of natural history (cfm), chicago, il; the american museum of natural history (amnh), new york, ny; the los angeles county museum of natural history (lacm), los angeles, ca; and the western australian museum, perth. specific measurements and data for the holotype and paratypes are given in the appendix. two of the paratypes, collected in the swain reefs area, were trawled between 90-105 fathoms. discussion l. indica queenslandica can be differentiated from l. indica indica by its generally more bulbous shape, by the background brown or grayish-brown color (in l. indica indica the background is generally white or light grayish white), and most strikingly, the markings on the spiral ribs. in l. indica queenslandica, the markings on the spiral ribs are lighter and tend to run axially in both the cords and in the intercord regions; in typical l. indica indica, a pattern of boldly, regularly maculated spiral cords is generally observed. many of the l. indica queenslandica paratypes included in the type series for the species were freshly dead-collected specimens from swain reefs—we have included these as paratypes because these have the most specific locality collection data (appendix); however, because these specimens were dead collected, they are lighter pinkish brown in color than the grayish brown or reddish brown of live-collected specimens. most of the live-collected specimens were from commercial dealers, and the precise collection data could not be definitively established, but were said to have been trawled in queensland, offshore in deeper water. we note that a recent publication figured a specimen reportedly collected in japan (okutani, 2000) that appears to be l. indica queenslandica identified as l. indica. some specimens in collections assignable to this species are labeled “taiwan”—however, many shells labeled “taiwan”, though purchased in taiwan, were actually collected off australia. if the northern pacific localities are verified, it would provide support for queenslandica being a separate species, rather than a subspecies of indica. distinctive forms in the lophiotoma indica complex of uncertain taxonomic status several very distinctive morphs are part of this species complex. these are compared to typical l. indica indica and l. indica queenslandica in fig. 2. these forms are described from a west to east gradient. l.c.f. indica, variant/form 1; arabian sea form this form can be differentiated from typical indica by its generally darker background, and broader shell with a proportionally shorter and broader canal. the peripheral carina is more prominently raised than in the typical form and shows a greater contrast in color pattern than is found in the rest of the shell (the background in the peripheral keel is distinctly whiter). a pattern of continuous dark axial flames in the body whorl is seen in most specimens, with each flame being much wider than in the typical variety. the subsutural fold has two equally strong, smooth spiral cords in contrast to one strong lower one in typical l. indica. this variant is shown in fig. 2a, specimens 3 and 4. most specimens of this form available to the author were obtained from fishing vessels in south india, and were probably collected by trawlers off kerala. the form may be found further west to the arabian peninsula (bosch et al., 1995). although the specimen figured from the gulf of oman in the book by bosch is consistent with this form, it appears to be dead collected. none of the specimens observed had an intact protoconch; therefore, it is possible that this is a group ii form. the distinctive shell morphology and the geographic distribution of this form are consistent with its being separable at least at a subspecific level. however, more specimens need to be examined to understand the relationship of this form to l. indica indica and l. indica queenslandica, as well as to some of the other morphs described below. 8 olivera l.c.f. indica, variant/form 2; western australian form in western australia, a distinct form that is smaller but more robust than typical l. indica indica is found (fig. 2a, specimens 5 and 6). one verified locality where this form has been collected is exmouth gulf; specimens were trawled in deeper water. the shells are much darker and smaller than typical l. indica, dominated by strong, dark brown axial markings rather than the maculations on the spiral cords. l.c.f. indica, variant/form 3; northwestern australian form a series of unusually light shells, collected in shallow water (intertidally in broome, australia) have a broader spire and relatively shorter canal with much larger brown maculations in the periphery, compared to typical l. indica indica (fig. 2a, specimens 7 and 8). l.c.f. indica, variant/form 4; northeastern luzon form two specimens of a broad gray form that is distinct from all other l. indica-like forms found in the philippines are in the los angeles county museum of natural history collection (#756481). these are shown in fig. 2a, specimens 9 and 10. the larger specimen (fig. 2a, specimen 10) is broader in outline, with less constricted sutures than other forms of l. indica. furthermore, the body whorl is light grayish, strongly maculated at the peripheral cord with an axial pattern that follows the growth line and continues through the body whorl to the canal in continuous zebra-like streaks. the canal has a brownish background color in most specimens. the subsutural region has a well-maculated cord, splitting into two cords separated by a canal in the body whorl. the juvenile specimen is very similar in general sculpture and coloration to some specimens of l. indica queenslandica, except that in overall shape it is more slender than comparably sized specimens of that species. this form also has affinity with the arabian sea form described from southern india. these specimens bear the label “1-5 fathoms on sand and coral bottom, escarpa point, cagayan province, northeast luzon island (collected 22 june 1964, norton collection).” one possibility to be considered further is that the l. indica queenslandica is in fact a distinct species, and that both this form and the arabian sea form (variant/ form 1) are variants of that species rather than of l. indica. group ii species: lophiotoma indica-like forms with polygyrate protoconchs the shells of species included in group ii are generally similar to forms in the l. indica complex in group i, and can be difficult to separate if protoconchs are not intact. however, all forms in group ii do not have the blunt paucispiral protoconch typical of forms in group i, but have polygyrate protoconchs (figs. 2c and 2d). we describe four new species that belong to this group, lophiotoma friedrichbonhoefferi, lophiotoma bisaya, lophiotoma tayabasensis, and lophiotoma dickkilburni, which are illustrated in fig. 3. the first three forms described as new species from the philippines comprise a discrete range of sizes: the relatively small l. friedrichbonhoefferi (35-50 mm), the medium-sized l. bisaya (60-75 mm), and the large l. tayabasensis (80-110 mm). these, as well as related variants, are illustrated in figs. 3 and 4. lophiotoma friedrichbonhoefferi, new species description this distinctive form, illustrated in fig. 3, is smaller and narrower than other group ii species, and has relatively larger and fewer maculations. the polygyrate protoconch (fig. 2d) distinguishes it from l. indica and other group i forms. in specimens where the spire is not corroded, the first postnuclear whorl shows a weakly gemmate structure that is visible in fig. 2d. most mature shells range in size from 35-50 mm (appendix). in addition to the polygyrate protoconch of ca. 3.5 whorls, there are about 11 teleoconch whorls. the spire is boldly maculated at both the peripheral rib and in the subsutural region; the subsutural cord is strong, and in later whorls is split into two, separated by a narrow canal. the area from the strong subsutural 9 larger forms in lophiotoma cord to the periphery is flat and almost perpendicular to the axis of the shell. the color of the shell is white with a light pinkish or purplish tone; the body whorl and base have about six primary cords that are more or less regularly maculated in the same purplish brown color as the peripheral cord and the subsutural region; these smaller maculations on the primary cords tend to be parallel to each other and to the growth line of the aperture. on the relatively long siphonal canal, the pattern of regular maculated ribs continues; there are about seven primary cords with additional weaker cords in some specimens. anterior to these cords is a distinctive purplish-brown band and the very tip of the siphonal canal is pure white. the maculations on l. friedrichbonhoefferi are bolder, and there is more contrast with the white background than in most related forms. most specimens of this form examined were from aligway island off n. mindanao, said to be collected by small trawls at depths of 30-100 fathoms. two deadcollected specimens, one relatively well preserved, in the paris museum (mnhn) are assignable to l. friedrichbonhoefferi. these are labeled, “13° 53’ n– fig. 3a. specimens illustrated not to scale, to be approximately the same size. dorsal and side view of lophiotoma friedrichbonhoefferi (a1 and a2); lophiotoma bisaya (a3 and a4); lophiotoma tayabasensis (a5 and a6); and lophiotoma dickkilburni (a7 and a8). fig. 3b. all specimens are shown to scale. b1 (holotype), b2 (paratype 1), b3 (paratype 8) and b4 (paratype 9) are lophiotoma friedrichbonhoefferi; b5 (holotype) and b6 (paratype 26) are lophiotoma bisaya; b7 (holotype) and b8 (paratype 26) are lophiotoma tayabasensis; while b9 (holotype) and b10 (paratype 1) are lophiotoma dickkilburni. 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 10 olivera 120° 09’ e, musorstom st. 56, 134 m, from near lubang island, philippines” (appendix). the polygyrate protoconchs are reasonably well-preserved in both specimens, which have been included in the type series. thus, the species is found from northern mindanao to lubang island, off western luzon in the philippines. type material the holotype will be deposited at the philippine national museum, manila. paratypes will be deposited at mnhn, paris; amnh, new york; usnm, washington; cfm, chicago; natal museum, south africa; lacm, los angles; and ansp, philadelphia. data on the types are given in the appendix. this species is named for a truly creative pioneer in several fields of science, friedrich bonhoeffer. the author has been a grateful beneficiary of friedrich bonhoeffer’s creativity, scientific insight and warm friendship. lophiotoma bisaya, new species description lophiotoma bisaya has a paucispiral protoconch and 1213 teleoconch whorls in typical mature specimens. the subsutural fold usually has a strong cord that is maculated, that often splits into two cords in the body whorl, with 2-3 similarly maculated primary cords between the subsutural and the peripheral cord. on the body whorl, there are usually 6 primary cords and 7-9 maculated cords on the canal. at the anterior end of the canal, there are approximately four additional cords with progressively decreasing intensity of maculation; the most anterior generally located in a distinct dark brown band. the tip of the siphonal canal is white and lacks any primary cords. the maculations are brown to maroon-brown, and in some specimens, there are axial patterns that result in a darker base color between the maculated cords; however, other specimens have no axial markings and the pattern on the body whorl is restricted to regular maculations on the primary cords. from northern cebu, bohol, and aligway island, specimens have a siphonal canal that is generally very straight, with the maculated cords giving way to a dark brown band, followed by the characteristic white tip (a feature that is also characteristic of l. friedrichbonhoefferi, as well as most specimens of l. unedo). type material the holotype will be deposited at the philippine national museum, manila. paratypes will be deposited at mnhn, paris; amnh, new york; usnm, washington; cfm, chicago; lacm, los angles; and ansp, philadelphia. data on the types are given in the appendix. discussion the shell pattern of the body whorl of l. bisaya is one of closely-spaced spiral cords, finely maculated with small squarish brown maculations; the slightly maroon tinge of these maculations gives philippine forms of l. bisaya a generally different color cast from l. indica. l. friedrichbonhoefferi can be differentiated from l. bisaya by the whiter background, fewer and bolder maculations, and generally smaller size of the former. although there are some members of the l. (unedogemmula) unedo complex that are very similar morphologically to l. bisaya, l. bisaya does not have the massive subsutural structure that is characteristic of philippine specimens of l. (unedogemmula) unedo. in l. bisaya, between the maculated subsutural cord and the periphery are typically 2-3 primary cords, decorated with maroon-brown maculations similar in intensity to the maculation on the cords on the body whorl. in l. friedrichbonhoefferi, the subsutural cord and periphery are strongly maculated—maculated cords in between are either completely absent or present only in the body whorl (always with much weaker maculations than in the subsutural cord). philippine specimens of l. unedo typically have a concave, deeply excavated region with fine unmaculated cords between the upper suture and the periphery. compared to specimens of l. indica indica, philippine specimens of l. bisaya have a more horizontal shoulder, perpendicular to the rest of the whorl, giving each whorl a distinctly squarish appearance. furthermore, the peripheral keel is much more depressed (in most specimens of l. indica, the peripheral keel comes to a 11 larger forms in lophiotoma sharp edge). the peripheral carina has a whiter background than the remainder of the whorl, and in some specimens the white area is extended, both above and below the actual sinus cord itself. a definitive characteristic is that l. bisaya has a polygyrate protoconch of approximately three whorls (fig. 2c), while all forms of the l. indica complex have a blunt paucispiral protoconch. l. bisaya appears to be closely related to l. friedrichbonhoefferi described above, as well as to l. dickkilburni as described below. shells of l. bisaya are generally smaller than l. tayabasensis (mature specimens 60-75 mm vs. 80-110 mm for l. tayabasensis); the maculations are a distinct maroonbrown color as opposed to the straw-brown maculations of l. tayabasensis. although there are some specimens of l. bisaya that begin to approach l. tayabasensis in shape and size, the differences in color, the generally squarish shape of the whorls, and the distinct dark maculations on the sinus cord in l. bisaya (as opposed to curved yellowish-brown markings on l. tayabasensis) will generally distinguish the two species. atypical varieties of l. bisaya unbanded variety a variation of l. bisaya apparently collected by trawlers working out of maqueda and carigara bays in samar island and tayabas bay in south luzon island is illustrated in fig. 4, specimen 2—these specimens are very similar to the type material, except that they lack the sharp demarcation provided by the dark brown band at the siphonal canal between the white anterior tip and the posterior brownish canal. a specimen in the los angeles county museum (lacm 75534) from tayabas bay (trawled at a depth of 22 fathoms in may 1965 by j. norton) has excellent preservation of the polygyrate protoconch. south indian variety a more distinctive form, also probably a variation of l. bisaya, that has a proportionally longer siphonal canal with generally lighter color streaks in the body whorl and canal has been collected in south india (fig. 4, specimen 3); the protoconchs were not preserved in any of the specimens examined. lophiotoma tayabasensis, new species this distinctive form is likely to be found in collections labeled lophiotoma unedo (or unedogemmula unedo or gemmula unedo), and sometimes lophiotoma indica. most specimens in collections were collected by trawlers in tayabas bay, luzon, philippines, in the late 1950s and the 1960s; this form is illustrated in the monograph by springsteen and leobrera as gemmula unedo (springsteen & leobrera, 1986; plate 76, #3). the misidentification of this previously unnamed philippine form probably came about because, for many years, this was the only large form of the turrinae at all similar to l. unedo routinely collected in philippine waters. the species appears to be common offshore, from southern luzon to northern mindanao. recent specimens have been collected in the cebu-bohol region by trawlers out of lapu-lapu city, mactan. description this is one of the largest philippine turrids, adult specimens reaching over 105 mm (appendix). l. tayabasensis has fine, regular straw-brown maculations along the spiral cords; the underlying axial streaks of yellow-brown to straw-brown vary considerably in intensity, and can be the dominant pattern in some specimens. the subsutural area typically has two cords in the later whorls, well-separated with the one further from the suture, strong and more boldly marked. there are 1-3 more maculated cords between the subsutural area and the peripheral keel. the sinus is deep and the peripheral keel is relatively flattened in the body whorl, with curved brown maculations that are parallel to the edge of the sinus. a characteristic feature of this species is that in many specimens, the early teleoconch whorls have sinus cord maculations that are more continuous and in earlier whorls of the spire, the sinus cord can be a uniform amber brown. below the peripheral sinus cord, there are 9-13 spiral cords on the body whorl that are finely-marked with 12 olivera brown, squarish maculations; at the siphonal canal, the spiral cords continue, but there is a transition to less prominent maculations (and in some specimens the maculations are completely obsolete). to varying degrees, the siphonal canal has a darker brown background which may either cover the entire canal, or be restricted to the anterior half; in almost all specimens, the very tip of the siphonal canal is significantly lighter in color. however, there is no distinctive dark brown border region characteristic of specimens of l. friedrichbonhoefferi or l. bisaya. in most specimens, the protoconch and early postnuclear whorls are either absent or extremely corroded; this form lacks gemmules in later whorls. the protoconch was not preserved in any specimens examined, but appears to be polygyrate from the few specimens with corroded remnants of the protoconch. one specimen showed evidence for weak gemmules in the first teleoconch whorl. type material the holotype will be deposited at the philippine national museum, manila. paratypes will be deposited at mnhn, paris; amnh, new york; usnm, washington; cfm, chicago; lacm, los angles; and ansp, philadelphia. data on the types are given in the appendix. the species is named for the type locality, tayabas bay, as well as the old philippine province of tayabas on luzon island, a traditional name for a long coastal province on luzon island. fig. 4. an illustration of various forms related to lophiotoma bisaya and lophiotoma dickkilburni. (top row) variations of l. bisaya. specimen 1, typical form from aligway, philippines; specimen 2, l. bisaya variant from tayabas bay; and specimen 3, l. bisaya variant collected from south india. (middle row) l. dickkilburni. specimen 4, paratype 1 from mozambique; specimen 5, holotype from south africa; specimen 6 is a south indian specimen tentatively assigned to l. dickkilburni. (bottom row) variant/forms related to l. dickkilburni. specimen 7, l.c.f. dickkilburni, variant/form 1, dark mottled variety from mozambique; specimens 8 and 9, l.c.f. dickkilburni, variant/form 2, longer canal form from natal, south africa (specimen 8) and from pondicherry (specimen 9), india; specimen 10, l.c.f. dickkilburni, variant/form 3, albinistic form from angoche, mozambique. 1 2 3 4 5 6 7 6 8 9 10 13 larger forms in lophiotoma discussion the lack of a massive subsutural cord distinguishes this species from most philippine specimens of the l. unedo complex; instead of a strongly concave region between the suture and the periphery, in l. tayabasensis the immediate subsutural area is slightly raised but otherwise the subsutural area has spiral cords that are decorated with light brown or purplish-brown maculations as in the rest of the body whorl, and the region is not deeply excavated or horizontal compared to the rest of the whorl, but instead is gently sloping towards the periphery. the continuous brown color of the sinus cord in the earlier spire whorl is another distinguishing characteristic. the species can be easily differentiated from l. friedrichbonhoefferi, l. bisaya, and other group ii species by its large size, more lightly colored maculations on the primary spiral cords, and more bulbous shape. lophiotoma dickkilburni, new species most of the specimens assigned to this species were collected in south africa and mozambique; a few deadcollected specimens from south india are possibly assignable to this taxon as well. the specimens examined show significant variability, and a larger series from a wider geographic range needs to be examined; it is probable that there is more than one group ii species in the western indian ocean. we have deliberately restricted our description and the type series to one specific form from south africa and mozambique. the relationship between the various species needs to be more definitively established. description this form has a polygyrate protoconch and 13-14 teleoconch whorls. the subsutural fold has two cords that are close together, and tends to form a distinct terrace, particularly in the earlier whorls. between the subsutural fold and the periphery there are usually three primary cords. in addition to the peripheral cord, the body whorl has 10-12 primary cords that are maculated in more or less parallel fashion with a variable number of cords continuing on the canal. the cords become weaker and disappear on the anterior end of the canal. an additional characteristic of many specimens is that on the later whorls, there is a white area surrounding the peripheral cord that is lighter in background color than the rest of the body whorl. the maculations are generally lighter brownish in color, with the color of the siphonal canal progressively lighter anteriorly. the anterior end of the siphonal canal is often curved towards the aperture end. type material the holotype will be deposited at the mnhn, paris. paratypes will be deposited at mnhn, paris; the natal museum, south africa; amnh, new york; usnm, washington; cfm, chicago; lacm, los angles; and ansp, philadelphia. data on the types are given in the appendix. this species is being named for richard (dick) kilburn, a preeminent authority on the taxonomy of turriform gastropods and of south african molluscs. dr. kilburn has been most generous in sharing his vast knowledge of turrids, and has been an invaluable resource for this work. discussion a large specimen collected in south india (fig. 4, specimen 6) seems to be conspecific with l. dickkilburni. l. dickkilburni appears most closely related to l. bisaya, but there appears to be a number of consistent differences. the tip of the siphonal canal in all specimens of l. bisaya examined from the type locality is white with a distinct darker brown border. the area from the suture to the periphery is much flatter in many l. bisaya specimens, a feature that gives the spire of l. bisaya a more pagoda-form aspect, instead of the more gradual curvature observed in l. dickkilburni. finally, a significant fraction of l. dickkilburni specimens have a curved siphonal canal, a feature not observed in l. bisaya. in general, the cords of l. dickkilburni are more finely maculated than l. bisaya; thus, the typical number of maculations 14 olivera per whorl on the peripheral cord of l. bisaya varies from 14-17, while in l. dickkilburni it is over 20 (generally around 25). the material from south india suggests that variants of both l. dickkilburni and l. bisaya may occur there. specimens 3 and 6 of fig. 4 are both from southern india; they do not appear to be conspecific, with one tentatively assigned to l. bisaya (specimen 3) and the other to l. dickkilburni (specimen 6). distinctive variants/forms possibly related to lophiotoma dickkilburni several distinctive forms that may or may not be conspecific with l. dickkilburni are shown in fig. 4. l.c.f. dickkilburni, variant/form 1 this form is larger and more heavily reticulated than l. dickkilburni; specimens examined were collected in mozambique. this differs from the type series of l. dickkilburni in having a darker purplish-brown color and a less prominent subsutural cord. l.c.f. dickkilburni, variant/form 2 another form possibly related to l. dickkilburni are slender lophiotoma indica-like specimens that have a purplish-brown background color, with long siphonal canals, collected in the south africa/ madagascar region. similar specimens have been found in india (fig. 4, specimens 8 and 9). in specimens the author has seen from the indian ocean, the protoconchs have been eroded—these are invariably simply labeled “lophiotoma indica” (although they are distinct from the forms described under group i above). there are a series of specimens morphologically similar collected by the mnhn, paris museum, from makassar, indonesia; although the color is slightly different—the specimens have a yellowish-brown instead of purplish-brown background—the sculpture and general shape are similar. these specimens have eroded protoconchs, but what remains suggests a polygyrate protoconch with an indication of gemmules on the first teleoconch whorl, observations consistent with these specimens being group ii forms. whether these are related to l. dickkilburni or any of the other group ii species described above, or whether these forms comprise a geographically widely distributed separate species can only be established when more material is examined. apparently, all of the specimens available to the author were dredged in deep water. the south african specimen had a label indicating that it was dredged off the natal coast at 200 m; the makassar specimens from indonesia were collected at a depth of 220 m. l.c.f. dickkilburni, variant/form 3 specimens from mozambique in which the shell is largely white (with some residual faint maculations, particularly in the earlier whorls) may also be related to the forms above. although the protoconch has not been preserved in any of the specimens examined, the general shape of these specimens differs from the similarly white “bulowi form” that belongs with the group i species, but which is much more slender. the general shape of these specimens is much more similar to l. dickkilburni. however, curiously the aperture of l. dickkilburni and related forms that all have a darker exterior color is whitish, while the aperture of these seemingly somewhat albinistic specimens is dark. these specimens are apparently being sold by dealers in mozambique material as “lophiotoma indica, albinos” or “lophiotoma indica, form bulowi,” but at this time seem more likely to be a local endemic in the l. dickkilburni complex. groups iii and iv species: overview of the “lophiotoma unedo” complex all forms included in groups iii and iv have been identified in recent publications (and in most collections) as lophiotoma unedo, or alternatively gemmula (unedogemmula) unedo—this name is occasionally applied to some of the species in group ii as well. this is one of the most confusing groups of the larger turrinae. the larger spectrum of specimens available has revealed some general patterns. 15 larger forms in lophiotoma in powell’s treatment of “gemmula unedo,” all of the specimens that he actually figured were from japan (powell, 1964), and the description of the species was largely based on japanese specimens. in the period before 1964, the great majority of specimens available of this group iii complex were collected at depths of about 50 fathoms from southern japan. powell observed: “the peripheral carina is weakly gemmate in the first few postnuclear whorls, although in some specimens the beading may persist to the penultimate whorl”; later, in the formal description: “the earlier postnuclear whorls bear distinct gemmules in the peripheral carina and in a few instances these persist over most of the remaining whorls.” the analysis of a wider suite of specimens from a broader geographic area indicates two distinct classes of specimens, based on whether the peripheral carina has gemmules in the earlier postnuclear whorls (group iii), or whether the gemmules persist to most of the remaining whorls (group iv). we believe that this character is reliable for separating almost all specimens into two taxonomically distinct groups. over the entire range of the complex, there are representatives of both groups iii and iv, including the japanese specimens assigned to l. unedo. the broader biogeographic survey indicates that the two groups are distinct from each other at all localities, with the morphological differences between the “early” (group iii) and “mostly” gemmate specimens (group iv) more marked in non-japanese material. this is particularly true in australia, where there is not only a more striking morphological differentiation, but some biogeographic segregation of the two groups (iii and iv) as well. the name pleurotoma (turris) unedo appears from kiener’s figure to apply to one of the “early” gemmate forms (group iii), and not to the “mostly” gemmate class (iv). the same appears to be true of the form pleurotoma (turris) invicta. group iii: lophiotoma (unedogemmula) unedo and related forms with early postnuclear gemmate whorls group iii forms comprise lophiotoma unedo and related forms that typically have gemmules similar to gemmula spp. on the first 3-4 postnuclear whorls, followed by a transition zone of 2-3 whorls where the gemmules become less distinct and may look like undulations in the peripheral keel. most group iii forms are conventionally assigned to l. unedo; several other species such as lophiotoma hastula (reeve, 1843), a small very distinct species, and lophiotoma deshayishii (doumet, 1839) also fit the description above for group iii species, but they are sufficiently distinctive from l. unedo or l. indica that they have long been recognized as different taxa, and will not be further discussed here. the forms assigned to l. unedo in most collections and publications also include group iv forms as noted above, but these are separable by the criteria described. however, even among “early gemmate” forms, there is still a very confusing array of morphologically diverse forms. most material labeled “lophiotoma unedo” in collections and recent books is the large japanese form. kiener’s type of the species is probably from indonesia and is one of the forms more poorly represented in most present-day collections; this form was not discussed nor figured when powell wrote his monograph on the turrinae. the author feels that there is uncertainty as to whether the typical japanese form is conspecific with the forms from indonesia that are most similar to the type illustration. although this is the form figured as l. unedo in most publications, the japanese form is at the minimum atypical (when compared to the available kiener figures) and may not even be the same species. we recognize one group iii form, lophiotoma capricornica, new species, as a local endemic collected offshore in queensland; the presence of fairly typical l. unedo in the same general locality justifies this separation. lophiotoma (unedogemmula) unedo lophiotoma unedo is a confusing taxon; even when all of the “mostly gemmate” forms are removed, a wide variety of forms are potentially assignable to l. unedo, extending geographically from southern japan to new south wales, and from fiji to southern india and the arabian peninsula. kiener’s type specimen, which apparently has not been located (r. kilburn, personal communication), is probably from indonesia. 16 olivera description shell large, fusiform with a deeply “u”-shaped posterior sinus. adult specimens have 12-13 teleoconch whorls in addition to the polygyrate protoconch (that is generally broken or corroded in most specimens). there are about three postnuclear whorls that are gemmate, and an additional 2-3 that are transitional (where the gemmules are bisected and get progressively weaker). on the spire whorls, there is a maculated subsutural rib (or fused pair of ribs), followed by a concave area with 4-5 unmaculated threads and a peripheral keel with two ribs, also strongly maculated. on the last whorls before the body whorl, 1-2 additional primary cords are emergent. on the body whorl, there are distinctive zones: the strongly maculated subsutural region, followed by an unmaculated or much more lightly maculated concave region, the maculated periphery, a darker area with 4-5 smaller maculated cords which comprise most of the body whorl. from the base through much of the siphonal canal, there is a generally lighter zone with 5-8 primary cords. the tip of the canal is usually white, bordered by a dark brown area, generally significantly darker than the rest of the canal. the canal is broad and proportionally shorter than in other closely related forms. there are well-developed, raised revolving threads visible within the aperture. the broader angle of the spire, the relatively shorter and wider canal, and the raised threads in the aperture are features that distinguish the forms that we assign to l. unedo from other forms in the complex. a number of specimens from indonesia seem close to kiener’s type figure. these are generally thinner shells —the example shown in fig. 5 (specimen 2) is from makassar, indonesia. similar shells but lighter in color with more prominent subsutural cords have been collected in aligway island, and by the french expedition to lubang island, philippines, in 180-200 m. these appear to be the closest morphological variant to the original kiener illustration. variations most recently collected specimens that we assign to this species have much thicker shells than the typical form. in some specimens, the subsutural rib is massive, and the sculpture of spire whorls is dominated by the subsutural rib and the immediately adjacent peripheral keel of the preceding whorl, both maculated (fig. 5, specimen 1). smaller specimens with massive subsutural cords have been collected by trawlers off kerala, india. some australian specimens examined seem intermediate between other forms of l. unedo and the description of unedogemmula binda from new south wales. the type of binda has the same welldeveloped, raised revolving threads visible within the aperture, and a “more obtusely rounded periphery with a weaker sinus rib and a shallower shoulder concavity, as well as more numerous basal spirals”. some australian specimens have more numerous basal spirals on the body whorl than other forms of unedo, but do not have the rounded periphery of the binda holotype specimen. until more material is available, we regard binda as an extreme form of lophiotoma unedo from southeastern australia. l.c.f. unedo, variant/form 1, “typical japanese form” the japanese specimens that powell figured as l. unedo are quite different from the kiener type, and may or may not be conspecific. since this form is so well represented in collections, we discuss it in more detail than other varieties. description (adapted from powell, 1964) adult shell, 75-105 mm in height, solid, fusiform with a tall spire, and long anterior canal. spire a little more than the height of the aperture + canal; spire angle 3035. sculptured, with two narrow, sharply raised spiral cords submargining the suture, followed by a bimarginate sinus rib which forms the peripheral angle at a little below median whorl height, then 2-3 primary spiral cords emergent between the sinus rib and the lower suture. on the base and neck there are about 17 primary spirals. on the concave shoulder area, there are from 4-7 crisp secondary spirals. all the interspaces carry from 1-3 spiral threads. whorls, 12-13, exclusive of a multispiral narrowly conic smooth protoconch of 3-3.5 whorls, terminating in a half whorl of brevic 17 larger forms in lophiotoma axials. the postnuclear whorls bear distinct gemmules on the peripheral carina. color pattern consisting of small reddish-brown dots, diffused into slightly larger maculations around the subsutural collar and the sinus rib, the whole loosely and irregularly connected axial by flexious, pale reddish-brown streaks that follow the successive grow lines. brown color in interior, aperture white. in occasional specimens, the sinus rib carries a third but weaker spiral than the two margining ones. operculum leaf-shaped with an apical nucleus. most specimens of this form are described from southern japan at depths of 50 fathoms. a few specimens of this variant (“japanese form”) are labeled as being collected from northern australian localities; these are all specimens from commercial dealers, and the localities need to be verified. however, until there is a more reliable characterization of these forms, we feel that it would be premature to separate them at the subspecific or specific level, or to conclude that they are conspecific. lophiotoma capricornica, new species this form seems most closely related to the “typical japanese form” described above. both have a more slender shell, with a proportionally longer aperture. like l. unedo, as well as l.c.f. unedo, variant/form 1 (“typical japanese form”), there is the typical white tip and brown marking bordering the white-tipped canal. however, l. capricornica is much thinner and distinctly different in its sculpture. description the shell is much whiter, thinner, and narrower than other related group iii forms conventionally assigned to the l. unedo complex, with 10-11 whorls. the subsutural area is defined by a rib that is faintly maculated some distance from the suture, followed by a slightly concave region with 3-4 white primary fig. 5. group iii forms of lophiotoma. (top row) l. unedo, different forms—specimen 1, variety with massive subsutural cords, pamilacan island, bohol, philippines; specimen 2, typical form, makassar, indonesia; specimen 3, kii, japan; specimen 4, large thick variety from sindangan, zamboanga del norte, philippines. (middle row) l.c.f. unedo, variant/form 1 (“typical japanese form”)—specimen 5, from tosa bay, japan; specimen 6, from the philippines. (bottom row) l. capricornica— specimen 7, holotype; specimen 8, paratype 6; specimen 9, paratype 2; and specimen 10, paratype 1. all specimens of l. capricornica were trawled in deep water off queensland, probably in the capricorn islands. 8 9 10 7 1 2 3 4 5 6 18 olivera threads. the peripheral keel has two cords and narrow brown axial markings. the primary cords are relatively fine and are mostly white. there is an area of two cords at the base that are more highly maculated than the other spiral cords on the body whorl. the remainder of the base and the canal is white except at the tip where a dark diagonal brown marking, typical of many forms in the l. indica/l. unedo complex, is found. in most specimens, this diagonal dark brown band is striking against the very white color of the background and the relatively sparse maculations in the rest of the shell. all of the specimens examined were collected off queensland, and trawled by fishing boats between 40120 fathoms. most were reportedly trawled near lady musgrave island. freshly collected specimens of l. capricornica have an attractive silky appearance. the species does not appear to have any raised lines inside the aperture, as described for other forms of group iii lophiotoma. type material the holotype will be deposited at the mnhn, paris. paratypes will be deposited at amnh, new york; usnm, washington; western australian museum, perth; cfm, chicago; lacm, los angles; and ansp, philadelphia. data on the types are given in the appendix. group iv: “mostly gemmate” l. unedo-like forms the group iv forms have gemmules beyond the early postnuclear whorls, typically up to the penultimate whorl. these comprise a confusing set of morphologically diverse deep-water forms, and there is almost certainly more speciation in the group than will be formally recognized here. some varieties in this group are very similar to the species in group iii, but distinguishable by the presence of gemmules on the peripheral carina of most whorls. a characteristic feature is that markings on the peripheral carina are restricted to the area between gemmules; these can be very regular in the earlier spire whorls, but invariably become irregular (and sometimes absent) in the last two whorls. another general feature of all forms in this group is the presence of irregular maculations in the subsutural area. forms in this group have been collected in japan, the philippines, western australia, new caledonia, madagascar, and reunion. there is considerable morphological diversity, and how the various forms are related to each other requires additional analysis of more specimens. we describe a new species that is represented by many specimens collected from the central philippines, l. panglaoensis, and have deliberately restricted the type material to a narrow morphological range from a narrow geographic locality. three other distinctive forms described below may or may not be conspecific with l. panglaoensis. the group iv specimens from deep water off madagascar appear sufficiently distinct to merit separation at the species level, and are of particular interest since they suggest a close relationship between group iv lophiotoma and certain gemmula spp., and thus provide the basis for a hypothetical evolutionary scenario (discussion). although this group is relatively poorly represented in most collections, the deep water expedition carried out by the paris museum, and gill-net collecting in the central philippines suggest that group iv forms are common at depths of 200-600 m, and that there may be a large number of yet-undiscovered species belonging to this general division of lophiotoma. lophiotoma panglaoensis, new species description most specimens examined are medium-sized for the genus (45-65 mm); the protoconch is not preserved in any of the type specimens. there are 10-11 teleoconch whorls. the subsutural region has an irregularly maculated spiral cord (split into two in some specimens). a white concave area with about five white spiral cords separates the subsutural region from the peripheral carina. the peripheral carina has two cords running parallel and is characterized by the presence of numerous gemmules up to the penultimate whorl in most specimens. there is a distinctive brownish stain (varying from orange-brown to purplish-brown) in the interstices between the gemmules (which are whitish); this is typically on the edge of the gemmule away from the 19 larger forms in lophiotoma aperture. the stain between the gemmules is more regularly spaced in the earlier whorls, becoming more irregular, particularly in the body whorl. the frequency and intensity of interstitial gemmule coloration shows considerable variation; these can be much lighter in some specimens, and even absent. on the later teleoconch whorls, there are between 1-3 primary cords emergent. these whorls have axial brown markings, that originate at the interstitial brown area of the peripheral rib and run parallel to the growth line. in the body whorl, there are approximately 13-16 primary cords that continue to the siphonal canal with secondary threads in between; in many specimens, the base and posterior canal are generally lighter in color than the region of the body whorl next to the peripheral canal. the very tip of the canal is whitish, with a diffuse brownish band bordering it. the holotype and some paratypes are shown in fig. 6a, top row, and fig. 6b, specimens 1 and 2. type material the holotype and paratypes 1-10 were collected off panglao island, bohol, philippines, by tangle nets in about 200 fathoms (the major commercial product of this fishery is murex alabaster, an attractive ornamental species). one specimen in the los angeles county museum, collected by james norton in july 1966, belongs to this series (paratype 11). this specimen, la cmnh 75849 was collected between 52-60 fathoms on sand and mud bottom, east of talaga, batangas bay, southwest luzon, philippines. the other paratypes include a series (12-14) collected from lubang island, off mindoro by the mnhn, paris. thus, the type series includes specimens from the central philippines (panglao island) to southwest luzon island. discussion the type material was deliberately restricted to specimens that closely resemble the holotype specimen collected off panglao island. a specimen in the collection of the american museum of natural history from tosa, japan appears to be conspecific with l. panglaoensis, strongly suggesting that the species occurs to southern japan. the distribution of shell morphology found in the specimens from lubang expeditions of the paris museum suggests that there may be two distinctive forms of l. panglaoensis, or two sympatric species. the lubang form that appears to be typical l. panglaoensis (paratypes 12-14) was collected between 190-210 m; some morphologically distinct specimens (see l.c.f. panglaoensis, variant/ form 2 below) were apparently collected in the same general area. the material from panglao is presumably from even greater depths; most panglao island specimens are thinner, more whitish and slender compared to the shallower lubang island material. all of the type material for l. panglaoensis is in the smaller size range of group iv forms; some forms belonging to this group are the largest and heaviest lophiotoma yet found, particularly the western australian material. the forms potentially related to l. panglaoensis are summarized below. distinctive forms related to l. panglaoensis several forms collected in the same central philippines region as l. panglaoensis are potentially variants of l. panglaoensis, but were deliberately excluded from the type material because of the possibility that they may be taxonomically distinct; two of the most distinctive forms, l.c.f. panglaoensis, variant/forms 1 and 2, are described below. the western australian material (l.c.f. panglaoensis, variant/form 3) is very distinctive, but at this time it is not possible to judge whether it is separable at the species or subspecific level, or an unusually large albinistic variety, conspecific with l. panglaoensis. l.c.f. panglaoensis, variant/form 1, broad thin form this is an extremely thin shell, much finer but with a broader spire than the type material, which is shown in fig. 6a, specimen 5. l.c.f. panglaoensis, variant/form 2 this form has much darker, larger purplish-brown interstitial markings than the typical l. panglaoensis, 20 olivera fig. 6. group iv lophiotoma species and distinctive variants. fig. 6a. (all specimens figured approximately the same size, not proportional to natural size). a1-3 lophiotoma panglaoensis, new species. a1 and a2 are the holotype, a3 is paratype 12. a4-6 distinct variant/forms potentially related to l. panglaoensis. a4 is the dark brown form of l.c.f. panglaoensis, variant/form 2; a5 is the broad thin form of l.c.f. panglaoensis, variant/form 1; and a6 is the western australian form of l.c.f. panglaoensis, variant/form 3. a7-8 l. madagascarensis, two views of the holotype. fig. 6b. the same set of forms (with some different specimens) illustrated showing relative size. b1-4: b1 (holotype) and b2 (paratype 4) are l. panglaoensis; b3 and b4 are l.c.f. panglaoensis, variant/form 2, extreme morphs. note the much more prominent spots between gemmules in the peripheral carina compared to typical l. panglaoensis. b5-7 l.c.f. panglaoensis, variant/form 1 (same specimen as in a5). b6 and b7 are l. madagascarensis. b6 is the holotype from madagascar, b7 is a variety from reunion. b8-9 l.c.f. panglaoensis, variant/form 3, both collected off western australia by scampi fishermen in very deep water. b9 is from the american museum of natural history collection (amnh 223170). 1 2 3 4 5 6 1 2 3 4 5 6 7 7 8 98 with thicker, sturdier shells, deeper brown in background color in the body whorl and much broader (fig. 6a, specimen 4 and fig. 6b, specimens 3 and 4). shells of this form can be much larger than typical l. panglaoensis (fig. 6b) and are apparently also found in japan. in some museum collections, such specimens are labeled “unedogemmula unedoides”; it appears that this is a manuscript name by kuroda that was never 21 larger forms in lophiotoma published. this form can be morphologically very similar to some specimens of l. unedo, but can be distinguished by the persistence of gemmules to the penultimate whorl. it is possible that there is more than one taxon that we have included in this group. l.c.f. panglaoensis, variant/form 3 a number of large lophiotoma specimens were collected in deep water, typically 300-400 m off western australia by scampi fishermen. most of these differ from the typical l. panglaoensis in being larger, generally lighter in color (many specimens are pure white), and in the absence of strong subsutural sculpture. as described above, there is variation in coloration even in typical l. panglaoensis, but the much larger average size and the absence of sculpture in the subsutural region distinguishes most of the western australian material from typical l. panglaoensis. a few western australian specimens (fig. 6b, specimen 8 for an example) have the general outline of l. panglaoensis (albeit broader and larger); on these specimens, light interstitial maculations can be seen. however, the majority of specimens are much larger, whiter in color, and have a much weaker subsutural cord than typical l. panglaoensis (fig. 6a, specimen 6, and fig. 6b, specimen 9). we think it likely that the western australian form deserves at least subspecific separation from l. panglaoensis. there is considerable morphological variation in the western australian material, and it is conceivable that these comprise more than one species. lophiotoma madagascarensis, new species in addition to l. panglaoensis and the distinctive forms described above, there is a group iv species collected in the western indian ocean in relatively deep water. this form is quite distinct from all of the other variations and forms described above, and appears to be clearly separable from l. panglaoensis. description the protoconch is polygyrate, and is followed by 10 teleoconch whorls. there are two thin subsutural cords irregularly maculated followed by approximately 5 fine white threads, with the subsutural maculations extending out to the threads to a varying extent in different specimens. the peripheral carina has gemmules, and between the gemmules, the cord is stained regularly in the earlier whorls with a deep purple-brown color. on the body whorl, the gemmules become obsolete and the maculations on the peripheral cord become very irregular in most mature specimens. there are 13-14 primary cords on the body whorl with 1-2 threads in between each primary cord. the ground color is a light off-white, with the very tip of the canal somewhat whiter than the rest of the body whorl. the spiral cords are sparsely and irregularly maculated with the same purplish-brown color as the periphery, and are organized axially on the body whorl giving an intermittent pattern of purplish brown that is most intense between the third and fifth primary cord from the peripheral carina, and becomes much lighter at the base and siphonal canal. most of the specimens examined were collected off madagascar, in 300-460 m. a specimen collected in reunion from an expedition in 1982 in 450-480 m (md32/reunion, st. cp179, 21° 03 s 55° 10 e) is more finely sculptured than the specimens from madagascar, but is likely to be conspecific with l. madagascarensis, with the maculations on the peripheral carina narrower and less prominent than in the madagascar specimens. the protoconch is polygyrate and there are 13 teleoconch whorls, with the last two lacking gemmules. discussion l. madagascarensis can be distinguished from l. panglaoensis by the thinner shell, the more triangular shape of the whorls resulting in a broader spire angle coupled with a constricted suture, and by the bolder maculations on the peripheral carina. examples of this species are illustrated in fig. 6a, bottom row, and fig. 6b, specimens 6 and 7. discussion larger lophiotoma specimens are generally identified either as lophiotoma indica or lophiotoma 22 olivera (unedogemmula) unedo in most publications and collections. in this work we have examined all forms that were accessible to us, which are conventionally assigned to the two taxa. a total of 21 distinctive forms can be differentiated morphologically; all of these have previously been identified as “indica” or “unedo”. of the 21 forms, in the opinion of the author, at the present time nine are sufficiently distinctive to separate at the species level (seven new species are described above), and one new subspecies is proposed. the assignment of the 11 remaining forms needs further evaluation; molecular data may be particularly useful in these cases. any assessment of the evolutionary relationships between various forms of the lophiotoma spp. described in this manuscript must be clearly regarded as preliminary. however, there is a clear gradient from gemmula-like to true l. indica-like forms. thus, the deep-water forms related to l. panglaoensis (group iv) are the most gemmula-like of this series; the forms in group iii related to l. unedo are intermediate in having the first few postnuclear whorls gemmate, followed by a transitional region with the larger whorls having gemmules largely absent (this group roughly corresponding to unedogemmula). forms such as l. friedrichbonhoefferi (group ii) represent a further transition, in having relict gemmules in the first postnuclear whorl. finally, there are the forms in group i that all have blunt paucispiral protoconchs with gemmules absent from all postnuclear whorls (a group that corresponds to powell’s concept of lophioturris). the gradient from gemmula-like to lophiotoma-like forms roughly parallels the bathymetric range of these forms. the more highly gemmate forms such as l. panglaoensis are collected in deeper water, and only the group i forms such as l. indica are found in shallow water (in certain cases in intertidal habitats). like all generalizations in biology, there are exceptions: l. indica queenslandica is a group i form that is apparently only found in deeper water. this range of forms from gemmula-like to lophiotomalike suggests a working hypothesis for the evolution of the larger turrinae. the longer geological history of gemmula, and its much wider biogeographic distribution (with living forms in the caribbean and eastern pacific, and a richer early tertiary fossil record) compared to the two other major groups of indo-pacific turrinae (turris and lophiotoma, that only have a fossil record back to the miocene) makes a gemmula-like ancestor likely for both lophiotoma and turris. there are several reasons to suggest that the closest group within gemmula to this branch of lophiotoma is the species complex related to g. kieneri. the stained interstices between gemmules that is characteristic of group iv species (l. madagascarensis and l. panglaoensis), as well as the irregularity of maculations on the subsutural cord are features shared by l. madagascarensis, l. panglaoensis, and g. kieneri. some unusual specimens of g. kieneri even show the irregular maculations in both the peripheral carina and the primary cords on the body whorl characteristic of group iv lophiotoma. a putative common ancestor of the l. madagascarensis/ l. panglaoensis group iv complex and g. kieneri may have resembled certain forms in the g. kieneri complex. it is notable that the specimens of g. kieneri that are at the extreme edge of the range, such as northern mozambique and new caledonia, are much more similar to the form in hawaii given the name gemmula interpolata (powell, 1966), a species closely allied to g. kieneri. a comparison of these forms with l. madagascarensis is shown in fig. 7. it seems reasonable to suggest that an ancestral form similar to these morphs within the g. kieneri complex, as well as the deep-water l. madagascarensis could have been the progenitor of both the g. kieneri complex of species (including g. kieneri and g. interpolata) as well as the group iv lophiotoma species discussed above. some specimens of l. madagascarensis show a strong affinity for some group iii forms, particularly l. capricornica. thus, deriving group iii from the putative gemmula ancestor would involve a more extensive loss of gemmules from the peripheral carina with adult development. the group ii species above, with forms like l. bisaya and l. friedrichbonhoefferi, were postulated in the first paper of the series as being a potential ancestral link from gemmula to certain types of turris species (such as turris pagasa (olivera, 1999)). thus, one could imagine that the evolution of both lophiotoma and turris from a gemmula-like ancestor may have involved ancestral forms similar to living group ii, iii, 23 larger forms in lophiotoma and iv forms discussed above. although a progression would appear logical from gemmula spp. with gemmules, to group iv/iii forms with gemmules in some whorls, to group ii/i forms with gemmules mostly absent, we suspect that most group ii/i forms are more likely independently derived from a different g. kieneri/ interpolata-like ancestor. the regular body maculations of some forms of g. interpolata are consistent with this branch of gemmula being most closely related to group i and ii lophiotomas as well. clearly, further characterization of the biology, anatomy, and molecular biology of the living species will provide data that would be relevant to an examination of this general evolutionary scheme for the turrinae. this hypothesis makes several predictions: (1) that g. kieneri/g. interpolata will prove to be genetically closer to l. madagascarensis/l. panglaoensis and even l. friedrichbonhoefferi than any other clade in gemmula; fig. 7a. series illustrating one branch of gemmula related to group iii and group iv lophiotomas. the specimens are not figured to scale in panel a. from left to right: a1 l.c.f. unedo, variant/form 1 (“typical japanese form”); a2 l. capricornica from queensland, australia; a3 l. madagascarensis variety, reunion; a4 l. madagascarensis, holotype, madagascar; a5 g. interpolata, oahu, hawaii; and a6 g. kieneri, mozambique variety. fig. 7b. some of the same species shown to scale with additional varieties of some of the species illustrated. from left to right: b1 l.c.f. unedo, variant/form 1, “typical japanese form” (group iii); b2 l. capricornica, queensland, australia (group iii); b3 l. madagascarensis (group iv) madagascar; b4 l. madagascarensis variety (group iv), reunion; b5 and b6 g. interpolata, oahu, hawaii (note that the shape of these specimens is not dissimilar from some of the group iii lophiotomas); b7, b8, and b9 are various varieties of g. kieneri taken from mozambique, philippines, and japan, respectively. 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 24 olivera and (2) that these particular gemmula may even be genetically more allied to the group of lophiotoma species that is the subject of this paper than to some other forms presently included in gemmula. thus, this is an evolutionary hypothesis that in principle can be evaluated as more detailed morpho-anatomical, molecular and biochemical data become available for the relevant taxa. acknowledgments the author would like to thank dr. philippe bouchet and philippe maestrati for the loan of specimens from the muséum national d’historie naturelle (mnhn), paris; specimens used in this study are referred to frequently in the text of the paper. i would also like to thank jay cordiero, formerly collections manager of the american museum of natural history, and dr. donn tippett, who guided me through the turrid collection at the national museum of natural history, smithsonian institution, washington. the advice and input of richard kilburn were essential. i would like to thank dr. lourdes j. cruz and noel saguil for their help in specimen collection. many of the specimens examined were collected as part of an exploratory study supported by a program project grant on venoms, gm 48677 from the national institute of general medical sciences, united states public health service. specimens were photographed by kerry matz, who also laid out the color plates. the micrographs of the protoconchs shown in fig. 2 were taken by nancy kurtzeborn. i am grateful to her for this contribution, as well as her patience and expertise in typing the entire manuscript through innumerable drafts. references bosch, d.t., s.p. dance, r.g. moolenbeek, & p.g. oliver, 1995. seashells of eastern arabia. motivate publishing, dubai, united arab emirates. 296 pp. bouchet, p., 1990. turrid genera and mode of development: the use and abuse of protoconch morphology. malacologia. 32: 69-77. hedley, c., 1922. a review of the australian turridae. rec. austr. mus. 13: 213-259. higo, s., p. callomon, & y. goto, 1999. catalogue and bibliography of the marine shell-bearing mollusca of japan. elle scientific publications, osaka, japan. 1173 pp. kilburn, r.n., 1983. turridae (mollusca: gastropoda) of southern africa and mozambique. part 1. subfamily turrinae. ann. natal mus. 25: 549-585. olivera, b.m., 1999. the subfamily turrinae in the philippines: the genus turris (röding, 1798). phil. j. sci. 128: 295-318. olivera, b.m., 2002. conus venom peptides: reflections from the biology of clades and species. annu. rev. ecol. syst. 33: 25-42. olivera, b.m., 2002. the gastropod genus xenuroturris (iredale, 1929) evaluated and a new turrid, lophiotoma olangoensis, described from the central philippines. science diliman. 14(2): 40-50. powell, a.w.b., 1964. the family turridae in the indopacific. part 1. the subfamily turrinae. indo-pacific mollusca. 1: 227-346. powell, a.w.b., 1966. the molluscan families speightiidae and turridae. bulletin of the auckland institute and museum 5: 1-184, +23 plates. röding, p.f., 1798. museum boltenianum pars secunda continens conchylia. j.c. trapp, hamburg. springsteen, f.j. & f.m. leobrera, 1986. shells of the philippines. carfel shell museum, manila, philippines. 377 pp. taylor, j.d., y.i. kantor, & a.v. sysoev, 1993. foregut anatomy, feeding mechanisms, relationships, and classification of the conoidea (= toxoglossa) (gastropoda). bull. nat. hist. mus. lond. (zool.) 59: 125-170. 25 larger forms in lophiotoma lophiotoma indica queenslandica, new subspecies holotype 47.3 15.6 19.1 queensland, australia paratype #1 62.8 19.3 36.6 cape moreton, queensland, australia paratype #2 67.0 21.2 41.8 swain reefs, queensland, australia paratype #3 41.6 14.2 24.7 swain reefs, queensland, australia1 paratype #4 57.0 19.0 32.8 queensland, australia paratype #5 60.8 19.3 35.2 queensland, australia paratype #6 77.4 25.1 42.8 queensland, australia paratype #7 72.9 24.7 42.3 queensland, australia paratype #8 64.8 21.0 37.2 australia paratype #9 42.7 15.2 24.5 queensland, australia paratype #10 36.8 12.4 21.8 australia paratype #11 53.3 16.9 30.8 queensland, australia paratype #12 68.0 22.1 41.0 cape moreton, queensland, australia paratype #13 63.5 20.4 35.9 swain reefs, queensland, australia paratype #14 68.5 22.8 34.7 swain reefs, queensland, australia paratype #15 71.7 24.6 44.8 swain reefs, queensland, australia paratype #16 75.0 22.9 46.3 swain reefs, queensland, australia paratype #17 68.5 21.1 38.1 swain reefs, queensland, australia2 lophiotoma freidrichbonhoefferi holotype 35.8 10.8 18.9 aligway island, philippines paratype #1 39.0 11.4 20.0 aligway island, philippines paratype #2 35.6 10.4 18.9 aligway island, philippines paratype #3 39.0 11.6 20.1 aligway island, philippines paratype #4 35.4 10.3 19.0 aligway island, philippines paratype #5 41.8 12.2 21.6 aligway island, philippines paratype #6 31.0 10.2 16.1 aligway island, philippines paratype #7 47.7 13.4 24.5 aligway island, philippines paratype #8 44.1 12.3 23.7 aligway island, philippines paratype #9 48.2 13.1 24.3 aligway island, philippines paratype #10 52.3 14.5 26.8 aligway island, philippines paratype #11 35.9 11.1 18.5 aligway island, philippines paratype #12 34.7 10.7 18.8 aligway island, philippines paratype #13 28.7 11.3 20.0 aligway island, philippines paratype #14 36.6 10.9 18.5 aligway island, philippines paratype #15 31.9 9.7 16.9 aligway island, philippines paratype #16 18.7 6.6 10.0 aligway island, philippines paratype #17 37.8 11.3 19.4 aligway island, philippines paratype #18 36.0 11.1 17.7 aligway island, philippines paratype #19 35.2 10.5 17.7 aligway island, philippines paratype #20 34.6 10.6 17.4 aligway island, philippines paratype #21 32.7 9.9 17.0 aligway island, philippines paratype #22 33.6 10.1 17.6 aligway island, philippines paratype #23 30.6 9.9 16.3 aligway island, philippines paratype #24 29.9 9.5 15.4 aligway island, philippines paratype #25 24.2 8.1 13.0 aligway island, philippines paratype #26 35.6 10.7 18.1 aligway island, philippines paratype #27 35.7 11.5 18.6 aligway island, philippines paratype #28 36.6 10.4 19.1 aligway island, philippines paratype #29 34.2 10.6 17.4 aligway island, philippines paratype #30 30.8 10.1 16.7 aligway island, philippines paratype #31 33.0 10.3 17.5 lubang island, philippines (mnhn)3 paratype #32 24.9 8.3 13.4 lubang island, philippines (mnhn)3 summary of types of new lophiotoma species and subspecies. appendix (prepared by nancy kurtzeborn). species measurement (mm) height widtha apertureb locality 26 olivera lophiotoma bisaya holotype 67.9 21.0 35.6 aligway island, northern mindanao, philippines paratype #1 51.3 16.2 25.9 aligway island, northern mindanao, philippines paratype #2 54.5 16.1 27.5 aligway island, northern mindanao, philippines paratype #3 59.1 16.6 31.7 aligway island, northern mindanao, philippines paratype #4 58.1 19.4 32.2 aligway island, northern mindanao, philippines paratype #5 64.9 19.5 33.7 aligway island, northern mindanao, philippines paratype #6 47.1 14.6 24.4 aligway island, northern mindanao, philippines paratype #7 70.7 20.2 37.9 aligway island, northern mindanao, philippines paratype #8 63.3 18.8 33.4 aligway island, northern mindanao, philippines paratype #9 80.8 23.8 40.0 aligway island, northern mindanao, philippines paratype #10 61.2 18.2 33.5 aligway island, northern mindanao, philippines paratype #11 73.6 22.6 37.2 aligway island, northern mindanao, philippines paratype #12 61.3 17.7 32.9 aligway island, northern mindanao, philippines paratype #13 71.4 21.3 37.3 aligway island, northern mindanao, philippines lophiotoma tayabasensis holotype 85.0 24.8 44.7 tayabas bay, philippines paratype #1 73.3 22.0 40.4 tayabas bay, philippines paratype #2 83.8 25.5 46.5 tayabas bay, philippines paratype #3 99.2 28.4 52.3 probably from tayabas bay, philippines paratype #4 94.7 27.4 49.5 probably from tayabas bay, philippines paratype #5 88.1 27.7 49.1 probably from tayabas bay, philippines paratype #6 92.8 27.7 48.7 probably from tayabas bay, philippines paratype #7 98.1 28.4 54.5 probably from tayabas bay, philippines paratype #8 76.8 23.4 42.6 tayabas bay, philippines paratype #9 93.8 27.7 46.5 probably from tayabas bay, philippines paratype #10 96.3 28.9 52.8 probably from tayabas bay, philippines paratype #11 93.1 28.0 47.5 probably from tayabas bay, philippines paratype #12 95.4 27.8 48.3 probably from tayabas bay, philippines paratype #13 87.3 27.1 47.5 probably from tayabas bay, philippines paratype #14 98.8 29.8 53.6 probably from tayabas bay, philippines paratype #15 86.9 26.4 45.5 between mactan and bohol island, philippines paratype #16 65.4 19.2 37.6 between mactan and bohol island, philippines paratype #17 106.9 31.7 57.5 probably from tayabas bay, philippines paratype #18 91.0 27.7 48.2 probably from tayabas bay, philippines paratype #19 87.2 26.7 47.0 probably from tayabas bay, philippines paratype #20 87.1 27.3 47.9 sindangan, dipolog, philippines paratype #21 85.6 25.5 45.5 probably from tayabas bay, philippines paratype #22 93.6 28.0 51.3 probably from tayabas bay, philippines paratype #23 76.6 22.8 40.6 between mactan and bohol island, philippines paratype #24 97.0 28.5 52.6 between mactan and bohol island, philippines paratype #25 61.7 20.1 34.7 between mactan and bohol island, philippines paratype #26 83.2 23.9 45.9 between mactan and bohol island, philippines paratype #27 74.8 22.0 40.1 between mactan and bohol island, philippines paratype #28 69.0 22.0 39.8 between mactan and bohol island, philippines lophiotoma dickkilburni holotype 63.4 19.2 30.1 tongaat, natal, south africa paratype #1 69.8 21.4 34.5 angoche, northern mozambique paratype #2 56.0 18.0 27.2 tongaat, natal, south africa paratype #3 46.2 16.0 23.4 tongaat, natal, south africa (broken) paratype #4 43.8 13.2 11.6 tongaat, natal, south africa paratype #5 53.9 17.9 19.0 tongaat, natal, south africa species measurement (mm) height widtha apertureb locality 27 larger forms in lophiotoma paratype #6 69.6 20.3 34.3 mozambique (amnh 162582)4 paratype #7 73.0 24.3 35.8 mozambique (amnh 162582)4 paratype #8 54.2 18.1 28.3 natal, south africa (amnh 174780)5 lophiotoma capricornica holotype 60.7 19.3 33.9 queensland, australia paratype #1 73.3 21.3 41.9 queensland, australia paratype #2 60.9 21.1 33.5 queensland, australia (canal damaged) paratype #3 64.0 19.2 34.5 queensland, australia paratype #4 54.3 17.6 29.7 queensland, australia paratype #5 58.2 19.1 32.6 queensland, australia paratype #6 54.7 17.6 31.8 queensland, australia lophiotoma panglaoensis holotype 54.2 17.2 27.9 panglao, bohol island, philippines paratype #1 60.1 18.5 31.2 panglao, bohol island, philippines paratype #2 46.5 15.1 22.3 panglao, bohol island, philippines paratype #3 59.3 18.5 28.4 panglao, bohol island, philippines paratype #4 52.7 16.5 25.1 panglao, bohol island, philippines paratype #5 45.2 15.0 22.9 panglao, bohol island, philippines paratype #6 54.2 17.2 26.2 panglao, bohol island, philippines paratype #7 49.5 15.5 25.6 panglao, bohol island, philippines paratype #8 73.6 24.7 38.8 panglao, bohol island, philippines paratype #9 64.2 21.2 34.1 panglao, bohol island, philippines paratype #10 64.8 20.4 34.7 panglao, bohol island, philippines paratype #11 46.6 14.9 23.8 sw luzon island, philippines (lacm 75849)6 paratype #12 49.6 15.7 25.0 lubang island, philippines (mnhn)7 paratype #13 36.8 11.8 18.5 lubang island, philippines (mnhn)7 paratype #14 46.1 14.4 22.5 lubang island, philippines (mnhn)8 lophiotoma madagascarensis holotype 44.7 15.0 23.4 madagascar (mnhn)9 paratype #1 42.8 13.9 22.1 madagascar (mnhn)9 paratype #2 54.4 18.6 28.8 madagascar (mnhn)9 paratype #3 64.9 19.8 32.5 madagascar (mnhn)10 paratype #4 54.9 18.4 28.4 madagascar (mnhn)11 paratype #5 59.1 19.4 29.8 madagascar (mnhn)11 paratype #6 66.1 21.4 32.9 madagascar (mnhn)11 paratype #7 63.5 21.0 33.0 madagascar (mnhn)11 paratype #8 63.2 20.3 32.0 madagascar (mnhn)11 paratype #9 65.2 21.7 32.8 madagascar (mnhn)11 paratype #10 60.8 19.2 30.3 madagascar (mnhn)11 paratype #11 67.7 22.3 32.0 madagascar (mnhn)11 paratype #12 59.0 19.8 29.4 madagascar (mnhn)11 paratype #13 55.9 18.6 28.3 madagascar (mnhn)11 paratype #14 60.8 19.6 30.8 madagascar (mnhn)11 paratype #15 61.9 20.2 30.6 madagascar (mnhn)11 paratype #16 65.1 20.8 34.1 madagascar (mnhn)11 paratype #17 62.1 21.0 31.6 madagascar (mnhn)11 paratype #18 65.5 19.6 32.5 reunion12 awidth of shell at its widest point. blength from posterior of aperture to anterior tip of siphonal canal. species measurement (mm) height widtha apertureb locality 28 olivera 1s7e swain reefs, australia 5/98; trawled 104-105 fathoms between 22°35’ s 153°16’ e and 22° 26’ s 153° 22’ e. 2swain reefs, australia 6/98; trawled in 91 fathoms 22° s to 22° 18’ s. 3musorstom st. 56, lubang island, philippines; 134 m 14° 00’ n 120°20’ e. 420 miles off mouth of limpop river, mozambique; 200-300 m, mud bottom. 5off tugela river mouth, natal, south africa; 100 fathoms, mud bottom. 6east of talaga, batangas bay, batangas province, sw luzon island, philippines; 52-67 fathoms on sand and mud bottom. 7musorstom st. 10; 187-205 m 14° n 120° 19’ e. 8musorstom 2-philippines st. cp66, lubang island, philippines; 192-209 m 14° 00’ n 120°20’ e. 9madagascar; 300 m dragage 3 sables calco-quartzeux 12°36.0’ s 48° 17.3’ e. 10sw madagascar, campagne crevettiere 1986, st. 117; 370 m 22°15’ s 43°07’ e. 11sw madagascar, campagne crevettiere 1986, st. 57; 460 m 22°26’ s 43°06’ e. 12md32/reunion 1982, st. cp179; 450-480 m 21°03’ s 55°10’ e. 7-pee value.pmd “pee value”: storing urine for subsequent dna analysis 54 science diliman (january-june 2019) 31:1, 54-68 “pee value”: storing urine for subsequent dna analysis nelvie fatima jane a. sol iven maria lourdes d. honrado gayvell ine c. calacal maria corazon a. de ungria* natural sciences research institute university of the philippines diliman abstract drug abuse is a prevalent problem in the philippines. with the increased drive to apprehend individuals who partake in the use of illegal drugs, there is a need to re-examine the handling and storage procedures for urine samples, which may be tested to prove or disprove allegations of drug use. with the availability of forensic dna technology as the most powerful tool for human identif ication, the inclusion of dna testing in decision trees used by law enforcers and government laboratories during drug investigations is expected to improve the process of determining actual drug users while promptly addressing allegations of misconduct. because dna testing is a relatively novel procedure previously not considered in drug investigations in the philippines, there is a need to test whether storage procedures for urine that showed positive results allow for subsequent dna testing. samples that test positive for drugs are routinely stored at -20oc for up to one year prior to disposal. in this study, urine dna samples were extracted from 20 male individuals. the samples were subsequently stored at room temperature, 4°c, and -20°c for 2 months and 9 months. this was followed by dna prof iling using the powerplex® 21 system. overall, dna extracted from urine samples stored at cool temperatures (4°c and -20°c) were found to provide more consistent dna prof ile results compared to samples that were stored at room temperature. we propose here a decision tree for drug testing from start to end that should serve as a decision support tool for philippine government agencies engaged in drug investigations. keyword s: dna prof iling, shor t tandem repeats, storage duration, storage temperature, urine dna extraction issn 0115-7809 print / issn 2012-0818 online _______________ *corresponding author n.f.j.a. soliven et al. 55 introduction use of illegal drugs or substances, such as methamphetamine hydrochloride (known locally as “shabu”, “ice”, or “meth”) and cannabis (or “marijuana”), is a prevalent problem in the philippines. in 2017, the philippine dangerous drugs board (ddb 2019) reported 4,045 individuals who were positive for illegal drugs, with the majority of drug users being male (91%). to curb the use of illegal drugs, the philippine government passed republic act 9165 or the “comprehensive dangerous drugs act of 2002” which prescribes the testing of samples from persons applying for certain types of licenses and/or jobs, as well as suspected drug users. in addition, ra 9165 allows the routine testing of samples from students of secondary and tertiary schools, employees of private and public off ices, off icers and members of law enforcement agencies, and candidates for public off ice to be done by government forensic or accredited drug testing laboratories (comprehensive dangerous drugs act of 2002). because of the increased severity of punishment meted out to suspected drug users, there is a need to formulate a decision tree to cover all aspects of testing of human urine samples. the decision tree should start from collection to drug testing until the conduct of additional conf irmatory drug tests and dna testing, when required. institutions mandated by national governments to stop the illegal use of drugs follow different operating procedures. hence, the compatibility of procedures for handling and storing urine, including dna prof iling methods, must be tested in each jurisdiction. for example, in the us and europe, storage temperatures of urine in different laboratories vary from 4°c to -80°c (brinkmann et al. 1992, yasuda et al. 2003; castella et al. 2006; soltyszewski et al. 2006; zhang et al. 2012, devesse et al. 2015). in the philippines, urine samples that test positive for drugs are stored up to one year at -20ºc. this temperature is known to preserve metabolites that can be detected if urine samples undergo further testing in cases of contention (doh 2004). notably, -20ºc is also used for prolonged storage of most biological and dna samples because dna degradation and breakdown of biological material are reduced at this temperature. dna testing of urine samples to evaluate allegation of sample switching has been reported in other countries (junge et al. 2002; soltyszewski et al. 2006). while not all urine samples are required to be authenticated, storage of urine at an ideal condition is still being done should any contention arise (srisiri et al. 2017). we foresee the same happening in the philippines, particularly if the proposed death “pee value”: storing urine for subsequent dna analysis 56 penalty bill for drug users is passed into law. autosomal short tandem repeat (astr) dna prof iling is commonly used to identify human sources of urine samples (junge et al. 2002, marques et al. 2005, thevis et al. 2007), as well as other types of samples that may be submitted as evidence (butler 2010). this study reports the testing of urine samples stored at different temperatures (room temperature, 4ºc, and -20ºc) after two months and nine months, in order to determine if the present storage conditions will allow the future dna testing of urine to resolve allegations of sample switching and laboratory misconduct. materials and methods ethics statement this study was approved by the university of the philippines manila research ethics board (upmreb-2012-0271). urine samples twenty males and f ive females between 18 and 40 years provided urine samples for the study. female urine samples were included as positive controls for dna extraction. female urine is known to contain more cells than male urine (prinz et al. 1993) because of anatomical differences between their urogenital systems. hence, the inclusion of female samples which are expected to yield suff icient dna for dna prof iling served as positive control in case the extraction and genotyping of male urine samples provide negative results. volunteers who were not able to produce at least 160 ml of urine, those with kidney disease or urological conditions, as well as females that were menstruating, were excluded. blood from menstruation contains dna from white blood cells which may inflate dna yield readings. each donor provided at least 160 ml of urine, which was thoroughly mixed by inverting the tubes several times prior to aliquoting into 16 tubes with 10 ml of urine each. four urine aliquots were stored in each of the following conditions: room temperature (rt ), refrigerated temperature (reft; 4°c), and freezing temperature (ft; -20°c). storage temperatures are approximated. samples are stored in one compartment per storage temperature. dna extractions were performed on two aliquots of urine after two and nine months post-collection. n.f.j.a. soliven et al. 57 reference samples to generate the reference dna prof ile per individual, male and female volunteers provided blood samples that were blotted on whatman® fta® cards (ge life sciences). blood on fta® samples were extracted following manufacturer’s protocol. the samples were amplif ied using powerplex ® 21 (pp21) system (promega) and analyzed using applied biosystems® 3500 (ab3500) genetic analyzer (thermo fisher scientif ic) and genemapper® id-x v.1.2 (thermo fisher scientif ic) software. the astr dna prof iles from blood served as reference for comparison with urine dna prof iles in order to assess concordance and % allele recovery. processing of urine samples for each storage condition, 10 ml of urine samples were aliquoted into smaller tubes with approximately 2 ml of urine each then centrifuged at 8000 rpm for 10 minutes to collect the cells. the supernatant was discarded and the precipitate was washed thrice with 200 μl phosphate buffered saline (pbs). dna per volunteer was extracted in duplicate using qiaamp ® dna micro kit (qiagen) following manufacturer’s protocol and eluted using 20 μl of elution buffer. for all extraction events, a negative control without urine sample that served as reagent blank was included. measuring dna quantity and evaluating dna qual ity dna yield and presence of inhibitor were determined using plexor® hy quantitation kit (promega) and the applied biosystem ® 7500 real-t ime pcr system (thermo fisher scientif ic) following manufacturer’s instructions. the pp21 astr kit, ab3500 genetic analyzer, and genemapper® id-x v.1.2 software were used to generate the astr dna prof iles. whenever possible, 0.5 ng of input dna was amplif ied in a 10μl pcr reaction. the exact dna input amounts are shown in figures 1 and 2. to generate the dna prof ile per volunteer, one dna extract that did not show inhibition results from the real-time pcr assay was selected for amplif ication. a total of 120 amplif ications were performed using samples from 20 volunteers stored at three temperatures and at two periods. “pee value”: storing urine for subsequent dna analysis 58 locus drop out allele drop out no drop out figure 1. heat map of the dna amplif ication results using male urine samples after 2 months of storage. visual representation of the autosomal alleles generated using powerplex® 21. pp21 markers are arranged based on the increasing molecular weights (bp) of expected range of amplicon size for a given astr dna marker on the x-axis. storage temperature, sample name, and amount of input dna are shown on the y-axis. samples within a storage temperature group are listed in decreasing amount of dna. ut05, ut06, and ut16 stored at rt, either have no dna or have dna below the assay’s detection limit (approximately 0.001 ng). these samples were amplif ied to test whether the multiplex pp21 reaction is robust enough to amplify dna below the plexor® hy limit of detection. n.f.j.a. soliven et al. 59 locus drop out allele drop out no drop out figure 2. heat map of the dna amplif ication results using male urine samples after 9 months of storage. visual representation of the autosomal alleles generated using powerplex® 21. pp21 markers are arranged based on the increasing molecular weights (bp) of the expected range of amplicon size for a given astr dna marker on the x-axis. storage temperature, sample name, and amount of input dna are shown on the y-axis. samples within a storage temperature group are listed in decreasing amount of dna. ut20 stored at rt, either has no dna or has dna below the plexor® hy assay’s detection limit (approximately 0.001 ng). this sample was amplif ied to test whether the pp21 multiplex reaction is robust enough to amplify dna below the plexor® hy limit of detection. “pee value”: storing urine for subsequent dna analysis 60 dna quality was evaluated based on allele recovery (ar) expressed as percentage of alleles generated over the total number of expected alleles, and peak height ratio (phr) when the dna prof ile yields heterozygous alleles at that dna marker. phr in percent (%) was calculated by dividing the peak height of the allele with a smaller peak by the peak height of the allele with a larger peak, multiplied by 100. statistical data analysis the statistical data analyses were performed using graphpad prism® version 6 (graphpad software, inc). results and discussion y ield of human dna and presence of inhibitors female urine samples, known to have more epithelial cells per unit volume, which served as controls for the dna extraction procedure, produced suff icient amplif iable dna (data not shown). most male urine samples (96%) did not exceed 1 ng/ml of urine (figure 3). statistical analysis using a two-way anova showed that storage time (p = 0.1922) and storage temperature (p = 0.4338) did not independently and interactively affect dna yield. figure 3. average yield (ng dna/ml urine) of human dna extracted in duplicate using the qiaamp ® dna micro kit from 20 male urine samples stored at different conditions. differences in dna yield for samples stored for 2 and 9 months (p = 0.1922) and at different temperature conditions (p = 0.4338) were not signif icant. horizontal lines indicate the median for each data set. n.f.j.a. soliven et al. 61 in addition to estimating dna concentration in real-time, the plexor ® hy quantitation kit which contains an internal pcr control (ipc) was used to test for the presence of pcr inhibitors. inhibitors may result in failed amplif ication, underestimation of dna quantity in real-time pcr assays and/or reduced allele recovery during dna prof iling (krenke et al. 2008, alaeddini 2012). for example, urea in urine is known to inhibit dna amplif ication (khan et al. 1991). in this study, samples stored at rt and ft exhibit less inhibition after 9 months of storage, whereas samples stored at reft display less inhibition after 2 months (table 1). the decrease in the number of samples exhibiting inhibition at rt and ft after 9 months may be due to the breakdown of urea and urinary creatinine over time (spierto et al. 1997, panyachariwat and steckel 2014). however, it is unclear why there appears to be less inhibition at reft at 2 months but the reverse is true at 9 months. a closer study of the biochemical breakdown of inhibitors under different conditions and their effect on downstream dna testing is recommended. dna qual ity urine samples stored at reft and ft were genotyped more successfully compared to samples stored at rt (figure 4). based on a two-way anova, storage temperature was found to be a signif icant source of variation in allele recovery (p<0.0001). at two months and nine months of storage, samples stored at rt have the highest incidences of allele drop-outs. at rt, samples with less than 0.1 ng of input dna in 10 μl volume reaction resulted in <50% allele recovery (figure 4). at reft and ft, samples with 0.1-0.5 ng of input dna have relatively higher rate of recovery at > 80% after 2 months of storage. these results are consistent with the work of ng et al. (2018) that reported over 90% allele recovery from samples stored at 70 days (~2 months) for up to 100 days (~3 months) at 4ºc and -20ºc. after 9 months of storage, samples stored at reft have >70% allele recovery, with a lower allele recovery (> 50%) observed for samples stored in ft. table 1. percentage of samples showing pcr inhibition at 2-month and 9-month storage rt 20 7.5 4°c (reft) 12.5 15 -20°c (ft) 17.5 7.5 * total of 40 male urine extracts tested percentage (%) of samples showing pcr inhibitionstorage temperature 2 months storage* 9 months storage* “pee value”: storing urine for subsequent dna analysis 62 with the amplif ication of low-level target dna (<0.1 ng), more stochastic effects (i.e. , allelic and locus drop-out and gross peak imbalance (phr ≤ 50%)) were observed. allele drop-ins are non-repeated spurious alleles that are detected due to stochastic effects in amplif ication coupled with the sensitivity of the system used to detect alleles. allele drop-outs occur when there is preferential amplif ication of shorter dna regions, which are made more pronounced when the dna template has undergone degradation (caragine et al. 2009; cowen et al. 2011,;alaeddini 2012, gill et al. 2015). overall, allele drop-ins and allele dropouts result in erroneous dna prof iles; hence, the need to evaluate results more carefully. a locus drop-out, which is a no result for the particular dna marker, decreases the informativeness of a urine dna prof ile that will be compared with a reference dna prof ile (e.g. , blood or saliva from the person of interest). in this study, urine samples left at room temperature for 2 months (figure 1) and 9 months (figure 2) exhibited considerable degradation compared to urine samples stored at reft and ft. meanwhile, gross peak imbalance at heterozygous dna markers still provides correct genotype data but is already indicative of the low dna quality of the sample. a phr threshold of 60% is typically used to associate and pair alleles into heterozygote genotypes (butler 2014). we observed that dna from urine stored at reft for 2 months have better phr compared to other storage conditions (figure 5), whereas urine stored at rt exhibited extreme peak height imbalance. a summary of allele drop-outs and peak height ratios are presented in table 2. figure 4. allele recovery of dna from urine at different storage conditions. the colors represent the range of the amount of dna template used in pcr amplif ication (green: 0.5 ng, blue: 0.1-0.49 ng, red: <0.1 ng). dna input of less than 0.1 ng generally results in poor allele recovery regardless of storage condition. allele recovery greater than 50% is observed for reft particularly at 2 months of storage even at < 0.1 ng dna input. storage at ft also resulted in good allele recovery at dna input of 0.1 ng or higher. mean allele recovery for 2 months of storage is at 33.7%, 90.75%, and 60.3% for rt, reft, and ft, respectively. the mean allele recovery for 9 months of storage is 34%, 74%, and 59.3% for rt, reft, and ft, respectively. n.f.j.a. soliven et al. 63 figure 5. average peak height ratio per locus (arranged by increasing molecular weight) for samples stored at different storage conditions. a discontinuous line marks the 60% peak height balance threshold. hence, we support the continued storage of urine samples at ft, as prescribed by the philippine doh (doh 2004), not only to preserve metabolites that serve as targets for drug testing, but also in the preservation of dna for subsequent genotyping, if the need arises. drug testing centers may also opt to store urine samples at refrigerator temperatures, if freezers are unavailable or when funding is limited, provided that drug metabolites are also preserved at this temperature. dna from urine samples stored at 9 months, regardless of temperature, exhibited highly imbalanced phr (< 40%). in forensic investigations, dna prof iles showing variable phr are more diff icult to interpret. our data shows that the storage of urine samples at reft for 2 months is the best condition for preserving the amount and quality of dna for genotyping. hence, there is need for law enforcement and table 2. average allele drop-outs and peak height ratio at each storage temperature and storage period. 2 months rt 66.3 12.0 reft 9.25 62.6 ft 39.7 32.1 9 months rt 66.0 17.6 reft 26.0 36.3 ft 40.7 29.7 storage period storage temperature average allele drop-out (%) average peak height ratio (%) “pee value”: storing urine for subsequent dna analysis 64 forensic drug testing laboratories to evaluate if a shortened storage period for urine samples is warranted. overall, the conduct of all drug tests and dna testing, when needed to evaluate allegations of sample switching or laboratory misconduct, should be performed as soon as possible. this study demonstrates the applicability of dna testing for the identif ication of the source of urine samples in drug use cases when necessary. the result of this evaluation was included in the proposed decision tree for the testing of urine in drug investigations (figure 6). figure 6. proposed decision tree for urine testing in drug investigations. n.f.j.a. soliven et al. 65 conclusion low amounts of dna exhibiting characteristics of inhibition and degradation were extracted from urine samples stored at varying conditions. useful dna prof iles were generated for most of the samples, regardless of storage condition. however, limited dna input (<0.1 ng) was observed to affect allele recovery. male urine contains a small amount of dna; thus, the quality of generated dna prof ile is affected by stochastic effects, inhibition, and degradation brought about by prolonged storage. while storage duration and temperature were not found to be signif icant sources of variation in dna yield, storage temperature evaluated using % allele recovery and peak height balance, was a signif icant source of variation in dna quality. dna extracted from male urine samples stored at cooler temperatures amplif ied better especially at two months of storage. thus, we recommend the storage of at least 10-ml aliquots of urine samples submitted for local drug testing at 4°c or lower and the generation of dna prof iles within this prescribed 2-month period, in order to generate good quality dna prof iles. acknowledgements this research was supported by the outright research grant of the off ice of the vice chancellor for research and development of the university of the philippines diliman (121211 pnse) and the natural science research institute (nsri). the authors thank the staff, particularly ms. miriam ruth dalet, ms. jazelyn salvador, and mr. paul ryan sales, of the nsri dna analysis laboratory for providing technical support. the authors also recognize ms. angelica rose sagum, rn for her assistance in the collection of reference blood samples. lastly, the authors acknowledge the volunteers who provided their samples for this study. references alaeddini r. 2012. forensic implications of pcr inhibition—a review. forensic science international: genetics. 6(3):297-305. brinkmann b, rand s, bajanowski j. 1992. forensic identif ication of urine samples. international journal of legal medicine. 105:59-61. butler j. 2010. fundamentals of forensic dna typing. san diego: academic press. b u t l e r j . 2 0 1 4 . a d v a n c e d t o p i c s i n f o r e n s i c d n a t y p i n g : i n t e r p r e t a t i o n . 1 s t e d . gaithersburg: academic press. p. 38. “pee value”: storing urine for subsequent dna analysis 66 caragine t, mikulasovich r, tamariz j, bajda e, sebestyen j, baum h, prinz m. 2009. validation of testing and interpretation protocols for low template dna samples using ampflstr® identif iler®. croatian medical journal. 50:250-267. castella v, dimo-simonin n, brandt-casadevall c, robinson n, saugy m, taroni f, mangin p. 2006. forensic identif ication of urine samples: a comparison between nuclear and mitochondrial dna markers. international journal of legal medicine. 120:67-72. cowen s, debenham p, dixon a , kutranov s, thomson j, way k. 2011. an investigation of the robustness of the consensus method of interpreting low-template dna prof iles. forensic science international: genetics. 5:400-406. 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philippines. 2002. republic act no. 9165: comprehensive dangerous drugs act of 2002. republic of the philippines. soltyszewski i, pepinski w, dobrzynska-tarasiuk a , janica j. 2006. dna typeability in liquid urine and urine stains using ampflstr sgm plus. advance medical sciences. 51:36-38. spier to f, hannon w, gunter e, smith s. 1997. stability of urine creatinine. clinica chemica acta. 264(2):227-232. srisiri k, jaroenwattana r, panvisavas n, bandhaya a. 2017. optimisation of dna recovery and analysis of urine samples stored on fta card. forensic science international: genetic supplemental series. 6:e520–e522. thevis m, geyer h, mareck u, sigmund g, henke j, henke l, schanzer w. 2007. detection of manipulation in doping control urine sample collection: a multidisciplinary approach to determine identical urine samples. analytical bioanalytical chemistry. 388(7):15391543. yasuda t, iida r, takeshita h, ueki m, nakajima t, kaneko y, mogi k, tsukahara t, kishi k. 2003. a simple method of dna extraction and str typing from urine samples using a commercially available dna/rna extraction kit. journal of forensic sciences. 48(1):1-3. zhang sh, zhao s, zhao zm, li c. 2012. genotyping of urinary samples stored with edta for forensic applications. genetics and molecular research. 11(3):3007-3012. _____________ nelvie fatima jane a. sol iven is a university research associate at the dna analysis laboratory, natural sciences research institute, university of the philippines diliman. she is a graduate student of the institute of biology, up diliman and obtained her bachelor of science degree from the same institute. her research is focused on forensic dna analysis and applications including ancestry informative markers. maria lourdes d. honrado is a graduate of bachelor of science in biology major in biotechnology from rizal technological university in mandaluyong city. she worked as a university research associate in the dna analysis laboratory, natural sciences research institute, up diliman in 2011. “pee value”: storing urine for subsequent dna analysis 68 gay vell ine c. calacal is a senior scientist working in the f ield of forensic dna typing/forensic genetics at the dna analysis laboratory, natural sciences research institute, up diliman. she obtained her master of science degree in microbiology from the institute of biology, up diliman and completed a forensic genetics and dna database technology course from the graduate school of biomedical sciences, university of north texas health science center at for t worth texas. usa. she is credited as one of the pioneers in the development of forensic dna typing research in the country, in the validation of analytical procedures for handling different types of biological samples for forensic applications, developing a system for the collection, handling and analysis of dna evidence, human remains identif ication and generation of the philippine reference population genetic databases. dr. maria corazon a. de ungria <madeungria@up.edu.ph> heads the dna analysis laboratory of the natural sciences research institute, up diliman. her research thrusts include the development of molecular procedures and studying human genetic variations for forensic applications. she currently holds a university researcher v position and is a scientist 2 of the dostcsc scientif ic career system. 4tanguilig-danao.pmd n.r.p. tanguilig and l.a .m. danao 5 science diliman (july-december 2017) 29:2, 5-31 a hybrid cfd-bem analysis of the aerodynamic performance of a cut-out hollow pipe horizontal axis w ind turbine blade neil richard p. tanguil ig* university of the philippines diliman louis angelo m. danao university of the philippines diliman abstract this study investigated the performance of a cut-out hollow pipe blade prof ile in small horizontal axis wind turbines (hawt). although this type of blade was expected to have losses in eff iciency, such blade prof ile can be easily manufactured locally, and could potentially have a lower cost compared to conventional blades with aerofoil prof iles. numerical simulations using unsteady reynolds averaged navier stokes (urans) w e r e u s e d t o d e r i v e t h e a e r o d y n a m i c c h a r a c t e r i s t i c s o f t h e c u t o u t hollow-pipe sections. the blade element momentum (bem) method was then used to investigate the performance of the hawt’s rotor. numerical results show that cut-out hollow-pipe sections have poor aerodynamic c h a r a c t e r i s t i c s d u e t o t h e i r s i m p l e g e o m e t r y a n d c r u d e d e s i g n . b e m demonstrate that rotor with cut-out hollow pipe blades can still extract the kinetic energy of the wind but only at low tip speed ratios. parametric studies show that the performance can be improved by altering the pitch of the blades and by adding additional blades to the rotor. keywords: wind energy, cfd, bem, blade analysis, wind turbine, pipe blade _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 6 figure 1. distribution of installed units of small wind turbine generators worldwide (gasenger and pitteloud 2014). introduction according to the global climate risk index of 2015, the philippines is the f ifth most affected country by extreme weather events in the past two decades. in fact, in 2013, the philippines topped the list because of typhoon haiyan, which is the strongest tropical cyclone on record to hit land. because of the effect of climate change in the country, the philippines has been actively participating in international mitigation programs for climate change. one of the remarkable steps of the philippine government in addressing the issue of climate change is the enactment of ra 9513 or the renewable energy act of 2008. the said law promotes the development, utilization, and commercialization of renewable energy (re) resources in the philippines (congress of the philippines 2008). re resources are energy resources that are naturally replenished very rapidly, such that their availability is existent over an indef inite period of time, which includes, but are not limited to, biomass, solar, wind, ocean, and hydropower (congress of the philippines 2008). these resources are considered as “clean” energy because they do not emit harmful pollution during their operational stage. since the implementation of the said law, 11242.6 mw of grid-connected re project contracts have been awarded by the department of energy (doe) to potential developers. this includes 1608.9 mw of wind energy potential and 1858.43 mw of solar energy potential. unlike solar-photovoltaic, installation of small wind turbine generators is still in its infancy stage as most of these installations happen only in three countries, namely, china, usa, and united kingdom (gasenger and pitteloud 2014). figure 1 shows the distribution of small wind turbine installation worldwide. n.r.p. tanguilig and l.a .m. danao 7 small wind turbine generators, like their large scale counterparts, can either be horizontal axis wind turbine (hawt) or vertical axis wind turbine (vawt). currently, hawts are more popular than vawts in terms of manufacturing production and research mainly because hawts can be more eff icient and have simpler aerodynamic characteristics compared to vawts. hawt is a lift-type machine which uses an aerofoil prof ile as cross-section of its rotor blades to help generate a torque enough to make the rotor turn. one of the main causes that affects the dissemination of small wind turbine generators is the cost of commercially available machines, which cost an average of us$6040/kw (gasenger and pitteloud 2014). in the philippines, this value is still too high for the small communities or even the local government (baranggay and/or municipal) to shoulder. another factor that affects the mobilization of small wind turbine generators is the diff iculty of manufacturing it locally as highly eff icient wind turbines require great craftsmanship to create an optimized rotor that can harness the energy of the wind eff iciently. optimized rotor is composed of blade sections with aerofoil prof ile and twisted accordingly to maximize the aerodynamic characteristics of the aerofoil. this is usually made from molded f iber glass or carbon f ibers to ensure the accuracy of the design. wood can also be used to signif icantly reduce the cost but it requires a highly skilled craftsman to do so. a good example of this is the hugh piggott machine which was named after its creator. this machine uses a flat bottom aerofoil prof ile without any twist to reduce the diff iculty of carving the wood; however, this makes it less eff icient than its commercially available counterparts. although this machine has a great potential of entering the global market for small wind turbine generators, it has only been marketed worldwide as a do-it-yourself wind turbine as mass production seems impossible because of the carving-skill requirement for labor. another rotor alternative for small wind turbine generator is blades created from hollow pipe section. unlike the hugh piggott machine, this does not require great craftsmanship, as the blades with its desired twist can just be cut-out completely from the hollowpipe. the downside of this is that the eff iciency of the rotor will signif icantly suffer as blade cross-section is not the usual rounded leading and pointed trailing edges aerofoil prof ile. a small wind turbine generator with rotor made from cut-out hollow pipe is not a new concept. in fact, the online resource wind and wet (www.windandwet.com) provides its visitors a blueprint of a blade made from hollow pipe section by requiring certain variables, such as length and diameter of hollow pipe. they also detailed the geometry computations of the cut-out hollow pipe blade. the blade a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 8 design is created by assuming a coeff icient of lift of 0.85 regardless of the geometry of the cut-out hollow pipe. unfortunately, they did not mention any literature that explains how they arrived at such a value. this could pose a problem as the resulting rotor could perform poorly against its expected production if the actual lift coeff icient is below the assumed value. the technical contribution of this study is to present baseline knowledge of the aerodynamics and performance of cut-out hollow pipe rotor by developing a computational fluid dynamics (cfd)-based numerical model of the aerofoil prof ile from cut-out hollow pipe, and to eventually design and evaluate the performance of a cut-out hollow pipe rotor through the well-established blade element momentum (bem) theory method. materials and methods numerical modell ing of aerofoil hawt a two-dimensional cfd model was used to represent the aerofoil and the freestream condition. the basis for the 2-d model is that the spanwise velocity component of the wind turbine blade is much lower than that of the streamwise component (hansen 2008). this is also supported by the review of relevant literature which show that the 2-d model is suff icient for this type of study (hills and sládek 2005; menter and langtry 2006; castelli et al. 2012; esfahanian et al. 2013; lanzafame et al. 2013). moreover, the lift, drag, and moment coeff icients for the 2-d models are def ined by the following equations: where u is the incoming wind velocity; c is the chord length of the aerofoil; ρ is the density of air; a is the projected aerofoil area (chord x span); and c l , c d , and c m are the lift, drag, and moment coeff icients, respectively. 2 l 1 l= ρu cc 2 2 d 1 d= ρu cc 2 2 m 1 m= ρu acc 2 , (1) (2) (3) n.r.p. tanguilig and l.a .m. danao 9 figure 2 shows the resulting blade geometry based on a blade radius (r) of two meters and tip speed ratio (λ) of six. it has a maximum chord length (c) of 308 mm at the base and a minimum chord length of 100 mm at the tip. based on this geometry, the authors selected the chord lengths of 100, 150, 200, 250, and 308 mm for this study with maximum camber ratios of 0.01, 0.08, 0.11, 0.19, and 0.36, respectively. aerofoils a200, a250, and a308 can be classif ied as high-cambered aerofoils because their respective maximum camber ratios exceed 0.10 (milgram 1 9 7 1 ) . 2-d numerical flow field for this study, the calculation of the reynolds number (re) was based on a wind speed of 12 m/s and tip-speed ratio (λ) of six. moreover, the standard condition for the thermophysical properties of air was considered (air density ρ = 1.225 kg/m3, kinematic viscosity μ = 1.7894x10-5 m2/s). the calculated re for the aerofoils were 4.99×106 (a100), 3.07×10 6 (a150), 2.70×106 (a200), 2.60×10 6 (a250), and 2.64×106 (a308). however, for the density based solver with far-f ield conditions, the mach number (ma) must be def ined instead of the re number. ma is the ratio of the relative velocity to the speed of sound (340.29 m/s). the calculated ma for the above re were 0.2145, 0.0878, 0.0579, 0.0447, and 0.0368, respectively. structured, unstructured, and hybrid grids were used to create all the meshing requirements of this study. the near-wall region is basically divided into three parts, namely the viscous sublayer, the buffer zone, and the fully-turbulent layer, as shown in numerous experimental studies (ansys 2011a). in this study, modeling the near-wall region correctly will signif icantly increase the accuracy of the simulation, since it is where the solution variables have large gradients (ansys 2011b). in order to correctly model the near-wall region, f iner grids should be figure 2. aerofoil geometries from the hollow pipe. a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 10 used to resolve the solution up to the wall (including the viscous sublayer). therefore, choosing the f irst cell height of the grid from the wall is very important because it can signif icantly impact the accuracy of the solution. an important parameter to consider in correctly modeling the computational domain is the node density around the aerofoil. the node density study avoids the overprediction of the node spacing of the computational grid, which can result in a longer computational time of the solver, without sacrif icing the accuracy of the solution. initial value of the number of nodes in each upper and lower surfaces of the aerofoil was spaced at 1% of the chord length of the aerofoil, while the number of nodes on both blunt leading and trailing edges was 20. this initial value in the upper and lower surfaces was then halved and doubled to consider the effect of decreasing and increasing the node spacing, respectively. table 1 shows the resulting number of nodes for the a100 aerofoil used in the node density study. table 1. number of nodes around the a100 aerofoil 1% c 2% c 0.5% c upper and lower surfaces nodes 200 100 400 blunt leading and trailing edge surfaces nodes 40 40 40 total 240 140 440 figure 3 shows the results of the lift, drag, and moment coeff icients for different node densities along the surface of the aerofoil. it can be observed that increasing the node density had little effect on the drag and moment coeff icients, especially in low angles of attack (α). in terms of the lift coeff icient (c|), the f inest node density deviated to a smaller value at α = 80° and 120° with a maximum δc| = 0.1 at α = 80° from the coarsest node density. however, the maximum deviation of the f inest node density to mid-level node density was δc| = 0.03 at α = 80° which can be deemed as very small and negligible. therefore, the chosen node density for the rest of the simulation runs was computed based on a spacing of 1% of the chord length of the aerofoil to reduce simulation time while still having adequate spatial resolution to accurately solve the problem. a structured o-type grid was adapted for a100 (figure 4), a150, a200, and a250 (figure 5), and was created by extruding the cells normally from the face of the aerofoils. the o-type grid is more suitable for aerofoils with blunt trailing edges (cooperman et al. 2010) compared to the conventional c-type grid, and minimizes n.r.p. tanguilig and l.a .m. danao 11 skewness especially for surfaces with high curvature. the f irst cell height for the grid was estimated such that the y+ value does not exceed 1, which is a requirement to accurately model the viscous sublayer of the boundary layer. (a) (b) (c) figure 4. a100 aerofoil grid: (a) computational domain, (b) leading edge mesh, (c) surface mesh. (a) (b) (c) figure 3. results of the node density study: (a) lift coeff icient c l , (b) drag coeff icient c d , (c) moment coeff icient c m . a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 12 the hybrid o-type mesh was adapted for the a308 aerofoil because of the diff iculty in creating a high quality structured o-type mesh, particularly with regards to the skewness of the cells. the main factor for this is the very high camber characteristics (max camber ratio = 0.36) of the a308 aerofoil. basically, the hybrid mesh is composed of a structured mesh from the surface of the aerofoil up to a certain height in which the skewness is tolerable, and an unstructured mesh for the rest of the computational domain. similar to the structured o-type mesh, the f irst cell height distance was predicted by using a value of y+ equal to 1. the succeeding cells were generated by using a growth factor of 1.1 up to a grid height of 0.025 which produces cells with skewness of already up to 0.65. the unstructured section figure 5. a250 aerofoil grid: (a) computational domain, (b) leading edge mesh, (c) surface mesh. (a) (b) (c) aside from the node density discussed above, the nodes were also distributed such that the f irst and last cells on the upper and lower surfaces of the leading and trailing edges have a maximum spacing of 0.0005 m. this is to ensure a higher spatial resolution on the regions where higher gradients of pressure and flow are expected. moreover, the growth rate was set at 1.1 and extruded up to a total length of 20 times the chord length of the aerofoil for both the inlet and outlet boundaries. table 2 shows the f irst cell height and the total number of cells generated for each aerofoil type. aerofoil type first cell height (m) number of cells a100 0.000004 26,904 a150 0.00001 36,624 a200 0.00002 48,832 a250 0.00002 57,352 table 2. first cell height and number of cells for each aerofoil n.r.p. tanguilig and l.a .m. danao 13 i i u 0 x    (4) (5)jii i j j i j tu u p u , t x x x              (6) i j i jt 2 s , (7) ji i j j i uu1 s = + . 2 x x        (8)   i i j i j j i j u p + u u = + 2 s t x x x             of the mesh was created by connecting isotropic triangle cells to the outer surface of the structured mesh, continuing up to a total grid height (structured plus unstructured) of 20 times the chord length of the aerofoil. the length of the connecting triangle cells is equal to the length of the outer quadrilateral cells to preserve the smoothness of the growth rate of the cells towards the outermost region of the domain. the total number of structured cells was 38,532, whereas the total number of unstructured cells was 21,928. to ensure proper modeling, equiangle skewness, a mesh quality parameter, can be checked. equiangle skewness is def ined as the maximum ratio of the cell's included angle to the angle of an equilateral element. cells with highly skewed angles can decrease the accuracy and destabilize the solution. skewness value can range from 0 (good) to 1 (bad), and for quadrilateral cells, the value is recommended to be limited to 0.8 to qualify as a good grid. for this study, the grid skewness was limited to 0.7 due to the high-camber characteristics of the aerofoils. 2-d numerical simulation in this study, the problem is well within the incompressible region. the equations for conservation of mass and momentum for incompressible flow are where u i is velocity, x i is position, t is time, p is pressure, ρ is density, and t ij is the viscous stress tensor def ined by where μ is the molecular viscosity and s ij is the strain-rate tensor, rewriting and simplifying the previous equations yield the navier-stokes equation in conservation form. a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 14 t ime averaging equations (4) and (6) yields the reynolds averaged equations of motion in conservation form, rewriting equation (8) in its reverse yields its most recognizable form. equation (11) is usually referred to as the reynolds-averaged navier-stokes equation, where the quantity is known as the reynolds-stress tensor. the averaging process effectively introduces unknowns through the reynolds-stress components without any additional equations. the closure problem of turbulence is essentially devising approximations for the unknown correlations in terms of flow properties that are known, so that a suff icient number of equations exist. ansys fluent 14.5 was the cfd package used in all simulations performed in this study. it implements 2-d reynolds-averaged navier-stokes equations by using a f inite-volume-based solver. a density-based solver with second order implicit formulation was selected for all flow computations, since most flows are assumed to be not in line with the mesh (ansys 2011b). residuals were set to 10 -6 as a global convergence criterion for improved accuracy. generally, the ideal time-step δti is calculated as 1/10 of the time the flow crosses the aerofoil, as shown in the following equation: however, to prevent any loss of unsteady physics of the flow f ield, suff icient temporal resolution is necessary. therefore, additional time-step sizes with values of half and twice of the calculated ideal time-step were also tested. the a100 aerofoil in this particular study was used with time-step sizes of 0.0001 (ideal time-step size δti), 0.00005 (0.5δti), and 0.0002 (2δti). the simulation for each case was performed for up to 1500 time-steps or until a periodic convergence is achieved. moreover, the number of iterations for each time-step (internal loop) was set at 50. all three cases were tested for angles of attack α from 0° to 20° in 2° intervals. i i u 0 x    (9) (10)   i j i j i ji j i j u p u u + u u 2 s t x x x                 (11) i ij ji j i j i j u u p u 2 s u u t x x x                   j i u u   i r c t 10u   (12) n.r.p. tanguilig and l.a .m. danao 15 figure 7 shows the resulting lift, drag, and moment coeff icients simulated from the different time-step sizes. the values resulting from the largest time-step size δt highly deviated from the two smaller δt, especially in terms of the lift coeff icient. on the other hand, the results from the two f iner δt are in good agreement, particularly in the lower ranges of angle of attack in which the design of the blade figure 7. results of the time-step size study: (a) lift coeff icient, (b) drag coeff icient, (c) moment coeff icient. (a) (b) (c) figure 6. a308 aerofoil grid: (a) computational domain, (b) leading edge mesh, (c) surface mesh. a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 16 in this study is limited to. based on these results, the time-step size adopted for the rest of the simulation runs was computed from the ideal time-step size δti equation above in order to minimize simulation time without sacrif icing the accuracy of the solution. bem method the blade element momentum (bem) theory is an old but widely used method in predicting the performance of a hawt rotor. it was developed by glauert in 1935 by combining momentum theory and the events taking place at the actual blades (blade element theory) to analyze airplane propeller performance (glauert 1935). momentum theory uses the conservation of momentum to derive the forces and flow conditions acting on the hawt rotor. applying the conservation of linear and angular momentum will result to the expressions for the differential thrust and differential torque, respectively: where r is the local radius; dr is the annular section thickness; ω is the blade rotational speed; and a and a' are the axial and tangential induction factors, respectively. the axial induction factor is def ined as the fractional decrease of wind velocity between the free stream and the rotor plane: where u 1 is the velocity at free stream, u 2 is the velocity right before the rotor plane, and u 4 is the downstream velocity. on the other hand, the blade element theory expresses the forces on the blade in terms of the lift and drag coeff icients and the angle of attack (manwell et al. 2009). the procedure subdivides the span length of the blade into n elements which are assumed to act independently of each other. the total force and moment acting on the blade is then computed by summing up the resulting forces and moments acting on each individual n element. based on these procedures and assumptions, the derived expressions for the normal force and differential torque are as follows:  2dt ρu 4a 1 a πrdr    3dq 4a' 1 a ρuπr ωdr,  (13) (14) 1 2 1 u u a u    4 1u u 1 2 a ,  (15) (16) n.r.p. tanguilig and l.a .m. danao 17 where ϕ is the angle of relative velocity of the wind, and σ' is the local solidity ratio. for a rotor with b number of blades, the solidity ratio is def ined by: by equating equations (13) and (17) and (14) and (18), the expressions for the axial and tangential induction factors can be derived: where λ r is the local tip speed ratio, and f is the prandtl's tip loss factor given by: moreover, the power coeff icient cp can be derived by taking the ratio of the power produced by the rotor to the power derived from the wind: where λh is the tip-speed ratio at the hub. qblade is an open source bem software used for the horizontal and vertical axis wind turbine design and optimization. it is based on the same bem discussed above and is integrated with xfoil to enable the user to design and compute the performance of a custom aerofoil. however, in this study, the aerofoil performance was predicted using the numerical methods discussed earlier. in general, the pre-stall lift and drag coeff icients data (α ≈ ±15°) of aerofoils were used in the design of hawt blade, since turbines nowadays are stall-regulated to maximize rotor eff iciency, and in some cases, limit the produced power. in the (17) (18)   2 2 n l d2 u (1 a) d f σ'πρ c cosφ c sinφ rdr sin φ      2 2 2 l d2 u (1 a) d q σ 'πρ c sinφ c co sφ r d r, sin φ    b c σ . 2πr  (19)  l d 2 σ c s inφ c c o sφa 1 a 4 f co s φ    (20)  l d 2 r σ c c o sφ c sinφa' , 1 a 4 fλ c o s φ    (21)   1 b r12 2 rf c o s e x p . rπ co sφ r                     (22) (23)    h λ 3 d 2p r r l λ c8c fλ a 1 a 1 co tφ dλ cλ ,            a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 18 qblade iteration process, angles of attack in the post-stall range may occur temporarily, and thus, polar data for all possible 360° angles of attack should be made available to ensure continuation of the bem algorithms (marten and wendler 2013). the polar data for all 360° angles of attack can actually be obtained in the cfd simulations but it will not be computationally economical because of the much longer time required to complete the simulation. for this study, an extrapolation algorithm embedded in qblade was used to obtain the full 360° angle of attack range. table 3 details the blade prof ile based on the guidelines from wind and wet (www.windandwet.com). since polars were only created for aerofoils a250, a200, a150, and a100, the polars of the other aerofoil prof iles were adopted from the next smaller aerofoil with available polar, except for a143.75 and a137.5 polar, which were adopted from the a150 polar, since these aerofoils differ very slightly (in terms of geometry) with the latter. table 4 lists the variables and their corresponding values used in the rotor simulation. aerofoil name chord (mm) twist (degrees) position (mm) a250 250 34.0181 0 a250 250 34.0181 260 a200 200 20.9124 440 a175 175 15.238 600 a150 150 9.92682 760 a143.75 143.75 8.64081 915 a137.5 137.5 7.37031 1070 a131.25 131.25 6.11335 1225 a125 125 4.8689 1380 a118.75 118.75 3.63603 1535 a112.5 112.5 2.41386 1690 a106.25 106.25 1.20158 1845 a100 100 0 2000 table 3. blade section characteristics table 4. variables used in rotor simulation variables values fluid density, ρ 1.225 kg/m3 kinematic viscosity, μ 1.7894×10-5 m2/s number of blade elements, n 50 maximum number of iterations 100000 maximum ε for convergence 0.00001 relaxation factor, ω relax 0.35 n.r.p. tanguilig and l.a .m. danao 19 (a) (b) figure 8. periodic convergence plots of lift coeff icient: (a) a100 at α = 0°, (b) a150 at α = 20°, (c) a200 at á = 2°, (d) a250 at α = 6°. (d)(c) results the aerodynamic coeff icients derived for different cut-out hollow pipe aerofoil prof iles were based on the procedure described earlier. the simulations were performed at an initial number of time-steps of 1500 and were increased accordingly until periodic convergence was achieved. the internal loop or number of iterations per time-step was set at 50 in order to achieve a global convergence criterion of 1×10 -6. all simulations were performed at an α range of -20° to 20° with re values from 2.64×106 (for a250) up to 4.99×106 (for a100). in steady simulations, a flat line of convergence is expected as more iteration is consumed. however, in unsteady or transient simulations, fluctuating force coeff icients are expected, and periodic convergence is achieved when the peaks and troughs of the curve are starting to settle into a f inal value. this periodic convergence is indicative of highly transient flow behavior with time-dependent flow separation and reattachment on the blade surface. figure 8 shows different unsteadiness behaviors of the lift coeff icient relative to time. figure 8a shows that convergence was already achieved within the f irst 1500 time-steps as there was already a constant fluctuation of the values of the lift coeff icient. initial run of the 1500 time-steps for a single aerofoil at 10 different α's took approximately f ive days to f inish in an intel i7 machine. on the other hand, the remaining three plots needed additional time-steps before a periodic or cyclical behavior of the coeff icients was achieved. shown f inite values of the lift coeff icients were computed by taking the average during the time range in which periodic convergence was already apparent. values of other force coeff icients (i.e. , drag and moment) at different α's were derived using the same flow process. a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 20 (b)(a) figure 9. numerically predicted force coeff icients of the different aerofoil sections: (a) lift, (b) drag. figure 9 shows the lift coeff icient cl and drag coeff icient cd of the four aerofoils at different α. for regularly shaped aerofoil prof iles, cl usually increases linearly with increasing α until a maximum value is reached. hereafter, cl decreases and the aerofoil is said to have stalled. on the other hand, cd is often predicted to be relatively constant at low α, and then increases abruptly as the aerofoil begins to stall. however, in the case of the subject aerofoils, cl and c d behaved differently with increasing α, because, among other things, of the geometric characteristics of the aerofoils having blunt leading and trailing edges, as well as being symmetric with respect to the vertical. it can be seen from the graphs that both c l and cd tended to increase when the aerofoils rotate clockwise (i.e. , increasing positive α). on the other hand, while they were rotating counter-clockwise (i.e. , increasing negative α), cl increased f irst then started to drop at higher values, while cd tended to increase from the beginning. it can also be seen that shorter aerofoils tended to produce lower lift but higher drag than their longer counterparts. considering that the thickness of each aerofoil is the same, shorter aerofoils appear to be “thicker” overall and flatter. this results in a much drastic flow separation from the wall compared to longer and “thinner” aerofoils, as shown in figure 10. flow separation on the lower side of the aerofoil decreases the pressure on that side, thereby increasing the drag and lowering the lift. (a) (b) (c) (d) figure 10. streamtraces of the four different aerofoils at α =10°: (a) a100, (b) a150, (c) a200, (d) a250. n.r.p. tanguilig and l.a .m. danao 21 figure 12. blade geometry created from cut-out hollow pipe. figure 11 shows the cl/cd plotted against α. it can be observed that the cl to cd ratio was very small, especially for aerofoils a100 and a150. for a100 aerofoil, in particular, the ratio was almost unity in positive angles of attacks, which means that while cl was increasing, cd was also increasing. this is very important to note, as aerofoil performance, in general, depends on its capacity to generate high lift while minimizing the effects of drag. for this case, the drag could overwhelm the effects of the lift, hence degrading the performance of the blade made from these aerofoils. this characteristic could be improved by either choosing a much smaller pipe thickness to make the shorter aerofoils appear “thin” and minimize separation in the flow regime, or by altering the geometry itself of the subject aerofoils by rounding off its leading edge and thinning its trailing edge. however, these methods are not within the scope of this study. the blade geometry was based on the wind and wet (www.windandwet.com) procedure but was slightly altered at the base due to the non-convergence of the a308 aerofoil in the static aerofoil studies. figure 12 shows the geometry of the blade and the location of the different aerofoils along the span. the twist angle at the hub was 34.018° and was uniformly reduced to get a 0° twist at the tip. figure 11. lift/drag ratio at different angles of attack. a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 22 figure 13. output power at different tip speed ratios. the initial analysis was performed in the rotor bem simulation module of qblade using the simulation variables in table 4. the simulation was run in a wind speed of 12 m/s and tip speed ratio range of 0 to 8. at each λ, the radial rotor experiences different angles of attack because of the different relative velocities and wake inductions. hence, considering the initial pitch of the blades, there is a certain λ in which the rotor will perform optimally. in this case, the optimum λ was at 0.5 which produces a power of 24.12 watts (figure 13). it can also be observed that, at other λ, the power is negative which could only mean that the drag force acting on the blades was much higher than the lift force being experienced by the blades. a closer look at this phenomenon is presented in figure 14 which shows the angle of the relative velocity of the wind or inflow angle and the tangential blade force coeff icient at different blade positions. the thick green line represents the plot for the tip speed ratio of 0.5. for high tip speed ratios, the inflow angle was almost parallel to the plane of rotation which makes the direction of the drag vector to point almost directly opposite the direction of rotation. because of this phenomenon and the fact that the blade has aerofoils with low lift to drag ratio, the resulting tangential blade force coeff icients in almost two-thirds of the span of the blade were negative and served to counter the positive tangential force coeff icients acting on the remaining one-thirds of the blade. parametric stud ies parametric studies were conducted to investigate how the variation of certain parameters will affect the performance of the rotor. the wind speed, blade pitch, and number of blades of the rotor were the parameters investigated. similar to the analysis in the initial study, all simulations were conducted in a λ range of 0 to 8 at 0.5 interval. n.r.p. tanguilig and l.a .m. danao 23 (a) (b) figure 14. results for the simulation at 12 m/s wind at different tip speed ratios: (a) inflow angles, (b) tangential blade force coeff icients. unlike in conventional blades with regular aerofoil sections, the twist angles of the aerofoil sections for the blade created from cut-out hollow-pipe section were f ixed. because of this, optimum twist angles for the aerofoil sections to produce maximum lift cannot be obtained. the only option left for designing the blade in order to achieve optimum performance is to vary the pitch in which the resultant of the force coeff icients acting on each aerofoil section will produce maximum lift and minimum drag forces. for this study, the investigated pitch range is from 0° to 90° at 5° interval. figure 15 shows the power produced by the rotor at different λ when the blades were pitched at different angles. the performance of the rotor can be seen as very dependent on the pitch angle of the blades. it can also be observed that the rotor is operating only at low λ range. the highest operating λ was only at 2.5 when the blades were pitched at 20°. moreover, the highest power output was 202.65 w (cp is 0.013) when the blades were pitched at 25° and operating at a λ of 2.0. this behavior of the rotor can be explained by looking at the axial induction factors and tangential blade force coeff icients along the span of the blade. at higher values of a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 24 λ, the rotor is partially acting like a propeller, as can be observed in the negative values of the axial induction factors (figure 16). instead of decelerating the flow behind the rotor, the energy from the rotor's rotation is converted into kinetic energy which then accelerates the wake rotation behind the rotor, resulting in a much less energy extraction of the rotor. on the other hand, the blades are experiencing positive values of tangential blade force coeff icient along most of the span of the blade at the lower λ levels compared to levels at higher λ (figure 17). the overall effect of the lift vector is higher than that of the drag vector at lower λ which makes the rotor harvest the kinetic energy of the flow, and thus, produce power. figure 16. values of axial induction factors at different blade positions pitched at 25° (heavy line is at ë = 2.0). figure 15. effects of varying the pitch angle on the performance of the rotor. n.r.p. tanguilig and l.a .m. danao 25 figure 17. tangential blade force coeff icient at different positions along the blade span pitched at 25° (heavy line is at ë = 2.0). typically, horizontal axis wind turbines are limited to three-bladed rotors. while increasing the number of blades can also increase the power, the difference is minimal or of no practical importance for high-speed machines. however, since previous studies showed that the rotor is only operating at low λ or at slow rotations, adding additional blades to the rotor can help increase the torque, and thus, the power output signif icantly. figure 18 shows the power output of the f ive-bladed rotor. similar to the previous analysis with three blades, the maximum power output was achieved when the blades were pitched at 25°. however, in this case, the value for maximum power was 317.20 watts (cp is 0.02), over a hundred watts more compared to the maximum output of the three-bladed rotor. similar to the three-bladed rotor, when the blades were pitched at 30°, the maximum power output was almost similar to that when pitched at 25°, although at a much slower rotation (at λ of 1.5). one of the reasons for the increase in power output is the increase in the axial induction factor of the blades in the f ive-bladed rotor, as can be observed in figure 19 for the blades pitched at 25°. this indicates that an increase in the induced velocity of the rotor contributes to the rotor's rotation. another parameter that was affected by increasing the number of blades is the tip loss correction factor. as the rotor of the wind turbine rotates, multiple helical structures in the wake were created due to tip vortices, which can signif icantly affect the induced velocity distribution at the rotor. the tip loss correction factor addresses this influence of tip vortices shed into the wake on the induced velocity f ield. the spanwise distribution of the tip loss correction factor was much higher in a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 26 figure 18. power performance of the f ive-bladed rotor at different pitch angles. figure 19. difference in the axial induction factors for f iveand three-bladed rotors with blades pitched at 25°. rotor with more blades (figure 20). the effect of tip vortices shed is reduced by increasing the number of blades because the wake screw downstream are kept closer together, making it rigid and thus containing the flow in the cylindrical slipstream (branlard 2011). figure 21 shows the lift and drag coeff icients of the a250 aerofoil section compared to a similar cambered plate section with rounded leading and pointed trailing edges. the aerodynamic coeff icients of the cambered plate section were derived from xfoil which imposes a steady state analysis (time-independent). although the lift values were comparable, the drag values for the blunt aerofoil were signif icantly larger than that of the rounded leading edge and pointed trailing edge aerofoil. such low drag values can have signif icant improvements in overall n.r.p. tanguilig and l.a .m. danao 27 performance as the tangential component of drag will be lower, resulting in higher positive torque values. figure 21. force coeff icients of a250 aerofoil derived from cfd analysis and a similar cambered plate derived from xfoil: (a) lift coeff icient, (b) drag coeff icient. figure 20. tip loss factors for f iveand three-bladed rotors with blades pitched at 2 5 ° . (a) (b) a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 28 conclusions an investigation of the aerodynamic performance of a crude design of cut-out hollow-pipe blade for hawt rotor was conducted. a combination of cfd and bem analysis was used for the investigation of the performance. a 2-d unsteady rans model was used to derive the aerodynamic coeff icients of the cut-out hollow pipe aerofoils. the derived aerodynamic coeff icients were then used as inputs to bem to compute the overall performance of the rotor made from cut-out hollow pipe blades. the unsteady periodic convergence of the flow is indicative of highly transient flow behavior with time-dependent flow separation and reattachment on the blade surface. due to the geometric characteristics of the subject aerofoils, the force coeff icients at different angles of attack behaved differently. while the cl increased, the cd also increased with positive angles of attack, which is in contrast with the aerodynamic characteristics of conventional aerofoils in which the cl is maximized and the effect of c d is minimized. this suggests that these aerofoils have a poor aerodynamic performance if used in a wind turbine blade. the bem simulations were conducted using the open-source software qblade. the force coeff icients, which were derived through cfd, were used as polars to def ine the aerofoils' aerodynamic characteristics. as expected, the crude design of the cutout hollow pipe blade performed poorly compared to its conventional counterparts, on account of the drag acting on the blade and overcoming the effects of lift. moreover, for higher tip speed ratios, the values of the axial induction factors along the span of the blade were negative, indicating that the rotor is acting like a propeller, wherein the energy from the rotor's rotation is converted into kinetic energy which then accelerates the wake rotation behind the rotor. parametric studies were also conducted to observe how certain variables affect the performance of the rotor. the f irst parameter investigated was the pitch angle of blade. adjusting the pitch angle resulted in higher power output. the maximum output power was obtained when the blade was pitched at 25°. the other parameter that was investigated is the number of blades. increasing the number of blades (from 3 to 5) also resulted in a much higher power output at low tip speed ratios. the methods presented in this study can serve as baseline knowledge in the investigation of the aerodynamics of cut-out hollow pipe turbine blades. although n.r.p. tanguilig and l.a .m. danao 29 such methods have been validated and used before in the study of conventional hawt blades, this is the f irst time, to the author's knowledge, that such methods have been extensively used to study cut-out hollow pipe blades. thus, to further advance the study of the subject, the following are suggested: • an experimental study with the use of wind tunnel can be performed to further validate the numerical results of the 2-d unsteady rans model. in literature, these rans models were used to investigate conventional aerofoils with rounded leading edge and pointed trailing edge. however, in this study, the aerofoil had blunt leading and trailing edges which can greatly affect the flow physics of the solution. hence, doing an experimental study can lessen the exposure of using rans models. • the crude design of the cut-out hollow pipe blade can be further improved by rounding the leading edge and pointing the trailing edge of the aerofoil. as seen in the xfoil steady simulation, doing such design alteration can drastically improve the aerodynamic coeff icients of such aerofoils, and hence, the overall performance of the rotor. this could also result in a much lower cost per power produced since no additional materials for the blade will be needed. • full three-dimensional cfd model of the rotor and flow regime will eliminate the simplifications brought about by the two-dimensional model and the assumptions in the bem. however, it is computationally expensive because millions of cells are required for creating a good quality mesh and time-steps will be much smaller in order to not overlook the effects of unsteady physics. a supercomputer is usually used to do such simulations. acknowledgments the authors would like to thank the department of science and technology (dost)engineering research and development for technology (erdt) and the off ice of the vice chancellor for research and development (ovcrd), university of the philippines diliman for the funding provided for this work. a hybrid cfd-bem analysis of the pipe horizontal axis w ind turbine blade 30 references airfoil tools [internet]. 2015. airfoil tools; [cited: 2015 november 15] available from: http://airfoiltools.com/airfoil/details?airfoil=cp-180-050-gn. 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applications. journal of turbomachinery. 128(3):423434. milgram jh. 1971. section data for thin, highly cambered airfoils in incompressible flow. massachusetts (ma): massachusetts institute of technology. sørensen nn. 2009. cfd modelling of laminar-turbulent transition for airfoils and rotors using the model. wind energy. 12(8):715-733. wind and wet [internet]. 2015. wind and wet; [cited: 15 november 2015]. available from www.windandwet .com. _____________ neil richard p. tanguil ig <nrptanguilig@gmail.com> is presently assistant project site manager for alternergy wind one corporation. he was involved in the predevelopment, construction, and commissioning of the 54 mw pililla wind farm in pililla, rizal. currently, his responsibilities include wind resource assessment, wind and power forecasting, and project management. he obtained both his bs civil engineering and ms energy engineering degrees from the university of the philippines diliman. louis angelo m. danao <louisdanao@coe.upd.edu.ph> is presently an associate professor of the department of mechanical engineering, university of the philippines diliman. he obtained his ph.d. in 2012 from the university of sheff ield, sheff ield, uk, where he conducted experimental and numerical work related to the performance and aerodynamics of vertical axis wind turbines. prior to this, he was involved in several solid mechanics research including the analysis of abdominal aortic aneurysms and the deformation of external f ixator for tibial fractures using f inite element analysis. he also developed mathematical models for torsion of solid and hollow rectangular sections using analytical and fea approach. presently, he is carrying out work on design and performance analysis of horizontal axis tidal turbines. he is a member of the american society of mechanical engineers, the philippine society of mechanical engineers, and the international association of engineers. sdinside front cover-jan.-june2018.pmd january-june 2018 • vol. 30 no. 1 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief irene m. villaseñor, ph.d. university of the philippines associate editors jose maria p. balmaceda, ph.d. university of the philippines louis angelo m. danao, ph.d. university of the philippines carlos primo c. david, ph.d. university of the philippines christian n. della, ph.d. university of glasgow singapore alonzo a. gabriel, ph.d. university of the philippines arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines dennis i. merino, ph.d. southeastern louisiana university jonas p. quilang, ph.d. university of the philippines arnel a. salvador, ph.d. university of the philippines terence p. tumolva, d.eng. university of the philippines managing editor gonzalo a. campoamor ii, ph.d. university of the philippines editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor sarah mae u. penir on the cover: images from the researches of metillo et al. , montaño et al. , penuliar et al. and bernardo and de leon. teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth buckle, ph.d. food science and technology group school of chemical sciences and engineering the university of new south wales, australia jose b. cruz, jr., ph.d. department of electrical and computer engineering the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. centre for scientif ic computing and department of physics university of warwick, united kingdom raul k. suarez, ph.d. department of ecology, evolution and marine biology university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. 8thoron exposure-iwaoka.pmd k. iwaoka et al. 87 science diliman (july-december 2018) 30:2, 87-95 prel iminary development of thoron exposure system in the phil ippines kazuki iwaoka* hirosaki university, japan national institutes for quantum and radiological sciences and technology, japan health physics research section, atomic research division philippine nuclear research institute department of science and technology (pnri-dost) republic of the philippines lorna jean h. palad el iza b. enriquez fe m. dela cruz christopher o. mendoza juanario u. ol ivares ryan joseph aniago christian l. dela sada health physics research section, atomic research division philippine nuclear research institute department of science and technology (pnri-dost) republic of the philippines masahiro hosoda shinji tokonami hirosaki university, japan abstract the influence of 220rn (thoron) which is a gaseous radioisotope like 222rn (radon) has been the focus of attention of recent studies concerning the protection of the general public from natural radiation. hence, it is necessary to investigate the possibility of exposure to thoron in the philippines. passive d e t ec t o r s , w h i c h d o n o t n e ed ex t e r n a l p ow e r, a r e oft e n u s e d fo r measurements for thoron concentration in the environment. however, it is necessary to check if the passive detectors can appropriately work by being exposed to thoron at several thoron concentrations before conducting the investigation. in this study, a thoron exposure system was developed in the philippines to validate the passive detectors for thoron measurement and to test its performance. the thoron exposure system in this study can control the thoron concentration at the range of 5.9 x 104 to 1.5 x 105 bq m-3. the thoron exposure system will be utilized to validate the passive detectors for the investigation of thoron exposure in the philippines in the future. keywords: t horon, radiation measurement, quality assurance, natural radionuclides _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online preliminary development of thoron exposure system 88 introduction it is well known that more than half of public exposure from natural radiation is due to 222rn(radon) and 220rn(thoron). radon is generated by the alpha decay of 226ra, which is derived from radioactive decays of 238u as an initial parent nuclide. thoron is generated by the alpha decay of 224ra, which is derived from radioactive decays of 232th as an initial parent nuclide. the half-life of radon (3.82 days) is longer than that of thoron (55.6 seconds). it is also believed that radon inhalation increases the risk of lung cancer and is regarded as the second risk factor (who 2009). in the current international commission on radiological protection (icrp) publication 126, an annual average radon activity concentration of 300 bq m-3 is recommended as the reference level in workplaces and all other buildings (icrp 2014). in the philippines, a national survey for indoor radon concentration has been performed in order to investigate the risk of exposure of the general public to radon. until now, indoor radon concentration above the icrp reference levels has not been reported (dela cruz et al. 2012). on the other hand, the influence of thoron on human health impacts has been the subject of recent studies (hosoda et al. 2014). although thoron and radon are exhaled from natural substances, such as soil, rock, and building material, and cause internal exposure, it has been regarded that the influence of thoron is too small compared to that of radon. however, it was found that thoron exposure is equal to or exceeds radon exposure, and is thus not negligible for dose estimation in some areas in asia, such as india and china (kudo et al. 2015; omori et al. 2016). therefore, it is necessary to investigate the possibility of thoron exposure in the philippines. pa s s i ve d e t ec t o r s , w h i c h d o n o t n eed exte r n a l p ow er, a r e ofte n u s ed fo r measurements of thoron concentration as well as radon concentration in the environment . to test whether the detectors can appropriately work before conducting the actual investigation, thoron exposure facilities are needed. although there are thoron exposure facilities in other countries, such as the hirosaki university in japan (pornnumpa et al. 2016; pornnumpa et al. 2017; janik 2017), there are no similar facilities in the philippines. hence, there are no means for any domestic research organization in the philippines to validate the devices for thoron measurement. in this study, a thoron exposure system was developed in the philippines to easily validate the devices for thoron measurement in the country. the performance of the system was tested. k. iwaoka et al. 89 materials and methods the thoron exposure system was developed at the philippine nuclear research institute of the department of science and technology with reference to literature (pornnumpa et al. 2016; pornnumpa et al. 2017). a schematic diagram of the system is shown in figure 1. the system consists of three units: thoron generator unit; exposure unit; and monitoring unit. air in the room goes to the thoron generator unit using an air-pump with a flow rate of 1l min-1, which then goes to the thoron exposure unit. air in the thoron exposure unit goes outside the monitoring unit with a flow rate of 1l min-1. in the thoron generator unit, the amount of thoron exhaled from the thoron source depends on the humidity of the flowing air (pornnumpa et al. 2016; pornnumpa et al. 2017). therefore, air that has stable humidity goes to the thoron source through a humidif ier. a sectional view of the humidif ier is shown in figure 2. dry air from the dryer goes to two pathways (aira and airb). the aira bubbles water to generate the humidity. the airb just passes through without any signif icant event. aira and airb join together eventually to go through the thoron source. the stable humidity can be maintained by controlling the flow rates of aira and airb using the regulators. the amount of thoron generated (i.e. , thoron concentration in the thoron exposure chamber) can be controlled by changing the humidity of the flowing air or amount of thoron source. in the exposure unit, thoron gas in the thoron exposure chamber can be homogeneously mixed by a circulator inside the exposure chamber. dimensions of the thoron exposure chamber are as follows: 20 cm in length; 30 cm in width; and 20 cm in height. thoron gas in the thoron exposure chamber goes to the monitoring unit. in the monitoring unit, continuous measurements of thoron concentration in the thoron exposure chamber can be performed. the humidity and temperature in the thoron exposure chamber can be continuously monitored using an environment monitor. figure 1. a schematic diagram of the thoron exposure system. preliminary development of thoron exposure system 90 the performance of the thoron exposure system developed in this study was tested as follows. humidity for the thoron source and thoron exposure chamber was maintained at 71-85%, which is within the range of typical relative humidity (rh) in manila (pagasa 2017), by controlling the humidity in the thoron generator unit. temperature for the thoron exposure chamber was set at approximately room temperature. a recorder (tr-73u, t&d corporation) was used for monitoring rh and temperature. a tube f illed with a gas mantle, which has high activity for thoron and very low activity for radon, was used as thoron source (i.e. , tube source). thoron gas generation was performed on three situations (f irst: utilization of one tube source; second: utilization of two tube sources; third: utilization of three tube sources). humidities of the thoron exposure chamber and tube sources were stabilized in advance before thoron gas started to flow through the thoron exposure chamber. a detector with electrostatic collection (rad7, durridge company inc.) was used for thoron gas monitoring. the rad7 can collect radon and thoron decay products on a silicon ion-implanted semiconductor using a high electric eld, and alpha particles released from the products can be measured with energy discrimination (burnett et al. 2001). because the rad7 is affected by humidity, the dryer was installed before the rad7. monitoring by the rad7 was performed for 8 hours at 30-minute intervals at a flow rate of 1l min-1. before thoron gas in the thoron exposure chamber reaches the rad7, thoron concentration decreases because of its short half-life. therefore, the indicated values of thoron concentration in the rad7 were corrected to the actual values of thoron concentration in the thoron exposure chamber using the following equations: figure 2. a sectional view of the humidif ier in the thoron exposure system. k. iwaoka et al. 91 f t = 1/{e(-λ × v/r)} (1) c = c r × f t × f d × f c (2) where f t is the correction factor for decay compensation during the gas’ flow from the thoron exposure chamber to the rad7’s dryer (-); λ is the decay constant of thoron (s-1); v is the volume of tubes from the thoron exposure chamber to the rad7’s dryer (cm3); r is the flow rate(cm3 s-1); c is the thoron concentration in the thoron exposure chamber; c r is the indicated value of thoron concentration in rad7; f d is the correction factor for decay compensation during the gas’ flow from the rad7’s dryer to the rad7 (-); and f c is the correction factor for responsivity of rad7(-). v was set at 10.8 cm3. r was set at 1.0 l min-1 (17 cm3 s-1). f d was assumed to be 2 on the basis of the rad7’s manual. f c was set as 1.0 as on the basis of the references (pornnumpa et al. 2016; pornnumpa et al. 2017). results and discussion the values of thoron concentration, rh, and temperature in the thoron exposure chamber with two tube sources are shown in figure 3 as a representative for the three situations of the tube source. the thoron concentration in the thoron exposure chamber was stably maintained within a constant temperature and humidity range. the values of thoron concentration in three situations of the tube source are shown in figure 4 and table 1. the thoron concentration, based on the average of 16 measurements, was maintained at 5.9 × 104 bq m -3 (relative standard deviation (rsd): 1.6 %) for one tube source, 1.1 × 105 bq m-3 (rsd: 1.7 %) for two tube figure 3. values of thoron concentration, rh, and temperature in two tube source. preliminary development of thoron exposure system 92 sources, and 1.5 × 105 bq m-3 (rsd: 1.6 %) for three tube sources. it was found that the thoron exposure system developed in this study can control the thoron concentration by adjusting the amount of thoron source. these values for thoron concentration are higher than those in the facilities in other countries, which range from 4.1 × 103 to 2.6 × 104 bq m-3 (germany) and 4.2 × 103 to 4.6 × 104 bq m-3 (japan) (janik 2017). although high concentration may be a benef icial function for shortening the exposure time, the f irst advantage of the exposure system in this study over thoron exposure facilities in other countries is its small structure (janik 2017) which can be easily developed. therefore, methods in this study can be useful for scientif ic research institutions in countries wherein f inancial resources are a constraint for construction of such calibration systems. figure 4. values of thoron concentration in each condition for the tube source. table 1. thoron concentration in each cond ition for tube source the values of concentration were obtained by averaging 16 measurements. the parentheses mean the relative standard deviation (1 sigma) based on 16 measurements. source cond ition thoron concentration (bq m-3) one tube source 5.9 × 104 (0.46) two tube sources 1.1 × 105 (0.43) three tube sources 1.5 × 105 (0.46) k. iwaoka et al. 93 conclusions in summary, a thoron exposure system was developed in the philippines to easily validate the devices for thoron measurements in the country. the performance for the system was tested. the thoron exposure system in this study can control the thoron concentration at the range of 5.9 × 104 to 1.5 × 105 bq m-3 by adjusting the amount of thoron source. in future studies, prolonged working periods will be made to ensure the stability of the thoron exposure system. in addition, inter-comparison studies for performance improvement of the thoron exposure system will be conducted in collaboration with other research institutes for method validation. acknowledgement this work was partially supported by jsps kakenhi grant numbers jp16k16234. author contributions kazuki iwaoka designed the study and wrote the manuscript. lorna jean h. palad contributed to the analysis and interpretation of the data, and assisted in the preparation of the manuscript. eliza b. enriquez and fe m. dela cruz contributed to installation of the chamber. all authors contributed extensively in the discussion of the work and in the review of the manuscript. references burnett wc, kim g, lane-smith d. 2001. a continuous monitor for assessment of 222rn in the coastal ocean. journal of radioanalytical and nuclear chemistry. 249:167-172. dela cruz fm, garcia ty, palad ljh, cobar mlc, duran eb. 2012. national indoor radon survey in filipino homes. philippine nuclear journal. 17:9-16. hosoda m, kudo h, iwaoka k, yamada r, suzuki t, tamakuma y, tokonami s. 2016. characteristic of thoron (220rn) in environment. applied radiation and isotopes. 120:710. [ i c r p ] i n t e r n a t i o n a l c o m m i s s i o n o n r a d i o l o g i c a l p r o t e c t i o n . 2 0 1 4 . r a d i o l o g i c a l protection against radon exposure. icrp publication 126. janik m. intercomparisons exercises of radon and thoron monitors provided by four laboratories: a review. japanese journal of health physics. 52:114-121. preliminary development of thoron exposure system 94 kudo h, tokonami s, omori y, ishikawa t, iwaoka k, sahoo sk, akata n, hosoda m, wanabongse p, pornnumpa c, sun q, li x, akiba s. 2015. comparative dosimetry for radon and thoron in high background radiation areas in china. radiation protection dosimetry. 167:155-159. omori y, prasad g, sorimachi a, sahoo sk, ishikawa t, sagard dv, ramola rc, tokonami s. 2016. long-term measurements of residential radon, thoron, and thoron progeny concentrations around the chhatrapur placer deposit, a high background radiation area in odisha, india. journal of environmental radioactivity. 162-163:371-378. [ pa g a s a ] p h i l i p p i n e a t m o s p h e r i c g e o p h y s i c a l a n d a s t r o n o m i c a l s e r v i c e s administration [internet]. 2017. climate of the philippines; [cited 2017 july 5]. available from: http: //www1.pagasa.dost .gov.ph/index.php/climate-of-the-philippines. po r n n u m p a c , i w a o k a k , h o s o d a m , toko n a m i s . 2 0 1 6 . d eve l o p m e n t of r a d o n a n d thoron exposure facilities in hirosaki university. presented at: jhps 2016: the 49th conference of japan health physics society; hirosaki, japan. pornnumpa c, oyama y, iwaoka k, hosoda m, tokonami s. 2018. development of radon a n d t h o r o n ex p o s u r e s y s te m s i n h i r o s a k i u n i ve r s i t y. ra d i a t i o n e n v i r o n m e n t a n d medicine. 7(1):13-20. [who] world health organization. 2009. who handbook on indoor radon: a public health perspective. geneva, switzerland: who press. _____________ kazuki iwaoka <iwaoka@hirosah-u.ac.jp> is a visiting scientist at the health physics research section, atomic research division, philippine nuclear research institute department of science and technology (pnri-dost), republic of the philippines, and an assistant professor of institute of radiation emergency medicine, hirosaki university. lorna jean h. palad is a senior researcher at the health physics research section, atomic research division, philippine nuclear research institute department of science and technology (pnri-dost), republic of the philippines. she graduated with the degree of bachelor of science in chemistry from the university of the philippines. she is currently the leader of the norm (naturally-occurring radioactive materials) project of the pnri-dost. she is mainly involved in the assessment of natural radioactivity in norm industries in the philippines. el iza b. enriquez graduated with the degree bachelor of science in fisheries from the university of the philippines diliman in 1981. she is currently the leader of k. iwaoka et al. 95 the marine radioactivity monitoring project of the philippine nuclear research institute, department of science and technology (pnri-dost). she is mainly involved in the assessment of natural and ar tif icial radioactivity in seawater, sediment and biota from various marine areas in the philippines. she is the focal person of the regional database in marine radioactivity in the asia-pacif ic region. fe m. dela cruz is a senior researcher at the health physics research section, atomic research division, philippine nuclear research institute depar tment of science and technology (pnri-dost), republic of the philippines. she graduated with the degree of bachelor of science in chemistry from the far eastern university. she is currently the leader of the radon and thoron monitoring project of the pnrid o s t. christopher o. mendoza is a registered chemist and a science research specialist ii at the health physics research section, atomic research division, philippine nuclear research institute department of science and technology (pnri-dost), republic of the philippines. he is also the designated pollution control off icer of the institute. masahiro hosoda is a lecturer at the department of radiological life sciences, hirosaki university graduate school of health sciences. shinji tokonami is a director at the institute of radiation emergency medicine, hirosaki university. juanario u. ol ivares is a graduate of bachelor of science in biology from the college of arts and sciences, cagayan state university in 2006. he is currently employed at health physics research section, atomic research division, philippine nuclear research institute department of science and technology (pnri-dost), republic of the philippines and holding a position of science research assistant. his research interests include radiation biology, environmental radiation monitoring and biological sciences. ryan joseph aniago is a science research specialist at the health physics research section, atomic research division, philippine nuclear research institute department of science and technology (pnri-dost), republic of the philippines. christian l. dela sada is a project assistant ii at the health physics research section, atomic research division, philippine nuclear research institute department of science and technology (pnri-dost), republic of the philippines. 1 layman’s abstracts main articles crustal deformation of luzon and its implications on the stability of the philippine survey network gerald a. galgana, teresito c. bacolcol, elliot c. klein, charisma victoria dela cruz-cayapan, rosalie b. reyes, and wilfredo m. rada the island of luzon is situated in a broad region of intense geologic deformation, called a plate boundary zone. such regions are dotted with tectonic faults that produce periodic, earthquake-related motions, as well as rotations and internal deformation of small tectonic plates with respect to one another. these dynamic environments are characterized by continuous strain or deformation in the crust, which in turn affects the natural environment including man-made structures— among these are plat or property boundary monuments, benchmarks, horizontal control/triangulation stations and other survey control stations. when such survey networks are moved or deformed, this disrupts the integrity of the fundamental reference system that is being used to provide coordinates or positions of reference markers for land surveys and mapping systems. essentially, this work partly identifies and resolves an important component of a persistent problem that has plagued the old philippine survey network: deformation based on geologic or tectonic motion. this research focuses on identifying the deformation patterns of luzon, then presents a two-step modeling approach that estimates the deformation specifically due to locking of the faults and to rotation and internal deformation of the tectonic plates that compose luzon. the implications as to how and where resurveys should be implemented are then briefly discussed. science diliman (january-june 2020) 32:1, 1-4 2 layman’s abstracts the jordan canonical form of a product of elementary s-unitary matrices erwin j. gonda and agnes t. paras a generalization of a unitary matrix is the s-unitary matrix for nonsingular and hermitian s. a complex matrix q is said to be s-unitary if q*sq = s. an s-unitary q is called elementary if rank (q – l ) = 1. the multiplicative group of s-unitary matrices is generated by elementary matrices. we determine all the possible jordan canonical forms of a product of two elementary s-unitary matrices. mineralization, biodegradation, and antagonistic activities of gut-associated bacteria and fungi of african nightcrawler, eudrilus eugeniae (kinberg, 1867) maria reynalen f. mapile and marie christine m. obusan earthworms and their interactions with microorganisms offer beneficial effects that can improve organic matter decomposition, enhance nutrient availability, and suppress pathogens in the soil. in this study, bacteria and fungi isolated fromthe gut of eudrilus eugeniae (kinberg 1867), commonly known as african nightcrawler or anc, were found to exhibit activities that add value to its casts and compost. six fungal isolates inhibited the growth of bacteria. two bacterial isolates demonstrated the ability to fix nitrogen, solubilize phosphate, and use polyethylene as carbon source. the activities of these bacteria and fungi must be further explored to optimize the use of anc’s casts and compost for agricultural, medical, and other applications. 3 layman’s abstracts body size, habitat, and diet of freshwater crabs isolapotamon mindanaoense and sundathelphusa miguelito (crustacea: brachyura) in the municipality of lake sebu, south cotabato, philippines ziljih s. molina, jemateo b. neri, rizza may p. cañete, and ephrime b. metillo two species of edible freshwater crabs, namely isolapotamon mindanaoense (rathbun 1904) and sundathelphusa miguelito (mendoza and sy 2017), are known to be found only in mindanao island, but the biology of these species is poorly investigated. the authors studied the body size, microhabitat, and feeding habits of these two crab species collected by hand in the vicinity of three waterfall sites in south cotabato, southern philippines. both species feed on a variety of food composed of plant fragments, insect and fish fragments, and unidentified food items, but the larger sized i. mindanaoense fed more on animal fragments compared to the smaller sized s. miguelito that mainly ate plant fragments and unidentified food items. apart from differences in body size and feeding habits, the two species differ in habitats with the larger species found in areas with big boulders while the smaller species inhabit sand and gravel substrates. these differences may help minimize competition, allowing the coexistence of the two species. tracing the source of the non-native philippine population of the greenhouse frog eleutherodactylus planirostris (cope, 1862) through dna barcodes gerard clinton l. que, emerson y. sy, perry s. ong†, and ian kendrich c. fontanilla the greenhouse frog (eleutherodactylus planirostris) is a small amphibian native to cuba and outlying islands that has recently been accidentally introduced into the philippines. following its first detection in davao in 2013, it has since spread to eight islands in the country, including negros, cebu, and luzon island. the authors of this paper wanted to find out where the frogs came from and how they were introduced. several frogs were collected from luzon and one from negros. 4 layman’s abstracts using dna barcoding, a tool that relies on a segment of dna that can reliably discriminate between various groups of organisms, we were able to narrow down the source population to florida, usa or western cuba. all collected frogs had similar nucleotide sequences with each other, with almost no genetic diversity among them. importation of plants from the usa is the probable mode of introduction, since the greenhouse frog lays its eggs in soil rather than water and is often found in bromeliad plants. 5-masangcay-feeding habits.pmd feeding habits of mobula japanica 24 science diliman (january-june 2018) 30:1, 24-44 feed ing habits of mobula japanica (chondrichthyes, mobul idae) in butuan bay, mindanao island, phil ippines shirlamaine irina g. masangcay caraga state university ephrime b. metillo* mindanao state university-iligan institute of technology ken-ichi hayashizaki kitasato university satoru tamada kitasato university shuhei nishida university of tokyo abstract the diet of the spinetail devil ray mobula japanica müller and henle 1841 from butuan bay, philippines was investigated from january to may 2016 using data on its stomach contents, and c and n stable isotope analyses, in order to contribute to the scarce information on the feeding biology of the threatened tropical populations of the mobula species. e x a m i n a t i o n o f 1 6 m . j a p a n i c a s t o m a c h s r e v e a l e d i n g e s t i o n o f t h e euphausiid pseudeuphausia latifrons, sergestid shrimps acetes intermedius and lucifer spp. , copepods, and other rare prey items. the tropical krill p. l a t i f r o n s w a s t h e m o s t co m m o n , ofte n t h e s o l e fo o d , t h a t i n c r e a s e s body length of individuals towards the warmer months of april and may, w h i c h co i n c i d e w i t h t h e p e a k s e a s o n of m . j a p a n i ca f i s h e r i e s . re s u l t s f r o m δ 1 3c a n d δ 1 5n s t a b l e i s o t o p e a n a l y s i s a r e c o n s i s t e n t w i t h t h e assimilation of large zooplankton and micronektonic crustaceans. this study is the f irst repor t on the feeding of m. japanica in tropical waters and the identif ication of euphausiid p. latifrons as its dominant prey. keywords: stomach content, mobula, pseudeuphausia latifrons, population structure, tropical _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online s.i.g. masangcay et al. 25 introduction the spinetail devil ray mobula japanica from the family mobulidae is a large marine f ish with cartilaginous skeleton (couturier et al. 2012). locally known as pantihan, mobulids or devil rays are pelagic f ishes found in shallow and deep waters in the tropical and temperate regions (cortes and blum 2008; bizzarro et al. 2009; scacco et al. 2009; canese et al. 2011; metillo and masangcay 2015; croll et al. 2016; francis and jones 2016). in new zealand, the species is a common bycatch in skipjack tuna purse seine f isheries (francis and jones 2016). most mobula, particularly m. japanica, vary in size with disk width ranging 1–3 meters (paulin et al. 1982; notarbartolo di sciara 1987; white et al. 2006b), and are commonly known to be very fast swimmers that feed on zooplankton (couturier et al. 2012). they have low natural rate of mortality, slow-growth with long life span, late sexual maturation, and have few but large offspring (dulvy et al. 2003; musick and ellis 2005; garcia et al. 2008; croll et al. 2016). mobula japanica is currently listed as near-threatened by the international union for conservation of nature (iucn) red list of threatened species (white et al. 2006a), yet they are still being actively f ished in bohol sea, central philippines via the traditional harpoon and purse seine methods for sale and local consumption in the coastal area (alava et al. 2002; rayos et al. 2012; acebes 2013; freeman 2014; croll et al. 2016). fishermen (galdo and sanchez, personal communication) conf irmed that m. japanica are seasonally observed in butuan bay, mindanao island during the northeast monsoon when coastal enrichment is highest in eastern bohol sea (cabrera et al. 2011). mobula species are pelagic megafauna yet they subsist on a primary diet of zooplankton and ichthyoplankton (couturier et al. 2012). they grow very large due to the direct feeding on abundant zooplankton and ichthyoplankton in the second trophic level much closer to abundant primary producers (shwenk 2000). zooplanktivorous devil rays are indispensable in the marine ecosystem since they tap the lower trophic levels (couturier et al. 2012; jaine et al. 2012), and become important indicator species of climate change, as their planktonic food source is highly susceptible to environmental changes (e.g. , ocean acidif ication and warming waters) (hays et al. 2005; richardson 2008; weeks et al. 2015). studies on the biology and ecology of mobulidae started since the 17th century, but information on its feeding habits is limited (willoughby 1686; stewart et al. 2016). aggregations of devil rays are generally linked with local productivity and food availability (celona 2004; sleeman et al. 2007; dewar et al. 2008; marshall et al. 2009; anderson et al. 2011; couturier et al. 2011; marshall et al. 2011) . zooplanktivory is their generic feeding habit (weeks et al. 2015), but speciesfeeding habits of mobula japanica 26 specif ic differences occur. for instance, m. thurstoni is known to feed on mysid shrimps and euphausiids, and dwells in non-overlapping microhabitats with other species (notarbartolo di sciara 1988). examination of the stomach contents, and c and n stable isotope techniques have shown varying feeding habits among subtropical and temperate sub-populations u n d e r t h e g e n u s m o b u l a, s u c h a s m . m o b u l a r, m . h y p o s t o m a , m . r o c h e b r u n e i , m. tarapacana, m. birostris, m. alfredi, m. japanica, and m. munkiana, which generally feed on small f ishes and crustaceans like krill (meganyctiphanes norvegica, nyctiphanes simplex), the mysid mysidium sp., and other planktonic organisms (notarbartolo di sciara 1988; sampson et al. 2010; couturier et al. 2012). the diets of the near threatened species m. kuhlii and m. eregoodootenkee are unknown thus far (pierce and bennett 2003; bizzarro et al. 2009). current knowledge of the prey preference of the tropical mobula japanica based on its stomach contents is limited to the studies of notarbatolo di sciara (1988) and sampson et al. (2010) conducted off the coast of california, usa. analyzing feeding habits of tropical populations will determine diet preference and allow inference on habitat use and feeding behavior, which are both very impor tant to the conservation and management of the ray (stewart et al. 2016). hence, this work investigated the feeding habits of m. japanica in butuan bay, northeastern part of mindanao island, philippines with specif ic aims of analyzing the composition of its stomach contents, and indirectly inferring its feeding habits using c and n stable isotope analysis. materials and methods study area butuan bay is located in the northeastern area of mindanao (figure 1). its coastline is connected to the north with bohol sea or mindanao sea, which is known to have extreme southwest movement of surface currents coming from the pacif ic ocean (cabrera et al. 2011). the entire bay has an average depth of 100 meters and a maximum depth of 800 meters (nga nautical chart 1996). numerous river tributaries flow directly into the bay, including the agusan river, the third longest river in the philippines, carrying water discharges from interconnecting rivers, channels, and lakes (primavera and tumanda 2008). a biologically enriched estuarine frontal plume usually occurs near the mouth of the large agusan river in the bay (cabrera et al. 2011). climatic condition in butuan bay includes signif icant amount of rainfall throughout the year even in the driest month. the bay is exposed to strong trade winds and storms during the northeast monsoon (december-april). s.i.g. masangcay et al. 27 buenavista is a f ishing coastal municipality located at the southern portion of the bay (figure 1) (indab and suarez-aspilla 2004). the area is considered as a major f ishing ground with rich f ishery resources (bfar 2015). personal interviews in the locality validated the regular landing of mobula in buenavista, but f isherfolks report catching of rays at other locations in the bay, particularly off the coast of carmen, nasipit, cabadbaran, and tubay (figure 1) (metillo and masangcay 2015). field sampl ing of ray stomach, muscle tissue, and prey items field collection of ray stomach samples, potential prey, and tissues for c and n stable isotopes was conducted from 18 january to 13 may 2016 to coincide with the f ishing season in butuan bay. sixteen specimens of m. japanica (table 1) were purchased from local f ishermen who caught the f ish as bycatch from sardine and skipjack tuna gill net fishing during the day in f ive locations in butuan bay (figure 1). fishermen from buenavista and other locations in butuan bay consistently stressed that their devil ray collection sites are just within the bay (metillo and masangcay 2015). during specimen collections of this study, a standard procedure of asking f ishermen where they captured the rays revealed that they can be found in the deep portions of butuan bay. the digestive tract of the 16 individuals was removed by cutting the most anterior end of the esophagus and the most posterior end of the intestine. the length and outer diameter of the stomach were measured to estimate the stomach volume that will be used in the stomach content analysis. figure 1. geographical location of butuan bay in northeastern mindanao, and collection sites of landed mobula japanica and plankton samples off the municipality of carmen (triangle), nasipit (square), buenavista (dot), cabadbaran (diamond), and tubay (star) in the province of agusan del norte. inset is the map of the philippines with the study site enclosed in a square. feeding habits of mobula japanica 28 afterwards, the stomach and intestine (figure 2) were longitudinally dissected, spread apart, and the stomach contents were thoroughly flushed into clean plastic containers and preserved in 10% buffered formalin in f iltered seawater. muscle tissue samples from f ive mobula japanica individuals were obtained near the ventroposterior area of the pectoral f ins using a sharp scalpel, and were immediately placed in a clean vial and labeled properly. t issue samples were brought to the laboratory in an ice chest, and immediately dried in an oven at a temperature of 60 oc for 48 hours. a female 148 72 19 166 18-jan-16 b male 130 68 23 160 25-jan-16 c male 120 55 5 148 12-feb-16 d male 137 67 16.4 146 27-feb-16 buenavista area e 11-mar-16 buenavista area f male 134 60 8 143 18-mar-16 buenavista area g male 18-mar-16 cabadbaran area h 30-mar-16 cabadbaran area i male 129 59 17 150 31-mar-16 buenavista area j male 146 69 22 142 31-mar-16 tubay area k 02-apr-16 buenavista area l female 135 60 18 154 09-apr-16 carmen area m 10-apr-16 carmen area n male 130 66 12 140 09-may-16 buenavista area o male 134 63 17 148 11-may-16 buenavista area p male 157 73 22 178 13-may-16 buenavista area note: “-”, undetermined # sex disk width (d w ) disk length (c f ) cephal ic fin length (c f ) tail length (t l ) date collected site collected table 1. body measurements (cm) of the 16 mobula japanica ind ividuals collected in butuan bay, northeastern mindanao, phil ippines figure 2.the digestive tract of mobula japanica from butuan bay, philippines. s.i.g. masangcay et al. 29 p l a n k t o n s a m p l e s w e r e c o l l e c t e d o n 2 5 j a n u a r y 2 0 1 6 a t t h e l o c a t i o n w h e r e m. japanica were caught (table 2), particularly off buenavista. conical nets with mesh sizes of 100 μm and 20 μm were towed horizontally at sub-surface depths to collect zooplankton and particulate organic matter (pom), respectively (metillo et al. 2015). night sampling involving several 3-minute tows was performed until the desired amounts of plankton and pom triplicate samples were collected. zooplankton samples were size-fractionated using a series of sieves with nylon gauzes of different mesh sizes (<100 μm, 100–200 μm, 200–335 μm, 335–1000 μm, >1000 μm), and were viewed under a dissecting stereo microscope to remove any debris in the sample. zooplankton taxa (e.g. crab megalopa, decapod shrimp, hydrozoa) were sorted from the bulk samples to represent extra-large zooplankton (zxl). samples from the 20-μm mesh plankton net were f iltered, and particles trapped in the 20-μm sieve were regarded as pom (table 3). large zooplankton samples (zxl, zl, zm) were carefully handpicked using f ine forceps, placed in foil, and dried in an oven at a temperature of 60oc for 48 hours. smaller size fractions (zs) of zooplankton and pom samples were separately f iltered onto pre-combusted glass f iber f ilters (gf/f), dried in the same manner as large zooplankton, and placed inside eppendorf tubes until stable isotope analysis. table 2. tabulated coord inates of each plankton tow for stable isotopes analysis collected on 2016 january 25 in butuan bay, northeastern mindanao, phil ippines 1 100 125.401405° 8.987764° 2 100 125.399261° 8.988452° 3 100 125.397559° 8.988784° 4 100 125.396298° 8.988920° 5 20 125.394506° 8.989511° 6 20 125.393166° 8.989122° 7 20 125.391468° 8.988398° 8 20 125.389072° 8.988368° 9 20 125.386612° 8.988413° 10 20 125.384991° 8.988234° 11 20 125.383185° 8.988025° tow no. net mesh size μm coord inates longitude latitude feeding habits of mobula japanica 30 stomach content analysis the stomach content of each individual ray was removed and placed in a beaker for subsampling. the entire sample was divided into 10 parts with each tenth regarded as a subsample. three sub-samples were then thoroughly identif ied using a stereomicroscope for large particles and a compound microscope for smaller ones. the contents of the intestine were also inspected, but the materials were already heavily digested and unidentif iable; hence, they were not included in the analysis. the index of relative importance (iri) of each food item category was computed using the formula of pinkas et al. (1971): iri = (cn + cv) x f , where cn is the percentage numerical count of each food item relative to the total count of all food items; c v is the percentage volume (assuming cylindrical shape of the ray cardiac stomach) of each food item (estimated from the product of the proportion 18-jan-16 mobula japonica (female) mj day 1 fish landing 18-jan-16 pseudeuphausia latifrons ka n/a 30 net towing 25-jan-16 m. japonica (male) mj day 1 fish landing 12-feb-16 m. japonica (male) mj day 1 fish landing 27-feb-16 m. japonica (male) mj day 1 fish landing 18-mar-16 m. japonica (male) mj day 1 fish landing 25-jan-16 lucifer spp. lu night 6 net towing 25-jan-16 acetes intermedius ac night 15 net towing 25-jan-16 clupeidae fj night 4 net towing 25-jan-16 exocoetidae fj night 1 net towing 25-jan-16 blennidae f l night 4 net towing 25-jan-16 carangidae f l night 6 net towing 25-jan-16 gobidae f l night 4 net towing 25-jan-16 crab megalopa zxl night 2 net towing 25-jan-16 decapod shrimp zxl night 2 net towing 25-jan-16 hydrozoa zxl night 1 net towing 25-jan-16 macrosetella sp. zxl night 2 net towing 25-jan-16 acartia sp. zl night 18 net towing 25-jan-16 labidocera sp. zl night 7 net towing 25-jan-16 calanid copepods zl night 22 net towing 25-jan-16 parthenope sp. (zoea) zl night 3 net towing 25-jan-16 paracalanus sp. zm night 18 net towing 25-jan-16 copepods (200-335 μm) zm night 1 g net towing 25-jan-16 mesozooplankton zs night 1 g net towing (100-200 μm) 25-jan-16 particulate matter pom night 1 g net towing (20-100 μm) legend: *specimen means individual organism, except the bottom three rows where specimen is expressed in gram (g) sampl ing dates taxa/size group code period of collection no. of specimen* source table 3. list of specimens used for c and n stable isotope analysis. s.i.g. masangcay et al. 31 of space occupied by each food item and the volume of the cylindrical cardiac stomach) relative to the volume of all food item combined; and f is the percentage occurrence of each food item in the stomachs of all f ish individuals analyzed. the use of the iri (pinkas et al. 1971) reduces biased description of animal dietary data. this method has been widely used in studying diet composition of large marine animals and proved eff icient in determining a snapshot view of prey items in the stomach (notarbatolo di sciara 1988; alonso et al. 2001; moura et al. 2008; schluessel et al. 2010). analysis of c and n stable isotopes dried samples were pulverized using acid-washed mortar and pestle. powdered samples were aseptically placed in eppendorf tubes and properly labelled. all samples were analyzed through dual c and n stable isotopes technique using the thermo stable isotopes analyzer coupled with the thermo finnigan delta plus xp isotope ratio mass spectrometer via a conflo-iii continuous flow interface (metillo et al. 2015). samples with elemental c and n ratio > 4 were corrected for effects of lipids (post 2002). data treatment isotopic values between the two m. japanica individuals were compared using student homoscedastic t-test (spss 2002). results of the stable isotope analysis (sia) were plotted and interpreted using omnigraphsketcher version 1.1.4. relationships between prey and predator were calculated using the trophic enrichment factor values 3.2±0.43‰ for δ15n and 1.8±0.29‰ for δ13c (mccutchan et al 2003). trophic levels (tl) of all samples were determined based on nitrogen isotopic values using the equation (vander zanden and rasmussen 2001): tl consumer = [(δ 15n consumer —δ 15n baseline )/3.4 + 2], where δ 15n consumer is the mean value of the predator, δ15n baseline is the isotopic δ15n values from the microplankton (z1,100– 200 μm) samples, and a trophic eff iciency factor value of 3.4. results stomach content the state of the 16 m. japanica individuals only allowed sexing 12 which comprised ten males and two females (table i). body size as disk width (d w ) ranged from 120– 157 cm, while disk length (d l ) ranged from 55–73 cm. the stomach of all 16 feeding habits of mobula japanica 32 individuals examined contained ingested food composed of intact identif iable prey items mixed with few digested food items. prey organisms identif ied were mostly planktonic euphausiids, sergestid shrimps, copepods, and other categories as minor food items (table 4). stomach contents from all rays consisted almost exclusively of adult and eggs of the krill pseudeuphausia latifrons g.o. sars 1883, which exhibited iri values of 15,180.28 and 4,537.11, respectively. the other prey items in decreasing order of iri values were the sergestoid shrimp lucifer > sergestid shrimp acetes intermedius > copepods > flatworm > plant fragments > polychaete larvae = mollusc veligers. although a. intermedius was found to have a low iri value of 10.65, it dominated the stomach content of one m. japanica. pseudeuphausia latifrons 588,413 76.594 75.209 100.00 15,180.28 76.66 krill egg 175,866 22.893 22.479 100.00 4,537.11 22.91 lucifer sp. 3,348 0.436 0.638 68.75 73.85 0.37 acetes intermedius 540 0.070 1.634 6.25 10.65 0.05 copepods 47 0.006 0.017 31.25 0.72 0.00 flatworm 5 0.001 0.018 6.25 0.12 0.00 mollusc veligers 1 0.000 0.001 6.25 0.01 0.00 polychaete larvae 1 0.000 0.001 6.25 0.01 0.00 plant fragments 1 0.000 0.003 6.25 0.02 0.00 prey number % n % v % fo iri % iri table 4. diet analysis of mobula japanica based on 9 prey types collected from the stomachs of 16 ind ividuals. n, number; v, volume; fo, frequency of occurrence; iri, index of relative importance c and n stable isotope values we obtained muscle tissues from f ive m. japanica individuals with sizes ranging from 130–147 cm (d w ) (table 5). individual values of c and n stable isotopes for these individuals were not signif icantly different (t = 1.56, df = 3, p = 0.22). mean isotopic values for the f ive m. japanica were -16.07±0.52‰ for δ13c and 10.69± 0.34‰ for δ15n. the δ15n isotopic values were accordingly used to calculate and determine the trophic positions of m. japanica and its potential prey types. the 3.27 (female) and 3.16 (male) tl for the f ive m. japanica fall within those of secondary consumers. the stable isotope biplot displays the tl and carbon source of each taxon (figure 3). on the other hand, isotopic signals of m. japanica show an enriched 13c compared to potential preys which have mean values ranging from -19.46‰ (medium size zooplankton) to -17.15‰ (juvenile f ish), with the exception of f ish larvae (-13.83‰). mean δ13c values for ichthyoplankton (juvenile and larvae) differed with more depleted values for juveniles than those of larvae. sergestid s.i.g. masangcay et al. 33 shrimps a. intermedius had more enriched mean values (-18.47‰ for δ13c and 7.89‰ for δ15n) than those of lucifer spp (-19.03‰ for δ13c and 6.29‰ for δ15n). the mean δ13c values of both large zooplankton (zl 335-1000 μm, and zxl >1000μm) are more enriched at -18.65‰ to -18.40‰ in comparison to the value of smaller zooplankton (z1 100–200 μm: -19.27‰). the krill p. latifrons exhibited a mean δ13c value (-17.96‰) which is roughly similar to those of large zooplankton. figure 3. isotopic values of 13c and 15n for mobula japanica (mj) and potential prey from butuan bay, northeastern mindanao, philippines. zs, zooplankton (100–200 μm); zm, zooplankton (200–335 μm); zl, zooplankton (335–1000 μm); zxl, zooplankton (> 1000 μm); fj, f ish juvenile; fl, f ish larva; pom, particulate organic matter; ac, acetes intermedius; ka, adult pseudeuphausia latifrons; lu, lucifer spp. m. japanica (female) mj1 -16.20±0.16 10.69± 0.04 1 3.27 m. japanica (male) mj2 -15.68±0.35 10.44±0.25 4 3.16 acetes intermedius ac -18.45±0.10 7.89±0.23 3 2.44 lucifer spp. lu -19.03±0.27 6.29±0.30 3 2.44 p. latifrons ka -17.96±0.48 8.34±0.08 3 2.34 fish juvenile fj -17.15±0.55 7.37±1.00 2 2.29 fish larvae f l -13.83±4.67 7.11±0.47 2 2.22 macrozooplankton (>1000μm) zxl -18.40±1.36 6.40±0.19 3 2.01 mesozooplankton (100-200μm) zs -19.27±0.24 6.38±0.10 3 2.00 mesozooplankton (200-335μm) zm -19.46±0.12 6.71±0.12 3 2.10 mesozooplankton (335-1000μm) zl -18.65±0.21 6.47±0.80 3 2.03 microzooplankton (20-100μm) pom -15.96±1.32 2.78±0.80 3 1.82 taxa code mean δδδδδ13c mean δδδδδ15c n trophic position (tp) table 5. δδδδδ13c and δδδδδ15n isotopic values (mean±standard deviation) and trophic position of m. japanica and its potential prey. numbers in the species codes represent the repl icate used for analysis. feeding habits of mobula japanica 34 discussion diet of m. japanica all collected m. japanica (d w = 120–157 cm) in this study were immature (notarbatolo sciara 1988; white et al. 2006b) and dominated by males. according to sampson et al. (2010), m. japanica individuals that are <205 cm (d w ) are considered immature. the sizes of the m. japanica of the present study were def initely smaller compared to those of the new zealand population with individuals showing an average of 200 cm (d w ) and 100 cm (d l ) (francis and jones 2016). the predominance of immature individuals may reflect the movement of larger rays to other areas (white et al. 2006b). these rays were caught by f ishermen during the day, which validates the f indings of croll et al. (2012) that m. japanica is commonly observed at the surface waters (<5 m) during daytime and goes to deeper waters (>50 m) at night in search for food. devil rays in general are surface water dwellers that spend long periods in the surface during the day (gadig et al. 2003). it is suggested that surface aggregation of this species may be attributed to the daytime swarming of the krill prey p. latifrons (wilson et al. 2001) and nyctiphanes simplex (gendron 1992). generally, stomach content analysis (sca) provides information on prey items in l i m i t ed t i m e s c a l e s ( e . g . h o u r s to d a y s ) . i n t h i s s t u d y, s ca r e s u l t s co n f i r m e d m. japanica to have a strong feeding aff inity with planktonic organisms, which may be associated with its gill morphology (paig-tran et al. 2011). rays are eff icient in capturing zooplanktonic prey by funnelling microscopic plankton into their mouth and trapping them onto the gills which are made up of f ilter-like mesh of small bones (paig-tran et al. 2013). all 16 m. japanica actively fed since their stomachs contained quantif iable prey. by contrast, notarbatolo di sciara (1988) reported only 19 (24%) out of 78 m. japanica individuals had food in their stomach. in this study, the predominance of the tropical krill p. latifrons in almost all of the stomach contents of m. japanica was observed (masangcay et al. 2018). by comparison, the subtropical populations of m. japanica and m. thurstoni exclusively feed on the subtropical krill n. simplex, while m. munkiana feed on the mysid mysidium sp. (notarbatolo di sciara 1988; sampson et al. 2010). mobula thurstoni primarily feeds on euphausiids (gadig et al. 2003), but it was also observed to intensively feed on mysid shrimps (notarbartolo di sciara, 1988). manta birostris (wilson et al. 2001) and rhincodon typus (wilson and newbound 2001; jarman and wilson 2004) are also reported to prey on p. latifrons at daytime. these f indings are in close agreement with our study, which identif ies the krill species p. latifrons s.i.g. masangcay et al. 35 as the most important prey of m. japanica from butuan bay. in addition, our local plankton net tows did yield a few p. latifrons at the locations off buenavista where fishermen capture devil rays. the few p. latifrons collected is attributable to the inefficiency of the conical plankton net in catching micronektonic krill (nemoto 1983; wiebe et al. 2005). aside from adult krill, other prey items include krill eggs, whose abundance is due to the large number of egg-carrying female p. latifrons that can bear up to 164 eggs or more per individual (wilson et al. 2003a). although it is possible that the eggs would have been ingested as freely suspended eggs in the water column, we believe these were most likely dislodged from the mother krill’s brood pouch as a result of the peristalsis of the ray stomach. interestingly, the shallow water sergestid shrimp a. intermedius dominated one stomach of m. japanica, indicating ingestion of other shallow water/estuarine micronektonic crustaceans (jarman and wilson 2004) and a possible switch to alternative prey (notarbatolo di sciara 1988; wetherbee and cortés 2004). past studies on ray stomach content report the importance of pelagic micronektonic crustaceans like euphausiids (true krill), sergestid shrimps like acetes spp. , and other planktonic species (couturier et al. 2012). micronektonic crustaceans form dense swarms and f ilter-feeding devil rays might have evolved to track large aggregations of micronektonic crustaceans whose sizes ensure energy to support the activities of these pelagic megafauna (sampson et al. 2010; couturier et al. 2013). population structure analysis of p. latifrons showed changes in the size-structure, reflecting individual growth from january to may (masangcay et al. 2018). breeding season of this species appears to be during the warm and dry months of march to may, which coincides with the decrease in number of juvenile individuals and the increase in abundance of large egg-carrying females during these months. incidentally, the f ishing season of m. japanica in bohol sea (alava et al. 2002; acebes 2013; freeman 2014) and butuan bay (metillo and masangcay 2015) is from september to may with the peak season lasting from february to april. however, sightings and f ishing of devil rays could extend up to june in butuan bay (metillo and masangcay 2015) and bohol sea (freeman 2014). we are convinced that the preponderance of m. japanica individuals during these months could be linked with the availability of swarms of p. latifrons. high primary production in butuan bay drives the abundance of p. latifrons, which prefer habitats with high abundance of zooplankton depending on dense phytoplankton (wilson et al. 2003b). however, p. latifrons are reported to also feed on detritus (hirota and nemoto 1989). the peak of surface chlorophyll α is often o b s e r v e d i n b u t u a n b a y a t a r o u n d 2 0 – 3 0 m d e p t h ( v i l l a n oy, p e r s o n a l feeding habits of mobula japanica 36 communication). the primary production in butuan bay is at maximum during december to february when heavy rainfall causes highest river discharge (as indicated by highest chromophoric dissolved organic matter or cdom) (figure 4 in cabrera et al. 2011) and a pronounced plume from agusan river, the second largest river in the philippines (villanoy et al. 2011). another primary production enhancement mechanism in butuan bay is the “double estuarine type circulation”, which is mostly driven by the large inflow of waters from the pacif ic ocean passing through surigao strait and entrains large amounts of deep, nutrient-rich waters to the surface (cabrera et al. 2011). this mechanism is strongest during the months of december to march when the northeast monsoon winds generate the westbound surface current bohol jet in the bohol sea (cabrera et al. 2011). this regular circulation pattern, together with the highest freshwater discharge from the large agusan river, eventually leads to nutrient enrichment and phytoplankton bloom, which in turn fuel high zooplankton abundance that feed the p. latifrons population. c and n stable isotope values stable isotope analysis allowed the determination of a consumer-food relationship between m. japanica and its potential prey in butuan bay. the isotopic signature of δ15n reflects an organism’s trophic position, while isotopic differences among δ13c values can trace the original dietary carbon source of the consumer, whether it originated from a marine, freshwater, or terrestrial environment (shiffman et al. 2012). furthermore, δ13c gradients may also reflect the food web relationship between coastal or benthic, and offshore or pelagic regions (dahl et al. 2003; hussey et al 2011). depleted δ13c values (-22‰ to -17‰) denote pelagic feeding, whereas enriched δ13c values (> -17‰) imply coastal and/or benthic foraging (france 1 9 9 5 ) . here, we report the c and n stable isotopes values of one female and four male m. japanica individuals from butuan bay. values among m. japanica individuals did not differ, which agrees with the study of sampson et al. (2010) who repor ted no difference in δ13c and δ15n values between stage of maturity, between sex, among monthly values, and between species (m. thurstoni and m. japanica). similarly, couturier et al. (2013) found similar δ13c and δ15n stable isotopes in manta alfredi from both lady elliot island and north stradbroke island in queensland, australia. less stable isotope variation in these large organisms may be explained by the long-term (months) turnover rates of c and n stable isotopes in muscle tissues of mobulids (sampson et al. 2010). the low variability in stable isotope values (0.16— s.i.g. masangcay et al. 37 0.35 for δ13c and 0.04—0.25 for δ15n in this study) is also indicative of a highly specialized diet (sweeting et al. 2005). mean δ13c isotopic value (-16.07‰) of m. japanica falls within the enriched category which implies that its diet would most likely be composed of prey from offshore marine and planktonic habitat (france 1995). the present study reports comparable δ13c values reported in other mobulidae studies: m. thurstoni (-16.74‰) and m. japanica (-16.78‰) (sampson et al. 2010); m. diabolus (-16.02‰) (borell et al. 2011); and m. alfred i (-17.4‰) (couturier et al. 2013). 13c values of potential prey are enriched. the difference in values for juvenile and larval f ishes may be related with migration, wherein larvae are spawned in deep spawning ground (more enriched 13c signature) but juveniles move to shallow nursery habitats (more depleted 13c signature) (tanaka et al. 2008). therefore, the 13c values of the juvenile f ish acetes spp. , p. latifrons, and zooplankton are reflective of shallow neritic and estuarine organisms. the stomach content analysis in this study reveals that m. japanica preys on zooplankton, par ticularly micronektonic shrimps euphausiid (p. latifrons) and sergestid (a. intermedius) in butuan bay. the calculated trophic position based on mean values of m. japanica suggests the species is a low trophic level secondary consumer that assimilates nitrogen of primary consumers, concurring with the f indings of the stomach analysis. however, following the mean trophic enrichment factors (3.2‰ for δ15n and 1.8±0.29‰ for δ13c) of mccutchan et al. (2003), m. japanica would primarily eat not only the krill p. latifrons and the sergestid acetes intermedius, but also juvenile f ish. this is not surprising as other mobulids are reported to ingest ichthyoplankton, proving plasticity in its feeding habits (stewart et al. 2016). conclusion stomach contents of 16 m. japanica individuals were dominated by adults and eggs of pseudeuphausia latifrons euphausiid, followed by a much lesser amount of sergestid shrimps (lucifer sp. and acetes intermedius), copepods, and planktonic remains. larger female p. latifrons was observed to be the most dominant in the diet of m. japonica, which coincided during the peak of the reproductive cycle of the krill. stable isotopes of c and n in muscle tissues of f ive m. japanica individuals and potential preys conf irm the strong feeding aff inity of m. japanica with micronektonic crustaceans. this study is the f irst formal report on the feeding of m. japanica in tropical philippine waters. although the current f indings are useful feeding habits of mobula japanica 38 input to local conservation and management of m. japanica, we recommend that longer period of study should be made to include other ray species in the philippines. acknowledgements the authors gratefully acknowledge the department of research, msu-iligan institute of technology; department of science and technology (dost)-advance science and technology human resources development program; department of science and technology (dost )-science education institute (sei); and the japan society for the promotion of science (jsps) (the asian core and the core-to-core programs) for their financial and technical support. we also thank dr. mtrd sanchezmetillo for copyediting the manuscript. references acebes jmv. 2013. hunting “big fish”: a marine environmental history of a contested f ishery in the bohol sea [doctoral disser tation]. per th, western australia: murdoch university. alava mnr, dolumbalo erz, yaptinchay aa, trono rb. 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university-iligan institute of technology. she graduated m.s. marine biology from mindanao state university-iligan institute of technology on time as a dostasthrdp scholarship in 2016, and was hired immediately after graduation as instructor at the caraga state university, butuan city, philippines. ephrime b. metillo <ephrime.metillo@g.msuiit.edu.ph> is b.s. zoology graduate at the mindanao state university marawi city. he was university research assistant at the marine science institute of the university of the philippines diliman before doing a straight ph.d. program at the university of tasmania at hobart, australia under the australian international development assistance bureau (aidab) equity and merit scholarship scheme. he is now professor at the mindanao state university-iligan institute of technology, iligan city, philippines. ken-ichi hayashizaki is a marine scientist, an expert on stable isotope analysis and currently associate professor at kitasato university, sagamihara, japan. satoru tamada is a recent graduate of master of science marine bioscience at kitasato university, japan with focus on the use of stable isotope analysis in the ecology of the japanese flounder. shuhei nishida is a recently retired professor at the atmosphere and ocean research institute of the university of tokyo, japan. he has published more than 110 papers in the f ield of marine biology and became editor of the springer journal marine biology. his main f ield of interest is marine zooplankton biology and ecology. assessment-olavides1-12 corrected with better figures.pmd assessment of the sea cucumber resource 1science diliman (july-december 2010) 22:2, 1-12 assessment of the sea cucumber resource and fishery in the bolinao-anda reef system ronald dionnie d. olavides*, christine mae a. edullantes, marie antonette juinio-meñez the marine science institute, university of the philippines, diliman, quezon city 1101 *corresponding author email: olavides.ronald@gmail.com phone: +63 2 922 3959 abstract fishery-independent and -dependent surveys were conducted to assess the status of the sea cucumber resource and fishery in bolinao and anda, pangasinan. thirty-five species of sea cucmbers were recorded in 25 sampling stations within seagrass beds, coral reefs and mixed habitats. combined with previous studies in the area, there about 49 species of sea cucumbers in the bolinao-anda reef system. the estimated total population density of all aspidochirote sea cucumber species is 63 ind. has-1. the artisanal multi-species fishery is at present primarily based on holothuria scabra, stichopus horrens and bohadschia marmorata although there are indications that other high-value species were fished to local extinction. taken together, the small sizes (<15 cm body length) of the majority of aspidochirote sea cucumbers, their low population densities, and the continuous decrease in catches are clear signs of an overexploited fishery that will likely collapse without management intervention. adaptive management strategies for bolinao and anda are recommended based on the findings of this survey. keywords: biodiversity; holothurians; resource assessment; resource management; sea cucumbers; trepang introduction sea cucumbers are among the most important and highly priced marine invertebrate resources in the philippines. its fishery served as a significant source of livelihood for many of the coastal communities in the archipelago (domantay, 1934; trinidad-roa, 1987; nievales, 2007; choo, 2008), and forms the basis of a multi-million dollar export industry of trepang or dried sea cucumbers (gamboa et al., 2004). the insatiable market demand and unsustainable fishery practices have led to a rapid decline in high-value sea cucumber resources throughout the philippines and in many parts of the world (lawrence et al., 2004; battaglene & bell, 2006). although the philippines is the second largest exporter of tropical sea cucumbers in the world, there has been no specific effort to effectively regulate and manage the fishery on a national scale (casilagan & juinio-meñez, 2007). the scarcity of useful fishery baseline information in most regions is often cited as an obstacle in the formulation of a management plan (gamboa et al., 2004). this paper aims to address information gaps in the municipalities of bolinao and anda, both in pangasinan, luzon island, philippines. transect surveys were done to identify the sea cucumber species present in the area, their relative abundance, densities and size structure, and distribution in major marine habitats. interviews with sea cucumber collectors, processors and traders were undertaken to document local fishery methods and knowledge/perceptions, and characterize socioolavides, r.d. et al. 2 economic aspect of the fishery. this paper presents the results of the baseline surveys conducted in october 2007 to february 2009 and recommends adaptive management strategies for the sea cucumber fishery in bolinao and anda. materials and methods site selection and survey method sampling stations were selected from three broad habitat types, particularly seagrass, coralline and mixed habitats using a satellite map of the bolinao and anda reef system superimposed with remote sensing data. manta tows were undertaken to ground-truth preselected sites. gps coordinates of sampling stations are plotted in mapping software. a total of 25 sampling stations covering 37,500-m2 were surveyed in the bolinao-anda reef: 25,500 m2 in bolinao (19,500 m2 in fishing grounds and 6,000 m2 inside protected areas) and 12,000 m2 in anda (figure 1). three replicate 500 m2 (100 m x 5 m) belt transects per sampling station were laid perpendicular to the shore and surveyed by pairs of observers for sea cucumbers during daytime and within 1-20 m depth range. identification to genus and species level was done through examination of external morphology, microscopic dermal ossicles from tissue samples and using identification keys (e.g. domantay, 1960; tan tiu, 1981; reyes-leonardo et al., 1985; cannon & silver, 1986; conand, 1998; schoppe, 2000; desurmont, 2003; and kerr et al., 2006). aspidochirotes or those species belonging to families holothuriidae and stichopodidae were measured in its relaxed state either in situ or in the boat. as sea cucumbers contract when disturbed, many specimens were placed in 90l bins with seawater in the boat for a few minutes to let it regain its “relaxed” length and width. total body length (from mouth to anus) and maximum width were measured to the nearest cm by tracing a tape measure in its body contour. specimens were then taken out of the water for a few figure 1. location map of sampling stations in the bolinao-anda reef system science diliman (july-december 2010) 22:2, 1-12 assessment of the sea cucumber resource 3 minutes (to let it expel some water from its gut) and weighed to the nearest gram using a digital scale for individuals below 100 g and to the nearest 10 g using a weighing scale more than 100 g. meetings with stakeholders were held in coordination with the local government to identify stakeholders based on their involvement in the fishery (i.e. traders, processors and fishers). key informants near traditional sea cucumber fishery areas (e.g. barangays dewey, victory and pilar) were interviewed using stratified survey questionnaires and unstructured discussions on site. focus group discussions (fgd) were undertaken with a group of young sea cucumber collectors, old fishermen, and the middlemen traders of bolinao and anda. one-year price records dated june 2006–june 2007 from the sales receipts of a major sea cucumber trader in anda were analyzed. wholesale and retail buyers in binondo (manila’s chinatown) were also surveyed to gather and confirm information on product grading, pricing and insights on trepang trade. data handling and analysis the following formulae were used in the calculation of population parameters: population density per species (d): d = n i / a where: n i = total number of individuals per species a = total area covered in hectares relative abundance per species (% ab): % ab = d / d  the shannon index of general diversity was calculated using the formula: h = -s n i /n log n i /n where: n i = importance value for each species (i.e number of individuals) n = total of importance values results and discussion species richness, distribution, abundance and density a total of 35 sea cucumber species were found in the bolinao-anda reef system during the surveys, adding 15 new records to the taxonomic study done in bolinao, which reported 28 species (reyes-leonardo et al., 1985). the species inventory based on the surveys conducted and available literature shown in table 1 indicates that there are at least 49 species in bolinaoanda reef system. table 1 also shows the common names, local names and commercial values of the sea cucumbers listed. note that thelenota ananas, t. anax, and stichopus chloronotus were only found as dried products and four species under the order dendrochirotida are still being identified with the help of taxonomists. there are over 170 sea cucumber species in the philippines (clark & rowe, 1971; tan tiu, 1981; reyesleonardo, 1984; reyes-leonardo et al., 1985; lane et al., 2000; kerr et al., 2006). of these, over 137 species are present in the south china sea biogeographic region including those that are found in bolinao-anda reef system (reyes-leonardo et al., 1985; lane et al., 2000; this study). compared to the neighboring reef, the hundred islands in alaminos, has around 30 species (domantay, 1960; juinio-meñez et al., unpublished) and share at least 15 species in common with the present study. taxonomic works for sea cucumbers in calatagan, batangas (pacific side) reported 28 species (reyes-leonardo, 1984), while in mactan and the other islands off cebu (visayan sea) reported 27 species (tan tiu, 1981). in terms of species diversity, the shannon index for pooled sampling stations indicated high species diversity across habitats (1.98-2.50), as well as in the overall index for bolinao and anda, with 2.67 and 2.38, respectively (table 2). most of the sea cucumber surveys in the philippines, however, report only number of species and vary greatly in the total area surveyed whenever indicated. the number of species of all aspidochirotid sea cucumbers (order aspidochirotida) varied per station science diliman (july-december 2010) 22:2, 1-12 olavides, r.d. et al. 4 from 1-12 species with an average of 5 species. the most species-rich station was brgy. victory seagrass station (12 species), closely followed by brgy. pilar coralline station (11 species) and panacalan island mixed habitat station (11 species). from pooled stations per habitat, the most species-rich habitats were coralline stations, closely followed by seagrass stations (table 2). the mpa sampling stations were not any better compared to open-access coralline stations in terms of the number of species and individual sea cucumbers. in order to clearly establish patterns of species richness and elucidate occurences of species in the different habitat types there is a need to employ quantitative methods of selecting and categorizing sampling stations in future surveys. science diliman (july-december 2010) 22:2, 1-12 scientific name common names a local names value b order aspidochirotida 1 actinopyga echinites deepwater redfish khaki m 2 actinopyga lecanora + stonefish buli-buli m 3 actinopyga miliaris + blackfish khaki m 4 bohadschia argus leopardfish matang-itik, leopard l 5 bohadschia koellikeri + mottled sc lawayan nc 6 bohadschia marmorata brownspotted sandfish bi-ker, lawayan l 7 bohadschia similis + brownspotted sandfish bi-ker, lawayan l 8 bohadschia vitiensis brown sandfish lawayan-taba l 9 bohadschia sp. “spots” eye-spot sc matang-itik l 10 holothuria (acanthotrapeza) coluber snakefish balat-aso, patola rig vl 11 holothuria (cystipus) inhabilis nc 12 holothuria (halodeima) atra lollyfish black beauty l-vl 13 holothuria (halodeima) edulis pinkfish red beauty, hotdog vl 14 holothuria (halodeima) pulla + nc 15 holothuria (lessonothuria) pardalis nc 16 holothuria (lessonothuria) verrucosa nc 17 holothuria (mertensiothuria) leucospilota whitethreadsfish brown beauty, balat uwak vl 18 holothuria (metriatyla) albiventer marten’s sc rotong, batunan nc 19 holothuria (metriatyla) scabra sandfish putian, bokloden, kurtido h-m 20 holothuria (microthele) nobilis black teatfish susuan, kiskisan h-m 21 holothuria (microthele) fuscogilva white teatfish susuan, kiskisan h-m 22 holothuria (platyperona) difficilis nc 23 holothuria (selenkothuria) erinacea + nc 24 holothuria (stauropora) fuscocinerea variegated sc labuyo, puyos vl 25 holothuria (stauropora) pervicax stubborn sc sunlot, sunlutan vl 26 holothuria (thymiosycia) arenicola borrowing sc rotong nc 27 holothuria (thymiosycia) hilla tigertail sc rotong, batuli vl 28 holothuria (thymiosycia) aff. hilla epi’s sc rotong nc 29 holothuria (thymiosycia) impatiens impatient sc sunlot nc 30 pearsonothuria graeffei flowerfish flower vl 31 stichopus chloronotus* greenfish kwatro kantos h 32 stichopus hermanni curryfish gadul, hanginan h-m 33 stichopus horrens dragonfish, warty sc gadul, hanginan, daremusak h-m 34 stichopus variegatus + variegated sc gadul, hanginan h-m 35 stichopus sp. gadul, rotong h-m 36 thelenota ananas* prickly redfish pinya-pinya m 37 thelenota anax * amberfish legs m order dendrochirotida 38 actinocucumis typicus + nc 39 cladolabes schmeltzii + nc 40-43 4 unidentified dendrichirotid species nc order apodida 44 opheodesoma glabra + medusan sc rokosan 45 opheodesoma grisea medusan sc rokosan nc 46 pendekaplectana nigra + medusan sc rokosan nc 47 polyplectana kefersteini medusan sc rokosan nc 48 synapta maculata medusan sc rokosan nc 49 synaptula media sponge sc nc + reported in reyes-leonardo et al. (1985) but not found during the survey; * found as processed samples only; a sc=sea cucumber; b h(high)=p1,000-4000/kg, m(medium)=p500-1,000/kg, l(low)=p100-500/kg, vl(very low)=<p100/kg or rejected; nc=non-commercial table 1.sea cucumber species inventory for bolinao and anda, pangasinan with local names and trade value. assessment of the sea cucumber resource 5 the most frequently observed species were synapta maculata, holothuria impatiens, opheodesoma grisea, h. leucospilota and h. albiventer (table 2). holothuria scabra, h. fuscocinerea and h. impatiens were present in all the habitats surveyed. other species were observed to have narrow distributions with respect to habitat type, specifically pearsonothuria graeffei in coralline sites, bohadschia argus, h. inhabilis, and h. aff. hilla in seagrass sites, and h. coluber in macroalgae-dominated sites. holothuria fuscogilva, which was earlier thought to be depleted to local extinction, was observed only once in a coralline site in brgy. balingasay mpa. synaptula media and 4 species of dendrochirotid sea cucumbers (order table 2. presence, frequency of observation, species richness and species diversity of sea cucumbers in bolinao and anda sampling stations (excluding individuals and species found outside the transects) science diliman (july-december 2010) 22:2, 1-12 species name* bolinao anda p r e s e n c e i n s a m p l in g s t a t io n s ( % ) s e a g r a s s (5 s ta ti o n s ) c o r a l r e e f (6 s ta ti o n s ) m a c r o a l g a e (4 s ta ti o n s ) s e a g r a s s (4 s ta ti o n s ) m a c r o a l g a e (4 s ta ti o n s ) 1 actinopyga echinites ++ ++ ++ 25 2 bohadschia argus + 4 3 b. marmorata + + + ++ 21 4 b. vitiensis + + 8 5 bohadschia sp. “spots” + 4 6 holothuria (acanthotrapeza) coluber + + 8 7 h. (cystipus) inhabilis + 4 8 h. (halodeima) atra ++ + 8 9 h. (halodeima) edulis + 4 10 h. (lessonothuria) verrucosa + + 8 11 h. (mertensiothuria) leucospilota ++ ++ + +++ 33 12 h. (metriatyla) albiventer ++ ++ +++ + 33 13 h. (metriatyla) scabra + ++ + +++ 29 14 h. (microthele) fuscogilva + 4 15 h. (stauropora) fuscocinerea +++ + + ++ + 33 16 h. (stauropora) pervicax + + + 13 17 h. (thymiosycia) arenicola ++ + + + 21 18 h. (thymiosycia) hilla + ++ 13 19 h. (thymiosycia) aff. hilla + 4 20 h. (thymiosycia) impatiens + +++ +++ +++ ++ 50 21 pearsonothuria graeffei ++ 8 22 stichopus hermanni + 4 23 s. horrens + +++ + + 25 24 dendrochirotid sp. 1 + 4 25 dendrochirotid sp. 2 ++ 8 26 dendrochirotid sp. 3 + 4 27 dendrochirotid sp. 4 ++ 8 28 opheodesoma grisea +++++ ++ + + 38 29 polyplectana kefersteini ++++ + + 25 30 synapta maculata +++++ ++++ ++++ +++ +++ 79 31 synaptula media + + 8 species richness 19 17 10 12 17 species diversity (shannon index) of aspidochirotes 2.50 2.01 2.13 1.98 2.19 2.67 2.38 * of the 35 species found in bolinao-anda reef system, 3 species (stichopus chloronotus, thelenota ananas and t. anax) were found only as processed samples. olavides, r.d. et al. 6 dendrochirotida) were found to be aggregating in a small area in tomasa point, bolinao and were rarely found elsewhere during the survey. these species of dendrochirotids tend to aggregate in large numbers, have limited distribution, and small adult sizes. the number of aspidochirotid sea cucumber individuals per species in a site and even in pooled habitats was generally very low that density estimates per species were pooled for the whole reef (table 3). estimates of population densities ranged from 0.3 to 9 ind.ha-1 per species and a total of 63 ind.ha -1 for all aspidochirotes in the bolinao-anda reef. anda stations have denser populations compared to bolinao. the densities in fished coralline areas and coralline mpas were almost identical (46 and 44 individuals has-1, respectively). while mpas were expected to have higher population densities by providing refuge from fishing, the mpas sampled varied in the level of enforcement (e.g. from not guarded to strictly guarded by stakeholders), and as such, poaching could have negated the potential effect of closed access habitats on sea cucumber populations. among aspidochirotid sea cucumbers, holothuria impatiens, h. albiventer, s. horrens, h. scabra, h. fuscocinerea and h. hilla have the highest density with a range of 4-9 ind.ha-1 (table 3). although the minimum population densities for broadcast spawning sea cucumber species to reproduce effectively have not been established, densities less than 100 ind.ha-1 is considered low, and less than 30 ind.ha -1 may be within a critical level at which populations will fail to repopulate (purcell et al., 2009). the population densities of sea cucumbers in the bolinao-anda reef may be too sparse to achieve a high probability of table 3. actual counts of aspidochirote sea cucumbers (order aspidochirotida), estimates of density and relative abundances in bolinao-anda transect surveys. science diliman (july-december 2010) 22:2, 1-12 rank species number of ind. density (ind. has-1) r e l a t iv e a b u n d a n c e (% ) b o l in a o a n d a p o o l e d b o l in a o a n d a p o o l e d 1 holothuria impatiens 18 17 35 7.1 14.2 9.3 14.9% 2 h. albiventer 10 22 32 3.9 18.3 8.5 13.6% 3 stichopus horrens 9 17 26 3.5 14.2 6.9 11.1% 4 h. scabra 10 13 23 3.9 10.8 6.1 9.8% 5 h. fuscocinerea 11 6 17 4.3 5.0 4.5 7.2% 6 h. hilla 14 1 15 5.5 0.8 4.0 6.4% 7 pearsonothuria graeffei 15 0 15 5.9 0.0 4.0 6.4% 8 h. leucospilota 9 4 13 3.5 3.3 3.5 5.5% 9 a. echinites 4 6 10 1.6 5.0 2.7 4.3% 10 h. arenicola 8 1 9 3.1 0.8 2.4 3.8% 11 s. hermanni 0 8 8 0.0 6.7 2.1 3.4% 12 bohadschia marmorata 3 3 6 1.2 2.5 1.6 2.6% 13 h. edulis 0 5 5 0.0 4.2 1.3 2.1% 14 h. coluber 2 2 4 0.8 1.7 1.1 1.7% 15 h. pervicax 3 1 4 1.2 0.8 1.1 1.7% 16 h. atra 2 1 3 0.8 0.8 0.8 1.3% 17 h. rigida 2 1 3 0.8 0.8 0.8 1.3% 18 b. vitiensis 0 2 2 0.0 1.7 0.5 0.9% 19 h. verrucosa 2 0 2 0.8 0.0 0.5 0.9% 20 b. argus 1 0 1 0.4 0.0 0.3 0.4% 21 bohadschia sp. “spots” 1 0 1 0.4 0.0 0.3 0.4% 23 h. fuscogilva 1 0 1 0.4 0.0 0.3 0.4% 125 110 235 49.4 91.7 62.9 100 assessment of the sea cucumber resource 7 fertilization success during spawning events, thus, affecting contribution the larval supply of sea cucumbers. at present, the vulnerability of the sea cucumber stocks to decline is exacerbated by its unregulated fishery. size structure the majority of the aspidochirotid sea cucumbers found have mean lengths less than 15 cm (figure 2). the legal minimum length imposed in queensland, australia is 15 cm for all species (bruckner, 2005). this, however, may not provide enough chance for sea cucumbers to reproduce before being harvested and did not consider studies on species-specific size-at-first-maturity (e.g. conand, 1993). based on the species-based size limits in papua new guinea for 17 major species (bruckner, 2005), the majority of the sea cucumbers in bolinao and anda are undersized and should not be collected, processed or sold. the low abundance of large, sexually mature individuals for the main commercial species h. scabra, s. horrens, a. echinites, and b. marmorata reflects strong fishing pressure in the area, and indicates that reproductive potential is low relative to the reported population density. despite the low densities of adult sea cucumbers, the presence of some juvenile h. scabra in cangaluyan station, and numerous s. horrens in tondol station suggest that recruitment persist at least in these areas, although the source of larvae may be outside the area covered in the survey. catch composition, fishing techniques and fishery profile there are about 41 commercial species in the philippines (nfrdi, unpublished), and 25 of these are figure 2. lengths of commercial sea cucumbers species in bolinao and anda, pangasinan science diliman (july-december 2010) 22:2, 1-12 olavides, r.d. et al. 8 regularly collected for the trepang or dried sea cucumber product (schoppe, 2000). based on interviews with local processors and traders, 26 species are considered commercial in bolinao and anda: 7 of high-value, 5 of medium-value and the rest of minor or no value (table 1). the combined percentage of “low value species” (bohadschia and holothuria species with thin body wall), “medium value species” (actinopyga and thelenota species), and “high value species” (holothuria species with thick body walls and stichopus species) is only 23% (figure 3). most of the sampled commercial species belong to the “very low value” category, which refers to the “worm-like” species previously considered non-commercial. fishing high-value sea cucumber species such as h. scabra and s. horrens is mainly done by free-diving or gleaning. groups of gleaners typically collect shallow-water sea cucumbers in addition to shellfishes, crabs and seaweeds during low tide in intertidal flats often by wading or by raking the sediments. free-divers and spearfishers equipped with bamboo rafts (balsa), makeshift goggles and wooden fins, dive to depths of around 2-5 m to scan the bottom area, overturn rocks and pick any sea cucumbers in addition to target figure 3. relative abundance of sea cucumbers in bolinao, pangasinan per species and value category (non-aspidochirote species and species with very low abundance not shown but included in the computation) organisms. before the widespread depletion of sea cucumber stocks, air compressors allowed fishers to efficiently harvest deeper water species such as thelenota ananas, t. anax and h. whitmaei. dedicated nighttime harvests are also done monthly or seasonally for predominantly nocturnal species (e.g. stichopus spp., bohadschia spp., and actinpyga lecanora). this multi-species fishery is an important supplemental livelihood for 90% (n=29) of the respondents in bolinao, of which 57% are young males, 29% are adult males, and 14% are adult females. the young males, mostly primary school leavers, are free-divers whereas the adult males are engaged in more varied fishery activities often using balsa or motorized bangka. the women augment the income of the family by weaving buri (palm leaves) sleeping mats and gleaning. historical trends in the local fishery interviews and focused group discussions with fishermen in bolinao (n=16) and anda (n=18) who collected sea cucumbers since 30 years ago revealed major changes in the fishery (figure 4; paña et al., science diliman (july-december 2010) 22:2, 1-12 assessment of the sea cucumber resource 9 unpublished data). in the early 1970’s, there were only around 25 fishing households who regularly gleaned and trawled for sea cucumbers, primarily targeting b. marmorata, h. fuscopunctata, h. scabra, h. atra, and s. horrens. chinese traders who visited the area stimulated local interest in the fishery, and also taught some fishers how to process sea cucumbers into trepang the traditional way. fishing efforts soon intensified reaching an average of 100 kg day-1 per person during its peak in the 1980’s, which coinciding with the “boom” in sea cucumber fishery exports of the philippines (trinidad-roa, 1987). over 35 families in brgy. victory alone regularly harvested high-value sea cucumbers by the boatload, often by means of compressor-diving. the target species now included a. echinites, h. fuscogilva, h. nobilis, t. ananas and t. anax. in the 1990’s, however, there was a sharp decline in catch to an average of 25 kg day-1 per person, and collection was done twice or thrice a week. based on catch landings in brgy. pilar in 2002, the average daily catch per gleaner was 2.55 kg day-1, and total catch for all gleaners was only 27 kg day-1 (nievales et al., unpublished data). the average monthly catch for h. scabra in the municipality of anda, peaked at 109 kg in 2002 but sharply declined to around 20 kg in 2004, and has not been able to recover since (pastor et al., 2007). five more lower value species were added to the list of targeted species, namely: h. leucospilota, h. coluber, h. fuscocinerea and a. lecanora. longtime fishers also note the remarkable decline in average size of sea cucumbers caught, and their need to cover more area to make the trip worthwhile. many coastal barangays that traditionally collect sea cucumbers like brgy. pilar and brgy. victory reported that income derived from sea cucumber fishing has become less significant as a means of livelihood. overall, the fishery has downgraded from commercial to artisanal level and is characterized by the collection of smaller-sized sea cucumbers in an expanding fishing ground, increasing number species, increasing proportion of low value types, and a diminishing catch per effort. despite the decline of sea cucumber stocks due to unsustainable fishing practices, the fishery remains largely unmanaged. recent trend in sea cucumber trade the traditional market flow in bolinao and anda is that fishers sell their catch (for as low as php 40 kg-1 for assorted species and size) to local processors-traders who, in turn, sell dried sea cucumbers to a middlemen or the satellite branch of a marine products company in alaminos city. the price of the product depends on the species, and “grade” according to size and specific product standards for appearance, texture, odor, and hardness based on cursory assessment by traders (akamine 2001). the market testing done for bolinao and anda products revealed that most of the mediumsized h. scabra products were assessed as “class b” and priced at php1,300 kg-1 because of inadequate cleaning and drying (“class a” is priced at php 2,900 kg-1). although the binondo, manila market offers much higher profit margins for the fishers (table 4), most of the local stakeholders who sell directly to wholesale buyers in binondo also lose considerable potential income due to poor product quality. poorly processed and small-sized trepangs are especially prone to downpricing. alternative direct markets in binondo include the authentic chinese restaurants along ongpin st., arranque wet market where “rehydrated” trepang is bought anywhere around php 280-500 kg-1, and chinese grocery stores that sell prime grade trepang for php 6,000 kg-1. the price of the high-value species typically fluctuates every month but eventually leads to a yearly increase. based on sales records for stichopus spp, prices increase almost monthly for all size grades, with a peak between february and march coinciding with the chinese new year. small but steady price increases are also apparent for low-value species such as holothuria coluber, h. edulis, h. fuscocinerea, h. leucospilota, and h. atra. strong market pressure on the unregulated fishery is further reflected by the multilevel categorization even for the small-sized products (e.g. sizes “s”, “xs” and “xxs” for h. scabra and a. echinites) and the shift of proportion of the products towards the lower value species that are always sold for less than php 1,000 kg-1 regardless of processing quality and size. in any case, premium prices are paid for large individuals while prices for smaller products declines disproportionately (battaglene & bell, 2006). science diliman (july-december 2010) 22:2, 1-12 olavides, r.d. et al. 10 figure 4. trend of catch per unit of effort for commercial sea cucumbers in bolinao and anda, pangasinan from 1970’s to present based on 16 respondents from bolinao and 18 respondents from anda. science diliman (january-june 2010) 22:1, 1-12 species grade price per kg (php) scientific name trade name size grade size or no./kg alaminos, pangasinan binondo, manila stichopus horrens dudlo, hanginan, gadul, daremusak l 3.1 in. 2,280 2,600 m 2.5-3.0 2,080 2,300 s 3.0-2.5 1,750 1,700 xs 1.5-2.0 1,250 1,250 xxs <1.5 1,250 class b, xxs <1.5 700 holothuria scabra putian, kurtido xxl 15 pcs. 4,800 xl 16-26 2,480 4,500 l 26-35 1,750 3,800 m 36-45 1,400 2,900 s 46-60 950 2,300 xs 61-80 780 2,100 xxs >80 1,400 class b, l 26-35 1,500 class b, m 36-45 950 1,300 class b, s 46-60 1000 1,200 class b, xs 61-80 1,100 bohadschia marmorata lawayan l 730 950 m 700 750 s 500 500 xs 200 holothuria coluber patola orig assorted 540 600 holothuria atra black beauty m-l 400-600 s or assorted 180 180 holothuria leucospilota brown beauty assorted 250 410 250-420 pearsonothuria graeffei tres cantos s-l 70-240 assorted 170 holothuria fuscocinerea labuyo, puyos l 180 s or assorted 100 115 130 table 4. price list of common commercial sea cucumbers from wholesale buyers in alaminos and binondo. assessment of the sea cucumber resource 11 conclusions and recommendations as in many parts of the philippines where surveys have been conducted, the sea cucumber resources in the bolinao-anda reef system is characterized by high species diversity. thirty-five species recorded in this study, and when combined with previous species inventories in the area, a total of 49 species can be found in bolinao-anda reef system. new species or records are likely to be found in often overlooked or less known taxa such as the dendrochirotids. species distributions in the habitat types suggest habitat association in several species such as pearsonothuria graeffei, holothuria inhabilis and the dendrochirotids. on the other hand, population densities of aspidochirotid species including high-value species (e.g. holothuria scabra and stichopus horrens) fall critically below the level for fertilization success during spawning, which could indicate that the area is a poor larval source for sea cucumbers. size structure of major commercial species indicates strong fishing pressure. interviews provided corroborating anecdotal accounts of the fishery’s decline since the 1980s due to overfishing. furthermore, potential income is not realized due to poor post-harvest and trade practices. taken together, these are clear signs of an overexploited fishery that is likely to collapse without proper management intervention. we, therefore, recommend the following management strategies based on the findings and insights from the surveys undertaken in bolinao and anda: 1) protection and monitoring of critical nursery areas where juveniles of high-value species were found (e.g. brgy. tondol and cangaluyan); 2) maintenance of the pilot sea cucumber ranching sites established in bolinao and anda as reproductive reserves for sea cucumbers and supplemental livelihood for the stakeholders; 3) explore sea cucumber restocking using wild stocks in the bolinao seagrass demonstration site to help rebuild a multi-species community of sea cucumbers; 4) restrict and sanction the gathering or processing of any live sea cucumber below 350 g or 20 cm in length, and the trading of less than 5 cm dried sea cucumber (or over 60 pieces per kilogram); 5) establish a registry, fishing permit and catch monitoring system in the municipal level in order to gather long-term fishery data, and encourage compliance to regulations; 6) improve product value of trepang through postharvest support and quality control at the municipal level for fishers and processors; 7) integration of the recommended strategies for sea cucumber management into municipal fisheries management plan and municipal fisheries ordinance following the adaptive management approach. acknowledgment we are grateful for the cooperation of the municipality of bolinao and anda, and smmv and sbma people’s organization; dr. miguel fortes and the bsds project for the satellite-derived habitat map; dr. steven purcell and dr. ruth gamboa for the helpful review and comments; tirso catbagan and christopher ragos for assistance in the field; marie antonette paña for providing inputs on the socio-economic aspects; dr. alexander kerr for providing taxonomic references; and dost-pcamrd for funding this study. this paper was presented at the 10th pams national symposium held in davao city in october 2009. this is marine science institute contribution no. 398. references akamine, j., 2001. trepang exploitation in the philippines: updated information. spc bêche-de-mer information bulletin 17:17–21. battaglene, s.c., & j.d. bell, 2006. the restocking of sea cucumbers in the pacific islands. p. 109–132. in bartley d.m., & k.l. leber (eds.). case studies in marine ranching. fao fisheries technical paper no. 429. bruckner, a., 2005 (ed). the proceedings of the technical workshop on the conservation of 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october 1987. 15– 17 pp. science diliman (july-december 2010) 22:2, 1-12 ocr document 5pajarito-effect of zeolite.pmd effect of zeolite treatment on the blooming behavior of paraff i n wax 34 science diliman (january-june 2016) 28:1, 34-53 effect of zeol ite treatment on the blooming behavior of paraffin wax in natural rubber composites bryan b. pajarito* university of the philippines diliman nico v. berba university of the philippines diliman jadreign keisheen c. par to university of the philippines diliman raechel anne v. yabut university of the philippines diliman abstract the blooming behavior of paraff in wax in natural rubber (nr) composites w a s s t u d i e d a s f u n c t i o n o f z e o l i t e t r e a t m e n t . t h r e e t y p e s o f z e o l i t e treatment were treated as factors: acid activation using hydrochloric acid ( h c l ) s o l u t i o n , i o n e x c h a n g e u s i n g t e t r a d e c y l d i m e t h y l a m i n e ( t d a ) c h l o r i d e s a l t , a n d o r g a n i c m o d i f i c a t i o n u s i n g g l yce r o l m o n o s t e a r a te (gms). the zeolite was treated according to a 23 full factorial design of ex p e r i m e n t . at te n u a ted to t a l r e f l ec t a n ce – f o u r i e r t r a n s fo r m i n f r a r ed (atr-ftir) spectroscopy was used to characterize the chemical structure of treated zeolite. treated zeolite was applied as f iller to nr composites deliberately compounded with high amount of paraff in wax. the amount of bloomed wax in surface of nr composite sheets was monitored with time at 50oc. results show the bloom amount to be linear with the square root of time. nr composites reinforced with untreated, acid-activated, a n d i o n exc h a n g ed zeo l i te f i l l e r s i n d i c a te r ed u c t i o n i n w a x b l o o m i n g as compared to unf illed nr. the bloom rate (slope) and initial bloom (y-intercept) were determined from the experimental plots. analysis of v a r i a n ce ( a n ova ) s h ow s t h e b l o o m r a te to b e s i g n i f i c a n t l y i n c r e a s ed when zeolite f illers are treated with gms. meanwhile, initial bloom was signif icantly enhanced when zeolite f illers are treated with tda chloride _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online b.b. pajarito, et al 35 salt and gms. the signif icant increase in bloom rate and initial bloom can be attributed to the softening of the nr matrix at high amounts of tda chloride salt and gms. keyword s: natural rubber, composite, paraff in wax, blooming, zeolite introduction blooming is a phenomenon wherein soluble and oversaturated additives migrate from the bulk matrix and form a deposit at the rubber surface. the deposit can be solid precipitates or exudates of the migrated additives. a large number of additives are known to bloom in vulcanized rubber, such as sulfur, activators, antidegradants, various accelerators, process oils, and plasticizers (venable and greene 1922; auerbach and gehman 1954; nah and thomas 1980; wake et al. 1983; keen et al. 1992; sugiura et al. 1996; choi 1997, 1998, 1999a, 1999b; jurkowski and jurkowska 1998; ciesielski 1999; parra et al. 2000; bielinski et al. 2005; mark et al. 2005; bart 2006; dick 2009, 2014; basak et al. 2010; saeed et al. 2011, 2012a, 2012b; torregrossa-coque et al. 2011a, 2011b; ). while antidegradants, such as wax and paraphenylenediamines, are designed to purposely migrate at the rubber surface and create a passive barrier against ozone attack, the appearance of bloom is usually undesirable. unwanted bloom can cause unaesthetic appeal, decreased building tack and interfered adhesion to material surfaces, skin irritation (bart 2006), and reduced fatigue life of rubber (saeed et al. 2011). it also increases manufacturing cost and lowers labor productivity. bloomed surfaces of rejected rubber articles are usually washed with solvents or flushed with a steam jet (pedretti 1967) for possible recycle or reuse. however, most cases of blooming are persistent and require further surface treatments. research works on additives that can reduce blooming in rubber are very few. pedretti (1967) claimed additives from a group consisting of dialcohols, such as alkylene glycols and polyakylene oxides; aliphatic polyalcohols, such as glycerine and erythrite; and mixtures of aliphatic alcohols in equal parts by weight with silicic acids, alkylene glycols, and polyalkylene oxides, as additives capable of lowering and inhibiting bloom. sugiura et al. (1996) worked on the use of sepiolite on preventing bloom in ethylene-propylene-non-conjugated diene terpolymer (epdm). fujiki and tanaka (2002) patented a bloom inhibitor, which comprises an alkylene oxide adduct of a saponif ied ethylene-saturated carboxylic acid vinyl ester copolymer and polyether compound. effect of zeolite treatment on the blooming behavior of paraff i n wax 36 this work employed zeolites as potential additives for reducing and inhibiting bloom in rubber. zeolites are compounds of naturally occurring aluminosilicate minerals with symmetrically stacked alumina and silica tetrahedral, resulting to an open and stable three-dimensional crystalline structure (similar to a honeycomb) with a negative charge. they are often used as adsorbent and ion-exchange materials (siriyong and keawwattanna 2012). while inclusion of zeolite in rubber may yield to bloom reduction, its application does not always result to improved mechanical properties (al-ghamdi and mark 1988; siriyong and keawwattanna 2012) due to its inherent hydrophilic nature and poor dispersion in the rubber matrix. chemical treatments, such as acid activation, (christidis et al. 2003; bukit and frida 2013), ion exchange with amine salts containing hydrophobic functional groups (chakraborty et al. 2009), and organic modif ication with surfactants (das et al. 2011; rooj et al. 2012), can improve the compatibility of zeolite with rubber molecules. these treatments for inorganic mineral f illers are often cited for improving the physical and material proper ties of rubber composites; however, their effect on blooming behavior is yet to be explored. this work studies the effect of different zeolite treatments on the blooming behavior of paraff in wax in natural rubber (nr) composites. paraff in wax was chosen as a model compound to deliberately bloom in nr and its composites. specif ically, this work has the following objectives: (1) investigate the blooming behavior of paraff in wax with time; (2) determine the effect of reinforcing nr with raw and treated zeolite f illers to blooming behavior; and (3) determine how chemical treatment of raw zeolite affects blooming in nr composites. three chemical treatments of raw zeolite were considered as factors in a full factorial design of experiment: acid activation using hydrochloric acid (hcl) solution, ion exchange using a tertiary amine chloride salt, and organic modif ication using a non-ionic surfactant. attenuated total reflection – fourier transform infrared (atr-ftir) spectroscopy was used to characterize the chemical structure of treated zeolite f illers. amount of bloom in surface of rubber sheet specimens was monitored gravimetrically with time. from the experimental results, analysis of variance (anova) was utilized to determine the zeolite treatments that have signif icant effects on the blooming behavior. main and interaction effects of signif icant zeolite treatments were computed and discussed. b.b. pajarito, et al 37 materials and methods materials zeolite powder (hscas, saile industries) was used as received. the untreated powder has a cation exchange capacity (cec) of 171.55 meq per 100 g of zeolite. hcl (ar1107-g2.5l, rci labscan limited) was used for the acid activation of zeolite. tetradecyldimethylamine tda (farmin d4098, pilipinas kao, inc.) and glycerol monostearate gms (cerin gms 100 se, chemrez technologies, inc.) were utilized for ion exchange and organic modif ication of zeolite, respectively. figure 1 shows the chemical structures of tda and gms. figure 1. chemical structures of (a) tda and (b) gms. acid activation two liters of hcl solution at specif ied concentration (1.5 or 3 m) were prepared in a 4-liter glass beaker at 80oc. zeolite (166.7 g) was added to the hot solution and stirred occasionally for 40 minutes. after stirring, the hot slurry was cooled at room temperature, allowing zeolite to settle at the bottom of the beaker. excess liquid was f iltered using a buchner setup to recover acid-activated zeolite. after f iltration and decantation, acid-activated zeolite was washed with tap water until the ph of wash water is greater than six. treated zeolite was then recovered by f iltration and oven dried at 95oc for at least 24 hours. dried acid-activated zeolite was further ground using a mortar and pestle before storage in a sealed plastic container. effect of zeolite treatment on the blooming behavior of paraff i n wax 38 ion exchange with amine salt sixty grams of zeolite was dispersed in three liters of distilled water in a 4-liter beaker using a disperser (ika t50 digital ultra-turrax) for one minute at 5000 rpm. mixing temperature was maintained at 80oc using a silicone rubber heating tape (briskheat) controlled by a digital thermostat (dt-6, uplift accessories). a required amount of tda (1.5 or 3 equivalent times the cec of zeolite) was stirred with an equivalent amount of hcl solution in 500 ml water at 80oc. the hot amine salt solution was then added to the zeolite slurry, with the stirring continuing for an hour at 80oc and 1250 rpm using a digital overhead stirrer (ika rw20). after stirring, the hot slurry was cooled at room temperature. ion-exchanged zeolite was recovered by sedimentation, decantation, and buchner f iltration. the collected zeolite was oven dried at 95oc and ground with mortar and pestle before storage. organic mod if ication with surfactant one hundred twenty grams of zeolite was mixed with specif ied amounts of gms (1.5 or 3 wt% of zeolite) using mortar and pestle. the mortar, along with the mixture, was kept in an oven at 100oc for 15 minutes. the mixture was then homogenized thoroughly at hot condition with the pestle. the process was repeated three times before storage. design of experiment table 1 shows the 23 full factorial design (lazic 2004) employed during the chemical treatment and preparation of zeolite f illers. sample l is the raw and untreated l 0 0 0 a 3 0 0 b 0 3 0 c 0 0 3 a b 3 3 0 a c 3 0 3 bc 0 3 3 abc 3 3 3 0 1.5 1.5 1.5 label a-hci concentration, m b-amount of tda salt, nx the cec of zeolite c-amount of gms, wt% of zeolite table 1. full factorial design of experiment employed during chemical treatment and preparation of zeol ite fillers b.b. pajarito, et al 39 zeolite, while samples a, b, and c are the acid-activated (3 m of hcl solution), ionexchanged (tda chloride salt at 3x the cec of zeolite), and organo-modif ied (gms at 3 wt% of zeolite) zeolites, respectively. zeolites from varied combinations of treatments (samples ab, ac, bc, and abc) were also prepared as required by the design of experiment. sample 0 was treated with 1.5 m of hcl solution, tda chloride salt at 1.5x the cec of zeolite, and gms at 1.5 wt% of zeolite. a total of nine samples of zeolite f illers (95 g each sample) were prepared and oven dried at 120oc for at least 48 hours before sending to rhodeco rubber processing services, inc. (caloocan city) for the compounding and vulcanization of the nr composites. atr-ftir spectroscopy of treated zeol ite chemical changes on the treated zeolite samples were studied using an ftir spectrometer (thermo nicolet 6700) coupled with a macro diamond atr crystal plate accessory. a total of 32 scans were co-added at 2 cm-1 resolution from midinfrared range of 400 to 4000 cm-1. background spectra were obtained without zeolite samples prior to actual analysis. nr composite formulation table 2 shows the formulation of the nr composite used during compounding. the formulation was derived from previous work (pajarito et al. 2014) with additives, such as reclaimed rubber, calcium carbonate (caco 3 ), kaolin clay, asphalt, and used oil, replaced by zeolite f iller. moreover, the amount of paraff in wax in the formulation was deliberately increased from 1.50 to 7.50 phr to induce blooming. natural rubber and zeolite f iller were f irst mixed in a two roll mill, followed by zinc oxide, stearic acid, and paraff in wax. the resulting master batch was aged for 24 hours at ambient conditions before roll milling again with sulfur, mercaptobenzothiazole disulf ide (mbts), mercaptobenzothiazole (mbt ), and diphenylguanidine (dpg). nr composites were vulcanized using a heated compression moulding press at 160 oc for 20 minutes. rubber sheets have f inal dimensions of 300 x 300 x 3 mm. it should be noted that all nr specimens (unf illed nr and 9 types of nr composites) were compounded for two days before being subjected to vulcanization (~ 0.5 day). all vulcanized nr sheets were cooled at ambient conditions for at least an hour, packed, and delivered to polymer research laboratory for testing. accelerated blooming experiment was immediately performed after receipt of nr sheets. effect of zeolite treatment on the blooming behavior of paraff i n wax 40 accelerated blooming experiment specimens of 50 x 50 mm were cut from the nr composite sheets. initial weights of the specimens (w i ) were recorded before the experiment. the blooming experiment was performed using a laboratory oven maintained at 50oc for 120 hours. specimens were stored in individual aluminium foil pouches before they were placed in the oven. the amount of bloom was determined by periodically removing the bloomed additives from the rubber surface using f ive passes of adhesive tape (scotch tape), and measuring the resulting weight loss in an analytical balance (nah and thomas 1980). bloom amount (m t in wt%) in rubber specimens was calculated by: 100   i ti t w ww m (1) where w t is the weight of specimen after bloom removal using adhesive tape at time t. three replicate nr composite specimens per zeolite sample were used to report the average bloom amount. results and discussion figure 2a shows the atr-ftir spectra of the zeolite samples. all spectra show distinct peaks at 439 – 447 cm-1 and 1018 – 1034 cm -1, indicating si-o or al-o bending mode and asymmetric tetrahedral stretching, respectively (christidis et al. 2003). decrease in the intensity and broadening of peak at 1018 – 1034 cm -1 for table 2. formulation of nr composite used during compound ing ingred ient phr natural rubber (spr 20) 100.00 zinc oxide 6.00 stearic acid 1.50 sulfur 2.50 mercaptobenzothiazole disulf ide (mbts) 1.50 mercaptobenzothiazole (mbt) 1.50 diphenylguanidine (dpg) 1.50 paraff in wax 7.50 zeolite f iller 4.75 b.b. pajarito, et al 41 acid-activated zeolite samples indicate the destruction of free linkages and sitetrahedra by dealumination. dealumination by acid activation is also indicated by the slight shift of peak at 1018 – 1034 cm-1 to higher wave numbers (holmberg et al. 2004). figure 2. atr-ftir spectra of (a) zeolite samples and (b) tda and gms. effect of zeolite treatment on the blooming behavior of paraff i n wax 42 zeolite samples ion exchanged with tda chloride salt display specif ic peaks at 1427 – 1460, 2846 – 2854, and 2916 – 2924 cm-1. the peaks at 2846 – 2854 and 2916 – 2924 cm-1 are prominent in samples b and bc, par tially visible in sample abc, and faint in sample 0. a small peak at 1427 – 1460 cm-1 is also observed in samples b, bc, and abc. these peaks can also be found in the atr-ftir spectrum of tda (figure 2b), which can be attributed to c-h, c-c, and n-c vibrations in the tertiary amine salt (nezamzadeh-ejhieh and badri 2011). zeolite samples organo-modif ied with gms (samples c, ac, bc, abc, and 0) do not exhibit the broad o-h peak at 3300 cm-1 and the c=o stretch at 1735 cm -1 for carbonyls, carboxylic, and its derivatives, which are functional groups present in gms (figures 1 and 2b). this result is expected due to the small amount of gms used during organic modif ication (1.5 or 3 wt% of zeolite). it should be noted that the chemical treatments also change the particle size of zeolite f illers. for instance, raw and untreated zeolite has a median diameter (measured by sieve analysis) of 52.9 μm. acid activation decreases the measured particle size (e.g. median diameter of sample a is 37.5 μm), while ion exchange with tda chloride salt and organic modif ication with gms increase the par ticle size (e.g. median diameter of sample bc is 78.3 μm). the decrease in particle size after acid activation is due to dealumination, while the increase in particle size after ion exchange and organic modif ication can be attributed to the aggregation of treated zeolite f illers. it is noticeable during preparation that the acid-activated zeolite f illers are visually f iner than raw zeolite, while ion-exchanged and gmstreated zeolite f illers are sticky and tend to form clumps during drying. figure 3a shows an optical image of nr composite surface covered with bloomed paraff in wax. bloomed paraff in wax was removed and collected from the rubber surface using adhesive tape (figure 3b). the resulting weight loss is equivalent to bloom amount. figure 4 shows the bloom amount in terms of cumulative weight loss in nr composite specimens as a function of the square root of time at 50oc. all specimens show increasing bloom amount with time. as predicted by nah and thomas (1980), bloom amount was also found to be linear with the square root of time, which indicates diffusion by fickian mechanism. the experimental plots have non-zero bloom amount at t = 0 h, indicating the presence of wax bloom at the specimen surface even before the blooming experiment was initiated. nr composites reinforced with raw (sample l), acid-activated (sample a), ion-exchanged (sample b), and both acid-activated and ion-exchanged (sample ab) zeolite f illers exhibit decrease in wax blooming as compared to nr without zeolite. in terms of b.b. pajarito, et al 43 reducing bloom in nr composites, the zeolite f illers can be ranked as follows: l > a > ab > b. the observed reduction in blooming due to the inclusion of zeolite in nr can be attributed to the inherent porosity of the f illers. meanwhile, zeolite sample c has the same blooming behavior as nr without zeolite (figure 4a), while samples ac, bc, abc, and 0 enhance wax blooming (figure 4b). zeolite ac has the highest bloom amount among the samples (ac > bc > 0 > abc > c). while zeolite f illers are expected to impart rigidity in the rubber matrix and impede the migration of paraff in wax to the rubber surface, the treatment with gms could have lowered the inherent porosity of natural zeolite, with gms molecules occupying and limiting figure 3. optical images of (a) nr composite surface covered with bloomed paraff in wax, and (b) bloomed paraff in wax removed and collected from the rubber surface using adhesive tape. surface of nr composite after 5 passes of adhesive tape bloom of paraffin wax bloomed paraffin wax collected by adhesive tape effect of zeolite treatment on the blooming behavior of paraff i n wax 44 the availability of the pores for wax adsorption. unreacted gms (molecules of gms not attached to zeolite) could also have plasticized the rubber matrix, negating the rigidity offered by zeolite f illers and allowing paraff in wax to diffuse through the soft rubber matrix. note that combining gms treatment with acid activation (samples ac, 0, and abc) and ion exchange with tda salt (samples bc, 0, and abc) result to an increase in bloom amount when compared to nr without zeolite and nr f illed with sample zeolite c. the reduction in bloom amount observed in nr containing zeolite samples a, b, and ab can be considered ineffective after the addition of the gms treatment. the effect of gms treatment on bloom amount is more pronounced as compared to other zeolite treatments. figure 4. bloom amount in terms of cumulative weight loss in nr composite specimens as function of the square root of time at 50oc. b.b. pajarito, et al 45 a line was f itted with the experimental data to determine the bloom rate (slope of the line in %-h-0.5) and initial bloom (y-intercept in %). table 3 shows the average bloom rate and initial bloom determined from the experimental plots. the coeff icient of determination r2 calculated from 30 data sets (8 samples from 23 full factorial design, sample 0, and control; 3 replicates each zeolite sample) ranges from 0.952 to 0.999. in terms of bloom rate, raw zeolite (sample l) shows the lowest value at 0.030 %-h-0.5, while zeolite activated with 1.5 m hcl, ion exchanged with tda chloride salt at 1.5x the cec of zeolite, and organo-modif ied with gms at 1.5 wt% of zeolite (sample 0) yields the highest value at 0.040 %-h-0.5. for the initial bloom, acid-activated zeolite (sample a) exhibits the lowest at 0.789%, while acid-activated and organo-modif ied zeolite (sample ac) shows the highest value at 1.537%. l 0.030 0.816 a 0.036 0.789 b 0.034 1.232 c 0.037 1.354 a b 0.037 1.044 a c 0.037 1.537 bc 0.039 1.438 abc 0.039 1.400 0 0.040 1.434 nr without zeolite 0.031 1.380 bloom rate, %-h-0.5 initial bloom, % label table 3. average bloom rate and initial bloom determined from the experimental plots using bloom rate and initial bloom as responses, anova was executed in minitab 17 to determine the signif icant main and interacting factor treatments affecting blooming behavior in nr composites. the following model was utilized during anova: cbaabccbbccaacbaabccbbaa xxxxxxxxxxxxy   0 (2) where y is the response (bloom rate or initial bloom), â 0 , â a , … , â c are the regression coeff icients, x a is hcl concentration during acid activation (0 or 3 m), x b is amount of tda chloride salt (nx the cec of zeolite) during ion exchange (0 or 3), and x c is the amount of gms during organic modif ication (0 or 3 wt% of zeolite). after data analysis, response functions obtained for the bloom rate (y 1 ) and initial bloom (y 2 ) are given by equations 3 and 4, respectively: effect of zeolite treatment on the blooming behavior of paraff i n wax 46 (3) (4) tables 4 and 5 show the anova results for bloom rate and initial bloom, respectively. for bloom rate, only the organic modif ication using gms was found to be statistically signif icant at 95% conf idence level (p = 0.035). figure 5 shows the main effect of acid activation (a), ion exchange with tda chloride salt (b), and organic modif ication with gms (c) on bloom rate of nr composites reinforced with zeolite. the treatment with gms surfactant at 3 wt% of zeolite signif icantly increased the bloom rate from 0.034 to 0.038 %-h-0.5. though not statistically significant, acid activation and ion exchange with tda chloride salt are also observed to increase bloom rate in nr composites. in the case of the initial bloom, the main and interaction effects of ion exchange with tda chloride salt and organic modif ication with gms are found to be statistically signif icant. figure 6 shows the main effect of acid activation (a), ion exchange with tda chloride salt (b), and organic modif ication with gms (c) on initial bloom of nr composites reinforced with zeolite. ion exchange treatment signif icantly increased the initial bloom from 1.124% to 1.279% (p = 0.028). organic modif ication with gms has the same direction of effect, enhancing the initial bloom from 0.970% to 1.432% (p = 0.000). in terms of magnitude, organic modif ication with gms has higher effect on increasing the initial bloom than ion exchange with tda chloride salt. while statistically insignif icant, acid activation is observed to decrease the initial bloom from 1.210% to 1.193%. figure 7 shows the interaction effect of ion exchange with tda chloride salt (b) and organic modif ication with gms (c) on the initial bloom of nr composites reinforced with zeolite (p = 0.012). the increase in initial bloom due to ion exchange from 0.802% to 1.138% is observed when zeolite is not organo-modif ied with gms. however, the effect of ion exchange on initial bloom is severely diminished when zeolite is treated with gms. the magnitude of increase in initial bloom due to organic modif ication with gms is also reduced when zeolite is treated with tda chloride salt. cbacbca bacba xxxxxxx xxxxxy 0001.00003.00007.0 0004.00024.00015.00022.00298.0 1   cbacbca bacba xxxxxxx xxxxxy 0022.00368.00233.0 0179.01793.01386.00088.08157.0 2   b.b. pajarito, et al 47 table 4. anova result for bloom rate model 7 0.000197 0.000028 0.145 linear 3 0.000157 0.000052 0.042 a – acid activation 1 0.000040 0.000004 0.124 b – ion exchange 1 0.000036 0.000036 0.142 c – gms modification 1 0.000081 0.000081 0.035 2-way interactions 3 0.000036 0.000012 0.520 a*b 1 0.000006 0.000006 0.549 a*c 1 0.000030 0.000030 0.182 b*c 1 0.000001 0.000001 0.856 3-way interaction 1 0.000005 0.000005 0.587 a*b*c 1 0.000005 0.000005 0.587 error 16 0.000243 0.000015 total 23 0.000440 source degree of freedom adjusted sum of squares adjusted mean squares p value table 5. anova result for initial bloom model 7 1.72666 0.24667 0.000 linear 3 1.42664 0.47555 0.000 a – acid activation 1 0.00178 0.00178 0.791 b – ion exchange 1 0.14383 0.14383 0.028 c – gms modification 1 1.28102 1.28102 0.000 2-way interactions 3 0.29865 0.09955 0.025 a*b 1 0.05471 0.05471 0.154 a*c 1 0.04817 0.04817 0.180 b*c 1 0.19577 0.19577 0.012 3-way interaction 1 0.00137 0.00137 0.816 a*b*c 1 0.00137 0.00137 0.816 error 16 0.39141 0.02446 total 23 2.11807 source degree of freedom adjusted sum of squares adjusted mean squares p value the increase in bloom rate and initial bloom of paraff in wax in nr composites reinforced with zeolite can be attributed to the softening of the nr matrix. during chemical treatment of zeolite, tda chloride salt and gms are applied in excess to ensure complete exchange of inorganic cations and successful hydrophobization of zeolite surface. however, excess and unreacted tda salt and gms in treated zeolite f iller can act as a lubricating or softening agent (chakraborty et al. 2009). plasticized nr matrix lowers the diffusion resistance for wax blooming. to check if chemical effect of zeolite treatment on the blooming behavior of paraff i n wax 48 treatments of zeolite f iller result to nr matrix softening, shore a hardness of composite specimens was measured. computation of relative factor effects (pajarito et al. 2014; pajarito 2015; pajarito and arabit 2015) shows that the ion exchange treatment of zeolite f iller reduces the hardness of the nr composites by 3.627% as compared to the untreated zeolite. organic modif ication with gms also lowers the hardness of nr composites by 4.982%. the results of the anova and figure 7 suggest the synergistic effect between tda chloride salt and gms in reducing initial bloom in nr composites. the addition of gms after ion exchange treatment of zeolite f iller with tda chloride salt increases the concentration of long ch 2 moieties in the nr matrix. other than solubilizing and plasticizing the nr matrix, these functional groups can also interact with paraffin wax and reduce its migration due to aff inity in chemical structure. the observed decrease in magnitude of factor effects to initial bloom after combining ion exchange and organic modif ication treatments can be attributed to this interaction. figure 5. main effect of (a) acid activation, (b) ion exchange with tda chloride salt, and (c) organic modification with gms on the bloom rate of nr composites reinforced with zeolite. b.b. pajarito, et al 49 figure 6. main effect of (a) acid activation, (b) ion exchange with tda chloride salt, and (c) organic modif ication with gms on the initial bloom of nr composites reinforced with zeolite. figure 7. interaction effect of ion exchange with tda chloride salt (b) and organic modif ication with gms (c) on the initial bloom of nr composites reinforced with zeolite. effect of zeolite treatment on the blooming behavior of paraff i n wax 50 conclusions the amount of paraff in wax blooming at the surface of unf illed nr and nr composites reinforced with raw and treated zeolite f illers varies linearly with the square root of time. this behavior suggests the fickian diffusion of paraff in wax in the nr matrix. reinforcing nr with raw, acid-activated, ion-exchanged with tda chloride salt, and both acid-activated and ion-exchanged zeolite f illers reduces the amount of paraff in wax blooming in nr. raw and acid-activated zeolites are the most effective in decreasing the amount of bloom. meanwhile, the amount of bloomed wax is increased when nr is reinforced with gms-treated zeolite f illers. among the chemical treatments, organic modif ication of raw zeolite with gms signif icantly increases the bloom rate and initial amount of bloom in nr composites. zeolite treatment by ion exchange with tda chloride salt also enhances initial amount of bloom in nr composites. acknowledgments the authors acknowledge the off ice of the chancellor of the university of the philippines diliman, through the off ice of the v ice chancellor for research and development, for funding support through the ph.d. incentive awards (project no. 141417 phdia). the authors also 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chemical curatives on the cyclic fatigue life of natural rubber f illed with a silanized silica nanof iller. j appl polym sci. 120(5):2497-2507. saeed f, ansarifar a , ellis rj, hailemeskel y. 2012. measuring effect of the blooming of chemical curatives on the rate of cyclic fatigue crack growth in natural rubber f illed with a silanized silica nanof iller. j appl polym sci. 124(2):1372-1383. b.b. pajarito, et al 53 _____________ bryan b pajarito <bryan.pajarito@gmail.com> is an associate professor and researcher at the department of chemical engineering, university of the philippines diliman. he obtained his meng and deng in chemical engineering at tokyo institute of technology (japan). his current research interests involve optimizing formulations of oxo-biodegradable pe f ilms for commercial use, developing clay f illers for rubber compounding, and investigating novel thermoset-based composites for barrier applications. nico v. berba, jadreign keisheen c. par to, and raechel anne v. yabut were undergraduate research advisees of dr. pajarito under polymer research laboratory, department of chemical engineering, university of the philippines diliman. s a e e d f , a n s a r i f a r a , e l l i s r j , h a i l e m e s k e l y. 2 0 1 3 . a s s e s s i n g e f f e c t o f t h e reagglomeration and migration of chemical curatives on the mechanical properties of natural rubber vulcanizate. adv polym tech. 32(s1):e153-e165. siriyong t, keawwattana w. 2012. utilization of different curing systems and natural zeolite as f iller and absorbent for natural rubber/nitrile rubber blend. kasetsar t j nat sci. 46(6):918-930. sugiura m, horii m, hayashi h, sasayama m. 1996. application of sepiolite to prevent bleeding and blooming for epdm rubber composition. appl clay sci. 11(2):89-97. to r r eg r o s a co q u e r , a l v a r ezg a r c i a s , m a r t í n m a r t i n ez j m . 2 0 1 1 . m i g r a t i o n of l ow molecular weight moiety at rubber–polyurethane interface: an atr-ir spectroscopy study. int j adhes. 31(6):389-397. torregrosa-coque r, alvarez-garcia s, martin-mar tinez jm. 2011. effect of temperature on the extent of migration of low molecular weight moieties to rubber surface. int j adhes. 31(1):20-28. venable cs, greene cd. 1922. solubility of sulfur in rubber. ind eng chem. 14(4):319320. wake wc, tidd bk, loadman mjr. 1983. analysis of rubber and rubber-like polymers. london: applied science. ssd-sample article social science diliman, vol. 9, number 1, january-june 2013 humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) analyn salvador-amores, myles capareda & fenelyn nabuab call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 01_device compaction rates and paleo-sea levels 39 compaction rates and paleo-sea levels along the delta complex north of manila bay, luzon island, philippines janneli lea a. soriaa*, fernando p. siringanb and kelvin s. rodolfob anational institute of geological sciences, university of the philippines, diliman, quezon city a, bdepartment of earth and environmental sciences, university of illinois at chicago atel. no.: (632) 925-82-89; e-mail: jsoria@nigs.upd.edu.ph date received: march 2, 2006; date accepted: july 7, 2006 abstract science diliman (july-december 2005) 17:2, 39-45 *corresponding author uncontrolled groundwater extraction has been proposed as the main cause of accelerated subsidence in the delta region north of manila bay. however, natural autocompaction of deltaic sediment and other anthropogenic factors also enhance subsidence, amplifying global sea-level rise and aggravating land loss, flooding, and tidal inundation. here, we report how we determine the longer-term subsidence rates and paleo-sealevel history of the delta plain using sediment cores. four sediment cores 3 to 10.7 m long taken in bocaue and malolos, bulacan and lubao, pampanga all display shoaling-upward sequences that consist of, from bottom to top: basal shallow-marine clays comprising nearly half of each core; mangrove peat; beach sand; fluvial sand and mud; and uppermost floodplain clays. porosities of the deltaic sediments range from 0.3 to 0.8. peat has the highest porosities, from 0.7 to 0.8. calculations indicate about 2 to 6 m of compaction for the whole sediment sequence. wood fragments at 7 m and 8.4 m depths in the shallow-marine section of the pampanga core respectively yielded radiocarbon ages of 1800 ± 40 and 1730 ± 40 years. if around 1,000 years ago is when the surface 10 m of sediments started compacting, they would have done so at rates of 0.2 to 0.6 cm/y. natural compaction in similar environments such as in po delta, italy and mississippi delta are comparable, ranging from 0.09 to 0.37 cm/y. the small values acquired in this study imply that large human-induced components may account for as much as 97 percent of the subsidence in pampanga. keywords: autocompaction, subsidence, paleo-sea level, delta introduction local, relative sea-level rise from both natural and human-induced land subsidence, well recognized in other countries, can be orders of magnitude more rapid than global sea-level rise. neither, however, has yet been understood and accepted in the philippines. deltaic lowlands are very vulnerable to ecological change and economic damage due to marine invasion and floods exacerbated by sea level rise. worsening floods in the region north of manila bay continue to draw much attention as they affect this densely populated and highly developed agriand aquaculture area. studies by siringan and rodolfo (2003) and rodolfo and siringan (2006) indicate that aggravated flooding there is due to local, relative sea-level changes greatly enhanced by accelerated sediment compaction and ground subsidence caused by excessive groundwater withdrawal. additional land subsidence can also be expected from natural compaction caused by loading of delta sediments during deposition. soria, siringan and rodolfo 40 this ongoing work uses delta-plain sediment core data to establish the lateral and vertical response of the delta complex to long-term sea level fluctuations, and to determine the contribution of natural compaction to relative sea-level changes. the results provide a more complete picture, and thus a better understanding of the role of subsidence in the worsening floods, and should greatly aid in mitigating subsidence with appropriate and cost-effective measures. physiographic and geologic setting the southern end of the central luzon basin (fig. 1a) is a broad tidal-river delta complex, formed by the sediments delivered to northern manila bay by the pampanga, angat, bulacan-meycauayan rivers within the pampanga river basin, the largest of the 26 catchment areas (siringan and ringor, 1997). covering about 2,700 km2 (siringan and rodolfo, 2003), the delta complex is occupied by the coastal towns of bataan, pampanga, bulacan, and the kamanava area of northwestern metro manila. the delta surface has a very gentle gradient, with elevations rising from mean sea level (msl) to 10 meter above msl 10 km to 25 km inland (sandoval and mamaril, 1969; nepc, 1987). to the west, the delta is flanked by the eastern zambales mountains of eocene ophiolites and the recent pinatubo, natib, and mariveles volcanoes. to the east, the delta is contained by the foothills of the southern sierra madre mountains. these consist of creteceous to eocene metavolcanics, metasediments and ophiolites, oligocene to miocene carbonates, marine clastics and volcaniclastics, and pliocene to pleistocene shallow marine to terrestrial sedimentary and pyroclastics (bmg, 1982; bed, 1986; encarnacion, 2004). quaternary alluvium underlying the delta plain typically consists of consolidated silt or clay, and poorly cemented sand and gravel derived from the pleistocene guadalupe formation (bmg, 1982; jica, 1982). the young fluvio-marine deposits are highly susceptible to compaction. borehole data in the kamanava cities of kaloocan, malabon, navotas and valenzuela show the holocene marine and river deposits vary laterally in thickness from 1-31m (cti engineering co., ltd., 2001). lineaments in satellite images of the region are most likely active faults that might be responsible for some figure 1 a) major geologic structures of central luzon basin modified from maleterre (1989) and nelson et al., (2000). b) coastal morphology and local tectonic features compaction rates and paleo-sea levels 41 of the vertical motions (siringan and rodolfo, 2003). the most prominent lineament passes northeastward through hermosa, bataan to bacolor, pampanga and bounds wetlands to the southeast and dry alluvial plains to the southwest (fig. 1b). paleoshoreline positions at least nine chenier ridges in a 1991 radar image along the navotas-obando coastal plain are sand accumulations heaped by waves on a muddy substrate along former shorelines. the most inland and oldest of these ridges, near the bulacan river, is approximately 7 km north of the present shoreline (siringan and ringor, 1997). chenier ridges are absent, however, along the western coast. instead the landward limit of wetlands in hagonoy, bulacan and macabebe, pampanga are marked by paleo-delta lobes 5 to 10 km inland. gaillard, et al. (2005) defined the ca. 500 800 y bp paleo-shoreline by using core data and the landward limit of wetlands. apparently, the area on which the pampanga towns lubao, sasmuan, guagua, and minalin are situated (fig. b) was an embayment until it was rapidly filled with volcaniclastic pinatubo sediments generated by the eruptive episode of 500800 y bp (the buag event of newhall et al., 1996). figure 2. core sites location across the delta complex. precipitation and tides the region has two pronounced seasons, dry from november to april and wet during the rest of the year. precipitation records of the philippine atmospheric, geophysical and astronomical administration (pagasa) from 1961 to 1997 document that southwest monsoon winds and typhoons deliver approximately 1,000-3,000 mm of annual rainfall, about 325 to 425 mm during the wettest months of july to september. tides in the bay are predominantly diurnal, with a microtidal range of 1.25 m along the metro manila coast (siringan and ringor, 1997). human induced compaction a number of aquifers 15 to 30 meters deep in the delta plain sediments are tapped by shallow water wells for domestic, agriand aquacultural uses (sandoval and mamaril, 1969). fishponds use large volumes of groundwater to replace water fouled by overfeeding. in addition, the near-surface sediments are rapidly dewatered when the ponds are dried up twice a year to prepare for the next cropping. to some extent, this practice is similar to the drainage practices in the peat lands of netherlands that accelerate natural compaction soria, siringan and rodolfo 42 rates of 1.7 to 6 mm/yr to several centimeters or more (stephens, et al., 1984). there, however, the enhanced rates eventually decrease to a constant rate equal to or even lower than the pre-disturbance values. both surface and shallow dewatering processes enhance compaction of the deltaic sediments due to loss of original volumes of pore waters trapped in clays and organic-rich soils such as peat. enhanced compaction in the deeper sections of the delta plain can be attributed to withdrawal from wells as deep as 110 m (siringan and rodolfo, 2003). methods seven sediment cores ranging in lengths from 3.0 to 10.7 m (fig. 2) were taken using a manual mud corer at locations associated with paleo-shoreline features such as beach ridges and paleo-deltas delineated from a satellite image. data acquired from four of these cores (pam 4, bul 1, bul 7, and bul 16) are presented in this report. color and grain size were logged, and paleoenvironments were deduced from lithologies, sedimentary structures, and organic matter and molluscan contents. porosities and compaction ratios were calculated by measuring water contents and bulk densities. subsamples from the peat layers are currently being processed for radiocarbon dating. previous age dates of wood fragments at 7 m and 8.4 m depths in the shallow-marine section of core pam 4 yielded radiocarbon ages of 1800 ± 40 and 1730 ± 40 y bp, respectively (gaillard et al., 2005). precise elevations of each core site still need to be determined. figure 3. shallow subsurface stratigraphy of the delta complex north of manila bay. compaction rates and paleo-sea levels 43 results and discussion paleoenvironments and paleo-sea levels most of the cores are complete upward sequences consisting of basal shallow-marine clay, transitioning into a mangrove-peat that in turn is overlain by beach sand capped by fluvial sand and mud transitioning to floodplain clays (fig. 3). such sequences represent deposition in successively shallower environments; the beach sand on top of the peat documents deepening by relative sea level rise, followed by fluvial progradation. the shoaling upward sequence from marine clay to mangrove peat indicates shoreline progradation. subsequently, as evidenced by beach sand overlying peat layers, the coastline retreated, possibly due to either regional sea-level rise or a local seismic event affecting the entire delta plain. the buag eruption of mt. pinatubo, or fault movements along one or more of the many lineaments in the delta plain may have caused co-seismic subsidence. finally, natural and human-induced compaction lowered the beach sands to their present depths. importantly, the peat deposits overlain by beach sand in other core sites indicate a baywide event, not localized erosion caused by delta shifts. the beach sands deposited soon after the eruption of pinatubo 500 to 800 years ago record a pre-eruption shoreline more landward than the present one. high sediment input after the eruption rapidly filled the paleopampanga bay (gaillard et al., 2005), prograding fluvial sediments translating the shoreline seaward close to its present position. porosity and natural compaction natural compaction, caused by the squeezing out of pore water by the accumulating weight of overlying sediments, is recorded in the loss of porosity with depth. in the uppermost 10 m of deltaic sediments, porosities range from 0.3 to 0.8. peats have the highest values of 0.7 to 0.8, and sands are the least porous, at only 0.3 to 0.4. to reconstruct the initial thickness of each sedimentary layer, these porosity values were compared to initial porosities of recent deltaic sediments along manila bay, as well as from atkins and mcbride (1992). compaction ratios were determined with the equation cr= h1/h2= 1-n2/1-n1 where h1= initial thickness, h2 = present thickness, n1 = initial porosity, n2 = present porosity (einsele, 1992). each compaction ratio yields a magnitude of the reduction in sediment thickness (table 1) due to loss in porosity owing to natural and or anthropogenic causes. in lubao, the 10-m surface sedimentary sequence has been compacted about 1.6 to 5.6 m; about 0.25-2.42 and 0.49-1.36 m respectively for the peat and peaty clay layers, sediment types easily compacted due to their very high water and organic contents. as in the thessaloniki delta plain in greece (stiros, 2001), oxidation of peat soils in the vadose zone may also contribute to subsidence. table 1. magnitudes of compaction of the sediments in pampanga and bulacan. sediment type present thickness, m initial thickness, m decrease in thickness, m pam 4 bul 16 pam 4 bul 16 pam 4 bul 16 fluvial sand 0.95 1.07 1.30 0.12 0.35 beach sand 0.65 0.74 0.1 mangrove peat 1.75 1.05 2.00 4.17 1.66 3.33 0.25 2.42 0.61 2.28 subtidal peaty clay 1.70 0.95 2.19 3.06 1.36 1.90 0.49 1.36 0.41 0.95 shallow marine clay 4.20 2.20 4.88 5.60 2.44 2.84 0.68 1.40 0.24 1.06 total 9.25 4.20 10.88 14.87 5.46 8.07 1.64 5.63 1.26 4.29 soria, siringan and rodolfo 44 assuming that the top of the core is at present mean sea level (msl), 1.6 to 5.6 m of compaction indicates that the peat layer presently 3.5 m below msl was deposited in the depth zone from 1.9 below to 2.1 m above present msl. if co-seismicity caused the sudden deepening represented by the peat being overlain by beach sand, an even higher elevation is required. reconstructions of regional paleosealevel by berdin and others (2003) and siringan and others (2003) indicate that sea level reached it present position in the philippines about 1,000 years bp. this implies that the middle value of estimated compaction is more realistic. assuming that compaction started about 1,000 years ago, the deltaic sediments could have been compacted at rates of 0.16-0.56 cm/yr, 0.36 cm/yr on the average, accounting for 2 to 8 percent of the estimated 2 to 8 cm/yr typical subsidence rates from 1991 – 2001 (rodolfo and siringan, 2006). the difference between the calculated compaction rates and the observed subsidence is most probably due to compaction of the sediments below the sampled surface sections, but other factors yet to be recognized may be lowering the deltaic surface. our natural compaction rates are higher than the 0.09 to 0.37 cm/yr calculated by kuecher and others (1993) in a similar mississippi delta environment, and the 0.1 cm/yr in po delta, italy (pirazzoli, 1996) but longer durations and sequences there might account for the difference. conclusion the compaction ratio of near-surface sediments indicate that of the 2 – 8 cm/yr of subsidence, about 2 to 8 percent can be attributed to natural compaction of the near surface sediments. this implies that enhanced dewatering of the upper 30 m of the sediment column can potentially account for almost 98 % of the subsidence rates during the past decade. acknowledgements this study was funded by research grant 040415 tnse of the university of the philippines-office of the vicechancellor for research and development to j.l.a. soria. logistics and support funds were provided to f.p. siringan by the national institute of geological sciences of the university of the philippines and the bureau of agricultural research, department of agriculture. we are grateful to gerald quiña, nathaniel baluda, gerardo sumat, pepito cortez, and joan reotita for providing assistance in the field; peter zamora for drafting some of the figures. references atkins, j.e. and e.f. mcbride, 1992. porosity and packing of holocene river, dune, and beach sands. the american association of petroleum geologists bulletin, 76(3): 339355. berdin, r.d., f.p. siringan, and y. maeda, 2003. holocene relative sea-level changes and mangrove response in southwest bohol, journal of coastal research, 19(2): 304313. bureau of energy development, 1986, sedimentary basins of the philippines: their geology and hydrocarbon potential, vol. ii-a: basins of luzon; volume vii: well summary charts. bureau of mines and geosciences, 1982. geology and mineral resources of the philippines: manila: 406 pp. cti engineering co., ltd. 2001. sectoral reportb, soil mechanical investigation. unpublished report submitted to kamanava area flood control and drainage improvement project, republic of the philippines department of public works and highways, manila: 28 pp. einsele, g., 1992. sedimentary basins: evolution, facies, and sediment budget. springer-verlag berlin heidelberg. 628 p. encarnacion, j. 2004. multiple ohiolite generation preserved in the northern philippines and the growth of an island arc complex. tectonophysics. 392(1-4): 103-130. gaillard, j.c., f.g. delfin, jr., e.z. dizon, j.a. larkin, v.j. paz, e.g. ramos, c.t. remotigue, k.s. rodolfo, f.p. siringan, j.l.a. soria, j.v. umbal, 2005. anthropogenic dimension of the eruption of mount pinatubo, philippines, between 800 and 500 years bp. l’anthropologie. 102(2): 249-266. compaction rates and paleo-sea levels 45 japan international cooperation agency (jica), 1982. feasibility report on the pampanga delta development project. the republic of the philippines, ministry of public works and highways, national irrigation administration. kuecher, g.j., n. chandra, h.h. roberts, j.h. suhayda, s.j. williams, s.p. penland, and w.j. autin, 1993. consolidation potential in southern louisiana: coastal zone ’93. proceedings of the 8th symposium on coastal and ocean management. am. shore and beach preservation assoc. am soc. civ. eng: 1197-1214. maleterre, p. 1989. histoire sedimentaire, magmatique, tectonique et metallogenique d’un arc cenezoic deforme en regime de transpresion: la cordillere centrale de luzon, a l’extremite de la faille philippine, sur les transects de baguio et de cervantes-bontoc, contexte structural et geodynamique des mineralisations epithermales auriferes. brest, france: l’universite de bretagne occidentale, ph.d. thesis: 304 pp. national environmental protection council (nepc), 1987. philippine groundwater salinity intrusion control study. quezon city, nepc: 188 pp. nelson. a.r., s.f. personius, r.e. rimando, r.s. punongbayan, n. tuñgol, h. mirabueno, a. rasdas. 2000. multiple large earthquakes in the past 1500 years on a fault in metropolitan manila, the philippines. seismological society of america. 90: 73-85. newhall, c.g., daag, a.s., delfin jr., f.j., hoblitt, r.p.,mcgeehin, j., pallister, j.s., et al., 1996. eruptive history of mount pinatubo. in: newhall, c.g., and punongbayan, r.s. (eds.), fire and mud: eruption and lahars of mount pinatubo, philippines. university of washington press and phivolcs, seattle and quezon city: 165–195. pirazzoli, p.a., 1996. sea-level changes: the last 20, 000 years. john wiley & sons, ltd., england: 211 pp. rodolfo, k.s., and f.p. siringan (2006). ignoring groundwater overuse, the main cause of regional subsidence and worsening flood at the head of manila bay, philippines. disasters. special issue on climate change and disasters. 30 (1). sandoval, m.p. and f.b.mamaril, 1969. hydrogeology of central luzon. unpublished report. 171 p. siringan, f.p. and c.l. ringor, 1997. predominant nearshore sediment dispersal patterns in manila bay, science diliman 9 (1 & 2):2940. siringan, f.p., y.maeda, and r.d. berdin, 2003. multiple holocene highstands in the philippines. poster presented in puglia final conference quaternary coastal morphology and sea level changes, otranto/tarantopuglia (italy), september 22-28, 2003. siringan, f.p. and rodolfo, k.s., 2003. relative sea-level changes and worsening floods in the western pampanga delta: causes and some possible mitigation measures. science diliman 15(2):1-12. stephens, j.c., l.h. allen, and e. chew. 1984. organic soil subsidence. american geological society reviews in engineering geology. in: holzer, t.l. (ed.) 6: 107-122. stiros, s.c., 2001. subsidence of the thessaloniki (northern greece) coastal plain, 1960-1999, engineering geology. 6: 1243-256. sd inside back cover.pmd 1 reviewers for this issue july-december 2013 • volume 25 no. 2 zenaida g. baoanan, phd department of biology university of the philippines baguio edwino s. fernando, phd college of forestry and natural resources university of the philippines los baños ian kendrich c. fontanilla, phd institute of biology university of the philippines diliman william sm. gruezo, phd institute of biological sciences university of the philippines los baños francisco m. heralde iii, phd department of biochemistry and molecular biology university of the philippines manila hilton y. lam, phd department of clinical epidemiology university of the philippines manila ambrocio melvin a. matias, msc institute of biology university of the philippines diliman s. suzanne mingoa-licuanan, phd marine science institute university of the philippines diliman milagros p. navarro, phd institute of mathematics (ret.) university of the philippines diliman preciosa corazon b. pabroa, phd philippine nuclear research institute department of science and technology leni l. quirit, phd institute of chemistry university of the philippines diliman marian p. roque, phd institute of mathematics university of the philippines diliman raquel o. rubio, msc natural sciences research institute university of the philippines diliman benjamin m. vallejo, phd institute of environmental science and meteorology university of the philippines diliman 10info for authors.pmd 101 1. science diliman is a journal of pure and applied sciences. considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); 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fax no.: (632)927-7516 e-mail: hedreyda@laguna.net date received: april 14, 2003; date accepted: may 17, 2005 six vibrio isolates identified biochemically as vibrio campbellii from southeast asian fisheries development center (seafdec) in tigbauan, iloilo, were characterized by 16 rdna sequence, total protein profile, and dna profile analyses. genomic dna from the isolates were subjected to pcr using four sets of primers targeting gene fragments of hemolysin and toxr based on sequences from reference vibrio harveyi (ifo15634), v. campbellii (ifo1563), and local isolates identified as v. harveyi. total protein profile could not distinguish the isolates from one another and from the reference v. harveyi (ifo15634) and v. campbellii (ifo15631). analysis of 16s rdna sequences revealed high degree of sequence similarity (96% 99%) of the six local isolates with other vibrio species including v. campbellii and v. parahaemolyticus, indicating that this analysis will not be useful in resolving their identity. all six isolates exhibited characteristic reference v. harveyi pcr profile when a primer set designed to amplify a 308-bp fragment of hemolysin gene in that species was used. however, no amplicons were generated for these isolates using primers that amplify toxr gene fragments in v. harveyi. this suggests that the six isolates were not bonafide v. harveyi strains. the isolates also did not exhibit v. campbellii characteristics since the primer designed to target the toxr gene in v. campbellii could not amplify dna from any of the six isolates, suggesting that they were not bonafide v. campbellii strains either. the toxr gene from the six isolates could be amplified using a primer based on toxr gene sequences from a seafdec isolate previously identified as v. harveyi (pn-9801). these data suggest that the six isolates previously identified as v. campbellii as well as pn-9801 may be classified in one group separate from bonafide reference v. harveyi and reference v. campbellii strains, based on the identical results in the molecular analyses performed in this study. keywords: vibrio, toxr, hemolysin, protein profile, pcr *corresponding author cortado et al. 24 introduction penaeid shrimp farming, especially of the giant tiger prawn, penaus monodon dominates crustacean production not only in the philippines but also in the entire southeast asian region. in fact in 1995, a production of 0.48 million metric tons of black tiger prawn was recorded from southeast asia (subasinghe et al., 1997). in the philippines, production from tiger prawn totaled 36,798 metric tons, accounting for 20% of total yield in 1998 (philippine fisheries profile– bfar, 1998). one major concern in philippine shrimp farming industry however, is the emergence of diseases, which results to significant reduction in production. diseases in cultured shrimps, which have been causing great production loss in philippine aquaculture industry, are attributed mainly to bacterial pathogens v. harveyi and v. campbellii. these pathogens were shown to be among the most dominant species associated with mortalities in black tiger shrimp in the philippines (de la peña et al., 2001). biochemical tests were used to identify and classify the pathogens after heavy mortalities due to luminescent vibriosis have been observed among pond-cultured shrimps. in the same study (de la pena et al., 2001), it was also demonstrated that several strains, including the six isolates used in this study, were pathogenic when injected intramuscularly to shrimps. however, identity of the local isolates should be confirmed because biochemical tests were not sufficient to distinguish the local isolates from each other or from reference strain v. harveyi and v. campbellii. the use of 16s rdna sequences may not be suitable for the purpose, since these sequences in closely related species such as v. harveyi and v. campbellii have high homologies (about 96% to 99%). in this study, some molecular approaches were performed to characterize and confirm the identity of the six previously mentioned local isolates, identified as v. campbellii. the study was conducted with the following objectives: (1) to compare total protein profiles of the six seafdec local isolates with those of reference v. campbelli and v. harveyi strains. (2) evaluate if indeed 16s rdna sequences will not be able to resolve their identity. (3) to compare pcr profiles of the six local isolates with those of reference v. harveyi and reference v. campbellii strains using four sets of primers namely vhhemo which targets hemolysin gene fragments of v. harveyi, vhtoxr and vctoxr which amplifies toxr gene fragments of reference v. harveyi and v. campbellii, respectively, and pntoxr designed to amplify toxr gene fragments of local isolates previously identified as v. harveyi. materials and methods bacterial isolates used the six local pathogenic vibrio isolates, which this study aims to characterize, were obtained from southeast asian fisheries development center (seafdec) in tigbauan, iloilo (table 1). this research also utilized two reference strains: v. harveyi (ifo 15634) and v. campbellii (ifo 15631), both from the institute of fermentation osaka (ifo) japan. another local isolate (pn-9801) identified as v. harveyi based on biochemical tests was also used in this study. vibrio cultures were continuously maintained at 30°c incubation in nutrient agar media containing 1.5% nacl. dna extraction genomic dna from all vibrio isolates used was extracted using the nucleospin dna extraction kit commercially available from clontech laboratories, inc. (palo alto, ca, usa). vc 1 vc 2 vc 3 vc 4 vc 5 vc 6 crw-9807 piz-9824 nrw-9805 br-9807 brw-9804 crwp-9805 v. campbellii v. campbellii v. campbellii v. campbellii v. campbellii v. campbellii designation id number biochemical identification (dela peña et al., 2001) table 1. local isolates used in this study. local vibrio isolates 25 16s rdna sequence analysis the 16s rdna fragments of the six isolates were amplified using specific primers targeting this gene. the pcr products were gel-purified and sequenced. sequence homology of the isolates was then compared with known 16s rdna sequences of various species deposited in the database using blast sequence homology search, to determine the degree of relatedness of the isolates with the reference strains. extraction of total proteins total proteins were extracted from the six local vibrio isolates, two reference strains: v. harveyi and v. campbelli and a local isolate (pn-9801) identified as v. harveyi. cells were collected from a 10 ml overnight bacterial culture in liquid broth by centrifugation at 10,000 x g for 2 minutes at room temperature. the resulting pellet was resuspended in sterile distilled deionized water after which 200 ml of 2x treatment buffer was added. the treatment buffer consist of 0.125m tris-cl, 4% sodium dodecyl sulfate (sds), 20% glycerol, 0.2m dithiothreitol (dtt) and 0.02% bromphenol blue. the cells in treatment buffer were boiled for 10 minutes and then centrifuged at 10,000 x g for 5 minutes at room temperature to pellet the debris and insoluble materials. the supernatant containing total protein extracts were transferred to fresh 1.5-ml tubes. the concentrations of the extracted proteins were determined by spectrophotometry at a wavelength of 280 nm (ausubel, 1997). bovine serum albumin (bsa) was used as protein standard. sample concentration was standardized using 1x tb. sds-polyacrylamide gel electrophoresis (page) of total proteins from the isolates total protein extracts of each isolate were run in a 7.5% acrylamide gel using the discontinuous laemmli sdspage method. this was done in a hoefer se400 sturdier vertical unit [amersham] setup with 1.5 mm gel spacers. for each sample, 100 mg protein extract was loaded onto each well. along with the samples, a broad-range protein ladder from biorad served as molecular weight marker. proteins run in the gels were stained using the vorum silver staining protocol (mortz et al., 2001). pcr-based characterization of vibrio isolates four sets of primers namely vhhemo, vhtoxr, vctoxr and pntoxr, which were designed in earlier studies to target specific regions of the hemolysin and toxr genes of reference v. harveyi and v. campbellii strains as well as two local vibrio isolates, were used in this study. vhhemo, vhtoxr and pntoxr primers were designed by conejero (2002) for the specific detection of different strains of v. harveyi. the vctoxr primer set was designed to amplify a fragment of toxr from v. campbellii (sobrepeña et al., 2002). sequences of each primer set used appear in table 2. pcr using hemolysin primer vhhemo dna extracted from the isolates and reference strains, v. harveyi and v. campbellii were used as templates for pcr with primers originally designed for a hemolysin gene fragment. the vhhemo primer set targets a unique 308-bp region in the hemolysin gene of the reference v. harveyi (ifo 15634) and a local vibrio harveyi strain (pn-9801). dna samples were subjected to pcr in perkin elmer gene amp system (perkin elmer corporation, singapore). conditions for pcr with vhhemo primers were as follows: 30 cycles at 94°c for 1 min, 53°c for 1 min, and 72°c for 1 min with an initial denaturation at 94°c for 5 minutes and a final extension step at 72°c for 7 minutes. vhhemo vhtoxr vctoxr pntoxr table 2. pcr primer sequences used in the study (conejero & hedreyda, 2003). forward reverse forward reverse forward reverse forward reverse tca gtgcct ctc aag taa ga gct tga taa cac ttt gcg gt ttc tga agc agc act cac tcg act ggt gaa gac tca ttc tga agc agc act cac ttc tga agc agc act cac agc agc tgc tcc agt tga ctg ctc aat tga tgg cag primer sequence 5’ – 3’ cortado et al. 26 pcr using vhtoxr primer vhtoxr primers were used to amplify genes from the six local isolates and from reference strains, v. harveyi and v. campbellii. the primer was based on partial toxr gene sequence of a reference v. harveyi strain (ifo 15634). conditions for pcr with vhtoxr primers were as follows: 30 cycles at 94°c for 1 min, 63°c for 1 min, and 72°c for 1 min with an initial denaturation at 94°c for 5 minutes and a final extension step at 72°c for 7 minutes. pcr using the vctoxr primer vctoxr primer set was previously designed to amplify a 230-bp fragment of the toxr from v. campbellii. this primer set was used in pcr using dna templates from the six isolates and from reference strains, v. campbellii and v. harveyi. pcr of the samples with vctoxr was accomplished using the following conditions: 30 cycles of denaturation at 94°c for 1 min, annealing at 57°c for 1 min, and extension at 72°c for 1 min with an initial denaturation at 94°c for 5 min and a final extension step at 72°c for 7 min. pcr using the pntoxr primers pntoxr primers were based on partial toxr sequence of a local vibrio isolate (pn-9801) previously identified as v. harveyi. the primer set was designed to target the 226-bp fragment of toxr from local vibrio isolates. dna from the six local isolates, reference v. campbellii (ifo-15631) and a local isolate (pn-9801) were used as template in the following run: 30 cycles at 92°c for 45 secs, 63°c for 1 min, and 72°c for 1.5 min with an initial denaturation at 94°c for 5 min and a final extension step at 72°c for 7 min. the pcr mix without the dna template was used as a negative control for all runs. the components for each 20-ml reaction mix are as follows: 13.9 ml sterile distilled water, 2 ml buffer, 0.6 ml mgcl2, 0.4 ml dntps, 1 ml forward and 1 ml reverse 10mm primers, 0.1 ml taq polymerase, and 1 ml template. pcr products along with a 50-bp or 100-bp molecular weight marker were run in 1.5% agarose gels, stained with ethidium bromide and visualized under ultraviolet light. results and discussion the six vibrio isolates used in this study (crw-9807, piz-9824, nrw-9805, br-9807, brw-9804, crwp9805) were previously identified by traditional biochemical tests as v. campbellii (de la peña et al., 2001). however, variable responses to biochemical tests are often observed in vibrio species. these tests are also seldom adequate to distinguish between vibrio species especially closely related ones. in a species characterization study of de la peña and co-workers (2001) for example, only the ability to decarboxylate ornithine differentiates v. harveyi from v. campbellii. similar characteristics were exhibited by both strains in all other reactions tested. so to confirm whether the six local seafdec isolates are strains of bonafide v. campbellii, molecular tests were performed. a molecular approach offers the speed and ease of method without compromising accuracy of identification. the isolates can be characterized based on their 16s rdna sequences as well as protein and dna profiles using powerful molecular tools such as sds-page and genetargeted pcr for discrimination between various strains. characterization using total protein profile the isolates were first characterized based on the protein profiles generated after resolution of total soluble protein in an sds-page. using protein profile analysis, relationships between bacteria can be determined based on the presence of unique and shared bands. this was demonstrated by que (2002) to deduce genetic relatedness in several vibrio reference strains where several protein bands were found to be characteristic of each species. isolates were therefore expected to exhibit distinct protein profiles that could identify them with any of the reference strains used based on the presence of similar protein bands. surprisingly, similar total protein profiles were generated by the six isolates (fig.1), the two reference strains, v. harveyi (ifo 15634) and v. campbellii (ifo 15634) and a local isolate identified as v. harveyi (pn9801). all were characterized by the presence of two heavily stained bands near the 45 kda marker and four lightly stained high molecular weight bands between the 97.4 and 66 kda markers. compared to the rest, local vibrio isolates 27 one slight difference though, can be observed with the profile of v. harveyi, which is the presence of a distinct band just below the 31 kda marker. these observed similarities in total protein profiles indicate the close relatedness of the six local isolates with v. harveyi and v. campbellii reference strains and with a locally isolated v. harveyi strain. protein profile analysis was not able to sufficiently distinguish the isolates from each other nor identify them with any of the two reference strains used. analysis of 16s rdna sequence another way to elucidate genetic relatedness among bacteria is the use of 16s rdna sequence analysis. dorsch and co-researchers (1992) have shown the close relationship of v. alginolyticus, v. campbellii, v. harveyi, v. proteolyticus, v. parahaemolyticus, and v. natriegens by analyzing conserved and variable sequences in this gene. an available database containing known 16s rdna sequences of various bacterial species allow alignment of new sequences with those already recorded in the database. the use of blast sequence homology search to analyze newly obtained 16s rdna sequences of the six local isolates [genbank accession nos. dq017901 (crw-9807), dq017902 (piz-9824), dq017903 (nrw-9805), dq017904 (br-9807), dq017905 (brw-9804), and dq017906 (crwp9805)] revealed high degree of sequence similarity (96%-99%) in 16s rdna of the six local isolates with other vibrio species including v. campbellii and v. parahaemolyticus (table 3). the high interspecies homology observed in 16s rdna was also confirmed by phylogenetic analysis using the mega v2.1 software (fig. 2). these observations suggest the need for other molecular typing methods that target specific gene sequences in vibrio species for more accurate classification. pcr targeting hemolysin gene fragments the hemolysin gene, found to be present in several vibrio species codes for an important virulence factor in bacteria. pathogenic activity of hemolysins includes erythrocyte disruption in various species and toxicity for cells and small experimental animals (nishibuchi & kaper, 1995). to detect the presence of hemolysin gene and to test if primers designed based on v. harveyi would work with the six local isolates, vhhemo primers were used in pcr. these primers were shown to generate a characteristic 308-bp amplicon from v. harveyi reference strains as well as from a variant local isolate (pn-9801) identified as v. harveyi (conejero & hedreyda, 2004). amplification of the 308-bp hemolysin gene fragment using dna templates from the five of six local isolates was observed (fig. 3), indicating that a gene homologue is present in the five local isolates and the sequences in the primer annealing m 1 2 3 4 5 6 7 8 9 200 kda 97.4 66.2 45.0 31.0 fig. 1. total protein profile of the vibrio isolates. lanes 1-6: crw-9807, piz-9824, nrw-9805, br-9807, brw-9804, crwp-9805; lanes 7 and 8 are reference strains v. harveyi (ifo15634) and v. campbellii (ifo15631), respectively; lane 9 is a local isolate (pn-9801) identified as v. harveyi. m is a broad-range protein ladder from biorad. table 3. 16s rdna sequence analysis. crw-9807 piz-9824 nrw-9805 br-9807 brw-9804 crwp-9805 98% v. campbellii, vibrio sp, v. parahaemolyticus 96% v. campbellii, vibrio sp, v. parahaemolyticus 99% v. campbellii, vibrio sp, v. parahaemolyticus 99% v. campbellii, vibrio sp, v. parahaemolyticus 99% v. parahaemolyticus, 98% v. campbellii 99% v. campbellii, v. parahaemolyticus isolate id number 16s rdna sequence similarity cortado et al. 28 sites were significantly homologous to that of the v. harveyi reference strain. at 53°c annealing temperature, five local isolates were able to produce distinct 308-bp amplicons with the vc2 and vc4 (piz9824 and br-9807, respectively) both exhibiting an additional band of about 600-bp in their profiles which could not be explained at this point. one isolate, vc6 (crwp-9805), gave only a 600-bp product instead of the expected 308-bp fragment. the absence of the 308-bp band in the profile of reference strain v. campbellii indicates that hemolysin gene sequence variation in the primer annealing sites are present in v. campbellii. data also suggest that hemolysin gene sequences of the six local isolates being studied maybe exhibiting greater homology with reference strain v. harveyi compared to the hemolysin sequences of v. campbellii. this adds to the confusion in species confirmation of these local isolates. although they were previously identified by biochemical means as v. campbellii, these results show that they are not exhibiting bonafide v. campbellii characteristics. pcr targeting toxr gene fragments vhtoxr primers the toxr gene is considered a main regulator for coordinating expression of various virulence genes in 100 54 67 100 0.04 0.020.03 92 61 36 61 0.01 0.00 vc5 brw-9804 v. campbellii v. harveyi v. parahaemolyticus v. natriegens v. alginolyticus vc6 crwp-9805 vc1 crw-9807 vc3 nrw-9805 vc2 piz-9824 vc4 br-9807 v. anguillarum v. fluvialis v. vulnificus v. mimicus v. cholerae m 1 2 3 4 5 6 7 8 9 600 bp 308 bp100 bp fig. 3. pcr profile of the six vibrio isolates using vhhemo primers. lanes 1-6 contains the local isolates: crw-9807, piz-9824, nrw-9805, br-9807, brw-9804, crwp-9805; lanes 7 and 8 are products from the reference strains v. harveyi (ifo 15634) and v. campbellii (ifo 15631), respectively; lane 9 is the negative control (no template). m is the 100-bp molecular weight marker. fig. 2. unrooted tree constructed by the neighbor-joining method based on the 16s rrna gene. percent bootstrap values from 1,000 replicates generated are given at the branching points. bars indicate genetic distance. local vibrio isolates 29 bacteria. it is also hypothesized to be part of the conserved genes in the ancestral chromosome of members of the family vibrionaceae (osorio et al., 2000). sequencing of the toxr gene revealed the presence of variable sequences that can be utilized for the identification of specific species of vibrio. a toxr homologue from a reference strain v. harveyi (ifo 15634) was demonstrated (conejero, 2002) to be useful for designing primers (vhtoxr) specific for v. harveyi reference strains. based on this homologue another primer pntoxr was designed for the detection of toxr fragments in locally derived vibrio isolates pathogenic to p. monodon. another set of primers (vctoxr) targeting a toxr gene fragment in v. campbellii was designed (sobrepeña et al., 2002) based on the partial toxr sequence of a reference v. campbellii strain (ifo 15631). all the above-mentioned primers were used to test which primer will result in amplification of the toxr gene fragment and consequently to test if indeed they will exhibit the characteristic v. campbellii results, that is producing amplicons only when using the primers vctoxr designed based on v. campbellii sequences. pcr using dna templates from the six local isolates with vhtoxr primers did not result in the amplification of the 390-bp toxr fragment that is produced in pcr using v. harveyi reference strain dna template. although the vhhemo primer generated the unexpected 308-bp amplicons for the isolates previously identified as v. campbellii, these six isolates did not exhibit profiles characteristic of v. harveyi reference strain in pcr using the vhtoxr primers. this further complicates the analysis because the use of two different primer set in pcr resulted in conflicting result, one suggesting closer relatedness to v. harveyi and the other does not. vctoxr primers when the dna from six local isolates were subjected to pcr using vctoxr primers, designed based on sequences of toxr gene fragments of reference v. campbellii,, no amplicons were observed either (data not shown). interestingly, data reveals that the toxr gene fragment in the six local isolates may have significant sequence variation particularly at the primer annealing sites from those of reference strains v. campbellii. therefore, the six isolates are not exhibiting bonafide v. campbellii characteristics. these findings point out to the possibility that the local isolates could be very close relatives of vibrio harveyi or vibrio campbellii but may later be classified as an entirely different group separate from these two species. pntoxr primers another set of primers (pntoxr) based on partial sequences of the toxr gene isolated from a local isolate pn-9801, previously identified as v. harveyi, could generate a 226-bp band in pcr using dna template from that local isolate (pn-9801). when dna from the six isolates used in this study were subjected to pcr using pntoxr primers, the 226-bp amplicon was also produced in all six samples (fig. 4). no amplicon was produced in pcr using dna templates from reference v. harveyi and v. campbellii. these data strongly support the hypothesis that the local isolates previously identified as v. harveyi and the local isolates used in this study previously identified as v. campbellii, could be belonging to the same classification. preliminary total protein profile analysis of these local isolates revealed almost identical profiles. the hemolysin gene fragment from these isolates could not be amplified by three primers used to amplify hemolysin gene fragments from bonafide v. harveyi isolates but the primer set vhhemo that was able to produce a 308-bp amplicon in reference strain v. m 1 2 3 4 5 6 7 8 9 850 bp 226 bp 350 bp fig. 4. pcr profile of the six vibrio isolates using pntoxr primers. lanes 1-6 contains the six local isolates: crw-9807, piz-9824, nrw-9805, br-9807, brw-9804, crwp-9805; lanes 7 and 8 are products from the reference strains v. campbellii (ifo 15631) and a local isolate pn-9801, respectively; lane 9 is the negative control (no template). m is the 50-bp molecular weight marker. cortado et al. 30 harveyi as well as the all the local isolates in this study. dna templates from 6 local isolates in this study, could not be amplified by the vhtoxr and the vctoxr primers that are used to amplify toxr gene fragments from v. harveyi and v campbellii, respectively. the molecular data from this study provide preliminary evidence for a proposed separate classification of a local vibrio isolates from previous studies and from this present study, distinct and separate from bonafide v. harveyi and v. campbellii. where they are currently included based on biochemical tests. one approach to resolve this classification problem involves dna sequence analysis of additional genes that are present in all vibrios in order to provide additional data for grouping isolates together or discriminating members of different but closely related vibrio species. candidate gene markers that are being considered include the gyrase b gene, toxr gene, and the gene for hemolyisn (conejero & hedreyda, 2003; conejero & hedreyda, 2004). acknowledgments the authors would like to thank mia judith u. conejero-viray for the use of her toxr and hemolysin gene primers. references conejero, m., 2002. identification of gene sequences for the specific detection of vibrio harveyi. a masteral thesis submitted to the national institute of molecular biology and biotechnology, university of the philippines, diliman, quezon city. conejero, m.j.u. & c.t. hedreyda, 2003. isolation of partial toxr gene of vibrio harveyi and design of toxr-targeted pcr primers for species detection. j. appl. microbiol. 95(3): 602611. conejero, m.j.u. & c.t. hedreyda, 2004. pcr detection of hemolysin (vhh) gene in vibrio harveyi. j. gen. appl. microbiol. 50: 137-142. de la peña, l., c. lavilla-pitogo, & m. paner, 2001. luminescent vibriosis associated with mortality in pondcultured shrimp penaeus monodon in the philippines: species composition. fish path. 36(3): 133-138. dorsch, m., d. lane, & e. stackebrandt, 1992. towards a phylogeny of the genus vibrio based on 16s rrna sequences. int. j. sys. bacteriol. 42: 58-63. mortz, e., t. krogh, h. vorum, & a. gorg, 2001. improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ ionization-time of flight analysis. proteomics. 1: 1359-1363. nishibuchi, m. & k. james, 1995. thermostable direct hemolysin gene of vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. infect. immun. 63(6): 20932099. osorio, c. & k. klose, 2000. a region of the transmembrane regulatory protein toxr that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among vibrio species. j. bacteriol. 182: 526528. philippine fisheries profile, 1998. bureau of fisheries and aquatic resources. retrieved april 2, 2003, from http:// www.prc.dircon.co.uk/philippine_aquaculture_industry.htm. que, c., 2002. molecular characterization of closely-related vibrio species through protein and dna profile analysis. a masteral thesis submitted to the national institute of molecular biology and biotechnology, university of the philippines, diliman, quezon city. sobrepeña, k. & c. t. hedreyda, 2002. dna sequence analysis of partial toxr gene fragment of vibrio campbellii. poster paper presented at the annual convention of the phillippine society for biochemistry and molecular biology, searca auditorium, uplb, december 2002. subasinghe, r., m. phillips, & a. tacon, 1997. review of the state of world aquaculture. fao fisheries technical paper. no. 886, http://www.fao.org/docrep/003/w7499e/ w7499e07.htm. 12journal policy.pmd 99 journal pol icy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the off ice of the v ice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 101 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientif ic misconduct policy. in attendance were: d r. co r a zo n d. v i l l a r e a l , r d u o d i r ec to r, p r e s i d i n g ; d r h e n r y j . ra m o s , p m rg o director and professor, nip; atty. vy va v ictoria aguirre, ovcrd legal consultant; e d i to r s i n c h i e f d r. m a r i co r s o r i a n o (science dil iman) a n d d r. m a r i a m a n g a h a s (s o c i a l s c i e n c e d i l i m a n ) . m s . n a n i e d o m i n g o a n d m s . d e r c y m a r a r a c , e d i t o r i a l assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. sd-sample article r.a. magbitang and others 1 science diliman (january-june 2013) 25:1, 1-14 issn 0115-7809 print / issn 2012-0818 online determination of cd and pb in fruit juice, bottled tea, condiments and dried fish samples using icp-ms riza a. magbitang1 , melanie a. bucsit2, eugene s.f. t ia2, rowena grace rumbaoa3, lowela lou m. cervas3, danica angel ine dimaya2 and irene b. rodriguez 1,2* 1natural sciences research institute, college of science 2institute of chemistry, college of science 3department of food science and nutrition, college of home economics university of the philippines diliman, quezon city, 1101 philippines abstract metals like cadmium (cd) and lead (pb) are introduced in the environment through natural processes and anthropogenic activities and may end up b e i n g p r e s e n t i n f o o d , w h e r e t h e s e m e t a l s m a y p o s e h e a l t h r i s k s . a m e t h o d s u i t a b l e f o r t h e s i m u l t a n e o u s d e t e r m i n a t i o n o f c d a n d p b i n v a r i o u s m a t r i c e s o f f o o d s t u f f s w a s v a l i d a t e d a n d a p p l i e d t o d i f f e r e n t s a m p l e s i n c l u d i n g f r u i t j u i c e , b o t t l e d i c e d t e a , s e v e r a l t y p e s o f condiments, and in edible tissues of dried f ish locally produced in the philippines. fruit juice and bottled iced tea samples were f iltered prior t o q u a n t i f i c a t i o n o f m e t a l s u s i n g i n d u c t i v e l y co u p l e d p l a s m a m a s s s p e c t r o m e t r y ( i c p m s ) . co n d i m e n t s a n d d r i e d f i s h s a m p l e s w e r e m i n e r a l i z e d u s i n g m i c r o w a v e a s s i s t e d n i t r i c a c i d d i g e s t i o n b e f o r e subsequent metal detection with icp-ms. the method was validated using ce r tif ied reference materials dorm 3 and nist 1643e, and evaluation o f r e c o v e r y o f s p i k e d s a m p l e s . t h e m e t h o d w a s l i n e a r i n t h e concentration range 0.01 to 500µg l -1 with correlation coeff icients of 0.999 for both analytes. the estimated detection limits were 0.060 µgl-1 and 0.186 µgl-1 for cd and pb, respectively. the determined levels of cd in fruit juice were in the range 0.06 ± 0.01 to 0.67 ± 0.01 µgl -1, and pb was detected in only one sample at 0.37 ± 0.02µgl -1. for the bottled iced tea samples, cd was detected in only one sample (0.13 ± 0.02 µgl1) w h i l e n o n e o f t h e s a m p l e s h a d d e t e c t a b l e p b c o n c e n t r a t i o n . f o r t h e condiments, the determined cd levels were in the range 0.83 ± 0.06 to _______________ *corresponding author determination of cd and pb in fruit juice 2 306.13 ± 2.52µgl-1, whereas the determined pb levels were in the range 2.14 ± 0.38 to 67.45 ± 7.76µgl-1. for the dried f ish samples, the cd levels determined were in the range 2.00 ± 0.21 to 231.67 ± 5.32 µg kg-1 and that for pb were in the range 2.38 ± 0.70 to 113.29 ± 2.25 µg kg-1. these determined levels in different foodstuffs highlight the need for routine monitoring of these contaminants. keywords: tr a ce m e t a l , f r u i t j u i ce s , co n d i m e n t s , d r i ed f i s h , m i c r ow a ve digestion, icpms introduction commonly consumed beverages in the philippines include commercial fruit juices and iced tea, which are quite popular not only due to their palatable flavors but also because of their perceived nutritive value. over the years, businesses manufacturing these beverages have continued to increase and the market has demanded for other flavors to be introduced. fruit juices are available in various packages (tetra packs, bottled, etc.) and flavors (apple, orange, or mixtures of flavors, etc.). aside from fruit juice, filipinos are also consumers of condiments which are mostly fermented products such as soy sauce (from soy beans), vinegar (from sugar cane and coconut nectar), f ish sauce and f ish paste, and shrimp paste (from krill). these are often used as seasonings in main dishes, as ingredients in marinade, and as dipping sauces. dietary guidelines worldwide increasingly recommend f ish consumption because of their nutritional benef its such as proteins, vitamins, minerals, and omega-3 polyunsaturated fatty acids (pufas) which exhibit protective effect against coronary heart disease and stroke (kris-etherton and others 2002, joint who/fao expert consultation 2003, domingo 2007). however, concerns have been raised about f ish consumption due to the potential health risks associated with environmental contaminants such as presence of metals that exhibit toxicity (domingo 2007). all of these food products may be sources of metal contaminants when these have not passed rigorous quality checks. cadmium (cd) and lead (pb) are ubiquitous in the environment and may enter the food chain due to their various applications and the improper handling of wastes. cd is used as pigment in paints, in batteries, as stabilizers for pvc and can also be used in alloys (jarup and others 1998). likewise, pb is used in batteries, alloys, pigments, cable sheathing, ammunition and as petrol additives (garcia-lestün and others 2010, jarup and others 1998, ). improper handling of wastes containing these metals and the indiscriminate use of products containing these may lead to the release of these contaminants in the environment. consequently, they can then r.a. magbitang and others 3 enter the food chain. the presence of these metals in food is a concern because of possible negative health effects. adverse health effects of cd in the body include kidney, skeletal and reproductive def iciencies (jarup and others 1998). pb may cause poor intellectual performance in children and may induce renal and cardiovascular diseases in adults (fewtrell and others 2004, garcia-lestün and others 2010). the adverse health effects of these metals prompted the joint fao/who food standard programme codex committee on contaminants in foods (cccf) to release, in march 2011, a new list of maximum levels (mls) for contaminants and toxins. for cd, the committee set a provisional tolerable monthly intake (ptmi) of 25 µg kg-1 body weight. in the case of pb, the committee estimated that the previously established ptwi of 25 µg kg-1 body weight was no longer suff icient to prevent the effects of pb. consequently, a maximum level of 0.50 mg kg-1 of pb in fruit juice was established. the joint fao/who expert committee on food additives suggested maximum levels for cd of 2 mg kg-1 in marine bivalve mollusks and provisional tolerable weekly intake (ptwi) of 0.007 mg kg -1 body weight, and maximum levels for pb of 0.3 mg kg-1 in f ish and ptwi of 0.025 mg kg-1 body weight (jecfa 1993). likewise, the task group on contaminants in f ish and other seafood of the marine strategy framework directive (msfd) of the european union has set maximum values of these metals in muscle tissues of f ish. according to the report, cd should not exceed 0.050 mg kg-1 based on the wet weight while pb should not exceed 0.30 mg kg-1 wet weight (swartenbroux and others 2010). to ensure the quality of products, especially food items, there has been a steady development in analytical techniques and instrument capabilities for monitoring metal concentration. the importance of analyzing trace contaminants has called for methodologies which are robust, able to detect trace concentrations while increasing the sample throughput and sensitivity in the analysis of varied sample matrices. conventional techniques employed by most laboratories include the use of openvessel acid digestion, dry ashing or a combination of both as pre-treatment steps to elemental analysis using flame atomic absorption spectroscopy (f-aas). more advanced laboratories utilize graphite furnace aas (gf-aas) or plasma-based techniques such as inductively coupled plasma mass spectrometry (icpms) or optical emission spectroscopy (icpoes). in this study, cd and pb were determined in fruit juice, bottled iced tea, condiment samples and dried f ish samples that are locally available in the philippines after performing a suitable pre-treatment step, either microwave digestion or simple f iltration and dilution, using icpms. determination of cd and pb in fruit juice 4 methodology chemicals and reagents all chemicals and reagents used in this study were of analytical reagent grade unless otherwise specif ied. nitric acid was obtained from merck (darmstadt, germany). single-element standards of cd, pb, indium (in) and rhenium (re) with concentrations equivalent to 10,000 ± 30 µg ml-1 were purchased from cpi international (santa rosa, ca , usa). the reference material nist 1643e (trace elements in water) used in this study was obtained from the national institute of standards and technology (maryland, usa). another reference material used was dorm 3 (f ish protein certif ied reference material for trace metals), which was obtained from the national research council canada – institute for national measurement standards (ontario, canada). all dilutions and solution preparations were done using ultrapure water prepared using a barnstead system (18.2 m cm resistivity, thermo fisher scientif ic, selangor darul ehsan, malaysia). instrumentation a multiwave 3000 microwave digestion system (anton paar, graz, austria) f itted with a 16-position rotor for high digestion performance was used for the complete mineralization of the samples. an agilent 7500cx icpms (agilent technologies, germany) equipped with a micromist glass concentric nebulizer and an integrated autosampler (i-as with type a vials, 89 x 6 ml capacity) was used as the elementselective detector. the monitored masses were m/z 111 for cd and m/z 208 for pb. the internal standards used were monitored at m/z equal to 115 for in and 185 for re. the optimum conditions used for the analyses were as follows: rf power = 1550 w, carrier gas flow rate = 0.85 0.87 l min -1, make-up gas flow rate = 0.21 – 0.24 l min-1, nebulizer pump = 0.1 rps, sampling depth = 7.1 7.6 mm and the spray chamber temperature = 2.0 °c. the calibration standards were prepared in the range 0.01 µg l-1 to 500 µg l-1 which had correlation coeff icients of 0.999 or better for both analytes. the method detection limits obtained were 0.060 µg l-1 for cd and 0.186 µg l-1 for pb. these were estimated by getting the standard deviation of the results from the analysis of seven replicate solutions containing 1 µg l-1 of cd and pb, then subsequently multiplying the sd by the student’s t value (for n = 7, t value is equal to 3.143; ripp 1996). a drift standard, composed of 1.0 µg l-1 of both cd and pb, was measured repeatedly throughout the analysis sequence to determine the precision of the measurements within a day. the determined relative standard deviations of the repeated measurements of the drift standard were less than 1% and 2% for cd and ω r.a. magbitang and others 5 pb, respectively. these values indicate a stable instrument performance and good precision of measurements. sampl ing all samples (fruit juice and condiments) included in the study were locally produced by different manufacturers and represent common products in the market. the samples were bought from a local supermarket which carries varied products. a total of 17 fruit juices and 5 bottled iced tea samples were included in the analysis. various flavors of fruit juices were included: apple, grape, mango, orange, strawberry, calamansi, and guyabano. for the bottled iced tea samples, different brands that carry lemon flavor were included. eighteen condiment samples were analyzed in the study, which included cane and spiced coconut vinegars, f ish and shrimp pastes, f ish sauce, soy sauce, and liquid seasoning. the dried f ish samples were collected from various wet and dry markets in quezon city. they were identif ied to the nearest family by a research scientist at the institute of biology, college of science, university of the philippines diliman. the identif ication of the exact species of the dried f ish samples was hampered because of the drying procedure that the samples have been subjected to, which led to the f ish samples losing most of the scales and f ins necessary for the identif ication procedure. sample preparation the fruit juice and iced tea samples were analyzed for the metal content after a simple f iltration and dilution process. samples were f iltered using nylon syringe f ilters (pore size: 0.45 µm, diameter: 25 mm; obtained from membrane solutions llc, shanghai, china) immediately after opening. three replicates were prepared for every sample. samples were diluted 10-fold, acidif ied to 1% hno 3 , and then analyzed using icp-ms. accuracy of the method was evaluated by analyzing nist 1643e prepared in the same way and by spiking some samples with known amounts of cd and pb standards, which were then subjected to the same procedure. the condiment samples were mineralized by microwave-assisted acid digestion after addition of 2.0 ml of hno 3 and 6.0 ml of water for solid samples (250 mg weighed to the nearest 0.01 mg) or 2.0 ml of hno 3 and 5.0 ml water for liquid samples (volume of sample = 1.0 ml). the digestion step was carried out by following this optimized program (modif ied from magbitang and rodriguez 2012): the system was ramped to 600w for 5 min, kept at this condition for 30 min, decreased to 500 w and held at this condition for another 10 min, before allowing determination of cd and pb in fruit juice 6 the system to cool. after digestion, the volume was adjusted to 10 ml. for the dried f ish analysis, the edible portions of the dried f ish samples were carefully scraped using acid-washed plastic knives. triplicate aliquots (250 mg weighed to the nearest 0.1 mg) of each sample were weighed and subjected to the optimized mineralization procedure prior to icpms analysis. triplicate aliquots of reference materials dorm-3 (250 mg) and nist 1643e (1.0 ml) were digested along with the condiment samples and dried f ish samples. results and discussion the results for the validation of the pre-treatment methods and icpms parameters using certif ied reference materials are summarized in table 1. nist reference material 1643e was used for both the validation of the f iltration step used for juice and tea samples, and the microwave-assisted digestion step used for the condiment samples. the reference material dorm 3 was also used for the validation of the microwave-assisted digestion step to simulate the complex matrix of some of the condiment samples that were derived from f ish. this was also the main reference material for the dried f ish samples. the results for nist 1643e analyzed after simple f iltration reflected good accuracy, with 103.3% recovery for cd and 92.4% for pb. this was comparable to the results for nist 1643e obtained after the microwave-assisted digestion with 104.2% recovery for cd and 97.1% for pb. as for the more complex matrix, the values obtained were 88.0% recovery for cd and 84.6% recovery for pb in dorm 3 after the complete mineralization by microwave digestion. these results suggest that the microwave digestion step would be a suff icient pre-treatment step for the condiment samples and dried f ish samples prior to icpms analysis. table 1. determined values for cd and pb in the certified reference materials processed using simple filtration or microwave-assisted acid d igestion prior to icpms analysis (expressed as mean concentration ± sd, n = 3) apre-treatment step includes simple filtration followed by dilution. bpre-treatment step includes microwave-assisted acid digestion followed by dilution. certified element certified value obtained value % recovery reference material nist 1643ea 111cd 6.568 ± 0.073 µg l-1 6.78 ± 0.16 µg l-1 103.2 208pb 19.630 ± 0.210 µg l-1 18.14 ± 1.17 µg l-1 92.4 nist 1643eb 111cd 6.568 ± 0.073 µg l-1 6.85 ± 2.66 µg l-1 104.2 208pb 19.630 ± 0.210 µg l-1 19.05 ± 2.16 µg l-1 97.1 dorm 3b 111cd 290.000 ± 20.00 µg kg-1 255.17 ± 4.13 µg kg-1 88.0 208pb 395.000 ± 50.00 µg kg-1 334.27 ± 17.72 µg kg-1 84.5 r.a. magbitang and others 7 for further validation of the f iltration step used for juice and tea samples, known concentrations of the standards were spiked in some of the samples and the results are presented in table 2. for the recovery test, juice and tea samples (one each) were spiked with 0.5, 5, and 50 µg l-1 concentrations of cd and pb standards. as shown above, good recovery values for the three different concentrations were obtained for cd, which ranged from 100.3% to 106.0%. recovery of pb at the lowest concentration was observed to be low, which were at 57.6% and 72.6% for juice and tea samples, respectively. for the higher concentrations, 5 and 50 µg l-1, recoveries were from 95.4% to 98.9%. these recovery values are within the acceptable values as suggested by taverniers and others (2004) and may show the influence of the matrix of the sample on the recovery of the target analytes. the good recoveries of the spiked samples indicate that the simple f iltration and dilution method used in the study is reliable in quantifying the amount of these metals in juice and bottled iced tea samples. analysis of these samples can be done successfully without any further sample pretreatment. the results of the analysis of cd and pb in actual samples of fruit juice and bottled iced tea samples, as well as their respective brand codes, are summarized in table 3. the different brands are coded from a to l because they carry various products and flavors. from the data obtained, it is evident that most of the juice samples analyzed have pb content lower than the method detection limit while some have detectable cd concentrations. currently, there is no maximum limit set for cd in fruit juices. the determined levels of cd and pb in these samples indicate that there is no signif icant level of the metal contaminants in these food products but the detectable levels of cd suggest the need for routine monitoring of these contaminants in these products. juice 0.5 0.50 ± 0.02 100.3 0.36 ± 0.22 72.6 5 5.09 ± 0.05 101.9 4.77 ± 0.15 95.4 50 51.38 ± 0.87 102.8 48.60 ± 0.22 97.2 tea 0.5 0.53 ± 0.02 105.3 0.29 ± 0.14 57.6 5 5.16 ± 0.06 103.2 4.94 ± 0.36 98.9 50 53.01 ± 0.81 106.0 48.38 ± 0.54 96.8 concentration of cd and pb (µg l-1) addedsample obtained value (µg l-1) pb% recoverycd % recovery table 2. percent recovery values for juice and tea samples spiked with known concentrations of standard cd and pb solutions (expressed as mean concentration ± sd, n = 3) determination of cd and pb in fruit juice 8 apple a < mdl 0.37 ± 0.02 b < mdl < mdl mango a 0.25 ± 0.05 < mdl b 0.23 ± 0.05 < mdl c 0.67 ± 0.01 < mdl e < mdl < mdl orange a < mdl < mdl b < mdl < mdl c 0.12 ± 0.01 < mdl f < mdl nd g 0.35 ± 0.02 < mdl grape a 0.10 ± 0.02 < mdl b < mdl < mdl c 0.06 ± 0.01 < mdl strawberry c < mdl < mdl calamansi d < mdl < mdl guyabano e 0.12 ± 0.03 < mdl bottled iced tea h < mdl nd i 0.13 ± 0.02 nd j < mdl nd k < mdl nd l < mdl nd nd: no detectable value indicates that the signals from icpms measurements were not significantly higher than the blank. <mdl: below the method detection limit. table 3. determined concentration of cd and pb in d ifferent juice and bottled iced tea samples (expressed as mean concentration ± sd, n = 3) samples concentration (µg l-1) fruit juice brand cd pb the different condiment samples were subjected to the mineralization procedure by microwave digestion prior to elemental analysis. the determined concentration of cd and pb in the condiment samples, with the corresponding brand codes, are presented in table 4. of all the condiments included in the study, only shrimp paste is available in solid form. the determined levels of cd in shrimp paste were 9.68 ± 2.25 and 88.69 ± 6.58 µg kg-1 for the two brands. for the liquid condiment samples, the determined levels of cd ranged from 0.83 ± 0.06 to 306.13 ± 2.52 µg l-1. on the other hand, the determined levels of pb were 50.68 ± 14.9 and 63.91 ± 6.77 µg kg-1 in the two brands of shrimp paste. the obtained values for pb in liquid condiment samples were in the range 2.14 ± 0.38 to 67.45 ± 7.76 µg l-1. in general, shrimp and f ish paste samples contain elevated levels of cd and pb, which may have been due to the raw material itself. seafood and seafood products are widely known to contain elevated levels of metal contaminants. r.a. magbitang and others 9 for the dried f ish, the samples chosen for this study were dried f ish types preferred by consumers and widely available in all the major wet and dry markets in the most populated city in metro manila. the determined cd and pb levels for all samples are summarized in table 5, including information about the sources. from the data, all determined pb levels and most of cd levels were below the suggested limit by the eu-msfd (swartenbroux and others 2010). all of the determined values for cd and pb were below the suggested maximum limits of the jecfa for marine-derived food products (jecfa 1993). the cd levels determined were in the range 2.00 ± 0.21 to 231.67 ± 5.32 µg kg-1 and that for pb were in the range 2.38 ± 0.70 to 113.29 ± 2.25 µg kg -1. the determined levels of cd and pb in the samples studied show that there is a need to monitor these contaminants in these types of food products. although the concentrations in the fruit juice and tea samples are relatively low, routine monitoring is still necessary as the effects of these contaminants are dependent on the extent of the exposure. compared with the other types of condiments, the table 4. determined concentration of cd and pb in d ifferent condiment samples (expressed as mean concentration ± sd, n = 3) shrimp paste* 1 88.69 ± 6.58 50.68 ± 14.9 7 9.68 ± 2.25 63.91 ± 6.77 fish paste 1 21.07 ± 2.43 37.59 ± 1.67 2 116.44 ± 10.52 67.45 ± 7.76 fish sauce 2 306.13 ± 2.52 4.24 ± 0.98 3 1.94 ± 0.10 2.14 ± 0.38 8 106.53 ± 1.40 18.07 ± 3.30 soy sauce 3 0.83 ± 0.06 <mdl 4 6.24 ± 0.15 2.61 ± 0.74 6 8.83 ± 0.32 6.66 ± 1.66 spiced coconut vinegar 5a 2.45 ± 0.27 10.24 ± 3.33 5b 3.22 ± 0.20 9.81 ± 2.46 white vinegar 4 <mdl <mdl 6 <mdl <mdl cane vinegar 4 <mdl <mdl 6 <mdl <mdl liquid seasoning 9 0.89 ± 0.05 3.32 ± 1.14 10 1.94 ± 0.47 3.33 ± 0.65 *concentrations are expressed as µg kg-1 <mdl = below detection limit a spicy variant; b sweet variant type of condiment mean concentration (µg l-1) brand cd pb determination of cd and pb in fruit juice 10 table 5. determined concentrations of cd and pb in dried fish samples expressed as mean ± sd (n = 3). common names, scientific names in some and sample codes are shown (sample codes are designated with respect to source market) m1fa purse-eyed scad selar sp. 26.93 ± 1.31 6.35 ± 0.69 m1fb deep body sardinella sardinella brachysoma 25.82 ± 1.49 3.44 ± 0.78 m1fc beltf ish family trichiuridae 3.53 ± 0.15 35.29 ± 1.34 m1fd parrot f ish scarus sp. 32.93 ± 0.78 8.90 ± 1.13 m1fe croaker family sciaenidae 6.84 ± 0.35 47.42 ± 1.56 m2fa yellow tail fusilier caesio caerulaurea 57.23 ± 2.49 23.10 ± 0.93 m2fb deep body sardinella sardinella brachysoma 22.23 ± 0.86 15.32 ± 1.16 m2fc short-bodied family carangidae 28.24 ± 1.58 66.22 ± 1.85 mackerel m2fd flying f ish cypselurus sp. 55.27 ± 1.45 113.29 ± 2.25 m3fa long-jawed mackerel rastrelliger sp. 7.35 ± 1.29 2.38 ± 0.70 m3fb deep body sardinella sardinella brachysoma 5.68 ± 0.54 67.78 ± 4.88 m3fc short-bodied mackerel family carangidae 45.01 ± 2.06 < mdl m3fd tilapia oreochromis sp. < mdl 13.94 ± 1.09 m3fe threadf in bream nemipterus sp. 16.34 ± 1.28 < mdl m4fa cavalla f ish carangoides sp. 46.45 ± 1.76 28.67 ± 0.82 m4fb deep body sardinella sardinella brachysoma 2.00 ± 0.21 < mdl m4fc purse-eyed scad selar sp. 137.75 ± 2.75 < mdl m4fd ponyf ish leiognathus sp. 19.81 1.33 10.14 ± 1.15 m4fe barracuda family sphyraenidae 7.70 ± 0.53 12.00 ± 1.48 m5fa lizard f ish family synodontidae 121.08 ± 1.89 4.14 ± 1.10 m5fb deep body sardinella sardinella brachysoma 2.92 ± 0.30 < mdl m5fc goat f ish family mullidae 54.28 ± 1.95 27.60 ± 1.21 m5fd cavalla f ish carangoides sp. 41.38 ± 1.45 46.59 ± 1.32 m6fa threadf in bream nemipterus sp. 19.38 ± 1.31 5.46 ± 0.69 m6fb deep body sardinella sardinella brachysoma 3.16 ± 0.37 < mdl m6fc tilapia oreochromis sp. < mdl < mdl m6fd yellow tail fusilier caesio caerulaurea 59.27 ± 1.38 64.51 ± 1.55 m6fe round scad decapterus sp. 22.14 ± 0.91 4.48 ± 1.22 m7fa round scad decapterus sp. 231.67 ± 5.32 < mdl m7fb deep body sardinella sardinella brachysoma 4.32 ± 0.44 < mdl m7fc long-jawed mackerel rastrelliger sp. 15.27 ± 1.18 2.42 ± 1.18 m7fd ponyf ish leiognathus sp. 25.09 ± 0.93 5.41 ± 4.55 sample code and source common name scientific name cd concentration µg kg-1 pb concentration µg kg-1 r.a. magbitang and others 11 f ishand shrimp-derived condiments contain elevated levels of both cd and pb, which indicate that the contamination may come from the source material. for these types of condiments, further work should be done to ascertain where the source of contamination is from. moreover, the obtained values for the analytes in these condiment samples show that stricter regulations should be in place to ensure that these food products are safe for human consumption. the data presented here show that cd and pb are present in dried f ish samples and although most of the determined levels were below the suggested limits by the eu-mfsd and the jecfa, these detected values merit routine monitoring of cd and pb levels in edible f ish tissues especially those types that are consumed regularly. conclusion and recommendations different sample preparation methods suitable for the pre-treatment of fruit juice, b o t t l ed te a , co n d i m e n t s a n d d r i ed f i s h s a m p l e s p r i o r to m e t a l s e l ec t i ve determination by icpms were validated and applied to actual samples. the methods were found to be simple and suitable for different matrices. application of the simple f iltration and subsequent analysis by icpms to different fruit juice and bottled tea samples showed that cd and pb were detectable in fruit juice samples while these were not detected in the bottled tea samples. different types of condiments and the edible tissues of dried f ish samples were subjected to the mineralization by microwave-assisted acid digestion and icpms analysis. the results showed that cd and pb were present in most types of condiments, with the exception of white and cane vinegar, and are also present in edible tissues of dried f ish. the results also revealed that the sample pre-treatments steps were suitable for the different samples and robust enough to allow for the simultaneous determination of cd and pb by icpms. the presence of these metal contaminants in these food products necessitates for a wider scope of study, which should cover larger sample size and longer time frame, to assess if there is indeed a need for stricter regulations to ensure safety of the consumers. the samples included in this study are commonly consumed by the majority of the population and hence, the presence of these contaminants in the food products may mean signif icant exposure, which warrants the need for the wider scope of sampling area, size and duration. a study which focuses on the differences in the daily intake of varying types of consumers and the associated intake of the contaminants may also provide meaningful data for proper assessment of risk. determination of cd and pb in fruit juice 12 acknowledgments the authors would like to thank the natural sciences research institute (up diliman) and the university of the philippines system for funding this work. the authors would also like to thank dr. luis maria garcia of the institute of biology, college of science, university of the philippines diliman for his help in this work. references domingo jl. 2007. omega-3 fatty acids and the benef its of f ish consumption: is all that glitters gold? environ int 33: 993–998. efsa panel on contaminants in the food chain (contam). 2010. scientif ic opinion on lead in food. efsa journal 8: 1570. available from: doi:10.2903/j.efsa.2010.1570 efsa panel on contaminants in the food chain (contam). 2011. scientif ic opinion on tolerable weekly intake for cadmium. efsa journal 9: 1975. available from doi:10.2903/ j.efsa.2011.1975 fewtrell lj, prüss-üstün a, landrigan p, ayuso-mateos jl. 2004. estimating the global b u r d e n o f d i s e a s e o f m i l d m e n t a l r e t a r d a t i o n a n d c a r d i o v a s c u l a r d i s e a s e s f r o m environmental lead exposure. environ res 94: 120-133. garcia-lestün j, m éndez j, pásaro e, laffon b. 2010. genotoxic effects of lead: an updated review. environ int 36: 623-636. jarup l, berglund m, elinder cg, nordberg g, vahter g. 1998. health effects of cadmium exposure—a review of the literature and a risk estimate. scand j work environ health 24: 1-52. jecfa. 1993. evaluation of certain food additives and contaminants. forty-f irst repor t of the joint fao/who expert committee on food additives. world health organization, technical repor t series 837. joint who/fao expert consultation. 2003. diet, nutrition and the prevention of chronic diseases. geneva, switzerland: world health organization. kris-ether ton pm, harris ws, appel lj. 2002. fish consumption, f ish oil, omega-3 fatty acids, and cardiovascular disease. j am hear t assoc, 106: 2747-2757. magbitang ra , rodriguez ib. 2012. cadmium and lead determination by icpms: method optimization and application in carabao milk samples. science diliman, 24: 1-11. mol s. 2011. levels of selected trace metals in canned tuna f ish produced in turkey. j food compos anal 24: 66-69. ripp j. 1996. analytical detection limit guidance & laboratory guide for determining m e t h o d d e tec t i o n l i m i t s . w i s co n s i n d e p a r t m e n t of n a t u r a l re s o u r ce s , la b o r a to r y cer tif ication program. r.a. magbitang and others 13 swar tenbroux f, albajedo b, angelidis m, aulne m, bar tkevics v, besada v, bignert a , bitterhof a, hallikainen a, hoogenboom r, jorhem l, jud m, law r, licht cederberg d, mcgovern e, miniero r, schneider r, velikova v, verstraete f, v inas l, vlad s. 2010. marine strategy framework directive – task group 9 repor t contaminants in f ish and other seafood. scientif ic and technical research series – issn 1018-5593. isbn 97892-79-15652-6. available from doi 10.2788/86934 taverniers i, de loose m, van bockstaele e. 2004. trends in quality in the analytical laboratory. ii. analytical method validation and quality assurance. trends anal chem 23: 535-552. vanhaecke f, vanhoe h, dams r, vandecasteele c. 1992. the use of internal standard in icp-ms. talanta 39: 737-42. who. 2011. codex committee on contaminants in foods (cccf) f ifth session. joint fao/who food standards programme. _______________ irene b. rodriguez <ibrodriguez@upd.edu.ph> is currently a postdoctoral research fellow at the research center for environmental changes in academia sinica, taiwan. her work is centered on how trace metals interact with light to control the nitrogen cycle in marine biogeochemical systems. she is involved in research to better understand the role of trace metals in an ocean undergoing acidif ication, and how phytoplankton utilize these metals and accumulate these in the cells before eventually passing the metals up the trophic levels. riza a. magbitang <riza.magbitang@gmail.com> is a university research associate i (ura i) at the natural sciences research institute, university of the philippines diliman. she is currently enrolled in the ms chemistry program of the institute of chemistry of the same university, where she also obtained her bs chemistry degree. her research interests, among others, include environmental analytical chemistry, particularly optimization and validation of methods for the determination of environmental pollutants. melanie a. bucsit is currently an instructor at the institute of chemistry, university of the philippines diliman, where she also obtained her ms chemistry degree. rowena grace o. rumbaoa <rorumbaoa@yahoo.com> is a faculty member at the department of food science and nutrition, at the college of home economics, university of the philippines diliman. she primarily handles courses in food chemistry and analysis, and food processing. she received her ms food science degree from the same university, where she is also currently pursuing her phd degree. she has co-authored some works on algal polysaccharides, antioxidants, and determination of cd and pb in fruit juice 14 proximate composition of foods. she has also completed researches on rootcrop antioxidants, processing and fortif ication of juices, modif ied atmosphere packaging and algal polysaccharides. eugene s.f. t ia and danica angel ine dimaya are graduate students at the institute of chemistry, college of science, university of the philippines diliman. lowela lou m. cervas <lowelacervas@yahoo.com> is an instructor at the university of the philippines los baños where she handles courses on food analysis, food processing, food packaging and labeling, food hygiene and sanitation, and practicum i– pilot scale. 