8-viability of phytoplankton-austero.pmd N.M. Austero et al. 69 SCIENCE DILIMAN (JANUARY-JUNE 2019) 31:1, 69-78 V iabil ity of Phytoplankton from Ballast Waters of International Vessels Ber thing at Por ts of Cebu and Subic Bay, Phil ippines Nero M. Austero* Marine Science Institute University of the Philippines Diliman Subhash S. Sawant CSIR-National Institute of Oceanography India Rhodora V. Azanza National Academy of Science and Technology Department of Science and Technology ABSTRACT T h e v i a b i l i t y o f b a l l a s t w a t e r p h y t o p l a n k t o n w a s a s s e s s e d t h r o u g h incubation experiment. Leptocylindrus sp. and Thalassionema spp. were found to be viable when incubated in port water and ballast water media showing some increase in cell numbers. Bloom-forming diatom taxa, such as Chaetoceros spp. and Coscinod iscus spp. , potentially harmful diatom species Pseudo-nitzschia spp. , and dinoflagellates, Gambierd iscus spp. and P r o r o c e n t r u m s p p . w e r e a l s o i d e n t i f i e d i n b a l l a s t w a t e r s f r o m international vessels. These results further suggest possible successful t r a n s p o r t o f t h e s e o r g a n i s m s v i a s h i p p i n g , w h i c h c o u l d f a c i l i t a t e t h e introduction and lead to bioinvasion in the local aquatic environment. Keyword s: Ballast water, diatoms, dinoflagellates, phytoplankton, por ts _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online Viability of phytoplankton from ballast waters 70 INTRODUCTION Ballast water essentially provides stability and maneuverability to ships, thereby assuring safe operating conditions (David 2015). In ports, ballast water operations are usually conducted either via gravity or pumps to take in or discharge ballast water (David 2015), which may contain potentially harmful algal bloom species that will be transported from one port to another. Ballast water has been identif ied as one of the major vectors for the global dispersal of alien invasive species, toxic harmful algal bloom species and pathogens causing ecological, economic, and health problems (Hallegraeff and Bolch 1991; Bolch and de Salas 2007; Drake et al. 2007; Eames et al. 2008; Simkanin et al. 2009; Klein et al. 2010). The present study was aimed at assessing the viability of organisms in ballast water tanks of international vessels berthed at Cebu and Subic ports in Philippines. Attempts to identify common organisms found in ballast waters have also been made. MATERIALS AND METHODS Collection of ballast water samples Four international vessels were boarded for onboard collection of ballast water samples in the months of May and July 2015 in an opportunistic sampling method only. Vessel 1 (V1) is a 4,718-ton general cargo vessel registered from Panama. Its last port of call was in Sumbawa, Indonesia. Previously visited ports include: (1) Makassar, Indonesia, (2) Singapore, (3) Chiba and Yokohama, Japan, (4) Busan, Korea, and (5) Qingdao, China. In addition, vessel 2 (V2) is a general cargo vessel with 6,150 tons gross tonnage under the flagship of Panama. Prior to berthing in Cebu International Port (CIP), its last port of call was in Zhanjiang, China. Its previously visited ports include: (1) Haiku and Hongkong, China, (2) Chiba and Funabashi, Japan, (3) Jingtang, China, (4) Incheon, Korea, (5) Hon Gai, Vietnam, (6) Bangkok, Thailand, (7) Kuantan, Malaysia, and (8) Kaohsiung and Guang Zhou, China. Moreover, vessel 3 (V3) is a 33,990-ton bulk carrier vessel registered from Singapore. Its last port of call was in Taboneo, Indonesia. Its previous visited ports include: (1) Makassar, Indonesia, (2) Singapore, (3) Port Klang, Vietnam, (4) Cai Mep, Vietnam, (5) Washington, USA, (6) Okkye, S. Korea, (7) Vostochny, Russia, (8) Qingdao, China, (9) Busan and Kunsan, S. Korea, and (10) Tamatave, Madagascar. Lastly, vessel 4 (V4) is a 31,753-ton bulk carrier vessel under the flagship of Panama. Its last port of call was in Balboa, N.M. Austero et al. 71 Panama. Its previously visited ports include: (1) Cristobal, Panama, (2) New Orleans, USA, (3) Bayovar, Peru, (4) Buenaventura, Colombia, (5) Houston, Texas, USA, (6) Altamira, Mexico, (7) Yeosu, S. Korea, (8) Shanghai, China, (9) Kaohsiung, Taiwan, (10) Susaki, Japan and (11) Port Luis, Mauritius. Ballast water sample access points were via the (1) main engine pump for V1, (2) manholes for V2 and V4, and (3) sounding pipes for V3 based on the availability (Table 1). Table 1. List of international vessels sampled in Cebu International