82 journal of multidisciplinary applied natural science vol. 2 no. 2 (2022) research article antagonism activity of phosphate solubilizing bacteria against ganoderma philippii and fusarium oxysporum of acacia plants muhammad asril*, yuni lisafitri, and bayo alhusaeri siregar received : february 16, 2022 revised : march 30, 2022 accepted : april 03, 2022 online : april 03, 2022 abstract ganoderma philippii and fusarium oxysporum 0148c are the primary pathogenic fungi that causes root rot and damping-off in young acacia plants. the best treatment to date is the use of biological control agents. phosphate solubilizing bacteria (psb) isolated from acid soil is a bacterial isolate classified as plant growth-promoting bacteria (pgpb). pgpb has an indirect function as a biocontrol agent for fungal pathogens. this study aimed to determine the potential of psb isolate ef.nap 8 in inhibiting g. philippii and f. oxysporum 0148c from acacia plants. the method used is a dual culture antagonism test and observation of abnormal hyphae after the antagonism process. the results showed that the isolate ef.nap 8 inhibited g. philippii by 34.44% and f. oxysporum 0148c by 33.33%. the abnormality of hyphae after antagonistic activity results in hyphal malformations such as hyphae lysis and hyphae coiling. the antagonistic activity of psb ef.nap 8 isolate is one of part of the ability of a bacterium classified as pgpb in the form of biocontrol activity against pathogenic fungi. this provides information regarding the opportunity to utilize ef.nap 8 as a candidate agent for controlling fungal pathogens on acacia plants. keywords acacia, f. oxysporum, g. philippii, plant growth-promoting bacteria, phosphate solubilizing bacteria 1. introduction control of pathogenic fungi on plantations, especially industrial plants, is essential. pathogenic fungi can damage plant growth and production, and control can be done using chemicals or biologically. the use of bacteria as agents for controlling pathogenic fungi in plants is currently a priority. one of which is pathogenic fungi that attack acacia plants such as ganoderma philippii and fusarium oxysporum. g. philippii is a fungal pathogen that infects young plants acacia mangium and eucalyptus pellita, causing root rot in both types of plants [1]. root rot caused by g. philippii is an essential disease in acacia plants. the threat of this disease increases in the planting of a. mangium [2]. acacia spp. including plant species widely developed for industrial plantation forests in several hti areas in sumatra and kalimantan, developed by pt. arara abadi, riau. acacia plants are susceptible to root diseases, causing an increase in the area of attack and damage caused from time to time. a. mangium plants in south sumatra, riau, east kalimantan, and several other areas were attacked by root rot disease [3]. besides g. philippii, f. oxysporum is also a significant problem in acacia seedlings. this pathogenic fungus causes sprouting or root rot during the seedling process. if it is not appropriately handled in the early stages of its growth, it will spread throughout the planting area [4]. various studies have been reported regarding the attack of f. oxysporum on various types of acacia plants such as a. koa [5], a. nilotica [6], even capable of causing the germination of a. mangium to fall six days after the germination process [7]. this disease control strategy is currently being developed, particularly in applying microbes to biological control agents [8][9]. in 2020, asril et al. [10] has succeeded in isolating phosphate solubilizing bacteria from acid soil in the institut teknologi sumatera area, one of which is ef.nap 8 isolate. phosphate solubilizing bacteria is one of the criteria for bacteria classified as plant growth-promoting bacteria (pgpb). pgpb provide many benefits to host plants directly or indirectly. the direct role of pgpb in plants includes dissolving phosphate, nitrogen, other minerals and hormone production, while the indirect mechanism is by suppressing the growth of plant pathogens [11]. pgpb has been known as a plant disease biocontrol agent. bacillus species are copyright holder: © asril, m., lisafitri, y., and siregar, a. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.118 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.118 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.118&domain=pdf&date_stamp=2022-04-08 j. multidiscip. appl. nat. sci. 83 antagonistic against fungal pathogens such as f. oxysporum. bacillus amyloliquefaciens is a phosphate-solubilizing bacterium that can inhibit the growth of f. oxysporum by producing secondary antifungal metabolites, 1aminocyclopropane carboxylic acid deaminase, chitinase and cellulase enzymes [12]. chitinase enzyme was able to inhibit the attack of f. oxysporum on chilli sprouts [13], g. boninense in oil palm plantations [14] and g. philippii, which causes the red root of acacia plants [15]. another species, b. megaterium, as a biocontrol agent, ralstonia solanacearum, can produce plant growth promoters (pgp) components, phosphate solubilization [16]. this study aims to test the inhibitory ability of the fungal pathogenic isolate of phosphate solubilizing bacteria ef.nap 8 and to determine the production of other secondary metabolites that are part of the ability of the pgpb group of bacteria. 2. materials and methods 2.1 materials the material used is phosphate solubilizing bacteria isolated from the institut teknologi sumatera acid soil, which is coded ef.nap 8 [10]. bacterial isolates were inoculated on nutrient agar from merck supplemented with 5% ca3(po4)2 medium and incubated at 30 °c. isolates of pathogenic fungi f. oxysporum 0148c and g. philippii were collected from pt. arara abadi, riau. pathogenic fungi isolates were inoculated on potato dextrose agar (pda) media from hi-media and incubated at 28 °c. 2.2 methods 2.2.1 preparation of bacterial isolate and antagonism testing phosphate solubilizing bacteria isolates ef.nap 8 were subjected to gram staining and biochemical tests include motility test in sulfide indole motility (sim) medium, catalase, oxidase, mr-vp, citrate, and urease test before antagonism testing. the method used to test antagonism against fungal pathogens using the dual culture method. pathogenic fungi in circular blocks measuring 6 mm were grown on the test pda media. meanwhile, the test bacteria isolates were grown on pda media with a streak technique of 3 cm. the distance between the bacterial isolates and the test fungus was 3 cm each (petri dish diameter = 9 cm) (figure 1). the treatment test was repeated three times. the antagonism test treatment was observed for nine days and incubated at room temperature (28 °c). the percentage of inhibition (p) of the pathogenic fungus was calculated by the formula: [100% x (r1-r2)/r1], where r1 is the length of mycelium growth of the pathogenic fungus towards the edge of the petri (3 cm) and r2 is the length of the mycelium towards the bacterial streak (3 cm) [17][18]. 2.2.2 abnormal hyphae observation microscopic observation of the abnormal hyphae structure of pathogenic fungi was carried out by observing the tip of the mycelium in the zone of inhibition after the antagonism test. the mycelium ends of the pathogenic fungi f. oxysporum and g. philippi from pda media were cut into block squares and placed on a glass object. hyphae abnormalities were observed under a microscope (light binocular microscope, olympus, japan) figure 1. schematic of dual culture antagonism testing . no test results 1 gram stain gram negative 2 cell shape bacil 3 motility motile 4 catalase + 5 oxidase + 6 indole 7 mr-vp 8 citric + 9 urease + table 1. characteristics of ef.nap 8 isolates. j. multidiscip. appl. nat. sci. 84 include bending of hyphae tips, hyphae coiling, hyphae lysis, hyphae splitting, branching, and dwarf hyphae [19]. 3. results and discussions 3.1 characteristic of isolate and ability of its antagonistic activity based on the results of gram staining and bacterial biochemical tests, isolate ef.nap 8 was a gram-negative rod-shaped bacterium and was motile. these bacteria can also produce catalase and oxidase enzymes and are classified as aerobic bacteria. ef.nap 8 isolate was also able to use citrate as a carbon source and produce urease (table 1). based on the results of the antagonism test of ef.nap 8 isolates against g. philippii and f. oxysporum 0148c showed that the phosphate solubilizing bacteria isolates from acid soil could inhibit the growth of both types of fungal pathogens with varying percentages of inhibition. ef.nap 8 isolate inhibited both types of fungi by 34.44% and 33.33%, respectively (figure 2). these results indicate that the bacterial isolate ef.nap 8 can be a biological control agent for acacia plant pathogenic fungi. the percentage of inhibition in vitro was carried out as an initial indication to determine the ability of ef.nap 8 phosphate solubilizing bacteria isolates in inhibiting fungal pathogens. the ability of phosphate solubilizing bacteria ef.nap 8 in inhibiting both types of fungal pathogens is thought to be due to the ability of pgpb indirectly to produce antibiotics and lytic enzymes [20]. ef.nap 8 isolate formed a strong inhibition zone against fungal pathogens. as a result, the growth of g. philippii and f. oxysporum 0148c was inhibited to above 30%. the percentage of inhibition of ef.nap 8 isolates against f. oxysporum was higher than the inhibition of b. amyloquefaciens pcfs against f. oxysporum, figure 2. percentage of inhibition of ef.nap 8 isolate against fungal pathogens after nine days of incubation. figure 3. antagonism of ef.nap 8 isolates against g. philippii and f. oxysporium 0148c on the ninth day of incubation. j. multidiscip. appl. nat. sci. 85 which was only able to inhibit 28% [12]. various antibiotics produced by pseudomonas pgpb have been identified, including the compounds amphicin, 2,4-diacetylphloroglucinol (dapg), oomycin a, phenazine, pyoluteorin, pyrrolnitrin, tensin, tropolone and cyclic lipopolypeptides [21]–[23]. the synthesis of various antibiotics/antifungals is the main characteristic of pgpb, which is most often associated with the ability of bacteria to prevent the proliferation of plant pathogens, especially fungi [23][24]. in addition to antibiotic/ antifungal activity, bacteria can produce enzymes such as chitinase, cellulase, β-1,3 glucanase, protease and lipase, which can partially degrade cell walls of pathogenic fungi. pgpb can synthesize one or more enzymes that are often found to have biocontrol activity against various fungal pathogens such as f. oxysporum, rhizoctonia solani, botrytis cinerea, phytophthora sp. and phytium ultimum [25]. the cell wall components of ganoderma spp and f. oxyporum hyphae are dominated by chitin and glucan [13][14][26] so that the lytic enzyme of chitinase and glucanase can easily degrade the cell wall components of pathogenic fungi. in addition, competition for nutrients and niches is one of the ways in the process of inhibiting pathogenic fungi [11]. although it is difficult to demonstrate directly, competition for nutrients can be observed in the test of ef.nap 8 isolates against g. philippii. in this case, isolate ef.nap 8 experienced significant growth. it can be seen that the growth of the isolate on the part that was scratched on the medium was getting thicker, the growth was getting wider and longer compared to the growth in the test with f. oxysporium 0148c (figure 3). 3.2 hyphae abnormal formation based on microscopic observation of abnormal hyphae structure of g. philippii and f. oxysporum 0148c after the antagonism test, it was shown that the inhibition of ef.nap 8 bacterial isolates against both fungal pathogens were in the form of inhibition of mycelium and thinning of hyphae walls. due to antagonistic activity, hyphae undergo changes in shape or malformations such as coiled hyphae, coiled hyphae and hyphae lysis (figures 4 and 5). hyphal abnormalities were dominated by the formation of lysed hyphae, both in g. philippii and in f. oxysporum 0148c. the presence of hyphae malformations of pathogenic fungi is thought to be due to a hyperparasitism mechanism of bacterial isolates against pathogenic fungi so that the fungal cell walls are degraded. the contact between bacteria and fungal pathogens causes isolates of figure 4. abnormality of g. philippii hyphae after the antagonism process, (a) coiled hyphae, (b), (c) lysis hyphae, (d) normal hyphae. j. multidiscip. appl. nat. sci. 86 antagonistic bacteria to produce compounds or secondary metabolites in the form of antimicrobials, causing damage to the hyphae of pathogenic fungi [27]. compounds produced by bacterial isolates will cause shortening, swelling of hyphae or other forms of malformations [28][29]. in addition to secondary metabolites, lytic enzymes such as chitinase and glucanase also play a role in the process of necrosis and hyphae lysis [14]. bacterial lysis activity is one of the mechanisms that have implications for disease biocontrol. in order to address the shortcomings of microscopic analysis, we propose the performance of molecular analysis techniques on the different strains [30]–[34]. 4. conclusions phosphate solubilizing bacteria isolated from acid soil ef.nap 8 had antagonistic activity against acacia plant pathogenic fungi, namely g. philippii and f. oxysporum 0148c, with inhibition percentages of 34.44% and 33.33%, respectively. inhibition of the fungus is indicated by the presence of hyphae abnormalities such as hyphae lysis, hyphae coiling and coiling as a form of indication of antagonistic activity in the form of production of metabolic compounds or lytic enzymes. this antagonistic activity provides information about the opportunity to use ef.nap 8 isolate as a candidate for biological control agent for acacia plant pathogens. molecular fingerprinting presents a powerful tool to seek the outmost information regarding antagonist effect. author information corresponding author muhammad asril — department of biology, institut teknologi sumatera, bandar lampung35365 (indonesia); orcid.org/0000-0001-8637-4190 email: m.asril@bi.itera.ac.id authors yuni lisafitri — department of environmental engineering, institut teknologi sumatera, bandar lampung-35365 (indonesia); orcid.org/0000-0002-9367-0200 bayo alhusaeri siregar — department of plant protection, pt. arara abadi sinar mas forestry, riau-28685 (indonesia); acknowledgement the authors express their gratitude to the institut teknologi sumatera for the funding support of this work through the smart itera research scheme with contract number b/306/it9.c1/pt.01.03/2019 to muhammad asril. figure 5. the abnormality of the hyphae of f. oxysporum 0148c after the antagonism process, (a) coiled hyphae, (b) twisted hyphae, (c-e) lysis hyphae, (f) normal hyphae. https://orcid.org/0000-0001-8637-4190 mailto:m.asril@bi.itera.ac.id https://orcid.org/0000-0002-9367-0200 j. multidiscip. appl. nat. sci. 87 references [1] w. gill, a. eyles, m. glen, and c. mohammed. 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(2017). “analysis of molecular proxies of a peat core by thermally assisted hydrolysis and methylation -gas chromatography combined with multivariate analysis”. journal of analytical and applied pyrolysis. 124 : 726–732. 10.1016/j.jaap.2016.11.014. https://doi.org/10.3390/polym14010194 https://doi.org/10.1016/j.orggeochem.2016.12.005 https://doi.org/10.1016/j.orggeochem.2016.12.005 https://doi.org/10.1016/j.chemgeo.2020.119644 https://doi.org/10.1016/j.chemgeo.2020.119644 https://doi.org/10.1016/j.jaap.2018.05.011 https://doi.org/10.1016/j.jaap.2016.11.014 18 journal of multidisciplinary applied natural science vol. 1 no. 1 (2021) research article received : august 27, 2020 revised : september 1, 2020 accepted : november 16, 2020 online : november 19, 2020 climate change increases the concentration of the greenhouse effect, this was caused by the lack of trees as a function of carbon sequestration. therefore, this study aims to map the vegetation distribution in the streets of the city of malang and to measure its carbon stocks. the used method was vegetation analysis, to calculate the estimation of biomass, carbon storage and co2 absorption using the allometric equation brown; brown and lugo; and morikawa. the study was conducted at the street lots of traffic activity, there are six stations representing the city of malang, those are tlogomas street, north of ahmad yani street, letjend sutoyo street, panglima sudirman street, sudanco supriadi street and kolonel sugiono street. the results of this study are that the most carbon-absorbing tree is albizia saman with a value of 287,656 kg and the region that absorbs the most carbon is panglima sudirman street, that located in the middle of the city. copyright holder: © madapuri, g. n., azwar, h. n., and hasyim, m. a. (2021) first publication right: journal of multidisciplinary applied natural science this article is licensed under: https://doi.org/10.47352/jmans.v1i1.5 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i1.5 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i1.5&domain=pdf&date_stamp=2021-01-09 j. multidiscip. appl. nat. sci. 19 figure 1. malang city map and research location (scale 1:20.000 km) [24]. the tools used in this study include a tape measure, rope, lux meter, gps, writing instruments, identification books, digital cameras.the material used is a shade tree along the arterial road which is the object of research and the organ of the shade tree. 2.2. inventaritation the inventory in this study aims to determine tree conditions, composition and dominance. the inventory is carried out through a vegetation j. multidiscip. appl. nat. sci. 20 type biomass (kg) stock carbon (kg) co2 absorption (kg) albizia saman 156888,85 78444,43 287655,71 annona muricata 72796,40 36398,20 133472,20 artocarpus elasticus 47208,69 23604,35 86557,14 barringtonia asiatica 45834,25 22917,13 84037,10 aridelia sp. 41124,16 20562,08 75401,14 leucaena leucocephala 37273,03 18636,52 68340,1 felicium decipiens 36942,79 18471,4 67734,61 cerbera manghas 13235,72 6617,86 24267,69 mangifera indica 7835,56 3917,78 14366,50 sterculia foetida 6911,027 3455,51 12671,37 chrysophyllum cainito 3894,22 1947,11 7140,05 polyalthia sp. 3264,58 1632,29 5985,62 ficus benjamina 3147,21 1573,61 5770,42 bauhinia purpurea 2969,67 1484,84 5444,90 ficus vens 2803,66 1401,83 5140,52 hibiscus tiliaceus 1872,50 936,25 3433,22 dimocarpus longan 1389,50 694,75 2547,64 lagestroemia speciosa 1254,99 627,50 2301,039 morinda citrifolia 772,56 386,28 1416,49 spathodea campanulata 735,05 367,53 1347,72 mimusops elengi 659,01 329,50 1208,2 mangifera ordonata 539,06 269,53 988,36 psidium guajava 506,82 253,41 929,26 pterocarpus indicus 445,53 222,76 816,88 terminalia catappa 409,67 204,83 751,14 persea americana 381,56 190,78 699,59 pinus merkusii 216,54 108,27 397,03 roystonea regia 124,10 62,05 227,54 peltophorum pterocarpum 93,69 46,84 171,78 melaleuca leucadendra 50,01 25,00 91,71 swietenia macrophylla 45,34 22,67 83,15 erythrina cristagalli 37,38 18,69 68,53 syzigium sp. 30,41 15,20 55,76 tamarindus indica 30,41 15,20 55,76 gliricidia sepium 24,68 12,344 45,27 table 1. biomass; stock carbon; and co2 absorption of plants in malang city roads. analysis, the following is an outline of the vegetation analysis procedure: 1. determined the location of the sampling using the principle of purposive sampling, which is based on the spacing. 2. this study uses 6 stations. each station has a length of 1,000m × 2m based on the guidelines for the provision and utilization of rth in urban areas. 3. each station has 5 substations with a size of 200 m × 2 meters. 4. the coordinates of each station are determined by gps. 5. shade tree species are identified, if the species name is not known, identification is carried out in the laboratory. j. multidiscip. appl. nat. sci. 21 6. measuring the circumference of the stem diameter at breast height (dbh) is carried out. 7. criteria for road shade trees to be used include a minimum trunk diameter of 15 cm and a fork of at least 2 meters from the top of the ground. 8. measuring the temperature, light intensity, and air humidity. 9. enter all field data into the observation form and then analyz vegetation data [25][26]. 2.3. estimation of co2 absorption, biomass, and carbon deposit calculation of co2 absorption, estimation of biomass and carbon deposits. estimation procedure for co2 sequestration and carbon storage based on ning et al. [27]: 1. record the local name and latin name of the tree to be measured. 2. the diameter of the shade trees is measured along with the vegetation analysis measurement procedure (to make it easier to use a 1.3 meter long wooden stick). if the ground surface and trunk are uneven, it can be seen in the dbh measurement rules. 3. the biomass of shade trees was measured using the allometric equation. 4. measurement of co2 absorption, biomass and carbon deposits is carried out by entering the biomass value of shade trees in the equation. 3. results and discussions the results of carbon calculations on arterial road in malang showed in the table 1. based on the result showed that the plant that had the highest absorption and carbon storage was albizia saman. the albizia saman plant has a wide cover and wide tree diameter. this was confirmed in the research that said the greater base area, that the greater carbon stock. this carbon deposit will make the tree bigger [22]. based on experiment results by setiawan [24]. st. tlogomas has carbon absorption 269.837 kg and carbon stock 73.585 kg, st. ahmad yani has carbon absorption 352.727 kg and carbon stock 96.189 kg, st. jendral sutoyo has carbon absorption 263.262 kg and carbon stock 71.792 kg, st. panglima sudirman has carbon absorption 285.642 kg and carbon stock 77.895 kg, st. sudanco supriadi has carbon absorption 284.318 kg and carbon stock 77.534 kg, st. kolonel sugiono has carbon absorption 118.007 kg and carbon stock 32.181 kg. by compare with data we got on the below, carbon absorption and carbon stock on every street has decreased cause many trees are cut down for road repair, construction and land conversion for industry. the highest carbon deposits and uptake is in the st. panglima sudirman (figure 2). this is because this area has high density vegetation. according of the literature states that the amount of carbon stored between land varies, depending on the diversity and density of existing plants, soil types and how they are managed [28]–[30]. 4. conclusions from this research, we conclude that carbon stocks and uptake in malang city has increased because there are many new plants planted. the largest carbon stocks and uptake are in the panglima sudirman road area and the plants that store the most carbon stock and absorption are albizia saman. author information corresponding author gita niken madapuri — department of biolost . p an gl im a su di rm an st . l et je n su to yo st . t lo go m as st . n or th a hm ad y an i st . k ol on el s ug io no st . s . s up ri ad i 0 50000 100000 150000 200000 250000 300000 350000 biomass (kg) carbon deposits (kg) co2 absorption (kg) figure 2. regional observation result. j. multidiscip. appl. nat. sci. 22 gy, maulana malik ibrahim state islamic university of malang, malang-65144 (indonesia); email: gitaniken1203@gmail.com authors haidar nazarudin azwar — department of biology, maulana malik ibrahim state islamic university of malang, malang65144 (indonesia); muhammad asmuni hasyim — department of biology, maulana malik ibrahim state islamic university of malang, malang65144 (indonesia); acknowledgement this research supported by science and technology faculty, maulana malik ibrahim malang islamic state university which has lent research tools and green tech for give us research funding. references [1] r. j. keenan. 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(2020). “carbon accumulation in agroforestry systems is affected by tree species diversity, age and regional climate: a global meta-analysis”. global ecology and biogeography. 29 (10): 1817–1828. 10.1111/geb.13145. http://doi.org/10.1111/gcb.13791 http://doi.org/10.1016/j.foreco.2019.03.053 http://doi.org/10.1111/geb.13145 13 journal of multidisciplinary applied natural science vol. 1 no. 1 (2021) research article structural, morphological and elemental analysis of selectively etched and exfoliated ti3alc2 max phase received : september 12, 2020 revised : september 21, 2020 accepted : october 19, 2020 online : october 20, 2020 in the present research major focus is on the synthesis of materials that can be easily used in small portable devices and as energy storage devices. here we focused on a new family of 2d materials ti3c2 (mxenes). ti3alc2 (max phase) was intercalated using selective etching of aluminium present in the max phase. the etching was done using hf in combination with hcl followed by delaminated in dmso medium using ultrasonication. the synthesized samples were physically characterized via xrd, sem and edx. the xrd diffractogram confirms the formation of mxene through its characteristic plane (002) arising at 2 θ~9°. the morphological study revealed the stacked layered sheet like structure obtained through sem. the elemental confirmation of removal of aluminium was done as indicated by edx spectroscopy. the 2d materials like graphene are widely known for their distinctive properties that are not found in their bulk counterparts, thus making them ideal for a vast range of applications extending from electronic and optoelectronic devices to electrochemical catalysis generally, 2d materials are produced by exfoliating layered 3d materials with weak van der waals-like coupling between layers recent technological advancements have allowed the synthesis of various 2d materials by chemical exfoliation or mechanical cleavage of layered 3d precursors. mxene are newly discovered 2d ceramic materials belonging to the family of transition metal carbides, carbonitrides and nitrides naguib et al. was the pioneer in synthesizing a 2d nanocrystalline material by exfoliation of layered solids in 2011 max phases are the precursors of mxene, they are a large family of ternary carbides or nitrides with a general chemical formula of mn+1axn, where m stands for early transition metal, a is an element generally from group iiia or iva, x is either carbon or nitrogen and n =1,2,3 and so on max phases have layered structure in which mx units and a layers are alternately stacked. because the m-x bonds are much stronger than ma bonds, the a layer can be selectively removed from max phases by hf etching, thus creating mxenes the mxene made by hf etching are attached with functional groups like oxygen (=o), hydroxyl (-oh) or fluorine (-f) or a combination of these, due to its high surface energi thus, the general formula of mxenes can be summarized as mn+1xntx, where t represents surface termination groups and x is the number of termination groups per formula unit mxenes exist in a multi-layered structure, which appears as planar sheets stacked in flakes. these multi-layered structures can be delaminated into single-layer flakes by sonication mxenes have hydrophilic surfaces, good chemical stability, fabulous electrical conductivity, and environment friendly. they have plenty of exciting mechanical, electronic, magnetic, and electrochemical properties which have drawn a huge research interest in this area mxene have a flexible and layered structure. owning to their 2d morphology, they can easily form composites with other materials, so they can integrate the properties of different materials in a complementary way. due to their low vapour pressure, excellent conductivity, non-flammability, and outstanding electrochemical activity, mxene and its composites copyright holder: © jahan, n., hussain, s., rahman, h. u., manzoor, i., pandey, s., habib, k., ali, s. k., pandita, r., and upadhyay, c. (2021) first publication right: journal of multidisciplinary applied natural science this article is licensed under: https://doi.org/10.47352/jmans.v1i1.6 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i1.6 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i1.6&domain=pdf&date_stamp=2021-01-08 j. multidiscip. appl. nat. sci. 14 can operate at very high voltages compared to the other electrolytes. therefore, they can find use as high-performance electrodes in energy storage devices, catalysis, supercapacitors, and pseudocapacitors they have cations intercalation capability so they can also work as anode material for li-ion batteries researchers are also looking to use mxene as reinforcement for other composites, to increase their mechanical properties. recently mxenes and mxene-based composites find prominent use in the environment related fields. they have been used as efficient catalysts or co-catalysts for electro/photocatalytic water splitting and photocatalytic reduction of carbon dioxide (co2). they can remove contaminants in water including heavy metal ions, organic dyes, eutrophic substances, and nuclear waste. they have also been applied to biosensors and gas sensors, and exhibiting excellent performances. many theoretical papers have synthesized and mxene and predicted their properties and applications. herein, we have analyzed the mxene ti3c2 which was selectively etched and exfoliated from ti3alc2 max phase. a complete analysis on its structural, morphological, and elemental properties has been done. the reports suggest that the synthesized mxene samples could be used for various sensing, electromagnetic shielding and energy harvesting, and storage applications. for the synthesis of mxenes, max phase ti3alc2 powder (sigma aldrich), the etchants which include hydrofluoric acid (hf) and hydrochloric acid (hcl), and dimethyl sulfoxide (dmso) for delamination were used. all chemicals are analytical grade and used directly without further purification. prior to the experiment, teflon and glass beakers were cleaned with aqua regia for 3 hours, soap solution, and de-ionized water, respectively. 2.2. synthesis of ti3c2tx mxenes synthesis was done through selective etching of aluminium layers from the bulk max phase at room temperature. with a combination of hf and hcl of different concentrations of etchant is used to etch out of the max phase powder. ti3c2tx was synthesized by etching al from ti3alc2 of 1 gram appropriated from teflon beaker and 20 ml of etchant in which 30%wt hf and 9m hcl is further blend for 48 hours. as the etching is concluded, the figure 1. x-ray diffraction spectra of ti3alc2 (max phase) and ti3c2tx (mxene). figure 2. (a) represents the sem pictograph of ti3alc2 (max phase) and (b) represents the exfoliated accordion type morphology of stacked layers of ti3c2tx (mxene) flakes. a b j. multidiscip. appl. nat. sci. 15 solution is cleaned numerous times via centrifugation at 7,000 rpm for 10 minutes for the respective cycle until the ph value reached approx 6. the etching is not sufficient for complete removal of aluminium, so for further exfoliation, the above-exfoliated powder is intercalated with dmso for 8 hours. then, the intercalated sample is sonicated in deionized followed by washing several times at 7000 rpm to remove the residual. then, the obtained powder sample is treated with 1 m naoh and further washed away. after washing, the obtained sample is dried in an oven at 60 °c for 48 hours for obtaining flakes mxenes powder. 2.3. material characterization techniques x-ray diffraction (xrd) analysis was carried out for structure characterization with a powder diffractometer (bruker, ecod8 advance) with cu kα radiation (wavelength=1.5418 å). a scanning electron microscope, sem, (jeol jsm 7600f) equipped with an energy dispersive spectrometer, exs was cast-off for getting surface morphological and elemental confirmation. 3. results and discussions 3.1. structural analysis figure 1 shows the x-ray diffraction plot of the prepared ti3alc2 max phase and ti3c2tx mxene. in this plot, the dominant peak of ti3alc2 max phase appears at 2θ~9° while for the ti3c2tx mxene appears at 2θ ~11°. those peaks correspond to the (002) planes of hexagonal crystal structure. the multiple peaks show that the max phase powder and mxene powder were polycrystalline in nature. the max phase powder had excellent crystallinity compared to mxene powder. the crystallite size of max phase powder and mxene powder were calculated by using the debyescherrer formula: (1) where λ is the wavelength of x-ray, θ is the bragg’s angle, and β is the full width at half maxima (fwhm) of the dominating peak corresponding to the (002) plane. the average crystallite size of ti3alc2 max phase was 37.56 nm and ti3c2tx mxene was 14.68 nm as shown in table 1. it is observed that the crystallite size decreases after etching. in comparison of ti3alc2 max phase and ti3c2tx mxene, most of the diffraction peaks disappeared in ti3c2tx mxene. it shows the depletion of ti3alc2 max phase into ti3c2tx mxene phase (i.e. removal of al). the intensity of the peak at 39° almost decreased, which shows the removal of al [12]. in addition, the shift of ti3c2tx mxene peaks from 9.74° to 11.11° shows the compression of the interlayer distance from 9.07å to 8.03å. 3.2. morphological study through sem the surface topography image analysis of obtained synthesize mxenes flakes were carriedtable 1. average crystallite size and interplanar spacing of max phase and mxene. parameters ti3alc2 (maxphase) ti3c2tx (mxene) avg. crystallite size (nm) 37.56 14.68 interlayer spacing (å) 9.07 8.03 figure 3. typical edx spectra of ti3c2tx after the selective etching of aluminium layers from the max phase. j. multidiscip. appl. nat. sci. 16 out via sem. the tightly bonded layer of the max phase flakes are found be exfoliated following hf and hcl etchings. in general, the stacked layers of mxene resemble to the shape of accordion with irregular profile bounded by weak van der waals force. the layers could be more exfoliated via the delamination process. figure 2 shows the sem image of ti3alc2 (max phase) and ti3c2tx (mxene). 3.3. elemental analysis from edx spectroscopy the information about the elemental composition in the exfoliated mxene sample is given by the energy-dispersive x-ray (edx) spectroscopy in figure 3. it is evident from the plot that the traces of aluminium in the sample are negligible, i.e. below 0.5%. the presence of chlorine is in small amount but it is assumed to be as a result of hcl used in the process of etching of the sample and is proportionately higher than aluminium. a greater intensity peak of silicon is noticed because of the silicon substrate upon which the mxene sample is drop casted for various analyses. carbon is present in large quantity whereas titanium is comparatively low in concentration. table 2 gives a tabular representation of the concentration in weight percentage of each element present in the sample. 4. conclusions 2d materials like mxene are newly discovered ceramic materials, which have the features to incorporate the properties of different materials. this paper highlights the mxene synthesis with a fusion of etchants like hf in combination with hcl that precisely etched the aluminium layer. the xrd demonstration of phase represents the excellent crystallinity and the most prominent peak arises in the range of 9-11° representing the (002) plane. sem displays non-uniform profile of mxene having accordion like structure after further delamination process. the edx plot reveals that most of the aluminium are easily etched from the max phase, although the negligible traces of aluminium < 0.5% is still present. mxene synthesized by the selectively etching process can be best suited for applications in energy storage devices where the high voltage operation of its composites can swiftly carry out the faradaic reactions. author information corresponding author chandramani upadhyay — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0002-7307-6968 email: cupadhyay012@gmail.com authors nushrat jahan — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0001-7888-4166 salim hussain — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0002-1775-8630 huzef ur rahman — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0002-6041-1034 insha manzoor — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0001-5629-6347 shashank pandey — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0001-6963-1271 kamran habib — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0001-9891-0042 syed kaabir ali — centre for nanoscience and table 2. the percentage of elements present in the sample after the formation of mxene. element series atomic % silicon k-series 36.48 titanium k-series 13.69 carbon k-series 48.49 chlorine k-series 0.85 aluminium k-series 0.49 total 100 https://orcid.org/0000-0002-7307-6968 mailto:cupadhyay012@gmail.com https://orcid.org/0000-0001-7888-4166 https://orcid.org/0000-0002-1775-8630 https://orcid.org/0000-0002-6041-1034 https://orcid.org/0000-0001-5629-6347 https://orcid.org/0000-0001-6963-1271 https://orcid.org/0000-0001-9891-0042 j. multidiscip. appl. nat. sci. 17 nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0002-8111-2244 reetika pandita — centre for nanoscience and nanotechnology, jamia millia islamia, new delhi-110025 (india); orcid.org/0000-0003-0316-5647 references [1] b. anasori, c. shi, e. j. moon, y. xie, c. a. voigt, p. r. c. kent, s. j. may, s. j. l. billinge, m. w. barsoum, and y. gogotsi. 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availability; demand and distribution. optimization of production by producers and optimization of the ordering of goods by distributors are important steps to maintain price stability for necessities. optimization of production and ordering of staple goods will have a significant impact on the financial sector because it is closely related to the prediction of the number of raw materials used, production costs, storage costs, and also distribution costs of goods. one of the fuzzy inference methods that can be used for this optimization is the mamdani method. to get the output on the application of the fuzzy logic of the mamdani method, four stages are needed, formation of fuzzy sets; application of implication functions; composition of rules and defuzzification. fuzzy logic mamdani method can be used to predict the amount of national rice that must be produced. if it is known that the need is 21,908,784 tons of rice and the supply is 65,457,456 tons, the amount of national rice that must be produced is 14,624,592 tons. seen on a global scale, food security remains a serious problem, especially in developing countries where challenges of sustainable food supply are exacerbated by rapid population increases, limited access to food intake, vulnerability, price volatility, protective measures imposed by governments, and so on [1]. several studies have shown that food insecurity is influenced by various factors, including population growth, availability of fertile land and water resources, and climate change [2]. indonesia is one of the developing countries that have fairly good food security. as an indicator, the people in indonesia are guaranteed a sustainable supply of food and other necessities. even so, there is often instability in the prices of necessities, especially before the religious holidays. as a consequence, people's purchasing power decreases and the fulfillment of primary needs is limited, even though their availability is abundant this is certainly material for self-evaluation for policymakers in finding solutions so that prices of staple goods are always stable so that it has an impact on the welfare of the community [3]. it must be admitted that rice and other staple goods, such as meat, cooking oil, salt, and sugar, are the most important commodities in indonesia because of their role as a staple food, which the majority of every indonesian population consumes every day as nutritional intake. not only that, various staple goods are also a dominant strategic commodity in the indonesian economy because they are closely related to monetary policy and involve socio -political issues. to meet these needs, many things affect the stability of availability and price, starting from climatic conditions, logistics systems, and domestic market conditions as well as global market conditions. when examined further, price movements of staple goods, especially rice, are strongly influenced by three factors [4]. the first factor is the factor of the availability of the goods themselves, which comes from the crops of the farmers for rice and corn; the production of meat, eggs, and milk from the farmers and the production from the company for other necessities such as sugar and cooking oil. the second factor is the demand factor from consumers. consumer demand is often influenced by several factors, for example an increase in demand ahead of national religious holidays; panic or consumer concerns about the scarcity of various commodities in the market and changes in consumption copyright holder: © wawan, w., zuniati, m., and setiawan, a. (2021) first publication right: journal of multidisciplinary applied natural science this article is licensed under: https://doi.org/10.47352/jmans.v1i1.3 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i1.3 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i1.3&domain=pdf&date_stamp=2021-01-10 j. multidiscip. appl. nat. sci. 37 patterns, preferences, and diversification of staple foods consumer. the third factor is the distribution factor that can trigger an increase and decrease in the price of necessities. the distribution process incurs several costs, such as the number of distribution costs, the distance from the production center to the consumption center, and the disruption in the distribution process. indonesia has no longer been able to be rice self -sufficient since 1984. the deficit forced the government to import rice from the philippines and thailand as a result of great demand. it is difficult to know so many variables that cause rice selfsufficiency. it could be induced, for example, by weak agricultural infrastructure in indonesia, government policies, a lack of land to open up new fields, as well as a lack of labor. given that indonesia is a vast tropical area and also has ample human capital, this is rather ironic. the government is still trying to find a solution so that it is possible to solve the rice self-sufficiency problem and increase the welfare of the citizens of indonesia. therefore any policy determined whether it will work in the rice self-sufficiency program must be re-evaluated by the government. the performance of government policy would have an effect on the increase in the amount of rice production. one way to quantify uncertainty in forecasting using the fuzzy logic of the mamdani method is to calculate the amount of rice production that is uncertain [4]. taking into account these few things, there are at least four things that need to be considered in maintaining the price stability of staple goods, namely: production; availability; demand and distribution. furthermore, in this article, we will see how fuzzy logic contributes to controlling the price of staple goods. in this article, we will also provide an example of how to optimize production using the mamdani method of fuzzy logic application. this procedure is an important part of providing a reference for producers and distributors of necessities in managing the supply chain to minimize the costs of production and distribution of goods. as a final implication, the stability of prices for necessities on the market is maintained. based on this explanation, the purpose of writing articles are as follows providing knowledge related to the use of the fuzzy mamdani method in the optimization of production and ordering of national rice. fuzzy logic fuzzy can be interpreted as fuzzy or vague. fuzzy logic is a set of mathematical principles for the representation of knowledge based on the degree of membership fuzzy logic is a form of many logical values; relates to the use of reasoning for approximations. compared to traditional or classical binary sets (where variables can take true or false values), fuzzy logic variables have truth values that range in levels between 0 and 1. fuzzy logic itself has been extended to deal with the concept of partial truth, where the truth of value can range between completely true and completely false linguistic variables if a variable uses words in the colloquial language as its value, then the variable is called a linguistic variable, where words are characterized by a vague set defined in a defined universe of variables linguistic variables are defined as variables whose value is predictive statements, economic planning, and management, in natural or artificial language j. multidiscip. appl. nat. sci. 38 ∪ 3. results and discussion it has been mentioned earlier that controlling the prices of basic needs involves four things, namely: (1) the production process, (2) the availability of goods, (3) the amount of market demand or comj. multidiscip. appl. nat. sci. 39 munity needs, and (4) the cost of distributing goods. producers and distributors contribute significantly to the price of necessities in the market. for several important commodities such as rice, the amount of agricultural production, availability in bulog, and community needs greatly affect the price of rice. for producers providing other staple goods such as cooking oil, sugar, milk, and other staples, the optimization of production will have major implications for the selling price in the market. optimization of the production of staple goods will affect the financial sector because it is closely related to estimates of raw materials, production costs, storage costs, and also distribution costs of goods. as part of controlling the prices of necessities, the production optimization process is important. on the other hand, distributors also play a crucial role in maintaining the price stability of necessities. distributors must be careful in ordering products from producers. of course, ordering products must also be adjusted to the availability of goods and market demand. if the distributor is not careful in this matter, it can have implications for the distributor's loss. in this regard, fuzzy logic can be used in optimizing various sectors to control prices for necessities in the market. for producers, fuzzy logic can contribute to the optimization of production, while distributors can be used in optimizing orders from producers. the fuzzy approach can be effectively utilized to deal with imprecision and uncertainty [11]. this approach can be used for performance evaluation that allows organizations, producers, or distributors to carry out professional assessments in evaluating the supply chain of staple goods. in real problems in the field, performance evaluation techniques with fuzzy logic can be used to handle cases such as subjectivity, obscurity, and imprecise information. the application of fuzzy set theory in evaluation systems can improve evaluation results [12]. when viewed historically, the initial idea of applying fuzzy sets to control problems is presented more explicitly by chang and zadeh [13]. in subsequent developments, research has been carried out on real problems initiated by mamdani and assilian [14] and mamdani [15]. until now, there have been many studies using fuzzy logic in the case of controlling the prices of necessities, for example, research [16] has provided an application for the problem of cryptic production inventory planning using the optimality equation. many studies have included distortion factors in preparing the optimization of production quantities. for example [17] developed and investigated the economic order quantity (eoq) model in the fuzzy sense by considering stochastic demand, partial backlogging, and the degree of fuzzy damage. the proposed model is also extended to the case where the partial backlogging factor is assumed to be a fuzzy number. the total minimum cost and optimal order quantity are obtained by defuzzifying the total cost through the signed distance method. nagoor gani and maheswari [18] discussed the retailer's ordering policy of two levels of late payment taking into account the selling price per item being higher than the purchase cost per item. the demand and selling price per item is taken as triangular fuzzy numbers. the function principle is used to calculate the optimal cycle times and the economical order quantity. the method of representation of the tiered average integration is used for defuzzification in this case. another study, lee and yaou [19] developed the economic production quantity (epq) model in which demand and production quantity are assumed to be vague. then lo et al. [20] presented the epq model which includes uncertain factors such as unreliable machines, defective products, or imperfect goods. they have used the epq model to explore the most economical choice of production cost factors based on the uncertainties that may exist. furthermore, chen and chang [21] developed an epq model with irreparably defective production yields. chang [22] discusses how to get the economic order quantity, when the quantity demanded is uncertain. while lee and yao [19] and lin and yao [23] have also written several articles on cryptic production models, they have not yet developed an inventory model with imperfect products. an inventory model with imperfect products was carried out by chen, et al. [24] who proposed a fuzzy economic production quantity (fepq) model with imperfect products that can be sold at a discount. in this model, costs and quantities are expressed as trapezoidal fuzzy numbers. these various studies have provided important references regarding the contribution of fuzzy logic in controlling the prices of necessities. optimization of production and ordering of goods by considering various things such as machine reliability, imperj. multidiscip. appl. nat. sci. 40 fections in production, availability of imperfect or defective products, and uncertain market demand is urgent considerations in planning the supply of staple goods. fuzzy logic has made a valuable contribution in controlling the prices of necessities. an important part of fuzzy logic is represented by fuzzy logic control (flc), derived from control theory based on the mathematical model of an open-loop process to control. flc has been successfully applied to a variety of practical problems such as control of warm water, robots, heat exchange, traffic at intersections, car speed, automotive engineering, washing machines, and so on [25]. in connection with the optimization of production and ordering of staple goods, several methods can be used in the application of fuzzy logic in this field, for example, the mamdani method, the tsukamoto method, and the sugeno method. as presented iancu [25], the most commonly used fuzzy inference technique is the mamdani method proposed by mamdani and assilian [14], as a first attempt to control a steam engine and boiler combination by synthesizing a set of linguistic rule controls obtained from experienced human operators. the work was inspired by the equally influential publication of [8]. interest in fuzzy control has continued since then, and the literature on the subject has grown rapidly to date. in the mamdani method, the fuzzy implications are modeled by the mamdani minimum operator, the conjunction operator is min, the t-norm of the min composition rule, and for rule aggregation, the max operator is used. according to iancu [25] there are four steps in using the mamdani method, namely: (1) fuzzification, (2) rule evaluation, (3) aggregation of the rule output, and (4) defuzzification. the first step is to take a sharp input, x_0dany_0, and determine the extent of where these inputs belong to each of the corresponding fuzzy sets. in the second step, if the given fuzzy rule has multiple antecedents, the fuzzy operator (and or or) is used to get a single number that represents the antecedent evaluation result. to evaluate the separation of the rule's antecedents, one uses the fuzzy or operation. next in step three, the membership functions of all consequent rules previously truncated or scaled are combined into a single fuzzy set. as for step four, the most popular defuzzification method is the centroid technique. more easily, [10] said that to get the output from the mamdani method, four stages are needed, formation of fuzzy sets; application of implication functions; composition of rules and 4) defuzzification. furthermore, a simple example will be given related to the use of the mamdani method to predict the amount of production of one of the basic needs, namely rice, if it is known that the community's rice needs and availability in bulog. the data used for this example is data from bps relating to the availability of rice commodities in indonesia in the period 2006 to 2017 [4]. based on data from bps, information is obtained that the lowest amount of production in that year was 54,454,947 tons (2006), while the highest production amount occurred in 2017 with a total of 79,175,945 tons [26]. the lowest rice availability occurred in 2006 with 54,506. 