4experiments and pilot study-pascual.pmd r.m. pascual and r.c.l. guevara 5 science diliman (january-june 2017) 29:1, 5-36 experiments and pilot study evaluating the performance of read ing miscue detector and automated read ing tutor for filipino: a children’s speech technology for improving literacy ronald m. pascual* far eastern university manila rowena cristina l. guevara university of the philippines diliman abstract the latest advances in speech processing technology have allowed the development of automated reading tutors (art ) for improving children's literacy. a n art is a computer-assisted learning system based on oral reading fluency (orf) instruction and automated speech recognition (asr) technology. however, the design of an art system is language-specif ic, and thus, requires developing a system specif ically for the filipino language. in a previous work, the authors have presented the development of the children's filipino speech corpus (cfsc) for the purpose of designing an art in filipino. in this paper, the authors present the evaluation of the art in filipino which integrates a reference verif ication (rv)and word duration analysis-based reading miscue detector (rmd), a user interface, and a feedback and instruction set. the authors also present the performance evaluation of the rmd in offline tests, and the effectiveness of the art as shown by the results of the intervention program, a month-long pilot study that involved the use of the art by a small group of students. offline test results show that the rmd's performance (i.e. , fa rate ≈ 3% and mderr rate ≈ 5%) is at par with those from state-of-the-art rmds reported in the literature. the results of the art intervention experiment showed that the students, on the average, have improved in their words correct per minute (wcpm) rate by 4.66 times, in their orf-16 scores by 6.0 times, and in their reading comprehension exam scores by 4.4 times, after using the art. key words: reading miscue detector, automated reading tutor, reference verif ication, word duration analysis, filipino speech _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online a children’s speech technology for improving literacy 6 introduction the latest advances in the speech processing technology, coupled with the current problems that the country's primary education system are facing, such as the poor reading performance of the students and the shortage of teachers, inspired the authors to focus on the development of an automated reading tutor (art) for improving filipino children's literacy. an art is a computer-assisted learning system based on oral reading fluency (orf) instruction and automated speech recognition (asr) technology. the main task that an art performs is the automatic detection of reading miscues or disfluencies in an input speech. through the reading miscue detector (rmd), the art is capable of “listening” to the reader and spot reading errors so that it may offer help (e.g. , by modeling the correct pronunciation of a text passage) whenever necessary. figure 1 presents an overview of the art system and its basic components. it must be noted that, unlike a conventional automatic speech recognizer, the rmd knows in advance the desired or target speech pattern that should be uttered by the learner or user. the objective is to identify possible deviations (miscue, error, or disfluency) from the target speech pattern using a certain method. the designs of art and rmd systems however are language-specif ic. for instance, the project listen's reading tutor (mostow et al. 1994), the colorado literacy tutor (hagen et al. 2003), and the system presented by black et al. (2011) were all designed for the english language. the rmd systems presented by liu et al. (2008) and by duchateau et al. (2006) were designed for the chinese mandarin and dutch figure 1. overview of the automated reading tutor (art ) system and its basic components. r.m. pascual and r.c.l. guevara 7 languages, respectively. recently, rahman et al. (2014) made an effort to develop an asr system that can be used for arts for malay-speaking children, while rayner et al. (2014) developed a ruleand grammar-based computer-assisted language learning (call) system for german-speaking children. in this study, the authors focused on the development of a system for filipino, the national language of the philippines and a language used in the philippine basic education system. features and orthography of filipino are very distinct from other languages, and thus, there is an apparent need to develop a system specif ically designed for the language. for instance, according to speech rhythm, it has been shown that filipino is generally classif ied as a syllable-timed language (guevara et al. 2010). in syllable-timed languages, and unlike in stress-timed languages, such as english, every syllable is perceived as taking up roughly the same amount of time, except for small variations due to the prosody. moreover, the authors decided to develop a system specif ically designed for children in the early grade levels because, according to educators and reading experts, intervention programs, such as reading tutorials done at early grades, are most effective and likely ensure reading success at later grades (wasik and slavin 1993; national reading panel 2 0 0 0 ) . however, a diff iculty in developing systems for children is the unavailability of appropriate children's speech corpus that can be used for a particular application in a particular language (gerosa et al. 2009; russell 2010). it was noted by russell (2010) and suggested by gerosa et al. (2009) that, although an asr system trained on adults' speech can employ an adaptation technique that improves its performance in processing children's speech, it is unlikely that its performance will exceed that of a counterpart system trained on children's speech. in this study, the authors also present the development of a children's filipino speech corpus or the cfsc (pascual and guevara 2012a) that was used for the design and implementation of the rmd system for filipino. most of the rmd systems that were reported in the literature have generally used either of the two baseline systems: (1) a conventional asr; or the (2) reference verif ication (rv) method. the rmd systems presented by mostow et al. (1994), liu et al. (2008) and duchateau et al. (2006) all employed the f irst type of baseline system (i.e. , a conventional asr) for decoding what the reader has said. in the conventional asr baseline system type, the recognition results are compared with the "reference" (i.e. , the target or expected sound/s in the text to be read) to check whether there are any deviations (i.e. , reading miscues or disfluencies). moreover, a suitable language model (lm) based on the reading text is typically used together with the asr baseline system, in order to improve the recognition performance. a children’s speech technology for improving literacy 8 by contrast, other rmd systems, such as by black et al. (2011) and by bolaños et al. (2009), employ the second type of baseline system (i.e. , the rv method) and do not use an lm. under the rv framework, the reader's speech data and the reference are usually forced-aligned while the likelihood of the sounds is calculated. the system then classif ies whether an input speech sound, in comparison with the reference, is accepted or rejected. thus, the rv method may be regarded as a speech classif ication method rather than a speech recognition method. the rv method may also similarly be seen as a form of speech verif ication or pronunciation verif ication. an obvious advantage of the rv method over the conventional asr baseline system is that it avoids the problems caused by using a complex or dynamic lm (bolaños et al. 2009). the rv method, however, suffers from relatively lower miscue detection rate due to its observed tendency to usually ignore single phone or syllable deletion, insertion, or substitution, or even repetitions and selfcorrections. thus for practical rmd systems, an additional process is usually integrated to the baseline rv method, in order to achieve a better performance. in this paper, the authors present the performance evaluation of the rmd system for the automatic detection of reading miscues in children's filipino speech. the rmd system is the core of the art system that the authors have developed for filipino (pascual and guevara 2012b). the architecture of the rmd system consists of two levels: (1) a phone-level rv method on the f irst level; and, (2) a word-level alignment and word duration analysis (wda) method on the second level. an interesting related study by duong et al. (2011) discusses about the use of word duration-based template models for automatically assessing children's oral reading prosody in english. the focus of this study is the use of wda for automatic detection of reading miscues and disfluencies for application in art system for filipino. over the past few years, a number of f ield or pilot studies regarding the implementation and evaluation of these arts in various languages and countries have been published. for instance, mills-tettey et al. (2009) conducted a f ield study in selected schools in ghana and zambia in africa to investigate the viability and effectiveness of the use of the project listen's reading tutor, in order to improve the reading skills of children in english as a second language (l2). using the same art system, mostow et al. (2013) made a 7-month study that involved 178 students, and claimed that the use of the art resulted to improvements expected from guided oral reading, such as higher gains in fluency and reading comprehension. duchateau et al. (2009) presented the evaluation of an art for fluency instruction in dutch as a f irst language (l1), and claimed that the specif ic art works satisfactorily for children, even for those with reading disabilities, in a real school environment. tsau (2012) conducted a f ield study, which was participated r.m. pascual and r.c.l. guevara 9 in by students in central taiwan, and reported that their art system named "my english tutor" have successfully enhanced the oral reading fluency (orf) of the efl learners. similarly, reeder et al. (2015) employed an art system for english in their study conducted in a public elementary school in vancouver, canada, and concluded that the art system successfully contributed to the reading development of young learners of english as additional language (eal). in this paper, the authors present the evaluation of an art for filipino that integrates an rmd, a user interface, and a feedback and instruction set. the next sections present a discussion of the results of the art intervention program, a month-long pilot study that involved the use of the art by a small group of students. the program aims to evaluate the effectiveness of the art in improving the reading skills of the learners. design and evaluation method for the reading miscue detector for filipino children's fil ipino speech corpus and models some studies in the past few years, such as by kazemzadeh et al. (2005), batliner et al. (2005), cleuren et al. (2008), and gao et al. (2012), have focused on the development of children's speech corpora in languages, such as english, dutch, italian, german, swedish, and mandarin. the absence of a speech corpus that can be used for the development of an rmd and art for filipino has motivated the authors to develop a medium-scale, genderand age-balanced cfsc (pascual and guevara 2012). nearly all of the speakers in the cfsc are native speakers of filipino. the cfsc consists of two parts: (1) a part containing good reading pronunciations; and, (2) a part containing examples of actual reading miscues and disfluencies. the cfsc provides the following data sets: training data set for the generation of speech models, reference speech features (such as word durations) set extracted from good pronunciations, offline test set for the evaluation of the rmd system, and data set for the analysis of actual reading miscues found in children's filipino speech. the f irst part of the children's speech corpus (i.e. , the good pronunciations) contains about f ive hours of continuous read speech collected from a total of 37 grades 2 to 5 students (ages ranging from 7 to 12 years). out of the 37 students, 17 are girls and 20 are boys. the second part of the children's speech corpus (i.e. , the part containing reading miscues) contains about three hours of continuous read speech a children’s speech technology for improving literacy 10 collected from a total of 20 grades 1 to 3 students (ages ranging from about 6 to 9 years). of these students, 11 are girls and 9 are boys. nearly the entire cfsc contains orthographic transcriptions for the speech data. to further make the cfsc useful for the rmd design, the authors transcribed a part of the cfsc at phoneme level. note that the smallest possible sound unit where a reading miscue may occur is in a single phone or syllable, thus suggesting the need for phone-level transcriptions. the phone-level transcription process, which is one of the most expensive parts of the design, was executed in a semi-automated method. that is, the speech data were initially machine-transcribed through phoneme forced-alignment method using a hidden markov modelor hmm-based speech modeling toolkit htk (young et al. 2006), and the machine transcriptions were then manually re-aligned afterwards if needed. a bootstrap data set (about 20 minutes of hand-transcribed speech) was used to facilitate the automated transcription method. the phoneme set used for this study includes a total of 35 phones and diphones as listed in table 1. phone class phones / diphones stop /p/, /b/, /t/, /d/, /k/, /g/, /q/ (glottal stop) fricative /f/, /v/, /s/, /z/, /sh/ affricate /j/ nasal /m/, /n/, /ng/ lateral liquid /l/ retroflex liquid /r/ glide /w/, /y/ vowels /a/, /e/, /i/, /o/, /u/ diphones /ha/, /he/, /hi/, /ho/, /hu/, /at/, /aw/, /ay/, /oy/ pause / silence /pau/ table 1. phoneme set used for children’s fil ipino speech corpus (cfsc) transcriptions a total of nine filipino text passages, six of which are short stories while the other three are expository-type texts, were used for the cfsc recordings. adarna house, a publisher of filipino short stories for children in the philippines, provided the six short stories while the other three expository-type texts were adopted from various school textbooks. all the text passages are age-appropriate and have been suggested by research collaborators from the college of education at the university of the philippines diliman. r.m. pascual and r.c.l. guevara 11 design of the reference verif icationand word duration analysis-based reading miscue detector for fil ipino for an rmd, the desired or target speech pattern (reference) that should be uttered by the reader is known in advance, and the objective is to identify possible deviation (miscue or error) from the reference. as mentioned in section i, the rv method for detecting reading miscues aligns the input speech with the reference, and decides through a certain similarity measure whether or not the input speech sounds are the same as the reference sounds. there are generally two possible approaches in estimating the likelihood or similarity of the expected sounds in the reference to those sounds found in the input speech. the f irst approach is through a time-domain template matching, while the second approach is through speech model (hmm) comparison using parametric representation of speech features, such as the mel frequency cepstral coeff icients (mfccs). while template matching-based asr offers simplicity, it also suffers from several diff iculties and limitations, such as inability to generalize features from many speakers and example utterances, low computational eff iciency for larger number of test/reference patterns, and the inability to incorporate statistical features from a given data set. the advantages of hmm-based asr over template matchingbased systems have influenced us in selecting the hmm-based approach for designing the rv-based rmd in filipino. in particular, the hmm-based approach allowed us to practically use all the information available in the training data and to design an rmd that is speaker-independent. furthermore, the hmm-based approach also permitted us to implement a computationally eff icient and practically realizable rmd. in this study, the authors' initial approach is to design a baseline system that uses phone-level rv method. to do this, the authors f irst generated the hidden markov models (hmms) for all the phones in the phoneme set used in this study by training the system with 1.5 hours of phone-level transcribed children's speech in the cfsc. the hmms consist of three states with 39 mfccs comprising 13 static coeff icients plus 13 delta coeff icients plus 13 acceleration coeff icients. an overview of the markov model is illustrated in figure 2. it is worth noting that the 3-state hmm prototype shown in figure 2 appears to actually have f ive states. this is only due to the speech modeling toolkit convention. the f irst and the last states are non-emitting states, and thus, are part of the network of hmms, but do not describe any of the input data. only the middle three states emit observation vectors. a children’s speech technology for improving literacy 12 given the reference text passage and an input speech data, the phone-level rv method then proceeds as follows: 1. form the reference (or expected) phone symbol sequence (including symbols for expected short pauses/silences for proper phrasing) from the reference text passage. 2. perform an hmm viterbi-forced alignment process between the reference phones and the phones found in the input speech data. (this process produces the log likelihood scores for each phone in the reference). 3. using the output likelihood scores ρ v for each phone v from step 2, perform a threshold-based classif ication to decide whether or not a reading miscue has occurred. that is, where p = log likelihood score threshold. figure 2. three-state left-to-right hidden markov model (hmm) used in this study. states 1 and 5 are non-emitting. observation vectors consist of 39 mel frequency cepstral coeff icients (mfccs). the aij’s are the transition probabilities. 1 ( ), min [ ] 0 ( ), min [ ] v all v rv v all v miscue present p md miscue absent p         (1), r.m. pascual and r.c.l. guevara 13 figure 3 graphically illustrates the rv method through a specif ic example. in this example, the reference text is a four-word filipino phrase /maraming prutas at gulay/ (many fruits and vegetables). the rv process is initiated by phonetically spelling out the reference, thus producing the phone sequence: /m/, /a/, /r/, /a/, /m/, /i/, /ng/, /p/, /r/, /u/, /t/, /a/, /s/, /q/, /at/, /g/, /u/, /l/, and, /ay/. the reference phone sequence is then stored as a text f ile. during the execution of the rv process, the reference phones are all initially assigned to a starting position and have equal durations. the viterbi-forced alignment process proceeds by taking the reference phones one-by-one and f inding the best alignment (i.e. , the alignment that produces the maximum likelihood score) with the input speech data. the numbers alongside the phone symbols in the last tier shown in figure 3 are the log likelihood scores. intuitively, we may decide that there has been a reading miscue (i.e. , deviation from the expected or reference phones) if a phone likelihood score goes below the lower threshold. while the previous condition generally applies, it is now worth noting that due to the nature of the viterbi-forced alignment and likelihood scoring, there is also a need to set another threshold (i.e. , an upper threshold) for the purpose of detecting a miscue. the upper threshold is necessary for the detection of cases wherein the likelihood scores become too high due to certain types of miscues or disfluencies, such as vowel elongation or pause/silence prolongation. figure 3. illustration of the application of the reference verif ication (rv) process used in this study to a specif ic utterance of a sentence in the filipino reading text. a children’s speech technology for improving literacy 14 as mentioned in the previous section, the phone-level rv method alone is expected to give a relatively low miscue detection rate at an acceptable false alarm rate due to its poor ability to detect single-phone or single-syllable errors, as well as disfluencies like brief hesitation pauses, self-corrections, and repetitions. to address this problem, the authors' approach was to integrate a second-level (i.e. , wordlevel) process that uses a duration-based prosodic feature (i.e. , word durations) to the baseline phone-level rv process, in order to obtain a better detection of reading miscues. the idea was based on an initial observation that, in many cases of actual reading miscues and disfluencies found in the cfsc, the effective word durations highly deviated from the expected word durations, which are based on good pronunciation examples. figure 4 illustrates such a case of a reading miscue found in a certain f ive-word sentence in the cfsc. figure 4 shows the speech waveforms of a good pronunciation example (upper plot) and an utterance containing a reading miscue (lower plot) for a particular sentence. the machine-detected word boundaries, indicated by the rectangle edges, are shown beneath the respective plots. the orthographic transcriptions of the speech data are also shown below each plot. note that the duration of word number 3 in the lower plot is signif icantly longer than that of the upper plot. the observed word duration deviation from that of the good pronunciation is due to the reading miscue (which is a substitution of the word "ito'y" for "itong", followed by a self-correction) found in the lower plot. figure 4. illustration of the machine-detected word duration deviation (word number 3) from the reference (upper plot) due to a reading miscue found in the speech data shown in the lower plot. r.m. pascual and r.c.l. guevara 15 in order to implement the wda for the purpose of reading miscue detection, the authors initially extracted the average sentence-normalized word durations from the good pronunciation examples in the cfsc. the reference word durations were initially stored in a look-up table. after the initialization process, the main wda process then proceeds as follows: 1. given a certain sentence, form the reference word sequence (i.e., a static word decoding network where word-ends are connected only to the fanin nodes of the alternative pronunciations of the next word appearing in the reference). for the purpose of this step, the authors used a pronunciation dictionary of 650 unique words, including alternative pronunciations and a silence/pause model, found in the reading text passages. 2. perform a word-level viterbi-forced alignment process between the reference and the input speech data. (this process produces the machinedetected word boundaries for each word in the reference sentence.) 3. from the machine-detected word boundaries in step 2, calculate the sentence-normalized durations for each word in the sentence. (normalization with respect to local sentence duration is necessary to compensate for different reading rates.) 4. calculate the relative deviations (from the normalized reference word durations) of the normalized word durations found in step 3. 5. using the relative word-duration deviations δ v for each word v in step 4, perform a decision process (of whether or not there was a reading miscue) as follows: (2), where δ = word duration deviation threshold. 1 ( ), max [ ] 0 ( ), max [ ] v all v wda v all v miscue present md miscue absent           a children’s speech technology for improving literacy 16 figure 5 shows the combined phone-level rv and wda method, which is simply referred to here as the "rv-plus-wda" method, for the f inal design of the rmd for filipino. for the rv-plus-wda method, the miscue detection is now given by the following classif ication scheme: where mdrv and mdwda are given respectively in equations (1) and (2). 1 ( ), [ ] 0 0 ( ), [ ] 0 rv wda rv wda miscue present md md md miscue absent md md       (3), figure 5. overview of the combined methods, reference verif ication (rv) and word duration analysis (wda), for the reading miscue detector (rmd) design. input speech signal feature extraction (mfcc) and preprocessing hmm-based phonelevel reference alignment and likelihood scoring reference phone sequence acoustic model (hmm definitions) likelihood scores >threshold? word duration and phrasing analysis (via world-level reference alignment) word/pause duration within bounds? no reading miscue / disfluency detected reading miscue / disfluency detected word pronunciation dictionary word/ pauseduration reference (“good readers”) n n y y r.m. pascual and r.c.l. guevara 17 read ing miscue detector performance evaluation method in order to evaluate the performances of the rmd systems presented in the previous section, the authors performed two sets of offline tests that employ various threshold values. the f irst set of tests was performed to evaluate the performance of the phone-level rv-based rmd, while the second sets of tests evaluate the performance of the two-level rv-plus-wda-based rmd. for both sets of tests, the authors used an offline test set containing 100 sentences or a total of 1,030 words that were randomly selected from the cfsc. the authors considered selecting the test f iles, such that there is a fairly balanced representation in terms of gender and of age. among the 100 sentences in the aforementioned test set, 50 contained at least one reading miscue or disfluency, while the other 50 contain good pronunciations. analysis of the test set showed that there are seven types of reading miscues found in children's filipino speech: (1) par tial word; (2) hesitation pause; (3) insertion; (4) repetition; (5) substitution; (6) deletion; and, (7) elongation. table 2 summarizes the relative frequencies of occurrences of the aforementioned reading miscues in the test set. table 2. read ing miscue occurrences in the test set type of miscue/disfluency relative frequency hesitation pause 30.1% partial word 20.4% insertion 18.4% repetition 13.6% substitution 7.8% deletion 6.8% elongation 2.9% the performances of rmd systems were evaluated using the two measures commonly used in literature: the false alarm (fa) rate; and, the reading miscue detection error (mderr) rate (duchateau et al. 2006; liu et al. 2008; black et al. 2 0 1 1 ) . the fa rate is def ined as the number of words erroneously detected as read incorrectly divided by the total number of correct pronunciations. that is, fa = fp / (tn + fp) (4), a children’s speech technology for improving literacy 18 where fp = number of false positives (i.e. , false detections of a miscue), and tn = number of true negatives (i.e. , correct detections of the absence of a miscue). the mderr rate, also referred to as misdetection (md) rate, is def ined as the number of miscues that were not detected divided by the total number of miscues. that is, md = fn / (tp + fn) (5), where fn = number of false negatives (i.e. , undetected miscues), and tp = number of true positives (correctly detected miscues). reading miscue detector performance: test results and discussion to commence with the f irst set of offline tests that evaluate the performance of the phone-level rv-based rmd, the authors performed offline tests using the test set described in the previous section for various upper threshold values. for the offline tests, an initial f ixed lower threshold value of -1000 (log likelihood score) was employed based on the observation that this setting did not introduce any false alarm. figure 6 shows the results of the test runs in terms of fa and mderr rates for various upper threshold values. figure 6 shows that false alarms vanished at around an upper threshold value of 550. since the upper threshold generally imposes a less strict condition than that of the lower threshold, the authors decided to employ the aforementioned value as a f ixed upper threshold value for all the figure 6. false alarm (fa) and miscue detection error (mderr) rates as functions of the upper threshold values for the reference verif ication (rv)-based reading miscue detector (rmd). r.m. pascual and r.c.l. guevara 19 succeeding tests and system operations. thus, for the rest of this article, the term "threshold" for log likelihood score actually pertains to the lower threshold. in addition, we may note that the plots in figure 6 contain fluctuations due to the f inite amount of data in the offline test set. for the error rate curves in the succeeding f igures, the authors minimized the fluctuations, in order to predict the general behavior of the system as the size of the test set increases. that is, the fa and mderr rate curves were modeled based on the widely used assumption in literature that the probability distribution function (pdf) of the reading miscue characteristics in the test data follows a normal (gaussian) distribution. thus, the authors f it the error rate curves to an approximation of a gaussian cumulative distribution function (cdf) in the least-squares sense. after setting the upper threshold value for the rmd constant, the authors performed another set of offline tests for various lower threshold values. figure 7 shows the resulting performance of the of phone-level rv-based rmd in terms of fa (dashed curve) and mderr (solid curve) rates for various phone likelihood score threshold (i.e. , lower threshold) values. the trends in figure 7 show a generally increasing fa rate and decreasing mderr rate as the threshold is increased. since rmds are never perfect, it has been customary for art systems to be biased towards having lower fa rates at the expense of having higher mderr rates. this is done, in order to avoid frustration on the reader with too many unnecessary interventions (mostow and aist 1999). typically, an fa rate equal to or higher than 10% for reading tutors is not a good performance compared to state-of-the-art systems that usually have figure 7. false alarm (fa) and miscue detection error (mderr) rates of the phonelevel reference verif ication (rv)-based reading miscue detector (rmd). a children’s speech technology for improving literacy 20 lower fa rates. taking into consideration figure 7, for instance, the probable best case is to adjust the threshold, such that the fa rate is approximately 5%, while the mderr rate is approximately 22.5%. however, an mderr rate of 22.5% may generally be seen as still an unsatisfactory performance by an rmd. thus, the succeeding parts of this section present how much improvement for the rmd's performance can be achieved by incorporating a second level process for reading miscue detection. investigation of the misdetection cases revealed that the previously presented baseline method is generally unable to detect the following miscues and disfluencies: (1) single-syllable or single-phone deletion, insertion, or substitution that has durations of 150-250 milliseconds; (2) brief hesitation pause that is less than 500 milliseconds; and, (3) some restarts or self-corrections. the second set of offline test results shown in figure 8 presents the performance of the two-level rv-plus-wda-based rmd in terms of fa (dashed) and mderr (solid) rates for various word duration deviation threshold values. note that, unlike with those from figure 7, the trends in figure 8 show a generally decreasing fa rate and increasing mderr rate as the word duration deviation threshold is increased. note that higher word duration deviation threshold values result to a less strict miscue detector, while the opposite is true for higher likelihood score threshold values. figure 8. false alarm (fa) and miscue detection error (mderr) rates for the twolevel reference verif ication and word duration analysis (rv-plus-wda)-based reading miscue detector (rmd). r.m. pascual and r.c.l. guevara 21 as discussed in the previous section, the two-level rv-plus-wda method combines two different methods that use two different thresholds. the results shown in figure 8 imply that the word duration deviation threshold varied while the likelihood score threshold remained f ixed. the authors have attempted the use of different combinations of the two thresholds. the results of the aforementioned experiments showed that the best results (i.e. , lowest overall fa and mderr rates) were obtained by making the f irst detector level (i.e. , the phone-level rv method) less strict while allowing the second detector level (i.e. , the wda method) catch the misdetection cases in the former. specif ically, the authors have f ixed the phone likelihood score threshold, such that the phone-level rv method alone has an fa rate of about 2% at an mderr rate of roughly 30%. the f inal threshold values used for the f irst and for the second rmd levels, respectively, are: p = -650 (log likelihood score), and δ = 70% (word duration deviation). this combination seems to give the lowest overall fa and mderr rates while having a good fa-to-mderr rate ratio. in particular, the approximate error rates for the threshold combination are fa rate = 3% and mderr rate = 5%. the fa and mderr rates for the selected threshold combination may also be deduced from the tabular summary of the various combinations of the threshold values. table 3 may also be used as a guide in predicting how the rmd system will perform in case another threshold combination is desired. table 3. false alarm (fa) and miscue detection error (mderr) rates for various combinations of two thresholds, p and δ p = 1000 20% 4 % 2 % 8 % 0 % 12% p = 650 20% 2 % 2 % 6 % 0 % 10% p = 570 20% 0 % 6 % 2 % 4 % 8 % p = 500 26% 0 % 12% 0 % 10% 6 % fa rate mderr rate fa rate fa ratemderr rate mderr rate δδδδδ = 50% δδδδδ = 755% δδδδδ = 100% note. p = phone-level log likelihood score threshold; δ = word duration deviation threshold in order to obtain a better comparison (independent of threshold) of the system performances, the receiver operating characteristic (roc) graphs, as shown in figure 9, were generated by plotting the fa rates versus the mderr rates. figure 9 shows the roc graphs of the phone-level rv-based rmd (dashed curve) and the two-level rv-plus-wda-based rmd (solid curve). compared with the phone-level rv method a children’s speech technology for improving literacy 22 alone, the combined rv-plus-wda method has signif icantly improved the rmd's performance. specif ically, figure 9 shows that, at an fa rate of about 3%, the rv method alone obtained an mderr rate of about 25%, while the rv-plus-wda method obtained an mderr rate of about 5%. thus, at this fa rate, the combined rv-pluswda method provided an mderr rate absolute improvement of 20% over the rv method alone. figure 9. receiver operating characteristic (roc) graphs or error rates trade-off curves for the phone-level reference verif ication (rv) method (dashed), and for the the two-level reference verif ication and word duration analysis (rv-plus-wda) method (solid) for the reading miscue detector (rmd) design. table 4 summarizes and compares the performances of the phone-level rv and the rv-plus-wda method at selected operating points. operating points 1 and 2 are where both methods obtained the same fa rates, namely 3% and 10%, respectively. operating point 3 is known as the equal error rate (eer), where fa and mderr rates are equal for a certain method. phone-level rv fa 3 % 10% 18% mderr 25% 20.5% 18% rv-plus-wda fa 3 % 10% 4.25% mderr 5 % 3 % 4.25% rmd method error rate operating point 1 operating point 2 operating point 3 table 4. false alarm (fa) and miscue detection error (mderr) rates for the phone-level reference verification (rv), and for the two-level reference verification and word duration analysis (rv-plus-wda) methods at selected operating points r.m. pascual and r.c.l. guevara 23 the previous discussions have made clear that the rv-plus-wda method has a more superior performance than the phone-level rv method. nearly about 70% of the reading miscues that were missed by the phone-level rv method were successfully detected by the rv-plus-wda method. one reason that could explain this result is the inability of the phone-level rv method to detect deviations from the expected sounds when the deviations happen only for a short period of time. in particular, the phone-level rv method was observed to have diff iculties in detecting syllable insertions, deletions, or substitutions, and word restarts or immediate selfcorrections. upon further investigation, the authors found out that about 57% of the misdetection cases are syllable or phone insertions, substitutions, and deletions. moreover, the durations of the undetected insertions and substitutions mostly range from about 150 to 250 milliseconds. brief hesitation pauses, ranging from about 250 to 400 milliseconds, constitute about 28% of the misdetection cases. selfcorrections, restarts, and repetitions all together make up about 14% of the misdetection cases. with these reading miscues, the behavior of the phone alignment method is to align a reference phone to within a beam of few phones in sequence found in the input speech. in the process of seeking alignment, the system has the tendency to either skip some inserted phones or ignore missing phones in the input speech. figure 10 shows an example of a reading miscue that was undetected by the phonelevel rv method. as we can see from the f irst plot in figure 10, the reference phones that were forced-aligned with the input speech are for the filipino phrase /tinatawag din itong/. the transcription of the actual input speech shown in the f irst plot however is given as /tina(ta)wag din (itoy-) itong/, which contains a syllable deletion (i.e. , syllable /ta/ in /tinatawag/) and a self-correction for a miscue (i.e. , /itoy/). examination of the log likelihood scores, shown at the bottom of the f irst plot, reveals that the miscues were undetected (i.e. , the likelihood scores were all above the threshold). in particular, we can observe that the reference phones for the word /itong/ were forced-align within the span of the uttered phrase /(itoy-) itong/ without generating a score below the threshold. the second plot of figure 10 shows the result of the word-level forced-alignment process for implementing the wda, wherein the reference text consists of the three words /tinatawag/,/din/, and /itong/. note that the second plot shows the same input speech as that of the f irst plot. the word boundaries shown in the second plot are system-generated and are used by the system to calculate the word durations. we may observe that the reference word /itong/ was forced-aligned within the span of the uttered phrase /(itoy-) itong/ which contain a miscue. consequently, the detected duration of the word /itong/ became signif icantly higher than normal. the normal range for word a children’s speech technology for improving literacy 24 duration is based on measurements made from the good pronunciations in the cfsc. the third plot in figure 10 shows the result of word-level alignment process for an input speech corresponding to a good pronunciation. a signif icant difference may be observed when the detected relative word duration for the word /itong/ in the second plot is compared to that in the third plot. in fact, the system was able to detect that the word /itong/ from the input speech, shown in the second plot, has a 136% deviation from the normal. since a 136% deviation is above the threshold set for the system, the reading miscue is therefore detected by the wda method. the effectiveness of the wda method in detecting reading miscues in filipino may further be explained by two main reasons: (1) the suitability of the wda method to the nature of reading miscues in children's filipino read speech; and, (2) the suitability of the wda method to the nature of the filipino language. table 2 in the previous section has listed the different types of reading miscues/ disfluencies found in children's filipino read speech taken from the cfsc as: partial figure 10. a specif ic example of a case wherein a reading miscue, undetected by the phone-level reference verif ication (rv) method, was detected by the word duration analysis (wda) method. r.m. pascual and r.c.l. guevara 25 word; hesitation pause; insertion; repetition; substitution; deletion; and, elongation. an examination of the nature of these reading miscues would show that all of them, except substitution, are time-dependent. that is, these reading miscues would affect the effective word durations as measured by the system. moreover, the authors have observed from the test set that hesitation pauses, partial words (mostly followed by a short pause or a restart), and repetitions are the miscue types that usually cause the largest word duration deviations. since the aforementioned miscue types constitute the majority of the miscues found in the test set, the wda method therefore generally becomes an effective way of detecting the reading miscues. since filipino is a syllable-timed language, the insertion or deletion of syllables or words, as well as pauses, definitely affects the effective word durations, as measured by the rmd system. the wda method for the rmd may therefore be shown to be especially effective for syllable-timed languages, such as filipino. design of the automated reading tutor for filipino the art for filipino presented in this paper has the following major components: (1) the rmd; (2) the oral/visual feedback and instruction set; and, (3) the graphical user interface. the rmd for filipino, which is the core of the art, employs a two-level rv-pluswda architecture as presented in the previous section. the performance measures for the rmd are the fa rate and the mderr rate. the best result of the offline performance evaluation tests shows that the rmd's operating point may be calibrated, such that the fa rate is approximately 3% while the mderr rate is approximately 5%. the fa and mderr rates that the authors obtained for the rmd for filipino prove that it is at par with the state-of-the-art rmds (duchateau et al. 2006; liu et al. 2008; black et al. 2011) reported in the literature. for comparison, table 5 summarizes the performance of the two-level rv-plus-wda-based rmd presented in this paper, together with those from other systems reported in the literature. the oral/visual feedback and instruction set allows active interaction between the machine and the child (user or learner). the orf instruction set used in this study is a set of pre-recorded speech of a human tutor, who is a reading expert and an education sector research collaborator from the college of education at the university of the philippines diliman. any desired sentence within the instruction set can automatically be played-back by the art system whenever there is a need to model the correct pronunciations of the words in the text passages. a children’s speech technology for improving literacy 26 as briefly discussed in the previous section, nine filipino text passages were used for both the speech database collection and the art system development. the six short stories were provided through a non-disclosure agreement by adarna publishing house, a leading publisher of filipino short stories for children. the three expository texts were adapted from various grade school textbooks used in the philippines. each of the text passages adapted was provided with a corresponding reading age recommendation by its publisher. moreover, the text passages have been selected through the suggestions of research collaborators. the nine text passages all together have a total of 2,169 words (about 650 of which are unique) and 290 sentences. the art also provides audible comments and visual animations as positive feedback or "praise" (mostow and aist 1999) in response to a perceived good reading performance of the learner. according to suggestions in the literature, giving positive feedback is a powerful motivation and it demonstrates that the art system is a perceptive and responsive audience for the learner's efforts. in the art presented in this study, the authors employed the audible comments: "mahusay!" (excellent!), "magaling!" (good!), and "kahanga-hanga!" (admirable!). the positive comments are alternately played-back by the system whenever no reading miscues were detected from the input speech. aside from audible comments, the art also displays an animated icon whenever the aforementioned comments are being played-back. figure 11 shows the graphical user interface of the art in filipino. the graphical user interface of the art allows the reader to select a story and navigate through the sentences within the selected story. an important design consideration for the interface is its simplicity because the intended user may be as young as a grade 1 student (typically aged 5 to 7). black et al. (2011) 8.1% 11.5% english liu et al. (2008) 5.82% 9.07% chinese mandarin duchateau et al. (2006) 2.1% 8.4% 23.1% 16.9% dutch two-level rv-plus-wda 3 % 5 % filipino state-of-the-art rmds false alarm (fa) rate miscue detection error (mderr) rate language table 5. summary of specifications of various state-of-the-art read ing miscue detectors (rmd) r.m. pascual and r.c.l. guevara 27 automated reading tutor (art) intervention program: a pilot study design of the art intervention program the art intervention program or the pilot study, a pioneering experiment in computer-assisted orf instruction in filipino, is basically a reading tutorial program that makes use of the art presented in the previous section. the within-subjects experiment involved a group of six grade-2 students from the university of the philippines integrated school (upis), and consists of two separate one-month periods. during the f irst period of the experiment, the experiment group depended only on regular classroom instruction for improving their reading skills. during the second period, the art was used by the experiment group, in addition to the regular classroom instruction. the art intervention program allowed the group to use the art for about 45 minutes per day, three days per week. in order to evaluate the effectiveness of the art in providing reading skill improvement to the students, three sets of oral reading fluency assessments (orfa) were administered to the experiment group. figure 12 graphically summarizes the pilot study experiment and its timeline. as we can verify from figure 12, the f irst orfa was given prior to the start of period 1, the second orfa at the end of period 1, and the third orfa at the end of period 2. all three orf assessments were administered by an expert, a filipino teacher who also has a research experience in automated filipino essay evaluation. as suggested figure 11. the graphical user interface of the automated reading tutor (art ) for filipino. a children’s speech technology for improving literacy 28 by research collaborators, the orfa employed both familiar and unfamiliar text passages, in order to obtain a more complete observation regarding the effects of the use of the art in student's learning for both text structures. the orfa also included a comprehension exam that has an objective-type and an essay-type components. in this study, the authors employed the following three commonly used orfa measures in the literature: (1) number of words correct per minute (wcpm), (2) 16-point multi-dimensional orf score; and, (3) reading comprehension scores. group gain score analysis, also known as difference score analysis (smolkowski 2013), was selected because it provides a simple and precise, yet unbiased and reliable way of interpreting the true change (ragosa 1983). for instance, the wcpm gain scores clearly and meaningfully tell educators whether the experiment group improved, retained, or deteriorated, and by precisely how much, in their reading skills (smolkowski 2013). results of the art intervention program and the oral read ing fluency (orf) assessments the primary result of the art intervention program presented in this study is expressed in terms of wcpm, the most widely used measure for orfa (rasinski 2004). figure 13 summarizes the average or group wcpm improvements for all the students in the experiment group. an important pattern that may be noted in figure 13 is that the group wcpm slope for the second period became abruptly higher compared to the slope for the f irst period. thus, there was a signif icantly higher improvement in group wcpm in period 2 than that in period 1. it therefore suggests that, on the average, the reading tutor had a positive effect of accelerating the improvement of the students’ orf. figure 12. pilot study experiment and timeline. r.m. pascual and r.c.l. guevara 29 figure 13. group average number of words correct per minute (wcpm) for the three oral reading fluency assessments (orfa). in table 6, the signif icantly higher overall average wcpm gain for period 2 compared to that for period 1 clearly shows that the experiment group signif icantly improved their orf after using the art. the normal growth (due to the usual classroom instruction) of the group in reading fluency for one month is shown in table 6 to be only 3.53 words per minute. after using the art for a month however, the improvement of the group suddenly jumped up to 16.46 words per minute, an improvement which is 12.93 words per minute higher than the normal. to have a better idea on the magnitude of improvement in the reading fluency of the group due to the use of the art, we may calculate the wcpm gain ratio (i.e. , group gain for period 2, divided by the group gain for period 1). the overall wcpm gain ratio was calculated to be 4.66. this means that the fluency improvement rate in period 2 (i.e. , when the art was used) improved by 466% than the normal period 1 2.01 5.05 3.53 (without using the art) (sd=3.88) period 2 18.75 14.17 16.46 (using the art) (sd=8.56) note. sd = standard deviation group gain (wcpm) famil iar text unfamil iar text familiar text table 6. words correct per minute (wcpm) group gains for the two experiment periods a children’s speech technology for improving literacy 30 learning rate. in other words, after the art was used for a month, the experiment showed that the reading fluency of the group has been accelerated by an amount of time roughly equivalent to three and a half months. in order to show that the larger wcpm group gain in period 2 is indeed attributable to the use of the art, the authors calculated the correlations between score gains in period 1, the score gains in period 2, and the score gains in the whole experiment period. the correlation of the score gains in period 2 and the score gains in the whole experimental period (i.e. , periods 1 and 2 combined) shows a strong and signif icant relationship (i.e. , with a coeff icient of 91.17% at p < 0.00005) between the score gains in period 2 and the whole two-month experimental period. therefore, the observed overall improvement of the students in their reading fluency can indeed be attributed to the use of the art. to further illustrate that it is less likely that the reading fluency improvement of the students in the experiment group is due to their normal learning rate trends or to random chance, the authors also referred to the orf norms used for english (hasbrouck 2006). in doing this, the authors emphasize that their purpose is not to directly compare the wcpm levels observed from their experiment to the normal wcpm levels in english. the normal wcpm levels for english are expected to be different from normal wcpm levels in filipino due to the differences in features and orthography between the two languages. thus, the authors only referred to the orf norms in english for the purpose of generally comparing their experimentally observed reading fluency improvement trend to the expected normal reading fluency trend in english. the aforementioned wcpm trend comparison for grade 2 level is given in figure 14. a simple graphical analysis on the wcpm trends shown figure 14. comparison of group average number of words correct per minute (wcpm) trends between us norm (english) and experiment observations (filipino). r.m. pascual and r.c.l. guevara 31 in figure 14 would suggest that the reading fluency improvement observed from the experiment group during period 2 is high relative to english orf norm, and significantly higher than the reading fluency improvement observed during period 1. moreover, the expected english orf improvement throughout the entire school year is fairly linear. by contrast, the reading fluency improvement trend observed from the experiment group for the entire two-month experiment period highly deviated from the linear trend. thus, simple trend analysis clearly shows that the orf improvement of the students in the experiment group during period 2 is unusually high, and this may be attributed to the treatment made during the period (i.e. , the use of the art ).the second set of orfa results is based on the measure known as the orf-16 score obtained through the use of a 16-point multi-dimensional orf rubric proposed by rasinski (2004). the four dimensions considered in the orf-16 rubric are: (1) expression and volume; (2) phrasing; (3) smoothness; and, (4) pace. the trend in the orf-16 group scores shown in figure 15 also seem to agree with that of the wcpm group scores presented earlier in this section. we may observe from figure 15 that there was a sudden increase in the reading fluency of the students in the experiment group during period 2, the period when the art was used by the group. thus, in a similar way that was shown earlier in this section, it follows that the sudden improvement in the student's reading fluency may be attributed to the use of the art. the overall orf-16 group gain ratio, which is calculated to be 6.0, means that the observed reading fluency improvement of the students in the experiment group during the time that they were using the art is about six times better compared to the time that they were not using the art. figure 15. sixteen-point multi-dimensional oral reading fluency (orf-16) group scores for the three oral reading fluency assessments (orfas). a children’s speech technology for improving literacy 32 i n o r d e r t o s e e h o w t h e r e a d i n g f l u e n c y d e v e l o p m e n t h a s a f f e c t e d t h e comprehension of the students in the experiment group, the third set of orfa results was based on the comprehension exam scores. the plots in figure 16 show that, on the average, the student's comprehension also abruptly improved during period 2, the time when they were using the art. the overall comprehension exam score gain ratio, computed to be 4.43, indicates that the students have improved in their comprehension by more than four times after using the art. figure 16. comprehension exam group scores for the three oral reading fluency assessments (orfas). conclusion and future directions in this paper, the authors presented a two-level rmd for the design of an art for filipino that uses phone-level rv and wda methods. the results of offline tests showed that the rmd's performance (i.e. , false alarm rate ≈ 3% and misdetection rate ≈ 5%) is at par with those from state-of-the-art rmds (duchateau et al. 2006; liu et al. 2008; black et al. 2011) reported in the literature. the advantages of the rv-plus-wda rmd are design simplicity (i.e. , it did not require building a complex language model or using an adaptation technique) and low training cost (i.e. , it only required 1.5 hours of training data). the authors of this study have discussed the design of the art prototype that integrates the two-level rv-plus-wda rmd, the user interface, and the feedback and instruction sets. the authors suggest the following design considerations: (1) user interface simplicity on account of very young users; (2) minimal interventions r.m. pascual and r.c.l. guevara 33 to avoid children's frustration; and, (3) the use of positive feedback or "praise" that most children seem to appreciate. moreover, it is suggested that all model pronunciations in the instruction set should contain the "correct" or acceptable prosodic features because it has been observed that students have the tendency to adopt these features. the authors have presented in this paper an experimental procedure for evaluating the effectiveness of an art for filipino that involves an art intervention program and a set of orfa for a small group of students. the results of the art intervention experiment clearly showed the art's effectiveness in improving the students' orf in terms of wcpm, orf-16 scores, and comprehension scores. specif ically, the results of the orfas showed that, after using the art, the students, on the average, have improved in their wcpm by 4.66 times compared to the period when they were not using the art. correlation and trend analysis undoubtedly implies that the improvement of the students in their reading fluency was indeed attributable to the use of the art. similarly, after using the art, the students have improved in their orf-16 scores by 6.0 times compared to the period when they were not using the system. the results of experiment have also shown that, after using the art, the students, on the average, were 4.4 times better in reading comprehension than when they were not using the art. with all the positive results that the authors obtained from the study, the art in filipino seems to be a promising and important filipino speech technology to further develop and implement for the primary education system in the philippines. future directions for this study include the development of an automated oral reading assessment system for children's filipino speech, adaptation of the rmd system for nonnative speakers of filipino (or those who speak filipino as their second or third language), and adaptation of the system design methods for other philippine languages and other related applications. acknowledgments the authors would like acknowledge the up digital signal processing laboratory, up integrated school, up college of education, up department of linguistics, and adarna house for lending their support to the study. this research was funded by the chedsegs grant from the commission on higher education of the philippines, in collaboration with the off i ce of the v i ce-chancellor for research and development of university of the philippines diliman. a children’s speech technology for improving literacy 34 references batliner a, blomberg m, d'arcy s, elenius d, giuliani d, gerosa m, hacker c, russell m, steidl s, wong m. 2005. the pf_star children's speech corpus. in: proceedings of interspeech; lisbon, portugal. p. 2761-2764. black m, tepperman j, narayanan s. 2011. automatic prediction of children's reading ability for high-level literacy 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[internet]. 2013. gain score analysis. oregon: oregon research institute; [cited 2016 dec]. available from http://homes.ori.org/keiths/tips/stats_gainscores.html. tsau s. 2012. the effects of an automatic speech analysis system on enhancing efl learners’ oral reading fluency. procedia-social and behavioral sciences. 64(2012):141150. wasik b, slavin r. 1993. preventing early reading failure with one-to-one tutoring: a review of f ive programs. reading research quar terly. 28(2):178-200. yo u n g s , e ve r m a n n g , g a l e s m , wo o d l a n d p. 2 0 0 6 . t h e h t k b o o k [ i n t e r n e t ] . u k : cambridge university engineering department; [cited 2010 nov 19]. available from http://htk.eng.cam.ac.uk. _____________ dr. ronald m. pascual <ronaldmpascual@gmail.com> is an associate professor and assistant director of the electronics and electrical engineering department of feu institute of technology, manila. he received his ph.d. in electrical and electronics engineering from university of the philippines diliman as a ched scholar, his m.s. in electronics and communications engineering from de la salle university, manila as a dost scholar, and his b.s. in electronics and communications engineering from pamantasan ng lungsod ng maynila. his research interests include speech signal processing, and speech technology development. dr. rowena cristina l. guevara is the undersecretary for research and development of the department of science and technology (dost) and a professor of the digital signal processing laboratory of the university of the philippines diliman. she was a former executive director of dost-philippine council for industry, energy, and emerging technology research and development, and was a former dean of the college of engineering of the university of the philippines diliman. she received her ph.d. in electrical engineering from university of michigan, ann arbor as a dost scholar, and her m.s. and b.s. in electrical engineering from the university of the philippines diliman. her research interests include speech signal processing, and audio and communications signal processing. 2editor's note-jan.-june2018.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online irene m. v illaseñor, ph.d. editor-in-chief welcome to the f irst issue of science diliman for 2018. let me share the good news that science diliman is now included in the asean citation index database. science diliman is also indexed in the emerging sources citation index and is a recipient of the journal incentive program (jip) by the commission on higher education. this issue includes four main articles and a short communication. the f irst article by authors bernardo and de leon considers a cellular system design deployment called hetnets (heterogeneous network) as viable solutions to meet the demands of 5g, the next generation of cellular system. hetnets are networks connecting computers and other devices with different operating systems and/or protocols. in the second article, authors masangcay et al. studied the feeding biology of spinetail devil ray (mobula japanica) belonging to the genus mobula. species of this genus are commonly called devil rays or flying rays. they feed mainly on tropical krills, locally known as alamang. in the short communication by the same authors, the focus of the study is on the krills and their spawning habits. the article by narsico et al. determined that the brown macroalgae sargassum spp. is the most widely distributed source of fucoidan and has higher content of partially purif ied fucoidan. fucoidan is commercially available, and is known for its nutritional and health benef its. the last article by authors daquioag et al. investigated the presence of s. aureus and methicillin-resistant s. aureus (mrsa) in computer service providers, computer peripheral, and handrails of public utility jeepneys (pujs). interestingly, handrails of pujs have the least carriage of s. aureus, while mrsa carriage is relatively low. staph infections are treated by antibiotics and treatment becomes more complicated in mrsa infections. thank you to our authors and reviewers for their contributions to the increasing quality of articles in science diliman! assessment-fidelino.pmd acute toxicity of thiamethoxam to achatina fulica 21science diliman (july-december 2012) 24:2, 21-27 abstract assessment of acute toxicity of thiamethoxam (actara® 25wg) to achatina fulica and its potential ecological applications maria kim feliz n. abog, jose jaime lorenzo c. de rivera, sarah christina w. estacio, jay s. fidelino*, orlie john y. lavilla, john christopher a. pilapil, and ma. dolores c. tongco institute of biology, college of science, university of the philippines diliman *corresponding author: institute of biology, university of the philippines tel.: (02) 981 8500 loc. 5713; e-mail: jsfidelino@gmail.com achatina fulica is considered as one of the world’s worst invasive species and is known to cause ecological disruption as well as agricultural and health problems in the philippines. the study evaluated the use of the insecticide thiamethoxam (actara® 25wg) in the control of a. fulica populations. this was done by performing an acute toxicity test of thiamethoxam to a. fulica. six individuals each were exposed to thiamethoxam concentrations of 0, 50, 100, 200, and 400 µg/l for 72 hours. the percent mortality was then determined after the exposure period. probit analysis was used to determine the ld 50 of thiamethoxam to a. fulica. the ld 50 was found to be 662.95 ± 172.98 µg/l. using the mean body weight of the snails, the ld 50 per body weight of a. fulica was determined to be 90.09 ± 23.60 µg/kg. this is 70 times lower than the recommended application of thiamethoxam on field. thus, normal application would eliminate a. fulica. however, because the ld 50 to a. fulica is higher than that for other beneficial non-target species such as honey bees (0.03 µg/bee), the use of thiamethoxam in the control of a. fulica populations is only recommended when in conjunction with the control of target pest insects. keywords: ecotoxicology, acute toxicity fidelino, j.s. and others 22 science diliman (july-december 2012) 24:2, 21-27 introduction the prevalence of invasive alien species is a global trend and is considered to be one of the major causes of biodiversity loss, economic damage (lowe and others 2000), and potentially detrimental environmental changes (leung and others 2002). one such species is achatina fulica (achatinidae), the giant african snail, a terrestrial gastropod with a widespread distribution in the humid tropics (fontanilla 2010). the international union for conservation of nature lists a. fulica as one of the 100 world’s worst invasive species (lowe and others 2000). a. fulica originated in east africa but now occupies the indian subcontinent, southeast asia, the pacific, and the caribbean (fontanilla 2010). its introduction into non-native habitats has been documented to be both intentional (i.e. as a food source) and accidental (venette and larson 2004). the reproductive biology of a. fulica has largely aided its success as an invasive species, with the snail reaching maturity at five to eight months and producing 10 to 400 eggs per clutch and up to 1800 eggs per year (fontanilla 2010). it is also a voracious herbivore and its wide host range makes it a threat to native flora as well as agricultural crops (venette and larson 2004). a. fulica may also spread diseases, as it is known to be an intermediate host of the rat lungworm angiostrongylus cantonensis, which can infect humans in its third juvenile stage. a. cantonensis can cause eosinophilic meningoencephalitis (eme) or angiostrongyliasis (fontanilla 2010). black pod disease, a plant disease caused by phytophthora palmivora, may also be spread through the snail’s feces (venette and larson 2004). in the philippines, a. fulica populations are known to attack and feed on grown crops, gardens and agricultural farms of ampalaya (momordica charantia), arrowroot (canna edulis), banana (musa paradisiaca), cabbage (brassica spp.), canna sp., cassava (manihot esculenta), citrus sp., coffee (coffea spp.), sunflower (cosmos spp.), cucumber (cucumis sativus), eggplant (solanum melongena), upo (gourd: lagenaria leucantha), gumamela (hibiscus spp.), malunggay (moringa olifeira), papaya (carica papaya), ramie (boehmeria nivea), squash (cucurbita sp.), sweet potato (ipomoea batatas), patola (luffa sp.), and yam (dioscorea alata) among others (sherley 2000, raut and barker 2002, fontanilla 2010). various strategies for controlling a. fulica populations have been proposed and attempted, but so far, no single control measure has been successful in the eradication of the pest. one strategy that has been attempted in the philippines involved the purposeful introduction o f the platydemus manokwari (turbellaria: rhynchodemidae), a non-native invertebrate enemy of a. fulica, to bugsuk island. although the giant african snails were eradicated in some areas, the introduction of p. manokwari was said to have adverse effect on indigenous gastropod fauna (sherley 2000). a more common strategy involves chemical control with the use of toxicants, repellents, or pesticides. despite the development of new molluscicidal agents, very few are targeted against a. fulica (raut and barker 2002). however, even non-target pesticides such as brodifacoum, which is aimed at rodents and vertebrate pests, have been found to be toxic to a. fulica (booth and others 2001, hoare and hare 2006). thus, using non-target pesticides as chemical control agents for a. fulica may have great potential. the median lethal dose (ld 50 ) is the most frequently used measure of the acute toxicity of a substance. expressing toxicity as ld 50 provides a relative measure that can be used to compare substances with different mechanisms based solely on their lethal effect. the measurement of ld 50 also assumes that it estimates the toxicity of the most hazardous constituent in the mixture (spencer and colonna 2003). a smaller ld 50 value means relatively greater toxicity, indicating that a smaller amount of the substance is required for the death of the test organism (girard 2010). this study aimed to determine the acute toxicity, expressed as median lethal dose (ld 50 ), of thiamethoxam (actara® 25wg) to achatina fulica, which is a non-target species of the insecticide. the study also assessed whether thiamethoxam would be effective in the pest control of the snail. acute toxicity of thiamethoxam to achatina fulica 23science diliman (july-december 2012) 24:2, 21-27 materials and methods achatina fulica ninety achatina fulica individuals were obtained from around the university of the philippines diliman campus, weighed, and then placed in 15 identical glass containers (30 cm x 30 cm x 45 cm), with six individuals in each container. nylon tulle netting was used as lids to contain the samples. three centimeters of loamy soil were added to the container and kept moist throughout the study by spraying with distilled water. ten grams of tomato leaves were also placed per container to serve as food for the snails for the entire duration of the study. the snails were given a 24-hour acclimatization period before the experiment proper. thiamethoxam thiamethoxam, 3-(2chloro-thiazol-5-ylmethyl)-5methyl-{1,3,5}oxadiazinan-4-yldene-n-nitroamine, was obtained from syngenta philippines, inc. (makati city, philippines). formulated granules of thiamethoxam (actara® 25wg) were dissolved in distilled water and thiamethoxam solutions were prepared in four concentrations of 50, 100, 200, and 400 µg/l each. the concentrations were calculated based on the formulation of the granules used (250 g thiamethoxam per kg). the ph and temperature of the solutions were then measured. toxicity test toxicity tests were performed to determine the ld 50 of thiamethoxam to a. fulica . ten ml of the thiamethoxam solutions was applied by spraying on the soil surface and the sides of the containers. the snails were left to feed on tomato leaves during the 72-hour toxicity test period. concentrations tested were 50, 100, 200, and 400 µg/l of thiamethoxam in the insecticide solutions applied per container. a control treatment was also made, with distilled water used as control. after 72 hours, percent mortality was determined by examining the containers for dead snails. three replicates of the toxicity test were performed. using the mean weight of a. fulica used per treatment, the thiamethoxam concentrations were also expressed in treatment mean weight (kg) thiamethoxam concentration (µg/kg) control 0.0130 ± 0.0545 0 50 µg/l 0.0109 ± 0.0519 7.678532 100 µg/l 0.0137 ± 0.4650 13.79039 200 µg/l 0.0149 ± 0.0264 22.33958 400 µg/l 0.0121 ± 0.3497 59.18589 table 1. concentration of thiamethoxam applied per body weight of achatina fulica µg/kg (table 1). using ibm spss statistic v.19, the ld 50 (in µg/l and µg/kg) was determined by probit analysis. chi-square goodness-of-fit test was also performed in spss to check if the probit model fits the data adequately. results the temperature of the insecticide solutions ranged from 23.6-23.7°c, while a ph of 6.87 was measured for all the solutions. the percent mortality of a. fulica in the different treatments is shown in table 2. the probit transformation output from spss statistic v.19 is shown in figure 1 (thiamethoxam concentration expressed in µg/l) and figure 2 (thiamethoxam concentration expressed in µg/kg). from the probit analysis graph, the 72-hr median lethal dose (ld 50 ) of thiamethoxam to a. fulica was determined to be 662.95 ± 172.98 µg/ l (90.09 ± 23.60 µg/kg). a chi-square goodness-of-fit test was also performed with a null hypothesis that the probit analysis model fits the dose response data (table 3). discussion thiamethoxam is a neonicotinoid that is capable of mimicking acetylcholine. it acts selectively on the nicotinic acetylcholine receptors of insects, ultimately damaging the nervous system and leading to the death of the organism (nauen and others 2003). neonicotinoids are popular insecticides because the neural pathway affected is more common in invertebrates than in other animal groups (kindemba 2009). fidelino, j.s. and others 24 science diliman (july-december 2012) 24:2, 21-27 the toxicity (ld 50 ) of thiamethoxam has been previously determined in other species. the 24-hr ld 50 was found to be 0.46 µg/g (460 µg/kbw) in houseflies (musca domestica), 10.84 µg/g (10,840 µg/kbw) in german cockroaches (blatella germanica) (eremina and lopatina 2005), 0.03 µg/bee in honey bees (apis mellifera) (iwasa and others 2004), and 0.024 µg/g (24 µg/kbw) in bumblebees (bombus terrestris) (nra 2001). for mice and rats, the ld 50 values were 1,563 mg/kbw (1,563,000 µg/kbw for 59-day exposure) in rats, 783 mg/kbw (783,000 µg/kbw for 14-day exposure) in male mice, and 964 mg/kbw (964,000 µg/kbw for 14day exposure) in female mice (nra 2001). thiamethoxam applied by contact or ingestion is particularly highly toxic to honey bees and bumblebees, and has been implicated along with other pesticides as possible cause of colony collapse disorder (kindemba 2009, nra 2001). ld 50 chi-square significance 662.95 ± 172.98 µg/l 0.980 90.09 ± 23.60 µg/kg 0.983 table 3. the 72-hr median lethal dose (ld 50 ) of thiamethoxam to achatina fulica percent treatment mortality control 0 50 µg/l 5.555556 100 µg/l 16.66667 200 µg/l 22.22222 400 µg/l 38.88889 table 2. percent mortality of achatina fulica in the five treatments of thiamethoxam figure 1. probit transformation output for the determination of the 72-hour median lethal dose (ld 50 ) (µg/l) of thiamethoxam to a. fulica acute toxicity of thiamethoxam to achatina fulica 25science diliman (july-december 2012) 24:2, 21-27 the recommended application of actara® 25wg for tomatoes, potatoes, and sugar beets is 20 g/hl (syngenta 2011), which is effectively 50,000 µg/l thiamethoxam. degradation of thiamethoxam is primarily caused by aqueous photolysis, while photolytic degradation of thiamethoxam absorbed into the soil is not significant. in soil/water systems, thiamethoxam accumulates in the sediment phase while degradation is continuous (nra 2001). the half-life of thiamethoxam in soil ranges from 7 to 109 days, with longer persistence under dry conditions. thiamethoxam also has the potential to leach down under heavy rainfall conditions (nra 2001, gupta and others 2008 ). the ld 50 of thiamethoxam to a. fulica obtained was not within the range of concentrations used in the study (50 to 400 µg/l). typically, a range-finding experiment is performed to find a more specific range of concentrations prior to the toxicity test proper. the range-finding experiment was not performed because of scarcity of test animals due to the dry weather during the entire study period. this was also the reason why only six individuals were used per dose of thiamethoxam. instead of a range-finding experiment, previous literature on the toxicity of thiamethoxam and other similar insecticides on other land snails and mollusks were used as the basis for the range of concentrations prepared (booth and others 2001, salama and others 2005, minakshi and mahajan 2012). the recommended application is about 70 times the ld 50 determined in this study. this prescribed application would be able to eliminate a. fulica pests, if present. however, the use of thiamethoxam solely figure 2. probit transformation output for the determination of the 72-hour median lethal dose (ld 50 ) (µg/kg) of thiamethoxam to a. fulica per body weight (µg/kg) fidelino, j.s. and others 26 science diliman (july-december 2012) 24:2, 21-27 as a molluscicidal to a. fulica is not recommended due to the lower ld 50 of the insecticide to other non-target organisms and to the persistence of thiamethoxam, especially in dry soil. furthermore, since thiamethoxam may potentially leach under heavy rainfall, contamination of nearby water bodies and other ecosystems becomes a concern. the potential effect on other non-target organisms, especially native and beneficial species, should also be considered. thus, the use of thiamethoxam in the control of a. fulica populations is only recommended in conjunction with the control of target pest insects. acknowledgments the researchers would like to thank the late mang oca, for whom this paper is written, for the snail supply, and the institute of biology, for use of equipment and of the ecology and taxonomy laboratory. references booth lh, eason ct, spurr eb. 2001. literature review of the acute toxicity and persistence of brodifacoum to invertebrates. in: department of conservation, editor. literature review of the acute toxicity and persistence of brodifacoum to invertebrates and studies of residue risks to wildlife and people. science for conservation 177. p 1-9. eremina o, lopatina y. 2005. investigation of neonicotinoid insecticides against house fly musca domestica (diptrea: muscidae) and german cockroach blatella germanica (blattodea: blattelidae). in: proceedings of the fifth international conference on urban pests [cited 2012 march 27]. available from http://www.icup.org.uk/reports/ icup090.pdf fontanilla ikc. 2010. achatina (lissachatina) fulica bowdich: its molecular phylogeny, genetic variation in global populations, and its possible role in the spread of the rat lungworm angiostrongylus cantonensis (chen) [phd thesis]. uk: the university of nottingham. 633 leaves. girard je. 2010. principles of environmental chemistry. 2nd ed. sudbury, ma: jones and barlett publishers. 687 p. gupta s, gajbhiye vt, gupta rk. 2008. soil dissipation and leaching behavior of a neonicotinoid insecticide thiamethoxam. bull environ contam toxicol 80: 431-37. hoare jm, hare km. 2006. the impact of brodifacoum on non-target wildlife: gaps in knowledge. new zealand journal of ecology 30(2): 157-67. iwasa t, motoyama n, ambrose jt, roe mr. 2004. mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, apis mellifera. crop prot. 23: 371-78. kindemba v. 2009. the impact of neonicotinoid insecticdes on bumblebees, honey bees and other non-target invertebrates. uk: buglife – the invertebrate conservation trust. 52 p. leung b, lodge dm, finoff d, shogren jf, lewis ma, lamberti g. 2002. an ounce of prevention or a pound of cure: bioeconomic risk analysis of invasive species. proc. r. soc. lond. b. 269: 2407-13. lowe s, browne m, boudjelas s, de poorter m. 2000. 100 of the world’s worst invasive alien species – a selection from the global invasive species database. switzerland: invasive species specialist group, species survival commission, world conservation union. 12 p. mikashi r, mahajan ay. 2012. toxicity evaluation of thiamethoxam and triazophos to the freshwater bivalve lamellidens marginalis (lamark). trends in life sciences 1(3): 29-33. national registration authority (nra). 2001. evaluation of the new active thiamethoxam in the product cruiser 350 fs insecticide seed treatment. canberra, australia: national registration authority for agricultural and veterinary chemicals. 51 p. nauen r, ebbinghaus-kintscher u, salgado vl, kaussmann m. 2003. thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants. pesticide biochemistry and physiology 76: 55-69. acute toxicity of thiamethoxam to achatina fulica 27science diliman (july-december 2012) 24:2, 21-27 raut sk, barker gm. 2002. achatina fulica bowdich and other achatinidae as pests in tropical agriculture. in: barker gm, editor. molluscs as crop pests. wallingford; cabi publishing. p. 84-87. salama ak, osman ka, saber na, soliman sa. 2005. oxidative stress induced by different pesticides in the land snails helix aspersa. pakistan journal of biological sciences 8(1): 92-96. sherley g. 2000. invasive species in the pacific: a technical review and draft regional strategy. samoa: south pacific regional environment programme. 149 p. spencer ab, colonna gr. 2003. nfpa pocket guide to hazardous materials. massachusetts: national fire protection association, inc. 220 p. syngenta. 2011. actara  25wg fact sheet [cited 2012 march 27]. available from http://www.syngetna.com/country/eg/ e n / c r o p p r p t e c t i o n / o u r p r o d u c t s / i n s e c t i c i d e s / p a g e s / actara25 wg.aspx venette rc, larson m. 2004. mini risk assessment: giant african snail, achatina fulica bowdich [gastropoda: achatinidae] [cited 2012 march 2]/ available from http:// www.aphis.usda.gov/plant_health/plant_pest_info/ pest_detection/downloads/pra/afulicapra.pdf 5 characterization of open water explosions from confiscated explosives in the philippines – possible implications to local marine mammals archie i. veloria institute of environmental science and meteorology university of the philippines diliman daniella t. hernandez giovanni a. tapang national institute of physics university of the philippines diliman lemnuel v. aragones* institute of environmental science and meteorology and natural sciences research institute university of the philippines diliman abstract underwater noise poses serious threats to marine mammals, which rely on underwater sound primarily for communication, orientation, and foraging. in this study, underwater noise from dynamite fishing was analyzed to infer possible effects on local marine mammals, particularly cetaceans. simulated explosions were performed on 9 july 2018 using confiscated explosives from illegal fishers in san fernando, la union. the acoustic properties of blasts from single pulse explosions were characterized using sound recordings captured by a hydrophone. dominant frequencies from the sound recordings showed that the noise generated by the explosions can be perceived by marine mammals sensitive to the auditory bandwidth of 7 hz to 180 khz. blast charge weights were estimated to determine sound pressure levels generated by the explosions at varying distances from the source. these results imply that marine mammals within 150 m of the explosion will experience debilitating injuries (e.g., acoustic trauma, disorientation) even from a single pulse. by characterizing the acoustic properties of these local explosives, its potential impacts to local marine mammals and other marine organisms can be elucidated. these acoustic calculations can be further enhanced by considering backscattered waves and determining the actual chemical composition of these explosives. keywords: marine mammals, cetaceans, underwater noise, underwater explosion, dynamite fishing, hydrophone * corresponding author science diliman (january-june 2021) 33:1, 5-21 characterization of open water explosions from confiscated explosives in the philippines 6 introduction many marine lifeforms can generate and perceive sound (au et al. 1974; popper et al. 2004; henninger and watson 2005; bailey et al. 2010). some of these are marine mammals that rely on underwater sound for communication, orientation, and foraging (tyack and clark 2000; finneran 2015). exposure to anthropogenic noise may pose serious threats to aquatic species such as marine mammals. because of this, various research efforts have been conducted to determine the effects of underwater noise on marine mammals (e.g., southall et al. 2007). research programs have also been developed worldwide on investigate noise impacts to different marine mammal species (e.g., erbe 2012). however, most of these studies examined impacts from large explosive loads. characterization of the acoustic properties of small explosive loads like those from illegal dynamite fishing has not yet been conducted. noise may affect marine mammals in many ways. at higher sound levels, noise may interfere with marine mammal communication and hinder acoustic signal detection (erbe 2012). prolonged exposure can also affect the auditory system and may induce a shift in its hearing threshold (southall et al. 2008; pacini et al. 2017). underwater noise may also pose physical threats such as concussive effects, damage to tissues and organs, and bubble formation (erbe 2012). noise can also induce stress and eventually cause health problems to marine mammals. given the possible effects of noise to marine mammals, government departments in many countries now regulate underwater noise emission from industries. in the philippines, the bureau of fisheries and aquatic resources (bfar) of the department of agriculture (da) has the mandate to protect and manage cetaceans (i.e., dolphins and whales), while the biodiversity management bureau (bmb) of the department of environment and natural resources (denr) has jurisdiction over the dugongs. one of the threats to marine mammals is dynamite fishing and similar underwater noise that may induce acoustic trauma (wahlberg 2002; mccauley et al. 2003; aragones et al. 2010; pacini et al. 2017). the amended philippine fisheries code of 1998 explicitly criminalizes the conduct of dynamite fishing (ra 10654). however, despite the existence of regulatory law prohibiting dynamite fishing, this practice has persisted. dynamite fishing, although possibly more common in shallow waters (~100 m), is also conducted in deep waters mainly to avoid being caught by authorities (ignacio 2018) and to target larger schools of fish (2018 focus group discussion with region 1 fishers conducted by aragones l; unreferenced). in region 1 of the philippines, reported incidents of dynamite fishing during 2017– a.i. veloria et al. 7 2020 ranged from 44 m up to 12 km away from the shoreline (2021 may 13 letter from bfar 1 to aragones l; unreferenced). illegal fishers usually use ammonium nitrate from crop fertilizers as their main explosive compound. formulations of explosives vary among fishers, but they often use 350 ml glass bottles. while detonations from these explosives are generally small, the persistence of this illegal practice may induce debilitating injuries among nearby marine mammals and fish as well as destroy coral reefs. this study aimed to characterize the blasts from confiscated explosives in the philippines and infer, based on the acoustic properties of a single pulse, the possible impacts on marine mammals, particularly cetaceans. this study utilized confiscated explosives from different fishermen. as such, the magnitude of the explosives is not uniform throughout the trials. moreover, the exact formulations of the explosives were unknown; there were variations in the compounds and total amounts used. analyses were limited to the assumption that the explosion experiments occurred in open water with no influence of obstructions producing backscattered waves and that the explosives detonated at a fixed vertical distance upon falling into the water. lastly, following regulations on such experiments, representatives from bfar and the philippine coast guard (pcg) ensured that no marine animals were present near the detonation site. materials and methods simulating and recording underwater explosions the confiscated underwater explosives used were provided by the northern command of the pcg and the bfar region 1 office. four explosives in separate 350 ml bottles were used in simulating dynamite fishing in open waters off san fernando, la union on 9 july 2018. the experiment was conducted in open waters (~200 m deep) in coordination with the pcg and bfar 1 offices who provided personnel and equipment to ensure safety in the field. four explosions were detonated approximately 12 m below surface water at varying lateral distances from the hydrophone (see table 1). a cetacean researchtm cr1a hydrophone with a spectradaq-200 sound card was used for recording underwater sound generated from these explosions. table 1. time of recording and actual distance of explosion from the hydrophone setup. trial time distance from source (m) 1 10:18 am 195.46 2 10:37 am 267.25 3 10:50 am 424.01 4 10:54 am 773.47 characterization of open water explosions from confiscated explosives in the philippines 8 analyzing the acoustic pressure of the blasts an explosion occurs when there is an outburst of energy in the form of light, heat, sound, and shock wave. a shock wave is compressed air or strong pressure that radially travels greater than the speed of sound (ling et al. 2016). it happens when sound waves are constructively interfering with other similar sound waves arriving simultaneously, creating a violent change in stress, density and temperature. shock waves are commonly produced by explosions, supersonic aircrafts, lightning, or other phenomena that cause sudden change in pressure. the release of strong pressure is not limited to air; it can also happen to other elastic mediums like water (ridah 1988) and solids (thurston 1974). in an underwater explosion, a shock wave also creates large underwater sound pressure that rapidly decays through time (sayapin et al. 2006; veksler et al. 2009; liu et al. 2018). the acoustic pressure of the blast was analyzed for each simulated explosion. to determine peak underwater pressure, sound recordings were calibrated using the spectraplus software of the hydrophone, utilizing a transducer sensitivity value of -198 db re 1v/ μpa and a transfer factor of 0.1259 mv/pa. after calibration, plots of pressure (in pa) over time were generated. peak pressures were extracted from each plot per trial and summarized in table 2. table 2. estimated charge weight in grams of tnt. trials distance from source (m) peak pressure (pa) charge weight (g tnt) charge weight (g nh4no3) 1 195.46 52938.86 83.30 198.33 2 267.25 37238.66 83.68 199.24 3 424.01 30059.17 189.25 450.60 4 773.47 20039.45 391.85 932.98 backscattered waves generated in the explosion may have produced peak pressures following the detonation. backscattered waves can be in the form of scattered waves or reflected waves. scattered waves result from waves that are incident on rough surfaces (ainslie 2010) such as rocks, corals, soft bottom, etc. on the other hand, reflected waves result from those incident on smooth surfaces (ainslie 2010). in this study, only the peak pressures depicting each explosion trial were considered. peak pressures from backscattered waves were not analyzed as these are brought by external factors aside from the explosion itself. estimating the charge weight the confiscated explosives come in different sizes and composition depending on the formulation of the illegal fisher involved. as such, estimating the charge a.i. veloria et al. 9 weight of the explosion was performed by assuming that the explosives can be represented by the amount of trinitrotoluene (tnt) present in the blast charge. the charge weight was then estimated using the equation for shock wave pressure (samareh salavati pour and alizadeh 2012; soloway and dahl 2014) generated by the explosion, p 0 = 52.4 × 106 r w 1/3 –1.13 (1) where p0 is the peak incident pressure in pascals, w is the charge weight in kg of tnt, and r is the distance between the hydrophone setup and the source of explosion in meters. the estimated charge weight, w, is expressed in kg of tnt because this compound has been used as a reference for the relative effectiveness of other explosive compounds (soloway and dahl 2014). ammonium nitrate (nh 4 no 3 ), the most common explosive used by illegal fishers in the philippines, has a relative effectiveness of 0.42 kg tnt (soloway and dahl 2014). this factor can be used to estimate the actual charge weight of the explosive in terms of nh 4 no 3 . analyzing peak frequency and sound pressure levels from raw recordings the raw recordings were processed to identify dominant frequencies and determine sound pressure levels (spls) at peak frequencies. first, the recordings were clipped to 30 seconds before and after detecting the explosion to minimize the noise in post-processing the data. fast fourier transform (fft) was done using the raw sound measurements to identify the peak frequency (dominant frequency). theoretical peak pressures and spls were calculated using the estimated charge weight of the explosion and the actual distance between the hydrophone and detonation source. using equation 1, peak incident pressure per distance was estimated and then converted to spl using the equation spl re 1 μpa = 20 log 10 p 0 p r (2) where p 0 is the peak incident pressure in pa and p r is the reference pressure in μpa. analyses on marine mammal sensitivity are usually done using parameters derived from the generated shock wave, such as peak incident pressure, spl, sound exposure level, and shock wave impulse. moreover, the auditory bandwidth of select marine mammals are considered for specific sensitivity to sound generated by the explosion. in this study, analyses focused on interpreting acquired peak incident pressures and spls generated by the simulated explosions. characterization of open water explosions from confiscated explosives in the philippines 10 results and discussion any experiment on explosives, particularly to simulate illegal dynamite fishing, is a challenge. the explosion experiment conducted involved recording the simulated explosion using a cr1a hydrophone with spectradaq-200 sound card. recordings were initially analyzed as raw voltage measurements against time as shown in figure 1. based on the figure, the absolute peak voltage measurements in all trials indicate the start of the explosion, representing the events of highest pressure change. succeeding peaks of voltage measurements following the explosion were pressure changes from backscattered waves formed along the boundaries of the explosion. however, these succeeding peak voltages, which can be converted to peak pressures, were not analyzed. figure 1. raw signals (in volts) recorded by the hydrophone during the simulated explosions. a.i. veloria et al. 11 fft was implemented on the absolute voltage measurements from the recordings. subsequently, the power spectra were obtained as shown in figure 2. the power spectra show the frequency space of the raw signals after applying fft. the amount of energy in every frequency emitted by the explosions was shown in these plots. the evident increase of power in every trial signified the considerable energy emitted by the confiscated explosives. in this experiment, the average frequency with the highest power was 12.1 khz. the average frequency of the explosions is within the auditory bandwidth of any cetacean group, as summarized in table 3, suggesting that the blast from these types of explosions may be perceived by marine mammals and may cause direct and even debilitating impacts (e.g., auditory injury or acoustic trauma) to individuals in the vicinity of the explosion. figure 2. power spectra of the explosions in db. characterization of open water explosions from confiscated explosives in the philippines 12 table 3. cetacean criteria for injury from single pulse explosion (after southall et al. 2008 and mitchell et al. 2009). marine mammal group (cetaceans) genera represented estimated auditory bandwidth threshold for injury from single pulses (explosion) low-frequency balaena, caperea, eschrichtius, megaptera, balaenoptera 7 hz to 22 khz 230 db spl db peak re: 1μpa or 23 psi peak pressure mid-frequency steno, sousa, sotalia, tursiops, stenella, delphinus, lagenodelphis, lagenorhynchus, lissodelphis, grampus, peponocephala, feresa, pseudorca, orcinus, globicephala, orcaella, physeter, delphinapterus, monodon, ziphius, berardius, tasmacetus, hyperoodon, mesoplodon 150 hz to 160 khz high-frequency phocoena, neophocaena, phocoenoides, platanista, inia, kogia, lipotes, pontoporia, cephalorhynchus 200 hz to 180 khz underwater explosions produce anthropogenic sound that may pose threats and induce injury and acoustic trauma to aquatic species (wahlberg 2002; mccauley et al. 2003; martin and popper 2016). a set of criteria for marine mammal noise exposure was proposed by southall et al. (2008), as summarized in table 3. for a single pulse or explosion, a spl of 230 db re 1 μpa is set as the threshold for the onset of temporary threshold shift (tts) and auditory injury to cetaceans (southall et al. 2008). criteria for other marine mammal groups were provided by southall but only those for cetaceans were considered in this study. spl from the recordings was obtained to analyze the strength of the acoustic waves generated from the simulated explosions and relate it to possible injuries to marine mammals. figure 3 shows the spl obtained per trial in db with respect to 1 μpa. the maximum peak values represent the pressure levels of the explosions per trial. the average spl obtained at the dominant frequency of 12.1 khz is 137.94 db re 1 μpa. this spl is more than the spl range classified by erbe (2012) using audiograms of various marine mammals. these marine mammals include representatives from families monodontidae, delphinidae, phocoenidae, ziphiidae, phocidae, otariidae, odobenidae, and sirenia, which have minimum hearing thresholds of around 50 to 70 db re 1 μpa at 10 khz frequency (erbe 2012). these marine mammals can therefore perceive the generated sound from these explosions. note that philippine marine mammals include delphinids, ziphiids, and a sirenian, the dugong (aragones et al. 2010). the average spl from the explosion is also close to 150 to 160 db re 1 μpa, the spl generated by multiple pulse explosions that caused changes in behavioral responses of humpback whales (todd et al. 1996; southall et al. 2008). a.i. veloria et al. 13 behavioral changes demonstrated by humpback whales on these pressure levels were individual alertness, prolonged orientation, minor changes in locomotion speed and direction, moderate change in respiration rate, and minor cessation or modification of vocal behavior (southall et al. 2008). higher spl is expected at closer distances to the explosion, which can be detrimental to marine animals in the area. the average spl is also less than 230 db re 1 μpa, the previously stated threshold for injury and onset of tts on marine mammals caused by single pulse explosions (southall et al. 2008). these results suggest that while the maximum spl is less than the injury threshold, the dominant frequency and spl values show that the sounds generated by this type of explosive are within the hearing bandwidth of cetaceans and may induce possible masking of sounds and changes to their behavior, to mention a few. most of the groups of cetaceans mentioned in table 3 (i.e., those using middle frequency bandwidth) are present throughout the philippines (aragones et al. 2010; aragones et al. 2017; aragones and laggui 2019). transmission loss of the acoustic pressure as it travels from the source up to the hydrophone setup may have reduced the energy of the generated shock wave upon reaching the hydrophone. thus, it is important to further analyze the simulated explosions for possible effects to cetaceans based on the acoustic charge weight of the blast at closer distances from the explosion. figure 3. sound pressure level (db re 1 μpa) from the simulated explosions. characterization of open water explosions from confiscated explosives in the philippines 14 the simulated explosions were characterized based on its estimated charge weight. because of the non-uniform composition of the explosives depending on the fisher, the exact explosive compounds used remained undetermined in this study. instead, the explosives were assumed to be composed of ammonium nitrate (nh 4 no 3 ). moreover, the charge weight of each explosion was estimated based on available literature (samareh salavati pour and alizadeh 2012; soloway and dahl 2014), which assume the blast charge weight in kilograms of tnt. the peak pressure values directly obtained from calibrated measurements in spectraplus were used to estimate blast charge weight. table 3 shows the estimated charge weight of each explosion both in terms of tnt and nh 4 no 3 . trials 1 and 2 have almost similar charge weights while trial 4 has the highest charge weight of 391.85 g of tnt. using the estimated charge weights, theoretical peak pressures were calculated. for the intended purpose of explosives used by illegal fishers, keevin and hempen (1997) established that mortality and internal damages in bluegill fish abruptly increased at 500 kpa peak pressure generated by a 2 kg charge of t-100 explosive detonated at 2 m depth. theoretical peak incident pressures at varying lateral distances from the explosion were calculated from the estimated charge weight, as shown in figure 4. peak pressures from the simulated explosions reach 500 kpa at distances around 25 m to 45 m from the detonation site. this means that fishes within almost 50 m of the explosion will experience internal damages such as ruptured swim bladder or damages to the kidney, liver, spleen, and heart that may eventually lead to death. therefore, the fish are easily collected by these illegal fishers upon explosion. peak pressures of 23 psi or around 160 kpa cause physiological disruptions to all kinds of marine mammals (mitchell et al. 2009). a range of about 73 m to 123 m away from the explosion trials generated peak pressures of approximately 160 kpa. within this range, tts may be observed in present marine mammals. marine mammals experiencing tts lose their hearing sensitivity, leading to failure in communication, orientation, and foraging (tyack and clark 2000). this may help explain the findings of aragones and his colleagues in 2010, wherein they reported a relatively large proportion of live stranders (60%) in the philippines. a.i. veloria et al. 15 figure 4. theoretical peak incident pressure based on estimated charge weight. spls generated by the blast were then estimated from pressure values and plotted with respect to distance from the source as shown in figure 5. demonstrated in the figure is the spl generated when using g of tnt (in blue line) and g of nh 4 no 3 (in red line). spls from charge weights using relative effectiveness of nh 4 no 3 to tnt were projected in the blue line. note that a slight difference in charge weight generated a different spectrum of spls. the plots show the decrease in spl farther from the explosion. moreover, orange lines depict the actual distance of the hydrophone setup to the blast. also, from these plots, the maximum distance with an spl value of less than 230 db re 1 μpa was determined. this was estimated to project a minimum lateral distance a cetacean will be safe from injury caused by a single pulse explosion like dynamite blast. characterization of open water explosions from confiscated explosives in the philippines 16 figure 5. theoretical sound pressure levels based on estimated charge weight. orange lines indicate the distance between the source of explosion and the hydrophone setup. table 4 summarizes the minimum distance from the explosion site where marine mammals may be safe from injuries caused by single pulse explosions. the criteria for the injuries were based on the 230 db re 1 µpa spl threshold by southall et al. (2008) and 23 psi peak pressure by mitchell et al. (2009). as shown in the table, the trial 4 explosion has the farthermost distance (~123 m) that may be hypothetically declared a safe zone for cetaceans. given the precautionary principle, this hypothetical distance will be extended to 150 m. therefore, the safe zones for all trials were less than 150 m, suggesting that the dynamite blasts may affect fishes, dolphins and whales, and dugongs that are close to the explosion site. lastly, the results are all true given the assumption that backscattered waves do not affect the measurements conducted and that the trials were done in open water far from obstructions. this study provides key results to elucidate the impacts of small explosive loads underwater. most of the studies worldwide come from the united states navy and army and private institutions that all looked at large explosions, low frequency sounds, and the like. a.i. veloria et al. 17 table 4. identified safe zones for marine mammals during simulated explosions. trials distance from source (m) peak pressure (pa) charge weight (g tnt) distance from source not safe for marine mammals based on 230 db re 1µpa (m) distance from source not safe for marine mammals based on 23 psi peak pressure (m) 1 195.46 52938.86 83.30 40.19 73.45 2 267.25 37238.66 83.68 40.25 73.56 3 424.01 30059.17 189.25 52.83 96.55 4 773.47 20039.45 391.85 67.34 123.07 evidence of acoustic trauma from dynamite blasts have been reported in the philippines (pacini et al. 2017). the hearing loss due to dynamite fishing recorded in spinner (stenella longirostris) and rough-toothed dolphins (steno bredanensis) were based on hearing measurements using non-invasive auditory brain stem responses. pacini and her colleagues (2017) concluded that this hearing loss was reflective of exposure to blasts and related impulsive sounds. this implies that these animals might have been just outside of the safe zone as they ended up stranded. we are just starting to understand impacts of dynamite blasts on marine mammals in the philippines. the simulated single pulse explosion in open water is the minimum requirement to analyze possible effects of underwater noise on local marine mammals. obstructions that affect sound, which are more predominant nearshore because of surface texture, are expected to produce intensified blast and prolonged multiple pulses from backscattered waves. the estimated 150 m safe zone may not necessarily be harmless. the dugongs, which are less agile than the cetaceans and often more nearshore, may be very vulnerable to the debilitating effects of these types of blasts, especially explosions done in shallow water. the dugong is one of the most common stranders in the philippines (aragones et al. 2017; aragones and laggui 2019) indicates the possible contribution of dynamite blasting. further studies are required to elucidate the finer impacts of this illegal dynamite fishing practice on our vulnerable and charismatic marine vertebrates and coral reefs. summary and conclusions marine mammals can create and detect underwater sounds that they use for communication, orientation, and foraging. underwater noise such as dynamite blasts may pose serious threats to marine mammals in the form of behavioral responses and physical injuries (e.g., acoustic trauma). in the philippines, illegal dynamite fishing persists even with enhanced law enforcement (e.g., establishment of the bfar-fisheries resource protection group). characterization of open water explosions from confiscated explosives in the philippines 18 sound recordings were generated from simulated blasts using typical confiscated explosives. the sound recordings were post-processed to depict pressure, power level, and spl acquired from an underwater explosion. while it was determined that the dominant frequency of peak pressure is within the hearing bandwidth of marine mammals, the spl obtained from the explosions may not necessarily imply imminent damaging injury caused by single pulse explosions. however, the low spl obtained may be caused by transmission loss as the sound travels from the source to the hydrophone. thus, further analyses were done regarding the charge weight of the blast at varying distances from the source. different charge weights were obtained from the four simulated explosions supporting the observed differences in explosive size and composition. using the estimated charge weights (in kg of tnt), theoretical spls were plotted with respect to distance from the source of the explosion. results showed that at closer distances (<150 m), the explosions may pose serious injury to marine mammals. it is important to note that these safe zones assume that the explosion does not produce backscattered waves. therefore, animals outside this zone may still be impacted. evidence of this comes from pacini and her colleagues (2017) who reported hearing loss in two stranded cetaceans in the philippines allegedly due to dynamite blasts. it is then recommended that future simulations and analyses consider surface reflection that may induce significant changes in acoustic pressure from backscattered waves. this can be done through explosion trials in shallow waters, which produce scattered and reflected waves based on surface texture. lastly, the characterization of compounds present in the explosives may be helpful in accurately estimating the charge weight of the explosion. acknowledgments the study was funded through the philippine council for industry, energy and emerging technology – natural sciences research institute joint research program grant (awarded to lv aragones). this study would not have been possible without the support (patrol boats and personnel) and permission granted by national director eduardo gongona of da-bfar; regional director nestor domenden of dabfar regional fisheries office 1; and captain joseph coyme, commander of the northwestern luzon district of the philippine coast guard. thanks also to jamaica caras, ehmir cristobal, and the many men in uniform of bfar and pcg for assisting us in the field work. many thanks to the two anonymous reviewers who helped improve this manuscript. a.i. veloria et al. 19 references ainslie m. 2010. principles of sonar performance modelling. berlin: springer. aragones l, roque m, flores m, encomienda r, laule g, espinos b, maniago f, diaz g, alesna e, braun r. 2010. the philippine marine mammal strandings from 1998 to 2009: animals in the philippines in peril? aquat mamm. 36(3):219–233. aragones l, laggui hl. 2019. marine mammal strandings in the philippines from 2017 to 2018: initial biennial analysis. quezon city, philippines: philippine marine mammal stranding network. technical report no. 2; [accessed 2020 august 6]. http://pmmsn. org/resource-center/. aragones l, laggui hl, amor a. 2017. the philippine marine mammal strandings from 2005 to 2016. quezon city, philippines: philippine marine mammal stranding network. technical report no. 1; 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[date unknown]. science of sound. kingston (ri): the university of rhode island; [accessed 2020 august 26]. https://dosits.org/science/. veksler d, sayapin a, efimov s, krasik ye. 2009. characterization of different wire configurations in underwater electrical explosion. ieee trans plasma sci. 37:88–98. walhberg m. 2002. the acoustic behavior of diving sperm whales observed with a hydrophone array. j exp mar biol ecol. 281(1–2):53–62. ______ lemnuel v. aragones <laragones@iesm.upd.edu.ph> is a professor and the current director of the institute of environmental science and meteorology (up-iesm), university of the philippines diliman. he received his ph.d. in tropical environmental studies (marine ecology) from james cook university, australia. he is the head of the marine mammal research and stranding laboratory at up-iesm and is working on marine biology, ecological sustainable development and conservation and management of marine mammals and other large marine vertebrates. archie i. veloria is a senior science research specialist at up-iesm and is working on instrumentation physics, satellite remote sensing and extreme weather monitoring. he is a member of the predictions for the environment and applications of remote sensing laboratory at up-iesm. he finished his bachelor of science in applied physics at university of the philippines los baños and master of science in meteorology at university of the philippines diliman. daniella t. hernandez finished her bachelor of science in physics at the university of the philippines diliman. she was a member of the instrumentation physics laboratory at the national institute of physics (nip), university of the philippines diliman and worked on underwater acoustic instrumentation and signal analysis. giovanni a. tapang is a professor at nip and the current dean of the college of science, university of the philippines diliman. he received his ph.d. in physics from the university of the philippines diliman. he is the head of the synchronization, biology, and optics research subgroup of the instrumentation physics laboratory at nip. he specializes in instrumentation physics, complex networks, optics and computational physics. 01_device a survey of macro-invertebrate gleaning 11 a survey of macro-invertebrate gleaning in the banate bay intertidal area, eastern panay island annabelle g.c. del norte-campos1, wilfredo l. campos2 and karen a. villarta1, 2 1marine biology laboratory and ocean bio laboratory, division of biological sciences, college of arts & sciences (cas), university of the philippines in the visayas miagao, iloilo tel. no. (033) 315-9636; fax no. (033) 315-9271; e-mail: wlcampos@mozcom.com date received: january 18, 2006; date accepted: may 30, 2006 abstract science diliman (july-december 2005) 17:2, 11-20 *corresponding author the gleaning fishery on the intertidal areas of banate bay, eastern panay was surveyed monthly from february 2002 to january 2003, to derive information on species composition, catch, catch rates, and annual value. total biomass, gleaning and turnover rates were determined from a fishery-independent survey conducted in june 2005. catches of the fishery consisted of a total of 17 species, comprised of mollusks, crustaceans and a brachiopod. the bivalves katelysia hiantina, scapharca inaequivalvis, and gafrarium tumidum were the top three species, together comprising 88.79 % of the total catch. the total mean daily catch per gleaner for all species was equivalent to 73.75 g/m-2/gleaner-1. catch rate and catch volume for the mollusks were highest between may-july and november-december, coinciding with the southwest and northeast monsoons, respectively. the large riverine inputs to the area, together with the mangrove-derived organic matter, periodically resuspended by the tidal fluctuations, are seen as responsible for increasing organic matter content of the substrates and abundance of the species. total annual catch of the fishery is estimated to range from 20,988.7 to 43, 527.62 kg, with a median value of 31,205.6 kg. this latter value divided by the estimated total biomass in the area of 2,441. 03 kg gives a turnover rate of 12.8. the total annual catch for the entire fishery is equivalent to a total value of php 421t to 897t/yr-1. the latter correspond to an annual income of php 14,043.90 to 29,904.67/gleaner-1/ yr-1, small amounts which may be sustainable due to the high turnover rate of the system. keywords: gleaning, benthos, species composition, catch, catch rates, biomass, turnover rate del norte-campos, campos and villarta 12 introduction macro-invertebrates make up a significant portion of different kinds of fisheries in the country. many of them are commercially important species (del norte-campos et al. 2000), that have become the target of many fishing gears such as the trawl (del norte-campos et al. 2003a). one of the most important methods of gathering invertebrates is gleaning on intertidal areas. gleaning on these highly-accessible fishing grounds is a form of fishing requiring the least of implements, and as such, is a highly favored fishing method for coastal communities. despite its importance, there is a general lack of information in the country on this type of fishing method except for the works of de guzman (1990) on the reef flats of bolinao, pangasinan, schoppe et al. (1998) on the cuatro islas, leyte and del norte-campos et al. (2003a) on malalison island, antique. these papers cover areas that are on or contiguous with coral reefs. however, aside from reefs, gleaning is also conducted in other types of habitats, such as sandymuddy intertidal areas with nearby mangrove stands. further, gleaning can also be of two types: 1) general, where species are collected as encountered, and 2) specific, where there is/are certain target species. one example of a gleaning fishery on sandy-muddy intertidal (non-reef) areas is that existing on banate bay, eastern panay island, which has not been documented at all. this paper aims a) to characterize the general gleaning fishery on the sandy-muddy intertidal zone of banate bay in terms of species composition, volume of catch (kg) by species, and catch rate (kg/gleaner/hr), and b) to derive preliminary estimates of biomass (kg/km2) and gleaning rate. gathering these baseline data is the first step in formulating proper utilization guidelines to ensure sustainable harvesting of the resources. materials and methods the study was conducted for one year, from february 2002 to january 2003, on the intertidal areas of bgy. tinurian, banate bay, eastern panay island (fig. 1). the area is fully exposed during ebb tides and inundated at high tide. the seaward part of the area has a predominantly sandy substrate, interspersed with small to medium-sized rocks and boulders. the landward part which has a muddy substrate, is bordered with mangroves particularly sonneratia alba, avicennia rumphiana and rhizopora apiculata. the monthly gleaning fishery survey consisted of following/accompanying 3-4 gleaners at work for the purpose of determining the total area covered by each gleaner. gleaning by the observed group was observed to be restricted to a very specific area, i.e. in the remains of what used to be a fishpond dike with measurable dimensions of about 900 x 10 m. the gleaners in the area usually work in groups that pool their catches at the end. from these pooled catches, species composition, along with numbers and weight (kg) of the catch by species were determined. type specimens were also collected and brought to the laboratory for proper identification, using fao (1978) as basis, except for one crab species which was sent to the national museum, carcinology division. data collection was supplemented with informal interviews with gleaners, specifically to gather information on gleaning practices, gleaner profiles and markets for species. prices for each species were determined by getting the corresponding weights (in kg) of piles ("tumpok"), normally used in selling. to determine biomass, (bs) a fishery-independent sampling was conducted in june 2005. all the macroinvertebrate biota from a total of 15 2 x 2 m quadrats (4 m2), dug 5-6 inches into the sand/mud were sampled and sorted. the quadrats were laid in 4 transect lines parallel to the general orientation of the gleaned area, with 1 line (transect 4) located 3-5 m away from the remains of the dike, and the other 3 lines (transects 1-3) located within a 5-10 m belt covering the entire length of the remains of the dike. the biota were sorted into species and weighed. the total gleaning area was measured with the use of a gps. during this sampling date, the catch (cs in g/m -2) of the gleaners was also averaged and then divided by the mean biomass (bs in g m2) taken from the quadrats to estimate gleaning rate (gr in %). a survey of macro-invertebrate gleaning 13 figure 1. map showing the study area, sandy-muddy flats of banate bay (inset). 1 0º59 ’ 1 22º4 3’ 4 5 ’ 45’ 1 005 5’ 5 7’ 4 7’ 1 0 05 9 ’ 1 005 7’’ 10 055 ’ 1 22 043 ’ 4 7’ del norte-campos, campos and villarta 14 volume of the catch (g/m-2/gleaner-1) was computed by month and averaged for the entire year by species. individual mean catches by species were summed to get the mean catch (cf in g m -2 gleaner-1) over the year. species importance was then based on the corresponding percentages of the mean daily catch volume values. the mean annual standing stock (bann) was computed by dividing the mean catch (cf) computed from the fishery by the gleaning rate (gr). this resulting (bann) value was multiplied by the area of gleanable habitat (a in m2) to get the total biomass (btot in kg). total annual catch (ctot in kg) was derived by multiplying the mean catch (cf) per gleaner with the number of gleaners in the area, the number of gleaning days over the entire year and the area covered per gleaner. the estimated total annual catch (ctot in kg) was then divided by the total biomass (btot in kg) to get the turnover rate (t). total annual catches (kg) per species were multiplied with the corresponding selling price (php kg-1) to derive the total annual value (php) by species. total annual value (php) was then derived by adding the total annual values (php) for all species. catch rates (kg/gleaner-1/ hr-1) of the most abundant group were also plotted against months. results gleaning practices/profile gleaning in banate comes in two forms: general and specific. the latter involves collection primarily of the small short razor clam azorinus abbreviatus, which is mostly found in the muddy substrates of the mangrove areas. both forms of gleaning on the banate intertidal flats are conducted daily, during daytime low tide, i.e. when the area is either partially or fully exposed. the gleaners are usually entire families, consisting of the father and mother, in general ranging from 30-50 yrs. old, as well as their children usually in their early teens. they are mostly residents of bgy. tinurian, barotac nuevo, iloilo, from where they usually travel on nonmotorized boats down the river to the gleaning site, a distance requiring a travel time of ~1 hr. for most of these groups, gleaning is the main source of livelihood, although usually supplemented with fishing in the river for shrimps and crabs, using net traps. species composition and importance a total of 17 species was recorded during the survey (tab. 1). mollusks constituted the most abundant group with 13 species (12 bivalves and 1 gastropod), whereas the rest of the catch was composed of 3 crustacean species, and 1 brachiopod. the top three species are the hiant venus katelysia hiantina ("punaw"), the inequivalve ark scapharca inaequivalvis ('litob"), and the tumid venus gafrarium tumidum ("bug-atan"), together comprising 88.79% of the total catch volume. aside from these three, other bivalve species likewise gathered were the rock edible oyster (crassostrea echinata), small short razor (azorinus abbreviatus), the boxlike tellin (merisca capsoides), the decussate ark (barbatia foliata), the granular ark shell (anadara granosa), the pill ark shell (a. pilula), the mussels perna viridis and modiolus metcalfei and the japanese dosinia (dosinorbis japonica). these species belong to 6 bivalve families namely, veneridae (3 spp.), arcidae (4 spp.), mytilidae (2 spp.), and 1 species each for family solecurtidae, ostreidae, and acropagiinae. aside from the bivalves, the three species of crabs (malacostraca: decapoda) represented in the catch were, the blue swimming crab portunus pelagicus, the crenate swimming crab thallamita crenata and the eriphiid stone crab ozius rugulosus. the dog conch strombus canarium (gastropoda), as well as the lamp shell lingula unguris of phylum brachiopoda make up the rest of the catch. catch rates, volume and annual value catch rates of the most abundant group (mollusks) showed the highest values in april, july, and november 2002 and january 2003 (fig. 2). mean catch rates for the individual species likewise follow the trend shown by species importance. thus, catch rates for the top three species were also the highest, namely: k. hiantina = 0.548 kg/hr, s. inaequivalvis = 0.366 kg/hr, and 0.193 kg/hr for g. tumidum. trends in the catch volume of the abundant group (fig. 3) appear to coincide with those of the catch rates (fig. 2), i. e., values were higher between may-july 2002 and also between november 2002 and february 2003. a survey of macro-invertebrate gleaning 15 0 1 2 3 4 f eb ru ar y 02 m ar ch a pr il m ay j un e j ul y a ug us t s ep te m be r o ct ob er n ov em be r d ec em be r j an ua ry 0 3 m onths c at ch r at e (k g/ gl ea ne r/ hr ) mollusk b rachiopod c rustacean figure 2. catch rate (kg/gleaner/hr) of the most abundant groups gleaned on the intertidal areas of banate bay, february 2002 to january 2003. there are reportedly 40 gleaners in the area, although it is unlikely that all of them are always active. thus, the annual catch (ctot) ranges from 20,988.7 kg for 20 gleaners and 264 days of gleaning to a maximum of 43,527.62 kg for 30 gleaners and 365 days. the median value of 31,205.6 kg is taken to be the most reasonable estimate of annual catch. most of the 14 species have commercial value, whereby the prices are higher when sold in the city (tab. 2). this is especially the case for a. abbreviatus, thus explaining the existence of the specific type of gleaning primarily for this species. the total annual value of the annual catch is equivalent to php 421,047.11 when species are merely sold in the barangay, and php 897,140.00 when sold in the city. if computed for a total of 30 gleaners this brings a total annual income of php 14,034 gleaner-1 yr-1 and php 29,904.67 gleaner-1 yr-1, respectively. gleaning rate, biomass and turnover rate the gleaning survey showed that the mean overall biomass (bs) estimates are more consistent when only 12 out of the 15 quadrats are taken (table 3). the 12 quadrats are those situated within the dike remains (transects 1-3) or the ones that are truly representative of the biomass of the gleaned area, as this is where gleaning is mostly confined. using the mean biomass (38.38 g m-2) from these 12 quadrats, the estimated gleaning rate (gr) is 62.7% (tab. 4). at this rate, the total biomass (bott) is estimated to be 2,441.03 kg. using the median annual catch value (ctot) of 31,205.6 kg, the turnover rate (t) is 12.8. discussion the importance of gleaning in the country has generally been underestimated (schoppe et al., 1998). this is due to several factors, foremost of which is the difficulty in monitoring catches in such non-organized artisanal fisheries (lopez 1986), and the generally lower regard for invertebrates which form the bulk of the catches in this type of fishing. however, this form of fishing being highly accessible and of low capital investment (mcmanus 1989) may actually play a bigger role in the livelihood of fishers considering the decline of most artisanal/municipal fisheries in the country (campos et al. 2002; barut et al. 2004). species composition the mollusks, specifically bivalves, dominate the catch of the gleaning fishery in the banate sandy intertidal areas, as these represent a more suitable habitat for these burrowing and suspension-feeding species. azorinus abbreviatus, on the other hand, is largely del norte-campos, campos and villarta 16 table 1. species composition of gleaned macro invertebrates ranked according to mean catch (g/m2/gleaner) in banate bay, eastern panay, sampled from feb 2002-jan 2003. (identification and names of most species were based on fao, 1998). species mean catch scientific name common name local name (g/m2 gleaner) % 1. katelysia hiantina (mollusca: bivalvia) hiant venus "punaw" 31.74 43.04 2. scapharca inaequivalvis (mollusca: bivalvia) inequivalve ark "litob" 22.82 30.94 3. gafrarium tumidum (mollusca: bivalvia) tumid venus "bug-atan" 10.92 14.81 4. crassostrea echinata (mollusca: bivalvia) rock edible oyster "sisi" 2.44 3.31 5. azorinus abbreviatus (mollusca: bivalvia) small short razor "tikhan" 1.50 2.03 6. merisca capsoides (mollusca: bivalvia) boxlike tellin "agrutan" 1.49 2.02 7. barbatia foliata (mollusca: bivalvia) decussate ark "kiwi-kiwi" 1.30 1.76 crabs (crustacea: malacostraca): 0.53 0.72 8. portunus pelagicus flower crab, blue "kasag" 9. thallamita crenata crenate swimming crab "dawat" 10. ozius rugulosus stone crab "kumong" 11. lingula unguris (brachipoda) lamp shell "ugpan" 0.29 0.39 12. strombus canarium (mollusca: gastropoda) dog conch "sikad" 0.22 0.30 13. anadara granosa (mollusca: bivalvia) granular ark shell "bacalan" 0.18 0.25 14. anadara pilula (mollusca: bivalvia) pill ark shell "litog-litog" 0.10 0.14 15. modiolus metcalfei (mollusca: bivalvia) yellow-banded horse mussel "bahong" 0.09 0.12 16. dosinorbis japonica (mollusca: bivalvia) japanese dosinia "agihis" 0.08 0.11 17. perna viridis (mollusca: bivalvia) asian brown mussel "green shell" 0.05 0.07 total(cf) 73.75 100.00 limited to the mangrove areas, thereby preferring the muddy/silty substrate type. this species thus shares the same habitat and habit as the mud clam anodontia edentula (primavera et al. 2002). in these intertidal areas as in others, distribution of species is closely correlated with substrate characteristics and variability, as well as the dominant vegetation (de guzman 1990). the relationship of species distribution to the observed distribution patterns can further be elucidated by zonation studies. compared to the species composition of gleaned species on the malalison reef flats in antique where gastropods predominate (del norte-campos et al. 2003a), the burrowing habit of the bivalves is appropriate in the banate intertidal areas which are openly subjected to tidal exposure. the malalison gastropods on the other hand, are able to take shelter provided by the existing coral cover. the blue swimming crab portunus pelagicus is poorly represented in the catch, as it is caught more by traps and trawl (del norte-campos et al. 2004), than by gleaning. the ones found here are smaller-sized juveniles, utilizing these shallower areas as nursery grounds. lingula unguris ("ugpan"), the brachiopod, turns out to be a commercially important species in panay: it was seen sold in markets, e.g. pontevedra, capiz (del norte-campos et al. 2000), and also harvested in malalison island (del norte-campos et al. 2003b). more are however, caught in capiz since the species is usually found along river bank systems a survey of macro-invertebrate gleaning 17 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 f eb ru ar y 02 m ar ch a pr il m ay j un e j ul y a ug us t s ep te m be r o ct ob er n ov em be r d ec em be r j an ua ry 0 3 months c at ch v ol um e (k g/ gl ea ne r) figure 3. volume of catch (kg gleaner-1) of the most abundant group (mollusks) on the intertidal table 2. total annual catch (kg), price (php/kg) when sold in the barangay and in iloilo city by species, and total annual value (php) of gleaned macro-invertebrates from the intertidal flats of tinurian, banate bay, eastern panay. species total annual price total annual value catch (php/kg-1) (php) (kg) bgy. city bgy. city 1. katelysia hiantin 13,430.48 15.00 40.00 201,457.20 537,219.20 2. scapharca inaequivalvis 9,655.55 13.00 30.00 125,522.15 289,666.50 3. gafrarium tumidum 4,621.04 8.00 a 36,968.32 4. crassostrea echinata 1,031.35 18.00 30.00 18,564.30 30,940.50 5. azorinus abbreviatus 632.86 20.00 60.00 12,657.20 37,971.60 6. merisca capsoides 630.39 b b 7. barbatia foliata 550.47 13.00 a 7,156.11 8. portunus pelagicus 224.12 60.00 b 13,447.20 9. thallamita crenata b b 10. ozius rugulosus 11. lingula unguris 121.43 20.00 a 2,428.60 12. strombus canarium 94.69 b b 13. anadara granosa 77.70 18.00 a 1,398.60 14. anadara pilula 42.31 15.00 a 634.65 15. modiolus metcalfei 36.71 13.00 a 477.23 16. dosinorbis japonica 34.12 c c 17. perna viridis 22.37 15.00 60.00 335.55 1,342.20 total 31,205.60 421,047.11 897,140.00 (ctot) a = not sold in the city b = not sold due to low abundance c = not eaten del norte-campos, campos and villarta 18 in addition, catches of some species have been noted to dwindle over the years. foremost of these species are the windowpane shells placuna sella and p. placenta, which used to be abundant in the area. nowadays, p. sella, locally known as "bay-ad" is still collected in banate bay and in northeastern panay (declarador & del norte-campos 2004) but primarily through compressor diving in subtidal areas. p. placenta on the other hand, has not been observed in any part of panay, although seafdec has had restocking efforts for this species in some parts of panay. table 3. biomass estimates (g/m-2) from the quadrats sampled along 4 transects during the fishery-independent survey on the gleaned intertidal area in tinurian, banate bay, june 2005. quadrat no. biomass (g m-2) overall mean overall mean biomass biomass (bs) transects (g m-2) (g m-2) 1 2 3 4 1 17.8 20.5 28.1 4.7 2 8.5 23.5 13.7 2.3 3 34.7 99.4 49.0 4.6 4 110.1 19.9 35.4 transects 1-4 transects 1-3 mean 42.75 40.79 31.56 3.87 31.47 38.38 sd 46.17 39.13 14.73 1.32 32.5 31.52 n 4 4 4 3 15 12 table 4. data parameters, formula input values and computations to calculate the turnover rate (t). parameters input values remarks mean catch (cf) 73.75 g m-2 gleaner-1 (see tab. 1) total annual catch (ctot) 31,205.6 kg. (see tab. 2) mean biomass (bs) 38.38 g m-2 (see tab. 3) area of gleaning habitat (a) 20,753.5 m-2 (actual measure using a gps) mean catch (cs) 24.07 g m-2 (observed and computed) computations gleaning rate (gr) = cs/bs = 24.07 g m-2 / 38.38 g m-2 gr = 0.627 or 62.7% mean annual biomass (bann) = cf/gr = 73.75 g m-2 gleaner-1/0.627 (bann) = 117.62 g m -2 total biomass (btot) = bann x a = 117.62 g m2 x 20,753.5 m2 (btot) = 2,441,026.6 g or 2,441.03 kg turnover rate (t) = ctot/btot = 31,205.6 kg/2,441.03 kg (t) = 12.8 species value marketing of the species in the city is dictated by two factors, namely: market appeal and abundance. the higher price of the small short razor a. abbreviatus ("tikhan") in the city explains the existence of specific gleaning for this species. according to the gleaners, higher prices for this species in the city are best achieved when they are peddled to the chinese who favor them more. due to the lower catches of blue crabs, on the other had, they are not sold anymore in a survey of macro-invertebrate gleaning 19 the city. in addition, due to the low quantities of the ark shells anadara pilula and a. granosa, they are in practice mixed with s. inaequivalvis when sold and thus, prices shown here are if and when sufficient quantities of the 2 species are available. biomass and turnover rates though the gleaners claim that there are no seasons in the catches, it appears that there are two periods with higher catch rates and resultant catches. these higher catches can be attributed to the higher abundances of the species. the two peaks in the volume of the catch and catch rates for the top three species may be related to their reproductive cycles. for scapharca inaequivalvis, for example, the two observed peaks in aug 2002 and jan 2003 coincide with what has been reported as the main periods of gonad activity of the species in the area (ledesma-fernandez & del nortecampos 2004). gonad activity was correlated with the monsoon periods, especially the southwest monsoon that coincides with the rainy months and thus, higher levels of terriginous inputs and food. the biomass and resulting turnover rate estimates are here considered preliminary until a more extensive survey over a longer duration can be conducted. just the same, how meaningful is a gleaning rate of almost 13 x that translate to a biomass replenishment (i.e. turnover) of about a month? although this turnover rate appears to be high, such rates may be possible considering factors such as the inherent location of the area and the high growth rates of tropical mollusks, which make up the bulk of the catches. these intertidal areas are subjected to periodic tidal fluctuations that serve to provide the means to transport and distribute a high organic load from the bordering mangrove areas, as well as the adjacent river system (odum 1968). the growth rate of the ark shell s. inaequivalvis, one of the top two species of the harvested bivalves in the area, for example, has been initially estimated to be 0.21 mm day-1(del norte-campos & villarta, unpubl.), a rate consistent with high turnover rates, esp. when considered that the species matures at small sizes (ledesma-fernandez & del norte-campos 2004), and thus at a young age. in a study on the snail telescopium telescopium, the similar organically-enriched fishpond environments were pointed out as responsible for an even higher growth rate of 0.69 mm day-1 (del nortecampos et al. 1998), thus making the derived turnover rates quite possible. thus the estimated turnove rate indicates a stronger trend towads the sustainability of these gleaned resources. together with the present study, more studies on the reproduction and feeding patterns of these species, as well as the ecological impact of this activity are clearly necessary. above all, there is a need to examine the growth of organisms in more detail, and to derive seasonal estimates of biomass and gleaning rates. acknowledgements this work was partially funded by the da/afma/ bar project "assessment of commercially-important invertebrates of inter-island waters (panay)" and the unep/gef project "biodiversity indicators for national use (binu)". fishery sampling assistance was provided by rheza beldia, marilu declarador, jenny panes and leah l. fernandez. g. genito, a. mequila, m.m. noblezada, a. santillan, dm estremadura, f. nabuab, c. acabado, j. asis, f. and m. tajolosa, p. beldia ii assisted in the fisheryindependent survey. ms. marivene manuel-santos (national museum) kindly identified the crab species ozius rugulosus, while dr. resureccion sadaba (div. bio. sci., cas, upv) confirmed the identity of the mangrove species. we also thank the gleaners of bgy. tinorian, barotac nuevo, specifically dolores belicena and family for giving access to their catches. references barut, n.c., m.d. santos & l.r. garces. 2004. overview of philippine marine fisheries, pp. 22-31. in: da-bfar (department of agriculture-bureau of fisheries and aquatic resources). in turbulent seas: the status of philippine marine fisheries. coastal resource management project, cebu city, philippines: 378 p. campos, w.l., n.p. evano, p.d. beldia ii & a.s. santillan. 2002. prospects from improved protection in marine reserves. upv j. nat. sci. 7(1 & 2): 146-157. del norte-campos, campos and villarta 20 declarador, m.b. & a.g.c. del norte-campos 2004. the diving fishery of bancal bay, northeastern panay. upv j. nat. sci. 9: 244-252. del norte-campos, a.g.c., j.h. apistar & i. a. arboleda. 1998. growth, production, and reproduction of the snail telescopium telescopium (linné) in brackishwater ponds in iloilo, philippines. upv j. nat. sci. 3(2): 83-95. del norte-campos, a.g.c., r.a. beldia, k.a. villarta, & m.a. tad-y. 2000. an inventory and market survey of commercially-important invertebrates around panay island and use of the data to prioritize research. upv j. nat. sci. 5(1): 9-11. del norte-campos, a.g.c., k.a. villarta, j.b. panes. 2003a. invertebrate trawl fishery of pilar and capiz bays, northern panay, west central philippines. upv j. nat. sci. 8 (1 & 2): 115-128. del norte-campos, a.g.c., m.b. declarador, r.a. beldia. 2003b. catch composition, harvest and effort estimates of gleaned macroinvertebrates in malalison island, northwestern panay. upv j. nat. sci. 8 (1 & 2): 129-141. del norte-campos, a.g.c., k.a. villarta, j.b. panes & m. declarador. 2004. catch and catch rates of the blue swimming crab (portunus pelagicus l.) in various fishing grounds in panay island. upv j. nat. sci. 9(1): 79-86. del norte-campos, a.g.c. & k.a. villarta. unpubl. population biology of the bivalves katelysia hiantina (lamarck, 1818), scapharca inaequivalvis (bruguiere, 1789) and azorinus abbreviatus (gould, 1861) from the banate bay intertidal area, eastern panay. de guzman, a. b. 1990. community structure of macrobenthic invertebrates on exploited reef flats of santiago island, bolinao, pangasinan. m.s. mar. bio. thesis, up diliman, quezon city: 145 p. freire, f., a.g. diola & f.b. sotto. 1998. commerciallyimportant species in bantayan island, cebu based on market survey and gleaning activities. (abstract only). philipp. sci. 35: 188. fao.1978. fao species identification guide for fishery purposes. the living marine resources of the western pacific. vol. 1 (seaweeds, corals, bivalves, & gastropods): 123-646 and vol. 2 (cephalopods, crustaceans, holothurians & sharks): 1045-1154. in: carpenter, k.e. and v.h. niem (eds.). food and agriculture organization, rome. ledesma-fernandez, l. & a.g.c. del norte-campos. 2004. reproductive cycle of the ark shell scapharca inaequivalvis (bruguiere, 1789) (mollusca, pelecypoda: arcidae) in banate bay, west central philippines. upv j. nat. sci. 9(1): 111-123. lopez, m.d.g. 1986. an invertebrate resource survey of lingayen gulf, philippines. pp. 402-409. in: g.s. jamieson & n. bourne (eds.). north pac. wrkshp. stock assess. mgt. inverts. can. spec. publ. fish aquat. sci. 92: 402-409. mcmanus, l.t. 1989. the gleaners of northwest lingayen gulf, philippines. naga. the iclarm quarterly. 12: 13. odum, e. 1968. energy flow in ecosystems: a historical review. 8: 11-18. primavera, j.h., m.j.h.l. lebata, l.f. gustilo & j.p. altamirano. 2002. collection of the clam anodontia edentula in mangrove habitats in panay and guimaras, central philippines. wetlands ecol. mgt. 10: 363-370. schoppe, s., j. gatus, p. milan & r. a. seronay. 1998. gleaning activities on the islands of apid, digyo, and mahaba, inopacan, leyte, philippines. philipp. scient. 35: 130-140. 3layman'sabstracts.pmd layman’s abstracts 1 science diliman (january-june 2019) 31:1, 1-3 layman’s abstracts benthic macroinvertebrates of the university of the phil ippines dil iman campus waterways and their variation across land use in an urban, academic landscape francis s. magbanua, john claude renan b. salluta, danielle dominique d. deborde and maria brenda m. hernandez issn 0115-7809 print / issn 2012-0818 online u r b a n d eve l o p m e n t a f fec t s s t r e a m s t h r o u g h c h a n g e s i n s t r e a m f l ow, shape, and water quality which influence the aquatic communities. the university of the philippines diliman campus has gone through numerous l a n d s c a p e a n d i n f r a s t r u c t u r e d e v e l o p m e n t s . u n l i k e t h e t e r r e s t r i a l environment, the extent to which these developments have impacted the campus waterways is unknown. thus, our research aims to assess the o v e r a l l c o n d i t i o n o f t h e w a t e r w a y s i n t h e c a m p u s u s i n g t h e b e n t h i c m a c r o i n v e r t e b r a t e c o m m u n i t i e s . a t o t a l o f 1 9 s t r e a m r e a c h e s w e r e s a m p l e d i n n o v e m b e r 2 0 1 5 a n d 2 0 1 6 i n t h e f o l l o w i n g l a n d u s e c a t e g o r i e s : a c a d e m i c u n i t s ( 6 s i t e s ) , c a m p u s c o r e ( 8 s i t e s ) , a n d p a r k s a n d o p e n s p a c e s ( 5 s i t e s ) . o u r s t u d y r e v e a l s t h a t a l l s a m p l e d s t r e a m reaches, regardless of their land use categories, are under poor to severe pollution conditions. all macroinvertebrate-based metrics and indices i n d i c a t e d e g r a d e d w a t e r q u a l i t y a n d s t r e a m h e a l t h . o u r r e s u l t s a r e consistent with urban stream studies elsewhere, which suggest that landb a s e d a c t i v i t i e s c a n b e s t r e s s f u l f o r s o m e a q u a t i c o r g a n i s m s , a n d a t t i m e s , r e s u l t i n r e d u c e d a b u n d a n c e a n d e v e n r e d u c t i o n i n s p e c i e s composition. layman’s abstracts 2 val idation of two extraction methods for human dna from cigarette butts paul ryan l. sales, dorothy emma c. ferrer, gay vell ine c. calacal, jazelyn m. salvador and maria corazon a. de ungria cigarette butts found in crime scenes may be used to identify persons and link them to a crime through dna analysis on evidentiary materials. the analysis of cigarette butts is challenging because these items are often exposed to chemical and environmental contaminants which can damage the dna . in this study, several factors were tested to compare the amount and quality of dna obtained from cigarette butts using two methods for extracting dna . results show that exposure to an outside environment has signif icant effects on dna yield and recovery for both extraction procedures. prolonged storage of cigarette butts of up to six months affected the amount of dna that can be obtained using the kitbased method. however, complete dna prof iles can be generated from cigarette butts stored for six months provided that these samples are stored indoors under controlled temperature conditions and with minimal exposure to contaminants. fol iar fungal endophytes of selected med icinal plants from the province of albay, phil ippines jonathan jaime g. guerrero, mheljor a. general and jazzlyn t. imperial fungi residing within plants known as endophytes were isolated from t h e l e a v e s o f t h e t e n m o s t f r e q u e n t l y u s e d m e d i c i n a l p l a n t s i n t h e p r o v i n c e o f a l b a y a t t h r e e d i f f e r e n t l o c a t i o n s : u p l a n d , l o w l a n d , a n d co a s t a l a r e a s . t h e o cc u r r e n ce , f r eq u e n c y, a n d i s o l a t i o n r a te o f t h e s e fungi were compared. a total of 120 isolates belonging to 17 species were identif ied. glomerella cingulata and colletotrichum gloeosporioides were the most frequent fungi occurring in 10 and nine plants, respectively. layman’s abstracts 3 t h e t o t a l n u m b e r o f i s o l a t e s a n d u n i q u e s p e c i e s d i d n o t v a r y signif icantly across sampling sites. blumea balsamifera (sambong) hosted the most endophytes with 16 isolates, while banana leaves yielded the l e a s t w i t h e i g h t i s o l a t e s . s o m e s p e c i e s o f f u n g i w e r e f o u n d i n a l l sampling sites, while a few occurred only in one site. the collection of additional samples from other sites within the province and the testing of the biological properties of the isolates are recommended. “pee value”: storing urine for subsequent dna analysis nelvie fatima jane a. sol iven, maria lourdes d. honrado, gay vell ine c. calacal and maria corazon a. de ungria t h e p h i l i p p i n e g o v e r n m e n t i m p l e m e n t s m a n d a t o r y a n d r a n d o m d r u g testing to monitor cases of drug abuse and trading. due to the possible repercussions of testing positive for drug use, def inite identif ication of t h e s o u r ce of t h e u r i n e i s c r u c i a l . d n a p r of i l i n g h a s r e s o l ve d i s s u e s r eg a r d i n g i d e n t i f i c a t i o n of t h e s o u r ce of u r i n e i n m a n y c a s e s a b r o a d . t h e i n c l u s i o n o f d n a t e s t i n g d u r i n g d r u g i n v e s t i g a t i o n s i n t h e p h i l i p p i n e s c a n i m p r o v e t h e p r o c e s s o f i d e n t i f y i n g d r u g u s e r s a n d address allegations of misconduct. since dna testing is previously not considered in drug investigations in the philippines, there is a need to test if storage procedures for urine that showed positive results allow for subsequent dna testing. in this study, dna from male individuals were extracted from urine stored at room temperature, 4°c, and -20°c for 2 months and 9 months followed by dna prof iling. overall, dna extracted from urine samples stored at cool temperatures (4°c and -20°c) were fo u n d to p r ov i d e b e t te r d n a p r of i l e s co m p a r ed to s a m p l e s t h a t we r e stored at room temperature. a decision tree for drug testing that should serve as support tool for philippine government agencies engaged in drug investigations was proposed. 8potential cholesterol-lowering-isah.pmd r.r. isah and c.l. chichioco-hernandez 83 science diliman (july-december 2016) 28:2, 83-91 potential cholesterol-lowering activity of selected plant extracts raff i r. isah university of the philippines diliman christine l. chichioco-hernandez* university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online cholesterol is an essential component of cell membranes and a precursor of steroid hormones and bile acids (nelson and cox 2008). its homeostasis is maintained by a complex regulatory network that balances its biosynthesis, consumption, and transport, preventing its accumulation (voet et al. 2013). increased levels of circulating cholesterol, known as hypercholesterolemia, is a strong risk factor in the development of cardiovascular diseases, such as atherosclerosis (voet et al. 2013). cholesterol biosynthesis involves several enzymatic reactions (bloch 1965). one e n z y m a t i c r e a c t i o n i s t h e n a d p h d e p e n d e n t r e d u c t i o n o f β h y d r o x y β methylglutaryl-coenzyme a (hmg-coa) to mevalonate by the enzyme hmg-coa reductase (ec 1.1.1.34). this reaction is considered as the rate-limiting step and the point of regulation in the pathway. the rate of cholesterol synthesis is regulated by subjecting hmg-coa reductase to competitive inhibition, allosteric effects, and hormonal control (voet et al. 2013). one way to lower cholesterol levels is to inhibit the activity of hmg-coa reductase with statins (endo and hasumi 1993). however, adverse effects from statin usage, such as liver damage, have been reported (golomb and evans 2008), underscoring the need for new cholesterol-lowering drugs that are more potent but with minimal adverse effects. plants are excellent sources of bioactive compounds due to their natural abundance and availability. the determination of plant-derived compounds and their pharmacological screening provide a basis for drug discovery (palvai and urooj 2014). previous studies have shown that plant extracts are potential sources of hmg-coa reductase inhibitors (gholamhoseinian et al. 2010; reddy et al. 2012; iqbal et al. 2014). short communication potential cholesterol-lowering activity of selected plant extracts 84 pouteria campechiana from the family sapotaceae is commonly known as canistel and locally referred to as tiesa. ethyl acetate extracts of p. campechiana contain six stilbenes and six flavonoid gylcosides (chichioco-hernandez et al. 2008). the fruit extracts exhibited antioxidant and hepatoprotective properties (aseervatham et al. 2014). antioxidant properties were also reported for the pulp and peel extracts of p. campechiana leaves (kong et al. 2013). barringtonia asiatica of family lethycidaceae or f ish poison tree is locally known as botong. the uses of b. asiatica in traditional medicine include the treatment of stomachache and rheumatism with its heated leaves, and the removal of intestinal worms with its seeds (wild fact sheets 2013). the whole tree is known to contain poisonous saponin. two major saponins and a triterpene ester saponin were isolated from the seeds, while a new triterpene was isolated from a freeze-dried bark (herlt et al. 2002; rumampuk et al. 2003; ragasa et al. 2012). using brine shrimp hatchability and lethality assay, which may correlate with cytotoxic activity, the fruit extracts exhibited higher activity than the leaf extracts (mojica and micor 2007). the saponins showed high antifeedant activity toward epilachna sp. (herlt et al. 2002). the freeze-dried bark extract also exhibited slight antimicrobial activities against candida albicans, staphylococcus aureus, and pseudomonas aeruginosa (ragasa et al. 2012). vitex parviflora of family verbenaceae is commonly known as molave. the distribution of v. parviflora is documented in the philippines and southeast asia. traditional uses of v. parviflora include the use of its bark extract in wounds, poison, diarrhea, jaundice, and dropsy (orwa et al. 2009). phytochemical screening of its methanol extract yielded positive results for alkaloids, flavonoids, terpenoids, phenolic compounds, and saponins. moreover, the extract also exhibited gastroprotective properties in ethanol and aspirin-induced models in mice (tarin and chichiocohernandez 2012). antimutagenic properties were also reported for phytol, lupeol, β-amyrin, sitosterol, and stigmasterol isolated from its ethyl acetate extract (ragasa et al. 2003). antidesma bunius from the family euphorbiaceae or currant tree is locally known as bignay. the a . bunius tree grows in china, india, southeast asia, and australia. the leaves of a. bunius are sudorif ic and used to remedy snakebites (morton 1987). the fruits of a. bunius were reported to contain three flavonoids, namely catechin, procyanidin b1, and procyanidin b2 (butkhup and samapito 2008). high phenolic content and antioxidant and antimicrobial activities were observed in its mature fruits (lizardo et al. 2015). methanol extracts of its fruits and leaves were active in r.r. isah and c.l. chichioco-hernandez 85 brine shrimp and hatchability assay (micor et al. 2005). the leaves were found to contain the polyphenols gallic acid, ferrulic acid, ellagic acid, corilagin, vicinin ii, and amentoflavone, and its extracts were shown to possess antioxidant and hepatoprotective properties (kassem et al. 2013). the aqueous ethanol (80%) extracts of the leaves also inhibited α-glucosidase activity (lawag et al. 2013). the leaves and bark were also reported to contain dammara-20,24-dien-3β-ol, friedelan-3β-ol,friedelin, and β-sitosterol (hui and sung 1968). diospyros blancoi of family ebenaceae or velvet apple is locally known as mabolo (fruit) and kamagong (tree). the tree is native to the philippines, and was introduced in southeast asian islands, as well as in the united states. in folklore, the mabolo fruit has been used for the treatment of diarrhea and wounds, while the other parts have been utilized for the treatment of respiratory diseases and skin ailments (morton 1987). the volatile components of the mabolo fruit were studied via gc and gc/ms and 96 compounds were identif ied, including benzyl butyrate, butyl butyrate, and cinnamyl butyrate (pino et al. 2008). the ethyl acetate acetate extract of the leaves yielded bioactive terpenes—isoarborinol methyl ether, α-amyrin palmitate, α-amyrin palmitoleate, β-amyrin palmitate, β-amyrin palmitoleate, and squalene. isoarborinol methyl ether displayed signif icant antimicrobial activity, whereas β-amyrin palmitoleate showed signif icant antimicrobial, analgesic, and anti-inflammatory activities (ragasa et al. 2009). phytochemical screening of the ethanol extract of the leaves revealed the presence of tannins and alkaloids. the study also reported significant antioxidant, antidiarrheal, antimicrobial, and cytotoxic activities for the ethanol extract of the leaves (howlader et al. , 2012b). a similar study also reported the presence of tannins, alkaloids, reducing sugars, and gums in the ethanol extract, which also possessed antidiarrheal activity (hossain et al. 2012). in another study, the methanol extract of the leaves revealed positive results for the presence of alkaloids, flavonoids, tannins, sugars, and gums, and possessed signif icant free radical scavenging activity and dose-dependent antidiarrheal activity (howlader et al. 2012a). another report also suggested signif icant dpph and hydroxyl-scavenging activities of the ethanol extract of mabolo leaves (lee et al. 2006). methanol extract of the leaves exhibited signif icant anti-asthma effects in murine model of allergic airway inflammation (lee et al. 2 0 1 2 ) . in this study, the hmgcr inhibitory actions of p. campechiana, b. asiatica, v. parviflora, a. bunius, and d. blancoi extracts were evaluated using an established assay protocol. potential cholesterol-lowering activity of selected plant extracts 86 collection of samples the leaves of p. campechiana, b. asiatica, v. parviflora, a. bunius, and d. blancoi were collected within the university of the philippines diliman campus and submitted to the dr. jose vera santos herbarium, institute of biology, up diliman for verif ication. preparation of plant extracts leaves of good condition were washed with water and air-dried. the leaves were homogenized using a blender, weighed, and soaked in distilled methanol. the methanol extracts were f iltered and concentrated in vacuo at 40°c using a rotary evaporator (ika® rv 10). the resulting methanol extracts were dissolved in 200 ml or 250 ml distilled water (depending on the weight of extract), and exhaustively partitioned between distilled water and hexane at a volume ratio of 1:2. the layers were allowed to settle and the hexane extract was collected. the aqueous layer was partitioned with ethyl acetate (1:2) and the procedure was repeated. the hexane and ethyl acetate extracts were concentrated in vacuo at 40°c. the aqueous layer was lyophilized to concentrate the extract. hmg-coa reductase inhibitory assay the hmg-coa reductase assay kit was purchased from sigma-aldrich®. the kit includes the enzyme hmg-coa reductase, the substrate hmg-coa, nadph, and the inhibitor solution (pravastatin). the enzyme, substrate, and nadph were prepared with the buffer to make stock solutions of 0.12μm, 150 μm, and 405 μm, respectively. in 1.5 ml eppendorf tubes, 1.5 mg each of the methanol, hexane, ethyl acetate, and aqueous extracts were dissolved in 10% dmso and 90% buffer (ph 7.4 0.1 m phosphate buffer with 0.12 m kcl, 0.001 m edta , and 0.05 m dithiothreitol) to make 1 mg/ml samples. two trials of four replicates for each extract, including the blank control, solvent control, and positive control, were prepared in a 96-well plate. each reaction well contains the following: 185 ml buffer, 50 ml nadph, 50 ml hmg-coa, 10 ml of enzymes, and 5 ml of either the plant sample, solvent control, or positive control. the f inal concentrations of the sample/pravastatin, enzyme, substrate, and nadph were 0.0167 mg/ml, 0.004 μm, 25 μm, and 67.5 μm, respectively. r.r. isah and c.l. chichioco-hernandez 87 in each designated well, either the plant sample, pravastatin, or solvent was f irst added, followed by the buffer, nadph, and hmg-coa. the microplate was incubated at 37°c for 5 minutes, after which 10 μl of hmg-coa reductase was added. the absorbance was read at 340 nm for every 15 seconds (up to 10 minutes) using the thermo scientif ic multiskan™ go microplate spectrophotometer. percent inhibition was calculated using the equation below, where is the rate of change in absorbance. average values and standard deviations were reported. (1) results and discussion the yields of the methanol extracts of the f ive plants are repor ted in table 1. it should be noted, however, that yields are inconclusive since the plants were not exhaustively rewashed with methanol. % ∆ ∆ δ δ ∆ ∆ 0   p. campechiana 302.54 36.67 12.12 71.22±0.30 b. asiatica 265.56 26.88 10.12 32.61±9.21 v. parviflora 253.96 23.45 9.23 41.40±4.73 a. bunius 191.29 9.50 4.96 93.21±2.13 d. blancoi 92.98 22.77 24.49 96.17±4.69 pravastatin (positive control) 90.94±0.37 plant/control sample (g) methanol extract (g) yield (%) % inhibition table 1. percent yield of methanol extracts and their respective percent inhibition results of the hmg-coa reductase inhibitory assay using the methanol extracts are shown in table 1. p. campechiana, a . bunius, and d. blancoi exhibited signif icant inhibition of hmg-coa reductase activity since their percent inhibition values are greater than 50%. a. bunius, and d. blancoi were selected for further studies due to the greater degree of inhibition they displayed. table 2 shows the percent inhibition of hexane, ethyl acetate, and aqueous extracts of a. bunius and d. blancoi. the par titioned extracts exhibited signif icant inhibition of hmgcr, with values ranging from 89.19% to 97.74%. the extracts of a . bunius and d. blancoi had potential cholesterol-lowering activity of selected plant extracts 88 inhibition values greater than the positive control (pravastatin). the inhibitory activity of the two plants may be attributed to the secondary metabolites found in the extracts. the a. bunius extracts have been reported to contain flavonoids and phenolic compounds (butkhup and samappito 2008; lizardo et al. 2015), while d. blancoi extracts were shown to contain alkaloids and flavonoids (howlader et al. 2012). phenolic compounds identif ied from citrus maxima peels have been shown to inhibit hmg-coa reductase activity (ademosun et al. 2016). similarly, phenolic compounds from grapefruit peels inhibited hmg-coa reductase activity (ademosun et al. 2015). citrus bergamia juice contains a high percentage of flavonoids and the rare flavonoids brutieridin and melitidin (janda et al. 2016). brutieridin and melitidin were subjected to computational studies, which revealed that the two molecules bind eff iciently at the catalytic site of hmg-coa reductase (leopoldini et al. 2010). plant extracts are great sources of bioactive compounds. it is recommended that the phytochemicals from of a. bunius and d. blancoi responsible for the hmg-coa reductase inhibitory activity be isolated and identif ied for fur ther studies. additionally, cell lines, such as caco-2 cell lines, and in vivo animal models should be used to predict absorption. a. bunius 89.19±0.55 95.54±0.35 96.53±0.55 d. blancoi 91.10±0.17 97.74±0.78 95.15±0.15 pravastatin 84.38±0.28 sample hexane extract ethyl acetate extract aqueous extract table 2. average percent inhibition ± sd of hexane, ethyl acetate, and aqueous extracts of a. bunius, and d. blancoi acknowledgement this research was partially funded by the department of science and technology through the philippine council for health research and development. references ademosun ao, oboh g, passamonti s, tramer f, ziberna l, boligon aa , athayde ml. 2015. phenolics from grapefruit peels inhibit hmg-coa reductase and angiotensin-i converting enzyme and show antioxidative properties in endothelial ea.hy 926 cells. food science and human wellness. 4(2):80-85. r.r. isah and c.l. chichioco-hernandez 89 ademosun ao, oboh g, passamonti s, tramer f, ziberna l, boligon aa. 2016. modulation of hmg-coa reductase and glutathione-linked enzymes and protection against prooxidant induced oxidative damage in colon (caco-2) cells and rat colon homogenates b y p h e n o l i c e x t r a c t s f r o m s h a d d o c k ( c i t r u s m a x i m a ) p e e l s . j o u r n a l o f a p p l i e d biomedicine [internet]. 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[ i n t e r n e t ] 2 0 1 3 . [ c i t e d 2 0 1 5 j u n e 1 ] . a v a i l a b l e f r o m : h t t p : / / w w w.wildsingapore.com/wildfacts/plants/coastal/barringtonia/asiatica.htm. _____________ raffi r. isah is a bs chemistry graduate of the college of science, up diliman. christine c. hernandez <cchernandez@up.edu.ph> is an associate professor and a principal investigator of the bioorganic and natural products laboratory of the institute of chemistry, university of the philippines diliman. 96 information for authors 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the office of the vice chancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles and short communications may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out. the letter to the editor usually contains these: that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1" margins on all sides. each page of the manuscript must include continuous line numbers in the margin. all pages should be numbered consecutively on the upper right hand corner of the page. 7. page 1 should contain the article title, author(s), affiliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 97 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. the author must also submit a layman’s abstract of not more than 200 words. 9. the paper should be organized as follows: abstract and layman’s abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, figures, tables, and figure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be defined at first mention 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the first author’s name plus “et al.” need to be given. references in the text should follow the council of science editors (cse) scientific style and format, 7th edition, 2006. examples: articles from journals: print ( section 29.3.7.1 p. 518-527) format: author(s). date. article title. journal title. volume(issue):location example: smart n, fang zy, marwick th. 2003. a practical guide to exercise raining for heart failure patients. j card fail. 9(1):49-58. articles from journals: online (section 29.3.7.13 p. 557-558) format: author(s) of article. date of publication. title of article. title of journal (edition) [medium designator]. 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102 • short communications are reports of significant new data arising from problems with narrow, well defined limits before broader studies are completed; and results have not been published in print elsewhere, except as partial communications or posters in conference proceedings; • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; • abstracts will be required on submission but not to be part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages) in length with simple layout, a maximum of two tables and two figures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors may opt to submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor-in-chief science diliman office of the vice-chancellor for research and development university of the philippines lower ground floor, phivolcs bldg., c.p. garcia avenue up campus, diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. 8-shortcom-masangcay-pop structure.pmd population structure of the krill prey 74 science diliman (january-june 2018) 30:1, 74-81 population structure of the krill prey of the spinetail devil ray mobula japanica (chondrichthyes, mobul idae) from southeastern bohol sea, phil ippines shirlamaine irina g. masangcay caraga state university ephrime b. metillo* mindanao state university-iligan institute of technology shuhei nishida university of tokyo _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online in our recent study on the feeding biology of the spinetail devil ray mobula japanica müller and henle, 1841 from butuan bay, southeastern bohol sea (figure 1) (masangcay et al. 2018), we observed the predominance of euphausiids or krill in the stomach content of the ray. the norwegian term ‘krill’ refers to holoplanktonic shrimp-like crustaceans that belong to the order euphausiacea (mauchline 1980). figure 1. geographical location of butuan bay in northeastern mindanao (southeastern bohol sea) and collection sites of landed spinetail devil ray mobula japanica off the municipality of carmen (triangle), nasipit (square), buenavista (dot), cabadbaran (diamond), and tubay (star) in the province of agusan del norte. inset is the map of the philippines with the study site enclosed in a square. s.i.g. masangcay et al. 75 having an adult body size range of about 1 to 6 cm long, the 86 known species and 11 genera of krill (baker et al. 1990) usually form dense swarms, and dwell exclusively in oceans and seas at depths of 73 to 220 m (mauchline 1980). they generally graze upon phytoplankton and detritus, and prey on small zooplankton at surface layers, and through their instinctive diel vertical migration, krill help shunt tons of carbon down to the greater depths of the ocean (sameoto et al. 1987). krill are widely known as food of baleen whales, seals, f ish, and marine birds (mauchline 1969, 1980), but recent studies include whaleshark and manta and devil rays in the list of animals feeding on krill (notarbatolo-di-sciara 1988; wilson et al. 2001; wilson and newbound 2001; jarman and wilson 2004; sampson et al. 2010; masangcay et al. 2018). population structure analysis, which is a major aspect of demography, provides data on the proportions of different life stages, sex ratio, individual size-frequency, and reproductive state of individuals—information that have important ramif ications on the species interactions and the population itself (ranta et al. 2006). demographic studies of tropical krill species are very limited, but most warm water species are small, mature and breed in 10-12 months, reach 16 to 20 mm in total length of inf inity, and a life span of 1 year (mauchline 1980). krill are diff icult to collect using conventional conical plankton nets (masangcay 2016), but their high abundance in the stomach of m. japanica provided the opportunity to characterize the size structure of intact krill individuals from butuan bay. the objective of this study is to describe the population structure of these ingested krill from january to may 2016, during the peak season period of m. japanica f ishing in butuan bay, southeastern bohol sea, philippines. krill samples were obtained from the stomachs of bycatch m. japanica individuals (n = 16; for ray body lengths, see masangcay et al. 2018) caught monthly from january to may 2016 in butuan bay, southeastern bohol sea, philippines (figure 1). krill were carefully identif ied following the descriptions of weigmann (1971) and brinton (1975). in order to determine krill size-structure, a sub-sample was randomly collected from the entire m. japanica stomach content samples. since stomach samples contain >1000 individuals per stomach, 1/10th sub-sample was obtained for krill size-structure analysis (brinton 1975). intact individuals of juvenile and adult male and female stages were meticulously sorted out since partially digested individuals were common in the sub-samples. we made sure that, apart from juveniles, at least 50 males and 50 females were obtained from the sub-sample, with the total number of sorted individuals greater than the 100 mentioned by watkins (1986). krill were measured individually based on their total length (from the medial tip of the rostral plate to the end of the telson excluding any setae) using a vernier caliper with an accuracy of 0.01 mm (juáres et al. 2017). the size of every krill individual was recorded, and size-frequency histograms were constructed population structure of the krill prey 76 for different body length categories. variation of sizes of pseudeuphausia latifrons (p. latifrons) in the stomach population structure analysis based on the influence of size, sex, and sampling month was examined using multifactorial anova (spss 2 0 0 2 ) . we identif ied the krill as pseudeuphausia latifrons (g.o. sars, 1883) (figure 2). the population structure of ingested p. latifrons from each of the 16 ray individuals are shown in figure 3. krill total lengths ranged between 4.0 mm and 10.9 mm for figure 2. p. latifrons (g.o. sars, 1883), the krill species dominating the ingested prey of the spinetail devil ray mobula japanica from butuan bay, southeastern bohol sea. a: adult female (f ) and male (m) individuals. lateral view of anterior cephalothorax of male (b) and female (c) showing the frontal plate (white arrows). d: lateral view of the lappet (l) on the left antennular peduncle of an adult female. e: dorsal view of frontal plate (r) in female. f: left adult male petasma with processes (black arrows) on the inner lobe (il); ol – outer lobe. g: right adult male petasma with processes on the inner lobe (il); ol – outer lobe. s.i.g. masangcay et al. 77 both sexes. individuals that were within the range of 4.0 mm – 6.9 mm were classif ied as juveniles, while those larger than 7.0 mm were adults. sizes of prey differed according to the monthly collection of predator (f = 5.52, df = 24, p = 0.00), with males larger than females (f = 2.25, df = 6, p = 0.04). ingested krill between january to early march ranged between 4.0 mm and 9.9 mm, and the juveniles were abundant until late march. towards the end of march, the sizes of krill were def initely larger by 1 mm for both sexes (f = 21.83, df = 6, p = 0.00), and a switch in frequency figure 3. length-frequency distribution of p. latifrons (g.o. sars, 1883) collected from the stomach contents of the spinetail devil ray mobula japanica individuals (a-p) from butuan bay, southeastern bohol sea, philippines. solid bar males; open bar females. dates indicate day of collection of the spinetail devil ray. population structure of the krill prey 78 values with less juvenile krill and more adult krill was observed (f = 20.72, df = 1, p = 0.00). by april and may, both adult male and female prey were noticeably larger, ranging between 5.0 mm and 10.9 mm, than the preceding months which visibly show fewer juvenile prey (f = 18. 06, df = 1, p = 0.05). the length-frequency histograms clearly show changes in the size-structure of p. latifrons, reflecting the individual growth from january to may (figure 3). except in january when there were no signif icant differences in krill sizes between sexes, we observed that male krill were generally larger than female, which agree with the f indings of hanamura et al. (2003) on stranded p. latifrons in western japan. the histograms show a decrease in the number of juveniles as evidenced by the absence of krill of the smallest length interval and only a few second to the smallest length interval during the warm months of april and may. there was also an increase in the number of adult individuals from the largest length interval in the same warm months. the data on krill lengths show that, towards the warm month of april, body size of krill was bigger and the number of egg-carrying females was greater. this can be related to reproduction, as the tropical krill p. latifrons tends to be smaller before warmer months (april and may in butuan bay) and becomes larger in warmer months due to mature females carrying eggs (wilson et al. 2003). if we assume that the minimum maturity length of p. latifrons is at 8mm (wilson et al. 2003), our data indicate that the breeding of this krill species occurs from january to may, with the peak of breeding season occurring in the warmer months of april and may, on account of the many large egg-carrying females during these months. moreover, for p. latifrons, larger females were found to carry more eggs (wilson et al. 2003). our f indings seem to slightly differ from the report that p. latifrons peak in spawning at the end of the northeast monsoon in vietnam waters in the south china sea (brinton 1975). our study provides evidence that spawning can also occur during the intermonsoon months of april and may for p. latifrons in butuan bay. the temporal change in the size structure of spinetail devil ray-ingested populations of p. latifrons indicates that juvenile and adult male and female individuals are present from january to may in butuan bay. while juveniles became rare, the largest male and female individuals appeared during the warmer months of april and may. these females bore eggs, indicating spawning in april and may. krill species p. latifrons dominated the ingested food of m. japonica from january to may in butuan bay. the january to may window is within the fishing season of the spinetail devil ray m. japanica in bohol sea (alava et al. 2002; acebes 2013; freeman 2014) and butuan bay (metillo and masangcay 2016), which fall on s.i.g. masangcay et al. 79 september to may, with peak season during february to april. it remains to be studied if there is a link between the temporal pattern of p. latifrons abundance and the upwelling events associated with a strong northeast monsoon and the estuarine plume formation during highest river discharge in butuan bay (cabrera et al. 2011; villanoy et al. 2011). acknowledgements we are very grateful for the f inancial support of the department of science and technologyscience education institute, the off ice of the vice-chancellor for research and extension of mindanao state university-iligan institute of technology, the manta trust uk, and the japan society for the promotion of science (jsps) (the asian core and the core-to-core programs); the f ield assistance by local fisherfolks of buenavista, agusan del norte; the copyediting help of dr. mtrd sanchez-metillo; and constructive comments of anonymous reviewers. references acebes jmv. 2013. hunting “big fish”: a marine environmental history of a contested f ishery in the bohol sea [doctoral disser tation]. western australia: murdoch university. alava mnr, dolumbalo erz, yaptinchay aa , trono rb. 2002. fishery and trade of whale sharks and manta rays in the bohol sea, philippines. in: fowler sl, reed tm, dipper fa , ed i to r s . e l a s m o b r a n c h b i o d i v e r s i t y, co n s e r v a t i o n a n d m a n a g e m e n t : pr o ceed i n g s of the international seminar and workshop; sabah, malaysia: occasional paper of the iucn species survival commission no. 25. p. 132-148. baker a, de c, boden bp, brinton e. 1990. a practical guide to the euphausiids of the world. london: british museum (natural history). 96 pp. brinton e. 1975. euphausiids of the southeast asian waters. scientif ic results of marine investigations of the south china sea and the gulf of thailand 1959-1961, volume 4. usa: university of california. naga repor t . p. 1-287. cabrera oc, villanoy cl, david lt, gordon al. 2011. barrier layer control of entrainment and upwelling in the bohol sea, philippines. oceanography. 24:130-141. f r e e m a n a l . 2 0 1 4 . m o b u l i d a e f i s h e r y i n b o h o l , p h i l i p p i n e s : a n a s s e s s m e n t of i t s sustainability [mres thesis]. uk: swansea university. h a n a m u r a y, s a i to n , h a y a s h i k i . 2 0 0 3 . s h o r e s t r a n d i n g o f t h e n e r i t i c e u p h a u s i i d pseudeuphausia latif rons (g.o. sars, 1883) in western japan. crustaceana. 76(9):11471152. population structure of the krill prey 80 jarman sn, w ilson sg. 2004. dna-based species identif ication of krill consumed by whale sharks. journal of fish biology. 65:586-591. juáres ma, casaux r, corbalán a, blanco g, pereira ga, perchivale pj, coria nr, mercedes santos mm. 2018. diet of adélie penguins (pygoscelis adeliae) at stranger point (25 de mayo/king george island, antarctica) over a 13-year period (2003–2015). polar biology. 4(2):303-311. masangcay si. 2016. feeding biology of devil rays (mobulidae, chondrichthyes) in butuan bay, nor theastern mindanao, philippines [msc thesis]. iligan city, philippines: mindanao state university-iligan institute of technology. masangcay si, metillo eb, hayashizaki k, tamada t, nishida s. 2018. feeding habits of mobula japanica (chondrichthyes, mobulidae) in butuan bay, mindanao is. , philippines. science diliman. 30(1):24-44. mauchline j, fisher lr. 1969. the biology of euphausiids. advances in marine biology. 7:1-454. mauchline j. 1980. the biology of mysids and euphausiids. advances in marine biology. 18:1-681. metillo eb, masangcay sig. 2016. rapid assessment of rays (mantas and mobulids) in sulu sea and bohol sea. technical report submitted to the manta trust fund, uk; save our seas and mindanao state university-iligan institute of technology. notarbartolo-di-sciara g. 1988. natural history of the rays of the genus mobula in the gulf of california. fishery bulletin. 86(1):45-66. ra n t a e , l u n d b e r g p, ka i t a l a v. 2 0 0 6 . e co l o g y of p o p u l a t i o n s . lo n d o n : c a m b r i d g e university press. 373 p. sameoto dl, guglielmo l, lewis mk. 1987. day/night ver tical distribution of euphausiids in the eastern tropical pacif ic. marine biology. 96:235-245. sampson l, galván-magaña f, de silva-dávila r, aguíñiga-garcía s, o’sullivan jb. 2010. diet and trophic position of the devil rays mobula thurstoni and mobula japanica as inferred from stable isotope analysis. journal of the marine biological association of the united kingdom. 90(5):969-976. spss. 2002. spss for windows version 11. chicago, il: spss inc. v illanoy c, cabrera o, yniguez a, camoying m, de guzman a, david l, flament p. 2011. m o n s o o n d r i v e n c o a s t a l u p w e l l i n g o f f z a m b o a n g a p e n i n s u l a , p h i l i p p i n e s . oceanography. 24(1):156-165. watkins jl. 1986. variations in the size of antarctic krill, euphausia superba dana, in small swarms. marine ecology progress series. 31(1):67-73. weigmann r. 1971. eine isolierte population von pseudeuphausia latifrons (crustacea: euphausiacea) im persischen golf. marine biology. 8(4):351-355. s.i.g. masangcay et al. 81 wilson sg, newbound dr. 2001. two whale shark faecal samples from ningaloo reef, western australia. bulletin of marine science. 68(2):361-362. wilson sg, meekan m, carleton j, stewar t t, knott b. 2003. distribution, abundance and reproductive biology of pseudeuphausia latifrons and other euphausiids on the southern nor th west shelf, western australia. marine biology. 142(2):369-379. wilson sg, pauly t, meekan mg. 2001. daytime surface swarming by pseudeuphausia latif rons (crustacea, euphausiacea) o ff ningaloo reef, western australia. bulletin of marine science. 68(1):157-162. _____________ shirlamaine irina g. masangcay is a graduate of b.s. marine biology from mindanao state university-iligan institute of technology. she graduated m.s. marine biology from mindanao state university-iligan institute of technology on time as a dostasthrdp scholarship in 2016, and was hired immediately after graduation as instructor at the caraga state university, butuan city, philippines. ephrime b. metillo <ephrime.metillo@g.msuiit.edu.ph> is b.s. zoology graduate at the mindanao state university marawi city. he was university research assistant at the marine science institute of the university of the philippines diliman before doing a straight ph.d. program at the university of tasmania at hobart, australia under the australian international development assistance bureau (aidab) equity and merit scholarship scheme. he is now professor at the mindanao state university-iligan institute of technology, iligan city, philippines. shuhei nishida is a recently retired professor at the atmosphere and ocean research institute of the university of tokyo, japan. he has published more than 110 papers in the f ield of marine biology and became editor of the springer journal marine biology. his main f ield of interest is marine zooplankton biology and ecology. fishery-villarta.pmd fishery of the short-necked clam paphia undulata 43 fishery of the short-necked clam paphia undulata in southern negros occidental, central philippines* karen a. villarta* and annabelle g.c. del norte-campos marine biology lab, college of arts & sciences university of the philippines visayas, miagao, iloilo *corresponding author: karenvillarta@yahoo.com abstract introduction like many finfish and invertebrates, shellfish are known to be good sources of protein for most of the coastal communities in the country. they also contribute to the total fisheries production in the country. however, these resources, including bivalves, are harvested at an increasing rate in most of our coastal areas, an obvious result of the swelling population in coastal areas as well as the constant demand of fishery resources in both the local and export markets. consequently, bivalve aquaculture experiments are being attempted in various places to offset the exhaustion of natural beds by overexploitation (fao, 1998). it is known that some bivalve species are popular delicacies served not only this study documents the fishery of the short-necked clam paphia undulata in coastal waters of southern negros occidental. catch and effort estimates were determined based on daily records of compressor divers gathered between february-july 2008 in himamaylan city and july 2008-may 2009 in the town of hinigaran. fishing and marketing practices in both areas were also documented and population biology information noted. compared to earlier conditions, present fishing patterns show a worsened stage of overexploitation primarily characterized by collection of predominantly small and immature (mostly <45 mm shell lengths) sizes. intensity/duration and location of fishing also varied due to both abundance and demand factors. the difference in sizes of clams and the varying fishing durations in each area suggest a non-uniform pattern of settlement resulting most likely from differential larval recruitment, the likely factors causing the local boom and bust fishery. the larger and long term extent of the effect of these factors can only be further investigated by parallel 2-3 year fishery-dependent and -independent surveys. keywords: paphia undulata, catch, catch rates, central philippines locally but in neighboring countries as well. the shortnecked clam paphia undulata (figure 1), locally known as “nylon shell” in western visayas is a commercially important invertebrate resource in the philippines and is in fact one of the most sought after bivalve species in the region. it is usually sold live or chilled/frozen in the local markets while its meat is processed for the export trade. fao (1988) reported that paphia undulata constituted about 4.1% of the total world and asian landings (excluding japan) of the most important mollusk species in asia for the year 1986 alone. however, there is a lack of parallel information for recent years thus the values can not updated. in negros occidental, particularly in the towns of hinigaran and himamaylan, p. undulata is harvested from mudflats through compressor diving. agasen et al. (1998) did an assessment of the species in the same science diliman (january-june 2010) 22:1, 43-51 *this paper is part of the proceedings of the 10th national symposium on marine science held in davao city in october 2009. villarta, k.a. and a.g.c. del norte-campos 44 area and concluded that it was overexploited. the fishery is still on-going in the area although there is a rather irregular seasonality in fishing due to dependence on stock abundance. a re-assessment of its status after more than a decade is needed considering that the importance of management interventions, on top of its over-exploitation, is still not properly recognized. thus the objective of this paper was to examine the current status of the fishery by determining catch volume, catch rates, and their seasonality, as well as annual income, and to use these as bases for the assessment. a separate paper (del norte-campos & villarta, 2010) compares the status of the stocks 13 years ago (agasen et al. 1998) and the present, using population biology information. materials and methods the study was conducted in himamaylan and hinigaran, southern negros occidental, central west visayas, philippines (fig. 2). compressor diving operations for nylon shell are not continuous throughout the year, but shift from one locality to the other, depending on availability of marketable sized shells, without regular seasonality. hence catch data were collected only during the months of operation in the respective localities, i.e. february to july 2008 in himamaylan and july 2008 to may 2009 in hinigaran. for both areas, estimates of the total volume of catch (kg) and the corresponding fishing effort (hours spent fishing) per figure 1. the short necked clam paphia undulata (born, 1778) (mollusca, pelecypoda: veneridae), locally known in western visayas as the “nylon shell”. fisher were determined based on daily records of compressor divers, while information on selling prices (php), manpower and other fishing practices were obtained through informal interviews with the traders, vessel owners/operators and divers. the catch per unit effort (kg hr-1) was computed by dividing the catch (kg) with the fishing effort (h). daily catches were averaged and then multiplied by the number of days fished per month to get estimates of the total monthly catch (kg) per diver. total monthly catches were then multiplied by the actual number of months of diving operations to get the annual catch (kg) for each diver. this was then multiplied by the total number of divers to estimate the total annual catch of the fishery in both areas. to determine the annual value (php) of the nylon shell catch for each area, the total annual catches were multiplied by the corresponding lower and upper limit of their price range during the time of survey in himamaylan and hinigaran, respectively. from this, the income per diver was also estimated by dividing the annual value by the number of divers in each area and dividing this further with the number of days fished for the year. figure 2. location of the study area in southern negros occidental. science diliman (january-june 2010) 22:1, 43-51 fishery of the short-necked clam paphia undulata 45 results and discussion overview of the fishery nylon shell burrow deep into the muddy substrate and are harvested by compressor divers in depths ranging from 4 to 8 fathoms (7 to 14 meters) along the coast of negros. compressor diving, also known as hookah, is also used in other parts of the country for gathering paphia spp, particularly in leyte, leyte (cesar et al., 2003) and bolinao, pangasinan (pastor and juiniomeñez, 2003). in southern negros occidental compressor diving for p. undulata is conducted during the daytime. majority of the motorized boats leave around 0600h and return to land their catches around noon to early afternoon. each boat usually has 3 divers and 1 compressor operator or lineman. the landing site in himamaylan is located in sitio batang, brgy talaban. in hinigaran, the 2 landing sites are located in the coastal barangays of tagda and gargato. buyers typically finance the operations and dictate the selling price of the nylon shells from as low as php 40 to as high as php 80 per kilo in himamaylan, and from php 10 to 30 per kilo in hinigaran. the disparity in selling price is discussed below. nylon shells bought from the divers are sorted mostly by women in the landing sites. shells 2 inches (50mm) in length are packed live and shipped for export to taiwan. a taiwanese importer accepts up to 4 tons daily (except sundays) of live nylon shells for export. the amount in excess of 4 tons, if any, is processed as table 1. mean daily diving hours (per diver), number of diving days per month, mean daily catch (kg diver-1), mean catch rate (kg diver-1hr-1) and mean monthly catch (kg diver-1) in himamaylan, negros occidental. meat along with the meat of shells < 2 inches in length for export to japan. the compressor diving fishery in himamaylan started operations in february 2008 and lasted for only six months. the same group of divers and buyers then moved north to a neighboring town, hinigaran, where diving operations started in july 2008 and ended in may 2009 (11 months). catch and catch rates himamaylan based on daily records of compressor divers from february to july 2008, mean daily catch ranged from 4.7 to 17.7 kg per diver, with an average of 4.3 diving hrs per day (table 1).the highest computed catch rate (3 kg diver-1 hr-1) was recorded in february while the lowest was observed in june (1.2 kg diver-1 hr-1). on the average, compressor divers go out to harvest nylon shells 27 days in a month. from these, the computed mean monthly catch per diver (kg) was also highest in february (459.5) and lowest in june (116.5). catch and catch rates decreased after february and continued to decline until june (figure 5). the fishery stopped sometime in july although total catch for this month increased from the previous month (june). this rise in total catch may be attributed to the smaller number of boats (lower fishing effort) fishing in july (figure 6), reducing competition for area covered by diving and increasing individual catch rates. science diliman (january-june 2010) 22:1, 43-51 > months mean # of divers surveyed mean daily diving hours diving days mean daily catch (kg diver-1) mean cpue (kg diver-1hr-1) mean monthly catch (kg diver-1) feb '08 41.0 6.1 26 17.7 3.0 459.5 mar 36.6 4.0 31 10.9 2.9 337.2 apr 40.0 3.8 30 8.0 2.2 239.9 may 27.8 3.8 22 5.6 1.5 122.8 jun 26.4 3.6 25 4.7 1.2 116.5 jul 19.5 4.1 27 9.8 2.2 264.0 overall mean 31.9 4.2 26.8 9.4 2.2 256.7 sd 8.63 0.95 3.31 4.68 0.69 130.78 villarta, k.a. and a.g.c. del norte-campos 46 catch of the same species (locally called kabloy) was also observed during the survey period. catch and value estimates based on the mean monthly catch of 256.7 kg/diver (table 1) and a total of six months of fishing operations in himamaylan, the total “annual” catch of p. undulata per diver in the area was estimated to be 1.5 mt (table 3). information provided by local enumerators indicates that there were about 150 divers operating in himamaylan during the months monitored. with this number of divers, the total annual catch for himamaylan during the study was 231.0 tons. using the lower and upper limits of the price range the total value of the catch per year would be between php 9.2 – 18.5 m. this translates to a gross income of between php 382.00 – 766.00 per diver per day. however, this would still be subject to at least 30% deduction for the boat operator’s share. fishing operations in hinigaran lasted for eleven months with a mean monthly catch of 572.1 kg/diver. this gave a total annual catch per diver of 6.3mt (table 3). information from enumerators provides a conservative hinigaran computed catch and catch rates from july 2008 to may 2009 for hinigaran are shown in table 2. mean daily catch and cpue were highest in october-december 2008 and lowest in april to may 2009. mean daily diving hours ranged from 4.0 to 5.4 with an overall mean of 4.9 hrs diving per day. diving days per month varied throughout the duration of the fishery and this ranged from 12 to 30 with an average of 25 ± 6.2 days per month. table 2 also shows the mean monthly catch per diver whereby highest values were also observed in the months of october, november and december 2008. lowest mean monthly catch was observed in april and may 2009. a decline in the catch was observed after december which continued decreasing in the following months until all local fishing operations ceased after may 2009 (figure 7). this period of operation indicates the absence of regular seasonality in the harvesting of nylon shell, and that effort is highest when other pressures, like demand for export, are present. this pattern was also observed in the study of agasen et al. (1998) in the same area, wherein a decline in catch rates was observed from july 1996 to february 1997. in a similar study in bolinao, pangasinan (pastor and juinio-meñez, 2003), a continued decline in the figure 3. size distribution of paphia undulata from data collected during compressor diving surveys in hinigaran and himamaylan, southern negros occidental, april 2008 – march 2009. science diliman (january-june 2010) 22:1, 43-51 fishery of the short-necked clam paphia undulata 47 (minimum) estimate of 120 divers. total annual catch for hinigaran during the study was about 755.2 tons, with a corresponding value ranging from php 7.6 – 22.7 m. this would result to an estimated gross income per diver of php 230 – 692 per day. the current estimate of total annual catch for hinigaran is 44.3% of the previous estimate (1356.3 mt) from agasen et al.(1998). this decline suggests a more serious situation of stock depletion since the negros stock was already considered overfished 13 years ago. this is further supported by estimates of exploitation rate (e=0.75) for recent years (del norte-campos & villarta, 2010). in addition, a comparison of the size distribution of shells clearly shows a shift to smaller sizes (mode ~ 45mm) in recent landings compared to those from 13 years ago (mode ~ 60mm) (agasen et al. 1998) (figure 4). this is especially significant since p. undulata attains sexual maturity at a size of about 45mm (nabuab et al. this volume). clearly, a substantial portion of landings from the current fishery consist of immature shells which have not been able to spawn yet. this is a condition that typically leads to reduced recruitment. limiting catches only to shells larger than 45mm will allow most of the stock to spawn, maintaining table 2. mean daily diving hours (per diver), number of diving days per month, mean daily catch (kg diver-1), mean catch rate (kg diver-1hr-1) and mean monthly catch (kg diver-1) in hinigaran, negros occidental. natural recruitment levels. these results provide guidelines that are of critical importance in saving what is left of the nylon shell fishery in the study area. the reason for the disparity in the price of nylon shell in the two localities is a result of the difference in sizes of the harvested shells in the two areas. figure 3 shows that the modal size of nylon shells harvested from himamaylan were from 55-60mm, while those from hinigaran were much smaller with a modal size from 40-50mm. hence the higher price commanded by larger shells in himamaylan outweighed the 3-fold difference in estimated total landings in hinigaran in terms of value (table 3). furthermore, an asymptotic length (l¥) of 79 mm was estimated by del norte-campos and villarta (2010) from the present data, which is lower than that (81.5 mm) estimated by agasen et al. (1998). this reflects the scarcity of bigger sizes in recent catches (figure 4) which again is consistent with worsening conditions in the fishery. apart from larger individuals in himamaylan, monthly catches from a parallel fishery-independent dredge survey (palla et al., in prep) showed that mean overall abundance of nylon shell was almost 12 times higher science diliman (january-june 2010) 22:1, 43-51 months mean # of divers surveyed mean daily diving hours diving days mean daily catch (kg diver -1) mean cpue (kg diver-1 hr-1) mean monthly catch per diver(kg) jul '08 14 5.1 22.0 21.5 4.2 472.6 aug '08 5 5.1 12.0 15.0 2.9 179.5 sep '08 7 4.0 17.0 23.8 6.1 404.5 oct '08 34 5.0 30.0 35.4 7.0 1062.4 nov '08 39 5.4 29.0 33.7 6.2 977.6 dec '08 34 5.4 30.0 33.4 6.2 1001.4 jan '09 36 5.1 22.0 25.9 4.9 570.2 feb '09 39 4.8 28.0 22.7 4.8 635.4 mar '09 45 4.5 31.0 18.4 4.1 571.7 apr '09 37 4.4 29.0 9.5 2.1 274.5 may '09 7 4.7 23.0 6.2 1.3 142.9 overall mean 27.1 4.9 24.8 22.3 4.5 572.1 sd 15.41 0.44 6.18 9.66 1.84 325.27 villarta, k.a. and a.g.c. del norte-campos 48 figure 4. shell length (mm) comparisons between agasen et al., (1998) and present data, covering only the months wherein fishing occurred. figure 5. mean daily catch (kg diver-1) and mean monthly catch (kg diver-1) in himamaylan, negros occidental from february to july 2008. science diliman (january-june 2010) 22:1, 43-51 fishery of the short-necked clam paphia undulata 49 figure 6. average number of boats that operated in himamaylan, negros occidental, from april to july 2008. 0 10 20 30 40 jul '08 a ug sep o ct nov d ec jan '09 feb mar a pr may months 0 200 400 600 800 1000 1200 mean daily catch m ean monthly catch m e a n d a il y c a tc h m e a n m o n t h ly c a t ch 0 10 20 30 40 jul '08 a ug sep o ct nov d ec jan '09 feb mar a pr may months 0 200 400 600 800 1000 1200 mean daily catch m ean monthly catch m e a n d a il y c a tc h m e a n m o n t h ly c a t ch figure 7. mean daily catch (kg diver-1) and mean monthly catch (kg diver-1) in hinigaran, negros occidental from july 2008 to may 2009. science diliman (january-june 2010) 22:1, 43-51 villarta, k.a. and a.g.c. del norte-campos 50 along the coast of hinigaran (mean = 76.9 ind/100m2) than in himamaylan (mean = 6.5 ind/100m2). this suggests that while p. undulata in the two localities belong to the same stock, recruitment is not spatially uniform and this eventually influences how the fishery operates in these localities. the much higher abundance of smaller (= younger) individuals in hinigaran indicates higher settlement (recruitment) in these waters. once shells reach marketable size, their much higher abundance in hinigaran attracts more fishing to the area, resulting in a shift of operations from himamaylan, as was observed in july 2008. heavy fishing may then exhaust this portion of the stock (in hinigaran) if allowed to continue without regulation. observations during the study period indicate that this is indeed what happens. when abundances in hinigaran become too low and unprofitable, the return of operations to himamaylan results in catching larger (=older) shells which had been allowed to grow the previous season. this explains the disparity in sizes and their corresponding prices as earlier discussed and also suggests that it is this portion of the stock that likely contributes to the bulk of spawning and recruitment the following year. thus, the extent and duration of fishing in hinigaran, may determine whether or not what is left in himamaylan is able to maintain a fishable stock every year. when heavy fishing disrupts reproduction and recruitment in all portions of the stock, this leads to extreme interannual variability in abundance, resulting in “boom and bust” fisheries. in this context, the uneven impact of fishing table 3. catch and value estimates for the short-necked clam paphia undulata in himamaylan and hinigaran, negros occidental. on nylon shell along the coast of southern negros occidental results in the shifting of operations from one locality to another, which is more preferable to situations with extreme highs and lows. because the above scenario implies a cycle of at least 2-3 years, there is a need for both fishery-dependent and – independent investigations covering such durations if we are to understand the dynamics of and to properly manage such targeted fisheries. acknowledgments this study was funded by the dost/pcamrd project “studies on the biology and fishery of the short-necked clam paphia udulata born, 1778 (mollusca, pelecypoda: veneridae) in negros occidental waters”. f. nabuab, l. manalo, r. palla and m. burlas provided field and lab assistance. w.l. campos and two anonymous referees gave valuable comments and provided insights for the improvement of the manuscript. references agasen, e.v., c.m. del mundo and g.o. matias. 1998. assessment of paphia undulata in negros occidental/ guimaras strait waters. j. shellfish res. 17(5): 1613-167. cesar, s.a. b.p. germano and j.l.f. melgo. 2003. preliminary results on the population, reproductive and fishery biology of the nylon shell, paphia textile (gmelin 1791), in leyte. upv j. nat. sci. 8:83-95 science diliman (january-june 2010) 22:1, 43-51 area mean monthly catch (kg/diver) months of operation total annual catch per diver (mt) total no. of divers total annual catch for the area (t) price (php lower & upper limit) total value of catch (m php) total no. of days fished income per diver per day (php) himamaylan 256.66 6 1.54 150 231 40-80 9.24-18.48 161 382-766 hinigaran 572.08 11 6.29 120 755 10-30 7.6-22.7 273 230-692 fishery of the short-necked clam paphia undulata 51 del norte-campos, a. and k.villarta. 2010. use of population paramters in examing changes in the status of short-necked clam paphia undulata born, 1778 (mollusca, pelecypoda: veneridae) in coastal waters of southern negros occidental. science diliman 21(1):pp-pp. fao species identification guide for fishery purposes. 1998. carpenter, k.e. and v.h. niem (eds.). the living marine resources of the western central pacific. vol. 1. seaweeds, corals, bivalves and gastropods. rome, fao: 1-686. fao status of mollusk culture in selected asian countries. 1988. http://www.fao.org/docrep/field/003/ab718e/ ab718e00.htm nabuab, f.m., fernandez l.f. and a.g.c. del norte-campos. in prep. reproductive biology of the short-necked clam paphia undulata (born 1778) from negros occidental waters. science diliman (in press) pastor, d.s and m.a. juinio-menez. 2003. boom and bust kabloy fishery – the bolinao experience. the philippine scientist. 40: 88-100. science diliman (january-june 2010) 22:1, 43-51 sdinside front cover-july-dec2018.pmd july-december 2018 • vol. 30 no. 2 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june and december) by the university of the philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief irene m. villaseñor, ph.d. university of the philippines associate editors jose maria p. balmaceda, ph.d. university of the philippines louis angelo m. danao, ph.d. university of the philippines carlos primo c. david, ph.d. university of the philippines christian n. della, ph.d. university of glasgow singapore alonzo a. gabriel, ph.d. university of the philippines arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines dennis i. merino, ph.d. southeastern louisiana university jonas p. quilang, ph.d. university of the philippines arnel a. salvador, ph.d. university of the philippines terence p. tumolva, d.eng. university of the philippines managing editor gonzalo a. campoamor ii, ph.d. university of the philippines editorial assistant narita e.c. de las alas layout artist dercylis g. mararac copyeditor sarah mae u. penir on the cover: teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus department of food science and technology school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus department of electrical and computer engineering the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheff ield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. department of physics university of warwick, united kingdom raul k. suarez, ph.d. department of zoology university of british columbia myra o. villareal, ph.d. graduate school of canada life and environmental sciences university of tsukuba, japan contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. panels of photos of the devastated reefs wiped-out by yolanda, and the various examples of coral fragments which showed fast growth rates after re-attachment. 3phil.reef fish-go.pmd ja anticamara and others 1 science diliman (january-june) 27:1, 1-47 national patterns of phil ippine reef fish diversity and its impl ications on the current municipal-level management jonathan a. anticamara university of the philippines diliman kevin thomas b. go* university of the philippines diliman stevenson s. ongsyping university of the philippines diliman francesco antonio t. valdecañas university of the philippines diliman ryan gabriel s. madrid university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract recent national-level assessments of philippine reef f ish diversity have b e e n m a i n l y b a s e d o n s p e c i e s r i c h n e s s s u r v e y s , b u t g e n e r a l l y d o n o t a c c o u n t f o r r e e f f i s h a b u n d a n ce a n d b i o m a s s — m e t r i c s t h a t b e t t e r d e s c r i b e f i s h co m m u n i t y a s s e m b l a g e s . g i v e n t h a t t h e p h i l i p p i n e s i s considered a major biodiversity hotspot and is heavily reliant on coastal r e s o u r c e s , t h e r e i s a g r e a t n e e d t o q u a n t i f y t h e c u r r e n t s t a t u s o f i t s r e e f f i s h d i v e r s i t y u s i n g s t a n d a r d i ze d m e t h o d s . h e r e , s t a n d a r d i z e d underwater visual census (uvc) belt transect sampling methods were u s ed to q u a n t i f y c u r r e n t l eve l s of r ee f f i s h s p ec i e s r i c h n e s s , r e l a t i ve a b u n d a n ce, a n d r e l a t i ve b i o m a s s t h r o u g h o u t t h e p h i l i p p i n e s . re s u l t s showed that most surveyed municipalities were still species-rich (22.2 ± 0.8 reef f ish species per 100 m 2), but appeared depleted in terms of r e e f f i s h a b u n d a n c e a n d b i o m a s s . pa r t i t i o n i n g a n a l y s i s r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s i n r ee f f i s h s p e c i e s r i c h n e s s p a t t e r n s a c r o s s municipalities, suggesting the presence of a few restricted-range and r a r e s p e c i e s p e r s i te . h o w ev e r, p a r t i t i o n i n g a n a l y s i s a cco u n t i n g f o r national patterns of philippine reef fish diversity 2 r e l a t i v e a b u n d a n c e s h o w e d t h a t r e e f f i s h d i v e r s i t y w a s g e n e r a l l y h o m o g e n o u s a c r o s s s t u d y s i t e s , s u g g e s t i n g t h e d o m i n a n c e o f a f e w highly-abundant species. simper analysis revealed that philippine reefs were generally dominated by small and medium-bodied species, rather than large-bodied species—the latter of which are especially vulnerable to f i s h i n g d u e to ce r t a i n l i fe h i s to r y t r a i t s (e.g. , l a te a g e a t m a t u r i t y a n d s l o w g r o w t h r a t e ) a n d c o m m e r c i a l e x p l o i t a t i o n . w h i l e c u r r e n t municipal-level management may be suff icient for restricted-range f ish species, large-scale conservation efforts (i.e. , in the form of collaborative marine reserve networks) are needed for wide-range and l a r g e b o d i e d s p e c i e s t h a t a r e n o t c o n f i n e d t o p o l i t i c a l l y d e f i n e d m u n i c i p a l b o u n d a r i e s . i n a d d i t i o n , l o n g t e r m a n d n a t i o n w i d e e f f o r t s t o systematically monitor philippine reef diversity are needed to provide up-to-date knowledge of the status of philippine reef diversity that will h e l p s u p p o r t s c i e n c e b a s e d r e e f m a n a g e m e n t a n d r e c o v e r y e f f o r t s throughout the country. keywords: conservation, coastal management, marine reserves, philippine r e e f s , r ee f f i s h e r i e s layman’s abstract recent national-level assessments of philippine reef f ish diversity are mainly based on the number of species present, but generally do not account for the abundance and biomass of these species—metrics that b e t t e r d e s c r i b e t h e f i s h co m m u n i t y co m p o s i t i o n . u n d e r s t a n d i n g t h e current status of reef f ish in the philippines is impor tant , considering that the country is a marine biodiversity hotspot, and is greatly reliant on marine resources for food and livelihood. to address this, we conducted underwater reef f ish surveys throughout t h e c o u n t r y, r e c o r d i n g f i s h a b u n d a n ce a n d s i z e u s i n g s t a n d a r d i ze d methods. we found that most surveyed municipalities still held a high n u m b e r o f s p e c i e s , b u t a p p e a r e d d e p l e t e d i n t e r m s o f r e e f f i s h a b u n d a n c e a n d b i o m a s s . f u r t h e r a n a l y s i s s u g g e s t e d t h a t m o s t m u n i c i p a l i t i e s w e r e h o m e t o s o m e r e s t r i c t e d r a n g e a n d r a r e s p e c i e s , b u t w e r e d o m i n a t e d b y a f e w h i g h l y a b u n d a n t s p e c i e s . f u r t h e r m o r e , philippine reefs were generally dominated by smalland medium-bodied s p e c i e s , r a t h e r t h a n l a r g e b o d i e d s p e c i e s . l a r g e b o d i e d f i s h a r e e s p e c i a l l y v u l n e r a b l e to f i s h i n g d u e to t h e i r h i g h co m m e r c i a l v a l u e , w h i c h m a k e s t h e m d e s i r a b l e f i s h e r i e s t a r g e t s . i n a d d i t i o n , ce r t a i n ja anticamara and others 3 characteristics, such as slow growth and low reproductive rate, also add to the vulnerability of large-bodied species. a l t h o u g h c u r r e n t m u n i c i p a l l eve l m a n a g e m e n t m a y b e s u f f i c i e n t f o r r e s t r i c t e d r a n g e f i s h s p e c i e s , l a r g e s c a l e c o n s e r v a t i o n e f f o r t s a r e n e e d e d f o r w i d e r a n g e a n d l a r g e b o d i e d s p e c i e s t h a t t r a v e r s e l a r g e areas, often across politically-def ined municipal boundaries. an example o f l a r g e s c a l e c o n s e r v a t i o n e f f o r t s w o u l d b e c r e a t i n g n e t w o r k s o f “marine reserves,” which are areas of protected sea where f ishing and other exploitative activities are not allowed. in addition, long-term and nationwide efforts to systematically monitor philippine reef diversity are needed to provide up-to-date knowledge on the status of philippine reef diversity. this knowledge is key in helping suppor t science-based reef management and recovery effor ts throughout the country. introduction the philippines, an archipelagic nation located in the coral triangle of the indopacif ic region, is considered to be one of the global centres of marine f ish diversity (carpenter and springer 2005). the country is also a known biodiversity hotspot— a place that is rapidly losing biodiversity in a short amount of time due to extensive habitat destruction and exploitation (licuanan and gomez 2000; roberts and others 2002; possingham and wilson 2005; allen 2008). over the past two decades, pressure from overexploitation and destructive human activities have contributed to the degradation of philippine reefs and the deterioration of coastal resources (gomez and others 1994; gomez 1997; white and vogt 2000; nanola and others 2006; briones 2007). declines in coastal resource production, particularly in the f isheries sector, is a matter of concern for the filipino people, since many filipinos rely heavily on f isheries products for both food and livelihood (gjertsen 2005; bfar 2012). therefore, it is important to ask what is left of reef f ish diversity in the philippines, considering the country’s growing population and the potential increase in demands for f isheries-related products that may follow. most recent national or regional analyses of reef f ish diversity in the philippines have been based on species presence or absence data obtained through a variety of sources, including underwater visual census (uvc) assessments, museum collections, published literature, and expert opinion (carpenter and springer 2005; allen 2008; nañola jr. and others 2011). these studies used presence-absence data from various sources to create species distribution maps that allow assessments of biodiversity patterns over large areas, and pinpoint hotspots of conservation national patterns of philippine reef fish diversity 4 importance. other assessments have used vulnerability scores (often put together by experts based on information on species population trends, distribution, life history, ecology, threats, and existing conservation measures) (comeros-raynal and others 2011) or local ecological knowledge (lavides and others 2009) to map areas at risk of potential species loss. while useful, these assessments do not account for f ish abundance and biomass—metrics that are needed in estimating potential f isheries production or yield, the effectiveness of management schemes (e.g. , marine reserve [mr] enforcement), and describing f ish community assemblages in greater detail than species presence-absence data. to date, only a few studies have presented estimates of reef f ish diversity in the philippines that account for not only species richness, but also species relative abundance and biomass (go and others in press; nanola and others 2006). however, many of these publications, generally included only a few sites in the country (allen 2002; stockwell and others 2009; anticamara and others 2010), were limited to a few commercial species (alcala 1988; russ and alcala 2004; russ and others 2005), or were mainly focused on studying the effectiveness of select no-take marine reserves (mrs). thus, there is still a great need to quantify the current status of reef f ish diversity in representative sites throughout the philippines by gathering reef f ish diversity data that not only reflects estimates of species richness, but also show the relative abundance and biomass of reef f ish species. collecting such data is vital in providing up-to-date knowledge for science-based decision making and marine resource management in the country (walton and others 2014). in the philippines, marine resource management began with a centralized, topdown, and use-oriented structure (alcala and russ 2006). such early philippine policies encouraged greater use of natural resources, which lead to depletion and habitat degradation. with the top-down approach, management responsibility often fell upon the central government, or government bureaucracies centred around large cities such as manila and cebu (pomeroy and carlos 1997; alcala and russ 2006). unfortunately, in the case of the philippines, this top-down approach to management was mostly ineffective, as the governing bodies were unable to properly manage resource exploitation and the expansion of f isheries (which included destructive and illegal f ishing methods) in the country (alcala and russ 2006). however, in recent times, the responsibilities and power to establish marine resource policy in the philippines has since shifted towards community-based comanagement, which involves the municipal lgus and, more importantly, the primary resource users themselves—the local f ishers and coastal communities. a number of well-enforced mrs built on community co-management and collaboration have been documented in the philippines, although these have only covered specif ic localities throughout the country (white and courtney 2002; alcala and russ 2006; ja anticamara and others 5 arceo and others 2008; cabral and others 2014). resource co-management tends to have better continuity over human generations, particularly if the local communities enforcing these policies are convinced of its effectiveness and have a strong desire to participate (alcala and russ 2006). conversely, a lack of belief and participation in the management system could easily lead to non-compliance and resistance (oracion and others 2005). there is generally a lack of standards in managing mrs among philippine municipal governments, and the quality of management can vary with the skills and interests of local off icials (white and courtney 2002). inconsistencies in enforcement may be limiting the effectiveness of marine resource policies across the country, and need to be addressed, especially with the turnover of jurisdictions with every change in local government administration after elections. current national policy devolves biodiversity conservation and management effort in the philippines to the municipal level. for example, the local government code (lgc) of 1991 provided municipal local government units (lgus) with authority to carry-out specif ic functions, including the establishment of policies regarding the conservation and management of natural resources, such as the establishment of reserves and protected areas (philippine government 1991). in addition, the republic of the philippines fisheries code republic act (ra) 8550 states that all philippine municipalities must allocate about 15% of its municipal waters (i.e. , coastal waters from foreshore to 15 km away from the coasts) as mrs (department of agriculture 1998). however, while the number of well-enforced mrs has increased over the years (maypa and others 2012), recent estimates suggest that only about 1% of philippine coral reef areas are well-protected (white and others 2014), and 90% of the 1,000+ mrs currently existing in the philippines are small or < 1km2 (weeks and others 2010). despite the pressing need to improve coastal resource management in the country (weeks and others 2014), the current status of philippine reef f ish biodiversity remains largely unmeasured, except for surrogate data from a few sites, or select (usually commercial) families and species (russ and alcala 2004; russ and others 2005; stockwell and others 2009). the main goal of this paper is to present the results of a recent and systematic assessment of philippine reef f ish diversity, accounting for reef f ish species richness, relative abundance, and relative biomass across representative sites throughout the country. in addition, this paper will explore patterns of reef f ish diversity and dominance across the philippines. it is not within the scope of this paper to quantify the effects of municipal-level management on philippine reef f ish diversity, but rather to discuss how diversity patterns revealed in the study are potentially related to the existing municipal-level “devolution of power” of marine resource national patterns of philippine reef fish diversity 6 management in the country to date. therefore, much of the analysis in this paper on philippine reef f ish biodiversity patterns will be conducted at the municipal level (e.g. , comparing biodiversity between and across municipalities). specif ically, the paper seeks to answer the following questions: (1) what is the general picture of reef f ish biodiversity throughout the philippines to date, based on different biodiversity metrics (e.g. , species richness, evenness, abundance, and biomass)?; (2) how does reef f ish biodiversity vary across philippine municipalities based on these metrics?; (3) what patterns can be observed in reef f ish assemblages throughout the country?; and (4) what types of f ish species dominate philippine coral reefs to date? methods study site selection and survey methods using google earth satellite images, we selected sites that most likely had coral reefs close to shore. in addition, study sites were selected to represent the philippines’ three major island groups (e.g. , luzon, visayas, and mindanao) and the six marine biogeographic regions proposed by aliño and gomez (1994), while accounting for budget and logistical constraints such as travel time, costs, issues of site accessibility, traveling with lots of equipment, and safety. surveyed reef sites within each municipality included areas inside and outside mrs (where mr boundary demarcation was clearly established), and were often referred by local f ishers, boatmen, or local government unit (lgu) off icers, whom we asked to direct us to reef areas where we could record as much of the local f ish diversity as possible. due to budget and logistical limitations, the number of surveyed transects varied per municipality and biogeographic region (appendix 1). to quantify philippine reef f ish diversity, standardized underwater visual census (uvc) belt transects surveys were conducted throughout the philippines. a total of 420 belt transects, belonging to 119 reef sites, forty-nine municipalities, and six philippine marine biogeographic regions were surveyed from march 2012 to june 2014 (approximately two-year period), spanning north to south of the philippines (figure 1). the uvc belt transect method used is an established non-destructive reef f ish survey method, for quantifying reef f ish species diversity (brock 1982; samoilys 1997; samoilys and carlos 2000). to conduct uvc surveys, a diver (j. anticamara) swam along a 20 x 5 m transect and recorded all size (cm) and abundance estimates of non-cryptic reef f ish species with a minimum length of 1 cm encountered within the transect boundaries. ja anticamara and others 7 typically, a minimum length of 10-11 cm is recommended to avoid errors in length and abundance estimates (bellwood and alcala 1988). however, we initially observed that many of our survey sites were dominated by small-bodied species and individuals, so setting the minimum length of our methods to 10 cm would exclude a signif icant portion of the reef f ish community. therefore, we decided to include all reef f ish species down to a minimum length of 1 cm, to appropriately represent the true status of reef f ish diversity throughout our survey sites. the estimated length of recorded individual f ish species was later converted into weight using length-weight (lw) relationships available in fishbase (froese and pauly 2014). in cases where the lw relationship of a particular f ish species was not figure 1. map of surveyed reef sites throughout the philippines. the broken lines represent demarcations of the philippine marine biogeographic regions as proposed by aliño and gomez (1994). the number of transects and municipalities surveyed per biogeographic region can be found in appendix 1. national patterns of philippine reef fish diversity 8 available in fishbase, the lw of the congener or family member of similar shape and maximum total length was used (anticamara and others 2010). all surveyed transects were conducted at depths ranging from 3–6 m to capture as much f ish diversity as possible at a manageable depth, since reef f ish diversity and abundance are often high at this depth range relative to other depth ranges (friedlander and parrish 1998; friedlander and others 2003). to avoid variation due to surveyor’s error, we only analyzed uvc survey data obtained by one of the authors (j. anticamara), who has had nearly twenty years of experience conducting underwater surveys in philippine coral reefs (samoilys and others 2007; anticamara 2009; anticamara and others 2010). all surveys were conducted during daylight hours, and each transect was surveyed for approximately 20 minutes. our choice of transect dimensions (20 x 5 m), number of transect replicates (3-4 transect replicates per reef site, or 8–10 transects per municipality) and total surveyed reef area per site (300–400 m2total reef area per site, or 800–1,000 m2 per municipality) is comparable to uvc methods used in other studies quantifying reef f ish diversity (brock 1982; friedlander and parrish 1998; t issot and others 2004; nakamura and tsuchiya 2008; shibuno and others 2008; honda and others 2 0 1 3 ) . the uvc belt transect method underestimates the abundance of cryptic reef f ish species (e.g. , blennies, gobies, dottybacks, and eels) and nocturnal species (e.g. , sweepers, soldierf ishes, and priacanthids), since such species may remain hidden from census divers (willis 2001). on the other hand, highly-mobile species may be overestimated, due to their conspicuous movements in the diver’s f ield of vision (smith 1988). to address these limitations, we conducted uvc surveys at slow swim speeds of about 5 m2 min-1 (or roughly 20 min per 100 m 2 transect), which improves counting accuracy, search eff iciency (samoilys and carlos 2000), and avoids scaring away skittish f ish, while taking care not to double-count individuals that re-enter the transect area. in addition, photographs of all encountered reef f ish species were taken for identif ication verif ication using a number of references (allen and others 2003; kuiter and debelius 2006; froese and pauly 2014). data analysis first, to present the adequacy of our current sampling effort in capturing philippine reef f ish diversity, we constructed species accumulation curves (sacs) for each of the six sampled biogeographic regions. a sac is a graph of recorded number of ja anticamara and others 9 species as a function of sampling effort and allows for the estimation of the total number of species in a given area per increased sampling unit (colwell and others 2004). initially, the sac rises steeply as common and abundant species are recorded, then more slowly as rare species are recorded (ugland and others 2003). to examine general patterns of reef f ish diversity across the philippines, histograms of reef f ish species richness, abundance, and biomass per municipality were constructed. then, to examine potential differences in reef f ish diversity between municipalities across the country, bar plots showing mean (and standard errors se) species richness, abundance, and biomass per municipality were also produced. examining spatial trends at the municipal level coincides with the paper’s objectives to discuss our research f indings in relation to the current municipal-level policy of philippine coastal management. to explore patterns of reef f ish assemblages throughout the country, non-metric multidimensional scaling (mds) analysis was performed to help visualize potential grouping-by-similarity of reef f ish assemblages at various spatial scales, namely: transects, reef sites, municipalities, or marine biogeographic regions. mds analysis uses a constructed bray-curtis similarity matrix to visually map the similarities of the 420 sampled transects—i.e. , transects that are more similar are plotted closertogether, while transects that are dissimilar are plotted further apart. for example, if transects from a given biogeographic region grouped more closely-together than with transects from other biogeographic regions, this would suggest that reef f ish assemblages in that biogeographic region are distinct from the other biogeographic regions. similarly, if transects from a given municipality grouped more closely together than with transects from other municipalities, this would suggest that reef f ish assemblages in sampled transects within that municipality are similar and are distinct from transects sampled from the other municipalities. mds analysis would therefore allow us to determine if transects grouped at certain spatial scales or did not show any clear grouping at all. mds analysis was performed separately for reef f ish assemblage similarity based on reef f ish abundance and biomass data. to further examine reef-f ish assemblages at multiple spatial scales, additive diversity partitioning was performed. in additive diversity partitioning, total diversity (�) is the sum of the mean local diversity or the mean diversity within samples or transects ( ), and the diversity between samples (�) at various def ined scales (e.g. , between transects, reef sites, municipalities, or biogeographic regions). in an unbalanced, hierarchal sampling design, such as in our case, each sample level can be represented as hierarchal spatial scales (veech and others 2002). specif ically, in this study, represents mean within-transect reef f ish diversity, �1 represents � _ � _ national patterns of philippine reef fish diversity 10 1 —∑ pijklnpijk 2 between-transec t d i versity, � 2 represents between-reef site d i versity, � 3 represents between-municipality diversity, �4 represents between-biogeographic region diversity, and � represents the estimated total philippine reef f ish diversity based on all our samples, as summarized in the following equations: where is the mean diversity for each hierarchal spatial scale. estimated total philippine diversity (�) based on all our samples can be expressed as: (5) therefore, diversity partitioning can be used to determine the proportional contributions (percentage) of each level of and �-diversity to �-diversity, wherein total diversity � = 100% (lande 1996). however, the interpretation of and � varies depending on the particular diversity index used. the general equation for is presented below: (6) based on the above equation, the sample weight q ij is the proportion of the total number of individuals found in each sample j, and sampling level i. in addition, can be calculated using different diversity indices d ij . when partitioning is based on species richness index, d ij is the number of species in transect j, at sampling level i. when par titioning is based on shannon’s diversity index, d ij = , where p ijk is the proportional abundance of species k in transect j. similarly, when partitioning is based on simpson’s diversity index, d ij = (crist and others 2 0 0 3 ) . partition v2 freeware (veech and crist 2007) was used to run additive diversity partitioning analysis, and to test for signif icant differences between our data (e.g. , “observed diversity”) and randomly-generated null models (e.g. , “expected diversity”) (veech and crist 2007). null models were generated based on 999 � _ � _ �1transects = dsites — transects� __ �3municipalities = dbiogeographic—dmunicipalities _ _ �4biogeographic = � dbiogeographic _ � = � + �1 + �2 + �3 + �4 • � = ∑ _ n i t = 1 d ij q ij 1 —∑ pijklnpijk �2sites = dmunicipalities—dsites _ _ _ d (1) (2) (3) (4) 0 � _ � _ ja anticamara and others 11 individual-based randomizations. a signif icant difference between the observed data portioning and randomized data partitioning means that the observed hierarchical patterns of diversity are real and unlikely to be produced by a randomized assignment of species to samples at various def ined scales. finally, to examine dominance patterns in reef f ish assemblages, a similarity percentage (simper) analysis was run separately for reef f ish species abundance and biomass. simper determines the contributions of each reef f ish species to the pair-wise bray-curtis similarities of all surveyed transects within each municipality. the species with the highest contributions are usually the most abundant or have the highest total biomass and therefore are generally the most dominant species. both mds and simper analyses were run using primer v6 (clarke and gorley 2006). the body sizes (maximum total length (max tl)) of the top f ive dominant reef f ish species for all municipalities was obtained from fishbase and categorized as small-bodied (max tl < 10 cm), medium-bodied (max tl = 10.1 30 cm), and large-bodied (max tl > 30.1 cm). results overall, we identif ied a total of 375 non-cryptic reef f ish species, belonging to forty-eight families, across the 420 transects that we surveyed throughout the entire philippines. species accumulation curves per biogeographic region showed increasing number of species with every additional transect (figure 2). however, the rate of increase in the number of species per additional transect started to plateau or visibly slow-down at around 20-30 transects per biogeographic region, at which point over 100 reef f ish species had been recorded. this suggests that most common or dominant species in each biogeographic region have been recorded after surveying about 20–30 100 m2 transects, and additional species detected by surveying more transects may be rare or cryptic species. patterns in the frequency distribution of transects with respect to mean reef f ish species richness (22.2 ± 0.8 species), abundance (387.7 ± 66.1 f ish), and biomass (2.5 ± 0.3 kg) differed. transects were normally distributed in terms of species richness (figure 3a). on the other hand, transects were skewed to the left in terms of both abundance and biomass, indicating that most transects had abundance and biomass values way below the mean for both metrics (figure 3d-e). bar plots on mean species richness, abundance, and biomass per municipality showed generally lower variation in species richness within and among municipalities, but 0 national patterns of philippine reef fish diversity 12 higher variation in terms of both abundance and biomass (figure 4). most municipalities (61% of forty-nine municipalities) had high species richness (i.e. , “high” def ined as having values within or above the range values of the mean ± se across all municipalities, and “low” def ined as having values below it). on the other hand, most municipalities had low abundance (63%) and biomass (59%). mds analysis of the bray-curtis similarity in reef f ish species assemblages with respect to species relative abundance or relative biomass did not show clear grouping of surveyed transects according to either biogeographic regions or municipalities (appendix 2). however, some transects from the same municipality tended to group together, indicating within-municipal similarity of reef f ish assemblages, at least for those municipalities. additive par tition of reef f ish diversity in terms of species richness and species evenness (e.g. , shannon’s diversity and simpson’s diversity) showed different spatial patterns of philippine reef f ish biodiversity. in terms of species richness, observed �1 and �2-diversity did not signif icantly differ from the expected null model, while observed �3 and �4-diversity were signif icantly higher than the expected figure 2. species accumulation curves (sacs) per biogeographic region, for the visayas sea (a), northern philippine sea (b), sulu sea (c), southern philippine sea (d), south china sea (e), and celebes sea (f ). sac curves show the cumulative number of f ish species found in every additional transect based on 1,000 permutations of transect ordering. ja anticamara and others 13 null model. furthermore, most of species richness � -diversity was accounted for by �3 (37.7%) and �4 (41.8%). in contrast, accounted for much of shannon’s (50.0%) and simpson’s (81.13%) �-diversity. dominance analysis using simper indicated that each municipality generally had different sets of top f ive dominant species in terms of species’ relative abundance (appendix 3a) and biomass (appendix 3b). the top f ive dominant species within each municipality generally accounted for about 73.7 ± 1.6% of the total abundance figure 3. histograms showing per-transect frequency distributions for reef f ish species richness (a), shannon’s diversity (b), pielou’s evenness (c), abundance (d), and biomass (e). the broken line on each graph represents the mean value per transect for that respective metric. � _ national patterns of philippine reef fish diversity 14 and 65.6 ± 2.0% of the total biomass. of the 375 reef f ish species recorded in our study, only 66 and 68 species comprised the top f ive dominant species in terms of abundance and biomass for all municipalities, respectively. certain reef f ish families tended to dominate the top f ive species per municipality. for example, of the 66 species included in the dominant species listed for all municipalities based on figure 4. bar plots showing per-municipality mean ± se bars for reef f ish species richness (a), shannon’s diversity (b), pielou’s evenness (c), abundance (d), and biomass (e), arranged by biogeographic region, and from north to south of the philippines. municipality codes can be found in appendix 1. ja anticamara and others 15 abundance, 58% were damself ishes and 13% were wrasses. in terms of biomass, 18% of the 68 top f ive dominant species were wrasses, 16% were damself ishes, and 13% were butterflyf ishes. fur thermore, many of these dominant species in terms of both abundance and biomass were small to medium-bodied species. in terms of abundance, small-bodied, medium-bodied, and large-bodied species made up 36%, 58%, and 6% of the top f ive dominant species, respectively. in terms of biomass, small-bodied, medium-bodied, and large-bodied species made up 9%, 57%, and 34% of the top f ive dominant species, respectively. however, most of the medium to large-bodied species that appeared as top f ive dominant in terms of biomass were mainly omnivores, herbivores, and corallivores, suggesting the decline of most large-bodied carnivorous reef f ish species throughout the philippines. discussion overall, results from this research show that many coral reef areas throughout the philippines are still highly diverse, only if reef f ish species richness is used as the sole measure of biodiversity. however, while still considerably species-rich (i.e. , most municipalities having at least 21-23 species per 100 m2), many areas throughout the philippines appear to be exhibiting signs of depletion in terms of f ish abundance and biomass, and in fact, previous studies have described the depletion of species richness as well (lavides and others 2009; nañola jr. and others 2011). diversity partitioning analysis revealed that philippine reef f ish assemblages can be characterized as having high variations in species richness across municipalities, but generally low species evenness (e.g. , shannon’s and simpson’s diversity indices). differences in species richness rather than evenness best explained differences in reef f ish assemblages between municipalities, suggesting the presence of restricted-range species (i.e. , species found in only a few of the surveyed municipalities) in each municipality (go and others in press). on the other hand, shannon’s and simpson’s diversity were best explained by withintransect diversity, suggesting that most surveyed reefs were dominated by a few, highly-abundant reef f ish species. further investigation of dominance patterns via simper analysis revealed that most surveyed reef sites were dominated by abundant small and medium-bodied reef f ish species. however, there was also a general rarity of large-bodied species throughout most philippine reefs—a f inding which suggests overexploitation due to f ishing, considering that large-bodied, high trophic-level species are particularly targeted by f isheries (pauly and others 1998), and are especially vulnerable to f ishing due to particular life history traits (abesamis and others 2014). these f indings suggest that the current “municipality-bynational patterns of philippine reef fish diversity 16 municipality” policy to biodiversity conservation in the philippines may be affecting reef f ish assemblages throughout the country (but see qualif ied discussions and elaborations of this point below). although previous work accounting for philippine reef f ish abundance, biomass, and functional diversity has been done (carpenter and others 1981; russ and alcala 1998a; russ and alcala 1998b; nanola and others 2006), to date, the most common measure of reef f ish diversity in the philippines is species richness (allen 2002; carpenter and springer 2005; allen 2008; nañola jr. and others 2011). species richness or species presence-absence data is useful in estimating species range, restriction or expansion of range, and potential local extirpation (lavides and others 2009; nañola jr. and others 2011). however, results of the current study show that species richness alone is not always a good indicator of reef f ish diversity status in the country. for example, while reef f ish species richness remains high in most places throughout the philippines, examination of the other metrics reveals that most places in the country actually have low reef f ish abundance and biomass. indeed, other studies have also found that reef f ish species richness may exhibit less-obvious changes than reef f ish species abundance, in response to disturbances such as exploitation and habitat degradation (alcala 1988; harmelin and others 1995; jones and others 2004)—human-induced disturbances that are common in many coastal areas of the philippines. therefore, the effects of such disturbances on reef f ish assemblages may be underestimated, if only species richness is taken into account. many species still exist, but most in very low population size or abundance throughout the sampled municipalities. patterns of phil ippine reef fish d iversity: restricted-range species and the dominance of a few highly-abundant species results of additive partitioning analysis suggest two main findings regarding spatial patterns of reef f ish assemblages throughout the country: (1) the presence of restricted-range species influences the differences in species richness between municipalities; and (2) only a few, abundant species tend to dominate reef f ish assemblages throughout the country, and greatly influence species evenness. with regards to our f irst f inding—additive diversity partitioning analysis showed that between-municipality (�3) and between-biogeographic region (�4) diversity species richness was signif icantly greater than that predicted by the null models, and also accounted for a relatively large portion of � -diversity. this means that differences in reef f ish species richness between municipalities and between biogeographic regions may reflect real variations in species richness at these spatial scales ja anticamara and others 17 (belmaker and others 2008). the high contribution of �3-diversity to �-diversity in terms of species richness indicates that the presence of restricted-range and rare species may account for the difference in species richness between municipalities (rodríguez-zaragoza and others 2011). indeed, many of the reef f ish species included in our study exhibited restricted ranges (go and others in press). however, we suspect that the restricted ranges of many of these species is not due to evolutionary or ecological factors, considering the nationwide distributions of most of these species based on previous records (carpenter and springer 2005; nañola jr. and others 2011; froese and pauly 2014), but rather due to the high rates of exploitation and reef degradation in the philippines to date. with regards to our second f inding of diversity partitioning analysis—it is possible to infer that most surveyed areas were dominated by a few, highly-abundant species, because -diversity accounted for much of shannon’s and simpsons’ � -diversity, but not for species richness’ � -diversity (rodríguez-zaragoza and others 2011). high -diversity when accounting for species relative abundance (e.g. , evenness metrics like shannon’s and simpson’s indices) can be interpreted as homogeneity of species assemblages across surveyed transects, because the very high abundance of a few species common to all sites overwhelms the small amounts of betweensite (�) variation contributed by the non-abundant species (rodríguez-zaragoza and others 2011). these f indings suggest that surveyed reef f ish assemblages per municipality are generally characterized by high species richness, but low evenness (rodríguez-zaragoza and others 2011). the differences in observed patterns from diversity partitioning analysis between species richness and evenness again highlights the importance of measuring biodiversity using different metrics (gering and others 2003). dominance patterns in phil ippine reef fish assemblages: the abundance of small and med ium-bod ied species analysis of reef f ish species assemblages based on bray-curtis simper showed that most of the dominant species in surveyed reefs were small and mediumbodied species such as wrasses, damself ishes, and butterflyf ishes, in terms of abundance. although some large-bodied species were among the top f ive dominant species in terms of biomass, this does not necessarily mean that these species are abundant in philippine reefs—indeed, large-bodied reef f ish species such as emperors, groupers, jacks, snappers, and sweetlips were rarely dominant in terms of abundance across all surveyed reefs. this may be due to the fact that: (1) larger maximum body size—along with other life history traits such as slower growth � _ � _ national patterns of philippine reef fish diversity 18 rate, longer lifespan, later age at maturity, and lower rates of natural mortality—has been associated with increased vulnerability to f ishing (abesamis and others 2014); and (2) large-bodied f ish species are especially targeted by f ishers for their higher commercial value than small-bodied species (russ and alcala 1996; pauly and others 1998). shifts in f ish assemblages from dominance of larger-bodied species and individuals towards dominance of smaller-bodied species and individuals have been documented in the past, following high levels of exploitation (greenstreet and hall 1996; bianchi and others 2000; rogers and ellis 2000; levin and others 2006). thus, high exploitation rates in the philippines, accompanying increasing demands for f ish production and a growing human and f ishing population, may be threatening most commercially-important, large-bodied reef f ish species in the country. the exploitation-induced depletion of large-bodied reef f ish species may have negative implications on philippine f isheries production and the food security of many filipinos, who are largely-dependent on f ish products as a dietary protein source, and actually prefer to consume large-bodied f ish species (bfar 2012). however, formal assessments on the threatened status of many reef f ish species in the philippines are limited by the lack of available species abundance and distribution data in the past and recent years. this makes assessment criteria commonly used by internationally-recognized conservation organizations like the iucn (such as population decline and range contraction) diff icult to apply for many reef f ish species in the philippines. as a result, many philippine reef f ish species remain under-assessed or totally unassessed (go and others in press; iucn 2014)—an issue that should be addressed by conservation and management efforts in the country. caveats and limitations the main caveat of the current study is that the number of surveyed transects varied across municipalities and biogeographic regions. this could lead to underrepresentation of reef f ish diversity for biogeographic regions that had a disproportionally fewer number of surveyed transects than the other surveyed regions (e.g. , celebes sea, in our study). nañola jr. and others (2011), who presented reef f ish species richness patterns across philippine marine biogeographic regions, showed with sacs that the number of species recorded per additional transect surveyed increased rapidly until about 40 to 50 transects per biogeographic region, after which the addition of new species recorded per additional transect slowed down. this suggests that around 40 to 50 surveyed transects are required to account ja anticamara and others 19 for most of the common or abundant species in each biogeographic region. based on this estimate, reef f ish diversity in the celebes sea biogeographic region is underrepresented in our study. however, based on our own sacs and data, our sampling effort adequately captured reef f ish diversity for all biogeographic regions, as all of our sacs per biogeographic region approached asymptotic patterns. in addition, we surveyed reef sites and municipalities that were geographically far apart, and selected sites referred by local f ishers, boatmen, or lgu off icers to capture as much representative reef f ish biodiversity per municipality and per biogeographic region as possible. however, despite these efforts to survey as much reef f ish diversity as possible, none of our sacs approached the 350–500 reef f ish species recorded per biogeographic region at 40-50 transects reported by nañola jr and others (2011), even after we re-ran the sac construction using jackknife 2 estimators —e.g. , the estimator used by nañola jr. and others (2011), which is based on species presence-absence data (smith and pontius 2006). this may suggest a general depletion of reef f ish diversity throughout the philippines (lavides and others 2009; nañola jr. and others 2011), considering that nañola jr. and others (2011) included reef f ish survey data from 1991 to 2008. to account for the differences in the number of transects per municipality and biogeographic region when conducting diversity partitioning analysis, we used an unbalanced sampling design in partition v2 (veech and crist 2007). unbalanced sampling in diversity partitioning has been used in previous studies on reef f ish diversity patterns as well, where sampling effort was not uniform across study areas (rodríguez-zaragoza and arias-gonzalez 2008; francisco-ramos and ariasgonzález 2013). impl ications for management the observed patterns in reef f ish assemblages throughout the country may be affected by the municipal-level organization of coastal management in the philippines today. for example, signif icant differences in diversity metrics (particularly abundance and biomass) between municipalities, as well as the presence of restricted-range species in each municipality, may be potentially due to the variations in management effectiveness (e.g. , mr enforcement) between these municipalities (although this is not tested in the current study). the positive effect of well-enforced mrs on local f ish diversity has been documented in previous studies (russ and alcala 1999; walmsley and white 2003; samoilys and others national patterns of philippine reef fish diversity 20 2007; maypa and others 2012; bergseth and others 2013), and it is highly possible that surveyed municipalities that exhibited high diversity metrics were also those municipalities that had well-enforced mrs. however, quantifying the effects of varied management on reef f ish diversity across municipalities is diff icult given available datasets, since only 14 of the 49 municipalities included in our study had mrs with available enforcement ratings on the recently established philippine marine protected area network (cabral and others 2014). in addition, management effectiveness may be linked to the interest and support of stakeholders. for example, the distribution of mrs in the philippines is concentrated in the visayas region (weeks and others 2010)—a region where academic institutions and non-government organizations (ngos) continue to support mr establishment (pollnac and others 2001), and where the f irst efforts of philippine mr establishment began (alcala and russ 2006). while much has been done to quantify the extent of mr establishment and enforcement throughout the country (weeks and others 2010; maypa and others 2012), the effectiveness in terms of biological indicators (e.g. , reef f ish species abundance, biomass, and f ish yield) of most philippine mr’s is still largely unknown. maypa and others (2012) presented the most recent analysis of philippine mr effectiveness on coral reef health, but only included a few (n = 56) mrs from the visayas region that had available biophysical data. thus, there is still a great need to monitor biological indicators of mr effectiveness throughout the philippines. to date, the coral triangle marine protected area system (ctmpas), created by the coral triangle initiative (cti) in 2009, hopes to achieve well-managed mpas throughout the six coral triangle countries by integrating the aforementioned ecological, social, and governance factors through a consistent and science-based system of mr establishment (walton and others 2014). however, there is still a great need to improve the enforcement, monitoring, socioeconomic accountability, governance, and f inancial support of many mrs in the philippines (white and others 2014). in addition, facilitating collaboration and communication between multiple stakeholders, increasing local capacity to manage mrs, and developing learning networks across mr managers are invaluable in achieving successful mr enforcement (weeks and others 2014). finally, it is important to account for ecological factors in mr design, such as adequate habitat representation, protection of critical areas used in a species’ different life history stages (e.g. , spawning grounds, nurseries), ensuring connectivity between protected habitats, accounting for resilience or vulnerability to climate change, and minimizing local anthropogenic threats (e.g. , land-based runoff and siltation) (green and others 2014). for example, while current small-scale municipal-level management may be suff icient for restricted-ranged species, implementing large-scale (e.g. , across networks of mrs ja anticamara and others 21 rather than at select, individual mrs) and long-term management and monitoring of reef f ish diversity would help ref ine and adjust marine biodiversity conservation strategies for the country, effectively manage species that traverse large spatial units beyond municipal boundaries (e.g. , large-bodied species such as groupers, snappers, sharks, and whales, etc.), and ensure proper connectivity of reef f ish diversity throughout the country (kramer and chapman 1999; beets and others 2003; lowry and others 2009; matias and others 2013; green and others 2014). results from this research provide the most recent analysis on the current status of reef f ish diversity throughout the philippines using a standardized or systematic survey strategy. the results and conclusions from this research suggest that there is a great need to fully enforce the current marine biodiversity conservation policies of the philippines, to conduct national coral reef assessments that are systematic, scientif ically-sound, well-organized (licuanan and aliño 2014), and to mitigate reef degradation and the depletion of valuable marine biodiversity resources. by ensuring that 15% of philippine municipal waters receive effective protection from further overexploitation and destructive f ishing (e.g. , dynamite f ishing and the use of poison), the remaining reef areas of the philippines will have some chance of recovery, which will allow them to continue to provide benef its to philippine f isheries and food security, and maintain the high levels of diversity in the country (russ and others 2004; russ and others 2005; anticamara and others 2 0 1 0 ) . acknowledgments we would like to thank the mayors and local government units and agencies of all the municipalities included in this research for their support. we also would like to thank the local people’s organizations in bolinao (kisahan ng mga samahan alay sa kalikasan [kaisaka]), masinloc (samahang pangkaunlarang san salvador [spss]), and mabini (samahang pangkaunlarang san teodoro [spsti]). special thanks to the state universities who helped in this research: ateneo de naga (joanaviva plopeni and shane bimeda), bicol state university (karina luth discaya, antonino mendoza, meek salvador), mariano marcos university (wilnorie rasay), palawan state university (arselene bitara and dr. michael pido), and university of eastern philippines (saula gabona). we also thank two research assistants who helped in the f ield work: ambrosio melvin matias and justin albert de ramos. funding for this research comes from the following: foundation for the philippine environment (fpe), off ice of the vice chancellor for research and development up ovcrd (111104 phdia), natural sciences research institute up nsri (project code: bio-11-1-08), and the center for integrative and development studies up cids. national patterns of 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ongsyping is a medical student at the university of santo tomas faculty of medicine and surgery. before his graduation from the institute of biology, up diliman in 2013, he worked at the ja lab group for his undergraduate thesis on reef f ish diversity. francesco antonio t. valdecañas is a medical student at the university of the philippines college of medicine. before his graduation from the institute of biology, up diliman in 2013, he worked at the ja lab group for his undergraduate thesis on coral and reef f ish relationships. ryan gabriel s. madrid is an environmental consultant at berkman international inc. before his graduation from the institute of biology, up diliman in 2013, he worked at the ja lab group for his undergraduate thesis on coral diversity and distribution. white at, a l i ñ o p m , cros a , and others. 2014. marine protected areas in the co r a l triangle: progress, issues, and options. coast manag. 42:87–106. white at and cour tney ca . 2002. experience with marine protected area planning and management in the philippines. coast manag. 30:1–26. white at, vogt hp. 2000. philippine coral reefs under threat: lesson learned after 25 years of community-based reef conservation. mar pollut bull. 40:537–550. willis tj. 2001. visual census methods underestimate density and diversity of cryptic reef f ishes. j fish biol. 59:1408–1411. ja anticamara and others 29 1.01 pagudpod 2 south china sea 1.02 burgos 2 south china sea 1.03 curimao 6 south china sea 1.04 sinait 4 south china sea 1.05 bolinao 19 south china sea 1.06 alaminos 25 south china sea 1.07 masinloc 11 south china sea 1.08 el nido 5 south china sea 1.09 quezon 6 south china sea total 80 south china sea 2.01 sta. ana 18 north east philippine sea 2.02 baler 8 north east philippine sea 2.03 caramoan 8 north east philippine sea 2.04 tabaco 7 north east philippine sea 2.05 lavesarez 4 north east philippine sea 2.06 catarman 6 north east philippine sea 2.07 laoang 4 north east philippine sea total 61 north east philippine sea 3.01 mabini 17 visayas sea 3.02 puerto galera 9 visayas sea 3.03 bongabong 5 visayas sea 3.04 romblon 6 visayas sea 3.05 san fernando 5 visayas sea 3.06 mandaon 6 visayas sea 3.07 cataingan 6 visayas sea 3.08 malay 3 visayas sea 3.09 buruanga 6 visayas sea 3.10 inopacan 11 visayas sea 3.11 getafe 5 visayas sea 3.12 tubigon 6 visayas sea 3.13 calape 2 visayas sea 3.14 panglao 6 visayas sea 3.15 mambajao 5 visayas sea 3.16 mahinog 5 visayas sea total 97 visayas sea municipality code municipality number of transects biogeographic region appendix 1 all surveyed municipalities, with corresponding codes and biogeographic regions national patterns of philippine reef fish diversity 30 4.01 anini-y 10 sulu sea 4.02 nueva valencia 8 sulu sea 4.03 puerto princesa 8 sulu sea 4.04 bataraza 10 sulu sea 4.05 bongao 14 sulu sea 4.06 simunul 5 sulu sea total 55 sulu sea 5.01 lawaan 16 southern philippine sea 5.02 balangiga 6 southern philippine sea 5.03 giporlos 10 southern philippine sea 5.04 quinapondan 8 southern philippine sea 5.05 salcedo 6 southern philippine sea 5.06 guiuan 31 southern philippine sea 5.07 surigao 11 southern philippine sea 5.08 mati 23 southern philippine sea total 111 southern philippine sea 6.01 parang 4 celebes sea 6.02 glan 6 celebes sea 6.03 sarangani 6 celebes sea total 16 celebes sea municipality code municipality number of transects biogeographic region appendix 1 all surveyed municipalities, with corresponding codes and biogeographic regions (cont’n.) ja anticamara and others 31 appendix 2 mds plots of bray-curtis similarity among municipalities in terms of species abundance (a) and species biomass (b). transects with the same shape denote transects from withinthe same biogeographic region. municipality codes can be found in appendix 1 national patterns of philippine reef fish diversity 32 1.01 thalassoma amblycephalum 34.48 labridae 16.0 medium similarity: 39.7 chromis margaritifer 34.48 pomacentridae 9.0 small top 5: 93.1 pomacentrus bankanensis 17.24 pomacentridae 9.0 small others: 6.9 chaetodon kleinii 3.45 chaetodontidae 15.0 medium centropyge vroliki 3.45 pomacanthidae 12.0 medium 1.02 ctenochaetus striatus 20.33 acanthuridae 26.0 medium similarity: 15.0 chromis margaritifer 16.26 pomacentridae 9.0 small top 5: 70.7 thalassoma amblycephalum 16.26 labridae 16.0 medium others: 29.3 plectroglyphidodon dickii 9.76 pomacentridae 11.0 medium chromis vanderbilti 8.13 pomacentridae 4.5 small 1.03 pomacentrus philippinus 25.09 pomacentridae 10.0 small similarity: 16.9 ctenochaetus striatus 17.89 acanthuridae 26.0 medium top 5: 66.02 thalassoma hardwicke 9.16 labridae 20.0 medium others: 33.98 neoglyphidodon nigroris 8.19 pomacentridae 13.0 medium ctenochaetus cyanocheilus 5.69 acanthuridae 13.7 medium 1.04 plectroglyphidodon lacrymatus 28.91 pomacentridae 10.0 small similarity: 40.1 pomacentrus philippinus 17.56 pomacentridae 10.0 small top 5: 78.9 chromis margaritifer 13.19 pomacentridae 9.0 small others: 21.1 pomacentrus lepidogenys 10.59 pomacentridae 9.0 small neoglyphidodon nigroris 8.62 pomacentridae 13.0 medium 1.05 chromis margaritifer 25.38 pomacentridae 9.0 small similarity: 12.1 thalassoma hardwicke 12.72 labridae 20.0 medium top 5: 64.3 plectroglyphidodon lacrymatus 10.20 pomacentridae 10.0 small others: 35.7 ctenochaetus striatus 9.35 acanthuridae 26.0 medium coris batuensis 6.61 labridae 17.0 medium 1.06 pomacentrus chrysurus 22.66 pomacentridae 9.0 small similarity: 12.6 neoglyphidodon melas 18.94 pomacentridae 18.0 medium top 5: 60.2 plectroglyphidodon lacrymatus 8.01 pomacentridae 10.0 small others: 39.8 macropharyngodon meleagris 5.98 labridae 15.0 medium stethojulis trilineata 4.64 labridae 15.0 medium appendix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) municipality code species contributory % family max tl (cm) body size ja anticamara and others 33 1.07 ctenochaetus striatus 37.47 acanthuridae 26.0 medium similarity: 27.8 chromis margaritifer 15.63 pomacentridae 9.0 small top 5: 80.4 thalassoma hardwicke 14.48 labridae 20.0 medium others: 19.6 stegastes fasciolatus 6.63 pomacentridae 15.0 medium plectroglyphidodon dickii 6.15 pomacentridae 11.0 medium 1.08 plectroglyphidodon lacrymatus 49.18 pomacentridae 10.0 small similarity: 19.4 abudefduf sexfasciatus 7.94 pomacentridae 16.0 medium top 5: 78.1 hemiglyphidodon plagiometopon 7.38 pomacentridae 18.0 medium others: 21.9 thalassoma lunare 6.85 labridae 25.0 medium labroides dimidiatus 6.78 labridae 11.5 medium 1.09 plectroglyphidodon lacrymatus 35.52 pomacentridae 10.0 small similarity: 12.0 neoglyphidodon nigroris 30.56 pomacentridae 13.0 medium top 5: 81.5 amblyglyphidodon curacao 7.14 pomacentridae 11.0 medium others: 18.5 apogon griffini 4.21 apogonidae 13.5 medium thalassoma lunare 4.08 pomacentridae 25.0 medium 2.01 ctenochaetus striatus 43.81 acanthuridae 26.0 medium similarity: 22.0 chrysiptera rex 6.12 pomacentridae 7.0 small top 5: 66.1 plectroglyphidodon lacrymatus 5.98 pomacentridae 10.0 small others: 33.9 zanclus cornutus 5.96 zanclidae 23.0 medium pomacentrus coelestis 4.23 pomacentridae 9.0 small 2.02 ctenochaetus striatus 23.65 acanthuridae 26.0 medium similarity: 39.3 chrysiptera rex 21.73 pomacentridae 7.0 small top 5: 83.8 plectroglyphidodon lacrymatus 19.81 pomacentridae 10.0 small others: 16.2 pomacentrus lepidogenys 14.23 pomacentridae 9.0 small pomacentrus bankanensis 4.35 pomacentridae 9.0 small 2.03 abudefduf sexfasciatus 14.18 pomacentridae 16.0 medium similarity: 21.1 chaetodon octofasciatus 13.87 chaetodontidae 12.0 medium top 5: 56.0 pomacentrus bankanensis 13.27 pomacentridae 9.0 small others: 44.0 chrysiptera rex 7.93 pomacentridae 7.0 small amblyglyphidodon curacao 6.74 pomacentridae 11.0 medium appendix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size national patterns of philippine reef fish diversity 34 2.04 pomacentrus moluccensis 21.31 pomacentridae 9.0 small similarity: 23.1 pomacentrus lepidogenys 18.72 pomacentridae 9.0 small top 5: 68.8 amblyglyphidodon curacao 13.84 pomacentridae 11.0 medium others: 31.2 pomacentrus bankanensis 9.04 pomacentridae 9.0 small plectroglyphidodon lacrymatus 5.85 pomacentridae 10.0 small 2.05 chromis atripectoralis 66.22 pomacentridae 12.0 medium similarity: 55.5 pomacentrus lepidogenys 6.48 pomacentridae 9.0 small top 5: 87.9 pomacentrus moluccensis 5.68 pomacentridae 9.0 small others: 12.1 amblyglyphidodon curacao 5.40 pomacentridae 11.0 medium neoglyphidodon nigroris 4.08 pomacentridae 13.0 medium 2.06 chrysiptera rex 25.25 pomacentridae 7.0 small similarity: 36.9 pomacentrus lepidogenys 24.04 pomacentridae 9.0 small top 5: 78.3 pomacentrus bankanensis 12.58 pomacentridae 9.0 small others: 21.7 thalassoma hardwicke 8.28 labridae 20.0 medium ctenochaetus striatus 8.15 acanthuridae 26.0 medium 2.07 pomacentrus simsiang 25.64 pomacentridae 7.0 small similarity: 11.3 thalassoma hardwicke 13.85 labridae 20.0 medium top 5: 68.1 pomacentrus opisthostigma 10.85 pomacentridae 6.5 small others: 31.9 labrichthys unilineatus 10.04 labridae 17.5 medium neoglyphidodon nigroris 7.72 pomacentridae 13.0 medium 2.08 scarus rivulatus 23.48 scaridae 40.0 large similarity: 19.5 pomacentrus alexanderae 14.93 pomacentridae 9.0 small top 5: 67.0 pomacentrus moluccensis 12.52 pomacentridae 9.0 small others: 33.0 amblyglyphidodon curacao 11.23 pomacentridae 11.0 medium pomacentrus simsiang 4.87 pomacentridae 7.0 small 2.09 pomacentrus chrysurus 71.09 pomacentridae 9.0 small similarity: 42.0 chrysiptera rex 9.73 pomacentridae 7.0 small top 5: 89.2 thalassoma hardwicke 3.11 pomacentridae 20.0 medium others: 10.8 scolopsis lineatus 2.72 nemipteridae 23.0 medium naso unicornis 2.59 acanthuridae 70.0 large appendix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size ja anticamara and others 35 2.10 pomacentrus chrysurus 40.35 pomacentridae 9.0 small similarity: 16.9 scarus rivulatus 11.85 scaridae 40.0 large top 5: 77.1 chrysiptera rex 9.11 pomacentridae 7.0 small others: 22.9 pomacentrus moluccensis 8.20 pomacentridae 9.0 small pomacentrus alexanderae 7.58 pomacentridae 9.0 small 2.11 chromis ternatensis 34.07 pomacentridae 10.0 small similarity: 24.7 chrysiptera cyanea 17.41 pomacentridae 8.5 small top 5: 77.7 pomacentrus burroughi 9.89 pomacentridae 8.5 small others: 22.3 pomacentrus alexanderae 8.93 pomacentridae 9.0 small hemiglyphidodon plagiometopon 7.39 pomacentridae 18.0 medium 2.12 pomacentrus moluccensis 16.84 pomacentridae 9.0 small similarity: 28.8 scarus rivulatus 16.61 scaridae 40.0 large top 5: 62.5 amblyglyphidodon curacao 14.49 pomacentridae 11.0 medium others: 37.5 chromis ternatensis 7.75 pomacentridae 10.0 small dischistodus prosopotaenia 6.80 pomacentridae 17.0 medium 2.13 pomacentrus coelestis 15.08 pomacentridae 9.0 small similarity: 17.4 scarus rivulatus 11.61 scaridae 40.0 large top 5: 53.1 pomacentrus chrysurus 11.03 pomacentridae 9.0 small others: 46.9 pomacentrus moluccensis 8.29 pomacentridae 9.0 small pomacentrus burroughi 7.07 pomacentridae 8.5 small 3.01 pseudanthias huchti 22.52 serranidae 12.0 medium similarity: 19.1 pomacentrus moluccensis 21.86 pomacentridae 9.0 small top 5: 59.6 pomacentrus brachialis 6.05 pomacentridae 8.0 small others: 40.4 chromis viridis 4.94 pomacentridae 8.0 small centropyge vroliki 4.26 pomacentridae 12.0 medium 3.02 acanthochromis polyacanthus 39.84 acanthuridae 14.0 medium similarity: 23.0 pomacentrus moluccensis 14.6 pomacentridae 9.0 small top 5: 67.5 chaetodon kleinii 4.71 chaetodontidae 15.0 medium others: 32.5 amblyglyphidodon curacao 4.63 pomacentridae 11.0 medium chaetodon lunulatus 3.71 chaetodontidae 14.0 medium appendix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size national patterns of philippine reef fish diversity 36 3.03 centropyge vroliki 30.32 pomacanthidae 12.0 medium similarity: 20.3 pomacentrus bankanensis 26.1 pomacentridae 9.0 small top 5: 91.4 thalassoma hardwicke 25.7 labridae 20.0 medium others: 8.6 plectroglyphidodon lacrymatus 5.24 pomacentridae 10.0 small pomacentrus coelestis 4.06 pomacentridae 9.0 small 3.04 ctenochaetus striatus 18.01 acanthuridae 26.0 medium similarity: 21.0 pomacentrus moluccensis 13.27 pomacentridae 9.0 small top 5: 53.3 dascyllus trimaculatus 9.85 pomacentridae 11.0 medium others: 46.7 centropyge vroliki 6.36 pomacanthidae 12.0 medium pomacentrus lepidogenys 5.76 pomacentridae 9.0 small 3.05 pomacentrus moluccensis 55.51 pomacentridae 9.0 small similarity: 18.1 pomacentrus bankanensis 9.45 pomacentridae 9.0 small top 5: 80.6 pomacentrus brachialis 5.30 pomacentridae 8.0 small others: 19.4 abudefduf vaigiensis 5.19 pomacentridae 20.0 medium pomacentrus chrysurus 5.12 pomacentridae 9.0 small 3.06 thalassoma lunare 28.15 labridae 25.0 medium similarity: 46.8 pomacentrus chrysurus 18.45 pomacentridae 9.0 small top 5: 78.2 scarus rivulatus 15.27 scaridae 40.0 large others: 21.8 pomacentrus simsiang 11.61 pomacentridae 7.0 small dascyllus trimaculatus 4.77 pomacentridae 11.0 medium 3.07 pomacentrus moluccensis 43.24 pomacentridae 9.0 small similarity: 32.6 pomacentrus chrysurus 14.21 pomacentridae 9.0 small top 5: 76.1 amblygliphidodon ternatensis 6.82 pomacentridae 10.0 small others: 23.9 neoglyphidodon melas 6.68 pomacentridae 18.0 medium amblyglyphidodon curacao 5.18 pomacentridae 11.0 medium 3.08 chaetodon kleinii 54.25 chaetodontidae 15.0 medium similarity: 26.2 plectroglyphidodon lacrymatus 20.13 pomacentridae 10.0 small top 5: 94.2 dascyllus trimaculatus 13.29 pomacentridae 11.0 medium others: 5.8 scarus rivulatus 4.51 scaridae 40.0 large dascyllus reticulatus 2.04 pomacentridae 9.0 small append ix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size ja anticamara and others 37 3.09 pomacentrus coelestis 58.18 pomacentridae 9.0 small similarity: 19.4 pomacentrus bankanensis 12.49 pomacentridae 9.0 small top 5: 87.0 chrysiptera cyanea 9.24 pomacentridae 8.5 small others: 13.0 centropyge vroliki 4.20 pomacanthidae 12.0 medium ctenochaetus striatus 2.94 acanthuridae 26.0 medium 3.10 plectroglyphidodon lacrymatus 27.83 pomacentridae 10.0 small similarity: 27.2 pomacentrus moluccensis 24.62 pomacentridae 9.0 small top 5: 75.2 amblyglyphidodon curacao 13.03 pomacentridae 11.0 medium others: 24.8 zebrasoma scopas 4.99 zanclidae 20.0 medium ctenochaetus striatus 4.77 acanthuridae 26.0 medium 3.11 thalassoma lunare 30.08 labridae 25.0 medium similarity: 26.0 pomacentrus chrysurus 19.54 pomacentridae 9.0 small top 5: 75.5 pomacentrus simsiang 12.87 pomacentridae 7.0 small others: 24.5 plectroglyphidodon lacrymatus 6.94 pomacentridae 10.0 small chromis ternatensis 6.07 pomacentridae 10.0 small 3.12 pomacentrus moluccensis 34.66 pomacentridae 9.0 small similarity: 35.0 pomacentrus burroughi 24.03 pomacentridae 8.5 small top 5: 74.3 chromis ternatensis 5.36 pomacentridae 10.0 small others: 25.7 sphaeramia nematoptera 5.23 apogonidae 8.5 small chaetodon octofasciatus 5.03 chaetodontidae 12.0 medium 3.13 dascyllus aruanus 41.73 pomacentridae 10.0 small similarity: 14.5 pomacentrus moluccensis 28.78 pomacentridae 9.0 small top 5: 82.8 pomacentrus alexanderae 5.76 pomacentridae 9.0 small others: 17.2 amphiprion clarkii 3.60 pomacentridae 15.0 medium amblyglyphidodon curacao 2.88 pomacentridae 11.0 medium 3.14 pomacentrus moluccensis 34.64 pomacentridae 9.0 small similarity: 19.6 pseudanthias tuka 14.82 serranidae 12.0 medium top 5: 83.2 pseudanthias huchti 13.63 serranidae 12.0 medium others: 16.8 caesio caerulaurea 12.67 caesionidae 35.0 large pomacentrus alexanderae 7.47 pomacentridae 9.0 small append ix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size national patterns of philippine reef fish diversity 38 appendix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size 3.15 scarus rivulatus 29.36 scaridae 40.0 large similarity: 33.2 chromis weberi 17.67 pomacentridae 13.5 medium top 5: 68.9 dascyllus trimaculatus 11.68 pomacentridae 11.0 medium others: 31.1 plectroglyphidodon lacrymatus 5.20 pomacentridae 10.0 small centropyge vroliki 5.03 pomacanthidae 12.0 medium 3.16 pomacentrus moluccensis 42.01 pomacentridae 9.0 small similarity: 33.9 caesio caerulaurea 23.14 caesionidae 35.0 large top 5: 90.7 amblyglyphidodon curacao 22.6 pomacentridae 11.0 medium others: 9.3 pomacentrus brachialis 1.83 pomacentridae 8.0 small neoglyphidodon nigroris 1.14 pomacentridae 13.0 medium 4.01 abudefduf vaigiensis 14.50 pomacentridae 20.0 medium similarity: 25.7 plectroglyphidodon lacrymatus 14.04 pomacentridae 10.0 small top 5: 61.4 ctenochaetus striatus 12.55 acanthuridae 26.0 medium others: 38.6 pomacentrus vaiuli 10.93 pomacentridae 10.0 small thalassoma hardwicke 9.34 labridae 20.0 medium 4.02 plectroglyphidodon lacrymatus 37.35 pomacentridae 10.0 small similarity: 14.12 halichoeres hortulanus 8.30 labridae 27.0 medium top 5: 62.7 pomacentrus moluccensis 6.69 pomacentridae 9.0 small others: 37.3 pomacentrus coelestis 5.62 pomacentridae 9.0 small thalassoma lunare 4.71 labridae 25.0 medium 4.03 pomacentrus simsiang 18.08 pomacentridae 7.0 small similarity: 11.3 plectroglyphidodon lacrymatus 8.57 pomacentridae 10.0 small top 5: 49.5 dascyllus reticulatus 8.2 pomacentridae 9.0 small others: 50.5 dischistodus prosopotaenia 7.62 pomacentridae 17.0 medium apogon griffini 7.0 apogonidae 13.5 medium 4.04 pomacentrus moluccensis 26.72 pomacentridae 9.0 small similarity: 34.3 pomacentrus adelus 21.94 pomacentridae 8.5 small top 5: 71.5 plectroglyphidodon lacrymatus 14.4 pomacentridae 10.0 small others: 28.5 thalassoma hardwicke 4.22 labridae 20.0 medium amblyglyphidodon curacao 4.2 pomacentridae 11.0 medium ja anticamara and others 39 appendix 3a table showing the top 5 dominant species in terms of abundance, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size 4.05 pomacentrus moluccensis 26.79 pomacentridae 9.0 small similarity: 20.2 chromis margaritifer 9.98 pomacentridae 9.0 small top 5: 62.8 pomacentrus simsiang 9.74 pomacentridae 7.0 small others: 37.2 dascyllus reticulatus 8.21 pomacentridae 9.0 small ctenochaetus striatus 8.06 acanthuridae 26.0 medium 4.06 cirrhilabrus cyanopleura 53.02 labridae 15.0 medium similarity: 22.7 pomacentrus lepidogenys 11.76 pomacentridae 9.0 small top 5: 83.9 scolopsis bilineatus 7.79 nemipteridae 23.0 medium others: 16.1 ctenochaetus striatus 6.7 acanthuridae 26.0 medium thalassoma lunare 4.65 labridae 25.0 medium 5.01 pomacentrus moluccensis 52.49 pomacentridae 9.0 small similarity: 27.0 pomacentrus chrysurus 10.42 pomacentridae 9.0 small top 5: 86.4 amblyglyphidodon curacao 10.34 pomacentridae 11.0 medium others: 13.6 chromis viridis 8.1 pomacentridae 8.0 small neoglyphidodon nigroris 5.02 pomacentridae 13.0 medium 5.02 acanthochromis polyacanthus 40.12 pomacentridae 14.0 medium similarity: 29.9 pomacentrus moluccensis 18.8 pomacentridae 9.0 small top 5: 74.3 pomacentrus lepidogenys 7.24 pomacentridae 9.0 small others: 25.7 plectroglyphidodon lacrymatus 4.17 pomacentridae 10.0 small ctenochaetus striatus 4 acanthuridae 26.0 medium 6.01 thalassoma lunare 24.91 labridae 25.0 medium similarity: 29.2 scarus rivulatus 20.96 scaridae 40.0 large top 5: 78.5 chlorurus sordidus 17.74 pomacentridae 40.0 large others: 21.5 halichoeres melanurus 7.55 labridae 12.0 medium chaetodon octofasciatus 7.37 chaetodontidae 12.0 medium 6.02 dascyllus reticulatus 20.63 pomacentridae 9.0 small similarity: 24.7 plectroglyphidodon lacrymatus 17.27 pomacentridae 10.0 small top 5: 67.1 ctenochaetus striatus 12.58 acanthuridae 26.0 medium others: 32.9 plectroglyphidodon dickii 9.35 pomacentridae 11.0 medium pomacentrus moluccensis 7.25 pomacentridae 9.0 small 6.03 pomacentrus lepidogenys 19.13 pomacentridae 9.0 small similarity: 34.3 acanthochromis polyacanthus 15.01 pomacentridae 14.0 medium top 5: 65.2 ctenochaetus striatus 12.12 acanthuridae 26.0 medium others: 34.8 plectroglyphidodon lacrymatus 11.63 pomacentridae 10.0 small centropyge vroliki 7.28 pomacanthidae 12.0 medium national patterns of philippine reef fish diversity 40 1.01 zanclus cornutus 38.46 zanclidae 23 medium similarity:20.7 chaetodon ornatissimus 25.62 chaetodontidae 20 medium top 5: 94.5 chaetodon kleinii 25.52 chaetodontidae 15 medium others: 5.5 sufflamen chrysopterus 2.79 balistidae 30 medium centropyge vroliki 2.14 pomacanthidae 12 medium 1.02 ctenochaetus striatus 28.29 acanthuridae 26 medium similarity: 37.4 chlorurus sordidus 20.20 scaridae 40 large top 5: 85.4 cheilinus chlorourus 13.92 labridae 45 large others: 14.6 zanclus cornutus 11.70 zanclidae 23 medium chaetodon kleinii 11.31 chaetodontidae 15 medium 1.03 halichoeres hortulanus 28.41 labridae 27 medium similarity: 17.9 ctenochaetus striatus 23.04 acanthuridae 26 medium top 5: 75.4 parupeneus multifasciatus 10.23 mullidae 35 large others: 24.6 thalassoma hardwicke 8.50 labridae 20 medium chlorurus sordidus 5.18 scaridae 40 large 1.04 epinephelus merra 16.80 serranidae 31 large similarity: 28.6 thalassoma lunare 14.72 labridae 25 medium top 5: 59.9 plectroglyphidodon lacrymatus 10.28 pomacentridae 10 small others: 40.1 halichoeres melanurus 9.33 labridae 12 medium labracinus cyclophthalmus 8.72 pseudochromidae 20 medium 1.05 ctenochaetus striatus 34.43 acanthuridae 26 medium similarity: 12.2 thalassoma hardwicke 24.79 labridae 20 medium top 5: 72.8 plectroglyphidodon lacrymatus 6.04 pomacentridae 10 small others: 27.2 thalassoma lunare 4.09 labridae 25 medium lutjanus decussatus 3.45 lutjanidae 35 large 1.06 dischistodus prosopotaenia 23.73 pomacentridae 17 medium similarity: 9.6 neoglyphidodon melas 21.73 pomacentridae 18 medium top 5: 69.2 dascyllus trimaculatus 10.46 pomacentridae 11 medium others: 30.8 choerodon anchorago 7.41 labridae 38 large plectroglyphidodon lacrymatus 5.86 pomacentridae 10 small appendix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) municipality code species contributory % family max tl (cm) body size ja anticamara and others 41 1.07 ctenochaetus striatus 38.67 acanthuridae 26 medium similarity: 13.3 thalassoma hardwicke 11.74 labridae 20 medium top 5: 72.3 balistapus undulatus 10.28 balistidae 30 medium others: 27.7 epinephelus merra 5.94 serranidae 31 large stegastes fasciolatus 5.69 pomacentridae 15 medium 1.08 thalassoma lunare 19.78 labridae 25 medium similarity: 17.5 ctenochaetus striatus 19.51 acanthuridae 26 medium top 5: 70.5 scolopsis margaritifer 12.77 nemipteridae 28 medium others: 29.5 arothron nigropunctatus 9.73 tetraodontidae 33 large thalassoma hardwicke 8.75 labridae 20 medium 1.09 lutjanus decussatus 10.22 lutjanidae 35 large similarity: 9.2 dischistodus prosopotaenia 9.35 pomacentridae 17 medium top 5: 44.1 plectroglyphidodon lacrymatus 8.86 pomacentridae 10 small others: 55.9 cheilinus chlorourus 8.18 labridae 45 large neoglyphidodon nigroris 7.49 pomacentridae 13 medium 2.01 ctenochaetus striatus 42.90 acanthuridae 26 medium similarity: 17.5 zanclus cornutus 13.85 zanclidae 23 medium top 5: 71.7 chaetodon vagabundus 7.50 chaetodontidae 23 medium others: 28.3 halichoeres hortulanus 4.40 labridae 27 medium thalassoma hardwicke 3.07 labridae 20 medium 2.02 ctenochaetus striatus 46.36 acanthuridae 26 medium similarity: 23.0 chlorurus sordidus 13.45 scaridae 40 large top 5: 76.7 parupeneus multifasciatus 5.99 mullidae 35 large others: 23.3 hemigymnus fasciatus 5.61 labridae 80 large plectroglyphidodon lacrymatus 5.31 pomacentridae 10 small 2.03 chlorurus sordidus 24.39 scaridae 40 large similarity: 16.5 scarus flavipectoralis 23.29 scaridae 40 large top 5: 70.1 lutjanus decussatus 10.55 lutjanidae 35 large others: 29.9 ctenochaetus striatus 6.29 chaetodontidae 26 medium zanclus cornutus 5.58 zanclidae 23 medium append ix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size national patterns of philippine reef fish diversity 42 2.04 lutjanus decussatus 15.06 lutjanidae 35 large similarity: 17.0 zanclus cornutus 13.37 zanclidae 23 medium top 5: 57.5 ctenochaetus striatus 12.83 acanthuridae 26 medium others: 42.5 halichoeres hortulanus 10.30 labridae 27 medium scolopsis bilineatus 5.94 nemipteridae 23 medium 2.05 zanclus cornutus 17.01 zanclidae 23 medium similarity: 25.8 chaetodon lunulatus 7.48 chaetodontidae 14 medium top 5: 42.6 hemigymnus fasciatus 6.71 labridae 80 large others: 57.4 chaetodontoplus mesoleucus 6.09 pomacanthidae 18 medium labrichthys unilineatus 5.27 labridae 17.5 medium 2.06 ctenochaetus striatus 38.67 acanthuridae 26 medium similarity: 18.7 thalassoma hardwicke 13.10 labridae 20 medium top 5: 77.6 chlorurus sordidus 11.99 scaridae 40 large others: 22.4 lutjanus decussatus 8.58 lutjanidae 35 large chaetodon citrinellus 5.27 chaetodontidae 13 medium 2.07 choerodon anchorago 31.69 labridae 38 large similarity: 12.3 thalassoma hardwicke 24.77 labridae 20 medium top 5: 80.0 pomacentrus simsiang 10.24 pomacentridae 7 small others: 20.0 siganus unimaculatus 7.52 siganidae 20 medium amblyglyphidodon curacao 5.33 pomacentridae 11 medium 2.08 scarus rivulatus 24.38 scaridae 40 large similarity: 17.6 chlorurus sordidus 20.28 scaridae 40 large top 5: 63.3 lutjanus decussatus 10.05 lutjanidae 35 large others: 36.7 hemigymnus melapterus 4.73 labridae 90 large chaetodon octofasciatus 3.89 chaetodontidae 12 medium 2.09 thalassoma hardwicke 24.86 labridae 20 medium similarity: 12.3 lutjanus decussatus 12.78 lutjanidae 35 large top 5: 68.1 cheilinus chlorourus 10.75 lutjanidae 45 large others: 31.9 pomacentrus chrysurus 10.59 pomacentridae 9 small scolopsis lineatus 9.14 nemipteridae 23 medium appendix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size ja anticamara and others 43 2.10 scarus rivulatus 24.85 scaridae 40 large similarity: 15.6 choerodon anchorago 18.49 labridae 38 large top 5: 67.3 thalassoma hardwicke 13.17 labridae 20 medium others: 32.7 halichoeres melanurus 6.91 labridae 12 medium coris batuensis 3.88 labridae 17 medium 2.11 hemiglyphidodon plagiometopon27.14 labridae 18 medium similarity: 20.4 chaetodontoplus mesoleucus 15.78 pomacanthidae 18 medium top 5: 72.5 scarus rivulatus 14.19 scaridae 40 large others: 27.5 hemigymnus melapterus 8.20 labridae 90 large chaetodon octofasciatus 7.15 chaetodontidae 12 medium 2.12 scarus rivulatus 27.12 scaridae 40 large similarity: 28.4 dischistodus prosopotaenia 22.69 pomacentridae 17 medium top 5: 70.9 lutjanus decussatus 8.91 lutjanidae 35 large others: 29.1 halichoeres chloropterus 6.90 labridae 19 medium choerodon anchorago 5.24 labridae 38 large 2.13 scarus rivulatus 32.13 scaridae 40 large similarity: 15.2 hemigymnus melapterus 10.07 labridae 90 large top 5: 61.5 choerodon anchorago 8.92 labridae 38 large others: 38.5 scolopsis bilineatus 5.19 nemipteridae 23 medium hemiglyphidodon plagiometopon 5.17 labridae 18 medium 3.01 thalassoma lunare 9.07 labridae 25 medium similarity: 13.2 chaetodon baronessa 7.44 chaetodontidae 16 medium top 5: 63.6 zebrasoma scopas 7.17 zanclidae 20 medium others: 36.4 pomacentrus moluccensis 6.40 pomacentridae 9 small chaetodon kleinii 6.31 chaetodontidae 15 medium 3.02 chaetodon lunulatus 22.15 chaetodontidae 14 medium similarity: 19.8 chaetodon baronessa 8.29 chaetodontidae 16 medium top 5: 50.6 ctenochaetus striatus 7.24 acanthuridae 26 medium others: 49.4 halichoeres hortulanus 6.84 labridae 27 medium thalassoma lunare 6.03 labridae 25 medium appendix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size national patterns of philippine reef fish diversity 44 3.03 thalassoma hardwicke 54.44 labridae 20 medium similarity: 11.7 centropyge vroliki 17.48 pomacanthidae 12 medium top 5: 94.0 pomacentrus bankanensis 11.98 pomacentridae 9 small others: 6.0 halichoeres hortulanus 8.18 labridae 27 medium bodianus mesothorax 1.94 labridae 25 medium 3.04 ctenochaetus striatus 17.56 chaetodontidae 26 medium similarity: 18.7 thalassoma lunare 12.39 lutjanidae 25 medium top 5: 55.2 chaetodon vagabundus 10.3 chaetodontidae 23 medium others: 45.8 parupeneus multifasciatus 8.37 mullidae 35 large zebrasoma scopas 6.60 acanthuridae 20 medium 3.05 thalassoma lunare 14.11 scaridae 25 medium similarity: 9.9 chaetodon baronessa 8.90 chaetodontidae 16 medium top 5: 46.9 plectorhinchus vittatus 8.56 haemulidae 72 large others: 53.1 halichoeres melanurus 8.48 labridae 12 medium neoglyphidodon nigroris 6.84 pomacentridae 13 medium 3.06 thalassoma lunare 53.96 labridae 25 medium similarity: 40.6 scolopsis bilineatus 18.61 nemipteridae 23 medium top 5: 85.9 cephalopholis boenak 5.70 serranidae 30 medium others: 14.1 halichoeres melanurus 3.91 labridae 12 medium scarus rivulatus 3.70 scaridae 40 large 3.07 labracinus cyclophthalmus 25.50 pseudochromidae 20 medium similarity: 19.9 thalassoma hardwicke 8.02 labridae 20 medium top 5: 53.7 chaetodontoplus mesoleucus 7.95 chaetodontidae 18 medium others: 46.3 halichoeres chloropterus 6.59 labridae 19 medium thalassoma lunare 5.64 labridae 25 medium 3.08 chaetodon kleinii 21.83 chaetodontidae 15 medium similarity: 11.3 plectroglyphidodon lacrymatus 13.36 chaetodontidae 10 small top 5: 64.3 dascyllus trimaculatus 11.14 chaetodontidae 11 medium others: 35.7 scarus rivulatus 10.27 scaridae 40 large dascyllus reticulatus 7.73 chaetodontidae 9 small appendix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size ja anticamara and others 45 3.09 centropyge vroliki 17.0 pomacanthidae 12 medium similarity: 11.2 thalassoma lunare 13.44 labridae 25 medium top 5: 63.7 chaetodon kleinii 12.92 chaetodontidae 15 medium others: 36.3 dascyllus trimaculatus 10.52 pomacentridae 11 medium pomacentrus bankanensis 9.84 pomacentridae 9 small 3.10 balistapus undulatus 30.72 balistidae 30 medium similarity: 25.5 ctenochaetus striatus 11.77 chaetodontidae 26 medium top 5: 67.5 chaetodon baronessa 9.09 chaetodontidae 16 medium others: 32.5 chaetodon lunulatus 8.32 chaetodontidae 14 medium zebrasoma scopas 7.59 acanthuridae 20 medium 3.11 thalassoma lunare 40.76 labridae 25 medium similarity: 16.8 halichoeres chloropterus 13.99 labridae 19 medium top 5: 85.3 halichoeres melanurus 13.64 labridae 12 medium others: 14.7 pomacentrus chrysurus 8.68 pomacentridae 9 small pomacentrus simsiang 8.23 pomacentridae 7 small 3.12 neoglyphidodon melas 22.17 pomacentridae 18 medium similarity: 19.1 chlorurus sordidus 14.38 scaridae 40 large top 5: 57.4 scarus quoyi 8.25 scaridae 40 large others: 42.6 scarus dimidiatus 6.36 scaridae 40 large chaetodontoplus mesoleucus 6.21 pomacanthidae 18 medium 3.13 thalassoma lunare 29.49 labridae 25 medium similarity: 43.1 parupeneus multifasciatus 10.86 mullidae 35 large top 5: 66.7 scolopsis bilineatus 9.96 nemipteridae 23 medium others: 33.3 chaetodon baronessa 8.56 chaetodontidae 16 medium centropyge vroliki 7.81 pomacanthidae 12 medium 3.14 thalassoma hardwicke 17.82 labridae 20 medium similarity: 13.4 scarus niger 9.63 scaridae 40 large top 5: 48.7 acanthurus lineatus 7.72 acanthuridae 38 large others: 51.3 chlorurus sordidus 7.51 scaridae 40 large melichthys vidua 6.06 balistidae 40 large appendix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size national patterns of philippine reef fish diversity 46 append ix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size 3.15 ctenochaetus striatus 27.04 acanthuridae 26 medium similarity: 24.2 chaetodon kleinii 13.11 chaetodontidae 15 medium top 5: 63.5 thalassoma lunare 10.32 labridae 25 medium others: 36.5 dascyllus trimaculatus 6.74 pomacentridae 11 medium scarus rivulatus 6.31 scaridae 40 large 3.16 pygoplites diacanthus 20.12 pomacanthidae 25 medium similarity: 13.1 chlorurus bleekeri 13.20 scaridae 49 large top 5: 63.5 chlorurus sordidus 12.29 scaridae 40 large others: 36.5 platax boersii 10.82 ephippidae 40 large hemigymnus fasciatus 7.05 labridae 80 large 4.01 ctenochaetus striatus 35.59 acanthuridae 26 medium similarity: 19.9 acanthurus lineatus 13.87 acanthuridae 38 large top 5: 67.3 thalassoma hardwicke 10.03 labridae 20 medium others: 32.7 parupeneus multifasciatus 4.61 mullidae 35 large chaetodon vagabundus 3.25 chaetodontidae 23 medium 4.02 acanthurus lineatus 22.18 acanthuridae 38 large similarity:14.6 ctenochaetus striatus 12.58 acanthuridae 26 medium top 5: 61.6 epinephelus merra 10.70 serranidae 31 large others: 38.4 halichoeres hortulanus 8.09 labridae 27 medium chaetodon baronessa 8.02 chaetodontidae 16 medium 4.03 plectorhinchus chaetodonoides 12.89 haemulidae 72 large similarity:13.0 hemiglyphidodon plagiometopon 9.65 pomacentridae 18 medium top 5: 44.3 dischistodus prosopotaenia 7.69 pomacentridae 17 medium others: 55.7 acanthurus auranticavus 7.38 acanthuridae 35 large pentapodus bifasciatus 6.72 nemipteridae 18 medium 4.04 ctenochaetus striatus 15.95 acanthuridae 26 medium similarity:18.0 thalassoma hardwicke 11.24 labridae 20 medium top 5: 44.0 thalassoma lunare 6.69 labridae 25 medium others: 56.0 chlorurus sordidus 5.87 scaridae 40 large lutjanus decussatus 4.29 lutjanidae 35 large ja anticamara and others 47 append ix 3b table showing the top 5 dominant species in terms of biomass, based on bray-curtis simper analysis within municipalities, for each municipality, arranged from north to south of the philippines. names of each municipality and biogeographic region can be found in appendix 1. also shown are the average within-municipality similarity % (“similarity”), the total contributory % of the top 5 dominant species for each municipality (“top 5”), and the total contributory % of all other species not included in the top 5 (“others”) (cont’n.) municipality code species contributory % family max tl (cm) body size 4.05 ctenochaetus striatus 30.01 acanthuridae 26 medium similarity:13.1 chaetodon lunulatus 12.9 chaetodontidae 14 medium top 5: 59.1 zebrasoma scopas 6.64 acanthuridae 20 medium others: 40.9 balistapus undulatus 5.85 balistidae 30 medium pomacentrus moluccensis 3.74 pomacentridae 9 small 4.06 ctenochaetus striatus 49.75 acanthuridae 26 medium similarity:18.2 scolopsis bilineatus 19.54 nemipteridae 23 medium top 5: 89.0 chaetodon kleinii 8.7 chaetodontidae 15 medium others: 11.0 thalassoma lunare 7.24 labridae 25 medium centropyge bicolor 3.72 pomacanthidae 15 medium 5.01 pomacentrus chrysurus 17.35 pomacentridae 9 small similarity:15.7 pomacentrus moluccensis 16.27 pomacentridae 9 small top 5: 64.9 thalassoma lunare 11.6 labridae 25 medium others: 35.1 neoglyphidodon nigroris 10.22 pomacentridae 13 medium chaetodon octofasciatus 9.49 chaetodontidae 12 medium 5.02 balistapus undulatus 15.29 balistidae 30 medium similarity:20.9 ctenochaetus striatus 12.64 chaetodontidae 26 medium top 5: 43.7 thalassoma hardwicke 6.5 labridae 20 medium others: 56.3 parupeneus multifasciatus 4.79 mullidae 35 large naso lituratus 4.52 acanthuridae 46 large 6.01 chlorurus sordidus 55.2 scaridae 40 large similarity:28.7 thalassoma lunare 22.12 labridae 25 medium top 5: 95.6 ctenochaetus striatus 11.16 acanthuridae 26 medium others: 4.4 cephalopholis argus 5 serranidae 60 large chaetodon octofasciatus 2.17 chaetodontidae 12 medium 6.02 ctenochaetus striatus 37.91 acanthuridae 26 medium similarity:22.3 balistapus undulatus 6.7 balistidae 30 medium top 5: 60.4 thalassoma hardwicke 5.67 labridae 20 medium others: 39.6 plectroglyphidodon dickii 5.38 pomacentridae 11 medium plectroglyphidodon lacrymatus 4.74 pomacentridae 10 small 6.03 ctenochaetus striatus 22.99 acanthuridae 26 medium similarity: 29.0 balistapus undulatus 12.09 balistidae 30 medium top 5: 56.6 parupeneus multifasciatus 8.28 mullidae 35 large others: 43.4 zanclus cornutus 7.16 zanclidae 23 medium thalassoma hardwicke 6.07 labridae 20 medium 2editor's note.pmd 1 from the editor in this issue, we are featuring four articles from different branches of the sciences. from the f ield of environmental science, we have an ar ticle on cetacean watching in the southern tañon strait protected seascape and how the participatory process has guided the environmental management efforts of the various stakeholders in the cetacean watching industry. the second article is a contribution from mathematics and focuses on a method in solving certain non-linear singular partial differential equations. the third a r t i c l e , f r o m c h e m i s t r y, d e m o n s t r a t e s t h e u s e f u l n e s s o f t h e i o n chromatographic method in the determination of monochloroacetic acid in swimming pool waters. finally, from biology, we have the ar ticle on accessioned specimens in the jose vera santos memorial herbarium, which is located at the institute of biology in up diliman. we a r e a l s o fe a t u r i n g o n e co m m u n i c a t i o n s p a p e r a n a l y z i n g t h e characteristics of typhoon yolanda (international name: haiyan) vis-à-vis data on the characteristics of other tropical cyclones archived in the ibtracs database, to indicate the importance of science in disaster mitigation. finally, we are adding a compilation of the abstracts of the patents, trademarks, industrial designs, and other intellectual property registrations awarded to up diliman inventors, researchers, and creative workers. it is envisioned that these information will encourage the up community to f ile more patents and other means of intellectual property protection. in this regard, the off ice of the vice-chancellor for research and development is on hand to assist up diliman constituents. we wish for 2014 to be a more productive year for everyone. happy holidays. marco nemesio e. montaño, phd editor-in-chief issn 0115-7809 print/issn 2012-0818 online sdinside front cove-december2013-revised.pmd july-december 2013 • volume 25 no. 2 international advisory board science diliman issn 0115-7809 science diliman is published semi-annually (june a n d d e c e m b e r ) b y t h e u n i v e r s i t y o f t h e philippines diliman through the off ice of the vice-chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor-in-chief marco nemesio e. montaño, phd associate editors ma. patricia v. azanza, phd jose maria p. balmaceda, phd carlos primo c. david, phd joel joseph s. marciano jr. , phd jonas p. quilang, phd arnel a . salvador, phd irene m. villaseñor, phd managing editor violeda a. umali, phd editorial assistants epifania m. domingo dercylis g. mararac on the cover: a group of spinner dolphins (stenella longirostris) leaping of the water surface in the southern tañon strait protected seascape. photo taken by apple kristine s. amor of the institute of environmental science and meteorology, university of the philippines diliman. rigoberto c. advincula, phd department of chemistry university of houston alfonso m. albano, phd department of physics bryn mawr college, bryn mawr, pennsylvania kenneth buckel, phd food science and technology group school of chemical sciences and engineering the university of new south wales sydney, australia jose b. cruz, phd department of electrical and computer engineering ohio state university flor crisanta f. galvez, phd quality assurance and technical manager kerry ingredients and flavours (americas region) 7989-82nd st. , delta, vc v4g 1l7, canada victor c. gavino, phd department of nutrition university of montreal, canada kelvin s. rodolfo, phd department of earth and environmental sciences university of illinois, chicago, illinois rudolf a. roemer, phd centre for scientif ic computing and department of physics university of warwick luis g. sison, phd electrical and electronics engineering institute university of the philippines diliman raul k. suarez, phd department of ecology, evolution and marine biology university of california, sta. barbara contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement 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(adapted with permission from the editors of ieee sensors journal) short term assessment of phytoplankton in cebu and subic bay 32 science diliman (july-december 2018) 30:2, 32-53 short-term assessment of phytoplankton composition and abundance in cebu and subic bay ports, phil ippines nero m. austero* maritime and underwater cultural heritage division national museum of the philippines rhodora v. azanza marine science institute college of science university of the philippines national academy of science and technology department of science and technology abstract a short-term study to evaluate the composition and abundance of marine d i a t o m s a n d d i n o f l a g e l l a t e s i n t w o m a j o r p o r t s i n t h e c o u n t r y w a s c o n d u c t e d i n m a y 2 0 1 5 a n d j u l y 2 0 1 5 . p s e u d o n i t s z c h i a s p p . b l o o m c o m p r i s i n g a b o u t 4 2 . 3 % o f t h e t o t a l p h y t o p l a n k t o n p o p u l a t i o n w a s o b s e r v e d i n c e b u i n t e r n a t i o n a l p o r t i n m a y 2 0 1 5 . f u r t h e r m o r e , c h a e t o c e r o s s p p . c o m p r i s e d a b o u t 5 3 . 5 8 % o f t h e t o t a l p h y t o p l a n k t o n p o p u l a t i o n i n n a v a l s u p p l y d e p o t ( n s d ) t e r m i n a l i n m a y 2 0 1 5 , w h i l e t h a l a s s i o n e m a s p p . a n d l e p t o c y l i n d r u s s p p . a c c o u n t e d f o r 5 0 . 1 6 % a n d 34.78%, respectively, of the total phytoplankton population in july 2015. bloom-forming and potentially harmful species including diatoms, such a s co s c i n o d i s c u s s p p. , n i t z s c h i a s p p . , a n d p s e u d o n i t z s c h i a s p p . , a n d d i n of l a g e l l a t e s , s u c h a s ce r a t i u m s p p. , ce r a t i u m f u r ca , g o n ya u l a x s p p. , g y m n o d i n i u m s p p. , l i n g u l u d i n i u m s p p. , p h a l a c r o m a s p p. , p r o r o ce n t r u m m i ca n s , p r o r o ce n t r u m s p p. , a n d t h e i o cu n e s co l i s ted h a r m f u l a l g a l bloom (hab) species dinophysis caudate, were also recorded. the results o f t h i s s t u d y c o n t r i b u t e t o t h e e s t a b l i s h m e n t o f b a s e l i n e d a t a f o r phytoplankton composition and abundance, which are necessary for the identif ication of potentially toxic/harmful microalgae which pose risks of ballast water inclusion and transport. keywords: p h y t o p l a n k t o n , p o r t s , h a r m f u l a l g a l b l o o m s , d i a t o m s , dinoflagellates _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online n.m. austero and r.v. azanza 33 introduction phytoplankton are predominant aquatic microalgae and play important role in the marine and estuarine ecosystem. they signif icantly contribute to biological productivity (arumugam et al. 2005; sharma et al. 2015) and are the f irst biological community to respond to environmental changes (kozak et al. 2015). however, excessive growth and accumulation of phytoplankton in response to increased favorable conditions attributed to anthropogenic and chemical pollutions have caused recurrent phytoplankton blooms known as “red tides,” which have had negative impacts in many coastal waters (corrales and maclean 1995; azanza and taylor 2001; wong and wong 2003; yap et al. 2004; chang et al. 2009; furio et al. 2012; yñiguez et al. 2012). the inevitable impacts of the worldwide harmful algal bloom occurrences on biodiversity, economics, and human health, have drawn the attention of the scientif ic community and have prompted the use of mitigation and control methods (padilla et al. 2010). harmful algal blooms (habs) have been a recurring phenomenon causing havoc in the many affected areas in the philippines and the rest of the southeast asian region (corrales and maclean 1995; azanza and taylor 2001; hansen et al. 2004; wang et al. 2008; yñiguez et al. 2012). associated with paralytic shellf ish poisoning (psp) and even fatalities, hab events in the country have been primarily caused by the toxic dinoflagellates pyrodinium bahamense var. compresum and alexandrium species, which greatly affected manila bay and other 31 bays/coastal areas in the country from 1983 to 2013 (azanza et al. 2004; sombrito et al. 2004; azanza and benico 2013). microalgal researches in the country have been mainly focused on major bays affected by habs and less focalized on major ports with high vessel traff ic where risk of ballast water inclusion and transport of hab species are more likely. the inclusion and survival of phytoplankton in ballast tanks of vessels have already been well documented in many studies all over the world. in this study, two of the country’s major ports were studied to evaluate the composition and abundance of marine diatoms and dinoflagellates, which are necessary baseline data for the identif ication of potentially toxic/harmful microalgae that pose risks of being transported from one port to another via ballast water movement. n.m. austero and r.v. azanza 35 250-ml seawater samples were also collected to measure chlorophyll-a values and nutrient (po 4 , sio 3 , no 3 , no 2 and nh 3 ,) content using the modif ied method described by strickland and parsons (1972). biological parameters phytoplankton samples were collected from 10-m depths by towing vertically from the water column using a plankton net with a 0.35-m mouth-diameter and 25-µm mesh size. to ensure the capture of rare taxa, the plankton net was towed three times (equivalent to three subsamples). for the quantitative analysis, a standard rotor flow meter was also attached to determine the f iltered water volume. the samples were subsequently preserved with lugol’s solution and used for qualitative analysis. enumeration and counting of phytoplankton was performed using sedgwick-rafter counting chamber based on microscopic methods for quantitative phytoplankton analysis following the techniques of corrales et al. (1995) and azanza (1997). phytoplankton taxonomic identif ication was based on tomas (1997) and yamaji (1984) up to the lowest taxonomic level possible. images of phytoplankton species were also captured using the dinolite eye-piece and carl zeiss axio-cam. statistical analysis prior to various univariate and multivariate analysis, all data were tested for normality with shapiro – wilks normality test available in r v. 3.2.2. non-normal data were transformed (e.g. , sine, cosine, square root, etc.) (table 1). table 1. shapiro-wilks normality tests temperature none 0.1819 salinity none 0.09651 chl-a log 0.07606 cip inorganic n none 0.165 po 4 none 0.06551 sio 3 none 0.1794 temperature none 0.484 salinity sin 0.3055 chl-a none 0.2549 nsd inorganic n none 0.7597 po 4 none 0.6599 sio 3 none 0.4319 inorganic n transformations ρρρρρ-value short term assessment of phytoplankton in cebu and subic bay 38 table 2. abundance (cells/l iter) and composition of phytoplankton taxa identified from port water sampled in cip, cebu and nsd terminal, subic in may 2015 and july 2015 bacillariophyceae amphora ehrenberg ex kutzing 1844 22 119 auricula castracane 1873 120 bacillaria gmelin 1791 4 13 310 bacteriastrum shadbolt 1854 119 43 3,494 cerataulina peragallo ex schutt 1896 217 12 120 472 chaetoceros ehrenberg 1844* 1,606 242 8,198 17,247 cocconeis ehrenberg 1837 251 102 59 136 corethron castrane 1886 7 coscinodiscus ehrenberg 1839** 15 266 98 1,651 cyclotella (kutzing) brebisson 1838 77 cylindrotheca rabenhorst 1859 15 cymbella agardh 1830 31 diploneis ehrenberg 1854 4 ditylum bailey 1861 248 57 144 fragilaria lyngbye 1819 47 1,986 ethmodiscus castracane 1886 21 guinardia peragallo 1892 128 96 419 helicotheca ricard 1987 54 93 himiaulus heiberg 1863 30 87 leptocylindrus cleve 1889* 4,232 149,426 licmophora agardh 1827 10 98 196 26 lithodesmium ehrenberg 1839 39 37 melosira agardh 1824 67 129 237 386 navicula bory de st. vincent 1822 32 117 15 102 nitszchia hassall 1845** 25 193 105 41 odontella agardh 1832 147 33 26 21,792 planktoniella schutt 1892 6 pleurosigma smith 1852 822 487 117 pseudo-nitszchia peragallo 1900** 42,246 179 15 119 rhizosolenia brightwell 1858* 627 71 52 707 skeletonema greville 1865* 3,788 296 237 stephanopyxis ehrenberg 1845 6 surirella turpin 1828 34 11 thalassionema grunow ex mereschkowsky 6 229 486 215,487 thalassiosira cleve 1873* 4,840 121 235 11,397 triceratium ehrenberg 1839 12 15 dinophycea ceratium schrank 1793** 81 19 203 78 ceratium furca claparede and lachmann 1859** 110 23 107 151 dinophysis caudata saville-kent 1881*** 15 62 diplopsalis berg 1881 42 51 814 495 gonyaulax diesing 1866** 361 253 gonyaulax polygramma stein 1883 16 21 232 61 cip cebu nsd subic may july may july taxonomy n.m. austero and r.v. azanza 39 in addition, cip recorded 46 phytoplankton taxa comprising 31 diatoms, 15 dinoflagellates, and one blue green alga. twenty-three diatoms and 13 dinoflagellates were identif ied in may, while 24 diatoms and 13 dinoflagellates were recorded in july. the most abundant taxa pseudo-nitzschia spp. accounted for 42.3% of the total phytoplankton population during the month of may. commonly known taxa identif ied include cerataulina spp. , chaetoceros spp. , dytilum spp. leptocylindrus spp. , rhizosolenia spp. , skeletonema spp. , thalassiosira spp.; common diatoms, such as chaetoceros spp. , coscinodsicus spp. , navicula spp. , nitzschia spp. , pleurosigma spp. , pseudo-nitzschia spp. , rhizosolenia spp. , thalassionema spp.; and dinoflagellates prorocentrum spp. and protoperidium spp. (table 2). on the other hand, 47 phytoplankton taxa comprising 31 diatoms and 16 dinoflagellates were identif ied in the nsd terminal (table 2). seventeen diatoms and 14 dinoflagellates were identif ied in may, which consisted mainly of chaetoceros spp. (53.58% of the total phytoplankton population). diatoms melosira spp. , thalassionema spp. , thalassiosira spp. , and dinoflagellates ceratium spp. , diplopsalis spp. , gonyaulax spp. , pyrophacus spp. , prorocentrum spp. , protoperidinium spp. were cip cebu table 2. abundance (cells/l iter) and composition of phytoplankton taxa identified from port water sampled in cip, cebu and nsd terminal, subic in may 2015 and july 2015 (cont’n.) nsd subic may july may july taxonomy gymnodinium stein 1878** 146 gyrodinium kofoid and swezy 1921 4 27 linguludinium wall 1967** 48 15 oxyphysis kofoid 1926 211 peridinium ehrenberg 1832 4 184 76 169 phalacroma stein 1883** 10 6 61 prorocentrum micans ehrenberg 1833** 18 42 99 179 prorocentrum ehrenberg 1883** 10 140 1,002 237 protoperidinium berg 1881* 542 50 1,863 1,461 pyrophacus stein 1883 28 21 262 126 scrippsiella balech ex loebelich iii 1965 89 41 scrippsiella trochoidea (stein) loeblich iii 1976* 59 218 cyanophyceae trichodesmium ehrenberg ex gomont 1892 298 22 26 total number of taxa bacillariophyceae 23 24 17 30 dinophyceae 13 13 14 13 cyanophyceae 1 1 1 note: (*) bloom-forming species, (**) potentially habs, (***) ioc unesco listed habs. short term assessment of phytoplankton in cebu and subic bay 40 also present. moreover, 30 diatoms and 13 dinoflagellates were identif ied in july. thalassionema spp. accounted for 50.16%, while leptocylindrus spp. accounted for 34.78% of the total phytoplankton population. diatoms cerataul ina s p p. , chaetoceros spp. , coscinodiscus spp. , fragillaria spp. , odontella spp. , thalassiosira spp. , and dinoflagellates diplopsalis spp. and protoperidinium spp. were also present. shannon diversity index and simpson dominance index in cip, the shannon diversity index (h) value was highest in stations 2 and 3 (kruskal, p= 1.83e-02), while the highest dominance (d) value was observed in station 1 (kruskal, p= 1.83e-02) in may 2015. on the other hand, in july 2015, the h values measured from station 1 to station 3 are 3.12, 2.20, and 2.86, respectively (table 2). in addition, high h values of 0.81, 0.69, and 0.64 for station 1 to station 3, respectively, were also observed in nsd terminal during the month of may. in addition, the h values measured from station 1 to station 3 were 1.37, 1.21, and 1.06, respectively, in july. the highest d value was measured in station 3 (table 3). table 3. variation in phytoplankton d iversity ind ices in d ifferent stations (stn01-stn03) in two sampl ing ports (cip, cebu and nsd, subic) in may 2015 and july 2015 shannon (h) stn01 0.65 3.12 2.15 1.37 stn02 1.90 2.20 1.86 1.21 stn03 1.93 2.86 1.70 1.06 simpson (d) stn01 0.72 0.06 0.19 0.34 stn02 0.22 0.27 0.31 0.39 stn03 0.24 0.09 0.36 0.59 note: diversity index values were measured using past3 (ver. 1.0). cebu international port naval supply depot may july may july principal component analysis (pca) in cip, four of the f ive principal components (pcs) with eigenvalues greater than one account for 95.47% variability in may and july (table 4). about 73.57% of the variation was explained by the f irst two axes (figure 4). the f irst pc (dim1) explains 58.69% of the variation that revealed high loadings for sio 3 , nh 3 , no 3 po 4 , temperature, and salinity with abundant taxa dytilum, rhizosolenia, thalassionema, chaetoceros, pleurosigma, and cerataulina. the second pc (14.88%) obtained high loadings for sio 3 with pseudonitzschia, skeletonema, and odontella. pc3 (12.41%) short term assessment of phytoplankton in cebu and subic bay 42 in nsd, on the other hand, four of the f ive pcs that exhibited eigenvalues greater than one accounted for 96.58% variability in may and july (table 5). around 69.47% of the variation was explained by the f irst two pcs (figure 5). the f irst pc (dim1) explains 38.15% of the variation that revealed high loadings for temperature, salinity, no 2 , nh 3 , po 4 , and sio 3 with abundant taxa thalassionema and coscinodiscus. the second pc (31.35%) obtained high loadings for abundant taxa odontella, chaetoceros, protoperidinium, fragillaria, and thalassiosira. pc3 (18.25%) had the highest loadings for leptocylindrus, protoperidinium, and cerataulina, whereas the highest loadings in pc4 (8.86%) was for no 3 . eigenvalue 6.481 5.328 3.103 1.506 0.582 % of variation 38.125 31.345 18.250 8.861 3.421 cum. % or variation 38.125 69.468 87.718 96.579 100.000 component loadings temperature 0.86592 -0.44676 0.14010 -0.16179 0.06922 salinity -0.96457 -0.04542 0.01823 -0.25923 0.00024 no 2 0.92640 0.27654 0.10119 -0.21296 0.09854 no 3 -0.57761 0.11300 0.20498 -0.70465 0.33917 nh 3 0.67018 0.45869 0.51562 -0.24921 -0.11173 po 4 0.81348 -0.07143 0.47928 -0.18012 -0.04427 sio 3 -0.93228 0.32153 0.14050 -0.07588 0.26644 leptocylindrus 0.30362 0.59502 -0.74174 0.00518 0.05962 thalassionema -0.84456 0.35621 -0.33028 0.02809 -0.22352 odontella 0.17453 -0.93818 0.20461 0.20198 -0.08181 chaetoceros -0.33899 0.81073 0.14595 0.42296 -0.16612 coscinodiscus 0.66902 0.53795 -0.49476 -0.03567 0.13027 protoperidinium -0.18876 0.65360 0.71367 0.09521 -0.13705 diplopsalis 0.24206 0.41979 0.46947 0.64451 -0.35971 cerataulina 0.27101 0.41564 -0.86572 0.05977 0.02739 fragillaria -0.01500 -0.97752 -0.01239 0.18260 -0.10355 thalassiosira -0.25074 -0.82393 0.26151 0.29603 0.31975 table 5. important pcs (eigenvalues >1) from variables describing nsd, subic with high component load ings shown in bold variables dim. 1 dim. 2 dim. 3 dim. 4 dim. 5 environmental cond itions summary of the multivariate analysis of variances (manova) of physico-chemical parameters, such as temperature, salinity, chlorophyll-a, and nutrients, between months in cip are shown in table 6. average ambient water temperature (29.50oc and 27.88oc, p=0.0009643) (figure 6a), salinity (34.38 ppt and 35.14 ppt, p=0.0007995) (figure 6b), chlorophyll-a (0.43 µg/l short term assessment of phytoplankton in cebu and subic bay 46 temperature 1 2.1961 2.19615 36.756 0.003737 salinity 1 0.21373 0.21373 22.237 0.009201 chl-a 1 0.0024 0.0024 0.0615 0.8163 inorganic n 1 38.913 38.913 119.82 0.0003957 po 4 1 0.104017 0.104017 7.3945 0.05303 sio 3 1 21.3948 21.3948 33.457 0.004438 dfvariables sum sq mean sq f value ρρρρρ-value table 7. summary of manova among variables in nsd, subic in may 2015 and july 2015 discussion the results of this study are essential for the establishment of the baseline data of the most common phytoplankton found in the two major ports in the philippines. results revealed diatoms to be the most common and dominant taxa in both cebu and subic ports as indicated by high chlorophyll-a concentrations. this result supports wang and wang (2011) who proposed that chlorophyll-a concentration is indicative of phytoplankton biomass in response to certain environmental factors. in manila bay, diatoms have also been observed as the most dominant taxa in all seasons from 1997 to 1998 (azanza and miranda 2001), while algal bloom of diatom stephanophyxis was recorded in 2008 which could be attributed to the eutrophicated waters of the bay (chang et al. 2009). diatom abundance was also reported in cape bolinao, pangasinan in all seasons from 1998 to 2000 (yap et al. 2004), in 2001 before the onset of prorocentrum minimum bloom in 2002 (azanza et al. 2005), during the summer periods in 2004 (azanza et al. 2006), and during the alexandrium bloom events from 2010 to 2011 (azanza and benico 2013) associated with eutrophication (san diego mcglone et al. 2008). other studies abroad also noted that diatoms, such as skeletonema costatum, are most common in harbor areas, similar to what has been observed in visakhapatnam harbour, india (rao and mohanchand 1988) and jiaozhou bay, china (hou and shu 2005; liu et al. 2 0 0 5 ) . this abundance could be associated with several parameters, such as temperature, salinity, and nutrients, which influence phytoplankton diversity and community distribution (shridhar et al. 2006; shapoori and gholami 2014). this was apparent in the study when, for example, diatom pseudo-nitzschia spp. , recorded the highest cell densities, while other diatoms, such as leptocylindrus, pleurosigma, thalassiosira, and rhizosolenia, were also common in cip when temperature, salinity, and inorganic po 4 were abundant in may. previous studies have revealed that an increase in n.m. austero and r.v. azanza 47 temperature enhances aggregation and optimal growth of diatoms at higher temperature and salinity in harbor and bay area (hemalatha et al. 2012; araujo and garcia 2005; thornton and thake 1998; mortensen et al. 1988). shridhar et al. (2006) and heneash et al. (2015) also mentioned salinity as one of the main physical parameters that could explain plankton diversity and community variations. in addition, using pca, the algal abundance of the common taxa in cip was revealed to have been greatly influenced by the ambient physico-chemical parameters at 58.7% and 14.9% variation. the pc loadings of physical parameters suggest that the abundance of leptocylindrus and pleurosigma were influenced by the highly correlated temperature and po 4 , whereas highly correlated salinity, no 3 , and nh 3 may also have significant impacts on the abundance of thalassiosira and rhizosolenia. moreover, the high cell abundance of thalassionema, leptocylindrus, thalassiosira, coscnodiscus, chaetoceros, odontella, and fragillaria were also recorded in nsd terminal when temperature and inorganic nutrients, such as inorganic nitrogen, phosphates, and silicates, were higher in july. this result further demonstrates the essential influence of both temperature and nutrients to phytoplankton diversity and abundance (nowrouzi and valabi 2011; shapoori and gholami 2014). aside from the fact that diatoms need inorganic silicates to form their shells (turner et al. 1998), the experiments of egge and aksnes (1992) also indicate that diatoms dominate when sio 3 is above 2 µm/l and tend to aggregate in areas with relatively high silicate concentration (sommer 1998). more importantly, both inorganic phosphates and inorganic nitrogen are essential nutrients that stimulate the growth and activities of phytoplankton (bizsel et al. 2001; ho et al. 2008; martin-jezequel et al. 2015). pca further supports the influence of these parameters in the variation of the algal abundance in nsd terminal. the loads of physical parameters suggest that coscinodiscus was influenced by highly correlated parameters, such as temperature, no 2 , nh 3, and po 4 , whereas the abundance of thalassionema was greatly influenced by the highly correlated salinity, no 3 , and sio 3 . interestingly, pseudo-nitzschia spp. was recorded as the most abundant diatom in cip in may, whereas leptocylindrus spp also exhibited high cell abundance in nsd terminal in july; however, both taxa did not respond to any physico-chemical parameters according to pca. this result could either be species-specif ic or due to combined effects of other factors, thereby warranting further investigation. it is also important to note that phytoplankton diversity and other physico-chemical processes determine the distribution of inorganic nutrients (webber et al. 2003; longhi and beisner 2009). taking into consideration the combination of factors, such as land runoffs, nutrient uptake, excretion, and other physiological aspects of short term assessment of phytoplankton in cebu and subic bay 48 the dominant organisms, which are known to influence nutrient concentration and availability in the marine environment (liu et al. 2005), will also be more essential in future studies. in summary, the main f indings of this study are the data of the assemblages and composition of diatoms and dinoflagellates in two of the major ports in the country. diatoms were the most dominant and abundant component among all stations in both ports being investigated, which could be attributed to physical parameters, such as water salinity, temperature, and nutrients, as suggested by the pca results. however, other factors, such as nutrient loadings from land runoffs, nutrient uptake, excretion, and other physiological aspects of dominant species that could influence nutrient availability, were not examined in this study. thus, it is highly recommended to carry out a thorough research regarding those parameters in the future. further studies on the temporal distribution of phytoplankton assemblages and composition in both ports should also be considered in the future. lastly, the presence of bloomforming, potentially harmful and toxic species in cebu and subic ports could impose risks in the possible uptake of these organisms in ballast tanks and transport. results of this study would contribute to and serve an essential part in the creation of baseline data for port states, which could be utilized as an important tool in complying with the international maritime organization – ballast water management convention 2004 (imo-bwm-2004). acknowledgements we would like to thank the department of science and technology (dost) – philippine council for agriculture, aquatic and natural resources research and development (pcaarrd) for the funding support received in line with the project “invasive marine organisms transferable by ships in selected areas in the philippines”. we would also like to extend our appreciation and acknowledgement to the philippine coast guard ports state control manila and coast guard district central visayas cebu station command who had extended their support and assistance during the conduct of this study, and to the subic bay metropolitan authority 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p o s i t i o n a n d production of phytoplankton in f ish pens of cape bolinao, pangasinan: a f ield study. marine pollution bulletin. 49(9-10):819-832. yñiguez at, ca yetano a , v i l l a n oy cl, alabia i, fernandez i , pa l e r m o j d, benico g a , siringan fp, azanza rv. 2012. investigating the roles of intrinsic and extrinsic factors in the blooms of pyrod inium bahamense var. compressum using an individual-based model. procedia environmental sciences. 13:1462-1476. n.m. austero and r.v. azanza 53 _____________ rhodora v. azanza, ph.d., professor emeritus of the marine science institute, university of the philippines diliman and academician, national academy of science and technology. she has served the university in various positions (including being dean of the college of science) and other international organizations. her researches and publications have been on biology and ecology of seaweeds, and microalgae including environmental impacts and management of algal blooms. nero m. austero, msc. <nerodgreat@gmail.com>, former senior research assistant of dr. rhodora v. azanza. his previous research involvements were on harmful algal blooms or red tides where he successfully cultured ciguatera f ish poisoning (cfp) causing dinoflagellates, harmful/invasive marine organisms in ballast water of international vessels, and biofouling organisms established in wharves and other pier facilities. he obtained his master’s degree in marine science from the marine science institute, university of the philippines diliman. 01_device becira, eunice 46 introduction the philippines, an archipelago of some 7,100 islands with an area of 300,838 km2 had approximately 450,000ha of mangroves in 1918 (brown & fischer 1920 as cited by pcarrd 1991). in 1980, about 220,242ha of mangrove forests borders 17,360km of philippine coastline (gomez 1980). of these mangrove stands 146,000ha were with closed canopy representing primary and secondary growth conditions dominated by true mangrove species (gomez 1980). mangroves of large and contiguous extent can be found state of mangroves in tiniguiban cove, puerto princesa bay, puerto princesa city, palawan eunice m. becira palawan state university puerto princesa city e-mail: c/o j_becira@yahoo.com date received: march 2, 2006; date accepted: may 30, 2006 abstract *corresponding author the study state of mangroves in tiniguiban cove, puerto princesa bay, puerto princesa city was documented and assessed using the following parameters: total area covered, diversity, population structure and diversity and threats to mangrove (cutting). the study was conducted in may 2003 using plot-quadrat method. ten plots were non-randomly distributed in the study area. fourteen major and one associated mangrove species were found in the study area (h=0.85). the three dominant species were rhizophora apiculata (41% ), sonneratia alba (39%) and r. stylosa (13%). these species also accordingly, they had the highest importance values at 192, 146, and 80, respectively. considering the small size of the mangrove stand, diversity was relatively high compared in other areas of palawan. however, continuous expansion of residential areas as well as the development of various students poses a threat to the survival of mangroves. thus, demarcation of the present mangrove area in tiniguiban cove is needed. in panay, biliran, samar, leyte, palawan and mindanao (gomez 1980). ninety-five percent of the remaining mangroves are secondary growth and only 5% are old or primary mangroves. the later are mainly found in palawan (melana 1994 as cited by melana et al. 2000). with an estimated national deforestation rate of 4,432ha/year between 1951 and 1988 (denr 1990), palawan and the rest of region iv had a total mangrove area of 51,000ha representing 43 % of the country's total mangrove areas. according to the denr 1995 statistics, conversion to fishponds, prawn farms, salt ponds, reclamation and other forms of industrial development have reduced the mangrove area to 117,700ha (melana et al. 2000).the steady reduction in mangrove areas during science diliman (july-december 2005) 17:2, 46-51 state of mangroves in tiniguiban cove 47 is characterized by mangroves, which narrowly (50 100m) fringe the coastline. in order to assess the size of the area covered by mangroves and to identify the location of structures that might influence the occurrence of mangroves, the area was assessed and mapped using the global positioning system garmin ii. all mangrove species encountered were recorded at species level using the following references: calumpong and meñez (1997), tomlinson (1986) and a dichotomous identification key developed by schoppe (undated). to assess the community structure in terms of density, dominance and frequency of mangroves, a total of ten plots were non-randomly established. the size of the plots was 100m2 each. this is the usual plot size suggested for mangrove assessment having at least 40 trees (english et al. 1997). in each plot, the species were identified, stems per species were counted and the girth of each tree was measured at breast height (gbh) approximately 1.3 m above the ground using a measuring tape. seedlings (height 1m or less) and saplings (girth less than 4cm and height more than 1m) were identified. the number of individuals per species was determined by actual counts. the past several decades is attributed to harvesting of mangroves for charcoal or firewood production, and to forest clearing for fishponds establishment. expansion of coastal communities also played a role in reduction of mangrove areas (pcarrd 1991). in tiniguiban cove, the establishment of a power barge, fish cages and expansion of residential areas in the coastal communities contributed to the reduction of mangrove vegetation. this led to this study on the state of the mangroves in tiniguiban cove. this paper reports the status of mangroves in tiniguiban cove in terms of: a) total area covered, b) species composition, c) community structure, and d) cutting commencing may 1 to july 4, 2003. methodology this study was conducted in the eastern part of the tiniguiban cove, puerto princesa city located at the north-eastern part of puerto princesa bay (figure 1). the study area is situated east of the palawan state university and west of camp h. mendoza, 4 km from the city proper (ca. 09o46'04" north latitude and 118o43'26" east longitude, salva et al. 1996). the area figure 1. map of palawan showing the location of puerto princesa bay and the tiniguiban cove in puerto princesa city, palawan, philippines. becira, eunice 48 for each mangrove species encountered in any of the plots, notes were taken on the maximum inundation level based on the watermark on the bark. to assess threats to mangroves, cut branches, cutting of whole trees and dead trees were counted per plot. results and discussion site specifications the major adjacent and nearby components which the researcher thought to be the major factors influencing the existence of mangrove in the locality were identified. these include the residential area (colored yellow), tiniguiban elementary school, pc compound, city agricultural seed farm and the palawan state university (figure 2). among them, the continuous development of the residential area seems to pose the major threat to the mangrove stand. the total mangrove area (colored dark green) is about five hectares including approximately 0.18ha reforested area (figure 2). species composition the area was composed of 14 major mangrove species and one mangrove associate. these includes avicennia marina, a. officinalis, bruguiera cylindrical, b. figure 2. map of the study area showing the location of sampling plots. sexangula, ceriops tagal, excoecaria agallocha, lumnitzera littorea, l. racemosa, rhizophora apiculata, r. mucronata, r. stylosa, sonneratia alba, xylocarpus granatum, nypa fruticans and pandanus sp. the number of major mangrove species found in tiniguiban cove is higher (14 species) compared to recorded in snake island, honda bay (8 species; schoppe 2003); benito marcelo beach (7 species; batin et al. 2001) and in sabang, puerto princesa city (10 species; batin 2003) but comparable in bancaobancao, puerto princesa city (spcp-asti 1999) though with different species. the recorded species richness in tiniguiban cove is low compared to bataraza and balabac (southern palawan) where 22 and 21 major mangroves were found (conservation international 2004), respectively (table 1). this study showed that even a small area like tiniguiban cove can harbor a diverse mangrove flora. community structure mangroves in the study area showed a typical zonation based on inundation level (per observation). species found in the landward zone (0-10m from the shoreline; inundation, 6 cm) were l. littorea, l. racemosa, b. cylindrica, b. sexangula, e. agallocha and x. state of mangroves in tiniguiban cove 49 table 1. number of mangrove species and approximate area in puerto princesa city, southern palawan, romblon and mindoro. puerto princesa city southern palawan romblon mindoro site t in ig ui ba n c ov e (t hi s st ud y) sa ba ng (b at in 2 00 3) m an gi ng is da (s pc pa st i 1 99 9) b an ca ob an ca o (s pc pa st i 1 99 9) sn ak e is la nd (s ch op pe 2 00 3) b at ar az a (c on se rv at io n in te rn at io na l 2 00 4) b al ab ac (c on se rv at io n in te rn at io na l 2 00 4) r om bl on (s pc pa st i 1 99 9) o cc id en ta l m in do ro (s pc pa st i 1 99 9) number of species 14 10 15 14 8 9 9 22 21 area (ha) 5 4 182 51 na 440 60 na na na = not available granatum. in the central part of the forest (inundation: 2.53 32.2cm) are a. marina, a. officinalis and c. tagal. in the seaward zone (inundation: >60cm) were the species of rhizophora apiculata, r. mucronata, r. stylosa, and s. alba occurred. among mangrove species, r. apiculata has the highest basal area (10.47 m2ha-1) followed by s. alba (10.00 m2ha-1) (table 2). rhizophora apiculata has the highest stem density (5130 stems/ha) followed by r. stylosa (2803 stems/ha). sonneratia alba ranked third based on stems density (1720 stems/ha), despite having equal mean basal area as r. apiculata. this shows that s. alba had larger trunks and therefore was comparable with rhizophora species when it comes to dominance in the community. the other species of mangrove were rather inconspicuous in terms of basal area and stem number (table 2). rhizophora apiculata occurred in all plots. it was followed by s. alba (90%), r. stylosa (40%) and r. mucronata (30%). other identified species occurred only once or twice in all sampling plots (table 2). r. apiculata and s. alba dominated the community in terms of size (table 2) and may have the highest contribution to energy cycle of the ecosystem (smith 1992). the relative dominance of the species in the entire study area ranged from 0.06% to 41.11%. rhizophora apiculata dominated all ten plots with a mean relative dominance of 41.11% followed by s. alba (39.27%) and r. stylosa (13.53%). others have less than 5% relative dominance. relative density of the species ranged from 0.19 to 51.4%. rhizophora apiculata had the highest density followed by r. stylosa (26.8%) and s. alba (16.6%). other mangrove species had less than 1% relative density. the domination of rhizophora may be probably due to the muddy substrate in the site which is favorable for its growth (hogarth 1999). in tiniguiban cove, the most important species was r. apiculata followed by s. alba and r. stylosa. avicennia marina is accounted for the lowest importance value (table 2). the diversity of mangroves in tiniguiban cove using shannon index was 0.851. the diversity results signify that the area was dominated by only a few species particularly r. apiculata, s. alba and r. stylosa. diversity index in tiniguiban cove was however higher compared to barangay mangingisda (0.502) and barangay bancao-bancao (0.584) of puerto princesa city and in the nearby provinces of palawan particularly romblon (0.528) and mindoro (0.607) becira, eunice 50 (spcp-asti 1999). in comparison with the other sites, tiniguiban cove is a good mangrove site but should be managed immediately since disturbances and threats are besetting the site. mangrove seedlings and saplings of the three dominant species (r. apiculata, r. mucronata and s. alba) contributed to the recruitment in tiniguiban cove. the number of seedlings and saplings of r. apiculata was 320 and 1070, respectively. it was followed by s. alba with 150 seedlings and 480 saplings. r. mucronata ranked third with a total of 410 seedlings and saplings per hectare. other species had no or only few recorded seedlings and saplings. threats to mangroves the fringing mangroves in tiniguiban cove are subjected to human disturbances. in fact, part of the area had been already cleared and most houses were made of light and/or concrete materials existing along the seashore. a portion of the mangrove forest has also been converted into a fishpond with two compartments of approximately 0.01 hectare each. domestic animals are also raised by the communities. domestic wastes (wastewater, cellophane and the like) are directly discharge into the immediate environment. within the forest, cut trees were observed. rhizophora apiculata, r. mucronata and s. alba had the highest number of cut trees (1070, 650 and 510 cut trees per hectare, respectively). being the most common trees in the area it seems that these species are more susceptible to exploitation for as fence enclosure and posts. conclusion based on the study, it was concluded that a possibility of having a diverse mangrove ecosystem may not be limited in large areas though threats to mangrove stand is greater in a small areas. construction, improper waste disposal and cutting activities especially affect the stands of r. apiculata, r. mucronata, and s. alba. recommendations for management purposes, demarcation of the present mangrove stand in the tiniguiban cove should be avicennia marina 0.02 20 0.1 10.0 0.06 0.19 10.25 a. officinalis 0.06 50 0.2 20.00 0.23 0.47 20.71 bruguiera cylindrica 0.08 20 0.1 10.00 0.32 0.19 10.50 b. sexangula 0.03 50 0.1 10.00 0.10 0.47 10.57 ceriops tagal 0.05 60 0.1 10.00 0.20 0.57 10.77 excoecaria agallocha 0.09 20 0.1 10.00 0.35 0.38 10.73 lumnitzera littorea 0.62 20 0.1 10.00 2.45 0.47 12.93 l. racemosa 0.3 70 0.2 20.00 1.19 0.76 21.95 rhizophora apiculata 10.47 5130 1 100.00 41.11 51.37 192.48 r. mucronata 0.03 80 0.3 30.00 0.13 0.76 30.89 r. stylosa 3.45 2830 0.4 40.00 13.53 26.82 80.35 sonneratia alba 10 1720 0.9 90.00 39.27 16.68 145.95 xylocarpus granatum 0.27 120 0.2 20.00 1.06 0.85 21.92 total 25.48 10190 na na na na na species of mangrove m ea n ba sa l ar ea (m 2 /h a) m ea n nu m be r of st em s/ ha f re qu en cy r el at iv e fr eq ue nc y r el at iv e do m in an ce r el at iv e de ns it y im po rt an ce v al ue table 2. basal area, mean number of individuals per hectare and frequency of the mangrove species in the study area. state of mangroves in tiniguiban cove 51 conducted by the proper authority particularly the denr for monitoring purposes. to increase the level of awareness of the residents of barangay tiniguiban on the ecological importance of mangroves, educational and information campaigns should be conducted. likewise, a follow-up or continuous study on mangroves particularly on economic aspects should also be done. acknowledgment the author would like to acknowledge dr. sabine schoppe, dr. edgardo castillo, the palawan state university administration and the western philippines university-puerto princesa campus, puerto princesa city. references batin, g, becira, j., kaiser, d. and m. ledesma 2001. species composition and community structure of the fringing mangroves of benito marcelo beach, puerto princesa city, palawan. unpublished report. state polytechnic college of palawan-puerto princesa campus, sta. 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palawan, philippines. unpublished report. state polytechnic college of palawanpuerto princesa campus, 9 pp. schoppe, s. undated. key to the major and minor mangroves of the philippines. unpublished identification key. state polytechnic college of palawanpuerto princesa campus, 9 pp. smith,t.j. iii 1992. forest structure. in: robatson,a. i and d.m. alongi (eds.). tropical mangrove ecosystems. american geophysical union,washington dc,usa. 329 pp. spcp-asti, 1999. a report on the rapid resource assessment in some coastal areas of north and west sulu sea. unpublished report. state polytechnic college of palawan-aquatic science and technology institute, 402 pp. tomlinson, p.b. 1986. the botany of mangroves. cambridge university press, 413 pp. 8-viability of phytoplankton-austero.pmd n.m. austero et al. 69 science diliman (january-june 2019) 31:1, 69-78 v iabil ity of phytoplankton from ballast waters of international vessels ber thing at por ts of cebu and subic bay, phil ippines nero m. austero* marine science institute university of the philippines diliman subhash s. sawant csir-national institute of oceanography india rhodora v. azanza national academy of science and technology department of science and technology abstract t h e v i a b i l i t y o f b a l l a s t w a t e r p h y t o p l a n k t o n w a s a s s e s s e d t h r o u g h incubation experiment. leptocylindrus sp. and thalassionema spp. were found to be viable when incubated in port water and ballast water media showing some increase in cell numbers. bloom-forming diatom taxa, such as chaetoceros spp. and coscinod iscus spp. , potentially harmful diatom species pseudo-nitzschia spp. , and dinoflagellates, gambierd iscus spp. and p r o r o c e n t r u m s p p . w e r e a l s o i d e n t i f i e d i n b a l l a s t w a t e r s f r o m international vessels. these results further suggest possible successful t r a n s p o r t o f t h e s e o r g a n i s m s v i a s h i p p i n g , w h i c h c o u l d f a c i l i t a t e t h e introduction and lead to bioinvasion in the local aquatic environment. keyword s: ballast water, diatoms, dinoflagellates, phytoplankton, por ts _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online viability of phytoplankton from ballast waters 70 introduction ballast water essentially provides stability and maneuverability to ships, thereby assuring safe operating conditions (david 2015). in ports, ballast water operations are usually conducted either via gravity or pumps to take in or discharge ballast water (david 2015), which may contain potentially harmful algal bloom species that will be transported from one port to another. ballast water has been identif ied as one of the major vectors for the global dispersal of alien invasive species, toxic harmful algal bloom species and pathogens causing ecological, economic, and health problems (hallegraeff and bolch 1991; bolch and de salas 2007; drake et al. 2007; eames et al. 2008; simkanin et al. 2009; klein et al. 2010). the present study was aimed at assessing the viability of organisms in ballast water tanks of international vessels berthed at cebu and subic ports in philippines. attempts to identify common organisms found in ballast waters have also been made. materials and methods collection of ballast water samples four international vessels were boarded for onboard collection of ballast water samples in the months of may and july 2015 in an opportunistic sampling method only. vessel 1 (v1) is a 4,718-ton general cargo vessel registered from panama. its last port of call was in sumbawa, indonesia. previously visited ports include: (1) makassar, indonesia, (2) singapore, (3) chiba and yokohama, japan, (4) busan, korea, and (5) qingdao, china. in addition, vessel 2 (v2) is a general cargo vessel with 6,150 tons gross tonnage under the flagship of panama. prior to berthing in cebu international port (cip), its last port of call was in zhanjiang, china. its previously visited ports include: (1) haiku and hongkong, china, (2) chiba and funabashi, japan, (3) jingtang, china, (4) incheon, korea, (5) hon gai, vietnam, (6) bangkok, thailand, (7) kuantan, malaysia, and (8) kaohsiung and guang zhou, china. moreover, vessel 3 (v3) is a 33,990-ton bulk carrier vessel registered from singapore. its last port of call was in taboneo, indonesia. its previous visited ports include: (1) makassar, indonesia, (2) singapore, (3) port klang, vietnam, (4) cai mep, vietnam, (5) washington, usa, (6) okkye, s. korea, (7) vostochny, russia, (8) qingdao, china, (9) busan and kunsan, s. korea, and (10) tamatave, madagascar. lastly, vessel 4 (v4) is a 31,753-ton bulk carrier vessel under the flagship of panama. its last port of call was in balboa, n.m. austero et al. 71 panama. its previously visited ports include: (1) cristobal, panama, (2) new orleans, usa, (3) bayovar, peru, (4) buenaventura, colombia, (5) houston, texas, usa, (6) altamira, mexico, (7) yeosu, s. korea, (8) shanghai, china, (9) kaohsiung, taiwan, (10) susaki, japan and (11) port luis, mauritius. ballast water sample access points were via the (1) main engine pump for v1, (2) manholes for v2 and v4, and (3) sounding pipes for v3 based on the availability (table 1). table 1. list of international vessels sampled in cebu international port (cip), cebu, central visayas and naval supply depot (nsd) in subic bay, zambales, phil ippines in may and july 2015 1 gen. cargo main engine buckets 3 5.2015 cip indonesia 2 gen. cargo manhole buckets 5 7.2015 cip china 3 bulk carrier sounding pipe pipe sampler 1 5.2015 nsd indonesia 4 bulk carrier manhole plankton net 5 7.2015 nsd panama * sample volume is per bucket (1 bucket = ~ 2.5-3 liters of ballast water) vessel type access point sampl ing gears sample volume* sampl ing date sampl ing port last port of sample collection was peformed using buckets, plankton nets, and pipe sampler. in the present study, the main emphasis was on phytoplankton only; hence, the samples were f ixed with lugol’s solution and brought to the shore laboratory for further analysis. these samples were then analyzed for phytoplankton cell abundance and identif ied to genus or species level using identif ication keys (yamaji 1984; tomas 1997). in addition, samples were also collected and brought to the shore laboratory without preservative and used for incubation experiments. the unpreserved samples were pooled accordingly and passed through a 60-μm sieve to remove larger organisms, and the f iltrate, containing smaller cells, were cultured in various media. med ia preparation and incubation experiment three types of culture media were used for this experiment: (1) diluted f/2mediaprepared following the methods of guillard and ryther (1962), guillard (1975), corrales et al. (1995), and azanza (1997); (2) port water; and (3) ballast water media; which were prepared by f iltering through a 0.45-μm whatman filter paper following kang et al. (2010) and were then sterilized by autoclaving. viability of phytoplankton from ballast waters 72 approximately 10-ml f iltered ballast water samples were inoculated into each 100-ml media in triplicate and were incubated for 6 days. three 1-ml aliquots were drawn from each flask on alternate days for phytoplankton analysis and identif ication. cultures were maintained at 30oc and 33±1psu in a 12:12 light/dark cycle with 143.88±27.62-μmol photon m-2s-1. results and discussion in this study, a total of 16 diatoms, three dinoflagellates and four zooplankton taxa were identif ied from the ballast water samples. highest diatom count was observed in v4, moderate in v2, and minimal in v1 and v3. the presence of potentially toxic diatom pseudo-nitzschia sp. (figure 1a), and dino-flagellates gambierdiscus sp. (figure 1b), prorocentrum sp. (figure 1c), and protoperidinium sp. (figure 1d) in ballast water conf irms that ships can harbor these species in ballast water (hallegraeff and bolch 1991; gollasch et al. 2000; drake et al. 2007; burkholder et al. 2007; klein et al. 2010). these species may have been carried into vessel tanks during the process of ballast water exchange. the presence of potentially toxic gambierdiscus sp. in ballast water, on the other hand, supports the theory of hallegraeff (1992) and bomber et al. (1988) that, in spite of being not known to produce resistant cysts, these species are well capable of surviving and getting dispersed as epiphytes attached to drifting macro-algae. the main f indings of the incubation experiment show that, among the vessels sampled, only v2 and v4 recorded viable cells (figures 2a and 2b). -no skeletonema spp. and nitzschia sp. cells were recorded from v2; however, they appeared as viable cells after the 6th day of incubation in port water media (figure 2a), which suggests that these cells were present in the ballast water, but were absent in the subsample taken for counting. this was also true in the case of leptocylindrus sp. , figure 1. potentially harmful phytoplankton taxa in ballast tanks sampled as light micrographs (lms): (a) pseudo-nitzschia sp. , (b) gambierdiscus sp. , (c) prorocentrum sp. , and (d) protoperidinium sp. n.m. austero et al. 73 which was not found in ballast water sample, but exhibited considerable increase in cell number after six days in port water media. on the contrary, thalassiosira sp. , which is a bloom-forming diatom, was found to grow in all three types of media with maximum growth in port water media after six days (32 times). these results could be attributed to the capability of diatoms to produce viable resting spores that possess the ability to germinate even after a long period of time and that can withstand adverse ballast tank conditions (doucette and fryxell 1985; mcquoid et al. 2002; harnstrom et al. 2011; montresor et al. 2013). experiments of carney et al. (2011), for instance, also revealed the survival of phytoplankton cells and the successful germination of macro-alga e. flexuosa spores (kolwalkar et al. 2007) even after prolonged exposure in the dark. figure 2. phytoplankton composition in ballast water samples from (a) v2 and (b) v4, and viable diatom cells (*) following six days of incubation period. (note: bw – ballast water, pw – port water, f10 – diluted f media, d00 – day zero, d02 – day 2, d04 – day 4, d06 – day 6) viability of phytoplankton from ballast waters 74 these results suggest that these diatoms can possibly survive if discharged into new environments, leading to possible introduction of new species and/or invasion. studies of steichen and quigg (2015) and kang et al. (2010) have also reported that ballast water-borne diatoms exhibit viability and growth after exposure to changing salinity and nutrient regimes. studies have also shown that even the use of modern technologies and techniques implemented in most ocean-going vessels as part of their ballast water management systems do not guarantee 100% eff icacy (grob and pollet 2015). certain photosynthetic organisms were able to grow within 4 to 20 days when released back into favorable conditions even after treatment (stehouwer et al. 2010; van der star et al. 2011; liebich et al. 2012; martínez et al. 2013). on the other hand, analysis of phytoplankton composition and abundance in ambient waters of cebu international port (cip) and naval supply depot (nsd) terminal identif ied at least 53 phytoplankton taxa with 36 diatoms and 16 dinoflagellates. ballast water-borne diatoms, such as skeletonema spp. , nitzschia sp. , leptocylindrus sp. , and thalassiosira sp. , were among the identif ied taxa from the ambient waters of cebu and subic bay ports. these organisms were also identif ied in the waters of panama, china, and indonesia (d’croz et al. 1991; liu et al. 2005; serihollo et al. 2015; effendi et al. 2016). these f indings imply that these species could be cosmopolitan and may have been traveling between these ports. the use of modern and new approaches could be very helpful in inferring these species’ geographical sources in future studies. this preliminary investigation suggests that the foreign vessels involved in trading with the philippines, especially those which arrive for picking up of cargo, could pose potential risk of transport of harmful or bloom-forming organisms. additional studies, if conducted in the future, would provide a database useful for assessing ballast water risk for philippine ports by using a suitable model or tool, which in turn, will provide a decision support system (dss) to the concerned authority in line with the international maritime organization – ballast water management convention 2004. acknowledgements we would like to thank the department of science and technology (dost ) – philippine council for agriculture, aquatic resources research and development (pcaarrd) for the funding support received in line with the project “invasive n.m. austero et al. 75 marine organisms transferable by ships in selected areas in the philippines”. we would also like to extend our appreciation and acknowledgement to the philippine coast guard ports state control manila and coast guard district central visayas cebu station command who extended their support and assistance during the conduct of this study. we would also like to thank the subic bay metropolitan authority (sbma) ecology center and seaport department for their f ield assistance. upmsi pimo project also received assistance and support from the following people: jayson orpeza, estrelita flores, and julius mendoza. references azanza rv. 1997. contributions to the understanding of the bloom dynamics of pyrod inium bahamense var. compressum; 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malmö, sweden. sweden: wmu publications. p. 233–240. steichen jl, quigg a. 2015. assessing the viability of microorganisms in the ballast w a t e r o f v e s s e l s t r a n s i t i n g t h e n o r t h a t l a n t i c o c e a n . m a r i n e p o l l u t i o n b u l l e t i n . 101(1):258-266. tomas cr. 1997. identifying marine phytoplankton. california: academic press. van der star i, liebich v, stehouwer pp. 2012. the forgotten fraction: the impor tance of organisms smaller than 10 ìm when evaluating ballast water treatment systems. in: o l g u n a , ka r a ko ç f t, h a a g f , ed i to r s . pr o ceed i n g s o f t h e g l o b a l r & d f o r u m o n compliance monitoring and enforcement; istanbul, turkey. turkey: tübýtak – mrc publications. p. 41-49. yamaji i. 1984. illustrations of the marine plankton of japan. 3rd ed. osaka: hoikusha publishing co. , ltd. viability of phytoplankton from ballast waters 78 _____________ nero m. austero, msc. <nerodgreat@gmail.com> former senior research assistant of professor rhodora v. azanza. his previous research involvements were on harmful algal blooms or red tides -where he successfully cultured ciguatera f ish poisoning (cfp) causing dinoflagellates, harmful/invasive marine organisms in ballast water of international vessels, and biofouling organisms established in wharves and other pier facilities. he obtained his master’s degree in marine science from the marine science institute, university of the philippines diliman. subhash s. sawant, ph.d., chief scientist at the csir-national institute of oceanography, goa, india has vast experience in the f ield of marine sciences. his areas of interest are biofouling and ships’ ballast water management. he has developed a new model for conducting ballast water risk assessment, which can be used for controlling transfer and introduction of aquatic alien species by ships in any port. rhodora v. azanza, ph.d. <rhodaazanza@yahoo.com> professor emeritus of the marine science institute, up diliman and president of the national academy of science and technology (nast). she has served the university in various positions (including being dean of the college of science) and other international organizations. her researches and publications have been on biology and ecology of seaweeds, and microalgae including environmental impacts and management of algal blooms. 