Port (CIP), Cebu, Central Visayas and Naval Supply Depot (NSD) in Subic Bay, Zambales, Phil ippines in May and July 2015 1 Gen. Cargo Main Engine Buckets 3 5.2015 CIP Indonesia 2 Gen. Cargo Manhole Buckets 5 7.2015 CIP China 3 Bulk Carrier Sounding pipe Pipe sampler 1 5.2015 NSD Indonesia 4 Bulk Carrier Manhole Plankton Net 5 7.2015 NSD Panama * Sample volume is per bucket (1 bucket = ~ 2.5-3 Liters of Ballast Water) Vessel Type Access Point Sampl ing Gears Sample Volume* Sampl ing Date Sampl ing Port Last Port of Sample collection was peformed using buckets, plankton nets, and pipe sampler. In the present study, the main emphasis was on phytoplankton only; hence, the samples were f ixed with Lugol’s solution and brought to the shore laboratory for further analysis. These samples were then analyzed for phytoplankton cell abundance and identif ied to genus or species level using identif ication keys (Yamaji 1984; Tomas 1997). In addition, samples were also collected and brought to the shore laboratory without preservative and used for incubation experiments. The unpreserved samples were pooled accordingly and passed through a 60-μm sieve to remove larger organisms, and the f iltrate, containing smaller cells, were cultured in various media. Med ia preparation and incubation experiment Three types of culture media were used for this experiment: (1) Diluted F/2Media- prepared following the methods of Guillard and Ryther (1962), Guillard (1975), Corrales et al. (1995), and Azanza (1997); (2) port water; and (3) ballast water media; which were prepared by f iltering through a 0.45-μm Whatman Filter paper following Kang et al. (2010) and were then sterilized by autoclaving. Viability of phytoplankton from ballast waters 72 Approximately 10-mL f iltered ballast water samples were inoculated into each 100-mL media in triplicate and were incubated for 6 days. Three 1-mL aliquots were drawn from each flask on alternate days for phytoplankton analysis and identif ication. Cultures were maintained at 30oC and 33±1psu in a 12:12 light/dark cycle with 143.88±27.62-μmol photon m-2s-1. RESULTS AND DISCUSSION In this study, a total of 16 diatoms, three dinoflagellates and four zooplankton taxa were identif ied from the ballast water samples. Highest diatom count was observed in V4, moderate in V2, and minimal in V1 and V3. The presence of potentially toxic diatom Pseudo-nitzschia sp. (Figure 1a), and dino-flagellates Gambierdiscus sp. (Figure 1b), Prorocentrum sp. (Figure 1c), and Protoperidinium sp. (Figure 1d) in ballast water conf irms that ships can harbor these species in ballast water (Hallegraeff and Bolch 1991; Gollasch et al. 2000; Drake et al. 2007; Burkholder et al. 2007; Klein et al. 2010). These species may have been carried into vessel tanks during the process of ballast water exchange. The presence of potentially toxic Gambierdiscus sp. in ballast water, on the other hand, supports the theory of Hallegraeff (1992) and Bomber et al. (1988) that, in spite of being not known to produce resistant cysts, these species are well capable of surviving and getting dispersed as epiphytes attached to drifting macro-algae. The main f indings of the incubation experiment show that, among the vessels sampled, only V2 and V4 recorded viable cells (Figures 2a and 2b). -No Skeletonema spp. and Nitzschia sp. cells were recorded from V2; however, they appeared as viable cells after the 6th day of incubation in port water media (Figure 2a), which suggests that these cells were present in the ballast water, but were absent in the subsample taken for counting. This was also true in the case of Leptocylindrus sp. , Figure 1. Potentially harmful phytoplankton taxa in ballast tanks sampled as light micrographs (LMs): (a) Pseudo-nitzschia sp. , (b) Gambierdiscus sp. , (c) Prorocentrum sp. , and (d) Protoperidinium sp. N.M. Austero et al. 73 which was not found in ballast water sample, but exhibited considerable increase in cell number after six days in port water media. On the contrary, Thalassiosira sp. , which is a bloom-forming diatom, was found to grow in all three types of media with maximum growth in port water media after six days (32 times). These results could be attributed to the capability of diatoms to produce viable resting spores that possess the ability to germinate even after a long period of time and that can withstand adverse ballast tank conditions (Doucette and Fryxell 1985; McQuoid et al. 2002; Harnstrom et al. 2011; Montresor et al. 2013). Experiments of Carney et al. (2011), for instance, also