436 tons, while the highest rice availability occurred in 2017 with a total of 79,682,632 tons. on the other hand, based on bps data, it is also known that the smallest national demand for rice occurred in 2007 with a total of 20,587,547 tons, while the largest amount occurred in 2017 with the total demand for rice reaching 22,847,706 tons. in this case, there are three variables, namely: 2 input variables, including the need variable and the supply variable, while for output there is 1 variable, namely: rice production. the need variable has 2 linguistic values, namely up and down, the inventory variable has 2 linguistic values, namely a lot and a little, while the production variable has 2 linguistic values, namely increasing and decreasing. rice production uses the following four fuzzy rules. • [r1] if demand down and supply a lot, then production is less • [r2] if demand is down and inventory is little, then production is less • [r3] if demand increases and supply is many, then production increase • [r4] if demand increases and supply is little, then production increase the solution for this example using the mamdani method of fuzzy logic is as follows: j. multidiscip. appl. nat. sci. 41 1. fuzzy set formation the formation of fuzzy sets is the first step taken when using the mamdani method. there are three fuzzy variables to be modeled, namely: · requirement: consists of two fuzzy sets, namely: down and up. · inventory: consists of two fuzzy sets, namely: little and many. · production: consists of two fuzzy sets, namely: reduce and add. 2. application function implications for example, it is known that the total national rice demand in 2018 is 21,908,784 tons and the total supply is 65,457,456 tons of rice. the application used in this example is the min rule. · [r1] if the need down and supply a lot, then production is less. · [r2] if demand is down and inventory is little, then production is less. obtained · [r3] if supply increases and supplies are many, then increased production · [r4] if needs up and supply is little, then production increase. obtained 3. composition of rules the method used to compose all rules is the max method. the membership function for the results of this composition is as follows. 4. defuzzifikasi the defuzzification method used is the centroid method with a continuous domain, using the following formula. with this formula, the following results are obtained. so for the example given gives results that the amount of national rice that must be produced if it is known that the need is 21,908,784 tons of rice and the supply is 65,457,456 tons is 14,624,592 tons of rice. 4. conclusions optimization of rice production by producers and optimization of the ordering of goods by distributors are important steps to maintain price stability for necessities in the market. optimization of production and ordering of staple goods will have a significant impact on the financial sector because it is closely related to the prediction of the number of raw materials used, production costs, storage costs, and also distribution costs of goods. one of the fuzzy inference methods that can be used for this purpose is the mamdani method. to get the output on the application of the fuzzy logic of the mamdani method, four stages are needed, formation of fuzzy sets; application of implication functions (rules); composition of rules and defuzzification. fuzzy logic mamdani method can be used to predict the amount of national rice that must be produced. if it is known that the need is 21,908,784 j. multidiscip. appl. nat. sci. 42 tons of rice and the supply is 65,457,456 tons, the amount of national rice that must be produced is 14,624,592 tons. author information corresponding author wawan wawan — department of mathematics education, institute for islamic studies ma’arif nahdlatul ulama (iaimnu), metro-34111 (indonesia); https://orcid.org/0000-0002-5573-623x email: awanwawan0215@gmail.com authors mai zuniati — department of english education, institute for islamic studies ma’arif nahdlatul ulama (iaimnu), metro-34111 (indonesia); https://orcid.org/0000-0002-1231-1426 agus setiawan — department of mathematics education, institute for islamic studies ma’arif nahdlatul ulama (iaimnu), metro-34111 (indonesia); https://orcid.org/0000-0002-9712-5461 acknowledgement this work was supported by lembaga pengembangan penelitian dan pengabdian kepada masyarakat (lp3m) institute for islamic studies ma’arif nu (iaimnu) metro lampung with grant number 108/lp3m/iaim-nu/x/2019 references [1] v. erokhin. 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18, 2022 accepted : october 21, 2022 online : october 28, 2022 abstract the aim of this study was to identify and describe the euglenids (euglenophyceae, euglenophyta) from the peat waters of palangka raya, central kalimantan, indonesia. this study revealed that 8 species of euglenids were found in the study sites which belong to 3 genera, namely euglena, lepocinclis, and phacus. the euglenid species identified include euglena gracilis g.a. klebs 1883, euglena mutabilis f. schmitz 1884, lepocinclis acus (o.f.müller) b.marin & melkonian 2003, lepocinclis ovum (ehrenberg) lemmermann 1901, lepocinclis spirogyroides b.marin & melkonian 2003, phacus cordatus (pochmann) zakryś & lukomska 2015, phacus helikoides pochmann 1942, and phacus orbicularis hübner 1886. the eight euglenid species found in this study have never been reported before, thus these findings provide additional new data regarding algae diversity in peat waters of central kalimantan, indonesia. keywords euglena, lepocinclis, microalgae, peat waters, phacus, phytoplankton 1. introduction research on algae in central kalimantan, indonesia, especially freshwater algae from peat waters has been intensively carried out since the last few years [1][2]. these studies have contributed the scientific data regarding the diversity of algae in the peat waters habitat. adam [2] reported 10 species of desmids from the peat waters in palangka raya, consisting of 5 cosmarium species and 5 euastrum species respectively. the most recent study is related to the morphological study of coelastrum cambricum, a microalgae species commonly found in peat waters. this study revealed the distinctive morphological characteristics of c. cambricum where 6 cells were observed in the center of the coenobium forming a pentagonal pattern that has the potential to be studied further as a key feature of coelastrum species identification [1]. similar studies still need to be done to develop scientific data related to the diversity of algae in indonesia, especially central kalimantan which has extensive peat water habitats. euglenophyceae is a group of flagellate green algae found in a variety of freshwater and marine environments [3][4]. members of the euglenophyceae are distinctive with red eyespots that are part of the eyespot apparatus [5]. the eyespot apparatus consists of the paraflagellar body, which connects the eyespot to the flagellum. the function of eyespot apparatus is as a photoreceptive organelle [6]–[8]. according to algaebase [9], there are 1,007 algal species belonging to euglenophyceae which lack references that provide data on their distribution in indonesia, especially in central kalimantan. recent studies regarding the diversity of microalgae in indonesia have revealed several species of euglenophyceae that are distributed in bengkulu (sumatra) [10], south sumatra [11], east java [12], central lombok [13], and west kalimantan [14] which belong to the genera euglena, trachelomonas, lepocinclis and phacus. the aim of this study was to identify and describe the euglenids (euglenophyceae, euglenophyta) from the peat waters of palangka raya, central kalimantan, indonesia, to contribute to the development of scientific data on algae diversity. 2. materials and methods 2.1. study site and sample collection publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2022 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.145 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.131 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.145&domain=pdf&date_stamp=2022-10-28 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 82 table 1. the list of published works used to assist the identification phase. this research was conducted in shallow peat waters located in palangka raya, central kalimantan, indonesia (figure 1). water samples were collected directly from peat waters along with sediment from the bottom of the trench, then stored in collection bottles. 2.2. microscopic observation and microphotography the samples were observed microscopically using olympus cx21 at 400× (10× ocular; 40× objective) magnification in the laboratory of biology education program, university of palangka raya. microphotography method explained as follows: specimen objects found that are suspected to be euglenid species are immediately photographed using 64megapixel cellphone camera directly to the eyepiece of the microscope for further identification. 2.3. image processing the photographed specimen image was then processed for color and detail enhancement using adobe photoshop cc. scale bar was added using imagej software version 1.53g [15] to estimate the object length. scaling on imagej uses the method of estimating cell size by knowing the diameter of the field of view of the microscope [2][16], which in this study used a 400× magnification with a diameter of 0.4 mm or 400 μm. 2.4. euglenid identification the identification of euglenid was carried out by analyzing the observed morphological characteristics (e.g., the presence of red eyespot & flagella; shape of the cells, and cell size) both qualitatively and quantitatively. morphotaxonomic identification was done up to the species level using all available information. the following published works were used to assist the identification phase (table 1). 3. results and discussions this study revealed 8 euglenid microalgae species found in the peat waters of palangka raya, indonesia, consisting of 3 genera, e.g., euglena (2 species), lepocinclis (3 species), and phacus (3 species) as presented in the table 2. all euglenid species were found in shallow peat waters with the depth ranges from 30-50 cm and ph of 5. these characteristics of peat waters are in accordance with several references which describe that the color of peat water usually appears in blackish brown color, has acidity range between ph 3-5 and the organic matter content is relatively high [21][22]. 3.1. euglena ehrenberg, 1830 euglena is spindle-shaped unicellular flagellate green algae with cylindrical or oval body; has a single flagellum embedded at the anterior end of the cell; red eyespots are clearly visible on the anterior area of the cells [5][9][20]. currently, euglena consists of 156 taxonomically accepted species names and 52 accepted varieties [9]. taxonomic enumeration euglenozoa class euglenophyceae order euglenida family euglenidae genus euglena ehrenberg 1830 no. published works ref. 1 algaebase. world-wide electronic publication. national university of ireland, galway. [9] 2 a short guide to common heterotrophic flagellates of freshwater habitats based on the morphology of living organisms [17] 3 algae identification lab guide: accompanying manual to the algae identification field guide [18] 4 a beginner’s guide to freshwater algae [5] 5 easy identification of the most common freshwater algae [19] 6 how to know the freshwater algae [20] j. multidiscip. appl. nat. sci. 83 3.1.1. euglena gracilis g.a. klebs 1883 taxonomy: e. gracilis g.a. klebs 1883 is currently taxonomically accepted species name according to algaebase. first published in a paper entitled “über die organisation einiger flagellatengruppen und ihre beziehungen zu algen und infusorien” by klebs in 1883 [9]. description: e. gracilis were observed to have bright green color; a clearly visible red eyespot; a single flagellum; and known to be the largest species of euglena found in this study. the shapes of the cell are dynamic due to the metabolic movement, can vary from oval or spherical to highly elongated shape. cell length from anterior to posterior end ranged from 93-267 μm. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in site 1 (2°13'03.7"s 113°53'38.9"e) and site 2 (2° 12'55.2"s 113°53'46.9"e). 3.1.2. euglena mutabilis f. schmitz 1884 taxonomy: e. mutabilis f. schmitz 1884 is currently taxonomically accepted species name according to algaebase. first published in a paper entitled “beitrage zur kenntniss der chromatophoren” by schmitz in 1884 [9]. description: e. mutabilis were observed as small euglena cells with a single flagellum, narrow ends in the posterior and anterior areas of the cell, and clearly visible red eyespot. cell length from anterior to posterior end ranged from 73-89 μm. previously published work described e. mutabilis cells 57–65 µm long and 10–11 µm wide; cylindrical with narrowed ends; cells may be attached to substrate at posterior end; cells bend while swimming [23]. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in site 1 (2°13'03.7"s 113°53'38.9"e) and site 3 (2° 12'58.0"s 113°53'48.8"e). figure 1. research map: palangka raya, central kalimantan, indonesia (map source: openstreetmap & bing aerial, qgis 3.16.3 hannover). j. multidiscip. appl. nat. sci. 84 3.2. lepocinclis perty, 1849, nom. cons. lepocinclis is photothropic flagellates; ovoid or spindle-shaped; with red eyespots that are clearly visible on the anterior region of the cells as in euglena [4][9][20][24]. the cells are completely rigid and unflattened which distinguishes this genus from the genera euglena and phacus [9]. currently, lepocinclis consists of 88 taxonomically accepted species names and 66 accepted varieties [9]. taxonomic enumeration euglenozoa class euglenophyceae order euglenida family phacaceae genus lepocinclis perty, 1849, nom. cons. 3.2.1. lepocinclis acus (o.f.müller) b.marin & melkonian 2003 taxonomy: lepocinclis acus (o.f.müller) b.marin & melkonian 2003 is currently taxonomically accepted species name according to algaebase. published in a paper entitled “phylogeny and taxonomic revision of plastidcontaining euglenophytes based on ssu rdna sequence comparisons and synapomorphic signatures in the ssu rrna secondary structure” by marin et al. in 2003 [9]. this species has 4 homotypic synonyms and 10 heterotypic synonyms [9]. description: l. acus were observed to have elongated spindle-shaped cells with narrow ends in the posterior and anterior areas of the cell; cell length around 200 μm. according to kim et al. [24], l. acus cells 125-163 μm long and 9-77 μm wide; almost rigid; long-fusiform; the anterior and posterior ends are narrowed. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found only in site 2 (2°12'55.2"s 113°53'46.9"e). 3.2.2. lepocinclis ovum (ehrenberg) lemmermann 1901 taxonomy: l. ovum (ehrenberg) lemmermann 1901 is currently taxonomically accepted species name according to algaebase. first published in a paper entitled “beiträge zur kenntniss der planktonalgen. xii. notizen über einige schwebealgen. xiii. das phytoplankton des ryck und des greifswalder boddens” by lemmermann in 1901 [9]. this species has 3 homotypic synonyms and 2 heterotypic synonyms [9]. description: l. ovum were observed in the form of elliptical or spherical cells with clearly visible red eyespot; short caudal process on the posterior. l. ovum is the smallest euglenid species found in this study with up to 25 μm long in diameter. previously published works described l. ovum cells are citriform, fusiform, sub-globose, ovate to elliptical, and elliptical in shape [23]. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in all three sampling sites: site 1 (2°13'03.7"s 113° 53'38.9"e), site 2 (2°12'55.2"s 113°53'46.9"e), and site 3 (2°12'58.0"s 113°53'48.8"e). 3.2.3. lepocinclis spirogyroides b.marin & melkonian 2003 taxonomy: l. spirogyroides b.marin & melkonian 2003 is currently taxonomically accepted species name according to algaebase. table 2. euglenid species found in this study. no familia genera species status of species name* 1 euglenidae dujardin 1841: 347 euglena ehrenberg, 1830 e. gracilis e. mutabilis accepted taxonomically accepted taxonomically 2 phacaceae j.i. kim, triemer & w. shin 2010: 1280 lepocinclis perty, 1849, nom. cons. l. acus l. ovum l. spirogyroides accepted taxonomically accepted taxonomically accepted taxonomically phacus dujardin, 1841, nom. et typ. cons. p. cordatus p. helikoides p. orbicularis accepted taxonomically accepted taxonomically accepted taxonomically *based on algaebase data 2022 [9] j. multidiscip. appl. nat. sci. 85 published in a paper entitled “phylogeny and taxonomic revision of plastid-containing euglenophytes based on ssu rdna sequence comparisons and synapomorphic signatures in the ssu rrna secondary structure” by marin et al. in 2003 [9]. this species has 4 heterotypic synonyms [9]. description: l. spirogyroides were observed to have that makes l. spirogyroides appear to have helical striated lines along its cells. according to dawson et al. [25], the ornamentations on the cell wall named pellicular warts, iron-enriched and mineralized structures of the extracellular matrix. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in site 1 (2°13'03.7"s 113°53'38.9"e) and site 2 (2° 12'55.2"s 113°53'46.9"e). 3.3. phacus dujardin, 1841, nom. et typ. cons phacus is a genus of green flagellates with rigid, compressed and flattened cells, and most appear in a leaf-shaped, folds or grooves running helically or longitudinally, giving the cells irregular or triradiate cross-sections; some species twisted into flat corkscrews [5][9]. phacus consists of 167 taxonomically accepted species names and 73 accepted varieties [9]. taxonomic enumeration euglenozoa class euglenophyceae order euglenida family phacaceae genus phacus dujardin, 1841, nom. et typ. cons. figure 2. euglena: (a-h) e. gracilis; and (i-j) e. mutabilis. j. multidiscip. appl. nat. sci. 86 3.3.1. phacus cordatus (pochmann) zakryś & lukomska 2015 taxonomy: p. cordatus (pochmann) zakryś & lukomska 2015 is currently taxonomically accepted species name according to algaebase. published in a paper entitled “delimiting species in the phacus longicauda complex (euglenida) through morphological and molecular analyses” by lukomska-kowalczyk et al. in 2015 [9]. this species has 2 homotypic synonyms that are phacus longicauda subsp. cordatus pochmann 1942 and phacus longicauda f. cordatus (pochmann) popova 1955 [9]. description: p. cordatus were observed to have a leaf-shaped; slightly shaped like a heart; red eyespots are clearly visible on the anterior region of the cells; long tail; cell length from the anterior to the caudal end is 133 μm and around 70 μm long from the anterior to posterior base. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in site 1 (2°13'03.7"s 113°53'38.9"e) and site 2 (2° 12'55.2"s 113°53'46.9"e). 3.3.2. phacus helikoides pochmann 1942 taxonomy: p. helikoides pochmann 1942 2015 is currently taxonomically accepted species name according to algaebase. first published in a paper entitled “synopsis der gattung phacus” by pochmann in 1942 [9]. this species has 1 homotypic synonym (phacus tortus var. helikoides (pochmann) huber-pestalozzi 1955) and 2 heterotypic synonyms (phacus tortus var. tortuosus skvortsov 1928 and phacus sesquitortus pochmann 1942) [9]. description: p. helikoides were observed to have cells with helical folds; flagella and red eyespots; anterior to posterior (caudal end) length ranges from 62-89 μm. p. helikoides is actually helical in shape throughout the entire cell in figure 3. lepocinclis: (a-b) l. acus; (d) l. ovum; and (d) l. spirogyroides. j. multidiscip. appl. nat. sci. 87 contrast to most phacus species which are flattened and leaf-shaped. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in site 1 (2°13'03.7"s 113°53'38.9"e) and site 2 (2° 12'55.2"s 113°53'46.9"e). 3.3.3. phacus orbicularis hübner 1886 taxonomy: phacus orbicularis hübner 1886 is currently taxonomically accepted species name according to algaebase. first published in a paper entitled “euglenaceen-flora von stralsund” by hübner in 1886 [9]. this species has 14 heterotypic synonyms [9]. description: p. orbicularis were observed to have ovoid cell shape; short curved caudal process; clearly visible red eyespot; anterior to posterior length ranges from 67-89 μm. kosmala et al. [26], kim et al. [24], and łukomska-kowalczyk [27] described that p. orbicularis are characterized by wide, ovoid cells ending with a sharp curved tail. localities: shallow peat waters in palangka raya, central kalimantan, indonesia. found in all three sampling sites: site 1 (2°13'03.7"s 113° 53'38.9"e), site 2 (2°12'55.2"s 113°53'46.9"e), and site 3 (2°12'58.0"s 113°53'48.8"e). 4. conclusions this study revealed that in the peat waters of palangka raya, central kalimantan, indonesia, 8 species of euglenids (euglenophyceae, euglenophyta) were found which belong to 3 genera. the euglenid species identified include euglena gracilis, euglena mutabilis, lepocinclis acus, lepocinclis ovum, lepocinclis spirogyroides, phacus cordatus, phacus helikoides, and phacus orbicularis. in this study, there were several other specimens that were suspected to be euglenid species but could not be identified due to lack of information needed to determine the species name, further research needs to be done using molecular identification methods for more accurate results. author information corresponding author chaidir adam — biology education program, university of palangka raya, palangka raya – 74874 (indonesia); center for development of figure 4. phacus: (a-b) p. cordatus; (c-e) p. helikoides; and (f-h) p. orbicularis. j. multidiscip. appl. nat. sci. 88 science, technology and peatland innovation (ppiig), university of palangka raya, palangka raya – 74874 (indonesia); orcid.org/0000-0002-8907-1117 email: chaidir.adam03@gmail.com author agus haryono — biology education program, university of palangka raya, palangka raya – 74874 (indonesia); center for development of science, technology and peatland innovation (ppiig), university of palangka raya, palangka raya – 74874 (indonesia); orcid.org/0000-0003-3725-201x author contributions c. a. study conception and design; sample collection, observation, and identification; and draft manuscript preparation. a. h. analysis and interpretation of results. all authors reviewed the results and approved the final version of the manuscript. conflicts of interest the author(s) declared no conflict of interest references [1] c. adam and a. haryono. 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[9] observed that the plant height (54.0 to 66.0 cm); number of leaves per plant (208-240); leaf size (4.5 to 8.5 cm); plant spread (19.0 to 32.0 cm); number of branches (4 to 12); number of flowers (25-40); stalk length (8.8 to 13.3 cm) and days of first flowering (55 to 70 days) varied; respectively in t7 (100% rice husk) to t3 (100% cocodust). the different color of leaves and flowers in chrysanthemum flower crop and also the maximum flower period was observed early decemberfebruary in germplasms. taweesak et al. [10] observed that the irrigation effect on plant height of chrysanthemum. pinching is one of the most suitable tactics for effective cultivation of cut flowers. it is act of cutting or nipping off force to new development branching in the plants so that the eventual number of flowers is increased [11]. if the growing tips are copyright holder: © ehsanullah, m., tarapder, s. a., maukeeb, a. r. m., khan, a. u., and khan, a. u. (2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.15 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.15 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.15&domain=pdf&date_stamp=2021-07-02 j. multidiscip. appl. nat. sci. 63 pinched out, adjusts are diverted into lateral buds and branching occurs. modification of plant planning by means of pinching has been done in several commercial flower crops. while ma et al. [12], stated that pinching leads to late flowering in the c. indicum plant, as well as its effect on the branch’s length. it is preferable to pinch the small plants when 10 cm height to encourage production side branches, and then pinched these branches when reached 10 cm in length [13]. ahmade [14] observed that the twice pinching gave a significant reduction in the plant height, the inflorescence diameter, the plant dry weight and the seeds weight (100 seeds). this study also noted that the increased the branches number, chlorophyll intensity in the leaf, the period from planting to inflorescence bud formation, the period to opening 50% of inflorescences, and the total number of inflorescences, the plant aesthetic value (degree) and the plant aesthetic period. the commercial cultivation of chrysanthemum with superiority flowers and higher yield is needed for consumption in local market and to offer livelihood specially to the marginal and small farmers. thus, the pinching can play a vital role in the improvement of flowering and yield of chrysanthemum. keeping in view the above points the present investigation, pinching time has been evaluated on the growth, and flower yield in chrysanthemum crop. the general objective of this study is to assess the effect of pinching in the chrysanthemum crop. 2. materials and methods 2.1. experimental site the present investigation was carried out at the experimental farm of landscape, ornamental and floriculture division, hrc, bari, gazipur, bangladesh during the period from july 2007 to june 2008. the study area situated in 23.9917° n longitude and 90.4137° e latitude at an altitude of 9 meter above the sea level. 2.2. planting material seed of genotype of cm-022 were used in the experiment during the period from july 2007 to june 2008. 2.3. methods 2.3.1. design of the experiment and treatments the experiment was laid out in randomized complete block design (rcbd) with three replications. one plant was planted in a pot, containing the potting media according to the treatments and five plants were constituted the unit of treatment. there were six treatments in the experiment, comprising different pinching in table 1. 2.3.2. pot preparation the experiment was conducted in earthen pots of 12 cm size. the pots were washed and cleaned thoroughly before filling up of potting media. 2.3.3. seedling raising, transplanting and fertilization primarily cuttings of chrysanthemum genotype (cm-022) were prepared for planting in the sand in mid-august, 2007. immediately after rooting, the mini plantlets were transferred to the pot containing media that consists of one-part coarse sand, one part garden soil, one part cocodust, one-part cowdung, a quarter part of wood ashes and two tables spoonfuls of bone meal in mid-september, 2007. subsequently, 10 g tsp and 3 g mp per pot were applied. urea @ 2, 3 and 3 g per pot was applied at 20, 30 and 40 days after transplanting respectively for getting the best growth and flowering of plants [15]–[17]. 2.3.4. irrigation and weeding weeding and mulching were done in the pots whenever it was necessary to keep the pots free from weeds. chrysanthemum plants need frequent irrigation. the pots were irrigated every alternate day to keep the media moistened. table 1. treatments and different pinching composition. treatments different pinching t0 no pinching t1 once 40 days t2 once 50 days t3 once 60 days t4 twice 40 and 50 days t5 thrice 40, 50 and 60 days j. multidiscip. appl. nat. sci. 64 2.3.5. staking of plant each plant was supported by 40 cm long bamboo stick to facilitate the branches of the plant to keep erect. the plant in each pot was fastened loosely with the bamboo stick by jute string to prevent the plant from lodging. 2.3.6. pest and disease control a 2 g/l of ridomil and 2 ml/l of malathion in water was sprayed once fortnight to the plants as protective measures against diseases and insect attack. 2.3.7. harvesting of flowers the spikes were harvested when the flower attained commercial stage flower open before shedding of pollens from the outer row of the disc florets. 2.3.8. collection of data data were collected on the following parameters for interpretation of the result of the experiment. the parameters were plant height (cm), days to flowering, branch number, leaf number, plant spread (cm), flower size (cm) and flower number. plant height (cm): plant height refers to the length of the plant from ground level to tip of erect leaf. height of 5 plants was measured and the mean was calculated. it was measured in cm. number of leaves plant: number of leaves per plant was recorded by counting all the leaves from 5 plants and the mean was calculated. plant spread (cm): the plant spread was measured in cross way (north -south and east-west) by measuring scale. the average of the two measurements was done and expressed in cm. leaf size (cm): the length and breadth of leaf was measured by a measuring scale and the average of the two measurements was done and expressed in cm for a single leaf. later on, the mean of individual leaf size from 5 selected plants was calculated. number of branches plant: number of branches per plant was recorded by counting all the main branches from 5 plants and the mean was calculated. days to flowering: it was recorded by counting the days from planting to first visibility of flower bud in the plant from each pot. number of flowers plant: number of flowers produced per plant was counted and recorded. flower size: flower size was measured in cross way following north-south and east-west position by a measuring scale and the average of the two measurements was done and expressed in cm for a single flower. later on, the mean of individual flower size from 5 selected plants was calculated. 2.3.9. statistical analysis the data recorded on different plant and floral parameters were statistically analyzed through analysis of variance with the help of ‘mstat’ software. the difference between treatment means were compared by duncan’s multiple range test (dmrt) according to baniasadi et al [18]. 3. results and discussions 3.1. plants height (cm) height of chrysanthemum plant (cm-022) was significantly influenced by pinching in table 2. table 2. plant and floral character of chrysanthemum as influenced by pinching. treatment plant height (cm) days to flowering branch number leaf number plant spread (cm) flower size (cm) t0 60a 57d 05c 200d 17.0d 6.9a t1 57ab 62c 07bc 214cd 19.0cd 6.9a t2 55b 63c 07bc 218c 21.0c 7.0a t3 52bc 63c 09b 224bc 23.0bc 7.1a t4 49c 68ab 10ab 228b 25.0b 7.2a t5 45d 70a 12a 235a 30.0a 7.3a cv (%) 12.40 10.80 16.30 13.00 11.72 8.14 note: t0no pinching, t1once 40 days, t2once 50 days, t3once 60 days, t4twice 40 and 50 days, t5thrice 40, 50 and 60 days. means followed by similar letter(s) inside the column do not vary significantly (p = 0.05). j. multidiscip. appl. nat. sci. 65 thus, the highest plant height (60 cm) was observed under no pinching and lowest (45 cm) was recorded by pinching the plants thrice (t5). the plant varied from 40-120 cm in the field condition [17]. this was due to repetitive removal of apical portion of main branch; axillary buds become free from correlative inhibition of apical dominance and started growing. this resulted into more branching and spread of plants. thus, height was reduced in pinched plants. similar results observed by susila et al. [19] in chrysanthemum flower crop. 3.2. days required for flowering it is evident from the table 2 that the increased number of pinching resulted into significant delay in the flowering of chrysanthemum. thus, the earliest flowering (57 days) was observed where no pinching was followed. there was no significant difference between pinching once (t1, t2 and t3) which took 62, 63 and 63 days respectively, but further significant delay in flowering (68 days) was recorded by pinching the plants twice followed by pinching the plants thrice (70 days). the delay in flowering by pinching was due to removal of physiological mature portion and the new shoots which emerged out from the pinched plants took more time to become physiological inductive to produce flowers than non-pinched plants. similar results have been observed by rajan et al in chrysanthemum [20]. 3.3. number of branches the number of branches was quite variable in different treatments in table 2. the highest number of branches (12) was observed in t5 treatment followed by t4 treatment (10). the lowest number of branches was recorded in t0 treatment (05). this was due to repetitive removal of apical bud which leads to enhanced branch number observed in t5 treatment. the above findings are in agreement with that of in marigold [21]. 3.4. number of leaves maximum number of leaves (235) was recorded in t5 treatment (pinching thrice) followed by t4 treatment (228) in table 2. kumar et al [22] observed increased leaf number in carnation plants while pinched thrice. adequate numbers of leaves are essential for normal growth and production. an increase in number of leaves causes the accumulation of greater photosynthesis leading to better growth parameters. 3.5. plant spread it has been observed that plant spreads were significantly affected by the different treatments (table 2). the treatment t5 attained maximum plant spread (30 cm). this was due to higher the branch number with high leave content under pinched thrice ultimately increased plant spread. chandel et al [23] also observed increased plant spread while pinched twice or thrice in chrysanthemum. 3.6. flower size (cm) the flower size of chrysanthemum was not significantly improved by various treatment of pinching (table 2). the results are in agreed with yao et al [24] in carnation. 3.7. number of flowers perusal of figure 1 show that by increasing the number of pinching, there was an increase in the number (45) of flower per plant. the lowest number of flowers (28) was recorded under no pinching. number of flowers was affected by pinching was due to increased number of branches. similar results observed by sharma et al. [25] and findings were varied from 19.73-42.26 in chrysanthemum plant. 4. conclusions the observations recorded from the present investigation revealed that the pinching effect in the morphological and flower characters in the figure 1. flower number of chrysanthemums. j. multidiscip. appl. nat. sci. 66 chrysanthemum flower crop. timely planting and pinching effect in the plant growth and production of quality flowers in the field. pinching carried out in chrysanthemum improved in number of flowers per plant, plot and yield per hectare. among the six treatment, pinching the plants thrice showed the highest efficacy and it could be used as treatment in cultivation of chrysanthemum indicum for growth and quality flower production. author information corresponding author ahasan ullah khan — department of agroforestry and environmental science, sylhet agricultural university, sylhet-3100 (bangladesh); department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh) ; orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors md. ehsanullah — department of entomology, govt. shahid akbar ali science and technology college, thakurgaon-5120 (bangladesh); shofiul azam tarapder — department of agronomy and haor agriculture, sylhet agricultural university, sylhet-3100 (bangladesh); abu rashed md. maukeeb — department of agricultural chemistry, khulna agricultural university, khulna-9100 (bangladesh); anayat ullah khan — department of mathematics, jashore university of science and technology, jashore-7408 (bangladesh); references [1] m.-c. song, h.-j. yang, t.-s. jeong, k.-t. kim, and n.-i. baek. 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oral cancer”. a multidisciplinary approach would be effective, with a broadening of the knowledge of health professionals to identify cancerous lesions. a timely diagnosis of oscc is crucial and its management is fundamental also for dental specialists. with the aim to anticipate the diagnosis, it will be desirable to set prevention campaigns also by the assistance in telemedicine. keywords covid-19, sars-cov-2, oral cancer, oral squamous cell carcinoma, public health 1. introduction sars-cov-2 infection leads to a severe acute respiratory syndrome that can present with various clinical manifestations that fall within the definition of "coronavirus disease 2019" (covid-19) [1]. the infection has spread very rapidly since december 2020, bringing much concern to global public health. in europe it has spread since february 2020, where several national health systems have faced and continues to face enormous difficulties in the fight against the pandemic with seasonal periods of lockdown. this significant spread has led to shortages of health personnel and adequate facilities to deal with the most serious cases of the covid-19 disease [2][3]. there are important consequences on the social, economic and employment level, also due to the need for social distancing, the blocking of many workers and the closure or time limit of numerous companies during the confinement periods. the post-lockdown situation has imposed maximum prudence in the reopening of work activities and in the reintegration of the worker in public offices and companies, favoring the working methods in working from home. this pandemic period poses further challenges in the area of cancer diagnosis and treatment [4][5]. oral carcinoma is the most common of the malignant tumors of the oral cavity that tends to appear in the fifth or sixth decade of life, generally the diagnosis is made when the neoplasm is in an advanced clinical phase and therefore with a subsequent prognosis with high morbidity and high mortality. diagnostic delay for this condition is common. oral cavity squamous cell carcinoma (oscc) is the most common malignancy of the head and neck; in the united states alone, the american cancer society estimated that there were 48,330 new cases of oral cavity and pharyngeal cancer in 2016 [6]. worldwide 350,000–400,000 new cases are diagnosed each year [7]. in 2018, 354,900 cases of oral scc were estimated worldwide, with 177,400 deaths [8]. the incidence of oral cancer in western areas is about 3-6% which also reaches 30% in eastern-indian countries, this significant difference is attributable to the customs and traditions of the various populations involved [8][9]. squamous cell carcinomas (sccs) make up over 90% of all oral cancers. the remaining 10% of neoplasms arises from epithelium, connective tissue, minor salivary glands, lymphoid tissue and melanocytes or from distant tumor metastases. nonsquamous cell carcinomas of the oral cavity are rare [10]. minor salivary gland carcinomas account for copyright holder: © pulcini, r., d’agostino, s., dolci, m., bissioli, a., caporaso, l., and iarussi, f. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.110 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.110 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.110&domain=pdf&date_stamp=2022-04-04 j. multidiscip. appl. nat. sci. 66 less than 5% of oral cancers. they frequently occur on the hard palate (60%), on the lips (25%) and on the buccal mucosa (15%). mucoepidermoid carcinoma is the most common type (54%), followed by low-grade adenocarcinoma (17%) and adenoid cystic carcinoma (15%) [11]. risk factors for oral cancer are tobacco, betel quid chewing, alcohol, infectious agents (hpv), alterations in nutritional and dietary factors, oral hygiene, chronic trauma, genetic factors [12]. prevention of oral cancer requires better education of professional profiles such as dentists and dental hygienists on lifestyle-related risk factors and a better understanding of the interpretation of signs and symptoms to achieve an early diagnosis. they can be considered sentinels in the interpretation of oral lesions. a very important consideration in the fundamental role of these two figures concerns the periodicity with which patients carry out six-monthly preventive checks for oral diseases such as: caries, periodontitis and gingivitis, therefore they would have a better chance of meeting patients with potentially cancerous lesions, taking into account that 30% of patients delay seeking help about 3 months after self-discovery for symptoms attributable to oral cancer [13]–[15]. to obtain effective prevention measures, a multidisciplinary approach by different health professionals is therefore necessary. 2. materials and methods this research is based on the prisma statement (preferred reporting items for systematic reviews and meta analysis) updated by page et al. in 2020. an electronic search was performed on pubmed considering the modern period of the last 2 years from 2019 to 2021, the following mesh was used: “covid-19 oral cancer”. studies eligibility criteria are description of oral cancer during the covid-19 pandemic in adult patients, reviews and letters to the editor including free full text and abstracts exclusively in english. 3. results and discussions the digital investigation has provided 54 articles, but only 18 were positive for inclusion criteria, while other were excluded because regarding other pathologies with covid-19. the data contained in the eligible articles were developed in order to understand and analyze any shortcomings of the various types of prevention on oral cancer patients in the ongoing covid-19 pandemic. five articles have been shown to be highly relevant to the delay in oral cancer diagnosis during covid-19 (figure 1). the main results are summarized in table 1. ferlay, et al. [8], has collected data from 185 countries for the cancer situation in the world reporting that in 2018, it was estimated that 354,900 cases of oral scc occurred worldwide, with 177,400 deaths. from the study by scott, et al. [13] it was observed that 54% of participants with symptoms or potential malignancies delayed seeking help and 39% waited more than 3 months before visiting a healthcare professional. so, it is possible to speculate how these data may actually deteriorate in the context of a covid-19 pandemic. various decrees have limited the movement of people, movements only in case of work-related reasons, reasons of urgency or health reasons, bringing further inconvenience to fragile subjects such as the elderly. people appear to be reluctant to go to hospitals and facilities such as dental offices for check-ups despite the provisions and aids used [17]. covid-19 has also led to a decrease in the influx of patients to health and hospital facilities figure 1. prisma statement flow chart [16]. j. multidiscip. appl. nat. sci. 67 with regard to attention to prevention (primary, secondary and tertiary) leading to a worsening of global public health. the goal of the primary preventive measure is to raise public awareness of risk factors and aims to change public behavior. secondary prevention aims at detecting malignant lesions early through screening techniques. tertiary prevention aims to prevent the retraining of oral tumors in patients. diagnostic delay reduces both surgical and radiotherapy therapeutic success [18]. this delay results in therapies that become highly invasive, as the adjacent tissues are compromised by altering the oral cavity from a functional, morphological and psychological point of view. surgical services for oral cancer patients need a contingency plan to keep surgical care in a pandemic or post-pandemic phase [17][18]. world health organization (who) defines quality of life as the perception of the individual as an individual's perception of his or her position in life in the cultural context and value systems in which he lives, in relation to his goals and his expectations. the clinical manifestations of a late intervention against oral cancer also leads to negative effects in the quality of life of patients who may have post-surgical treatment dysfunctions in communication, swallowing, functional aesthetic alterations. in a historical period like this can be perceived in current conditions and which has changed many lifestyles, also reducing their quality of life, a right priority should be given to patients who could have further collateral damage if preventive measures are not implemented. 4. conclusions oral cancer involves many people and many of them fearful of contracting covid 19, neglecting the signs and symptoms and further delaying the diagnosis of any cancerous lesions, seriously affecting the prognosis. on these bases, the result from this search seems to point out the delay in oscc diagnosis during the pandemic. multi-level prevention will be the key to ensure patient health. an expansion of knowledge for the recognition of these injuries in the dental sector could anticipate the diagnosis. furthermore, it is logical to think of a valid support that can come from technology, and it could be represented by telemedicine services available for patients of surgical interest and other patients. however, further future studies are needed to better interpret the data of a possible increased mortality of patients with oral cancer in the covid -19 era. author information corresponding author silvia d’agostino — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); orcid.org/0000-0002-4778-4768 email: silviadagostino00@gmail.com authors riccardo pulcini — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); orcid.org/0000-0001-8334-0577 marco dolci — department of medical, oral table 1. summary of significant articles from the search. authors and year results da cruz perez, et al. (2020) [15] dentists should be more aware of oscc symptoms during the pandemic for a timely diagnosis villani, et al. (2020) [17] telephone triage is crucial to avoid loss of follow-up during the pandemic day, et al. (2020) [18] covid-19 delays oscc diagnosis causing reduced therapeutical success søreide, et al. (2020) [19] oscc diagnosis delay means more invasive treatments ferrari, et al. (2021) [20] oral oncology needs specific protocols during the pandemic in order to reach a wider cluster of patients https://orcid.org/0000-0002-4778-4768 mailto:silviadagostino00@gmail.com https://orcid.org/0000-0001-8334-0577 j. multidiscip. appl. nat. sci. 68 and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); orcid.org/0000-0001-5495-5660 alessandra bissioli — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); lucia caporaso — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); francesca iarussi — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); references [1] y. shi, y. wang, c. shao, j. huang, j. gan, x. huang, e. bucci, m. piacentini, g. ippolito, and g. melino. 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(2021). “covid-19 screening protocols for preoperative assessment of head and neck cancer patients candidate for elective surgery in the midst of the pandemic: a narrative review with comparison between two italian institutions”. oral oncology. 112 : 105043. 10.1016/ j.oraloncology.2020.105043. https://doi.org/10.1016/j.biopha.2018.07.114 https://doi.org/10.1016/j.biopha.2018.07.114 https://doi.org/10.1016/j.biopha.2018.07.114 https://doi.org/10.1016/j.biopha.2018.07.114 https://doi.org/10.1136/bmj.n71 https://doi.org/10.3390/ijerph17124609 https://doi.org/10.1016/j.oraloncology.2020.104684 https://doi.org/10.1016/j.oraloncology.2020.104684 https://doi.org/10.1002/bjs.11670 https://doi.org/10.1016/j.oraloncology.2020.105043 https://doi.org/10.1016/j.oraloncology.2020.105043 19 journal of multidisciplinary applied natural science vol. 2 no. 1 (2022) review article the importance of the dental hygienist in implantology: a narrative review lucia caporaso, alessandra bissoli, francesca iarussi, riccardo pulcini, marco dolci, and silvia d’agostino* received : may 28, 2021 revised : september 4, 2021 accepted : september 19, 2021 online : september 19, 2021 abstract dental implants have become a standard treatment in the replacement of missing teeth. if maintaining good oral health in a mouth without prosthetic elements requires proper self oral hygiene and professional maintenance, preserving implant health requires even more accuracy and consistency. dental hygienists are fundamental in the management of the bacterial biofilm, in the context of prevention, education and professional treatment of the patient. this review explores the importance and the role of the dental hygienist in oral implantology. keywords biofilm, dental hygienists, dental implants, oral health, peri-implantitis 1. introduction oral implantology employs osseointegrated implants used as support for fixed or removable prosthetic rehabilitation. it is universally recognized as a safe clinical methodology, able to ensure lasting results in oral therapy. dental implants are similar but different from teeth. they lack some basic characteristic structures such as root cementum, periodontal ligament, and bundle bone. the implant doesn’t have dento-alveolar and the dento-gingival fiber bundles that connect the soft tissues with the tooth (root cementum) and it is rigidly anchored (ankylosed) to the bone, whereas the tooth is mobile within its socket [1]. before starting any dental implant therapy, clinicians must be thoroughly familiar with the patient's medical and oral condition and assess all the possible risks. implant failure is not only related to poor osseointegration but also to the presence of some risk factors and conditions that influence the outcome and prognosis of implant therapy in the short, medium and long term [2]. peri-implantitis is surely one of these conditions. as the 2017 world workshop of chicago 4th group stated, “peri-implantitis is a plaque-associated pathological condition occurring in tissues around dental implants, characterized by inflammation in the periimplant mucosa and subsequent progressive loss of supporting bone” [2]. scientific evidence suggests that peri-implantitis may be secondary to periimplant mucositis resulting from poor home and professional maintenance [3]. so the main cause of this pathological condition, as for periodontitis, is the bacterial plaque also known as biofilm. biofilm can be defined as a multi-microbial community immersed in a self-produced polymeric matrix, which associates with biotic or abiotic surfaces wetted by a liquid and it is the result of a multistage process. if in the early stages of bacterial colonization we have commensal bacteria, the biofilm composition changes as the plaque ages: in the deeper regions appear anaerobic bacteria. even if biofilm associated with peri-implant disease has not been widely documented, the microbiome of healthy and diseased implants sites have been compared and peri-implantitis was associated with species of the red complex such as porphyromonas gingivalis and tannerella forsythia [4]. other studies indicated that peri-implantitis was most frequently related to opportunistic pathogens such as pseudomonas aeruginosa and staphylococcus aureus [5][6], fungal organisms (e.g., candida albicans) [5][7][8] and viruses (e.g., human cytomegalovirus, epstein-barr virus) [9], suggesting a more complex and heterogeneous infection [10][11]. achieving satisfactory control over bacterial biofilm in an environment that is typically colonized by a range of both harmful and copyright holder: © caporaso, l., bissoli, a., iarussi, f., pulcini r., dolci, m., and d’agostino, s. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.94 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.98 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.94&domain=pdf&date_stamp=2022-01-14 j. multidiscip. appl. nat. sci. 20 non-harmful bacteria is not easy. it requires the commitment of various professionals and most importantly the total compliance of the patient, who must feel part of a project aimed at achieving lasting oral health. in particular dental hygienists work is about prevention and oral health maintenance. in fact, the dental hygienist is the key provider of preventive oral care (primary, secondary and tertiary prevention) in order to promote and improve the oral health of individuals, families and groups in society. he/she possesses the necessary professional qualifications, depending on the state, and is able to provide patient-centered, holistic and evidence based preventive oral care independently. dental hygienists are routinely responsible for the patient's oral health education and maintenance of dental implants. hygienists are asked to perform an educational and instructional task in order to achieve good compliance and to improve the patient's self hygiene behavior, a fundamental condition for maintaining optimal oral health. the aim of the study is to explore the importance and the role of the dental hygienist in the management of dental implants’ biofilm. 2. materials and methods the literature search was performed on the pubmed database. studies published in the last 5 years from 2015 to 2021, with free full text available, were considered for inclusion. the following keywords as mesh terms or free words were used: “dental hygienists” or “dental hygiene” and “dental implants” or “dental hygienists” and “dental implants”. both review articles and research articles considered relevant for discussion were selected and analyzed. studies in other languages than english were excluded. evidence was presented in the form of a narrative review. study eligibility criteria were the role of the dental hygienist in the maintenance of dental implants through the connection between professional oral hygiene, motivation for home oral care, both performed by dental hygienists, and outcomes on implant maintenance. 3. results and discussions the selection process resulted in a total 101 potentially relevant articles. after review of titles and abstract, 97 articles were excluded because they didn’t meet the eligibility criteria; the majority of the works didn’t include the figure of the dental hygienist, so a total of 4 articles were reviewed. the work of yong-keum choi et al. considered agestratified associations between dental health behaviors related to tooth brushing and oral hygiene product use in korean adults with implants [12]. authors found that self-performed oral hygiene practices combining tooth brushing, at least twice per day, and dental floss were found to be significantly related to a low prevalence of periodontitis in people with implants. two other studies have focused on dental implant treatment and maintenance [13][14]; both of them showed that mechanical debridement and oral hygiene instructions, performed by dental hygienists, were crucial in the treatment of peri-implant mucositis. in particular the second study suggests that clinicians need to check if the prosthetic construction allows access for optimal hygiene and it also underlines the importance of the follow up in patients with dental implants. finally only one article by schneider et al. discussed the frequency of visits to the dental hygienist and taylored oral hygiene measures in switzerland [15]. no articles were found specifically addressing the role of the dental hygienist in the maintenance of dental implants. oral implantology is a young discipline, a field in which research still has a lot to do, especially in the maintenance of implant health and in the treatment of peri-implant pathologies. as studies have shown, maintaining good self-performed oral hygiene reduces the risk of peri-implant mucositis and peri-implantitis [12]–[14]. the explanation is that by frequently and carefully disaggregating the biofilm the most aggressive bacterial species are not allowed to proliferate, organize themselves and colonize the various niches of the oral cavity. a dental hygienist plays a key role in this: his goal should be to maintain peri-implant health even before therapeutic resolution. success can be achieved through the awareness of the different types of implants, of the various oral hygiene tools, and of the patient's cleaning and maintenance skills. it is also necessary for the dental hygienist to become an integral part of the professional team composed by the implantology surgeon and the j. multidiscip. appl. nat. sci. 21 prosthetist to ensure that the prosthetic construction allows access for optimal hygiene. this allows the dental hygienist to discriminate and select the most suitable techniques and tools for patients and recommend tailored oral hygiene measures to increase the compliance. it is equally important that the patient understands, even before undergoing surgery, that the implant or the implants will require constant professional as well as self maintenance. finally, there is an important factor that should not be underestimated in the study by c. schneider et al. the visits to the dentist decreased slightly over the years, while the visits to the dental hygienist increased. the combination of both indicates a small increase in oral health care utilisation in switzerland between 1992 and 2012. these observations point to a trend towards a more specialised and tailored approach in oral healthcare provision, which is underlined by the increasing ratio of dentist to non-dentist oral health care workers in other european countries [16][17]. 4. conclusions dental hygienists play a key role in maintaining implant health, managing patients' oral habits, and keeping patients’ compliance high. however the lack of scientific studies investigating their valuable work on osseointegrated implants does not lead to definite conclusions. there is a need for further studies to evaluate the role of dental hygienists in oral implantology. author information corresponding author silvia d’agostino — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); orcid.org/0000-0002-4778-4768 email: silviadagostino00@gmail.com authors lucia caporaso — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); alessandra bissoli — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); francesca iarussi — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); riccardo pulcini — department of innovative technologies in medicine & dentistry, university g. d’annunzio, chieti 65013 (italy); orcid.org/0000-0001-8334-0577 marco dolci — department of medical, oral and biotechnological sciences, university g. d’annunzio, chieti 65013 (italy); orcid.org/0000-0001-5495-5660 references [1] m. g. araujo and j. lindhe. 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(2015). skillmix in preventive dental practice will it help address need in the future?. bmc oral health. 15 (1). 10.1186/1472-6831-15-s1s10. https://doi.org/10.1111/j.1600-051x.2010.01666.x https://doi.org/10.1111/j.1600-051x.2010.01666.x https://doi.org/10.1186/s40729-015-0010-6 https://doi.org/10.1186/s40729-015-0010-6 https://doi.org/10.1111/clr.12387 https://doi.org/10.1111/clr.12387 https://doi.org/10.1016/j.ijom.2010.11.004 https://doi.org/10.11607/jomi.4150 https://doi.org/10.5037/jomr.2016.7310 https://doi.org/10.5051/jpis.1904700235 https://doi.org/10.1111/jcpe.13295 https://doi.org/10.1111/jcpe.13295 https://doi.org/10.1111/idj.12490 https://doi.org/10.1111/idj.12490 https://doi.org/10.1922/idj-2530widstrom06 https://doi.org/10.1922/idj-2530widstrom06 https://doi.org/10.1186/1472-6831-15-s1-s10 https://doi.org/10.1186/1472-6831-15-s1-s10 24 journal of multidisciplinary applied natural science vol. 3 no. 1 (2023) research article effective recovery of palladium(ii) ions using chitosanbased adsorbent material rokiy alfanaar, krisfian tata aneka priyangga, arif cahyo imawan, jumina jumina, and yehezkiel steven kurniawan* received : june 6, 2022 revised : august 1, 2022 accepted : august 13, 2022 online : august 22, 2022 abstract chitosan is one of the naturally abundant, biodegradable, and low-cost adsorbent materials for metal adsorption purposes. in this work, we evaluated the application of chitosan materials derived from seafood wastes in depok beach, yogyakarta, for an effective recovery of the palladium(ii) ions. first of all, the seafood wastes were treated to obtain chitin and then followed by the deacetylation process to produce chitosan material with a deacetylation degree of 78.42%. the chitosan material was characterized using fourier transform infrared (ftir) spectrophotometer. it was found that chitosan gave high adsorption percentage (90%) for palladium(ii) ions due to the complexation with hydroxyl, amino and carbonyl functional groups. the palladium(ii) adsorption onto chitosan material followed the pseudo-second-order (r 2 = 0.9978) and langmuir (r 2 = 0.9979) models for kinetic and isotherm experiments, respectively, with a maximum adsorption capacity value of 0.70 mmol g -1 . the palladium(ii) ions could be easily desorbed in 90% percentage using 1.0 m hcl solution from metal-laden chitosan to regenerate the adsorbent material. the chitosan -based adsorbent material did not lose its adsorption capability after three consecutive cycles with no significant structural change as revealed from the ftir data. these results showed the potential application of natural chitosan materials derived from seafood wastes for the effective recovery of palladium(ii) ions. keywords adsorption, chitosan, palladium, recovery, selective 1. introduction palladium is a precious metal that is widely applied in batteries, catalysts, and other advanced materials in our daily life [1][2]. however, the supply of palladium metals is unstable due to global maldistribution and limited resources. because of that, the palladiums’ recovery from electronic wastes and wastewaters shall be seriously considered as an alternative resource of palladium metals in the future. efforts on the palladiums’ recovery have been given over the past several years [3]-[5]. effective recovery of precious metal ions from electronic wastes has been reported by sathuluri et al. [6] in 2018 using calixarene derivatives. they reported the presence of nitrogen functional groups was crucial for palladium(ii) complexation [6]-[9]. however, calixarenes are expensive materials thus limiting their usage for palladium(ii) recovery in a commercial application. therefore, the search for a cheap and convenient recovery process of palladium ions needs to be established. chitosan is a nitrogenated biomaterial that is naturally abundant, biocompatible, and biodegradable. chitosan is composed of a mixture of n-acetylglucosamine and d-glucosamine monomers as minor and major building blocks, respectively [10]. chitosan materials can be obtained through a deacetylation reaction of chitins derived from crustacean shells [11]. nowadays, chitosan materials have been extensively investigated as adsorbent materials for metal ions adsorption through chemical interactions. from the molecular point of view, chitosan contains hydroxyl, ether, carbonyl, amide and amino groups that could act as lewis bases to form a stable complex with positive charge metal ions as lewis acids. this phenomenon could be exploited for both heavy metal removal and precious metal recovery [12]-[14]. to the best of our knowledge, an investigation on the chitosan production from seafood wastes in depok beach, yogyakarta, for palladium(ii) ions recovery has not been reported yet. because of that, the utilization of crustacean shell wastes to produce chitosan materials for precious metal ions recovery through the adsorption process is an excellent solution to increase the economic value of seafood wastes. herein, we reported the preparation of chitosan material through the deacetylation of publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2022 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.131 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.131 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.131&domain=pdf&date_stamp=2022-08-22 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 25 figure 1. ftir spectrum of chitosan material. chitins derived from seafood wastes obtained at depok beach. the produced chitosan material was applied for palladium(ii) adsorption from an individual metal system and then the adsorption kinetics and isotherm models were studied. the palladium(ii) ions were recovered under the acidic condition to regenerate the chitosan-based adsorbent materials for the other three consecutive cycles of the adsorption process. 2. materials and methods 2.1. materials seafood wastes were kindly donated by a fisherman from depok beach, yogyakarta, while sodium hydroxide (naoh), hydrochloric acid (hcl), nitric acid (hno3), sulfuric acid (h2so4), and palladium(ii) nitrate were purchased from merck in pro analytical grade. 2.2. instruments the fourier transform infrared (ftir) spectra of chitosan materials were recorded from a shimadzu ir prestige-21 spectrophotometer. meanwhile, the adsorption percentage (% adsorption) was calculated by measuring palladium (ii) ions concentration using an atomic absorption spectrophotometer (aas) apparatus. the mathematical equation to calculate the % adsorption is mentioned as follows. (1) 2.3. methods 2.3.1. preparation of chitosan material from seafood wastes the chitin isolation was conducted in a similar manner to the previous report [11] with some modifications. at first, the seafood wastes were mashed with the aid of a mortar and subjected to deproteination, demineralization, and depigmentation processes. the deproteination method was conducted to remove the proteins from the chitin by refluxing the seafood waste powders in 0.1 m naoh in 1:10 mass to volume ratio for 2 hours. the residue was rinsed with distilled water until the ph of the filtrate was neutral. afterward, the demineralization process was performed by mixing the residue in 1 m hcl in 1:20 mass to volume ratio at room temperature for 3 hours. the solid was then washed with distilled water until the ph of the filtrate reached 7. furthermore, the depigmentation process was carried out by mixing the residue in 0.05 m naocl in 1:10 mass to volume ratio at room temperature for 1 hour. the produced chitin material was washed with distilled water and dried. the chitosan material was prepared by refluxing chitin in 1.5 m naoh in 1:20 mass to volume ratio for 3 hours. the produced chitosan was rinsed with distilled water until the ph of the filtrate was neutral. then, the chitosan material was dried and characterized by ftir analysis, while the deacetylation degree was estimated using moore and robert method [15]. the mathematical equation to estimate the deacetylation degree is j. multidiscip. appl. nat. sci. 26 mentioned as follows. (2) 2.3.2. palladium(ii) adsorption from aqueous solution a stock solution of palladium(ii) ions was prepared by dissolving 230 mg (1 mmol) palladium (ii) nitrate in 100 ml distilled water to give a final concentration of 10 mm. chitosan material (200 mg) was added into 50 ml of palladium(ii) solution and stirred at room temperature. the kinetics of palladium(ii) adsorption was studied by changing the contact time by 15, 25, 50, 75, and 100 minutes, while the palladium(ii) adsorption isotherm was investigated by varying the initial concentration of palladium(ii) ions of 0.1, 1, 2.5, 5, 7.5, and 10 mm. the palladium(ii) ions concentration after the adsorption process was then determined by aas analysis. 3. results and discussions 3.1. preparation of chitosan material from seafood wastes isolation of chitin from seafood wastes was performed through three consecutive steps, i.e., deproteination, demineralization and depigmentation. these consecutive processes were required to remove protein, mineral and pigment impurities from chitin material, respectively. protein is easily denaturated under alkaline conditions while inorganic minerals (mostly calcium carbonate) are highly soluble under acidic conditions. the pigments from crustacean shells are commonly bleached with the help of naocl through reduction-oxidation reactions [11]. the deproteination, demineralization, and depigmentation processes produced each targeted material in 54, 48, and 91% yield, respectively. figure 2. effect of contact time on the palladium(ii) ions adsorption using chitosan-based material. mass of the adsorbent = 200 mg. volume of the aqueous solution = 50 ml. [palladium(ii)] = 0.1 mm. shaking speed = 150 rpm. kinetic model mathematical equation r 2 pseudo-first-order log (qe-qt) = -0.0238 t – 1.3472 0.7750 pseudo-second-order t/qt = 47.414 t + 1404.2 0.9978 elovich qt/t = -2.52×10 -6 (ln t)/t + 0.0005 0.9135 intraparticle diffusion qt = 0.0031 t 0.5 + 0.0060 0.9015 liquid film diffusion ln(1-qt/qe) = -0.0548 t – 0.6921 0.9780 table 1. mathematical models of palladium(ii) adsorption kinetics using chitosan-based material. j. multidiscip. appl. nat. sci. 27 then, chitosan material was prepared from the deacetylation reaction of chitin. the amide functional groups are hydrolyzed under alkaline conditions to give free amino functional groups which are useful for metal ion complexation. the deacetylation reaction yielded chitosan in 20% yield. these data showed that the preparation of chitosan material from seafood wastes was achieved in 4% overall yield in this work. the ftir spectrum of chitosan (figure 1) showed characteristic signals of o–h and –nh2, c–h sp 3 , c=o, n–h, and c–o functional groups at 3382, 2877, 1654, 1584, and 1079 cm -1 , respectively. it was found that the deacetylation degree of the obtained chitosan material was 78.42% according to moore and roberts’ calculation. 3.2. palladium(ii) adsorption from aqueous solution the adsorption of palladium(ii) ions onto the chitosan material was evaluated. the time dependence adsorption profile is depicted in figure 2 by varying contact times. the equilibrium of palladium(ii) ions adsorption was achieved after 75 minutes to give around 90% adsorption percentage with an experimental amount of palladium(ii) ions adsorbed per gram of chitosan (qe) value of 0.0225 mmol g -1 . the kinetic analysis of palladium(ii) ions adsorption was studied by implementing pseudofirst-order, pseudo-second-order, elovich, intraparticle diffusion, and liquid film diffusion models. the kinetics data are listed in table 1 in which the palladium(ii) ions adsorption fits well with the pseudo-second-order mathematic model as indicated by the highest r 2 value (0.9978) with a reaction rate constant of 1.60 g mmol -1 min -1 . furthermore, the theoretical qe value (0.02109 mmol g -1 ) was close to the experimental qe value (0.02250 mmol g -1 ) demonstrating the validity of the pseudo-second-order kinetic model in this work. figure 3. isotherm adsorption for palladium(ii) ions using chitosan-based material. mass of the adsorbent = 200 mg. volume of the aqueous solution = 50 ml. shaking speed = 150 rpm. adsorption time = 100 min. isotherm adsorption model mathematical equation r 2 langmuir ce/qe = 1.4267 ce + 0.3730 0.9979 freundlich ln qe = 0.5410 ln ce – 1.0877 0.9375 temkin qe = 0.1048 ln ce + 0.5134 0.9561 dubinin-radushkevich ln qe = 8.3×10 -7 [rt ln (1 + 1/ce)] 2 – 2.6011 0.6867 table 2. isotherm adsorption model of palladium(ii) ions using chitosan-based material. j. multidiscip. appl. nat. sci. 28 the isotherm adsorption of palladium(ii) ions was studied by varying the initial concentration of palladium(ii) ions in the aqueous phase. the isotherm adsorption profile of palladium(ii) ions is depicted in figure 3. the equilibrium state of palladium(ii) ions adsorption was achieved at a qe value of 0.70 mmol g -1 . the isotherm adsorption analysis of palladium(ii) ions was further studied by implementing langmuir, freundlich, temkin, and dubinin-radushkevich models. the isotherm adsorption data are listed in table 2 in which the palladium(ii) ions adsorption perfectly matches with the langmuir model indicated by the highest r 2 value (0.9979). based on the langmuir model, the maximum adsorption capacity (qmax) and langmuir constant (kl) of palladium(ii) ions adsorption using chitosan-based material were 0.70 mmol g -1 and 3.82 l mmol -1 , respectively. the separation constant (rl) is a critical parameter to determine whether the adsorption is favorable or not. the rl value of palladium(ii) adsorption using chitosan-based material was 0.025 demonstrating that the adsorption process was highly favorable (0 < rl < 1). furthermore, it was estimated from the temkin model that the heat of adsorption was around 23.80 kj mol -1 indicating chemisorption of palladium(ii) ions on the surface of chitosan material. since the chitosan is composed of nacetylglucosamine and d-glucosamine monomers in 22.58 and 78.42%, respectively, thus the chitosan could interact with palladium(ii) ions through hydroxyl, amino and carbonyl functional groups. to elucidate the adsorption sites of chitosan, ftir analysis was performed. the ftir spectra of chitosan before and after palladium(ii) loading are shown in figure 4. the signal of o–h and –nh2 functional groups was shifted from 3382 to 3433 cm -1 indicating that the interaction of chitosan with palladium(ii) ions broke the intermolecular and intramolecular hydrogen bonds of chitosan. the signal of c–h sp 3 was shifted from 2877 to 2908 cm -1 due to conformational change of figure 4. ftir spectra of chitosan-based material (a) before and (b) after palladium(ii) loading. [palladium (ii)] = 10 mm. figure 5. plausible chemical interactions for palladium(ii) adsorption on the chitosan-based material. j. multidiscip. appl. nat. sci. 29 tetrahydropyran heterocyclic of glucose after palladium(ii) adsorption which was in agreement with the previous report [16]. the signal of c=o shifted from 1654 to 1620 cm -1 indicating that dipole-cation interaction was observed between the oxygen atom of carbonyl (acted as lewis base) and palladium(ii) ions (acted as lewis acid). the signal of n–h bending shifted from 1584 to 1620 cm -1 and overlapped with the signal of c=o due to dipolecation interactions which agreed to the shift of o–h and –nh2 signals. meanwhile, the signal of c–o slightly changed from 1079 to 1078 cm -1 due to conformational change in glucose. it meant that the main mechanism of palladium(ii) adsorption happened through electrostatic and dipole-cation interactions as supported by the langmuir and temkin isotherm data. the plausible adsorption site of palladium(ii) ions is depicted in figure 5. on the other hand, table 3 shows the qmax parameters for palladium(ii) adsorption using other reported adsorbent materials. compared to the commercial chitosan material (qmax = 0.59 mmol g 1 ), the prepared chitosan in this work gave a slightly higher qmax value (qmax = 0.70 mmol g -1 ) due to the variety in the bio-composition of crustacean shell sources in the seafood wastes at depok beach. the qmax value of chitosan in this work still can be improved by several materials modifications such as crosslinking, impregnation and copolymerization methods (see table 3 no. 5-17). this result demonstrates that chitosan material derived from seafood wastes is a promising adsorbent material for palladium(ii) ions adsorption. effective recovery of precious metal ions not only depends on the high adsorption capability but also on the ease of the desorption stage. in this work, acid reagents were used to recover palladium (ii) ions from the metal-laden chitosan material. since the adsorption mechanism depends on the electrostatic and dipole-cation interactions thus the no adsorbent material qmax (mmol g -1 ) reference 1 dimercapto-thiodiazole-chitosan 0.13 [16] 2 activated carbon-coated-chitosan 0.41 [17] 3 chitosan 0.59 [17] 4 chitosan from seafood wastes 0.70 present work 5 carboxymethylcellulose-crosslinked-chitosan 0.83 [18] 6 l-lysine-crosslinked-chitosan 1.03 [19] 7 thiourea-chitosan 1.06 [20] 8 glycine-crosslinked-chitosan 1.13 [21] 9 thiocarbamoyl-chitosan 1.24 [22] 10 bipyridinedicarbaldehyde-crosslinked-chitosan 1.45 [13] 11 glutaraldehyde-crosslinked-chitosan 1.69 [23] 12 ethylenedisulfide-chitosan 2.41 [24] 13 phenantrolinedicarbaldehyde-crosslinked-chitosan 2.47 [13] 14 glutaraldehyde-8hydroxyquinoline-crosslinked-chitosan 3.20 [13] 15 rubeanic acid-chitosan 3.31 [25] 16 chitosan-beads gel 3.38 [26] 17 polyethyleneimine-chitosan 3.75 [27] table 3. maximum adsorption capacities of reported adsorbent materials for palladium(ii) ions. j. multidiscip. appl. nat. sci. 30 palladium(ii) ions shall be easily desorbed by protonation of hydroxyl, amino and carbonyl functional groups [28]. as expected, acidic reagents were able to recover palladium(ii) ions in 65–90% as shown in figure 6. the recovery percentage of palladium(ii) ions using 0.1 m h2so4 (71%) was higher than 0.1 m hno3 (65%) due to a stronger acidic property of h2so4. meanwhile, the recovery percentage of palladium(ii) ions using 0.1 m hcl (76%) was higher than 0.1 m h2so4 (71%) due to the complexation of palladium(ii) ions with chloride anions to form [pdcl4] 2. a higher concentration of hcl reagent (i.e., 1.0 m) yielded a higher recovery percentage (90%), which was remarkable. furthermore, the chitosan was reprecipitated by adjusting the ph value to neutral and then filtered for a reusability study. the reusability of chitosan-based material was performed by subjecting the regenerated chitosanbased material after the desorption process to another adsorption process. the reusability data of palladium(ii) ions adsorption using chitosan-based material are shown in figure 7. it was found that the chitosan-based material did not lose its adsorption capability (87–90%) even after three consecutive cycles. furthermore, the ftir spectra of reused chitosan-based material (see figure 8) showed no significant change in the chitosan structure demonstrating the high stability of chitosan-based material for palladium(ii) ion recovery purposes. this work successfully demonstrated the excellent solution for the utilization of seafood wastes in depok beach as the chitosan materials resource for palladium(ii) ions recovery through an adsorption process. figure 6. recovery of palladium(ii) ions from metal-laden chitosan-based materials. [loaded palladium(ii)] = 0.09 mm. figure 7. reusability of chitosan-based materials for palladium(ii) recovery. figure 8. ftir spectra of recycled chitosan-based material for each cycle. j. multidiscip. appl. nat. sci. 31 4. conclusions chitosan material has been successfully prepared through chemical processes from seafood wastes in depok beach, yogyakarta in a 4% overall yield. the chitosan-based material showed effective palladium(ii) ions adsorption from the aqueous solution. the adsorption required 75 minutes to reach a plateau in which the adsorption kinetics followed the pseudo-second-order model with a reaction rate constant of 1.60 g mmol -1 min -1 . on the other hand, the isotherm adsorption of palladium(ii) ions fit well with the langmuir model giving qmax, kl, and rl values of 0.70 mmol g -1 , 3.82 l mmol -1 , and 0.025, respectively, demonstrating a favorable chemical adsorption process. the estimated heat of palladium(ii) ions adsorption was 23.80 kj mol -1 indicating a chemisorption process as supported by the ftir data. employing 1.0 m hcl gave a high desorption percentage (90%) thus the chitosan-based material can be reused for three consecutive cycles without losing its adsorption capability and structural stability, which was remarkable. these results demonstrate the potential application of seafood wastes as an alternative resource of bio-adsorbent material for effective palladium(ii) ions recovery in the future. author information corresponding author yehezkiel steven kurniawan — department of chemistry, faculty of mathematics and natural science, universitas gadjah mada, yogyakarta55281 (indonesia); orcid.org/0000-0002-4547-239x email: yehezkiel.steven.k@mail.ugm.ac.id authors rokiy alfanaar — department of chemistry, faculty of mathematic and natural sciences, universitas palangka raya, palangka raya73111 (indonesia); orcid.org/0000-0002-6690-2758 krisfian tata aneka priyangga — department of chemistry, faculty of mathematics and natural science, universitas gadjah mada, yogyakarta-55281 (indonesia); orcid.org/0000-0002-4119-2470 arif cahyo imawan — graduate institute of applied science and technology, national taiwan university of science and technology, taipei-10607 (taiwan); orcid.org/0000-0003-1910-7572 jumina jumina — department of chemistry, faculty of mathematics and natural science, universitas gadjah mada, yogyakarta-55281 (indonesia); orcid.org/0000-0003-2604-7838 author contributions conception and design: y.s.k. acquisition data: y.s.k., r.a., and k.t.a.p. analysis and interpretation of data: j. and a.c.i. drafting the article, review, and editing: y.s.k., k.t.a.p., and a.c.i. all authors have read and approved to the final version of the manuscript. conflicts of interest the author(s) declared no conflict of interest acknowledgement financial support from the ministry of education, culture, research, and technology republic of indonesia is greatly acknowledged. references [1] t. t. v. phan, t. c. huynh, p. manivasagan, s. mondal, and j. oh. 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[2], the density of sea urchins in lemukutan island, bengkayang ranged from 273–453 ind/ha. meanwhile, the density of sea urchins on penata kecil island ranged from 167–347 ind/ha and on penata besar island has a range from 307–387 ind/ ha. intertidal zones of sancang coasts were known to have marine organism diversity ranging from seagrass [4] that functioned as food resources for marine fauna, gastropods [5], echinodermata, and particular holothuroidea [6]. despite extensive studies on sancang’s intertidal zones, there is a paucity of sea urchin communities and which sides of sancang’s intertidal zones should be protected and conserved. here, this study aims to estimate suitable habitats for supporting the conservation of sea urchins in sancang’s intertidal zones. 2. materials and methods 2.1. study site this research was conducted at intertidal zones of sancang coast, garut district, west java, indonesia on july 19-21, 2020. the research location was a part of leuweung sancang natural reserve. the research location consists of 3 stations, namely station 1 located in ciporeang on publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.162 open access received : january 19, 2023 https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.162 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.162&domain=pdf&date_stamp=2023-02-27 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 114 the east side with rock and coral substrates, station 2 located in cikujangjambe in the middle with a sandy substrate overgrown with algae, and station 3 located in cibako in the west side which is dominated by seagrass and algae (see table 1). these three stations were chosen because the three locations have quite different ecological characteristics, so they are assumed to have different sea urchin species diversity. all stations were bordered by a natural reserve on the north side and indian ocean on the south side. the research location is shown in figure 1. 2.2. methods 2.2.1. sea urchin sampling and surveys sea urchin sampling was carried out using the belt transect method which a transect was drawn perpendicularly from the coast to the sea for 100 m. sampling was carried out at 3 stations (table 1) and each station contained 100 plots with a size of 1×1 m for each plot in sequence. identification of sea urchins was carried out by comparing the morphology of the sea urchin species obtained with the morphological characteristics identified by raghunathan et al. [7]. moreover, the scientific articles based on the shape, color, spines, and patterns of sea urchins were also reviewed. 2.2.2. environmental variable measurements measurements of the environmental variable at each station were carried out around 07.00-09.00 am (gmt+7). measured environmental variables were including ph, salinity (‰), dissolved oxygen (do, mg/l), and water temperature (°c). all variables were measured in situ using digital multiparameter devices. 2.2.3. suitable habitat estimation analysis estimations of suitable habitats for sea urchins followed the workflow (figure 2) and methods reported by meixler and bain [8] and boitt et al. [9]. the used suitability method was the scoring and overlay of environmental variables and sea urchin individual numbers mapped into an intertidal zone map. all environmental variable values including ph, salinity, dissolved oxygen, and water temperature were scored from the lowest and the highest value. the lowest score of the variable was considered the least suitable and the highest score of the variable was considered more suitable. then, all the score values were tabulated into the geographical information system (gis) table along with their geocoordinates. the tabulated gis tables containing environmental variables and sea urchin individual numbers were used and interpolated to create gis layers and vector shapes. all the layers were overlayed and score values in each environmental variable layer and vector shape were summed to create composite layers. the final step was to classify score values in the composite layers and vector shapes with the highest score values. they were classified as the most suitable habitat classified as high-very high classes. while the vector shapes with the lowest score values were classified as the least suitable habitat classified as low-very low classes. this process was conducted figure 1. three station (st. 1, st. 2, st. 3) locations at intertidal zones of sancang coast, leuweung sancang natural reserve, west java, indonesia. j. multidiscip. appl. nat. sci. 115 by using gis software (arcview 3.3). 2.2.4. statistical analysis statistical analysis of chi-square was used to calculate the significant differences in compositions of areas designated as the most and the least suitable habitats for stations 1, 2 and 3. the significant level used was p < 0.05. 3. results and discussions 3.1. sea urchin community based on the results, there were 3 sea urchin species observed at the intertidal zones of the sancang coast. those species were diadema setosum, heterocentrotus trigonarius and stomopneustes variolaris. mean individuals based on the species (figure 3.a) shows that s. variolaris has the highest mean individuals up to 6.3 individuals (95%ci: 0-13.9), followed by h. trigonarius up to 1.6 individuals (95%ci: 0-4.94), and the lowest mean was observed in d. setosum with values of 1.3 individuals (95%ci: 0-3.94). while mean individuals of sea urchins based on the locations (figure 3.b) shows that the cibako site has the highest sea urchin individual mean with values of 7.6 individuals (95%ci: 1.43-13.9). figure 4 depicts the spatial distributions and species compositions of individual numbers of sea urchin species such as d. setosum, h. trigonarius and s. variolaris at the sancang coast, leuweung sancang natural reserve, west java, indonesia. it can be seen that cibako site has more species in comparison to ciporeang and cikujangjambe sites. besides that, s. variolaris is the most common species since it has a wider distribution in comparison to other species. this species has dominated both the ciporeang and cikujangjambe sites. 3.2. environmental variables figure 5 shows the spatial pattern and distribution of measured environmental variables. based on the result, the water temperature was increasing towards the ciporeang site, or station 1, on the east side. the water with the highest temperature (28.1 °c) was observed at station 1. a similar pattern can be observed for the dissolved oxygen parameter. the dissolved oxygen was also rising along the sancang coast's intertidal zones. the highest water dissolved oxygen was observed in station 1 was equal to 5.64 mg/l. in contrast, ph and salinity showed the opposite pattern. according to the result, ph was observed declining towards the ciporeang site, or station 1 on the east side, with the lowest ph value at 8.34. similarly, salinity was also observed declining towards the ciporeang site, or station 1 on the east side, with the lowest salinity values at 32.65. this condition makes the seagrass and algal dominant sites at cibako in the west have colder water, alkali water, high salinity, and less dissolved oxygen. while the rocky ciporeang on the east side has warmer water, acidic water, low salinity yet oxygen-rich water. 3.3. suitable habitat estimations figure 6 shows the habitat suitability for sea urchin species at intertidal zones using the presence of sea urchins and environmental variables as inputs. it can be seen that the suitability was increasing from the east to the west sides, and this made the intertidal zones on the east side not suitable, while the intertidal zones on the west side were the most suitable (chi-square test, x 2 = 338.221, p < 0.05). up to 55% of intertidal zones (figure 7) at ciporeang were classified as not suitable, and only 45% of the zones were moderately suitable. the cikujangjambe sites in the middle have up to 70% of the intertidal zones table 1. characteristics of study sites. stations latitude longitude substrates and remarks 1. ciporeang-east 7°44ʹ19ʺ s 107°52ʹ9ʺ e rock and coral substrates. bordered directly by a river mouth. fragmented and patchy seagrass covers are observed here. 2. cikujangjambe-middle 7°44ʹ19ʺ s 107°52ʹ38ʺ e a sandy substrate overgrown with algae. 3. cibako-west 7°44ʹ24ʺ ls 107°52ʹ26ʺ e dominated by seagrass and algae. the bottom substrates consist of rocky substrates. j. multidiscip. appl. nat. sci. 116 classified as suitable. while all intertidal zones at cibako sites were suitable for sea urchins with 70% classified as very high and 30% as high suitable. 3.4. discussions this research highlights the importance of sea urchin conservation by elaborating on the suitable habitats for conserving the sea urchin community in the intertidal zones of the sancang coasts. this is in agreement with matsiori et al. [10] previous studies that highlight current threats to sea urchins and the importance of designating conservation areas by determining suitable habitats. the determination of suitable habitat will lead to the establishment of marine protected areas (mpa) that have proven to be a successful tool for the conservation of marine forests and the control of sea urchin populations [11][12]. in the intertidal zones, d. setosum individuals were very rare in comparison to other sea urchin species. according to an interview with the local community, d. setosum was frequently consumed. the d. setosum sea urchin is edible and desirable as food by locals and is a promising target for potential fishing [13]. s. variolaris, on the other hand, was abundant and widespread throughout the intertidal zone, with the highest individual numbers observed at the ciporeang site. the presence of s. variolaris in varied substrates was related to the adaptation ability of this species [14]. sea urchins belonging to stomopneustidae can use their sharp teeth to carve sedimentary rocks and create pits in which their spheroid-shaped bodies can fit, and this adaptation ability explains s. variolaris's presence across intertidal zones of the sancang coast [15]. the cibako site was selected as the most suitable habitat considering several environmental variables that can favour the life of sea urchins. this is very important since differences in the distribution and abundance of sea urchins may be attributed to the variability of habitats available across sites [16]. besides that, since one of the urchin species, d. setosum, is very rare and threatened by harvesting, designating cibako as a conservation zone will increase the d. setosum density. on the contrary, ciporeang site was considered suitable. this condition was related to the location of ciporeang figure 2. workflow and method for suitable habitat analysis. j. multidiscip. appl. nat. sci. 117 near the river mouth. proximity to the river mouth causes the intertidal zones in ciporeang to receive more freshwater in comparison to other sites, thus, reducing the water salinity and making these sites unsuitable to fulfil the salinity levels for sea urchins. according to allen et al. [17], reduced salinity has strong negative effects on fertilization, development, and hatching in sea urchins. lower salinity as observed in the ciporeang site will reduce the percentage of eggs that will be fertilized. this explains why the ciporeang site was not suitable. besides declining in salinity, the ciporeang site also has a ph decline, which makes the water in this site more acidic and not suitable for sea urchins. despite the fact that the differences in environmental variables were actually quite small, they could have an effect on the species, considering that the urchin is an invertebrate organism that may have less metabolism and mechanisms to cope with environmental variable differences. according to sato et al. [18], declines in ph will likely affect the ecology and fitness of sea urchins. besides that, the urchins will grow more slowly in acidic water with declining ph [19]. acidic water as observed in ciporeang will increase sea urchin metabolism costs for calcification or cellular homeostasis, and this will impact growth and result in increased mortality of sea urchin larvae during the pelagic life stage [20]. the acidic water at the ciporeang site has been influenced by its proximity to the river mouth. the river mouth is a site that receives wastewater discharge from a nearby settlement. domestic wastewater discharged into rivers is the primary contributor to degraded water quality [21][23] and acidification in downstream areas [24]. figure 3. mean individuals of sea urchins based on species (a) and stations (b) at intertidal zones of sancang coast, leuweung sancang natural reserve, west java,indonesia. figure 4. compositions of individual numbers of sea urchin species (diadema setosum, heterocentrotus trigonarius, stomopneustes variolaris) at intertidal zones of sancang coast, leuweung sancang natural reserve, west java, indonesia. j. multidiscip. appl. nat. sci. 118 figure 5. spatial estimations of environmental variables include water temperature, ph, salinity, and do at intertidal zones of sancang coast, leuweung sancang natural reserve, west java, indonesia. j. multidiscip. appl. nat. sci. 119 4. conclusions in the intertidal zones of the sancang coast, the most abundant and common sea urchin species was stomopneustes variolaris. the west side of the coast at cibako has the most diverse sea urchin species. this site was also characterized by colder, alkaline and saline water. on the contrary, the eastern side of the coast at ciporeang near the river mouth has fewer sea urchin species and is characterized by warmer, acidic and fresh water. it is estimated that the west side at cibako, followed by the middle side of the intertidal zones with higher than 50% of their intertidal zones classified as high and very suitable, were more suitable than the east side. then, the conservation of the sancang coast's sea urchin community should prioritize the west side, with particular attention to the cibako sites. authors information corresponding author vita meylani — department of biology education, universitas siliwangi, tasikmalaya46115 (indonesia); orcid.org/0000-0003-4386-3298 email: vibriovita@unsil.ac.id authors purwati kuswarini suprapto — department of biology education, universitas siliwangi, tasikmalaya-46115 (indonesia); orcid.org/0000-0001-5037-4666 ika raymita husna — department of biology education, universitas siliwangi, tasikmalaya46115 (indonesia); orcid.org/0009-0005-6196-2582 andri wibowo — ecology laboratory, universitas indonesia, depok-16424 (indonesia); orcid.org/0000-0001-7787-5735 figure 6. habitat suitability for sea urchin species at intertidal zones of sancang coast, leuweung sancang natural reserve, west java, indonesia. figure 7. area compositions of suitable habitats (chi-square test, x 2 = 338.221, p < 0.05) for sea urchin species in stations 1, 2 and 3 at intertidal zones of sancang coast, leuweung sancang natural reserve, west java, indonesia. https://orcid.org/0000-0003-4386-3298 mailto:vibriovita@unsil.ac.id https://orcid.org/0000-0001-5037-4666 https://orcid.org/0009-0005-6196-2582 https://orcid.org/0000-0001-7787-5735 j. multidiscip. appl. nat. sci. 120 author contributions v.m. designed the research and supervised all the processes, p.k and i.r.h. collected the field data, and a.w analysed and wrote the manuscript. finally, a.b. and e.n. proofread the manuscript. conflicts of interest the authors declare no conflict of interest. acknowledgement we wish to express our thanks to the students for the field and survey assistance (nurfauzi ahmad, ika ramdana bachin, chestalita oktaviana, pujiati nurachman, rina maryani, taufik, nurul, dedi koswara, virdan, sendi and eri). references [1] e. elmasry, h. a. omar, f. a. abdel razek, and m. a. el-magd. 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(2019) concluded that poor access to water, sanitation, and hygiene practices of caregivers directly relates to the prevalence of malaria among their children [7]. a mathematical model of malaria dynamics was developed with naturally acquired transient immunity in the presence of protected travellers [3]. a non-autonomous model was also developed to assess the impact of different microclimate conditions on the transmission dynamics of malaria [8]. a mathematical model has been proposed for the transmission dynamics of malaria by incorporating change via education as a control strategy [9]. the human population follows the susceptible-protected-exposed-infectious-recovered (speir) pattern and the mosquito population follows susceptible-exposed-infectious (sei) patterns. an analytical study was carried out to investigate the local stability of the system while the basic reproduction number was obtained using copyright holder: © oluwafemi, t., and azuaba, e. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.97 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.97 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.97&domain=pdf&date_stamp=2022-01-13 j. multidiscip. appl. nat. sci. 2 the next-generation matrix method. the result shows that the disease-free equilibrium of the system is locally asymptotically stable if .. the impact of temperature in malaria disease transmission dynamics was mathematically studied [10]. the seir model was suitable for the human population and lsei compartment model was suitable for mosquito population. it was observed that temperature affects the transmission dynamics of malaria significantly. the impact of drug resistance in malaria transmission was also modelled [11]. many mathematical models have been developed to study malaria dynamics but none has been discussed to study the impact of hygiene on malaria transmission dynamics as proposed in this work. in this work, we propose a deterministic mathematical model for assessing the impact of hygiene on malaria transmission dynamics. the basic reproduction number is computed and the local and global stability of the disease-free equilibrium are established. furthermore, the sensitivity analysis of the parameters is also evaluated. 2. materials and methods 2.1. model formulation in this model, the total human population denoted by ( ) is subdivided into unhygienic susceptible human population ( ), hygienic susceptible human population ( ), unhygienic infected human population ( ), hygienic infected human population ( ) and the recovered human population ( ). the mosquito population denoted by ( ) is subdivided into susceptible mosquitoes ( )and infected mosquitoes ( ). see the equations 2.1 and 2.2. (2.1) (2.2) let be the recruitment rate of the human population. a fraction enters unhygienic susceptible human class while the remaining fraction ( ) enters the hygienic susceptible human class. the unhygienic susceptible class is increased by the rate at which unhygienic human class lose immunity after recovery given as , and reduced by the rate of progression to hygienic class ( ), the force of infection for the unhygienic class ( ) and natural human death rate ( ). the hygienic susceptible human compartment is increased by the , the rate at which hygienic human loss immunity after recovery at , while the compartment is reduced by natural human death rate and the force of infection for the hygienic class . the is increased by and reduced by , rate of progression from to given as . malaria induced death for unhygienic human class and recovery for unhygienic human are denoted as and . the is increased by and then reduced by the recovery rate for a hygienic human class given as . malaria induced death for hygienic human class is denoted as . the human recovery class ( ) is increased by and , then reduced by , , and . the is increased by the mosquito recruitment rate given as , reduced by the mosquitoes death rate , and force of infection for mosquito given as . meanwhile, the is increased by and . given the above description and definitions of variables and parameters in table 1 and 2, the following are the model equations: (2.3) (2.4) (2.5) (2.6) (2.7) (2.8) (2.9) “where” (2.10) j. multidiscip. appl. nat. sci. 3 2.2. invariant region the invariant region can be obtained by the following theorem. theorem 1 the solutions of the model are feasible for all if they enter the invariant region (2.11) proof: let (2.12) be any solution of the system with non-negative initial conditions. hence, all feasible solution set of the human population of the malaria model enters the region (2.13) similarly, the feasible solution set of the vector population enter the region (2.14) therefore, the region is positively invariant i.e. the solution remains positive for all initial values. thus, the model is biologically meaningful and mathematically well-posed in the domain . 