123 information for authors 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the office of the vice chancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); 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• short communications are reports of significant new data arising from problems with narrow, well defined limits before broader studies are completed; and results have not been published in print elsewhere, except as partial communications or posters in conference proceedings; • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; 130 • abstracts will be required on submission but not to be part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages) in length with simple layout, a maximum of two tables and two figures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors should submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor in chief science diliman office of the vice chancellor for research and development university of the philippines lower ground floor, phivolcs bldg., c.p. garcia avenue up campus, diliman, quezon city 1101, philippines frequency distribution of blood groups abo, mn and rh factor in philippine cosmopolitan, regional, and the national populations ruth marian s. guzman1*, ricardo noel r. gervasio1, ian kendrich c. fontanilla1, ernelea p. cao1,2 1institute of biology, college of science, university of the philippines, diliman 1101, quezon city 2natural sciences research institute, university of the philippines, diliman 1101, quezon city telefax: (63-2) 920-5471 *corresponding author: maan.guzman@gmail.com received: 28 feb 2009; revised: 20 january 2010; accepted: 13 july 2010 abstract frequency distribution of blood groups is important as it is used in modern medicine, genetic research, anthropology, and tracing ancestral relations of humans. blood groups include the abo, rh and the mn red cell antigens. the frequency distribution of these three blood groups were obtained and assessed for differences from three populations: (1) a regional population from the town of cabagan located in isabela province; (2) a cosmopolitan population from the university of the philippines’ roster of students; and (3) the national population’s data obtained from blood banks all over the philippines. this study sought to determine the frequency distribution of abo, mn, and rh factor blood groups to establish whether there exist differences in distribution among the three population categories. standard blood agglutination sampling was conducted in these populations to determine blood types. chi-square tests on the genetic frequencies reveal that there is no significant difference in the distribution of blood groups abo and rh. the blood group mn, however, displayed twice as many m blood type in the regional population than in the cosmopolitan population. this suggests a localized segregation of alleles responsible for the mn blood type within distinct populations in the philippines. computation of the allelic frequencies also revealed that both populations are not at hardy-weinberg equilibrium based on the distribution of the different mn blood types. keywords: genetic frequencies, abo, mn, rh factor, hardy-weinberg equilibrium introduction among the various factors that contribute to a person’s individuality are antigens attached to surface of red blood cells and naturally occurring antibodies that circulate in the serum. the abo blood group and the rhesus factor or rh blood group are two of the most notable type groups in humans due to their importance and association with blood transfusion (khattak, et al. 2008). the mode of inheritance of the abo blood group follows the multiple allelic mode of inheritance and is quite stable to be used to exclude paternity in paternity issues. the rh antigen is named after the rhesus monkey, macaca mulatta (zimmerman) where it was initially detected. the mode of inheritance of these antigens is complex, and there are two science diliman 21(2):43-49 43 mailto:maan.guzman@gmail.com guzman, gervasio, fontanilla & cao theoretical models that attempt to explain the pattern of inheritance. the wiener system postulates a single gene locus with a series of at least ten multiple alleles. the fisher system assumes the existence of at least three closely linked loci designated as c, d, and e. both are currently in use and are still being studied. however, only the presence of the d antigen in the fisher system serves as the basis for classification of the rh blood group; this way, the mode of inheritance is simply single gene inheritance with accompanying dominance. the most notable medical importance of this blood group system is the occurrence of rh incompatibility between mother and fetus, which is a major factor in the development of erythroblastosis fetalis or hemolytic disease of the newborn (dennis et al., 1998). m and n blood group antigens are presented by glycophorins a (gpa) and b (gpb) of the erythrocyte membrane. gpa expresses m or n blood group antigen depending on the allelic gene (gpam gene or gpan gene), while gpb expresses only the n antigen. m or n blood group is specified by the first and fifth nh2-terminal amino acid residues in the mature proteins that are encoded by the second exon of these genes (kudo & fukuda, 1994). the agglutinogens m and n are inherited as a single pair of allelomorphic genes (hyman, 1942), and the m and n alleles are codominant to one another. this allows determination of the allelic frequency of the m and n alleles with relative ease. many studies of human genetics have used the mn system because it is possible to distinguish the heterozygote mn from both homozygotes mm and nn. additionally, there does not appear to be any selection pressure against either allele. thus, the mn system is a good test of the hardy-weinberg law. these three blood groups have their genetic components confined to different regions. the abo locus is located on chromosome 9, specifically, in the segment 9q34.1-q34.2 (narahara et al. 1986). the human blood group rh polypeptide has been used to map the rh locus, by in situ hybridization, to the region p34.3-p36.1 of chromosome 1 (cherif zahar, 1991). lastly, the locus of the gene responsible for the mn antigen activity is confined to chromosome 4, in the 4q28.2–4q31.1 segment (wakui, 1990). since these genes are not linked, it is assumed that the behaviour of the genes responsible for the blood groups will not affect the other blood groups’ expressivity in individuals. the hardy-weinberg model describes a mathematical relationship that allows the prediction of the frequency of offspring genotypes based on parental allele frequencies. it also predicts that allele frequencies will not change from one generation to the next, indicative of non-evolution (klug & cummings, 2002; mayo, 2008). for a population to be in hardy-weinberg equilibrium, the following assumptions are required to hold: random mating, no mutation, no migration, no stochastic effects or genetic drift due to small population size, and equal fertility for all genotype groups so that no selection is occurring (minelli et al. 2008). violation of any of these assumptions can result to evolutionary change in terms of allelic frequency distribution (mayo, 2008). these conditions, however, seldom occur simultaneously, resulting to most populations not exhibiting hardy-weinberg equilibrium and are therefore evolving. all human populations share the same blood group systems, differing only in the frequencies of specific types. the incidence of abo, rh snd mn groups varies in different parts of the world and in different races (khattak et al. 2008, thamaria et al. 1972). assessing blood group frequency distribution is multipurpose, as beside their importance in evolution, their relation to disease and environment is being increasingly sought out in modern medicine (khattak et al. 2008). blood group antigens are not only crucial in the medical field, but can also be utilized in genetic research, anthropology, and tracing ancestral relation of human (khan et al. 2004). it is predicted that the inhabitants of a rural town with a regional population and considerably less human migration in and out of the area than a cosmopolitan location, would maintain significantly different genetic frequencies. to test this, we performed blood type tests for abo, mn and rh blood groups on individuals from a rural town and compared them with results taken from metro manila, capital city of the philippines, as well as national data collected from blood banks across the country. the main objective of this paper was to identify frequency distribution differences in the abo, rh and mn blood groups among a local population, a “mixed” population and the collective 44 science diliman frequency distribution of blood groups abo, mn and rh factor national data. patterns of local distribution were then compared to the global data available. materials and methods sample sites description blood samples for a regional population were obtained from the rural town of cabagan, located in the province of isabela. adjacent towns include santa maria, san pablo and tumauini. cabagan is a relatively small town approximately 465 kilometres from metro manila with a population size of 43,562 individuals according to the 2007 government census. the population is comprised mostly of people belonging to the ybanag ethnolinguistic minority. for the cosmopolitan population, undergraduate and graduate students in the university of the philippines in diliman were requested to donate blood samples. the working assumption is that university students come from different provinces, and are thus distributed in a random fashion contributing to a mixed cosmopolitan population. the collective national data on abo and rh blood group distribution was acquired through the philippine national red cross main office located in gen. luna cor. victoria st., intramuros, manila. data were obtained from blood bank databases. blood sample collection sample collection was standardized to at least 50 donors in order to get statistically sound data. a total of 110 students from two participating universities were screened for their respective abo, mn, and rhesus (rh) factor blood types. fifty-one blood samples were taken from 31female and 20 male students of the university of the philippines-diliman (upd). fifty-nine blood samples were taken from 20 female and 49 male students of the isabela state university (isu), garita heights campus, cabagan, isabela. to comply with the requirement that blood sample donors be genuine ybanag, each student from isu was interviewed for his/her ethnicity. the interview clarified questions of ethnicity by tracing the residence of the students’ parents and grandparents. blood typing five drops of blood were obtained from each donor by pricking the tip of the index finger with a sterile lancet. each drop of blood was placed on a spot plate containing a blood typing anti serum. the following monoclonal antibodies were used: eryclonetm anti-a and anti-b of tulip diagnostics ltd. (india) for the abo blood type; epiclone-2 anti-d of csl-australia for the rh group; and anti-m and anti-n of cypress diagnostics (belgium) for the mn blood group. agglutination of the blood drop with the five test sera was then assessed by gently probing through the mixture using a lancet. blood drops exhibiting a clotting reaction with the test sera were considered positive for that particular blood grouping reagent. each reaction and corresponding blood types for each blood donor was recorded and subjected to pooling and statistical analysis. statistical analysis to determine the degree of association, if present, among the different categories in each blood type, genotypic frequencies from the cosmopolitan (upd), regional (isu) and national (pnrc intramuros chapter) populations were subjected to chi-square tests. all statistical tests were conducted using the statistical package for social sciences (spss) version 15.0 (chicago, il, usa). significant differences were determined at p ≤ 0.05. for the mn blood group, allelic frequencies were computed, and then factored in the chi-square test that was used to verify whether the participating populations are in hardy-weinberg equilibrium. likewise, p values less than 0.05 were considered statistically significant. results the blood group abo is randomly and independently distributed in the cosmopolitan (upd), regional (isu) and national populations. a common pattern on the distribution of blood types among the three populations is seen in figure 1 wherein blood type o is most prominent, followed by blood type a, blood type b, then the least common blood type ab. pearson chi-square values comparing the upd and the isu populations to that of the national confirmed no positive association between the frequency distribution of blood groups and the location of sampling. science diliman 45 guzman, gervasio, fontanilla & cao however, verification by allelic frequencies for the blood types cannot be carried out, as it is not known whether blood types a and b are homozygous or heterozygous. there is no significant difference in the frequency distribution of the blood group rhesus factor among cosmopolitan (upd), regional (isu) and national populations. a seen in figure 2, the distribution of the rh types in the three populations are all consistent, with rh+ being the more dominant allele and rh expression being almost negligible. the two smaller populations (upd and isu) corroborate the pattern set by the national population in that filipinos, in general, are rh+. there is a significant difference in the frequency distribution of blood group mn in cosmopolitan (upd) and regional (isu) populations. the distribution of the mn blood groups is shown in figure 3. blood type mm is seen as the predominant blood type in the regional population (72.88%) as opposed to the cosmopolitan population (37.25%) wherein the distribution of the three blood types is more closely associated. allelic frequencies were also computed; with the m allele occurring at 78.8% in the isu population, far higher than 52.0% for the upd population. statistical analysis showed that, using the computed allelic frequencies, the cosmopolitan and the regional populations were both not in hardy-weinberg equilibrium with p values less than 0.05 and 0.01, respectively (table 1). discussion the immune system is accountable for the success or failure of blood transfusions. it is of utmost importance that the donor blood cells match that of the recipient; otherwise, donor blood cells may be destroyed by antibodies present in the plasma of the recipient. the abo system is the main blood group considered in such procedures (adeyemo & soboyejo, 2006). the results of this study present data indicative of similar, random segregation of the different blood types of this system in regional, cosmopolitan and the overall population. chi-square testing confirmed no positive association of abo genotypic frequency between populations in metro manila, cabagan town and nationwide philippine population. this was despite the fact that only 16% of subjects from isabela were type b, as compared to 26% for the nationwide data. the o blood type has the highest percentages in the three data sets, followed by a and b types, and the ab type having the smallest proportion (figure 1). interestingly, the same trend of abo blood group distribution is also seen in the american indian population in cherokee, usa, in the african american population in st. louis, usa, and in the chinese population in hong kong (mourant, 1976). this trend, however, is not representative of the majority of populations surveyed (americans, french canadians, japanese, 46 science diliman figure 1. percentage distribution of abo blood group in three populations. upd = university of the philippines, diliman; isu = isabela state university, cabagan, isabela; pnrc = philippine national red cross national data. general trend for all three populations consist of blood type o having the highest frequency, followed by type a, then type b then lastly, type ab. figure 2. percentage distribution of rhesus factor blood group in cosmopolitan, regional and national populations. upd = university of the philippines, diliman; isu = isabela state university, cabagan, isabela; pnrc = philippine national red cross national data. occurrence of type rhamong the three population data is negligible. frequency distribution of blood groups abo, mn and rh factor palestinians, jews and eskimos), with blood type a having the greatest proportion followed by type o, type b, and type ab (mourant, 1976). most antigens from the blood group rh are weak and do not really elicit antibody production. the exception is the d antigen, which is strong and is likely to cause transfusion problems. thus, the classification of being rh+ or rh is dependent on the presence of this particular antigen (klug & cummings, 2002). as such, this blood group is considered to be a case of single gene inheritance with dominance. the three sample populations yielded similar results; all subjects sampled in cabagan and up diliman were rh+ and the national data showed an insignificant percentage of rh individuals (figure 2). these outcomes indicate that the recessive allele for the d antigen is very rare in the philippine population. when compared to global data presented by mourant (1976) and khattak (2008), the rh factor data from this study follows the same pattern found in all the populations surveyed, with a minor rh type representation with a range of 0% to 17%. these populations include american indians, arabs, bengalis, africans, chinese, eskimos, mexicans, and americans. the mn blood group system is of little apparent importance in cases of transfusion reactions or maternal-fetal incompatibilities because few people produce anti-m or anti-n even after repeated exposures to the antigens (weder et al. 1991). data from the mn blood group illustrate different results from that found in abo and rh blood groups; the frequencies of the mn genotypes of the populations in cabagan, isabela (isu) and manila (upd) were found to have positive association (figure 3), suggesting that the two populations are distinct (klug & cummings, 2002). moreover, comparing the calculated gene frequencies for the m and n alleles for both populations reinforce the positive association. the data taken from the isu population, with the allelic frequency of m tripling that of the allelic frequency of n, is similar to the mn data obtained from the more isolated populations such as the apache indians in the usa and some groups in mexico city and iran (mourant, 1976). the data obtained from the cosmopolitan setting of manila produced similar proportions of allelic frequencies of m and n with respect to those of the african-americans, caucasians and chinese populations in the usa (mourant, 1976). to assess the state of the population in terms of the hardy-weinberg equilibrium, the m and n allelic frequencies of the regional and cosmopolitan populations were subjected to the hardy-weinberg law. computations indicate that both populations were not in equilibrium (table 1). for the cosmopolitan population, it is postulated that this condition is accounted for by gene flow facilitated by migration. residents of the cosmopolitan population tend to originate from different places in the country, giving the population the characteristic of an increased rate of migration that leads to hybridization. new genes are passed into the gene pool of the parental populations. this intogression results in gene flow, hence the violation of the hardy-weinberg law (minelli et al. 2008). the regional population, on the other hand, displays little migration. however, compared to the cosmopolitan population with a population of about 20 million, the regional population is much smaller in size. hardy-weinberg equilibrium might not have been achieved due to possible interbreeding in the members of the local population. the frequency distribution of m and n alleles, with the m allele being more dominant, further reinforces this idea of genetic drift, as stochastic effects usually leads to the frequency alleles drifting toward higher or lower values (wang et al. 1998; trikalinos et al. 2008). both the cosmopolitan and regional populations' violation of the hardy-weinberg law indicate science diliman 47 figure 3. percentage distribution of mn blood group in cosmopolitan (upd) and regional (isu) populations. upd = university of the philippines, diliman; isu = isabela state university, cabagan, isabela; pnrc = philippine national red cross national data. frequency distribution of the mn blood group types differs significantly between the regional and cosmopolitan populations. guzman, gervasio, fontanilla & cao table 1. chi square tests for significant difference from the expected blood group distributions of populations at hardyweinberg equilibrium (hwe). both populations are not at hwe equilibrium. upd (m=52.0%; n=48.0%) isu (m=78.8%; n=21.2%) observed (o) expected (e) (o-e)2/e observed (o) expected (e) (o-e)2/e mm 19 13.79 1.97 43 36.64 1.11 mn 15 25.46 4.3 7 19.71 8.2 nn 17 11.75 2.35 9 2.65 15.2 total 51 χ2 = 8.6102 59 χ2 = 24.5021 conclusion p<0.05; significantly different from expected hwe p<<0.01; significantly different from expected hwe evolution at some level but is likely to be caused by different reasons. studies have shown some medical implications of the different blood types in the mn blood group system. the mn blood group is most commonly associated and is a good candidate for future studies on the genetic basis of human essential hypertension (miller et al. 1979; heise et al. 1987). moreover, weder et al. (1991) reported results that indicate the possible relationship of the mn blood group antigen to systolic blood pressures but with effects that are significantly different and oppositely directed in men and women. lastly, the study by delanghe et al. (1995) links the distribution of the mn phenotypes according to age with the diagnosis of essential hypertension, suggesting that the mn phenotype, indeed, is a genetic factor associated with early detection of essential hypertension. we report in this paper the blood type frequencies for multiple blood groups of a regional population in isabela province in northern luzon, philippines. no significant differences were found in the abo and rh blood groups’ frequency distribution compared to the cosmopolitan and national data. based on data gathered for the mn blood group, however, we conclude that this population is distinct from the population in the capital city of manila. both populations showed departure from the hardy weinberg equilibrium, which could be attributed to migrations for the upd population and genetic drift for the isu population. it is recommended that further surveys on the distribution of the mn blood group system be conducted on the different populations to determine if there is a trend in the regional variation of the m and n alleles across the country in order to shed light on the behaviour of these genes at the population level. data on hypertension and age of diagnosis should also be obtained from the corresponding populations to confirm or contradict the medical implication of the mn blood group. acknowledgements the authors would like to thank ms. rose ragamat of pnrc main office (intramuros, manila) for her generous help. the authors would also like to thank ms. tatiana abano and ms jocelyn serrano for their contributions in the initial phase of this project. we are also grateful for the participation of the institute of biology in the university of the philippines, diliman and isabela state university in this study. references adeyemo, o.a. & o.b. soboyejo, 2006. frequency distribution of abo, rh blood groups and blood genotypes among the cell biology and genetics students of university of lagos, nigeria. african journal of biotechnology 5 (22): 2062-2065. chérif-zahar, b., m.g. mattéi, c. le van kim, p. bailly, j.p. cartron & y. colin, 1991. localization of the human rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization. human genetics 86(4): 398-400. delanghe, j., d. duprez, m. de buyzere, d. robbrecht, b. bergez, g. leroux-roels & d. clement, 1995. mn blood group, a genetic marker for essential arterial hypertension in young adults. european heart journal 16(9): 1269-1276. dennis, y.m., n.m. hylem & c. fidler, 1998. prenatal diagnosis for fetal rhd status by molecular analysis of 48 science diliman frequency distribution of blood groups abo, mn and rh factor material plasma. new england journal of medicine 337: 1734–1738. heise, e.r., m.a. moore, q.b. reid & h.o. goodman, 1987. possible association of mn locus haplotypes with essential hypertension. hypertension 9: 634-640. hyman-parker, h., 1942. the development of the agglutinogens m and n in newborn infants. the journal of 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companies, inc. usa. 1098 pp. trikalinos, t.a., salanti, g., khoury, m.j., ioannidis j.p.a. 2006. impact of violations and deviations in hardy-weinberg equilibrium on postulated gene-disease associations. american journal of epidemiology 163(4):300-309. wakui, k., t. nishida, j. masuda, t. itoh, d. katsumata, t. ohno & y. fukushima, 1991. de novo interstitial deletion of 4q[46,xx,del(4)(q27q28.2)] with intact blood group-mn locus, confining its locus to 4q28.2–4q31.1. journal of human genetics 36(2): 149-153. wang, j., caballero, a., hill, w.g. 1998. the effect of linkage disequilibrium and deviation from hardyweinberg proportions on the changes in genetic variance with bottlenecking. heredity 81:174-186. weder, a.b., n.j. schork & s. julius, 1991. linkage of mn locus and erythrocyte lithium-sodium countertransport in tecumseh, michigan. hypertension 17: 977-981. science diliman 49 7marine macroalgae-villasenor.pmd i.m. villaseñor 61 science diliman (january-june 2016) 28:1, 61-67 marine macroalgae: a review irene m. v illasenor university of the philippines diliman issn 0115-7809 print / issn 2012-0818 online introduction there is an abundance of marine algae along the philippine coastline (36, 289 km) (hurtado et al. 2013). these algae are used for food, feeds, and medicine, among others. the most popular seaweeds that are utilized as food products are those belonging to the caulerpa, euchema, and gracilaria species (montaño 2002). sargassum sp, brown seaweed, is traditionally used as a f ish wrapper, vegetable, fertilizer, flower inducer, insect repellant, animal feed, and as a drink with reported health benef its (montaño et al. 2006). some philippine seaweed exhibit antimicrobial (mabugay et al. 1994) and cytotoxic activities against selected human cancer cell lines (tantengco et al. 2015). kappacarrageenan gel can also be used to sequester paralytic shell f ish poison (psp) (cañete and montaño 2002). the potential of seaweed resources as biofuel was also explored with the identif ication of the seaweed species sargassum spp. , turbinaria spp. , hydroclathrus spp. , caulerpa spp. , and ulva spp. as possible sources of biomass for biofuel production (marquez et al. 2014). agar the worldwide production of the gelling agent agar mainly relies on the red algae of the order gracilariales and gelidiales for raw material (villanueva et al. 2010). chemical property the alkali-modified agar from gracilaria edulis has a basic repeating unit of alternating 3-linked 6-o-methyl-beta-d-galactopyranose and 4-linked 3,6-anhydro-alpha-lgalactopyranose with partial methylation at o-2 of the anhydrogalactose and partial m a r i n e m a c r o a l g a e : a rev i ew 62 sulfation at the o-4of the methylated galactose residue (villanueva and montaño 1999). agar with the regular agarobiose repeating unit was also isolated from gracilaria arcuata and gracilaria tenuistipitata. agar from g. tenuistipitata has partial methylation at the 6-position of the d-galactosyl residues. both agars from g. arcuata and g. tenuistipitata exhibit sulphate substitution at varying positions in the polymer (montaño et al. 1999). another potential source of agar is the red algal order ceramiales. the native agar from laurencia flexilis has the same basic repeating unit with minor sulfation at 4-position of the 3-linked galactose residues. it has low sulfate and high 3, 6-anhydrogalactose levels (villanueva et al. 2010). physical property g. arcuata produces a soft agar gel, while the agar from g. tenuistipitata exhibits gel qualities typical of most gracilaria agars (montaño et al. 1999). alkali modif ication enhanced agar gel strength and syrenesis. the gel strength of g. edulis was considerably enhanced with the addition of sodium, potassium, and calcium ions (villanueva and montaño 1999). the native agar from l. flexilis formed a gel with moderate gel strength and higher gel syneresis (villanueva et al. 2010). compared with gracilaria spp. and gracilariopsis bailinae, gracilaria firma exhibited the highest growth rate and agar gel strength, and a high resistance to epiphytes when grown under controlled flow-through culture conditions (araño et al. 2000). in another study, the gel strength and the gelling and melting temperatures of gels prepared from gracilaria eucheumoides, gracilaria firma, gracilaria salicornia, l. flexibilis, and gracilariopsis heteroclada increased, whereas the syneresis index decreased upon the addition of sucrose (romero et al. 2000). gel strength of agar from g. eucheumoides was optimum when extracted at 900c, 10% alkali (naoh) concentration, and 2-hour duration. however, the agar yield was higher when the extraction was performed at a lower temperature, higher alkali concentration, and shorter treatment time. higher 3,6-anhydrogalactose content and lower sulphate level were obtained at higher temperature and alkali concentration, and longer duration of the treatment (villanueva et al. 1997). a seasonal assessment generally showed that the highest biomass and maximum agar yields from gelidiella acerosa (roleda et al. 1997a), g. eucheumoides (villanueva i.m. villaseñor 63 et al. 1999), and g. edulis (romero et al. 2007) are obtained during the rainy season. the three studies, however, have different conclusions regarding the quality and chemistry of the agars obtained. the overall gel quality (gel strength, viscosity, gelling, and melting temperatures) in g. acerosa was highest during the dry season (roleda et al. 1997a), while the agar from g. edulis exhibited the highest gel strength, deformation, cohesiveness, and melting temperature when collected during the onset of the rainy season (romero et al. 2007). both agars from g. eucheumoides and g. acerosa exhibited the strongest gels in july (villanueva et al. 1999). signif icant seasonal variations were also observed in the gelling and melting temperatures of agar from g. eucheumoides. the sulphate content of agar from g. acerosa was the lowest during the dry season (roleda et al. 1997a), which is in contrast to the results obtained by villanueva et al. (1999). g. acerosa exhibited a higher sulphate content and lower 3,6-anhydrogalactose during the dry season, while the sulphate content in agar samples from g. eucheumoides varied slightly. the agar from g. edulis contained the lowest amount of sulfate and mono-o-methylated residues (romero et al. 2007). vegetative plants of g. acerosa yielded higher agar content with high gel strength compared to tetrasporic plants (roleda et al. 1997b). pressure cooking, compared to the traditional method of boiling, extracted more agar from g. acerosa but lowered its quality (villanueva et al. 1998). agar samples from g. acerosa pretreated with acetic acid (0.5% for 1 hour at 16-200c) and autoclaved at 15-20 psi for one hour gave the highest agar yield and strength (roleda et al. 1997c). gamma irradiation increased the yield but decreased gel strength in agar samples from g. acerosa (villanueva et al. 1998). irradiation did not signif icantly change the sulphate level but decreased the 3,6-anhydrogalactose content of agar. carrageenan the carrageenophytes include four genera, six species, and 21 morphotypes/ varieties/cultivars under the family solieriaceae (hurtado et al. 2013). kappaphycus alvarezii, eucheuma denticulatum, and kappaphycus sp. sacol variety are the carrageenan-containing red seaweeds currently farmed in the philippines (aguilan et al. 2003). vegetative regeneration is the only farming method for eucheuma and kappaphycus, thereby necessitating the development of an alternative method of generating sporelings (azanza-corrales et al. 1996). m a r i n e m a c r o a l g a e : a rev i ew 64 chemical property the polysaccharide extracted from the seaweed kappaphycus striatum (schmitz) doty (sacol variety) is composed mainly of 3-linked beta-d-galactopyranosyl-4sulfate residues alternating with 4-linked 3,6-anhydro-alpha-d-galactopyranosyl (kappa carrageenan), 3,6-anhydrogalactopyranosyl-2-sulfate (iota-carrageenan), and 6-o-methylgalactopyranosyl-4-sulfate (methylated carrageenan) (villanueva and montaño 2003), and mu-precursor residues (aguilan et al. 2003) as minor components. by contrast, e. denticulatum predominantly contains iota-carrageenan with signif icant amounts of nu-precursor residues (aguilan et al. 2003). another seaweed varietry locally called “endong” has a similar appearance to k. alvarezii (doty) doty ex silva var. tambalang doty a . however, ‘’endong’’ mostly contains carrageenan of the iota type (villanueva et al. 2009). it was subsequently named as e. denticulatum (burman) collins & hervey var. endong trono & ganzonfortes var. nov. (ganzon-fortes et al. 2012). seaweed farmers are advised to separate their harvests of “endong” and “tambalang”. physical property among the carrageenophytes, kappaphycus sp. sacol variety is fast growing and has improved resistance against the “ice-ice” disease (aguilan et al. 2003). ‘’ice-ice’’ disease causes the depolymerization of kappa-carrageenan, leading to decreased average molecular weight, carrageenan yield, gel strength and viscosity, and increased syneresis index (mendoza et al. 2002). as an alternative to industrial alkali treatment, postharvest batch culture with low nutrient concentrations produced native carrageenans in e. denticulatum (“spinosum”) that had significantly higher gel strengths (villanueva and montaño 2014). in another study, k. alvarezii, kappaphycus sp. , and k. striatum were cultivated in tanks containing f ish farm effluent. fish farm effluent has high ammonium content (villanueva et al. 2005). all three carrageenophytes reduced the ammonium content of the f ish farm effluent and showed improved carrageenan content. however, the carrageenan quality was not signif icantly enhanced (rodriquez and montaño 2007). a study by mendoza et al. (2006) showed that the mature tissues of k. striatum sacol green variety yielded greater amounts of carrageenan; by contrast, the young tissues exhibited higher gel strength, cohesiveness, and viscosity, and lower average i.m. villaseñor 65 molecular weight. the minor iota carrageenan and methlylated carrageenan units in the major kappa-carrageenan decreased in quantity with age. villanueva et al. (2011) recommended that the harvest time for k. alvarezii var. alvarezii to be after eight weeks of culture, while for k. striatum var. sacol, kappaphycus sp. “aring-aring” and kappaphycus sp. “duyan” to be nine weeks. the highest optimization index, in terms of biomass, carrageenan yield, and gel strength, was observed during these weeks. gigartinacean and solieriacean are hybrids of kappa-iota carrageenans with a cooccurrence of kappa and iota structures in a chain in the former and as separate chains in the latter. gigartinacean hybrid has a lower molecular weight and produced inferior gels compared to solieriacean. both hybrids exhibited similar functional performance as viscosity enhancing/stabilizing and build-up agents (villanueva et al. 2005). references aguilan jt, broom je, hemmingson ja, dayrit fd, montaño mne, dancel mca, niñonuevo mr, furneaux rh. 2003. structural analysis of carrageenan from farmed varieties of philippine seaweeds. botanica marina. 46:179-192. araño kg, trono jr. gc, montaño ne, hur tado aq, villanueva rd. 2000. growth agar yield and quality of selected agarophyte species from the philippines. botanica marina. 23:517-524. az a n z a co r r a l e s r , a l i a z a t t, m o n t a ñ o n e . 1 9 9 6 . rec r u i t m e n t o f e u c h e u m a a n d kappaphycus on a farm in tawi-tawi, philippines. hydrobiologia. 326/327:235-244. cañete sjp, montaño mne. 2002. κ-carrageenan gel as agent to sequester paralytic shellf ish poison. marine biotechnology. 4:565-570. ganzon-fortes et, trono jr. gc, villanueva rd, romero jb, montaño mne. 2012. ‘endong’, a rare variety of the farmed carrageenophyte eucheuma denticulatum (burman) collins & hervey from the philippines. journal of applied phycology. 24:1107-1111. h u r t a d o a q , m o n t a ñ o m n e , m a r t i n e z g o s s m r . 2 0 1 3 . c o m m e r c i a l p r o d u c t i o n o f carrageenophytes in the philippines: ensuring long-term sustainability for the industry. journal of applied phycology. 25:733-742. mabugay ec, santos ps, montaño mne. 1994. screening for antimicrobial activities from selected philippines seaweeds. acta manilana. 42:31-37. m a r i n e m a c r o a l g a e : a rev i ew 66 marquez gpb, santiañez wje, trono jr. gc, montaño mne, araki h, takeuchi h, hasegawa t. 2 0 1 4 . s e a w ee d b i o m a s s of t h e p h i l i p p i n e s : s u s t a i n a b l e f ee d s t o c k fo r b i o g a s production. renewable and sustainable energy reviews. 38:1056-1068. mendoza wg, ganzon-for tes et, villanueva rd, romero jb, montaño mne. 2006. t issue age as a factor affecting carrageenan quantity and quality in farmed kappaphycus striatum (schmitz) doty ex silva. botanica marina. 49:57-64. mendoza wg, montaño nme, ganzon-for tes et, v illanueva rd. 2002. chemical and gelling prof ile of ice-ice infected carrageenan from cultivated kappaphycus striatum ( s c h m i t z ) s a c o l s t r a i n ( s o l i e r i a c e a e , g i g a r t i n a l e s , r h o d o p h y t a ) . j o u r n a l o f a p p l i e d phycology. 14:409-418. montaño mne, rodriquez mrc, balitaan rl. 2006. ethnobotany of sargassum spp in the philippines. coastal marine science. 30(1):222-225. m o n t a ñ o m n e . 2 0 0 2 . s e a w e e d s i n p h i l i p p i n e f o o d : tr a d i t i o n a l u s e s a n d r e c e n t developments. fisheries series. 68 supplement ii:1457-1459. montaño ne, v illanueva rd, romero jb. 1999. chemical characteristics and gelling pro pe rti e s o f agar fro m two philippine gracilaria spp. (gracilariales, rhodophyta). journal of applied phycology. 11:27-34. ro d r i q u e z m r c a n d m o n t a ñ o m n e . 2 0 0 7. b i o r e m e d i a t i o n p o t e n t i a l o f t h r e e carrageenophytes cultivated in tanks with seawater from f ish farms. journal of applied phycology. 19:755-762. roleda my, ganzon-for tes et, montaño ne, de los reyes fn. 1997a. temporal variation in the biomass, quantity and quality of agar from gelidiella acerosa (forsskal) feldmann et hamel rhodophyta: gelidiales) from cape bolinao, nw philippines. botanica marina. 40:487-495. roleda my, ganzon-fortes et, montaño ne. 1997b. agar from vegetatiove and tetrasporic gelidiella acerosa (gelidiales, rhodophyta). botanica marina. 40:501-506. ro l e d a m y, m o n t a ñ o n e , g a n z o n f o r t e s e t, v i l l a n u e v a r d . 1 9 9 7c . a c e t i c a c i d pretreatment in agar extraction of philippine gelidella acerosa (forsskaal) feldmann et hamel (rhodophyta, gelidiales). botanica marina. 40:63-69. romero jb, villanueva rd, montaño mne. 2008. stability of agar in the seaweed gracilaria eucheumatoides (g raci l ari al e s, rhodopyta) during postharvest storage. bioresource technology. 99:8151-8155. romero jb, montaño mne, merca fe, v illanueva rd, liao ml, bacic a . 2007. seasonal variations in the composition and gel quality of agar from gracilaria edulis in the philippines. botanica marina. 50:191-194. romero jb, montaño mne, merca fa , rumbaoa rg o, v illanueva rd. 2000. effect of sucrose on some physical properties of different philippine agars. philippine journal of science. 129(1):7-13. i.m. villaseñor 67 tantengco oag, limbo ca, montaño mne, jacinto sd. 2015. cytotoxic activity of crude extract and fractions from sargassum siliquosum (jg agardh) and other seaweeds against selected human cancer cell lines. journal of biosciences. 7(2):207-215. villanueva rd, montaño mne. 2014. enhancement of carrageenan gel quality in the commercially important tropical seaweed eucheuma denticulatum (rhodophyta), with postharvest treatment in low-nutrient conditions. botanica marina. 57(3):217-223. villanueva rd, romero jb, montaño mne, dela peña po. 2011. harvest optimization of four kappaphycus species from the philippines. biomass and bioenergy. 35:1311-1316. villanueva rd, romero jb, ragasa alr, montaño mne. 2010. agar from the red seaweed, laurencia flexilis (ceramiales, rhodophyta) from northern philippines. phycological research. 58:151-156. villanueva rd, montaño mne, romero jb. 2009. iota-carrageenan from a newly farmed, rare variety of eucheumoid seaweed—”endong”. journal of applied phycology. 21:2730. villanueva rd, mendoza wg, rodriguez mrc, romero jb, montaño mne. 2005. structure a n d f u n c t i o n a l p e r f o r m a n c e o f g i g a r t i n a c e a n k a p p a i o t a h y b r i d c a r r a g e e n a n a n d solieriacean kappa-iota carrageenan blends. food hydrocolloids. 18:283-292. v illanueva rd, yap ht, montaño mne. 2005. survivorship of coral juveniles in a f ish farm environment. marine pollution bulletin. 51:580-589. villanueva rd, montaño mne. 2003. fine chemical structure of carrageenan from the commercially cultivated kappaphycus striatum (sacol variety) (solieriaceae, gigartinales, rhodophyta). journal of phycology. 39:513-518. villanueva rd, montaño mne. 1999. highly methylated agar from gracilaria edulis (gracilariales, rhodophyta). journal of applied phycology. 11:225-227. v illanueva rd, montaño mne, romero jb, aliganga aka , enriquez ep. 1999. seasonal variations in the yield, gelling properties, and chemical composition of agars from gracilaria euchemoides and gelidiella acerosa (rhodophyta) from the philippines. botanica marina. 42:175-182. v illanueva rd, rumbaoa ro, gomez av, loquias mm, dela rosa am, montaño mne. 1998. γ-irradiation in the extraction of agar from gelidiella acerosa (forsskaal) feldmann et hamel. botanica marina. 41:199-202. villanueva rd, pagba cv, montaño mne. 1997. optimized agar extraction from gracilaria eucheumoides harvey. botanica marina. 40:369-372. editor’s note humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> journal cover images courtesy of (l-r) vargas museum & anticamara and tan call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 3layman'sabstracts.pmd layman’s abstracts 1 science diliman (january-june 2018) 30:1, 1-3 layman’s abstracts low-complexity physical layer security scheme for heterogeneous cellular networks based on coord inated precod ing design and artif icial noise generation neil irwin m. bernardo and franz de leon issn 0115-7809 print / issn 2012-0818 online heterogeneous network (hetnet) deployment is a cellular system design approach in which multiple low power access nodes are underlaid in a t r a d i t i o n a l m a c r o c e l l u l a r n e t w o r k . h e t n e t s a r e a b l e t o p r o v i d e s u b s t a n t i a l i n c r e a s e i n ce l l u l a r c a p a c i t y a n d e n e r g y e f f i c i e n c y t h u s making it a viable solution to meet the demands in the next generation of cellular system, also known as 5g. in this study, we investigated how security can be incorporated into the downlink transmission (i.e. from telecommunications network to mobile phones) of hetnets while still m a i n t a i n i n g t h e i r h i g h ce l l u l a r c a p a c i t y a n d h i g h e n e r g y e f f i c i e n c y proper ties. our approach is to integrate information security, user data r a t e r e q u i r e m e n t , a n d p o w e r co n s u m p t i o n o f t h e h e t n e t ’s d o w n l i n k transmission into a computationally-tractable convex optimization model. f r o m t h e f o r m u l a t e d m o d e l , w e p r o p o s e a s e c u r i t y t e c h n i q u e w i t h s u b o p t i m a l p e r f o r m a n c e b u t w i t h a c o m p u t a t i o n a l c o m p l e x i t y t h a t i s feasible for real-time implementation. feed ing habits of mobula japanica (chond richthyes, mobul idae) in butuan bay, mindanao island, phil ippines shirlamaine irina g. masangcay, ephrime b. metillo, ken-ichi hayashizaki, satoru tamada and shuhei nishida we studied the feeding habits of the spinetail devil ray mobula japanica, l o c a l l y k n ow n a s pa n t i h a n , f r o m b u t u a n b a y, e a s te r n b o h o l s e a f r o m january to may 2016 using data on its stomach contents, and carbon a n d n i t r o g e n s t a b l e i s o t o p e a n a l y s e s . s m a l l s h r i m p l i k e k r i l l pseudeuphausia latifrons, known locally as alamang, contributed almost 100% to the devil ray’s ingested food. stable isotope analysis conf irmed the specialized feeding and assimilation of the krill food. this study is the f irst to identify the swarming krill p. latifrons as the major food of the spinetail devil ray in butuan bay. layman’s abstracts 2 fucoidan content in phil ippine brown seaweeds joemark t. narsico, joyce a. nieva, alper james g. alcaraz, elad io g.m. anino v, norchel corcia f. gomez and marco nemesio e. montaño the philippines is home to hundreds of seaweed species that serve as sources of high-value natural products, such as fucoidan. fucoidan is a s u l f a t e d p o l y s a c c h a r i d e t h a t c a n b e e x t r a c t e d f r o m t h e c e l l w a l l s o f brown seaweeds and is reported to have a wide range of bioactivities for possible medicinal applications. in this study, we assessed philippine brown seaweeds as sources of fucoidan by determining which species or genera among local brown algae has the highest content of partially p u r i f i e d f u co i d a n a n d w h e r e t h e s e s p ec i e s c a n b e f o u n d w i t h i n t h e country. fucoidan content from different species of brown seaweeds were determined in f i fty sites across four teen provinces in nor thern luzo n ( c a g a y a n , i l o c o s ) , w e s t l u z o n ( p a n g a s i n a n ) , t h e e a s t e r n s e a b o a r d o f l u z o n ( q u e z o n p r o v i n c e , c a m a r i n e s , s o r s o g o n ) , c e n t r a l a n d e a s t e r n visayas (bohol, cebu, negros oriental, negros occidental), and northern m i n d a n a o ( c a m i g u i n , l a n a o d e l n o r t e , m i s a m i s o r i e n t a l , m i s a m i s occidental). sargassum spp. , the most abundant in all sites, and turbinaria o r n a t a, fo u n d o n l y i n 1 1 s i te s , b o t h h a ve s i g n i f i c a n t l y h i g h e r co n te n t compared to the other samples. similarly, higher content of semi-pure f u c o i d a n w e r e o b s e r v e d i n b r o w n s e a w e e d s f r o m b o h o l , c e b u , pangasinan, quezon province, camiguin, and cagayan. staphylococcus aureus and methicill in-resistant s. aureus (mrsa) carriage in publ ic computer service providers and util ity jeepneys in up dil iman jann eldy l. daquioag, ricardo bened ict c. almirol, mary grace b. ayala, ma. socorro edden p. subejano and gil m. penul iar staphylococcus aureus is a bacterium that can cause serious infections. i t i s o f t e n f o u n d i n s o l i d o b j e c t s , s u c h a s c o m p u t e r p e r i p h e r a l s o f computer service providers (csps) and handrails of public utility jeepneys (pujs). s. aureus infections are often treated without complications, except layman’s abstracts 3 in cases where a particular strain called methicillin-resistant s. aureus (mrsa) is involved. in this study, the prevalence of s. aureus and mrsa in csps, computer peripherals, and handrails of pujs inside up diliman, and associated risk factors for contamination were determined. s. aureus a n d m r s a w e r e i d e n t i f i e d u s i n g m o r p h o l o g i c a l , b i o c h e m i c a l , a n d molecular methods from 162 computer peripherals from 27 csps, and 196 puj handrails. s. aureus was identif ied in 92.6% of csps, 36.4% of computer peripherals, and 7.1% of pujs, whereas mrsa was present in 3.1% of csps and 2% of pujs. no signif icant associations between s. aureus/ m r s a a n d t h e a s s e s s ed r i s k f a c to r s we r e o b s e r ved ( p > 0 . 0 5 ) . re s u l t s indicate that, while s. aureus prevalence is relatively high, mrsa carriage is low in csps and pujs in up diliman. population structure of the krill prey of the spinetail devil ray mobula japanica (chondrichthyes, mobul idae) from southeastern bohol sea, phil ippines shirlamaine irina g. masangcay, ephrime b. metillo and shuhei nishida while investigating the feeding habits of the spinetail devil ray mobula japonica in butuan bay, we found true krill (known locally as alamang) a s t h e m a i n , oft e n t h e o n l y fo o d i te m i n t h e s to m a c h o f t h e r a y. we identif ied the krill species as pseudeuphausia latifrons. information about the population of this krill species is very limited, thus this study was aimed at analyzing the size-composition of individuals collected from the stomach of the ray from january to may 2016. the total lengths of intact krill ranged between 4.0–6.9 mm for juveniles and 7.0–10.9 mm for adults. in general, males were larger than females. juveniles were dominant until late march, and adults dominated by april and may. the largest male and egg-carrying female individuals also appeared during t h e w a r m m o n t h s of a p r i l a n d m a y, i n d i c a t i n g s p a w n i n g d u r i n g t h e s e months. this study provides evidence that individuals of the krill p. latifrons e a t e n b y r a y s g r o w f r o m j u v e n i l e s t o a d u l t s f r o m j a n u a r y t o m a y i n butuan bay. 31 the jordan canonical form of a product of elementary s-unitary matrices erwin j. gonda agnes t. paras* institute of mathematics and natural sciences research institute university of the philippines diliman abstract let s be an n-by-n, nonsingular, and hermitian matrix. a square complex matrix q is said to be s-unitary if q*sq = s. an s-unitary matrix q is said to be elementary if rank(q — i) = 1. it is known what form every elementary s-unitary can take, and that every s-unitary can be written as a product of elementary s-unitaries. in this paper, we determine the jordan canonical form of a product of two elementary s-unitaries. keywords: elementary s-unitary matrix, hermitian matrix, jordan canonical form introduction let m n be the set of all n-by-n matrices with entries in the complex field ℂ and let gl n be the set of all nonsingular matrices in m n . let s ∈ gl n be hermitian. a q ∈ m n is said to be s-unitary if q*sq = s, where q* is the conjugate transpose of q (gohberg et al. 2005). if s = i, then the set of s-unitary matrices in gl n coincides with the set of unitary matrices. let u s be the set of all s-unitary matrices. observe that u s is nonempty since i ∈ us. if q ∈ u s , then q -1 is similar to q*, |det q| = 1, and αq ∈ u s for all α ∈ ℂ with modulus 1. moreover, u s is a group under multiplication and consists of all matrices in m n that preserve the scalar product [u,v] s = u*sv for all u, v ∈ ℂn. an h ∈ us is called elementary if rank(h – i) = 1. let h s be the set of all elementary s-unitary matrices. when s is hermitian, h s = k s ∪ l s , where k s = {kx,r = i + irxx*s ∶ x ∈ ℂ n\{0}, x*sx = 0, and r ∈ ℝ\{0}} and ls = {lx,φ = i + (eiφ – 1) x*sx xx*s: x ∈ ℂn, x*sx ≠ 0, φ ∈ ℝ, and eiφ ≠ 1} * corresponding author science diliman (january-june 2020) 32:1, 31-41 the jordan canonical form of a product of elementary s-unitary matrices 32 (catbagan 2015). if v ∈ ℂn such that v*sv ≠ 0, the λ s -householder matrix sv = i – 2 v*sv vv*s generalizes the householder matrix of v, which is equal to lv,π for s = i (merino et al. 2011; horn and johnson 2013). if kx,r ∈ ks, then kx,r –1 = kx,–r and kx,r is similar to in–2 ⊕ j2 (1). if lx,φ ∈ ls, then lx,φ–1 = lx,–φ and lx,φ is similar to i n–1 ⊕ [eiφ]. hence h ∈ h s if and only if h–1 ∈ h s . thus, i is a product of two elements of h s . moreover, if a ∈ u s , then a can be written as a product of elements of h s (catbagan 2015). thus, the elements of h s generate the group u s . since there are two types of elements of h s , there are three types of products of two elements of h s up to similarity. we wish to determine which jordan canonical forms arise for each possibility, since the jordan canonical form of a matrix reveals a lot of information such as its rank, nullity, eigenvalues, and their algebraic and geometric multiplicities. analogous results for symplectic matrices and j-householder matrices can be found in de la rosa et al. (2012). preliminaries if s = p*p for some p ∈ gl n , then x*sx > 0, when 0 ≠ x ∈ ℂn; and q ∈ u s if and only if pqp–1 ∈ u i . hence when s is positive definite, h s = l s , and every s-unitary is similar to a unitary matrix. since u s = u –s , from now on we only consider s that is *-congruent to i k ⊕–i n–k for 0 < k < n, that is s = p * (i k ⊕ –i n–k ) p, for some p ∈ gl n . let n be a positive integer such that n ≥ 2, and t ⊆ ℂn be nonempty. let ts be the s-perpendicular subspace of t defined by ts = { z ∈ ℂn | x * sz = 0, for all x ∈ t } . then dimts = n – dim(spant ) , since ts = (s(spant))⊥, which is the orthogonal complement of s(spant ) with respect to the usual inner product on ℂn, and ℂn = w ⊕ w⊥ for any subspace w of ℂn. let hx, hy ∈ hs and a = hx hy . then hx = i + αxx*s and hy = i + βyy*s, for some nonzero α, β ∈ ℂ. if {x, y} is linearly dependent, then y = δx, for some δ ∈ ℂ. this implies a = i + αxx*s + βyy*s + αβxx*syy*s = i + (α + β|δ|2 + αβ|δ|2 x*sx)xx*s. hence a = i + μxx*s, where μ = α + β|δ|2 + αβ|δ|2 x*sx ∈ ℂ. if μ = 0, then a = i, which implies hx = hy –1. if μ ≠ 0, then rank(a – i) = 1, and since a ∈ u s , we have a ∈ h s . e.j. gonda and a.t. paras 33 suppose {x, y} is linearly independent. let z ∈ ℂn be given. suppose z ∈ ker (a – i), that is, az = z. then 0 = (a – i)z = (αx*sz)x + (βy* sz)y + αβ(x*sy)(y*sz)x. since {x, y} is linearly independent, and α, β are nonzero, we have y*sz = 0 and it follows that x*sz = 0. thus, z ∈ {x, y}s. conversely, suppose z ∈ {x, y}s. then x*sz = y*sz = 0 and so (a – i)z = α(x*sz)x + β(y*sz)y + αβ(x*sy)(y*sz)x = 0, that is, z ∈ ker(a – i). therefore ker(a – i) = {x, y}s. lemma 1. let s ∈ gl n be hermitian and let x, y ∈ ℂn be nonzero. suppose h x , h y ∈ h s and a = h x h y . if {x, y} is linearly dependent, then a = i or a ∈ h s . if {x, y} is linearly independent, then ker(a – i) = {x, y}s. if {x, y} is linearly independent, an immediate consequence of lemma 1 is that dim(ker(a – i)) = dim({x, y}s) = n – 2. thus, there are n – 2 jordan blocks corresponding to 1 in the jordan canonical form of a. for completeness, we include the following lemma, which is used several times in the paper and can be readily verified. if a = [a ij ] ∈ m n , the trace of a is defined to be tra = σn j = 1ajj. lemma 2. let a, b ∈ m 2 be given such that neither is a scalar matrix. then a and b are similar if and only if tra = trb and det a = det b. let {x, y} be a linearly independent subset of ℂn. we consider each of the three possibilities (i) h x , h y ∈ k s , (ii) h x , h y ∈ l s , or (iii) h x ∈ k s and h y ∈ l s , and determine the jordan canonical form of the product h x h y . hx, hy ∈ ks let {x, y} be a linearly independent subset of ℂn such that h x , h y ∈ k s , i.e., h x = i + ir x xx*s and h y = i + ir y yy*s, where x*sx = y*sy = 0, and rx, ry are nonzero real numbers. if a = h x h y , then a = i + ir x xx*s + ir y yy*s – r x r y (x*sy)xy*s. either x*sy = 0 or x*sy ≠ 0. the jordan canonical form of a product of elementary s-unitary matrices 34 case 1: if x*sy = 0, then a = i + ir x xx*s + ir y yy*s. note that {x, y}s = {x}s ∩ {y}s, which is of dimension n – 2. if n > 2, then there exists z ∈ {y}s but z ∉ {x}s. hence, (a – i ) z = ir x (x*sz)x ≠ 0. since x*sx = y*sy = x*sy = 0, we have (a – i ) 2 = 0. since a – i ≠ 0, the minimal polynomial of a is (x – 1)2 and so the largest jordan block corresponding to 1 is of size 2. the number of jordan blocks corresponding to 1 of size 1 is rank(a – i ) 0 –2 rank(a – i ) + rank(a – i )2 = n – 2(2) + 0 = n – 4. since 1 is the only eigenvalue of a and there are n – 2 jordan blocks corresponding to 1, a is similar to i n – 4 ⊕ j 2 (1) ⊕ j 2 (1). if n = 2, then x*sy ≠ 0, otherwise x*sx = y*sy = x*sy = 0 and {x, y} linearly independent imply ℂ2 = {x, y}s is of dimension n – 2 = 0, which is a contradiction. case 2: suppose x*sy ≠ 0. we find any remaining eigenvalues of a. the images of x and y under a are ax = x + ir x (x*sx)x + ir y (y*sx)y – r x r y (x*sy)(y*sx)x = (1 – r x r y |x*sy|2) x + ir y (y*sx)y and ay = y + ir x (x*sy)x + ir y (y*sy)y – r x r y (x*sy)(y*sy)x = y + ir y (x*sy)x . hence span{x, y} is invariant under a. consider the restriction of a to span{x, y} and its matrix representation m = 1 – rx ry |x*sy| 2 ir y (x*sy) iry (y*sx) 1 with respect to the ordered basis {x, y}. since x*sx = y*sy = 0 and x*sy ≠ 0, we have ℂn = span{x, y} ⊕ {x, y}s. thus a is similar to m ⊕ i n–2 and 1 is not an eigenvalue of m. note that det(m) = 1 and tr(m) = 2 – r x r y |x*sy|2 ∈ ℝ. since a ∈ u s has determinant 1 and m is not a scalar matrix, we see that m is similar to one of the following: diag(eiθ, e–iθ), where θ ∈ ℝ such that eiθ ≠ ±1; j 2 (–1); or diag(λ, λ–1), where λ ∈ ℝ and |λ| > 1. we determine if the preceding three possibilities for the jordan canonical form of m occur. let θ ∈ ℝ such that eiθ ≠ ±1. if we choose rx, ry ∈ ℝ such that rx ry = 2(1–cosθ) |x*sy|2 ≠ 0, then det (m) = 1 = det (diag(eiθ, e–iθ)) and tr (m) = 2 cosθ = tr (diag(eiθ, e–iθ)). by lemma 2, m is similar to diag(eiθ, e–iθ). e.j. gonda and a.t. paras 35 if we choose rx, ry ∈ ℝ such that rx ry = 4 |x*sy|2 , then tr(m) = –2 = tr(j 2 (–1)) and det (m) = 1 = det (j 2 (–1)). by lemma 2, m is similar to j 2 (–1). let λ ∈ ℝ such that |λ| > 1. if we choose rx, ry ∈ ℝ such that rx ry = (2–λ–λ–1) |x*sy|2 ≠ 0, then we have det (m) = 1 = det (diag(λ, λ–1)) and tr(m) = –2 = tr(diag(λ, λ–1)). since λ ≠ λ–1, we have that m is similar to diag(λ, λ–1). theorem 3. let s ∈ gl n be indefinite hermitian and x, y ∈ ℂn be given. if {x, y} is linearly independent and hx, hy ∈ ks, then the product hxhy is similar to one of the following: a. i n–4 ⊕ j 2 (1) ⊕ j 2 (1) b. i n–2 ⊕ j 2 (–1) c. i n–2 ⊕ diag(eiθ, e–iθ), where θ ∈ ℝ such that eiθ ≠ ±1 d. i n–2 ⊕ diag(λ, λ–1), where |λ| > 1 and λ ∈ ℝ. hx, hy ∈ ls we now consider the product of two elements of l s . let x, y ∈ ℂn such that {x, y} is linearly independent and hx, hy ∈ ls, that is, hx = i + eiα –1 x*sx xx*s and h y = i + eiβ –1 y*sy yy*s, where x*sx and y*sy are nonzero, and α, β ∈ ℝ such that eiα ≠ 1 and eiβ ≠ 1. since hy = hay for all nonzero a ∈ ℂ, we can assume that x*sx, y*sy ∈ {1, –1}. if a = hx hy, then a = i + eiα –1 x*sx xx*s + eiβ –1 y*sy yy*s + eiα –1 x*sx eiβ –1 y*sy (x*sy)xy*s. case 1: if x*sy = 0, then a = i + eiα –1 x*sx xx*s + eiβ –1 y*sy yy*s. observe that ax = x + (eiα –1)x = eiαx. hence, x is an eigenvector of a corresponding to eiα. similarly, y is an eigenvector of a corresponding to eiβ. since x*sx and y*sy are nonzero and x*sy = 0, we have ℂn = span{x, y} ⊕ {x, y}s. hence a is similar to i n–2 ⊕ diag(eiα, eiβ). the jordan canonical form of a product of elementary s-unitary matrices 36 case 2: suppose x*sy ≠ 0. we find any remaining eigenvalues of a. the images of x and y under a are ax = x + (eiα –1)x + eiβ –1 y*sy (y*sx)y + eiα –1 x*sx eiβ –1 y*sy (x*sy)(y*sx)x = �eiα + eiα –1 x*sx eiβ –1 y*sy |x*sy|2�x + � eiβ –1 y*sy (y*sx)�y and ay = y + eiα –1 x*sx (x*sy)x + eiβ –1 y*sy (y*sy)y + eiα –1 x*sx eiβ –1 y*sy (x*sy)(y*sy)x = eiβy + �eiβx*sy eiα –1 x*sx �x. hence span{x, y} is invariant under a. consider the restriction of a to span{x, y} and its matrix representation l = eiα + eiα –1 x*sx eiβ –1 y*sy |x*sy|2 eiβx*sy eiα –1 x*sx eiβ –1 y*sy ( y * s x ) eiβ with respect to the ordered basis {x, y}. note that ax + by ∈ {x, y}s for some a, b ∈ ℂ if and only if x*s(ax + by) = 0 and y*s(ax + by) = 0, that is x*sx x*sy y*sx y*sy � � a b = 0 0 . since x*sx, y*sy ∈ {1, –1}, we have {x, y}s ∩ span{x, y} = {0} if and only if x*sx and y*sy have opposite signs or |x*sy| ≠ 1. if x*sx = y*sy ∈ {±1} and |x*sy| = 1, then x, y ∉ {x, y}s and {x, y}s ∩ span{x, y} = span{(x*sy)x – (x*sx)y}. e.j. gonda and a.t. paras 37 hence span{x} ⊕ {x, y}s is of dimension n–1 and contains span{x, y}. now ax can be rewritten as ax = ei(α+β) x – (eiβ –1)(y*sx)[(x*sy)x – (x*sx)y]. this implies that span{x} ⊕ {x, y}s is invariant under a. since deta = ei(α+β) and rank(a – i) = 2, we have that a is similar to i n–3 ⊕ j 2 (1) ⊕ [ei(α+β)], if ei(α+β) ≠ 1; or i n–3 ⊕ j 3 (1), if ei(α+β ) = 1. if ℂn = span{x, y} ⊕ {x, y}s, then a is similar to i n–2 ⊕ l and 1 is not an eigenvalue of l. observe that det l = ei(α+β) and tr l = eiα + eiβ + eiα –1 x*sx eiβ –1 y*sy |x*sy|2. since a ∈ u s and l is not a scalar matrix, then l is similar to one of the following: diag(eiθ, eiϕ), where θ, ϕ ∈ ℝ such that eiθ, eiϕ are distinct and both are not equal to 1; j 2 (λ), where |λ| = 1 but λ ≠ 1; or diag (λ, λ–1), where |λ| > 1. it suffices to determine whether the last two possibilities for the jordan canonical form of l occur. but first we need to determine the possible nonzero values of x*sy, when x*sx, y*sy ∈ {1, –1} and {x, y} is linearly independent. let e i ∈ ℂn denote the column with ith entry 1 and 0 elsewhere. suppose c ∈ ℂ is nonzero and s = p*(i k ⊕ – i n–k )p, for some nonsingular p and integer 0 < k < n. if |c| > 1, we can take x, y ∈ ℂn such that px = e 1 and py = ce 1 + |c|2 –1e k+1, so that x*sx = 1, y*sy = |c|2 – (|c|2 – 1) = 1, and x*sy = c. thus, if |c| > 1, there exists a linearly independent set {x, y} such that x*sx = y*sy and x*sy = c. if c ∈ ℂ is nonzero and we take x, y ∈ ℂn such that px = e 1 and py = ce 1 + |c|2 –1e k+1, then x*sx = 1, y*sy = |c| 2 – (|c|2 + 1) = –1 and x*sy = c. hence every nonzero c ∈ ℂ can be realized as x*sy by a linearly independent set {x, y} such that x*sx = –y*sy, when s is *-congruent to i k ⊕ –i n–k . let α = β ∈ ℝ such that α ≠ kπ, for all k ∈ ℤ. if a = re(eiα), then –4eiα (eiα – 1)2 = 2 1–a > 1. if we take x, y ∈ ℂn such that x*sx = 1 = y*sy and |x*sy|2 = –4eiα (eiα – 1)2 , then tr l = 2eiα + (eiα–1)2 |x*sy|2 = –2eiα and det l = ei2α. since l is not a scalar matrix, it follows from lemma 2 that l is similar to j 2 (–eiα), where eiα ≠ ±1. if we take eiα = e–iβ = i, and x, y ∈ ℂn such that x*sx = 1 = –y*sy and |x*sy| = 1, then tr l = –2 and det l = 1. since l is not a scalar matrix, l is similar to j 2 (–1). let λ = reiθ, where r > 1 and θ ≠ 2kπ for all k ∈ ℤ. then – eiθ(r –1)2 (eiθ–1)2r is positive. � � the jordan canonical form of a product of elementary s-unitary matrices 38 if we take α = β = θ, and x, y ∈ ℂn such that x*sx = 1 = –y*sy and |x*sy|2 = – eiθ(r –1)2 (eiθ–1)2r , then tr l = 2eiθ– (eiθ–1)2 |x*sy|2 = eiθ (r + r–1) = λ + λ–1 and det l = ei2θ = λ λ–1 . hence l is similar to diag(λ, λ–1). let λ = r, where r > 1. let β = –α and α ∈ ℝ such that re(eiα) = r–1. since (r –1)2 r > 0, we have r –r –1 2(1–r –1) > 1. if we take x, y ∈ ℂn such that x*sx = 1 = y*sy and |x*sy|2 = r –r –1 2(1–r –1) , then tr l = 2r –1 + 2(1–r –1) |x*sy|2 = r + r –1 and det l = 1. hence l is similar to diag(r, r –1). theorem 4. let s ∈ gl n be indefinite hermitian and x, y ∈ ℂn be given. if {x, y} is linearly independent such that hx, hy ∈ ls, then the product hxhy is similar to one of the following: a. i n–2 ⊕ diag(eiθ, eiϕ), where θ, ϕ ∈ ℝ such that eiθ, eiϕ ≠ 1 b. i n–3 ⊕ j 2 (1) ⊕ [eiθ], where θ ∈ ℝ and eiθ ≠ 1 c. i n–3 ⊕ j 3 (1) d. i n–2 ⊕ j 2 (λ), where |λ| = 1 and λ ≠ 1 e. i n–2 ⊕ diag(λ, λ–1), where |λ| > 1. hx ∈ ks and hy ∈ ls lastly, we consider the product of an element of k s and of l s . if x, y ∈ ℂn are nonzero such that hx ∈ ks and hy ∈ ls, then hx = i + irxx*s, where r ∈ ℝ ∖ {0}, and x*sx = 0, and hy = i + e iα–1 y*sy yy*s, where eiα ≠ 1. note that {x, y} is linearly independent since x*sx = 0 ≠ y*sy. if a = hx hy, then a = i + irxx*s + e iα–1 y*sy yy*s + ir e iα–1 y*sy (x*sy) xy*s. case 1: if x*sy = 0, then a = i + ir xx*s + e iα–1 y*sy yy*s, ax = x, and ay = eiαy. since x*sy = 0 and x*sx = 0, we have (a – i )2 = (e iα–1)2 y*sy yy*s and (a – i )3 = (e iα–1)3 y*sy yy*s . e.j. gonda and a.t. paras 39 observe that rank(a – i ) = 2 and rank (a – i )2 = rank(a – i )3 = 1 , which imply that 2 is the size of the largest jordan block corresponding to 1, and the number of jordan blocks of size 2 corresponding to 1 is rank(a – i ) – 2rank(a – i )2 + rank(a – i )3 = 2 – 2(1) + 1 = 1. since there are n–2 jordan blocks corresponding to 1 and det a = eiα, we have that a is similar to i n–3 ⊕ j 2 (1) ⊕ [eiα]. case 2: suppose x*sy ≠ 0. the images of x and y under a are ax = x + e iα–1 y*sy (y*sx)y + ir e iα–1 y*sy |x*sy|2x = �1+ir e iα–1 y*sy |x*sy|2� x + e iα–1 y*sy (y*sx)y and ay = y + ir(x*sy)x + (eiα –1)y + ir(eiα – 1)(x*sy)x = ireiα (x*sy)x + eiαy. hence span{x, y} is invariant under a. consider the restriction of a to span{x, y} and its matrix representation k = 1 + ir eiα –1 y*sy |x*sy|2 ireiα(x*sy) eiα –1 y*sy ( y * s x ) eiα with respect to the ordered basis {x, y}. since ℂn = span{x, y} ⊕ {x, y}s, a is similar to i n–2 ⊕ k and 1 is not an eigenvalue of k. note that det k = eiα ≠ 1 and tr k = eiα + 1 + ir eiα –1 y*sy |x*sy|2. since a is s-unitary, k is similar to one of the following: diag(eiθ, eiϕ), where θ, ϕ ∈ ℝ such that eiθ, eiϕ are distinct with both not equal to 1, and ei(θ + ϕ) = eiα; or diag(λ, λ–1 ), where |λ| > 1 and λ ≠ ±1. we now determine whether the three possibilities for the jordan canonical form of k occur. the jordan canonical form of a product of elementary s-unitary matrices 40 let θ, ϕ ∈ ℝ such that eiθ, eiϕ, and ei(θ+ϕ) are not equal to 1, and eiθ ≠ eiϕ. if α = θ + ϕ, choose r ∈ ℝ such that r(eiα –1) = (y*sy)(1–e iθ)(eiϕ–1) i|x*sy|2 . this has a solution since (1–eiθ)(eiϕ–1) eiα –1 = –1 + e iθ + eiϕ–2 eiα –1 is nonzero and the real part of eiθ + eiϕ–2 eiα –1 is 1. then det k = ei(θ + ϕ) = det(diag(eiθ, eiϕ)) and tr k = eiθ + eiϕ = tr(diag(eiθ, eiϕ)). thus k is similar to diag(eiθ, eiϕ). let λ = teiγ, where t, γ ∈ ℝ such that t > 1 and ei2γ ≠ 1. choose α = 2γ and r ∈ ℝ such that r(eiα –1) = (y*sy) (1–te iγ) (t–1eiγ– 1) i|x*sy|2 . this has a solution since (1–teiγ) (t–1eiγ– 1) eiα–1 = –1 + (t + t –1) eiγ– 2 ei2γ–1 is nonzero and the real part of (t + t –1) eiγ– 2 ei2γ–1 is 1. then det k = λ λ–1 = det(diag(λ, λ–1 )) and tr k = (t + t–1) eiγ = tr (diag(λ, λ–1)). by lemma 2, k is similar to diag(λ, λ–1). let λ = eiβ, where β ∈ ℝ and λ ≠ ±1. choose α = 2β and r ∈ ℝ such that r = (1–λ) y*sy i(λ + 1)|x*sy|2 . this has a solution since 1–λ λ + 1 = –1 + 2 λ + 1 and the real part of 2 λ + 1 is 1. then det k = λ2 = det j 2 (λ) and tr k = 2λ = tr j 2 (λ). since k is not a scalar matrix, k is similar to j 2 (λ). theorem 5. let s ∈ gl n be indefinite hermitian and x, y ∈ ℂn be given. if {x, y} is linearly independent such that hx ∈ ks and hy ∈ ls, then the product hx hy is similar to one of the following: a. i n–3 ⊕ j 2 (1) ⊕ [eiα], for some α ∈ ℝ such that eiα ≠ 1 b. i n–2 ⊕ diag(eiθ, eiϕ), where θ, ϕ ∈ ℝ such that eiθ, eiϕ, ei(θ + ϕ) are all not equal to 1, and eiθ ≠ eiϕ c. i n–2 ⊕ diag(λ, λ–1), where |λ| > 1 and λ ∉ ℝ d. i n–2 ⊕ j 2 (λ), where |λ| = 1 but λ ≠ ±1. e.j. gonda and a.t. paras 41 acknowledgments the work of a.t. paras was supported by the natural sciences research institute (nsri) project mat-18-1-05. references catbagan k. 2015. on the λs-polar decomposition and the λ s -elementary matrices [b.s. thesis]. philippines: university of the philippines diliman. de la rosa kl, merino di, paras at. 2012. the j-householder matrices. linear algebra appl. 436(5):1189-1194. gohberg i, lancaster p, rodman l. 2005. indefinite linear algebra and applications. basel: birkhäuser. horn ra, johnson cr. 2013. matrix analysis. 2nd ed. new york: cambridge university press. merino di, paras at, teh td. 2011. the λ s -householder matrices. linear algebra appl. 436:2643-2664. ______ erwin j. gonda is a b.s. mathematics graduate of university of the philippines diliman and is currently working in the insurance industry. agnes t. paras <agnes@math.upd.edu.ph> is a professor of mathematics at university of the philippines diliman 10call for papers-new.pmd social science diliman, vol. 9, number 1, january-june 2013 humanities diliman, science diliman and social science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> journal cover images courtesy of (l-r) vargas museum & magdalita et al. call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 13info for authors.pmd 103 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the off ice of the vicechancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles and short communications may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); 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[photo from agno, pangasinan by biodiversity research laboratory] contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. science diliman july-december 2019 • vol. 31 no. 2issn print 0115-7809 issn online 2012-0818 international advisory board teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus university of illinois, usa university of california, irvine, usa the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheffield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. department of physics university of warwick, united kingdom raul k. suarez, ph.d. professor emeritus university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences graduate school university of tsukuba, japan sd-sample article p.n.y. young and i.k.c. fontanilla 53 science diliman (january-june 2014) 26:1 53-76 biodistribution of the informal group basommatophora in the philippines patrick noel y. young* ian kendrich c. fontanilla university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract b a s o m m a t o p h o r a i s a n i n f o r m a l g r o u p w i t h i n t h e m o l l u s c a n s u b c l a s s p u l m o n a t a c o m p r i s i n g o f a i r b r e a t h i n g f r e s h w a t e r s n a i l s t h a t a r e t y p i c a l l y c h a r a c t e r i z e d b y e y e s p o t s l o c a t e d a t t h e b a s e o f t w o n o n contractile tentacles and two external genital orif ices. they also have varied shell structures and habitats, not only within the group but also within families. families of the basommatophora are highly ubiquitous and may play a role in the life cycles of various parasites of humans and animals. basommatophora has a worldwide geographical distribution across freshwater, terrestrial and marine habitats. however, little is known on their distribution in the philippines. this report focuses on describing t h e b i o g e o g r a p h i c a l d i s t r i b u t i o n o f t h e b a s o m m a t o p h o r a n s i n t h e p h i l i p p i n e s t h r o u g h d a t a g a t h e r e d f r o m m u s e u m c o l l e c t i o n s , f o r e i g n d a t a b a s e s a c c e s s e d o n l i n e , a n d i d e n t i f i c a t i o n o f s p e c i e s f o u n d i n various literatures. a qualitative description of the distribution of each basommatophora family in the philippines is given by distribution maps, indicating locations where specimens were collected and/or identif ied. a t o t a l o f 3 3 6 c o u n t s o f b a s o m m a t o p h o r a n s f r o m 2 2 g e n e r a w e r e encountered from available literature, museums and public databases. the majority of the occurrences are from the genera siphonaria. the data and maps generated describe most of the distribution to be in luzon, with visayas and mindanao having close counts with each other. the philippines has the third most occurrences and genera of basommatophorans of all tropical countries in the world. however, the true diversity of the group c o u l d b e h i g h e r i f a m o r e s y s t e m a t i c s a m p l i n g o f t h e a r c h i p e l a g o i s conducted. keywords: basommatophora, philippines, biogeographical distribution biodistribution of the informal group basommatophora 54 introduction basommatophora is currently regarded as an informal group of the molluscan subclass pulmonata, containing about 2500 species that are grouped into the superfamilies amphiboloidea and siphonarioidea as well as the clade hygrophila. there are nine recognized families under this group (bouchet and rocroi 2005). however, it was found recently that basommatophora is polyphyletic, and all its members were moved into a new taxon, panpulmonata (jörger and others 2010). the most distinct observable characteristics of this group are the eyespots, which are normally located at the base of two non-contractile tentacles, and the two external genital orif ices. other features seem to be shared with other pulmonate superorders, though specializations may occur if there are major changes in habitat or body form (solem 2008). basommatophora has a worldwide geographical distribution, with species inhabiting predominantly freshwater but may also thrive in marine and terrestrial habitats (fretter and peake 1978). like other invertebrates, freshwater gastropods present an overall pattern of high diversity in the tropics, with low levels of species richness and endemicity at higher latitudes. small oceanic islands are noteworthy for generally low levels of freshwater gastropod species richness and endemism (starmühlner 1979). most species are capable of self-fertilization, so it is possible to populate a new body of water; however, cross-fertilization is the normal mode of reproduction (solem 2008). egg-laying is the norm for the basommatophorans, although two species of protancylus have been observed to brood eggs (albrecht and glaubrecht 2006). generation time is usually short, so enormous numbers of snails can build up very quickly during favorable conditions. as a result, basommatophoran snails show minor variation among populations (solem 2008). families in this suborder inhabit a great variety of freshwater habitats, living in water of less than 12 ft depth. only rarely have there been live species observed at relatively great depths. these species must depend upon cutaneous respiration or air bubbles for oxygen exchange while species living in shallow waters come to the surface at regular intervals to breathe. in addition, variations in shell structure and form exist (solem 2008). the forms of the shell vary widely not only within basommatophora but also within the individual families. family siphonariidae has shells that are cap-shaped, with an irregular bulge on the right side and a secondary gill in the mantle cavity. amphibolidae has high-spired and dextral shells and are also unique in having operculum as adults. gills are lacking but amphibolids possess an osphradium (olfactory organ) in the mantle cavity. a high-spired dextral shell also commonly characterizes the families chilinidae and most of lymnaeidae. p.n.y. young and i.k.c. fontanilla 55 planorbidae often has a planispiral shell that coils sinistrally, with their spire reduced and all whorls being arranged into a single plane. a sinistral shell is also always present in physidae. reduction in the elevation of the spire and the number of whorls also vary. partial reduction of the whorls has occurred in latiidae. a vestigial spire is also present in some species of planorbidae (fretter and peake 1978, pechenik 2005). because of these and other variations, it is diff icult to f ind structures that are common to all taxa that can def ine the group (solem 2008). families of basommatophora are usually highly visible and are ecologically signif icant both for serving as a food resource for vertebrates and for browsing on the shallow-water encrusting organisms such as algae, fungi, and protozoa. certain snails of this order are also of medical signif icance by serving as intermediate hosts for trematode parasites of both humans and domestic animals. some planorbids transmit schistosomiasis in africa, the west indies, and south america while lymnaeids are involved usually in both sheep and cattle liver fluke life cycles (solem 2008). previous data identif ied f ive basommatophoran families present in the philippines, namely acroloxidae, lymnaeidae, physidae, planorbidae, and siphonariidae (pagulayan 1995, springsteen and leobrera 1986). ancylidae, formerly a distinct family within basommatophora, was placed under siphonariidae (bouchet and rocroi 2005). however, there is no consolidated data on the biogeographical distribution of the group in the philippines. this study, therefore, aims to survey available literature and journals, museums and online databases in order to address this problem. materials and methods data for the distribution of basommatophorans in the philippines were gathered from various databases, using known basommatophoran families in the modern taxonomy scheme provided by bouchet and rocroi (2005). the online network of global biodiversity information facility (http://data.gbif.org/ welcome.htm), which compiled various datasets around the world, was accessed through the internet. a spreadsheet of basommatophoran occurrences was downloaded (http://data.gbif.org/occurrences/downloadspreadsheet.htm?c[0].s=20 &c[0].p=0&c[0].o=1491) and formatted to show the country and location where the species were collected and which database they were obtained from. a listing of basommatophoran occurrences in the philippines found in relevant literature was also included. basommatophoran occurrences in the philippines found in both listings are tabulated in table 1. biodistribution of the informal group basommatophora 56 family acroloxidae acroloxus sp. tapul group, lapac island, vicinity of siasi, usnm sulu archipelago (1) family lymnaeidae amphipeplea sp. marinduque (1) mcz amphipeplea cumingiana camarines sur (1), * (1) ucm amphipeplea luzonica zamboanga (1) ucm amphipeplea quadrasi donsul, sorsogon (1); ermita, manila (1); ansp manila, manila (1) bullastra cumingiana solano (1) nbcnl bullastra velutinoides * (1) zmb/moll lymnaea sp. abra de ilog, qcc, mindoro (1); cebu (1); usnm laguna de bay, luzon (1); mabilangan, mt. data, mt. province, luzon (1); panay island, jaro river (1); tapul group, lapac island, vicinity of siasi, sulu archipelago (6) sulu islands (1) ansp lymnaea blaisei * (1) ucm lymnaea cumingiana bayninan, banaue, ifugao (1); sinebaran creek, nmp matnog, sorsogon (1) lymnaea monticola * (1) mncn lymnaea pereger trinidad mountain, benguet (1) ansp lymnaea rubiginosa cebu (1) nmp lymnaea swinhoei manila (1); trinidad valley (1) mcz trinidad valley (1) nmp myxas sp. calamianes (1) mncn myxas cumingiana (=myxas * (1) flmnh cumingianus and * (3) lmd m. cumungi) catanduanes (1) mncn * (1); bukid river, tacloban, palo, leyte (1); nmp candaba swamps, candaba, pampanga (1); dagami, guinarona, leyte (1); macalajar (1); table 1. basommatophoran genera and species found occurring in the phil ippines. no d istribution data are ind icated for species marked with (*) because of no specificity in location. sources for each specimen are the following: usnm (nmnh invertebrate zoology collections), mcz (museum of comparative zoology, harvard university), ucm (cumnh mollusc collection), ansp (malacology), nbcnl (naturalis biodiversity center (nl) – mollusca), zmb/moll (systax – zoological collections), mncn (museo nacional de ciencias naturales, colección de malacología), flmnh (invertebratezoology), nbcnl (naturalis biodiversity center (nl) – mollusca), toya (mollusca specimens of toyama science museum), nmr (natural history museum rotterdam (nl) – mollusca collection), nmp (national museum of the philippines), springsteen and leobrera’s shells of the philippines (sp), pagulayan’s studies on the biodiversity of the molluscan fauna of lake taal, batangas (p), boragay’s a survey of gastropods at the university of the philippines diliman campus (b), and bequaert and clench’s philippine lymnaeidae and planorbidae (plp). ind ividual (species) location (count) dataset p.n.y. young and i.k.c. fontanilla 57 table 1. basommatophoran genera and species found occurring in the phil ippines. no d istribution data are ind icated for species marked with (*) because of no specificity in location (cont’n.). ind ividual (species) location (count) dataset negros occidental (1) mcz musuan, bukidnon, mindanao (1) b * (1) nbcnl myxas imperialis batangas, lipa (1); cebu (1); nagcarlang, palayan, mcz laguna (1); san juan river, calamba, laguna (1); manila (1) myxas luzonica * (1) mncn myxas quadrasi * (4) flmnh porac (1) mncn radix sp. tapul group, lapac island, vicinity of siasi, sulu usnm archipelago (1) radix auricularia (=lymnaea batang creek, san victor, tacloban, leyte (1); nmp auricularia) bukid river, tacloban, palo, leyte (1); quilot river, palo, leyte (1); tacloban, leyte (1); tributary of bukid river, tacloban, leyte (2); tributary of quilot river, guingawan, tacloban, leyte (2); lake taal (1); daral-og river, tacloban, leyte (1) plp radix philippinensis bohol, vilar id. , barrio toog (1); calbiga, mcz (=lymnaea philippnensis samar (1); cebu (1); binan, laguna (1); and lymnaea philippinica) mahaihai, laguna (1); albuero, leyte (2); caridad, leyte (1); ormoc, leyte (1); palo, leyte (2); tarragona, leyte (1); manila (2) cagbatan island, coron, palawan (1); nmp carmen-sagbayan-bacani road (1) cebu (1); mangaldan (1) plp cebu (1); guihulngan (1); manila (1) mncn bilar, bohol island (1) ucm maynit, laguna (1); manila (1) ansp * (4) flmnh radix quadrasi (=lymnaea * (1) flmnh quadrasi and bulinus up diliman (1) nmp quadrasi) agus river, manila (1) ucm mindanao, macajalar (1) mcz radix swinhoei macajalar (1) mcz stagnicola wyomingensis tapul group, lapac island, vicinity of siasi, usnm sulu archipelago (1) family physidae limnophysa sp. tapul group, lapac island, vicinity of siasi, ansp sulu archipelago (3) tapul group, lapac island, vicinity of siasi, mcz sulu archipelago (2) tapul group, lapac island, vicinity of siasi, mncn sulu archipelago (3) tapul group, lapac island, vicinity of siasi, zmb/moll sulu archipelago (1) biodistribution of the informal group basommatophora 58 physa sp. * (1) ansp * (2); panay island usnm tapul group, lapac island, vicinity of siasi, plp sulu archipelago (1) tapul group, lapac island, vicinity of siasi, zmb/moll sulu archipelago (2) physa heterostropha tapul group, lapac island, vicinity of siasi, zmb/moll sulu archipelago (1) physa hungerfordiana aparri, cagayan (2); claveria, cagayan (1); usnm malabon, manila (2); porac (1) claveria, cagayan (1); morong (1) lmd manila (1) mcz manila (1) mncn physa philippina * (3) usnm bosoboso near manila (1) ansp physa semperi * (1) usnm family planorbidae amerianna quadrasi lake mainit (1) mncn amerianna sulcifera * (1) flmnh anisus convexiusculus ilocos sur, rio del pueblo de sinay (1); mcz san juan del monte (1); surigao (1) anisus corinna leyte, baybay (2) mcz anisus quadrasi leyte, palo (1); mindoro (1); san juan river, mcz manila (1) bulinus sp. sulu archipelagi, tapul group, lapac island, usnm vicinity of siasi (1) bulinus boholensis bohol, baclayon (3) mcz bulinus bullulus mindoro (1) mcz bulinus camelopardalis * (1) mcz bulinus hungerfordianus * (1); fcmnh (b. hungerfordiana) dagami, digabonegan, leyte (1); palo, leyte (2); mcz tarragona, leyte (1); tolosa, leyte (1); manila (1); irrigation canal, bo. dita, cabuyao, laguna (2); laguna bay, laguna (1); alabang river, manila (1); muntinglupa river, manila (1); virac, catanduanes (1) bulinus luzonicus * (1) mcz bulinus mindoroensis mindoro (1) mcz bulinus ustulatus mindoro (1) mcz cyclophinus canaliferous mindoro (1) mcz gyraulus chinensis * (2) smf lake taal (1) p up diliman (1) b gyraulus compressus * (1) flmnh gyraulus convexiusculus * (2) flmnh aparri, cagayan (1); boac (1); pasig river, mncn manila (1); visita palanas, lacy (1) table 1. basommatophoran genera and species found occurring in the phil ippines. no d istribution data are ind icated for species marked with (*) because of no specificity in location (cont’n.). ind ividual (species) location (count) dataset p.n.y. young and i.k.c. fontanilla 59 gyraulus prashadi zambales prov., san antonio (2) mcz gyraulus quadrasi * (1) flmnh hippeutis umbilicalis palo, leyte (1) mcz mearnsii indoplanorbis exustus up diliman (1) b pettancylus manillensis lake taal (1) p physastra hungerfordiana * (5) flmnh lake taal (1) p up diliman (1) b physastra quadrasi lake mainit (1) plp planorbis sp. * (1); surigao (1); bohol (1) mcz * (1) mncn sulu archipelagi, tapul group, lapac island, usnm vicinity of siasi (3) planorbis compressus * (1) ucm alrededores, manila (1); baclayon, bohol (1); mcz isabela, luzon (1); lacy, siquijor (1); paco, manila (1) ermita, manila (1); isabela, luzon (1); ansp loey, siquijor (1); manila (1); marinduque (1) planorbis corneus * (1); zamboanga (1) mncn planorbis lugubris batangas, lipa (1) mcz planorbis mindanensis cotobato, rio grande valley, lake baluan (1) mcz cotobato, rio grande valley, lake baluan (1) usnm planorbis philippinarum * (1) mncn planorbis planorbis * (1) flmnh planorbis quadrasi inopacan (1) mncn montalban (1) mcz planorbis umbilicalis cagayan, buguey (1) mncn segmentina umbilicalis cagayan, buguey (1); san antonio, zambales (1) mcz family siphonariidae siphonaria sp. mambajao, camiguin island (1) nmp ambulony island (1) cas maubu beach, jojo island, sulu archipelago (1) ansp muso, siasi island, sulu archipelago (1) turnina island (2) ypm cabra island, mindoro (1); cuyo, palawan (2); bacuit, palawan (1) mcz siphonaria acuta palawan, bacuit (1) mcz * (1) flmnh siphonaria atra balite beach, puerto galera, mindoro (1); nmp bohol (1); buenavista, marinduque (1); cabcaban, bataan (1), cagayan de sulu (1); ibabang pulo, pagbilao, grande island, pagbilao, quezon (1); manlumod, mogpog, marinduque (1); puntod, gaspar island, tres reyes islands, marinduque (1); tawi-tawi island, sulu (1) table 1. basommatophoran genera and species found occurring in the phil ippines. no d istribution data are ind icated for species marked with (*) because of no specificity in location (cont’n.). ind ividual (species) location (count) dataset biodistribution of the informal group basommatophora 60 butas, mariveles point, bataan (1); cabra island, mcz mindoro (1); candelaria, zambales (1); bacuit, palawan (1); cuyo, palawan (1) little santa cruz, mindoro (1); * (1) ucm * (3) flmnh siphonaria cornuta balabac, mangsee del sur (1); palawan (1) mcz siphonaria corrugata semirara island (1) nmp siphonaria diemenensis candelaria, zambales, luzon (1) mcz siphonaria japonica * (2) flmnh siphonaria javanica barangay tawog, bulusan, sorsogon (1); nmp batangas bay, batangas (1); bulalasas bay, or. mindoro (1); cata-an river, san joaquin, iloilo (1); malitpalit islet, pandan, catanduanes (1); morongborongan island, sorsogon (1); muelle bay, puerto galera, or. mindoro (1); pandan, catanduanes (1); sinugbuan, san joaquin, iloilo (1); sulangan, guiuan, eastern samar (1); tabugoe, pandan, catanduanes (1) zamboanga (1) mcz siphonaria kurrachiensis palawan, bacuit (1) mcz siphonaria laciniosa batangas bay, batangas (1); punta canomay, nmp calveria, burias island, masbate (1); subic, calimtaan island, sorsogon (1) butas, mariveles pt., bataan, luzon (1); mcz cabra island, mindoro (1); calapan (2); candelaria, zambales, luzon (2); dapitan bay (1); lubang island, mindoro (1); mindoro (1); santa margarita, w. samar (1) * (3) flmnh * (3) toya siphonaria normalis balite beach, puerto galera, mindoro (1); nmp bobon (1); bolauos, narvacan, ilocos sur (1); cabuyo, torrijos, marinduque (2); dapdap, tagum, sta. cruz, marinduque (2); honda bay, palawan (1); imelda park, ilocos sur (1); lusong-bagac, bataan (1); manlumod, mogpog, marinduque (1); melchor island, tres reyes island, gasan, marinduque (1); parpatong, bangui, ilocos norte (1); sulvec, narvacan, ilocos sur (1); suyo, bangui, ilocos norte (1) * (2) flmnh siphonaria sipho * (1) ucm palawan (2) mcz * (1) lmd siphonaria siquijorensis siquijor (1) ansp baler, quezon (1); lubang island, mindoro (1) nmp * (1) nbcnl * (1) nmr table 1. basommatophoran genera and species found occurring in the phil ippines. no d istribution data are ind icated for species marked with (*) because of no specificity in location (cont’n.). ind ividual (species) location (count) dataset p.n.y. young and i.k.c. fontanilla 61 siphonaria sirius angas point, otavi, bulan, sorsogon (1); nmp balbagon island, carles, iloilo (1); barangay tawog, bulusan, sorsogon (1); canlubi, pandan, catanduanes (1); cata-an river, san joaquin, iloilo (1); dayhagan, suchan, panay island (1); catanduanes (1); macalanhog island, gigmoto, catanduanes (1); malitpalit islet, pandan, catanduanes (1); morongborongan island, sorsogon (1); punta nasio, taloto-an, pan de azucar island, concepcion, iloilo (1); sinugbuan, san joaquin, iloilo (1); sorsogon (1); subic, calimtaan island, sorsogon (1) * (1) ucm table 1. basommatophoran genera and species found occurring in the phil ippines. no d istribution data are ind icated for species marked with (*) because of no specificity in location (cont’n.). ind ividual (species) location (count) dataset the number of basommatophoran families and genera occurring in tropical countries in the world were also obtained from the online listing and are tabulated in table 2. endemicity was also checked by comparing the occurrences between other countries. the website discoverlife (http://www.discoverlife.org/) was also used to check for further consistencies. endemicity was also conf irmed if there were no clear sources refuting it for certain species. endemic species are tabulated in table 3. the national museum of the philippines in manila was visited to document and list down the specimens in their collection with the help of ms. vivian ang. the documentation of the specimens are found in appendix 1. all data were tabulated using a spreadsheet computer program (microsoft excel 2007). locations where each species was found were marked using adobe photoshop cs5 on an outline map of the philippines, from free us and world maps.com (http://www.freeusandworldmaps.com). results and discussion a total of 336 recorded occurrences, 21 genera, and 75 species from the listings indicated above were found to occur in the philippines. these records reveal basommatophorans from f ive families out of nine total based on bouchet and rocroi’s (2005) classif ication scheme: acroloxidae (1 occurrence, 1 genus, 1 species), lymnaeidae (99 occurrences, 6 genera, 20 species), physidae (32 occurrences, 2 genera, 6 species), planorbidae (95 occurrences, 11 genera, 35 species), and siphonariidae (111 occurrences, 1 genus, 13 species). this is mostly consistent biodistribution of the informal group basommatophora 62 table 2. a l ist of tropical countries defined as being in the region between the tropic of cancer (23° 26’ 16” n) and tropic of capricorn (23° 26’ 16” s) found in the world. listed also are the number of basommatophoran occurrences and genera that are found occurring in each country. (accessed through gbif data portal, data.gbif.org, 2014-01-29) mexico 623 31 congo 339 19 philippines 336 21 cuba 238 20 indonesia 190 13 brazil 188 23 india 172 17 panama 171 17 jamaica 122 17 costa rica 113 16 uganda 112 11 guatemala 107 20 peru 102 17 venezuela 97 19 honduras 94 1 cape verde 83 5 thailand 74 13 haiti 66 13 kenya 58 12 nicaragua 54 12 bolivia 45 7 sudan 42 8 dominican republic 41 11 oman 40 5 bermuda 37 7 ecuador 36 9 madagascar 35 9 colombia 35 12 puerto rico 35 15 cayman islands 33 5 fiji 33 5 vietnam 27 6 liberia 26 5 el salvador 25 8 new caledonia 24 7 sri lanka 21 6 suriname 21 7 senegal 20 4 tropical country number of occurrences number of genera p.n.y. young and i.k.c. fontanilla 63 papua new guinea 20 7 malaysia 16 3 yemen 16 6 netherlands antilles 16 8 tanzania 15 7 seychelles 14 2 cameroon 13 5 trinidad and tobago 13 10 angola 12 3 singapore 12 8 sierra leone 11 4 malawi 10 3 vanuatu 10 3 barbados 10 6 antigua and barbuda 9 6 zambia 8 4 zimbabwe 6 5 cocos islands 5 1 laos 5 3 belize 5 5 american samoa 4 1 nigeria 4 4 equatorial guinea 3 1 hong kong 3 1 ghana 3 2 namibia 3 2 gabon 2 1 gambia 2 1 dominica 2 2 french guiana 2 2 mozambique 2 2 botswana 1 1 montserrat 1 1 niger 1 1 tropical country number of occurrences number of genera table 2. a list of tropical countries defined as being in the region between the tropic of cancer (23° 26’ 16” n) and tropic of capricorn (23° 26’ 16” s) found in the world (cont’n.). listed also are the number of basommatophoran occurrences and genera that are found occurring in each country. (accessed through gbif data portal, data.gbif.org, 2014-01-29) biodistribution of the informal group basommatophora 64 family lymnaeidae amphipeplea cumingiana camarines sur amphipeplea luzonica zamboanga amphipeplea quadrasi donsul, sorsogon; ermita, manila; manila, manila bullastra cumingiana solano bullastra velutinoides * myxas cumingiana catanduanes; bukid river, tacloban, palo, leyte; candaba swamps, candaba, pampanga; dagami, guinarona, leyte; macalajar, negros occidental; musuan, bukidnon, mindanao myxas imperialis batangas, lipa; cebu; nagcarlan, palayan, laguna; san juan river, calamba, laguna; manila myxas luzonica * myxas quadrasi * radix philippinensis bohol, vilar id., barrio toog; calbiga, samar; cebu; biñan, laguna; mahaihai, laguna; albuero, leyte; caridad, leyte; ormoc, leyte; palo, leyte; tarragona, leyte; manila; cagbatan island, coron, palawan; carmen-sagbayan-bacani road, cebu); mangaldan, cebu; guihulngan, bilar, bohol island; maynit, laguna; manila radix quadrasi up diliman; agus river, manila; mindanao, macajalar family physidae physa hungerfordiana aparri, cagayan; claveria, cagayan; malabon, manila; porac; morong, manila physa philippina bosoboso near manila physa semperi * family planorbidae anisus corinna leyte, baybay anisus quadrasi leyte, palo; mindoro; san juan river, manila amerianna quadrasi lake mainit amerianna sulcifera * bulinus boholensis bohol, baclayon bulinus bullulus mindoro genus location table 3. a list of endemic basommatophoran genera and species found in the phil ippines with the distribution found in other countries, as can be gleaned from the gbif spreadsheet, wherein lymnaeidae, planorbidae and siphonariidae were also the most commonly occurring families. figure 1 presents the families found in the philippines and their relative numbers to each other. figure 2 shows the relative numbers of the genera that can be found in the philippines to each other. p.n.y. young and i.k.c. fontanilla 65 genus location table 3. a list of endemic basommatophoran genera and species found in the phil ippines (cont’n.). bulinus camelopardalis * bulinus hungerfordianus dagami, digabonegan, leyte; palo, leyte; tarragona, leyte; tolosa, leyte; manila; irrigation canal, bo. dita, cabuyao, laguna; laguna bay, laguna; alabang river, manila; muntinglupa river, manila; virac, catanduanes bulinus luzonicus * bulinus mindoroensis mindoro cyclophinus canaliferous mindoro gyraulus prashadi * physastra hungerfordiana lake taal; up diliman planorbis mindanensis cotobato, rio grande valley, lake baluan planorbi sphilippinarum * planorbis quadrasi inopacan; montalban segmentina umbilicalis cagayan, buguey; san antonio, zambales family siphonariidae siphonaria cornuta balabac, mangsee del sur; palawan siphonaria (family siphonariidae) has the most occurrences in the philippines, accounting for 33% of the total. the genus has a widespread distribution, with increasing diversity towards the tropics (vermeij 1973). hodgson (1999) speculated that the success of the siphonariids in general could be due partly to their resistance to increased temperature and desiccation through physiological, morphological or behavioral adaptations. furthermore, siphonariids are generalist grazers, feeding on a wide range of microalgae, f ilamentous algae, foliaceous algae, and macrophytous corticated algae (underwood and jemakoff 1981, jara and moreno 1984, santelices and correa 1985, godoy and moreno 1989, hodgson 1999), as well as lichens (borland 1950) and cyanobacteria (chan 2003). siphonaria can also influence the settlement, growth and survival of algae, as well as barnacle recruitment and survival (jara and moreno 1984, hodgson 1999) because of their occurrence in large numbers. some species change their vertical distribution according to seasons. it is widely assumed that the vertical distribution of siphonariids is affected by both biotic – e.g. , interspecif ic competition for space (black 1979, hodgson 1999) – and wave action (allanson 1958, voss 1959, hodgson 1999). however, more research is needed as little is known about the ecology of tropical species (hodgson 1 9 9 9 ) . many basommatophoran snails have a widespread and cosmopolitan distribution due to their biology. for instance, the north american ancylid ferrissia fragilis has biodistribution of the informal group basommatophora 66 been spreading in europe probably due to its many biological attributes such as its ability to aestivate and survive in stagnant waters, its small size, and even its hermaphroditic life cycle (walther and others 2006). this may also be the case for the siphonariids. furthermore, the wide distribution of certain invertebrates between unconnected habitat patches could depend on passive dispersal mechanisms (bilton and others 2001). for snails like siphonariids, passive dispersal could be done through being carried on the feet or feathers of birds from one body of water to another. perhaps serving as evidence is basommatophoran snails appearing in the geological record at the jurassic-cretaceous boundary 145.5 million years ago, shortly after the origin of birds (solem 2008). other forms of dispersal are rafting on aquatic vegetation, marine/brackish larval dispersal phase, stream capture and even by air (e.g. , cyclonic storms) (purchon 1977). however, there is no clear material linking this to be the reason for the multitude of occurrences of siphonaria globally or in the philippines. figure 1. the basommatophoran families found in the philippines. figure 2. the frequencies of basommatophoran genera in the philippines. p.n.y. young and i.k.c. fontanilla 67 figure 3 presents the distribution maps of the basommatophoran families found in the philippines. the maps indicate where the shells were collected or found. it should be noted that some specimens encountered did not have their localities properly identif ied and were therefore excluded. there were 138 individuals (52.07%) from luzon, accounting for majority of the specimens. meanwhile, 68 individuals (25.66%) were found in the visayas and 59 individuals (22.26%), in mindanao. the data thus demonstrate that the basommatophoran species are distributed all throughout the philippine archipelago. the distribution is noted to be heterogeneous, with clustering in the luzon region but still dispersed almost evenly among the island groups. it is noted that the patchy distribution could be due to the limited and sporadic sampling of the basommatophorans in the philippines. amongst the tropical countries in the world, the philippines, with 336 occurrences and 21 genera, ranks third in basommatophoran occurrences, behind mexico and congo. mexico has 623 occurrences and 31 genera while congo has 339 genera and 19 genera. the philippines accounts for 8% of the worldwide basommatophoran occurrences out of the 72 countries listed in the gbif spreadsheet. the philippines also has 32 endemic species (table 3); that is, 42.67% of basommatophoran endemic species can be found in the philippines. of these, majority are from planorbidae ( 4 6 . 6 7 % ) a n d ly m n a e i d a e ( 2 6 . 6 7 % ) . w i t h t h i s m u c h p r o p o r t i o n o f t h e basommatophoran occurrences and endemic species, a more systematic sampling is needed to assess the full biodiversity of the basommatophora in the philippines and account for its evolution. one of the endemic species from lymnaedae is radix quadrasi. remigio and blair (1997) suggested that it may be conspecif ic with r. rubiginosa from malaysia on the basis of nearly identical mitochondrial 16s rrna gene sequences and that they are subspecies of the eurasian r. auricularia, which is also present in the philippines. however, correa and others (2010) evaluated 50 lymnaeid taxa using the nuclear its1 and its-2 sequences in addition to the 16s rrna gene, and they found that, though both r. quadrasi and r. rubiginosa clustered together, they gave very distinct sequences to be considered the same species. furthermore, both taxa clustered separately from r. auricularia, clearly indicating that they are distinct from r. auricularia. summary survey of basommatophora in the philippines based on literature and existing collections demonstrates high diversity in comparison to other tropical countries, with 21 known genera and at least 32 endemic species. these values, however, biodistribution of the informal group basommatophora 68 figure 3. the distribution map of basommatophorans in the philippines. p.n.y. young and i.k.c. fontanilla 69 could be a gross underestimate of the true diversity of the group as sampling is sporadic. this warrants further systematic sampling to address this issue. acknowledgments we thank the national museum of the philippines for allowing us to use their specimens for this paper. this manuscript also benef ited from the contributions of the many databases to the gbif website. references allanson br. 1958. some aspects of the ecology of the molluscan genus siphonaria in south africa. port acta biol b 6: 179-212. a l b r e c h t c , g l a u b r e c h t m . 2 0 0 6 . b r o o d c a r e a m o n g b a s o m m a t o p h o r a n s : a u n i q u e r e p r o d u c t i v e s t r a t e g y i n t h e f r e s h w a t e r l i m p e t p r o t a n c y l u s ( h e t e r o b r a n c h i a : protancylidae), endemic to ancient lakes on sulawesi, indonesia. acta zool 87: 49-58. bilton dt, freeland jr, okamura b. 2001. dispersal in freshwater inver tebrates. ann rev ecol syst 32: 159-181. bequaer t jc, clench wj. 1939. philippine lymnaeidae and planorbidae. phil j sci 69(1): 7-21. black r. 1979. competition between intertidal limpets: an intrusive niche on a steep resource gradient. j animal ecol 48: 401-411. boragay ab. 2004. a survey of gastropods at the university of the philippines diliman ca m p u s ( q u ezo n c i t y, m e t r o m a n i l a ) [ b s t h e s i s ] . q u ezo n c i t y : u n i v e r s i t y of t h e philippines diliman. p 45. b o r l a n d c . 1 9 5 0 . e c o l o g i c a l s t u d i e s o f b e n h a m i n a o b l i q u a t a ( s o w e r b y ) , a basommatophorous pulmonate in otago harbour. transactions of the royal society of new zealand 78: 385-393. b o u c h e t p, ro c r o i j p. 2 0 0 5 . c l a s s i f i c a t i o n a n d n o m e n c l a to r o f g a s t r o p o d f a m i l i e s . malacologia 47(1-2): 397. chan ks. 2003. variation in cyanobacteria-dominated biof ilms: consequences for the diet , growth and reproduction of an inter tidal grazer, siphonaria japonica, on hong kong shores [phd thesis]. hong kong: the university of hong kong. p 256. chim ck, tan ks. 2009. ver tical distribution, spawning and recruitment of siphonaria guamensis (gastropoda: pulmonata) on a seawall in singapore. raffles b zool s 22: 269-278. co r r e a ac , e s co b a r j s , d u r a n d p, re n a u d f , d a v i d p, j a r n e p, po i n t i e r j p, h u r t r ezb o u s s e s s . 2 0 1 0 . b r i d g i n g g a p s i n t h e m o l ec u l a r p h y l o g e n y of t h e ly m n a e i d a e (gastropoda: pulmonata), vectors of fascioliasis. bmc evol biol 10: 381. biodistribution of the informal group basommatophora 70 c u k i n g n a n a j , p a g u l a y a n r c . 1 9 9 5 . a n a t o m y o f t h e d i g e s t i v e s y s t e m o f radix s p . ( b a s s o m a to p h o r a : ly m a n a e i d a e ) f r o m lake ta a l , b a t a n g a s . s c i e n ce d i l i m a n ( 7 & 8 ) : 48-52. faustino la . 1928. summary of philippine marine and fresh-water mollusks. manila: bureau of printing. 382 p. fretter v, peake j. 1978. pulmonates: volume 2a, systematics, evolution and ecology. london: academic press inc. ltd. p 1-44. godoy c, moreno ca. 1989. indirect effects of human exclusion from the rocky intertidal in southern chile: a case of cross-linkage between herbivores. oikos 54: 101-106. grande c, templado j, zardoya r. 2008. evolution of gastropod mitochondrial genome arrangements. bmc evol biol 8: 61. h o d g s o n a n . 1 9 9 9 . t h e b i o l o g y o f s i p h o n a r i i d l i m p e t s ( g a s t r o p o d a : p u l m o n a t a ) . oceanogr mar biol ann rev 37: 245-314. jara hf, moreno ca. 1984. herbivory and structure in a midlittoral rocky community: a case in southern chile. ecol 65(1): 28-38. jörger km, stöger i, kano y, fukuda h, knebelsberger t, schrödl m. 2010. on the origin of acochlidia and other enigmatic euthyneuran gastropods, with implications for the systematics of heterobranchia. bmc evol biol 10: 323. pagulayan rc. 1995. studies on the biodiversity of the molluscan fauna of lake taal, batangas. up research digest ii(2): 26-27. pechenik ja. 2005. biology of the invertebrates. 5th ed. new york: mc-graw hill. 590 p. purchon i. 1977. the biology of the mollusca. 2nd ed. new york: pergamon press. 560 p. re m i g i o e a , b l a i r, d. 1 9 9 7. m o l ec u l a r s y s t e m a t i c s of t h e f r e s h w a t e r s n a i l f a m i l y lymnaeidae (pulmonata: basommatophora) utilizing mitochondrial ribosomal dna sequences. j moll stud 63: 173-185. s a n t e l i c e s b , c o r r e a j . 1 9 8 5 . d i f f e r e n t i a l s u r v i v a l o f m a c r o a l g a e t o d i g e s t i o n b y intertidal herbivore molluscs. j exp mar biol ecol 88: 183-191. solem ga. 2008. basommatophora. available from: http://accessscience.com/content/ basommatophora/074200. accessed 2011 jul 15. springsteen f, leobrera fm. 1986. shells of the philippines. philippines: carfel seashell museum. p. 377. starmühlne r f. 1979. d i s tri bu ti o n o f fre s hwater molluscs in mountain streams of tropical indo-pacif ic islands. malacologia 18: 245-255. underwood aj, jernakoff p. 1981. effects of interactions between algae and grazing gastropods on the structure of a low-shore intertidal algal community. oecologia 48: 221-233. p.n.y. young and i.k.c. fontanilla 71 ve r m e i j g j . 1 9 7 3 . m o r p h o l o g i c a l p a t te r n s i n h i g h i n te r t i d a l g a s t r o p o d s : a d a p t i ve strategies and their limitations. mar biol 20: 319-346. voss na . 1959. studies on the pulmonate gastropod siphonaria pectinata (linnaeus) from the southeast coast of florida. bull mar sci 8: 84-99. walther ac, lee t, burch jb, foighil do. 2006. conf irmation that the nor th american ancylid ferrissia fragilis (tyron, 1863) is a cryptic invader of european and east asian freshwater ecosystems. j moll stud 72: 318-321. appendix documentation of the basommatophoran specimens found in the national museum of the philippines. scale is in inches (1 inch = 2.54 cm). 1. radix auricularia 2. radix philippinensis biodistribution of the informal group basommatophora 72 5. myxas cumingiana 3. lymnaea rubiginosa 4. lymnaea sp. p.n.y. young and i.k.c. fontanilla 73 6. indoplanorbis exustus 7. physastra hungerfordiana 8. siphonaria atra biodistribution of the informal group basommatophora 74 10. siphonaria javanica 11. siphonaria laciniosa 9. siphonaria corrugata p.n.y. young and i.k.c. fontanilla 75 14. siphonaria sirius 13. siphonaria siquijorensis 12. siphonaria normalis biodistribution of the informal group basommatophora 76 _______________ patrick noel y. young <patnyoung@yahoo.com> is a graduate from the institute of biology, university of the philippines diliman, where he received his bs in biology. ian kendrich c. fontanilla <ianfontanilla@hotmail.com> is an assistant professor and member of the invertebrate museum at the institute of biology, university of the philippines diliman. he received his phd in genetics from the university of nottingham, united kingdom. he is also the president of the malacological society of the philippines. 15. siphonaria sp. 2editor's note-july-dec.2018.pmd 1 from the editor issn 0115-7809 print/issn 2012-0818 online irene m. v illaseñor, ph.d. editor-in-chief welcome readers! in this july to december 2018 issue of science diliman, i am pleased to present four full papers and a short communication. authors anticamara and tan re-attached coral fragments to sturdy natural substrates such as dead massive corals. the coral fragments were an aftermath of the devastation of coral reefs in eastern samar by super-typhoon haiyan. after a year of monitoring, 88% of the re-attached coral fragments survived, 43% of which exhibited high growth rates. the results will help in the restoration of damaged coral reefs and will benef it many reef f ishes. in another paper, authors austero and azanza studied the composition and abundance of marine diatoms and dinoflagellates in cebu international port (cip) and naval supply depot (nsd). the results of this short-term study established baseline data for the identif ication of potentially toxic and harmful microalgae. chakraborty and co-authors fabricated a stable and non-cytotoxic scaffold consisting of a polyelectrolyte complex of two biopolymers. the properties of the scaffold promote cell growth and facilitate the healing process of bones and tissues. authors mernilo-tutanes and caga-anan def ined fuzzy on ideal sets and proved a hahn-banach theorem using fuzzy on ideal sets. there is much we do not know about thoron in the environment, which makes it opportune for author kazuki-iwaoka and co-authors to develop a thoron exposure system for the validation of the use of passive detectors for thoron measurement. the details are presented in the f ive manuscripts while layman’s abstracts will help the non-specialists. 6magdalita-morphological.pmd e.m.n. cabarrubias et al. 51 science diliman (july-december 2017) 29:2, 51-81 morphological characterization, evaluation and selection of hibiscus (hibiscus rosa-sinensis l) hybrids elena may n. cabarrubias university of the philippines los baños pabl ito m. magdal ita* university of the philippines los baños antonio g. lalusin university of the philippines los baños norma g. med ina university of the philippines los baños abstract f i f t y s e v e n h i b i s c u s h y b r i d p r o g e n i e s f r o m d i f f e r e n t c r o s s e s w e r e characterized and evaluated for morphological traits to select hybrids with unique color and form. a total of 14 progenies with the following pedigrees were selected: 22xdt-9, (llxefa)xgc-2, (llxefa)xgc-8, dsxgc-7, 20xgc-5, (gcxbgb)xhp-4, gcxds-4, abaxmdm-1, abaxmdm-3, 23xgc-2, cvxnb-1, cvxnb-2, cvxmp-4 and cvxnb-6. phenotypic data were analyzed f o r p r i n c i p a l c o m p o n e n t a n a l y s i s ( p c a ) a n d a g g l o m e r a t i v e c l u s t e r analysis. correlation using pca revealed signif icant positive association between flower size and leaf size, and between petiole length and leaf size. pca depicted three major pcs with eigenvalue >1 contributing 78% of t h e to t a l c u m u l a t i ve v a r i a b i l i t y a m o n g d i f fe r e n t h y b r i d s . t h e pci showed positive factor loadings for all the traits. the contribution of flower size, leaf size and style length was highest in pc-i. cluster analysis g r o u p ed t h e 5 7 h y b r i d s i n to f i ve c l u s te r s . c l u s t e r i h a d t h e h i g h e s t number of members (16), consisting of yellow-orange and purple flowers with a mean size of 131.09 mm. cluster-ii had 15 members, possessing white and red-purple hybrids with a mean size of 140.54 mm. cluster-iii was composed of f ive yellow members with a mean size of 131.12 mm. cluster-iv had 13 members, comprising yellow and yellow-orange hybrids whose flowers are small and have a mean size of 115.20 mm. clusterv consists of eight redand red-purple-colored hybrids with mean size of 130.21 mm. the study revealed that hybrids with large flowers and l o n g e r p e t i o l e s t e n d t o h a v e w i d e r l e a v e s , a n d t h e s e r e s u l t s w e r e i n agreement with the dendrogram groupings of the 57 hybrids. keyword s: agglomerate cluster analysis, hibiscus rosa-sinensis l. , hybrids, principal component analysis _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online morphological characterization, evaluation and selection of hibiscus 52 introduction hibiscus is a genus of the family malvaceae or mallow family composed of more than 400 species of flowering plants (edmonds 1991). it is a native of the tropical and sub-tropical regions of the world, and exists as a small tree, shrub, or herb (mcmullen 1999). the double red hibiscus rosa-sinensis had a broad distribution in china, india, south-east asia and the pacif ic islands in pre-european discovery days. the other forms from asia and the islands of the south indian ocean were introduced into the green houses of europe in early decades of the 19th century (howie 1980). it is thought that hibiscus was f irst introduced to the philippines by the chinese traders. new forms and cultivars were introduced into the country by the american colonizers in the 1890s (magdalita et al. 2016; magdalita and pimentel 2010a). in the philippines, hibiscus has become a popular ornamental plant because of its attractive flowers. in addition, some varieties are used as food, medicine, feed, ingredients of industrial products, symbol for religious purposes and national emblem in some countries (magdalita et al. 2011). to date, various new varieties have been developed through cross breeding, and these new varieties are getting popular with home gardeners, landscapers and other hibiscus enthusiasts. cross breeding in hibiscus aid in the creation of new varieties with improved traits, especially color, form, size, number of flowers, longevity, continued blooming, disease resistance and growth habit (magdalita and pimentel 2013). various hibiscus hybrids between local and foreign varieties were developed, resulting in 44 varieties that were named after filipina achievers (magdalita and pimentel 2010b). traditionally, plant breeders have selected plants based on their visible or measurable traits, such as flower color, seed color, leaf shape, fruit shape, stem length, etc. (jiang 2013). characterization descriptors pertain to those traits that tend to be highly heritable, ranging from morphological to molecular markers, and are expressed in all environments, in order to establish differences or similarities in phenotypic traits of each accession. characterization and evaluation based on phenotypic traits is a quick, easy and practical guide in selection of parents for use in hybridization (brown and caligari 2008). this plant selection technique can be enhanced by utilizing principal component analysis (pca) and cluster analysis. pca is a multivariate analysis technique used to determine the relative signif icance of different variables, f inding patterns in data of high dimension, prior to cluster analysis (jackson 1991). furthermore, pca is used to reduce the dimension of the data set and leads to the understanding of variables by determining how much of the total e.m.n. cabarrubias et al. 53 variance is contributed by each data set. on the other hand, cluster analysis is used to determine patterns and relationship between objects in the data set, and to group objects into homogenous and well-def ined groups, wherein observations within each cluster are similar to one another and the complete set of clusters contain all individuals (everitt et al. 2010). for this study, determination of the relationship between the different hybrid progenies is an important factor for the selection of parents in the hybridization program. the objectives of the study were as follows: (1) characterize, evaluate, and select promising progenies from the 57 hybrids based on qualitative and quantitative traits; (2) categorize or group the hybrid progenies into f ive major clusters; and, (3) determine the correlation of the different quantitative characters of the hybrids. materials and methods plant materials hibiscus hybrids developed by the breeder-scientists of the institute of crop science and institute of plant breeding, college of agriculture and food science, university of the philippines los baños were used in the study. fifty-seven hibiscus hybrid progenies from 10 different crosses established in 2013 were characterized and evaluated phenotypically. the hibiscus hybrids were planted in single rows in the hibiscus breeding block and given the standard cultural practices. five to ten progeny plants per cross were characterized and evaluated based on their growth habit, and leaf and flower traits. plant characters the height and width of 5-10 progenies of every cross were measured using a meterstick from the base to the top of the plant, and across the widest portion of the canopy, respectively. leaf characteristics ten leaves from each plant of the 57 hybrid progenies were used for evaluation. the following qualitative leaf traits of each of the hybrid progenies were characterized: leaf arrangement, type, form or outline, margin, apex, base, attachment morphological characterization, evaluation and selection of hibiscus 54 and color. the descriptions of most leaf characteristics were based from simpson (2006). the quantitative traits, such as leaf length, width and petiole length were measured using a ruler. flower characteristics characterization and evaluation of 10 fully opened flowers from each hybrid progeny was performed in the morning. the qualitative traits evaluated included inflorescence type, bloom type, flower color, stigma color, style and stigma type and calyx color. the flower and leaf colors were determined using the royal horticultural society color chart (rhs) of london 5 th ed. (rhs 2007) and were matched with the rhs color coordinate. descriptions of the flower characteristics were based on the standards of the australian hibiscus society (2007). the quantitative traits evaluated were bloom size/diameter, length and width of each corolla, angle of display of flower, pedicel length, receptacle diameter, ovary length and width, calyx lobe length and width, stigma length and width, and style length. selection selection of the hybrid progenies with potential for variety development was conducted in 10 different crosses. identif ication of selections was primarily based on new flower color and form. the other traits considered in selection were nonfolding of the petals, retention of flower color for the day, prolif icacy of blooming, bushiness, and plant vigor. in each cross, one to three hybrid progenies possessing good flower characteristics and plant growth habit were selected.this study was conducted at the institute of plant breeding, college of agriculture and food science, university of the philippines los baños from april 2013 to may 2015. statistical design and analysis the experimental hybrid progenies were planted in single rows in the hibiscus breeding blocks. in each cross, f ive to eight hybrid progenies were used for evaluation. thirty-two morphological characters consisting of nine qualitative and three quantitative traits for leaves, and six qualitative and 13 quantitative traits for flowers were gathered. they were analyzed using the statistical tool for agricultural research (star) software (irri 2014). nine selected quantitative traits were subjected to principal component analysis (pca), in order to reduce the e.m.n. cabarrubias et al. 55 number of variables in the dataset while maintaining the variability in the data. eigen values and eigen vectors plus relative and cumulative proportion of the total variance were calculated to identify the signif icant traits that will be used for cluster analysis. traits with a correlation value of e ≥ 0.3 were considered relevant for the component to be used for clustering of hybrid progenies. agglomerative cluster analysis was performed using the complete clustering method and gower value as the distance determinant method to assess the level of similarity between the hybrid progenies for the dendrogram grouping. results and discussion morphological characterization, evaluation and selection characterization and evaluation of the morphological traits of hibiscus hybrids prior to selection and variety release was performed. this strategy has been implemented to identify elite progenies from different crosses of hibiscus whose parents are genetically diverse (pimentel 1999, san pascual 2015, magdalita et al. 2016). parallel to this study is the characterization of hibiscus sabdariffa, locally known as roselle, to identify varieties with good bast f iber qualities (mwasiagi et al. 2014). in the present study, the morphological characteristics of the 13 crosses and the selections identif ied from these crosses were presented as follows: 22xdt is a cross between hibiscus rosa-sinensis ‘accession 22’ (yellow) and hibiscus rosa-sinensis ‘domini m. torrevillas’ (dark orange). six hybrid progenies of this cross with pedigrees 22xdt-1, 22xdt-2, 22xdt-5, 22xdt-6, 22xdt-9 and 22xdt-10 had different flower colors ranging from different shades of yellow and orange. 22xdt-1 is persimmon orange (rhcc 28 a) with an orange-red (rhcc n 34 b) eye zone surrounded by grayed orange (rhcc 163 c) halo, and yellow-orange (rhcc 21 c) edges with yellow vein markings radiating from the center to the petal. 22xdt-2 is canary yellow (rhcc 9 a) with a red (rhcc 47 b) eye zone and saturn red (rhcc 30 b) vein markings radiating from the center to the petal. 22xdt-5 is indian yellow (rhcc 17 a) with a scarlet red (rhcc 46 b) eye zone. 22xdt-6 had two color forms: orange-red and yellow-orange. the orange-red corolla (rhcc n30 a) had a scarlet red (rhcc 46 b) eye zone surrounded by grayed orange (rhcc n 170 b) halo and yellow vein markings radiating from the center to the petal. the yelloworange or indian yellow (rhcc 17 a) corolla had a scarlet red (rhcc 46 b) eye zone. 22xdt-9 is china rose (rhcc 58 d) with a currant red (rhcc 46 a) eye zone and white (rhcc 155 a) edges. 22xdt-10 is straw yellow (rhcc 13 c) with yellow and orange (rhcc 28 c) eye zone. the red eye expressed in each hybrid morphological characterization, evaluation and selection of hibiscus 56 progeny was inherited from the male parent, h. rosa-sinensis ‘domini torrevillas’. this suggests that the red color for the eye could be dominant to yellow. based on the criteria for selection, 22xdt-9 (figure 1f) was selected. it expressed a unique flower color: red-purple or china rose (rhcc 58 d) and white petal edges, which are not observed in other hybrid progenies of the cross (table 1). it has good quality blooms and the white edges of the petals are undulating. this selection has a similar flower color to hibiscus rosa-sinensis ‘arlene b. arcillas’ (magdalita and pimentel 2013) and h. rosa-sinensis ‘obdulia f. sison’ (magdalita and pimentel 2010b). in addition, its tufted petal edges are similar to the foreign variety h. rosasinensis ‘lillian amy’ whose parents are h. rosa-sinensis ‘miss liberty’ and ‘inspiration’ (australian hibiscus society 2007). figure 1. hybrids of accession 22 x hibiscus rosa-sinensis ‘domini torrevillas’. (a) 22xdt-1; (b) 22xdt-2; (c) 22xdt-5; (d-e) 22xdt-6; (f) 22xdt-9; and, (g) 22xdt-10. (llxefa)xgc is a cross between hibiscus rosa-sinensis ‘loren legarda’, hibiscus rosasinensis ‘estrella f. alabastro’ (light whitish pink), and hibiscus rosa-sinensis ‘gelia castillo’ (golden yellow). five hybrid progenies of this cross with pedigrees (llxefa)xgc-2, (llxefa)xgc-3, (llxefa)xgc-5, (llxefa)xgc-7 and (llxefa)xgc-8 had flower colors with different shades of yellow, orange and red-purple. (llxefa)xgc-2 is orange-red (rhcc n 30 b) with a red (rhcc 46 b) eye zone. (llxefa)xgc-3 is lemon yellow (rhcc 14 c) with a claret rose (rhcc 50 a) eye surrounded by two layers of grayed purple (rhcc 186 c) and persimmon orange (rhcc 28 a) halos. (llxefa)xgc-5 is spinel red (rhcc 54 c) with a neyron rose (rhcc 55 a) eye radiating from the center to the petal. (llxefa)xgc-7 is chinese yellow (rhcc 16 a) with a white (rhcc n 155 b) eye and orpiment orange (rhcc 25 a) vein markings on the petals. (llxefa)xgc-8 is roseine purple (rhcc 68 c) with white spots. its eye is spinel red (rhcc 54 a) surrounded with a purple (rhcc 76 c) halo. e.m.n. cabarrubias et al. 57 selected hybrids flower characteristics fol iation growth habit solitary, simple, regular, arranged alternately, 1.31 tall and 146.8 mm in diameter simple and ovate leaf 0.53 m wide, and the angle of display form. margin is entire, semi-erect of the flower on the base is obtuse and growth habit plant is 74 degrees. leaf tip is acute. leaf and fast grower is 71.6 mm long and on its own root. red purple or china 53.2 mm wide. rose (rhcc 58 d) petals with currant red (rhcc 46 a) eye and white (rhcc 155 a) petal edges. solitary, simple, regular, arranged alternately, 2.07 m tall and 130.1 mm in diameter simple and cordate/ 0.79 m wide, and the angle of display cordiform leaf form. shrubby, semiof flower on the margin is crenate, erect growth plant is 74 degrees. base is cordate and habit and fast leaf tip is obtuse. leaf grower on its orange red (rhcc n is 80.7 mm long and own root. 30 b) petals with red 82.6 mm wide. (rhcc 46 b) eye. solitary, simple, regular, arranged alternately, 1.92 m tall 131.6 mm in diameter simple and cordate/ and 0.86 m and the angle of display cordiform leaf form. wide, shrubby, of flower on the plant margin is crenate, base semi-erect is 80 degrees. is cordate and the growth habit leaf tip is acute. and fast grower red purple or roseine leaf is 71.1 mm long on its own purple (rhcc 68 c) and 59.1 mm wide. root. petals with white and pink splashes on the petals. eye is spinel red (rhcc 54 a) surrounded by purple (rhcc 76 c) halo. solitary, simple, regular, arranged alternately, 1.19 m tall 141.9 mm in diameter simple and cordate/ and 0.51 m and the angle of display cordiform leaf form. wide, semiof flower on the plant margin is serrate, erect growth is 83 degrees. base is cordate, and habit and leaf tip is acute. leaf fast grower grayed purple (rhcc is 110.1 mm long and on its own root. 186 b) petals with red/ 94.1 mm wide. neyron rose (rhcc 55 b) eye. it has bluish white vein markings radiating from the eye to the petals. table 1. selected hybrids and their important characteristics morphological characterization, evaluation and selection of hibiscus 58 selected hybrids flower characteristics fol iation growth habit solitary, simple, regular, arranged alternately, 1.71 m tall 136 mm in diameter simple and cordate/ and 1.30 m and the angle of display cordiform leaf form. wide, semiof flower on the plant margin is crenate, erect growth is 80 degrees. base is cordate and habit and fast leaf tip is acute. grower on its pastel pink or red purple leaf is 101.4 mm own root. (rhcc 69 c) with red long and 85.6 mm purple or ruby red wide. (rhcc 59 a) eye surrounded by red purple or spiraea red (rhcc 63 c) halo that extends to the petals. solitary, simple, regular, arranged alternately, 1.55 m tall and 142.9 mm in diameter simple and cordate/ 1.50 m wide, and the angle of display cordiform leaf form. shrubby, semiof flower on the plant margin is serrate, erect growth is 88 degrees. base is cordate and habit and fast leaf tip is acute. grower on its yellow orange (rhcc leaf is 72.8 mm long own root. 14 b) petals with red and 68 mm wide. purple (rhcc n 57 a) eye surrounded by red purple or spiraea (rhcc 63 c) halo that extends to the petals. solitary, simple, regular, arranged alternately, 1.88 m tall 131.4 mm in diameter simple and cordate/ and 1.24 m and the angle of display cordiform leaf form. wide, shrubby, of flower on the plant margin is crenate, semi-erect is 81 degrees. base is cordate and growth habit leaf tip is acute. and fast neyron rose (rhcc 56 a) leaf is 83.6 mm long grower on its petals with cardinal red and 85.1 mm wide. own root. (rhcc 53 a) eye and light neyron rose (rhcc 55 b) vein markings. solitary, simple, regular, arranged alternately, 1.58 m tall and 114 mm in diameter simple and cordate/ 1.09 m wide, and the angle of display cordiform leaf form. semi-erect of flower on the plant margin is crenate, growth habit is 69 degrees. base is cordate and fast grower and leaf tip is on its own root. orange (rhcc n 25 c) rounded. leaf is petals with neyron 89.1 mm long and rose (rhccf 55 a) eye 78 mm wide. surrounded by white halo. table 1. selected hybrids and their important characteristics (cont’n.) e.m.n. cabarrubias et al. 59 table 1. selected hybrids and their important characteristics (cont’n.) selected hybrids flower characteristics fol iation growth habit solitary, simple, regular, arranged alternately, 1.91 m tall and 113.4 mm in diameter simple and cordate/ 1.09 m wide, and the angle of display cordiform leaf form. semi-erect of flower on the plant margin is entire, base growth habit is 80 degrees. is cordate and leaf and fast grower tip is retuse. leaf is on its own root. buttercup yellow (rhcc 66.7 mm long and 15 a) petals with red/ 58.2 mm wide. carmine rose (rhcc 52 c) eye surrounded by two layers of grayed purple or magenta rose (rhcc 186 d) and red purple (rhcc 62 c) halos that extend to the petals. solitary, simple, regular, arranged alternately, 2.87 m tall and 117.9 mm in diameter simple and cordiform/ 0.91 m wide, and the angle of cordate leaf form. shrubby, semidisplay of flower on margin is serrate, erect growth the plant is 67 degrees. base is cordate and habit and fast leaf tip is acute. leaf grower on its scarlet red (rhcc 43 c) is 69.4 mm long and own root. petals with orange 66.7 mm wide. red or saturn red (rhcc 30 c) petal edges and cardinal red (rhcc 53 a) eye. solitary, simple, regular, arranged alternately, 1.88 m tall 131.4 mm in diameter simple and cordate/ and 1.24 m and the angle of display cordiform leaf form. wide, shrubby, of flower on the plant margin is crenate, semi-erect is 81 degrees. base is cordate and growth habit leaf tip is acute. and fast neyron rose (rhcc 56 a) leaf is 83.6 mm long grower on its petals with cardinal red and 85.1 mm wide. own root. (rhcc 53 a) eye and light neyron rose (rhcc 55 b) vein markings. solitary, simple, regular, arranged alternately, 1.58 m tall and 137.4 mm in diameter simple and cordiform/ 1.02 m wide, and the angle of display cordate leaf form. shrubby, semiof flower on the plant margin is crenate, erect growth is 78 degrees. base is cordate and habit and fast leaf tip is acute. leaf grower on its spinel red (rhcc 54 c) is 97.6 mm long and own root. petal with yellow orange/ 88.1 mm wide. cadmium orange (rhcc 23 c) edges and red (rhcc 46 b) eye. morphological characterization, evaluation and selection of hibiscus 60 selected hybrids flower characteristics fol iation growth habit solitary, simple, regular, arranged alternately, 1.12 m tall and 140.7 mm in diameter simple and ovate leaf 0.97 m wide, and the angle of display form. margin is shrubby, semiof flower on the plant crenate, leaf base is erect growth is 81 degrees. obtuse and leaf tip habit and fast is acute. leaf is grower on its neyron rose (rhcc 55 b) 80.8 mm long and own root. petals with rhodonite 57.3 mm wide. red (rhcc 51a) eye and white vein markings radiating from the eye to the petals. solitary, simple, regular, arranged alternately, 1.58 m tall and 135.6 mm in diameter simple and cordiform/ 1.20 m wide, and the angle of cordate leaf form. shrubby, semidisplay of the flower on margin is serrate, erect growth the plant is 75 degrees. base is cordate and habit and fast leaf tip is acute. leaf grower on its carmine rose (rhcc is 77.8 mm long and own root. 52 c) petals with 73.8 mm wide. spanish orange (rhcc 26 b) petal edges and currant red (rhcc 46 a) eye. table 1. selected hybrids and their important characteristics (cont’n.) based on the criteria for selection, two progenies were identif ied as candidates, namely (llxefa)xgc-2 and (llxefa)xgc-8 (figures 2a and 2e). (llxefa)xgc-2, which is orange-red (rhcc n 30b), had a similar flower color to h. rosa-sinensis ‘ledivina v. cariño’ (magdalita et al. 2016) ‘cynthia a. villar’ (magdalita and pimentel 2013) including ‘loren b. legarda’ (magdalita et al. 2011). (llxefa)xgc-2 inherited the deep red eye from gc and (llxefa)xgc-8 inherited its color from llxefa . (llxefa)xgc-8 had splashes of white and pink on its petals (table 1). on the other hand, the red eye of gc was expressed in different shades in all progenies except for (llxefa)xgc-7. this suggests that the red eye could be dominant to white and pink. dsxgc is a cross between hibiscus rosa-sinensis ‘diamond star’, a creamy white hybrid, and the golden yellow h. rosa-sinensis ‘gelia castillo’. six hybrid progenies of this cross with pedigrees dsxgc-1, dsxgc-2, dsxgc-3, dsxgc-6, dsxgc-7 and dsxgc-8 had flower colors with different shades of white, yellow, yellow-orange, and grayed purple. dsxgc-1 is aureolin (rhcc 12 a) with a cardinal red (rhcc 53 a) eye surrounded by a white halo extending to the petals. dsxgc-2 is white (155 a) e.m.n. cabarrubias et al. 61 with a red/rhodonite red (rhcc 51 a) eye. dsxgc-3 is nasturtium orange (rhcc 25 b) with a red (rhcc 44 b) eye surrounded by a grayed orange (rhcc 165 b) halo. dsxgc-6 is white (rhcc nn155 d) with a neyron rose (rhcc 55 a) eye. dsxgc-7 is grayed purple (rhcc 186 b) with a neyron rose (rhcc 55 b) eye and bluish white vein markings radiating from the eye to the petals. dsxgc-8 is aureolin (rhcc 12 a) with a cardinal red (rhcc 53 a) eye surrounded by a white halo extending to the petals. figure 2. hybrids of [hibiscus rosa-sinensis ‘loren legarda’ x hibiscus rosa-sinensis ‘estrella f. alabastro’] x hibiscus rosa-sinensis ‘gelia castillo’. (a) (llxefa)xgc-2; (b) (llxefa)xgc-3; (c) (llxefa)xgc-5; (d) (llxefa)xgc-7; and, (e) (llxefa)xgc-8. the selected hybrid progeny in this cross was dsxgc-7 (figure 3e). this selected hybrid has traits different from the parents, such as the grayed purple petals (table 1). it has a flower color similar to h. rosa-sinensis ‘patricia b. licuanan’ (magdalita et al. 2016) and ‘kristie anne kenney’ (magdalita et al. 2011). in this cross, the red eye of both parents was expressed in different shades in f ive out of six (83.33%) progenies, namely dsxgc-1, dsxgc-2, dsxgc-3, dsxgc-6 and dsxgc-8. this suggests that the red eye is dominant to white and yellow. figure 3. hybrids of hibiscus rosa-sinensis ‘diamond star’ x h. rosa-sinensis ‘gelia castillo’. (a) dsxgc-1; (b) dsxgc-2; (c) dsxgc-3; (d) dsxgc-6; (e) dsxgc-7; and, (f) dsxgc-8. morphological characterization, evaluation and selection of hibiscus 62 20xgc is a cross between the red-purple hibiscus rosa-sinensis ‘accession 20’ and the golden yellow h. rosa-sinensis ‘gelia castillo’. five hybrid progenies in this cross with pedigrees 20xgc-3, 20xgc-4, 20xgc-5, 20xgc-6 and 20xgc-9 had flower colors with different shades of purple and orange.the progeny 20xgc-6 was redpurple (rhcc 58 c) with a red-purple (rhcc 59 a) eye extending to the petals. 20xgc-3 is grayed purple (rhcc 186 b) to spiraea red (rhcc 63 b) with a ruby red (59 a) eye and pink vein markings extending to the petals. 20xgc-4 is grayed orange (rhcc 169 a) with a cardinal red (rhcc 53 a) eye surrounded by a neyron rose (rhcc 55 c) halo extending to the petal. 20xgc-5 is red-purple (rhcc 69 c) with a ruby red (rhcc 59 a) eye zone surrounded by a spiraea red (rhcc 63 c) halo that extends to the petals. 20xgc-9 is phlox pink (rhcc 62 b) with pink blushes on the edges of the petals and light pink vein markings radiating from the eye to the petals. based on the criteria for selection, 20xgc-5 (figure 4d) was selected out of the f ive progenies. its unique properties include semi-overlapping pastel pink petals and striking cartwheel red-purple eye (table 1). in addition, the dark red eye of gc was inherited by three out of f ive (60%) hybrid progenies, suggesting again that the red eye is dominant to yellow and purple, while the purple petal is dominant to yellow. (gcxbgb)xhp is a cross between the yellow hybrid hibiscus rosa-sinensis ‘gelia castillo’ x hibiscus rosa-sinensis ‘betty go belmonte’ and the red-purple variety hibiscus rosa-sinensis ‘hot pink’. six hybrid progenies generated in this cross with pedigrees (gcxbgb)xhp-1, (gcxbgb)xhp-2, (gcxbgb)xhp-4, (gcxbgb)xhp-7, (gcxbgb)xhp-8 and (gcxbgb)xhp-9 had flowers with different hues of red-purple. (gcxbgb)xhp-1 is red-purple (rhcc 61 b) with a red-purple (rhcc n 57 a) eye figure 4. hybrids of hibiscus rosa-sinensis ‘accession 20’ and h. rosa-sinensis ‘gelia castillo’. (a)20xgc-6; (b) 20xgc-3; (c) 20xgc-4; (d)20xgc-5; and, (e) 20xgc-9. e.m.n. cabarrubias et al. 63 zone. (gcxbgb)xhp-2 is amaranth rose (rhcc 65 a) with a tyrian purple (rhcc n 57 a) eye. (gcxbgb)xhp-4 is rhodamine pink (rhcc 62 a) with a currant red (rhcc 46 a) eye and pink vein markings extending to the petals. (gcxbgb)xhp-7 is amaranth rose (rhcc 65 a) with a rose red (rhcc 58 b) eye and pink vein markings radiating from the eye to the petals. (gcxbgb)xhp-8 is red-purple (rhcc 61 b) with a strong red purple eye. (gcxbgb)xhp-9 is a light red purple hybrid (rhcc n 57 b) with a dark red purple (rhcc n 57 a) eye. out of the six progenies in this cross, only one hybrid progeny plant, (gcxbgb)xhp-4, (figure 5c) was selected. the hybrid has a unique combination of pastel pink or rhodamine pink petals and a dark red eye (table 1).the pastel pink or red purple or rhodamine pink (rhcc 62 a) flower color of this selection is similar to h. rosasinensis ‘pia s. cayetano’ (magdalita et al. 2016). in this cross, the red eye from the f e m a l e p a r e n t g c x b g b w a s i n h e r i t e d b y f o u r o u t o f s i x ( 6 6 . 6 % ) , n a m e l y (gcxbgb)xhp-1, (gcxbgb)xhp-2, (gcxbgb)xhp-4 and (gcxbgb)xhp-7. this suggests that the red eye is dominant to yellow in this particular cross. gcxds is a cross between hibiscus rosa-sinensis ‘gelia castillo’ (golden yellow) and hibiscus rosa-sinensis ‘diamond star’ (creamy white). five hybrid progenies generated in this cross with pedigrees gcxds-1, gcxds-2, gcxds-4, gcxds-5 and gcxds-6 had flower colors with different shades of yellow (3), red (1), and white (1). gcxds-1 is white (rhcc n 155 a) with a red (rhcc 55 d) eye. gcxds-2 is turkey red (rhcc 46 c) with a red (rhcc n 34 a) eye. gcxds-4 is yellow-orange (rhcc 14 b) with a red purple (rhcc n 57 a) eye surrounded by a spiraea (rhcc 63 c) halo extending to the petals. gcxds-5 is yellow-orange (rhcc 16 b) with a neyron rose (rhcc55 c) figure 5. hybrids of hibiscus rosa-sinensis ‘gelia castillo’ by h. rosa-sinensis ‘betty go belmonte’ and h. rosa-sinensis ‘hot pink’. (a) (gcxbgb)xhp-1; (b) (gcxbgb)xhp2 ; ( c ) ( g c x b g b ) x h p 4 ; ( d ) ( g c x b g b ) x h p 7 ; ( e ) ( g c x b g b ) x h p 8 ; a n d , ( f ) (gcxbgb)xhp-9. morphological characterization, evaluation and selection of hibiscus 64 eye surrounded by white streaks that extend to the petals. gcxds-6 is buttercup yellow (rhcc 15 a) with a neyron rose (rhcc 55 a) eye surrounded by white streaks that extend to the petals. out of the f ive progenies, only gcxds-4 (figure 6c) was selected because of its unique cartwheel eye pattern surrounded by a white halo and its yellow-orange ruffled semi-overlapping petals. the dark red eye from both parents was inherited by four out of f ive progenies (80%). again in this cross, the red eye is dominant to yellow and white. this hybrid is a prolif ic bloomer with good quality of blooms. in addition, this yellow-orange flower (rhcc 14 b) is similar to h. rosa-sinensis ‘ vilma abaya-dimacuha’ (magdalita et al. 2016), ‘marilyn d. marañon’ (magdalita and pimentel 2013), ‘mercedes b. concepcion’, and ‘betty go-belmonte (magdalita et al. 2 0 0 9 ) . abaxmdm is a cross between hibiscus rosa-sinensis ‘arlene b. arcillas’ (carmine rose) and hibiscus rosa-sinensis ‘marilyn d. marañon’ (lemon yellow). seven hybrid progenies in this cross with pedigrees abaxmdm-1, abaxmdm-2, abaxmdm-3, abaxmdm-4, abaxmdm-5, abaxmdm-6 and abaxmdm-7 had flower colors with different shades of orange, red, yellow and white. abaxmdm-1 is neyron rose (rhcc 56 a) with a cardinal red (rhcc 53 a) eye and neyron (rhcc 55 b) vein markings. abaxmdm-2 is f ire red (rhcc 33 b) with a spinel red (rhcc 54 a) eye and orange (rhcc 32 c) petal edges. abaxmdm-3 is orange (rhcc n 25 c) with a neyron rose (rhcc 55 a) eye surrounded by a white halo. abaxmdm-4 is persimmon orange (rhcc 28 a) with a neyron rose (rhcc 55 a) eye. abaxmdm-5 is buttercup figure 6. hybrids of hibiscus rosa-sinensis ‘gelia castillo’ and hibiscus rosa-sinensis ‘diamond star’. (a) gcxds-1; (b) gcxds-2; (c) gcxds-4; (d) gcxds-5; and, (e) gcxds-6. e.m.n. cabarrubias et al. 65 yellow (rhcc 15 a) with a red purple (rhcc n 57 a) eye. abaxmdm-6 is white (rhcc n155 c) with a red-purple (rhcc n 57 a) eye and red-purple (rhcc 62 d) vein markings extending from the eye to the petals. abaxmdm-7 is neyron rose (rhcc 56 a) with a cardinal red (rhcc 53 a) eye and neyron (rhcc 55 b) vein markings extending from the eye to the petals. out of the seven progenies, two were selected, namely abaxmdm-1 and abaxmdm3 (figures 7a and 7c). abaxmdm-1 was selected for its pastel pink or neyron rose petals with distinct cardinal red eye and semi-ruffled petal edges, while abaxmdm3 was selected because of its attractive light orange petals with semi-ruffled edges (table 1). three progenies out of seven (42.86%) inherited the pink color of the maternal parent aba, while four (57.1%) possessed the orange color not present in either parents. the ruffled edges of the female parent mdm was inherited by f ive (71.43%) out of seven progenies. six out of seven (85.71%) progenies had the red eye inherited from the female parent aba. this suggests that the red eye is dominant to yellow in this cross. figure 7. hybrids of hibiscus rosa-sinensis ‘arlene b. arcillas’ and hibiscus rosasinensis ‘marilyn d. marañon’. (a) abaxmdm-1; (b) abaxmdm-2; (c) abaxmdm-3; (d) abaxmdm-4; (e) abaxmdm-5; (f) abaxmdm-6; and, (g) abaxmdm-7. 23xgc is a cross between the light yellow hibiscus rosa-sinensis ‘accession 23’ and the golden yellow hibiscus rosa-sinensis ‘gelia castillo’. five hybrid progenies generated in this cross with pedigrees 23xgc-2, 23xgc-5, 23xgc-6, 23xgc-8 and 23xgc-10 had flower colors with different shades of yellow. 23xgc-2 is buttercup yellow (rhcc 15 a) with a carmine rose (rhcc 52 c) eye surrounded by two layers of magenta rose (rhcc 186 d) and red purple (rhcc 62 c) halos that extend to the petals. 23xgc-5 is lemon yellow (rhcc 13 b) with a red (rhcc 45 b) eye inherited from gc and surrounded by a white halo that extends to the petals. 23xgc-6 is saffron yellow (rhcc 21 a) with a neyron rose (rhcc 55 a) eye and persimmon morphological characterization, evaluation and selection of hibiscus 66 orange (rhcc 28 a) vein markings extending from the eye to the petals. 23xgc-8 is buttercup yellow (rhcc 15 a) with a dawn pink (rhcc 49 a) eye surrounded by a white halo that extends to the petals. 23xgc-10 is buttercup yellow (rhcc 15 a) with a carmine rose (rhcc 52 c) eye surrounded by two layers of magenta rose (rhcc 186 d) and red purple (rhcc 62 c) halos that extend to the petals. four out of f ive (80.0%) progenies inherited the dark red eye from gc. in this cross, the dark red eye was dominant to the yellow. based on the criteria for selection, 23xgc-2 (figure 8a) was selected out of the f ive progenies because of its bright and striking red eye surrounded by a white halo. cvxnb is a cross between the orange hibiscus rosa-sinensis ‘cynthia villar’ and the white hibiscus rosa-sinensis ‘new bangkok’. six hybrid progenies generated in this cross with pedigrees cvxnb-1, cvxnb-2, cvxnb-3, cvxnb-4, cvxnb-5 and cvxnb-6 had flower colors ranging from red-orange to yellow (figure 9). cvxnb-1 is scarlet (rhcc 43 c) with saturn red (rhcc 30 c) petal edges and a cardinal red (rhcc 53 a) eye. cvxnb-2 is spinel red (rhcc 54 c) with cadmium orange (rhcc 23 c) petal edges and a red (rhcc 46 b) eye. cvxnb-3 is cadmium orange (rhcc 23 b). cvxnb-4 is buttercup yellow (rhcc 15 a) with a cardinal red (rhcc 53 a) eye surrounded by a white halo extending to the petals. cvxnb-5 is carmine red (rhcc 52 b) with jasper red (rhcc 39 a) petal edges and a cardinal red (rhcc 53 a) eye.cvxnb-6 is carmine rose (rhcc 52 c) with spanish orange (rhcc 26 b) petal edges and a currant red (rhcc 46 a) eye. figure 8. hybrids of hibiscus rosa-sinensis ‘accession 23’ and h. rosa-sinensis ‘gelia castillo’. (a) 23xgc-2; (b) 23xgc-5; (c) 23xgc-6; (d) 23xgc-8; and, (e) 23xgc-10. e.m.n. cabarrubias et al. 67 figure 9. hybrids of hibiscus rosa-sinensis ‘cynthia villar’ and h. rosa-sinensis ‘new bangkok’. (a) cvxnb-1; (b) cvxnb-2; (c) cvxnb-3; (d) cvxnb-4; (e) cvxnb-5; and, (f) cvxnb-6. out of the six hybrid progenies, three hybrid progenies, namely cvxnb-1, cvxnb-2, and cvxnb-6, were selected (figure 9) because of their unique red eye that radiates to the scarlet red petals inherited from the male parent ‘new bangkok’ (table 1). cvxnb-6, which has carmine rose (rhcc 52 c) petals with spanish orange (rhcc 26 b) petal edges and currant red (rhcc 46 a) eye (table 1), is similar to the foreign variety h. rosa-sinensis ‘erin rachel’ (australian hibiscus society 2004). four hybrid progenies out of six (66.6%) inherited the red eye, which is dominant to white and orange in this cross. cvxmp is a cross between hibiscus rosa-sinensis ‘cynthia villar’ (orange) and hibiscus rosa-sinensis ‘marjorie pink’ (pink). five hybrid progenies generated from this cross with pedigrees cvxmp-1, cvxmp-2, cvxmp-3, cvxmp-4 and cvxmp-5 had flower colors ranging from orange, light pink, yellow to light purple (figure 10). cvxmp-1 is vermilion (rhcc 41 a) with a rhodonite red (rhcc 51 a) eye.cvxmp-2 is buttercup yellow (rhcc 15 a) with a cardinal red (rhcc 53 a) eye surrounded by neyron rose (rhcc 55 b) vein markings that extend to the petals. cvxmp-3 is crimson (rhcc 52 a) to azalea pink (rhcc 41 c) with a currant red (rhcc 46 a) eye. cvxmp-4 is neyron rose (rhcc 55 b) with a rhodonite red (rhcc 51 a) eye and white vein markings radiating from the eye to the petals. cvxmp-5 is neyron rose (rhcc 55 b) with a rhodonite red (rhcc 51 a) eye. out of the f ive progenies, only the cvxmp-4 (20%) was selected (figure 10d). it has a unique rhodonite red eye with a light purple halo radiating to the neyron rose petals and ruffled edges (table 1). the red eye from both parents was inherited by the f ive hybrid progenies, indicating that the red eye is dominant to orange and light pink. one progeny, cvxmp-2, had a crest which is a unique trait not present in both parents (figure 10b). morphological characterization, evaluation and selection of hibiscus 68 principal component and agglomerative cluster analyses of phenotypic traits the principal component analysis (pca) and agglomerative cluster analysis (aca) are two multivariate approaches that can be utilized to analyze relationships within and between samples. each can be used to complement each other to generate more precise information. pca is known to be less sensitive to distances between clusters, while cluster analysis generally reproduces distances between close neighbors faithfully but shows distortion among members of large clusters (sneath and sokal 1973). to study the patterns of variation within the data matrix, the pca based on the correlation matrix of the sample means for the nine floral traits was carried out using the eigen program (jacob and guennebaud 2016). pca is also a tool for selection of variables according to the degree of their variances in each principal component generated. pca generates principal components, eigenvalues, proportion of variance, cumulative proportion and eigenvectors. the variation shown in the nine floral traits of the 57 hybrids can be explained using pca which grouped these traits into nine pcs (table 2).the principal components generated were ranked by importance through variances. in this study, out of nine principal components (pcs), three pcs, namely: pc-i; pc-ii; and pc-iii had eigenvalues >1, accounting for 78% of total cumulative variability of the various traits among the 57 different hybrid progenies (table 2). this suggests medium correlation among the floral traits studied. similarly, in jackfruit (artocarpus heterophyllus lam.), an figure 10. hybrids of hibiscus rosa-sinensis ‘cynthia v illar’ and h. rosa-sinensis ‘marjorie pink’. (a) cvxmp-1; (b) cvxmp-2; (c) cvxmp-3; (d) cvxmp-4; and, (e) cvxmp-5. e.m.n. cabarrubias et al. 69 eigenvalue greater than 1 was obtained using 41 morphological traits in 88 trees studied, subsequently generating 12 pcs. these 12 pcs accounted for 72.5% of the total variability of the jackfruit genotypes studied (dayap 2000), which is not distant to the variability (78%) obtained for the hibiscus hybrids. i 3.93 0.44 0.44 bloom size, corolla length, corolla width, style length, leaf length, leaf width ii 1.85 0.21 0.64 style length, petiole length, leaf length, leaf width iii 1.24 0.14 0.79 pedicel length, receptacle length iv 0.69 0.08 0.86 pedicel length, receptacle length v 0.50 0.06 0.91 petiole length, leaf width vi 0.41 0.05 0.96 bloom size, corolla with, style length vii 0.18 0.02 0.98 corolla length, corolla width, leaf length viii 0.13 0.02 0.99 corolla length, leaf length, leaf width ix 0.07 0.01 1.00 bloom size, corolla length principal components (pcs) eigenvalues proportion of variance cumulative proportion principal characters represented table 2. eigenvalues, proportion of the variabil ity, and the characters represented by the nine principal components of 57 hibiscus experimental hybrid progenies the contribution of pc-i to the variability in the data set was highest (43.69%), followed by pc-ii at 20.56% and pc-iii at 13.72%. pca results revealed patterns of correlation through the eigenvectors. traits with eigenvectors of ≥0.3 were considered relevant for the component (table 3). the f irst principal component (pc-i) showed positive variable loadings for all the traits, including bloom size (ev=0.45), corolla length (ev=0.44), corolla width (ev=0.42), pedicel length (ev=0.15), style length (ev=0.31), petiole length (ev=0.30), leaf length (ev=0.35), leaf width (ev=0.32), and receptacle diameter (ev=0.087). the characteristics that had positive signif icant eigenvectors in pc-i were bloom size, corolla length, corolla width, style length, leaf length, and leaf width. this suggests that hybrids with larger flower size tend to have longer and wider leaves. pc-ii indicated positive variable loadings for petiole length (ev=0.38), leaf length (ev=0.45) and leaf width morphological characterization, evaluation and selection of hibiscus 70 (ev=0.47) except for style length (ev=-0.40). petiole length had a signif icant positive correlation with leaf length and leaf width. however, it had signif icant negative association with style length, suggesting that flowers with longer petiole tend to have shorter style. in the third principal component (pc-iii), receptacle length (ev=0.70) had a significant negative correlation with pedicel length (ev=-0.66), suggesting that flowers with larger receptacle tend to have shorter pedicel. the pca reduced the 16 quantitative traits to nine that still contain important information about the data set. bloom size, corolla length, corolla width, style length, leaf width, leaf length, petiole length, pedicel length, and receptacle diameter obtained high variances, were considered important traits in the first three principal components, namely: pc-i; pc-2; and, pc-3. the same characteristics had the largest contribution to the total variation in the data set. they were used to group the 57 hibiscus hybrid progenies into f ive distinct clusters, thus providing a better clustering of the hybrid progenies in a dendrogram (figure 11). as a whole, while pca and cluster analysis are both useful in analyzing the relationships across and among the 57 hibiscus hybrids, they both complemented each other in generating more precise information in the selection of 14 hybrid progenies out of the 57. clustering generated a highly branched structure (figure 11), suggesting the existence of a highly variable group, wherein selection of desirable hibiscus hybrid progenies can be performed effectively. in addition, clustering was able to optimally capture the representative progenies of the different crosses having similar floral traits like petal color and bloom size, thereby facilitating the selection of the 14 desirable hybrid progenies from the various crosses. on the other hand, pca effectively showed the patterns of variation in the bloom size 0.45* -0.25 0.06 -0.03 0.03 -0.32* 0.28 -0.09 0.74* corolla length 0.44* -0.28 0.001 0.02 0.02 -0.05 0.50* -0.31* -0.61* corolla width 0.42* -0.25 0.17 0.09 0.09 -0.39* -0.67* 0.27 -0.23 ped icel length 0.15 0.23 -0.62* -0.66* -0.16 -0.27 -0.08 0.005 -0.07 style length 0.31* -0.40* 0.21 -0.13 -0.16 0.77* -0.19 0.12 0.12 petiole length 0.29 0.38* -0.18 0.07 0.82* 0.22 -0.06 -0.08 0.04 leaf length 0.35* 0.45* 0.01 0.24 -0.28 0.06 0.30* 0.67* -0.05 leaf width 0.32* 0.47* 0.14 0.19 -0.42* 0.11 -0.30 -0.59* 0.06 receptacle 0.09 0.16 0.70* -0.67* 0.08 0.15 0.06 0.07 -0.03 length *significant eigenvector (≥0.3) variables pc-i pc-ii pc-iii pc-iv pc-v pc-vi pc-vii pc-viii pc-ix table 3. eigenvectors of the characters in each principal component (pc) e.m.n. cabarrubias et al. 71 data matrix of the various floral traits studied and separated the nine distinct principal components based on the derived eigenvalues of the primary characters represented in each component (table 2). for instance, pc-i with eigenvalue of 3.93 accounted for 78.0% of the variability of different hybrid progenies in terms of bloom size, corolla length, corolla width, style length, leaf length and leaf width. since many of the useful traits are included in this principal component, particularly bloom size, corolla length and width, and style length, which are indeed necessary in the rigorous selection of the 14 desirable hybrid progenies (table 1), the process of selection was implemented effectively and eff iciently in this set of hibiscus hybrids. selection was executed through the combined information derived from both cluster analysis and pca. agglomerative cluster analysis and correlation among phenotypic traits based on a priori knowledge, f ive clusters were arbitrarily selected to form the dendrogram, which was automatically cut at a gower distance between 0.3 and 0.4 using the statistical tool for agricultural research (star) software (irri 2014). the 57 hibiscus hybrids clustered into f ive groupings (table 4) based on the clustering analysis of 16 phenotypic characters (figure 11).the dendrogram was based on selected qualitative traits, including flower color, eye color, stigma color, leaf form, leaf margin, leaf apices, and leaf base and quantitative traits, namely: bloom size; corolla length; corolla width; style length; leaf width; leaf length; table 4. distribution of hibiscus hybrids in d ifferent clusters i 22xdt-1, 22xdt-10, (llxefa)xgc-2, (llxefa)xgc-3, 16 (llxefa)xgc-5, (llxefa)xgc-8, gcxds-2, gcxds-4 abaxmdm-1, abaxmdm-2, abaxmdm-4, abaxmdm-5, abaxmdm-7, 23xgc-10, cvxnb-3, cvxnb-6 ii 22xdt-2, 22xdt-5, dsxgc-1, dsxgc-2, dsxgc-6, 15 dsxgc-7, dsxgc-8, 20xgc-3, 20xgc-4, 20xgc-5, 20xgc-9, gcxbgb)xhp-1, gcxds-1, gcxds-6, cvxnb-2 iii 22xdt-6, 22xdt-9, (llxefa)xgc-7, 23xgc-6, 23xgc-8 5 iv dsxgc-3, 20xgc-7, (gcxbgb)xhp-8, (gcxbgb)xhp-9, 13 abaxmdm-3, abaxmdm-6, 23xgc-2, cvxnb-1, cvxnb-4, cvxnb-5, cvxmp-1, cvxmp-2, cvxmp-3 v 20xgc-6, (gcxbgb)xhp-2, (gcxbgb)xhp-4, 8 (gcxbgb)xhp-7, gcxds-5, 23xgc-5, cvxmp-4, cvxmp-5 cluster ped igree of experimental hybrids no. of experimental hybrids in a cluster morphological characterization, evaluation and selection of hibiscus 72 petiole length; pedicel length; and, receptacle diameter of the progenies. it was constructed by employing the complete clustering method and gower distance as a measure of dissimilarity. 2 3 x g c 1 0 (llxefa)xgc-8 gcxds 2 2 2 x d t1 0 c v x n b 6 (llxefa)xgc-3 g c x d s 4 (llxefa)xgc-5 (llxefa)xgc-2 abaxmdm-4 abaxmdm-2 c v x n b 3 abaxmdm-5 abaxmdm-7 abaxmdm-1 2 2 x d t1 (gcxbgb)xhp-9 (gcxbgb)xhp-8 d s x g c 3 c v x m p 1 c v x n b 5 c v x n b 4 c v x n b 1 abaxmdm-6 c v x m p 3 2 0 x g c 7 c v x m p 2 2 3 x g c 2 abaxmdm-3 2 0 x g c 4 d s x g c 7 2 0 x g c 3 (gcxbgb)xhp-1 2 0 x g c 9 g c x d s 1 d s x g c 6 g c x d s 6 2 2 x d t2 d s x g c 2 c v x n b 2 2 2 x d t5 2 0 x g c 5 d s x g c 8 d s x g c 1 c v x m p 5 2 3 x g c 5 (gcxbgb)xhp-7 (gcxbgb)xhp-2 (gcxbgb)xhp-4 2 0 x g c 6 c v x m p 4 g c x d s 5 2 3 x g c 8 2 3 x g c 6 2 2 x d t9 0 . 5 0 . 4 0 . 3 0 . 2 0 . 1 0 . 0 d en d ro gr am u si n g a gg lo m er at iv e c lu st er in g m et h od figure 11. the clustering of the 57 hibiscus hybrids. e.m.n. cabarrubias et al. 73 the 57 hybrid progenies grouped into f ive clusters based on agglomerate clustering (figure 11). the cluster number, specif ic hybrid progenies and number of progenies in each cluster are indicated in table 4. the hybrids in cluster-i were from six different crosses, namely 22xdt, (llxefa)xgc, gcxds, abaxmdm, 23xgc and cvxnb, which consist of 16 hybrid progenies (table 4). the hybrid progenies in this cluster have flower sizes that ranged from 115.10 mm to 143.30 mm, with a mean of 131.09 mm. the flowers of hybrids in this group had an average style length of 61.38 mm, petiole length of 27.22 mm and pedicel length of 37.90 mm (table 4). cluster-i comprises yellow to orange-red hybrids, most of which have red eye (figure 11). the 15 members of cluster-ii were from the crosses of 22xdt, dsxgc, 20xgc, (gcxbgb)xhp, gcxds, and cvxnb. the progenies had flower sizes that ranged from 118.40 mm to 152.30 mm, with a mean of 140.54 mm. in this group, the flowers of the hybrid progenies had an average style length of 61.57 mm, petiole length of 39.53 mm and pedicel length of 39.79 mm. cluster-ii was represented by white and red-purple hybrids mostly with red eye (figure 11). cluster-iii contained f ive yellow hybrids from the crosses of 22xdt, (llxefa)xgc, and 23xgc. their flowers had sizes ranging from 125.0 mm to 146.80 mm, with a mean of 131.12 mm. flowers of hybrids in this group had an average style length of 58.08 mm, petiole length of 22.80 mm and pedicel length of 24.86 mm. thirteen hybrid progenies from the crosses of dsxgc, 20xgc, (gcxbgb)xhp, abaxmdm, 23xgc, cvxnb and cvxmp formed cluster-iv. flowers of hybrids in this cluster were small compared to other hybrids belonging to clusters-i, ii, iii and v. the bloom size ranged from 95 mm to 126.00 mm, with a mean of 115.20 mm. flowers of hybrids belonging to this cluster had an average style length of 52.76 mm, petiole length of 24.07 mm and pedicel length of 29.44 mm. cluster-iv contained yellow and yellow-orange hybrid progenies with red eye. the members of cluster-v are 8 hybrid progenies from the crosses of 20xgc, (gcxbgb)xhp, gcxds, 23xgc and cvxmp. their flowers had sizes ranging from 105.70 mm to 160.80 mm, with a mean of 130.21 mm. flowers of the hybrids in this group had an average style length of 52.71 mm, petiole length of 27.80 mm and pedicel length of 39.82 mm. the hybrids in cluster-v had pinkish flowers with red eye (figure 11). morphological characterization, evaluation and selection of hibiscus 74 the mean and standard deviation of the quantitative traits of the hibiscus hybrid progenies belonging to the different clusters are presented in table 5. the correlation matrix showed different levels of relationship among the traits (table 6). bloom size is moderately correlated with corolla length (r=0.91) and width (r=0.86) and style length (r= 0.60), suggesting that the size of the bloom or flower is associated with its overall length and width, and the length of the style. similarly, correlation studies conducted in other hibiscus varieties with oneand two-day retention on the plant indicated that peduncle diameter, length and petal thickness correlated strongly with the retention of the flower in planta (valdoz et al 2017). in addition, the length of the style is correlated with the length of the corolla (r=0.70) and leaf width is correlated with leaf length (r=0.84). cluster 16 mean 131.09 150.76 112.30 61.38 27.22 80.40 71.33 37.90 9.47 i sd 7.58 8.26 6.62 7.43 7.43 9.53 10.79 9.63 1.53 cluster 15 mean 140.54 159.77 117.93 61.57 39.53 106.42 86.35 39.79 9.37 ii sd 8.67 7.89 8.56 5.92 7.39 8.78 9.28 10.60 0.81 cluster 5 mean 131.12 148.24 109.42 58.08 22.80 68.92 58.54 24.86 9.63 iii sd 8.87 11.81 7.20 10.42 6.57 6.42 8.02 10.03 1.18 cluster 13 mean 115.20 138.56 101.65 52.76 24.07 77.59 64.45 29.44 9.07 i v sd 7.88 11.58 6.49 11.42 7.95 14.54 10.46 7.44 0.86 cluster 8 mean 130.21 146.91 108.13 52.71 27.80 83.15 62.98 39.82 8.84 v sd 21.75 18.48 16.47 12.52 5.08 10.79 11.07 11.71 0.76 cluster number n statistics morphological characters bloom size corolla length corolla width style length petiole length leaf length leaf width ped ical length receptacle length table 5. mean and standard deviation (sd) of quantitative characters of hibiscus hybrids belonging to the five clusters bloom size 1.00 corolla length 0.91** 1.00 corolla width 0.86** 0.80** 1.00 style length 0.60* 0.70* 0.53* 1.00 petiole length 0.31 0.31 0.28 0.11 1.00 leaf length 0.40 0.37 0.35 0.10 0.60* 1.00 leaf width 0.33 0.30 0.33 0.05 0.52* 0.84** 1.00 ped icel length 0.15 0.13 0.01 0.16 0.34 0.28 0.22 1.00 receptacle length 0.13 0.06 0.16 -0.09 0.07 0.15 0.26 -0.14 1.00 ** high correlation *medium correlation bloom size corolla length corolla width style length petiole length leaf length leaf width ped icel length receptacle length table 6. correlation matrix of nine qual itative characters of hibiscus hybrids e.m.n. cabarrubias et al. 75 the results also show that bloom size had low correlation with petiole length (r= 0.31), leaf size (r=0.40), pedicel length (r=0.15), and receptacle length (r=0.13). a similar pattern was also observed in corolla length, corolla width, and style length. petiole length had moderate correlation with leaf length (r=0.60) and leaf width (r=0.52). pedicel length had low positive correlation with flower size (r=0.15), leaf length (r=0.28), leaf width (r=0.22), style length (r=0.16) and petiole length (r=0.34). however, pedicel length had low negative correlation with receptacle length (r=-0.14). receptacle length had low negative correlation with style length (r=-0.09) and had low positive correlation with flower size (r=0.13), leaf length (r= 0.15), leaf width (r= 0.26) and petiole length (r= 0.07). a dendrogram with a cophenetic correlation value of 1 gives a perfect picture of the pattern of dis/similarities of the data. low values indicate low correlation between the dendrogram and the original similarities or dissimilarities. the cophenetic coeff icient gives a measure of how well the original data match the hierarchical clustering through comparisons of the resemblance values from the similarity or dissimilarity matrix with those implied from the dendrogram. for a dendrogram to be a reasonably good reflection of a matrix of association, values of 0.85 or higher are desirable (stuessy 2009). the cophenetic correlation coeff icient obtained in this study was 0.667, which is relatively lower than the desirable value, indicating that the dis/similarities of the data may not be well presented in the dendrogram generated. a similar cophenetic correlation coeff icient of 0.766 was also obtained in clustering 88 genotypes of jackfruit, another highly heterozygous species (dayap 2000). however, a low cophenetic correlation coeff icient does not necessarily mean that the dendrogram is not acceptable or that the relationships expressed are lacking in taxonomic value; instead, it may indicate that distortion has taken place during clustering (stuessy 2009). the dendrogram showed a highly branched structure, suggesting a considerable degree of variability and divergence within the 57 hibiscus hybrid progenies studied. this observed variability can be attributed to the genetic differences of the hybrid progenies. these differences are expected because the parents used for hybridization are highly heterozygous. a similar dendrogram also suggesting high genetic variability of samples for different horticultural traits was generated for 22 pineapple hybrids and cultivars (ines et al. 2009), whose parents were also heterozygous. morphological and rapd markers effectively revealed this high degree of variability in pineapple (ines et al. 2009). however, the variability observed among the 57 hybrid progenies cannot be solely attributed to genetic differences but also to environmental conditions. therefore, the interaction of the morphological characterization, evaluation and selection of hibiscus 76 genotype and the environment can contribute to the total variability among the 57 hybrid progenies. as exhibited by the dendrogram, the agglomerative clustering method demonstrated its eff iciency in clustering the 57 hybrid progenies. however, a decision has to be made on how many variables will be used to group the hybrid progenies. in this study, quantitative and qualitative characters, including bloom size, corolla length, corolla width, style length, leaf width, leaf length, petiole length, pedicel length and receptacle length, were used to group the hybrid progenies into f ive clusters. however, classifying the 57 hybrid progenies into groups is a subjective procedure because there is no def inite algorithm that can be used in deciding at what specif ic coeff icient distance the dendrogram should be cut (brown 1991). in some cases, a priori knowledge can facilitate the determination of the number of clusters to be formed. for instance, cena (1995) used the three populations of cacao, namely ferastero, criollo, and trinitario, as the basis for grouping the university of southern mindanao agricultural research center (usmarc) cacao collections. the study found out that most of the studied clones with known varietal group fall in the same cluster. one common practice in clustering is to examine the tree generated and then designate similarity and difference levels above which the individuals are considered grouped. representative flower samples for each of the clusters are shown in figure 11. conclusion this characterization study showed a wide range of variation in both qualitative and quantitative characters between the crosses. for some characters like flower color and flower size, variation was also observed among hybrid progenies within a specif ic cross. based from the evaluation of morphological traits, 14 hybrid progenies were selected from the 10 different crosses. the 14 hybrid progenies are as follows: 22xdt-9; (llxefa)xgc-2; (llxefa)xgc-7; (llxefa)xgc-8; dsxgc-7; 20xgc-5; (gcxbgb)xhp-4; gcxds-4; abaxmdm-1; abaxmdm-3; 23xgc-2; cvxnb-1; cvxnb-2; cvxnb-6; and, cvxmp-4. 22xdt-9 is china rose with a currant red eye zone and white edges. (llxefa)xgc-2 is orange-red with a red eye zone. (llxefa)xgc-7 is chinese yellow with a white eye zone and orpiment orange vein markings. (llxefa)xgc-8 is roseine purple with white spots and spinel red eye color surrounded by purple halo. dsxgc-7 is grayed purple with a neyron rose eye and bluish white e.m.n. cabarrubias et al. 77 vein markings radiating from the center to the petal. 20xgc-5 is red-purple with a ruby red eye zone surrounded by a spiraea red halo extending to the petal. (gcxbgb)xhp-4 is rhodamine pink with a currant red eye zone and pink vein markings extending to the petal. gcxds-4 is yellow-orange with a red-purple eye zone surrounded by a spiraea halo extending to the petal. abaxmdm-1 is neyron rose with a cardinal red eye zone and neyron vein markings. abaxmdm-3 is orange with a neyron rose eye zone surrounded by a white halo. 23xgc-2 is buttercup yellow with a carmine rose eye zone surrounded by two layers of magenta rose and redpurple halo extending to the petals. cvxnb-1 is scarlet with saturn red edges and a cardinal red eye. cvxnb-2 is spinel red with cadmium orange edges and a red eye zone. cvxnb-6 is carmine rose with spanish orange edges and a currant red eye zone. cvxmp-4 is neyron rose with a rhodonite red eye zone and white vein markings radiating from the center to the petal. the height of the hybrids ranged from 1.17-2.87 m, while their width or canopy spread of 0.53-1.50 m classif ied them as semi-dwarf plants. in addition to the hybrids’ desirable floral traits, plant height and width are equally important since they are useful traits for consideration in maintaining a good architecture of the varieties as a potted ornament and a landscaping material. these selected hybrid progenies could be used for variety release. moreover, the selected hybrids were propagated asexually either by marcotting or air-layering and cleft grafting, propagation methods that will make them breed true-to-type. these methods guarantee the genetic stability of their desirable floral characteristics in succeeding asexual generations. results of present study suggest that red could be dominant to yellow as observed in the hybrids of 20xgc, (gcxbgb)xhp, and cvxmp. majority of the hybrids expressed different hues of red. the study also revealed that yellow could be dominant to white, as observed in the hybrids of gcxds. cluster analysis based on pca using the f irst three components, namely pc-i, pc-ii and pc-iii, grouped the 57 hibiscus hybrid progenies into f ive different clusters. the dendrogram showed a highly branched structure, suggesting the high degree of variability among the different hibiscus hybrid progenies studied. this high variability suggests that there is a wide window for selection of unique genotypes in the different hybrid progenies evaluated both by pca and cluster analysis. this information can be used as a basis for selecting specif ic hybrid progenies for the development into varieties. in addition, for future studies of similar type of research, it is recommended that further quantitative characterization should be performed using morphometric analysis. morphological characterization, evaluation and selection of hibiscus 78 acknowledgements the hibiscus breeding programme is supported by the off ice of the former uplb chancellor luis rey i. velasco, and the crop science cluster-institute of plant breeding, college of agriculture and food science, university of the philippines los baños. this research paper was based from the project “mass production and propagation of the hibiscus rosa-sinensis ‘st. bridget college’ for beautif ication purposes” funded by st. bridget college, batangas city (january 2012-june 2013).the authors would like to thank mr. marcelino t. gregorio, mr. jessie v. silverio, mr. renato pabalate, ms. maria fe h. cayaban and ms. juliana a . balogo for the assistance they provided to this study. references [ahs] australian hibiscus society. 2004. hibiscus growing guide. 2nd ed. queensland, australia: the australian hibiscus society inc. p. 40. [ahs] australian hibiscus society. 2007. hibiscus growing guide. 4thed. queensland, australia: the australian hibiscus society inc. p. 42. brown js. 1991. principal component and cluster analyses of cotton cultivar variability across the us cotton belt. crop science. 31(4):915-922. brown j, caligari ds. 2008. an introduction to plant breeding. oxford: blackwell publishing ltd. p. 209. cena rl. 1995. classif ication of cacao germplasm using morphological characters and isozyme analysis [disser tation]. los baños, laguna: university of the philippines los baños. dayap ft. 2000. morphological and isozyme characterization of jackfruit (artocarpus heterophyll u s lam.) collections [disser tation]. los baños, laguna: university of the philippines los baños. p. 118. edmonds jm. 1991. the distribution of hibiscus l. section furcaria in tropical east a f r i c a . i n : s y s t e m a t i c a n d e co g eo g r a p h i c s t u d i e s o n c r o p g e n e p o o l s 6 . ro m e : international board for plant genetic resources. p. 60. everitt bs, landau s, leese m, stahl d. 2011. cluster analysis. 5th ed. oxford: john wiley and sons. p. 348. howie j. 1980. hibiscusqueen of the flowers. 1st ed. brisbane, australia. ines mbc, magdalita pm, dela viña cd, dela cruz jr fs, villegas vn. 2009.randomly amplif ied polymorphic dna (rapd) analysis to reveal genetic relationships among pineapple (ananas comosus (l.) merr.) genotypes. philippine journal of crop science. 34(3):1-10. e.m.n. cabarrubias et al. 79 [irri] international rice research institute. 2014. statistical tool for agricultural research software. los baños, laguna: international rice research institute. jackson ej. 1991. a user’s guide to principal components. new york: john wiley and sons, inc. p. 563. jacob b, guennebaud g. [internet]. 2016. eigen is a c++ template library for linear algebra: matrices, vectors, numerical solvers, and related algorithms. [updated 2017 j u n 1 6 ; c i t e d 2 0 1 7 j u n 2 3 ] . a v a i l a b l e f r o m : h t t p : / / e i g e n . t u x f a m i l y. o r g / index.php?title=main_page. jiang gl. 2013. molecular markers and marker-assisted breeding in plants. in: anderson sb, editor. plant breeding from laboratories to fields. croatia: intech. p. 45-83. magdalita pm, gonzales-lee vrc, pimentel rb. 2009. hibiscus hybrids oblation series: a tribute to outstanding university of the philippines alumnae for the university centennial year. philippine journal of crop science. 34(1):113-118. magdalita pm, pimentel rb. 2010a. hibiscus hybrids and philippines’ women achievers. n a t i o n a l ac a d e m y of s c i e n ce a n d tec h n o l o g y p h i l i p p i n e s . ta g u i g , m e t r o m a n i l a : national academy of science and technology philippines. p. 55. magdalita pm, pimentel rb. 2010b. the philippine hibiscus hybrids. los baños, laguna: crop science cluster-institute of plant breeding (csc-ipb), college of agriculture, university of the philippines los banos (uplb). p. 71. magdalita pm, gonzales-lee vrc, pimentel rb. 2011. development and hor ticultural characteristics of hibiscus hybrids “ women in public service series”. philippine journal of crop science. 36(2):56-62. magdalita pm, pimentel rb. 2013. development of hibiscus hybrids ‘ women in public service series ii’ and propagation studies on hibiscus rosa-sinensis ‘cynthia a . villar’. philippine science letters. 6(1):39-55. m a g d a l i t a p m , c a y a b a n m f h , g r e g o r i o m t, s i l v e r i o j v. 2 0 1 6 . d e v e l o p m e n t a n d characterization of nine new hibiscus hybrids. philippine journal of crop science. 41(2):31-45. mcmullen ck. 1999. flowering plants of the galápagos. new york: cornell university press. p. 370. m w a s i a g i j i , yu c w, p h o l o g o l o t, wa i t h a k a a , k a m a l h a e a n d o c h o l a j r. 2 0 1 4 . characterization of the kenyan hibiscus sabdariffa l. (roselle) bast f ibre. fibres and textiles in eastern europe. 3(105):31-34. pimentel rb. 1999. new cultivars and germplasm hibiscus (hibiscus rosa-sinensis l.) centennial series. philippine agricultural scientist. 82(2):238-244. [rhs] royal hor ticultural society of london. 2007. rhs colour chart . 5th ed. unpublished standard reference for colours. london: flower council of holland. morphological characterization, evaluation and selection of hibiscus 80 s a n p a s c u a l a o . 2 0 1 5 . p h e n o t y p i c e v a l u a t i o n a n d p r o p a g a t i o n o f h i b i s c u s h y b r i d s (hibiscus rosa-sinensis l.) [bs thesis]. los baños, laguna: crop science cluster, college of agriculture, university of the philippines los baños. p. 65. simpson mg. 2006. plant systematics. netherlands: elsevier inc. p. 579. sneath pha , sokal rr. 1973.numerical taxonomy. california: w.h. freeman. stuessy tf. 2009. plant taxonomy: the systematic evaluation of comparative data. new york: columbia university press. p. 568. valdoz jc, magdalita pm, absulio wl, sotto rc. 2017. morpho-anatomical characters a n d e t h y l e n e p r o d u c t i o n i n h i b i s c u s r o s a s i n e n s i s l i n r e l a t i o n t o t w o d a y f l o r a l retention. philippine agricultural scientist. 100(2):168-177. _____________ elena may n. cabarrubias holds a bachelor of science in agriculture (agronomy) from the university of philippines los baños. she is a former university research associate at the institute of plant breeding and at present she is a research assistant at the ecosystems research and development bureau-department of environment and natural resources. she is an active member of the up painters’ club where she participates in various art exhibitions. pabl ito m. magdal ita <pabsmagdalita@gmail.com> holds a phd degree in agricultural science (plant breeding and biotechnology) and a post-doctoral training (plant genetic engineering) from the university of queensland in brisbane, australia. he is a professor i of plant breeding and plant biotechnology and up scientist i at the institute of crop science, and project leader of fruit (papaya, banana, avocado, soursop, chico, rambutan, pummelo, etc.) and ornamental (hibiscus, croton, bougainvilla, sampaguita, ixora, etc.) breeding at the institute of plant breeding, college of agriculture and food science, university of the philippines los baños. he was an outstanding young scientist (plant breeding) of the national academy of science and technology in the year 2000, the fcssp achievement award for research in 2011 and gawad saka outstanding agricultural scientist-finalist in 2011, among others. norma g. medina holds a master of science degree from the university of philippines los baños. she is an assistant professor 2 of ornamental horticulture and division head at the crop production and management division at the institute of crop science, college of agriculture and food science, university of the philippines los baños. also, she is involved as project staff in various projects such as edible landscaping, utilization of landscape organic waste materials and commercialization of indigenous and endemic plants. e.m.n. cabarrubias et al. 81 antonio g. lalusin holds a phd degree in bioresource engineering from the university of tsukuba in japan. he is an associate professor 5 in conventional and molecular plant breeding at the institute of crop science, college of agriculture and food science, university of the philippines los baños. also, he is a project leader in the varietal improvement of feeds and industrial crops (cassava, abaca, sugarcane, coconut and sweet potato) using conventional and biotechnology-assisted techniques. he was a recipient of the outstanding young scientist award (genetics and plant breeding) of the national academy of science and technology in 2009 and, the fcssp achievement award for teaching in 2007, among others. 01_device focal benthic mollusks 1 focal benthic mollusks (mollusca: bivalvia and gastropoda) of selected sites in tubbataha reef national marine park, palawan, philippines roger g. dolorosa* and sabine schoppe western philippine university-puerto princesa campus, santa monica heights, 5300 puerto princesa city, palawan, philippines tel. no.: (048) 434-398; telfax: (048) 433-4367; e-mail: rogerdolorosa@yahoo.com date received: march 2, 2006; date accepted: july 6, 2006 abstract science diliman (july-december 2005) 17:2, 1-10 the study was conducted at tubbataha reef national marine park from may 6-11, 2005. seven preestablished stations with survey sites at 5 and 10 m depth and one intertidal area were assessed using 150 m permanent belt transects. focal benthic mollusks found one meter to the left and right of transects were identified and counted. a total of 19 species belonging to eight families were recorded, of which 15 species are univalves. in the intertidal area a total of 12 species were noted, 13 species at the shallow (5 m) and five species at deeper (10 m) areas. species belonging to the family tridacnidae and trochidae were the most abundant. among the subtidal stations, the highest number of individuals was noted at a shallow reef flat (station vi). in terms of density, the intertidal area had the highest (213,310 ind. km-2) followed by the shallow (72,870 ind. km-2) and the deep with 5,720 ind. km-2. the densities of tridacna crocea (133,330 ind. km-2) and hippopus hippopus (3,330 ind. km-2) at the intertidal area were found to be higher than in most other survey sites in palawan but previous density records at the park indicate a stiff decline. on the contrary, the first record on the density of t. squamosa (950 ind. km-2) at the park is much lower compared to that from other parts of palawan. large and commercially valuable gastropods like, trochus niloticus, tectus maculatus and t. pyramis that are rarely encountered at the intertidal areas were abundant at the trnmp. other important species like tridacna gigas, charonia tritonis and cassis cornuta were not encountered at the study sites. to fully assess the abundance of focal mollusks, permanent transects should be established in the same seven sites but in shallow reef flat of about 2 m deep, in the lagoon and in the intertidal of north and south islets where species composition, density and growth could be monitored on an annual basis which could be used to evaluate the management effectiveness at the trnmp. key words: bivalves, focal species, gastropods, palawan, philippines, tubbataha reef *corresponding author dolorosa and schoppe 2 coral reefs are the most biologically diverse of shallow water marine ecosystems but are being degraded worldwide by human activities and climate warming (roberts et al., 2002). they are critically important for the ecosystem goods and services they provide to maritime tropical and subtropical nations (souter & linden, 2000). yet reefs are in serious decline; an estimated 30% are already severely damaged, and close to 60% may be lost by 2030 (wilkinson, 2000). there are no pristine reefs left (jackson et al., 2001). local successes at protecting coral reefs over the past 30 years have failed to reverse regional scale declines, and global management of reefs must undergo a radical change in emphasis and implementation if it is to make a real difference (hughes et al., 2003). in the 1980s, the previously pristine and unique tubbataha reef of palawan started to show evidence of serious destruction and over-exploitation that has continued even after its declaration as a natural marine park in 1988 (arquiza & white, 1999). with the creation of the tubbataha management foundation in 1990, the declaration as world heritage site in 1993, and finally the establishment of the protected area management board in 1999, conditions did improve under the impact of resource management strategies with national and international support. the current tubbataha reef national marine park (trnmp) management has the goal to preserve the globally significant biological diversity and ecological processes of tubbataha and to manage them and the surrounding areas on a sustainable basis. while regular monitoring of coral reef conditions and associated fish fauna, has been conducted for the past eight years (sabater et al., 2004), the assessment of focal species was only in november 2003 during the trnmp management effectiveness workshop recognized as a gap. focal species are organisms of ecological importance and/or human value that are of priority interest for management through the marine protected area. aside from top predators, benthic mollusks were identified as important indicators of habitat health and proper resource management and protection. since species abundance of focal species is one of the most widely used biological ‘success’ measures of management effectiveness (pomeroy et al., 2003) the present study intended for introduction fig. 1. map showing tubbataha reef national marine park. trnmp is part of the municipality of cagayancillo, an oceanic island in the philippines (inset). (source: sabater et al., 2004). tubbataha reef national marine park cagayancillo puerto princesa province of palawan, philippines sulu sea focal benthic mollusks 3 fig. 2. location of the seven monitoring stations (triangles) in tubbataha reef national marine park. (source, sabater et al., 2004). i ii iii iv v vi vii dolorosa and schoppe 4 the first time to determine the species composition and density of focal benthic mollusk species in trnmp. materials and methods tubbataha reef is located in the sulu sea, 150 km southeast of puerto princesa city, palawan. it is under the political boundaries of the municipality of cagayancillo, located about 80 km northeast of tubbataha (fig. 1). seven sites were surveyed within the reef area. geographic locations of the sites are provided in sabater et al. (2004). four sites (1-4) are in the north atoll, and three sites (5-7) in the south atoll (fig. 2). site selection around the atoll is based on the various coral reef structures found representative of the monsoonal exposure effects. the reef structure ranges from monospecific stands of acropora brueggemanni and sand-reef patch areas located at the sheltered reef areas to spur-and-groove habitats with mixed coral assemblage dominated by robust corals located at the exposed areas (sabater et al., 2004). each site has two permanent transects of 150 m length; one at about 5 m and one at about 10 m depth. these transects have been established since 1997 for the regular monitoring of benthic communities (especially corals) and reef-associated fishes. shallow transects are typically along slightly sloping parts of the reef characterized by branching corals while deep transects are located along steep slopes (walls). in the present study, focal bivalve and gastropod species were assessed alongside these transects. transect width was limited to 1-m belts right and left of each transect. all except two transects were assessed during day time. the number of species abundance of each focal species such as flagship, indicator (e.g. triton shell), keystone, target (commercially valuable) and threatened species (according to iucn 2004) were assessed. in addition to the shallow and deep transects, the intertidal area near stn. v at the south islet was assessed. the intertidal was characterized by few live corals at the seaward margin followed by massive coral rocks and rubble intercepted by sandy-muddy substratum. a 150 m transect line was laid during low tide from the exposed reef margin towards the light house. the shells were identified using the following references: carpenter & niem (1998), knop (1996), allen & steen (1994), sabelli (1991), garcia (1986), springsteen & leobrera (1986) and oliver (1975). diversity and abundance data were analyzed per site, with intertidal transects separated from shallow and deep transects. density per square meter was obtained by dividing the number of individuals recorded with the area (300 m2) surveyed per transect, then it was extrapolated into number of individuals per square kilometers (ind. km-2). results species composition a total of 19 species belonging to eight families were recorded, of which 15 species are univalves. of these, tridacnidae and conidae had the highest number of species comprising 21 % for each family. this was followed by trochidae and cypraeacide (both with 16 %). lowest percent share belong to families fasciolariidae, mitridae and turbinidae (fig. 3). density a total of 12 focal species belonging to six families were recorded at the intertidal area at the south islet. bivalves were only represented by the family tridacnidae. among all species tridacna crocea was the most abundant and dense (133,330 ind. km-2) followed by conus lividus (20,000 ind. km-2) and trochus niloticus (166,670 ind. km-2). the calculated density of focal mollusks within that intertidal area was 213,310 ind. km-2 (table 1). at the shallow area, a total of 13 focal species under eight families were recorded. gastropods dominated with seven families and 10 species. in terms of abundance, t. crocea ranked first with an average of 30,480 ind. km-2, followed by tectus pyramis (15,710 ind. km-2), and t. maxima (12,860 ind. km-2) (table 1). tridacna squamosa was so rare (950 ind. km-2) that it was only encountered in stations iv and vi. the calculated density of all focal mollusks in shallow areas was 72,870 ind. km-2. highest abundance was recorded at stn. vi which is a reef flat dominated by massive focal benthic mollusks 5 tridacnidae 21% trochidae 16% turbinidae 5% strombidae 11% cypraeacidae 16% mitridae 5% fasciolariidae 5% conidae 21% fig. 3. percent species composition of focal benthic mollusks at trnmp. family species intertidal* shallow (5m) deep (10m) tridacnidae hippopus hippopus 33,330 tridacna crocea 133,330 30,480 tridacna maxima 12,860 950 tridacna squamosa 950 total no. of bivalve species 2 3 1 trochidae tectus pyramis 3,330 15,710 1,910 trochus maculatus 3,810 1,430 trochus niloticus 16,670 3,330 turbinidae turbo chrysostomus 3,330 1,430 strombidae lambis chiragra chiragra 480 lambis sp. (juvenile) 3,330 cypraeacidae cypraea annulus 13,330 cypraea moneta 6,670 cypraea tigris 1,430 950 fasciolariidae pleuroploca trapezium 480 conidae conus lividus 20,000 conus miles cf 3,330 950 conus sp. 3,330 480 conus virgo 3,330 480 mitridae mitra papalis 480 total no. of gastropod species 10 10 4 total density 213,310 72,870 5,720 *one transect only table 1. density (ind. km-2) of focal benthic mollusks at trnmp. dolorosa and schoppe 6 corals. other stations were covered with branching corals or were located at the drop off so that mollusk abundance was relatively lower compared to that in stn. vi and the intertidal area. broken shells of trochus and turbo were noted in many stations indicating the presence of mollusk predators. large (<60 cm long) holothurians (bohadschia argus, pearsonothuria graeffei, holothuria sp., thelenota ananas, stichopus horrens, s. chloronotus) were common along the shallow areas in stn. iii. very few focal mollusks were noted in the deep transects. they were represented by five species under four families. in terms of abundance, t. pyramis had a calculated density of 1,910 ind. km-2, followed by t. maculatus (1,430 ind. km-2). tridacna maxima and cypraea tigris both had a density of 950 ind. km-2 (table 1). the density of all focal mollusks at 10 m depth was only 5720 ind. km-2. empty and or broken shells of trochus and turbo were noted in some stations. generally, mollusks in trnmp were more abundant in shallow waters, and had the highest density along the intertidal transect (fig. 4). discussion among the 19 focal species, only the giant clams are listed under the iucn red list of threatened species (iucn, 2004). tridacna squamosa, t. maxima and h. hippopus are under lower risk/conservation dependent (lr/cd ver 2.3 1994) while t. crocea falls under the lower risk/least concern (lr/lc ver 2.3 1994) category. however, under the fisheries administrative order no. 208 series of 2001, all seven species of giant clams are listed as endangered. trochus niloticus is also listed as threatened (bfar, 2006). for a world heritage site like trnmp, the recorded number of threatened and endangered mollusks is quite low. the observed pattern of increasing density of focal mollusks from subtidal to intertidal sites is substrateand depth-related. deep transects were either along steep reef walls or on reef slopes dominated by branching corals. in most of the sites surveyed, the reef starts sloping at about 3 m, hence most shallow transects were on sloping areas, too often covered with branching coral formations. only shallow reef platforms dominated by massive corals (such as stn. vi,) as well 0 50000 100000 150000 200000 250000 i ii iii iv v vi vii s. islet stations in di vi du al s/ sq . k m shallow deep intertidal fig. 4. density (ind. km-2) of focal mollusks in the different stations. focal benthic mollusks 7 as intertidal areas with coral rocks and partly sandmuddy substrate (such as the one in south islet) provide the habitat preferred by the mollusks under study. this was especially obvious for t. crocea which needs hard substrate to bore into and sunlight for photosynthesis. the niche of t. crocea, living embedded in massive corals, had been observed by various authors (see alcala, 1986; juinio et al., 1988; bolen, 2005), and explains its high density in the intertidal and shallow reef flat areas in this study. yamaguchi (1996) also found t. crocea and t. maxima to be common in coral rocks around seagrass beds in the north reef of tubbataha. in terms of t. crocea density, trnmp ranks second after survey sites in northern palawan (alcala, 1986), followed by snake island, honda bay (bolen, 2005), binaluan bay, taytay (sayson, 2003) and roxas (condesa, 2005) (table 2). density patterns among those sites are not expected to be related to resource management and protection of giant clams since t. crocea is little if at all exploited. high density of t. crocea seems solely substratum related, reflecting the availability of coral heads into which the species burrows. in the intertidal areas of roxas surveyed by condesa (2005) e.g. such coral heads were almost completely absent, resulting in only 7,000 and 15,000 ind. km-2 (table 2). although density of tridacnids, especially t. crocea in trnmp was found to be higher than in most other survey sites in palawan, previous density records of t. crocea and t. maxima from trnmp in 1993-1995 indicate a stiff decline (calumpong & cadiz, 1993; ozoa, 1995). tridacna crocea for example decreased from 2,200,000 ind. km-2 in 1993 (calumpong & cadiz, 1993) to 1,000,000 in 1995 (ozoa, 1995), and to 133,330 in 2005 (table 2). the much lower density at intertidal area at south islet, however, is not enough to conclude that there is a continuous decline of tridacnids, considering that the present data were only taken from a single transect. hence, permanent mollusk transects should be established in the same seven sites but in shallow reef flats of about 2 m deep. in addition, permanent transects should be established in the lagoon and in the intertidal of north and south islets where species composition, density and growth could be monitored along these transects on an annual basis. unlike t. crocea which was still relatively abundant, the other giant clam species, especially non-boring and heavily exploited giant clam were rather rare. very low numbers of t. squamosa and h. hippopus were noted during the present study, as well as earlier by yamaguchi (1996). although yamaguchi (1996) reports dead shells of h. hippopus in seagrass beds, he believes that this was not necessarily attributed to harvesting by fishermen. in papua new guinea, however, fishermen, as well as poachers, collect giant clams by extracting the meat leaving the shell behind (kinch, 2002). white et al. (2003) affirms that large scale extraction of precious and common shells and giant clams has been done in the past in the tubbataha reef. and still, recently, hundreds of giant clams were confiscated from chinese fishermen who poached in trnmp. records by ticke (2002) state 200 confiscated t. gigas while benavent-villena & pido (2004) are referring to 30 sacks of dried giant clams confiscated that time. although no t. gigas was encountered during the present study, tubbataha park rangers mentioned that there are still some t. gigas in the park. the current status of the giant clams in trnmp might lead to similar conditions as in the mu ko surin marine national park in thailand where t. crocea is abundant, t. maxima is rare, t. squamosa is nearing extinction, and t. gigas and h. hippopus are considered extinct (talaythai 2001). according to kinch (2002) giant clams are highly vulnerable to stock depletion, for the stocks will become non-sustaining when densities fall below the critical mass. aside from that, the low settlement, survival and growth of t. squamosa on live coral substrate (calumpong et al., 2003) would take hundreds of years for the re-establishment of a stock particularly in isolated areas (munro, 1993 in kinch, 2002). these may be the reasons that despite protecting trnmp for more than a decade, populations of commercially valuable giant clam species at the seven established stations remain low. this condition requires the establishment of specific sites for non-boring tridacnid species to ensure fertilization and larval recruitment. the stability or an increase in the population of the focal benthic mollusks especially the following giant clam species: t. gigas, t. squamosa and h. hippopus over the next few years would indicate success of the conservation measures employed by the trnmp. dolorosa and schoppe 8 other large and commercially valuable species such as the gastropods, trochus niloticus, tectus maculatus and t. pyramis which are nowadays rarely encountered in intertidal areas were abundant at the trnmp. intertidal areas near coastal residents of palawan are usually overexploited as density data indicate. condesa (2005) for example recorded a mean density of only 1,000 ind. km-2 of t. niloticus in green island, roxas. still lower is the density of the species in the cartier reef, australia where it only ranged from 133-333 ind. km-2 (smith et al., 2002). it is expected that tubbataha’s previously overexploited gastropods stocks have recovered under the past years of protection. since gastropods mate, fertilization and therewith recruitment and increase of stocks are easier achieved compared to broadcast spawning in bivalve species, whose gametes are easily dispersed in water by currents. the very low number of other commercially important gastropods like lambis spp. and the absence of charonia tritonis tritonis and cassis cornuta both in shallow and deeper areas may again be attributed to the substrata of the survey sites. cassis cornuta for example is common on sandy substrate between coral formations (carpenter & niem 1998) which was rarely encountered along the surveyed transects. over exploitation in previous years might also have led to the absence of the species in the survey sites, which were easily accessible to poachers before. park rangers claimed that c. cornuta and c. tritonis are still present in the lagoon, an area that was always difficult to access by poachers. therefore, it is recommend that in order to better assess the species composition and density of focal species in the trnmp, more survey transects per site need to be established at the intertidal and shallow reef flats. acknowledgments this study was funded by conservation international palawan as part of the biophysical monitoring efforts of tubbataha management office and tubbataha protected area management board. thanks are also due to tubbataha management office and the crew of the m/bca minerva of the wwf and to marla chassels of conservation international. the comments and suggestions of the two reviewers are greatly acknowledged. table 2. density (ind. km-2) of commercially exploited benthic mollusks at trnmp in comparison with other studies. palawan, philippines tridacna crocea 15,000 7,000 120,000 55,833 18,069 328,619 1,400,000 2,200,000 1,000,000 133,330 30,480 t. maxima 100,000 25,509 2,667 500,000 200,000 333,300 12,860 t. squamosa 20,000 3,050 1,240 2,714 950 t. gigas hippopus hippopus 300 1,110 111,100 3,330 all giant clam spp. 15,000 7,000 240,300 59,993 44,818 344,000 1,900,000 2,400,000 1,444,400 136,660 44,290 author & location condesa (2005) bolen (2005) sayson (2003) alcala (1986) calumpong and cadiz (1993) ozoa (1995) this study (2005) green island, roxas, intertidal caramay, roxas, intertidal snake island, ppc, 1-5m binaluan bay, taytay, 1.5-3.5m cagayan island, 0.5-5m northern palawan, 0.5-5m trnmp north islet, 6-10 m trnmp south islet, intertidal trnmp intertidal trnmp intertidal 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(ed.) 2000. status of coral reefs of the world. australian institute of marine science, townsville, australia: 363 pp. yamaguchi, m., 1996. shallow water molluscan assemblages of the tubbataha reef, republic of the philippines. in: a report of the project for resources survey and conservation of tubbataha reefs national marine park. denr and marine parks center of japan, p. 61-79. university of the philippines diliman office of the vice chancellor for research and development call for papers humanities diliman, science diliman, and social science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> journal cover images courtesy of (l-r) vargas museum & biodiversity research laboratory (agno, pangasinan) 9guidelines.pmd 79 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) 04_bacillus with adjusted p33_11 nov comparison of conventional plate 31 abstract science diliman (january-june 2005) 17:1, 31-36 comparison of conventional plate assays with dna-based screening protocols for protease and cellulase production from putative bacillus isolates diana rose e. rañoa, candice y. lumibao, jennifer l. roxas, boris b. san luis, and cynthia t. hedreyda* national institute of molecular biology and biotechnology college of science, university of the philippines 1101 diliman, quezon city, philippines tel. no.: (632)981-8500 local 3953; fax no.: (632)927-7516 e-mail: hedreyda@laguna.net date received: april 14, 2003; date accepted: june 29, 2005 *corresponding author putative bacillus isolates obtained from mud and soil samples of mt. makiling, los baños, laguna were screened for the production of either cellulase or alkaline protease using cellulose and casein plate assays, respectively. five out of eight isolates assayed for cellulase activity tested positive. dna from these eight samples were extracted using the modified rose method for slot blot hybridization with cellulase gene probe. out of the five samples positive for the cellulose plate assay, only three exhibited hybridization results. dna from the eight isolates were used as templates for pcr amplification using primers specific for b. subtilis cellulase gene. out of the eight isolates, two produced the expected 350-bp cellulase amplicon. another set of ten putative bacillus isolates were screened for the production of alkaline protease using casein plate assay. four isolates exhibited protease activity. genomic dna extracted from ten isolates were subjected to slot blot hybridization using a fragment of alkaline protease gene from b. pumilus. five isolates, of which four previously tested positive for protease activity, showed positive hybridization signals. pcr analysis using primers designed based on alkaline protease gene of b. pumilus showed that only one isolate produced the expected 320-bp pcr amplicon. these data suggest that biochemical plate assays may be advantageous for isolating bacteria that produce different types of cellulase and alkaline protease, while dna-based methods are useful in detecting specific target genes but may therefore miss out on novel or variant enzymes. keywords: bacillus, alkaline protease, cellulase, slot blot hybridization, polymerase chain reaction (pcr) introduction various bacillus species have been found to excrete extracellular enzymes, which are very important for the degradation and hydrolysis of several biomass polymers (rao et al., 1998; ferrari et al., 1993; pero & sloma, 1993). the two major enzymes that were included in this study are alkaline protease and cellulase; both of which are essential components of a wide range of products in the detergent, food, and chemical industry (ferrari et al., 1993). rañoa et al. 32 identification of potential enzyme-producing bacteria is an important step in searching for useful and novel enzymes. traditional biochemical methods of screening for enzymes involve growing the microorganisms on an agar plate containing the appropriate enzyme substrate. on the other hand, dna-based methods, such as dna hybridization and polymerase chain reaction (pcr), are based on the detection of the particular gene coding for the target enzyme. this study aimed to compare the conventional plate assay with pcr and dna hybridization as screening methods for protease and cellulose producing bacteria. specifically, putative bacillus isolates were screened for the production of alkaline protease and cellulase using plate assays. the genomic dna of these bacterial isolates was then tested for the presence of genes homologous to bacillus alkaline protease and cellulase genes using polymerase chain reaction (pcr) and slot blot hybridization. materials and methods bacterial samples eighteen putative bacillus isolates were obtained from soil and mud samples of mt. makiling, los baños, laguna. those obtained from soil were designated as 1sa-23, 3sa-9, 1sm-9, 1sm-18, 3sa-13, 1sm-8, 4sm-8, and 5sa-17, while those from mud were 3ma-11, 6ma-6, 6mm-4, 7mm-30, 4ma-13, 7mm12, 3ma-13, 4mm-33, 5ma-14, and 7mm-17. the isolates were routinely grown in nutrient agar slants at 37oc overnight and were stored at 4oc for further use. traditional biochemical assay bacillus isolates were screened for their ability to excrete either alkaline protease and cellulase using traditional plate assay techniques. the casein plate assay was used as test for the presence of alkaline protease. the isolates were line-inoculated onto a luria-bertani (lb)–agar medium containing 1% casein and incubated overnight at 37oc. the cellulase assay used the modified procedure of yeoh et al. (1985) for screening cellulolytic fungi. isolates were streaked on plates containing 0.1% (w/v) nano3, 0.5% (w/v) nah2po4, 0.5% (w/v) kcl, 0.1% (w/v) mgso4, 0.14% (w/v) yeast extract, 0.1% (w/v) k2hpo4, 0.3% (w/v) carboxymethyl cellulose and 2% (w/v) agar. after incubation at 37oc overnight, the plates were flooded with deep blood-red cellulose stain (0.8% i2 crystals, 0.8% (w/v) ki, 8 mm kcl) for 5 to 10 minutes or until clearing zones appeared around the periphery of bacterial colony that excreted cellulase. dna extraction total dna was extracted from overnight nutrient-broth cultures of the isolates following a procedure based on the rose protocol of steiner et al. (1995). bacterial cells were pelleted at 20,800 rcf for one minute (eppendorf centrifuge 5415 c), washed with 1 ml 0.85% nacl, and treated with 300 µl modified rose buffer [10 mm tris-hcl (ph 8.0), 100 mm edta (ph 8.0), 1% (w/v) sarkosyl, and 1% (w/v) polyvinylpolypyrrolidone (pvpp)]. samples were incubated at 90oc for 20 minutes in a water bath. tubes were then placed on ice or stored at 4oc. the supernatant containing the genomic dna, was collected after centrifugation at 10,600 rcf for 5 minutes. pcr amplification the genomic dna of all bacterial isolates were used as template for pcr amplification using primers specific for alkaline protease and cellulase genes, respectively. rose genomic dna extracts were diluted 1:54 with sterile water prior to pcr amplification. pcr was performed using the perkin-elmer geneamp pcr system 2400 thermocycler (perkin elmer corp., singapore) with a reaction volume of 20 µl containing 1x pcr buffer, 1.0 mm mgcl2, 0.2 mm dntps, 0.5 µm each of the forward and reverse primers, 0.025 u/ µl taq dna polymerase, and 1 µl (50-100 ng) diluted dna extract. comparison of conventional plate 33 pcr conditions for alkaline protease gene amplification include an initial denaturation at 94oc for 5 minutes; 40 cycles of denaturation at 94°c for 1 minute, annealing at 53°c for 40 seconds, extension at 72oc for 45 seconds; and a final extension at 72°c for 7 minutes. the same conditions were used for the amplification of cellulase gene, except for the annealing temperature, which was set at 51oc for 40 seconds. dna hybridization template dna preparation dna extracted from the selected bacterial isolates were boiled for 10 minutes, and immediately placed on ice for 1-2 minutes. a volume of 50 µl of the dna template was applied onto a nylon membrane presoaked in 1x tris-edta (te) buffer using a pr 648 slot blot filtration manifold (amersham pharmacia biotech; california, usa). the membrane blotted with dna was soaked in a denaturation solution 1 (0.5m naoh, 1.5 m nacl) for 10 minutes and then transferred into solution 2 (0.1m tris ph 7.5, 1.5m nacl) for another 10 minutes. the blotted membrane was airdried and subjected to uv cross-linking using gs gene linker (bio-rad laboratories, usa) before prehybridization with a solution containing 5x ssc, 1% blocking reagent, 0.1% sarkosyl, 0.02% sds) at 68oc for one hour. hybridization with labeled probe pcr amplification products containing fragments of either b. pumilus alkaline protease gene or b. subtilis cellulase gene, were labeled with digoxigenin, following the protocol described by roche molecular biochemicals (mannheim, germany) to produce the probe. the labeled gene probes were allowed to hybridize with the dna templates on the membrane at 68oc overnight. detection of hybridization after hybridization and prior to detection, the membrane was washed twice in a solution containing 2x ssc and 0.1% sds at room temperature for 5 minutes, with shaking. the membrane was further washed twice in a solution containing 0.1x ssc and 0.1% sds at 65oc for 5 minutes. hybridization signals were detected using an enzyme-linked immunoassay with anti-dig-ap (digoxigenin-alkaline phosphatase) fab fragments and nbt-bcip (nitroblue tetrazolium chloride-5-bromo-4-chloro indoyl phosphate toluidine salt) substrate following the manufacturer’s procedure (roche molecular biochemicals; mannheim, germany). results and discussion biochemical assay screening for either cellulase or alkaline protease production of the 18 putative bacillus isolates was done by cellulose and casein plate assay, respectively. bacterial isolates with positive cellulase activity are detected by the formation of a clearing zone around the bacterial colony after the addition of a cellulose stain onto the plate. these isolates are said to utilize cellulose as carbon source, and convert the substrate into cellobiose and glucose sub-units. on the other hand, casein assay is a general method used to screen for microorganisms that excrete alkaline proteases in the surrounding medium. incorporating casein as the sole carbon source into an agar medium will detect alkaline protease-secreting organisms by the presence of a clear halo of hydrolyzed casein around the growing colony. among the eight putative bacillus isolates used for cellulose plate assay, four were obtained from soil (1sa-23, 1sm-9, 1sm-18, and 3sa-9) and the other four were taken from mud (7mm-30, 3ma-11, 6ma6, and 6mm-4). out of these eight samples, two isolates from soil and three isolates from mud were shown to have cellulase activity (table 1). for casein plate assay, only four out of 10 bacterial samples were positive for alkaline protease activity. two out of the four putative bacillus isolates from soil exhibited alkaline protease activity, while only two of the six screened soil isolates were found to secrete alkaline protease. rañoa et al. 34 dna hybridization all hybridization assays require careful probe selection, optimization of hybridization conditions, and strict attention to quality control to achieve the sensitivity and specificity required for routine screening. slot blot hybridization assays permit qualitative detection of the presence of a cellulase and alkaline protease gene in a dna template using specific probes prepared from bacillus species. optimized hybridization parameters that gave a signal for the positive control (dna template from bacillus subtilis) and no hybridization for the negative control (dna template from bacillus thurigiensis) was used in the screening for target genes from unknown isolates. out of the five samples positive for the cellulose plate assay, only three exhibited hybridization with the cellulase gene probe prepared from b. subtilis (fig. 1). however, two other samples that were negative for the plate assay gave positive hybridization results. the possible explanations for this are: (1) cellulase gene is present but the expressed protein is not secreted; and (2) a gene with a different function is homologous to b. subtilis cellulase gene. on the other hand, all isolates positive for the casein plate assay also showed positive results in slot blot hybridization using a fragment of an alkaline protease gene from b. pumilus as probe (fig. 2). this suggests that the isolates possess an alkaline protease gene homologue. however, another isolate negative in the casein plate assay also demonstrated positive hybridization signal, suggesting that enzyme may be produced but not excreted to the medium. polymerase chain reaction pcr involves multiple cycles of template denaturation, primer annealing, and primer elongation to amplify a specific gene that is present in the template dna. table 1. summary of protein and dna-based screening for cellulase performed on eight initial bacterial isolates. 7mm-30 1sa-23 6mm-4 3ma-11 1sm-18 1sm-9 6ma-6 3sa-9 + + + + + + + + + + + + not tested sample cellulose assay slot blot hybridization pcr a b 5 61 2 3 4 fig. 1. slot blot hybridization of dna templates prepared using modified rose method with a probe prepared from a fragment of b. subtilis cellulase gene. lanes a1 to a5 and b2 to b5 contain dna extracted from unknown bacterial isolates: 7mm30, 1sa-23, 3sa-9, 1sm-9, 6mm-4, 3ma-11, 6ma-6, and 1sm-18, respectively. lanes a6 and b6 contain the positive (b. subtilis) and negative (b. thuringiensis) genomic dna controls, respectively. lane b5 contains cellulase gene fragment from b. subtilis, while lane b1 contains only te buffer. a b 5 61 2 3 4 7 fig. 2. slot blot hybridization of dna templates prepared using modified rose method with a probe prepared from a fragment of b. pumilus alkaline protease gene. lanes a1 to a5 and b1 to b5 contain dna extracted from unknown bacterial isolates: 7mm-12, 4sm-8, 4mm-13, 1sm-8; 7mm-12, 3sa-13, 5sa17, 3ma-13, 4mm-33, 5ma-14, respectively. lanes a7 and b7 contain the positive (b. pumilus) and negative (b. thuringiensis) genomic dna controls, respectively. lane b6 contains alkaline protease gene fragment from b. pumilus, and lane a6 contains only te buffer. comparison of conventional plate 35 optimized parameters that produced the expected amplicon using a positive control (dna template of bacillus subtilis for cellulase and dna from bacillus pumillus for protesase) that carries the target gene, were used in pcr screening for target genes from unknown test isolates. using dna templates prepared by modified rose method, two isolates produced the expected 350 bp cellulase amplicon (fig. 3). on the other hand, only one out of four isolates positive for casein plate assay produced the expected 320 bp alkaline protease gene fragment (fig. 4). comparison of the different screening methods tables 1 and 2 show the summarized results for all screening methods for cellulase and alkaline protease, respectively. the number of samples that were positive for amplification of target gene was found to be less than those positive for dna hybridization. this suggests that pcr is a more specific method in detecting target genes. pcr is largely dependent on the annealing of primers designed from bacillus species. under stringent conditions and in the absence of a specific dna template sequence that is complementary to the primers used, no amplification of target genes will occur. however, it is possible that the samples may have homologous alkaline protease genes, and that the pcr primers were not able to anneal to their target sites due to some sequence variation. the considerably less number of bacterial isolates that were positive for both dna-based screening methods, as compared to the original number of samples positive for the plate assays, suggests that some isolates may possess cellulase and alkaline protease genes with significant sequence variation from those of the gene probes or primers which were based on bacillus enzymes. 5 61 2 3 4 7 2642 bp m m x y 350 bp 100 bp 8 9 fig. 3. amplified fragments of b. subtilis cellulase gene generated from dna templates extracted using modified rose. lanes 1-8: 7mm-30, 1sa-23, 6mm-4, 3ma-11, 1sm18, 1sm-9, 6ma-6, and 3sa-9, respectively. lane 9 contains amplified cellulase gene fragment of b. subtilis (m4-11). lane m contains the 100-bp dna ladder. lanes x and y contain the negative controls b. thuringiensis and pcr mix, respectively. 5 61 2 3 4 7 2642 bp m x 320 bp fig. 4. amplified fragments of b. pumilus alkaline protease gene generated from dna templates extracted using modified rose. lanes 1-6: 4mm-33, 5ma-14, 4sm-8, 5sa-17, 1sm8, and 7mm-17, respectively. lane 7 contains amplified alkaline protease gene fragment of b. pumilus (m3-8). lane m contains the 100-bp dna ladder (invitrogen life technologies). lane x contains the negative control (pcr mix). 3sa-13 4ma-13 7mm-12 3ma-13 1sm-8 4mm-33 5ma-14 4sm-8 5sa-17 7mm-17 + + + + + + + + + not tested not tested not tested not tested + sample cellulose assay slot blot hybridization pcr table 2. summary of protein and dna-based screening for alkaline protease performed on ten additional bacterial isolates. rañoa et al. 36 an ideal detection or screening system should be applicable to a vast majority of microbial strains, show good reproducibility over a long period of time, and yield consistent results. moreover, the technique should be simple, inexpensive, and require less labor. among the three screening procedures used, the traditional biochemical assay is the cheapest and easiest method to perform. it requires cheap and easily accessible reagents, and only basic microbiological and aseptic techniques are needed to culture microbial strains. however, biochemical assays do not specifically detect certain target enzymes. dna-based screening protocols may be more expensive and labor-intensive to perform as compared to biochemical assays, but they are more specific in detecting particular genes. slot blot hybridization can be used to screen large numbers of dna samples. a drawback for this method is the fact that several steps have to be performed before a result is obtained. moreover, it is difficult to distinguish crosshybridization of the probe to other genes since complementarity between the probe and the dna template need not be 100% homologous for a signal to be produced. pcr, on the other hand, is a very sensitive method. however, the presence of even minute amount of contaminating dna and inhibiting factors could affect pcr results. moreover, pcr relies on an optimized pcr parameters and a properly designed pair of primers. designing of primers specific for a particular gene requires prior knowledge of the gene’s sequence. as reflected on table 3, slot blot hybridization is more labor intensive, but it is relatively cheaper than pcr and can analyze a large number of samples in one batch, thereby making it more favorable for use in routine screening. references hedreyda, c.t., m.j. conejero, & b.b. san luis, 2002. dna-based detection of enzyme-producing bacterial isolates. terminal project report submitted to the university of the philippines, diliman, quezon city. ferrari, e., a. jarnagin, & b. schmidt, 1993. commercial production of extracellular enzymes. in sonenshein, a.l., j.a. hoch, & r. losick (eds.) bacillus and other grampositive bacteria: biochemistry, physiology, and molecular genetics. american society for miocrobiology, usa. 917937. pero, j. & a. sloma, 1993. proteases. in sonenshein, a.l., j.a. hoch, & r. losick (eds.) bacillus and other grampositive bacteria: biochemistry, physiology, and molecular genetics. american society for miocrobiology, usa. 939952. rao, m., a. tanksale, m. ghatge, & v. deshpande, 1998. molecular and biotechnological aspects of microbial proteases. microbiol. mol. biol. rev. 62: 597-635. steiner, j., c. poklemba, r. fjellstrom, & l. elliott, 1995. a rapid one-tube genomic dna extraction process for pcr and rapd analyses. nucleic acids res. 23: 2569-2570. yeoh, h., e. khew, & g. lim, 1985. a simple method for screening cellulolytic fungi. mycologia. 77: 161-162. table 3. comparison of pcr and slot blot hybridization in terms of cost effectivity and labor intensiveness. p c r (using perkin-elmer geneamp pcr system 2400 thermocycler) slot blot hybridization php 230.00 php 90.00 5 48 24 48 screening procedure cost per sample (inclusive of a positive and a negative control) total time of screening (hour) number of samples analyzed per batch sd-sample article a.i. mabilangan and others 15 science diliman (january-june 2013) 25:1, 15-28 introduction porous silicon is a two-phase composite material with a refractive index that can vary from air to silicon (si). porous silicon, among other porous materials, is nontoxic and is relatively more affordable to produce because of the abundance of silicon. porous silicon has been shown to produce photoluminescence despite its base material’s indirect bandgap (canham 1990), which paved the way for applications in optoelectronics (bisi and others 2000). its high surface-to-volume ratio and high reactivity to chemicals makes it a good candidate for gas (naderi and others 2012) and chemical sensors (ozdemir and others 2010). furthermore, its absorbance has fabrication and characterization of porous sil icon for photonic appl ications arvin i. mabilangan*, niel gabriel e. saplagio, eloise p. anguluan, neil irvin f. cabello, rhona ol ivia m. gonzales, armando s. somintac and arnel a. salvador university of the philippines diliman abstract porous silicon (psi) thin f ilms from p-type silicon (100) substrates were fabricated using a simple table top electrochemical etching setup with a 1:1 hf:etoh electrolyte solution. porous silico n f ilms with different m o r p h o l o g i e s a n d o p t i c a l p r o p e r t i e s w e r e a c h i e v e d b y v a r y i n g t h e e t c h i n g p a r a m e t e r s , s u c h a s h f c o n c e n t r a t i o n , e t c h i n g t i m e a n d a n o d i z a t i o n c u r r e n t . i t w a s o b s e r v ed t h a t t h e f i l m t h i c k n e s s of t h e fabricated psi increased with etch time and hf concentration. the etch r a t e i n c r e a s e d w i t h t h e a p p l i e d a n o d i z a t i o n c u r r e n t . re f l e c t i o n spectroscopy at normal incidence was used to determine the refractive i n d i c e s o f t h e f a b r i c a t e d f i l m s . u s i n g t h e s e l l m e i e r e q u a t i o n , t h e c h r o m a t i c d i s p e r s i o n o f t h e f i l m s w a s o b t a i n e d f o r d i f f e r e n t h f concentrations and anodization currents. keywords: silicon, anodization, porous materials, photonic applications issn 0115-7809 print / issn 2012-0818 online ____________ *corresponding author fabrication and characterization of porous silicon 16 made it a suitable material for photodetectors (garcia and others 2008) and solar cell devices (ramizy and others 2011). aside from the abundance of silicon, the simplicity of its fabrication and its variable refractive index due to its controllable porosity make it possible to create components or materials for photonic applications without the use of vacuum chambers and expensive deposition techniques. these applications include wave guides (jelenski and others 2005), dichroic mirrors (diener and others 2001), rugate f ilters (lorenzo and others 2005), and fabry-perot f ilters (vinegoni and others 2000). the structure of porous silicon is dependent on the hf concentration, current density, etch rate, substrate doping, type of electrolyte solution, and type of electrochemical cell (halimaoui and others 1995), among other parameters. the exact process of formation is not yet clear; however, there are many proposed models as to how it is hole injection and attack on a si-h bond by fluoride ion second attack by a fluoride ion with hydrogen evolution and electron injection into the substrate hf attack the si-si backbonds. the remaining si surface atoms are bonded to the h atoms and a silicon tetrafluoride (sif4) molecule is produced. figure 1. mechanism of the electrochemical dissolution of si in an hf based electrolyte proposed by gösele and lehmann. adapted from o. bisi and others (2000). a.i. mabilangan and others 17 formed. the model of gösele and lehmann (1991) posits that electronic holes present on the surface of silicon cause the fluorine ions (f -) to attack the silicon hydride (si-h) bonds, forming hydrogen gas and sif 4 & h 2 sif 6 which are dissolved in the solution. the etched silicon loses the holes in the process preventing further reaction with the fions (see figure 1). in this work, psi was fabricated from p-type (100) silicon by means of a simple tabletop electrochemical etching setup. etching parameters such as hf concentration, etch time and current were varied to characterize their effect on the thickness and refractive index of the psi. these results are important in further exploration of psi for photonic applications. (b) figure 2. actual setup (a) and schematic diagram of the anodization cell (b) of the electrochemical etching setup used in fabricating psi f ilms. (a) fabrication and characterization of porous silicon 18 results and discussion photonic materials greatly depend on the physical dimension and refractive index of the material used. the variability of the morphology such as thickness and porosity of psi is essential. psi film thickness figure 3 shows the dependence of f ilm thickness on the hf concentration used in the electrolyte. the increasing trend means that the production of psi is faster for higher hf concentrations. this can be attributed to the abundance of fions that attack the si-h bonds in the electrolyte. the increase in the fion population in the etching solution lessens the time between reactions, thus hastening the etching and producing psi much faster. from the trend, we can predict that for concentrations methodology the substrate used was a 500µm thick, polished on one side, monocrystalline p-type silicon wafer with (100) orientation cut into 1x1.5cm samples. the silicon substrates were subjected to standard degreasing procedures and the unpolished surface was covered with an hf-resistant polymer before electrochemical etching. the electrochemical etching was done using a simple tabletop setup shown in figure 2. the sample was attached to a plexiglass sample holder with a silver metal cathode and the silicon sample as the anode. the sample holder was lowered into the single tank anodization cell so that 1x1cm of the silicon was submerged into a 1:1 solution of hydrofluoric acid (hf) and absolute ethanol. a tektronix pws4721 programmable current source was used to drive the electrochemical process in the system. after etching, the samples were rinsed with absolute ethanol to reduce surface tension between the pores. hf concentrations were varied (6%, 9%, 12%, and 24%), as well as the current supplied (1ma, 5ma, 10ma, 15ma, and 20ma) and etch times (from 3mins to 20mins). each parameter was varied while keeping the other two constant to establish the effects of each on the morphology of the fabricated psi and the corresponding effect on its optical properties. a philips xl 30 feg scanning electron microscopy (sem) was used to determine the thickness and pore dimensions of the psi. reflectance spectroscopy was also used to determine the wavelength-dependent refractive index of the samples through a sellmeier equation f it. the reflectivity of the samples was characterized in the range 400-1100nm. a.i. mabilangan and others 19 lower than 5%, psi formation may not be possible because the applied anodic current may not be suff icient to drive the etching reaction through the resistive solution. (a) (b) figure 3. thickness dependence of psi f ilms on hf concentration (a) and their corresponding cross sectional sem micrographs (b). all psi f ilms are etched at 10ma for 10mins. fabrication and characterization of porous silicon 20 figure 4 shows the thickness variation of the psi f ilms etched at different times for two different anodization currents. for both psi etched at 5ma and 15ma, it was observed that as the etching time was increased, the thickness of the psi f ilm also increased but at different rates. the thickness of the f ilms fabricated with (a) (b) figure 4. cross-sectional sem micrographs of psi etch at 5ma (a) and 15ma (b) for different etching times. all psi was etched with 12% hf concentration. a.i. mabilangan and others 21 varying etch time and anodization current was plotted (figure 5a) and the etch rate was calculated. for each anodization current, the thickness depends linearly with the etch time. the etch rate, however, depends non-linearly with the anodization current (figure 5b). the obtained results agree with the results of berger and others (1997). (a) (b) figure 5. thickness dependence of fabricated psi f ilms on etching time for different anodization currents (a); etch rate vs. anodization current density (b). fabrication and characterization of porous silicon 22 where a, b, c, and d are f itting parameters. (1) pore size and refractive index being able to tune the refractive index of porous silicon is one of its most important characteristics in order for it to act as a waveguide. the results obtained show that the refractive index of porous silicon can be tuned by changing one or more of its etching parameters to effectively vary the pore size. the observed increase in pore size as the hf concentration decreases (figure 6) is due to the favored chemical etching (lateral) rather than electrochemical etching (vertical) because of the consequent increase in the resistivity of the solution. therefore, lower refractive index is expected for psi etched at lower hf concentrations because of the decrease in optical density (higher air-to-silicon ratio because of larger pore size). the refractive indices of psi f ilms were obtained using reflectance spectroscopy at normal incidence. using the local minima and maxima observed in each reflectivity spectrum, we can compute the refractive indices through (2) where i is the number of complete cycles between two local minima or maxima (ë 0 and ë i ) and d is the f ilm thickness (schroder 2006). the reflectivity spectra for f ilms with small d are expected to have fewer oscillations over the chosen scan range of 400-1100 nm, and thus less computed n values. from the calculated refractive indices, a sellmeier f it (2) is used to obtain the wavelength-dependent refractive index or dispersion curve of the samples, figure 6. pore size dependence of psi etched for different hf concentration. all psi f ilms are etched at 10ma for 15mins. a.i. mabilangan and others 23 %hf figure 7. computed refractive ind ices of psi f ilms fabricated at different hf concentrations. each dot represents the refractive index n computed from equation (1) for each of the 3 samples, fabricated at 10ma for 15mins using different hf concentrations (6%, 12%, 24%). since psi is considered a two-phase composite material of air and silicon, we expect that its refractive index varies between air and silicon (1.00 to 3.44). we can describe the quantity of air and silicon in the psi by the porosity, formally def ined as the volumetric ratio between the air in the pores and the bulk silicon before it was etched. however, since si exhibits wavelength-dependent refractive index or chromatic dispersion, we also expect psi to exhibit this property. figure 7 shows the dependence of the refractive index of porous silicon on the hf concentration used. it can be noted that higher concentrations produce higher ranges of refractive indices. this result agrees with the decrease in pore dimensions at higher concentrations as shown earlier. since pores are much larger in less concentrated solutions, their refractive indices are expected to approach that of air; the samples etched in higher concentrations form smaller pores and are expected to have a refractive index closer to that of silicon. the f igure also shows the 24%hf samples reaching up to a refractive index twice of that obtained for the 6%hf concentration, where the dispersion curves are shifted upward as concentration is increased. fabrication and characterization of porous silicon 24 figure 8 shows the computed refractive indices for different etch times. the computed refractive indices for varying etch times follow roughly the same dispersion curves for both 5ma and 15ma anodization currents (figure 8). this result supports the assumption that as etch time is increased, the porosity of the sample remains fairly constant. the pore size is unaffected by longer etch times. (a ) (b) figure 8. computed refractive indices of psi f ilms fabricated at 5ma (a) and 15ma (b) for different etching times in a 12% hf solution. a.i. mabilangan and others 25 figure 9. chromatic dispersions obtained through sellmeier equation for psi etched at different anodization currents. figure 9 shows the f itted dispersion curves for different anodization currents. the refractive index was observed to decrease as current density increased due to the increase in porosity of the psi layer. this agrees with the result regarding the pore dimensions as discussed earlier. when higher anodization currents are used, an increase in the electron hole population in the si surface occurs. as a result, sites for pore formation are increased, thereby increasing the average number of pores. this, in effect, increases the ratio of air-tosilicon of the sample, which translates to the lower refractive indices for higher current densities used. summary porous silicon was successfully fabricated from p-type silicon (100) substrates using a simple tabletop electrochemical etching setup with hf as the electrolyte. fabrication parameters such as hf concentration, etch time, and anodization current were varied to establish a relationship with these parameters to the thickness and refractive index of porous silicon. fabrication and characterization of porous silicon 26 psi f ilm thickness increases for longer etch times and higher hf concentrations. constant etch rates have been observed for different anodization currents. etch rates follow a nonlinear increase as the anodization current is increased. the refractive index of the fabricated psi depends on the pore size, which is dependent only on the hf concentration and anodization current. lower hf concentration produces larger pore sizes that decrease the refractive index due to higher air-tosilicon ratio, producing lower dispersion curves. psi fabrication with high anodization currents tends to have lower dispersion curves due to the increase in the sites for pore formation. photonic devices such as distributed bragg reflectors and photonic crystals can thus be made from porous silicon using a simple electochemical etching setup due to the ease in controlling f ilm thickness and refractive index. acknowledgments we would like to acknowledge the following institutions for supporting the study: a) university of the philippines diliman – off ice of the vice-chancellor for research and development, b) department of science and technology – national research council of the philippines, c) department of science and technology – philippine council for industry, energy and emerging technology research and development, and d) department of science and technology – grants-in-aid program. we would also like to thank lorenzo lopez jr. , angela faustino, and michaelrey cainglet for their assistance. references badoz pa, bensahel d, bomchil g, ferrieu f, halimaoui a , perret p, regolini j, sagnes i, vincent g. 1992. 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silicon-based material and devices. san diego, california, academic press. p 124-188. _______________ arvin i. mabilangan <arvinmabilangan@gmail.com>, niel gabriel e. saplagio, eloise p. anguluan, neil irvin f. cabello, and rhona ol ivia m. gonzales are graduate students aff iliated with the condensed matter physics laboratory of the national institute of physics, university of the philippines diliman. arnel a. salvador, phd <asalvadornip@gmail.com> is a professor at the national institute of physics, university of the philippines diliman. dr. salvador earned his fabrication and characterization of porous silicon 28 bachelor’s degree in physics, cum laude, from the university of the philippines diliman. he earned his phd in physics from the university of illinois at urbanachampaign in the united states. armando s. somintac, phd <somintac@gmail.com> is an assistant professor and currently the program coordinator of the condensed matter physics laboratory at the national institute of physics, university of the philippines diliman. sc i e n c e di l i m a n is published semi-annually (june and december) by the university of the philippines diliman through the office of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief jonas p. quilang, ph.d. associate editors jose maria p. balmaceda, ph.d. university of the philippines louis angelo m. danao, ph.d. university of the philippines carlos primo c. david, ph.d. university of the philippines christian n. della, ph.d. university of glasgow singapore arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines dennis i. merino, ph.d. southeastern louisiana university arnel a. salvador, ph.d. university of the philippines terence p. tumolva, d.eng. university of the philippines irene m. villaseñor, ph.d. university of the philippines managing editor conchitina r. cruz, ph.d. university of the philippines editorial assistant narita e.c. de las alas layout artist mirriam m. velasco copyeditor jean lau wang on the cover: the cover image shows the diagram of sensing uric acid (ua) using a fabricated sensor based on urate oxidase (uox) and copper (ii/i) oxide (cuo/cu2o) on the surface of a carbon paste electrode (cpe). the uox-cuo-cpe sensor may be developed into low-cost sensing material for health monitoring applications. photo from allan christopher yago. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. science diliman july-december 2020 • vol. 32 no. 2issn print 0115-7809 issn online 2012-0818 international advisory board teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus university of illinois, usa university of california, irvine, usa the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheffield, united 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(subscription price is subject to change without prior notice.) i/we would like to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php 650) humanities diliman science diliman social science diliman grand total method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman ovcrd)  money remittance (payable to anna angelica p. angala, c/o ovcrd research dissemination office, with office address as indicated above and telephone no. 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927-2309; 436-87-20 telfax: (632) 927-2568 e-mail: rduo.ovcrd@up.edu.ph website: http://www.ovcrd.upd.edu.ph science diliman a journal of pure and applied sciences teofilo a. abrajano jr., ph.d. department of earth and environmental sciences rensselaer polytechnic institute troy, new york abrajt@rpi.edu alfonso m. albano, ph.d. department of physics bryn mawr college bryn mawr, pennsylvania aalbano@brynmawr.edu angel c. alcala, ph.d angelo king center for research and environmental management silliman university dumaguete city, philippines sumanila@psdn.org.ph; suakcrem@philwebinc.com felixberto a. buot, ph.d. naval research laboratory washington, d.c. buot@estd.nrl.navy.mil; fab@cfdrc.com josefino c. comiso, ph.d. laboratory for hydrospheric processes nasa goddard space flight center greenbelt, maryland comiso@joey.gsfc.nasa.gov lourdes j. cruz, ph.d. the marine science institute university of the philippines diliman quezon city, philippines luly@upmsi.ph edgardo d. gomez, ph.d. the marine science institute university of the philippines diliman quezon city, philippines edgomez@upmsi.ph per juel hansen, ph.d. marine biological laboratory university of copenhagen helsingor, denmark pjhansen@zi.ku.dk jorgen hylleberg, ph.d. department of biological sciences and marine ecology university of aarhus aarhus, denmark hylleberg@biology.aau.dk chris m. ireland, ph.d. department of medicinal chemistry university of utah salt lake city, utah cireland@deans.pharm.utah.edu international advisory committee emil q. javier, ph. d. institute of plant breeding university of the philippines los baños college, laguna, philippines emil.javier@cgiar.org amador muriel, ph.d. data transport system new york, new york dtsny@msn.com baldomero m. olivera, ph.d. department of biology university of utah salt lake city, utah olivera@biology.utah.edu eduardo a. padlan, ph.d. kensington, maryland edpadlan@aol.com william g. padolina, ph.d. international rice research institute college, los baños, laguna w.padolina@cgiar.org kelvin s. rodolfo, ph.d. department of earth and environmental sciences university of illinois chicago, illinois krodolfo@uic.edu gary e. rodrick, ph.d. department of food science and human nutrition university of florida gainesville, florida gerodrick@mail.ifas.ufl.edu francis j. schmitz, ph.d. department of chemistry and biochemistry university of oklahoma norman, oklahoma fjschmitz@chemdept.chem.ou.edu gavino c. trono jr., ph.d. the marine science institute university of the philippines diliman quezon city, philippines trono@upmsi.ph ocr document 07_variational chan and villanueva 22 a variational pertubation approach l. c. chan* and a. villanueva national institute of physics, university of the philippines diliman, quezon city 1101 e-mail: lchan@nip.upd.edu.ph abstract science diliman (july-december 2004)) 16:2, 22–25 *corresponding author the paper discusses how the variationally inspired perturbation theory (vipt) scheme of approximation in quantum mechanics can be improved convergencewise if one uses for variational trial functions the perturbative series with variational parameter, so that one is effectively doing variational calculations directly on the high-order perturbative series for energy. the result optimizes the high-order energy directly and thus represents a significant improvement over the vipt procedure. when applied to a double potential in which even the vipt is badly divergent, we saw that the result is still very much convergent. introduction in a recent article, a new approximation scheme for time-independent problems was presented by you et al. (1998) which improves upon the regular perturbation approach by making use of a variational calculation to obtain a more convergent split up of the hamiltonian into the unperturbed hamiltonian and the perturbation. they used this method to get the ground and first excited states of the anharmonic oscillator problem and the ground state of the helium problem. this scheme was called the variationally improved perturbation theory (vipt) by aitchison and dudek (2002), who applied it to the coulomb plus linear potential . the results are very encouraging, especially for large perturbations for which the regular perturbation approach has convergence problems. it is also especially relevant for systems in which a good soluble unperturbed hamiltonian with a small perturbation cannot be found. it is to these systems in which the vipt is divergent that we address the present paper. in this paper, we shall pursue an alternate way of perturbing the physical system with which we calculate the coefficients of the perturbative series for wave functions by a variational approach. we shall introduce a system for which the vipt diverges very badly and show that the present technique, called variational perturbation theory (vpt), allows us to calculate a real eigenvalue of energy which is still very close to the correct value. this paper is organized as follows: in sec. 2, we introduced the anharmonic oscillator potential with a negative harmonic term so that it represents a doublewell potential. the vipt is then applied to this problem for three different assignments of the strengths of the strengths of the harmonic term, corresponding to different depths of the double well. in sec. 3, we apply the vpt with three variational parameters corresponding to the strength of the mother hamiltonian, the harmonic perturbation, and the anharmonic strengths. in sec. 4, we summarize and comment on what we have achieved. ( )v r r r α β= − +v variational perturbation 23 variationally inspired perturbation theory consider a system under the hamiltonian , (1) where the part h 0 +v 1 is soluble and v 2 is normally treated as a perturbation but the strength of v 2 is so large that the normal perturbation approach is inapplicable. the vipt handles the problem by using hp(s) = h 0 + (1–s)v 1 as the unperturbed hamiltonian, with s chosen variationally, treating (2) as the new perturbation. this judicious choice of s gives a better first approximation to energy so that the new perturbation approach will improve in convergence property. the vipt is done as follows: first, we evaluate the expectation value of the total hamiltonian using eigenfunctions of hp(s), , (3) as trial functions to get the optimal value of s. note here that the unperturbed eigenvalues and eigenfunctions actively depend on s. the meaning of the optimal parent hamiltonian means that we are choosing the optimal value of the potential (1–s)v 1 to best approximate the true potential v 1 +v 2 for the particular eigenstate. thus the optimal parent hamiltonian can be used as the unperturbed hamiltonian and the regular schrodinger perturbation theory can be applied with the perturbation given by eq. (2). the variational calculation actually gives the energy up to first order. higher-order perturbative calculations for energy are calculated by regular perturbation techniques. for the potential we will investigate, we choose a double well potential , (4) with the parameter w < 0 and l = 1. this form of the potential is a double-well potential whose depth is controlled by the value of w, i.e., the more negative w is, the deeper the double well. in particular, we investigate the potential for three values of the parameter w, w = –1, –4, –10. the potential in eq. (4) is chosen because the matrix elements of the potential is particularly simple. the results of perturbation calculations, done to first and third orders, are given in tables 1 and 2 as follows: 0 1 2h h v v= + + 1 2'h v vσ= + ( ) ( ) ( ) ( )0n n nhp eσ φ σ σ φ σ= ( ) 2 3 4 2 21 2 m x v x m x ω ω λ= ω + h table 1. first-order vipt results for ground state. ωωωωω σσσσσ0 e0,calc e0,exact error -1 -4 -10 -3.6712 -5.2876 -10.3370 0.5364 -0.0151 -1.9359 0.5148 -0.1304 -4.1358 0.0216 0.1253 2.1990 table 2. third-order vipt results for ground state. ωωωωω σσσσσ 0 e 0,calc e 0,exact error -1 -4 -10 -3.6712 -5.2876 -10.3370 0.5364 0.3820 34.3614 0.5148 -0.1304 -4.1358 0.0226 0.5124 37.50 we notice that when the wells deepen, i.e., as w becomes more negative, convergence becomes bad. in fact the case w = –10 is an extremely divergent case. variational perturbation theory if perturbation theory converges, the results can be obtained in a quick fashion by calculating the expectation value of the hamiltonian h with respect to the normalized perturbative series for wave function , (5) where the perturbation operator k can be obtained in series form from the equation (speisman, 1957) . (6) it was shown that using k to order n gives an energy to order 2n+1. thus, using (7) ( ) ( )1 2/ 1n n n nk kkψ φ φ φ φ= + + ( )1 0 0 ' ' n q k h p e h = − ( ) ( )0 0 ' n q k q k h p k e h = − + − chan and villanueva 24 will give energy up to the third order. since we are dealing with the expectation value of h, we can optimize the results by regarding the parameters s and l as variational parameters. this modification can be very advantageous if the results of applying vipt turns out to be divergent, the optimization process in vpt could still give convergent results. the first-order vpt is the same as the first-order vipt since in both cases it is s that is used as the variational parameter. for third-order vpt, we use two variational parameters s and l for the term h’ in eq. (5) so that now the trial wave functions depend on three parameters: (8) (9) with . (10) substituting, we get we noticed that instead of diverging, this third-order result represents a tremendous improvement over the first-order results. we shall also investigate the situation for the first excited state in the next section. higher excited states in this section, we give the corresponding results for the first excited state. for first-order vipt and vpt results we have: ( ) ( )( ) ( ) ( ) ( ) ( ) 1 0 1/ 2 1 1 0 0 1 1 k k k φ σ ψ σ φ σ φ + = ⎡ ⎤+ ⎢ ⎥⎣ ⎦ ( ) ( ) ( ) 00 0 1/ 2 0 020 0 1 ' 1 ' ' p p q h e h q h h e h φ σ φ σ φ σ ⎛ ⎞ +⎜ ⎟⎜ ⎟−⎝ ⎠= ⎡ ⎤ ⎢ ⎥+ ⎢ ⎥⎡ ⎤−⎣ ⎦⎢ ⎥⎣ ⎦ 2 3 2 2 41' 2 m h m x x ω ω= σ + λ h ( )( )( ) ( )( ) ( ) 1 1 0 0 0 020 0 1 ' 1 1 ' ' p p k h h k h q h h e h φ φ φ φ + + + = + − the results of third-order vpt is given in table 3 as follows: table 3. third-order vpt results for ground state. ωωωωω σσσσσ 0 e 0,calc e 0,exact error -1 -4 -10 -5.254 -6.569 -14.000 0.5151 -0.1263 -4.0475 0.5148 -0.1304 -4.1358 0.0003 0.0041 0.0883 σσσσς -5.657 -7.577 44.522 λλλλλ 1.0075 1.0321 -29.7200 table 4. first-order vipt and vpt results for first excited state. ωωωωω σσσσσ0 e0,calc e0,exact error -1 -4 -10 -5.0000 -6.4235 -10.8490 2.0625 0.7878 -3.0317 2.0206 0.6614 -4.1191 0.0419 0.1264 1.0874 table 5. third-order vipt results for first excited state. ωωωωω σσσσσ 0 e 0,calc e 0,exact error -1 -4 -10 -5.0000 -6.4235 -10.8490 2.0334 0.9110 10.8930 2.0206 0.6614 -4.1191 0.0419 0.2494 15.0840 the third-order vipt results are given in table 5 as follows: notice again that the result is very highly divergent for w = –10. on the other hand, when the third-order vpt is performed the results are as given in table 6: table 6. third-order vpt results for first excited state. ωωωωω σσσσσ 1 e 1,calc e 1,exact error -1 -4 -10 -6.1425 -7.9329 -12.733 2.0154 0.6660 -3.9138 2.0206 0.6614 -4.1191 0.0052 0.0046 0.2053 σσσσς -6.5036 -7.4540 -7.1704 ∆∆∆∆∆ 1.0622 1.0321 0.0000 again, the convergence is much improved over first order results. variational perturbation 25 discussions and conclusions we have observed in one particular example, in which the ordinary perturbation or the improved vipt results were not convergent, that the use of vpt in which the perturbed wavefunctions were used as variational trial functions with variational parameters inserted for each term in the perturbation series has been able to render convergence to the results. the reason for the good convergence property of the vpt lies in the fact that it is a variational method. in a variational method, as one increases the number of parameters, one is sampling over greater numbers of trial functions, with the consequence of always increasing the accuracy, and therefore, in general, the higher the order of perturbation, the more perturbation parameters would be used, and the more accurate the result becomes. references aitchison, i.j.r. & j.j. dudek, 2002. variationally improved perturbation theory and the potential –(a/r)+br. eur. j. phys. 23: 605–614. speisman, g., 1957. phys. rev. 107: 1180. you, s.k., k.j. jeon, c.k. khu, & k. nahm, 1198. a new approximation scheme in quantum mechanics. eur. j. phys. 19: 179-86. 5chua-fontanilla.pmd genetic comparison of oncomelania hupensis quadrasi 32 science diliman (july-december 2017) 29:2, 32-50 genetic comparison of oncomelania hupensis quad rasi (möllendorf, 1895) (gastropoda: pomatiopsidae), the intermediate host of schistosoma japonicum in the phil ippines, based on 16s ribosomal rna sequence james christopher c. chua* ian kim b. tabios pebbles grayle p. tamayo lyd ia r. leonardo university of the philippines manila ian kendrich c. fontanilla raffy jay c. fornillos university of the philippines diliman emmanuel ryan c. de chavez university of the philippines los baños takeshi agatsuma kochi medical school mihoko kikuchi nagasaki university naoko kato-hayashi yuichi chigusa dokkyo medical university abstract schistosomiasis japonica is a water-borne trematode infection transmitted by different subspecies of oncomelania hupensis. as parasites may either co-evolve or locally adapt with their hosts, snail diversity, as revealed by morphometric and genetic studies, may reflect parasite diversity and elucidate snail susceptibility and transmission patterns. this study aimed t o c o m p a r e i s o l a t e s o f o . h . q u a d r a s i b a s e d o n a 3 4 2 b p f r a g m e n t o f _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online j.c. c. chua et al. 33 the 16s ribosomal rna gene. o. h. quadrasi isolates were collected from nine provinces known to have s. japonicum in the philippines, namely cagayan valley, bohol, negros occidental, leyte, davao, davao del sur, mindoro oriental, nor thern samar, and sorsogon. o. h. hupensis and o. h. nosophora isolates were also collected from china and japan, respectively. t h e 1 6 s r i b o s o m a l r n a g e n e o f e a c h s p e c i m e n w a s a m p l i f i e d a n d sequenced. phylogenetic and network analyses based on the 221 16s rrna gene sequences revealed that o. h. quadrasi clustered as a distinct clade from the two other subspecies. of the four identif ied haplotypes for o. h. quadrasi, two haplotypes were from negros oriental (ohq2 and ohq3), and one haplotype was from bohol (ohq4). the isolates from the r e m a i n i n g s e v e n p r o v i n c e s s h a r e d a c o m m o n h a p l o t y p e ( o h q 1 ) . t h e c u r r e n t s t u d y w a s a b l e t o s h o w t h e r e l a t i o n s h i p a m o n g o . h u p e n s i s s u b s p e c i e s a n d d e m o n s t r a t e t h e l i m i t e d a b i l i t y o f m i t o c h o n d r i a l 1 6 s ribosomal molecular marker in differentiating o. h. quadrasi geographic strains in the philippines. k e y w o r d s : o n c o m e l a n i a h u p e n s i s q u a d r a s i , s c h i s t o s o m a j a p o n i c u m , schistosomiasis japonica, snail intermediate host, haplotype, 16s r i bo s o mal rna gene introduction oncomelania hupensis (gastropoda: pomatiopsidae) is an amphibious freshwater snail that acts as the intermediate host of schistosoma japonicum, a parasitic worm of great public health concern. nine geographical subspecies found throughout asia have been recognized for the o. hupensis species complex (who 1993). the four subspecies in china are o. h. hupensis (yangtze basin), o. h. robertsoni (sichuan and yunnan), o. h. tangi (fujian), and o. h. guangxiensis (guangxi). in other asian countries, the subspecies present are o. h. nosophora in japan, o. h. lindoensis in indonesia, o. h. chiui and o. h. formosana in taiwan, and o. h. quad rasi in the philippines (davis et al. 1995a). the subspecies mainly differ in shell dimensions, electromorphic and antigenic patterns, reproductive potential, growth rate, and compatibility with various geographic strains of s. japonicum (davis et al. 1995b; zhao et al. 2010). oncomelania hupensis quad rasi in the philippines was f irst recognized as the intermediate host of s. japonicum by tubangui, based on observations made in palo, leyte (blas 1988; leonardo et al. 2016). an adult o. h. quadrasi shell is genetic comparison of oncomelania hupensis quadrasi 34 colored light brown to black, usually measures 3 to 5 mm, and has 5 to 7 whorls (figure 1). these snails are dioecious and thrive in many different kinds of wet shaded environments. these habitats include flood plains, swampy areas, and grass lands. although considered transient, rice f ields and other man-made habitats, such as irrigation canals and burrow pits, also support these snails. they prefer areas with dense vegetation where flow of water is sluggish, and rivers that are not very deep or too wide. o. h. quadrasi colonies have been known to occur over most of mindanao and samar, eastern leyte, bohol, in small areas in southeastern luzon, and eastern mindoro (pesigan et al. 1958). o. h. quadrasi colonies have been recently discovered in cagayan valley in northern luzon and in negros occidental (leonardo et al. 2015). figure 1. oncomelania hupensis quadrasi (mollendorf 1895) collected from the province of leyte in eastern philippines. juvenile (right) snails measure a maximum of 3 mm while adult snails reach more than 3 mm in size. 3 mm genetic studies on the intermediate host are important in determining the relationship of the intermediate host with the parasite, as the presence or absence of co-evolution may signif icantly affect transmission patterns. co-evolution implies the restriction of the parasite to certain snail hosts, limiting transmission to areas where snails are compatible with the parasite (davis et al. 1999), whereas the absence of co-evolution, as evidenced by recent studies, may result in host-switching or acquisition, making it possible for the parasite to spread in other areas (attwood et al. 2015). previous studies have reported the different compatibilities of snail populations from different geographic locations with strains of s. japonicum, and variation in susceptibility to sodium pentachlorophenate (napcp), a molluscicidal agent (cross et al. 1984; hong et al. 1995; rachford 1997). determination of genetic variants within geographical isolates of o. h. quadrasi is necessary for the evaluation of their potential in transmitting s. japonicum. past research revealed only minor variation among geographic isolates of o. h. quad rasi in the philippines (viyanant et al. 1987; woodruff et al. 1988); however, j.c. c. chua et al. 35 these studies only involved techniques that tested phenotypic characters, which are subject to environmental influences. some enzyme-coding genes are only expressed under certain environmental conditions (davis et al. 1995a). hope and mcmanus (1994) used polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) to directly examine the internal transcribed spacer (its) gene, and found minor variation among philippine snails. a recent paper of saijuntha et al. (2014) studied 12s ribosomal rna gene sequences of o. h. quadrasi from three provinces with s. japonicum, and they were able to show population substructuring associated with geographical origin. to further investigate the phylogeography of o. h. quadrasi in the philippines, this study examined genetic variation among snail populations collected from 12 localities in seven islands based on the 16s rrna gene. snail samples from two newly identif ied endemic foci for s. japonicum in cagayan valley and negros occidental were included in the analysis. the 16s rrna gene was selected since previous studies have demonstrated its utility to assess genetic variation in populations in the aquatic snail potamopyrgus antipodorum (stadler et al. 2005), the slug arion subfucus (pinceel et al. 2005), the giant african snail achatina fulica (fontanilla et al. 2014), and the pomatiopsid snail neotricula aperta (attwood et al. 2008). materials and methods snail collection geographic isolates of o. h. quadrasi were collected from nine provinces across seven islands of the philippines: cagayan and sorsogon on luzon island; mindoro oriental on mindoro island; negros occidental on negros island; bohol on bohol island; leyte on leyte island; samar on samar island, and davao and davao del sur on mindanao island (table 1). thirty individual snails from each locality were examined. snails collected were identif ied as o. h. quadrasi by comparison with previously identif ied voucher specimens archived in the department of parasitology, college of public health, university of the philippines manila. samples of o. h. hupensis from china and o. h. nosophora from japan were also included in the study. snails were washed with 0.9% nacl and placed in a vial with 70% ethanol before being transported to the laboratory. the head-foot muscle was dissected under the microscope, and this was used in the dna extraction. genetic comparison of oncomelania hupensis quadrasi 36 mcag magraf il cagayan valley luzon philippines o. h. quadrasi 27 tcag tapel cagayan valley luzon philippines o. h. quadrasi 3 tsor tulay sorsogon luzon philippines o. h. quadrasi 30 bdmin batong dalig mindoro oriental mindoro philippines o. h. quadrasi 30 bneg bonbon negros occidental negros philippines o. h. quadrasi 22 hneg hinag-ongan negros occidental negros philippines o. h. quadrasi 6 mboh magsaysay bohol bohol philippines o. h. quadrasi 30 pley palo leyte leyte philippines o. h. quadrasi 13 dley dagami leyte leyte philippines o. h. quadrasi 11 sam catarman northern samar samar philippines o. h. quadrasi 18 dav compostela valley davao mindanao philippines o. h. quadrasi 29 das davao del sur mindanao philippines o. h. quadrasi 2 wanh wuwei anhui mainland china china o. h. hupensis 4 tanh tongling anhui mainland china china o. h. hupensis 4 hanh hexian anhui mainland china china o. h. hupensis 4 xanh xiuzhou anhui mainland china china o. h. hupensis 4 lanh lingjiang anhui mainland china china o. h. hupensis 4 danh dongzhi anhui mainland china china o. h. hupensis 4 sanh shitai anhui mainland china china o. h. hupensis 4 jhun jiangshan hunan mainland china china o. h. hupensis 4 czhe chizhou zhejiang mainland china china o. h. hupensis 4 nyam nirasaki yamanashi honshu japan o. h. nosophora 5 kyam kofu yamanashi honshu japan o. h. nosophora 2 table 1. source organisms, local ities, and sample sizes of 16s sequences analysed sample code local ity province island country subspecies no. j.c. c. chua et al. 37 dna extraction, pcr ampl if ication, and dna sequencing dna extraction was carried out using the qiagen® genomic dna extraction kittm (usa) following the manufacturer’s protocol. dna was quantif ied using thermo scientif ic®nanodrop tm 2000c (usa). pcr amplif ication of the 16s rrna gene was performed in a total volume of 50 μl containing 20mm buffer (tris-hcl, mgcl 2 , and kcl), 10 pmol each of forward and reverse primers, 2.5 mm dntp, 1.25 units roche® taq dna polymerase, and 8.0 μl dna template. the forward and reverse primers used were 16s1ionco5 5’–tgaccgtgcgaaggtagcat–3’, which was designed for this study, and 16sscp2i 5’– cctagtccaacatcgaggtc–3’, which was obtained from fontanilla et al. (2010). this primer pair amplif ies a 342-bp fragment of the 16s rrna gene that corresponds to its secondary structure’s domain iv (lydeard et al. 2000), which is also the same fragment used in other previous studies (stadler et al. 2005; pinceel et al. 2005; attwood et al. 2008; fontanilla et al. 2014). initial denaturation temperature was 94°c for 3 minutes succeeded by 43 cycles of denaturation at 94°c for 30 seconds, annealing at 45°c for 30 seconds, and extension at 65°c for 1 minute, which was followed by one f inal extension step at 72°c for 5 minutes. pcr products were purif ied from agarose using qiagen® qiaquick gel extraction kit tm (usa) following the manufacturer’s protocol. purif ied pcr products were sent to 1st base pte. ltd. in malaysia for dna sequencing. dna sequence analysis sequences were aligned using bioedit version 7.0.9.0 (hall 2008). aligned sequences were inspected manually and distinct haplotypes were identif ied using the data analysis in molecular biology and evolution (dambe) version 5.2.79 software (xia and xie 2001). consensus sequences were submitted to ncbi’s g e n b a n k ( n a t i o n a l c e n t e r f o r b i o t e c h n o l o g y i n f o r m a t i o n ; h t t p s : / / www.ncbi.nlm.nih.gov/) for the assignment of accession numbers (table 2). for the phylogenetic analyses, three methods of tree construction, two requiring a model of dna substitution and one method that did not, were employed. for the model-based methods, the best model with optimized parameters was determined using the bayesian inference criterion (bic) in jmodel test version 0.1.1 (posada 2008). once the optimal model was identif ied, the maximum likelihood (ml) tree (cavalli-sfroza and edwards 1967; felsenstein 1981) and neighbor joining tree (saitou and nei 1987) were constructed using phyml version 2.4.4 (guindon and gascuel 2003) and paup version 4.0b10 package (swofford 2002), respectively. genetic comparison of oncomelania hupensis quadrasi 38 for the non-model based method, the maximum parsimony (mp) tree (eck and dayhoff 1966; fitch 1977) was constructed using mega6 (tamura et al. 2013). all three methods rooted the trees on o. minima, and bootstrap resampling (felsenstein 1985) with 1000 replicates was also undertaken. an ml tree that includes the bootstrap supports for the clades based on the ml, nj, and mp analyses was consequently generated. ohq1 philippines: palo, leyte l6 ky432666 o. h. quadasi ohq2 philippines: calatrava, negros occidental n28 ky432668 o. h. quadrasi ohq3 philippines: calatrava, negros occidental n15 ky432667 o. h. quadrasi ohq4 philippines: magsaysay, bohol b1 ky432669 o. h. quadrasi ohh1 china: wuwei, anhui 1ahwuwei ky432670 o. h. hupensis ohh2 china: lingjiang, anhui 1ahlingjiang ky432671 o. h. hupensis ohh3 china: hexian, anhui 1ahhexian ky432673 o. h. hupensis ohh4 china: xiuzhou, anhui 1ahxiuzhou ky432674 o. h. hupensis ohh5 china: shitai, anhui 1ahshitai ky432675 o. h. hupensis ohh6 china: chizhou, zhejiang 1zjchizhou ky432676 o. h. hupensis ohh7 china: jiangshan hunan 1hnjiangshan ky432672 o. h. hupensis ohn1 japan: kofu, yamanashi 1kofu ky432677 o. h. nosophora sequence id country isolate genbank accession subspecies table 2. genbank accessions of 12 haplotypes of the 16s rrna gene of various oncomelania spp. a median joining network of the distinct haplotypes was also constructed using the network version 4.502 program (bandelt et al. 1999), in order to elucidate the relationships of the different o. hupensis subspecies, and in particular, the philippine o. hupensis quadrasi haplotypes. dnasp5 was utilized to compute for the haplotype number (slatkin and hudson, 1991; librado and rozas, 2009). results analysis of o. hupensis subspecies a total of 221 16s rrna sequences were obtained for o. h. quadrasi, 36 for o. h. hupensis, and seven for o. h. nosophora (table 1). alignment revealed 20 polymorphic sites consisting of 19 transitions and one insertion/deletion (table 3). the variable j.c. c. chua et al. 39 sites yielded 12 haplotypes: four haplotypes of o. h. quadrasi, seven haplotypes of o. h. hupensis, and a single haplotype of o. h. nosophora (tables 2, 3, and 5). the hky85+g model was identif ied to be the best model for sequence evolution with optimized parameters using the bayesian inference criterion (bic). median-joining network analysis supports the ml tree f indings as four similarly distributed clusters were identif ied. the o. h. quadrasi haplotypes clustered in one group and were assigned to be closely related to an o. h. robertsoni haplotype (figure 2). the haplotype network analysis suggests that philippine haplotypes share one, or possibly two putative haplotype ancestors (mv5 and mv6; figure 3), which split into two lineages. the f irst lineage includes the common haplotype (ohq1) found in majority of the sampled areas, which in turn gave rise to the bohol haplotype (ohq4). the other lineage gave rise to the two haplotypes in negros occidental, the hinabo (or hinab-ongan) haplotype (ohq2) and the bonbon haplotype (ohq3). there were no common or shared haplotypes among the three subspecies. the ml tree also showed that o. h. quadrasi formed a highly supported (92% ml, 95% mp, and 90% nj bootstraps) cluster that was distinct from the other subspecies (figure 3). the phylogenetic tree contradicts current subspecies classif ication based on haplotype nucleotide position 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 5 5 9 0 0 0 1 2 2 2 2 3 4 4 4 5 9 9 1 5 1 5 2 0 2 6 9 1 2 4 8 5 3 5 6 8 7 8 ohq1 g a a t g c c g g c a t a g a a a t a ohq2 . . . . . . . . a . . . . . . . t . . . ohq3 . . . . . . . . a . . . . . . . t . c . ohq4 . . . . . . t . . . . . . . . . . . . ohh1 a . . c . t t . a . . . g a . g g . g ohh2 a . . c . t t . a . . . g a . g . . g ohh3 a . . c . t t . a . . . g a g . . . g ohh4 a . . c . t t a a . . . g a . g . . g ohh5 a . . c . t t . . . . . g a . g . . g ohh6 a . . c . t t . a . g c g a . g . . g ohh7 a g . c . t t . a . . . g a . g . . g ohn1 a . g c a t t . a t . . g a . g g . g table 3. variable positions of the 16s rrna gene sequences of o. h. quad rasi (ohq), o. h. hupensis (ohh), and o. h. nosophora (ohn) haplotypes. nucleotide position number is based on o. h. hupensis isolate scms complete mitochondrial genome sequence [genbank: nc_012899] genetic comparison of oncomelania hupensis quadrasi 40 geographic origin as o. h. hupensis subspecies clustered with o. h. formosana from taiwan and o. h. nosophora from japan. o. h. robertsoni and o. h. quad rasi were observed to be monophyletic. figure 2. maximum likelihood tree of o. hupensis based on 342 nucleotides of the 16s rrna gene and using the hky85+g model of dna substitution. haplotypes with (*) represent samples from this study while the rest were obtained from genbank. the tree was rooted on o. minima. values on nodes represent bootstrap support percentage (out of 1000 bootstrap samples) for ml/mp/nj; values less than 50% are not shown. bootstraps for nj were likewise based on the hky85+γ model. bootstraps for mp were based on six equally parsimonious trees. scale bar represents one nucleotide substitution for every 100 nucleotides. j.c. c. chua et al. 41 genetic diversity of o. h. quad rasi genetic distance ranged from 0.30 to 7.69% among subspecies haplotypes, and 0.30 to 0.95% among o. h. quadrasi haplotypes (table 4). of the four haplotypes of o. h. quadrasi, two haplotypes were from bonbon and hinab-ongan in negros occidental (ohq2 and ohq3), and a single haplotype was from magsaysay, bohol (ohq4). the isolates from the remaining seven provinces formed a single shared haplotype (ohq1) (table 5). the haplotype and nucleotide diversities for the philippine population were 0.429 ± 0.037 and 0.00230 ± 0.00024, respectively (table 4). there was no substantial genetic subdivision formed among the nine provinces, suggesting that high rates of gene flow occur among these regions. figure 3. median-joining haplotype network of three subspecies of o. hupensis based on 16s rrna gene sequences. the size of the circles represents the frequency of each haplotype (found inside parenthesis) among the isolates. red dots represent putative haplotypes. ohq = o. h. quadrasi; ohh = o. h. hupensis; ohn = o. h. nosophora. table 4. molecular variations of the o. h. quadrasi from the phil ippines grouped accord ing to island of origin. n = number of samples; h = number of haplotype; μμμμμ = number of non-overlapping haplotype; hd = haplotype d iversity; πππππ = nucleotide d iversity population n h μμμμμ hd ± sd πππππ ± sd luzon 60 1 0 0.000 ± 0.000 na mindoro 30 1 0 0.000 ± 0.000 na leyte 24 1 0 0.000 ± 0.000 na samar 18 1 0 0.000 ± 0.000 na negros 28 2 2 0.349 ± 0.090 0.00125 ± 0.00001 bohol 30 1 1 0.000 ± 0.000 na mindanao 31 1 0 0.000 ± 0.000 na all populations 226 4 4 0.429 ± 0.037 0.00230 ± 0.00024 genetic comparison of oncomelania hupensis quadrasi 42 discussion the study was able to show that o. h. quadrasi is genetically different from the other subspecies of o. hupensis found in other asian countries based on the 16s rrna sequences. however, only limited genetic substructuring existed among the geographical isolates of o. h. quadrasi in the nine provinces of the philippines. two major clades were observed in the ml tree. o. h. hupensis, o. h. tangi, o. h. formosana, and o. h. nosophora formed clade 1. o. h. robertsoni isolates grouped with o. h. quadrasi in clade 2. the four haplotypes of o. h. quadrasi clustered together to form a distinct subclade. these results agree with those of a recent comparative phylogenetic study of oncomelania hupensis and schistosoma japonicum using mitochondrial genes, in which o. h. tangi and o. h. formosana also clustered with o. h. hupensis, suggesting that they are not valid subspecies but rather are o. h. hupensis variants (attwood et al. 2015). the same study likewise found o. h. robertsoni to be not monophyletic (attwood et al. 2015). the phylogenetic tree in this study closely resembles the trees previously constructed using the mitochondrial 12s ribosomal gene (okamoto et al. 2003), and combined mitochondrial coi and 16s genes (wilke et al. 2006). in the 12s tree, o. h. hupensis also clustered with o. h. nosophora and o. h. formosana, while o. h. quad rasi also formed a monophyletic clade. similarly, they identif ied a bohol haplotype and a shared haplotype from mindoro and sorsogon. the previous study also identif ied two haplotypes from different sampled areas in davao, digos, and asuncion, while the current study using the 16s genetic marker identif ied the davao representative from compostela valley to be identical with the common haplotype from the remaining provinces. it is therefore possible that increased sampling effort from other areas in davao could lead to the discovery of other 16s rrna gene haplotypes; this may also be the case for the other provinces. cagayan valley and negros occidental have only been recently identif ied as endemic for schistosomiasis in 2002 and 2005, respectively (leonardo et al. 2015). the current hypothesis for these two areas is that o. h. quadrasi had always been present in these places, and recent migration of schistosome-infected human and/ or animal hosts resulted in sustained transmission. however, results of the network analysis suggest the possibility of negros occidental snail isolates evolving independently from other geographic isolates. therefore, the same apparent barriers to dispersal that led to the snails from negros occidental being genetically distinct from the other populations could lead to similar isolation and divergence of the schistosomes present there. this may likely have been the result of allopatric j.c. c. chua et al. 43 speciation, in which new strains arise in non-overlapping geographic locations. this suggests that negros occidental had always been endemic for schistosomiasis but clinical cases were unreported or misdiagnosed. for cagayan valley, the founder effect seems more likely as the local snails were discovered to be identical with the isolates from other schistosomiasis-positive provinces, which also raises the possible endemicity of the o. h. quadrasi haplotypes in other luzon provinces. performing a molecular clock analysis could be applied to conf irm either hypothesis. this type of analysis has been successfully accomplished for o. h. robertsoni (hauswald et al. 2011) and japanese pomatiopsids (kameda and kato 2001). the presence of fewer o. h. quadrasi haplotypes compared to the chinese populations may be explained by isolation by distance, or either founder effect or population bottlenecking. genetic differentiation among individuals increases as geographical distance increases, which is brought about by isolated populations being prevented from mating with other populations (wright 1943). this could explain why china, which is much larger than the philippines, has more snail haplotypes. founder effect occurs when populations originate from only a few individuals (freeman and herron 2004), such as in achatina fulica populations (fontanilla et al. 2014). this may be the case if o. h. quadrasi were introduced by migratory birds from chinese o. hupensis ancestors. population bottlenecking, the other possibility, may have been the result of early schistosomiasis control efforts, wherein the use of molluscicides successfully reduced snail populations. however, when the use of molluscicides was halted due to adverse environmental effects, the surviving snail populations repopulated the affected areas, resulting in reduced genetic variation among progenies. the use of molluscicides could also have acted as a selection pressure that caused the appearance of new strains, which is a possible explanation of the emergence of the bohol haplotype from the shared haplotype. the founder effect seems to be more likely since negros occidental and cagayan valley were recently discovered to be schistosomiasis endemic areas and were previously not subjected to control efforts. however, o. h. quadrasi populations in these two areas may have been unintentionally reduced together with crop pests as they co-inhabit rice f ields (freeman and herron 2004). with most isolates under a common haplotype, effects of existing snail control measures (e.g. , clearing of vegetation and exposure of habitats to direct sunlight) on these haplotypes would unlikely vary and are expected to have the same impact as in previous instances. however, more detailed studies on tolerance ranges, habitat preferences, and molluscicide susceptibility, particularly on other haplotypes, are needed to ascertain this assumption. genetic comparison of oncomelania hupensis quadrasi 44 t ab le 5 . d is tr ib u ti o n o f 1 6 s r r n a g e n e h ap lo ty p e s at v ar io u s lo ca li ti e s sa m p le d f o r o . h . q u a d ra si , o . h . h u p en si s an d o . h . n os op h or a. o h q 1 o h q 2 o h q 3 o h q 4 o h h 1 o h h 2 o h h 3 o h h 4 o h h 5 o h h 6 o h h 7 o h n 1 m c a g 2 7 2 7 tc ag 3 3 t s o r 3 0 3 0 b d m in 3 0 3 0 b n eg 2 2 2 2 h n eg 6 6 m b o h 3 0 3 0 p le y 1 3 1 3 d le y 1 1 1 1 s a m 1 8 1 8 d av 2 9 2 9 d a s 2 2 w a n h 4 4 ta n h 4 2 2 h a n h 4 4 x a n h 4 4 la n h 4 4 d a n h 4 2 2 s a n h 4 4 jh u n 4 2 2 c z h e 4 4 k o fu 7 7 1 6 3 2 2 6 3 0 6 8 4 4 6 6 2 7 to ta l : 2 6 4 2 2 1 3 6 7 lo ca li ty n n u m b er o f sa m p le s in e ac h h ap lo ty p e o f o . h . qu ad ra si n u m b er o f sa m p le s in e ac h h ap lo ty p e o f o . h. h up en si s n u m b e r o f sa m p le s in e ac h h ap lo ty p e o f o . h . no so ph or a j.c. c. chua et al. 45 the use of additional markers as concatenated sequences is recommended to further characterize the different haplotypes present in the philippines. the lack of genetic substructuring among the o. h. quadrasi isolates based on the 16s gene in this study is in contrast to what saijuntha et al. (2014) observed. combining the markers may provide greater phylogenetic signal and more variable positions. it should be noted that the evolutionary histories of taxa are reflected by the evolutionary histories of their genomes rather than by single genes, which may have different evolutionary histories that interact with each other and with the environment (morrison 2006). conclusions the current study was able to demonstrate o. h. quadrasi’s genetic diversity using the 16s rrna gene. o. h. quadrasi is a distinct subspecies with four haplotypes, two from negros occidental (ohq2 and ohq3), a single haplotype in bohol (ohq4), and one from the remaining provinces in the study (ohq1). there might be a need for the re-evaluation of the o. hupensis subspecies classif ication as chinese isolates spread into the two clades. competing interests the authors do not have any competing interests. authors’ contributions jc, if, mk, nh, yc, and ll conceptualized the project. jc, pt, it, and ll performed snail collection in the philippines, while ta provided samples from china and japan. jc, if, erdc, pt, it, rjf, and ta performed pcr, sequence alignment, and data analysis. jc, if, ll, it, and rjf wrote the manuscript with the suggestions of all the other authors. acknowledgments we thank the f ield and technical staff of the municipal health off ice and barangay health units in the provinces where we collected the samples. this research was supported by the philippine council for health research and development (pchrd) of the department of science and technology (dost) and the natural sciences research institute of the university of the philippines, diliman. genetic comparison of oncomelania hupensis quadrasi 46 references attwood sw, faith fa , campbell i, upar tham es. 2008. the distribution of mekong schistosomiasis, past and future: preliminary indications from an analysis of genetic evolution in the intermediate host. parasitology international. 57(3):256-270. attwood 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63:1-124. okamoto m, lo ct, t iu wu, qui d, hadidjaja p, upatham s, sugiyama h, taguchi t, hirai h, saitoh y, habe s, kawanaka m, hirata m, agatsuma t. 2003. phylogenetic relationships of snails of the genera oncomelania and tricula inferred from the mitochondrial 12s rna gene. japanese journal of tropical medicine and hygiene. 31(1):5-10. genetic comparison of oncomelania hupensis quadrasi 48 pesigan tp, hairston ng, jauregui jj, garcia eg, santos at, santos bc, besa aa. 1958. studies on schistosoma japonicum infection in the philippines. 2. the molluscan host. bulletin of the world health organization. 18(4):481-578. pinceel j, jordeans k, van houtte n, bernon g, backeljau t. 2005. population genetics and identity of an introduced terrestrial slug: arion subfuscus s.l. in the north-east usa (gastropoda, pulmonata, arionidae). genetica. 125:155-217. posada d. 2008. jmodeltest: phylogenetic model averaging. molecular biology and evolution. 25:1253-1256. rachford fw. 1977. oncomelania hupensis quadrasi from mindoro (victoria), leyte (palo), and mindanao (davao del norte) of the philippines: susceptibility to infection with philippine isolates of schistosoma japonicum. journal of parasitology. 63(6):1129-1130. saijuntha w, jarilla b, leonardo a , sunico l, leonardo l, andrews r, sithithaworn p, petney t, kirinoki m, kato-hayashi n, kikuchi m,chigusa y, agatsuma t. 2014. genetic structure inferred from mitochondrial 12s ribosomal rna sequence of oncomelania quadrasi, the intermediate snail host of schistosoma japonicum in the philippines. the american journal of tropical medicine and hygiene. 90(6):1140-1145. saitou n, nei m. 1987. the neighbor-joining method: a new method for reconstructing evolutionary trees. molecular biology and evolution. 4:406-425. slatkin m, hudson rr. 1991. pairwise comparisons of mitochondrial dna sequences in stable and exponentially growing populations. genetics. 129:555-562. stadler t, frye m, neiman m, lively cm. 2005. mitochondrial haplotypes and the new zealand origin of clonal european potamopyrgus, an invasive aquatic snail. molecular ecology. 14(8):2465-2473. swofford, dl. 2002. paup* 4.0b10. sunderland (ma): sinauer associates. ta m u r a k , s t ec h e r g , pe t e r s o n d, f i l i p s k i a , ku m a r s . 2 0 1 3 . m e g a 6 : m o l e c u l a r evolutionary genetics analysis version 6.0. molecular biology and evolution. 30:27252729. v iyanant v, upatham es, blas bl, yuan hc. 1987. analysis of allozymes by electrofocusing in schistosome snail hosts (oncomelania hupensis) from china and the philippines. malacological review. 20:91-96. [who] world health organization. 1993. who technical report series. the control of s c h i s t o s o m i a s i s : 2 n d r e p o r t o f t h e w h o e x p e r t c o m m i t t e e . g e n e v a : w o r l d h e a l t h organization. w ilke t, davis gm, dongchuan q, spear rc. 2006. extreme mitochondrial sequence diversity in the intermediate schistosomiasis host oncomelania hupensis robertsoni: another case of ancestral polymorphism? malacologia. 48:143-157. j.c. c. chua et al. 49 _____________ james christopher c. chua <jameschristopher.chua@gmail.com> is a full time faculty at the college of medical technology of the chinese general hospital in manila. he obtained his bs medical technology at far eastern university and ms public health at the college of public health, up manila. he specializes in medical laboratory science, public health and parasitology. ian kim b. tabios is a medical student at the college of medicine, up manila under the md-ph.d. (molecular medicine) program. he obtained his bs. biology at the institute of biology, up diliman. he specializes in parasitology and molecular medicine. pebbles grayle p. tamayo is a science research specialist at the research institute for tropical medicine, department of health and an ms public health student at the college of public health, up manila. she obtained her bs biological sciences at west visayas state university, iloilo city. she specializes in public health and parasitology. lydia r. leonardo is a professor of parasitology at the department of parasitology, college of public health, up manila. she is a recognized expert in schistosomiasis research in the philippines. she obtained her bs zoology at up diliman, ms marine biology at the marine science institute, up diliman, and doctor of public health at the college of public health, up manila. she specializes in marine biology, public health and parasitology. ian kendrich c. fontanilla is an associate professor and head of the dna barcoding laboratory at the institute of biology, up diliman. he received his ph.d. in genetics from the university of nottingham, united kingdom. he specializes in malacology, molecular genetics and phylogenetics. woodruff ds, staub kc, upatham es, viyanant v,yuan, hc. 1988. genetic variation in oncomelania hupensis: schistosoma japonicum transmitting snails in china and the philippines are distinct species. malacologia. 29:347-361. wright s. 1943. isolation by distance. genetics. 28(2): 114-138. xia x, xie z. 2001. dambe: software package for data analysis in molecular biology and evolution. journal of heredity. 92(4):371-373. zhao pz, jiang ms, littlewood dtj & nie p. 2010. distinct genetic diversity of oncomelania hupensis, intermediate host of schistosoma japonicum in mainland china as revealed by its sequences. plos neglected tropical diseases. 4(3):e611. genetic comparison of oncomelania hupensis quadrasi 50 raffy jay c. fornillos is a university research associate at the natural science research institute, up diliman and a mscience biology (genetics) student at the institute of biology, up diliman. he obtained his bs biology at leyte normal university, tacloban city. he specializes in molecular genetics, phylogenetics and parasitology. emmanuel ryan c. de chavez is an assistant professor and research coordinator at the institute of biological sciences, up los baños. he obtained his ph.d. in life sciences at the graduate of school of life sciences, tohoku university in tohoku, japan. he specializes in malacology, community ecology and evolutionary biology. takeshi agatsuma is a special assistant professor at kochi university school of medicine and a former professor at the department of medicine, kochi medical school in kochi, japan. he specializes in parasitology, including sanitary zoology. mihoko kikuchi is a senior assistant professor at the department of immunogenetics, institute of tropical medicine (nekken), nagasaki university, nagasaki, japan. she specializes in immunogenetics and infectious diseases. naoko kato-kayashi is an assistant professor at the school of medicine, dokkyo medical university, tochigi, japan. she specializes in public health, health science, and parasitology. yuichi chigusa is a professor at the school of medicine of dokkyo medical university, tochigi, japan. he specializes in public health, sociology and history of science and technology, and parasitology, including sanitary zoology. lubricant-adewuyi.pmd adewuyi, a. and oderinde, r.a. 12 science diliman (july-december 2012) 24:2, 12-20 adewale adewuyi a,b*and rotimi a. oderinde c adepartment of chemical sciences, redeemer’s university, mowe, ogun state, nigeria bcentre for lipid research, india institute of chemical technology, hyderabad – 500 007 cindustrial unit, department of chemistry, university of ibadan, ibadan, oyo state, nigeria corresponding author*: phone*: +2348035826679; email: walexy62@yahoo.com abstract oil was extracted from the seed of the lonchocarpus sericeus using hexane in a soxhlet extractor. the oil was characterized and used for the synthesis of polyol via epoxy ring opening reaction with 2-ethylhexanol. the structural characterization of the polyol was confirmed using ftir and 1hnmr. the gc analysis of the oil of l. sericeus revealed c18:3 and c18:1 fatty acid as the dominant fatty acids present in the oil. the polyol had hydroxyl value of 182.10 ± 0.20 mg koh/g, a copper strip corrosion value of 1a and a flash point of 280.00 ± 1.20oc. the synthesis and lubricant properties exhibited by the polyol suggested that the seed oil of l. sericeus can be chemically modified and employed as a starting material for lubricant production. keywords: epoxidation, fatty acids, lonchocarpus sericeus, lubricant, polyol lubricant properties of the polyol from the seed oil of lonchocarpus sericeus lubricant properties of polyol from lonchocarpus sericeus 13science diliman (july-december 2012) 24:2, 12-20 introduction unsaturated fatty compounds have found new applications as renewable raw materials as the use of renewable resources in industrial application is of great importance to the oleochemical industry (kirk-othmer 1996). these compounds can be functionalized at the c-c double bond by electrophilic addition reactions to give oleochemicals potentially new and interesting properties. vegetable oil, for example, has been found to be a renewable resource for industrial applications that can be used as base stock for the production of environmentally friendly and rapidly biodegradable lubricants (ghazali and others 2006). vegetable oil normally consists of a triglyceride mixture of fatty acids with the individual fatty acids characterized by the number of carbon atoms in the hydrocarbon chain, ranging generally from c8 to c22 and additionally by the number of double bonds (unsaturated bonds) in the chain. lubricants are a substance used to reduce friction between moving surfaces. they may also serve the function of transporting foreign particles and of distributing heat. typically, lubricants are 90% composed of a base oil (most often petroleum fractions, called mineral oils) with the remaining 10% being additives (bartels and others 2003). vegetable oils or synthetic liquids such as hydrogenated polyolefins, esters, silicones, fluorocarbons and many others are sometimes used as base oils. additives deliver reduced friction and wear, increased viscosity, improved viscosity index, and resistance to corrosion and oxidation (boughton and horvath 2004). in addition to industrial applications, lubricants are used for many other purposes which include bio-medical applications on humans (e.g. lubricants for artificial joints), ultrasound examinations, internal examinations for males and females, and the use of personal lubricants for sexual purposes. however, the application of vegetable oils as a lubricant is limited due to their low oxidation stability and high melting point, which is due to the â-carbon in the glycerol molecule and the unsaturation or double bonds in the acyls of the fatty acids (kaya and others 2009). chemical modification of vegetable oils by addition reactions to the double bonds constitutes a promising way of improving the property and quality of vegetable oil (wagner and others 2001, birova and others 2002). this modification can be achieved by introducing a different functional group to the unsaturation or double bonds in the acyls of the fatty acids. polyols are compounds containing more than one hydroxyl group (oh). each hydroxyl is attached to separate carbon atoms of an aliphatic skeleton. polymeric polyols are mainly used as reactants to make other polymers. they have several applications such as in the production of polyurethanes, lubricants, fibers, foam insulators, adhesives and protective coatings (john and others 2002, narine and others 2007). the lowtemperature behavior, stability, evaporation tendencies, ageing resistance, and compatibility with technical materials present polyols of vegetable oils as promising lubricants (sharma and others 2006, marchetti and errazu 2008). the lonchocarpus sericeus is a shrub or small tree that can grow from 10 to 16 meters high. it flowers with dense hanging racemes of purple flowers, mainly when leafless, which makes it perfect for display purposes. it is frequently planted in villages as a shadetree and in gardens and commentaries. the wood is clear yellow, sometimes marbled, with heart-wood and olive-green. the bark strips easily and is a good source of fiber. the flowers have a marked smell similar to vanilla. the fruit and seeds, however, are considered to be violently poisonous (burkill 1994). the seed of this plant is often discarded as waste in nigeria and there is no specific use for either the seed or oil from the plant. this present work evaluated the properties of polyol synthesized from the seed oil of l. sericeus by epoxy ring opening reaction with 2-ethylhexanol. materials and methods materials the mature seeds of the lonchocarpus sericeus were collected from the trees grown at the garden of the university of ibadan in oyo state, nigeria. they were adewuyi, a. and oderinde, r.a. 14 identified at the herbarium unit of the botany department of the university of ibadan. formic acid (100%), hydrogen peroxide (30%), and 2-ethylhexanol were purchased from merck (darmstadt, germany). all solvents and chemicals used in this study were of analytical grade and were purchased from s.d. fine chemicals, mumbai. physicochemical analysis of lonchocarpus sericeus oil from the dried seeds of the l. sericeus were extracted with n-hexane for 10 hours using soxhlet extractor (adewuyi and others 2009). the oil was analyzed for iodine value, saponification value, and free fatty acid content by methods described by the association of official analytical chemists (aoac 1994). the percentage oxirane value of the epoxidised l. sericeus seed oil was determined by aocs method (aocs 1997a). fatty acid composition of lonchocarpus sericeus fatty acid methyl esters of the oil were prepared by refluxing the samples at 70oc for 3 hours in 2% sulphuric acid in methanol. the esters were extracted into ethyl acetate, washed free of acid, and passed over anhydrous sodium sulphate. the ethyl acetate extracts were further concentrated using a rotary evaporator. the fatty acid composition was analyzed using an agilent 6890 n series gas chromatography equipped with a flame ionization detector (fid) on a split injector. a fused silica capillary column (db-225, 30 x 0.32 m i.d., j & w scientifics, usa) was used with the injector and detector temperature maintained at 230oc and 250oc, respectively. the oven temperature was programmed to 160oc for 2 minutes and finally increased to 230oc at 4oc/min. the carrier gas was nitrogen at a flow rate of 1.5 ml/min. the area percentages were recorded with a standard chemstation data system. epoxidation of the oil of lonchocarpus sericeus the epoxidation was carried out in 150-ml threenecked round-bottom flask equipped with a thermometer sensor and a mechanical stirrer. the whole apparatus was kept in an oil bath to maintain the desired temperatures. 0.0482 mol of the methyl esters and 0.106 mol of 100% formic acid were placed in the flask and cooled to a temperature of 15oc while stirring. 0.407 mol of hydrogen peroxide was added, drop-wise, with continuous stirring for about 30 minutes. the temperature was later raised to 70oc and maintained at this temperature for 3 hours. after the formation of epoxide, the mixture was cooled to room temperature and the epoxidised oil was extracted with ethyl acetate, washed with water until free of acid, and passed over sodium sulfate. this was later concentrated using a rotary evaporator. the equation of reaction is shown in figures 1a and b. synthesis of polyol from the epoxidised oil of lonchocarpus sericeus polyol was synthesized from the epoxidised oil of the l. sericeus by oxirane ring opening using 2ethylhaxanol in the presence of tetrafluoroboric acid as catalyst (1 % by weight of 2-ethylhexanol and epoxidised oil). the molar ratio of the epoxyl group to the oh group was 1:10. alcohol and catalyst were placed in a 500 ml three-neck flask equipped with a refluxing column, a mechanical stirrer, and a thermometer. the flask was heated using an oil bath. epoxidised oil was then added to the mixture of the alcohol and the catalyst. the reaction mixture was kept at 80oc for 3 hours. after cooling to room temperature, ammonia (30% in water) was added to neutralize the catalyst. the reaction mixture was washed with water several times and passed over sodium sulphate. the resulting product was later concentrated using a rotary evaporator. the equation of reaction is shown in figure 1c. h2 o 2 + hcooh hcoooh + h 2o -ch=ch + hcoooch -ch-hc + hcooh o c h c h o c c y o h y y= o-ch2-(ch2)3-ch-ch3 ch2ch3 figure 1c. epoxy ring opening with 2-ethylhexanol figure 1a. formation of peroxyacid (rangarajan and others 1995, okieimen and others 2002) figure 1b. epoxidation (rangarajan and others 1995, okieimen and others 2002) science diliman (july-december 2012) 24:2, 12-20 lubricant properties of polyol from lonchocarpus sericeus 15 fourier transform infrared spectroscopy (ftir) the ftir spectra of the oil and polyol were recorded using a perkin elmer ftir system spectrum bx lr64912c. the samples were spread over nacl cells, and their spectra were recorded in the range of 4000400 cm-1. nuclear magnetic resonance spectroscopy (nmr) 1hnmr spectra of the oil and polyol were obtained using a 300 mhz brucker nmr spectrophotometer in cdcl 3 containing some amount of tms as internal standard. properties of polyol from lonchocarpus sericeus a copper corrosion test was carried out using the method described by the standard method of the american society for testing and materials (astm) (2004). the four-ball weld load test of the polyol was estimated following the astm method (2010a). viscosity was determined as described by the standard method of the astm (1998). the oxidative stability was determined using the standard test for oxidation stability of steam turbine oils by rotating pressure vessel as described by the astm (2002), while the hydroxyl value of the polyol was established as described by the standard method of aocs (1997c). density was determined using the anton paar density meter (dma 450m model) as described by the astm 4052 (2009). the flash point was evaluated using the aocs modified method for closed cup flash point determination (1997b). emulsion stability was evaluated using the scavini apparatus equipped with a tachometer, speed variator, and thermostated heater according to astm d 1401 (2010b) while pour point was determined using the astm d 5949 method (2010c). results and discussion physicochemical analysis and fatty acid composition of lonchocarpus sericeus seed oil table 1 presents the physicochemical characterization of l. sericeus seed oil. the percentage oil content of the seed of l. sericeus was found to be 28.00 ± 0.50 %. the free fatty acid was 1.61±0.10 %. the color of the oil was light green, while the iodine value was found to be 166.88±0.80 g iodine/100g. the oil was liquid at room temperature with a saponification value of 195.20±0.50 mgkoh/g. the dominant fatty acid found in the oil were c18:1 (27.80 ± 0.20 %) and c18:3 (26.30 ± 0.20 %) as shown in table 2. c24:0 was found as 4.60 ± 0.10 % while c22:0 was 13.90 ± 0.40 %. fatty acids l. sericeus 16:0 7.50 ± 0.10 18:0 4.30 ± 0.10 18:1 27.80 ± 0.20 18:2 11.30 ± 0.50 18:3 26.30 ± 0.20 20:0 1.60 ± 0.50 20:1 2.30 ± 0.10 22:0 13.90 ± 0.40 22:1 0.40 ± 0.20 24:0 4.60 ± 0.10 unsaturated 68.10 ± 0.30 saturated 31.90 ± 0.40 values are mean + standard deviation of duplicate determinations table 2. fatty acid compositions (wt%) of l. sericeus seed oils epoxidation and hydroxylation of lonchocarpus sericeus seed oil the presence of an unsaturated functional group was confirmed in the seed oil of l. sericeus by ftir and 1hnmr as shown in figures 2a, 2b, and 3. the unsaturation of the fatty acid was found to be 68.10 ± 0.30 %, as shown in table 2. the epoxidation reaction science diliman (july-december 2012) 24:2, 12-20 table 1. physicochemical characterization of the oils from l. sericeus parameter l. sericeus oil yield (%) 28.00 ± 0.50 colour light green free fatty acid (%) 1.61±0.10 iodine value(g iodine/100g) 166.88±0.80 state at room temperature liquid saponification value(mgkoh/g) 195.20±0.50 values are mean + standard deviation of triplicate determinations adewuyi, a. and oderinde, r.a. 16 was characterized by two main reactions involving the formation of peroxoacid (peroxoformic acid) and formation of epoxides. the first step is the acidcatalyzed formation of peroxoformic acid from formic acid, while the second step is the uncatalyzed epoxidation of the l. sericeus seed oil with the peroxoformic acid. the percentage oxirane oxygen content of the epoxidised l. sericeus seed oil was found to be 5.10 ± 0.40 %. the ftir spectra of the oil and polyol are shown in figures 2a and b. the c-h stretching of c=c-h in the oil, which suggests the presence of unsaturated functional groups, was detected at 3010 cm-1. the unsaturated peak disappeared in the polyol indicating that the unsaturated bonds have been modified. bands were noticed at 1736 and 730 cm-1, which could be accounted for as being the c=o stretching frequency of ester. the peak at 2930 cm-1 was also found in the spectra, which may be attributed to the c-h stretching of –ch 3. the peak at 3452 could be accounted for as being the vibrational frequency of the oh functional group suggesting the formation of the polyol. figures 3a and 3b show the 1hnmr spectra of the oil and polyol, respectively. the ethylene protons were observed only in the oil at 5.3 ppm confirming the unsaturation observed at 3010 cm-1 in the ftir spectrum. the methane proton of the ch backbone of the â-glycerol carbon was observed at 4.4 ppm. the methyl groups of the esters in the oil and polyol were seen at 2.3 ppm and 1.9 ppm, respectively. the saturated methylene groups were observed at about 1.1-1.4 ppm, while terminal methyl groups exhibited a chemical shift between 0.5 and 1.0 ppm in the oil and polyol. the formation of polyol was confirmed by a signal at 2.8 ppm, which was assigned to the contribution from the hydroxyl functional groups. properties of polyol from lonchocarpus sericeus the hydroxyl value of the polyol from l. sericeus was determined to be 182.10 ± 0.20 mg koh/g as presented in table 3. the hydroxyl value is higher than what was reported for canola based polyol (152.4 ± 0.3 mg koh/ g), castor oil (165.2 ± 3.8 mg koh/g), and in the range of that of soybean based polyol (narine and others 2007). this is important in quality control and it gives an idea of the molecular weight and the functionality of the polyol, especially when it is to be used in the creation of other products such as in the case of polyurethane. a copper strip corrosion test indicated some clues on the possibility of corrosion or corrosion tendency of figure 2a. ftir spectra of the oil of l. sericeus science diliman (july-december 2012) 24:2, 12-20 lubricant properties of polyol from lonchocarpus sericeus 17 figure 2b. ftir spectra of the polyol of l. sericeus 0.00.00.50.51.01.01.51.52.02.02.52.53.03.03.53.54.04.04.54.55.05.05.55.56.06.06.56.5 0 . 50 . 51 .01 .01 .51 .52 .02 .02 .52 .53 .03 .03 .53 .54 . 04 . 04 .54 .55 . 05 . 05 .55 .56 . 06 . 06 .56 .57 .07 .07 . 57 . 58 .08 .0 figure 3a. 1hnmr spectra of the oil of l. sericeus figure 3b. 1hnmr spectra of the polyol of l. sericeus science diliman (july-december 2012) 24:2, 12-20 adewuyi, a. and oderinde, r.a. 18 the polyol. the copper strip corrosion test of l. sericeus polyol gave a result of 1a indicating the stability of this polyol towards corrosion. this is also an indication that the oil of l. sericeus may be suitable as a feed stock for the production of lubricants since the 1a value reflects the ability of this polyol to withstand corroding conditions . the four-ball wear test was indicative of the relative wear-preventive properties of lubricating fluids in sliding contact under the prescribed test conditions. the value of the four-ball weld load test was found as 140.00 ± 0.00 kg for the polyol of l. sericeus. the flash point was determined to be 280.00 ± 1.20oc; the flash point is the lowest temperature at which the polyol can vaporize to form an ignitable mixture in air, which helps in characterizing and classifying such products. a value of 221oc has been recommended by international organization for standardization (iso) for grade 32 oil, while the density was 0.857 g/m3 (rexnord 1998, brhrtsg 2004). the flash point in the study was higher than the iso specification, which indicated that l. sericeus polyol can be easily handled and transported since flammability will not be a serious issue; moreover, the use of such as a lubricating agent in high temperature systems will be an advantage. part of iso specifications also included a viscosity of 32 cst at 40oc, and 5.4 cst at 100oc. the viscosity in the present study was noticed to decrease as temperature increases as this was found to be 32.62 ± 0.50 cst at 25oc, 17.04 ± 0.20 cst at 40oc and 3.00 ± 0.30 cst at 100oc; these values suggest the flow of this polyol at the examined temperatures and the conditions at which they could be used. the density was also found to follow this trend by reducing as temperature increases just as the pour point was found to be 2.50 ± 0.20oc. the oxidative stability was found to be 85.00 ± 0.50 min with an iodine value of 0.30 ± 0.10 g iodine/100g while the emulsion stability was 9.00 ± 0.20 min. apart from the iso, other specifications have been recommended by various bodies such as the american petroleum institute (api) and the american society for testing and material (astm). conclusion the lubricant properties of polyol synthesized from the seed oil of lonchocarpus sericeus was studied. the structural characterization of the synthesized polyol was confirmed using ftir and 1hnmr. the properties such as copper strip corrosion test, hydroxyl value, flash point, viscosity and four-ball wear exhibited by the polyols in the present study showed that the seed oil of l. sericeus can be chemically modified and employed as a starting material for lubricant. acknowledgments adewuyi adewale thanks the third world academy of sciences (twas) for awarding him the research fellowship that allowed him to carry out this work at the indian institute of chemical technology (iict). thanks also to dr j. s. yadav, director of the iict, for his support and encouragement. references parameter l. sericeus iodine value (g iodine/100g) 0.30 ± 0.10 hydroxyl value (mg koh/g) 182.10 ± 0.20 viscosity (25oc) (cst) 32.62 ± 0.50 viscosity (40oc) (cst) 17.04 ± 0.20 viscosity (100o) (cst) 3.00 ± 0.30 copper strip corrosion 1a pour point (oc) 2.50 ± 0.20 oxidative stability (min) 85.00 ± 0.50 four ball test (kg) 140.00 ± 0.00 emulsion stability(min) 9.00 ± 0.20 density (25o) (g/cm3) 0.993 ± 0.10 density (40o) (g/cm3) 0.981 ± 0.10 density (90o) (g/cm3) 0.929 ± 0.10 flash point (oc) 280.00 ± 1.20 values are mean + standard deviation of triplicate determinations table 3. lubricant properties of the polyol from l. sericeus science diliman (july-december 2012) 24:2, 12-20 adewuyi a, oderinde ra, ajayi ia. 2009. the metal composition, proximate properties and the effect of refining on the physicochemical characterization of baphia nitida and gliricidia sepium seed and seed oil. j. food technol. 7: 43-49. lubricant properties of polyol from lonchocarpus sericeus 19science diliman (july-december 2012) 24:2, 12-20 association of official analytical chemists (aoac). 1997c. aocs official method for the determination of hydroxyl value [cd 13-60]. in: official methods of analysis, 16th ed. va: aoac. p 1-2. bartels t, bock w, braun j, busch c, buss w, dresel w, freiler c, harperscheid m, heckler r, hörner d, kubicki f, lingg g, losch a, luther r, mang t, noll s, omeis j. 2003. lubricants and lubrication. ullman’s encyclopedia of industrial chemistry, 6th ed. wiley-vch. birova a, pavlovicova a, cvengros j. 2002. lubricating oils based on chemically modified vegetable oils. j. synth. lubr. 18: 291-99. boughton b, horvath a. 2004. environmental assessment of used oil management methods. environ. sci. technol. 38: 353-58. bureau of reclamation hydroelectric research and technical services group. 2004. lubrication of powerplant equipment: facilities, instructions, standards, and techniques. volume 2-4. colorado: u.s. department of the interior, bureau of reclamation. burkill hm. 1994. the useful plants of west tropical africa, 2nd ed. royal botanic gardens, kew. ghazali z, wan nik wb, ku bulat kh, ani fn, xian lf. 2006. the effect of light on the oxidative stability of palm olein. in: proceedings of the 1st international conference on natural resources engineering & technology; 2006 july 24-25; putrajaya, malaysia. p 631-37. john j, bhattacharya m, turner rb. 2002. characterization of polyurethane foams from soybean oil. j. appl. polym. sci. 86: 3097-3107. kaya cc, hamamci a, baysa o, akba s, erdogan a, saydut a. 2009. methyl ester of peanut (arachis hypogeal l.) seed oil as a potential feedstock for biodiesel production. renewable energy 34: 1257-60. kirk re, othmer df. 1996. kirk-othmer encyclopedia of chemical technology, 4th ed, vol 21. ny: john wiley. american society for testing and materials (astm). 1997. standard test method for kinematic viscosity of transparent and opaque liquids [astm d445-97]. pennsylvania (pa): astm international. american society for testing and materials (astm). 2002. standard test method for oxidation stability of steam turbine oils by rotating pressure vessel [astm d2272-02]. pennsylvania (pa): astm international. american society for testing materials (astm). 2004. copper strip corrosion test d396 specification for fuel oils. pennsylvania (pa): astm international. american society for testing materials (astm). 2009. standard test method for density, relative density, and api gravity of liquids by digital density meter [astm d405209]. pennsylvania (pa): astm international. american society for testing materials (astm). 2010a. standard test method for measurement of extreme-pressure properties of lubricating grease (four-ball method) [astm d2596-10]. pennsylvania (pa): astm international. american society for testing materials (astm). 2010b. standard test method for water separability of petroleum oils and synthetic fluids [astm d1401-10]. pennsylvania (pa): astm international. american society for testing materials (astm). 2010c. standard test method for pour point of petroleum products (automatic pressure pulsing method) [astm d5949-10]. pennsylvania (pa): astm international. association of official analytical chemists (aoac). 1994. official methods of analysis, 14th ed, vol 67. va: aoac. p 503-15. association of official analytical chemists (aoac). 1997a. american oil chemists’ society (aocs) official method cd 9-57. in: official methods of analysis, 16th ed. va: aoac. p 1-2. association of official analytical chemists (aoac). 1997b. aocs official method for flash point closed cup method (modified closed cup method, astm d93-80)[cc 9b-55]. in: official methods of analysis, 16th ed. va: aoac. adewuyi, a. and oderinde, r.a. 20 science diliman (july-december 2012) 24:2, 12-20 marchetti jm, errazu af. 2008. esterification of free fatty acids using sulfuric acid as catalyst in the presence of triglyceride. biomass and bioenergy 32: 892-95. narine ss, kong x, bouzidi l, sporns p. 2007. physical properties of polyurethanes produced from polyols from seed oils: i. elastomers. j. am. oil chem. soc. 84: 55-63. okieimen fe, bakare io, okieimen co. 2002. studies on the epoxidation of rubber seed oil. ind. crops prod. 15: 139-44. rangarajan b, havey a, grulk ea, culnan pd. 1995. kinetics parameters of a two-phase model for in-situ epoxidation of soybean oil. j. am. oil chem. soc. 72: 1161-69. rexnord industries, llc, gear group. 1998. lubrication recommendations. wi: rexnord industries. sharma bk, adhvaryu a, liu z, erhan sz. 2006. chemical modification of vegetable oils for lubricant applications. j. am. oil chem. soc. 83: 129-36. wagner h, luther r, mang t. 2001. lubricant based fluids based on renewable raw materials, their catalytic manufacture and modification. appl. catal. 221: 429-42. 9guidelines.pmd 82 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) 05_palero palero and garcia 66 science diliman (january-june 2001) 13:1, 66-72 abstract the third-harmonic 355 nm output of a pulsed nd-yag laser is converted into uv, vis, and nir laser light by stimulated raman scattering in high pressure hydrogen gas. laser lines in the 223 to 309 nm and 416 to 865 nm spectral regions are generated by anti-stokes and stokes raman shifting, respectively. experimental results on the energy output, conversion efficiencies, spectral profile, and temporal behavior of the various stokes and anti-stokes raman laser lines are presented. the first and second stokes shifted wavelength with wavelengths of 416 nm and 503 nm yielded a maximum energy conversion efficiency of 60.4% and 58.8% , respectively. key words: stimulated raman scattering, raman shifting, frequency conversion. frequency conversion of the 355 nm nd:yag laser via stimulated raman scattering in hydrogen j.a. palero* and w.o. garcia photonics research laboratory, national institute of physics college of science, university of the philippines diliman, quezon city 1101, philippines e-mail: jonathan@nip.upd.edu.ph introduction stimulated raman scattering (srs) is a powerful technique for shifting the wavelength of laser radiation into another spectral region. it is capable of simultaneously generating multiple laser lines ranging from vacuum ultraviolet (vuv) to far infrared (fir). compared to other frequency conversion methods, it is simple, economical, robust, and capable of high conversion efficiency. srs has been used in a wide range of applications, such as generation of short laser pulses, time-gated low light level imaging, and lidar measurements. in this work, the third harmonic 355 nm output of a pulsed nd:yag laser is shifted into the uv, vis, and nir region of 223 to 865 nm through srs in high pressure hydrogen gas. the energy, spectral profile, and temporal behavior of the rayleigh and srs lines are measured as a function of the 355 nm laser excitation energy and hydrogen gas pressure. srs conversion efficiencies are calculated and presented. spontaneous and stimulated raman scattering a simplified diagram of the spontaneous raman effect is illustrated in fig. 1. an incident photon (ωp) excites a molecule from the ground state to a virtual state. as the molecule relaxes to the first excited state, it emits a photon (ωs) with a frequency lower than the incident photon. this frequency down-conversion process (ωp > ωs) is called stokes shifting. if the incident photon (ωp) is absorbed by a molecule initially in the first excited state, the emitted photon (ωas) will have a higher frequency as the molecule relaxes to the ground state. the upconversion of the photon frequency (ωp < ωas) is *corresponding author frequency conversion of the 355 nm nd:yag laser 67 referred to as anti-stokes shifting. one of the characteristic parameters in this phenomenon is the energy difference between the first excited state and the ground state called the raman frequency of the medium. for hydrogen gas, this quantity is equal to 4155.25 cm-1 corresponding to the energy between the ground state and the first vibrational state. at a sufficiently high incident photon flux, a process known as stimulated raman scattering (srs) can occur. it is a nonlinear optical process arising from the interaction of an intense laser beam with a raman active medium such as caco3, sicl4, ch4, and h2. the stimulated raman scattering process is shown in fig. 2. in fig. 2a-c, a pump photon ωp excites a molecule to the virtual state and interacts with a stokes photon ωs to stimulate the emission of another stokes photon ωs. fig. 2d-f show the stimulated emission of an anti-stokes photon by the interaction of an anti-stokes photon and a molecule in a virtual state. in contrast to the incoherent radiation produced through the spontaneous raman effect, srs leads to the generation of coherent light that has the same wavelength and phase as the incident fig. 2. the stimulated raman scattering process. fig. 1. a simplified diagram of the spontaneous raman effect virtual state ( a ) ( b ) ( a ) ( b ) ( c ) ( d ) ( e ) ( f ) palero and garcia 68 photon. furthermore, the srs output is much more intense than the weakly scattered radiation from the spontaneous raman effect. if the intensity of the first stokes-shifted frequency (s1) is high enough, another srs interaction can occur between s1 and the medium. this process lead to the generation of a second stokes-shifted frequency (s2). higher-order stokes wavelengths are produced by this process known as srs cascade. on the other hand, generation of higher order anti-stokes wavelengths is also possible by a nonlinear process known as fourwave mixing (fwm). it involves the interaction of three waves to form a fourth wave. fig. 3 shows the srs cascade and fwm processes involved in the generation of stokes and anti-stokes wavelengths. unlike the srs process, the generation of radiation by fwm is sensitive to the phase mismatch between the wave vectors of the interacting waves. these leads to the low efficiency of the fwm process at high pressures where the phase mismatch is greatest. fwm also contribute to the generation of higher-order stokes often to a greater extent than the srs cascade. table 1 shows the antistokes and stokes wavelengths generated by stimulated raman scattering in hydrogen using a 355 nm laser excitation. fig. 3. the srs cascade and fwm processes involved in the generation of stokes and anti-stokes wavelengths in the case of srs in gases, the plane-wave steadystate raman gain coefficient gr is given by: gr = 2 λ2s ∆n dδ ____ ______ ___ , hcνs πc∆νr dω where λs is the stokes wavelength (cm), νs is the stokes or raman frequency (cm-1), c is the speed of light (cm/ s), h is planck’s constant (js), ∆n is the difference in population between the initial and final states (cm-3), ∆vr is the raman linewidth (cm -1), and dσ/dω is the differential cross section for raman scattering (cm2/ sr). in the case of hydrogen, ∆vr = 11.2/p + 1.58p, where p is the gas pressure given in atm. an analysis ω p ωs1 ωs2 ω s1 ω s2 ωs3 ωp ωs1 ωas1 ωp ωp ωs1 ωas2 ωas1 (a) (b) (c) (d) (e) raman order wavelength as4 222.9 as3 245.8 as2 273.8 as1 309.0 s1 415.9 s2 502.9 s3 635.9 s4 864.5 table 1. anti-stokes and stokes wavelengths generated by stimulated raman scattering in hydrogen using a 355 nm laser excitation. ( a ) ( b ) ( c ) ( d ) ( e ) frequency conversion of the 355 nm nd:yag laser 69 of the gain coefficient as a function of pressure shows that it saturates for pressures greater than 20 atm. the stokes intensity varies exponentially with the interaction length (z) as is(z) = is(o) exp (grilz) where il and is are the incident laser intensity and the stokes intensity. according to these equations, the stokes intensity and the srs conversion efficiency depend on laser beam intensity, laser beam geometry, and raman active gas and its pressure. experimental setup & methodology fig. 4 shows the schematic diagram of the raman cell and the gas handling system. the raman cell is constructed from stainless steel hollow cylinders that are welded together. both ends of the cell are sealed with uv grade fused silica windows and o-rings. the cell is provided with inlet and outlet gas ports. a mechanical vacuum pump is used to evacuate the cell. upon evacuation, ultra high purity hydrogen (99.9999% purity) is introduced into the cell from a gas cylinder through a gas regulator and shut-off valve. a mechanical pressure gauge monitors the hydrogen pressure. a schematic diagram of the experimental setup is shown in fig. 5. the laser is a q-switched linearly polarized nd:yag laser (spectra physics gcr-230-10) which operates at 10 hz pulse repetition rate, a wavelength of 1064 nm, and 5–8 ns (fwhm) pulse width. 355 nm output is obtained by the use of potassium dideuterium phospate (kd*p) crystals. the laser beam is passed through a diaphragm (d) set at an aperture of 1.5-mm diameter. a glan laser polarizer (gp) is used to vary the laser pulse energy. the laser beam then is focused into a raman cell using a 50 cm lens (l1). a 30 cm lens (l2) placed at the exit port of the cell is used to collimate the raman output into a pellin-broca prism (pb) for separation into the rayleigh, srs stokes, and anti-stokes components. a pyroelectric detector (molectron j25hr) is used to measure the pulse energies of the 355 nm excitation laser, rayleigh, srs stokes, and anti-stokes lines. the spectral profile of the excitation, rayleigh, srs stokes, and anti-stokes lines are measured with a computercontrolled monochromator (spex 1000m). the temporal behavior is monitored with an ultra-fast biplanar phototube (hamamatsu r1328u-5) and a 500 mhz digitizing oscilloscope. results and discussion the spectral profile of the raman output at a maximum input pump energy of 6.5 mj and hydrogen pressure of 80 psi is shown in fig. 6. there are eight raman-shifted laser lines: four stokes and four anti-stokes, covering the ultraviolet to the near-infrared wavelength region. spectral line-widths (fwhm) ranging from 0.02 nm to 0.08 nm are observed. stokes lines are stronger than nd:yag laser (355 nm) hydrogen raman celld gp l1 l2 pb rayleigh, stokes and anti-stokes output fig. 5. schematic diagram of the experimental setup raman cell vacuum pump h2 pressure gauge pressure gauge end caps vent fig. 4. the schematic diagram of the raman cell and the gas handling s y s t e m . palero and garcia 70 the anti-stokes lines, and the intensity tends to weaken with increasing raman order. fig. 7 shows the temporal behavior of the raman output at different 355 nm laser excitation energy. the input laser pulse, which has a gaussian profile, is truncated at half its maximum to show the temporal behavior of the scattered pulses more clearly. at an excitation energy of 2.27 mj, generation of the first (s1) and second stokes (s2) are observed. the 355 nm rayleigh scattering has a lower energy than the input laser energy and a distortion in the pulse shape is observed due to the energy conversion into s1 and s2. a delay time between the rayleigh and s1 is evident in the figure due to the required pump threshold intensity to generate a stokes pulse. s2 also propagates with a time delay in respect to s1 by the same reason, which also caused a distortion on the pulse shape of s1. the observed time delay in the generation of higher-order stokes is a clear evidence of the srs cascade process. at an input laser energy of 4.45 mj, the increase in intensity of s1 and s2 is accompanied by the generation of s3 and as1. although s2 increases in intensity, distortion in its pulse shape becomes apparent because of the onset of s3. the first anti-stokes as1 is observed to propagate earlier than s2 or s3 which can be explained by the fact that anti-stokes wavelengths are generated not by the srs cascade but fwm. for instance, as1 is produced by the interaction of s1 and two photons of the pump, and although it cannot propagate earlier than either s1 or the rayleigh, it may be generated earlier than the higher order stokes. an increase in intensity for all raman-shifted pulses and the generation of a second anti-stokes shifted pulse as2 are observed at 6.45 mj of input laser energy. at this point, distortion of the stokes pulses which may be due to the increased efficiency of fwm at high laser intensity becomes very apparent. the parasitic behavior of the anti-stokes pulses with the stokes is due to the 0 5 10 15 r e lat iv e time (ns) 0 0. 1 0. 2 0. 3 0. 4 0 5 10 15 n or m al iz e d i n te n si ty 0 5 1 0 15 l a s e r ray le ig h s 1 s 2 s 3 a s1 a s2 e = 2. 2 7 mj e = 4. 4 5 mj e = 6. 4 5 mj relative time (ns) n o rm al iz ed i n te n si ty fig. 7. the temporal behavior of the raman output at different 355 nm laser excitation energy r e s u lt s 0 0.04 0.08 0.12 0.16 in te ns ity ( a. u. ) center wavelength (nm) 223.0 245.8 273.8 309.0 415.9354.6 635.9502.9 864.5 as4 as3 as2 as1 rayleigh s1 s2 s3 s4 fig. 6. the spectral profile of the raman output at a maximum input pump energy of 6.5 mj and hydrogen pressure of 80 psi center wavelength (nm) in te n si ty ( au ) frequency conversion of the 355 nm nd:yag laser 71 fact that generation of higher-order anti-stokes through fwm is not restricted to the interaction of one set of waves. in fact, when n stokes and anti-stokes beams are present (total number, including the pump), there is a total of (n–1) (n–2)/2 distinct fwm processes among them. fig. 8 shows the energy conversion efficiencies of the stokes and anti-stokes on the incident 355 nm laser energy at a hydrogen pressure of 80 psi. the threshold for the generation of the first stokes (s1), s2, s3, and anti stokes (as1) occur at 2.0 mj, 2.7 mj, 3.9 mj, and 2.3 mj, respectively. the dependence of the stokes and anti-stokes conversion efficiencies with hydrogen pressure at 6.5 mj of input laser energy is shown in fig. 9. the conversion efficiency is defined as the ratio between the output energy and the laser input energy. the peak conversion efficiency of s1 of 38% occurs at 300 psi. for s2, the peak conversion efficiency of 55% is attained at 500 psi. the conversion efficiency of the anti-stokes reaches a maximum at around 80 psi. anti-stokes and higher-order stokes are generated primarily by fwm processes at low pressures during which the phase mismatch of the interacting waves is minimum. at pressures greater than 150 psi, the phase mismatch is increased such that fwm is reduced. fig. 10 shows the optimum energy conversion efficiencies of the stokes and anti-stokes. conclusions stimulated raman scattering in hydrogen is investigated as a method for generating laser radiation in the region of 223 to 865 nm (uv to nir). a cascade process of 0 2 4 6 8 10 12 14 16 18 20 0 1 2 3 4 5 6 input pulse energy (mj) c on ve rs io n e ffi ci en cy ( % ) s1 s2 s3 s4 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 input pulse energy (mj) c on ve rs io n e ffi ci en cy ( % ) as1 as2 as3 as4 stokes anti-stokes fig. 8. energy conversion efficiencies of the stokes and anti-stokes on the incident 355 nm laser energy at 80 psi hydrogen pressure 0 10 20 30 40 50 60 70 0 150 300 450 600 hydrogen pressure (psi) c on ve rs io n e ffi ci en cy ( % ) s1 s2 s3 s4 0 1 2 3 4 5 6 0 150 300 450 600 hydrogen pressure (psi) c on ve rs io n e ffi ci en cy ( % ) as1 as2 as3 as4 stokes anti-stokes fig. 9. the dependence of the stokes and anti-stokes conversion efficiencies with hydrogen pressure 60.4 58.8 9.8 2.2 5.9 2.4 1.1 0.6 0 10 20 30 40 50 60 70 c on ve rs io n e ffi ci en cy ( % ) s1 (700psi) s2 (700psi) s3 (150psi) s4 (90psi) as1 (80psi) as2 (80psi) as3 (80psi) as4 (70psi) fig. 10. optimum energy conversion efficiencies of the stokes and anti-stokes input pulse energy (mj) input pulse energy (mj) hydrogen pressure (psi) hydrogen pressure (psi) c o n ve rs io n e ff ic ie n cy c o n ve rs io n e ff ic ie n cy c o n ve rs io n e ff ic ie n cy ( % ) c o n ve rs io n e ff ic ie n cy ( % ) stokes anti-stokesstokes anti-stokes c o n ve rs io n e ff ic ie n cy ( % ) palero and garcia 72 generation of the raman components are observed from the temporal behavior of the output. the energy conversion efficiencies of the stokes and anti-stokes are observed to increase with increasing energy and to exhibit saturation. anti-stokes and higher-order stokes are observed to have optimum conversion efficiencies at low pressures due to fwm generation while s1 and s2 are optimum at high pressures. energy conversion efficiencies of 60%, 59%, 10%, and 2% are measured for s1, s2, s3, and s4 stokes wavelengths, respectively. on the other hand, 6%, 2%, 1%, and 0.6% conversion efficiencies are achieved for as1, as2, as3, and as4 anti-stokes wavelengths, respectively. these results are comparable to the results of papayannis et al. (1998) who reported the generation of uv and vis laser light in h2 using a third harmonic 355 nm nd:yag laser pump with conversion efficiencies as high as 50% at the first stokes shifted frequency. acknowledgments the authors acknowledge the assistance provided by mr. jose omar s. amistoso and ms. marian f. baclayon, the support of the philippine department of science and technology through the engineering and science education project and the advise given by dr. caesar a. saloma and dr. marlon h. daza. references carnuth, w. & t. trickl, 1994. a powerful eyesafe aerosol lidar: application of stimulated raman backscattering of 1.06 mm radiation. rev. sci. instrum. 65: 3324-3330. chu, z., u.n. singh, & t.d. wilkerson, 1991. multiple stokes wavelength generation in h2, d2, and ch4 for lidar aerosol measurements. appl. opt. 30: 4350-4357. duncan, m.d., r. mahon, l.l. tankersley, & j. reintjes, 1992. low-light-level, quantum-noise-limited amplification in a stimulated raman amplifier. j. opt. soc. am. b. 9: 2107-2121. milton, m.j.t., g. amcellet, a. apituley, j. bosenberg, w. carnuth, f. castagnoli, t. trickl, h. edner, l. stefanutti, t. schaberl, a. sunesson, & c. weitkamp, 1998. raman-shifted laser sources suitable for differential-absorption lidar measurements of ozone in the troposphere. appl. phys. b. 66: 105-113. papayannis, a.d., g.n. tsikrikas, & a.a. serafetinides, 1998. generation of uv and vis laser light by stimulated raman scattering in h2, d2, and h2/he using a pulsed nd:yag laser at 355 nm. appl. phys. b. 67: 563-568. yamada, t., t. nakata, & f. kannari, 1995. subnanosecond pulse generation in the vuv by dualwavelength pumped stimulated rotational raman scattering. ieee j. quantum electron. 31: 1274-1284. sd-sample article 77 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions  if changes are made, choose a new title for the paper.  use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality.  reference your conference paper in the appropriate locations.  include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.”  indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed.  provide the original conference paper (upload a pdf f ile during the submission process).  if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions  expand the background section and include additional references.  include novel scientif ic content and expanded descriptions of procedures.  provide data that was not published at the conference.  revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission f rom the editors of ieee sensors journal) sc i e n c e di l i m a n is published semi-annually (june and december) by the university of the philippines diliman through the office of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief jonas p. quilang, ph.d. associate editors jose maria p. balmaceda, ph.d. university of the philippines louis angelo m. danao, ph.d. university of the philippines carlos primo c. david, ph.d. university of the philippines christian n. della, ph.d. university of glasgow singapore alonzo a. gabriel, ph.d.† university of the philippines arnold m. guloy, ph.d. university of houston gil s. jacinto, ph.d. university of the philippines dennis i. merino, ph.d. southeastern louisiana university arnel a. salvador, ph.d. university of the philippines terence p. tumolva, d.eng. university of the philippines irene m. villaseñor, ph.d. university of the philippines managing editor conchitina r. cruz, ph.d. university of the philippines editorial assistant narita e.c. de las alas layout artist mirriam m. velasco copyeditor jean lau wang on the cover: crustal deformation of southwestern luzon and its implications to the stability of the philippine survey network this is a deformation map of the luzon region, showing spatially-varying deformation rates (residual strain rate values) plotted together with the direction of deformation (normalized residual strain rate axes). credits: dr. elliot klein. contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. science diliman january-june 2020 • vol. 32 no. 1issn print 0115-7809 issn online 2012-0818 international advisory board teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus university of illinois, usa university of california, irvine, usa the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheffield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. department of physics university of warwick, united kingdom raul k. suarez, ph.d. professor emeritus university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences graduate school university of tsukuba, japan sd-sample article e.c. capinpin jr. 51 science diliman (january-june 2013) 25:1, 51-70 status of abalone fishery and experiential mariculture as a resource conservation strategy in carot, anda, pangasinan emmanuel c. capinpin jr. pangasinan state university abstract the study describes the abalone f ishery in carot , anda, pangasinan to develop mariculture and to reseed a part of the harvest as a resource conservation strategy. the abalone f ishery of anda is ar tisanal or smallscale, typif ied by f ishers gleaning or free-diving on shallow rocky areas w h i c h a r e t h e t y p i c a l h a b i t a t of a b a l o n e . low d e n s i t i e s of 1 . 