revealed the survival of phytoplankton cells and the successful germination of macro-alga E. flexuosa spores (Kolwalkar et al. 2007) even after prolonged exposure in the dark. Figure 2. Phytoplankton composition in ballast water samples from (A) V2 and (B) V4, and viable diatom cells (*) following six days of incubation period. (Note: BW – ballast water, PW – port water, F10 – diluted F media, D00 – day zero, D02 – day 2, D04 – day 4, D06 – day 6) Viability of phytoplankton from ballast waters 74 These results suggest that these diatoms can possibly survive if discharged into new environments, leading to possible introduction of new species and/or invasion. Studies of Steichen and Quigg (2015) and Kang et al. (2010) have also reported that ballast water-borne diatoms exhibit viability and growth after exposure to changing salinity and nutrient regimes. Studies have also shown that even the use of modern technologies and techniques implemented in most ocean-going vessels as part of their ballast water management systems do not guarantee 100% eff icacy (Grob and Pollet 2015). Certain photosynthetic organisms were able to grow within 4 to 20 days when released back into favorable conditions even after treatment (Stehouwer et al. 2010; van der Star et al. 2011; Liebich et al. 2012; Martínez et al. 2013). On the other hand, analysis of phytoplankton composition and abundance in ambient waters of Cebu International Port (CIP) and Naval Supply Depot (NSD) terminal identif ied at least 53 phytoplankton taxa with 36 diatoms and 16 dinoflagellates. Ballast water-borne diatoms, such as Skeletonema spp. , Nitzschia sp. , Leptocylindrus sp. , and Thalassiosira sp. , were among the identif ied taxa from the ambient waters of Cebu and Subic Bay ports. These organisms were also identif ied in the waters of Panama, China, and Indonesia (D’Croz et al. 1991; Liu et al. 2005; Serihollo et al. 2015; Effendi et al. 2016). These f indings imply that these species could be cosmopolitan and may have been traveling between these ports. The use of modern and new approaches could be very helpful in inferring these species’ geographical sources in future studies. This preliminary investigation suggests that the foreign vessels involved in trading with the Philippines, especially those which arrive for picking up of cargo, could pose potential risk of transport of harmful or bloom-forming organisms. Additional studies, if conducted in the future, would provide a database useful for assessing ballast water risk for Philippine ports by using a suitable model or tool, which in turn, will provide a Decision Support System (DSS) to the concerned authority in line with the International Maritime Organization – Ballast Water Management Convention - 2004. ACKNOWLEDGEMENTS We would like to thank the Department of Science and Technology (DOST ) – Philippine Council for Agriculture, Aquatic Resources Research and Development (PCAARRD) for the funding support received in line with the project “Invasive N.M. Austero et al. 75 Marine Organisms transferable by ships in selected areas in the Philippines”. 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Austero, MSc. former Senior Research Assistant of Professor Rhodora V. Azanza. His previous research involvements were on harmful algal blooms or red tides -where he successfully cultured ciguatera f ish poisoning (CFP) causing dinoflagellates, harmful/invasive marine organisms in ballast water of international vessels, and biofouling organisms established in wharves and other pier facilities. He obtained his Master’s Degree in Marine Science from the Marine Science Institute, University of the Philippines Diliman. Subhash S. Sawant, Ph.D., Chief Scientist at the CSIR-National Institute of Oceanography, Goa, India has vast experience in the f ield of Marine Sciences. His areas of interest are Biofouling and Ships’ Ballast Water Management. He has developed a new model for conducting Ballast Water Risk Assessment, which can be used for controlling transfer and introduction of aquatic alien species by ships in any port. Rhodora V. Azanza, Ph.D. Professor Emeritus of the Marine Science Institute, UP Diliman and President of the National Academy of Science and Technology (NAST). She has served the University in various positions (including being Dean of the College of Science) and other international organizations. Her researches and publications have been on biology and ecology of seaweeds, and microalgae including environmental impacts and management of algal blooms.