2.3. disease free equilibrium (dfe) the dfe of the model equations can be found by setting the right hand of the model (2.3) – (2.9) to zero, i.e. which gives (2.3) (2.4) figure 1. model flow diagram. symbols description su unhygienic susceptible human sn hygienic susceptible human iu unhygienic infected human in hygienic infected human r recovered human sv susceptible mosquitoes iv infected mosquitoes table 1. variables. j. multidiscip. appl. nat. sci. 4 (2.5) (2.6) (2.7) (2.8) (2.9) at dfe, so we have, table 2. model parameters. symbols description recruitment rate of human population recruitment rate of mosquitoes progression from su to sn progression from iu to in disease—induced death for the unhygienic human class disease—induced death for the hygienic human class biting rate of mosquito for unhygienic human class biting rate of mosquito for hygienic human class transmission probability of infection from mosquito to human transmission probability of infection from human to mosquitoes the force of infection for unhygienic human class the force of infection for hygienic human class force of infection for mosquitoes biting rate of mosquitoes rate of reduction of infection for hygienic class modification parameter rate of recovery for unhygienic human class rate of recovery for hygienic human class rate at which recovered human become susceptible hygienic rate natural human death rate natural death rate of mosquitoes total human population j. multidiscip. appl. nat. sci. 5 after computing simultaneously, we have therefore, the dfe point of the model is given by (2.15) 2.4. basic reproduction number (r0) the r0 is defined as the number of secondary malaria infections produced by one infected individual in a completely susceptible community. the next-generation method [12] will be employed to compute r0. the f(x) is the rate of new infection appearance while v(x) is the rate of transfer of individuals into compartments. therefore, (2.16) (2.17) whereas (2.18) (2.19) the r0 is the largest eigenvalue or spectral radius of fv -1 . hence, (2.20) theorem 2 the dfe for model system is locally asymptotically stable if and unstable otherwise. proof: at dfe, the jacobian matrix is given by symbols sensitivity index -1 1 -0.00013 -0.000041 -0.000041 -0.29 0.00022 1 1 1 1 -0.087 1 -0.000015 -0.71 -0.00011 1 -2 table 3. indices of sensitivity. j. multidiscip. appl. nat. sci. 6 the eigenvalues are: it is observed that all the eigenvalues are negative, this implies that at the dfe point is locally asymptotically stable, this means that malaria infection can be eliminated from the population. 2.5. global stability of the disease free equilibrium (dfe) theorem 3 the dfe of the model system is globally asymptotically stable if . proof: consider the following lyapunov function: (2.21) differentiating yield (2.22) symbols values source 100 [13] 1000 [14] 0.25 (assumed) 0.5 (assumed) 0.13 (assumed) 0.06 (assumed) 0.17 (assumed) 0.1 (assumed) 0.03 [2] 0.09 [2] 0.12 [15] 0.08 (assumed) 0.5 (assumed) 0.05 (assumed) 0.15 (assumed) 0.7902 [14] 0.46 (assumed) 0.00004 [13] 0.0000569 [14] table 4. parameter values of model. j. multidiscip. appl. nat. sci. 7 at dfe, it was found that (2.23) from the equation above, if (2.24) hence, the dfe is globally asymptotically stable. 2.6. sensitivity analysis in this section, sensitivity analysis is carried out to identify the parameters that have a great influence on the r0. the sensitivity index of r0 to a given parameter p is given by the relation (2.25) table 3 shows the sensitivity indices of the basic reproduction number to the parameters. the parameters with positive indices indicate that the basic reproduction number increases as their values increase. while the parameters with negative sensitivity indices indicate an increase in these parameters will result in the decline of the basic reproduction number and vice-versa. 3. result and discussions first, this system of model is biologically meaningful and mathematically well-posed in the given domain . the r0 of the model is computed using the next-generation method. the existence of the disease-free equilibrium of the system is established and the condition for the local stability of the disease-free equilibrium and global stability of the disease-free equilibrium follows using the lyapunov function. the dfe is locally asymptotically stable if and globally asymptotically stable if . sensitivity analysis of the model equation is carried out as illustrated in table 3. from the table, it shows that the natural death rate of mosquitoes ( ) is most sensitive to the basic reproduction number. 4. conclusions in this work, the mathematical model to assess the impact of hygiene on malaria transmission dynamics was proposed and analyzed. the model is divided into the human population and vector (mosquito) population, the human population is further subdivided into the susceptible unhygienic human population, susceptible hygienic human population, infected unhygienic human population, infected hygienic human population and recovered human, while the vector population is subdivided into the susceptible vector and infected vector. we proved that the model equation is biologically meaningful and mathematically well-posed. the disease-free equilibrium (dfe) is established and it was observed that dfe is locally asymptotically stable if while globally asymptotically stable if using lyapunov function. sensitivity analysis of the model parameters is carried out and it shows that the natural death rate of mosquitoes is most sensitive to the basic reproduction number. this implies that individuals must continue to engage in activities that promote both personal hygiene and environmental hygiene so as reduce the growth of mosquito hence curbing the spread of malaria also government and other nongovernmental organizations (ngos) must continue to intensify campaigns on hygienic practices at individual and community levels. future studies can be carried out on this model such as: establishing and proving the existence of the unique endemic equilibrium point, analyzing the stability (local and global stability) of the endemic equilibrium point, and solving the model equations using any analytic method available. author information corresponding author temidayo oluwafemi — general studies department, newgate college of health technology, minna 920211 (nigeria); email: dayofemi985@gmail.com mailto:dayofemi985@gmail.com j. multidiscip. appl. nat. sci. 8 author emmanuel azuaba — department of mathematics, bingham university karu, nassarawa 962101 (nigeria) ; references [1] e. azuaba, j. m. orverem, y. m. kura and u. j. dahiru. 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(2013). “mathematical model for malaria transmission dynamics in human and mosquito populations with nonlinear forces of infection”. international journal of pure and applied mathematics. 88 (1): 125-56. https://doi.org/10.2174/1874279301810010166 https://doi.org/10.1080/17513758.2020.1722265 https://doi.org/10.1007/978-3-030-46020-4 https://doi.org/10.5897/ijps10.542 https://doi.org/10.5897/ijps10.542 https://doi.org/10.1098/rsif.2009.0386 https://doi.org/10.2478/bile-2019-0013 https://doi.org/10.2478/bile-2019-0013 https://doi.org/10.1007/s40819-017-0394-5 https://doi.org/10.1007/s40819-017-0394-5 j. multidiscip. appl. nat. sci. 9 10.12732/ijpam.v88i1.10. https://doi.org/10.12732/ijpam.v88i1.10 100 journal of multidisciplinary applied natural science vol. 1 no. 2 (2021) review article limitations in pigeon keeping: a review m ashraful kabir received : april 4, 2021 revised : june 9, 2021 accepted : june 19, 2021 online : july 13, 2021 abstract pigeons are showing remarkable progress with more profit. this sector has many potential markets in the country. after visiting some pigeon houses, many limitations have been come out. many rearers collect pigeons based on only physical appearance, and in the long run, they cannot get pure offspring. king pigeon is considered a table breed in the world but in bangladesh rearers keep them as a common fancy item. pigeons do not get their nutritious food from the owners. result suggests that many rare pigeons are caught by predators and due to unwanted dirt in those lofts birds are affected by many diseases. some remarkable farms are showing quality environment in their lofts and getting desirable profit. keywords limitations, pigeon, keeping, rearing 1. introduction pigeon keeping is a primitive hobby all over the world. many famous people of the world kept pigeons for their amusement [1]–[3]. activities of pigeon club and research are increasing in the country and this the best way for overcoming many limitations in this pigeon sector [4]–[6]. in the year 2020, 29 february, the national pigeon association of bangladesh (npab) organized an international pigeon fair in dhaka [7] for strengthening the bondage of pigeon rearers all over the world due to commercial purpose. mulyani et al. [8] completed several works on the moulting of birds. at this time, female pigeon needs special care and extra protein added food is recommended. genetically, pigeons have very strong navigation power as a whole [9]– [11]. the only chromosomal method is the reliable indicator for sex determination of pigeon and he mentioned 14 points for identification of purebreds of pigeon [12]. secondly, haunshi [13] described some good points for pigeon identification. lapiedra et al. [14] studied the genetic behaviour of pigeons. any crossbred have been considered an excellent foster pigeon in bangladesh [15][16]. in bangladesh many pigeon markets and pet shops are available. when pigeon lovers purchase their racing pigeon they observe flight muscle, flying capability in their home, feathers, and eyes. in the case of the fantail pigeon, they see only the number of rectrices, large-size, crest, and tremule [17]. rats and mice are available in most pigeon farms in bangladesh which are the main source of typhoid bacterium [18][19]. many pigeon keepers provide huge poultry feed to the pigeons and for this pigeons got obese and ultimately the production slows down. naturally, pigeons are very intelligent and normally do not get fear to see domestic animals [20]. the objective of this review is to perceive the major limitations with their solutions in the pigeon farms of bangladesh as a whole. 2. characteristics of purebreds each breed of pigeon has some salient features. before starting a pigeon farm need to understand those grading points of the pigeons. body structure, disease-free condition, legs, tail, breast, head-beakcere-wattle-eye, neck, etc. all are the points for collecting a pigeon [17][21]–[23]. in addition, need to know their weight, leg feather, number of rectrices, rump width, and position of wing [24]– [26]. 3. commercial meat-bred king pigeon is the best for meat purpose [7]. females will have a useful breeding life of up to 10 years and males 5 years. pigeon meat has some copyright holder: © kabir, m. a. (2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.86 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.86 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.86&domain=pdf&date_stamp=2021-07-13 j. multidiscip. appl. nat. sci. 101 remedic values like depression, muscle pain, thyroid problems, skin problems, hair loss, nail problems, lower immunity and this meat is enriched with more zinc, phosphorus, iron, sodium, selenium, and copper. chinese people take pigeons’ meat as their medicinal value [27]. 4. food and nutrition in the life of pigeons we see their growing, moulting, breeding, laying and hatching stages. home-made healthy grits are good for the bone formation all the year round. for controlling fungal growth in feed need to dry feed by sunlight. sometimes, long bristles of paddy can attach to the pigeons’ gullet which causes abscess. supplied water will be clean always. in pre-laying/hatching/ suckling stagescorn 6 kg, wheat 4 kg, black-pea 2.5 kg, japanese millet 0.5 kg, broiler feed 1 kg, green-pea 0.5 kg (figure 1), and mustard 250 g are suitable [20]. abd el-hack et al. [28] mentioned that the feed consumption is about one-tenth of the pigeon’s body weight. 5. pigeons’ predators a study in bangladesh suggested that out of 12 predators’ mammals were the highest number [29]. in israel and bangladesh, golden jackals were seen as scavengers [30]–[32]. poison bait is used for controlling this golden jackal [33][34]. sometimes rock-pigeons are killed by predator birds. there are 17 species of raptors are found in bangladesh consisted of three familiesfalconidae, accipitridae, and pandionidae [35]. indeed, falcon does not breed in bangladesh. a piece of mirror on the long trees beside the farm and irregular flying of tumbler and racing pigeons makes puzzle of the raptors [35]. snakes can be controlled by using thallium sulfate. to make fear, scarecrow and water spray are useful for protecting such pigeons [29]. monitor-lizards may come near the loft for carcasses. for protecting pigeons, farmers should know the breeding season of crows. pit bull, fox terrier, and airedale terrier dogs are good for patrolling the loft [36]. treepie (dendrocitta spp.) consumes 64% insect in the crop field and only attacks 0.1% to columba livia nestling [37]. one inch mesh size is sufficient for controlling sparrows within the loft. white feathered pigeons are always caught by the predators. 6. diseases of pigeons and cage sanitary out of 20 diseases of pigeons in bangladesh 30% were for bacterium, 20% viruses, 15% rearing related, protozoan and vitamin deficiency 10%, 5% for fungal, and mycoplasma and food related 5% [38] (figure 2). in the country most pigeon keepers are not conscious about the diseases of their pigeons. some mycotic, bacterial, protozoal, chlamydial, rickettsial, and parasitic diseases are considered as zoonotic diseases and their numbers are approximately thirty [39]. pigeons are occasionally treated as zoonotic harbour. pigeons waste carries aspergillus fungus and 2% salmonella bacterium. dirty water mainly after pigeons’ bath is the great source of fluke and lots of mites. runt, king, and strasser are higher intensity of parasites and other study showed that older pigeons can be affected by trichomonas galllinae. coccidiosis/blood dysentery is second common problem of all farms. disinfection by using savlon, figure 1. food stuffs for pigeons. 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 a m o u n t (k g ) green pea japanese millet broiler feed black pea wheat corn 0.5 0.5 1.0 2.5 4.0 6.0 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 p e r c e n ta g e ( % ) mycoplasma and food fungi protozoa and vitamin rearing virus bacteria 5 % 5 % 10 % 15 % 20 % 30 % 15 % others figure 2. percentages of disease incidents. j. multidiscip. appl. nat. sci. 102 dettol, potash, and bleaching powder is good at all. excessive number of birds in a narrow space and poor ventilation causes the ammonia toxicosis that enhances ocular discharge. newly fledged pigeons’ showed the highest mortality and time between fledging and settlement in a feeding flock is crucial [40]. 7. conclusions bangladesh is an excellent harbour for many fancy pigeons. all birds have true markings but need to ensure those characteristics by the keepers by keen observation. we can establish a pigeon farm only for human meat consumption after fulfilling our hobby. in this regard, king pigeon, strasser, and maltese breeds can be good perspective bangladesh. without nutritious food, it is quite impossible to get sufficient meat from the squabs and nice pairs for the fulfillment of our hobby. different stages of pigeons need different food ratios with sufficient home-made grits. we should increase our knowledge for understanding purebreds either they are fancy or table breed. in addition, we need to overcome major limitations such as cleanliness and balanced diet for achieving positive impact in our economy. author information corresponding author m. ashraful kabir — department of biology, cantonment public college, saidpur cantonment 5311 (bangladesh); orcid.org/0000-0001-7093-2741 email: ashraful.mission@gmail.com conflict of interest the authors declare that there is no conflict of interest. references [1] m. a. kabir, t. j. hawkeswood, and d. makhan. 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(2020). “genetic diversity and population structure in beninese pigeon pea [cajanus cajan (l.) huth] landraces collection revealed by ssr and genome wide snp markers”. genetic resources and crop evolution. 67 (1): 191–208. 10.1007/s10722019-00864-9. https://doi.org/10.1080/01652176.2018.1482028 https://doi.org/10.2979/pjhs.3.1.04 https://doi.org/10.1007/s10722-019-00864-9 https://doi.org/10.1007/s10722-019-00864-9 131 journal of multidisciplinary applied natural science vol. 3 no. 2 (2023) research article an analytic solution to the inhomogeneous verhulst equation using multiple expansion methods agus salim*, albert sulaiman, and mishima kenji received : february 3, 2023 revised : march 31, 2023 accepted : april 1, 2023 online : april 17, 2023 abstract the present study aims to obtain an analytic solution for the inhomogeneous verhults equation using multiple expansion methods. this study identifies the external factors represented by the inhomogeneous term that determine optimal variable conditions for ecosystem population growth. the simulation involves scenarios that utilize constant growth rates, periodic growth rates, constant external factors, and periodic external factors. it is found that external factors increase population growth, whereas constant external factors prevent growth under saturation conditions. periodic external factors cause fluctuations in the amplitude of growth regions. the present study will highlight and discuss the development and application of the solution. keywords environmental factor; inhomogeneous verhults equation, multiple scale expansion 1. introduction the analytical solution describing the evolution of population growth was proposed by pierre francois verhulst, which has a sigmoid curve [1]. ecologists and population scientists use population change models to describe the behavior of a population [2]. this model predicts how the population as a system experiences the dynamics of change. the model for population change generally consists of an exponential curve and a logistic curve. the main differences between the two models include three things: 1) exponential growth is j-shaped while logistic growth is sigmoid (sshaped), 2) exponential growth only depends on population size but logistic growth depends on several factors including population size, competition, and a number of resources, and 3) exponential growth applies to populations that do not have growth restrictions while logistical growth applies to each population with carrying capacity [3]-[6]. the exponential growth model describes certain data patterns that increase over time. the data graph reflects the exponential function and creates a j-shape. with respect to population change, exponential growth occurs when an unlimited amount of resources are available to the population. a clear example is the pest population which will grow exponentially if there is no limit to how much food the pest can eat from the garden. another example is the current human population growing at an exponential rate. the logistic growth model describes data patterns where the growth rate gets smaller as the population approaches a certain maximum (known as the carrying capacity). the logistic growth graph is a sigmoid curve. with respect to population changes, logistic growth occurs when available resources are limited or when there is competition between animals. the pest population will increase until the introduction of pesticides and its carrying capacity allows the garden to thrive [7]. the behavior of the solution is exponential at the initial stage, followed by saturation, and finally, growth stops at maturity time. this equation has received a lot of attention from many researchers and has been shown to have various implications [8]. although the equation has been known for a long time, many studies are still conducted to examine its general properties. further investigation is carried out by considering the effect of the environment on growth [9][10]. the process of nature is irreversible, as manifested in an open system where there is an exchange of energy and matter between the system and the environment. the verhulst or logistics equation is a closed system, making it interesting to consider the environmental effect on the system. in a closed system, the parameters of the models are publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.176 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.176 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.176&domain=pdf&date_stamp=2023-04-17 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 132 assumed to be constant. however, in an open system, this assumption of constancy is not always true since the dynamics of such models can be influenced by environmental changes such as food availability, birth rates, disasters, etc. this means that the environmental effect should be taken into account in growth modeling. recently, the effect of a slowly varying environment has also been discussed [10][11]. the inhomogeneous verhulst model has been used in determining pandemic curves, such as covid-19 because it can determine the unpredictable peaks of second, third, and fourthwave contamination. taking into account the statistical inhomogeneity of age groups, it can be demonstrated quantitative understanding of the different behavior rules to flatten the covid-19 pandemic curve and over time multi-peak periods simultaneously. the simulation is based on the verhulst model with general analytical logistic equations for limited growth. the exponential growth for the first wave is faster than for the next wave [12]. this paper investigates the environmental effect on logistic processes with an external effect, i.e., the slowly varying effect of natural growth. the slowly varying effect is represented by an inhomogeneous term in the verhulst equation. the multiple expansion methods will be used to solve the equation, and the behavior of the solution will be discussed. 2. materials and methods 2.1. the proposed model in this chapter, the inhomogeneity of the verhults equation will be derived. it begins with the standard of the verhulst equation that usually can be written in eq.(1) [13]. (1) where p(t) is called the population function, r defines the growth rate and k is called the carrying capacity (limitation of population, i.e., the maximum population density that the environment can carry). eq. (1) have the basic structure in the form of dp=dt= g(p) r(p) where g(p) is called the growth rate and r(p) is the removal rate. in many cases, the growth rate and the carrying capacity are time-dependent. the quadratic term represents the competition between members of the same population for resources, i.e., intraspecific competition. the analytical solution of eq. (1) with initial condition p(0) = p0 and limt→∞ p(t) = k is given by goswami et al [14]. (2) recently, the generalization of the verhulst equation with environmental effect was done by adding the new term in eq.(1) as shown in eq.(3) [11]. (3) where f(p; t) is a function of p and t. miskinis and vasilliauskiene studied a special case of the environment called the influence of harvesting of the population where the verhulst equation is given by eq.(4) [10]. (4) where c(t) is the harvesting population that is time-dependent. this is a homogeneous verhulst equation with a variable coefficient (varying the growth rate). the analytic solution is given by eq. (5) [10]. (5) where the new parameter θ=r-c(t) represents differences between growth rate and harvesting. the variation of solution based on the harvesting function c(t) can be obtained by solving the integral. it is not difficult to show that for c(t) = 0, then the standard verhuls equation was obtained. we modify eq.(4) by taking into account the time dependent on the growth rate so that eq.(4) yields eq.(6). (6) where the new coefficient now are given by θ(t) =r(t)-c(t) and γ(t)=r(t)/k, respectively. it is clear, if j. multidiscip. appl. nat. sci. 133 the growth rate a constant then this yields the miskinis and vasilliauskiene model [10], i.e., eq (4). the equation is the focus of the study. 2.2. an analytic solution in this section, we discuss a slowly varying environment that is represented by the timedependent coefficient in the inhomogeneous verhulst equation. assuming that the variation of the model parameters is slow relative to other quantities, we can apply the analytic multi-scaling technique to obtain solutions [15]. this method is expected to successfully represent the variation of the population over time. by assuming that the influence of the environment is weak, we can use the multiple time scale expansion method to find an analytical solution. the solution takes the form of eq 7., (7) where ϵ is a small parameter, τ = ϵt and the initial condition is the same as eq (1). the differential operation satisfies the chain rule for example (dg (t,τ)/dt= ∂g/∂t+∂g/∂τ×dτ/dt) the multiple scale expansion means that the nonlinear term is weak so that eq. (6) become eq.(8). (8) substituting this solution eq. (7) into eq. (6) then we should solve the equation order by order. the equation for zero order is given by eq.(9), (9) where p0 is the zero order of the solution. then the solution is given by eq. 10. (10) next, the first order is given by eq. 11, (11) where p1 is the first order of the solution this is an inhomogeneous coefficient variable differential equation of first order. the solution yield eq. 12, (12) due to ϵ being a small parameter then we can stop the calculation until the second order. thus, the second order is given by eq. 13, (13) the solution is given by eq. 14, (14) where p10 and p20 are an integration constant. figure 1. regression between data (random sample) and solution of homogenous logistic equation. j. multidiscip. appl. nat. sci. 134 3. results and discussions we will begin with a simple system. in realworld data, we should use curve fitting to understand how the model behaves relative to the properties of the data. since we do not have realworld data available, we will use simulated data instead. first, we normalize the data and the solution of the logistic equation as follows eq. 15, (15) the data are generated by a random variable with 50 samples. curve fitting between the data and logistic function is depicted in fig. 1. if the deviation of the data from the solution is small, then the system satisfies logistic homogeneity. otherwise, if the variation is greater, then we fit the solution with the effect of the environment taken into account. to further study the environmental effect, let us use the same simulation parameters as in miskinis et al. [10] where k = 52.0 mm, r = 3.19×10 -2 h -1 , ϵ is about 0.1, and then initial condition p0 is 0.27 mm and c=0.015 h -1 . we simulate the solution for four scenarios. in the first scenario, c(t) is zero, and r(t) is a constant. in the second scenario, c(t) and r(t) are constant, and in the third scenario c(t) is a periodic function and r(t) is a constant. in the last scenario, c (t) and r(t) both are periodic functions with frequencies 3.3 π and 2.38 π, respectively. the simulation is depicted in fig.2. the red color is a condition when r is constant and c is zero. this is nothing else than a standard verhulst solution that is the same as miskisnis et al. [10]. the blue color represents a simulation with a constant growth rate and constant external factors. this simulation shows that the positive constant external factor tends to increase growth. this result agreed with the previous works by idlango et al. [9] and tsoularis and wallace [16]. the external factor will lead to growth, and the system will not become saturated. these results also show that the growth factor will dominate in the long run. when the growth rate tends to be periodic and the external factor remains constant, there is a fluctuation in the growth region, which eventually becomes constant after 15 days. this can be seen in the black color. a similar pattern can also be found when the growth rate and external factors are periodic. the fluctuations occur with a higher amplitude, which can be seen in the green color. they show the saturation condition for up to 350 days. in general, logistic equations, including inhomogeneous ones, are typically solved numerically due to the availability of adequate numerical tools such as finite differences, among others, that are based on approximations [17]. figure 2. simulation of verhults solution with varying variable coefficients. the red color represents constant r and c = 0, the blue color represents constant r and c, the black color represents periodic r and constant c, and the green color represents periodic r, and c. j. multidiscip. appl. nat. sci. 135 however, an analytic solution is a closed-form mathematical expression that provides a complete and exact solution to a problem. it can offer a deeper understanding of the problem's behavior and properties and can also be used to develop theories, validate numerical methods, and guide experimental investigations. in some cases, we can easily calculate and predict how a system behaves when we apply external forces using simple computational tools that do not require largecapacity computers. however, for more complex cases, numerical studies may be necessary. for instance, the evolution of many species to ecosystem conditions with eutrophication and complex environment [18]-[20] and chaos phenomena [21] serve as an example. despite its drawbacks, this method provides an in-depth look at the capabilities of logistic equations with their variations as one of the fundamental processes in nature. 4. conclusions the environmental effect in logistic processes, which is represented by variable coefficients in the growth rate and removal rate, has been investigated. the multiple expansion method was used to find an analytic solution of the variable coefficient of the inhomogeneous varhults equation. we simulated scenarios with constant growth rate, periodic growth rate, constant external factor, and periodic external factor. it was found that the external factor tends to increase the growth of the population, whereas the constant external factor prevents growth in saturation conditions. meanwhile, the periodic external factor tends to fluctuate the amplitude in the growth region. we will carry out simulations with ecological measurement data for the next research. authors information corresponding author agus salim — department of biology, universitas islam negeri syarif hidayatullah, jakarta-15412 (indonesia); orcid.org/0000-0002-2599-8925 email: agus_salim@uinjkt.ac.id authors albert sulaiman — research center for climate and atmosfer, badan riset dan inovasi nasional (brin), bandung-40173 (indonesia); orcid.org/0000-0002-0306-7003 mishima kenji— department of chemical engineering, fukuoka university, fukuoka81401 (japan); orcid.org/0009-0002-5700-6297 author contributions project leader dan write paper a. s. 1; develop analytic model and write paper, a. s. 2; analysis result, m. k. conflicts of interest the authors declare no conflict of interest. acknowledgement this research funded by dipa 2018 the ministry of religious affairs republic indonesia. references [1] m. m. a. khater. 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https://doi.org/10.1007/978-0-387-48918-6 https://doi.org/10.1007/978-0-387-48918-6 https://doi.org/10.1016/s0025-5564(02)00096-2 https://doi.org/10.1016/s0025-5564(02)00096-2 https://doi.org/10.1002/mma.7323 https://doi.org/10.1002/mma.7323 https://doi.org/10.1007/978-94-017-2464-7_1 https://doi.org/10.1007/978-94-017-2464-7_1 https://doi.org/10.1007/s11356-020-12081-4 https://doi.org/10.1007/s11356-020-12081-4 j. multidiscip. appl. nat. sci. 137 397. https://doi.org/10.1111/ele.14156. https://doi.org/10.1111/ele.14156 44 journal of multidisciplinary applied natural science vol. 1 no. 1 (2021) research article antioxidant and antibacterial activities of magnesium oxide nanoparticles prepared using aqueous extract of moringa oleifera bark as green agents hanif amrulloh , awalul fatiqin, wasinton simanjuntak, hapin afriyani, annissa annissa received : october 13, 2020 revised : january 11, 2021 accepted : january 25, 2021 online : january 30, 2021 abstract in this research, magnesium oxide nanoparticles (mgonps) was prepared from mgcl2 solution using aqueous extract of moringa oleifera (m. oleifera) bark as green agent. preparation procedure involved mixing of mgcl2.6h2o solution and the aqueous extract of m. oleifera bark, followed by drop wise addition of naoh solution. the formation of mgonps in this study was confirmed using uv-vis absorption. the spherical crystal structure of mgonps was confirmed by xrd analysis. the average particle size of the synthesized mgonps was found between 60—100 nm using sem and tem images and psa results. the mgonps synthesized showed good antioxidant activity, as well as antibacterial activity against s. aureus, e. faecalis, e. coli, and s. dysenteriae bacteria. keywords antibacterial, antioxidant, bark extract, green synthesis, magnesium oxide nanoparticles 1. introduction green method is an experimental procedure that uses extracts from plants as agents to form nanoparticles. this method provides a variety of resources in the process of nanoparticle synthesis [1]–[3]. in addition, there are many other advantages in the use of natural materials in the synthesis of nanoparticles. the use of biodegradable raw materials for the synthesis of nanoparticles has no harmful impact on the environment, leads to a green technology policy, and is first and foremost environmentally friendly. second, the efficiency of synthesis, which promotes further production through the availability of abundant natural raw materials. third, the nanoparticles produced are biocompatible in comparison with conventionally seized nanoparticles [4]. magnesium oxide nanoparticles (mgonps) are multipurpose metal oxide nanoparticles which have many applications in various fields. mgonps have been widely used as catalysts [2][5][6] and catalyst supports for various organic reactions [7], adsorbents [8]–[10] and electrochemical biosensors [11]. in biomedical applications, there are evidence showing mgonps have antioxidant [12], antibacterial [13]–[15], antifungal [12][16], and anticancer [16] activities. moringa oleifera (m. oleifera) is a multipurpose tropical plant from the moriceae family which is spread throughout india, asia, and sub-saharan africa [17]. the use of m. oleifera extract has been widely used in synthesizing metal nanoparticles and metal oxides. moringa flower extract can be used to prepare palladium [18], silver [19], and hydroxyapatite [20] nanoparticles. on other hand, moringa leaf extract is useful to prepare silver [21], zinc oxide [22], titanium oxide [23] and nickel oxide [24] nanoparticles. phytochemical analysis of water extracts of m. oleifera bark shows that moringa wood bark contains compounds such as alkaloids, phenolic acids, terpenoids, and flavonoids [25]. these compounds play a role in the chelation process and they are able to reduce metal ions to nanoparticles [26]. the effect of m. oleifera bark water extract for preparation of mgonps was examined in this research. this research has been confirmed since the special use of these plant components for nanoparticle preparation is still limited. characterization of the mgonps was performed through uv-vis absorption spectroscopy, x-ray diffraction (xrd), electron scanning microscopy (sem), electron transmission microscopy (tem), and particle size analyzer (psa). the bioactivity of mgonps was subsequently examined as antioxidant and antibacterial agents. copyright holder: © amrulloh, h., fatiqin, a., simanjuntak, w., afriyani, h., annissa, a. (2021) first publication right: journal of multidisciplinary applied natural science this article is licensed under: https://doi.org/10.47352/jmans.v1i1.9 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i1.9 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i1.9&domain=pdf&date_stamp=2021-01-30 j. multidiscip. appl. nat. sci. 45 2. materials and methods 2.1. materials fresh m. oleifera bark were collected from the plants that grow naturally around the city of metro, lampung, indonesia during september 2019. laboratory grade magnesium chloride hexahydrate (mgcl2.6h2o), folin-ciocalteu reagents, sodium carbonate (na2co3), gallic acid, catechin, aluminum chloride (alcl3), sodium nitrite (nano2), and sodium hydroxide (naoh) were purchased from merck sigma-aldrich reagent pte, singapore. 2.2. methods 2.2.1. plant extract a fresh, m. oleifera sample washed by floating water, dried and then poured into powder and stored at room temperature under direct sunlight. in 100 ml of distilled water, four grams of m. oleifera bark powder was soaked and heated for 20 minutes at 60 ° c. the mixture was then filtered 1 hour with whatman filter paper 1 to isolate the extract from the residue. the mixture was left overnight. 2.2.2 phytochemicals analysis the aqueous extract was subjected to phytochemical analysis to detect the presence of carbohydrates, amino acids, glycosides, polyphenols, saponins, steroids, flavonoids, tannins, and alkaloids. total phenolic was estimated using the follin-ciocalteu test [27], and the result was expressed in µg/mg gallic acid equivalent (gae) unit. total flavonoid content was determined by the colorimetric alcl3 method employing catechin as standard and expressed as µg/mg equivalent of catechin (ce) [28]. 2.2.3. preparation of mgo nanoparticles (mgonps) to synthesize mgonps, 50 ml of an aqueous extract of m. oleifera barks was mixed with 50 ml mgcl2.6h2o solution 1 mm in a beaker at 90 °c and stirred at 600 rpm. the 1 m naoh solution was added drop wise until the color of the mixture faded out and precipitate was formed. the mixture was left for 3 hours to maximize the synthesis process. the mgonps synthesized was centrifuged at 7,500 rpm at room temperature and re-dispersed in deionized water and methanol (99%) to remove biological residues. the process was repeated twice, table 1. phytochemicals analysis of m. oleifera bark aqueous extract. chemical constituents testing methods m. oleifera bark aqueous extract alkaloids dragendroff's test + flavonoids shinoda test + saponins foam test + carbohydrate anthrone test + polyphenols puncal-d proteins ninhydrin test amino acids millon's test phenolics ferric chloride test + triterpens salkowski test anthraquinones borntragges test + = present, = absence figure 1. total phenolic and flavonoid content in m. oleifera bark aqueous extract, notes: value is mean ± sd. j. multidiscip. appl. nat. sci. 46 and the solid was dried at 100 °c. the solid was subjected to calcination at 600 °c for 5 hours was used to optimize the formation of oxides. 2.2.4. characterizations of mgo nanoparticles (mgonps) several techniques were used to characterize mgonps. the confirmation of mgonps synthesis was based on the change in the color of the mixture during the reaction and recorded with uv-visible spectroscopy (analytic jena specord 200 plus) by scanning the spectrum in the range of 200 800 nm. the morphology of mgonps was studied by sem fei inspect-s50. the size and morphology of the mgonps were investigated by tem jeol jem 1400) and the average particle size by psa horiba sz 100z). the crystal structure of the synthesized mgonps nanoparticles was confirmed by xrd pananalytical expert pro. 2.2.5. antioxidant activity antioxidant activity of m. oleifera bark aqueous extract (bem) and mgonps was evaluated through 2,2-diphenyl-1-picrylhydrazyl (dpph) radical assay in accordance with the procedure described, using ascorbic acid as reference [29]. a dpph 0.1 mm solution was prepared by dissolving dpph in ethanol. as much as 1 mg of ascorbic acid was dissolved in 1 ml of methanol. dilution was carried out to make a standard solution of ascorbic acid with different concentrations (50-500 μg/μl). for each tube containing a standard solution of ascorbic acid (200 μl), 1 ml of 0.1 mm dpph solution was added and followed by the addition of 800 μl 50 mm tris-hcl buffer (ph7.4). the final volume was adjusted to 4 ml using ethanol. stock solutions for bem and mgonps were prepared by dissolving 1 mg of each sample in 1 ml of methanol and dimethylsulfoxide, respectively. different aliquots of stock solution (50-500 μg) were added to separate tubes, and the final volume was adjusted to 2 ml using ethanol. a total of 1 ml of 0.1 mm dpph solution and 800 μl 50 mm trishcl buffer (ph7.4) was added to each tube. the control was made by mixing 1 ml dpph 0.1 mm, 800 μl 50 mm tris-hcl buffer (ph7.4), and 2 ml ethanol. absorbance was recorded after incubation for 30 minutes at room temperature, measured by uv-vis spectrophotometer at 517 nm. the percentage of antioxidant activity (% inhibition) was calculated using the following equation: (1) the mean and standard deviation (sd) were calculated based on triplicate measurements by repeating three times. 2.2.6. antibacterial activity 2.2.6.1. microorganism and inoculum preparation the antibacterial activity of bem and mgonps nanoparticles was evaluated against both grampositive (s. aureus and e. faecalis) and gramnegative (e. coli and s. dysenteriae) obtained from the microbiology laboratory of airlangga university. bacterial cultures for testing were cultivated on nutrient agar (na) tilted by selecting a colony from the mueller-hinton agar plate (mha) after 24 hours. a single bacterial or fungal colony is selected and transferred to the mueller-hinton (mhb) broth using a sterilized loop, followed by shaking at 100 rpm at 37 °c overnight for a normalized population. the optical density of bacterial or fungal suspense was maintained at the standard 0.5 for macfarland by addition of sterilized mhb for the test of antibacterial and antifungal activities. the inoculum is therefore composed in about 106-107 cfu / ml of several fungi or bacteria. figure 2. uv-vis spectrum of m. oleifera aqueous extract and mgonps. j. multidiscip. appl. nat. sci. 47 2.2.6.2. minimum inhibition concentration (mic) determination the resazurin microtiter test was used to assess the minimum concentration of inhibition. this approach has been chosen as the easiest and most economical way to simultaneously scan for multiple microorganism isolates and produces satisfactory performance. a 270 mg tablet of resazurin in 40 ml of sterile distilled water was prepared to dissolve the resazurin solution. the test was conducted in aseptic conditions in 96-well plates. the samples were transferred into the plate well with a volume of 100 μl containing 600 mg/ml. subsequently, the checked sample was applied to all other wells by 50 μl of bacterial suspense and diluted severely. each well was subsequently supplemented with 10 μl resazurin solution. the plate was covered with film to avoid dehydration and incubated for 24 hours at 37 °c. the change in color was noticed visually. a change in blue to rose color signaling growth of the cells was considered. in case of a color change, the mic was registered at the lowest concentration. the mixtures between the sterile distilled water and the dimethylsulfoxide solvents and the nutrient broth were used as adverse controls by streptomycin (antibacterial) at 0.02 μg/ μl concentration. 3. result and discussions 3.1. phytochemical screening of m. oleifera bark aqueous extract different methods are used to qualitatively test phytochemical compounds contained in m. oleifera (bem) extracts. the test method used refers to the report das et al. [29] and the qualitative evaluations of several chemical contents are shown in table 1. table 1 shows bem extract contains phytochemical compounds such as alkaloids, flavonoids, saponins, and phenolics. these compounds play a role in the chelation process and are able to reduce metal ions to nanoparticles [26]. total phenolic and flavonoid levels in bem extract 21.65 ± 4.25 µg/mg gae and 55.31 ± 3.82 figure 3. (a) diffraction pattern, (b) sem image, (c) tem image and (d) particles size distribution of synthesized mgonps. j. multidiscip. appl. nat. sci. 48 µg/mg ce respectively, as shown in figure 1. extracts of some parts of the plant contain different constituents with distinct functional groups, which can act as reducing or chelating agents in the formation of nanoparticles. total phenolic and flavonoid levels in bark water extracts are lower than total phenolic and flavonoid levels in water extracts of other m. oleifera plants that have been previously reported. siddhuraju and becker [30] reported total phenolic content in leaves is 74.30 ± 9.00 µg/mg gae, while mohammed and manan [31] reported total phenolic content in ore is 101.79 ± 2.89 µg/mg gae. the total flavonoid content in leaf water extract was reported by okumu et al. [32] amounted to 79.13 ± 13.04 (µg / mg ce). 3.2. characterization 3.2.1. uv-vis spectroscopy of bem and mgonps synthesized synthesis of mgonps using moringa aqueous extract followed by a change in color during the synthesis process. the color of the solution changes from clear (mgcl2 solution) to dark brown when added with moringa extract. after addition of naoh, the color of the solution changes to brighter, indicating the formation of mgo and mg(oh)2 complexes in the solution. adsorption spectrum of mgonps measured in the range of 200—800 nm. figure 2 shows the uv spectrum with a sharp peak at around 290 nm, which confirms the formation of mgo nanoparticles [28]. besides, the precursor ion mg 2+ , mgcl2 salt does not show a spectrum at the specified wavelength. the existence of a peak of about 280—290 nm can be attributed to the formation of metal oxide nanoparticles after the addition of plant extracts and naoh solution [28]. 3.2.2. size and morphology of mgonps synthesized in order to correctly assess the atomic position in the lattice structure, a crystalline stage and structure of the synthesized mgonps were studied using xrd technology. the xrd patterns of mgonps are shown in figure 3(a). the mgonps showed a high intensity peak with two peaks at 42.915 and 62.304 and a low intensity of 31.636, 74.729 and 78.629. the obtained results have been verified using xrd data (no: 78-0430) from jcpds. a high purity of synthesized mgo nanoparticles does not appear as major peaks from mg or other impurities observed on the diffractogram. the average crystalline (d) diameter was calculated with the formula of scherrer (equation 2) for (200) planes of 20–30 nm. (2) where k is a constant dimension depending on the particular geometry of the target, λ is the wavelength of x-ray radiation, β is the full width at half maximum (fwhm) of the significant peaks in radians, and θ is the bragg's angle. sem has been used to carry out morphological tests of mgonps synthesized with extract. figure 3(b) shows a micrograph image of the mgonps. the resulting mgonps are in the form of spherical particle of 20 to 80 nm. figure 3 (c) and (d) shows tem and psa of mgonps prepared. the results of psa show that the mgo with particle size in the range of nanometer with relatively narrow distribution was produced. the samples obtained have the particle size in the range of 60—100 nm. the existence of spheres particles with a size ranging from 60—100 nm was supported by the results of tem. based on these results, mgo nanoparticles have been successfully produced using aqueous extracts of bark of m. oleifera plant, providing an alternative method for synthesizing mgo nanoparticles. amrulloh et al. [33] reported the use aqueous extract of moringa leaves as a green agent in synthesizing mgonps. the water extract sample of moringa leaves contains phytochemical compounds such as alkaloids, saponins, carbohydrates, polyphenols, proteins, and amino acids with figure 4. antioxidant activity of bem and mgonps. j. multidiscip. appl. nat. sci. 49 phenolics and flavonoids contents of 34.75 ± 4.03 μg/mg gae and 74.28 ± 4.82 μg/mg ce, respectively. the structure of the synthesized mgonps was confirmed by the spherical structure. the average particle size of the synthesized mgonps measured between 40—70 nm. the mgonps synthesized from leaf aqueous extract have narrower particle size distribution than that of mgo produced using bark aqueous extract, which was attributable to the presence of antioxidants-rich compounds (e.g. flavonoids, phenolic acids, reducing sugars, etc.) [34][35]. these biomolecules play a vital role as outstanding bio-reducing and/or bio-capping agents towards generation of the nanoparticles [36]. 3.3. antioxidant the antioxidant activity of bem and mgonps was assessed by dpph test using ascorbic acid as a positive control. the free radical capture activity of dpph bem and mgonps is directly related to their concentration. dpph is a stable compound and accepts hydrogen or electrons from bem or mgo nanoparticles. this test is often used for the antioxidant activity of compounds present in medicinal plant extracts [37]. the antioxidant activity of mgonps tends to be higher than that of bem (figure 5). researchers have reported similar observations of antioxidant activity by mgo [28], zno [38], and cuo [39] nanoparticles. 3.4. antibacterial the potential antibacterial activity of bem and mgonps was evaluated against gram-positive (s. aureus and e. faecalis) and gram-negative (e. coli and s. dysenteriae) bacteria clinically isolated in vitro. mic values of the presence of bem against s. aureus, e. faecalis, e. coli, and s. dysenteriae are in the range 300—550 g/ml (figure 5). the antibacterial activity of mgonps nanoparticles was observed with mic values (300—550 μg/ml). if the susceptibility between the bacteria tested is compared, s. dysenteriae is very susceptible to bem and mgonps compared to other test bacteria. medicinal plants, including m. oleifera, are traditionally used to treat various diseases because of their antibacterial activity [40]. the antibacterial activity of mgonps against gram-positive and gram-negative test bacteria showed different results. the difference between gram-positive and gram-negative bacteria is mainly in the structure of their cell walls. gram-positive bacteria have a thick layer of peptidoglycan without an outer membrane and contain teichoic acid. in contrast, gram-negative bacteria have a thin layer of peptidoglycan with an outer membrane that contains lipopolysaccharides. because of this difference, each type of bacteria shows a different sensitivity [41]. 