6 7 t o 8 individuals per 250 m2 were observed. local f ishers have knowledge of productive f ishing areas. hence, cage culture of abalone in these areas c o u l d b e a v i a b l e r e s o u r c e c o n s e r v a t i o n s t r a t e g y a s t h e y s e r v e a s reproductive reserves to supply larvae for continued productivity of the f ishing grounds. abalone mariculture following the farmer field school (ffs) concept was explored to address both resource management and e c o n o m i c n e e d s . a s a r e s o u r c e e n h a n c e m e n t a c t i v i t y, m a r i c u l t u r e guarantees that cultured abalone are allowed to grow to maturity before harvested, while some are retained to restock a marine sanctuary. since mariculture makes possible the aggregation of individuals, the probability that fertilization would take place is increased. as supplemental source o f l i v e l i h o o d , a b a l o n e i s a h i g h v a l u e c o m m o d i t y a n d i t s c u l t u r e c a n h e l p a u g m e n t t h e i n co m e of f i s h e r s . s m a l l a b a l o n e ( 3 4 c m ) c a n b e c u l t u r e d f u r t h e r f o r 3 4 m o n t h s t o i n c r e a s e t h e i r s i z e a n d w e i g h t . mariculture should be done from november to may to avoid the rainy s e a s o n a n d i m p r o v e s u r v i v o r s h i p . t h e e x p e r i e n t i a l a c t i v i t y w a s s u cce s s f u l b e c a u s e i t b ec a m e a m e a n s f o r t h e f i s h e r s to ex p e r i e n ce resource management. under the ffs, the researcher became a facilitator a n d m e n t o r e d t h e c o o p e r a t o r s i n l e a r n i n g f r o m t h e i r e x p e r i e n c e . t h e l e s s o n s s h a r p e n ed t h e f i s h e r s ’ s k i l l s i n o b s e r v a t i o n , p r o b l e m s o l v i n g , d e c i s i o n m a k i n g , a n d c r i t i c a l t h i n k i n g . t h i s e n a b l e d t h e m t o g a i n a n appreciation of their resource. keywords: abalone f ishery, mariculture, farmer field school, conservation, resource management, experiential activity issn 0115-7809 print / issn 2012-0818 online status of abalone fishery and experiential mariculture 52 introduction fisheries of all kinds in the philippines have surpassed their sustainable levels of catch or are overf ished (green and others 2003). the sea urchin f ishery in bolinao, pangasinan is one example showing the disastrous effects of unregulated harvesting of marine invertebrates (juinio-meñez and others 2001). commercial harvesting started in the 1970s but lasted only a few years due to heavy exploitation. the scenario was aggravated by the practice of collecting small urchins even before they had the opportunity to reproduce. the university of the philippines marine science institute embarked on seed production to enhance the recovery of wild urchin populations through reseeding in marine protected areas, and the promotion of community-based grow-out culture. results were promising, with juvenile recruitment pulses recorded beginning in 1999 (juinio-meñez and others 2008a, b). at present, the sea urchin industry in bolinao is seen to have been revived and is thriving very well. this strategy could also work well with other highly exploited species like abalone. abalone can be found in carot, anda in pangasinan but is now believed to be overf ished. the area is being degraded due to overharvesting and other destructive f ishing practices such as destroying rocks to collect cryptic abalone and cyanide f ishing, among others. abalone is a high-value commodity with a big potential for export. the life history traits of abalone make them especially vulnerable to overf ishing, which poses a great challenge to f ishery management. in areas where the abalone are found to be very few and widely apart, chances of successful fertilization are low. unless the direct users and the policy makers are educated about the status and proper management of the abalone f ishery, it may eventually collapse. hence, sustainable alternatives to overharvesting need to be explored and promoted in coastal f isher communities to mitigate the prevalent trend of ever-decreasing catches. one possible option is to educate coastal f isherfolk communities to help them become resource managers and promote community-based abalone mariculture as a resource conservation strategy (capinpin 2012). the abalone f ishery of anda is artisanal or small-scale, typif ied by f ishers gleaning or free-diving on shallow rocky areas which are the typical habitat of abalone. in promoting resource management to the coastal f isherfolk, it must be harmonized with their immediate need for daily subsistence (bangi and juinio-meñez 2001). if the aim is to reduce pressure on marine resources, the f isherfolk must have other means of livelihood. thus, mariculture was explored to address both resource e.c. capinpin jr. 53 management issues and the need for development of sea-based livelihood. as a resource enhancement activity, mariculture guarantees that the cultured organisms with economic value can be allowed to grow to sexual maturity before they are harvested, while retaining some for reseeding. since mariculture makes possible the aggregation of individuals, the probability that fertilization would take place is increased. as supplemental source of livelihood, abalone is a high-value commodity that can help augment the income of the communities. moreover, by taking care of the abalone stocks in cages, the f isherfolk will gain an appreciation of, and eventually care for, their unique but important resource. this study is envisioned to become an entry point in heightening the ecological awareness of local communities on sustainable management through hands-on, experiential, resource management activities. the present study aims to assess (1) the status of the abalone f ishery in carot, anda, pangasinan to justify the establishment of abalone mariculture primarily as a resource conservation strategy and as a supplemental source of livelihood; (2) the growth and survival of abalone in cages; and (3) the efforts to reseed a part of the harvest of the mariculture cooperators for resource conservation. materials and methods study area the study was conducted in barangay carot in anda, pangasinan, northern philippines where the target species is naturally available. only about 30 f ishers — both daytime and night-time f ishers — are actively f ishing for abalone in carot. this small number is due to the fact that it requires experience to be able to harvest the cryptic abalone. to these f ishers, abalone f ishing comprises a large part of their income. assessment of abalone fishery group and ind ivid ual interviews a focus group discussion was conducted among abalone gatherers to collect data on abalone gathering and collection methods, abalone species caught, average catch per day, time of collection, marketing, biology and ecology, and historical trends in f ish catch. additional information was collected via individual interviews with abalone gatherers who were also identif ied as possible cooperators in the experiential activity/farmer field school (ffs) activity. status of abalone fishery and experiential mariculture 54 transect and actual catch data transect surveys were done in carot (banlag and purod wilda) and in barangay tondol on december 18 and 19, 2012, respectively. tondol was included as a basis for comparison as abalone can also be found in that area. three replicate 50m x 5m belt transects were established in each site and all abalone within the belt were counted. in addition, on march 14, 2011, the catch composition of two cooperators was examined to validate data gathered from the interviews and actual survey. abalone mariculture using the farmer field school (ffs) concept the ffs is a group-based learning process that involves actual interactions with and among gatherers/farmers (gallagher 2003). in this process, the participants carry out experiential learning activities that help them understand the ecology of abalone. in this particular study, these activities involved growth experiments, regular f ield observations, and group analysis. likewise, the local research partners/ cooperators were involved in this undertaking through experiential (participatory) experimentation concerning culturing abalone in cages as a way to enhance the recovery of abalone stocks and as a possible supplemental source of livelihood. identification of cooperators six abalone gatherers were chosen to implement the project based on the following criteria: 1. must be an active member of the community, preferably a group leader; 2. has attended a basic seminar on the biology and culture of abalone; 3. interested in conducting experimental/trial grow-out of abalone in cages; 4. has time for the activity; and 5. has time to attend monthly regular meetings as well as emergency meetings, especially when problems arise during the course of the activity. the tasks of the cooperators included: participation in the mentoring sessions; setting up of cage culture sites; stocking, monitoring the growth and development of stocked individuals; cleaning and maintenance of the cages; feeding; and recording any unusual observations. cage materials were provided by the researcher. the labor and maintenance were provided by the cooperators as their contribution in the experiential activity. it was also agreed with the cooperators that they should harvest only stocks that had attained a shell length of 50 mm — mature abalone that have already spawned many times and have already contributed e.c. capinpin jr. 55 to the replenishment of the population. it was made clear to them that there should be no immediate expectations of prof it, but it was hoped that the practice could serve as reproductive reserves to further replenish natural stocks. it was emphasized that abalone or h. asinina attains sexual maturity at 35 mm and spawns at 13-15 day intervals (capinpin and others 1998). it was also an agreed policy to leave or retain a part of the harvest in the cages to be released in a nearby marine protected area. source of abalone for stock conservation and resource management purposes, it was best to use native stocks rather than those coming from another region since the latter might have a different genetic structure that could pose a threat to the genetic diversity of the local stocks. however, local stocks were diff icult to f ind in large numbers so only a few small wild abalone were used. grow-out experiments net cages measuring 50 x 50 x 20 cm were constructed using polyvinyl chloride (pvc) pipes f itted together and covered with nets similar to that used by capinpin and others (1999). two pieces of pvc gutter were placed inside each cage as shelters. the cages were suspended and tied to bamboo poles set at depths of about 1.5 m. it was ensured that the cages were not exposed during the lowest tides. two trials were conducted, with each trial consisting of three replicate cages. the stocking densities, initial sizes, and date of stocking of small wild abalone are shown in table 1. trial stocking density initial size initial weight date of (abalone/cage) (cm) (g) stocking 1 25 42.74±0.54 16.60 april 8, 2011 2 50 40.99±0.80 14.39 may 30, 2011 mean of 3 replicates mean±se table 1. stocking densities, initial sizes and date of stocking of abalone grown in sea cages the abalone mariculture was conducted for 3-4 months or up to 120 days. feeding was done at weekly intervals by providing a pre-determined amount of the algae found in the area, preferably hydropuntia edulis (=gracilaria coronopifolia) using the established daily feeding scheme as a guide (capinpin and others 1999). status of abalone fishery and experiential mariculture 56 the grow-out area (n 16°20’52.8" e 119°59’38.9") for pilot culture was accessible to the cooperators and visible from their residences, allowing them to guard the site against poaching. the site is also a natural seagrass area, dominated by enhalus acoroides and thalassia hemprichii. water depth is about 1.5 m during the lowest tide, and water circulates freely. there are no freshwater tributaries in the vicinity. growth eval uation abalone length and weight were measured every 15 days. shell lengths were measured using a vernier caliper and weights were measured using a kitchen weighing balance. mean weights were determined by taking the biomass (total weight of the sample) divided by the total number of animals. mean shell lengths were taken from all the stocked individuals. daily growth rates in terms of weight (dg w ) and shell length (dg sl ) were also calculated as follows: dg w (g day -1) = g w /n dg sl (µm day-1) = g sl /n where g w is increase in weight (g) calculated as f inal weight minus initial weight, g sl is increase in shell length (µm) calculated as f inal length minus initial length, and n is days of culture. physico-chemical parameters physico-chemical parameters such as temperature, salinity, ph, ammonia, and nitrite were recorded weekly. assessment of reseeding as a resource conservation strategy to reiterate, it was agreed upon at the start of the mariculture activity that a portion of the harvest will be set aside for reseeding in a nearby marine protected area. prior to the release, the abalone were tagged using dymo and glue and placed in cut pvc pipes adapted from mccormick and others (1994). this method allows the easy transport of abalone and affords protection from predation immediately after transfer. upon reaching the reseeding site, one end of each pipe was lodged in crevices or between boulders where the abalone can avoid exposure and exit safely. the site was revisited after one day to observe movement and/or presence of empty shells. e.c. capinpin jr. 57 statistical treatment and data analysis frequencies, means and percentages were computed to summarize and describe the results. additional data were presented in descriptive form as narrated by the subjects. cost and return analysis a simple cost and return analysis was done based on the following assumptions: 1. the farmers would use indigenous materials such as bamboo for cages instead of pvc materials, which was provided to them in the present study; and 2. the sale of abalone would be at php 400/kg, which is the current farm gate price (encena and bayona 2010). results and discussion assessment of abalone fishery there are two abalone species found in anda, pangasinan, namely haliotis asinina and h. planata. of the two species, only h. asinina was gathered as h. planata was rare. h. asinina has the potential for culture because of its large size and body weight and fast growth rate (capinpin and corre 1996, capinpin and others 1999). there were two types of abalone gatherers: the daytime and the night-time gatherers. the daytime collectors used a face mask and an improvised hook to collect abalone. the night-time collectors used a face mask, hook, and a lighting device (petromax). both types of f ishers used rafts in order to move around the f ishing ground and improvised nets (nylon or bamboo) to hold collected abalone. of the 12 abalone gatherers interviewed, 67% were regular day time gatherers and 33% were regular night-time gatherers. they also gleaned other commercially important invertebrates such as sea cucumbers, sea urchins, and other shellf ishes. all of them have more than ten (10) years experience in abalone gathering. interviewees claimed that night-time gatherers collected more abalone than daytime gatherers. night-time gatherers harvested up to 10 kg each of abalone. this is not surprising as abalone are nocturnal animals. they hide under rocks and in crevices at daytime and move out at night to feed on seaweeds. night divers need not overturn the rocks since abalone are out searching for food or spawn when they are gravid during new and full moon periods (capinpin and hosoya 1995, status of abalone fishery and experiential mariculture 58 figure 1. size frequency distribution of abalone catch (n=34) of cooperator #1 off the coast of cabungan near the sanctuary in a 4-h f ishing operation. counihan and others 2001). ready-to-spawn abalone are active, with their foot relaxed and flabby (breen and adkins 1981, setyono 2006). setyono (2006) observed that they do not retract their foot when touched, and would even crawl freely onto one’s hand when handled. night divers noted that abalone were easy to catch at this time; describing them (abalone) as maamo (tame) and madulas (loosely attached to the substratum). night divers go f ishing during the new and full moon periods because they can collect more abalone during these times. the divers would f ish for about 2-3 consecutive nights without missing any particular night. depending on sea conditions, all the night divers would f ish until dawn, i.e. up to 6-7 hours at a time. for h. asinina, spawning episodes coincide with new and full moons for recently captured abalone held in tanks (capinpin and hosoya 1995, counihan and others 2001). actual catch data figures 1 and 2 show the size distribution of the catch of the two f isher-cooperators. for the f irst cooperator, the 34 pieces of abalone caught in a 4-h f ishing operation had a total weight of 1.2 kg with an average weight of 35.29 g. this corresponds to a catch per unit effort (cpue) of 300 g/h. the biggest number (41%) of the catch fall under the 5.1-5.5 cm size category. overall, 68% of the catch was over 5 cm in size and within the agreed legal size limit among the cooperators (fig. 1). on the e.c. capinpin jr. 59 figure 2. size frequency distribution of abalone catch (n=29) of cooperator #2 off cangaluyan island in a 3-h f ishing operation. other hand, the total catch of the second cooperator (fig. 2) in a 3-h fishing operation off cangaluyan island consisted of 29 pieces for a total weight of 1.15 kg or a mean weight of 39.65 g. the biggest number (45%) of the catch fell in the 6.1-6.5 cm category. the computed cpue was 383 g/h. overall, 83% of the total catch was over 5 cm in size, which was considered as legal size limit. sungthong and others (1993) surveyed the distribution of wild h. asinina around samet island in thailand in 1989 and reported the number of abalone collected by divers per hour (cpue) from 5.79-6.00 in hin khan na and 9.5-10.25 in ao thien reefs. the cpues of the two f ishermen of 8.5-9.7 abalone per hour in the present study was comparable to that in ao thien reef (9.5-10.25 abalone per hour) in samet island. abalone smaller than 5 cm represented 25% (16/63) of the combined catches. fishers typically include small abalone (<5 cm) when they sell their catch. as much as 1/4 of abalone caught in local waters may be grown further to a larger size for better prof it, as well as allowing them to reproduce many times before harvest. transect surveys table 2 shows the estimated density from the transect surveys at carot (banlag and purod wilda) and tondol. there were more abalone collected in banlag than in purod wilda and tondol. abalone were cryptic during daytime when the survey was done and the distribution was highly dependent on the presence of suitable rocky substrates. status of abalone fishery and experiential mariculture 60 the observed densities of abalone were 8.00, 1.67, and 3.33 per 250 m2 of surveyed area in banlag, purod wilda, and tondol, respectively. the observed densities in banlag (3.2 per 100 m2) were similar to the study of maliao and others (2004) on selected open access areas in sagay, negros occidental. the densities in purod wilda and in tondol were lower (0.67-1.33 per 100 m2). maliao and others (2004) reported higher mean abalone densities in mpa areas in sagay, implying that a properly enforced no-take mpa can promote recovery of local stocks. site coord inates number (per 250 m-2) banlag in carot transect 1 n 16° 21’33.2" e 120°00’06.6" 10 transect 2 n 16° 21’34.3" e 120°00’06.4" 10 transect 3 n 16° 21’45.6" e 120°00’06.6" 4 mean 8.00 purod in carot transect 1 n 16° 21’26.5" e 120° 00’04.4" 1 transect 2 n 16° 21’23.9" e 120° 00’01.6" 0 transect 3 n 16° 21’17.5" e 120° 00’01.5" 4 mean 1.67 tondol transect 1 n 16°19’24.8" e 120° 00’04.7" 1 transect 2 n 16°19’09.2" e 120°01’21.7" 3 transect 3 n 16°19’12.5" e 120°01’23.0" 6 mean 3.33 table 2. actual density of abalone inside the 250 m2 belt transect catch data based on interview based on interviews, the current catch of daytime f ishers ranges from 1 to 2 kg in a typical f ishing trip lasting about 4-6 h, from about 8 am to about 2 pm. on the other hand, night-time gatherers can collect as high as 7-10 kg each during the whole night but at a much limited time, depending on sea conditions as well as on the phase of the moon. comparing the actual catch data and what the f isherfolk revealed in the interview, it can be inferred that the data from interviews were valid and approximated the actual data. this implies that, with validation, the information gathered from interviews can be a useful companion to standard scientif ic approaches. local trade and market abalone f ishers currently have two buyers in the area. one of them buys their catch at php130/kg. the other buyer, a korean, buys abalone at a higher price of php190-200/kg. the korean reportedly transferred to balingasay, bolinao and entrusted the buying e.c. capinpin jr. 61 of abalone to a local resident from barangay cabungan. however, this person would not pay cash upon delivery. hence, some f ishers are forced to sell their catch to the local buyer in carot at a much lower price in exchange for immediate cash. thus, there is the need to f ind an alternative market for abalone collected in anda, as abalone is an expensive food item in luxury restaurants. the f ishers stated that they sell their abalone catch live or fresh to the buyers. the buyer from carot processes the abalone catch by salting and boiling them before these are delivered to manila. the korean sells live abalone directly to restaurants in nearby dagupan city, angeles city, or baguio city or directly to other koreans. abalone has a traditional place in asian societies particularly china, japan, and korea as an item of prestige, and is considered customary in banquets and traditional feasts (oakes and ponte 1996). growth and survival of cultured abalone first trial the f irst batch of small wild abalone was stocked on april 8, 2011 and the culture ended on july 8, 2011. within three months of culture, the mean shell length increased from 42.74 ± 0.54 mm to 49.55 ± 0.30 mm (table 3). on the other hand, the mean total weight increased from 16.60 ± 0.70 g to 24.78 ± 1.61 g. the harvest of the stocks was done after reaching an agreed size of about 50 mm. all of the harvested stocks were mature and had ripe gonads. capinpin and others (1998) identif ied that sexual maturity of this species is reached at a size of 35 mm for both male and females and it is assumed they have spawned every 2 weeks following a lunar cycle. thus, the cultured stocks had contributed to repopulating the days mean shell length mean weight survival rate (mm) (g) (%) 0 42.7±0.5 16.6 100.0±0.0 15 43.3±0.6 17.2 100.0±0.0 30 44.2±0.6 18.9 100.0±0.0 45 44.4±0.6 19.8 100.0±0.0 60 45.3±1.1 20.2 96.0±2.3 75 47.2±0.8 21.2 92.0±4.0 90 49.6±0.3 24.8 92.0±4.0 mean of 3 replicates mean±se table 3. mean shell length (mm), mean weight (g) and survival rates (%) of the first batch of wild abalone cultured in net cages for 90 days (trial 1) status of abalone fishery and experiential mariculture 62 surrounding areas. the survival rate of cultured abalone was high, ranging from 88100% with a mean of 92.0 ± 4.0 %. a total of about 1.7 kg was harvested from the 3 cages utilized in the f irst batch. the growth rates, however, were very low (table 4) compared to the results obtained by capinpin and others (1999), wherein the growth rates were above 100 µm d-1 in shell growth and above 0.20 g d-1 in weight for abalone stocked in sea cages with similar stocking densities. likewise, the survival rates were lower at 92% in the present study compared to >95% in the previous study at similar stocking densities (capinpin and others 1999). in contrast, the results of the present study were comparable to abalone cultured in tanks with limited water exchange (capinpin and corre 1996). in the latter, the researchers reported mean daily growth rates of 55.8 µm d-1 in shell length and 0.09 g d -1 in weight during the later part of the culture period when the abalone were larger. further, they observed a decreasing trend in growth rates during the latter part of the feeding experiment. this reduction in growth rates of abalone was possibly due to the requirement for energy during gonad maturation. the slowing of growth rate following sexual maturity in abalone is well known and has been attributed to the channeling of energy into gonad development (capinpin and corre 1996, mercer and others 1993). similar results were observed by estepa and meñez (2001)in their study on sea cucumbers. they observed the slowing of growth of sea cucumbers during the latter part of their 8-month culture in pens in pilar, bolinao. they speculated that growth slowed down when the samples reached mean weights of over 240 g, possibly as an indication that the species had reached its maturity stage. repl icate dgw (g d -1) dgsl (µm d -1) 1 0.12 79.44 2 0.11 88.89 3 0.04 58.67 mean 0.09 75.67±8.93 mean±se table 4. mean growth rates in length (dgsl) and weight (dgw) of the first batch of wild abalone cultured for 90 days (trial 1) e.c. capinpin jr. 63 second trial table 5 shows the mean growth in length, weight, and survival rates of small wild abalone cultured for 105 days. the second batch of abalone grew from 40.99 mm to 49.71 mm in shell length and from 14.39 g to 23.17 g in weight after 105 days. the culture period took a longer time to complete because of the smaller initial size of the second batch (40.99±0.80 mm) compared to the f irst batch (42.74±0.54 mm). the computed growth rate was 83.02 µm day-1 in terms of shell length and 0.084 g day-1 in terms of weight (table 6). these were comparable to that observed in the f irst batch, but also lower than earlier results of capinpin and others (1999). trial 2 survival rates were also lower compared to the f irst batch due to prolonged heavy rains. it began after one month from stocking up to the end of the culture period. repl icate dgw (g d -1) dgsl (µm d -1) 1 0.06 78.00 2 0.08 82.48 3 0.12 88.57 mean 0.08 83.02±3.06 mean±se table 6. mean daily growth rates in terms of length (dgsl) and weight (dgw) of small wild abalone cultured in sea cages for 105 days (trial 2) days mean shell length mean weight survival rate (mm) (g) (%) 0 41.0 ± 0.8 14.4 100.0 ± 0.0 15 41.4 ± 1.2 14.6 93.3 ± 4.7 30 41.7 ± 1.4 15.6 84.7 ± 3.7 45 43.3 ± 1.4 16.1 71.3 ± 2.4 60 45.4 ± 2.2 19.7 62.0 ± 7.0 75 46.0 ± 2.1 20.2 58.7 ± 8.7 90 48.8 ± 1.1 22.2 54.0 ± 10.1 105 49.7 ± 1.1 23.2 41.3 ± 15.5 mean of 3 replicates mean±se table 5. mean shell length (mm), mean weight (g) and survival rates (%) of the second batch of wild abalone cultured in net cages for 105 days (trial 2) status of abalone fishery and experiential mariculture 64 it is also a well-known fact that the growth of abalone and other shellf ish decreases as stocking density increases. consequently, it takes a longer time to reach harvest size (capinpin and others 1999). similar mortalities were observed for sea urchins cultured in pens in bolinao (bangi and meñez 2001). the death of balding sea urchins in cages was attributed partly to the occurrence of a phenomenon they called “kulaba” or water poisoning observed to occur annually during the rainy months, and partly due to high stocking density. physico-chemical properties of water water temperature and ph were in normal conditions and ranged from 29-33°c and from 8.0-8.6, respectively, throughout the culture period; whereas ammonia and nitrite were undetected and were always 0 mg/l and <0.3 mg/l, respectively, throughout the culture period. on the other hand, salinity ranged from 29.2 to 34.5 ppt (specif ic gravity, 1.022 to 1.026) during the whole of april up to june 14. it dropped to 23.9 from 26.6 ppt from june 29 to july 15 (specif ic gravity, 1.018 to 1.020) which corresponded to the time when typhoons falcon and goring ravaged northern luzon. even without typhoons, the continuous rain during this period lowered salinity levels that affected the cultured abalone. normal seawater salinity is about 32-35 ppt. salinity normalized from july 15 onwards until the end of the culture period, which allowed some of the abalone in trial 2 to recover despite being exposed to prolonged heavy rains in an enclosed environment. cost and return analysis table 7 shows a simple cost and return analysis based on several assumptions such as the use of indigenous materials (e.g. bamboo) for the manufacture of cages and the increase of abalone price to php 400/kg, the current farm gate price for the species (encena and bayona 2010). although a small net income was attained during the 3-month culture period, prof itability can be increased on a larger scale (encena and bayona 2010) by improving survival rates and searching for new market opportunities. reseeding into a protected area thirty six (36) abalone from the f irst harvest were tagged for reseeding; the rest were processed and sold. the tagged abalone were released into the panacalan mpa (n 16° 16’ 07.0" e 120° 01’ 42.7") in macaleeng, anda on july 11, 2011. macaleeng is about an hour of boat-ride from carot. e.c. capinpin jr. 65 anda has f ive mpas situated in carot (13.3 ha), cabungan (18 ha), caniogan (9.8 ha), magsaysay (14.8 ha), and panacalan (48.59 ha). the mpas in carot and cabungan were the earliest established (1998) while panacalan was the most recent (2003) (salmo and others 2005). however, according to the cooperators, the sanctuary in carot is no longer operational. even in 2004, salmo and others (2005) reported that there had been problems in enforcement due to the weakening of people’s organizations (pos) in the area, which is attributed to changes in leadership of these pos. the panacalan mpa was chosen as the reseeding site because it is the most well-enforced mpa to date in the area. the 36 tagged abalone reflect the commitment made by the cooperators at the start of the mariculture activity. the use of cut pvc pipes in reseeding minimizes handling stress and protects abalone from predation during and after their placement on the ocean floor. tegner and butler (1985) noted that abalone handled excessively produce mucus which then attracts predators. high mortalities often occur within hours of transplantation. protection during the initial hours following placement allows the abalone to recover from handling and transport stress and become acclimated to the release site. in addition to providing a safe refuge during the initial acclimation period, the conf iguration of the planting module is such that one end can be lodged in crevices or between boulders. the abalone can thus avoid exposure and can exit safely. the site was revisited after one day and it was observed that all tagged abalone left the pvc pipes and took refuge in the surrounding crevices. a search for empty shells in the surrounding areas revealed no mortality due to reseeding and/or predation. it is assumed that the abalone were able to disperse into the surrounding areas safely. production (kg) 1.7 survival rate (%) 92 farm gate price (php/kg) 400 cost of seed (php 2/abalone x 75)* 150 cost of cage (php 75 x 3 cages)** 225 gross revenue (php) 680 net income (php) 305 *cost of small abalone weighing an average of 15 g at php 130/kg, the actual price f ishers sell their catch in the area **cage made of bamboo frame covered with net table 7. a simple cost and return analysis of abalone mariculture based on the results of trial 1 using 3 cages with a stocking density of 25 abalone per cage and cultured for 90 days status of abalone fishery and experiential mariculture 66 studies on the effect of abalone size at the time of release have indicated that, at least for some species, there is an optimal size for maximum survival. for instance, the survival of juvenile madaka (h. gigantea) abalone after one year in the ocean increased from 10 to 70% as the size of the abalone planted was increased from 10 to 30 mm (inoue 1976 as cited in mccormick and others 1994). in this study, though the number released was low (only 36), the abalone released was large (about 5 cm) and came previously from the wild, and had a strong chance to survive. experiential mariculture activity the mariculture activity following the ffs concept was very interesting and meaningful to the local cooperators. it is because the ffs is particularly adapted to f ield study, where specif ic hands-on management skills are required (gallagher 2003, gallagher and others 2006). the cooperators were more comfortable in the f ield than in classrooms as there were no lectures and all activities were based on experiential, participatory, hands-on work. in the experiential activity/ffs, the f ield itself became the “teacher” as it provided most of the training materials such as the cultured animal, seaweeds as its food, its predators, pests and other real problems. the activities included in the experiential activity followed the natural cycle of their subjects. in this case, the cycle was from “abalone seed” to a “mature abalone” that contributed to the replenishment of its population. as much as possible, the approach allowed all aspects of the subject to be covered, in parallel with what was happening in the f ield. the lessons sharpened the cooperators’ skills in the areas of observation, problem-solving, and decision-making, and helped develop their critical thinking (gallagher 2003, gallagher and others 2006). it was ensured that the activities were participatory and hands-on so as to encourage learning. being a hands-on activity, it was a new experience for the cooperators specif ically on how to address both their resource management and economic needs. this activity was envisioned to contribute in enhancing abalone populations while heightening the f isherfolk’s ecological knowledge of the life history of abalone and its culture. at the same time, it was viewed as a potential means to provide supplemental source of livelihood. the experimental activity helped the cooperators to understand the importance of choosing a suitable site for the cultured species. they learned that mariculture should be done during fair weather (i.e. november to april or may) and that the cages are best located further out in the sea to avoid the lowering of the salinity during heavy rains. the experiments, though limited, reinforced what they learned in their lecture. they learned the importance of monitoring the growth and survival e.c. capinpin jr. 67 of the organisms as well as water quality parameters. this gave them an idea about how long it would take to culture the abalone and to determine the optimal time to harvest. the activity heightened their environmental awareness and understanding of the ecological principles and the rationale for resource management (capinpin 2 0 1 2 ) . in the experiential activity, the researcher became a facilitator who assisted the cooperators to learn from their experience towards sustainable practices and resource management. this is opposed to technology transfer wherein an extension staff is expected to be an expert conveying lessons from the research to farmers in a top-down approach, particularly when there are no hands-on activities. during the experiments, several problems arose such as the low survival of abalone in trial 2, and the need to reevaluate task and time allotment of each member for the different maintenance activities. using the ffs concept in mariculture, the learners advanced because they were given a free hand to learn and to decide which problem-solving steps they would to take during the mariculture activity. in the process, the cooperators participated in providing solutions as well as ref inements and adjustments to the techniques and time allotment schemes. they also suggested ways on how to grow the abalone faster based on their own observations and experiences. thus, the activity provided them with a sense of ownership over the resource and responsibility towards their actions. conclusion mariculture is an excellent tool for the conservation of f ishery resources particularly abalone. it is also a sustainable supplemental source of livelihood. the viability of abalone mariculture is favored by the biological attributes of this species. specif ically, the abalone has fast development and growth rates. likewise, the use of cages entails low capital outlay and maintenance cost. the growing of small abalone in sea cages can increase their size and weight, which translate to better prices when they are sold. it is recommended to replicate the mariculture of abalone in other areas to create dense breeding populations which can help in enhancing the existing breeders and the periodic release of abalone in sanctuaries (capinpin 2012). through actually performing the culture of abalone in cages, the cooperators gained technical skills in selecting suitable sites for the culture of these organisms as well as in choosing cultured species that feed low in the food chain (e.g. abalone). the cooperators also gained capability in monitoring the development of stocked individuals, and improved their knowledge on the biology of the cultured organism. status of abalone fishery and experiential mariculture 68 overall, the experiential mariculture was considered as a successful activity in terms of providing experience to the cooperators in resource management and of providing a venue for the study of abalone mariculture as a supplemental source of livelihood. acknowledgments the author wishes to thank ms. elizabeth tomas, the municipal agriculturist, and the honorable municipal mayor aldrin cerdan for allowing the researcher to conduct this study in anda, pangasinan, for providing all the necessary documents, maps, unpublished reports, etc. , and for their whole-hearted support during the entire conduct of the study. the author also acknowledges the par ticipation of the two cooperators, rudy raquiem and pepito versoza; the advice of dr. ruth guzman of the rizal technological university and drs. phares parayno, angelina galang, donna paz reyes, and ma. lourdes baybay of miriam college before and during the conduct of the study; the assistance of the author’s students during the transect surveys; and prof. ceferino toledo of psu and two anonymous reviewers for reviewing this manuscript. references bangi hgp, juinio-meñez ma. 2001. sea urchin grow-out culture: assessment of potential f o r i m p l e m e n t i n g c o m m u n i t y b a s e d c o a s t a l r e s o u r c e m a n a g e m e n t ( t h e c a s e i n arnedo). in: mcmanus lt, meñez maj, aliño pm, ferrer em, dizon jca, editors. paving the wa y f o r co a s t a l re s o u r ce s m a n a g e m e n t : t h e b o l i n a o e x p e r i e n ce ( 1 9 9 3 1 9 9 7 ) . university of the philippines marine science institute, up-college of social work and community development, and haribon foundation for the conservation of natural re s o u r ces, i n c . i n te r n a t i o n a l d eve l o p m e n t re s e a r c h ce n t r e – ca n a d a , co m m u n i t y based coastal resources management program. p 28-36. breen pa , adkins be. 1981. spawning in a british columbian population of nor thern abalone, haliotis kamtschatkana. the veliger 23: 177-179. capinpin ec jr. 2012. using local ecological knowledge and environmental education in resource management of abalone in carot, anda, pangasinan. science diliman 24: 43-55. capinpin ec jr. , hosoya m. 1995. spawning and larval development of a tropical abalone haliotis asinina linné. philipp. j. sci. 124: 215-232. capinpin ec jr. , corre kg. 1996. growth rate of the philippine abalone, haliotis asinina fed an ar tif icial diet and macroalgae. aquaculture 144: 81-89. capinpin ec jr. , toledo jd, encena vc ii, doi m. 1999. density dependent growth of the tropical abalone haliotis asinina in cage culture. aquaculture 171: 227-235. e.c. capinpin jr. 69 capinpin ec jr. , encena vc ii, bayona nc. 1998. studies on the reproductive biology of the donkey’s ear abalone haliotis asinina linné. aquaculture 166: 141-150. co u n i h a n rt, m c n a m a r a d c , s o u te r d c , j e b r ee n e j , pr e s to n n p, d eg n a n b m . 2 0 0 1 . pattern, synchrony and predictability of spawning of the tropical abalone haliotis asinina from heron reef, australia. mar. ecol. prog. ser. 213: 193-202. encena v.c. ii, bayona nc. 2010. farming of the tropical abalone h a l i o t i s a s i n i n a . aquaculture extension manual no. 49. southeast asian fisheries development center aquaculture department. estepa ng, juinio-meñez ma. 2001. a community-based experimental culture of sea cucumbers and siganids as tool for resource enhancement . in: mcmanus lt, meñez maj, aliño pm, ferrer em, dizon jca , editors. paving the way for coastal resources management: the bolinao experience (1993-1997). university of the philippines marine science institute, up-college of social work and community development, and haribon foundation for the conservation of natural resources, inc. international development research centre – canada, community-based coastal resources management program. p 11-27. gallagher kd. 2003. fundamental elements of a farmer f ield school. leisa magazine 19(1): 5-6 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(http://www.farmerf ieldschool. net/document_en/ 05_06.pdf ). gallagher kd, braun ar, duveskog d. 2006. demystifying farmer field school concepts. wa g e n i n g e n u n i v e r s i t y. [ c i t e d 1 2 j a n u a r y 2 0 1 1 ] . ( h t t p : / / w w w. j a t r o p h a . p r o / p d f bestanden/ ffsdemystif ication.pdf ). green sj, white at, flores jo, carreon mf iii, sia ae. 2003. philippine f isheries in crisis: a framework for management. coastal resource management project of the depar tment of environment and natural resources, cebu city, philippines. 77 p. juinio-meñez ma , malay mcd, bangi hgp. 2001. sea urchin grow-out culture. coastal resources management tools. marine environment resources foundation, inc. the marine science institute, university of the philippines, diliman, quezon city. 34 p. juinio-meñez ma, pastor d, bangi hg. 2008a. indications of recruitment enhancement in the sea urchin tripneustes gratilla due to stock restoration efforts. proceedings of the 11th international coral reef symposium, ft. lauderdale, florida, 7-11 july 2008. p 1017-1021. juinio-meñez ma, bangi hg, malay mcd, pastor ds, 2008b. enhancing the recovery of depleted tripnuestes gratilla stocks through grow-out culture and restocking. rev. fish. sci. 16: 35-43. maliao rj, webb el, jensen kr. 2004. a survey of stock of the donkey’s ear abalone, haliotis asinina l. in the sagay marine reserve, philippines: evaluating the effectiveness of marine protected area enforcement . fisheries research 66: 343-353. status of abalone fishery and experiential mariculture 70 mccormick tb, herbinson k, mill ts, altick j. 1994. a review of abalone seeding, possible signif icance and a new seeding device. bull. mar. sci. 55: 680-693. mercer jp, mai ks, donlon j. 1993. comparative studies on the nutrition of two species of abalone haliotis tuberculata linnaeus and haliotis discus hannai ino. i. effects of algal diets on growth and biochemical composition. inver t. reprod. dev. 23: 75-88. oakes fr, ponte rd. 1996. the abalone market: opportunities for cultured abalone. aquaculture 140: 187-195. salmo sg iii, arceo ho, pacif ico kp, aurellado eb, alano hg. 2005. marine protected areas of anda, pangasinan. reefs through t ime: 2004. biennial repor t on the status of philippine coral reefs. coral reef information network of the philippines (philreefs) and the marine science institute, university of the philippines, diliman, quezon city. p 21-29. setyono ded. 2006. induction spawning for the tropical abalone (haliotis asinina) in the laboratory. indonesian aquaculture journal 1(1): 17-27. sungthong sv, ingsrisawang v, fujiwara s. 1993. sudden decrease in abundance of donkey’s ear abalone. hal iotis asinina around samet island during 1989-1990. thai mar. fish. res. bull. 4: 9-100. tegner mj, butler ra. 1985. survival and mor tality of seeded and native red abalones, haliotis rufescens, on the palos verdes peninsula. california fish game 71: 150-163. _______________ emmanuel c. capinpin jr. <manny_capinpin@yahoo.com> is currently an assistant professor iv at the pangasinan state university, binmaley campus, binmaley, pangasinan. university of the philippines diliman office of the vice chancellor for research and development call for papers humanities diliman, science diliman, and social science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> cover image credits: vargas museum (humanities diliman) and biodiversity research laboratory (science diliman) 5-validation-de ungria.pmd p.r.l. sales et al. 25 science diliman (january-june 2019) 31:1, 25-36 val idation of two extraction methods for human dna from cigarette butts paul ryan l. sales dorothy emma c. ferrer gay vell ine c. calacal jazelyn m. salvador maria corazon a. de ungria* dna analysis laboratory natural science research institute university of the philippines diliman abstract cigarette butts found in crime scenes may be used to identify persons and link them to a crime through dna prof iling of epithelial cells from saliva stains on these materials. downstream analysis of cigarette butts p o s e s s o m e c h a l l e n g e s b e c a u s e t h e s e a r e o f t e n e x p o s e d t o c h e m i c a l c o n t a m i n a n t s a n d e n v i r o n m e n t a l c o n d i t i o n s w h i c h l e a d t o d n a degradation. in this study, s everal factors were tested to compare the amount and quality of dna obtained from cigarette butts extracted using an organic procedure and the qiaamp® dna micro kit (qiagen). results show that exposure to an outside environment had a signif icant effect o n d n a y i e l d a n d a m p l i f i a b i l i t y f o r b o t h e x t r a c t i o n p r o c e d u r e s . prolonged storage of cigarette butts of up to six months affected the amount of dna that can be extracted using the qiaamp® dna micro kit. however, complete dna prof iles can be generated from cigarette butts stored for six months provided that these samples are stored indoors under controlled temperature conditions and with minimal exposure to contaminants. keywords: cigarette butts, dna typing, dna yield, forensic science _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online validation of two extraction methods for human dna from cigarette butts 26 introduction dna analysis of evidentiary materials is a powerful tool for linking an individual to a victim or a crime scene (hochmeister et al. 1991; walsh et al. 1992). proper crime scene collection and handling are essential to preserve the integrity of the evidence. saliva may be found on various surfaces found at the crime scene, such as cigarette butts (hochmeister et al. 1991; watanabe et al. 2003), leftover food (sweet and hilderbrandt 1999; abaz et al. 2002), and bite marks on human skin (pretty and sweet 2001; chávez-briones et al. 2015). dna from epithelial cells present in saliva may be used for dna analysis. however, saliva stains on most surfaces, once dry, are invisible to the naked eye. in addition, dna preparations can potentially contain co-extracted inhibitors that could affect downstream dna analysis. in a report by watanabe et al. (2003), certain kinds of dyes in cigarette butts inhibit pcr amplif ication. thus, a key determining step in the success or failure of obtaining a dna prof ile relies on the extraction method used to recover amplif iable dna. with the availability of multiplex short tandem repeat (str) dna marker systems targeting autosomal chromosomes (astrs) or gonosomal chromosomes (xstrs and ystrs), generating dna prof iles with a high capacity to differentiate human sources was made possible (hochmeister et al. 1991; balogh et al. 2003). in a 2009 survey, about 28.3% of filipinos aged 15 years and older, or about 17.3 million individuals smoked cigarettes in the philippines (who 2009). hence, cigarette butts are likely to be present in crime scenes that may contain dna from (1) the perpetrator who smoked while waiting for the opportune moment to commit the crime; (2) the victim/s, including those whose location remains unknown; and (3) the witness who could provide vital information for the investigation. many crimes in the philippines are located outdoors where the average temperature is 28.3°c, and the humidity ranges from 71 85% (pagasa 2016). when dna contained in crime scene samples are not recovered soon after a crime, these outdoor conditions promote microbial growth which leads to the degradation of dna. in the present study, we compared the utility of an organic procedure and a silicabased dna extraction method for handling cigarette butt samples. we also tested the effect of storage time and conditions, and cigarette type, on the generation of human dna prof iles from these samples. p.r.l. sales et al. 27 materials and methods institutional review clearance to conduct the study was provided by the natural sciences research institute, university of the philippines diliman. sample collection samples were collected from two volunteers who are regular smokers. both volunteers were asked to smoke three types of cigarettes, namely (1) regular-type; (2) light or less nicotine; and (3) light with menthol, following their normal routines. for the 24-hour stored samples, three regular-type cigarette butts from each volunteer were collected and stored for 24 hours indoors at room temperature (25°c 27°c). volunteers were asked to smoke three cigarettes in one day and each cigarette was smoked at a different time of the day. each cigarette butt was split into two slices with the cut parallel to the seam of the cigarette. each half was placed in separate sterile tubes, suspended, and incubated in 12 ml phosphate buffered saline (pbs) solution for three hours with shaking. for every cigarette butt, one half was subjected to organic dna extraction, while the other half was subjected to dna extraction using the qiaamp® dna micro kit. for the six-month storage samples, four cigarettes for each cigarette type (regular, lights, and lights-menthol) were collected from each volunteer. for each cigarette type, volunteers were asked to smoke four cigarettes per day with two cigarettes smoked in the morning and two cigarettes smoked in the afternoon. volunteers were also asked to leave some time before smoking the second cigarette. samples were stored at room temperature prior to extraction. each cigarette butt was split into two slices with the cut parallel to the seam of the cigarette. one half was subjected to organic dna extraction, while the other half was subjected to dna extraction using the qiaamp® dna micro kit. for the indoor storage set-up, four cigarettes for each cigarette type (regular, lights, and lights-menthol) were collected from each volunteer and stored for six months indoors. as for the outdoor storage set-up, four cigarettes for each cigarette type (regular, lights, and lights-menthol) were collected from each volunteer and left outdoors for six months. volunteers smoked each cigarette in intervals. validation of two extraction methods for human dna from cigarette butts 28 cigarettes left outdoors were deposited directly on soil and were exposed to external elements, such as direct sunlight and rain. cigarettes were also exposed to any animal, insect, and microbial activity that may occur in the area the cigarettes were deposited on. dna extraction qiaamp® dna micro kit extraction was carried out using the manufacturer’s protocol with some modif ications. modif ications include the use of 640 μl of lysis solution to submerge the cigarette butt samples and incubating these in lysis solution for two hours. organic extraction was performed using the protocol described by budowle et al. (2000) with some modif ications. the modif ied protocol made use of a 1000-μl cell lysis solution containing 835 μl of ten buffer (10 mm tris, 10 mm edta , and 100 mm nacl), 100 μl of 20% sds (invitrogen), 40 μl of 1 m dtt (roche), and 25 μl of 20 mg/ml proteinase k (novagen). samples were incubated with shaking at 56°c for ~24 hours in an eppendorf thermomixer® (eppendorf ). an equal volume of 25:24:1 mix of phenol (invitrogen), chloroform (merck millipore), isoamyl alcohol (j.t.baker), and a maxtract® low density (qiagen) phase-lock gel was used to separate organic and aqueous layers. dna extracts were purif ied using a microcon® 100 centrifugal f ilter (merck millipore) and was eluted using 40 μl of te-4 buffer. dna ampl if ication dna was quantif ied using the powerquant® system (promega corporation) (ewing et al. 2016), and quantitation results were analyzed using the powerquant® analysis tool following manufacturer’s instructions. a 0.50-ng dna template was amplif ied using half-volume reactions (12.5 μl) of the powerplex® 16 hs system (promega corporation) kit. polymerase chain reaction (pcr) conditions were as follows: initial denaturation at 96°c for 2 minutes, followed by 10 cycles of denaturation at 94°c for 30 seconds, annealing at 60°c for 30 seconds, and extension at 70°c for 45 seconds. this was followed by 22 cycles of denaturation at 90°c for 30 seconds, annealing at 60°c for 30 seconds, and extension at 70°c for 45 seconds. pcr amplif ication was completed with a f inal extension at 60°c for 30 minutes and a f inal hold at 4°c. p.r.l. sales et al. 29 fragment detection and profile analysis detection of the amplif ied dna fragments was carried out in the applied biosystems® 3500 genetic analyzer (thermo fisher scientif ic). generated prof iles were analyzed using the genemapper® id-x software version 1.2 (thermo fisher scientif ic). an analytical threshold of 50 relative fluorescence units (rfu) was used to distinguish true peaks from background noise. allele recovery computation blood samples from volunteers were collected and were used to generate their reference dna prof iles for comparison with the dna prof iles generated from the cigarette butts. the number of alleles in a volunteer’s reference prof ile was considered as the volunteer’s “expected” number of alleles. the number of alleles in the dna prof ile from the cigarette butt replicate was considered as the “observed” number of alleles for that sample replicate. allele drop-ins detected in cigarette butt samples were not considered part of the “observed” alleles. to determine the percentage allele recovery for each sample replicate, the observed allele number was divided by the expected number of alleles, then multiplied by 1 0 0 . statistical analysis data in the comparison of dna yield from the two dna extraction methods, storage environment, and storage time were analyzed using the unpaired student’s t-test. data from the comparison of dna yield from different cigarette types were analyzed using one-way analysis of variance (anova). all statistical analyses were performed using the graphpad prism 7 software (graphpad software, inc.). validation of two extraction methods for human dna from cigarette butts 30 results and discussion comparison of dna yield and allele recovery between the standard organic procedure and the qiaamp ® dna micro kit method for dna extraction for regular-type cigarettes extracted after a 24-hour storage period, there was no signif icant difference (p=0.078) between the dna yields of the two extraction methods (mean org =719.97±303.16; mean qia =1188.33±500.35) (table 1). complete dna prof iles were generated from all dna extracts of 24-hour stored samples. in samples stored indoors for six months prior to processing, difference in dna yield (p=0.008) was signif icant (mean org =1013.55±565.03; mean qia =139.45±229.31) (figure 1a and table 2). allele recovery across extraction methods (p=0.073) was not signif icant. table 1. dna recovery from regular-type cigarette butt samples extracted using organic qiaamp® dna micro kit extraction methods after a 24-hour indoor storage period. dna extraction method dna yield (ng dna/g cigarette butt) mean ± sd organic extraction 719.97 ± 303.16 qiaamp® dna micro kit 1188.33 ± 500.35 figure 1. comparisons of dna yield across different factors. (a) comparison of dna yields between the organic and the qiaamp® dna micro kit extraction methods from regular-type cigarette butt samples stored indoors for six months prior to extraction. difference in dna yields between the two extraction methods is signif icant (p=0.008). (b) comparison of dna yields from cigarette butt samples extracted using the organic method with respect to indoor storage times. difference in dna yield (p=0.877) between the two storage durations is not signif icant. (c) comparison of dna yields from cigarette butt samples extracted using the qiaamp® dna micro kit with respect to indoor storage times. difference in dna yield between the two storage durations is signif icant (p=0.0001). p.r.l. sales et al. 31 after six months of storage prior to processing, dna extracts from the qiaamp® dna micro kit have lower dna yields compared to those obtained via the organic extraction procedure. this observation illustrates the limitation of a silica membranebased dna extraction method, such as the qiaamp® dna micro kit. when the lysis solution is transferred to the qiaamp® minelute column, dna binds to the silica particles in the membrane while other molecules remain in the lysis solution. since the amount of silica particles of the qiaamp® minelute column is limited, the amount of dna that may be recovered from the lysis solution is also limited. competitive binding among human dna, non-human dna, and other substances occurring in the silica membrane contributes to the decrease in human dna yield using silica membrane-based dna extraction. competitive binding occurs when negatively-charged non-human dna or other molecules bind to the positivelycharged silica surface instead of the target human dna. furthermore, human dna bound to the silica surface could be displaced by molecules with a stronger aff inity to the silica surface. mundorff and davoren (2014) noted that pcr inhibitors and non-human dna may also co-isolate with human dna during extraction. allele recovery data show that the amount of amplif iable dna recovered during silica column extraction generated partial (>80% alleles recovered) to full prof iles. effect of storage environment on dna yield and allele recovery dna yield in cigarette butt extracts exposed outdoors for six months was lower compared to samples stored indoors at room temperature. samples extracted using the organic extraction method exhibited a signif icant difference in dna yield with respect to storage environments (p=0.0001). similarly, the difference in dna yield between samples stored in different environments and extracted using the qiaamp® dna micro kit was signif icant (p=0.001). poor allele recovery was observed in samples exposed outdoors (0 – 17%), while better allele recovery was remarked in dna extraction method dna yield (ng dna/g cigarette butt) mean ± sd organic extraction 1013.55 ± 565.03 qiaamp® dna micro kit 139.45 ± 229.31 table 2. dna recovery from regular-type cigarette butt samples extracted using organic and qiaamp dna micro kit extraction methods after a 6-month indoor storage period validation of two extraction methods for human dna from cigarette butts 32 samples stored indoors (27 – 100%). poor allele recovery of samples left outdoors was supported by dna quantitation results, wherein the powerquant® analysis tool flagged 33% of samples as degraded, while 54% had a zero quantitation value. this result is consistent with the study of casey et al. (2013) who reported a lower count of nucleated cells from cigarette butts left outdoors compared to those stored indoors. effect of storage time on dna yield and allele recovery dna extracts obtained using the qiaamp® dna micro kit exhibited a signif icant difference in dna yield with respect to storage times (p=0.0001). for these samples, higher dna yield was observed after storage for 24-hours compared to samples stored for six months. no signif icant difference was observed in the dna yields of samples extracted using the organic method and stored for 24 hours versus those stored for six months (p=0.877) (figure 1b). by contrast, the dna yields of 24-hour and 6-month samples extracted using the qiaamp® dna micro kit were signif icantly different (p=0.0001) (figure 1c). dna left on cigarette butts is highly prone to oxidation from reactive oxygen species (ros), such as nicotine present in cigarette smoke (ginzkey et al. 2012). exposure of dehydrated (matsuo et al. 1995) and lyophilized (molina and anchordoquy 2008; bonnet et al. 2010) dna to atmospheric oxygen at room temperature can also lead to dna oxidation. in the oxidized state of dna, the dna molecule becomes less negatively charged, which in turn makes the binding of dna to silica weaker. thus, the prolonged exposure to oxidative stresses during long-term storage can decrease the adsorption eff iciency of silica columns in dna extraction. it is therefore recommended that evidentiary samples that may have been exposed to harsh environmental conditions, such as cigarette butts left in a crime scene, must be processed immediately to decrease dna loss during storage. no signif icant difference in allele recovery with respect to storage durations was observed in both organic (p=0.408) and qiaamp® dna micro kit (p=0.051) extraction methods. p.r.l. sales et al. 33 effect of cigarette type on dna yield and allele recovery smoking habits differ from person to person. smokers would have preferences on the type of cigarette based on the amount of nicotine and presence of other ingredients (e.g. , menthol). three cigarette types, namely regular, less nicotine (lights), and less nicotine with menthol (lights-menthol), were tested to determine the effect of cigarette type on the amount and quality of dna recovered. for both extraction procedures, the results showed that cigarette type had no significant effect on dna yield (p org = 0.465; p qia = 0.748) nor on allele recovery (p org = 0.613; p qia = 0.297) (tables 3 and 4). conclusions this study shows that the amount and quality of human dna on cigarette butts deteriorate over time, and the extent of dna loss is more pronounced outdoors under warm and humid conditions. a suitable extraction method for a certain type of cigarette butt sample would differ on a case-to-case basis. for samples collected table 3. dna yield and allele recovery from cigarette butt samples of three types of cigarettes extracted using the organic extraction method after a six-month indoor storage period. regular 760.17 ± 528.53 99.58 ± 1.18 light 1653.89 ± 2003.18 99.58 ± 1.18 light with menthol 1272.32 ± 1013.93 100.00 ± 0 % allele recovery mean ± sd cigarette type dna yield (ng dna/g cigarette butt) mean ± sd table 4. dna yield and alele recovery from cigarette butt samples of three types of cigarettes extracted using the qiaamp® dna micro kit after a six-month indoor storage period. regular 104.59 ± 214.50 81.64 ± 26.17 light 176.64 ± 245.51 88.33 ± 20.39 light with menthol 178.21 ± 141.56 97.32 ± 7.58 % allele recovery mean ± sd cigarette type dna yield (ng dna/g cigarette butt mean ± sd validation of two extraction methods for human dna from cigarette butts 34 indoors and stored for a short period of time, it is more practical to use the qiaamp® dna micro kit because the process is faster, makes use of less hazardous chemicals, and involves less tube transfers, which in turn decreases the risk of contamination during processing. if possible, extraction of dna from cigarette butts that are submitted as evidence should be processed immediately to maximize dna recovery. for samples collected outdoors and stored indoors for a long period of time, the use of the organic extraction method is advised, since it can yield more dna, allowing increased number of analysis despite low copies or low quality of dna. storage of cigarette butt samples in laboratories with controlled temperature ranging from 22oc to 25oc and with reduced humidity slows but does not completely stop dna degradation. this is particularly important in the philippines where delays in the collection and submission of evidence to dna laboratories result in the prolonged outdoor exposure of samples, including cigarette butt samples, in locations that are subject to warm and humid conditions. acknowledgements the authors would like to acknowledge the off ice of the vice chancellor for research and development (ovcrd) of the university of the philippines diliman for providing the outright research grant (ovcrd project no. 111106 pnse) that funded this work. the authors would also like to thank the volunteers for their participation and miriam ruth m. dalet, of the dna analysis laboratory, for support. references abaz j, walsh sj, curran jm, moss ds, cullen j, bright j, crowe ga, cockerton sl, power te. 2002. comparison of the variables affecting the recovery of dna from common drinking containers. forensic science international. 126:233-240. balogh mk, burger j, bender k, schneider pm, alt kw. 2003. str genotyping and mtdna sequencing of latent f ingerprint on paper. forensic science international. 137:188195. bonnet j, colotte m, coudy d, couallier v, por tier j, morin b, tuffet s. 2010. chain and conformation stability of solid-state dna: implications for room temperature storage. nucleic acids research. 38(5):1513-1546. budowle b, smith j, moretti t, dizinno j. 2000. dna typing protocols: molecular biology and forensic analysis. natick (ma): eaton publishing. p. 20-22, 228. casey l, engen s, frank g. 2013. quantitative analysis of the dna distribution on p.r.l. sales et al. 35 cigarette butt f ilter paper. journal of forensic science. 58(2):470-473. chávez-briones ml, hernández-cortés r, jaramillo-rangel g, ortega-martínez m. 2015. relevance of sampling and dna extraction techniques for the analysis of salivary evidence from bite marks: a case repor t . genetics molecular research. 14(3):1016510171. ewing mm, thompson jm, mclaren rs, purpero vm, thomas kj, dobrowski pa, degroot g a , ro s m o s e l , s to r t s d r . 2 0 1 6 . h u m a n d n a q u a n t i f i c a t i o n a n d s a m p l e q u a l i t y a s s e s s m e n t : d e v e l o p m e n t a l v a l i d a t i o n o f t h e p o w e r q u a n t . f o r e n s i c s c i e n c e international: genetics. 23:166-177. ginzkey c, stueber t, friehs g, koehler c, hackenberg s, richter e, hagen r, kleinsasser nh. 2012. analysis of nicotine-induced dna damage in cells of the human respiratory tract. toxicology letters. 208:23-29. hochmeister mn, budowle b, jung j, borer uv, comey ct, dirnhofer r. 1991. pcr-based typing of dna extracted from cigarette butts. international journal of legal medicine. 104:229-233. matsuo s, tokoyumi s, osaka m, hamazaki s, sugiyama t. 1995. degradation of dna in d r i e d t i s s u e s b y a t m o s p h e r i c o x y g e n . b i o c h e m i c a l a n d b i o p h y s i c a l re s e a r c h communications. 208(2):1021-1027. molina mc, anchordoquy tj. 2008. degradation of lyophilized lipid/dna complexes during storage: the role of lipid and reactive oxygen species. biochemica et biophysica acta. 1778:2119-2126. mundorff a, davoren jm. 2014. examination of dna yield rates for different skeletal elements at increasing post mortem intervals. forensic science international: genetics. 8:55-63. philippine atmospheric, geophysical and astronomical services administration. climate of the philippines [internet]. manila: pagasa; [cited 2016 october 17]. available from http: //pagasa.dost .gov.ph/index.php/climate-of-the-philippines. pretty ia, sweet d. 2001. a look at forensic dentistry – par t 1: the role of teeth in the determination of human identity. british dental journal. 190(7):359-366. sweet d, hilderbrandt d. 1999. saliva from cheese bite yields dna prof ile of burglar: a case repor t . international journal of legal medicine. 112:201-203. walsh dj, corey a, cotton rw, forman l, herrin jr gl, word cj, garner dd. 1992. isolation of deoxyribonucleic acid (dna) from saliva and forensic science samples containing saliva. journal of forensic science. 37(2):387-395. watanabe y, takayama t, hirata k, yamada s, nagai a , nakamura i, bnai y, ohya i. 2003. dna typing from cigarette butts. legal medicine journal. 5:s177-s179. world health organization [internet]. 2009. geneva, switzerland. philippines’ global adult tobacco survey; [cited 2017 may 29]. available from https://www.who.int/tobacco/ surveillance/2009_gats_report_philippines.pdf?ua=1. validation of two extraction methods for human dna from cigarette butts 36 _____________ paul ryan l. sales <plsales@up.edu.ph> is a university research associate at the dna analysis laboratory of the natural sciences research institute, up diliman. he earned his bachelor of science in biology degree at the institute of biology, up diliman and is currently enrolled as a graduate student in the ms biology program at the institute of biology, up diliman. his research interests are in the use of dna technology for forensic applications. dorothy emma c. ferrer is a chemist at the forensic division of the investigation off ice of the commission on human rights of the philippines. she earned her master of forensic sciences in crime scene investigation at the george washington university and her bachelor of science in chemistry degree at the institute of chemistry, up diliman. gay vell ine c. calacal is a senior scientist working in the f ield of forensic dna typing/forensic genetics at the dna analysis laboratory, natural sciences research institute, up diliman. she obtained her master of science degree in microbiology from the institute of biology, up diliman and completed a forensic genetics and dna database technology course from the graduate school of biomedical sciences, university of north texas health science center at fort worth texas. usa. she is credited as one of the pioneers in the development of forensic dna typing research in the country, in the validation of analytical procedures for handling different types of biological samples for forensic applications, developing a system for the collection, handling and analysis of dna evidence, human remains identif ication and generation of the philippine reference population genetic databases. jazelyn m. salvador is currently a university researcher i at the dna analysis laboratory, natural sciences research institute, up diliman. she finished her bachelor of science in biology degree from the up manila and her master of science in microbiology degree at the up diliman. her research interest includes molecular population genetics and forensic dna applications. dr. maria corazon a. de ungria <madeungria@up.edu.ph> heads the dna analysis laboratory of the natural sciences research institute, university of the philippines diliman. her research thrusts include the development of molecular procedures and studying human genetic variations for forensic applications. she currently holds a university researcher v position and is a scientist 2 of the dostcsc scientif ic career system. 10-call for papers-new.pmd editor’s note humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> journal cover images courtesy of (l-r) vargas museum & anticamara and tan call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development 80 journal policy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the office of the vice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document defines research misconduct, specifies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identifies appropriate disciplinary actions. definitions scientific misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsification, or plagiarism in proposing, performing, or reviewing research in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 81 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research are concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “note to contributors” stipulates that “all articles must have a high degree of scholarhip,” the “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a certification from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right...” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as defined above and; b.) is specific and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the base for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specific advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the author is affiliated as well as the funding agency that supported the research. 82 the board shall ensure correction of the literature in the succeeding issue through various methods as defined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refeering a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and suppport appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. endnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientific misconduct policy. in attendance were: dr. corazon d. villareal, rduo director, presiding; dr. henry j. ramos, pmrgo director and professor, nip; atty. vyva victoria aguirre, ovcrd legal consultant; editors-in-chief dr. maricor soriano (science diliman) and dr. maria mangahas (socia l science diliman). ms nanie domingo and ms. dercy mararac, editorial assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these definitions of the forms of research misconduct are qouted verbatim from the policy of the office of research integrity of the united states public health service. similar phrasings of definitions are adopted in the references listed at the end of this document. 83 references council of science editors. “white paper on promoting integrity on scientific journal publications, as approved by the cse board of directors on september 3, 2006.” www.councilscienceeditors.org. accessed on january 26, 2009. “policy on scientific misconduct: university of southern california. http://policies.usc.edu/ policies/scientificmisconduct070108.pdf “scientific misconduct policy: new york university, the office of sponsored programs. https:// www.nyu.edu/osp/policies/scientificmisconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/physics/ optics/journal/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ajp/ submit.html editorial 1 guest editorial the enterprise called science has been likened to the construction of an edifice. there has to be a solid foundation from which a concrete and metal framework is made. the framework then holds together the individual blocks that form the bulk of the structure. the foundation and the framework of philippine marine science were and are being laid by the pioneers such as angel alcala, edgardo gomez, flor lacanilao, francisco nemenzo, gavino trono, jr., gregorio velasquez, among many others. much of the subsequent work would not have risen to great heights if these pioneers have not allowed us to build on their work. others among us today have started building new walls, annexes, and structures altogether, and will likely be pre-eminent in the future. whatever the structure, it starts with individual elements of metal and concrete – the scientific paper. this volume (fourth in a series) contains some of the scientific papers presented at the 8th national symposium in marine science (2005), the largest symposium ever held on marine science in the philippines. the symposium was marked by greater participation from the regional marine science institutions and non-governmental organizations. we are happy to see them contribute to this edifice that is philippine marine science. funding for the pams proceedings was provided by the bureau of agricultural research (bar) and the philippine council for aquatic and marine research and development (pcamrd). wilfredo “al” licuanan immediate past president philippine association of marine science 01_device comparison between videographic and photographic 7science diliman (january-june 2007) 19:1, 7-13 *corresponding author continuous degradation of coral reefs creates a need for techniques that can assess reefs condition rapidly and efficiently. the video transect survey is commonly used to monitor benthic communities because it is rapid, provides a permanent historical record of the data, and can help minimize observer bias. but this technology is not readily available to most research institutions because of its high cost. in this study, a low cost photographic method was used to survey benthic communities in the subtidal flat inside caniogan marine sanctuary, tondol, anda, pangasinan. results from this method were then compared with those from videographic methods. for the low cost photographic method, ten regularly spaced shots were directly taken from each 5m transect, totaling to 100 frames. ten 5m x ~0.25m video transects were also run over each of the twenty selected patch reefs, covering the whole demarcated area. ten regularly spaced frames were then taken from the videotape in each transect, totaling to 100 frames in each patch reef. in the laboratory, all frames were analyzed using the systematic 5-point method. both methods yielded comparable time in field data collection. however, videographic method demanded more time in post-collection computer analysis and it is more costly due to the required additional computer software and hardware. pairwise t-tests and analysis of similarities (anosim) revealed that both methods gathered similar results in terms of the diversity (p>0.05) and in terms of percentage composition (p>0.05) of life forms recorded, suggesting that both can be used interchangeably in benthic community surveys. keywords: transect methodology; coral reefs; point intercept techniques; video transects comparison between videographic and photographic methods in assessing coral reef benthic communities patrick c. cabaitan1*, wilfredo y. licuanan1, 2 and edgardo d. gomez1 1the marine science institute, university of the philippines diliman, quezon city 1101, philippines 2bro. alfred shields, fsc marine station, and biology department de la salle university, 2401 taft avenue, manila 1004, philippines tel.: +63 02 9223959; fax: +63 02 9247678 email address: pcabaitan@yahoo.com date submitted: april 7, 2006; date accepted: october 3, 2006 abstract cabaitan, licuanan, gomez 8 introduction reefs around the world are continuing to decline due to constant disturbance brought about by both anthropogenic and natural disturbance (wilkinson, 2004). rapid and efficient techniques that can be used to assess reefs condition are needed. the philippines is one of the pioneer countries that carried out a nationwide assessment of its reefs condition (gomez, 1991; gomez et al., 1994). during the 1980's, only professional marine biologists conducted surveys. at present, with the widespread establishment of marine protected areas (mpa) to reverse the trend of reef degradation (aliño et al., 2002), non-scientists like recreational divers and fisherfolks are becoming interested in conducting reef assessment by themselves. thus, a low-cost technique that can record and produce data rapidly and efficiently in the field and in the laboratory; and can be used even with less supervision by field biologists, should be considered. video transect surveys are commonly used by reef scientists to monitor reef benthic communities because of its advantages over the line intercept transect (lit) method (carleton and done 1995). video transect survey is more advantageous over the lit method because: 1) it is rapid; 2) it can reduce time required for field data collection; 3) it provides a permanent historical record of the data; 4) it allows comparison between observers in the laboratory to minimize observer bias; 5) field data can be collected by people not trained in lifeform identification; and resampling on other biotas can be done later (osborne and oxley, 1997). similar to the lit method, video transects are undertaken along a transect line with known length is laid along a depth contour. however, instead of having a researcher do the identification of the benthic community in situ, a video recording is taken along the entire transect and the identification is done in the laboratory with the use of frames or pictures extracted from the video tapes (english 1997). but video transects also have disadvantages. the main constraint is that this technology is not readily available to most research institutions because of its high cost, which is at least 7 times more expensive. it requires regular maintenance because there are more moving parts and needs additional computer hardware and software. also, reduced time in field data collection translates to more time in post-collection computer analysis. video transects often produce low resolution pictures or frames that may limit identification (uychiaco et al., 1992), though this may be improved with the availability of more advance technology in video processing and high definition cameras. the said disadvantages of video transects can be addressed by using an alternative technique such as photographic method. instead of swathing a video on laid transects and then extracting frames or photos from the video transects in the laboratory, the photographic method directly takes frames from the laid transect in the field. in this way, the time-consuming extracting of frames from the videos will be omitted. photographic method can also provide pictures with better resolution that may allow identification of biota to finer taxonomic resolution (aronson et al., 1994). this study aimed to compare videographic and photographic methods in assessing reef benthic communities, more specifically to find out whether both can record the same number and percentage composition of life forms. the study also intended to compare the cost-effectiveness and the time spent in the field data collection and in data post collection between the two methods. materials and methods coral reef benthic communities in the subtidal flat inside caniogan marine sanctuary, tondol, anda, pangasinan (see figure 1) were surveyed using videographic and photographic methods. twenty patch reefs were identified and demarcated with permanent 5mx5m-plots. patch reefs are at least 20 m away from each other and are interspersed among numerous other reefs. all surveys were only done inside the plots, to minimize the variability due to the movements of transects. each permanent plot serves as the sampling unit. for the videographic method, ten 5m x ~0.25m video transects were run over each of the twenty selected patch reefs, covering the whole demarcated area. this comparison between videographic and photographic 9 is equivalent to 100m transect per patch reef. in the laboratory, ten regularly spaced frames were then taken from the videotape of each transect, totaling to 100 frames in each patch reef. in this study, sony pc110 minidv video camera was used. all video transects were first transferred to the computer via firewire® link, with the use of pinnacle 7® software. virtualdub® was then used to extract frames from each video transect and was also used to automatically overlay 5 sampling points on all frames.the transfer of video transects to the computer is the part where time spent in post-collection computer analysis increases. for the photographic method, 100 shots were directly taken from ten regularly spaced points along each 5m transect with the use of a sony p8 digital camera. all frames were instantly transferred to the computer once the usb cable was connected. all frames with poor color balance and lighting were enhanced using adobe® photoshop® elements by means of auto levels command. both the video camera and the still camera were positioned ~0.25m away and parallel to the substratum during recording. the benthic characteristics under each of the 5 marks on a frame were identified according to the life forms defined by english et al. (1997). parameters such as 1) number of benthic life forms and 2) percentage composition of life forms recorded with the use of videographic methods were compared with the parameters recorded from photographic methods, using pairwise t-test and analysis of similarity (anosim) respectively. percentage composition of lifeforms were graphically represented in two-dimensional ordination plots by non-metric multidimensional scaling (nmds) using the bray-curtis measure of similarity. data were transformed to fourth root so that each lifeform contributed fairly evenly to each analysis. primer (plymouth routines in multivariate ecological research) v5® software was used to run anosim and nmds. results both videographic and photographic method yielded comparable time, 5.94+0.64 and 5.57+1.07 minutes respectively, in field data collection for every patch 120.00 16.29 figure 1. map showing study site in the subtidal flats off tondol, anda, pangasinan (northwestern philippines). cabaitan, licuanan, gomez 10 reef (see table 1). but a substantial difference in time was experienced during the data post collection. videographic method demanded additional time, about sixty-seven minutes, in the transfer of video transects from the video camera to the computer. in terms of cost, videorgraphic method is more expensive by about php 200,000 due to the additional computer software & hardware, and the video camera and housing itself. both videographic and photographic method identified similar number of lifeforms, 14.1 + 1.5 and 14.65 + 2.6 respectively (see table 2). by means of pairwise t-test, a p-value of 0.49 was obtained when the two methods were compared. the similarity of the methods in terms of percentage composition of lifeforms was also analyzed (see table 3 for benthic attributes). no differences were found in the community composition or levels of abundance of lifeforms between the two methods as shown in the mds plot (see figure 2). the anosim resulted in a global r value of 0.01 (p = 0.37). the near zero r value shows a very high level of similarity between the two methods. discussion both univariate (t-test) and multivariate (anosim) analyses showed that both methods can record similar number and percentage composition of lifeforms, which are commonly used to characterize the condition of reefs (loya, 1978; carleton & done, 1995). this suggests that both methods may be used interchangeably in benthic community surveys. thus, photographic method can be used as an alternative to the videographic method as it retains the advantages of video transects over lit: rapid, objective, can keep permanent record (osborne and oxley, 1997). constraints of videographic method are resolved by the photographic method: photographic method can save about sixty-seven minutes per patch reef in postcollection computer analysis (see also uychiaco et al., 1992). using the photographic method can reduce the cost of the field equipment by seven times cheaper and the cost of computer post-data collection. the only limitation of the photographic method is that resampling of frames is not possible. on the other hand, videographic method allows further resampling of frames from each video transect. this means more frames can be collected in future, in addition to the initial number of frames intended to be extracted, for other purpose like analysis of other biota (preskitt et al., 2004) or examination of increased sample size (ryan, 2004). the videographic method does collect more images, albeit at lower resolution. the patch reef photographic videographic method method 1 15 18 2 13 18 3 14 15 4 16 18 5 12 12 6 14 13 7 12 14 8 11 12 9 14 14 10 13 11 11 14 11 12 14 15 13 17 15 14 14 13 15 13 13 16 16 18 17 15 19 18 16 14 19 14 12 20 15 18 average 14.1 ± 1.5 14.65 ± 2.6 table 2 abundance of lifeform categories recorded per patch reef variables videographic photographic method method time underwater 5.94 ± 0.64 mins 5.57 ± 1.07 mins data post collection 92 mins 25 mins cost of hardware and software 220,000 to 230,000 php 30,000 to 35,000 php table 1 comparison between videographic and photographic methods in terms of time in the field data collection and laboratory post-data collection, and cost estimate comparison between videographic and photographic 11 highest dimension of pictures that can be extracted from the video clips is 640x480 while digital camera used in this study can take pictures with a dimension of up to 2048 x 1536. however, recent advancement of digital video may allow the extraction of pictures with higher dimension or resolution.videographic method sometimes produces distorted pictures, especially when abrupt movements of video transects happened in the field. pictures with higher dimension can be enlarged or magnified that allows identification of corals or other biota to finer taxonomic resolution (aronson 1994). before the collection of data in the field, environmental condition of the sites should be considered because there is greater chance of getting a photo with poor quality from both video and still camera, when water turbulence and sedimentation is high and light is low figure 2. non-metric multidimensional scaling (nmds) ordination on fourth root transformed lifeform percentage cover data collected using videographic (empty square) and photographic (inverted fill triangle) method. patch reef lifeforms methods p v p v p v p v p v p v p v p v p v p v ta 42.4 41.8 63.6 55.2 50.8 53.6 67.0 62.2 71.4 67.0 70.8 63.8 62.6 70.2 57.4 51.2 42.4 29.4 39.0 31.0 s 38.8 37.8 13.4 18.0 37.0 31.0 6.0 11.6 12.4 17.8 12.0 20.4 22.0 13.4 31.4 35.0 45.2 53.2 44.4 56.6 cm 6.0 4.6 6.8 7.2 3.0 4.2 6.6 5.4 7.4 6.0 6.2 3.8 3.8 4.0 2.0 3.6 4.4 6.8 5.8 3.8 ce 5.6 6.0 8.2 5.2 3.0 3.4 9.4 9.0 2.8 1.6 4.6 5.4 6.0 3.6 3.8 1.8 3.4 3.6 4.2 3.2 act 1.6 1.8 1.4 0.6 0.4 0.0 0.6 0.4 0.0 0.0 0.4 0.8 0.8 0.0 0.4 0.0 0.4 0.4 0.0 0.0 cb 1.2 0.4 0.8 1.8 0.2 0.4 0.6 2.0 1.6 0.8 1.0 0.4 0.4 0.4 0.0 1.0 1.0 1.2 0.4 0.6 ddd 1.2 0.8 0.0 1.4 0.0 2.0 0.0 0.8 0.0 0.8 0.2 0.2 0.0 2.0 0.2 1.2 0.4 1.0 0.0 1.0 dca 0.8 1.0 1.8 3.0 0.0 0.8 0.2 1.4 0.0 0.0 0.6 0.0 1.0 0.0 0.0 0.0 0.0 0.0 1.6 2.0 r 0.6 0.8 0.0 1.2 0.8 1.0 2.8 1.6 0.0 0.4 0.0 0.4 0.2 0.6 0.4 1.8 0.0 1.0 0.8 0.4 cs 0.6 0.8 0.8 1.4 0.2 0.0 1.0 0.0 0.4 0.0 0.6 0.8 0.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0 ma 0.4 1.4 0.6 0.8 0.2 1.6 0.4 0.4 1.2 1.4 0.6 2.2 0.4 0.4 0.2 0.0 0.2 0.2 0.2 0.0 acd 0.4 0.4 0.0 0.2 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ca 0.2 1.4 0.0 0.8 0.6 0.2 1.8 0.4 0.0 0.2 0.0 0.0 0.4 0.6 0.2 0.2 0.2 0.4 0.6 0.0 acb 0.2 0.4 0.6 0.4 0.4 0.0 0.0 2.2 1.2 2.8 1.4 1.2 0.0 2.0 0.0 0.8 0.4 0.2 0.0 0.0 rck 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.4 0.0 0.2 0.0 0.0 0.0 0.0 0.2 0.2 0.2 0.0 0.2 0.2 cme 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 acs 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 cbt 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 cf 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.2 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 chl 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 cmr 0.0 0.0 0.2 0.2 0.2 0.0 0.8 0.6 0.2 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.2 0.2 0.0 ot 0.0 0.2 0.2 0.2 0.0 0.0 0.6 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 sc 0.0 0.2 1.6 2.0 3.0 1.2 1.8 1.0 0.8 1.0 1.2 0.4 2.2 2.2 3.8 3.0 1.2 2.0 1.6 1.2 sp 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.4 1.0 0.0 total percentage 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 abundance of lifeform 14 17 13 18 14 13 16 17 12 12 14 13 12 13 11 12 14 14 13 10 3 8 9 104 5 6 71 2 table 3. percentages of life forms identified using the photographic (p) and videographic (v) methods for every patch reef. life forms are arranged from highest to lowest in terms of percentage cover. acb=acropora branching, acd=acropora digitate, acs=acropora submassive, act=acropora table, cb=coral branching, cbt=coral tubipora, ce=coral encrusting, cf=coral foliose, chl=coral heliopora, cm=coral massive, cme=coral millepora, cmr=coral mushroom, cs=coral submassive, ca=coralline algae, ma=macroalgae, ta=turf algae, dca=dead coral with algae, ot=other biota, sc=soft coral, sp=sponge, rck=rock, s=sand, r=rubble, ddd=unidentified cabaitan, licuanan, gomez 12 although, post processing of digital images can overcome some of this. poor quality of frames may alter the results of the analysis. the availability of this low cost photographic method provides an alternative technique to local resource management projects by non-government and local government units. this can allow rapid assessment and monitoring of reef condition by non-biologists without losing the scientific value of the collected data. the information that can be provided by the photographic method would be important to stakeholders and decision makers to better manage and conserve their reefs. acknowledgements we would like to thank conservation international for the student's travel grant and pams for the opportunity to present and publish this paper. much of the data used in this paper are from the baseline surveys of the project, "coral reef habitat and productivity enhancement through coral transplantation and giant clam restocking" of prof. edgardo d. gomez, a pew fellow in marine conservation. references aliño, p.m, e.f.b. miclat, c.l. nanola jr., h.a. roaquiaouit, and r.t. campos (eds). 2002. atlas of philippine coral reefs. goodwill trading co., inc. manila, philippines. aronson, r.b., p.j. edmunds, w.f. precht, d.w. swanson, and d.r. levitan. 1994. large-scale, long-term monitoring of caribbean coral reefs: simple, quick, inexpensive techniques. atoll research bulletin 421: 1-19. carleton, j.h. & t.j. done. 1995. quantitative video sampling of coral reef benthos: large-scale application. coral reefs 14:35-46. english, s., c. wilkinson & v. baker (eds.). 1997. survey manual for tropical marine science, 2nd ed., australian institute of marine science, townsville. gomez, e.d. 1991. coral reef ecosystems and resources of the philippines. canopy international (1991) 16(5):1, 67,10-12. gomez ed, aliño pm, yap ht and licuanan wy. 1994. a review of the status of the status of the philippine reefs. marine pollution bulletin 29(1-3): 62-68 table 3. cont’d patch reef lifeform s m ethods p v p v p v p v p v p v p v p v p v p v ta 33.6 49.2 36.2 52.4 44.6 61.2 65.4 69.0 46.6 47.0 75.8 70.6 70.0 62.0 61.8 58.2 74.0 62.0 62.6 54.4 s 51.8 39.2 40.4 25.2 34.8 20.8 19.6 12.6 29.4 37.2 8.4 8.6 12.4 16.0 18.2 25.0 12.8 23.6 25.6 30.8 c m 5.0 5.4 4.6 5.0 6.6 6.8 6.2 7.8 6.8 4.2 5.0 5.8 1.6 3.6 7.4 7.6 2.4 4.0 2.8 4.0 c e 3.2 3.4 6.8 5.6 4.8 4.0 3.0 3.0 7.2 2.8 3.4 5.6 8.2 8.4 7.0 4.0 3.4 3.8 2.4 1.6 a c t 0.8 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 c b 1.0 0.4 1.6 0.4 0.6 0.0 0.6 0.2 0.2 0.0 0.6 0.6 1.0 2.4 0.0 0.0 0.4 0.4 1.0 0.8 d d d 0.2 0.6 0.6 1.2 0.2 1.0 0.0 0.2 0.0 0.8 0.2 1.2 0.0 0.6 0.4 0.6 0.4 0.2 0.2 0.6 d c a 0.0 0.0 1.6 1.8 0.8 1.2 0.0 1.4 1.0 0.8 0.2 0.8 0.0 0.6 0.4 0.0 0.0 0.0 0.2 0.6 r 1.4 0.8 1.2 3.2 0.4 1.6 0.8 1.8 2.0 1.2 0.2 0.8 1.0 1.0 0.6 0.4 0.6 1.2 0.0 1.2 c s 0.2 0.2 0.2 0.4 0.4 0.0 0.2 1.2 0.2 0.2 0.6 0.6 0.6 0.2 0.4 0.0 0.4 0.2 0.0 0.0 m a 0.2 0.4 0.0 0.0 0.4 0.2 0.6 0.0 0.4 0.2 0.0 0.8 0.2 0.0 0.2 0.2 0.4 0.0 0.0 0.0 a c d 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 c a 0.4 0.0 0.8 0.0 0.4 0.0 0.2 0.2 0.2 0.0 0.4 0.4 0.0 0.2 0.2 0.2 0.8 0.0 0.2 0.2 a c b 0.0 0.0 0.0 0.2 1.0 0.0 0.4 0.0 0.2 0.8 1.0 1.4 1.6 0.2 1.2 0.8 0.6 1.2 0.2 0.6 r c k 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.2 0.0 0.2 0.2 0.0 0.0 0.0 0.0 0.0 0.0 c m e 0.0 0.0 0.2 1.2 0.0 0.0 0.2 0.2 0.0 0.2 0.0 0.0 0.2 0.2 0.2 0.2 0.6 0.0 0.4 0.4 a c s 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.2 0.4 0.6 0.0 0.2 0.0 0.4 c b t 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 c f 0.4 0.0 1.2 0.4 0.2 0.2 0.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 1.0 0.8 c h l 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.2 c m r 0.0 0.2 0.2 0.0 0.4 0.2 1.0 0.6 0.4 0.0 1.2 0.8 0.6 0.2 0.4 0.0 1.0 1.4 0.8 0.6 o t 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.2 0.4 0.0 0.4 0.0 0.2 0.0 0.0 0.2 0.2 sc 1.6 0.2 4.4 3.0 3.2 2.6 1.6 1.8 5.4 4.4 1.8 1.2 1.6 2.6 1.0 1.8 2.2 1.8 2.2 2.6 sp 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.2 0.2 0.2 0.0 0.0 0.0 0.0 0.2 0.0 total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 abundance of 14 11 14 13 17 12 14 13 13 13 16 17 15 19 16 14 14 12 15 17 11 12 13 14 15 20 16 17 18 19 comparison between videographic and photographic 13 loya, y. 1978. plotless and transect methods. in: stoddart, d.r. & r.e. johannes (eds.) coral reefs: research methods, unesco monographs on oceanographic methodology, 197-217. osborne, k. & w.g. oxley. 1997. sampling benthic communities using video transects. in: english, s., c. wilkinson & v. baker (eds.). survey manual for tropical marine science, 2nd ed., australian institute of marine science, townsville. preskitt, l.b., p.s. vroom, and c.m. smith. 2004. a rapid ecological assessment (rea) quantitative survey method for benthic algae using photoquadrats with scuba. pacific science 58 (2): 201-209. ryan, d.a.j. 2004. point sampling strategies for estimating coverage from benthic video transects. environmetrics, 15, 193-207. uychiaco, a.j., p.m. aliño & m.p. atrigenio. 1992. video and other monitoring techniques for coral reef communities. proc 3rd asean science and technology week conference, marine science: living coastal resources 6:35-40. wilkinson, c. 2004. status of coral reefs of the world: 2004. australian institute of marine science. from the editor it has been almost one year since the first case of coronavirus disease 2019 (covid-19) has been reported in china, which eventually led to the ongoing pandemic. as of 16 december 2020, the world health organization reported 72,196,732 confirmed cases of covid-19 and 1,630,521 deaths globally, and 451,839 confirmed cases and 8,812 deaths in the philippines (https://covid19.who.int/). although these numbers continue to increase every day, there is light at the end of the tunnel because the us food and drug administration has recently issued emergency use authorization for one of the vaccines that have been developed to combat this dreaded disease. other vaccines are expected to be authorized for emergency use in the coming days. these vaccines have been developed and approved for use at an unprecedented speed – barely one year. normally, it takes about 10 years for vaccines to be developed and approved. we hope that these vaccines will reach the shores of the philippines soon so that we can already put an end to this pandemic and get back to our normal lives. in this issue, we feature four research articles. the first article is by torres et al. who used geometric morphometrics to uncover body shape differences among populations of the striped snakehead (locally known as dalag) from three sites in laguna de bay and to correlate these shape differences with the physicochemical characteristics of water in those sites. the authors wanted to find out if there were selective pressures within each site that would lead to different morphotypes in the species. the authors found that shape variation was greatest in the head region, which was correlated with differences in dissolved oxygen content and ph of water in the three sampling sites. the second article is by bacaoco et al. who designed a low-cost differential optical absorption spectroscopy (doas) for air quality monitoring in urban areas. the authors demonstrated that the doas set-up that they developed is capable of measuring trace amounts of atmospheric nitrogen dioxide (no 2 ) concentration and aerosol optical thickness (aot). in addition to its reliable sensitivity and it being a cheaper alternative for environmental monitoring of pollutants, the set-up can also be made portable so that it can be easily transported to the field. the third article is by buenaventura and yago who developed a selective and sensitive electrochemical biosensor for uric acid determination using carbon paste electrode (cpe) modified with copper (ii) oxide (cuo) particles and urate oxidase (uox) enzyme. the authors also demonstrated that their uox-cuo-cpe biosensor is reusable, reproducible, and stable even after five weeks of storage. uric acid is the end-product of purine catabolism in humans and other primates. abnormal levels of uric acid can lead to various diseases; hence, uric acid levels are routinely analyzed in clinical laboratories during physical examination. the last article is by fornillos et al. who used dna barcoding to establish the identity of bivalves collected from two freshwater environments in the philippines. the bivalves were found to be chinese pond mussel (sinanodonta woodiana), which are known invasive exotic species. genetic analyses of dna sequences of these bivalves from the philippines and from other countries revealed that the s. woodiana that were included in the authors’ study could have originated from indonesia or malaysia. the authors surmised that introduced fish species carrying the larvae of this exotic bivalve species could have been the most likely route of introduction into the philippines. jonas p. quilang, ph.d. editor-in-chief 77 science diliman (july-december 2020) 32:2, 77-96 molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) from mindoro and leyte islands, philippines raffy jay c. fornillos* gerard clinton l. que rogel victor d. mendoza ian kendrich c. fontanilla dna barcoding laboratory, institute of biology college of science university of the philippines diliman perry s. ong† biodiversity research laboratory, institute of biology college of science university of the philippines diliman abstract the chinese pond mussel sinanodonta woodiana (lea, 1834) is a large freshwater bivalve species of the family unionidae and a known invasive alien species. proper verification of its identity as well as its source population is crucial for the control of its spread. however, its high plastic shell morphology that resembles other non-invasive species of unionids can be an obstacle. the distribution and ecological impact of this invasive unionid is not fully understood and should be further investigated to prevent further spread in the philippines. in this study, we used the cytochrome oxidase i (cox1) gene to verify the identity of putative s. woodiana samples collected from bato creek in oriental mindoro and lake danao in leyte, philippines and elucidate their source populations. eighteen cytochrome oxidase subunit 1 (cox1) barcodes were generated from samples collected from lake danao, leyte (n=13) and bato creek, oriental mindoro (n=5). these barcodes were subjected to basic local alignment search tool (blast) analysis, which showed that the cox1 sequences from the philippine samples matched with those of s. woodiana (>94%) found in genbank. the sequences were then aligned with cox1 sequences of s. woodiana and other unionid representatives from genbank. * corresponding author molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 78 phylogenetic and haplotype network analyses also showed three haplotypes (hap 1, 2, and 4) of s. woodiana samples from lake danao and bato creek. hap 1 and 2 are distinct haplotypes observed in lake danao samples while hap 4 is shared between lake danao and bato creek samples and have clustered with conspecific specimens from malaysia and indonesia, suggesting their potential island southeast asian origin. keywords: unionidae, dna barcoding, invasive alien species, sinanodonta woodiana introduction the study of invasive alien species (ias) cannot be overemphasized, and the negative impact of these organisms cannot be underestimated as they often affect the agricultural sector and cause significant public health problems (andersen et al. 2004). ias have been introduced either accidentally or purposely, and many are now uncontrolled in newly colonized ecosystems where they thrive and continually cause significant economic and ecological damage (anderson 1993). a common example is the introduction of the golden apple snail pomacea canaliculata in the philippines, which was originally imported as an alternative protein source for farmers but are now considered an agricultural pest due to their uncontrolled proliferation in agricultural lands (anderson 1993; halwart 1994). another freshwater mollusk, the invasive unionid chinese pond mussel sinanodonta woodiana (lea, 1834), is now considered a major problem in many countries in europe, north america and other parts of southeast asia (kraszewski and zdanowski 2007; popa and murariu 2009; lajtner and crnčan 2011; colomba et al. 2013; kamburska et al. 2013; soroka et al. 2014). this bivalve species, which closely resembles its non-invasive relatives in the genus anodonta, negatively affects local anodontine populations including a. anatina and other unionids in europe (guarneri et al. 2014). s. woodiana’s invasive capacity is primarily attributed to its high filtering capacity, and the crossresistance induction of its parasitic glochidium larva to its fish host (donrovich et al. 2017; douda and čadková 2017). these characteristics show that these invasive mussels can become “masters of invasion” due to their high tolerance of various environmental factors such as changes in temperature, moisture, humidity, and utilization of hosts, thus making them able to survive and reproduce in many types of habitats (mwatawala et al. 2006; davidson et al. 2011). r. c. fornillos et al. 79 sinanodonta woodiana is a large freshwater dioecious bivalve species of the family unionidae. this family is a large group of freshwater mussels with several member species endemic in freshwaters of north america, europe and east asia. s. woodiana’s native distribution include the freshwater basins of amur river, hanka lake, china, hong kong, taiwan, cambodia, thailand, and japan but were also reported in other non-endemic territories in europe where they are now widespread and have established stable populations (kraszewski and zdanowski 2007; popa and murariu 2009; latjner and crnčan 2011; colomba et al. 2013; kamburska et al. 2013; soroka et al. 2014). the invasion of s. woodiana in european freshwaters was due to the introduction of fish stocks infested with the bivalve’s parasitic larval glochidium which primarily attaches to the gills of its fish host (kraszewski 2006; kraszewski and zdanowski 2007; guarneri et al. 2014). s. woodiana produces large biomass both in natural and colonized areas, and has the ability to tolerate a wide range of physical and chemical factors which contribute to its capacity to invade new aquatic habitats. this bivalve is also a broad host generalist, often able to develop in both co-invasive and native fish hosts (douda et al. 2012). moreover, its invasiveness is also associated with its ability to grow fast and produce high numbers of offspring, and to its long life span ranging from 10-15 years (dudgeon and morton 1983; afanasjev et al. 2001; kraszewski and zdanowski 2007; guarneri et al. 2014). aside from food and fish host competition, s. woodiana can quickly dominate both natural and pre-colonized habitat by establishing a strong benthic-pelagic coupling that may result in changes in the biocenoses of organisms affecting the physical characteristics of freshwater systems (kraszewski and zdanowsk 2001; kraszewski and zdanowski 2007; guarneri et al. 2014). proper identification and detection of s. woodiana have been a challenge since its shell morphology resembles that of the native species of anodontines such as anodonta anatina (guarneri et al. 2014). in fact, the taxonomy of s. woodiana was originally assigned to the genus anodonta but was later grouped to the more appropriate genus sinanodonta to standardize differences in nomenclature and classification especially for inland water mollusks where anodontines and s. woodiana coexist (kraszewski and zdanowski 2007; guarneri et al. 2014). morphology-based analysis and molecular tools for species identification have been used for s. woodiana in the past. linear and geometric morphometrics were used for s. woodiana samples from europe and other parts of asia such as the philippines (demayo et al. 2012; guarneri et al. 2014). results showed that s. woodiana’s phenotype is highly plastic, and the variations in shell dimensions measured were due to the effects of various environmental factors. in the study molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 80 of guarneri et al. (2014), populations of s. woodiana in two of italy’s largest lakes, po and maggiore, indicated that the species exhibits variation even between populations and may potentially lead to species misidentification. similar results were reported on geometric morphometrics of s. woodiana specimens collected from two separate freshwater bodies (lake lanao and lawis stream) in mindanao, philippines. variations observed in its shell were attributed to allometry within populations and varying environmental factors were hypothesized to play a major role. molecular analysis for species identification was also carried out in european s. woodiana samples using the general barcode gene cytochrome c oxidase subunit 1 (cox1) (guarneri et al. 2014), confirming 99.84-100% similarity of the samples compared using s. woodiana accessions in the barcode of life database (bold). a more comprehensive analysis using various s. woodiana cox1 gene accessions in genbank (https://www.ncbi.nlm.nih.gov/) was generated using bayesian inference, which indicated two major lineages detected, namely temperate and tropical, in which samples from the philippines clustered together with other s. woodiana from indonesia and malaysia to form the tropical lineage (vikhrev et al. 2017). the status of s. woodiana distribution in the philippines is limited and is only known in few locations based on previous reports such as in mindanao (demayo et al. 2012). another introduced unionid species with shell form and shape similarity with s. woodiana is cristaria plicata, which is also present in the country but is used for pearl farming (guerrero et al. 2002). the possibility of introducing the wrong unionid species for pearl farming is highly likely due to the striking resemblance and plastic shell morphology of the two species. just like s. woodiana, c. plicata is considered an invasive exotic species and may pose occupational hazard such as injuring workers due to its sharp shell, but is also a source of food and income to people who culture it (cagauan et al. 2007). c. plicata was detected in taal lake (mutia et al. 2017), lake oro in agusan del sur (sularte and jumawan, 2016), and has been reportedly cultured in nueva ecija and laguna for pearl farming (guerrero 2002). in this study, we conducted molecular identification through dna barcoding by using the cox1 gene from putative s. woodiana samples collected from bato creek and lake danao in the absence of a useful morphological key to confirm species identity. these aforementioned sites have no prior reports on the occurrence of s. woodiana. this study also utilized neighbor-joining and maximum likelihood (ml) algorithms and median-joining haplotype network analysis to further reveal the relationships of the two subpopulations in the philippines with other specimens of s. woodiana from other countries accessed from genbank. r. c. fornillos et al. 81 materials and methods sample collection s. woodiana samples were bought from local vendors along lake danao in the municipality of ormoc, leyte and handpicked from the waters along bato creek in the municipality of victoria, oriental mindoro (figure 1). the difference in sampling methodology arose due to a serendipitous discovery of s. woodiana in oriental mindoro during fieldwork for another unrelated study. samples were stored in re-sealable plastic bags, placed in a styrofoam chest filled with ice cubes and then transported to the dna barcoding laboratory at the institute of biology of the university of the philippines diliman in quezon city. photographs of the samples were likewise taken. morphometrics of the bato creek, oriental mindoro specimens were also taken (table 1). figure 1. location of the (a) study sites in bato creek, mindoro and lake danao, leyte. sinanodonta woodiana sample collected from (b) bato creek and (c) lake danao. map adapted from arcgis base map downloaded from http://www.geoportal.gov.ph/viewer. molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 82 table 1. measurements of the dorsal shell length of 18 s. woodiana specimens presented in this study as well as their corresponding genbank accession numbers. specimen genbank accession number dorsal length (cm) mindorobv1 mn322559 7.51 mindorobv2 mn322558 4.20 mindorobv3 mn322555 4.33 mindorobv4 mn322557 6.28 mindorobv5 mn322554 4.90 un1 kx424967 11.5 un2 kx424968 10.3 un4 kx424969 11.5 un5 kx424970 10.0 un6 kx424976 11.0 un8 kx424977 11.0 un10 kx424978 10.0 un12 kx424979 12.0 sw3 kx424971 * sw4a kx424972 * sw5b kx424973 * sw7a kx424974 * sw8b kx424975 * *no data dna extraction and polymerase chain reaction foot muscle tissue from 13 individuals of s. woodiana samples from lake danao and 5 individuals from bato creek were used. dna extraction was performed using a commercial dna extraction kit (purelink® genomic dna extraction kit, invitrogen life technologies or purelink™ genomic dna mini kit, thermofisher scientific) following the manufacturer’s protocol with a final elution of 150 microliters (ul). for dna quantitation using nanodrop 2000c (thermo scientific), 1 ul was used for each sample. two primer sets were used for cox1 amplification, namely hco-lco (hco: 5’ taaacttcagggtgaccaaaaaatca -3’, lco: 5’ggtcaacaaatcataaagatattgg -3’) (folmer et al. 1994) and styhcostylcoii (styhco: 5’gaattaaaaatatatacttctgggtg -3’, stylcoii: 5’ acgaatcataaggatattggtac -3’) (fontanilla et al. 2017). either primer pair, which targets the same region of cox1, was used for amplification. r. c. fornillos et al. 83 for lake danao samples, polymerase chain reaction (pcr) was done by preparing a master mixture with the following components: 5 ul 10x pcr buffer (-mgcl 2 ), 0.5 ul 0.1875 mm dntp, 1.25 ul for each primer (styhco, stylcoii, respectively), 5 ul q buffer, 1 ul 25 mm mgcl 2 , 0.125 ul taq polymerase (5u/ul), 8.875 ul nuclease-free dh 2 o, and 2 ul dna for a total of 25 ul per sample using a pcr condition described by ma et al. (2012) and run in a 96-well thermocycler (multigene optimax). for the bato creek samples, the pcr master mix consisted of 5 ul 10x mytaq™ pcr buffer (with 5 mm dntps and 15 mm mgcl 2 ), 1 ul of forward and reverse primers (lco, hco, respectively), 1 ul of 50 mm mgcl 2 , 0.125 ul of mytaq™ dna polymerase (5u/ul) (bioline, united kingdom), 14.875 ul nuclease dh 2 o, and 2 ul of dna template for a total of 25 ul. pcr conditions consisted of an initial denaturation step at 95°c for 5 minutes, followed by 36 cycles of denaturation at 92°c for 30 seconds, annealing at 51°c for 30 seconds, extension at 68°c for 2 minutes, and a final extension step at 68°c for 5 minutes run in a 96-well thermocycler (simpliamp, applied biosystems). the difference in reagents is not expected to affect results since specificity for the target area of cox1 is dependent on the primers. agarose gel electrophoresis, purification and sequencing each pcr product was loaded in a 1% agarose gel stained with 1% ethidium bromide (etbr), submerged in 0.5x tbe (1 l 5x tbe = 54 g tris, 27.5 g boric acid, 20 ml of 0.5 m edta ph 8.0) and exposed to 100 volts of electricity for 30 minutes to separate amplicons (~650-700 bp) using a horizontal gel electrophoresis apparatus (gel xl ultra v-2 labnet). the gel was visualized in an ultraviolet transilluminator. visible bands were cut carefully using sterile scalpel blades and were stored in 2 ul microcentrifuge tubes until purification. the purification process was done using a commercial gel extraction kit (qiaquick® gel extraction kit, qiagen and thermo scientific™ genejet gel extraction kit) following the manufacturer’s protocol. a final elution volume of 50 ul was prepared per sample. samples were sent to 1st base malaysia and macrogen south korea together with the primers used for single pass sequencing for both forward and reverse sequences. dna sequences in silico analyses forward and reverse sequences were assembled using staden package version 4.0 (staden et al. 2000), and each consensus sequence was subjected to nucleotide blast® (altschul and koonin 1998) for determining related sequences. one hundred ninety cox1 sequences of unionids were downloaded from genbank (http://blast. molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 84 ncbi.nlm.nih.gov/) and included in the analyses. s. woodiana cox1 sequences and other unionid cox1 sequences were downloaded and aligned in bioedit sequence alignment editor 7.0.9.0 (hall 1999) using the feature clustalw (gibson et al. 1996). uniform gaps were deleted and the shortest sequence was used as a reference for cutting the edges of the alignment. the sequence alignment file was converted to nexus (.nex) format using dambe v. 6.4.81 (xia 2013, 2017), a format readable by paup* version 4.01b10 (swofford 2002) for tree construction and genetic distance calculation. the substitution model was determined using jmodeltest v.2.1.10 (darriba et al. 2012), and the model with the best log-likelihood score was chosen using the akaike information criterion (aic) (akaike 1973, 1974; hurvich and tsai 1993). neighbor-joining (saitou and nei 1987) and maximum likelihood (ml) (felsenstein 1981) tree construction methods were applied. bootstrap replication was executed to determine branch support and reliability (bootstrap nreps=1000) (felsenstein 1985). the generated ml tree was visualized and rooted using tree explorer (tamura 1999), with the bootstrap supports for both tree construction methods incorporated. the tree was rooted on two species under margaritiferidae, a sister family of unionidae (combosch et al. 2017), margaritifera margaritifera (jn243891, dq060171) and margaritifera auricularia (kc703969, jx046574). unique haplotypes were then subjected to median-joining haplotype network analysis using network v5.0.1.1 (bandelt et al. 1999) to determine the possible origin of philippine s. woodiana. haplotype network analysis aimed to determine the relationship between observed haplotypes in a dataset depending on a number of single nucleotide polymorphisms (snps) observed in the cox1 gene. results a total of 18 cox1 sequences obtained from s. woodiana individuals collected from bato creek, mindoro and lake danao, leyte (figure 1, table 1) were generated in this study with 21 identified haplotypes from the generated cox 1 alignment (figure 3, table 2). blast results (table 3) showed that the cox1 sequences matched those of s. woodiana found in the genbank (>94%). ml tree based on trn+i model of dna substitution as determined by jmodeltest showed the distinct clustering of the philippine specimens together with other s. woodiana specimens from malaysia and indonesia with 91% ml bootstraps (figure 2). r. c. fornillos et al. 85 figure 2. maximum likelihood tree of s. woodiana geographical isolates using the mitochondrial cytochrome oxidase subunit 1 gene (cox1) tested in paup*. bootstrap replication was done to test branch reliability (bootstrap nreps = 1000) to nodes with molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 86 bootstrap support < 50 were only shown. the tree was rooted on four cox1 sequences of two species under the margaritiferidae family [margaritifera margaritifera (genbank accession no. jn243891, dq060171)] [margaritifera auricularia (genbank accession no. kc703969, jx046574)]. ml tree on the right is an inset that shows the branches and bootstrap supports of the tropical invasive lineage. figure 3. median-joining network of s. woodiana haplotypes of the cytochrome c oxidase subunit i gene (cox1) from genbank and sequences generated in this study. a total of 21 haplotypes were observed from the generated 520 bases long cox1 dna alignment. red dots are putative haplotypes. sequences from lake danao split into three haplotypes (hap 1, hap 2, hap 4), while those from bato creek, mindoro oriental grouped with one of the lake danao haplotypes and other s. woodiana samples from malaysia and indonesia (hap 4). table 2. composition of the 21 distinct haplotypes used in the median-joining haplotype network analysis. hap 1, 2, and 4 are haplotypes associated with s. woodiana samples collected from bato creek and lake danao in the islands of mindoro and leyte, respectively. samples with missing locations have no sampling details indicated in genbank. haplotype no. genbank accession number of sequences species id location 1 kx424968 sinanodonta woodiana lake danao, leyte, philippines 2 kx424979, kx424978, kx424973, kx424972, kx424971 sinanodonta woodiana lake danao, leyte, philippines 3 kx051325, kx051324, kx051316 sinanodonta woodiana malaysia r. c. fornillos et al. 87 4 kx424977, kx424976, kx424975, kx424974, kx424970, kx424969, kx424967 sinanodonta woodiana lake danao, leyte, philippines mn322554 mn322559, mn322558, mn322555, mn322557, sinanodonta woodiana bato creek, mindoro, philippines kx051326, kx051320, kx051318, kx051321 sinanodonta woodiana malaysia ku891642, ku891641 sinanodonta woodiana indonesia 5 kx051323, kx051319 kx051317 sinanodonta woodiana malaysia 6 kx051315 sinanodonta woodiana malaysia 7 kx051328 sinanodonta woodiana malaysia 8 kj434487 sinanodonta woodiana china 9 ky561633 anemina sp. russia 10 gq451870 anodonta archaeformis south korea 11 gq451869 anodonta archaeformis south korea 12 ab055627 sinanodonta woodiana japan 13 ky561635 sinanodonta sp. vietnam 14 kx822668 sinanodonta woodiana vietnam 15 km272949 sinanodonta woodiana china 16 kj434486 sinanodonta woodiana china 17 gq451868 sinanodonta woodiana south korea 18 gq451867 sinanodonta woodiana south korea 19 ku853269, ku853268 ku853267, ku853266 sinanodonta woodiana russia 20 kj434489, kj434488, kj434490 sinanodonta woodiana china 21 kj125079 sinanodonta woodiana ukraine jq253894 anodonta woodiana ukraine kj125078 sinanodonta woodiana poland ef440349, af468683 anodonta woodiana poland kf731775, kf731776 anodonta woodiana italy kj434483, kj434482, kj434485, kj434484 sinanodonta woodiana jq253893, hq283345, hq283344 anodonta woodiana table 2. composition of the 21 distinct haplotypes used in the median-joining haplotype network analysis. hap 1, 2, and 4 are haplotypes associated with s. woodiana samples collected from bato creek and lake danao in the islands of mindoro and leyte, respectively. samples with missing locations have no sampling details indicated in genbank. (cont'n.) molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 88 table 3. blastn results of 18 s. woodiana sequences generated from this study. accession number blastn result query cover percent identity kx424977 kx424976 kx424975 kx424970 kx424974 kx424969 kx424967 mn322559 mn322558 mn322555 mn322557 mn322554 mh319868 sinanodonta woodiana ugsb 19578 isolate 24480 cytochrome c oxidase subunit i (coi) 100% 100% kx424968 mh319868 sinanodonta woodiana ugsb 19578 isolate 24480 cytochrome c oxidase subunit i (coi) 99% 100% kx424972 kx424971 kx424979 kx424978 kx424973 mg742232 sinanodonta woodiana cytochrome oxidase subunit i (coi) gene, partial cds; mitochondrial 100% 100% a total of three haplotypes were observed in all s. woodiana cox1 sequences generated from this study, two were from lake danao (hap 1 and hap 2) and one was shared between lake danao and bato creek (hap 4). these unique cox1 sequences were used for haplotype network analysis (figure 3). results showed that the lake danao and bato creek samples of s. woodiana probably came from a population of s. woodiana from malaysia introduced to the lake, though the mode of introduction is uncertain and still a subject for investigation. among all haplotypes, s. woodiana from malaysia and indonesia are closest to the lake danao and bato creek specimens based on the level of support for branch reliability on the node where the philippine specimens diverged from the chinese s. woodiana and on the minimal snps observed among philippine, chinese, and other island southeast asian (malaysia and indonesia) specimens as compared to european specimens (figure 2). r. c. fornillos et al. 89 discussion the use of molecular data is crucial for species delineation if traditional taxonomy, which is based on morphological traits, is insufficient. in this study, the presence of the s. woodiana is confirmed using dna barcoding using cox1 (hebert et al. 2003). phylogenetic and median-joining haplotype network analyses are useful tools for visualizing the relationships of samples with different geographical distributions. the ml tree of cox1 of unionid taxa showed that the lake danao and bato creek specimens are conspecific with s. woodiana. median-joining haplotype network analysis visualizes the possible origin of the philippine s. woodiana populations using all distinct haplotypes of s. woodiana and creates a network based on mutation events that occurred among haplotypes. the philippine specimens probably originated from malaysia. the low haplotype diversity observed from the lake danao and bato creek samples might be due to founder effect; the same observation has been reported by soroka et al. (2014) on their analysis of s. woodiana samples from hungary and poland. dna barcoding for species identification is proven useful for s. woodiana as the high plasticity of the morphology of s. woodiana hinders accurate identification of the species (kraszewski 2006; guarneri et al. 2014). in addition, other molecular markers and even the use of microsatellites have been suggested, which may provide useful information on its source and path of invasion and better resolution on the phylogenetic relationship of s. woodiana to other unionids (bogan and roe 2008; popa et al. 2011). the potential host fish species of s. woodiana within lake danao and bato creek system is also a promising area of research. its possible interaction with local fish stocks such as oreochromis niloticus (nile tilapia), chanos chanos (milkfish), as well as its known host cyprinus carpio may be of particular interest due to the mussel being a generalist towards its host (douda et al. 2012). identification of the host fish species responsible for its invasion success by bearing its glochidium larva could reveal how the species was introduced in the philippines. such was the case for europe where the bivalve utilized aristichthus nobilis (bighead carp), ctenopharyngodon idella (grass carp) and hypophthalamichthys molitrix (silver carp) for its dispersal and propagation (colomba et al. 2013). the introduction of many species for aquaculture could be the most likely route of introduction of the mussel in the philippines. therefore, a survey of freshwater habitats, particularly those utilized for aquaculture using introduced species, is necessary to assess the distribution and spread of this invasive species. molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 90 moreover, further surveys on other endemic unionids with detailed descriptions on morphology, molecular identity, and life history is encouraged so that accurate comparative assessments could be made such as on how invasive exotic unionids like s. woodiana impact their distribution and dispersal. this is the case of rectidens sumatrensis, a native unionid in borneo, for which s. woodiana is a major competitor and a significant threat that has dominated freshwater habitats in the island and is likely associated with intentional introductions made for food source and ornamental purposes (zieritz et al. 2018). conclusions invasive alien species are an emerging threat to global biodiversity. due to the increase in mobility and access to agricultural goods and aquaculture products, opportunistic transport of organisms highly associated with these commodities may likewise occur. in this study, s. woodiana was detected from two sites in the philippines. phylogenetic and haplotype network analyses revealed that the philippine populations are most closely related to other island southeast asian haplotypes, such as those from indonesia and malaysia, and may even suggest their likely origin from these areas. introduced fish species bearing the glochidium larva may be the most likely route of introduction in the philippines. the mussel’s resemblance to other native unionid species could also facilitate its further spread. other potential freshwater areas in the philippines can be surveyed for the presence of s. woodiana to determine its distribution and potential effect to local unionid species. moreover, s. woodiana’s resemblance to its relatives may facilitate its further spread as it could be mistaken for another unionid, such as the bivalve cristaria plicata, which also belongs to family unionidae and resembles s. woodiana morphologically. like s. woodiana, it is native to east asia and has been introduced in the philippines. recommendations increasing sample size and adding more locations can be performed to provide more comprehensive data on the distribution of s. woodiana in the philippines. population genetic analysis can also shed light on the potential origin and route of spread in the philippines. detection and survey of the presence of the glochidium larva in fish hosts could aid in mitigating the spread of the ias in the philippines through its host. areas with reported c. plicata should be prioritized to confirm species identity through barcoding and to survey other native unionid species inhabiting the sites. r. c. fornillos et al. 91 additionally, further investigations should be made on how s. woodiana was dispersed in these non-native territories such as in lake danao and bato creek. around lake danao, for example, s. woodiana is being sold and consumed as a major shellfish commodity. short informal interviews conducted by the authors with the vendors reported that s. woodiana was purposely introduced in the lake primarily as a source of food and income for the locals, though these reports should be carefully interpreted and further confirmed by implementing more scientific and systematic methods of extracting information from the locals such as interviews using questionnaires, focus group discussions, and key informant interviews. this can be explored in the future and information from these surveys will supplement biological data in tracing the origin and path of invasion of s. woodiana. furthermore, examining the distribution network of both s. woodiana and its potential host fish species in the locality will provide significant information on the path and spread of invasion most especially if there are restocking practices being made using s. woodiana or of fish hosts infected with the mussel’s glochidium to other freshwater bodies, thus contributing to the further spread. in the study, s. woodiana samples collected from lake danao were bought from vendors in the same clump, making it difficult to trace the specific spot in the lake where these mussels were collected. a geographic information systems approach will help us understand its dispersion by mapping sites with stable s. woodiana populations in freshwater habitats and in sites where they are recently reported. in this manner, authorities will be able to manage s. woodiana’s invasion, protecting more freshwater habitats from colonization. acknowledgements this study was funded by the emerging interdisciplinary research (eidr) program of the office of the vice president for academic affairs of the university of the philippines provided to pso and the department of science and technologyaccelerated science and technology human resources development program (dost-asthrdp) provided to rjcf. moreover, the authors would like to express their gratitude to all research assistants and principal investigators of dna barcoding laboratory, molecular population genetics laboratory, and biodiversity research laboratory of the institute of biology, university of the philippines diliman for their support in the undertaking of this study. consent for publication all authors gave their consent for the publication of this work. molecular 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[cited 2019 july 11]; 108(4):431-437. http://dx.doi.org/10.1093/jhered/ esx033. zieritz a, bogan ae, rahim kaa, sousa r, jainih l, harun s, abd razak nf, gallardo b, mcgowan s, hassan r, lopes-lima, m. 2018. changes and drivers of freshwater mussel diversity and distribution in northern borneo. bio conserv. 219:126-137. molecular identification of the chinese pond mussel sinanodonta woodiana (lea, 1834) 96 ______ raffy jay c. fornillos is an instructor at the institute of biology, up diliman. he obtained his ms biology at the same institution. he specializes in molecular parasitology. rogel victor d. mendoza is currently a research associate under the dna barcoding laboratory at the institute of biology, up diliman. he obtained his ms in biology from the institute of biology, up diliman with his thesis focusing on the population and molecular genetics of bat species. gerard clinton l. que is a member of the molecular population genetics laboratory of the institute of biology, up diliman. he did his thesis work for his bachelor of science and master of science in biology at the same institute under his adviser, dr. ian kendrich c. fontanilla. his research interest is in molecular phylogenetics and molecular ecology. perry s. ong† was a professor and head of the biodiversity research laboratory at the institute of biology, up diliman. he obtained his ph.d. at the monash university department of ecology and wildlife biology. his expertise was in philippine wildlife biology. he passed away on 2 march 2019. ian kendrich c. fontanilla is a professor and head of the dna barcoding laboratory at the institute of biology, up diliman. he received his ph.d. in genetics from the university of nottingham, united kingdom. he specializes in molecular genetics, phylogenetics, and malacology. optimized-casas.pmd casas, e.v. and others 28 science diliman (july-december 2012) 24:2, 28-49 abstract optimized drying parameters of water hyacinths (eichhornia crassipes. l) edgardo v. casas1*, jemar g. raquid2, kevin f. yaptenco3, and engelbert k. peralta4 agricultural and bioprocess division, institute of agricultural engineering college of engineering and agro-industrial technology university of the philippines los baños, college, laguna, philippines *corresponding author: e-mail: evcasas04@gmail.com �� = �. �� ± . � � − � + �. � �−� + ( . ���−� )� the study investigated the optimum drying conditions of water hyacinth to contribute in the improvement of present drying processes. the effects of independent parameters (drying temperature, airflow rate, and number of passes) on the responses were determined using the response surface methodology. the response parameters were composed of (1) final moisture content, (2) moisture ratio, (3) drying rate, (4) tensile strength, and (5) browning index. box and behnken experimental design represented the design of experiments that resulted in 15 drying runs. statistical analysis evaluated the treatment effects. drying temperature significantly affected the drying rate, moisture ratio, and browning index. airflow rate had a significant effect only on the drying rate, while the number of passes significantly affected both the drying rate and browning index. the optimized conditions for drying the water hyacinth were at drying temperature of 90ˆc, airflow rate of 0.044m3/s, and number of passes equivalent to five. the best model that characterizes the drying of water hyacinth is a rational function expressed as: keywords: box-behnken design, desirability function approach, drying, multiple response optimizations, response surface methodology, water hyacinth optimized drying parameters of water hyacinths (eichhornia crassipes. l) 29science diliman (july-december 2012) 24:2, 28-49 introduction background of the study the water hyacinth (eichhornia crassipes), popularly known to filipinos as the water lily, is a free-floating plant growing in freshwater. this invasive species, native to south america, has fibrous tissue, high energy and protein content, and a moisture content of 95%. it is also primarily used as an ornament in garden ponds. however, due to its large population in water bodies, it has become undesirable and harmful to other aquatic species as well as aquatic ecosystems. it also causes the clogging of waterways, which effectively reduces the flow of water. moreover, it is also decreasing the dissolved oxygen level in the water, which is needed by many aquatic species like fish. nowadays, there are different practical applications to utilize the abundance of the water hyacinths so that they become more useful. for example, paper and rope productions use water hyacinth fibers. in many parts of asia, it is used as an animal feed for pigs, ducks, and fish. in addition, water hyacinths are also used as fertilizers and aids in water purification either for drinking or for liquid effluent from sewage systems. in the philippines, the most common use for the plant is for handicrafts like baskets, matting, and furniture. according to abella (2010), the water hyacinth handicraft industry in laguna is now in its growth stage since it is in the period of rapid market acceptance and shows substantial profit improvement. moreover, these handicraft products are exported to other countries in europe and asia, like japan and korea. in order to use the water hyacinth as a raw material for handicraft making, its water content must be lowered through a drying process. currently, sun drying is the most common practice for drying. after gathering the plants from water bodies, their stems are subjected to sun drying for one week. however, this present drying system has many drawbacks. sun drying does not properly dry the stalks (to desired moisture levels) and it is time-consuming. (2007) stated that sun drying is not a good method for drying because it is prone to losses primarily due to insect infestation and microbial attacks. he also reported that sun-drying method lacks control in the quality of the product. another problem with sun drying is that it is highly dependent on weather, making it very inefficient and unreliable. significance of the study in any handicraft industry, the major factors that impact on the success of this business are high quality of product and labor productivity. with the growing demand of water hyacinth handicraft products in the international market, production of these products must possess a good quality. one way to achieve this is by improving the present process of drying. proper drying will prevent rotting and mold growth because it lessens the moisture content of the product. determining the right combination of drying parameters will be helpful in achieving the satisfactory quality of the product, as well as making the drying operation more economical. this study determined the optimum heated-air drying condition for water hyacinths. results of this study will be helpful in the development of more effective drying procedures for water hyacinths. objectives of the study the main objective of this study was to determine the optimum drying parameters for water hyacinth. specifically, this study aimed to: (a) determine the effects of air temperature, air flow rate, and dewatering on the responses such as drying rate, moisture ratio, browning index, tensile strength, and final moisture content; (b) find the optimum drying condition for the heatedair drying of water hyacinths; and (c) develop a drying model equation for water hyacinths. this study investigated the heated-air drying process that considered air temperature, airflow rate, and the number of passes in the roller (for dewatering) as independent parameters. a metal roller, normally used for forming metal sheets into smooth cylinders, dewatered the samples. the clearance between the rollers was maintained constant at 3.5mm, though pressures exerted by the roller would be more telling in influencing the water removal during drying. casas, e.v. and others 30 science diliman (july-december 2012) 24:2, 28-49 this study was conducted from march 2011 to march 2012 at the agricultural bio-processing division, institute of agricultural engineering, college of engineering and agro-industrial technology, university of the philippines los baños. water hyacinth the water hyacinth (eichhornia crassipes) is an aquatic weed belonging to the floating-type category. water hyacinths are normally found in freshwater areas of temperate and tropical regions of the world. they grow in water environments having a ph of 7 with a high abundance of nitrogen, phosphorus, and potassium. the optimum temperature for the growth of this plant is between 28oc to 30oc. the composition of water hyacinths is dominated by a high percentage of water. according to the national academy of science (1977), a water hyacinth usually has 5% dry matter. in addition to that, it has a high nitrogen content with at least 80% of it in the form of protein. based on data from the bureau of animal industry of the republic of the philippines, the leaves and roots of the water hyacinth have protein contents of 18.7% and 11.8%, respectively. the fiber contents of leaves and roots of the water hyacinth are 17.1% and 7.9%, respectively (patent storm 2011). water hyacinth in handicraft making as a material for handicraft making, the water hyacinth generates livelihood programs for many filipinos. agribusiness week writes that members of the buhi ecumenical development association, inc. (bedai) of buhi, camarines sur use this plant in many handicraft items. among the products made out of this plant are table runners, placemats, canisters, bags, slippers, and wall décor. in laguna, one of the livelihood projects of the government is the comprehensive livelihood and emergency program. one of its sub-programs is the water hyacinth development program that aims to provide employment through water hyacinth gathering and semi-processing. moreover, this program also intends to develop a barangay-based livelihood enterprise that utilizes and transforms water hyacinths into useful products. since this program’s implementation, 15 towns of laguna have participated. nine of them were engaged and trained in water hyacinth handicraft making, while the rest focused on green fertilizer and charcoal making (abella 2010). another livelihood project has been developed in taguig city. morelos (2008) reports that each barangay in taguig process water hyacinths into different products. christmas decors, lanterns, and novelty items are some of the examples. furthermore, the las piñas city government is also active in utilizing the water hyacinth as source of livelihood (echeminacla 2011). abella (2010) has written about the status, problems, and prospects of the water hyacinth handicraft produced in laguna. she notes that there is a great opportunity for water hyacinth handicrafts due to their high demand in the world market and the increase in the preference for environment-friendly products. however, she reports that the climatic condition in the philippines is one of the problems that hinder the development of the water hyacinth handicraft industry. another problem, as cited by ararat (2009), is improper drying and preservation of water hyacinth: a businessperson from laguna named cesar pasco exported several container vans of water hyacinth handicrafts but after just a week, all those products were sent back to him because they had molds. improper drying and preservation of water hyacinth stems is considered as the main reason for the mold formation. as such, the drying process is vital in attaining good quality water hyacinth products. however, no data are currently available regarding the standard quality attributes of a dried water hyacinth fibers used for handicraft making. drying henderson and perry (1987) define drying as the process of removing moisture from the material until it reaches a desired level and it achieves equilibrium with the storage environment. this process is commonly used in food preservation since the shelf-life of a material is highly dependent on its moisture content. a high moisture content increases microbial activity hence deteriorating the quality of a material. theory of drying. heat transfer and mass transfer are the two fundamental mechanisms that occur in the drying process of solid materials. heat, in the form of latent heat of vaporization, is brought to the material optimized drying parameters of water hyacinths (eichhornia crassipes. l) 31science diliman (july-december 2012) 24:2, 28-49 mainly via convection with a little fraction of radiation and conduction. air is used as a means for the heat to flow from its surroundings to the actual material. the mass transfer is in liquid or vapor form from the interior of the material and as vapor from the surface (henderson and perry 1987). factors affecting drying there are several factors that affect the rate of heat and mass transfer influencing the rate of drying. these include drying air temperature, relative humidity, airflow rate, and thickness. temperature. drying air temperature has the most significant effect on the rate of drying. the drying rate has a directly proportional relationship with temperature. a higher temperature results in faster drying rate. this is due to the temperature dependence of the material properties related to drying. relative humidity. relative humidity is a significant factor in the determination of the final or equilibrium moisture content of the material. moreover, it also has a great influence on total drying time. since the evaporation rate of water is dependent on the partial pressure of water vapor in the air surrounding the product and the pressure on the product’s surface, at constant air speed, a lower relative humidity can be used for faster evaporation (fernandez 2007). airflow. airflow rate is only important during the first stages of drying. many researchers do not consider this parameter in the determination of drying characteristics of a product due to the negligible effect of surface moisture movement resistance compared to the internal resistance. however, there are studies showing that airflow rate has a considerable effect on the product. jose (2000) concludes that airflow rate has a great effect on the drying of carabao mangoes, since it is needed to transfer energy to the product, to evaporate the water, and to carry the water vapor away. thickness. according to candelaria (1991 cited by jose 2000), one of the reasons that hasten the drying is that thinner layers reduce the distance travelled by heat in the center of the material. solpico (2007) found that thickness has no significant effect on drying of cocoa trinitario variety. drying curve the amount of residual moisture of a material at any time during drying operation is illustrated by a drying curve. typically, there are four stages of drying. the first stage is the initial induction stage, which corresponds to the initial unsteady state heating period. after this, the drying process continues to the second stage, which is the constant rate period. in the constant rate period, the drying occurs at the surface. drying is similar to the evaporation of water from a free surface. in addition to that, there is a linear relationship between moisture content and time during this stage. the surface temperature of the material is constant since heat gained by the material is lost through evaporation. furthermore, the drying rate is greatly influenced by the drying environment, which includes drying air temperature, relative humidity, and air velocity. the mode of heat transfer is mainly via convection. at the end of this period, critical moisture content is reached; this can be defined as the moisture content at which surface saturation cannot be maintained and the drying rate begins to fall. falling rate period constitutes the last two stages of drying. it is divided into two zones: the first falling rate, where evaporation occurs from a saturated surface of decreasing area, and the second falling rate period or the sub-surface drying. in this stage, the property of the material has a great influence on the drying rate or moisture movement. tensile strength according to joseph (1986), tensile strength is obtained by determining the required force to break a fiber crosssectional mass equivalent to one unit of the measure used. the term designated to the individual strength of fibers is called tenacity. this defines how strong a material is. dewatering and crushing crushing is a type of size reduction that deals with the application of force to a unit being reduced in excess of its strength. failure results in the rupturing of the material in many directions. a new surface and particle casas, e.v. and others 32 science diliman (july-december 2012) 24:2, 28-49 characteristic depends upon the material property and the force application method, a form of size reduction commonly applied in sugarcane juice extraction, as well as in breaking the forage crops structure to hasten up drying. one of the most common crushing equipment is a rigid roll or bed, which can be seen in sorghum mill. in addition, a double roller, with or without serrated surfaces, produces uniform products (henderson and perry 1987). browning browning is considered acceptable if it boosts the appearance and flavor of foods. the browning reaction rate is dependent on several factors such as drying temperature, ph, moisture content of the product, heat treatment period, and the nature and concentration of the reactants. this reaction affects the color of the product and causes textural change (unido 2011). for water hyacinths as handicraft materials, browning is desirable since it is an indicator that the product is already dried (sun drying). materials and methods the materials and equipment used in the study comprise of the following: 1. water hyacinth 2. dryer 3. electronic balance 4. carbolite™ oven 5. knife 6. drying tray 7. ruler 8. feeler gauge 9. steel tape 10. manometer 11. konica minoltatm cr-10 color reader 12. instron universal testing machine 13. roller 14. resealable plastic bags 15. sling psychrometer sample preparation water hyacinth samples were collected from laguna de bay. the leaves and roots of the plant were cut off. only the stem of the plant was acquired. then, the stems were cut into a length of 40 cm each. dewatering the prepared samples underwent dewatering using a sheet metal roller. the roller, as shown in figure 2, was prepared with a clearance of 3.5 mm. before passing through the roller, the weight of the prepared samples was determined. each sample was passed through the roller at a specified number of passes (4, 6, and 8). after passing, the samples were weighed again and placed in the pre-weighed drying tray. the following equation computed the percentage of water removed by dewatering: (1) where: w i is the weight of the sample before passing through the roller w f is the weight of the sample after passing through the roller initial moisture content determination the air oven method determined the initial moisture content of the sample. three replicates of fresh dewatered samples, each weighing 25 grams, were prepared. the carbolite oven dried the prepared samples for 72hours as shown in figure 3. equation (2) below computed the initial moisture content: where: mc i is the initial moisture content w i is the initial weight of the sample w f is the final weight of the sample %mci = �� − ���� ∗ 100% %water removed = �� − ���� ∗ 100% (2) optimized drying parameters of water hyacinths (eichhornia crassipes. l) 33 science diliman (july-december 2012) 24:2, 28-49 airflow rate determination the orifice flow calculation was used to measure the airflow rate. using equation (3), the airflow rate at different blower opening setting was determined (gasho.org 2010). where: c is the orifice coefficient = 0.65 (as cited in palunday 2007) %mci = �� − ���� ∗ 100% %water removed = �� − ���� ∗ 100% k = constant = 4,005 when p is expressed in in. of water p is the pressure differential across the orifice q is the flow rate in cubic feet per minute (cfm) a is the total orifice area expressed in square feet (orifice diameter = 8.5cm) a fluid passing through an orifice constriction (figure 1) will experience a drop in pressure across the orifice. this change can be used to measure the flow rate of the fluid. the default calculation involves air passing through a medium sized orifice 4" pipe. figure 2. the dried water hyacinth inside the resealable plastic bag downstream pressure sensor upstream pressure tap p1 p2 d1 do flow direction orifice plate ∆) = )1 − )2 figure 1. orifice setup drying the samples were dried using a laboratory dryer that operated at desired drying conditions. the drying temperatures used were 70°c, 80°c, and 90°c. on the other hand, the airflow rates were 0.041m3/s, 0.047m3/s, and 0.053m3/s. the dryer was first conditioned with a dummy sample for 30 minutes to 1 hour until the specified drying setup stabilized. then, the prepared samples were placed inside the dryer. the weight of the samples were measured at 30-minute intervals. the drying of the samples continued until no change in weight had occurred for two successive readings. the moisture content, at which the product had stopped losing water and no change in weight had occurred, is the dynamic equilibrium moisture content. the dried samples were placed inside the resealable plastic bags as shown in figure 2. (3) casas, e.v. and others 34 science diliman (july-december 2012) 24:2, 28-49 experimental design the box and behnken (1960) design of experiment matrix was used in this study (montgomery 2001). a three-level fractional factorial three-parameter experiment design resulting in 15 drying runs was used. table 1 lists the parameters used for this experiment that resulted in the 15 runs shown in table 2. response surface methodology (rsm) is a collection of mathematical techniques that has been successfully used for developing, improving and optimizing processes (myers and others 2009). rsm enables the reduction in the number of experimental trials needed to evaluate multiple parameters and their interactions thus requiring less time and labor. rsm has been widely applied for optimizing processes in the food industry (kumar and others 2009, wang and others 2010). optimization procedure numerical optimization was carried out using statistica 7.0 software. multiple responses were optimized simultaneously using a desirability function that combines all the responses into one measurement maximizing most of the responses except final moisture contents that was minimized. table 2. combination of the three independent parameters for the 15 drying runs r u n temperature airflow no. of o c m 3/s p a s s e s 1 8 0 0.053 8 2 9 0 0.047 4 3 8 0 0.041 8 4 8 0 0.047 6 5 9 0 0.047 8 6 8 0 0.041 4 7 8 0 0.047 6 8 7 0 0.047 8 9 9 0 0.053 6 1 0 8 0 0.047 6 1 1 8 0 0.053 4 1 2 7 0 0.053 6 1 3 9 0 0.041 6 1 4 7 0 0.041 6 1 5 7 0 0.047 4 table 1. the independent and dependent parameters explored in the experiment i n d e p e n d e n t coded parameter d e p e n d e n t p a r a m e t e r symbol used p a r a m e t e r temperature, °c x 1 7 0 8 0 9 0 final moisture content(y 1 ) air flow, m3/s x 2 0.041 0.047 0.053 moisture ratio (y 2 ) drying rate (y 3 ) x 3 4 6 8 tensile strength (y 4 ) browning index (y 5 ) 1 0 1 number of passes through the roller optimized drying parameters of water hyacinths (eichhornia crassipes. l) 35 response parameters drying rate. equation (4) calculated the drying rate for the complete drying period represented as: where: mc i is the initial moisture content mc f is the final moisture content moisture ratio. equation (5) estimated the moisture ratio that represents the proportion of moisture removal at a given time interval (casas 2010): where: mc is the moisture content at time t mc 0 is the initial moisture content (dry basis) mc e is the dynamic equilibrium moisture content analysis of the drying data collected a number of theoretical, semi theoretical, and empirical drying models were reported in the literature. the most frequently used model for thin layer drying is the lumped parameter type, such as the newton equation (liu and bakker-arkema 1997, kingsly and others 2007). the moisture ratio during drying is determined by equation (5). for the mathematical analysis, it is assumed that the moisture gradient driving force during drying is a liquid concentration gradient. the effect of heat transfer is neglected as a simplifying assumption. for all experimental conditions, the value of (m-me)/(mi-me) expressing dimensionless moisture content obtained are similarly expressed by akintunde and afon (2009). drying curve and model equations the subsequent list presents some of the drying model equations used by some authors to describe the drying characteristics of materials. science diliman (july-december 2012) 24:2, 28-49 +,-�./ ,012 = 34� − 34�1�52 56�718,2 ,01�6(39) = (3 − 32 )(36 − 32 ) equations describing drying characteristics model no. model name model equation reference 39 = exp (−<1) 39 = exp (−<1. ) 39 = 02=> (−<1) 3 9 = 02=>(−<1) + ? 39 = 02=>(−</1) + @2=>(−<1 1 39 = 1 + 01 + @12 1 = 0a.(39 ) + @a.(39 )]2 39 = 02=>(−<1) + (1 − 0)exp (−/1) 39 = 02=>(−<1) + (1 − 0)exp (−/1) 39 = 02=>(−<1) + @2=>(−/1) + ?2=>(−<1) 39 = 02=>(−? c 1 d2 e] 39 exp (−< c 1 d2 e . ] 39 = 02=>(−<1) + (1 − 0)exp (−<01) 39 =exp [(−<1). ] 39 = 02=>(−<1. + @1 1 lewis o’callaghan 1971 liu and others 1997 2 page zang 1991 agrawal and singh 1997 3 modified page overhults 1973 white and others 1981 4 henderson and pabis westerman and white 1973 chinnan 1984 5 yagcioglu and others yagcioglu and others 2001 6 two-term henderson 1974 rahman and others 1998 7 two-term exponential sharaf-elden and others 1980 8 wang and singh wang and singh 1978 9 t h o m s o n thomson and others 1968 paulsen and thomson 1973 1 0 diffusion approach kassem 1998 1 1 verma and others verma and others 1985 1 2 modified henderson and pabis karathamos 1999 1 3 simplified fick’s diffusion (sffd) equation diamante and munro 1991 1 4 modified page equation ii diamante and munro 1993 1 5 midilli and kucuk midilli and others 2002 (4) (5) casas, e.v. and others 36 (a) (b) browning index. konica minoltatm cr-10 color reader measured the l, a, b values of the samples after drying. three measurements taken at random locations for each sample represented the values that calculated browning index (bi) using equation (6) (de vela 2011). where: tensile strength. the instron universal testing machine, as illustrated in figure 3, was used to determine the tensile strength of the dried samples. cutting the dried samples in a dumbbell-shaped form prepared the samples for testing. no standard has been established on the tensile specimen shape for water hyacinth. equation (9) determined the linear density presented as: linear density = weight of the samplelength of the sample where: length of the sample is in meters weight of the sample is in milligram the test is valid if the break occurred at the middle section of the sample (figure 4a), otherwise the test is invalid (figure 4b). the length and weight of the sample was also measured using the electronic balance. this data was used for strength computation. 2cm 10cm 1cm figure 3. the tensile specimen (b) (a) (a) (a) (b) figure 4. (a) valid test sample and (b) invalid test sample science diliman (july-december 2012) 24:2, 28-49 vt = 100(= − 0.31)0.17 = = 0 + 1.75d5.645d + 0 − 3.012@ the samples, properly aligned and placed between the two grips in the machine, were subjected to tensile tests at a preset crosshead speed of 10mm/min (as cited in opiña 2008) until they broke. equation (8), as opiña (2008) used, computed the tensile strength or tenacity of the dried samples represented by samples represented by: where: maximum load is in newton linear density is in g/km or mg/m tenacity = maximum load linear density (6) (7) (9) (8) optimized drying parameters of water hyacinths (eichhornia crassipes. l) 37 linear density = weight of the samplelength of the sample final moisture content determination. (equation 10) estimated the final moisture content (casimina 2010): mcf = mct ∗ wfwt where: mc f = final moisture content mc t = moisture content at time t w f = final weight, g w t = weight at time t, g statistical analysis response surface analysis of the experimental data was carried out using a commercial statistical package, sas 8 response surface regression program, that did a regression analysis as well as an analysis of variance (anova). the response surface analysis of the experimental data, shown in table 4, was also done by fitting equation (11) to the experimental data to determine the regression coefficients and statistical significance of the model terms. significance of the model term was assessed by f-ratio at probability (p) of 0.05. model adequacies were determined by model analysis, lack of fit test, coefficient of determination (r2), predicted error of sum of squares (press), and coefficient of variation (cv). the analysis fit the data to the second order quadratic model for all the responses using rsreg program of illustrated as: o = p6 + q p�r � < 1 + q p�s < 1 r� 2 + q < 1 q p�s < 1 rt ru the response surface regression of sas v8 software performed the anova, regression analysis, and regression coefficient calculations. in determining the optimum values for temperature, airflow, and number of passes, the response surface regression of statistica v7 was used. additionally, curve expert 1.4 was used to generate the model drying equations from the calculated moisture ratio (mr). results and discussion the study determined the optimum heated-air drying condition of water hyacinth. fifteen (15) drying runs were performed based on the box and behnken design of experiment. the resulting data were analyzed to find whether the independent parameters (drying air temperature, airflow rate, and number of passes in the roller) affect the response considered. dewatering and initial moisture content dewatering as a pre-drying treatment is primarily done to decrease the water content of the water hyacinth samples so as to speed up the drying process. in this study, the crushing method using a roller was done. the number of passes through the roller is considered as an independent parameter. the amount of water reduced and initial moisture content at each of the 15 runs is summarized in table 4. the percentage of water removal due to crushing ranged from 2.42% to 9.49%. the highest reduction of water was attained at run no. 14 while the lowest was attained at run no. 11. however, there is no direct relationship established between the number of passes and the percent of water removed. aside from reducing the water content, the physical properties of the sample are also affected by this process. the thickness of the sample was reduced in size and the surface area was flatteened. in addition, the clearance between the rollers remained constant (3.5mm) resulting in the lessening of the variability of thickness among the samples. from this, the effect of thickness in the drying process was diminished. however, the presence of damage or breakage in the samples after passing in the roller existed. science diliman (july-december 2012) 24:2, 28-49 (10) (11) casas, e.v. and others 38 determining the initial moisture content of the samples at each set-up, run no. 10 obtained the highest initial moisture content of 94.43% while run no. 12 attained the lowest initial moisture content of 92.21%. this result may be attributed to the crushing method. effects of the independent variables on response variables the summary of data on the responses at each drying set-up is presented in table 4. as shown in figure 8, run no. 9 achieved the highest drying rate of 0.322 %mc/min while run no. 12 obtained the lowest drying rate of 0.216 %mc/min. this was expected since run no. 9 possessed the highest drying air temperature of 90ˆ c as well as highest airflow rate of 0.053m3/s. on the other hand, run no. 12 has the lowest drying air temperature of 70oc but highest airflow rate of 0.053m3/s. drying at high temperature decreases the drying time; in this way, the drying rate increases. this only shows that temperature affects the drying rate. moisture ratio (mr) was another response investigated. the obtained values of this response at each drying run are illustrated in figure 9. the highest value was 0.132 attained by run no. 13, while the lowest value is 0.096, attained by run no. 8. it could be noted that science diliman (july-december 2012) 24:2, 28-49 run mc initial %water removed (% wb ) 1 93.45 5.49 2 92.60 7.32 3 93.20 4.46 4 92.81 9.38 5 92.87 3.66 6 93.40 3.05 7 94.32 2.42 8 94.22 3.07 9 94.16 4.88 10 94.43 2.42 11 93.57 2.40 12 92.21 8.64 13 93.35 3.13 14 93.84 9.49 15 92.80 4.40 table 3. the percentage of water removed and initial moisture content at each drying run table 4. summary of data showing the responses at each drying set-up 1 80 0.037 8 8.44 0.12312 0.28336 0.00980 77.4191 2 90 0.03 4 6.61 0.13118 0.31826 0.01237 73.3673 3 80 0.023 8 6.21 0.12240 0.28996 0.00978 75.3889 4 80 0.03 6 8.96 0.12475 0.27950 0.01340 49.9864 5 90 0.003 8 7.64 0.12900 0.31566 0.01060 43.388 6 80 0.023 4 5.29 0.11178 0.24476 0.00992 77.9319 7 80 0.03 6 7.62 0.12319 0.28901 0.00702 56.5011 8 70 0.003 8 5.96 0.00619 0.22630 0.00925 64.9468 9 90 0.037 6 7.24 0.1215 0.32191 0.01055 55.8929 10 80 0.03 6. 8.80 0.12092 0.28544 0.01105 53.0061 11 80 0.037 4 8.61 0.12370 0.28320 0.00811 59.4689 12 70 0.037 6 7.83 0.11874 0.21635 0.01378 45.547 13 90 0.023 6 7.93 0.13240 0.28473 0.01038 53.8275 14 70 0.023 6 7.87 0.11377 0.22042 0.01028 53.1614 15 70 0.03 4 7.33 0.10965 0.21916 0.01265 50.5459 run independent parameters responses temperature airflow no. of passes mc final (%wb) moisture ratio drying rate (%wb/min) tensile strength (n/tex) browning index optimized drying parameters of water hyacinths (eichhornia crassipes. l) 39 run no. 13 has the highest drying temperature of 90oc set-up. on the other hand, run no. 8 possessed the lowest drying temperature of 70oc set-up. this implies that drying air temperature influences the moisture ratio. table 4 summarizes the data on the response variables investigated in drying water hyacinth and discussed in the following sections final moisture contents. the final moisture contents (figures 5 and 10) attained by each drying run ranged from 6.21% wb in run no. 3, due probably to the highest number of passes in the roller that squeezed out most of the initial water prior to drying the samples, to run no. 4, which obtained the highest final moisture content of 8.96% wb at the end of drying probably due to the high initial moisture content at the initial stage or prior to passing to the roller for squeezing the water from the samples (table 4 and figures 9-17). average drying rates. the average drying rate (figure 9) measured the speed of moisture removal that ranged from 0.22 % min-1 to 0.32 %min-1 exhibited by runs 15 and 2, respectively. moisture ratio (mr). figure 9 illustrates the average moisture ratio calculated from the 15 drying runs. run 8 exhibited the lowest mr while run 13 showed the highest value of mr. tensile strengths. as shown in figure 15, run no. 12, subjected to 70oc drying temperature, an airflow rate of 0.053m3/s and number of passes equivalent to 6, exhibited the highest tensile strength of 0.0137n/tex. on the other hand, run no. 7, having temperature of 80oc, airflow rate of 0.047m3/s, and 8 passes through the drying set-up, attained the lowest tensile strength of 0.007n/tex. figure 10 shows the final moisture content of 15 drying treatments. the highest value of final moisture content that the dried sample exhibited was 8.96%, which occurred at run no. 4, while the lowest value was 5.29%, which was obtained by run no. 6. browning index. browning index, which evaluates the browning color of the sample, was also considered in this study. the value for 15 drying treatments is illustrated in figure 12. run no. 6, conducted at 80oc, 0.041m3/s airflow rate, and 5 passes, obtained the highest browning index among the entire drying runs. the lowest value is obtained at run no. 5 in spite of conducted at temperature of 9oc, airflow rate of 0.047m3/s, and number of passes equivalent to 8. this may be due to other factors contributing to browning such as oxidation and residual enzyme activity (unido 2011). run 5 showed the lowest browning index while run 6 had the highest browning index among experimental runs conducted in optimizing the drying parameters of water hyacinths (figure 11). this shows that samples dried at higher temperatures (above 80o) will have higher probability of having brownish color than those dried at lower temperatures due to the effects of temperatures on color. additionally, browning index is significant at 95% level while drying rate is significant at 90% level in crossproduct model. the lack of fit test measured the failure of the model to represent the data obtained from the experiment. it was found that the lack of fit was not significant for all of the response variables except in moisture ratio. the non-significant lack of fit is good since it indicates that the data in the experiment fit the generated models. effects of the independent parameters on the responses the effects of airflow rates, air temperatures, and number of passes on the roller are summarized in table 5. in each experimental run, drying air temperatures significantly affected the average drying rates, moisture ratio, and browning index of the dried samples of water hyacinths but had no pronounced effects on their final moisture contents and tensile strengths. the airflow rates were only significant in the average drying rates of the dried samples. on the other hand, the number of passes on the roller prior to drying the samples had significant contributions in the average drying rates and browning index. science diliman (july-december 2012) 24:2, 28-49 casas, e.v. and others 40 table 5. anova test results for the significance of the independent parameters airflow rate significantly affected the drying rate at 95% level of significance but failed to influence the rest of the responses. since air is the medium of heat transfer, different rates of airflow might affect the time of drying. the number of passes also significantly affects drying rate and browning index. the dewatering process might also result in the opening of the porous spaces that have an effect on the process of drying. meanwhile, the effect of the number of passes in browning may be attributed to the physical damage acquired during the dewatering process (passing through the roller). according to unido (2011), the color of the plant changed when the tissue is bruised, cut, or exposed to any number of abnormal conditions. this injured tissue darkens on exposure to air. figures 5 to 9b depict the surface plots for each response as affected by temperature, airflow rate, and number of passes. figure 5. surface plot for final moisture content vs. airflow rate and drying air temperature figure 6. surface plot for moisture ratio vs. airflow rate and drying air temperature science diliman (july-december 2012) 24:2, 28-49 the anova results (see table 5), show that temperature significantly affected drying rate and browning index at the 95% level. moreover, moisture ratio was also significantly affected by this parameter at 90%. the result for the effect of temperature in drying rate is expected. this is because drying temperature and drying rate have a directly proportional relationship. in addition, more moisture is absorbed when the temperature is high. in terms of the effect in browning, this may be due to temperature on the pigment production of plants. according to unido (2011), higher temperatures can reduce the amount of pigment resulting in browning phenomena. on the other hand, browning’s effect is not established with regard to the final moisture content and tensile strength. this result is confirmed by the data in this investigation. as opiña (2010) concluded, temperature had no significant effect in the final moisture content and tensile strength of the product. temperature 0.01729** 2.482607ns 8.510e-04* 1.008e-05ns 757.78750** airflow 0.00177** 3.528331ns 9.792e-05ns 5.084e-05ns 355.3798ns passes 0.000842* 6.04483ns 1.290e-03ns 4.292e-04ns 1190.233** parameter drying rate final mc moisture ratio tensile strength browning index sum of squares * * significant at 95% * significant at 90% ns not significant optimized drying parameters of water hyacinths (eichhornia crassipes. l) 41 figure 7a. surface plot for drying rate vs. airflow rate and drying air temperature 3d surface plot (wat er hyacint h 7v*15c) 0.3275 0.32 0.31 0.3 0.29 0.28 0.27 0.26 0.25 0.24 0.23 0.22 0.21 0.2 figure 7b. surface plot of drying rates as affected by drying air temperature and no. of passes through the roller 3d surface pl ot (wat er hyacint h 7v*15c) 90 80 70 60 50 figure 9b. surface plot of browning index as affected by drying air temperature and no. of passes the roller 80 70 60 50 40 figure 9a. surface plot for browning index vs. airflow rate and drying air temperature 3d surface plot (wat er hyacint h 7v*15c) 0.012 0.01 0.008 0.006 0.004 figure 8b. surface plot of tensile strength as affected by drying air temperature and no. of passes through the roller 0.015 0.014 0.013 0.012 0.011 0.01 0.009 figure 8a. surface plot for tensile strength vs. airflow rate and drying air temperature science diliman (july-december 2012) 24:2, 28-49 casas, e.v. and others 42 table 6 presents the anova results of the regression analysis as analyzed by sas 8. results showed that drying rate is significant at the 95% level in terms of linear model. furthermore, drying rate and browning index are significant in both quadratic and total model. additionally, browning index is significant at the 95% level while drying rate is significant at the 90% level in cross-product model. the lack of fit test measured the failure of the model to represent the data obtained from the experiment. it was found that the lack of fit was not significant for all of the response variables except in moisture ratio. the non-significant lack of fit is good since it indicates that the data in the experiment fit the generated models. coefficient of variation (cv) determines the data variability relative to the mean. a low value of cv is desirable since it signifies more consistency of data values. if the value is more consistent then the variation is less. the anova test showed that drying rate obtained the lowest cv value of 2.84%, indicating some degree of accuracy of the data gathered (especially weights of samples at particular designated time intervals). on the other hand, tensile strength has more variation among the responses since it obtained the highest cv value of 24.02% possibly due to the high sensitivity of the instron machine during tensile force measurements. coefficient of determination (r2) is another parameter used to analyze the regression model. it measures how well the model represents the data. among the responses, drying rate generates the highest value with 0.9845. this means that 98% of variation in the drying rate can be explained by the model generated (gomez and gomez 1984). in contrast, tensile strength attained the lowest value with 0.3286. it could be noted that tensile strength is not significantly affected by the independent parameters, whereas, the drying rate is greatly affected by the independent parameters. table 7 presents the values of regression coefficients of the second order polynomials model for each of the responses. the generated model is based on the coded data in of the form of: y= ß k0 + ß k1 x 1 + ß k2 x 2 + ß k3 x 3 + ß k11 x 1 2 + ß k21 x 2 2 + ß k22 x 3 2 + ß k31 x 1 x 2 + ß k32 x 1 x 3 + ß k33 x 2 x 3 where: ß k = coefficient y = response parameter x 1 (drying temperature), x 2 (airflow rate), x 3 (passes) = independent variables science diliman (july-december 2012) 24:2, 28-49 (9) linear 3 2.948175ns 0.000761* 0.016889** 0.00000261ns 79.237108ns quadratic 3 4.990845ns 0.00009483ns 0.00104** 0.000008974ns 1028.441736** cross product 3 1.84265ns 0.000114ns 0.000956** 0.000004274ns 620.815352** total model 9 9.781668ns 0.00965ns 0.018884** 0.000015858ns 1728.494196** lack of fit 3 5.355825ns 0.000241** 0.000252ns 0.000011576ns 146.701531ns pure error 2 1.07126 0.000007418 4.62e-05 0.000020823 21.258213 total error 5 6.427025 0.000248 2.98e-04 0.00003299 167.959745 coefficient of variation, cv 15.13 5.8573 2.83389 24.0236 9.7641 r2 0.6035 0.7955 0.9845 0.3286 0.9114 sum of squares source df final mc moisture ratio drying rate tensile strength browing index table 6. anova test results for the significance of response surface regression model optimized drying parameters of water hyacinths (eichhornia crassipes. l) 43 optimum drying conditions the optimum combination of the three independent parameters was determined and analyzed by statistica v7.0, which employed 100 iterations to arrive at the optimum condition for drying. based on the result, the optimum drying condition for water hyacinths is at a temperature of 90oc, an airflow rate of 0.044m3/s, and a number of passes equivalent to 5. with 68.89% desirability, the following predicted values for each response could be attained as shown in table 8. highlighted values show the predicted values at optimum conditions. figure 10 presents the plot of responses at optimum conditions of the independent parameters tested with desirability profile. (14) the regression equation for each response, based on table 7 and following equation (9), is presented below: y1= -55.601+ 0.74867x 1 + 1093.06x 2 + 1.92542x 3 -0.005x 1 2 -2.7083x 2 2 6805.6x 3 2+ 0.03x 1 x 2 -22.708x 1 x 3 -0.2694x 2 x 3 y2= -0.2821+ 0.00665x 1 + 2.64991x 2 + 1.92542x 3 -0.005x 1 2 -2.7083x 2 2 -6805.6x 3 2 + 0.03x 1 x 2 -22.708x 1 x 3 -0.2694x 2 x 3 y3= -1.3332+ 0.02055x 1 + 17.1542x 2 + 0.05747x 3 -0.0015x 1 2 +0.17188x 2 2 254.48x 3 2 -0.0001x 1 x 2 —0.9383x 1 x 3 -4e05x 2 x 3 y4= 0.02527-0.0016x 1 + 2.30104x 2 -0.002x 3 +1.3e-05x 1 2 -0.0139x 2 2 -14.688x 3 2 +2e05x 1 x 2 +0.38125x 1 x 3 -0.0001x 2 x 3 (10) (11) (12) (13) science diliman (july-december 2012) 24:2, 28-49 y5= -324.83+14.8829x 1 -13578x 2 -4.6229x 3 0.077x 1 2 +34.5707x 2 2 +137070x 3 2 -0.5548x 1 x 2 +365.95x 1 x 3 +3.16781x 2 x 3 coefficient ß k0 -55.601 -0.2821 -1.3332 0.02527 -324.83 ß k1 0.74867 0.00665 0.02055 -0.0016 14.8829 ß k2 1093.06 2.64991 17.1542 2.30104 -13578 ß k3 1.92542 0.01166 0.05747 -0.002 -4.6229 ß k11 -0.005 -2e-05 -0.0015 1.3e-05 -0.0777 ß k21 -2.7083 -0.0593 0.17188 -0.0139 34.5707 ß k21 -6805.6 38.9815 -254.48 -14.688 137070 ß k31 0.03 0.00014 -0.0001 2e-05 -0.5548 ß k32 -22.708 -0.2333 -0.9383 0.38125 365.95 ß k33 -0.2694 -0.001 -4e-05 -0.0001 3.16781 response parameter final mc (y1) mr (y2) drying rate (y3) tensile strength (y4) browning index (y5) table 7. regression coefficients of the second order polynomials casas, e.v. and others 44 figure 10. predicted responses at optimum conditions with desirability science diliman (july-december 2012) 24:2, 28-49 optimized drying parameters of water hyacinths (eichhornia crassipes. l) 45 table 8. predicted responses at optimum conditions of independent parameters temp 70 7.2519 0.1100 0.2157 0.011873 50.0879 0.00000 temp 75 7.6497 0.1166 0.2490 0.010781 56.6854 0.46367 temp 80 7.8001 0.1221 0.2751 0.01033 59.396 0.50572 temp 85 7.7031 0.1263 0.2938 0.010522 58.21972 0.57325 temp 90 7.3586 0.1294 0.3052 0.011355 53.15656 0.68893 airflow rate 0.041 6.8755 0.1299 0.2943 0.01084 59.56775 0.65168 airflow rate 0.044 7.3586 0.1294 0.3052 0.011355 53.15656 0.68893 airflow rate 0.047 7.7206 0.1296 0.3116 0.011605 50.10358 0.68760 airflow rate 0.05 7.9614 0.1304 0.3134 0.011589 50.40883 0.66245 airflow rate 0.053 8.0812 0.1320 0.3106 0.011307 54.07229 0.62155 no. of passes 4 6.5234 0.1267 0.3020 0.011162 62.67069 0.65749 no. of passes 5 7.3586 0.1294 0.3052 0.011355 53.15656 0.68893 no. of passes 6 7.6546 0.1301 0.3084 0.011269 49.97804 0.68226 no. of passes 7 7.4116 0.1287 0.3115 0.010902 53.13515 0.67572 no. of passes 8 6.6296 0.1253 0.3145 0.010256 62.62789 0.63171 predicted fmc predicted mr predicted drying rate predicted tensile strength predicted browning index desirability value factor* factor level *highlighted values represented the optimum conditions for drying water hyacinth verification of the optimum drying conditions after determining the optimum values, three validation runs were performed to compare and verify the validated response against the predicted response. table 9 shows the average responses obtained by the three verification runs at optimum conditions. as table 9 depicts, the verification run obtained an average final moisture content of 7.34%wb, a moisture ratio of 0.122, a drying rate of 0.281%wb/min, a tensile strength of 0.011n/tex, and a browning index of 54.25. the table also shows that final moisture content attained the lowest percent error of 0.32%. in contrast, the highest percent error value was obtained by drying rate with 8.11%. these verification results validated the established optimum conditions within the range of independent parameter values tested. response predicted verified error (%) final moisture content 7.3586 7.335005 0.320 moisture ratio 0.129437 0.121972 5.767 drying rate 0.305295 0.280532 8.111 tensile strength 0.011355 0.010899 4.017 browning index 53.15656 54.25077 2.058 table 9. comparison between the predicted and verified values at each response science diliman (july-december 2012) 24:2, 28-49 casas, e.v. and others 46 thin layer drying modeling for water hyacinth curve expert 1.4 analyzed the drying data and generated the model equation that best represents the data for 15 drying runs. table 10 presents the best regression model describing the mr over time. it also includes the standard error(s) and correlation coefficient(r), which depict the goodness of fit of the model. low s value, in particular approximating zero, is desirable. on the other hand, high r-value approaching 1 is desired. based on these criteria, run no. 6 obtained the best model described by rational function model shown below. where: mr = moisture ratio (dimensionless) and x = drying time, minutes this shows that not all drying models of agricultural materials fall in the exponential, logarithmic, and page models. in addition, figure 11 illustrates the generated rational model. figure 11. moisture ratio vs. time of run no. 6 the selection of a suitable drying model entails analyzing the mr data and drying time using curve expert 1.4. to find the best expression to represent the data. the coefficient of determination r2 and standard deviation were the main criteria for selecting the best equation. in addition to the coefficient of determination, the goodness of fit was determined by various statistical parameters such as reduced mean square of the deviation and root mean square error rmse. for quality fit, r2 value should be higher, and mean deviation, and rmse values should be lower (togrul and pehlivan 2002), (goyal and others 2008). summary and conclusion the study determined the optimum drying condition of water hyacinths. investigations and analyses used three independent parameters that include drying temperature, airflow rate, and number of passes through a roller. the study investigated and analyzed their effects on the following responses: (1) final moisture content (2) moisture ratio, (3) drying rate, (4) tensile strength, and (5) browning index. the study employed box and behnken design of experiment that resulted in 15 experimental drying treatments. science diliman (july-december 2012) 24:2, 28-49 39 = 1.00 − 3.072 −3 = 1 + 9.872−2= + (3.842−4=)2 (15) optimized drying parameters of water hyacinths (eichhornia crassipes. l) 47 *evolved as the best-fit model for mr run model coefficient r s 1 harris modely=1/(a+bx^c) a =1.00005523042e+0 b =3.30183500112e-002 0.999775 0.00702 c =1.46668439402e+0 2 harris modely=1/(a+bx^c) a =1.00004473005e+000 b =1.30928391619e-002 0.999842 0.00625 c =1.72426998311e+000 3 harris modely=1/(a+bx^c) a =1.00005215880e+000 b =2.39857648115e-002 0.999811 0.00642 c =1.54992776612e+000 4 harris modely=1/(a+bx^c) a =1.00005652566e+000 b =2.26207802164e-002 0.999822 0.00623 c =1.54628769281e+000 5 harris modely=1/(a+bx^c) a =9.99974113763e-001 b =4.11336454945e-002 0.999597 0.00997 c =1.42897052299e+000 6 rational function a =1.00000413749e+000 y=(a+bx)/(1+cx+dx^2) b =-3.07074708753e-003 0.999996* 0.00088 c =9.87235806713e-002 d =3.84339630684e-004 7 harris modely=1/(a+bx^c) a =1.00005859539e+000 b =3.57582990477e-002 0.999739 0.00753 c =1.44622888295e+000 8 rational function a =9.99973074346e-001 y=(a+bx)/(1+cx+dx^2) b =-2.96250878191e-003 0.999969 0.00236 c =1.78121101892e-001 d =-5.00855559474e-005 9 harris modely=1/(a+bx^c) a =1.00002252503e+000 b =3.55091977548e-002 0.999835 0.00639 c =1.55481149148e+000 1 0 harris modely=1/(a+bx^c) a =1.00005195841e+000 b =2.88889379873e-002 c =1.51673974815e+000 0.999788 0.00681 1 1 harris modely=1/(a+bx^c) a =1.00006073191e+000 b =2.30702158390e-002 0.999784 0.00687 c =1.54812594422e+000 1 2 harris modely=1/(a+bx^c) a =1.00040497838e+000 b =3.55494618261e-002 0.998872 0.01358 c =1.29290400827e+000 1 3 harris modely=1/(a+bx^c) a =1.00011096476e+000 b =1.26392023222e-002 0.999759 0.00726 c =1.63096327027e+000 1 4 rational function a =9.99924438129e-001 y=(a+bx)/(1+cx+dx^2) b =-2.89792856124e-003 0.999972 0.00225 c =9.11988779985e-002 d =4.98218837768e-005 1 5 harris modely=1/(a+bx^c) a =1.00015477627e+000 b =3.60377446995e-002 0.999542 0.00865 c =1.34811348987e+000 table 10. the best-fit model for 15 drying runs experimental results revealed that drying temperature significantly affected drying rate, moisture ratio, and browning index. airflow rate has a significant effect on the drying rate only, while the number of passes significantly affected both the drying rate and browning index. the optimum drying condition for water hyacinth is at a temperature of 90oc, an airflow rate of 0.044m3/s, and a number of passes equivalent to 5, with a desirability of 68.69%. the predicted responses are 7.36%wb for final moisture content, 0.129 for moisture ratio, 0.305%mcwb/min for drying rate, 0.0113n/tex for tensile strength, and 53.16 for browning index. science diliman (july-december 2012) 24:2, 28-49 casas, e.v. and others 48 verification runs showed that the percent error for all of the responses ranged from 0.32% to 8.11% with final moisture content having the lowest and drying rate obtaining the highest. regression analysis resulted in model equations that sufficiently illustrated moisture ratio with time. rational function is the best model that characterizes the drying of water hyacinth with an equation expressed as: where: mr is the moisture ratio; and x is the time in minutes the model was very much different from most models developed for agricultural products that normally come in the form of exponential or exponential association. recommendations future studies should consider the following: 1. relative humidity should be considered as an independent parameter if the dryer has the capability to control this parameter since it is one of the factors affecting drying. 2. the clearance between the rollers can also be used as an independent parameter by adjusting it into different ranges. also, the force applied by the rollers during pressing should be included in future studies. references science diliman (july-december 2012) 24:2, 28-49 39 = 1.00 − 3.072 −3 = 1 + 9.872 −2 = + (3.842 −4 =)2 (16) abella aas. 2010. status, problems, and prospects of water hyacinth handicraft produced in laguna [undergraduate thesis]. laguna: university of the philippines los baños. 134 leaves. agrawal yc, sing rp. 1977. thin-layer drying studies on short-grain rough rice. asae paper no. 3531. 13 p. ararat rk. 2009. fprdi helps water hyacinth industry [cited 2011 august 22]. available from http://diaryongtagalog.com casas ev. 2010. drying simplified [lecture handout in aeng 31fundamentals of crop processing]. located at: agricultural and bioprocess division, institute of agricultural engineering, college of engineering and agroindustrial technology, university of the philippines los baños. casimina ha. 2010. reduction of hcn level in cassava (manihot esculenta crantz) leaves at the optimum drying conditions obtained through heated 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drying characteristics of laurel leaves. ege university research final report. turkey: agricultural machinery department, faculty of agriculture, ege university. ocr document 22 impacts of probiotics on water quality and milkfish production (chanos chanos) grown in polluted ponds of marilao and meycauayan, bulacan john vincent r. pleto* environmental biology division, institute of biological sciences college of arts and sciences university of the philippines los baños mark dondi m. arboleda school of environmental science and management university of the philippines los baños veronica p. migo department of chemical engineering college of engineering and agro-industrial technology university of the philippines los baños jessica f. simbahan institute of biology, college of science university of the philippines diliman abstract the marilao-meycauayan-obando river system is known to be heavily polluted with organics and heavy metals, thus affecting the ecosystem. this study used probiotics as an ecological approach to improve environmental quality, with a focus on determining the impacts of probiotics on fish health and survival as well as water quality. probiotics are microbial feed supplements that can improve the survival and health of organisms. probiotics were applied at the start and after two months of culture period. physico-chemical water quality parameters were recorded. growth parameters such as fish body weight, feed conversion ratio (fcr), and survival rate were determined. polymerase chain reaction-denaturing gradient gel electrophoresis (pcr-dgge) method was used to determine the microbial community present in the guts of milkfish (chanos chanos) grown in polluted water treated with probiotics. the results showed that ponds treated with probiotics had higher dissolved oxygen and lower biochemical oxygen demand (bod) and nitrate and phosphate levels, which are beneficial for the growth of milkfish. however, higher ammonia and chemical oxygen demand (cod) were observed in the probiotic ponds. higher survival rate (95.3%) was obtained in treated ponds compared to non-treated ponds (74.1%). the fcr * corresponding author science diliman (january-june 2021) 33:1, 22-39 j.v.r. pleto et al. 23 was less in probiotic-treated ponds (0.74) than non-treated ponds (1.35), which is beneficial for fish production. the study showed that the probiotic strains (bacillus) were not able to establish in the milkfish gut. instead, strains related to cetobacterium, clostridium, conexibacter, cyanobium, cyanothece, cylindrospermum, helicobacter, romboutsia, synechococcus, and vibrio were detected in the guts of milkfish. overall, the probiotics had an impact on water quality and fish health through improvement of growth and survival rate. keywords: probiotics, water quality, chanos chanos, pcr-dgge (polymerase chain reaction-denaturing gradient gel electrophoresis), microflora introduction the marilao-meycauayan-obando river system (mmors) is one of the most polluted rivers in the philippines. in 2007, mmors was included in the list of “dirty thirty” rivers in the world by then blacksmith institute (now pure earth), an environmental group in new york (blacksmith institute 2007). the river system was found to be heavily polluted with organic matter and heavy metals coming from different sources such as untreated municipal wastewater and industries in used lead acid battery recycling and gold refineries. unfortunately, there are aquaculture ponds along the river system that are considered to be one of the primary sources of livelihood of fisherfolk in bulacan. since water from aquaculture ponds come from the river system, the ponds’ ecosystem and people who consume the fishes are at high health risk. the ecosystem, including microorganisms, is greatly affected due to the pollutants present in the environment. in order to restore the ecosystem and make it conducive for fish growth, the use of probiotics was explored. the first application of probiotics was to test the ability to increase the growth of organisms that live in water. then, it was used to improve water quality and to treat bacterial infections (cruz et al. 2012). the aquaculture industry employs microorganisms, termed probiotics, as live adjunct to the diet of fishes. these probiotics have beneficial effects on the host by boosting the utilization of feeds or enhancing their nutritional value, strengthening host response against diseases or improving the quality of the environment (verschuere et al. 2000). the application of probiotics is an eco-friendly bioremediation technology for aquaculture that can improve water quality. during the culture period, water quality has the tendency to deteriorate due to the accumulation of metabolic wastes, decomposition of unused feeds, and decay of biotic materials. probiotics can utilize or decompose the organic matter and toxic material in water, thus improving water quality. the use of probiotics in aquaculture can also inhibit pathogens and promote the growth of farmed fish. it controls pathogens through a variety of mechanisms that serve as an alternative impacts of probiotics on water quality and milkfish production (chanos chanos) 24 to antibiotics (padmavathi et al. 2012). in this study, probiotics were applied in fishponds in two sites located along the mmors to assess their performance in improving water quality and growth and survival of milkfish (chanos chanos). the diversity of microflora in the fish gut was also assessed. to date, no study has been undertaken to determine the impact of probiotics on the survival of fishes growing in heavily polluted sites in the philippines. the composition and identification of fish microflora has been investigated using culture-dependent methods that relied on phenotypic and biochemical characteristics. also, only a low percentage of intestinal microflora can be cultured in laboratory media and identified using phenotypic and biochemical approaches. the analysis of polymerase chain reaction (pcr)-amplified 16s rrna genes from gut samples through denaturing gradient gel electrophoresis (dgge) allows for the visualization of the bacterial community through the dna fingerprint of the presumed dominating microorganisms present in the sample. microbial identification and clustering through pcr-dgge without the need for classical culturable substrates allowed the direct use of the samples and a faster and simpler protocol (manzano et al. 2012). pcr-dgge has become a popular method for studying gut microbiota of humans and animals because it provides a rapid survey of the microbial community regardless of their ability to grow in cultivation media (possemiers et al. 2004; kim et al. 2007). however, one of the main limitations of dgge is that it does not have the resolution to detect organisms that represent less than 1% of the overall community (muyzer et al. 1993). in this study, probiotics were tested on fishponds in two sites located along the mmors to assess their performance in improving water quality and fish health. there have been no studies conducted on the use of probiotics in aquaculture ponds that get water from a polluted source. the use of probiotics has been proven to be positive promoters of aquatic animal growth, survival and health (hai 2015). the objective of the study was to determine the impacts of probiotics on milkfish growth and survival and water quality. the study also determined the microflora diversity of milkfish gut using the pcr-dgge method. materials and methods study site fishponds along the marilao and meycauayan rivers of mmors were the sites for this study. the two sampling sites were located in barangay nagbalon, marilao, bulacan and barangay liputan, meycauayan, bulacan. three replicates of probioticj.v.r. pleto et al. 25 treated and non-treated ponds were prepared in each site. the ponds used in the study are those owned by farmers who agreed to participate in the study. table 1 shows the physical characteristics of the ponds used in the study. table 1. physical characteristics of the ponds used in the study. pond location treatment size (sq.m.) depth (m) 1 nagbalon treated with probiotics 1400 1.0 2 nagbalon treated with probiotics 780 1.5 3 liputan treated with probiotics 300 1.0 4 nagbalon non-treated 670 1.5 5 nagbalon non-treated 300 1.0 6 liputan non-treated 300 1.0 pond treatment, preparation and application of probiotics the probiotic used was purchased from charoen phokphand (cp) feeds in samal, bataan and consisted of super biotic® and ph fixer®. super biotic and ph fixer both consist of a strain of bacillus microorganism with a concentration of 109 cfu/ ml. super biotic was applied once during pond preparation and ph fixer was also applied once after two months of culture. super biotic was applied at a dosage of 3 l per ha and ph fixer at a dosage of 150 g per ha. super biotic was mixed with molasses and water at a rate of 500 ml molasses and 100 l of water for every 1 l super biotic and was incubated for 24 hours in a drum before application to the ponds. the viability of probiotics was not conducted since the counts were already given on the product label and the probiotics were already tested and validated by the manufacturer. monitoring of physico-chemical parameters dissolved oxygen (do), temperature, ph, salinity, ammonia, phosphates, biochemical oxygen demand (bod), and chemical oxygen demand (cod) of the ponds were monitored throughout the four months of culture. the in situ parameters such as do, temperature, ph, and salinity were determined on a daily basis during the morning and the afternoon. the do, temperature, and ph were measured using a do meter (hach hq30d) and ultrameter iii 9p. salinity was determined using the atago s-mill refractometer. ex situ parameters such as ammonia, phosphates, bod, and cod were determined monthly. stocking of milkfish milkfish fingerlings were obtained from a farm in binmaley, pangasinan. these were conditioned and acclimatized for one week in the reservoir pond before stocking at 1.5 fish/m2. a 27% allowance of milkfish was added for mortality and destructive impacts of probiotics on water quality and milkfish production (chanos chanos) 26 sampling every month. the average initial weight of fingerlings was 4.4 g for the nagbalon site and 5.2 g for the liputan site. the average initial length of fingerlings were 7.7 cm and 8.3 cm for nagbalon and liputan, respectively. the feeding program and the feeds used for the milkfish were adopted from cp. feeding of milkfish was done every morning. table 2 shows the feeding management program from cp feeds. table 2. feeding program for the milkfish using cp feeds. day of culture average body weight (abw) (g) % feed feed code 1 2 10.0 cp 9041 30 35 6.0 cp 9910s 60 90 4.0 cp 9991 90 170 2.7 cp 9991 120 300 2.3 cp 9992 sample preparation six milkfish were randomly collected from each replicate pond during the second and fourth months of culture. milkfish samples were collected after feeding. the milkfish were placed in an ice chest and transported to the laboratory. the exterior of each fish was cleaned with 95% ethanol prior to dissection. dissection of the gut was done using a sterile scalpel/blade. microbes attached to the intestinal wall were part of the natural gut microflora (ringo et al. 2001), thus the whole intestine and gut contents were used for all extractions. pooled gut samples for each pond were homogenized in a blender in te buffer then kept in -80 °c until further use. total bacterial count samples from the homogenized gut were obtained and dilutions of these samples were spread on nutrient agar plates. drops of nystatin were added to the agar to prevent fungal growth. the plates were incubated at room temperature (28–30 °c) for 24–48 hours. colonies that developed on the plates were counted and reflected as colony forming units per gram of sample. dna extraction and dna amplification by polymerase chain reaction dna was extracted from the homogenized sample using the protocol of zr 96 soil microbe dna kit™ and analyzed by electrophoresis for confirmation. the 16s rdna were amplified by pcr using the bacterial primers prbac-338fgc (5’-cgcccgccgcgcgcggcgggcggggcgggggcacggggggactcctacgggag gcagcag-3’) and 518r (5’attaccgcggctgctgg) (muyzer et al. 1993). each mixture j.v.r. pleto et al. 27 (final volume of 50 µl) contained about 3.0 µl of template dna, forward and reverse primers (prbac-338fgc and 518r) at 0.2 µm, the deoxyribonucleotide triphosphate (dntps) and mgcl 2 at 200 µm each, 5 µl of 10x pcr buffer mgcl 2 free and 0.4 µl of taq polymerase. the pcr conditions were an initial denaturation at 94 °c for 1 min, then annealing at 65 °c (with an increasing temperature 1 °c per cycle) for 1 min and extension at 72 °c for 3 min followed by 20 cycles of 94 °c for 1 min, 55 °c for 1 min, and 72°c for 3 min. pcr products were analyzed in 1% (w/v) agarose gel in 0.5x tae buffer using mupid gel electrophoresis set-up and observed on a uv transilluminator (dcodetm). denaturing gradient gel electrophoresis (dgge) pcr products were analyzed using the dcodetm universal mutation detection system (bio-rad laboratories) using the method described by muyzer et al. (1993) and improved by leesing (2005). samples containing approximately equal quantities of amplicons (dna concentration was not determined) were loaded into 8% w/w polyacrylamide gel with a denaturing gradient urea formamide spreading 35 to 65%. electrophoresis was done at 60 °c in tae buffer (2m sodium-acetate, 0.05m edta, ph 8.3) at 20 v for 10 min and then 80 v for 12 hours. after electrophoresis, the gels were stained with ethidium bromide (50 mg/l) for 1 hour and rinsed in distilled water for 20 min. the gels were observed under a uv transilluminator. the bands were excised, sliced into small pieces and incubated in pcr grade water 37 °c for 30 min before storing at -80 °c until further use. sequencing and identification the excised bands were subjected to pcr for amplification. the same conditions were used except for the annealing temperature of 62 °c. each mixture contained about 0.5 µl of template dna, the primers (prbac-338fgc and 518r) at 0.2 µm each, the deoxyribonucleotide triphosphate (dntps) and mgcl 2 at 200 µm each, 5.0 µl of 10x pcr buffer mgcl 2 free and 0.4 µl of taq polymerase. pcr products were analyzed by electrophoresis as previously mentioned. the pcr amplicons were sent to aitbiotech, singapore for sequencing. the results were viewed using finchtv. the identities of the organisms were determined using basic local alignment tool (blast). multiple sequence alignment was done using the software bioedit™ version 7.1 (hall 2011). the phylogenetic tree was constructed using the phylip software (felsenstein 2005). impacts of probiotics on water quality and milkfish production (chanos chanos) 28 results physico-chemical water quality parameters of ponds table 3 shows the mean physico-chemical values of pond water for the non-treated and treated ponds for the whole monitoring period. the do is relatively higher in ponds treated with probiotics for morning and afternoon monitoring. the water temperature varied from 29.33 ± 0.52 to 31.93 ± 0.15 ˚c. the ph of the ponds was slightly alkaline. the ammonia levels of treated ponds have a higher mean at 2.72 ± 1.33 ppm compared to non-treated ponds at 1.98 ± 0.98 ppm. the ammonia levels exceeded the recommended level of denr at 0.5 ppm. the nitrate and phosphate levels of ponds treated with probiotics were lower than those of the non-treated ponds. the nitrate levels of ponds were within the recommended level of 7 ppm while phosphate levels exceeded the 0.5 ppm recommended level. the bod of non-treated ponds (12.17 ± 1.61 ppm) were higher than the levels in treated ponds (10.67 ± 1.76 ppm). the cod of treated ponds (650.93 ± 97.93 ppm) were much higher than those in non-treated ponds (502.00 ± 65.98 ppm). both bod and cod exceeded the denr recommended limits of 7 ppm and 100 ppm, respectively. table 3. mean physico-chemical parameters of the ponds for the whole monitoring period. treatment ponds monitoring time do (mg/l) temp. (˚c) ph ammonia (ppm) nitrates (ppm) phosphates (ppm) bod (ppm) cod** (ppm) non-treated ponds am 6.34 ± 1.23 29.36 ± 0.63 8.05 ± 0.28 1.98 ± 0.98 0.42 ± 0.13 1.81 ± 1.49 12.17 ± 1.61 502.00 ± 65.98 pm 11.85 ± 0.85 31.71 ± 0.20 8.65 ± 0.15 treated ponds am 7.13 ± 0.51 29.33 ± 0.52 8.03 ± 0.06 2.72 ± 1.33 0.30 ± 0.10 0.85 ± 0.05 10.67 ± 1.76 650.93 ± 97.93 pm 12.76 ± 0.71 31.93 ± 0.15 8.44 ± 0.20 recommended level* 5.0 mg/l 25 31°c 6.5 – 8.5 0.05 ppm 7 ppm 0.5 ppm 7 ppm 100 ppm * the recommended level for each parameter is based on the water quality guidelines and general effluent standards of 2016 (dao 16-08) by the department of environment and natural resources (denr) **there was a significant difference for the chemical oxygen demand (cod) of the nontreated and treated ponds (p<0.05). average body weight, total bacterial count and percent survival of milkfish the average body weight and the gut bacterial counts were determined from milkfish gathered from all the ponds in the nagbalon and liputan sites. j.v.r. pleto et al. 29 the initial microbial counts of the fingerlings and probiotics used were 4.67 x 104 and 3.05 x 105 cfu/ml, respectively. table 4 shows the average body weight of milkfish and the total viable count of bacterial isolates from the guts of the samples after two and four months of culture in the ponds. ponds treated with probiotics during the second month have milkfish with relatively higher body weight compared to those in the non-treated ponds. during the harvest, the average body weight of milkfish from treated ponds was 169.17 g while that in the non-treated ponds was only 129.57 g. the feed conversion ratio (fcr) is the rate measuring the efficiency by which fish convert feed into the desired output. the lower the fcr, the less feed is being used to produce 1 kg of fish. in terms of the survival rate, it was noted that fish grown in ponds treated with probiotics have higher survival rate (95.3%) compared to those in non-treated ponds. based on the results, treated ponds have relatively higher microbial population during the 4th month. table 4. average body weight, feed conversion ratio, and total viable count of bacterial isolates of milkfish from the two ponds. treatment ponds second month fourth month feed conversion ratio (fcr) percent survival* average body weight (g) total bacterial count (cfu/ml) average body weight (g) total bacterial count (cfu/ml) non-treated ponds 48.97 ± 19.03 3.08 x 105 ± 3.15 x 105 129.57 ± 68.00 2.21 x 105 ± 2.80 x 105 1.35 ± 1.31 74.1% treated ponds 61.33 ± 14.99 5.19 x 104 ± 2.62 x 104 169.17 ± 54.05 3.48 x 105 ± 2.75 x 105 0.74 ± 0.10 95.3% p value 0.403 0.481 0.536 0.724 0.22 *based on the number of milkfish that survived denaturing gradient gel electrophoresis, sequencing and identification dgge analysis was conducted through the amplification of the v3 variable region of bacterial 16s rdna. figure 1 shows the dgge pattern of the amplified dna from milkfish gut during the 2nd and 4th months for both sites as well as those of the fingerlings and probiotics. the identities of the marker organisms are as follows: a lactobacillus plantarum lp1; b lactobacillus casei 4e5; c pediococcus acidilactici; d – bacillus sp.; and e lactobacillus acidophilus 1900w. impacts of probiotics on water quality and milkfish production (chanos chanos) 30 a b figure 1. (a) result of the denaturing gradient gel electrophoresis from different ponds during the 2nd month of collection and the fingerlings and probiotics used in the study. (b) result of the denaturing gradient gel electrophoresis from different ponds during the 4th month of collection. dgge analysis shows the presence on the second month of synechoccus rubescens (bands 13, 20, and 23) and cyanothece sp. (bands 12, 16, 18, and 19). none of the fishes, especially those from ponds treated with probiotics, showed the presence of the probiotic strains bacillus pumilus (band 8) and chlorobaculum tepidum (band 9). strains with closest identities to helicobacter sp. (band 1) and clostridium sp. (band 3) were found in the guts of fingerlings. cetobacterium somerae, corresponding to bands 29, 34, 40, 48, and 51, was present in the 4th month in almost all ponds. the presence of pathogenic organisms related to vibrio cholera (band 35) and clostridium ghonii (band 36) was also noted on the 4th month. the dgge profile of the probiotics used contained strains of bacillus, chlorobium and chlorobaculum. a phylogenetic tree (figure 2) was constructed to determine the relationship of the strains’ sequences among each other and with other type strains. the phylogenetic tree identifies seven major clusters to which the gut strains shared closest identity. they are the actinobacteria-like, bacillus-like, chlorobia-like, clostridialike, cyanobacteria-like, epsilonproteobacteria-like, and fusobacteria-like clusters. sequences 7, 33, 35, 36, and 38 did not cluster with a known microorganism and could be an entirely different species. j.v.r. pleto et al. 31 fi gu re 2 . p hy lo ge ne ti c tr ee o f 16 s rr n a se qu en ce s fr om g ut o f m il kf is h cu lt iv at ed in p on ds . t he s eq ue nc e le ng th is 1 40 b as e pa ir s. th e re su lt an t tr ee w as e va lu at ed b y bo ot st ra p an al ys is b as ed o n 10 00 r ep li ca te s to r ep re se nt t he e vo lu ti on ar y hi st or y of t he t ax a an al yz ed . t he p er ce nt ag e of r ep li ca te tr ee s in w hi ch a ss oc ia te d ta xa c lu st er ed t og et he r in t he b oo ts tr ap t es t is s ho w n ne xt t o th e br an ch es . t he t re e is d ra w n to s ca le , w it h br an ch l en gt hs m ea su re d in t he n um be r of s ub st it ut io ns p er s it e. r hi zo bi um le gu m in os ar um w as t he o ut gr ou p or ga ni sm . impacts of probiotics on water quality and milkfish production (chanos chanos) 32 discussion impacts of probiotics on water quality in aquaculture, the common problem in water quality is the increase in organic matter, phosphorus and nitrogen levels due to the feeds and fertilizers being applied in ponds. high concentrations of nitrate can reduce animal growth and survival in aquaculture (davidson et al. 2014). studies showed that probiotics improve the quality of water and the pond bottom sediment, thereby creating a stress-free environment for the animals and thus improving their health (moriarty et al. 2005). probiotics have proven their effectiveness in improving water quality by enhancing decomposition of organic matter, reducing nitrogen and phosphorus concentrations, and controlling ammonia, nitrite, and hydrogen sulphide (boyd and massaut 1999; ma et al. 2009; cha et al. 2013). in the study of mahmud et.al. (2016), ponds treated with probiotics had higher oxygen levels, better water transparency, less ammonium and fewer cyanobacteria. based on the water quality assessment, probiotic treated ponds had relatively higher do and lower ph, nitrates, phosphates, and bod levels, which is beneficial to fish growth. however, ammonia and cod levels were higher in treated ponds. this might be due to the lower frequency of application of probiotics throughout the culture period. it was suggested that maintaining high levels of probiotics in production ponds minimizes the accumulation of dissolved and particulate organic carbon (balcázar et al. 2006). impacts of growth and survival of milkfish several studies have found that feeding probiotic bacteria can increase the growth rate, weight gain and feed efficiency (fcr) of several aquaculture species. the increased growth performance produced by the use of probiotics is achieved by enhancing metabolic pathways by contributing vitamins, short-chain fatty acids and enzymes that are either not normally produced by the host or sufficiently included in their diet (merrifield et al. 2010). application of probiotics can improve aquatic animal growth rates, feed utility by influencing digestive enzyme processes and survival rates (hai 2015). this study showed that milkfish grown in probiotic treated ponds have relatively higher average body weight and survival rate and lower fcr. a lower fcr means that it takes less feed to produce 1 kg of fish; hence, production of milkfish is more cost-efficient. the use of probiotics as growth promoters was applied to nile tilapia (oreochromis niloticus) using the streptococcus strain, significantly increasing crude protein and lipid content in the fish. also, average weight increased from 0.154 g to 6.164 g in 9 weeks of culture (lara et al. 2003). the results of the study agreed with related literature that application of probiotics would increase the weight and survival rate of fish as well as lower the fcr. j.v.r. pleto et al. 33 microflora of milkfish the common microflorae of milkfish include bacillus sp., bifidobacterium sp., carnoacterium sp., eubacterium sp., lactobacillus sp., lactococcus sp., micrococcus sp., photobacterium sp., pseudomonas sp., shewanella sp., staphylococcus sp., and vibrio sp. (prayitno et al. 2015). acinetobacter, aeromonas, flavobacterium, lactococcus, and pseudomonas, obligate anaerobes bacteroides, clostridium, and fusobacterium, and members of family enterobacteriaceae dominate the guts of freshwater species (gómez and balcázar 2008). these microorganisms play important roles in immunity mechanism and provide a positive influence on the health of the fishes. lactobacillus fermentum, lactobacillus gasseri, lactobacillus delbrueckii, and micrococcus lylae were potentially used as probiotics in aquaculture (prayitno et al. 2015). based on the results obtained, no probiotic strains were found in the guts of the milkfish after the application of probiotics. dgge analysis was used to analyze the microbial community of milkfish gut. the dgge profile of the commercial probiotics used in the study contained bacillus, chlorobium, and chlorobaculum species. these probiotics can diminish the growth of pathogens and increase the growth of beneficial bacteria, leading to improved fish quality (ninawe and selvin 2009; chen and hu 2011). the probiotics species failed to establish in the guts of the milkfish due to the frequency of application. according to balcázar et al. (2006), probiotic microorganisms are able to colonize gastrointestinal tracts when administered over a long period of time because they have a higher multiplication rate than the rate of expulsion; so, as probiotics are constantly added to fish cultures, they adhere to the fishes’ intestinal mucosa, developing and exercising their multiple benefits. dgge is a useful tool in monitoring spatiotemporal changes in the microbial community structure. it can provide full sequences that can be used for further analysis and can easily compare microbial diversity between samples. however, this technique can only detect dominant species with relative abundance of > 0.1% (muyzer et al. 1993). dgge mostly targets abundant taxa present (jiang et al. 2018). in the study, the dominant taxa might be the species present in the environment and the bacteria present in the probiotics were not that abundant. another limitation of using dgge is that the relationship among nucleotide sequence, phylogenetic affiliation and the melting point is not well established and the retardation of the fragment in the gel matrix may not properly indicate phylogenetic relatedness at higher resolution, like species level (kisand and wikner 2003). strains related to cetobacterium, clostridium, conexibacter, cyanobium, cyanothece cylindrospermum, helicobacter, romboutsia, synechococcus, and vibrio were detected in the guts of the milkfish. helicobacter is a major water-borne pathogen but impacts of probiotics on water quality and milkfish production (chanos chanos) 34 its occurrence in fishes is still unknown. several clostridia inhabit the anoxic environment of the intestinal tract and could cause severe diseases. synechococcus is a phototrophic microbe fundamental in carbon and nutrient cycling. it often constitutes the bulk of the photosynthetic biomass and is responsible for a significant proportion of primary production in oligotrophic water. however, synechococcus blooms may occur, which can produce harmful toxins that could cause fish kill (seymour et al. 2010). most vibrios and related bacteria are found in aquatic environments. these vibrio group bacteria can emit light through the process called bioluminescence (madigan et al. 2012). vibrio parahaemolyticus and vibrio vulnificus are commonly present in fish gut microbiome and could be transferred to other environmental reservoirs, implicating fish in the persistence and dispersal of potential pathogens. these vibrio species are often found to be the dominant bacteria in and on marine fish and are common members of the gut microflora in both farmed and wild fish (givens 2012). studies have been conducted on determining the adherence and colonization of microorganisms in gastrointestinal tracts. according to conway (1996), microorganisms can colonize the intestinal tract when they can persist for a long time due to their multiplication rate. also, another important aspect identified by nikoskelainen et al. (2001) is that mucosal adhesion is an important criterion for selection of probiotics to be applied for fish. hence, it is essential to first determine the gut microbiota of milkfish grown in the aquaculture ponds to determine which can colonize longer in the gut. some yeast strains have a strong adhesion potential to fish guts that could compete with other microorganisms (li et al. 2018). also, it was suggested that one approach to evaluate gut microbiome is to follow a 24hour starvation period (zhang et al. 2016). studying the colonization of probiotics in fish guts is very complex due to the different environmental and stochastic factors and high influx of microorganisms in water (li et al. 2018). according to li et al. (2018), colonization should be replaced with populate or passive colonization when describing the probiotic content isolated from guts. conclusions and recommendation probiotics is a supplemental feed that could improve the growth and survival of fishes as well as the water quality of ponds. the research study focused on determining the impacts of the use of probiotics on fish health and water quality. it also determined the microflora diversity of milkfish guts using the pcr-dgge method. the results showed that probiotics had a positive impact on water quality. ponds treated with probiotics had higher do levels and lower ph, nitrates, j.v.r. pleto et al. 35 phosphates and bod levels, which are beneficial for growth and survival. milkfish grown in probiotic ponds had higher weight and survival rate and better fcr, which is beneficial for production. however, probiotic species (bacillus) failed to establish in the guts of the milkfish due to the frequency of application. in the study, the probiotics contain strains of bacillus, chlorobium, and chlorobaculum, which are beneficial for fishes. strains related to helicobacter, cetobacterium, romboutsia, synechococcus, vibrio, cylindrospermum, cyanothece, conexibacter, clostridium, and cyanobium were detected in the guts of the milkfish. the frequency of application of the probiotics might be the reason why the strains were not able to establish. microbiota data of water and pond sediments should be included to determine how the environment has been altered by the probiotics. this could determine if the probiotic strains used were able to thrive after application. another recommendation would be mixing the probiotics mixture to the feeds of fishes for establishment in the gut. other strains of probiotics, such as lactic acid bacteria (lactobacillus sp. and carnobacterium sp.), vibrio sp., and pseudomonas sp., could be tested and utilized as probiotics. isolating, screening, and culturing of probiotic strains from the river system that can survive in a polluted environment could help in successful introduction to the aquatic organisms. acknowledgments the authors would like to express their deepest gratitude to the pure earth (blacksmith institute) philippines team for its trust, encouragement, and full support in the conduct of this research, and also to university of the philippines los baños – national institute of molecular biology and biotechnology (uplb-biotech) for allowing us to conduct the laboratory analysis. references balcázar jl, blas id, ruiz-z i, cunningham d, vendrell d, múzquiz jl. 2006. the role of probiotics in aquaculture. vet microbiol. [accessed 2020 sep 28];114(3-4):173–186. https:// doi.org/10.1016/j.vetmic.2006.01.009. blacksmith institute. 2007. top 10 most polluted places: 2007. new york: pure earth. 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[accessed 2020 dec 8];454:72–80. https://doi.org/10.1016/j.aquaculture.2015.12.014. j.v.r. pleto et al. 39 ______ assistant professor john vincent r. pleto <jrpleto@up.edu.ph> is a faculty member at the institute of biological sciences, college of arts and sciences, university of the philippines los baños. his research interests include aquatic ecology, specifically water quality and bioremediation technology, and environmental impact assessment studies. dr. veronica p. migo is a professor at the department of chemical engineering, college of engineering and agro-industrial technology, university of the philippines los baños. her research interests include wastewater treatment, recycling of agro-industrial wastewater, global inventory of pollutants, validation of analytical methods, design of analytical laboratories for food and feed, soil, plant and fertilizer analysis, and water and wastewater analysis for iso 17025 accreditation. dr. mark dondi m. arboleda is an associate professor at the school of environmental science and management, university of the philippines los baños. his research interests include aquatic ecology, microbiology, molecular biology and biotechnology, marine protected areas and sustainable community development. dr. jessica f. simbahan is an associate professor at the institute of biology, university of the philippines diliman. her research interests include microbial ecology and biotechnology. 3layman'sabstracts.pmd layman’s abstracts 1 science diliman (july-december 2018) 30:2, 1-3 layman’s abstracts survival and growth of re-attached storm-generated coral fragments post super-typhoon haiyan (a.k.a. yolanda) jonathan a. anticamara and barron cedric a. tan issn 0115-7809 print / issn 2012-0818 online after super-typhoon haiyan (a.k.a. yolanda) hit eastern samar, many of the coral reefs in the area were devastated due the strong waves and c u r r e n t b r o u g h t a b o u t b y t h e s t o r m . i n f a c t , s o m e o f t h e s h a l l o w branching coral reefs were broken into pieces and in some areas vast expanse of reefs were transformed i n to co r a l r u b b l e o r f r a g m e n t s . to h e l p r e v i v e a n d r e c o v e r s o m e o f t h e d y i n g c o r a l f r a g m e n t s , w e r e a t t a c h ed t h o s e t h a t a r e s t i l l a l i v e o n t o s t u r d y s u b s t r a t e ( e . g . , d e a d massive corals) using concrete nails and cable ties. we monitored the survival and growth of these re-attached coral fragments for about a year and we found that most of these fragments star ted to cement (i.e. , bio-mineralize) themselves onto the substrate and survived. in addition, w e f o u n d t h a t m a n y o f t h e s p e c i e s t h a t w e r e a t t a c h e d s h o w e d f a s t g r o w t h r a t e s . t h e s e r e s u l t s p r o v i d e h o p e a n d a f f o r d a b l e s o l u t i o n f o r a c t i ve r ecove r y of s to r m d ev a s t a ted r e e f s . we r eco m m e n d t h a t lo c a l government units invest in active recovery of degraded coral reefs using t e c h n i q u e s d e m o n s t r a t e d i n t h i s s t u d y i n o r d e r t o p r e v e n t t h e l o c a l extinction of many reef f ishes that are dependent on live coral cover for food and habitat. short-term assessment of phytoplankton composition and abundance in cebu and subic bay por ts, phil ippines nero m. austero and rhodora v. azanza in may 2015 and july 2015, two major ports in the country were evaluated for composition and abundance of marine diatoms and dinoflagellates. f o r t h e m a y 2 0 1 5 s a m p l i n g , t h e d i a t o m s p s e u d o n i t s z c h i a s p p . a n d chaetoceros spp. were the most abundant in cebu international port (cip) and in the naval supply depot (nsd), respectively. moreover, diatoms like layman’s abstracts 2 t h a l a s s i o n e m a s p p . a n d l e p t o c y l i n d r u s s p p . a l s o r e c o r d e d h i g h c e l l d e n s i t i e s i n n s d te r m i n a l i n j u l y 2 0 1 5 . o t h e r m i c r o a l g a e o b s e r v ed i n c l u d e d i a t o m s l i ke co s c i n o d i s c u s s p p . , n i t z s c h i a s p p. , a n d p s e u d o nitzschia spp. , and dinoflagellates, such as ceratium spp. , ceratium furca, gonyaulax spp. , gymnod inium spp. , lingul ud inium spp. , phalacroma spp. , prorocentrum micans, prorocentrum spp. ,and dinophysis caudata. the results o f t h i s s t u d y c o n t r i b u t e t o t h e e s t a b l i s h m e n t o f b a s e l i n e d a t a f o r phytoplankton composition and abundance, which are necessary for the identif ication of potentially toxic/harmful microalgae which pose risks of ballast water inclusion and transport. polyelectrolyte complex of chitosan and -carrageenan as potential scaffold for t issue engineering soma chakrabor ty, kristina angel ica m. yatco, edward king lim chua and modesto t. chua s c a f f o l d s a r e s u p p o r t s t h a t a r e e x p e c t e d t o a i d i n c e l l g r o w t h i n s i d e the body to facilitate the healing process of fractured bones or any other ruptured tissue. they should be nontoxic, have suff icient space within (porous) to allow the cells to expand, and should not collapse during the growth of the cells. they should undergo degradation inside the body once optimal cell growth takes place. in this study, two biopolymers, namely chitosan (can be extracted from t h e s h e l l o f c r u s t a c e a n s ) a n d c a r r a g e e n a n ( c a n b e o b t a i n e d f r o m r e d s e a w e e d s ) , w e r e u s e d t o m a k e t h e s c a f f o l d s . t h e t w o p o l y m e r c h a i n s we r e a l l owed to fo r m a co m p l ex w i t h e a c h o t h e r, a n d t h e i r p o s i t i o n s w e r e l o c k e d b y t e t h e r i n g ( c r o s s l i n k i n g ) t h e c a r r a g e e n a n c h a i n s w i t h calcium chloride. the scaffolds were further strengthened through the addition of nano-size hydroxyapatite (a fortifying component of bones). t h e r e s u l t i n g s c a f f o l d s w e r e p o r o u s w i t h d e s i r a b l e s t r e n g t h , a n d underwent very slow degradation under human physiological conditions while providing suff icient time for promoting cell growth. overall, the system has the potential to heal bones and damaged tissues. κκκκκ layman’s abstracts 3 fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem lezel n. mernilo-tutanes and randy l. caga-anan in set theory, an ideal is a collection of sets that are considered to be small or negligible. on the other hand, a fuzzy set is a class of objects that can be used to mathematically represent uncertainty and to provide a formal tool to deal with imprecisions present in many problems. we u s e i d e a l s t o d e f i n e f u z z y o n i d e a l s e t s , w h i c h c a n b e s e e n a s a g e n e r a l i z a t i o n o f t h e f u z z y s e t s . w e e s t a b l i s h s o m e o f t h e b a s i c properties. we also state and prove an extension theorem involving fuzzy on ideal sets. prel iminary development of thoron exposure system in the phil ippines kazuki iwaoka, lorna jean h. palad, el iza b. enriquez, fe m. dela cruz, christopher o. mendoza, masahiro hosoda, shinji tokonami, juanario u. ol ivares, ryan joseph aniago and christian l. dela sada the influence of 220rn (thoron) which is a gaseous radioisotope like 222rn ( r a d o n ) h a s b e e n t h e f o c u s o f a t t e n t i o n o f r e c e n t s t u d i e s c o n c e r n i n g the protection of the general public from natural radiation. hence, it is n e c e s s a r y t o i n v e s t i g a t e t h e p o s s i b i l i t y o f e x p o s u r e t o t h o r o n i n t h e p h i l i p p i n e s . pa s s i ve d e tecto r s , w h i c h d o n o t n eed exte r n a l p owe r, a r e o f t e n u s e d f o r m e a s u r e m e n t s f o r t h o r o n c o n c e n t r a t i o n i n t h e environment . however, it is necessary to check if the passive detectors can appropriately work by being exposed to thoron at several thoron concentrations before conducting the investigation. in this study, a thoron exposure system was developed in the philippines to validate the passive d e t e c t o r s f o r t h o r o n m e a s u r e m e n t a n d t o t e s t i t s p e r f o r m a n c e . t h e thoron exposure system in this study can control the thoron concentration at the range of 5.9 x 104 to 1.5 x 105 bq m”3. the thoron exposure system will be utilized to validate the passive detectors for the investigation of thoron exposure in the philippines in the future. draft-rapid (2).pmd a rapid method for simultaneous determination 1 a rapid method for simultaneous determination of arsenic, cadmium and lead in drinking water by inductively coupled plasma mass spectrometry joshua rey p. torres1, ma. krystell g. banaag2, irene b. rodriguez*1,3 1natural sciences research institute, 2national institute of geological sciences, 3institute of chemistry, college of science, university of the philippines diliman, quezon city 1101, philippines *corresponding author: email: ibrodriguez@up.edu.ph, tel./fax: +632 9205427 abstract the raw water source of drinking water in most areas in the philippines is typically river water and in some cases groundwater. these sources are prone to elevated levels of metals and metalloids that may cause exposure of the general population when the treatment of the water is inadequate. this work presents a simple method based on epa method 200.8 for the determination of total concentrations of arsenic (as), cadmium (cd) and lead (pb) in drinking water using inductively coupled plasma-mass spectrometry (icp-ms) as the elementselective detector. this was applied in the determination of these elements in the water supply in metro manila, philippines. the method detection limits were 0.095 μg l-1, 0.043 μg l-1, and 0.114 μg l-1 for total as, cd and pb, respectively. the method was validated using national institute of standards and technology (nist) 1643e certified reference material for trace elements in water and determined values were 60.4 ± 0.5 μg l-1, 6.7 ± 0.1 μg l-1, and 19.6 ± 0.5 μg l-1 for as, cd and pb, respectively. these determined values were in good agreement with the certified values in the reference material. analysis of actual drinking water samples showed that most samples did not exceed the limit of the philippine drinking water standard for the elements. keywords: as, cd, pb, icpms, metro manila, drinking water science diliman (january-june 2010) 22:1, 1-8 rodriguez, ib, et al 2 introduction consumption of drinking water contaminated with toxic metals such as as, cd and pb has deleterious effects on human health (kazi et al. 2009; jain and ali 2000; massó et al. 2007; satarug et al. 2003). arsenic, which exists in different oxidation states, causes acute and chronic effects and is a suspected carcinogen that is responsible for skin and lung cancer (hughes 2002). it attacks the nervous, cardiovascular, hepatic, endocrine, skin, renal and hematological systems which may cause skin lesions, encephalopathy, cirrhosis, hepatomegaly, diabetes and bone marrow depression (hughes 2002). bangladesh is said to have the world’s biggest as contamination where high levels of as species have been found in groundwater in around 80% of the country’s area and has placed an estimated 40 million people at risk (karim 2000). cadmium, for its part, may cause renal tubular dysfunction in kidneys and hepatotoxicity in livers after chronic exposure to it (fowler 2009). in southeast asia, the highest cadmium exposure has been shown to be through dietary intake with rice as the major pathway (ikeda et al. 2000). the possible mechanisms for pb toxicity includes generation of reactive oxygen species, alteration of lipid metabolism, damage to the mitochondria, disruption of calcium homeostasis and substitution for zinc in various zinc-mediated processes. these may eventually result to neurological disorders, cancer, birth defects, and growth retardation especially in children (ahameda and siddiqui 2007). recent trends have made people more aware of the risks associated with trace metal toxicity and this led various regulatory agencies to set maximum allowable concentrations of these contaminants in the water to evaluate quality (haider et al. 2002; roccaro et al. 2005). these guidelines define the mandatory quality standards of water intended for human consumption. the guideline values in drinking water being implemented by the world health organization (who) are as follows: 10.0 μg l-1 for as, 3.0 μg l-1 for cd, and 10.0 μg l-1 for pb (who 1996). in the philippines, standard values of these trace metals in drinking water have been established by the government’s department of health (doh). according to the philippine national standards for drinking water 2007, the threshold limits in water intended for human consumption are 50.0 μg l-1 for as, 3.0 μg l-1 for cd, and 10.0 μg l-1 for pb (doh 2007). equally important as setting the allowable concentration level for these metals is the development of techniques and methods that accurately quantify them at trace concentrations. standard methods used for trace metal determination such as those from the us environmental protection agency (us-epa) include graphite furnace atomic absorption spectroscopy (gf-aas, epa method 213.2, 1994), inductively coupled plasma optical emission spectroscopy (icp-oes, epa method 200.7, 1994) and icp-mass spectrometry (icp-ms, epa method 200.8, creed et al. 1994). each of the methodologies employed offer unique advantages over others but also come with disadvantages and for this study, icp-ms was used as the element selective detector due to the wide linear range (up to nine orders of magnitude), ultra trace and multi-elemental capability, and very low detection limits offered by the instrument (thomas 2001). this paper presents a simple, rapid and more sensitive method utilizing icp-ms for element detection in drinking water which was applied to survey the occurrence of as, cd and pb in the drinking water of all major cities comprising metro manila. the method described is simpler compared to epa method 200.8 (creed et al. 1994) which is the standard method for determination of waters and wastes by icpms because of the absence of any pre-treatment step in the current method as opposed to the acid digestion step necessary in epa method 200.8. the lack of a pre-treatment step in the optimized method limits its applicability to determination of total concentrations of the analytes in drinking water samples due to possibility of higher dissolved solids in other matrices. this direct technique, however, is very useful for a direct and rapid determination of as, cd and pb in drinking water samples with minimal waste generated during the analysis of these contaminants for routine monitoring. experiment standard solutions and reagents all reagents used in this work were of analytical grade. single element standards of as, cd, pb and germanium (ge) were purchased from cpi international (santa rosa, ca, usa). a certified reference material (nist science diliman (january-june 2010) 22:1, 1-8 a rapid method for simultaneous determination 3 1643e, trace elements in water) by making appropriate dilutions of the stock solutions with ultrapure water and were acidified to have an acid concentration of 2% hno 3 (v/v) in polyethylene tubes. the material nist 1643e was diluted 10-fold and was also acidified prior to validation experiments. analytical blanks were prepared using ultrapure water in the same manner as the sampled tap water. sample collection metro manila, also called national capital region, is composed of sixteen cities and one municipality (shown in fig. 1) and is home to about 12 million inhabitants. tap water was sampled from all the sixteen cities and municipality of metro manila as described by the philippine national standards for drinking water (doh, 2007). samples were collected in 250 ml polyethylene containers and kept at about 4°c during transport. the samples were acidified to have an acid concentration of 2% hno 3 (v/v) and maintained at 4°c before analysis. in total, 50 tap water samples were taken from households and establishments all of which are connected to one of the two major drinking water treatment plants supplying the cities. the sampling was conducted in august 2009. the samples were collected directly from the tap after allowing the water to flow freely for one minute. field fortified samples were prepared using ultrapure water. aliquots of the acidified samples were then placed in the autosampler vials and subjected to analysis to determine the total concentrations of as, cd and pb. instrumentation an agilent 7500cx icpms (agilent technologies, germany) equipped with a micromist glass concentric nebulizer and an integrated autosampler (i-as with type a vials, 89 x 6 ml capacity) was used as the elementselective detector. the instrument was tuned to get the maximum sensitivity for all the analytes and reduce the interferences from oxides and doubly-charged species. the optimum operating conditions are summarized in table 1. instrument drift was monitored by analyzing test solutions containing 1.00 μg l-1 of as, cd, and pb at repeated intervals throughout the analysis sequence. ge (100.0 μg l-1 ge in 2% hno 3 ) was added online using the peristaltic pump and used as internal standard for as. the analysis of one sample figure 1. map of metropolitan manila showing the sixteen cities and one municipality comprising the metropolitan area. table 1. optimum instrument conditions for the agilent 7500cx icp-ms used in the analysis. instrument parameters rf power 1550 w carrier gas flow rate 0.85 l/min makeup gas flow rate 0.25 l/min nebulizer pump 0.01 rps sampling depth 8.2 mm s/c temp 2.0 °c peripump 30 rps science diliman (january-june 2010) 22:1, 1-8 rodriguez, ib, et al 4 took about 3 min from drawing the sample until the rinses were finished prior to drawing of the next sample. results and discussion spectral interference potential spectroscopic interferences that may be encountered were corrected using the built-in interference equations in the agilent icpms chemstation system (g1834b). the primary disinfection process employed in the philippines is chlorination, thus the drinking water samples may have quantities of chlorine that may introduce spectral interferences in the form 40ar35cl and influence accuracy of 75as determination. during analysis, counts for m/z 75 (as), 77 (se) and 82 (se) were monitored. comparing raw counts (n=3) for m/z = 75, 77 and 82 from samples of drinking water showed that the average detectable counts for 77 and 82 were 2.84% and 4.41% of the counts corresponding to m/z 75. these results indicate that interference from chloride was corrected using the mathematical equations in the chemstation software as these are capable of correcting signals of the analyte if the interfering element contributes <20% of the total signal (d’ilio et al. 2008). method validation linearity, internal standard response, detection limits, accuracy and repeatability were evaluated to assess method performance. linearity was demonstrated for all the investigated elements (as, cd, pb) in the range 0.01 to 100.0 μg l-1, with correlation coefficients of at least 0.999 for all three elements. the method detection limits were estimated by analyzing 7 replicates of a solution containing 1μg l-1 of all three elements. the detection limits were calculated by multiplying the standard deviation of the calculated elemental concentrations in the test solutions by the student’s tvalue (3.143 for seven replicates, wisconsin department of natural resources laboratory, 1996). the estimated values were 0.095 μg l-1, 0.043μg l-1, and 0.114 μg l-1 for as, cd and pb, respectively. these values are better compared to the reported detection limits in epa method 200.8: 1.4 μg l-1 for as, 0.5 μg l-1 for cd and 0.6 μg l-1 for pb (creed et al. 1994). for validation, the method was applied to determine the total as, cd and pb in nist 1643e reference water. the results of repeated analysis of the reference material in different days showed good agreement against the certified values for all elements investigated (values summarized in table 2). ge was used as internal standard for as determination and was found to be ineffective for correction of cd and pb levels. this was expected since internal standard correction requires that the analyte and internal standard used should have relatively close m/z ratios for effective correction (eickhorst and seuber 2004). concentrations of pb and cd, therefore, were determined by external calibration without the use of the internal standard. the signals for ge (taken as counts per second) were monitored throughout the run and showed a relative standard deviation (rsd) of 1.22% indicative of a stable response and hence, improving the analysis for as. the rsd for ge signals also indicates stable signals for the three analytes and this was validated by the rsd determined for the drift standard (1.0 μg l-1 measured at repeated intervals throughout the analysis sequence) which was determined to be in the range 1.8% to 2.3% for the three analytes. analysis of actual samples the determined total as, cd and pb in drinking water samples are summarized in table 3. the analysis of the field, laboratory and calibration blanks reflected element measured value (μg l-1) certified value (μg l-1) day 1 day 2 day 3 as 60.4 ± 0.5 60.6 ± 0.5 60.1 ± 0.7 60.45 ± 0.72 cd 6.7 ± 0.1 6.8 ± 0.2 6.6 ± 0.1 6.568 ± 0.073 pb 19.6 ± 0.5 19.3 ± 0.4 19.7 ± 0.6 19.63 ± 0.21 table 2. determined total concentrations of as, cd and pb in certified reference material nist 1643e (mean ± sd, n=3) in different days. science diliman (january-june 2010) 22:1, 1-8 a rapid method for simultaneous determination 5 city/ sample municipality number as (ìg l-1) cd (ìg l-1) pb (ìg l-1) marikina 1 1.47 ± 0.01 0.05 ± 0.02 0.68 ± 0.02 2 0.38 ± 0.02 0.07 ± 0.02 0.30 ± 0.07 3 0.76 ± 0.03 < mdl 0.25 ± 0.02 pasig 4 0.65 ± 0.04 < mdl 0.55 ± 0.02 5 0.49 ± 0.02 < mdl 0.26 ± 0.04 6 0.45 ± 0.01 < mdl < mdl pateros 7 0.41 ± 0.03 < mdl 0.28 ± 0.01 8 0.38 ± 0.02 < mdl 0.66 ± 0.01 makati 9 0.35 ± 0.01 < mdl 0.24 ± 0.01 10 0.36 ± 0.01 < mdl 0.75 ± 0.02 11 0.37 ± 0.02 < mdl 0.41 ± 0.01 taguig 12 0.32 ± 0.02 < mdl 1.35 ± 0.01 13 0.33 ± 0.04 < mdl 0.34 ± 0.01 muntinlupa 14 5.47 ± 0.16 < mdl < mdl 15 12.46 ± 0.06 < mdl 0.60 ± 0.01 16 4.61 ± 0.15 < mdl 0.67 ± 0.01 parañaque 17 5.06 ± 0.06 < mdl < mdl 18 3.93 ± 0.15 < mdl 0.28 ± 0.01 19 0.31 ± 0.02 < mdl 0.25 ± 0.01 las piñas 20 3.27 ± 0.04 < mdl 1.77 ± 0.01 21 4.07 ± 0.14 < mdl 0.12 ± 0.01 pasay 22 3.18 ± 0.04 < mdl 0.12 ± 0.01 23 0.32 ± 0.02 < mdl 0.60 ± 0.01 24 0.29 ± 0.01 < mdl 0.25 ± 0.01 mandaluyong 25 0.31 ± 0.05 < mdl 0.71 ± 0.01 26 0.29 ± 0.02 < mdl 0.49 ± 0.02 27 0.29 ± 0.02 < mdl 1.15 ± 0.01 quezon 28 0.26 ± 0.03 0.11 ± 0.01 8.62 ± 0.04 29 0.27 ± 0.01 < mdl 0.53 ± 0.01 30 0.68 ± 0.02 0.05 ± 0.01 0.68 ± 0.02 31 0.37 ± 0.02 < mdl 0.48 ± 0.01 valenzuela 32 0.34 ± 0.01 < mdl 1.51 ± 0.02 33 0.54 ± 0.05 < mdl 0.69 ± 0.01 34 0.50 ± 0.04 < mdl 0.43 ± 0.01 malabon 35 0.39 ± 0.02 < mdl 0.59 ± 0.01 36 0.28 ± 0.03 < mdl < mdl 37 0.27 ± 0.03 < mdl 0.44 ± 0.01 navotas 38 0.25 ± 0.03 < mdl 0.51 ± 0.00 39 0.28 ± 0.01 < mdl 0.65 ± 0.01 40 0.26 ± 0.01 < mdl 0.17 ± 0.01 caloocan 41 0.30 ± 0.04 < mdl 0.14 ± 0.01 42 0.25 ± 0.02 < mdl 0.16 ± 0.01 43 0.23 ± 0.01 < mdl 1.28 ± 0.01 manila 44 0.27 ± 0.02 < mdl 0.13 ± 0.01 45 0.23 ± 0.03 < mdl 1.15 ± 0.01 46 0.21 ± 0.03 < mdl 0.64 ± 0.01 47 0.25 ± 0.02 < mdl 1.04 ± 0.01 san juan 48 0.30 ± 0.02 < mdl 1.07 ± 0.01 49 0.27 ± 0.04 < mdl 18.98 ± 0.08 50 12.96 ± 0.12 0.05 ± 0.01 6.72 ± 0.05 table 3. determined total concentrations of as, cd and pb in drinking water samples (mean ± sd, n=3). (the method detection limits were 0.095 μg l-1, 0.043 μg l-1, and 0.114 μg l-1 for total as, cd and pb, respectively.) science diliman (january-june 2010) 22:1, 1-8 rodriguez, ib, et al 6 elemental concentrations below the method detection limits. the validation of the method using the certified reference material and drift standard was carried out alongside the analysis of actual samples (results discussed in previous sections). for the actual samples, except for the measured pb in a tap water sample taken from san juan city, the determined concentrations of all elements monitored in all the samples passed the standard limits prescribed for drinking water in the philippines. notably, one sample from muntinlupa city was above the guideline value set by the who for as in drinking water. concentrations of as in the samples ranged from 0.21 ± 0.03 μg l-1 to a maximum of 12.96 ± 0.06μg l-1. also, it can be observed that compared to most of the cities in metro manila, a relatively high as concentration resulted from samples that were taken in a set of neighboring cities (las piñas, parañaque and muntinlupa). it is suspected that possible sources of contamination would be intrusion from groundwater in which natural levels of as largely depends on the area’s geochemical features as what was observed in similar studies (cavar et al. 2005, peters et al. 2006). however, it is deemed that our observation on the slightly elevated as in these neighboring cities, certainly, warrants further investigation. most of the measured cd concentrations were either below the method detection limit or were in the range 0.05 ± 0.01 to 0.11 ± 0.01 μg l-1. cd was only detected in several sampling sites. the pb concentrations were measurable but were low compared to the allowable concentration except for one sample (san juan city, site 49). although pb may be naturally occurring in certain areas, it rarely enters the water supply as a result of its dissolution from natural sources. pb contamination in drinking water is largely caused by corrosion in pipes that make up the distribution network or household pipelines (conio et al. 1996). this is probably the reason why elevated levels of this trace metal were observed in two samples from san juan (18.98 ± 0.08 μg l-1 and 6.72 ± 0.05 μg l-1) and one in quezon city (8.62 ± 0.04 μg l-1). in recent years, massive rehabilitation of the distribution lines were carried out by the water concessionaires but some areas may still be using old pipes. there is little information about the occurrence of trace metals in the drinking water in most cities in southeast asia. in the philippines, one research reported on the influence of the presence of a dumpsite on the quality of the water (sia su 2007). total cd was included as a parameter to be considered in the study but the focus was entirely on the probable relationship of the occurrence of cd, sulfate and coliform to the observed incidence of diarrhea in the population nearby the dumpsite. another research looked at the exposure of women living in metro manila toward pb and cd as reflected in the results of the analysis in blood and urine samples taken from the volunteers (zhang et al. 1998). the results showed that pb exposure was through inhalation of air contaminated by vehicular exhausts and that cd exposure through air accounts for only 15% of the body burden suggesting other pathways of exposure. recent studies have reflected that the main source of as exposure is via the drinking water. but rice, which is the staple food of most people in southeast asia, has been shown to accumulate as and is now posing an increased risk to the population consuming it (meharg 2004). cd has also been reported to be accumulated in rice (ikeda et al. 2000). the major pathway for pb exposure is still through inhalation of air but recent regulations on the use of pb as anti-knock agent in fuels have reduced the pb concentration in air significantly (suk et al. 2003). the results on the total concentrations of as, cd and pb in the drinking water supply in metro manila show that the population inhabiting this metropolitan capital is at least safe from exposure to these elements via the drinking water source. these results also validate that the optimized method may be used for routine monitoring of as, cd and pb in the drinking water supply. conclusion the study presented in this paper demonstrates a simple, rapid and accurate method based on epa method 200.8 for the simultaneous determination of as, cd and pb in drinking water using inductively coupled plasma mass spectrometry as element selective detector. the estimated detection limits are very low suggesting that the method may be applied for trace analysis of these analytes in drinking water. the method may be used for routine monitoring or random analysis of these contaminants in the drinking water supply. application science diliman (january-june 2010) 22:1, 1-8 a rapid method for simultaneous determination 7 of the method in the analysis of drinking water from all cities in metro manila indicated that all except for one sample passed the drinking water standards for these trace contaminants set by the regulatory agencies in the philippines. acknowledgements the authors would like to thank the natural sciences research institute, university of the philippines diliman for funding this work. references ahamed, m, k.j. siddiqui. 2007, environmental lead toxicity and nutritional factors. clin. nutr. 26: 400-408. cavar , s, t. klapec, r.j. grubešiæ, m. valek. 2005. high exposure to arsenic from drinking water at several localities in eastern croatia. sci. total environ.. 339: 277-282. conio, o., m. ottaviani, v. formentera, c. lasagna, f. palumbo. 1996. evaluation of the lead content in water for human consumption. microchem. j. 54: 355-359. creed, j.t., c.a.brockhoff, t.d. martin. 1994. us environmental protection agency (epa), method 200.8: determination of trace elements in 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philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document def ines research misconduct, specif ies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identif ies appropriate disciplinary actions. definitions scientif ic misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsif ication, or plagiarism in proposing, performing, or reviewing research or in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsif ication is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is 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such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specif ic advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the 84 author is aff iliated as well as the funding agency that supported the research. the board shall ensure correction of the literature in the succeeding issue through various methods as def ined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientif ic misconduct policy. in attendance were: d r. co r a zo n d. v i l l a r e a l , r d u o d i r ec to r, p r e s i d i n g ; d r h e n r y j . ra m o s , p m rg o director and professor, nip; atty. vyva v ictoria aguirre, ovcrd legal consultant; e d i to r s i n c h i e f d r. m a r i co r s o r i a n o (science dil iman) a n d d r. m a r i a m a n g a h a s (s o c i a l s c i e n c e d i l i m a n ) . m s . n a n i e d o m i n g o a n d m s . d e r c y m a r a r a c , e d i t o r i a l assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these def initions of the forms of research misconduct are quoted verbatim from the policy of the off ice of research integrity of the united states public health service. similar phrasings of def initions are adopted in the references listed at the end of this document. 85 references council of science editors. “ white paper on promoting integrity in scientif ic journal pu b l i c a t i o n s , a s a p p r oved by t h e c s e b o a r d of d i r ec to r s o n s e p te m b e r 3 , 2 0 0 6 .” www.council scienceeditors.org. accessed on january 26, 2009. “ po l i c y o n s c i e n t i f i c m i s c o n d u c t : u n i v e r s i t y o f s o u t h e r n c a l i f o r n i a . h t t p : / / policies.usc.edu/plicies/scientif ic misconduct070108.pdf “scientif ic misconduct policy: new york university, the off ice of sponsored programs. https: //www.nyu.edu/osp/policies/scientif ic misconduct .php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/optics/journall/11082 “manuscript submission procedures.” american journal of physics. http: //www.kzoo.edu/ ajp/submit.html sd-sample article 78 humanities diliman, social science diliman and science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice-chancellor for research and development (ovcrd). papers are accepted year-round. authors must submit their works on or before 15 may for publication consideration in the december issue, and on or before 15 october for publication consideration in the june issue. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> photos courtesy of (l-r) analyn salvador-amores, myles capareda & fenelyn nabuab call for paperscall for paperscall for paperscall for paperscall for papers university of the philippines diliman office of the vice-chancellor for research and development ocr document 4quercetin-toralba.pmd rp-hplc analysis of quercetin in the extract of sambong 48 science diliman (january-june) 27:1, 48-63 rp-hplc analysis of quercetin in the extract of sambong (bl umea bal samifera (l) dc) leaves joanna v. toralba* university of the philippines manila noel s. quiming university of the philippines manila jocelyn sb palacpac university of the philippines manila _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online abstract blumea balsamifera (l) dc, known in the philippines as sambong, is an h e r b v a l u ed fo r i t s h e a l t h b e n e f i t s e s p ec i a l l y i n t h e m a n a g e m e n t of u r o l i t h i a s i s . va r i o u s p h y t o c h e m i c a l s , i n c l u d i n g f l a v o n o i d s s u c h a s quercetin, have been determined in sambong leaves. a reversed-phase h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c m e t h o d ( r p h p lc ) w a s developed for the quantitative determination of quercetin in the methanol ex t r a c t of s a m b o n g l e a ve s o b t a i n ed f r o m ley te, co t a b a to, a n d n u ev a ecija, philippines. the methanol extracts of sambong were prepared by maceration followed by rotary evaporation. the solid phase extraction (spe) for the sample cleanup involved the use of a c18 spe packing, a 0.5-ml sample load (50 mg/ml solution), and elution with 4-ml of 80:20 m e t h a n o l : 0 . 5 % h 3 po 4 . t h e h p lc c o n d i t i o n s f o r t h e d e te r m i n a t i o n of q u e r c e t i n i n v o l v e d t h e u s e o f a c 1 8 4 . 6 m m x 2 5 0 m m c o l u m n m a i n t a i n e d a t 3 0 ° c , 2 5 4 n m u v d e t e c t i o n , a n d a m o b i l e p h a s e composition of 25 parts methanol and 75 parts mixture of 0.5% h 3 po 4 and 0.2% triethylamine with a 1 ml/min flow rate in gradient elution. a g o o d l i n e a r i t y a t t h e c o n c e n t r a t i o n r a n g e o f 3 . 7 2 – 1 2 4 µ g / m l o f quercetin standard (r2=0.9989) was observed with the limits of detection (lod) and quantitation (loq) at 0.68 ng/ml and 2.28 ng/ml, respectively. t h e i n t r a d a y ( n = 5 ) a n d i n t e r d a y ( n = 3 ) p r e c i s i o n v a l u e s w e r e s a t i s f a c t o r y ( % r s d < 2 % ) . t h e r ecov e r y e f f i c i e n cy of t h e s p e s a m p l e c l e a n u p s t e p , w h i c h w a s c h e c k e d b y s p i k i n g s a m b o n g s o l u t i o n w i t h jv toralba and others 49 quercetin standard, was 102.41%. the quercetin contents are 0.2337mg, 0 . 1 3 5 0 m g , a n d 0 . 2 9 4 0 m g p e r g r a m o f t h e p o w d e r e d d r i e d l e a v e s o f sambong from nueva ecija, cotabato, and leyte, respectively. this is the f i r s t r e p o r t of q u e r ce t i n co n te n t i n t h e l e a ve s of s a m b o n g co l l ected from the philippines. ke y w o r d s : s a m b o n g , bl u m e a b a l samifera , q u e r ce t i n , h p lc, s o l i d p h a s e extraction layman’s abstract blumea balsamifera (l) dc, known in the philippines as sambong, is an h e r b v a l u ed fo r i t s h e a l t h b e n e f i t s e s p ec i a l l y i n t h e m a n a g e m e n t of u r o l i t h i a s i s . va r i o u s p h y t o c h e m i c a l s , i n c l u d i n g f l a v o n o i d s s u c h a s quercetin, have been determined in sambong leaves collected from other asian countries using instrumental techniques such as high performance liquid chromatography (hplc). an hplc method can be used to generate a c h e m i c a l f i n g e r p r i n t u s e f u l i n t h e a c c u r a t e a u t h e n t i c a t i o n a n d identif ication of herbal medicines, in the comparison of plant materials g r o w n i n d i f f e r e n t r e g i o n s , a n d d e t e r m i n a t i o n o f a m o u n t o f t h e chemically characteristic or pharmacologically active components. this s t u d y f o c u s e d o n t h e d e v e l o p m e n t o f a n h p lc m e t h o d f o r t h e d e t e r m i n a t i o n o f q u e r c e t i n i n s a m b o n g l e a v e s c o l l e c t e d f r o m n u e v a e c i j a , co t a b a to, a n d ley te. t h e m e t h a n o l ex t r a c t s of s a m b o n g l e a ves were prepared by maceration and by a solid phase extraction technique developed in this study. a reversed phase hplc method was optimized and used in the determination of quercetin in the prepared extracts. the quercetin contents determined using the developed methods are 0 . 2 3 3 7 m g , 0 . 1 3 5 0 m g , a n d 0 . 2 9 4 0 m g p e r g r a m o f t h e p o w d e r e d d r i e d leaves of sambong from nueva ecija, cotabato, and leyte, respectively. this is the f irst repor t of quercetin content in the leaves of sambong collected from the philippines. introduction blumea balsamifera (l) dc, commonly known in the philippines as sambong, is an herb that can grow anywhere in the philippines and is also abundant in other asian countries like india, china, and malaysia (quisumbing 1978). the leaves of sambong are extensively used in folk medicine to address various conditions including arthritis, rheumatism, headache, chest pains, diarrhea, dysentery, stomach pain, cough, and fever relief. the decoction is used as a diuretic in edema, in expelling kidney rp-hplc analysis of quercetin in the extract of sambong 50 stones, and in the management of urolithiasis (quisumbing 1978; doh bfad 2005). the philippine national drug formulary (pndf) essential drugs list (doh-nfc 2005) includes sambong in its list of drugs under diuretics, taken orally as a 250 mg or as a 500 mg tablet. alternative preparations include capsules and bags for infusion. the leaf infusions and dosage forms were used to provide evidence of the pharmacologic activities of b balsamifera including intraocular pressure lowering effect (arroyo and others 1990), antimutagenic activity (lim-sylianco and others 1987), and dissolution of calcium/urinary tract stones (rico 1992). phytochemical investigation of sambong leaves reveals a diverse list of chemical constituents present which includes the flavonoids. the high amount of flavonoid content of the crude methanol extracts was attributed for the xanthine oxidase inhibitory and antioxidant activities of b. balsamifera (fazilutan and others 2003; fazilutan and others 2004). among the flavonoids found in the leaves of sambong are quercetin, velutin, luteolin, luteolin-7-methyl ether, rhamnetin, tamarixetin, ombuin, 3,3’-dimethyoxyquercetin, 3,7-dimethylquercetin, quercetin-3,7,3.-trimethyl e t h e r, 3 , 7, 4 ’t r i m e t h y l q u e r ce t i n , p e r s i co g e n i n , a n d 3 ’, 4 ’, 5 t r i h yd r oxy 3 , 6 , 7trimethoxyflavone (barua and sharma 1992; fazilutan and others 2004; ali and others 2005). the use of hplc has been documented for the determination of flavonoids in extracts of plant materials and food products (hertog and others 1993; crozier and others 1997; zu and others 2006; andres-lacueva and others 2008; lin and others 2008). the research of fazilutan and others (2005) focused on the hplc quantitative determination of f ive major flavonoids in the methanol extract of sambong leaves from f ive different sources in malaysia. the flavonoids included in their study are q u e r ce t i n , d i h yd r o q u e r ce t i n 7, 4 'd i m e t h y l e t h e r, b l u m e a t i n , 5 , 7, 3 ', 5 'te t r a hydroxyflavanone, and dihydroquercetin-4'methyl ether. an hplc method can be used to generate a chemical f ingerprint useful in the accurate authentication and identif ication of herbal medicines, in the comparison of plant materials grown in different regions, and the determination of amount of the chemically characteristic or pharmacologically active components. in this study, hplc was used in the determination of quercetin (figure 1) in the extracts of sambong leaves collected from leyte, cotabato, and nueva ecija. at the time of the writing of this manuscript, these are the f irst repor ted values of quercetin in sambong samples cultivated in the philippines. jv toralba and others 51 materials and methods chemicals, reagents, and materials methanol (rci labscan® limited) and acetonitrile (jt baker®) solvents used were of hplc grade. the phosphoric acid 85% (rci labscan® limited) and triethylamine (jt baker®) used were of analytical reagent (ar) grade. distilled water was used in the aqueous solutions that were prepared. the quercetin standard was from sigmaaldrich®. solvents used as mobile phase were f iltered using a 0.45-µm 47-mm (diameter) nylon membrane f ilter discs (whatman®) while solutions injected to the hplc system were f iltered using a 0.45-µm 25-mm (diameter) puradisctm 25 nyl nylon membrane with polypropylene housing f ilter (whatman®). the solid phase extraction (spe) was done using a 6-ml capacity spe tube packed with 500-mg octadecylsilane (c-18) packing (varian® bond elut-c18). instruments the spectrophotometer used in the method developed is a genesys 10uv scanning thermospectronic® spectrophotometer. the hplc analysis was done using a perkinelmer® s200 hplc system equipped with a quaternary pump, manual injector, column oven, uv detector, and totalchrom workstation® software. the hplc column used was a 4.6-mm internal diameter, 250-mm length brownlee analytical perkinelmer® column with an octadecylsilane (c1-8) 5-µm particle size packing. figure 1. structure of the flavonoid quercetin determined in blumea balsamifera (l) d c rp-hplc analysis of quercetin in the extract of sambong 52 plant materials the healthy and mature leaves of sambong were bought from farms located in three provinces of the philippines: nueva ecija, leyte, and cotabato. the drying and comminution of the sambong leaves were done in the processing facility of each area. a certif icate of analysis for the sambong samples bought from leyte and cotabato was provided by philippine institute of traditional and alternative heath care (pitahc). a voucher specimen for the authentication of the sample from nueva ecija has been deposited in the botany division of the national museum. sample preparation crude extraction of 50 grams of powdered dried leaves was done by maceration for 18 hours using 500 ml methanol as solvent, with prior heating to 40°c in a water bath for 6 hours. the extract was collected and f iltered. the f iltrate was dried at 45°c in vacuo using a rotary evaporator. sample extraction and cleanup different solvent volumes and the sonication times for the preparation of the sample solutions were investigated in this study. the optimized conditions of 50 mg/ml sambong solution in methanol, prepared with the aid of sonication for 15 minutes, were used. different volumes of methanol extract to be loaded, composition and volume of eluting solvent, and total volume of eluate to be collected were examined. the optimized conditions, described in the succeeding sentences, were used in the sample cleanup. the spe tube was preconditioned by sequentially passing 6 ml of methanol and 6 ml of 0.5% h 3 po 4 . a 0.5 ml portion of the sambong solution was loaded to the same spe tube and eluted with 4.00 ml of 80:20 methanol:0.5% h 3 po 4 . the eluate was collected. another 0.5 ml portion of the sambong solution was loaded to another separate spe tube and treated in the same manner. the eluates of each of the two spe tubes (about 8 ml) were pooled and diluted to 10.0-ml with methanol. the sambong solution was f iltered through a 0.45-µm nylon membrane f ilter and used in the hplc analysis. the recovery eff iciency of the spe as a cleanup method was determined by adding 124 µl of 1.26 mg/ml quercetin standard solution to a 1.00 ml aliquot of a 50.12 jv toralba and others 53 mg/ml sambong solution. the spiked sambong solution was equally divided and delivered to two spe tubes as described previously, to achieve a f inal concentration of 15.62 µg/ml of quercetin standard in the pooled eluate. the quercetin standard solution and unspiked sambong solution were prepared in the same manner. the solutions were f iltered and injected to the hplc system. quercetin standard solution preparation standard stock solution (1.24 mg/ml) of quercetin was prepared in methanol. aliquots of the stock solution were diluted with methanol to prepare six concentrations of quercetin standard solution (3.72-124.00 µg/ml). solutions were f iltered through a 0.45-µm nylon membrane f ilter and used in the hplc analysis. chromatographic cond itions the conditions that were optimized for the hplc determination of quercetin were wavelength of detection, and the composition and gradient program of the mobile phase. the optimized procedure, described in the succeeding sentences, were used in the analysis of the sambong solution. chromatographic separation was carried out using an octadecylsilane (c18) 5 µm particle size stationary phase packed in a 4.6-mm x 250-mm column maintained at 30°c. the uv detector was operated at 254 nm. the volume of solutions injected to the hplc was 20 µl. the mobile phase was run with a flow rate of 1 ml/min in a gradient elution (table 1). the mobile phase was f iltered through a 0.45-µm nylon membrane f ilter and sonicated prior to use. the chromatographic peak of quercetin in the sambong solution was conf irmed by comparing the retention time with those of the quercetin standard solutions. quantif ication was made according to the linear calibration curves of the quercetin standard solutions. using the data generated by the injection of the six (6) quercetin standard solutions and methanol as blank solution (n=5), an evaluation of the peak areas as a function of the concentration of the solutions was done by calculating the parameters slope, intercept, and coeff icient of correlation. the limit of detection (lod) and limit of quantitation (loq) were computed from the same set of data. the intra-day precision rp-hplc analysis of quercetin in the extract of sambong 54 (n=3) for the peak area and retention time of quercetin were reported as % relative standard deviation (%rsd). the intra-day (n=5) and inter-day (n=3) precision of the methods were also tested using the sambong solution and expressed as %rsd. statistical analysis the quercetin content of the extracts was expressed as mean ± %rsd. a t-test (p< 0.05) was performed to determine the signif icance of the difference between means of the quercetin content of sambong from the different sources. results and discussions optimization of the sample extraction and cleanup effect of solvent and sonication different solvent ratios were tested for the preparation of the sample solution from the sambong extracts prepared from maceration and rotary evaporation (table 2). although the quercetin standard dissolved in solvents containing 80% or 20% methanol, an incomplete dissolution was observed when these solvents were applied to the sambong extract. even with the aid of sonication, the amount of insoluble components did not appreciably decrease (table 3). the sambong solution prepared in pure methanol that was subjected to sonication for 15 minutes was used in this study. *0.5% h 3 po 4 , 0.2%tea: this was prepared by adding 1.75 ml of 85% phosphoric acid to water, swirling thoroughly, then adding 1.38 ml of triethylamine (tea), and diluting to 500.0-ml with water table 1. mobile phase composition and grad ient program for the hplc analysis of solutions t ime (minutes) mobile phase composition elution type 0.0 25:75 methanol:0.5% h 3 po 4 , 0.2%tea* gradient 0.0 – 10.0 45:55 methanol:0.5% h 3 po 4 , 0.2%tea gradient 10.0 – 30.0 55:45 methanol:0.5% h 3 po 4 , 0.2%tea gradient 30.0 – 40.0 100% methanol gradient 40.0 – 50.0 100% methanol isocratic jv toralba and others 55 effect of vol ume load ing and el uting solvent in spe the spe cleanup procedure in this study was modif ied from the work of chen and others (2001) wherein flavonoids and other phenolic compounds in cranberry juice were extracted in a c18 spe car tridge and analyzed by hplc. the aim of the spe in this study was to remove most of the unwanted components including the pigments in the methanol extract while allowing the quercetin to be collected completely. when 0.3 ml and 0.5 ml volumes of 50 mg/ml sambong solution were loaded to separate spe tubes, no peak corresponding to quercetin retention time was observed in the hplc analysis of the eluate. however, at 0.8 ml and 1 ml volumes of the same concentration of sambong solution, the eluates showed a peak corresponding to the retention time of quercetin. the eluates of two spe tubes (about 4 ml each) loaded with 0.5 ml of the sambong solution were pooled to achieve a quantif iable amount in the hplc analysis. solvent description of solution observed at 5-minute time intervals 0 min 5 min 10 min 15 min 20 min 25 min 100% small portion small portion very small complete complete complete methanol undissolved undissolved portion solubility solubility solubility undissolved 80:20 large portion large portion large portion large portion large portion large portion methanol: (lumps) (lumps) (lumps) (lumps) (dispersed) (dispersed) 0.5% h 3 po 4 undissolved undissolved undissolved undissolved undissolved undissolved 50:50 large portion large portion large portion large portion large portion large portion methanol: (lumps) (lumps) (lumps) (lumps) (lumps) (dispersed) 0.5% h 3 po 4 undissolved undissolved undissolved undissolved undissolved undissolved table 3. effect of sonication on the solubil ity of sambong extracts in various solvents table 2. solubil ity of quercetin standard and sambong extract in d ifferent solvents solvent quercetin standard sambong extract 100% methanol soluble soluble 80:20 methanol:0.5% h 3 po 4 soluble; precipitation was large portion remained observed after one week insoluble 50:50 methanol:0.5% h 3 po 4 soluble; precipitation was large portion of remained observed after two days insoluble 20:80 methanol:0.5% h 3 po 4 insoluble large portion of remained insoluble 100% 0.5% h 3 po 4 insoluble insoluble rp-hplc analysis of quercetin in the extract of sambong 56 methanol or acetonitrile can remove quercetin completely from the spe packing. however, the use of pure organic solvents caused the elution of more undesired compounds. the addition of 0.5% h 3 po 4 to methanol increased the polarity of the eluting solvent which enhanced the solubility of the polar quercetin to the eluting solvent. the sambong solution in the spe tube was eluted with 4.00 ml of 80:20 methanol: 0.5% h 3 po 4 . a second 4.00 ml of eluting solvent added to the same spe tube did not result to additional elution of quercetin. recovery eff iciency the eff iciency of extraction of quercetin of the sambong solution treated by spe was demonstrated by spiking a sambong extract solution with quercetin standard solution and allowing this to pass through the spe tube (figure 2). the solutions were analyzed using the optimized hplc method. a mean recovery of 102.41% (± 1.15) for quercetin was achieved in this study. figure 2. overlay of chromatograms of solutions that were treated in the spe tube in the same manner: (a) methanol; (b) quercetin standard solution (15.62 µg/ml); (c) sambong solution spiked with 15.62 µg/ml quercetin standard; and (d) unspiked sambong solution optimization of the chromatographic cond itions effect of wavelength of detection using the uv spectrophotometer, a spectral scan from 200 nm to 350 nm of a quercetin standard solution was done. the three wavelengths that registered jv toralba and others 57 maximum absorbance values were 254 nm, 263 nm, and 274 nm. the wavelength chosen for quercetin determination in this study was 254 nm because quercetin standard exhibited the highest value of area under the curve at this wavelength when tested using the hplc-uv detector (table 4). table 4. peak area of quercetin standard tested using an hplc-uv detector wavelength mean peak area (n=3, ± %rsd) 255 nm 11,342,168.100 ± 2.56 270 nm 7,001,168.213 ± 1.62 360 nm 9,525,722.357 ± 5.27 effect of composition and grad ient program of the mobile phase the hplc conditions used in this study were modif ied from the work of fazilutan and others (2005) where f ive major flavonoids, including quercetin, were quantitatively determined in the methanol extract of sambong collected from the different areas in malaysia. their study used a c18 (250x 4.6-mm, 5 ìm particle size) column as the stationary phase and a mobile phase consisting of 50:50 methanol: 0.5% h 3 po 4 in an isocratic mode and delivered at 0.9 ml/min. the flavonoids were detected at 285 nm. quercetin, a polar molecule, can be eluted from a c18 stationary phase by using a polar mobile phase. this study explored gradient elution of various proportions of methanol and 0.5% h 3 po 4 to increase the resolution of quercetin from adjacent peaks and to improve the peak shape of the compound. the flow rate for all tests was held constant at 1.00 ml/min. eventually, a gradient that starts with 25:75 methanol: 0.5% h 3 po 4 and the methanol content increasing to 100% in a 50-minute program yielded a satisfactory separation of quercetin (figure 3a). the addition of triethylamine was also investigated to improve the peak shape of quercetin in the sambong sample. the concentration of triethylamine in the 25:75 methanol: 0.5% h 3 po 4 mobile phase was varied from 0.1% to 1%. a 0.2% concentration of triethylamine resulted to an acceptable resolution of quercetin from adjacent peaks, as well as an improved peak shape (figure 3b). triethylamine, at a suitable concentration, acts as an ion pairing reagent for the stationary phase, which reduced the tailing of quercetin. when the concentration of triethylamine was increased, peak breaking and band broadening resulted (figure 3c). rp-hplc analysis of quercetin in the extract of sambong 58 figure 3. chromatograms of sambong solutions run in different mobile phase compositions. the mobile phase composition was changing to 100% methanol in a 50-minute gradient program starting with the following solvent combinations: (a) 25:75 methanol:0.5% h 3 po 4 ; (b) 25:75 methanol:0.5% h 3 po 4 with 0.2% triethylamine; and (c) 25:75 methanol:0.5% h 3 po 4 with 0.5% triethylamine. the following hplc conditions were then applied in the analysis of quercetin in the sambong solution: c18,5 µm particle size stationary packed in a 4.6-mm x 250-mm column and maintained at 30°c, 254-nm wavelength of detection, 1.00 ml/min mobile phase flow rate, and a mobile phase and gradient condition as outlined in table 1. linearity, limit of detection and limit of quantitation, precision a series of six quercetin standard solutions (3.72-124.00 µg/ml) was tested to determine the linearity between the concentration and peak areas. a high correlation coeff icient of 0.9989 and regression equation of y=102789x-159435 was jv toralba and others 59 generated from the hplc analysis of the quercetin standard. the chromatograms of the six quercetin standard solutions are provided in figure 4. the limits of detection and quantitation were evaluated on the basis of a signal-to-noise ratio of 3 and 10, respectively. the detection limit was determined as 0.68 ng/ml, while limit of quantitation was at 2.28 ng/ml. this indicates that the hplc method is suff iciently sensitive for the determination of quercetin in sambong. figure 4. overlay of chromatograms of the quercetin standard solutions analyzed using the optimized hplc method: (a) 124.00 µg/ml; (b) 62.00 µg/ml; (c) 31.00 µg/ml; (d) 15.50 µg/ml; (e) 7.44 µg/ml; and (f) 3.72 µg/ml rp-hplc analysis of quercetin in the extract of sambong 60 the %rsd of the peak areas and retention times of the quercetin standards are both quite low which indicated that precision is good (table 5). the precision was also measured using the peak areas of the quercetin in the sambong solutions. both the intra-day (%rsd = 1.52%, n=5) and inter-day (%rsd = 1.73%, n=3) precision studies also showed that the method is both repeatable and reproducible. quantitation of quercetin in sambong leaves the optimized spe sample preparation and cleanup and hplc method were applied to the sambong leaves collected from leyte, cotabato, and nueva ecija. the corresponding chromatograms of the sambong samples are shown in figure 5. the summary of results on the quantitation is presented in table 6. the quercetin content ranged from 0.135 to 0.294 mg per gram of powdered dried leaves. at the time of the writing of this article, these are the f irst reported values of quercetin in sambong samples grown in the philippines. these results are close to the values reported in the study of fazilutan and others (2005) where quercetin was reported to be present in the range of 0.021 to 0.958 mg per gram of dried b. balsamifera leaves cultivated in the different regions of malaysia. the quercetin content of sambong from cotabato differed signif icantly (t-test, p<0.05) from that found in leaves from nueva ecija and leyte. the quercetin content of samples from leyte and nueva ecija did not differ signif icantly from each other. the variation in the quantity of quercetin may be due to various factors such as cultivation conditions (soil, temperature, moisture) and agricultural practices (use of fertilizers and pesticides). table 5. %rsd of the retention time and peak area response of quercetin standard solutions 124.00 0.07 0.89 62.00 0.13 0.16 31.00 0.65 1.02 15.50 0.88 1.30 7.44 0.64 0.99 3.72 0.21 1.48 quercetin standard concentration (µg/ml) intra-day %rsd for retention t ime intra-day %rsd for peak area jv toralba and others 61 table 6. quercetin retention time, peak area, and content as determined by hplc analysis of the sambong leaves from leyte, cotabato, and nueva ecija leyte 20.67 ± 0.65 1,345,645.36 ± 1.27 0.2940 cotabato 20.59 ± 0.82 698,866.84 ± 1.05 0.1350 nueva ecija 20.57 ± 0.80 1,334,535.34 ± 1.24 0.2337 source retention t ime (minutes, n=3, mean ±%rsd) peak area (au, n=3, mean ±%rsd quercetin in mg per gram powdered leaves figure 5. overlay of chromatograms of the sambong from (a) leyte, (b) cotabato, and (c) nueva ecija analyzed using the optimized hplc method acknowledgments the main author would like to thank the college of pharmacy. appreciation is also extended to dr. noel s. quiming for generously providing the quercetin standard used in this study. rp-hplc analysis of quercetin in the extract of sambong 62 references ali dms, wong kc, lim pk. 2005. flavonoids from blumea balsamifera. fitoterapia. 76:128-30. andres-lacueva c, monagas m, khan n, izquierdo-pulido m, urpi-sarda m, permanyer j, lamuela-raventós. 2008. flavanol and flavonol contents of cocoa powder products: influence of the manufacturing process. journal of agricultural and food chemistry. 56:3111-3117. arroyo mh, fajardo rv, agulto mb. 1990. comparison of the intraocular pressure lowering effects of acetazolamide and blumea balsamifera. philippine journal of ophthalmology. 19(3):101-04. barua nc, sharma rp. 1992. 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[doh-nfc] department of health–national formulary committee. 2005. philippine n a t i o n a l d r u g fo r m u l a r y e s s e n t i a l d r u g s l i s t . vo l . 1 , 6 t h ed . m a n i l a : t h e n a t i o n a l formulary committee national drug policy pharma 50 project management unit (pmu 50) doh. fazilutan n, zhari i, nornisah m. 2003. antioxidant activity of extracts from the leaves of blumea balsamifera dc and their major flavonoids with the α-carotene-linoleic acid model system. malaysian journal of pharmaceutical sciences. 1:51-64. fazilutan n, zhari i, nornisah m, mas rosemal hmh. 2004. free radical-scavenging activity of organic extracts and of pure flavonoids of blumea balsamifera dc leaves. food chemistry. 88:243-252. fazilutan n, zhari i, sundram k, mohamed n. 2005. rp-hplc method for the quantitative analysis of naturally occurring flavonoids in leaves of blumea balsamifera dc. journal of chromatographic science. 43(8):416–20. h e r t o g m g l , h o l l m a n p c h , a n d v a n d e p u t t e b . 1 9 9 3 . c o n t e n t o f p o t e n t i a l l y a n t i c a r c i n o g e n i c f l a v o n o i d s o f t e a i n f u s i o n s , w i n e s , a n d f r u i t j u i c e s . j o u r n a l o f agricultural and food chemistry. 41:1242-1246. l i m s y l i a n c o cy, b l a n c o f r b , l i m c m . 1 9 8 7. m u t a g e n i c i t y, c l a s t o g e n i c i t y a n d a n t i m u t a g e n i c i t y of m ed i c i n a l p l a n t t a b l e t s p r o d u ced by t h e n s ta p i l o t p l a n t i i . sambong tablets. philippine journal of science. 116(1):13-18. jv toralba and others 63 lin jk, weng ms. 2006. flavonoids as nutraceuticals. in: grotewold e, editor. the science of flavonoids. new york: springer science, inc. p. 213-238. quisumbing e. 1978. medicinal plants of the philippines. quezon city: katha publishing co. rico f. 1992. sambong (blumea balsamifera): its effect on calcium stone. philippine journal of urology. 2(1):9-13. zu y, li c, fu y, zhao c. 2006. simultaneous determination of catechin, rutin, quercetin, kaempferol and isorhamnetin in the extract of sea buckthorn (hippophae rhamnoides l.) leaves by rp-hplc with dad. journal of pharmaceutical and biomedical analysis. 41(3):714-19. _______________ joanna v. toralba <jvtoralba@post.upm.edu.ph> is an assistant professor of the department of pharmaceutical chemistry, college of pharmacy, university of the philippines manila, where she is currently teaching and advising undergraduate research on pharmaceutical analysis and methods development. she graduated from the university of the philippines manila with degrees in bs pharmacy and ms pharmacy (pharmaceutical chemistry). noel s. quiming is an associate professor of the department of physical sciences and mathematics, college of arts and sciences, university of the philippines manila. he has published articles on chromatography and its application to analysis of natural products in international peer reviewed journals. he received his doctor of engineering degree from toyohashi university of technology in japan and ms chemistry degree from university of the philippines diliman. jocelyn sb palacpac is an associate professor of the department of industrial pharmacy, college of pharmacy, university of the philippines manila, where she served as chair of the department and as dean of the college. she is currently the assistant director of the institute of herbal medicine, national institutes of health, university of the philippines manila. she is the project leader of the research project on the formulation of dosage forms of philippine medicinal plant constituents. 27 cytotoxic and genotoxic potential of the money tree (pachira aquatica) stem and leaf extracts jordan ferdin a. halili* institute of biology college of science university of the philippines diliman jane nicole n. catacutan melissa c. gaudario maries ann r. silvestre mary lorraine f. lorido juan r. liwag memorial high school gapan city rich milton r. dulay department of biology central luzon state university abstract there is a global demand for the discovery of anticancer drugs. this study was designed as an anticancer prescreening to evaluate the cytotoxic and genotoxic potential of stem and leaf extracts of money tree, pachira aquatica, one of the plant species with limited scientific studies. bioactivity of p. aquatica extracts was initially assessed using brine shrimp lethality assay (bsla). plant and animal models of cell proliferation were used to investigate cytostatic and cytocidal effects. onion root tip chromosomal aberration assay (ortcaa) was conducted to examine antimitotic and genotoxic activities. embryotoxicity and teratogenicity were determined using zebrafish developmental toxicity assay (zdta). using bsla, the p. aquatica leaf extract had an estimated lc 50 value of 11.87 µg/ml, which indicated that it was bioactive and toxic. ortcaa revealed that all stem extract concentrations reduced mitotic indices, which were comparable to 5 mg/l of maleic hydrazide (positive control) while all leaf extract concentrations induced mitotic block at prophase/metaphase boundary. prominent chromosomal aberrations observed were bridges and stickiness suggesting genotoxicity of extracts. zdta showed 100% embryonic death at 20, 100 and 200 µg/ml of both extracts after 12-hour post-treatment application. moreover, cytological abnormalities in onion cells and early zebrafish embryonic death implied the activation of apoptosis. based on the results, money tree extracts have promising cytostatic (inhibition of growth, division and differentiation) and cytocidal (lethal) effects, which are important qualities of an anticancer drug. the money tree is therefore a potential source of a nature-based chemotherapeutic compound. keywords: anticancer prescreening, brine shrimp, cytotoxic, genotoxic, pachira aquatica, zebrafish * corresponding author science diliman (july-december 2019) 31:2, 27-48 cytotoxic and genotoxic potential of the money tree (pachira aquatica) 28 introduction cancer is among the leading causes of mortality globally. according to the american cancer society (2015), one out of seven deaths worldwide is caused by cancer. this global health burden accounted for 8.2 million deaths in 2012 (world health organization 2015). in the philippines, 98,200 individuals are affected by cancer each year and more than half of these patients died in 2014 (asian cancer institute 2015). despite the technological advancements in the development of treatments for cancer (us national cancer institute 2007), there is no existing ideal anticancer mechanism until now that can kill only cancer cells and spare healthy cells (amara 2013). in particular, chemically-synthesized drugs used in chemotherapies result in a wide range of detrimental effects and/or even total destruction of non-targeted sites in the human body (seideil et al. 2012). nowadays, most anticancer drugs target one or more stages of the cell cycle and the structure and function of deoxyribonucleic acid (dna). high-level damages must be inflicted on the dna to increase the error recognition of the “relaxed” cell cycle checkpoints (mostly in mitotic and synthesis phases) of cancer cells to produce irreparable damages to induce cell death (cheung et al. 2013). thus, the search for chemotherapeutic agents with novel modes of cytotoxic action has become more important than ever (american cancer society 2015; coseri 2009). through the years, plants and their secondary metabolites have been abundant sources of anticancer agents since these nature-based compounds exhibit a wide range of cytotoxic activities and promising selectivity (cragg and newman 2005; newman 2008). in this study, we used the money tree, pachira aquatica, which is considered an understudied plant species since it has limited ethno-pharmacological records (lawal et al. 2015; shibatani et al. 1999). the study aimed to investigate the cytotoxic and genotoxic properties of p. aquatica. specifically, this study evaluated the cytostatic (i.e., the ability to stop cell growth, division and differentiation) and cytocidal (lethal) effects of p. aquatica extracts on onion root tips and zebrafish embryos as plant and animal models of cell proliferation. the study contributes baseline data important to developing naturebased chemotherapeutic drugs and informs the general public regarding the possible risks and adverse effects of using the money plant as traditional/herbal medicine. j.f.a. halili et al. 29 materials and methods experimental procedures for the brine shrimp lethality assay and onion root tip chromosomal aberration assay were done in the institute of biology, university of the philippines, diliman, quezon city. zebrafish developmental toxicity assay (iacuc paf-ib-2016-16) was conducted at the department of biological sciences, central luzon state university, science city of muñoz, nuevaecija. preparation of crude ethanol extracts and phytochemical analysis the p. aquatica stems and leaves were used since this is a benchmark study on the evaluation of its phytocompounds and biological activities. the p. aquatica samples were collected from san nicolas, gapan city, nueva ecija and were identified and authenticated (authentication number 15-7-018) as pachira aquatica aublet of malvaceae family by mr. danilo n. tandang, museum researcher ii of the botany division, national museum in ermita, manila. five kilograms each of fresh stems and leaves were used for the preparation of crude ethanol extracts, which was done at the industrial technology development institute (itdi), department of science and technology (dost), maimpis, city of san fernando, pampanga. the stems and leaves were washed and air-dried. the plant samples were then powdered through a blender and were soaked separately in 95% ethanol (1:5 w/v) at room temperature for 72 hours. the extracts were filtered and evaporated under reduced pressure using a rotary evaporator at 55°c (souza et al. 2014). the extracts from rotary evaporation were air-dried in separate petri dishes for 24 hours, producing a solid precipitate. phytochemical analyses of the p. aquatica stem (pa-se) and leaf (pa-le) crude ethanol extracts were also done at itdi-dost. preparation of treatments for the brine shrimp lethality assay, various dilutions of pa-se and pa-le were prepared following the modified dilution procedure of sarah et al. (2017), giving the final concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 µg/ml. negative control (artificial sea water with 1% dmso) and positive control (0.3 mg/l of potassium dichromate) were also prepared in six replicates in the two trials of this bioassay. for the onion root tip chromosomal aberration assay, a total of four experimental treatments (100, 250, 500 and 1000 µg/ml) were prepared, each in three replicates. positive (5 mg/l of maleic hydrazide) and negative (1% dmso in distilled water) controls were also included. cytotoxic and genotoxic potential of the money tree (pachira aquatica) 30 for the zebrafish developmental toxicity assay, the final concentrations were 2, 10, 20, 100 and 200 µg/ml. embryo medium (294 mg cacl 2 , 123.25 mg mgso 4 , 64.75 mg nahco 3 , and 5.75 mg kcl dissolved in 1 l of distilled water) was used as the negative control. a total of six treatments were prepared, each in three replicates. brine shrimp lethality assay (bsla) three milligrams of brine shrimp (artemia salina) eggs were hatched in a beaker with 400 ml of artificial sea water with 3.8% sodium chloride solution under constant aeration and illumination of fluorescent lamp for 48 hours. after hatching, 10-15 brine shrimps were transferred into each well of a 24well plate containing different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/ml) of pa-se and pa-le. potassium dichromate and 1% dmso in artificial seawater served as the positive and negative controls, respectively. after 24 hours, the dead a. salina were counted manually. the percentage mortality (%m) in each well was calculated by dividing the number of dead brine shrimps by the total number of brine shrimps and multiplying by 100. abbott’s mortality formula was used to correct the percentage mortality of brine shrimps to ensure the bioactivity of extracts (meyer et al. 1982). the abbot’s mortality formula is as follows: corrected % mortality = y – x x × 100 (1) where x = % mortality of brine shrimps in control y = % mortality of brine shrimps in treatments the median lethal concentration (lc 50 ) was computed using the generated equation of the line in aprobit analysis done in microsoft excel 2013 (microsoft corp. 2013). median lethal concentration with a value less than 1000 µg/ml was considered toxic while lc 50 value greater than 1000 µg/ml was non-toxic (meyer et al.1982). onion root tip chromosomal aberration assay (ortcaa) forty onion bulbs (allium cepa) of common variety and equal sizes were obtained from a local market in muñoz, quezon city. a scalpel was used in peeling the outer scales of the bulbs and old roots to expose the root primordia. eighteen out of forty onions were selected randomly after examining for uniformity in size and condition. each extract was prepared in different concentrations at 100, 250, 500 and 1000 µg/ml including the positive (5 mg/l maleic hydrazide) and negative controls (1% dmso in distilled water). selected onions were transferred into bottles containing the treatments with three replicates each for 48 hours. j.f.a. halili et al. 31 onion root tips were then cut and fixed in farmer’s fluid (one part glacial acetic acid and three parts ethanol) in plastic micro-tubes and placed in the refrigerator after 24 hours. root tips were then cut to 1-2 mm, hydrolyzed with a drop of 1n hydrochloric acid (hcl) for three minutes, dried with tissue paper and macerated using a scalpel. aceto-orcein (2%) was used to stain the onion cells for three minutes, then the glass slide was passed over the flame of an alcohol lamp twice, ensuring that the stain did not boil. a cover slip was used to squash the cells and then excess stain was removed. the preparation was then sealed with a natural nail polish. the slides were examined using a zeiss compound light microscope under 10x objective then switched to 40x objective to take closer observations of the morphological structure and mitotic phase of cells. this procedure with some modifications was used following el-shahaby et al. (2003). the mitotic index, mitotic phase indices, and percentage of aberrant cells were calculated using the following formulas (fiskesjo 1985): mitotic index = total number of dividing cells total number of cells counted × 100 (2) mitotic phase index = total number of dividing cells at a specific phase total number of dividing cells × 100 (3) % aberrant cells = total number of aberrant cells total number of cells counted × 100 (4) in addition, chromosomal aberrations such as lagging, loss and bridging were identified. investigations of other structural abnormalities in the nucleus and cytoplasm were also done. zebrafish developmental toxicity assay (zdta) dechlorinated water with oxygen saturation was usedin a glass aquarium for the spawning of zebrafish (danio rerio) at 1:2 ratio between mature females to males. adult (10-12 months of age) zebrafish were placed in a plastic mesh and were submerged in the aquarium. the aquarium was covered with black plastic bag for 12 hours and then well-lighted (500-540 lux) for another 12 hours to trigger spawning. fertilization of the eggs typically occurs within 30 minutes after light exposure. the fertilized eggs were siphoned out of the aquarium using a hose and aspirator bulb to isolate it from the water. embryos were rinsed thrice using distilled cytotoxic and genotoxic potential of the money tree (pachira aquatica) 32 water and were placed in a watch glass with an embryo medium and observed under the compound microscope to select embryos at blastula stage (50% epiboly) (halili and quilang 2011). four selected embryos were transferred to 24-well plates, each with 3 ml of concentration of pa-se and pa-le (2, 10, 20, 100, 200 µg/ml and embryo water as negative control), and incubated at 26°c ± 1°c. observations were made after 12, 24, 36 and 48 hours of incubation using a compound microscope. hatchability, growth retardation, malformations and mortality rates were observed and recorded (nagel 2002). data analysis lc 50 values and 95% confidence intervals for bsla were determined using the probit analysis method described by finney (1952). statistical differences between mitotic index, mitotic phase index, and % aberrant cells for ortcaa and hatchability, growth retardation, malformations, and mortality rates for zdta were determined using one-way analysis of variance (anova) followed by a post-hoc analysis (duncan multiple range test) in spss version 22.0 (ibm corp, 2013). differences with p<0.05 between experimental and control groups were considered. waste disposal chemicals and solvents used were disposed in appropriate organic and inorganic waste containers. solid wastes were collected in a waste container and disposed according to the laboratory health, safety and environment management system of up diliman. brine shrimps exposed to different treatments were treated with 10% bleach solution for 24 hours before their disposal (national nanotechnology infrastructure network 2013). zebrafish embryos were subjected to euthanasia by means of submersion in ice water for 20 minutes prior to disposal (national institutes of health 2013). results phytochemical analysis a qualitative phytochemical analysis of p. aquatica stem and leaf extract (pa-se and pa-le) secondary metabolites exhibited the presence of the same compounds, namely, alkaloids, glycosides, and tannins (condensed), which were already recorded to have promising anticancer and antitumor activities (fattorusso and taglialatela-scafati 2007; nandakumar et al. 2008; menger et al. 2012). table 1 shows the result of screening and methods used to determine the presence of phytocompounds. j.f.a. halili et al. 33 table 1. phytochemicals present in p. aquatica extracts. test parameter test method leaf stem alkaloids mayer/meyer test + + anthraquinones borntrager test glycosides keller-killiani test + + flavonoids bate-smith and metcalf test tannins ferric chloride test + + saponins froth test legend: + present; absent brine shrimp lethality assay (bsla) bioactivity was determined by the lethality of brine shrimp exposed to the extracts for 24 hours. pa-se was most active against a. salina nauplii at 100 µg/ml (81.88%) and lowest at 25 µg/ml (75.40%) while pa-le recorded its maximum nauplii mortality at 25 µg/ml (92.44%) and lowest at 3.125 µg/ml (83.20%). the ability of the bioactive compounds of pa-le to cause mortality in brine shrimp nauplii was exhibited in a dose-dependent manner with the estimated median lethal concentration (lc 50 ) of 11.87 µg/ml (95% ci: 0.11 14.29) (figure 1). on the other hand, pa-se did not show dose-dependence with an lc 50 > 100 µg/ml. figure 1. fitted regression line and equation to determine the lethal concentration 50 (lc 50 ) of p. aquatica leaf (pa-le) crude ethanol extract in the percentage mortality of brine shrimp nauplii. data are the probit transformed values of the corrected % mortalities against the log of the concentration of treatments. mean percent mortality was determined from six replicates for each concentration in each of two trials of brine shrimp lethality assay (bsla). (regression line: slope= 0.04485, b=4.5181, r2=0.9254). cytotoxic and genotoxic potential of the money tree (pachira aquatica) 34 onion root tip chromosomal aberration assay (ortcaa) figure 2a shows the rate of mitosis of a. cepa root tip cells after 24 hours. the mitotic indices of root meristems treated with 100 µg/ml, 250 µg/ml, 500 µg/ml and 1000 µg/ml stem extract were 7.47%, 8.00%, 8.13%, and 9.07%, respectively, which are not significantly different. the mitotic activity of onion root tip cells a 100 mg/ml 250 mg/ml 500 mg/ml 1000 mg/ml 5 mg/ml maleic b 100 mg/ml 250 mg/ml 500 mg/ml 1000 mg/ml 5 mg/ml maleic figure 2. the percentage of dividing onion cells after exposure to p. aquatica stem (pa-se) crude ethanol extract(panel a) and p. aquatica leaf (pa-le) crude ethanol extract (panel b). mitotic index was computed by dividing the total number of dividing cells by the total number of cells counted x 100. for each treatment, data are means of the mitotic indices of three replicates and, for each replicate, 1000 onion cells were counted. treatments having the same lowercase letter data labels are not significantly different. statistical differences were determined by one-way anova (p<0.05) followed by duncan’s multiple range test. j.f.a. halili et al. 35 inhibited by the extract was statistically comparable to the positive control (6.20%) and significantly lower compared to the negative control (21.87%). the pre-prophase inhibition of 1000 µg/ml leaf extract (8.47%) was comparable with the positive control (6.20%), and all pa-le extract levels were noted with significantly lower mitotic indices than the negative control (21.87%) (figure 2b). moreover, the number of onion cells entering mitosis decreased as concentration increased, exhibiting the concentration dependence of the antimitotic action of pa-le. table 2. prophase, metaphase, anaphase and telophase indices in onion root cells exposed to different concentrations of p. aquatica stem crude ethanol extract (pa-se), negative control (1% dmso), and positive control (5 mg/l maleic hydrazide). mitotic phase indices  treatments prophase metaphase anaphase telophase 1% dmso solution 36.59b 2.44b 14.63c 46.34c 100 µg/ml 19.64a 0.00a 1.79a 78.57e 250 µg/ml 20.00a 3.33bc 6.67b 70.00d 500 µg/ml 87.70d 2.46b 2.46a 7.38b 1000 µg/ml 89.71d 2.94b 2.94ab 4.41a 5 mg/l maleic hydrazide 80.65c 5.38c 5.38b 8.60b for each column, means followed by the same lowercase bold letter are not significantly different. statistically significant differences were determined by one-way anova (p<0.05) followed by duncan’s multiple range test. mitotic phase index was computed by dividing the total number of cells at a specific stage by the total number of dividing cells x 100. data are means of the mitotic phase indices of three replicates per treatment and, for each replicate, 1000 onion cells were counted. table 2 shows that there were higher percentages of cells in telophase for root meristem cells treated with 100 µg/ml (78.57%) and 250 µg/ml (70%) pa-se. prophase accumulation was observed in allium cepa cells exposed to 500 µg/ml (87.70%) and 1000 µg/ml (89.71%) extract, which were even higher than the positive control. there was also a drastic decrease in the percentage of actively proliferating cells with 100, 500 and 1000 µg/ml pa-se at metaphase and anaphase, which only ranges from 0.0-3.3%. prominent mitotic block at the prophase/metaphase boundary was seen in onion cells treated with pa-le. more than 45% of the dividing cells were accumulated in this stage (table 3). cytotoxic and genotoxic potential of the money tree (pachira aquatica) 36 table 3. prophase, metaphase, anaphase and telophase indices in onion root cells exposed to different concentrations of p. aquatica leaf crude ethanol extract (pa-le), negative control (1% dmso), and positive control (5 mg/l maleic hydrazide). mitotic phase indices  treatments prophase metaphase anaphase telophase 1% dmso solution 36.59a 2.44a 14.63c 46.34d 100 µg/ml 71.43e 9.52c 9.52b 9.52a 250 µg/ml 64.94d 11.69cd 12.99c 10.39a 500 µg/ml 62.25c 7.28cd 7.28ab 23.18c 1000 µg/ml 47.24b 15.75e 23.62d 13.39b 5 mg/l maleic hydrazide 80.65f 5.38b 5.38a 8.60a for each column, means followed by the same lowercase bold letter are not significantly different. statistically significant differences were determined by one-way anova (p<0.05) followed by duncan’s multiple range test. mitotic phase index was computed by dividing the total number of cells at a specific stage by the total number of dividing cells x 100. data are means of the mitotic phase indices of three replicates per treatment and, for each replicate, 1000 onion cells were counted. the percentage of chromosomal aberrations in allium cepa root tip cells exposed tothe lowest to highest concentration (0.63-3.17%) of pa-se were statistically similar to maleic hydrazide (1.3%), which suggests the genotoxic action of the phytocompounds present in the extract. figure 3a further reveals that the effects of 100 µg/ml (4.20%), 500 µg/ml (4.93%) and 1000 µg/ml (6.20%) in inducing cytological anomalies were comparable with the positive control (6.17%). moreover, the over-all occurrence of abnormalities in all extract levels (4.97-6.83%) has no significant difference with maleic hydrazide (7.47%), which further shows the potent cytotoxic mechanisms of mt extracts even at its lowest concentration. a 100 mg/ml 250 mg/ml 500 mg/ml 1000 mg/ml 5 mg/ml maleic j.f.a. halili et al. 37 b 100 mg/ml 250 mg/ml 500 mg/ml 1000 mg/ml 5 mg/ml maleic figure 3. the percentage of cytological and chromosomal damages in onion cells caused by p. aquatica stem (pa-se) crude ethanol extract (panel a) and p. aquatica leaf (pa-le) crude ethanol extract (panel b). percent abnormality was computed by dividing the total number of aberrant cells by the total number of dividing cells x 100. for each treatment, data are means of % abnormalities for chromosomal, cytological and over-all (combined chromosomal and cytological abnormalities) of three replicatesand, for each replicate, 1000 onion cells were counted. means having the same lowercase letter data labels are not significantly different. statistical differences were determined by one-way anova (p<0.05) followed by duncan’s multiple range test. means with asterisk (*) as data labels are significantly different from the positive control. root meristems exposed to 100 µg/ml (0.93%) and 250 µg/ml (0.4%) of pa-le were also statistically similar to the effects of maleic hydrazide. figure 3b shows that the cytological and over-all abnormalities induced by the 100 (3.27%) and 1000 µg/ml (11.27%) pa-le were comparable with the positive control. the results also revealed that the cell anomalies induced by 500 µg/ml pa-le were statistically higher than the positive control. morphological irregularities in cytoplasm (condensation and shrinkage) and nucleus (condensation and margination) were also seen (figure 4). cytotoxic and genotoxic potential of the money tree (pachira aquatica) 38 figure 4. the mitotic cell aberrations triggered by p. aquatica stem (pa-se) crude ethanol extract and p. aquatica leaf (pa-le) crude ethanol extract.mitotic cells at 40x showing aberrant cells (indicated by arrows) observed in allium cepa root tips exposed to p. aquatica stem and leaf extracts: astrap nucleus; bgiant cell, disintegrated cytoplasm; cchromosomal bridge; dnuclear lesions; esticky metaphase; fgiant nucleus; gghost cell, sticky anaphase; hnuclear erosion. zebrafish developmental toxicity assay (zdta) results showed that the mortality rate triggered by pa-se was dependent on dose and time of exposure (figure 5a). the pa-le was noted with 100% mortality rate in 20, 100 and 200 µg/ml concentrations at 12 hour post-treatment application (hpta) (figure 5b). similarly, the 10 µg/ml pa-le led to the death of all embryos after 36 hours. on the other hand, over-all growth retardation was seen in all zebrafish embryos treated with 2 and 10 µg/mlpa-le that survived after 24 hours. moreover, a 12-hour hatching delay in d. rerio embryos treated with 2 µg/mlpa-le was recorded compared to normal hatching of embryos in negative control at 48 hpta. a 2 mg/ml 10 mg/ml 20 mg/ml 100 mg/ml 100 mg/ml j.f.a. halili et al. 39 b 2 mg/ml 10 mg/ml 20 mg/ml 100 mg/ml 100 mg/ml figure 5. the zebrafish embryonic deaths triggered by p. aquatica stem (pa-se) crude ethanol extract (panel a) and p. aquatica leaf (pa-le) crude ethanol extract (panel b). data are means of percentage mortalities of the three replicates of each concentration (hpta – hour posttreatment application). means with asterisk (*) as data labels are significantly different from the negative control. morphological abnormalities caused by exposure to 2 µg/ml pa-le at 60 hpta include bent tail, bent spine, yolk sac edema, pericardial edema and ocular edema. embryos exposed to 2 µg/ml pa-se after 36 and 48 hours showed coagulation, unformed tail, unformed head, detached tail, yolk sac edema and yolk sac not depleted (figure 6). figure 6. phenotypic changes of zebrafish embryos treated with p. aquatica stem (pa-se) crude ethanol extract (bottom row) and p. aquatica leaf (pa-le) crude ethanol extract (top row) at 36-60 hpta. abbreviations: bsbent spine (scoliosis); btbent tail; cocoagulation; dtdeformed tail; oeocular edema; ppigmentation; pepericardial edema; ufh, unformed head; uftunformed tail; yndyolk sac not depleted; and yseyolk sac edema cytotoxic and genotoxic potential of the money tree (pachira aquatica) 40 discussion lethality of p. aquatica on brine shrimp brine shrimp (artemia salina) lethality assay was used to examine the bioactivity of the money tree phytocompounds (lieberman 1999). this arthropod assay is internationally recognized as a prescreening for antitumor activity (meyer et al. 1982) and for its positive correlation with human cancer line tests (mclaughlin and rogers 1998; carballo et al. 2002; naidu et al. 2014). of all animal models, brine shrimps are known to be sensitive to the biological activities of phytocompounds, thus, the gradual increase in the percentage mortality and its dose-independent activity could be due to the antagonistic relationship among the three phytochemicals (alkaloids, glycosides and condensed tannins) present in both extracts, which resulted in suboptimal bioactivity (milugo et al. 2013). in a cytotoxic analysis by pourfraidon and sharma (2009) using 19 plants with prominent anticancer properties, 42% of the extracts exhibited 61-100% brine shrimp inhibition, 32% of the investigated plants killed 31-60% of the nauplii while 26% had weaker cytotoxic activity resulting in 0-30% a. salina lethality after 24 hours. the pa-le therefore has a potent toxic behavior since it resulted in 83-92% mortality against brine shrimps in a dose-dependent manner. according to meyer’s toxicity index (1982), lc50<1000 µg/ml is considered toxic while lc50>1000 µg/ml is non-toxic. moreover, substances with lc50<200 µg/ml in bsla could be subjected to further cell culture to detect its antitumor activity (romeo 2012). meyer et al. (1982) investigated 12 physiologically active components using bsla. three of these, podophyllotoxin, barberine chloride and strychnine sulfate, resulted in lc 50 values less than 100 µg/ml while other compounds have lc 50 values greater than 150 µg/ml. moreover, bagya et al. (2011) tested vincristine, a nature-based chemotherapeutic drug, using brine shrimp assay and recorded a median lethal concentration value of 380 µg/ml. in this study, the computed lc 50 value for p. aquatica leaf extracts was 11.87 µg/ml, which suggests that the plant could be an abundant source of bioactive compounds and a candidate for further confirmatory testing of cytotoxic mechanisms that could be exhibited in cell growth, function, division and differentiation (goodman et al. 1980) and the ability to activate its self-destruction machineries or cytocidal effect (foster 1991). j.f.a. halili et al. 41 cytostatic and genotoxic effects of p. aquatica on onion root cells onion (allium cepa) root tip chromosomal aberration assay is considered a reliable system since the patterns of division of onion cells, cancer cells and human somatic cells are similar. ortcaa was widely considered a prescreening for anticancer/ antimitotic compounds (william and omoh 1996) as plant cells were noted to be 1000 times more resistant to colchicine, a carcinogenic substance and well-known mitotic inhibitor (kihlman 1996). furthermore, this assay was proven to be practical since one rootlet can show a range of dna damage (tedesco and laughinghouse iv 2012). the mitotic index is an indicator of cell proliferation (gadano et al. 2002) wherein low index could be due to the inhibition of dna/protein synthesis (chandra et al. 2005), blockage of the g2 phase of cell cycle, which causes delay in the cell cycle kinetics (rojas et al. 1993), and mitotic arrest (kumar et al. 2006), which could eventually lead to cell death (el-ghamery et al. 2000). the results suggest that pa-se in varying concentrations have the same effects in inducing the accumulation of interphase cells with maleic hydrazide, which is a known plant growth depressant (gubb and mactavish 2002). extracts of common medicinal plants, such as azadirachta indica, morinda lucida, cymbopogon citratus and carica papaya, that were tested on onion root cells have 10-19% mitotic indices on their highest concentrations (akinboro and bakare 2007). vincristine, a plant-derived anticancer drug was noted with 0-3% mitotic indices. in this present study, the mi of the onion meristem cells treated with the lowest concentration (100 µg/ml) of pa-se and pa-le were 7% and 17%, respectively, which signifies inhibition of the mitotic process and delay in the cell cycle kinetics (udo et al. 2015). specific antimitotic action of money tree extracts was further revealed in the results of mitotic phase indices. prominent mitotic block at the prophase/metaphase boundary could be attributed to the action of the pa-le to cause interruptions in nuclear membrane disintegration due to the inhibitory effects carried from the earlier phases of cell cycle, (wilson 1965) delay or blockage in the formation of spindle fiber (ilbas et al. 2011) and prophase arrest (njoku et al. 2015). similar effects of prophase accumulation in the dividing cells were also documented in the studies using telfairia occidentalis (udo et al. 2015), aloe vera (ilbas et al. 2011) and a. indica (akaneme and amaefule 2012), which is an indicator of anti-spindular effects at the early stage of mitosis. results suggest the promising antimitotic cytotoxic and genotoxic potential of the money tree (pachira aquatica) 42 activity induced by both extracts and its capability to build up cells at prophase, which is similar to the antimitotic action of taxol and taxotere, two clinically used plant-derived anticancer drugs (agnieszka et al. 2008). to investigate the ability of the money tree phytocompounds to interact with dna and cause chromosomal damages, a microscopic analysis of onion cells was done. morphological (cytological) abnormalities, particularly in the nucleus and cytoplasm, were also examined to check for apoptotic markers.in the study of atoyebi et al. (2015) on the genotoxicity of luffa cylindrica, nymphaea lotus and spondias mombin extracts, results showed the frequency of cellular aberrations that range from 0.8-3.5%. percentage of abnormalities induced by a. indica, m. lucida, c. citratus, m. indica and c. papaya on meristem cells ranged from 0.0-0.14% (akinboro and bakare, 2007). in this study, the frequencies of cell anomalies were noted at 2.47-13.67% in both extracts, thus exhibiting prominent genotoxic mechanisms. exposure of the onion root tip meristem cells to different concentrations of money tree extracts resulted in chromosomal aberrations including fragmentation, chromosome bridges and stickiness (figure 4), which was caused by dna-damaging activities on its structure and function (darlington 1942; el-ghamery et al. 2000) and suggest that both extracts could be genotoxic.the observed formation of micronuclei in the onion root tip cells were early indicators of its genotoxicity (shahsavan and samadi 2001). furthermore, according to pennel and lamb (1997), morphological irregularities observed in the cytoplasm and nucleus indicate that onion meristematic cells were undergoing apoptosis. the results suggest that the a. cepa cells treated with pa-se and pa-le exhibited cytoplasmic and nuclear abnormalities that activated apoptosis to prevent genetic disruptions. embryotoxicty and teratogenicity of p. aquatica on zebrafish zebrafish (danio rerio) developmental toxicity assay is a reliable vertebrate model and highly efficient tool for cancer drug prescreening due to its high correlation with the results of mtt cell proliferation assay (li et al. 2012) since embryo genesis and carcinogenesis have similar processes of progression and most known anticancer drugs are teratogens and vice versa (blagosklonny 2005). zebrafish embryo lethality was indicated by coagulation (milky white appearance), lack of heartbeat, tail detachment and absence of somites (hood 2011). it can be noted that as the concentration of mt stem extract increases, the percentage mortality of d. rerio embryo also increases since bioactive compounds are often toxic at j.f.a. halili et al. 43 higher concentrations (moshafiet al. 2008). the findings imply the dosage and time dependence of the lethal effects of the phytocompounds present in pa-se and pa-le. in addition, both extracts were remarkable with 100% mortality rate after 12 hours post-treatment application (hpta) at 20, 100 and 200 µg/ml, while most proven anticancer drugs induce zebrafish embryo death within 24 hours (li et al. 2012). sulik et al. (1988) suggested that this early and high mortality rate among zebrafish embryos was due to the activation of the cells’ apoptotic feature. high frequency of embryonic death previously discussed serves as an early marker of teratological action (kusumi and dunwoodie 2009). teratogenic indicators include growth retardation and malformations in tail, head, heart, notochord, yolk sac and spine (hood 2011). the malformations observed in d. rerio embryos exposed to both extracts were validation of the activation of apoptosis (sulik et al. 1988). moreover, the teratogenic endpoints induced by pa-le were found to be similar with the embryo malformations caused by retinoic acid, coumarin, valproic acid and warfarin, which are very potent teratogens and potential anticancer agents (wang et al. 2014; weigt et al. 2012; aluru et al. 2013). results suggest that money tree phytocompounds possibly triggered one or more teratogenic mechanisms, which include disruptions and alteration in dna sequence and synthesis, chromosomal abnormalities, mitotic disturbance that often results in reduced cell proliferation, cell death and malformations (wilson et al. 1973), which were seen in the zebrafish embryos. future research directions for p. aquatica though the results could not confirm whether the extracts could be genotoxic or cytotoxic or both, the extracts have promising cytostatic (inhibition of growth, division and differentiation) and cytocidal (lethal) effects: two important fates of an anticancer drug. further studies on optimization, dosage and delivery could show that p. aquatica could be a potential source of a nature-based chemotherapeutic compound. however, standardization and control measures must be applied in using the plant as traditional herbal medicine. excessive and prolonged intake could produce adverse effects to humans especially to pregnant women. the use of the other plants parts (i.e., bark, roots, flowers) of p. aquatica should be exploredas potential sources of cytotoxic and genotoxic compounds as well. isolation, purification and identification of specific bioactive compounds that are responsible for the optimal cytotoxic and genotoxic activities should be done. also, the effect of the money tree extracts on human cancer cell lines and other mammalian test systems may be further investigated. cytotoxic and genotoxic potential of the money tree (pachira aquatica) 44 acknowledgement we wish to thank the department of education of the republic of the philippines for the funding to do this research. the researchers would also like to send their sincerest thanks and gratitude to the teachers and administrators of juan r. liwag memorial high school for the unending support, encouragement and guidance throughout this research project. mostly, this piece of work is highly dedicated and offered unto the lord. to god be the glory! references abbott w. 1987. a method of computing the effectiveness of an insecticide. j econ entomol. 3(2):302-303. agnieszka m, sliwinska e, wierzbicka m, kuras m. 2008. comparative investigations of cytotoxic action of anticancer drugs taxol and taxotere on meristematic cells of allium cepa. caryologia. 61(1):26-44. akaneme i, amaefule f, comfort c. 2012. evaluation of the cytotoxicity and genotoxicity of aqueous leaf extracts of azadirachta indica a. juss using the allium test. j med plants res. 6(22):3898-3907. akinboro a, bakare, a. 2007. cytotoxic and genotoxic effects of aqueous extract of five medicinal plants on allium cepa linn. j ethnopharmacol. 112(3):470-475. aluru n, deak k, jenny m, hahn, e. 2013. developmental exposure to valproic acid alters the expression of micrornas involved in neurodevelopment in zebrafish. neurotoxicol teratol. 40:46-58. amara a. 2013. methods other than experimental animals for screening antitumor compounds. am j cancer sci. 1(1):1-27. 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[cited 2015 february 16]. available from: www.who.int/mediacentre/factsheets/fs297/en/ ______ jordan ferdin a. halili <jf_halili@yahoo.com> is an m.s. in biology (major in genetics) graduate at the institute of biology, university of the philippines, diliman. he is currently an instructor 7 and taking up his ph.d. in biology at the same institute. his research interests include molecular phylogenetics of land snails and various biomonitoring assays as well as the zebrafish as a model for diseases and behaviour. mary lorraine f. lorido is currently a research adviser and secondary school master teacher 1 at juan r. liwag memorial high school junior high school. her research interests include natural products and microbiology. rich milton dulay is a b.s. biology graduate and associate professor 3 of central luzon state university. he obtained his m.s. biology degree at de la salle university. he is currently finishing his doctoral degree in biology at de la salle university. his research interests include mushroom, biotechnology and teratology. jane nicole n. catacutan, melissa c. gaudarioand maries ann r. silvestre were high school research advisees of mr. halili under the cytotoxicity and genotoxicity laboratory, institute of biology, university of the philippines diliman. 01_device salmo, torio, esteban 24 science diliman (january-june 2007) 19:1, 24-34 *corresponding author evaluation of rehabilitation strategies and management schemes for the improvement of mangrove management programs in lingayen gulf severino g. salmo iii*a, dante d. torioa and janalezza morvenna a. estebanb *the marine science institute, college of science university of the philippines, diliman 1101 quezon city tel. (02)523-4143, (0918)226-0206; email: jon@upmsi.ph date submitted: march 2, 2006; date accepted: february 27, 2007 a u.p. marine science institute – bolinao marine laboratory guiguiwanen, luciente i 2406 bolinao, pangasinan email: jon@upmsi.ph b – candidate, master of arts in development policy de la salle university manila 2401 taft avenue, 1004, manila abstract we evaluated the mangrove rehabilitation strategies and management schemes in five municipalities in lingayen gulf (bolinao, anda, bani, alaminos and san fernando). mangrove planting appears to be the first and only option used in the area, ignoring other recommended management strategies, e.g. conservation, landscaping, and sustainable production. all planting sites were located in coastal fringes and are mostly monospeficic stands of the species rhizophora mucronata. the planted mangroves were constrained by low seedling survival and stunted growth as probably caused by poor species-substrate matching, mono-species planting and pest infestations. three management schemes were noted: community-managed (bolinao and anda), local government unit (lgu)-managed (alaminos and san fernando), and co-managed between the lgu and the community (bani). the community-managed mangrove areas have the benefits of voluntary efforts from community-based organizations in conducting daily management activities but were constrained with budgetary and logistical concerns. in contrast, both lgu-managed and co-managed areas received institutional and logistical supports from their respective municipal governments, but lacking community participation made mangrove management difficult. almost two decades of mangrove management indeed helped improved the mangrove forest condition, at least in terms of forest structure. these projects demonstrated some level of success but also encountered several setbacks. several lessons can be derived from these areas that can help improve the mangrove rehabilitation and management approaches in lingayen gulf. among the recommendations are: (1) provide ordinance enacting the remaining natural secondary growth mangroves as marine protected areas, (2) promote planting in former mangrove areas by reverting abandoned, idled and unproductive aquaculture ponds to mangroves; (3) improve management schemes by formulating resource management plan, institutionalizing annual budget allocation, enhancing community participation, and enhancing tenurial instrument; and (4) incorporate periodic project evaluation. keywords: mangrove, planting, rehabilitation, management, community participation, tenurial instrument evaluation of rehabilitation strategies and management schemes 25 introduction the philippine mangrove forests declined from 500,000 has in the early 1900s (brown and fisher, 1920) to only about 117,700 has in 1995 (denr statistics, 1998), and further decreased to 109,700 has in 2003 (fao, 2003). mangrove destruction was aggravated in the 1960s due to the adoption of government’s policy intensifying aquaculture production that paved way for clearing vast mangrove areas (primavera, 2000). such destruction may have aggravated the decline of coastal fisheries that consequently contributed to poverty especially among marginal fishers. coastal resources management programs including mangrove rehabilitation projects were initiated by various institutions in the 1980s aiming to restore the natural ecological structure and functions of mangroves. most mangrove rehabilitation projects however failed due to various technical, social and institutional concerns. among these constraints are lack of technical knowledge and expertise in mangrove management, lack of awareness and poor community participation in project management, and lacking policy support on institutionalization and financial sustainability of the project (fortes, 1995). in lingayen gulf for example, various groups implemented mangrove rehabilitation projects since the late 1980s. most projects however, have low success as manifested by low survival and stunted growth of seedlings. similar with most reforestation cases in the philippines, the mangrove planting projects in the area was constrained by inappropriate technical design and lacking socio-institutional support mechanisms. this case study therefore aims to evaluate the rehabilitation strategies and management schemes, and provide recommendations for the improvement of mangrove rehabilitation programs in lingayen gulf. materials and methods site information lingayen gulf is a semi-circular embayment in the northwestern coast of luzon and is one of the most important fishing grounds in northern philippines. it is comprised of 18 municipalities with a total population of about 1,000,000 (nso, 2000), of which 40% are concentrated in coastal villages (mcmanus and chua, 1990). the mangrove forests in lingayen gulf are largely secondary growth stands of avicennia, sonneratia and rhizophora in coastal fringes with nipa swamps in some riverine areas. in pangasinan, mangrove forests were reduced from about 9.9 km2 in 1978 to 4 km2 in 2002 (msi, 2002). the decline in mangrove forests can be attributed to conversion into fishponds, pollution from mine tailings, and cutting for domestic uses. in anda for example, mangroves were drastically reduced from 394 has in 1932 to only 22 has in 2003 (table 1; slgp, 2003). presently, the only relatively intact remaining secondary growth mangroves in lingayen gulf are in anda (tondol) and alaminos (pangapisan-mona) having 22 has and 11 has, respectively (table 1; figure 1; slgp, 2003). among the recorded species are avicennia marina, a. officinalis, aegiceras floridum, bruguiera cylindrica, b. gymnorrhiza, ceriops tagal, excoecaria agallocha, bolinao 3 32 25 8 11 anda 394 22 48 18 10 9 bani 1 42 10 0 19 alaminos 11 9 22 0 7 san fernando 1 4 10 0 5 * namria, 1932 ** merf sagip lingayen gulf project, 2004 table 1 mangrove plantation profile of five selected municipalities in lingayen gulf. municipality/ city old growth natural mangroves (ha)* secondary natural mangroves (ha)** reforested area (ha) proposed rehabilitation area (ha) cbfma areas (#) age of plantation (yrs) salmo, torio, esteban 26 figure 1. mangrove distribution map in western lingayen gulf (bolinao, anda, bani and alaminos) showing the location of remaining natural secondary growth mangroves, location of rehabilitation sites, and proposed rehabilitation sites (inset: map of the philippines). rhizophora apiculata, r. mucronata, r. stylosa, sonneratia alba, sonneratia caseolaris and nypa fruticans. the mangrove plantation in bani have a basal area of 4 m2.ha-1 with mean stem density of 1,500 trees.ha-1 (slgp, 2005). these mangroves support a total of 88 species of birds: 47 species are residents, 36 species are either winter or passage visitors, and 5 species are both migratory and resident populations in the philippines (manamtam, 2005; unpublished data). the largest and oldest mangrove planting site is located in bani (42 has; 19 years) and is being co-managed by the municipal government and people’s organizations (po) since the late 1980s (municipal government of bani, 2003). the community-managed mangrove rehabilitation projects in the municipalities of bolinao and anda (municipal government of bolinao, 1999; municipal government of anda, 2002) started in 1995 and 1997, respectively. most sites in bolinao and anda are primarily managed by pos who are comprised mostly of marginal fishers. the mangrove planting site in pilar, bolinao earns the distinction of the first area awarded with community-based forest management agreement (cbfma) on mangrove areas in lingayen gulf (salmo et al., 1999). mangrove rehabilitation projects in alaminos and san fernando are primarily managed by the respective municipal governments and were initiated in 1999 and 2001, respectively (table 2). assessment of mangrove rehabilitation strategies and management schemes mangrove rehabilitation strategies and management schemes from each municipality were documented and reviewed through focus group discussions in 2003 (slgp, 2003). periodic ocular inspections in all planting sites were conducted (at least once every three months) from march 2003 to january 2006. this was evaluation of rehabilitation strategies and management schemes 27 complemented with reviews and evaluations of municipal fisheries ordinances, coastal resources management plans and cbfma documents. to assess community participation, a survey questionnaire was randomly distributed among mangrove managers composed of members of pos, lgus, ngos and ngas in all sites to determine their roles in the establishment, maintenance and development in mangrove management (20 respondents per municipality). results and discussion mangrove rehabilitation strategies since the late 1980s, mangrove planting was used as the main management strategy, involving an estimated costs ranging from p10,000 – p40,000.ha-1. the respective coastal management plan of each municipal government indicated that planting would likely remain as the main management strategy for at least until five years. mangrove planting appears to be the primary option and the only management strategy used in the area. however, planting is only one of four recommended management strategies, others are conservation, landscaping, and sustainable production (see field, 1999). but these options were not yet seriously considered in the area. lewis (2005) further suggested that mangrove planting should be considered as the last option and only upon carefully determining that natural regeneration does not occur. municipality/ no. of no. of pos % active % fishers city ngos involved member in pos ship in pos bolinao 2 8 50 70 anda 2 14 50 70 bani 2 3 43 50 alaminos 2 1 30 40 san fernando 1 1 20 30 table 2 number of non-government organizations (ngo) and people’s organizations (po) engaged in mangrove management in five selected municipalities of lingayen gulf. all planting sites are mono-specific stands located in coastal fringes, using almost exclusively rhizophora mucronata species. planting distance between seedlings is usually at 1.5 x 1.5m and 2 x 2m. from late 1980s to 1990s, rhizophora mucronata propagules was preferred since it is easier to collect and a lot cheaper (p1-2.propagule-1) compared to nursery-grown seedlings (p10-15.seedling-1). the planted seedlings were observed to have higher longevity compared to propagules. only the municipalities of bani and san fernando ventured into planting in riverine and creek areas. planting period almost occurred all throughout the year. seedlings were tied to bamboo sticks of at most 1m height to help enhance vigor and reduce the dragging effects of coastal currents. except in bani (and partly in san fernando), all planting sites have bamboo fences with nets that aims to reduce the entry of debris that could strangle the seedlings. the local communities undertook cleaning and other maintenance activities, albeit quite irregular and inconsistent (i.e. once a week). the growth and survival of mangroves particularly the younger and newly planted seedlings are constrained by gleaning activities and frequent passing of boats. monitoring of growth and survival of seedlings were initially conducted in bolinao, anda and bani but were not subsequently sustained. in all planting sites, seedlings suffered high mortalities (sometimes reaching >90% as early as six months after planting) and stunted growth. in addition, mangroves planted along coastal fringes are subjected to frequent stresses caused by coastal currents and wave actions and smothering by marine algae (e.g. sargassum spp. and ulva spp.) that strangles the seedlings (pers. obs.). seedlings planted during high tidal conditions (e.g. march – august) usually have higher mortalities. the high mortalities and stunted growth could be attributed to poor substrate-species matching, pest infestations, and failure to recognize natural species zonation of mangroves (i.e. as proposed by agaloos, 1994). trying to avert high seedling mortality, multi-species planting was practiced starting in year 2003 incorporating the species avicennia marina, sonneratia alba, ceriops decandra, and nypa fruticans, among others. survival improved a bit at least one year after planting, but afterwards, the problems on high mortalities and stunted growth again recurs. salmo, torio, esteban 28 among sites, only bani enacted its mangrove reforestation site as a marine protected area (municipal government of bani, 2001) prohibiting poaching and harvesting of marine resources. it is the only plantation that has an effective patrolling system conducting regular surveillance and arrest of poachers. several livelihood projects were pilot-tested at experimental scale in bani that aims to provide supplemental income to local mangrove managers. these projects have shown potential good harvests but they are not yet fully implemented at a scale that could significantly increase the income of the communities. in 2005, apiculture was introduced in selected mangroves in anda. preliminary reports showed high yields and could potentially demonstrate a suitable livelihood project in the long run. mangrove management schemes three management schemes were noted: communitymanaged (bolinao and anda), local government unit (lgu)-managed (alaminos and san fernando), and co-managed between the lgu and the community (bani). all mangrove sites were part of the integrated coastal resources management program of each municipal government. the community-managed and co-managed mangrove areas capitalized on voluntary efforts from community-based organizations in conducting daily management activities despite budgetary and logistical constraints. in contrast, lgumanaged areas received institutional and logistical supports from their respective municipal governments, but weak community participation made mangrove management difficult (figure 2). the community-led mangrove management scheme highlights the voluntary engagement of the communities in doing regular maintenance activities (salmo and torio, 2004). however, the downside of having the po members of the community exclusively leading the project is the limited representation of other community stakeholders particularly those that are not members of the pos. community-led mangrove management projects also exhibit poor linkage with other institutions, as well as weak capabilities in deterring mangrove forest violators since there was no legitimate patrolling and enforcement mechanisms. on the other hand, lgu-led scheme have centralized management. this scheme could realize some level of success as demonstrated in the visayas (primavera and altamirano, 2001). but because of its diverse and broad management objectives, lgu-managed mangrove management programs are difficult to monitor. there may also be lack of sense of community ownership largely due to its highly government-centralized nature. in this case study, the most beneficial scheme could be the co-management mode that shows collaboration between the communities and the lgu (salmo and torio, 2004). it was observed that there was also a more legitimized collective decision making process and a strong sense of ownership by the communities. both communityand co-management schemes may also be more resilient, since the continuity of mangrove management program is relatively more assured should changes in political leadership occur. institutional linkages were also formed by both the communities and the lgu. the factors that could help enhance success in resource management projects were listed in several case studies (see primavera, 2000; baticados, 2004; walters, 1997; ellison, 2000; lewis, 2005; field, 1999). for this study, we identified and focused on three aspects that could contribute to a successful mangrove management in lingayen gulf. these were: (a) resource management plans, (b) community participation, and (c) tenurial instrument. resource management plan resource management plans are practically lacking in all municipalities. among sites, only bani have the de facto mangrove management plan by virtue of its action plan emanating from the enactment of the mangrove protected area. but such plan was not yet comprehensive and systematic enough to effectively use as management guide. a detailed and systematic resource management plan would help the proper implementation of daily management activities. at the least, the plan should contain project objectives, strategies, time frame, estimated budget and expected outcome. the local agenda 21 planning guide (iclei, 1996) particularly the sections on the planning process and development of performance indicators could be adapted. moreover, the formulated resource evaluation of rehabilitation strategies and management schemes 29 0 10 20 30 40 50 po lgu ngo nga 0 10 20 30 40 50 0 10 20 30 40 50 e st ab lis hm en t d ai ly m ai nt en an ce l iv el ih oo d p ro je ct s anda alaminos/ san fernando bolinao bani figure 2. relative degrees of involvement expressed in percent (%) of mangrove managers (po people's organization; lgu local government unit; ngo non-government organization; nga national government agencies) in the establishment, maintenance and development of mangrove plantation. bani salmo, torio, esteban 30 management plan should be complemented by an institutionalized and allocated annual budget. rather than acquiring funds on a per activity basis as have been practiced, an institutionalized allocated budget could help systematize regular operations and maintenance activities. moreover, having a resource management plan and allocated budget could help in periodically evaluating the progress and costeffectiveness of the mangrove management projects. community participation the role of the communities in the planning and implementation contributes to the success of resource management projects (ferrer et al., 1996; field, 1999; pollnac, 1994; wells, 1996, iirr, 1998; baticados, 2004). in bolinao and anda, the communities through the po have been the primary institution in charge of doing the daily maintenance activities (e.g. replanting, cleaning, monitoring) since 1995. their voluntary engagements may have been the result of community organizing and capability building programs facilitated by the cbcrm programs sponsored by the marine science institute marine environment and resources foundation (msi – merf) in bolinao and the university of the philippines social action for research and development foundation, inc. (upsardfi) in anda since 1995. if given monetary value, these voluntary community efforts are of considerable amount and highlight the need for evening of the social equity gap in sharing the costs of management by local governments for the public welfare benefits derived from the environmental services of the mangroves. sustaining such level of shared responsibilities pose a challenge to the mangrove managers. tenurial instrument like community participation, tenurial instrument such as community-based forest management agreement (cbfma) could contribute success in mangrove management projects (farnsworth and ellison, 1997). the cbfma encourages communities to participate and assume responsibilities in conducting regular management activities (katon et al., 1998). bacalla (2006) stressed the importance of cbfma in attaining sustainable forest management. eighteen sites were under cbfma (slgp, 2005) which are all located in bolinao and anda, coincidentally where relatively stronger pos exists. these sites showed the critical role of the communities as caretakers and resource managers doing regular maintenance activities. in 2006, the mangrove site in macaleeng, anda was recognized as the best cbfm site in region i. while cbfma is a good tenurial instrument, sustaining financial and technical supports from municipal government and concerned national government agencies (nga) remains to be a challenge. after the ceremonial awarding of cbfma, the communities are left on their own initiatives in managing the project. fortunately, the communities are resourceful enough in soliciting financial and technical assistance. most sites in bolinao were able to solicit at least p 5,000 per year from their respective barangay councils. in addition, sites with cbfma have been recipients of considerable financial support from external funding institutions. recently, the acquisition of cbfma has been one of the requirements of the provincial government in selecting recipients of multi-species mangrove seedlings. summary, lessons learned and recommendations almost two decades of mangrove management in lingayen gulf yielded 130 hectares of planted mangroves in 23 sites. there were indeed improvements in mangrove forest conditions, at least in terms of forest structure. likewise, there were also perceptions from the communities that harvests from fisheries and gleaning activities improved due to the planted mangroves. the mangrove plantation in bani have high populations of wetland birds and includes the endemic philippine duck (anas luzonica). because of these avifauna, the mangrove plantation became a popular tourist destination site. similarly, the mangrove sites in bolinao and anda were favorite learning destination areas among environmental groups that wanted to draw lessons on mobilizing communities to participate in resource management projects. most sites already earned local and national recognitions because of their impressive mangrove management. for example, bani was granted likas yaman award in 1996, anda was cited as the best cbfma in region i evaluation of rehabilitation strategies and management schemes 31 in 2006, and alaminos as the most outstanding coastal local government for mangrove development initiative in pangasinan in 2006. clearly, these projects demonstrated some level of success but also encountered several setbacks most notably the low survival and stunted growth of the planted seedlings. several lessons can be derived from these areas to improve the mangrove rehabilitation and management in lingayen gulf that could potentially serve as management model in region i and in northwestern luzon. improving rehabilitation strategies mangrove planting have been the primary if not the only management strategy used in all sites that ignores other recommended strategies (i.e. as proposed by field, 1999). instead of focusing on planting, priority attention should be given in immediately declaring the remaining secondary natural mangrove stands in anda and alaminos as a marine protected area which should be properly guarded against encroachers. these stands were already subjected to small-scale cutting and other forms of disturbances. failure or delays in providing protection could significantly reduce the only remaining and most intact mangrove cover in lingayen gulf and may also limit the viable sources of propagules and seedlings for future mangrove planting. in addition, mangrove planting should only be conducted as the last strategy (see lewis, 2005) after correcting hydrological problems (to determine causes of blocking of propagule dispersal that’s limits natural seedling recruitment) and determining that natural regeneration does not occur. unfortunately, these two basic information were not available. but despite lacking information, the communities are still doing perpetual planting, thereby almost regularly repeating management failures. it is therefore imperative to determine and establish the hydrological patterns and natural regeneration rate prior to continuing any mangrove planting activities. the information could also eventually help in estimating the total plantable area and locating viable plantation sites. planting sites were mostly located in coastal fringes except in few instances where plantings are conducted along riverbanks (in bani and san fernando). for almost fifteen years, the sites have practiced monospecies planting using rhizophora mucronata species. the species used for planting should have been chosen following the natural mangrove zonation pattern (see agaloos, 1994). the inappropriate choice of species could have caused high seedling mortalities. attempts to use multi-species seedlings (i.e. avicennia sp., sonneratia sp., etc.) only slightly increased the survival rate and prolonged the lifespan of the planted seedlings. however, seedlings still died a year after planting as probably caused by barnacle infestation, strong currents, and disturbances from gleaning activities. considering these setbacks and huge costs incurred in the past, the rehabilitation strategies should be carefully evaluated. it is recommended to abjure planting in coastal fringes, at least temporarily, and instead prioritize planting in former mangrove areas that are in higher elevation which may have higher chances of survival. compared to coastal fringes, former mangrove areas may offer freely available propagules and seedlings, have relatively more stable substrates, and have natural defense against coastal currents and debris which could save significant amount that could have been otherwise spent for growing seedlings and establishing fences. aside from saving costs, planting in former mangrove areas could also enhance the natural regeneration of mangroves. however, like in most parts of the country, former mangrove areas were converted to aquaculture ponds (primavera, 1995). reverting idled, abandoned, and unproductive ponds to mangroves have long been advocated in the past (see primavera, 2005; lewis et al., 2005) and is also legally mandated in the philippines (sec. 46 and 49; r.a. 8550). reverting aquaculture ponds into mangroves are rarely undertaken in the country and posed a great challenge to mangrove managers. in lingayen gulf, only the municipal government of anda have taken the initial step by taking an inventory of all existing and abandoned fishponds in preparation for mangrove reversion. improving management schemes the mangrove management schemes varies among municipal governments. the municipal governments of alaminos and san fernando have more pro-active roles thus ensuring financial sustainability. conversely, the municipal governments of bolinao and anda rely on active community participation of people’s salmo, torio, esteban 32 organizations in conducting establishment and maintenance activities. in bani, there was a comanagement arrangement between the municipal government and the communities. as demonstrated in this case study, and as proposed in several studies (farnsworth and ellison, 1997; primavera, 2000; walters, 1997; field, 1999; katon et al., 1998), community participation and tenurial instruments could facilitate the success of mangrove management programs. if these two aspects can be incorporated in alaminos and san fernando, the mangrove management may likely contribute in realizing success. it is therefore a challenge for the mangrove managers to sustain such level of participation and optimize the benefits provided under the cbfmas. sustaining community’s interests may be made possible when they realize tangible benefits, e.g. in terms of food and cash. in terms of gleaning harvests, the macrofaunal abundance and biomass in reforested mangroves may start to provide food benefits 7-10 years after planting (e.g. 2,750 kg.ha-1 of macroinvertebrates; salmo, 2005). thus, it is important that while waiting for that period, some forms of livelihood projects or financial support should be provided to the communities. the municipal government of bani employed few mangrove caretakers as a form of incentive. this approach could also be use in bolinao and anda whose community members have been voluntarily taking care of the planted mangroves for already more than ten years. project evaluation the success (or failure) of a mangrove management program should be periodically monitored and evaluated. unfortunately, most mangrove management programs do not have a monitoring and evaluation component (ellison, 2000; bosire et al., 2003; ha et al., 2003; crona and ronnback, 2005). ellison (2000) suggested that mangrove management should be quantitatively evaluated containing at the least the following minimum parameters: tree stand structure, tree abundance, species richness and diversity, invertebrate abundance, species richness and diversity, primary production (biomass and litter), nutrient export, and hydrologic patterns. the biophysical parameters should be complemented with socioeconomic parameters to help determine if the living status of the communities have improved with improving mangrove forest conditions (e.g. see salmo et al., 2004). lastly, the importance of the active participation of the important stakeholders and decision makers are crucial to the success of rehabilitation from selection of sites and the choice of their strategies. a systematic evaluation would help track the progress of the program, draw lessons, and ultimately incorporate the lessons as management and policy inputs and contribute to the overall success of the mangrove management program. acknowledgments we thank the people’s organizations, local governments, ngos and ngas in lingayen gulf for providing information and cooperating with the authors during the conduct of this study. dr. perry alino of upmsi gave valuable insights, comments and editorial assistance in improving the manuscript. the sagip lingayen gulf project provided travel expenses for s. salmo 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(abstract) presented in the 8th philippine association of marine scientist (pams) symposium held on 20-22 october 2005 in palawan state university, puerto princesa city, palawan. salmo, s.g. iii and d. d. torio. 2004. comparative study of mangrove management approaches in lingayen gulf, northwestern philippines. a case study presented in the 6th coastal zone canada conference, st. johns, newfoundland canada, june 26-30, 2004. salmo, s.g. iii, a.j. uychiaoco, e.p. batungbacal, m. dela cruz, m. lavidez, m. nightingale, e. enderez, 2004. costs and benefits of maintaining community-based marine protected areas. in bacal et al. (ed.), our cbcrm story in the making, cbcrm resource center and oxfam great britain, pp 75-113. walters, b.b., 1997. human ecological questions for tropical restoration: experiences from planting native upland trees and mangroves in the philippines. forest ecology and management 99: 275-290. wells, d. 1996. environmental policy: a global perspective for the twenty-first century prentice hall: nj. research dissemination office office of the vice chancellor for research and development lower ground floor, phivolcs bldg., c.p. garcia ave., up diliman 1101 quezon city (632) 3436-8720 fax (632) 8927-2568 rdo.ovcrd@up.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice chancellor for research and development (ovcrd) – humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is phpp650.00. 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(adapted with permission f rom the editors of ieee sensors journal) 01_device rate of loaded sediments in honda bay, puerto princesa city 53 *corresponding author science diliman (january-june 2006) 18:1, 53-58 introduction honda bay is one of the bays of puerto princesa city. surrounded by 18 coastal barangays, it is a major fishing ground of both artisanal and commercial fishers (figure 1.) the bay has an area of approximately 280km2 and located in the northeast of mainland palawan between 9o50' to 10o00' n latitude and 118o44' to 119o00' e longitude. six major rivers namely: bacungan, tandayak, babuyan, tanabag, langugan, rate of loaded sediments in honda bay, puerto princesa city, palawan, philippines joel g. becira western philippines university puerto princesa campus p.o box 93, pueerto princesa city 5300 palawan, philippines tel. no. (048) 433-4367 e-mail: j_becira@yahoo.com abstract a study in honda bay, puerto princesa city, palawan, philippines was conducted to determine and compare the rate of loaded sediments of and among six sites in honda bay and during two seasons, and to discuss sedimentation rate in relation to selected environmental parameters commencing on april 2003 to november 2003. results showed that the babuyan river significantly contributed to the amounts of sediments being loaded to honda bay, puerto princesa city of which the amount of sediments loaded between the two sampling season and among the six sampling events had no significant difference. sediment's color and texture affirmed the land-based activities in babuyan and mauyon watersheds, which eventually carried into the bay during storm weather condition. the amount of trapped sediments in all stations is probably affected by the river's discharge capacity and the river's water velocity, although the duration sediment traps were mounted could also affect the measurement of sedimentation rate. keywords: sedimentation rate, loaded sediments, honda bay, puerto princesa city, philippines and tapul and numerous small tributaries drain into the bay (figure 1). during rainy season, these rivers carry high amounts of sediments into the bay (personal observation). siltation is considered to be a problem on a national scale (upmsc 1979 as cited by aliño 1983) particularly in the coastal resources. in fact, dead corals in the northeast interior part of puerto princesa bay were attributed to siltation and sedimentation coming from household wastes and/or the degrading watershed (cruz et al 1988 as cited by frmp 2001) which finally affects fish catch. in palawan, devastating land-based activities in the watershed of honda bay have caused becira, joel g. 54 erosion and siltation may have likewise contributed to a decrease of fish catch per unit effort from 36.5kg in 1985 to 8.4kg in 1989 (sandalo 1994) and to 5kg in 1996 (iclarm 1996). this study was conducted to describe the status of sedimentation in honda bay with the following specific objectives: 1.) to determine and compare the rate of loaded sediments of and among the six rivers in honda bay and during two seasons, and 2.) to discuss sedimentation rate in relation to selected environmental parameters. materials and methods a total of six sampling sites were selected. these are in bgy. tagburos, sta. lourdes, bacungan, sta. cruz, tandayak and babuyan (figure 1). in the mouth of each of these rivers, a sampling station was established. all stations were assessed in terms of sedimentation rate, water temperature, salinity, water velocity, ph, total dissolved solids and transparency. sampling of physical and chemical parameters was conducted thrice during the dry season (april, may, and june 2003) and thrice during rainy months (august, september and november 2003). sampling of all stations was conducted within one day except during the first sampling in april 2003 when two days were needed. three sets of sediment traps were installed in all 6 stations with 3 replicates per set (a set is composed of 3 individual traps measuring 11.5cm in length and 5cm in diameter) (english et al 1997) on the same day. for each established stations, sediment traps were mounted 100 meters away from the mouth of the river towards the sea. the three replicates were mounted close (0.3m) to each other. results and discussion in babuyan river, the sedimentation rate ranged from 57 to 168 mg/cm2/day; in tandayak it ranged from 13 to 63mg/cm2/day; in sta. cruz it ranged from 9 to 52mg/ cm2/day; in bacungan it ranged from 31 to 56mg/cm2/ puerto princesa bay ulugan bay bacungan tandayak babuyan tanabag langugan tapul 9o40’ 9o50’ 10o00’ 10o10’ 118 o40’ 119 o00’ 118 o70’ puerto princesa city m ap of palawan sta. cruz sta. lourdes tagburos stn 1 stn 2 stn 3 stn 4 stn 5 stn 6 honda bay map of palawan figure 1. map of palawan (lower right) showing the study area and the location of the sampling sites in honda bay, puerto princesa city. rate of loaded sediments in honda bay, puerto princesa city 55 day; in sta. lourdes it ranged from 9 to 49mg/cm2/ day; and in tagburos sedimentation rate ranged from 21 to 111mg/cm2/day. the average rate of sedimentation in the six stations established near the mouth of the rivers ranged from 27 to 125mg/cm2day (figure 2). the amount of sediment in babuyan river was higher than in all other stations. this was true for both seasons. the sedimentation rate of babuyan river is significantly differs (p<0.05) from the rates of all other stations. t-test and anova showed that sedimentation in each of the six sites is significantly different from each other (p<0.05)). furthermore, the test revealed that sedimentation during dry season was significantly different from sedimentation during the wet season. sedimentation was also significantly different between the six sampling events in the rivers of babuyan, tandayak, bacungan, sta. lourdes, and tagburos. significant differences in sedimentation during sampling seasons (wet & dry) were established in the rivers of tandayak, bacungan, tagburos. among the six river stations, mean sedimentation was highest in babuyan river, 125mg/cm2/day. noticeable was that the sedimentation in babuyan river was higher during the dry season (136mg/cm2day) than during the wet season (115 mg/cm2/day). this could be attributed to the fact that during the dry season only data for the month of may were available. the sediment traps set up in april and june were lost. similar pattern of higher sedimentation rate during the dry season was observed in bacungan, sta. lourdes and tagburos. also in the former two of these sites sediment traps were lost. the higher sedimentation rate during the dry season at these sites might be due to the duration of sediment entrapment. the sediment traps were set up for one month during the dry season, while during the wet season they were set up over a day only. the lowest river sedimentation rate was recorded in sta. cruz with a mean value of 27mg/cm2/day. sedimentation in babuyan and bacungan river are perceived to be attributed to the intense land use in the area. the two barangays had intensive agricultural figure 2. mean sedimentation rate in the studied river discharging honda bay during dry and wet season. becira, joel g. 56 practices in vast areas. sediments collected in these rivers were characterized by very fine particles and reddish colored sediments, a characteristic of a fluvial type of soil. the fine grain size of the collected sediments and its brownish to reddish color indicate that this is an eroded soil which is carried by the storm water during heavy rains. sedimentation in sta. lourdes was probably attributed by making a site for quarrying during road construction. collected sediments are composed of sands and organic particles. in tagburos, though fishpond in the coastal part of the barangay was present, high sedimentation is probably attributed by the road construction, which just finished during the collection period. these findings conformed to the statement of winger (1981) that infusion of the eroded sediments into the river systems is caused by improper land use. in sta. cruz and tandayak, sedimentation was probably influence by the presence of mangroves in the mouth of the river and in along the shore and the planting of fruit trees in the hillsides as well. agricultural activities were not much observed in these barangays. of the six rivers understudied, babuyan and bacungan were the one having the wider river mouth (120m and 50m) and still the deeper ones (both had an average depth of 3.5m), respectively which may also influence the siltation and sedimentation processes. average rate of loaded sediments in honda bay was much higher (50.55mg/cm2/day) compared to dona paula bay, west coast of india (bhaskar et al. 2000), which only ranged from 0.11 to 1.34mg/cm2/day with an average of 0.54mg/cm2/day. however, direct comparison may not be advisable due to differences in trap designs (pvc pipe used had a diameter of 20cm), exposure periods, and differences in the hydrological aspects of the different marine environments. a study conducted by aliño (1983) in three areas (matab-ang, bato, and looc) in toledo city, cebu using 2" x 8" pvc pipe had an average sedimentation of 16.20, 10.10 and 33.5mg/cm2/day, respectively. these values are similar to those measured in honda bay (27 to 36mg/cm2/day). only babuyan river, bacungan river and tagburos river (125, 48, and 47mg/cm2/day, respectively) had higher values. in singapore, increasing coastal development has led to increased sedimentation from 3-6mg/cm2/day in 1979 (chan 1980) to 5.45mg/cm2/day (lane 1991, low & chow 1994), which is lower than the loaded sediments in honda bay. the water transparency in the 6 rivers during all the sampling events was similarly low (figure 3). for all rivers the mean transparency was 1.65m (± 0.4) during the dry season and 0.98m (± 0.6) during the wet season (table 1). the lowest transparency (0.98 ± 0.21) was measured in sta. lourdes. the water velocity in bacungan ranged from 29.41 to 52.63cm/sec while in babuyan it ranged from 16.98 to 45.45cm/sec, which is consistently lower than in bacungan river. the lowest mean water velocity was in sta. lourdes with a value of 13.86 ± 6.17cm/sec (table 2).the high mean water velocities were observed in bacungan and babuyan river with 40.59 ±12.44 and 32.96 ± 12.13cm/sec, respectively. total dissolved solids were high in sta. lourdes (24.58 ± 0.74). lowest total dissolved solids were taken in babuyan (12.03 ± 5.81) and bacungan (14.67 ± 6.25) rivers (table 5). in all stations, temperature did not vary much from the mean (ranges from 28.5 ± 1.64 to 30.43±1.25). the highest reading was taken in sta. cruz and the lowest reading was taken in babuyan river (table 2). the mean ph reading in all stations was almost neutral to slightly basic. the lowest ph reading was taken in bacungan river (7.99 ± 0.44). total dissolved solids had a high correlation with the sedimentation rate in babuyan river (r = -0.90), tandayak (r = 0.79), sta. cruz (r = -0.85) and bacungan (r = -0.97). in sta. lourdes (r = 0.47), a moderate correlation was established. water velocity in bacungan and tandayak river had a high positive correlation with the sedimentation rate. a substantial relationship between these two parameters was established in tagburos (r = -0.61). in this site, sedimentation might have been affected by incoming high tide. the turbulences caused when sea and river waters meet during incoming high tide might have affected the settling of particulate matter, which then remained suspended in the water column. rate of loaded sediments in honda bay, puerto princesa city 57 recommendations for further study, inorganic contents of the collected sediments should be measured and the relationship between total dissolved solid and sedimentation rate should be examined and be evaluated as for the total dissolved solids be an indicator of sedimentation rate. acknowledgments the author wishes to acknowledge the western philippines university-puerto princesa campus and dr. sabine schoppe for the untiring support. figure 3. water transparency in six river stations in honda bay, puerto princesa city. station/ physicochemical parameters salinity (ppt) temperature (oc) ph tds (mg/l) transparency (m) velocity (cm/sec) \babuyan 8.90-27.30 26.80-31.25 6.88-10.21 634-18.36 0.50-2.50 16.98-45.45 tandayak 8.10-33.10 28.60-33.10 7.82-10.00 7.02-25.20 0.50-3.75 8.00-33.33 sta. cruz 25.50-32.90 29.60-32.90 7.81-9.30 20.10-25.10 1.00-1.90 11.11-29.41 bacungan 7.30-32.20 28.40-33.10 7.31-8.43 6.36-23.60 0.50-2.50 29.41-52.63 tagburos 13.30-32.50 28.40-30.80 7.58-8.69 11.06-24.90 1.00-2.70 11.00-27.87 sta. lourdes 30.80-33.70 29.50-31.00 7.90-8.58 23.70-25.70 0.75-1.25 8.06-22.22 table 1. range of the physico-chemical parameters in all six stations in honda bay, puerto princesa city from april 2003 to november 2003. becira, joel g. 58 references aliño, p.f., 1983. the effects of mine tailings on the structure of coral communities in toledo, cebu. unpublished ms thesis. university of the philippines . 105 pp. bhaskar, d.v, cardozo, e, giriyan, a, garg, a., & n b. bhosle. 2000 sedimentation of particulate matter in the dona paula bay, west coast of india during november to may 1995 to 1997. estuaries. 23(5): 722 734 calow, p., 1998. the encyclopedia of ecology and environmental management. blackwell science ltd. united kingdom. 805 pp. el-swaify, s.a. 2000. operative processes for sedimentbased watershed degradation in small, tropical volcanic island ecosystem. pages 35-49 in: r. lal (ed.), integrated watershed management in the global ecosystem. soil and water conservation society. crc press. usa. pp. 395. frmp, 2001. resource and social assessment of honda bay and puerto princesa bay: a terminal report. philippines denr english, s., wilkinson, c. & v. baker. 1997. survey manual for tropical marine resources. 2nd edition. australian institute of marine science. townsville, australia. 390 pp. gonzales, b.j. 2000. puerto princesa city integrated coastal resource management network. a concept paper. frmp-puerto princesa city. 9 pp. gonzales, b.j. 2003. fisheries management in honda bay, p.xxx. in da-bfar (department of agriculture-bureau of fisheries and aquatic resources) in turbulent seas: the status of marine fisheries. coastal resource management project, cebu city, philippines. xxx pp. gonzales, b.j. in press. puerto princesa bay and honda bay, palawan: an ecological profile. 15 pp. iclarm (international center for living aquatic resources management). 1996. resource and ecological assessment of honda bay, palawan, philippines, iclarm, makati city, philippines lalli, m.c. & t.r. parsons, 1997. biological oceanography. 2nd edition. butterworthheinemann. woburn, great britain. 314 pp. lane, d.j.w. 1991. growth of scleractinian corals on sediment -stressed reefs in singapore. in: alcala, a.c. (ed.) proceedings of the regional syposium in living resources in coastal areas. university of the philippines, manila. pp. 97-106 low, j.k.y. & l.m. chow. 1994a. sedimentation rates in singapore waters. in: sudara, s., wilkinson, c.r. & l.m. chow (eds.). proceedings 3rd asean-australia symosium on living coastal resources. vol: 2 research papers. chulalongkorn university, bangkok, thailand, may 1994 in press. winger, p.v. 1981. physical and chemical characteristics of warmwater streams. pages 32-44 in: l.a. krumholtz (ed.), the warmwater stream symposium. southern division, american fisheries society, bethesda, maryland. 256 pp. v from the editor this june 2020 issue of science diliman is memorable in a way because it is being released in the midst of the coronavirus disease 2019 (covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). to date, there are over 14 million confirmed cases of covid-19 and more than 600,000 deaths worldwide. in the philippines, as of this writing, there have been 70,764 confirmed cases of covid-19 with 1,837 deaths. these numbers continue to increase as scientists are scrambling to develop vaccines for this dreaded disease. this disease has caused so much anxiety and misery as lives and livelihoods have been lost and as our normal lives have been disrupted in very profound ways. our hope rests in science to stem the tide of the spread of the virus, to find cures for this disease, and eventually, to bring our lives back to normalcy. as we are struggling with anxiety and uncertainty amid the covid-19 pandemic, science diliman deeply mourns the passing of one of its associate editors, dr. alonzo a. gabriel, on march 31, 2020. he was a professor in the department of food science and nutrition at the college of home economics, university of the philippines diliman. he was a prolific and multi-awarded food scientist. his research focused on food processing and preservation, microbiological food safety, and traditional and innovative food technologies. he was a tremendous loss, indeed, not just to science diliman and the university, but also to the scientific community. in this issue, we feature five research articles. the first article is by galgana et al. who determined the effects of the tectonic deformation of luzon on existing survey networks in the philippines. their block and continuum modeling experiments revealed the amount and spatial extent of deformation within the island of luzon. understanding the nature of this deformation is needed so that appropriate corrections can be applied to minimize transient errors affecting the philippine survey network. their study, however, is limited to examining the contribution of tectonic deformation to the overall error field. the authors recommend the application of corrections for other systematic error sources, such as global reference system corrections, rapid horizontal and vertical crustal motions, volcano-induced deformation, and hydrological effects. the second article is by gonda and paras who determined the jordan canonical form of a product of two elementary 𝑆-unitaries. the third article is by mapile and obusan who isolated bacteria and fungi from the gut of the earthworm, african nightcrawler, and screened these microorganisms for their ability to fix nitrogen, solubilize phosphate, and utilize polyethylene, as well as for the antagonistic potential of fungal isolates against bacterial isolates. the authors hope to harness the earthworm’s casts and compost for agricultural, medical, and other applications. the fourth article is by molina et al. who examined the body size, microhabitat, and feeding ecology of two species of edible freshwater or semi-terrestrial crabs that are endemic to mindanao island in the philippines. the authors hope to contribute to a better understanding of the biology of these two economically important crabs so that appropriate conservation strategies can be formulated for the two species. the fifth and last article is by que et al. who used dna barcoding to trace the possible source of the non-native philippine population of the greenhouse frog. their analysis revealed that the dna sequences of the samples they collected from the philippines are genetically identical to dna sequences of samples from hawai’i and florida, usa and are closely related to the dna sequence of an individual from cuba. the authors surmised that importation of plants from the usa is the most likely mode of introduction because this frog species lays its eggs in soil and is often found in bromeliad plants that are commonly imported from the usa into the country. jonas p. quilang, ph.d. editor-in-chief 71 evaluation of low molecular weight bis-urea derivatives as antimicrobial agents frances abygail f. genio natural sciences research institute institute of chemistry, college of science university of the philippines diliman monissa c. paderes* institute of chemistry university of the philippines diliman abstract antibiotic resistance against common microbes is an ongoing concern worldwide. this warrants continuous studies that aim to discover new compounds with antimicrobial properties. in this study, sixteen low molecular weight bis-urea derivatives were screened for their in vitro antimicrobial properties using agar well diffusion method. the structure of the bis-urea compounds is comprised of cyclic and aromatic linkers and a variety of symmetric end groups such as aliphatic chains and heteroaromatic groups. significant antimicrobial activity against strains of escherichia coli, staphylococcus aureus, and klebsiella pneumoniae was observed for compounds with long aliphatic chains compared to those with benzyl and heteroaromatic end groups. further studies on the minimum inhibitory concentration and cytotoxicity can aid in the development of these compounds as antimicrobial agents as well as for other possible biomedical and environmental applications. keywords: antimicrobial activity, bis-urea derivatives, agar well diffusion, antimicrobial agents * corresponding author science diliman (january-june 2022) 34:1, 71-81 evaluation of low molecular weight bis-urea derivatives as antimicrobial agents 72 introduction misuse and abuse of antibiotics have been prevalent in the philippines for a long time due to misconceptions about their proper use and the availability of the drugs in local stores (barber et al. 2017). this attitude towards antibiotics, among other factors, contributes to the emergence and spread of antibiotic-resistant strains, making this a global health concern (nys et al. 2004). common bacteria such as escherichia coli (e. coli) are already resistant to antibiotic drugs such as cefazolin, gentamicin, ciprofloxacin, and older drugs including ampicillin, oxytetracycline, trimethoprim, and chloramphenicol (nys et al. 2004). in other studies, staphylococcus aureus (s. aureus) was also found to exhibit resistance against chloramphenicol, tetracycline, and ciprofloxacin (juayang et al. 2014) while drugs such as ceftazidime and avibactam have been ineffective against klebsiella pneumoniae (k. pneumoniae) strains (chou et al. 2016). the growing antibiotic resistance is a serious threat to modern society, so there has been a necessity to discover new antimicrobial agents that would be effective against strains yet to be discovered (moellering 2011; kumar b et al. 2016). some of the more diverse antibiotic agents discovered to aid this problem are low molecular weight compounds, which include peptides (baquero et al. 1978; marshall and arenas 2003), steroids (moore et al. 1993), and other compounds isolated from natural sources (höltzel et al. 2000). synthetic low molecular weight compounds have also been reported as antimicrobial agents (rufián-henares and morales 2007; keche et al. 2012; poonia et al. 2020). specific examples of these synthetic compounds include quinoline derivatives which contain urea groups (keche et al. 2012) and bis (urea-1,2,3-triazole) compounds (poonia et al. 2020). urea moieties have been incorporated into molecules used as a scaffold for bioactive molecules with both organic and medicinal applications (gündüz et al. 2020). specifically, urea derivatives have been known to possess both antimicrobial and antiviral properties (sun et al. 2006; abdel-rahman and morsy 2007; džimbeg et al. 2008). although more specific mechanisms explaining the antimicrobial properties of urea derivatives are yet to be studied, there are few studies that have been reported. for instance, gündüz et al. (2020) observed that 1,3-disubstituted urea derivatives support the quorum sensing inhibition of pseudomonas aeruginosa which decreased the biofilm formation of the bacteria. in another study by lal et al. (2020), urea-1,2,3-triazole-amide hybrid compound with a good antimicrobial activity was observed to have significant docking interactions with the active site of s. aureus dna gyrase complexed with dna. f.a. f. genio and m. c. paderes 73 in this study, sixteen (16) readily synthesized low molecular weight bis-urea compounds were screened for antimicrobial activity against three organisms, e. coli, k. pneumoniae, and s. aureus using agar well diffusion method. the linkers used for the bis-urea compounds are cyclic and aromatic moieties, whereas the end groups are either aliphatic chains, benzyl, or picolyl (pyridyl-containing) groups. pyridine derivatives are well-studied for their anti-hypertensive, anti-neoplastic, and anti-inflammatory properties (lednicer and mitscher 1977) and have also been previously reported to exhibit antimicrobial properties (patel et al. 2010; kumar s et al. 2016). moreover, the incorporation of long aliphatic chain lengths enhances the antimicrobial properties as observed by akedo et al. (1977), violette et al. (2006), and yong et al. (2007). materials and methods synthesis of bis-urea compounds a series of bis-urea compounds were synthesized following the representative scheme shown below (genio and paderes 2021). following the method used by rutgeerts et al. (2019), various aromatic and cyclic diisocyanates and amines containing aliphatic, aromatic, and heteroaromatic groups were used to synthesize the bis-urea compounds. the diisocyanates and amines were stirred in anhydrous tetrahydrofuran (thf) at room temperature for an hour. the reaction mixture was kept under a nitrogen atmosphere and anhydrous thf was used because of the sensitivity to moisture of the starting reagents. products were precipitated out, filtered, washed with diethyl ether, and dried under a vacuum. scheme 1. representative procedure for the synthesis of bis-urea compound 1a. the detailed method and characterization of the compounds used in this study were previously reported by genio and paderes (2021). 1h nmr, 13c nmr, mass spectrometry, and ftir spectroscopy were among the methods performed to confirm the synthesis of the compounds. evaluation of low molecular weight bis-urea derivatives as antimicrobial agents 74 agar well diffusion method screening of the antimicrobial properties was performed by the microbial research and services laboratory (mrsl) of the natural sciences research institute (nsri), university of the philippines diliman using the agar well diffusion method. samples were all prepared in 98% ethanol using a 5 µg/ml concentration. ethanol, which is the solvent used for the sample solutions, was used as the negative control. for the positive controls, commercially available antibiotics cloxacillin (clox) and cefalexin (cef) were used. both compounds were prepared in ethanol at 5 µg/ml concentration to compare their antimicrobial activities. the organisms used were escherichia coli upcc 1195, klebsiella pneumoniae upcc 1360, and staphylococcus aureus upcc 1143. using peptone water as suspending medium, suspensions of the test organisms were prepared from 24 h old culture of microbes. these microbial suspensions were introduced and swabbed onto the surface of about 3 mm thick, pre-poured nutrient agar plates. the swab was distributed evenly twice over the agar surface. portions of 200 µl samples were placed on the three equidistant wells of 10 mm diameter made on the agar plate. the agar plates were left for 24 h at 35 °c and clearing zones were measured and used to calculate the antimicrobial index (ai) following the formula: ai = diameter of clearing zone – diameter of well diameter of well (1) formula for calculating antimicrobial index (ai) results and discussion as shown in scheme 2, the synthesis of low molecular weight bis-urea compounds was performed through condensation of various diisocyanates and amines. the reaction produced white powdery precipitates at 49-97% yields and were easily filtered off from the mixture (genio and paderes 2021). the solid products were washed several times with organic solvents such as diethyl ether to remove any unreacted or excess starting materials and ensure the purity of the bis-urea compounds. spectra obtained from the characterization of these compounds confirmed the formation of the urea groups and showed only peaks that correspond to the predicted product, implying high purity. more specifically, the absence of the diisocyanate peaks and the appearance of carbonyl peaks were noted in the ir spectra. proton peaks at a chemical shift of around 4-6 ppm were also observed in the 1h nmr spectra, indicating the successful formation of urea groups. f.a. f. genio and m. c. paderes 75 scheme 2. general scheme for the synthesis of bis-urea compounds. the synthesized bis-urea compounds, shown in figure 1, were tested for antimicrobial activity against gram-positive bacteria, s. aureus, and gram-negative bacteria, e. coli and k. pneumoniae. examples of the resulting wells from the assay using compound 6b are shown in figure 2. three wells in the agar plate were observed to have clearing areas where the microbial organisms did not grow. these areas were measured to calculate the antimicrobial index per compound against specific organisms. the results of the clearing zones in millimeters and the calculated antimicrobial indices of all sixteen compounds against each of the three organisms are summarized in table 1. for comparison, results for the antimicrobial activity of the commercial antibiotics, cloxacillin and cefalexin, prepared at 5 µg/ml using ethanol were included in table 1. the results show that all compounds, except 1d and 1e, exhibited zones of inhibition when tested against e. coli. however, only the antimicrobial indices of 2b, 4b, and 6b are significantly larger than those with the other derivatives. among the three compounds, 2b was observed to have the largest ai value, which is at 2.1. for the bacteria k. pneumoniae, most compounds displayed a zone of inhibition except for compounds 1a and 1d. similarly, good activity was observed for compounds 2b, 4b, and 6b, with 2b and 4b showing significantly larger ai (2.6 and 2.2, respectively). moreover, compounds 2b, 4b, 5b, and 6b also displayed notable ai values against s. aureus. generally, compounds containing long aliphatic end groups (2b, 4b, 5b, and 6b) demonstrated potential antimicrobial activity against the three organisms. the antimicrobial activities seemed to be dependent both on the linker and the alkyl end groups. for instance, bis-urea compounds with dicyclohexyl group as the linker (compounds 1) did not exhibit good activity against all the organisms tested. compounds with cyclohexyl group (2b) and aromatic linkers (4b, 5b, and 6b) on the other hand, displayed significant antimicrobial activity. evaluation of low molecular weight bis-urea derivatives as antimicrobial agents 76 n h n h n h n h o o n n n h n h n h n h o o n n n h n h n h n h o o n h n h n h n h o o 1a 1b 1c 1d 1e ch3(ch2)5 (ch2)5ch3 n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 compounds 1 n h n h n h n h o o n n n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 2a 2b compounds 2 compounds 3 n h n h n h n h oo nn n h n h n h n h oo (ch2)5ch3ch3(ch2)5 n h n h n h n h oo (ch2)11ch3ch3(ch2)11 3a 3c 3b compounds 4 compounds 5 n h n h n h n h o o n n 4a n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 4b compounds 6 o n h n h n h n h o o n n o n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 5a 5b n h n h n h n h o o n n n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 6a 6b figure 1. structures of synthesized bis-urea compounds. a b c figure 2. images of the clearing zones of compound 6b in agar well diffusion antimicrobial assay against a) e. coli, b) k. pneumoniae, and c) s. aureus. f.a. f. genio and m. c. paderes 77 table 1. results of antimicrobial activity assay samplea e. coli k. pneumoniae s. aureus clearing zone, mmb ai clearing zone, mmb ai clearing zone, mmb ai 1a 11.0 ± 0.0 0.1 0 12.0 ± 0.0 0.2 1b 11.0 ± 0.0 0.1 11.0 ± 0.0 0.1 12.0 ± 0.0 0.2 1c 11.0 ± 0.0 0.1 11.0 ± 0.0 0.1 12.0 ± 0.0 0.2 1d 0 0 11.0 ± 0.0 0.1 1e 0 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 2a 11.7 ± 0.6 0.2 12.7 ± 0.6 0.3 12.7 ± 0.6 0.3 2b 30.7 ± 0.6 2.1 36.0 ± 0.0 2.6 25.0 ± 0.0 1.5 3a 13.3 ± 0.6 0.3 14.0 ± 0.0 0.4 12.0 ± 0.0 0.2 3b 12.3 ± 0.6 0.2 14.0 ± 0.0 0.4 12.7 ± 0.6 0.3 3c 11.0 ± 0.0 0.1 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 4a 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 4b 21.0 ± 0.0 1.1 32.3 ± 0.6 2.2 20.0 ± 0.0 1 5a 12.3 ± 0.6 0.2 12.7 ± 0.6 0.3 12.0 ± 0.0 0.2 5b 15.0 ± 0.0 0.5 11.0 ± 0.0 0.1 25.3 ± 0.6 1.5 6a 12.0 ± 0.0 0.2 13.0 ± 0.0 0.3 12.7 ± 0.6 0.3 6b 24.3 ± 0.6 1.4 29.3 ± 0.6 1.9 16.3 ± 0.6 0.6 cloxc 13.3 ± 1.2 0.3 11.3 ± 0.6 0.1 25.7 ± 1.5 1.6 cefd 16.3 ± 1.2 0.6 12.3 ± 1.2 0.2 12.7 ± 1.2 0.3 etohe 12.0 ± 0.0 0.2 13.0 ± 0.0 0.3 12.0 ± 0.0 0.2 asamples were prepared at 5 µg/ml in ethanol; bclearing zone is the average of three trials; cclox = cloxacillin (positive control) at 5 µg/ ml in ethanol; dcef = cefalexin (positive control) at 5 µg/ml in ethanol; e etoh = ethanol; (-) no clearing zone was observed. the observed difference in the antimicrobial activity between compounds with the heteroaromatic end group (2a, 4a, 5a, and 6a) versus compounds with the aliphatic end groups (2b, 4b, 5b, and 6b) may be due to increased hydrophobicity brought by the long alkyl chain lengths (li et al. 2013). the hydrophobic nature of compounds that were previously analyzed from membrane binding and efficacy can be a factor affecting the compounds’ antimicrobial activity (kuroda et al. 2008). some of the antimicrobial indices obtained in this study are either comparable to or higher than the observed values for the antibiotics cloxacillin and cefalexin. compounds 2b, 4b, and 6b have significantly higher ai values than both clox and cef against the gram-negative bacteria, e. coli and k. pneumoniae. on the other hand, only 2b and 5b have comparable ai values with clox against the gramevaluation of low molecular weight bis-urea derivatives as antimicrobial agents 78 positive bacteria, s. aureus. these results imply the potential of these compounds as antimicrobial agents with properties that could be readily fine-tuned through structural modifications. however, further studies may be required to explore this potential antimicrobial property. conclusions sixteen bis-urea compounds were evaluated for their antimicrobial activity against e. coli, k. pneumoniae, and s. aureus. bis-urea compounds with long aliphatic side chains (2b, 4b, 5b, and 6b) showed significant activity against the tested organisms. compound 2b was found to be effective against all bacteria with ai values of 2.1 (e. coli), 2.6 for (k. pneumoniae), and 1.5 (s. aureus). compounds 4b and 5b were both selective with 4b being the most effective against k. pneumoniae and 5b against s. aureus. compound 6b on the other hand, showed activity with both e. coli and k. pneumoniae. further analysis of the potential of these bis-urea compounds as antimicrobial agents through antimicrobial assays against different organisms, cytotoxicity tests, and minimum inhibitory concentration assays can be conducted to explore more applications of these compounds as drugs or compatible ingredients to formulations. 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lamour k, chaloin o, frisch b, briand j, monteil h, guichard g. 2006. mimicking helical antibacterial peptides with nonpeptidic folding oligomers. chem biol. 13(5):531-538. https://doi.org/10.1016/j.chembiol.2006.03.009. yong y, xiao-li y, xue-gang l, jing z, baoshun z, lujiang y. 2007. synthesis and antimicrobial activity of 8-alkylberberine derivatives with a long aliphatic chain. planta med. 73(6):602604. https://doi.org/10.1055/s-2007-967180. ______ frances abygail f. genio earned her b.s. and m.s. in chemistry from the institute of chemistry, university of the philippines diliman. she is currently working as a university research associate i under projects funded by the natural sciences research institute (nsri), university of the philippines diliman. her research studies include synthesis and characterization of bis-urea molecules and their rheological and biological applications. monissa c. paderes <mcpaderes1@up.edu.ph> obtained her b.s. degree in chemistry at university of the philippines diliman, m.s. degree in chemistry at university of south carolina, columbia, and doctorate degree at state university of new york, buffalo. she worked as a research fellow at national university of singapore and as research scientist at ku leuven and procter & gamble in belgium. her expertise includes developing metal and organocatalyzed methods for the synthesis of organic compounds with biological applications and design and functionalization of polymers as rheology modifiers for hydrophobic and hydrophilic systems. sc i e n c e di l i m a n is published semi-annually (june and december) by the university of the philippines diliman through the office of the vice chancellor for research and development. the journal features articles of pure and applied sciences. editorial board editor in chief jonas p. quilang, ph.d. associate editors jose maria p. balmaceda, ph.d. university of the philippines diliman louis angelo m. danao, ph.d. university of the philippines diliman carlos primo c. david, ph.d. university of the philippines diliman christian n. della, ph.d. university of glasgow singapore arnold m. guloy, ph.d. university of houston marie antonette j. meñez, ph.d. university of the philippines diliman dennis i. merino, ph.d. southeastern louisiana university arnel a. salvador, ph.d. university of the philippines diliman terence p. tumolva, d.eng. university of the philippines diliman casiana blanca j. villarino, ph.d. university of the philippines diliman irene m. villaseñor, ph.d. university of the philippines diliman managing editor conchitina r. cruz, ph.d. university of the philippines editorial assistant narita e.c. de las alas layout artist mirriam m. velasco copyeditor jean lau wang on the cover: actual dynamite blast/ fishing (from da-bfar, location unknown), the spl of trail 4 (the loudest), and a small group of spinner dolphins (stenella longirostris) off southern tanon strait protected seascape (photo by lv aragones). contents of this journal may not be reproduced without the publisher’s written permission except for fair use, i.e. , for personal, educational and research purposes, in accordance with copyright law. reprinting and republication in any other journal or compilation is likewise prohibited except as provided in the publication agreement when the author reprints his/her article for inclusion in any publication where he/she is the author or editor, subject to giving proper credit to the original publication of the article in the journal. the views expressed in the articles are those of the authors and do not necessarily reflect the views of the publisher and the editors. science diliman january-june 2021 • vol. 33 no. 1issn print 0115-7809 issn online 2012-0818 international advisory board teofilo a. abrajano, jr., ph.d. king abdullah university of science and technology kingdom of saudi arabia rigoberto c. advincula, ph.d. case school of engineering case western reserve university, usa kenneth a. buckle, ph.d. professor emeritus school of chemical engineering the university of new south wales, australia jose b. cruz, jr., ph.d. professor emeritus university of illinois, usa university of california, irvine, usa the ohio state university, usa john p. encarnacion, ph.d. department of earth and atmospheric sciences saint louis university, usa mihali a. felipe, ph.d. gerstein lab, bioinformatics yale university, usa robert j. howell, ph.d. department of mechanical engineering the university of sheffield, united kingdom jeanmaire e. molina, ph.d. department of biology long island university, brooklyn, usa rudolf a. roemer, ph.d. department of physics university of warwick, united kingdom raul k. suarez, ph.d. professor emeritus university of california, sta. barbara, usa myra o. villareal, ph.d. life and environmental sciences graduate school university of tsukuba, japan 8ahernandez-singson-short com.pmd d.b. b. singson and c.l. chichioco-hernandez 97 science diliman (july-december 2017) 29:2, 97-101 angiotensin-converting enzyme inhibitory action of selected plants dionisio bong b. singson university of the philippines diliman christine l. chichioco-hernandez* university of the philippines diliman _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online hypertension is the fourth leading cause of morbidity in the philippines affecting more than 20% of filipino adults (doh 2013). recognized as the “silent killer”, it gradually destroys the body without any symptoms. untreated hypertension may lead to stroke, blindness, heart attack, and kidney and heart failure (chobanian et al. 2 0 0 3 ) . angiotensin-converting enzyme (ace) is a key metalloprotease in the reninangiotensin-aldosterone system. it catalyzes two reactions: conversion of the inactive decapeptide angiotensin i into the vasoconstrictor and salt-retaining octapeptide angiotensin ii, and the hydrolysis of the vasodilator bradykinin which is contributory in lowering blood pressure. these lead to the elevation of blood pressure in an individual (ferrario 2010). inhibition of ace is central in preventing hypertension (cushman et al. 1977). one way to lower blood pressure is through the use of ace inhibitors (chobanian et al. 2003).however, its usage is associated with unwanted side effects, such as nephrotoxicity and congenital malformations (adhiyaman et al. 2001; cooper et al. 2006).it is necessary to look for other blood pressure lowering compounds that are equally effective but with minimal side effects. several studies have tried to look for other potential ace inhibitors from various plant sources (adsersen and adsersen 1997; tsutsumi et al. 1998; duncan et al. 1999; somanadhan et al. 1999; oh et al. 2002; loizzo et al. 2007). in this study, extracts from bixa orellana (bixaceae), artocarpus heterophyllus (moraceae), morus alba (moraceae), nymphaea pubescens (nymphaeacea) and syzygium samarangense (myrtaceae) were evaluated for their anti-hypertensive potential using an aceinhibitory colorimetric assay. angiotensin-converting enzyme inhibitory action of selected plants 98 the yields of the different extracts from the f ive plants are reported in table 1. results of the ace inhibitory assay are shown in figure 1. table 2 shows the outcomes of the phytochemical screening of the different methanol extracts. figure 1. average percent inhibition of the various extracts. a. heterophyllus 184.56 3.41 34.27 19.66 b. orellana 177.19 8.95 24.58 20.38 m. alba 171.21 6.61 17.31 5.77 n. pubescens 104.93 11.58 19.93 4.86 s. samarangense 162.3 5.67 16.54 16.54 plant weight of dried leaves (g) yield methanol extract (%) yield hexane extract (%) yield ethyl acetate extract (%) table 1. percent yield of leaves and extracts a. heterophyllus + + + + + + b. orellana + + + + + m. alba + + + + + n. pubescens + + + + + + s. samarangense + + + plant saponins flavonoids card iac glycosides phenol ic compounds alkaloids terpenoids tannins table 2. phytochemical profile of the methanol extracts d.b. b. singson and c.l. chichioco-hernandez 99 the hexane and ethyl acetate extracts of b. orellana and s. samarangense exhibited signif icant inhibition of ace activity, with their percent inhibition values exceeding 50%. it is notable that these two plants are positive for the presence of flavonoids. this is consistent with previous f indings attributing effective ace inhibitory activity to flavonoids (lacaille-dubois et al. 2001; perez-viscaino et al. 2009; jimenezferrer et al. 2010; balasuriya and rupasinghe 2011; balasuriya and rupasinghe 2012). flavonoids were previously identif ied from the leaves of s. samarangense (nair et al. 1999). other syzygium species were found to contain flavonoids (samy et al. 2014). flavonoid bisulphates were previously identif ied from the extracts of b. orellana (harborne 1975). several ace inhibitory flavonoids were isolated and identif ied from the ethanol extract of erythroxylum laurifolium leaves (hansen et al. 1996). flavan-3-ols and procyanidins displayed inhibitory activity against ace and the effect was observed to be dependent on the number of the epicatechin units forming the procyanidin (actis-goretta et al. 2003) epidemiological studies have shown the link between food consumption and protective effect of compounds against cardiovascular disease. several lines of evidence support that dietary intake of flavonoids from cocoa have a wide range of health benef its including endothelial function, blood pressure, inflammation, and oxidative stress (grassi and ferri 2014). in a double-blind, placebo-controlled, parallel trial, chokeberry flavonoids were shown to have a clinical potential in preventing ischemic heart disease because of their ability to reduce the severity of inflammation (naruszewicz et al. 2007). isolation and identification of the compounds responsible for the inhibition of ace activity are ongoing. the activities of the b. orellana and s. samarangense extracts in hypertensive-induced mice will be evaluated using the non-invasive tail-cuff blood pressure measurement method. supplementary material review of literature pertinent to the plants and experimental details relating to this paper are available online. acknowledgements this work was partially funded by the department of science and technology (dost) through the philippine council for health research and development (pchrd). angiotensin-converting enzyme inhibitory action of selected plants 100 references ac t i s g o r e t t a l , o t t a v i a n i j i , ke e n c l , f r a g a c g . 2 0 0 3 . i n h i b i t i o n of a n g i o te n s i n co n ve r t i n g e n z y m e ( ac e ) a c t i v i t y by f l a v a n 3 o l s a n d p r o cy a n i d i n s . f e b s le t te r s . 555(3):597-600. adhiyaman v, asghar m, oke a, white ad, shah iu.2001.nephrotoxicity in the elderly due to co-prescription of angiotensin converting enzyme inhibitors and nonsteroidal anti-inflammatory drugs. journal 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[ i n te r n e t ] . 2 0 0 3 . le a d i n g c a u s e s o f m o r b i t y. m a n i l a : depar tment of health.[cited 2016 november 23].available from:http: //www.doh.gov.ph/ node/1482. d u n c a n a c , j ä g e r a k , v a n s t a d e n j . 1 9 9 9 . s c r e e n i n g o f z u l u m e d i c i n a l p l a n t s f o r angiotensin conver ting enzyme (ace) inhibitors. journal of ethnopharmacology. 68(13):63-70 ferrario c.2010. addressing the theoretical and clinical advantages of combination therapy with inhibitors of the renin-angiotensin-aldosterone system: antihypertensive effects and benef its beyond bp control. life science.86(9-10):289-299. grassi d, ferri c. 2014.chapter 78 – cocoa, flavonoids and cardiovascular protection. polyphenols in human health and disease. 2:1009-1023. hansen k, adsersen a , smitt u w, n y m a n u , c h r i s t e n s e n s b , s c h w a r t n e r c , wa g n e r h.1996. angiotensin converting enzyme (ace) inhibitory flavonoids from erythroxylum d.b. b. 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krishnan s, ravikrishna c, madhusudanan kp. 1999.new and rare flavonol glycosides from the leaves of syzygium samarangense. fitoterapia.70(2):148-151. narusze wicz m , la n i ew s k a i , m i l l o b , d l u z n i e w s k i m . 2 0 0 7. co m b i n a t i o n t h e r a py of s t a t i n w i t h f l a v o n o i d s r i c h e x t r a c t f r o m c h o k e b e r r y f r u i t s e n h a n c e d r e d u c t i o n i n cardiovascular risk markers in patients after myocardial infarction (mi). atherosclerosis. 194(2):e179-e184. oh h, kang dg, lee s, lee sh.2002.angiotensin conve r ting enzyme inhibitors from cuscuta japonica choisy. journal of ethnopharmacology.83(1-2):105-108. perez-vizcaino f, duarte j, jimenez r, santos-buelga c, osuna a. 2009.antihypertensive effects of the flavonoid quercetin. pharmacological repor ts. 61(1):67-75. samy mn, sugimoto s, matsunami k, otsuka h, kamel ms. 2014.one new flavonoid glycoside and one new natural triterpene rhamnoside from the leaves of syzygium grande. phytochemistry letters. 10:86-90. somanadhan b , varughese g, palpu p, sre edharan r, gudiksen l, smitt uw, nyman u.1999.an ethnopharmacological survey for potential angiotensin converting enzyme inhibitors from indian medicinal plants. journal of ethnopharmacology.65(2):103-112. tsutsumi y, shimada a , miyano a , nishida t, mitsunaga t.1998.in vitro screening of a n g i o t e n s i n i c o n v e r t i n g e n z y m e i n h i b i t o r s f r o m j a p a n e s e c e d a r ( c r y p t o m e r i a japonica).journal of wood science.44(6):463-468. _____________ dionisio bong b. singson is a bs chemistry graduate of the university of the philippines diliman and a former instructor of the institute of chemistry. christine l. chichioco-hernandez <cchernandez@up.edu.ph> is an associate professor and director of the institute of chemistry, university of the philippines diliman. her research area focuses on bioactive compounds from terrestrial plants. ocr document guidelines-revised.pmd guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions: ü if changes are made, choose a new title for the paper. ü use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality. ü reference your conference paper in the appropriate locations. ü include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.” ü indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed. ü provide the original conference paper (upload a pdf file during the submission process). ü if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions: ü expand the background section and include additional references. ü include novel scientific content and expanded descriptions of procedures. ü provide data that was not published at the conference. ü revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission from the editors of ieee sensors journal) ocr document ocr document 01_device survivorship and growth performance of red spiny lobster 11 survivorship and growth performance of red spiny lobster panulirus longipes longipes reared in floating netcages fed with sardinella spp at different feeding rates *corresponding author joel g. becira and marivel orcajada western philippines university-puerto princesa campus puerto princesa city email: j_becira@yahoo.com abstract this study was conducted to determine the survivorship and growth performance of panulirus longipes longipes fed with sardinella spp. in floating netcages at different feeding rates and to determine which feeding rate provides the best feed conversion ratio. results showed that a feeding rate of 20% gave the highest growth (final average weight) followed by 15% and 10% feeding rates, respectively. in terms of length increment, 20% feeding rate likewise promoted the highest increment. differences in feed conversion ratio among treatments was significant (p<0.05). among the different treatments however, stocks fed at 15% body weight attained better growth increment and trend in feed conversion ratio. keywords: feeding rates, panulirus longiceps longiceps, cage culture, sardinella spp. introduction panulirus longipes, a long-legged spiny lobster (ravago-gotanco et al. 2003) had pointed spines on their carapace and antenna (shokita 1991). the body of p. longipes longipes is red, walking legs are red with many white spots; each abdominal somite has many small white spots (gonzales and taniguchi 1995). lobsters are favored food in many countries because of their fine flavor. even in this age of abundant food supply, demand for luxury food like this exceeds their supply (shokita et al. 1991). lobsters have excellent market demand and price. live lobsters commands an even higher price compared to frozen lobsters (james and marian 2003) as they are important export items and expensive delicacies (pcarrd 1981). lobster business today is a lucrative fishing enterprise because of its unlimited demand (campomanes 1992). according to bardach et al. (1972), lobster p. japonicus can be cultured in ponds and shallow bays. in guimaras, philippines, p. ornatus, p. versicolor and p. longipes longipes were cultured in pens (tambuli 1988). in palawan, lobsters were temporarily stocked in submerged cages to keep the animals' alive (gonzales and taniguchi 1995) before marketing. according to gonzales and taniguchi (1995), lobster > 100g should not be sold to any fishery market to properly benefit man. they should be released in its science diliman (january-june 2006) 18:1, 11-17 becira and orcajada 12 natural habitat to grow further or grown in cages or in pens until they reach a marketable size (150 grams or more). however, knowledge on the biology of many crustacean species is still inadequate and it needs to be extended through further information dissemination and experimentation (gonzales and taniguchi 1995). since this species of lobster is a high value and their population is relatively low compared with the other species of lobster (ravago-gotanco et al. 2003), they are vulnerable to overexploitation. to lessen population pressure in the wild, this study was conducted to determine the feasibility of culturing panulirus longipes longipes. specifically, this study was conducted to determine the growth performance of red spiny lobster (panulirus longipes longipes) reared in floating netcages fed with chopped sardinella spp. at different feeding rates, and to determine which feeding rate can provide the best feed conversion ratio. methodology the lobster culture was conducted near the western philippines university puerto princesa campus fish cage project in tiniguiban cove, puerto princesa city (figure 1) located 09°46.464 n and 118° 43.898 e. red spiny lobsters were collected from the offshore of sabang, puerto princesa city. forty five (45) individuals were collected and soaked in iced seawater for 3 to 5 seconds until they stopped moving. the animals were then wrapped individually by a piece of newspaper, and systematically arranged inside the styrofoam box. the lobsters were transported early in the morning to the study area. upon arrival, the box was opened and the experimental animals were unwrapped individually inside the cage. lobster was held at surface of water, until they swam freely in the prepared cages. the floating netcages made up of bamboo poles (3.5 m. in length) were divided into 9 (1m x 1m) cage frames. a polyethylene b-net 1m x 1m x 1.5m with a mesh size of 0.25 cm was used as netcages. these netcages were firmly attached to the cage frame. reinforcement bar measuring 1 x 1 meter was placed inside the cage as sinker (plate 1). for protection of cage net, perimeter net (2.50cm mesh size) was installed. figure 1. map of puerto princesa bay showing the experimental site. plate 1. installation of sinker in the floating net cages used in the study. upon arrival in the experimental site, the box was opened and the experimental animals were unwrapped individually inside the cage. lobsters were held at surface of water until they swam freely at a stocking density of 5 pieces per cage. initial weight and carapace length of lobsters to be experimented ranged from 43.13 to 45.80g and 3.45 to 3.56cm, respectively. three feeding rates (20%, 15% and 10%: n=5) with three replicates were used in the study. replicates for each feeding rate were assigned in the netcages using completely randomized design. the red spiny lobster was fed with fresh or frozen chopped sardinella spp. feed was given once a day at 5 to 6 pm. individual weight and carapace length (pascual et al. 1980) of experimented red spiny lobsters were survivorship and growth performance of red spiny lobster 13 measured using tanita™ weighing scale (500g capacity) and a vernier caliper, respectively at the start of the rearing period and every 25 days thereafter for 75 days. sampling was done early in the morning. water temperature and salinity were determined daily. the mean average body weight and carapace length among feeding rate were analyzed using analysis of variance (gomez and gomez 1984). feed conversion ratio (fcr) in each treatment was computed by dividing the total feed consumed with the weight gain (pascual 1989) after 75 days culture period, growth increment (weight and carapace length) and survival rate in every 25 days culture period were also computed. results and discussion survivorship one hundred percent survival were obtained from 15% and 10% feeding rates while 20% feeding rate had a survival rate of only 86.67% as an effect of a mortality of two lobsters in its two replicates, although differences among survival rates in the different feeding rates was not significant (p>0.05). in spite of it, the average survival rate in this study (95.56%) was higher compared to the 90% expected survival rate of lobsters grown in netcages (tambuli 1998). though survival and growth of lobsters are affected by temperature, salinity and pollutants, temperature is the most important environmental factor that affects the growth rate of lobsters (phillips et al. 1980). the water temperature in experimental site ranged from 28.17 to 31.07oc. water salinity within the cages ranged from 29.43 to 30.83ppt while dissolved oxygen ranged from 4.77 to 5.5mg/l. the values obtained in the experimental site for temperature and salinity was almost similar to the criteria established by phillips et al. (1980), aiken (1980) and van olst et al. (1980) in which water temperature in the culture cage must range from 29 to 32oc and the salinity should be 30 to 34ppt for better production. temperature higher than 32°c is detrimental for lobster's growth and survival (aiken 1980); however, van olst et al. (1980) and phillips et al. (1980) stated that lobsters can tolerate temperature as high as 32°c and as low as 18oc provided salinity must ranged from 30-35ppt (lee and wickins 1992). although lobster could adjust to a wide range of salinity (stewart 1980), salinity fluctuations are naturally occurring and causing environmental stress in cultured lobster, even lobster is a euryhaline and osmoconformer species. in relation to this, phillips et al. (1980) further stated that seven percent salinity differential from 30 and 34ppt would cause high mortality. mortality in cultured lobsters is also caused by the amount of dissolved oxygen (radhakrishan and vijayakirmaran 1984). juinio-menez and ruinta (1996) as cited by salvacion, (2000) stated that mortality occurred when dissolved oxygen is less than 4.0 mg/li which is conforming to the statement of booth and kittaka (1994), that lethal level of dissolved oxygen for lobster culture ranging from 0.5 to 3.0 mg/li though this was not observed in the study area. thus, the high survival rate of cultured red spiny lobsters in tinguiban cove will probably due to the favored conditions in the experimental site though cannibalism of molted lobster was observed in one of the replicates lobster at 20% feeding rate. growth performance weight and length the lobster fed with chopped sardinella spp. at 20% feeding rate obtained the highest average weight gain (table 1). a final average body weight of 66.13g, 65.87g, and 63.73g were obtained from 20%, 15% and 10%, respectively. during 50 days of culture, p. longipes longipes fed at 20% and 10% body weight were almost similar in weight (59.22g. and 59.33g.). however, it was then observed that body weight of stocks fed at 15% body weight rapidly increased towards the end of the 75 days culture period (table 1), although it had the lowest initial average body weight (43.13 g) while stocks at 20% feeding rate constantly gain weight (22.87g) until it reached 75 days of culture period. an apparent increase in average weight was observed in 15% feeding rate until 75 days reaching 65.87 grams. on the other hand, the 10% feeding rate had the highest body weight in the first 25 days and slows down its growth thereafter while the lobsters fed at 20% feeding rate obtained the highest final average weight (66.13g) becira and orcajada 14 in 75 days culture period. differences in gain and final weights among feeding rates were not significant (p>0.5), which is consistent with the condition at the starts of study. initial carapace length of lobsters being cultured ranged from 3.45cm to 3.56cm (table 1). lobster fed at 15% feeding rate obtained the highest final average carapace length of 4.06cm followed by 20% (4.03cm) and 10% (3.95cm), respectively. these results in carapace length (0.052-0.054cm/day carapace length increment) were comparable and even higher with the findings of smale (1978) regarding the growth of the juveniles of p. homarus in south africa which had a carapace length increment of 0.01cm/day. the total weight gained of animals in the cages which were fed equivalent to 20% of total biomass was 22.87g; followed by 15%, 22.73g. feeding rate of 10% only attained a gained in weight of 17.93g which could be reflected to the highest metabolic rate of organisms at the early age where feeds given were not enough to sustain lobsters' physiological processes. the total carapace length gained in experimental lobster was almost similar in 20% (0.58cm) and 15% (0.55cm). the lowest carapace length gain was with 10% feeding rate (0.39cm). the differences in lobsters' carapace length gains were not significant at 5% level of significance. weight and carapace length increment feeding rate of 20% body weight had the highest average weight increment (0.1016g/day). it was followed by the lobster fed at 15% (0.1011g/day). lobster fed at 10% had the lowest average weight increment with only 0.0797g/day (table 2). lobster fed at 20% feeding rate got the highest weight increment (0.0544 and 0.1582g/day) for the first 25 and 50 days culture period. it was followed by the lobsters given 10% feeding rate (0.0436 and 0.1368g/ day). however, after 75 days culture period, lobsters fed at 15% feeding rate showed a consistent increase in weight (table 2) unlike with the lobsters which fed at 20% and 10% feeding rate, respectively. lobster fed at 20% feeding rate had the highest average carapace length increment (0.0077cm/day), followed by 15% (0.0073cm/day) and 10% (0.0017cm/day). during the initial 50 days culture period, highest growth increment in carapace length was observed in 20% (0.0092cm/day). lobsters fed at 10% feeding rate obtained a low carapace length increment towards the end of the culture period probably due to molting (two lobsters molted when the study ended). likewise, consistent increase was observed at 15% feeding rate and continuously increases towards the end of 75 days culture period resulting to the highest increase in carapace length which was probably attributed to the amount of feeds intake (table 2). table 1. average body weight and carapace length of panulirus after 75 days culture period. feeding rate 20% 15% 10% days of culture carapace carapace carapace weight length weight length weight length (g) (cm) (g) (cm) (g) (cm) 43.27 3.45 43.13 3.51 45.8 3.56 0 ±1.15 ±0.11 ±2.37 ±0.11 ±2.31 ±0.02 47.35 3.62 45.53 3.54 49.07 3.71 25 ±2.48 ±0.09 ±2.80 ±0.13 ±2.76 ±0.05 59.22 3.85 51.93 3.73 59.33 3.91 50 ±3.20 ±0.08 ±5.49 ±0.12 ±3.41 ±0.06 66.13 4.03 65.87 4.06 63.73 3.95 75 ±2.68 ±0.06 ±3.41 ±0.00 ±5.84 ±0.17 gain 22.87 0.58 22.73 0.55 17.93 0.39 survivorship and growth performance of red spiny lobster 15 the results in growth of p. longipes longipes showed that a feeding rate of 10% and 20% respectively obtained an almost similar faster growth at the start while 15% feeding rate slowly improved during the 50 days culture period. as cultured lobster became older the feeding rate of 15% obtained a higher growth in carapace length and body weight followed by 20% and slow growth rate was observed in 10% which could be reflected to the amount of food being intake. for lobster fed at 20% feeding rate, more leftovers had been collected. amount of leftovers were enough to affect the water quality and probably the cause of mortality in two replicates of 20% feeding rate. on the other hand, lobsters fed at 10% were very much active during the first culture period. since the feeding rate is low, the amount of feeds given to the stocks when they become older probably may be not enough, resulting in the reduction of growth increment on both weight and carapace length. feed conversion ratio cultured lobsters fed at 20% feeding rate consumed the highest amount of feeds after 75 days of culture (table 3). it was followed by the lobsters fed at 15% and 10% with 22.73kg and 17.93kg, respectively. as a result, lobster fed at 20% body weight obtained the poor feed conversion ratio (table 3). although, pascual (1989) stated that the lower the fcr, the better it is because less feed is required per unit weight gain as shown in 10% feeding rate (table 3), the feeding rate of 15% showed the best trend in feed conversion ratio (figure 2) which also showed better weight and carapace length increment (table 2).figure 2. trend of feed conversion ratio among three feeding rates. fe ed c on ve rs io n r at io 0.3 0.6 0.9 1.2 0-25 26-50 51-75 days of culture 20% 15% 10% table 2. growth increment of panulirus longipes longipes at tiniguiban cove, puerto princesa bay. feeding rate 20% 15% 10% days of culture carapace carapace carapace weight length weight length weight length increment increment increment increment increment increment (g/day) (cm/day) (g/day) (cm/day) (g/day) (cm/day) 0 25 0.0544 0.0068 0.0320 0.0012 0.0436 0.0020 50 0.1583 0.0092 0.0853 0.0076 0.1368 0.0027 75 0.0921 0.0072 0.1859 0.0132 0.0587 0.0005 total 0.3048 0.0232 0.3032 0.0220 0.2391 0.0052 average 0.1016 0.0077 0.1011 0.0073 0.0797 0.0017 becira and orcajada 16 differences in feed conversion ratio (fcr) between and among feeding rates were significant (p<0.05), and significant difference (p<0.01) was also observed between 20% and 10% feeding rates. conclusion and recommendations based on this study, the differences in weight among feeding rates were not significant though significant difference was found on their feed conservation ratio. weight and carapace length increment is useful in determining the feasibility of designed feeding rates. to get better results, this study should be conducted for a minimum of 6 months to determine how efficient the feeds be converted into flesh of lobster taking different feeding rates. acknowledgements the authors would like to acknowledge the faculty of aquaculture section, college of fisheries and aquatic sciences of the western philippines university puerto princesa campus for their untiring support. likewise, efforts of the two reviewers of the science diliman is gratefully acknowledged. references aiken, d. e., 1980. molting and growth. in j. s. cobb and b.f. phillip (editors). the biology and management of lobster. vol. i. press, new york. pp 91-164. bardach, j. e, j.h. ryther, and w. o. mclarney, 1972. aquaculture: the farming and husbandry of freshwater and marine organisms, wiley interscience, a division of john wiley and sons new york 1000 pp. campomanes, r.m., 1992. nesmarrdec newsletter. lobster can be raised in fish pen transfer. gomez, k.a & a.a. gomez, 1984. statistical procedures for agricultural research. 2nd ed. john wiley and sons, inc. canada. 680 pp. gonzales, b.j. and n. taniguchi. 1995. spiny lobsterfishery in palawan, philippines with considerations on its conservation and management. research journal for lobster. pp. 123-124. james, c. m. and marian, p., 2003. lobster fattening and fishery in india. infofish international. aquaculture. pp 9-11. lee, d. o'c., and j. f. wickens, 1992. crustacean farming. blackwell science ltd. pp.17. lellis, w. 1991. "spiny lobster: a mariculture candidate for the caribbean". as cited in: mohan, r. 2001. tropical spiny lobster a new mariculture species for the sultanate of oman and the arabian gulf states. in goddard, s., aloufi, h., mcilwain, j., and claereboudt, m. eds. proc. 1st international conference on fisheries, aquaculture and environment in the nw indian ocean, sultan qaboos university, muscat, sultanate of oman, pp. 158-164. pascual, f. p. 1989. nutrition and feeding of penaeus monodon, aquaculture extension manual no. 3 seafdec aquaculture department, tigbauan, iloilo, philippines, pp.15 pcarrd. 1981. state of the art. marine invertebrates research. los baños, laguna. pp 3-4. phillips, e.s., j.s. cobb and r.w. george. 1980. general biology. in: j.s., cobb and e.s. phillips (editors). the biology and management of lobster. vol. i. press, new york. pp. 1-82. ravago-gotanco, r. and m.a.r. juinio-menez. 2003. phylogenetic position of the striped-legged forms of panulirus longipes (a. milne-edwards, 1868) (decapoda,palinuridae) inferred from mitochondrial dna sequences. crustaceana., 75:1047-1059. days of culture feeding rate 20% 15% 10% 0 25 3.24525 2.42606 1.71750 26 50 3.55125 2.56106 1.84013 51 75 4.44150 2.92106 2.22488 total 11.238 7.908 5.78251 total weight gain (g) 22.87 22.73 17.93 fcr 0.491 0.348 0.322 table 3. total feeds consumed in kg after 75-day culture survivorship and growth performance of red spiny lobster 17 salvacion, r.c., 2000. feed conversion efficiency of growth of bamboo green lobster (panulirus versicolor) with different types of feed cultured in floating netcages, in tiniguiban cove, puerto princesa city. unpublished undergraduate thesis. western philippines university puerto princesa campus, puerto princesa city shokita, s., k. kakazu, a. tomori, and t. toma 1991. aquaculture in tropical areas. toshima ku, tokyo, japan. pp. 204-210. smale, m.j. 1978. migration, growth and feeding in the natal rock lobster panulirus homarus (linnaeus). as cited in: mohan, r. 2001. tropical spiny lobster a new mariculture species for the sultanate of oman and the arabian gulf states. in goddard, s., al-oufi, h., mcilwain, j., and claereboudt, m. eds. proc. 1st international conference on fisheries, aquaculture and environment in the nw indian ocean, sultan qaboos university, muscat, sultanate of oman, pp. 158-164. stewart, j.e., 1980. disease. in: j.s cobb and b.f phillips (editors). the biology and management of lobsters. vol. i. press new york. pp. 307-342. tambuli. 1998. enterprise alternative: lobster farming no. 4 a publication for coastal practitioners pp. 28-32. van olst, j.c., carlberg, j.m. and hughes, j.t., 1980. aquaculture. in: j.s cobb and b.f phillips (editors). the biology and management of lobsters. vol. ii. press new york. pp. 333-384. research dissemination office office of the vice chancellor for research and development lower ground floor. phivolcs bldg., c.p. garcia ave., up diliman 1101 quezon city (632)3436-8720 fax (632) 8927-2568 research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice chancellor for research and development (ovcrd): humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is php 650.00. (subscription price is subject to change without prior notice.) i/we would like to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php 650) humanities diliman sciencediliman social science diliman grand total method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman ovcrd)  money remittance (payable to anna angelica p. angala, c/o ovcrd research dissemination office, with office address as indicated above and telephone no. (632) 3436-8720. subscriber details name/institution contact person (for institutional subscribers) mailing address email address telephone no. fax no. please send accomplished subscription form to the rdo-ovcrd via email or fax (please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. 84 information for authors 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the office of the vice chancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out. the letter to the editor usually contains these: that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1” margins on all sides. each page of the manuscriptmust include continuous line numbers in the margin. all pages should be numbered consecutively on the upper right hand corner of the page. 7. page 1 should contain the article title, author(s), affiliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 85 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. the author must also submit a layman’s abstract of not more than 200 words. 9. the paper should be organized as follows: abstract and layman’s abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, figures, tables, and figure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be defined at first mention. 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the first author’s name plus “et al.” need to be given. references in the text should follow the council of science editors (cse) scientific style and format, 8th edition, 2014. examples: articles from journals: print format: author(s). date. article title. journal title. volume(issue):location. example: smart n, fang zy, marwick th. 2003. a practical guide to exercise training for heart failure patients. j card fail. 9(1):49–58. 86 articles from journals: online format: author(s) of article. date of publication. title of article. title of journal (edition). [date updated; date accessed];volume(issue):location. notes. example: savage e, ramsay m, white j, beard s, lawson h, hunjan r, brown d. 2005. mumps outbreaks across england and wales in 2004: observational study. bmj. [accessed 2005 may 31];330(7500):1119–1120. http://bmj.bmjjournals.com/ cgi/reprint/330/7500/1119. doi:10.1136/bmj.330.7500.1119. articles from newspapers: print format: author(s). date. title of article. title of newspaper (edition). section:beginning page of article (column no.). example: weiss r. 2003 apr 11. study shows problems in cloning people: researchers find replicating primates will be harder than other mammals. washington post (home ed.). sect. a:12 (col. 1). books: print format: author(s). date. title. edition. place of publication: publisher. extent. notes. example: schott j, priest j. 2002. leading antenatal classes: a practical guide. 2nd ed. boston (ma): books for midwives. books: online format: author(s). date of publication. title of book. edition. place of publication: publisher; [date updated; date accessed]. notes. example: griffiths ajf, miller jh, suzuki dt, lewontin rc, gelbart wm. c2000. introduction to genetic analysis. 7th ed. new york (ny): w. h. freeman & co.; [accessed 2005 may 31]. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.view.. showtoc&rid=iga. toc. 87 book chapter with editors format: author(s). date of publication. chapter title. in: editor(s) of book. title of book. edition. place of publication: publisher. pages of the chapter. example: allen, c. 2007. bacteria, bioterrorism, and the geranium ladies of guatemala. in: cabezas al, reese e, waller m, editors. wages of empire: neoliberal policies, repression, and women’s poverty. boulder (co): paradigm press. p. 169-177. without editors format: author(s). title of article. in: title of book.  edition. place of publication: publisher; date of publication. notes. example: hazeltine wa. aids. in: the encyclopedia americana. international ed. danbury (ct): grolier incorporated; 1990. p. 365–366. conference proceedings/papers published without author(s) format: editor(s). date. title of book. number and name of conference; date of conference; place of conference. place of publication: publisher. extent. notes. example: callaos n, margenstern m, zhang j, castillo o, doberkat ee, editors. c2003. sci 2003. proceedings of the 7th world multiconference on systemics, cybernetics and informatics; orlando, fl. orlando (fl): international institute of informatics and systematics. published with author(s) format: author(s) of paper. date. title of paper. in: editor(s). title of book. number and name of conference; date of conference; place of conference. place of publication: publisher. location. notes. 88 example: lee dj, bates d, dromey c, xu x, antani s. c2003. an imaging system correlating lip shapes with tongue contact patterns for speech pathology research. in: krol m, mitra s, lee dj, editors. cbms 2003. proceedings of the 16th ieee symposium on computer-based medical systems; new york. los alamitos (ca): ieee computer society. p. 307–313. unpublished format: author(s). date of the conference. title of paper. paper presented at: title of conference. number and name of the conference; place of the conference. example: antani s, long lr, thoma gr, lee dj. 2003. anatomical shape representation in spine x-ray images. paper presented at: viip 2003. proceedings of the 3rd iasted international conference on visualization, imaging and image processing; benalmadena, spain. technical reports format: author(s). date. title of report. edition. place of publication: publisher. extent. report no.: notes example: feller ba. 1981. health characteristics of persons with chronic activity limitation, united states, 1979. hyattsville (md): national center for health statistics (us). report no.: vhs-ser-10/137. available from: ntis, springfield, va; pb88-228622. dissertations and theses format: author(s). date. title of dissertation or thesis [content designator]. place of publication: publisher. extent. notes. example: lutz m. 1989. 1903: american nervousness and the economy of cultural change [dissertation]. [stanford (ca)]: stanford university. 89 group/corporate author format: [abbreviation of group] name of group (country). date. title. place of publication. publisher. notes. example: [iom] institute of medicine (us). 1975. legalized abortion and the public health: report of a study by a committee of the institute of medicine. washington (dc): national academy of sciences (us). other internet materials homepage format: title of homepage. date of publication. edition. place of publication: publisher; [date updated; date accessed]. notes. example: apsnet: plant pathology online. c1994–2005. st paul (mn): american phytopathological association; [accessed 2005 jun 20]. http://www.apsnet. org/. for more detailed examples please refer to the cse manual 8th edition. 12. the list of references at the end of the paper should include only works mentioned in the text and should be arranged alphabetically by the name of the author. 13. responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. 14. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 15. figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each figure or table on a separate page. at the lower hand corner, the name of the author and the figure number should be indicated. high resolution images must accompany the manuscript. 90 16. illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2” x 11” size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructed photo-graphic prints (not photocopies), trimmed at right angles and in the final size desired by the author.. 17. when possible, all organisms must be identified by the scientific binomen. 18. mathematical equations should be clearly presented so that they can be interpreted properly. 19. obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 20. all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc., especially those adopted by the council of science editors (cse) scientific style and format, 8th edition, 2014. 22. less common abbreviations may be printed as footnotes. 23. short communications must be guided by the following points: • short communications are reports of limited data or important findings that warrant publication before the completion of the study; • short communications are reports of significant new data arising from problems with narrow, well defined limits before broader studies are completed; and results have not been published in print elsewhere, except as partial communications or posters in conference proceedings; • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; 91 • abstracts will be required on submission but not to be part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages) in length with simple layout, a maximum of two tables and two figures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors should submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor in chief science diliman office of the vice chancellor for research and development university of the philippines lower ground floor, phivolcs bldg., c.p. garcia avenue up campus, diliman, quezon city 1101, philippines sd-sample article 74 1. science diliman is a journal of pure and applied sciences. considered for publication are primary and original papers. review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out; that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1" margins on all sides. all pages should be numbered consecutively on the upper right hand corner of the page. 7. page 1 should contain the article title, author(s), aff iliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. information for authors 75 9. the paper should be organized as follows: abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, f igures, tables, and figure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be def ined at f irst mention. 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the f irst author’s name plus “and others” need to be given. references in the text should follow the council of biology editors (cbe) style system (cbe manual, 6th ed.). 12. the list of references at the end of the paper should include only works mentioned in the text and should be arranged alphabetically by the name of the author. 13. responsibility for the accuracy of bibliographic references rests entirely with the author, who is requested to use as few “in press” citations as possible. “in press” citations must include the name of the journal that has accepted the paper. 14. footnotes in the text should be numbered consecutively. footnotes to the title or authors of the article are marked by asterisks and placed on the title page. 15. figures and graphs should always be mentioned in the text and numbered with arabic numerals. a brief descriptive caption should be provided for each f igure or table on a separate page. at the lower hand corner, the name of the author and the f igure number should be indicated. 1 6 . illustration hard copy should comprise: line drawing should be of good quality and should not exceed 8 1/2" x 11" size paper, with clearly legible inscriptions, even if reduced to 85% of their size. photographs/illustrations: well-constructed photo-graphic prints (not photocopies), trimmed at right angles and in the f inal size desired by the author. 76 17. when possible, all organisms must be identified by the scientif ic binomen. 18. mathematical equations should be clearly presented so that they can be interpreted properly. 19. obscure primes, symbols, and dots must be brought to the attention of the printer. distinguish very clearly number 1 and letter l. use fractional exponents instead of root signs and the solidus (/) for fractions wherever their use will save vertical space. 2 0 . all equations must be numbered sequentially in arabic numerals in parentheses on the right-hand side of the equations. 21. the authors should follow internationally accepted abbreviations, symbols, units, etc. , especially those adopted by the council of biology editors style manual (6th ed. , 1994) and the journal of american chemical society. 2 2 . less common abbreviations may be printed as footnotes. 23. manuscript templates in word, open off ice and latex are now available online at www.sciencediliman.edu.ph. authors may opt to submit their typeset manuscripts as an email attachment to <rduo.ovcrd@upd.edu.ph> and <rduo.ovcrd2012@gmail.com>. manuscripts may also be submitted in hard copy (3 copies) to: the editor-in-chief science diliman off ice of the vice-chancellor for research and development lgf phivolcs bldg. , c.p. garcia ave. university of the philippines diliman, quezon city 1101, philippines camera-ready illustrations (original plus one copy) must accompany the manuscript. 1 layman’s abstracts main articles the influence of vegetation and insect abundance on insectivorous bat activity during dusk emergence in an urban green space in metro manila, philippines jay s. fidelino and jelaine l. gan bats provide benefits to humans and ecosystems—like regulating insect populations—and are sensitive to changes in the environment. as such, bat activity is increasingly being used to study the effects of urbanization and other land use changes. however, to be effectively used as a bioindicator, baseline information on bat activity patterns must first be established. in this study, we aimed to determine patterns in the abundance and kinds of insect-eating bats present within an urban green space in the philippines' capital region, and how they are related to habitat type, insect abundance, and environmental conditions during emergence at dusk. we measured bat activity using a portable recorder at five time intervals from 5:30 pm to 7:30 pm, and compared between 10 open and 10 forested sites. we classified calls into distinct kinds called “sonotypes” based on five variables. we found no difference in bat activity between forested and open sites, but more sonotypes were recorded in open sites. both bat activity and number of sonotypes peaked between 6:00 pm and 6:30 pm, representing a short bout of foraging activity at dusk emergence. however, we did not observe significant relationships between bat activity and insect abundance, air temperature, and relative humidity. our study found considerable bat activity and diversity in an urban ecosystem, a poorly explored field of research in the philippines. additional studies are necessary to understand the impact of land use changes on philippine bats, and to inform their conservation and management in habitats modified by humans. science diliman (july-december 2019) 31:2, 1-4 2 layman’s abstracts cytotoxic and genotoxic potential of the money tree (pachira aquatica) stem and leaf extracts jordan ferdin a. halili, jane nicole n. catacutan, melissa c. gaudario, maries ann r. silvestre, mary lorraine f. lorido, and rich milton r. dulay this study is a preliminary screening of the anticancer potential of stem and leaf extracts of money tree, pachira aquatica, one of the plant species with very few studies done. the toxic effects of p. aquatica extracts were initially checked using brine shrimp lethality assay (bsla). onion root tip chromosomal aberration assay (ortcaa) was conducted to examine the effects on cell division and the chromosomes of onions. the ability to cause deformities to the developing embryo of zebrafish was determined using zebrafish developmental toxicity assay (zdta). using bsla, the p. aquatic leaf extract was seen as toxic. ortcaa revealed that all stem extract concentrations reduced cell division when compared to the positive control, while all leaf extract concentrations showed cells that stopped dividing during prophase/ metaphase boundary. zdta showed 100% embryonic death starting from 20 μg/ml of both extracts after 12 hours of treatment applications. abnormalities in onion cells and early zebrafish embryonic death implied the activation of programmed cell death.  thus, money tree extracts have promising cytostatic (inhibition of growth, division and differentiation of cells) and cytocidal (lethal) effects, important qualities of an anticancer drug, and is therefore a potential source of a naturebased chemotherapeutic compound. proliferative activities of benguet legume cultivars on a breast epithelial cell line cielo mae d. marquez, john carlo f. dela cruz, conrad allan c. chong, leomir a. diaz, harold m. carag, dhennis t. versoza, noel s. quiming, marilou g. nicolas, and michael c. velarde legumes are commonly grown around the globe and consumed as part of the human diet. legumes have components that mimic the action of human estrogens, which may disturb normal body processes or promote diseases. since legume consumption has many nutritive benefits but is also associated with estrogenic 3 layman’s abstracts activity, this study investigated the effects of six benguet legumes on the growth of an estrogen-responsive breast cancer cell line. among the six legumes, extracts from the common beans and cowpeas slightly promoted the growth of breast cancer cells at a low dose but decreased its growth at a higher dose. this suggests that legumes may contain similar compounds associated with growth-inducing and inhibitory effects on breast cancer cells. insights on the scientific publications of the faculty of the college of science, up diliman: 1998–2017 carlos primo c. david and mart cyrel m. geronia the publication record of faculty members reflects a research institution’s scientific productivity and impact. the quantity of papers published, where it is published, and its citation counts have also become standard metrics in assessing for grants, promotions, and tenure for faculty members. in consideration of these factors, we compiled and analyzed the trends of the 20-year (1998–2017) publication record of the up college of science (up science) spread across 10 institutes and programs. we included 2,295 published papers indexed in scopus and web of science databases by faculty members and researchers affiliated with up science from 1998-2017. overall, the total number of papers has increased in the last few years and, on average, at least one paper is published per faculty member annually. collaboration with other institutions is up and still increasing, with foreign partnerships outpacing others. up science has also increased its papers published in leading journals. we found that during their second decade of service, faculty members begin training younger researchers, shifting roles from being primarily researchers to mentors. these trends may serve as guide on how we can improve scientific productivity not only of the institution but also the faculty and research community of up science. 4 layman’s abstracts health risk assessment: total mercury in canned tuna and in yellowfin and frigate tuna caught from leyte gulf and philippine sea arvin u. pacoma and leni g. yap-dejeto tuna, both canned and locally caught, were analyzed for mercury content. locally caught tuna, frigate (auxist hazard) and yellowfin tuna (thunnus albacores), and canned tuna had average concentrations of 0.024 ug/g, 0.002 ug/g, and 0.07 ug/g, respectively. the results suggest that health risk is low in eating any locally caught tuna from eastern visayas. whereas, canned tuna is safe to eat as long as not more than one can per day is consumed by an adult. lower amounts should be consumed by vulnerable segments of the population such as children and childbearing women. fish kill-pp1-3.pmd fish kill in the philippines 1 gil s. jacinto marine science institute, university of the philippines, diliman, quezon city fish kill in the philippines—déjà vu science diliman (july-december 2011) 23:2, 1-3 almost ten years ago today, the country woke up to screaming headlines— “massive fish kill in pangasinan” or something akin to that. the fish kill phenomenon, familiar to fishers in freshwater and coastal bodies of water where fish farming was being pursued, was suddenly manifested at a scale that had heretofore not been experienced. in order to provide a context, a definition of terms is useful. a fish kill is “a significant and sudden death of fish or other aquatic animals such as crabs or prawns. these events are characterized by large numbers of aquatic animals dying over a short time, usually in a clearly defined area” (queensland dept of environment & heritage, 1998). estimates of the revenues lost in the february 2002 milkfish catastrophe were in the order of php 500m. whatever the numbers were or the value of resources lost, what became clear was that the losses were huge and the incident affected not only the fish culture operators and the caretakers but also the coastal communities in the vicinity of the catastrophe. the impacts of the fish kill cascaded even to the fishing industry beyond the locality as prices of fish plummeted; and consumers stayed away from eating fish, in general, not just milkfish from pangasinan. obviously, the phenomenon stirred to action the executive and legislative branches of government at the local and national levels. academic institutions and research organizations hastened efforts to understand the problem. the house of representatives and the senate conducted their investigations “in aid of legislation.” workshops were held at the municipal and provincial level, and a national fish kill workshop was conducted at the marine science institute, university of the philippines diliman, in february 2002. more than 100 individuals representing virtually all sectors that had a stake or interest in the finfish aquaculture industry participated in the national fish kill workshop. the assessment from the workshops then was that the immediate cause of the fish kill was the bloom of the dinoflagellate prorocentrum minimum, coupled with hypoxia (<2.8 mg/l dissolved oxygen) in the water column, resulting in asphyxiation of fish in the cages and pens. the plight of the fish may have been exacerbated by the release from the sediments of toxic hydrogen sulfide and ammonia, compounds produced following the decomposition of organic material in the water column and sediments (unused feed, fish waste products, and dead plankton from the bloom). however, the intermediate or root cause of the fish kill could be traced to the proliferation and intensification of finfish farming in the coastal waters of bolinao and anda (pangasinan), activities that went well beyond the carrying capacity of the nearshore environment. the assessment of carrying capacity of coastal waters for a particular type of activity, let alone a multitude of uses, is by no means fully understood. however, relying on acquired data and best information available then, researchers of the marine science institute (up diliman) had proposed an optimum number of structures (pens and cages) that should be allowed in the contiguous waters of bolinao and anda and the sites where these structures should be located. the number of structures were not to exceed 500, and areas that were naturally constricted (e.g., caquiputan channel) were to be left open. concurrence by the local executives of bolinao was manifested by the adoption of these recommendations into the municipality’s coastal management plan and the passage of a municipal ordinance in 1999. unfortunately, the ordinance was not adhered to. by the end of 2001, just months prior to the massive fish jacinto, g. s. 2 science diliman (july-december 2011) 23:2, 1-3 kill, ~1170 pens and cages had been built, with most of these stocked well beyond the optimum stocking density (san diego-mcglone et al. 2008). a greater number of cultured fish meant more feeds used and wasted, especially as cage and pen operators also employed cheaper (but low quality) feeds. the coastal waters of bolinao turned eutrophic (nutrient rich and enhanced the bloom of phytoplankton), and the proliferation of structures reduced flushing rates of the coastal waters. what followed, as they say, is history. in the intervening years, several projects and forums involving groups from norway and scotland, the bureau of fisheries and aquatic resources, and research institutions in the country were conducted. among these were: environmental monitoring and modeling of aquaculture in risk areas of the philippines (emma); mitigating impact from aquaculture in the philippines (philminaq); and sagip lingayen gulf, which developed and implemented prototypes of a marine emergency response system (mersys). capability building of bfar and local government units staff for environmental monitoring in aquaculture sites was implemented, better practices for aquaculture were discussed and documented, zoning plans were formulated and ordinances were enacted, technical forums were organized, a handbook for sustainable aquaculture for lgus was completed and circulated, and a joint administrative order (jao) on sustainable aquaculture was signed by the heads of the departments of agriculture, environment, and interior and local governments. fast forward to 2011, the country was again besieged by a string of fish kill events that not only affected the “traditional” coastal finfish growing areas in the country (e.g., pangasinan) but was also happening in freshwater lakes (i.e., taal lake) at a scale that worried people from all sectors. because taal lake is relatively near metro manila, media coverage was extensive; so, too, was the attention given by the legislative branch of government. once more a senate inquiry was held. local and national government agencies, academic institutions, people’s organizations, ngos, and the private sector attended. a moratorium on the stocking of fish in taal lake was to be implemented, illegal and inappropriately sited structures were to be dismantled, water quality monitoring was to be intensified, and a review of aquaculture policies and guidelines was called for. a fish kill forum was held at up diliman in august 2011 and attended by various stakeholders, many of whom were present in february 2002. speakers tackled the (same) basic question “what caused the fish kill” and reviewed information from past workshops on what should be done. once more the speakers echoed the cause of the problem—the carrying capacity of the culture areas had been exceeded, even if the proximate cause may have been asphyxiation of the fish because of very low dissolved oxygen in the water column. a distinction needs to be made on what may be the immediate cause of fish kills from the intermediate and root causes. by way of analogy, a person’s immediate cause of death may be respiratory failure. in turn, the underlying cause of death may be infection with the human immunodeficiency virus that may have occurred several years before the person’s demise, bringing about, for example, acquired immunodeficiency syndrome (aids) and followed by pneumonia a few days before the person’s death. not long after the taal lake fish kills this year, media reports cited “authorities” blaming the fish kills on a temperature change, which purportedly brought down dissolved oxygen levels in the lake and caused the fish kills. there was no evidence to support this suggestion. morever, even if a 3 degree celsius water temperature change had taken place (which would have been unlikely), the solubility of oxygen in water would not have decreased significantly and would not have been critical for the fish (<4 mg/l). others even invoked “climate change”, an assessment that showed how little appreciation some people have on the temporal scale associated with climate change processes. recommendations from the august 2011 fish kill forum echoed those made during the february 2002 meeting of stakeholders: 1) a national fish kill quick response is needed; 2) an effective monitoring and surveillance system has to be established; 3) accountability and compliance is key; 4) a two-stage licensing system fish kill in the philippines 3science diliman (july-december 2011) 23:2, 1-3 for mariculture activities in order to provide checks and balances is important; 5) lgus need to review and further strengthen existing sanctions and monitoring mechanisms; 6) empowerment and education are essential; 7) fishers and stakeholders must be aware of and apply only the proper technologies and practices in mariculture; 8) a comprehensive national framework on sustainable aquaculture must be put in place; and 9) regular forums on mariculture must be conducted for the benefit of all stakeholders. it is convenient to blame nature for disasters that ultimately are caused (or at the very least exacerbated) by human actions or inaction; and fish kills are no exception. we experience today the fish kill problem that we encountered a decade ago. we knew then what had to be done because we had already identified the causal factors and the steps required to address these. some progress has been made but at a pace that is, seemingly, unable to keep up with the proliferation of the problem. moreover, and as articulated many years ago, accountability of public officials and aquaculture practitioners is key along with their compliance with laws and good practices. the food and agriculture organization defined sustainable aquaculture as “the management and conservation of the natural resource base and the orientation of technological and institutional change in such a manner as to ensure the attainment and continued satisfaction of human needs for present and future generations.” (bfarphilminaq, 2008). thus, aquaculture is not to “cause irreversible damage; harm human health or the safety of human beings; jeopardize the future productive base for short-term economic benefit; adversely affect biodiversity or sensitive habitats; and, adversely affect essential ecological processes.” stakeholders in the aquaculture industry echo this perspective, although if one were candid, it seems that we only provide lip service to this principle. a decade ago i remarked at the national forum on fish kills that “mariculture will have to be addressed now because of the likelihood of unbridled expansion of the industry and associated dire consequences.” as in déjà vu, we have a sense that we have already witnessed or experienced a current situation. but unlike a déjà vu experience, we do know the exact circumstances of the previous encounter, and these circumstances were not imagined. today, that observation and perspective remains. my fear, however, is that for some aquaculture areas in the country, we may already have reached the “tipping point” with the negative consequences, and impacts may possibly be irreversible. action and responses to address unsustainable aquaculture were needed from government and other stakeholders in the aquaculture industry many years ago; if we wait another decade, it will undoubtedly be too late! references bfar-philminaq. 2007. managing aquaculture and its impacts: a guidebook for local governments. bureau of fisheries and aquatic resources (bfar)-philminaq project, diliman, quezon city, 80 queensland. dept.of environment and heritage (1998) fish kill reporting and investigation manual: for use in investigation of possible breaches of the environmental protection act 1994 and fisheries act 1994. dept. of environment & heritage. san diego-mcglone, m.l., azanza, r.v., villanoy, c.l., jacinto, g.s. (2008) eutrophic waters, algal bloom and fish kill in fish farming areas in bolinao, pangasinan, philippines. mar pollut bull 57: 295–301. 13info for authors.pmd 86 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the off ice of the vicechancellor for research and development (ovcrd). considered for publication are primary and original papers. review articles and short communications may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities at the institute where the work has been carried out. the letter to the editor usually contains these: that, if and when the manuscript is accepted for publication, the authors agree to the automatic transfer of the copyright to the publisher; that the manuscript will not be published elsewhere in any language without the consent of the copyright holders; that written permission of the copyright holder is obtained by the authors for material used from other copyrighted sources; and that any costs associated with obtaining this permission are the authors’ responsibility. 5. authors must submit electronically prepared manuscripts in microsoft word. 6. manuscripts should be formatted for a4 paper, double-spaced, with 1" margins on all sides. each page of the manuscript must include continuous line numbers in the margin. all pages should be numbered consecutively on the upper right hand corner of the page. information for authors 87 7. page 1 should contain the ar ticle title, author(s), aff iliation(s), and the name and complete mailing address (and telephone number, fax number, and e-mail) of the person to whom correspondence should be sent. 8. page 2 should contain a short abstract of not more than 250 words. the abstract should contain facts and conclusions, rather than citation of the areas and subjects that have been treated or discussed. the abstract should start with the hypothesis or a statement of the problem to be solved, followed by a description of the method or technique utilized to solve the problem. the abstract should end with a summary of the results that were obtained and their implications. it is to be followed by a maximum of six key words. the author must also submit a layman’s abstract of not more than 200 words. 9. the paper should be organized as follows: abstract and layman’s abstract introduction materials and methods results and discussion (or results separate from discussion) acknowledgments references 10. reference lists, f igures, tables, and f igure/list captions should all be on separate sheets, all of which should be double-spaced, and numbered. standard nomenclature should be used. unfamiliar terms, abbreviations, and symbols must be def ined at f irst mention. 11. references to the literature citations in the text should be by author and year; where there are two authors, both should be named; with three or more only the f irst author’s name plus “et al.” need to be given. references in the text should follow the council of science editors (cse) scientif ic style and format, 7th edition, 2006. 88 examples: articles from journals: print ( section 29.3.7.1 p. 518-527) format: author(s). date. article title. journal title. volume(issue):location. example: smar t n, fang zy, marwick th. 2003. a practical guide to exercise raining for heart failure patients. j card fail. 9(1):49-58. ar ticles from journals: onl ine (section 29.3.7.13 p. 557-558) format: author(s) of article. date of publication. title of article. title of journal (edition) [medium designator]. 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communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages ) in length with simple layout, a maximum of two tables and two f igures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 24. authors may opt to submit their typeset manuscripts as an email attachment to <science.updiliman@up.edu.ph>. submissions should be addressed to: the editor in chief science diliman off ice of the vice-chancellor for research and development university of the philippines lower ground floor, phivolcs bldg. , c.p. garcia avenue up campus, diliman, quezon city 1101, philippines camera-ready illustrations must accompany the manuscript. 01_device detection and tracking of tropical cyclones 35 abstract *corresponding author science diliman (july-december 2007) 19:2, 35-48 a regional climate model is used to detect tropical cyclones (tc) and simulate their tracks for a fourmonth (june-july-august-september) wet season in the philippine region. the model, run at 45-km resolution, is forced along the boundaries with 6-hourly reanalyses data (era-40 with about 250-km resolution). three experiments are devised which varied the size of the domain and placement of the boundaries a detection and tracking algorithm is developed using 850-mb vorticity threshold, minimum sea level pressure and the presence of a warm core aloft as criteria. the tracks extracted from the era-40 field, herein called analyses track, are compared with jtwc best track to test the performance of the tracking algorithm. of the fourteen (14) tc that entered the domain, ten were formed in the pacific ocean and four in the south china sea. the algorithm detected all tc and skillfully captured the jtwc best track. from the 417 cases (6-hourly positions of the 14 tc), the mean zonal and meridional errors are -164, -23 km, respectively, where the analyses tracks are on the average moving faster westward and southward than the best track. the relatively small magnitude of errors indicates skill of the tracking method. the regional model is able to detect all 14 tc but with tracks that are farther displaced north of analyses. simulation of track was enhanced as domain size is decreased. the intensity simulation is improved as more typhoons otherwise not found in the forcing data are generated by the regional model. this study demonstrates that a regional model forced by "perfect" boundary conditions can reasonably simulate the tracks and intensity of tropical cyclones on a seasonal scale. the importance of the use of the proper domain configuration is also shown. keywords: regional climate modeling, tropical cyclone tracks detection and tracking of tropical cyclones on a seasonal scale in the philippines josefina c. argete1* and raquel v. francisco2 1associate professor institute of environmental science and meteorology college of science, university of the philippines 1101 diliman, quezon city, philippines e-mail: argetej@yahoo.com 2scientist i, pagasa-dost date received: may 11, 2006; date accepted: april 19, 2007 argete and francisco 36 introduction the northwest pacific basin spawns the maximum number of tropical cyclones (tc) globally, about 35 per year. the typical track of these tc is northwestward, with about 20 passing the philippine area of responsibility (par). because the philippines lies right in the path of tc, reports of staggering damage to life and property due to this natural disaster is an annual event. as a mitigating measure, accurate forecasting of track and intensity is given the highest priority. these are derived as short-range (3-5 day) forecasts from mesoscale models. seasonal or climate forecasts, on the other hand, are predictions on the time scale of months. these are very important for mediumrange planning in government and industry. for instance, it is observed that the frequency, tracks and intensity of tc are influenced by the warm (el nino) and cold (la nina) phases of the enso the consequences of which impact on crop production, water supply and even the incidence of pests and diseases. empirically-based (statistical analyses of past data) methods search for predictors such as sst and other nino indices and are limited to stating that tc expected in the next season are higher or lower than normal (chan et al, 2001). dynamical forecasts of tc activity are produced at major weather centers running global circulation models (gcm). because of its global scope, however, the resolution is low (bengtsson et al, 1995). furthermore, gcm run on mainframes. local research institutions in developing / transitioning economies do not have the computational capacity at present to run gcm, and yet there is a pressing need for them to improve regional forecasting capacity using currently viable methods. an economical solution for improving the resolution is to nest a regional climate model within a global model. in this scheme, the regional model which is configured to run on a personal computer is forced at the boundaries by the global model outputs for the region which may be requested from centers running gcm. the cyclones produced by the regional models have been found to be weaker than observed but are more realistic than the vortices generated by gcm (walsh and waterson, 1997; nguyen and walsh, 2001; vitart et al, 2003; landman et al, 2005). gcms are able to create tropical vortices similar to tropical cyclones. the coarse resolution however leads to lack of the presence of the eye and eyewall and are weaker than observed (bengtsson et al, 1995) which then influence the simulated intensity and track. the mean number of tropical storms simulated by gcm is only about ½ the number observed. different gcms showed variabilities but in general the tracks were found to be located too far to the east over the indian ocean (vitart et al, 1997) and the western north pacific (camargo and zebiak, 2002a). as mentioned, an alternative and economical solution for improving the resolution is to nest a regional climate model within a global model. the basic question of whether regional climate models can generate meaningful small-scale features that are absent in the initial and boundary conditions supplied by global models has often been asked. some argue that predictability is limited by turbulence (de elia and laprise, 2002). it would be too optimistic to say that the formation and movement of a mesoscale system such as a tropical cyclone which are defined by synoptic conditions which have a predictability of a few days at most can be predicted months in advance. however, tropical cyclones are also influenced by large-scale flow. and since large-scale fields of the atmosphere are predictable on a seasonal scale, these can be "downscaled" using regional climate models. regional model runs over the southwestern indian ocean (landman, et al, 2005) and australian region (walsh and waterson, 1997) simulated more realistic vortices but found the tc to weaken faster than observed in their westward track. experiments on the proper choice of domain size and the positioning of the lateral boundaries for the regional model improved their results in capturing the life cycle of tc. the domain must be large enough to encompass the large-scale atmospheric fields provided at the boundaries that are relevant to tropical cyclone formation and movement and allow model internal processes to develop with minimal constraints from the boundaries (seth and giorgi, 1998, landman et al 2005). in this study, the performance of a regional climate model in detecting and tracking tropical cyclones for a four-month (jjas) wet season period in the philippine science diliman (july-december 2007) 19:2, 35-48 detection and tracking of tropical cyclones 37 region is investigated. the 2005 version of regcm3 is used, details of which are presented in section iia. the model is forced along the lateral boundaries with time-dependent reanalyses data with about 250-km resolution. the use of reanalyses data allows evaluation of the regional model performance given "perfect" boundary conditions. a detection and tracking algorithm is developed using meteorological variables discussed in section iic. the skill of the algorithm is tested by comparing the tracks extracted from reanalyses with the best track data of jtwc. three model experiments are devised by varying the domain size and placement of lateral boundaries. model tracks are then compared with reanalyses tracks. finally, the influence of domain size in simulating tropical cyclone intensity is assessed. materials and methods a. the regional climate model the international centre for theoretical physics (ictp) regional climate model version released in february 2005 and herein called regcm3 is used in this study. it was developed at the national center for atmospheric research (ncar) and described in detail by giorgi et al (1993, a,b). the dynamical component is essentially the same as the standard pennsylvania state university mesoscale model (mm4). since then, as new physics schemes have become available, refinements were done on the model. it is a hydrostatic, compressible, primitive equation, terrain following sigma coordinate model with 18 vertical levels. since the domain is not of a global nature, the lateral boundaries require periodic forcing. a one-way nesting technique (giorgi et al, 1994) uses ecmwf reanalyses (era-40) meteorological fields as driving initial and time-dependent lateral conditions, with sea surface temperature (sst) data used as surface boundary condition. the regcm3 model is composed of four components: terrain, initial and boundary conditions (icbc), regcm (main program) and postprocessor. terrain and icbc are the two components of the preprocessor. terrain information was obtained from an elevation dataset of the us geological survey at 3-min resolution (usgs gtopo30_3min). the land use information is taken from the global land cover characterization of vegetation or land cover at 3-min resolution (usgs glcc_3min). the initial and boundary condition datasets for the icbc were obtained from era-40, a 40-yr (up to 2002) 6hourly 2.5o resolution global reanalyses of atmospheric fields. surface boundary information is obtained from weekly optimal interpolation sea surface temperature (oisst) data at 1o resolution. the datasets were requested from ictp (http://ictp.trieste.it/~pubregcm). b. model experiments the simulation of tc tracks was performed on three domains shown in fig.1. the largest domain (designated as d1) consists of 148 grids along x and 85 grids along y and covers the area 100oe to 160oe and 0o to 33on. the eastern boundary is placed at 160oe to capture the area of maximum tc genesis for storms affecting the philippines (elsberry et al, 1987). the eastern fig. 1. the regional model domains (labeled d1, d2 and d3). d1 is the largest while d2 is 10 longitude degrees smaller than d1. the smallest domain d3 is embedded within d1 and d2. the topography is represented as height contours in meters. science diliman (july-december 2007) 19:2, 35-48 argete and francisco 38 boundary is then moved westward to 150oe , with the other three boundaries unchanged, to create a smaller domain d2 with 122 grids along x and 85 grids along y. the smaller third domain d3 with 98 grids along x and 51 grids along y is embedded within d1 and d2. the horizontal resolution for all experiments is set at 45 km at a time step of 120 s. each model run is initialized on may 25, 1996 and allowed to proceed until october 1, 1996, with the first 6 days of integration discarded to allow for model spin-up. the 6-hourly model outputs of atmospheric fields therefore cover the june-julyaugust -september (jjas) tc season in 1996 in the philippine region. c. tracking tc the detection and tracking algorithm is based on methods described in three papers (walsh and waterson,(1997), vitart (1997) and camargo (2002)). these methods have similarities as well as differences. the similarities are basically on the use of certain criteria in determining whether a vortex is considered a model storm. these criteria were found to be basindependent. in camargo and zebiak (2002), they used a detection and tracking algorithm which applies basindependent threshold criteria to low-level vorticity, surface wind speed and vertically integrated temperature anomaly to determine the position of the storms every 6 hrs. the tracking algorithm was applied to an ensemble of general circulation models. in vitart (1997), differences with those of camargo (2002) are the inclusion of minimum sea level pressure as detection parameter and a different approach in determining the warm core aloft. in walsh (1977), the three detection criteria are 850-mb vorticity maximum, the warm core aloft and the 10-m windspeed. detection and tracking algorithm the following description of the detection method is largely based on the work of vitart (1997). the algorithm first locates the position of intense vortices with a warm core for the initial time step as follows: 1. locate the local maximum of relative vorticity greater than 3.6 x 10-5 s-1 at 850 mb. this is the lowlevel vorticity threshold of camargo for the west north pacific basin. here relative vorticity ζ is defined as (3.1) the center of vorticity is manually located on the vorticity map of the regcm3 (hereafter referred to as model vorticity) on the date of the first day of a storm. this first day of storm is based on the observed best track data of jtwc*. for example, one jtwc storm starts on june 19 and ends july 27. the model vorticity on june 19 is displayed and the cursor is pointed at the center of vorticity close to the coordinates observed by jtwc. this gives the initial coordinates of the model storm. 2. locate the minimum sea level pressure as the center of the storm. define a box (10 x 10 grids) centered around the model maximum vorticity. the coordinates of the minimum sea level pressure within the box is now defined as the center of the model storm. 3. test whether the model storm has a warm core aloft. * define a box 8o x 8o (lat x lon) centered at the storm center between 200 and 500 mb and determine the volume-average temperature (tave) * define a box 2o x 2o centered at the storm center between 200 and 500 mb and calculate the anomalous temperature per grid ( tanom = t tave) * get the coordinates of the center of the maximum tanom . this is the center of the warm core. * from the center of the warm core, the temperature must decrease by at least 0.5oc in all directions within the 8o x 8o box. * test whether the center of the warm core and the center of the storm are within 2-degrees (lat/lon) of each other. if the warm core is too far from the storm center, it is not considered a model storm. *best-track dataset for northwest pacific ocean. this is a past analysis of typhoons provided by the joint typhoon warning typhoon centre (jtwc), hawaii. http://www.npmoc.navy.mil y u x v ∂ ∂ − ∂ ∂ =ζ science diliman (july-december 2007) 19:2, 35-48 detection and tracking of tropical cyclones 39 the above criteria must be satisfied to define a model storm. after the initial storm center is located, the 6-h track of the storm is tracked as follows: 4. test if on the next time step, there are storms within 800 km of the previous storm's center. if there is none, the trajectory is stopped. otherwise, the storm in the present time step must belong to the trajectory of the initial storm. in the west north pacific where more than one storm may occur within 800 km of each other, preference is given to the storms located in the northwestern quadrant. 5. the algorithm is run for the duration of the storm as observed by jtwc or when the storm crosses the domain boundaries. the algorithm outputs the date of analysis, the coordinates of the storm center, and the wind speed. the model and jtwc trajectories are then plotted and compared. d. validating model tracks the difference between model and observed tracks is measured as displacement error. this is defined as the distance between the forecast (x f , y f ) position and the observed (x o , y o ) position of the storm, x and y being longitude and latitude, respectively. the displacement error (e) in km is estimated from the zonal (e z ) and meridional (e m ) errors: a positive e z indicates a forecast track that is moving slower than the observed track in the westward migration. a positive e m means that the forecast track is north of the observed position. results and discussion a. assessment of the tracking algorithm the detection and tracking method is tested on era40 data interpolated to the same horizontal resolution as the model grid. the resulting 6-h tracks for each tc, herein called the analyses tracks, are then compared with the 6-h jtwc tracks. it is to be noted that while both data sets (era-40 and jtwc) are derived from observations, a disparity is expected because the nature and the manner by which they are produced are very different. jtwc best tracks are post-analyzed tracks and are point estimates of minimum sea level pressure and maximum sustained winds. era-40 is a description of the large-scale atmospheric fields at a coarser resolution. it is a global analysis of atmospheric circulation produced by threedimensional variational (3-dvar) 6-hourly data assimilation of an atmospheric model for 45 years from 1957 to 2002 (kallberg et al, 2005). it was produced to foster international research by making observations and analyses widely available. the era-40 data product available from ecmwf public data server and used in this study has 2.5o resolution. here, gridded atmospheric variables u, v, t, p and q at 23 pressure levels and 2.5-degree (about 255 km) horizontal resolution are regridded to 45 km within the domain. using this "observed" gridded data, the tracking algorithm is applied over the domain to produce the analyses tracks. this comparison of tracks aims to calibrate the tracking algorithm. the difference in 6hourly positions of the analyses and jtwc tracks expressed as zonal and meridional errors is considered a measure of the performance of the tracking algorithm. if the tracking algorithm has skill, the analyses tracks should capture the jtwc best tracks. a divergence of the two tracks may in part be due to the inherent difference of the two data sets as mentioned above but for lack of basis in quantifying this, the error obtained is attributed to the performance of the tracking algorithm. the calibration thus accounts for two sources of error the inherent difference between era-40 and jtwc datasets and the limitations of the tracking algorithm the sum of which is considered a measure of the skill of the tracking. 111) 2 yy (cos)xx( ofofz + −=e 111)yy( ofm −=e 2/122 )( mz eee += science diliman (july-december 2007) 19:2, 35-48 (3.3) (3.2) (3.4) argete and francisco 40 based on the jtwc tracks, a total of 14 tc entered the largest domain in jjas 1996: 0 in june, 5 in july, 4 in august and 5 in september. these are numbered 1 to 14 in table 1. the 6-h jtwc and analyses tracks for 5 selected tc are shown in fig 2 (a-e). each tc is identified by the time (yyyymmddhh) it first attained tropical depression stage according to jtwc data. a tc is classified as a tropical depression when the maximum sustained wind near the center (u max ) is less than 17 m/s, a tropical storm when u max is between 17 and 33 m/s and a typhoon when u max is greater than 33 m/s. of the 14 tc that entered the domain, 10 were formed in the pacific ocean (pac) and 4 in the south china sea (scs). the algorithm was able to detect and track all the 14 tc in the era-40 fields. the 5 cases shown in fig. 2 are chosen to represent two cases for storms that formed in the pacific ocean (fig 2a and fig 2c), two from the south china sea (fig. 2b and fig. 2e) and one for a case when two storms exist at a distance close enough to cause interaction for a given time period (fig. 2d). it is to be noted that the jtwc best tracks are skillfully simulated by the tracking algorithm regardless of the tc's area of genesis, except for two cases (represented as tc #5 and #11 in table 1) one of which is shown in fig 2d. here, the analyses track of tc with duration july 27-31 (tc #5) diverged from tc number and date e z e m e remarks 1. jul 11-17 1996 -476 99 592 pac 2. jul 19-27 1996 -27 39 179 pac 3. jul 19-25 1996 24 7 140 scs 4. jul 24-31 1996 1 -32 145 pac 5. jul 27-31 1996 -1119 37 1233 pac / twins with tc#4 6. aug 01-14 1996 -17 -32 160 pac 7. aug 04-07 1996 110 47 226 scs 8. aug 11-16 1996 146 -38 195 scs 9. aug 14-23 1996 10 -130 262 pac 10. sep 02-10 1996 169 -189 285 pac 11. sep 06-14 1996 -1199 175 1285 pac / twins with tc#10 12. sep 09-23 1996 -173 -87 273 pac 13. sep 16-23 1996 67 -13 141 scs 14. sep 22-30 1996 -19 -81 178 pac weighted average -164 -23 378 std deviation 514 241 476 the jtwc track and followed closely the track of tc in fig. 2c with duration july 24-31 (tc #4). with this case of having twin storms (or pairs) in a region, it is seen that the algorithm may have to be improved in the proper control of identifying which tc is to be tracked. the same situation of having storm pairs is seen in another case (not shown) wherein tc with duration september 6-14 (tc #11) interacts with the tracks of tc on september 2-10 (tc #10). the errors in terms of differences between analyses and best track are shown in table 1. the errors are calculated using eqn 3.2 3.4 with era-40 as the forecast position and jtwc as the observed position. negative values of the zonal and meridional errors indicate that the analyses tracks are on the average moving faster westward and southward than the best track. the errors are (weighted) averaged over the corresponding 6-h positions. the largest errors are incurred for the two cases with storms (twins) whose tracks approach each other during a certain period in their duration. these are tc #5 and tc #11. from the 417 counts of 6 h positions for the 14 tc, the mean displacement error is about 378 km. a scatterplot of the zonal and meridional errors for the 417 cases is shown in fig. 3. based on the mean zonal (-164 km) and meridional (-23 km) errors, the analyses tracks are biased towards the left (fast to the west) and south of table 1 zonal (ez), meridional (em) and displacement error (e) in km of era-40 track referenced against jtwc best track science diliman (july-december 2007) 19:2, 35-48 detection and tracking of tropical cyclones 41 a fig. 2. six-hourly position and intensity of five selected cyclones from jtwc best-track and era-40. science diliman (july-december 2007) 19:2, 35-48 argete and francisco 42 the best track position. the ellipse in the figure, centered on (-164, -23) indicates the range of the zonal and meridional errors which accounts for 68% of the population, or 1 standard deviation. the bias is largely due to the twin storms tc #5 and tc #11. if the two cases having twin or interacting storms are excluded, the zonal and meridional errors are drastically reduced to 15 and 34 km, respectively, where the analyses tracks are slightly east and northward of the jtwc track. with a 45-km grid size, the average errors translate to 0.3 and 0.8 grid displacements from the jtwc track. this is a relatively small magnitude of error. however, twin storms often occur in the nw pacific basin and excluding them from analysis is not appropriate. for this calibration, a zonal error of -678 km (mean +/1s) or (-164 514) and meridional error of -264 km (-23 241) are established. these errors are of the same order of magnitude as that found by camargo and sobel (2003) on the eastward bias of echam4.5 with 2.50 horizontal resolution. they note that while the 2000 km zonal displacement may seem large, it is not significantly greater than the horizontal extent of a typical tc and is therefore modest from a dynamical point of view. fig. 3. six-hourly zonal (x-axis) and meridional (y-axis) errors of analyses tracks referenced against jtwc tracks. the ellipse indicates position errors of 1 standard deviation centered on the mean. given the competent performance of the tracking, the same algorithm is used in the subsequent tracking of tc simulated by the regional model. the question now to be addressed is whether the regional model if fed by 6-h era-40 fields at the lateral boundaries is able to simulate the atmospheric variables in the domain that define a tropical cyclone. if the model simulates the era-40 fields, then the errors between model and era40 tracks should be minimal, or of similar order of magnitude as the errors used in the calibration phase. errors much larger than these may be attributed to limitations of the regional model. b. effect of domain size on model tracks the model and analyses tracks are compared in fig. 4(a-e) for the same 5 selected storms in fig.2(a-e). the regional model detected and tracked all the 14 tc that are observed in the era-40 fields but only 5 representative cases are shown. the large domain d1 extends to 160oe while d2 covers up to 150oe, 10 degrees shorter. the tracks for domain d3 are not shown in this paper for the main reason that they cause science diliman (july-december 2007) 19:2, 35-48 detection and tracking of tropical cyclones 43 fig. 4. model track and intensity for domains d1 and d2 for 5 selected cyclones. the reference era-40 track and intensity are also shown. science diliman (july-december 2007) 19:2, 35-48 argete and francisco 44 fig 5. six-hourly zonal (x-axis) and meridional (y-axis) errors of model tracks referenced against jtwc (left) and era-40 (right) for the three domains. the basins of tc origin (pacific ocean and south china sea) are also indicated. science diliman (july-december 2007) 19:2, 35-48 detection and tracking of tropical cyclones 45 unnecessary clutter in the tc track diagrams. however, the summary for the d3 experiments are mentioned to reinforce the conclusions drawn on the influence of domain size on tc track and intensity simulation. the date above each figure denotes the time the tc became a tropical depression as reported by jtwc. the 6-h zonal and meridional errors of the model tracks referenced against jtwc best track (mod-jtwc) and era-40 (mod-era) track are shown as scatter plots in fig.5. the errors from tc that developed in the pacific ocean (pac) and south china sea (scs) are also indicated. for all domain sizes, the model is able to capture the tc tracks regardless of its basin of origin but the magnitude of errors is larger for pacific tc. tc coming from the pacific travel over a wide expanse of ocean, have longer tracks and are reportedly easier to track. tc in the scs are historically difficult to forecast due to the short erratic tracks, slow movement and the complicating influence of topography during landfall. the small zonal error may be due to the smaller spatial area covered by the scs storms. this also suggests the sensitivity of the model to topography. the large displacement of the model tracks from observations as seen in d2 is again due to the presence of twin tc in the pacific ocean. this is evident in fig. 4d for tc#5. the best track for tc#5 (shown in fig. 2d) is better captured by the model in the large domain d1 than in d2. the main difference between d1 and d2 is the position of the eastern part of the domains. for tc#5 which developed close to the eastern boundary of d2, boundary effects may have influenced the simulation of the tc. the tracking method is also a source of error. since the location of the warm core is determined from the volume-averaged temperature of a box of size 80 centered on the tc and the location of the tc center is determined from the minimum sea level pressure in a box 0f 10 grids, a tc very close to the boundary may have an erroneously determined warm core and tc center position. a comparison of the zonal and meridional errors among analyses tracks and model tracks at three domain sizes is summarized in table 2 and presented as scatter plots in fig. 6. as was done for fig. 3, the ellipses represent the spread within 1 standard deviation from the mean zonal and meridional error. the errors from analyses tracks are shown in bold. of the four ellipses, that of the analyses tracks is the smallest because the errors here are derived from a comparison of two observed data sets. in this calibration step, the errors are considered to arise from the inherent difference of the fig. 6. positions of the mean zonal and meridional errors of the model tracks relative to era-40 (left) and jtwc (right) for the three domains. the corresponding ellipses of 1 standard deviation centered on the means are also shown (d1 solid, d2 dashed and d3 dotted). bold solid ellipse represents that of the analyses tracks shown in fig.3. science diliman (july-december 2007) 19:2, 35-48 argete and francisco 46 data sets and on the skill of the tracking algorithm. the other three ellipses are measures of the errors due to the regional model, the tracking method and the bias between data sets combined. the differences of the means of the four ellipses were found to be statistically significant at 1% using a standard t-test, except for the zonal errors of d2 and analyses and meridional errors of d3 and analyses, which were significantly different at 10% probability level. the shape of the ellipses indicates larger east-west errors. the model errors are smallest for the small domain d3. this is expected since error growth for small domains is constrained by the boundaries. a regional model forced with reanalyses at the boundaries gives a better hindcast simulation for a smaller domain (chouinard et al, 1994). the use of a very small domain results to small errors because the tracks are artificially controlled by the boundaries and the model physics are not allowed to develop. a very small domain however misses some tc that may be important for the region. for this case, 4 tc whose tracks were outside the domain but whose influence was felt in the philippines were missed. increasing the domain size from d3 to d1 led to tracks that are displaced northwards (em positive). the westward bias of model tracks in d2 is in part due to tc#5 which developed very close to the eastern boundary of d2 and to the twin storms tc#5 and tc#11 which were not properly identified by the tracking algorithm. basing largely on the standard deviation, d1 is considered the optimal domain configuration in this study. the size and placement of d1 allow the capture of the most number of tc affecting the philippine region, avoid the constraints of boundaries and minimize propagation of errors. when a very large domain is used (not done here), it is possible that the model physics is not sufficient to generate a vortex on its own. it is also possible that even with "perfect" model physics, the vortex remains weak because of the limitations imposed by chaos (landman et al, 2005). these issues cannot be settled in this study since many sensitivity studies on the internal variability of the model need be done and are beyond the scope of this work. c. effect of domain size on model storm intensity it is expected that a tc is much weaker in the analyses (era-40) than in the best track because winds in the former are area-averaged while those in the latter are point estimates. the weak intensity of storms in the analyses is evident in the plots in fig.2. the graphical representation of intensity is quantified in table 3 and presented as a bar plot in fig.7. consider first the columns for d1 in the table (which is the data plotted in fig. 2). of the 417 cases of 6-h positions of tc, there are 160 jtwc typhoons but no tc was measured in era-40 that has a wind speed greater than 33 ms-1. the same is observed for d2 and d3. there are too many tropical depressions and tropical storms in era40 indicating the much weaker winds in the dataset. mean std dev ez em ez em analyses -164 -23 514 240 d1 125 307 731 463 d2 -249 157 972 485 d3 -19 9 503 309 table 2. zonal and meridional errors of analyses tracks and model tracks at varying domain sizes. table 3. counts of 6-h tc intensity from jtwc best track, era-40 and model output for the domain experiments. d1 d2 d3 jtwc era-40 model jtwc era-40 model jtwc era-40 model depression 156 296 248 156 290 138 129 237 94 tropical storm 101 121 160 97 117 241 73 53 190 typhoon 160 0 9 154 0 28 88 0 6 no. of cases 417 417 417 407 407 407 290 290 290 science diliman (july-december 2007) 19:2, 35-48 detection and tracking of tropical cyclones 47 to assess whether the regional model is able to better simulate the winds and hence the intensity of tc, the frequency of the 6-h model tc is compared with jtwc and era-40 for the three domains. the regional model is able to generate more tropical storms and typhoons: 28 of the observed 154 typhoons from jtwc (18%) were detected in d2 and 6 of the 88 (7%) in d3. the increase in intensity is least in the large domain d1 with 9 typhoons of 160 (6%) observed. this indicates that the physically-based downscaling of the regional model has improved the wind simulation over the domain. the size and placement of domain boundaries influence not only the simulation of the tracks but also feedback on a more realistic simulation of the winds in a tc. while d1 gave a better performance in terms of smaller zonal and meridional errors, d2 gave a better simulation of tc intensity. conclusions this study shows the potential of regional climate models to detect tropical cyclones and simulate their tracks on a seasonal scale (jjas) in the philippine region. the value added to the simulation is attributed to the increased resolution and model physics and is shown to be a function of domain size and placement of boundaries. a very small domain simulates the tracks reasonably well but the seemingly good simulation may be due to artificially constrained errors due to the boundary. for all domains, model tracks were found to be displaced northwards, fast in the westward migration and are generally slow to intensify. basing on the zonal and meridional errors, d1 is considered the optimal domain configuration in this study. the size and placement of d1 allow the capture of the most number of tc affecting the philippine region, avoid the constraints of boundaries and minimize propagation of errors. in terms of wind simulation, the model was able to generate typhoons which are absent in the forcing data, indicating the skill of the downscaling. more typhoons were formed in d2 than in d1. these results indicate that the size and placement of domain boundaries influence not only the simulation of the tracks but also the simulation of the winds in a tc. the regional model in this study is run at a resolution of 45 km and is forced at the boundaries by era-40 data which is globally observed data at a resolution of about 250 km. the use of reanalyses data allows evaluation of model results given "perfect" boundary conditions. if gcm integrations are used as boundary forcing for the regional model and if the quality of gcm outputs approach that of reanalyses, tracks and intensities of cyclones can be forecast on a seasonal scale. this study is a pioneering initiative on investigating the feasibility of making seasonal forecast fig 7. frequency of 6-h model tropical cyclone intensity for the three domains relative to jtwc and era-40. science diliman (july-december 2007) 19:2, 35-48 argete and francisco 48 of tc from regional models in the philippine region. follow-up studies must be done to build on the methodology developed for this study. these include further improvement of the tracking algorithm particularly in tracking interacting storms, sensitivity studies on the influence of the land surface / terrain on storms, and the influence of the simulated tc structure and intensity on the choice of convective parameterizations. acknowledgements this study was funded by the office of the vice chancellor for research and development (ovcrd), up diliman as project no. 040402. the authors are grateful for the technical support extended by the international centre for theoretical physics on the use of regcm3 and for providing all data sets required by the model. references bengtsson, l, et al. 1995. hurricane-type vortices in a general circulation model. tellus, 47a. 175-196. camargo and zebiak. 2002. improving the detection and tracking of tropical cyclones in atmospheric general circulation models. wea and forecasting. 17, 1152-1162. _______ and sobel. 2004. formation of tropical storms in an atmospheric general circulation model. tellus. 56a, 5667. chan, j, j. shi and k liu. 2001. improvements in the seasonal forecasting of tropical cyclone activity over the western north pacific. wea and forecasting. 16, 491-498. chouinard, c., j.mailhot, h.l. mitchell, a. staniforth and t. hogue. 1994. the canadian regional data assimilation system: operational and research applications. mon wea rev, 122,1306-1325. de elia r. and r. laprise. 2002. forecasting skill limits of nested, limited-area models: a perfect model approach. mon wea rev. 130, 2006-2023. elsberry, r (ed). et al. 1987. a global view of tropical cyclones. 185 pp. giorgi f, et al. 1993a. development of a second generationregional climate model (regcm2). part i. boundary layer and radiative transfer processes. mon wea rev, 121, 2794-2813. ______ et al. 1993b. part ii. convective processes and assimilation of lateral boundary conditions. mon wea rev 121, 2814-2832. kallberg, p., p. berrisford, b. hoskins, a. simmons, s. uppala, s. lamie-thepaut and r. hine. 2005. ecmwf re-analysis project report series: 19. era-40 atlas. reading, england, 191 pp. landman, w. , a. seth and s. camargo. 2005. the effect of regional climate model domain choice on the simulation of tropical cyclone-like vortices in the southwestern indian ocean. j climate, 18, 1263-1274. nguyen and k. walsh. 2001. inter-annual, decadal and transient greenhouse simulation of tropical cyclone-like vortices in a regional climate model of the south pacific. j climate, 14, 3043-3054. seth, a. and f. giorgi. 1998. the effects of domain choice on summer precipitation and sensitivity in a regional climate model. j climate, 11, 2698-2712. vitart, f. 1997. simulation of interannual variability of tropical storm frequency in an ensemble of gcm integrations. j climate. 10, 745-760. _______, d. anderson and t. stockdale. 2003. seasonal forecating of tropical cyclone landfall over mozambique. j climate, 16, 3932-3945. walsh, k. and i. watterson. 1997. tropical cyclone-like vortices in a limited area model: comparison with observed climatology. j climate, 10:2240-2259. science diliman (july-december 2007) 19:2, 35-48 40 on the propagation of chaos for recombination models on the propagation of chaos for recombination models lu kevin ong institute of mathematics university of the philippines diliman abstract we consider binary interactions in an n-particle system. in particular, we use probability distributions known as recombination models to describe these interactions. chaos propagates when the stochastic independence of two random particles in a particle system persists in time, as the number of particles tends to infinity. the concept of propagation of chaos was first introduced by kac in connection with the boltzmann equation, while modeling binary collisions in a gas. we obtain a development of kac’s program in the framework of recombination models. specifically, our aim is to prove the relevant propagation of chaos phenomenon for our particle system. we first show that the solution for the master equation of our time-continuous process converges. then, we use this solution together with the concepts of marginal measure and chaos to prove our desired result. our main theorem for this study says that if a sequence of measures on our defined particle system is chaotic, then the resulting sequence of measures that had undergone the recombination process is also chaotic. this implies that the study of one particle after recombination gives information on the behavior of a group of particles in our particle system. keywords: particle system, recombination, master equation, propagation of chaos introduction in 1956, kac investigated the probabilistic foundations of kinetic theory and introduced the concept of propagation of chaos for the boltzmann equation. gottlieb (1998) stated in his thesis that kac invented a class of interacting particle systems wherein particles collide at random with each other while the density of particles evolves deterministically in the limit of infinite particle number. he also described the concept of propagation of chaos in general, and stated that chaos propagates when the stochastic independence of two random particles in a particle system persists in time, as the number of particles tends to infinity. several topics in propagation of chaos play a major role in the field of probability, as discussed by sznitman (1991). in particular, he wrote that one motivation for science diliman (january-june 2021) 33:1, 40-56 l.k. ong 41 the subject was to investigate the connection between a detailed and a reduced description of the particles’ evolution. he also stated that propagation of chaos deals with symmetric evolution of particles. that is, the study of one individual provides information on the behavior of the group. another research related to this topic includes the paper of mckean (1967), where he introduced propagation of chaos for interacting diffusions and analyzed what are now called mckean-vlasov equations. more recently in 2018, thai studied a discrete version of the particle approximation of the mckean-vlasov equations, and she proved the propagation of chaos property as well. many authors are concerned with proving that specific systems propagate chaos. in this study, we observe the propagation of chaos property for recombination models. rabani et al. (1995) stated that random matings occur between parental chromosomes via a mechanism known as “crossover”; that is, children inherit pieces of genetic material from different parents according to some random rule. recombination models are probability distributions that represent this randomness. in 2016, caputo and sinclair stated that recombination models based on random mating have a number of applications in the natural sciences and play a significant role in the analysis of genetic algorithms. moreover, they used quadratic dynamical systems to study the rate of convergence to equilibrium in terms of relative entropy for recombination models. let ω n = {-1,1}n for a fixed natural number n. for each natural number n, we consider the state space ω = ω n n . we think of each state as a sequence of n particles in the system, where each particle is represented by a string of bits in ω n . our main result in this paper is on the propagation of chaos phenomenon for recombination models that is represented by the interaction in this particle system. materials and methods recombination models let [n] denote the set {1, ..., n}. given a ⊂ [n] and σ ∈ ω n , we write σ a for the a-component of σ, that is, the subsequence (σ j , j ∈ a). for example, suppose n = 5 and let σ = (−1, −1, 1, −1, 1), a = {2, 3, 5}. then σ a = (−1, 1, 1). definition 1. (recombination at a set). given a ⊂ [n] and (σ, η) ∈ ω n × ω n , the recombination of (σ, η) at a consists in exchanging the a-component of σ with the a-component of η. this defines the map (σ, η) → (η a σ a c, σ a η a c ), on the propagation of chaos for recombination models 42 on the propagation of chaos for recombination models where ac is the complement of set a, and the components of η a σ a c ∈ ω n is defined by (η a σ ac ) j = η j if j ∈a, σ j if j ∈ac . ⎧ ⎨ ⎪ ⎩⎪ ⎫ ⎬ ⎪ ⎭⎪ we have a similar definition for the components of σ a η a c. for example, suppose n = 5 and let σ = (−1, −1, 1, −1, 1), η = (−1, 1, 1, 1, −1), a = {2, 3, 5}. then η a σ a c = (−1, 1, 1, −1, −1) and σ a η a c = (−1, −1, 1, 1, 1). the following process is motivated by the discussion of carlen et al. (2010). definition 2. (recombination walk). let s = (σ(1), σ(2), ..., σ(n)) denote a state in ω, where σ(i) ∈ ω n for each i ∈ [n ]. s represents a set of n particles in our system. recombination walk on ω is a process that illustrates the evolution of an initial state s by applying recombinations on a random pair of particles at each step. we describe the process as follows: 1. randomly pick a pair (k, l) of distinct indices in [n], uniformly chosen from among all such pairs. 2. choose a subset a ⊂ [n] at random according to some probability distribution ν. 3. update s by leaving σ(i) unchanged for i ≠ k, l, and updating the particles σ(k) and σ(l) via the recombination at a. let r k,l,a s denote the new state in ω. that is, if we assume k < l, the mapping r k,l,a on ω is given by (σ(1), σ(2), ..., σ(n)) → (σ(1), ..., σ(l) σ(k) , ..., σ(k) , σ(l) , ..., σ(n)). here are some examples of recombination models represented by ν, from caputo and siclair (2016): 1. single sire recombination: v(a) 1 n 1(a = {i} 1=1 n ∑ ), where 1 is an indicator function on subsets of [n]. that is, 1(a = {i}) = 1 if a = {i} for some i ∈[n], 0 otherwise. ⎧ ⎨ ⎪ ⎩⎪ ⎫ ⎬ ⎪ ⎭⎪ 2. one-point crossover: v(a) = 1 n+ 1 1(a = j i i=0 n ∑ ), where j0 = {} and ji = [i] for i ≥ 1. a a c a a c l.k. ong 43 3. uniform crossover: v(a) = 1 2n , for all a ⊂ [n]. 4. the bernoulli (q) model: for some q ∈ [0, 1/2], v(a) = q |a|(1−q)n−|a|. master equation let s j denote the position after the jth step of the walk and φ ∈ rω be a bounded borel measurable function on ω. we let t : φ → q n φ be the markov transition operator on rω represented by the transition matrix q n . based on the defined process above, the function (q n φ) ∈ rω is given by (q n ϕ)(s) = e[ϕ(s j+1 ) | s j = s] = n 2 ⎛ ⎝⎜ ⎞ ⎠⎟ −1 v(a)φ(r k ,l,a s). a ∑ 1≤k<l≤n ∑ let p i be any probability measure on {−1, 1} for i ∈ [n] and let π n be the direct product of the p i ’s, that is, π n = p 1 ⊗ p 2 ⊗ ... ⊗ p n . it is not difficult to see that the product measure π = π n n on ω gives us a reversible process, as defined by levin et al. (2009): indeed, for any s = (σ(1), σ(2), ..., σ(n)), we have π(r k ,l,a s) = π n (σ ( j) ) j≠k ,l ∏ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥πn(σ a (l)σ ac (k ) )π n (σ a (k )σ ac (l) ) = π(s). since r k,l,a (r k,l,a s) = s, the transition matrix q n is symmetric, and so we have π(s)q n (s, r k,l,a s) = π(r k,l,a s)q n (r k,l,a s, s), where q n (x, y) = p[s j+1 = y|s j = x]. the following definition is adopted from carlen, degond and wennberg (2013). definition 3. (time-continuous master equation). the time-continuous master equation associated to a discrete markov process with transition matrix q is given by d dt f (n )(t,s) = l*f (n ) (t,s) with f (n ) (0,s) = f 0 (n )(s), on the propagation of chaos for recombination models 44 on the propagation of chaos for recombination models where l* = n [q* − i] with q* the adjoint of q, i is the identity matrix, and f o (n) is the initial probability density function. since the q n is symmetric, we have q* n = qn . we now give our time-continuous master equation in the following proposition, which is motivated by carlen et al. (2010). proposition 4. if the law µ 0 (n) of the initial state s 0 has a density f 0 (n) with respect to π, then for all t > 0, the law µ t (n) of s t has a density f t (n) with respect to π, where f t (n) is the solution of the following equation d dt f n( ) = l n µ(n ) t,s( ) with f (n ) (0,s) = f0 n( ) s( ),                      (1) where l n = n (q n − i) and i is the identity matrix. remarks: 1. note that l n is self-adjoint, since q n is symmetric. 2. as defined in the proposition, we have µ t (n)(s) = f (n)(t, s)π(s) and thus (1) is equivalent to d dt µ n( ) = l n µ(n ) t,s( ) with µ(n ) (0,s) = µ0(n ) s( ). (2) 3. the solution µ t (n) of (2) is given by µ t (n ) = etln µ 0 (n ) = t j j!j=0 ∞ ∑ ln j µ 0 (n ). propagation of chaos we now present some known definitions needed for our theorem. the following definition is taken from carlen et al. (2010). definition 5. (marginal measure). let µ(n) be a probability measure on ω and let k be a positive integer with k < n. the marginal measure of µ(n) for the first k particles on a⊂ ωk n is defined as p k (µ(n))[a] = µ(n)[{(σ(1), ..., σ(k)) ∈ a}]. we adopt the following definition from sznitman (1991). l.k. ong 45 definition 6. (chaos). let µ be a probability measure on ω n . let {µ(n)} ∞n =1 be a sequence of symmetric probability measures on ω, i.e., the value is the same no matter the order of its arguments. we say that {µ(n)} ∞n =1 is µ-chaotic if for {g k : 1 ≤ k < n } ⊂ c b (ω n ), where c b (ω n ) is the set of bounded functions from ω to r, we have lim n→∞ g(s)µ(n ) (s) s∈ω ∑ = gt (σ )µ σ ∈ω n ∑ (σ ). ⎛ ⎝⎜ ⎞ ⎠⎟t=1 k ∏ , (3) where g = g 1 ⊗ g 2 ⊗ ... ⊗ g k ⊗ 1 ⊗ ... ⊗ 1 with n − k copies of the 1. we adopt the following definition from gottlieb (1998). definition 7. (propagation of chaos). a sequence {q n } ∞n =1 whose n -th term is a markov transition function on ω propagates chaos if, whenever a sequence of measures {µ(n)} ∞n =1 ⊂ ω is µ-chaotic for some measure µ ∈ ωn, then for any t ≥ 0, the sequence {µ t (n)} ∞n =1 ⊂ ω which satisfies (2) is µ̄ t-chaotic for some measure µ̄ t ∈ ω n . results and discussion we now present our main result for this study in the following theorem. statement of the main result theorem 8. (propagation of chaos for recombination models). let µ(n) be the probability measure on ω with probability density f (n) with respect to π. suppose that {µ(n)} ∞n =1 is a µ-chaotic family, where µ is some probability measure on ω n . for each natural number n, let f (n)(t, ·) denote the solution of (1) at time t, starting from the initial data f (n). let µ(n)(t, ·) be the measure on ω with probability density f (n)(t, ·), with initial measure µ(n) at t = 0. then for any t ≥ 0, {µ(n)(t, ·)} ∞n =1 is µ(t, σ)-chaotic, where µ(t, σ) is the solution of the following problem: µ(0,⋅) = µ d dt µ(t,σ ) = v(a)[µ a (t,σ a )⊗ µ ac (t,σ ac )− µ(t,σ )], a ∑ ⎧ ⎨ ⎪ ⎩ ⎪ (4) where µ a (t,σ a ) = µ(t,σ ). σ ac ∑ on the propagation of chaos for recombination models 46 on the propagation of chaos for recombination models remarks: 1. for each natural number n, we will use the notation µ(n)(·) instead of µ(n)(0, ·). 2. from the discussion of hauray and mischler (2014): suppose µ(n) is symmetric. define the empirical measure of the system as m n (t,∑) = 1 n δ σ (1)(t ) t=1 n ∑ (∑), where ∑ is a subset of ω n and δ is the dirac measure. that is, δ σ (1)(t ) (∑) = 1, if σ (i )(t)∈∑, 0, otherwise. ⎧ ⎨ ⎪ ⎩⎪ then µ(n) is µ-chaotic is equivalent to saying that lim n→∞ m n (t,σ) = µ(t,σ). moreover, it is also equivalent to condition (3) with k = 2. we will use this later in the proof of our theorem. 3. the differential equation given in (4) has a unique solution, as shown by baake et al. (2016) in the general case. in our theorem, this solution is given by µ(t, σ), which we will choose appropriately in our proof. we will first prove three lemmas that will be needed for the proof of our theorem. three lemmas the first lemma that we will prove concerns the convergence of the series that we will get from the solution of (2). lemma 9. let g ∈ c b (ω) that depends only on σ(1), ..., σ(k). if t < 1/4, for any natural number n, the power series etlng = t j j!j=0 ∞ ∑ ln j g, (5) absolutely converges for t ∈ [0, t]. l.k. ong 47 proof: suppose that g depends only on one particle and, without loss of generality, we can set this to be the first particle. that is, for some g : ωn → r, we have g(s) = g(σ(1), ..., σ(n)) = g(σ(1)). since g ∈ c b (ω), this implies that g ∈ c b (ω n ), that is for all σ ∈ ω n , |g(σ)| < m for some m ∈ r. let n be a natural number. since l n = n (q n − i), by using the definition of q n , we have for any s = (σ(1), σ(2), ..., σ(n)) that l n g(s) = n n 2 ⎛ ⎝⎜ ⎞ ⎠⎟ −1 v(a)[g(r k ,l,a s a ∑ 1≤k<l≤n ∑ ) − g(s)] = 2 n − 1 v(a)[g a ∑ l=2 n ∑ (σ a (l)σ ac ( 1) ) − g(σ ( 1) )] so that l n g(s) < 2 n −1 v(a) (2m) = 4m a ∑ l=2 n ∑ . (6) define g 2 by g 2 (σ ( 1) ,σ (l) ) = v(a)[g a ∑ (σ a (l)σ ac ( 1) ) − g(σ ( 1) )], for l ∈{2,...,n}. then l n g(s) = 2 n −1 g 2 ( l=2 n ∑ σ (1),σ (l) ). in general, we will prove by induction that l n j g(s) < j!4 jm, (7) for any natural number j. on the propagation of chaos for recombination models 48 on the propagation of chaos for recombination models indeed j = 1 is true by (6). suppose l n j g(s) < j!4 jm, for some natural number k. let g k+1 = l n k g, where g k+1 : ω n k+1 →!. l n k+1g(s) = l n g k+1 (s (k+1) ) = 2 n − 1 v(a) [g 1≤l 1 <l 2 ≤k+1 ∑ a ∑ (rl 1 , l 2 ,as (k+1) ) − g(s (k+1) )] + 2 n − 1 v(a)[g a ∑ j=k+2 n ∑ l=1 k+1 ∑ (σ ( 1) ,...,σ a ( j)σ ac (l) ,...,σ (k+1) ) − g(s (k+1) )] so that l n k+1g(s) < 2 n −1 k +1 2 ⎛ ⎝⎜ ⎞ ⎠⎟ 2g(s (k+1)) + 2 n −1 (k +1)(n −k −1)2g(s (k+1)) = 4 g(s (k+1)) (k +1)( 2n −k −2 2n −2 ) < 4(k!4km)(k +1) = (k +1)!4k+1m now, for 0 ≤ t < 1/4, the series for etlng absolutely converges since t j j!j=0 ∞ ∑ ln j g < t j j!j=0 ∞ ∑ j!4 jm = m (4t)j . j=0 ∞ ∑ in general, if we start with g, a functional of p particles where p ≥ 2, (7) is changed into |lj n g(s)| < (j + p – 1)!4jm. (8) for all natural numbers n, s ∈ ω. furthermore, for any natural number j, j + p −1( )!4 jm = j! j + p−1( )! j!  4 jm < j! j + p−1( )p−1 4 jm. so that for 0 ≤ t < 1/4 and any natural number n, we have t j j!j=0 ∞ ∑ ln j g < (4t)j( j + p−1)p−1m. j=0 ∞ ∑ then l.k. ong 49 thus, etlng is absolutely convergent for 0 ≤ t < 1/4. this ends the proof of the lemma. before we proceed to the second lemma, we first look at the following observation. for each k ∈ [n − 1], we have p k (µ(n )(t,s (k ) )) = µ(n )(t,s). σ (k+1) ,...,σ (n ) ∑ let g be a function that depends on σ(1) and define g 2 as we have defined before. then l n g(s)µ(n ) s ∑ (s) = 2 n −1 g 2 (σ (1),σ (l) )µ(n )(s) l=2 n ∑ σ (3) ,...σ (n ) ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ σ (1) ,σ (2) ∑ . so that, lim n→∞ l n g s∈ω ∑ (s)µ(n )(s) = 2 µ2(s (2))g 2 (s (2)) s(2)∈ω(2) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥, where µ 2 (σ (1),σ (2)) = lim n→∞ p 2 (µ(n )(0,σ (1),σ (2))). we now prove a more general result in the following lemma. lemma 10. let g be a function of one variable, say σ(1). then for any natrual number k, lim n→∞ l n k s∈ω ∑ g(s)µ(n )(s) = 2k µk+1(s (k+1))g k+1 (s (k+1)) s(k+1)∈ω(k+1) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥, (9) where µ k +1 (s (k+1)) = lim n→∞ p k +1 (µ(n )(0,s (k+1))),g 1 = g, and g k+1 (s (k+1)) = v(a) g k (σ (1),...,σ (t−1),σ a (k+1)σ ac (t ),...,σ (k ) )− g k (s (k ) )⎡⎣ ⎤ ⎦ a ∑ t=1 k ∑ . (10) proof: note that for k ≥ 2 we have on the propagation of chaos for recombination models 50 on the propagation of chaos for recombination models l n k g(s) = 2k n −1 v(a) a ∑ [gk−1( 1≤l 1 <l 2 ≤k ∑ rl 1 ,l 2 ,a s (k−1))− g k−1 (s (k−1))] + 2k n −1 g k+1 ( j=k+1 n ∑ σ (1),...,σ (k ),σ ( j) ) = α(s)+ β(s), where α(s) and β(s) represent the 1st and 2nd set of summations in the right-hand side, respectively. since the summations in α(s) involve a finite number of terms, we can write it as α(s) = c n −1 for some constant c. so that, lim n→∞ α s∈ω ∑ (s)µ(n )(s) = lim n→∞ c n −1 µ s∈ω ∑ (n ) (s) = lim n→∞ c n −1 = 0. now, by symmetry we have β s∈ω ∑ (s)µ(n )(s) = 2 k(n −k) n −1 g k+1 (s (k+1))µ(n )(s) σ (k+2) ,...,σ (n ) ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥. σ (1) ,...,σ (k+1) ∑ so that lim n→∞ β s∈ω ∑ (s)µ(n )(s) = 2k µk+1(s (k+1))g k+1 (s (k+1)) s(k+1)∈ω(k+1) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥. this ends the proof of the lemma. finally, we prove the third lemma that will be essential to the proof of our theorem. lemma 11. let g 1 and h 1 be functions of a single variable. let γ 2 be a function of two variables, say σ(1) and σ(2), such that γ 2 (σ(1), σ(2)) = g 1 (σ(1))h 1 (σ(2)). then for 0 ≤ t < 1/4, l.k. ong 51 (2t)l l!l=0 ∞ ∑ γ l+2(s (l+2)) s( l+2) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) ) t=1 l+2 ∏ = (2t) j j!j=0 ∞ ∑ gj+1(s ( j+1)) s( j+1) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) ) t=1 j+1 ∏ × (2t)k k!k=0 ∞ ∑ hk+1(s (k+1)) s (k+1) ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) ) t=1 k+1 ∏ , (11) where g j+1 , h k+1 , γ l+2 are defined inductively as in (10) for j, k, l ≥ 0. proof: let rhs demote the hand side of equation (11). distributing the terms in the rhs gives us. rhs = (2t)l l!l=0 ∞ ∑ l m ⎛ ⎝⎜ ⎞ ⎠⎟m=0 l ∑ s( l+2) ∑ gm+1(σ (1),σ (3),...,σ (m+2))⎡⎣ ⎤⎦ × h (l−m)+1 (σ (2),σ (m+3),...,σ (l+2))⎡⎣ ⎤ ⎦ µ1 t=1 l+2 ∏ (σ (t ) ). the result is proved if we show that for each l ≥ 0, γ l+2 (s (l+2))g(s (l+2)) s( l+2) ∑ = l m ⎛ ⎝⎜ ⎞ ⎠⎟m=0 l ∑ s( l+2) ∑ gm+1(σ (1),σ (3),...,σ (m+2))⎡⎣ ⎤⎦ × h (l−m)+1 (σ (2),σ (m+3),...,σ (l+2))⎡⎣ ⎤ ⎦g(s (l+2)), (12) for any symmetric function g. the case l = 0 is true by the definition of γ 2 , that is g 1 s(2) ∑ (σ (1))h1(σ (2))g(s (2)) = γ 2 s(2) ∑ (s (2))g(s (2)). the proof readily follows by using induction on this initial condition. on the propagation of chaos for recombination models 52 on the propagation of chaos for recombination models proof of the theorem we now proceed with the proof of our theorem. proof theorem 4: again, we consider the case when g ∈ c b (ω) that depends only on one variable, say σ(1). since l n is self-adjoint, we have g(s)µ(n )(t,s) s∈ω ∑ = g(s)etln µ(n )(s) s∈ω ∑ = t k k! l n k k=0 ∞ ∑ s∈ω ∑ g(s)µ(n )(s). since we are assuming that {µ(n)} n∈! is µchaotic, we have from (9) lim n→∞ lk s∈ω ∑ g(s)µ(n )(s) = 2k gk+1(s (k+1)) s (k+1)∈ω(k+1) ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) ), t=1 k+1 ∏ where µ 1 (0) = lim n→∞ p 1 (µ(n )(0,σ )). note also that g(s)µ(n )(t,s) = g(σ (1) µ(n )(t,s) σ (2) ,...,σ (n ) ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ σ (1) ∑ s∈ω ∑ = g(σ (1))p1(µ(n )(t,σ (1))). σ (1) ∑ from (5), it follows that for 0 ≤ t < 1/4, g(σ (1))µ 1 (t,σ (1)) σ (1) ∑ = (2t) k k!k=0 ∞ ∑ gk+1(s (k+1)) s(k+1)∈ω(k+1) ∑ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) ), t=1 k+1 ⨿ (13) where µ(t, σ (1)) = lim n→∞ p 1 (µ(n )(t,σ (1))). starting now from a function γ 2 (σ (1), σ(2)) = g(σ(1))h(σ(2)) are defining γ k (s(k)) inductively as in (10), we obtain again for 0 ≤ t < 1/4, g σ (1) ,σ (2) ∑ (σ (1))h(σ (2))µ2(t,σ (1),σ (2)) = (2t)k k!k=0 ∞ ∑ γ k+2(s (k+2)) s(k+2)∈ω(k+2) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) ), t=1 k+2 ∏ l.k. ong 53 where µ 2 (t, σ (1),σ (2)) = lim n→∞ p 2 (µ(n )(t,σ (1),σ (2))). from (11) and (13) it follows that g σ (1) ,σ (2) ∑ (σ (1))h(σ (2))µ2(t,σ (1),σ (2)) = g(σ (1))µ 1 (t,σ (1) σ (1) ∑ )⎡ ⎣ ⎢ ⎤ ⎦ ⎥ h(σ (2))µ 1 (t,σ (2) σ (1) ∑ )⎡ ⎣ ⎢ ⎤ ⎦ ⎥. (14) since g, h are arbitrarily chosen, we have 0 ≤ t < 1/4, µ 2 (t,σ (1),σ (2)) = µ 1 (t,σ (1))µ 1 (t,σ (2)). we only need to show that µ1(t,σ (1)) is the solution of (4). similar computations will lead to g 2 σ (1) ,σ (2) ∑ (σ (1),σ (2))µ1(t,σ (1)),µ 2 (t,σ (2)) = (2t)k k!k=0 ∞ ∑ gk+2(s (k+2)) s(k+2)∈ω(k+2) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) t=1 k+2 ∏ ). (15) now differentiating (13), we get g σ (1) ∑ (σ (1)) d d t µ 1 (t,σ (1)) = (2t)k−1 (k −1)!k=1 ∞ ∑ gk+1(s (k+1)) s(k+1)∈ω(k+1) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) t=1 k+1 ∏ ). on the propagation of chaos for recombination models 54 on the propagation of chaos for recombination models using a change of variable for the summation in the right hand side, g σ (1) ∑ (σ (1)) d d t µ 1 (t,σ (1)) = (2t)k k!k=0 ∞ ∑ gk+2(s (k+2)) s(k+2)∈ω(k+2) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ µ1(σ (t ) t=1 k+2 ∏ ) = g 2 (σ (1),σ (2))µ 1 (t,σ (1))µ 1 (t,σ (2)) σ (1) ,σ (2) ∑ = (q 2 − i)g(σ (1))µ 1 (t,σ (1))µ 1 (t,σ (2)) σ (1) ,σ (2) ∑ . again, by using the self-adjoint property, we have g σ (1) ∑ (σ (1)) d d t µ 1 (t,σ (1)) = g(σ (1))(q 2 − i)µ 1 (t,σ (1))µ 1 (t,σ (2)) σ (1) ,σ (2) ∑ = g(σ (1)) σ (2) ∑ σ (1) ∑ v(a)[µ1(t,σ a (2)σ ac (1) − µ 1 (t,σ (1))] a ∑⎡ ⎣⎢ ⎤ ⎦⎥ µ(t,σ (2)) = g(σ (1)) σ (1) ∑ v(a) a ∑ µ1(t,σ a (2)σ ac (1))(µ 1 (t,σ (2))( )− µ(t,σ (1)) σ (2) ∑⎡ ⎣ ⎢ ⎤ ⎦ ⎥ = g(σ (1)) σ (1) ∑ v(a) a ∑ [µ1a(t,σ a (1))⊗ µ 1ac (t,σ ac (1))− µ 1 (t,σ (1))] finally, we need to remove the restriction on t < 1/4. we can start with some t 0 , where 0 ≤ t 0 < 1/4, and we repeat the argument to extend the proof to time t where t 0 ≤ t < t 0 + 1/4. we can see that by proceeding in this manner, we can cover the whole time range, that is, µ(n)(t, ·) is µ(t, σ)-chaotic for any natural number n and t ≥ 0. this ends the proof of the theorem. l.k. ong 55 conclusion in this study, we introduced some definitions and ideas used to prove propagation of chaos for a particle system where the binary interactions are represented by recombination models. specifically, we showed that if a sequence of measures {µ(n)}∞ n =1 on our defined particle system ω is chaotic, then for any t ≥ 0, {µ t (n)}∞ n =1 , is also chaotic, where each measure µ t (n) i s obtained after applying recombinations on µ(n) for some time t. acknowledgment the author thanks the department of mathematics and physics of roma tre university for its hospitality during his research in italy. he also thanks the support of the office of the vice president for academic affairs, university of the philippines, during his research in italy. references baake e, baake m, salamat m. 2016. the general recombination equation in continuous time and its solution. discrete contin dyn syst. 36:63-95. caputo p, sinclair a. 2018. entropy production in nonlinear recombination models. bernoulli. 24(4b):3246-3282. carlen ea, carvalho mc, le roux j, loss m, villani c. 2010. entropy and chaos in the kac model. kinet. relat. models. 3:85-122. carlen ea, degond p, wennberg b. 2013. kinetic limits for pair-interaction driven master equations and biological swarm models math models methods appl sci. 23:1339-1376. gottlieb ad. 1998. markov transitions and the propagation of chaos [dissertation]. lawrence berkeley national laboratory. hauray m, mischler s. 2014. on kac’s chaos and related problems. j func anal. 266:6055-6157. kac m. 1956. foundations of kinetic theory. in:  neyman j. proceedings of the third berkeley symposium on mathematical statistics and probability; 1954 december and 1955 july august; statistical laboratory of the university of california, berkeley. berkeley, california: university of california press. 3:171-197. levin d, peres y, wilmer e. 2009. markov chains and mixing times. american mathematical society. on the propagation of chaos for recombination models 56 on the propagation of chaos for recombination models mckean hp. 1967. propagation of chaos for a class of nonlinear parabolic equations. lecture series in differential equations. 7:41-57. rabani y, rabinovich y, sinclair a. 1995. a computational view of population genetics. in proc 36-th ieee symp found comp. sci. 83-92. sznitman as. 1991. topics in propagation of chaos. in ecole d’ et’e de probabilit’es ’de saintflour xix-1989. berlin: springer. p. 165-251. thai mn. 2015. birth and death process in mean field type interaction. https://arxiv.org/ abs/1510.03238. ______ lu kevin ong <lkong@math.upd.edu.ph> is currently a ph.d. student and an instructor at the institute of mathematics, university of the philippines diliman. his research interests include probability theory, mathematical statistics, actuarial mathematics, and dynamical systems. he aims to contribute to the development of mathematics in the philippines through research and mentorship. 97 information for authors to submit your paper, please create an account at https://mc04.manuscriptcentral. com/scidil. 1. science diliman is a journal of pure and applied sciences published by the university of the philippines through the office of the vice chancellor for research and development (ovcrd). considered for publication are primary and original papers. short communications and review articles may occasionally be accepted. in all other cases, papers should present new and previously unpublished material. 2. contributions must be in english and should not have been submitted for publication elsewhere. 3. manuscripts are selected for publication according to editorial assessment of their suitability and reviews of independent referees. they will be sent to two or three reviewers, chosen for their expertise. contributors may suggest reviewers. 4. submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, review, or thesis); 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(049)536-2203; fax no. (049)536-2205 e-mail: vlbarraquio@yahoo.com date submitted: july 18, 2006; date accepted: january 11, 2007 abstract lactic acid and probiotic bacteria were enumerated and isolated from commercially available yoghurt and probiotic milk products. lactobacillus delbrueckii ssp. bulgaricus were enumerated and isolated using mrs agar incubated anaerobically at 37oc for 72 hrs. m17 agar was used for the enumeration and isolation of streptococcus thermophilus incubated aerobically at 37oc for 48 hrs. mrs agar and modified mrs agar (mrs + l-cysteine + licl + na propionate) were used for the enumeration and isolation of probiotic bacteria. both were incubated anaerobically at 37oc for 72 hrs. morphological, physiological and biochemical reactions were used to characterize the isolates. str. thermophilus counts ranged from 2.6 x 1011 to 2.9 x 1020 cfu/g with fruit yoghurt (fy) having the highest count and yoghurt natural (yn) with the lowest count. highest lactobacillus delbrueckii ssp. bulgaricus count was obtained in duo yoghurt (dy), 1.1 x 109 and lowest in yoghurt drink (yd), 8.0 x 107 cfu/g. the highest probiotic bacterial count of 2.3 x 108 was obtained in yakult (yk) and neslac (nes) showed the lowest, 1.6 x 102 cfu/g. the viable counts of all the products examined met the prescribed minimum viable count of 105 to 106cfu/g for the claimed health benefits for the consumer except for chamyto plain (cp), nes and nan-2 (nan). morphological, physiological and biochemical characteristics showed that the following genera and species were present pediococcus acidilactici (yn), p. pentosaceus (fy), lactobacillus delbrueckii delbrueckii and l. brevis in non fat high calcium yoghurt (nc), l. acidophilus and l. delbrueckii delbrueckii (dy, yd), p. damnosus and p. pentosaceus in chamyto orange (co), l. delbrueckii bulgaricus, l. acidophilus, and l. delbrueckii delbrueckii (cp), l. para. paracasei (yk) and bifidobacterium ssp. (nes and nan).of the 28 isolates characterized in this study, 15 were lactobacillus (5 species), 5 were pediococcus (3 species), 6 were bifidobacterium (species not identified), and 2 were actinomyces israelii (1 species). key words: lab, lactic acid bacteria, probiotic, fermented dairy products barraquio, karna, emata 24 and activity in the carrier food before consumption (gilliland, 1989). the health and nutritional benefits ascribed to probiotics can be generalized under the following categories: maintenance of normal intestinal microflora balance in infant and old age, improvement of lactose tolerance and digestibility of the milk products, antitumorogenic activity, reduction of serum cholesterol levels, synthesis of b-complex vitamins, and absorption of calcium (david & dauas, 1991). the commonly used probiotic strains in different dairy products are: l. acidophilus, l. casei shirota, l. casei immunitas, l. crispatus, l. gasserei, l. johansonii, l. plantarum, l. reuterii, b. adolescentis, b. animalis, b. bifidum, b. breve, b. infantis, b. lactis, b.longum, e. faecium, and e. faecalis (holzapfel et al., 1998). this study deals with the isolation and identification of lactic acid and probiotic bacteria from locally available fermented and probiotic dairy products with the ultimate objective of preserving the isolates for their future potential use in the development of new fermented and probiotic foods, feeds, pharmaceuticals, and other applications. materials and methods lactic acid bacteria (lab) locally available fermented dairy products, namely: nestle yoghurt natural (yn), fruit yoghurt (fy), non fat high calcium yoghurt (nc), yoghurt drink (yd), and duo yoghurt (dy), which were all claimed to contain lactic acid bacteria (lab), were used in this study as sources of lab. the products were collected from the supermarkets as fresh as possible. their manufacturing date, use by date and composition were carefully recorded. three (3) samples of each product were obtained and as much as possible, it was ensured that the triplicates were within the same or very close manufacturing dates. all samples were maintained at a temperature range of 4 to 60c prior to immediate microbiological examination. plating and isolation of lab were done as shown in figure 1 (idf, 1988). anaerobic incubation was done using bbl gas pak (code no.270308, becton dickinson and co., usa). number of colonies equal to the square root of the total colony count were isolated from the plates counted. morphological characterization was done by introduction lactic acid bacteria (lab), which are primarily used by the dairy industry, are extensively utilized in the fermentation of wide variety of food products and are known for their preservative and therapeutic effects (gourama, 1995). streptococcus thermophilus and lactobacillus bulgaricus are used for manufacturing of yogurt. lactococcus lactis, lactococcus cremoris, lactococcus diacetylactis, leuconostoc cremoris, leuconostoc lactis, lactobacillus helveticus, lactobacillus acidophilus, lactobacillus casei, and streptococcus sp. are used for the production of ripened cheese, cultured milk, cream and ripened butter and l. acidophilus and l. casei are widely used as probiotic bacteria in human and animal health. in meat and fish fermentation, lactobacillus plantarum and pediococcus acidilactici are used. lactobacillus sanfrancisco, l. brevis, l. plantarum, l. delbrueckii, l. lechmannii, l. casei and l. brevis are used for the production of soda crackers (gilliland, 1990). metchnikoff first hypothesized the importance of lactobacilli for human health and longevity at the beginning of 19 th century. he considered the gut microbes as detrimental rather than beneficial and suggested that desirable effects might only be expected from their substitution by yogurt bacteria. since then attempts have been made, especially during the last two to three decades to improve the health status by modulating the indigenous intestinal microflora by live microbial adjunct, now called "probiotics" (holzapfel et al., 1998). the word "probiotic" was derived from the greek word which means "on be half of ". the concept was introduced by lilly & stillwell (1965) and was intended to stimulate substances produced by one microorganism to enhance the growth of another. probiotic therefore is the exact opposite of antibiotic. the word probiotic was used later to refer to animal feed supplements and was defined as a live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance (fuller, 1989). the probiotic bacterial culture upon passage through the upper digestive tract must be capable of surviving and growing in the intestine and maintain their viability lactic acid and probiotic bacteria 25 examining colony growth, gram reaction and cell size measurement (benson, 1998). catalase test was done according to harrigan & mccance (1976). biochemical characterization of the isolates was done following holt et al. (1994) and using api 50 ch identification system (biomeriux sa, 69280 marcy l'etoile, france). the scheme for phenotypic identification is presented in figure 2. figure 1. plating and isolation of lab (idf, 1988). (l. bulgaricus) lab fermented dairy products yogurt natural (yn), fruit yogurt (fy), non fat high calcium yogurt (nc), yogurt drink (yd) and duo yogurt (dy) m 17 agar (str. thermophilus) mrs agar aerobic (37oc/48 hrs) anaaerobic (37oc/72 hrs) colonies colonies isolation isolation mrs agar stabs anaerobic (37oc/72 hrs) m 17 agar slants aerobic (37oc/48 hrs) barraquio, karna, emata 26 stab / slant cultures catalase (ve) catalase (+ ve) (discard) gram reaction / morphology gram ( + ve ) gram ( ve ) (discard) api test (sugar fermentation) figure 2. phenotypic identification of lab and probiotic bacteria stab/slant cultures catalase (ve) catalase (+ ve) (discard) gram reaction / morphology gram (+ ve) gram (ve) (discard) api test (sugar fermentation) probiotic bacteria locally available probiotic dairy products from different supermarkets were used as the sources of probiotic bacteria. the probiotic dairy products and microorganisms claimed to be present in them are given in table 1.their manufacturing date, use by date and composition were carefully recorded. all samples were maintained at a temperature range of 4 to 6o c during transit to the laboratory for immediate microbiological examination. the scheme for plating and isolation of probiotic bacteria is presented in figure 3. for chamyto and yakult, the diluents used were as recommended by idf (1988). the diluents for neslac and nan were as specified in idf (1999). colonies were picked from the plate such that the number of isolates taken was equal to the square root of the total colony count. morphological, physiological and biochemical characterization were done similarly as for lab, figure 2. identified lab and probiotic bacteria were preserved as described by kisworo & barraquio (2003) and deposited at the philippine network of microbial culture collections (pnmcc), biotech, uplb. statistical analysis completely randomized design (crd) with three replications per treatment was used. fermented dairy products were the treatments to evaluate the presence of bacteria, their types, numbers and characteristics in the products (gomez & gomez, 1984). analysis of variance (anova) in crd using the sas soft ware was done on the data on bacterial counts. the least significant difference (lsd) test was used to determine differences among fermented dairy product means. results and discussion lab and probiotic counts of dairy products the mean lab and probiotic bacterial counts of different yogurt samples are given in table 2. the data showed that the mean str. thermophilus counts of fy was highest, 2.9 x 1020 cfu/g (p ≤ 0.01) and yn was the lowest, 2.6 x 1011 cfu/g. the reason for the dairy products probiotic bacteria manufacturer claimed to be present chamyto plain lactobacillus sp. nestlephilippines, (cp) cabuyao, laguna, (nestle,switzerland) chamyto orange do do (co) neslac (infant bifidobacterium bifidus do dried milk, nes) nan-2 (infant bifidobacterium bifidus do dried milk, nan yakult l. casei strain yakult philippine fermented milk, shirota inc., laguna yk) (collab. yakult, japan) table 1 probiotic dairy product samples lactic acid and probiotic bacteria 27 higher mean putative count of fy may be due to the differences in manufacturing dates of samples used. the mean lactobacillus delbrueckii ssp. bulgaricus counts of different yogurt samples are also given in table 2. the data showed that the mean counts of dy, nc, fy and yn were statistically at par with each other but significantly higher than the yd.highest lactobacillus delbrueckii ssp. bulgaricus count was obtained in dy, 1.1 x 109 cfu/g, while yd was lowest, 8.0 x 107 cfu/g (p ≤ 0.05), which may be due to the loss of nutrients in the yogurt and accumulation of waste with age (three weeks) of the sample (kozaki et al., 1992). the data also showed that the mean putative probiotic count of yk was significantly higher than the other samples. probiotic bacterial count of yk was 2.30 x 108 cfu/g and nes showed the lowest, 1.6 x 102 cfu/ g. the reason for the differences in mean counts may be due to the nature of the product, nes being a powder. the amount of water needed for growth of microorganisms varies. this water requirement is best expressed in terms of available water or water activity, aw. most bacteria grow well in a medium with aw approaching 1.00. liquid milk has an aw of about 0.98 while dried or powdered milk's aw is below 0.60 (frazier and westhoff, 1988). the mean probiotic count of nan (1 year-old) was significantly higher than the nes (2 probiotic dairy products neslac and nan-2 cham yto and yakult m odified m rs agar m rs agar (m rs +l-cysteine +licl +na-prop.) (for lab) (for bifidobacterium) anaerobic anaerobic 370 c / 72 hrs 370 c / 72 hrs colonies colonies m odified m rs stabs m rs stabs anaerobic, 370 c / 72 hrs figure 3. plating and isolation of probiotic bacteria (idf, 1999) table 1 probiotic dairy product samples barraquio, karna, emata 28 years old), which may be due to the age of the sample, nes having earlier manufacturing date. shah (2000) recommended a minimum lab count of not less than 106 cfu/g in fermented dairy product. according to robinson (1987), for the consumers to obtain the claimed health benefit, the minimum number of lab and probiotic bacteria should be maintained at 105 cfu/g of cultured product. cp, nes and nan did not meet the prescribed minimum viable counts mentioned. colony characteristics of lab and probiotic bacterial isolates colony characteristics of lab and probiotic isolates were studied by picking up the typical, well isolated and representative colony that appeared on the plate. a single colony was aseptically picked-up and transferred to stab/slant for study of the growth pattern of isolates on the solid media. growth of isolates was observed and cultural characteristics were described (table 3). in this study, five isolates namely, yn-6, fy4, fy-5, co-5 and co-6 have colony characteristics that resembled the genus, pediococcus as described by sneath et al (1986). the colonies appeared grayish white in color, round, smooth entire and filiform, smooth and regular growth on stab. fifteen isolates namely, nc4, nc-6, dy-1, dy-2, dy-3, dy-6, yd-1, yd-2, yk-1, yk-2, yk-3, yk-7, cp-4, cp-5 and cp-6 were found to be typical of genus lactobacillus colonies of which generally appeared white to yellowish in color, round, spindle, triangular, star-like structure with effuse, filiform, irregular and arborescent on slant streak and stab. regarding probiotic bacteria, six (6) isolates namely, nes-1, nan-1, nan-2, nan-4, nan-5 and nan-6, showed colony characteristics that resembled bifidobacterium sp. holt et al. (1994) described the colonies as creamy white glistening with soft consistency, raised and convex elevation, round, triangular and spindle shaped. isolates nes-2 and nan3 resembled actinomyces, which were creamy white in color, round, smooth, soft to mucoid which also resembled more or less bifidobacterium (sneath et al., 1986; waksman,1967). morphological characteristics of lab and probiotic bacterial isolates the lab and probiotic isolates were characterized morphologically by gram staining. all 70 isolates were gram positive. the morphological characteristics of lab and probiotic isolates are presented in the table 4. cell measurements were based on 48 hr-old culture in case of str. thermophilus and 72 hr-old culture in case of l. delbrueckii ssp. bulgaricus and probiotic bacterial isolates. cell measurements of isolates were done under the high power objective (400 x). cell measurements of lab isolates ranged from 0.4 to 1.0 by 2 to 10 µm whereas the cell measurements of pediococcus isolates ranged from 1.0 to 4.0 µm in diameter. the lab belonging to the genustable 2 mean lab and probiotic bacterial counts sample mean counts (n=3, cfu/g) for lab str. lacto. thermophilus1 bulgaricus1 fruit yogurt (fy) 2.9 x 1020 a 2.6 x 108 a yogurt drink (yd) 1.4 x 1019 b 8.0 x 107 b non fat high ca yogurt (nc) 2.6 x 1014 c 3.1 x 108 a duo yogurt (dy) 1.8 x 1013 d 1.1 x 109 a yogurt natural (yn) 2.6 x 1011 e 2.3 x 108 a for probiotic bacteria2 yakult (yk) 2.3 x 108 a chamyto, orange (co) 3.6 x 106 b chamyto, plain (cp) 3.4 x 104 c nan 2 (nan) 1.6 x 103 d neslac (nes) 1.6 x 102 e 1 means with the same superscript are not significantly different at p ≤ 0.01 (s.thermophilus) and p ≤ 0.05 (l.bulgaricus). 2 means with the same superscript are not significantly different at p ≤ 0.01 n = number of samples examined lactic acid and probiotic bacteria 29 lactobacillus have rod shaped cells, usually regular but sometimes they are almost coccoid, commonly in short chains with cell size of 0.5 to 1.2 x 1.0 to 10 µm while the genus pediococcus has spherical cells, 1.0 to 2.0 µm in diameter in pairs or tetrads (holt et al., 1994). out of 28 isolates, fifteen (15) isolates namely: nc-4, nc-6, dy-1, dy-2, dy-3, dy-6, yd-1, yd-2, yk-1, yk-2, yk-3, yk-7, cp-4, cp-5 and cp-6 resembled the genus lactobacillus as described by holt et al., (1994). five (5) isolates, namely yn-6, fl-4, fl-5, co-5 and co6, were found to be typical of the genus no. isolates identity colony characteristics 1 yn 6 pedio.acidilactici colonies round, smooth, white on solid media, surface growth on broth and arborescent growth on stab 2 nc 4 lact.delb.delb colonies round, smooth, raised and filiform and entire growth on solid media 3 nc 6 lact. brevis colonies round, raised, surrounded by transparent area on solid media and punctiform on slant. 4 f y 4 pedio.pentosaceus colonies round, regular, flat and yellow color and effuse growth on solid media. 5 f l -5 pedio pentosaceus colonies round, white, regular, raised and effuse growth on solid media. 6 dy -1 lact.delb delb colonies round, smooth, raised and filiform entire growth on solid media 7 dy-2 lact. acidophilus colonies round, white raised& smaller size effuse and entire growth on slant 8 dy3 lact. acidophillus colonies round, smooth, flat, entire and white filliform, raised and entire growth on slant 9 dy -6 lact. acidophillus colonies round, white raised and non-transparent filiform, raised and entire growth on slant 10 yd-1 lact. acidophilus colonies round, white, convex & non transparent, regular & entire and slant growth is filiform 11 yd-2 lact.delb.delb.. colonies round, smooth white and flat on plate the growth is arborescent, white and rose 12 co-5 pedio. damnosus colonies round, yellow flat and filiform, smooth & regular growth on stab 13 co-6 pedio pentosaceus colonies small dot structure, white, raised, punctiform & beaded type growth on stab 14 yk-1 lact. paracasei colonies round, light yellow, raised regular & punctiform growth on plates & filiform and irregular growth on stab 15 yk-2 lact.paracasei colonies triangular, light yellow, raised, and punctiform on plate & filiform growth on stab 16 yk-3 lact. paracasei colonies yellow, spindle, raised, regular and dot like on plate, arborescent and irregular on stab 17 yk-7 lact.paracasei colonies triangular, white, raised, regular,star like on plate and arborescent and irregular on stab 18 cp-4 lact.delb bulg. colonies small, raised, regular, non-transparent, arborescent and irregular growth on stab 19 cp-5 lact acidophilus colonies triangular,irregular,white raised on plate and arborescent and irregular growth on stab 20 cp-6 lact. delb.delb. colonies round, transparent, small, flat on plate arborescent and irregular growth on stab 21 nes-1 bifidobacterium sp. colonies round, regular, white glistening convex soft on plate and arborescent growth on stab 22 nes-2 actinomyces israelii colonies spindle, entire, white glistening, convex, smooth and filiform growth on stab 23 nan-1 bifidobacterium sp. colonies round, transparent, convex, smooth & entire and arborescent growth on stab 24 nan-2 bifidobacterium sp. colonies spindle, non transparent, convex, entire, smooth and cream color & filiform growth on stab 25 nan-3 actinomyces israelii colonies round creamy, convex, entire and arborescent growth on stab 26 nan-4 bifidobacterium sp. colonies triangular, cream color, convex, entire and arborescent growth on stab. 27 nan-5 bifidobacterium sp. colonies spindle, cream color, convex, entire and filiform growth on stab. 28 nan-6 bifidobacterium sp. colonies spindle, cream color, very small size, convex, entire and filiform growth on stab. ( ) not a lab nor probiotic bacteria table 3. colony characteristics of lab and probiotic bacteria table 3. colony characteristics of lab and probiotic bacteria barraquio, karna, emata 30 pediococcus in gram reaction, cell shape, cell size and occurrence. six (6) of the probiotic isolates, namely, nes-1, nan-1, nan-2, nan-4, nan-5 and nan-6 appeared as thin, gram positive rods, branched, arranged singly and pairs in v and y arrangements, in chains, typical of bifidobacterium as described by holt et al. (1994). actinomyces israelii was also isolated from nes-2 and nan-3 which might have come from the processing plant environment. waksman (1967) explained that actinomyces occurs virtually in every natural substrate such as fresh water basins, foodstuffs and the atmosphere and multiply most abundantly in various depths of soil and compost in temperate and tropical regions. sugar fermentation of lab and probiotic bacterial isolates out of 70 isolates, only 28 isolates were selected and subjected to sugar fermentation test due to budgetary constraints. the sugar fermentation patterns of lab and probiotic bacterial isolates are presented in table 5. the summary of api output of lab and probiotic bacterial isolates is presented in tables 6 and 7, respectively, while the sources of lab and probiotic isolates are shown in table 8. in general, in case of lab, majority of the isolates were lactobacillus with 5 different species identified followed by pediococcus with 3 different species. damelin et al. (1995), in his study on the biodiversity of lab from food related ecosystem also reported that the lactobacillus strain dominated all ecosystems and consisted 65% of lactobacillus isolates. kisworo and barraquio (2003), also found in their study of raw milk and white soft cheese that the most predominant genus isolated was lactobacillus, which comprised fourteen (14) out of twenty-three (23) isolates. another work conducted by tzanetakis and litopoulou-tzanetaki (1992), showed that lactobacillus was the predominant genus over enterococcus and pediococcus in cheese. rodrigues et al. (1995), reported that majority of species found in raw cow milk cheese were lactobacillus casei ssp. casei followed by lactococcus lactis ssp. lactis. table 4 morphological characteristics of lab and probiotic bacterial isolates identity shape average cell size pediococcus acidilactici cells spherical, in tetrads, also in pairs 1.0 2.0 µm pediococcus pentosaceus cells spherical, in tetrads, also pairs 1.0 2.0 µm pediococcus damnosus cells spherical, tetrads, some are in pairs 1.0 4.0 µm lact. brevis cells rod shaped, regular rounded ends, occur singly/chains 0.6-1.0 x 4 6 µm lact. delb. delbrueckii cells rod shaped, regular, occurring in short chains 0.4-1.0 x 4-8 µm lact. acidophilus cells rod shaped, regular, occurring in short chains. 0.6-1.0 x 2-8 µm lact. delb.bulgaricus cells rod shaped, occur in pairs or chains with square ends 0.5-1.0 x 4 -10 µm lact. para. paracasei cells thin rods, with square ends and in chains 0.4 1.0 x 2-4 µm bifidobacterium sp. cells thin rods, branched, v and y arrangements in chains. 0.5-1.3 x 1.5-8 µm (actinomyces israelii) cells short rods with clubbed ends occurring in v and y 0.5-1.x 24µm arrangements in chains ( ) not a lab nor probiotic bacteria lactic acid and probiotic bacteria 31 table 5. sugar fermentation patterns of lab and probiotic bacterial isolates isolates sugar 1 2 3 4 5 6 7 8 9 10 glycerol + + l-arabinose + + + + d-arabinose + + ribose + + + + d-xylose + + + + + d-glucose + + + + + + + + + + d-fructose + + + + + + + + d-mannose + + + + + + + + + l-sorbose + sorbitol + rhamnose + + + n-a.glucosamine + + + + + amygdalin + + + + arbutine + + + + + esculin + + + + + + + + + salicine + + + + + + + + manitol + + + maltose + + + + + + lactose + + + + + + α-m.d-glucoside + galactose + + + + + + gelatin trehalose + + + + + + cellobiose + + + + + + + + saccharose + + + + + + melezitose + + inulin + d-raffinose + + b-gentiobiose + + + + + + d-turanose + + d-tagatose + + + + + darbitol gluconate + adonitol + indol urease 1lactobacillus acidophilus 2lactobacillus para.paracasei 3 -lactobacillus delb. bulgaricus 4 -lactobacillus delb. delbrueckii 5lactobacillus brevis 6pediococcus pentosaceus 7 -pediococcus damnosus 8 -pediococcus acidilactici 9 -bifidobacterium spp. 10actinomyces israelii barraquio, karna, emata 32 no. isolates identity % identification 01 y n-6 pediococcus acidilactici 99.9 02 nc -4 lact. delb delb rueckii 96.8 03 nc-6 lact. brevis 99.7 04 fy -4 pedio.pentosaceus 85.1 05 fy -5 pedio. pentosaceus 99.9 06 dy -1 lact. delb. delbrueckii 90.7 07 dy 2 lact. acidophilus 99.1 08 dy 3 lact. acidophilus 99.9 09 dy 6 lact. acidophilus 98.2 10 yd 1 lact. acidophilus 95.8 11 yd 2 lact. delb.delbrueckii 99.9 12 co-5 pedio. damnosus 99.8 13 co-6 pedio. pentosaceus 99.9 14 yk 1 lact. para. paracasei 99.9 15 yk 2 lact. para. paracasei 99.9 16 yk 3 lact. para. paracasei 99.5 17 yk 7 lact.para. paracasei 92.5 18 cp 4 lact. delb. bulgaricus 99.7 19 cp 5 lact. acidophilus 99.7 20 cp 6 lact. delb.delbrueckii 98.2 summary and conclusions cultural, morphological, physiological and biochemical characteristics showed that the following genera and species of lab and probiotic bacteria were present in the dairy products examined: pediococcus acidilactici (yn), pediococcus pentosaceus (fy), lactobacillus delbrueckii delbrueckii and lactobacillus brevis (nc), lactobacillus acidophilus and lactobacillus delbrueckii delbrueckii (dy), lactobacillus delbrueckii delbrueckii and lactobacillus acidophilus (yd), pediococcus damnosus and pediococcus pentosaceus (co), lactobacillus delbrueckii bulgaricus, lactobacillus acidophilus and lactobacillus delbrueckii delbrueckii (cp), lactobacillus para. paracasei (yk), and bifidobacterium sp. (nes and nan). of the twenty-eight (28) isolates, five (5) were lactobacillus acidophilus, four (4) each of l. table 6 summary of api identification output for lab no. isolates identity % identification 1 nes-1 bifidobacterium sp. 98.2 % 2 nes-2 (actinomyces israelii) 95.2 % 3 nan-1 bifidobacterium sp. 90.2 % 4 nan-2 bifidobacterium sp. 94.5 % 5 nan-3 (actinomyces israelii) 95.2 % 6 nan-4 bifidobacterium sp. 96.5 % 7 nan-5 bifidobacterium sp. 95.7 % 8 nan-6 bifidobacterium sp. 96.5 % ( ) not a lab nor probiotic bacteria table 7 summary of api identification output for probiotic bacterial isolates table 8 sources of lab and probiotic bacterial isolates sources lab and probiotic no. of bacteria isolates yogurt natural pedio. acidilactici 1 non fat high ca yogurt lact.delb.delbrueckii 1 lact. brevis 1 fruit yogurt pedio. pentosaceus 2 duo yogurt lact.delb.delbrueckii 1 lact.acidophilus 3 yogurt drink lact. acidophilus 1 lact.delb.delbrueckii 1 chamyto (orange) pedio. damnosus 1 pedio. pentosaceus 1 chamyto (plain) lact.delb. bulgaricus 1 lact. acidophilus 1 lact. delb. delbrueckii 1 yakult (plain) lact. para. paracasei 4 neslac (powder milk) bifidobacterium sp. 1 (actinomyces israelii) 1 nan-2 (powder milk) bifidobacterium sp. 5 (actinomyces israelii) 1 total isolates 28 ( ) not a lab nor probiotic bacteria lactic acid and probiotic bacteria 33 para.paracasei and l. delbrueckii delbrueckii, one (i) each of l. brevis and l. delbrueckii bulgaricus. there were three (3) pediococcus pentosaceus and one each of p. damnosus and p. acidilactici. of the probiotic bacterial isolates there were six (6) bifidobacterium sp. found and two (2) actinomyces israelii. the viable counts of all the products met the prescribed minimum viable count of 105 to 106 cfu/g for the claimed health benefits for the consumer except for chamyto plain, neslac and nan-2. acknowledgments the senior author is grateful to the arep/world bank project for the financial support. references benson, h.j., 1998. gram staining. microbiological applications. laboratory manual in general microbiology. new york, mc graw-hill: 468. damelin, l.h., g.a. dykes, & a. von hoy, 1995. biodiversity of lactic acid bacteria from food related ecosystem. microbios 83 (334): 13-22. david, b.h. & g.h. dauas, 1991. bifidobacteria their potential for use in american dairy products. food technol. 45 (4): 74-80. frazier, w.c. & d.c. westhoff, 1988. food microbiology, 4th ed. new york, mcgraw-hill book co. 539. fuller, r,. 1989. probiotics in man and animals: a review. j. appl. bacteriol. 66 (5): 365-368. gilliland, s.e., 1989. acidophillus milk products: a review of potential benefits to consumers, j. dairy sci. 72 (10): 24832494. gilliland, s.e., 1990. health and nutritional benefits from lactic acid bacteria. fems microbiology reviews 87: 175-188. gomez, k.a. & a.a. gomez,1984. statistical procedure for agricultural research, 2nd ed. singapore, john wiley and sons, inc.:680. gourama, h.& l b. bullerman, 1995. antimycotic and antiaflatoxigenic effect of lactic acid bacteria. a review. j. food protection 58 (11): 1275-1280. harrigan, w.f. & m.e. mc cance, 1976. laboratory methods in food and dairy microbiology. london, academic press inc.: 452 pp. holt, j.g., n.r. krieg, p.h.a. sneath, j.t. staley, & s.t. williams, 1994. bergey's manual of determinative bacteriology. baltimore, williams and wilkins: 787 pp. holzapfel, w.h., p. haberer, j. snel, u. schillinger, & j. huis int veld, 1998. over view of gut flora and probiotic. int. j. of food microbiol. 41 (2): 85-101. idf. 1988. yogurt: enumeration of characteristic microorganisms. colony count techniques at 37 0c. international dairy federation bulletin 117 a, brussels, belgium: 1-5 idf. 1999. guidelines for the enumeration of bifidobacteria in fermented dairy products. international dairy federation bulletin 340, brussels, belgium: 19-23. kisworo, d. & v.l. barraquio, 2003. characteristics of lactic acid bacteria from raw milk and white soft cheese. the philippine agricultural scientist 86 (1): 56-64. kozaki, m., t. uchimura, & s. okada, 1992. manual for lactic acid bacteria. tokyo university of agriculture, tokyo, asakura book shop: 164. lilly, d.m. & r. stillwell, 1985. probiotics: growth promoting factors produced by microorganisms in: golden, b.r, 1998. health benefits of probiotics. british j. of nutr. 80 suppl. 2: s 203-207. metchnikoff, f., 1908. the prolongation of life. in: n.p.shah, 1994. lactobacillus acidophilus and lactose intolerance. a review. asian food journal 9(2): 47-54. robinson, r.k., 1987. survival of lactobacillus acidophilus in fermented products. in: ravula, r.r. & n.p shah, 1998. biotechnology techniques 12(11): 819-822. barraquio, karna, emata 34 rodrigues-medina, m.l., m.e.tarnodijo, j. carballo, & r.m. sarmineto, 1995. microbiological study of leon raw cow milk cheese, a spanish craft variety. j. food protection 58 (9): 998-1006. shah, n.p., 2000. probiotic bacteria: selective enumeration and survival in dairy foods. j. dairy sci. 83 (4): 894-907. sneath, p.h.a., n.s. mair, m.e. sharpe, & j.g. holt, 1986. bergey's manual of systematic bacteriology (vol 2). baltimore, williams and wilkins: 965-1599. tznetakis, n. & e. litopoulou-tznetaki, 1992. changes in numbers and kinds of lactic acid bacteria in feta and teleme, two greek cheese from ewes' milk. j. dairy sci. 75(6): 13891393. waksman, s.a., 1967. the actinomycetes (a summary of current knowledge). new york, the ronald press co.: 280. call for papers-new.pmd ocr document 01_device fernandez, pepito r. 18 the relevance of governance institutions in marine protected area design and management: lessons from northeastern iloilo, philippines pepito r. fernandez jr. phd candidate department of human geography research school of pacific and asian studies australian national university, canberra act 0200 australia telephone: +61 2 6125 0403 facsimile: +61 2 6125 4896 email: sonny_fernandez@up.edu.ph and assoc. prof. in political science (on study-leave) division of social sciences-cas university of the philippines in the visayas, miagao, iloilo abstract science diliman (january-june 2006) 18:1, 18-34 the experience of the philippines on decentralized marine protected area (mpa) management can provide an instructive purview of nature-society processes and politics of scale in a post-colonial and tropical marine fisheries setting. this paper examines and analyzes the comparative advantage and limitations in adopting government regulation, community-based initiatives and co-management arrangements (i.e., rules) in designing and implementing mpas to meet conservation and livelihood goals. the theoretical discussion will be enriched by providing relevant contextual factors (i.e., biophysical setting, community attributes and institutional setting) from secondary literature and social science field data gathered from march to december 2005 in various coastal municipalities in northeastern iloilo province, philippines. the study site contains 17 mpas established between 1994 to 2004 with diverse backgrounds and profiles, and are governed by various alliances (state and/or non-state actors) in different scales (i.e., local to international). the paper will argue that no single institutional arrangement is likely to be effective in addressing all the circumstances surrounding mpa design and implementation. but in the milieu of a depleted natural resource base, and the continued deterioration of the livelihood and health of poor people, environmental protection of mpas and municipal fishing grounds of subsistence fishers should be prioritized by various actors and policy networks. the relevance of governance institutions in marine protected area 19 introduction a significant proportion of the social and economic welfare of an archipelagic country like the philippines depends directly or indirectly on the availability of environmental goods and services provided by productive coastal and fisheries resources. the countries tropical coastal areas1 are characterized by highly diverse ecosystems and equally diverse related functions such as providing a source of income and food. the philippines is the 8th larges producer of fish and other marine products in 2004 and its 13.6 million metric tons output is 2.5 percent of world total (fao statistics division website). the country is also the 11th largest fish harvester in the world with fisheries contributing 22.59 percent of agricultural export (fao 2003). although estimates reveal that only 1.7 million of the 85 million of the population are directly earning a living from fisheries2 , 40.7 per cent of per capita daily animal protein intake of the country comes from fisheries products (green et al. 2003, world bank 2005: 30). philippine coastal and marine resources, aside from contributing vast quantities of food and supports an economy based on nature, also functions as a trade and transportation route. coasts also provide tourism dollars, by attracting people who want to explore these unique environments and engage in various recreational activities. since coastal and marine resource systems are environmentally, economically, socio-culturally and politically important "managing" and "protecting" them has always been an important goal for government (i.e. local and national levels). but the conventional approach of optimizing economic and biological yield of a few species thru maximum sustainable yield (msy) calculations is unsuited in tropical countries like the philippines with a multi-species marine resource (pomeroy 1995). moreover, in these tropical and less-well-managed economy contexts where target species are often highly vulnerable, ecological data are incomplete, fish landings often undocumented, with ill-defined property rights (roberts & polunin 1991, roberts 1997, johannes 1998), important public interest issues such as sustainable fisheries and environmental justice can be compromised. since the 1980s coastal and marine policy is increasingly adopting various kinds of marine protected area (mpa)3 strategies to achieve multiple objectives (russ & alcala 1999). but a large number of mpas are not effective and do not achieve their goals (mcclanahan 1999). a lack of efficacy can occur because of the lack of community acceptance and institutional support by concerned actors/stakeholders in various scales (white & voght 2000, dietz et al. 2003). since the late 1980s environmental awareness and calls for "sustainable development" (i.e., economic growth coupled with environmental protection and social equity) from multilevel locations, have encouraged the participation of civil society (e.g. people's organizations or pos, nongovernmental organizations or ngos, academe, etc.) and market forces (e.g. business enterprises, financial institutions, multinational corporations, etc.) in the 1is technically “... a band of dry land and adjacent ocean space (water and submerged land) in which terrestrial processes and uses directly affect oceanic processes and uses and vice versa; its geographic extent may include areas within a landmark limit of one (1) kilometer from the shoreline at high tide to include mangrove swamps, brackish water ponds, nipa swamps, estuarine rivers, sandy beaches and other areas withhin a seaward limit of 200 meters isobath to include coral reefs, seagrass beds and other soft-bottom areas” (the philippine fisheries code of 1998, 5). 2approximately 1.3 million are municipal fishers while 375,000 are commercial fishers. but fish catch is skewed in favor of the latter with a share of 54.3 percent of total annual catch. the contribution of aquaculture and marine ranching or fish pen culture to total philippine fish production, on the other hand, is on the rise and currently employs 16,497 individuals (green 2003:33, rosario 2006:3). 3mpas can be generally described as coastal or marine environments that are established or constructed for conservation and protection, and where activities are managed based on specific rules and guidelines that are imposed by individuals and/or groups through technologies and methods of socio-cultural, political and economic institutions. the work of foucault (1991) provides insights on how the governance of mpas involves not only what people can do (rules), but also what goals and behaviors are considered socially desirable (norms/expectations). fernandez, pepito r. 20 discourse of coastal area management and the remaking and recomposition of governance regimes. this outcome was institutionalized through devolution and decentralization of marine and coastal area governance. in the philippines, following the enactment of the 1987 philippine constitution4 , the promise and trend of increased people's participation in multilevel development processes and natural resources management was legitimated by enabling national legislation such as the local government code of 1992 and fisheries code of 1998. in terms of legislation, the philippines is now one of the few countries in the world that: transfer decisionmaking authority to local communities; shifts decisionmaking and fiscal powers to local branches of government, and; holistically incorporates the fisheries sector in coastal resources management or crm (fernandez et al. 2000, meltzer 1998). recently, scientific research has adjudged the philippines as the epicenter of marine biodiversity per unit area in the world (carpenter and springer 2005). the study also provides ample evidence that the marine and coastal resources of the country, and elsewhere, are threatened and overexploited. some attribute these problems to flawed science, as well as the interrelated socio-economic problems (i.e., population increase and poverty) putting pressure on fragile natural resources. others argue that fisheries management fails to change incentive structures, and promotes inefficient fishing practices that are also inconsistent with community values. still others observe that even though political will may exist to manage coastal fisheries, various stakeholders often lack the data, skills and resources necessary for effective management (see roberts 1997 and wb 2005 for an overview). also, crm in tropical contexts, also presents a complex social problem or classic collective action problem (sandler 1992), coupled with intense competition over scare marine resources. it is therefore expected that governing and managing such "heritage" will increasingly become an international agenda spurring diverse and complex political and power relationships across scale. but dominant and apolitical perspectives do a poor job of analyzing issues related to institutional design (means) and performance (ends) of coastal management systems in the country. the paper begins with a brief overview of three major types of institutional arrangements used to manage coastal zones and mpas in the philippines in order to identify their characteristics and illustrate the importance of considering institutional design and performance indicators. the experience of coastal municipalities in northeastern iloilo province (nip) in institutionalizing mpa efforts are then weaved into the discussions to support arguments and insights. the paper concludes by calling for the adoption of an approach to institutional analysis that considers the context and varied outcomes of relational politics in designing and performing coastal management and development. methods this paper is based on research results from field data gathered in coastal municipalities of northeastern iloilo (ni), particularly those organized under the northern iloilo alliance for coastal development (niacdev)5, 4the constitution provides an explicit call for active grassroots and civil society participation in democratic and distributive processes in development work for the welfare of the country (art. ii, sec. 23). it recognizes the obligation of the state to “protect, develop and conserve marine resources (art. xii, sec. 7) and “protect the right of subsistence of fishermen, especially local communities (art. ii, sec. 22). 5the niacdev is a registered non-profit decisionmaking and management council composed of 10 municipalities (seven have coastal areas) and led by local chief executives (i.e., mayors). founded in 1998 by six municipalities (seven have coastal areas) and led by two volunteers (i.e., a municipal councilor and a crm officer). the niacdev coastal area has island barangays. subsistence and commercial fishing activities, as well as fish processing, abound in its shallow (below 100 feet) fishing ground, dotted with patches of coral reefs, mangrove areas, and seagrass beds. the relevance of governance institutions in marine protected area 21 from march to december 20056. although proximate with one another, the biophysical and socio-economic context of the members of niacdev is diverse (see figure 1 and tables 1 & 2). data were collected, organized and analyzed using qualitative research techniques and include the following: 1. analysis of secondary data, mostly from municipal and government offices, and published and unpublished reports of experts/researchers. 2. key informant interviews with actors from government, non-government organizations, and local communities to verify and enrich the secondary information. 3. participation and attendance in various crm-based planning, implementation and monitoring exercises at the barangay, municipal and intermunicipal levels. 4. participant observation further enhanced the collection and analysis of data. due to the sensitivity of enforcement and compliance issues that were encountered in the field, the exact location of key events and identity of key informants are not revealed in the text. the various data sources for this study were reviewed and analysed together so that findings were based on convergence of information from different origins. the development of converging lines of inquiry through the process of triangulation, and the comparison of case study sites, allowed for the corroboration of evidence. this paper was also enriched by a multidisciplinary perspective (i.e., political science and human geography) in analysing socio-ecological systems at various scales. the use of triangulation and multidisciplinarity in the research process attempts to reduce biased conclusions and overcome the limitation of looking at the topic using a specific research method or academic perspective. 6part of thesis fieldwork results for the phd program of the department of human geography-rspas. a travel grant was provided by the australian national university, with support from the doctoral studies fund of the university of the philippines. table 1. bio-physical context of coastal municipalities in northeastern iloilo (ni). land area (km2) 175.52 57.3 52.61 103.52 97.2 30.55 127.07 surface area of 250 0.6 8 368 320 10 7.6 mun. waters (km2) hectarage for --; --; 13.5; -27; 33; ---; 200.27; ---; 17.43; -2.5; 5; 6.5 3; 13; 14 coral reefs, mangrove and seagrasses* length of 74.83 3.5 8.2 94.7 120 28.51 23.7 shoreline (km) no. of islands 8 0 2 30 17 3 1 (0-10; 10-15; (8; 0; 0) (2; 0; 0) (16; 4; 10) (11; 4; 2) (3; 0; 0) (1; 0; 0) 15+ kms. from shore) ajuy balasan batad carles concepcion estancia san dionisio nis municipal waters are relatively shallow (less than 60 meters). anchovies, goatfish, mackerel, sardine, herrings, slipmouth, nemiptepids, crevalles, whitings, therapons, as well as blue crabs, squids, and shrimps, lobsters, seashells are targeted species. milkfish, tilapia, prawns, and seaweeds are cultured. fernandez, pepito r. 22 results and discussions coastal area and marine fisheries management and conservation programs in the philippines, and in less well-managed economies, are often based on three types of institutional arrangements: (1) bureaucracybased (fernandez et al. 2000), (2) community-based (charles 1992, ferrer et al. 2001), and co-management (kuperan et al. 2003). the history of natural resource management and conservation reflects elements of control and coercion by government and state institutions. the perceived failures and shortcomings of centralized state institutions, however, drew together disparate communities and interests into collective awareness and action to challenge or reconfigure existing institutions for natural resources management and conservation. bureaucratic institutions for bureaucracy-based institutional frameworks a common assumption is that an external leviathan or hobbesian leader is necessary to prevent the "tragedy of the commons", an assumed condition where all individuals seek personal benefits in environmental systems. since costs of extraction (i.e., overexploitation) are shared by the entire members of the community, accelerated individual extraction is pursued and inevitably leads to environmental destruction (gordon 1954, ostrom 1990). institutional arrangements based on centralized national or local government control can be considered as the dominant or default position in managing coasts and related resources (bryant & bailey 1997). analysts do not discuss the need for state control over coastal resources but on how to efficaciously decentralize state control of the existing regulatory system. effort is made to examine the development and implementation of various coastal and fisheries management plans and regulations (cite government backed studies). since it occupies the dominant position, bureaucracy-based arrangements are widely criticized by those favoring different arrangements (e.g., ferrer and de la cruz 2001). table 2. socio-demographic context of coastal municipalities in northeastern iloilo. coastal brgy. 18 (53%) 2 (8.6%) 6 (25%) 32 (97%) 18 (72%) 16 (64%) 9 (31%) (% of total brgy.) coastal popu27,174 1,543 6,815 18,920 28,742 27,351 10,917 lation (% of total (60%) (6%) (40%) (40%) (84%) (72%) (43%) pop. 2000) registered mun. 3,185 120 142 4,500 (est.) 3,211 1,296 462 fishers, 2005 (1,182) (5 motor (200 motor (2943 motor (932 motor (232 motor (251 motor (# of vessels) 46 non) 144 non) 3623 non) 704 non) 165 non) 176 non) commercial fish --1,608.4 783.76 21.23 1,144 968.4 prod. ‘003, (5; 0) (--; 0) (--; 0) (--; 1) (318; 1) (420; 2) (36; 1) mt/year (# of vessels, ports) fishpond area 1,159 has. 836.3 has. 88 has. 1,539 (--) 220 has. 260.3 has. 281.86 has. (# of operators) (148) (45) (23) (15) (2) (14) fish processing 1 for crabs - 1 for crabs 2 for crabs 2 for blue 4 for crabs 4 for squids, plants (prod’n.) (1.5) (10.5-14) (3.5) crabs (7-21) & shellfish shrimps, in metric tons (3.5) anchovies ajuy balasan batad carles concepcion estancia san note: sixty percent of ni municipalities are in the top 10 percent of the poorest in the province, while its annual population growth rate is higher than the national average at 2.8 percent. child malnutrition in the coastal and island barangays of ni is 32 to 44 percent of the 0-6 year old bracket (source: municipal health offices in ni) the relevance of governance institutions in marine protected area 23 figure 1. location of coastal municipalities of niacdev in northeastern iloilo province fernandez, pepito r. 24 in bureaucracy-based arrangements, property rights to fish and exploit coastal resources are held by government on behalf of the public and the focus is on developing regulations that maintain stocks of resources at sustainable levels. other socio-economic and conservation goals, however, may be embedded in these programs. in the philippines, government uses coastal and fisheries policy to promote the twin goals of coastal management for community (i.e., 1998 fisheries code) and national (i.e., agriculture and fisheries modernization act of 1998). but in the context of “depleted state of resources” (stobutzki et al. 2006:113), and the continued deterioration of the livelihood and health of poor people (wri 2005), environmental protection to enable resilient and viable local communities should be prioritized. at the national level determining the maximum sustainable yield (msy) or total allowable catch (tac)7 is considered officially important in bureaucracy-based arrangements because the information is used to control fishing effort by adopting rules that limit entry (e.g. thru a fisherfolk identification system, and boat licensing of vessels below 3 gross tons), creating closed seasons, restricting illegal gear and imposing volume and size limits of fish caught etc.8 it is assumed that when and where there is some stability in these rules, it improves accountability, lowers administrative costs, and improves equity, since the rules often apply to all fishers or user groups. although the national government endorses the use of data on msy or tac to guide policy, there is no evidence that the strategy is being implemented. in the municipality of concepcion, and the rest of the coastal-based members of the niacdev, such figures are not utilized in local policy. but enabling local 8determining msy or “best available scientific data” is a key guide to manage coastal areas as explained in the philippine fisheries code or republic act 8550. see israel and banzon (no date) for a sample of how to derive msy for the philippines. 7it should be noted that conventional fisheries management system is heavily influenced by the temperate scientific method of calculating maximum sustainable yield (msy) of a few species. msy attempts to model and relate level of fishing effort with the biological optimum that can sustain fish species. as mentioned earlier, authoritative studies show that these models have limited usefulness in tropical fisheries with its multi-species nature (pomeroy 1995), and in complex large-scale ecosystems (gunderson and pritchard 2002; 251264). legislation, efforts at patrolling coastal areas and application of other strategies mentioned earlier (save for the imposition of volume and size limits of fish caught) are being instituted to limit fisher and vessel entry to municipal waters. the process of developing fishery management plans in bureaucratic systems can easily be politicized and subject to capture by subsistence and commercial fishers, politicians, or even conservation groups (smith et al. 2003). conflicts among competing stakeholders can make policy change costly, time-consuming, and increase information costs. consequently, the system's ability to rapidly adapt to catch declines, change in fishing technology, or changing social, political, and economic conditions may be reduced. such dilemma can be observed in nip. key informant interviews and participant observations reveal persistent conflicts over management plans and strategies between and among subsistence fishers, commercial fishing operators, politicians and their pressure groups, fish processing plants, barangays with marine protected areas (mpas), and non-governmental organizations (ngos) that implement various development programs/projects. recent decline in the harvest of crabs in nip has ignited an on-going debate on conservation policy and mitigating measures. on the other hand, the adoption of new fishing technology, such as that of fishpots (submerged fish traps that is attached to buoys with flags/markers), for example, has led to intense conflicts between smallscale fishers and commercial fishers. the latter complain that fishpot of artisenal fishers block their passageway towards the open sea, while former accuse commercial fleets of dragging and destroying their fishing gears. problems can also occur in any institutional arrangement when the system is ''chaotic'' and rules are changed or bended frequently in response to political pressures before fishers adapt or decisionmakers can tell if rules are working. the relevance of governance institutions in marine protected area 25 violation of size limits for fish caught, fishing during closed seasons or in closed areas, underor unreported catch (sterner2003), and even bribery and corruption. thus, all these can lead to high enforcement costs and the danger of driving out law-abiding fishers when they cannot compete with lawbreakers and their politically powerful allies and patrons. in nip, municipal governments are regularly challenged to keep up with reports (usually texted via cellphone by local residents to elected officials, coastal patrol groups, and local police forces) and apprehend many types of violations listed above committed by local fishers and outsiders. the municipality of ajuy experiences these kinds of inefficiencies. the municipality of ajuy, for example, spends approximately php 5,000 a day on fuel cost and food to enable its two patrol boats and crew to operate 24 hours a day. even when operations are successful, apprehending officers or local residents are hesitant to file court cases against offenders due to the lengthy and expensive process of litigation. in an interview, a former barangay captain of ajuy decried the lack of financial and legal support provided by the municipal government in her attendance of court hearings as a complainant against the owner of a trawling vessel apprehended two years ago. consequently, difficulties in implementing a statecentered approach in coastal and fisheries management/ conservation have created interest in other institutional arrangements. in the philippines and elsewhere, there are three main reasons to account for interest in nonbureaucratic arrangements in natural resources management. the first reason has more to do with attempts by government bureaucracies to ensure their continued survival as they face rising debt, declining terms of trade, economic liberalization and market integration. these internal and external economic processes are forcing nation-states to reduce the size of their civil services and thus their capacities for direct service provision (boer and rooimans ed. 1994). in the drive to 'do more with less', governments forge, at times reluctantly, new partnerships with civil society groups (i.e., private organizations that are non-profit making and are non-political parties) and adopt participatory approaches in development activities which presumably give local people more control over research and development processes (hulme &shepherd 2003). the asian financial crisis of 1997, controversy over the extent of municipal territorial waters, poaching in mpa sites, and the role of provincial governments in fishery law enforcement are some noteworthy examples in the complex islandbased context of nip. the situation is that the niacdev municipalities previously adopted the department of environment and natural resources' (denr) administrative order 17 or dao 17 in delineating their municipal waters. under the archipelagic doctrine of dao 17, the extent of municipal waters was set 15 kilometers from the farthest island territory. under such delineation scheme three municipalities have a municipal territory that is more than 20 kilometers from its mainland shore. latching on to the promise of increased revenue, and exploiting a loophole in the local government code of 1991, registered municipal commercial fishers in one of the three archipelagic municipalities are allowed (thru a municipal ordinance) to exploit offshore fishing areas from a 10.1 to 15-kilometer radius from the mainland. but the provincial government of iloilo, which set up their own "mobile patrol groups" in nip, argues that providing municipal access rights to commercial fishing vessels is inconsistent with stipulations of the 1998 fisheries code that ban all active fishing gears within municipal waters. this lack of common interpretation of the law, and political conflict between the governor's office and the niacdev coalition, has therefore caused enforcement and compliance problems, as well as the continued violation of the territorial integrity of municipal waters and the 17 municipality-endorsed mpa sites (mostly located near offshore barangays) in nip. recent reports reveal that the national government has endorsed the operation of a navy gunboat in the wider visayan sea area to guard against all forms of illegal fishing. as a consequence, at least three enforcement teams are now operating independently to protect the highly exploited and contested area of nip and the visayan sea. bureaucracy-based arrangements can also be inefficient. increase in fisheries regulations and its operations may increase the effort required to catch the same quantity of fish. high discards of fish result in additional wasted effort and undocumented stock loss. rules also create incentives to engage in various forms of cheating behavior such as using illegal gears, fernandez, pepito r. 26 caused by highly mobile flows of money and speculative assaults on vulnerable economies, enhanced the need to devolve and decentralize most government service functions to conserve scarce resources. the second factor is the international community itself, which has been instrumental in stimulating third world government's growing interest in participatory and decentralized approaches in governance. there is a growing tendency for donor countries and institutions to place conditions on grants and loans to governments that require them to support participatory and democratic initiatives. some donors claim to be linking participatory development directly to state accountability, empowerment of local groups and transparency in decisionmaking (grounder 1994). development-oriented non-governmental organizations (ngos), which have been at the forefront in enhancing political participation in the philippines, have been heavily supported by foreign donors to strengthen pro-democracy causes (clarke 1998), that includes natural resources-based management and conservation issues and concerns. third, and perhaps the most important, the "hollowingout" of the state (i.e., increased inclusion of non-state actors in policymaking and service delivery) in managing society and nature (see rhodes 1996) is the result of the dissatisfaction with the theories and practices that dominated development and management thinking from the end of world war ii, when the united states of america (us) and other developed countries took increased interest about the problems of "underdevelopment" in former colonies. earlier development paradigms tended to focus on capital formation and technology transfer rather than upgrading of labor, and on industrial rather than agricultural development and thus were inappropriate to the conditions of less-developed countries. consequently, most development and management approaches adopted during the 1950s and 1960s involved a passive role for the majority of the people concerned whose participation was limited to adoption of the new technology and to resource commitments through the payments of taxes and the consumption of imported and domestic goods. on the other hand, decision and policymaking were vested in highly trained technocrats and were to be implemented by rationally organized bureaucracies (dubsky 1993). community-based initiatives in development and management perspectives have since been enshrined in the 1987 constitution and policy pronouncements. community-based frameworks community-based (cb) arrangements are more diverse than the bureaucratic counterpart and go by various names such as community-based management or common property resources management (ferrer et al. 2001, wade 1987). the framework rests on the understanding that coastal (and other ecosystems) resources over which struggles occur are traditionally managed as collective or common property. local management structures, often based on local knowledge of such environmental systems, commonly provide rules of use that can maintain subsistence or renewal of these community resources. community rights to common property are therefore more important for poor and underprivileged people in countries like the philippines since they have little or no other property, except for their social capital and experiential knowledge of the environment. in the light of continued cycle of environmental destruction and lack of food security in coastal areas, community-based management regimes are usually necessary for the regular protection of sensitive and marginal ecosystems, cost-efficiency, and the inclusion of objectives to uplift people's quality of life (dasgupta 1995, cohen & uphoff 1980). at a theoretical level a cb perspective was a response to the "tragedy of the commons" metaphor that depicted community-led or collective use of resources as tending towards abuse and degradation with actors maximizing individual benefits to the detriment of all. it further argued that centralized bureaucratic regulation or privatization could solve the dilemma of sustainably managing collective resources (gordon 1954). on the contrary, some radical perspectives argue that the increasingly capitalized or globalized economies profoundly altered (and still altering) the social and political circumstances of actors that manage common resources. this leads to the entrance of more coercive the relevance of governance institutions in marine protected area 27 states and new markets into basic social economies which results in the appropriation of communal capital away from local communities into the hands of elites, non-residents and other distant parties (muldavin 1996). this marxist perspective has been further enriched by related institutional frameworks from green materialism (e.g., o'connor 1996), peasant studies (blaikie & brookfield 1985, scott 1985), postcolonial theory (said 1985), political ecology (bryant and bailey 1997) and feminism (shiva 1998). nevertheless, most responses to statist or centralized management systems took the form of proving empirically that collective or community-based management of commonly held resources can be successful if conditions allow for negotiation and iterative observation of outcomes (ciriacy-wantrup and bishop 1975). failure of community-based management, by contrast, merely represents failures in the specific structures of rules that govern a collective property. recovery of sustainable management is a painstaking and time-consuming task of crafting new and better rules, rather than imposing central authority or slicing up the commons into bits of private property or harvest rights (ostrom 1990). in community-based institutional arrangement, the "community" (or subgroups within it) holds property rights to fish and the emphasis is on encouraging fishing communities to develop rules to regulate themselves or to maintain existing self-governance systems such that social norms, rules, and sanctions are used to allocate fishing rights or govern fisher behavior (ostrom 1990). rules take a variety of forms, including gear limits, restrictions on effort or fishing seasons, and total or partial ban on certain fishing grounds (particularly in protected areas). social sanctions rather than administrative penalties are the primary enforcement tool, although monetary or material sanctions may also be used (ostrom 1990). different goals and values are embedded in community-based arrangements. these include resource user control (rather than centralized government control and private property arrangements), the preservation of community culture, internal accountability, and preservation of small fishers and communities (mccay & jentoft 1996). compared to bureaucracy and private property/access arrangements, there are few critics of community-based institutional approaches. analysis tends to explain gains made by focusing on ''success stories'' that examine socio-economic and biophysical patterns in small fishing or island communities in countries like the philippines (russ & alcala 1996, ferrer at al. 2001). as a result, analysts interested in large-scale commercial fisheries or integrated management initiatives may not view it as a viable institutional arrangement (leal 2002). a major problem though is the resilience, sustainability and capacity of dominantly community-based institutions (cbi) and initiatives. arguments exist that cbis can breakdown because of the temptation to "free ride." individuals tend to vary their behaviour depending on circumstances. some adopt narrow selfinterested behaviour, others behave selfishly only on certain occasions, still others rely on reciprocity and are able to overcome the tendency to free ride (sterner 2003). a related problem is capture by local leaders and the resulting social inequalities that will prevail (davis & bailey 1996). there is also conflicting evidence on whether norms change rapidly enough to respond to changing ecosystems or exploitation levelsparticularly when the changes are driven by outside forces (rose 2002). the experience of communities with mpas in nip indeed shows some weaknesses in cb regimes. interviews reveal that fishers from neighboring barangays with no mpas indeed poach on mpa sites. moreover, the incursion of commercial fishers to protected zones (where sustainable fishing such as hook-and-line is allowed) creates a sense of helplessness among the local barangay protectors such that they end up violating their own rules to ensure that they benefit from their own management efforts. based on further interviews, however, the continued violation of mpa regulations by "big names" and the ineffective punishment of "influential personalities" who violate mpa rules is the most challenging problem fernandez, pepito r. 28 that needs to be addressed to promote sustained efforts towards local management and conservation.9 the absence of central state control can also lead to a lack of public accountability and even "illegal" behavior. in some cases, the rules developed to govern fishers in community-based arrangements are inefficient, unclear and operates on a case-to-case basis. field data in northeastern iloilo indicate for example that local community enforcers do not abide by common guidelines in apprehension procedures or in determining the level of punishment or fines for those that infringe mpa ordinances. community-based arrangements also have problems controlling distant or commercial fishing fleets whose fast crafts easily slip away from protected areas, and evade detection at night. the problem is complicated by the fact that local community members who enforce the protection of coastal areas (usually the area 200 meters from the coastline) are discouraged from doing their job due to verbal threats from influential operators of commercial fishing vessels, and their backers from local government and local police. finally, it should be acknowledged that local communities are embedded in a variety of institutions. community-based institutions do not have unimpeded control of the coastal zone. municipal/city governments are legally empowered to manage/ develop coasts, set-up regulatory structures, legislate ordinances, and muster the resources to enforce rules. co-management design co-management has now emerged as a third institutional arrangement that is used in developing institutional arrangements for coastal areas (kuperan et al. 2003). co-management refers to various levels of institutional partnership between localand statelevel management systems. generally speaking, cooperation or co-management refers to a mode of interaction among various sectors, agencies, or groups to achieve common goals or visions while maintaining their own institutional autonomy. institutional partnership regimes take on various forms and mechanisms. such relationships are nurtured and developed depending on the degree of urgency to respond to a particular need, level of trust, organizational culture, target clientele/area, or commonality of mandate. it revolves around the sharing of vision, resources, expertise, and systems to create a greater and meaningful impact on for natural resources management at various management levels or scales. in this perspective cooperation or comanagement involves levels or rungs of institutional participation in sustainable development activities and the enabling policy and legislation. using typologies described by pomeroy (1994) and, sen & nielsen (1996) levels of partnership between/among pos, ngos and government organizations may be described as: 1. consultative in nature where institutions establish new relations with other organizations for information exchange. regular venues such as consultations or dialogues are organized to serve as initial mechanisms through which various institutions know each other by sharing experiences, ideas and opinions. 2. coordinative in nature where efforts are extended to avoid duplication of activities and where attempts are made to synchronize separate institutional initiatives for greater efficiency and effectiveness in field operations. as starting point for coordination, interagency committees and activities are usually organized to do a checklist or inventory of project interventions in communities and their resource base. 3. complimentary in nature where institutions conduct separate initiatives guided by a common program framework characterized by purposive efforts to support each other. 4. collaborative in nature where institutions agree to work together, sharing common vision, establishing common objectives, and plans of action on a program level. mechanisms are institutionalized 9initial reports of the post-solar i/petron “oil-spill” disaster off guimaras province (that deposited oil slick in the coasts of ajuy and concepcion, iloilo), however, indicate a swifter and more responsive action of cb and non-state actors at mitigating the disaster (thru the use of indigenous oil spill boom), as compared to the slow and chaotic national government response. the relevance of governance institutions in marine protected area 29 to facilitate delivery of services to target communities and their resource base. 5. critical in nature and perhaps the highest form and level of cooperation where institutions consider each other as indispensable partners in pursuing broad development goals and visions. sectors work together on a more strategic long-term arrangement on various aspects of the socio-economic and political life of the community and its resource base. resources are shared and all partners participate actively in policy formulation and decision making process. in coastal area concerns co-management is a hybrid institutional arrangement that emphasizes sharing responsibility for fisheries management between government and user groups to manage resources in order to reduce costs to government and improve decision making. fisher involvement improves the quality of the time and place information used to craft management systems by tapping local knowledge. it also results in a greater congruence between local conditions and the institutional arrangement, thus reducing transaction costs. a key characteristic of co-management is the distribution of property rights. if property rights are viewed as a bundle of rights and responsibilities, then co-management splits the property rights bundle between users and government. the distribution of property rights is important because resource users must, at a minimum, have access, withdrawal, and management rights to have sufficient incentive to manage resources over the long-term (ostrom & schlager 1996). each co-management arrangement is somewhat unique and uses a combination of policy instruments employed by the aforementioned arrangements that is tailored to fit local conditions. accordingly, enforcement ranges from government penalties to social sanctions and it is problematic when fishers are unwilling to sanction fellow fishers. cheating behavior still exists and tends to reflect the combination of policy instruments used. these arrangements are subject to capture when the commercial industry is unwilling to reduce catch when necessary. it can be difficult for noncommercial interests or small fishers to participate in these systems (kuperan et al. 2003). moreover, while co-management may reduce costs to government, user groups must have the financial, technical, and administrative capacity to perform their management responsibilities. mpas in nip are mostly established as a co-managed common-pool resource. various institutional arrangements help frame mpa governance in nip. once a barangay council passes a petition for the creation of an mpa, an ordinance is framed and endorsed by the municipal sandigan bayan (local legislative body). although municipal waters are effectively under the jurisdiction, control and protection of the municipal government, day-to-day mpa management effectively becomes a community-based or barangay endeavor that is temporarily financed or supported by state (national and international) or nonstate actors. area coverage is either 200 meters from a barangay's shoreline or coral reef, or within a 2kilometer radius enclosure off islands/islets. enclosures or area coverage are plotted using techniques of convenience (i.e., easy to monitor or facing barangay community) that do not follow environmental features. core or no-take zones are uncommon (but do exist in a minority of cases) as mpas are primarily designed for food security and not biodiversity conservation. mpas in nip are designed as a type of property right reserved for sustainable exploitation (i.e., hook and line fishing) by community stewards. the goal of regeneration of corals and seagrasses, promotion of breeding area for fish, and enhancement of fishery stocks are of secondary interest. based on interviews with state and non-state actors on the structure of the intermunicipal management body (i.e., niacdev), and assessment of its resources (i.e., personnel and budget for municipal coastal management efforts), a number of organizational weaknesses can be noted (see table 3). these include: the absence of full-time staff to administer various committee functions of the management council; low annual municipal budgets for coastal management that range from php 100,000 to pphp 250,000 (the council itself has no funds and relies on donations from state and non-state sources); low levels of support from fisherfolk groups, and; weak communication systems to disseminate performance indicators and policy outcomes. fernandez, pepito r. 30 10since last year, the municipal members of niacdev focused its attention on health issues after getting a multi million peso grant from the united states agency for international development (consult http: // pdf.usaid.gov / pdf_docs /pdacg085.pdf # search = %22anihead%2bhealth%22). ironically, a niacdev-member municipality recently endorsed a highly controversial plan (i.e., seen as anti-health and anti-mpa by sections of the local community and various environmental and non-profit groups) to set up a 100 megawatt coal-fired power plant in its coastal community. nevertheless, the intermunicipal management council has achieved some important milestones, such as: meeting regularly (i.e., at least once in two months), thru municipal representatives (i.e. agricultural/crm/ environmental officials or personnel) to keep pace with each others' activities10; drafting of a unified fishery law (although not implemented across the region), and; the effective representation of the management body as a dynamic force, enabling the group to secure multimillion funding from national and foreign sources to create various crm-related "development" programs and projects (i.e., health improvement, population control, environmental management/conservation). moreover, municipal governments like concepcion have passed legislation to strengthen crm initiatives by creating a fisheries and aquatic resources management office (supported by the municipal fisheries and aquatic resources management council or mfarmc) management council of selected representatives from coastal barangays), and passing a municipal ordinance calling for barangays to allot php 5,000 annually to crm projects/programs. the table3. major features of the crm and mpa governance system in northeastern iloilo crm budget 100,000 100,000 200,000 100,000 970,000 100,000 75,000 (% of total mun. (0.2%) (0.31%) (1.0%) (0.31%) (2.9%) (0.29%) (0.27%) budget), 2003 crm actors niacdev, niacdev, niacdev niacdev, niacdev niacdev niacdev mun. enro, fisheries mao, fisheries crmo, volunteer, fisheries mfarmc, technician, mfarmc, technician, mfarmc, 6 mfarmc, technician, 18 mfarmc, bfarmcs mfarmc bfarmcs, 12 bfarmc, mfarmc, bfarmcs/ 1coop. pos ngos 1 ngo, fisherfolk, pos, 3 pos orgs. 4 ngos wardens & 65; 2 boats 0 60; 3 boats 100; 3 boats 160; 6 boats 45; 1boats inactive patrol boats (3-5 days) (7 days) (7 days) (7 days) (7 days) (frequency of patrol per week) year mpas 1994; -1997 1996 2000, 2002, 2004 -established 2002 2003 # of mpas 3 (1 with - 1 1 9 + 1 sea 1 -(brgy.-based) core zone) scape (some w/ core zones) ajuy balasan batad carles concepcion estancia san dionisio types of mpa/ crm initiatives mangrove & upland refo.; ar deployment; stock assessment, participatory coastal assessment; crm, livelihood, paralegel and law enforcement trainings, deployment of markers, buoys, guardhouses in most mpa sites in last two years. concepcion is showcase with mayor winning national awards as best lce in the country in 2004. in 2005, concepcion won the national galing pook award for its “convergence” program. the relevance of governance institutions in marine protected area 31 local chief executive of concepcion is also lobbying for the passage of an ordinance to allocate an unspecified amount of money in support of the annual operating expenses of the mfarmc. conclusions the analysis of these institutional arrangements, enriched by experiences from nip, demonstrates the importance of understanding governance/institutional design and performance in mpa and crm initiatives. it was illustrated that decisions over access and management rules can be taken by central bureaucratic government, local communities, or even in co-managed modes. it was observed, however, that institutions from a top-down nature, whereby government agencies have a dominant decision-making role, are divergent with that of locally led community initiatives. these two contrasting views illustrate that too top-down institutional arrangements raises the risks of imposition which may be manifested by apathy, objections and non-cooperation by other actors. on the other hand, a dominantly community-based approach raises the risks of parochialism, where local resource exploitation interests and free rider beahviour may crop up. it is noted, however, that the tension between bureaucratic and community-based institutional arrangements is, to a degree, a manifestation of the divergent aims that they may harbour. top-down strategies tend to prioritize the goal of using coastal resources for economic development that may or may not lead to equitable distribution of gains, responsibilities or risks. in the context of coastal resource degradation and destruction, however, more bottom-up frameworks have gained more currency and local community support for purposes of low-cost conservation and protection activities, as well as the promise of social equity. due to the continued institutional dilemma faced by bureaucratic and community-based approaches, there is now increased call for a more balanced approach, with government agencies working in partnership with fishers and other interest groups in a co-management arrangement. the coalition of the municipal governments of nip with selected coastal barangays in setting up mpas is a case in point. but the question remains as to what balance of power will be appropriate in such partnerships so as to sustain, on the one hand, the continued success of wider-scale, strategic goals (e.g., increased fisheries production and revenue), and on the other, the fulfillment of more local priorities (e.g., mpa protection, livelihood, and food security). i argue that the balance will, necessarily, be dependent on the context (i.e., actors and their varied biophysical, cultural and organizational setting), aims, and costs of addressing a coastal resource problems or issues. but dryzek (1987) observes that as the geographic scale and scope of resource exploitation interdependencies increases (such as in the coastal and fishery context of nip); the need for some central authority to coordinate negotiations and enforce agreements also increases. whatever stand or policy that the niacdev member municipalities take, one can only hope that the mpa and crm objectives of cb groups can be prioritized, and not marginalized due to the existence of new funding opportunities that focus on other goals (i.e., public health). the issues and problems confronting coastal areas are diverse and no institutional arrangement is likely to be effective in addressing them in all circumstances. effective coastal governance and service delivery, particularly in mpa sites, requires much more than designing some theoretically optimal policy. it raises institutional, social, and moral issues that must be clarified through deliberation and negotiation. ultimately, the selection of policies and the institutional arrangements used to implement them is a political decision. scholars need to help clarify and define problems and then help decision makers identify appropriate goals, objectives, and values to achieve. this requires understanding how a program/project works, who benefits and loses, how it changes incentives, whether the intervention is likely to accomplish what was intended, and how it can be improved or discontinued. sound policy analysis must also remain focused on trying to determine which institutional arrangement will perform best in a particular setting. analysts should consider important contextual factors affecting institutional design and examine the full range of costs that influence institutional performance. given the multiple and competing policy objectives that underlie the general management of contiguous coasts in the fernandez, pepito r. 32 philippines, it is important to use various criteria to assess overall institutional performance and understand the trade-offs that exist between them so as to construct a relevant, site-specific and resilient governance regime. “but in the milieu of a depleted natural resource base, and the continued deterioration of the livelihood and health of coastal dwellers, environmental protection of mpas and municipal fishing grounds of subsistence fishers should be prioritized by various actors and policy networks. acknowledgements i am indebted to the department of human geographyrspas, australian national university and the university of the philippines system for the financial assistance in the conduct of fieldwork in nip. this paper could not have been produced without the cooperation and support of public officials, municipal personnel, ngos and local communities of northeastern iloilo. through a travel grant from the philippine association of marine science an earlier version of the manuscript was presented at the 8th national symposium in 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last august 2019 to early february 2020 on selected study grids on campus. to determine habitat associations, the species richness of each vertebrate class (i.e., amphibia, reptilia, and mammalia) was analyzed with habitat characteristics of the grid using regression analysis. based on the surveys and recent records (2015 onwards) in literature, a total of 33 species were recorded: seven amphibians, 15 reptiles, and 11 mammals. comparison with historical records from 1998 revealed that an additional two amphibian species, seven reptile species, and six mammalian species have been sighted within the area since 2015. however, a fork-tongued frog, falling under the genus fejervarya, and four reptilian species that had previously been recorded within the study sites were not observed. habitat association analysis revealed that building area is correlated with species richness, with reptilian species richness being positively correlated with it. overall, this study shows that the up diliman campus supports considerable urban biodiversity despite recent developments. keywords: urban biodiversity, amphibian diversity, reptile diversity, mammal diversity * corresponding author science diliman (january-june 2022) 34:1, 53-70 sd june2022_pasumbal.indd 53 7/29/2022 4:12:49 pm an updated inventory and habitat association analysis of the non-avian vertebrates 54 introduction the global increase of the human population lends itself to rapid urbanization, with urbanized areas becoming the most rapidly expanding habitat type worldwide (faeth et al. 2011). currently, 55% of the world’s population resides in urban areas, and this number is expected to increase to 68% by 2050 (united nations 2019). this rapid urbanization entails the conversion of natural green spaces into manmade infrastructures (e.g., roads, houses) and fragmentation of habitats, which are associated with biodiversity loss and environmental degradation (müller et al. 2013). amidst these threats, pockets of green spaces serve as refuges where flora and fauna can thrive within urban landscapes (pickett et al. 2001). urban ecosystems, along with their associated biodiversity (nilon 2011), provide numerous ecosystem services (edwards et al. 2020). these services are derived from the interactions between different species and between species and their environment, such as seed dispersal, pollination, pest control, and the like (montoya et al. 2012). with most people now living in cities, these biodiversity-derived services are influenced by anthropogenic activities, with alterations to species composition, abundances, richness, and evenness tied to changes in their habitat (faeth et al. 2011; nilon 2011). the university of the philippines diliman (upd) campus is one of the last remaining green spaces in metro manila. the 493 hectares of land within its bounds provide habitats for a diversity of plant and animal species within the surrounding ‘sea’ of urban infrastructure. a survey conducted by ong et al. (1999) revealed the campus to be home to over 50 unique vertebrate fauna including six amphibian species, nine reptilian species, 38 bird species, and eight mammalian species. after more than a score, these numbers have become outdated given new species records and potential species loss (vallejo and aloy 2014). much of the landscape has changed as well, with many new infrastructures and other campus developments. this study aims to provide an updated inventory of non-avian vertebrate fauna found within the up diliman campus, as well as to determine general specieshabitat associations. this would highlight the importance of green spaces for wildlife as well as provide information that can guide land-use management and maintenance practices of the campus to better conserve urban biodiversity. sd june2022_pasumbal.indd 54 7/29/2022 4:12:49 pm r. v. pasumbal jr. et al. 55 methodology the 493-hectare upd campus is located in quezon city, national capital region. the city ranks as the most populous among the 16 highly urbanized cities in the region based on the 2020 census (philippine statistics authority 2021). the campus was acquired in 1939 but was officially used a decade after when the administration was transferred from the old campus in manila (university of the philippines diliman n.d.a). since then, numerous buildings have been built to support the university’s functions, and the campus now has more than 900 buildings (university of the philippines diliman n.d.b). development has been guided by the 2012 land use plan (appendix 1) wherein 28% of the campus’ land area has been dedicated to academic or academic support units (137.70 ha), 22.5% has been dedicated for residential use (110.87 ha), 4.4% for the campus core (21.66 ha), and 3.7% designated as a protected forest area (18.25 ha) (university of the philippines diliman n.d.b). the campus map was divided into study grids measuring 250 m by 250 m in qgis (figure 1). selected grids were surveyed for the presence of non-avian vertebrate figure 1. dominant habitat map of up diliman campus (campus boundary outlined in dark red lines). the 58 study grids (in 250 m by 250 m spacing) were color-coded based on the dominant plant and building features observed within: built area (grey), grassland (yellow), grassy open forest (pale green), layered open forest (dark green), urban open forest (brown), cropland (pale yellow). white lines signify roads. landmark areas are indicated by color coded pins: up lagoon (blue), palma hall (orange), romulo hall (red), up msi (pale yellow), binhi arboretum (purple), and national science complex (green). sd june2022_pasumbal.indd 55 7/29/2022 4:12:49 pm an updated inventory and habitat association analysis of the non-avian vertebrates 56 fauna and classified into habitat types based on vegetation composition and built area coverage. the grids located across commonwealth avenue (i.e., up arboretum, up-ayala land technohub) and katipunan avenue (up town center) were not included due to time and logistic constraints. sampling events were conducted from august 2019 to early february 2020. habitat sampling in each grid, the dominant plant feature (e.g., grasses, shrubs, and trees) was determined through on-site surveys and inspection of satellite images. additional information on each grid’s canopy cover and building area was measured using satellite imagery through google earth (google earth pro 2017). these were then used to classify the grids into the following vegetation cover classification types: built area – heavily modified areas with high building footprint (i.e., 1-2 story infrastructures) and with minimal vegetation (e.g., potted plants, scattered trees) around the infrastructure cropland – agricultural spaces mostly planted with low crops (i.e., rice, vegetables) grassland – open spaces with scattered small shrubs and extensive grass cover grassy open forest – vegetated areas dominated by large trees with a few understory layer/s and abundant groundcover layered open forest – vegetated areas with multiple structural layers consisting of trees, shrubs, and groundcover urban open forest – vegetated areas with multiple layers of trees, shrubs, and groundcover interspersed with low-level infrastructures wildlife survey amphibian, reptilian, and mammalian species records were obtained through a series of grid surveys. these were supplemented with data collected through collating recent wildlife records dating from 2015 to 2021. the sources included student thesis studies, class field exercises (i.e., biology courses), social media (i.e., the up wild facebook page), and other citizen science platforms (e.g., inaturalist). such reports were verified through photographs and/or specimen examination. grids lacking in data from these supplemental sources were prioritized in the survey effort, leading to a total of 26 grids sampled for amphibians, 23 grids for reptiles, and 15 grids for mammals. sampling was only done during the wet season, sd june2022_pasumbal.indd 56 7/29/2022 4:12:49 pm r. v. pasumbal jr. et al. 57 as the covid-19 pandemic prevented us from conducting the dry season sampling activities. amphibians and reptiles were surveyed using time-constrained opportunistic searches at night (bennett 1999). at least one man-hour of survey effort was spent in each grid. on the other hand, bats were sampled using mist nets (hoffmann et al. 2010). two mist nets were opened in each grid from 1800h to 2100h for a total of six net hours per grid. on the other hand, non-volant mammals were captured using cage traps. five cage traps per grid were baited with bread with peanut butter. traps were left deployed for two nights, for a total of 10 trap nights per grid, but were checked and rebaited as needed daily. captured mammals were released after species identification. species encounters outside of the survey period were also noted as off-survey data. identification was aided using field guides (alcala and brown 1998; das 2015; heaney et al. 2016). experts from the up diliman institute of biology verified for uncertain identification. species-habitat association species richness per grid derived from these records was then compared against the grids’ respective habitat type, canopy cover, and building area through glm analysis with poisson error distribution using rstudio (rstudio team 2020). additionally, the species richness per class (i.e., amphibia, reptilia, mammalia) was also analyzed in relation to canopy cover and building area. results & discussion a total of 33 unique non-avian vertebrate species comprising six amphibian families, seven reptilian families, and five mammalian families were present across 58 grids of six different habitat classifications within the campus. habitat sampling the study site was a mosaic of habitat types (figure 1). the majority of the 58 grids were classified as layered open forests (29.3%), grassy open forests (22.4%), or built areas (22.4%). there were relatively few grids representing grasslands (14.0%), urban open forest (6.9%), and cropland (5.2%). sd june2022_pasumbal.indd 57 7/29/2022 4:12:49 pm an updated inventory and habitat association analysis of the non-avian vertebrates 58 wildlife inventory this study provided an updated campus inventory of non-avian vertebrate species (table 1). a total of 33 species was recorded, which includes seven amphibians, 15 reptiles, and 11 mammals. compared to the ong et al. (1999) study, 20 new species records were added while seven species were not recorded here (table 2). field surveys alone revealed a total of 16 non-avian vertebrate species within the campus. additional records were obtained from the literature review (table 2). table 1. compiled list of non-avian vertebrate species found within the upd campus from survey efforts, conducted from august 2019 to early february 2020 (indicated by asterisk), and literature review from 2015-2021 (items without an asterisk) family common name scientific name amphibians bufonidae cane toad * rhinella marina dicroglossidae chinese edible frog * hoplobatrachus rugulosus common puddle frog * occidozyga laevis ‎eleutherodactylidae greenhouse frog * eleutherodactylus planirostris microhylidae banded bullfrog * kaloula pulchra ranidae common green frog * hylarana erythraea rhacophoridae common tree frog * polypedates leucomystax reptiles trionychidae chinese softshell turtle pelodiscus sinensis ‎emydidae red-eared slider trachemys scripta elegans geoemydidae southeast asian box turtle cuora amboinensis gekkonidae tender-skinned gecko gehyra mutilata brooke’s house gecko * hemidactylus brookii common house gecko * hemidactylus frenatus flat-tailed house gecko hemidactylus platyurus tokay gecko gekko gecko scincidae common sun skink eutropis multifasciata northern philippine sun skink eutropis borealis ‎typhlopidae brahminy blind snake * indotyphlops braminus colubridae gervais’ worm snake * calamaria gervaisii island wolf snake * lycodon capucinus sd june2022_pasumbal.indd 58 7/29/2022 4:12:49 pm r. v. pasumbal jr. et al. 59 northern triangle-spotted snake cyclocorus lineatus philippine rat snake coelognathus erythrurus mammals pteropodidae cave nectar bat * eonycteris spelaea geoffroy’s rousette rousettus amplexicaudatus greater musky fruit bat * ptenochirus jagori lesser short-nosed fruit bat * cynopterus brachyotis long-tongued fruit bat macroglossus minimus ‎emballonuridae black-bearded tomb bat taphozous melanopogon vespertilionidae java pipistrelle pipistrellus javanicus lesser asiatic yellow bat scotophilus kuhlii sciuridae finlayson’s squirrel callosciurus finlaysonii muridae asian house rat * rattus tanezumi asian house shrew suncus murinus table 2. a compiled list of amphibians, reptiles and mammals found within up diliman from 1997-1998 (ong et al. 1999), 2019-2020 survey data, and 2015-2021 literature data species oct 1997 aug 1998 (ong et al. 1999) this study aug 2019 feb 2020 surveys 2015-2021 literature data amphibians rhinella marina x x hylarana erythraea x x fejervarya sp. x hoplobatrachus rugulosus x x polypedates leucomystax x x occidozyga laevis x x eleutherodactylus planirostris x kaloula pulchra x reptiles table 1. compiled list of non-avian vertebrate species found within the upd campus from survey efforts, conducted from august 2019 to early february 2020 (indicated by asterisk), and literature review from 2015-2021 (items without an asterisk) (cont’n.) sd june2022_pasumbal.indd 59 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 60 gekko gecko x off census encounter 2021, class field exercise 2018 hemidactylus frenatus x x hemidactylus stejnegeri x class field exercise 2018 hemidactylus platyurus x class field exercise 2018 eutropis multifasciata x class field exercise 2019 naja philipinensis x rhabdophis spilogaster x pelodiscus sinensis class field exercise 2018 trachemys scripta elegans class field exercise 2018 cuora amboinensis class field exercise 2018 gehyra mutilata off census encounter 2021 eutropis borealis class field exercise 2018 cyclocorus lineatus class field exercise 2019 coelognathus erythrurus verified via photograph 2018 hemidactylus brookii x indotyphlops braminus x calamaria gervaisii x lycodon capucinus x mammals cynopterus brachyotis x x ptenochirus jagori x x rousettus amplexicaudatus x class field exercise 2017 eonicteris spelaea x x suncus murinus x off census encounter 2021, class field exercise 2019 rattus norvegicus x rattus exulans x macroglossus minimus thesis study (abdao, 2019) taphozous melanopogon class field exercise 2018 pipistrellus javanicus class field exercise 2018 scotophilus kuhlii class field exercise 2018 callosciurus finlaysonii verified via video 2020 rattus tanezumi x *previously misidentified as limnonectes macrocephalus table 2. a compiled list of amphibians, reptiles and mammals found within up diliman from 1997-1998 (ong et al. 1999), 2019-2020 survey data, and 2015-2021 literature data (cont’n.) sd june2022_pasumbal.indd 60 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 61 amphibians there are seven amphibian species found within the campus. all species were observed during the surveys and they belong under the families of true toads (bufonidae), forked-tongue frogs (dicroglossidae), rain frogs (eleutherodactylidae), narrow-mouthed frogs (microhylidae), true frogs (ranidae), and tree frogs (rhacophoridae). introduced species dominated the campus amphibian fauna. out of the seven species, only the common tree frog (p. leucomystax) and the common puddle frog (o. laevis) are native to the country. two new exotic species, the banded bullfrog (k. pulchra) and the greenhouse frog (e. planirostris) (diesmos et al. 2015) have successfully established populations on campus and were encountered frequently in different sites. the former was first reported in luzon in 2003 and was believed to have been brought by the pet trade or as a cargo stowaway (pili et al. 2019). its presence is conspicuous, especially during breeding season with its loud calls. on the other hand, the e. planirostris was first reported in quezon city in 2014 as a stowaway in the plant trade (sy et al. 2015), and by 2018 it was already common in the campus. this influx of new species was accompanied by a loss of a previous, potentially endemic, species. ong et al. (1999) reported the presence of the luzon fanged frog (limnonectes macrocephalus) within the up arboretum. however, the accompanying photograph of the species was later identified as fejervarya sp. (p. kim, personal communication with author, february 2021). two frogs under this genus, f. moodiei and f. vittigera, are known to occur in freshwater marshes and wetlands in luzon (diesmos et al. 2015). regardless, neither of the two fejervarya species nor l. macrocephalus were encountered in our surveys or in other recent amphibian surveys (roño 2015). the loss of this species may be attributable to a loss of suitable habitat within the campus. a study by villasenor et al. (2017) similarly correlates a decrease in frog species richness with habitat disturbance brought about by urban development. changes in their aquatic habitat (water body size, aquatic vegetation), as well as urbanization of the terrestrial environment within one kilometer of that aquatic habitat, have been shown to impact the presence of different frog species. the streams around which they would usually be found could have been cemented over with the construction of ripraps, leading to the species’ eventual disappearance. reptiles the reptilian community had the highest species richness (15 sp.) among the vertebrate classes. it is composed of three turtles, five geckos, two skinks, and five snakes. of which, three snakes and two geckos were observed during the surveys. sd june2022_pasumbal.indd 61 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 62 of the three turtle species in the campus found in habitats near water bodies, only the southeast asian box turtle (c. amboinensis) is native. it was photographed from a man-made pond at the palma hall and near a stream at the ib-edc binhi threatened species arboretum. meanwhile, the red-eared slider (t. scripta elegans) and the chinese softshell turtle (p. sinensis) were recorded within the national science complex. the presence of the t. scripta elegans in the wild was verified from a specimen that was unexpectedly caught in a pitfall trap last 2018, while the p. sinensis was recorded as a dead specimen in 2018 and alive in 2019. both turtles are introduced species, with the former most likely introduced from the pet trade and the latter from the food trade (sy 2015). for the geckos, all five species are known to be commensals or associated with human settlements (bowles et al. 2019; lwin et al. 2019; lwin et al. 2021; wogan et al. 2021a; wogan et al. 2021b). they were abundant in roadside trees and artificial structures, particularly on surfaces near lighting fixtures. these areas served as their hunting grounds for insects that were attracted to the light (zozaya et al. 2015). of the five, only two species, the brooke’s house gecko (h. brookii) and the common house gecko (h. frenatus), were verified during survey efforts. the h. brookii was identified by its numerous tubercles on the dorsum, while the h. frenatus was distinguished by its cylindrical tail with serrations. outside of the surveys, the authors also identified the tender-skinned gecko (g. mutilata) based on its smooth-looking skin and relatively wider tail base. the presence of the tokay gecko (g. gecko) was confirmed through its distinct call and visual encounter at the up arboretum in 2021. during the day, diurnal lizards were represented by two native skink species. the endemic northern philippine sun skink (e. borealis) was added to the list, confirmed through visual encounters and specimen examination. along with the many-lined sun skink (e. multifasciata), the two skinks were often encountered while basking. lastly, a diversity of snakes inhabits the campus. three species were recorded during the field survey, while the rest were confirmed from chance visual encounters and photographic evidence. both the brahminy blind snake (i. braminus) and the gervais’ worm snake (c. gervaisii) were seen under flowerpots or plant debris on the ground. they were found at vegetated sites near up lagoon and at the binhi arboretum. the blind snake, as well as the oriental wolf snake (l. capucinus), are non-endemic species that are common in urban areas (wogan and chan-ard, 2012; leviton et al. 2018). on the other hand, the campus is also home to two endemic snakes, the philippine rat snake (c. erythrurus) and the northern triangle-spotted snake (c. lineatus). the former was seen near buildings and houses (e.g., area 2, institute of sd june2022_pasumbal.indd 62 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 63 biology), while the latter was seen at the layered open forest site beside the marine science institute (msi). all snakes recorded on campus were either non-venomous or mildly venomous. a few reptiles were not recorded again since the 1998 inventory. the philippine cobra (naja philippinensis) and the northern water snake (rhabdophis spilogaster) were neither encountered nor reported within the last decade. for lizards, the stejneger’s leaf-toed gecko (hemidactylus stejnegeri) and skinks under the genus sphenomorphus were similarly absent. geckos are difficult to distinguish without close examination of features. more species are possibly found on campus that have yet to be included in this list. mammals the mammalian community of the campus is composed of eight volant and three non-volant small mammal species. of which, three fruit bat species and one invasive non-volant mammal species were captured during the field survey. nevertheless, all previously known mammal species were recorded again in recent literature except for the invasive norwegian rat (rattus norvegicus). all bats recorded on campus were native species. among the fruit bat family (pteropodidae), only the greater musky bat (p. jagori) is endemic to the country. this species and the lesser short-nosed fruit bat (c. brachyotis) are the two most captured bats on campus. they were recorded in various habitats and even in areas of high human disturbance for as long as there were fruits and figs available. both species have also been seen roosting in old buildings and trees. the other frugivorous and/ or nectarivorous bats, however, were captured less frequently. the cave nectar bat (e. spelaea) and geoffroy’s rousettes (r. amplexicaudatus) are cave-dwelling species (waldien et al. 2019; waldien et al. 2020). between the two, the r. amplexicaudatus was rarer and last recorded in 2017 during a class exercise in ecology at the binhi arboretum. given the absence of caves on campus, the encountered individuals were likely passing by or foraging on campus. the long-tongued fruit bat (m. minimus) was caught a handful of times at the binhi arboretum during a student thesis study (abdao 2019). the presence of fruiting trees on the campus explains the presence of these bat species. maintaining and improving plant diversity can further promote urban bat diversity (threlfall et al. 2016). the four insect bat species are likewise native and represent two families (emballonuridae and vespertilionidae). although the surveys failed to capture any of the insect bats, the authors have captured the java pipistrelle (p. javanicus), the lesser asiatic yellow bat (s. kuhlii), and the black-bearded tomb bat (t. melanopogon) sd june2022_pasumbal.indd 63 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 64 during ecology and vertebrate biology class exercises (table 2). a small colony of t. melanopogon was also observed to be roosting at an old building, the romulo hall. in contrast, the non-flying mammals in the list were all introduced species. only the asian house rat (r. tanezumi) was recorded in the surveys. the presence of the shrew was confirmed from a few dead specimens at the national science complex, while the finlayson’s squirrel (c. finlaysonii) was caught in a video near the marine science institute by the security guard. the squirrel is native to thailand, cambodia, laos, and vietnam but was introduced to other countries, including the philippines, through the pet trade (bertolino 2009). due to their high adaptability to urban habitats, these squirrels are considered ‘high-risk’ species and have the potential to become invasive once populations have been established (bertolino and lurz 2013). species-habitat association the 16 species encountered during the survey period were found across five different habitat types, namely built areas, grasslands, grassy open forests, layered open forests, and open urban forests (table 3). table 3. abundance of non-avian vertebrate species encountered during survey efforts within up diliman across different habitat types species habitat types built area grassland grassy open forest layered open forest open urban forest amphibians e. planirostris 4 0 47 21 1 h. rugulosus 1 0 0 0 1 h. erythraea 0 1 1 3 0 k. pulchra 9 10 7 10 0 o. laevis 0 16 0 0 0 p. leucomystax 14 1 1 13 1 r. marina 22 69 92 108 1 reptiles c. gervaisii 0 0 0 0 1 h. brookii 10 2 7 8 2 h. frenatus 26 7 34 40 7 i. braminus 1 0 0 1 0 l. capucinus 0 0 1 0 0 mammals c. brachyotis 4 15 6 14 11 e. spelaea 1 1 0 0 0 p. jagori 3 17 1 2 3 r. tanezumi 2 0 0 2 1 sd june2022_pasumbal.indd 64 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 65 overall species richness per grid was analyzed across the different habitat types. built areas and grassy open forests, on average, show lower species richness compared to the other habitat classifications (figure 2). however, results revealed no significant differences on the total vertebrate species richness of the grid between habitat types (kruskal-wallis test, p-value = 0.9021). this also suggested that all habitat types are equally important in supporting urban vertebrate diversity. figure 2. comparison of species richness among amphibians, reptiles, and mammals encountered during the august 2019 – february 2020 survey efforts per grid across the five habitat classifications of the up diliman study site. species richness was also examined against grid characteristics, canopy area, and building area. the number of species per grid was not influenced by canopy cover (kruskal-wallis test; p-value = 0.88). examining species richness against building area, however, revealed a positive association (glm; p-value<0.0001). upon further analysis at the taxonomic class level, only the reptilian species richness was affected by building area (glm; p-value= 0.0033). this may be attributed to the high abundance of h. frenatus around buildings (ota and whittaker 2010). both amphibian and mammalian species richness showed non-significant relationship with building area (p-value = 0.1 and p-value = 0.44, respectively). this lack of a significant correlation between habitat type and species richness may be explained by several factors. the assemblage of species found within the campus, particularly the non-native and invasive species, may be generalists which occupy a high diversity of habitats and are less sensitive to habitat disturbance than species restricted to a smaller number of habitats (segura et al. 2007). it is also possible other factors have a greater effect on species richness within the campus. sd june2022_pasumbal.indd 65 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 66 a study by vallejo et al. from 2008 for example, has found that bird abundance is more affected by the number of spatial entities (i.e., the number of buildings and trees) as compared to spatial area (i.e., building area and canopy cover), which was used in this study. a similar pattern may be observed in the abundance of other non-avian vertebrate taxa. conclusions with globally increasing urbanization rates, green urban spaces are important centers of biodiversity that are increasingly becoming threatened. the up campus has undergone and is continuing to undergo a transformation in the form of further urban development projects. this has consequently impacted the different species residing within the campus. a frog under the fejervarya genus, the endemic snakes naja philippinensis, and rhabdophis spilogaster, as well as the gecko hemidactylus stejnegeri and skinks under the sphenomorphus genus, are all previously recorded species that were not observed in this study. change is part and parcel of infrastructural development but should not come at the cost of valuable wildlife resources. light pollution from bright streetlights (holker et al. 2010), the cementation of grassy patches (klaus 2013), and the construction of ripraps around streams (bernhardt and palmer 2007) are a few examples of construction projects that can negatively impact the wildlife present in the area. moving forward, the preservation of this wildlife resource should be a constant consideration. care must be taken to ensure that the resulting space is beneficial to the residents of the area, humans and wildlife alike. in order to attain a future wherein wildlife thrives alongside urban development, it is imperative that the continued monitoring of our wildlife resources and how they change over time is maintained. in future studies, it is also recommended to examine seasonality, an aspect that was excluded from this study as a result of covid-19 restrictions, which may reveal a different set of observable species. lastly, it is also recommended to examine spatial entities, in addition to spatial area and habitat types, when determining species habitat associations. sd june2022_pasumbal.indd 66 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 67 references abdao drl. 2019. correlation of diet and prey abundance of philippine scops owl (otus megalotis: walden, 1875) in the threatened species arboretum, [undergraduate thesis]. 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[accessed 2022 jan 08]. https://dx.doi.org/10.2305/ iucn.uk.2021-3.rlts.t99156022a1434103.en. zozaya s, alford r, schwarzkopf l. 2015. invasive house geckos are more willing to use artificial lights than are native geckos. austral ecol. 40(8):982-987. ______ romel v. pasumbal jr. <rpasumbal@gmail.com > is an m.s. biology student from the institute of biology, up diliman. his research interests lie in the biology and ecology of vertebrates, particularly those of herpetofauna. jelaine l. gan is an instructor at the institute of biology. her research interests vertebrate biology and ecology, urban ecosystems, and conservation biology. geoffrey jules n. solidum is a cum laude graduate of b. landscape architecture in up diliman. a professional painter, filmmaker, and graphic designer, his works focus on the creation of visual displays and commentaries tackling societal, political, and environmental issues. nappy l. navarra is an associate professor at the up college of architecture. his research interests are in landscape architecture, landscape ecology urban ecosystems, and resilience. sd june2022_pasumbal.indd 70 7/29/2022 4:12:50 pm 01_goss a checklist of navicula 1 a checklist of navicula (class bacillariophyceae) of the philippines milagrosa r. martinez-goss professor, institute of biological sciences and curator, museum of natural history university of the philippines los baños college, laguna 4031, philippines abstract a unified account of all known diatoms in the genus navicula, class bacillariophyceae, of the philippines from 1886 to 1987 is presented. a total of 226 taxa are listed. included herewith are the references that originally described or cited the taxon and the ecological/habitat records for each taxon. as much as possible, a list of exsciccata materials is given together with their repository places. keywords: navicula, bacillariophyceae, philippines, diatoms introduction this compedium presents a unified account of all known diatoms under the genus navicula from 1886 t o 1 9 8 7 o r w i t h i n a s p a n o f o v e r 1 0 0 y e a r s . referencing was based mainly on available literature that deals in whole or in part on philippine materials. in some cases philippine exsciccata materials (diatom slides) that were found stored in the different diatom herbaria in the western world were also noted. a wealth of these specimens were discovered at the diatom section of the british museum of natural history (bmnh) in london, england, which include the collections of adam, bess, comber, etc. the collections of mann from the uss albatross were also observed at the smithsonian institution of the u.s. museum of natural history in washington, d.c., u.s.a. i have not had a chance to see the holotypes of hustedt and skvortzow’s collections. but according to dr. charles w. reimer of the academy of natural sciences of philadelphia (ansp), there are some collections of skvortzow that are in hustedt’s collection, and the latter are stored in the herbarium of the alfred wegener institute for polar and marine research in bremerhaven, germany. the list includes 21 that are reported as new taxa (table 1) and four species found only in mexico (campeche bay) and in the marine waters of the philippines and nowhere else according to m a n n ( 1 9 2 5 ) , i . e . , n . a p p ro x i m a t a v a r. substauroneiformis grun., n. carinefera grun., n. h e n n e d y i v a r. c a l i f o r n i c a ( g r e v. ) c l . a n d n . margarita schmidt (?). there are 226 taxa recorded here that are recognized by patrick and reimer (1966), round et al., (1990), and vanlandingham (1975). unidentified specific epithets are recorded but not enumerated. the scope and use of the list is as in the previous paper (martinez-goss, 1995). science diliman (january-june 2001) 13:1, 1-32 2 martinez-goss navicula biggemata n. caeca n. clavata f. corpulenta (mann) hust n. delecta mann n. elegantoides hust. n. funiculata mann n. glabrissima mann n. halophila f. tenuirostris hust. n. hustedtii f. philippina skv. n. imitans mann n. insignita hust. n. luzonensis hust. n. mendica mann n. mucicoloides hust. n. philippina skv. n. philippinarum mann n. pulverulenta (pulvulenta) mann n. pugio mann n. retrostauros mann* n. spiculifera mann* n. transluscens mann marine marine marine marine laguna de bay; camarines sur, buhi lake marine marine rizal, manila quezon city, balara marine laguna de bay, rizal, lyon (ponds) camarines sur, buhi lake (rare) marine rizal, lyon (ponds) quezon city, balara marine marine marine marine marine marine mann, 1925 mann, 1925 hustedt, 1964 mann, 1925 hustedt, 1942a mann, 1925 mann, 1925 hustedt, 1942a skvortzow, 1937 mann, 1925 hustedt, 1942a hustedt, 1942a mann, 1925 hustedt, 1942a skvortzow, 1937 mann, 1925 mann, 1925 mann, 1925 mann, 1925 mann, 1925 marine table 1. new taxa of navicula reported from the philippines *not recognized by vanlandingham the list navicula bory, p. 562, 1824; patrick & reimer, p. 439, 1966. abrupta (greg.) donkin donkin, p. 13, 2/6, 1870; mann, p. 94, 1925. bottom marine samples taken near the islands by the united states steamer (uss) albatross, mann (1.c.). abruptoides hust. hustedt, p. 515, f. 1557, 1964; podzorski & hakansson, p. 79, 31/8, 1987. luzon: palawan, pto. princesa (white beach mangrove, root scrapings from rhizophora), podzorski & hakansson (l.c.). americana ehr. ehrenberg, p. 129 (417), 1841 (1843); hustedt, p. 280, f. 464, 1930; _______, p. 64, 1942a. luzon: laguna, los baños (ponds, laguna de bay); camarines sur, buhi (river), hustedt (1942a). mindanao: lanao (uyaan lake), hustedt (1942a). anglica ralfs ralfs in pritchard, p. 900, 1861; hustedt, p. 303, f. 530-531, 1930; _______, p. 73, 1942a. mindanao: lanao (ponds), hustedt (1942a). angusta grun. grunow, 10:528, 5/19, 1860. = navicula cari var. angusta grun. grunow in van heurck, 7/17, 1885; hustedt, p. 72, 1942a; patrick & reimer, p. 514-515, 49/5, 1966. luzon: laguna, los baños (ponds), hustedt, 1942a. taxon source reference a checklist of navicula 3 visayas: leyte, danao lake, hustedt, 1942a. approximata grev. greville, p. 28, 4/4, 1859; mann, p. 94, 1925. = n. lyra var. approximata (grev.) cl. cleve, p. 4 (1/1) 1878. bottom marine samples taken near the islands by the uss albatross, mann (1.c.); british museum (bmnh), adam’s slide #45.1/2; ts 60/64. _______ f. morei (moreii) (o’ meara) hust. hustedt p. 416, f. 1491, 1964. = navicula hennedyi var. niceaensis h. perag. peragallo, p. 47, 5/39 e.p., 1888. bmnh, comber slide #31898. visayas: cebu. _______ var. substauroneiformis (grun.) cl. grunow in schmidt, et al., 2/20, 21, 1874; mann, p. 94, 1925. bottom marine samples, mann (l.c.). _______ var. ? bmnh, adam’s-bess # ts497 3/2. arvensis hust. hustedt, p. 249, 20/19, 20, 1937b; _______, p. 60, 1942a; patrick & reimer, p. 483, 46/1-2, 1966. luzon: batangas, taal (crater lake), hustedt (1942a). bacillum ehr. ehrenberg, p. 130, 1838, (1839); _________, p. 418 (130), 4/5, f. 8, 1841 (1843); hustedt, p. 64, 1942a. luzon: manila (reservoir); laguna, los baños; camarines sur, buhi (lake), hustedt (1942a). baileyana grun. grunow in schmidt, et al, p. 88, 1/31, 1874; castracane, p. 812, 1886. visayas: cebu, castracane (l.c.) = navicula polysticta grev. greville, p. 247,5/2, 1859. visayas: cebu, bmnh, comber # 31892. barbitos schmidt schmidt, et al., 129/5, 1874; mann, p. 95, 1925. bottom marine samples taken around the islands by the uss albatross, mann (l.c.). biformis (grun.) mann mann, p. 95, 20/6-7, 1925. bottom marine samples taken around the islands by the uss albatross, mann (1.c.). = mastoneis biformis (grun.) cl. cleve, p. 194, 1894. luzon: manila, bmnh, comber # 31904. visayas: cebu, bmnh, comber # 31895. bigemmata mann mann, pp. 95-96, 21/1, 1925. bottom marine samples taken around the islands by the uss albatross. type-cat. no. 43639 deposited in the united states national museum of natural history (usnm), mann (1.c.). 4 martinez-goss bipunctata o’ meara o’ meara, p. 286, 13/5, 1872; de toni, pp. 78-79, 1891. mindanao: brackishwaters coming from the volcanic mountain of the island in cagayan, sulu archipelago (chimmo), o’ meara (l.c.); de toni, (l.c.). borneoensis hust. hustedt, p. 735, f. 1712, 1966; podzorski & hakansson, p. 84, 33/7, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted on cove, marine), podzorski & hakansson (1.c.). brasiliensis grun.* grunow, p. 152, 14/10, 1863; schmidt, atlas, 6/19-21, 23/25, 31/33, 1874; mann, p. 97, 1925. bottom marine samples taken around the islands by the uss albatross, mann (1.c.). bmnh, adam’s # f19.29, 40 2/18.883; ts 262 1/10-12, 6/7, ts 60/60. visayas: cebu, bmnh, comber’s #31898, 31895, 31894. brasiliensis var. _______cstr. castracane, p. 812, 1886. bmnh, adam’s-bess #706, ts 60/66. visayas: cebu, castracane (l. c.) brekkaensis peters. petersen, p. 389, f. 16, 1928; hustedt, p. 58, 1942a. luzon: rizal, pancipit river, hustedt (l.c.). bruchii grun. grunow in cleve, p. 13, 1928; cleve, p. 36, 1895; de toni, pp. 177-178, 1891. luzon: manila (marine), (deby collection, bmnh). bryophila peters. petersen, p. 388, f. 13, 1928; hustedt, p. 60, 1942a. mindanao: lanao lakes of dagiangan and uyaan, hustedt (1.c.). caeca mann mann, p. 97, 21/3, 1925. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43641, usnm, mann (1.c.). capitata ehr.ehrenberg, p. 185, 13/20, 1838; van heurck, p. 187, 25/719, 1896; patrick & reimer, pp. 536-537, 52/1-2, 1966; podzorski & hakansson, p. 81, 32/10, 1987. luzon: palawan, taytay, langen (malapakan cove, algae encrusted on the cove, marine), podzorski & hakansson (1.c.). cari ehr. ehrenberg, p. 83, 1836; _________, p. 174, 1838; hustedt, p. 299, fig. 512, 1930; _______, p. 72, 1942a. luzon: rizal, malabon (dagatan swamp), hustedt (1942a). mindanao: uyaan lake, hustedt (1942a). carinifera grun. grunow in schmidt, et al., 2/1, 1874; mann, p. 98, 1925; hustedt, p. 754, f. 1730, 1966. *not recognized by vanlandingham, 1975. a checklist of navicula 5 bottom marine samples taken near the islands by the uss albatross, mann (1.c.). cincta (ehr.) ralfs van heurck, p. 178, 3/105a, 1896; schmidt, et al., 299/26-30, 1874; hustedt, p. 298, f. 510, 1930; pantastico, p. 194, 17/4, 1977; patrick & reimer, p. 516, 49/8, 1966. luzon: laguna, los baños, mayondon (on shoreline of laguna de bay), pantastico (1.c.). circumsecta (grun.) grun. grunow in schmidt, et al., 3/26-27, 1874; cleve et grunow, p. 42, 1880; mann, p. 98, 1925; hustedt, p. 409, f. 1487a-c, 1964. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). = navicula polysticta var. circumsecta (grun.) grun. grunow in schmidt, et al., p. 89, 1/36, 42, 1874. bmnh, adam’s # gc 2089. circumtexta meister meister in schmidt, et al., 394/33-35, 1874; podzorski & hakansson, pp. 81-82, 32/4, 1987. luzon: palawan, taytay, langen (malapakan cove, scrapings from sand at low tide, marine), podzorski & hakansson (1.c.). citrus krasske krasske, p. 199, f. 14, 1923; hustedt, p. 303, f. 529, 1930; _______, p. 66, 1942a. luzon: laguna de bay, hustedt (1942a). clavata greg. gregory, p. 46, 5/17, 1856. bmnh, adam’s # f83 1/2; 4/3; 4. 120, 3/2.5. luzon: manila (on shell), bmnh, comber # 30925, 31903. visayas: cebu, bmnh, comber # 31900, 31898. = navicula clavata var. caribaea cl. cleve, p. 61, 1895. visayas: cebu, bmnh, comber # 31895. = navicula caribaea cl. cleve in schmidt, et al., 2/17, 70/48, 1874; mann, pp. 99-100, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). = navicula wrightii o’ meara luzon: manila, bmnh, comber # b. 31903. _______ f. corpulenta (mann) hust. hustedt, p. 445, f. 1510-1511, 1964. = n. corpulenta mann mann, p. 99, 22/4, 1925. bottom marine samples taken around the islands by the uss albatross. type-cat. no. 43642, usnm, mann (1.c.). _______ f. elliptica (schmidt)hust. schmidt, et al., 3/13, 1874; hustedt, p. 449, 1964. luzon: manila, bmnh, comber # 31903. visayas: cebu, bmnh, comber # 31898. _______ var. indica (grev.) cl. cleve, p. 62, 1895; schmidt, et al., 204/12, 1874. bmnh, adam’s # gc 1865.6.1976; bess #920; ts #497.2/3.4.522.2/4, f885. 6 martinez-goss = n. indica grev. (non ehr.) greville, p. 95, 9/13, 1862; cleve, p. 62, 1895; mann, p. 104, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s # j 4201, m 68/4. luzon: manila (marine), bmnh, rae’s collection. visayas: cebu, bmnh, comber # 31898, rae’s collection. _______ var. producta ? sensu adam bmnh, adam’s # f886. luzon: manila, bmnh, comber # 31904. _______ var. ? bmnh, adam’s # ts60/61.657. clipeiformis könig könig, p. 51, 59, 2/3, 3/6, 1959; hustedt, p. 552, f. 1589, 1964; podzorski & hakansson, p. 76, 31/9, 1987. luzon: palawan, taytay, langen (malapakan cove, surface scrapings from sand at low tide, marine), podzorski & hakansson (1.c.). cluthensis greg. gregory, p. 478, 9/2, 1857; hustedt, p. 651, f. 1653a-d, 1966. = n. erythraea grun., p. 539, 5/17 (3/17), 1860; mann, p. 101, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). confervacea (ktz.) grun. grunow in van heurck, 14/36, 1880; woltereck, p. 37, 1941; hustedt, p. 63, 1942a; prowse, p. 41, 12/n-o, 1962; hustedt, p. 205, f. 1324a-d, 1962; podzorski & hakansson, pp. 77-78, 1987. luzon: benguet, banaue (ponds), woltereck, (1942); rizal, malabon, dagatan (swamp), marulay water reservoir, lyon ponds; laguna, los baños, laguna de bay; batangas, taal (taal lake, yambocrater lake, crater lake); camarines sur, buhi (lake, river); oriental mindoro, naujan (river), calapan lake, hustedt (1942a); palawan, roxas (caruray river, alt., c. 50 m, from rock surface), taytay, lake manguao, (algae growing on floating grass roots, alt. c. 45 m), podzorski & hakansson, (1987). visayas: leyte, danao lake, hustedt (1942a). mindanao: lanao lake, hustedt (1942). = n. confervacea f. nipponica skv. skvortzow, p. 34, 2/3, 4/23, 1936; hustedt, p. 61, f. 23, 1942a luzon: camarines sur, buhi lake, hustedt (l.c.). consors sch. schmidt, et al., 48/24-27, 1874; mann, p. 98, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). contenta grun. grunow in van heurck p. 109, 1885; van heurck, p. 230, 5/239, 1896; hustedt, p. 277, f. 458a, 1930; skvortzow, p. 291, 1/40, 1937; hustedt, p. 58, 1942a. a checklist of navicula 7 luzon: batangas, taal (crater lake), hustedt (1942a). ______ var. biceps (arn. ms. grun. in v.h.) cleve van huerck, p. 230, 5/240, 1896. = n. contenta f. biceps (arn. grun in v.h.) hust. hustedt, p. 277, f. 458c, 1930; _______, p. 58, 1942a. luzon: pancipit river, hustedt (1942a). _______ f. parallela (peters.) hust. hustedt, p. 277, f. 458b, 1930; _______, p. 58, 1942a. luzon: camarines sur, buhi (lake), hustedt (1942a). cryptocephala ktz. kützing, p. 95, 3/20, 26, 1844; donkin, p. 37, 5/14, 1871; van heurck, p. 180, 3/122, 1896; hustedt, p. 295, f. 496, 1930; skvortzow, p. 291, 2/3, 1937; hustedt, p. 67, 1942a; podzorski & hakansson, p. 87, 1987. luzon: rizal, manila (water reservoir ii), ponds in the bureau of science grounds; laguna, laguna de bay; camarines sur, buhi lake; oriental mindoro, naujan lake, hustedt (1942a); rizal, quezon city (balara filters, skvortzow, (l.c.); palawan, brooke’s point (scrapings from a limestone rock in tumarabong river, alt. c. 10 m), podzorski & hakansson (1987). cf. cryptolyra brockmann brockmann, p. 19, 3/22, 23, 1950; hustedt, p. 534, f. 1570, 1964; podzorski & hakansson, p. 79, 32/5, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted on the cove, marine), podzorski & hakansson (1.c.). cuspidata (ktz.) ktz. kützing, p. 94, 3/24, 37, 1844; w. smith, p. 47, 16/131, 1853; van heurck, p. 214, 4/190, 1896; mann, p. 99, 1925; hustedt, p. 268, f. 433, 1930. freshwater sample as detritus from the bottom marine samples taken by the uss albatross, mann (1.c.). _______ var. ambigua (ehr.) cl. cleve, p. 110, 1894; hustedt, p. 51, 1942a. luzon: rizal (ponds in the bureau of science grounds); laguna, los baños, laguna de bay (lake and ponds); camarines sur, buhi lake, hustedt (1942a); palawan, roxas, pagdanan range, ibangley brookside hill (streamside pool, alt. c. 40 m), podzorski & hakansson (1.c.). mindanao: cagayan de oro, singnan lake; lanao (lake), hustedt (1.c.). cyclops mann mann, pp. 99-100, 21/5, 1925. bottom marine samples taken near the islands by the uss albatross; frequently observed. typecat. no. 43643, usnm, mann (1.c.). delecta mann mann, p. 100, 24/5-7, 1925. bottom marine samples taken around the islands by the uss albatross; frequently observed. type-cat. no. 43644, usnm, mann (1.c.). bmnh, adam’s # m58/31. dicephala ehr. ehrenberg, p. 45, 1837; _________, p. 185, 1838; van heurck, p. 188, 3/138, 1896; hustedt, p. 302, f. 526, 1930; _______, p. 73, 1942a. 8 martinez-goss luzon: laguna, los baños (freshwater ponds), hustedt (1942a). _______ var. undulata östr. östrup, p. 25, 3/33, 1918 (1920); hustedt, p. 73, 1942a. luzon: camarines sur, buhi lake, hustedt (1942a). diffluens schmidt = navicula diffusa schmidt schmidt, et al., 2/15, 2/28, 1874; mann, p. 101, 1925. bottom marine samples taken near the islands by the uss albatross, mann (l.c.). directa (w. sm.) ralfs ralfs in pritchard, p. 906, 1861. luzon: manila, bmnh, comber # 31903, 31904. distans (w. sm.) ralfs ralfs in pritchard, p. 907, 1861; schmidt, et al., 46/11-14, 1874. luzon: manila (on shell), bmnh, comber # 30925, 30926. durandii (durrandii) kitt. kitton in schmidt, et al., 129/1, 1874; castracane, p. 812, 1886; mann, p. 101, 21/6, 1925; hustedt, p. 485, f. 1540, 1964. bottom marine samples taken near the islands by the uss albatross, mann (1.c.); bmnh, adam’s # ts522.1/1. vanlandingham (1975) does not think the specimen is in accordance to kitton. visayas: cebu, castracane (l. c.) _______ var. rhomboidea (cstr.) schmidt schmidt, et al., 129/2, 1874. = navicula bullata norman var. rhomboides cstr. castracane, p. 30, 30/8, 1886. visayas: cebu, castracane (l.c.). _______ var ? bmnh, adam’s # m 65/5. elegantoides hust. hustedt, p. 76, f. 142, 1942a; woltereck, p. 52, 1941; prowse, p. 42, 8/d, 1962. luzon: benguet, banaue (ponds), woltereck (l.c.); rizal, manila (lyon ponds, ponds in bureau of science grounds); laguna, los baños, laguna de bay; buhi lake, hustedt (1.c.). exigua (greg.) grun. grunow in van heurck, 8/32, 1880; hustedt, p. 303, f. 538, 1930; _______, p. 73, 1942a. luzon: camarines sur, buhi lake, hustedt (1942a). mindanao: uyaan lake, hustedt (1942a). forcipata grev. greville, p. 83, 6/10-11, 1859; schmidt, et al., 70/17, 1874; cleve, p. 65, 1895; van heurck, p. 203, 4/163, 1896; peragallo & peragallo, p. 130, 21/28, 1897-1908. bmnh, adam’s # j 413. visayas: cebu, bmnh, comber # 31898. _______ var. densestriata schmidt schmidt, et al., 70/14-16, 1874; mann, p. 102, 1925. a checklist of navicula 9 bottom marine samples taken near the islands by the uss albatross, mann (1.c.). _______ cf. forcipata var. elongata perag & perag. peragallo & peragallo, p. 131, 21/24, 1897-1908; podzorski & hakansson, p. 79, 33/1, 1987. luzon: palawan, pto. princesa, pto. princesa bay (table head, on sandy bottom at 15 m depth), podzorski & hakansson (1.c.). _______ var. versicolor (grun.) grun. grunow in cleve et möller, no. 208-210, 1879. visayas: cebu, bmnh, comber # 31894, 31902. _______ var ? bmnh, adam’s # g-211. funiculata mann mann, p. 102, 22/1-2, 1925. bottom marine samples taken around the islands by the uss albatross. type-cat. no. 43645, usnm, mann (1.c.). bmnh, adam’s # ts 60/2.76. genifera schmidt schmidt, et al., 2/6, 1874; hustedt, p. 435, f. 1505, 1964. bmnh, adam’s # w 935. glabrissima mann mann, p. 103, 22/3, 1925. mindanao: jolo, sulu, sulu (bottom marine sample, rare). type-cat. no. 43646, usnm, mann (1.c.). gracilis ehr. ehrenberg, p. 64, 69, 1830 (1832); _________, p. 155, 1854; van heurck, p. 179, 3/109, 1896; hustedt, p. 299, f. 514, 1930. luzon: rizal, manila (tabaco river), sample taken by the ship princess louise, ehrenberg (1854). granulata bailey bailey, p. 10, f. 16, 1854 (1853); cleve, p. 48, 1895; schmidt, et al., 244/7, 1874; hustedt, p. 702, f. 1696, 1966; podzorski & hakansson, p. 84, 33/14, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from the sand at low tide, by the cove), podzorski & hakansson (1.c.). cf. gregaria donk. donkin, p. 10, 1/10, 1861; van heurck, p. 181, 3/125, 1896; hustedt, p. 269, f. 437, 1930; podzorski & hakansson, p. 78, 1987. =navicula granulata cf. gregaria donk. donkin, p. 10, 1/10, 1861; van heurck, p. 181, 3/125, 1896; hustedt, p. 269, f. 437, 1930; podzorski & hakansson, p. 78, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted on the cove, marine), podzorski & hakansson (1.c.). aff. grevillei (ag.) heiberg heiberg, p. 83, 1863; van heurck, p. 232, 5/243, 1896; prowse, p. 43, 12/6, 1962; podzorski & hakansson, p. 77, 30/9, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted on the cove, marine), podzorski & hakansson (1987). grimmii (grimmei) krasske krasske, p. 45, 1/14, 1925; woltereck, p. 52, 1941; hustedt, p. 274, f. 448, 1930; hustedt, p. 55, 1942a. luzon: benguet, banaue (ponds), woltereck (l.c.); rizal, lyon (ponds); camarines sur, buhi (river), hustedt (1942a). mindanao: lanao (lake), hustedt (1942a). halophila (grun.) cl. cleve, p. 109, 1894; hustedt, p. 268, f. 436, 1930; _______, p. 51, 1942a; woltereck, p. 52, 1941. luzon: mt. province, banaue (ponds) woltereck (l.c.); rizal, lyon (ponds); laguna, los baños (pond), hustedt (1942a). mindanao: lanao, uyaan (lakes), hustedt (1942a). _______ f. tenuirostris hust. hustedt, pp. 51-52, f. 76, 1942a. luzon: rizal, manila (ponds in the bureau of science grounds, rare), hustedt (1.c.). hamulifera grun. grunow in cleve and grunow, p. 44, 1880; cleve, vol. 1, p. 154, 3/16-19, 1894; peragallo & peragallo, p. 65, 8/16, 1897-1908; mann, p. 104, 1925; hustedt, p. 312, f. 1430, 1962. bottom sediments taken near the islands by the uss albatross, mann (1.c.). hassiaca krasske krasske, p. 47, 2/26, 1925; hustedt, p. 279, f. 462, 1930; _______, p. 216, f. 1332, 1962; podzorski & hakansson, p. 81, 32/2, 1987. luzon: palawan, taytay, lake manguao (danao), podzorski & hakansson (l.c.). hennedyi (henedii) w. sm. w. smith, p. 93, 1856; gregory, p. 40, 5/3, 1856; van heurck, p. 93, 9/14, 1880-1885; cleve, p. 57, 1895; mann, p. 104, 1925. bottom sediments taken near the islands by the uss albatross, mann (1.c.); bmnh, adam’s # e 530, m 58/14, f119.1/14.3/3. luzon: manila, bmnh, comber #30925, 31903, 31904, 30926 (on shell). visayas: cebu, bmnh, comber #31900. =navicula hennedyi var. niceaensis (niceaeensis) h. perag. h. peragallo, p. 55, 5/39,1888. bmnh, adam’s # m 58/22-3. ______ f. bacillifera (pantoc.) cl. cleve, p. 58, 1895. = navicula rugosa janisch janisch, 15/10, 11, 1888; cleve, p. 59, 1895. bmnh, adam’s # e 530. _______ var. californica (grev.) cl. greville, p. 29, 4/5, 1859; cleve, p. 58, 1895; mann, p. 97, 1925. = navicula californica grev. greville, p. 29, 4/5, 1859; mann, p. 97, 1925. bottom marine samples taken near the islands by the uss albatross, mann (l.c.); bmnh, adam’s # m58/20-1; 24.30. =navicula californica var. campechiana grun. grunow in schmidt, et al., 3/19, 1874; mann, p. 97, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). 10 _______ var. furcata perag. & perag. peragallo & peragallo, p. 141, 24/16, 17, 1897-1908. bmnh, adam’s # f 18 2/9.10. _______ f. granulata (grun.) hust. grunow in schmidt, et al., 3/3, 1874; hustedt, p. 458, f. 1519, 1964. =navicula hennedyi var. circumsecta cl. cleve p. 58, 1895 (non grunow, 1874 in schmidt, et al., 1874). it means cleve thought the specimen is not the same as in grunow in schmidt, et al., but vanlandingham says they are the same. bmnh, adam’s # ts 519/3. _______ var. neopolitana cl. cleve, p. 58, 1895; podzorski & hakansson, p. 80, 31/12, 1987. luzon: palawan, taytay (beach on washedashore seaweed), podzorski & hakansson (1.c.). _______ f. ? bmnh, adam’s # f 120.3/7. hennedyi hust.* podzorski & hakansson, p. 79, 30/1, 1987. luzon: palawan, pto. princesa, table head, south of pto. princesa bay (from surface at 15 m depth), podzorski & hakansson (1.c.). howeana hagelstein hagelstein, p. 385, 7/51, 1939; podzorski & hakansson, pp. 76-77, 30/6, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from the sand at low tide in malapakan cove), podzorski & hakansson (1.c.). hustedtii krasske krasske, 3:198, f. 3, 1923; patrick & reimer, p. 527, 50/8, 1966. = navicula hustedtii f. philippina skv. skvortzow, p. 290, 1/44, 1937a. luzon: rizal, quezon city, balara (water filter no. 8), skvortzow (l.c.). imitans mann mann, p. 104, 22/5, 6, 1925. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43643, usnm, mann (1.c.). indicatrix vanlan. vanlandingham, nom. nov. = n. alpha chol. cholnoky, p. 173, fig. 57, 1955 (non cl. 1893). luzon: manila, bmnh, comber #31903. inexacta mann mann, p. 105, 1925. = n. (alloioneis) gruendleri (cl. et grun.) cl. mann, p. 104, 105, 22/7, 1925. bottom marine samples taken near the islands by uss albatross. no type designated, mann (1.c.). inhalata schmidt schmidt, et al., 2/30, 1874; cleve, p. 57, 1895; mann, p. 107, 23/2, 1925; hustedt, p. 390, f. 1475, 1964. bottom marine samples taken near the islands by uss albatross, mann (1.c.); marine, cleve (l.c.). insignita hust. hustedt, p. 73, f. 126, 139-141, 1942a; woltereck, p. 52, 1941; patrick & reimer, p. 447, 40/3, 1966. 1 1 12 martinez-goss luzon: mt. province, bontoc, (benguet, banaue, ponds); lyon (ponds), woltereck (1.c.), hustedt (1.c.); rizal; laguna, laguna de bay, hustedt (1.c.). insociabilis krasske krasske, p. 114, 3/17, 1932; schmidt, et al., 40/103-105, 1874; hustedt, p. 63, 1942a; _______, p. 181, f. 1315a-h, 1962. luzon: rizal, pancipit river, hustedt (1942a). iota (jota) cl. cleve, p. 134, 5/22, 1894. visayas: cebu, bmnh, comber # 31898. irrorata var. substauroneiformis (grun.) cl. cleve, p. 56, 1895 =navicula approximata var substauroneiformis (grun.) cl. grunow in schmidt, et al., 2/20, 21, 1874; mann, p. 94, 1925. bottom marine samples taken near the islands by the uss albatross, mann (l.c.). jejuna schmidt schmidt, (1876) in schmidt, et al., 46/76, 1874; mann, p. 107, 1925; skvortzow, p. 280, 4/7, 1932. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). laevissima ktz. kützing, p. 96, 21/14, 1844; patrick & reimer, p. 497, 47/13, 1966. bmnh, adam’s # gc 1994. = navicula bacilliformis grun. grunow in cleve & grunow, p. 44, 2/51, 1880; hustedt, p. 55, 1942a. luzon: camarines sur, lake buhi, hustedt (l.c.). lagerheimii cl. cleve, p. 131, 1894; _____, p. 101, 7/1, 1894; woltereck, 41:37-176,1941; hustedt, in a. s. atlas, t. 370, f. 19-21, 1874; hustedt, p. 54, 1942a. luzon: mt. province, bontoc (banaue, ponds), woltereck (l.c.); laguna de bay, los baños (ponds), pancipit river; camarines sur, lake buhi, river, hustedt (1942a). mindanao: lanao, lanao lake, uyaan lake, hustedt (1942a). _______ f. intermedia hust. hustedt, in a. s. atlas, t. 370, f. 22, 1874; luzon: oriental mindoro, calapan lake, hustedt (l.c.). lanceolata (ag.) ktz. kützing, p. 94, 28/38, 30/48, 1844; van heurck, p. 186, 3/139, 1896; hustedt, p. 305, f. 540, 1930; _______, p. 72, 1942a. mindanao: lanao, uyaan lake, hustedt (1942a). latissima greg. gregory, p. 40, 5/4, 1856. luzon: manila, bmnh, comber #31904. longa (greg.) ralfs ralfs in pritchard, p. 906, 1861; donkin, p. 55, 8/3a-b, 1872; van heurck, p. 185, 25/716, 1896; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). a checklist of navicula 13 lorenzii (grun.) hust. hustedt, p. 29, f. 1188, 1961; podzorski & hakansson, p. 78, 3/1, 1a, 2, 2a, 1987. = stichodesmis australis grev. greville, p. 535, 13/1-4, 1863. bottom marine samples taken near the islands by the uss albatross, mann (l.c.). luzon: palawan, pto. princesa, table head (from sand surface at 15 m deep of pto. princesa bay), podzorski & hakansson (1.c.). = navicula scopulorum sensu cl. cleve, p. 151, e.p., 1894. visayas: cebu, bmnh, comber # 31892. lunula cl. cleve, 5/5,1894; ______, p. 51, 1895. bmnh, adam’s # g 211. luzonensis hust. hustedt, p. 59, f. 106, 1942a. luzon: camarines sur, buhi lake, hustedt (1.c.). lyra ehr. ehrenberg, p. 419, 1/1, f. 9a, 1841 (1843); bailey, p. 431, 1853; schmidt, et al., 2/16, 129/11-14, 1874; cleve, p. 63, 1895; mann, p. 108, 1925; podzorski & hakansson, p. 80, 31/10, 1987. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s-bess #715, f 121, 2/7, m 58/ 15.16, ts 497.2/2. luzon: manila, bmnh, comber #31904; palawan, taytay, langen, malapakan is. (algae encrusted in malapakan cove, marine), podzorski & hakansson (1.c.). = navicula gregoryana grev. greville, p. 10, 3/7, 1857. luzon: manila, (on sponge), bmnh, comber #33424. visayas: cebu, bmnh, comber # 31894. _______ var. cuneata temp. & perag. tempere & peragallo, p. 473, no. 988, 1914. visayas: cebu, bmnh, comber # 31894, 31898. _______ var. elliptica schmidt schmidt in schmidt, et al., 2/29, 2/34, 1874; cleve, p. 64, 1895; skvortzow, p. 272, 3/9, 1931; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.); marine, cleve (l.c.). bmnh, adam’s #f 60.1/5. visayas: cebu, bmnh, comber #31898. = navicula lyra var. subelliptica cl. cleve, p. 64, 1895. marine, deby’s collection, cleve (l.c.). _______ var. elliptica f. bullata (norman) cl. cleve, p. 63, 1895; schmidt, et al., 3/8-9, 1874. = n. bullata norman norman, p. 8, 2/7, 1861; mann, p. 97, 1925. 14 martinez-goss bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s # f 40, 2/12.17. visayas: cebu, bmnh, comber # 31894, 31897, 31898 (2), 31900. _______ var. granulata perag. & perag. peragallo & peragallo, 23/5, 1897; schmidt, et al., 258/1,1874___. bmnh, adam’s # f 45 1/10. _______ var. indica, sensu adam bmnh, adam’s # w 405. _______ var. insignis schmidt schmidt in schmidt, et al., 2/27, 257/4, 1874; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). = navicula lyra var.? intermedia perag. & perag. peragallo & peragallo, p. 136 (141), 23/6 (11), 24/18, 1897. bmnh, adam’s # f 20.2/8. _______ var. recta grev. greville, p. 28, 4/3, 1859; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s # ts 497-2/5. = n. lyra var. recta f. typica perag. peragallo, 22/8, 1891. bmnh, adam’s # ts 522 1/4.5. = n. lyra var. recta f. subelliptica perag. & perag. peragallo & peragallo, 22/7 (1897). bmnh, adam’s # f 40 2/15.45.1/9.60 1/8. ts 497 2/10. _______ var. seductilis (schmidt) o’meara = navicula seductilis schmidt schmidt in schmidt, et al., 2/35, 1874; mann, p. 119, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). _______ var. signata schmidt schmidt in schmidt, et al., 2/4, 1874; castracane, p. 33, 30/13, 1886. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). frustules found in the vicinity of the philippines, castracane (l.c.). visayas: cebu, bmnh, comber # 31893, 31895. _______ var. subtropica schmidt schmidt in schmidt, et al., 2/24.25, 1874; wolle, 16/14, 1890. bmnh, adam’s # f 40 2/15.45.1/9.60 1/8. ts 497 2/10. _______ var. ? bmnh, adam’s # f 20.2/7.45/12-14.119.3/8. g211; ts 262.1/6-9.6/3.522 1/2. maculata var. caribaea (cl.) cl. cleve, p. 46, 1895. = navicula caribaea cl. cleve in schmidt, et al., 6/10-12, 1874; mann, p. 97, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). maculosa donk. donkin, p. 25, 5/1, 1871; schmidt, et al., 259/25, 1874. a checklist of navicula 15 visayas: cebu, bmnh, comber # 31899. = navicula diffusa schmidt schmidt (1874) in schmidt, et al., 2/28, 1874; mann, p. 101, 1925. = navicula madagascarensis cl. cleve, p. 23, 4/2, 1891; mann, p. 108, 25/4, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). malombensis otto müller* otto müller, p. 84, 1/13, 1910. = navicula mammalis cstr. castracane, p. 30, 20/2, 1886. bottom marine samples taken near philippine waters obtained by h.m.s. challenger, castracane (1.c.). mammalis cstr. castracane, p. 30, 20/2, 1886. neighborhood of the philippines, castracane (l.c.). manokwariensis choln. cholnoky, p. 181, f. 71, 1963. = navicula margarita schmidt schmidt (1892) in schmidt, et al., 174/17, 1874; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). margaritifera truan y luard et witt truan y luard et witt, p. 17, 4/10, 1888. = navicula marginata lewis lewis, p. 64, 2/1, 1861; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). mauriciana v.h. van heurck, p. 11, 2/182, 1909. = n. maxima greg. gregory, p. 41, 4/19, 1855; mann, pp. 108-109, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). mendica mann mann, p. 109, 23/3, 1925. bottom marine sample taken near the islands by the uss albatross. type-cat. no. 43650, usnm, mann (1.c.). menisculus schum. schumann, p. 56, 2/33, 1867; skvortzow, p. 292, 2/8, 1937; hustedt, p. 70, 1942a. luzon: rizal, quezon city, balara, skvortzow (l.c.); manila (ponds, bureau of science grounds); batangas, taal (lake); camarines sur, buhi (lake); hustedt (1.c.). minicans hanna hanna, p. 200, 13/4, 1932. = navicula mimula mann mann, p. 110, 23/4, 1925. bottom sediments taken near the islands by the uss albatross, mann (1.c.). minima grun. grunow in van heurck, p. 107, 14/15, 1880-1885. *not recognized by vanlandingham, 1975. 16 martinez-goss = navicula minima var. atomoides (grun.) cl. cleve, p. 128, 1894; hustedt, p. 56, 1942a. luzon: rizal, manila (ponds, bureau of science grounds); camarines sur, buhi lake, hustedt (l.c.). mindanao: lanao (lake), hustedt (1.c.). minuscula grun. grunow in van heurck, 14/3, 1880; skvortzow, p. 291, 1/41, 1937; hustedt, p. 59, 1942a. luzon: rizal, quezon city (balara water filters), skvortzow (1.c.). mindanao: lanao (lake, ponds), hustedt (1.c.). mira pant. & greguss greguss in greguss, p. 212, 6/8, 1913; = navicula mirabilis leud.-fortm. leuduger fortmorel, p. 31, 2/21, 1879; mann, p. 110, 1925. bottom marine sediments taken near the islands by the uss albatross, rare, mann (1.c.). modica hust. hustedt, p. 916, 41/21-23, 1945. = navicula molesta mann mann, p. 110-111, 23/5, 1925. bmnh, adam’s # f 119, 7/3. mindanao: jolo, sulu. type-cat. no. 43651, usnm, mann (1.c.). monilifera f. constricta (perag. & perag.) hust. hustedt, p. 712, f. 1699b, 1966; podzorski & hakansson, pp. 84-85, 34/1, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from the sand in the cove, at low tide); podzorski & hakansson (1.c.). mucicoloides hust. hustedt, p. 59, f. 98-102, 1942a. luzon: benguet, bontoc; rizal, lyon (ponds), hustedt (1.c.). muralis grun. grunow in van heurck, 14/27, 1880; hustedt, p. 59, 1942a. luzon: camarines sur, buhi lake, hustedt (1.c.). mutica ktz. kützing, p. 93, 3/32, 1844; woltereck, p. 52, 1941; hustedt, p. 52, 1942a; podzorski & hakansson, p. 85, 33/8, 1987. luzon: benguet, banaue (ponds), woltereck (1.c.); laguna, los baños (ponds, laguna de bay); batangas, taal lake, crater lake; camarines sur, buhi lake, hustedt (1.c.); palawan, taytay, langen, malapakan is. (algae in the cove, marine); taytay, lake manguao (danao), (alt. c. 45 m, at the end of dry season, hence, lake at its shallowest); roxas, pagdangan range, ibangley brookside hill (alt. c. 40 m, on epiphyllous bryophyte beside a river), podzorski & hakansson (1.c.). visayas: leyte, danao lake, hustedt (1.c.). mindanao: lanao, uyaan lake, hustedt (1.c.). _______ var. cohnii (hilse) grun. grunow in van heurck, 10/17, 1880; hustedt, p. 52-53, 1942a. luzon: rizal, lyon; laguna, los baños (ponds), hustedt (1.c.). mindanao: leyte, uyaan lake, hustedt (1.c.). a checklist of navicula 17 _______f. intermedia (hust.) hust. hustedt, p. 586, f. 1593, 1966; podzorski & hakansson, p. 85, 33/9, 1987. luzon: palawan, roxas, pagdangan range, (ibangley brookside hill, from leaf surfaces), podzorski & hakansson (1.c.). = navicula lagerheimii var. intermedia hust. hustedt in schmidt, et al., 370/22, 1874; hustedt, p. 54, 1942a. luzon: oriental mindoro, calapan lake, hustedt (1.c.). _______ var. nivalis (ehr.) hust. hustedt, p. 290, 1911. = navicula mutica var. tropica hust. hustedt, p. 233, 17/6, 1937b. luzon: laguna, los baños (ponds); camarines sur, buhi lake, buhi river; oriental mindoro, naujan (river), hustedt (1.c.). nebulosa greg. gregory, p. 480, 9/8, 1857; mann, p. 111, 1925. bottom sediments taken near the islands by the uss albatross, mann (l.c.). visayas: cebu, bmnh, comber #31900. nippon skv. skvortzow, p. 277, 5/17, 1936. = navicula nitescens (greg.) ralfs* ralfs in pritchard, p. 898, 1861; mann, p. 111, 1925. bottom sediments taken near the islands by the uss albatross, mann (1.c.). northumbricaeformis m. perag m. pergallo in heribaud, et al., p. 15, 1/19, 1920; = navicula notabilis grev. greville, p. 18, 1/19, 1863; mann, p. 111, 1925. bottom sediments taken near the islands by the uss albatross, mann, (l.c.). nummularia grev. greville, p. 249, 5/6, 1859; de toni, p. 96, 1891; mann, p. 111, 1925. bottom sediments taken near the islands by the uss albatross, mann (1.c.). = navicula forcipata var. suborbicularis grun. grunow in van heurck, 10/5, 1880. cleve, p. 66, 1895. mindanao:sulu sea(deby coll.). aff. ny cl. cleve, p. 75, 1/24, 1894; podzorski & hakansson, p. 75-76, 31/4, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from sand at low tide in the cove, marine), podzorski & hakansson (l.c.). nympharum hust. hustedt, 1936, in schmidt, et al., 401/85-87, 1874. = navicula oamaruensis grun.* grunow, 1888, in schmidt, et al., 129/9, 1874__; mann, p. 111, 1925. bottom sediments taken near the islands by the uss albatross, mann (l.c.). obesa o. müll. o. müller, p. 84, 2/27, 1910. *not recognized by vanlandingham, 1975. 18 martinez-goss = navicula obesa (grev.) mann mann, pp. 111-112, 23/6, 24/1, 1925. bottom marine samples taken near the islands by the uss albatross. no type designated, mann (1.c.). occulta krasske krasske, p. 353, f. 12a-b, 1929. = navicula ocellata mann* mann, p. 112, 24/2, 1925. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43652, usnm, mann (1.c.). oscitans schmidt schmidt, 1875, in schmidt, et al., 6/41, 1874; mann, p. 112, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). ______ var. subundulata cl. & grove grove, p. 67, 10/10, 1891. bmnh, adam’s # ts 523/5.8. = navicula suboscitans mann mann, p. 122, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). pacifica (cstr.) mann* castracane p. 23, pl. 20, f. a, 1886; mann, p. 113, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). palpebralis var. semiplena (greg.) cl. cleve, p. 70, 1895. = navicula angulosa var. beta greg. gregory, p. 42,5/8,1856; mann, p. 94, 1925. bottom sediments, mann (l.c.). palustris var. obesa zanon zanon, p. 111, 2/10, 1949. =navicula pandura breb. schmidt, et al., 11/1, 2, 4, 8, 9, 1874; mann, p. 113, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). perrotettii (grun.) cl. cleve, p. 110, 3/12, 1894; hustedt, p. 51, 1942a. diatom samples from the philippines of slightly brackish waters derived from dr. rae’s collection, cleve (1.c.). luzon: rizal, manila (ponds, bureau of science grounds); laguna, los baños (laguna de bay, ponds); camarines sur, buhi (lake, river), hustedt (l.c.). philippina skv. skvortzow, p. 291, 1/25, 45, 1937a. luzon: rizal, quezon city, balara (water filter no. 8), skvortzow (l.c.). phillippinarum mann mann, pp. 114-115, 25/1, 1925. bottom marine samples taken near the islands, including those from the sulu group, taken by the uss albatross. type-cat. no. 43655, usnm, mann (1.c.). bmnh, adam’s # f 83, 6/57; ts 523/9. platyventris meist. meister, p. 95, f. 33, 1934 (1935); podzorski & hakansson, p. 81, 32/3, 1987.*not recognized by vanlandingham, 1975. a checklist of navicula 19 luzon: palawan, langen, malapakan is. (surface scrapings from the sand at low tide in the cove), podzorski & hakansson (1.c.). plicatula grun. cleve & möller, slide #154-155; grunow ms. in cleve, p. 155, 3/28, 1894; mann, pp. 115-116, 24/8-9, 1925; podzorski & hakansson, p. 78, 30/8, 1987. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). luzon: palawan, taytay, langen, malapakan is. (algae from the cove, marine), podzorski & hakansson (1.c.). visayas: cebu, bmnh, comber # 31894. plutonia o’ meara* o’meara, p. 286, 13/6, 1872; de toni, p.78, 1891. mindanao: sulu, cagayan, (brackishwaters from the volcanic mountain), de toni (l. c.). praetexta ehr. ehrenberg, p. 214, 1840 (1841); castracane, p.812, 1886; mann, p. 116, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s # gc 2094; f 119 3/9; ts 497.1/10; ts 523/11; ts 60/81. luzon: manila (on shell), bmnh, comber # 30925. visayas: cebu, castracane (l.c.). _______ var. abundans schmidt schmidt, et al., 129/8, 1874; cleve, p. 55, 1895. visayas: cebu, bmnh, comber # 31898, 31895, 31894. _______ var. ? bmnh, adam’s # f 45.2/16. protracta (grun.) cl. cleve, p. 140, 1894; podzorski & hakansson, p. 77, 33/13, 1987. luzon: palawan, taytay (lake manguao, alt. c. 45 m., end of the dry season when it was at its shallowest, surrounded by semi-deciduous rainforest), podzorski & hakansson (1.c.). pseudobryophila hust. hustedt, p. 60, 1942a; _______, p. 114, 1942b. visayas: leyte (danao lake), hustedt (1942a). pseudoclavata mann mann, p. 116, 25/2, 1925. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43656, usnm, mann (1.c.). pseudoforcipata hust. hustedt, pp. 119-120 f. 11, 1942b; podzorski & hakansson, p. 80, 31/67, 1987. luzon: palawan, taytay, langen, malapakan is. (from the cove, marine); pto. princesa (south of pto. princesa bay, from the rapidly deepening zone of the sulu sea, hand collected from corals at 10 m depth), podzorski & hakansson (1.c.). pseudony hust. hustedt, p. 23, 8/11, 1955; podzorski & hakansson, p. 80, 30/2, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted from the cove, marine), podzorski & hakansson (1.c.). *not recognized by vanlandingham, 1975. 20 martinez-goss pugio mann mann, p. 117, 26/4, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). pulverulenta (pulvulenta) mann mann, pp. 117-118, 25/3, 1925. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43659, usnm, mann (1.c.). pupula ktz. kützing, p. 93, 30/40, 1844; woltereck, p. 52, 1941; hustedt, p. 64, 1942a; podzorski & hakansson, p. 76, 30/3, 1987. luzon: benguet, banaue (ponds), woltereck, 1941; rizal, malabon, dagatan (swamp), manila (ponds in the bureau of science grounds, pancipit river, reservoir ii); laguna, los baños (ponds, laguna de bay), san pablo (bunot lake, bato lake); batangas, taal (yambo crater lake, crater lake); camarines sur, buhi (lake, river); payren lake; oriental mindoro, naujan lake, hustedt (1.c); palawan, taytay (lake manguao, alt. 45 m, near public works’ resthouse, at the end of the dry season, when lake was shallowest; small pool in stream bed flowing into nw side of the lake, 1.8 km from public work’s resthouse, alt. 130 m), podzorski & hakansson (1.c.). mindanao: lanao (uyaan lake, lanao lake, river); jolo, sulu (seit lake), hustedt (1.c.). _______ var. rectangularis (greg.) cl. & grun. cleve & grunow, p. 45, 1880. = navicula f. rectangularis (greg.) cl. & grun. hustedt, pp. 64-65, 1942a; podzorski & hakansson, p. 76, 30/4, 1987. luzon: palawan, roxas, pagdanan range (ibangley brookside hill, alt. 40 m, streamside pool), podzorski & hakansson (1.c.). visayas: leyte (danao lake), hustedt (1.c.). mindanao: lanao (uyaan lake), hustedt (1.c.). _______ f. rostrata (hust.) hust. hustedt, p. 291, 3/39, 1911; _______, p. 64, 1942a; _______, p. 282, 1957. luzon: laguna, los baños (laguna de bay, pond); batangas, taal (taal lake); camarines sur, buhi (lake), hustedt (1942a). mindanao: lanao (pond), hustedt (1942a). pygmaea ktz. kützing, p. 77, 1849; woltereck, p. 52, 1941; hustedt, pp. 66-67, 1942a; podzorski & hakansson, p. 80, 31/11, 1987. luzon: benguet, banaue (ponds), woltereck (1.c.); rizal, manila (bureau of science grounds, ponds), pancipit river, lyon (ponds); laguna, los baños (ponds, laguna de bay); camarines sur, buhi (river), hustedt (1.c.); palawan, pto. princesa (south of pto. princesa bay, hand collected from sand surface at 15 m depth); taytay, langen, malapakan is. (surface scrapings from sand at low tide), podzorski & hakansson (1.c.). mindanao: lanao (lanao lake), jolo, sulu (seit lake), hustedt (1.c.). quarnerensoides hust.* hustedt, p. 49, f. 1203, 1961; (doubtful-fide: vanlandingham, p.2759,1975); podzorski & hakansson, 78, 1987. luzon: palawan, pto. princesa, table head (hand collected from sand surface at 15 m depth of sulu sea), podzorski & hakansson (1.c.). *not recognized by vanlandingham, 1975. a checklist of navicula 21 radiosa ktz. kützing, p. 91, 4/23, 1844; woltereck, p. 52, 1941; hustedt, pp. 69-70, 1942a; podzorski & hakansson, p. 82, 33/2, 3. luzon: benguet, banaue (ponds); rizal, malabon (dagatan swamp), manila (water reservoir iii, ponds in bureau of science grounds, lyon ponds), hustedt (1.c.); brooke’s point (tumarabong river over oligocene-miocene reef limestones, alt. 10 m, scrapings from the surface of a limestone rock), podzorski & hakansson (1.c.). _______ var. tenella (breb. ex ktz.) cl. et möll. (non grun. fide vanlandingham, 1975); cleve et möller slide no. 224, 1881; hustedt, pp. 69-70, 1942a; podzorski & hakansson, p. 82, 33/4, 1987. luzon: batangas, taal (volcano lake), hustedt (1.c.); palawan, pto. princesa (irawan river over mafic and mafic-plutonic rocks, alt. c. 150 m; scrapings from a bryophyte behind a waterfall), podzorski & hakansson, (1.c.). raena (cstr.) de t. de toni, p. 30, 1891; mann, p. 118, 1925. bottom marine samples taken near the islands by the uss albatross. type species from the philippines (fide: castracane below), mann (1.c.). = pinnularia raena cstr. castracane, p. 25-26, 15/3, 1886. visayas: cebu, from dr. rae’s collection, castracane (1.c.). ramosissima (ag.) cl. cleve, p. 26, 1895; podzorski & hakansson, p. 82, 34/3, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted from the cove, marine), podzorski & hakansson (1.c.). retracta meist. meister, p. 262, 4/6, 1937; = navicula retrostauros mann* mann, p. 118, 25/2, 26/1, 2, 1925. bmnh, adam’s #m 59.1/8; ts 60/59. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43660 usnm, mann (l.c.). rhaphoneis (ehr.) grun. grunow, p. 19, 91/17, 1867; podzorski & hakansson, pp. 82-83, 32/6, 7, 1987. luzon: palawan, taytay, langen, malapakan is. (algae scraped from the cove), podzorski & hakansson (1.c.). rhombica greg. gregory, p. 40, 4/16, 1855; mann, p. 119, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). _______var. japonica (brun.) cl. cleve, p. 152, 1894. bmnh, adam’s #ts 523/2. rhynchocephala (rhynococephala) ktz. kützing, p. 152, 30/35, 1844; hustedt, p. 67, 72, 1942a; delos reyes, p. 59, pl. 4, f. 17, 1972. luzon: laguna, los baños (laguna de bay); batangas, taal (crater lake); hustedt (1.c.). visayas: (tigbao water supply), delos reyes (1.c.). riparia hust. hustedt, p. 52, f. 77-78, 1942a. *not recognized by vanlandingham, 1975. 22 martinez-goss luzon: rizal, malabon (dagatan swamp), hustedt (1.c.). robertsiana (robertsoniana) grev. greville, p. 235, 3/9, 1865. visayas: cebu, bmnh, comber #31895. _______ var. cuneata (schmidt) amosse’ amosse’, p. 111, 1924. = n. prodiga mann mann, p. 252, 53/4, 1907; ______, p. 116, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1925). ruttneri hust. hustedt, 1936, in schmidt, et al., 402/30-38, 49-52, 1874; _______, p. 55, 1942a. luzon: camarines sur, buhi (lake), hustedt (1942a). _______ var. capitata hust. hustedt, 1936, in schmidt, et al., 402/47, 48, 1874; _______, p. 55, 1942a. luzon: manila (reservoir ii), oriental mindoro, naujan (river), hustedt (l.c.). _______ var. rostrata hust. hustedt, 1936, in schmidt, et al., 402/39-45, 1874; ______, p. 56, 1942a. luzon: rizal, pancipit river; oriental mindoro, naujan (naujan lake), hustedt (1942a). mindanao: lanao (pond), hustedt (1942a). salinarum grun. grunow in cleve & möller, slide no. 107, 1878; grunow in cleve & grunow, p. 33, 2/34, 1880; pantastico, p. 195, 17/3, 1977. luzon: laguna, los baños, mayondon, pantastico (1.c.). schaarschmidtii pantocsek pantocsek, p. 28, 14/121, 1886. visayas: cebu, bmnh, comber #31900. schoenfeldii hust. hustedt, p. 301, f. 520, 1930; _______, p. 70, 1942a. luzon: oriental mindoro, naujan (river), hustedt (1942a). schroeteri meist. meister, p. 38, f. 100, 1932; hustedt, p. 72, 1942a. luzon: laguna, los baños (laguna de bay); batangas, taal (taal lake); oriental mindoro, naujan (river), hustedt (1.c.). _______ var. escambica patr. patrick, p. 104, 7/3, 1959; podzorski & hakansson, p.83, 33/5, 1987. luzon: palawan, roxas, pagdanan range, (ibangley brookside hill streamside freshwater pool, alt. c. 40 m.), podzorski & hakansson (l.c.). scutelloides w. sm. smith, p.9, 1856; _____, in gregory, p. 4, 1/15, 1856; hustedt, p. 66, 1942a. luzon: batangas, taal (crater lake); camarines sur, buhi (lake), hustedt. (l.c.) mindanao: lanao (lanao lake, pond), hustedt (1.c.). seminulum grun.grunow, p. 552, 4/3(2/3), 1860; hustedt, p. 56, 1942a. luzon: camarines sur, buhi (lake); batangas, taal (crater lake). a checklist of navicula 23 mindanao: lanao (lanao lake, pond); hustedt (l.c.). semistauros mann* mann, 119-120, 26/5, 1925. bottom marine samples taken near the islands by the uss albatross, type-cat. no. 43661, usnm, mann (l.c.). sigma brun brun, p. 39, 16/5, 1891; brun later homonym, fide vanlandingham p. 2799, 1975; podzorski & hakansson, p. 75, 29/12, 1987. luzon: palawan, pto. princesa, table head (from sand surface at 15 m depth of sulu sea), podzorski & hakansson (1.c.). spectabilis greg. gregory, p. 481, 9/10, 1857; cleve, p. 60, 1895; mann, p. 121, 27/1-2, 1925; podzorski & hakansson, pp. 80-81, 32/1, 1987. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). diatom samples (presumably from dr. rae’s collection), cleve, 1894, p. 60, 1895. bmnh, adam’s # j 4006; bess 1709/3; g 211; f 40.2/16.45.1/11. luzon: palawan, pto. princesa, table head (from the sand surface at 15 m depth of sulu deep sea), podzorski & hakansson (1.c.). visayas: cebu, bmnh, comber #318931. _______ var. controversa (schmidt) cl. schmidt, et al., 3/5, 1874; cleve, p. 60, 1895. bmnh, adam’s # ts 60/4. _______ var. emarginata cl. cleve p. 60, 1895. = navicula excavata var. ________ schmidt schmidt in schmidt, et al., 3/22-25, 1874. visayas: cebu, bmnh, comber #31899. _______ var. excavata (grev.) cl. cleve, p. 61, 1895; = n. excavata grev. greville, p. 130, 12/15, 1866; mann, p. 102, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s # f 83.1/9.119.2/4; ts 522.2/2. = n. oswaldii janisch janisch, 1, 15/12, 1888; mann, p. 113, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). _______ var. minuta cl. mereschkowsky, p. 320, 2/9-12, 1902. = navicula hennedyi var. minuta cl. cleve, p. 7, 1/15, 1881. visayas: cebu, bmnh, comber #31894. _______ var. ? bmnh, adam’s # f 931; bess 1710/3; ts 497. 2/7-9. spiculifera mann* mann, pp. 121-122, 26/8, 1925. *not recognized by vanlandingham, 1975. 24 martinez-goss bottom marine samples taken near the islands by the uss albatross. type-cat. no. 4663, usnm, mann (1.c.). spiralis o’ meara* de toni, p. 74, 1891. mindanao: saline waters from the volcanic crater, cagayan, sulu archipelago, de toni (l.c.). studerri sensu comber* luzon: manila (sponge), bmnh, comber # 30924. subarvensis hust. hustedt, p. 60, f. 107-108, 1942a. luzon: camarines sur, buhi (buhi lake), hustedt (1.c.). = n. lyra var. subcarinata grun. grunow, 1874, in schmidt, et al., 2/5, 1874; mann, p. 108, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, bess 62; f 40 2/19; ts 497.2/6, 522, 2/6, 3/8. luzon: manila, bmnh, comber #31903. subdecipiens de t. de toni, p. 147, 1891. = n. decipiens cstr. castracane, p. 32, 27/17, 1886. from philippine marine diatom collection of dr. rae, castracane (l.c.). subdecussis hust. hustedt, pp. 75-76, f. 136-138, 1942a. luzon: rizal, manila (reservoir ii), hustedt (1942a). subforcipata hust. hustedt, p. 533, f. 1569, 1964; podzorski & hakansson, p. 75, 31/51, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from sand at low tide), podzorski & hakansson (1.c.). subrhynchocephala hust. hustedt, p. 156, 1/11, 1935; _______, p. 67, 1942a. luzon: rizal, manila (reservoir ii, pancipit river); laguna, los baños (pond, laguna de bay, payren lake); batangas, taal (crater lake); camarines sur, buhi (buhi lake, river), hustedt (1942a). visayas: leyte (danao lake), hustedt (1942a.). suluensis o’ meara* de toni, p. 80, 1891. mindanao: brackishwaters in the crater of a volcanic island, cagayan, sulu, de toni (l.c.). takoradiensis hendey hendey, p. 67, 1/8, 1958; podzorski & hakansson, p. 77, 30/7, 1987. luzon: palawan, pto. princesa, table head (from sand surface at 15 m depth of the sulu sea), podzorski & hakansson (1.c.). tenera hust. hustedt, p. 259, 18/11-12, 1937a; _______, p. 67, 1942a. luzon: rizal, manila (pancipit river); batangas, taal (crater lake); hustedt (1942a). terminata hust. hustedt, p. 589, f. 1594, 1966; *not recognized by vanlandingham, 1975. a checklist of navicula 25 = n. mutica var. tropica hust. hustedt, 1936 in schmidt, et al., 405/39-42, 1874; _______, pp. 53-54, 1942a. luzon: laguna, los baños (pond); camarines sur (buhi lake); oriental mindoro, naujan (naujan river), hustedt (1942a). translucens mann mann, p. 123, 26/9, 1925. bottom marine samples taken near the islands by the uss albatross. type-cat. no. 43665, usnm, mann (1.c.). tripunctata (o.f. müll.) bory* bory, p. 128, 1822; podzorski & hakansson, p. 83, 33/6, 1987. luzon: palawan, taytay, langen, malapakan is. (scrapings from sand surface at low tide), pto. princesa, honda bay (scraping from coral reefs at the edge of an atoll), podzorski & hakansson (1.c.). uniseriata östrup östrup, p. 8, 1/9, 1913; podzorski & hakansson, pp. 77, 33/12, 1987. luzon: palawan, taytay, langen, malapakan is. (algae encrusted from cove), podzorski & hakansson (1.c.). variostriata krasske krasske, p. 197, f. 12, 1923; hustedt, p. 55, 1942a. luzon: laguna (laguna de bay), hustedt (1.c.). venusta janisch janisch, 15/17, 1888; mann, p. 124, 1925. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). venustissima kitt. kitton, p. 101, 1889; kitton in leuduger-fortmorel., p. 17, 2/3, 1892. bmnh, adam’s # gc 2177. viminea hust. hustedt, 1934 in schmidt, et al., 397/10-11, 1874; podzorski & hakansson, p. 83, 32/8-9, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from sand at low tide), podzorski & hakansson (1.c.). viridula (ktz.) ehr. ehrenberg, p. 53, 1836. = navicula viridula ktz. kützing, p. 23, 13/12, 1833; hustedt, p. 72, 1942a. luzon: laguna, los baños (pond); camarines sur, buhi (buhi lake), hustedt (1.c.). werestschangini (werestschangini) skv. & meyer skvortzow & meyer, p. 20, 1/64, 1928; podzorski & hakansson, p. 85, 34/5, 1987. luzon: palawan, taytay, langen, malapakan is. (surface scrapings from sand at low tide), podzorski & hakansson (1.c.). yarrensis (jarrensis) grun. grunow 1876, in schmidt, et al., 46/1-6, 1874; mann, p. 126, 1925; podzorski & hakansson, p. 79, 31/3, 1987. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). bmnh, adam’s # gc 2190; ts 262.1/ 4.5.18.6/1.2.89. *not recognized by vanlandingham, 1975. 26 martinez-goss luzon: palawan, taytay, langen, malapakan is. (algae scraped from the cove), podzorski & hakansson (1.c.). zanzibarica grev. var. zebuana cstr. greville, p. 129, 12/22, 1866; castracane, p. 31, 28/8, 1886; a. schmidt, 1888 in a. schmidt, et al., 129/4, 1874. visayas: cebu, castracane (1.c.). zostereti (zosteretii) grun. grunow, p. 528, 4/23, (2/23) 1860; mann, p. 125, 1925; podzorski & hakansson, pp. 83-84, 33/10, 1987. bottom marine samples taken near the islands by the uss albatross, mann (1.c.). luzon: palawan, taytay, langen, malapakan cove (scrapings from the cove), pto. princesa, white beach (on mud surface in a mangrove), honda bay (coral scrapings from a bay), podzorski & hakansson (1.c.). sp. (miniscula) podzorski & hakansson, p. 81, 19/11, 1987. luzon: palawan, taytay, langen, malapakan cove (scraping from sand surface at low tide), podzorski & hakansson (1.c.). sp. 1 esguerra, p. 174, 1951; villadolid, p. 21, 1957. sp. 2 bmnh, adam’s-bess # m64-66; ts 80; bess 22/ 6.714; ts 522.2/3.7.3/3. visayas: cebu, castracane, p. 812, 1886. sp. 3 white chalk collected by the ship princess louise to manila, under capt. wendt (1830-34). identification by dr. meyer. ehrenberg, atlas, p. 177, pl. 1, 1854. acknowledgments this checklist was completed thru the financial assistance of the jessup-mchenry fund grant of the academy of natural sciences of philadelphia (ansp), u.s.a. and the british council of england; the kind hospitality in making available their references and specimens of the ansp and the british museum of national history; the patience in encoding and revising the paper by c.m. nuñez, m.v. marasigan and w.f. fabelllore. references amosse, a., 1924. diatomees de la cote orientale d’ afrique. bulletin du museum national d’ histoire naturalle. t.20 (1924), p.109-116, 159-166, 247-254, 329-335. bailey, j.w., 1853. list of diatomaceae collected by the united states exploring expedition under capt. wilkes, u.s.n. proc. nat. acad. sci. phila. 6:431-432. bory de saint-vincent, j.b.m, 1822-1831. collaborator in “dictionaire classique d’ histoire naturelle,” 17 vol. paris. atlas et illustration des planches t. 17, 1831. (diatomaceae are scattered throughout the work alphabetically). brockman, c., 1950. die watt-diatomeen der schleswig-holsteinischen westküste. abhandlungen d e r s e n c k e n b e r g i s c h e n n a t u r f o r s c h e n d e n gesellschaft. 478: 1-26, 6 taf. brun, j., 1891. diatomees especes nouvelles marines, fossiles ou pelagiques, geneve, imprimerie aubertschuchardt. 47p., 12 pl. castracane, c.a.f., 1886. report on the scientific results of the voyage of h.m.s. challenger during the years 1873-1876; botany, london, her majesty’s stationary office, vol. 2, 178p., 30 pl. cholnoky, b.j., 1955. diatomeen aus salzhaltigen binnengen binnengewässer der westlichen kaapprovincz in südafrika. berichte der dentschen botanischen gesellschaft, bd. 68, s. 11-23. a checklist of navicula 27 cholnoky, b.j., 1963. ein beitrag zur kenntnis der diatomeen flora von hollan disch new guinea. nova hedwigia. 5(1-4): 157-198, 3 taf. cleve, p.t., 1878. diatoms erom the west indian archipelago. bihang till kongl. svenska vetenskapsakadmiens handlinger. 5(8):1-22, pl. 1-5. cleve, p.t., 1881. on some new and little known diatoms. konglinga svenska vetenskaps-akadmiens handlinger. 18(5): 1-28, pl. 1-6. cleve, p.t., 1891. diatomees rares on nonvelles. le diatomiste. 1: 75-78. cleve, p.t., 1894. sur quel ques especes nouvelles ou peu connues. le diatomiste. 2(17):99. cleve, p.t., 1895. vetens kaps-akademiens handlingar, bd. 26, p. 1-194, pl. 1-5 (part i (1894)); bd. 27, p. 1219, pl. 1-4 (part ii (1895)). cleve, p.t. & j.d. moller, (eds), 1877-1882. diatoms (exsiccata). upsala, sweden, esatas edquists boktryckeri (this series of slides consists of 324 slides and were all examined by a. grunow). cleve, p.t. & a. grunow, 1880. beitrage zur kenntriss der artischen diatomeen. kongl. svenska veten.akadem. handl. stockholm, sweden, kongl boktryckeriet. 17(2):121p., 7 pl. de los reyes, p., 1972. the algae of the tigbao water supply. leyte-samar studies 6:48-60. de toni, g.b., 1894. sylloge algarum omnium hucusque cognitarum, vol. ii, bacillarieae. section i, r a p h i d e a e , p . 1 4 9 0 ( 1 8 9 1 ) ; s e c t i o n i i , pseudoraphideae, p. 491-817 (1892); section iii, cryptoraphideae, p. 818-1556. donkin, a. 1861. on marine diatomeceae of northumberland with a description of several new species. quart. j. microsc. sci. 1 (new series): 1-15, pl. 1 donkin, a. 1870-1873. the natural history of the british diatomaceae. part 1, p. 1-24, pl. 1-4 (1870); part 2, p. 25-48, pl. 5-8 (1871). london. ehrenberg, c.g., 1830 (1832). beiträge zur kenntniss der organization der infusorien und iher geographischen verbreitung besonders in sibirien. (preussische physikalische) abhandlungen der königlichen akademie der wissenschaften zu berlin, s. 1-88, 8 taf. ehrenberg, c.g., 1830 (1839). uber die bildung der kreidefelsen und des kreidemergels durch unsichtbare organismen. abhandlungen der königlichen akademie der wissenschaften zu berlin, s. 59-147, taf. 1-4. ehrenberg, c.g., 1836. nachrichten uber das vorkommen fossiler infusorien. bericht ü die zur bekanntmachung geeigneten verhandlungen der koniglichen preuss. akademie der wissenschaften zu berlin, erster jahrgang, 1836, s. 83-86. ehrenberg, c.g., 1837. mittheilungen aus den verhandlungen der gaselllschaft naturforschender freunde zu berlin 31 marz 1837, etc. ehrenberg, c.g., 1838.die infusionsthierchen als vollkommende organismen. ein blick in das tiefere organiche leben der natur. s. i-xvii, 1-548, taf. 164 (atlas). leopold vass, leipzig. ehrenberg, c.g., 1841(1843sic).verbreltung und einfluss des mikroskopischan lebens in sud-und nordamerika. bericht uber die zur bekanntmachung gee igneten verhandlungen der koniglichen akademie da wissonschaften zer berlin, theil 1, s. 291-445, 4 taf. ehrenberg, c.g., 1840 (1841). uber ausgezeichnete jetzt leberde peruaniche und mexikanische meeresinfusorien, welche mit zur erlauterung rathselhaften fossilen formen der kreidebildung dienen. bericht über die zur bekanntmachung geeigneten verhandlungen der königl. preuss. akademie der wissenschaften zu berlin, s. 157-162. ehrenberg, c.g., 1854. die systematische charakteristik der neuen mikroskopichen organismen des tiefen atlantischen ocean für den monatsbericht zum druck zu übergeben, deren verzeichniss in monat februar bereits mitgetheilt worden ist. berucht über die zur bechantmachung geeigneten verhandlungen der 28 martinez-goss königlichen preuss. akademie der wissenschaften zu berlin, s. 236-252. esguerra, r.s., 1951. enumeration of algae in philippine bangos fishponds and in the digestive tract of the fish with notes on conditions favorable for their growth. phil. j. fish , 1: 175-196. gregory, w., 1855. on a post-tertiary lacustrine s a n d c o n t a i n i n g d i a t o m a c e o u s e x u v i a e f r o m glenshina near inverness. quart. j. microsc. sci. 3: 30-43, pl. 4. gregory, w., 1856. notice of some new species of british freshwater diatomaceae. quart. j. of microsc. sci. 4:1-14 (for the figure see: vol. iv, pl. 1, f. 1-42). gregory, w., 1857. on new forms of marine diatomaceae found in the firth of clyde and in loch fyne. (drawn by r.k. greville) trans. roy. soc. of edinb. 21:473-542, pl. 9-14. greguss, p. 1913. die kieselalgen der meeraugen von surian. (a surianitengerszemek kovamoszatai.) botanikai közlemenyek, bd. 12, s. 202-225. greville, r.k., 1857. description of some new diatomaceous forms from the west indies. quart. j. microsc. sci. 5: 7-12, pl.3 greville, r.k., 1859. descriptions of some new species and varieties of naviculae, etc., observed in california guano. edinb. new philo. j. n.s. 10:25-30, pl. 4, 8 fig. greville, r.k., 1862. descriptions of new and rare diatoms. series vi. trans. microsc. soc. 10:89-96, pl. 9, (pp. 1-7). greville, r.k., 1863. descriptions of new genera and species of diatoms from the south pacific. part i. edinb. new philo. journ. 18:34-43, pl. 1, 43 fig. greville, r.k., 1865. description of new and rare diatoms. trans. microsc. soc. 13 (n.s.): 1-10, 2 plates (i-ii), series xv, p. 24-34, 2 plates (v-vi), series xvii< p. 97-105, 2 plates (viii-ix). greville, r.k., 1866. descriptions of new and rare diatoms. series 20. trans. microsoc. soc. 14 (n.s.):121-130, pl. 11 & 12, 25f. grove, e., (many dates). collection (exsiccatae) of e. grove. preston, brighton. grunow, a., 1860. weber neue oder ungeniigend gekannte algen. aus den verhandlungen der k.k. zoologisch-botanischen gesellschaft in wien [jahrgang 1860] besconclers abgedruckt. pp. 1-80, pl. 3-7. grunow, a., 1863. uber einige neue und ungenugend bekannte arten und gattungen von diatomaceen. zweite folge. verhandl. der kaiserlich-konigl. zoologisch-botanischen gesselschaft in wien. 13:137-162, pls. 13-14. grunow, a., 1867. diatomeen auf sargassum von honduras, gesammelt von lindig. hedwigia. 6(1-3): 1-8, 17-37. grunow, a., 1880. vorläufige bemerkungen zu einer systematischen anordung der schizonema – und berkeleya – arten, mit bezug auf die in van heurch’s “diatocaceenflora von belgien” veröffetlichten abbildungen der frusteln auf taf. xv, xvi, and xvii. botanisches centralblatt. 4(47/48): 1506-1520, 15851598. hagelstein, r., 1938. the diatomaceae of puerto rico and the virgin islands. new york academy of sciences, survey of porto rico and the virgin islands. 8(3): 313-450, 9 plates. hagelstein, r., 1938(1939). the diatomaceae of puerto rico and the virgin islands. new york academy of science survey of puerto rico and virgin islands. 8(3): 313-450, 9 pls. hanna, g.d., 1930. the dates and publication of tempe’re and peragallo’s diatomees du monde entier, edition 2. j. paleontology 4(3):296-297, 1-68p. hanna, g.d., 1932. pliocene diatoms of wallace country, kansas. university of kansas science bulletin 20(21): 369-394, pls. 31-34. a checklist of navicula 29 heiberg, p.a.c., 1863. conspectus criticus diatomacearum danicarum. wilhelm priors forlag, kjobenhavn: 135 p. 6 pls. hendey, n.i. 1958. marine diatoms from some west african ports. journ. of the royal microsc. soc. 77(3): 28-85. heribaud, j., et al., 1920. les diatomees des travertins d’ auvergue. bruxelles, imprimerie de scintifique: 206 p., 7 pl. hustedt, f., 1911. beiträge zur algenflora von bremen. iv. bacillariaceen dus der wumme. abh. naturw. ver. brem. 209(2): pp. 257-315, 3 pl. hustedt, f., 1930. bacillariophyta (diatomeae). in a. pascher “die susswasser-flora mitteleuropas”, heft 10, 466 s., 875 fig., jena, gustav fischa verlag. h u s t e d t , f. , 1 9 3 0 1 9 6 6 . d i e k i e s e l a l g e n deutschlands, õsterreichs und der schweiz unter berucksichtigung der übrigen länder europas sowie der angrenzenden meeresgebiete. in: rabenhorst l., “kryptogamen-flora von deutchland, õsterreich und der schweiz”, 7. leipzig, akademische verlagsgesellschaft. hustedt, f., 1935. die fossile diatomeenflora in den ablagerungen des tobasees auf sumatra. 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(planches et phototypie d’ aprés les photographies de c. janisch). 16 lichtdruck-tafeln (325 figuren) mit tafelerklärungen von 16 seiten. kitton, f., 1889. new species of navicula (n. venustissima). jour. royal microsc. soc., p. 101. könig, d. 1959. diatomeen der bucht von arcachon (dep. gironde frankreich). zeitschrift der deutschen geologischen gesellschaft. 111: 33-61. krasske, g., 1923. die diatomeen des casseler beckens und seiner randgebirge, nebst einigen wichtigen funden aus niederhessen. botanisches archiv. 3(4): 185-209. krasske, g., 1925. die bacillariaceen–vegetation niederhessens. abhandalungen und bericht. (56 des) veriens für naturkunde, zu casul, 84-849 vereinsjahr 1919-1926, s. 1-119, 2 taf. krasske, g., 1929. beiträge zur diatomeenflora sachsens. botanisches archiv. 27 (3/4): 348-380, 25 fig. krasske, g., 1932. diatomeen aus dem oberpliocän von willershausen. archiv. fur hydrobiol. 24(3): 430448, taf. 16. kützing, f. t., 1833. synopsis diatomacearum oder versuch einer systematischer zusammenstellung der 30 martinez-goss diatomeen. linnaea. 8: 529-620, taf. 13-19. berlin (sonderabdruck, s. 1-92, 1834). kützing, f.t., 1844. die kieselschaligen bacillarien oder diatomeen. nordhausen, w. kohne. 152p., 30 pl. kützing, f.t., 1849. species algarum. lipsiae, f.a. brockhaus., 922s. leuduger-fortmorel, g., 1879. catalogue des diatomees de l’ile ceylon. mémoires de la société d’ emulation des côtes du nord.saint-brieuc, libraire francisque guyon, imprimeur, 73p., 9 pl. leuduger-fortmorel, g., 1892. diatomees de la malaisie. annales du jardin botanique de buitenzorg. 11: 1-60, pl. 1-7. lewis, f.w., 1861. notes on new and rarer species of diatomaceae of the united states seaboard. proc. acad. nat. sci. of phila. 13:61-71, 2 pls. also, quart. j. microsc. sci. 2( new series): 155. lewis, f.w., 1865. on extreme and exceptional variations of diatoms in some white mountain localities, etc. proc. acad. nat. sci. phila. 7-18, pl. 1-2. mann, a., 1907. report on the diatoms of the albatross voyages in the pacific ocean, 1888-1904. contributions from the united states national herbarium, vol. 10, part 5, pp. 221-419, 11 pl. mann, a., 1925. marine diatoms of the philippine islands. smithsonian institution, united states national museum bull. #100: vol. 6, part 1, 182p., 39 pl. martinez-goss, m.r., 1995. a checklist of nitzschia and tryblionella (class bacillariophyceae) of the philippines. phil. j. sci. 124(1):75-99. meister, f.r., 1912. die kieselalgen der schweiz. in beitrage zur kryptogamenflora der schweiz. band iv, heft i. bern, druck und verlag von j.k. wyss.: 254 s. 48 taf. meister, f.r., 1932. keilalgen aus asien. berlin, verlag von gebriider borntraeger: 56 s., 19 taf. meister, f.r., 1934 (1935). seltene und nene kieselalgen. bericht der schweizerische botanische gessellschaft.44: 87-108. meister, f.r., 1937. seltine und nene kieselalgen. ii. berichte der schweiserischen botanischen gessellschaft. 47: 258-276, tafeln 3-13. mereschkowsky, c., 1902, note sur quelques diatomées de la mer noire. journal de botanique (morot, ed., paris). 16: 319-324, 358-360, 416-430. muller, o., 1903-1910. bacillariaceen aus dem nyassal and und einigen benachbarten gebieten. 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(632) 3436-8720. subscriber details name/institution contact person (for institutional subscribers) mailing address email address telephone no. fax no. please send accomplished subscription form to the rdo-ovcrd via email or fax(please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. hd inside back cover-july-dec2018.pmd 1 s e l e c t e d f o r c o v e r a g e i n t h e emerging sources citation index of c l a r i v a t e a n a l y t i c s ( f o r m e r l y t h o m s o n r e u t e r s i n t e l l e c t u a l property and science business). science diliman: a philippine journal of pure and applied sciences listed in asean citation index, www.asean-cites.org/index.php?r= journal%2fpublic-view&id=620 awardee of the journal challenge grant through the journal incubation program of the commission on higher education (ched). 