4. conclusions green synthesis of mgonps that were prepared using m. oleifera bark aqueous extract was successfully carried out. the formation of mgonps in this synthesis was confirmed using uv-vis absorption. the spherical crystal structure of mgonps was confirmed by xrd analysis. the average particle size of the synthesized mgo nanoparticles measured between 60—100 nm using sem and tem images and psa results. our study shows that mgo nanoparticles synthesized exhibit a good antioxidant activity, which is higher than ascorbic acid as a positive standard. we also demonstrated that water extracts of m. oleifera bark and mgonps have medium antibacterial activity against s. aureus, e. faecalis, e. coli, and s. dysenteriae with mic values in range of 300—550 g/ml. author information corresponding author hanif amrulloh — department of mathematic figure 5. antibacterial activity of bem and mgonps. j. multidiscip. appl. nat. sci. 50 education, institute for islamic studies ma’arif nu (iaimnu), metro-34111 (indonesia) ; orcid.org/0000-0001-7458-9258 email: amrulloh.hanif@iaimnumetrolampung.ac.id authors awalul fatiqin — department of biology, islamic state university raden fatah, palembang -30126 (indonesia); orcid.org/0000-0001-7799-2835 wasinton simanjuntak — department of chemistry, lampung university, bandar lampung3514 (indonesia); orcid.org/0000-0001-8152-5084 hapin afriyani — department of chemistry, lampung university, bandar lampung3514 (indonesia); orcid.org/0000-0002-6092-127x annissa annissa — department of public health, faletehan university, serang-42161 (indonesia); acknowledgement this research supported by the ministry of religious affairs republic indonesia through collaboration research boptn uin raden fatah palembang no: b-383/un.09/pp.06/05/2019. furthermore, acknowledgment also expressed for the full support from laboratorium sentral mineral & material maju universitas negeri malang, direktorat riset & pengabdian masyarakat universitas indonesia, and laboratorium tem jurusan kimia universitas gajah mada for technical contributions on the research projects. references [1] l. katata-seru, t. moremedi, o. s. aremu, and i. bahadur. 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husk silica 2 3 sri elfina1,a*); kamisah delilawati pandiangan2,b); novesar jamarun3,c); fejri 4 subriadi1,d); hafnimardiyanti hafnimardiyanti1,e); roswita roswita1,f) 5 6 1 chemical analysis study program, politeknik ati padang, padang-25771 (indonesia) 7 2 department chemistry, lampung university, bandar lampung-35145 (indonesia) 8 3 department chemistry, andalas university, padang-26163 (indonesia) 9 a)correspondence: srielfina73@gmail.com 10 b)kamisah.delilawati@fmipa.unila.ac.id 11 c) novesar62@yahoo.com 12 d)fejri.subriadiatip@gmail.com 13 e)hafnimardiyanti11@gmail.com 14 f)roswitacaem@gmail.com 15 16 17 orcids: 18 first author : https://orcid.org/0009-0003-0788-3010 19 second author : https://orcid.org/0000-0001-6347-2361 20 third author : https://orcid.org/0000-0001-8284-145x 21 fourth author : https://orcid.org/0009-0005-1485-3252 22 fifth author : https://orcid.org/0009-0000-2706-3296 23 sixth author : https://orcid.org/0009-0005-5905-7946 24 25 acknowledgement 26 27 this research supported by badan pengembangan sumber daya manusia industri 28 (bpsdmi) the ministry of industry republic indonesia with contract number 29 8/bpsdmi.3/spkp/i/2022. 30 31 author contibutions 32 33 for research articles with several authors, a short paragraph specifying their individual 34 contributions must be provided. the following statements should be used "conceptualization, 35 s. e.; k. d. p. and n. j.; methodology, s. e. and k. d. p.; formal analysis, s. e.; k. d. p.; 36 investigation, f. s.; resources, n. j.; f. s. and h.; data curation, n. j.; writing – original 37 draft preparation, k. d. p. and s. e.; writing – review & editing, k. d. p..; visualization, f. 38 s.; project administration, r.”. 39 40 41 a cc ep te d m a n u sc ri pt mailto:kamisah.delilawati@fmipa.unila.ac.id mailto:novesar62@yahoo.com https://orcid.org/0009-0003-0788-3010 https://orcid.org/0000-0001-8284-145x https://orcid.org/0009-0005-1485-3252 https://orcid.org/0009-0000-2706-3296 https://orcid.org/0009-0005-5905-7946 conflict of interest 1 2 the authors declare no conflict of interest. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 a cc ep te d m a n u sc ri pt transesterification of palm oil catalyzed by cao/sio2 1 prepared from limestone and rice husk silica 2 3 abstract. in this study, cao/sio2 composites were prepared from rice husk silica (rh-sio2) 4 and limestone from a local company. the composites with different mass ratios of cao to sio2 5 (1:1, 1:2, 1:3, 1:5, and 1:10) were synthesized using the sol-gel technique and characterized 6 using xrf, xrd, and sem. the composites were then used as catalysts for the 7 transesterification of palm oil, with the main purpose to investigate the effect of catalyst 8 compositions on the percentage of conversion of the oil. the results of xrd and sem confirm 9 the existence of rh-sio2 as an amorphous material, and cao as crystalline material, while the 10 composites are a mixture of amorphous and crystalline phases. the catalysts were then used in 11 transesterification experiments and the percentage of oil conversion was calculated. to 12 confirm the successful conversion of palm oil into fatty acid methyl esters, the products of the 13 reactions were analyzed using gc-ms. the experimental results demonstrated that the 14 composites prepared exhibit catalytic activity, with the highest conversion (60%) achieved 15 using the catalyst with the cao to sio2 ratio of 1:3. 16 17 keywords: composite; catalyst; limestone; rice husk silica; palm oil; biodiesel 18 19 1. introduction 20 21 in the realm of renewable energy, biodiesel is a non-fossil fuel that has reached a 22 commercial level. biodiesel has been utilized in several countries in the form of a mixture with 23 petrochemical diesel in a certain ratio, depending on the policy implemented by the government 24 of the countries. as an example, the blend of 20% biodiesel and 80% petrochemical diesel and 25 known as b20, has been used in india [1]. the use of b20 has also been implemented in 26 indonesia since the year of 2018 and it is projected to use b30 in the year 2030 [2]. chemically, 27 biodiesel is a mixture of fatty acid methyl esters (fame) produced from the reaction between 28 vegetable oil and methanol in the presence of a catalyst. 29 apart from its increasing role as a fuel, higher price than that of fossil diesel remains a 30 fundamental challenge faced by the biodiesel industry. in this regard, previous workers have 31 suggested that catalyst has a significant role in the reduction of production cost [3][4]. to 32 overcome this problem, the search for low-cost catalyst that works effectively and is 33 a cc ep te d m a n u sc ri pt environmentally friendly has become a priority of many workers involved in biodiesel studies. 1 in this respect, there has been a shift from homogeneous catalysts to heterogeneous catalysts, 2 leading to the development of various types of solid composites which are mainly composed 3 of metal oxide as active sites supported on porous solids. 4 one of the metal oxides that has been widely used as site active is cao. in previous 5 studies, this metal oxide has been used as a pure compound to catalyze transesterification of 6 soybean oil [5][6], and waste cooking oil [7]. this oxide has been supported on various solids 7 and applied for transesterification of various vegetable oils, such as cao/sio2 prepared from 8 eggshell and na2sio3 for transesterification of palm oil [8] and cao/sio2 prepared from 9 eggshell and sio2 for transesterification of palm oil [9]. in another study, pandiangan et al. 10 [10] also reported the use of cao/sio2 for transesterification of rubber seed oil. the use of 11 cao/al2o3 as a catalyst has also been reported for transesterification of nannochloropsis 12 oculata microalga’s lipid [11] and biodiesel production from corn oil [12]. the cao 13 composites with the use of other supports have also been reported, such as nay zeolites for 14 transesterification of soybean oil [13] and natural zeolite for transesterification of rapeseed oil 15 [14]. the wide utilization of cao as an active site of heterogeneous catalyst is based on its 16 strong alkaline strength. this particular oxide is known to have higher alkalinity than mgo and 17 also availability since can be obtained from various sources, limestone, mollusc shells, and 18 eggshells [15][16]. 19 in this study, cao/sio2 composites with different compositions were synthesized using 20 a sol-gel technique from rice husk silica and limestone as raw materials, with the main goal to 21 investigate the effect of composition on the catalytic activity of the composites for 22 transesterification of palm oil. for this purpose, the catalysts with the mass ratios of cao to 23 sio2 of 1:1, 1:2, 1:3, 1:5, and 1:10 were prepared and then characterized using xrf, xrd, and 24 sem. the catalysts were then used in transesterification experiments and the percentage of oil 25 conversion was calculated. to confirm the successful conversion of palm oil into fame, the 26 products of the reactions were analyzed using gc-ms. 27 28 2. materials and methods 29 30 2.1. materials and equipments.limestone was obtained from cv. aikes tanjung 31 mandari, a local company in the city of halaban, west sumatra. the chemicals of analytical 32 grade sodium hydroxide (naoh), nitric acid (hno3), and methanol (ch3oh) were purchased 33 from merck. rice husk silica was collected from a local source in bandar lampung. palm oil 34 a cc ep te d m a n u sc ri pt was collected from a local company in pesisir selatan, west sumatra. equipments used were 1 analytical balance (aes 104 120-4), ph meter (metrohm model), oven (memmert un 2 universal 321 model), electrical heater (stuart am 500c), furnace (thermolyne muffle 3 thermolyne 1100), hotplate stirrer, thermometer, and reflux apparatus. instruments used were 4 xrf (panalytical epsilon 3), xrd (bruker d8 advance), sem/eds (s50 type edax 5 ametek), and gc-ms (gcms-qp2010 se shimadzu). 6 7 2.2. methods 8 9 2.2.1. extraction of rh-sio2. extraction of rh-sio2 was carried out following the 10 previously reported procedure [10]. rice husks were cleaned of impurities by soaking in hot 11 water and then allowed at room temperature overnight to separate the floating and sinking 12 husks. the sinking husks, presumably containing high silica content, were collected while the 13 floating husks were discharged. to extract the silica, a sample of 500 g of rice husk was soaked 14 in 500 ml of 1.5% naoh solution. the mixture was boiled and allowed to stand for 30 min. 15 the sample was then filtered and the filtrate containing dissolved silica was collected. to 16 precipitate silica, a 10% hno3 solution was added gradually to the filtrate. the gel was then 17 separated and washed with hot distilled water to remove excess acid. the silica obtained from 18 this treatment was then dried in an oven at 100 °c for 24 h to remove the water content. 19 20 2.2.2. preparation of cao. to obtain cao, limestone (caco3) was subjected to 21 calcination treatment at 600 °c for 5 h. the obtained cao solid was ground into powder and 22 then sieved with a 200 mesh sieve. 23 24 2.2.3. preparation of cao/sio2 composites. in this study, the cao/sio2 composites with 25 different mass ratios of 1:1, 1:2, 1:3, 1:5, and 1:10 were prepared using the sol-gel procedure. 26 a specified mass of rh-sio2 was dissolved in naoh 1.5% solution and a specified mass of 27 cao was dissolved in concentrated hno3. after both raw materials were completely 28 dissolved, the solutions were mixed by slow addition of cao solution into rh-sio2 solution 29 and allowed to stand for the gel formation. the gel was oven dried at 100 °c for 8 h, and then 30 ground into powder and sieved using a 200 mesh sieve. the composites were then characterized 31 using xrf, xrd, and sem. 32 33 a cc ep te d m a n u sc ri pt 2.2.4. catalytic activity test. the activity of the catalyst samples as heterogeneous 1 catalysts was then evaluated through the transesterification of palm oil using methanol, to 2 convert the oil into methyl esters. each cao/sio2 catalyst was tested for the transesterification 3 reaction. all experiments were run at fixed oil-to-methanol ratio of 1:8 and a catalyst load of 4 10% relative to the mass of the oil. the experiments were run for 6 h at 70 °c in a 500 ml 5 round-bottom flask connected to a water condenser. after the completion of reaction time, the 6 reaction mixture was allowed to cool and then filtered into a separatory funnel and allowed at 7 room temperature for 24 h to allow the separation between the biodiesel and excess methanol 8 (upper layer) and the remaining oil (bottom layer). the excess methanol was removed from the 9 upper layer by evaporation, and the volume of biodiesel was measured to calculate the 10 percentage of conversion of the oil, according to the equation (1) reported by pandiangan et al. 11 [17]. 12 13 % 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 = 𝑉𝑖− 𝑉𝑓 𝑉𝑖 𝑥 100 % (1) 14 15 where vi is the initial volume of oil (ml) and vf is the volume of unreacted oil (ml). 16 17 3. results and discussions 18 19 3.1. xrf analysis. the chemical composition of sio2, cao, and cao/sio2 composites 20 was determined using the xrf technique. the main components, in the form of oxide, are 21 shown in table 1. 22 23 table 1. chemical composition of the samples investigated 24 sample oxide content (%) sio2 cao al2o3 p2o5 others cao (from limestone) 1.018 95.943 1.205 0.969 2.865 sio2 (from rice husk) 97.863 0.246 0.540 0.904 0.447 cao/sio2 1:1 61.152 31.28 1.357 3.047 3.164 cao/sio2 1:2 73.377 22.929 1.714 0.920 1.060 cao/sio2 1:3 81.796 12.041 0.939 2.850 2.374 cao/sio2 1:5 82.507 11.468 0.980 3.456 1.589 cao/sio2 1:10 83.981 9.161 1.149 3.727 1.982 25 a cc ep te d m a n u sc ri pt the data presented in table 1 display that the cao and sio2 from rice husk (rh-sio2) 1 used in this study have a purity of 95.943 and 97.863 %, respectively, suggesting that the 2 characteristics and the catalytic activity of the cao/sio2 composites synthesized are practically 3 determined by these two main chemical components, although some minor components were 4 also detected. 5 6 3.2. xrd characterization. to investigate the phase composing the samples, the cao, 7 rh-sio2, and the composites prepared were characterized using xrd technique. the xrd 8 diffractograms of the samples are presented in figure 1. 9 10 11 figure 1. x-ray diffraction pattern of cao (a) and rice husk silica (rh-sio2) (b). 12 13 the xrd diffractogram of the cao sample is characterized by the existence of sharp 14 peaks and agrees with the pattern for cao standard recorded in pcpdf-win database (icdd 15 04-0777 and 82-1690). the xrd diffractogram of rh-sio2, which is characterized by a broad 16 peak at 2θ = 22.6°, is also in agreement with the pattern for sio2 standard provided in pcpdf-17 win database (icdd 01-0424) with diffraction peaks around 2θ = 22–24°. 18 to investigate the effect of composition on the structure of the composites, the samples 19 were characterized using xrd, and the diffractograms of the composites are shown in figure 20 2. as can be seen in figure 2, the diffractograms of the samples are very similar and resemble 21 the pattern observed for cao. the only quite significant difference between the diffractograms 22 a cc ep te d m a n u sc ri pt is the relative intensity of the peaks which tends to decrease with increasing amount of silica 1 in the composite material. 2 3 4 figure 2. the x-ray diffraction patterns of composites at different compositions. 5 6 3.3. sem characterization. to investigate the surface morphology, which is another 7 important characteristic of solid materials, the samples of rh-sio2, cao, and the composites 8 were characterized using sem. the micrographs of the rh-sio2 and cao obtained are shown 9 in figure 3. as displayed in figure 3, the rh-sio2 is characterized by heterogeneous surface 10 features, in terms of particle sizes and distribution of the particles on the surface. in addition, 11 the sample is marked by the irregular shapes of the particles, justifying the existence of the 12 sample as amorphous material, as has been demonstrated by the xrd diffractogram in figure 13 a cc ep te d m a n u sc ri pt 1(b). the heterogeneous surface morphology is also displayed by the micrograph of cao, 1 however, the existence of rectangular particles can be observed, although the particles are not 2 well separated but tend to agglomerate to form large particles. 3 4 5 figure 3. sem micrographs of rh-sio2 with 1000x magnification (a), 15000x 6 magnification (b), and micrographs of cao with 1000x magnification (c), and 15000x 7 magnification (d). 8 9 the composites were also characterized using sem and the micrographs obtained are 10 compiled in figure 4. as can be seen in figure 4, the surface morphologies observed suggest 11 the existence of all samples as a mixture of amorphous and crystalline materials, forming 12 agglomerates as has also been observed by others [18]. in addition, the heterogeneity of the 13 samples in terms of particle sizes and distribution of the particles on the surface of the samples 14 is very evident, as displayed by the micrographs. related to the application of the composites 15 as catalyst, the amorphous phase, presumably the rh-sio2 is the component to play the role 16 as the host for the reaction while the cao as the crystalline component acted as an active site 17 of the catalyst, as depicted in reaction mechanism in figure 5 [19]. 18 19 a cc ep te d m a n u sc ri pt 1 figure 4. sem micrographs of the composites with different compositions: (a) cao/sio2 1:1, 2 (b) cao/sio2 1:2. (c) cao/sio2 1:3, (d) cao/sio2 1:5, and (e) cao/sio2 1:10. 3 4 5 6 7 8 a cc ep te d m a n u sc ri pt 1 figure 5. transesterification mechanism of vegetable oil using cao as an active site of 2 catalyst. 3 4 3.4. catalytic activity test. a typical example of a transesterification reaction is shown 5 in figure 6. the upper layer is the biodiesel layer mixed with excess methanol and the bottom 6 layer is unreacted oil. the volume of unreacted oil was measured to determine the percentage 7 of conversion using the equation presented in the experimental section. the results obtained 8 are presented in table 2. 9 10 11 figure 6. typical example of transesterification product obtained in this study. 12 13 14 a cc ep te d m a n u sc ri pt table 2. conversion of oil using catalyst with different compositions 1 no. catalyst composition (cao/sio2 ratio) oil conversion (%) 1 1:1 48 2 1:2 52 3 1:3 60 4 1:5 43 5 1:10 36 2 as can be seen in table 2 there is no evident trend that shows the effect of composite 3 compositions on the oil conversion achieved. however, it can be noted that the highest oil 4 conversion (60%) was achieved with the use of the cao/sio2 composite with aratio of cao to 5 sio2 of 1:1. based on these results, it should be noted that the oil conversion achieved in this 6 study relatively lower compared to those reported by others for the same oil [20][21]. in this 7 respect, it should be acknowledged that more study is required to optimize the performance of 8 the cao/sio2 composites, in recognition of the higher performance of this catalyst reported by 9 other works [3][19], together with the existence of the limestone and rice husk as low-cost raw 10 materials. 11 12 4. conclusions 13 14 the experimental results obtained in this study demonstrated that the cao/sio2 15 composites with different cao to sio2 ratios prepared from limestone and rice husk silica exist 16 as a mixture of amorphous and crystalline phases according to xrd characterization. 17 according to sem results, the surface morphology of the samples is characterized by 18 heterogeneous features in terms of particle size and shape, as well as particle distribution on 19 the surface of the samples. the transesterification experiments revealed that the highest oil 20 conversion achieved is 60% with the use of composite with the cao to sio2 ratio of 1: 3. this 21 conversion is relatively higher than the results for the same oil with the use of different 22 catalysts, but relatively lower compared to the results reported by others. in this respect, it 23 should be acknowledged that more study is required to optimize the performance of the 24 cao/sio2 composites. despite this relatively low performance, this type of composite is still 25 a promising catalyst system since the better performance was reported by other researchers. in 26 addition, both limestone and rice husk are abundantly available and categorized as low-cost 27 raw materials. 28 a cc ep te d m a n u sc ri pt references 1 2 [1] s. kour and w. k. balwan. 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"biodiesel production from palm olein: a sustainable bioresource for 8 nigeria". heliyon. 6 (4): e03725. 10.1016/j.heliyon.2020.e03725. 9 10 a cc ep te d m a n u sc ri pt https://doi.org/10.1016/j.fuel.2007.10.019 https://doi.org/10.19053/01217488.v11.n2.2020.11142 https://doi.org/10.1016/j.heliyon.2020.e03725 90 journal of multidisciplinary applied natural science vol. 3 no. 1 (2023) research article antibacterial activity and bioautography of the chloroform fraction of morel berry (physalis angulata l.) root against staphylococcus epidermidis and pseudomonas aeruginosa pratika viogenta*, laila susanti, and latifah megasari received : october 6, 2022 revised : january 4, 2023 accepted : january 5, 2023 online : january 11, 2023 abstract the urgency of finding novel sources of active compounds to overcome infectious diseases is encouraged. morel berry ( physalis angulata l.) is a traditional herbal plant that can be used as an antimicrobial because of its unique chemical content. this study aims to find compounds that have antibacterial activity from the chloroform fraction of morel berry roots. this study used the kirby -bauer method with concentrations of 20%, 40%, 60%, 80%, and 100% against staphylococcus epidermidis and pseudomonas aeruginosa bacteria. determination of antibacterial compounds and their functional groups was performed using thin-layer chromatography (tlc) technique and reagent spray test, tlc-bioautography and fourier transforms infrared (ftir) studies. the chloroform fraction of morel berry showed a zone of inhibition with the highest diameter of 11.50 and 12.00 mm against s. epidermidis and p. aeruginosa, respectively. phytochemical screening revealed the presence of alkaloids, flavonoids, tannins, and saponins in the chloroform fraction. the relative retention stain of 0.54 on the tlc plate inhibited the growth of the two tested bacteria and indicated the presence of functional groups o-h phenol, c-h aliphatic, c=o ester, c=c aromatic, c-oh alcohol, and c-h aromatic. this study found that tannins in the roots of morel berries could be used as a natural antibacterial agent to treat s. epidermidis and p. aeruginosa. keywords physalis angulata l, bioautography, tannin, antibacterial, chloroform fraction 1. introduction the use of natural materials derived from plants as traditional medicine has long been practised to treat various health problems in tropical countries including indonesia. this ethnomedical technique is quite profitable because the raw materials are easily obtained or cultivated, relatively cheap, can be prepared at home, have low toxicity, efficient, and are rarely accompanied by side effects [1]. plants that have been selected for medicinal use for thousands of years are the most obvious starting point for effective new medicines. recently, the use of medicinal plants has increased in spite of advances in synthetic drugs. there is also an increasing use of medicinal plants in industrialized countries for galenic preparations and herbal remedies [2]. the morel berry plant (physalis angulata l.) belongs to the solanaceae family, known in indonesia as “ceplukan” or “ciplukan” (figure 1). p. angulata l. is known to be rich in benefits as medicinal plants and has been shown to have antiparasitic, anti-inflammatory, antinociceptive, antimicrobial, antimalarial, antileishmanial, immunosuppressive, antiasthmatic, diuretic, and anticancer properties [3]. this ability is inseparable from the active compounds present in morel berry plants. morel berry contains bioactive compounds like flavonoids (leaves and shoots), alkaloids (roots), tannins (fruit), saponins (shoots), polyphenols and physalin (leaves), and vitamin a and vitamin c (fruit) [4]. other studies stated that this plant also contains flavonoids, saponins, fisalin a and b, witafisalin a and b, and also terpenes [3]. it is known that the composition of the active compounds in morel berry leaves is rich in flavonoids, alkaloids and physalin b, which has been conducted several times in vitro and in vivo studies showing the biological and pharmacological activities of flavonoid compounds are very diverse, one of which is having antibacterial activity [5]. various research results state that morel berry root has shown antibacterial activity. the ethanol extract of the morel berry root was screened for phytochemicals positive for flavonoids, alkaloids and saponins and showed bactericidal ability against pseudomonas aeruginosa bacteria at a concentration of 20% with an inhibition zone publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.148 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.148 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.148&domain=pdf&date_stamp=2023-01-11 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 91 figure 1. morel berry plant (physalis angulata l.) diameter of 10.01 mm [6]. the n-hexane, ethyl acetate and water fractions of morel berry herbal have antibacterial activity against staphylococcus epidermidis [7]. the essential oil of the aerial and root parts of morel berry has antibacterial and antifungal activity with a mic value of 4 mg/ml [8]. the results of the phytochemical screening showed that the ethanol extract of morel berry contained flavonoids, saponins, tannins, and alkaloids [9]. however, the ethanol extract still contains chemical compounds which have not been separated based on their polarity. therefore, fractionation is carried out to separate the active compounds based on their polarity level. the solvent used for the morel berry root fraction is chloroform because the use of chloroform facilitates the process of separating compounds because only compounds that are semi-polar to those that are polar can dissolve in chloroform solvent. this is adapted to the basic principle of extraction, namely a compound will be dissolved only with a solvent that is relatively the same in polarity and besides that chloroform has a relatively low boiling point so that it is easily evaporated [10]. the bioautography method can be used as a detection method to find an antimicrobial compound that has not been identified by localizing the antimicrobial activity on a chromatogram resulting from thin-layer chromatography (tlc). the basis of this method is the agar diffusion technique, and the antibacterial compounds transfer from the tlc plate to the agar medium which already contains bacteria [11]. so far, the method for detecting antibacterial from morel berry plants uses the agar diffusion method. so, this study was done to see if the chloroform fraction of morel berry roots could suppress the growth of s. epidermidis and p. aeruginosa bacteria using bioautography methods and to find out what secondary metabolites were in the chloroform fraction of morel berry roots. 2. materials and methods 2.1. materials morel berry roots (p. angulata l.) were collected from bandar lampung, lampung, indonesia in march 2019. the two test bacteria used were staphylococcus epidermidis and pseudomonas aeruginosa. meanwhile, nutrient agar (na), nutrient broth (nb), amikacin, aquadest (h2o), ethanol 70% (c2h6o), ethanol pa, chloroform pa (chcl3), n-hexane pa (ch3(ch2) 4ch3), methanol, fecl3, ammonia (nh3), liebermann-burchard, and bourchadat reagents were purchased and used without further purification. 2.2. methods 2.2.1. morel berry root extract fresh morel berry roots, as much as 3 kg, were cleaned with flowing water to remove impurities. the roots were cut and dried without direct sunlight to obtain simplicia. a total of 500 g of simplicia morel berry roots were macerated with 70% ethanol. the solvent was changed every 24 hours until the solvent was clear (six times). then, 250 ml of liquid extract was made by letting the extract evaporate in a rotary evaporator. 2.2.2. morel berry root fraction a total of 100 ml of liquid extract of morel berry root was fractionated using a separating funnel and added to 100 ml of n-hexane, then shaken slowly and allowed to stand until the ethanol and n-hexane fractions were separated. the nhexane fraction was separated and then repeated up to three times. the n-hexane fraction was collected. next, 100 ml of chloroform was added to the ethanol fraction and shaken slowly and then allowed to stand until a layer of the ethanol fraction and chloroform fraction was obtained. the chloroform fraction was separated and then j. multidiscip. appl. nat. sci. 92 repeated up to 3 times. the chloroform fraction was collected and evaporated with a rotary evaporator and then the obtained fraction was stored in the refrigerator. 2.2.3. antibacterial analysis an antibacterial activity test using the agar well diffusion method. in a sterile petri dish, a 100 µl suspension of p. aeruginosa and s. epidermidis bacteria was added and followed by the addition of 25 ml of unsolidified na medium. the mixture was homogenized and allowed to solidify. several holes were made in the substrate using a 6 mm corkborer. the chloroform fraction of morel berry roots was injected with concentrations of 20%, 40%, 60%, 80%, 100%, as well as amikacin as a positive control and dmso 1% as a negative control. each hole received up to 100 µl of chloroform fraction of morel berry root, amikacin, and distilled water. all petri dishes were incubated for 24 hours at 37 ℃. furthermore, observations and measurements of the inhibition zone formed around the diffusion hole were carried out using a caliper. according to the classification of antimicrobial activity, it is classified into three levels: strong activity (diz > 20 mm), moderate activity (12 mm < diz < 20 mm), and weak activity (diz < 12 mm) [12]. 2.2.4. thin layer chromatography the separation of compounds from the chloroform fraction was carried out using a silica gel plate g60f254 with a size of 7 × 2 cm. next, a line was marked on the top and bottom edges of the plate with a distance of 1 cm to indicate the initial position of the spots and the top edge as a boundary sign of the elution process. furthermore, the plate was activated by heating at a temperature of 100 ℃ for 1 hour to remove the water content on the tlc plate. prior to elution, the eluent used was chloroform fractionate was left to be saturated first. afterward, each mobile phase mixture was inserted into the chamber and then tightly closed and saturated using filter paper for 10 minutes. this saturation was done to equalize the vapour pressure in the entire vessel. the chloroform fraction was spotted on the tlc plate at a distance of 5 cm from the bottom edge of the plate using a capillary tube and then dried by aeration. then, it was put into a saturated chamber and allowed to elute up to the specified chromatogram plate limit. the plate was removed from the chamber and observed with a 254 nm and a 366 nm uv lamp. visible stains were detected with bouchardat spray reagent for alkaloids (brown color), ammonia for flavonoids (yellow brown/ green/brown/pink color), fecl3 for tannins (strong green, red, purple, blue or black color) and table 1. inhibition zone of morel berry root chloroform fraction against s. epidermidis and p. aeruginosa bacteria concentration inhibitor zone diameter (mm) s. epidermidis p. aeruginosa k. 20% 0 a ± 0.00 0 a ± 0.00 k. 40% 8.04 c ± 0.65 8.56 c ± 1.45 k. 60% 8.76 cd ± 0.28 8.82 cd ± 0.98 k. 80% 9.94 cd ± 0.92 9.95 cd ± 1.20 k. 100% 11.50 de ± 0.63 12.00 de ± 0.88 k 0 a ± 0.00 0 a ± 0.00 k + 30.03 b ± 0.38 30.16 b ± 0.38 * numbers followed by the same letter in the same column were not significantly different based on the 5% lsd test. ** k -: sterile distilled water; k +: amikacin j. multidiscip. appl. nat. sci. 93 liebermann-burchard for saponins (purple color) [6]. furthermore, the measurement of the retention factor (rf) spots in the chromatogram from the tlc results was carried out. 2.2.5. bioautography test as much as 20 ml of nutrient agar was poured into petridishes and left to solidify. on solid media, 100 µl suspensions of s. epidermidis and p. aeruginosa bacteria were spread. the chromatogram of the compounds separated by tlc was placed on the medium. the media was allowed to stand for 3 hours in the refrigerator, the chromatogram plate was removed from the medium. it was then incubated for 24 hours at 37 ℃. the inhibition zone formed was calculated for its rf value. rf with a value equal to or close to the rf value in tlc was thought to be the most effective active compound as an antibacterial. 2.2.6. functional group characterization with ftir spectrophotometer identification of functional groups was carried out by scraping the tlc plate which had the same rf as tlc-bioautography from the chloroform fraction of morel berry roots. subsequently identified using an ftir spectrophotometer (agilent/cary 630) with the inhouse method. 3. results and discussions 3.1. extraction and fractionation in this study, 3 kg of fresh morel berry roots were used and processed to obtain 500 g of simplicia. the drying process was done to reduce the amount of water in simplicia. the extraction of the active compounds contained in the morel berry roots was carried out by the maceration method. this method is included in the cold extraction method which can be carried out without using heating to avoid damage to the active substance. in the extraction process, 250 ml of morel berry root liquid extract was obtained. ethanol 70% was selected as a solvent because the sample tested is a dry material so 30% of the water content serves to open the pores of the simplicia which facilitates the process of withdrawing the compound at the time of extraction [13]. soaking the sample in maceration can make the cell wall of the sample break and make the compounds present in the sample in the cytoplasm attracted by the solvent. the cell wall breaks due to the difference in concentration inside and outside the cell. the concentration outside the cell is higher than the concentration inside the cell which is low enough that the cell wall breaks because it cannot withstand the pressure of the difference in figure 2. the cross-sectional results of the tlc test of the chloroform fraction of morel berry roots with the mobile phase of chloroform (5) (v)). a: visible light, b: uv 254, c: uv 366, d: ammonia, e: fecl3, f: bouchardat reagent and g: liebermann-burchard reagent. j. multidiscip. appl. nat. sci. 94 concentration [14]. the sample was re-macerated by changing the solvent until the solvent was colorless which indicated that the maceration was completed as the maceration is more efficient to do repeatedly than just once [15]. the maceration results are then filtered and concentrated. concentration is carried out with the help of a rotary evaporator at a temperature of 60 ℃. substances that are thermolabile must be concentrated at a temperature below their boiling point so that the content of secondary metabolites contained in the solvent is not damaged by high temperatures [16]. in this study, fractionation was carried out by the liquid-liquid fractionation method, which is a separation method using two immiscible liquids. the solvent with a higher density is in the lower layer while the solvent with lower density is in the upper layer. the fraction that was found was dried out with a rotary evaporator and then used for the test of antibacterial activity. 3.2 antibacterial analysis testing the antibacterial activity of the chloroform fraction using the agar diffusion method (well diffusion). the test bacteria used were s. epidermidis representing gram-positive bacteria and p. aeruginosa representing gram-negative bacteria. the use of these bacteria was aimed at determining whether the chloroform fraction of morel berry roots had specific or broad-spectrum antibacterial activity. antibacterial compounds will diffuse into solid media inoculated by bacteria and inhibit bacterial growth which is indicated by the formation of a clear area around the hole [11]. this method was chosen because it was simple, easy to work with, accurate, does not require special equipment, was versatile for all fast-growing pathogenic bacteria, and was often used in antibiotic susceptibility testing in quality control programs. the results indicated the presence of an inhibition zone around the well which is marked by a clear zone. this shows that the tested bacteria were sensitive to the chloroform faction of the morel berry roots and the antibiotics that were used as positive controls. in this test, it was carried out at various concentrations of 20%, 40% 60%, 80% and 100% to determine the effect of giving different fraction concentrations to the test bacteria. the results of the average diameter measurement of the antibacterial power of the chloroform fraction against the test bacteria s. epidermidis and p. aeruginosa can be seen in table 1. in this study, the chloroform fraction was more effective in inhibiting the growth of p. aeruginosa bacteria than s. epidermidis bacteria. the different responses of these two bacteria to antibacterial compounds were caused by differences in the composition and structure of their cell walls. differences in the cell wall structure of gramnegative bacteria and gram-positive bacteria affect the sensitivity to bacteria. the cell wall of grampositive bacteria consists of about 40 layers of peptidoglycan so that peptidoglycan makes up 70% of the dry mass of the cell wall. this causes the cell walls to become thick and stiff. in contrast to gramnegative bacteria, the cell wall of peptidoglycan is only about 10% of the dry mass of the cell wall, table 2. tlc test results chloroform fraction of morel berry root compound detection positive result research result commentary rf flavonoid ammonia brownish yellow, orange, red brownish yellow + 0,36; 0,44; 0,60; 0,84 tannin fecl3 green, red, purple, blue or black black + 0,54; 0,68; 0,82; 0,92 alkaloid bouchardat reagent brown brown + 0,3; 0,58; 0,72; 0,96 saponin lieberman burchard purple no changes – water 50 ℃ foam lasts 10 minutes foam was formed for 10 minutes + j. multidiscip. appl. nat. sci. 95 thus causing the cell wall of gram-negative bacteria to be thinner [17]. gram-negative bacteria have a large lipid content and have a protein porin that acts as a channel for the entry of active substances into bacterial cells. the entry of these active substances impairs enzyme activity in cells and causes cell damage. when there are a lot of lipids in cells, it makes it easier for active substances to get into cells [18]. the results obtained from the average inhibition zone measurement showed that the chloroform fraction of morel berry roots was not active against s. epidermidis and p. aeruginosa. these fractions with concentrations of 40, 60% and 80% against the test bacteria s. epidermidis and p. aeruginosa were classified as weak category. for a concentration of 100%, it is classified as moderate activity against p. aeruginosa bacteria. based on the results, it shows that the antibacterial properties of the compounds in the chloroform fraction of morel berry roots are broad-spectrum. this means that they work against both gram-positive and gram-negative bacteria. the ability of an antibacterial agent to eliminate the ability of living organisms depends on the concentration of the material used [14]. based on the results of the antibacterial activity test in table 1, the diameter of the inhibition zone for the growth of s. epidermidis and p. aeruginosa bacteria formed at a concentration of 100% was greater than the concentrations of 40, 60%, and 80%. the higher concentration of an antibacterial agent, the greater the antibacterial activity as the extract has more phytochemical compounds with antibacterial properties. 3.2. phytochemical screening by tlc morel berry roots were subjected to tlc analysis aiming to determine the compound content of the chloroform fraction. the mobile phase used for the chloroform fraction, namely chloroform (5) (v)), was chosen because it was adapted to the solubility properties of the compound being analyzed because it is semipolar [19]. the selection of this eluent is based on the results of the orientation of the eluent that has been carried out which in this eluent produces the best separation with the highest number of stains after detection of spots on uv lamps at 254 and 366 nm. according to saifudin (2014) [20], the characteristics of a good eluent are marked by the number of spots that appear, the spots formed are tailless, and the distance between one spot and another is clear. the results of the cross-sectional test of the chloroform fraction of morel berry roots on the tlc plate showed that there were flavonoids, tannins, and alkaloids (figure 2 and table 2). the saponin test based on liebermen-burchard's reagent did not show positive results, but with detection with warm water, a long-lasting foam was formed which proved that the chloroform fraction of morel berry roots was positive for saponins. morel berry roots contain active compounds including alkaloids, flavonoids, and saponins [6]. spots on the chloroform fraction of morel berry roots by looking at the results of the ammonia spray test can be stated to contain flavonoid compounds with rf 0.36, 0.44, 0.60, and 0.84. one of the stains on this fraction rf 0.44 has the same rf as group flavonoids so the ethanol fraction of morel berry roots may contain flavonoid compounds. previous research on the identification of flavonoids in alchemilla species showed the presence of figure 3. results of bioautography of the chloroform fraction of morel berry roots with chloroform 5 (v/v) mobile phase against a.) staphylococcus epidermidis and b.) pseudomonas aeruginosa. j. multidiscip. appl. nat. sci. 96 flavonoid compounds that had the same rf value as flavonoids and rutin with rf of 0.72 and 0.44, respectively [21]. phytochemical results and tlc analysis from other studies showed that the results of spraying using fecl3 on the e. hirta l. extract produced a black color in the tannins [22]. the cross-sectional results of tlc of the chloroform fraction of morel berry roots showed positive results containing tannins after being sprayed with fecl3 which was indicated by the formation of a black color on the plate. the formation of a black color after being added to fecl3 is due to the fact that tannins will form complex compounds with fe 3+ ions [23]. the chloroform fraction of morel berry roots produced an rf of 0.54, 0.68, 0.82, and 0.92. one of the spots from this fraction with an rf of 0.54 is almost the same as the rf in general, so it can be said that the chloroform fraction of the plant roots contains tannin compounds. research on the polar fraction of murraya panicullata l. jack leaf extract stated a rf value of 0.55 which is a tannin compound [24]. phytochemical results and tlc analysis from other studies showed that the results of spraying using fecl3 on the e. hirta l. extract produced a black color in the tannins [22]. the cross-sectional results of tlc of the chloroform fraction of morel berry roots showed positive results containing alkaloids after being sprayed with bouchardat reagent which was indicated by the formation of a brown color on the plate. in the manufacture of bouchardat reagent, iodine reacts with i ions from potassium iodide to produce i 3 ions which are brown in color. in the bouchardat test, metal ions k + will bind with nitrogen in the alkaloids to form a precipitated potassium-alkaloid complex [23]. the most common rf values for alkaloids were 0.07–0.62 and the chloroform fraction of morel berry roots produced rf values of 0.30, 0.58, 0.72, and 0.96, respectively. the spots with rf 0.30 and 0.58 for the chloroform fraction were in accordance with the rf in general so it can be said that the chloroform fraction of morel berry roots contained alkaloid compounds [25]. 3.3. bioautography analysis the bioautography test was carried out by tlc method. this test aims to determine the natural compounds contained in the chloroform fraction of morel berry roots on the chromatogram by looking at the inhibition zone formed on the agar medium. in this study, the contact bioautography method was used due to its simplicity of the work and the results are more clearly visible. the eluted plates were contacted on the surface of solid media containing s. epidermidis and p. aeruginosa bacteria inoculated for 3 hours, respectively. the plate was then removed from the medium and the agar medium was incubated, in order to obtain a zone of figure 4. results of the ftir spectrum for the chloroform fraction of morel berry roots j. multidiscip. appl. nat. sci. 97 inhibition on the surface of the media where the plates were contacted indicating the location of the compound responsible for the antibacterial activity. the results of tlc-bioautography showed that there was an inhibition zone in the two tests (figure 3). the largest inhibition zone that appeared was in the spots with an rf value of 0.54 against both s. epidermidis and p. aeruginosa. from the results of the tlc test, the spots on the chloroform fraction rf 0.54 were tannins, indicating that these active compounds had the most role as antibacterial agents. 3.4. ftir the functional groups of antibacterial compounds were identified to elucidate which functional groups that exhibit antibacterial activity against s. epidermidis and p. aeruginosa bacteria. the chloroform fraction of morel berry roots was re -eluted on the tlc plate with chloroform 5 (v/v) as eluent and the stain with a rf value of 0.54 was scraped off. the results of the scrapings were analyzed by ftir and shown in figure 4. the results of the ftir analysis (table 3) showed that the o-h group with a broad but not sharp band at a wavelength of 3265.1 cm -1 is a phenol group. this assumption was confirmed by absorption with a wavelength of 1051.1 cm -1 , indicating the presence of a c-o alcohol group. absorption at a wavelength of 2937.1 cm -1 indicates the presence of a c-h aliphatic group. the absorption at a wavelength of 1729.5 cm -1 indicates the presence of a c=o ester group. absorption at a wavelength of 1654.9 cm -1 indicates the presence of a c=c aromatic group. this assumption is reinforced by absorption at a wavelength of 790.2 cm -1 which indicates the presence of an aromatic ch group [26]. the rf stain of 0.54 from the chloroform fraction of morel berry roots showed the presence of functional groups o-h phenol, c-h aliphatic, c=o ester, c=c aromatic, c-o alcohol, and c-h aromatic. this functional group leads to the tannin group. characterization of the bark of ceriops tagal proved that the functional groups of o-h phenol, ch aliphatic, c=c aromatic, c-o alcohol and c-h aromatic belong to the condensed tannin compounds [27]. 4. conclusions the chloroform fraction of morel berry (physalis angulata l.) root was proven to inhibit the growth of s. epidermidis and p. aeruginosa bacteria while the extract was only able to inhibit the growth of p. aeruginosa. the chloroform fraction of morel berry root contains potential bioactive substances including flavonoids, alkaloids, saponins, and tannins. the rf stain 0.54 from the chloroform fraction of morel berry roots showed the presence of functional groups o-h phenol, c-h aliphatic, c=o ester, c=c aromatic, co alcohol, and c-h aromatic. tannins with an rf value of 0.54 at tlc in morel berry roots have been shown to have antibacterial activity against s. epidermidis and p. aeruginosa. from that perspective, they contain broad-spectrum antimicrobial agents that can be developed as an effective antibacterial treatment to combat typical antibiotic-resistant pathogens. author information corresponding author pratika viogenta — department of pharmacy, lambung mangkurat university, banjarbaru— 70714 (indonesia); orcid.org/0000-0002-9443-0874 email: pratika.viogenta@ulm.ac.id authors laila susanti — department of pharmacy, tulang bawang university, bandar lampung— 35128 (indonesia); orcid.org/0000-0002-4440-6606 latifah megasari — department of pharmacy, wave number (cm -1 ) functional groups 3265.1 o-h phenol 2937.1 c-h aliphatic 1729.5 c=o ester 1654.9 c=c aromatic 1051.1 c-o alcohol 790.2 c-h aromatic table 3. ftir absorption pattern of morel berry root chloroform fraction https://orcid.org/0000-0002-9443-0874 mailto:pratika.viogenta@ulm.ac.id https://orcid.org/0000-0002-4440-6606 j. multidiscip. appl. nat. sci. 98 tulang bawang university, bandar lampung— 35128 (indonesia); orcid.org/0000-0003-0164-5997 author contributions conceptualization, p.v. and l.s.; methodology, p.v. and l.s.; software, p.v.; validation, p.v. and l.s.; formal analysis, p.v. and l.s.; investigation, l.m.; resources, p.v. and l.s.; data curation, l.m.; writing – original draft preparation, p.v.; writing – review & editing, p.v. and l.s.; visualization, l.m.; supervision, p.v.; project administration, p.v. and l.s.; funding acquisition, p.v. conflicts of interest the author(s) declared no conflict of interest references [1] f. bawamenewi, m. bintang, m. safithri, and f. h. pasaribu. 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"characterization of tingi (ceriops tagal) bark as vegetable tanning material". majalah kulit, karet, dan plastik. 30 (2): 71–78. 10.20543/ mkkp.v30i2.128. https://doi.org/10.35799/pha.8.2019.29320 https://doi.org/10.4314/ajtcam.v9i3.18 https://doi.org/10.36733/medicamento.v3i2.891 https://doi.org/10.53342/pharmasci.v2i1.63 https://doi.org/10.53342/pharmasci.v2i1.63 https://doi.org/10.36733/medicamento.v3i2.891 https://doi.org/10.36733/medicamento.v3i2.891 journal of multidisciplinary applied natural science accepted manuscript • open access integration of rational functions to cite this article before publication: l. rathour, d. obradovic, k. khatri, s. k. tiwari, l. n. mishra, and v. n. mishra. 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; 4,d*)tiwari kant shiv ;3,c)khatri kejal ;2,b)obradovic dragan ;1,a)rathour laxmi3 6,f); vishnu narayan mishra 5,e)lakshmi narayan mishra 4 5 (india) 796012technology, mizoramdepartment of mathematics, national institute of 1 6 )serbia( 12208-elementary school "jovan cvijic", pozarevac2 7 )india( 314403dungarpur simalwara, college, government mathematics, of department3 8 )india(363642 -department of mathematics, lukhdhirji engineering college, morbi4 9 dia)in(632014 -vellore of technology, institute vellore mathematics, of department5 10 )india(484887 -amarkantak university, tribal gandhi national mathematics, indira of department6 11 laxmirathour817@gmail.com a) 12 dragishaobradovic@yahoo.comb) 13 kejal0909@gmail.comc) 14 shivkant.math@gmail.com :correspondence d) 15 lakshminarayanmishra04@gmail.come) 16 vishnunarayanmishra@gmail.comf) 17 18 orcids: 19 first author : https://orcid.org/0000-0002-2659-7568 20 second author : https://orcid.org/0000-0001-5871-6958 21 third author : https://orcid.org/0000-0002-3425-1727 22 fourth author : https://orcid.org/0000-0003-0942-3467 23 fifth author : https://orcid.org/0000-0001-7774-7290 24 sixth author : https://orcid.org/0000-0002-2159-7710 25 26 27 author contributions 28 29 all authors equally contributed to this paper. 30 31 conflict of interest 32 33 the authors declare that there is no conflict of interest. 34 35 36 37 38 39 40 41 42 43 44 45 46 47 a cc ep te d m a n u sc ri pt mailto:laxmirathour817@gmail.com mailto:b)dragishaobradovic@yahoo.com mailto:c)kejal0909@gmail.com mailto:shivkant.math@gmail.com mailto:e)lakshminarayanmishra04@gmail.com mailto:f)vishnunarayanmishra@gmail.com https://orcid.org/0000-0002-2659-7568 https://orcid.org/0000-0001-5871-6958 https://orcid.org/0000-0002-3425-1727 https://orcid.org/0000-0003-0942-3467 https://orcid.org/0000-0001-7774-7290 journal of multidisciplinary applied natural science integration of rational functions 1 2 abstract. a rational function can always be integrated, that is, the integral of such a function 3 is always an elementary function. the integration procedure is complex and consists of four 4 steps: elimination of the common zero-points of the numerator and denominator, reduction to 5 a true rational function, decomposition into partial fractions and integration of the obtained 6 expressions using direct integration, substitution method or partial integration method. 7 integrating rational functions is important because integrals of rational functions of 8 trigonometric functions as well as integrals of some irrational functions are reduced to 9 integrals of rational functions by appropriate transformations. 10 11 key words: mathematics; rational functions; polynomial functions; partial fractions. 12 13 1. introduction 14 15 the tasks of teaching higher mathematics are to demonstrate to students the operation of 16 the laws of materialistic dialectics, the essence of the scientific approach, the specifics of 17 mathematics and its role in the realization of scientific and technological progress, using 18 examples of mathematical concepts and methods. it is necessary to teach students the 19 methods of research and solving mathematical formalized tasks, to develop in students the 20 ability to analyze the obtained results, to inculcate in them the skills of independent study of 21 literature on mathematics and its application. 22 every rational fraction is integrated into elementary functions, but studying the general 23 integration algorithm and alternative approaches is quite a difficult task. from lectures and 24 seminars, it is impossible to sufficiently consider all the details, then the main part of the 25 material is usually discussed in the classroom, and the rest is given to students for 26 independent study. this paper was created within the educational and methodological 27 complex of the discipline "mathematical analysis" and is dedicated to the topic "integration 28 of rational fractions". it considers those cases and methods that cannot be considered in their 29 entirety in the classroom. 30 integration of rational fractions. expression of the form 𝑃𝑚(𝑥) 𝑄𝑛(𝑥) , where pm(x) and qn(x) 31 polynomials m-th and n-th degree, is called a rational fraction. a rational fraction is called 32 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science proper if mn and improper if m  n . the problem of integrating a rational fraction can be 1 reduced to the problem integration of a proper rational fraction, since any improper rational 2 fraction can be represented by division by a column in as the sum of a polynomial and a 3 proper rational fraction. we recite some research papers and books [1]-[7] for the right 4 direction of integration of rational functions and its applications. 5 6 2. representation of a rational functions as a sum of 7 polynomials 8 9 those who say that trigonometry is not needed in real life are not needed on the road. so, 10 what are its common applied tasks? measure the distance between inaccessible objects. the 11 triangle technique is of great importance, which enables the measurement of distances to 12 nearby stars in astronomy, between benchmarks in geography, control satellite navigation 13 systems. the use of trigonometric techniques should also be noted, such as navigation 14 techniques, music theory, acoustics, optics, financial market analysis, electronics, probability 15 theory, statistics, biology, medicine (ultrasound and computed tomography), pharmacology, 16 chemistry, number theory (and, as a result, cryptology, seismology, meteorology, 17 oceanology, cartography, many parts of physics, topography and geodesy, architecture, 18 phonetics, economics, electronic equipment, mechanical engineering, computer graphics, 19 crystallography etc. 20 a rational function is a ratio of polynomials where the polynomial in the denominator 21 should not be equal to zero. doesn't that look like the definition of a rational number (which 22 is of the form p/k, where k = 0)? 23 polynomial functions play an important role in mathematics. they are generally simple to 24 compute (requiring only calculations that can be performed manually) and can be used to 25 model many real-world phenomena. in fact, scientists and mathematicians often simplify 26 complex mathematical models by substituting a polynomial model that is "close enough" for 27 their purposes. 28 let it be 𝑓(𝑥) = 𝑃𝑛(𝑥) 𝑄𝑚(𝑥) rational function. if , it is 𝑛 ≥ 𝑚 , i.e. not a real rational function, 29 then by division 𝑃𝑛 (𝑥) with 𝑄𝑚 (𝑥), 𝑓 can be represented as a sum of polynomials and real 30 rational functions in the form 31 𝑓(𝑥) = 𝑃(𝑥) + 𝑅(𝑥) 𝑄𝑚 (𝑥) 32 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science let's say it is 𝑓(𝑥) = 𝑃𝑛(𝑥) 𝑄𝑚(𝑥) true rational function (𝑛 < 𝑚). then it can be decomposed into 1 a sum of partial fractions of the form 2 3 𝐴 (𝑥−𝑎)𝑘 that is 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐)𝑘 4 5 where the first type of fraction comes from real, and the second from the type of complex 6 zero-points of polynomials in the denominator of the function f. therefore, the problem is 7 reduced to solving the integral of the form 8 9 ∫ 𝐴 (𝑥−𝑎)𝑘 𝑑𝑥 and ∫ 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐)𝑘 𝑑𝑥 10 11 where a and b are some real constants, is a natural number, and is a quadratic trinomial 12 𝑥2 + 𝑏𝑥 + 𝑐 has complex-conjugate zero-points. 13 14 integrals of the form ∫ 𝐴 (𝑥−𝑎)𝑘 𝑑𝑥 15 1. if it is 𝑘 = 1, he gives ∫ 𝐴 (𝑥−𝑎) 𝑑𝑥 = 𝐴 ln|𝑥 − 𝑎| + 𝐶. 16 2. if it is 𝑘 > 1, then by changing the variable 𝑥 − 𝑎 = 𝑡 ⟹ 𝑑𝑥 = 𝑑𝑡, we have it 17 ∫ 𝐴 (𝑥−𝑎)𝑘 𝑑𝑥 = { 𝑥 − 𝑎 = 𝑡 𝑑𝑥 = 𝑑𝑡 } = 𝐴 ∫ 𝑡 −𝑘 𝑑𝑡 = 𝐴 𝑡−𝑘+1 −𝑘+1 + 𝐶 = 𝐴 (1−𝑘)(𝑥−𝑎)1−𝑘 + 𝐶. 18 19 3. reduction to true rational function 20 21 a rational function is a true rational function if the degree of the numerator is less than the 22 degree of the denominator. if the degree of the numerator is greater than or equal to the 23 degree of the denominator, we can divide the polynomials, so we have 24 25 𝑓(𝑥) = 𝑝(𝑥) 𝑞(𝑥) = 𝑠(𝑥) + 𝑟(𝑥) 𝑞(𝑥) 26 27 where r and s are polynomials. the polynomial is the remainder when dividing, and the 28 degree of r is ᶴ s (x) dx less than the power of q. of course, it is solved by direct integration, 29 so we can conclude the following: 30 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science integrating rational functions is reduced to integrating real rational functions of the form 1 2 𝑓(𝑥) = 𝑝(𝑥) 𝑞(𝑥) 3 4 where p and q have no common zero-points and the degree of p is less than the degree of 5 q. 6 example: dividing a polynomial give 7 8 𝑥3+𝑥 𝑥2−1 = 𝑥 + 2𝑥 𝑥2−1 , 9 10 so, it is ∫ 𝑥3+𝑥 𝑥2−1 𝑑𝑥 = ∫ (𝑥 + 2𝑥 𝑥2−1 ) 𝑑𝑥 11 = ∫ 𝑥 𝑑𝑥 + ∫ 2𝑥 𝑥2−1 𝑑𝑥 = 𝑥2 2 + ∫ 𝑑𝑡 𝑡 , putting { 𝑥2 − 1 = 𝑡, 2𝑥 𝑑𝑥 = 𝑑𝑡 } 12 = 𝑥2 2 + ln|𝑡| + 𝐶 = 𝑥2 2 + ln|𝑥2 − 1| + 𝐶. 13 14 4. integration by partial fractions 15 16 integration by partial fractions is a method used to decompose and then integrate a rational 17 fraction that has complex terms in the denominator. by using a partial fraction, we calculate 18 and decompose the expression into simpler terms so that we can easily calculate or integrate 19 the resulting expression. 20 the basic idea in integration by partial fractions is to factor the denominator and then 21 factor them into two different fractions where the denominators are the factors and the 22 numerator is calculated accordingly. let's learn more about the different forms used in 23 integration by partial fractions, as well as the different methods. 24 25 4.1. we decompose the sub integral function into partial fractions 26 27 𝑥−3 𝑥3−𝑥 = 𝑥−3 𝑥(𝑥2−1) = 𝑥−3 𝑥(𝑥−1)(𝑥+1) = 𝐴 𝑥 + 𝐵 𝑥−1 + 𝐶 𝑥+1 28 29 using the polynomial similarity method, we get that 𝐴 = 3, 𝐵 = −1, 𝐶 = −2 30 31 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science ∫ 𝑥−3 𝑥3−𝑥 𝑑𝑥 = ∫ 𝑥−3 𝑥(𝑥−1)(𝑥+1) 𝑑𝑥 = ∫ [ 3 𝑥 − 1 𝑥−1 − 2 𝑥+1 ] 𝑑𝑥 1 = ∫ 3 𝑥 𝑑𝑥 − ∫ 1 𝑥−1 𝑑𝑥 − ∫ 2 𝑥+1 𝑑𝑥 = 3 ln|𝑥| − ln|𝑥 − 1| − 2ln|𝑥 + 1| + 𝐶 2 = ln | 𝑥3 (𝑥−1)(𝑥+1)2 | + 𝐶 3 4 integrals of the form ∫ 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐) 𝑑𝑥. we have already solved integrals of this form using 5 the shift method 6 7 ∫ 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐) 𝑑𝑥 = ∫ 𝐴(2𝑥+𝑏)+(𝐵− 𝐴𝑏 2 ) 𝑎𝑥2+𝑏𝑥+𝑐 𝑑𝑥 8 = 𝐴 2 ∫ 2𝑥+𝑏 𝑎𝑥2+𝑏𝑥+𝑐 𝑑𝑥 + (𝐵 − 𝐴𝑏 2 ) . ∫ 𝑑𝑥 𝑎𝑥2+𝑏𝑥+𝑐 . 9 10 where in ∫ 2𝑎𝑥+𝑏 𝑥2+𝑏𝑥+𝑐 𝑑𝑥 = ∫ (𝑎𝑥2+𝑏𝑥+𝑐) 𝑎𝑥2+𝑏𝑥+𝑐 𝑑𝑥 = ln|𝑎𝑥2 + 𝑏𝑥 + 𝑐| + 𝐶 and 11 12 ∫ 𝑑𝑥 𝑥2+𝑏𝑥+𝑐 = ∫ 𝑑𝑥 (𝑥+ 𝑏 2 )2+(𝑐− 𝑏2 42 ) putting { 𝑥 + 𝑏 2 = 𝑡, 𝑐 − 𝑏2 42 = 𝑘2 𝑑𝑥 = 𝑑𝑡 } 13 = ∫ 𝑑𝑡 𝑡2+𝑘 2 = 1 𝑘 𝑎𝑟𝑐𝑡𝑔𝑥 𝑡 𝑘 + 𝐶 = 1 √𝑐− 𝑏2 42 𝑎𝑟𝑐𝑡𝑔𝑥 𝑥+ 𝑏 2 √𝑐− 𝑏2 42 + 𝐶. 14 15 integrals of the form ∫ 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐)𝑘 𝑑𝑥, wherein 𝑘 ≥ 2, 𝑏2 − 4𝑐 < 0. let's perform the 16 transformation of the sub integral function 17 18 ∫ 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐)𝑘 𝑑𝑥 = ∫ 𝐴(2𝑥+𝑏)+(𝐵− 𝐴𝑏 2 ) (𝑥2+𝑏𝑥+𝑐)𝑘 𝑑𝑥 19 = 𝐴 2 ∫ 2𝑥+𝑏 (𝑥2+𝑏𝑥+𝑐)𝑘 𝑑𝑥 + (𝐵 − 𝐴𝑏 2 ) . ∫ 𝑑𝑥 (𝑥2+𝑏𝑥+𝑐)𝑘 . 20 we can solve the first integral by shifting, where's he from 𝑎𝑥2 + 𝑏𝑥 + 𝑐 = 𝑡. so, we get 21 (2𝑥 + 𝑏)𝑑𝑥 = 𝑑𝑡. 22 23 𝐼0 = ∫ 𝐴𝑥+𝐵 (𝑥2+𝑏𝑥+𝑐)𝑘 𝑑𝑥 = ∫ 𝑑𝑡 𝑡𝑘 = 1 (1−𝑘)𝑡 𝑘−1 + 𝐶 = 1 (1−𝑘)(𝑥2+𝑏𝑥+𝑐)𝑘−1 + 𝐶. 24 25 let's denote the second integral by 𝐼𝑘 = ∫ 𝑑𝑥 (𝑎𝑥2+𝑏𝑥+𝑐)𝑘 and perform the transformation 26 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science 1 𝐼𝑘 = ∫ 𝑑𝑥 (𝑎𝑥2+𝑏𝑥+𝑐)𝑘 = ∫ 𝑑𝑥 [(𝑥+ 𝑏 2 ) 2 +(𝑐− 𝑏2 42 )] 𝑘 2 3 introducing a shift 𝑥 + 𝑏 2 = 𝑡, 𝑑𝑥 = 𝑑𝑡 and label 𝑐 − 𝑏2 42 = 𝑙2 and 𝐼𝑘 we get 4 5 𝐼𝑘 = ∫ 𝑑𝑥 𝑡2+𝑙2 . 6 7 by combining the variable change method and partial integration, after a series of 8 relations, a recursive formula for calculating the integral is obtained, which we will not derive 9 here, but give it in its finished form 10 11 𝐼𝑘 = 𝑡 2𝑙(𝑘−1)(𝑡2+𝑙2)𝑘−1 + 2𝑘−3 2𝑙2(𝑘−1) ∙ ∫ 𝑑𝑡 (𝑡 2+𝑙2)𝑘−1 , 𝐼1 = ∫ 𝑑𝑡 𝑡2+𝑙2 = 1 𝑙 𝑎𝑟𝑐𝑡𝑔𝑥 𝑡 𝑙 + 𝐶 12 𝐼𝑘−1 13 14 example: let's calculate the integral ∫ 𝑥−1 (𝑥2+2𝑥+3)2 15 how it is 𝑝 = 2 and 𝑞 = 3, it is 𝑝2 − 4𝑞 = −8 < 0, so the denominator has no real zero-16 points. let's transform the integral 17 𝐼 = ∫ 𝑥−1 (𝑥2+2𝑥+3𝑐)2 𝑑𝑥 = ∫ 1 2 (2𝑥+2)+(−1−1) (𝑥2+2𝑥+2)2 𝑑𝑥 = 1 2 ∫ 2𝑥+2 (𝑥2+2𝑥+3)2 𝑑𝑥 − 2 ∙ ∫ 𝑑𝑥 (𝑥2+2𝑥+3𝑐)2 . 18 19 𝐼0 = ∫ 2𝑥+2 (𝑥2+2𝑥+3𝑐)2 𝑑𝑥 = { 𝑥2 + 2𝑥 + 3𝑐 = 𝑡 (𝑥2 + 2𝑥 + 3𝑐)𝑑𝑥 = (2𝑥 + 2)𝑑𝑥 = 𝑑𝑡 } 20 = ∫ 𝑑𝑡 𝑡2 = − 1 𝑡 + 𝐶 = 1 𝑥2+2𝑥+3𝑐 + 𝐶. 21 𝐼2 = ∫ 𝑑𝑥 (𝑎𝑥2+𝑏𝑥+𝑐)2 = ∫ 𝑑𝑥 [(𝑥+1)2+(3−1)]2 = { 𝑥 + 1 = 𝑡 𝑑𝑥 = 𝑑𝑡 } = 𝑑𝑡 [𝑡2+2]2 . 22 23 by substituting into the recurrent formula, we get 24 𝐼2 = 𝑡 2∙2(2−1)(𝑡2+2)2−1 + 2∙2−3 2∙2(2−1) ∙ ∫ 𝑑𝑡 (𝑡2+2)2−1 , where 𝐼1 = ∫ 𝑑𝑡 𝑡2 +2 = 1 √2 𝑎𝑟𝑐𝑡𝑔 𝑡 2 + 𝐶, 25 𝐼2−1=1 26 27 so, it is 𝐼2 = 𝑡 4(𝑡2+2) + 1 4 ∙ 1 √2 𝑎𝑟𝑐𝑡𝑔 𝑡 √2 + 𝐶 = {𝑡 = 𝑥 + 1} 28 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science = 𝑥+1 4(𝑥2+2𝑥+3) + 1 4√2 𝑎𝑟𝑐𝑡𝑔 𝑥+1 √2 + 𝐶. 1 2 so, it is the final solution 3 𝐼 = ∫ 𝑥−1 (𝑥2+2𝑥+3𝑐)2 𝑑𝑥 = 1 2 𝐼0 − 2𝐼2 4 = 1 𝑥2+2𝑥+3𝑐 + 𝑥+1 4(𝑥2+2𝑥+3) + 1 4√2 𝑎𝑟𝑐𝑡𝑔 𝑥+1 √2 + 𝐶 5 = 𝑥+2 4(𝑥2+2𝑥+3) + 1 4√2 𝑎𝑟𝑐𝑡𝑔 𝑥+1 √2 + 𝐶. 6 7 5. contribution to the integration of rational 8 functions 9 10 mathematical thinking and reasoning are the main goal of mathematics education. it is 11 known from research that tasks involving problem solving, modelling and argumentation 12 provide excellent opportunities to realize students' mathematical thinking [8]. many topics in 13 secondary education are already taught through tasks of this type (e.g. elementary algebra, 14 exponential functions). however, there are still topics that are traditionally taught formally 15 and mechanically, which makes it difficult for students to think and communicate in a 16 conceptual sense. this is the case for the integral concept, which was introduced in most 17 european countries in the last two years of secondary education and taught in a procedural 18 way. 19 the integration of rational functions involves a number of cases that require different 20 procedures. these procedures are based on operations such as calculating derivatives, 21 dividing polynomials, factoring, and solving equations; everything is applied in order to 22 decompose to the original integral in simpler cases that can be approached using already 23 known techniques. 24 to find the rational part, we first need to know about square-free factorization. an 25 important result in algebra is that every polynomial with rational coefficients can be uniquely 26 factored into irreducible polynomials with rational coefficients, up to multiplication by a 27 nonzero constant and rearrangement of factors, just as every integer can be uniquely factored 28 into prime up to multiples of 1 and -1 and redistribution of factors (technically, it is with 29 coefficients from the unique domain of factorization, for which rationales are a special case, 30 up to the multiplication of unity, which for rationales is every constant except zero). 31 a cc ep te d m a n u sc ri pt journal of multidisciplinary applied natural science to integrate the corresponding rational function, we can apply the method of partial 1 fractions. this method allows converting the integral of a complicated rational function into a 2 sum of integrals of simpler functions. the denominators of partial fractions can contain 3 unrepeated linear factors, repeated linear factors, unrepeated irreducible quadratic factors, 4 and repeated irreducible quadratic factors. 5 6 6. conclusion 7 8 the integration of rational functions includes a variety of cases requiring different 9 procedures. these procedures are based on operations such as derivative calculation, division 10 of polynomials, factorization and resolution of equations; all applied in order to decompose to 11 the original integral in simpler cases that can be approached using the techniques already 12 known. 13 14 references 15 16 [1] c. canuto and a. tabacco. (2015). "mathematical analysis i". springer cham, 17 switzerland. 10.1007/978-3-319-12772-9. 18 [2] d. lazard and r. rioboo. (1990). "integration of rational functions: rational 19 computation of the logarithmic part". journal of symbolic computation. 9 (2): 113-115. 20 10.1016/s0747-7171(08)80026-0. 21 [3] r. h. c. moir, r. m. corless, m. m. maza, and n. xie. (2019). "symbolic-numeric 22 integration of rational functions". numerical algorithms. 83 (4): 1295-1320. 23 10.1007/s11075-019-00726-6. 24 [4] m.-t. noda and e. miyahiro. (1992). "a hybrid approach for the integration of a 25 rational function". journal of computational and applied mathematics. 40 (3): 259-26 268. 10.1016/0377-0427(92)90182-w. 27 [5] m. t. noda and e. miyahiro. (1990). "on the symbolic/numeric hybrid integration". 28 proceedings of the international symposium on symbolic and algebraic computation. 29 10.1145/96877.96973. 30 [6] b. m. trager. (1976). "algebraic factoring and rational function integration". 31 proceedings of the third acm symposium on symbolic and algebraic computation. 32 10.1145/800205.806338. 33 a cc ep te d m a n u sc ri pt https://doi.org/10.1007/978-3-319-12772-9 https://doi.org/10.1016/s0747-7171(08)80026-0 https://doi.org/10.1007/s11075-019-00726-6 https://doi.org/10.1016/0377-0427(92)90182-w https://doi.org/10.1145/96877.96973 https://doi.org/10.1145/800205.806338 journal of multidisciplinary applied natural science [7] k. gürlebeck, k. habetha, and w. sprößig. (2007). "holomorphic functions in the 1 plane and n-dimensional space". springer science & business media, basel. 2 [8] j. lithner. (2007). "a research framework for creative and imitative reasoning". 3 educational studies in mathematics. 67 (3): 255-276. 10.1007/s10649-007-9104-2. 4 5 a cc ep te d m a n u sc ri pt https://doi.org/10.1007/s10649-007-9104-2 1. introduction 3. reduction to true rational function 4. integration by partial fractions 6. conclusion 58 journal of multidisciplinary applied natural science vol. 2 no. 1 (2022) research article isolation of indigenous fungi in river containing ammonia from rubber industry waste in jember dwi nur rikhma sari* and septarini dian anitasari received : november 15, 2021 revised : january 26, 2022 accepted : january 27, 2022 online : january 27, 2022 abstract indigenous fungi are fungi that can degrade organic compounds and make them a source of nutrition for metabolism and life so that they can to survive in various environments including environments polluted by ammonia waste from the rubber factory industry. this study was conducted to obtain fungal isolates that can survive in river water contaminated with ammonia from rubber industry waste in jember. isolation was obtained from river water contaminated with rubber factory waste containing ammonia and isolat ed using the media of potatoes dextrose agar (pda). the method used in this research is descriptive exploration, namely isolatin g and culturing fungi using the dilution method. the results of the isolation will identify the genus of fungi based on macroscopic and microscopic morphological characters. data analysis was done descriptively. the results obtained 4 isolates of indigenous fun gi that can degrade ammonia, namely aspergillus sp., fusarium sp., penicillium sp., and yeast groups. keywords indigenous fungi, ammonia waste, rubber waste 1. introduction liquid waste is one type of waste whose presence is often a problem in people's lives [1]. liquid waste originating from industry generally contains chemical compounds, heavy metals, hazardous and toxic materials (b3), as well as various organic compounds in high concentrations [2]. indonesia is one of the largest rubber producers in the world, with a total area of 3.6 million hectares (ha) with annual rubber production reaching 3.6 million tons [3]. in addition to having a positive value, if the rubber factory is not processed according to standards, it will produce negative impacts, one of which is as a contributor to ammonia waste in river waters. the high ammonia content is caused by ineffective sewage treatment and still using the anaerobic pond method to treat waste so that only phosphorus, nitrogen, and carbon compounds will be reduced, while ammonia levels are still stored in it [4]. from various wastewater treatment that contains ammonia, biological treatment of ammonia waste can also be carried out. waste treatment by utilizing microorganisms that can decompose organic substances in the waste. one of the microorganisms that can degrade ammonia waste is the taste of fungal microorganisms. rubber waste containing ammonia contains various species of indigenous fungi that have the potential to degrade the ammonia compound. these indigenous fungi can be obtained by isolating them from ammonia waste which can later be used as a method in treating ammonia waste in the water. indigenous microorganisms originating from their substrate habitat have a very high ability to degrade their substrate [5]. several studies on the isolation of indigenous fungi from water contaminated with the organic matter have been carried out, one of which has succeeded in obtaining 11 isolates of fungi from textile industry waste that have ligninolytic abilities [6]. in addition, other studies were also conducted on the isolation of indigenous fungi from palm oil waste as many as 16 isolates which have the ability as lignocellulolytic agents in the waste [5]. research on the isolation of indigenous fungi in industrial waste has been carried out, but in addition, research on the isolation of macroscopic and microscopic properties of indigenous fungi in river waters containing ammonia from rubber factory waste in jember has never been carried out. based on this background, research on the isolation of indigenous fungi capable of utilizing and degrading ammonia in rubber industry waste needs copyright holder: © sari, d. n. r. and anitasari, s. d. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.109 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.109 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.109&domain=pdf&date_stamp=2022-01-30 j. multidiscip. appl. nat. sci. 59 to be researched. the purpose of this study was to determine the genus of indigenous fungi that can degrade ammonia waste in rivers contaminated with rubber factory waste in jember. 2. materials and methods 2.1. materials in this study, the tools used were petri dishes, glass beaker, measuring pipette, bunsen, ph meter, ammonia level measuring instrument, and autoclave. while the materials used in this study were sterile aquadest, potatoes dextrose agar (pda) media, industrial rubber liquid waste, spirit, cotton, aluminum foil, markers, label paper, wood paper [6]. this research was carried out in july 2021, at the biology laboratory of pgri argopuro jember university. the sample used came from a river that is close to the waste from the ptpn xii rubber factory, glantangan gardens, jember. samples were taken at three points that are closest to the location of the rubber industry waste disposal. 2.2. methods 2.2.1. sampling the sample was obtained from the river behind the residents' house, which contains ammonia from the waste processing of the rubber factory, ptpn xii jember. a sampling of 100 ml at each point (3 sampling points). 2.2.2. preparation of pda as for making 2.8 g/l pda media: weighing as much as 2.8 gram, and putting it into a glass beaker. after that, the media was added with 100 ml of distilled water and heated on a heater until homogeneous. the media of pda that has been made, then put into erlenmeyer and tightly covered with cotton and coated with aluminum foil. the media was then sterilized using an autoclave at 1 atm, 121 ºc for 20 minutes. next, each petri dish is filled with 5 ml of media and wait for it to solidify [7]. 2.2.3. sterilization of tools and materials all tools and materials to be used must be washed and dried beforehand. the mouth of the pipette, beaker glass, and erlenmeyer was first covered with cotton, while the petri dishes were wrapped in newspaper. then all the tools are wrapped with aluminum foil and wooden paper/ brown paper. the tools that we will use in this study are sterilized by autoclave 1 atm, 121 ºc for 120 minutes [8]. 2.2.4. isolation of indigenous fungi fungal isolation was carried out by pouring 1 ml of sample into a petri dish containing 5 ml of pda media, with a dilution of 10 -4 . after mixing until homogeneous, then rotate the petri dish slowly on the table to form a figure of eight. after the media and water samples solidified completely, the petri dish was then inverted so that the condensed water did not fall onto the lid of the cup. all petri dishes were then wrapped in paper and stored at room temperature for 3-7 days. after the isolation of indigenous fungi was completed, the fungal isolates were purified using media of potato dextrose agar (pda). fungal colonies growing in petri dishes were isolated for each different colony [9]. 2.2.5. characterization of indigenous fungi identification of fungi refers to reference books, figure 1. the results of the isolation of indigenous fungi in river water waste contaminated with ammonia. (a) isolation of fungi at point 1; (b) isolation of fungi at point 2; (c) isolation of fungi at point 3. j. multidiscip. appl. nat. sci. 60 namely the identification of fungi: an illustrated introduction with keys, glossary, and guide to literature spi edition. macroscopic observations included the shape, texture, and color of fungal colonies growing on pda media. for microscopic characters, observations were made using a microscope. 2.2.6. data analysis data analysis was carried out descriptively based on the morphological characteristics and characteristics of the fungus, which was then identified by referring to the book the identification of fungi: an illustrated introduction with keys, glossary, and guide to literature spi edition [10]. 3. results and discussions based on the results of the isolation of fungi at three sample locations grown on media of potatoes dextrose agar (pda) (figure 1), 6 types of fungal isolates were obtained. furthermore, from the isolated fungi, pure isolation was carried out on pda media which was then identified based on their morphological characters. based on the identification of the morphological characters, both macroscopic and microscopic, of the isolated fungi (figure 2, table 1), it can be seen that the 6 isolates belonged to the aspergillus, penicillium, fusarium, and yeast groups. the results of the isolation and identification of fungi from water samples containing ammonia showed that the 6 isolates belonged to the ascomycota division with different families. fusarium sp. into the family netriacceae with fusarium sp., aspergillus sp., and penicillium sp. into the family trichomaceae [11]–[14]. these four genera of fungal isolates are known to be able to survive in water containing ammonia and other organic materials that are used as food sources during cell metabolism. indigenous fungi are species of fungi that are capable of degrading several organic compounds that will be used as a source of nutrients in the process of cell metabolism [15]. in places polluted by several organic compounds, including water contaminated with paper mill waste containing ammonia, several species of fungi function as bioremediation agents or as bioremediation agents or mycoremediation. mycoremediation is one method that uses biological agents, namely fungi, to reduce waste from the environment. the presence of fungi in organic compound waste has enzymatic activity that can degrade pollutant waste [16]. in this research, the genus aspergillus was isolated from river water which became the waste disposal of the rubber industry, at the age of 2 x 24 hours. based on macroscopic characteristics, colonies were white-brown with a velvety texture and radial stripes (fig. 3 a.1, table 1). the results of this isolation are supported by research that reports that the macroscopic characteristics of aspergillus fumingatus grown on media of pda appear white filamentous colonies that then turn green with white edges and the lower surface of the colonies is yellow to brown [17]. the microscopic character of aspergillus sp. has septate hyphae and hyaline, spherical conidia (fig. 3 a.2, table 1). this microscopic observation is supported by other studies which show the presence of conidia a round to semi-spherical shape with smooth walls, conidiophytes having thick walls and having septate hyphae [18]. figure 2. fungi isolate at point 1 (a) isolate 1, (b) isolate 2; at point 2 (c) isolate 3, (d) isolate 4; and at point 3 (e) isolate 5, (f) isolate 6; (1 : front view; 2 : back view). j. multidiscip. appl. nat. sci. 61 aspergillus sp. can dissolve insoluble inorganic compounds by secreting organic acids. aspergillus sp. produces protease enzymes that function in the transformation of organic nitrogen (in the form of protein) in the soil as well as other organic wastes [19]. the ammonia reduction concentration is related to the sulfide reduction concentration in the effluent containing aspergillus sp. under aerobic conditions which will oxidize nitrite to nitrate in a non-toxic form [20]. fungi of the genus aspergillus sp. is known to be widely used by various studies in degrading waste containing high organic compounds [9]. isolate penicillium sp. which was isolated for 3 x 24 hours had a dartmouth green colored colony character with slightly white edges, turquoise cotton -like structure and white edges (fig. 3 b.1, table 1). the results of this isolation are supported by research that shows the macroscopic character of the isolate penicillium sp. the cotton-like structure is greenish-white in color and changes color to bottle green with beige edges [17]. in addition, isolates of penicillium sp. the other shows a small green color in the middle and white edges [17]. microscopic characters of penicillium sp. (fig. 3 b.2, table 1), showed that there were smooth-walled conidia, branching conidiophores, metules, stipes, and had septate hyphae. this research is supported by other studies that state that the genus penicillium sp. has hyaline septate hyphae, rounded conidia, and phialid [21]. penicillium sp. is a group of fungi that can dissolve phosphate by excreting organic acids produced to release the p bonds from the binding metal [22]. the existence of penicillium sp., in rivers containing ammonia, cannot be separated from its ability to live and survive in various types of environments. in addition, river water which is used as a rubber factory waste disposal site contains a lot of inorganic and organic substances which make it the best place for the growth of microorganisms including penicillium sp. [23]. species of fusarium sp., which had been isolated for 3 x 24 hours showed mycelium colony characters like cotton and brownish-white, not pigmented to the media and showed no radial lines (fig. 3 c.1, table 1). this study is supported by observations made by other researchers who isolated fusarium sp. on media of pda with colonies that have different colors because this fungus is unstable and its dna is very easy to mutate [24]. in addition, the growth of fusarium sp. on media of pda and isolated results showed that the macroscopic character of the colonies was brownish white (broken white) [8]. for microscopic characters (fig. 3 c.2, table 1) of fusarium sp., indicating that this fungus had branched or unbranched conidiophores, conidia had many septate and shaped hyphae ovoid. this research was supported by other researchers who observed the fungi fusarium sp. have multiseptal hyphae and figure 3. isolate of indigenous fungi that degrades ammonia in 3 x 24 h, a) isolates of aspergillus sp.; b) isolates of penicillium sp.; c) isolates of fusarium sp.; and d) isolate yeast group; (1 : macroscopic, 2 : microscopic). j. multidiscip. appl. nat. sci. 62 monophyalid conidiophores, long and unbranched [9][24]. the existence of fusarium sp. on water containing ammonia from industrial processing of the rubber factory showed that this fungus was able to utilize various organic compounds as a source of metabolism. the fungi fusarium sp., are commonly found in wastewater treatment sediments which are beneficial in increasing the decomposition of organic matter [24]. in addition, this fungus is a heterotrophic microorganism that assists the nitrification process by converting ammonia into nitrate and nitrite, thereby reducing the concentration of ammonia in a bioreactor [25]. the last isolate that was successfully isolated using media of pda was from the yeast/yeast group, which is a polyphyletic group of basidiomycota and ascomycota fungi. this fungus has unicellular characteristics and produces buds. species included in the fungi group are saccharomyces cereviceae and candida albicans. the macroscopic character of this yeast group has a circular shape with convex-shaped elevation, entire margin and milky-white color (fig. 3 d.1, table 1). this study was supported by other studies that isolated yeasts with macroscopic characters of circular shape, convex elevation, rough surface, entire margin, milky white and measuring 0.01 0.5 m [26]. microscopic characters are round, elongated ovoid, not pseudohyphae and budding (fig. 3 d.2, table 1). based on the results of research that has been carried out, it shows that saccharomyces cereviceae which belongs to the yeast group in the anaerobic biofiltration process shows a decrease in bod, a decrease in total ammonia levels and an increase in cod in polluted water [27]. while for other yeast species, candida albicans can live in the environment. water containing ammonia and has potential as a bioremediation agent [28]. 4. conclusions based on the results of the study, four indigenous fungi isolates were obtained that were able to live in environmental conditions containing ammonia. the four isolates were thought to have the ability to act as bioremediation agents for wastewater containing ammonia from paper mill processing waste, in jember. most of the isolates belonged to the ascomycota and basidiomycota divisions, namely aspergillus sp., fusarium sp., penicillium sp., and the yeast group. author information corresponding author dwi nur rikhma sari — department of biology, university of pgri argopuro jember, jember – 68121 (indonesia); email: rikhmasari.dnrs@gmail.com author septarini dian anitasari — department of biology, university of pgri argopuro jember, jember – 68121 (indonesia); orcid.org/0000-0002-2790-7986 references [1] a. w. satria, m. rahmawati, and a. prasetya. 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(2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.90 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.90 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.90&domain=pdf&date_stamp=2022-01-16 j. multidiscip. appl. nat. sci. 42 used wcc with a dose of 6% to convert cooking oil waste in 82% biodiesel yield [17]. alptekin et al. conducted a reduction of ffa content using acid catalysts and then produced biodiesel using alkaline catalysts [18]. dang et al. convert wco from canteen source to biodiesel [19]. xiong et al. convert alcohol made from used cooking oil waste into biodiesel [20]. marchetti conducted a study on the comparison of biodiesel products produced using raw materials for new cooking oil and used cooking oil, the results obtained were 40% of biodiesel products [21]. agarwal et al. produced biodiesel by converting used cooking oil into biodiesel using a koh catalyst in 98.2% yield [22]. considering some of the aspects described above, the study aims to produce biodiesel from used cooking oil waste taking into account the advantages offered by the heterogeneous catalyst cao made from pure breed chicken eggshells. 2. materials and methods 2. 1. materials used cooking oil and breed chicken eggshell samples were collected from the restaurant in bandar lampung city, lampung, indonesia. the other chemicals used include: methanol, n-hexane, cerium sulfate, and h2so4 purchased from merck sg. 2.2. methods 2.2.1. synthesize cao heterogeneous catalysts pure breed chicken eggshells were washed several times and then boiled. the membrane layer was separated from the shell and then dried at room temperature. furthermore, the eggshell was crushed into small particles and then filtered. after being crushed, then the material was calcined and sintered using a muffle furnace at 900 0 c for 10 h to form a cao structure [23]. the synthesis scheme of the cao heterogeneous catalyst is shown in figure 1. 2.2.2. preparation of biodiesel as much as 200 g of used cooking oil was weighed, added with 60 g of methanol and 4 g of cao catalyst, and then stirred at 60 ± 4 0 c for 6 h. after that, the mixture was allowed to stand for 24 h until 3 phases are formed, namely biodiesel (top layer), glycerol (middle layer), and the catalyst material (bottom part). crude biodiesel and glycerol along with the catalyst are separated and placed in another container. a certain amount of water was then added into the biodiesel mixture for a purification purpose. the crude biodiesel was washed repeatedly until the aqueous phase no longer contained soap and appeared as a clear solution. finally, the biodiesel was heated at 60 0 c for 10 min to evaporate the water which may still be left. figure 1. the synthesis scheme of cao heterogeneous catalysts from pure breed chicken eggshells. figure 2. preparation scheme of biodiesel made from waste cooking oil using cao heterogeneous catalyst. j. multidiscip. appl. nat. sci. 43 3. results and discussions based on the screening test using tlc, the obtained rf values were 0.66 and 0.88 as shown in figure 3. these results indicate that there were various types of methyl ester compounds with varying carbon chain lengths according to the degree of polarity where the value of 0.88 is relatively more non-polar and has a longer carbon chain than the compounds with an rf value of 0.66. furthermore, the results of the characterization using gc-ms analysis obtained chromatogram data. a total of 18 peaks were observed in the chromatogram, and the first 2 peaks could not be identified because the intensity was too low to be considered satisfactory. the chromatogram of identified compounds showed in figure 4 and the composition was presented in table 1. from figure 4 and table 1, the identification of methyl esters indicates that the transesterification of fatty acids in wco took place already. the data also showed that methyl octadecanoate was found as the most prominent component of the sample, which was corresponded to the presence of palmitic acid as the most abundant component in wco [24]. the information presented in table 2 was an additional analysis to determine the three basic characteristics of biodiesel. biodiesel standard values according to the indonesian standard (sni 7182-2015) are included in table 2 for comparison. compared to standard values, all three characteristics are acceptable. in the future, the determination of other parameters included in the standard is required for further commercialization of the produced biodiesel in this study [25]–[27]. 4. conclusions the cao-based heterogeneous catalyst derived from chicken eggshells can be used as a heterogeneous catalyst to produce biodiesel fuel from waste cooking oil as biodiesel raw material. the results of the tlc screening test and gc-ms analysis showed the formation of several methyl ester compounds. author information corresponding author rodhiansyah djayasinga — medical figure 4. chromatogram of typical biodiesel produced. figure 3. rf values of methyl ester produced. j. multidiscip. appl. nat. sci. 44 laboratory technology, health polytechnics tanjungkarang, bandar lampung 405308 (indonesia); orcid.org/0000000320890795 email: rodhiansyah@poltekkes-tjk.ac.id authors andi setiawan — department of chemistry, university of lampung, bandar lampung 35145 (indonesia); orcid.org/0000-0002-0731-6417 agus purnomo — medical laboratory technology, health polytechnics tanjungkarang, bandar lampung 405308 (indonesia); ahmad zakaria amien — medical laboratory technology, health polytechnics tanjungkarang, table 1. the composition of biodiesel produced in this study based on gc-ms analysis. peak number retention time (min) mw m/z compound name relative percentage 1 13.670 no hits 0.250 2 16.912 no hits 2.910 3 21.634 444 73 dodecamethyl-ester 0.320 4 22.757 186 74 decanoic acid, methyl ester 1.440 5 25.947 518 73 tetradecamethyl-ester 0.570 6 27.934 214 74 undecanoic acid, 10-methyl-, methyl ester 9.890 7 29.757 592 355 hexadecamethyl-ester 0.310 8 32.564 242 74 tridecanoic acid,12-methyl-,methyl ester 16.06 9 34.717 256 74 pentadecanoic acid,methyl ester 0.870 10 36.323 268 74 9-hexadecenoic acid, methyl ester, (z)1.240 11 36.741 270 74 pentadecanoic acid,14-methyl-,methyl ester 21.04 12 36.901 270 74 pentadecanoic acid,14-methyl-,methyl ester 16.00 13 38.715 284 74 hexadecanoic acid, 14-methyl-,methyl ester 0.890 14 39.967 294 67 9,12-octadecadienoic acid (z,z)-, methyl ester 1.110 15 40.099 294 67 8,11-octadecadienoic acid,methyl ester 1.320 16 40.277 296 264 9-octadecenoic acid,methyl ester, e4.200 17 40.589 298 74 octadecanoic acid, methyl ester 20.10 18 44.108 326 74 eicosanoic acid, methyl ester 1.470 total 100.0 parameter analysis result standard value (sni) density at 40 0 c 855 kg/m 3 850-890 kg/m 3 (astm d-1298) kinematic viscosity at 40 0 c 3.74 mm 2 /s (cst) 2.3-6.0 mm 2 /s (cst) (astm d 445) flash point (bowl closed) 135 0 c > 100 0 c (astm d 93) table 2. comparison of produced biodiesel with indonesian standard for biodiesel. http://orcid.org/0000000320890795 mailto:rodhiansyah@poltekkes-tjk.ac.id http://orcid.org/0000-0002-0731-6417 j. multidiscip. appl. nat. sci. 45 bandar lampung 405308 (indonesia); hertanti hertanti — medical laboratory technology, health polytechnics tanjungkarang, bandar lampung 405308 (indonesia); acknowledgement the author would like to thank the ministry of health of the republic of indonesia for the research funds provided through the decree of the director of the tanjungkarang health polytechnics number. hk.02.03/1.2/2712/2019 references [1] f. nadeem, i. a. bhatti, a. ashar, m. yousaf, m. iqbal, m. mohsin, j. nisar, n. tamam, 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[18] demonstrates that dual-phase lto/tio2 has a high potential as anode material for high-rate application of libs. the synthesized dual phase lto/tio2 nanowire arrays have been applied as integrated anodes for high-rate libs [18]. the facile hydrothermal and ion exchange route developed to synthesize dual phase lto/tio2 nanowire arrays. the resulting lto/tio2 has a ratio of about 1:2. the introduction of tio2 to li4ti5o12 increases the specific capacity copyright holder: © he, y. f., chu, d. y., and zhuo, z. (2021) first publication right: journal of multidisciplinary applied natural science this article is licensed under: https://doi.org/10.47352/jmans.v1i1.8 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i1.8 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i1.8&domain=pdf&date_stamp=2021-01-31 j. multidiscip. appl. nat. sci. 55 and creates a dual-phase nanostructure with a high grain boundary density that facilitates electrons and li-ions transport. the resulting dual-phase nanowire electrode has good rate capability compared to pure lithium titanate and tio2. the results obtained also explain that the dual-phase lto/tio2 produced has good cycle stability. in this work, the lto-tio2 dual-phase nanowires synthesized using tio2 nanowires as precursors through a hydrothermal process at various times lithiation of 10, 24 and 48 h. sem and xrd tests were carried out to characterize samples. the electrochemical tests were done through cycling and rate test to study the cycle stability. 2. materials and methods 2.1. materials laboratory grade titanium (iv) butoxide (97 %), glucose (≥99.5 %), ethanol, sodium hydroxide, hydrochloric acid and lithium hydroxide monohydrate purchased from merck sigma-aldrich reagent pte, singapore. 2.2. methods 2.2.1. hydrothermal synthesis lto-tio2 nanowires tio2 nanowires synthesized by a hydrothermal method. in this approach, 2 g titanium (iv) butoxide dissolved in 12 g ethanol. sodium hydroxide (1.2 g) and 0.5 g glucose dissolved in 15 g water. these two solutions then mixed to form a white suspension. after stirring and ultrasonication, the suspension transferred to autoclave and heated to 260 ºc for 24 h. afterwards, 2.5 ml hydrochloric acid (35 %) and 200 ml water added to the suspension. the suspension stood 24 h and washed with water and ethanol. after drying the product, the powders collected and calcined at 600 ºc with ar protection for one h. the product then noted as tio2 nanowires. to synthesize lto/tio2 nanowires, the previous synthesized tio2 nanowires used as precursors. in this process, 0.05 g tio2 nanowires dispersed in water with the aid of ultrasonication. then, 2.5 g lithium hydroxide monohydrate dissolved in the suspension. the suspension was transferred to an autoclave and heated to 80 ºc for figure 1. sem image of lto/tio2-10 (a), lto/tio2-24 (b), and lto/tio2-48 (c) sample phase composition (wt. %) rp rwp χ 2 lto/tio2-10 tio2 85.10 17.55 25.38 2.14 lto 14.90 lto/tio2-24 tio2 52.26 19.46 26.14 2.75 lto 45.74 lto/tio2-48 tio2 3.54 18.65 26.75 2.98 lto 78.50 li2tio3 17.96 table 1. results of phase composition and structural parameters obtained from the rietveld analysis. j. multidiscip. appl. nat. sci. 56 10 h, 24 h, and 48 h. after that, the product washed with water several times and then dried. finally, the collected, dried white product annealed at a temperature of 600 ºc for 2 h and noted as lto/ tio2-10, lto/tio2-24, and lto/tio2-48. 2.2.2 lto-tio2 nanowires characterization lto-tio2 nanowires that have produced are characterized using different instrument techniques. the surface morphology of nanoparticles studied using scanning electron microscopy (sem, fei nova nanosem 450). the nanoparticles' crystal structure studied using x-ray diffraction (xrd, panalytical xpert multipurpose x-ray diffraction system) techniques. the range of 2θ set from 10 to 100, and the material for the target was cupper. 2.2.3. electrochemical performance study li/li2o electrodes chosen as the counter electrode for the tested electrodes. 25 µm thick glass fibers used as the separators. the 1m lithium hexafluorophosphate in ethylene carbonate and ethyl methyl carbonate (5/5 by volume) selected as the electrolyte. all coin cells assembled in an ar filled dry glove box. the electrical properties analyzed by netware ct-4008-5v10ma-164 batteries testing system, and the charge-discharge cycling performed between 1-3 v (vs li/li + ) at room temperature using different c rates (1 c=175 ma/g). figure 2. xrd patterns and peak patterns of ltotio2 nanowires with different lithiation time. figure 3. charge-discharge curve of the initial 3 cycles of lto/tio2-10 (a), lto/tio2-24 (b), and lto/ tio2-48 (c). j. multidiscip. appl. nat. sci. 57 3. result and discussions 3.1. sem and xrd characterization the morphology of the nanowires examined with sem. as shown in figure 1 (a), after 10 h lithiation process, there were little small particles grown on the wires' surface. the mean diameter of nanowire samples determined from sem imaging was around 100 nm. with the increasing of lithiation time, the amount of the small particles grew. after a 24 h reaction, the nanowires' structure can still be recognized (figure 1 b). after 48 h reaction, most wires fully covered by the small lithiated particles (figure 1 c). this process caused most of the separated nanowires to be connected and increased the nanowires diameter to around 200 nm. this phenomenon changed the morphology of the nanowires to more nanosheets-like or spongefigure 4. first, 50 th and 100 th voltage-specific capacity curves at 0.1 c of lto/tio2-10 (a), lto/tio2-24 (b), and lto/tio2-48 (c) figure 5. cycling performance of lto-tio2 nanowire. (a) lto/tio2-10, (b) lto/tio2-24, (c) lto/tio2-48. j. multidiscip. appl. nat. sci. 58 like structure [19]. xrd used to identify the crystallographic structure. as shown in figure 2, after 10 h reaction, there a small number of li4ti5o12 produced. however, a large number of tio2 phase remained. all major bragg peaks well matched with those of anatase tio2 (icdd no. 00-004-0477) [20] and cubic spinel li4ti5o12 (icdd no. 00-049-0207) [21] respectively. as the reaction time extends to 24 h, more tio2 phase consumed, and more lto produced. the main components for the nanowires were still anatase tio2 and spinel li4ti5o12 (figure 2). after 48 h lithiation process, most tio2 reacted to form lto. because excessive lioh was used as a lithium source during the hydrothermal reaction, the prolonged reaction enabled more lithium ions to be involved in the process [22]. consequently, li2tio3 (icsd no. 01-075-1602) [23] with a higher li to ti ratio than li4ti5o12 was formed (figure 2). the observed major peaks at 18.4437° and 43.2608° representatives for spinel li4ti5o12, while the peak at 43.6510° contributed by (4 0 0) face of monoclinic li2tio3. compared with 10 and 24 h samples, the major peak intensity for anatase tio2, which located at around 25.2872°, severely weakened, indicating the low content of tio2 in the nanowires. also, the excessive lithium ions can be inserted into the lattice gaps in both li2tio3 and li4ti5o12 to form lto with different li to ti ratio. as a result, some miscellaneous peaks were observed in the xrd pattern (figure 2). unmatched peaks belongs to lto with different phases. these xrd results confirm the sem results where with increasing lithiation time, nanosheetslike or sponge-like structures are formed. the structure formed indicates the li4ti5o12 produced during the process. 