42 mineralization, biodegradation, and antagonistic activities of gut-associated bacteria and fungi of african nightcrawler, eudrilus eugeniae (kinberg, 1867) maria reynalen f. mapile* marie christine m. obusan institute of biology college of science university of the philippines diliman abstract earthworms and their interactions with microorganisms offer beneficial effects that can improve organic matter decomposition, enhance nutrient availability, and suppress pathogens in the soil. in this study, microorganisms from the gut of eudrilus eugeniae (kinberg, 1867), commonly known as african nightcrawler or anc, were isolated through pour plate method and screened for their activities using assays to confirm nitrogen fixation, phosphate solubilization, polyethylene utilization, and antagonistic potential. the identifications of eight bacterial and six fungal isolates were confirmed based on nearest phylogenetic affiliations. fungal isolates aspergillus aculeatus, aspergillus japonicus, fomitopsis sp., and penicillium citrinum exhibited antagonistic activity against bacillus subtilis, escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus. bacterial isolates aeromonas caviae and bacillus xiamenensis utilized lowand high-density polyethylene as carbon sources. these isolates were also found to have high phosphate solubilization index (2.55-2.67) with high amount of phosphate solubilized (a. caviae: 0.799; b. xiamenensis: 0.778) at decreasing ph (i.e. ph 7.0 to 4.0). a. caviae and b. xiamenensis also showed nitrogen-fixing activity which is supported by the detection of nifh gene (>300 bp) and high nitrogen content (50 kg/ha no3-n) of vermicasts. the activities of these gut-associated bacteria and fungi must be further explored to optimize the use of anc’s casts and compost for agricultural, medical, and other applications. keywords: antagonistic activity, earthworm, microorganisms, nitrogen fixation, phosphate solubilization, polyethylene utilization * corresponding author science diliman (january-june 2020) 32:1, 42-67 m.r.f. mapile and m.c.m. obusan 43 introduction earthworms are considered “ecosystem engineers” owing to their role in nutrient cycling through vermicomposting (lavelle and martin 1992; chapuis-lardy et al. 1998). vermicomposting facilitates degradation of a wide variety of materials and produces products for agricultural applications. for example, compost products of earthworm species eisenia fetida (savigny, 1826), eudrilus eugeniae (kinberg, 1867), lampito mauritii kinberg, 1867, perionyx ceylanensis michaelsen, 1904, and perionyx excavatus perrier, 1872 were proven to improve the growth and yield of bell pepper, cucumber, marigold, strawberry, tomato, and ornamental plants (atiyeh et al. 2000; azarmi et al. 2008; singh et al. 2008; karmegam and daniel 2009; zhao et al. 2017; rekha et al. 2018). the contribution of microbial interactions of earthworms in nutrient cycling through mineralization and organic matter decomposition has been reported (lavelle and martin 1992; chapuis-lardy et al. 1998; bohlen et al. 2004; aira et al. 2009). the presence of acinetobacter spp., azotobacter spp., bacillus spp., clostridium spp., halobacterium spp., micrococcus lylae, pseudomonas aeruginosa, spirocheata spp., staphylococcus aureus, and streptococcus mutans in the gut and casts of libyodrilus violaceus beddard, 1891 was associated with high rate of organic matter decomposition (idowu et al. 2006). bacteria capable of phosphate solubilization were detected in the gut of allolobophora chlorotica, aporrectodea longa, and e. fetida (maheswari and sudha 2013). acinetobacter baumanni, lactobacillus pantheries, virigibacillius chiquenigi, and several species of bacillus were isolated from epigeic e. fetida that was proven to efficiently degrade and convert paper cups into vermicompost (arumugam et al. 2014). paper, garden, and kitchen wastes were also degraded through the action of e. fetida (wani et al. 2013; amita and joseph 2017). the antagonistic potential of microorganisms associated with earthworms was also studied. the casts of pheretima posthuma were found to harbor actinomycetes with antagonistic activity against human bacterial pathogens including b. subtilis, escherichia coli, p. aeruginosa, and s. aureus (kumar et al. 2012). other studies on antagonistic activity involved testing extracts of earthworms against bacteria. the growth of e. coli, klebsiella pneumoniae, staphylococcus epidermidis, proteus vulgaris, p. mirabilis, p. aeruginosa, and s. aureus was inhibited by l. mauritii and p. excavatus powder (prakash and gunasekaran 2011). the antagonistic activity of l. mauritii extracts against aeromonas hydrophila, b. subtilis, salmonella typhi, s. aureus, and vibrio parahaemolyticus was also confirmed (bhorgin and uma 2014; kathireswari et al. 2014). the extracts of lumbricus rubellus hoffmeister, 1843 mineralization, biodegradation, and antagonistic activities 44 were effective against the human pathogen porphyromonas gingivalis (dharmawati et al. 2019), while wegeneriona sp. extracts were effective against serratia marcescens (dhanam et al. 2016). the extracts of p. excavatus and p. posthuma showed inhibitory activity against fish pathogens including a. hydrophila, enterobacter aerogenes, e. coli, micrococcus luteus, p. aeruginosa, pseudomonas fluorescens, and s. aureus (bansal et al. 2015). diverse bacteria and fungi were found to inhabit the gut of anc (bamidele et al. 2014) and other earthworm species such as aporrectodea caliginosa (savigny, 1826), eisenia andrei bouché, 1972, e. fetida, l. violaceus, lumbricus terrestris linnaeus, 1758, and p. excavatus (toyota and kimura 2000; pižl and nováková 2003; idowu et al. 2006; chowdhury et al. 2007; byzov et al. 2009; owa et al. 2013; bamidele et al. 2014). the gut environment, characterized by different ph levels, moisture content, oxygen concentrations, and nutrient levels, affects the composition and metabolic activities of gut-associated microorganisms (karsten and drake 1995; horn et al. 2003; idowu et al. 2006). in turn, these microorganisms contribute to primary production, microclimate regulation, pollution remediation, and nutrient cycling of earthworms in the soil environment (blouin et al. 2013). anc is an epigeic species that is commonly used in vermicomposting. the species is popular due to its fast growth (40-49 days to reach sexual maturity), voracious feeding, consumption of high volume of wastes, rapid decomposition of organic matter, and tolerance to adverse environmental conditions (viljoen and reinecke 1989; reinecke et al. 1992; dominguez et al. 2001; monebi and ugwumba 2013). in the philippines, anc was first introduced in the 1980s, and is currently being promoted by the department of agriculture for vermicomposting as the species prefers temperature ranging from 25 °c to 30 °c that is common in the tropics (dominguez et al. 2001; blakemore 2015). composts processed by anc are being used as fertilizer, such as for lowland and upland rice (guerrero and guerrero 2014; blakemore 2015) and as feed, such as for nile tilapia (oreochromis niloticus) (guerrero and guerrero 2014). despite the prevalence and high rate of utilization of anc for vermicomposting, there is generally lack of information on the composition, diversity, and activities of microorganisms associated with this species in the philippines. this study aimed to isolate bacteria and fungi from the gut of anc, and screen these microorganisms for nitrogen fixation, phosphate solubilization, polyethylene utilization, and antagonistic potential. the findings of this study may contribute to better understanding of the utilization of anc for vermicomposting in relation to the beneficial activities of their gut-associated bacteria and fungi. m.r.f. mapile and m.c.m. obusan 45 materials and methods collection of earthworm samples earthworms, identified as e. eugeniae (african nightcrawler) following the description of blakemore (2015) (nonillon aspe, personal communication), were collected along with their soil substrate from the vermicompost facility of task force solid waste management (tfswm), university of the philippines diliman (upd), quezon city, philippines. twenty adult earthworms, characterized by the presence of clitellum, were individually handpicked and placed in a container (17.3 cm x 11.8 cm x 3.8 cm) made of polypropylene (owa et al. 2013). soil samples were collected from the uppermost 10-40 cm of the vermicompost bed (horn et al. 2003; idowu et al. 2006). samples were immediately transported to the laboratory for processing. earthworms were stored in a container provided with aeration and moist sterile filter paper. fresh vermicasts were collected from the containers after 12-15 hours (bityutskii and kaidun 2008). earthworms were then starved for 48-72 hours, surface sterilized with 70% ethanol for 30 seconds, washed three times with sterile distilled water, and kept frozen for 3-4 hours at –16 °c (horn et al. 2003; byzov et al. 2009; mudziwapasi et al. 2016). gut contents were obtained through dissection following the protocol of owa et al. (2013). isolation and purification of bacteria and fungi from anc gut bacteria and fungi were isolated from the gut samples through pour plate method (byzov et al. 2009; mudziwapasi et al. 2016). briefly, 0.5 g of gut contents was suspended in 2.5 ml sterile distilled water (1:5 ratio) and vortexed until homogenized. serial dilutions up to 10-7 were performed by adding 1 ml of the homogenized sample into 9 ml sterile distilled water. from the last two dilutions (10-6 and 10-7), 1 ml aliquot was inoculated onto nutrient agar (na) supplemented with nystatin for bacterial isolation and potato dextrose agar (pda) supplemented with chloramphenicol for fungal isolation. na plates were incubated for 18-72 hours at 37 °c and for 7 days in anaerobic condition at room temperature while pda plates were incubated for 5 days at 25-27 °c. for purification, colonies with distinct morphologies were selected and repeatedly sub-cultured (idowu et al. 2006; byzov et al. 2009; owa et al. 2013; bamidele et al. 2014). mineralization, biodegradation, and antagonistic activities 46 extraction of bacterial and fungal dna pure bacterial isolates were subjected to dna extraction using boil lysis method (dashti et al. 2009; barbosa et al. 2016). one ml of overnight culture of bacteria in nutrient broth (nb) were centrifuged and re-suspended in 100 μl sterile distilled water in a 1.5 ml sterile tube, vortexed for 15 seconds, and centrifuged at 13,100 rpm for 5 minutes. the supernatant was discarded and 100 μl sterile distilled water was added followed by centrifugation for 10 minutes. pellets re-suspended in 5 μl sterile distilled water were boiled at 100 °c in a dry bath for 15 minutes and then centrifuged for 2 minutes. supernatant containing the dna was transferred into a new sterile tube. fungal dna extraction was carried out following the protocol of liu et al. (2000). a lump of mycelia grown in pda was inoculated onto a sterile 1.5 ml tube with 500 μl lysis buffer and then left at room temperature for 10 minutes. after adding 150 μl potassium acetate solution, the tube was then vortexed, and centrifuged at 13,200 rpm for 1 minute. supernatant was transferred into a new tube with equal volume of isopropyl alcohol, mixed, and centrifuged for 2 minutes. pellets were washed with 300 μl 70% ethanol, and centrifuged at 10,000 rpm for 1 minute. after air-drying, pellets were dissolved in 50 μl 1x tris-edta. molecular identification of isolated bacteria and fungi polymerase chain reaction (pcr) was performed using the universal primers 27f (5'-agagtttgatcmtggctcag-3') and 1392r (5'-acgggcggtgtgtrc-3') for bacteria (furlong et al. 2002) and its1 (5'-tccgtaggtgaacctgcgg-3') and its4 (5'-tcctccgcttattgatatgc-3') for fungi (martin and rygiewicz 2005). for bacteria, the reaction mixture (25 μl) consisted of 12.5 μl of 2x taq master mix, 1 μl of each primer, 2 μl bacterial dna (control excluded dna), and nuclease-free water. for amplification of 27f and 1392r (>500 bp), the pcr conditions were: initial denaturation for 2 minutes at 94 °c followed by 25 cycles of denaturation for 30 seconds at 94 °c, annealing for 30 seconds at 60 °c, extension for 45 seconds at 72 °c, and final extension for 7 minutes at 72 °c. for fungi, the reaction mixture (25 μl) consisted of 12.5 μl of 2x taq master mix, 1 μl of each primer, 3 μl fungal dna (control excluded dna), and nuclease-free water. for amplification of its1 and its4 (>500 bp), the pcr conditions were: initial denaturation for 5 minutes at 95 °c followed by 35 cycles of denaturation for 30 seconds at 95 °c, annealing for 1 minute at 55 °c, extension for 1 minute at 72 °c, and final extension for 6 minutes at 72 °c. pcr products were electrophoresed on 1.5% agarose gel with gelred in tae buffer for 30 minutes at 80 v using a 100-bp molecular weight dna marker and m.r.f. mapile and m.c.m. obusan 47 then submitted to macrogen (korea) for purification and sequencing. the basic local alignment search tool (blast) was used for sequence identification. sequences were aligned and trimmed using bioedit prior to construction of bayesian inference (bi) tree using mrbayes version 3.1.2 and treeview version 1.6.6. in vitro screening of anc gut-associated fungi for antagonistic activity the antagonistic activity of isolated fungi against b. subtilis, e. coli, p. aeruginosa, and s. aureus was evaluated using the antagonism test following the protocol of suárez-estrella et al. (2007). briefly, a block from a 5d-old culture of fungi in pda was placed at the center of mueller-hinton agar (mha) plate. then, 100 μl of test organisms cultured in nutrient broth were spot inoculated approximately 2.5 cm from the block. plates were incubated for at least 48 hours at 30-37 °c and checked for inhibition indicated by the absence of any contact between fungal isolates and test organisms. in vitro screening of anc gut-associated bacteria for polyethylene utilization screening for polyethylene utilization was done by inoculating 100 μl of 18-24 h-old bacterial cultures from nutrient broth into 10 ml sterile bushnell haas (bh) broth supplemented with 0.3% low-density polyethylene (ldpe) and high-density polyethylene (hdpe) powder. all broth tubes were incubated for 7 days in a shaker at 37 °c with 180 rpm agitation and observed daily for turbidity to confirm the growth of bacteria that were able to utilize polyethylene as carbon source for bacterial growth. in vitro screening of anc gut-associated bacteria for nutrient mineralization activity ten μl of 18-24 h-old bacterial cultures from nutrient broth were spot inoculated onto nitrogen-free malate media supplemented with bromothymol blue (btb), pikovskaya’s agar (himedia m520), and aleksandrow agar (himedia m1996), and incubated for 5 days at 37 °c. cultures were observed every 24 hours for nitrogen fixation activity indicated by a blue coloration zone (gothwal et al. 2008). phosphorus and potassium solubilizations were indicated by a clearing zone. phosphorus solubilization index (si) was calculated based on colony and zone diameters (shanware et al. 2014; sharon et al. 2016). mineralization, biodegradation, and antagonistic activities 48 isolates positive for nitrogen fixation were subsequently subjected to molecular detection of nifh gene (>300 bp) (szymanska et al. 2016a). pcr amplification was carried out using the nifh gene primers 19f (5'-gcxwtytayggxaarggxgg-3') and 388r (5'-aaxccrccrcaxacxacrtc-3'). the reaction mixture (23.5 μl) consisted of 10 μl of 2x taq master mix, 0.125 μl of each primer, 13 μl of nuclease-free water, and 0.375 μl bacterial dna (control excluded dna). the pcr conditions were: initial denaturation for 5 minutes at 94 °c followed by 40 cycles of denaturation for 30 seconds at 94 °c, annealing for 1 minute at 50 °c, extension for 1 minute at 72 °c, and final extension for 5 minutes at 72 °c (szymanska et al. 2016b). pcr products were electrophoresed on 1.5% agarose gel with gelred in tae buffer for 30 minutes at 80 v using a 100-bp molecular weight dna marker. isolates positive for phosphate solubilization were further subjected to murphy and riley (1962) method for phosphate quantification. one μl of fresh bacterial culture in nb was inoculated into 50 ml of nbrip (national botanical research institute’s phosphate) medium at ph 7.0 supplemented with calcium phosphate (ca 3 (po 4 ) 2 ) as sole source of phosphorus while medium without inoculum served as the control. all flasks were incubated for 72 hours at 24 ºc under constant agitation at 120 rpm (matos et al. 2017). after centrifugation and filtration, the ph of the filtrate was measured using ph paper while phosphate content based on absorbance values was measured using spectrophotometer at 880 nm (watanabe and olsen 1965). measurement of macronutrients in soil and vermicasts twenty grams each of soil and vermicast samples were air-dried overnight and sieved prior to analysis (zhang and schrader 1993; aira et al. 2003; hmar and ramanujam 2014). the nitrogen (n), phosphorus (p), and potassium (k) content of these samples were measured using npk soil test kit (himedia k054m) following manufacturer’s instructions. results and discussion anc gut-associated bacteria and fungi a total of eight bacteria and six fungi were isolated from the gut of anc (table 1). the sequences of bacterial isolates showed highest similarities to aeromonas caviae (99.37%), bacillus xiamenensis (99.86%), bacillus thuringiensis (98.54% 99.86%), and paenibacillus xylanilyticus (98.24%) (figure 1). the sequences of fungal isolates showed highest similarities to aspergillus aculeatus (99.12% 99.82%), aspergillus japonicus (99.17%), fomitopsis sp. (99.23% 99.83%), and penicillium citrinum (99.02%) (figure 2). m.r.f. mapile and m.c.m. obusan 49 table 1. bacteria and fungi isolated from the gut of african nightcrawler or anc, eudrilus eugeniae (kinberg, 1867) nearest phylogenetic affiliation accession number nearest phylogenetic affiliation accession number 1 aeromonas caviae mg737573 1 aspergillus aculeatus jx291165 2 bacillus xiamenensis nr_148244 2 aspergillus aculeatus mh892845 3 bacillus thuringiensis jx994097 3 aspergillus japonicus kc128815 4 bacillus thuringiensis mn108016 4 fomitopsis sp. jq067652 5 bacillus thuringiensis mg722793 5 fomitopsis sp. fj372677 6 paenibacillus xylanilyticus kj023382 6 penicillium citrinum mh427065 7 paenibacillus xylanilyticus jx281766 8 paenibacillus xylanilyticus hf585011 m.r.f. mapile and m.c.m. obusan 49 table 1. bacteria and fungi isolated from the gut of african nightcrawler or anc, eudrilus eugeniae (kinberg, 1867) nearest phylogenetic affiliation accession number nearest phylogenetic affiliation accession number 1 aeromonas caviae mg737573 1 aspergillus aculeatus jx291165 2 bacillus xiamenensis nr_148244 2 aspergillus aculeatus mh892845 3 bacillus thuringiensis jx994097 3 aspergillus japonicus kc128815 4 bacillus thuringiensis mn108016 4 fomitopsis sp. jq067652 5 bacillus thuringiensis mg722793 5 fomitopsis sp. fj372677 6 paenibacillus xylanilyticus kj023382 6 penicillium citrinum mh427065 7 paenibacillus xylanilyticus jx281766 8 paenibacillus xylanilyticus hf585011 bacillus thuringiensis bacillus thuringiensis bacillus thuringiensis bacillus subtilis (ay030331) paenibacillus xylanilyticus paenibacillus xylanilyticus paenibacillus xylanilyticus paenibacillus lentimorbus (af071861) paenibacillus sp. (ab041720) aeromonas caviae aeromonas media (x60410) aeromonas hydrophila (x60404) aeromonas eucrenophila (x60411) aeromonas sobria (x60412) aeromonas veronii (x60414) flavobacterium johnsoniae (fn298315) bacillus xiamenensis bacillus megaterium (ay030338) 1.00 1.00 0.97 0.96 0.81 1.00 0.99 0.80 figure 1. bayesian inference tree of earthworm gut-associated bacteria based on 484 nucleotides. the tree is rooted on the bacteroidetes f. johnsoniae. the number of generations and heating temperature used were 10,000,000 and 0.1, respectively. numbers on nodes represent posterior probability values. values less than 0.7 are not shown. mineralization, biodegradation, and antagonistic activities 50 b. xiamenensis was also isolated from the gut of anc in india (utekar and deshmukh 2019). other species isolated from the gut of anc in india include bacillus pumilus (shankar et al. 2011), b. aerius, b. licheniformis, b. safensis, b. subtilis, b. tropicus (utekar and deshmukh 2019), b. cereus, and b. subtilis (emperor and kumar 2015; govindarajan and prabaharan 2015a, 2015b). published studies on the isolation of a. caviae from earthworm gut are limited, but its occurrence in seafood, aquafarms, and mangroves (joseph et al. 2013) as well as association with diarrhea/ gastroenteritis (dwivedi et al. 2008) were reported. the gut of anc was also found to be inhabited by aspergillus flavus, a. fumigatus, a. nidulans, a. niger, and a. ochraceous (parthasarathi et al. 2007; bamidele et al. 2014; emperor and kumar 2015) as well mineralization, biodegradation, and antagonistic activities 50 aspergillus sp. (gu595031) aspergillus indologenus (mh861245) aspergillus uvarum (nr_135330) penicillium sp. (ab728537) penicillium citrinum penicillium janthinellum (kx138426) fomitopsis meliae (kt718002) fomitopsis sp. (kt194148) mortierella sp. (kt964847) fomitopsis sp. fomitopsis sp. aspergillus aculeatus aspergillus aculeatus aspergillus japonicus 0.83 0.97 1.00 1.00 1.00 1.00 0.94 1.00 0.78 figure 2. bayesian inference tree of earthworm gut-associated fungi based on 587 nucleotides. the tree is rooted on the mucoromycota mortierella sp. the number of generations and heating temperature used were 10,000,000 and 0.125, respectively. numbers on nodes represent posterior probability values. values less than 0.7 are not shown. b. xiamenensis was also isolated from the gut of anc in india (utekar and deshmukh 2019). other species isolated from the gut of anc in india include bacillus pumilus (shankar et al. 2011), b. aerius, b. licheniformis, b. safensis, b. subtilis, b. tropicus (utekar and deshmukh 2019), b. cereus, and b. subtilis (emperor and kumar 2015; govindarajan and prabaharan 2015a, 2015b). published studies on the isolation of a. caviae from earthworm gut are limited, but its occurrence in seafood, aquafarms, and mangroves (joseph et al. 2013) as well as association with diarrhea/ gastroenteritis (dwivedi et al. 2008) were reported. the gut of anc was also found to be inhabited by aspergillus flavus, a. fumigatus, a. nidulans, a. niger, and a. ochraceous (parthasarathi et al. 2007; bamidele et al. 2014; emperor and kumar 2015) as well m.r.f. mapile and m.c.m. obusan 51 as penicillium sp. (bamidele et al. 2014; sahoo et al. 2015) while its vermicompost was found to have p. citrinum (emperor and kumar 2015). antagonistic activity of anc gut-associated fungi the antagonistic activity of gut-associated fungi against gram-positive b. subtilis and s. aureus and gram-negative e. coli and p. aeruginosa was confirmed (table 2). all fungal isolates showed activity against b. subtilis, e. coli, and p. aeruginosa. growth of s. aureus was also inhibited by the fungal isolates except a. aculeatus. there is lack of information on the antagonistic activity of fungi associated with the gut of anc, with most of the studies reporting the activity of its paste. anc paste was reported to inhibit the growth of b. subtilis, e. coli, k. pneumoniae, and s. aureus (vasanthi et al. 2013; chauhan et al. 2014; sethulakshmi et al. 2018). table 2. antagonistic activity of fungi isolated from the gut of african nightcrawler or anc, eudrilus eugeniae (kinberg, 1867) test organisms a. aculeatus a. aculeatus a. japonicus fomitopsis sp. fomitopsis sp. p. citrinum bacillus subtilis + + + + + + escherichia coli + + + + + + pseudomonas aeruginosa + + + + + + staphylococcus aureus + + + + (+) with antagonistic activity, (-) without antagonistic activity the antagonistic activity of aspergillus, fomitopsis, and penicillium isolated from organisms other than earthworms was also reported. a. aculeatus isolated from avicennia marina (black mangrove along red sea) and a. japonicus isolated from tridax procumbens (coat button or tridax daisy) inhibited the growth of b. subtilis, e. coli, k. pneumoniae, p. vulgaris, salmonella typhimurium, s. aureus, and streptococcus pyogenes (aharwal et al. 2018; basheer et al. 2018). the activity of a. aculeatus against gram-positive and -negative bacteria was associated with its secondary metabolites namely ergosterol, ergosterol peroxide, secalonic acid d and f, variecolactone, and variecolin (yodsing et al. 2017). fomitopsis feei, f. lilacinogilva, and f. rosea collected from india, australia, and philippines respectively, were tested to be effective against the above mentioned bacteria as well as e. aerogenes, m. luteus, and p. mirabilis (bala et al. 2011; nidadavolu et al. 2011; gaylan et al. mineralization, biodegradation, and antagonistic activities 52 2018). p. citrinum isolated from marine and soil samples inhibited the growth of b. subtilis, e. coli, s. typhi, and s. aureus (christophersen et al. 1998; gharaei-fathabad et al. 2014). the inhibition was attributed to the production of mycotoxin citrinin, which was also found to be effective against b. cereus, b. pumilus, b. subtilis, e. coli, k. pneumoniae, lactobacillus arabinosus, p. mirabilis, s. typhi, s. typhimurium, shigella boydii, s. dysenteriae, s. sonnei, s. aureus, streptococcus pneumoniae, and vibrio cholerae (mazumder et al. 2002). polyethylene utilization of anc gut-associated bacteria a. caviae and b. xiamenensis utilized both low-density polyethylene (ldpe) and highdensity polyethylene (hdpe) after 120 hours of incubation (figure 3). members of bacillus (b. mycoides and b. subtilis) isolated from mangrove soil were reported to degrade ldpe and hdpe (ibiene et al. 2013) while b. megaterium isolated from plastic dumpsite soil was reported to degrade polyethylene in general (mahalakshmi and siddiq 2015). other types of polymers such as polyethylene terephthalate (pet), polypropylene (pp), and polystyrene (ps) were degraded by bacillus species from mangrove sediments and soil samples (asmita et al. 2015; auta et al. 2018). reduction of polymer mass by 4% confirmed the utilization of pp by bacillus sp. for growth after 40 days of incubation (auta et al. 2018). bioremediation of soil polluted with diesel was also associated with anc action, along with the reduction of the concentrations of arsenic, cadmium, chromium, copper, lead, mercury, nickel, and vanadium (ekperusi and aigbodion 2015). another earthworm species, l. terrestris, reduced 60% of ldpe particle size within four weeks through the action of bacteria (firmicutes and actinobacteria) associated with its gut (lwanga et al. 2018). mineralization, biodegradation, and antagonistic activities 52 2018). p. citrinum isolated from marine and soil samples inhibited the growth of b. subtilis, e. coli, s. typhi, and s. aureus (christophersen et al. 1998; gharaei-fathabad et al. 2014). the inhibition was attributed to the production of mycotoxin citrinin, which was also found to be effective against b. cereus, b. pumilus, b. subtilis, e. coli, k. pneumoniae, lactobacillus arabinosus, p. mirabilis, s. typhi, s. typhimurium, shigella boydii, s. dysenteriae, s. sonnei, s. aureus, streptococcus pneumoniae, and vibrio cholerae (mazumder et al. 2002). polyethylene utilization of anc gut-associated bacteria a. caviae and b. xiamenensis utilized both low-density polyethylene (ldpe) and highdensity polyethylene (hdpe) after 120 hours of incubation (figure 3). members of bacillus (b. mycoides and b. subtilis) isolated from mangrove soil were reported to degrade ldpe and hdpe (ibiene et al. 2013) while b. megaterium isolated from plastic dumpsite soil was reported to degrade polyethylene in general (mahalakshmi and siddiq 2015). other types of polymers such as polyethylene terephthalate (pet), polypropylene (pp), and polystyrene (ps) were degraded by bacillus species from mangrove sediments and soil samples (asmita et al. 2015; auta et al. 2018). reduction of polymer mass by 4% confirmed the utilization of pp by bacillus sp. for growth after 40 days of incubation (auta et al. 2018). bioremediation of soil polluted with diesel was also associated with anc action, along with the reduction of the concentrations of arsenic, cadmium, chromium, copper, lead, mercury, nickel, and vanadium (ekperusi and aigbodion 2015). another earthworm species, l. terrestris, reduced 60% of ldpe particle size within four weeks through the action of bacteria (firmicutes and actinobacteria) associated with its gut (lwanga et al. 2018). a b c d ldpe hdpe figure 3. low-density polyethylene (ldpe) and high-density polyethylene (hdpe) utilization by two bacteria, aeromonas caviae (a, c) and bacillus xiamenensis (b, d), shown by turbidity (left tube) compared with the negative control (right tube). nutrient mineralization activities of anc gut-associated bacteria a. caviae and b. xiamenensis were found to solubilize phosphate with high si values of 2.67 and 2.55, respectively. after 22 hours of incubation, clearing zones were first nutrient mineralization activities of anc gut-associated bacteria a. caviae and b. xiamenensis were found to solubilize phosphate with high si values of 2.67 and 2.55, respectively. after 22 hours of incubation, clearing zones were first m.r.f. mapile and m.c.m. obusan 53 observed on pikovskaya’s agar medium, with maximum activity detected at 96 hours (figure 4). the amounts of phosphate solubilized by a. caviae and b. xiamenensis were higher than the control (figure 5) with observed decrease in ph (from 7.0 to 4.0). the two phosphate solubilizing isolates were also able to fix nitrogen on nitrogen-free malate medium as indicated by blue colored zones first observed after 96 hours of incubation, with maximum activity at 120 hours (figure 4). the target nifh gene was detected in these isolates (figure 6). m.r.f. mapile and m.c.m. obusan 53 observed on pikovskaya’s agar medium, with maximum activity detected at 96 hours (figure 4). the amounts of phosphate solubilized by a. caviae and b. xiamenensis were higher than the control (figure 5) with observed decrease in ph (from 7.0 to 4.0). the two phosphate solubilizing isolates were also able to fix nitrogen on nitrogen-free malate medium as indicated by blue colored zones first observed after 96 hours of incubation, with maximum activity at 120 hours (figure 4). the target nifh gene was detected in these isolates (figure 6). figure 4. diameter of clearing zones and blue colored zones produced by bacteria isolated from the gut of anc (n=3). colony diameter = diameter of bacterial growth; zone diameter = diameter of clearing/blue colored zone. figure 5. amount of phosphate solubilized by two bacteria, aeromonas caviae and bacillus xiamenensis. mineralization, biodegradation, and antagonistic activities 54 the presence of phosphate solubilizing bacteria (psb) and nitrogen-fixing bacteria (nfb) in the gut of anc was previously reported in india (albasha et al. 2014; sequeira and chandrashekar 2015; khobragade and more 2016). the mechanisms involved in phosphate solubilization include ion-exchange, chelation, acidification, and organic acid production (chen et al. 2006). in this study, the confirmed mineralization activities exhibited by the bacterial isolates might be associated with the high p and n concentrations in vermicasts (lee 1992; zhang et al. 2000; shamini and fauziah 2014; prabha et al. 2015). bacteria capable of phosphate solubilization and nitrogen fixation were also isolated from the gut of other earthworm species. epigeic e. fetida was found to be inhabited by gut-associated psb and nfb (hussain et al. 2016). the nitrogenase activity of earthworms in the gut of anecic l. terrestris as well as endogeic aporrectodea rosea and a. caliginosa (umarov et al. 2008) was evaluated. as it is in the present study, the maximum mineralization activity of bacteria associated with endogeic metaphire posthuma was observed at 96 hours of incubation, the period at which bacteria might have reached exponential phase (biswas et al. 2018). likewise, aeromonas salmonicida and a. caviae were reported elsewhere to show phosphate solubilization activity on pikovskaya’s medium after 5 days of incubation (chen et al. 2012). aeromonas vaga showed solubilization efficiency when subjected to varying temperatures (15, 25, 35, and 45 °c) and 8% sodium chloride (nacl) at mineralization, biodegradation, and antagonistic activities 54 500 bp 400 300 200 100 ladder a. caviae b. xiamenensis (-) control figure 6. molecular detection of nifh gene in two bacterial isolates, aeromonas caviae and bacillus xiamenensis. the presence of phosphate solubilizing bacteria (psb) and nitrogen-fixing bacteria (nfb) in the gut of anc was previously reported in india (albasha et al. 2014; sequeira and chandrashekar 2015; khobragade and more 2016). the mechanisms involved in phosphate solubilization include ion-exchange, chelation, acidification, and organic acid production (chen et al. 2006). in this study, the confirmed mineralization activities exhibited by the bacterial isolates might be associated with the high p and n concentrations in vermicasts (lee 1992; zhang et al. 2000; shamini and fauziah 2014; prabha et al. 2015). bacteria capable of phosphate solubilization and nitrogen fixation were also isolated from the gut of other earthworm species. epigeic e. fetida was found to be inhabited by gut-associated psb and nfb (hussain et al. 2016). the nitrogenase activity of earthworms in the gut of anecic l. terrestris as well as endogeic aporrectodea rosea and a. caliginosa (umarov et al. 2008) was evaluated. as it is in the present study, the maximum mineralization activity of bacteria associated with endogeic metaphire posthuma was observed at 96 hours of incubation, the period at which bacteria might have reached exponential phase (biswas et al. 2018). likewise, aeromonas salmonicida and a. caviae were reported elsewhere to show phosphate solubilization activity on pikovskaya’s medium after 5 days of incubation (chen et al. 2012). aeromonas vaga showed solubilization efficiency when subjected to varying temperatures (15, 25, 35, and 45 °c) and 8% sodium chloride (nacl) at m.r.f. mapile and m.c.m. obusan 55 ph 10 (jha et al. 2013). aeromonas allosaccharophila, a. hydrophila, and a. media isolated from rhizospheric soil were also confirmed to be psb (aarab et al. 2015). the si values for the phosphate solubilization activity of a. caviae and b. xiamenensis ranged from 2.55 to 2.67, which is comparatively higher than the reportedly high si of 2.0 for aeromonas sp. isolated from rhizospheric soils of cabbage fields in iran (motamedi et al. 2016). species of bacillus such as b. cereus, b. megaterium, b. simplex, and b. subtilis are also known phosphate solubilizers (bahadir et al. 2018; saeid et al. 2018; zheng et al. 2018). bacillus sp. and b. pumilus isolated from banana tree roots (matos et al. 2017) as well as b. subtilis and b. tequilensis isolated from lentil rhizosphere in ethiopia (midekssa et al. 2015) solubilized calcium phosphate. bacillus spp. capable of phosphate solubilization were isolated from anc gut and vermicasts (albasha et al. 2014). as what was observed in the present study, there was a decrease in ph with increased amount of p solubilized by rhizospheric aeromonas (kundu et al. 2009) and bacillus species (matos et al. 2017; mohamed et al. 2018). the decrease in ph was asserted to be directly proportional to increased p solubilization due to acidification from secretion of organic acids (mohamed et al. 2018). nitrogen-fixing a. hydrophila and aeromonas sp. were isolated from rice fields (xie et al. 2003) and from the rhizosphere of cabbage (motamedi et al. 2016), respectively. the genus bacillus is known to have nitrogen-fixing species namely b. azotoformans, b. brevis, b. cereus, b. licheniformis, b. megaterium, b. pumilus, and b. subtilis (xie et al. 2003). b. subtilis isolated from the rhizosphere of ground nut exhibited nitrogenfixing activity (satapute et al. 2012). the detection of nifh in aeromonas sp. (flores mireles et al. 2007) and in bacillus alkalidiazotrophicus, b. arseniciselenatis (sorokin et al. 2008), and b. cereus (emmyrafedziawati and stella 2018) was done to support the findings on their nitrogen fixation activity. the gene has been the biomarker of choice for nfb as it encodes for the nitrogenase reductase subunit of nitrogenase enzyme involved in nitrogen fixation (emmyrafedziawati and stella 2018). the amount of p was higher in vermicasts (56-73 kg/ha) than in the soil substrate (22-56 kg/ha). this is consistent with previous reports noting high p content of vermicomposts processed by anc (shamini and fauziah 2014; prabha et al. 2015) and in vermicasts of allolobophora caliginosa (sharpley and syers 1976), l. terrestris (le bayon and binet 2006), metaphire tschiliensis tschiliensis (teng et al. 2012), drawida sp., eutyphoeus mizoramensis, metaphire houlleti, p. excavatus, and p. macintoshi (hmar and ramanujam 2014). the release of p in vermicasts is attributed to solubilization of microorganisms during gut passage (lee 1992; zhang et al. 2000). mineralization, biodegradation, and antagonistic activities 56 likewise, nitrogen content, measured as nitrate nitrogen (no 3 -n) in vermicasts, was found to be more than twice (50 kg/ha) that of the soil substrate (20 kg/ha). several studies confirmed the same observation in the vermicasts of allolobophora molleri, a. caliginosa (aira et al. 2003), l. violaceus (idowu et al. 2006), l. terrestris, octolasion cyaneum (buck et al. 1999), and m. tschiliensis tschiliensis (teng et al. 2012) and in vermicomposts processed by anc (prabha et al. 2015), e. fetida, and p. excavatus (mistry et al. 2015). higher nitrogen content in vermicasts was associated with the activity of microorganisms that promote mineralization process (mistry et al. 2015). the degradation of organic wastes through vermicomposting is essential for nutrient cycling (yi-wei et al. 2012). wastes reported to be efficiently degraded by anc through vermicomposting include rice straw (yi-wei et al. 2012), coir pith (nattudurai et al. 2014), and biogas plant slurry (bps) (rajeshkumar and ravichandran 2015). degradation of rice straw was completed by anc in a shorter time (134 days) compared to p. excavatus (171 days), resulting to higher nutrient content in vermicasts (yi-wei et al. 2012). moreover, it only takes 60 days for anc to degrade coir pith, which usually takes longer time to degrade due to its lignincellulose complex (nattudurai et al. 2014). degradation of bps was found to be enhanced by anc as indicated by the decrease of total organic carbon (rajeshkumar and ravichandran 2015). composts processed by anc caused increase in plant height and weight, as well as increase in the length of shoots, roots, leaves, and root hairs of agricultural crops cyamopsis tetragonoloba (cluster bean) (nattudurai et al. 2014) and vigna radiata (mung bean) (rajeshkumar and ravichandran 2015). improved growth and yield of plants treated with vermicomposts were attributed to increased concentrations of npk (nattudurai et al. 2014; rajeshkumar and ravichandran 2015). conclusions eight bacteria and six fungi were isolated from the gut of anc. the fungi identified as a. aculeatus, a. japonicus, fomitopsis sp., and p. citrinum exhibited antagonistic activity against b. subtilis, e. coli, p. aeruginosa, and s. aureus. among gut-associated bacteria identified as a. caviae, b. xiamenensis, b. thuringiensis, and p. xylanilyticus, the first two were found to utilize ldpe and hdpe as carbon sources for bacterial growth, indicating plastic biodegradation potential. both isolates yielded high phosphate solubilization index and showed nitrogen fixation activity supported by the presence of nifh gene. high concentrations of nitrogen and phosphorus in the vermicasts of anc may be associated with the confirmed mineralization activities. m.r.f. mapile and m.c.m. obusan 57 acknowledgments this work, conducted at the microbial ecology of terrestrial and aquatic systems (metas) laboratory of the institute of biology, university of the philippines diliman, was supported by the commission on higher education (ched) through the k to 12 dare to research grant. conflicts of interest mrfm and mcmo declare that they have no conflicts of interest. contributions of individual authors mrfm and mcmo conceived the study, designed the experiment, and analyzed data. mrfm performed most of the procedures. mcmo wrote the proposal and received the funding for the research. both authors contributed to manuscript writing. references aarab s, ollero fj, megías m, laglaoui a, bakkali m, arakrak a. 2015. isolation and screening of bacteria from rhizospheric soils of rice fields in northwestern morocco for different plant growth promotion (pgp) activities: an in vitro study. int j curr microbiol appl sci. 4(1):260 269. aharwal rp, kumar s, thakur y, deshmukh l, sandhu ss. 2018. evaluation of antibacterial activity of endophytic 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a greenhouse. j soils sediments. 17(12):2718-2730. zheng bx, ibrahim m, zhang dp, bi qf, li hz, zhou gw, ding k, peñuelas j, zhu yg, yang xr. 2018. identification and characterization of inorganic-phosphate-solubilizing bacteria from agricultural fields with a rapid isolation method. amb express. 8:1-12. ______ maria reynalen f. mapile <mariareynalenfrigillanamapile@gmail.com> is a ph.d. biology student at the institute of biology, university of the philippines diliman and is currently affiliated with the microbial ecology of terrestrial and aquatic systems (metas) laboratory. she earned her bachelor of science in biology degree at the university of the philippines baguio and her master of science in biology degree at the saint louis university. her current research interests include gut microbial diversity of invertebrates and their beneficial interactions and contributions in soil nutrient cycling. marie christine m. obusan is an assistant professor and head of the aquatic division of the microbial ecology of terrestrial and aquatic systems (metas) laboratory, institute of biology, university of the philippines diliman. she received her ph.d. in environmental science from the university of the philippines diliman. she specializes in microbial diversity and interactions in sentinel species and habitats. 6-guerrero-foliar fungal.pmd j.j.g. guerrero et al. 37 science diliman (january-june 2019) 31:1, 37-53 fol iar fungal endophytes of selected medicinal plants from the province of albay, phil ippines jonathan jaime g. guerrero* mheljor a. general jazzlyn t. imperial college of science bicol university abstract f u n g a l e n d o p h y t e s w e r e i s o l a t e d f r o m t h e l e a v e s o f t h e 1 0 m o s t frequently used medicinal plants in the province of albay, philippines at three different locations: upland, lowland, and coastal areas. their occurrence, frequency, and isolation rates were compared. a total of 120 i s o l a te s b e l o n g i n g to 1 7 s p ec i e s we r e i d e n t i f i ed. g l o m e r e l l a c i n g u l a t a (stoneman) spauld. & h. schrenk and colletotrichum gloeosporioides m.b. dickman were the most frequent fungi occurring in 10 and nine plants, respectively. no signif icant difference in the total number of isolates, a s w e l l a s t h e t o t a l n u m b e r o f u n i q u e s p e c i e s f r o m a m o n g s a m p l i n g s i t e s , w a s d e t e c t e d . b l u m e a b a l s a m i f e r a ( l . ) d c h a r b o r e d t h e m o s t endophytes with 16 isolates, while banana leaves yielded the least with eight isolates. there were species of fungi that cut across all sampling sites, while a few occurred only in one site. the collection of additional s a m p l e s f r o m o t h e r s i t e s w i t h i n t h e p r o v i n c e a n d t h e t e s t i n g o f t h e biological properties of the isolates are recommended. keyword s: albay, endophytes, glomerella cingulata, medicinal plants, upland issn 0115-7809 print / issn 2012-0818 online introduction traditionally, fungal endophytes are species of fungi residing within their hosts without causing apparent harm, emerging only during host-tissue senescence (rodriguez et al. 2009) or when some physiological changes happen in the host tissue. endophytism has long been regarded to be a form of mutualism. however, saikkonen et al. (1998) strongly suggested that this forms a continuum of _______________ *corresponding author foliar fungal endophytes of selected medicinal plants 38 interaction, and that endophytism simply refers to where the fungus resides and not exactly how it interacts with its plant host. all plants, therefore, would host at least one fungal endophyte throughout its life cycle. the continuous documentation of fungal endophytes of plants emerges from numerous objective endpoints. it can be towards the practical application of their natural products because of the superiority of natural selection over combinatorial chemistry in novel substance discovery (schulz et al. 2002). moreover, an ecological standpoint examines the changing roles the fungi play in relation to its plant host (carroll 1988; saikkonen et al. 1998; rodriguez et al. 2009). diversity, taxonomy, and phylogeny also are important drivers for endophyte research because of their ubiquity and geographical distribution (arnold and lutzoni 2007). more importantly, endophytes are signif icant in estimating overall fungal diversity (arnold et al. 2 0 0 0 ) . regardless of the objective, it remains clear that endophyte documentation is a common denominator of nearly every, if not all, fungal endophyte research. plant grouping is the basis for many processes, such as bioprospecting, ecological inferences, and diversity analysis. these groupings could be formal taxonomic grouping, or an informal grouping based on phenotypic, functional, or geographical similarities. the absence of species identif ication, or at least characterization, may be an obstacle and can substantially limit future prospects. medicinal plants form an informal but relatively cohesive group. the extent of the members of this group is unclear as their medicinal uses vary from place to place. in the philippines, plants with medicinal value were already documented as early as the 1950s. comprehensive ethnobotanical surveys were also carried out in different parts of the country (tantiado 2012; abe and ohtani 2013). several research have elucidated various properties of medicinal plants, such as antibacterial (penecilla and magno 2011), anti-hyperglycemic (villasenor and lamadrid 2006) and antioxidant properties (peteros and uy 2010), among others. the diversity of plants and their medicinal properties may mirror the diversity of fungal endophytes residing within them, and may even extend to the potential properties of the fungi themselves. huang et al. (2008) for instance, have shown that certain fungal taxa were more likely to coexist with plants producing certain phenolic compounds. radu and kqueen (2002) posed the question of whether medicinal properties are produced by plants themselves or are consequences of j.j.g. guerrero et al. 39 the fungal-plant association. from a biological standpoint, this may be hard to answer because growing a plant without any endophyte may be challenging. however, the fungi, even without the plant, may be harnessed to produce the same benef its as that of its host. therefore, this research is an initial species assessment of the fungal endophytes of ten medicinal plants located in the province of albay, philippines based on a previous medicinal plant survey by mirandilla and abalon (2013). the fungal e n d o p h y t e s o f t h e m e d i c i n a l p l a n t s u s e d i n t h i s r e s e a r c h h a v e m i n i m a l documentation. for instance, cassava (manihot esculenta crantz.) and coconut (cocos nucifera l.) only record bacterial endophytes (melo et al. 2009; rajendran et al. 2015), while research on the fungal endophyte of mango was limited only to colletotrichum spp. (vieira et al. 2015). likewise, literature comparing the occurrence of foliar fungal endophytes in upland, lowland, and coastal areas are limited. the ten medicinal plants included in this study are the ten most used in the province, and thus, their occurrence in the sampling sites are guaranteed. materials and methods med icinal plants ten medicinal plants were collected in this study based on the previous assessment of their diversity and use in the province of albay, philippines (mirandilla and abalon 2013). the 10 medicinal plants shown in table 1 are the most commonly used in the province of albay. common name scientific name code used in this study papaya carica papaya l. cp cassava manihot esculenta crantz. m e mango mangifera indica l. mi jackfruit artocarpus heterophyllus lam. ah gabi colocasia esculenta (l.) schott. ce coconut cocos nucifera l. cn malunggay moringa oleifera lam. m o oregano origanum vulgare l. ov banana musa paradisiaca l. mp sambong blumea balsamifera (l.) dc bb table 1. med icinal plants in albay, phil ippines used in this study foliar fungal endophytes of selected medicinal plants 40 sampl ing sites sampling was performed from august to september 2016. four sampling areas were selected within the province of albay to represent sites with adjacent upland, lowland, and coastal areas, namely the cities of ligao, legazpi, tabaco, and the municipality of pio duran. sites were qualitatively classif ied as upland, lowland, and coastal based on the characteristics of the area and on elevation. upland areas refer to sites with hilly or inclined terrains, lowland areas refer to those close to residences and human settlements, and coastal areas refer to those proximate to the sea. one individual of each medicinal plant from each area was chosen. leaf samples were collected from each of the medicinal plants and were taken immediately to the laboratory for plating. for those with big leaves, such as banana and coconut, a random portion of the entire compound leaf was cut out and placed in a sterile plastic bag. isolation of fungal endophytes ten mature leaves without apparent symptoms of disease, such as necrosis, chlorosis, and presence of external wounds and deformities, were selected from the fresh samples. the leaves were washed thoroughly with running water to remove any debris on the surface. after blot drying, a 0.64-cm diameter puncher was used to create one circular leaf disc per leaf at the mid-section of the blade not including the midrib. each disc was sterilized following the methods of torres and dela cruz (2015) with modif ications. leaf discs were soaked in 95% ethanol for 1 minute, then transferred to a 0.5% naoh (zonrox®) solution for 2 minutes, and washed twice in sterile distilled water. leaf discs were then blot dried prior to plating. leaf discs were plated on potato dextrose agar (pda, himedia). a leaf print, done by touching the leaf disc on the pda, was made to ensure only endophytes were isolated. plates with growth on the leaf print were discarded and not used in the study. identification of fungal endophytes identif ication of isolates were f irst based on morpho-cultural characteristics and compared to existing taxonomic keys, specif ically that of watanabe (2010). isolates similar in cultural and morphological characteristics were grouped and a reference isolate was duplicated in tubes. isolates or groups that exhibited differences in j.j.g. guerrero et al. 41 morpho-cultural characteristics were separated into subgroups, and one reference isolate per subgroup was also sent for the sequencing of the its gene. extraction of the genomic dna from the culture and the sequencing of the its gene was performed by macrogen, korea. the primers its 1 5'-tccgtaggtgaacctgcgg-3' and its 4 5’-tcctccgct tattgatatgc-3' were used for the amplif ication of the its gene, which is the universal dna barcode marker for fungi (schoch et al. 2012). the pcr reaction was performed with 20 ng of genomic dna as the template: initial denaturation at 95ºc for 2 minutes; 35 cycles of 95ºc for 1 minute, 55ºc for 1 minute, and 72ºc for 1 minute; and a f inal extension at 72ºc for 10 minutes. the resulting nucleotide sequences were then cleared of noises and aligned using chromaspro and mega7 (kumar et al. 2015). the identities of the isolates were determined by homology against the national center for biotechnology information (ncbi) database using basic local alignment search tool (blast) (altschul et al. 1990). only hits with homology greater than 97% were considered. for isolates which obtained hits with less than 97% homology, their identities were assigned based on their morpho-cultural characteristics. all isolates are maintained at the bicol university college of science department of biology with duplicate deposits at the university of the philippines – los baños museum of natural history microbial culture collection. data analysis the isolation rate (ir) of each fungal species was calculated using the following equation: % 100   % 100  (1) (2) where [plant–site] is def ined as the number of medicinal plants sampled multiplied by number of locations (upland, lowland, and coastal). differences in the number of unique isolates per location were statistically analyzed by the nonparametric kruskall-wallis test using the r programming software (r core team 2013). signif icant difference was determined at an alpha value of 0.05. foliar fungal endophytes of selected medicinal plants 42 results a total of 120 isolates belonging to 17 fungal species were identif ied. representative isolates are listed in table 2 and are shown in figure 1. because of some morphocultural differences observed during the growth stage of the isolates, some species have numerous reference cultures. the counts of the isolates were then combined when appropriate to produce the succeeding tables in this study. ref-iso 1 kp132299.1 hormographiella aspergillata strain ihem 14649 97 hormographiella aspergillata ref-iso 2 kf679356.1 fusarium solani strain ihb f 2353 99 fusarium solani ref-iso 3 jx535014.1 fusarium solani strain mml4012 97 fusarium solani ref-iso 4 kp724996.1 coprinopsis cinerea isolate 21l06i2 99 coprinopsis cinerea ref-iso 5 kp900278.1 colletotrichum gloeosporioides strain ss1-cjs12 99 colletotrichum gloeospioides ref-iso 6 jf710555.1 colletotrichum gloeosporioides isolate omc4 18s 98 colletotrichum gloeospioides ref-iso 7 jx902434.1 colletotrichum gloeosporioides isolate oorc30 99 colletotrichum gloeospioides ref-iso 8 kt342872.1 colletotrichum gloeosporioides strain ds01 18s 97 colletotrichum gloeospioides ref-iso 9 fj172224.1 colletotrichum gloeosporioides isolate cg0305 99 colletotrichum gloeospioides ref-iso 10 kf706658.1 aspergillus unguis strain fs95 99 aspergillus unguis ref-iso 11 fj654485.1 aspergillus sp. sv/09-11 18s 98 aspergillus sp.1 ref-iso 12 kp686465.1 aspergillus sp. bab-4665 98 aspergillus sp.1 ref-iso 13 jf436891.1 aspergillus sp. ymca 11 97 aspergillus sp.2 ref-iso 14 kp686456.1 aspergillus sp. bab-4649 1 96 aspergillus sp.3 ref-iso 15 kf923855.1 glomerella cingulata isolate uom p 99 glomerella cingulata ref-iso 16 jx868760.1 glomerella cingulata strain lvpei.b431_11 99 glomerella cingulata ref-iso 17 kf848941.1 phomopsis sp. fz100 98 phomopsis sp. ref-iso 18 kt208382.1 phomopsis sp. gxglb-3 99 phomopsis sp. ref-iso 19 gu066650.1 phomopsis sp. 76cg/l 99 phomopsis sp. ref-iso 20 gu066617.1 phomopsis sp. 27ld/t 97 phomopsis sp. ref-iso 21 km362394.1 diaporthe sp. c53-134 98 diaporthe sp. ref-iso 22 fj799937.1 diaporthe sp. sab-2009a strain q3310 18s 99 diaporthe sp. ref-iso 23 kp217184.1 daldinia sp. p4.2 99 daldinia sp. ref-iso 24 jx559584.1 guignardia mangiferae strain lgmf1163 99 guignardia mangiferae ref-iso 25 lm652417.1 microascus cinereus 96 microascus cinereus ref-iso 26 ln482516.1 aspergillus flavus 99 aspergillus flavus ref-iso 27 jq763433.1 aspergillus flavus 97 aspergillus flavus ref-iso 28 kf577897.1 aspergillus flavus strain a0628 97 aspergillus flavus ref-iso 29 kf221065.1 aspergillus flavus strain bp5 97 aspergillus flavus ref-iso 30 jn709035.1 aspergillus nomius strain sge19 96 aspergillus nomius ref-iso 31 ab976023.1 aspergillus fumigatus 97 aspergillus fumigatus table 2. representative fungal endophytes isolated from med icinal plants in albay, phil ippines as identified through the sequencing of their its gene reference isolate code genbank accession number most closely related fungal species based on its sequence homology % homology identity j.j.g. guerrero et al. 43 numerically, the upland area had the highest total endophytic count of 45 isolates among the sampling sites (table 3). this, however, is not statistically signif icant compared to the coastal and lowland sampling areas. medicinal plants from the upland, lowland, and coastal sampling sites constitute 37.5%, 35.8%, and 26.7% of the total isolates, respectively. no signif icant difference was detected in the number of fungal endophytes isolated from the same medicinal plants across different locations. figure 1. representative fungal endophytes in medicinal plants from albay, philippines grown on potato dextrose agar (pda) for eight days: (a) aspergillus fumigatus, (b) aspergillus sp.1, (c) aspergillus unguis, (d) aspergillus sp.2, (e) aspergillus sp. 3, (f) colletotrichum gloeosporioides, (g) phomopsis sp. , (h) diaporthe sp. , and (i) fusarium solani. lowland 5 3 7 4 4 5 3 5 2 5 43 35.8 coastal 5 1 2 2 4 3 5 3 3 4 32 26.7 upland 3 7 4 7 2 2 4 6 3 7 45 37.5 total 13 11 13 13 10 10 12 14 8 16 120 100 p-value = 0.2344 ns no significant difference at 0.05 level of confidence cp m e m i a h c e cn m o o v m p bb totalns %location total count table 3. number of fungal endophytic isolates from med icinal plants in albay, phil ippines across d ifferent sampl ing locations foliar fungal endophytes of selected medicinal plants 44 the total number of unique species (table 4), which refers to the number of unique individual fungus regardless of the number of times it was isolated from the hosts, is highest in the upland area and lowest in the coastal area. the obtained values for the total number of unique species do not signif icantly vary from each other. most species of fungi had overlapping occurrence both in sampling sites and the host plant. lowland 3 3 7 4 4 5 3 3 2 4 12 coastal 4 1 2 2 3 3 4 3 3 3 10 upland 2 5 3 5 2 1 4 4 2 7 14 total 6 7 9 7 6 8 7 8 6 8 17 p-value = 0.3107 ns no significant difference at 0.05 level of confidence cp m e m i a h cw cn m o o v m p totalnslocation table 4. number of unique species of fungal endophytes from med icinal plants in albay, phil ippines across d ifferent sampl ing sites bb glomerella cingulata was the most frequent among the fungal endophytes, having been isolated from all the medicinal plants sampled, and in 70% of the sampling areas (table 5). the total isolation rate is 10%, 2.33% of which is contributed by g. cingulata. colletotrichum gloeosporioides had an isolation rate of 1.67%, while the genus aspergillus collectively forms 1.32% of the isolation rate. isolation rate represents the probability of encountering the same species from a pool of isolates. a higher isolation rate means that the species is grown more frequently relative to the total number of isolates. most isolates are from the phylum ascomycota, although many are of the anamorphic form, such as aspergillus spp. , fusarium solani ( m a r t . ) s a c c . , a n d co l l e t o t r i c h u m g l o e o s p o r i o i d e s . t h e b a s i d i o m y c e t e s hormographiella aspergillata and coprinopsis cinerea (schaeff.) redhead, vilgalys & moncalvo may be anamorph and teleomorph of the same species (surmont et al. 2002). should this be the case, in combination, they account for 1.16% of the isolation rate. seven species of fungi were absent from the coastal areas, while f ive and three species were not found in the lowland and upland areas, respectively. six species of fungi, namely hormographiella aspergillata, coprinopsis cinerea, aspergillus unguis weill & l. gaudin, aspergillus sp.1, glomerella cingulata, colletotrichum gloeosporioides, and fusarium solani, were consistently isolated from all sampling sites. j.j.g. guerrero et al. 45 table 5. fungal endophytes of med icinal plants in albay, phil ippines hormographiella basidiomycota 4 10.00 0.00 30.00 13.33 0.33 aspergillata coprinopsis cinerea basidiomycota 10 30.00 30.00 30.00 30.00 0.83 aspergillus unguis ascomycota 6 20.00 30.00 10.00 20.00 0.50 aspergillus sp. 1 ascomycota 3 10.00 10.00 10.00 10.00 0.25 glomerella cingulata ascomycota 28 50.00 70.00 90.00 70.00 2.33 colletotrichum ascomycota 20 70.00 10.00 50.00 43.33 1.67 gloeosporioides phomopsis sp. ascomycota 9 0.00 40.00 20.00 23.33 0.75 diaporthe sp. ascomycota 12 50.00 40.00 10.00 36.67 1.00 daldinia sp. ascomycota 5 20.00 0.00 30.00 16.67 0.42 guignardia ascomycota 1 10.00 0.00 0.00 3.33 0.08 mangiferae a.j. roy microascus ascomycota 4 20.00 0.00 20.00 13.33 0.33 cinereus curzi aspergillus sp. 2 ascomycota 1 10.00 0.00 0.00 3.33 0.08 aspergillus sp. 3 ascomycota 1 10.00 0.00 0.00 3.33 0.08 aspergillus ascomycota 3 0.00 10.00 20.00 10.00 0.25 flavus link aspergillus nomius ascomycota 1 10.00 0.00 0.00 3.33 0.08 kurtzman, b.w. horn & hesselt aspergillus ascomycota 1 0.00 10.00 0.00 3.33 0.08 fumigatus fresenius fusarium solani ascomycota 11 30.00 30.00 40.00 33.33 0.92 total 120 100.00 10.00 upland coastal lowland total species p h y l u m number of isolates isolation rate (%) total number of leaf discs plated = 1,200 total number of [plant–site] = 30 discussion med icinal plants as source of endophytes the fungi g. cingulata and c. gloeosporioides, the two most isolated species in this study, are common fungal endophytes of plants as they are also latent pathogens of many important fruits, such as mango, papaya, and avocado. in particular, for papaya, the c. gloeosporioides isolated in this study may be in its latency stage because no symptoms manifested on the leaf samples and symptoms of normal anthracnose disease often occur on the fruit. f. solani, likewise, was previously reported to be an endophyte of the medicinal plants alpinia calcarata, bixa orellana, calophyllum inophyllum, catharanthus roseus, and aquilaria sinensis, all of which are plants known for their anticancer properties (cui et al. 2011; sunitha et al. 2013). interestingly, f. solani isolated from a. sinensis exhibited antitumor properties, somehow mirroring the biological property of its host plant (cui et al. 2011). other endophytes isolated in this study form the f irst record of their isolation from specif ic medicinal plants. foliar fungal endophytes of selected medicinal plants 46 it is now well known that all plants are in a symbiotic relationship with at least one fungal endophyte (arnold et al. 2000; rodriguez and redman 2008; rodriguez et al. 2009), translating to fungal species diversity (arnold and lutzoni 2007). rodriguez et al. (2009) grouped together endophytes that primarily reflect differences in evolutionary relatedness, taxonomy, plant hosts, and ecological functions. because of the importance of medicinal plants, especially in the philippine context, it becomes an ar tif icial plant group from where to isolate endophytic fungi. the medicinal benef its that may be derived from the plant may also theoretically be exhibited by the endophytes residing in them. based on a survey by mirandilla and abalon (2013), the ten medicinal plants included in this study are the top ten medicinal plants used by households in the same sampling sites. because of the importance of these medicinal plants to locals, their occurrence in the selected sampling areas is assured. potential endophytes from these medicinal plants may also provide similar medicinal applications. the diversity of fungal endophytes cannot be oversimplif ied. just as there are a diverse group of plants to act as hosts, a variety of fungal groups could also grow in nearly all parts of the plant. aside from the stem and leaves, the roots have been noted to also harbor fungal endophytes which can form mutualistic associations functionally similar to mycorrhizal symbiosis (jumpponen 2001). a wide host population for fungal endophytes has been observed from the salt-tolerant species of mangroves (suryanarayanan et al. 1998; kumaresan and suryanarayanan 2001; a n a n d a a n d s r i d h a r 2 0 0 2 ) , t r o p i c a l f o r e s t s ( s u r y a n a r a y a n a n e t a l . 2 0 0 2 ; suryanarayanan et al. 2003), medicinal plants (huang et al. 2008), and important agricultural crops including rice (naik et al. 2009) and wheat (dingle and mcgee 2003). environmental factors also affect fungal endophyte diversity as seen in studies on altitudinal and grazing gradients (granath et al. 2007), as well as precipitation (herrera et al. 2011). medicinal plants are important hosts to fungal endophytes because of their biological applications. numerous studies have already explored medicinal plants and their associated fungal endophytes (wiyakrutta et al. 2004; li et al. 2005; tejesvi et al. 2007), and almost all of these tackle their medicinal potentials. whether the plants’ medicinal value is a product of the symbiosis with its endophyte or is independent of the relationship is relative. it is also possible that the plants’ medicinal value can be mirrored by the fungal endophyte, and thus, when isolated, the endophyte can also produce by itself the same active compounds in plants. the latter has already been shown to be true for many plants. for instance, taxomyces andreanae strobel, a. stierle, d. stierle & w.m. hess produces taxol and taxane similar with its host, the pacif ic yew (stierle et al. 1993). j.j.g. guerrero et al. 47 considerations in sampl ing area location may be a big factor in determining the number of species, as well as the uniqueness of the fungal community. location can alter the microclimate experienced by the host plant, thereby directly affecting the presence or absence of plant species, and their distribution and habit. although not statistically observed in this research, many other studies suggest that geographical location can determine the species composition of fungal endophytes among plants. for instance, mishra et al. (2012) noted that some species of fungi occur mostly exclusively at a particular season and in locations where emissions of certain gases and particulates are present. similar observations were noted by göre and bucak (2007) when the number of fungal species that were isolated in leaves differed between sampling sites and the dominant fungi were site-dependent. this was not observed in this study primarily because of the small number of samples and the proximity of sampling sites. specif ic weather conditions that can support any correlation were not recorded. sánchez-márquez et al. (2008) suggested that increasing the number of plants or locations would most likely reveal new endophytic species. endophytic relationship with plants any symbiotic relationship with the plant can be fluid, and therefore, fungal endophytes cannot be clear-cut mutualists all the time. species such as f. solani, c. gloeosporioides, and g. cingulata are known pathogens of plants, and yet, are considered endophytes because of their location in the plant. endophytism, by itself, should only be regarded as the location of the fungus and does not fully depict the fungus’ interaction with the host. according to faeth (2002), the predominant defensive mutualism perspective is a product of a long list of research involving agronomic grass cultivars and may not be the case among native grasses. the preference to look at plant-fungal endophyte interactions in the light of defensive mutualism may be because of the low genetic diversity and altered growing environment of domesticated grasses, and this concept should not be generalized. this defensive mutualism is rare even in introduced and domesticated grasses. models suggest that host and fungus genotypes, as well as the environmental conditions, affect the direction of interactions (faeth and fagan 2002). faeth and sullivan (2003) also challenged defensive mutualism by showing that mutualistic asexual endophytes of native grasses are usually parasitic because of the negative effects on plant reproduction (number and mass of seeds and germination rate). moreover, sieber (2007) mentioned that evidence for such is mostly circumstantial but agreed that plants would probably not survive many environmental stresses foliar fungal endophytes of selected medicinal plants 48 without the symbiosis with fungal endophytes. to prove unequivocally the positive impact of endophytes to its host, an endophyte-free control must be developed which, according to sieber (2007), is a major problem. in addition, fungi can switch from quiescence to pathogenicity when factors are favorable for the fungi and unfavorable for the host. this switch is genetic, as observed in the genus colletotrichum which causes anthracnose in cucurbits (freeman and rodriguez 1993). t h e s w i t c h l a t e r o n l e a d s t o t h e m a i n t e n a n c e o f c o m p a t i b i l i t y b e t w e e n colletotrichum spp. and cucurbits, similar to that of a pathogen, as well as the survival of the host in what is known to be a balanced plant-endophyte status (kogel et al. 2006). colletotrichum spp. can switch from being pathogens to mutualists based on the host genotype, and thus, its ability to be an endophyte is not strictly conf ined (rodriguez et al. 2005). potential biological appl ications of fungal endophytes g. cingulata and c. gloeosporioides, two of the most isolated endophytes in this study, are known to have anticancer properties (gangadevi et al. 2008), while phomopsis spp. are known to produce terpenoids and isoflavonoids acting as antimicrobials (redko et al. 2006; nithya and muthumary 2010). there is no literature stating the biological applications of hormographiella aspergillata which is known to cause disease in humans (surmont et al. 2002). h. aspergillata isolated from this study was not well characterized for this reason. f. solani, although known to be a plant pathogen, is also known to produce taxol, an anticancer agent (kusari et al. 2009). because biological properties often differ due to location and host, the applications of the isolates from this research may be the subject of future investigations. conclusion and recommendations medicinal plants are good sources of fungal endophytes from which metabolites can be obtained with wide spectrum of applications. from this research, fungal endophytes from pre-selected medicinal plants were isolated and identif ied. a comparison of their occurrence across sampling sites and host plants displayed no signif icant difference. it is yet to be established 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e n d o p h y t e s associated with the philippine endemic tree, canarium ovatum. mycosphere. 6:266273. vieira wa , michereff sj, de morais ma, hyde kd, câmara mp. 2014. endophytic species of co l l e t o t r i c h u m a s s o c i a ted w i t h m a n g o i n n o r t h e a s te r n b r a z i l . f u n g a l d i ve r s i t y. 67(1):181-202. j.j.g. guerrero et al. 53 villaseñor im, lamadrid mra. 2006. comparative anti-hyperglycemic potentials of medicinal plants. journal of ethnopharmacology. 104(1-2):129-131. watanabe t. 2010. pictorial atlas of soil and seed fungi: morphologies of cultured fungi and key to species. florida: crc press. wiyakrutta s, sriubolmas n, panphut w, thongon n, danwisetkanjana k, ruangrungsi n, meevootisom v. 2004. endophytic fungi with anti-microbial, anti-cancer and antimalarial activities isolated from thai medicinal plants. world journal of microbiology and biotechnology. 20(3):265-272. _____________ jonathan jaime g. guerrero is a faculty of the department of biology, college of science, bicol university. his research interest includes mycology, environmental science, and biodiversity conservation. he is currently f inishing his master’s degree in plant pathology at the university of the philippines los baños and kasetsart university – bangkok. mheljor a. general is a b.s. biology graduate of bicol university, currently aff iliated with the environmental management bureau of the department of environment and natural resources. he was a research assistant at the bicol university college of science. he is currently f inishing his m.s. biology degree at bicol university as a ched scholar. jazzlyn t. imperial is an m.s. biology degree holder from the university of the philippines diliman, majoring in genetics. prof. imperial is currently aff iliated with bicol university as a full-time professor handling genetics subjects for b.s. biology students. her work includes bioactive compounds of fungal endophytes. 06_multicultural multicultural teaching 17 a multicultural teaching framework for physics s. l. m. carreon university of the philippines integrated school, katipunan avenue, diliman, quezon city 1101 e-mail: sher_carreon@yahoo.com abstract science diliman (july-december 2004) 16:2, 17–21 *corresponding author this paper presents a framework for integrating varying cultural elements in the teaching of physics concepts. the framework is based on a study on the effects of a multicultural physics teaching approach on student concept understanding and attitude towards physics. introduction the unesco has identified that one of the pillars of learning for the 21st century is an education system that develops one’s understanding of other people and their culture (science education of asian countries, 1999). in the 1999 conference on science education of asian countries, it was cited that cultural diversity must be one of the considerations during the process of science curriculum development. some science education experts also support that scientific literacy for all students can be achieved if the science curriculum reflects the diversity of the society (madrazo & rhoton, 2001). however, there are science educators who have contrary views and consider multicultural education irrelevant to science and mathematics but more appropriate for social studies, language, music, and arts (boutte, 1999). similarly, rosenthal reported that there are scientists and educators who believe that there is no need for multicultural education in science because science is culturally neutral, inherently universal, and global in perspective (morey & kitano, 1997). in 1998, samuels developed and evaluated a collection of multicultural materials for teaching mathematics in middle schools in bermuda. the study revealed that the materials were favorably evaluated and found to be interesting, motivational, and appropriate for the bermudian context. samuels’ study also established that it was possible to develop materials that satisfy both multicultural education and mathematics teaching (samuels, 1999). in the philippines, reyes-matipo (1997) conducted a study on ethnic diversity and physics education. the study revealed that there were significant differences in the attitude and achievement in physics among four different ethnic groups. based on the findings of the study, it was recommended that physics instruction should be infused with customs and traditions of the groups to attain optimal science learning (reyesmatipo, 1997). in 2002, the department of education (deped) in cooperation with the college of education of the university of the philippines created the cultureresponsive curriculum for indigenous peoples (ccip) to show its commitment in providing a curriculum that is relevant and suitable to the way of life and needs of indigenous peoples. the ccip proposed a restatement of the goal of science and health in the revised basic education curriculum; that is, “demonstrate understanding of how science, technology, and health relate to the comprehension of the local environment and culture, and application of skills, attitudes, and carreon 18 values in dealing with varied life situations (deped, 2002).” consolidating the aforementioned global and national perspectives and studies on multicultural education in science has led to the conceptualization of a study on a multicultural teaching approach in physics. this paper presents the method and results of a study on the effects of a multicultural physics teaching approach on student concept understanding and attitude towards physics. it culminates in a multicultural teaching framework that can be used by other physics educators in their own attempts to explore multicultural education in physics. effects of a multicultural physics teaching approach on student concept understanding and attitude towards physics (carreon, 2004) in 2003, the researcher conducted a study on the effects of a multicultural physics teaching approach on student concept understanding and attitude towards physics. varying cultural elements of the five dominant ethnic groups in isabela were integrated in the teaching of physics concepts, principles, and laws. subjects the study involved sixty-one 15-year-old students who were incoming seniors in a private high school in santiago city, isabela. thirty students were exposed to the multicultural physics teaching approach (mpta) and 31 students were exposed to the nonmulticultural teaching approach (nmta). procedure prior to the instructional phase, the students answered a physics concept understanding test and an attitude scale towards physics which were both developed by the researcher. the concept understanding and attitude towards physics of the students from the two classes were found to be comparable prior to the treatment. cultural elements coming from the five dominant ethnic groups of the locality (ilocano, ibanag, gaddang, yogad, and tagalog) were used to teach physics concepts. the cultural elements were composed of ethnic games, myths and legends, important places, and musical instruments derived from books and interaction with the cultural coordinator of the locality. the cultural elements were integrated using the inclusion strategy and artifacts strategy. the mpta class was exposed to teaching/learning activities such as games, inquiry, role play, reading activity, challenge activities, poe (predict, observe, explain), lecture-discussion, demonstration, exploration activity, musical presentation, creative problem solving, puzzle, illustrating scenes, quizzes, and creating toys. the researcher taught both classes and requested a science teacher from the host school to observe regularly. the other class, the nmta class, was only handled differently from the mpta class in terms of cultural integration. the learning activities for the nmta class were almost parallel to those of the mpta class. the physics concept understanding test and attitude scale towards physics were administered again after the instructional phase. results using the one-tailed t-test for independent samples, the mean post-test scores in the physics concept understanding test of the mpta and nmta classes were compared and found to have no significant difference at a = 0.05. the same statistical test was used to analyze the mean post-test attitude rating in the attitude scale towards physics of the two classes. the analysis revealed that the mean post-test attitude rating of the two classes did not differ significantly at a = 0.05. table 1 shows the summary of the tests of the difference between the mean post-test scores of the two classes in the physics concept understanding test and the mean post-test attitude ratings in the attitude scale towards physics. further analysis of the concept understanding score of the two classes in each item showed that the mpta and nmta classes significantly differed in seven items. the mpta group had a higher mean score in five out multicultural teaching 19 of seven items in which the two groups differed significantly. the concepts that were tested in the five test items were the final velocity of an object (thrown upward) at maximum height, acceleration of an object (thrown upward) at maximum height, total energy of a system after energy transformation without heat loss, change in speed of a fluid as it flows into a smaller pipe, and relation of loudness of sound to amplitude and pitch to frequency. the attitude rating of the two classes in each item of the attitude scale was analyzed and found to significantly differ in only one item. the item was “physics is not needed in our society.” since the item was negatively stated, the scoring was reversed. the mpta class had a mean rating of 4.63 in that item, while the nmta class had 4.32. the highest possible rating per item was 5. discussion the multicultural physics teaching approach had no general effect on student concept understanding and attitude towards physics. however, it could improve student understanding of certain physics concepts. it could also help students recognize that physics is needed in the society. the researcher noted two factors that might have affected the results of the study: the cultural awareness of the mpta students and the time the study was conducted. the cultural elements that were used in the study were researched from books, based on the interaction with the cultural coordinator of the locality, and drawn from the cultural experiences of the researcher who grew up in the locality. although the students could identify to which ethnic group they belong to, they did not recognize the cultural elements of the dominant ethnic groups. they appreciated the elements when they were told that the inputs were part of their culture. the other factor that could have affected the results of the study was the time it was conducted. the study was conducted during summer break. one of the recommendations was to duplicate the study during a regular school year. multicultural teaching framework for physics from the experience and results of implementing a multicultural physics teaching approach, the researcher proposes a multicultural teaching framework that can be used by other educators who may wish to explore the integration of cultural elements of other ethnic groups that are not cited in this study. figure 1 shows the multicultural teaching framework for physics. preparation the first step in the preparation for multicultural teaching begins with identifying the dominant ethnic groups represented in the class. this can be done by fielding a questionnaire that can probe on the students’ parentage, identification with an ethnic group, length of stay in the locality, and spoken dialect. second, search for the cultural elements of the dominant ethnic groups that are represented in the class. local plants and animals, important places, natural resources, beauty spots, local literature, local materials, local costume, local music, beliefs and practices, indigenous knowledge, local food, local history, ethnic games, and local musical instruments can be considered as cultural elements (deped, 2002). search for supporting literature and consult with the cultural authorities of the locality to validate the identified cultural elements. table 1. tests of difference between the mean post-test scores and mean post-test attitude ratings of the mpta and nmta classes. physics concept understanding test attitude scale towards physics mpta nmta mpta nmta 15.97 15.45 77.83 78.87 5.007 4.760 10.164 8.523 0.341 0.334 instruments class mean score/ rating sd significance level (one-tailed) *perfect score in the concept understanding test = 40. **perfect attitude rating = 100. carreon 20 third, determine the cultural awareness of the students involved in the study. provide a list of the cultural elements of the dominant ethnic groups and ask the students to identify the elements that they practice or are quite familiar with. fourth, based on the cultural elements that are familiar to the students, prepare the list of the classified elements that will be used in the study. fifth, align the identified cultural elements with the physics topics/concepts. the cultural elements should naturally blend in the content. they should not divert the class from the track of the lesson, rather, they should serve as the catalyst to the development of the lesson or they should further enrich the lesson. sixth, select appropriate integrative strategies. the ccip has identified the following integrative strategies: inclusion strategy and artifacts strategy. the inclusion strategy uses indigenous knowledge and cultural elements as examples to demonstrate concepts and make students realize the connection between their culture and science learning (deped, 2002). ethnic games, myths and legends, and local beliefs and practices are some of the cultural elements that can be considered for the inclusion strategy. the artifacts strategy relies on the use of actual objects that are indigenous to the place or are created by the local people (deped, 2002). local musical instruments, plants, products, materials, and ethnic costumes can be used for this strategy. seventh, identify the evaluation strategies. use strategies that are suitable, learner-centered, and that enhance active learning, critical and creative thinking, problem solving, and cooperative learning. eight, organize the cultural elements, physics topic, integrative strategy, and evaluation strategy in a cohesive lesson plan. implementation the implementation of the multicultural lesson plan should be done objectively. present both the similarities and differences of the cultural elements of different ethnic groups. emphasize that there should be no issue regarding superiority or inferiority of the cultural elements. also, look into the inconsistency of certain cultural elements with physics concepts, principles, and laws. objective learning should prevail over appreciation of cultural heritage. evaluation evaluate the different aspects of implementation of the multicultural lesson plan such as students’ reaction to the integration, facilitation of the lesson plan, and students’ scores and outputs in the evaluation activities. revision revise the multicultural lesson based on the results of the evaluation. strive to implement the revised multicultural lesson plan. preparation 1. identifying the dominant ethnic groups represented in the class; 2. identifying the cultural elements of the dominant ethnic groups; 3. determining the cultural awareness of the students involved in the study; 4. deciding on the cultural elements to be used; 5. aligning the cultural elements with the physics topics/concepts; 6. choosing appropriate integrative strategies; 7. identifying evaluation strategies; and 8. writing the multicultural lesson plan implementation evaluation revision fig. 1. multicultural teaching framework for physics. multicultural teaching 21 implications to physics education multicultural physics teaching may not have a general effect on student concept understanding and attitude towards physics, but it can improve the understanding of certain concepts and can also help students realize that physics is needed in our society. in this essence, multicultural physics teaching has considerable impact in making science more relevant and meaningful to the students, which is one of the primary objectives of education today. the framework presented in this paper describes how physics educators can further study the integration of varying cultural elements in teaching physics concepts, principles, and laws. only five ethnic groups from the northern region of the country were explored in the study. as there are 77 major ethnic groups in the philippines, the study is an initial step towards exploring the potential of multicultural physics teaching in our country. its effects on other aspects of the physics learning process are also possible areas of study for other researchers. although this framework is drawn from a multicultural teaching experience in physics, the steps of the multicultural teaching framework can also be adapted by other disciplines. acknowledgment this research was supported by a grant from the dostscience education institute (sei). references boutte, g., 1999. multicultural education: raising consciousness. wadsworth publishing co., belmont, california. carreon, s.l.m., 2004. effects of multicultural physics teaching approach on student concept understanding and attitude towards physics. paper presented in the parallel sessions of the apptea conference, april 2004. department of education, 2002. third elementary education project: culture-responsive curriculum for indigenous peoples. up college of education. madrazo, g.m. & j. rhoton, 2001. principles and practices in multicultural science education: implications for professional development. in bowers, p. & j. rhoton (eds.) issues in science education: professional development leadership and the diverse learner. national science teachers association press. morey, a. & m.k. kitano (eds.), 1997. multicultural course transformation in higher education: a broader truth. allyn & bacon, massachussetts. reyes-matipo, s.c., 1997. differences in attitude among four ethnic groups in cagayan (unpublished m.s. thesis). university of the philippines, diliman, quezon city. samuels, a.e., 1999. multicultural mathematics materials for bermudian middle schools. columbia university teachers college: dai-a59/07, p. 2408. science education of asian countries: following up of unesco world conference on science, 1999. 85 journal policy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the office of the vice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document defines research misconduct, specifies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identifies appropriate disciplinary actions. definitions scientific misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsification, or plagiarism in proposing, performing, or reviewing research in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 86 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research are concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “note to contributors” stipulates that “all articles must have a high degree of scholarhip,” the “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a certification from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right...” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as defined above and; b.) is specific and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the base for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specific advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the author is affiliated as well as the funding agency that supported the research. 87 the board shall ensure correction of the literature in the succeeding issue through various methods as defined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refeering a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and suppport appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. endnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientific misconduct policy. in attendance were: dr. corazon d. villareal, rduo director, presiding; dr. henry j. ramos, pmrgo director and professor, nip; atty. vyva victoria aguirre, ovcrd legal consultant; editors-in-chief dr. maricor soriano (science diliman) and dr. maria mangahas (socia l science diliman). ms nanie domingo and ms. dercy mararac, editorial assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these definitions of the forms of research misconduct are qouted verbatim from the policy of the office of research integrity of the united states public health service. similar phrasings of definitions are adopted in the references listed at the end of this document. 88 references council of science editors. “white paper on promoting integrity on scientific journal publications, as approved by the cse board of directors on september 3, 2006.” www.councilscienceeditors.org. accessed on january 26, 2009. “policy on scientific misconduct: university of southern california. http://policies.usc.edu/ policies/scientificmisconduct070108.pdf “scientific misconduct policy: new york university, the office of sponsored programs. https:// www.nyu.edu/osp/policies/scientificmisconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/physics/ optics/journal/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ajp/ submit.html 08_flux blanca et al. 26 flux creep investigation in bi2sr2cacu2o8+δ high-temperature superconductor g. r. blanca*, j. ronulo, g. dumlao, and r. sarmago condensed matter physicslaboratory, national institute of physics, university of the philippines, diliman, quezon city, 1101 e-mail: gsblanca@up.edu.ph abstract science diliman (july-december 2004) 16:2, 26–30 *corresponding author the flux creep process in a c-axis bi2sr2cacu2o8+δ thin film was investigated at different temperatures and applied fields using the kim-anderson (ka) approach. the peaked behavior shown in the magnetoresistance profile was attributed to the competing mechanisms of flux motion and sample-intrinsic transition near tc.within the temperature range where the competition occurs, u increases with temperature and consequently a decrease in the superconducting volume corresponds to a decrease in the flux creep. moreover, the flux creep potential barrier varies with applied current i at all temperatures consistent with the ka model. introduction investigations of the flux-creep process in type-ii superconductors reveal important information about the interaction of vortices with pinning centers and among the vortices themselves (landau & ott, 2001). studies of this type are essential in controlling the properties in the mixed state as well as in the realization of highcurrent applications. the magnetic flux motion in hightemperature superconductors (htscs) is extremely complex and this presents a problem in the understanding of dissipation mechanism in these materials (poole et al., 1995). however, after reconsideration of the available data, landau and ott (2001) recently showed that low-temperature flux creep and vortex motion close to the superconducting critical temperature (tc) may very well be explained using the kim-anderson description if a realistic profile of the pinning potential was taken into account. furthermore, they showed that the specific features of vortex dynamics, such as the irreversibility line and a vortexglass transition, not only may be explained by employing the kim-anderson model, but are a direct consequence of the simple model. this paper investigates the flux creep process in bi2sr2cacu2o8+d. an analysis of the magnetoresistance was initially made to determine the exclusive flux regime. the current dependence of the potential barrier was obtained and analyzed using the kim-anderson model. direct transport measurements were performed on the sample to obtain resistance and i-v profiles. methodology resistance measurements at varying fields were done on a c-axis bi2sr2cacu2o8+d thin film grown via combined liquid-assisted powder deposition and liquidphase sintering process (ambanta, 2004). standard four-point probe method was utilized in the transport measurements with pressed indium solder on gold wires flux creep investigation 27 as contacts. isothermal current-voltage (i-v) measurements were also obtained at different temperatures with magnetic field applications of 0.1 and 1.25 t and a transport current of 10 ma. magnetoresistance profiles are presented in this work using , (1) where r(b) corresponds to resistance measurements with an applied magnetic field of certain magnitude b and transport current i, and r(b=0) pertains to zerofield resistance measurements with transport current i. results and discussion the resistance-temperature profiles of the sample shown in fig. 1 reveals the sample’s superconducting critical temperature tc indicated at 87 k. upon the application of increasing magnetic field, a very pronounced broadening of the transition region was observed. this is expected due to the increasing number of mobile flux lines, corresponding to a higher applied magnetic field. the magnetoresistance profiles were obtained to determine the flux creep regime. the magnetoresistance exhibits a peaked behavior which depicts the nature of dissipation in the mixed state of the high temperature superconducting sample. we define four distinct region of the magnetoresistance profile as illustrated in fig. 2. region i corresponds to the regime of zero dissipation, which may include the meissner state at low temperature, and the vortex glass state, the regime of immobile flux lines due to insurmountable potential barrier, at higher temperature. region ii, the region of increasing magnetoresistance with temperature, should correspond to the flux creep regime, where vortices hop out of pinning sites via thermal activation, causing dissipation that increases with temperature. the temperature-coinciding maxima of the magnetoresistance profiles, the boundary between regions ii and iii, occur at 80 k, the exact same temperature when the bi2sr2cacu2o8+d sample begins its intrinsic transition to the normal state. this transition is characterized by a progressive phase decoupling at the sample’s weak links resulting effectively to decreasing total superconducting volume and, correspondingly, increasing normal regions where the flux lines can “leak” into. in region iii, this competition is manifested by a decreasing magnetoresistance in the approach to tc. this means that, in this region, the competition results to an effective decrease of vortex motion. in region iv, the sample is in the normal state where full penetration of the electric field is permitted and the applied current is dissipated according to the sample’s normal state properties. the isothermal i-v profiles obtained with varying applied fields are shown in fig. 3. four i-v measurements were obtained at temperatures chosen within region ii for each magnetic application of 1.25 and 0.1 t. for an unpinned but damped flux-line lattice, the average velocity of the flux lines vf is proportional ( ) ( ) ( )magnetoresistance , 0,r b i r b iω = − = fig. 1. the broadening of the transition region with increasing applied magnetic field perpendicular to the transport current. temperature (k) 20 40 60 80 100 120 140 160 r es is ta nc e ( ωωωω ω ) 0.0 0.2 1.6 1.4 1.2 1.0 0.8 0.6 0.4 fig. 2. the increasing magnetoresistance with temperature in region ii corresponds to the flux creep regime. temperature (k) 20 40 60 80 100 120 140 160 r es is ta nc e ( ωωωω ω ) 0.00 0.05 0.30 0.25 0.20 0.15 0.10 blanca et al. 28 to the current density j = jc, and one obtains a linear relationship between v and i, noting that the electric field e due to vortex motion causes the measurable voltage drop v. when pinning is important, the average velocity associated with the thermally activated jumps of flux lines, so e is also exponentially dependent on the barrier u(j) as is v [7]. that is, (2) where (3) here, lhop is the vortex hopping distance, b is the applied magnetic field, lcreep is that length of the sample, which contributes to the flux creep and c is the speed of light. thus, flux creep is generally associated with a highly nonlinear i-v relationship, as the profiles in fig. 3 show, dictated by the specific dependence of u on i and by the exponential dependence of e (or v) on u. as further depicted by the profiles, the voltage drop measured with the same magnitude of current increases with increasing temperature, consistent with the magnetoresistance data. from eq. (2), the current dependence of the potential barrier was obtained from the isothermal i-v data using (4) where a is current independent and equal to . (5) figures 4 shows, in (u–a) against i plot, the current dependence of the barrier u at different temperatures in the flux creep regime with (a) 1.25 and (b) 0.1 t applied magnetic fields. with temperature, u–a decreases for both applied magnetic fields. this behavior is consistent with the ka thermally activated mechanism. all profiles, however, depict nonlinearity of the barrier u on i. this dependence is different from the original kim-anderson flux creep model assumption of a linear u(j). but, as first recognized by fig. 3. the i-v plots at different temperature values with applied magnetic fields of (a) 1.25 and (b) 0.1 t show nonlinearity associated with flux motion. current (a) vo lta ge d ro p (v ) vo lta ge d ro p (v ) (a) 1.25 t (b) 0.1 t 79 k 77 k 76 k 73 k 79 k 77 k 76 k 73 k 0.00 0.02 0.04 0.06 0.08 0.10 current (a) 0.00 0.02 0.04 0.06 0.08 0.10 -0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.000 0.001 0.002 0.003 0.004 0.005 ( )0 exp bv v uk t= − 0 hop creep 0 v l bl v c = ( ) ( )lnbu i k t v i α= − ⎡ ⎤ +⎣ ⎦ ( )0lnbk t vα = fig. 4. the potential barrier u diverges as current i approaches zero for both applied fields. current (a) u – αααα α 0.00 0.02 0.04 0.06 0.08 0.10 current (a) 0.00 0.02 0.04 0.06 0.08 0.10 7.0e-21 8.0e-21 9.0e-21 1.0e-20 1.1e-20 1.2e-20 1.3e-20 79 k 77 k 76 k 73 k (a) 1.25 t 6.0e-21 5.0e-21 (b) 0.1 t 79 k 77 k 76 k 73 k u – αααα α 8.0e-21 1.0e-20 1.2e-20 1.4e-20 1.6e-20 6.0e-21 flux creep investigation 29 beasley et al. [8] and recently strongly put forward by landau and ott [9], the linearity of u with j is only a first approximation and a more realistic barrier should exhibit a nonlinear dependence on current. there is, further, no physical reason in the ka description for anticipating a linear u(j). the divergence of the flux creep potential barrier u as i approaches zero is explained by the collective creep theory in terms of a state of immobility of an infinitely large flux bundle. the vortex-glass model relates this immobility to a thermodynamic phase transition from an initially mobile state. a divergent u from eq. (4), however, corresponds to a zero measured voltage drop, which may be expected of a moving vortex system producing zero net electric field due to random hopping. in the presence of an applied current, an effective nonzero electric field can only be expected because flux creep is facilitated towards the direction of the driving lorentz force. furthermore, the electrodynamics of the collective creep theory predicts an inverse power-law dependence of the flux creep potential barrier u on j, with a temperature-dependent universal exponent. figures 5(a) and 5(b) show, however, that the barriers obtained at 79 k with 1.25 t and 73 k with 0.1 t, respectively, fit perfectly to the logarithmic function (6) to further investigate the rate of change of the potential barrier with respect to current, plot of du/di versus current was determined. the collapse to a single curve of d(u–a)/di, for both with 1.25 t in fig. 6(a) and 0.1 t in fig. 6(b), shows that at all temperatures, u varies with i in the same manner. moreover, this collapse is consistent with the simple anderson-kim thermally activated mechanism with (7) where u(j) is the function determining the dependence of u on applied current j and u(t) determines u with temperature t. it was noted earlier that in the temperature regime of the observed mixed state where the sample undergoes ( )lny a b x= + ( ) ( ) ( ),u j t u j u t= fig. 5. the potential barrier u behaves logarithmically with current i at (a) 79 and (b) 73 k with 1.25 and 0.1 t applied fields, respectively. current (a) u – αααα α 0.00 0.02 0.04 0.06 0.08 0.10 current (a) 0.00 0.02 0.04 0.06 0.08 0.10 7.0e-21 8.0e-21 9.0e-21 1.0e-20 1.1e-20 1.2e-20 (a) 1.25 t 6.0e-21 5.0e-21 u – αααα α 8.0e-21 1.0e-20 1.2e-20 1.4e-20 1.6e-20 6.0e-21 79 k log i (b) 0.1 t 73 k log i an intrinsic transition to the normal state—region iii in fig. 2—an increasing potential barrier with temperature could be anticipated where the magnetoresistance profiles obtained without prior use of the andersonkim model, manifests a decreasing behavior with temperature. it was, however, pointed out that this barrier could not be the true flux creep barrier since an increasing barrier with temperature only relates to a decreasing superconducting volume or an increasing normal region. figure 7 shows the apparent barrier at 82 and 85 k, obtained through the same procedure using the anderson-kim flux creep equation. indeed, the barrier increases rather than decrease with temperature, consistent with the decreasing magnetoresistance. conclusions the flux creep process in bi2sr2cacu2o8+d can be described and analyzed using the anderson-kim approach. magnetoresistance profiles exhibit peaked blanca et al. 30 fig. 7. the potential barrier u decreases with increasing temperature in the region of decreasing magnetoresistance. current (a) 0.00 0.02 0.04 0.06 0.08 0.10 u – αααα α 2.0e-21 4.0e-21 1.0e-20 1.2e-20 1.4e-20 0.0 85 k 82 k 8.0e-21 6.0e-21 fig. 6. the potential barrier u behaves in the same manner for all temperature values at (a) 79 and (b) 73 k with applied fields of 1.25 and 0.1 t, respectively. current (a) du /d i 0.00 0.02 0.04 0.06 0.08 0.10 current (a) 0.00 0.02 0.04 0.06 0.08 0.10 -2.0e-18 -1.5e-18 -1.0e-18 -5.0e-19 0.0 (a) 1.25 t -2.5e-18 du /d i -2.0e-18 -1.5e-18 -1.0e-18 -5.0e-19 0.0 -2.5e-18 (b) 0.1 t 73 k 76 k 77 k 79 k 73 k 76 k 77 k 79 k barrier varies with i at all temperatures consistent with the kim-anderson description. references ambanta, r.i., 2004. c-axis oriented b1-2212 superconducting thin film using liquid powder deposition and growth by partial melting (undergraduate thesis), march 2004. landau, i.l. & h.r. ott, 2001. phys. rev. b. 63: 184516. landau, i.l. & h.r. ott, 2002. phys. rev. b. 65: 064511. poole, c.p., et al., 1995. superconductivity. san diego, academic press. yeshurun, y., a.p. malozemoff, & a. shaulov, 1996. rev. mod. phys. 68: 912. behavior due to competing mechanisms of flux motion and sample-intrinsic transition to the normal state near tc. in the temperature range where this competition occurs, the potential barrier increases with temperature since the increasing normal regions where vortices peak into, and simultaneously, the decreasing superconducting volume should correspond to an effective decrease in flux creep. flux creep potential sd-sample article 1 dahlia c. apodaca, phd mines and geosciences bureau department of environment and natural resources johnrob y. bantang, phd national institute of physics university of the philippines diliman wilfredo l. campos, phd college of arts and sciences university of the philippines diliman relicardo m. coloso, phd southeast asian fisheries development center/aquaculture department tigbauan, iloilo maria phythias b. espino, phd institute of chemistry university of the philippines diliman edna g. fortes, phd marine science institute university of the philippines diliman alipio t. garcia, phd department of physics university of the philippines baguio luis b. maria b. garcia, dfsc institute of biology university of the philippines diliman paul john l. geraldino, phd department of biology university of san carlos, cebu city francisco m. heralde iii, phd department of biochemistry and molecular biology university of the philippines manila reviewers for this issue january-june 2013 • volume 25 no. 1 vincent v. hilomen, phd institute of biological sciences university of the philippines los baños hilton y. lam, phd department of clinical epidemiology university of the philippines manila lawrence liiao, phd graduate school of biosphere science hiroshima university, japan jose ernie c. lope, phd institute of mathematics university of the philippines diliman hildie maria nacorda, phd marine science institute university of the philippines diliman minerva s.d. olympia, phd institute of fish processing and technology university of the philippines visayas perry s. ong, phd institute of biology university of the philippines diliman gil nonato santos, phd department of physics de la salle university benjamin m. vallejo, phd institute of environmental science and meteorology university of the philippines diliman 92 journal policy on research misconduct1 (final march 13, 2009)2 principles the journals 3 published by the office of the vice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document defines research misconduct, specifies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identifies appropriate disciplinary actions. definitions scientific misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsification, or plagiarism in proposing, performing, or reviewing research or in reporting research results. 4 fabrication is making up data or results and recording or reporting them. 5 falsification is manipulating research materials,equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 93 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research area concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “notes to contributors” stipulates that “all articles must have a high degree of scholarship,” that “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a certification from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right….” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as defined above and; b.) is specific and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the bases for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specific advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the author is affiliated as well as the funding agency that supported the research. 94 the board shall ensure correction of the literature in the succeeding issue through various methods as defined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refereeing a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and support appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. footnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientific misconduct policy. in attendance were: dr. corazon d. villareal, rduo director, presiding; dr henry j. ramos, pmrgo director and professor, nip; atty. vyva victoria aguirre, ovcrd legal consultant; editors-in-chief dr. maricor soriano (science diliman) and dr. maria mangahas (social science diliman). ms. nanie domingo and ms. dercy mararac, editorial assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these definitions of the forms of research misconduct are quoted verbatim from the policy of the office of research integrity of the united states public health service. similar phrasings of definitions are adopted in the references listed at the end of this document. 95 references council of science editors. “white paper on promoting integrity in scientific journal publications, as approved by the cse board of directors on september 3, 2006.” www. council scienceeditors.org. accessed on january 26, 2009. “policy on scientific misconduct: university of southern california. http:// policies.usc.edu/ plicies/scientific misconduct070108.pdf “scientific misconduct policy: new york university, the office of sponsored programs. https:// www.nyu.edu/osp/policies/scientific misconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/ physics/ optics/journall/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ ajp/ submit.html ocr document 01_device noblezada and campos 14 science diliman (january-june 2007) 19:1, 14-23 *corresponding author abstract composition, abundance and distribution of chaetognaths along the pacific coast and adjacent internal waters of the philippines mary mar p. noblezada and wilfredo l. campos oceanbio laboratory, division of biological sciences, college of arts and sciences, university of the philippines in the visayas, miagao, iloilo 5023 e-mail: zoea21st@yahoo.com/oceanbio2002@yahoo.com telefax: 033 3159271 chaetognath species composition, distribution and relative abundance along the pacific coast and the internal waters of the philippines are presented based on the analysis of 12 samples collected from two oceanographic cruises in april-may 2001. the occurrence of species in relation to water masses is noted and results are analyzed with regards to the main hydrologic features of the surveyed area. nineteen species of chaetognaths belonging to 3 genera were identified from the samples. in order of relative abundance, these include: sagitta enflata, s.bipunctata, s. bedoti, s. neglecta, s. robusta, s. ferox, s. pacifica, s. hexaptera, s. serratodentata, s. decipiens, s. regularis, s. macrocephala, s. minima, s. oceanica, s. pulchra, pterosagitta draco, krohnitta subtilis, s. nagae, and s. johorensis. the overall mean density of all arrow worms was 4.85 ind.·m-3 (range 0.90-30.89). sagitta enflata was the most abundant and frequent species in all stations analyzed and comprised 49.7%. based on their respective distributions reported in the literature, the results indicate a strong influx of oceanic water from the pacific ocean into the visayan sea. keywords: chaetognaths, epiplanktonic, mesoplanktonic, bathyplanktonic, oceanic, and neritic. composition, abundance and distribution of chaetognaths 15 introduction the phylum chaetognatha is comprised of vermiform marine invertebrates commonly called "arrow-worms". at recent estimate, they include about 150 species from 24 genera and 10 families (johnson 2005). they are exclusively marine organisms and majority of them are planktonic. they can be found in all oceans from the surface to great depths, and are often second or third in abundance after copepods (kehayias 2003). although chaetognaths are eaten by numerous larger carnivorous organisms, they are themselves active predators (casanova 1999) and are believed to be major predators on copepods (reeve 1970 and feigenbaum 1991). they also prey on other small crustaceans, larval fish and other chaetognaths. they thus play an important role in the transfer of energy from copepods to higher trophic levels. their role as predators and competitors of larval fish has been evaluated in recent years (baier and purcell 1997; brodeur and terazaki 1999). the impact of chaetognath predation on fish larvae may be exaggerated due to the relative scarcity of fish larvae in the plankton. they nevertheless contribute to reduction of larval abundance during periods of fish production (casanova 1999). chaetognaths are also known as excellent indicators of water masses because of their close relationships with certain environmental variables (e.g. salinity, temperature and dissolved oxygen) as well as their species-specific horizontal and vertical distribution (terazaki and miller 1986). as a result, chaetognaths are often categorized according to the type of water mass they are best adapted to. warm water species are generally characteristic of tropical regions, while coldwater ones are generally characteristic of temperate regions. those characteristic of mixed waters tend to occur in greater abundance where cold and warm water mix, though they may be found in cold or warm water occasionally. inspite of their abundance and importance, many aspects of chaetognath biology are not yet known. these include morphological variation, distribution and other ecological aspects. a better understanding of chaetognath assemblage distribution could be a significant "tool" in determining water mass movement and circulation, and for further validation of certain oceanographic phenomena such as upwelling events and frontal formation. furthermore, information on their abundance and distribution can lead to the appreciation and, eventually, quantification of their impact on the abundance of fish larvae and other marine fauna, of which they are believed to be major predators and competitors for food. few studies on chaethognaths have been conducted in the philippines. among the pioneer studies are michael (1919), bieri (1959), alvariño (1967), and rottman (1978), which deal with descriptions of the different species and their observed distributions. estudillo (1980) investigated the abundance & distribution of arrow worms in the visayan sea, but limited his identifications to genus level only. other studies, including jumao-as and von westernhagen (1978), and johnson (2006), have been limited to specific water basins. this study examined the species composition, abundance and distribution chaetognaths along the pacific coast and adjacent internal waters and provides inputs on how these are related to local hydrography. materials and methods samples were collected during two oceanographic cruises covering: (1) 34 stations on and off the bicol shelf from april 1-11, 2001 on board the r/v dabfar; and (2) 46 stations from san bernardino strait into the visayan sea and further west into sibuyan sea from april 26may 2, 2001 on board the trv sardinella of the university of the philippines in the visayas (fig. 1). from this grid of stations, 12 were chosen to represent the transition from open oceanic to internal waters: 6 in bicol shelf, 3 in san bernardino strait and 3 in the visayan sea. samples were collected by means of double oblique tows to a maximum depth of 100 m, using a net of 60 cm mouth diameter and 335 µm mesh size. a flow meter was mounted at the mouth to measure the volume of water filtered. since sampling during both cruises was continuous, both day and night samples in each of the 3 sub-areas were chosen. plankton samples were preserved in 10% noblezada and campos 16 buffered seawater-formalin solution in the field and brought to the laboratory for processing and identification. biomass in each station was determined by measuring the displaced volume of each sample and expressing this as ml.·m-3 water filtered. chaetognaths were sorted out and identified to species under compound and dissecting microscopes, using the keys of michael (1911, 1919), alvariño (1967), michel (1984), pierrot-bults (1988), bieri (1991) and casanova (1999). results and discussion species composition a total of 2,963 specimens were examined. from these, 19 species from 3 genera were identified. table 1 lists the species recorded, their mean densities and relative abundance in the samples examined. sagitta enflata was the most dominant species and comprised 49.7 % of all chaetognaths recorded. among the top ten species were s. bipunctata, s. bedoti, s.neglecta, s. robusta, s. ferox, s. pacifica, s. hexaptera, s. serratodentata and s. decipiens. together, these species comprised 96.4% of the total. in general, the observed composition is consistent with those reported in previous investigations in various areas in the country (alvariño 1967, jumao-as and von westernhagen 1978, rottman 1978 and johnson et al.., 2006). in hilutungan channel, cebu, jumao-as and von westernhagen (1978) reported 13 species of chaetognaths, all of which have also been recorded in the present study. sagitta enflata was the most common and abundant species. of the thirteen, 12 were described as epiplanktonic, while only sagitta decipiens was described as mesoplanktonic. similarly, a total of 22 species from 4 genera were recently reported from the sulu sea and celebes sea (johnson et al., 2006). again, s. enflata dominated the chaetognath assemblages, comprising about 39% of all arrow worms recorded. all 22 species have been reported in the present as well as in previous studies (alvariño 1967, jumao-as and von westernhagen 1978, rottman 1978), although the number of species recorded varied in each of these studies. such differences attributed to differences in sampling methods and areas investigated. for example, both jumao-as and von westernhagen (1978) and the present study were confined to sampling the upper 100m of the water column, although the present study had a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 44 40 41 42 43 46 45 47 6 7 8 9 10 11 12 17 20 21 23 25 27 28 29 31 33 34 35 36 36a 37 38 43 44 45 46 47 48 49 50 51 52 n pacific ocean celebes sea sulu sea south china sea 120 122 124 16 figure 1. map showing two oceanographic cruises: (+) denoting stations surveyed during the bicol shelf cruise and (o) internal waters. the encircled stations were samples used for this study. composition, abundance and distribution of chaetognaths 17 much more extensive spatial coverage. johnson et al. (2006), on the other hand, employed a multiple opening-closing net to collect samples up to 1000m deep. abundance figure 2 shows the density distribution (ind.·m-3) of chaetognaths in the study area. the overall mean density was 5.0 ind.·m-3, with values ranging from 0.90-30.9. the highest concentration was recorded in station b36 in bicol shelf, with a density of 30.9 ind.·m-3. the lowest concentration was also observed in bicol shelf, while stations in the internal waters (san bernardino strait and visayan sea) showed low to moderate concentrations. on average, densities in bicol shelf were almost two times higher than those in san bernardino strait and three times higher than those in the visayan sea (table 2). species mean sd % bicol shelf san bernardino visayan sea strait b7 b9 b10 b11 b36 b38 s24 s27 s30 v33 v37 v39 sagitta enflata 2.41 4.22 49.73 x x x x x x x x x x x x sagitta bipunctata 0.46 0.68 9.54 x x x x x x x x x sagitta bedoti 0.52 1.22 10.78 x x x x x x x x x x sagitta neglecta 0.37 0.70 7.55 x x x x x x x x x sagitta robusta 0.26 0.52 5.41 x x x x x x x x x x sagitta ferox 0.22 0.44 4.63 x x x x x x x x x x x sagitta pacifica 0.10 0.16 2.15 x x x x x x x sagitta hexaptera 0.08 0.12 1.72 x x x x x sagitta serratodentata 0.09 0.19 1.84 x x x x x x x sagitta decipiens 0.13 0.35 2.60 x x x x x sagitta regularis 0.04 0.06 0.90 x x x x x x x sagitta macrocephala 0.11 0.33 2.18 x x x x x x x sagitta minima 0.01 0.02 0.14 x sagitta oceanica 0.01 0.02 0.21 x x sagitta pulchra 0.01 0.02 0.15 x pterosagitta. draco 0.01 0.02 0.13 x x x krohnitta. subtilis 0.01 0.03 0.23 sagitta nagae 0.00 0.01 0.08 x x sagitta johorensis 0.00 0.00 0.01 sagitta sp. 0.00 0.00 0.02 x x x x 4.85 9.12 100.00 mean density (ind/m3) 0.05 0.12 0.05 0.10 1.54 0.14 0.10 0.26 0.14 0.18 0.10 0.14 sd 0.14 0.44 0.13 0.20 3.42 0.28 0.16 1.09 0.37 0.25 0.33 0.27 no spp recorded 5 5 4 11 11 12 10 6 15 15 7 11 table 1 mean densities, relative abundance and frequency of occurrence of the species collected in the pacific coast (bicol shelf) and internal waters of the philippines. x denotes present 122 124 126 10 12 14 16 figure 2. density (ind.·m-3) distribution of chaetognaths in the surveyed area. (marker size increases as density increases with the following categories: <1, <5, <15, and >35). noblezada and campos 18 bicol shelf san visayan bernardino sea strait mean density 6.66 3.33 2.75 sd 11.90 1.66 0.82 cumulative no. of sp. 14 17 18 table 2 species diversity (cumulative no. of sp.), standard deviation (sd), and density (ind.•m-3) of chaeotognaths in the surveyed areas: bicol shelf, san bernardino strait and visayan sea distribution of the nineteen species recorded, twelve (12) were at least moderately abundant and frequently occurring, and were observed both in the more open waters outside (i.e., bicol shelf) and in internal waters (i.e., san bernardino strait & visayan sea). of these, six (6) species showed comparable abundances within and outside of internal waters, including the ubiquitous species s. enflata and s. ferox (fig. 4), which is consistent with their being reported as oceanic species 0.001-0.05 0.05-1 absent + 1-16 122 124 126 10 12 14 16 s. pacifica 122 124 126 10 12 14 16 s. hexaptera 122 124 126 10 12 14 16 s. decipiens 122 124 126 10 12 14 16 s. serratodentata figure 3. horizontal distribution of s. pacifica, s. hexaptera, s. decipiens, and s. serratodentata. frequently occurred at lowmoderate densities composition, abundance and distribution of chaetognaths 19 in previous studies (johnson et al., 2006, jumao-as and von westernhagen 1978, rottman 1978, alvariño 1967, bieri 1959, michael 1919). the occurrence of primarily oceanic species in internal waters may be brought about by water exchange across channels and into adjacent waters, such as san bernardino strait and the visayan sea (campos et al., 2002). in such cases, the resulting geographical distribution would likely be similar outside and inside internal waters, as was observed for s. enflata, s. ferox, s,serratodentata, s. pacifica, s. macrocephala & s. decipiens (fig. 3 ), or with higher abundances and or frequencies outside. the latter was observed for another five (5) species, namely s. bipunctata,s. robusta, s. hexaptera, s. bedoti & s. neglecta (fig. 4), although the latter two have been previously reported as neritic (johnson et al., 2006 and jumao-as and von westernhagen 1978). while the use of the terms "oceanic" and "neritic" is without exclusivity, it should indicate where higher abundances and or frequencies are observed. hence, the present results do not agree with the reported neritic distribution for these two species, although more samples from other areas should be examined for a more definitive description of their distribution. similarly, s. regularis is reported as oceanic, although our results show higher abundances in internal waters (fig. 5). of the seven (7) rare species, those with <25% frequency of occurrence, five (5) (s. johorensis, p. draco, s. pulchra, minima & k. subtilis) were observed only in internal waters, although they are reported as oceanic in previous studies. while our current results suggest that these 5 species are neritic only (fig. 5), higher abundances outside of internal waters (e.g., south china sea) cannot be discounted. oceanic waters from the south china sea enter central philippines via channels north and south of mindoro, although the visayan sea is still several water basins further inside. these species have been reported as oceanic in studies covering the pacific ocean (johnson 2005), south china sea (alvariño 1967) and sulu sea (johnson et al., 2066 and michael 1919). the other two species, s. nagae and s. oceanica, are reported as oceanic (johnson et al., 2006, jumao-as and von westernhagen 1978 and michael 1919) and showed distributions consistent with the definition provided above. diversity while chaetognath density increased from internal waters towards the pacific (bicol shelf), diversity (cumulative number of species) decreased in the same direction (i.e., towards oceanic waters) (table 2). the dominance of s. enflata was not consistent with any of these trends. s. enflata comprised 43.7% of all chaetognaths in the visayan sea, 67.9% in the san bernardino strait and 46.4% in the bicol shelf. while overall concentrations were low in the visayan sea, there were several species showing similar levels of abundance. the resulting low dominance implies that the area provides a wide range of tolerable environmental conditions for more species to exist. this high diversity in the visayan sea might be attributed to such factors as high productivity, as reflected by chl a concentrations which are 3 times higher than in the bicol shelf (campos et al., 2002 and primavera et al., 2002), and coastal topography, which allows water exchange with several adjacent water basins thereby adding to the heterogeneity of source-habitats for plankton, in general, and arrow worms in particular. in contrast, the bicol shelf area investigated is oceanic, with comparatively little, if any, topographical heterogeneity. high overall density in this area is primarily attributed to high overall chaetognath concentrations in two stations, b36 and b38, which are both located in the vicinity of an upwelling zone (amedo et al., 2002). it is also in the bicol shelf area where the lowest arrow worm diversities were observed (table 1). similarly, highest dominance (of s. enflata) was observed in station b36 also located within the upwelling zone where only a moderate number of species were recorded in the sample. the occurence of mesopelagic and mesobathypelagic species (s. decipiens, s. serratodentata, s. macrocephala, s. minima and krohnitta subtilis) in samples collected from the upper layer of water could be explained by vertical transport effected by the upwelling, in which deep-living organisms are carried to the epipelagic layer. most of the chaetognaths found in the studied area are epiplanktonic species, and others are migratory noblezada and campos 20 122 124 126 10 12 14 16 s. robusta 122 124 126 10 12 14 16 s. ferox 122 124 126 10 12 14 16 s. bipunctata 122 124 126 10 12 14 16 s. neglecta 122 124 126 10 12 14 16 s. bedoti 122 124 126 10 12 14 16 s. enflata 0.001-0.05 0.05-1 absent + 1-16 figure 4. horizontal distribution of s. robusta, s. ferox, s. bipunctata, s. neglecta, s. bedoti and s. enflata. frequently occurred at high densities composition, abundance and distribution of chaetognaths 21 figure 5. horizontal distribution of s. ocenica, p. draco, s. regularis, s. macrocephala, s. pulchra,, s. johorensis, s. minima, s. nagae and k.. subtilis. occurred at low densities and low frequency. 0.001-0.05 0.05-1 absent + 1-16 122 124 126 10 12 14 16 s. oceanica 122 124 126 10 12 14 16 p. draco 122 124 126 10 12 14 16 s. regularis 122 124 126 10 12 14 16 s. macrocephala 10 12 14 16 122 124 126 s. pulchra 122 124 126 10 12. 14 16 s. johorensis 122 124 126 10 12 14 16 s. minima 122 124 126 10 12 14 16 s. nagae 122. 124 126. 10 12 14 16 k. subtilis noblezada and campos 22 elements with a wide range of vertical movements in the water column. in practice, chaetognaths have been categorized by where they are most concentrated on a regular basis over a large scale. the ocean is a dynamic moving environment so species can be moved around and displaced to areas where they are otherwise uncommon. it is not clear whether those species recorded only inshore are truly neritic only, but it is clear that these species are rare, if at all found in the pacific oceanic waters. what is more certain is that the many connections to the visayan sea of various internal water basins, in addition to the potential influence from the south china sea and the sulu sea, contribute to high plankton species richness in this area. the distribution of chaetognaths has often been directly related to hydrographic factors (michael, 1911 and alvariño, 1964). hydrographic factors are important in determining which oceanic species live successfully in the shallow-water environment. in this study, oceanic species were observed in large numbers in almost all the stations surveyed. this results from the direct strong influence of water exchange between open waters and internal waters through the san bernardino strait (campos et al., 2002). this phenomenon, together with the hydrologic influence of upwelled waters, enhances the local abundance and diversity of chaetognaths in the areas surveyed. this initial report on the chaetognaths of the pacific coast and internal waters of the philippines is a good starting point for further continuing the study to include samples collected from other internal basins adjacent to the visayan sea as well as the scs and sulu sea, and from other months to examine seasonal variation. this will contribute to a better understanding of water movement and mixing between waters from various sources and, within a larger context, provide more insights on the sources of planktonic propagules whose stocks are heavily exploited in internal waters. acknowledgements this study forms part of the pacific seaboard r & d program funded by the department of science and technology. we would like to thank the captains & crews of the trv sardinella and m/v da bfar for assistance in field sampling. we are grateful to the oceanbio and marine bio laboratory people for their support and assistance. references alvariño a. 1967. the chaetognatha of the naga expedition (1959-1961) in the south china sea and the gulf of thailand. part 1_systematics. scripts inst. naga, 4-2. la jolla, 197 pp. amedo, c.l.a., c'l. villanoy and m.j. udarbe-wlaker. 2002. indicators of upwelling at the northern bicol shelf. upv j. nat. sci. 7 (1&2):42-52. baier, c.t. and purcell j.e. 1997. trophic interactions of chaetognaths, larval fish and zooplankton in the south atlantic bight. mar. ecol. prog. ser. 146: 43-53. bieri, r. 1959. the distribution of planktonic chaetognatha and their relationship to water masses. limnol. oceanogr. 4: 1-28. bieri, r. 1991. systematics of the chaetognatha. in bone, q., h. kapp, pierrot-bults, a.c. (ed.).the biology of chaetognaths. oxford university press, oxford, 122-136 pp. brodeur, r. d. and terazaki, m. 1999. springtime abundance of chaetogntahs in the shelf region of the northern gulf of alaska, with observations on the vertical distribution and feeding of sagitta elegans. fish. oceanogr. 8: 93-103. campos, w.l., estremadura, dm and canto r., 2002. composition and abundance of major zooplankton taxa in the eastern central philippines inter-island waters. upv j. nat. sci. 7 (1&2): 66-80. casanova, j. p.1999. chaetognatha. in, south atlantic zooplankton, edited by d. boltovskoy, backhuys publishers, leiden. 1353-1374 pp. feigenbaum, d. 1991. food and feeding behavior. in bone, q., h. kapp, pierrot-bults, a.c. (ed.).the biology of chaetognaths. oxford university press, oxford, 45-54 pp. composition, abundance and distribution of chaetognaths 23 johnson, t.b., nishikawa, j and terazaki, m. 2006 community structure and vertical distribution of chaetognaths in the celebes and sulu seas. coastal marine science 30(1): 360-372 johnson, t.b. 2005. ecological study of pelagic chaetogntahs in the pacific ocean. the university of tokyo, tokyo japan. unpublished doctoral dissertation: 171 pp. johnson, t.b. and terazaki, m. 2003. species composition and depth distribution of chaeognthas in a kuroshio warmcore ring and oyashio water. journal of plankton research. 25(10): 1279-1289. jumao-as mdb and von westernhagen, h. 1975. vertical distribution of epiplanktonic chaetognaths in the upper 100m of the hilutungan channel, cebu, philippnes. mar. biol. 29: 2001-210. kehayias, george. 2003. quantitative aspects of feeding of chaetognaths in the eastern mediterranean pelagic waters. j. mar. biol. ass. u. k. 83, 559-569. michel, h.b. 1984. noaa technical report nmfs 15: chaetognaths of the caribbean sea and adjacent areas. noaa, 1-33 pp. michael, e.l. 1911. classification and vertical distribution of the chaetognatha of the san diego region. univ. calif. publ. zool. 8:20-186. michael, e.l. 1919. report on the chaetognatha collected by the united states fisheries steamer "albatross" during the philippine expedition, 1970-1910. buluu. u.s. natn. mus 100 (1): 235-277. primavera, k., san diego-mcglone, l. and jacinto, g. 2002. chemical hydrography and primary production. upv j. nat. sci. 7 (1&2): 32-41. reeve, m. r., 1970. the biology of chaetognatha. quantitative aspects of growth and egg production in sagitta hispida. in steele j.h. (ed). marine food chains. edinburg: 168-189 pp. rottman, m.l. 1978. ecology of recurrent groups of pteropods, euphausiids, and chaetognaths in the gulf of thailand and the south china sea. mar biol. 48:63-78. terazaki, m. and miller, c.b. 1986. life history and vertical distribution of pelagic chaeognaths at ocean station p in the subartic pacific. deep-sea res. 33: 323-337. inside front cover-19-1 editorial board editor in chief rhodora v. azanza associate editors flerida a. cariño biomolecular sciences florecita s. de guzman organic chemistry caesar a. saloma computer science, engineering, mathematics, and physics fernando p. siringan earth sciences (geology) cesar l. villanoy earth sciences (oceanography) corazon d. villareal managing editor dercylis g. mararac editorial assistant science diliman (issn 0115-7809) is published semi-annually by the research dissemination and utilization office (rduo) of the office of the vice chancellor for research and development (ovcrd), university 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university dumaguete city, philippines sumanila@psdn.org.ph; suakcrem@philwebinc.com felixberto a. buot, ph.d. naval research laboratory washington, d.c. buot@estd.nrl.navy.mil; fab@cfdrc.com josefino c. comiso, ph.d. laboratory for hydrospheric processes nasa goddard space flight center greenbelt, maryland comiso@joey.gsfc.nasa.gov lourdes j. cruz, ph.d. the marine science institute university of the philippines diliman quezon city, philippines luly@upmsi.ph edgardo d. gomez, ph.d. the marine science institute university of the philippines diliman quezon city, philippines edgomez@upmsi.ph per juel hansen, ph.d. marine biological laboratory university of copenhagen helsingor, denmark pjhansen@zi.ku.dk jorgen hylleberg, ph.d. department of biological sciences and marine ecology university of aarhus aarhus, denmark hylleberg@biology.aau.dk chris m. ireland, ph.d. department of medicinal chemistry university of utah salt lake city, utah cireland@deans.pharm.utah.edu international advisory committee emil q. javier, ph. d. institute of plant breeding university of the philippines los baños college, laguna, philippines emil.javier@cgiar.org amador muriel, ph.d. data transport system new york, new york dtsny@msn.com baldomero m. olivera, ph.d. department of biology university of utah salt lake city, utah olivera@biology.utah.edu eduardo a. padlan, ph.d. kensington, maryland edpadlan@aol.com william g. padolina, ph.d. international rice research institute college, los baños, laguna w.padolina@cgiar.org kelvin s. rodolfo, ph.d. department of earth and environmental sciences university of illinois chicago, illinois krodolfo@uic.edu gary e. rodrick, ph.d. department of food science and human nutrition university of florida gainesville, florida gerodrick@mail.ifas.ufl.edu francis j. schmitz, ph.d. department of chemistry and biochemistry university of oklahoma norman, oklahoma fjschmitz@chemdept.chem.ou.edu gavino c. trono jr., ph.d. the marine science institute university of the philippines diliman quezon city, philippines trono@upmsi.ph 01_device some aspects of the population biology 1 some aspects of the population biology of the green tiger prawn penaeus semisulcatus (de haan, 1844) from pilar and capiz bays, northern panay, west central philippines science diliman (january-june 2006) 18:1, 1-10 *corresponding author karen a. villarta*1,2, annabelle g.c. del norte-campos1 & wilfredo l. campos2 1marine biology laboratory and 2ocean bio laboratory, division of biological sciences, college of arts and sciences (cas), university of the philippines in the visayas, 5023 miag-ao, iloilo tel. no. +63 33 3159636, telefax no. +63 33 3159271 upvmarbio@yahoo.com, karenvillarta@yahoo.com date submitted: january 27, 2006; date accepted: september 1, 2006 abstract this study is a first report on the population biology of the green tiger prawn penaeus semisulcatus (de haan) from northern panay, west central philippines. the study was conducted for eight months (may to december 2002) whereby total lengths of both male and female p. semisulcatus of various sizes were measured monthly from the catches of municipal trawlers operating at pilar and capiz bays. based on the bhattacharya method, a mean growth rate of 0.78 ± 0.28 and 1.45 ± 0.39 mm/day were estimated for males and females, respectively. using the elefan i method, growth parameters derived for males were l∞ = 263 mm, k = 0.7/yr and a growth index (ø') of 4.69. on the other hand, growth parameters derived for females were l∞ = 271 mm, k = 1.6/yr and a growth index (ø') of 5.07. based on length-converted catch curve analysis, the total mortality (z) of the male population is estimated to be 3.61/yr while that of the females is 5.65/yr. male prawns showed a higher exploitation rate (0.53) compared to that of the females (0.35) indicating the susceptibility of males to fishing. this study also revealed that trawlers in pilar and capiz bays are already getting small sizes of prawns, without allowing them to reach sexual maturity. hence, there is a need to increase the present mesh size (2.5 cm) of the cod end of trawls in order to avoid growth overfishing, which may occur with continued increase in fishing effort. furthermore, the recruitment pattern showed two pulses of unequal strengths and time, dividing the year into a 7-5 month pattern. the said pattern, especially for females, may have resulted from a major and minor spawning peak of the said species during the months of june-september and january. keywords: penaeus semisulcatus, growth, recruitment pattern, mortality, pilar and capiz bays villarta, del norte-campos, campos 2 introduction the green tiger prawn, penaeus semisulcatus de haan (1844), locally known in panay as "bulik", is known to inhabit sandy or muddy-sand bottoms and is caught by trawls in depths down to 130 meters (dore and frimodt, 1987; fao, 1998). in pilar and capiz bays, northern panay the trawl fishery exists mainly for the invertebrates (del norte-campos et al. 2003). p. semisulcatus ranks high among commercially important invertebrates in panay and demands a high price in the local market making it one of the target species of trawl operators (del norte-campos et al. 2000). in the philippines, agasen and del mundo (1988) studied the growth, mortality and exploitation rates of the white prawn penaeus indicus in manila bay. in spite of their significance, there is no other account on the quantitative assessment of shrimp resources here in the country. as for p. semisulcatus, studies that have been conducted in the philippines focused on the food and feeding habits (tiews et al. 1976; del nortecampos et al. 2004) and the reproductive cycle (villarta and del norte-campos, 2004) of the species. outside the country, however, several studies on some aspects of the population dynamics of the species have been undertaken (thomas 1975, garcia and le reste 1981, mohamed et al. 1981, kirkwood and somers 1984, siddeek 1991, xu et al. 1995, ye and mohammed 1999, and mehanna 2000). here in the country, studies on population dynamics have focused mainly on fish while the arguably equally important marine invertebrates have received less attention. penaeid shrimps, for example, are less studied although they are one of the most valuable fishery resources. these commercially exploited species are not only consumed locally but exported outside the country as well. in bfar statistics (2003), shrimp/ prawn ranked 2nd among major fishery exports in terms of value. this strong demand may result to a further increase in the fishing effort in the trawl fishery which may affect the total production of shrimp in the country. in this line, it is important to study not only their reproductive activity but also the effect of the rate of exploitation to the dynamics of its population. hence, there is a need for studies on the biology and stock assessment of the species to ensure that these resources continue to be sustainable. the present paper is the first account of some aspects of the population biology of p. semisulcatus from pilar and capiz bays, northern panay, as an endeavor to contribute information to its general biology. this study aims to estimate some basic parameters needed for establishing a management plan to ensure that this resource continue to be viable. materials and methods study area and survey the sampling was conducted for eight months (may to december 2002) in libas port, brgy. libas, roxas city. total length of shrimps, measured from the tip of rostrum to the tip of the telson, was determined to the nearest millimeter using a ruler. measurements were done monthly for both male and female p. semisulcatus of various sizes taken from the catches of municipal trawlers, which operate in pilar and capiz bays, northern panay, located between 122°40'-55' e and 11°36'-43' n (figure 1), and dock at libas port. the depth of trawling operation for these vessels is between 5-25 fathoms (9-46 m) with the net used for fishing having a mesh size at the cod end of 2.5 cm. laboratory and data analyses monthly length measurements (tl) were grouped into 10 mm size classes (50-50.99, 60-69.99, 70-79.99, etc. mm) and analyzed using the bhattacharya (1967) method, incorporated in the fisat software (fisat, 1997). to compute growth rates (mm/day) by cohort, increments between the modal lengths (mm) derived using the bhattacharya method, were divided with their respective time increments (days). mean growth rates for each cohort were averaged to estimate the annual mean growth rate for all cohorts. the growth parameters l∞ and k were derived using the elefan i. the growth index ø' was computed according to the equation of pauly and munro (1984) as follows: some aspects of the population biology 3 nnn antiq ue aklan capiz ilo ilo antiq ue aklan capiz ilo ilo antiq ue aklan capiz ilo ilo l i . cu a s fo r p tc a p i z b a y 122040’ 122054.8’ 11035’ 11040’ 11045’ l i . cu a s fo r p tc a p i z b a y 122040’122040’ 122054.8’122054.8’ 11035’11035’ 11040’11040’ 11045’11045’ figure 1. map showing the study area, pilar and capiz bays, northern panay. numbers indicate depths in fathoms. villarta, del norte-campos, campos 4 ø ' = log k + 2 log l∞ where k is the growth constant per year and l∞ is the asymptotic length (tl). the values derived for growth were then compared with those of other studies on penaeid shrimps. the mean recruitment patterns, as well as the instantaneous total mortality (z per year) were determined using the elefan ii program. in this method, the recruitment pulses were reconstructed from the length-frequency data to determine the number of pulses per year and the relative strength of each pulse. elefan ii outputs the points in percent of the recruitment in one year; from this a recruitment graph is made. the peaks show the number of recruitment seasons. total mortality z was derived using the lengthconverted catch curve analysis. this was estimated from the slope of the catch curve derived from the length frequency data. the catch curve is the graph of the natural logarithm of the number of prawns of a given age against their age. natural mortality (m) was estimated by computing an average m/k ratio from shrimp literature (agasen and del mundo 1988, sumiono 1988, mehanna 2000 and campos and berkeley 2003) then multiplying this value by the k derived from the length frequency data for the species. fishing mortality (f) was then computed based on the definition: f = z m. exploitation rate, i.e. the rate of fishing mortality to total mortality was computed from: e = f/z. the derived estimates were then compared with other studies to allow further assessment. results and discussion growth the total number of samples ranged from 90 to 209 for females and 194 to 220 per month for males. size frequency data, for both female and male, collected from may to december 2002 are plotted in figures 2 and 3, respectively. for females, the smallest individuals (75 mm tl) were observed in the months of may, june, august and september, while the largest figure 2. length frequency distribution of the green tiger prawn penaeus semisulcatus (female), may-december 2002, collected from pilar and capiz bays, northern panay. fr eq ue nc y (% ) 0 10 20 30 40 jun n=209 0 10 20 30 40 jul n=230 0 10 20 30 40 aug n=203 0 10 20 30 40 sept n = 209 0 10 20 30 40 oct n = 113 6 3 5 4 12 0 10 20 30 40 may '02 n=209 0 10 20 30 40 dec n = 907 nov n = 144 0 10 20 30 40 fr eq ue nc y (% ) 0 10 20 30 40 jun n=209 0 10 20 30 40 jul n=230 0 10 20 30 40 aug n=203 0 10 20 30 40 sept n = 209 0 10 20 30 40 oct n = 113 6 3 5 4 12 0 10 20 30 40 may '02 n=209 0 10 20 30 40 dec n = 907 nov n = 144 0 10 20 30 40 0 10 20 30 40 jun n=209 0 10 20 30 40 jul n=230 0 10 20 30 40 aug n=203 0 10 20 30 40 sept n = 209 0 10 20 30 40 oct n = 113 6 3 5 4 12 0 10 20 30 40 may '02 n=209 0 10 20 30 40 dec n = 907 nov n = 144 0 10 20 30 40 total length (mm) fr eq ue nc y (% ) total length (mm) sizes (> 215 mm) were found in all months (figure 2). most individuals, however, fell within the range of 145195 mm tl. the smallest male individual (65 mm tl) was found in the month of june while large sizes (> 215 mm) were found in the month of may (figure 3). most male prawns fell within the range of 115-155 mm tl. these results reveal that females are generally larger than the males. villarta and del norte-campos (2003) reported that the minimum sizes of sexual maturity for male and female green tiger prawn from pilar and capiz bays are 123 may ‘02 n=209 jun n=209 123 4 56 7 jul n=230 aug n=203 sept n=209 oct n=113 nov n=144 dec n=90 some aspects of the population biology 5 and 145 mm tl, respectively. furthermore, partially spawned gonads were already observed at smaller sizes, based on histology, (i.e. 104 mm for males and 143 mm for females) indicating that this species can be mature at sizes smaller than what were recorded. using this as basis, it was computed from the lengthfrequency data that 1.25% of male and 24.4% of female samples were not yet sexually mature. this indicates that trawlers in pilarand capiz bays are already getting smaller sizes of prawns, without allowing them to reach sexual maturity. a further increase in fishing effort may result in growth overfishing, which takes place when resources are fished at an average size that is smaller than the size that would produce the maximum yield per recruit. to avoid this, it is important that p. semisulcatus be caught at sizes larger than the recorded size at sexual maturity. hence, there is a need for an increase in the currently used mesh size (2.5 cm) of the cod end of trawls operating in the area. based on the bhattacharya method, a total of 7 cohorts were derived for females (figure 2). daily growth rates ranged from 0.51 to 1.98 mm/day, with a mean annual growth rate of 1.45 ± 0.39 mm/day (n=14) (table 1a). on the other hand, 4 cohorts were derived for male prawns (figure 3), with daily growth rates ranging from 0.49 to 1.03 mm/day and a computed mean annual growth rate of 0.78 ± 0.28 mm/day (table 1b). the relatively higher growth rate of the female green tiger prawn is in agreement with the larger sizes they attain as also pointed out by thomas (1975). this result also agrees with mehanna (2000) who also reported a higher growth rate of female p. semisulcatus from the gulf of suez, egypt. the von bertalanffy growth function parameter estimates derived for females using elefan i were l∞ = 271 mm and k = 1.6 per year. the growth index (ø') computed from these parameters was 5.07. vbgf parameters for males were l∞ = 263 mm and k = 0.7 per year and a growth index (ø') of 4.69. it has been observed that the growth constant (k) was higher in females confirming the high growth rate drawn from the bhattacharya analysis. however, this estimated k for males differed markedly from other estimates reported in other studies. this may have resulted from the high value for male asymptotic length (l∞) derived in this study compared to those obtained from other figure 3. length frequency distribution of the green tiger prawn penaeus semisulcatus (male), may-december 2002, collected from pilar and capiz bays, northern panay. fr eq ue nc y (% ) 1 2 3 4 0 10 20 30 40 may '02 n = 194 0 10 20 30 40 jun n = 219 0 10 20 30 40 jul n = 209 0 10 20 30 40 aug n = 217 0 10 20 30 40 sept n = 209 0 10 20 30 40 oct n = 203 0 10 20 30 40 nov n = 208 0 10 20 30 40 dec n = 220 fr eq ue nc y (% ) 1 2 3 4 0 10 20 30 40 may '02 n = 194 0 10 20 30 40 jun n = 219 0 10 20 30 40 jul n = 209 0 10 20 30 40 aug n = 217 0 10 20 30 40 sept n = 209 0 10 20 30 40 oct n = 203 0 10 20 30 40 nov n = 208 0 10 20 30 40 dec n = 220 1 2 3 4 0 10 20 30 40 may '02 n = 194 0 10 20 30 40 jun n = 219 0 10 20 30 40 jul n = 209 0 10 20 30 40 aug n = 217 0 10 20 30 40 sept n = 209 0 10 20 30 40 oct n = 203 0 10 20 30 40 nov n = 208 0 10 20 30 40 dec n = 220 total length (mm)total length (mm) fr eq ue nc y (% ) may ‘02 n=194 jun n=219 jul n=209 aug n=217 sept n=209 oct n=203 nov n=208 dec n=220 1 2 3 4 areas. nevertheless, the growth performance indices (ø') for both male and female obtained from this study compare well with the study of mehanna (2000) for the same species. to further allow comparisons with the derived estimates, growth parameters for other penaeid species from other areas are shown in table 2. the growth parameters derived using the bhattacharya analysis and the von bertalanffy growth formula were found to agree quite well. the estimated values from villarta, del norte-campos, campos 6 both methods gave similar trends in the growth rates of both sexes of green tiger prawn. vibhasiri (1988) also stressed the aptness of the use of the said methods for metapenaeus affinis in the gulf of thailand. since molts are separated by intermolt periods in the growth pattern of penaeid shrimps (garcia, 1988), the modal progression analysis of carapace length or body length is usually used for age estimation. also, it has been well reported that both male and female penaeids attain their highest growth rate during the first three months of life, after which, the increment in length gradually decreases with further increase in age. based on the data, the oldest calculated ages for p. semisulcatus in the sample are 2.4 yrs for females and 3.8 yrs for males. these values may be assumed to be the longevity (tmax) periods for both the male and female. for the females, this result compare well with that of mehanna (2003) for the same species in the gulf of suez. however, longevity for males in this study was found to be double that of the male population in gulf of suez. this may be attributed to the low k value obtained for males. it may be that the growth formula used in this study was not appropriate for the male samples since it may have been biased towards smaller male individuals which occurred in higher frequencies, thus resulting in the low k value. recruitment pattern the derived recruitment pattern for both female and male p. semisulcatus using elefan ii consists of two peaks of unequal strengths and durations, dividing the year into a 7-5 month pattern (figure 4). major peak table 1. growth rates of the derived cohorts of female (a) and male (b) penaeus semisulcatus sampled from may-december 2002 in pilar-capiz bays, northern panay. a. a. cohort no. lt + 1 lt lt + 1 lt t+1 t growth rate (mm) (mm) (mm) (days) (mm/day) i 219.23 203.84 15.39 30 0.51 ii 183.49 154.52 28.97 30 0.97 iii 222.87 175.10 47.77 30 1.59 175.10 138.28 36.83 30 1.23 138.28 88.08 50.20 30 1.67 iv 220.00 171.70 48.30 30 1.61 171.70 141.43 30.26 30 1.01 141.43 88.15 53.28 30 1.78 v 221.89 177.65 44.24 30 1.47 177.65 128.16 49.50 30 1.65 vi 236.96 190.24 46.72 30 1.56 190.24 136.09 54.15 30 1.81 136.09 93.33 42.76 30 1.43 vii 205.95 146.42 59.53 30 1.98 mean 188.63 145.21 43.42 30.00 1.45 sd 33.94 37.05 11.83 0.0 0.39 n 14 14 14 14 14 b. cohort no. l2 l1 lt + 1 lt t+1 t growth rate (mm) (mm) (mm) (days) (mm/day) i 168.05 137.87 30.18 30 1.01 ii 144.28 129.55 14.74 30 0.49 iii 152.21 121.30 30.91 30 1.03 iv 136.50 118.97 17.53 30 0.58 mean 150.26 126.92 23.34 30.00 0.78 sd 13.48 8.59 8.40 0.0 0.28 n 4 4 4 4 4 b. some aspects of the population biology 7 for females accounted for 92.04% and the remaining 7.96% for the minor peak. on the other hand, the major recruitment peak for males accounted for 90.86% and 9.14% for the minor peak. the length-frequency data for female individuals show highest recruitment during the months of may, june, august and september, whereby, small individuals occur in significant number. based on the reproductive data of females (villarta and del norte-campos, 2003), spawning activity peaks during the southwest monsoon (june to september), which could be taken to correspond to this major recruitment pulse. the minor pulse, on the other hand, occurred in january corresponding to individuals with developing gametes. crocos and van der velde (1995) also described a bimodal spawning pattern for penaeids. however, despite the 2 peaks recruitment occurs yearround since mature females can be found at any time of the year (villarta and del norte-campos, 2003). the case is also true for males wherein they are reported to have a gametogenic activity the entire year; hence, recruitment also occurs the entire year. mortality based on length-converted catch curve analysis the total mortality (z) for both female and male p. semisulcatus is equivalent to 5.65 and 3.61, respectively. as estimated from m/k values averaged from literature (table 3) and subsequently multiplied with the estimated k for male and female prawns in this study, the values for natural mortality (m) are 3.65 for females and 1.70 for males. these values when subtracted from the z values give estimate fishing mortality (f) values of 2.00 and 1.91 for females and males, respectively. exploitation rates for both sexes were then computed to be 0.35 for females and 0.53 for males. as shown in table 3, results from other studies on penaeid prawns show higher mortality values in males than in females contrary to what was obtained from this study. nonetheless, a similar trend in exploitation rate (e) was observed wherein males obtained higher values compared to those of the females. as suggested by gulland (1971), if natural mortality is less than or equal to fishing mortality (i.e., if e > 0.5) then the stock is said to be overexploited. in this study, it was observed that the exploitation rate (e) for male green tiger prawn was greater than 0.5, indicating that the male stock is exploited. this may be attributed to the differences in behavior between sexes. as in the case of penaeus table 2. growth parameters of some penaeid species in various areas of study (ø' + log k + 2 log l∞∞∞∞∞ ). species cl” (mm) tl” (mm) k (per yr) area and year literature penaeus 271.0 263.0 1.60 0.70 5.07 4.69 pilar-capiz bays, this study semisulcatus philippines, 2002 268.0 224.0 1.56 1.77 5.05 4.95 gulf of suez, egypt, mehanna 1997/1998 (2000) 48.00 39.50 1.69 1.33 3.59 3.32 southern kuwaiti waters, siddeek and 1982-1983 abdul-ghaffar, 1991, in siddeek (1991) p. orientalis 201.3 163.5 po hai sea, china jingyao, 1981 in ye (1984) p. indicus 41.50 40.50 210.0 205.0 1.00 1.20 2.64 2.70 manila bay, 1982 agasen and del mundo (1988) 44.70 43.40 226.0 220.0 1.00 1.20 2.71 2.75 punnaikkayal, india, 1978 agasen and del mundo (1988) 42.34 40.70 214.0 206.0 1.10 2.71 manappad, india, 1978 agasen and del mundo (1988) p. merguiensis 53.10 43.80 1.15 1.60 3.51 3.49 south coast of java sumiono (1988) 1977-1979 53.20 42.40 0.90 1.50 3.41 3.43 south coast of java sumiono (1988) 1982-1984 p. duorarum 43.40 33.20 187 146.1 1.72 1.61 3.70 3.46 biscayne bay, florida campos and 1986 berkeley (2003) + + + + ø ' villarta, del norte-campos, campos 8 merguiensis from the south coast of java (sumiono, 1988), high mortality and exploitation rates observed in male p. semisulcatus from pilar and capiz bays could be a result of their vulnerability to the fishery than females of the same size. it may be that females have different migration patterns and move out of the fishing grounds for spawning. p. semisulcatus which has a type 3 life cycle spawns offshore and the larvae develop there, while the juveniles develop in inshore waters in seagrass or algal beds (haywood et al., 1995). this movement of adults from shallow coastal waters to offshore to mate and spawn is observed generally in penaeids (baldock, 1999; fao, 1998) although it is also reported that penaeid prawns, in some areas, undertake a short shoreward migration before spawning (dall et al. 1990; garcia, 1988; ye, 1984). in one study, george and rao (1967) attributed variation in sex ratios of different penaeid prawns to the movement of females out of the fishing grounds to deeper waters for spawning. rao (1989) also suggested a sex-wise figure 4. recruitment pattern of male and female p. semisulcatus from pilar and capiz bays, northern 0 5 10 15 20 25 1 2 3 4 5 6 7 8 9 10 11 12 relative time (months) % r ec ru itm en t male female table 3. comparison of mortality indices in different species of prawns from various areas of study. species z (per yr) m (per yr) k (per yr) m/k f f/z area and year literature penaeus 5.65 3.61 3.65 1.70 1.60 0.70 2.00 1.91 0.35 0.53 pilar-capiz bays, this study 2002 semi6.77 8.18 2.20 2.52 1.56 1.77 1.41 1.42 4.57 5.66 0.68 0.69 gulf of suez, egypt, mehanna (2000) sulcatus 1997/1998 p. indicus 4.34 4.90 1.94 2.20 1.00 1.20 1.94 1.83 2.40 2.70 0.55 0.55 manila bay, 1982 agasen and del mundo (1988) 3.34 5.37 1.83 2.08 1.00 1.20 1.83 1.73 1.51 3.29 0.45 0.61 punnaikkayal, india, agasen and 1978 del mundo (1988) 2.94 1.98 1.10 1.80 0.96 0.33 manappad, india, agasen and 1978 del mundo (1988) p. mer4.50 11.10 3.40 6.50 1.15 1.60 2.96 4.06 1.10 4.60 0.24 0.41 south coast of java sumiono (1988) merguiensis 1977-1979 4.90 9.50 3.40 6.50 0.90 1.50 3.78 4.33 1.50 3.00 0.31 0.32 south coast of java sumiono (1988) 1982-1984 p. duo3.84 3.88 3.00 2.99 1.72 1.61 1.75 1.86 0.84 0.89 0.22 0.23 biscayne bay, campos and rarum florida, 1986 berkeley (2003) mean 4.76 6.18 2.77 3.31 1.28 1.33 2.28 2.43 1.99 2.88 0.40 0.46 + + + + + + some aspects of the population biology 9 segregation of the brown prawn metapenaeus monoceros in the fishing grounds of kakinada coast, india, as an explanation to significant differences in sex ratio, while ramamurthy et al. (1978) ascribed it to breeding movements. furthermore, this difference in sex ratio may be due to differential growth and mortality between the sexes as pointed out by devi (1987) for the indian white prawn p. indicus from kakinada, east coast of india. hence, gear efficiency of the trawl may be dependent on the behavior differences between sexes since trawlers in pilar and capiz bays operate only within 5-25 fathoms albeit year-round. acknowledgements this work is part of the afma-cfn program "assessment of commercially-important invertebrate resources in inter-island waters (panay)". the authors are grateful for the support and field assistance of noemi, solomon and regina alisasis of roxas city who made data collection possible. the authors are also thankful for the field and laboratory assistance of m. declarador, j. panes and r. beldia. references agasen, e.v. and c.m. del mundo. 1998. growth, mortality and exploitation rates of penaeus indicus in manila bay, philippines and 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(1988). an assessment of jinga shrimp, metapenaeus aiffinis (penaeidae), in ban don bay, gulf of thailand. in venema, s.c., j. möller-christensen and d. pauly (eds.). fao fisheries report 389. rome. villarta, k.a. and a.g.c. del norte-campos. 2004. reproductive cycle of the green tiger prawn penaeus semisulcatus (de haan, 1844) from pilar-capiz bays, northern panay. upv j. nat. sci. 9(1): 147-156. xu, x., j.m. bishop, h.m.a. mohammed, and a.h. alsaffar. estimation of the natural mortality rate of green tiger prawns penaeus semisulcatus (de haan, 1844) in kuwait waters using relative abundance data. j. shellfish res. 14(1): 179184. ye, c.c. 1984. the prawn (penaeus orientalis kishinouye) in pohai sea and their fishery. in j.a. gulland and b.j. rothschild (eds.), penaeid shrimps: their biology and management. fishing new books, farnham, england, p. 49-59. ye, y. and h.m.a. mohammed. 1999. an analysis in variation in catchability of green tiger prawn, penaeus semisulcatus, in waters off kuwait. fish. bull. 97: 702 712. 01_device institutional arrangements and processes in marine fishery reserves 47science diliman (july-december 2006) 18:2, 47-56 *corresponding author institutional arrangements and processes in marine fishery reserves-sanctuaries establishment in lagonoy gulf raul g. bradecina*1 and plutomeo nieves2 1partido state university nato, sagñay, camarines sur email: rgbradecina@yahoo.com 2bicol universitytabaco campus2 tayhi, tabaco, albay date submitted: april 18, 2006; date accepted: september 13, 2006 abstract this paper described the processes and institutional arrangements of mfr-s’ establishment in lagonoy gulf from period 1993 to 2004. the analysis made use of primary and secondary data mainly derived from key informant interviews and participatory resource assessment (pra). results showed that the establishment of marine fisheries reserve-sanctuary in lagonoy gulf started in 1993. during the ten-year period between 1993 and 2004, a total of 8 mfr-s were established with majority in albay and the least in camarines sur. two categories of institutional arrangements were identified in the gulf, namely: lgu-initiated and community-initiated, commonly facilitated by external agents during the project's incipient stage. three types of institutional partnership emerged namely: local government unit-non-government organizations/ national government agencies (lgu-ngos/ ngas) and people's organization (po)-academe partnerships. the institutional arrangement and type of partnership determine the process and mechanism of implementation of mfr-s in lagonoy gulf. an academe-facilitated process focuses on research as starting point. ngo-facilitated process starts generally with community organizing, while nga mostly proceed with policy implementation. the communityacademe partnership is strong in the preparatory and planning phase. the lgu-ngo/nga partnership on the other hand, is strong in the ordinance adoption phase. some factors could derail or facilitate the development of the establishment process in the logical order, these are: the capability of the lgu, the agenda and the long-term commitment of external agents, the interplay of these hindering and facilitating factors in the establishment process of mfr-s developed a 3-phase logical establishment process model for lagonoy gulf consisting of the following phases: (1) preparation and planning; (2) ordinance approval; and (3) implementation and management. this typology evolved two distinct types of mechanism of implementation, namely: (i.) a mechanism where the mfrs planning preempts the ordinance for its establishment; and (ii.). a mechanism where the mfr-s planning precedes the ordinance for its establishment. the former type of implementation mechanism featured an enactment of ordinance establishing the mfr-s followed by a community consultation and bradecina and nieves 48 introduction lagonoy gulf is the largest and the most resource-rich fishing ground in the bicol region. it is bordered by the three provinces of camarines sur, catanduanes and albay, comprising 486 barangays in 14 coastal municipalities. existing literature described in dramatic terms the issues of poverty and degradation of coastal environment in lagonoy gulf. in the last decade, several coastal resource management projects have been introduced in the gulf to comprehensively address these issues. marine fishery reserve-sanctuary (mfr-s) establishment is an effective tool for resource generation within the framework of coastal resource management (hermes, 2003). most of these mfr-s serves as a management tool and entry point for coastal resource management in lagonoy gulf to effect sustainable development. as one crm practitioner puts it, "sustainable development entails more than the environmental protection of the coastal systems. it is a process in which policies are designed to bring development that is economically viable, socially just, politically participatory and ecologically sustainable. it depends among others, on the effectiveness of the institutional arrangements as well as interactions among entities involved in the management area". this necessitates institutional analysis to determine the way in which these resources can be better managed to meet the needs of the present generation without compromising the future generation of their own needs. management planning. the scheme resulted to the non-integration of the mfr-s management plan into the ordinance that established it. while the latter type of implementation mechanism featured a community consultation and mfr-s establishment (barangay crm) followed by the enactment of an ordinance establishing an mfr-s by the municipal government. the scheme resulted to the integration of the mfr-s management plan into the ordinance that established it. a more appropriate and effective mfr-s establishment process could be evolved from the lagonoy gulf experience by placing the different institutional arrangements, phase sequence and process typologies at appropriate periods capitalizing on their respective strengths. keywords: institutional arrangements, marine fisheries-reserve establishment process, coastal resource management, lagonoy gulf the passing of the local government code in 1992 devolved the authority as well as the responsibility of managing municipal waters to lgus. as a tool for coastal resource management, the establishment of mfr-s has been supported by some lgus, research institutions, ngos and by multilateral and bilateral donor organizations, employing various strategies and approaches. the fisheries code enacted in 1998 which granted the lgus the jurisdiction and authority to manage, conserve, develop, protect and utilize and dispose all fish and fishery aquatic resources within their respective municipal waters provided coastal communities in lagonoy gulf greater autonomy in mfr-s management. the new code institutionalized the role of local fishermen and resource users in the community based planning and implementation of policies and programs in coastal resource management by the creation of barangay fisheries and aquatic resources management councils (bfarmcs). some mfr-s implementers capitalized on this development in pursuing mfr-s management. the impetus for mfr-s establishment in lagonoy gulf was further complemented by the agriculture and fisheries modernization act (afma) that upheld the principle of sustainable development and people empowerment, among others, in fisheries development. the implementation of the fishery sector program (fsp 1) from 1991 to 1997 in 12 bays in the country with the support of asian development bank (adb) generated and initiated crm plan for lagonoy gulf. the program pursued as a strategy, the generation of institutional arrangements and processes in marine fishery reserves 49 bay wide crm plans through the involvement of fishing communities by contracting non government organizations (ngos). the gulf wide project together with the national policy efforts helped establish the first generation of mfr-s in the gulf. a continuation of the fsp, the fishery sector resource management program (frmp) in 1998-until present year, succeeded these efforts to achieve sustainable development of the fisheries sector and the reduction of poverty among municipal fishermen in the gulf through a series of interventions to reverse the trend of fisheries resource depletion in municipal waters and address the issues on poverty. fisheries resource management as one of the components of the program supported mfr-s establishment as a key crm strategy. these developments together with their respective socio-political and geographic conditions shaped the institutional arrangements and processes of mfr-s establishment in lagonoy gulf in the last ten years. the progress, the institutional arrangements and the processes involved in the course of the establishment and management of this mfr-s were not documented. with their potential as an effective tool for resource management, there is a need to gain insights from various mfr-s establishment experiences to improve the process and develop models that will ensure a relative degree of success. this paper described the process and institutional arrangements involved in establishing mfr-s in lagonoy gulf from period 1993 to 2004. methodology participatory resource assessments using focus group discussion and key informant interview were conducted in six mfr-s sites in lagonoy gulf to gather primary data. document analysis using technical reports, municipal ordinances, project evaluation papers, brochures and similar publications covering mfr-s established from 1993 to 2004 were used to enhance the quality of the data. case studies of establishment processes and institutional arrangement of mfr-s establishment in atulayan island, san miguel island and agojo representing the provinces of camarines sur, albay and catanduanes respectively were done. results and discussion marine fishery reserves-sanctuaries established in lagonoy gulf table 1 shows the historical sequence and provincial locations of mfr-s in lagonoy gulf from 1993 to 2002. the first to be established were the mfr-s in atulayan island, sangay, camarines sur, agojo in san andres, catanduanes and gaba, in rapu-rapu, albay. the latest is the mfr-s in bato, batalay, catanduanes. three mfr-s were established in 1993, while only one was established in 1998. between 2001 and 2002, three mfr-s were established (david, et al. 2004). from 1993 to 2002, a total of 8 mfr-s projects have been implemented in lagonoy gulf. five of these are in albay, two are in catanduanes and one is in camarines sur. the pioneer mfr-s such as atulayan island and agojo were established within the implementation period of the fsp 1 in lagonoy gulf. in contrast, the younger mfr-s were established at the height of the frmp implementation between 2001 and 2002. only one mfr-s (malinao) was implemented through the community-based coastal resource management table 1.time line and provincial locations of mfr-s in lagonoy gulf year program existing legal framework mfr-s province 1993 fsp pd 704, lgc 1992 gaba, rapu-rapu albay 1993 fsp pd 704, lgc 1992 atulayan island, sangay camarines sur 1993 fsp pd 704, lgc 1992 agojo, san andres catanduanes 1998 fsp ra 8550, afma, lgc 1992 sagurong, san miguel island albay 2002 frmp ra 8550, afma, lgc 1992 tiwi albay 2002 cbcrmp ra 8550, afma, lgc 1992 malinao albay 2002 frmp ra 8550, afma, lgc 1992 ponco-baranca, bacacay albay 2001 frmp ra 8550, afma, lgc 1992 bato, batalay catanduanes bradecina and nieves 50 project (cbcrmp) of the department of finance within the same period. the existing legal framework and program interventions shaped the institutional arrangement and process of the mfr-s in lagonoy gulf. the presidential decree (pd) 704 and the local government code of 1992 were the existing institutional mechanisms when the mfr-s in atulayan island in sangay, camarines sur, agojo in san andres catanduanes and gaba in rapu-rapu, albay were established. environmental protection is the more influential agenda that shaped the establishment process of these pioneer mfr-ss. the implementation of fishery ordinances, policy implementation and lguinitiation of the project characterized the establishment process of most of these mfr-ss. when the local government code of 1992 devolved the right to manage the municipal fisheries to the local government units (lgus) from bureau of fisheries and aquatic resources (bfar), the lgu of sagñay, at the initiative of bfar, established the first mfr-s in lagonoy gulf in atulayan island. the implementation of the fisheries sector program (fsp 1) in 1995 and the fisheries resource management project (frmp) in 1998 by the department of agriculture (da) -bfar in lagonoy gulf which viewed coastal resource management as an approach to address the problems faced by the marine resources had brought to fore the concept of multisectoral collaboration between ngo, academe, people's organization (po) and the establishment of mfr-s as one of the strategies in coastal resource management (pelea, et al, 2004). project components of these programs included fisheries resource management and community-organizing activities in coastal communities carried out by non-government organizations (ngos). environmental governance is the more influential agenda that shaped the establishment process of the most recent mfr-s in albay such as those in san miguel island in tabaco, tiwi, malilipot and malinao. these were established when the fisheries code (fc) of 1998 and the agriculture and fisheries modernization act (afma), aside from the local government code (lgc) of 1992 were the existing legislative mechanisms. community participation in mfr-s establishment characterized the establishment process of most of these mfr-ss. mfr-s establishment institutional arrangements and processes the establishment of mfr-s in lagonoy gulf fall under two categories -lgu-initiated or communityinitiated and commonly facilitated by external agents. the establishment process of mfr-s in the gulf was facilitated by external agents during the incipient stage. these external agents take the form of the national government agencies (nga) i.e. the bureau of fisheries and aquatic resources (bfar) and the department of environment and natural resources (denr) among others, the academe or research institutions i.e. bicol university tabaco campus (butc) and non-government organizations i.e. development research and resources productivity, inc. (drrp), community empowerment and resources development (care-cerd), bicol cooperative development center (bcdc) and aquinas university of legaspi-center for extension and research (aul-cres) (table 2). majority, or four out of six of the mfr-s in lagonoy gulf have been established through the initiatitive of the community or the fisherfolks themselves. while only two were initiated by the lgu or the municipal government. community-initiated mfr-s establishment process appeared to be a common feature in albay province. on the other hand, lgu-initiated mfr-s establishment seemed to be a common arrangement table 2 institutional arrangements of mfr-s establishment in lagonoy gulf albay cam. sur catanduanes total smi tiwi malinao bacacay atulayan agojo leading entity community 1 1 1 1 4 lgu 1 1 2 external agent nga 1 1 ngo 1 1 1 1 4 academe 1 1 institutional arrangements and processes in marine fishery reserves 51 in the provinces of catanduanes and camarines sur. as to the external agents that facilitated these mfr-s in the incipient stage, the ngos dominated in most of the mfr-s establishment in albay particularly under the frmp implementation period. while one out of the six mfr-s in lagonoy gulf was established with the nga and the academe as external agents. for instance, the bfar, a national government agency, facilitated the lgu-initiated mfr-s establishment in atulayan island, camarines sur, and the bicol university, an academe, served as the external agent that facilitated the community-initiated mfr-s establishment in san miguel island, tabaco, albay. along with these establishment processes emerged three categories of institutional collaborations in mfrs establishment in lagonoy gulf namely: poacademe, lgu/nga-po and lgu/ngo-po collaborations. the po-academe partnership, represented by the san miguel island-marine fishery reserve council (smi-mfrc) and bicol university, tabaco campus tie-up in albay proceeded by way of community-initiated activity implemented with the help of the academe (butc) as an external agent. the lguinitiated activity featured the lgu/nga-po partnership represented by sagñay lgu/ bfar and fisherfolks tie-up in atulayan island, sagñay camarines sur, with an nga (bfar) serving as external agent. similarly, a process of establishment where the lgu initiated the establishment of an mfr-s with an ngo (care) as external agent and in partnership with the fisherfolks featured the lgu/ ngo-po partnership represented by the san andres lgu/ care philippines and agojo community mangrove development organization (acomdo) tieup in agojo, san andres, catanduanes. phases and mechanisms of implementation the institutional arrangement and type of partnership determine the processes and mechanisms of implementation of mfr-s in lagonoy gulf. the processes could be summarized into three general phases: (i.) preparation and planning; (ii.) adoption/ ordinance proposal and endorsement; and (iii.) management and monitoring. these phases and their respective activities that often overlap together are presented in table 3. the phases in the establishment process of the six mfr-s studied involved one or combinations of activities. in these activities, the facilitation of external agents varies in terms of degree of involvement. the community-initiated and academeled partnership as represented by smi-mfr-s, pobutc tie-up was more extensive in the preparation and planning phase in terms of activities in the mfrs establishment process. the involvement of the academe was seen all throughout the phases. on the other hand, lgu-initiated and ngo/nga-facilitated partnerships have relatively less extensive preparatory activities, usually dominated by public hearings of proposed ordinance, community organizing and lgunga coordinative works. the involvement of lgu and ngos as external agents was seen more particularly in the incipient stage and in the preparatory phase. the lgu-initiated process featuring either an ngo or nga tie-up appeared to be characterized by relatively extensive activities in the ordinance adoption phase. both processes however, did not differ much in the number of activities in the management phase. some activities such as resource assessment, creations of management plan in the preparatory phase and communication of results to stakeholders in the management and monitoring phase were more pronounced in the academe-facilitated mfr-s establishment process. sequencing the activities involved in the mfr-ss establishment showed that resource and socioeconomic assessments were the most common activities under the preparation phase of communityinitiated, academe-facilitated institutional arrangement. meanwhile, community organizing was the most common starting point of lgu-initiated ngofacilitated arrangement. an lgu-initiated, ngafacilitated arrangement was characterized by ordinance enactment as a common activity in the preparation phase (table 4). the academe-facilitated process focused on research as starting point. on the other hand, the ngo-facilitated process usually starts with community organizing. the lgu-initiated process proceeds with enactment of ordinances and enforcement. the preparation and planning phase in san miguel island mfr in tabaco, albay started with a field study on coastal habitats, capture fisheries and socioeconomic condition of the island by the bicol university college bradecina and nieves 52 of fisheries in 1995 and 1996. after the field activities, the results of the study were presented formally to the community and the management plan was conceived with the community. within the same year, the barangay council approved the management plan. a year later, more than 233 residents of the island in a general assembly meeting approved the management plan. the second phase was carried out through the enactment of barangay resolution no.4, s. 1997 establishing the mfr-s in barangay sagurong by the barangay council. this was followed by the signing of a memorandum of agreement between the barangay council of sagurong, the butc and the municipal council of tabaco for the collaborative management of the mfr-s. a series of activities followed that resulted in the organization of the mfr-s council, a multi-sectoral governing body of the mfr-s. the representative to the mfr-s council, which served as the core group was chosen by the community. the management and monitoring phase in the mfr-s consisted of passing of municipal resolution to allocate fund for the mfr-s markers, the establishment of a philippine national police (pnp) detachment in barangay sagurong and a dialogue with the community for its maintenance in 1997. the physical construction of markers in the mfr-s and complementary mechanism for managing the mfr-s were accomplished. other mfr-s in albay such as in tiwi, malilipot and bacacay were installed recently and are in progress. installed in the light of the frmp and cb-crm programs, their establishment features a communityinitiated process through the ngos working within the context of the frmp and cbcrmp project objectives. the preparation phase of mfr-s in these municipalities included the following steps or activities, namely: the conduct of a participatory resource table 3. phases and activities in two types of mfr-s institutional arrangements in lagonoy gulf phases and activities institutional arrangements community-initiated lgu-initiated (po-academe (lgu-ngo, lgu-nga partnership) partnership involved involved external external agent agent preparation and planning resource and socio-economic assessment (rsa) x academe information and education campaign (iec) x academe x ngo barangay consultation/public hearings x academe x community organizing/ selection of representatives of mfr council (co) x x ngo mfr management planning and presentation (mp) x academe coordination, exploratory conference to create mfr nga drafting and endorsement of barangay resolutions x academe x capability building seminars academe ngo adoption/ordinance proposal and endorsement adoption of barangay resolutions x x sb session and enactment of ordinance (mo) x x sb selection and creation of mfr council representatives, induction; creation of executive committee to implement ordinance x academe x publication of ordinance and information dissemination x management and monitoring provision of fund and logistics (me) x x operationalization of bantay dagat x x establishment of pnp detachment x x enactment of management mechanisms e.g. mfr-s regulated activities x academe x provision of physical markers (me) x academe x communication of result of socio-economic surveys to stakeholders x academe institutional arrangements and processes in marine fishery reserves 53 appraisal in the sites; crm (and mfr-s) planning; and enactment of ordinance for the final establishment of the mfr-s. the implementation of strategies for mfr-s establishment in agojo, san andres, catanduanes, started from community organizing. two strategies were employed by the ngos in community organizing, namely: (1) for barangays without existing organizations, the normal process of consciousness raising and core group formation was adopted; (2) for barangays with existing organization, reorganization and strengthening were made through orientation and values development activities. the preparation phase of mfr-s establishment in atulayan, sangay, camarines sur proceeded from the encouragement of the bureau of fisheries and aquatic resources upon sangay lgu in pursuance of the provisions of ra 1790 in 1993 (lauraya et al, 2003). heeding to the agency's insinuation to promote coastal resource protection in the municipal waters of sangay, the sagñay lgu conducted general assemblies at the barangay level to illicit issues from the barangay as an input for the draft ordinance that established the mfrs. a series of public hearings were made at the barangay level which was done prior to the submission of proposed ordinance to the sangguniang bayan. after the hearings, the municipal council passed the ordinance marking the completion of the second phase in the process. the management phase consisted of activation of the bantay dagat patrol to monitor the compliance of the ordinance and the allocation of fund. based on a published literature, the process of establishing and implementing mfr-s must proceed from the following logical model (balgos et al, 2000; berkes, et al, 2001): 1. community entry and preparation 2. planning and ordinance formulation 3. ordinance approval 4. implementation and adjustment each phase of the establishment process is associated with specific activities: phase 1 involves appraisal (issue identification and baseline assessment) and core group formation; phase 2 involves public education, capacity building; community consultation, ordinance formulation and drafting of management plan; phase 3 involves mfr-s establishment, mfr-s program preparation and budgeting, and; phase 4 involves management and monitoring, and establishment. the omission of some supporting factors in the establishment process of most mfr-s throughout the three provinces of camarines sur, albay, and catanduanes prevented them from pursuing the logical model. these were manifested in the lgu-initiated mfr-s establishment process' absence of appraisal (baseline assessment) and core group formation and management planning which derailed phases 1 and 2 in the ideal logical model. some of the factors that hindered the lgu from pursuing the process include the lack of capability of lgu personnel to conduct baseline assessment and the agenda of the key agencies table 4. sequencing of mfr-s establishment processes in lagonoy gulf provinces albay camarines sur catanduanes smi atulayan agojo process sequencing rsa>iec>co>mp> o>mo>rsa>iec> co>iec>o>m0>mp* o>mo* mp* partnership community-initiated lgu-initiated initiating institution community lgu lgu external agent academe nga ngo *rsa=resource and socioeconomic assessment; iec=information and education campaign; co=community organizing; o=ordinance proposal and enactment; mp= mfr planning; mo=provision of fund and logistics, monitoring and evaluation bradecina and nieves 54 involved. most lgu personnel have not had proper training on the conduct of scientific assessment of coastal habitats and do not have the know-how to interpret and infer implications for management from the findings. on the other hand, the communityinitiated process in san miguel island mfr-s manifested the mfr-s establishment process in lagonoy gulf that almost identically followed the logical model. the process was facilitated by a technically capable external agent i.e. bicol university, tabaco campus, that provided a long -term commitment to the project as well as the sharing of responsibility and authority between the lgu and the community. the interplay of these hindering and facilitating factors in the establishment process of mfr-s developed a 3phase logical model for lagonoy gulf consisting of the following: phase 1, preparation and planning; phase 2, ordinance approval; and phase 3, implementation and management. this typology of mfr-s establishment process in the gulf showed two distinct types of mechanism of implementation, namely: (i.) a process where the mfr-s planning preempts the ordinance for its establishment; and (ii.). a process where the mfr-s planning precedes the ordinance for its establishment. in lgu-initiated and nga facilitated mfr-s establishment process, e.g. atulayan mfr-s, sagñay, camarines sur which was established when the old fisheries law (pd 704) was in effect, the phase on mfrs adoption and proposal of an ordinance for its establishment preempts the preparation and planning phases. the mechanism of implementation characteristic of this type of establishment process featured an enactment of ordinance establishing the mfr-s followed by a community consultation and an mfr-s management planning. this resulted to the nonintegration of the mfr-s management plan into the ordinance that established it. the municipal government had assumed the main responsibility of establishing and operating the mfr-s while the fisherfolks and the barangay council in the area that hosts the mfr-s had only a supporting role. the nonfunctional status of atulayan mfr-s, sagñay camarines sur in the previous years has been attributed for the absence of organized community effort to implement the fishery ordinances at the barangay level (lauraya, et al, 2003) (table 5). however, one outstanding feature of the lgu-initiated process is that the establishment of mfr-s was achieved in a very expeditious manner. this was attributed to the administrative power and existing resources of the lgu that obliged local community officials to establish the mfr-s and to implement law enforcement in their area of jurisdiction. in the community-initiated and academe facilitated establishment process, e.g. san miguel island, tabaco, albay (smi-mfr-s), and to some extent, the rest of the most recent ngo-facilitated mfr-s in albay which were established during the frmp and cbcrmp periods, the phase on mfr-s preparation and management planning precedes the mfr-s establishment and ordinance adoption phases.. the mechanism of implementation of this type of establishment process featured a community consultation and mfr-s establishment (barangay crm) followed by the enactment of an ordinance establishing an mfr-s by the municipal government this resulted to the integration of the mfr-s management plan into the ordinance that established it. a participatory process allowed the community a full say in developing the management plan of smimfr-s in tabaco albay. the presence of organized community effort that resulted from participation in management planning of the stakeholders was attributed to the functional status for the mfr-s. conclusion the description of institutional arrangements and process in establishing mfr-s in lagonoy gulf provided some insights that will help in improving mfr-s establishment activities in the future. in lagonoy gulf, the type of the initiating entity and the external agent that is going to be involved more or less influenced the direction of the mfr-s establishment process. the lgu-initiated arrangement displayed a top-bottom process of mfr-s establishment using its bureaucratic resources and administrative power. the community-initiated activity exhibited a bottom-top process developing community responsibility through power sharing between the lgu and the fisherfolks. institutional arrangements and processes in marine fishery reserves 55 among the external agents, the academe provides the most extensive involvement. the technical capability of the academe and its pioneering spirit provided the greater advantage as an initiating partner institution. external agents differed in their approaches to facilitate mfr-s establishment process in the gulf. the institutional mandate and the technical expertise of the external agent determine their process of entry and the sustainability of the mfr-s. lgu-initiated and nga/ngo facilitated process is strong in the ordinance adoption phase. its administrative power demonstrated greater capability to facilitate organization and mobilization of available political and financial resources to institutionalize the project. community-initiated and academe facilitated process is strong in the preparatory and planning phase. it manifested a strong grassroots support that emanated from an understanding of community context offering great potential for sustaining the project. while the activities in the process are situation-dependent and do not necessarily require being sequential, the conduct of baseline assessment that is primarily responsible in eliciting collective reflection of necessity and support for the establishment of the project is a key preliminary activity in the preparation and planning phase. the lack of technical capability of lgu personnel and the focus for policy implementation of the institutions involved could derail the implementation of some important phases and activities. but the presence of a technically competent external partner with long-term commitment could facilitate the implementation of most of the critical preparatory phases. given the dynamic political and socio-cultural context and diverse institutional mandates of collaborating external agents in lagonoy gulf, relevant institutional arrangements and phase sequence could be developed from its mfr-s establishment experience. a more appropriate and effective mfr-s establishment process could be evolved from the lagonoy gulf experience by placing the different institutional arrangement and process typologies at appropriate periods capitalizing on their respective strengths. the appropriateness of a community-initiated and academe-facilitated institutional arrangement with nga support was table 5 mechanisms involved in the implementation and process outcomes of mfr-establishment in lagonoy gulf typology of mfr institutional establishment arrangement mechanism process outcomes and process participatory core group implementation institutionalization process in development process of mfr-s fund management process planning community-initiated ordinance presence of core group mfr-council presence of preempts community internally and sbc tasked enabling mechanism planning involvement in facilitated with overall through municipal management management ordinance planning of the mfr-s; presence of community effort to manage mfr-s lgu-initiated ordinance lack of core group the lgu tasked absence of precedes management externally with the main enabling planning plan, facilitated; implementation; mechanism; absence of technical the role of fund direct community staff of the barangay from the mayor’s involvement lgu assigned council relefund in management the role of gated to support planning core group role bradecina and nieves 56 highlighted during the preparatory phase when community support is still weak and the need to strengthen their capability and encourage participation is crucial. the lgu-initiated and ngas/ngos facilitated process was highlighted when law enforcement is weak and the need for the institutionalization of management mechanisms are critical. acknowledgments we wish to acknowledge the following for their support: bureau of fisheries and aquatic resourcesfisheries resource management project (bfarfrmp), bureau of fisheries and aquatic resources, region v office, prof. vic soliman, project leader, lagonoy gulf socioeconomic and resource assessment project. references aquinas university of legaspi-center for research and extension services (aul-cres) 2003. final report, fisheries resource management project, caramoan and presentacion cluster. balgos, m.t., bayer, b. crawford, c. padilao, j. tulunen and a. white, 2000. proceedins:philippines_-indonesia workshop on community-based marine sanctuaries. berkes, f. r. mahon, p.mcconney, r. pollnac, and r. pomeoy 2001. managing small scale fisheries: alternative directions and methods. care philippines and cerd, inc. 2001. final report: community organizing and livelihood development component-frmp catanduanes. david, n.d., a.p.camaya, r. buella, a. mendoza and v. soliman. 2004. status of key coastal habitats of the marine fishery reserves in lagonoy gulf, bicol region: in rapid resource assessment phase of the lagonoy gulf postresource and socioeconomic assessment (inception report). development research and resource productivity, inc.(drrp) ______. 6th quarter accomplishment report fisheries sector program-coastal resources management hermes, r. 2003. marine protected area-sustaining mechanisms for effective implementation and desirable impacts, in: acedera, m.m., e.m. redera and c.r. pagdilao, (eds.) integrated coastal management experiences in the philippines, proceedings of the integrated coastal management practitioners convention in the philippines, 10-12, november 1998. lauraya, f.m., n. dullesco and a.p. candelaria. 2003. status of implementing marine fishery resource related ordinances in the municipality of sagñay, camarines sur, bicol. pelea, n., s. borbe, and m.j. pelea, 2004. socioeconomic status of lagonoy gulf: in rapid resource assessment phase of the lagonoy gulf post-resource and socioeconomic assessment (inception report). pomeroy, r.s. and carlos, m.b. 1997. community-based coastal resource management in the philippines: a review and evaluation of programs and projects, marine policy, vol. 21, no.5 pp.445-464. 5 geometric morphometric analysis of channa striata (striped snakehead) populations from laguna de bay, philippines reveals shape differences in relation to water quality shenna kate m. torres* institute of biology, college of science university of the philippines diliman natural sciences research institute, college of science university of the philippines brian s. santos francis s. magbanua institute of biology, college of science university of the philippines diliman abstract channa striata, locally known as dalag, constitute a major aquaculture resource in laguna de bay. owing to its popularity as a food source, threats such as overfishing may potentially place this species at risk. however, studies regarding its status within the lake is lacking. one way to address this gap is through population studies using geometric morphometrics. in this study, a total of 82 specimens were collected across three areas of the lake, namely, binangonan, calamba, and tanay. these areas were assessed using secondary data for physicochemical parameters, which revealed significantly higher ammonium-nitrogen levels in binangonan compared to the other areas. geometric morphometrics was then used to determine whether shape variation existed among c. striata populations. results showed that shape variation was greatest in the cranial region, with fish from binangonan and tanay having the greatest variation in shape. on the other hand, specimens from calamba had the highest morphometric values. lastly, these findings were then correlated with water quality data using canonical correlation analysis. results indicated that shape variation in the cranial region was correlated with differences in dissolved oxygen and ph content of the lake. the weight and length of fish were inversely correlated to the levels of ammonium-nitrogen and total dissolved solids, with specimens from binangonan displaying a high sensitivity to ammonium-nitrogen. keywords: dalag, freshwater fish, shape variation, laguna lake, physicochemical parameters * corresponding author science diliman (july-december 2020) 32:2, 5-24 geometric morphometric analysis of channa striata (striped snakehead) populations 6 introduction laguna de bay, located at the eastern part of metro manila, philippines, is the largest lake in the country, with a surface area of 900 km2 and an average depth of 2.5 m (llda 2016). the lake is one of the top producers of freshwater fish, providing a source of food and income and contributing heavily to the economic growth of the country (cuvin-aralar 1990; aquino et al. 2011). among the fishes present in the lake are introduced cultured species (milkfish chanos chanos, bighead carp aristichthys nobilis, tra catfish pangasianodon hypophthalmus and nile tilapia oreochromis niloticus), native and other species (silvery theraponid leiopotherapon plumbeus, manila sea catfish arius manillensis, gobies glossogobius giuris, giuris margaritacea, and striped snakehead channa striata) (cuvin-aralar 2016). however, despite the management plans and policies implemented for the lake, a decrease in fish catch (tamayo-zarafalla et al. 2002) as well as recent fish kills (angeles 2019; cinco 2020) were noted to have been occurring at the lake. various factors can be attributed to these events, such as overfishing, intensive aquaculture (santos-borja and nepomuceno 2006), illegal use of destructive fishing gear (i.e., spear and drag seine) (palma et al. 2002), rapid land use, and water pollution (e.g., organic waste, solid waste) (kosmehl et al. 2008). these problems pose harm to the biodiversity in the area while also negatively affecting local aquaculture enterprises. among the economically important fish species with an observed decline in catch data is the dalag (channa striata). c. striata, commonly known as striped snakehead, is regarded to be of high economic value in the country as it contributed a total value production cost of 1,043,474.86 philippine pesos in 2015, placing this species at the third spot among species contributing to inland water capture production (psa 2018). it is a popular fish due to its high-quality meat, low-fat content, few intramuscular spines, tasty flavor, and relatively cheaper price compared to other fishes (song et al. 2013). perhaps due to the increasing demand for the species, a decline in c. striata catch data was noted by the bureau of agricultural statistics (2012; 2015; 2018; 2019) from 10,469.58 metric tons in 2010 to 9,512.3 metric tons in 2017, or an average decline of 7% in nine years. given the high value of this fish, especially as a top target fish that is prone to overfishing and population decline, it is imperative to study their population. one way to manage and assess the population of this fish is through phenotypic variation using morphometric identification. changes in the growth and development of a fish often creates a difference in body shape within a species and may be influenced by the interplay among the environment, genetics, and selection s.k.m. torres et al. 7 on the life history of a species (cadrin 2000). a widely used method to study shape variation is geometric morphometrics (gm) (santos and quilang 2012), which is unlike traditional morphometric tools that make use of linear measurements, counts, and ratios (adams et al. 2004) to differentiate between populations. landmark points of a species, which are defined as the anatomical points in the species (richtsmeier et al. 2002), are commonly used in gm. santos and quilang (2012) studied populations of catfish (arius manillensis and arius dispar) in laguna de bay that were observed to be in decline likely due to local overfishing. in their study, the left side body and dorsal head view of the two species were subjected to gm analysis, which revealed that most of the shape variation came from body size rather than the dorsal head of the fish, which suggests influence by various factors such as species diet, movement, and habitat. the objective of the study was to examine the shape variation between populations of c. striata found in laguna de bay, specifically in the northwest, south, and central regions of the lake, using gm which can be used to provide information regarding the current condition of c. striata in the lake. the different areas within the lake were chosen according to the differences in land use, specifically: the northwest bay (binangonan), which is the closest bay to metro manila and is surrounded by highly urbanized communities, industrialized sites, and ports for small boats; the south bay (calamba), which is mostly surrounded by residential areas; and the central bay (tanay), which is mainly surrounded by agricultural areas and farmland (johnson and iizuka 2015). likewise, these areas were the major landing sites (rizal and laguna) for c. striata found in laguna de bay (bas 2018). in addition, water quality conditions in these three areas, using the physicochemical parameters measured by the laguna lake development authority, were assessed to know whether a significant change in the physicochemical parameters happened in the lake. the correlation between the shape variation found in c. striata and possible changes in physicochemical parameters were also investigated. this information can be used to reveal whether certain selective pressures within the area could favor certain phenotypic structures that could result in different morphotypes of c. striata. materials and methods specimen collection channa striata specimens were obtained from binangonan (14°27’47.36” n, 121°11’34.98” e), calamba (14°12’38.93” n, 121°09’53.15” e), and tanay (14°29’33.79” n, 121°17’17.54” e) areas of laguna de bay (figure 1). a total of 82 fish geometric morphometric analysis of channa striata (striped snakehead) populations 8 specimens were collected on 20 january 2018 and 17 march 2018 through the help of local fishermen around the lake. specifically, 30 specimens from binangonan, 27 specimens from calamba, and 25 specimens from tanay were collected. the fishing gear used to collect the specimens were a combination of a fish shelter set at the lake bottom that served as an aggregating site and a manual seine to catch the fish (locally called takibo) (palma et al. 2002; 2017 personal communication with fishermen in the sampling areas). across the three areas, the same set of fishing gear (takibo) was used by the fishermen. in addition, the total weight and length measurements (total length, body depth, and pectoral length) were measured using a weighing scale and metric ruler, respectively. figure 1. map of laguna de bay showing the three sampling areas in binangonan, calamba, and tanay. s.k.m. torres et al. 9 water quality data the following water quality data were obtained from the laguna lake development authority (llda): water temperature (in °c) and total dissolved solids (tds) (in mg/l) (from 2013-2016), dissolved oxygen (do) (in mg/l) and ph (from 2015-2017), and ammonium-nitrogen (in mg/l) and nitrate-nitrogen (mg/l) (from 2016-2017). each data on physicochemical parameters was specific among the three sites. the do, ph, ammonium-nitrogen, and nitrate-nitrogen of the lake were measured monthly, while the temperature and tds were measured yearly. analysis on length, weight, condition factor, and water quality data the condition factor of each specimen, which was calculated by dividing the total weight (in grams) by the cube of the total length (in centimeters) and multiplying the quotient by 100, was recorded. next, the weight and length measurements, as well as the condition factor, were subjected to one-way analysis of variance (anova) with post-hoc tests (tukey’s hsd) using the ibm spss statistics version 26 (ibm corporation2019). similarly, the water quality data across the three sampling sites were subjected to one-way anova followed by post-hoc tests (tukey’s hsd) using the ibm spss statistics version 26 (ibm corporation2019). these tests were used to detect the differences in specimen morphometry and physicochemical parameters across the three sampling sites. likewise, the condition factor was used to assess the well-being and degree of fatness of the fish (zelditch et al. 2004). geometric morphometrics and data analysis the left side body of each specimen was pinned in place on a white background with a standard metric ruler at the bottom in order to provide scale. each specimen was photographed using a canon eos 700d dslr camera. the ten landmarks, serving as anatomical points, chosen for this study were based on those used by song et al. (2013), namely: (1) anterior tip of the snout; (2) posterior aspect of the neurocranium; (3) origin of dorsal fin; (4) insertion of dorsal fin; (5) anterior attachment of dorsal membrane from caudal fin; (6) posterior end of vertebrae column; (7) insertion of anal fin; (8) original of anal fin; (9) origin of pelvic fin; and (10) posterior end of lower jaw (figure 2). the landmarks were plotted digitally on each image using the tpsdig2 software (rohlf 2010). the raw landmark coordinates were superimposed as shape variables using the coordgen8 software through the generalized procrustes analysis (gpa). gpa was used to ensure that differences in shape would be independent of size, position, or orientation of the fish (slice 2007) and to check for possible outliers (sotola et al. 2019). the corrected coordinates geometric morphometric analysis of channa striata (striped snakehead) populations 10 generated from gpa were used for subsequent analysis. the centroid size, which is the square root of the sum of squared distances of the landmarks in a configuration to the average location (slice 2007), of each specimen was calculated using the coordge8 software. figure 2. landmarks of c. striata used in the study: (1) anterior tip of the snout; (2) posterior aspect of the neurocranium; (3) origin of dorsal fin; (4) insertion of dorsal fin; (5) anterior attachment of dorsal membrane from caudal fin; (6) posterior end of vertebrae column; (7) insertion of anal fin; (8) origin of anal fin; (9) origin of pelvic fin; and (10) posterior end of lower jaw. principal component analysis (pca) was performed using the pcagen8 software to examine overall shape variability among all specimens collected from the three localities. to test whether the observed shape variations were not dependent on the size of each specimen, multivariate analysis of covariance (mancova) was performed using ibm spss statistics version 26 (ibm corporation 2019). in this test, the cv 1 and cv 2 scores were treated as the dependent variable, the standard length as the covariate, and the study sites as the fixed factor (zelditch et al. 2004). to further validate the results of mancova, regression of the pc 1 scores on the logarithm of the centroid size was performed to determine the growth pattern of c. striata using microsoft® excel®for microsoft 365. the mancova and regression analysis were both used to identify whether c. striata exhibits isometric or allometric patterns of growth. multivariate analysisof variance (manova) was performed using the cvagen8 to differentiate the three localities. results were summarized using canonical variate analysis (cva). deformation grids and vector plots were generated to visualize the shape variation among the population. in addition, pairwise comparisons between populations were conducted using cvagen8 and twogroup 8 software. on the other hand, twogroup8 software was used to calculate the goodall’s f-test. manova and goodall’s f-test were performed to detect the differences or similarities among the three populations. s.k.m. torres et al. 11 correlation of morphometric values to water quality data the correlation between the morphometric values of the c. striata populations and the water quality data using the physicochemical parameters measured were examined using canonical correlation analysis (cca). separate cca were used to describe the correlation between fish morphometric values (weight, total length, body depth, and pectoral length) and physicochemical parameters of the lake (total dissolved solids and ammonia content). the other one was done between the 10 anatomical points of the fish and physicochemical parameters of the lake (ph and dissolved oxygen). the canonical variate (cv) scores, canonical correlation coefficients, and wilk’s test of significance were generated through the cca package of r studio version 1.1.442 (torres 2020). these data were used to examine whether environmental factors were correlated with the shape variation observed among c. striata populations. results length, weight, and condition factor the descriptive statistics of the measured weight and length of each specimen from the three sampling sites within laguna de bay were summarized in table 1. the measured parameters were as follows: weight ranged from 76.00 g to 1040.00 g, total fish length ranged from 20.80 cm to 52.10 cm, body depth ranged from 2.90 cm to 7.50 cm, and pectoral fin length ranged from 2.60 cm to 8.20 cm. the calculated condition factor ranged from 0.30 to 1.60, while the calculated centroid size ranged from 22.72 to 53.37. when these measurements were subjected to one-way anova with post hoc tests, it was observed that of the six morphometric variables, only the condition factor (p=0.092) did not differ across the sampling sites (table 1). results of the post-hoc tests revealed that the total weight (p=0.003), total length (p=0.001), body depth (p<0.001), and centroid size (p<0.001) of specimens collected from calamba statistically differ from those obtained in binangonan and tanay. geometric morphometric analysis of channa striata (striped snakehead) populations 12 table 1. summary (means ± se, f-values, and p-values) of the anovas comparing water quality variables across three areas in laguna de bay. do – dissolved oxygen, tds – total dissolved solids. ranking for post hoc tests in cases with significant effects are given. p-values <0.05 are in bold print. parameter binangonan calamba tanay f-value p-value ranking water temp (°c) 28.70 ± 0.08 28.50 ± 0.05 28.20 ± 0.14 2.923 0.105 do (mg/l) 8.27 ± 0.40 8.32 ± 0.19 8.80 ± 0.42 0.752 0.474 ph 8.20 ± 0.08 8.36 ± 0.79 8.40 ± 0.10 1.403 0.251 tds (mg/l) 385.25 ± 100.68 352.25 ± 102.90 386.50 ± 128.50 0.030 0.970 nitrate-n (mg/l) 0.39 ± 0.11 0.22 ± 0.08 0.22 ± 0.07 1.265 0.289 ammonium-n (mg/l) 0.13 ± 0.04 0.04 ± 0.01 0.05 ± 0.01 3.965 0.024 binangonan>(calamba=tanay) water quality the summary of data for the physicochemical parameters measured by the laguna lake development authority were summarized in table 2. across the sampling sites, no significant difference was found in water temperature, do, ph, tds, and nitratenitrogen (table 2). however, ammonium-nitrogen levels across sampling sites (p=0.024) statistically differed, with a higher mean concentration in binangonan (0.13 mg/l) compared to calamba and tanay (table 2). table 2. summary (means ± se, f-values, and p-values) of the anovas comparing morphometric variables of c. striata across the three areas in laguna de bay. ranking for post hoc tests in cases with significant effects are given. p-values <0.05 are in bold print. morphometric variable binangonan (n=30) calamba (n=27) tanay (n=25) f-value p-value ranking total weight 285.63 ± 25.99 448.63 ± 53.97 294.56 ± 17.25 6.444 0.003 calamba > (binangonan =tanay) total length 30.58 ± 0.94 36.37 ± 1.54 32.43 ± 0.68 7.093 0.001 calamba > (binangonan =tanay) condition factor 0.92 ± 0.01 0.85 ± 0.04 0.85 ± 0.02 2.463 0.092 centroid size 31.53 ± 1.00 37.04 ± 1.82 31.43 ± 0.67 6.367 0.003 calamba > (binangonan =tanay) body depth 4.13 ± 0.12 5.10 ± 0.23 4.22 ± 0.06 11.808 <0.001 calamba > (binangonan =tanay) pectoral fin length 4.43 ± 0.14 5.31 ± 0.23 3.93 ± 0.06 15.980 <0.001 calamba < binangonan <tanay s.k.m. torres et al. 13 geometric morphometrics the principal component analysis constructed a rotation of 16 shape variables derived from the set of 10 landmarks used in the study. the principal component 1 (pc 1) and principal component 2 (pc 2) scores explain 39.63% and 16.61% of the variance, respectively. these pcs do not discriminate among the populations (figure 3). three other pcs had variance greater than 5%. altogether, these first five pcs account for 84.94% of the variance (table 3). figure 3. principal component analysis (pca) plot for 82 specimens of c. striata sampled from three areas in laguna de bay. pc1 (x-axis) accounts for 39.63% of total variance among individuals and pc2 (y-axis) accounts for 16.61% of total variance among individuals. table 3. eigenvalues and corresponding percentage of variance explained by each principal component for specimens of c. striata principal component* 1 2 3 4 5 eigenvalue 0.0007 0.0003 0.0002 0.0002 0.0001 percent variation 36.67 16.63 12.25 9.90 6.56 *only the principal component with percent variance above 5% were shown. the results of the mancova performed on the cv 1 and cv 2 scores, as these account for the greatest variation in shape, yielded a significant wilks λ value of 0.88 (p=0.050). similarly, to further validate the results of the mancova test, regression of the pc 1 scores against the logarithm of the calculated centroid size for each specimen was done. regression analysis yielded a non-statistically significant p-value of 0.148 for allometric test, supporting the claim that shape variation was independent of the size of the specimens. geometric morphometric analysis of channa striata (striped snakehead) populations 14 analysis of shape variation through cva was done as pca did not differentiate the three populations clearly. the cva plot generated in this study showed that canonical variate 1 (cv 1) accounts for 70.24% of the total variance, while canonical variate 2 (cv 2) accounts for 29.76% of the total variance (figure 4). cv 1 was able to differentiate the tanay population from other areas, while cv 2 differentiated the calamba population from other areas. the shape variation along the x-axis (cv 1) accounted for differences in the posterior aspect of the neurocranium of the specimens, wherein the more positive end is correlated with a longer neurocranium as compared to the more negative end. on the other hand, shape variation along the y-axis (cv 2) correspond to the disparities in the posterior end of the lower jaws of the specimens, wherein the more positive end is correlated with a larger and more elongated head as compared to the more negative end where a more forward lower jaw was observed (figure 5). figure 4. canonical variate analysis of shape variables of 82 specimens of c. striata sampled from three areas in laguna de bay. cv1 (x-axis) accounts for 70.24% of total variance among group means and cv2 (y-axis) accounts for 29.76% of total variance among group means. s.k.m. torres et al. 15 figure 5. deformation grids showing the procrustes deformation and vector diagrams showing the direction and magnitude of shape variations along cv 1 and cv 2 in 82 specimens of c. striata across three study sites. in terms of accuracy of the generated cva a posteriori assignment, correct classification rates of the specimens based on mahalanobis distances ranged from 70.37% to 80.00% (table 4). the groupings showed that binangonan had the lowest percentage of misclassification, wherein only 25.00% (6 out of 24) of the specimens were misclassified under calamba and tanay. on the other hand, specimens from calamba showed the highest percentage of misclassification, wherein 42.10% (8 out of 19) were misclassified under binangonan and tanay. finally, 72.00% of the specimens from tanay were classified correctly, with 8.00% and 20.00% misclassified as specimens from binangonan and calamba, respectively. table 4. canonical variate analysis (cva) classification for c. striata a priori a posteriori binangonan calamba tanay binangonan 24 (80.00%) 2 (6.67%) 4 (13.33%) calamba 5 (18.52%) 19 (70.37%) 3 (11.11%) tanay 2 (8%) 5 (20.00%) 18 (72%) geometric morphometric analysis of channa striata (striped snakehead) populations 16 based on the pairwise comparison between populations, shape variation between specimens from each study site was significant (p<0.001; figure 4), with the greatest differentiation observed between binangonan and tanay specimens (highest goodall’s f value of 10.64) and least differentiation observed between binangonan and calamba specimens (lowest goodall’s f value of 2.78) (table 5). table 5. pairwise comparisons among sampling areas using goodall’s f-test and canonical variate analysis-multivariate analysis of variance (cva-manova). b = binangonan, c = calamba, t = tanay. areas compared n goodall’s f-test cva-manova f-value dist df p-value wilk’s x df p-value b t 55 10.64 0.04 16 848.00 <0.001 0.375 44.16 16 <0.001 c t 52 5.63 0.03 16 0.03 <0.001 0.361 42.75 16 <0.001 b c 57 2.78 0.01 16 880.00 <0.001 0.493 33.26 16 0.007 correlation of morphometric values and water quality data the results of the canonical correlation analysis between the measured morphometric values of the 82 specimens of c. striata and physicochemical parameters from laguna de bay revealed that the two cvs were statistically significant (table 6). cv 1 was correlated with total length and pectoral length, while cv 2 was correlated with total weight and total length (table 8). specifically, cv 1 correlated total length and pectoral length with total dissolved solid content, while cv 2 correlated total weight and pectoral length with ammonia levels (table 8). the cca plot generated revealed an inverse relationship between the morphometric values and physicochemical parameters (figure 6). figure 6. canonical correlation analysis (cca) plot between measured morphometric values from 82 specimens of c. striata and ammonium-nitrogen and tds content of laguna de bay. s.k.m. torres et al. 17 table 6. canonical correlation analysis between the measured morphometric values from 82 specimens of c. striata and physicochemical parameters of laguna de bay canonical variate canonical correlation f-value p-value 1 0.6140 7.66 <0.001 2 0.4302 5.83 <0.001 table 7. canonical correlation analysis between landmark points from 82 specimens of c. striata and measured physicochemical parameters of laguna de bay canonical variate canonical correlation f-value p-value 1 0.7778 2.99 <0.001 2 0.6049 1.85 <0.001 table 8. canonical variate loadings showing the correlation between the two cvs (cv 1 and cv 2) and the original variables (morphometric value and physicochemical parameter). values in bold indicate very high or low cv. variables cv 1 cv 2 morphometric variable 1. weight -0.28 1.17 2. total length 1.25 -1.57 3. body depth -0.38 -0.96 4. pectoral length -1.31 0.67 physicochemical parameter 1. total dissolved solids 1.00 0.05 2. ammonia 0.05 1.00 similarly, canonical correlation analysis between the shape variables of the 82 specimens of c. striata and the physicochemical parameters from laguna de bay revealed that the two cvs were statistically significant (table 7). cv 1 was correlated with landmark 1 (anterior tip of the snout), landmark 2 (posterior aspect of the neurocranium), landmark 3 (origin of dorsal fin), and landmark 9 (origin of pelvic fin). on the other hand, cv 2 was mostly correlated with landmark 1 (anterior tip of the snout), landmark 2 (posterior aspect of the neurocranium), landmark 3 (origin of dorsal fin), and landmark 10 (posterior end of lower jaw) (table 9). the cca plot generated using the shape variables and physicochemical parameters revealed that the cranial region was sensitive to the do and ph content of the lake (figure 7). geometric morphometric analysis of channa striata (striped snakehead) populations 18 table 9. canonical variate loadings showing the correlation between the two cvs (cv 1 and cv 2) and the original variables (landmark points and physicochemical parameters). values in bold indicate very high or low cv. variables cv 1 cv 2 shape coordinates 1. anterior tip of the snout (x) -2512.09 -1953.39 2. anterior tip of the snout (y) -27539.89 2769.75 3. posterior aspect of the neurocranium (x) -6538.51 -3083.47 4. posterior aspect of the neurocranium (y) -10271.17 1284.84 5. origin of dorsal fin (x) -4512.86 -2021.76 6. origin of dorsal fin (y) -11927.67 1685.98 7. insertion of dorsal fin (x) -1225.03 -681.14 8. insertion of dorsal fin (y) -1469.71 601.33 9. anterior attachment of dorsal membrane from caudal fin (x) -1480.41 -817.74 10. anterior attachment of dorsal membrane from caudal fin (y) -1626.45 762.76 11. posterior end of vertebrae column (x) -722.23 -977.11 12. posterior end of vertebrae column (y) -1688.77 769.83 13. insertion of anal fin (x) -518.49 -715.78 14. insertion of anal fin (y) -1765.35 681.30 15. origin of anal fin (x) -450.85 -1420.32 16. origin of anal fin (y) -8755.59 1622.57 17. origin of pelvic fin (x) -333.18 -1109.35 18. origin of pelvic fin (y) -11672.64 1609.75 19. posterior end of lower jaw (x) -849.79 -1781.21 20. posterior end of lower jaw (y) -8733.12 1071.93 physicochemical parameters 1. ph 2.00 -2.96 2. dissolved oxygen (do) -2.75 2.29 figure 7. canonical correlation analysis (cca) plot between landmark points from 82 specimens of c. striata and do and ph content of laguna de bay. s.k.m. torres et al. 19 discussion channa striata continues to be a top freshwater species, contributing significantly to freshwater fish production in the philippines. despite this, detailed information regarding their population remains limited (jamaluddin et al. 2011). the present study investigated the population of c. striata through geometric morphometric analysis. it was revealed that phenotypic variation was present among the populations of c. striata within laguna de bay. the morphological differences were concentrated on the cranial region, specifically at the posterior aspect of the neurocranium and the posterior end of lower jaw. similarly, aside from the shape variation observed in the study, differences in the morphometric characteristics of the specimens were also observed. in particular, specimens from the calamba area had a statistically significant value for total weight, total length, body depth, pectoral length, and centroid size, in contrast with the specimens from binangonan and tanay. in terms of the growth pattern, the study yielded a statistically significant wilks λ value for allometric growth pattern and may indicate that size does not play a notable role in the shape variation of the specimens. morphological differences in fishes often account for selection and geographic barriers (cadrin 2000); however, studies have shown that environmental constraints may also play a part in this process (su et al. 2018). variations accounting for environmental differences may reveal insights regarding the ecological strategies of a species, such as feeding habits, movement patterns, and interaction with other species (santos and quilang 2012; yen et al. 2019). the results of the study regarding the higher morphological variation in the cranial region of c. striata found in laguna de bay were similar to the study done by yen et al. (2019) for c. striata found in mekong delta, malaysia. yen et al. (2019) explained that usually c. striata with a larger head tends to be advantageous, especially when competition for food is present; aggressive behavior in c. striata tends to arise when competition is higher, favoring a larger head phenotype. in terms of the differences in the physicochemical parameters measured among the areas in laguna de bay, a statistically significant difference was observed in the ammonium-nitrogen (mg/l) content in the binangonan area as compared to those in calamba and tanay. the observed statistically significant high level of ammonium-nitrogen content in binangonan might be affected by the rapid land use near the area. binangonan is mainly surrounded by urbanized and industrialized areas, thus it is possible that waste from these areas, such as organic materials and heavy metals, may have affected the physicochemical parameters near binangonan (kosmehl et al. 2008). however, due to the limitations of the study, wherein only secondary data were used, further validation of these results may still be needed. geometric morphometric analysis of channa striata (striped snakehead) populations 20 canonical variate analysis done on the specimens revealed that the posterior aspect of the neurocranium separated the specimens obtained from the tanay areas from those obtained from the other areas. figure 4 shows that the specimens from tanay clumped together in the positive x-axis, indicating that the specimens from this area have a longer posterior aspect of the neurocranium. on the other hand, calamba specimens were separated from the other areas through the posterior end of lower jaw, with the specimens clumping together at the negative y-axis. this indicates that the specimens from calamba had shortened heads and more forward lower jaws compared to the others. pairwise comparisons among the three populations using goodall’s f-test showed that specimens from binangonan and tanay had the greatest variation in shape, and specimens from binangonan and calamba had the least variation in shape. the findings indicate that specimens from binangonan had the largest head region compared to specimens from other areas. these results might help us to better understand the behavior of c. striata across the three areas as a response to their environment. the sensitivity of the head region of the c. striata to do content and ph of the lake (figure 7) may be suggestive of a compensatory mechanism. when the lake has low do content (hypoxic water), the c. striata tends to burrow itself in the mud to keep their skin and breathing apparatus moist (courtenay and williams 2004). in this study, although no statistical difference was noted in terms of do across the areas, still, not so high value of do with a range of 8.02 to 8.93 mg/l—compared to the 5.00 mg/l minimum standard of the denr water quality guidelines for class c waters—was calculated. the longer neurocranium and elongated head of the fish in tanay may indicate constant use of this part to burrow and breathe in mud. likewise, shape variation within the population of c. striata in the lake may explain the aquaculture practices found across the three areas. in a study conducted by santos and quilang (2012), they noted a higher density of fish pens in the binangonan area compared to the calamba and tanay areas. fish feeds that diffuse out of the pens may provide nutrients to the nearby waters, giving a greater fitness advantage to fishes near the pens as compared to specimens without, which may translate to a variation in shape. because of this practice, they found out that arius sp. from binangonan had the highest correct classification based on the mahalanobis approach. this result is similar to the results found in this study, where c. striata obtained in the binangonan area had the highest correct classification. in terms of the morphometric values, length and weight measurements were inversely correlated with high levels of ammonia and total dissolved solids using the cca. it can be inferred that the elevated ammonia levels in binangonan may s.k.m. torres et al. 21 have influenced the morphology of the c. striata in the area since it was observed that the average length and weight of c. striata were less than the average length and weight of specimens in either calamba or tanay. a study by li et. al in 2014 showed that decreased growth was observed in fish that were exposed to high ammonia levels. another study by shin et al. in 2016 showed that elevated ammonia exposure resulted in a decrease in hematological factors, such as red blood cell count, white blood cell count, and hematocrit, as well as in indicators of growth performance such as daily length gain and daily weight gain. the results from these studies mirror the observed pattern in specimens from binangonan, where a higher ammonia level resulted in decreased growth. in summary, analysis of the c. striata population revealed that variation in morphometric values was observed among specimens across three areas of laguna de bay. among these specimens, c. striata from the binangonan and tanay areas were the least similar in morphology, with the greatest variation observed in the cranial region. likewise, specimens from calamba were found to have the highest morphometric values in terms of length and weight measurements. assessment of the sampling sites in terms of physicochemical parameters revealed that the ammonium-nitrogen levels in binangonan was statistically significant compared to the calamba and tanay areas. lastly, the obtained morphometric values were then correlated with the physicochemical parameters of the lake using canonical correlation analysis. cca reveals that dissolved oxygen and ph content may play a significant role in the cranial region of the fish. on the other hand, weight and length measurements were inversely correlated to high levels of ammonium-nitrate and total dissolved solid content. for future studies, it is recommended to perform geometric morphometrics in the dorsal head region of the fish to further investigate the variation in head region observed in this study. likewise, other methods to study the population of c. striata is recommended, such as through the use of molecular methods. genetic analysis may provide a deeper understanding regarding the population structure of this species, as well as the extent of their adaptability to the environment, which can be helpful for management and conservation purposes. acknowledgments this study was supported by the office of the vice chancellor for research and development, up diliman (project code: 191910 phdia). the authors would like to thank the laguna lake development authority for sharing their data, dr. jonas p. quilang for his valuable insights, and paolo gabriel c. alcantara for technical assistance. we also thank angelo joshua c. luciano for his help with figure 1. geometric morphometric analysis of channa striata (striped snakehead) 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[accessed 2020 june 28]; https:// github.com/smtorres04/2020_channastriata_scencediliman. turan cd, erguden m, gurlek n, basusta-turan f. 2004. morphometric structuring of the anchovy (engraulisencrasicolus l.) in the black, aegean and northeastern mediterranean seas. turk j vet anim sci. 28:865-871. yen td, duyen vn, hien ttt, pomeroy r, hillary e. 2019. variation in morphometric characteristics between cultured and wild striped snakehead (channa striata) populations in the mekong delta. can tho univ j sci. 11(1):70-77. zelditch ml, swiderski dl, sheets hd, fink wl. 2004. geometric morphometrics for biologists: a primer. 2nd ed. usa: elsevier. ______ shenna kate m. torres <smtorres@up.edu.ph> is a university research associate at the natural sciences research institute, university of the philippines diliman working on population studies of economically important fish. she is also a member of the molecular population genetics laboratory, institute of biology, up diliman. she finished her bachelor of secondary education major in biology and master of science in biology in the same university. brian s. santos is an assistant professor and a principal investigator from the molecular and population genetics laboratory, institute of biology, university of the philippines diliman. he received his ph.d. in biology from the university of the philippines diliman. he specializes in population genetics of commercially important fishery species. francis s. magbanua is an assistant professor and head of the aquatic biology research laboratory, institute of biology, university of the philippines diliman. he received his ph.d. in zoology from the university of otago, dunedin, new zealand. he specializes in freshwater ecology and biomonitoring using fish and benthic macroinvertebrates. v from the editor we are finally adapting to the new normal of the covid-19 pandemic, realizing how a global health issue can disrupt every aspect of society. it showed us how scientists can step up and create vaccines in record time to manage a pandemic, when the process could very well have extended beyond two years. a few positives came out of our pandemic experience as reported by both local and international researchers, such as improvements in the natural environment as anthropogenic activities slowed down. this includes a temporary decrease in greenhouse gas emissions, improvement in air and water quality, and the general recovery of natural ecosystems. though the four articles in this issue—my first as editor-inchief of science diliman (sd)—are not directly related to covid-19, they all connect to health and biodiversity, two human concerns that were greatly affected by the pandemic. two very different ecosystems are highlighted in this sd issue. the first is discussed in the paper by que et al. about the latest phylogeny research on philippine cockatoos of the forests of palawan. this paper places the local species that are most related to tanimbar corellas of indonesia and the western corellas of southwest australia. the second kind of ecosystem is discussed in the paper by pasumbal et al. about the latest biodiversity survey of non-avian vertebrates found in the up diliman campus. this updates a similar survey in 1998, with the latest survey reporting the presence of additional animals: two amphibians, seven reptiles, and six new mammals. the survey affirms the high species richness found on our campus. tambago and amor of the up institute of chemistry present their findings on two chalcone derivatives extracted from the leaves of s. samarangense (blume) merr. (java apple) that show anti-inflammatory properties. this could very well lead to the development of a new class of anti-inflammatory drugs. similarly, the paper authored by genio and paderes (also of the up diliman institute of chemistry) reports the synthesis of white powder precipitates composed of bis-urea compounds. their anti-microbial properties were tested against different microorganisms with several identified compounds showing promising results. as universities go back to a sense of normalcy this year, we hope that local researchers are able to resume the work on projects put on hold as well as jumpstart new research studies. carlos primo c. david, ph.d. editor-in-chief ocr document 7fuzzy-caga-anan.pmd fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 70 science diliman (july-december 2018) 30:2, 70-86 fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem lezel n. mernilo-tutanes mathematics department bukidnon state university randy l. caga-anan* department of mathematics and statistics msu-iligan institute of technology and prism abstract in set theory, an ideal is a collection of sets that are considered to be small or negligible, such that every subset of an element of the ideal m u s t a l s o b e i n t h e i d e a l a n d t h e u n i o n o f a n y t w o e l e m e n t s o f t h e ideal must also be in the ideal. a fuzzy set is a class of objects with grades of membership in the interval [0, 1]. it is used to mathematically represent uncertainty and provide a formal tool to deal with imprecisions present in many problems. we use ideals to def ine fuzzy on ideal sets, which can be seen as a generalization of the fuzzy sets. we establish s o m e o f i t s b a s i c p r o p e r t i e s , a n d w e s t a t e a n d p r o v e a h a h n b a n a c h t h e o r e m w i t h t h e f u z z y o n i d e a l s e t s , w h i c h c a n b e s e e n a s a g e n e r a l i z a t i o n o f a f u z z y h a h n b a n a c h t h e o r e m , w h i c h i n t u r n , i s a fuzzif ied generalization of an analytic form of the classical hahn-banach theorem. mathematics subject classification (2010): 62b86 keyword s: fuzzy, ideal, hahn-banach theorem _______________ *corresponding author issn 0115-7809 print / issn 2012-0818 online introduction t h e co n ce p t of i d e a l s p a ce s w a s f i r s t s t u d i ed by ku r a tow s k i ( 1 9 6 6 ) a n d vaidyanathaswamy (1945). formally, given a set x, an ideal i(x) is a nonempty collection of subsets of x that satisf ies: i. a ∈ i (x) and b ⊆ a implies b ∈ i (x); and ii. a ∈ i (x) and b ∈ i (x) implies a ∪ b ∈ i (x). l.n. mernilo-tutanes and r.l. caga-anan 71 ideal spaces were then imported to topology. for instance, jankovic and hamlett (1990) investigated the notion of topological spaces with ideals. thereafter, ideal spaces found their way to other concepts in topology. in 1965, fuzzy sets were introduced by zadeh (1965) and klaua (1965) as extension of the classical notion of set. in classical set theory, the membership of elements in a set is assessed in binary terms: an element either belongs or does not belong to the set. that is, a set a on a universal set x can be identif ied as the characteristic function of a having only the values 0 or 1. on the other hand, a fuzzy set on x is formally def ined as a mapping from x into the unit interval [0, 1]. then in 1978, zadeh introduced possibility theory as an extension of his theory of fuzzy sets and fuzzy logic (zadeh 1978). possibility theory should not be confused with probability theory. it is an uncertainty theory trying to make sense of incomplete information and is viewed as a complement to probability theory. similar to a probability distribution, the theory uses a possibility distribution. a possibility distribution is a mapping π x from a set of states to a totally ordered set such as the unit interval [0, 1]. as one may easily notice, a possibility distribution can be used as an interpretation of the fuzzy sets. we provide the example given by zadeh (1978) to better understand a possibility distribution and differentiate it from a probability distribution. suppose we have the statement “hans ate x eggs for breakfast,” with x taking values in u = {1,2,3,...}. we may associate a possibility distribution with x by interpreting π x (u) as the “degree of ease with which hans can eat u eggs”. we may also associate a probability distribution with x by interpreting p x (u) as the probability of “hans eating u eggs for breakfast”. the values of π x (u) and p x (u) may look like as shown in the following table. u 1 2 3 4 5 6 7 8 π x (u) 1 1 1 1 0.7 0.5 0.4 0.2 p x ( u ) 0.2 0.7 0.1 0 0 0 0 0 one may easily notice that the sum of all values for p x (u) is equal to 1, but for π x (u), it is not. we may also observe that the possibility that hans may eat 3 eggs for breakfast is 1 but the probability that he may do so is quite small. hence, we can say that a high degree of possibility does not imply a high degree of probability, nor does a low degree of probability imply a low degree of possibility. however, if an event is impossible, then it should be improbable. fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 72 in this study, we want to capture statements like the following. suppose we want to complete the statement “tomorrow , ” we may have the following choices: a. will be sunny b. there will be rain c. will be cloudy d. there will be a storm we may give a value of 0.8 for the possibility that “tomorrow will be sunny”, 0.6 for the possibility that “tomorrow there will be rain”, 0.4 for the possibility that “tomorrow will be cloudy”, and 0.2 for the possibility that “tomorrow there will be a storm”. now, consider the possibility that “tomorrow will be sunny and there will also be rain.” this condition is very rare but not impossible. we cannot just give this possibility a value equal to the inf imum of the possibility that “tomorrow will be sunny” and the possibility that “tomorrow there will be rain”. we know that this possibility should be far less than any of these two possibilities. hence, we may give it a separate possibility value not dependent on the other two mentioned possibilities, say, 0.01. with this situation in mind, we def ine a fuzzy on ideal set in the next section. as an application of this new concept, we consider the hahn-banach theorem. the theorem is no doubt an important and powerful result in functional analysis. it was generalized in many directions. one of it is by fuzzy sets, like what rhie and hwang (1999) did. in a similar way, we generalize the theorem with fuzzy on ideal sets. fuzzy on ideal sets we now formally def ine a fuzzy on ideal set. def inition 2.1. let x be a nonempty set i(x) an ideal on x, and i be the unit interval [0, 1]. a function μ:i(x)→i called a fuzzy on ideal set provided: i. μ (∅) = 0 and ii. for nonempty sets a, b ∈ i (x), with a ⊆ b, we have μ (b) ≤ μ (a). l.n. mernilo-tutanes and r.l. caga-anan 73 we denote by i i(x) the set of all such μ. it is important to note here that the “reverse inequality”, μ (b) ≤ μ (a), of def inition 2.1 encapsulates our preceding idea that the possibility that “tomorrow will be sunny and there will also be rain” should not just be equal to the inf imum of the possibility that “tomorrow will be sunny” and the possibility that “tomorrow there will be rain” as it can be far less. remark 2.2. we may think of a fuzzy on ideal set as a generalization of a fuzzy set in x in the sense that the set of all fuzzy sets i x can be embedded in i p(x), where p(x) is the power set of x— the largest ideal on x . to see this, let μ : x → i be a fuzzy set in x. we can identify μ with μ ∈ip(x) def ined by example 2.3. let x = {a,b} and μ = {(a,1), (b,0.5)} be on x. we may identify μ with the fuzzy on ideal set μ def ined as μ = {({a},1), ({b},0.5), ({a,b},0), (∅,0)}. example 2.4. let x be a nonempty set and μ : x → i be a fuzzy set. we can def ine a fuzzy on ideal set π : p(x)→ i as π (a) = inf x∈a μ(x) and π (∅) = 0. this is called a guaranteed possibility in dubois and prade (2000). remark 2.5. it is important to note that def inition 2.1 does not def ine a measure. for a ⊆ b, a measure m should have m(a)≤m(b), not the reverse inequality, as in our def inition. next, we def ine some relational operators between fuzzy on ideal sets. def inition 2.6. let x be a nonempty set and μ1, μ2 ∈ i i(x). we say that μ1 ≤ μ2 , μ1 ≥ μ2 or μ1 = μ2 , provided that, for every a∈ i(x), we have μ1(a) ≤ μ2(a), μ1(a) ≥ μ2 (a), or μ1(a) =μ2(a), respectively. definition 2.7. let x be a nonempty set and μ ∈ i i(x). the complement of μ denoted by μ c: i (x) →i is def ined by μ c (∅) = 0, and for ∅ ≠ a∈ i(x) , μ c : (a) = inf {1– μ ({x})}. remark 2.8. for a = {x}, the preceding def inition coincides with the def inition of the complement of a fuzzy set. def inition 2.9. let x be a nonempty set and i(x) be an ideal on x. if {μ j | j∈ j} is a collection of fuzzy on ideal sets, then the union and the intersection of the μ j ’s are given by: ∼ ∼ μ (x), if a={x}, x∈x; ∼μ (a)= 0, otherwise. ∼ x∈a fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 74 i. (∨ j∈j μ j )(a) = sup {μ j (a)| j ∈ j}; and ii. (∧ j∈j μ j )(a) = inf {μ j (a)| j ∈ j}, respectively, for every a ∈ i(x). next, we show that the complement, union, and intersection of fuzzy on ideal sets are also fuzzy on ideal sets. proposition 2.10. let x be a nonempty set and i(x) be an ideal on x. if {μ j | j ∈ j} is a collection of fuzzy on ideal sets, then μ c j , ∨ j∈j μ j , and ∧ j∈j μ j are fuzzy on ideal sets. proof. let ∅ ≠ a, b ∈ i(x), such that a ⊆ b. then, {μ ({x}) : x∈a} ⊆{μ ({x}) : x ∈ b}, a n d s o , { 1 – μ ( { x } ) : x ∈ a } ⊆ { 1 – μ ( { x } ) : x ∈ b } . t h u s , f o r j ∈ j , μ c j ( a ) = inf{1–μ j ({x})} ≥ inf{1–μ j ({x})}= μ c j ( b). by def inition 2.7, μ c j (∅) = 0 for each j∈j, and hence, μ c j is a fuzzy on ideal set. now, since a ⊆ b and μ j ∈ ii(x), μ j (a) ≥ μ j (b) where j∈j. it follows that sup{μ j (a)| j ∈ j}≥ sup{μ j (b)| j ∈ j}, and so, (∨ j∈j μ j )(a) ≥ (∨ j∈j μ j )(b). note that (∨ j∈j μ i )(∅) = sup{μ j (∅) | j ∈ j}= 0, and hence, ∨ j∈j μ j is a fuzzy on ideal set. similarly, we can show that ∧ j∈j μ j is a fuzzy on ideal set.  mappings in this section, we show that, given only a mapping between nonempty sets (not a mapping between fuzzy on ideals sets), we can def ine the image and pre-image of fuzzy on ideal sets. the following are preparatory def initions and results. definition 3.1. let x and y be nonempty sets, and let f : x → y be a mapping. if i(x) and i(y) are ideals on x and y, respectively, we def ine f (i(x)) = {f (a) : a ∈ i (x)} and f –1(i(y)) = {a : a ⊆ f –1 (b), b ∈ i (y )}, where f (a) and f –1 (b) are the usual image and pre-image of a ⊆ x and b ⊆ y, respectively. theorem 3.2. let x and y be nonempty sets and let f : x → y be a mapping. if i(x) and i(y) are ideals on x and y, respectively, then f(i(x)) and f –1(i(y)) are ideals on y and x, respectively. proof. since i(x) is an ideal on x, i(x)≠∅. it follows that f(i(x)) ≠∅. let b 2 ∈f (i(x)). then, there exists a 2 ∈ i (x), such that f (a 2 ) = b 2 . let b 1 ⊆ b 2 . now, let a 1 = a 2 ∩ f –1 (b 1 ). then, a 1 ⊆ a 2 and f (a 1 ) = b 1 . note that a 1 ∈ i (x), since it is a subset of a 2 ∈ i (x). thus, x∈bx∈a l.n. mernilo-tutanes and r.l. caga-anan 75 b 1 ∈ f (i (x)). next, suppose that b 1 , b 2 ∈ f (i (x)), then there exist a 1 , a 2 ∈ i (x), such that f (a 1 ) = b 1 and f (a 2 ) = b 2 . now, a 1 ∪ a 2 ∈ i (x), since i (x) is an ideal. thus, b 1 ∪ b 2 = f (a 1 ) ∪ f (a 2 ) = f (a 1 ∪ a 2 )∈ f (i (x)). therefore, f (i (x)) is an ideal on y. since i (y) is an ideal on y, i (y) ≠ ∅. it follows that f –1 (i(y)) ≠ ∅. let a 2 ∈ f –1(i(y)). then, there exists b 2 ∈ i(y) such that a 2 ⊆ f –1(b 2 ) . let a 1 ⊆ a 2 . then, we also have a 1 ⊆ f –1(b 2 ), a n d t h u s , a 1 ∈ f –1(i(y)). n e x t , s u p p o s e t h a t a 1 , a 2 ∈ f –1(i(y)), t h e n t h e r e e x i s t b 1 , b 2 ∈ i(y ), such that a 1 ⊆ f –1(b 1 ) and a 1 ⊆ f –1(b 2 ). since b 1 ∪b 2 ∈ i(y), i(y) being an ideal on y, we have a 1 ∪a 2 ⊆ f –1(b 1 ∪b 2 ) , then a 1 ∪a 2 ⊆ f –1 (i(y)). thus, f –1 (i(y)) is an ideal on x .  the next def inition def ines the image and pre-image of fuzzy on ideal sets out of an ordinary mapping, and the next proposition proves that it is well-def ined. definition 3.3. let x and y be nonempty sets, and let f : x → y be a mapping. let μ ∈ ii(x) and σ ∈ ii(y) for some ideals i (x) and i (y) of x and y, respectively. def ine the image of μ, denoted by f [μ], and the pre-image of σ, denoted by f –1[σ], as follows: i. f [μ] : f (i(x)) →i , where for b ∈ f (i(x)), f [μ](b) = sup μ (a), where s b = {a∈ i(x)) : f (a) = b}; and ii. f –1[σ ]:f –1(i(y))→i, where for a ∈ f –1(i(y)), f –1[σ ] (a) = (σ ° f )(a). proposition 3.4. let x and y be nonempty sets, and let f : x → y be a mapping. let μ ∈ i i(x) and σ ∈ ii(y). then, f [μ] and f –1[σ ] are fuzzy on ideal sets on y and x, respectively. proof. we f irst note that, by theorem 3.2, f (i(x)) and f –1(i(y)) are ideals on y and x respectively. if b ≠ ∅, then s b = {∅}, and so, f [μ]∅ = sup a∈ sb μ(a) = 0. also, for ∅ = a ∈ f –1(i(y)), we have f (a) = ∅, and so, f –1[σ]:f –1(∅) = σ (f(a)) = 0. now, let ∅ ≠ b 1 , b 2 ∈ f (i(x)), such that b 1 ⊆ b 2 . then, for a 1 , a 2 ∈i(x) , such that f (a 1 )=b 1 , f (a 2 )=b 2 , and a 1 ⊆ a 2 , we have μ (a 2 ) ≤ μ (a 1 ) , since μ is a fuzzy on ideal set. hence, sup a∈sb 2 μ (a) ≤ sup a∈sb 1 μ (a), and so, f [μ](b 2 ) ≤ f [μ](b 1 ). now, for ∅ ≠ a 1 , a 2 ∈ f –1(i(y)), such that a 1 ⊆ a 2 , we have f (a 1 ) ⊆ (a 2 ) . hence, σ f (a 2 )) ≤ σ f (a 1 )), since σ is a fuzzy on ideal set. consequently, f –1[σ ](a 2 ) ≤ f –1[σ ](a 1 ). therefore, f [μ] and f –1[σ ] are fuzzy on ideal sets on y and x, respectively.  a∈sb fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 76 fuzzy on ideal hahn-banach theorem rhie and hwang (1999) fuzzif ied the analytic form of the hahn-banach theorem. they built the idea from the works of katsaras (1981, 1984) and katsaras and liu (1977) on fuzzy vector spaces and fuzzy seminorm, and the work of krishna and sarma (1991) on the generation of the fuzzy vector topology from an ordinary vector topology. we state and prove the analytic form of the hahn-banach theorem in the fuzzy on ideal setting. this can be seen as a generalization of the fuzzy hanhbanach theorem by rhie and hwang (1999). we follow the ideas and the flow of proof by rhie and hwang (1999). we recall f irst that for a vector space x over and a, b ⊆ x, we have a+b={a+b:a∈a and b∈b} and for , ta = {ta:a∈a}. let i(x) be an ideal on x. we def ine an associated set x 0 by x 0 = {a:{a} ∈ i(x)}. that is, x 0 is the set out of the singleton subsets of i(x). note that x 0 ⊆ x. for ∅ ≠ a∈i(x) , by the f irst property of an ideal, all singleton subsets of a are also in i(x), and so, a ⊆ x 0 . we want next that i(x) be closed under f inite addition and scalar multiplication. the next proposition shows that it is enough to assume that x 0 is a linear subspace of x , such that x 0 ∈ i (x). proposition 4.1. let x be a vector space over , and let x 0 be a linear subspace of x. if i (x) is an ideal of x, such that x 0 ∈ i (x), then for every a, b ⊆ x 0 and every , we have ta, a+b∈i(x). proof. let a, b ⊆ x 0 and . since x 0 is a linear subspace of x, it follows that ta = {ta : a∈a} ⊆ x 0 and a+b ={a+b : a∈a and b∈b} ⊆ x 0 . since x 0 ∈ i(x), then any subset of x 0 is in i(x). that is, ta and a+b are in i(x) .  to move forward we need to def ine f inite addition and scalar multiplication of fuzzy on ideal sets, such that the result is also a fuzzy on ideal set. def inition 4.2. let x be a vector space over , and i(x) be an ideal on x , such that x 0 is a linear subspace of x and x 0 ∈ i(x). for any μ, v ∈ ii(x), we def ine μ+v as follows: (μ+v)(∅)=0 and for ∅ ≠ a∈i(x),             ngleton)s not a si (or a iotherwise.axxv n)a singleto (or a is a={x}; ifxxxxvx av xxx },:}))({{(inf },,:})({})({{sup =))(( 02121 =21      ∈ ∈ ∈ l.n. mernilo-tutanes and r.l. caga-anan 77 in the case a = {x} in def inition 4.2, we note that, since x 0 is a linear subspace of x, we can always express x as x = 0 + x, and thus, the set under the sup is never empty. def inition 4.3. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). for a scalar and ∈ ii(x) , we def ine tμ as follows: (tμ)(∅) = 0 and for ∅ ≠ a∈i(x), ∈             a = {0 } .i f t= 0 a n dxyysu p { 0 } ; aif t = 0 a n d 0 ;if tat at ,:} )({ 0 , , =))(( 0 1    proposition 4.4. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈i(x). if and μ, v ∈ ii(x), then tμ and μ+v are in i i(x). proof. we note that, by def inition 4.3, we have (tμ)(∅) = 0. now, let ∅ ≠ a 1 , a 2 ∈i(x), such that a 1 ⊆ a 2 and . let t ≠ 0. then, and t-1 a 1 ⊆ t-1a 2 . note that t–1a 1 , a 2 ∈i(x) by proposition 4.1. it follows that μ (t–1a 1 ) ≥ μ (t–1a 2 ) since μ ∈ ii(x). suppose t = 0 and a 1 , a 2 ≠ {0}, we have (tμ)(a 1 ) = 0 = (tμ)(a 2 ). now, for t = 0 and a 1 , a 2 = {0}, we have (tμ)(a 1 ) = (tμ)(a 2 ) . if t = 0, a 1 = {0} and a 2 ≠ {0} , then (tμ)(a 2 ) = 0 ≤ (tμ)(a 1 ). thus, tμ ∈ ii(x). next, we show that μ + v ∈ii(x). by def inition 4.2, (μ + v)(∅)=0. let ∅≠ a 1 , a 2 ∈i(x), such that a 1 ⊆ a 2 . suppose a 2 is a singleton, then a 1 must be a singleton. then, a 1 = {x}=a 2 . notice that, (μ + v)(a 1 ) = (μ + v)(a 2 ). now, if a 2 is not a singleton, then a 1 is either a singleton or not. in any case, since a 1 ⊆ a 2 , we have{(μ+v)({x}): x∈a 1 }⊆{(μ+v )({x}): x∈a 2 }. thus, inf{(μ+v)({x}): x∈a 1 } ≥ inf{(μ+v)({x}): x∈a 2 }. consequently, (μ+v)(a 1 ) ≥ (μ+v)(a 2 ). therefore, μ + v ∈ i i(x). next, we need to def ine a fuzzy on ideal seminorm. we begin by def ining its properties. ∈ ∈   fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 78 def inition 4.5. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). a ρ ∈ i i(x) is said to be i. convex if ρ (ta+(1–t)b) ≥ min{ρ (a), ρ (b)} for every t∈ [0,1] and ∅ ≠ a, b ∈ i(x); ii. balanced if (tρ)(a) ≤ ρ (a) for every with | t |≤ 1 and ∅ ≠ a ∈ i(x); iii. absorbing if sup 1>0 (tρ)(a)=1 for every ∅ ≠ a ∈ i(x). def inition 4.6. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈i(x). a ρ ∈ i i(x) is called a fuzzy on ideal seminorm if it is convex, balanced, and absorbing. associated with a fuzzy on ideal seminorm, we def ine below an important mapping, and then we prove that it has the properties of an ordinary seminorm. def inition 4.7. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈i(x) . let ρ be a fuzzy on ideal seminorm. for each ε ∈ (0,1), we def ine pε : i (x) →[ 0 , +∞) by pε (∅) = 0 and for ∅ ≠ a ∈ i( x), pε (a)=inf{t>0:(tρ)(a)>ε}. observe that pε is well-def ined since ρ is absorbing. moreover, 1= sup1>0 (tρ)({0}) = sup 1>0 (ρ)(t-1{0})=sup 1>0 (ρ)({0}) implies that ρ ({0})=1. remark 4.8. for 0< ε 1 < ε 2 < 1 , we have {t>0:( t ρ (a) ε 2 }⊆{t >0:(t ρ) (a) > ε 1 }. hence, pε (a)=inf{t>0 : (tρ)(a)>ε 1}≤ inf{t>0 : (tρ)(a)>ε 2}=pε 2 (a). that is, {pε} is increasing in ε . theorem 4.9. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈i(x). if ρ is a fuzzy on ideal seminorm, then, for each ε ∈ (0,1), we have pε satisfying the following properties: i. , for all and ∅ ≠ a ∈ i(x) ; ii. , for ∅ ≠ a,b ∈ i(x) . ∈ ( ) =| | ( )p a p a   ( ) ( ) ( )p a b p a p b     l.n. mernilo-tutanes and r.l. caga-anan 79 proof. i. let ε ∈(0,1) and ∅ ≠ a ∈ i(x). if α = 0, then αa = {0}. observe that let α ≠ 0. since ρ is balanced, (tρ )(a) ≤ ρ (a) for | t |≤1. in particular, t = –1 implies (–ρ)(a) ≤ ρ (a), and so, . now, hence, . next, consider that . it follows that . thus, . consequently, then, ii. let ∅ ≠ a, b ∈ i( x), r ∈{t >0 : (tρ)(a) >ε}, and s ∈{t >0 : (tρ)(b) >ε}. then, (rρ)(a) >ε and (sρ)(b) >ε. now, by convexity of ρ, we have . it follows that and so, . . hence, we have 0 ∙ 0: 0 0: 1 0 0: 0 0:1 0 |0| . 1 1 1 . 0: 0: 0: | | | | ′ 0: ′ , | | ′ 0: ′ | | ′ 0: ′ | | 0: | | . by the preceding result where ′ | | 0: 0: ⊆ 0: . , , , ∈ 0: . fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 80 thus, therefore, .  the next theorem shows that the inf imum of the pε has properties similar to it. the orem 4.10. let x be a vector space over , and i( x ) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). let ρ be a fuzzy on ideal seminorm. then, the function p:i (x) → [0,+∞) def ined by p(∅)=0, and for ∅ ≠ a ∈ i( x), p(a) = inf{pε (a):ε ∈(0,1)} satisf ies the following properties: i . for all and ii. , for all . proof. the f irst property follows directly from the f irst proper ty in theorem 4.9. let ∅ ≠ a,b ∈i( x). since {p ε} is increasing in ε, for every ∅ ≠ a∈i(x ), p(a) = inf{pε (a):ε ∈(0,1)} = limε→0 pε (a). thus, p(a+b)=inf{pε (a+b):ε ∈(0,1)}≤ inf{pε (a)+pε (b):ε ∈(0,1)} = lim{pε (a)+pε(b)} = lim{pε (a)+lim pε(b)=p (a)+p (b).  the next two theorems give us the relationship between fuzzy on ideal seminorms and its associated mappings having the ordinary seminorm properties. it is our key to tap on the classical hahn-banach theorem that will be used in the proof of our fuzzy on ideal hahn-banach theorem. theorem 4.11. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). furthermore, let ρ 1 and ρ 2 be two fuzzy on ideal seminorms. if for every a∈i( x) , ρ 1 (a) ≤ ρ 2 (a), then for every ε ∈(0,1), p1(a) ≥ p 2(a) for all a∈i( x). proof. if for every a∈i (x), ρ 1 (a) ≤ ρ 2 (a), then for every a∈i(x) and t > 0, (tρ 1 )(a) = ρ 1 (t-1a) ≤ ρ 2 (t-1a) = (tρ 2 )(a). let ε ∈(0,1) and ∅ ≠ a∈i(x). observe that {t>0:(tρ 1 )(a) >ε} is a subset of {t > 0:(tρ 2 )(a)>ε}. hence, inf{t > 0:(tρ 1 )(a) >ε} ≥ {t > 0:(tρ 2 )(a)>ε}. thus, p1(a) ≥ p2(a).  0: 0: 0: . | | , ∈ ∅ ∈ ; ∅ , ∈ ε ε ε→0 ε→0ε→0 ε ε l.n. mernilo-tutanes and r.l. caga-anan 81 remark 4.12. the converse of theorem 4.11 does not always hold. to see this, let x = and i(x) = p( ). def ine ρ 1 and ρ 2 as follows: one can check that ρ 1 and ρ 2 are fuzzy on ideal seminorms and for ε ∈ (0,1), but ρ 1 ≤ ρ 2 and ρ 2 ≤ ρ 1 . the following *-property will give us a suff icient condition for the converse to hold. def inition 4.13. let x be a vector space over , and let i(x) be an ideal on x, such that x 0 is a linear subspace of x. let x 0 ∈ i(x). let ρ be a fuzzy on ideal seminorm. we say that ρ has the *-property if, for every ∅≠a∈ i(x), we have ρ (a) = inf{ρ (ta):0 < t < 1}. an example of a fuzzy on ideal seminorm with the *-property will be given later. it is a crucial part of our main theorem. in the meantime, let us prove that, with the *-property, the converse of theorem 4.11 will hold. lemma 4.14. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x. let x 0 ∈ i(x). let ρ be a fuzzy on ideal seminorm with the *-property. if ∅≠a∈ i(x) and ρ (a)<ε <1, then pε (a)>1. proof. let ∅≠a∈ i(x) and ρ (a)<ε <1. since ρ is balanced being a fuzzy on ideal s e m i n o r m , ( t ρ ) ( a ) ≤ ρ ( a ) < ε f o r | t | ≤ 1 . t h u s , pε ( a ) = i n f { t > 0 : ( t ρ ) ( a ) > ε } = inf{t>1:(tρ)(a)> ε}≥1. we are left to show that pε (a)≠1. suppose pε (a)=1. then, (tρ)(a)>ε for all t >1. s i n c e ρ h a s t h e *p r o p e r t y, ρ ( a ) = i n f { ρ ( t a ) : 0 < t < 1 } = i n f { ( t 1 ρ ) ( a ) : 0 < t < 1 } =inf{(tρ)(a):t >1}≥ε. however, this is a contradiction, since ρ (a) < ε. therefore, pε (a)>1.  2 1, ∈ 1,1 ; 1 3 , ∈ 5, 1 ∪ 1,5 ; 0, . 1 1, ∈ 1,1 ; 1 3 , ∈ 5, 1 ∪ 1,5 ; 0, . 1 2 | | fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 82 theorem 4.15. let x be a vector space over , and let i(x) be an ideal on x, such that x 0 is a linear subspace of x. let x 0 ∈ i(x). let ρ 1 and ρ 2 be two fuzzy on ideal seminorms, with ρ 2 having the *-property. if for every ε ∈ (0,1), we have for every a∈ i(x), then for every a∈ i(x). proof. if a = ∅, then by def inition, . suppose that for every ε ∈ (0,1), , and that there exists a b∈ i(x), such that ρ 2 (b) < ρ 1 (b) . let ρ 2 (b) < ε < ρ 1 (b). if t = 1, then tρ 1 (b) = ρ 1 (b) > ε, and so, . since ρ 2 is balanced, . by lemma 4.14, . thus, . this is a contradiction to our assumption that for every ε ∈ (0,1) , , . therefore, we must have , .  we now def ine and prove an important fuzzy on ideal seminorm with the *-property. we begin with the following def inition. def inition 4.16. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). let m be also a linear subspace of x and f:m→ be a linear functional. furthermore, let i(m) be an ideal of m, such that m 0 ∈ i(m) . we associate with f the function def ined by , and for , . let we def ine by χ bf (∅) = 0, and for ∅ ≠a∈ i(x), observe that, for t > 0, . for convenience, whenever we have , we let the and say that the . theorem 4.17. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). let m be also a linear subspace of x and f:m→ be a linear functional. furthermore, let i(m) be an ideal of m, such that m 0 ∈i(m) . then, χ bf is a fuzzy on ideal seminorm with the *-property . proof. we f irst show that χ bf has the *-proper ty. let ∅≠a∈ i(m). suppose that then, . then, for all 0 < t < 1, sup x∈α|f(tx)|= t sup x∈α|f(x)|≤ t<1. hence, χ bf (ta) = 1, for all 0 < t < 1, and so, inf{χbf(ta):0 < t < 1}= 1. suppose that χ bf (a) = 0. then, π f (a) = sup x∈α|f (x)| >1. assume inf{χbf(ta)|0 < t < 1}= 1. it follows that, for all 0<t<1, χ bf (ta) =1, implying that π f (ta) = sup x∈α| f(tx)| = t sup x∈α|f(x)|≤1. thus, for all 0< t <1, supx∈α| f(x)|≤ . consequently,   )()( 21 apap     )()( 21 aa   1 2 1 2 0   )()( 21 apap     )( xia    1}>)(:0>{=)( 1 1  bttinfbp   1}>)(:0>{=)( 2 2  bttinfbp   1>)(2 bp 2 1( ) > ( )p b p b    )()( 21 apap     )( xia    )( xia   )()( 21 aa   : → ∪ ∞   0=)(f   )(mia   |)(|sup=)( xfa axf  ∈ : ∅ : → 0,1   )()( atta ff      |)(|sup=)( xfa axf 1. ∈ | | 1 lim t→+∞πf (a)= 1 πf (a) =1t t 1 t ∈ | | 1 1 . 1, ∈ ; 0, . 0 0 1 l.n. mernilo-tutanes and r.l. caga-anan 83 for all η >0. since η is arbitrary, sup x∈α|f(x)| ≤ 1, which contradicts to χbf(a) = 0. hence, there exists 0 < t < 1, such that χ bf (ta) = 0, that is inf{χ bf (ta):0 < t < 1}= 0. therefore, χ bf has the *-property. next we show that it is a fuzzy on ideal set. it is enough to show the reverse inequality. let ∅ ≠ a,b ∈ i(m), such that a ⊆ b. if χχ bf (b) = 0, then the reverse inequality automatically follows. suppose that χ bf (b) = 1. then, π f (b) = sup x∈β | f (x)| ≤1. now, π f (a) = sup x∈α | f (x)| ≤ supx∈β | f (x)| ≤ 1. hence, χ bf (a) = 1. thus, χ bf (b) ≤ χ bf(a). we are left to show that χ bf is convex, balanced, and absorbing. let ∅ ≠ a,b ∈ i(m). if χ bf (a) = 0 or χ bf (b) = 0, then the inequality for convex is clearly satisf ied. suppose that a,b ∈ b f , that is, χ bf (a) = 1 and χ bf(b) = 1. then, s u p x ∈ β | f ( x ) | ≤ 1 and supx∈β |f (x)| ≤1. it follows that, for each 0 ≤ t ≤1, since f is linear, we have ≤ t + 1–t = 1. thus, χbf (ta+(1–t)b) = 1, and so χbf is convex. let ∅ ≠ a,b ∈ i(m) and | t| ≤ 1. if (tχχ bf)(a) = 0 , then the inequality for balanced is clearly satisf ied. suppose (t χ b f )( a) = 1 . then for each t with | t| ≤ 1, we have . hence, sup x∈α| f(x)|≤| f(x)|≤|t| ≤ 1, and so, χχ bf (a)= 1. consequently, χ bf is balanced. lastly, let ∅ ≠ a ∈ i (m). if πf (a) = supx∈α | f (x)| < +∞, t a k e t 0 = s u p x ∈ α | f ( x ) | ≤ | f ( x ) | . t h e n , . t h u s , (t 0 χ bf) (a)=1. therefore, supt >0 (tχ bf)(a)= 1. if πf (a) = supx∈α|f (x)| ≤ +∞, we have remarked prior to this theorem that supt >0 (tχ bf)(a)= 1. thus, χbf is absorbing.  finally, we have the fuzzy on ideal hahn-banach theorem. theorem 4.18. let x be a vector space over , and i(x) be an ideal on x, such that x 0 is a linear subspace of x and x 0 ∈ i(x). let m be also a linear subspace of x, and i(m) be an ideal of m, such that m 0 ∈ i(m) and m 0 ⊆ x 0 . let ρ ∈ ii(x) be a fuzzy on ideal seminorm. if f:m 0 → is a linear functional, such that χ bf (a) ≥ ρ (a) for all a ∈ i (m), then there exists a linear functional g:x 0 → , such that: i. f(x) = g (x), x ∈m0; and ii. χ bg (a) ≥ ρ (a) for all a∈i(x). ∈ 1 | | ∈ , ∈ | 1 | ∈ 1 ∈ | | 1 ∈ 0 1 0 ∈ | | 1 ∈ , ∈ | | 1 | | fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 84 proof. let f be a linear functional on m 0 , such that χ bf ( a) ≥ ρ (a) for all a ∈i (m) . note f irst that χ bf is a fuzzy on ideal seminorm with the *-property. in theorem 4.11, let χ bf=ρ2 and ρ=ρ1. then, the corresponding pε 2 is given by: for any ∅ ≠ a∈ i(m) and ε ∈(0,1), pε 2(a) =inf{t>0:tρ 2 (a) >ε } =inf{t>0:ρ 2 (a/t) >ε } =inf{t>0:ρ 2 (a/t) =1}, since ρ 2 is a characteristic function =inf{t>0:π f (a/t) ≤1}, that is (a/t) ∈ b f =inf{t>0:π f (a) ≤t}, since f is linear =π f (a). thus, by theorem 4.11, for all ε ∈(0,1), pε 2(a) = π f (a) ≤ pε 1(a) for all ∅ ≠ a ∈ i(m), where pε 1(a) =inf{t>0:tρ (a) >ε}for all ∅ ≠ a ∈ i(x). observe that we are considering here a ∈ i(x), instead of just i(m). this can be done because ρ ∈ ii(x ). by the last inequality, π f (a) = sup x∈α | f (x)| ≤ p(a) = inf{pε 1(a):ε ∈(0,1)} for all ∅ ≠ a ∈ i(m). in particular, |f (x)| ≤ p({x}) , for all x ∈ m 0 . note that by theorem 4.10, p restricted to the singletons can be seen as a sublinear functional on x 0 . hence, by applying the classical hahn-banach theorem, there exists a linear functional g:x 0 → such that: i. f(x) = g (x), x ∈ m0; and ii. |g (x)| ≤ p({x}), for all x ∈ x 0 . now, let ∅ ≠ a ∈ i(x). note f irst that, by the def inition of p and the reverse inequality satisf ied by ρ, if x ∈ a, then p ({x}) ≤ p(a). hence by (ii),  x ∈ a, |g (x)| ≤ p(a). thus, we have sup x∈α |g(x)| = πg (a)≤ p(a) for all ∅ ≠ a ∈ i(x). let χbg =ρ2 in theorem 4.15. then, the corresponding pε 2 is given by: for any ∅≠a∈ i(x) and ε ∈(0,1), pε 2(a) =inf{t>0:tρ 2 (a) >ε } =inf{t>0:ρ 2 (a/t) >ε } =inf{t>0:ρ 2 (a/t) =1}, since ρ 2 is a characteristic function =inf{t>0:π g (a/t) ≤1}, that is (a/t) ∈ b g =inf{t>0:π g (a) ≤t}, since g is linear =π g (a). l.n. mernilo-tutanes and r.l. caga-anan 85 thus, for all ε ∈(0,1), pε 2(a) ≤ p(a) = inf{pε 1(a):ε ∈(0,1)} for all ∅ ≠ a ∈ i(x). hence, for all ε ∈(0,1), pε 2(a) ≤ pε 1(a) = inf{t>0:(tρ )(a) >ε } for all ∅ ≠ a ∈ i(x). since χ b g has the *-proper ty, by theorem 4.15, we have χ b g (a) ≥ ρ (a) for all a ∈ i(x).  acknowledgement the f irst author was supported by the commission on higher education (ched) of the philippines and the second author by the premier research institute of science and mathematics (prism) of msu-iit. references dubois d, prade h. 2000. fundamentals of fuzzy sets. new york: springer us. j a n k o v i c d , h a m l e t t t r . 1 9 9 0 . n e w t o p o l o g i e s f r o m o l d v i a i d e a l s . a m e r i c a n mathematical monthly. 97(4):295-310. katsaras ak. 1981. fuzzy topological vector space i. fuzzy sets and systems. 6(1):85-95. katsaras ak. 1984. fuzzy topological vector space ii. fuzzy sets and systems. 12(2):143154. katsaras ak, liu db. 1977. fuzzy vector spaces and fuzzy topological vector spaces. journal of mathematical analysis and applications. 58(1):135-146. klaua d. 1965. über einen ansatz zur mehrwertigen mengenlehre. monatsberichte der königlichen preussische akademie des w issenschaften zu berlin. 7:859-876. krishna sv, sarma kkm. 1991. fuzzy topological vector spaces-topological generation and normability. fuzzy sets and systems. 41(1):89-99. kuratowski k. 1966. topology. new york: academic press. rhie gs, hwang ia. 1999. on the fuzzy hahn-banach theorem-an analytic form. fuzzy sets and systems. 108(1):117-121. vaidyanathaswamy r. 1944. the localisation theory in set topology. proceedings indian academy of sciences (mathematical sciences) 20(1):51-61. zadeh la . 1965. fuzzy sets. information and control. 8(3):338-353. za d e h la . 1 9 7 8 . f u z zy s e t s a s a b a s i s fo r a t h eo r y of p o s s i b i l i t y. f u z zy s e t s a n d systems. 1:3-28. fuzzy on ideal sets and a fuzzy on ideal hahn-banach theorem 86 _____________ randy l. caga-anan, ph.d. (randy.caga-anan@g.msuiit.edu.ph) is an associate professor at the department of mathematics and statistics, mindanao state universityiligan institute of technology. he took his ph.d. from the university of the philippines diliman, and is currently working on topology, graph theory, and partial differential equations. lezel n. mernilo-tutanes, ph.d. (l_mernilo@yahoo.com) is an instructor at the mathematics department, bukidnon state university. she took her ph.d. at the mindanao state universityiligan institute of technology with a dissertation on topology. 71 evaluation of low molecular weight bis-urea derivatives as antimicrobial agents frances abygail f. genio natural sciences research institute institute of chemistry, college of science university of the philippines diliman monissa c. paderes* institute of chemistry university of the philippines diliman abstract antibiotic resistance against common microbes is an ongoing concern worldwide. this warrants continuous studies that aim to discover new compounds with antimicrobial properties. in this study, sixteen low molecular weight bis-urea derivatives were screened for their in vitro antimicrobial properties using agar well diffusion method. the structure of the bis-urea compounds is comprised of cyclic and aromatic linkers and a variety of symmetric end groups such as aliphatic chains and heteroaromatic groups. significant antimicrobial activity against strains of escherichia coli, staphylococcus aureus, and klebsiella pneumoniae was observed for compounds with long aliphatic chains compared to those with benzyl and heteroaromatic end groups. further studies on the minimum inhibitory concentration and cytotoxicity can aid in the development of these compounds as antimicrobial agents as well as for other possible biomedical and environmental applications. keywords: antimicrobial activity, bis-urea derivatives, agar well diffusion, antimicrobial agents * corresponding author science diliman (january-june 2022) 34:1, 71-81 evaluation of low molecular weight bis-urea derivatives as antimicrobial agents 72 introduction misuse and abuse of antibiotics have been prevalent in the philippines for a long time due to misconceptions about their proper use and the availability of the drugs in local stores (barber et al. 2017). this attitude towards antibiotics, among other factors, contributes to the emergence and spread of antibiotic-resistant strains, making this a global health concern (nys et al. 2004). common bacteria such as escherichia coli (e. coli) are already resistant to antibiotic drugs such as cefazolin, gentamicin, ciprofloxacin, and older drugs including ampicillin, oxytetracycline, trimethoprim, and chloramphenicol (nys et al. 2004). in other studies, staphylococcus aureus (s. aureus) was also found to exhibit resistance against chloramphenicol, tetracycline, and ciprofloxacin (juayang et al. 2014) while drugs such as ceftazidime and avibactam have been ineffective against klebsiella pneumoniae (k. pneumoniae) strains (chou et al. 2016). the growing antibiotic resistance is a serious threat to modern society, so there has been a necessity to discover new antimicrobial agents that would be effective against strains yet to be discovered (moellering 2011; kumar b et al. 2016). some of the more diverse antibiotic agents discovered to aid this problem are low molecular weight compounds, which include peptides (baquero et al. 1978; marshall and arenas 2003), steroids (moore et al. 1993), and other compounds isolated from natural sources (höltzel et al. 2000). synthetic low molecular weight compounds have also been reported as antimicrobial agents (rufián-henares and morales 2007; keche et al. 2012; poonia et al. 2020). specific examples of these synthetic compounds include quinoline derivatives which contain urea groups (keche et al. 2012) and bis (urea-1,2,3-triazole) compounds (poonia et al. 2020). urea moieties have been incorporated into molecules used as a scaffold for bioactive molecules with both organic and medicinal applications (gündüz et al. 2020). specifically, urea derivatives have been known to possess both antimicrobial and antiviral properties (sun et al. 2006; abdel-rahman and morsy 2007; džimbeg et al. 2008). although more specific mechanisms explaining the antimicrobial properties of urea derivatives are yet to be studied, there are few studies that have been reported. for instance, gündüz et al. (2020) observed that 1,3-disubstituted urea derivatives support the quorum sensing inhibition of pseudomonas aeruginosa which decreased the biofilm formation of the bacteria. in another study by lal et al. (2020), urea-1,2,3-triazole-amide hybrid compound with a good antimicrobial activity was observed to have significant docking interactions with the active site of s. aureus dna gyrase complexed with dna. f.a. f. genio and m. c. paderes 73 in this study, sixteen (16) readily synthesized low molecular weight bis-urea compounds were screened for antimicrobial activity against three organisms, e. coli, k. pneumoniae, and s. aureus using agar well diffusion method. the linkers used for the bis-urea compounds are cyclic and aromatic moieties, whereas the end groups are either aliphatic chains, benzyl, or picolyl (pyridyl-containing) groups. pyridine derivatives are well-studied for their anti-hypertensive, anti-neoplastic, and anti-inflammatory properties (lednicer and mitscher 1977) and have also been previously reported to exhibit antimicrobial properties (patel et al. 2010; kumar s et al. 2016). moreover, the incorporation of long aliphatic chain lengths enhances the antimicrobial properties as observed by akedo et al. (1977), violette et al. (2006), and yong et al. (2007). materials and methods synthesis of bis-urea compounds a series of bis-urea compounds were synthesized following the representative scheme shown below (genio and paderes 2021). following the method used by rutgeerts et al. (2019), various aromatic and cyclic diisocyanates and amines containing aliphatic, aromatic, and heteroaromatic groups were used to synthesize the bis-urea compounds. the diisocyanates and amines were stirred in anhydrous tetrahydrofuran (thf) at room temperature for an hour. the reaction mixture was kept under a nitrogen atmosphere and anhydrous thf was used because of the sensitivity to moisture of the starting reagents. products were precipitated out, filtered, washed with diethyl ether, and dried under a vacuum. scheme 1. representative procedure for the synthesis of bis-urea compound 1a. the detailed method and characterization of the compounds used in this study were previously reported by genio and paderes (2021). 1h nmr, 13c nmr, mass spectrometry, and ftir spectroscopy were among the methods performed to confirm the synthesis of the compounds. evaluation of low molecular weight bis-urea derivatives as antimicrobial agents 74 agar well diffusion method screening of the antimicrobial properties was performed by the microbial research and services laboratory (mrsl) of the natural sciences research institute (nsri), university of the philippines diliman using the agar well diffusion method. samples were all prepared in 98% ethanol using a 5 µg/ml concentration. ethanol, which is the solvent used for the sample solutions, was used as the negative control. for the positive controls, commercially available antibiotics cloxacillin (clox) and cefalexin (cef) were used. both compounds were prepared in ethanol at 5 µg/ml concentration to compare their antimicrobial activities. the organisms used were escherichia coli upcc 1195, klebsiella pneumoniae upcc 1360, and staphylococcus aureus upcc 1143. using peptone water as suspending medium, suspensions of the test organisms were prepared from 24 h old culture of microbes. these microbial suspensions were introduced and swabbed onto the surface of about 3 mm thick, pre-poured nutrient agar plates. the swab was distributed evenly twice over the agar surface. portions of 200 µl samples were placed on the three equidistant wells of 10 mm diameter made on the agar plate. the agar plates were left for 24 h at 35 °c and clearing zones were measured and used to calculate the antimicrobial index (ai) following the formula: ai = diameter of clearing zone – diameter of well diameter of well (1) formula for calculating antimicrobial index (ai) results and discussion as shown in scheme 2, the synthesis of low molecular weight bis-urea compounds was performed through condensation of various diisocyanates and amines. the reaction produced white powdery precipitates at 49-97% yields and were easily filtered off from the mixture (genio and paderes 2021). the solid products were washed several times with organic solvents such as diethyl ether to remove any unreacted or excess starting materials and ensure the purity of the bis-urea compounds. spectra obtained from the characterization of these compounds confirmed the formation of the urea groups and showed only peaks that correspond to the predicted product, implying high purity. more specifically, the absence of the diisocyanate peaks and the appearance of carbonyl peaks were noted in the ir spectra. proton peaks at a chemical shift of around 4-6 ppm were also observed in the 1h nmr spectra, indicating the successful formation of urea groups. f.a. f. genio and m. c. paderes 75 scheme 2. general scheme for the synthesis of bis-urea compounds. the synthesized bis-urea compounds, shown in figure 1, were tested for antimicrobial activity against gram-positive bacteria, s. aureus, and gram-negative bacteria, e. coli and k. pneumoniae. examples of the resulting wells from the assay using compound 6b are shown in figure 2. three wells in the agar plate were observed to have clearing areas where the microbial organisms did not grow. these areas were measured to calculate the antimicrobial index per compound against specific organisms. the results of the clearing zones in millimeters and the calculated antimicrobial indices of all sixteen compounds against each of the three organisms are summarized in table 1. for comparison, results for the antimicrobial activity of the commercial antibiotics, cloxacillin and cefalexin, prepared at 5 µg/ml using ethanol were included in table 1. the results show that all compounds, except 1d and 1e, exhibited zones of inhibition when tested against e. coli. however, only the antimicrobial indices of 2b, 4b, and 6b are significantly larger than those with the other derivatives. among the three compounds, 2b was observed to have the largest ai value, which is at 2.1. for the bacteria k. pneumoniae, most compounds displayed a zone of inhibition except for compounds 1a and 1d. similarly, good activity was observed for compounds 2b, 4b, and 6b, with 2b and 4b showing significantly larger ai (2.6 and 2.2, respectively). moreover, compounds 2b, 4b, 5b, and 6b also displayed notable ai values against s. aureus. generally, compounds containing long aliphatic end groups (2b, 4b, 5b, and 6b) demonstrated potential antimicrobial activity against the three organisms. the antimicrobial activities seemed to be dependent both on the linker and the alkyl end groups. for instance, bis-urea compounds with dicyclohexyl group as the linker (compounds 1) did not exhibit good activity against all the organisms tested. compounds with cyclohexyl group (2b) and aromatic linkers (4b, 5b, and 6b) on the other hand, displayed significant antimicrobial activity. evaluation of low molecular weight bis-urea derivatives as antimicrobial agents 76 n h n h n h n h o o n n n h n h n h n h o o n n n h n h n h n h o o n h n h n h n h o o 1a 1b 1c 1d 1e ch3(ch2)5 (ch2)5ch3 n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 compounds 1 n h n h n h n h o o n n n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 2a 2b compounds 2 compounds 3 n h n h n h n h oo nn n h n h n h n h oo (ch2)5ch3ch3(ch2)5 n h n h n h n h oo (ch2)11ch3ch3(ch2)11 3a 3c 3b compounds 4 compounds 5 n h n h n h n h o o n n 4a n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 4b compounds 6 o n h n h n h n h o o n n o n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 5a 5b n h n h n h n h o o n n n h n h n h n h o o ch3(ch2)11 (ch2)11ch3 6a 6b figure 1. structures of synthesized bis-urea compounds. a b c figure 2. images of the clearing zones of compound 6b in agar well diffusion antimicrobial assay against a) e. coli, b) k. pneumoniae, and c) s. aureus. f.a. f. genio and m. c. paderes 77 table 1. results of antimicrobial activity assay samplea e. coli k. pneumoniae s. aureus clearing zone, mmb ai clearing zone, mmb ai clearing zone, mmb ai 1a 11.0 ± 0.0 0.1 0 12.0 ± 0.0 0.2 1b 11.0 ± 0.0 0.1 11.0 ± 0.0 0.1 12.0 ± 0.0 0.2 1c 11.0 ± 0.0 0.1 11.0 ± 0.0 0.1 12.0 ± 0.0 0.2 1d 0 0 11.0 ± 0.0 0.1 1e 0 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 2a 11.7 ± 0.6 0.2 12.7 ± 0.6 0.3 12.7 ± 0.6 0.3 2b 30.7 ± 0.6 2.1 36.0 ± 0.0 2.6 25.0 ± 0.0 1.5 3a 13.3 ± 0.6 0.3 14.0 ± 0.0 0.4 12.0 ± 0.0 0.2 3b 12.3 ± 0.6 0.2 14.0 ± 0.0 0.4 12.7 ± 0.6 0.3 3c 11.0 ± 0.0 0.1 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 4a 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 12.0 ± 0.0 0.2 4b 21.0 ± 0.0 1.1 32.3 ± 0.6 2.2 20.0 ± 0.0 1 5a 12.3 ± 0.6 0.2 12.7 ± 0.6 0.3 12.0 ± 0.0 0.2 5b 15.0 ± 0.0 0.5 11.0 ± 0.0 0.1 25.3 ± 0.6 1.5 6a 12.0 ± 0.0 0.2 13.0 ± 0.0 0.3 12.7 ± 0.6 0.3 6b 24.3 ± 0.6 1.4 29.3 ± 0.6 1.9 16.3 ± 0.6 0.6 cloxc 13.3 ± 1.2 0.3 11.3 ± 0.6 0.1 25.7 ± 1.5 1.6 cefd 16.3 ± 1.2 0.6 12.3 ± 1.2 0.2 12.7 ± 1.2 0.3 etohe 12.0 ± 0.0 0.2 13.0 ± 0.0 0.3 12.0 ± 0.0 0.2 asamples were prepared at 5 µg/ml in ethanol; bclearing zone is the average of three trials; cclox = cloxacillin (positive control) at 5 µg/ ml in ethanol; dcef = cefalexin (positive control) at 5 µg/ml in ethanol; e etoh = ethanol; (-) no clearing zone was observed. the observed difference in the antimicrobial activity between compounds with the heteroaromatic end group (2a, 4a, 5a, and 6a) versus compounds with the aliphatic end groups (2b, 4b, 5b, and 6b) may be due to increased hydrophobicity brought by the long alkyl chain lengths (li et al. 2013). the hydrophobic nature of compounds that were previously analyzed from membrane binding and efficacy can be a factor affecting the compounds’ antimicrobial activity (kuroda et al. 2008). some of the antimicrobial indices obtained in this study are either comparable to or higher than the observed values for the antibiotics cloxacillin and cefalexin. compounds 2b, 4b, and 6b have significantly higher ai values than both clox and cef against the gram-negative bacteria, e. coli and k. pneumoniae. on the other hand, only 2b and 5b have comparable ai values with clox against the gramevaluation of low molecular weight bis-urea derivatives as antimicrobial agents 78 positive bacteria, s. aureus. these results imply the potential of these compounds as antimicrobial agents with properties that could be readily fine-tuned through structural modifications. however, further studies may be required to explore this potential antimicrobial property. conclusions sixteen bis-urea compounds were evaluated for their antimicrobial activity against e. coli, k. pneumoniae, and s. aureus. bis-urea compounds with long aliphatic side chains (2b, 4b, 5b, and 6b) showed significant activity against the tested organisms. compound 2b was found to be effective against all bacteria with ai values of 2.1 (e. coli), 2.6 for (k. pneumoniae), and 1.5 (s. aureus). compounds 4b and 5b were both selective with 4b being the most effective against k. pneumoniae and 5b against s. aureus. compound 6b on the other hand, showed activity with both e. coli and k. pneumoniae. further analysis of the potential of these bis-urea compounds as antimicrobial agents through antimicrobial assays against different organisms, cytotoxicity tests, and minimum inhibitory concentration assays can be conducted to explore more applications of these compounds as drugs or compatible ingredients to formulations. 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lamour k, chaloin o, frisch b, briand j, monteil h, guichard g. 2006. mimicking helical antibacterial peptides with nonpeptidic folding oligomers. chem biol. 13(5):531-538. https://doi.org/10.1016/j.chembiol.2006.03.009. yong y, xiao-li y, xue-gang l, jing z, baoshun z, lujiang y. 2007. synthesis and antimicrobial activity of 8-alkylberberine derivatives with a long aliphatic chain. planta med. 73(6):602604. https://doi.org/10.1055/s-2007-967180. ______ frances abygail f. genio earned her b.s. and m.s. in chemistry from the institute of chemistry, university of the philippines diliman. she is currently working as a university research associate i under projects funded by the natural sciences research institute (nsri), university of the philippines diliman. her research studies include synthesis and characterization of bis-urea molecules and their rheological and biological applications. monissa c. paderes <mcpaderes1@up.edu.ph> obtained her b.s. degree in chemistry at university of the philippines diliman, m.s. degree in chemistry at university of south carolina, columbia, and doctorate degree at state university of new york, buffalo. she worked as a research fellow at national university of singapore and as research scientist at ku leuven and procter & gamble in belgium. her expertise includes developing metal and organocatalyzed methods for the synthesis of organic compounds with biological applications and design and functionalization of polymers as rheology modifiers for hydrophobic and hydrophilic systems. 05_tempering obias and banzon 12 tempering and annealing in a verdier-stockmayer polymer e. r. obias and r. s. banzon structure and dynamics group, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: erobias@up.edu.ph; rbanzon@nip.upd.edu.ph abstract science diliman (july-december 2004) 16:2, 12–16 two monte carlo methods, simulated annealing and parallel tempering, were applied to a verdierstockmayer polymer. the efficiency of the two algorithms in exploring the lowest energy state possible for the model polymers was measured by the number of energy-degenerate configurations (configurations that have the same energy but are structurally different). parallel tempering consistently explored more energy-degenerate configurations as compared with simulated annealing. introduction monte carlo (mc) methods are widely used in a number of simulations in statistical physics (liang & wong, 1997; marinari & parisi, 1992; hansmann, 1997). this paper considers the applications and limitations of two mc methods, simulated annealing (sa) (kirkpatrick et al., 1983) and parallel tempering (pt) (frenkel & smit, 2002), applied to a twodimensional (2d) polymer that follows the verdierstockmayer (verdier & stockmayer, 1962; gould & tobochnik, 1996) model. the main task of numerical simulations on this matter is to study the polymer’s low-energy conformations, which depend on how the phase space is comprehensively explored. however, systems of this type have energy landscapes characterized by many deep local minima separated by high-energy barriers. at low temperatures, traditional monte carlo and molecular-dynamics simulations tend to get “trapped” in one of these minima, thereby hampering simulations to explore the polymer’s ground-state configurations. the two algorithms discussed in this paper, which are modifications of the original metropolis algorithm (metropolis et al., 1953), hope to alleviate the problem by minimizing the probability of settling to these local minima and continue to search for the global minimum. in sa, the temperature is initially high so that a relatively large percentage of the random steps that result in an increase in the energy will be accepted, thus making the polymer move freely. the temperature is then gradually lowered, hence the term annealing, until the target temperature is reached and the polymer is able to relax at its most stable conformation. in pt, simulation is done over n systems at different temperatures. multiple copies of the polymer run in parallel until a “swap” is imposed, hence the term parallel tempering. temperature differences are relatively small enough to allow the occasional swapping of two neighboring systems with different temperatures. methodology the polymer used in the simulation follows a 2d verdier-stockmayer model. each monomer occupies one of the vertices in a 2d lattice. initially, the polymer is completely unfolded. local movement is one monomer at a time and self-avoiding. figure 1 tempering and annealing 13 illustrates the two types of movements: end move (monomers 1 and 8) and corner move (monomers 4 and 7). total energy will be computed as the sum of all nonbonded nearest-neighbor interactions. in both sa and pt, a trial move is done by randomly selecting a monomer and moving it to a valid site subject to the types of movements depicted in fig. 1. the move is accepted if a random number (0,1) is less than the boltzmann probability exp(–de/t ) {boltzmann factor set to unity}, where de is the change in total energy and t is the temperature. if the change in energy is small enough or the temperature is high enough, then there is a high probability of acceptance. in pt, an additional trial move attempts to “swap” neighboring temperature polymers. this trial move is done by randomly selecting two neighboring temperature polymers and attempting to swap their configurations. the swap move is accepted if a random number (0,1) is less than the probability exp[(ei–ej)(bi– bj)], where b = 1/t. if there is enough overlap between these two systems, then there is a high probability of accepting the swap. the annealing schedule for simulated annealing is linear, every 5x105 mc steps, while the swapping ratio adapted for pt is 90% mc moves: 10% swapping moves (frenkel & smit, 2002). for a fair comparison, both algorithms have the same steps per temperature and the same total steps. the experiment consists of two parts. the first part had the interaction energies randomly generated [–4, – 2] and assigned to each monomer type (i.e., a hypothetical polymer is generated by choosing a sequence of n integers at random from the range 1– 20, corresponding to the 20 different possible amino acids) and temperatures ranged [0.5,10] in increments of 0.5 (giordano, 1997) to determine the transition region using both methods. the second part had the interaction energies fixed to –2 and temperatures were limited in the range [1,5] to investigate further the ability of the two algorithms to explore the lowest energy state possible. the lengths of the polymers used were 15 and 30 monomer units. results and discussion determination of the transition region figures 2 and 3 show average energy and end-to-end length of the 15-monomer chain using sa and pt. a transition region is observed in the region around t~2, both for the energy and end-to-end length graphs, as seen in the sharp curves in the plots. each point is an average of 5x105 monte carlo steps. the polymer has its lowest energy state at the lowest temperature. average end-to-end length decreases with temperature as seen in fig. 3. this means that the polymer has a relatively greater degree of folding at low temperatures. note that in pt, each point in the graph, which is also an average of 5x105 mc and swapping steps, is in a different system. the fluctuations can be eliminated by averaging more mc steps and performing more experiments. 1’ 1 2 1’ 4 3 4 5 6 7 8’ 8 7’ fig. 1. possible movements of a polymer with eight monomers. initial position shown with solid lines and unprimed monomer numbers; possible moves are indicated by dotted lines and primed monomer numbers. 0 1 2 3 4 5 6 7 8 9 10 temperature a ve ra g e en er g y -22 -20 -18 -16 -14 -12 -10 -8 -6 -4 fig. 2. average energy as a function of temperature. obias and banzon 14 investigation in the transition region to further investigate the algorithm’s ability to explore the lowest energy state configurations, we must be able to easily identify different configurations in a region where, expectedly, a large number of configurations may occur. one way to do this is to fix the interaction energies to some number, in this case –2 so there is a high probability of acceptance, and limit the thermodynamical states in the vicinity of the transition region in the temperature range [1,5]. figure 6 shows a 1x103 point representative of energies using sa with interaction energies fixed to –2 and temperatures annealed from t = 5 to t = 1 in increments of 1 every 5x105 mc steps for a total of 2.5x106 mc fig. 3. average end-to-end length as a function of temperature. 0 1 2 3 4 5 6 7 8 9 10 temperature a ve ra ge e nd -t oen d le ng th 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 fig. 4. energy at t = 1 using sa. 0 1000 2000 3000 4000 5000 mc steps (x100) e n er g y -26 -24 -22 -20 -18 -16 -14 -12 -10 -8 -6 fig. 5. energy at t = 1 using pt. 0 1000 2000 3000 4000 5000 mc steps (x100) e n er g y -25 -20 -15 -10 -5 although the two methods produced very similar results, they are different in terms of their ability to explore the lowest energy states. figures 4 and 5 show a 5x103 points representative of the 5x105 energies sampled at a temperature equal to 1 for the two algorithms. each point represents one energy state. in fig. 4, it is noticeable that some states are visited repeatedly as shown by consistent values lined horizontally in the graph even though we had initially set the interaction energies randomly. the polymer is somehow “trapped” at some states that it tends to repeatedly fold at this configuration. this repeated visits in some states are probable since the probability that they are degenerate is small considering the short length of the polymer. this is not seen in fig. 5, as there are more energy states represented at the low-energy states. fig. 6. 1x103 point representative of energies when interaction is fixed to –2 using sa. temperature 1 2 3 4 5 -16 -14 -12 -10 -8 -6 -4 -2 0 e n er g y tempering and annealing 15 steps. one can clearly identify the nine possible energy states from the plot with –16 as the lowest possible energy. figure 7 shows a similar pattern for parallel tempering with –16 also as the lowest possible energy. in fig. 7, five systems with temperatures in the range [1,5] are represented with 5x105 mc steps (including the swaps) for each system. with no distinct criteria for comparison on their ability to explore the lowest energy states, we resort to counting structural differences called energy-degenerate configurations (edc) explored in the lowest energy state. figure 8 shows six examples of edcs in 15 monomers with eight nearest-neighbor interactions and energy = –16. using the same parameters, fig. 9 presents a comparison of 20 independent experiments on the number of edcs explored by the two algorithms at t = 1 in the simulation, where the energy of interaction is fixed to –2 and temperature ranged [1,5]. rotation and mirror images are considered edcs so the number may be overestimated by a factor of 8 assuming all types of configurations are sampled. as seen from the graph, pt is consistently able to explore more distinct configurations as compared with sa. to verify the consistency of the performance in exploring edcs at the lowest energy state, we simulated a longer polymer consisting of 30 monomer units. trial experiments showed, however, that the lowest energy state is difficult to achieve using the two algorithms as the polymer is considerably long, thus giving it a much larger energy landscape and more local minima to overcome. figures 10 and 11 show a comparison of two lowest energy configurations obtained from trial simulations, which are –38 and –40. in fig. 10, experiment 4 using sa did not reach the energy state equal to –38, while all experiments in pt did. in fig. 11, 9 out of 20 experiments in pt managed to find the lowest energy state possible with the energy fig. 7. 1x103 point representative of energies when interaction is fixed to –2 using pt. temperature 1 2 3 4 5 -16 -14 -12 -10 -8 -6 -4 -2 0 e ne rg y fig. 8. examples of energy-degenerate configurations (edc) in 15 monomers with energy = –16. experiment 2 0 4 300 6 450 8 10 12 14 150 16 18 20 n um be r of e d c s fig. 9. comparison on the number of edcs explored at the lowest possible energy state (t = 1) in 15 monomers with energy = –16. experiment 2 0 4 200 6 250 8 10 12 14 100 16 18 20 n um be r of e d c s fig. 10. comparison on the number of edcs explored at the “second” lowest possible energy state (t = 1) in 30 monomers with energy < –38. 150 50 obias and banzon 16 equal to –40 as compared with simulated annealing which only had three. this further supplements the advantage of pt as a better energy landscape explorer. conclusion information on the number of edcs demonstrate to be a useful criterion in comparing algorithms whose objective is to study details of the energy landscape of a system. parallel tempering is consistently able to search more edcs at the lowest energy state as compared with sa. however, both algorithms find it difficult to find the lowest energy state for longer polymers due to its larger energy landscape and more local minima to overcome. further investigations in the annealing schedule and swapping ratio will be made. references frenkel, d. & b. smit, understanding molecular simulation. 2nd ed. academic press, san diego: chap. 14. giordano, n., 1997. computational physics. prentice-hall inc, new jersey: chap. 11. gould, h. & j. tobochnik, 1996. an introduction to computer simulation methods. 2nd ed. addison-wesley publishing company inc., new york: chap. 12. hansmann, u., 1997. parallel tempering algorithm for conformational studies of biological molecules. chem. phys. lett. 281: 140–150. kirkpatrick, s., c.d. gelatt, jr., & m.p. vecchi, 1983. optimization by simulated annealing. science. 220: 671–680. liang, l. & w.h. wong, 1997. dynamic weighting in monte carlo and optimization. proceedings of the national academy of sciences. 94: 14220–14224. marinari, e. & g. parisi, 1992. simulated tempering: a new monte carlo scheme. europhys. lett. 19: 451–455. metropolis, n., a. rosenbluth, m. rosenbluth, a. teller, & e. teller, 1953. equation of state calculations by fast computing machines. j. chem. phys. 21: 1087–1092. verdier, p.h., & w.h. stockmayer, 1962. monte carlo calculations on the dynamics of polymers in dilute solution. j. chem. phys. 36: 227. fig. 11. comparison on the number of edcs explored at the lowest possible energy state (t = 1) in 30 monomers with energy = –40. experiment 2 0 4 5 6 6 8 10 12 14 3 16 18 20 n um be r of e d c s 4 2 1 09_calorimetric calorimetric measurements 31 calorimetric measurements of the output power of the 2.48 ghz commercial magnetron leo mendel d. rosario1 and roy b. tumlos2 plasma physics laboratory, national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: 1ldrosario@up.edu.ph and 2troyb@nip.upd.edu.ph abstract science diliman (july-december 2004) 16:2, 31–35 a 2.45 ghz magnetron from a domestic microwave oven, with a power output rating of 1.5 kw, is utilized as the microwave source in the design of a multipurpose electron cyclotron resonance plasma device in the plasma physics laboratory of the national institute of physics. measurements of the power output to a 2 liter water load and a dummy load were obtained using calorimetry. the experiment shows that it is possible to achieve a relatively stable maximum average power of 190 w delivered to the 2 liter water load continuously for at least 10 min. it is also shown that the magnetron can deliver a maximum power of about 2.2 kw. introduction in electron cyclotron resonance (ecr) plasmas, the electrons are heated in the inhomogeneous magnetic field by interaction with axially launched electromagnetic waves (kawai et al., 2001). the advantage of an ecr device unlike the dc or lowfrequency discharges is that the vessel walls are not bombarded by electrons that release more electrons and impurity atoms that can contaminate the plasma volume (macdonald & tetenbaum, 1978). the compact ecr device also reduces power consumption due to the use of permanent magnets to produce minimum-b structures for electron plasma confinement (schlapp et al., 1992). in some devices using microwave discharges, thin-film deposition and surface treatment are performed on substrates which are not heated by other means than the one delivered by the discharge itself. thin films of silicon carbide (sic) (shimada et al., 1997) and silicon nitride (sin) (sitbon et al., 1995) have been deposited in this manner. ecr technology is also utilized in the deposition of diamond thin films (barshilia et al., 1996) through microwave plasma enhanced chemical-vapor deposition (mpecvd). microwave plasmas are also used as ion sources for beam-assisted surface modification and deposition (wartski et al., 1996). high-power microwave sources are often expensive for the assembly of the ecr device so that 2.45 ghz commercial magnetrons used in microwave ovens were also used as altenative microwave sources. schlapp et al. (1992) and wutte et al. (1994) constructed ecr devices using a 2.45 ghz magnetron for the production of multiply charged ions (schlapp et al., 1992) and li+ ions (wutte et al., 1994), respectively. an increasing interest in the application of high-power microwaves led to the design of different methods of establishing the actual value of power delivered by the microwave source. piotrowski (1998) mentioned that there are three methods currently utilized for microwave power measurements. the first type is based on rosario and tumlos 32 sampling high-power microwaves via a directional coupler with precisely known directivity and using an accurrate power meter. this method is often expensive and requires special calibration. the second type is based on the calorimetric measurements of a heated flowing water load. the system consists of a glass tube filled with water, which is introduced into a waveguide at a small angle with respect to the incident radiation to reduce the reflection of microwaves. piotrowski himself used this method for designing a low-cost calibration system for high-power microwave sources. the third type is based on the comparison of temperature increase of a heated water load from a dcpowered heater. this method requires sophisticated heating and temperature control equipment. a dummy load is installed in the setup that would serve as a complementary load to the target load, in this experiment, a 2 l water load, and later would be the plasma in the ecr vacuum chamber. it would be used to calibrate the delivered power to the load by measuring the power delivered to it. it is therefore important to determine in this experiment the various power measurements to the load and dummy load for the different configurations of the setup. methodology the schematic diagram of the microwave source is shown in fig. 1. the microwave source has a basic rlc circuit. the high-voltage transformer steps up the voltage source to approximately 3–4 kv that will be supplied to the magnetron. the magnetron used in the experiment has a frequency of 2.45 ghz and an output power rating of 1.5 kw. it is enclosed by an aluminum casing with air inflow and outflow fans installed on its sides used specifically for cooling the magnetron. the diagram of the experimental setup is shown in fig. 2. the magnetron is coupled to a circulator, which splits the path of the microwave into two parts. the dummy load is comprised of two glass tubes where water would flow at a rate of 5.95 l/min. the top view and cross section of the dummy load is shown in fig. 3. the water for the dummy load is supplied by a cft75 recirculating chiller, which also controls the water flow rate and temperature. the excess reflected power would be dumped into the dummy load to avoid damaging the magnetron. the other side of the circulator is connected to the microwave oven case through a rectangular waveguide and tuner stubs assembly. the tuner stubs are used for matching the impedance of this branch of the circulator fuse ac source f an thermal fuse spark detector m ag ne tro n fig. 1. schematic diagram of the microwave source circuit. microwave oven casing (inside is the 2 l water load) converter rectangular waveguide tuner stubs circulator air inflow fan air outflow fan dummy load microwave source (inside is the 2.45 ghz magnetron) fig. 2. diagram of the experimental setup. (a) glass tubes water outflow water inflow (b) fig. 3. dummy load component: (a) top view and (b) topview cross section. calorimetric measurements 33 the 2 l water load is heated for different times of operation (2, 4, 6, 8, and 10 min) with converter stick settings. the tuner stubs are all kept at a setting where all the stubs are fully withdrawn. three runs are done for the same exposure time to the microwave radiation. the initial and final temperatures of the 2 l water load are measured using a bk toolkit 2706 as a digital thermometer within 0.01oc. the dummy load is heated for 10 min, while simultaneously measuring the temperature for 1 min intervals. the power absorbed by the 2 l load, p2 l water load is calculated from the following equation: , (1) where mw is the mass of the water load, cw is the specific heat of water, mc is the mass of the water container, cc is the specific heat of the water container, tf and ti are the final and initial temperatures of the 2 l water load, and t is the exposure time to microwave radiation. the power absorbed by the dummy load pdummy load is calculated from the following equation: , (2) with the microwave source. the microwaves are converted from rectangular to cylindrical volumes by adding a converter between the rectangular waveguide and the microwave oven casing. the microwave converter is shown in fig. 4. where f is the mass flow rate per second, c is the specific heat of water, tf and ti are the temperatures of the outflow and inflow water loads, respectively. the total power absorbed by the two loads ptotal is calculated from the following expression: (3) the heat lost from the 2 l water load to air and the heat from the conduction of the ceramic bowl to the microwave oven casing are negligible and they would be a small correction to the calculated total power. results and discussion the power absorbed by the 2 l water load and its average values for 10 min are shown in figs. 5 and 6, respectively. the converter stick settings correspond to the percentage of the length of the converter stick that is pushed down. the 0% converter stick setting means that the converter stick is fully pulled up while the 100% means that the converter stick is fully pushed down. the estimated errors for fig. 5 are in the 6 min exposure time. the total power absorbed by the two loads and its average values are shown in figs. 7 and 8, respectively. the maximum power absorbed by the 2 l water load was observed at the 50% converter stick setting while the minimum was observed at the 75% converter stick setting as seen in figs. 6 and 8. the maximum power p2 l water load = [ (mwcw + mccc )(tf – ti) / t ] pdummy load = [ fcw(tf – ti)] ptotal = p2 l water load + pdummy load figure 4. microwave converter with fully depressed converter stick: (a) front view and (b) side view. (a) converter stick (b) fig. 5. the power absorbed by the 2 l water load for an exposure time of 10 min with different converter stick settings. time (min) a ve ra ge p ow er ( w ) rosario and tumlos 34 absorbed by the 2 l water load has an average of 190 w as seen in fig. 6. the maximum total power absorbed by the 2 l water load and the dummy load was observed at the 100% converter stick setting while the minimum was observed at the 25% converter stick setting as seen in figs. 6 and 8. it is seen in figs. 6 and 8 that the average power delivered by the magnetron fluctuates and depends on the converter stick settings. conclusion the power delivered to the load is attenuated by the rectangular to cylindrical mode converter as shown in the calculated power for the different settings of the fig. 6. average power absorbed of the 2 l water load with with different converter stick settings. converter stick settings (%) fully pulled up converter stick fully pushed down converter stick a ve ra ge p ow er ( w ) fig. 7. the total power absorbed by the 2 l water load and the dummy load for an exposure time of 10 min with different converter stick settings. p ow er ( w ) time (min) fig. 8. average total power absorbed from the microwave source with different converter stick settings. converter stick settings (%) fully pulled up converter stick fully pushed down converter stick a ve ra ge t ot al p ow er ( w ) converter stick. this result could be used to vary the power delivered to the plasma chamber of the ecr device aside by the use of tuner stubs. even with mismatch in the geometry of the rectangular microwave case with the cylindrical waves, a relatively stable maximum power of 190 w is still delivered to the 2 l water load continuously for at least 10 min. the 190 w is enough to maintain the plasma for the generation of multiply charged ions in the ecr device. the large difference of the measured total power of 2.2 kw for some of the data compared to the rating of 1.5 kw may have resulted from the mismatch of the load characteristics with that of the magnetron circuit. this could have easily resulted in the change of the frequency of the microwaves and the power delivered to the load. evidently, from fig. 8, the total power obtained around 1.5 kw is near the condition of the maximum power delivered to the 2 l water load, that is at 50% setting of the converter stick. this could be the condition for optimized impedance matching between the magnetron circuit and load. the experiment have shown that it is possible to correlate the power delivered between the load and the dummy load. this correlation can be later used to estimate the power delivered to the plasma in the forthcoming ecr experiments without directly measuring the power delivered to it, but by monitoring the power delivered to the dummy load. calorimetric measurements 35 references barshilia, h.c., b.r. metha, & v.d. vankar, 1996. growth of diamond thin films by microwave plasma chemical vapor deposition process. j. mater. res. 11: 1019. kawai, y., d. meyer, a. nadzeyka, u. wolters, & k wiesemann, 2001. isotope effects in an electron cyclotron resonance ion source in mixtures of 15 n/14 n. plasma sources sci. technol. 10: 451–458. macdonald, a.d. & s.j. tetenbaum, 1978. high frequency and microwave discharge. academic press, new york: chap. 3. piotrowski, a., 1998. a low-cost calibration system for highpower microwave sources. j. microwaves optoelectron. 1: 45–52. schlapp, m., r. trassl, m. liehr, e. salzborn, & s.j. tetenbaum, 1992. a low-power low-cost 2.45 ghz ecris for the production of multipy charged ions. rev. sci. instrum. 63: 2541. shimada, m., t. ono, i. okada, & s. matsuo, 1997. sic x-ray lithography mask fabricated by electron cyclotron resonance plasma source coupled with divided microwaves. j. vac. sci. technol. b. 15: 736. sitbon, s., m.c. hugon, b. agius, f. abel, j.l. courant, & m. puesch, 1995. low-temperature deposition of silicon nitride films by distributed electron cyclotron resonance plasma-enhanced chemical vapor deposition. j. vac. sci. technol. a. 13: 2900. wartski l., c. schwebet, & j. aubert, 1996. radio frequency, microwave, and electron cyclotron resonance ion sources for industrial applications: a review. rev. sci. instrum. 67: 895. wutte, d., m. leitner, & h.p. winter, 1994. a 2.45 ghz electron cyclotron resonance multi-ma li+ ion gun for fusion plasma diagnostics. rev. sci. instrum. 65: 1094–1096. 01_device preliminary results on the use of clay to control pyrodinium bloom 35science diliman (january-june 2006) 18:1, 35-42 introduction among the dinoflagellate species that are known to cause harmful algal blooms (habs), pyrodinium bahamense var compressum poses one of the most alarming effects both to man and the environment. the first toxic paralytic shellfish poisoning in the philippines caused by this species was in 1983 in western samar and leyte which resulted in losses to the green mussel industry in the area and several loss of life and numerous poisonings to the locals who ingested the infected shellfish. (azanza, 1997; arafiles et al., 1984). preliminary results on the use of clay to control pyrodinium bloom a mitigation strategy larry v. padilla *, maria lourdes san diego-mcglone, rhodora v. azanza marine science institute, university of the philippines, diliman, quezon city, 1101 telephone no. 9223944 fax no: 9247678 email: larrypadilla@upmsi.ph, mcglonem@upmsi.ph, rhod@upmsi.ph abstract the frequent and expanded occurrence of pyrodinium bahamense var compressum blooms in the philippines since 1983 has prompted the need to find mechanisms to control the harmful effects of these toxic dinoflagellates. a promising method now being explored is the use of powdered clay minerals which when added to the growth media is capable of flocculating with the algal cells. in this study, the efficiency of ball clay, brown bentonite, and malampaya sound sediments to remove pyrodinium cells in seawater was tested. the addition of 1 g/l of suspended ball clay to 50 ml of cultured pyrodinium cells (~1.037 x106 cells/l) removed 99.56% of the algal cells after 2.5 hours. prolonging the exposure time to 5 and 24 hours showed no significant increase in flocculation. brown bentonite and malampaya sound sediments showed low to moderate removal efficiency not exceeding 70% and 50%, respectively. the effect of ball clay addition on seawater chemistry showed no change in ammonia concentration but nitrate decreased after 5 and 24 hours of clay addition. results for nitrite and phosphate were however more variable. various control measures have been proposed to help mitigate the toxic bloom caused by p. bahamense. these include use of chemical compounds such as hydrogen peroxides and copper sulfate that could directly kill the algal cells and the introduction of predatory species that would consume the target phytoplankton (boesch et al., 1997; cenr, 2000). the effectivity of these methods vary with some having negative impacts on the marine ecosystem (e.g. non species specific effect, persistent in seawater). a potentially effective control method is the use of clay minerals that could attach to the algal cells, increase their density, and force them to settle to the sediments where they will die or encyst. previous studies have shown that many types of clay can be effective in removing harmful algae. according*corresponding author padilla, mcglone, azanza 36 to sengco et al. (2000) 12 of the 25 types of clays that were tested against gymnodinium breve are effective. later studies showed that agitation could improve the cell removal efficiency of clay against specific type of algae (i.e. karenia brevis, heterocapsa triquetra) (sengco et al., 2004; archambault et al., 2003). furthermore, prolonging the exposure period of the clay treatment may help in the removal of the algal cells from the water column (hagstrom and grancli, 2004; sengco et al., 2004; yu et al., 2003; pierce et al., 2003). the use of various types of clay in mitigating habs has been widely explored in china (yu et al., 1994), south korea (bae et al., 1998; na et al., 1996), japan (maruyama et al., 1987) and the us (anderson et al., 2004; beaulieu et al., 2005). in the philippines, mitigation measures against algal blooms have not been explored. this study examines the use of clay to remove harmful algae. although the method is not new, this is the first attempt to control blooms of p. bahamense. the possible effect of using clay on water chemistry (nutrients n and p) was also determined. materials and methods clay preparation and pyrodinium cultures the clay mineral samples used in this study were purchased from industrial specialties co. inc., a local mining company in taytay, rizal. ball clay and brown bentonite materials came in powder form, and using wet sieving, grain size was determined to be less than 63µm (>99%). particle sizes of the clay material were also determined using pipette analysis of mud method (lewis et al., 1994). a clay stock was prepared by adding 100 g powdered clay to 1l of filtered (<0.2µm) and autoclaved seawater from bolinao, pangasinan. sediments obtained from malampaya sound, palawan were also tested for removal efficiency. strains of p. bahamense (04-25-95 pbc mz rva) were subcultured using f/2 media (gullard and rhyther, 1962) on autoclaved seawater from bolinao (pangasinan) as base and incubated at 28±2 °c. the cultured organisms were allowed to reach mid exponential growth phase before they were harvested and tested against the clay and sediment materials. the growth stages of the batch culture were monitored by cell counting every three days until the desired experimental cell density was reached (>1000 cells/ ml). clay flocculation with pyrodinium the clay removal experiment commenced when p. bahamense cell density reached >1000 cells/ml at its mid exponential growth phase. eighteen (18) autoclaved 50-ml erlenmeyer flasks were filled to the 50 ml mark with the cultured p. bahamense. initial cell count was determined in each flask before addition of the clay. prepared clay slurries with concentrations of 0.25, 0.5, 0.75, 1 and 2 g/l were added to the flasks. autoclaved seawater was used as control. upon clay addition, each flask was gently swirled 10 times to disperse and homogenize the clay throughout the culture media. the flasks were then placed in a welllighted area in the laboratory and arranged following a completely randomized block design. the exposure periods of the experiment were 2.5, 5 and 24 hours. the cell counting procedure involved pipetting 5 ml of sample from the upper ~2cm layer of the flask content and putting the sample in a 20-ml scintillation vial. samples were preserved by adding 0.02 ml of lugol's solution and the cells later counted using a sedgewick-rafter chamber with grid viewed under a light microscope. the removal efficiency of the clay and sediments was determined using the formula: removal efficiency = 1final cell count / initial cell count x 100% the results obtained were statistically analyzed using repeated measures design of anova for variation in exposure period, and one-way anova for variation in treatment. levene's test for homogeneity of variances was utilized to meet the anova assumption that the variances are nearly equal. when data is not homogenous, log transformation was used. statistical results were determined using the sas program. effect of clay on water chemistry the effect of clay on water chemistry was determined on the clay material that had the highest removal efficiency. seawater from bolinao, pangasinan was filtered using cellulose ester membrane filter (0.2 µm) and transferred to nine 500 ml erlenmeyer flasks. the preliminary results on the use of clay to control pyrodinium bloom 37 prepared clay stock was added dropwise to seawater to obtain a final clay concentration of 0, 0.5 and 1 g/l (all in triplicate). a control was added to the set up. 125 ml of aliquot was taken prior to and after 5 and 24 hours of clay addition. samples were filtered and later analyzed for ammonia, nitrate, nitrite and phosphate using methods described by strickland and parsons (1972) using a shimadzu uv-vis spectrophotometer. the data obtained was statistically analyzed using the same methods as in the clay flocculation experiment. results and discussion removal efficiency flocculation of pyrodinium bahamense with clay material is shown in figure 1. the removal efficiency of brown bentonite, ball clay and malampaya sediment with increasing clay slurry concentration is shown in figure 2. highest removal efficiency was 99.9% with ball clay, 69.3% with brown bentonite, and 48.4% with malampaya sediments. very high removal efficiency was obtained when ball clay was used under all exposure periods (figure 2a). removal efficiencies using 0.5 to 2 g/l clay concentration were not significantly different from 2.5 to 24 hours of exposure. thus, with even a low clay concentration of 0.5 g/l, high removal efficiency can be attained with ball clay. for brown bentonite, highest removal occurred after 5 hours at 0.75 g/l clay treatment although there was no significant difference from the 0.25 to 1 g/l treatment (figure 2b). increasing the clay treatment to 2 g/l decreased removal efficiency after 2.5 and 5 hours exposure. the malampaya sediments were less efficient in removing p. bahamense than clay, i.e 20% after 2.5 and 5 hours that improved to 48.42% at 2 g/l treatment after 24 hours (figure 2c). swirling could simulate turbulence thereby increase the rate of collision between the cell and clay particles. according to young-han and kim (2001), higher collision rate will be achieved if the clay particle size and cell size are similar. the clay-cell flocculation in low reynolds number non turbulent flow regime can be well described by equations including hydrodynamics and interparticle forces (van der waals force and electrostatic force). hence the larger the size range difference, the lower the collision frequency. approximately 88% of the ball clay used in this study fall under the 16-31 mm size fraction (table 1). there is an overlap with the diameter of pyrodinium bahamense which is about ~25-40 mm.this could explain the high removal efficiency using ball clay. unlike ball clay, the 16-31 µm size fraction in brown bentonite comprise only 8% of the clay material (table 1), thus the lower removal efficiency using brown figure 1. photomicrograph of pyrodinium bahamense var compressum cell without the clay attached at 250x (a), and clay flocculated with the cell at 100x (b). a. b. 10µm10µm cell clay 15µm padilla, mcglone, azanza 38 figure 2. removal efficiency of ball clay (a), brown bentonite (b), and malampaya sediments (c) with increasing clay treatment. -20% 0% 20% 40% 60% 80% 100% 0 0.5 1 1.5 2 clay treatment using malampaya sediment (g/l) re m ov al e ffi ci en cy 2.5 hours 5 hours 24 hours c -20% 0% 20% 40% 60% 80% 100% 0 0.5 1 1.5 2 clay treatment using brown bentonite (g/l) re m o va l e ff ic ie cy 2.5 hours 5 hours 24 hours b -20% 0% 20% 40% 60% 80% 100% 0 0.5 1 1.5 2 clay tr e atm e n t u s in g b all clay (g /l ) re m ov al e ff ic ie nc y 2.5 hours 5 hours 24 hours a preliminary results on the use of clay to control pyrodinium bloom 39 bentonite. from visual inspection, malampaya sediments have coarser particles, which may explain the low removal efficiency. the relatively uniformly distributed particle sizes in brown bentonite (table 1) could have hastened the settlement of the clay as a result of differential sedimentation. the small sized clays may have been intercepted by the denser large sizes, thus rapidly gaining size/mass for settlement and lessening the contact period between clay and the pyrodinium cells. differential sedimentation can result in increased effectiveness only when majority of clay and cells are near but not identical in sizes, as most likely the case for ball clay and pyrodinium cells. the results of this study suggest that even at 2 g/l, >99% removal efficiency is reached. in other studies, there was 100% removal of pyrmnesium parvum and 86.6% removal of aureococcus anophagefferens when 4 g/l of phosphatic clay was used (hagstrom and graneli, 2004; sengco et al., 2001). as much as 10 g/ l of yellow loess clay was used in the removal of 7080% of cochlodinium polykrikoides (choi et al., 1998). according to yu et al. (1994) 90% of prorocentrum minimum was removed using 0.1 g/l of kaolin with the coagulant pacs (polyhydroxy aluminum chloride) added.removal efficiency increased to >95% using 0.5 g/l kaolin and pacs. the concentration of ball clay slurry (2 g/l) in this study that produced the highest removal efficiency fall within the range of concentration reported by other studies. ball clay, brown bentonite, and the malampaya sediments were subjected to the same exposure periods and all these materials underwent immediate reaction with the cell on the first 2.5 hours of clay addition. prolonging the exposure of the pyrodinium cells to the 24th hour increased the removal efficiency of malampaya sediments but removal using ball clay and brown bentonite remained the same (not significantly different at 0.05 level). there is even a possibility that pyrodinium cells can be removed faster than 2.5 hours. the unchanging removal efficiency with ball clay and brown bentonite after 24 hours suggests the potential of clay addition as a mitigation strategy. in a study done by archambault et al. (2004), removal of karenia brevis was 40% after 14 days. after 48 hours of clay addition, the removal efficiency of the dinoflagellate heterocapsa triquetra was at 100% (pierce et al., 2003). further studies must be done to assess the removal efficiency of ball clay beyond 24 hours. there may also be a need to determine if flocculation is permanent or temporary and hence dependent on resuspension processes that can release pyrodinium back to the water column.temporary flocculation was observed with brown bentonite, an indication of weak attachment of the particles. weak compaction of the bottom clay material may also result to escape or detachment of the cell. effect of clay addition on seawater nutrient concentration an evaluation of the effect of clay addition on seawater nutrient content is necessary to determine any changes in water quality conditions since this may adversely affect marine organisms in the water column. there was a significant increase in phosphate concentration with increase in clay treatment (p= <0.0001 at α 0.05) and exposure periods (p= 0.0012 at α 0.05) during the 1st run with no significant difference observed in clay treatment (p= 0.2316 at α 0.05) and significant decrease (p=0.0112 at α 0.05) after 24 hours in a second run (table 2). a significant decrease in nitrite after 24 hours was observed during the 1st and 2nd run (p= 0.0013 and p= 0.0064 at α 0.05, respectively). a significant decrease in nitrite with increasing clay treatment during the 1st run (p= 0.0780 at α 0.05) was obtained but not in the 2nd run (p= 0.0047 at α 0.05). the difference in table 1. particle size analysis of ball clay (a) brown bentonite (b). particle size (µm) a) ball clay b) brown bentonite >63 0 % 14.31 % 31-63 8.6 % 2.81 % 16-31 88.17 % 8.10 % 8-16 0.2 % 15.90 % 2-8 0.16 % 31.13 % <2 2.87 % 27.75 % padilla, mcglone, azanza 40 results between the 1st and 2nd run for varying clay treatment concentrations may be due to the different seawater samples used in the two runs. seawater during the 2nd run had higher phosphate concentration. clay materials tend to adsorb or desorb phosphate depending on seawater phosphate levels (lake and macintyre, 1977). no significant change was observed with ammonia on both runs. the decrease in nitrate concentration was determined to be significant (p= 0.0020 at α 0.05) with increase in clay treatment.however, prolonged exposure periods showed no significant change (p=0.8319 at α 0.05) in the concentration of nitrate. different types of clay vary in their effects on the concentration of nutrients in seawater. anderson et al. (2004) showed that the cationic polymer-treated kaolinite material did not release any nutrient. however, the other two clays that were tested, bentonite and florida phosphatic clay, released nitrate and silicate and/or phosphate, respectively. moreover, the addition of polyaluminum chloride (a coagulant) to kaolinite helped remove phosphate and nitrate from seawater. according to black and waring (1976), addition of kaolinite and lowering the ph increased the adsorption of nitrate. in which case the nitrate attached to the clay particles are carried to the bottom upon settlement of the clay particles. the preliminary results of this study showed a promising method to control pyrodinium blooms using ball clay. it has the potential to remove more than 95% of the algal cells in the water column after 2.5 hours in controlled laboratory condition. however, a suitable technique for ball clay application in the field should be investigated. the impact of clays on benthic organisms must also be examined. acknowledgements this project received support from the philippine council for aquatic and marine research development (pcamrd) of the department of science and technology (dost). the authors would like to thank ms. estrelita flores for her assistance and dr. fernando siringan for providing the malampaya sound sediments. nutrients first run second run clay concentration 0 hours 5 hours 24 hours 0 hours 5 hours 24 hours phosphate 0 g/l clay 0.08 0.07 0.09 0.23 0.2 0.14 0.5 g/l clay 0.08 0.16 1.45 0.23 0.19 0.1 1 g/l clay 0.08 1.63 1.48 0.23 0.22 0.17 ammonia 0 g/l clay 4.18 3.58 2.67 2.82 2.69 2.26 0.5 g/l clay 4.18 3.8 2.73 2.82 3.09 2.85 1 g/l clay 4.18 4.19 3.33 2.82 3 2.74 nitrite 0 g/l clay 0.05 0.09 0.09 0.07 0.07 0.06 0.5 g/l clay 0.05 0.11 0.09 0.07 0.06 0.05 1 g/l clay 0.05 0.09 0.08 0.07 0.06 0.05 nitrate 0 g/l clay 0.52 0.63 0.57 0.5 g/l clay 0.52 0.5 0.46 1 g/l clay 0.52 0.33 0.3 table 2. phosphate, ammonia, nitrite, and nitrate concentrations at 0, 5 and 24 hours after addition of 0, 0.5 and 1 g/l of clay preliminary results on the use of clay to control pyrodinium bloom 41 references arafiles l., r. hermes, j. morales, 1984, lethal effect of paralytic shellfish poisoning (psp) from perna viridis with notes on the distribution of pyrodinium bahamense var compressum during a red tide in the philippines, proceedings on toxic red tides and shellfish toxicity in southeast asia, singapore, 43-51. archambault m., j. grant, m. bricelj, 2003, removal efficiency of the dinoflagellate heterocapsa triquetra by phosphatic clay, marine ecology progress series, 253, 97109. azanza r., 1997, contributions to the understanding of the bloom dynamics of pyrodinium bahamense var compressum: a toxic red tide causative organism, science diliman, 1 and 2, 1-6. bae h., h. choi, w. lee, s. yoon, 1998, control of the red tide by yellow loess dispersion, proceedings of korea-china joint symposium and harmful algal blooms, 53-60. beaulieu s., m. sengco, d. anderson, 2005, using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs, harmful algae, 4, 123-138. black a., s. waring, 1976, nitrate leaching and adsorption in a krasnozem from redland bay, qld. ii. soil factors influencing adsorption australian journal of soil research 14, 181 188. boesch d., d. anderson, r. horner, s. shumway, p. tester, t. whiteledge, 1997, harmful algal blooms in coastal waters: options for prevention, control and mitigation, noaa coastal ocean program, 10, 21-28. cenr, 2000, national assessment of harmful algal blooms in us waters, national science and technology council committee on environemnt and natural resources, washington, dc. choi h., p. kim, w. lee, s. yun, h. kim, h. lee, 1998, removal efficiency of cochlodinium polykrikoides by yellow loess, journal of korean fisheries society, 31, 109113. lake c., w. macintyre, 1977, phosphate and tripolyphosphate adsorption by clay minerals and estuarine sediments, virginia water resources research center. lewis d., d. mcconchie., 1994, analytical sedimentology, chapman and hall, new york, 103-107. maruyama t., r. yamada, k. usui., h. suzuki.,t. yoshida, 1987, removal of marine red tide plankton with acid treated clay, nippon suisan gakkaishi, 53, 1811-1819. na g., w. choi, y. chun, 1996, a study on red tide control with loess suspension, journal of aquaculture, 9, 239-245. pierce r., m. henry, c. higham, p. blum, m. sengco, d. anderson, 2003, removal of harmful algal cells (karenia brevis) and toxins from seawater culture by clay flocculation, harmful algae, 75, 1-8. sengco m., a. li, k. tugend., r. kulis, d. anderson, 2001, removal of red and brown tide cells using clay flocculation. i. laboratory culture experiments with gymnodinium breve and aureococcus anophagefferens., marine ecology progress series, 210, 41-53. sengco m., m. hagstrom, r. graneli, d. anderson, 2004, removal of pyrmnesium parvum (haptophyceae) and its toxins using clay minerals, harmful algae, 101, 1-14. sengco m., d. anderson, 2004, controlling harmful algal blooms through clay flocculation, journal of eukaryotic microbiology, 51, 169-172. usup g., r. azanza, 1998, physiology and bloom dynamics of the tropical dinoflagellate pyrodinium bahamense, nato asi series, g41. young-han m., w. kim, 2001, a theoretical consideration of algae removal with clays, microchemical journal, 682, 157-161. yu z., j. zou, x. ma, 1994, a new method to improve the capability of clays for removing red tide organisms, chines journal of oceanology and limnology, 25, 226-232. padilla, mcglone, azanza 42 yu z., m. sengco, d. anderson, 2003, flocculation and removal of the brown tide organisms, aureococcus anophagefferens (chrysophyceae), using clays, journal of applied phycology, 00, 1-10. yu z., j. zou, x. ma, 1995, study on the kinetics of clays removing red tide organisms, chinese journal of oceanology and limnology, 26, 1-6. yu z., j. zou, x. ma, 1994, application of clays to removal of red tide organisms i. coagulation of red tide organisms with clays, chinese journal of oceanology and limnology, 12, 193-200. 38 chalcone derivatives with cyclooxygenase inhibiting activity gian carlo v. tambago evangeline c. amor* terrestrial natural products laboratory institute of chemistry, college of science university of the philippines diliman abstract dimethylcardamonin (dmc) or (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone was isolated from syzygium samarangense (blume) merr. leaves using vacuum liquid chromatography and normal phase silica-gel column chromatography. dmc was purified from the fraction that eluted out of 9:1 (v/v) hexane: ethyl acetate to 7:3 (v/v) hexane: ethyl acetate. using a modified method from geissman (1948), dmc was derivatized via alkaline peroxidation from which compounds a and b were obtained. compound a was identified to be (2s)-7hydroxy-5-methoxy-6,8-dimethyl flavanone (c 18 h 18 o 4 ) while b had a methoxy group on its 4’ position instead of a hydroxyl group with respect to dmc. the flavanone derivative may have been formed due to substituent effects on the ring. dmc, a, and b were tested for their inhibitory activity against cyclooxygenase enzymes at 10 and 100 ppm. a and b gave at least 50% inhibitory activity at 100 ppm and were found to be cox-2 selective inhibitors. dmc was inactive at both 10 and 100 ppm. keywords: dimethylchalcone, flavanone, syzygium samarangense, alkaline peroxidation, cyclooxygenase inhibition * corresponding author science diliman (january-june 2022) 34:1, 38-52 sd june2022_tambago.indd 38 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 39 introduction the earliest remedy for inflammation is the extract of willow bark (salix sp.), which dates back to 1862 (cavaillon 2017; desborough and keeling 2017). willow bark extract is found to contain salicylic acid (2-hydroxybenzoic acid), which was derivatized in 1897 by acetylation to produce aspirin (2-acetyloxybenzoic acid) (jeffreys 2004; desborough and keeling 2017). aspirin was commercialized in 1905 and since then has been used to relieve pain, inflammation, and fever (desborough and keeling 2017). in 1963, a new drug called indomethacin was developed for people experiencing pain and inflammation due to rheumatoid arthritis (shen 1982). aspirin and indomethacin are examples of non-steroidal anti-inflammatory drugs (nsaids). however, these drugs are gastrotoxic and cause ulceration (marnett and kalgutkar 1998; marnett 2002; pasa et al. 2009). this side effect was related to the ability of nsaids to inhibit both cyclooxygenase-1 (cox-1) and cyclooxygenase-2 (cox-2)—enzymes mediating inflammation. thus, a new class of anti-inflammatory drugs called coxibs was developed. these drugs selectively inhibit cox-2 which means that they do not possess the gastric toxicity of nsaids (patrono and rocca 2009). however, it was later found that coxibs are hepatoand cardiotoxic (patrono and rocca 2009; badri et al. 2016). many coxibs including celecoxib, rofecoxib, and valdecoxib were banned from use (sun et al. 2007; hawboldt 2008; thomas et al. 2017). this led to a search for a new class of cox-2 selective inhibitors. among the interesting derivatized products from chalcones are the aurones and flavones, which are products of the oxidative cyclization of 2’-hydroxychalcones (masesane 2015). biologically, aurones are synthesized from chalcones with the aid of aureusidin synthase (nakayama et al. 2000; nakayama 2002; davies et al. 2006). however, not all plants have this enzyme to synthesize this subclass of phytochemicals. some researchers were able to synthesize aurones from chalcones in vitro and synthetic routes have been explored to hasten the study of aurones. one of the developed methods is the oxidative cyclization of 2’-hydroxychalcone using the acetate salt of metals at high oxidation states (e.g., mercury (ii) acetate) in an appropriate organic solvent such as dimethylsulfoxide (dmso) (detsi et al. 2002; agrawal and soni 2006), pyridine (agrawal and soni 2006), or acetic acid (sekizaki 1988). modifications to this process had been done, such as the use of copper (ii) bromide in dmso (detsi et al. 2002) and the use of gold (i) catalyst (harkat et al. 2008). another possible method is through the alkaline peroxidation of 2’-methoxy chalcones (geissman 1948). sd june2022_tambago.indd 39 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 40 the three chalcones (figure 1) isolated from s. samarangense (blume) merr. are known to inhibit the lipopolysaccharide (lps)-induced cellular production of nitric oxide (no) and prostaglandin e2 (pge2) (kim et al. 2010). however, it has not been confirmed whether dmc, the most abundant chalcone isolated from s. samarangense by amor et al. (2007), could also inhibit the production of prostaglandins through the direct inhibition of cyclooxygenases. this study re-isolated dmc from s. samarangense and derivatized it to determine the cyclooxygenase-inhibiting activity of the derivatives. figure 1. structure of cardamonin (r 1 , r 2 = h), stercurensin (r 1 = ch 3 , r 2 = h), and dimethylcardamonin (r 1 , r 2 = ch 3 ). materials and methods plant collection, extraction, and solvent partitioning s. samarangense leaves were collected from diliman, quezon city. the samples were authenticated, and a voucher specimen was deposited at the jose vera herbarium of the university of the philippines (up) diliman institute of biology with accession no. 14258. ground s. samarangense leaves (541 g) were soaked in single distilled technical grade (tg) methanol (~1.5 l) for 3-5 days done six times. after extraction, the resulting filtrate was evaporated in vacuo with a rotary evaporator (ika rv-10) at 40°c to obtain the crude methanol extract (46 g). the concentrated methanol extract (15 g) was suspended in distilled water (100 ml) and was extracted successively with tg hexane (600 ml) up to six times to obtain the hexane extract. sd june2022_tambago.indd 40 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 41 fractionation, isolation, and purification all solvents (methanol, hexane, and ethyl acetate) used for fractionation, isolation, and purification are of analytical grade except for those used for vacuum liquid chromatography (vlc) where tg solvents were used. the hexane extract (4.1 g) was fractionated using vacuum liquid chromatography (silica gel 60 g, thin-layer chromatography grade, merck) employing gradient elution from 100% hexane to 100% ethyl acetate at 10% (v/v) increments, and then 100% ethyl acetate to 100% methanol at 50% (v/v) increments. a total of twelve vlc fractions were collected, concentrated, and dried in vacuo at 40°c using a rotary evaporator. separation was monitored by thin layer chromatography (tlc) using silica gel 60 gf 254 (tlc grade, merck 105554). tlc plates (4.5 in x 4.5 in) were viewed under an ultraviolet (uv) lamp set at 254 nm (far uv) and 365 nm (near uv) and exposed to iodine crystals for visualization. yellow-orange crystals formed from the vlc fraction that eluted from 9:1 (v/v) hexane: ethyl acetate. this fraction was dissolved in 9:1 (v/v) hexane: ethyl acetate and was subjected to normal phase silica-gel column chromatography (npcc) using a wet-packed stationary phase (silica gel 60, column chromatography grade, merck 1.07734.1000). gradient elution was performed from 9:1 (v/v) hexane: ethyl acetate to 100% ethyl acetate at 10% (v/v) increments. subfractions were collected at 5-ml portions monitored with tlc. a pure compound was obtained from the subfraction which eluted at 7:3 (v/v) hexane: ethyl acetate. (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone (dmc): yellow-orange crystals; esi-ms positive mode c 18 h 18 o 4 [m+h]+ found m/z 299.1293 (calcd 299.1283, error 3.34 ppm). 1h-nmr (500 mhz, cdcl 3 , ppm) δ 2.15 (s, 3’-ch 3 ), 2.17 (s, 5’-ch 3 ), 3.67 (s, 6’-och 3 ), 5.42 (s, 4’-oh), 7.43 (m, h-3), 7.43 (m, h-4), 7.43 (m, h-5), 7.66 (dd, h-2), 7.66 (dd, h-6), 7.86 (d, β-h), 8.01 (d, α-h), 13.62 (s, 2’-oh); 13c-nmr (126 mhz, cdcl 3 , ppm) [dept] δ 7.57 (c-1) [c], 8.24 (3’-ch 3 ) [ch 3 ], 62.38 (6’-och 3 ) [ch 3 ], 106.54 (c-1’) [c], 108.85 (c-3’) [c], 109.07 (c-5’) [c], 126.71 (c-α) [ch], 128.42 (c-3) [ch], 128.42 (c-5) [ch], 128.93 (c-2) [ch], 128.93 (c-6) [ch], 130.2 (c-4) [ch], 135.36 (c-1) [c], 142.9 (c-β) [ch], 158.86 (c-6’) [c], 159.18 (c-4’) [c], 162.04 (c-2’) [c], 193.38 (c=o) [c]. alkaline peroxidation of chalcone the alkaline peroxidation procedure was adopted from geissman and fukushima (1948) with slight modifications. a total of 50 mg of the chalcone was dissolved in 2.5 ml cold methanol. this solution was kept in an ice bath (~5°c). subsequently, 0.75 ml of 16% cold aqueous naoh (18 equiv.) and 0.5 ml of cold 20% h 2 o 2 sd june2022_tambago.indd 41 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 42 (25 equiv.) were added. the mixture was left to stand overnight at 5°c. if any precipitation was observed, 3n hcl was added to neutralize the mixture. the resulting solution was then extracted with 13 ml ethyl acetate. afterward, another set of peroxidation was done using 10 ml cold methanol, 3.0 ml of cold aqueous 16% naoh (72 equiv.), and 2.0 ml of cold 20% h 2 o 2 (102 equiv.), which was followed by extraction using 50 ml ethyl acetate. the resulting ethyl acetate extracts were then dried, and the products were purified using dropper column chromatography. the first peroxidation procedure yielded compound a (8.0% yield), while the second produced compound b (18% yield). compound a ((2s)-7-hydroxy-5-methoxy-6,8-dimethyl flavanone): yellow crystals. 1h-nmr (500 mhz, cdcl 3 , ppm) δ 2.15 (s, 6-ch 3 ), 2.15 (s, 8-ch 3 ), 2.84 (dd, ha-3), 2.98 (dd, hb-3), 3.82 (s, 5-och 3 ), 5.42 (dd, h-2), 5.43 (s, 7’-oh), 7.42 (m, h-2’), 7.42 (m, h-3’), 7.42 (m, h-4’), 7.42 (m, h-5’), 7.42 (m, h-6’). compound b ((e)-2’-hydroxy-4’,6’-dimethoxy-3’,5’-dimethyl chalcone): faint yellow crystals. 1h-nmr (500 mhz, cdcl 3 , ppm) δ 2.12 (s, 3’-ch 3 ), 2.14 (s, 5’-ch 3 ), 3.48 (s, 4’-och 3 ), 3.65 (s, 6’-och 3 ), 7.40 (d, h-3), 7.40 (d, h-4), 7.40 (d, h-5), 7.64 (dd, h-2), 7.64 (dd, h-6), 7.83 (d, β-h), 7.98 (d, α-h), 13.62 (s, 2’-oh). cyclooxygenase inhibition assay the reagents used in the cyclooxygenase inhibition assay include 100 mm tris buffer (ph 8.0), 1 mm hematin cofactor in 0.1 m naoh, ampliflutm red or adph (10-acetyl-3,7-dihydroxyphenoxazine): 2 mm in dmso, 40 mm arachidonic acid in ethanol, indomethacin (sigma) positive control, cyclooxygenase-1 from sheep (sigma) and recombinant cyclooxygenase-2 from human (sigma). dmc and its derivatives were screened for their cyclooxygenase inhibiting activity at 10 ppm and 100 ppm using the method developed by the terrestrial natural products laboratory at the institute of chemistry, up diliman (opog et al. 2019). a 10,000 ppm stock sample solution was prepared in a microcentrifuge tube by dissolving 3 mg of the sample in 300 μl of dmso. working stock solutions with concentrations of 200 ppm and 20 ppm were prepared in microcentrifuge tubes from the 10,000 ppm stock. the assay was done in a closed chamber flushed with nitrogen gas, using indomethacin (sigma) with an effective concentration of 1,500 ppm as the positive control. from the working stock, 10 μl of the test sample was added to a mixture of 50 μl of tris buffer and 120 μl of cox-hematin solution. after incubation for 15 minutes at 25°c, 10 μl amplex red reagent and 10 μl arachidonic acid were added. the fluorescence was then monitored every 12 seconds for sd june2022_tambago.indd 42 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 43 3 minutes at 535 nm and 590 nm as the wavelengths of excitation and emission, respectively using a clariostar® microplate reader. each assay was done in two trials with two replicates per trial. the percent inhibition was calculated using the equation: %inhibition = v negative – v sample v negative x 100% (1) where v negative = reaction velocity of the negative control, and v sample = reaction velocity of the sample a sample is considered active when the cox-2 inhibition is greater than 50% and the ratio of the cox-2 and cox-1 inhibition is greater than 1. the assay data collected were subjected to several statistical methods, including the one-sample kolmogorov-smirnov test, levene’s test, anova, welch’s t-test, brown-forsythe test, and the post-hoc test (dunnett t3 or tamhane’s t2). liquid chromatography-mass spectrometry (lc-ms) the samples were analyzed using masslynxtm software v4.1 by waters and mzmine v2.6. the parameters used for the analysis are summarized in table 1. table 1. lc-ms parameters and conditions for sample analysis parameter condition unit xevo g2-xs qtof, acquity uplc® system column acquity uplc® csh tm fluoro-phenyl, 1.7μm (2.1 x 50mm), t=30°c mobile phase gradient of 100% acn-95% h2o-100% acn flow rate 0.3 ml/min data processing masslynxtm v4.1 nuclear magnetic resonance (nmr) isolates with enough amount for elucidation were subjected to 1h-nmr and 13c-nmr spectroscopy using 500-mhz agilent nmr spectrometer at the analytical services laboratory of the institute of chemistry, up diliman. the solvent used was deuterated chloroform (cdcl 3 ). data were processed using mestrenovatm ver 6.1© mestrelab research s.l. sd june2022_tambago.indd 43 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 44 results and discussion isolation of chalcone approximately 200 mg of yellow-orange needle-like crystals were collected from the vlc fraction that eluted out from 9:1 (v/v) hexane: ethyl acetate and purified using npcc with 7:3 (v/v) hexane: ethyl acetate. it has an rf value of 0.65 in 7:3 (v/v) hexane: ethyl acetate solution and is visible under uv (254 nm and 365 nm). the compound was identified to be (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone (dmc) and its structure (figure 2) was elucidated using nmr and lc-hrms data. the relative configuration was determined to be (e)based on the coupling constant of the αand β-hydrogens (15.7 hz). figure 2. dmc or (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone elucidated using 1d-nmr, 2d-nmr, dept, and lc-hrms spectra. 13c-nmr shifts are indicated in red while 1h-nmr chemical shifts are indicated in blue. drawn using chemdraw. derivatization of chalcones dmc was subjected to alkaline peroxidation and a red-orange solution was formed. two sets of derivatizations were performed for the oxidation of dmc-producing compounds a and b (figure 3). sd june2022_tambago.indd 44 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 45 figure 3. reaction schemes for the alkaline peroxidation of dmc yielding compounds a and b. from the first peroxidation procedure, four milligrams of yellow needle-like crystals (a) with an rf value of 0.53 in 7:3 (v/v) hexane: ethyl acetate was collected after purification. this was identified to be (2s)-7-hydroxy-5-methoxy-6,8-dimethyl flavanone (figure 4) and is visible under uv (254 nm) with a fluorescence observed at 365 nm. figure 4. (2s)-7-hydroxy-5-methoxy-6,8-dimethyl flavanone structure elucidated using 1h-nmr and cosy spectra. 1h-nmr chemical shifts are indicated in blue. drawn using chemdraw. sd june2022_tambago.indd 45 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 46 peroxidation of dmc was monitored through 1h-nmr. the doublet of doublet signals at δ 2.84, 2.98, and 5.42 is indicative of a flavanone skeleton. this was confirmed to be 7-hydroxy-5-methoxy-6,8-dimethyl flavanone when chemical shifts obtained were compared with published literature values (marinho et al. 2008; memon et al. 2015) and has an (s)relative configuration based on the coupling constants (3.0 and 12.9 hz). the flavanone derivative of dmc was obtained by direct nucleophilic attack by the anionic oxygen atom on the β-carbon atom of the oxide intermediate (geissman and fukushima 1948). the substituents of the ring did not provide substantial inhibition to the resonance in the system that would have led to the formation of the aurone. another set of alkaline peroxidation of dmc was carried out using excess reagents. nine milligrams of compound b (figure 5) were collected. figure 5. (e)-2’-hydroxy-4’,6’-dimethoxy-3’,5’-dimethyl chalcone structure elucidated using 1h-nmr and cosy spectra. 1h-nmr chemical shifts are indicated in blue. drawn using chemdraw. the collected compound b has an rf value of 0.6 in 7:3 (v/v) hexane: ethyl acetate and is visible under uv (254 nm). the 1h-nmr spectrum of b shows two signals for a methyl group (-ch 3 ) at δ 2.13 and δ 2.16, two methoxy (-och 3 ) groups at δ 3.49 and δ 3.66, aromatic protons (ar-h) at δ 7.34 – 8.00 [δ 8.00 (d, j = 15.7 hz, 1h), δ 7.84 (d, j = 15.7 hz, 1h), δ 7.66 (dd, j = 8.4, 6.3 hz, 2h)], and a signal at δ 13.63 for the hydroxyl (-oh) proton. compound b has a methoxy group instead of a hydroxyl group at the 4’ position with respect to dmc. sd june2022_tambago.indd 46 7/29/2022 4:11:42 pm g.c. v. tambago and e.c. amor 47 validation of the cyclooxygenase inhibition assay the cyclooxygenase inhibition assay is based on the action of the inhibitor on the cyclooxygenase and its substrate, arachidonic acid. to confirm that the assay is working and the data collected from it are valid, the activity of the enzyme and substrate was monitored by determining if the michaelis-menten constant (k m ) and v max are constant and within the acceptable range. acceptable range means the parameters collected are either in line with theoretical data based on previous studies or within a constant range of values established historically in the laboratory. the responses of varied concentrations of arachidonic acid against a constant amount of enzyme were recorded and plotted in figure 6. figure 6. michaelis-menten plot of cox-1 and cox-2 enzymes with arachidonic acid substrate. the k m and v max values obtained for cox-1 were 279.4 μm and 12.6 fi/s, respectively, while 488.0 μm and 20.2 fi/s, respectively, were calculated for cox-2. these values are consistent with the historically established data range (cox-1: k m = 200 550 μm; cox-2: k m = 200 – 600 μm) for the assay parameters (opog et al. 2019). the assay results for the cyclooxygenase inhibiting activity of dmc and its derivatives are shown in figure 7. sd june2022_tambago.indd 47 7/29/2022 4:11:42 pm chalcone derivatives with cyclooxygenase inhibiting activity 48 figure 7. cox inhibition summary for dmc and its derivatives. compound a gave 55.57 ± 1.43% inhibitory activity against cox-2 at 100 ppm and 45.54 ± 8.19% against cox-1. compound b gave 86.70 ± 2.25% inhibitory activity against cox-2 at 100 ppm and 81.22 ± 2.84% against cox-1. compounds a and b were found to be cox-2 selective inhibitors with cox-2 to cox-1 inhibition ratios of 1.22 and 1.07, respectively. conclusion alkaline peroxidation of (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone from the leaf extracts of syzygium samarangense (blume) merr. yielded two products, 7-hydroxy-5-methoxy-6,8-dimethyl flavanone and 2’-hydroxy-4’,6’-dimethoxy-3’,5’dimethyl chalcone. this is the first report of the peroxidation of dmc producing these flavonoids. flavonoids are plant-derived natural products that have long been studied for their anti-inflammatory properties (read 1995; zhang et al. 2006). some flavanones have been reported to exhibit in silico cox-2 selective inhibiting activity such as eriodictyol (3’,4’,5,7-tetrahydroxyflavan-4-one), hesperetin (3’,5,7-trihydroxy-4’methoxyflavan-4-one), and naringenin (4’-5,7-trihydroxyflavan-4-one) (akinloye et al. 2019). on the other hand, synthetic chalcone derivatives have also been reported as cox-2 selective inhibitors in several studies (razmi et al. 2013; jantan et al. 2014). sd june2022_tambago.indd 48 7/29/2022 4:11:42 pm g.c. v. tambago and e.c. amor 49 dmc, a, and b were tested for their anti-inflammatory activity using an in vitro cyclooxygenase inhibition assay. both derivatives show selective inhibition of cyclooxygenase-2 at 100 ppm. this is the first report of the selective cyclooxygenase-2 inhibiting activity of 7-hydroxy-5-methoxy-6,8-dimethyl flavanone and 2’-hydroxy4’,6’-dimethoxy-3’,5’-dimethyl chalcone. these flavonoids could potentially be lead candidates in the development of new anti-inflammatory drugs. acknowledgements this research was made possible with the funding from the office of the vice chancellor for research and development of the university of the philippines diliman and the facilities at the institute of chemistry, up diliman. we also thank mr. jacob noel inguito of the terrestrial natural products laboratory for helping with the assay. references agrawal n, soni, p. 2006. a new process for the synthesis of aurones by using mercury (ii) acetate in pyridine and cupric bromide in dimetyl sulfoxide. indian j chem. 45b, 1301-1303. akinloye o, metibemu d, akinloye d, onigbinde s, olaosebikan i, florence o, damilola b, bolarinwa a, olubunmi o. 2019. flavanones from sorghum bicolor selectively inhibit cox-2: in-silico and in-vivo validation. egypt j med hum. 20(34). amor e, villaseñor i, antemano r, perveen z, concepcion 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20(8):14212–14233. doi:10.3390/molecules200814212. sd june2022_tambago.indd 50 7/29/2022 4:11:42 pm g.c. v. tambago and e.c. amor 51 nakayama t. 2002. enzymology of aurone biosynthesis. j biosci bioeng. 94(6):487491. nakayama t, yonekura-sakakibara k, sato t, kikuchi s, fukui y, fukuchi-mizutani m, ueda t, nakao m, tanaka y, kusumi t, et al. 2000. aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration. science. 290(5494):1163-1166. opog, a, villones, l, amor, e. 2019. cyclooxygenase (cox) inhibition assay: fluorometric method. in: guevarra a, alvero r, editors. tuklas lunas® protocols for drug discovery and development volume iib: primary bioactivity assays. taguig: philippine council for health research and development. pasa s, bayan k, kucukoner m, tuzun y, altintas a, cil t, danis r, ayyildiz o. 2009. the effects of non-steroidal anti-inflammatory drugs on platelet function and severity of upper gastrointestinal haemorrhage. j thromb thrombolysis. 28(1): 83-89. patrono c, rocca b. 2009. nonsteroidal anti-inflammatory drugs: past, present and future. pharmacol res. 59(5):285-289. razmi a, zarghi a, arfaee s, naderi n, faizi m. 2013. evaluation of anti-nociceptive and anti-inflammatory activities of chalcone derivatives. iran j pharm sci. 12(suppl):153-159. read m. 1995. flavonoids: naturally occurring anti-inflammatory agents. am j pathol. 147(2):235-237. sekizaki h. 1988. synthesis of 2-benzylidene-3(2h)-benzofuran-3-ones (aurones) by oxidation of 2’-hydroxychalcones with mercury (ii) acetate. bull chem soc jpn. 61(4):1407-1409. shen t. 1982. the discovery of indomethacin and the proliferation of nsaids. semin arthritis rheum. 12(2):89-93. sun s, lee k, bertram c, godlstein j. 2007. withdrawal of cox-2 selective inhibitors rofecoxib and valdecoxib: impact on nsaid and gastroprotective drug prescribing and utilization. curr med res opin. 23(8):1859-1866. thomas d, ali z, zachariah s, sundararaj k, van cuyk m, cooper j. 2017. coxibs refocus attention on the cardiovascular risks of non-aspirin nsaids. am j cardiovasc drugs. 