3.2. electrochemical characterization half-cell batteries tests carried out with metal lithium as both the counter and the reference electrode to study the electrochemical performance of all three kinds of dual-phase nanowires. fig. 3 has shown the charge-discharge curves of the first three cycles of all three kinds of samples. during the charge and discharge process, the c rate was 0.5 c, so the current in this process a constant value for all three samples. all samples except lto/tio2-48 display two distinguishable gradient changes during their charge and discharge processes (figure 3 a, b). the turning points at around 2.0 v and 1.78 v referred to the insertion and extraction of lithium ions in the tio2 crystal. the turning points, which around 1.6 v and 1.5 v, on the other hand, were contributed by li4ti5o12. in the batteries tests, capacity calculated by multiplication of time, current and voltage, since the current a fixed value, the area under the charge-discharge curve had represented the electrode's capacity. naturally, a higher and longer plateau produces more capacity than a low and short plane [24]. in the case of lto/tio2-48, there were no tio2 charge and discharge planes observed in its curve due to low tio2 content (figure 3 c). li4ti5o12 provided almost all capacity. as for the lto/tio210, whose tio2 content was the highest, the curve shifted most (figure 3 a). both the charge and discharge planes of tio2 shortened while no significant variation happened to the li4ti5o12 plane. the reason lies in the difference in electrochemical stability between these two contents. the li4ti5o12 is known as zero strain materials in lithium-ion batteries, which means there is almost no volume change during the insertion and extraction of lithium ions [25]. its crystal structure can be preserved to the greatest extent in the cyclic test. on the other hand, compared with li4ti5o12, tio2 can store more lithium ions per formula unit. this property makes it inevitable that the crystal volume will change significantly in cycling. as a result, the crystal figure 7. rate performance at various current rate from 0.2 c to 20 c and then back to 0.5 c and 0.2 c of lto/tio2­-10, lto/tio2­-24, and lto/tio2­-48 j. multidiscip. appl. nat. sci. 59 structure is more likely to be damaged, and the capacity it provided is also reduced at a higher rate [26]. figure 4 show the voltage-specific capacity curves at 0.1 c. as the cycle number increased, each sample had a decrease in both charge and discharged specific capacity. considering the lto/ tio2-10 and the lto/tio2-24 (figure 4 a, b), in the first 50 th cycle, the tio2 charge and discharge planes have shrunk to about half of their initial length. the lto/tio2-48 had almost no tio2 content, so the tio2 charge plane was a slope and cannot be distinguished even at the first cycle (fig. 4 c) discharge plane of tio2 in the curve of lto/ tio2-48. in li4ti5o12, the lengths of charge and discharge plateaus also reduced in all three samples after the first cycles. since not all lithium ions inserted in the lattice could be extracted during the discharge process, the length of the plane decreased with cycling [27]. although the capacity of tio2 decayed relatively quicker compared with li4ti5o12, it still provided the valuable capacity to the electrode. as shown in fig. 5, the first discharge specific capacity of lto/ tio2-10 was 181.68 mah/g, while the lto/tio224 had 175.29 mah/g as initial. on the other hand, the lto/tio2-48, which short in tio2 content, have only 154.30 mah/g for its first cycle. after 100 cycle testing, the capacity for lto/tio2-10, lto/tio2-24, and lto/tio2-48 was 135.16 mah/ g, 142.65 mah/g, and 121.83 mah/g. the retentions of the first cycle were 74.7 %, 81.87 %, and 79.32 %, respectively. the rated capacity is also an essential consideration in lithium-ion batteries. to examine the effect of the different composite electrodes on rate capability, these three ti-based nanowires studied by charging/discharging at different current rates which were 0.2 c, 0.5 c, 1 c, 2 c, 5 c, 10 c, 20 c, 0.5 c, and 0.2 c. figure 6 shows lto/tio210 has the highest initial capacity, which is 184.75 mah/g. the initial capacity of lto/tio2-24 was slightly lower than the lto/tio2-10, and it was 177.35 mah/g. the lto/tio2-48 have the lowest initial capacity. however, at high current rates like 10 c or 20 c, the specific capacity of lto/tio2-10 and lto/tio2-24 seriously hindered, because when it at the high current rate, the spinel tio2 stepped to an unstable electrochemical situation and cannot be fully involved in the electrode reaction. furthermore, its crystal structure was more likely to be damaged. since the li4ti5o12 have good electrochemical stability, when the current rate raised to 10 c and 20 c, the lto/tio2-48 sample has a high specific capacity than the others. after the rate capacity testing, the lto/tio2-10, lto/tio2-24, and lto/tio2-48 have maintained 161.25, 165.25, and 152.53 mah/g separately. the retentions for each sample were 86.71 %, 92.86 % and 89.79 %, respectively. theoretically, the lto/ tio2-48 with the lowest tio2 content should have the highest retention after the rate test, but there are some other phases of lto produced during the hydrothermal reaction. these impurities are less electrochemically stable than the pure spinel li4ti5o12. as a result, the rate capacity performance of lto/tio2-48 was worse than it should be. 4. conclusions the lto/tio2 dual-phase nanowires synthesized by hydrothermal reaction. the lto/ tio2 nanowires had different morphologies due to various lithiation time. the nanowires initial thickness was around 100 nm, and the 48 h lithiated sample had a thickness of around 200 nm, and the particles almost entirely covered the wires. the xrd results showed that the longer the lithiation time, the lto levels increased. the first discharge specific capacity of lto/tio2-10, lto/tio2-24, and lto/tio2-48 was 181.68, 175.29 and 154.30 mah/g, respectively. after the rate capacity testing, the lto/tio2-10, lto/tio2-24, and lto/tio2-48 have maintained 161.25, 165.25, and 152.53 mah/g separately. the retentions for each sample were 86.71, 92.86 and 89.79 %. based on the results of electrochemical performance, increased lto content helped increase the samples cycle stability. however, at 48 h lithiation time also produced impurities, which reduced the cycle stability. author information corresponding author yillin fan he — school of materials science & engineering, university of new south wales, sydney-2052 (australia); email: hf.yillin@unsw.edu.au mailto:hf.yillin@unsw.edu.au j. multidiscip. appl. nat. sci. 60 authors dongzhi yang chu — school of materials science & engineering, university of new south wales, sydney-2052 (australia); zhensheng zhuo — school of materials science & engineering, university of new south wales, sydney-2052 (australia); references [1] k. wu, l. qian, x. sun, x. lei, n. wu, h. zhao, and y. zhang. 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(2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.80 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.80 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.80&domain=pdf&date_stamp=2021-07-05 j. multidiscip. appl. nat. sci. 70 distilled water were purchased from progo chemical distributor while methylene blue and concentrated sulfuric acid (98%) was purchased from merck in pro analytical grade. 2.2. instruments the fourier transform infrared (ftir) of the material was measured by a shimadzu ir prestige21 spectrophotometer while the morphological image of the material was recorded using a scanning electron microscope (sem) jeol jsm6510. the x-ray diffractogram of the material was obtained using shimadzu xrd s-6000. the surface analysis of the material was examined through a nitrogen adsorption-desorption method using a quantachrome nova station a apparatus. the concentration of methylene blue in the aqueous solution was measured from a standard curve with several concentration series (5, 10, 20, and 30 mg l -1 ) using a double beam shimadzu uv-vis spectrophotometer uv-1800. 2.3. methods 2.3.1. activation of kaolinite activation of kaolinite material was carried out in a similar procedure previously reported by kurniawan et al. [19]. briefly, as much as 10 g of raw kaolinite material was added into 100 ml sulfuric acid 1.0 m. the mixture was refluxed at 350 k for 6 h. after the reaction, the mixture was filtrated and the residue was washed until neutral. the residue was dried at 330 k and characterized by ftir, xrd, sem, and surface analysis. 2.3.2. adsorption of methylene blue methylene blue solution was prepared by dissolving 20 mg of methylene blue in 1 l of distilled water to obtain a concentration of 20 mg l 1 . as much as 200 mg of kaolinite material was added into 50 ml of methylene blue solution and then the mixture was stirred at 150 rpm. the concentration of methylene blue was measured using a uv-vis spectrophotometer. the adsorption kinetic study was conducted by varying the adsorption time form 50 ml of methylene blue 20 mg l -1 with 10 mg adsorbent material. meanwhile, the adsorption isotherm study was evaluated by varying the methylene blue concentration as the aqueous solution. figure 1. (a) ftir spectrum and (b) xrd pattern of activated kaolinite material. figure 2. (a) sem micrograph and (b) nitrogen adsorption-desorption isotherm plot of activated kaolinite material. j. multidiscip. appl. nat. sci. 71 3. results and discussions 3.1. activation of kaolinite acidic activation of kaolinite material was carried out to remove the impurities from its threedimensional structure [20]. the common impurities of clay materials are base metal ions that interacted with the tetrahedral silica and octahedral alumina framework through electrostatic interaction. the success of the activation process was elucidated using the ftir spectroscopy. figure 1(a) shows the ftir spectrum of the activated kaolinite material from 600-4,000 cm -1 . it was found that the o-si-o bending and si-o stretching signals at 696 and 800 cm -1 appeared as a sharp signal. the alo6 and sio4 absorption signals appeared as a broad signal at 960 -1,193 cm -1 . while the o-h and alo-h functional groups are appeared as a broad signal at 3,458 cm -1 and a sharp signal at 3,674 cm -1 , respectively. these spectral characteristics were similar to the other reported literature thus proving that the activation of kaolinite material was successfully carried out [21][22]. the xrd pattern of activated kaolinite is displayed in figure 1(b). since the kaolinite material was obtained from wediombo beach thus the several crystalline phases were found such as kaolinite (jcpds 00-005-0143), quartz (jcpds 00046-1045), halloysite (jcpds 00-029-1487), and cristobalite (jcpds 00-039-1425). the diffractogram signals at 2 = 10.06, 19.46, 26.96, 29.42, 39.82, 40.76, 42.78, 43.92, 46.20, 48.44, 55.30, 56.56, 63.48, and 75.80 correspond to the kaolinite crystal phase with hkl plane of (0 0 1), (0 2 0), (1 1 1), (-1 1 2), (0 1 3), (-1 -3 2), (-1 -2 3), (-2 2 2), (-2 1 3), (1 4 1), (3 0 0), (-1 4 3), (-2 5 1), and (-2 -6 2), respectively. the diffractogram signals at 2 = 26.96, 60.32, 68.40, and 73.78 correspond to the quartz crystal phase with (1 0 1), (2 1 1), (3 0 1), and (1 0 4) hkl planes, respectively. the diffractogram signals at 2 = 13.66, 20.06, 35.15, 54.27, and 62.64 correspond to the halloysite crystal phase with hkl plane of (0 0 1), (1 0 0), (1 1 0), (2 1 0), and (3 0 0), respectively. meanwhile, the diffractogram signals at 2 = 21.98 and 36.56 correspond to the cristobalite crystal phase with (1 figure 3. (a) visible spectra and (b) calibration curve of methylene blue standard solution. figure 4. (a) effect of adsorption time on the adsorption capability of activated kaolinite material. mass of the adsorbent = 200 mg. volume of the aqueous solution = 50 ml. [methylene blue] = 20 mg l -1 . shaking speed = 150 rpm. (b) adsorption isotherm for methylene blue using activated kaolinite material. mass of the adsorbent = 200 mg. volume of the aqueous solution = 50 ml. shaking speed = 150 rpm. adsorption time = 30 min. j. multidiscip. appl. nat. sci. 72 0 1) and (2 0 0) hkl planes, respectively. from the spectral assignment, the activated kaolinite material was mainly composed of kaolinite crystal phase as previously described [23]. the sem micrograph of activated kaolinite is shown in figure 2(a) with a scale bar of 200 mm. the morphology of activated kaolinite material shows agglomerated crystal materials in micrometer size. this morphology supported the xrd data that the activated kaolinite material composed of several crystal phases. the nitrogen adsorption-desorption isotherm plot is shown in figure 2(b). from the bet calculation, the activated kaolinite material has a surface area of 76.86 m 2 g -1 , a pore size of 8.39 nm, and a pore volume of 17.66 ml g -1 . the activated kaolinite material has a higher surface area, a bigger pore size, and a larger pore volume than the un-activated kaolinite material, i.e. 61.13 m 2 g -1 , 7.72 nm, and 0.12 ml g -1 , respectively [15]. because of that, the activated kaolinite material was further evaluated for methylene blue adsorption. 3.2. adsorption of methylene blue using activated kaolinite material before the adsorption experiment, a series of methylene blue standard solution was prepared at 5, 10, 20, and 30 mg l -1 concentration. the visible spectrum of a series of methylene blue standard solution is shown in figure 3(a) while figure 3(b) shows the calibration curve to calculate the figure 5. kinetic models of methylene blue adsorption using activated kaolinite material. mass of the adsorbent = 200 mg. volume of the aqueous solution = 50 ml. [methylene blue] = 20 mg l -1 . shaking speed = 150 rpm. j. multidiscip. appl. nat. sci. 73 concentration of methylene blue. methylene blue has two adsorption signals at 612 and 665 nm as previously reported. the calibration curve gave the equation of abs = 0.0872 [methylene blue] + 0.004 with r 2 value of 0.9944. the kinetic study of methylene blue adsorption using activated kaolinite material was evaluated and the results are shown in figures 4(a) and 5, as well as table 1. the required time to reach equilibrium was 20 min. from the kinetic models, it was found that the methylene blue adsorption followed ho and mckay model with an r 2 value of 0.9868. the theoretical qe (amount of methylene blue adsorbed per unit of activated kaolinite material) value (3.26 mg g -1 ) was near to the experimental qe value (3.15 mg g -1 ) demonstrating the validity of ho and mckay model in the present study [24]. on the other hand, the adsorption isotherm of methylene blue adsorption was studied and the results are shown in figures 4(b) and 6, as well as table 2. the qe value was reached a plateau at ce = 12 mg l -1 . from the adsorption isotherm models, it was found that the methylene blue adsorption followed the langmuir model with an r 2 value of 0.9961. according to the langmuir model, the maximum adsorption capacity (qmax) and langmuir constant (kl) of 3.40 mg g -1 and 1.36 l mg -1 , respectively. the separation constant (rl) value was 0.035 demonstrating that the adsorption of methylene blue using activated kaolinite material was favorable (0 < rl < 1) [24]. table 3 shows the qmax values of several figure 6. adsorption isotherm models of methylene blue adsorption using activated kaolinite material. mass of the adsorbent = 200 mg. volume of the aqueous solution = 50 ml. shaking speed = 150 rpm. adsorption time = 30 min. kinetic model mathematical equation r 2 lagergren log (qe-qt) = -0.0209 t – 0.0049 0.4639 ho-mckay t/qt = 0.3072 t + 1.1941 0.9868 elovich qt/t = -0.0037 (ln t)/t + 0.2271 0.9258 intraparticle diffusion qt = 0.1917 t 0.5 + 1.7474 0.7155 liquid film diffusion ln(1-qt/qe) = -0.0482 t – 1.1586 0.4639 tabel 2. isotherm adsorption model of methylene blue using activated kaolinite material. j. multidiscip. appl. nat. sci. 74 adsorbent materials. a different source of kaolinite material gave different qmax values (see table 3 lines 1-4). this phenomenon may be caused by different geochemical environment and crystal phases. the qmax value of wediombo kaolinite was lower than algerian and indian kaolinites but still higher than malaysian kaolinite. furthermore, the qmax value of wediombo kaolinite was higher than other adsorbent materials, such as fly ash, red mud, glass wool, fine ground wheat straw, activated carbons, living biomass, eggshell, chrome sludge, and magnetic nanoparticles, which was remarkable (see table 3 lines 5-15). it was reported that clay-based materials consist of tetrahedral silica and octahedral alumina layers with both bronsted and lewis acid sites. sandollah et al. reported methylene blue adsorption on activated kaolinite occurred through an ionexchange reaction [3]. meanwhile, rida et al. reported that methylene blue was adsorbed on the activated kaolinite surface through hydrogen bonding and electrostatic interactions [15]. therefore, the adsorption of methylene blue could happen through either hydrogen bonding or electrostatic interactions as shown in figure 7. such a moderate qmax value (3.40 mg g -1 ) may be caused by the agglomeration of several crystal phases as shown from the sem image in figure 2(a). 4. conclusions activation of wediombo kaolinite using acidic condition yielded a low-cost activated kaolinite material with efficient adsorption capability for methylene blue removal. the success of the activation process was proved by the ftir spectrum. the activated kaolinite contains kaolinite, quartz, halloysite, and cristobalite crystal phases as shown from the xrd data. the morphology of activated kaolinite material shows agglomerated crystal materials in micrometer size with high surface area and large pore volume. it was found that the methylene blue adsorption followed ho and mckay model with qe value of 3.15 mg g -1 . on the other hand, the adsorption isotherm of methylene blue adsorption followed the langmuir model with qmax, kl, and rl values of 3.40 mg g -1 , 1.36 l mg -1 , and 0.035, respectively. the qmax value of wediombo kaolinite was higher than other adsorbent materials demonstrating that efficient methylene blue removal from the aqueous phase has been successfully achieved. nevertheless, further modification of the kaolinite material is still required to improve its adsorption ability for a real commercial process. author information corresponding author yehezkiel steven kurniawan — ma chung research center for photosynthetic pigments, universitas ma chung, malang-65151 (indonesia); orcid.org/0000-0002-4547-239x email: yehezkiel.steven@machung.ac.id authors christyowati primi sagita — ma chung research center for photosynthetic pigments, universitas ma chung, malang-65151 isotherm adsorption model mathematical equation r 2 langmuir ce/qe = 0.2945 ce + 0.2166 0.9961 freundlich ln qe = 0.2097 ln ce + 0.5707 0.9513 temkin qe = -0.0037 ln ce + 1.9478 0.9503 dubinin-radushkevich ln qe = 2 x 10 -8 [rt ln (1 + 1/ce)] 2 + 0.2115 0.9187 tabel 2. isotherm adsorption model of methylene blue using activated kaolinite material. figure 7. plausible chemical interactions for methylene blue adsorption on the surface of activated kaolinite material. https://orcid.org/0000-0002-4547-239x mailto:yehezkiel.steven@machung.ac.id j. multidiscip. appl. nat. sci. 75 (indonesia); department of industrial chemistry, pukyong national university, busan-48513 (south korea); orcid.org/0000-0002-9626-091x limpat nulandaya — center for progressive materials-technology and innovation park, pavol jozef šafárik university, kosice-04011 (slovakia); 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https://doi.org/10.1016/j.dyepig.2003.10.008 122 journal of multidisciplinary applied natural science vol. 3 no. 2 (2023) research article the study on characteristics of heat treatment of the aa2024 aluminum alloys fatima ezzohra el garchani* and moulay rachid kabiri revised : february 28, 2023 accepted : march 2, 2023 online : march 11, 2023 abstract this study examined the behavior of aa2024 aluminum alloys, which received different heat treatments, i.e., homogenization, quenching, artificial aging, and recrystallization. the homogenization temperature is set at 495 °c for 5 hours and followed by slow cooling in the furnace. then a quenching treatment was done with cold water. artificially aged alloys were heated to 495 °c for 5 hours, followed by rapid water cooling, then heating to 140 °c for 2 hours, and followed by slow cooling in the furnace. the recrystallization treatment was also studied by heating the solution to 495 °c for 5 hours, followed by rapid cooling with cold water and heating to 310 °c for 3 hours in the furnace. in this study, we used an optical microscope and a scanning electron microscope to analyze the microstructures of the samples. x-ray fluorescence analysis was used to determine the proportion of each element on the surface of each alloy. phase and structural analyses were performed by x-ray diffraction while the mass loss was calculated after 60 hours of exposure for all samples in a climate chamber. our work revealed that the rate of mass loss could decrease with heat treatment of the alloy due to the emergence of additional phases and dispersoids. the result showed that the surface fraction of each element was changed due to heat treatment in an aggressive environment, in which the al2cu and al2cumg phases disappeared after the solution treatment as revealed from the xrd data. keywords aa2024, sem, heat treatment, weight loss, xrd, aluminum alloys 1. introduction high-strength aluminum alloys have a prominent role in the aviation industry. currently, these alloys account for approximately 80% of the weight of the empty frame of a civil transport aircraft. improvements in the alloys and the development of high strength, high toughness, and superior corrosion resistance grades have enabled them to maintain their leading position. the zn, cu and si elements were added to pure aluminum to enhance its physical qualities, however, these elements were corrosive. due to their high strength-to-weight ratio, simplicity of processing, and affordability, aluminum and its alloys are considered essential engineering materials [1]-[3]. they are widely used in the aerospace and construction sectors [4][5]. however, unlike other metal alloys, aluminum and its alloys exhibit a wide range of microstructures due to alloying components and impurities [6]. the high-strength and heat-treatable aluminum alloys of the 2xxx and 7xxx families are mainly used in heavy vehicles, buildings, and the aerospace industry. magnesium and copper are added as alloying components in the 2xxx series to improve their mechanical properties [7][8]. metal joining is an essential part of the manufacturing process for massive structures [9] [10]. the primary strengthening method for highstrength 2xxx aluminum alloys is artificial precipitation aging which is frequently used to increase the strength of the alloy. due to their exceptional resistance to stress corrosion cracking, these aluminum alloys are widely used in the automotive and aerospace industries [11]. alcoa first proposed this alloy as an "alclad" sheet with a t3 temper in 1931. al2024 is said to have better formability with slight variations in composition, such as hf-2024 created by pechiney [12]-[14]. heat treatment techniques are effective to improve the mechanical properties of 2xxx series aluminum alloy welds [15][16]. mechanical properties are frequently enhanced by solutions and aging processes. recently, heat treatment techniques have been used to improve the characteristics of 2xxx series waam aluminum alloy components. gu et al. [17] processed waam 2219 aluminum alloy using solution processing and artificial aging and discovered the immense potential of its strongest qualities. to treat the publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.166 open access received : january 16, 2023 https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.166 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.166&domain=pdf&date_stamp=2023-03-11 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 123 deposits of 2219 aluminum alloy, bai et al. [18] performed unique heat treatment, reporting that the final strength was increased. to treat waam 2024 aluminum alloy, qi et al. [19] used solution and natural aging (with varying solution treatment temperatures). the property development trend was also examined, and it was found that the properties showed an increasing trend with increasing solution processing temperatures, however, the research results are not thorough enough. thus, more organized research is needed [20]. since the heat treatment procedure considerably impacts the alloy's microstructure, mechanical properties, and corrosion resistance, it is crucial to study the heat treatment to prevent corrosion of 2024 aluminum alloy. in particular, heat treatment can increase the corrosion resistance of aluminum alloy 2024 by precipitating small particles of reinforcing phases, such as al2cumg and al2cu, which can increase the alloy's resistance to localized corrosion. in addition, the distribution and shape of intermetallic particles can be affected by the heat treatment procedure, which can substantially impact the corrosion resistance of the alloy [21]-[25]. in general, understanding how heat treatment affects corrosion resistance is essential to construct and optimizing the heat treatment procedure of 2024 aluminum alloy and improving its performance in aerospace and other industries. the study aims to test the impact of heat treatment on 2024 aluminum alloy. the microstructural results, mass loss, x-ray fluorescence and x-ray diffraction were discussed in detail. 2. materials and methods 2.1 material the studied alloy is al 2024 t3 of dimension (1 mm × 2,5cm × 2,5cm) is shown in figure 1 while the chemical composition is shown in table 1. 2.2 methods 2.2.1 heat treatment the alloy underwent various heat treatments using the nabertherm c440/450 type furnace: quenching, homogenization, artificial aging, and recrystallization. the homogenization temperature was 495 °c for 5 hours and followed by slow cooling in the furnace. for quenching, the samples were heated to the same temperature as the previous treatment and followed by cold water quenching. alloys that underwent artificial aging were heated to 495 °c for 5 hours, followed by rapid cooling with water; heating to 140 °c for 2 hours and followed by slow cooling in the furnace. the recrystallization treatment was performed at 495 °c for 5 hours, followed by rapid cooling with cold water and heating to 310 °c for 3 hours in the furnace. 2.2.2 micrography microstructure and phase observations were performed by olympus bx60 optical microscopy (om) and scanning electron microscopy (sem) model jsm-it500hr at an accelerating voltage of 10kv. the specimens were polished to 400, 600, 800, 1000 and 1200 grit, then with 6-micron diamond paste, and finally polished to 1 micron and then etched with flick's reagent (90 –100 ml water and 0.1–10 ml hydrofluoric acid). 2.2.3 x-ray fluorescence the phases of the treated samples were identified by x-ray fluorescence of type-bruker d8 advance. the specimens were polished to 400, 600, 800, 1000, and 1200 grit and then with 6-micron figure 1. aluminium alloy 2024 t3 element si fe cu mn mg cr zn ti al other elements composition% 0.5 0.5 3.8–4,9 0.3–0.9 1.2–1.8 0.1 0.25 0.15 90.7–94,7 0.05–0.15 table 1. characteristics of study sites. j. multidiscip. appl. nat. sci. 124 diamond paste. 2.2.4 x-ray diffraction analysis xrd is used to identify the crystalline phases formed after heat treatment for aluminum alloy aa2024, using the diffractometer (bruker d8 advance), equipped with the kα radiation of copper (λ = 0.15406 nm) produced at 40kv and the highscore plus (version: 3.0.0.123, developer panalytical b.v) software was used to identify the phases. 2.2.5 weight loss the mass loss was calculated by the difference between the mass of the sample before and after the climate chamber test; which is expressed by the following formula 1. mass loss = ∆m/m0= (mf-mi)/mi (1) loss of mass by immersion: the samples are polished with silicate papers, from 400 to 1000 grains, and then exposed to the climatic chamber type excal 1413-ha according to din 12880 for 60 cycles. each cycle consists of 55 minutes from – 30 °c to room temperature. after every six cycles, the samples were dried before weighing. sample arrangement: the samples must never contact each other or with any metal object or body that might react aggressively, and each sample must be arranged so that the mist can flow freely over it. 3. results and discussions 3.1. effect of temperature on the microstructure of aa2024 t3 the morphologies of aluminum alloys resulting from the heat treatment tests were observed to study the effect of temperature on grain development, which were shown in figures 2 and 3. figures (2) and (3) show that the 2024 t3 alloy (a) contains a large number of intermetallic figure 2. micrographs of aa2024t3 materials: (a) control sample, (b) quenching sample, (c) homogenization sample, (d) artificial aging sample, and (e) recrystallization sample. j. multidiscip. appl. nat. sci. 125 compound particles, which are divided into two main groups, feand mn-rich particles and mg-rich particles. another report by buchheit et al. [26] has already observed this morphology. after quenching (figures 2 and 3 (b)), we observe an increase in the number of particles and a high density of vacancies, as well as a coexistence of large and fine particles rich in cr, mn and cu, in the solid solution. the micrographic observations of the 2024 alloys after solution treatment reveals a granular structure in the presence of an important quantity of intermetallic particles. these particles are the consequence of impurities such as fe, si and mn as shown in figures 2 and 3 (c). ghosh et al. [27] observed the exposure of severe dendritic segregation and the formation of secondary eutectic phases in the form of mg (zn, cu, al). they also noticed a coarse arrangement of al2cu and al7cu2fe during casting and dissolution during homogenization. the presence of two eutectic phases of al2cumg and cu3sn was found during the six-hour homogenization and fine dispersoids [27]. these intermetallic particles are not only large (several microns), isolated and irregularly shaped particles figure 3. sem photomicrograph of aa2024 t3 alloys: (a) control sample, (b) quenching sample, (c) homogenization sample, (d) artificial aging sample, and (e) recrystallization sample. j. multidiscip. appl. nat. sci. 126 but also observed in clusters of several particles. fine particles were observed to be homogeneously distributed within the grains. whatever the homogenization treatment, some large particles already observed in the raw state remain insoluble. however, a significant increase in their size can be noticed. the eutectic phases evolve along with the microstructural and crystallographic textures. yang et al. [28] found that the average cooling rate has a significant impact on the morphology of the primary al; thus, a lower cooling rate can promote the transformation of the primary grain (al) from a dendrite or rosette to a spherical shape. for artificial aging and recrystallization, which are shown in figures 2 and 3 (d) and (e) respectively, we have unevenly distributed coherent and spherical dispersions that are quite absent in some regions. in contrast, they are heterogeneously distributed in other regions. in order to analyze the elements in the surfaces of each alloy, we performed x-ray fluorescence spectrometry and x-ray diffraction. 3.2. effect of temperature on the composition of aa2024 t3 the table 2 shows the composition of each element. table 2 shows the percentage of each element on the surface of each alloy studied. the percentages of magnesium and aluminum are modified. we notice a significant increase in the percentage of magnesium and a decrease in aluminum. this can be explained by a replacement of aluminum-rich with magnesium-rich phases due to the increase in temperature. this is consistent with the results of figures (2) and (3). table 2. xrf of heat treated 2024 aluminum alloys control homogenization quenching artificial aging recrystallization mg% 1.68 8.90 6.32 4.63 5.72 al% 92.77 85.63 88.54 90.15 88.98 cu% 4.51 4.20 4.23 4.24 4.25 si% 0.10 0.40 0.04 0.11 0.18 mn% 0.58 0.55 0.53 0.54 0.55 fe% 0.19 0.19 0.19 0.19 0.18 cr% 0.02 0 0.01 0 0.01 zn% 0.10 0.10 0.10 0.10 0.10 figure 4. xrd of heat treated 2024 aluminum alloys; (a) control sample, (b) quenching sample, (c) homogenization sample, (d) artificial aging sample, and (e)recrystallization sample. j. multidiscip. appl. nat. sci. 127 3.3 effect of heat treatment on the xrd of aa2024 t3 alloy figure 4 shows the xrd diagrams corresponding to the 2024 aluminum alloy after the heat treatments. the phase analysis of all materials shows the presence of the al phase and the cucontaining intermetallic compounds generated during the melting of the alloy (al cu, al2cu, and al2cumg). it is noticed that all the alloys present characteristic peaks of the al cu phase (monoclinic, c2/m, a = 0.9889 nm, b = 0.4105 nm, c = 0.6913 nm) [29]. the al2cu phase (tetragonal, 14/mcm, a = 0.6066 nm, c = 0.4878 nm) is present only in the recrystallization treatment, we also notice the absence of the al2cumg (s) phase (orthorhombic, cmcm, a = 0.400 nm, b = 0.923 nm, c = 0.580 nm) which suggests that they may have been dissolved during the solution heat treatment which is following the literature [30]-[33]. mu et al. [34] speculated that θ and s phases would form in the heat-treated samples; but would be very difficult to detect by xrd due to their tiny sizes and lowvolume fractions. 3.4 effect of heat treatment on the mass loss of aa2024 t3 alloy mass loss measurements were performed for each heat treatment. the samples were exposed to the climatic chamber in different cycles. each cycle consists of one hour and every six cycles mass loss measurement is performed. figure 5 shows the variation of mass loss as a function of immersion time for the different heat treatments. curve d, which represents the artificial aging treatment curve, is very different from the other curves. its mass loss is significantly large compared to the other treatments that underwent degradation of the solid-liquid interface resulting from oxidation of the alloy. therefore, the sample that underwent the artificial aging treatment has better corrosion resistance compared to samples a, b, c, and e. meyveci et al. [35] found that the wear resistance of aa 2024 aluminum alloys decreased with extreme age. samples that were aged at 220 °c showed the most loss in mass. on the other hand, the samples of aa 2024 aluminum alloy, which had the highest hardness values and were aged at 180 °c, showed the best wear resistance. the longer the aging period, the lower the rate of mass loss. this is the result of how aging works. 4. conclusions in this project, we have characterized the behavior of aa 2024 alloys and determined the effect of heat treatments on the alloys. from the sem and optical microscope observations, some microstructural textures were developed on the figure 5. mass loss of the alloy as a function of immersion time in the aa2024 t3 climatic chamber (a) control sample, (b) quenching sample, (c) homogenization sample, (d) artificial aging sample, and (e) recrystallization sample. file:///e:/in%20press/166%20el%20garchani%20et%20al/accepted%20manuscript%20166-anonymous%20manuscript%20file-920-4-15-20230228.docx#_enref_29#_enref_29 j. multidiscip. appl. nat. sci. 128 surface of aa 2024 alloys after heat treatment. the variation of each element on the surface of each alloy changes after heat treatment. furthermore, the role of heat treatment is to slow down the corrosion of the studied alloy in an aggressive environment. authors information corresponding author fatima ezzohra el garchani — laboratory of energetic, materials, and sustainable development, university moulay ismail, meknes -50500 (morocco); orcid.org/0000-0002-6655-6103 email: f.ezzohra.elgarchani@gmail.com authors moulay rachid kabiri — laboratory of energetic, materials, and sustainable development, university moulay ismail, meknes -50500 (morocco); orcid.org/0009-0009-3502-6001 author contributions f. e. e. g. validation, formal analysis, investigation, writing – original draft preparation, writing – review & editing, and project administration; m. r. k. conceptualization, methodology, visualization, and supervision. conflicts of interest the authors declare no conflict of interest. references [1] d. brough and h. jouhara. 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however, it was 1.0 in the case of the infected plot. to sum up, this study at tropical regions has presented the impacts of teak defoliator (hyblaea puera) on growth(height and diameter) and carbon accumulation on teak plantation area. keywords teak defoliator, carbon stocks, plantation forest 1. introduction the species teak (tectona grandis) is considered to be one of the most valuable timber trees in southeast asia due to its outstanding wood properties which rank third among the tropical hardwood species in plantation areas and constitute about 8 percent of the plantations worldwide [1]– [3]. this relatively fast-growing species of the family lamiaceae has an attractive natural color and is valuable for high-quality furniture and interior finishing [4]–[6]. the incidence of teak defoliator affected trees ranged from 5 to 10% across all inspected commercial fields in the world [7]. carbon sequestration and storage may be increased significantly if tree growth increases without any external disturbance and infection [8]. in the context of nepal, t. grandis was introduced in 1960 in chiliya, rupendehi followed by some block plantations in sagarnath, sarlahi, and ratuwamai by forest product development board [9]. the range and scale of teak (tectonagrandis) plantation on private land has tremendously increased in the terai region of nepal [10]. with the increase in plantation, diseases and infection from teak defoliator and root rot have also been increased [10]. from preliminary surveys conducted in plantation areas, teak defoliators i.e. hyblaea puera (lepidoptera: hyblaeidae) have greatly affected teak growth and development. so far, many studies have been conducted related to the growth pattern and distribution of tectona grandis in nepal but very few studies have been concentrated on the impacts of teak defoliator on carbon accumulation [10][11]. therefore, this research was objectively carried out to assess how teak defoliators make an impact on carbon accumulation quantitatively, and to study the intensity of infection and correlation between diameter and height growth in infected plantation areas and infection-free plantation area. 2. materials and methods 2.1. study area this study was conducted in the eastern tropical region of nepal in the letang municipality of morang district; the terai region of nepal as shown in fig. 1. the figure also depicts the research plots (both healthy and infected). in the study area, the mean annual temperature is higher than 16 °c and copyright holder: © bhandari, m. r. and bhattarai, s. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.127 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.127 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.127&domain=pdf&date_stamp=2022-07-12 j. multidiscip. appl. nat. sci. 132 figure 1. study area map on country, district and municipality map. means annual rainfall is over 1500 mm [12]. the study area consists of two patches of 2 ha (i.e., 1 ha each): a (infected) and b (healthy) of almost 800 teaks (tectona grandis) species each of which are separated by a buffer area of 300m between the patches. both the patches of teak (tectona grandis) were planted in 2013 a. d. these areas are registered as private forests by the division forest office. 2.2. methods 2.2.1. research plot establishment and design two plantation forest patches (research plots) of 1 ha each and 800 trees per plot were taken for research purposes. patch a consisted of the infected poles, whereas patch b was of healthy (noneinfected) ones. in both patches, the growth of the individual sample trees was assessed with the help of the two variables: height and dbh. these variables were considered appropriate for the study because they are easily. two plantation plots of the same locality, which were about 300 meters apart from each other's, were chosen for the research purpose. these sites were previously used as agricultural land and were converted into the private forest by the plantation of the tectona grandis species in the same year and number with prescribed methodology in the equal area of 1 ha each. furthermore, soil factors were not significantly distinct as they were under the same land-use system. additionally, physiographical factors such as slope, and elevation were also similar if not the same. hence, herbivorous (hyblaea puera) attack was the exclusively contrasting factor among the plots. with the help of gps, the boundary of the patches was delineated and the shape of the patches was not taken into consideration for the research work because the sites were taken in a readily available state and a total (1 ha) plantation area was used as a single patch. 2.2.2. sampling (selection of the sample tree) out of 800 teak species in each patch, 5% trees (40 trees) were taken as a sample tree which was selected based on a systematic method with the help of fishnet (arc gis tool). 40 random equidistance points (geo-coordinates) each were generated inside both patches a and b. then, gps was used to locate the aforementioned geo-coordinates in the field within3-meter accuracy. once the point was located, the next step was to select the sample tree for further measurement. to be biased-free, 4 nearby trees were selected in a clockwise direction from four cardinals (n,w,s, and e) with the help of a compass. after this, one sample tree was selected among the 4 trees according to the personal judgments regarding the representativeness of the individual trees in terms of health, dbh, and j. multidiscip. appl. nat. sci. 133 height. 2.2.3. measurement 2.2.3.1. growth of the individual tree the first measurement was carried out in 2019 january, when dbh (diameter at breast height) was measured at 1.3m with the help of a diameter tap, and height was measured with the help of clinometers (sunto). the measured trees were marked and numbered from 1 to 40 in each patch. the second measurement was carried out in 2021 december when the same 40 marked trees in each patch were again measured and height and diameter were measured and recorded. 2.2.3.2. intensity of pest attack visible symptoms of the pest (teak defoliator) such extensive areas of plantations being defoliated, and the ground is littered with fallen leaf skeletons. partially eaten leaves wither and fall off later were taken into the account for the evidence of the infection. analysis of the intensity of the pest attack in an individual tree was done instantly in the field by the analytical view of the observer. the intensity was categorized into three categories: 0, 1, 2, and 3. the intensity of the pest attack increases with the number from 0 to 3 [13]. 2.2.4. data analysis the obtained height and diameter data were used to obtain basal area and above-ground growing stocks of each tree from both measurements and a summation was done to obtain total above-ground stocks in 2 different periods of measurement shown in equation 1. above growing stocks (g.sabove) = basal area (b.a.) x height (h) x form factor (0.5) (1) the obtained growing stocks were used to calculate above-ground biomass (equation 2).the conversion factor adopted in this study is influenced by the contents of studies by pukkala [14], md is mean wood density i.e., 0.712. above ground biomass = above ground growing stocks x md (2) on the other hand, below-ground biomass was calculated considering 15% of the above-ground biomass [15]. total biomass (b) =above ground biomass + 15% of above-ground biomass (3) the obtained biomass was then converted into total carbon content assuming 48% of total biomass as carbon content [16]. change in carbon content in this 2-year study period was then calculated. change/addition of carbon stocks = obtained carbon stocks at 2021 measurement — obtained carbon stocks at 2019 measurement (4) similarly, total co2 sequestration in 2 different times period in two different patches was also calculated and differences in co2 sequestration in two different patches were obtained as shown in equation 5 [17]. figure 2.the growing stock of the patches. j. multidiscip. appl. nat. sci. 134 w (co2) =3.67 x w(carbon) (5) the obtained data and results were analyzed and presented using statistical tools, graphs, and charts. the fresh ginger roots used for this research (201 g) were obtained from the lafia main market, in nasarawa state. it was transported in a polyethylene bag to the department of chemistry laboratory, federal university of lafia. it was washed and rinsed severally with deionized water to remove dust and adhering soil particles. the thin outer covers were carefully peeled using stainless steel knife. the sample was washed again and sliced into bits and pounded using a ceramic mortar. the pounded sample was placed in a stainless basin, and 2 l of deionized water was added. the mixture was allowed to stand for 10 min, stirred and then filtered. the residue obtained after the juice extraction was re-soaked in 2 l of deionized water for another 10 min. this process was repeated for further three consecutive times and filtered after each soaking. the chaff obtained after this series of extraction is referred to as the ginger root waste. the waste was air-dried for 24 h and then ovendried for 72 h at 80 °c. the dried sample was ground into fine powder using ceramic mortar and pestle and sieved to less than 1 mm fine particle size with a sieve and stored in a plastic sealable bag ready for the chemical analysis. 3. results and discussions 3.1. change in growing stocks in these two years of measurements figure 3.total biomass and carbon content in two different measurement (health patch). figure 4. total biomass and carbon content in two different measurements (infected patch). the growth stocks (as shown in fig. 2) in the first year of measurement (i.e. 2019) in the healthy patches were found to be 5.69 m 3 which increased to 7.78 m 3 (i.e. 36.73 %) in the second measurement in 2021. where, in the context of the infected patch, first-year growing stocks were found at 4.09 m 3 which increased to 4.84 m 3 (i.e. 18.34 %). 3.2. change in biomass, carbon content, and co2 sequestration in the healthy patch the total biomass and carbon content in the healthy patch was found to be 4.65 tons and 2.2 tons respectively which increased to 6.35 tons and 3.04 tons in the second measurement (fig. 3). the change in carbon content in these two years of the study periods was found to be 38 %. whereas total carbon sequestration in these two years of the period was found to be 3.09 tons which shows a 38 % increase in respect to the previous measurement. 3.3. change in biomass, carbon stocks, and co2 sequestration in the infected patch as shown in fig. 4, total biomass and carbon content in the infected patches were found to be 3.33 tons and 1.6 tons respectively which increased to 3.94 tons and 1.89 tons in the second measurement. the change in carbon content in these two years of the study period was found to be 18%. whereas total carbon sequestration in these two years of the period was found to be 1.07 tons which shows an 18.23 % increase in respect to the previous measurement. 3.4. correlation between height and diameter correlations between dbh and the height of j. multidiscip. appl. nat. sci. 135 individual trees were established for both healthy and infected patches during the years 2019 and 2021 (fig. 5 to 8). a highly positive correlation was found between diameter and height in a healthy patch in both the measurement i.e., 0.88 and 0.89. whereas less positive correlation as compared to a healthy patch was found between diameter and height in an infected patch in both the measurements i.e.,0.64 and 0.69. 3.5. mean difference between the variables dbh and height data from 2019 and 2021 of the infected and healthy plots have been plotted in the scattered diagram which is illustrated in fig 9. the result from the scattered graph provides information about the difference between the two plots (healthy & infected) regarding the aforementioned variables. the trend line of the healthy plot depicts the consistent and steep rise from 2019 to 2021, whereas the trend line of the infected plot illustrates an inconsistent and slow rise. additionally, trend lines of infected plots lag behind that of healthy plots, during both years. all in all, the healthy plot showed a satisfactory result in comparison with the infected plot for the two variables: height and dbh. this increment of two variables was also evident in the mean difference of variables in two plots during the given period. the mean height increment of the healthy plot was 1.1, while it was 0.5 in the case of the infected plot. furthermore, the mean dbh increment of the healthy plot was 2.1; however, it was 1.0 in the case of the infected plot. so, the growth rate was high in the healthy plot in comparison with the infected plot. 3.6. infection intensity of teak defoliator (hyblaea puera) in the infected patch analysis of the intensity of the pest attack in an individual tree was done instantly in the field by the analytical view of the observer. among those 40 sampled trees; 5 trees have an infection intensity of 0. 16 trees with intensity 1, 11 trees with intensity 2, and 7 trees with intensity 3. trees under category figure 5. diameter and height of healthy patch in 2019. figure 6. diameter and height in the healthy patch in 2021. figure 7. diameter and height in the infected patch in 2019. figure 8. diameter and height in the infected patch in 2021. j. multidiscip. appl. nat. sci. 136 o had a 29 % increment in growth stocks in these 2 years, similarly, teak trees under categories 1, 2,3 had 21 %, 16 %, and 10 % respectively. 3.7. discussions this study has shown how infection and diseases in teak affect overall carbon accumulation in the plantation area. during these two years of the study period, we observed a 38% of increment in carbon stocks in healthy patches and only 18% in teak defoliator infected patches. the defoliation by h. puera in the infected patch was epidemic, with a decrease in growth percentage and carbon accumulation. a similar kind of observation was also found by camarero et al. [18]. several studies have evaluated the effects of insect defoliators on the plantation. wu et al. [19] has mentioned outbreak on older (6-12 years old) plantations with defoliation up to 90 % resulting in high mortality and significant diameter and height reductions. similarly, a highly positive correlation was observed in healthy patches in these two measurements between height and diameter (i.e., 0.88 and 0.89) whereas the correlation between height and diameter in infected patches was only 0.64 and 0.69. the effect and impacts of teak defoliator in teak have been seen in mean height and dbh increment in infected patches. mean height and dbh increment are observed in half of the healthy patches. a similar observation was observed by callister [20] where defoliation of teak leaves reduces the capability of producing plant food reserve which hampers the annual increment in girth and height. according to islam et al. [21], the population outbreak of h. puera and e. machaeralis has effectively suppressed the incidence of teak defoliator and leaf skeletonizer and subsequently triggered early leaf flushing in teak forests. the present finding of this research closely matches the past research, finding, and observation. 4. conclusions the growth of a healthy patch surpassed the growth of an infected one. growth was measured in terms of height, dbh, growing stock, and change in biomass. in all aspects, healthy patch showed better performance. hence, the present investigation has shown that the teaks defoliator hyblaea puera hurts carbon stocks accumulation and overall growth in plantation forests while making a comparison between healthy teak plantation forests without the impacts of teak defoliator and the infected teak plantation forest with visible symptoms of teak defoliator. author information corresponding author subodh bhattarai — personal laboratory, practical solutions, kathmandu-44600 (nepal); orcid.org/0000-0002-0769-5197 email: bhattaraisubodh888@gmail.com figure 9. dbh and height of all trees. https://orcid.org/0000-0002-0769-5197 mailto:bhattaraisubodh888@gmail.com j. multidiscip. appl. nat. sci. 137 author madan raj bhandari — research and development division, timber corporation nepal, kathmandu-56613 (nepal); orcid.org/0000-0001-5944-5694 acknowledgement this study would not have been possible without the generous direction, assistance, and collaboration of numerous persons and officials, all of whom deserve thanks. my mentor, mr. madan bahasyal (researcher at forestaction, nepal), who gave ongoing assistance and encouragement at various stages of my research endeavor deserves special mention. additionally, we would like to extend our gratitude to nepal ban nigam for supplying me with all of the necessary research instruments. mr. basanta raj shrestha, mr. sachin bhattarai, miss sanita shrestha, and all of our friends deserve eternal gratitude for their encouragement, interactions, recommendations, and unwavering support throughout the proposal writing, and data collection/analysis, and final report writing processes. also, i'd want to express my gratitude to the seminar attendees as well as those who provided feedback on my work. references [1] t. kenzo, w. himmapan, r. yoneda, n. tedsorn, t. vacharangkura, g. hitsuma, and i. noda. 