17(5):343-346. zhang x, hung t, phuong p, ngoc t, min bs, song ks, seong y, bae k. 2006. antiinflammatory activity of flavonoids from populus davidania. arch pharm res. 29(12):1102-1108. sd june2022_tambago.indd 51 7/29/2022 4:11:42 pm chalcone derivatives with cyclooxygenase inhibiting activity 52 ______ gian carlo v. tambago <gvtambago@up.edu.ph> finished his bachelor of science in chemistry at the university of the philippines diliman. he is currently an m.s. student at the institute of chemistry, university of the philippines-diliman, and a science research specialist at the terrestrial natural products laboratory from the same institute. he is working on the isolation and elucidation of anticancer compounds from philippine terrestrial plants. evangeline c. amor is a professor at the institute of chemistry, university of the philippines diliman. she received her ph.d. in chemistry from the same university. she heads the terrestrial natural products laboratory research group and specializes in natural products and organic chemistry with research focus on the isolation and structure elucidation of bioactive (analgesic, hypoglycemic, anticancer, anti-inflammatory) compounds from philippine plants, structure-activity relationship studies, mutagenicity, and clastogenicity studies on medicinal plant preparations, and enzyme inhibition studies. sd june2022_tambago.indd 52 7/29/2022 4:11:42 pm comparison-penuliar (2).pmd comparison of clinical and environmental isolates 9 abstract science diliman (january-june 2010) 22:1,9-18 comparison of clinical and environmental isolates of acanthamoeba based on morphology, protease and gelatinase activity, and the cysteine proteinase gene gil m. penuliar,1* ronald r. matias,2 and filipinas f. natividad2 1institute of biology, university of the philippines, diliman, quezon city 1101 philippines; 2research and biotechnology division, st. luke’s medical center, 279 e. rodriguez sr. boulevard, quezon city, 1102 philippines *corresponding author: email: gmp@nih.go.jp; fax: +81 3 5285 1173;tel.: +81 3 5285 1111 ext. 2733 acanthamoeba spp. are opportunistic pathogens that cause amebic keratitis and granulomatous amebic encephalitis in man. recent attempts to correlate pathogenicity with species have been proven difficult due to inconsistencies in morphology-based classification. the objectives of this study were: (1) to compare clinical and environmental isolates based on morphology, protease and gelatinase activity, and the cysteine proteinase (cp) gene, and (2) to determine whether these features can be used to differentiate the isolates. results show some degree of variation in trophozoite and cyst morphology. zymography, demonstrated gross differences in banding patterns, and the protease activity of clinical isolates was greater than the environmental isolates (p-value < 0.01). amplification of the cp gene yielded two bands in the environmental isolates, approximately 755 bp and 440 bp in length. in contrast, only one band, either the 755 bp or 440 bp band was amplified in the clinical isolates. the results confirmed the limitations of morphology in differentiating acanthamoeba species, and suggest that zymography, protease activity, and detection of the cp gene are useful reference tests to distinguish pathogenic from non-pathogenic isolates. key words: acanthamoeba, protease activity, cysteine proteinase gene penuliar, g.m., et al 10 introduction acanthamoeba is a free-living protozoan characterized by acanthapodia in the trophozoite stage and a doublewalled cyst stage. it is widely distributed in nature due to its simple nutritional requirement and ability to encyst. isolates have been obtained from soil and water samples, parts of plants and animals, contact lenses, and air-conditioning units (zaragoza, 1994; anderson, 1988). based on clinical and epidemiological information, acanthamoeba is considered to be an opportunistic pathogen of humans (marciano-cabral et al., 2000; khan et al., 2001). some species can cause fatal granulomatous amebic encephalitis (gae), but most amebic infections are associated with eye keratitis (armstrong 2000; niederkorn et al., 1999). a number of researches have been conducted to identify acanthamoeba isolates based on morphology, biochemical activity, and genetic composition. with the increasing number of medical cases associated with acanthamoeba, some of these studies have attempted to correlate their results with virulence. currently, pathogenicity in acanthamoeba is demonstrated only when an isolate is obtained from a diseased tissue of a patient, or if an isolate can infect an animal model system (vodkin et al., 1992). there is, therefore, a need to develop alternative methods that could effectively differentiate pathogenic from non-pathogenic isolates. it has been reported that acanthamoeba-conditioned medium (acm) produces cytophatic effects in epithelial cells and corneal tissues, suggesting that secreted proteases are involved in epithelial-cell disaggregation (khan et al., 2000; badenoch et al., 1995). in 1993, hadas and mazur showed two major proteases in the acm that could be separated electrophoretically. they reported a 35 kda acidic protease and a 65 kda alkaline protease that belonged to the cysteine type of proteases. studies have shown that cysteine proteinases (cps) are more active in pathogenic strains, and that pathogenicity is the result of proteolysis of the host’s extracellular matrix by cps (yun et al., 1999; tannich, 1998). khan and paget in 2002, on the other hand, used genus-specific primers for the pcr amplification of 18s rdna to detect and speciate acanthamoeba isolated from clinical and environmental sources. their results suggested that significant variation exists between and within morphologically defined acanthamoeba species. the objectives of this study were: (1) to compare 2 clinical and 3 environmental isolates of acanthamoeba based on morphology, protease and gelatinase activity, and the cysteine proteinase (cp) gene, and (2) to determine whether these characteristics can be used to effectively differentiate the isolates. materials and methods isolation, cloning, and culture of acanthamoeba (de jonckheere, 1980) soil samples collected from davao (dav), iloilo (ilo), and pangasinan (pan), were suspended in sterile distilled water. mixtures were allowed to stand for 15 min, and supernatants filtered with whatman filter paper no. 4. filter papers were inverted on 1.5% non-nutrient agar (nna) plates, previously seeded with heat-killed escherichia coli. plates were incubated at 37oc and observed daily for trophozoites and cysts. after 1 week of incubation, sections on the plates containing large numbers of cysts were excised and transferred to new nna plates. the two clinical isolates (rbd and nag) used in the study were obtained from the research and biotechnology division (rbd) of st. luke’s medical center, and the institute of tropical medicine, in nagasaki, japan. to axenize the environmental isolates, nna plates were flooded with 10 ml sterile phosphate buffer saline (pbs), ph 7.2, and the cysts gently scraped off the plates with a sterile lancet. suspensions were examined under a compound microscope, and diluted several times, until a single cyst of each isolate was obtained, and transferred to new nna plates. plates were incubated and observed daily for trophozoites and cysts. after one week, cysts were harvested as previously described, and suspensions centrifuged at 2,000 rpm for 10 min. pellets were resuspended in 1n hcl for 3 h at room temperature (rt). hcl was removed after centrifugation at 2000 rpm for 10 min and pellets were washed 3 times with pbs. cysts were resuspended in 25 ml proteose peptone yeast extract glucose solution (ppyg) and incubated at 37oc. science diliman (january-june 2010) 22:1,9-18 comparison of clinical and environmental isolates 11 trophozoite and cyst morphology acanthamoeba cultures were incubated for 10 min on ice and centrifuged at 700 rpm for 5 min. supernatants were discarded and pellets resuspended in 1 ml pbs. suspensions were examined under a 100x objective lens, oil immersion, and phase contrast microscope. one hundred trophozoites were measured and photographed with a nikontm digital camera. cultures were allowed to encyst in nna plates and the cysts harvested as previously described. the same number of cysts was measured and photographed. electron microscopy (yagita et al., 1995) cysts were harvested and fixed with 2.5% buffered glutaraldehyde for 4 h. samples were washed twice with 0.1m pbs for 10 min and centrifuged at 2,000 rpm for 5 min at rt. cysts were fixed with 2% osmium tetroxide for 1 h and washed twice with 0.1m pbs. pellets were transferred to 1.5 ml microcentrifuge tubes and dehydrated with 30% ethanol. cysts were agitated for 10 min and centrifuged at 2,000 rpm for 2 min. the dehydration step was repeated using 70% ethanol, 95% ethanol, and 95% acetone. samples were dehydrated 3 times with absolute acetone, placed in absolute propylene, and infiltrated with 1:1 propylene oxide and embedding media for 24 h. cysts were transferred to bean capsules and infiltrated with 1:3 propylene oxide and embedding media for 24 h. samples were centrifuged at maximum speed, and the supernatant replaced with absolute embedding media, and polymerized at 65oc for 24 h. cysts were viewed and photographed under a jeol jem 1010 tm transmission electron microscope. protease activity (sarah et., 1994) three-day old cultures of acanthamoeba were harvested as previously described, and counted. approximately 2 x 106 cells were transferred to new culture flasks and incubated for 1 h at 37oc. media were removed and replaced with 10 ml pbs. cultures were incubated for 24 h at 37oc, and the trophozoites harvested by centrifugation at 700 rpm for 10 min at rt. acm of the isolates were concentrated 8 to 10 folds by dialysis using polyethylene glycol. one hundred microliters of acm was incubated with 200 μl 1 mg/ml azocasein for 1 h at rt. reactions were stopped by adding 300 μl 10% trichloroacetic acid (tca). mixtures were shaken for 15 min and centrifuged at 3,000 rpm for 5 min. five hundred microliters of 1m naoh was added to 500 μl supernatant, and the absorbance read at 440 nm using a spectra max 190 tm spectrophotometer. in another set-up, the acm was pre-incubated with 1mm ethylenediamine tetra-acetic acid (edta) for 30 min before mixing with azocasein. gelatinase activity (itoh et al., 1998) five microliters of concentrated acm was mixed with 5 μl sample buffer, and electrophoresed in 10% sdspage with 0.1% gelatin. the gel was washed with 2.5% triton x-100 for 2 h and incubated in 0.1 m glycine, ph 7.4, overnight at rt. the gel was stained with 0.5% [w/v] coomassie brilliant blue r-250, overnight, and destained in acetic acid: methanol: water (10:30:60). bands were visualized on a light box and photographed. dna extraction (kilvington et al., 1991) trophozoites were harvested as previously described. cell pellets were transferred to 1.5 ml microcentrifuge tubes and resuspended in 300 μl lysis buffer (100 mm disodium edta, 100 mm naci, 10 mm tris hydrochloride [ph 8.0]), supplemented with 3 μl of rnase (1mg/ml). tubes were incubated for 30 min at 65oc, and centrifuged at 8,200 rpm for 10 min at 4oc. supernatants were transferred to new tubes, mixed with 500 μl of phenol: chloroform: isoamyl alcohol (pci), and centrifuged. aqueous phases were transferred to new tubes and re-extracted with 500 μl pci. after centrifugation, the aqueous phases were transferred to new tubes and mixed with 45 μl of 5 m ammonium acetate and 900 μl of cold absolute ethanol. samples were incubated for 12 h at -20oc. precipitated dna was collected by centrifugation at 12,000 rpm for 15 min at 4oc. pellets were washed with 70% ethanol, air dried for 30 min at rt, and resuspended in 30 μl sterile distilled water. pcr amplification a pcr mix for 5 samples was prepared composed of 108 μl sterile distilled water, 15 μl 10x pcr buffer, 4.8 science diliman (january-june 2010) 22:1,9-18 penuliar, g.m., et al 12 μl 50 mm mgcl 2 , 3 μl 20 mm dntps, 1.2 μl 250 u taq polymerase, 3 μl 50 pm wrg3 forward primer, and 3 μl 50 pm wrg4 reverse primer. twenty-three microliters of the pcr mix was loaded into five 0.5 ml pcr tubes and 10 ng of dna sample was added. pcr tubes were loaded into an eppendorf mastercylertm with the following conditions: initial denaturation for 10 min at 94oc, and subsequent denaturation for 1 min, annealing for 1.5 min at 52oc, extension for 1.5 min at 72oc, and denaturation for 1 min. annealing and extension were repeated 38 times, followed by a final extension of 7 min at 72oc, and 10 min at 4oc. pcr products were electrophoresed in 2% agarose gel. bands were visualized after ethidium bromide (1 μl/ ml) staining. phylogenetic analysis (felsenstein, 2000) a similarity matrix was prepared from all the data obtained, and the characters were encoded as 1 and 0 to indicate the presence or absence of a character state, respectively; or to signify measurements above or below the mean of a set of values, respectively. the matrix was analyzed using pars (discrete character parsimony) in phylip 3.6 and treeview 1.6.6. statistical analysis data were analyzed using the single-factor anova and correlation functions of microsoft excel statistical package (microsoft corp. redmond, wa, usa). probability levels (p) less than 0.01 were considered significant. results trophozoite and cyst morphology the trophozoites of all the isolates were characterized by acanthapodia (figure 1a). a clear separation of the hyaline ectoplasm and granular endoplasm was observed. the nucleus contained a centrally located nucleolus, and vacuoles were observed in the endoplasm. the diameter and nucleo-cytoplasmic (nc) ratio of the trophozoites varied (table 1). rbd had the largest diameter (20.8 ± 0.7 μm), while ilo had the smallest (18.9 ± 0.5 μm). ilo, however, had the largest nc ratio (0.008), while nag had the smallest (0.004). the diameter and nc ratio of the cysts also varied (table 1). rbd had the largest diameter (17.8 ± 3.1μm), figure 1: all of the isolates were identified as acanthamoeba based on trophozoite and cyst morphology. (a) trophozoites were characterized by the presence of acanthapodia projecting from the hyaline ectoplasm. (b) cysts were bilaminar, composed of a wrinkled ectocyst and a smoother inner endocyst. the cyst walls meet at the region of the ostiole. (c) electron photomicrograph of pan showing the different parts of the cyst. the ostiole was covered from the inside by an operculum. the nucleus was large with a dense centrally located nucleolus. bar = 5 μm. science diliman (january-june 2010) 22:1,9-18 comparison of clinical and environmental isolates 13 while nag had the smallest (14.3 ± 2.9 μm). rbd also had the largest nc ratio (0.071), while pan had the smallest (0.059). the endocyst was predominantly spherical for all the isolates (figure 1b), although a few cysts with polygonal and stellate-shaped endocysts were also observed (data not shown). the number of ostioles varied in number and diameter (table 1). rbd and dav had 1 to 3 ostioles, while nag, ilo, and pan had 1 to 2 ostioles. the ostioles of rbd had the largest diameter (1.93 ± 0.42 μm), while ilo had the smallest (1.61 ± 0.55 μm). opercula were observed to cover the ostioles from the inside (figure 1c). the cell membrane of the cysts was crenate in appearance and not closely apposed to the endocyst. a centrally located nucleolus was also observed (figure 1c). table 1. morphology-based measurements. the diameter and nucleo-cytoplasmic (nc) ratio were determined for the trophozoites and cysts (n = 100), while the number of ostioles and ostiole diameter were determined from the cyst electron photomicrographs (n = 5). figure 2. protease and gelatinase activity, and cp gene amplification. (a) the protease activity of the clinical isolates was significantly higher than the environmental isolates (p-value < 0.01). a reduction in protease activity was observed when the samples were pre-incubated with 1mm edta. (b) the acm of the isolates produced a total of 9 bands in 10% sds-page with 0.1% gelatin, indicating the presence of gelatinases. bands 1, 2, 6, and 9 were observed in nag and ilo. nag lacked band 4 while dav and pan lacked band 8. bands 3, 5, and 7 were observed only in nag, dav, and rbd, respectively. (c) amplification of the cp gene yielded two bands in the environmental isolates, approximately 755 bp and 440 bp in length. in contrast, only the 755 bp band was amplified from rbd, while nag produced only the 440 bp band. science diliman (january-june 2010) 22:1,9-18 penuliar, g.m., et al 14 protease and gelatinase activity the protease activity varied among the isolates (figure 2a). nag had the highest enzyme activity, while pan had the lowest. the enyzme activity decreased when the acm was pre-incubated with 1mm edta. the acm of the isolates produced different banding patterns on the zymogram (figure 2b). nine different bands were observed. bands 1, 2, 6, and 9 were observed in nag and ilo. nag lacked band 4, while dav and pan lacked band 8. bands 3, 5, and 7 were observed only in nag, dav and rbd, respectively. acm preincubated with 1mm edta produced the same number of bands in all the isolates (data not shown). pcr amplification of the cp gene amplification of the cp gene yielded two bands in the environmental isolates, approximately 755 bp and 440 bp in length (figure 2c). in contrast, only the 755 bp band was amplified from rbd, while nag produced only the 440 bp band. phylogenetic analysis based from the first few trees generated, nag separated early from the other isolates (data not shown). it was, therefore, omitted in the final analysis, to observe the relationship of rbd with the environmental isolates. ilo was observed to branch first, followed by rbd (figure 3). pan and dav were the last to separate. when morphological characters alone were used, however, pan and rbd separated from dav and ilo (data not shown). this indicated that the range of values obtained from the morphological features were not able to distinguish the isolates. discussion trophozoite and cyst morphology the trophozoite morphology of the isolates corresponded to the descriptions made by matias (1991) and anderson (1988). the diameters varied significantly (p-value < 0.01), especially between ilo and the other isolates. a few trophozoites from each isolate were observed to have smaller and larger diameters than most of the trophozoites. considering that the isolates were clonal, variations in diameter can be attributed to a transition from logarithmic growth to population growth deceleration. it was reported that marked changes in cell size and macromolecular composition are often observed during such transitions in acanthamoeba. in addition, it was also reported that during each cell cycle, trophozoites release 12-37% of their weight (anderson, 1988). when the shape of the cell and form of the pseudopodia are similar, taxonomic distinctions in amoeba are then based on the nucleus and occurrence of cysts. nc ratio is the ratio of the volume of the nucleus and cytoplasm. within isolates, the ratio is regulated by the activities of the nucleus, and variations in the ratio reflect differences in the age of the cell. it is reported to be high in dividing cells and low in old cells. differences in the nc ratio of the trophozoites were significant (pvalue < 0.01), but it did not differentiate the clinical from the environmental isolates. figure 3. phylogenetic analysis of rbd and the environmental isolates. nag was not included in the final analysis because earlier analyses showed the early branching of nag from the other isolates. the pars utility in phylip 3.6 and treeview 1.6.6 were used to generate the dendrogram. ilo was observed to branch first, followed by rbd. pan and dav were the last to separate. the early separation of ilo was consistent based on trophozoite diameter, while the branching of rbd correlated with the cyst diameter, protease activity, and cp gene amplification data. science diliman (january-june 2010) 22:1,9-18 comparison of clinical and environmental isolates 15 the morphology of the cysts reflected the studies made by yagita et al. (1995) and lasman (1977). the size and shape of the cysts fell under group iii (figure 1b,c). the group includes cysts with a mean diameter <18 μm, and an endocyst that is usually round (visvesvara, 1991). the few polygonal and stellateshaped cysts observed within isolates may be attributed to differences in the degree of desiccation, as this factor is reported to affect cyst morphology (russel, 1996; ma et al., 1990). differences in cyst diameters were significant (p-value < 0.01), although it failed to distinguish the clinical from the environmental isolates. rbd, however, was observed to have a subpopulation of cysts with a diameter of 20 μm (data not shown). variations in cyst diameter can also be due to the same factors affecting trophozoite diameter; but in conjunction with factors that lead to encystment in nna plates like cell density and desiccation (szenasi et al., 1998). it was, therefore, not surprising to observe some degree of correspondence (0.885) between the size of the cyst and trophozoite. differences in the nc ratio of the cysts were significant (p-value < 0.01) and it divided the isolates into 2 groups. the nc ratio of the clinical isolates was at least seven units greater than the environmental isolates, indicating that the clinical isolates had larger nuclei. the ectocyst was thicker than the endocyst for all isolates (figure 1b,c). this was not surprising since the ectocysts is the part exposed to the environment. the ectocyst, however, appeared less compact than the endocyst. in 1998, mehlotra tried to determine if pathogenic species of acanthamoeba had thinner cyst wall than non-pathogenic species. the results of this study provided no clear answer (data not shown). the number of ostioles varied between isolates. ostioles are passages through which the trophozoite leaves the cyst during excystment. it was, therefore, expected to observe some degree of correlation between the diameter of the ostiole with that of the trophozoites (0.6) and cysts (0.5). each ostiole was covered from the inside by an operculum that had the same electron density as the endocyst. the ostiole is reported to function as an environmental sensor (kilvington & white, 1994). protease and gelatinase activity a number of researchers have demonstrated through cytotoxicity and colorimetric assays that acanthamoeba secretes proteases (khan et al., 2000; he et al., 1990). in this study, azocasein was used as a substrate to detect and quantify protease activity (figure 2a). the results indicated that acm contained proteases. in addition, the protease activity of the 5 isolates varied significantly (p-value < 0.01). clinical isolates exhibited higher protease activity compared to environmental isolates. this result was consistent with previous findings that pathogenic species secrete more proteases than non-pathogenic species (khan et al., 2000; hadas & mazur, 1993). tannich (1998) suggested that the low amount of proteinases in nonpathogenic entamoeba spp. was due to a lower number of proteinase expressing genes. the present data suggest that differences in protease activity can be used as a marker for differentiating clinical and environmental isolates of acanthamoeba. results also showed that edta significantly inhibited acm protease activity (p-value < 0.01). edta is a general protease inhibitor that chelates bivalent metal ions in enyzmes. the finding, however, did not correlate with the work of mitro et al. (1994), who reported that no significant inhibition of protease activity was observed with 10 mm edta. in addition, hadas and mazur (1993), reported that 10 mm edta enhanced protease activity. the concentration used in their studies, however, was different from the concentration used in this study. edta works effectively at a concentration of 1 mm to 10 mm, and can be inhibitory or stimulatory depending on its concentration. alfieri et al. (2000) reported that the hydrolysis of azocasein was predominantly associated with the activity of cysteine proteinases. however, since the inhibitor used was not specific for cysteine proteinases, the observed acm protease activity may be due to other proteases. to visualize the protease activity, acm was electrophoresed in an sds-page with 0.1% gelatin (figure 2b). the bands observe indicated the presence of gelatinases. moreover, variations in banding pattern intensity, suggested that various gelatinases were science diliman (january-june 2010) 22:1,9-18 penuliar, g.m., et al 16 secreted in different amounts by the isolates. the finding correlated with the work of mitro et al. (1994), that acanthamoeba secretes different types of proteases. although the banding patterns did not differentiate the clinical and environmental isolates, the variations observed can still be used to characterize the isolates individually. pre-incubation of the acm with 1 mm edta produced the same number of bands, suggesting that gelatinases were not inhibited by edta. detection of the cp gene with the developments of new tools in molecular biology, several workers have attempted to identify markers at the nucleic acid level to distinguish pathogenic from non-pathogenic isolates (mirelman et al., 1996). in this study, primers wrg3 and wrg4 were used to detect the cp gene in the genomic dna of acanthamoeba. the primers were designed to amplify a 755 bp fragment of the cp (ehcp1) gene found in entamoeba histolytica. the primers were able to amplify certain regions in the dna of acanthamoeba, indicating the presence of conserved regions between e. histolytica and acanthamoeba (figure 2c). variations in the pcr products indicated that the clinical isolates differ not only from the environmental isolates but also from each other. furthermore, amplification of the 440 bp band in nag and environmental isolates, suggested the presence of incomplete repeats of the target sequence in the isolates. the amplification was specific, since the band consistently appeared in all the trials. the detection of the cp gene with primers designed for e. histolytica, was not surprising since it had been reported that closely related species contain the same gene for cp. the gene, however, may vary due to numerous nucleotide exchanges, insertions, and deletions (willhoeft et al., 1999; mirelman et al., 1996). phylogenetic analysis the pars utility in phylip 3.6 was used because the program allows users to analyze similarity matrices based on the assumptions that ancestral states are unknown and that characters evolve independently (felsenstein, 2000). nag was not included in the final analysis because earlier analyses showed the early branching of nag from the other isolates (data not shown). considering the geographical location where nag was isolated, and based on the pcr data, it was not surprising that nag was not as closely related to the environmental isolates compared to rbd. the early separation of ilo was consistent based on trophozoite diameter, while the branching of rbd correlated with the cyst diameter, protease activity, and cp gene amplification data. in conclusion, the results obtained from this study verified the limitations inherent in morphology-based methods of identification. while trophozoite and cyst morphology effectively differentiate acanthamoeba from other protozoa, morphological characters cannot distinguish the isolates based on pathogenicity. a practical approach to solve this problem is to include more stable characters like enzyme activity and amplification of conserved genes, which has been shown in this study, and by previous researchers, to be valuable tools in differentiating pathogenic from non-pathogenic isolates. acknowledgments the authors thank dr. gloria l. enriquez, dr. windell l. rivera, and the research and biotechnology division (rbd) of st. luke’s medical center. references alfieri, s., c. correla, s. motegi, & e. pral, 2000. proteinase activities in total extracts and in medium conditioned by acanthamoeba polyphaga trophozoites. j. parasitol. 86(2): 220-227. anderson, r., 1988. comparative protozoology: ecology, physiology, life history. new york: springer-verlag. armstrong, m., 2000. the pathogenesis of human acanthamoeba infections. infect dis. rev. 2(2): 65-73. badenoch, p., m. adams, & d. coster, 1995. corneal virulence, cytophatic effect on human keratocytes and genetic characterization of acanthamoeba. int. j. parasitol. 25(2): 229-239. science diliman (january-june 2010) 22:1,9-18 comparison of clinical and environmental isolates 17 de jonckheere, j., 1980. growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different acanthamoeba spp. appl environ microbiol. 39(4): 681-5. felsenstein, j., 2000. phylip 3.6: phylogeny inference package. available from url: http:// evolution.genetics.washington.edu/phylip/ hadas, e. & t. mazur, 1993. biochemical markers of pathogenicity and virulence of acanthamoeba sp. strains. parasitol. res. 79(8): 696-698. he, y., j. niederkorn, j. mcculley, g. stewart, d. meyer, r. silvany, & j. doughtery, 1990. in vivo and in vitro collagenolytic activity of acanthamoeba castellani. invest. opthal. vis. sci. 31:2235-2240. itoh, y., a. ito, k. iwata, k. tanzawa, y. mori, & h. nagase, 1998. plasma membrane-bound tissue inhibitor of metallopeptidases (timp)-2 specifically inhibits matrix metallopeptidase 2 (gelatinase a) activated on the cell surface. j. biol. chem. 273: 24360-24367. khan, n. & t. paget, 2002. molecular tools for speciation and epidemiological studies of acanthamoeba. curr. microbiol. 44(6): 444-4449. khan, n., e. jarroll, & t. paget, 2001. acanthamoeba can be differentiated clinically by the polymerase chain reaction and simple plating assays. curr. microbiol. 43: 204-208. khan, n., e. jaroll, n. panjawani, z. cao, & t. paget, 2000. proteases as marker for differentiation of pathogenic and non-pathogenic species of acanthamoeba. journal of clinical microbiology. 38(8): 2858-2861. kilvington, s. & p. white, 1994. acanthamoeba: biology, ecology, and human disease. review in medical microbiology. 5(1): 12-20. kilvington, s., j. beeching, & d. white, 1991. differentiation of acanthamoeba strains from infected corneas and the environment by using restriction endonuclease digestion of whole-cell dna. j. clin. microbiol. 29(2): 310-4. lasman, m., 1977. light and electron microscopic observations on encystment of acanthamoeba palestinensis reich. j. protozool. 24(2): 244-248. ma, p., g. visvesvara, a. martinez, f. theodore, d. pierremare, & t. sawyer, 1990. naegleria and acanthamoeba infections. rev. infect. dis. 12: 490-573. marciano-cabral, f., r. puffenbarger, & g. cabral, 2000: the increasing importance of acanthamoeba infections. j. eukaryot. microbiol. 47: 29-36. matias, r., j. schotteliu, c. raddatz, & r. michel, 1991. species identification and characterization of a acanthamoeba strain from human cornea. parasitology research. 77(6): 469-474. mehlotra, r., 1998. differentiation of pathogenic and nonpathogenic entamoeba: has the question been answered? indian society of gastroenterology. 17: 58-60. mirelman, d., y. michamowitzi, b. bohn-gloning, & b. walderich, 1996. a homologue of the cysteine proteinase gene (acp1) of pathogenic entamoeba histolytica in present in non-pathogenic e. dispar strains. parasitol. res. 36: 518-543. mitro, k., a. bhagavathiammi, g. bobbett, j. mckerrow, b. chokshi, & r. james, 1994. partial characterization of the proteolytic secretions of acanthamoeba polyphaga. expt. parasitol. 78(4): 377-378. niederkorn, j., y. alizadeh, h. lether, & j. mcculley, 1999. the pathogenesis of acanthamoeba keratitis. microbes infect. 1(6): 437-443. russel, a., 1996. the lethal effects of biguanides on cysts and trophozoites of acanthamoeba castellani. j. appl. bacteriol. 8(1): 73-77. sarah, g., s. de la motte, & w. wagner, 1994. protease methods, p. 28. in rj beynon and js bond (ed), proteolytic enzymes, a practical approach, 2nd ed. irl, oxford, uk. szensi, s., t. endo, k. yagita, & e. nagy, 1998. isolation, identification, and increasing importance of free-living amoeba causing human diseases. j. med. microbiol. 47: 516. science diliman (january-june 2010) 22:1,9-18 penuliar, g.m., et al 18 tannich, e., 1998. entamoeba histolytica and e. dispar: comparison of molecules considered important for host tissue destruction. transactions of the royal society of tropical medicine and hygiene. 92: 593-596. visvesvara, g., 1991. classification of acanthamoeba. rev infect dis. 13 suppl 5:s369-72. vodkin, m., d. howe, g. visvesvara, & g. mclaughlin, 1992. identification of acanthamoeba at the genetic and specific levels using the polymerase chain reaction. j. protozool. 39(3): 378-385. willhoeft, u., l. hamann, & e. tannich, 1999. a dna corresponding to the gene encoding cysteine proteinase 5 in entamoeba histolytica in present and positionally conserved but highly degenerated in e. dispar. infection and immunity. 67(11): 5925-5929. yagita, k., r. matias, t. yasuda, f. natividad, g. enriquez, & t. endo, 1995. acanthamoeba sp. from the philippines: electron microscopy studies on naturally occurring bacterial symbionts. parasitology research. 81: 98-102. yun, h., k. kim, s. park, u. hwang, k. hong, j. ryu, & d. min. 1999. cloning of a cysteine proteinase gene from acanthamoeba culbertsonii. mol. cells. 9(5): 491-496. zaragosa, s., 1994. ecology of free-living amoeba. crit. rev microbiol. 20(3): 225-241. science diliman (january-june 2010) 22:1,9-18 ocr document 70 undergraduate experiments on qualitative analysis of color and surface modifications and quantitative analysis using the tauc plot method through ultraviolet-visible relative specular reflectance marte c. villena* mona b. antivola janelle stephanie m. torrefiel rheo b. lamorena-lim institute of chemistry university of the philippines diliman abstract the study of the spectroscopy of solid materials is not as extensive compared to that of homogeneous liquid solutions. optical properties, such as reflectance, transmittance, and absorbance, may be used for the characterization of new materials. qualitative characterization of colored materials and quantitative determination of surface modification and band gap analysis through the tauc plot method were obtained using visible-reflectance spectroscopy. differentiation of reflectance peaks was observed in the spectra of colored papers and acrylic spray paints. expected trends of higher reflectance according to the ability of a color to reflect light were also observed. surface analysis through the reflectance method indicated that spectra of pristine and surface-modified samples showed a significant difference in signals, which is attributed to the roughening and contamination of the surface of the materials. using the tauc plot method on the absorbance spectrum data of an electrodeposited zinc oxide film, the calculated bge was 3.80 ev, with a 15.15% deviation from its literature value of 3.30 ev. in addition, zinc sulfide, which was chemically deposited on glass slides, had an average band gap of 3.0 ev, with an 8.82% error against its literature value of 3.4 ev. these experiments on specular reflectance of solid materials, particularly optical and new materials, may be incorporated in undergraduate laboratory courses. keywords: specular relative reflectance, surface analysis, color characterization, bandgap, tauc plot, solid analysis * corresponding author science diliman (july-december 2021) 33:2, 70-89 m. c. villena et al. 71 introduction reflected light is highly dependent on the nature of the surface of a sample. there are two kinds of reflection resulting from the difference in texture and composition of the surface. specular reflectance is observed when light is reflected from a smooth surface and at a definite angle while diffuse reflectance results from uneven and rough surfaces that tend to reflect light in all directions. (figure 1) (polato and masetti 1988) figure 1. specular reflectance happens when light is reflected from a smooth surface (left), whereas diffuse reflectance results from the uneven and rough surface of a sample, which reflects light in all directions (right). (figure illustrated by mona b. antivola.) reflectance, which may be either relative or absolute, has been traditionally measured using reflectometers or reflectance attachments to spectrophotometers (polato and masetti 1988). absolute reflectance calculates the true reflectance of material relative to a light source and assumes that air has 100% reflectance. relative reflectance involves the measurement of reflectance of a reference plate using barium sulfate or an aluminum-coated mirror as the usual reference materials. absolute specular accessories offer an option to vary the angle of incidence and may not require a calibrated mirror as the reference material, but they are more expensive than relative specular accessories. relative specular accessories, on the other hand, require a calibrated reference mirror for baseline correction. however, due to the fixed angle of incidence, polarization may be minimized and still yield a reflectance measurement, albeit relative. relative reflectance accessories offer a small sample compartment that allows the analysis of smaller-sized samples but has limitations on the sample properties in that a flat sample is needed. sample preparation is an easy step for reflectance measurements and this method of analysis prevents the sample from being destroyed, thus allowing it to be used in other parts of an experiment. instruments have been improved over time to accommodate reflectance measurements that do undergraduate experiments on qualitative analysis of color and surface modifications 72 not require the use of additional sampling techniques. reflectance data is useful in the study of various properties of materials and the process is easy enough even for undergraduate students to carry out (fernández-garcía 2017). the effect of surface modification such as surface roughening and surface treatment using spray paint will lead to expected changes in the reflectance values. another application of reflectance values, as well as transmittance, is to extract further information such as band gap energy (bge) as shown in figure 2. also called forbidden energy gap (e g ), bge quantifies the distance between the valence band, which is occupied by electrons in the ground state, and the conduction band, where the electrons move from the valence band due to excitation. this energy is simply the separation between the lowest conduction band and the highest valence band. it represents the minimum energy required for the excitation of electrons when in the state of conduction (harouna et al. 2015). figure 2. the band gap diagram shows the position of the valence and conduction bands. depending on the distance or e g , a material can be conducting or non-conducting. (figure illustrated by mona b. antivola.) the bge dictates whether a material is a metal, semiconductor, or insulator. bge, therefore, gives the distinct properties of a material. when there is a large distance between the valence and conduction bands, a material is insulating. the main purpose of this study is to develop analytical and qualitative experiments for undergraduate science and non-science students for the characterization of solid materials in the range of ultraviolet-visible (uv-vis) light using a uv-vis spectrophotometer with relative specular reflectance accessory. it is important to develop innovative laboratory experiments using spectroscopy of solids, which is increasingly valuable in material research and development (czegan and hoover m. c. villena et al. 73 2012). several undergraduate experiments using reflectance spectroscopy were previously designed (iyere 2000; cordon and lagorio 2007; bufaroosha et al. 2020) and different equipment have been used to measure reflectance (témun et al. 2006; isik et al. 2017; suli 2017; sattar 2019; song et al. 2019). reflectance spectroscopy has been used for the characterization of color in different paper mediums (havlínová et al. 2002; harouna et al. 2015; suli et al. 2017; tourniéa et al. 2017; pérez-arantegui et al. 2018; sattar 2019), for surface analysis (lermond and rogers 1955; evans and dennison 2012), and for the determination of bge (lopez and gomez 2011; ojeda and rojas 2013; vishwakarma et al. 2015; isik and gasanly 2017) and its quantitative analysis using the tauc plot method (tauc 1974; makuła et al. 2018). methodology instrumentation the uv-visible spectrophotometer used was a shimadzu uv-1700 double-beam spectrophotometer, which was connected to a desktop computer and operated using the uvprobe software. initialization of the instrument was done through the uvprobe software, after which the specular reflectance accessory (sra) with 5° incidence angle attachment was mounted onto the sample compartment. upon mounting the accessory, baseline correction was performed and sample analysis was done. undergraduate experiments for non-science majors reflectance of offset printed commercial colored paper offset printed commercial colored papers were obtained from the brochure of reference colors from davies® paints (davies paints philippines, inc. 2019), which allowed for similar thickness and gloss among all samples. the papers were cut into 1 inch x 1 inch sizes and analyzed in the sample compartment of the sra and were analyzed for three trials. the obtained spectra were overlaid in the uvprobe software and differences in spectra shape and %reflectance values were studied. the spectra were labeled according to the color corresponding to each spectrum for easier identification; for samples with three trials, the trials were separated by different shades of the color corresponding to it. using the uvprobe software, the peak heights and inflection wavelengths of each spectrum were determined. the spectra can be overlapped to show a comparison of %reflectance. undergraduate experiments on qualitative analysis of color and surface modifications 74 reflectance of acrylic paints glass cover slips were spray-painted to completely cover their surface. the spectra of three colors of spray paints (red paint, black paint, and white paint) were analyzed. the same procedure for the analysis of the papers was done for acrylic paints. the uvprobe software was also used to determine the peak heights and inflection wavelengths of each spectrum and an overlapped graph was made to show a comparison of %r. reflectance of aluminum foil after roughening and surface treatment with paint surface modification was achieved through crumpling of the foil. qualitative analysis of optical measurements was recorded for both pristine and roughened samples. the qualitative analysis included the alteration of pristine aluminum samples through surface treatment with acrylic paint. the aluminum foil was spraypainted evenly to change the material on its surface. the surface treatment using paint on an aluminum surface is widely done in the aluminum industry for anticorrosion. undergraduate experiment for science majors uv-vis data on absorbance was used for the calculation of bge. the optical bge of zinc oxide (zno) film was obtained from its absorbance spectrum using the tauc plot method. from the absorbance spectrum, the value of 1240 was divided by the wavelength. additionally, the value of (αhv)2—where hv is the incident photonic energy and α is the absorbance coefficient—was calculated by multiplying the absorbance by 2.303 and by the energy computed earlier. these two values were then used to generate the tauc plot by plotting (αhv)2 against energy. from this plot¸ a tangent line was drawn in an area where the value of α was zero. the point of intersection between the x-axis, which was the energy, and the tangent line was the value of direct optical bge (tauc 1974; makuła et al. 2018). the zno film was synthesized and provided by dr. leon m. payawan+. using chemical bath deposition (cbd) on thin films of zinc sulfide (zns), an inorganic semiconducting sulfide was produced. the method of cbd is particularly appropriate for use by students to produce thin films of semiconducting materials in a regular laboratory session and with standard equipment. the modified experiment is based mainly on experiments developed by ibanez et al. (1991). the glassware were immersed in dilute nitric acid. half of the slide was used for depositing the semiconducting film and the experiment was done in a wellm. c. villena et al. 75 ventilated hood. a water bath in a 100 ml beaker on a magnetic-stirring hot plate was set up. the reaction mixture for zns was prepared by adding 1.0 ml of 0.1 m thioacetamide (or thiourea) solution to 1.0 ml of 0.1 m zinc sulfate (znso 4 ), followed by 1.5 ml of a 6m ammonia (nh 3 ) solution. the reaction mixture was placed in a 10 ml beaker containing a stirring bar and was placed in the bath. the temperature was then allowed to reach approximately 80–85 °c and was maintained for 30 minutes. the solution was allowed to cool to 50 °c while being stirred continuously. a glass slide was then placed halfway into the solution while the temperature was maintained as constant as possible at 50 °c. after an hour, the heating was stopped and the bath was cooled at room temperature. the deposition was left overnight, after which the slide was cautiously removed from the beaker using forceps. it was then rinsed with deionized water and dried under an n 2 atmosphere in a desiccator since zns can be oxidized into znso 4 in the presence of moist air. after the slide had dried it was placed in the accessory and the specular reflectance spectrum was obtained. results and discussion qualitative experiments on offset printed colored papers the range of available colors for offset papers is so wide that it is worth expanding color analysis using this paper medium. three batches of colored papers were prepared to test the capability of the sra in differentiating colors. the reflectance of different color hues for the first batch, visually different colored papers were analyzed. the colors selected were those labeled azure blue, ivy green, chocolate brown, cathay red, and jolly orange in the davies® paints brochure (davies paints philippines, inc. 2019). multiple trials were done to ensure the repeatability of the reflectance measurement. the reflectance spectra were recorded from 900–200 nm. figure 3 illustrates the overlay of the obtained spectra of the colored paper samples and each line is an overlay of three trials. as observed from the spectra, an easily identifiable region is present in the 750– 900 nm range. in this region, it can be observed that cathay red has the highest %reflectance, followed by ivy green, azure blue, jolly orange, and chocolate brown. chocolate brown and jolly orange can be differentiated from the other colors due to their distinct spectra. cathay red, azure blue, and ivy green converge at 800–900 nm but later diverge at the 400–700 nm area. undergraduate experiments on qualitative analysis of color and surface modifications 76 figure 3. reflectance spectra of differently-colored paper samples. the corresponding spectra are colored accordingly: cathay red (red), azure blue (blue), ivy green (green), jolly orange (orange), and chocolate brown (brown). the lower figure shows reference colors for azure blue, cathay red, ivy green, jolly orange, and chocolate brown from the brochure provided by davies® paints. the characteristic peak heights of cathay red, azure blue, ivy green, jolly orange, and chocolate brown are shown in table 1. the reference colors were obtained from the brochure provided by davies® paints (davies paints philippines, inc. 2019) for easier color identification. table 1. characteristic peak heights of cathay red, azure blue, ivy green, jolly orange, and chocolate brown paper sample peak heights, nm cathay red 499, 630, 827 azure blue 451, 655, 818 ivy green 509, 681, 752, 826 jolly orange 508, 610, 830 chocolate brown 827 m. c. villena et al. 77 the reflectance of yellow and neutral color shades two different colors and their different tones were analyzed to show the characterization that may be obtained from the analysis of reflectance spectra. the main colors were yellow and neutral. variations of yellow are sunny day, calamansi, and yellow breeze, while neutral colors are ivory and beige. the spectra obtained from the analysis of these colored papers are shown in figure 4. figure 4. reflectance spectra of different shades of yellow and neutral colors. from top to bottom, the spectra are colored according to the corresponding colors: yellow breeze, sunny day, calamansi, ivory, heavy beige. the bottom figure shows the reference colors for yellow breeze, sunny day, calamansi, ivory, and heavy beige from the brochure provided by davies®paints. it is essential to identify the differences between two tones of the same color, and it can be observed that the tones of the same color can be differentiated at 522 and 835 nm with spectra having the same shape. among the yellows, yellow breeze can be differentiated from sunny day and, while only a small difference, calamansi exhibits a lower %r compared to sunny day as well. the neutrals, ivory and heavy beige, also show a distinction, albeit a smaller difference than the former two. it should also be noted that there is a significant difference between the “parent” undergraduate experiments on qualitative analysis of color and surface modifications 78 colors; the yellows can be grouped as all shades exhibit a higher %r reading, while the neutrals exhibit lower %r. reference colors may vary from the real-life samples due to differences in how computers display colors. however, the differences obtained from the spectra mirror what tonal differences the human eye can detect. the reflectance of shades of pink similar shades of pink were analyzed to test the effectiveness of the instrument in differentiating colors. four variations were selected for this section, namely ballerina, carnival pink, lovely blush, and pretty in pink, all of which fall under the “pink” color. the %r spectra obtained from analyzing these samples are displayed in figure 5. due to the difficulty posed by color-coding using similar shades of pink, the spectra are color-coded using distinctly different colors. figure 5. reflectance spectra of different shades of pink. from top to bottom, the spectra are colored according to the corresponding colors: lovely bush, ballerina, carnival pink and pretty in pink. the bottom figure shows the reference colors for ballerina, carnival pink, lovely blush, and pretty in pink from the brochure provided by davies® paints. m. c. villena et al. 79 generally, all spectra follow the same spectrum shape as the analyses done above. lovely blush exhibits a drastic change in %r as it goes from 900 nm to about 600 nm. it is important to note the differences in %r exhibited among the samples, as they are key to distinguishing different colors from one another. the colors are generally close to each other and would be classified by the average person as “pink”, but according to the spectra obtained, there is a significant difference that may be used to distinguish these colors from one another. ballerina and carnival pink are colors that an average person may have difficulty differentiating due to the similar shade they exhibit, but the spectra may be able to differentiate between them. it can also be seen that throughout the three sets of colored paper samples, all samples obtained %r that did not exceed 2% reflectance. this may be indicative of how the sra may be able to differentiate color between samples of similar composition, but may have difficulty analyzing samples that do not exhibit a significant amount of gloss. the reflectance of acrylic spray paints to test the ability of the sra in differentiating red paint, black paint, and white paint, the spectra of the three colors were analyzed (figure 6). figure 6. reflectance spectra of acrylic spray paints. from top to bottom, the spectra are colored corresponding to the color of its sample: white, red, and black. as observed in the spectra, the white paint exhibited the highest %r, followed by red and black. this matches the theory that white light is readily reflected, and that red and black absorb the light and thus reflect less light off its surface. in the 400–900 undergraduate experiments on qualitative analysis of color and surface modifications 80 nm range, which is the area of concern for analysis, a significant difference between the white paint spectrum and the red paint spectrum can be observed. it should be noted that the %reflectance obtained from these paint samples is significantly higher than those obtained from the two previous sections. this may be attributed to the gloss provided by the glass slide, as well as the gloss of the spray paint. as the length of application was approximately one second, it can be inferred that the glass slide still had a significant influence on the analysis of the samples. qualitative experiments on reflectance on modified surfaces the experimental results on surface roughening by scanning the %r of a sample in its pristine form versus one with surface modification was done on the surface of aluminum. another kind of modification was achieved by adding a layer of colored paint on aluminum foil, plastic, and mirror surfaces. surface modification by surface roughening surface roughening was done on aluminum foil by crumpling the sample, thereby producing folds, dents, and possible scratches. it can be seen in table 2 that expected changes in the measurements were brought about by changes on the surface of the material. table 2. readily available samples tested for the effect of surface modification on %r sample description initial reading reading after surface modification the front, shiny surface of a smooth piece of aluminum foil 19.434 %r 1.892 %r the back of a smooth piece of aluminum foil 11.877 %r 1.611 %r the %reflectance scans were recorded from 900–200 nm on the spectrum mode. the spectrum of wavelength versus %r for the mentioned samples is in figure 7 below. the difference in reflectance can be attributed to the more reflective surface of the front side of the aluminum foil. m. c. villena et al. 81 figure 7. an overlay of the %r spectra was obtained for pristine (blue) versus crumpled (red) aluminum foil. the reflectance of surfaces treated with acrylic paints figures 8–11 show the %r spectra obtained for both the pristine form and spraypainted surfaces of aluminum foil, plastic, and a mirror. the altered surfaces of samples treated with paint showed a decrease in %r. this was expected of a homogeneous sample because its reflecting surface was covered with a nonreflecting material. reflectance will depend on the new outer surface and its thickness. in this case, it can be said that the paint was not uv-active because of the decrease in %r. furthermore, it can be noted that this method using a uv-vis spectrophotometer with sra can qualitatively show the effect of surface changes on the optical properties of homogeneous samples. figure 8. the %r spectra were obtained for pristine (blue) versus stained with yellow paint (red) aluminum foil. the left pictures show the individual spectrum while the right is an undergraduate experiments on qualitative analysis of color and surface modifications 82 overlay of the two spectra. figure 9. the %r spectra were obtained for pristine (blue) versus stained with red paint (magenta) aluminum foil. the left pictures show the individual spectrum while the right is an overlay of the two spectra. figure 10. the %r spectra were obtained for pristine (green) versus stained with red paint (violet) thick plastic. the left pictures show the individual spectrum while the right is an overlay of the two spectra. m. c. villena et al. 83 figure 11. the %r spectra were obtained for a pristine (light blue green) versus stained with red paint (black) mirror. the left pictures show the individual spectrum while the right is an overlay of the two spectra. quantitative analysis for the calculation of band gap energy using tauc plot as an application, bge was calculated from the absorbance data generated using uv-vis with sra. the use of tauc plot as the method for solving bge limited the samples to semiconductors or nanomaterials such as zno doped polypyrrole nanocomposite (olada et al. 2018). figure 12 shows one of the absorbance plots of zno and, from this data, the tauc plot was generated as shown in figure 13. figure 12. the absorbance plot of the zno sample. undergraduate experiments on qualitative analysis of color and surface modifications 84 figure 13. the tauc plot with energy versus (ahv)2 for the zno film sample. the tauc plot, which is used to calculate the bge of films and nanomaterials, was generated by plotting (αhv)2 against energy. for the zno film samples, the average direct bge for three trials was calculated to be 3.80 + 0.04 ev with a 15.15 % error from its literature value of 3.37 ev (olada and sajedeh 2018). knowledge of bge has an industrial significance. as mentioned, the bge dictates whether a material is a metal, semiconductor, or insulator. such knowledge is used in industries, particularly those manufacturing electronic devices. the different synthetic methods of preparation may alter the band gap value of a zno semiconductor because structural faults may arise and alter the electronic structure (tahir et al. 2021). zno is considered an insulator but in photovoltaic applications it is considered a semiconductor with bge of 3.4 ev. studies on zno have shown that it is an active material for photodegradation, and with a fast reintegration of electrons and holes, which decreases its photoefficiency. in another activity, zns was chemically deposited on glass slides and its band gap was determined using an sra (5° incidence angle) attachment. the average bge for three trials was determined to be 3.0 ev ± 0 with an 8.82% error from its literature value of 3.4 ev (ibanez et al. 1991). the experiment introduced zns as one of the most important semiconductors and it may in principle support both nand p-doping. also, zns is a low-cost, inactive compound with convenient mechanical properties such as good fracture strength and hardness (d’amico et al. 2017). figure 14 shows the tauc plot of the deposited zns. m. c. villena et al. 85 figure 14. the tauc plot with energy versus (ahv)2 for the zns film sample deposited on a glass slide. conclusions and recommendations we have devised laboratory experiments for undergraduate science and nonscience majors to identify and quantify the properties of solids by measuring their relative specular reflectance using a uv-vis spectrophotometer with a relative sra and observing a specific interaction of light with solid surfaces. upon analysis of three different materials using the sra, it can be concluded that color is a property that may be qualitatively analyzed. the %reflectance range may vary depending on the parent color and material and may be key in differentiating colors for a specific material where color is the sole variable property. generally, distinct colors have different ranges of %reflectance, and varying color hues result in different %reflectance readings within the given range. it can also be concluded that an sra is an effective tool for observing surface modifications in solid samples. roughening causes folds, dents, and possible scratched surfaces, which showed a decrease in specular reflectance. surface alteration by adding paint was shown to be one method to decrease reflectance and alter reflectance properties. for the quantitative analysis, the tauc plot method proves to be an effective tool for the calculation of bge of thin films and nanomaterials. it is recommended to study using relative specular reflectance on materials to determine the band gap and compare with results using more expensive and advanced accessories such undergraduate experiments on qualitative analysis of color and surface modifications 86 the integrating sphere, which is used in the american society for testing and materials (astm) methods. qualitative analysis of colors using reflectance spectra was shown to differentiate color hues in offset printed paper and a quantitative analysis of the colors of materials using relative specular reflectance may be carried out. conversion of reflectance spectra to cielab color values and, extensively, to rgb color values has been done in previous studies using colorimeters. specular reflectance obtained using the sra may provide a cost-friendly alternative as colorimeters are focused mainly on color-related analyses, while sra provides different properties to be analyzed. while this study focused on the correlation of color to specular reflectance, other studies may also be carried out to test the capability of the sra, especially for industries that widely use reflectance data. currently, specular reflectance is used widely in the study of thin films. further studies may be done on nanomaterials for nanotoxicology studies and thin films for chemical sensor development. new methods of preparing the sample, such as pelletization of powdered materials, are also recommended for further studies. acknowledgements the authors would like to thank the office of the vice-chancellor for research and development (ovcrd), university of the philippines diliman (up diliman) for the full funding grant for the purchase of the accessory. we are grateful to the analytical services laboratory of the institute of chemistry, up diliman for the use of the uv-visible spectrophotometer. we would also like to acknowledge dr. leon m. payawan+ and his research assistant, mr. yasmin edañol, from the institute of chemistry, up diliman for the sample they had provided. references bufaroosha m, al neyadi s, alzamly a, toutounji m, saleh n, abuhattab by, al-hemyari a, alyammahi a, alzahmi s, altubji m, et al. 2020. undergraduate experiment using absorption and diffuse reflectance spectroscopies: theoretical and experimental bandgap calculations of porphyrins and metalloporphyrins. world journal of chemical education. 8(2):87–91. http://pubs.sciepub.com/wjce/8/2/4. cordon gb, lagorio mg. 2007. absorption and scattering coefficients: a biophysical chemistry experiment using reflectance. j chem educ. 84(7):1167–1170. czegan dac, hoover dk. 2012. uv−visible spectrometers: versatile instruments across the chemistry curriculum. j chem educ. 89:304−309. m. c. villena et al. 87 davies paints philippines, inc. 2019. davies® sun&rain® 100% acrylic waterproofing paint [brochure]. 2019 oct, issue no. 10. https://www.daviespaints.com.ph/wp-content/uploads/ davies-sun-_-rain-brochure.pdf evans a, dennison j. 2012. the effects of surface modification on spacecraft charging parameters. ieee transactions on plasma science. 40(2):305–310. fernández-garcía a, sutter f, martínez-arcos l, sansom c, wolfertstetter f, delord c. 2017. equipment and methods for measuring reflectance of concentrating solar reflector materials. solar energy materials and solar cells. 167:28–52. hanania j, stenhouse k, donev j. band gap. 2015 sep 10. calgary (ab): the university of calgary; [updated 2015 nov 28; accessed 2020 feb 2]. https://energyeducation.ca/ encyclopedia/band_gap harouna as,  bringier b,  khoudeir m. 2015. print spectral reflectance estimation using trichromatic camera. in meriaudeau f, aubreton o, editors. proceedings of the twelfth international conference on quality control by artificial vision 2015. 2015 june 3–5; le creusot, france. society of photo-optical instrumentation engineers (spie). https://doi. org/10.1117/12.2182751 havlínová b, babiaková d, brezová v, ďurovič m, novotná m, belányia f. 2002. the stability of offset inks on paper upon ageing, dyes and pigments. 54:173–188. ibanez j, solorza o, gomez-del-campo e. 1991. preparation of semiconducting materials in the laboratory production of cds thin films and estimation of their band gap energy. j chem educ. 68(10):872–875. isik m, gasanly n. 2017. composition-tuned bandgap energy and refractive index in gas x se 1-x layered mixed crystals. materials chemistry and physics. 190:74–78. https://doi.org/10.1016/j. matchemphys.2016.12.059 iyere pa. 2000. the development of innovative laboratory experiments with uv–visible spectrophotometer. j chem educ. 77(2):153–154. lermond c, rogers l. 1955. experiment on reflectance measurements. journal of chemical education. 32(2):92. lopez r, gomez r. 2012. band-gap energy estimation from diffuse reflectance measurements on sol-gel and commercial tio 2 : a comparative study. journal of sol-gel science and technology. 61(1):1–7. makuła p, pacia m, macyk w. 2018. how to correctly determine the band gap energy of modified semiconductor photocatalysts based on uv−vis spectra. the journal of physical chemistry letters. 9(23):6814–6817. doi: 10.1021/acs.jpclett.8b028 undergraduate experiments on qualitative analysis of color and surface modifications 88 ojeda c, rojas f. 2013. recent applications in derivative ultraviolet/visible absorption spectrophotometry: 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yoo de, kim yj, yoo yj, lee dw, siva v, kang is, song ym. 2019. reflective color filter with precise control of the color coordinate achieved by stacking silicon nanowire arrays onto ultrathin optical coatings. sci rep. 9(1):3350. doi: 10.1038/s41598-019-40001-1. suli w, liu b, xin s, zhang s. 2017. structural color patterns on paper fabricated by inkjet printer and their application in anticounterfeiting. j phys chem lett. 8:2835−2841. tahir n, zahida m, bhattia i , manshab a, naqvib s, hussainc, t. 2021. chapter 7: metal oxidebased ternary nanocomposites for wastewater treatment. aquananotechnology. https://doi. org/10.1016/b978-0-12-821141-0.00022-7 tauc j. 1974. optical properties of amorphous semiconductors. in: tauc j, editor. amorphous and liquid semiconductors. boston(ma):springer. https://doi.org/10.1007/978-1-46158705-7_4. témun a, mattsson l, heikkilä i. 2006. localizing micro-defects on rough metal surfaces. 4m 2006 second international conference on multi-material micro manufacture. 169–172. doi:10.1016/b978-008045263-0/50039-8 tourniéa a, carréb p, christine a, boustc c, lavédrinea b. 2017. identification of chromogenic colour photographic print brand by fiber optical reflectance spectroscopy and statistical analysis. journal of cultural heritage. 26:28–35. m. c. villena et al. 89 vishwakarma j, sabu b, bhotkar k, mehta s, muthurajan h. 2015. surface and band gap energy of zno nanoparticles to fine tune the optical properties. international journal of chemical and physical sciences. 5:28-35. ______ rheo b. lamorena-lim is currently a full professor at the institute of chemistry, university of the philippines diliman. she obtained her ph.d. degree in civil and environmental engineering in 2011 from korea advanced institute of science and technology, daejeon, republic of korea. research main interests include alternative analytical methods on metal determination in environmental samples (such as soil and sediment samples), mineralization techniques in co 2 sequestration, and measurements of chemical composition of ambient particulate matter. she applies her knowledge in analytical chemistry and physical chemistry for understanding the phenomena in the environment to provide solutions or remediation technologies on environmental problems. marte c. villena <mcvillena1@up.edu.ph> is a university researcher at the analytical services laboratory of the institute of chemistry, university of the philippines-diliman. he obtained his bachelor’s and master’s degree in chemistry at the same university. his current interests include analytical methods for food and environmental analysis. mona antivola earned her bachelor’s degree in chemistry from the university of the philippines diliman. she worked on her undergraduate thesis and did her on-the-job training in the analytical services laboratory of the institute of chemistry. janelle stephanie torrefiel finished her undergraduate studies in university of the philippines diliman, earning a degree in b.s. chemistry. now a laboratory analyst by profession, she believes that there is still a lot that may be learned about the world. tetrabromobisphenol a in indoor dust 5science diliman (july-december 2011) 23:2, 5-16 tetrabromobisphenol a in indoor dust from houses and internet cafes maria pythias b. espino* and jessica n. leon institute of chemistry and natural science research institute, college of science university of the philippines, diliman, quezon city, 1101 philippines telefax: +63-2-9205427 *corresponding author: mbespino@up.edu.ph abstract tetrabromobisphenol a or tbbpa is a widely used brominated flame retardant in modern materials such as electronic products, plastics and building materials. tbbpa can leach out of flame retardant-treated products during production, use, reuse, and final disposal of these consumer products. it has thus become a contemporary environmental contaminant. this study reports the levels of tbbpa in indoor dust from houses (n=4) and internet cafes (n=5) in angono and quezon city, in the philippines. tbbpa in indoor dust was analyzed by ultrasonication-assisted hexane extraction and hplc-uv determination. the instrument and method detection limits were 0.004 ng ul-1 and 1275 ng g-1, respectively. the standard calibration solutions ranged from 0.03 to 0.30 ng ul-1 (r2=0.9956). the extraction recovery was 78% (n=3). tbbpa was found in six of the nine indoor dust samples studied where the concentrations ranged from not detected to 4916 ng g-1. the occurrence and levels of tbbpa in indoor dusts are significant and suggest the need for further investigations especially in other microenvironments where people may be exposed to this contaminant. key words: tetrabromobisphenol a, indoor dust, ultrasonication, hplc-uv introduction tetrabromobisphenol a (tbbpa) belongs to a group of chemicals known as brominated flame retardants. the structure of tbbpa (4,4’-isopropylidenebis(2,6dibromophenol); cas no. 79-94-7) is shown in figure 1. brominated flame retardants are commonly found in modern-day materials such as electronic and electrical equipment, plastic products, upholstery and construction materials. these brominated flame retardant chemicals, including tbbpa as well as polybrominated biphenyls (pbbs), polybrominated diphenyl ethers (pbdes) and hexabromocyclodecanes (hbcds), are regarded as very efficient in reducing the flammability of modern consumer products. the world market for all types of flame retardants was reportedly over 2 billion us dollars in 2000 and was ch3 ch3 br b r b r b r ohoh figure 1. chemical structure of tbbpa expected to increase each year (tullo, 2000; morf et al., 2005; shaw et al., 2010). the commercial production of pbbs has ceased in the united states in the 1970s, while the higher molecular weight congeners were still in use until 1985 in germany and espino, m.p.b. and leon, j.n. 6 science diliman (july-december 2011) 23:2, 5-16 until 2000 in france (alaee et al., 2003). the formulations of pbdes particularly for pentaand octabromodiphenyl ethers were used in the united states until 2004, while decabromodiphenyl ether is still available until the scheduled phase-out in 2013 (us epa, 2011). the ban and restrictions imposed on pbde formulations consequently increased the demand for tbbpa and hbcds. for tbbpa, the estimates of use in 2001 were 89,400 tons y-1 in asia, 18,000 tons y-1 in the americas, and 11,600 tons y-1 in europe (covaci et al., 2009). currently, it is the widely used flame retardant in the market (bsef fact sheet, 2010). tbbpa is a reactive flame retardant such that it is covalently bonded to the polymeric material; it is also an additive flame retardant similar to pbdes and hbcds that are dissolved in the polymer. tbbpa is extensively used in electronic and electrical equipment including computers, televisions, vacuum cleaners, washing machines, office equipment and other high technology equipment (bsef fact sheet, 2010; watanabe and sakai, 2003). as a reactive flame retardant, it is commonly found in printed circuit boards. in small waste electronic and electrical equipment dismantled in australian treatment plants, for example, the average amount of tbbpa in printed circuit boards approximates those of tetra-, pentaand decabromodiphenyl ethers (salhofer & tesar, 2011). as an additive flame retardant, it is found in acrylonitrile-butadiene-styrene plastics used in casings for televisions or electronic devices. tbbpa, for instance, was among the flame retardants detected in rear and front covers of lcd tvs as well as in chassis, panels, adaptors, cooling fans or speakers of laptop computers in the japanese market in 2008 (kajiwara et al., 2011). in its foremost application as a reactive flame retardant, tbbpa is perceived to be stable and not easily released from the final product. despite having no known natural source, however, it has been detected in the different environmental compartments. tbbpa may leak into the environment throughout the lifecycles of flame retardant-protected consumer products. tbbpa may enter the environment during its production, during use and reuse of tbbpa-treated products, and when these products reach their endof-life and final disposal. it is not surprising that tbbpa and the other brominated flame retardants are now among the emerging global contaminants. these environmental contaminants are receiving similar attention, particularly in reviews and assessments, as the classic twelve persistent organic pollutants or pops (alaee et al., 2003; kemmlein et al., 2003; covaci et al., 2007; de wit et al., 2010). tbbpa has a melting point of 181oc, log k ow of 5.903, reported water solubilities of <0.5 ug/l, 0.001 mg/l and <0.08 mg/l, and vapor pressure of <1.19x10-5 pa at 20oc (us epa, 2001). shaw et al. (2010) summarized studies describing the effects and properties of tbbpa as a cytotoxicant, immunotoxicant, thyroid hormone agonist, or endocrine disruptor, among other effects. the occurrence and persistence of tbbpa in the environment have been documented. tbbpa has been detected in air, dust, soils, water, sediments, biota, mammalian tissues, and human serum and milk (covaci et al., 2009; jakobsson et al., 2002; johnson-restrepo et al., 2008; watanabe and sakai, 2003). like the pops compounds, tbbpa is now found in the arctic region (de wit et al., 2006; de wit et al., 2010), revealing its capability to undergo long-range transport. and analogous to the traditional pops, tbbpa is ana lyzed by standard chromatographic techniques such as high pressure liquid chromatography (hplc) with ultraviolet (uv) or mass spectrometri c detection and gas chromatography with diazomethane, n-methyltrimethylsilyltrifluo roacetamide or methyl chloroformate derivatization followed by mass spectrometry. the extraction of tbbpa from the different environmental matrices may involve methods such as soxhlet extraction, sonication, microwaveasssisted extraction, accelerated solvent extraction, supercritical fluid extraction, pressurized liquid extraction, liquid-liquid extraction, or solid-phase extraction. the choice of extraction method depends on the sample matrix. clean-up of the extracts may involve acid treatment, the use of alumina, silica or florisil columns, or in some instances, a solid-phase extraction step to remove extraneous interferents before instrumental analysis (covaci et al., 2003; covaci et al., 2009). the various techniques in the analysis of tbbpa and related compounds are described in review papers of van leeuven and de boer (2008) and covaci et al. (2003; 2007; 2009). tetrabromobisphenol a in indoor dust 7science diliman (july-december 2011) 23:2, 5-16 one of the causes of tbbpa contamination in the environment is the improper disposal of waste electronic and electrical equipment or electronic waste. in the philippines, there is limited information available on the disposal of these wastes (cardenas et al., 2006). filipinos tend to store their electronic wastes for a long period of time before eventually giving them away or disposing of them, in many cases, into the ordinary garbage bin (espino, 2008). this practice is common, perhaps due to the lack of guidelines on proper disposal or the expectation that a portion of the equipment’s cost is recovered when a buyer is found. if left unchecked, this practice may pose threat to human and ecological health. there is potential for exposure to tbbpa in indoor environments where tbbpatreated consumer products abound; there is also possible exposure to tbbpa in these places where filipinos keep their outdated or broken electronic products for extended periods of time. although tbbpa has been reported to be present in indoor air and dust elsewhere, mostly in developed countries, studies on tbbpa con tamination in indoor environments in asia are still scarce. in this paper, we present the first account on the occurrence of tbbpa in indoor places in the philippines, specifically inside houses and internet cafes, and the latent exposure of filipinos to this emerging environmental contaminant. experimental standards and solvents. the standards 3,5,3’,5’tetrabromobisphenol a (tbbpa, 98.8%) and pentachloronitrobenzene (pcnb, 96.4%, 5 mg ml-1 in methanol) were purchased from sigma-aldrich (st. louis, mo, usa). stock solutions of tbbpa and pcnb were prepared in methanol at 10 ng ul-1 each. these were diluted to appropriate concentrations in the same solvent using volumetric equipment to make the calibration and spiking solutions. the hplc-grade methanol (100.0%), acetonitrile (99.9 %), water (2 ppm maximum residues), and n-hexane (99.8% as c6-isomers) were pur chased from j t baker (philipsburg, nj, usa). the solvents used for figure 2. sampling sites in quezon city and in angono, rizal [maps adapted and modified from http://mapsof.net/ uploads/static-maps/metro_manila_politicalmap.png and http://www.mapcentral.ph/] espino, m.p.b. and leon, j.n. 8 preparing the mobile phase were passed through 0.45 um x 47 mm nylon membrane filters (whatman international ltd., maidstone, england) and degassed under vacuum in a buchner flask set-up. sampling, sample preparation and extraction of tbbpa in indoor dust. indoor dust samples were collected from filters of air conditioning units in five internet cafes near the university of the philippines diliman campus in quezon city and in four houses in angono, rizal during the period of february to september 2010. the choice of sampling sites was mainly based on easy access and permission granted by owners to enter their houses and internet cafes. figure 2 shows the locations of the sampling sites. the dust samples were carefully loosened and scraped from the filters using a 3.5 cm x 1 cm wire brush with a 13 cm length handle (home solutions, philippines). a new brush was used for each sample. the dust samples were placed in 20-ml vials (rpi corporation, mt. prospect, il, usa), covered with aluminum foil, and stored in the freezer at 0 to 4oc until analysis. the dust samples were homogenized by passing through a 500 um mesh sieve (w.s. tyler, mentor, oh, usa). triplicates of 0.1 g dust samples (duplicates for limited sample masses collected) were weighed accurately in 4-ml vials and extracted with 2 ml hexane using a power sonic 410 ultrasonicator (hwashin technology, gyeonggi-do, korea). the ultrasonicator has a 10-l bath capacity, 40 khz ultrasonic frequency and power consumption of 600 w. the ultrasonication-assisted hexane extraction was carried out for 15 min at room temperature. the hexane extract was filtered through a 0.2 um x 13 mm nylon membrane filter (whatman international ltd., maidstone, england). a 500 ul of the hexane extract was placed in a microvial, evaporated to almost dryness using a water bath at temperature below 80oc, and reconstituted in 500 ul methanol. added to this final extract was a 50 ng pcnb internal standard. pcnb was chosen as internal standard because it gave a high uv activity at the optimum wavelength of tbbpa and its use resulted in an acceptable analysis time with tbbpa. the dust samples used for recovery experiments were prepared by exhaustive extraction using 100% hexane followed by 100% methanol. these were air dried at room temperature, fortified with tbbpa, equilibrated and air dried overnight, and taken through the entire ultrasonication-assisted hexane extraction procedure. hplc-uv analysis . a shimadzu uv1700 spectrophotometer (kyoto, japan) was used to measure the wavelengths at which the tbbpa and pcnb registered maxi mum absorbances.the optimum wavelength suitable for the simultaneous determination of these compounds was 207 nm which was then used in the hplc-uv analysis of tbbpa in the indoor dust extracts. the system used for the analysis of tbbpa was a lc-10as/spd-10av chromatograph equipped with a uv detector (shimadzu, kyoto, japan). the analytical column was a thermohypersil c18 column 250 mm x 5 um x 4.6 mm i.d. (thermo fisher scientific, ma, usa). attached to this column was a phenomenex c18 guard column 4 mm x 3.0 mm i.d. (torrance, ca, usa). the following are the optimum hplc-uv conditions used in the analysis: the mobile phase was 70:30 acetonitrile:water; the flow rate was 1 ml min-1 which resulted in a total pressure of 77 kgf-cm2; the oven temperature was maintained at 30ºc; and the uv detector was set at 207 nm. a 25 ul of the standard solution or sample extract was manually injected into the 20-ul sample loop. data acquisition and processing were done using a class lc-10 shimadzu analysis system and software version 1.64. figur e 3 shows a hplc-uv chromatogram of tbbpa and the internal standard. method performance and validation. a solution of 0.03 ng ul-1 tbbpa in methanol was used to evaluate the reproducibility of the optimized hplc-uv detection method. the calibration range for the analysis was established using standard solutions in linear response intervals of the optimized detection method. the statistical detection limit of the instrument was obtained using the equation dl = student’s t x sd (equation 1) science diliman (july-december 2011) 23:2, 5-16 tetrabromobisphenol a in indoor dust 9 where the student’s t value is 3.1438 with n-1 degrees of freedom at 99% confidence, and sd is the standard deviation of the measured tbbpa concentrations (n=7) in the spike level of 0.03 ng ul-1 in methanol. the statistical method detection limit (mdl) was similarly obtained using equation 1 but based on the standard deviation of seven replicate measurements of 2000 ng g-1 tbbpa spiked in indoor dust. the extraction recovery of tbbpa in indoor dust was evaluated from measurements of spiked tbbpa in previously cleaned indoor dust. the indoor dust was spiked with 2000 ng g-1 tbbpa and allowed to stand overnight. three replicates of 0.1 g of this spiked dust were extracted with hexane by ultrasonication and the tbbpa was mea sured by hplc-uv determination. the recovery was calculated using the equation % recovery = [(tbbpa ex tr ac t /pcnb ex tr ac t ) / (tbbpa standard /pcnb standard )] x 100 (equation 2) where tbbpa extract is the area of tbbpa in the extract of indoor dust at 2000 ng g -1 spike concentration; pcnb extract is the area of the internal standard in the extract at 0.1 ng ul-1; tbbpa standard is tha area of tbbpa in the standard solution at 0.1 ng ul-1 concentration corresponding to the spiked tbbpa concentration in the indoor dust; and pcnb standard is the area of the internal standard in the standard solution at 0.1 ng ul-1 concentration. data analyses as well as calculations of concentrations, standard deviations and recovery were performed using excel microsoft office 2003. determination of tbbpa in indoor dust. the indoor dust samples were analyzed using the optimized ultrasonication-assisted hexane extraction and hplcuv determination. the tbbpa in the extracts was determined and quantified using calibration solutions of 0, 0.03, 0.05, 0.08, 0.1, 0.2 and 0.3 ng ul-1 tbbpa in methanol; the extracts and calibration solutions also contained 0.1 ng ul-1 pcnb. the tbbpa configure 3. hplc-uv chromatogram of tbbpa and pcnb measured at 207 nm. the mobile phase was 70:30 acetonitrile:water. science diliman (july-december 2011) 23:2, 5-16 espino, m.p.b. and leon, j.n. 10 centrations in the extracts were measured by linear regression analysis a nd the corresponding concentrations in the 0.1 g indoor dust samples were then calculated from these measurements. solvent and procedural blanks (pooled indoor dust cleaned by extraction using 100% hexane followed by 100% methanol) were likewise analyzed together with the indoor dust samples to check for interferences or background peaks from laboratory conditions and equipment. in these blanks, no peaks were detected at the retention times of the target analytes. results and discussion analytical determination of tbbpa in indoor dust the analysis of tbbpa by high pressure liquid chromatography and uv detection at 207 nm wavelength was optimized with 70:30 acetonitrilewater as mobile phase. after testing various twosolvent combinations of acetonitrile, methanol and water, the 70:30 acetonitrile-water eluent resulted in the best chromatographic conditions, i.e., having the optimal area response and shortest analysis time. this hplc-uv determination is very reproducible with less than 5% and 2% relative standard deviations for peak areas and retention times, respectively (table 1). the analytical parameters are summarized in table 2. the six-level calibration curves in the linear response range of 0.03 to 0.3 ng ul-1 have regression r2 greater than 0.99 for both area of tbbpa and area response of tbbpa against pcnb. the determination can thus be carried out with or without pcnb, i.e., by internal standard calibration or by external calibration, respectively. however, it should be noted that in the analysis of real samples and especially with manual injection, internal standard addition is preferred. the instrument detection limit is 0.004 ng ul-1 which can allow for trace analysis of tbbpa in an extract dissolved in methanol. this instrument detection limit is lower than the 1.1 ng ul-1 reported by pöhlein et al. (2005) in a similar hplc-uv determination of tbbpa but using aqueous buffered methanol as chromatographic eluent and 205 nm wavelength for detection. for limited amounts of samples such as indoor dust, an ultrasonication extraction procedure was deemed ideal because it can support small-volume glassware to contain small-size samples. in addition, ultrasonication extraction is less solvent-requiring, less time-consuming, and less costly. different solvents were tested and hexane was found to be the suitable extraction solvent for tbbpa in indoor dust. methanol and ethyl acetate were observed to extract extraneous compounds along with tbbpa from the indoor dust replicates (spike level =0.03 ng ul-1) tbbpa retention time, min pcnb retention time, min retention time ratio, tbbpa/ pcnb area of tbbpa area of pcnb area ratio, tbbpa/ pcnb 1 5.95 9.83 0.605 3524 19769 0.1783 2 5.90 9.81 0.601 3816 22251 0.1715 3 5.93 9.86 0.601 3565 20047 0.1778 4 6.18 10.27 0.602 3712 19516 0.1902 5 6.09 10.12 0.602 3414 20197 0.1690 6 6.07 10.08 0.602 3515 20460 0.1718 7 6.06 10.06 0.602 3383 20090 0.1684 mean 6.03 10.00 0.602 3561 20332 0.1752 sd 0.10 0.18 0.002 155 898 0.0076 % rsd 1.68 1.76 0.283 4.36 4.42 4.36 table 1. repeatability of tbbpa determination by hplc-uv detection science diliman (july-december 2011) 23:2, 5-16 tetrabromobisphenol a in indoor dust 11 samples. the coextractives imparted color in the extract thus requiring further clean-up. this was not the case with hexane where the extract was colorless. also, hexane evaporates easily when reconstituting the extract in methanol prior to instrumental analysis. several flame retardants measurements in indoor dust reported in literature do not provide recoveries of the extraction, likely because of the lack of reference materials. since a certified reference material for tbbpa in indoor dust was also not available in this study, the accuracy of the ultrasonication-assisted hexane extraction was assessed by replicate analyses of blank indoor dust spiked with 2000 ng g-1 tbbpa. this provided an estimation of the extraction performance and recovery which was defined as the percent ratio of the measured amount divided by the spiked or expected amount. an acceptable recovery of 78% was obtained which demonstrates the efficiency of the ultrasonication-assisted hexane extraction in tbbpa removal from the sample matrix. the method detection limit is 1275 ng g-1 which is relatively high for use in environmental monitoring of trace organic pollutants. this is expected because preconcentration was not done during the extract preparation and before instrumental analysis. nonetheless, this method detection limit is acceptable for the present purpose wherein the method was used in indoor dust samples anticipated to be highly contaminated with tbbpa. the method can thus be useful in the analysis of indoor dust from enclosed environments where tbbpa-laden consumer products are present. for tbbpa determination in less contaminated samples, such as in outdoor dust where tbbpa is more dispersed, a sample size greater than 0.1 g may be used or a twoto four-fold preconcentration may be carried out to significantly lower the method detection limit. overall, the analysis of tbbpa in indoor dust is fast, requires minimal solvent or sample preparation, and does not need rigorous clean-up prior to hplc-uv determination. concentrations and possible sources of tbbpa in the indoor dust samples dust and airborne particulates provide sinks for organic pollutants. tbbpa is also expected to adhere in this matrix. tbbpa contamination in dust collected in houses, pubs, offices, schools or cars has been reported (abb et al., 2011; abdallah, et al., 2008; d’hollander et al., 2010; geens et al., 2009; harrad et al., 2010; harrad et al., 2011; takigami et al., 2009). hence, these microenvironments are likely places for exposure to tbbpa. the tbbpa concentrations in parameters method performance tbbpa calibration range 0. 03 – 0.30 ng ul-1 pcnb internal standard spike level 0.10 ng ul-1 calibration equation using area ratio of tbbpa/pcnb y = 6.0714x – 0.0185 (r2 = 0 .9956) calibration equation using tbbpa area only y = 12.495x – 0.086 (r2 = 0 .9980) rsd of measured tbbpa concentration at spike level of 2000 ng g-1 3.96% (n=7) extraction recovery 78% (n=3; sd=24) instrument detection limit 0.004 ng ul-1 method detection limit 1275 ng g-1 table 2. analytical parameters for tbbpa determination and quantitation in indoor dust science diliman (july-december 2011) 23:2, 5-16 espino, m.p.b. and leon, j.n. 12 indoor dust samples from houses and internet cafes in the present study are summarized in table 3. tbbpa was detected in 67% of these samples. to our knowledge, this shows for the first time tbbpa contamination in indoor dust in the philippines particularly in internet cafes where young people spend considerable time. higher concentrations were found in internet cafe indoor dusts than in house indoor dusts. in the internet cafe dust samples where tbbpa was detected, the average concentration was 2934 ng g-1. of the dust samples taken from the houses, only one had tbbpa concentration above the method detection limit. since tbbpa determination in internet cafe indoor dusts has never been done before, only the tbbpa concentrations in house dusts were compared to those reported in other countries (table 4). the 1348 ng g-1 concentration in one of the house dust samples in this study is notably higher than those reported elsewhere. this concentration may be comparable to some samples collected in belgium (geens et al., 2009). the comparability, however, may be limited since different sampling techniques and analytical methods were followed. in addition, there are studies that involve only a small sample size such that the measurements of tbbpa contamination may not provide a general assessment of human exposure to tbbpa in houses. despite very few investigations on human exposure to brominated flame retardants, it has been proposed that this can be through food intake, dust ingestion and inhalation (abdallah et al., 2008; table 3. tbbpa concentrations in the indoor dust samples nd = not detected; a < mdl (tbbpa was detected; the calculated 388 ng g-1 is below the statistical mdl); baverage of duplicate analyses; ccorrected for recovery. science diliman (july-december 2011) 23:2, 5-16 dust samples site description tbbpa concentration, ng g-1 (n=3) correctedc tbbpa concentration, ng g-1 house ha bedroom: 1 tv, 1 pc nd nd hb bedroom: 1 pc, 1 laptop computer < mdla (388) < mdl hd bedroom: 1 tv 1348 1728 he bedroom: 1 tv, 1 dvd player nd nd internet cafe ia internet room: 9 pcs 2220 b 2846 ib internet room: 20 pcs 2235 2865 ic internet room: 15 pcs, 1 ph otocopier 2368 3036 ie internet room: 11 pcs 4916b 6300 if internet room: 8 pcs nd nd tetrabromobisphenol a in indoor dust 13 country samples range (ng g-1) collection/ sampling year reference philip pines house dust (n=4) nd – 1348 2010 this study japan house dust (n=2) 490 – 520 2006 takigami, et al., 2009 belgium house dust (n=45) < 3 – 419 2008 d’hollander, et al., 2010 belgium domestic dust (n=18) 0.85 – 1481 2008 geens, et.al., 2009 uk germany/ usa house dust (n=45) house dust (germany, n=24; usa, n=2) < mql – 382 nd – 470 2006-2007 not reported abdallah, et al., 2008 abb, et al., 2011 table 4. comparison of tbbpa concentrations in house dust in different countries nd = not detected; < mql = less than the reported method quantitation limit. geens et al., 2009; harrad et al., 2010). geens et al. (2009) illustrated that dietary intake is the major path of exposure to environmental contaminants including tbbpa, while contaminated dust is a minor contributor. kose et al. (2008) and jakobsson et al. (2002) demonstrated that electronic products are among the causes of brominated flame retardants contamination in ind oor environments and contamination increases when these flame retardanttreated products are present. in the houses and internet cafes where we collected the indoor dusts, we recorded information on the would-be sources of tbbpa. the house dust samples were taken from air-conditioned bedrooms which contained at least one television set or one computer. in the internet cafes, the number of computers ranged from 8 to 20. the higher concentrations of tbbpa and the frequency of tbbpa occurrence in indoor dust from the internet cafes may be associated with the more number of computers present in these places. it should be noted, however, that the collection of indoor dust was not controlled nor simultaneously timed so as to respect the private and business activities in these places. as such, caution should be made in interpreting the tbbpa concentrations as these relate to the number of computers inside the internet cafes. nevertheless, the data obtained here provide insights on the occurrence of tbbpa in indoor environments and the potential exposure of filipinos to tbbpa from electronic products which are commonplace in these modern times. the results of this study can also serve as basis or baseline for future studies. while there is no other information yet on tbbpa contamination in indoor environments in the philippines, the findings in this study prompt the need to look at tbbpa occurrence in other enclosed places (viz. offices, classrooms, libraries, malls and other public microenvironments) where filipinos stay for many hours. and while there is no health-based guideline against this contaminant, the effects of storage and disposal of electronic products should also be studied. a report on organohalogens in breast milk from mothers living in payatas dumpsite in the philippines showed higher levels of the flame retardants pbdes compared to those reported in other asian countries (malarvannan et al., 2009). tbbpa was not among the flame retardants they investigated. it would be an interesting further study to determine the occurrence science diliman (july-december 2011) 23:2, 5-16 espino, m.p.b. and leon, j.n. 14 and levels of tbbpa in breast milk and serum from this exposed group. in this country where there is still no clear guideline on electronic waste disposal, comprehensive studies on the levels of brominated flame retardants in the local environment is important. when brominated flame retardant-treated things end up in dumpsites, soil contamination may ensue. these wastes may emit brominated flame retardant chemicals including tbbpa which can contaminate not only the soil but also the air and water. scientific data on the environmental levels of these contaminants are important to the relevant government authorities as they formulate guidelines on the reduction of indoor or outdoor contamination and as they implement policies on electronic waste storage, handling, treatment and disposal. conclusions an ultrasonication-assisted hexane extraction and determination by hplc-uv detection were optimized and applied in the analysis of tbbpa in indoor dust. in six of the nine indoor dust samples analyzed, tbbpa was detected in concentrations up to 4916 ng g-1. this study presented primary measurements of tbbpa in indoor dust from houses and internet cafes in the philippines. the relatively high levels of tbbpa in most of the samples are believed to be caused by leaching of tbbpa from flame retardant-treated electronic products present in the sampling sites. furthermore, this study provided indication suggesting that indeed dust represents one of the potential sources of human exposure to tbbpa in these indoor environments. acknowledgements the up diliman natural science research institute provided the funding support (project no. che-102-03). we are very grateful to the owners of the houses and internet 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council. brominated flame retardant industry panel. http:/ /www.epa.gov, accessed in october 21, 2011. van leeuven s., & de boer, j. 2008. advances in the gas chromatographic determination of persistent organic pollutants in the aquatic environment. j. chromatogr. a 1186: 161-182. watanabe i., & sakai, s. 2003. environmental release and behavior of brominated flame retardants. environ. int. 29: 665-682. science diliman (july-december 2011) 23:2, 5-16 5 the influence of vegetation and insect abundance on insectivorous bat activity during dusk emergence in an urban space in metro manila, philippines jay s. fidelino* jelaine l. gan institute of biology college of science university of the philippines diliman abstract because of their importance in the maintenance of ecological processes and sensitivity to multiple stressors, bat activity is increasingly being used to study habitat associations, including the effects of urbanization and other landuse changes. however, to be effectively used as a bioindicator, baseline information on bat activity patterns must first be established. in this study, we aimed to determine patterns of insectivorous bat activity, richness, and assemblage within an urban green space in the philippines’ capital region in relation to habitat type, insect abundance, and environmental conditions, with particular focus on activity at dusk emergence. bat activity was measured as the number of bat passes per minute using a portable bat recorder at five time intervals from 5:30 pm to 7:30 pm, and compared between 10 open and 10 forested sites. bat calls were classified into sonotypes based on five call variables. there was no difference in bat activity between forested and open sites, but more sonotypes were recorded in open sites. both bat activity and sonotype richness peaked between 6:00 pm and 6:30 pm, representing a short foraging bout upon dusk emergence. however, we did not observe significant relationships between bat activity and insect abundance, air temperature, and relative humidity. our study found considerable bat activity and diversity in an urban ecosystem, a poorly explored field of research in the philippines. additional studies are necessary to understand the impact of landuse changes on philippine bats, and to inform their conservation and management in anthropogenically altered habitats. keywords: bioacoustic monitoring, urban ecosystems, insectivorous bats * corresponding author science diliman (july-december 2019) 31:2, 5-26 the influence of vegetation and insect abundance 6 introduction the rapid development of urban areas in response to growing human populations often involve little to no planning in developing countries like the philippines (vallejo et al. 2009; de araujo and bernard 2016). metro manila, or the national capital region, is considered one of the five largest urban areas in the world, with an estimated population of 13 million in 2015 and an annual growth rate of 1.58% (philippine statistics authority 2016 ). in the past 30 years, urbanization has also spilled over the fringes of metro manila, with rapid land use conversion resulting in the continued fragmentation and destruction of green spaces in the national capital region and its surrounding provinces (bravo 2017). despite the fragmentation and destruction of natural habitats due to urbanization, several studies have shown that remaining green spaces in urban landscapes still support considerable diversity, acting as refuge for different species amidst a rapidly changing environment (pickett et al. 2001). for example, an inventory of terrestrial vertebrates within two university campuses in the center of metro manila has documented the presence of six amphibian species, 13 reptile species, 47 bird species, and 10 mammal species, of which 11 are endemic to the country (ong et al. 1999). more recent records of bird occurrences in metro manila from birdwatching hobbyists and clubs such as the wild bird club of the philippines (wbcp) place the species richness of birds at about 140 (wbcp2015). compared to birds, significantly less monitoring and studies have been done on the diversity of other terrestrial vertebrates in metro manila and other urban areas of the philippines. flight allows bats to cross environmental matrices otherwise unfavorable to other animals and to persist in altered habitats. these make bats important components of urban ecosystems, and likely crucial in the maintenance of ecological processes in urban landscapes. ecosystem services provided by bats include pollination and seed dispersal of urban flora, and control of arthropod populations (jones et al. 2009; de araújo and bernard 2016). insectivorous bats, in particular, are considered excellent pest controllers, as their insect diet amounts to 25-50% of their body mass (kunz et al. 2011; boyles et al. 2013). they are also sensitive to a wide range of stressors, such as habitat alteration and fragmentation, climate change, agricultural expansion, and pollution, and thus have the potential to be excellent bioindicators (jones et al. 2009). in addition, because bats are not influenced by regular direct human interactions that may influence community composition, studies in urban areas may offer insights more directly related to the urbanized elements of the environment (krauel and lebuhn 2016). j.s. fidelino and j.l. gan 7 the insectivorous bat fauna of the philippines consists of at least 55 species from six families, at least 10 of which are endemic (ingle and heaney 1992; heaney et al. 2016). because of their echolocation, insectivorous bats are difficult to sample using mist nets and require more labor-intensive capture techniques such as harp traps and tunnel traps (heaney et al. 2016). as such, much less information is known on their taxonomy, distribution, and general ecology despite being more speciose than fruit bats (heideman and utzurrum 2003; mildenstein et al. 2005). the development of bioacoustic techniques to detect and assess insectivorous bats has helped fill this research gap in the past two decades (sedlock 2001; sedlock et al. 2014a; sedlock et al. 2014b). bats emit calls with frequencies ranging from 9 to 212 khz, with considerable diversity in duration, bandwidth, and use of harmonics (maltby et al. 2009). using a combination of echolocation call parameters, bat calls can be distinguished between species, although significant intraspecific variation exists due to sex, age, geography, and habitat variations (stathopoulos et al. 2018). regardless, acoustic monitoring has been used as a tool in conservation and biomonitoring schemes, particularly in determining the effect of anthropogenic activities on biodiversity (barlow et al. 2015; jung and kalko 2010; kelly et al. 2016; newson et al. 2017). bat activity based on bioacoustics is increasingly being used to study habitat associations, including the effects of urbanization and other land use changes on bat assemblages. generally, bat activity decreases with increased intensity of urbanization, with variations in degree across species and geography (gehrt and chelsvig 2003; schimpp et al. 2018). studies have shown, however, that urban green spaces, with abundant nocturnal invertebrates and tree hollows, may ameliorate the impact of urbanization on insectivorous bats (avila-flores and fenton 2005; basham et al. 2011). unfortunately, very few ecological studies have been done on the bats of the philippines, much less so those targeted towards urban assemblages.tanalgo and hughes (2018), in their review of philippine bat research in the last two decades, report that only 15% of all studies focus on ecology, majority of which looked at the seed dispersal ecology of frugivorous bats. in addition, the review found only seven studies conducted in urban and othermodified habitats. thus, the impact of urbanization and other land use changes on bat assemblages is poorly understood in the philippines and warrants investigation. the influence of vegetation and insect abundance 8 there is great untapped potential in the use of insectivorous bat acoustics for conservation and monitoring in the philippines. however, there is still insufficient information on the most basic aspects of insectivorous bat ecology, especially in anthropogenically altered landscapes. as such, assessing the effects of urbanization on insectivorous bats requires first an understanding of local activity patterns and the factors that influence these patterns in urban environments. in this study, we aimed to determine patterns of insectivorous bat activity, richness, and assemblage within an urban green space in the center of the philippines’ capital, with particular focus on activity at dusk emergence. we also assessed if habitat type, insect abundance, and environmental conditions influence these activity and richness patterns. materials and methods study area and site selection the study was conducted within a 40-day sampling period from february to march 2018 within the university of the philippines (up) campus in diliman district, quezon city (14°37’50.37” n, 121°4’42.48” e). the university campus has academic use areas, residential areas, parks, commercial areas, and protected forest areas encompassing a total of 493 hectares. over 900 buildings are scattered across the campus, majority of which are for housing and academic use (university of the philippines 2012). according to data from 2009, daytime population within the campus averages at around 40,000, composed of students, academics, staff, and residents (vallejo et al. 2009). based on the latest land use plan of the campus (university of the philippines 2012) and satellite images (google earth 2017), we randomly selected ten sampling sites each of forested and open sites (figure 1). based on a 50x50 m grid overlaid on satellite images of the campus, we selected forested sites as those with canopy cover greater than 60%, while open sites have less than 10% cover (di gregorio and jansen 2000). because the presence of built-up structures has an influence on measured bat activity as possible roosting areas, all sites selected were at least 150 m away from any buildings within the campus. all sampling sites were at least 100 m apart.the selected forested sites were located in two forested patches within the study area, about 0.16 km2 and 0.05 km2 in total area. on the other hand, the selected open sites were located in five open areas, 0.02 km2, 0.05 km2, 0.02 km2, 0.10 km2, and 0.04 km2 in total area. j.s. fidelino and j.l. gan 9 figure 1. ten open sites (yellow) and ten forested sites (green) within the university of the philippines diliman campus in quezon city were chosen as sampling sites for the study. bat recordings recordings were collected using a pettersson elektronik m500 usb ultrasound microphone attached to a laptop pc. the m500 has a sampling frequency of 500 khz and a frequency range of 10 to 210 khz. sampling was only done on nights without rain, and avoiding the two nights prior to and following a full moon. one to three sampling sites were sampled per night, alternating between forested and open sites. we recorded for three minutes at each point, following barros et al. (2014) and de araujo and bernard (2016), with 30-minute intervals from 5:30 pm to 7:30 pm. the m500 was held about 1.3 m above the ground, angled at 45° relative to the ground. for each sampling point and time interval, the number of bat passes per minute were counted and used as a measure of bat activity. only search calls were used in this study. for each search call, the following call variables were extracted using batsound 4.2.0: call duration (d, in ms), frequency with maximum energy (fmax, in khz), initial frequency (fi, in khz), terminal frequency (ft, in khz), and frequency range (r, in khz). the following settings were used for spectrograms: fft size = 1024; fft overlap = 0; hanning window). using these call variables, a euclidean distance matrix was generated and used to construct a non-metric multidimensional scaling (nmds) biplot using the function “metamds” of the community ecology package (“vegan” v. 2.5-3) (oksanen et al. 2019) in the r statistical software (v. 3.5.1) (r core team 2018). clustering in the nmds biplot was the influence of vegetation and insect abundance 10 used to classify calls into sonotypes. clusters of less than five calls were assessed as outliers and not classified into any sonotype. discriminant function analysis (dfa) was then performed using spss statistics v. 20 (ibm 2011) to evaluate validity of the classification into distinct sonotypes. insect sampling and environmental data collection insect sampling was conducted simultaneously with bat activity recording using a sweep net. systematic sweeping within a 20 m x 20 m quadrat centered on the sampling point was conducted to obtain an estimate of nocturnal insect abundance. the insect sampling protocol used covers only the lower strata of the habitats sampled. this reduces the likelihood of our sampling interfering with the foraging bats in the area, majority of which have been observed to fly at least 15 m above the ground. however, it presumes a positive, but not necessarily close, correlation between insect abundance at different vertical strata (taylor 1960). collected insects were preserved in 70% ethanol and then identified to order level and quantified as abundances per order. air temperature and relative humidity were recorded at every sampling session, simultaneous with bat recording and insect sampling. data analyses kruskal-wallis tests were conducted to test for differences in bat activity and sonotype richness across different time points, and between open and forested sites. variations in insect abundance, air temperature, and relative humidity were also compared using kruskal-wallis tests. nmds biplots from bray-curtis distances and permutational manova were used to assess differences in bat assemblage using the “vegan” package in r (oksanen et al. 2019). sonotypes were used as proxy for taxa and bat activity as proxy for abundance. generalized linear models were constructed with quasi-poisson distribution to analyze the influence of time, habitat type, air temperature, and insect abundance on bat activity and sonotype richness. relative humidity was not included in the model due to collinearity with air temperature. a quasi-poisson model was used to correct for overdispersion. the model with the smallest corrected akaike’s information criterion (aicc) was considered the best-fitting model. j.s. fidelino and j.l. gan 11 results a total of 824 bat passes were recorded over 300 minutes of recordings. of the 100 files analyzed, 39 did not contain search calls (24 from forested and 15 from open sites). majority of the empty recordings were sampled at 5:30 pm (n = 18) and at 7:30 pm (n = 11). the number of bat passes per recording ranged from 0 to 54 in forested sites (mean± sem = 6.84 ± 1.81) and from 0 to 57 in open sites (mean = 9.64 ± 2.05). across sampling times, the number of bat passes per recording was lowest at 5:30 pm (mean = 0.35 ± 0.30) and highest at 6:30 pm (mean = 19.75 ± 4.05). five calls with low detail were not used to measure call characters. based on five call characters, the nmds biplot constructed revealed seven potential sonotypes and 15 outliers. each bat pass was classified into these seven sonotypes (figure 2). dfa confirmed the validity of the grouping, with 64.4% of original grouped cases correctly classified. the first two canonical functions cumulatively account for 98.0% of the discriminating ability, while including the third function explains 99.9% of the characters’discriminating ability. the two functions with the highest eigenvalues are most correlated with call duration (cv = 0.95) and both maximum frequency (cv = 0.96) and terminal frequency (cv = 0.92). the third canonical function is most highly correlated with frequency range (cv = 0.99). figure 2. canonical discriminant functions biplot showing classification of 804 bat calls into seven distinct sonotypes. the influence of vegetation and insect abundance 12 majority of the calls were classified into sonotype 7 (n = 659), most distinctly characterized by the shortest call duration, the widest frequency range, and the highest initial frequency. the six other sonotypes were only represented by five to 49 search calls (table 1). six sonotypes were recorded from both open and forested sites. sonotype 5 was recorded only in the open sites. table 1. call characteristics of the seven distinct sonotypes recorded within the university of the philippines diliman campus. values are shown as mean ± sem. sonotype (number of samples) initial frequency, fi (khz) terminal frequency, fi (khz) frequency at maximum energy, fmax (khz) call duration, d (ms) frequency range, r (khz) 1 (8) 30.88 ± 2.47 29.00 ± 2.73 30.20 ± 2.60 19.29 ± 1.58 1.88 ± 0.61 2 (28) 39.48 ± 1.45 37.19 ± 1.43 38.31 ± 1.41 12.45 ± 0.81 2.30 ± 0.40 3 (49) 44.52 ± 0.78 41.20 ± 0.57 42.39 ± 0.55 9.99 ± 0.40 3.32 ± 0.47 4 (29) 39.84 ± 2.07 32.19 ± 2.04 34.56 ± 1.88 10.92 ± 1.07 7.65 ± 0.91 5 (5) 50.70 ± 2.80 43.36 ± 0.46 44.14 ± 0.37 85.22 ± 3.99 7.34 ± 2.43 6 (26) 32.02 ± 1.42 22.82 ± 1.14 24.52 ± 1.13 12.63 ± 1.58 9.20 ± 1.24 7 (659) 52.29 ± 0.31 43.09 ± 0.11 44.88 ± 0.10 8.15 ± 0.14 9.20 ± 0.27 bat activity, richness, and assemblage bat activity was not significantly different between open and forested sites (h = 1.60, df = 1, p = 0.11), with open sites having 3.21 ± 0.68 bat passes per minute and forested sites having 2.28 ± 0.60 bat passes per minute. however, bat activity differed significantly across time points, both when forested and open sites are combined (h = 44.23, df = 4, p < 0.00) and when analyzed separately (h = 23.32, df = 4, p < 0.00 and h = 23.80, df = 4, p < 0.00 for forested and open sites, respectively) (figure 3). peak bat activity within our sampling time period was from 6:00 pm to 6:30 pm, with 4.13 ± 4.41 and 6.30 ± 6.13 bat passes per minute in forested sites and 5.63 ± 6.08 and 6.87 ± 5.62 bat passes per minute in open sites. between open and forested sites within each time point, there was no significant difference in bat activity. significantly more sonotypes were detected in open (n = 7; mean = 1.50 ± 0.21) than forested (n = 6; mean = 1.04 ± 0.21) sites (h = 2.01, df = 1, p = 0.045). in addition, there was a significant difference among sonotype richness across time points (h = 35.23, df= 4, p = 0.00). within each time point, there was no significant j.s. fidelino and j.l. gan 13 difference in sonotype richness between forested and open sites except at 7:00pm, when sonotype richness was higher in open areas (h = 2.16, df = 1, p = 0.04) (figure 4). we did not find significant differences in assemblage between open and forested sites, based on both permutational manova (f = 1.03, p = 0.45) and nmds biplots. on the other hand, permutational manova showed significant differences in assemblage across time points (f = 2.04, p = 0.02), although this is not readily apparent in the constructed nmds biplot (figure 4). figure 3. bat activity, quantified as average number of bat passes per minute per time point from 5:30 pm to 7:30 pm in open and forested sites within the university of the philippines diliman campus. bars indicate standard errors. figure 4. average sonotype richness per time point from 5:30 pm to 7:30 pm in open and forested sites within the university of the philippines diliman campus. bars indicate standard errors. the influence of vegetation and insect abundance 14 insect abundance, air temperature, and relative humidity to determine whether prey availability is associated with bat activity, insect sampling was done simultaneously with bat recording. a total of 721 insects distributed among nine orders (lepidoptera, trichoptera, blattodea, psocoptera, orthoptera, coleoptera, diptera, hemiptera and hymenoptera) were captured. more than half (62.3%) were identified as members of diptera, followed in abundance by hemiptera (11.1%). insect abundance significantly varied across time (h = 11.21, df = 4, p = 0.02) and between forested and open sites (h = 2.23, df = 1, p = 0.03) (figure 5). figure 5. average insect abundance per time point from 5:30 pm to 7:30 pm in open and forested sites within the university of the philippines diliman campus. bars indicate standard errors. environmental conditions, specifically air temperature and relative humidity, were also taken into consideration (figure 6). between 5:30 pm and 7:30 pm, air temperature ranged from 26.77°c to 29.03°c in forested sites (mean = 27.86 ± 0.43), and from 26.34°c to 29.88°c in open sites (mean = 28.42 ± 0.62). relative humidity ranged from 73.73% to 76.76% in forested sites (mean = 75.14 ± 0.58), and from 67.04% to 73.5% in open sites (mean = 69.36 ± 1.15). average relative humidity in forested sites was significantly higher than in open sites (h=5.58, df =1, p = 0.00). j.s. fidelino and j.l. gan 15 figure 6. average values for environmental variables per time point from 5:30 pm to 7:30 pm in open and forested sites within the university of the philippines diliman campus. (air temperature– solid lines; relative humidity –dashed lines). bat activity and sonotype richness models the model that best explained bat activity included all predictor variables-habitat type, time, insect abundance, and air temperature, based on corrected akaike information criteria (aicc) (table 2). however, bat activity was significantly influenced only by time, being significantly higher at 6:00 pm, 6:30 pm, and 7:00 pm (table 3). the best model for sonotype richness, on the other hand, only included habitat type and time as explanatory variables (table 2). however, only time was a significant predictor. as with bat activity, sonotype richness was significantly higher at 6:00 pm, 6:30 pm, and 7:00 pm (table 3). while habitat type wasnot a significant predictor of sonotype richness in our best model, we note that kruskal-wallis tests of sonotype richness found a significant difference between open and forested sites (p = 0.045). the influence of vegetation and insect abundance 16 table 2. differences in corrected akaike information criterion (δaicc) scores between the top-ranked model and other models assessing the association of bat activity and sonotype richness with habitat type (habitat), time (time), insect abundance (insects), and air temperature (temp). model δaicc bat activity habitat + time + insects + temp habitat + time + insects time + insects + temp habitat + time + temp habitat + time 0.0 12.31 17.92 21.48 24.77 sonotype richness habitat + time habitat + time + insects time habitat + time + temp time + temp 0.0 1.69 1.92 2.31 3.73 table 3. summary of results of generalized linearmodels for bat activity and sonotype richness as a function of habitat type, time, insect abundance, and air temperature. estimate se t-value p bat activity 6:00 pm 3.766 1.324 2.844 0.0055 6:30 pm 4.247 1.332 3.188 0.0020 7:00 pm 2.879 1.387 2.076 0.0407 7:30 pm 2.302 1.472 1.563 0.1214 air temperature 0.152 0.138 1.099 0.2746 insect abundance 0.016 0.010 1.538 0.1274 open sites 0.338 0.261 1.294 0.1990 sonotype richness 6:00 pm 2.611 0.731 3.571 0.0006 6:30 pm 2.754 0.741 3.717 0.0003 7:00 pm 2.255 0.778 2.900 0.0047 7:30 pm 1.510 0.860 1.755 0.0826 air temperature 0.032 0.111 0.291 0.7718 insect abundance 0.008 0.011 0.742 0.4599 open sites 0.382 0.232 1.649 0.1025 j.s. fidelino and j.l. gan 17 discussion the results of our study demonstrated variation in bat activity, sonotype richness, and sonotype assemblage across time during dusk emergence. however, we did not find significant associations of bat activity with habitat type, insect abundance, air temperature, and relative humidity. bat activity and prey availability the peak in activity we observed after sunset is typical for insectivorous bats (hayes 1997; taylor and o’neill 1988). this peak presumably represents initial foraging at dusk. other studies have detected another rise in activity just before sunrise, likely representing foraging bouts and commuting back to day roosts, creating a bimodal activity pattern (hayes 1997). extended survey until dawn may detect other similarly short bouts of activity in our study area. unfortunately, limitations in equipment capability and manpower resources limited the focus of this study to the activity of insectivorous bats during dusk emergence. despite these limitations, our findings provide some baseline for future studies on the relationship of bat activity with insect abundance, air temperature, and relative humidity. the short foraging bout we observed at dusk emergence should be taken into account when conducting monitoring work, especially when resources are limited. the foraging period shortly after sunset is explained by kunz and brock (1975) to be a response to a peak in prey abundance. however, we did not observe this association between insect abundance and bat activity. support for such a relationship is variable in literature. rautenbach et al. (1996) and hayes (1997) found a positive correlation between bat activity and insect abundance, measured as number of individuals and dry mass, respectively. on the other hand, rydell et al. (1996) found a delay in the dusk emergence of two aerial-hawking species after the peak in insect activity, and presumed this to be a compromise between prey availability and predation risk (jones and rydell 1994). thus, the influence of insect abundance on bat activity may be species-specific, both on bats and on their preferred insect prey. indeed, muller et al. (2012) found that the response of insectivorous bats to prey availability differed between foraging guilds. openhabitat foragers, but not edge-and closed-habitat foragers, responded significantly to increasing prey abundance. while the insect sampling conducted was able to eliminate the possibility of interfering with bat foraging by positioning the sampling at ground level, the presumption of correlation between bat abundance across different vertical strata the influence of vegetation and insect abundance 18 may not always be true. patterns of vertical stratification vary widely among different insect groups and are also influenced by the habitat type (davis et al. 2011). to circumvent the possibility of affecting bat foraging activity during insect sampling, some studies do not sample insects the same night as bat data collection (i.e., threlfall et al. 2012) while some collect insects both prior to and during bat activity recording and use both sets of data separately (i.e., salvarina et al. 2017). in addition, the diversity of insects that bats prey on in our study area may not have been sampled extensively using only sweep nets. different types of traps have their own limitations, and several studies have suggested that multiple trap types should be employed for a better estimate of insect abundance in a given area (brigham and saunders 1990; sleep and brigham 2003). these biases may partly explain the variability in the observed influence of prey availability on bat activity especially in older literature, as no standardized methodology of insect sampling has been employed across studies. consequently, it is likely that the lack of relationship we found between bat activity and insect abundance may be affected by the same bias. bat activity, air temperature, and relative humidity environmental conditions could affect insectivorous bat activity either through direct effect on bat energetic costs or through indirect effect on prey abundance (bender and hartman 2015). previous studies have observed a positive relationship between air temperature and bat activity due to thermoregulation costs (lacki 1984; gaisler et al. 1998). we did not observe any significant relationship in our study, but this may be due to the small variation in air temperature (2.9°c) between the two hours our sampling covered. existing literature showed varying relationships between relative humidity and bat activity. lacki (1984) attributes an observed positive relationship to the high relative humidity creating a more equal vapor pressure between the respiratory tract of bats and the environment, leading to less water loss. amorim et al. (2012) also found a positive correlation between bat activity and relative humidity, which they link to an increase in insect activity due to the combination of humidity and temperature. on the other hand, gaisler et al. (1998) found a negative correlation between the activity of eptesicusserotinus and relative humidity, but attributed it only to the effect of temperature on humidity. similarly, smith and mcwilliams (2016) expect a negative correlation, but note that humidity is correlated with other environmental variables such as air temperature and atmospheric pressure. j.s. fidelino and j.l. gan 19 habitat preferences we did not find significant differences in bat activity between forested and open areas within the campus. our classification of habitat types was limited to only two discrete categories based on ranges of percentage of canopy cover—above 60% for forested sites and below 10% for open sites. this allowed for a simple way of classifying sampling sites and establishing contrast between the habitat types we were evaluating, as transition types between these two extremes were excluded from the study (i.e., built-up areas with scattered canopy). because of this limitation, however, association of the observed patterns of bat activity with different aspects of vegetation structure of the sampling area cannot be evaluated. bat foraging activity is influenced in varying ways by different aspects of a habitat’s vegetation structure, including canopy cover (threlfall et al. 2012), distance between trees (ghert and chelsvig 2003), vegetation clutter, and ground cover (suarez-rubio et al. 2018). in our study, the presence or absence of canopy cover did not significantly affect bat activity. our results were similar to that of threlfall et al. (2012), who found that vegetation cover, including canopy and ground cover, had no direct influence on both insect biomass and bat activity in an urban landscape. we recommend looking at other aspects of vegetation structure and their influence on bat activity in the philippines. we also recommend assessing the influence of other aspects of urban landscapes on bat activity, including proximity to built-up structures, distance from bodies of water, and the presence of artificial lighting. echolocation call characters and habitat preferences differences in foraging strategy, including habitat preferences, are ultimately linked to both the characteristics of each species’ echolocation calls and their wing morphology (norberg and rayner 1987; sedlock 2016). echolocation call characteristics have been associated with the habitat preferences of insectivorous bat species. for example, the echolocation calls of forest species are characterized by short durations and long intervals, which minimize call-echo and echo-echo overlaps caused by vegetation. on the other hand, open habitat species have calls with relatively long durations and low frequencies, which travel greater distances but are not as precise (sedlock 2016). the influence of vegetation and insect abundance 20 our analysis of five call characters identified seven distinct sonotypes, all of which were recorded from open sites and all but one from forested sites. it is likely that the bat detector has recorded open-adapted bats foraging above the canopy in forested sites. all of the search calls recorded within the campus had dominant frequency-modulated (fm) components, characteristic of species from the families vespertilionidae and molossidae. we did not record any calls characteristic of species from the families hipposideridae and rhinolophidae, both of which have characteristic constant frequency (cf) components (sun et al. 2008). this is expected, because of the latter families’ known habitat associations with caves and well-developed forests, both of which are absent in the study area. because there is no baseline library of calls recorded from captured insectivorous bats within the campus, identification to species level is not possible. however, de dios (2015) captured three species of insectivorous bats within the campus using mist nets: scotophilus kuhlii, pipistrellus javanicus, and myotis muricola. available call characters in literature do not match any of our sonotypes, including data for the aforementioned three species from mt. makiling, laguna, approximately 60 km south of the study site (sedlock 2001). variability in calls could be due to geographic variation, as populations from different localities may exhibit differences in bat call characters (conole 2000; chen et al. 2009; jiang et al. 2015). because of these geographic differences, and other sources of interspecific and intraspecific echolocation call variation, russo et al. (2017) suggest limiting identification to species with distinctive calls and combining acoustic surveys with capture sampling. our study on the activity of insectivorous bats in one of the last remaining green spaces in the philippines’ most urbanized region is one of the few in the country that has utilized bioacoustics to study this group’s ecology. furthermore, urban biodiversity and ecology in the philippines have often been overlooked fields of study, perhaps due to the already existing gaps in knowledge about philippine biodiversity and ecology in increasingly threatened natural habitats. indeed, majority of bat research has targeted forest habitats, followed by caves and karst areas (tanalgo and hughes 2018). this prioritization of natural habitats where more species are expected and are more threatened is unsurprising, especially given limited resources in a developing country. however, several studies have demonstrated that the green spaces of metro manila are able to harbor significant levels of biodiversity, including some endemic and threatened species (ong et al. 1999; vallejo jr. et al. 2008; vallejo jr. et al. 2009). because the impact of land use changes on philippine bats is poorly understood, especially in urban landscapes, more comprehensive and long-term monitoring of bat assemblages is needed. j.s. fidelino and j.l. gan 21 baseline studies on the activity patterns of insectivorous bats in urban ecosystems such as this thus contribute to filling this research gap, and provide useful starting points for future research. bioacoustics technology provides an opportunity to conduct studies on insectivorous bats passively, utilizing less manpower. however, especially for the philippines where sufficient baseline has yet to be established, acoustic surveys should be combined with capture sampling using harp traps or mist nets due to interspecific and intraspecific variations that make identification challenging (russo et al. 2017). building a bat call library using captured bats would be an important step to allow attribution of call recordings to species. we also recommend studying temporal variation over a 12-hour period to identify other peaks in bat activity post-dusk emergence. lastly, we recommend exploring how bat activity is influenced by other landscape elements such as presence of artificial light, distance to a water body or forest edge, and proximity to man-made structures, as these are important aspects of urban landscapes that have been assessed to influence activity of bats in other studies. understanding the influence of different elements in an urban landscape can help inform the conservation and management of biodiversity in these anthropogenically altered habitats. acknowledgments we would like to thank the university of the philippines diliman institute of biology’s biodiversity research laboratory and ecology and taxonomy teaching laboratory for the equipment used. we would also like to thank alyssa fontanilla for the ultrasound microphone used in this study, which is under project 162514194 of the mohamed bin zayed species conservation fund. lastly, we also thank dr. carmela española, dr. francis magbanua, and the academic year 2017-2018 2nd semester class of biology 260 for their valuable inputs. references amorim f, rebelo h, rodrigues l. 2012. factors influencing bat activity and mortality at a wind farm in the mediterranean region. acta chiropterol. 14(2):439-457. avila-flores r, fenton mb. 2005. use of spatial features by foraging insectivorous bats in a large urban landscape. j mammal. 86(6):1193-1204. barlow ke, briggs pa, haysom ka, hutson am, lechiara nl, racey pa, walsh al, langton sd. 2015. citizen science reveals trends in bat populations: the national bat monitoring programme in great britain. biol conserv. 182:14-26. the influence of vegetation and insect abundance 22 barros mas, pessoa dma, rul am. 2014. habitat use and seasonal activity of insectivorous bats (mammalia: chiroptera) in the grasslands of southern brazil. zoologia. 31(2):153-161. basham r, law b, banks p. 2011. microbats in a ‘leafy’ urban landscape: are they persisting, and what factors influence their presence? 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one.7(6):e38800. the influence of vegetation and insect abundance 26 available from: https://journals.plos.org/plosone/ article?id=10.1371/journal.pone.0038800 doi:10.1371/journal.pone.0038800 university of the philippines diliman. 2012. diliman campus land use plan. quezon city: office of the campus architect. vallejo b jr, aloy a, ong p, tamino a, villasper j. 2008. spatial patterns of bird diversity and abundance in an urban tropical landscape: the university of the philippines (up) diliman campus. sci diliman. 20(1):1-10. vallejo bm jr, aloy ab, ong ps. 2009. the distribution, abundance and diversity of birds in manila’s last greenspaces. landscape urban plan. 89(3-4):75-85. ______ jay s. fidelino <jsfidelino@gmail.com> is an m.s. biology student and research associate at the biodiversity research laboratory, institute of biology. he is interested in the ecology and biogeography of philippine terrestrial vertebrates, restoration ecology, and biodiversity science communication. jelaine l. gan is an m.s. biology student and an instructor at the institute of biology. her research interests are on vertebrate biology and ecology. 01_device feeding relationships of dominant fish species 35science diliman (january-june 2007) 19:1, 35-46 *corresponding author introduction studies on diet composition are important in community ecology because the use of resources by organisms has a major influence on population interactions within a community. studies of species resource requirements have been used in attempts to understand factors controlling the distribution and abundance of organisms (ross, 1986). information about the food habits of fishes is useful in defining predator-prey relationships because predator pressure has a pervasive influence on the evolution of a population. data on different food items consumed by fish may eventually result in identification of stable food preference and in creation of trophic models as a tool to understand complex ecosystems (lopez-peralta and arcila, 2002; bachok, 2004). feeding relationships of dominant fish species in the visayan sea annie trixie l. mequila* and wilfredo l. campos oceanbio laboratory, division of biological sciences, college of arts and sciences, university of the philippines in the visayas 5023 miag-ao, iloilo e-mail: atmequila@yahoo.com telefax: 033 3159271 abstract this study examines the diet composition of 15 fish species belonging to 11 families in the visayan sea. a total of 50 prey items were identified from 323 stomachs examined. cluster analysis was used to assess similarities in the composition of prey items in individuals of similar sizes but of different species, and in individuals of the same species but of different size categories. the results showed high similarity among individuals of the same species and low similarity between different species of similar size categories. two major clusters were formed, showing generalist feeding (high niche widths) and trophic segregation (low food niche overlap). these suggest that food resources in the visayan sea are not limited and that a wide range of habitats is available to the fish community. keywords: stomach analysis, food niche overlap, food niche width, diet composition the visayan sea is considered as one of the country’s most productive fishing grounds, with an average annual catch of about 200,000mt (bfar, 2002). however, recent reports show that there has been a decline in small scale and commercial fish production in the area due to increased fishing pressure (hermes et al., 2004). most of the studies that have been conducted in the visayan sea deal with stock assessment of commercially important species. noncommercially important species have received less attention and their biology is, to a large extent, poorly documented.this study provides empirical information on gut contents of the more abundant fish species caught by trawls in the visayan sea and examines the extent of feeding relationships among them. methods sampling area and field collection visayan sea is located in central philippines between 11o and 12o n and 123o and 124o e, and covers a total area of 5,184 km2 (bfar, 2002). it is bounded by mequila and campos 36 coastal areas of northeastern panay, northern negros occidental, bantayan island, and masbate (fig. 1). samples were collected during a research cruise in the visayan sea in july 2003 on board the rv da-bfar. a major activity of the cruise was a trawl survey covering 10 stations distributed within the study area. at each station, a single 1-hour tow was conducted during the daytime with the use of a two-seam trawl with a cod end mesh size of 5.08 cm. a cover net with a mesh size of 1 cm was attached to the cod end to examine gear selectivity (another study). the trawl was towed at an average speed of 4.5 knots. specimens representing the entire size range of the more abundant fish species caught in 5 of the 10 stations were collected for stomach content analysis and preserved on board in 10% buffered seawater-formalin solution. for larger fish, an incision was made along the belly to allow the formalin to penetrate the stomach to minimize possible post mortem digestion. laboratory processing in the laboratory, fish were sorted and identified to species level. prior to dissection, total length, standard length, and fork length of each individual were measured to the nearest 1mm using a ruler and total wet weight was measured to the nearest 0.01 g using an electronic balance. stomachs were removed from the digestive tract and weighed to the nearest 0.01 g. the contents were removed and preserved in 10% buffered formalin for later analysis. after the removal of its contents, the stomach sac was weighed. total stomach content was then calculated as the difference of stomach weight with contents minus the stomach sac. stomach contents were examined under the dissecting microscope and identified to the lowest taxonomic level possible. prey items were identified and grouped into major categories including: (a) microcrustaceans (amphipods, caperellids, cladocerans, cumaceans, euphausids, halocarids, isopods, mysids, lucifer, and ostracods), (b) larval crustaceans (crab 122.5 123.0 10.5 11.0 11.5 12.0 12.5 panay neg celebes sea sulu sea south china sea pacific ocean n 122.5 123.0 10.5 11.0 11.5 12.0 12.5 panay neg celebes sea sulu sea south china sea south china sea pacific ocean n figure 1. map of the visayan sea showing the 5 sampling stations where samples for stomach content analysis were collected during the visayan sea project cruise in june 2003. feeding relationships of dominant fish species 37 megalopa and zoea, shrimp mysis, and cypris larvae), (c) benthic worms (annelid worms, ribbon worms, flatworms, gnathostomulids, and sipunculids), (d) echinoderms (brittle star, bipinnaria, and pentactula), and (e) urochordates (ascidians and larvaceans). data analysis feeding intensity was determined by calculating the fullness index (fi) using the formula of hureau (1969) (as given in del norte-campos, 1995): fi (%) = weight of stomach contents (g) x 100 body weight (g) examination of stomach contents employed numerical analysis described by windell and bowen (1978), wherein food items present were identified and counted. the frequency of occurrence (foc) of identified food items was calculated using the formula, foc = no. of food item i x % identified total no. of food items where % identified = 100 % unidentified. unidentified materials were visually estimated as percentage of the total stomach content. the narrow size range of each fish species did not allow the categorization of size classes prior to the stomach content analysis. with the premise that prey differs with growth, sizing was done after prey identification using a mathematical tool. a two-step cluster analysis (using the program comm, piepenburg and piatkowski, 1992) was done on foc data, first, to determine if size categories (e.g. small, medium, and large) for each fish species could be identified on the basis of prey similarity. for example, “small” selaroides leptolepis, neopomacentrus filamentosus, and leiognathus bindus were assigned with the same sizes (<100mm) although “small” n. filamentosus and l. bindus were of shorter lengths. the second step was done on the various size categories of the different species to determine similarities in gut contents with respect to size. food niche overlap across length classes was computed using schoener’s index (as given in salgado,et.al, 2004), si xy = 10.5 (∑  p xi – p yi  ) where p xi = relative frequency of food type i in the stomach of species x and p yi = relative frequency of food type i in the stomach of species y. the index ranges from 0, for entirely dissimilar diets, to 1 when the composition of the diets is identical but values >0.6 can be considered biologically significant (wallace, 1981; wallace and ramey , 1983; salgado, et al., 2004). food niche width was also computed for each species using the shannon-wiener index on data across all length classes using the formula, h’ = ∑ (p i ) log (p i ) where p i = proportion of prey item i in the stomach of the predator. results and discussion fifteen species belonging to 11 families were examined in the study and their respective size groupings resulting from the initial cluster analysis are listed in table 1. some species did not form size classes due to their narrow length range. out of 323 stomachs examined, only 8 were empty. a total of 50 prey items were identified excluding chyme (unidentified foods), rubble, sediment, and fish scales and bones (counted in the absence of fish in the sample). shrimps were the most abundant prey in the stomachs of pentapodus setosus, upeneus asymmetricus, plotosus lineatus, saurida undusquamis, parapercis alboguttata, and lagocephalus. lunaris. calanoids were abundant in n. filamentosus, apogon notatus, and l. bindus; fish larvae for s. leptolepis, fish for synodus variegatus, polychaetes for leiognathus rivulatus, nematodes for arothron manillensis, amphipods for scolopsis affinis, and pelagic harpacticoids (mostly microsetella) for rastrelliger kanagurta. the assessment of possible dietary shifts in the different species showed that there was a general absence of variation in the pattern of prey preference with increase in size in all 15 of them. for example, l. bindus (fig. 2a) showed overlapping size groupings while clear groupings were observed in s. variegatus (fig. 2b). mequila and campos 38 species n no. of non-empty size range size classes (tl in mm) stomachs (tl in mm) small medium large demersal f. leiognathidae leiognathus bindus 38 37 56 122 <100 101-110 >110 leiognathus rivulatus 19 19 76 132 <100 >100 f. mullidae upeneus asymmetricus 28 27 95 142 <110 110-120 >120 f. nemipteridae pentapodus setosus 15 15 111 162 no size class scolopsis affinis 16 16 128 213 <155 155-175 >175 f. plotosidae plotosus lineatus 24 21 99 264 <120 120-200 >200 f. penguipeidae parapercis alboguttata 7 7 126 215 no size class f. synodontidae saurida undosquamis 18 18 144 268 <200 >200 synodus variegatus 16 16 124 245 <150 150-200 >200 f. tetraodontidae arothron manillensis 16 15 86 -119 <100 >100 lagocephalus lunaris 18 18 79 227 <200 >200 pelagic f. carangidae selaroides leptolepis 34 33 78 153 <100 100-130 >130 f. scombridae rastrelliger kanagurta 26 26 142 208 no size class reef associated f. gobiidae apogon notatus 15 14 71 85 no size class f. pomacentridae neopomacentrus filamentosus 33 33 66 139 <100 101-110 >110 total 323 315 table 1. list of fish species used for stomach content analysis from visayan sea. 98 ──┐ 102 ──┴──┐ 103 ─────┴┐ 58 ────┐ │ 65 ────┴─┴┐ 98 ─────┐ │ 56 ─────┴─┴────────────────────────────────┐ 100 ───┐ │ 95 ───┴────┐ │ 104 ─────┐ │ │ 106 ─────┴──┴┐ │ 112 ─────┐ │ │ 107 ─────┴───┴────┐ │ 73 ──────────────┴─────┐ │ 105 ──────┐ │ │ 115 ──────┴──────┐ │ │ 105 ─────────────┴──────┴──┐ │ 110 ────┐ │ │ 112 ────┴┐ │ │ 111 ─────┴───┐ │ │ 116 ─────────┴────┐ │ │ 101 ────┐ │ │ │ 107 ────┴──────┐ │ │ │ 99 ───────────┤ │ │ │ 104 ───────┐ │ │ │ │ 114 ───────┴─┐ │ │ │ │ 109 ─────────┴─┴──┴────────┴─────┐ │ 102 ─────┐ │ │ 104 ─────┴──────┐ │ │ 112 ────────────┴─┐ │ │ 106 ──────────┐ │ │ │ 100 ──────────┴───┴──────────────┴───┐ │ 122 ───────┐ │ │ 101 ───────┴──────────────────┐ │ │ 106 ──────────────────┐ │ │ │ 105 ──────────────────┴───┐ │ │ │ 58 ──────────────────┴ ──┴─────┴─ ───┘ m (101-110) m (101-110) s (<100) l (>110) 98 ──┐ 102 ──┴──┐ 103 ─────┴┐ 58 ────┐ │ 65 ────┴─┴┐ 98 ─────┐ │ 56 ─────┴─┴────────────────────────────────┐ 100 ───┐ │ 95 ───┴────┐ │ 104 ─────┐ │ │ 106 ─────┴──┴┐ │ 112 ─────┐ │ │ 107 ─────┴───┴────┐ │ 73 ──────────────┴─────┐ │ 105 ──────┐ │ │ 115 ──────┴──────┐ │ │ 105 ─────────────┴──────┴──┐ │ 110 ────┐ │ │ 112 ────┴┐ │ │ 111 ─────┴───┐ │ │ 116 ─────────┴────┐ │ │ 101 ────┐ │ │ │ 107 ────┴──────┐ │ │ │ 99 ───────────┤ │ │ │ 104 ───────┐ │ │ │ │ 114 ───────┴─┐ │ │ │ │ 109 ─────────┴─┴──┴────────┴─────┐ │ 102 ─────┐ │ │ 104 ─────┴──────┐ │ │ 112 ────────────┴─┐ │ │ 106 ──────────┐ │ │ │ 100 ──────────┴───┴──────────────┴───┐ │ 122 ───────┐ │ │ 101 ───────┴──────────────────┐ │ │ 106 ──────────────────┐ │ │ │ 105 ──────────────────┴───┐ │ │ │ 58 ──────────────────┴ ──┴─────┴─ ───┘ m (101-110) m (101-110) s (<100) l (>110) m (101-110) m (101-110) s (<100) l (>110) figure 2a. results of cluster analysis of l. bindus based on prey similarity by size class. s (<150) l (>200) m (150-200) m (150-200) 245 ┐ 196 ┴┐ 216 ─┴───┐ 212 ─────┴──────┐ 169 ─┐ │ 161 ─┴──────────┴──────┐ 156 ────┐ │ 149 ────┴────┐ │ 140 ─────┐ │ │ 141 ─────┴───┴─────────┴──────────┐ 181 ─────────┐ │ 124 ─────────┴──────┐ │ 162 ────────────────┴─────────────┘ s (<150) l (>200) m (150-200) m (150-200) s (<150) l (>200) m (150-200) m (150-200) 245 ┐ 196 ┴┐ 216 ─┴───┐ 212 ─────┴──────┐ 169 ─┐ │ 161 ─┴──────────┴──────┐ 156 ────┐ │ 149 ────┴────┐ │ 140 ─────┐ │ │ 141 ─────┴───┴─────────┴──────────┐ 181 ─────────┐ │ 124 ─────────┴──────┐ │ 162 ────────────────┴─────────────┘ figure 2b. result of cluster analysis of s. variegatus based on prey similarity by size class. feeding relationships of dominant fish species 39 in spite of differences in the clarity of clustering, there was a general similarity in prey items across all size classes within a given species (figs. 3a and 3b), thus showing the absence of a shift in diet. furthermore, when prey similarities between size classes of the various species were examined, the second step of the cluster analysis showed low similarities (fig. 4). hence, the results show that similarity in apparent prey preference is higher among different sizes of the same species, than between similar size classes of different species. the general absence of a dietary shift is attributed to the narrow range of lengths of specimens available to the study. this may not necessarily be an artifact of size selectivity of the trawl, since the sizes of fish retained in the cover net were no different from those retained in the main net (unpublished information). furthermore close to ¼ of the typical catch in the trawl was made up of relatively fast-swimming scombrids (rastrelliger spp) (unpublished report). hence, if there were larger, generally faster-swimming fish in the surveyed areas, a representative portion of them would 0 20 40 60 80 100 <100 101-110 size class (mm) f o c ( % ) chyme calanoid chaetognath microcrustaceans others figure 3a. diet composition of l. bindus according to size class. 0 2 0 4 0 6 0 8 0 1 0 0 < 1 5 0 1 5 0 2 0 0 s iz e c l a s s ( m m ) f o c (% ) c h ym e n e m a to d e m g a s tr o p o d fis h figure 3b. diet composition of s. variegatus according to size class. mequila and campos 40 have been caught by the net. their absence in the catches thus reflects the predominance of small and younger fish in the visayan sea. in most cases, the largest specimens observed from the catches were much smaller compared to their maximum reported sizes in the literature (table 4). this is a common observation in heavily-fished waters. fish are generally opportunistic feeders and show a relatively high degree of variability in prey preference either within or among species. the shift in feeding mode from larvae to early juveniles shows that they feed at different levels of the food chain during different stages of their life cycle. however, ontogenetic changes in feeding habits of fish as they grow do not depend on body size per se, rather these are correlated with changes in key aspects of feeding mechanisms (wainwright and richard, 1995). several factors affect feeding in fish and can be categorized into intrinsic and extrinsic factors (hourston et al., 2004). intrinsic factors include mode of feeding, differences in swimming ability, and location in the water column, while extrinsic factors include variation in prey composition among habitat types and susceptibility of prey to predation. the assemblages formed by the cluster analysis (fig.4) can also be related to their overall habits. the two major clusters formed clearly separate the strictly demersal species (assemblage 2) from the overlapping occurrence of pelagic, reef associated, and some demersal species (assemblage 1). assemblage 1 can be further separated into 3 sub-groups: 1 a , 1 b , and 1 c . the first sub-group (1 a ) is formed by the pelagic carnivores s. leptolepis and r. kangurta. a reefassociated species a. notatus, was grouped together with demersal species p. lineatus and l. rivulatus in the second sub-group (1 b ) while n. filamentosus and l. bindus formed the third subcluster (1 c ). sub-groups 1 a and 1 b had guts which typically contained large amounts of partially digested material (chyme), although planktonic prey such as fish larvae (1 a ) and calanoids (1 b ) were also common (table 2). for subgroup 1 c, other plankton groups like larvaceans and figure 4. cluster analysis of size classes of 15 fish species based on prey similarity. s denotes small, m for medium, and l for large. sl s ┐ sl m ┴┐ sl l ─┴───────────┐ lb s ────────┐ │ rk ────────┴────┴──────┐ pl s ──┐ │ pl l ──┴─┐ │ pl m ──┐ │ │ an ──┴─┴───┐ │ lr m ─────┐ │ │ lr l ─────┴──┴───────────┴────────┐ nf s ────┐ │ nf m ────┴─────────┐ │ lb m ────┐ │ │ lb l ────┴──┐ │ │ nf l ───────┴──────┴──────────────┴─────── ua s ──┐ ua m ──┴─┐ ua l ────┴─────────────┐ sa s ─────┐ │ sa l ─────┴────┐ │ sa m ──────────┴───────┴──────┐ pa ────────┐ │ ll l ────────┴─┐ │ ll m ──────────┴─────┐ │ ps ────────────────┴──┐ │ su m ─────────┐ │ │ su l ─────────┴─────────┴─────┴───────┐ sv m ────┐ │ sv l ────┴─────────┐ │ am m ──────┐ │ │ am l ──────┴───────┴─────┐ │ sv s -────────-───────┴─-─-──-──-─┴─-─ 1 a 1 b 2 a 1 c 2 b c lu s te r 2 c lu s te r 1 1 a 1 b 2 a 1 c 2 b c lu s te r 2 c lu s te r 1 feeding relationships of dominant fish species 41 t a b le 2 . t w o w a y c o in c id e n c e t a b le s h o w in g v a ri a ti o n i n g u t c o n te n ts o f th e d if fe re n t fi s h s p e c ie s e x a m in e d f ro m t h e v is a y a n s e a . n o te : t a b le r o w s a re a rr a n g e d a c c o rd in g t o p re y i te m a s s e m b la g e s a n d c o lu m n s a re a rr a n g e d a c c o rd in g t o s p e c ie s -s iz e c lu s te rs 1 a 1 b 1 c p re y i te m s s i s i s i l b p i p i p i l r l r n f n f l b l b n f (s ) (m ) (l ) (s ) r k (s ) (l ) (m ) a n (m ) (l ) (s ) (m ) (m ) (l ) (l ) ** p o ly ch a e te la rv a e 0 .1 3 s ili co fl a g e lla te s 0 .0 8 u ro ch o rd a te s 2 .2 2 5 .3 4 9 .6 3 0 .6 4 1 8 .0 3 1 5 .1 7 2 .6 1 ch a e to g n a th 0 .0 7 0 .1 4 5 .7 7 0 .0 2 1 .6 7 0 .3 3 0 .0 4 1 5 .0 3 1 2 .3 2 7 .6 9 fis h e g g s 0 .0 2 6 .4 6 a 1 p te ro p o d 1 .2 2 sa lp s 3 .4 1 3 .1 4 c n id a ri a n s 0 .3 9 co p . n a u p liu s 0 .7 9 0 .0 3 fi sh b o n e s 0 .7 8 1 .5 5 cy cl o p o id 1 .7 4 5 .1 4 1 .0 4 0 .1 0 0 .1 8 0 .3 3 0 .9 8 1 .5 8 0 .2 1 0 .7 9 1 .0 3 a 2 h a rp a ct ic o id 1 .2 0 1 4 .2 2 3 .4 4 0 .5 4 4 .0 0 1 .5 7 1 .1 1 0 .2 1 0 .4 8 d in o fl a g e lla te s 0 .0 7 0 .1 3 0 .4 4 8 .7 0 2 .5 0 d ia to m s 0 .5 3 1 .0 6 8 .6 1 5 .2 2 0 .7 1 a lg a e 0 .4 4 3 .0 7 2 .5 0 co p e p o d 3 .5 4 2 .5 0 b 2 -1 b e n th ic w o rm s 0 .2 0 0 .2 5 0 .8 7 6 .5 0 fi sh sc a le 0 .2 0 4 .2 3 sq u id l a rv a c ru st a ce a n s 1 .2 8 1 .3 3 1 .2 2 1 .7 8 0 .7 9 1 .5 6 2 .8 0 0 .0 7 0 .1 9 0 .3 4 b 2 -2 p o ly ch a e te 0 .0 2 0 .0 5 2 .0 8 0 .1 7 0 .0 7 0 .6 9 0 .1 2 0 .3 1 0 .9 5 0 .3 4 0 .1 8 b iv a lv e 0 .0 9 0 .1 7 0 .8 7 0 .1 8 0 .0 6 1 .1 7 g a st ro p o d 0 .1 6 0 .1 7 0 .1 4 0 .2 7 0 .8 7 0 .3 6 fo ra m s 0 .1 2 b 2 -3 ru b b le cr a b se d im e n t m ic ro cr u st a ce a n s 1 4 .8 0 11 .3 5 1 8 .2 5 4 .4 6 1 .3 6 0 .0 7 0 .2 8 3 .3 3 6 .2 4 7 .6 1 5 .0 6 3 .1 7 6 .2 2 9 .4 8 1 6 .6 3 sh ri m p 1 .0 5 0 .7 7 1 .3 2 3 .0 5 0 .0 2 2 .6 9 3 .5 8 8 .1 9 6 .3 6 0 .7 0 0 .2 1 0 .9 5 5 .3 8 c h ym e 4 7 .5 0 4 9 .0 0 4 6 .0 0 5 3 .7 8 5 1 .9 2 8 6 .8 8 9 4 .4 0 8 2 .8 8 8 2 .1 4 7 1 .6 3 6 4 .1 3 1 8 .6 4 3 0 .5 6 2 9 .8 9 2 8 .6 7 3 2 .6 2 b 1 c a la n o id 1 4 .6 0 1 6 .4 2 1 2 .7 8 8 .6 7 1 0 .9 9 3 .3 8 0 .3 7 0 .8 8 4 .6 0 4 .0 2 2 .1 5 6 1 .0 4 6 2 .2 6 1 7 .9 7 1 5 .0 2 3 1 .9 6 f il a 1 8 .1 0 1 7 .6 3 1 8 .5 8 0 .0 1 e ch in o d e rm la rv a 0 .8 7 8 .3 4 9 .7 5 0 .1 5 f is h 0 .0 0 2 .8 6 0 .0 8 0 .4 2 n e m a to d e 1 .7 4 1 .8 1 1 .6 6 0 .2 4 0 .0 2 0 .8 3 0 .5 4 0 .8 9 0 .3 1 4 .5 1 0 .1 4 1 .7 0 0 .6 3 ** d e p a u p e ra te f ee d in g r el at io n sh ip s o f d o m in an t f is h s p ec ie s mequila and campos 42 t a b le 2 ( c o n ti n u e d ) tw o w a y c o in c id e n c e t a b le s h o w in g v a ri a ti o n i n g u t c o n te n ts o f th e d if fe re n t fi s h s p e c ie s e x a m in e d f ro m t h e v is a y a n s e a . n o te : t a b le r o w s a re a rr a n g e d a c c o rd in g t o p re y i te m a s s e m b la g e s a n d c o lu m n s a re a rr a n g e d a c c o rd in g t o s p e c ie s -s iz e c lu s te rs 2 a 2 b 2 c p re y i te m s u a u a u a s a s a s a l i l i s u s u s v s v a m a m s v (s ) (m ) (l ) (s ) (m ) (l ) p a (l ) (m ) p s (m ) (l ) (m ) (l ) (m ) (l ) (s ) ** p o ly ch a e te la rv a e s ili co fl a g e lla te s u ro ch o rd a te s ch a e to g n a th 0 .9 1 0 .7 8 fis h e g g s a 1 p te ro p o d sa lp s c n id a ri a n s co p . n a u p liu s fi sh b o n e s cy cl o p o id 1 .3 0 a 2 h a rp a ct ic o id d in o fl a g e lla te s 1 .4 6 0 .3 2 d ia to m s 0 .5 6 3 .6 7 a lg a e 0 .8 4 4 .6 5 8 .3 5 2 .3 8 4 .3 9 2 .4 2 4 .2 3 co p e p o d 3 .0 1 1 5 .8 8 8 .7 5 2 .2 5 2 .8 4 6 .0 6 0 .9 4 b 2 -1 b e n th ic w o rm s 2 .0 4 1 .2 9 2 .1 1 fi sh sc a le 3 .4 3 2 .4 3 1 .0 4 1 0 .0 5 sq u id 2 .3 6 1 .3 5 1 3 .5 7 1 .5 3 l a rv a c ru st a ce a n s 3 .0 8 1 .1 1 6 .3 5 -. 0 .9 1 4 .5 2 5 .4 3 0 .1 5 b 2 -2 p o ly ch a e te 1 .1 9 1 .6 7 3 .1 3 1 .2 9 1 .1 7 0 .6 3 b iv a lv e 1 .8 3 3 .0 4 0 .1 4 g a st ro p o d 2 .7 2 0 .4 8 1 .1 9 3 .1 7 1 3 .7 3 4 .4 7 fo ra m s 1 .3 5 2 .3 6 b 2 -3 ru b b le 4 .2 9 0 .9 2 1 .0 4 cr a b 2 .5 0 0 .3 8 5 .7 1 8 .6 4 0 .8 8 0 .5 5 se d im e n t 0 .7 1 3 .6 4 4 .7 3 m ic ro cr u st a ce a n s 3 4 .0 0 3 3 .7 6 3 3 .5 0 4 9 .7 0 3 1 .7 5 5 6 .2 6 8 .9 4 0 .1 6 2 .0 8 0 .8 3 0 .0 1 sh ri m p 2 2 .1 0 2 2 .4 9 2 7 .8 4 2 0 .8 4 1 3 .8 0 1 0 .5 3 2 2 .0 2 1 8 .5 7 11 .2 1 1 6 .9 4 6 4 .6 2 3 8 .0 3 0 .0 0 0 .4 5 c h ym e 3 8 .8 9 3 1 .6 7 2 3 .3 3 4 .8 3 7 .1 7 6 .2 5 5 0 .7 1 5 2 .5 7 6 3 .0 0 3 4 .8 7 2 5 .3 8 2 9 .7 0 5 5 .0 0 4 3 .3 3 3 0 .0 0 3 6 .4 3 1 6 .6 7 b 1 c a la n o id 0 .7 4 2 .3 6 1 .6 7 1 .2 5 f il a e ch in o d e rm la rv a 1 5 .8 2 2 0 .1 0 1 9 .9 2 1 0 .1 9 4 .2 8 11 .0 0 2 0 .1 6 f is h 1 .1 1 0 .5 8 1 2 .7 3 0 .9 2 1 .4 8 1 2 .0 5 9 .6 7 1 0 .5 0 4 3 .0 2 n e m a to d e 2 .0 4 4 .1 8 2 .1 1 1 .7 1 0 .7 9 6 .6 3 2 .0 2 2 7 .8 9 3 3 .0 8 4 3 .1 4 5 0 .2 2 2 0 .1 6 ** d e p a u p e ra te m eq u il a an d c am p o s feeding relationships of dominant fish species 43 chaetognaths were also common in their guts. subgroups 1 a and 1 c can be considered as generally planktivorous. observations on 1 c are consistent with overlapping feeding habits of reef-associated and demersal species in the area, particularly in stations adjacent to shallow coral reefs. for example, the pomacentrid n. filamentosus is typically found over soft bottoms of lagoons and inshore reefs around coral outcrops, rocks, and debris (froese, and pauly, 2006). leiognathids in general are bottom living but small individuals feed mostly on copepods and phytoplankton, while large fish feed predominantly on benthic invertebrates. l. bindus in particular feeds on calanoid copepods, ostracods, chaetognaths, polychaete larvae, and fishes (fao, 2001). similarly, l. rivulatus searches for planktonic prey using a protrusible mouth or by sieving potential food through their gill rakers (froese.and pauly, 2006). the high incidence of chyme in subcluster 1 b , however, may not make them typical planktivores, in spite of the occurrence of planktonic prey in their guts as stated above. p. lineatus and a. notatus for instance are known to be carnivorous, feeding primarily on crustaceans, mollusks, and fishes (fao, 1999), while a. notatus are known to feed on fish and zoobenthos (froese and pauly, 2006). since trawling was done during the daytime, the large amounts of chyme suggest that p. lineatus and a. notatus fed during the night, making most of the ingested prey partially digested by the time trawling was done. the second assemblage can also be divided into three sub-groups 2 a , 2 b , and 2 c . these are largely demersal in habit because plankton (e.g., calanoids) are generally absent from their diet and they consume mostly benthic prey. sub-group 2 a showed a high occurrence of shrimps and microcrustaceans in their guts, while the others showed common occurrences of shrimps, crabs (2 b ), and nematodes (2 c ) in their diets (table 2). food niche overlap was computed only for 7 demersal species (out of a total of 15) because the objective was to quantify the degree of overlap between size classes of different species. only species with complete size classes (mostly from assemblage 2) (fig. 4) were included in this analysis. these include s. variegatus, u. asymmetricus, l. bindus, s. affinis, p. lineatus, and a. manillensis. generally, the values of niche overlap between size groups of various species were low (table 3) and inconsistent with the premise that resource overlap decreases with growth. very few species in this study showed such a trend in resource use. examples are l. bindus and p. lineatus, and u. asymmetricus and s. affinis. food niche overlap provides insights on the existence, nature and strength of competitive interactions (del norte-campos, 1995). the concept of resource overlap is usually related with the intensity of competition because of the use of common resources. competing species will necessarily show extensive niche overlap, although high niche overlap does not always mean that there is competition. in the case of s. variegatus and a. manillensis, (table 3), high niche overlap does not necessarily mean there is competition between them, unless food resources are limited. the presence of high prey density may result in high diet overlap between species because there is no need to partition available resources. although no simultaneous investigation on prey availability was conducted in this study, previous reports on benthic infauna (mequila et al., 2004) and zooplankton (campos et al, 2002) surveys in the area show that abundances of prey for demersal and pelagic fishes are relatively high in the area. low food niche overlap, on the other hand, may mean that food resources have become wellpartitioned as a result of competitive interactions. divergence over evolutionary time in feeding morphology and behavior of fish for example may result in utilization of different resources, thus lowering the niche overlap among previously competing species (labropoulou and markakis, 1998). the feeding behavior of fish examined in this study range from ram feeders (r. kanagurta and s.leptolepis) and suction feeders (l. bindus and l. rivulatus), to browsers (u. asymmetricus), and crushers (l. lunaris, a. manillensis and p.lineatus). whether this is a result of previous competition among these species is uncertain. the high values of food niche width (h’) in table 4, regardless of sizes, indicate that fish consumed a diverse diet, implying that they are generalists in terms of feeding. although this seems contradictory to the low overlap values, it may mean that fish occupy a wide range of habitats, thus favoring utilization of a large number of mequila and campos 44 resources. according to amundsen et al. (1996), a population with a broad niche width may be the outcome of either true generalist behavior of each individual of a population (high within-individual variation) or specialization of individuals of the population on different prey (high between-individual variation), as would be the case with ontogenetic changes in diet. the latter, however, was not observed in this study. high overall productivity in the visayan sea is evidenced by consistently high fisheries yield since at least the 1970s (aprieto and villoso, 1979), in spite of heavy fishing pressure (hermes et al., 2004). this is the result of many factors, including its shallow depth and central location, which in turn allows not only water and nutrient exchange with various basins adjacent to it (campos et al., 2002), but fish movement and recruitment as well. its being surrounded by deep (>200m) water adds to the many factors that would favor high natural fish abundance in the area and the likelihood of strong competitive interactions. the results of the study, however, are not consistent with strong competition. this is attributed to an overall relative availability of food resulting from a general reduction in the abundance of consumers and a corresponding reduction on predation (consumption) on food resources (mequila et al., 2004). constant cropping brought about by heavy fishing, particularly of trawls which have been used in the area since the 1950s (aprieto and villoso, 1979) allows this. thus, table 4. food niche widths (h') of fish species by size class in the visayan sea, july 2003 note: maximum total length attained as reported in the literatures are included in the table for comparison with the size observed in this study. those indicated with sl were species with maximum length reported in standard length. size range niche width (h') (tl in cm) l max species (cm) reference small medium large synodus variegatus 12.4 24.5 30 sl masuda, 1984 0.57 0.50 0.56 upeneus asymmetricus 9.5 14.2 30 allen, 1999 0.55 0.67 0.67 leiognathus bindus 5.6 12.2 12 allen, 1999 0.78 0.85 0.92 scolopsis affinis 12.8 21.3 30 randall et.al., 1996 0.62 0.82 0.57 selaroides leptolepis 7.8 15.3 17 masuda, 1984 0.63 0.63 0.62 plotosus lineatus 9.9 26.4 32 randall et.al., 1996 0.27 0.31 0.12 arothron manillensis 8. 11.9 45 sl masuda, 1984 __ 0.64 0.51 neopomacentrus filamentosus 6.6 13.9 11 froese.and pauly, 2006 0.53 0.42 0.70 saurida undosquamis 14.4 26.8 50 sl masuda, 1984 __ 0.45 0.73 leiognathus rivulatus 7.6 13.2 12 rau and rau, 1980 __ 0.48 0.57 lagocephalus lunaris 7.9 22.7 45 sl masuda, 1984 __ 0.51 0.63 rastrelliger kanagurta 14.2 20.8 35 randall et.al., 1996 __ __ __ 0.68 apogon notatus 7.1 8.5 10 masuda, 1984 __ __ __ 0.32 pentapodus setosus 11.1 16.2 20 allen, 1999 __ __ __ 0.86 parapercis alboguttata 12.6 21.5 22 allen, 1999 __ __ __ 0.64 small medium large sv/ua 0.17 0.35 0.30 sv/lb 0.18 0.32 0.32 sv/sa 0.21 0.14 0.19 sv/pl 0.18 0.56 0.44 sv/am __ 0.61 0.70 ua/lb 0.50 0.43 0.41 ua/sa 0.60 0.54 0.50 ua/pl 0.44 0.43 0.28 ua/am __ 0.35 0.28 lb/sa 0.16 0.16 0.24 lb/pl 0.64 0.34 0.33 lb/am __ 0.35 0.30 sa/pl 0.08 0.18 0.10 sa/am __ 0.13 0.07 pl/am __ 0.32 0.37 table 3. food niche overlaps between fish species by size class in the visayan sea, july 2003 note: sv synodus variegatus; sa scolopsis affinis; pl plotosus lineatus; ua upeneus asymmetricus; am arothron manillensis; lb leiognathus bindus feeding relationships of dominant fish species 45 continuous cropping together with natural high overall productivity promote generalist feeding (high niche widths) in the fish community over a wide range of habitats (low niche overlap). acknowledgements this study was conducted as part of the hydrological survey of the visayan sea july 2003: trawl, oceanography, and plankton survey (vsea tops) project funded by an in-house grant from the upv. the authors wish to thank the officers and crew of the rv da-bfar for the assistance during the cruise, the visayan sea program for conducting the cruise, and the conservation international for providing the student travel grant to annie trixie l. mequila during the pams 8 symposium. references allen, g.r. 1999. fishes of southeast asia. periplus editions ltd. 292 p. amundsen, p.a., h.m. gabler, and f.j. staldvik. 1996. a new approach to graphical analysis of feeding strategy from stomach contents data modification of the costello (1990) method. j. fish biol. 48: 607-614. aprieto, v.l. and e.p.villoso. 1979. catch composition and relative abundance of trawl-caught fishes in the visayan sea. fish. res. j. philpp., 4(1):9-18. bachok, z., m.i. mansor, and r.m. noordin. 2004. diet composition and food habits of demersal and pelagic marine fishes from terengganu waters, east coast of peninsular malaysia. naga. 27:41-47. bfar (bureau of fisheries and aquatic resources). 2002. commercial fishery stock assessment of the visayan sea in region vi for the year 1998 to 2002. bureau of fisheries and aquatic resources regional office vi, iloilo city. unpublished technical report. campos, w.l., r.f.c. canto, and d.g. estremadura. 2002. abundance, composition and distribution of major zooplankton taxa in the interisland waters of the eastern central philippines. upv j. nat. sci. 7 (1): 66-80. del-norte – campos, a.g. 1995. ecological studies on the coexistence of the brown shrimp, crangon crangon l. and the gobies pomatoschistus microps kroyer and p. minutus pallas in shallow areas of the german wadden sea. university of hamburg. unpublished dissertation: 265 p. fao. 2001. fao species identification guide for fishery purposes. the living marine resources of the western central pacific. vol.5. bony fishes part 3 (menidae to pomacentridae). carpenter,k.e. and v.h. niem. 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especially those adopted by the council of science editors (cse) scientific style and format, 8th edition, 2014. 19. less common abbreviations may be printed as footnotes. 20. short communications must be guided by the following points: • short communications are reports of limited data or important findings that warrant publication before the completion of the study; • short communications are reports of significant new data arising from problems with narrow, well defined limits before broader studies are completed; and results have not been published in print elsewhere, except as partial communications or posters in conference proceedings; • short communications should not be divided into conventional sections like introduction, methodology, etc. but should be provided with keywords, full names and addresses of all authors, current addresses, email addresses, and contact person to whom queries and proofs should be sent; • abstracts will be required on submission, not as part of the short communication but for potential reviewers; • author must also submit a layman’s abstract of not more than 200 words; 96 • short communications are 3 to 4 print pages (about 6 to 10 manuscript pages) in length with simple layout, a maximum of two tables and two figures, and a small number of citations; • authors should make it clear that their work is to be treated as short communication. 21. questions regarding submissions should be sent to <science.updiliman@ up.edu.ph>. 01_device abundance and distribution of phytoplankton 1 introduction this study forms part of a larger effort aimed at determining the most suitable sites to set up marine protected areas in various parts of the country (aliño et al., 2002). one of the emergent aspects in this effort is the productive potential of the site, which is determined by combinations of biological and physicochemical factors, and manifested by characteristics such as age structure, diversity and abundance of producer and consumer assemblages, nutrient dynamics, water movement and overall habitat structure and complexity. phytoplankton are among the primary producers in shallow coastal areas and factors abundance, composition and distribution of phytoplankton in calamianes, palawan jules jason c. asis*, wilfredo l. campos and fenelyn m. nabuab oceanbio lab, division of biological sciences, college of arts and sciences, university of the philippines in the visayas 5023, miag-ao, iloilo tel no: 033-3159271 julesjason_asis@yahoo.com date submitted: february 1, 2006; date accepted: august 10, 2006 abstract phytoplankton in coron bay of the calamianes islands, palawan were investigated from 27-29 may 2004. samples were collected from 33 stations by filtering five 10 l buckets of surface water through a net with a 20mm mesh bag. the phytoplankton consisted of four major groups. diatoms, showed the highest mean density of 1432.9indivl-1. silicoflagellates comprised the next most abundant group with a mean density of 132.3 indivl-1. dinoflagellates followed with a mean density of 94.8 indivl-1, while the cyanobacteria (blue-green algae) had a mean density of 19.4 indivl-1. the top three diatoms were chaetoceros, bacteriastrum and coscinodiscus. the genus peridinium was the most abundant dinoflagellate, while tintinnopsis dominated the silicoflagellates. among the cyanophytes, trichodesmium showed the highest density. high phytoplankton concentrations were observed in the vicinity of a pearl farm and in areas adjacent to mangrove forests. overall abundance and diversity in the study area are higher than in other similarly reef-dominated areas in the country. this may be attributed to factors on both large and local scales. *corresponding author science diliman (july-december 2006) 18:2, 1-9 that affect them will most likely affect overall productivity of a given site. hence, characterization of phytoplankton assemblages contributes to the determination of productive potential. this study was conducted to determine the abundance, species composition and distribution of phytoplankton in coron bay, calamianes, palawan. methodology a. study area and field collection phytoplankton samples were collected as part of a survey on coral reefs in the vicinity of coron bay, calamianes group of islands, palawan (fig. 1) from 27-29 may 2004. the study area consists of several islands and islets that effectively separate the inner portion of coron bay, where there is little water asis, nabuab and campos 2 movement but with apparently high terrigenous inputs, from the main island of busuanga. most stations were located within the semi-enclosed area between the islands and the main island, while other stations were found in more open and exposed waters. a couple of stations were located in the vicinity of pearl farms. fifty liters (50l) of surface water phytoplankton were collected in each of the 33 stations using a 10l bucket and were then filtered through a 20µm mesh plankton net. samples were then placed in properly labeled container bottles and preserved in 10% buffered formaldehyde solution. b. laboratory analysis the volume of collected samples from each station was standardized to 20ml. for the first few samples, a series of 1ml aliquots were examined with the use of a sedgwick-rafter counting cell. the genera cumulative curve constructed from these initial data showed a minimum of five (5) aliquots were needed to adequately represent each sample. the rest of the samples were thus identified using 5 aliquots. phytoplankton were 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 14 +02 5 +056 33 16 8 13 22 10 +04 34 7 28 11 20 18 29 26 +03 23a 30a 19 12 2 27 9 30 15 24 17 23 coron culion busuanga apo is. uson is. bugor is. 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 14 +02 5 +056 33 16 8 13 22 10 +04 34 7 28 11 20 18 29 26 +03 23a 30a 19 12 2 27 9 30 15 24 17 23 coron culion busuanga apo is. uson is. bugor is. figure 1. map of calamianes group of islands, palawan showing station locations. identified to genera (lowest level possible) and were further grouped into major taxa: blue-green algae, diatoms, dinoflagellates and silicoflagellates. the taxonomic identification was based on references including gran and angst (1931), shirota (1966), ferguson (1968), smith (1977) and yamaji (1962). results and discussions density and composition one hundred nineteen (119) phytoplankton genera were identified. the top 20 genera (table 1) are typical tropical phytoplankters (sverdrup et al., 1942). mean overall phytoplankton density for the area was 1679.4 indivl-1. the diatoms dominated all samples and showed a mean density of 1432.9 indivl-1, comprising 85.3% of all phytoplankton observed (table 2). among them, chaetoceros was the most abundant, with a mean density of 669.8 indivl-1. diatoms are generally abundant in nutrient-rich nearshore waters (valiela, 1984) and are considered as the chief component of phytoplankton in the marine environment (sverdrup et al., 1942). abundance and distribution of phytoplankton 3 the silicoflagellates were the next dominant in terms of density, comprising about 7.9% of the total phytoplankton density in the area. tintinnopsis was the most abundant silicoflagellate. on the whole, dinoflagellates comprised about 5.6% of the total phytoplankton identified, with peridinium as the most abundant, showing a mean density of 37.2 indivl-1. it is noteworthy that some dinoflagellates and silicoflagellates share similar characteristics. some genera of the dinoflagellates possess "cyst" stages that may look similar to silicoflagellates when viewed under the microscope (matsuoka and fukuyo 2000). dinoflagellates were the cause of a harmful algal bloom (hab) occurrence in 2000 in malampaya sound, just south of the calamianes (borja et al., 2000). in the present study, some potentially toxic dinoflagellates, like ceratium were observed with a mean density of 21.4 indivl-1. this genus has been reported to cause blooms that result in harmless water discoloration in sheltered bays or enclosed coastal areas. dense blooms, however, may cause fish and invertebrate kills due to oxygen depletion (yasumoto et al., 1990). in addition, the same authors note that the genus dinophysis may cause diarrheic shellfish poisoning (dsp), even at concentrations as low as 200 cells•l-1, although they rarely form dense blooms. in the present study, dinophysis showed a mean density of 1.1 indivl-1. the cyanophytes, or blue-green algae, included only seven genera and showed a mean density of 19.4 indivl-1. representatives of this group include richelia and trichodesmium, which are known to be nitrogen-fixing organisms in the marine environment. individuals of richelia were often observed within rhizosolenia or entangled in the setae of chaetoceros. trichodesmium was the most abundant cyanophyte. blooms of this genus are common in subtropical and tropical waters and are believed to contribute considerably to pelagic nitrogen fixation in the oceans (carpenter and capone, 1992). such blooms are formed under optimum light conditions and good vertical mixing of water layers, when low dissolve inorganic nitrogen concentrations restrict the growth and survival of competing phytoplankton populations. it is not known if such bloom-forming conditions develop in the study area. trichodesmium showed a mean density of 8.2 indivl-1. distribution the percentage composition of phytoplankton is presented in fig. 2.the highest mean density was recorded in station 15 (table 3), located in a semienclosed bay within the boundaries of a pearl farm. high phytoplankton densities in pearl farms have been reported in putemun channel, southern chile, where algal abundance was positively correlated with the pearl oyster growth (toro et al., 1999). while the high abundance serves as a significant source of food for the oysters, the accumulation of organic material from oyster fecal material and the accompanying nutrient table 1 mean density (indivl-1) and relative density (%) of the top 20 phytoplankton genera (n=119) in calamianes genera mean density sd relative density chaetoceros 669.84 928.86 45.34 bacteriastrum 318.11 471.49 21.53 coscinodiscus 102.44 112.19 6.93 rhizosolenia 64.94 76.68 4.40 nitzchia 46.78 68.62 3.17 peridinium 37.15 41.71 2.51 tintinnopsis 32.33 61.36 2.19 fragillaria 26.20 49.79 1.77 dytilum 25.54 137.38 1.73 ceratium 21.36 22.78 1.45 tintinidium 21.30 80.13 1.44 dactyliosolen 18.79 38.12 1.27 eutintinnus 16.86 15.72 1.14 thalassiothrix 16.29 25.38 1.10 favella 12.42 29.46 0.84 asterionella 10.81 36.23 0.73 guinardia 9.59 18.02 0.65 pyrophacus 9.08 16.80 0.61 biddulpphia 9.05 10.15 0.61 navicula 8.46 10.44 0.57 table 2 mean density (indivl-1) and percentage composition of major taxa of phytoplankton in calamianes major taxa mean density sd percentage composition blue-green algae 19.4 3.2 1.2 diatoms 1432.9 95.2 85.3 dinoflagellates 94.8 8.1 5.6 silicoflagellates 132.3 7.6 7.9 total 1679.4 asis, nabuab and campos 4 blue-green algae 1.16% diatoms 85.32% dinoflagellates 5.65% silicoflagellates 7.88% figure 2. percentage composition of the major taxa of phytoplankton in calamianes. regeneration within the immediate vicinity of the farm would tend to favor high phytoplankton densities in such areas. in general, overall phytoplankton densities were high in the vicinity of the pearl farm and along the northern portion, but low in the southern portion of the study area (fig. 3). diatoms showed a distribution closely corresponding to the overall distribution (fig. 4), although the dinoflagellates showed highest concentrations along the western portion of the study area (fig. 5). similarly, cyanophytes showed lowest densities in the more open stations located in the midportion of the study area (fig. 6). silicoflagellates showed a patchy distribution with little spatial pattern (fig. 7). it is possible that nutrient distributions are responsible for the observed spatial distribution pattern, since diatoms, dinoflagellates and cyanophytes would have different nutrient requirements. there are also other equally important factors in determining distribution, such as water exchange and currents. overall, there appears to be lower densities along the coast of culion island, except in the vicinity of the pearl farm near bugor island (fig. 3). there are other pearl farms along the coast of busuanga island, but these are located further east of the surveyed area. table 3 phytoplankton mean density (indivl-1) across stations in calamianes stn mean stn mean no. density no. density 1 739.7 23 61.7 2 6242.4 24 833.8 3 1653.8 25 121.7 4 843.7 26 963.0 5 4972.9 27 629.5 6 3379.6 28 1305.0 7 3254.9 29 1048.6 8 1398.4 30 336.8 9 1926.2 31 797.2 10 384.0 32 2534.0 11 1968.7 33 1646.6 12 1319.5 13 733.8 14 1922.9 n 33 15 6940.4 mean 1,679.4 16 197.2 med 1,181.8 17 2392.6 sd 1,651.2 18 81.3 min 61.7 19 1111.8 max 6,940.4 20 731.2 21 1181.8 22 1765.4 abundance and distribution of phytoplankton 5 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 coron culion busuanga apo is. uson is. bugor is. 0 to 500 500 to 1500 1500 to 3500 3500 to 6500 6500 to 7000 figure 3. density distribution (indivl-1) of phytoplankton in calamianes, palawan (size classes: <500, 1500, 3500, 6500, 7000) 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 coronbugor is. 0 to 500 500 to 1500 1500 to 3500 3500 to 6500 6500 to 7000 figure 4. density distribution (indivl-1) of diatoms in calamianes, palawan (size classes: <500, 1500, 3500, 6500, 7000) asis, nabuab and campos 6 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 coronbugor is. 0 to 10 10 to 20 20 to 30 30 to 40 40 to 100 figure 6. density distribution (indivl-1) of blue-green algae in calamianes, palawan (size classes: <10, 20, 30, 40, 100 ) 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 coronbugor is. 0 to 50 50 to 100 100 to 150 150 to 200 200 to 300 figure 5. density distribution (indivl-1) of dinoflagellates in calamianes, palawan (size classes: <50, 100, 150, 200, 300) abundance and distribution of phytoplankton 7 119.9 119.95 120 120.05 120.1 120.15 120.2 120.25 120.3 11.75 11.8 11.85 11.9 11.95 12 12.05 coronbugor is. 0 to 100 100 to 200 200 to 300 300 to 400 400 to 500 figure 7. density distribution (indivl-1) of silicoflagellates in calamianes, palawan (size classes: <00, 200, 300, 400, 500) mangrove stands and forests line the shores of the islands along the northern portion of the study area, particularly between mainland busuanga and the major islands of uson, apo and others nearby (fig. 1). their proliferations in this area is likely a combined result of several factors, including relative protection from rough conditions in more open areas and, as a consequence, slower water exchange and perhaps less water movement. these are typical of mangrove habitats (lugo and snedaker, 1974; mann, 1982). these same conditions, along with enhanced nutrient inputs from the mangroves (odum and heald, 1975), may favor higher phytoplankton biomass in this area. whether such conditions likewise prevail along the northeastern coast of culion island is not known, although this area seems to be more exposed and lies along the main channel of water exchange with the northern sulu sea where calm conditions with slow flushing are less likely to occur. species richness the number of species recorded, also termed species richness, is one way of presenting diversity in an assemblage, but its relevance and usefulness is dependent on the extent of the area surveyed or the amount of samples collected. table 4 shows mean density estimates and number of genera of phytoplankton from four reef areas in the country, surveyed in 2004 and 2005 using the same sampling methods. density estimates in the calamianes area are about 33% higher than those reported for reefs in internal water basins (danajon bank, bohol), but are from 2-3 times higher than those recorded in reef areas bordering open waters (surigao del sur and tawitawi). similarly, the number of genera recorded in the calamianes is much higher than those recorded in the latter. recent investigations in the south china sea show that the area west of northern palawan has higher phytoplankton biomass, as indicated by chlorophyll a concentration, than waters further north (bajarias 2000; furio and borja 2000). relatively high concentrations of chlorophyll a have also been reported for the shelf, shoal, and oceanic areas west of northern palawan as well as in the sulu sea (san diego-mcglone et al. 1999). hence the high diversity of phytoplankton asis, nabuab and campos 8 assemblages in the calamianes may be attributed to the relatively high productivity of waters bordering it, while the relatively high overall concentrations are results of factors such as current and nutrient inputs which act on a more local scale. acknowledgements this study forms part of a larger plankton survey funded by the project fisheries improved for sustainable harvest (fish). jja was awarded a student travel grant by the marine environment and resources foundation to present the study at the 8th national pams symposium held in puerto princesa city, palawan. the authors wish to thank the oceanbio and marine bio lab people for their support and assistance in both the field and laboratory. references alino, p.m., w.l. campos, v.v. hilomen and n.t. lasola. 2002. marine fishery reserves: how can mfrs sustain fisheries in the philippines? in: campos, w.l., p.d. beldia and p.m. alino (eds), workshop proceedings of the afmamarine fishery reserves program, u.p. in the visayas, iloilo, 165-168. bajarias, f.f.a. 2000. phytoplankton in the surface layers of the south china sea, area iii: western philippines. in: proceedings of the third technical seminar on marine fishery resources survey in the south china sea, area iii: western philippines. seafdec special paper no. sec/ sp/41. southeast asian fisheries development center, bangkok, 220-234. borja, v.m., furio, e.f. and rodriguez, a.k., 2000. horizontal and vertical distribution of pyrodinium bahamense cysts in sediments of malampaya sound, palawan, philippines, conference proceeding on the ninth conference harmful algae blooms, tasmania. carpenter, e.j. and capone, d.g. 1992. nitrogen fixation in trichodesmium blooms. in: carpenter, e.j., capone, d.g. & rueter, j.g. (eds), marine pelagic cyanobacteria: trichodesmium and other diazotrophs, nato asi series, series c: mathematical and physical series, vol 362, kluwer academic publishers, dordrecht, 211-217 p. fergusson, e.j. 1968. dinoflagellates of the caribbean sea and adjacent seas. univ. of miami press, florida. 143 p. furio, e.f. and v.m. borja. 2000. the primary productivity in the south china sea, area iii: western philippines. in: proceedings of the third technical seminar on marine fishery resources survey in the south china sea, area iii: western philippines. seafdec special paper no. sec/ sp/41. southeast asian fisheries development center, bangkok, 235-250. gran, h.h. and e.c. angst. 1931. plankton diatoms of puget sound. univ. wash. press, seattle, usa: 515 p. lugo, a.e. and s.c. snedaker. 1974. the ecology of mangroves. ann. rev. ecol. syst., 5: 39-64. mann, k.h. 1982. ecology of coastal waters: a systems approach. univ. calif. press, berkeley, ca, usa, 322p. matsuoka, k. and y. fukuyo. 2000. technical guide for modern dinoflagellate cyst study. westpac-hab/ westpac/ ioc. table 4 phytoplankton index of richness from different studies no. mean sampling no. of stns genera density richness site nearby basin dates sampled (s) (n) (s/√√√√√n) calamianes, palawan south china sea may, 2004 33 119 1679.4 2.93 cantilan-carascal, surigao del sur pacific ocean june, 2004 30 56 453.8 2.63 tawi-tawi sulu/celebes sea august, 2004 19 61 896.1 2.04 danajon bank, bohol camotes sea december, 2005 28 80 1259.3 2.25 abundance and distribution of phytoplankton 9 odum, w.e. and e. j. heald. 1975. the detritus-based food web of an estuarine mangrove community. in: cronin, l.e. (ed.), estuarine research, vol. 1, academic press, new york, 265-286 p. san diego-mcglone, m.l., g.s. jacinto, v.c. dupra, i.s. narcise, d.o. padayao and i.b. velasquez. 1999. a comparison of nutrient characteristics and primary productivity in the sulu sea and south china sea. acta oceanographica taiwanica 37(3):219-229. shirota, a. 1966. the plankton of south vietnam (freshwater and marine plankton). overseas tech. coop. agency, japan: 462 p. smith, d. 1977. marine coastal plankton and marine invertebrate larvae. kendall/ hunt publishing co., iowa, u.s.a. 161 p. sverdrup, h. u., m. johnson, and r. fleming. 1942. the oceans. prenticehall, inc., new york. 1087 p. toro, e. j., paredes, p. i. & villagra, d. j. 1999. phytoplankton distribution and oyster, ostrea chilensis (philippi 1845), growth at putemun channel, southern chile. new zealand journal of marine and freshwater research 33: 499513 p. yamaji, i. 1962. the plankton of japanese coastal waters. hoikusha publishing co., ltd., japan. 239 p. yasumoto, t. 1990. marine microorganisms toxins an overview. in: in: granéli, e. sundström, b., edler, l. & anderson, d. m. (eds), toxic marine phytoplankton. elsevier sci, publ. co., inc., new york, 3 -8 p. oshima, y. sugawara, w., fukuyo, y. oguri, h., igarashi, t. & fujita, n. 1980. identification of dinophysis fortii as the causative organism of diarrhetic shellfish poisoning. bulletin of the japanese society of scientific fishiries 46: 14051411. 01_device feeding, growth, and survival of post-larval abalone 49 abstract *corresponding author science diliman (july-december 2007) 19:2, 49-59 feeding, growth, and survival of post-larval abalone haliotis asinina on different benthic diatoms emmanuel c. capinpin jr. marine science institute, college of science university of the philippines 1101 diliman, quezon city pangasinan state university binmaley campus, 2417 binmaley, pangasinan tel: (075) 543-3012 e-mail: manny_capinpin@yahoo.com date submitted: january 3, 2007; date accepted: may 4, 2007 the feeding behavior, digestive efficiency, growth, and survival of post-larval abalone haliotis asinina fed with 5 species of locally isolated benthic diatom strains (navicula mollis, n. ramosissima, stauroneis sp., pleurosigma sp., and cocconeis sp.) were examined in the laboratory. two 15-day feeding trials using 1 mm post-larvae were conducted. no significant differences were observed in sizes of post-larval abalone after 15 days in all diatom treatments (p>0.05). however, in both trials, cocconeis sp. resulted in high survival rates (88.9±5.6% and 80.0±20.0% for trials 1 and 2, respectively). cocconeis sp. was efficiently digested by post-larval abalone, with most of the cells being ruptured during ingestion and/or passage through the gut. one diatom strain, pleurosigma sp., resulted to a high survival but produced the slowest growth rate (<10 ìm.d-1 sl). it was probably not ingested easily during the experiment due to its large size or mobility. for the other diatom strains, n. mollis and n. ramosissima, most cells passed through the gut with the cells left intact. stauroneis sp. is highly digestible, but did not result to high survival, although the remaining live post-larval abalone fed on this diatom as well as on n. mollis grew faster during the second week of both feeding trials. n. ramosissima resulted to poorest survival rate (<10%) due to its poor digestibility. only cocconeis sp. showed a fairly high growth rate, digestion efficiency, and survival rate. n. mollis which gave a fairly high survival rate and stauroneis may be added towards the later stages of post-larval rearing as well as other large diatoms. the digestion efficiency of diatom strains is considered an important factor determining its dietary value, but other factors may also be important such as volume contents, biochemical composition, and other physical characteristics. key words: digestive efficiency; growth; survival; post-larval abalone; haliotis asinina; diatoms capinpin, emmanuel 50 introduction benthic diatom films are traditionally used to induce settlement and for use as initial food in abalone hatcheries (hahn, 1989). a method commonly used is to establish a visible or dense film of diatoms to trigger settlement and to provide food for post-larvae. older films (i.e. higher density) of some diatom strains are considered better in inducing settlement (kawamura & kikuchi, 1992; moss & tong, 1992; daume et al., 1999) and for feeding (searcy-bernal et al., 2004; day et al., 2004). however, without control over the type and density of diatoms, fast-growing naviculoids become dominant, which may or may not be good for settlement and consequently, feeding of post-larvae. these films develop rapidly in a short time, becoming dense and often peeling as sheets. they may interfere with settlement and feeding and may cause strong fluctuations in water quality in the diffusive boundary layer (searcy-bernal, 1996) such as oxygen supersaturation at high diatom densities when light is available and the opposite during dark conditions (searcy-bernal et al., 2004), which could explain the rapid decline in survival a few weeks after settlement. also, the species combination on the settlement plate is unpredictable and varies with season which could lead to inconsistent and variable settlement rates and growth of post-larval abalone (daume et al., 2000). several studies have examined post-larval feeding and growth using different diatom species (kawamura et al., 1995, 1998a; roberts et al., 1999a; carbajalmiranda et al., 2005; gordon et al., 2006). kawamura et al. (1998b) showed that post-larvae 0.8-2 mm in shell length grew faster on ‘digestible’ diatoms than on ‘indigestible’ ones. in addition, diatom cell size, attachment strength, frustule strength, condition of algal culture, aside from post-larval size can influence diatom digestibility (kawamura et al., 1995, 1998b). also the control of food supply is often considered an important factor in affecting growth and survival during the postlarval period as they increase feeding rate and as they grow (roberts et al., 1999a; martínez-ponce & searcybernal, 1998). in fact, grazing and growth rates of older post-larvae increase linearly with diatom density (searcy-bernal et al., 2001) and under continuous dark conditions (gorrostieta-hurtado & searcy-bernal, 2004). it is the aim of this study to isolate different local diatom strains and test their suitability as food source for postlarval h. asinina. by isolating and culturing benthic diatoms, there is a much better chance of achieving consistent larval settlement and good post-larval growth and survival. the diatoms to be used should practically be easy to culture and have fast growth in order to be a good candidate food species for the fast-growing postlarval abalone. materials and methods algal cultures several diatom species with prostrate growth forms were isolated from the acrylic settlement plates at the integrated mollusk and echinoderm hatchery at the bolinao marine laboratory (bml) in bolinao, pangasinan. kawamura & kikuchi (1992) suggest that prostrate diatoms induce higher settlement success than three-dimensional diatom communities. also, young post-larvae are known to actively select small, prostrate species as food source (norman-boudreau et al., 1986; matthews & cook, 1995; siqueiros-beltrones & voltolina, 2000). hence, efforts were geared towards isolation and culture of diatom strains forming flat communities. diatoms were isolated by picking single cells with a microcapillary using an inverted optical microscope as described by brand (1990). isolated cultures were grown in plastic culture plates (costar, 12 wells) using modified jørgensen’s medium (jørgensen, 1962), supplemented with 0.05 ìg.l-1 vitamin b 12 and maintained under a light intensity of 150 ìe.m-1.s-1 at 12:12 l:d cycle in an air-conditioned room. the diatoms were grown and re-isolated for a number of times to make sure that a monospecific culture was attained. five isolated diatom strains were chosen to be used for the early feeding experiments (navicula mollis, n. ramosissima, stauroneis sp., pleurosigma sp. and cocconeis sp.). the diatom cultures were not axenic. the cell dimensions and density of diatoms used for the feeding experiments are shown in table 1. the diatom density was counted using a square grid inserted into the eyepiece of the inverted microscope. science diliman (july-december 2007) 19:2, 49-59 feeding, growth, and survival of post-larval abalone 51 preservation and identification of diatoms light microscopy and scanning electron microscopy were used for the examination of whole cells to identify the diatom species (round et al., 1990). the diatoms were scraped, washed, centrifuged, acid-cleaned (hasle, 1978), fixed with 3% glutaraldehyde, washed with 0.1 m phosphate buffer, post-fixed with 1% osmium tetroxide, buffer washed again, dehydrated in ascending alcohol series, stub mounted and coated with gold, and viewed up to 2,000 x using a scanning electron microscope (sem hitachi s-510). abalone post-larvae abalone larvae were obtained from controlled natural spawning and artificial fertilization at the bml hatchery. about 38-39 h after fertilization, larvae with creeping ability were placed in prepared settlement tanks with naturally occurring diatoms (capinpin & hosoya, 1995). after about a month, several post-larval abalone were dislodged from the settlement plates using fine needles. the needles were used to dislodge them at the posterior end of the shell. only healthy and undamaged animals were used for the feeding trials. feeding trials two feeding trials were carried out using petri dishes (90-mm diameter) containing 30 ml filtered seawater (fsw). in trial 1, six post-larval abalone (1,132.0±11.2 ìm sl) per dish were reared on each species of diatom at about 26-27°c under a light intensity of 120-150 ìe.m-2.s-1 with a 12 h l:d photoperiod. three replicates were made. in trial 2, five slightly larger post-larvae (1,392.1±12.7 ìm sl) per dish were used. two replicates were made under the same culture conditions. in both trials, the culture medium for the diatoms in the dishes was changed with 0.2 ìm fsw prior to addition of the post-larval abalone. the feeding behavior of the post-larvae was observed and shell length measured individually using a micrometer inserted in the eyepiece of the optical microscope. shell lengths were measured every 3 days over a period of 15 days. at the time of observations, the seawater in each of the dishes was also changed. the diatoms in all the dishes were abundant throughout the experiment. no antibiotics were used during the feeding experiments. the daily growth rates were calculated for each individual for the whole period of 15 d and between 19 d and 10-15 days (tables 3 and 4). only post-larvae that are alive were used to calculate the mean daily growth rates. post-larval survival was also observed throughout the experimental period. digestion efficiency digestion efficiency refers to the ability of post-larvae to digest diatoms which may result from disruption of cell wall during grazing or passage through the gut (kawamura, 1996). to measure the digestion efficiency of post-larval abalone fed with the five diatom species, five abalone (1,300 ìm sl) post-larvae were reared on each diatom species in a petri dish under the same table 1. mean cell sizes (length and width), initial density, and growth forms of 5 benthic diatom species used in post-larval feeding trials diatom species cell length cell width initial density growth forma (µm, mean±se) cells.cm-2, mean±se) trial 1 trial 2 type adhesive strength navicula mollis 36.45±0.70 7.18±0.17 (1.70±0.28) x104 (4.92±0.68) x104 a + stauroneis sp. 21.36±0.45 5.36±0.17 (2.78±0.00) x105 (2.34±0.39) x105 a + navicula ramosissima 18.63±0.59 7.00±0.23 (1.18±0.35) x105 (2.21±0.24) x105 a + pleurosigma sp. 77.72±1.19 19.00±0.49 (4.07±1.36) x103 (1.63±0.09) x104 a + cocconeis sp. 14.54±0.32 6.64±0.18 (2.37±0.50) x105 (3.46±0.04) x105 b +++ agrowth forms based on classification by kawamura (1996). they are classified into 7 types (a-g), based on mode of attachment, whether solitary or colonial, and by their motility and adhesive strengths. science diliman (july-december 2007) 19:2, 49-59 capinpin, emmanuel 52 culture conditions. before adding the post-larvae, the number of whole, live cells and broken ones in the petri dishes were counted using the inverted optical microscope. after 3 days of sufficient feeding, the postlarvae were transferred to individual disposable plastic petri dishes filled with 0.2 ìm fsw without diatoms. after 1-2 h, recently released feces excreted by the post-larval abalone in the individual petri dishes were picked up using a microcapillary pipette, transferred onto a glass slide, covered with a cover slip, and the number of whole, live cells and that of broken ones counted under the light microscope. the digestive efficiency (kawamura et al., 1995) was calculated using the formula below: digestion efficiency (%) = (1-l/lo) x 100 where l is the mean proportion of whole live cells to the total diatom cells in the fecal pellet and lo is the mean proportion of whole live cells to the total diatom cells at the bottom surface of the petri dish before grazing. data analysis data were analyzed using single classification analysis of variance (anova) followed by duncan’s multiple range test. data on survival rates were arcsine transformed prior to analysis (gomez & gomez, 1984). some data on n. ramosissima were excluded from statistical analysis due to low survival. results post-larval size all abalone post-larvae reared on each diatom species showed active feeding behavior at the start of the experiments as evidenced by their grazing activity and by the ginger color of their digestive glands. the ingestion of diatom cells was confirmed through direct observations of feeding using the inverted optical microscope. in both trials 1 and 2, no significant differences in sizes (shell lengths) of post-larval abalone were observed in all diatom treatments (p>0.05) until day 15, except for n. ramosissima which was excluded in statistical analysis because of poor survival (figure 1). post-larval survival in terms of survival of post-larval abalone, not all the diatoms offered resulted in high survival rates (tables 3 and 4, figure 2). in trial 1, significant differences were observed in survival rates beginning day 9, with both cocconeis sp. and n. mollis giving the highest survival rates of 100%, followed by pleurosigma sp. (88.89%), stauroneis sp. (72.22%), and n. ramosissima (27.78%). at the end of the culture trial (day 15), highest survival rates were consistently obtained from cocconeis sp. (88.89%) followed by navicula mollis (66.67%) and pleurosigma (66.67%), with no significant differences among the three species (p>0.05). significantly low survival was attained from stauroneis sp. (33.33%) and n. ramosissima (5.56%). in trial 2, closely similar results were observed with the highest survival rates on cocconeis sp. and pleurosigma sp. (both 80%) followed by n. mollis (40%) at the end of the 15-day feeding trial (p<0.05; figure 2, table 4). significantly low survival was attained on n. ramosissima (0%) on days 12-15. daily growth rates in trial 1, no significant differences in daily growth rates of post-larval abalone were observed on all diatoms n. mollis, stauroneis sp., cocconeis sp. and pleurosigma sp. during the entire culture period, except n. ramosissima which was excluded in statistical analysis due to lack of data (i.e., poor survival). however, significantly faster growth rates were observed on n. mollis and stauroneis sp. during the second week than the first week (p<0.05). in trial 2, no significant differences in dgrs were observed on h. asinina post-larvae fed cocconeis sp. (26.20 ìm.d-1), n. mollis (22.95 ìm.d-1), and stauroneis sp. (21.30 ìm.d-1) during the entire culture period (p>0.05). however, the daily growth rates observed on the 3 diatom strains were significantly different from pleurosigma sp. (9.05 ìm.d -1). significantly faster growth rates were also observed on n. mollis and stauroneis sp. during the second week than the first week (p<0.05). science diliman (july-december 2007) 19:2, 49-59 feeding, growth, and survival of post-larval abalone 53 tr ia l 1 1 1 .1 1 .2 1 .3 1 .4 1 .5 0 3 6 9 1 2 1 5 da ys o f c u lt u r e s h el l l en g th ( m m ) n . m o llis s t a u ro n e is n . ra m o s is s im a p le u ro s ig m a c o c c o n e is t r ia l 2 1 1 . 2 1 . 4 1 . 6 1 . 8 2 0 9 1 2 1 5 da y s o f c u lt u r e s h el l l en g th ( m m ) n . m o llis s t a u ro n e is n . ra m o s is s im a p le u ro s ig m a c o c c o ne is figure 1. shell lengths of post-larval abalone h. asinina cultured using different diatom strains for 15 days. science diliman (july-december 2007) 19:2, 49-59 capinpin, emmanuel 54 digestion efficiency in terms of digestion efficiency, the most digestible species were cocconeis (97.4%) and stauroneis (100%) with no significant differences between the two species (p>0.05, table 2), followed by pleurosigma (47.11%), n. mollis (28.65%), and n. ramosissima (7.81%). the digestion efficiencies of post-larval abalone fed on stauroneis sp. and cocconeis sp. were high compared to other diatoms (table 2). however, of the 2 species, only cocconeis sp. supported high survival size compared to other diatom treatments probably because of the short observation period (i.e., 15 days). highest survival was also attained using cocconeis and pleurosigma as compared to the other treatments. discussion a method commonly used in abalone hatcheries is to establish a visible (=dense) film of diatoms to trigger settlement and provide food for post-larvae. without control over the type of diatoms, fast growing naviculoids are usually dominant. these may or may not be good for settlement and may or may not produce good post-larval growth and survival. these films develop rapidly, becoming very dense and dark, and often peeling as sheets. these cause strong fluctuations in boundary layer water quality (searcy-bernal, 1996; gorrostieta-hurtado & searcy-bernal, 2004), and this could explain the sudden mortality episodes that hatcheries often experience 2-8 weeks after settlement. post-larval abalone begins feeding soon after metamorphosis and eats small diatoms cells. however, most of the diatoms that post-larvae eat are not ruptured during feeding, and will pass through the gut alive. some strains of diatoms are readily ruptured while others are not. young post-larvae grow at similar rates on digestible and indigestible diatoms, but older post-larvae (>0.6-0.8 mm shell length) grow much more rapidly when fed breakable diatoms. stauroneis sp. is easily broken because they have very weak cell walls and appears to be a good candidate species for early feeding of abalone. however, this species is very fast-growing, usually becoming dense in a few days, and often observed to be peeling as sheets. this could cause large fluctuations in boundary layer water quality (e.g. dissolved oxygen), which may not be beneficial for young post-larvae. because of their fast growth, they are observed to be growing on the shells of post-larvae. they may also interfere with proper settlement and metamorphosis as the larvae become entangled (personal observations) and as mentioned by gorrostieta-hurtado & searcy-bernal (2004). hence, they are best added during the later stages of postlarval period. indeed, ungrazed diatom plates induce table 2. digestibility (digestive efficiency) of 5 benthic diatom species used in post-larval feeding trials diatom species percentage intact cells digestion (%, mean±se) efficiency* before in the fecal grazing (lo) material (l) n. mollis 100.00±0.00 71.35±3.60 28.66±3.60c stauroneis sp. 100.00±0.00 0.00±0.00 100.00±0.00a n. ramosissima 100.00±0.00 92.19±1.62 7.81±1.62d pleurosigma sp. 94.63±2.47 52.93±5.46 44.35±4.80b cocconeis sp. 100.00±0.00 2.60±1.26 97.40±1.26a *digestion efficiency (%)=(1-l/lo)x100 throughout the culture trials (figure 2, tables 3 and 4). stauroneis sp. resulted to a high digestion efficiency, which was also confirmed by sem when most cells were destroyed during scraping and acid cleaning, but it did not necessarily supported a high survival rate. although it supported good growth rates during the second week, these growth data were taken only from few surviving ones. the formation of first respiratory pore (notch stage) was observed at a shell length of 1.70-1.80 mm, whereas the closed respiratory pore (juvenile stage) was observed at 1.85-1.90 mm. at the end of trial 2, most of the animals reared on cocconeis sp. were in the notch stage or have closed respiratory pores, whereas only a few reared on n. mollis and pleurosigma sp. are in the notch stage. post-larval h. asinina fed on cocconeis sp. were the largest, although no significant differences were observed in science diliman (july-december 2007) 19:2, 49-59 feeding, growth, and survival of post-larval abalone 55 trial 1 0 20 40 60 80 100 0 3 6 9 12 15 culture period (days) s ur vi va l r at e (% ) n. m ollis stauroneis n. ram osissima pleurosigma cocconeis trial 2 0 20 40 60 80 100 0 3 6 9 12 15 culture period (days) s ur vi va l r at e (% ) n. m ollis stauroneis n. ram osissima pleurosigma cocconeis figure 2. survival rates of post-larval abalone h. asinina fed various diatom strains for 15 days. science diliman (july-december 2007) 19:2, 49-59 capinpin, emmanuel 56 lower attachment than pre-grazed diatom plates (gallardo & buen, 2003). cocconeis sp. has high adhesive strength and cells are easily broken during ingestion. on the other hand, all the other diatoms are mobile with low adhesive strengths and easily ingested by post-larval abalone without destruction of siliceous frustules, with exception of stauroneis sp. kawamura et al. (1995; 1998a) suggested that it is important for larger post-larvae (similar size to that used in the present study) to split open the cell walls by action of radula during ingestion or passage through gut to enable them to utilize the contents. as shown in the present study, cocconeis sp. a highly digestible diatom resulted to good growth and high survival rates especially in actively feeding post-larvae, but there may be exceptions (e.g. stauroneis sp.). these results are in conformity with earlier studies on temperate abalone (kawamura et al., 1995, 1998a). the results of this study verified that the physical characteristics of a diatom species, whether it is ruptured or not during grazing, is one of the most important factors determining its dietary value for growth of 1-2 mm post-larval abalone. various other factors may also influence the nutritional value of diatoms. these include the nature and quantity of their extracellular substances and secretions, the health (stage) of diatom culture, associated microbial flora (kawamura et al., 1998b) and biochemical composition (brown & jeffrey, 1995; carbajal-miranda et al., 2005; gordon et al., 2006 ). significantly faster growth rates during the second week for n. mollis and stauroneis sp. could be due to their increased capability to digest diatoms as they grow. the digestion efficiency of stauroneis sp. was high, although it had relatively low attachment strength, which could be due to structurally weak silica frustules, similar to that observed by kawamura et al. (1995) for cylindrotheca closterium. the others, n. mollis and n. ramosissima, passed through the gut intact. both were easily ingested due to low adhesive strengths. the low digestive efficiencies of these diatoms as expected resulted to slow growth rates particularly during the first week. roberts et al. (1999b) and onitsuka et al. (2004) found several progressive changes in the radula as post-larval abalone grow. small post-larvae (h. iris, <1 mm sl and h. diversicolor aquatilis, <2 mm sl) had highly curved teeth and low clearance angles, meaning that their teeth function as “scoops” which slide across the surface capable of collecting small diatoms and other fine loose particles such as extracellular mucus of diatoms. on the other hand, larger post-larvae had higher clearance angles, which are more suitable for grazing substrata to efficiently remove strongly attached diatoms. post-larvae also develop digestive enzymes as they grow (takami et al., 1998). cocconeis sp. is a very tightly attached, prostrate species. they become probably energetically inadequate as post-larvae grow as shown by slowing of growth during the second week (tables 3 and 4). the relatively higher settlement rate (unpublished data) and food value of this diatom make it a favorable candidate in the initial stages of abalone culture. three dimensional diatom communities, larger species, as well as fast-growing diatoms (e.g. stauroneis sp.) provide a much higher biomass per unit area than low volume prostrate cells. hence, they are also needed as they grow and best added during the later stages of post-larval rearing. a combination of selected species may also be beneficial (gordon et al., 2006). the ability to maintain a suitable quantity of food is also important in abalone hatcheries. ingestion rates increase as they grow (martínez-ponce & searcybernal, 1998; roberts et al., 1999a; searcy-bernal et al., 2001), so rapid clearing of diatom films is common. post-larvae are also observed to feed less and grow slower at lower diatom densities (day et al., 2004). it is interesting to note that post-larvae are relatively tolerant of starvation, but should not be starved for period longer than a week (roberts et al., 2000). it is very important to provide constant supply of appropriate diatoms during various stages of post-larval rearing. this issue is important for hatchery management in order to plan activities such as the type of diatoms to be used for settlement, type of food suitable at a particular stage, and the time at which it should be added. the control of diatoms used for settlement and early feeding in abalone hatcheries science diliman (july-december 2007) 19:2, 49-59 feeding, growth, and survival of post-larval abalone 57 table 3. shell length, daily growth rate, and survival rate of post-larval h. asinina fed on 5 diatom strains (trial 1) table 4. shell length, daily growth rate, and survival rate of post-larval h. asinina fed on 5 diatom strains (trial 2) should improve juvenile production. the use of diatoms as early food source for abalone still remains as the most viable method, although a new system utilizing artificial diet bound with agar/alginate solution to the plastic plates have shown promising results (stott et al., 2003, 2004). cocconeis is easy to culture and has very fast growth rates. it should be provided during the initial stages of the culture period particularly during settlement and early feeding. as post-larval abalone grows, the addition of other diatom species (stauroneis sp., n. mollis and other larger diatoms) should be applied in order to support fast growth rates. acknowledgements many thanks to ma. jocelyn c. ramirez of the electron microscopy service laboratory of the national institute of molecular biology and biotechnology, university of the philippines at los baños, laguna for help in sem work, dr. tomohiko kawamura for advice before the conduct of the work and identification of diatoms, and prof. ceferino f. toledo for reviewing the original manuscript. this research was supported by a fellowship granted to the author from the department of agriculture bureau of agricultural research (dabar) institutional development grant. this work was done at the laboratory of dr. annette juinio-meñez at bml, marine science institute of the university of the philippines. diatom strain shell length daily growth rate survival rate (µm, mean±se) (µm.day-1, mean±se) (%) initial final whole period week 1 week 2 day 3 day 9 day 15 (15 days) (day 1-9) (day 10-15) n. mollis 1,363.2±53.7 1,707.22±26.1 22.9±1.8a 14.6±1.7b 35.4±2.1a 50.0±30.0a 40.0±20.0a, b 40.0±20.0a, b stauroneis sp. 1,358.2± 7.2 1,677.5±61.3 21.3±4.6a 10.0±3.5b 38.3±6.2a 50.0±10.0a 50.0±10.0a, b 20.0± 0.0b n. ramosissima 1,405.9± 0.9 8.4* 20.0±20.0a 0.0±0.0b 0.0± 0.0b pleurosigma sp. 1,405.2±27.2 1,540.9±85.0 9.0±3.9b 7.2±3.4b 11.9±4.5b 80.0±20.0a 80.0±20.0a 80.0±20.0a cocconeis sp. 1,427.7± 2.5 1,820.8±66.2 26.2±4.6a 25.6±2.0a 27.1±8.5a, b 90.0±10.0a 80.0±20.0a 80.0±20.0a trial 2 = 2 replicates means with the same superscript are not significantly different from each other (p>0.05) *for 1 replicate only diatom strain shell length daily growth rate survival rate (µm, mean±se) (µm.day-1, mean±se) (%) initial final whole period week 1 week 2 day 3 day 9 day 15 (15 days) (day 1-9) (day 10-15) n. mollis 1,143.6±29.4 1,403.2±15.5 17.3±3.0a 6.3±0.7a 33.8±8.0a 100.0±0.0a 100.0± 0.0a 66.7± 9.6a, b stauroneis sp. 1,136.0±19.0 1,408.3±38.6 18.2±3.7a 13.8±1.9a 24.7±7.6a, b 100.0±0.0a 72.2± 5.6c 33.3± 9.6b, c n. ramosissima 1,143.2±17.8 1,317.1* 10.1* 9.0±4.8 7.7* 100.0±0.0a 27.8± 5.6d 5.6± 5.6c pleurosigma sp. 1,139.7±28.7 1,249.1±14.9 7.3±2.8a 6.4±2.2a 8.6±6.1b 100.0±0.0a 88.9± 5.6b 66.7±16.7a, b cocconeis sp. 1,097.5±35.2 1,401.8±63.5 20.3±6.6a 23.2±9.5a 16.0±2.2a, b 100.0±0.0a 100.0± 0.0a 88.9± 5.6a trial 1 = 3 replicates means with the same superscript are not significantly different from each other (p>0.05) *for 1 replicate only science diliman (july-december 2007) 19:2, 49-59 capinpin, emmanuel 58 references brand, l.e., 1990. the isolation and culture of microalgae for biotechnological applications. in: labeda, d.p. 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(ed.) phytoplankton manual. unesco, pp. 136142. jørgensen, e.g., 1962. antibiotic substances from cells and solution of unicellular algae with special reference to some chlorophyll derivatives. physiol. plant. 15:530-545. kawamura, t., 1996. the role of benthic diatoms in the early life stages of the japanese abalone (haliotis discus hannai). in watanabe, y., y. yamashita & y. oozeki (eds.) survival strategies in early life stages of marine resources. a.a. balkema, pp. 355-367. kawamura, t. & s. kikuchi, 1992. effects of benthic diatoms on settlement and metamorphosis of abalone larvae. suisanzoshoku 40:403-409. kawamura, t., r.d. roberts, & c.m. nicholson, 1998a. factors affecting the food value of diatom strains for postlarval abalone haliotis iris. aquaculture 160:81-88. kawamura, t., r.d. roberts, & h. takami, 1998b. a review of the feeding and growth of post-larval abalone. j. shellfish res. 17:615-625. kawamura, t., t. saido, h. takami, & y. yamashita, 1995. dietary value of 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17:723-727. science diliman (july-december 2007) 19:2, 49-59 editorial.pmd editorial for the past thirty-two years of science diliman, the journal published a relatively low number of articles. despite this, the contents continually demonstrate the use of science to respond to problems found in the philippines. articles of foreign authors are sometimes published in the journal, the aim of which is to show the similarity among scientists in answering scientific questions in a third world environment like ours. the five articles in this issue dwell on topics in chemistry, toxicology, material science and mariculture. all of the articles show how filipino scientists respond to local problems, thus manifesting their love of country – a trait that the university of the philippines inclulcates among its constituents. moreover, one should not forget the contributions of the editors, referees and technical staff. all do their share in ensuring that the articles are worthy of publication. their contributions may well be considered as acts of nationalism. the uniqueness of this journal is in the underscoring of the works of local scientists and technologists, generally with up diliman, that help alleviate the problems of the filipino community. the journal also accentuates the promotion of science and literacy, which hopefully would lead to the economic uplifting of the philippines. with this perspective in mind, we hope that more manuscripts would be submitted to this journal for publication. i 90 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions 9 if changes are made, choose a new title for the paper. 9 use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality. 9 reference your conference paper in the appropriate locations. 9 include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.” 9 indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed. 9 provide the original conference paper (upload a pdf file during the submission process). 9 if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions 9 expand the background section and include additional references. 9 include novel scientific content and expanded descriptions of procedures. 9 provide data that was not published at the conference. 9 revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission from the editors of ieee sensors journal) 68 body size, habitat, and diet of freshwater crabs isolapotamon mindanaoense and sundathelphusa miguelito (crustacea: brachyura) in the municipality of lake sebu, south cotabato, philippines ziljih s. molina jemateo b. neri rizza may p. cañete ephrime b. metillo* department of biological sciences college of science and mathematics mindanao state university-iligan institute of technology abstract isolapotamon mindanaoense (rathbun 1904) and sundathelphusa miguelito mendoza and sy 2017 have narrow biogeographical distribution and are both regarded as endemic to mindanao island. they are common and publicly consumed freshwater or semi-terrestrial crabs inhabiting vicinities near the waterfalls of lake sebu municipality, south cotabato in mindanao, but both species are scarcely investigated. this study aimed to examine the body size, microhabitat and the feeding ecology of these freshwater crab species. sex and carapace width and length of individuals were determined from specimens collected by hand at three waterfall sampling sites. feeding and feeding niche overlap were respectively analyzed using the index of relative importance (iri) of prey items from individual crab stomachs and the schoener’s r o index. food items ingested include fish fragments, insect body parts, fragments of aquatic vascular plants, freshwater algae, sand grains and amorphous materials, and these items were similar between species. however, the larger i. mindanaoense appeared to ingest more fish fragments and other animal prey items compared to s. miguelito which ingested more amorphous materials that are derived from benthic plants. however, the r o value of 93% was high, suggesting very similar diet. the two species further partition niches, with s. miguelito being smaller in size and inhabiting sand and gravel substrate, while the larger i. mindanaoense inhabit areas with big boulders. hence, the crabs can be categorized as omnivorous and detrivorous, and exhibit feeding and habitat niche partitioning that alleviate possible resource competition between the two species. keywords: mindanao, feeding ecology, iri, omnivory, niche partitioning, diet * corresponding author science diliman (january-june 2020) 32:1, 68-87 z.s. molina et al. 69 introduction primary freshwater crabs are found in the tropics and subtropics and occur in a wide variety of aquatic and terrestrial habitats (cumberlidge 2016). these decapod crustaceans are present in tree holes and leaf axils and in almost all freshwater bodies (ng and ng 2018). a fair number of species are also adapted to live in caves and rice fields (yeo et al. 2008). freshwater crabs are primarily nocturnal, preferring to remain hidden during the day in sheltered places and foraging mostly at night (grinang et al. 2016). they are largely omnivorous scavengers, mainly feeding on plant matter, but some are opportunistic carnivores feeding either on live prey such as fish and prawns or on dead animals that they encounter, and cannibalism is not uncommon (ng and tan 1998; magalhães 2003; cumberlidge 2016). freshwater crabs are one of the most ecologically important macro-invertebrate groups in tropical inland waters worldwide (rodriguez and magalhães, 2005; yeo et al. 2008). like most freshwater decapod crustaceans, these crabs have very high level of endemicity, require good forest cover (yeo et al. 2008) and pristine water conditions to survive and are excellent indicators of good water quality (reynolds et al. 2013). crabs are never considered to be an important group in trophic webs, and this might be due to lack of knowledge about their trophic roles in aquatic ecosystems (grinang et al. 2016). previous studies have suggested that crabs show selectiveness in food preferences depending on the food’s nutritional values, varieties and accessibility (meziane et al. 2002; dahdouh-guebas et al. 2011). moreover, studies of the feeding ecology of animals contribute to the knowledge of the nutritional requirements of species, interactions with other organisms, and community organization patterns over evolutionary time (carvalho et al. 2016; cumberlidge 2016). according to giddins et al. (1986), crabs prefer decaying leaves to senescent or fresh leaves when given the choice. they also suggested that crabs allow leaves to decompose inside their burrows for many weeks before eating them. during this time, tannins are lost from the leaves through leaching, while nitrogen concentration increases through bacterial action, resulting in higher nutritional content. another advantage of taking leaves into their burrows is having a safe environment for crabs to eat without fear of predators, tidal inundation, high temperatures and low humidity (wolcott and o’connor 1992). as is characteristic of brachyurans, most of the food items were found to be highly crushed into small fragments and hence only those structures that could be identified were relied upon for determining food composition and evaluation (carvalho et al. 2017). body size, habitat, and diet of freshwater crabs 70 futuyma (2005) reported that physical conditions of freshwater crabs (substratum type, salinity, temperature) as well as ecological traits (competitive interactions, predation pressure and feeding behavior) may differ among various environments. all these factors have led to a high degree of endemism in freshwater crabs. one of the key processes driving freshwater crab diversification is allopatric speciation resulting from geographic isolation, relatively low fecundity and poor dispersal abilities (cumberlidge et al. 2009). these ecological conditions and resources that influence the survival and persistence of species in an ecosystem are within the purview of the ecological niche concept (holt 2009). an ecological niche of a species is an abstract mapping of population parameters onto multidimensional environmental space, the axes of which are abiotic and biotic factors that influence birth and death rates (holt 2009). speciation of freshwater crabs can be linked with niche segregation and diversification (marijnissen et al. 2008), but the present changing environments and habitat loss can also contribute to niche shifts (cumberlidge et al. 2009). the recent discovery of sundathelphusa miguelito adds to the four species of the same genus endemic to mindanao island (mendoza and sy 2017). holotypes of the species were collected from barangay lake seloton, municipality of lake sebu, south cotabato province. on the other hand, isolapotamon mindanaoense was first described in 1998, adding to the three mindanao island endemic species of the same genus (ng and tan 1998). the type locality of i. mindanaoense was along tibuan river that empties into davao gulf. the ecology and/or biology of both freshwater and/or semi-terrestrial species are poorly studied (ng and tan 1998; esser and cumberlidge 2008; cai et al. 2016; mendoza and sy 2017). the ecological role of many tropical freshwater crabs is least understood despite their importance in trophic structures including food chains that can be traced up to human consumption (yeo et al. 2008; williner et al. 2014; cumberlidge et al. 2015). the body of knowledge on the biology and trophic roles of freshwater crabs in aquatic ecosystems is inadequate (grinang et al. 2016). information on body size and the feeding ecology of freshwater crabs is crucial for the conservation (yeo et al. 2016) and aquaculture utilization of species (pan et al. 2020). examining the food and feeding habits or diet composition is considered very important in crustacean ecology, biology and evolution (felgenhauer et al. 1989) and in developing conservation strategies (yeo et al. 2016; cumberlidge 2016). this study aimed to quantify the natural diet of the two freshwater crab species through stomach content analyses of freshwater crabs inhabiting lake sebu municipality in the province of south cotabato, mindanao, philippines. z.s. molina et al. 71 materials and methods description of study area specimens were collected in the municipality of lake sebu, south cotabato, which is located south-central of mindanao island, southern philippines and lies geographically between 6° 10.45’n and 124° 43.95’e (figure 1), surrounded by rolling hills and mountains covered with thick rain forest. the municipality has three lakes with lake sebu as the biggest. the lake’s shores and the surrounding rainforest are home to the indigenous t’bolis, tirurays, ubos and manobos. the vegetation of lake sebu, south cotabato can be classified into two categories, forest land and cultivated land. the major crops are corn and rice, and the type of soil is sandy loam. there are few extensive wetlands within the province of south cotabato, the most significant being lake sebu. most of the landscape in the area has been modified for eco-tourism or is utilized for irrigated rice cultivation. 124o43'04.56" 124o42'38.31" 124o42'19.56" 124o43'47.48" 124o44'05.54" 6o 15 ' 1 0. 81 " 6o 14 ' 5 0. 37 " 6o 14 ' 2 9. 79 " 6o 14 ' 0 4. 80 " 6o 13 ' 5 5. 96 " 1 2 3 mindanao figure 1. map of the philippines showing lake sebu and sampling sites (1, 2, and 3) for freshwater crabs. body size, habitat, and diet of freshwater crabs 72 sampling sites all three sampling sites are located in barangay lahit, lake sebu (figure 1). the barangay is located 684 meters above sea level (masl). the vegetation cover in the sampling area includes many fruit trees, overshadow trees, grasses and vegetables. the fruit trees available are palm, citrus and mango. this area has several scenic waterfalls located along the main stream of a river originating from nearby lake seloton in the east. lake sebu is the source of these waterfalls and their water outflow pass through the nearby agricultural lands, municipalities and barangays. crabs were collected by hand at the vicinities of hikong alo falls (site 1), hikong bente falls (site 2), and hikong b’lebel falls (site 3). neither residential nor commercial establishments are found along the river of the falls. a b c figure 2. sampling localities for the two freshwater crab species in lake sebu, south cotabato: (a) hikong alo falls, (b) hikong bente falls and (c) hikong b’lebel falls. the first waterfall, dubbed hikong alo, which literally means passage falls in t’boli is an ecotourism site (part of lake sebu’s seven waterfalls and zipline adventure). this site is located 684 meters above sea level (masl) at 6°14’ 36.3” and 124°43’ 45.4”e (figure 2a). located approximately 1.3 kilometers from the main road, this is the widest and most accessible among the seven falls in the area. the river in the waterfalls is relatively shallow and fast flowing with a muddy to rocky substrate. z.s. molina et al. 73 during the sampling period, the water was muddy and dark because it rained the night before. about 1 m downstream of the area, the river deepens and substrate at this site is silt. hikong bente waterfalls (figure 2b) is located 659 masl at 6°24’ 42.3”n and 124°43’ 44.8”e. it is a 70-foot tall waterfall with sheer rock wall standing on its left while a menagerie of shrubs clings on the right wall. the third waterfall, hikong b’lebel (figure 2c), is situated 714 masl at 6°14’ 51.7”n and 124°43’ 41.6”e. it was not accessible and required passing through slippery rocks and steep cliffs while clinging to the roots of whatever plants or trees growing in its cliffs. the water was shallow and fast flowing through a rocky to sandy substrate. the water temperature in sites 1, 2 and 3 were 25°c, 26°c and 26°c, respectively; ph levels in sites 1, 2 and 3 were 4.19, 3.35 and 3.59, respectively; and the respective dissolved oxygen measured in mg per liter were 5.73, 5.6 and 5.69. moreover, the current velocity were 1.2, 1.42 and 1.28 m/s, respectively. these values are within quality standards according to the department of environment and natural resources [denr] (2016) and can support aquatic life. collection and processing of samples information about the freshwater crabs was assessed via face to face interviews across the sampling sites. calling them “kagang”, local communities harvest the crabs for household consumption during the fallow season of farming. the two freshwater crab species, isolapotamon mindanaoense and sundathelphusa miguelito, were found to co-occur under rocks and in burrows along river banks. the entire sampling period was for three days (10-12 march 2017) with one day spent per site. samples were collected during the day (0500h-1200h and 1300h-1700h), involving purposive walks of six persons near the plunge area and along the stream bank of the waterfall sampling site. freshwater crabs were caught by hand in soil burrows, after lifting large stones and checking boulders submerged in shallow, slow-moving sections of the stream. the freshwater crabs were then narcotized and subsequently killed by immersing them in icy water. fresh coloration of the collected crabs was recorded by photography before preserving them in 95% ethanol. preserved samples were then immediately (< 2 days) transported to the laboratory as prolonged (10 weeks) storage in alcohol would have influence on carapace width (rufino et al. 2004) and body weight (qureshi et al. 2008) of the crab. body size, habitat, and diet of freshwater crabs 74 in the laboratory, the sex of individual crab was determined. also measured were the carapace width (cw, the distance across the carapace at its widest point) and carapace length (cl, the distance along the midline, from the frontal to the posterior margin of the carapace) using a vernier caliper with accuracy of 0.01 mm. crabs were counted and segregated based on their sex and size. the sex of the crabs was characterized based on the morphology of the abdomen. the pleon (abdomen) in most male crabs is narrow and triangular in form, while females have a broader and rounded abdomen (lopretto 1976). collected samples were identified based on the taxonomic key provided by ng (2004). stomach content analysis the diet composition of the freshwater crabs was evaluated through the examination of their stomach contents. the carapace of a crab was removed, and the entire stomach was carefully extracted. the stomach was dorsally cut and spread apart. stomach content was flushed onto a petri dish with filtered water, and viewed under a binocular microscope. as suggested by sukumaran and neelakantan (1977) each prey item was identified to the lowest possible taxonomic level. algae were identified based on intact filaments. for vascular plants, presence of fragments of leaves was useful in detecting macrophytic remains. insects were identified based on fragments of legs and wings. fish was identified based on undigested fragments of scales, teeth and gills. amorphous and sand grains were identified as decayed organic matter and fine inorganic particles, respectively. the feeding ecology of freshwater crabs was analyzed using the index of relative importance (iri). the iri is a composite measure that reduces bias in descriptions of animal dietary data (hart et al. 2002). the percentage number (n%), volume (v%) and frequency occurrence (f%) of different types of food materials were recorded. the percentages of f, v, n and iri were calculated using the following formulae of hyslop (1980): percentage occurrence, f% = the number of stomachs in which a given food item is found × 100 number of stomachs examined percentage numbers, n% = the number of food items found in each stomach × 100 number of total food items in all specimens percentage volume, v % = volume of one food item found in all specimens × 100 the volume of all food items in all specimens index of relative importance (iri) = f % × (n % + v %) z.s. molina et al. 75 diet overlap between species among sampling sites was computed using the formula of schoener (1970): r o = 100 (1 -σ|px i py i |/2), where r o is the overlap index expressed as percentage, and px i and py i are the relative importance (ratio of the points) of each food item i in the stomachs of predator x (isolapotamon mindanaoense) and y (sundathelphusa miguelito). results and discussion the two crab species crab individuals collected from sites 1, 2, and 3, respectively, were 45, 40, and 30 individuals for sundathelphusa miguelito (n = 115; 56 females and 59 males), and 32, 32, and 45 individuals for isolapotamon mindanaoense (n = 109; 48 females and 61 males) (table 1). i. mindanaoense individuals were most common at the highest elevation (site 3) while s. miguelito dominated the two lower elevation sites 1 and 2 (table 1). the two species are regarded as endemic to mindanao island (ng and tan 1998; mendoza and sy 2017). this is the first account on the demography and ecology of both species in a specific location in the southern part of mindanao island. as in the study of cai et al. (2016), more males were collected for the freshwater crabs parathelphusa reticulata and p. maculata. the disparity in numbers could be related to females having slower growth than males, which is probably a consequence of the different reproductive effort between the sexes (hartnoll and gould 1988). but sampling bias and behaviour (colpo and negreiros-fransozo 2016) and the impact of predation and/or harvesting (cumberlidge 2011; yeo et al. 2008) may also explain the deviation from the 1:1 female to male sex ratio. table 1. number of individuals of freshwater crabs isolapotamon mindanaoense and sundathelphusa miguelito examined for their diet from the three sampling sites in lake sebu, mindanao, philippines number of individuals sampling sites species site1 site 2 site 3 total gecarcinucidae sundathelphusa miguelito 45 40 30 115 potamidae isolapotamon mindanaoense 32 32 45 109 total number of individuals 77 72 75 224 body size, habitat, and diet of freshwater crabs 76 morphological description following bott (1970) and ng and tan (1998), the male abdomen of i. mindanaoense is proportionately broader (figure 3). the larger female specimen possesses a distinctly higher and more convex carapace, and the dactylus of the last ambulatory leg is also proportionately longer. carapace ovoid, dorsal surface distinctly convex, smooth; anterolateral regions without granules. according to mendoza and sy (2017), the live coloration of the carapace of sundathelphusa miguelito ranges from pale yellow to pale brown (figure 4). legs and ventral surface of the body are lighter in color on all specimens. male pleon (figure 4d) inverted t-shaped, relatively broad for the genus; dorsal surface gently convex longitudinally. body size, habitat, and diet of freshwater crabs 76 morphological description following bott (1970) and ng and tan (1998), the male abdomen of i. mindanaoense is proportionately broader (figure 3). the larger female specimen possesses a distinctly higher and more convex carapace, and the dactylus of the last ambulatory leg is also proportionately longer. carapace ovoid, dorsal surface distinctly convex, smooth; anterolateral regions without granules. according to mendoza and sy (2017), the live coloration of the carapace of sundathelphusa miguelito ranges from pale yellow to pale brown (figure 4). legs and ventral surface of the body are lighter in color on all specimens. male pleon (figure 4d) inverted t-shaped, relatively broad for the genus; dorsal surface gently convex longitudinally. figure 3. isolapotamon mindanaoense female dorsal and ventral view (a & b) and male dorsal and ventral view (c & d). scale bar =20 mm.figure 3. isolapotamon mindanaoense female dorsal and ventral view (a & b) and male dorsal and ventral view (c & d). scale bar =20 mm. z.s. molina et al. 77 z.s. molina et al. 77 figure 4. sundathelphusa miguelito dorsal and ventral view of female crab (a & b) and male dorsal and ventral view (c & d). scale bar = 20 mm. body size and distribution both species were found in all sampling sites with individuals normally associated with burrows. a total of 224 individual specimens were collected with sizes ranging between 21-13 mm cl and 26.5-16 mm cw for s. miguelito, and 41-12 mm cl and 46-20 mm cw for i. mindanaoense. carapace width values regressed (r2 = 0.95, p< 0.05) with carapace length values of males and females of both species clearly show smaller s. miguelito individuals as against those of i. mindanaoense with an overlap of large size s. miguelito female crabs and the smaller i. mindanaoense individuals (figure 5). figure 4. sundathelphusa miguelito dorsal and frontal view of female crab (a & b) and male dorsal and ventral view (c & d). scale bar = 20 mm. body size and distribution both species were found in all sampling sites with individuals normally associated with burrows. a total of 224 individual specimens were collected with sizes ranging between 21-13 mm cl and 26.5-16 mm cw for s. miguelito, and 41-12 mm cl and 46-20 mm cw for i. mindanaoense. carapace width values regressed (r2 = 0.95, p< 0.05) with carapace length values of males and females of both species clearly show smaller s. miguelito individuals as against those of i. mindanaoense with an overlap of large size s. miguelito female crabs and the smaller i. mindanaoense individuals (figure 5). body size, habitat, and diet of freshwater crabs 78 y = 1.07x + 2.74 r2 = 0.95 n = 145 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 im, female im, male sm, female sm, male carapace length (mm) c ar ap ac e w id th (m m ) figure 5. carapace width and carapace length scatterplot for females and males of both isolapotamon mindanaoense (im) and sundathelphusa miguelito (sm) crabs from south cotabato, mindanao. the distribution of different sizes of male and female individuals of both species showed some degree of overlap (figure 6). the largest male and female individuals of i. mindanaoense were collected at the highest elevation site (site 3), but the distribution of these individuals does not seem to overlap with individuals of s. miguelito of both sexes at the same site. males of i. mindanaoense had an overlapping distribution with both sexes of s. miguelito at the lowest elevation (site 2). similarly, at the intermediate elevation (site 1), males of i. mindanaoense overlapped with the distribution of s. miguelito females, and some small-sized female i. mindanaoense overlapped with both sexes of s. miguelito. habitat partitioning was shown with most s. miguelito individuals inhabiting sand and gravel substrate, while the larger i. mindanaoense inhabit areas with big boulders. z.s. molina et al. 79 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 11 13 14 16 17 19 20 22 23 25 26 28 29 31 32 34 35 37 38 40 41 43 site 1 684 masl site 2 659 masl site 3 714 masl a b c size range (mm) fr eq ue nc y) female im male im female sm male sm legend: figure 6. size-frequency distribution histograms for males and females of both isolapotamon mindanaoense (im) and sundathelphusa miguelito (sm) in the three waterfall sampling sites. masl = elevation as meters above sea level. body size (schoener 1970) and spatial distribution (uzunmehmetoğlu et al. 2019) are good indicators of potential competition among species that belong to the same clade. similar body size of sympatric crab species may suggest stronger competition as the range of food items would overlap for individuals of these species (giri and loy 2008). smallest individuals of i. mindanaoense and the largest individuals of s. miguelito would be expected to compete on similar food items as their body sizes strongly overlap, and the overlap seems to be strong at the two lower elevation sites. however, possible competition would be minimized as both species inhabit different microhabitats as mentioned above. body size, habitat, and diet of freshwater crabs 80 stomach content and feeding niche overlap all 224 collected crab individuals of the two species were dissected for gut content analysis. the food items eaten by the two freshwater crab species showed that their trophic roles in the community are as omnivores and opportunistic predators that link several trophic levels from both aquatic and terrestrial communities. in total, six food categories (table 2) were identified from foregut contents in i. mindanaoense and s. miguelito. different food items were found in the stomach of the freshwater crabs, including fish, insects, plant, algae, sand grains and amorphous. the overall food composition in their gut was similar in both male and female species. however, differences in the trophic activity between juvenile and adult crabs were not observed. studies of the feeding ecology of animals contribute to the knowledge of the nutritional requirements of species, interactions with other organisms, and community organization patterns over evolutionary time (lampert et al. 1992). freshwater crabs generally emerge from their hideout burrows at night (nocturnal) and forage for food (yeo et al. 2008). they are primarily omnivorous; generally scavengers but some are primarily vegetarians while other species are able to capture live prey (grinang et al. 2016). table 2. frequency of occurrence (fo), number (n), volume (v), and the index of relative importance (iri) of the different stomach food items of freshwater crabs isolapotamon mindanaoense and sundathelphusa miguelito food item %fo %n %v iri % iri isolapotamon mindanaoense fish 37.60 27.60 18.25 1723.96 30.49 insects 17.60 7.23 9.51 294.62 5.21 vascular plants 29.50 20.50 20.34 1204.78 21.31 freshwater algae 21.60 11.56 15.40 498.62 8.82 sand grains 20.70 11.17 14.64 534.27 9.45 amorphous 31.92 21.94 21.86 1398.10 24.73 sundathelphusa miguelito fish 37.50 22.11 32.96 2065.13 24.47 insects 15.60 9.76 19.26 452.71 5.36 z.s. molina et al. 81 food item %fo %n %v iri % iri vascular plants 32.50 20.00 39.26 1925.95 22.82 freshwater algae 20.60 10.73 21.11 655.90 7.77 sand grains 22.70 11.54 22.96 783.15 9.28 amorphous 39.70 25.85 38.52 2555.49 30.28 the stomach contents of both crab species appeared to contain highly digested matter and hence identification of food organisms was found difficult. further, unlike in large-sized crabs, no crustacean or mollusc was found in the stomachs of small-sized crabs possibly due to their less developed chelae, which were not strong enough to crush the exoskeleton of arthropoda as well as molluscs (sukumaran and neelakantan 1997; carvalho et al. 2017). in the stomach of i. mindanaoense (table 2), amorphous materials comprised the bulk of the diet (21.86% volume, 24.73% iri). fish fragments (37.6% frequency occurrence, 27.36% number, 30.49% iri) formed its most important prey and the most abundant ingested prey of the crab species. insects (5.21% iri) were the least important prey of the crab. it was observed that there was no variation in food items consumed among crabs with different body sizes and also no variation in ingested food items between male and female crabs. in percentage frequency of occurrence of prey items, fish was the principal food item in the stomachs of i. mindanaoense (37.6%). the percentages of amorphous, plants, algae, and sand grains were the second highest (31.92%, 29.5%, 21.6% and 20.7%, respectively) while insects (17.6%) were found in smaller quantities (table 2). as for the percentage volume, major prey items in the stomach contents of i. mindanaoense were amorphous (21.86%), plants (20.34%), fish (18.25%), algae (15.4%), sand grains (14.64%) and insects (9.51%) (figure 5). the fish and insects in the stomachs could not be identified to species level due to the crushed and ground food items that occurred during the feeding behavior of the crabs. ranging from 5.21% to 30.49%, the iri of the food items in i. mindanaoense confirmed the predominance of principal prey items. the highest percentage iri was for fish fragments (30.49%), whereas the lowest value (5.21%) was for insects (table 2). a similar diet has been observed in the freshwater crab sundanonautes africanus. sodamola et al. (2016) reported that different food items were found in the guts of table 2. frequency of occurrence (fo), number (n), volume (v), and the index of relative importance (iri) of the different stomach food items of freshwater crabs isolapotamon mindanaoense and sundathelphusa miguelito (cont’n.) body size, habitat, and diet of freshwater crabs 82 s. africanus, irrespective of size, sex and season. they include insects, plant materials, detritus, algae and sand grains. sand grains in all the guts containing food of both s. miguelito and i. mindanaoense as the sand grains were probably ingested along with food items during feeding. sand grains or gastroliths are believed to help macerate food materials in the cardiac stomach of crustaceans (felgenhauer et al. 1989). in the stomachs of s. miguelito (table 2), plants composed the bulk of the diet (39.26% volume, 22.82% iri). amorphous (39.7% frequency occurrence, 25.85% number, 30.28% iri) formed its most important prey and the most abundant ingested prey of the crab. insects (5.36% iri) were the least important prey of the crab. the percentage volume showed that the major prey items in the stomach contents of sundathelphusa were plants (39.26%), amorphous materials (38.52%), fish (32.96%), sand grains (22.96%), algae (21.11%) and insects (19.26%), respectively. in percentage of frequency of occurrence, amorphous material was the principal food item in the stomachs (39.7%) of s. miguelito. the percentages of fish fragments, plant fragments, sand grains, and algae were the next highest (37.5%, 32.5%, 22.7% and 20.6%, respectively). insects (15.6%) were found in smaller quantities in the stomach of the crabs (table 2). freshwater crabs exhibited various feeding habits with many kinds of food items such as amorphous, fish, insects, plant materials, algae and sand grains. the iri values of the food items of s. miguelito ranged from 5.36% to 30.28%. the highest percentage iri was observed for amorphous materials (30.28%) whereas the lowest (5.36%) was for insects (table 2). a large amount of fish fragments was found in the stomach of the larger i. mindanaoense indicating that the animal is capable of ingesting both plant and animal matter along with dead and decaying matter, which points to its omnivorous nature. the large amount of animal matter ingested by the crabs suggest a way to enhance the energy supply derived from less nutritional food (williner et al. 2014). presently both small and large crabs were observed to consume algae, insects, fish, plant matter, sand grain and amorphous material. the large amount of amorphous materials in the stomach of s. miguelito may be attributed to the degree of digestion in the macerating cardiac stomach of the crab. the study showed that the animal is capable of ingesting both plant and animal matter along with dead and decaying matter that indicated the omnivorous nature of the animal. this is in agreement with the freshwater crabs v. litterata or herring bow crab (devi et al. 2013), maydelliathelphusa masoniana (sharma et al. z.s. molina et al. 83 2016), and trichodactylus borellianus (carvalho et al. 2017), which were all reported to be omnivores capable of ingesting both animal and plant tissues. this study also revealed that s. miguelito is a bottom feeder because examination of its stomach contents showed that the deposit feeding method was more usual. future studies should attempt to confirm the findings here by investigating the feeding activity of male and female and different-sized (adults and juveniles) i. mindanaoense and s. miguelito, both daily and seasonally, as well as looking at the feeding morphology of both species. conclusion this study provides some baseline information on body size, distribution and feeding habits of endemic freshwater crab species isolapotamon mindanaoense and sundathelphusa miguelito. body sizes of the specimens collected overlapped among males and females within each species, but habitat partitioning was evident with the large-sized i. mindanaoense that were more common in big boulder habitats than the smaller s. miguelito which were found in sand and gravel substrates. in this study, the stomach contents of the two freshwater crab species had similar types of food items like insects, sand grains, fishes, algae, plants and amorphous material. however, some differences were observed based on the higher frequency of fish and animal fragments in the stomachs of the larger i. mindanaoense than those in s. miguelito, which appeared to consume more plant-based amorphous materials and decayed matter. thus, the crabs under study are omnivorous and detritivorous, considering that they consume plant and decomposed matter as well as animal matter. all kinds of food consumed were readily available, showing that the crabs were not selective in the consumption of available food in the study area. acknowledgments we are deeply grateful to the ched scholarship and thesis grant of zs molina, jb 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(sudanonautes africanus milne-edwards, 1983) at the bank of asejire in south-west, nigeria. int j fish aquat stud. 4(3): 538-542. sukumaran kk, neelakantan b. 1996. relative growth and sexual maturity in the marine crabs, portunus (portunus) sanguinolentus (herbst) and portunus (portunus) pelagicus (linnaeus) along the southwest coast of india. indian j fish. 43(3): 215-224. uzunmehmetoğlu oy, buřič m, erol kg, özköa r, çınar ş, kozák p. 2019. habitat separation of the crab potamon potamios and the crayfish pontastacus leptodactylus in lake eğirdir, turkey. limnologica. 78:125692. williner v, collins p. 2014. feeding spectra and activity of the freshwater crab trichodactylus kensleyi (decapoda: brachyura: trichodactylidae at la plata basin. zool stud. 53:71. wolcott dl, o’connor nj. 1992. herbivory in crabs: adaptations and ecological considerations. am zool. 32: 370-381. yeo dcj, luz s, cai y, cumberlidge n, mcgowan pjk, ng djj, raghavan r, davison gwh. conservation first: strategic planning to save the critically endangered singapore freshwater crab johora singaporensis. in: kawai t, cumberlidge n, editors. a global overview of the conservation of freshwater decapod crustaceans. cham, switzerland: springer; c2016. p. 359 372. yeo dcj, ng pkl, cumberlidge n, magalhães c, daniels sr, campos mr. 2008. global diversity of crabs (crustacea: decapoda: brachyura) in freshwater. hydrobiologia. 595:275-286. ______ ziljih s. molina graduated with a master of science in biology from msu-iligan institute of technology in 2019. she is now an instructor at sultan kudarat state university, tacurong, sultan kudarat. jemateo b. neri graduated with a master of science in biology from msu-iligan institute of technology in 2018. at present, he is taking up medicine at matias h. aznar memorial college of medicine, inc., guadalupe, cebu city. rizza may p. cañete graduated with a master of science in biology from msu-iligan institute of technology in 2019. she is now an instructor at christian pilgrim college, cagayan de oro city. ephrime b. metillo <ephrime.metillo@g.msuiit.edu.ph> is currently a professor and the dean of the college of science and mathematics of msu-iligan institute of technology. 82 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions 9 if changes are made, choose a new title for the paper. 9 use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality. 9 reference your conference paper in the appropriate locations. 9 include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.” 9 indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed. 9 provide the original conference paper (upload a pdf file during the submission process). 9 if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions 9 expand the background section and include additional references. 9 include novel scientific content and expanded descriptions of procedures. 9 provide data that was not published at the conference. 9 revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission from the editors of ieee sensors journal) 10_experimental juanico and saloma 36 experimental verification of the allelomimesis clustering model dranreb earl juanico* and caesar saloma national institute of physics, university of the philippines, diliman, quezon city 1101 e-mail: dojuanico@up.edu.ph abstract science diliman (july-december 2004) 16:2, 36–40 *corresponding author the allelomimesis clustering model is based on only two parameters α and p, which represent the probability of nearest-neighbor copying and the fraction of unresponsive agents, respectively. the model results into the formation of clusters of agents, the sizes of which obey a distribution that is determined by the values of α and p. several experimental data are fitted by tuning the two parameters. in particular, the significance of the value of α that corresponds to an experimental data is discussed and justified according to ecological context. recommendations for possible extensions of the model are also enumerated. introduction the penultimate hallmark of complex systems is the principle of emergence—macroscopic regularity of the system arising from apparently irregular (disordered) microscopic interactions between the system’s constituents. among the most commonly observed of these emergent properties is clustering. clusters in nature exist in different sizes. an interesting and somewhat unexpected observation is the regularity of the statistical distribution of cluster sizes, which generally obeys what is known as a power law. if we denote by s the size of a cluster and by f(s) the frequency of occurrence of s (i.e., the number of times that a particular value of s is tallied), then one would witness that the plot of f(s) versus s in double logarithmic scale is a decreasing straight line. the slope of this line corresponds to the exponent of the power law. let us refer to the value of this slope as τ. power-law behavior is a well-known result in the area of complex systems. this article presents a simple, yet general, mechanism that leads to this power-law behavior. in sec. 2, a phenomenological model based on allelomimetic behavior is discussed. allelomimesis is the tendency of individuals to imitate its neighbors; hence, allelomimetic behavior is the best candidate as local interaction that could lead to the formation of clusters. the results of the model are discussed in sec. 3. a comparison of these results with several cluster systems found in nature is also presented. a few recommendations for further extension of this study are pointed out in sec. 4. the clustering model behavior of a single agent the probability that an agent “performs” a certain “action” is described as follows: , (1) wherein σ is the total stimulus received by an agent (both from its environment and its neighboring agents) and sc is an arbitrary threshold stimulus level. equation ( ) 1, if 0, if σ σ σ σ σ >⎧ = ⎨ ≤⎩ c c p experimental verification 37 (1) means that an agent actuates if its stimulus level exceeds the threshold. such characteristic is akin to neuronal stimulation (churchland & sejnowski, 1992). in reality, however, an agent may override the consequence of σ>σc by a complementary “choice” of not performing an action. let us assume that the probability the agent will not actuate if σ>σc is 1–β. hence, eq. (1) may be rewritten in the following form: . (2) equation (2) incorporates the ability of an agent to decide. generally, β and σc may vary among agents. but to make the model as simple as possible, it is assumed that β is a mean value over a population of agents; hence, β is a constant with respect to a particular agent population. however, β is allowed to vary between different populations. on the other hand, two cases are considered in assigning the value of σc—it is either fixed (σc = 4) for all agents or it varies within a range (2 < σc < 16) among agents. lattice of agents agents are distributed in a d-dimensional lattice of length l consisting of ld discrete cells. a cell may only accommodate a single agent. thus, ld also corresponds to the size of the agent population. a fraction p of the agent population is designated to be unresponsive. unresponsive agents are characterized by β = 0 such that p(σ,β) = 0 even if σ>σc. these agents may be thought of as impurities in the lattice. the value of p is varied between 0 and 1. dynamics of action propagation the lattice is constantly bombarded by external stimuli (e.g., environmental conditions or public information in the form of advertisements, and the like). the consequence of this is that at each time step of the numerical experiment, σ→σ+1 for all agents (i.e., stimulus levels are raised by a unit). an agent is then randomly chosen, say an agent at cell i,j in a d = 2 lattice, to behave according to eq. (1). hence, if σ(i,j) exceeds σc(i,j), then this agent outputs a particular action a. otherwise, nothing happens and another agent is randomly chosen. allow us to distinguish this chosen agent as a harbinger. the harbinger is the initiator of an action. once a harbinger is selected, the bombardment of external stimuli is momentarily paused to allow us to focus on the consequence of the harbinger’s action to the entire lattice. furthermore, we assume that there can only be one harbinger at a time, but any agent is a potential harbinger at any given time. by the performance of a the harbinger’s stimulus level decreases: σ(i,j)→σ(i,j)–2d, as though releasing tension. meanwhile, the stimulus level of each of the harbinger’s 2d nearest neighbors is increased by a unit due to their observance of a. these neighbors behave according to eq. (2) to decide whether or not to mimic the harbinger and actuate a. for a responsive neighbor, sufficient stimulation (>σc) resulting from the observance of a makes it actuate a with a probability β. let us assume that β is of a particular value α. in contrast, any amount of stimulation brought about by the observance of a will have no effect on an unresponsive neighbor. the harbinger’s neighbors in turn pass around the information to their corresponding neighbors by actuating a. this propagation continues up to the last agent that performs a without influencing its corresponding nearest neighbors. the parameter α is defined as the allelomimesis index. its value is tuned between 0 and 1. on one hand, α = 0 is equivalent to setting p=1, i.e., all agents in the population are unresponsive to their neighbors; hence, noncopying or nonallelomimetic. on the other hand, α = 1 implies a highly allelomimetic population of agents, wherein imitation of neighbors is a big factor that promotes clustering. the dynamics of action propagation can be summarized as two fundamental processes: (i) the selection of a harbinger that initiates an action and (ii) propagation of action through nearest-neighbor connections. ( ) , if , 0, if β σ σ σ β σ σ >⎧ = ⎨ ≤⎩ c c p juanico and saloma 38 cluster and cluster size process (ii) is repeated until the action initiated by the harbinger ceases to propagate. all agents that actuate or have actuated a are considered to belong to a cluster and the total number of these agents corresponds to the size of the cluster s. subsequently, the bombardment of external stimuli is resumed for the proceeding time step. process (i) results in the initiation of another action and process (ii) propagates this action through the lattice, hence, establishing the formation of another cluster. by repeating processes (i) and (ii) over several time steps, one generates different values of s. this allows one to deduce the statistical distribution f(s). results and discussion numerical simulations figure 1 illustrates the morphology of clusters at different settings of the parameters α and p. these clusters apparently exhibit fractal structures resembling those that were produced by models of urban growth through diffusion-limited aggregation (makse et al., 1995). let us first deal with the effect of varying α by setting p = 0. figure 2 plots the power-law cluster size distribution (csd) in double logarithmic scale for different values of α. notice how the lines steepen with increasing value of α, indicating that the scaling exponent τ is negatively correlated with the parameter α. it is expected that τ→∞ as α→0, consistent with a dirac-delta csd centered at s=1 for α=0 (i.e., no clusters are formed). to show the effect of the parameter p on the csd, we fix α to a value of 1. figure 3 exhibits a distortion of the csd at large values of s. the degree of such distortion intensifies with increasing p. considering that thresholds σc may vary among agents, we compare the set of csds (with different α and p = 0) for the case wherein 2≤σc ≤16 with the set for which σc = 4. figure 4 plots the csds as data points in the former case and as broken lines in the latter case. there is no observable difference and this implies that the exact value of σc of an agent does not affect the csd. hence, the csd is robust to variations of σc within an agent population.fig. 1. clusters that result for α=1and p=0. x y 128 96 64 32 0 0 32 64 96 128 fig. 2. csd for different values of α at p=0. s f( s) α = 1 α = 0.75 α = 0.5 α = 0.25 α = 0.1 α = 0.01 10-1 100 101 102 103 100 10-1 10-2 10-3 10-4 10-5 10-6 fig. 3. csd at α = 0 and different values of p. s f( s) 100 101 102 103 100 10-1 10-2 10-3 p = 0 p = 0.01 p = 0.1 p = 0.5 experimental verification 39 comparison with data for real systems we fit our model to different csds taken from experimental observations of actual clusters. the goodness-of-fit is measured in terms of the mean square error (mse) between the data and the curve generated from the model. figures 5 and 6 present data on four animal systems and four distinct human cluster systems, respectively. remarkably, α is high for animal systems (except for serengeti lions), implying that allelomimesis is strongly expressed in animals. according to wagner and danchin (2003), “conspecific copying” (or allelomimesis) is a ubiquitous mechanism behind the formation of aggregates such as leks and colonies. bonabeau and dagorn (1995) showed that “biosocial attraction” (another form of allelomimesis) promotes schooling in fishes. parrish and edelstein-keshet (1999) further proposed that allelomimesis is a generic mechanism that maintains the cluster as a cohesive unit. indeed, ecological evidence for a high value of α in the animal kingdom is compelling. the low α (=0.1) for serengeti lions is explained by the fact that the lions that were observed by schaller (1972) were nomadic. this means that they are likely to wander alone or in small groups, hence, a small value of α. the value of α for human cluster systems is low as compared with animal systems, implying that allelomimesis is only moderately expressed in human beings. this can be justified by considering that humans are generally more highly cognitive than animals, which consequently overrides their instinctive tendency to be allelomimetic. furthermore, telecommunication technology (which only humans are capable of) diminishes the requirement of information transfer through nearest-neighbor connections such as fig. 4. comparison between imposing uniform threshold (σc=4) and random threshold (2<σc<16) for different values of α at p=0. s f( s) 100 101 102 103 100 10-1 10-3 10-5 10-2 10-4 α = 1.0 α = 0.75 α = 0.5 α = 0.25 uniform thresholds random thresholds fig. 5. fitting the model to four different animal systems. (a) spotted dolphin, stenella attenuata (α = 0.75, p = 0.3, mse = 0.00381); (b) west indian manatee, trichecus manatus (α = 1, p = 0.45, mse = 0.00034); (c) wasp, ropalidia fasciata (α = 0.75, p = 0.35, mse = 0.00059); (d) serengeti lion, panthera leo (α = 0.1, p = 0, mse = 0.08661). 100 101 102 103 100 10-1 10-2 10-3 s f( s) (c) 100 101 102 103 100 10-1 10-2 10-3 s* = s/30 n (s *) /n (1 ) (a) 100 101 102 103 100 10-1 10-2 10-3 s n (s *) /n (1 ) (b) 100 101 102 103 100 10-1 s* = s/30 n (s )/n (1 ) (d) fig. 6. fitting the model to four distinct human cluster systems. (a) urban agglomerations of india, 1991 (α = 0.32, p = 0, mse = 0.00394); (b) major cities of japan, 1994 (α = 28, p = 0, mse = 0.00021); (c) households/barrios of metro cebu, 2001 (α = 0.31, p = 0.25, mse = 0.00179); (d) firms/clusters of employees of us, 1997 (α = 0.32, p = 0, mse = 6.16504). 100 101 102 103 100 10-1 10-2 s* = s/43 n (s *) /n (1 ) (a) 100 101 102 103 100 10-1 10-2 10-3 s*=s/100 n (s *) /n (1 ) (b) 100 101 102 103 100 10-1 10-2 10-3 s* = s/168 n (s )/n (1 ) (c) 100 101 102 103 100 10-3 s f( s) (d) 10-1 10-2 juanico and saloma 40 allelomimesis. interestingly, α is not significantly different among distinct human cluster systems (α~0.3). this result is quite expected because even though we consider clusters of cities, of households, or of employees to be distinct from one another, one fact remains common between them—these systems are all made up of human beings. it would be worthwhile to investigate the origin of such seemingly universal value of α from a psychological point of view. recommendations the model due to its inherent simplicity has cut down on details as much as possible so that it can be considered generic, hence, applicable to a wide variety of systems. here, we suggest some minor points of modification to allow a more realistic description. the stimulation on an agent due to constant bombardment of external factors may not necessarily be equal to unity (i.e., σ→σ+1). it can be expressed as σ→σ+η, where η represents a positive poisson number that appropriately describes the time fluctuation of the amount of external stimuli. furthermore, subsequent stimulation of neighboring agents may not necessarily decrease the stimulus level of the harbinger by an amount that is equal to the number of its nearest neighbors. that is, one can write σ→σ−ε, where ε is a positive number derived from a gaussian or a binomial probability distribution. it follows that the ensuing stimulation of responsive neighbors that observe the actuation of the harbinger can be expressed as σn→σn+δn, where the subscript n represents the nearest neighbor and ∑nδn = ε. note here that δn could either be positive or negative, implying that the stimulation is excitatory or inhibitory, respectively (churchland & sejnowski, 1992). conclusion a simple model of cluster formation is proposed to explain the cluster size distribution observed for various cluster systems in nature. the model consists of two mutually independent parameters, namely, α and p. the value of α represents the probability that an agent mimics the action of its nearest neighbors, whereas p is the fraction of unresponsive agents that characterizes the particular agent population. resulting csds are highly dependent on α and p. the model fits into experimental data corresponding to various cluster systems in nature. a high value of α generally characterizes animal systems, whereas α~0.3 distinguishes human cluster systems. references churchland, p.s. & t. sejnowski, 1992. the computational brain. mit press, cambridge. makse, h., s. havlin, & h.e. stanley, 1995. modeling urban growth patterns. nature. 377: 608. wagner, r.h. & e. danchin, 2003. conspecific copying: a general mechanism of social aggregation. anim. behav. 65: 405. bonabeau, e. & l. dagorn, 1995. possible universality in the size distribution of fish schools. phys. rev. e. 51: 5220. parrish, j.k. & l. edelstein-keshet, 1999. from individuals to aggregations: complexity, epiphenomena, and evolutionary trade-offs of animal aggregation. science. 284: 99-101. schaller, g.b., 1972. the serengeti lion: a study of predatorprey relations. university of chicago press, chicago. 01_device comparison of several methods for nitrogen dioxide 1 introduction nitrogen dioxide (no 2 ) and sulfur dioxide (so 2 ) play important roles in ecosystem acidification (nishikawa, 2004). these gases are two of the air pollutants commonly monitored to assess air quality in the philippines. the others are total suspended particulates (tsp), particulate matter less than 10 um diameter (pm10), ozone, carbon monoxide and lead (environmental management bureau (emb), 1990). comparison of several methods for nitrogen dioxide and sulfur dioxide in metro manila air *leni l. quirit1, 2, karen n. hernandez1 and brian j. lee3 1natural sciences research institute and 2institute of chemistry,university of the philippines, diliman, quezon city 1101 and 3emission technologies, inc. (eti), makati city, philippines *e-mail: lquirit@chem.upd.edu.ph date submitted: september 7, 2005; date accepted: june 23, 2006 abstract the pollutant gases nitrogen dioxide (no 2 ) and sulfur dioxide (so 2 ) are two of the commonly monitored parameters for air quality assessment in the philippines. in this study, several active and passive sampling methods for the analysis of the two gases were tested. of the methods for no 2 first tested indoors, the nai sorbent passive sampling method was found most promising and was tested for ambient air no 2 (using both ferm and korean passive samplers) against a collocated automated chemiluminiscence sampler. for so 2 , ambient levels were simultaneously measured using naoh sorbent in a ferm passive sampler and a collocated automated fluorescence sampler. correlation and t testing indicate a positive bias for the no2 ferm method and a negative one for the so 2 ferm method in comparison with their corresponding automated methods. the results of the study support the 1997 annual average so 2 and no2 findings of a up natural sciences research institute (nsri) study (quirit et al, 1999) which used ferm passive samplers in various department of environment and natural resources (denr) metro manila air sampling stations. key words: no 2 , so 2 , passive samplers, chemiluminiscence sampler, fluorescence sampler metro manila (mm) data for ambient air pollutants are usually presented as levels versus time to show annual trends. for no 2 and so 2 , data from 1975 to the present have been reported. however, the methods used for sampling and analysis of these two gases differ within this time period, introducing an uncertainty when trends are analyzed. table 1 enumerates these different methods and the corresponding year(s) each was used, together with the institutions that did the sampling and analysis. this study compared different sampling and analysis methods for no 2 and so 2 , using both passive and active samplers and indoor and ambient sites. in 1998, a similar study was done with a fewer number of*corresponding author science diliman (july-december 2007) 19:2, 1-11 quirit, hernandez and lee 2 methods and samples, for no 2 in an indoor site and so 2 in a vehicular loading and unloading campus site (quirit et. al., 2002). the availability of automated monitoring samplers in 2003 made possible the collection of a greater number of samples in an ambient sampling site for both gases. four general methods used for past and present mm no 2 and so 2 data collection were used in this study. these are: passive sampling, manual active sampling to a sorbent, and automated active chemiluminiscence and fluorescence methods. the results could provide a significant basis for relating some of the past and present mm data for no 2 and so 2 in ambient air. materials and methods a variety of active and passive sampling methods for no 2 were first tested in the indoor laboratory air of the up nsri room 238. past studies have shown that indoor and outdoor no 2 levels were generally comparable (quirit et. al 2002, quirit et. al. 1999). these methods were compared to the standard manual active method accepted by denr, the griesssaltzman method (lodge, 1989a). all no 2 methods tested for indoor air resulted in the same colored griess-saltzman analyte which was measured by visible light absorption at 540 nm. the best method was chosen for ambient air sampling and the results compared with the automated chemiluminiscence sampler results for no 2 . for so 2 , indoor levels were generally lower than ambient levels (quirit et. al. 2002, quirit et. al. 1999) so only ambient samples were taken, using the ferm method for so 2 (ferm, 1991). the results were compared with the automated fluorescence sampler results for so 2 . the sampling site for ambient samples was the manila observatory grounds, situated at the ateneo de manila university campus, loyola heights, quezon city. table 1. metro manila no 2 and so 2 methods (quirit et. al., 2005) asorbent not given in reference npcc national air pollution control commission, denr-ncr denr national capital region, adb asian development bank, jsps japan society for the promotion of science, jica japan international cooperation agency, ncts national center for transportation studies, kfem korean federation for environmental movement period (institution) no 2 method so 2 method 1975 1983 (npcc) automated air sampler from japan automated air sampler from japan (emb 1990, emp 1996) (emb 1990, emb 1996) 1986-1996 (denr-ncr) manual sampler (tcm bubblers) (emb, 2003) 1991-1992 (adb and emb-denr) automated chemiluminiscence monitor (adb, 1992) 1996-1998 (up nsri and denr-ncr) passive (ferm) sampler with passive (ferm) sampler w/ naoh naoh/nai sorbent (quirit et. al. sorbent (quirit et. al. 2002, quirit et. al. 2002, quirit et. al. 1999, ferm 1991) 1999, ferm 1991) 1996-2002 (jsps and jica w/ ncts) japanese passive sampler w/ griess-saltzman sorbent (ica 1999, jsps 1998, teodoro 1996) 2003 (kfem) korean passive sampler w/ tea korean passive sampler w/ sorbenta sorbent (lee, 2002) (lee 2002) 2004-2005 (*denr-ncr) automated chemiluminiscence automated fluorescence monitor monitor (usepa, 1996) (usepa, 1993) science diliman (july-december 2007) 19:2, 1-11 comparison of several methods for nitrogen dioxide 3 no 2 determination methods standard active sampler griess-saltzman method (lodge, 1989a) ten ml of the griess-saltzman absorbing solution was pipetted into a dry fritted impinger (skc 4225) attached to an air sampling pump (skc 224-pcxr4). flow rate was set to 0.4 l/minute (lpm), with the sampling duration varied from at least about 8 h to at most 24 h. at the end of the sampling period, the resultant pinkcolored solution was transferred to a vial and analyzed immediately. calibration standards of nano 2 equivalent to 0, 0.08, 0.16, 0.24, 0.40 and 0.56 µl no 2 /ml absorbing solution were used to determine the no 2 level in the samples. the absorbances were read in a uv-vis spectrophotometer at a wavelength of 540 nm. nai-ferm passive sampler method (quirit et. al. 2002, ferm 1991) nai-naoh-methanol coating solutions were made fresh for each batch preparation of ferm samplers. fifty µl of this coating solution were pipetted onto paper filters (whatman 40) loaded in the partially assembled samplers. the samplers are described in the reference for the method (ferm, 1991) and in a previous publication (quirit et. al, 2002). the assembled samplers were then kept in a refrigerator inside polythene storage containers before and after exposure. sampling durations ranged from at least 1 week to at most 1 month (4 weeks), wherein the samplers were taken out of storage and attached to frisbees secured on top of 1.83 m (6 ft) poles. prior to analysis, the paper filters were transferred into vials and extracted with 5 ml ultrapure water. fiveml standards (0, 5, 10, 20, 40, 60, 80 µm nano2) were prepared and equal volumes (5 ml) of griesssaltzman solution were added to both standards and samples. absorbances were read in a uv-vis spectrophotometer (? = 540 nm) after 15 minutes. tea-ferm passive sampler method (ferm 1991, lodge 1989b) the tea (triethanolamine) liquid absorber solution was prepared and used to coat paper filters, as described in the intersociety committee methods of air sampling and analysis (lodge, 1989b). samplers used were the same as those described in the nai-ferm method (ferm, 1991), except the paper filters were first dried using lint-free tissue to absorb the excess coating solution and further dried by n2 stream prior to loading into the samplers. five-ml standards having concentrations of 0, 0.08, 0.16, 0.24, 0.40 and 0.56 µl no 2 /ml were prepared. samples were extracted with 5 ml ultrapure water and equal volumes of the griess-saltzman absorbing solution were added to both standards and samples. absorbances were measured at a wavelength of 540 nm. tea-korean passive sampler method (lee, 2002) basically the same procedures as the tea-ferm sampler method were followed for the tea-korean method, except for the different sampler used. the korean sampler is made up of a small clear plastic tube (internal diameter: 10.016 mm, outer diameter 12.092 mm, height: 43.176 mm) covered by a white plastic (probably teflon) on one end, and removable rubber caps on both ends. whatman 40 paper filters (cut to fit inside the tube) were coated with a liquid absorber solution prepared with five times as much tea (triethanolamine) but the same amount of butanol and ultrapure water as that used in the tea-ferm method. samples were extracted with 2.0 ml ultrapure water and standards were prepared as described previously. nai-korean passive sampler method (quirit et. al. 2002, ferm 1991, lee 2002) basically the same procedures as the nai-ferm sampler method were followed, except that the sampler used was the korean passive sampler described above. science diliman (july-december 2007) 19:2, 1-11 quirit, hernandez and lee 4 tea-glass fiber filter active sampler method (lodge 1989b, sickles et. al. 1990) glass fiber filters (sickles et. al., 1990) were soaked in liquid tea absorbing solution (lodge, 1989b), pressed between sheets of lint-free tissue and dried in a n 2 stream to remove any excess coating solution. filters were then placed inside sampling cassettes (skc 22545). no 2 was absorbed by pulling air with an skc 224-pcxr4 sampling pump at 1 lpm. after exposure, the filters were transferred into vials. five-ml calibration standards (0, 0.2, 0.6, 1, 1.4 µg no 2 /ml) were prepared from a stock standard of 2 µg no 2 /ml and the liquid absorber solution. samples were likewise extracted with 5 ml of the liquid absorber solution. then, to both standards and samples, griesssaltzman reagents were added and the resulting pinkcolored solutions were measured for their absorbances at 540 nm. tea-molecular sieve active sampler method (lodge, 1989b) the solid absorber was prepared by soaking molecular sieve in tea absorber solution for 30 minutes then drying in a vacuum oven at 70º c. these were then packed in a glass tube (measuring 55 cm in length and 5 mm in diameter) through which air was pumped into for 8 h using an skc 224-pcxr4 sampling pump at a rate of 0.4 lpm. ten-ml calibration standards were prepared similarly to those in the glass fiber method while the absorbed no2 was extracted using 50 ml of the liquid tea absorbing solution. one ml of 0.02%(v/v) hydrogen peroxide, 10 ml sulphanilamide solution and 1.4 ml of 0.1%(w/v) (1-naphtyl ethylenediamine dihydrochloride) neda solution were then added to the calibration standards and to a 10-ml aliquot of the extract, after which absorbance readings were measured at 540 nm. chemiluminiscence automated sampler method (usepa, 1996) no x levels were measured using a horiba apna360ce. no x is the sum of nitric oxide (no) and nitrogen dioxide (no 2 ) in a gaseous sample. two fractions of ambient air are sampled one has all of its no 2 converted to no, with the no then made to react with ozone (o 3 ) to form no 2 + o 2 + light. the chemiluminescence generated is therefore proportional to the nox level. on the other hand, the other fraction is reacted with o3 immediately (without reducing no 2 to no) and since no 2 is unreactive towards o 3 , only the no reacts, giving the no level. the difference between the values from fraction 1 and 2 therefore gives the no 2 level. so 2 determination methods naoh-ferm passive sampler method (quirit et. al. 2002, ferm 1991) the same procedures as those for the nai-ferm method for no 2 determination were followed in preparing the so 2 samplers, except that a different coating solution, naoh-methanol, was used. the absorbed so 2 , in the form of so 4 2ion, was analyzed by ion chromatography. prior to analysis, the paper filters were transferred into vials and extracted with 2.5 ml of degassed ultrapure water. sample solutions were filtered with 0.45 µm millipore filters to remove particles that might clog or damage the column. standard solutions containing 0.5, 2, 5, 10 and 20 ppm of so 4 2 were prepared using degassed ultrapure water and, along with the samples, were analyzed using an ion chromatograph (dionex ion chromatograph 2000i). 12.5 mm h 2 so 4 was used as a regenerant and 1.36 mm nahco 3 /1.44 mm na 2 co 3 as eluant. eluant flow rate was set to 2 ml/minute and sensitivity to 30 µs. fluorescence automated sampler method (usepa, 1993) so 2 levels were obtained using a tei 43c so 2 analyzer, which gives so 2 levels in ambient air by measuring the fluorescent electromagnetic radiation emitted by the so 2 molecules when excited with ultraviolet light. the fluorescence emitted is proportional to the so 2 mixing ratio in the sampled ambient air. science diliman (july-december 2007) 19:2, 1-11 comparison of several methods for nitrogen dioxide 5 results and discussion comparison of methods (indoor air no 2 ) figure 1 shows how the different methods compared with the standard griess-saltzman method for no 2 , and with each other. the assumption for the comparison is 100 % no 2 collection efficiency for the standard method. sieve active method had the lowest average collection efficiency. comparison of methods (ambient air no 2 ) for ambient no 2 , the nai-ferm passive sampling method was chosen on the basis of its performance in the indoor sampling comparison. the korean passive sampler was also tried, but with the sorbent changed the nai-ferm passive sampler gave the best results, with all sample levels having ratios (with simultaneous standard method sample levels) close to 1, indicating approximately 100 % collection efficiency. correlation with standard method levels gave an r = 0.9954. the teakorean passive sampler method had the next average ratio value closest to 1, but had significantly lower precision for the collection efficiencies, compared to the nai-ferm passive sampler method. the same poor precision for the ratios is true for the tea-glass fiber filter method. tea was also tried as a sorbent for the ferm passive sampler. although ratio precision and correlation with the standard method (r = 0.9644) were good, sampling efficiency (around 40 %) was low. the tea-molecular to the nai-ferm coating solution. therefore, what was tried was the same sorbent, but in different passive sampler configurations. sampling (one week to one month duration per sample) was from february to december, 2004. figures 2 and 3 show how these two passive sampler types compare with the automated chemiluminiscence method for no 2 in ambient air. the regression equation for the nai-ferm method, when correlated with the chemiluminiscence method (without forcing the intercept to zero), gives a slope of 0.9763 and an intercept, 0.0015 ppm, which is about twice the average standard deviation, 0.0007 ppm, for the data points. this indicates an overall positive bias of the nai-ferm method relative to the automated method. when the intercept of the correlation line is figure 1. comparison of methods for indoor air no 2 (using ratio of method no 2 level with standard griess-saltzman method no 2 level) (quirit et. al., 2005) ratio with standard method 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 5 10 15 20 data point ra ti o science diliman (july-december 2007) 19:2, 1-11 quirit, hernandez and lee 6 forced to zero, the slope became 1.0689, as shown in figure 2. assuming 100% collection efficiency for the automated method, the collection efficiency of the naiferm method for no 2 , based on this slope, is around 107%. the correlation coefficients, 0.8981 and 0.8940 respectively, for the non-zero and zero intercept equations, are similar. the korean passive sampler (using tea as coating solution) was used by a korean non-government figure 3. comparison of nai-korean sampler method with automated chemiluminiscence method for ambient air no 2 (n = 6 data points) (quirit et. al., 2005) ambient no2 y = 1.0973x r = 0.8136 0 0.005 0.01 0.015 0.02 0.025 0.03 0 0.005 0.01 0.015 chemiluminiscence no k o re a n s a m p le r n o 2 (p p m ) ambient no2 y = 1.0689x r = 0.8940 0 0.005 0.01 0.015 0.02 0.025 0 0.005 0.01 0.015 chemiluminiscence no2 f e rm n o 2 ( p p m ) figure 2. comparison of nai-ferm method with automated chemiluminiscence method for ambient air no 2 (n = 30 duplicate data points, ferm sampler 4% average rsd, chemiluminiscence method, 2% rsd) (quirit et. al., 2005) science diliman (july-december 2007) 19:2, 1-11 comparison of several methods for nitrogen dioxide 7 organization (ngo), the korean federation for environmental movement (kfem), in cooperation with a philippine ngo, the concerned citizens against pollution (cocap), plus students and teachers, grassroot ngo activists and some local government officials (lee, 2002). they collected 24 h no 2 air samples in various locations in metro manila from january 18 to january 19, 2002. the authors of this study used the sampler, but with a different coating solution (nai/ naoh) as mentioned in the methodology above. interest in the sampler was due to its simple configuration, plus its use for recently collected data by kfem. data points were considerably less for the nai-korean sampler method, since the authors procured only one sampler from the local ngo, concerned citizens against pollution (cocap), which participated in the above mentioned exercise. duplicate data points could not be acquired, hence the absence of an average rsd. kfem reports the sampler's precision as 9.6 % rsd. the regression equation for the nai-korean sampler, when correlated with the chemiluminiscence method (without forcing the intercept to zero), gives a slope of 1.2032 and an intercept of -0.0017 ppm, which is similar in magnitude to the nai-ferm intercept. results shown in figure 3, when the intercept is forced to zero, indicates approximately 110% no 2 collection efficiency, relative to the chemiluminiscence method. the correlation coefficients, 0.8168 and 0.8136 respectively, for the non-zero and zero intercept equations, are similar. comparison of methods (ambient air so 2 ) for ambient so 2 , the naoh-ferm passive sampling method was compared to the automated fluorescence method, and the results are shown in figure 4. sampling (one week to one month duration per sample) was from february to december, 2004. only 21 duplicate data points are shown, since some of the samples were analyzed by baso 4 turbidimetry (instead of by ion chromatography). the turbidimetric method performed poorly, in terms of precision, compared to ion chromatography. the standard active manual method accepted by denr uses a mercury compound (tetrachloromercurate or tcm) in the sorbent. this method was not used due to its being both cumbersome and toxic. ambient so2 y = 0.8404x r = 0.8477 0 0.002 0.004 0.006 0.008 0.01 0 0.002 0.004 0.006 fluorescence so2 (ppm f e rm s o 2 ( p p m ) figure 4. comparison of naoh-ferm method with automated fluorescence method for ambient air so 2 (n = 21 duplicate data points, ferm sampler 8% average rsd, fluorescence method 1% rsd) (quirit et. al., 2005) science diliman (july-december 2007) 19:2, 1-11 quirit, hernandez and lee 8 the regression equation for the naoh-ferm method, when correlated with the fluorescence method (without forcing the intercept to zero), gives a slope of 0.8479 and an intercept, -0.1008 ppm, the absolute magnitude of which is higher than the average standard deviation, 0.0019 ppm, for the data points. this indicates an overall negative bias of the naoh-ferm method relative to the automated method. when the intercept of the correlation line is forced to zero, the slope became 0.8404, as shown in figure 4. assuming 100% collection efficiency for the automated method, the collection efficiency of the naoh-ferm method for so2, based on this slope, is around 84%. the correlation coefficients, 0.8479 and 0.8477 respectively, for the non-zero and zero intercept equations, are similar. t test aside from correlation with the automated methods, the t test was also used to find out if there is a statistical difference between the results obtained using the passive sampler and the automated methods (christian, 2004). for both no 2 and so 2 , the passive sampler method was considered the test method, and the automated method was the accepted method. the two automated methods use calibration gases (usepa 1996, usepa 1993), while passive sampler levels were calculated from the dimensions of the sampler and fick's first law, a theoretical diffusion equation (quirit et. al. 2002, ferm 1991). the results are shown in table 2. the number of degrees of freedom, other than 8, in the t table was only up to 25, so the calculated t was done overall (30 data points or 29 degrees of freedom) and per set of 6 data points (5 degrees of freedom) for nai-ferm no 2 . this was also to compare with the 6 data points of the nai-korean sampler. the t test results (magnitude of t calculated > magnitude of t table ) confirm the positive bias (107% collection efficiency) for the nai-ferm method for no 2 , and the negative bias (84% collection efficiecy) for the naohferm method for so 2 , compared to the automated methods. the opposite result (magnitude of tcalculated < magnitude of ttable), indicating no statistical difference between the nai-korean sampler results and the chemiluminiscence method for no 2 at the 95% confidence level, is true for the small number of data points collected. this is also true, however for the first 6 data points of the nai-ferm method for no 2 . figures 5 and 6 further illustrate the biases for the ferm passive sampler methods, using % deviations from the automated sampler results. the results were compared to deviations of the ferm passive samplers (one month samplings) from parallel 24 h active sampler data in sweden (ferm, 1991). % deviation of nai-ferm from chemiluminescence -10 -5 0 5 10 15 20 25 1 3 5 7 9 11 13 15 17 19 21 data point % d e v ia ti o n figure 5. % deviation for no 2 [[(nai-ferm data chemiluminiscence data)*100/chemiluminiscence data)] (quirit et. al., 2005) science diliman (july-december 2007) 19:2, 1-11 comparison of several methods for nitrogen dioxide 9 in this study, around 77 % of the data points have no 2 levels greater than the automated chemiluminiscence levels, 10 % have levels close to the automated method levels, and 3 % have levels lower than chemiluminiscence levels, as seen in figure 5. this is similar to the sweden results, where 67 % of the approximately 48 data points had levels above the active sampler levels, and the rest of the data points were about equally distributed between those close to and those below active sampler levels (ferm, 1991). figure 6 shows that around 80 % of the data points have so 2 levels below the automated fluorescence levels, 10 % have levels close to the automated method levels, and 10 % have levels higher than fluorescence levels. this is again similar to the sweden results, where 65 % of the approximately 46 data points had levels below the active sampler levels, 11 % close to and 24 % above active sampler levels (ferm, 1991). the active sampler no 2 method done in sweden used sintered glass filters impregnated with the same sorbent (ki) for no 2 as the passive samplers, while the active impinger method (with 0.03% h 2 o 2 ) was used for comparison with the so 2 ferm passive samplers (coating solution is k 2 co 3 ). the coating solutions used in sweden are chemically similar to those used in this study (naoh is quickly transformed to na 2 co 3 when exposed to co 2 in air) for the passive samplers. it is interesting to note that even for the same no 2 sorbent used in the active and passive samplers in sweden, the positive bias was observed for no2 in ambient air, for the passive relative to the active samplers. precisions of the ferm samplers in this study, 4% and 8% rsd respectively for no 2 and so 2 , are similar to precisions (5% and 10% rsd respectively for no 2 and so 2 ) of the ferm sampler in the sweden study. no explanation, as of now, could be offered for the higher levels predominantly observed for no 2 passive samplers, relative to the active automated chemiluminiscence method levels. a passive sampler reported in niosh 6700 method for no 2 (niosh, 1984) gave results for a field study (ambient air) where the passive sampler levels were 109 ± 9% of the reference method (jones et. al., 1979). this is similar to the 107 ± 5% results for the ferm no 2 sampler in this study. for so 2 , it is postulated that part of the sorbed so 2 was not transformed to so 4 2-, but remained as so 3 2-, hence the seemingly lower collection efficiency of the passive sampler compared to the automated fluorescence sampler. the corresponding author is currently part of a research where so 3 2ions are analyzed by a method called pervaporation. % deviation of naoh-ferm from fluore -40 -30 -20 -10 0 10 20 1 3 5 7 9 11 13 15 data point % d e v ia ti o n figure 6. % deviation for so 2 [[(naoh-ferm data fluorescence data)*100/fluorescence data)] (quirit et. al., 2005) science diliman (july-december 2007) 19:2, 1-11 quirit, hernandez and lee 10 preliminary results using this method on the naohferm sampler extracts show the presence of so 3 2ions in the samplers exposed to ambient air. conclusion ferm passive sampler no 2 and so 2 levels in ambient air of the manila observatory were found to be positively and negatively biased, respectively, relative to automated chemiluminiscence no 2 and automated fluorescence so 2 levels. no explanation, as of now, could be found for the positive no 2 bias. incomplete oxidation of the sorbed so 2 in the basic sorbent of the ferm passive sampler is postulated to be the reason for the sampler's negative so 2 bias. this is due to the nature of the analytical method used for oxidized so 2 , which analyzes the fully oxidized form (so 4 2-), but not the the partially oxidized so 3 2form. this systematic error can be remedied by fully oxidizing the sorbed so 2 prior to analysis by addition of h 2 o 2 . an alternative remedy is analyzing the sampler extract for both so 3 2and so 4 2ions. the results of the study support the 1997 annual average so 2 and no 2 findings in a up nsri study using ferm passive samplers in various denr metro manila air sampling stations (quirit et al, 1999). two sites (taft and valenzuela) exceeded annual average so 2 standards (84 and 86 ug/m3 respectively compared to the annual so 2 national standard of 77 ug/m3). the 1997 average levels would even be higher if the negative bias of the ferm so 2 sampler is true. on the other hand, all sites were found to have 1997 annual no 2 average levels below the national annual standard (150 ug/m3). the average levels would be even lower if the ferm no 2 sampler positive bias is considered. in spite of limited results, the simplicity of the korean passive sampler makes it a good candidate for relatively cheap local fabrication. the ferm sampler configuration is more complicated (quirit et. al. 2002, ferm 1991) and local plastic mold manufacturers have quoted quite a steep price for its fabrication (> p200,000.00 for the mold). the korean ngo have not replied as to the material of their sampler, but the configuration does not have the stainless steel mesh nor the precisely molded parts of the ferm sampler, hence the preliminary assessment of its cheaper fabrication, compared to the ferm sampler. once the molds are made, however, production of the samplers is projected to be relatively cheap, according to the plastics mold manufacturers (they did not quote actual prices, but projected less than p100 per sampler). passive samplers are lightweight, convenient to use and does not need expensive pumps and electricity. imported passive samplers, however, are also quite expensive (around four times projected local price, once the mold is made). it would be good to be able to fabricate and study various configurations of locally produced passive samplers. acknowledgements the authors are grateful to up nsri for funding this study, fr. jose ramon villarin, s.j., ph.d. of the manila observatory for allowing us to conduct the passive sampling experiments inside their compound and mr. wilhelm ludwig r. fe and mr. noli mendoza for providing us with needed literature and assisting us in sample collection. references asian development bank, 1992. vehicular emission control planning in metro manila final report. christian g.d., 2004. analytical chemistry, 6th ed. usa, john wiley & sons: 90-97pp. environmental management bureau, 2003. 2002 national air quality status report. environmental management bureau, 1990. the philippine environment in the eighties. environmental management bureau, 1996. the philippine environmental quality report, 1990-1995. ferm m., 1991. a sensitive diffusional sampler. goteborg 1991-03-13. swedish environmental research institute, pp. 1-27. japan international cooperation agency, 1999. metro manila urban transportation integration study (mmutis) final report. science diliman (july-december 2007) 19:2, 1-11 comparison of several methods for nitrogen dioxide 11 japan society for the promotion of science, 1998. jsps-ncts air pollution survey using filter badge in metro manila. jones w., e.d. palmes, c. tomczyk & m. millson, 1979. field comparison of two methods for the determination of no 2 concentration in air. am. ind. hyg. assoc. j. 40:437-438. lee j-h., 2002. a fundamental survey using passive samplers for the establishment of air pollution monitoring system in metro manila, philippines. korean federation for environmental movement (kfem), pp. 64. lodge j.p. (ed.), 1989a. method 406: determination of the nitrogen dioxide content of theatmosphere (griesssaltzman reaction). in methods of air sampling and analysis, 3rd ed. intersociety committee (apca, acs, aiche, apwa, asme, aoac, hps, isa). michigan, usa, lewis publishers inc.: 389-393pp. lodge j.p. (ed.), 1989b. method 818: determination of nitrogen dioxide in air. ). in methods of air sampling and analysis, 3rd ed. intersociety committee (apca, acs, aiche, apwa, asme, aoac, hps, isa). michigan, usa, lewis publishers inc.: 601-603pp. national institute for occupational safety and health, 1984. nitrogen dioxide: method 6700. niosh manual of analytical methods, 3rd ed. vol. 2. cincinnati, ohio: us dept. of health & human services. nishikawa, y., 2004. simultaneous measurement of nitric acid, sulfur dioxide and ammonia in air using a passive sampling polyamide filter. j envi chem 14(2):351-355. quirit l.l., e.c. llaguno, p.c.b. pabroa, f.b.b. bello, b.p. gonzales, 2002. comparison of methods for ambient air sulfur dioxide and nitrogen dioxide. kimika 18(1):19-22. quirit l.l., e.c. llaguno, b.p. gonzales, f.b.b. bello, p.c.b. pabroa, 1999. acid gases and acidic particulate species in metro manila air. proceedings of the 15th phil chem congress, pp. 130-133. sickles j.e., p.m. grohse, l.l. hodson, c.a. salmons, k.w. cox, a.r. turner & e.d. estes, 1990. development of a method for the sampling and analysis of so 2 and no 2 from ambient air. anal chem 62:338-346. teodoro r.v., 1996. empirical analysis on the relationship between air pollution and traffic flow parameters, m.s.c.e. thesis, college of engineering, university of the philippines. usepa, 1996. automated reference method #rfna-0196111. federal register 61:11404. usepa, 1993. automated equivalent method #eqsa-0193092. federal register 58:6984. science diliman (july-december 2007) 19:2, 1-11 4-benthic-magbanua.pmd f.s. magbanua et al. 5 science diliman (january-june 2019) 31:1, 5-24 benthic macroinvertebrates of the university of the phil ippines dil iman campus waterways and their variation across land use in an urban, academic landscape francis s. magbanua* john claude renan b. salluta danielle dominique d. deborde maria brenda m. hernandez institute of biology university of the philippines diliman abstract urban development impacts stream ecosystems primarily via changes in hydrological regime, geomorphology, and in water quality. these changes in turn have biological effects. the university of the philippines diliman campus, located at the heart of the highly urbanized quezon city, has gone through numerous developments in terms of landscape and infrastructure. unlike the terrestrial environment, the extent to which these developments have impacted the campus waterways is unknown. hence, our research aims to assess the overall condition of the waterways in the campus based on the benthic macroinvertebrate assemblages. a total of 19 stream reaches w e r e s a m p l e d i n n o v e m b e r 2 0 1 5 a n d 2 0 1 6 i n t h e f o l l o w i n g l a n d u s e categories: academic/academic support units (six sites), campus core (eight sites), and parks and open spaces (f ive sites). one-way analysis of variance (anova) detected signif icant spatial difference in several macroinvertebratebased metrics, stream physicochemistry, and in-stream habitat condition elements. our study reveals that all sampled stream reaches, regardless of their land use categories, are under poor to severe pollution conditions. all m a c r o i n v e r t e b r a t e b a s e d m e t r i c s a n d i n d i c e s i n d i c a t e d e g r a d e d w a t e r quality and stream health. our results are consistent with urban stream studies elsewhere, which suggest that land-based activities can be stressful for some aquatic organisms, and at times, result in reduced abundance and even reduction in species composition. keywords: biomonitoring, biotic indices, stream habitat assessment, urban land use, water quality issn 0115-7809 print / issn 2012-0818 online _______________ *corresponding author benthic macroinvertebrates of the up diliman campus waterways 6 introduction urbanization affects the natural environment worldwide (pickett et al. 2001; grimm et al. 2008). in particular, urban development impacts stream ecosystems primarily via changes in hydrological regime through increased magnitude and frequency of high flows or through reduced base flow due to increase in impervious surfaces; changes in geomorphology through channel alteration; and changes in water quality through contaminated runoff and from direct point source discharges (walsh et al. 2005; moggridge et al. 2014). as a consequence, these physical and chemical changes have biological effects. while urban areas, such as the university of the philippines diliman campus, can support a wide range of terrestrial biota (ong et al. 1999; vallejo et al. 2009), we do not know whether the same is true for streams flowing through the urban landscape particularly in developing and emerging economies (but see freitag (2013) wherein he described a new species of hydraenid beetle found in headwater creeks inside the ateneo de manila university campus). this is due to the fact that, for over the past 10 years, the observed marked increase in research on urban aquatic ecosystems is biased towards temperate regions and in developed countries (francis 2012). a recent study has documented that tropical streams are naturally flashy due to high precipitation and watershed features, and thus, do not signif icantly differ with urban streams (ramirez et al. 2009). moreover, roy et al. (2009) reported that biological responses to urbanization range from broadly consistent to highly variable or understudied. consequently, there is a need for fur ther research to understand mechanisms of response to urbanization in other regions, such as the tropics, where cities are larger and growing rapidly. the university of the philippines diliman (upd) campus, located at the heart of the highly urbanized quezon city, has gone through numerous developments in terms of landscape and infrastructure. however, unlike the terrestrial environment (vallejo and aloy 2014), the extent to which these developments have impacted the waterways in the campus is unknown as no baseline study was conducted to compare the current conditions. meanwhile, evidence that many freshwater species are being threatened with extinction by urban development are being discovered elsewhere (paul and meyer 2001; walsh et al. 2005, 2007; brown et al. 2009; ramirez et al. 2 0 1 2 ) . in 2012, upd formulated the master site development plan that serves as a framework for the university’s physical growth for the next 13 years and as a set of guidelines for all improvements in the campus, including, among others, land use allocation, and building and landscape designs (espina and espina 2013). in this f.s. magbanua et al. 7 master plan, eight land uses have been recognized: campus core, academic/academic support units, science and technology park, resource generation zone, residential, community services, parks and open spaces, and protected forest area. nonetheless, we do not know whether the waterways, if any, in these areas are in good condition to support aquatic biota. to address this knowledge gap, we investigated the stream macroinvertebrate biodiversity in upd campus. specif ically, we assessed the overall condition of the waterways based on the benthic macroinvertebrates, water quality, and physical instream habitats along stream reaches in the following land uses: campus core, academic/academic support units, and parks and open spaces. materials and methods study site the university of the philippines diliman campus located in quezon city (14° 38’ n, 121° 2’ e) is the flagship and one of the constituent units of the university of the philippines system. with an area of 493 ha, the campus is a fully functional community and a government unit as it hosts an array of facilities, such as academic units, parks, and residential and commercial areas. daytime population peaks at around 40,000 individuals, which are mainly composed of students, faculty, employees, and some informal settlers (ong et al. 1999; vallejo et al. 2008). quezon city climate is classif ied as tropical monsoonal with a pronounced dry season from november to april and wet season from may to october (figure 1). figure 1. mean rainfall (± standard error) values in science garden, quezon city for the period 2000-2014, and for years, 2015 and 2016. data are from the climatology and agrometeorology division of the philippine atmospheric geophysical and astronomical services administration (pagasa). benthic macroinvertebrates of the up diliman campus waterways 8 nineteen sampling sites within the campus were selected and sampled in november 2015 and 2016 (figure 2). these sites were located in the following land use categories: academic units (au; 6 sites), campus core (cc; 8 sites), and parks and open spaces (po; 5 sites). because of a strong dry spell prevailing in the country in november 2015 (mean rainfall ± standard error = 0.54 ± 0.27 mm; figure 1), several sites ran dry, and hence, were not sampled. these include preselected waterways located in other land use categories (e.g. , science and technology park). nonetheless, the average (± standard error) rainfall in november 2016 was 3.34 ±1.26 mm (figure 1). benthic macroinvertebrates a 50-m sampling reach was established within each land use. following the method of de jesus-crespo and ramirez (2011), three collectors handpicked for 15 minutes all macroinvertebrates from each of the four major habitats (leaf packs, margin vegetation, pools, and riffles) within the 50-m reach. this procedure was continued until three replicate samples per habitat (one from each collector) had been collected. for comparison among sites with different proportions of stream habitat, an overall habitat-weighted value per taxon per site was calculated (de jesuscrespo and ramirez 2011). figure 2. map of the university of the philippines diliman campus in quezon city showing land uses and the location of sampling sites. f.s. magbanua et al. 9 all samples were preserved in 95% ethanol and were brought to the aquatic biology research laboratory, institute of biology, upd for sorting and identif ication. in the laboratory, samples were washed and elutriated using a 250-μm sieve to separate macroinvertebrates from plants, sediment, and other inorganic materials. macroinver tebrates were counted and identif ied to genus level under a stereo microscope. identif ication was performed using the keys of dudgeon (1999), yule and yong (2004), and the mekong river commission (2006). using the macroinvertebrate-habitat weighted value, the following macroinvertebrate metrics were calculated: total invertebrate density (the number of individual organisms collected per m2); taxon richness (the number of taxa counted in a sample); richness of the pollution-sensitive insect orders ephemeroptera-plecopteratrichoptera (ept) and ephemeroptera-plecoptera-trichoptera-coleoptera (eptc); simpson’s index of diversity (d); and simpson’s measure of evenness (e). moreover, biotic indices used in stream bioassessment and biomonitoring were calculated to determine the current condition of the upd waterways: hilsenhoff’s family biotic index, a biotic index for assessing organic and nutrient pollution using tolerance values of arthropod families (hilsenhoff 1988); biological monitoring working party (bmwp), a standardized score system based on tolerance scores of macroinvertebrate families to organic pollution (mustow 2002); average score per taxa (aspt ), a biotic index which measures river status using the calculated bmwp score divided by number of taxa (mustow 2002); stream inver tebrate grade number – average level version 2 (signal 2), a biotic index for australian river macroinvertebrates (chessman 1995, 2003); singapore’s stream biotic index score (singscore), a newly developed biotic index for measuring the health of singapore’s streams using benthic macroinvertebrates (blakely et al. 2014); and average tolerance score per taxon (atspt), a biotic index for evaluating stream health integrity using site disturbance scores and benthic macroinvertebrate abundance (chessman and giap 2010). physicochemical and habitat parameters in the same stream reach where macroinvertebrates were sampled, various physicochemical parameters were measured on site at three randomly selected locations within the 50-m reach: water temperature (°c) and dissolved oxygen (do; mg l-1) were obtained using a do meter (ysi ecosense do200a; yellow spring instruments, ohio, usa), and conductivity (μs/cm) and total dissolved solids (tds; mg l-1) with a hand-held meter (ysi ecosense300a; yellow spring instruments, ohio, usa). in addition, stream width (m), depth (cm), flow rate (m s-1), and water benthic macroinvertebrates of the up diliman campus waterways 10 discharge (m3 s-1) were measured within each reach. these physicochemical parameters were considered in this study because they have been shown to i n f l u e n c e t h e a b u n d a n c e a n d d i s t r i b u t i o n o f b e n t h i c m a c r o i n v e r t e b r a t e s (narangarvuu et al. 2014; yazdian et al. 2014). to evaluate the riparian zones and instream habitats, the modif ied stream visual assessment protocol (magbanua et al. 2013) was used. the protocol is composed of 15 items describing stream environmental condition in relation to channel flow; depth regime; bank stability; vegetative protection and zone; canopy cover; water appearance; nutrient enrichment; streambed characteristics, such as sediment deposition, habitats, habitat complexity, and barriers to movement; and aquatic macroinvertebrate community. each item is scored from 1 to 20, and the sum of all items scored was divided by the number of items scored to assess a site’s habitat condition. hence, a site having a score of ≤ 5 is considered poor, 5-10 is marginal, 10-15 is suboptimal, and 16-20 optimal. data analyses differences in macroinvertebrate assemblage across land uses were evaluated using non-metric multidimensional scaling (nmds) ordination technique through braycurtis similarity matrix after fourth-root transformation of assemblage data, followed by a conf irmatory analysis of similarity (anosim). global r values less than 0.25 indicate similarity in macroinvertebrate communities (refer to maroneze et al. (2011) and novais et al. (2012)). all analyses were performed using the software primer 6.0 (primer-e ltd, plymouth, uk). moreover, we tested differences for the various macroinvertebrate metrics, biotic indices, and physicochemical and habitat parameters among waterways under different land uses using analysis of variance (anova) in ibm spss statistics 20.0 (ibm corp. , new york usa). in the model, land use (academic units, campus core, and parks and open spaces) was the f ixed main (between-subjects effects) factor. if analyses of the f ixed main factor showed signif icance, we performed pairwise comparisons using post hoc tests (tukey’s hsd). for all signif icant f indings, effect sizes (es = partial η2 values, range 0-1; refer to garson (2012)) were reported to compare the magnitudes of effects detected (nakagawa and cuthill 2007). where necessary, data were log 10 (x)or log 10 (x + 1)-transformed prior to analyses to improve normality and homoscedasticity (quinn and keough 2002). f.s. magbanua et al. 11 results and discussion stream physicochemistry, riparian zone and in-stream habitats our results showed that, except for water temperature, all measured physicochemical parameters had signif icant differences across different land use (p d ≤ 0.048 in all cases; table 1). other than do, all parameters were highest in the parks and open spaces land use categories. by contrast, among measured riparian and instream habitat parameters, only canopy cover, water appearance, sediment deposition, and aquatic macroinvertebrate community differed across land uses, with academic units obtaining the highest score in all four parameters (p ≤ 0.039 in all cases; table 1). table 1. summary of the one-way anovas comparing physicochemistry, habitat parameters, biological response metrics, and biotic ind ices across d ifferent land uses. rankings for post hoc tests or specific contrasts in cases with significant effects are given. p-values < 0.05 are in bold print. effect sizes (es = partial ηηηηη2 values; range 0-1; categories: weak > 0.1, moderate > 0.3, strong > 0.5; nakagawa and cuthill 2007) are given for all significant find ings (in bold). au = academic units; cc = campus core; po = parks and open spaces; hfbi = hilsenhoff family biotic index; singscore = singapore score; bmwpthai = biological monitoring working party thai version; asptthai = average score per taxon thai version; signal 2 = stream invertebrate grade number average level version 2; atspt = average tolerance score per taxon parameter au cc po p-value es ranking physicochemistry water temperature 26.74 (0.22) 27.09 (0.16) 27.45 (0.24) 0.064 0.048 dissolved oxygen 2.22 (0.26) 1.36 (0.14) 1.52 (0.21) 0.005 0.090 au > (cc = po) tds 160.45 184.55 209.83 0.008 0.083 po > au (11.40) (10.89) (10.47) conductivity 338.26 360.37 481.00 0.048 0.053 po > cc (10.22) (16.95) (27.64) stream width 1.36 (0.01) 1.84 (0.17) 2.26 (0.22) 0.001 0.124 po > au water depth 8.36 (0.77) 9.81 ( 0.78) 13.35 (0.88) <0.001 0.133 po > (cc = au) flow rate 0.09 (0.02) 0.10 (0.02) 0.19 (0.03) 0.003 0.100 po > (cc = au) stream discharge 0.01 (0.002) 0.03 (0.01) 0.05 (0.01) <0.001 0.131 po > (cc = au) riparian and channel flow 7.72 (0.90) 7.58 (0.87) 8.67 (0.91) 0.357 0.044 instream habitat channel alteration 10.06 (0.93) 8.65 (0.80) 9.40 (0.98) 0.102 0.082 depth regime 6.06 (0.77) 6.76 (0.83) 8.57 (0.95) 0.185 0.066 bank stability 7.67 (0.88) 8.50 (0.86) 8.90 (0.93) 0.129 0.075 bank vegetative 9.89 (0.88) 9.33 (0.84) 9.87 (1.11) 0.465 0.035 protection riparian vegetative 9.53 (0.96) 9.50 (0.92) 9.93 (1.05) 0.726 0.018 zone canopy cover 8.75 (0.97) 8.58 (0.88) 5.63 (1.03) 0.039 0.109 (au = cc) > po water appearance 7.69 (0.84) 4.77 (0.71) 4.80 (0.74) 0.006 0.156 au > (cc = po) nutrient enrichment 6.92 (0.80) 6.69 (0.77) 5.97 (0.80) 0.725 0.018 sediment deposition 6.58 (0.69) 4.90 (0.67) 6.50 (0.71) 0.018 0.129 (au = po) > cc riffle embeddedness 6.61 (0.73) 4.93 (0.61) 6.30 (0.74) 0.161 0.073 barriers to species 9.92 (0.98) 7.83 (0.85) 9.57 (1.10) 0.109 0.080 movement fish habitat 6.50 (0.68) 6.33 (0.65) 6.30 (0.82) 0.872 0.010 complexity aquatic macro8.22 (0.81) 8.00 (0.75) 7.90 (0.95) 0.947 0.005 invertebrate habitat aquatic macro3.72 (0.13) 2.29 (0.10) 1.80 (0.10) <0.001 0.527 au > cc > po invertebrate community overall habitat score 7.72 (0.61) 6.99 (0.56) 7.34 (0.70) 0.818 0.004 benthic macroinvertebrates of the up diliman campus waterways 12 parameter au cc po p-value es ranking table 1. summary of the one-way anovas comparing physicochemistry, habitat parameters, biological response metrics, and biotic ind ices across d ifferent land uses. rankings for post hoc tests or specific contrasts in cases with significant effects are given. p-values < 0.05 are in bold print. effect sizes (es = partial ηηηηη2 values; range 0-1; categories: weak > 0.1, moderate > 0.3, strong > 0.5; nakagawa and cuthill 2007) are given for all significant find ings (in bold). au = academic units; cc = campus core; po = parks and open spaces; hfbi = hilsenhoff family biotic index; singscore = singapore score; bmwpthai = biological monitoring working party thai version; asptthai = average score per taxon thai version; signal 2 = stream invertebrate grade number average level version 2; atspt = average tolerance score per taxon (cont’n.) biological response macroinvertebrate 306.36 (99.46) 121.47 (24.30) 215.77 (34.41) 0.154 0.033 metrics density taxon richness 10.47 (0.89) 8.12 (0.60) 8.50 (0.95) 0.344 0.019 ept taxa richness 0.86 (0.14) 0.27 (0.07) 0.20 (0.07) <0.001 0.160 au > (cc = po) eptc taxa richness 1.44 (0.17) 0.83 (0.11) 0.87 (0.15) 0.020 0.068 au > (cc = po) simpson’s diversity 3.02 (0.31) 3.26 (0.63) 2.12 (0.23) 0.352 0.019 index simpson’s evenness 0.36 (0.05) 0.43 (0.08) 0.34 (0.06) 0.803 0.004 biotic ind ices hfbi 7.76 (0.13) 7.97 (0.13) 8.14 (0.12) 0.095 0.044 singscore 67.68 (3.29) 62.02 (2.07) 61.26 (2.76) 0.409 0.016 bmwpthai 2.68 (0.29) 2.30 (0.29) 2.83 (0.36) 0.197 0.030 asptthai 4.12 (0.13) 4.10 (0.11) 3.89 (0.17) 0.097 0.043 signal 2 2.84 (0.07) 2.66 (0.05) 2.66 (0.06) 0.241 0.026 atspt 57.43 (0.22) 58.69 (0.28) 58.61 (0.39) 0.001 0.114 (cc = po) > au these f indings are consistent with most urban stream studies done in the past (e.g. , couceiro et al. 2007; de jesus-crespo and ramirez 2011; baltazar et al. 2016; docile et al. 2016). increasing loads of organic and inorganic carbon in urban stream decrease the amount of do (daniel et al. 2002; butman et al. 2015; tromboni and dodds 2017). moreover, high dissolved solid concentrations had been observed in upd streams. studies conducted by horn et al. (2017), taka et al. (2017), and toor et al. (2017) all noted that dissolved solids are known to accumulate in areas with higher rates of inorganic runoff (e.g. , industrial sites, residential sites) and contribute to an increased ion concentration annually. lastly, changes in land use and hydrological gradients altered stream channels, depth, flow rate, and discharge in the campus waterways due to continued habitat degradation, land cover modif ication, and subsurface drainage, which through time, may negatively affect local stream habitat and biodiversity (allan 2004; potter et al. 2014; walsh and webb 2016; baumgartner and robinson 2017). f.s. magbanua et al. 13 in habitat assessment, only canopy cover, water appearance, sediment deposition and aquatic macroinvertebrate community exhibited marked differences across different land use types (table 1). changes in riverine spatial gradients has been tagged as major driver in declines of stream biota. canopy cover is essential for maintaining lower stream temperature and for increasing the allochthonous source of energy which in turn promotes diverse stream biotic assemblages (sponseller et al. 2001; kominoski et al. 2011). in urban streams, the amount of detritus breakdown is lower, leading to a much poorer biotic assemblages (roy et al. 2005; mar tins et al. 2015). furthermore, uriarte et al. (2011) noted that in water appearance the increasing load of organic and inorganic materials in streams brought by continued urban runoff and riparian habitat degradation leads to its much poorer state. likewise, extence et al. (2013) reported that sediment deposition also increases in streams with low flow, modif ied habitat, and excessive sediment output from the catchment. the diversity of aquatic macroinvertebrate community heavily depends on the condition of its habitat, which determines the community that it can support (weijters et al. 2009). modif ied habitats (e.g. , high conductivity, eutrophic streams) tend to support pollution tolerant taxa, while undisturbed habitats (e.g. , high do, low water temperature) support diverse benthic communities comprised mainly of pollutionsensitive taxa (miserendino et al. 2011). benthic macroinvertebrate assemblages a total of 42,663 macroinvertebrates belonging to 45 families and 56 genera were collected in 19 stream reaches within the upd campus. of these 56 genera identif ied, 10 comprised 93.6% of the total: the non-biting midge chironomus spp. (67.2%), the segmented worm oligochaeta (genus 1) (11.2%), the non-biting midge cricotopus spp. (4.5%), the moth fly psychoda spp. (3.3%), the lymnaeid snail radix quadrasi (1.7%), the shore fly brachydentera spp. (1.7%), the dragonfly brechmorhoga spp. (1.2%), the freshwater leech helobdella spp. (1.0%), the mayfly labiobaetis spp. (1.0%), and the non-biting midge thienamannimyia spp. (0.9%) (figure 3). the results of the ordination analysis reveal weak clustering (2d stress=0.24) across different land uses (figure 4). this was further supported by the global r of anosim for land uses (global r = 0.070, p = 0.1), indicating no observable variation in the macroinvertebrate community. however, among the different biological metrics analyzed in this study, the richness of the pollution-sensitive insect orders ept and eptc exhibited signif icant differences across different land uses (table 1). benthic macroinvertebrates of the up diliman campus waterways 14 figure 3. ten most dominant macroinver tebrates across land use types in the university of the philippines diliman campus waterways. f.s. magbanua et al. 15 figure 4. two-dimensional non-metric multidimensional scaling (nmds) plot of the stream macroinvertebrate community structure across different land uses in the university of the philippines diliman, quezon city, philippines. the resulting similarity of identif ied macroinvertebrate community across different land uses is a general trend in urban streams due to higher rates of organic and inorganic solute contamination along altered riparian and stream reaches (cuffney et al. 2010; ricart et al. 2010). for instance, the presence of pollution-tolerant taxa (e.g. , chironomidae, oligochaeta) and absence of pollution-sensitive taxa (e.g. , perlidae, psephenidae) may contribute to the similar aquatic invertebrate communities across different land uses, since the former can thrive in urban streams due to higher rates of water stress, while the latter requires pristine environmental conditions (de paiva silva et al. 2010; chang et al. 2014; mehring et al. 2017). upd streams are dominated by several members of the family chironomidae (e.g. , chironomus spp. , cricotopus spp. , thienamannimyia spp.) and oligochaeta, both of which are pollution-tolerant. these organisms can tolerate a wide range of environmental conditions (e.g. , low do concentration, high dissolved solids); thus, allowing them to thrive in all types of habitat, ranging from pristine to heavily degraded streams (cortelezzi et al. 2011; frizzera and alves 2012; rosa et al. 2 0 1 4 ) . however, the marked differences in ept and eptc taxa richness across different land uses indicated the capacity of upd streams to be inhabited by these taxa. similar f indings had been observed in the studies of lenat and crawford (1994) benthic macroinvertebrates of the up diliman campus waterways 16 and violin et al. (2011), wherein these authors observed ept and eptc taxa in urban sites. nonetheless, it should be noted that these identif ied taxa (e.g. , baetidae, hydrophilidae) are considered mildly tolerant to pollution by others (e.g. , rizopatron et al. 2013; chang et al. 2014), similar to the ept and eptc taxa identif ied in upd streams. furthermore, higher rates of organic runoff in urban streams can signif icantly increase the density of pollution-tolerant invertebrates and prevent possible colonization of pollution-sensitive taxa (roy et al. 2003; niyogi et al. 2007; shin et al. 2011). stream cond ition based on macroinver tebrate biotic ind ices all measured biotic indices reveal that, across different land uses, only atspt (p = 0.001) showed marked difference (table 1). in addition, the high pollution tolerance score of collected and identif ied invertebrates in upd waterways led to poor stream condition ratings in all biotic indices across different land uses (table 2). changed atspt values depict the quality of upd streams across different land uses, indicating the importance of riparian habitats in supporting diverse biotic communities (poff and zimmerman 2010). nonetheless, our results underscored the poor water quality condition of upd streams, regardless of land use (table 2). biotic indices assign numerical value to a specif ic taxon with a corresponding tolerance score based on its tolerance to pollution (zimmerman 1993). in the case table 2. mean (± standard error) values of computed biotic ind ices and the correspond ing cond ition ratings across d ifferent land uses. hfbi = hilsenhoff family biotic index; singscore = singapore score; bmwpthai = biological monitoring working party thai version; asptthai = average score per taxon thai version; signal 2 = stream invertebrate grade number average level version 2; atspt = average tolerance score per taxon hfbi 7.76 (0.13) very poor 7.97 (0.13) very poor 8.14 (0.11) very poor singscore 67.68 (3.29) poor 62.02 (2.07) poor 61.26 (2.76) poor bmwpthai 2.68 (0.29) very bad 2.30 (0.29) very bad 2.83 (0.36) very bad asptthai 4.12 (0.13) bad 4.10 (0.11) bad 3.89 (0.17) bad signal2 2.84 (0.07) probable severe 2.66 (0.05) probable severe 2.66 (0.06) probable severe atspt 57.43 (0.22) unhealthy 58.69 (0.28) unhealthy 58.61 (0.39) unhealthy biotic index index score cond ition rating index score cond ition rating index score cond ition rating academic units campus core parks and open spaces pollution pollution pollution f.s. magbanua et al. 17 of upd’s macroinvertebrate assemblage, the abundance of tolerant taxa resulted in the streams’ poor condition ratings. lastly, the biotic indices used in this study are all derived from other countries and have failed to consider local taxon that has no pre-assigned tolerance value, and thus, may not provide a true picture of the streams in regions outside its origin (zeybek et al. 2014). conclusion and recommendation globally, urban streams generally have higher loads of organic and inorganic pollution, compromised stream and riparian areas, abundant pollution-tolerant taxa, and poor water and habitat quality. our results reveal poor to severe stream conditions across land uses. marginal habitat assessment scores and sub-optimal physicochemical parameters in all streams supported these f indings, reflecting the intensity of riparian and stream modif ication. similarly, water quality based on considered variables also indicated poor quality, which is consistent with the stream biota dominated by pollution-tolerant taxa. these resulted in lower biotic index scores, providing further support for the severity of the conditions of upd streams. our f indings reflect similar patterns observed in urban streams, which may persist if upd streams and riparian habitats are not protected and restored. therefore, we recommend a campus-wide restoration of streams and waterways, as well as improvement of the wastewater treatment facility in the campus. we also suggest monitoring the streams and waterways during wet and dry seasons to provide a complete picture of the conditions of these waterways. this bioassessment may provide additional knowledge on the benthic macroinvertebrate community structure and the possible effects of environmental flow on these urban communities. acknowledgments this project was funded by the off ice of the chancellor of the university of the philippines diliman, in collaboration with the off ice of the v ice chancellor for research and development (ovcrd), through ovcrd phd incentive award (project nos. 151503 and 161619 phdia) awarded to f.s. magbanua. special thanks to the instiute of biology, university of the philippines diliman for the research load credit (ib2016-fsm-9). we are most grateful to jelaine gan, joy emika balagtas, dina marie de dios, angelo joshua luciano, marjohn baludo, julie-an gregorio, kris benthic macroinvertebrates of the up diliman campus waterways 18 or tizo, paul palomares, and leocris batucan jr. for their help in the f ield. we also thank angelo joshua luciano for his help with figure 2, angelo joshua luciano and julie-an gregorio for the photos of the macroinvertebrates in figure 3, and three anonymous referees for their helpful comments on the manuscript. references allan jd. 2004. landscapes and riverscapes: the influence of land use on stream ecosystems. annual review of ecology, evolution and systematics. 35:257-284. baltazar des, magcale-macandog d, tan mfo, zafaralla mt, cadiz, nm. 2016. a river h e a l t h s t a t u s m o d e l b a s e d o n w a te r q u a l i t y, m a c r o i n v e r t e b r a te s a n d l a n d u s e f o r niyugan river, cabuyao city, laguna, philippines. journal of environmental science and management. 19(2):38-53. baumgar tner sd, robinson ct. 2017. changes in macroinver tebrate trophic structure a l o n g a l a n d u s e g r a d i e n t w i t h i n a l o w l a n d s t r e a m n e t w o r k . a q u a t i c s c i e n c e s . 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the aquatic biology research laboratory, institute of biology, university of the philippines diliman. he received his ph.d. in zoology from the university of otago, dunedin, new zealand. he specializes in freshwater ecology and biomonitoring using f ish and benthic macroinvertebrates. john claude renan b. salluta is a research associate at the aquatic biology research laboratory, institute of biology, up diliman and a m.sc. environmental science student at the institute of environmental science and meteorology, up diliman. he obtained his b.sc. biology at southern luzon state university, quezon. danielle dominique d. deborde is a graduate from the institute of biology, up diliman, where he received his b.sc. in biology. maria brenda m. hernandez is a former instructor at the institute of biology, up diliman. she is currently f inishing her ph.d. degree in the department of biology, university of waterloo, ontario, canada. she specializes in limnology and benthic communities (freshwater algae and macroinvertebrates). university of the philippines diliman office of the vice chancellor for research and development call for papers humanities diliman, science diliman, and social science diliman are peer-reviewed journals published biannually (in june and december) by the university of the philippines diliman through the office of the vice chancellor for research and development (ovcrd). papers are accepted year-round. for more information, please visit the ovcrd website <www.ovcrd.upd.edu.ph> cover image credits: vargas museum (humanities diliman) and biodiversity research laboratory (science diliman) olivera 1 taxonomy of gemmula science diliman (january-june 2005) 17:1, 1-14 evaluation of philippine gemmula: i. forms related to g. speciosa and g. kieneri abstract baldomero m. olivera department of biology university of utah 257 south 1400 east #201 salt lake city, utah 84112-0840 email: olivera@biology.utah.edu date received: may 27, 2004; date accepted: september 17, 2004 gemmula (weinkauff, 1875) is the largest genus in the subfamily turrinae (h. & a. adams, 1853). in this article, philippine forms of gemmula with morphological similarities to gemmula speciosa (reeve, 1843) and gemmula kieneri (doumet, 1840) are evaluated. two new species, gemmula sogodensis and gemmula sikatunai, are described. a group of gemmula specimens collected from the western atlantic appear to be a form of gemmula sikatunai. the similarity between philippine and atlantic forms suggests that gemmula sikatunai is remarkably stable, long-lived species. keywords: turrinae, gemmula, relict species introduction the turrinae (h. & a. adams, 1853) are a subfamily of venomous gastropods that belong to the family turridae (h. & a. adams, 1853), superfamily conoidea (ponder & warén, 1988). we focus in this article on philippine forms of the genus gemmula (weinkauff, 1875), the “gemmate turrids” that are related to gemmula speciosa or gemmula kieneri. most philippine forms in the subfamily turrinae fall into three major genera, turris (röding, 1798), lophiotoma (casey, 1904), and gemmula. the first three papers of this series (olivera, 1999; olivera, 2002; olivera, 2004) presented an overview of philippine species of turris and lophiotoma. both turris and lophiotoma are strictly indo-pacific, and appear to be of relatively recent geological origin, since these genera do not appear to be represented in the fossil record earlier than the miocene (powell, 1964). in contrast, the fossil record for gemmula extends back to the paleocene (powell, 1964; powell, 1966). the biogeography of living forms in each genus also supports this picture: gemmula is found not only within the indo-pacific, but in the eastern pacific and caribbean provinces as well. powell remarked in the last comprehensive monograph on the indo-pacific turrinae that the genus gemmula “is the most vigorous member of the turrinae, and undoubtedly represents the main stem of the subfamily” (powell, 1964). both in the past and at present, the genus has had a wider radiation than any other genus in the turrinae. because of the significant amount of material collected between 30 and 600 meters by shell-gathering fishermen in the philippines in recent years, a substantial series of specimens of gemmula have become available. several previously undescribed philippine gemmula can be clearly recognized from this recently collected trove of new philippine material. in this paper, two new species 2 olivera are proposed on the basis of differences in shell morphology. unfortunately, the soft parts of most forms in the turrinae are not available for analysis, and although it would have been far preferable to have diagnosed these species with an accompanying comparative description of the entire anatomy, we were restricted to using only conchological characters. this is a limitation of this work; in particular, as will be described below, specimens of one of the new species proposed are from two widely separated geographic localities, and a more extensive diagnosis of the anatomy of the whole animals may demonstrate differences between these populations that are too subtle to be discerned from the shells alone. in the earlier monograph on species of turris in the philippines, the origins of the species complexes in that genus were the subject of speculation (olivera, 1999). our study of the forms of philippine gemmula discussed in this paper, including an almost identical form from the caribbean, has provided intriguing insights into the evolution of the turrinae. materials and methods specimen collection gemmula specimens examined were mostly from the author’s collection, collected from various philippine localities. specimens more recently obtained were collected primarily by using tangle nets in deep water (80-600 m), by trawling, or by hookah in the localities indicated under each species. the caribbean material examined was collected by a. kerstitch in 1997 by dredging in deep water off the island of barbados. material collected in the philippines by the muséum national d’histoire naturelle, paris was examined and has proven to be of exceptional value for establishing the bathymetric distribution of several forms. the author also examined material from the following american museums: national museum of natural history, smithsonian institution, washington, dc; the field museum of natural history, chicago, il; the american museum of natural history, new york, ny; and the los angeles county museum of natural history, los angeles, ca. species characterization the primary diagnostic keys used were the monograph of powell (powell, 1964), the book of springsteen and leobrera (springsteen & leobrera, 1986) and the 1983 article by kilburn (kilburn, 1983). morphological observations were restricted to the shells of each species. however, we have also collected alcohol-preserved material wherever possible in anticipation of a comprehensive molecular analysis of the turrinae. the gemmula speciosa group overview gemmula speciosa (reeve, 1843) is a well-known species found over much of the indo-pacific and the red sea. most philippine specimens in older collections were obtained from trawlers, particularly those operating around samar island (carigara and maqueda bays) and southwestern luzon island (tayabas bay), where g. speciosa was collected in fair numbers in the late 1950s and 1960s, until trawling methods were changed. g. speciosa is a relatively large species (attains >75 mm), with golden brown spiral cords on a white background, a brown cog-like peripheral cord and a rounded body whorl. the continuous golden-brown color of primary spiral cords without interruption is one defining characteristic of typical shells of the species, although substantial variation is observed in the intensity of the brown coloration. more recently, similar specimens from other philippine localities have been collected by gill nets; these are noticeably more slender and elongate than the material collected by trawling. the author was given a collection of turrinae from the caribbean, personally collected by the late alex kerstitch. this collection included forms similar to the more-recently collected material from the philippines. the rather remarkable biogeographic separation of the caribbean material, despite its similarity to philippine material prompted a closer examination of whether there were any consistent differences between the caribbean material and g. speciosa from the philippines. the conclusion from examining a large number of specimens is that in fact, there are two distinct taxa in the philippine material 3 taxonomy of gemmula conventionally identified as g. speciosa; one of these is practically indistinguishable from the caribbean specimens. classical trawled material agrees with the standard reeve description of g. speciosa. the narrower specimens, apparently all collected from deeper water than true g. speciosa are a different taxon, described here as a new species, gemmula sikatunai. thus, the new species appears to have been found in two widely separated localities, the philippines and the caribbean. we also include in this group the recently described gemmula lululimi (olivera, 1999). the biogeographic range suggests that the present living forms in the g. speciosa complex may be derived from a more widely distributed, presumptive ancestral form in the tethys sea very similar to g. sikatunai that has been largely unchanged for tens-of-millions of years. a detailed description of each form follows, as well as a discussion of differences between the forms. the three species included in the g. speciosa complex are shown in fig. 1. gemmula speciosa (reeve, 1843) description (adapted from powell, 1964). adult shell 50-78 mm in height. fusiform, with tall spire, and long, tapered, and slightly flexed anterior canal. whorls angulate and carinate at just below middle whorl height; base rather suddenly contracted. peripheral carina, a square-cut prominent flange densely studded with narrow, laterally compressed and peaked nodules which give a regular cog-like effect. primary spirals plain, thin but sharply raised, usually three above the carina, one of which is on a moderate subsutural fold, one or two below the carina, and about six on the base exclusive of the canal. the secondary sculpture consists of from one to three plain weak threads in the interspaces of the primaries. the surface is crowded with weak, crisp axial threads. color pattern of light brown to darker golden brown spirals on a buff background. the peripheral carina is uniformly colored, and all of the primary spirals are similarly tinted light brown to golden brown. in typical specimens, there are no interrupted markings, dots or dashes. the light amber protoconch is typical of gemmula (fig. 2), and is followed by nine teleoconch whorls in adult shells. in most specimens, the brown primary spirals become obsolete in the anterior siphonal canal, which becomes pure white. collection records powell recorded three stations where the depth was noted (27 fathoms and 35 fathoms from two stations in the philippines, and 73 meters from the gulf of oman). more recently, the musorstrom 3 expedition by mnhn, paris, collected 18 specimens of g. speciosa off lubang island, philippines (11° 45 n, 120° 45 e). the collection station was cd 141, and the depth range was 40-44 meters. the 18 specimens of g. speciosa in the dredge haul varied in size from 20 mm to 51 mm; eight of the specimens were dead-collected with large drill holes. the only other turrinae were three small gemmulas, including one gemmula monilifera, and two of an unidentified species. g. speciosa is apparently collected by divers off batangas, luzon island, philippines, buried deep in sand (jose javier, personal communication). gemmula sikatunai, new species this new species has been generally identified by commercial dealers as g. speciosa, but compared to g. speciosa, the shells examined are smaller and narrower, and were collected from deeper water. we divide g. sikatunai into two forms that have a discontinuous geographic distribution, an indo-pacific and a caribbean form; dead specimens of the latter were collected off barbados. gemmula sikatunai, philippine form description adult shell, 40-51 mm in height. fusiform, with tall spire and a long, relatively broad canal. whorls strongly angulate and strongly carinate. peripheral carina is gemmate, with two brown cords at the margins, the more posterior being somewhat stronger. particularly 4 olivera in the larger whorls, the center of the carina and gemmae are lighter colored than the bordering cords; on the body whorl, the two brown cords on the carina are well separated by the lighter-colored central area in mature specimens (fig. 1). the primary spirals are sharply raised, with one brown primary cord in the subsutural region, somewhat offset from the suture itself. between the subsutural fold and the peripheral carina are 2-4 white spiral cords. the region between the subsutural fold and the peripheral carina is whiter than the rest of the shell and has a somewhat silky texture. anterior to the peripheral carina are usually six primary spirals on the body whorl and the base, uniformly light brown to golden brown in color. there are 5-7 additional primary spirals on the canal, becoming progressively lighter and weaker in some specimens, but remaining quite strong almost to the end of the canal in other specimens. the secondary sculpture consists of from 1-3 weak white threads in the interspaces; these become strong towards the base and in the canal, becoming light brown towards fig. 1. top row, from left: gemmula speciosa (leftmost two specimens), gemmula sikatunai (middle two specimens), and gemmula lululimi (rightmost two specimens). for both g. sikatunai and g. lululimi, the left specimen is the holotype, the right is a paratype. middle row shows a magnified view to compare subsutural and sinus cord areas. bottom row: the same specimens showing a top view. note the continuous brown color and cog-like sinus cord of g. speciosa, and the more brown-colored, marginal sinus cord and bead-like structure of the sinus cord in both g. sikatunai and g. lululimi. note the lighter central region of these sinus cords in contrast to the uniform brown of g. speciosa. 5 taxonomy of gemmula the anterior in some specimens. the protoconch is polygyrate with axial ribs, typical of gemmula, and there are ten teleoconch whorls in adult shells. nomenclature this species, found around the island of bohol in the philippines, is named after a 16th century rajah of bohol, sikatuna. in a famous event of philippine colonial history, sikatuna and the spaniard legaspi entered into a blood compact. collection data the holotype and paratypes 1-5 were collected off bohol island, philippines by gill nets in 100-300 fathoms. the mnhn expedition to lubang island, philippines provides more specific data. the most productive source of g. sikatunai was in the musorstrom 2 expedition, station cp 66, 14° 00 n, 120° e at a depth of 192-209 meters; 42 specimens of g. sikatunai were collected in a single dredge haul. some other stations in the mnhn expedition collections that yielded the new species were station dl 34, musorstrom 2, 13° 28 n, 120° 12 e at a depth of 155-167 meters (three specimens of g. sikatunai) and musorstrom 1, station fig. 2. top row: gemmula sikatunai, new species. the two rightmost specimens are dead-collected shells off barbados that are assigned to this species. bottom row, left two figures: protoconchs of gemmula speciosa compared to the protoconchs of g. sikatunai (these are protoconchs of paratype 13 (left) and the holotype (right)). note the amber color and larger size of the g. speciosa protoconchs compared to the smaller g. sikatunai. 6 olivera 11, 14° 00 n, 120° 18 e, at 213-230 meters (four specimens of g. sikatunai). seven mnhn specimens are included as paratypes (appendix). it is notable that in all of the musorstrom stations at depths >150 m, no specimens of g. speciosa were collected. in contrast, in shallower waters (cd station 141, 40-44 meters referred to above) only g. speciosa and no g. sikatunai were collected. also included in the paratype series are two specimens in the los angeles county museum (lacm 75849) collected in 52-67 fathoms by j. norton, near talaga, batangas, s.w. luzon; if these depth records are accurate, it would be the most shallow record for g. sikatunai. gemmula sikatunai, caribbean form this form is strikingly similar to specimens from the indo-pacific. however, in addition to the striking geographic separation, there are subtle morphological differences between the two forms. description the adult shell is 39-47 mm in length, with 12-13 whorls. the spire angle is 33-35°, and the siphonal canal is relatively long. all primary spiral cords are uniformly golden brown. on the body whorl, there is a single primary subsutural cord, followed by a white area with six fine white threads. the peripheral carina is made up of two parallel brown spiral cords with gemmules, and on the body whorl there are two pairs of brown primary cords, separated by a white area followed by six to seven less prominent primary spiral threads which cover the base and siphonal canal. the specimens seen by the author are generally more elongate, and somewhat smaller than typical adult specimens of g. sikatunai from the philippines. collection data all known specimens of this subspecies were collected by alex kerstitch in june, 1997, in ca. 450 feet off barbados by dredging. the major turrid collected at this site was a small unidentified polystira species. differences between g. speciosa, g. sikatunai, philippine form and g. sikatunai, caribbean form g. speciosa grows to a larger size than g. sikatunai (>75 vs. <55 mm for the largest specimens), the whorls of g. speciosa are proportionally wider, and the protoconch appears to be larger and smoother. there are a number of consistent differences in sculpture. g. speciosa typically has two brown-colored primary cords (range, 1-4) between the subsutural region (which has a primary brown-colored cord in all forms) and the peripheral carina. in g. sikatunai, this area is white in all specimens examined. a definitive differentiating feature is the structure of the peripheral carina. in g. speciosa, the gemmules are laterally narrow, giving the sinus cord a characteristic “cog-like” structure; furthermore, the entire peripheral carina is generally a uniform brown color, although the intensity of the color can vary markedly. in g. sikatunai, particularly in later whorls, the brown color is concentrated on the two edges of the peripheral carina, on raised cords that curve around the gemmules, with a lighter color in between; the gemmules are not narrow and raised, and do not give a cog-like impression. g. sikatunai is narrower in outline than g. speciosa. the whorls of g. speciosa are somewhat rounded and contracted at the base; the whorls of g. sikatunai are triangular, strongly carinate, with the shoulder area quite sharply angled with respect to the rest of the whorl. finally, in g. speciosa, the brown primary cords tend to become obsolete in the upper half of the siphonal canal, leaving much of the siphonal canal pure white; in many specimens of g. sikatunai, the primary brown cords continue almost to the end of the canal. the philippine and caribbean forms of g. sikatunai are very similar; all specimens of the latter were deadcollected. in caribbean specimens, anterior to the peripheral carina are two strong brown primary ribs, followed by a gap with a conspicuous white area lacking a brown cord, followed by more primary cords more or less evenly spaced (in certain specimens, another gap occurs between the cords on the base and the cords on the canal, but this is variable). in philippine specimens, the brown cords are either more or less regularly spaced, or show an alternating pattern of dark and. light brown primary cords. caribbean specimens are proportionally 7 taxonomy of gemmula narrower than the typical philippine form. in caribbean specimens, the subsutural primary brown cord is more diffuse at the anterior edge. the most intriguing feature of these forms is their remarkable geographic separation. gemmula lululimi (olivera, 1999) this species has developed a more turris-like shape, but has striking morphological similarities to the gemmula speciosa/sikatunai complex in a number of aspects. g. lululimi is similar to other forms in the g. speciosa group in the continuous golden-brown or blackish-brown color on the spiral cords, on the same whitish background. the number of spiral cords, and the presence of gemmules on the sinus cord are additional characteristics in common. description (adapted from olivera, 1999) the spire is high, cone-shaped; adult specimens have 15-17 whorls. spire angle is 25-27°. there are three primary spiral cords, continuous brown colored, in each spire whorl. the subsutural fold has a major primary cord which is raised; in some larger specimens, a second cord is found immediately adjacent to the suture. there is a pronounced depression between the subsutural primary cord and the sinus cord. the sinus cord has gemmules, and in later whorls the two margins are brown, with the central gemmae lighter in color, almost white in some specimens. the sinus cord is not located peripherally, but is posterior to the periphery. a strong brown cord is present at the periphery in all the spire whorls. on the body whorl, there are 5-7 brown primary cords anterior to the sinus cord, with 4-6 brown primary cords on the anterior canal. the very tip of the canal is white, lacking the brown spiral cords in most (but not all) specimens. the specimens the author has examined range in size from 37-87 mm. discussion g. lululimi is unusual among members of this complex in its distinctively different shell morphology, which has some features more consistent with turris rather than gemmula, notably that the sinus cord is not the peripheral carina as in most species of gemmula; in the case of g. lululimi the sinus cord is non-peripheral, located instead between the subsutural area and periphery. however, the reasons for including this form in the genus gemmula were discussed with the original description (olivera, 1999); basically, all other characteristics of the shell have a strong affinity to the g. speciosa group, and no apparent phenetic similarity to other species in the genus turris. it should be noted that the structure of the sinus cord, though at a nonperipheral position is more similar to the peripheral cord of g. sikatunai than to g. speciosa (fig. 1). recently, a number of specimens was collected that are much darker in the color of the primary cords and have a more robust shell than the types. this form has been described as a species within the genus turris, turris (annulaturris) munizi by vera-peláez et al. (vera-peláez et al., 2000), but appears to be conspecific with g. lululimi. this species remains relatively rare, and is represented in collections by relatively few specimens. the gemmula kieneri group gemmula kieneri is a widely distributed indo-pacific species, and it has become clear that there are two distinct forms in the philippine material conventionally assigned to g. kieneri. a new species, g. sogodensis is described. gemmula kieneri (doumet, 1840) description (adapted from powell, 1964) adult shell up to 73 mm in height, robust, fusiform, with tall spire and long, rather straight anterior canal. spire whorls with a strong square-cut keel situated below the middle, not prominently projecting and sculptured, with closed spaced rectangular gemmules that are laterally compressed. there are regular squarish spots between the gemmules. the strong complex subsutural fold is irregularly blotched with brown, and usually consists of a primary cord (sometimes two) and additional threads. between the subsutural fold and the peripheral keel, there are usually 3-4 sharply raised, 8 olivera slightly imbricate threads. between the primary keel and the lower suture there is one primary cord and several threads. one the base, exclusive of the anterior canal, are about 6 primary cords, with irregularly disposed brown spots, with between 1-3 interstitial threads. the surface is covered with dense lamellate axial growth threads that imbricate the secondary spiral threads. discussion philippine specimens seem to be generally smaller with a proportionately broader spire than specimens from the northern pacific. the range of variation in philippine specimens is primarily in the shape of the shell; the ratio of the canal to the spire can vary considerably in philippine specimens. specimens from japanese waters are much more solid, robust shells. this species can normally be differentiated from g. sogodensis (see next species) by the irregularly blotched, usually broader subsutural cords, which because they are more prominent and larger, make g. kieneri seem much less constricted at the sutures than is the case with g. sogodensis. there is also considerable variation in color. in some specimens the whole area from the upper suture to the sinus cord can be quite deeply colored; in other specimens this area is whitish. in a few specimens, the body whorl has axial patches of light brown. gemmula sogodensis, new species a previously unrecognized form, morphologically somewhat intermediate between g. kieneri and g. speciosa, has been collected in sufficient numbers to convincingly demonstrate that it is distinct from g. speciosa, g. kieneri and g. cosmoi that it can be mistaken for, based on a number of conchological characters. description adult shells are 36-49 mm in length, with 11-13 whorls in adult specimens. the spire is relatively high, spire angle typically 34-36°. the anterior canal is long, and the ratio of aperture plus canal to total length is approximately 0.5. a single subsutural cord is present of a continuous reddish-brown color, followed anteriorly by a white area with flattened spiral threads. in occasional specimens, one of the threads is light brown in color. the large peripheral, highly gemmate sinus cord is white in the early spire whorls, with the interspaces on the peripheral cord between gemmules becoming brown colored in later whorls. on the body whorl, these interspaces become brown to reddishbrown, with raised white gemmules. the body whorl has about six primary spiral cords in addition to the peripheral sinus cord, each with an irregular pattern of golden or reddish-brown dots and white raised dashes. the cords on the body whorl are much less prominent than the sinus cord. on the sixth spiral cord there is usually a second sinus that forms on the lip. the canal is covered by numerous white spiral cords which are more flattened and less colored than the body whorl cords. the combination of the prominently gemmate sinus cord with white gemmae and golden or reddishbrown interspaces which are white in early spire whorls and become more deeply colored as the shell whorl gets larger, the uniformly colored subsutural cord, and the presence of a second sinus are distinctive characteristics of the species. the holotype and several paratypes are shown in fig. 3. collection data the type locality for g. sogodensis is sogod, cebu, philippines, for which the species is named. most philippine specimens known to the author that have been collected recently were from the type locality. the specific collection site is off barangay, tabunok, sogod in the central philippine island of cebu. g. sogodensis specimens were obtained using tangle nets that are approximately 600 meters long, at a depth of 60-100 fathoms. the specimens were collected from a black mud bottom, with a sand-silt substrate with high organic matter, approximately 1 km offshore from tabunok. the specimens had a pitch black coating which is removed when the shells are soaked in bleach and brushed off. conus from the same locality including conus tribblei and conus pagodus are typically covered with a green algae, while some conus and turrids (conus sulcatus and lophiotoma bisaya) are collected much more cleanly, with only the periostracum. the origin of the dark coating on the shells of g. sogodensis 9 taxonomy of gemmula must have some specific biological determinants. g. sogodensis appears to be one of the most common turrids at this locality; the main commercial product of this shell fishery is murex troscheli, a large decorative murex species. the specific information regarding specimen collection at the sogod, cebu locality was kindly provided by frank heralde. specimens of gemmula sogodensis have been collected from n. mindanao island in the philippines to taiwan and southern japan. one immature specimen collected by the mnhn musorstrom 3 expedition to lubang island, philippines was collected at station cp 86, 14°00n, 120°18e in 187-192 meters. fig. 3. top row: specimens of gemmula kieneri and a protoconch of g. kieneri (extreme right). bottom row, from left to right: gemmula sogodensis, holotype, paratype 11 and a specimen from southern japan, as well as the protoconch of paratype 15. the bar is 1 mm. 10 olivera discussion and comparison superficially, g. sogodensis seems intermediate between philippine forms of g. speciosa and g. kieneri. the new species is easily differentiated from g. speciosa because the gemmae on the peripheral cord are mostly white; in g. speciosa, the peripheral carina has a continuous golden-brown color, including the gemmae. the spiral cords in g. sogodensis are not continuously colored but interrupted on the body whorl (there are exceptional specimens where the spiral cords are uniformly colored with light brown). in this respect, g. sogodensis resembles g. kieneri, but it is easily differentiated from that species by the continuous brown color of the primary cord in the subsutural area and the white peripheral carina in early spire whorls. in g. kieneri, the subsutural cord is usually broad, with a complex structure, and irregularly maculated. the brown or reddish-brown subsutural cord of g. sogodensis is sharp, crisp and continuously colored (in this respect, more similar to g. speciosa). philippine specimens of g. kieneri generally have a higher frequency of gemmules, and on the spire whorls the alternating light and dark color intervals are maintained even on the early spire cords. other differences are the generally narrower, more elongate spire of g. sogodensis compared to g. kieneri. although many specimens of g. sogodensis have shells with growth scars, and are often corroded, the best specimens are among the most elegant and attractive of all gemmula. g. sogodensis may also be confused with gemmula cosmoi, but the former has both brown color in the interspaces between the gemmules and the sinus cord, as well as dashed brown markings between white areas on the primary cords of the body whorl; both features are generally lacking in g. cosmoi. in addition, g. cosmoi is generally narrower in outline than g. sogodensis, and the spiral cords between the brown subsutural cord and the peripheral carina, although white in both species, are much stronger in g. sogodensis. finally, g. cosmoi has characteristic comma-like gemmae (powell, 1964), which are not found in g. sogodensis. nomenclature this species name is derived from the type locality, sogod, cebu, philippines. types the holotype will be deposited in the philippine national museum. paratypes will be deposited at the field museum of natural history, chicago; the national museum of natural history, washington, dc; the academy of natural sciences, philadelphia; and the american museum of natural history, new york city. the paratype series includes two lots from the la county museum of natural history from taiwan (lacm 69006) and japan (lacm 47060), and one specimen from the muséum national d’histoire naturelle, paris (paratype 46). distribution of species it seems likely that g. sogodensis will be found to be more widely distributed in the indo-pacific. the specimens collected from taiwan and southern japan are documented in the appendix; the japanese material has been confused with g. cosmoi. discussion a comparison between the gemmula species complex described above and the turris babylonia clade (olivera, 1999), the dominant group of species in the genus turris, is most instructive. these two groups of species in the turrinae show striking contrasts: as described above, both the biogeography of gemmula and the fossil record are consistent with an early tertiary origin for this group, and remarkable stability in g. sikatunai. in contrast, both the biogeography and fossil evidence are consistent with a late tertiary origin for the t. babylonia clade of turris. the general impression is that the latter is a relatively young, rapidly evolving species complex. the data suggest (but do not prove) that g. sikatunai, one of the new gemmula species described in this study, 11 taxonomy of gemmula may be an exceptionally stable form. the distinct gemmula species complex found in philippine waters which is described above has no corresponding living forms in the eastern atlantic or the mediterranean. this makes the near identity in conchological characters of the dead-collected caribbean specimens of g. sikatunai to its indo-pacific counterpart most striking, given the immense geographic separation, and because the caribbean tropical marine fauna has long been separated from tropical indo-pacific species (since the closure of the tethys sea). it is possible that there has been incomplete sampling of gemmula spp., particularly given that they live in relatively deep water. it is noteworthy that g. speciosa has been found in the red sea; do these now occur in the eastern atlantic, or did they in the past? however, the most straightforward explanation for the biogeography of g. sikatunai is that it is of early tertiary origin, and was widely distributed in the tethys. this hypothesis needs to be critically evaluated both from additional collection from the eastern atlantic and eastern pacific, and by an examination of fossil material. a priori, it would seem unlikely that in a highly evolved group such as the turrinae, there might be relict species that are essentially unchanged since the tethys sea closed. perhaps the species evolved after the tethys sea closed, since there were migrations east and west of europe after the late eocene. each of these explanations makes specific experimental predictions. the dna sequence divergence in the two forms of g. sikatunai should be subject to analysis by molecular methods; the composition of their venoms, and whether or not there are striking similarities should provide additional data consistent with one of these alternatives. thus, a comparison of living material should be of exceptional interest. however, given that all caribbean specimens were dead-collected, the possibility remains that this population is now extinct. in this report, we have included in the gemmula speciosa group the recently described species gemmula lululimi. other workers have described the same form, but instead included it in the genus annulaturris, because the sinus cord is not at a peripheral location, a character generally used to diagnose members of turris and annulaturris (the latter sometimes regarded as a subgenus of the former). given the very striking similarities to gemmula sikatunai, as is documented above, we believe it unlikely that these striking similarities arise from convergence. furthermore, the species has no phenetic affinities with other species of the genus turris or annulaturris. we previously hypothesized that turris probably evolved from a lophiotoma-like ancestor (lophiotoma bisaya (olivera, 2004) was suggested to be one possibility). this transformation of the sinus cord from a peripheral position to one more posterior in each whorl was termed the g-t (gemmula-turris) transformation. the assignment of gemmula lululimi to the gemmula species group therefore has an underlying evolutionary implication: that this g-t transformation did not occur only once. we suggest that this morphological transformation has occurred at least twice, once to a gemmula kieneri-like descendant that had lost most of its gemmules (to yield turris babylonia and relations) and in a separate event, to a gemmula sikatunai-like form (to yield gemmula lululimi). the hypothetical conceptual framework is that the tethys sea contained ancestral forms of gemmula very similar to gemmula speciosa/sikatunai and gemmula kieneri. the latter species complex included forms in which the gemmules were lost (unedogemmulaand lophiotoma-like forms), one of which had a g-t transformation event that was ancestral to most of the modern species in the genus turris. in the case of gemmula lululimi however, the g-t transformation event took place in a g. sikatunai-like form that had not lost its gemmules when the g-t transformation occurred. although this hypothesis is a conjecture at this point, a more thorough characterization of g. lululimi should either be consistent with this explanation, or alternatively, should support a closer affinity with other species in turris/annulaturris. acknowledgments i would like to acknowledge the essential contribution of the late alex kerstitch to this work; his gift of unique dredged material from the barbados stimulated one of the major themes of this article. the input and advice of drs. donn tippett and gary rosenberg are greatly 12 olivera appreciated. i am grateful to philippe bouchet and philippe maestrati for the opportunity to examine the mnhn material from the philippines. collection data for the new species described was kindly provided by frank heralde and jose javier. this work was supported in part by the venom core of program project gm 48677 from the national institute of general medical science, and by a fund for distinguished professors from the university of utah. many of the specimens used in this study were from material used to collect venom for the program project. photographs were taken by kerry matz. i thank nancy kurtzeborn for her patience in going through all of the drafts of this article, and for her careful measurements of the type specimens; she was solely responsible for the preparation of the appendix. references adams, h. & a. adams, 1853-54. the genera of recent mollusca: arranged according to their organization. 1: 1-256, pls. 1-32, (1853), 257-484, pls. 33-60 (1854). john van voorst, london. casey, t.l., 1904. notes on the pleurotomidae with description of some new genera and species. transactions of the academy of science st. louis 14:123-170. doumet, e., 1840. pleurotoma in mollusques. magasin de zoologie. series ii year ii: pl. 10+2pp; pl 11+2pp. kilburn, r.n., 1983. turridae (mollusca: gastropoda) of southern africa and mozambique. part 1. subfamily turrinae. ann. natal mus. 25: 549-585. olivera, b.m., 1999. the subfamily turrinae in the philippines: the genus turris (röding, 1798). phil. j. sci. 128: 295-318. olivera, b.m., 2002. the gastropod genus xenuroturris (iredale, 1929) evaluated and a new turrid, lophiotoma olangoensis, described from the central philippines. science diliman. 14(2): 40-50. olivera, b.m., 2004. larger forms in lophiotoma defined: four new species described from the philippines and three from elsewhere in the indo-pacific. science diliman. 16(1): 1-28. ponder, w.f. & a. warén, 1988. classification of the caenogastropoda and heterostropha–a list of the familygroup and higher category names (ponder, w.f., ed.). malacological review supp. 4: 288-326. powell, a.w.b., 1964. the family turridae in the indo-pacific. part 1. the subfamily turrinae. indo-pacific mollusca. 1: 227-346. powell, a.w.b., 1966. the molluscan families speightiidae and turridae. bulletin of the auckland institute and museum. 5:1-184 + 23 plates. reeve, l.a., 1843-46. monograph of the genus pleurotoma. conchologia iconica. 1: 1-369. röding, p.f., 1798. museum boltenianum pars secunda continens conchylia. j.c. trapii, hamburg. springsteen, f.j. & f.m. leobrera, 1986. shells of the philippines. carfel shell museum, manila, philippines. vera-peláez, j.l., r. vega-luz, & m.c. lozano-francisco, 2000. five new species of the genus turris röding, 1798 (gastropoda, turridae, turrinae) of the philippines and one new species of the southern south pacific. malakos: monografía. 2: 1-29. 13 taxonomy of gemmula gemmula sikatunai, new species holotype 41.9 13.4 22.0 bohol island, philippines paratype #1 45.5 15.4 25.2 bohol island, philippines paratype #2 52.0 17.2 26.6 bohol island, philippines paratype #3 50.9 15.2 27.2 panglao island, philippines paratype #4 41.4 13.8 21.9 bohol island, philippines paratype #5 37.1 12.7 20.2 bohol island, philippines paratype #6 46.3 14.9 24.9 bohol island, philippines paratype #7 59.9 16.1 25.5 manila bay, philippines paratype #8 39.2 13.2 20.9 tayabas bay, philippines paratype #9 38.6 12.8 21.5 lubang island, philippines (mnhn)1 paratype #10 41.9 13.6 22.1 lubang island, philippines (mnhn)1 paratype #11 39.4 13.7 19.9 lubang island, philippines (mnhn)1 paratype #12 35.0 11.7 19.4 lubang island, philippines (mnhn)1 paratype #13 33.7 11.5 18.5 lubang island, philippines (mnhn)1 paratype #14 33.7 11.3 18.3 lubang island, philippines (mnhn)1 paratype #15 26.2 9.5 14.5 lubang island, philippines (mnhn)1 paratype #16 46.8 14.4 25.4 luzon island, philippines (lacm #75849)2 paratype #17 33.7 10.7 18.5 luzon island, philippines (lacm #75849)2 gemmula sikatunai, barbados variety specimen #1 41.0 13.4 22.5 barbados, west indies3 specimen #2 39.4 12.6 21.2 barbados, west indies3 specimen #3 45.8 14.0 23.9 barbados, west indies3 specimen #4 42.8 13.4 22.6 barbados, west indies3 specimen #5 32.0 10.3 17.3 barbados, west indies3 specimen #6 14.7 5.7 7.4 barbados, west indies3 (juvenile) gemmula sogodensis, new species holotype 44.5 15.4 23.5 sogod, cebu, philippines paratype #1 44.6 15.0 24.3 sogod, cebu, philippines paratype #2 40.7 14.4 22.5 sogod, cebu, philippines paratype #3 38.4 14.1 21.0 sogod, cebu, philippines paratype #4 44.0 14.0 24.2 sogod, cebu, philippines paratype #5 46.9 15.3 24.8 sogod, cebu, philippines paratype #6 34.6 12.8 20.0 sogod, cebu, philippines paratype #7 44.5 16.3 25.7 sogod, cebu, philippines paratype #8 43.2 14.2 22.6 sogod, cebu, philippines paratype #9 45.6 17.1 24.2 sogod, cebu, philippines paratype #10 40.6 16.0 21.0 sogod, cebu, philippines paratype #11 48.6 15.3 24.7 dipolog, philippines paratype #12 50.8 16.6 27.2 dipolog, philippines paratype #13 45.5 17.3 24.5 bohol, philippines paratype #14 45.0 15.2 24.2 bohol, philippines paratype #15 47.4 15.7 23.7 sogod, philippines paratype #16 46.9 17.6 25.0 sogod, philippines paratype #17 47.1 15.9 27.0 dipolog, philippines paratype #18 43.8 14.4 24.3 dipolog, philippines paratype #19 40.7 15.0 22.2 dipolog, philippines paratype #20 44.1 15.8 25.7 dipolog, philippines gemmula species. measurements and locality data for holotypes and paratypes. appendix (prepared by nancy kurtzeborn) species height widtha apertureb localitymeasurement (mm) 14 olivera paratype #21 41.1 13.3 23.2 dipolog, philippines paratype #22 39.1 12.5 20.5 dipolog, philippines paratype #23 43.2 14.5 23.6 bohol, philippines paratype #24 30.3 11.4 15.7 bohol, philippines (juvenile) paratype #25 46.2 16.0 23.6 sw taiwan (lacm 69006)4 paratype #26 45.4 15.0 24.1 sw taiwan (lacm 69006)4 paratype #27 57.0 17.4 29.7 kii, japan (lacm 47060)5 paratype #28 56.5 18.2 26.8 kii, japan (lacm 47060)5 paratype #29 53.8 17.8 28.5 kii, japan (lacm 47060)5 paratype #30 54.9 18.7 27.6 sagami bay, japan (amnh 16519)6 paratype #31 47.4 16.7 26.9 sogod, cebu, philippines paratype #32 51.1 17.5 29.3 sogod, cebu, philippines paratype #33 50.3 17.9 27.2 japan paratype #34 36.2 13.8 18.1 bohol, philippines paratype #35 48.0 15.9 26.4 philippines paratype #36 43.5 16.0 21.9 philippines paratype #37 48.4 16.0 25.8 philippines paratype #38 44.5 15.3 23.5 philippines paratype #39 46.9 15.5 25.4 philippines paratype #40 48.6 16.9 17.8 philippines paratype #41 47.2 16.7 25.5 philippines paratype #42 48.6 15.6 26.5 philippines paratype #43 44.6 15.5 25.7 philippines paratype #44 44.2 15.5 24.4 philippines paratype #45 41.5 14.7 21.6 philippines paratype #46 30.0 11.1 16.5 philippines7 awidth of shell at its widest point. blength from posterior of aperture to anterior tip of siphonal canal. 1lubang island, philippines; musorstrom 2-philippines st. cp66, 192-209 m 14°00n, 120°20e; mnhn-paris-malacologie. 2east of talaga, batangas bay, batangas province, sw luzon island, philippines; 52-67 fathoms on sand and mud bottom, 7/26/66; los angeles county museum of natural history. 3off barbados, west indies; dredged 450 feet, alex kerstitch, 6/97. 4tap’ing ting chaio, sw taiwan; trawled, 50-100 fathoms, leg franz steiner, 8/68, ex thelma crow; los angeles county museum of natural history. 5kii, japan, birch collection; los angeles county museum of natural history. 6sagami bay, japan, divers 1952; american museum of natural history. 7off lubang island, philippines; n.o. “coriolis” musorstrom 3, st. cp86 187-192 m, 14°00n, 120°18e, bouchet & triclot coll, 31mai85; mnhn-paris-malacologie. species height widtha apertureb localitymeasurement (mm) 82 health risk assessment: total mercury in canned tuna and in yellowfin and frigate tuna caught from leyte gulf and philippine sea arvin u. pacoma* senior high school department leyte national high school,tacloban leni g. yap-dejeto division of natural sciences and mathematics university of the philippines visayas abstract the total mercury (thg) concentrations in commercially available canned tuna and in yellowfin tuna (thunnus albacores) and frigate tuna (auxis thazard) caught from the waters of eastern visayas, philippines were determined by inductively coupled plasma optical emission spectrometry. the average total mercury concentrations measured from nine frigate tuna, three yellowfin tuna, and four canned tuna were 0.024 ug/g, 0.002 ug/g, and 0.07 ug/g, respectively. values of estimated daily intake for locally caught tuna for different age groups and sex were calculated. calculated daily dose for all locally caught tuna in the study were well below the allowed concentration of mercury in fish consumed per day regardless of age and sex, and thus may not pose a health risk to consumers. the same calculations were done for canned tuna with results further explained in the paper. keywords: canned tuna, mercury, auxisthazard, thunnus albacores * corresponding author science diliman (july-december 2019) 31:2, 82-88 a.u. pacoma and l.g. yap-dejeto 83 introduction over the last few decades, there has been increasing concern about the quality of the food we eat. heavy metals, such as cadmium, mercury, tin, and arsenic, are among the toxic substances that enter the marine environment, and eventually the human body (emami khansari et al. 2005). the most common form of mercury in fish, methylmercury (mehg), is a potent neurotoxin that is highly dangerous to fetal development (costa 1988). methylmercury enters the human body via the gastrointestinal (gi) tract. while the gi tract is the primary route of absorption, methylmercury can be absorbed through the skin and the lungs as well (rothstein and hayes 1960). once absorbed, it goes into the various parts of our body by binding with hemoglobin (kerper et al. 1992). it eventually enters the brain, where it is demethylated to form the inorganic elemental mercury. considering the pervasive consumption of tuna in the philippines, this study was conducted to determine the levels of total mercury in locally caught tuna in eastern visayas and in commercially canned tuna. materials and methods samples of tuna were fished by hook-and-line from three stations in leyte gulf (stations 1-3, figure 1) and three stations in the philippine sea (stations 4-6, figure 1). station 1 was located between the towns of palo, tanauan, and tolosa (n 11°05’02.3” e 125°08’01.3”). station 2 was located past the municipal waters of dulag, leyte (n 10°56’42.6” e 125°08’42.2”), while station 3 was around the vicinity of macarthur, leyte (n 10°47’59.2” e 125°06’30.7”). station 4 was located 30 km from the shoreline of guiuan, eastern samar (n 10°59’47” e 126°0’19”). station 5 was located in hernani, eastern samar (n 11°21’21” e 126°5’51”). lastly, station 6 was located in borongan city, eastern samar (n 11°37’33” e 125°43’39.5”). only a few tuna individuals were caught due to weather disturbances and financial constraints. tuna samples were caught between 25 december 2017 and 22 february 2018. two brands of canned tuna that are commercially and widely available in tacloban city were selected. two cans were tested for hg per brand. a modified set of criteria by maqbool et al. (2016) was used to select samples for analysis. health risk assessment: total mercury in canned tuna and in yellowfin 84 figure 1. locations of the six fishing sites in eastern visayas samples were prepared following the aoac standard method 2013.06 (aoac 2016). these were then digested in closed vessels of 50 ml with 65% nitric acid and analyzed using an inductively coupled plasma optical emission spectrometer. we then compared the concentration measured in the tuna samples to the maximum allowable limits of mercury (thg) in fish. estimates of the daily intake of the tuna samples that is safe for human consumption for filipinos were also calculated. results and discussion in this study, all canned and fresh samples of tuna were found to contain traces of mercury. frigate and yellowfin tuna samples were shown to have an average concentration of 0.024 and 0.002 ug/g mercury, respectively (table 1). on the other hand, canned tuna samples of both brands had an average concentration of 0.07 ug/g. it was also noted that the mercury concentration in locally caught tuna was lower than the concentration in canned tuna. a.u. pacoma and l.g. yap-dejeto 85 table 1. total hg concentrations in locally caught tuna (in wet weight) from leyte gulf and the philippine sea species and area no. of wet weight fork length mercury (ug/g) specimens (kg) (m) range mean yellowfin tuna (thunnus albacores): philippine sea: guiuan, eastern samar 1 3 1 0.003 0.003 philippine sea: borongan, eastern samar 2 1.25-2 0.5-0.75 0.002-0.01 0.006 frigate tuna (auxis thazard): leyte gulf: palo leyte 2 0.062-0.067 0.021-0.03 0.031-0.034 0.0325 leyte gulf: dulag, leyte 2 0.070-0.080 0.027-0.032 0.026-0.031 0.0285 leyte gulf: tanauan, leyte 2 0.068-0.073 0.028-0.033 0.029-0.031 0.03 philippine sea: hernani, eastern samar 2 0.078-0.081 0.029-0.04 0.026-0.027 0.0265 philippine sea: guiuan, eastern samar 1 1 1 0.01 0.01 the allowed concentration of mercury in fish is calculated from the daily reference dose (rf d ) and the daily consumption of fish.the rf d for mercury is the daily dose that is considered safe or the dose that does not entail an appreciable risk of adverse effects of mercury (iris 2001). the usepa calculated an rfd of 0.1 ug/kg bodyweight per day for mercury based on the risk to an adult woman, the population sector most vulnerable to the adverse effects of mercury (iris 2001). the per capita fish consumption of the filipino adult as reported by fao (2016) was 81.09 g per day in 2016. however, the published average weight is 61.3 kg for adult males, 54.3 kg for adult females (fnri 2013), 29.1 kg for male children and 28.0 kg for female children (florentino et al. 1987). the health risk assessment was done on adult females and children as they are more at risk from mercury intoxication. health risk assessment: total mercury in canned tuna and in yellowfin 86 based on the 2016 data for fish consumption and average weights,a daily dose of 0.0388 to 0.0015 ug/kg bodyweight per day for adult women, 0.0725 to 0.0028 ug/kg bodyweight per day for male children, and 0.0753 to 0.0029 ug/ kg bodyweight per day for female children was estimated for locally caught tuna with the maximum mercury contamination of 0.024 to 0.002 ug/g. whereas for canned tuna, with the maximum mercury contamination of 0.07 ug/g, the estimated daily dose for adult women is at 0.1 ug/kg bodyweight per day, and 0.20 ug/kg bodyweight per day for male and female children. the calculated daily dose or the estimated daily exposure due to the consumption of locally caught tuna is less than the (reference dose) rf d of 0.1 ug/kg bodyweight per day as set by the who and, therefore, does not entail any risk. the risk assessment for canned tuna indicates otherwise. both the organic and inorganics states of hg are considered threats to public health due to high toxicity when absorbed by organisms. the risk of toxicity is determined by the concentration of hg in the edible tissues and the amount of tuna consumed (araújo & cedeño-macias 2016). it greatly affects vulnerable segments of the population, such as pregnant women and children. chronic exposure to hg by ingestion of contaminated food—in this case, the canned tuna—can cause neurological and psychological problems commonly associated with minamata disease. other effects include nephrotoxicity, pulmonary and gastrointestinal damage, genetic damage, cardiovascular diseases, diabetes, abnormalities in fetal development, and cancer (araújo & cedeño-macias 2016). with the calculated high edi for adult women and male and female children, it is suggested that they consume less than total daily fish consumption of 81.09 g per day (fao 2016). in brief, adult filipino men and women may consume more than the daily fish consumption of 81.09 grams without the risk of mercury poisoning for locally caught tuna. whereas, for the canned tuna, it has exceeded the reference dose set by the who, except in the adult male group, and thus pose a significant neurological threat to vulnerable sectors of society, such as pregnant women and children. the presence of elevated mercury in food increases probable health risks and may have profound impacts on the body. consuming food high in mercury as described does not mean that life is in immediate peril.transitory excursion beyond the estimated daily intake limit would have no health risk if the mean consumption over an extended period is not surpassed, as the emphasis of edi is lifetime exposure. nevertheless, since only the total mercury content was analyzed for tuna, both locally caught and canned, supplementary research is essential to evaluate the specific methylmercury level and to establish the health threat of this seafood. a.u. pacoma and l.g. yap-dejeto 87 aknowledgements this study is funded through the small budget in – house research grant (sbirg) of the up visayas office of the vice chancellor for research and extension (ovcre). the authors wish to acknowledge their friends and family for happiness in times of distress, for food in times of hunger, for sharing the pain of sleepless nights in the laboratories, and for their unconditional love and untiring support in every aspect of this research.the authors also thank mr. eugene abria for his help in analyzing the results; mr. zosimo yakit and his son for accompanying the researchers during the sampling of locally caught tuna in the philippine sea sites; and the family of mrs. erlinda martija for the sampling of locally caught tuna in the leyte gulf sites. references araújo cvm, cedeño-macias la. 2016. heavy metals in yellowfin tuna (thunnus albacares) and common dolphinfish (coryphaena hippurus) landed on the ecuadorian coast. sci total environ. 541:149-154. costa lg. 1988.interactions of neurotoxicants with neurotransmitter systems. toxicology. 49:359-366. emamikhansari f, ghazi-khansari m, abdollahi m. 2005. heavy metals content of canned tuna fish. food chem. 93:293-296. [fao] food and agriculture organization (italy). 2016. regional statistical analysis of responses by fao members to the 2015 questionnaire on the code of conduct for responsible fisheries implementation. rome: food and agriculture organization. florentino rf, flores eg, magbitang ja, mendoza om, mendoza ts. 1987. proposed weight and height standards for 0-19 year old filipino children. trans nat acad sci tech (phils). 9:495-521. [fnri] food and nutrition research institute (philippines). 2013. 2nd national nutrition summit: 8th national nutrition survey. manila: fnri-department of science and techology. integrated risk information system (internet). 2001. methylmercury (mehg); casrn 2296792-6. us: epa [internet]. epa; [cited 2018 may 16]. available from: https:// cfpub.epa.gov/ ncea/iris/iris_documents/documents/subst/0073_summary.pdf kerper le, ballatori n, clarkson tw. 1992. methylmercury transport across the blood-brain barrier by an amino acid carrier. am j physiol regul integr comp physiol. 262:r761-r765. health risk assessment: total mercury in canned tuna and in yellowfin 88 maqbool d, george mp, george sp, philip a, ngis-iban pl, saley rm, honrado vgls, kenduiwa s, naidoo sr. 2016. analysis of mercury content in canned tuna fish commercially available in the philippines. world sci res. 3(1):57-61. rothstein a, hayes ad. 1960. the metabolism of mercury in the rat studied by isotope technique. j pharmacol exp ther. 130:166-176. ______ arvin u. pacoma <arvin.pacoma@deped.gov.ph> received a bachelor of science in biology from the university of the philippines visayas-tacloban college and is currently teaching general biology and research subjects in leyte national high school senior high school in tacloban city. his current research interest is in the field of bioaccumulation of heavy metals in marine organisms. he has joined symposiums and conferences organized by the national research council of the philippines and the department of health-eastern visayas regional health research and development colloquium, where he was honored with the best oral presenter and best poster presentation awards, respectively. leni g. yap-dejeto <lgyapdejeto@up.edu.ph> received a doctorate degree in aquatic biology from the university of tokyo and is currently teaching in the university of the philippines visayas-tacloban college as an associate professor. her current research interests are in the ecology of tropical microalgae, macroalgae, harmful algal blooms, and pernaviridis. she took her postgraduate studies in the university of california, santa cruz, california, usa. 38 chalcone derivatives with cyclooxygenase inhibiting activity gian carlo v. tambago evangeline c. amor* terrestrial natural products laboratory institute of chemistry, college of science university of the philippines diliman abstract dimethylcardamonin (dmc) or (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone was isolated from syzygium samarangense (blume) merr. leaves using vacuum liquid chromatography and normal phase silica-gel column chromatography. dmc was purified from the fraction that eluted out of 9:1 (v/v) hexane: ethyl acetate to 7:3 (v/v) hexane: ethyl acetate. using a modified method from geissman (1948), dmc was derivatized via alkaline peroxidation from which compounds a and b were obtained. compound a was identified to be (2s)-7hydroxy-5-methoxy-6,8-dimethyl flavanone (c 18 h 18 o 4 ) while b had a methoxy group on its 4’ position instead of a hydroxyl group with respect to dmc. the flavanone derivative may have been formed due to substituent effects on the ring. dmc, a, and b were tested for their inhibitory activity against cyclooxygenase enzymes at 10 and 100 ppm. a and b gave at least 50% inhibitory activity at 100 ppm and were found to be cox-2 selective inhibitors. dmc was inactive at both 10 and 100 ppm. keywords: dimethylchalcone, flavanone, syzygium samarangense, alkaline peroxidation, cyclooxygenase inhibition * corresponding author science diliman (january-june 2022) 34:1, 38-52 sd june2022_tambago.indd 38 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 39 introduction the earliest remedy for inflammation is the extract of willow bark (salix sp.), which dates back to 1862 (cavaillon 2017; desborough and keeling 2017). willow bark extract is found to contain salicylic acid (2-hydroxybenzoic acid), which was derivatized in 1897 by acetylation to produce aspirin (2-acetyloxybenzoic acid) (jeffreys 2004; desborough and keeling 2017). aspirin was commercialized in 1905 and since then has been used to relieve pain, inflammation, and fever (desborough and keeling 2017). in 1963, a new drug called indomethacin was developed for people experiencing pain and inflammation due to rheumatoid arthritis (shen 1982). aspirin and indomethacin are examples of non-steroidal anti-inflammatory drugs (nsaids). however, these drugs are gastrotoxic and cause ulceration (marnett and kalgutkar 1998; marnett 2002; pasa et al. 2009). this side effect was related to the ability of nsaids to inhibit both cyclooxygenase-1 (cox-1) and cyclooxygenase-2 (cox-2)—enzymes mediating inflammation. thus, a new class of anti-inflammatory drugs called coxibs was developed. these drugs selectively inhibit cox-2 which means that they do not possess the gastric toxicity of nsaids (patrono and rocca 2009). however, it was later found that coxibs are hepatoand cardiotoxic (patrono and rocca 2009; badri et al. 2016). many coxibs including celecoxib, rofecoxib, and valdecoxib were banned from use (sun et al. 2007; hawboldt 2008; thomas et al. 2017). this led to a search for a new class of cox-2 selective inhibitors. among the interesting derivatized products from chalcones are the aurones and flavones, which are products of the oxidative cyclization of 2’-hydroxychalcones (masesane 2015). biologically, aurones are synthesized from chalcones with the aid of aureusidin synthase (nakayama et al. 2000; nakayama 2002; davies et al. 2006). however, not all plants have this enzyme to synthesize this subclass of phytochemicals. some researchers were able to synthesize aurones from chalcones in vitro and synthetic routes have been explored to hasten the study of aurones. one of the developed methods is the oxidative cyclization of 2’-hydroxychalcone using the acetate salt of metals at high oxidation states (e.g., mercury (ii) acetate) in an appropriate organic solvent such as dimethylsulfoxide (dmso) (detsi et al. 2002; agrawal and soni 2006), pyridine (agrawal and soni 2006), or acetic acid (sekizaki 1988). modifications to this process had been done, such as the use of copper (ii) bromide in dmso (detsi et al. 2002) and the use of gold (i) catalyst (harkat et al. 2008). another possible method is through the alkaline peroxidation of 2’-methoxy chalcones (geissman 1948). sd june2022_tambago.indd 39 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 40 the three chalcones (figure 1) isolated from s. samarangense (blume) merr. are known to inhibit the lipopolysaccharide (lps)-induced cellular production of nitric oxide (no) and prostaglandin e2 (pge2) (kim et al. 2010). however, it has not been confirmed whether dmc, the most abundant chalcone isolated from s. samarangense by amor et al. (2007), could also inhibit the production of prostaglandins through the direct inhibition of cyclooxygenases. this study re-isolated dmc from s. samarangense and derivatized it to determine the cyclooxygenase-inhibiting activity of the derivatives. figure 1. structure of cardamonin (r 1 , r 2 = h), stercurensin (r 1 = ch 3 , r 2 = h), and dimethylcardamonin (r 1 , r 2 = ch 3 ). materials and methods plant collection, extraction, and solvent partitioning s. samarangense leaves were collected from diliman, quezon city. the samples were authenticated, and a voucher specimen was deposited at the jose vera herbarium of the university of the philippines (up) diliman institute of biology with accession no. 14258. ground s. samarangense leaves (541 g) were soaked in single distilled technical grade (tg) methanol (~1.5 l) for 3-5 days done six times. after extraction, the resulting filtrate was evaporated in vacuo with a rotary evaporator (ika rv-10) at 40°c to obtain the crude methanol extract (46 g). the concentrated methanol extract (15 g) was suspended in distilled water (100 ml) and was extracted successively with tg hexane (600 ml) up to six times to obtain the hexane extract. sd june2022_tambago.indd 40 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 41 fractionation, isolation, and purification all solvents (methanol, hexane, and ethyl acetate) used for fractionation, isolation, and purification are of analytical grade except for those used for vacuum liquid chromatography (vlc) where tg solvents were used. the hexane extract (4.1 g) was fractionated using vacuum liquid chromatography (silica gel 60 g, thin-layer chromatography grade, merck) employing gradient elution from 100% hexane to 100% ethyl acetate at 10% (v/v) increments, and then 100% ethyl acetate to 100% methanol at 50% (v/v) increments. a total of twelve vlc fractions were collected, concentrated, and dried in vacuo at 40°c using a rotary evaporator. separation was monitored by thin layer chromatography (tlc) using silica gel 60 gf 254 (tlc grade, merck 105554). tlc plates (4.5 in x 4.5 in) were viewed under an ultraviolet (uv) lamp set at 254 nm (far uv) and 365 nm (near uv) and exposed to iodine crystals for visualization. yellow-orange crystals formed from the vlc fraction that eluted from 9:1 (v/v) hexane: ethyl acetate. this fraction was dissolved in 9:1 (v/v) hexane: ethyl acetate and was subjected to normal phase silica-gel column chromatography (npcc) using a wet-packed stationary phase (silica gel 60, column chromatography grade, merck 1.07734.1000). gradient elution was performed from 9:1 (v/v) hexane: ethyl acetate to 100% ethyl acetate at 10% (v/v) increments. subfractions were collected at 5-ml portions monitored with tlc. a pure compound was obtained from the subfraction which eluted at 7:3 (v/v) hexane: ethyl acetate. (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone (dmc): yellow-orange crystals; esi-ms positive mode c 18 h 18 o 4 [m+h]+ found m/z 299.1293 (calcd 299.1283, error 3.34 ppm). 1h-nmr (500 mhz, cdcl 3 , ppm) δ 2.15 (s, 3’-ch 3 ), 2.17 (s, 5’-ch 3 ), 3.67 (s, 6’-och 3 ), 5.42 (s, 4’-oh), 7.43 (m, h-3), 7.43 (m, h-4), 7.43 (m, h-5), 7.66 (dd, h-2), 7.66 (dd, h-6), 7.86 (d, β-h), 8.01 (d, α-h), 13.62 (s, 2’-oh); 13c-nmr (126 mhz, cdcl 3 , ppm) [dept] δ 7.57 (c-1) [c], 8.24 (3’-ch 3 ) [ch 3 ], 62.38 (6’-och 3 ) [ch 3 ], 106.54 (c-1’) [c], 108.85 (c-3’) [c], 109.07 (c-5’) [c], 126.71 (c-α) [ch], 128.42 (c-3) [ch], 128.42 (c-5) [ch], 128.93 (c-2) [ch], 128.93 (c-6) [ch], 130.2 (c-4) [ch], 135.36 (c-1) [c], 142.9 (c-β) [ch], 158.86 (c-6’) [c], 159.18 (c-4’) [c], 162.04 (c-2’) [c], 193.38 (c=o) [c]. alkaline peroxidation of chalcone the alkaline peroxidation procedure was adopted from geissman and fukushima (1948) with slight modifications. a total of 50 mg of the chalcone was dissolved in 2.5 ml cold methanol. this solution was kept in an ice bath (~5°c). subsequently, 0.75 ml of 16% cold aqueous naoh (18 equiv.) and 0.5 ml of cold 20% h 2 o 2 sd june2022_tambago.indd 41 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 42 (25 equiv.) were added. the mixture was left to stand overnight at 5°c. if any precipitation was observed, 3n hcl was added to neutralize the mixture. the resulting solution was then extracted with 13 ml ethyl acetate. afterward, another set of peroxidation was done using 10 ml cold methanol, 3.0 ml of cold aqueous 16% naoh (72 equiv.), and 2.0 ml of cold 20% h 2 o 2 (102 equiv.), which was followed by extraction using 50 ml ethyl acetate. the resulting ethyl acetate extracts were then dried, and the products were purified using dropper column chromatography. the first peroxidation procedure yielded compound a (8.0% yield), while the second produced compound b (18% yield). compound a ((2s)-7-hydroxy-5-methoxy-6,8-dimethyl flavanone): yellow crystals. 1h-nmr (500 mhz, cdcl 3 , ppm) δ 2.15 (s, 6-ch 3 ), 2.15 (s, 8-ch 3 ), 2.84 (dd, ha-3), 2.98 (dd, hb-3), 3.82 (s, 5-och 3 ), 5.42 (dd, h-2), 5.43 (s, 7’-oh), 7.42 (m, h-2’), 7.42 (m, h-3’), 7.42 (m, h-4’), 7.42 (m, h-5’), 7.42 (m, h-6’). compound b ((e)-2’-hydroxy-4’,6’-dimethoxy-3’,5’-dimethyl chalcone): faint yellow crystals. 1h-nmr (500 mhz, cdcl 3 , ppm) δ 2.12 (s, 3’-ch 3 ), 2.14 (s, 5’-ch 3 ), 3.48 (s, 4’-och 3 ), 3.65 (s, 6’-och 3 ), 7.40 (d, h-3), 7.40 (d, h-4), 7.40 (d, h-5), 7.64 (dd, h-2), 7.64 (dd, h-6), 7.83 (d, β-h), 7.98 (d, α-h), 13.62 (s, 2’-oh). cyclooxygenase inhibition assay the reagents used in the cyclooxygenase inhibition assay include 100 mm tris buffer (ph 8.0), 1 mm hematin cofactor in 0.1 m naoh, ampliflutm red or adph (10-acetyl-3,7-dihydroxyphenoxazine): 2 mm in dmso, 40 mm arachidonic acid in ethanol, indomethacin (sigma) positive control, cyclooxygenase-1 from sheep (sigma) and recombinant cyclooxygenase-2 from human (sigma). dmc and its derivatives were screened for their cyclooxygenase inhibiting activity at 10 ppm and 100 ppm using the method developed by the terrestrial natural products laboratory at the institute of chemistry, up diliman (opog et al. 2019). a 10,000 ppm stock sample solution was prepared in a microcentrifuge tube by dissolving 3 mg of the sample in 300 μl of dmso. working stock solutions with concentrations of 200 ppm and 20 ppm were prepared in microcentrifuge tubes from the 10,000 ppm stock. the assay was done in a closed chamber flushed with nitrogen gas, using indomethacin (sigma) with an effective concentration of 1,500 ppm as the positive control. from the working stock, 10 μl of the test sample was added to a mixture of 50 μl of tris buffer and 120 μl of cox-hematin solution. after incubation for 15 minutes at 25°c, 10 μl amplex red reagent and 10 μl arachidonic acid were added. the fluorescence was then monitored every 12 seconds for sd june2022_tambago.indd 42 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 43 3 minutes at 535 nm and 590 nm as the wavelengths of excitation and emission, respectively using a clariostar® microplate reader. each assay was done in two trials with two replicates per trial. the percent inhibition was calculated using the equation: %inhibition = v negative – v sample v negative x 100% (1) where v negative = reaction velocity of the negative control, and v sample = reaction velocity of the sample a sample is considered active when the cox-2 inhibition is greater than 50% and the ratio of the cox-2 and cox-1 inhibition is greater than 1. the assay data collected were subjected to several statistical methods, including the one-sample kolmogorov-smirnov test, levene’s test, anova, welch’s t-test, brown-forsythe test, and the post-hoc test (dunnett t3 or tamhane’s t2). liquid chromatography-mass spectrometry (lc-ms) the samples were analyzed using masslynxtm software v4.1 by waters and mzmine v2.6. the parameters used for the analysis are summarized in table 1. table 1. lc-ms parameters and conditions for sample analysis parameter condition unit xevo g2-xs qtof, acquity uplc® system column acquity uplc® csh tm fluoro-phenyl, 1.7μm (2.1 x 50mm), t=30°c mobile phase gradient of 100% acn-95% h2o-100% acn flow rate 0.3 ml/min data processing masslynxtm v4.1 nuclear magnetic resonance (nmr) isolates with enough amount for elucidation were subjected to 1h-nmr and 13c-nmr spectroscopy using 500-mhz agilent nmr spectrometer at the analytical services laboratory of the institute of chemistry, up diliman. the solvent used was deuterated chloroform (cdcl 3 ). data were processed using mestrenovatm ver 6.1© mestrelab research s.l. sd june2022_tambago.indd 43 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 44 results and discussion isolation of chalcone approximately 200 mg of yellow-orange needle-like crystals were collected from the vlc fraction that eluted out from 9:1 (v/v) hexane: ethyl acetate and purified using npcc with 7:3 (v/v) hexane: ethyl acetate. it has an rf value of 0.65 in 7:3 (v/v) hexane: ethyl acetate solution and is visible under uv (254 nm and 365 nm). the compound was identified to be (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone (dmc) and its structure (figure 2) was elucidated using nmr and lc-hrms data. the relative configuration was determined to be (e)based on the coupling constant of the αand β-hydrogens (15.7 hz). figure 2. dmc or (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone elucidated using 1d-nmr, 2d-nmr, dept, and lc-hrms spectra. 13c-nmr shifts are indicated in red while 1h-nmr chemical shifts are indicated in blue. drawn using chemdraw. derivatization of chalcones dmc was subjected to alkaline peroxidation and a red-orange solution was formed. two sets of derivatizations were performed for the oxidation of dmc-producing compounds a and b (figure 3). sd june2022_tambago.indd 44 7/29/2022 4:11:41 pm g.c. v. tambago and e.c. amor 45 figure 3. reaction schemes for the alkaline peroxidation of dmc yielding compounds a and b. from the first peroxidation procedure, four milligrams of yellow needle-like crystals (a) with an rf value of 0.53 in 7:3 (v/v) hexane: ethyl acetate was collected after purification. this was identified to be (2s)-7-hydroxy-5-methoxy-6,8-dimethyl flavanone (figure 4) and is visible under uv (254 nm) with a fluorescence observed at 365 nm. figure 4. (2s)-7-hydroxy-5-methoxy-6,8-dimethyl flavanone structure elucidated using 1h-nmr and cosy spectra. 1h-nmr chemical shifts are indicated in blue. drawn using chemdraw. sd june2022_tambago.indd 45 7/29/2022 4:11:41 pm chalcone derivatives with cyclooxygenase inhibiting activity 46 peroxidation of dmc was monitored through 1h-nmr. the doublet of doublet signals at δ 2.84, 2.98, and 5.42 is indicative of a flavanone skeleton. this was confirmed to be 7-hydroxy-5-methoxy-6,8-dimethyl flavanone when chemical shifts obtained were compared with published literature values (marinho et al. 2008; memon et al. 2015) and has an (s)relative configuration based on the coupling constants (3.0 and 12.9 hz). the flavanone derivative of dmc was obtained by direct nucleophilic attack by the anionic oxygen atom on the β-carbon atom of the oxide intermediate (geissman and fukushima 1948). the substituents of the ring did not provide substantial inhibition to the resonance in the system that would have led to the formation of the aurone. another set of alkaline peroxidation of dmc was carried out using excess reagents. nine milligrams of compound b (figure 5) were collected. figure 5. (e)-2’-hydroxy-4’,6’-dimethoxy-3’,5’-dimethyl chalcone structure elucidated using 1h-nmr and cosy spectra. 1h-nmr chemical shifts are indicated in blue. drawn using chemdraw. the collected compound b has an rf value of 0.6 in 7:3 (v/v) hexane: ethyl acetate and is visible under uv (254 nm). the 1h-nmr spectrum of b shows two signals for a methyl group (-ch 3 ) at δ 2.13 and δ 2.16, two methoxy (-och 3 ) groups at δ 3.49 and δ 3.66, aromatic protons (ar-h) at δ 7.34 – 8.00 [δ 8.00 (d, j = 15.7 hz, 1h), δ 7.84 (d, j = 15.7 hz, 1h), δ 7.66 (dd, j = 8.4, 6.3 hz, 2h)], and a signal at δ 13.63 for the hydroxyl (-oh) proton. compound b has a methoxy group instead of a hydroxyl group at the 4’ position with respect to dmc. sd june2022_tambago.indd 46 7/29/2022 4:11:42 pm g.c. v. tambago and e.c. amor 47 validation of the cyclooxygenase inhibition assay the cyclooxygenase inhibition assay is based on the action of the inhibitor on the cyclooxygenase and its substrate, arachidonic acid. to confirm that the assay is working and the data collected from it are valid, the activity of the enzyme and substrate was monitored by determining if the michaelis-menten constant (k m ) and v max are constant and within the acceptable range. acceptable range means the parameters collected are either in line with theoretical data based on previous studies or within a constant range of values established historically in the laboratory. the responses of varied concentrations of arachidonic acid against a constant amount of enzyme were recorded and plotted in figure 6. figure 6. michaelis-menten plot of cox-1 and cox-2 enzymes with arachidonic acid substrate. the k m and v max values obtained for cox-1 were 279.4 μm and 12.6 fi/s, respectively, while 488.0 μm and 20.2 fi/s, respectively, were calculated for cox-2. these values are consistent with the historically established data range (cox-1: k m = 200 550 μm; cox-2: k m = 200 – 600 μm) for the assay parameters (opog et al. 2019). the assay results for the cyclooxygenase inhibiting activity of dmc and its derivatives are shown in figure 7. sd june2022_tambago.indd 47 7/29/2022 4:11:42 pm chalcone derivatives with cyclooxygenase inhibiting activity 48 figure 7. cox inhibition summary for dmc and its derivatives. compound a gave 55.57 ± 1.43% inhibitory activity against cox-2 at 100 ppm and 45.54 ± 8.19% against cox-1. compound b gave 86.70 ± 2.25% inhibitory activity against cox-2 at 100 ppm and 81.22 ± 2.84% against cox-1. compounds a and b were found to be cox-2 selective inhibitors with cox-2 to cox-1 inhibition ratios of 1.22 and 1.07, respectively. conclusion alkaline peroxidation of (e)-2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethyl chalcone from the leaf extracts of syzygium samarangense (blume) merr. yielded two products, 7-hydroxy-5-methoxy-6,8-dimethyl flavanone and 2’-hydroxy-4’,6’-dimethoxy-3’,5’dimethyl chalcone. this is the first report of the peroxidation of dmc producing these flavonoids. flavonoids are plant-derived natural products that have long been studied for their anti-inflammatory properties (read 1995; zhang et al. 2006). some flavanones have been reported to exhibit in silico cox-2 selective inhibiting activity such as eriodictyol (3’,4’,5,7-tetrahydroxyflavan-4-one), hesperetin (3’,5,7-trihydroxy-4’methoxyflavan-4-one), and naringenin (4’-5,7-trihydroxyflavan-4-one) (akinloye et al. 2019). on the other hand, synthetic chalcone derivatives have also been reported as cox-2 selective inhibitors in several studies (razmi et al. 2013; jantan et al. 2014). sd june2022_tambago.indd 48 7/29/2022 4:11:42 pm g.c. v. tambago and e.c. amor 49 dmc, a, and b were tested for their anti-inflammatory activity using an in vitro cyclooxygenase inhibition assay. both derivatives show selective inhibition of cyclooxygenase-2 at 100 ppm. this is the first report of the selective cyclooxygenase-2 inhibiting activity of 7-hydroxy-5-methoxy-6,8-dimethyl flavanone and 2’-hydroxy4’,6’-dimethoxy-3’,5’-dimethyl chalcone. these flavonoids could potentially be lead candidates in the development of new anti-inflammatory drugs. acknowledgements this research was made possible with the funding from the office of the vice chancellor for research and development of the university of the philippines diliman and the facilities at the institute of chemistry, up diliman. we also thank mr. jacob noel inguito of the terrestrial natural products laboratory for helping with the assay. references agrawal n, soni, p. 2006. a new process for the synthesis of aurones by using mercury (ii) acetate in pyridine and cupric bromide in dimetyl sulfoxide. indian j chem. 45b, 1301-1303. akinloye o, metibemu d, akinloye d, onigbinde s, olaosebikan i, florence o, damilola b, bolarinwa a, olubunmi o. 2019. flavanones from sorghum bicolor selectively inhibit cox-2: in-silico and in-vivo validation. egypt j med hum. 20(34). amor e, villaseñor i, antemano r, perveen z, concepcion 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83-89. patrono c, rocca b. 2009. nonsteroidal anti-inflammatory drugs: past, present and future. pharmacol res. 59(5):285-289. razmi a, zarghi a, arfaee s, naderi n, faizi m. 2013. evaluation of anti-nociceptive and anti-inflammatory activities of chalcone derivatives. iran j pharm sci. 12(suppl):153-159. read m. 1995. flavonoids: naturally occurring anti-inflammatory agents. am j pathol. 147(2):235-237. sekizaki h. 1988. synthesis of 2-benzylidene-3(2h)-benzofuran-3-ones (aurones) by oxidation of 2’-hydroxychalcones with mercury (ii) acetate. bull chem soc jpn. 61(4):1407-1409. shen t. 1982. the discovery of indomethacin and the proliferation of nsaids. semin arthritis rheum. 12(2):89-93. sun s, lee k, bertram c, godlstein j. 2007. withdrawal of cox-2 selective inhibitors rofecoxib and valdecoxib: impact on nsaid and gastroprotective drug prescribing and utilization. curr med res opin. 23(8):1859-1866. thomas d, ali z, zachariah s, sundararaj k, van cuyk m, cooper j. 2017. coxibs refocus attention on the cardiovascular risks of non-aspirin nsaids. am j cardiovasc drugs. 17(5):343-346. zhang x, hung t, phuong p, ngoc t, min bs, song ks, seong y, bae k. 2006. antiinflammatory activity of flavonoids from populus davidania. arch pharm res. 29(12):1102-1108. sd june2022_tambago.indd 51 7/29/2022 4:11:42 pm chalcone derivatives with cyclooxygenase inhibiting activity 52 ______ gian carlo v. tambago <gvtambago@up.edu.ph> finished his bachelor of science in chemistry at the university of the philippines diliman. he is currently an m.s. student at the institute of chemistry, university of the philippines-diliman, and a science research specialist at the terrestrial natural products laboratory from the same institute. he is working on the isolation and elucidation of anticancer compounds from philippine terrestrial plants. evangeline c. amor is a professor at the institute of chemistry, university of the philippines diliman. she received her ph.d. in chemistry from the same university. she heads the terrestrial natural products laboratory research group and specializes in natural products and organic chemistry with research focus on the isolation and structure elucidation of bioactive (analgesic, hypoglycemic, anticancer, anti-inflammatory) compounds from philippine plants, structure-activity relationship studies, mutagenicity, and clastogenicity studies on medicinal plant preparations, and enzyme inhibition studies. sd june2022_tambago.indd 52 7/29/2022 4:11:42 pm 01_device growth performance and initial heritability estimates 1science diliman (january-june 2007) 19:1, 1-6 growth performance and initial heritability estimates for growth traits in juvenile sea urchin tripneustes gratilla ma. josefa r. pante1*, talna lorena p. dela cruz1, joy joseph j. garvida1 1the marine science institute university of the philippines diliman, quezon city tel. (63-2) 922-3921 (63-2) 981-8500 local 2914 fax (63-2) 924-7678 email: dosette.pante@upmsi.ph date submitted: april 7, 2006; date accepted: august 10, 2006 abstract genetic improvement of performance traits of maricultured species is becoming an important concern. improvement of performance traits is important for two reasons: it enhances the growth and survival of the animals and it translates to economic gains to the fish farmer. in the sea urchin, tripneustes gratilla, growth performance of the different families and heritabilities for wet weight, test diameter and test height were estimated from 1,020 offspring from a mating of each of the 15 males with 1 or 2 females. measurements were done monthly starting at the grow-out stage or four months after hatching. there were significant family differences for the performance traits in sea urchin reared in tanks at the bml hatchery as revealed by anova. estimates of heritabilities based on the sire component of variance were low for wet weight (0.027), test diameter (0.033) and zero for test height. heritabilities estimated from the dam component of variance were low for wet weight (0.063), moderate for test diameter (0.286) and test height (0.227). the results indicate that test diameter and wet weight have lowly heritable traits, which means that mass or individual selection may not be the best method for improving the traits for sea urchin populations in bolinao. other methods such as family and combined family selection should be explored. key words: genetic improvement; heritability; sea urchin; tripneustes gratilla; growth trait; mariculture, selective breeding *corresponding author pante, dela cruz, garvida 2 introduction sea urchin fishery in the philippines has been a major yet underdeveloped source of income for coastal fishers. since the 1970s, the value of the sea urchins' roe or gonads as a delicacy has been realized in the country. sea urchins have been collected for local consumption as well as the export market. unfortunately, constant exploitation due to the increasing demand of both local and export markets brought about the collapse of the sea urchin population in bolinao, pangasinan (northwestern luzon), during the early 1990s. this in turn resulted in the loss of a major livelihood for some fishers, who were dependent on the said fishery. the collapse of this fishery brought about the development of sea urchin culture to enhance the recovery of natural populations (juinio-meñez et al. 1998). since its development, aquaculture of sea urchins has been continuously done in the outdoor hatchery of the bolinao marine laboratory (bml) of the u.p. marine science institute. the maricultured sea urchins are used to restock wild populations. however, gonads of these cultured sea urchins are not at par with international quality standards and cannot usually compete with that of other countries for the international market. clear, bright yellow or orange roe that are firm, 4-5 cm long and free of leaking fluids are the ones preferred fresh and brings out the highest prices (price and tom 1995). on the other hand, locally produced roe in bolinao are usually off-color, with leaking fluids, easily broken and are used for making secondary products such as roe paste only. nonetheless, traditional aquaculture practices may be further improved to be able to meet the increasing demand for high quality sea urchin gonads. application of genetic improvement in aquaculture has gained popularity in the recent years, and estimates of heritability and genetic correlations for many aquatic species have been reported. studies with atlantic salmon (jonasson 1993), coho salmon (withler and beacham 1994), pink salmon (smoker et al. 1994), rainbow trout (fishback et al. 2002), and common carp (vandeputte et al. 2004) revealed moderate to high heritabilities for length and weight. values of heritability for shell length and weight of mollusks such as european oyster ostrea edulis (toro 1990), mercenaria mercenaria (hilbish et al. 1993), and red abalone haliotis rufescens (jonasson et al. 1999) were also reported. however, there are very few published information about heritabilities and genetic correlations for growth traits of sea urchins. the study by liu et al. (2005) revealed point estimates for heritabilities based on the sire components of variance were moderate to high for body weight (0.21-0.49), test diameter (0.21-0.47), and test height (0.22-0.37), while genetic correlations were found to be significant for body weight with test diameter (0.30~0.65) and test height (0.22~0.37), and test diameter with test height. the results obtained by liu's group imply that it is possible to improve certain sea urchin populations through selective breeding. if the local sea urchins can be improved and enhanced genetically to produce the necessary quality for the export market, the value of sea urchin gonads will definitely increase. genetic principles can be applied to improve both the productivity and product quality, particularly the growth rate and gonad quality of sea urchins, which may help increase the profitability for the fishers and may help the industry produce high quality sea urchins. our study aims (1) to document existing cultured sea urchin strain in bolinao for evaluation of their culture performance, and (2) to estimate genetic parameters (heritability, genetic and phenotypic variances) for growth traits in sea urchins. materials and methods experimental material broodstock of the sea urchin tripneustes gratilla was composed of cultured and wild breeders collected on the reef flat of lucero, bolinao, pangasinan. culture of t. gratilla is continuously done at the bolinao marine laboratory using the reproduction method developed by juinio-meñez et al. (1998). this method was followed for our experiment. growth performance and initial heritability estimates 3 spawning and fertilization growth traits (wet weight, test diameter, test height) of each parent were recorded before spawning. wet weight (weight of sea urchin after letting individuals stand out of water for at least 5 minutes) was measured by using a digital weighing scale (o haus model hs 200), while test diameter and height was measured with a vernier caliper. sea urchins were spawned artificially by injecting 2 ml of 0.5 m kcl into the gonads through the peristomial cavity. eggs of each of two females were fertilized with the sperm of a single male (~1000:1 sperm to egg ratio). a total of 30 full-sib (sibling group that has a common mother and father) and 15 half-sib (sibling group that has only one common parent, either a mother or a father) families were generated from a mating of each of the 15 males with one or two females. for this study, a family refers to a full-sib group of closely related individuals that share common genes from a common mother and father. rearing each family was kept in a jar equipped with motorized stirrers rotating at 30-36 rpm. larvae were fed daily with isochrysis galbana during the first week and chaetoceros calcitrans two weeks after hatching (~40,000 cells/ml). larvae were induced to metamorphose into juveniles approximately 40 days post hatching by transferring each family from jars to plastic bins with benthic diatom. when juveniles reached 4 months post hatching, 60 individuals per family were randomly chosen and growth traits of each sea urchin were measured. out of the 30 families generated, only 17 families had the minimum number of individuals needed for the experiment proper. these 17 families were then transferred to hatchery tanks (each family kept in separate compartments; in triplicates, 20 individuals per replicate) and were then fed with the same amount of sargassum sp. per compartment (~750g sargassum per compartment). another monitoring of each sea urchin's test diameter, test height, and wet weight was done on the fifth month. statistical and genetic analysis the statistical model used in this experiment is: yij = µ + si + dij + eijk where yij is the test diameter, test height or wet weight of the kth progeny or offspring of the jth dam (female parent) mated to the ith sire (male parent), and eijk are uncontrolled environmental and genetic deviations attributable to the individuals. the generalized linear models (glm) procedure in the sas system was used to determine significant growth differences among families of sea urchins, while the variance component (varcomp) procedure of sas was used to determine between sire variance component (σσσσσ2s), dam variance component ( σσσσσ 2d), and environmental variance component (σσσσσ2e). sire heritability was estimated as h2s=4σσσσσ 2 s/(σσσσσ 2 s + σσσσσ 2 d + σσσσσ 2 e), while dam heritability was estimated as h2d=4σσσσσ 2 d/(σσσσσ 2 s + σσσσσ 2 d + σσσσσ 2 e) (becker 1984; bowman 1974; falconer 1989). standard errors of heritability were estimated as in becker (1984). results and discussion means, standard deviations and coefficient of variations of the growth traits for each family is presented in table 1. there were significant family differences for each trait as revealed by anova (figure 1). family number 8 consistently attained the highest measures for the three growth traits, while performance of succeeding families varied for each trait. estimates of heritabilities (table 2) based on the sire component of variance were zero for test height and low for wet weight and test diameter. heritabilities estimated from the dam component of variance were low for wet weight and moderate for test diameter and test height. as expected, heritabilities estimated from the dam component of variance were slightly larger than the heritabilities estimated from the sire component. this indicates that the dam genetic variance component is confounded with common environmental effects, maternal effects or non-additive genetic effects in the growth traits being measured. however, it is pante, dela cruz, garvida 4 possible that the high estimate from dam component can be attributed to maternal effects as xiaolin and co-workers (2004) noted that in the juvenile phase, development is affected by the quality of yolk reserves, where yolk reserves pertain to maternal effect. heritability is a ratio rather than an absolute figure, and changes in any of the sources of variation such as sire and dam genetic variance, and environmental variance, will change the heritability value. thus, heritabilities for the traits being considered are specific to particular populations, in this case, to the t. gratilla populations in bolinao. the estimates for some of the traits from this study were lower than those of liu et al. (2003). to estimate genetic parameters and to initiate genetic improvement for a particular population of species usually require a minimum of 50 full-sib families per generation, but due to financial constraint and die-offs, this study was able to produce only 17 families. this may also explain the high standard error values (table 2) for heritability estimates for the three growth traits. this is the first study in the philippines to estimate genetic parameters in the sea urchin, t. gratilla. the results indicate that test diameter and wet weight have lowly heritable traits, which means that mass or individual selection may not be the best method for improving those traits for the sea urchin populations in bolinao. individual selection means that individuals are retained as future broodstock based solely on their individual performance. other methods such as family or combined family selections (falconer 1989) should be explored. for these two approaches, selection is based wholly or in part on the average performance of entire families. the initial results from this study indicate that there is sufficient genetic variation for growth traits of the sea urchin populations studied in bolinao that could respond to genetic improvement. future work this study is still ongoing and the data presented in this paper were just preliminary results for the initial monitoring period. final monitoring of the growth traits would be done 8 (eight) months after hatching, when the sea urchins are in their harvest stage and might also be sexually mature. computations for heritability estimates would be performed on all the measured traits. correlation analysis would also be performed to determine the relationship of the growth traits with the gonadosomatic index (gsi). moreover, families would be ranked based on their growth trait performance. the parents for the next generation of sea urchin offspring would be selected from the top ranking families. family test diameter (mm) test height (mm) wet weight (g) n mean st.dev. coeff.var mean st.dev. coeff.var mean st.dev. coeff.var 1 60 44.6 3.6 8.0 25.3 2.2 8.6 41.1 8.6 20.8 2 60 44.4 2.6 5.9 24.6 1.7 6.9 39.8 5.6 14.0 3 60 45.7 3.2 6.9 25.5 2.5 9.7 43.1 7.0 16.2 4 60 47.2 4.4 9.2 25.2 2.6 10.3 47.5 12.7 26.7 5 60 47.2 3.8 8.1 24.6 1.7 6.9 45.1 8.2 18.2 6 60 45.2 3.4 7.4 25.2 3.3 13.1 41.5 8.3 20.1 7 60 44.9 3.4 7.5 24.9 2.4 9.7 42.4 8.7 20.6 8 60 49.2 4.4 9.0 27.7 2.8 9.9 51.4 12.2 23.7 9 60 46.1 4.3 9.3 25.5 1.7 6.5 44.3 7.3 16.5 10 60 47.7 3.4 7.1 26.1 2.1 8.1 47.6 9.6 20.3 11 60 48.5 5.9 12.1 25.6 3.5 13.9 47.9 14.7 30.8 12 60 46.4 4.6 10.0 26.8 2.5 9.2 45.3 10.9 24.1 13 60 46.8 3.1 6.5 26.0 3.4 13.1 44.8 8.0 17.9 14 60 46.9 4.0 8.6 25.4 2.3 8.9 44.4 10.3 23.1 15 60 47.3 3.1 6.6 25.4 2.2 8.8 44.3 7.6 17.1 16 60 45.4 5.1 11.3 25.8 3.1 11.9 42.2 11.3 26.9 17 60 47.2 4.3 9.1 24.6 2.7 10.8 47.4 11.7 24.7 table 1 summary statistics of growth traits of each family for the first monitoring period, n is the total number of individuals measured for each family growth performance and initial heritability estimates 5 figure 1. test diameter, mm (a), test height, mm (b) and wet weight, g (c) means of each sea urchin family for the first monitoring period. each family number represents one full-sib family. families are arranged from highest to lowest in terms of their performance in each growth trait. a) b) c) pante, dela cruz, garvida 6 acknowledgments the authors would like to thank the office of the vice chancellor for research and development (ovcrd) of the university of the philippines diliman for project funding, and the resource management ii of the sagip lingayen gulf project for their kind support. references becker, w.a., 1984. manual of quantitative genetics. academic enterprises, pullman: 190pp. bowman, j.c., 1974. an introduction to animal breeding. camelot press ltd. southhampton, great britain: 76pp. falconer, d.s., 1989. introduction to quantitative genetics 3rd ed. longman scientific & technical, essex: 438pp. fishback, g.a., r.g. danzmann, m.m. ferguson, & j.p. gibson, 2002. estimates of genetic parameters and genotype by environment interactions for growth traits of rainbow trout (oncorhynchus mykiss) as inferred using molecular pedigrees. aquaculture. 206:137-150. hilbish, t.j., e.p. winn, & p.d. rawson, 1993. genetic variation and covariation during larval and juvenile growth in mercenaria mercenaria. mar. biol. 115:97-104. jonasson, j., 1993. selection experiments in salmon ranching: i. genetic and environmental sources of variation in survival and growth in freshwater. aquaculture. 109:225236. jonasson, j., s.e. stefansson, a. gudnason, & a. steinarsson, 1999. genetic variation for survival and shell length of cultured red abalone (haliotis rufescens) in iceland. j. shellfish res. 18:621-625. juinio-meñez, m.a., n.n.d. macarawis, & h.g.p. bangi, 1998. community-based sea urchin (tripneustes gratilla) grow-out culture as a resource management tool. in proceedings of the north pacific symposium on invertebrate stock assessment and management. edited by g.s. jamieson and a. campbell. can. spec. publ. fish. aquat. sci. 125:393-399. liu, x.l., y.q. chang, j.h. xiang, & x.b. cao, 2005. estimates of genetic parameters for growth traits of the sea urchin strongylocentrotus intermedius. aquaculture. 243:27-32. price, r.j. and p.d. tom 1995. sea urchins. sea grant extension program publication. http://seaurchin.org/seaurchins.html smoker, w.w., a.j. gharrett, m.s. stekoll, & j.e. joyce, 1994. genetic analysis of size in an anadromous population of pink salmon. can. j. fish. aquat. sci. 51:9-15. toro, j.e., 1990. response to selection, heritability and genetic correlation for live weight and shell height in the european oyster ostrea edulis linne. rev. biol. mar. 25:135146. vandeputte, m., m. kocour, s. mauger, m. dupont-nivet, d. de guerry, m. rodina, d. gela, d. vallod, b. chevassus, & o. linhart, 2004. heritability estimates for growth-related traits using microsatellite parentage assignment in juvenile common carp (cyprinus carpio l.). aquaculture. 235:223236. withler, r.e. & t.d. beacham, 1994. genetic variation in body weight and flesh colour of the coho salmon (oncorhynchus kisutch) in british columbia. aquaculture. 119:135-148. xiaolin, l., c. yaqing, x. jianhai, d. jun, c. xuebin. 2004. study on heritability of growth in the juvenile sea urchin stronggylocentrotus nudus. j. shellfish res. 23 (2): 593597. growth trait h2s s.e. (h 2 s) h 2 d s.e. (h 2 d) test diameter 0.033 0.26 0.286 0.43 test height 0 0.13 0.227 0.23 wet weight 0.027 0.06 0.063 0.07 table 2 initial heritability estimates of growth traits for the first monitoring period 88 tracing the source of the non-native philippine population of the greenhouse frog eleutherodactylus planirostris (cope, 1862) through dna barcodes gerard clinton l. que* perry s. ong† ian kendrich c. fontanilla institute of biology college of science university of the philippines diliman emerson y. sy philippine center for terrestrial and aquatic research abstract eleutherodactylus planirostris (cope 1862), commonly called the greenhouse frog, is an insectivorous, direct-developing frog native to the caribbean. it has been widely introduced outside of its native range and has been known to reach population densities of about 12,500 frogs per hectare, posing a potential ecological threat in areas of its introduction, especially to local insect populations. recently, the species has been detected on several islands throughout the philippines. samples for this study were obtained from two locations in quezon city (luzon island) and one location in bacolod city (negros island). dna barcoding using three genes (cytochrome b, 16s rdna, and cytochrome oxidase subunit 1) was performed with the objective of identifying the source population of philippine e. planirostris. our results indicate that e. planirostris samples in the philippines are identical genetically to populations in hawai’i and florida, usa and are closely related to an individual from matanzas, cuba. a haplotype network built using the fitch algorithm also supports the cuban origin of the philippine samples. moreover, the philippine specimens have nearly identical sequences for all three genes, which may have implications on its success as an introduced species. keywords: dna barcoding, eleutherodactylus planirostris, cytb, 16s, cox1, cuba * corresponding author science diliman (january-june 2020) 32:1, 88-115 g.c.l. que et al. 89 introduction eleutherodactylus planirostris, commonly known as the greenhouse frog, is a new world species native to a few islands in the caribbean, specifically to cuba, the bahamas, and the cayman islands, a british overseas territory. the holotype described by cope (1862) was collected in the island of new providence in the bahamas. the status of the florida keys population is unclear; genetic dating indicates that it is possible the greenhouse frog colonized the islands naturally when the keys formed 125,000 years ago, but an artificial introduction from matanzas, cuba cannot be ruled out (heinicke et al. 2011). it has been introduced to numerous areas (figure 1), including mainland florida and louisiana (usa), g.c.l. que et al. 89 introduction eleutherodactylus planirostris, commonly known as the greenhouse frog, is a new world species native to a few islands in the caribbean, specifically to cuba, the bahamas, and the cayman islands, a british overseas territory. the holotype described by cope (1862) was collected in the island of new providence in the bahamas. the status of the florida keys population is unclear; genetic dating indicates that it is possible the greenhouse frog colonized the islands naturally when the keys formed 125,000 years ago, but an artificial introduction from matanzas, cuba cannot be ruled out (heinicke et al. 2011). it has been introduced to numerous areas (figure 1), including mainland florida and louisiana (usa), figure 1. distribution map of eleutherodactylus planirostris. a: native areas in the caribbean (green) and areas of introduction in the americas (blue). b: areas of introduction in the asia pacific. figure 1. distribution map of eleutherodactylus planirostris. a: native areas in the caribbean (green) and areas of introduction in the americas (blue). b: areas of introduction in the asia pacific. tracing the source of the non-native philippine population 90 nicaragua (heinicke et al. 2011), mexico (cedeno-vazquez et al. 2014), panama (crawford et al. 2011), jamaica (pough et al. 1977), hawai’i (usa) (krauss et al. 1999), and guam (christy et al. 2007). it belongs to the family eleutherodactylidae, a family of terrestrial frogs that exhibit direct development of eggs, which are laid in moist soil, that hatch into froglets and skip the aquatic tadpole stage (heinicke et al. 2007; hedges et al. 2008). adults are small, less than 30 mm snout-vent length (svl) (olson et al. 2012a). it inhabits grasslands, shrublands, and nonaquatic subterranean habitats. its highest altitudinal record is 727 meters above sea level (masl). its diet consists primarily of leaf-litter invertebrates, predominantly ants, mites, and springtails (olson and beard 2012; olson et al. 2012b). since the species may reach high population densities (approximately 12,500 individuals per hectare in hawai’i), they may constitute a potential threat to insect populations in their areas of introduction, particularly to ants (olson and beard, 2012; olson et al. 2012a). two color variations of this species are known: a mottled tan and brown pattern, and a tan and brown pattern with two dorsolateral yellowish brown stripes (lynn 1940). the striped pattern is apparently the dominant coloration in the frog’s native range, with a ratio of three striped to one mottled frog being found in cuba (goin 1947). however, in areas outside of its native range, the mottled pattern becomes more common, with the introduced specimens in guam all being of the mottled pattern and only 14% of the individuals caught in hawai’i displaying the striped pattern (goin 1947; olson and beard 2012; olson et al. 2012a). moreover, phylogenetic studies have found that two phylogenetically distinct groups exist within the species: one found in eastern cuba, the bahamas, and the cayman islands, and another in western cuba. the species is also not monophyletic: eleutherodactylus guanahacabibes, a species endemic to the guanahacabibes peninsula in cuba, has been found to be nested within e. planirostris lineages based on analysis of cytb barcodes (heinicke et al. 2011). recently, e. planirostris has also been detected in the philippines, notably in cebu (sy and salgo 2015), luzon (sy et al. 2015a), mindanao (olson et al. 2014), and negros (sy et al. 2015b), and is now present in eight islands in the archipelago (pili et al. 2019) (see figure 2 for pictures). the mode of introduction is probably through the ornamental plant trade (christy et al. 2007; olson et al. 2012a; pili et al. 2019). evidence for this comes from observations in hawai’i and guam, where the frogs are commonly first found around greenhouses and establishments that import plants, especially bromeliads (family bromeliaceae) and dracaena spp. g.c.l. que et al. 91 (family asparagaceae) (krauss et al. 1999; christy et al. 2007). eleutherodactylus planirostris has been documented laying eggs in flowerpots (goin 1944) and partially burying its eggs in moist earth (iturriaga and dugo-cota 2018), and both adults and juveniles inhabit areas with moist earth (goin 1947; iturriaga and dugo-cota 2018), so introduction through the plant trade is not surprising. records from the bureau of plant industry (bpi) of the department of agriculture of the philippine government indicate that ornamental plants were imported from the united states between 2005 and 2014 (e.g. tillandsia sp., family bromeliaceae, was imported in 2013), though no exact location in the us was given, and that palm seedlings were imported directly from hawai’i in 2006. this time period coincides with the species’ discovery in davao in 2013 (olson et al. 2014). in fact, many of the philippine records of the species have been found in ornamental plant market stalls or in landscaped gardens featuring ornamental plants (sy et al. 2015a; olson et al. 2014) (see figure 3). g.c.l. que et al. 91 nicaragua (heinicke et al. 2011), mexico (cedeno-vazquez et al. 2014), panama (crawford et al. 2011), jamaica (pough et al. 1977), hawai’i (usa) (krauss et al. 1999), and guam (christy et al. 2007). it belongs to the family eleutherodactylidae, a family of terrestrial frogs that exhibit direct development of eggs, which are laid in moist soil, that hatch into froglets and skip the aquatic tadpole stage (heinicke et al. 2007; hedges et al. 2008). adults are small, less than 30 mm snout-vent length (svl) (olson et al. 2012a). it inhabits grasslands, shrublands, and nonaquatic subterranean habitats. its highest altitudinal record is 727 meters above sea level (masl). its diet consists primarily of leaf-litter invertebrates, predominantly ants, mites, and springtails (olson and beard 2012; olson et al. 2012b). since the species may reach high population densities (approximately 12,500 individuals per hectare in hawai’i), they may constitute a potential threat to insect populations in their areas of introduction, particularly to ants (olson and beard, 2012; olson et al. 2012a). two color variations of this species are known: a mottled tan and brown pattern, and a tan and brown pattern with two dorsolateral yellowish brown stripes (lynn 1940). the striped pattern is apparently the dominant coloration in the frog’s native range, with a ratio of three striped to one mottled frog being found in cuba (goin 1947). however, in areas outside of its native range, the mottled pattern becomes more common, with the introduced specimens in guam all being of the mottled pattern and only 14% of the individuals caught in hawai’i displaying the striped pattern (goin 1947; olson and beard 2012; olson et al. 2012a). moreover, phylogenetic studies have found that two phylogenetically distinct groups exist within the species: one found in eastern cuba, the bahamas, and the cayman islands, and another in western cuba. the species is also not monophyletic: eleutherodactylus guanahacabibes, a species endemic to the guanahacabibes peninsula in cuba, has been found to be nested within e. planirostris lineages based on analysis of cytb barcodes (heinicke et al. 2011). recently, e. planirostris has also been detected in the philippines, notably in cebu (sy and salgo 2015), luzon (sy et al. 2015a), mindanao (olson et al. 2014), and negros (sy et al. 2015b), and is now present in eight islands in the archipelago (pili et al. 2019) (see figure 2 for pictures). the mode of introduction is probably through the ornamental plant trade (christy et al. 2007; olson et al. 2012a; pili et al. 2019). evidence for this comes from observations in hawai’i and guam, where the frogs are commonly first found around greenhouses and establishments that import plants, especially bromeliads (family bromeliaceae) and dracaena spp. (family asparagaceae) (krauss et al. 1999; christy et al. 2007). eleutherodactylus figure 2. photos of e. planirostris specimens found in the philippines (not the ones used in this study). a: from negros occidental, negros island. b: from barangay diliman, quezon city, luzon island. c: from barangay ugong norte, quezon city, luzon island. d: from barangay mariana, quezon city, luzon island. photos by emerson y. sy. planirostris has been documented laying eggs in flowerpots (goin 1944) and partially burying its eggs in moist earth (iturriaga and dugo-cota 2018), and both adults and juveniles inhabit areas with moist earth (goin 1947; iturriaga and dugo-cota 2018), so introduction through the plant trade is not surprising. records from the bureau of plant industry (bpi) of the department of agriculture of the philippine government indicate that ornamental plants were imported from the united states between 2005 and 2014 (e.g. tillandsia sp., family bromeliaceae, was imported in 2013), though no exact location in the us was given, and that palm seedlings were imported directly from hawai’i in 2006. this time period coincides with the species’ discovery in davao in 2013 (olson et al. 2014). in fact, many of the philippine records of the species have been found in ornamental plant market stalls or in landscaped gardens featuring ornamental plants (sy et al. 2015a; olson et al. 2014) (see figure 3). figure 2. photos of e. planirostris specimens found in the philippines (not the ones used in this study). a: from negros occidental, negros island. b: from barangay diliman, quezon city, luzon island. c: from barangay ugong norte, quezon city, luzon island. d: from barangay mariana, quezon city, luzon island. photos by emerson y. sy. tracing the source of the non-native philippine population 92 tracing the source of the non-native philippine population 92 globally, the spread of invasive alien species (ias) has led to loss of biodiversity, damaged agriculture, and the introduction of both animal and human diseases (early et al. 2016). impacts on small island biodiversity are especially severe, since populations on these islands are small relative to continental populations (russell et al. 2017; simberloff 2000). the iucn categorizes a species as alien if it has become introduced to an area not within its natural past and present distribution; it becomes invasive if it becomes problematic, such as by spreading disease or outcompeting native species, in its areas of introduction. following this definition and given the lack of documented impacts on philippine biodiversity, we do not yet advocate classifying the greenhouse frog as an invasive alien species. biological invasions of anurans have occurred before in the philippines, such as the cane toad, rhinella marina (jabon et al. 2019; pili et al. 2019). a study in the university of the philippines mindanao campus indicated that invasive frogs such as r. marina and kaloula pulchra were the dominant anuran species on the campus, outcompeting native frog species and preying on local wildlife, mostly insects but also vertebrates such as the skink (jabon et al. 2019). further studies on the ecology of the greenhouse frog, especially its diet, are needed in order to inform an assessment of whether it should be considered an ias. however, the potential negative impact of e. planirostris on local prey items, such as ants, is a cause for concern and studies are needed to document the local biology of e. planirostris, including its origin and mode of introduction. dna barcoding, a method of characterizing species based on short, unique segments of their genomes, has proven useful in identifying newly discovered species, whether native or invasive (blaxter 2003; blaxter 2004; crawford et al. 2011). in animals, the mitochondrial gene cytochrome oxidase subunit 1 (cox1) has become the recognized standard dna barcode due to its ability to distinguish between related species across different lineages as well as detect possible cryptic species (hebert et al. 2003; hebert et al. 2004; ward et figure 3. an individual e. planirostris found in a potted bromeliad. photo by emerson y. sy.figure 3. an individual e. planirostris found in a potted bromeliad. photo by emerson y. sy. globally, the spread of invasive alien species (ias) has led to loss of biodiversity, damaged agriculture, and the introduction of both animal and human diseases (early et al. 2016). impacts on small island biodiversity are especially severe, since populations on these islands are small relative to continental populations (russell et al. 2017; simberloff 2000). the iucn categorizes a species as alien if it has become introduced to an area not within its natural past and present distribution; it becomes invasive if it becomes problematic, such as by spreading disease or outcompeting native species, in its areas of introduction. following this definition and given the lack of documented impacts on philippine biodiversity, we do not yet advocate classifying the greenhouse frog as an invasive alien species. biological invasions of anurans have occurred before in the philippines, such as the cane toad, rhinella marina (jabon et al. 2019; pili et al. 2019). a study in the university of the philippines mindanao campus indicated that invasive frogs such as r. marina and kaloula pulchra were the dominant anuran species on the campus, outcompeting native frog species and preying on local wildlife, mostly insects but also vertebrates such as the skink (jabon et al. 2019). further studies on the ecology of the greenhouse frog, especially its diet, are needed in order to inform an assessment of whether it should be considered an ias. however, the potential negative impact of e. planirostris on local prey items, such as ants, is a cause for concern and studies are needed to document the local biology of e. planirostris, including its origin and mode of introduction. dna barcoding, a method of characterizing species based on short, unique segments of their genomes, has proven useful in identifying newly discovered species, whether native or invasive (blaxter 2003; blaxter 2004; crawford et al. 2011). in animals, the mitochondrial gene cytochrome oxidase subunit 1 (cox1) has become the recognized standard dna barcode due to its ability to distinguish between related species across different lineages as well as detect possible cryptic species (hebert et al. 2003; hebert et al. 2004; ward et g.c.l. que et al. 93 al. 2005). however, dna barcoding relies on existing taxonomic databases founded on traditional morphological techniques; it is a method to discriminate between genetic lineages and cannot be used to replace taxonomy (moritz and cicero 2004; hajibabei et al. 2017). locally, barcoding of the native fish fauna in taal lake detected two new invasive species of cichlids while confirming the presence of five other invasive species, 15 native species, and one endemic species (sardinella tawilis) (aquilino et al. 2011). moreover, it has also proven useful in tracing the origin of introduced species and in determining the presence of multiple introductions (kolbe et al. 2004; kolbe et al. 2008). it has already been utilized to trace the origins of invasive e. planirostris in hawai’i, panama, and mexico using mitochondrial genes (crawford et al. 2011; heinicke et al. 2011; cedeno-vasquez et al. 2014). crawford et al. (2011) used dna barcoding of the mitochondrial large ribosomal subunit 16s to identify some specimens of eleutherodactylus in panama as e. planirostris. they conjectured that the panamanian specimens came from florida due to sequence identity. heinicke et al. (2011) performed a similar study using the mitochondrial gene cytochrome b (cytb) for specimens in florida, usa. sequence identity indicates that florida specimens originate from matanzas, cuba. this study aimed to generate mitochondrial gene barcodes for philippine specimens of e. planirostris and use these genes to trace the source population of this novel introduced species and to address the need for more cox1 barcodes for amphibian taxa (smith et al. 2008; crawford et al. 2011). materials and methods acquisition of samples eight samples in total were collected: seven from quezon city, luzon island, and one from bacolod city, negros island (table 1). morphometric data, namely hind limb length (hll), tibia length (tl), snout-vent length (svl), snout length (sl), head length (hl), eye diameter (ed), and tympanic diameter (td), were measured for each specimen (table 2). all specimens were deposited in the natural history museum of the philippines, manila city. eleutherodactylus planirostris liver tissue samples were obtained from euthanized frogs and preserved in 99% ethyl alcohol by one of the authors (eys). no local or national sampling permits were needed since sampling was done under the auspices of the philippine national museum (pnm), which is permitted to sample biodiversity. for similar reasons, institutional animal care and use committee (iacuc) approval was not obtained due to the pnm’s mandate to sample philippine biodiversity. tracing the source of the non-native philippine population 94 tracing the source of the non-native philippine population 94 table 1. collection data for specimens used in this study field code collector locality collection date time method eys 387 john martyr barangay villamonte, bacolod city, negros occidental, negros island; 10.67666°n, 122.96150°e 4 sep 2014 08:00 – 17:00 h opportunistic sampling eys 395 emerson y. sy benjamin eleazar iii barangay mariana, quezon city, ncr, luzon island; 14.618889°n, 121.033056°e 7 aug 2014 08:00 – 17:00 h opportunistic sampling eys 396 emerson y. sy benjamin eleazar iii barangay mariana, quezon city, ncr, luzon island; 14.618889°n, 121.033056°e 7 aug 2014 08:00 – 17:00 h opportunistic sampling eys 397 emerson y. sy benjamin eleazar iii barangay mariana, quezon city, ncr, luzon island; 14.618889°n, 121.033056°e 7 aug 2014 08:00 – 17:00 h opportunistic sampling eys 398 emerson y. sy benjamin eleazar iii barangay mariana, quezon city, ncr, luzon island; 14.618889°n, 121.033056°e 7 aug 2014 08:00 – 17:00 h opportunistic sampling eys 399 emerson y. sy benjamin eleazar iii barangay mariana, quezon city, ncr, luzon island; 14.618889°n, 121.033056°e 7 aug 2014 08:00 – 17:00 h opportunistic sampling eys 400 emerson y. sy barangay up-diliman, quezon city, ncr, luzon island; 14.65205°n, 121.04552°e 2 oct 2014 08:00 – 17:00 h opportunistic sampling eys 401 emerson y. sy barangay up-diliman, quezon city, ncr, luzon island; 14.65205°n, 121.04552°e 2 oct 2014 08:00 – 17:00 h opportunistic sampling table 2. morphometric measurement of e. planirostris specimens specimen hll* (mm) tl (mm) svl (mm) sl (mm) hl (mm) ed (mm) td (mm) eys 387 27.15 11.35 22.75 3.75 8.75 3.05 1.95 eys 395 35.25 11.00 20.50 4.30 8.75 3.10 1.35 eys 396 36.85 11.55 23.85 4.30 9.65 3.55 1.80 eys 397 34.30 10.60 20.40 4.00 8.55 2.70 1.85 eys 398 27.85 8.40 17.00 3.55 7.45 2.60 1.30 eys 399 29.25 9.00 17.40 3.65 7.85 2.50 1.70 eys 400 32.75 10.10 19.05 3.80 7.80 2.35 1.65 eys 401 26.45 7.80 15.00 2.70 6.30 2.35 1.25 *hll=hind limb length, tl= tibia length, svl= snout-vent length, sl= snout length, hl= head length, ed= eye diameter, td= tympanic diameter. table 2. morphometric measurement of e. planirostris specimens g.c.l. que et al. 95 dna extraction and processing dna extraction was done via a modified form of the naoh lysis or alkaline lysis method (bimboim and doly 1979; as modified in fontanilla et al. 2014), which relies upon manual and chemical disruption of the cell membrane to release the contents of the cytoplasm and membrane-bound organelles, including the mitochondria, and the separation of dna and contaminants in iso-amyl alcohol and chloroform, respectively. dna concentration and purity of extracts were measured using thermoscientific™ nanodrop 2000c spectrophotometer. dna extracted from specimens of e. planirostris were then subjected to amplification via pcr. the primers for each gene are detailed in table 3. g.c.l. que et al. 95 dna extraction and processing dna extraction was done via a modified form of the naoh lysis or alkaline lysis method (bimboim and doly 1979; as modified in fontanilla et al. 2014), which relies upon manual and chemical disruption of the cell membrane to release the contents of the cytoplasm and membrane-bound organelles, including the mitochondria, and the separation of dna and contaminants in iso-amyl alcohol and chloroform, respectively. dna concentration and purity of extracts were measured using thermoscientific™ nanodrop 2000c spectrophotometer. dna extracted from specimens of e. planirostris were then subjected to amplification via pcr. the primers for each gene are detailed in table 3. table 3. primers used for polymerase chain reaction (pcr) in this study gene primer name and sequence (5’-3’) primer size (bp) reference cytochrome b (cytb) mvz15l-mod (sense): aactwatggcccmcacmatmcgwaa 1000 moritz et al. 1992; as modified by vences et al. 2003 mvz 16 (anti-sense): aaataggaawtatcawtctggtttwat moritz et al. 1992 16s 16s ar (sense): cgcctgtttatcaaaaacat 650 palumbi et al. 200216s br (anti-sense): ccggtctgaactcagatcacgt cytochrome c oxidase subunit 1 (cox1) lco 1490 (sense): ggtcaacaaatcataaagatattgg 655 folmer et al. 1994 hco 2198 (anti-sense): taaacttcagggtgaccaaaaaatca vf1 (sense): ttctcaaccaaccacaargayatygg 658 ivanova et al. 2007 vr1 (anti-sense): tagacttctgggtggccraaraayca for all genes, a 25 μl pcr mix was prepared using the following components: 11.375μl of ultrapure water (invitrogen™, thermo fisher scientific, usa), 5 μl of q solution (qiagen®, germany), 2.5 μl of 10x pcr buffer (vivantis, malaysia or for all genes, a 25 μl pcr mix was prepared using the following components: 11.375μl of ultrapure water (invitrogen™, thermo fisher scientific, usa), 5 μl of q solution (qiagen®, germany), 2.5 μl of 10x pcr buffer (vivantis, malaysia or tracing the source of the non-native philippine population 96 promega, usa), 1.25 μl each of the appropriate forward and reverse primer (10 μm), 1.0 μl of 25 mm mgcl 2 (roche, switzerland), 0.5 μl of 10 mm dntp (invitrogen™, thermo fisher scientific, usa), 0.125 μl of taq dna polymerase (5 u/μl, vivantis, usa or promega gotaq® flexi dna polymerase 5u/μl, promega, usa), and 2 μl of dna template. the conditions for the pcr run differed for each gene. for cytb (using mvz15l-mod and mvz 16), the initial denaturation was at 94 °c for 5 minutes, followed by 38 cycles at 94 °c for 30 seconds, 41 °c for 30 seconds, 72 °c for 60 seconds, and a final extension at 72 °c for 7 minutes (modified from heinicke et al. 2011). for 16s (using 16ar and 16sbr) and cox1 (using vf1 and vr1), a two-stage procedure was followed. the first stage required an initial denaturation at 94 °c for 5 minutes, followed by 5 cycles at 94 °c for 30 seconds, 45 °c for 30 seconds, and 72 °c for 45 seconds. the second stage involved 35 cycles at 94 °c for 30 seconds, 51 °c for 30 seconds, and 65 °c for 45 seconds, followed by a final extension step at 72 °c for 5 minutes (modified from ivanova et al. 2007). for cox1 (using hco and lco primers), the initial denaturation step was at 94 °c for 5 minutes, followed by 36 cycles at 94 °c for 30 seconds, 45 °c for 30 seconds and 65 °c for 1 minute, followed by a final extension step at 72 °c for 5 minutes (folmer et al. 1992). pcr was performed using either a multigene optimax™ thermocycler (labnet inc., usa) or a tgradient thermocycler (biometra®, germany), depending on machine availability. the pcr products were run through a 1% agarose gel stained with 1% ethidium bromide for 30 minutes and visualized under a uv transilluminator. kapa™ universal ladder was loaded alongside dna samples in order to measure band molecular weight. gel extraction followed the qiaquick® gel extraction kit by qiagen® protocol. to ensure the presence of dna, 2 μl of extract was run again through agarose gel electrophoresis. dna concentration of gel extracts was likewise measured using thermo scientifictm nanodrop 2000c spectrophotometer. sanger sequencing was performed by 1st base in singapore. the purified dna product was packaged together with 10 μl of the primers used during pcr. phylogenetic analysis and sequence comparison the sequences obtained from 1st base were checked and assembled using pregap4 and gap4 (bonfield et al. 1995) of the staden package (v. 2.0.0b10) (staden et al. g.c.l. que et al. 97 2000). blastn (nucleotide basic local alignment search tool) (altschul et al. 1990) was used to find the closest matches in genbank (www.ncbi.nlm.nih.gov/genbank/) (sayers et al. 2019). the sequences obtained for all three genes for each specimen were registered with the national center for biotechnology information (http:// www.ncbi.nlm.nih.gov/). genbank accession numbers for each gene from each specimen are given in table 4. table 4. genbank accession numbers for each gene sequenced of each specimen specimen field code genbank accession number cytb 16s cox1 eys 387 kt151767 kt151783 kt151775 eys 395 kt151770 kt151786 kt151777 eys 396 kt151771 kt151787 kt151778 eys 397 kt151772 kt151788 kt151779 eys 398 kt151773 kt151789 kt151780 eys 399 kt151774 kt151790 kt151782 eys 400 kt151768 kt151784 kt151781 eys 401 kt151769 kt151785 kt151776 greenhouse frog sequences in genbank were downloaded along with outgroup species and aligned alongside the query sequences using the clustalw function (thompson et al. 1994) in bioedit (v. 7.0.9) (hall 1999). to achieve uniform sequence length, longer sequences were trimmed to match the size of the shortest sequences. the outgroup chosen for 16s and cox1 was e. johnstonei since sequences for this species for both genes were available in genbank and it belongs to a separate subgenus (eleutherodactylus) than e. planirostris (euhyas) (hedges et al. 2008) and branches clearly from e. planirostris and its sister taxa in the analysis of pyron and weins (2011). for cytb, e. inoptatus was used as outgroup based on the study by heinicke et al. (2011) since it also belongs to another subgenus (pelorius) (hedges et al. 2008; heinicke et al. 2011). saturation testing was performed using the xia test (xia et al. 2003; xia and lemey 2009) in dambe (v. 6.4.81) (xia 2015; xia 2017). model testing was then performed for each gene using jmodel test 2 (v. 2.1.4) (darriba et al. 2012), and the corrected akaike information criterion score (akaike 1973; akaike 1974; hurvich and tsai 1993) to select the best fit model for phylogenetic analysis. tree construction was done using the neighbor joining (nj) tree construction method (saitou and nei 1987) with 1000 bootstrap pseudoreplicates calculated for branch supports (felsenstein 1985) using paup* (swofford 2003), following the optimum models determined by jmodel test. tracing the source of the non-native philippine population 98 separate trees were constructed for cytb, 16s and cox1. neighbor-joining (nj) trees were constructed separately for each gene using the kimura 3-parameter model gamma correction (tpm1μf+g) (kimura 1981) for the cytb alignment (666 bp), generalized time reversible model (tavare 1986) with gamma correction (gtr+g) for the 16s alignment (464bp, including gapped sites), and the hasegawa, kishino, and yano model (hasegawa et al. 1985) with gamma correction (hky+g) for the cox1 alignment (611 bp). for further analysis, a maximum likelihood (ml) tree was constructed for cytb sequences using iq-tree with 1000 ultrafast bootstraps (ufb) calculated as branch support (minh et al. 2013; nguyen et al. 2014). the haplotype network was created using fitchi (matschiner 2015), a python script based on the fitch algorithm (fitch 1970). statistical dispersal-vicariance analysis (s-diva) was performed using rasp v 4.2 (yu et al. 2015; yu et al. 2020). results and discussion blast search results nucleotide blast (blastn) search results showed that all the eight cytb sequences generated in this study matched with eleutherodactlyus planirostris sequence in genbank (accession no. hq831590) with 100% sequence identity. all eight cox1 sequences from this study had 100% sequence identity with e. planirostris in genbank with accession no. jf69001. the eight 16s sequences from this study matched with e. planirostris genbank accession nos. dq283107 and km252680 with sequence identity of 99.8% to 100%. according to heinicke et al. (2011), the largest mean intraspecific distance for e. planirostris using cytb is 7.3%, between eastern and western lineages of the species while interspecific distances calculated for other eleutherodactlyus species revealed an uncorrected pairwise distance of greater than 12% (rodriguez et al. 2013). given the low percentage difference of our sequences from e. planirostris cytb sequences in genbank, we conclude that our specimens are e. planirostris. our identification is supported by 16s and cox1 sequence data, which have a 99.8% to 100% sequence identity with e. planirostris sequences in genbank. phylogenetic trees both the nj and ml trees constructed from cytb sequences (figures 4 and 5) show that the philippine population of e. planirostris groups with specimens from hawai’i, florida (including the florida keys), and one specimen from carbonera, matanzas, cuba. g.c.l. que et al. 99 g.c.l. que et al. 99 figure 4. phylogenetic tree of e. planirostris and related species using 666 bp cytb barcodes and the tpm1uf+g model of molecular evolution. specimens whose labels start with eys were obtained for this study while the remaining sequences were obtained from genbank. the nj tree is rooted on e. inoptatus based on the analysis of heinick et al. (2011). branches with 50% or greater bootstrap support (nj/ml) have the corresponding number of bootstraps placed on the node. the country of origin of each sequence is placed beside the species name. blue represents eastern e. planirostris, red and purple represent the matanzas lineage and other lineages of western e. planirostris, respectively, brown corresponds to philippine specimens of e. planirostris and green represents e. guanahacabibes. (cu=cuba, ky= cayman islands, jm=jamaica, bs=bahamas, ht=haiti, ni=nicaragua, do=dominican republic, rp=philippines). the scale bar represents two nucleotide substitutions for every 10 nucleotides. figure 4. phylogenetic tree of e. planirostris and related species using 666 bp cytb barcodes and the tpm1uf+g model of molecular evolution. specimens whose labels start with eys were obtained for this study while the remaining sequences were obtained from genbank. the nj tree is rooted on e. inoptatus based on the analysis of heinick et al. (2011). branches with 50% or greater bootstrap support (nj/ml) have the corresponding number of bootstraps placed on the node. the country of origin of each sequence is placed beside the species name. blue represents eastern e. planirostris, red and purple represent the matanzas lineage and other lineages of western e. planirostris, respectively, brown corresponds to philippine specimens of e. planirostris and green represents e. guanahacabibes. (cu=cuba, ky= cayman islands, jm=jamaica, bs=bahamas, ht=haiti, ni=nicaragua, do=dominican republic, rp=philippines). the scale bar represents two nucleotide substitutions for every 10 nucleotides. tracing the source of the non-native philippine population 100 tracing the source of the non-native philippine population 100 figure 5. subtree of the cytb tree (figure 4) showing only e. planirostris sequences. blue represents eastern e. planirostris, red and purple represent the matanzas lineage and other lineages of western e. planirostris, respectively, brown corresponds to philippine specimens of e. planirostris, and green represents e. guanahacabibes. philippine specimens of e. planirostris group with the matanzas lineage of western e. planirostris and e. guanahacabibes groups with western e. planirostris. branches with 50% or greater bootstrap support (nj/ml) have the corresponding number of bootstraps placed on the node. (cu=cuba, ky= cayman islands, jm=jamaica, bs=bahamas, ht=haiti, ni=nicaragua, do=dominican republic, rp=philippines). the scale bar represents one nucleotide substitution for every 100 nucleotides. these sequences represent the matanzas lineage of western e. planirostris (heinicke et al. 2011), showing that the ultimate source of the philippine population is matanzas, cuba and that it shares a common origin with the hawai’i and florida populations. the immediate source of the philippine population is probably florida or hawai’i. this conclusion is supported by the 16s (figure 6) and cox1 (figure 7) nj trees, which show the philippine population grouping with those from florida and panama city. the same is true of the haplotype network (figure 8), which shows figure 5. subtree of the cytb tree (figure 4) showing only e. planirostris sequences. blue represents eastern e. planirostris, red and purple represent the matanzas lineage and other lineages of western e. planirostris, respectively, brown corresponds to philippine specimens of e. planirostris, and green represents e. guanahacabibes. philippine specimens of e. planirostris group with the matanzas lineage of western e. planirostris and e. guanahacabibes groups with western e. planirostris. branches with 50% or greater bootstrap support (nj/ml) have the corresponding number of bootstraps placed on the node. (cu=cuba, ky= cayman islands, jm=jamaica, bs=bahamas, ht=haiti, ni=nicaragua, do=dominican republic, rp=philippines). the scale bar represents one nucleotide substitution for every 100 nucleotides. these sequences represent the matanzas lineage of western e. planirostris (heinicke et al. 2011), showing that the ultimate source of the philippine population is matanzas, cuba and that it shares a common origin with the hawai’i and florida populations. the immediate source of the philippine population is probably florida or hawai’i. this conclusion is supported by the 16s (figure 6) and cox1 (figure 7) nj trees, which show the philippine population grouping with those from florida and panama city. the same is true of the haplotype network (figure 8), which shows g.c.l. que et al. 101 g.c.l. que et al. 101 figure 6. neighbor-joining tree of e. planirostris and related species using a 464 bp alignment (including gaps) of 16s barcodes and the gtr+g model of molecular evolution. specimens whose labels start with eys were obtained for this study while the remaining sequences were obtained from genbank. the tree is rooted on e. johnstonei based on the analysis of pyron and weins (2011). branches with 50% or greater bootstrap support have the corresponding number of bootstraps placed on the node. the country of origin of each sequence is placed beside the species name. blue represents eastern e. planirostris, red and purple represent the matanzas lineage and other lineages of western e. planirostris, respectively, brown corresponds to philippine specimens of e. planirostris, and green represents e. guanahacabibes. (cu=cuba, bs=bahamas, pa=panama, hk=hong kong, rp=philippines). the scale bar represents five nucleotide substitutions for every 100 nucleotides. figure 6. neighbor-joining tree of e. planirostris and related species using a 464 bp alignment (including gaps) of 16s barcodes and the gtr+g model of molecular evolution. specimens whose labels start with eys were obtained for this study while the remaining sequences were obtained from genbank. the tree is rooted on e. johnstonei based on the analysis of pyron and weins (2011). branches with 50% or greater bootstrap support have the corresponding number of bootstraps placed on the node. the country of origin of each sequence is placed beside the species name. blue represents eastern e. planirostris, red and purple represent the matanzas lineage and other lineages of western e. planirostris, respectively, brown corresponds to philippine specimens of e. planirostris, and green represents e. guanahacabibes. (cu=cuba, bs=bahamas, pa=panama, hk=hong kong, rp=philippines). the scale bar represents five nucleotide substitutions for every 100 nucleotides. tracing the source of the non-native philippine population 102 tracing the source of the non-native philippine population 102 figure 7. neighbor-joining tree of e. planirostris and related species using 611 bp cox1 barcodes and the hky+g model of molecular evolution. specimens whose labels start with eys were obtained for this study while the remaining sequences were obtained from genbank. the tree is rooted on e. johnstonei based on the analysis of pyron and weins (2011). branches with 50% or greater bootstrap support have the corresponding number of bootstraps placed on the node. the country of origin of each sequence is placed beside the species name. specimens in red represent western e. planirostris, while brown corresponds to philippine specimens of e. planirostris. (cu=cuba, pa=panama, br=brazil, rp=philippines). the scale bar represents one nucleotide substitution for every 10 nucleotides. figure 7. neighbor-joining tree of e. planirostris and related species using 611 bp cox1 barcodes and the hky+g model of molecular evolution. specimens whose labels start with eys were obtained for this study while the remaining sequences were obtained from genbank. the tree is rooted on e. johnstonei based on the analysis of pyron and weins (2011). branches with 50% or greater bootstrap support have the corresponding number of bootstraps placed on the node. the country of origin of each sequence is placed beside the species name. specimens in red represent western e. planirostris, while brown corresponds to philippine specimens of e. planirostris. (cu=cuba, pa=panama, br=brazil, rp=philippines). the scale bar represents one nucleotide substitution for every 10 nucleotides. g.c.l. que et al. 103 that the philippine population is more closely related to the matanzas lineage of e. planirostris while divergence-vicariance analysis shows that the ancestor to the philippine population is likely from the florida keys or western cuba (yellow doughnut; figure 9). the 16s nj tree (figure 5) also shows that samples from hong kong group with the philippine and florida lineages, indicating a shared origin for all locations, though the bootstrap support is rather low (60%). s-diva (figure 9) further shows that the common ancestor of the philippine population is from luzon. in light of the lack of samples from mindanao and other islands where the species has been detected (pili et al. 2019), this cannot be considered conclusive. the dispersal of the greenhouse frog in the archipelago will require more extensive sampling of the philippine population. g.c.l. que et al. 103 that the philippine population is more closely related to the matanzas lineage of e. planirostris while divergence-vicariance analysis shows that the ancestor to the philippine population is likely from the florida keys or western cuba (yellow doughnut; figure 9). the 16s nj tree (figure 5) also shows that samples from hong kong group with the philippine and florida lineages, indicating a shared origin for all locations, though the bootstrap support is rather low (60%). s-diva (figure 9) further shows that the common ancestor of the philippine population is from luzon. in light of the lack of samples from mindanao and other islands where the species has been detected (pili et al. 2019), this cannot be considered conclusive. the dispersal of the greenhouse frog in the archipelago will require more extensive sampling of the philippine population. figure 8. haplotype network of various lineages of e. planirostris and e. guanahacabibes constructed using the python script fitchi and following the fitch algorithm. figure 8. haplotype network of various lineages of e. planirostris and e. guanahacabibes constructed using the python script fitchi and following the fitch algorithm. tracing the source of the non-native philippine population 104 tracing the source of the non-native philippine population 104 figure 9. statistical dispersal vicariance analysis tree for e. planirostris. the common ancestor of the hawai’i, florida, and philippine populations come either from the florida keys or western cuba. figure 9. statistical dispersal vicariance analysis tree for e. planirostris. the common ancestor of the hawai’i, florida, and philippine populations come either from the florida keys or western cuba. g.c.l. que et al. 105 it is suspected that the species was introduced from florida to hawai’i and from hawai’i to guam through the ornamental plant trade (christy et al. 2007; olson et al. 2012a). the same mode of transport is hypothesized for the philippine population. records from the bureau of plant industry (bpi) show that ornamental plants, including bromeliads, were imported from the united states between 2005 and 2014. since the species was first detected in davao in october 2013 (olson et al. 2014), it is conceivable that the species was introduced during this time period. the philippine population ultimately originates from cuba and shows little variation in the sequences of three mitochondrial genes. only one haplotype was recovered for both 16s and cytb. for cox1, there were two haplotypes, but only one individual (eys 399) out of eight had a different haplotype. in contrast, 18 haplotypes were found for cytb in the species’ native range (figure 8) based on a genbank search. though the specimens studied here cannot be considered representative of the entire philippine population, it suggests that the local populations might be vulnerable to the founder effect (frankham 2005; parisod et al. 2005), which might reduce the adaptability of the species to local conditions and hamper its spread in new territory (lee 2002; frankham 2005), especially if these populations are isolated from each other and from further waves of introductions or if future introductions are similar genetically. morphological measurements provide some support for the genetic evidence: the average size of philippine e. planirostris, based on snout-vent length (svl), is similar to those found in panama and hawai’i (table 5) (crawford et al. 2011; olson et al. 2012a). furthermore, measurements of three (3) specimens in mexico by cedeno vasquez et al. (2014) gave 16, 20, and 21 mm svl, though no distinctions between sex were made in their study. this suggests that, morphologically, philippine specimens are similar to those found in other countries where the species is invasive (crawford et al. 2011; olson et al. 2012a; cedeno-vasquez et al. 2014). in cuba and the bahamas, where the species is native (schwartz 1974; hedges et al. 2008), schwartz (1974) described e. planirostris as having a maximum svl of 27 mm for gravid cuban females, 20 mm for cuban males while non-gravid females from isla de pinos, cuba and new providence, bahamas measured 28 mm svl; however, no mean lengths were given. tracing the source of the non-native philippine population 106 table 5. snout-vent length (svl) in millimeters (mm) of e. planirostris specimens caught in other countries compared to philippine specimens sex philippines1 panama2 hawai ’ i3 male 16.47 ± 1.28 17.5 17 female 21.31 ± 1.94 22.4 22 1 figures computed by authors based on five female specimens and three male specimens. no distinction was made between gravid and non-gravid females. 2 mean svl for female computed by authors based upon two measurements given by crawford et al. (2011) based on one gravid female and one non-gravid female. figure for male is based upon one male specimen. 3 figures are based upon calculations made by olson et al. (2012a) based upon a sample size of 100 for males and 176 specimens for females. no distinction was apparently made between nongravid and gravid females. interestingly, philippine specimens might still roughly follow the 3:1 ratio of striped to mottled color pattern seen in native populations of e. planirostris in cuba based on field observations by one of the authors (eys). this can only be confirmed by more extensive observations but, if so, this might suggest either a more direct origin from florida or cuba. it must be noted that a low initial diversity does not always hamper invasions, as seen in the invasive populations of the american bullfrog (lithobates catesbeianus) in china (bai et al. 2012). it is therefore important to manage the spread of e. planirostris as early as possible. this is especially important since the species is a potential ecological threat, capable of reaching high densities (around 12,500 frogs/hectare) and consuming high numbers of insects (up to 129,000 individuals/ hectare/night) based on observations in hawai’i (olson and beard 2012; olson et al. 2012a). to reach a more definitive conclusion on the species’ origin and the genetic diversity present in the introduced philippine population, the sample base needs to be expanded. sequences of specimens from davao city, mindanao, cebu city, cebu, and other islands where the greenhouse frog has been found need to be obtained in order to get a more complete picture of the genetic diversity present in the introduced philippine population. other parts of the country should be surveyed for additional populations of e. planirostris, which should also be barcoded. barcoding could also be expanded to include nuclear genes, such as recombination-activating gene 1 (rag1), which has been sequenced for several eleutherodactylus species, including e. planirostris, based on a search in genbank. g.c.l. que et al. 107 the cytb (figures 4 and 5) and 16s (figure 6) trees, as well as the haplotype network (figure 8), also show that e. planirostris is not monophyletic since e. planirostris clades contain e. guanahacabibes, a species that shares its range with western e. planirostris (hedges and diaz 2004; heinicke et al. 2011), though this grouping has low bootstrap support (53/76 nj/ml, respectively, for cytb). the results here for cytb (figures 4 and 5) are similar to the results of heinicke et al. (2011), which also found two lineages of e. planirostris and possible paraphyly for the species. however, since most of the genbank cytb sequences used here are from heinicke et al. (2011), our results are not surprising. interestingly, the analysis of pyron and weins (2011; suppl. info.) of lissamphibia (five genes were sequenced and analyzed for e. planirostris: 12s, 16s, h3a, rhod, sia, and tyr) showed that e. guanahacabibes diverges from a clade formed by e. planirostris and e. casparii, contrary to the findings of this study and of heinicke et al. (2011) using cytb sequences. this suggests that sampling more genes might be needed to fully resolve the phylogeny of the e. planirostris species group. for 16s (figure 6), the phylogeny is more problematic. eleutherodactylus guanahacabibes is shown to cluster with sequences that represent eastern e. planirostris (crawford et al. 2011), albeit with low bootstrap support (42 % nj). also, the sequence from havana, cuba (jf769005) that is supposed to represent western e. planirostris (crawford et al. 2011) does not group with either western or eastern e. planirostris, and this divergence has moderate bootstrap support (60% nj). a greater sample size for 16s, with sequences coming from various geographic regions, might help clarify the lineages present in e. planirostris with respect to the 16s gene and verify the groupings found using cytb. eight cox1 sequences for e. planirostris will be added to genbank as part of this study. while of interest by themselves since they represent a new geographic location for e. planirostris, they also add to the database of cox1 barcodes of amphibian taxa currently present in genbank and other databases, such as the barcode of life database (bold). they can be used for further studies of amphibians, including studies similar to that presented here. in summary, the eight specimens of e. planirostris barcoded in this study showed minimal genetic diversity amongst themselves based on sequences of three mitochondrial genes (cytb, 16s, and cox1). analysis of the cytb phylogenetic tree showed that the philippine samples grouped with the genbank sequences of e. planirostris specimens from matanzas province of cuba, hawai’i, and florida. for 16s and cox1, the philippine population grouped with specimens from florida tracing the source of the non-native philippine population 108 and panama. the ultimate origin of the introduced philippine population is matanzas, cuba, though the immediate origin is more likely to be hawai’i, florida or neighboring asian regions with introduced e. planirostris populations such as hong kong, considering the mode by which this species is thought to have spread. the low genetic diversity of local populations of e. planirostris suggests that it may be vulnerable to the founder effect, especially if these populations become isolated. acknowledgments gclq would like to acknowledge the support of the dna barcoding laboratory of the institute of biology, college of science, university of the philippines diliman and benjamin eleazar iii and john martyr for obtaining specimens used in this study. all authors would like to acknowledge dr. arvin diesmos, curator of the herpetology section of the natural history museum of the philippines, for allowing access to specimens for morphological measurements, and the bureau of plant industry of the department of agriculture, republic of the philippines, for providing unpublished importation records of plants from the united states of america for the period 2005 to 2014. gclq and ikcf would also like to thank the department of environment and natural resources biodiversity management bureau for funding support for this study through the project entitled “wildlife forensics and dna barcoding of native flora and fauna of the philippines.” conflict of interest the authors declare no conflict of interest. contribution of individual authors eys obtained specimens and scored morphometric data. eys, ikcf, and gclq conceptualized the study and experimental procedure. gclq performed the laboratory work and computer analysis. all authors analyzed the results and wrote the manuscript. literature cited akaike h. c1973. information theory and an extension of the maximum likelihood principle. in: petrov bn, csaki f, editors. 2nd international symposium on information theory; 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biology at the same laboratory under his adviser, dr. ian kendrich c. fontanilla. his research interest is in molecular phylogenetics and the current work is the result of his undergraduate thesis. emerson y. sy is a herpetologist with research interests in natural history, introduced species, and wildlife trade. he is a member of the nrcp–dost and denr’s technical working group on amphibians and reptiles. currently, he is a consultant of traffic–the wildlife trade monitoring network, monitor conservation research society, and usaid protect wildlife project. perry s. ong† was a professor and head of the biodiversity research laboratory at the institute of biology, up diliman. he was also dean of the college of science, up diliman. he obtained his phd at the monash university department of ecology and wildlife biology. his expertise was on philippine wildlife biology. he passed away on 2 march 2019. ian kendrich c. fontanilla is a professor and head of the dna barcoding laboratory at the institute of biology, up diliman. he received his ph.d. in genetics from the university of nottingham, united kingdom. he specializes in molecular genetics, phylogenetics, and malacology. 02_herrera developmental biology of the supermale yy tilapia 33science diliman (january-june 2001) 13:1, 33-40 abstract histogenesis of the reproductive system of supermale (yy) tilapia and xy tilapia reared at the central luzon state university was analyzed with the use of paraffin sections. in the course of development, the primordial germ cells appeared at the same age in yy and xy males, i.e., at 8 days posthatch. these cells which were larger in the yy (1.85 µm) than in the xy male (0.9 µm) later established themselves in the gonadal anlage by days 9-22. the lobules appeared earlier in the yy at day 15. blastema of the reproductive duct appeared in the yy at day 23 and in the xy at day 27. by day 79, meiotically active cells were abundant in both groups. by day 95, the yy fish showed mature sperm cells in the fully differentiated testis while it was at day 105 in the xy fish. the supermale consistently demonstrated bigger testis, with thicker somatic tissues, more spermatogenic cells, and more advanced developmental stage than xy fish of the same age. germ cell and nuclear size in the yy and xy fish were not statistically significantly different, although the general trend was that spermatogenic cells were bigger in the supermale tilapia. anova (α = 05) showed significant difference in size of the testis, spermatocysts, and vas deferens. the study showed that with the same rearing conditions and same age, the larger supermale tilapia has superior reproductive capacity with its larger testis and ducts, faster histogenesis and spermatogenesis, and higher gonadosomatic index (gsi). keywords: developmental biology, supermale tilapia, histogenesis, reproductive system developmental biology of the supermale yy tilapia (oreochromis niloticus): histogenesis of the reproductive system annabelle a. herrera*a, rinella r. cruza, & the fish genetics breeding program genetic manipulation for improved tilapia projectb ainstitute of biology, college of science, university of the philippines, diliman, quezon city bfreshwater aquaculture center, central luzon state university, munoz, nueva ecija introduction the nile tilapia (oreochromis niloticus), an introduced species from israel, is an important source of protein for filipinos. it is generally the most important tilapia species in the philippines and has gained wide acceptance and great economic importance in freshwater aquaculture since it is highly adaptable to domestication and can tolerate a wide range of conditions. the ease of breeding, however, has been one of the major drawbacks in the widespread adoption of tilapia farming technology (guerrero & abella, 1976). excessive reproduction leads to superfluous recruitment, particularly in pond populations, resulting in competition for available food and sparse resources. consequently, up to 50% of the biomass at harvest are unmarketable recruits because of size constraints. several practical solutions have been proposed to solve this problem, including manual sexing, use of predators to cull fry, and prevention of spawning in cages (mair* corresponding author herrera et al. 34 et al., 1997). presently, the most practical and effective method is the production of monosex male populations. male tilapias are preferred for rearing since they have a faster growth rate than females, and less energy is diverted into gamete production. thus, hormonal sex reversal by oral administration of the androgen 17aamethyltestosterone to produce nearly all male population has been practiced in tilapia farming for more than a decade now (mair et al., 1997). recently, yy technology was introduced in the philippines to improve the production of all male tilapia population. yy technology is a joint research undertaking between the university of wales, swansea, uk and the freshwater aquaculture center of central luzon state university in the philippines. it concentrates on the genetic manipulation of sex. this is achieved through a combination of feminization and progeny testing to identify the novel yy genotype that sires only xy male progeny, referred to as genetically male tilapia (gmt). most likely, the immediate benefit of the application of this genetic manipulation technique in tilapia is the further development, adaptation, and extension of the technology for large-scale production of genetically allmale o. niloticus. this newly introduced technology is presently being disseminated in the philippines, where yy males are being mass produced. inspite of the success of the yy technology, the exact mechanism of sex determination and differentiation in o. niloticus is still uncertain. the monofactorial system of sex determination in nile tilapia does not explain the occasional deviations from expected sex ratios based on studies involving sex reversal and chromosomal set manipulation. dr. graham c. mair (pers. comm), the proponent and senior consultant of the fish genetic project based in freshwater aquaculture center in central luzon state university, munoz, nueva ecija, (fac-clsu) discussed the need for histological investigation which could be of use in evaluating the presently existing sex control technologies. this study analyzes the histogenesis of the reproductive system of yy tilapia in comparison with that of the xy male. review of related literature yy male the existence of yy male fish was first shown by yamamoto as early as 1958 in medaka, oryzias latipes using the technique of hormonal sex reversal and selective breeding. subsequently, yy males were also produced in guppy, poecilia reticulata (yamamoto 1963), in goldfish, carassius auratus (yamamoto 1975), and in tilapia, oreochromis niloticus and o. mossambicus (varadaraj & pandian, 1989). there have been several published reports on yy males in fish. however, the survival of yy male fish was very low. in salmo gairdneri, the viability of yy individuals was observed only until the ‘eyestage’ of development (johnstone et al., 1979). thus, the viability of these genotype males in salmonids is still in question (calhoun & shelton, 1983). pandian & sheela (1995) concluded that the presence of more than one y chromosome rendered the male or female fish less viable. however, mair et al. (1997) reported that the yy male genotype of nile tilapia (o. niloticus) was viable and fertile like the normal (xy) males. mair (1997) further demonstrated the feasibility of large scale production of all male tilapia through genetic manipulation of sexual phenotype. he elaborately described the distinct steps in the production of the novel genotype ‘yy’ (supermale) nile tilapia. yy male technology the development of yy male technology involves a series of stages of feminization and progeny testing. the model for the breeding program in the production of yy males by genetic manipulation of sex in o. niloticus was proposed based on the extensive research on the genetics of sex determination in tilapia species carried out at the university college of wales, swansea, uk. studies revealed that this species exhibits predominantly monofactorial genotypic system with male heterogamety (xy) and female homogamety (xx) similar to that of humans (penman et al., 1987; mair et al., 1990. developmental biology of the supermale yy tilapia 35 it was proposed that by sex reversal (male to female), followed by progeny testing (to determine the genotype of a fish by the sex ratio of its progeny), it would be possible to identify sexreversed female (^eeee) with male genotypes (xy). these ^eeee could then be crossed with normal males (effectively crossing two males). the progeny of this cross would contain approximately ¼ of the novel genotype yy. this can be identified by the all-male sex ratio of their progeny in crosses with normal females (yy and xx gives only xy male progeny). further crosses of yy males with ^eeee followed by a second generation with sex reversal could then be crossed with yy males for the production of allmale producing yy male broodstock. this simple monofactorial hypothesis, however, failed to explain some deviations from predicted sex ratios. it was hypothesized that these arise through environmental influences, such as temperature (mair et al., 1990; trombka & avtalion, 1993). the temperature effect on sex determination, however, still does not explain the occurrence of the small percentage of males in putative monosex female progeny reared at ambient temperature. these deviations were also suspected to be due to autosomal or polyfactorial genetic effects. the technique is now widely adopted in philippine aquaculture, particularly in the fac-clsu where the fish genetic research project, in collaboration with the university of wales, swansea, is based. continuous research and development is being undertaken to further improve the sex control methodologies practiced in tilapia fish farming. materials and methods the study was conducted in collaboration with the fish genetics breeding program-genetically male tilapia of the fac-clsu, and the natural sciences research institute (nsri), university of the philippines, diliman, quezon city. the egypt-swansea strain of oreochromis niloticus produced by the fish genetic research program was used in the study. the fry were secured and reared at the breeding facilities of fac-clsu. the tissues for histological study were processed at the nsri, u.p. diliman, quezon city. experimental set-up two groups of undifferentiated fry, the xy males, which served as the control, and the yy males, were grown for approximately 5 months. six hundred fry in each group were collected from the down-welling artificial incubator and transferred into well-aerated 30lrectangular glass aquaria maintained at ambient temperature 30±4°c. two (2) glass aquaria were used in each group with a stocking density of 300 fry per aquarium (10 fry per liter). the fry used were of the same age at the first feeding stage, following completion of yolk sac absorption (<10 mm). cleaning of waste by siphoning was done daily. deep well water was replaced every two days. the fry were fed with commercially available fry mash (tatch feed with 33% crude protein) during the first two months. before feeding fry mash was sieved with a 1 mm-mesh size screen to remove particles that were too large to be ingested by the fish. feeding was done four times a day (0800, 1100, 1400, and 1700 hrs) at the rate of 20% of the body weight for the first seven days, 15% for the second week, and 10% for the succeeding weeks of the experimental period. adjustment of the diet ration was calculated by collecting and weighing ten percent (10%) of the total population of the fry every week. the fingerlings (about 100) were then transferred to fine-meshed hapas, 1m x 2m x 1m in dimension, suspended in a fertilized earthen pond, and fed with tatch feeds (31% crude protein). the fish were harvested after 5 months, during the onset of gonadal maturation, and again during the appearance of fully formed sperm cells. herrera et al. 36 histological preparation preparation of juvenile and adult reproductive organs histological processing was done on specimens from the time of hatching until the attainment of full sexual differentiation. starting from the time of hatching, 10 fry in each group were collected and fixed in bouin’s fluid everyday for 20 days, then roughly every week for the succeeding weeks until gonadal maturation. the region between the pronephros and the anus (approximate location of the gonads of the immature or juvenile fish) and the testis of the maturing and mature tilapia were processed for light microscopy. light microscopy samples were fixed in bouin’s fluid, dehydrated in a series of graded ethyl alcohol (10%, 20%, 30%,40%, 50%,60%,70%,85%,95% and 100%), and cleared in cedarwood oil for at least 6 hours and in xylene for 12 hours. the samples were then infiltrated in soft-hard paraffin at a ratio of 2:1, 1:1, and 1:2, respectively, after which, tissues were embedded in pure hard paraffin, trimmed and cut using a rotary microtome. a series of transverse sections (7-8 µm) of the region between the pronephros and the anus (for the immature until day 53), and the central portion of the maturing and mature testis were made and stained with hematoxylin-eosin and mounted with entellan mounting medium. prepared slides were then examined and selected parts were analyzed and photodocumented. results and discussion results of light microscopy studies showed differences in the timing and pattern of differentiation and maturation of the testis of the normal male (xy) and yy male nile tilapia. generally, the testis of the yy males, differentiated and matured earlier, and had bigger components and cells compared to the xy (gmt) males. table 1 summarizes the development of the testis and gonadosomatic index (gsi) (gsi=wt. of testis/weight of fish x 100) of yy and xy fish at different ages of development based on significant histological changes in the testes. hatching to 22 days posthatch gonads of the fry of both xy and yy, about 8 days posthatching, showed the primordial germ cell (pgc) lying in the genital ridge. the pgcs are distinguishable from somatic cells based on shape and size. pgcs are large cells, of round to oval shape, with round and prominent nuclei, and lightly staining cytoplasm. they are much larger compared to somatic cells. the pgcs table 1. summary of testicular development and gonadosomatic index (gsi) of yy and xy male tilapias at different developmental ages developmental age (days post hatch) 8 15 23 27 54 80 95 105 yy • pgc in early genital ridge • bipotential gonad • gsi cannot be measured • early lobule formation • gsi cannot be measured • efferent duct primodium appears • gsi = 0.2 • bigger duct • gsi = 1.2 • substantial number of seminiferous tubules • gsi = 1.9 • substantial number of spermatocysts • gsi = 2.1 • sperm ready for spermiation • gsi = 2.9 • abundant sperm • gsi = 3.6 xy • smaller pgcs in genital ridge • smaller (0.9-1.0 µm) • gsi cannot be measured • no lobules yet • gsi cannot be measured • no efferent duct yet • gsi = 0.2 • efferent duct anlage • gsi = 1.2 • f e w seminiferous tubules • gsi = 1.1 • fewer spermatocysts • gsi = 1.5 • few sperm • gsi = 1.6 • sperm ready for spermiation • gsi = 2.6 developmental biology of the supermale yy tilapia 37 observed in this study and their localization were similar to the pgcs observed by boco (1977) in tilapia mossambica and by herrera (1984) in tilapia nilotica. yy gonads were larger in diameter (1.8-2.0 µm) than those of the xy (0.9-1.0 µm, see table 2). yy pgcs appeared generally bigger (1.85µm) than those of the xy (0.9), although statistical analysis showed no significant difference. the formation of the paired gonadal primordia at 8 days is parallel to the findings of eckstein & spira (1965) in t. aurea and nakamura & takahashi (1973) in o. mossambica. the gonad anlage consisted of primordial germ cells enveloped by somatic cells. in xy, pgcs with large nuclei were observed at 15 days posthatching. at this time, an early lobule was already observed in yy. gonads at this stage were still sexually indifferent histologically. 23 to 32 days posthatch from day 23 to 32 posthatching, gonads increased in size. both somatic cells and germ cells increased in number. initial testicular differentiation was observed to occur at this stage both for xy and yy males, although yy gonads differentiated earlier than xy gonads. a slit-like space, the lumina primordium of the efferent ducts, was observed in the stromal tissue in the central region of the xy gonad at day 27 and at day 23 in yy. the initial appearance of the blastema of the efferent duct was the primary criterion set by nakamura & takahashi (1973) for testicular differentiation in t. mossambica. the findings in this study are similar to the result obtained by nakamura & takahashi (1983) in the gonadal differentiation of the normal xy male in nile tilapia. herrera (1984, 1987, 1996) observed onset of testis differentiation in younger (16-20 days old) fish probably due to earlier transfer of fry to larger cement ponds instead of aquaria. 33 to 79 days posthatch at 33 to 46 days, clusters of germ cells and somatic cells in the stroma of the gonad (testis) were observed. there was a significant change in the size and shape of the gonads. yy gonad was bigger and had larger cells than xy gonad. seminiferous tubules can be seen distinctly at day 54, both in xy and yy. within the tubules were large pgcs, spermatogenic and somatic cells. the yy gonad was observed to be bigger, with more tubules and gonial cells than in xy. the testis of both xy and yy were in a state of very active spermatogenesis at this stage. although the general trend was bigger spermatogenic nuclear size in yy than in xy, statistical analysis showed no significant difference. 80 to 145 days posthatch spermatogenic cells increased rapidly in number in the testes by 80 day posthatching. more gonial cells and cysts were observed in yy than in xy. active spermatogenesis was observed and spermatogenic cells were at various stages, in the numerous large tubules both in xy and yy testes. the testis at this stage table 2. comparison of the reproductive system of yy and xy o. niloticus l. structure yy xy range 45.0 52.0 1.8 2.5 2.2 11.0 1.3 10.0 0.3 1.1 0.3 0.8 0.3 0.6 0.1 0.2 375.0 720.0 m e a n ±±±±± standard deviation 49.23 ± 2.34 2.07 ± 0.23 5.25 ± 0.55 3.73 ± 0.47 0.67 ± 0.05 0.41 ± 0.14 0.40 ± 0.03 0.17 ± 0.01 512.34 ± 22.51 range 35.0 37.0 1.6 2.0 2.2 11.0 1.0 7.0 0.3 1.3 0.3 0.8 0.3 0.6 0.1 0.2 245.0 405.0 m e a n ±±±±± standard deviation 36.47 ± 0.76 1.78 ± 0.16 4.00 ± 0.29 2.88 ± 0.26 0.66 ± 0.18 0.44 ± 0.03 0.35 ± 0.0.03 0.16 ± 0.00 323.15 ± 23.47 testes length (mm) testes diameter (mm) seminiferous tubule diameter (µm) spermatocyst diameter (µm) spermatogonia nucleus diameter (µm) primary spermatocyte nucleus diameter (µm) secondary spermatocyte diameter (µm) spermatid diameter (µm) vas efferens diameter (µm) herrera et al. 38 and spermatocysts (mean diameter 3.73±0.47 µm) were significantly larger than in the xy male. comparison of the data on the size of the nuclei of various spermatogonia cell types showed no significant difference using anova. nuclei of spermatogonia of the supermale and xy male had mean diameters of 0.67±0.05 µm and 0.66±0.18 µm, respectively. primary spermatocyte nuclei of yy measured 0.41±0.14 µm while xy had 0.44±0.03 µm. the secondary spermatocytes, spermatids, and sperm of the yy showed no significant difference in the statistical analysis of the sizes, although a general trend was larger sizes for the yy male (table 2). fig. 1. xy testis at 105 days posthatching (430x) had less sperm than yy testis fig. 2. yy testis at 105 days posthatching (430x) show the seminiferous tubules, gonial cells, spermatyocytes (sc) and spermatids (sd) undergoing spermatogenesis sd sc contained numerous large tubules filled with cells in varying stages of spermatogenesis. in yy testis, abundant sperms filled the efferent ducts and main ducts ready for spermiation at day 95. the sperms were later observed in xy testis at day 105 (fig. 1). more sperms were found in yy testis than in xy testis (fig. 2). fully mature xy and yy testis at day 145 are shown in figs. 3 and 4. by 145 days, the average length of the testis of the xy fish was 36.47±0.76 mm while the yy fish was 49.23±2.34 mm. statistical analysis using anova (aa=0.05) proved that in supermale, the seminiferous tubules (mean diameter 5.25±0.55 µm), fig. 4. yy testis at 145 days posthatching (100x) shows the spermatogenic cells (sg), efferent ducts (ed), and vas deferens (vd) vd sg ed sg fig. 3. xy testis at 145 days posthatching (100x) shows the efferent ducts (ed), vas deferens (vd), and sperm (s) ed vd s developmental biology of the supermale yy tilapia 39 in the whole process of differentiation of the reproductive system of yy and xy tilapia, a positive correlation was observed between testis size and degree of maturation. in yy and xy fish of the same age, the gonadal maturation and testis size increase were faster in yy than in xy. no gross morphological, as well as histological abnormalities were observed in the yy male. since testis size was bigger in the yy fish, there would also be more tubules, increased surface area of the seminiferous epithelium, and more spermatogenic cells. the presence of a bigger vas deferens in the yy could be necessary for the bigger spermiation bulk during spawning. the lack of significant differences between the sizes of the spermatogenic cells may mean the absence of a negative effect of the lack of an x chromosome, or the presence of 2 y chromosomes. instead, such condition of 2 ys and no x chromosome may be advantageous, as evidenced by the bigger size of the yy fish, its testis and ducts, and the formation of more spermatogenic cells. in human beings, x chromosome carries several genes necessary for life (hickman et al., 1993) and absence of this is fatal (umaly & roderos, 1985). scott et al. (1989) suggested that the x and y sex chromosomes of tilapia are poorly differentiated, which may explain why yy males are viable. ohno (1970) noted that x and y are nearly identical in genetic composition in many fishes and amphibians. wolforth & hulata (1981) observed no case of heteromorphic chromosomes that could be regarded as sex chromosomes in many teleost species. the double dose of y or lack of x chromosome may give the fish advantage as shown in this study. investigation on the gene composition of the tilapia chromosomes is would be very enlightening. conclusions the study showed that using the same rearing conditions and comparing same age specimens, the larger supermale tilapia has superior reproductive capacity with its larger testis and ducts, higher gonadosomatic index, and faster histogenesis, testicular differentiation, and spermatogenesis. since xy males with 1 y chromosome have superior growth over xx females with no y chromosome, there is the possibility that 2 y chromosomes may effect the growth superiority observed in supermales. more investigations are needed to clarify the role of x and y chromosomes in tilapia, possibly using the tools of molecular biology and biotechnology. recommendations electron microscope analysis of the spermatogenic cells of yy and xy fish is recommended to elucidate on possible differences. analysis of the gene composition, as well as gene expression of the x and y chromosomes would be of invaluable help in making conclusive statements about the definitive advantage of having 2 y chromosomes and no x chromosome. factors affecting sex determination and differentiation should also be studied to properly evaluate the seemingly advantageous presence of 2 y chromosomes. capacity of the supermale sperm in fertilization should be investigated to determine the reproductive superiority of yy males over the xy males. acknowledgments the authors wish to express their sincerest thanks to the university of the philippines office of research coordination (up-orc) for financial assistance, the up natural sciences research institute and the up institute of biology for the use of their facilities, and the freshwater aquaculture center of central luzon state university, munoz, nueva ecija, for the fish used in this study. references boco, a., 1997. gonadal sex differentiation in normal embryonic development in tilapia mossambica peters. [m.s. thesis]. quezon city, university of the philippines diliman. 21 p. herrera et al. 40 calhoun, w.e. & w.l. shelton., 1983. sex ratios from spawnings of sex-reversed broodstock of tilapia nilotica. aquaculture. 33: 365-371. eckstein, b. and m. spira, 1965. effect of sex hormones on gonadal differentiation in a cichlid, tilapia aurea. biol. bull. 129: 482489. guerrero, r.d. & t.a. abella, 1976. induced sex reversal in tilapia nilotica with methyltestosterone. fish. res. j. philipp. 1: 46-49. herrera, a.a., 1984. histogenesis of the pituitary in relation to gonadal differentiation in tilapia nilotica [ph.d. dissertation]. quezon city, university of the philippines diliman. 113 p. herrera, a.a., 1987. fine structure of the gonadotrophs and gonads of pre-and post-spawning tilapia nilotica linnaeus. nat. appl. sci. bull. 39: 151-159. herrera, a.a., 1996. histology of tilapia, oreochromis niloticus. quezon city, bureau of fisheries and aquatic resources. johnstone, r., t.h. simpson & 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reversal in sex differentiation of yy zygotes in medaka, oryzias latipes. genetics. 48:293-306. yamamoto, t., 1975. a yy male goldfish from mating estrone-induced xy female and normal male. j. hered. 662:2-4. comparative use of meristic and geomorphometric analyses 41science diliman (july-december 2011) 23:2, 41-53 abstract comparative use of meristic and geomorphometric analyses of vegetative and floral characters in studying intraspecific variation in portulaca grandiflora (hook) athena jan f. valenzuela1, brian s. santos1, jonas p. quilang1 and ernelea p. cao1,2 1institute of biology, college of science, university of the philippines, diliman, quezon city 2natural sciences research institute, university of the philippines, diliman, quezon city *corresponding author: ernelea p. cao, institute of biology, college of science, university of the philippines, diliman, quezon city 1101 meristic and geometric morphometric analyses were applied to 150 portulaca grandiflora (hook) specimens in order to examine shape variations in vegetative and floral characters between and among the morphotypes of the species. canonical variate analysis of both meristic and geomorphometric data show significant (p<0.05 and p<0.01, respectively) variation of leaf shape, specifically that of the petiole shape, between “single” flowered and “double” flowered varieties. plants with single flowers possess leaves that are generally wider, especially toward the apex, while those of plants with double flowers are narrower. differences were also observed in the size and shape of the petiole such that “double” flowered varieties had a more distinct petiole compared to the “single” flowered varieties. petal shape was also significantly variable between the two varieties. principal component analysis of both meristic and geomorphometric data also show significant variation within the individuals which may explain the occurrence of the different morphotypes of the plant. cluster analysis using meristic data again showed separation between “single” flowered and “double” flowered varieties and further separation between petal colors. in general, meristic and geomorphometric data showed comparable results. description of shape variation in the vegetative and floral characters studied was consistent in the different analyses used. cao, e.p., et al 42 science diliman (july-december 2011) 23:2, 41-53 introduction morphometrics is a tool in which morphological characteristics are transformed into generalized shapes and quantitative data so that variations in and among biological structures can be compared without the bias of an experimenter. the goal of morphometrics is to illustrate patterns of variation in morphological shape so that they may be available for analysis (macleod, 1991). traditional methods of morphometrics make use of meristis data, i.e., linear measurements, angles and ratios. several problems however are encountered in traditional morphometrics. first, it is very time consuming and usually requires killing a specimen. second, the question of actual shape variation is being asked since variables obtained in traditional morphometrics are non-indicative of the shape (zelditch et al., 2004). a relatively new type of morphometric analysis is geometric morphometrics (gm). it tries to solve problems of size and proportion that are encountered in traditional morphometrics by using landmark-based analysis. it makes use of landmarks so that topological analysis of the specimen may be studied and geometric relationships can be determined. true landmarks are usually chosen based on homological structures. portulaca grandiflora (hook), commonly known as rose-moss, moss-rose, purslane or portulaca, is a widely cultivated species all over the world. the plant usually self-pollinates instead of relying on insectmediation or cross pollination. although this genus has been studied for seed variations (matthews et al., 1994), no extensive studies have been done about its other morphological features. moreover, no studies have been found to correlate the different species and variants of this genus using molecular data. since molecular studies are relatively expensive to conduct, geomorphometrics offer a relatively cheaper and faster kind of analysis that may aid in the proper taxonomic treatment of this highly polymorphic species (mereda et al., 2008). this study determined patterns of variation within the different morphotypes of p. grandiflora by measuring significantly chosen vegetative and floral characteristics and compared the usefulness of meristic and geometric morphometric analyses in order to differentiate the morphotypes from each other. materials and methods collection of samples five morphotypes of p. grandiflora, namely, double orange, single pink, double pink, double pink-white and single yellow, were obtained from a single site in bicol, philippines to eliminate variations due to ecological conditions. thirty individuals were sampled for each morphotype. data processing and digitization a total of sixteen (16) vegetative and floral characters were collected for each individual in triplicate measurements using a vernier calliper or count method. the six (6) vegetative and ten (10) floral characters considered were: stem width of base, stem width of first leaf, internode between first and second leaf, maximum leaf length, maximum leaf width, petiole length, number of inflorescence, number of petals, maximum petal length, maximum petal width, number of sepals, maximum sepal length, maximum sepal width, floral diameter, pedicel length, and number of stamens, these characters were chosen based on the study of mereda and co-workers (2008) which also involved intraspecific variation in a single species. the first leaf, a sepal and the first petal behind the sepal were also collected for each individual. these were photographed using either a nikon d60 or scanned using a brother dcp-165c scanner and the digitized specimens were assigned with landmarks using tpsdig2 (by f.j. rohlf, available at http:// life.bio.sunysb.edu/morph/). for the leaves, ten (10) landmarks were assigned as seen in figure 1. the landmarks used were based on type i or homologous landmarks (2, 5, 8 and 10) of jensen and co-workers (2002) and were added with comparative use of meristic and geomorphometric analyses 43science diliman (july-december 2011) 23:2, 41-53 type ii or pseudo landmarks (1, 3, 4, 6, 7, 9) which show maximum curvatures of the leaf (volkova, et al., 2005). for the sepals, four (4) landmarks were assigned as seen in figure 2. the following landmarks used were type ii. landmarks 1, 2 and 3 are the bases of the primary parallel veins while landmark 4 shows the apex of the sepal. figure 1. a sample leaf from a plant of single pink morphotype with ten (10) landmarks. figure 3. a sample petal from a plant of single pink morphotype with thirteen (13) landmarks. for the petals, thirteen (13) landmarks were assigned as seen in figure 3. the following landmarks used show the maximum curvatures of the petal. the tps files produced for each specimen were processed in coordgen6f to apply generalized procrustes analysis (gpa) which was needed to eliminate differences in shape, size and orientation, maintaining only shape differences (imp, by h.d. sheets, available at http://www2.canisius.edu/~sheets/ morphsoft.html). data analyses ordination analysis was done to examine shape variations. landmark-based analysis made use of cva-manova (canonical variate analysismultivariate analysis of variance) and pca (principal component analysis) using cvagen61 and pcagen6n, respectively (imp, by h.d. sheets, available at http://www2.canisius.edu/~sheets/ morphosoft.html). ordination analysis of traditional continuous data made use of cva-manova followed up by discriminant analysis and pca using the spss 19 software. clustering was also done for traditional continuous data using hierarchical clustering in spss 19 software. figure 2. a sample sepal from a plant of single pink morphotype with four (4) landmarks. cao, e.p., et al 44 results and discussion five (5) different morphotypes of p. grandiflora considered in the study were: double orange, single pink, double pink, double pink-white and single yellow, as seen in figure 4. thirty (30) individuals for cv, difference seen in the multivariate analysis was accounted for by petiole length. leaf ratio and petiole length gave the most contribution in the observed variations between groups. eigenvectors of petiole length and floral diameter are 1.149 and 0.205, respectively, while absolute pooled correlations are 0.817 and 0.146, respectively, for each variable. a plot of the first versus the second canonical variant in figure 5 also showed that “double” varieties gravitate toward the negative side of canonical variate 1 while “single” varieties clump together toward the positive side of canonical variate 1. no distinct separation can be made between groups based on canonical variate 2, as expected. canonical variate analysis-multivariate analysis of variance was applied to the landmarks generated for all 151 individual samples to observe shape variation in the leaves, petals and sepals of the five science diliman (july-december 2011) 23:2, 41-53 each morphotype and an added double pink individual (for gm analysis) were obtained amounting to one hundred fifty (150) individuals all in all. each individual was measured for sixteen (16) vegetative and floral meristic and nominal characters. figure 4. samples of each morphotype of p. grandiflora (hook). (a.1.) double orange petal, (b.1.) single pink petal (c.1.), double pink petal (d.1.), double pink-white petal (e.1.), single yellow petal (a.2.), double orange sepal (b.2.), single pink sepal (c.2.), double pink sepal (d.2.), double pink-white sepal (e.2.), single yellow sepal (a.3.), double orange leaf (b.3.), single pink leaf (c.3.), double pink leaf (d.3.), double pinkwhite leaf (e.3.), single yellow leaf. a.1 b.1 c.1 d.1 e.11 2 a.2 b.2 c.2 d.2 e.21 2 a.3 b.3 c.3 d.3 e.31 shape variation between different morphotypes of portulaca grandiflora multivariate analysis was applied to the meristic data obtained through measurement of vegetative and floral characters. variables were first transformed using natural logarithm or through ratios since initial data as enumerated were not normalized. also, variables which were not normalized through such transformations were discarded. multivariate analysis using wilk’s lambda (p<0.05) showed that differences within the five groups were significant. follow-up analysis was done through discriminant analysis. four (4) eigenvalues were generated from this analysis with only the first eigenvalue considered significant accounting for 67.6% of the observed variance while the next eigenvalue only accounted for 25.9% of total variation. looking at the first distinct 5mm 10mm comparative use of meristic and geomorphometric analyses 45 figure 5. plot of first canonical discriminant function vs second canonical discriminant function of each individual from five (5) p. grandiflora morphotypes. figure 6. canonical variance analysis on shape of the leaves of five (5) morphotypes (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow) of one hundred fifty-one (151) individuals of p. grandiflora. science diliman (july-december 2011) 23:2, 41-53 cao, e.p., et al 46 science diliman (july-december 2011) 23:2, 41-53 morphotypes. in comparing leaf shape of all five morphotypes, one (1) distinct canonical variate (cv) was generated by the cva program. a p-value of 2.22 x 10-16 (p<0.001) shows that there is a significant difference in shape between all five morphotypes. figure 6 shows two (2) distinct groups formed, that of the “single” flower varieties, when plotting the first two canonical variates of all individuals. generated procrustes deformation grids seen in figure 9a showed that variation is due to differences in shape toward the petiole area. variation in petal shape can also be observed when comparing all five morphotypes, giving two (2) distinct canonical variates with the first cv giving a p-value of 2.62 x 10-7. figure 7 shows the cv1 vs cv2 plot for petal shape variation but does not exhibit distinct groupings as with the leaves despite significance of p-value. overlapping between the groups can be seen. the procrustes deformation grids (figure 10a) that were generated also showed that variation can be accounted for by differences in shape toward the basal and laminar area. shape analysis for sepals generated one (1) distinct cv but did not show significant shape variation between the five morphotypes with a p-value of 1.26 x 10-3. this can also be seen in figure 8 wherein the cv1 vs cv2 plot showed severe overlapping between members of the groups. furthermore, no procrustes deformation grid could be generated for sepal shape. when comparing shape variance between leaves of “single” flowered and “double” flowered varieties, results showed that there is a significant difference in the leaf shape between the two groups (p-value = 2.22 x 10-16). shape variation can also be accounted for by differences in petiole shape as seen in figure 9b. variance in petal shape was also found to be significant (p-value = 2.29 x 10-5) when comparing between figure 7. canonical variance analysis on shape of the petals of five (5) morphotypes (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow) of 1one hundred fifty-one (151) individuals of p. grandiflora. comparative use of meristic and geomorphometric analyses 47science diliman (july-december 2011) 23:2, 41-53 “single” flowered and “double” flowered varieties with most of the variation coming from the basal and laminar area. in comparing sepals of “single” flowered and “double” flowered varieties, a significant shape variation can be seen. cva produced one distinct cv with p-value of 1.03 x 10-4 but no deformation procrustes grid can be produced. canonical variate analysis using both meristic continuous data and land-mark based data agree that there is a significant difference between the morphotypes most especially between “single” flowered varieties and “double” flowered varieties. using traditional data, a follow-up discriminant analysis showed that there is one distinct eigenvalue accounting for most of the observed variation to petiole length followed by floral diameter. land-mark based data also accounted for most of the observed variation to petiole length as shown by procrustes deformation grids of leaf land-marks. significant deformation toward the basal end could also be observed in grids produced by petal land-marks while no deformation grid could be made for sepals. in land-mark based cva, comparison of “single” and “double” flowered varieties was also made which only reaffirmed clusters made by comparison of the five different morphotypes. “single” flowered variants formed into one cluster towards the positive end of the first cv axis while “double” flowered variants formed another cluster toward the negative end in terms of leaf shape. shape variation in petals was accounted for by differences in the basal end and sepals did not show any significant distinction between the two groups. the results of the different analyses clearly showed that the “single” flowered and “double” flowered variants are distinct from each other. leaf size decreases as the number of petals increase in the floral figure 8. canonical variance analysis on shape of the sepals of five (5) morphotypes (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow) of one hundred fifty-one (151) individuals of p. grandiflora. cao, e.p., et al 48 whorl such that “single” flowered varieties showed leaves that are wider and “double” flowered varieties possessed leaves that are narrower. this agrees with the suggestion of ashman and majetic (2006) that there is a possible correlation in the genetic determination of floral and vegetative morphotypes, especially in actinomorphic (radially symmetric) flowers. figure 10. procrustes deformation grids from canonical variate analysis of petal shape. (a) five morphotypes, (b) single and double varieties, (c) single varieties, (d) double varieties science diliman (july-december 2011) 23:2, 41-53 figure 9. procrustes deformation grids from canonical variate analysis of leaf shape. (a) five morphotypes, (b) single and double varieties ,(c) single varieties, (d) double varieties a b1 c d2 -0.4 -0. 2 0 0.2 0.4 0.6 0.8 1 1.2 1. 4 1.6 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 -0. 6 -0. 4 -0. 2 0 0. 2 0. 4 0. 6 -0.4 0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 -0. 5 0 0.5 1 1. 5 -0. 6 -0. 4 -0. 2 0 0. 2 0. 4 0. 6 a b1 c d2 -3 -2 -1 0 1 2 3 4 5 6 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 -2 -1 0 1 2 3 4 5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 -1 0 1 2 3 4 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 -1 0 1 2 3 4 2 -1. 5 1 -0. 5 0 0. 5 1 1. 5 comparative use of meristic and geomorphometric analyses 49 shape variation within the species of portulaca grandiflora principal component analysis of merisitc data showed distinct variation within groups with bartlett’s test for spherecity having a significant value of 0.00. factor extraction produced three distinct principal components cumulatively explain 60.63% of total variance. all variables included showed contribution to the first component, with leaf width to width length followed by petiole length having the most absolute contribution. principal component analysis of landmark-based specimen using the leaf as a factor in figure 11 showed distinction between the “single” flowered and “double” flowered varieties with the “single” varieties concentrated towards the positive end while the “double” varieties concentrated toward the negative end. no further distinction can be made within the two groups formed. one distinct eigenvalue was generated which had a value of 2.32 x 10-5 showing figure 11. principal component analysis on leaf shape of 5 morphotypes. (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow ) of one hundred fifty-one (151) individuals of p. grandiflora. distinct variation within the groups. a procrustes deformation grid in figure 12 show variance is accounted for by shape difference in petiole area. using petal as a factor in figure 13, most of the individuals grouped together toward the positive end of principal component 1. however, several members from each of the five morphotypes deviated from the group and clumped together toward the negative end of principal component 1. two distinct eigenvalues were generated having a value of 0.00133 and 0.00153, respectively, both of which suggest distinct variation within the groups. the generated procrustes deformation grid in figure 12 accounts for variation from all sides of the petal. principal component analysis on sepals produced one distinct eigenvalue of 0.002526 showing distinct variation within groups as well. the principal component plot in figure 15 shows completely scattered individuals. no deformation grid was generated for this analysis. science diliman (july-december 2011) 23:2, 41-53 cao, e.p., et al 50 figure 12. procrustes deformation grids from principal component analysis on leaf shape of 5 morphotypes (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow ) of one hundred fifty-one (151) individuals of p. grandiflora. figure 13. principal component analysis on petal shape of 5 morphotypes. (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow) of one hundred fifty-one (151) individuals of p. grandiflora. science diliman (july-december 2011) 23:2, 41-53 comparative use of meristic and geomorphometric analyses 51 figure 15. principal component analysis on sepal shape of 5 morphotypes. (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow) of one hundred fifty-one (151) individuals of p. grandiflora. figure 14. procrustes deformation grids from principal component analysis on petal shape of 5 morphotypes. (30 double orange, 30 single pink, 31 double pink, 30 double pink-white and 30 single yellow) of one hundred fifty-one (151) individuals of p. grandiflora. science diliman (july-december 2011) 23:2, 41-53 cao, e.p., et al 52 principal component analysis of both traditional meristic data and geometric landmark-based data both showed significant variation within the groups. variation at the individual level is an important factor which can explain the high level of morphological variability between morphotypes as variation should first be present within the population before other selective forces such as pollinator preferences and other environmental forces can act on shaping the species (galen, 1999). however, results of pca do not fully agree with cva as expected. continuous data showed that the first principal component explains only 26.71% of variance and this variance could be accounted primarily to the ratio of leaf width to leaf length rather than petiole length. in landmark-based data, leaf landmarks still show that variation is due to differences in petiole shape of “single” and “double” flowered varieties. petal landmarks, on the other hand, show variation not only between morphotypes but among its members and the procrustes deformation grid accounts this to differences in the basal end of the petal as well as the margins of the petal. still, shape variation in sepal landmarks among the individuals cannot be clearly seen. figure 16. dendrogram using squared euclidean distances of between groups using meristic data of one hundred fifty-one (150) individuals of p. grandiflora (1: double orange 2: single pink 3: double pink 4: double pink/white 5: single yellow). science diliman (july-december 2011) 23:2, 41-53 comparative use of meristic and geomorphometric analyses 53 cluster analysis hierarchical cluster analysis using the means of each variable clearly showed that there is a separation between the “single” and “double” flowered plants with the two groups having a distance of 1.019. the two “single” flowered varieties were further separated having a distance of 0.136 between the two groups. between the three “double” flowered varieties, double-orange was first separated from the group having a distance of 0.135 between the other two “double” varieties. double-pink and double-pink/ white had the least distance of 0.072 among all groups. a dendrogram of the analysis can be seen in figure 16. although cluster analysis results should always be interpreted with caution due to the fact that there is a hierarchical assignment given to each group, it should be noted that results of the cluster analysis reiterate the fact that there is distinction between the “single” and “double” flowered varieties. separation between the different floral colors could be due to the fact that individuals were assigned in such grouping and hierarchy was given to each group since earlier analyses were not able to discriminate between such factors. it is of interest however that between “double” flowered varieties, pink and pink/white varieties were closer to each other than that of the orange variety. conclusion although continuous data from traditional methods did not necessarily coincide with landmark data from geometric morphometrics, it is noteworthy to mention that results were fairly comparable across analyses. the “single” flowered varieties could be clearly distinguished from the “double” flowered varieties using canonical variate analysis, principal component analysis and cluster analysis. given more time and resources, dna studies can be done in order to correlate the data obtained with molecular genetic variation between and within morphotypes of this plant of interest. science diliman (july-december 2011) 23:2, 41-53 references ashman, t.l. and c.j. majetic, 2006. genetic constraints on floral evolution: a review and evaluation of patterns. heredity. 96: 343-352. galen, c. 1999. why do flowers vary. bioscience. 49(8): 631-640. jensen, r.t.,, k.m. ciofani and l.c. miramontes, 2002. lines, outlines and landmarks: morphometric analyses of leaves of acer rubrum, acer saccharium (aceracaeae) and their hybrid. taxon. 51(3): 475-492. macleod, n., 1991. phylogenetic signals in morphometric data. retrieved in 15 november 2010 from http:// www.nhm.ac.uk/hosted_sites/paleonet/macleod/pdfs/ signals.pdf. matthews, j.f., d.w. ketron and s.f. zane, 1994. the seed surface morphology and cytology of six species of portulaca (portulacaceae). southern appalachian botanical society. 59(4): 331-337. mereda, p., i. hodalova,, p. martfoni, j. kucera and j. lihova, 2008. intraspecific variations in viola suvaris in europe: parallel evolution of white-flowered morphotypes. annals of botany. 102:443-462. volkova, p.a., v.v. choob and a.b. shipunov, 2005. the flower organ transition in water-lily (nymphaea alba l. s.l., nymphaceae) under the cross-examination of different morphological approaches. the materials of the white sea expedition of moscow south-west high school. vol 5. retrieved on 15 november 2010 from http://herba.msu.ru/ shipunov/belomor/english/2005/nymd.htm zelditch, m.l., d.l. swiderski,, h.d. sheets and w.l. fink, 2004. geometric morphometrics for biologists: a primer. elsevier inc. 42 urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode for electrochemical detection of uric acid angelo gabriel e. buenaventura allan christopher c. yago* institute of chemistry university of the philippines diliman natural sciences research institute university of the philippines diliman abstract this study presents an electrochemical biosensor developed for uric acid (ua) determination using carbon paste electrode (cpe) modified with copper (ii) oxide (cuo) particles and urate oxidase (uox) enzyme. base cpe is prepared using a multi-walled carbon nanotube (mwcnt) and a polydimethylsiloxane (pdms) binder. the main sensing process is based on the oxidation of ua into 5-hydroxyisourate (hiu) as catalyzed by uox, forming h 2 o 2 as byproduct, and then the h 2 o 2 reduction-oxidation (redox) reaction converts cuo to form cu 2 o; the amount of h 2 o 2 and hence ua in the sample is measured by the oxidative current measured for the conversion of cu 2 o back to cuo. cyclic voltammetry (cv) measurements revealed that the activity of uox was retained with an apparent michaelis constant (k m app) to be equal to 41.46 µm. differential pulse voltammetry (dpv) measurements of ua using uox-cuo-cpe showed a linear response ranging from 10 µm to 79.4 µm ua with a limit of detection (lod) determined to be equal to 8.82 µm. uox-cuo-cpe was shown to be selective towards ua even in the presence of creatinine, xanthine, and glucose. furthermore, uox-cuo-cpe was shown to be reusable (3.28% rsd), and its fabrication is repeatable using single factor analysis of variance (anova) [f(1.396) < f critical (5.143)]. uox-cuo-cpe was also shown to be stable even after five weeks of storage using the two-sample t-test [t(0.156) < t critical (4.303)]. based on a recovery test using synthetic urine sample, this study showed the applicability of uox-cuo-cpe in the detection of ua in human urine with 90.27%–102.03% recovery (n = 3). keywords: urate oxidase, copper (ii) oxide, carbon paste electrode, uric acid * corresponding author science diliman (july-december 2020) 32:2, 42-76 a.g. e. buenaventura and a.c. c. yago 43 introduction uric acid (ua) is the end-product of purine catabolism of higher primates including humans (walker et al. 1990). normal ua levels in blood for premenopausal females range from 2.6-6.0 milligram per deciliter (mg/dl) and 3.5-7.2 mg/dl for males and postmenopausal females (desideri et al. 2014). excess ua is normally excreted in urine. in normal adults consuming an average diet, the normal ua secretion in urine is 800 mg or less over 24 hours (suki and massry 2012). abnormal levels of ua have been associated with several diseases such as gout (perez-ruiz et al. 2015), hypertension (feig and johnson 2003; masuo et al. 2003), metabolic syndrome (matsuura et al. 1998; ishizaka et al. 2005), diabetes (nakanishi et al. 2003), kidney disease (obermayr et al. 2008), cardiovascular disease (bickel et al. 2002), hodgkin’s disease (kay and gottlieb 1973), fanconi syndrome (ben-ishay et al. 1961), and medullary thyroid cancer (puig et al. 1984). due to these associations of ua level with various diseases, ua analysis has become a routine test in clinical laboratories. two clinical methods are currently accepted for ua determination (walker et al. 1990; watts 1974). one is the colorimetric method which involves the reduction of chromagen such as sodium tungstate with ua to produce a measurable color change. this method is generally considered to give an overestimate of the true value of ua. another method is the uv differential absorption of ua using uricase or urate oxidase (uox), and then uox catalyzes the conversion of ua to allantoin. this method is more expensive because of the one-way use of uox. therefore, there is need to develop a selective and sensitive sensor that can be reused to maximize its cost efficiency. several sensing methods have been utilized for sensor development for ua determination including electrochemical (rafati et al. 2014), fluorescencebased (zhang et al. 2011), colorimetric (wu et al. 2015), and chemiluminescence (chaudhari et al. 2012). among these methods, the electrochemical method is the most widely used for sensor development of ua and other biologically important compounds due to high sensitivity and selectivity, portable field-based size, rapid response time and low cost (wang et al. 2008). one major problem in the electrochemical detection of ua is the coexistence of electroactive interference in biological fluids such as ascorbic acid (aa), which has similar oxidation potential, e 1/2 ≈ 200mv versus sce, at graphite electrodes (bravo et al. 1998). biological samples like blood and urine also contain various non-electroactive biochemical compounds that may interfere in ua measurements. thus, a selective recognition element is required to ensure high selectivity for ua detection. one strategy that can be used is to modify an electrode with an enzyme that has specific interaction with urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 44 the analyte. the group of usman ali et al. used uox and zno nanorods to fabricate an enzymatic biosensor for ua (usman ali et al. 2011). uox was electrostatically immobilized on gold substrate with chemically grown zno nanorods. in another study, an amperometric biosensor for ua was fabricated by immobilizing uox via glutaraldehyde crosslinking on polyaniline-polypyrrole (pani-ppy) composite film on the surface of a platinum electrode (arslan 2008). a similar study was conducted where semiconductor cuo particles and uox were utilized in the fabrication of an electrochemical biosensor for ua determination (jindal et al. 2012). in that study, cuo was deposited via pulse laser deposition (pld) onto a pt-coated glass substrate. the study reported the biocatalytic property of the immobilized uox by having low apparent michaelis constant (k m app) measured at 0.12 mm. however, the utilized technique of cuo deposition and substrate is complicated and more expensive than the electrodeposition method presented in this paper. our results showed that a simple ua electrochemical biosensor can be prepared using a uoxcuo-cpe system with low detection limit (8.82 µm) and highly selective towards ua. the biosensor showed to have high recovery values (90.27%–102.03%) using synthetic human urine, which indicates its applicability to human urine. the main goal of the study is to fabricate a selective and sensitive electrochemical biosensor for ua determination using cpe modified with cuo particles and uox. the specific objectives were as follows: 1) fabricate uox-cuo-cpe and evaluate its electrochemical response; 2) optimize the sensing parameters for ua determination and evaluate the sensing performance of uox-cuo-cpe; and 3) perform ua determinations in synthetic urine using uox-cuo-cpe. materials and methods chemicals and instrumentation multi-walled carbon nanotube powder (mwcnt) was purchased from chengdu organic chemicals co. ltd., chinese academy of sciences (purity: >95%; length 10–30 µm; internal diameter 5–10 nm and outer diameter 10–20 nm). copper chloride (cucl 2 ) (purity: ≥98.0%) was purchased from techno pharmchem. boric acid was purchased from unichem (purity: ≥99.5%). sodium hydroxide pellets (naoh) were purchased from univar (purity: ≥97.0%). sodium phosphate dibasic (na 2 hpo 4 ) was purchased from duksan reagents (purity: ≥99.0%). potassium dihydrogen orthophosphate (kh 2 po 4 ) was purchased from himedia (purity: ≥99.5%). potassium chloride (kcl) was purchased from alfa aesar (purity: ≥99.0%). sodium chloride a.g. e. buenaventura and a.c. c. yago 45 (nacl) (purity: ≥99.0%) and potassium ferrocyanide trihydrate {k 4 [fe(cn) 6 ] 3h 2 o} (purity: ≥98.5%) were purchased from jt baker. polydimethylsiloxane liquid (pdms) with 500 centistokes (cst) viscosity, uricase or urate oxidase from arthrobacter globiformis (uox) (15–30 units/mg), uric acid (ua) (purity: ≥99.0%), xanthine (purity: ≥99.5%), creatinine (purity: ≥98.0%), and d-(+)-glucose (purity: acs reagent) were purchased from sigma aldrich. all solutions were prepared using deionized water. blank and ua sensing solutions were all buffered using borate buffered saline (bbs) composed of 0.1 m borate and 0.2 m nacl. uox solutions were prepared by dissolving uox with bbs at ph 9.0. all electrochemical measurements, including cyclic voltammetry (cv), chronoamperometry (ca), electrochemical impedance spectroscopy (eis), and differential pulse voltammetry (dpv), were performed using autolab pgstat 302n with a three-electrode system. the fabricated uox-cuo-cpe was used as the working electrode; an ag/agcl (3.0 m kcl) electrode as the reference electrode; and a platinum (pt) coated rod as the counter electrode. preparation of uox-cuo-cpe a cpe composed of mwcnt and 500 cst pdms liquid polymer was used as the working electrode. the percentage composition as well as electrochemical pretreatment of the resulting electrode were already optimized in the previous studies of our group (buenaventura et al. 2016; buenaventura and yago 2018). figure 1 shows the scheme for fabrication of uox-cuo-cpe. in brief, 0.10 g of mwcnt powder was mixed with 0.90 g of pdms in an agate mortar and pestle. the resulting composite was packed in a 1-ml plastic tube. a copper wire was inserted in the plastic tube up to about 1 cm from surface, which served as the electrical contact. the surface of the resulting cpe was polished using a glass slide. the cpe then underwent anodization electrochemical pretreatment via cv, which was performed in 0.1 m naoh solution using the following parameters: scan rate: 100 mv/s, potential window: -0.3 v to +1.5 v, and number of cycles: 30. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 46 figure 1. sensor fabrication scheme for uox-cuo-cpe. the fabricated cpe was then further modified in order to fabricate uox-cuo-cpe. cpe was electrodeposited with cuo to produce cuo-cpe using the same procedure as our previous study on cuo-cpe (buenaventura and yago 2020). copper (cu) particles were first electrodeposited on the surface of cpe via ca. using cpe as working electrode in 20 mm cucl 2 with 0.1m kcl solution, -0.5 v was applied for 20 s. the resulting cu-cpe was anodized via ca. in 0.1 m naoh solution, +0.7 v was applied on cu-cpe for 60 s. the resulting cuo-cpe was then modified with uox. onto the surface of cuo-cpe, 45 µl of 0.3 mg/ml of uox in bbs (ph 9.0) was dropcasted and was allowed to air-dry for at least 12 hours. the modified electrode was washed with bbs (ph 9.0) then with deionized water to remove loosely bound enzymes. the resulting uox-cuo-cpe was stored in a refrigerator with a temperature of around 4 °c when not in use. electrochemical characterization of cpe, cuo-cpe and uox-cuo-cpe electrochemical measurements were done in order to determine the electrochemical signals towards various solutions using the fabricated biosensor. electrochemical measurements that were done include cyclic voltammetry (cv) and electrochemical impedance spectroscopy (eis). the parameters for cv measurements that were used are as follows: potential range -0.5 to +0.8 v and 10 mv/s scan rate. the parameters for eis measurements that were used are as follows: frequency range 0.1 hz to 20 khz; applied potential +0.45 v for bbs (ph 8.6) solution and +0.35 v for fe(cn) 6 4(aq) in bbs (ph 8.6) solution. a.g. e. buenaventura and a.c. c. yago 47 optimization of parameters for electrochemical determination of uric acid different parameters were optimized for uox-cuo-cpe, including enzyme loading (amount of enzyme dropcasted on the surface of the cuo-cpe), sensing solution ph (ph value of the sensing solution), and equilibration time (amount of time the biosensor was immersed into the sensing solution under stirring condition). for all the optimizations, univariate optimizations were done, i.e. one parameter (which is being optimized) was varied, while the rest of the parameters were held constant. the optimizations were all based on the measured signal towards 79.4 µm ua in bbs (ph 9.0) through cv measurements. the parameter value that gave the highest average peak current (ave. ip) was chosen as the optimized value. the optimization of enzyme loading was done via cv measurements using different uox-cuo-cpes with different enzyme loading (0.02–0.5 mg/ml uox in bbs). the optimized value was used for the subsequent optimizations. for the optimization of sensing solution ph, cv measurements of different ua (79.4 µm) sensing solutions with different ph values (ph 8.2–9.4) were done using uox-cuo-cpes; one biosensor was used for each ph value. the optimized value was used for the next optimization. for the optimization of equilibration time, uox-cuo-cpes were equilibrated in ua sensing solution at different equilibration times (1–5 mins) then cv measurements of ua (79.4 µm) in bbs (ph 8.6) were done afterwards; one biosensor was used for each equilibration time. uric acid sensing procedure ua sensing using uox-cuo-cpe involves two steps. uox-cuo-cpe was first dipped into the sensing solution for an optimized period of time under stirring condition. after that, the solution immediately underwent electrochemical measurement for ua sensing. differential pulse voltammetry (dpv) was used as the electrochemical detection technique for ua sensing using the fabricated uox-cuo-cpe biosensor. the parameters used for dpv measurements were as follows: initial potential -0.1 v, end potential 0.8 v, scan rate 0.01 v/s, step potential 5 mv, modulation amplitude 25 mv, and modulation time 50 ms. the calibration curve was obtained by measuring the peak height (ip) from dpv measurements of sensing solutions with varying ua concentrations (10.0–79.4 µm). three (3) measurements were done for each concentration. after each dpv measurement, an oxidation potential (+0.7 v) via chronoamperometry (ca) in bbs (ph 9.0) was applied in order to re-oxidize remaining cu 2 o to cuo. this was done in order to ensure repeatable measurements. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 48 selectivity study a selectivity study was conducted in order to evaluate the uox-cuo-cpe response towards ua in the presence of possible interfering species. this was done by measuring uox-cuo-cpe response towards different solutions with varying compositions as follows: 39.6 µm ua, 39.6 µm ua with 39.6 µm creatinine, 39.6 µm ua with 39.6 µm xanthine, and 39.6 µm ua with 39.6 µm glucose. measurements were also done using cpe for comparison. ua analysis in synthetic urine the uox-cuo-cpe is intended to be used as a ua sensor in human urine. however, this study is limited to using synthetic urine as representative of human urine. synthetic urine was prepared with constituents similar to the formulated artificial human urine in a separate study (khan et al. 2017), and was used for ua analysis. in brief, the prepared synthetic urine has the following components: 9.3g/l urea, 0.670 g/l creatinine, 0.2 g/l kcl, 8 g/l nacl, 1.14 g/l na 2 hpo 4 , and 0.2 g/l kh 2 po 4 . 1 ml of synthetic urine sample was mixed with 50 ml 0.2 m borate buffer (ph 8.6) in 0.4 m nacl, then diluted with deionized water to produce 100 ml synthetic urine sensing solution. the resulting solution underwent ua measurement using the uox-cuo-cpe sensor. separate synthetic urine sensing solutions were spiked with ua at three different spiking levels (7.4 µm, 30 µm, and 55.8 µm). these synthetic urine sensing solutions were prepared by adding an appropriate amount of 10 mm ua in 0.1 m naoh (stock ua solution) to 1 ml of synthetic urine solution, just before dilution using deionized water. spiked samples also underwent ua measurement. a recovery study was done in order to assess the matrix effect towards ua sensing using uox-cuo-cpe. it was done by measuring the percent recovery in the spiked synthetic urine samples using the equation below. (1) a.g. e. buenaventura and a.c. c. yago 49 results and discussion fabrication and optimization of uox-cuo-cpe and its ua sensing performance the uox-cuo-cpe biosensor reported in this study was fabricated using cpe that was previously studied and optimized by our group (buenaventura and yago 2018). cuo particles were electrodeposited onto the surface of cpe, producing cuo-cpe. previous experiments revealed that cuo-cpe can respond towards h 2 o 2 via dpv measurements (buenaventura and yago 2020). this is because h 2 o 2 can reduce cuo to cu 2 o spontaneously. the cu 2 o can be oxidized back to cuo upon application of suitable oxidation potential. for the cuo-cpe to selectively respond with ua, the surface of cuo-cpe was further modified with uox. the resulting electrode will be termed here as uox-cuo-cpe. as shown in figure 2a, uox can catalyze the oxidation of ua, where h 2 o 2 is a byproduct. figure 2b shows the sensing mechanism of uoxcuo-cpe for ua detection. the main sensing process is based on the oxidation of ua into 5-hydroxyisourate (hiu) as catalyzed by uox, forming h 2 o 2 as byproduct, and then the h 2 o 2 redox reaction converts cuo to form cu 2 o; the amount of h 2 o 2 and hence ua in the sample is measured by the oxidative current measured for the conversion of cu 2 o back to cuo. figure 2. (a) reaction scheme of enzymatic oxidation of ua by uox. (b) sensing mechanism for ua detection using uox-cuo-cpe. electrochemical response of uox-cuo-cpe towards ua in order to assess the viability of uox-cuo-cpe for ua determination, cv measurements were done. figure 3 shows the cv measurements of ua using cuocpe and uox-cuo-cpe. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 50 figure 3. cv measurements of 79.4 µm ua in bbs (ph 9.0) using uox-cuo-cpe (cu deposition time: 25 s; cu oxidation time: 125 s; uox enzyme loading: 0.1 mg/ml) and cuo-cpe (cu deposition time: 25 s; cu oxidation time: 125 s). cv measurements were done at 10 mv/s scan rate. insets show the whole cv curves. the cv curve of ua using uox-cuo-cpe showed two oxidation peaks at ~ +0.225 v (peak i) and at ~ +0.425 v (peak ii). presence of two peaks indicates that ua is oxidized in two different routes, i.e. non-enzymatic (direct electrochemical oxidation) and enzymatic. the non-enzymatic oxidation is known since reduced ua is an electroactive species with an oxidation half-reaction shown in figure 4. on the other hand, cuo-cpe showed only one oxidation peak for ua positioned at ~ +0.225 v (peak iii), which has similar peak position with peak i using uox-cuo-cpe. thus, peak i can be assigned to the direct electrochemical oxidation of ua. peak ii can then be assigned to enzymatic oxidation of ua. figure 4. oxidation half-reaction of ua. a.g. e. buenaventura and a.c. c. yago 51 optimization of uox-cuo-cpe fabrication and sensing parameters in order to obtain the highest sensitivity for ua sensing, different parameters were optimized including: (1) enzyme loading, (2) sensing solution ph, and (3) equilibration time. for the optimization of uox enzyme loading, uox-cuo-cpes with different uox enzyme loading were used for cv measurements of ua in bbs solution. appendix figure a1 shows the optimization of enzyme loading uox-cuocpe and the cv curves of ua using fabricated uox-cuo-cpes with different enzyme loading. as shown in the figure, an increasing trend can be observed for ave. ip (for oxidation peaks corresponding to enzymatic oxidation of ua positioned at ~ +0.410 v) with increasing enzyme concentration from 0.02 mg/ml uox to 0.3 mg/ ml uox. this can be attributed to the increase in the number of uox units present on the electrode surface. further increasing the uox concentration dropcasted onto the electrode causes a significant drop in peak currents both for electrochemical oxidation of ua and cu 2 o. this can be due to an increase in non-conducting enzyme units on the electrode surface which hinders not only the direct electron transfer from ua, but also the diffusion of enzymatically produced h 2 o 2 to the nearest cuo particle. from these observations, the optimized uox concentration for the fabrication of uox-cuo-cpe biosensor is 0.3 mg/ml. for the optimization of sensing solution ph, equally fabricated uox-cuo-cpes were used to measure for cv measurements of ua in bbs solutions. appendix figure a2 shows the optimization graph for the sensing solution ph optimization and the cv curves of ua at different solution ph values using uox-cuo-cpe. as shown in the figure, the response of the uox-cuo-cpe towards sensing solutions with ph 8.2 and ph 8.4 showed significantly small ave. ip for cu 2 o oxidation, indicating that at these ph values, the biosensor has low activity to produce h 2 o 2 . further increasing the ph to 8.6, the biosensor showed a significant increase in ave. ip for cu 2 o oxidation. further increasing the sensing solution ph led to a significant decrease in peak current for cu 2 o oxidation. these observations are due to the fact that ua molecules are at monoionic form at ph 8.6, which are a natural substrate of uox (gabison et al. 2008). increasing the ph will further deprotonize the ua, converting it to 3,9 – dianion form of ua, which is not the natural substrate of uox. for the optimization of equilibration time, equally fabricated uox-cuo-cpes were used to measure ua in bbs (ph 8.6) at different equilibration times via cv. appendix figure a3 shows the optimization graph for the equilibration time optimization and the cv curves of ua at different equilibration time using uox-cuo-cpe. as shown in the figure, 3 minutes equilibration time showed the highest ave. ip. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 52 shorter equilibration times were observed to have low ave. ip, indicating low h 2 o 2 production. longer equilibration times resulted in decreased ave. ip, which was caused by increased production of allantoin that may competitively inhibit ua at the active site of uox (gabison et al. 2006). with these observations, the optimized equilibration time was chosen to be equal to 3 minutes. table 1 summarizes the obtained value for different parameters that were obtained in this study. table 1. optimized parameters for uox-cuo-cpe optimized parameter optimized value cu deposition time 20 s cu oxidation time 60 s enzyme loading 0.3 mg/ml sensing solution ph ph 8.6 equilibration time 3 mins electrochemical characterization of cpe, cuo-cpe, and uox-cuo-cpe in the fabrication of uox-cuo-cpe, uox and cuo were used as modifiers on cpe and both can affect the overall surface conductivity of the resulting biosensor. in order to assess the effect of both cuo and uox on the rate of electron transfer at the electrode–solution interface, cv and eis measurements were done using cpe, cuo-cpe, and uox-cuo-cpe. figure 5 shows the cv curves, nyquist plots, and circuit models using cpe, cuo-cpe, and uox-cuo-cpe, on bbs (ph 8.6) with ferrocyanide [fe(cn) 6 4-] redox probe. figure 5 shows the cv and nyquist plots using the cpe, cuo-cpe, and uox-cuocpe on bbs (ph 8.6) with fe(cn) 6 4redox probe. figure 5a shows the cv curves of fe(cn) 6 4using the three electrodes. using cpe, typical reversible redox peaks with similar peak heights were observed owing to the conducting nature of mwcnt on the electrode surface. for cuo-cpe, a quasi-reversible redox peak was observed. the oxidation of fe(cn) 6 4to fe(cn) 6 3is more favored as indicated by a significantly lower reduction peak height as compared to oxidation peak height. for uox-cuocpe, a significant decrease in peak heights for both reduction and oxidation peaks was observed. the decrease in peak heights can be attributed to the decrease in surface conductivity of the electrode due to the non-conducting nature of uox. a.g. e. buenaventura and a.c. c. yago 53 figure 5. (a) cv curves and (b) nyquist plots using cpe, cuo-cpe, and uox-cuo-cpe. the solution used was 5 mm k 4 [fe(cn) 6 ] in bbs (ph 8.6). cv measurements were done at 10 mv/s scan rate. eis measurements were done at frequency range 20 khz–0.1 hz at 0.35 v applied potential. (c) circuit model from eis measurements using cpe, cuo-cpe, and uox-cuo-cpe. chi-squared (χ2) values for the curve fitting are shown. as shown in figure 5b, nyquist plots of the three electrodes indicate similar electrochemical cell properties in the presence of a redox probe. all of the electrodes were observed to have both electrochemical circles with linear portion at lower frequencies. such pattern of a nyquist plot can be modelled using randles cell with warburg impedance element as shown in figure 5c. the diameter of the electrochemical circle is equal to the charge transfer resistance (r ct ) or polarization resistance (r p ), which is directly related to the rate of electron transfer at the electrode-solution interface. based on the circuit modelling, the r ct of cpe, cuo-cpe, and uox-cuo-cpe were determined to be at 5.90 kω, 7.01 kω, and 7.52 kω, respectively. the r ct of cuo-cpe is significantly higher to the r ct of cpe. this observation is in conformity with the cv measurements in figure 5a, where fe(cn) 6 4/fe(cn )6 3redox couple showed a quasi-reversible redox reaction using cuo-cpe. for uox-cuo-cpe, its r ct is significantly higher than the r ct of both cpe and cuo-cpe. this observation can again be attributed to the non-conducting nature of uox, which resulted in hindered direct electron transfer between fe(cn) 6 4 and uox-cuo-cpe. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 54 assessment of biosensor activity in order to assess the biosensor activity, the apparent michaelis constant (k m app) was determined via cv measurements of ua solutions with different ua concentrations using the optimized uox-cuo-cpe. the k m app of uox on the electrode surface was estimated using the hanes-woolf plot. appendix figure a4 shows the hanes-woolf plot for k m app determination and the cv curves for different ua concentrations used for the hanes-woolf plot. the k m app was determined at 41.5 µm. the calculated k m app for uox-cuo-cpe is much lower than some of the previous reports on ua biosensor involving uox (jindal et al. 2012; verma et al. 2019). lower k m app is more favorable for an enzyme-based biosensor as it ensures enhanced affinity of the substrate to the immobilized enzyme which leads to enhanced response. this is due to the inverse relationship of k m app and reaction rate (ν). aside from k m app, maximum velocity (v max ) of the biosensor was also determined using the hanes-woolf plot. the v max gives information on the rate of enzymatic catalysis. the measured v max of the uox-cuo-cpe is at 6.93 x 10-5 µm ua detected/ min. this is much lower than the reported uox activity in free solution (for the same amount of uox dropcasted onto the cuo-cpe surface) which is at 2.14 x 10-1 µm ua converted to allantoin/min. a possible reason for this observation is because ua molecules are limited only to diffuse towards the active sites of uox that are facing the electrode–solution interface. thus, uox in free solution is more active than adsorbed uox. determination of analytical merits for ua sensing using uox-cuo-cpe and differential pulse voltammetry (dpv) as sensing technique dpv measurements for ua were done using the optimized value for different parameters as previously shown in table 1. a calibration curve was done by measuring different solutions of ua with varying ua concentrations. figure 6 shows the calibration curve for ua detection using the fabricated uox-cuo-cpe and the dpv curves of ua solutions with different concentrations using optimized uoxcuo-cpe. a.g. e. buenaventura and a.c. c. yago 55 figure 6. (a) calibration curve for ua measurements using uox-cuo-cpe. (b) dpv curves of ua in bbs (ph 8.6) with different ua concentration using uox-cuo-cpe. three (3) measurements were done for each ua concentration. error bars represent standard deviation for three measurements. ip for each dpv measurement was measured at around +0.370 v. as shown in figure 6a, a linear correlation was found between ua concentration and ave. ip ranging from 10.0 µm to 79.4 µm (r2 = 0.9926). the limit of detection (lod) for ua detection using the uox-cuo-cpe was determined at 8.82 µm. table 2 shows the figures of merit for the ua measurement using uox-cuo-cpe. linear correlation analysis of the calibration curve was done. as shown in table 2, the r2 value for the linear regression is equal to 0.9926, indicating linear correlation of ave. ip with ua concentration. table 2. figures of merit for ua measurement using uox-cuo-cpe figures of merit value linear range 10.0 µm – 79.4 µm sensitivity 1.348 ± 0.067 na/ µm linearity (r2) 0.9926 limit of detection (lod) 8.82 µm limit of quantification (loq) 29.39 µm assessment of uox-cuo-cpe selectivity uox is the molecular recognition element of the uox-cuo-cpe biosensor for ua. thus, the main purpose of uox is to enhance the selectivity of the biosensor. in order to assess the selectivity of the fabricated biosensor, the electrochemical response of uox-cuo-cpe towards possible interferences must first be studied. different possible interferences were studied including the following: creatinine, xanthine, and glucose. creatinine was included in this selectivity test because of urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 56 its presence in human urine as well as having a slight similarity in structure with ua. xanthine was included in this selectivity test because of its high structural similarity with ua. lastly, glucose was included in this selectivity test also because of its presence in urine. urea was not included in this selectivity test because of its non-electroactive nature and it was shown in a previous study that urea did not significantly interfere with h 2 o 2 measurements using cuo-cpe measurements (buenaventura and yago 2020). figure 7 shows the cv curves for the mentioned compounds using uox-cuo-cpe. from the figure, cv measurements of the studied interferences using uox-cuo-cpe showed no appreciable oxidation peak at ~ +0.4 v. among the studied interferences, only xanthine gave a significant oxidation peak located at ~ +0.65 v, which is far from the measurement potential (~ +0.4v) for ua. figure 7. (a) cv curves of 39.6 µm ua, creatinine, xanthine, and glucose in bbs (ph 8.6), using uox-cuo-cpe. (b) chemical structures of ua, creatinine, xanthine, and glucose. scan rate at 10 mv/s. inset in (a) shows the whole cv curves. to further assess the selectivity of uox-cuo-cpe towards ua, different biomolecules (creatinine, xanthine, and glucose) were added into separate ua sensing solutions and were measured using uox-cuo-cpe. figure 8 shows the selectivity study for uox-cuo-cpe in comparison with cpe. based on figure 8a, uox-cuo-cpe showed a.g. e. buenaventura and a.c. c. yago 57 no significant difference in its electrochemical response with ua when added with other biomolecules. this is confirmed by single factor analysis of variance (anova) with calculated f (3.120) less than the f critical (4.066). appendix table b1 shows the biosensor responses and the anova table. for comparison, a selectivity study of cpe was also done. figure 8b shows that there is a significant difference with the response of cpe towards ua alone as compared with added biomolecules. this is confirmed by single factor anova with calculated f (16.355) larger than the f critical (4.066). appendix table b2 shows the cpe responses and the anova table. therefore, the presence of creatinine, xanthine, and glucose do significantly affect the response of cpe towards ua. these results thus proved the effectiveness of the uox enzyme as a molecular recognition element in the final biosensor. appendix figure a5 shows the dpv curves of ua, and ua with interferences, using uox-cuocpe and cpe. figure 8. selectivity study of ua determination via dpv using (a) uox-cuo-cpe and (b) cpe. three measurements (n=3) were done for each sensing solution combination. measurement values are shown above the bars with the format: average peak current (ave. ip) ± standard deviation for three measurements. ip for each dpv measurement was measured at around +0.370 v. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 58 assessment of uox-cuo-cpe reusability and fabrication repeatability using uoxcuo-cpe in a chemical sensor/biosensor research, it is important also to assess the reusability as well as the fabrication repeatability of the sensor being developed. in this study, a sensor reusability test was done by measuring ua over several repetitions (n=5) using uox-cuo-cpe. as shown in appendix table b3, the average response of uox-cuo-cpe towards 79.4 µm ua is at 113.6 ± 3.7 na. the % relative standard deviation (% rsd) was determined at 3.28%. for the fabrication repeatability, ua measurements were done using three separate uox-cuo-cpes. figure 9 shows the fabrication repeatability graph for the uox-cuo-cpes. as shown in the figure, the biosensor responses did not differ significantly. this is confirmed by single factor anova with calculated f (1.396), which is less than f critical (5.143). appendix table b4 shows the biosensor responses and anova table. figure 9. biosensor fabrication repeatability study for uox-cuo-cpe biosensors. measurements for 79.4 µm ua in bbs (ph 8.6) were done using three separate uox-cuo-cpe biosensors (labelled as a, b, and c). three measurements (n = 3) were done for each biosensor. measurement values are shown above the bars with the format: average peak current (ave. ip) ± standard deviation for the three measurements. ip for each cv measurement was measured at around +0.420 v. assessment of uox-cuo-cpe biosensor stability the stability of uox-cuo-cpe was also studied to provide information about its usability after a certain period of storage. the uox-cuo-cpe biosensor was tested for five-week stability in order to evaluate if it can give a similar response after storing it at a cold temperature (~ +4°c). figure 10 compares the mean responses of the biosensor before (0th week) and after five weeks (5th week) of storage. as shown in the figure, uox-cuo-cpe responses before and after five weeks of storage did a.g. e. buenaventura and a.c. c. yago 59 not differ significantly. this is confirmed by a two-sample t-test with calculated t equals to 0.156, which is smaller than the t crit (df = 2, α = 0.05) which is equal to 4.303. appendix table b5 shows the biosensor responses as well as t-test summary. figure 10. five-week biosensor stability test. three (3) measurements for 79.4 µm ua in bbs (ph 8.6) were done for each data set. measurement values are shown above the bars with the format: average peak current (ave. ip) ± standard deviation for three measurements. ip for each cv measurement was measured at around +0.450 v. assessment of applicability of uox-cuo-cpe on human urine using synthetic human urine in order to evaluate the effect of the matrix of human urine towards the ua measurement using uox-cuo-cpe, a recovery test was performed. table 3 shows the results of the recovery test for three spiking levels (7.4µm, 30µm, and 55.8µm). as shown in the table, the recoveries for three spiking levels range from 90.27% to 102.3% with an rsd of not more than 2.55%, indicating that uox-cuo-cpe is applicable for the quantification of ua in human urine. appendix figure a6 shows the dpv measurements of non-spiked and spiked synthetic urine. table 3. recovery test for uox-cuo-cpe using synthetic urine sample detected (µm) added (µm) found (µm) recovery rsd (n=3) 1 0.00 ± 0.00* 7.40 7.55 ± 0.19 102.03% 2.55% 2 0.00 ± 0.00* 30.00 27.08 ± 0.28 90.27% 1.05% 3 0.00 ± 0.00* 55.80 52.01 ± 0.30 93.21% 0.59% *no observable peaks urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 60 comparison of uox-cuo-cpe with other reported uox-based biosensors table 4 shows the analytical performance of other reported uox-based ua biosensors as compared with the fabricated uox-cuo-cpe. comparing uox-cuo-cpe with other reported biosensors, the uox-cuo-cpe biosensor has relatively low lod and k m app. in particular, uox-cuo-cpe showed a significant improvement in performance compared to uox/cuo/pt (jindal et al. 2012). it also has lower k m app compared to uox/au-rgo/ito (verma et al. 2019) despite having a more sophisticated electrode modifier. this improved performance of uox-cuo-cpe can be attributed to several factors including: 1) the use of cpe that is based on mwcnt (a highly conducting carbon allotrope (jacobs et al. 2010)), and 2) the use of the dropcast method of enzyme depositing rather than covalent attachment of enzyme which can lower the activity of an enzyme (feng and ji 2011). thus, the advantage of uox-cuo-cpe, aside from having facile steps of fabrication, is good analytical performance for ua determination. table 4. analytical performance of reported uox-based electrochemical biosensors for ua compared to uox-cuo-cpe enzyme-based electrochemical biosensor electrochemical technique of detection linear range limit of detection apparent michaelis constant (km app) reference uox/cuo/pt amperometry 50 μm – 1 mm 140 μm 0.12 mm (jindal et al. 2012) nafion/uox-hrpmesoporous silica/gce amperometry 2 μm – 12 μm 0.33 μm -(mundacauribe et al. 2014) nafion/uox/zno nanosheets/ag/si amperometry 50 μm – 2 mm 0.019 μm 0.026 mm (ahmad et al. 2015) graphene oxide – uox/gce amperometry 20 μm – 491 μm 3.45 μm -(omar et al. 2016) uox/poly(4aminosalicylic acid)/ prussian blue/ carbon graphite electrode amperometry 10 μm – 200 μm 3.0 μm -(da cruz et al. 2017) uox/bull serum albumin (bsa)/blgmwcnts-ptnps/glassy carbon (gc) electrode amperometry 0.02 mm – 0.5 mm 0.8 μm -(han et al. 2019) uox/au-rgo/ito differential pulse voltammetry 50 μm – 800 μm 7.32 μm 51.75 μm (verma et al. 2019) uox-cuo-cpe differential pulse voltammetry 10.0 μm – 79.4 μm 8.82 μm 41.46 μm (0.0415 mm) this work a.g. e. buenaventura and a.c. c. yago 61 conclusion an electrochemical enzyme-based biosensor for ua determination was developed and optimized in this study. a carbon paste electrode (cpe) and its pretreatment, which were previously optimized by our group in separate studies (buenaventura et al. 2016; buenaventura and yago 2018), was modified further with cuo particles and a uox enzyme to fabricate a biosensor that is sensitive and selective towards ua. cv measurements proved the retention of the capability of the uox enzyme to catalyze ua oxidation after being dropcasted on the surface of cuo-cpe. the presence of uox on the uox-cuo-cpe resulted in an increase in r ct as compared to cuo-cpe and cpe due to the non-conducting nature of uox. several parameters, including uox enzyme loading, sensing solution ph, and equilibration time, were optimized. dpv measurements of ua using uox-cuo-cpe showed a linear correlation between ave. ip and ua concentration. the lod for ua measurement using uox-cuo-cpe was determined at 8.82 µm. uox-cuo-cpe showed improved performance over some reported studies, which mainly could be due to the use of mwcnt-based cpe and the use of a non-covalent approach of immobilizing the uox onto the electrode surface. because of the presence of uox as a selective modifier, uox-cuocpe was also proven to be selective towards ua even in the presence of creatinine, xanthine, and glucose. furthermore, uox-cuo-cpe was also proven to be reusable, reproducible, and stable even after five weeks of storage. results of a recovery test confirmed the applicability of uox-cuo-cpe for ua determination in human urine. acknowledgments this research was supported by the natural sciences research institute (nsri) of the university of the philippines diliman [che-17-1-01] and the department of science and technology (dost), philippines. references ahmad r, tripathy n, jang nk, khang g, hahn y-b. 2015. fabrication of highly sensitive uric acid biosensor based on directly grown zno nanosheets on electrode surface. sens actuators b chem. 206:146-151. arslan f. 2008. an amperometric biosensor for uric acid determination prepared from uricase immobilized in polyaniline-polypyrrole film. sensors (basel, switzerland). 8(9):5492-5500. ben-ishay d, dreyfuss f, ullmann td. 1961. fanconi syndrome with hypouricemia in an adult: family study. am j 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buenaventura and a.c. c. yago 65 ______ allan christopher c. yago <acyago@up.edu.ph>is currently an associate professor in the institute of chemistry up diliman. he took his b.s., m.s., and ph.d. in chemistry in the institute of chemistry, up diliman, from 2001 to 2014. his main research interests are on the preparation and characterization of sensor materials from polymers, nanoparticles, and composites, electrochemistry and electro-analytical methods, molecular imprinting on polymers, and anti-corrosion coatings. he heads the sensor materials development (smd) lab located in ic research building room 321-322. angelo gabriel e. buenaventura is currently a research assistant in the institute of chemistry up diliman. he took his m.s. chemistry degree in the institute of chemistry, up diliman, from 2015 to 2020. he got his b.s. chemistry degree in the college of science, university of santo tomas, from 2010 to 2014. his main research interest is on the development and fabrication of chemical sensors and biosensors for various chemicals including environmental pollutants and important health – related biochemical compounds. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 66 appendix figure a1 (a) optimization graph for uox loading optimization. three (3) measurements were done for each enzyme concentration. error bars represent standard deviation for three measurements. peak currents (ip) were measured at ep = ~ +0.410 v. (b) cv curves of 79.4 µm ua in bbs (ph 9.0) using uox-cuo-cpes which were fabricated by drop casting uox solution with different uox concentrations; scan rate at 10 mv/s, 3 minutes equilibration time, 20 s cu deposition time, 60 s cu oxidation time. inset shows the whole cv curves. a.g. e. buenaventura and a.c. c. yago 67 appendix figure a2 (a) optimization graph for sensing solution ph. three (3) measurements were done for each ph. error bars represent standard deviation for three measurements. ip for each cv measurement was measured at around +0.400 v. (b) cv curves of 79.4 µm ua in bbs (varying ph) using uox-cuo-cpe; scan rate at 10 mv/s, 3 minutes equilibration time, 20 s cu deposition time, 60 s cu oxidation time, 0.3 mg/ml enzyme loading. inset shows the whole cv curves. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 68 appendix figure a3 (a) optimization graph for equilibration time. three (3) measurements were done for each equilibration time. error bars represent standard deviation for three measurements. ip for each cv measurement was measured at around +0.400 v. (b) cv curves of 79.4 µm ua in bbs (ph 8.6) using uox-cuo-cpe; scan rate at 10 mv/s, varying equilibration time, 20 s cu deposition time, 60 s cu oxidation time, 0.3 mg/ml enzyme loading. inset shows the whole cv curves. a.g. e. buenaventura and a.c. c. yago 69 appendix figure a4 (a) hanes-woolf plot for determination of k m app. three (3) measurements were done for each ua concentration. error bars represent standard deviation for three measurements. ip for each cv measurement was measured at around +0.400 v. (b) cv plots of solutions with different ua concentration using uox-cuo-cpe; scan rate at 50 mv/s. inset shows the whole cv curves. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 70 appendix figure a5 dpv curves of blank (bbs; ph 8.6), ua, ua + creatinine, ua + xanthine, and ua + glucose, using uox-cuo-cpe (a) and cpe (b). a.g. e. buenaventura and a.c. c. yago 71 appendix figure a6 dpv curves of non-spiked synthetic urine sensing solution and synthetic urine sensing solutions spiked with varying amounts of ua; uox-cuo-cpe was used as working electrode. urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 72 appendix table b1 uox-cuo-cpe electrochemical response values for different sensing solution combinations and single factor anova table uox-cuo-cpe electrochemical responses sensing solution trial 1 trial 2 trial 3 average ua 5.06e+01 5.81e+01 5.82e+01 55.62033 ua + creatinine 6.05e+01 6.03e+01 5.98e+01 60.19333 ua + xanthine 6.43e+01 6.03e+01 5.98e+01 61.47167 ua + glucose 5.37e+01 5.69e+01 5.86e+01 56.384 single factor anova source of variation ss df ms f p-value f critical between groups 73.32234 3 24.44078 3.119790847 0.088047 4.066181 within groups 62.67287 8 7.834109 total 135.9952 11 a.g. e. buenaventura and a.c. c. yago 73 appendix table b2 cpe electrochemical response values for different sensing solution combinations and single factor anova table cpe electrochemical responses sensing solution trial 1 trial 2 trial 3 average ua 1.58e+02 1.39e+02 1.52e+02 149.3333 ua + creatinine 4.42e+01 5.48e+01 5.86e+01 52.55067 ua + xanthine 1.09e+02 4.33e+01 3.38e+01 62.14367 ua + glucose 4.41e+01 3.05e+01 3.07e+01 35.06833 single factor anova source of variation ss df ms f p-value f critical between groups 23367.11 3 7789.036 16.355 0.0008956 4.066181 within groups 3809.983 8 476.2479 total 27177.09 11 urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 74 appendix table b3 peak current (ip) for several trials of ua measurement using uox-cuo-cpe trials ip (na) 1 111.7 2 111.4 3 115.8 4 119.0 5 110.0 ave ip 113.6 sd (n=5) 3.7 % rsd 3.28 *cv technique was used **ip was measured for each cv measurement at around +0.410 v a.g. e. buenaventura and a.c. c. yago 75 appendix table b4 uox-cuo-cpes electrochemical responses towards ua and single-factor anova table uox-cuo-cpe electrochemical responses biosensor trial 1 trial 2 trial 3 average a 9.40e+01 1.13e+02 1.01e+02 96.95367 b 9.51e+01 9.72e+01 9.61e+01 103.8063 c 1.02e+02 1.02e+02 9.44e+01 97.32 single factor anova source of variation ss df ms f p-value f critical between groups 89.16576 2 44.58288 1.396001 0.317827 5.143253 within groups 191.6169 6 31.93614 total 280.7826 8 urate oxidase (uox)-copper oxide (cuo)-carbon polymer composite electrode 76 appendix table b5 uox-cuo-cpe electrochemical responses for ua before and after 5 weeks of storage and t-test summary uox-cuo-cpe responses trial 0th weekip (na) 5th week ip (na) 1 2.40e+02 1.90e+02 2 2.06e+02 2.13e+02 3 2.00e+02 2.11e+02 t-test summary 0th week ip (na) 5th week ip (na) difference average 2.15e+02 2.05e+02 1.07e+01 sd 21.44285 12.6609281 sd2 459.7956 160.2991 sd2/n 153.2652 53.4330333 df 2 t 0.747 t crit (95%) 4.303 11subscription form.pmd method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman-ovcrd)  money remittance (payable to narita e.c. de las alas, c/o ovcrd research dissemination and utilization office, with office address as indicated above and mobile phone no. 09209605857) subscriber details name/institution _________________________________________________________________________________________________________ contact person (for institutional subscribers) _____________________________________________________________________________________________ mailing address _____________________________________________________ email 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(adapted with permission from the editors of ieee sensors journal) 89 guidelines for expanding conference papers for submission to science diliman science diliman welcomes conference paper submissions provided they have been updated and expanded. below is a checklist of required and suggested actions for authors. mandatory actions 9 if changes are made, choose a new title for the paper. 9 use feedback obtained at the conference to update, revise, and rewrite the paper as appropriate to improve its overall quality. 9 reference your conference paper in the appropriate locations. 9 include a footnote in the submitted manuscript stating, e.g., “an earlier version of this paper was presented at the 20xx technical conference and was published in its proceedings.” 9 indicate in a letter (upload as a supporting document during the submission process) whether the conference paper was peer-reviewed and clearly state what has been changed. 9 provide the original conference paper (upload a pdf file during the submission process). 9 if the conference organizers or professional society hold the copyright for your conference paper, obtain permission to reprint figures and tables that are used in the expanded paper. recommended actions 9 expand the background section and include additional references. 9 include novel scientific content and expanded descriptions of procedures. 9 provide data that was not published at the conference. 9 revise and update figures and text to avoid exact duplication of the conference proceedings. (adapted with permission from the editors of ieee sensors journal) 53 an updated inventory and habitat association analysis of the non-avian vertebrates of the university of the philippines (up) diliman romel v. pasumbal jr.* jelaine l. gan institute of biology, college of science university of the philippines diliman geoffrey jules n. solidum nappy l. navarra college of architecture, college of science university of the philippines, diliman abstract an inventory of the non-avian terrestrial vertebrate species found within the 493-hectare land area of the up diliman campus is presented. visual encounter surveys for amphibians and reptiles, as well as mist-netting and trapping for mammals, were conducted last august 2019 to early february 2020 on selected study grids on campus. to determine habitat associations, the species richness of each vertebrate class (i.e., amphibia, reptilia, and mammalia) was analyzed with habitat characteristics of the grid using regression analysis. based on the surveys and recent records (2015 onwards) in literature, a total of 33 species were recorded: seven amphibians, 15 reptiles, and 11 mammals. comparison with historical records from 1998 revealed that an additional two amphibian species, seven reptile species, and six mammalian species have been sighted within the area since 2015. however, a fork-tongued frog, falling under the genus fejervarya, and four reptilian species that had previously been recorded within the study sites were not observed. habitat association analysis revealed that building area is correlated with species richness, with reptilian species richness being positively correlated with it. overall, this study shows that the up diliman campus supports considerable urban biodiversity despite recent developments. keywords: urban biodiversity, amphibian diversity, reptile diversity, mammal diversity * corresponding author science diliman (january-june 2022) 34:1, 53-70 sd june2022_pasumbal.indd 53 7/29/2022 4:12:49 pm an updated inventory and habitat association analysis of the non-avian vertebrates 54 introduction the global increase of the human population lends itself to rapid urbanization, with urbanized areas becoming the most rapidly expanding habitat type worldwide (faeth et al. 2011). currently, 55% of the world’s population resides in urban areas, and this number is expected to increase to 68% by 2050 (united nations 2019). this rapid urbanization entails the conversion of natural green spaces into manmade infrastructures (e.g., roads, houses) and fragmentation of habitats, which are associated with biodiversity loss and environmental degradation (müller et al. 2013). amidst these threats, pockets of green spaces serve as refuges where flora and fauna can thrive within urban landscapes (pickett et al. 2001). urban ecosystems, along with their associated biodiversity (nilon 2011), provide numerous ecosystem services (edwards et al. 2020). these services are derived from the interactions between different species and between species and their environment, such as seed dispersal, pollination, pest control, and the like (montoya et al. 2012). with most people now living in cities, these biodiversity-derived services are influenced by anthropogenic activities, with alterations to species composition, abundances, richness, and evenness tied to changes in their habitat (faeth et al. 2011; nilon 2011). the university of the philippines diliman (upd) campus is one of the last remaining green spaces in metro manila. the 493 hectares of land within its bounds provide habitats for a diversity of plant and animal species within the surrounding ‘sea’ of urban infrastructure. a survey conducted by ong et al. (1999) revealed the campus to be home to over 50 unique vertebrate fauna including six amphibian species, nine reptilian species, 38 bird species, and eight mammalian species. after more than a score, these numbers have become outdated given new species records and potential species loss (vallejo and aloy 2014). much of the landscape has changed as well, with many new infrastructures and other campus developments. this study aims to provide an updated inventory of non-avian vertebrate fauna found within the up diliman campus, as well as to determine general specieshabitat associations. this would highlight the importance of green spaces for wildlife as well as provide information that can guide land-use management and maintenance practices of the campus to better conserve urban biodiversity. sd june2022_pasumbal.indd 54 7/29/2022 4:12:49 pm r. v. pasumbal jr. et al. 55 methodology the 493-hectare upd campus is located in quezon city, national capital region. the city ranks as the most populous among the 16 highly urbanized cities in the region based on the 2020 census (philippine statistics authority 2021). the campus was acquired in 1939 but was officially used a decade after when the administration was transferred from the old campus in manila (university of the philippines diliman n.d.a). since then, numerous buildings have been built to support the university’s functions, and the campus now has more than 900 buildings (university of the philippines diliman n.d.b). development has been guided by the 2012 land use plan (appendix 1) wherein 28% of the campus’ land area has been dedicated to academic or academic support units (137.70 ha), 22.5% has been dedicated for residential use (110.87 ha), 4.4% for the campus core (21.66 ha), and 3.7% designated as a protected forest area (18.25 ha) (university of the philippines diliman n.d.b). the campus map was divided into study grids measuring 250 m by 250 m in qgis (figure 1). selected grids were surveyed for the presence of non-avian vertebrate figure 1. dominant habitat map of up diliman campus (campus boundary outlined in dark red lines). the 58 study grids (in 250 m by 250 m spacing) were color-coded based on the dominant plant and building features observed within: built area (grey), grassland (yellow), grassy open forest (pale green), layered open forest (dark green), urban open forest (brown), cropland (pale yellow). white lines signify roads. landmark areas are indicated by color coded pins: up lagoon (blue), palma hall (orange), romulo hall (red), up msi (pale yellow), binhi arboretum (purple), and national science complex (green). sd june2022_pasumbal.indd 55 7/29/2022 4:12:49 pm an updated inventory and habitat association analysis of the non-avian vertebrates 56 fauna and classified into habitat types based on vegetation composition and built area coverage. the grids located across commonwealth avenue (i.e., up arboretum, up-ayala land technohub) and katipunan avenue (up town center) were not included due to time and logistic constraints. sampling events were conducted from august 2019 to early february 2020. habitat sampling in each grid, the dominant plant feature (e.g., grasses, shrubs, and trees) was determined through on-site surveys and inspection of satellite images. additional information on each grid’s canopy cover and building area was measured using satellite imagery through google earth (google earth pro 2017). these were then used to classify the grids into the following vegetation cover classification types: built area – heavily modified areas with high building footprint (i.e., 1-2 story infrastructures) and with minimal vegetation (e.g., potted plants, scattered trees) around the infrastructure cropland – agricultural spaces mostly planted with low crops (i.e., rice, vegetables) grassland – open spaces with scattered small shrubs and extensive grass cover grassy open forest – vegetated areas dominated by large trees with a few understory layer/s and abundant groundcover layered open forest – vegetated areas with multiple structural layers consisting of trees, shrubs, and groundcover urban open forest – vegetated areas with multiple layers of trees, shrubs, and groundcover interspersed with low-level infrastructures wildlife survey amphibian, reptilian, and mammalian species records were obtained through a series of grid surveys. these were supplemented with data collected through collating recent wildlife records dating from 2015 to 2021. the sources included student thesis studies, class field exercises (i.e., biology courses), social media (i.e., the up wild facebook page), and other citizen science platforms (e.g., inaturalist). such reports were verified through photographs and/or specimen examination. grids lacking in data from these supplemental sources were prioritized in the survey effort, leading to a total of 26 grids sampled for amphibians, 23 grids for reptiles, and 15 grids for mammals. sampling was only done during the wet season, sd june2022_pasumbal.indd 56 7/29/2022 4:12:49 pm r. v. pasumbal jr. et al. 57 as the covid-19 pandemic prevented us from conducting the dry season sampling activities. amphibians and reptiles were surveyed using time-constrained opportunistic searches at night (bennett 1999). at least one man-hour of survey effort was spent in each grid. on the other hand, bats were sampled using mist nets (hoffmann et al. 2010). two mist nets were opened in each grid from 1800h to 2100h for a total of six net hours per grid. on the other hand, non-volant mammals were captured using cage traps. five cage traps per grid were baited with bread with peanut butter. traps were left deployed for two nights, for a total of 10 trap nights per grid, but were checked and rebaited as needed daily. captured mammals were released after species identification. species encounters outside of the survey period were also noted as off-survey data. identification was aided using field guides (alcala and brown 1998; das 2015; heaney et al. 2016). experts from the up diliman institute of biology verified for uncertain identification. species-habitat association species richness per grid derived from these records was then compared against the grids’ respective habitat type, canopy cover, and building area through glm analysis with poisson error distribution using rstudio (rstudio team 2020). additionally, the species richness per class (i.e., amphibia, reptilia, mammalia) was also analyzed in relation to canopy cover and building area. results & discussion a total of 33 unique non-avian vertebrate species comprising six amphibian families, seven reptilian families, and five mammalian families were present across 58 grids of six different habitat classifications within the campus. habitat sampling the study site was a mosaic of habitat types (figure 1). the majority of the 58 grids were classified as layered open forests (29.3%), grassy open forests (22.4%), or built areas (22.4%). there were relatively few grids representing grasslands (14.0%), urban open forest (6.9%), and cropland (5.2%). sd june2022_pasumbal.indd 57 7/29/2022 4:12:49 pm an updated inventory and habitat association analysis of the non-avian vertebrates 58 wildlife inventory this study provided an updated campus inventory of non-avian vertebrate species (table 1). a total of 33 species was recorded, which includes seven amphibians, 15 reptiles, and 11 mammals. compared to the ong et al. (1999) study, 20 new species records were added while seven species were not recorded here (table 2). field surveys alone revealed a total of 16 non-avian vertebrate species within the campus. additional records were obtained from the literature review (table 2). table 1. compiled list of non-avian vertebrate species found within the upd campus from survey efforts, conducted from august 2019 to early february 2020 (indicated by asterisk), and literature review from 2015-2021 (items without an asterisk) family common name scientific name amphibians bufonidae cane toad * rhinella marina dicroglossidae chinese edible frog * hoplobatrachus rugulosus common puddle frog * occidozyga laevis ‎eleutherodactylidae greenhouse frog * eleutherodactylus planirostris microhylidae banded bullfrog * kaloula pulchra ranidae common green frog * hylarana erythraea rhacophoridae common tree frog * polypedates leucomystax reptiles trionychidae chinese softshell turtle pelodiscus sinensis ‎emydidae red-eared slider trachemys scripta elegans geoemydidae southeast asian box turtle cuora amboinensis gekkonidae tender-skinned gecko gehyra mutilata brooke’s house gecko * hemidactylus brookii common house gecko * hemidactylus frenatus flat-tailed house gecko hemidactylus platyurus tokay gecko gekko gecko scincidae common sun skink eutropis multifasciata northern philippine sun skink eutropis borealis ‎typhlopidae brahminy blind snake * indotyphlops braminus colubridae gervais’ worm snake * calamaria gervaisii island wolf snake * lycodon capucinus sd june2022_pasumbal.indd 58 7/29/2022 4:12:49 pm r. v. pasumbal jr. et al. 59 northern triangle-spotted snake cyclocorus lineatus philippine rat snake coelognathus erythrurus mammals pteropodidae cave nectar bat * eonycteris spelaea geoffroy’s rousette rousettus amplexicaudatus greater musky fruit bat * ptenochirus jagori lesser short-nosed fruit bat * cynopterus brachyotis long-tongued fruit bat macroglossus minimus ‎emballonuridae black-bearded tomb bat taphozous melanopogon vespertilionidae java pipistrelle pipistrellus javanicus lesser asiatic yellow bat scotophilus kuhlii sciuridae finlayson’s squirrel callosciurus finlaysonii muridae asian house rat * rattus tanezumi asian house shrew suncus murinus table 2. a compiled list of amphibians, reptiles and mammals found within up diliman from 1997-1998 (ong et al. 1999), 2019-2020 survey data, and 2015-2021 literature data species oct 1997 aug 1998 (ong et al. 1999) this study aug 2019 feb 2020 surveys 2015-2021 literature data amphibians rhinella marina x x hylarana erythraea x x fejervarya sp. x hoplobatrachus rugulosus x x polypedates leucomystax x x occidozyga laevis x x eleutherodactylus planirostris x kaloula pulchra x reptiles table 1. compiled list of non-avian vertebrate species found within the upd campus from survey efforts, conducted from august 2019 to early february 2020 (indicated by asterisk), and literature review from 2015-2021 (items without an asterisk) (cont’n.) sd june2022_pasumbal.indd 59 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 60 gekko gecko x off census encounter 2021, class field exercise 2018 hemidactylus frenatus x x hemidactylus stejnegeri x class field exercise 2018 hemidactylus platyurus x class field exercise 2018 eutropis multifasciata x class field exercise 2019 naja philipinensis x rhabdophis spilogaster x pelodiscus sinensis class field exercise 2018 trachemys scripta elegans class field exercise 2018 cuora amboinensis class field exercise 2018 gehyra mutilata off census encounter 2021 eutropis borealis class field exercise 2018 cyclocorus lineatus class field exercise 2019 coelognathus erythrurus verified via photograph 2018 hemidactylus brookii x indotyphlops braminus x calamaria gervaisii x lycodon capucinus x mammals cynopterus brachyotis x x ptenochirus jagori x x rousettus amplexicaudatus x class field exercise 2017 eonicteris spelaea x x suncus murinus x off census encounter 2021, class field exercise 2019 rattus norvegicus x rattus exulans x macroglossus minimus thesis study (abdao, 2019) taphozous melanopogon class field exercise 2018 pipistrellus javanicus class field exercise 2018 scotophilus kuhlii class field exercise 2018 callosciurus finlaysonii verified via video 2020 rattus tanezumi x *previously misidentified as limnonectes macrocephalus table 2. a compiled list of amphibians, reptiles and mammals found within up diliman from 1997-1998 (ong et al. 1999), 2019-2020 survey data, and 2015-2021 literature data (cont’n.) sd june2022_pasumbal.indd 60 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 61 amphibians there are seven amphibian species found within the campus. all species were observed during the surveys and they belong under the families of true toads (bufonidae), forked-tongue frogs (dicroglossidae), rain frogs (eleutherodactylidae), narrow-mouthed frogs (microhylidae), true frogs (ranidae), and tree frogs (rhacophoridae). introduced species dominated the campus amphibian fauna. out of the seven species, only the common tree frog (p. leucomystax) and the common puddle frog (o. laevis) are native to the country. two new exotic species, the banded bullfrog (k. pulchra) and the greenhouse frog (e. planirostris) (diesmos et al. 2015) have successfully established populations on campus and were encountered frequently in different sites. the former was first reported in luzon in 2003 and was believed to have been brought by the pet trade or as a cargo stowaway (pili et al. 2019). its presence is conspicuous, especially during breeding season with its loud calls. on the other hand, the e. planirostris was first reported in quezon city in 2014 as a stowaway in the plant trade (sy et al. 2015), and by 2018 it was already common in the campus. this influx of new species was accompanied by a loss of a previous, potentially endemic, species. ong et al. (1999) reported the presence of the luzon fanged frog (limnonectes macrocephalus) within the up arboretum. however, the accompanying photograph of the species was later identified as fejervarya sp. (p. kim, personal communication with author, february 2021). two frogs under this genus, f. moodiei and f. vittigera, are known to occur in freshwater marshes and wetlands in luzon (diesmos et al. 2015). regardless, neither of the two fejervarya species nor l. macrocephalus were encountered in our surveys or in other recent amphibian surveys (roño 2015). the loss of this species may be attributable to a loss of suitable habitat within the campus. a study by villasenor et al. (2017) similarly correlates a decrease in frog species richness with habitat disturbance brought about by urban development. changes in their aquatic habitat (water body size, aquatic vegetation), as well as urbanization of the terrestrial environment within one kilometer of that aquatic habitat, have been shown to impact the presence of different frog species. the streams around which they would usually be found could have been cemented over with the construction of ripraps, leading to the species’ eventual disappearance. reptiles the reptilian community had the highest species richness (15 sp.) among the vertebrate classes. it is composed of three turtles, five geckos, two skinks, and five snakes. of which, three snakes and two geckos were observed during the surveys. sd june2022_pasumbal.indd 61 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 62 of the three turtle species in the campus found in habitats near water bodies, only the southeast asian box turtle (c. amboinensis) is native. it was photographed from a man-made pond at the palma hall and near a stream at the ib-edc binhi threatened species arboretum. meanwhile, the red-eared slider (t. scripta elegans) and the chinese softshell turtle (p. sinensis) were recorded within the national science complex. the presence of the t. scripta elegans in the wild was verified from a specimen that was unexpectedly caught in a pitfall trap last 2018, while the p. sinensis was recorded as a dead specimen in 2018 and alive in 2019. both turtles are introduced species, with the former most likely introduced from the pet trade and the latter from the food trade (sy 2015). for the geckos, all five species are known to be commensals or associated with human settlements (bowles et al. 2019; lwin et al. 2019; lwin et al. 2021; wogan et al. 2021a; wogan et al. 2021b). they were abundant in roadside trees and artificial structures, particularly on surfaces near lighting fixtures. these areas served as their hunting grounds for insects that were attracted to the light (zozaya et al. 2015). of the five, only two species, the brooke’s house gecko (h. brookii) and the common house gecko (h. frenatus), were verified during survey efforts. the h. brookii was identified by its numerous tubercles on the dorsum, while the h. frenatus was distinguished by its cylindrical tail with serrations. outside of the surveys, the authors also identified the tender-skinned gecko (g. mutilata) based on its smooth-looking skin and relatively wider tail base. the presence of the tokay gecko (g. gecko) was confirmed through its distinct call and visual encounter at the up arboretum in 2021. during the day, diurnal lizards were represented by two native skink species. the endemic northern philippine sun skink (e. borealis) was added to the list, confirmed through visual encounters and specimen examination. along with the many-lined sun skink (e. multifasciata), the two skinks were often encountered while basking. lastly, a diversity of snakes inhabits the campus. three species were recorded during the field survey, while the rest were confirmed from chance visual encounters and photographic evidence. both the brahminy blind snake (i. braminus) and the gervais’ worm snake (c. gervaisii) were seen under flowerpots or plant debris on the ground. they were found at vegetated sites near up lagoon and at the binhi arboretum. the blind snake, as well as the oriental wolf snake (l. capucinus), are non-endemic species that are common in urban areas (wogan and chan-ard, 2012; leviton et al. 2018). on the other hand, the campus is also home to two endemic snakes, the philippine rat snake (c. erythrurus) and the northern triangle-spotted snake (c. lineatus). the former was seen near buildings and houses (e.g., area 2, institute of sd june2022_pasumbal.indd 62 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 63 biology), while the latter was seen at the layered open forest site beside the marine science institute (msi). all snakes recorded on campus were either non-venomous or mildly venomous. a few reptiles were not recorded again since the 1998 inventory. the philippine cobra (naja philippinensis) and the northern water snake (rhabdophis spilogaster) were neither encountered nor reported within the last decade. for lizards, the stejneger’s leaf-toed gecko (hemidactylus stejnegeri) and skinks under the genus sphenomorphus were similarly absent. geckos are difficult to distinguish without close examination of features. more species are possibly found on campus that have yet to be included in this list. mammals the mammalian community of the campus is composed of eight volant and three non-volant small mammal species. of which, three fruit bat species and one invasive non-volant mammal species were captured during the field survey. nevertheless, all previously known mammal species were recorded again in recent literature except for the invasive norwegian rat (rattus norvegicus). all bats recorded on campus were native species. among the fruit bat family (pteropodidae), only the greater musky bat (p. jagori) is endemic to the country. this species and the lesser short-nosed fruit bat (c. brachyotis) are the two most captured bats on campus. they were recorded in various habitats and even in areas of high human disturbance for as long as there were fruits and figs available. both species have also been seen roosting in old buildings and trees. the other frugivorous and/ or nectarivorous bats, however, were captured less frequently. the cave nectar bat (e. spelaea) and geoffroy’s rousettes (r. amplexicaudatus) are cave-dwelling species (waldien et al. 2019; waldien et al. 2020). between the two, the r. amplexicaudatus was rarer and last recorded in 2017 during a class exercise in ecology at the binhi arboretum. given the absence of caves on campus, the encountered individuals were likely passing by or foraging on campus. the long-tongued fruit bat (m. minimus) was caught a handful of times at the binhi arboretum during a student thesis study (abdao 2019). the presence of fruiting trees on the campus explains the presence of these bat species. maintaining and improving plant diversity can further promote urban bat diversity (threlfall et al. 2016). the four insect bat species are likewise native and represent two families (emballonuridae and vespertilionidae). although the surveys failed to capture any of the insect bats, the authors have captured the java pipistrelle (p. javanicus), the lesser asiatic yellow bat (s. kuhlii), and the black-bearded tomb bat (t. melanopogon) sd june2022_pasumbal.indd 63 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 64 during ecology and vertebrate biology class exercises (table 2). a small colony of t. melanopogon was also observed to be roosting at an old building, the romulo hall. in contrast, the non-flying mammals in the list were all introduced species. only the asian house rat (r. tanezumi) was recorded in the surveys. the presence of the shrew was confirmed from a few dead specimens at the national science complex, while the finlayson’s squirrel (c. finlaysonii) was caught in a video near the marine science institute by the security guard. the squirrel is native to thailand, cambodia, laos, and vietnam but was introduced to other countries, including the philippines, through the pet trade (bertolino 2009). due to their high adaptability to urban habitats, these squirrels are considered ‘high-risk’ species and have the potential to become invasive once populations have been established (bertolino and lurz 2013). species-habitat association the 16 species encountered during the survey period were found across five different habitat types, namely built areas, grasslands, grassy open forests, layered open forests, and open urban forests (table 3). table 3. abundance of non-avian vertebrate species encountered during survey efforts within up diliman across different habitat types species habitat types built area grassland grassy open forest layered open forest open urban forest amphibians e. planirostris 4 0 47 21 1 h. rugulosus 1 0 0 0 1 h. erythraea 0 1 1 3 0 k. pulchra 9 10 7 10 0 o. laevis 0 16 0 0 0 p. leucomystax 14 1 1 13 1 r. marina 22 69 92 108 1 reptiles c. gervaisii 0 0 0 0 1 h. brookii 10 2 7 8 2 h. frenatus 26 7 34 40 7 i. braminus 1 0 0 1 0 l. capucinus 0 0 1 0 0 mammals c. brachyotis 4 15 6 14 11 e. spelaea 1 1 0 0 0 p. jagori 3 17 1 2 3 r. tanezumi 2 0 0 2 1 sd june2022_pasumbal.indd 64 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 65 overall species richness per grid was analyzed across the different habitat types. built areas and grassy open forests, on average, show lower species richness compared to the other habitat classifications (figure 2). however, results revealed no significant differences on the total vertebrate species richness of the grid between habitat types (kruskal-wallis test, p-value = 0.9021). this also suggested that all habitat types are equally important in supporting urban vertebrate diversity. figure 2. comparison of species richness among amphibians, reptiles, and mammals encountered during the august 2019 – february 2020 survey efforts per grid across the five habitat classifications of the up diliman study site. species richness was also examined against grid characteristics, canopy area, and building area. the number of species per grid was not influenced by canopy cover (kruskal-wallis test; p-value = 0.88). examining species richness against building area, however, revealed a positive association (glm; p-value<0.0001). upon further analysis at the taxonomic class level, only the reptilian species richness was affected by building area (glm; p-value= 0.0033). this may be attributed to the high abundance of h. frenatus around buildings (ota and whittaker 2010). both amphibian and mammalian species richness showed non-significant relationship with building area (p-value = 0.1 and p-value = 0.44, respectively). this lack of a significant correlation between habitat type and species richness may be explained by several factors. the assemblage of species found within the campus, particularly the non-native and invasive species, may be generalists which occupy a high diversity of habitats and are less sensitive to habitat disturbance than species restricted to a smaller number of habitats (segura et al. 2007). it is also possible other factors have a greater effect on species richness within the campus. sd june2022_pasumbal.indd 65 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 66 a study by vallejo et al. from 2008 for example, has found that bird abundance is more affected by the number of spatial entities (i.e., the number of buildings and trees) as compared to spatial area (i.e., building area and canopy cover), which was used in this study. a similar pattern may be observed in the abundance of other non-avian vertebrate taxa. conclusions with globally increasing urbanization rates, green urban spaces are important centers of biodiversity that are increasingly becoming threatened. the up campus has undergone and is continuing to undergo a transformation in the form of further urban development projects. this has consequently impacted the different species residing within the campus. a frog under the fejervarya genus, the endemic snakes naja philippinensis, and rhabdophis spilogaster, as well as the gecko hemidactylus stejnegeri and skinks under the sphenomorphus genus, are all previously recorded species that were not observed in this study. change is part and parcel of infrastructural development but should not come at the cost of valuable wildlife resources. light pollution from bright streetlights (holker et al. 2010), the cementation of grassy patches (klaus 2013), and the construction of ripraps around streams (bernhardt and palmer 2007) are a few examples of construction projects that can negatively impact the wildlife present in the area. moving forward, the preservation of this wildlife resource should be a constant consideration. care must be taken to ensure that the resulting space is beneficial to the residents of the area, humans and wildlife alike. in order to attain a future wherein wildlife thrives alongside urban development, it is imperative that the continued monitoring of our wildlife resources and how they change over time is maintained. in future studies, it is also recommended to examine seasonality, an aspect that was excluded from this study as a result of covid-19 restrictions, which may reveal a different set of observable species. lastly, it is also recommended to examine spatial entities, in addition to spatial area and habitat types, when determining species habitat associations. sd june2022_pasumbal.indd 66 7/29/2022 4:12:50 pm r. v. pasumbal jr. et al. 67 references abdao drl. 2019. correlation of diet and prey abundance of philippine scops owl (otus megalotis: walden, 1875) in the threatened species arboretum, [undergraduate thesis]. 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[accessed 2022 jan 07]. https://upd.edu.ph/about/facts-at-a-glance/. vallejo b, aloy ab. 2014. responses of the bird community in the university of the philippines diliman after campus redevelopment and the decline of two common urban bird species. philipp sci lett. 7(1):55-61. vallejo b, aloyab a, ong ps, tamino a, villasper j. 2008. spatial patterns of bird diversity and abundance in an urban tropical landscape: the university of the philippines (up) diliman campus. sci diliman. 20(1):1-10. villasenor nr, driscoll da, gibbons p, calhoun ajk, lindenmayer db. 2017. the relative importance of aquatic and terrestrial variables for frogs in an urbanizing landscape: key insights for sustainable urban development. landsc urban plan. 157:26-35. sd june2022_pasumbal.indd 69 7/29/2022 4:12:50 pm an updated inventory and habitat association analysis of the non-avian vertebrates 70 waldien dl, adleson s, wilson z. 2020. eonycterisspelaea. the iucn red list of threatened species 2020: e.t7787a22128326. 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(2021b). hemidactylus frenatus. the iucn red list of threatened species 2021: e.t99156022a1434103. [accessed 2022 jan 08]. https://dx.doi.org/10.2305/ iucn.uk.2021-3.rlts.t99156022a1434103.en. zozaya s, alford r, schwarzkopf l. 2015. invasive house geckos are more willing to use artificial lights than are native geckos. austral ecol. 40(8):982-987. ______ romel v. pasumbal jr. <rpasumbal@gmail.com > is an m.s. biology student from the institute of biology, up diliman. his research interests lie in the biology and ecology of vertebrates, particularly those of herpetofauna. jelaine l. gan is an instructor at the institute of biology. her research interests vertebrate biology and ecology, urban ecosystems, and conservation biology. geoffrey jules n. solidum is a cum laude graduate of b. landscape architecture in up diliman. a professional painter, filmmaker, and graphic designer, his works focus on the creation of visual displays and commentaries tackling societal, political, and environmental issues. nappy l. navarra is an associate professor at the up college of architecture. his research interests are in landscape architecture, landscape ecology urban ecosystems, and resilience. sd june2022_pasumbal.indd 70 7/29/2022 4:12:50 pm 93 journal policy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the office of the vice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document defines research misconduct, specifies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identifies appropriate disciplinary actions. definitions scientific misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsification, or plagiarism in proposing, performing, or reviewing research in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 94 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research are concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “note to contributors” stipulates that “all articles must have a high degree of scholarhip,” the “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a certification from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right...” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as defined above and; b.) is specific and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the base for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specific advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the author is affiliated as well as the funding agency that supported the research. 95 the board shall ensure correction of the literature in the succeeding issue through various methods as defined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refeering a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and suppport appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. endnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientific misconduct policy. in attendance were: dr. corazon d. villareal, rduo director, presiding; dr. henry j. ramos, pmrgo director and professor, nip; atty. vyva victoria aguirre, ovcrd legal consultant; editors-in-chief dr. maricor soriano (science diliman) and dr. maria mangahas (socia l science diliman). ms nanie domingo and ms. dercy mararac, editorial assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these definitions of the forms of research misconduct are qouted verbatim from the policy of the office of research integrity of the united states public health service. similar phrasings of definitions are adopted in the references listed at the end of this document. 96 references council of science editors. “white paper on promoting integrity on scientific journal publications, as approved by the cse board of directors on september 3, 2006.” www.councilscienceeditors.org. accessed on january 26, 2009. “policy on scientific misconduct: university of southern california. http://policies.usc.edu/ policies/scientificmisconduct070108.pdf “scientific misconduct policy: new york university, the office of sponsored programs. https:// www.nyu.edu/osp/policies/scientificmisconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/physics/ optics/journal/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ajp/ submit.html research dissemination office office of the vice chancellor for research and development lower ground floor. phivolcs bldg., c.p. garcia ave., up diliman 1101 quezon city (632)3436-8720 fax (632) 8927-2568 research.dissemination1@upd.edu.ph journal subscription form note: this subscription form is for the three journals published by up diliman through its office of the vice-chancellor for research and development (ovcrd): humanities diliman, science diliman, and social science diliman. each journal is published twice a year. the subscription price for each journal (vols. 1 and 2) is phpp650.00. (subscription price is subject to change without prior notice.) i/we would like to subscribe to the following journal/s: journal title (please check) number of subscriptions (for each journal, please indicate desired number of copies) total amount (number of subscriptions x php650) humanities diliman sciencediliman social science diliman grand total method of payment (please check one)  pay cash at the ovcrd (see address above)  pay in check (please make check payable to the university of the philippines diliman ovcrd)  money remittance (payable to anna angelica p. angala, c/o ovcrd research dissemination and utilization office, with office address as indicated above and telephone no. (632) 3436-8720. subscriber details name/institution contact person (for institutional subscribers) mailing address email address telephone no. fax no. please send accomplished subscription form to the rdo-ovcrd via email or fax(please see above for contact details). if mode of payment is through money remittance, please send proof of remittance together with the accomplished subscription form. 01_device development of a simple biological model 43 development of a simple biological model of vertical phytoplankton distribution *corresponding author karlo primavera*, maria lourdes san diego-mcglone, cesar villanoy marine science institute, university of the philippines, 1101 diliman, quezon city, philippines e-mail: bilbo@upmsi.ph, mcglone@upmsi.ph, cesarv@upmsi.ph abstract science diliman (january-june 2006) 18:1, 43-52 introduction phytoplankton is the base of the oceanic food web. these are consumed by bigger plankton, that are in turn eaten by organisms in the higher trophic levels. phytoplankters, much like terrestrial plants, grow in biomass by utilizing carbon (in the form of carbon dioxide) through the process of photosynthesis. although much smaller than their terrestrial counterparts, phytoplankton have greater surface area to volume ratios and much quicker turnover rates, making the amount of carbon dioxide that they consume quite significant. thus they could play an important role in the sequestration of atmospheric carbon dioxide, the most significant greenhouse gas contributing to global warming. phytoplankton in tropical waters aggregate and form a maxima below the surface where the common limiting materials for growth (light from the surface, nutrients from the depths) are at optimal levels. the location of optimum growth conditions is dependent on various physical, chemical and biological factors. the formation of phytoplankton maxima was simulated through a coupled physical and biological model for vertical chlorophyll distribution in philippine waters. this paper evaluates biological models and the significance of 1) different forms of phytoplankton response to irradiance and nutrient uptake, 2) rate of nutrient uptake, and 3) light and/or nutrient limitation determining nutrient uptake. phytoplankton response-to-irradiance form is less important than rate of light extinction in affecting the deep chlorophyll maximum (dcm) depth. the michaelis-menten form of nutrient uptake gives a bigger and deeper dcm but only under certain conditions. temperature does not significantly affect nutrient uptake gives bigger and deeper dcms. these findings will come in handy in future work of simulating empirical chlorophyll profiles. light is used as energy source in photosynthesis through the chlorophyll pigments, with chlorophyll α as the principal photosynthetic pigment common to all phytoplankton. a high correlation of chlorophyll-a and phytoplankton biomass distributions (akbulut, 2003) would then allow the use of chlorophyll α as a measure of phytoplankton biomass (parsons & strickland, 1963; engelsen et al., 2004; cloern & dufford, 2005). any difference in the distribution of chlorophyll and phytoplankton biomass may be attributed to sinking velocity and increasing chlorophyll to biomass ratios at low light levels (fennel and boss, 2003). aside from light, nutrients are required in the photosynthetic reaction as shown by the equation below (stumm & morgan, 1995). primavera, mcglone and villanoy 44 nutrients usually determine the rate of photosynthesis since their concentrations are found in limiting amounts (nitrogen for oceanic systems) compared to the abundance of light and carbon dioxide in surface waters (falkowski, 1997). the amount of light available for photosynthesis decreases with depth due to attenuation (combined absorption and scattering) from particles in the water as well as the water itself. inversely, nutrient gradients increase with depth. somewhere in between is where a combination of both factors will be optimum for photosynthesis and phytoplankton growth. phytoplankters tend to aggregate in this area giving rise to a phytoplankton maximum, commonly referred to as the deep chlorophyll maximum (dcm). models have been developed to explain phytoplankton dynamics. the npz (nutrient-phytoplanktonzooplankton) model by franks (2002) is the simplest model that describes oceanic plankton dynamics. + biological (1) dynamics c is the concentration of the state variable (n, p or z), kh and kv are the horizontal and vertical eddy diffusivities, u, v and w are the horizontal and vertical water velocities, and ws is the vertical sinking or swimming speed of the state variable. the biological dynamics of the npz model is shown as: ( ) ( ) ( ) ( )ppizphpngif dt dp −−= ( ) ( )zzjzph dt dz −= γ ( ) ( ) ( ) ( ) ( ) ( )zzjppizphpngif dt dn ++−+−= γ1 f(i) is phytoplankton response to irradiance, g(n) is phytoplankton nutrient uptake, h(p) is zooplankton grazing, i(p) is phytoplankton loss, j(z) is zooplankton loss, and the constant g is the grazing assimilation efficiency of zooplankton. the npz model is a very general form of ecosystem model and has been modified. hadfield and sharples (1996) and sharples (1999) added an internal cell nutrient variable to account for chlorophyll to phytoplankton biomass ratio variations with depth. zakardjian and prieur (1994) included oxygen as to deal with oxidation of reduced forms of nitrogen. fennel and boss (2003) differentiated phytoplankton biomass maxima and chlorophyll maxima. different parameterizations have also been added, such as effect of photoadaptation and sinking due to phytoplankton aggregation (doney et al., 1996) and nutrient exudation during respiration (bahamon and cruzado, 2003). varela et al. (1992) and hodges and rudnick (2004) focused on the dcm. varela et al. (1992) used several variables in his dcm model, these are 2 phytoplankton, 2 nutrients, and 2 heterotrophs. although the model results were consistent with empirical data, hodges and rudnick (2004) noted that the fundamental constraint of nitrogen conservation was not satisfied in varela et al.'s (1992) model. they then suggested a simple nutrientphytoplankton model, where zooplankton grazing was factored into phytoplankton loss. model results of hodges and rudnick (2004) showed that removal of nitrogen from the surface through surface boundary conditions and sinking of phytoplankton are necessary conditions in the formation of the dcm, and that addition of more variables do not significantly affect phytoplankton distribution. because of the advantage of having less variables and satisfying balance of nitrogen in the system, the model of hodges and rudnick (2004) was adapted in this study. the objective of the study is to examine the degree to which nutrients (specifically nitrate) determine the vertical distribution of chlorophyll a using a coupled physical-biological model, with { } 216110263106 138opnohc + (algal protoplasm) +−− ++++ hohhponoco 1812216106 2 2 432 (+ trace elements and energy) ( ) z c sww y c v x c u z c vk y c x c hk t c ∂ ∂ +− ∂ ∂ − ∂ ∂ − ∂ ∂ + ∂ ∂ + ∂ ∂ = ∂ ∂       2 2 2 2 2 2 development of a simple biological model 45 emphasis on the biological model. this could enhance understanding of phytoplankton dynamics and provide insights on fisheries potential of philippine waters. materials and methods the biological model used in this study is based on the nutrient-phytoplankton model of hodges and rudnick (2004). the coupled physical-biological model is shown below. um is the maximum nutrient uptake rate and kz is the vertical eddy diffusion coefficient. the boundary conditions are: p = 0, n = constant, at the bottom hodges and rudnick (2004) used only basic mechanisms to demonstrate the formation of the dcm. in doing so, complex formulations of parameters and relationship of variables that may be necessary to reproduce empirical chlorophyll profiles were not considered. since this study will eventually attempt to duplicate empirical chlorophyll data, related literature were reviewed to examine parameters for irradiance, nutrient uptake, and phytoplankton loss (tables 1 and 2). in the model of hodges and rudnick (2004), the growth term (last term) for phytoplankton utilizes both nutrient and light influence at the same time. an alternative is liebig's law, which uses either nutrient or light, or whichever is more limiting at a given time. also, the maximum nutrient uptake rate can be either constant or variable. eppley (1972) suggested a temperature-dependent maximum phytoplankton growth rate: um(t) and um(t0) are the maximum uptake rate at reference temperature t and reference temperature t0, respectively. q10 is the factor by which the uptake rate changes with every 10ºc change in temperature. ( ) ( ) 10/)(100 0 tt mm qtutu −= ( ) ( ) ( )pngifuppi z p w z p k t p msz +−      ∂ ∂ −      ∂ ∂ = ∂ ∂ 2 2 ( ) ( ) ( )pngifuppi z p k t n mz −+      ∂ ∂ = ∂ ∂ 2 2 0=      ∂ ∂ −= ∂ ∂ z p kpw z n zs , at the surface response-toirradiance, f(i) terms remarks source 1. f(i)=i/i0 linear; edwards et al. (2000), not dependent on franks (2002), surface irradiance hodges & rudnick (2004) 2. f(i)=i/(i0+i) saturating franks (2002) 3. f(i)=1-exp(-i/ i0) saturating; franks (2002) when photo-inhibition is insignificant 4. f(i)=tanh(i/ i0) saturating franks (2002) 5. f(i)= (i/io)(exp(1-i/ i0) saturating; franks (2002), when photoinhibition steele (1962) is significant 6. f(i)=i/(ki+i) ki=irradiance michaelis-menten form; varela et al. (1992), half-satn constant for multiple nutrients zakardjian & prieur (1994), & phytoplankton models gecek & legovic (2001), bahamon & cruzado (2003) 7. f(i)=qchl(αi-rb) α=slope of photohadfield & sharples (1996), synthesis-irradiance sharples (1999) curve; qchl=chlorophyll: biomass ratio; rb=respiration table 1. list of functional forms for phytoplankton response-to-irradiance primavera, mcglone and villanoy 46 0.0 0.2 0.4 0.6 0.8 1.0 response to irradiance 0 50 100 150 200 de pt h (m ) form 1 form 2 form 3 form 4 form 5 form 6 figure 1. phytoplankton profiles when nutrient uptake rate is constant (identical with profiles when nutrient uptake rate is variable). table 2. list of functional forms for nutrient uptake and phytoplankton loss nutrient uptake, g(n) terms remarks source 1. g(n)=n n=nutrient concentration edwards et al. (2000), hodges & rudnick (2004) 2. g(n)=n/(ks+n) ks=nutrient uptake michaelis-menten uptake varela et al. (1992), zakardjian half-saturation constant & prieur (1994), gecek & legovic (2001), bahamon & cruzado (2003) 3. g(n)=1-(kq/q) q=n/chl=internal droop's internal cell hadfield & sharples (1996), nutrient pool; quota model sharples (1999), franks (2002) kq=minimum q required by cell phytoplankton loss, i(p) terms remarks source 1. i(p)=d/p d=loss rate, constant linear franks (2002) 2. i(p)=d non-linear; doney et al. (1996), edwards et density-dependent al. (2000), franks (2002), hodges & rudnick (2004) development of a simple biological model 47 the parameter forms in tables 1 and 2 were tested on the model using the parameter values given in table 3. irradiance was calculated using beer's law, where i is irradiance at depth z, i0 irradiance at the surface, and χ the extinction coefficient. the first phytoplankton response-to-irradiance form in table 1 is often used in very simple biological models where the magnitude of surface irradiance is not important. forms # 2 4 are forms that have photosynthetically saturating response to irradiance. form # 5 is also saturating but takes into account possible photoinhibition by phytoplankton. the michaelis-menten response-to-irradiance form is often used in multiple nutrient and variable models. the profiles of these response-to-irradiance forms are shown in figure 1. the last response-to-irradiance form was not used because it distinguishes phytoplankton chlorophyll and biomass. only two of the three forms of phytoplankton nutrient uptake in table 2 were used since there was no empirical data available on internal nutrient pool that is required for the third form. the first form is commonly used in simple coupled models that only have one nutrient variable while the michaelis-menten form is often used in more complex models with multiple nutrient variables. the constant phytoplankton loss form (#2) in table 2 was not used because it does not conform to the closed system suggested by hodges and rudnick (2004). thus the model runs for the study dealt primarily with 1) comparison of the different response-to-irradiance and nutrient uptake forms, 2) use of variable (temperature-dependent) nutrient uptake rate against a constant rate, and 3) application of liebig's law compared to simultaneous light and nutrient influence in phytoplankton growth. results and discussion the model was run to determine response of phytoplankton to light and nutrients separately, and with simultaneous influence of these two parameters. different parameter forms of irradiance response and nutrient uptake were examined. variable and constant nutrient uptake rates were also tested. the resultant phytoplankton profiles when nutrient uptake rate is constant are shown in figure 2. as discussed below, figure 2 can also represent phytoplankton profiles resulting from a variable nutrient uptake rate, making the figure representative of phytoplankton profiles for all runs. comparison of parameter forms response to irradiance phytoplankton profiles using the different response to irradiance forms are differentiated among the columns in figure 2. model runs using the first four forms gave identical phytoplankton profiles (rows 1 4, figure 1) even though response to irradiance profiles were different close to the surface (lines with solid blocks, figure 1). the response to irradiance profiles converged at lower depths and could be the reason for the identical phytoplankton profiles. profiles using response-toirradiance forms # 5 and 6 have significantly slower light extinction rates thereby allowing for deeper light penetration (figure 1) and thus the deeper dcm (rows 5 and 6, figure 2). nutrient uptake there is no significant difference in the phytoplankton profiles from the two nutrient uptake forms using liebig's law (columns 3 and 4, figure 2). in contrast, the dcm is bigger, deeper and more defined with the michaelis-menten nutrient uptake form when there is simultaneous nutrient and light influence on nutrient uptake (columns 1 and 2, figure 2). since nutrient concentration tends to be low (<1µm nitrogen) due to model constraints and the half saturation constant ks is small (0.05), nutrient uptake will increase when the michaelis-menten form is used. with the forms using liebig's law, nutrient uptake increase occurs where light is limiting (below intersection of light factor and nutrient factor in figure 3). since the light factor (smaller than the nutrient factor) determines nutrient uptake in this region the increase in nutrient uptake is unable to influence the phytoplankton profile. when there is simultaneous influence of light and nutrients, the increase in nutrient uptake would result in a bigger, deeper and more defined dcm (columns 1 and 2, figure 2). ( )zeii χ−= 0 primavera, mcglone and villanoy 48 0 1 nitrogen units (µm) 0 100 200 de pt h (m ) 0 100 200 de pt h (m ) 0 100 200 de pt h (m ) 0 100 200 de pt h (m ) 0 100 200 de pt h (m ) 0 100 200 de ot h (m ) 0 1 nitrogen units (µm) 0 1 nitrogen units (µm) 0 1 nitrogen units (µm) simple nutrient uptake form michaelis-menten nutrient uptake form nutrient and light influence nutrient or light influence (liebig's law) re sp on se to ir ra di an ce fo rm 1 re sp on se to ir ra di an ce fo rm 2 re sp on se to ir ra di an ce fo rm 3 re sp on se to ir ra di an ce fo rm 4 re sp on se to ir ra di an ce fo rm 5 re sp on se to ir ra di an ce fo rm 6 simple nutrient uptake form michaelis-menten nutrient uptake form figure 2. response-to-irradiance profiles. development of a simple biological model 49 variable and constant nutrient uptake rate the phytoplankton profile resulting from use of a temperature-dependent nutrient uptake rate did not differ significantly from the profile resulting from the use of a constant nutrient uptake rate. although nutrient uptake rate is higher at higher temperature especially for the upper 100 m of the water column, there was no significant change in the dcm. this indicates that temperature variation does not affect phytoplankton growth based on the biological model and the nutrient uptake rate formula of eppley (1972). parsons et al. (1984) stated that photosynthesis of phytoplankton in tropical/sub-tropical communities is more likely to be limited by nutrients rather than temperature. valiela (1984) also suggests that temperature is not a primary limiting factor in primary production in the sea and may have an effect only under certain situations. nutrient uptake based on liebig's law and simultaneous nutrient-light influence the use of liebig's law in phytoplankton nutrient uptake consistently gave bigger, deeper and more defined dcms (columns 3 and 4, figure 2) compared to using light and nutrient influence simultaneously (columns 1 and 2, figure 2). this may be so because with liebig's law nutrient available for uptake is situated deeper in the water column (figure 4) and just below the nutricline nutrient availability is bigger, which could account for the bigger and deeper dcm. conclusion the light extinction rate inversely affects the depth of the dcm and is more important than the form of phytoplankton response-to-irradiance. compared to the 0 7 14 21 0 40 80 120 160 200 de pt h (m ) light factor nutrient factor (m-m) nutrient factor (simple) figure 3. nutrient and light factors when liebig's law determines phytoplankton nutrient uptake. primavera, mcglone and villanoy 50 0 7 14 21 0 40 80 120 160 200 de pt h (m ) light factor nutrient factor (w/o liebig's law) nutrient factor (w/ liebig's law) figure 4. nutrient and light factors when light and nutrient factors simultaneously influence phytoplankton nutrient uptake. parameter symbol value unit surface irradiance io 100 µem-2s-1 irradiance half-saturation constant ks 12 µem -2s-1 light extinction coefficient c 0.05 m-1 eddy diffusion coefficient kz 1 x 10 -4 m2s-1 maximum nutrient uptake rate um 20 d -1 sinking velocity ws 0.5 d -1 phytoplankton loss rate d 0.1 d-1 maximum uptake rate at reference temperature (20ºc) um(20) 20 d -1 uptake rate change factor q10 1.884 no units table 3. parameter constants used in all runs development of a simple biological model 51 simple nutrient uptake form the michaelis-menten form gives a bigger dcm depth and magnitude but only when there is simultaneous nutrient and light influence. nutrient uptake rate does not seem to be temperaturedependent and might be due to temperature being less of a limiting factor in primary production especially in the tropics. the use of liebig's law in phytoplankton nutrient uptake situates nutrients available for uptake deeper and results to deeper and bigger dcms. these findings will be most useful when the study will try to duplicate unique chlorophyll profiles among the different basins in philippine waters as reported by cordero et al. (unpublished report). rate of light extinction and application of liebig's law will be considered in differences in dcm depth while the michaelis-menten nutrient uptake form and liebig's law may be useful when there are differences in dcm magnitude. however, the need to increase the complexity of the biological model to include other nutrients, phytoplankton and zooplankton variables could arise. in which case the parameter forms used should be specific to such models (e.g. greek and legovic, 2001) and phytoplankton growth will follow liebig's law, since this is typical of multiple-variable models (varela et al., 1992; zakardjian & prieur, 1994; gecek and legovic, 2001; bahamon & cruzado, 2003). future work will be to find and develop a suitable physical model to couple with these biological models that will properly take into account the influence of hydrodynamics on the profiles of the variables studied. acknowledgments this study is partly funded by the thesis grant from the office of the vice-chancellor for research and development. references akbulut, a., 2003. the relationship between phytoplanktonic organisms and chlorophyll a in sultan sazligi. turk. j. bot. 27: 421-425. bahamon, n. & a. cruzado, 2003. modelling nitrogen fluxes in oligotrophic environments: nw mediterranean and ne atlantic. ecological modelling 163: 223-244. cloern, j.e. & r. dufford, 2005. phytoplankton community ecology: principles applied in san francisco bay. mar. ecol. prog. ser. 285: 11-28. cordero, k. s. a., c. l. villanoy, & l. t. david. comparison of chlorophyll distribution in the water basins around the philippines. (unpublished report) doney, s. c., d. m. glover, & r. g. najjar, 1996. a new coupled, one-dimensional biological-physical model for the upper ocean: application to the jgofs bermuda atlantic time-series study (bats) site. deep-sea research ii 43(23): 591-624. engelsen, o., h. hop, e.n., hegseth, e. hansen, & s. falkpetersen, 2004. deriving phytoplankton biomass in the marginal ice zone from satellite observable parameters. int. j. remote sensing 25(7-8): 1453-1457. eppley, r. w. 1972. temperature and phytoplankton growth in the sea. fishery bulletin 17: 15-24. fennel, k. & e. boss, 2003. subsurface maxima of phytoplankton and chlorophyll: steady-state solutions from a simple model. limnol. oceanogr. 48(4): 1521-1534. falkowski, p.g., 1997. evolution of the nitrogen cycle and its influence on the biological sequestration of co2 in the ocean. nature 387:272-275. franks, j., s., 2002. npz models of plankton dynamics: their construction, coupling to physics, and application. journal of oceanography 58: 379-387. franks, j. s., j. s. wroblewski, & g. r. flier, 1986. behavior of a simple plankton model with food-level acclimation by herbivores. marine biology 91: 121-129. gecek, s. & t. legovic, 2001. nutrients and grazing in modelling the deep chlorophyll maximum. ecological modelling 138: 143-152. primavera, mcglone and villanoy 52 hadfield, m., j. & j. sharples, 1996. modelling mixed layer depth and plankton biomass off the west coast of south island, new zealand. journal of marine systems 8: 1-29. hodges b. a. & d. l. rudnick, 2004. simple models of steady deep maxima in chlorophyll and biomass. deep-sea research i 51: 999-1015. mann, k.h. & j.r.n. lazier. 1996. dynamics of marine ecosystems: biological-physical interactions in the oceans 2nd edition. massachusetts, blackwell science inc.: 394 pp. parsons, t.r. & j.d.h. strickland, 1963. discussion of spectrophotometric determination of marine plant pigments, with revised equations for ascertaining chlorophylls and carotenoids. j. mar. res. 21: 155-163. sharples, j., 1999. investigating the seasonal vertical structure of phytoplankton in shelf areas. marine models 1: 3-38. steele, j.h., 1962. environmental control of photosynthesis in the sea. limnol. oceanogr. 7: 137-150. stumm, w. & j. j. morgan, 1981. aquatic chemistry an introduction emphasizing chemical equilibria in natural waters 2nd edition. new york, john wiles & sons: 780pp. valiela, i. 1984. marine ecological processes. new york, springer-verlag inc.: 546 pp. varela, r. a., a. cruzado, j. tintore, & e. g. ladona, 1992. modelling the deep-chlorophyll maximum: a coupled physical-biological approach. j. mar. res. 50: 441-463. zakardjian, b. & l. prieur, 1994. a numerical study of primary production related to vertical turbulent diffusion with special reference to vertical motions of the phytoplankton cells in nutrient and light fields. journal of marine systems 5: 267-295. 02_saccharomyces a possible role of peptides 15 abstract science diliman (january-june 2005) 17:1, 15-22 a possible role of peptides in the growth enhancement of an industrial strain of saccharomyces sp. dino paolo a. cortes1, albert francis e. domingo1, alexes c. daquinag2, and cynthia t. hedreyda1* 1national institute of molecular biology and biotechnology college of science, university of the philippines 1101 diliman, quezon city, philippines 2molecular physiology and biophysics baylor college of medicine, houston, texas, usa tel. no.: (632)981-8500 local 3953; fax no.: (632)927-7516 e-mail: hedreyda@laguna.net date received: may 7, 2003; date accepted: april 20, 2005 individual addition of a commercially available nutritional supplement and a methanol extract from an industrial saccharomyces sp. strain smc resulted in the enhanced growth of saccharomyces sp. strain smc in minimal medium. isolation of the growth enhancing components from aqueous extracts of the supplement and the cellular extract was performed using reversed-phase, gel filtration, and ion exchange chromatography. reversed-phase chromatography using sep-pak® vac c18 yielded aqueous washes which elicited increased yeast growth. gel filtration chromatography of the aqueous washes in a group separation mode using sephadex g25 gave three distinct groups for the nutritional supplement, and four distinct groups for the cellular extract. fraction groups that exhibited growth enhancing activity also exhibited high absorbances at all three wavelengths of 214, 260, and 280 nm. two major fractions which tested positive for growth enhancing activity in succeeding experiments were obtained after passing each of the active gfc groups through a toyopearl sp 550c cation exchanger column. the active component from the cellular extract did not bind to the cation exchanger. the absorbance data at 214 nm (peptide bond experimental absorbance maximum wavelength), the bradford assay (showing the presence of proteinaceous matter), and the active component’s inclusion in the sephadex g25 fractionation range of 1-5 kda (characteristic of small peptides) suggest that the growth enhancing components of the nutritional supplement and methanol cell extracts are peptides. keywords: brewer’s yeast, peptides, organic extraction, chromatography, bradford assay, nutritional supplement *corresponding author introduction in the alcoholic beverage industry, the biochemical cycle by which yeast metabolizes simple sugars into ethanol and other flavor compounds has always been closely watched for opportunities in which the brewer may intervene, either by manipulation of biological components or changes in process parameters. differences between what is known as the traditional brewing process as compared to modern methods are fast increasing in number. the progression of the cortes et al. 16 industry as a whole from small cottage breweries to the large factories has been brought about by increasing competition in a lucrative market wherein consumers demand the highest quality product at the most affordable price (iserentant, 1995). yet, even with the demand for change, brewers are cautious as to how they may alter their time-honored recipes, given consumer response to product changes. of current concern not only in alcoholic beverage but also in all other food industries are the advantages and disadvantages of using molecular techniques to improve current procedures and products. to the uninformed consumer, molecular biology almost always translates to genetic manipulation, a controversial issue that is currently negative in public perception. the industry is, thus, reluctant to employ molecular techniques given the above premise. however, deoxyribonucleic acid (dna) is not the only biomolecule that may be manipulated for process or product improvement; there still are the downstream components of molecular biology’s central dogma– rnas, peptides, and proteins. improvement of the production medium is one way to increase the product. we have been interested on the possible role of peptides as enhancers to the growth and fermentation performance of a saccharomyces sp. industrial strain used primarily for beer production. of several nitrogenous sources peptides are the most promising of all alterable factors in sugar substrate biochemistry (da cruz et al., 2002; patterson & ingledew, 1999). the next step would be to test its activity on the actual industrial sugar substrates used by brewers, prior to possible inclusion in the standard brewer’s recipe. this study deals with a potential biological parameter that may be exploited for industrial application. the study was conducted with the following specific objectives: (i) to test the commercially-available supplement and the yeast cell extract for growth enhancing activity. (ii) to examine the chemical nature of the supplement and yeast cell extract. (iii) to test the isolated fractions for growth enhancing activity. materials and methods saccharomyces growth and maintenance an industrial saccharomyces sp. brewer’s yeast strain was provided by san miguel corporation’s beer division (smcbd, mandaluyong city, metro manila, philippines) and referred here as saccharomyces sp. strain smc. upon receipt of the yeast it was subcultured onto a yepd-agar (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose, 2% w/v agar agar) plate. the plate culture was then stored at 4oc for a maximum of one month, after which subculturing onto a new plate was done. inoculum for the liquid culture was obtained from the yepd agar plate culture. each colony was suspended in 1 ml sterile distilled deionized water (sddh2o) before inoculation into the media. preparation of crude nutritional supplement and yeast methanol extract a liquid sample of yeast “peptide supplement” (as advertised) was provided by smcbd in a sealed dark brown bottle at room temperature. the liquid supplement was diluted with sddh2o in a 1 part supplement: 2 parts sddh2o ratio. the sample was then centrifuged for 15 minutes at 10,000 x g and 4°c. supernatant was then filtered using a 0.22 µm membrane filter. the clarified supernatant was stored in sealed, sterile conical tubes at 4ºc until further use. freshly-prepared industrial brewer ’s yeast (saccharomyces sp. strain smc) slurry obtained from smcbd was washed with distilled deionized water (ddh2o) until the supernatant was colorless. whole yeast cells were pelleted and collected from the slurry after centrifugation at 10,000 x g for 15 minutes at 4ºc. the yeast cells were allowed to freeze at -70ºc and were lyophilized until fully dry. organic extraction was then performed by stirring 7:3 methanol:water solution (70% methanol) into the dried yeast cells until a homogeneous suspension was obtained. the yeast a possible role of peptides 17 suspension was centrifuged at 10,000 x g for 15 minutes at 4ºc and the supernatant was collected and stored as the crude extract. the extraction process was performed twice on the same batch of samples. the crude yeast extract was dried by centrifugation under vacuum with heating at 45ºc. the samples were resuspended in sddh2o, sterilized using a 0.2 µm filter and stored at 4ºc. sample purification and analysis the nutritional supplement and the crude yeast cell extract were individually purified and analyzed as follows: reversed-phase chromatography: sep-pak® c18 sep-pak® vac 6 cc cartridges (waters corporation, massachusetts, usa) containing a c18 matrix were used in a solid phase extraction (spe) mode relying on reversed-phase chromatography (rpc) principles. an aqueous wash fraction (using ddh2o as eluent) and an organic wash fraction (using 70% v/v methanol as eluent) were separately obtained from the samples. the two fractions were then lyophilized and individually resuspended in ddh2o and later assayed for growth enhancing activity. gel filtration chromatography: sephadex g25 gel filtration chromatography (gfc) in a batch separation mode was performed using a sephadex g25 solid matrix, having a fractionation range of 1-5 kda. a glass column with 50 cm length x 1.5 cm internal diameter was packed with sephadex g25 superfinegrade resin in a 4ºc cold cabinet. column efficiency and void volume were checked and measured using blue dextran 2000 (amersham biosciences, sweden). three ml of the active fraction from the rpc run was then loaded onto the gfc column and eluted with ddh2o. the run proceeded at 0.5 ml/minute. four mlsized fractions were collected and monitored for absorbances at 214, 260, and 280 nm. upon analysis of absorbance data from the gfc run, fraction groups were selected and pooled according to distinctions in absorbance peaks. the resultant groups were then lyophilized and resuspended in ddh2o, before being assayed for growth enhancing activity. ion exchange chromatography: toyopearl sp 550c ion exchange chromatography (iex) using a toyopearl sp 550c cation exchanger resin was performed using a bio-rad econo system fast protein liquid chromatography (fplc) setup (bio-rad laboratories, inc., usa). a glass column with 9 cm length x 1.5 cm internal diameter was packed with resin at room temperature. five hundred µl of the active gfc group was loaded onto the iex column and eluted initially with ddh2o (0% salt). a linear gradient was then formed by the fplc, from 0% to 100% of 0.5 m nacl salt solution. gradient start and end points were recorded and are noted in the iex chromatogram. the run proceeded at 1.5 ml/minute, and fractions were collected at 3 ml/ fraction. the fractions were monitored for absorbances at 214, 260, and 280 nm. protein assay the bradford assay (bradford, 1976) was utilized in order to verify the presence of proteins and/or peptides. aliquots of the test samples in volumes from 5 to 50 µl were each brought to 100 µl sample volume using 0.15 m nacl solution. one ml of bradford reagent (sigma) was added to each sample in microcentrifuge tubes. the tubes were then vortexed and incubated at room temperature for at least two minutes, after which sample color change was evaluated and compared to a negative control. influence on growth of saccharomyces sp. smc strain the influence of nutritional supplement and yeast methanol extract on the growth of saccharomyces sp. strain smc was examined using a liquid minimal medium (0.67% w/v yeast nitrogen base without amino acids and ammonium sulfate, 2% w/v glucose). five hundred µl of the test sample was added into cottonplugged 250 ml erlenmeyer flasks, each containing cortes et al. 18 100 ml sterile liquid minimal medium. inoculation with a single colony of the yeast followed. the increase in cell mass was measured over time by monitoring the absorbance at 660 nm (dillemans et al., 1999) of 1 ml aliquots of the culture. the purity of the culture after the addition of the supplement was determined by spread plating and inspection of colony morphology. results and discussion growth of saccharomyces sp. industrial strain smc with or without the supplements the addition of the nutritional supplement resulted in considerable growth enhancement of the industrial strain saccharomyces sp. (smc) as shown by its growth curve compared to that of the negative control (fig. 1). similarly, the cellular extract enhanced the growth of the same yeast strain in minimal medium compared to the control (fig. 2). the cell growth enhancement is most likely caused by nitrogenous additives, because the culture medium is nitrogen-deficient. the water used was distilled and deionized, lessening the likelihood that ammonium salts could have been introduced via the water component. previous work by patterson and ingledew (1999) showed that mixtures of nitrogen sources, amino acids, ammonia, allantoin, and urea were preferred by yeast and appeared to inhibit the utilization of dipeptide from culture medium. single amino acids were better sources of nitrogen than were single sources of each homodipeptide. in the same study, it was observed that complex nitrogen mixtures (3 amino acids and 3 dipeptides), the yeast saccharomyces cerevisiae ncyc 1324 simultaneously used both amino acids and peptides as sources of nitrogen. these previous reports suggest that in the absence of other preferred nitrogen sources, dipeptides enhance yeast growth. in this study, addition of putative peptides present in the nutritional supplement and cell extracts, probably served as a crucial source of nitrogen because the yeast cells were grown in minimal medium. the presence of the putative peptides as n source resulted in significant growth enhancement compared to the control. sample analysis the methods used in this study are standard protocols for the isolation, purification, and characterization of proteins or peptides. whereas larger proteins are commonly rendered inactive by the methanol wash because they are denatured by the same hydrophobic properties of methanol, methanol extraction is generally the preferred way to extract peptides, because their small sizes keep their structure from being disrupted by the hydrophobic interaction of methanol. the purification strategies were devised taking into fig. 2. effect of cell extract on the growth of saccharomyces sp. smc strain. liquid minimal medium was with ( ) or without ( ) crude yeast extract. x axis = time (hours); y axis = absorbance (660 nm). o pt ic al d en si ty a t 66 0 nm 0 0.5 1 20.51.5 22.5 25 44.5 46.5 49 hours post-inoculation 0 0.2 0.4 0.6 0.8 1.2 1.0 1.4 fig. 1. effect of nutritional supplement on the growth of saccharomyces sp. smc strain with (\\) or without ( ) the prepared minimal supplement. x axis = time (hours); y axis = absorbance (660 nm). o pt ic al d en si ty a t 66 0 nm 0.00 1.42 2.42 27.01 29.01 45.51 47.51 50.51 hours post-inoculation 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 a possible role of peptides 19 consideration the novel nature of the peptide(s) in question and the relevance of each procedure to the biomolecule’s presumed structural complexity. protein assay of the samples aliquots from the prepared nutritional supplement all qualitatively registered a brick red to blue colorimetric reaction upon addition of the bradford reagent, suggesting the presence of proteinaceous matter. the same brick red to blue color change was manifested using the yeast cell extract fractions in the protein assay, thus, also indicating the presence of proteinaceous matter. the data using the bradford assay, however, could not accurately quantify the protein contents of the samples because different amino acid compositions have different absorbance readings depending on the binding of the coomassie® dye. selection of an appropriate standard protein is necessary to obtain an approximate amount of proteins in an unknown sample. this study began with a totally unknown active component, and a correct standard for quantitation could not be selected at this point (tal et al., 1985). sample purification and fraction analysis liquid chromatography techniques employed as part of the purification strategy include reversed-phase chromatography, gel filtration chromatography, and ion exchange chromatography. component elution was monitored by taking the absorbance values (at 214, 260, and 280 nm) of the fractions from the chromatography steps (except the rpc step since it was in a batch separation mode). the three wavelengths were used based on theoretical absorbance profiles for proteins in general, certain amino acids with aromatic side chains, and the peptide backbone (280, 260, and 214 nm, respectively; mathews & van holde, 1996). reversed-phase chromatography (rpc) and gel filtration chromatography (gfc) fractions were assayed for growth enhancement activity and tested for the presence of proteinaceous matter using the bradford assay. ion exchange chromatography was performed on the active gfc fraction of the nutritional supplement and the active component in the cell extract, revealing two major molecular groups present according to positive or negative charge in the first and that the active component did not bind to the cation, in the latter. reversed-phase chromatography: sep-pak® c18 solid phase extraction (spe)-rpc using the sep-pak® c18 cartridges gave two fractions from a batch mode: an aqueous (ddh2o) wash and an organic (70% methanol) wash, for both commercial nutrition supplement and methanol extracted samples. upon bradford analysis, the two washes of each sample retained the brick red to brown color of the original clarified supernatant. it was observed, however, that only the aqueous wash for both samples exhibited considerable growth enhancing activity turbidimetrically (figs. 3 and 4) and by viable plate count (data not shown). the active components for both samples did not adsorb to the hydrophobic c18 matrix of the sep-pak® cartridge, suggesting that they must have weak hydrophobicity, or that they may even be hydrophilic. based on the sep-pak® product literature, an analyte can either adsorb to the matrix with contaminants eluting in the aqueous wash, or an analyte can elute with the aqueous wash with contaminants adsorbing to the matrix. it is likely that the second case holds in this study, because the organic 70% methanol wash appears fig. 3. effect of nutritional supplement’s sep-pak® fractions on the growth of saccharomyces sp. smc strain. the samples tested were the aqueous sep-pak® wash ( ), the prepared nutritional supplement (\\), the 70% methanol wash (|||), and the control ( ). x axis = time (hours); y axis = absorbance (660 nm). o pt ic al d en si ty a t 66 0 nm 0.00 1.42 2.42 27.01 29.01 45.51 47.51 50.51 hours post-inoculation 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 cortes et al. 20 not to contain significant active component and exhibited only a low level of growth enhancement. gel filtration chromatography: sephadex g25 the gfc run resulted in three distinct fraction groups for the nutritional supplement (fig. 5) and four distinct fraction groups for the cell extract (fig. 6). fractions from each of the major groups were pooled together on the basis of the continuity of their absorbances at 214, 260, and 280 nm. only the second gfc groups of the nutritional supplement and of the cellular extract exhibited growth enhancing activity turbidimetrically (figs. 7 and 8) and by viable cell count (data not shown). ion exchange chromatography: toyopearl sp 550c iex was performed to investigate if the ionic interaction-based chromatography step could further purify the second gfc groups of the two samples. as fig. 5. sephadex g25 gfc chromatogram (batch separation mode) of the nutritional supplement. fraction absorbance values were read at 214 ( ), 260 (•), and 280 nm (x). fractions were pooled into three groups (g1, g2, g3). i = sample injection point, vo = void volume, vt = total column volume. volume eluted (ml) a b so rb an ce i vo vt g1 g2 g3 0 20 40 60 80 100 120 140 160 180 0.0 1.0 2.0 3.0 4.0 5.0 fig. 6. sephadex g25 gfc chromatogram (batch separation mode) of the cell extract. the absorbance of the fractions were read at 214 (o), 260 (♦), and 280 nm ( ). g1 g2 g3 g4 i vo vt a b so rb an ce fraction number (4 ml/fraction) 0 10 20 30 40 50 60 0.0 1.0 2.0 3.0 4.0 5.0 fig. 7. effect of nutritional supplement gfc fractions on the growth of saccharomyces sp. smc strain. the samples tested were the active sep-pak® fraction (\), gfc groups g1 (=), g2 (|||), g3 ( ), and negative control ( ). x axis = time (hours); y axis = absorbance (660 nm). a bs or ba nc e at 6 60 n m 0 1 21 2725 46 48 hours post-inoculation 0 0.1 0.2 0.3 0.4 0.6 0.5 0.7 fig. 4. effect of cell extract sep-pak® fractions on the growth of saccharomyces sp. smc strain. the samples tested were the aqueous rpc (=), organic rpc fraction (|||), the crude yeast extract ( ), and the control ( ). x axis = time (hours); y axis = absorbance (660 nm). o pt ic al d en si ty a t 66 0 nm 0 0.5 18.5 22.520.5 24.5 42.5 44.5 46.5 hours post-inoculation 0 0.2 0.4 0.6 0.8 1.2 1.0 1.4 1.6 a possible role of peptides 21 fig. 8. effect of cell extracts gfc fractions on the growth of saccharomyces sp. smc strain. the samples tested were the negative control (=), yeast cultures with aqueous rpc fraction (/), gfc groups g1 (\), g2 (♦), g3 ( ), and g4 ( ). x axis = time (hours); y axis = absorbance (660 nm). o pt ic al d en si ty a t 66 0 nm 0 0.5 1 182 20 23 42 44 hours post-inoculation 0 0.2 0.4 0.6 0.8 1.2 1.0 1.4 47.5 fig. 9. ion exchange chromatogram of gfc group g2 (nutritional supplement). the linear gradient’s progression from 0% to 100% 0.5 m nacl is marked by the straight line. the absorbances were read at 214 nm ( ), 260 nm (x), and 280 nm (=). a b so rb an ce volume eluted (ml) 0 20 40 60 80 100 0 0% 1 100% 2 3 fig. 11. effect of cell extract iex fractions on the growth of saccharomyces sp. smc strain. the samples tested were the yeast cultures with iex fraction 1 ( ) and 2 ( ) and the control (|||). x axis = time (hours); y axis = absorbance (660 nm). o pt ic al d en si ty a t 66 0 nm 0 1 2 96 13 15 42 44 hours post-inoculation 0 0.2 0.4 0.6 0.8 1.2 1.0 46 may be seen in the chromatograms, there were only two distinct peaks (figs. 9 and 10). this suggests that iex may be used as a purification step to clean the sample. furthermore, both iex groups 1 and 2 of the cellular extract showed growth enhancement activity compared to that of the negative control (fig. 11). only the enhancement brought about by iex group 1 is comparable to the crude extract’s activity. the enhancement caused by group 2, though not as fig. 10. ion exchange chromatogram of gfc group g2 (cell extract). the absorbances were read at 214 nm ( ) and 280 nm (•). the profile of the salt gradient used for elution is shown as the heavy line (–). a b so rb an ce fraction number (4 ml/fraction) 0 2 4 6 8 10 0 1 2 3 active fractions 0% 100% 12 14 16 18 4 5 significant, may have been brought about by other positively charged molecules. these data from the chromatographic separation suggests that the active components of the cellular extract are negatively charged molecules. previous work by patterson and ingledew (1999) showed that mixtures of nitrogen sources, amino acids, ammonia, allantoin, and urea were preferred by yeast cortes et al. 22 and appeared to inhibit the utilization of dipeptide from culture medium. single amino acids were better sources of nitrogen than were single sources of each homodipeptide. in the same study, it was observed that complex nitrogen mixtures (3 amino acids and 3 dipeptides), the yeast saccharomyces cerevisiae ncyc 1324 simultaneously used both amino acids and peptides as sources of nitrogen. these reports suggest that in the absence of other nitrogen sources, dipeptides enhance yeast growth. in this study, putative peptides probably served as crucial source of nitrogen because the yeast cells were grown in minimal medium, resulting in significant growth enhancement when nutritional supplements or cell extracts were added compared to the control. conclusion enhanced growth of saccharomyces sp. strain smc in minimal medium was observed upon the addition of either commercially available nutritional supplement or a methanol extract from an industrial saccharomyces sp. strain smc. analysis of the nutritional supplement and yeast methanol extract revealed significant absorbance at 214 nm wavelength (peptide bond experimental absorbance maximum wavelength), the presence of proteinaceous matter based on protein assay, and the active component’s inclusion in the sephadex g25 fractionation range of 1-5 kda (characteristic of small peptides), suggesting that the growth enhancing components of the nutritional supplement and methanol cell extracts are peptides. acknowledgments the authors are grateful to san miguel corporation for sponsoring this work. the authors also appreciate the help of the following people: dr. nilo o. juliano, dr. alberto d. rivera, and mr. nestor b. cadeliña. references bradford, m.m., 1976. a rapid and sensitive method for the quantization of microgram quantities of proteins utilizing the principle of protein-dye binding. anal. biochem.72: 248-254. da cruz, s.h., e.m. cilli, & j.r. ernandes, 2002. structural complexity of the nitrogen source and influence on yeast growth and fermentation. j. inst. brew. 108(1): 54-61. dillemans, m., l. van nedervelde, & a. debourg, 1999. characterization of a novel yeast factor increasing yeast brewing performances. proc. congr. eur. brew. conv. 27: 711-718. dillemans, m., l. van nedervelde, & a. debourg, 2001. an approach to the mode of action of a novel yeast factor increasing yeast brewing performance. j. am. soc. brew. chem. 59(3): 101-106. iserentant, d., 1995. beers: recent technological innovations in brewing. in lea, a.g.h. & j.r. piggott (eds.) fermented beverage production. london, blackie academic: 45-65. mathews, c.k. & k.e. van holde, 1996. introduction to proteins: the primary level of protein structure. in biochemistry, 2nd ed. california, benjamin/cummings: 129157; 204-206. matthews, t.m. & c. webb, 1991. culture systems. in tuite, m.f. & s.g. oliver (eds.) saccharomyces. new york, plenum press: 249-278. patterson, c.a. & w.m. ingledew, 1999. utilization of peptides by a lager brewing yeast. j. am. soc. brew. chem. 57(1): 1-8. tal, m., a. silberstein, & e. nusser, 1985. why does coomassie brilliant blue r interact differently with different proteins? a partial answer. j. biol. chem. 260(18): 99769980. 01_device noche and araneta 12 abstract *corresponding author science diliman (july-december 2007) 19:2, 12-22 an asynchronous ieee floating-point arithmetic unit joel r. noche* affiliation when work was started and completed: department of electrical and electronics engineering college of engineering, university of the philippines, diliman joel.noche@up.edu.ph present affiliation: department of mathematics and natural sciences college of arts and sciences, ateneo de naga university, naga city, camarines sur jrnoche@adnu.edu.ph date submitted: july 29, 2005; date accepted: may 11, 2006 jose c. araneta (deceased) department of electrical and electronics engineering college of engineering, university of the philippines, diliman an asynchronous floating-point arithmetic unit is designed and tested at the transistor level using cadence software. it uses cmos (complementary metal oxide semiconductor) and dcvs (differential cascode voltage switch) logic in a 0.35 µm process using a 3.3 v supply voltage, with dual-rail data and single-rail control signals using four-phase handshaking. using 17,085 transistors, the unit handles single-precision (32-bit) addition/subtraction, multiplication, division, and remainder using the ieee 754-1985 standard for binary floating-point arithmetic, with rounding and other operations to be handled by separate hardware or software. division and remainder are done using a restoring subtractive algorithm; multiplication uses an additive algorithm. exceptions are noted by flags (and not trap handlers) and the output is in single-precision. previous work on asynchronous floating-point arithmetic units have mostly focused on single operations such as division. this is the first work to the authors' knowledge that can perform floating-point addition, multiplication, division, and remainder using a common datapath. key words: asynchronous logic circuits, floating point arithmetic, calculation times an asynchronous ieee floating-point arithmetic unit 13 introduction asynchronous circuits, digital logic circuits that do not use a global clock signal, have attracted attention this past decade due to their potential advantages over synchronous circuits (hauck, 1995), (van berkel et al., 1999), (sutherland & ebergen, 2002). asynchronous circuits automatically adapt to changing physical conditions, operating faster when the temperature is lower or when the supply voltage is higher. they consume power only when and where performing computations. they allow robust mutual exclusion of signals, making them ideal for handling external inputs. they have better noise and electromagnetic compatibility properties. they exhibit no clock skew, and can thus be designed modularly. they are thus ideal for portable, low-power, wireless applications that are activated by external signals. asynchronous circuits have been used for some parts of a digital hearing aid (nielsen & sparsø, 1999) and a pager (kessels & marston, 1999), among others. some applications require accurate calculations to be made quickly. although real numbers can be handled by integer arithmetic hardware (grehan, 1988), implementing the format known as floating-point in hardware greatly improves performance. a binary floating-point standard proposed by ieee (1985) is widely adopted, enabling software developers to create easily-portable, highly reliable code. the standard defines four different formats; the one with the least number of bits (32) is called single-precision. floatingpoint numbers are represented as (-1)s × f × 2e, where s is the sign bit, f is the significand, and e is the exponent. if the floating-point number is normalized, (i.e., 1 ≤ f < 2), then the most significant bit of the significand is always 1 and can be removed to save space (packed). for single-precision, the result is a 23bit fraction f. the signed exponent is encoded as an unsigned number e called the exponent field using a bias representation, with a bias of 127 for singleprecision (i.e., e = e 127). the standard also defines special quantities: denormal numbers (values which are less than the smallest normalized values), 'not a number's (results of invalid operations), positive and negative zeroes, and positive and negative infinities. previous work on asynchronous floating-point arithmetic units have mostly focused on single operations such as division (williams & horowitz, 1991), (matsubara & ide, 1997), (won & choi, 2000). the work described in this paper is the first to the authors' knowledge that can handle ieee floating-point addition/subtraction, multiplication, division, and remainder using a common datapath. to achieve this goal with the available computing resources, we chose to use single-precision arithmetic, with rounding and other operations to be handled by separate hardware or software. to minimize circuit size, division and remainder are done using a restoring subtractive algorithm and multiplication uses an additive algorithm (noche, 2003). thus, the architecture is 'serial.' exceptions are noted by flags (and not trap handlers) and the output is in singleprecision. the datapath uses dual-rail dcvs (differential cascode voltage switch) logic, and the control unit uses cmos (complementary metal oxide semiconductor) logic. a transistor-level design of the unit using a 0.35 µm process and a 3.3 v supply voltage is designed and tested using cadence software (ic4.46 package for sun solaris 5.8). virtuoso schematic editing and virtuoso symbol editing are used to create the transistor-level schematics. testing is done using the affirma analog circuit design environment. materials and methods basic operation the unit has the following inputs: two 32-bit data inputs (the operands), four arithmetic control (request) signals (one for each operation), four rounding mode control signals, and five flag reset signals. the outputs are: a 32-bit data output (the result), a signal acknowledging correct receipt of the operands (ain), a signal indicating that the output is ready (aout), five flags, and the signals acknowledging their resets. the external system first makes the operands active, then waits for ain to become active. (when the external system later makes the data inputs inactive, ain will become inactive.) it then requests the operation to be performed by making the corresponding arithmetic science diliman (july-december 2007) 19:2, 12-22 noche and araneta 14 control signal active. the unit processes the data and any exceptions set the corresponding flags. after the result is computed, aout becomes active. the external system then makes the arithmetic control signal inactive. this deactivates the unit, making the result and aout inactive. it is now ready for the next set of operands and the next arithmetic operation. the flags are always active and once set, they remain set until explicitly cleared. they can only be set by the unit, and can only be reset by the external system. overview of the datapath figure 1 shows a block diagram of the unit's datapath, with the control unit and some control signals hidden for simplicity. the complete circuit schematics are in noche (2003). operands x and y, and result z are each composed of a sign bit, an exponent field, and a fraction (e.g., x = s x , e x , f x ). the main building blocks of the datapath are the registers and the adders. sr latches are used in the control unit and also for the exception flags. registers for the exponent calculations do not require any shifting, so sr latches (with completion signals) are also used there. although one 9-bit register e is enough for the addition, multiplication, and division operations, the remainder operation requires two additional 9-bit registers e 1 and e 2 in its initial operand normalization. significand calculations involve left and right shifting, so significands are stored in shift registers (modified versions of those in kishinevsky et al. (1994)). p is a 25-bit bidirectional shift register connected to a, a 24bit bidirectional shift register with an additional round bit. b is a 25-bit shift-left register. a 9-bit carry look-ahead (cla) adder (ruiz, 1998), (ruiz, 2000) is used for exponent calculations, and a 25-bit cla adder handles all the required significand calculations for all operations. special signals indicate if the result is zero or -1, and these were used in certain cases as completion signals. for example, the unit's remainder algorithm checks for the case where e x e y = -1. dcvs multiplexers are used to select the inputs to the registers and adders. the multiplexers do not have completion signals because the blocks their outputs are connected to work correctly whether the outputs are early or late. when two dual-rail signal paths merge into one and both will never be active at the same time, or gates are used instead of multiplexers to minimize the number of control signals needed. for example, the output of the 25-bit adder is connected to or gates because the control signals to enable the 25-bit adder and multiplexer 5 will never be high at the same time. control building blocks the rest of the building blocks are used in the control unit. these perform handshaking, counting, and conditional branching. the c-element is a basic asynchronous circuit building block, a device whose output changes to a value (logic 0 or 1) only when all its inputs are that value. aside from c-elements (shams et al., 1998), sr-latches, and boolean logic gates, most of the control circuitry of the unit uses building blocks well-suited for four-phase signaling and dual-rail data: decide, do, twice, and thrice. the last three are described by their signal transition graphs (kondratyev et al., 1998) and implemented as complex cmos gates using the software petrify (cortadella et al., 1997). petrify is also used to create the control circuitry for the shift registers, details of which are in noche (2003). figure 1. block diagram of the datapath e x s x f x s y e y f y x = 0? x = nan? x = science diliman (july-december 2007) 19:2, 12-22 an asynchronous ieee floating-point arithmetic unit 15 the decide building block the decide building block has two variants: the decide early and the decide late. their symbols are shown in figure 2. whenever the request on becomes active, the first of the inputs ti or fi to go high decides which of the outputs (t or f) will go high. only one of the outputs can go high at any time, and it goes and remains high only when on is active. once the 'decision' is 'made,' it cannot be changed or taken back. for example, if ti and on go high, then t goes high and remains high even if ti goes low, or fi goes high, or both (as long as on remains active). figure 2. symbols of (a) decide early, (b) decide late transistors driven by input signals that are the last to change are placed nearer the outputs to improve performance. the decide early block assumes that the data input becomes active and valid before on becomes active; decide late assumes that the data input becomes active and valid after on becomes active. figure 3 shows the transistor-level schematics of both versions. the inputs ti and fi (and their corresponding outputs t and f) are interchangeable. figure 4. symbols of (a) do unique, and (b) do guarded; (c) structure of do guarded figure 5. stg of do unique figure 6. symbols of (a) twice, (b) thrice figure 3. transistor-level schematics of (a) decide early, (b) decide late the do building block the do building block has two variants: the do unique and the do guarded. their symbols are shown in figures 4a and 4b. the do guarded block is a do unique block with an attached c-element as shown in figure 4c. figure 5 shows the stg of the do unique building block. the do unique building block is the same as the q-element of martin (1990). the twice and thrice building blocks the symbols and stgs of the twice and thrice building blocks are shown in figures 6, 7, and 8. a control circuit example an example illustrates how these building blocks are used to implement algorithms. the algorithm for division is as follows. the sign of the result is the exclusive-or of the operand signs. special cases (those with operands or results of zero, infinity, or 'not a number') are handled first. for ordinary cases, the second exponent is subtracted from the first and the bias is added to this. register a is set to zero. significand f x is placed in register p, and f y is placed in b. the science diliman (july-december 2007) 19:2, 12-22 noche and araneta 16 figure 7. stg of twice operands are then normalized. thus, register b is shifted left (and the preliminary exponent is incremented accordingly) until its most significant bit is 1. the contents of p are shifted left (and the preliminary exponent is decremented accordingly) until the significand there is normalized. the connected registers p and a are then shifted left 24 times. for each iteration, if the difference p b is positive, then it is written to p and the rightmost bit of a (bit 0) is set to 1. this step is done 25 times. after this, if the significand in a is not normalized, then a is shifted left once and the preliminary exponent decremented. if the difference p b is positive, then it is written to p and bit 0 of a is set to 1. the result (with the significand in a, bit 0 of a as the guard bit, and the round bit deduced from p) is then passed to the rounding unit. figure 8. stg of thrice figure 9 shows a part of the division algorithm and figure 10 shows one implementation. the example shows sequential and parallel operations; while-loop, for-to-do-next, and if-then constructs; and how subroutines are handled. as line number n in the algorithm is reached, signal nextn goes high. when the algorithm is completed, all the nextn signals go low in succession. note that the circuitry generating signals did2 and did4 are the same (a 2-input c-element whose inputs are ab and ce). these two c-elements can be replaced by a single c-element generating the signal did2or4, which replaces all instances of did2 and did4. if so, then a transition on did2or4 would be interpreted as transitions on both did2 and did4, resulting in incorrect behavior. the do building blocks generating the signals do2 and do4 should thus be do guarded blocks, so only the block that made the request would be acknowledged. results and discussions the unit uses variable-latency algorithms that are implemented using variable-latency circuits. completion times are thus expected to be data dependent because limited resources prevented the implementation of the rounding unit, the completion times reported here do not include the time needed to correctly round the result. the algorithms for the four operations and for rounding are in noche (2003). what affects completion times arithmetic operations start by unpacking denormals and checking for 'not a number's. this takes the same time t un for all operations. next, cases with operands or results of zero or infinity are checked. the different operations have different special cases, so this step takes different times: t as for addition, t ms for multiplication, t ds for division, and t rs for the remainder figure 9. sample algorithm science diliman (july-december 2007) 19:2, 12-22 an asynchronous ieee floating-point arithmetic unit 17 figure 10. implementation of sample algorithm science diliman (july-december 2007) 19:2, 12-22 noche and araneta 18 operation. the completion times of special cases are around 8 to 9 ns. for addition, the next step, the determination of the larger operand, takes a time t ad . adjusting the significand so that the exponents are equal takes a time dependent on the exponent difference: xt aa , where x = |e x e y |. the last steps take a time t al which is small if the result is zero, and large if the result is negative. but its effect on the completion time is negligible when compared with the effect of the exponent difference. thus, t al can be approximated as a constant equal to its average value. for multiplication, the next step is a significand addition for every bit in the first operand's significand that is a 1. this step takes a time n x t mad , where n x is the number of 1's in f x . there is also a fixed time taken for shifting the significands, t mss . the last steps take a variable time t ml depending on the result. but since it has little effect on the completion time, it can be treated as a constant. for division, the next step is to normalize the operands if they are denormal. this takes a time st n , where s is the number of shifts to normalize the operands. there is then a fixed loop, where a register write occurs when the temporary significand of the first operand is greater than or equal to the second operand's significand. this condition is noted by a 1 in the unrounded significand of the result f z '. this takes a time n z t dsb , where n z is the number of 1's in f z '. shifting the significands takes a fixed time t dss . the last steps take a variable time t dl depending on the result. its effect on the completion time is small, and it can be treated as a constant to simplify matters. for the remainder operation, the next step, normalizing the operands if they are denormal, takes a time st n . the remaining steps are more complicated, with many conditional branches. when e x < e y , the calculation is quick and this can be treated as a special case. when e x > e y , the algorithm may go through a loop that executes a shift up to e x e y times. however, if the significands are multiples of each other, this loop is exited. one easy way to quantify 'being multiples of each other' is to use the difference r x r y , where r x is the number of bits from the most significant 1 bit of f x to the least significant 1 bit of f x , and r y is defined similarly for f y . if each iteration in the loop takes a time trsb, this step cannot take longer than (e x e y ) t rsb , but can take a shorter time if r x r y < e x e y . thus, this step takes a time zt rsb , where z is either e x -e y or r x r y , whichever is the smaller positive number. when e x = e y , the completion time is t rl and depends highly on the significands. the effect of t rl is not negligible, but may be ignored for simplicity. note that operands having e x > e y also pass through the circuitry for e x = e y . table 1 shows that the completion times are mostly functions of the number of shifts or of additions. using improved adders or shifters will greatly shorten the completion times. experimental results there are 232 different single-precision floating-point values. thus there are 232 × 232 ≈ 1.8 × 1019 different possible test vectors for each operation. due to time constraints, only 248 test vectors were simulated. the simulated temperature was 25 ºc and each output was connected to a 5 ff capacitive load. these are typical simulation conditions; higher temperatures and larger loads would lengthen the completion times. the test inputs were all active and valid from the start. a reset pulse was applied to reset all flags, and then the arithmetic control signal was set high. once aout went high, the test circuit set the arithmetic control signal low, causing aout to go low. the time from when the arithmetic control signal went high to when aout went low is the completion time recorded for the test vector. the selection of test vectors and the results of the simulations are in noche (2003). figure 11 shows the completion time t (in ns) for ordinary case addition as a function of |e x e y |. figure 12 shows t for ordinary case multiplication as a function op detailed approximate + t un + t as + t ad + xt aa + t al a a x + b a × t un + t ms + n x t mad + t mss + t ml a m n x + b m ÷ t un + t ds + st n + n z t dsb + t dss + t dl a d n z + b d + c d s rem t un + t rs + st n + zt rsb + t rl a r z + b r + c r s table 1 expressions for completion times science diliman (july-december 2007) 19:2, 12-22 an asynchronous ieee floating-point arithmetic unit 19 of the number of 1's in the first operand's significand. figure 13 shows t for ordinary case division (no denormal operands) as a function of the number of 1's in the result's unrounded significand (including the guard, round, and sticky bits). figure 14 shows t for ordinary case division as a function of the number of shifts needed to normalize both operands (the test vectors here all have one 1 in f z '). figure 15 shows t for ordinary case remainder (no denormal operands, e x ≥ e y ) as a function of z (defined in the previous section). figure 16 shows t for ordinary case remainder as a function of the number of shifts needed to normalize both operands (if denormal) (the test vectors here all have x = y so that z = 0). the least squares line is shown for each graph. estimation of completion times 300 350 400 450 500 550 600 650 0 5 10 15 20 25 figure 12. multiplication completion times (ordinary cases) in ns as a function of the number of 1's in fx 0 500 1000 1500 2000 2500 3000 0 20 40 60 80 100 120 140 figure 11. addition completion times (ordinary cases) in ns as a function of |ex ey| 500 550 600 650 700 750 800 850 0 5 10 15 20 25 figure 13. division completion times (ordinary cases, without denormal operands) in ns as a function of the number of 1’s in fz’ 500 600 700 800 900 1000 1100 1200 0 10 20 30 40 50 figure 14. division completion times (ordinary cases, with one 1 in fz') in ns as a function of the number of shifts to normalize operands 0 100 200 300 400 500 600 700 0 5 10 15 20 25 figure 15. remainder completion times (ordinary cases, w/ o denormal operands, ex >= ey) in ns as a function of z science diliman (july-december 2007) 19:2, 12-22 noche and araneta 20 ns (nz = 1, s = 0) to 1107.0 ns (n z = 24, s = 22) for the completion times. for the remainder operation, 0 ≤ z ≤ 23 and 0 ≤ s ≤ 44, leading to a best-case estimate of 70.6 ns (z = 0, s = 0), and a worst-case estimate of 1126.6 ns (z = 23, s = 22) for the completion times. note that the predicted remainder completion time has a relatively large standard error. while these variable latency algorithms can result in very long completion times, in other cases the times are much shorter. for example, operations with zero operands finish very quickly in this work. this could prove useful in some applications. for example, taking advantage of the relative occurrence of zero-valued discrete cosine transform coefficients in compressed video led to fewer operations and reduced power consumption in (xanthopoulos & chandrakasan, 1999). power and energy consumption the power and energy consumptions of this work for a few test cases are shown in table 3. table 2 shows the predicted completion times t and the standard error in the predicted t based on the simulation results. for single precision, the absolute value of the exponent difference ranges from x = 0 to 254. the approximate range for addition completion times would thus be from 59.0 ns to 5850.2 ns. cases where x is large are quite rare. in oberman (1996), ten applications from the specfp92 benchmark suite yielded the following distribution for double-precision addition and subtraction operations: around 23% of them had x = 0 and around 20% had x = 1; 52% of the operations had x < 3 and around two-thirds had x < 6. using this distribution, the average addition completion time would be around 127.4 ns. 0 200 400 600 800 1000 1200 0 10 20 30 40 50 figure 16. remainder completion times (x = y) in ns as a function of the number of shifts to normalize operands the number of 1's in a single-precision significand ranges from n x = 0 to 24. multiplication completion times might thus range from 342.5 ns to 618.5 ns. for division, n z can vary from 1 to 26, while s can vary from 0 to 44, resulting in an estimated range of 554.0 conclusions an asynchronous single-precision floating-point arithmetic unit is designed and tested at the transistor level using cadence software. building blocks wellsuited for four-phase handshaking and dual-rail data are used to implement the algorithms. a serial architecture is chosen to keep the design small: only 17,085 transistors are used. provision for a rounding unit is included, which enables the unit to follow the ieee 754-1985 standard for binary floating-point arithmetic. due to limited time and resources, the op t (ns) standard error + 22.8x + 59.0 5.0 × 11.5n x + 342.5 10.1 ÷ 11.8n z + 542.2 + 12.8s 5.3 rem 26.4z + 70.6 + 20.4s 42.1 table 2 predicted completion times test vector (ns) (mw) (nj) 4195835 + 3145727 79.0 4.08 0.32 4195835 × 3145727 465.1 4.07 1.89 4195835 ÷ 3145727 703.1 3.87 2.72 4195835 rem 3145727 101.5 4.19 0.43 table 3 completion times, power consumption, and energy consumption of a few test cases science diliman (july-december 2007) 19:2, 12-22 an asynchronous ieee floating-point arithmetic unit 21 transistor-level design of the rounding unit is left for future work. previous work on asynchronous floating-point arithmetic units have mostly focused on single operations such as division. this is the first work to the authors' knowledge that can perform floating-point addition, multiplication, division, and remainder using a common datapath. the algorithms used in this work are designed to minimize area (and possibly cost) requirements. while current designs focus on improving speed, the recent trend toward mobile devices might make area-efficient designs more attractive. acknowledgments this study was granted financial support by the office of the vice chancellor for research and developmentuniversity of the philippines, diliman under grant no. 00010.1 nset. louis alarcón and anastacia ballesil provided useful information during the revision of this manuscript. j.r.n. thanks the family of j.c.a. for their support, and the anonymous referees for their suggestions. references 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evaluation of three 32-bit cmos adders in dcvs logic for self-timed circuits. ieee journal of solidstate circuits 33(4): 604-613. ruiz, g., 2000. addition to "evaluation of three 32-bit cmos adders in dcvs logic for self-timed circuits". ieee journal of solid-state circuits 35(10): 1517. shams, m., j. ebergen, and m. elmasry, 1998. modeling and comparing cmos implementations of the c-element. ieee transactions on very large scale integration (vlsi) systems 6(4): 563-567. science diliman (july-december 2007) 19:2, 12-22 noche and araneta 22 sutherland, i. and j. ebergen, 2002. computers without clocks. scientific american 287(8): 46-53 van berkel, c., m. josephs, and s. nowick, 1999. applications of asynchronous circuits. proceedings of the ieee 87(2): 223-233. williams, t. and m. horowitz, 1991. a zero-overhead selftimed 160-ns 54-b cmos divider. ieee journal of solidstate circuits 26(11): 1651-1661. won, j.-h. and k. choi, 2000. low power self-timed floatingpoint divider in 0.25µm technology. in proceedings of the 26th european solid-state circuits conference, stockholm, sweden. xanthopoulos, t. and a. chandrakasan, 1999. a low-power idct macrocell for mpeg-2 mp@ml exploiting data distribution properties for minimal activity. ieee journal of solid-state circuits 34(5): 693-703. science diliman (july-december 2007) 19:2, 12-22 119 journal policy on research misconduct1 (final march 13, 2009)2 principles the journals3 published by the office of the vice-chancellor for research and development, university of the philippines diliman (ovcrd, up diliman) uphold the highest standards of excellence and ethics in the conduct of research. these being publications of the flagship campus of the only national university of the philippines, the editorial boards consider the maintenance of such standards part of their commitment to public trust and the pure pursuit of new knowledge. as such, research misconduct shall never be tolerated. purpose this document defines research misconduct, specifies the internal controls the journals have formulated to prevent such misconduct, describes the process for responding to allegations of research misconduct, and identifies appropriate disciplinary actions. definitions scientific misconduct or research misconduct (henceforth these shall be used interchangeably) is the fabrication, falsification, or plagiarism in proposing, performing, or reviewing research in reporting research results.4 fabrication is making up data or results and recording or reporting them.5 falsification is manipulating research materials, equipment or processes, or changing or omitting data or results such that the research is not accurately represented in the research record. plagiarism is the appropriation of another person’s ideas, processes, results or words without giving appropriate credit. research misconduct does not include honest error or differences of opinion. 120 internal controls appointments to the editorial boards are based on track records of scholarship and research integrity. the journals strictly follow a double-blind refereeing process in which at least two experts in the research are concerned review any manuscript submission. three mechanisms ensure adequate safeguards against research misconduct. the “note to contributors” stipulates that “all articles must have a high degree of scholarhip,” the “all articles must be original” and that “all allegations of research misconduct shall be pursued assiduously.” the “manuscript submission form” includes a certification from the corresponding author on the veracity of the presentations of the co-authors. the publication agreement which the author signs before the article is published includes among others, a provision allowing wide latitude in responding to research misconduct: “the author warrants that the articles is original and does not infringe upon any proprietary or intellectual property right...” response to allegations of research misconduct upon receipt of a written allegation of research misconduct, the editor-in-chief shall convene the editorial board to review the allegation. the editorial board shall seek to establish if the complaint a.) is an instance of research misconduct as defined above and; b.) is specific and substantiated. if these requirements are not met, the editor-in-chief writes the complainant of the board’s decision to dismiss the complaint and the base for such dismissal. if these are met, the board consults with the referees of the article and may opt to consult with another expert in the research area concerned, to further determine the substance of the allegation. in both instances, the respondent shall be advised in writing of the receipt of such allegation and shall be allowed to respond. if the manuscript in question has not yet been published in the journal, the board shall return the article to the author with the specific advice on how to rework the article; the author is also given the option to withdraw the manuscript. if the manuscript has already been published in the journal, and research misconduct is proven, the editor-in-chief shall notify the author and the institution to which the author is affiliated as well as the funding agency that supported the research. 121 the board shall ensure correction of the literature in the succeeding issue through various methods as defined by the board. these may include errata, retractions, and apologies to be written by the author concerned. moreover, the board can opt to impose the following sanctions: 1. disallow the contributor concerned from refeering a manuscript submission; 2. ban the contributor from publishing in the journal for a period the board shall determine. disciplinary action the editorial board does not consider it within its purview to impose disciplinary sanctions against the contributors concerned. however, in the case of faculty, researchers, and students from up diliman, it shall adhere to the protocol in processing written complaints against the faculty and employees and suppport appropriate disciplinary action stipulated in the rules and regulations on the discipline of faculty members and employees of the up diliman faculty and administrative manual. endnotes 1 based on discussions in the meetings held on february 2, 2009 and february 24, 2009 at the ovcrd conference room in response to dean saloma’s request for science diliman to formulate a scientific misconduct policy. in attendance were: dr. corazon d. villareal, rduo director, presiding; dr. henry j. ramos, pmrgo director and professor, nip; atty. vyva victoria aguirre, ovcrd legal consultant; editors-in-chief dr. maricor soriano (science diliman) and dr. maria mangahas (socia l science diliman). ms nanie domingo and ms. dercy mararac, editorial assistants for ovcrd journals took down the minutes. 2 as approved in the meeting of the above discussants on february 24, 2009 at the ovcrd conference room. 3 science diliman, social science diliman, and humanities diliman 4 federal policy on research misconduct, united states of america. 5 these definitions of the forms of research misconduct are qouted verbatim from the policy of the office of research integrity of the united states public health service. similar phrasings of definitions are adopted in the references listed at the end of this document. 122 references council of science editors. “white paper on promoting integrity on scientific journal publications, as approved by the cse board of directors on september 3, 2006.” www.councilscienceeditors.org. accessed on january 26, 2009. “policy on scientific misconduct: university of southern california. http://policies.usc.edu/ policies/scientificmisconduct070108.pdf “scientific misconduct policy: new york university, the office of sponsored programs. https:// www.nyu.edu/osp/policies/scientificmisconduct.php “manuscript submission.” optical and quantum electronics. http://www.springer.com/physics/ optics/journal/11082 “manuscript submission procedures.” american journal of physics. http://www.kzoo.edu/ajp/ submit.html from the editor in this december 2019 issue of science diliman, we feature three research articles and one short communication from various disciplines in the biological sciences, and one research article on an analysis of the scientific publications of the college of science (cs), university of the philippines diliman (upd). ecologists jay fidelino and jelaine gan from upd determined the patterns of insectivorous bat activity, richness, and assemblage vis-à-vis habitat type, insect abundance, and environmental conditions within an urban green space in metro manila, the philippines’ capital region. although bats are not as charismatic as other animals, they are important to ecosystems. bats provide indirect benefits to humans, too. in recent years, there has been a growing interest in the use of bat activity to study the effects of urbanization and other land use changes. the study done by fidelino and gan is among the very few studies on bats in an urban setting in the philippines. developmental biologist jordan ferdin halili from upd and his co-workers evaluated the cytotoxicity and genotoxicity of the money tree (pachira aquatica) on plant and animal models as a preliminary screening for the money tree’s anticancer potential. over the years, there have been a number of studies conducted to discover compounds from plants that can be used to treat cancer and other diseases. halili and his co-workers found promising cytostatic and cytocidal effects of stem and leaf extracts from the money tree, which, according to the authors, indicate that the plant is a potential source of a nature-based chemotherapeutic compound. cell biologist michael velarde from upd and his co-workers studied the effects of extracts from seeds of six species of legumes on the growth of estrogen-responsive breast cancer cell line. their study is important because these legumes are grown for human consumption. although we derive nutritional benefits from consuming legumes, some species may have components that mimic human estrogens, which may disrupt normal body processes or promote diseases, according to the authors. velarde et al. found that low doses of extracts from common beans and cowpeas slightly promoted the growth of breast cancer cells, but high doses decreased the cancer cells’ growth. this suggests that certain legumes have potential estrogenic activities at certain doses. the fourth research article in this issue is an analysis done by geologist carlos primo david and his research associate mart cyrel geronia from cs, upd on the scientific productivity of the different institutes within cs, upd over a period of 20 years, i.e., from 1998 to 2017. the authors only included in their analysis scientific publications from thomson reuters’ web of science (now maintained by clarivate analytics) and scopus. the authors derived valuable insights from their data such as the possible drivers of scientific productivity, the need for research collaborations with other local and foreign institutions, and the challenge of publishing in prestigious and high-impact journals. the last article, authored by arvin pacoma and leni yap-dejeto from the university of the philippines visayas-tacloban college, is an analysis of the total mercury content of canned tuna and yellowfin tuna (thunnus albacores) and frigate tuna (auxist hazard) caught from the waters of eastern visayas, philippines. the authors also estimated the daily intake for locally caught tuna and accompanying total mercury concentrations; they found that these values were way below the allowed concentration of mercury in fish consumed per day regardless of sex and age, which suggests that there is no health risk to consumers. as for commercially available canned tuna, the authors recommend that no more than one can per day should be consumed by adults and that children and pregnant women should consume less. jonas p. quilang, ph.d. editor-in-chief