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[21] w. islam, m. adnan, a. shabbir, h. naveed, y. s. abubakar, m. qasim, m. tayyab, a. noman, m. s. nisar, k. a. khan, and h. ali. 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january 21, 2022 online : january 22, 2022 abstract climate change is one of the biggest threats for the new millennium, and bangladesh is considered as "poster child" as an impact on climate change in the world. the main focus of this study is to investigate the changing pattern of climate parameters, particularly temperature, rainfall, humidity, cloud coverage, and wind speed in two coastal islands of the southeastern coast of bangladesh from 1977 to 2017. the linear regression model described that the temperature in kutubdia and sandwip islands was 0.0298 and 0.0444 times increased from 1977 to 2017. besides, rainfall patterns in kutubdia decreased by 0.4083, and sandwip islands increased by 0.875 every year from its previous counterpart. the humidity level also increased due to the rise of temperature and water availability for evaporation from irrigation. moreover, significant changes in wind speed and the cloud coverage rate in the island areas increased with the increasing value of temperature and humidity. it also means the rainfall rate increases with cloud cover in the sky. however, the study found decreasing rates of bright sunshine in the island areas. the declining rate of sunshine is very high and is a matter of great concern for the agriculture and health sectors in particular areas. therefore, the community's concept of climate parameters, association, and extremes is well apparent. identify poor land use planning as the primary anthropogenic driver of the change, and they advocated boosting the capacity of linked fields that are in danger owing to climate change. to conclude, the coastal inhabitants recommended that taking preparedness would be easier if the information transmitted through quicker and reliable sources such as news broadcasts, phone messages, or the internet. keywords climate change, climate parameters, coastal islands, coastal inhabitants, linear regression model 1. introduction climate, as opposed to weather, is the long-term (usually about 30 years) generalized pattern of weather conditions, including temperature, rainfall, humidity, wind, pressure, and cloudiness, in a particular area. however, in addition to the averages, the deviation from the averages, or seasonal oscillations, as well as the weather extremes, are essential components of climate. the seasonal oscillations and deviations are very pronounced in bangladesh because of the monsoon type of climate prevailing here. in the study, 40 years long term average of the seasonal cycle of climate parameters is used for assessing the changing pattern of climate data in the three coastal islands. forty years of data are considered long enough to calculate an average that is not affected by year-to-year variability [1]–[3]. climate change poses a significant threat for bangladesh, particularly the projected climate change effects include sea-level rise, higher temperature, enhanced monsoon precipitation, and run-off, potentially reduced dry season precipitation, and increase in cyclone intensity in the coastal region. those threats would induce severe impediments to the socio-economic development of bangladesh including coastal areas. a subjective ranking of key climate change effects for coastal bangladesh identifies cyclone and sealevel rise as being of the highest priority in terms of severity, certainty, and urgency of impact. among the 64 districts of bangladesh, 26 districts are evidence of climate displacement in bangladesh. climate displacement is a major consequence of climate change in bangladesh because due to climate change-induced natural disasters, people are losing housing and land from their origin and are forced to be displaced in new areas to survive [4]– [6]. bangladesh ranked 7 th in the global climate risk index 2021, which said it was the seventhworst hit by climate change-induced natural disasters between 2000 and 2019, where the country lost 15,000 people, damaged the economic losses worth $ 3.72 billion, and experienced 185 extreme weather events because of climate change [7]. at copyright holder: © barua, p., rahman, s. h., and molla, m. h. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.107 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.107 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.107&domain=pdf&date_stamp=2022-01-25 j. multidiscip. appl. nat. sci. 48 present, bangladesh has been called the “ground zero” of climate change. however, the impact of ‘climate change’ is reasoning an increase in the frequency and severity of these disasters adversely affecting agriculture, water and sanitation, infrastructure, and health. bangladesh is having a coastal area of 47,211 sq. km, which is 32 % of its entire land. the coast of bangladesh is approximately 710 km long and has very low-lying flat land. 62 % of the land has an elevation of fewer than three meters, and 86% have less than five meters [8]–[10]. the coastal zone of bangladesh is comprised of 19 administrative districts stretching into 147 upazilas, delineated based on the tidal fluctuations, cyclone, storm surge risk, and salinity intrusion. among the upazilas, 48 from 12 districts face the coast or lower estuary and are known as exposed coast, and the rest 99 upazilas that are behind the exposed coast is known as interior coast [11]–[13]. the climate of bangladesh is conquered by seasonal reversal of winds from the southwest during summer and from the northwest during winter, consequences for wet southwest monsoon and dry north-west monsoon respectively. the south-eastern coastal area of bangladesh is comparatively susceptible to cyclones, tidal floods, coastal erosion, heavy rainfall that varies from year to year, both in terms of intensity and duration. several studies were conducted on climatic trends and climate change impacts in the coastal region of bangladesh. analysis of global observations of surface temperature shows that there has been a warming of about 0.6 °c over the past hundred years [14]. the trend is toward a more substantial increase in minimum than in maximum daily temperatures. the reason for this difference is linked to associated increases in low cloudiness and aerosol effects as well as the enhanced greenhouse effect. changes in precipitation and other components of the hydrological cycle are determined more by changes in the weather systems and their tracks than by changes in temperature. because such weather systems are so variable in both space and time, patterns of change in precipitation are much more complicated than patterns of temperature change. precipitation has increased over land in the high latitudes of the northern hemisphere, especially during the cold season [15][16]. from the record of temperature data over the last 100 years in bangladesh, it is found that the rate of temperature increment found higher than the present which is 0.5 °c. another study shows the mean annual temperature of bangladesh has increased between 1895 and 1980 at 0.3 °c over the past two decades and from 1900 to 2017, the rate increased by nearly 0.80 °c. in general, the associated degree of the increasing trend is found in both summer and winter temperatures [17]–[19]. there was no significant trend within the annual rainfalls of bangladesh. the study on the long-term monsoon rainfall pattern at 12 stations in bangladesh found no overall trend in seasonal total figure 1. rainfall pattern. j. multidiscip. appl. nat. sci. 49 rainfall but there detected some trends in monthly rainfalls [20]. climate analysis results are also dependent on the quality of the datasets, above all on their homogeneity. this study intends to understand the trend of climate parameters such as temperature, rainfall, humidity, cloud coverage, sunshine, and wind speed over the 40 years (1977-2017) for the coastal islands of the south-eastern coast of bangladesh through regression analysis to help farmers in increasing crop yields, minimize crop failures and equip them in adapting to erratic weather patterns and natural disasters triggered by global warming and climate change. detection of trends in long-time series of hydrological data is of paramount scientific and practical significance. 2. materials and methods 2.1. selection of the study area to conduct the study, researchers selected the three coastal islands of the southeastern coast of bangladesh which included sandwip and kutubdia for analysis of climate parameters. climate susceptibility, the trend of climate parameters, displacement rates, land erosion, recurrent disaster has been considered during the selected study areas. regionally the study area includes the southeastern coastal margin of bangladesh having the complex nature of physico-chemical condition which deals with the day-to-day lifestyle of the region. sandwip island belongs to the chittagong district with an area of 762.42 km 2 . besides, kutubdia island belongs to cox’s bazar district with an area of 215.8 km 2 which is bounded by the bay of bengal. 2.2. data acquisition and analysis data of climate parameters (temperature, rainfall, humidity, wind speed, cloud coverage, bright sunshine) in kutubdia and sandwip islands were collected from the bangladesh meteorological department (bmd) from 1977 to 2017. for statistical analysis of climate parameters, the researcher used average yearly data from the time series data which were conducted on the monthly average value for the last 40 years (1977-2017). the equation of a linear regression line is given as eq.1, where y is the observation on the dependent variables is the observation on the independent variable ‘a’ is the intercept of the line on the vertical axis, and ‘b’ is the slope of the line. the estimate of intercept ‘a’ and the regression coefficient ‘b’ by the least square method: (1) i.e. (2) and (3) coefficient of determination, (4) to fit regression lines of the rainy season monthly average rainfall, humidity, temperature, figure 2. changing pattern of temperature. j. multidiscip. appl. nat. sci. 50 cloud coverage, wind speed, humidity level (dependent variables) against time (independent variable) in years were plotted. linear regression lines were then fitted to determine the trends of rainfall. the drawing of the diagrams and the fitting of the regression lines were done in microsoft excel. the correlation coefficient determines the strength of the linear relationship between two variables. it always takes a value between –1 and +1, with 1 or –1 indicating a perfect correlation (all points would lie along a straight line in this case and have a residual of zero). a correlation coefficient close to or equal to zero indicates no relationship between the variables. the correlation coefficients between rainfall and time were calculated as follows. given the pairs of values (x1, y1), (x2, y2), ……….(xn, yn), the formula for computing the correlation coefficient is given by the regression equations, and the coefficient of determination (r 2 ) have been obtained through scattering diagrams by taking two indices at a time [21]–[23]. 3. results and discussions 3.1. rainfall bangladesh meteorological department (bmd) provided the climate data for kutubdia and sandwip upazila over the period 1977-2017. the climate data comprised monthly, seasonal, and annual average maximum, minimum climate parameters over the period. the annual total rainfall of the study was observed. the pattern of the total rainfall for the period of 1977-2017 was irregular but increased significantly from 2015 to 2017. for time-series data analysis of climatic components, particularly rainfall, the three climatic hotspot area's data were intended and analyzed. the following section has been depicted the real scenarios of rainfall variability in three different geographical locations. figure 1 shows the annual mean rainfall pattern in kutubdia upazila. here the fixed linear regression model was y = -0.4083x + 243.34 and r 2 = 0.0107. where, y = year; x, outcome variable = rainfall; independent variable. here, the intercept term 243.34 means that every year the constant maximum rainfall was 243.34 in the kutubdia area. the regression co-efficient -0.4083 means that the rainfall decreased by 0.4083 mm/year. figure 1 indicates the annual mean rainfall pattern in sandwip upazila. here the fixed linear regression line was y= 0.8572x + 278.23 and r 2 = 0.0342. where, y= year; x, outcome variable = rainfall; independent variable. here, the intercept term 278.23 means that every year the constant maximum rainfall was 278.23 in the sandwip area. the regression coefficient of 0.8572 means the rainfall was increased by 0.8572 mm/year. 3.2. temperature change figure 2 indicates the annual mean temperature pattern in kutubdia upazila. here the fixed linear regression line was y = 0.0298x – 29.62 and r 2 = 0.5882. where, y= year; x, outcome variable = figure 3. changing pattern of relative humidity. j. multidiscip. appl. nat. sci. 51 temperature; independent variable. here, the intercept term 29.62 means that every year the constant maximum temperature was 29.62 in the kutubdia area. the regression co-efficient 0.0298 means that the temperature was increased by 0.0298 times every year. the annual mean temperature pattern in sandwip upazila. here the fixed linear regression line was y = 0.0444x – 58.595 and r 2 = 0.6605. where, y= year; x, outcome variable = temperature; independent variable. here, the intercept term 58.595 means that every year the constant maximum temperature was 58.595 in the sandwip area. the regression co-efficient 0.044 means that the temperature was increased by 0.044 °c per year (figure 2). thus, the changing pattern of temperature in the two study areas was positively changed. last 42 years (from 1977 to 2017) climatic years, the temperature was changing nearly (+) 1.25 °c in kutubdia upazila, (+) and 1.85 °c in sandwip upazila respectively. among the 2 study locations, sandwip upazila was increased temperature among these regions. 3.3. humidity level figure 3 illustrates the annual mean humidity pattern in kutubdia upazila. here the fixed linear regression line was y = 0.075x + 80.686 and r 2 = 0.2066. where, y= year; x, outcome variable = humidity; independent variable. here, the intercept term 80.686 means that every year the constant maximum humidity was 80.686 in the kutubdia area. the regression co-efficient 0.075 means that the humidity level increased by 0.075% per year. the annual mean humidity pattern in sandwip upazila. here the fixed linear regression line was y = -0.0027x + 83.229 and r 2 = 0.0003. where, y= year; x, outcome variable = humidity; independent variable. here, the intercept term 83.229 means that every year the constant maximum humidity was 83.229 in the sandwip area. the regression coefficient -0.0027 means that the humidity level decreased 0.0027 % per year. 3.4. wind speed figure 4 indicates kutubdia has a special and potential natural resource which is wind for electricity production. figure 7 indicates the annual mean wind speed pattern in kutubdia upazila. here the fixed linear regression line was y = 0.0643x + 1.422 and r 2 = 0.627. where, y= year; x, outcome variable = wind speed; independent variable. here, the intercept term 1.422 means that every year the constant maximum wind speed was 1.422 m/s in the kutubdia area. the regression co-efficient 0.064 means that the wind speed was increased by 0.064 m/year. figure 4 indicates the annual mean wind speed pattern in sandwip upazila. here the fixed linear regression line was y = 0.068x + 0.705 and r 2 = 0.3731. where, y= year; x, outcome variable = wind speed; independent variable. here, the intercept term 0.705 means that every year the constant maximum wind speed was 0.705 m/s in sandwip upazila. the regression co-efficient 0.068 means the wind speed was increased by 0.068 m/ year. figure 4. changing pattern of wind speed. j. multidiscip. appl. nat. sci. 52 3.5. cloud coverage figure 5 indicates the annual mean cloud coverage pattern in kutubdia upazila. here the fixed linear regression line was y = 0.021x + 3.417 and r 2 = 0.2428. where, y= year; x, outcome variable = cloud coverage; independent variable. here, the intercept term 3.417 means that every year the constant maximum cloud coverage was 3.417 okta/year in kutubdia upazila. the regression co-efficient 0.021 means the cloud coverage was increased by 0.021 okta/year. figure 5 explores the annual mean cloud coverage pattern in sandwip upazila. here the fixed linear regression line was y = 0.0203x 36.979 and r 2 = 0.2738. where, y= year; x, outcome variable = cloud coverage; independent variable. here, the intercept term 3.078 means that every year the constant maximum cloud coverage was 3.078 in sandwip upazila. the regression coefficient of 0.020 means the cloud coverage increased by 0.020 okta/year. 3.6. sunshine figure 6 indicates the mean bright sunshine pattern in kutubdia upazila. here the fixed linear regression line was y = -0.062x + 8.540 and r 2 = 0.6094. where, y= year; x, outcome variable = bright sunshine; independent variable. here, the intercept term 8.540 means that every year the constant maximum bright sunshine was 8.540 in kutubdia upazila. the regression coefficient -0.062 means the bright sunshine decrease by 0.062 every year. figure 6 shows the mean bright sunshine pattern in sandwip upazila. here the fixed linear regression model was y = -0.067x + 8.270 and r 2 = 0.6967. where, y= year; x, outcome variable = bright sunshine; independent variable. here, the intercept term 8.270 means that every year the constant maximum bright sunshine was 8.270 in sandwip upazila. the regression coefficient -0.067 means the bright sunshine decrease by 0.067 every year. the study found the increasing trend of temperature for kutubdia and sandwip (+) 0.029 °c and (+) 0.044 °c from 1977 to 2017 respectively. besides, ghosh et al. [22] mentioned that the average minimum temperature in the kutubdia area was recorded from november to february and varied generally from 6.2 °c to 13.4 °c; while the maximum temperature of 39.5°c is observed in may. thus, the correlation coefficient between year and temperature was positive for all the study areas. rainfall variability in space and time is one of the most relevant characteristics of the climate of bangladesh. this will cause a cruel combination of more extreme floods and longer periods of droughts. bangladesh has been termed as one of the most vulnerable countries in the world due to climatic change [24]. the trend of rainfall (19772017) is calculated to envisage the temporal pattern of rainfall in bangladesh. the authors found the increasing pattern of rainfall in kutubdia was 1.957 mm/year and 0.875mm/year at sandwip. the correlation coefficient between year and rainfall was positive for the study areas. during 1961-1991 figure 5. changing pattern of cloud coverage. j. multidiscip. appl. nat. sci. 53 bangladesh faced 19 droughts. major drought occurred in 1973, 1976, 1978, 1979, 1980, 1981, 1982, 1984, 1986 and 2000. it was noticeable that in 1979 rainfall was abruptly decreased and severe drought caused widespread damage to crops. the consecutive droughts of 1979 directly affected about 42% of cultivated land, 44% of the population, and reduced rice production by an estimated 2 million tons and it was one of the severest in recent times [25]. the increasing trends in annual maximum rainfalls in kutubdia and sandwip upazila are eventually the path of traverse of the south-western monsoon wind. this indicates that the intensity of heavy rainfall may have increased along the main route of the monsoon wind. relative humidity (rh) is another important climatic factor and is also responsible for the formation of any kind of unsteady condition. study on this parameter is also important because temperature and rh relate good influence on the formation of the cyclone [26]–[28]. the study found significant changes in wind speed over the study areas. during the initiation time of the study area in 1977, the wind speed of kutubdia and sandwip were 1.9 m/s and 1.45 m/s. during 2017, the average wind speed of the 2 study areas was now 5.5 m/s and 5.7 m/s in kutubdia and sandwip respectively. hoque et al., [29] assess the wind speed of sandwip for the design of a wind farm in the coastal island sandwip. they calculated the data for one year 2015 round and found that the average wind speed is 3.56 m/s at sandwip. the recent development of wind rotor aerodynamics makes it feasible to extract energy from wind speed as low as 2.0 m/s. considering the per capita demand, the electricity demand of kutubdia was 138250 mwh in 2011 and 378 mwh per day. on the other hand, the southern and eastern part of kutubdia emerges to be gifted for wind electricity generation using large turbines where wind power density at the height of 50 m or higher is found to be above 200 w/m 2 annually over the year date of occurrence nature of phenomenon maximum wind speed (km/hr) tidal surge height (ft) death people displaced 15.10.83 cyclonic storm 93 3 50 5,000 09.11.83 severe cyclonic storm 136 5 300 15,000 24.05.85 severe cyclonic storm 154 15 12000 45,000 18.12.90 cyclonic storm (crossed as a depression) 115 5-7 400 20,000 29.04.91 severe cyclonic storm with a core of hurricane wind 225 12-22 150,000 450,000 02.05.94 severe cyclonic storm with a core of hurricane wind 278 5-6 400 15,000 25.11.95 severe cyclonic storm 140 10 650 30,000 19.05.97 severe cyclonic storm with a core of hurricane wind 232 15 150 15,000 27.09.97 severe cyclonic storm with a core of hurricane wind 150 10-15 30 12,000 20.05.98 severe cyclonic storm with a core of hurricane winds 173 3 45 20,000 16.05.13 cyclonic storm (mahasen) 100 5 25 70,000 30.07.15 cyclonic storm (komen) 100 5-7 132 120,000 21.05.16 cyclonic storm (ruano) 120 7-8 25 100,000 29.05.17 cyclonic storm ( mora) 130 5-7 20 70,000 table 1. list of major cyclonic storm’s heat in south-eastern coast from 1977 to 2017. source: barua et al. [4] j. multidiscip. appl. nat. sci. 54 september 1996 to august 1997. it is found that at 30 m height, the coast side of kutubdia should be sustainable for small turbines [27]. during the study, the authors found that cyclonic storm surge responsible for enormous life and property losses for the inhabitants of the kutubdia and sandwip islands. from the literature, it is found that 31 cyclones occurrence in the bay of bengal and responsible for disruption of significant damages to the assets and human life in the study areas (table 1). from the statistics of cyclone history of 1960 to 2017, approximately, 574,000 inhabitants of the south-eastern coast of bangladesh experienced displacement from their home and living land. inhabitants of kutubdia and sandwip islands stated that 350,000 inhabitants of kutubdia and sandwip islands have been forced to displaced and migrated from their living places from 1980 to 2018 because of natural disasters such as cyclones, tidal flood, coastal erosion, and waterlogging problem (table 2). one of the researchers found that bangladesh higher rate of cloud cover from june to august and in july it is maximum. he mentioned that the cloud cover of different locations in bangladesh is not the same. geological position, humidity variation, plant density, wind speed variation, temperature changes, etc. are important parameters to form clouds and causes for cloud cover variation [28]. during the investigation, the authors found average cloud coverage in kutubdia, and sandwip in 2017 was 5.2 okta/sec and 4.7 okta/sec while the cloud coverage was 3.56 okta/sec and 3.52 okta/sec in 1977 on kutubdia and sandwip respectively. besides, the annual average cloud coverage from 1953 to 2011 in kutubdia and sandwip was 3.45 okta/sec and 3.27 okta/sec respectively [29][30]. the correlation coefficient (r) between cloud cover with temperature, humidity, and rainfall is large and positive. it means the cloud cover rate is increased with the increasing value of temperature and humidity. it also means rainfall rate increases with the increase of cloud cover in the sky. on the other hand, the author found the decreasing rate of bright district upazila reason for displacement displaced peoples destination chittagong sandwip cyclone, erosion, tidal inundation, and water logging. 150,000 chittagong and cox’s bazar cox’s bazar kutubdia cyclone, erosion, tidal inundation, sudden flood, and water logging. 200,000 cox’s bazar total 350,000 table 2. numbers of climate displaced peoples of the study areas from 1977 to 2017. source: barua et al. [6] figure 6. changing pattern of bright sunshine level. j. multidiscip. appl. nat. sci. 55 sunshine levels in kutubdia and sandwip was 0.0216 m/sec times and 0.039 m/sec per year from the 1977 to 2017 timeline. 4. conclusions the finding of the study describes the changing pattern of annual climate parameters like temperature, rainfall, humidity, bright sunshine, cloud coverage, and wind speed for kutubdia and sandwip island from 1977 to 2017. long-term year -to-year anomalies over 40 years show that the increasing pattern of kutubdia and sandwip upazila are not the same in a consecutive year. of all climate parameters, rainfall is the single most crucial variable which has been widely considered as one of the starting points towards the apprehension of climate change courses. most of the island dwellers frequently complained that they might not survive very long if government agencies would not do anything for them. for protection from coastal erosion, some households have applied hard structures in parallel with the coast to protect their house/ land which is heightening of the dike, bamboo revetment, and concrete-pole breakwater. the function of such construction is to lessen the impact of waves and storms. the individual options cannot substitute for others but rather support other options. so, from the finding of the study, it was found the somewhat increase in temperature, rainfall, humidity, cloud coverage, wind speed but decreased in the bright sunshine level over the coastal islands of the south-eastern coast of bangladesh has been observed during the last forty years. however, the data and the analysis presented in the paper are inadequate to remark about the global climate change impact on climate trends in the south-eastern islands of bangladesh. increased temperature, rainfall, and monsoon precipitation are probably responsible for the frequent tropical disease outbreak and raised sternness and incidence of hydrological different disasters. on the other hand, amplified rainfall could assist to remain the groundwater in balance and agriculture production in the coastal areas. this is anticipated that the climate trend maps and the research finding will support to outline of the planning of climate change policy perspective in bangladesh and to understand the regional climate changes to realize the broad features of the asian coupled-land-atmospheric system. author information corresponding author prabal barua — department of environmental sciences, jahangirnagar university, dhaka 1342 (bangladesh); orcid.org/0000-0003-1372-0042 email: prabalims@gmail.com authors syed hafizur rahman — department of environmental sciences, jahangirnagar university, dhaka 1342 (bangladesh); orcid.org/0000-0003-0112-9124 morshed hossan molla — department of geography and environmental studies, university of chittagong, chittagong 4331 (bangladesh); orcid.org/0000-0001-6012-3238 acknowledgement the authors express the highly acknowledged to the bangladesh meteorological department officials for providing the climate parameters statistics over the mentioned period 1977 to 2017 which help to conduct the research study successfully. references [1] s. hastenrath, a. nicklis, and l. greischar. 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(2010). “dam safety effects due to human alteration of extreme precipitation”. water resources research. 46 (3): 10.1029/2009wr007704. https://doi.org/10.3126/jhm.v7i1.5612 https://doi.org/10.3126/jhm.v7i1.5612 https://doi.org/10.2307/2310140 https://doi.org/10.3390/su9050805 https://doi.org/10.1371/journal.pone.0018581 https://doi.org/10.1007/s10584-018-2139-9 https://doi.org/10.1007/s00704-003-0001-z https://doi.org/10.1016/j.microrel.2021.114418 https://doi.org/10.1007/s00382-021-05840-7 https://doi.org/10.1016/j.jhydrol.2020.125847 https://doi.org/10.1029/2009wr007704 138 journal of multidisciplinary applied natural science vol. 3 no. 2 (2023) research article processing of biodegradable waste from ceremonial activities in bali with black soldier fly (bsf) larvae mega mutiara sari, i wayan koko suryawan*, and iva yenis septiariva received : january 31, 2023 revised : march 27, 2023 accepted : march 29, 2023 online : may 3, 2023 abstract the conducted study aimed to analyze the waste processing process from traditional ceremonies in bali by examining the composition of the waste generated. since most of the waste generated during these ceremonies is a biodegradable organic material, it is feasible to use sustainable bioconversion techniques such as black soldier fly (bsf) larvae to convert this waste into useful products. in the study, a feeding trial was conducted using a garbage blender consisting of banana fruit, rice, banana leaf, coconut leaf, and flower wastes. the bsf larvae were fed at a rate of 40 mg/larvae per day with a total of 200 larvae. the researchers measured the key processing parameters including overall degradation (od), waste reduction index (wri), and feed conversion efficiency (ecd) which resulted in values of 41.27±0.42%, 3.30±0.03 g/day, and 7.82±0.43%, respectively. the study found that the dry residue produced during the process was around 30-31% and met the minimum standards set for compost or biomass fuel. additionally, the total mass of bsf larvae produced was around 16-18% in one cycle, which is a high protein source that can be used to feed animals. keywords bsf larvae, waste conversion, waste reduction 1. introduction the composition of traditional ceremonial waste, which is dominated by organic matter, is indeed very the potential to be used as compost. the waste contains almost 79.13% of wet waste that has the potential to be processed into compost. in addition, there are also other compositions, such as plastic, cans, and paper [1]. however, efforts to reduce waste have not been extensively carried out except for waste collection and sorting activities. thus, all waste is directly transported to the landfill. on the other hand, the use of waste from religious ritual activities, which consists of leftover canangs and ceremonies, is also one of the contributors to waste in landfills or water bodies [2]. on the other hand, the island of bali has many temples and performs ritual activities regularly and generates waste from traditional ceremonial activities [3]. various methods have been used to process traditional ceremonial waste, especially components of ceremonial waste. waste left over from the ceremony can usually be converted by the bioconversion method. several previous studies have recycled ceremonial waste into compost through the vermicomposting technique [4]-[6], as well as natural coloring agents, manures, and biofuels [7]. however, this technique is difficult to apply because of the complex nature of microorganisms. one alternative developed by the researchers is the processing offered by using black soldier fly (bsf) larvae as an organic waste bioconversion agen [8,9]. bsf larvae are very suitable for a large volume reducer of organic waste due to their ability to reduce the pungent odor of waste decomposition [10]. other studies have shown that bsf fly larvae are efficient as organic waste reducers because they can convert protein and lipid-rich biomass from their food substrate [11]. there are at least three products that are produced from the empowerment of bsf larvae, namely (1) larvae or pre-pupae bsf as an alternative source of protein for animal feed, (2) liquid resulting from the larval activity which functions as liquid fertilizer and (3) residual or dry organic waste that can be used as a fertilizer [12]. previous studies have shown that bsf composting can effectively reduce the amount of waste and convert it into a valuable resource [10] [13][14]. for example, a study conducted in malaysia found that bsf larvae could effectively degrade food waste, producing compost with high nitrogen and phosphorus content [15]. another publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.175 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.175 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.175&domain=pdf&date_stamp=2023-05-03 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 139 study conducted in china demonstrated that bsf composting could effectively reduce the volume of pig manure while producing high-quality compost [16]. the use of bsf for waste degradation has several advantages over traditional composting methods [13]. firstly, the bsf larvae are highly efficient at breaking down organic material, resulting in faster composting times [17]. secondly, bsf composting can reduce odors and attract fewer pests compared to traditional composting methods [18]. finally, bsf larvae are a valuable source of high-quality protein and can be used as animal feed, reducing the reliance on soy and fish meal [19]. in this study, the aim was to investigate the potential of using bsfl to biologically convert the waste generated from traditional ceremonies in bali. the present work aimed to identify the effectiveness of bsf larvae in converting waste into valuable products, such as compost and animal feed, which can be further utilized. by conducting this study, the researchers aimed to provide a reference for waste management practices in bali and promote the use of sustainable methods for waste disposal. therefore, the results of this study can contribute to the development of more effective and sustainable waste management practices in bali. 2. materials and methods 2.1. preliminary research preliminary research was conducted to obtain supporting data needed in the study. the data are waste characteristics in the form of waste composition data, water content, calorific value, initial ph, and the value of carbohydrate, protein, and fat content of ceremonial waste. the water content of the waste was obtained from the measurement of the dry weight of the waste. the moisture content needs to be known to calculate the wet weight of larval food requirements for each type of waste. dry weight measurement was carried out by heating in an oven for 1 day at a temperature of 105 °c. measurement of the initial ph of the waste is needed to determine the effect of the decomposition of bsf larvae on the ph of the waste. it is necessary to test the carbohydrate, protein, and fat content of the initial waste to determine the effect of the decomposition of bsf larvae. 2.2. experiment set-up in this study, the waste from traditional ceremonies in bali was collected and homogenized, and then placed into plastic containers with small holes for air circulation. to prepare the substrate mixed with waste from traditional ceremonies, a mixture of organic waste such as vegetables, fruits, and rice was collected from the market and mixed with the waste generated from traditional ceremonies. the waste from traditional ceremonies included offerings made from organic materials figure 1. composition of ceremonial waste in bali used in processing with bsf larvae. parameters quality moisture content 54.2±0.4% caloric value 2413±64.1 kcal/kg ph 6.8±0.6 carbohydrate 8.19% protein 3.85% fat 0.39% ash 0.41% table 1. initial characteristics of ceremonial waste in bali in processing with bsf larvae. j. multidiscip. appl. nat. sci. 140 such as flowers, leaves, and fruits, as well as burnt incense and ceremonial leftovers. the organic waste and traditional ceremony waste were mixed in a ratio of 1:1 (based on weight) to obtain a homogenous substrate. the substrate was then placed in plastic containers with a perforated lid and left for 3 days to allow microbial activity to begin. bsf eggs were introduced into each container, and the containers were then covered with a net to prevent the escape of the larvae. the containers were placed in a laboratory at room temperature (± 28 °c) and were regularly checked and maintained for moisture content. the samples were collected at regular intervals for analysis of physical and chemical parameters, such as ph, moisture content, and nutrient content. the experiment was conducted in triplicate to ensure the consistency and accuracy of the results. thorough mixing is an important step to ensure that the sample of waste for bsfl breeding is homogenous. thorough mixing of the waste material helps to ensure that all parts of the sample have a similar composition, which is important for obtaining reliable and consistent results in replicate trials. when preparing the waste material for bsfl breeding, it is recommended to mix the waste thoroughly to ensure that it is evenly distributed. this can be done using different methods such as shaking, stirring, or blending. for solid waste materials, a blender or other grinding device can be used to create a uniform consistency. without thorough mixing, there is a risk of uneven distribution of nutrients and other components in the waste material, which can result in inconsistent growth and development of the bsfl. regular monitoring of the moisture content of the waste material is necessary to ensure that it remains within the optimal range. moisture content can be monitored using a moisture meter or by conducting simple moisture tests such as the squeeze test or visual inspection. thorough mixing also can help distribute moisture evenly throughout the material. this can be achieved using mechanical hand mixing. 2.3. bsf larva breeding bsf breeding is performed by providing a reactor where eggs grow, a reactor where larvae grow, and cages for the reproduction of adult flies. the reactor volume used is 1 l with a media height figure 2. increasing bsf larvae mass in bali used in processing with bsf larvae (t1, t2 and t3 indicated trial 1, 2, and 3 respectively). figure 3. change of ph and temperature of feed-in processing with bsf larvae (t1, t2 and t3 indicated trial 1, 2, and 3 respectively). j. multidiscip. appl. nat. sci. 141 of 5 cm. as a medium for eggs to be provided, wooden sticks are provided with holes on the sides or by using cardboard that is pasted in several layers. eggs that have been laid by mature bsf in the media are then placed in an empty reactor and marked based on the time of collection. media eggs are checked every day, to check when the eggs have hatched. bsf larvae begin to be fed when the eggs that hatch in the reactor have hatched into larvae. each reactor used is filled with larvae with the same hatching date so that the larval age of each reactor can be known. feeding the bsf larvae was adjusted to the food needs based on dry weight, which was an average of 40 mg/larva per day [20], the daily waste requirement could be calculated based on the wet weight of the waste from the ceremony. the number of larvae put into the reactor is 200 tails. so that the dry weight of the waste from the ceremony that is needed every day is 8 g. however, because the experiment will be carried out with three replications, the food portion, and the number of bsf larvae are three times that amount. 2.4. measurement of bsf larval mass growth, ph, temperature, and moisture content measurement of larval body weight was carried out every three days from the beginning to the end of the running time. at the beginning and end of the experiment, the total growth weight of the larvae was weighed (200 larvae). measurements were carried out every 3 days on 10% of the number of larvae (20 tails) only [21], as a representation of the increase in overall larval weight in one reactor. the samples left in the reactor until the last day of the experiment will be used as residual data. the residual data obtained will be used to calculate the level of waste reduction that has been carried out by bsf larvae during the experimental period [20]. during the study, it was not certain that all larvae would survive to the end, where some larvae may die before the larvae reach the prepupa stage [22]. if this happens, the dead larvae do not need to be removed from the reactor. however, when mortality reached more than 50% (within a few days of the study) the experiment was stopped, as this indicated that the food provided was not suitable for the larvae. the death of larvae during the experiment can also be caused by too high temperatures in the study site or the presence of toxic substances in the food given [20]. the results of the mass measurement can also produce data that shows the mass balance of processing waste from ceremonies with bsf. the parameters are overall degradation (od) waste reduction index (wri) and feed conversion efficiency (ecd), which are calculated using equations 1-3. overall degradation (od) = (1) waste reduction index (wri) = (2) figure 4. change of moisture content of feed in processing with bsf larvae (t1, t2 and t3 indicated trial 1, 2, and 3 respectively). note: t1, t2 and t3 indicated trial 1, 2, and 3 respectively. parameters unit t1 t2 t3 average standard deviation total dry feed offered g 248.00 248.00 248.00 248.00 ±0 dry residue remained g 144.50 145.90 146.50 145.66 ±1.04 od % 41.73 41.15 40.92 41.27 ±0.42 wri g/day 3.34 3.29 3.27 3.30 ±0.03 ecd % 7.61 7.53 8.31 7.82 ±0.43 table 2. parameters of waste processing results with bsf larvae. j. multidiscip. appl. nat. sci. 142 the efficiency of conversion of digested feed (edc) = (3) temperature, ph, and moisture content are essential parameters that can affect the growth of bsf larvae. thus, periodic measurements are needed following the measurement of the body weight of bsf larvae. this measurement is carried out with calibrated tools such as ph meters, digital thermometers, and moisture meters. 3. results and discussions the composition of the waste leftover from ceremonies in bali is dominated by organic matter, which has the potential to be used as compost [23] [24]. the results of measurements were carried out by random sampling and proportionally at one of the temples in denpasar city which produces the highest waste generation. this study only uses organic materials that are easily degraded. therefore only 5 major compositions were used as feed for bsf larvae. the waste contains as much as 79.13% wet waste that has the potential to be processed into compost (figure 1). in addition, there are also other compositions, such as plastic, cans and paper. people's behaviour in using disposable items when going to the temple, such as plastic, crackle, or paper is still very high, so the waste must be separated again if it is to be processed or recycled [25]. the data on carbohydrate, protein, and fat content obtained in this study are presented in table 1. gobbi et al. stated that the quality and quantity of food ingested by bsf larvae have an important influence on the growth and timing of larval development, survival, mortality and ovarian development of adult insects and determine the physiological and morphological development of adult bsf [26]. the content of protein, carbohydrates, and fat needed by bsf larvae have to be fulfilled in the body so that if these nutrients are not met, the body's health will be disturbed. nutrition itself functions for the growth and development of organisms. the growth of bsf begins when the eggs hatch into larvae. the results showed that more than 95% of the eggs hatched on the 3rd day after laying the eggs in the culture medium and entered the active feeding phase on the 7th day. therefore, the mass measurement in this study began on the 7th day, when the weight of each larva was different. the larval phase lasts 12-18 days. during the feeding phase, the larvae grow in length and width, so that the mass in the processing of ceremonial waste until the pupa phase continues to increase (figure 2). ceremonial waste is an excellent medium to support the growth of bsf larvae because it has the figure 5. mass composition in waste processing with bsf larvae (t1, t2 and t3 indicated trial 1, 2, and 3 respectively). j. multidiscip. appl. nat. sci. 143 right nutritional content. the temperature and ph of the media play an important role in the bioconversion process of organic waste by larvae in accelerating composting and supporting larval growth [27]-[29]. measurements of the temperature and ph of the media began when the larvae were transferred to the treatment container. the temperature of the media in the three treatments ranged from 29.1-34.1 o c, and the ph of the media was 6.9-7.7 still in the neutral ph range (figure 3). during the degradation process of organic waste by bsfl, the ph trend observed is also influenced by the activity of microorganisms. the ph initially decreases because of organic acids being produced during the early stages of degradation [30]. these microorganisms produce organic acids as a byproduct of their metabolism [31]. in the case of your study, the ph reached its minimum value (became more acidic) during days 15-17, which corresponds to the early stages of the degradation process. this suggests that the microorganisms responsible for the degradation of organic matter were active during this period and were producing organic acids, leading to a decrease in ph. as the degradation process continues and more organic matter is consumed by the bsfl, the production of organic acids slows down and the ph starts to increase. this increase in ph is due to the production of ammonia and other basic compounds because of microbial activity. these basic compounds come from the breakdown of proteins and amino acids in the waste material. this can make the environment more alkaline and lead to the ph reaching its maximum value (becoming more basic) during day 22, which corresponds to the later stages of the degradation process. it is important to monitor ph during the bsfl degradation process to ensure that the optimal conditions for microbial activity are maintained. a ph range of 7-8 is considered optimal for the bsfl gut microbiota, which will ensure efficient degradation of organic matter. the moisture content of the waste media left over from the ceremony during the decomposition process has decreased continuously from around 70% to 50% on day 28 (figure 4). in addition, in the composting process with bsf larvae, microorganisms can utilize organic matter if the organic material dissolves in water. a moisture content of 40–60% is the optimum range for microbial metabolism. if the moisture content is less than 40%, microbial activity will decrease and will be even lower at 15% moisture content. in contrast, if the moisture content is greater than 60%, the nutrients will be leached, and the air volume will decrease, as a result, microbial activity will decrease, and anaerobic fermentation will occur which causes an unpleasant odor. since the moisture content in the research process was well maintained, an unpleasant odor did not appear, and the decomposition process of organic matter was achieved. moisture content is an important factor that affects the degradation process of organic waste by microorganisms, including those present in the gut of bsf larvae. if the moisture content is too low, microbial activity can be inhibited, and if it is too high, the waste can become waterlogged and anaerobic conditions can occur, leading to the production of unpleasant odors. in our study, the moisture content was well maintained, which means that the waste material provided an optimal environment for microbial activity, including the activity of the bsf larvae gut microbiota. this led to the efficient degradation of organic matter by the bsf larvae, and the production of an unpleasant odor was prevented. maintaining an optimal parameters unit average value stdev standard ph 6.25 ±0.9 4-8 c-organic % 40.7 ±2.1 ≥12 n-organic % 2.015 ±0.2 c/n 20.32 ±2.8 10-25 water content % 17.3 ±1.4 13-20 higher heating value (hhv) kcal/kg 4163.5 ±81.3 table 3. characteristic of dry residue. j. multidiscip. appl. nat. sci. 144 moisture content also ensures that the bsfl larvae have access to water for hydration and digestion, which is important for their growth and development. additionally, an optimal moisture content helps to regulate the temperature within the waste material, preventing overheating or cooling, which can also affect microbial activity. the wri value in this study shows a similar value where the standard deviation is only ±0.03 (table 2). consumption of excessive bait can cause the value of the percentage of bait consumed to the total bait to be lower. the test results show that the ecd value in each repetition is also not significantly different. the low amount of bait consumed resulted in a decrease in the amount of bait converted into bsf larvae biomass. the low value of ecd on the growth of insect larvae is related to the quality of the available bait. the composition of the processing mass consisting of total dry feed offered, dry residue remaining, and total larval biomass from processing can be seen in figure 5. the amount of processing results show a high dry residue composition of 3031% while the total mass of bsf larvae can reach 16-18% in one cycle. both products can be reused into materials of economic value. therefore, it is necessary to further measure the quality of the residue product. the compost produced by bsf larvae has the same characteristics, namely black, coarse texture, granular and has a distinctive odor of larval. while the crude residue is part of the organic waste that has a hard texture so that it cannot be digested by the larvae during the feeding phase. larvae prefer organic waste that has a hard-to-soft texture [32]. the characteristic of dry residue is listed in table 3. the characteristic of bsf larvae mass is shown in table 4. the protein content is a macro parameter contained in the body of bsf larvae. in this study, protein can reach 43.85%. protein is a polypeptide macromolecule composed of several amino acids and connected by peptide bonds [33]. protein consists of essential amino acids and nonessential amino acids. protein functions as a building block and protective substance for the body and can also be a source of energy [34]. the energy contained in bsf larvae can reach 3303 kcal/kg. energy itself is also formed by the presence of organic carbons in the feed [35]-[37]. the results of the calculations on the carbohydrate content test that have been carried out, the percentage of nutrients can be seen in table 3, which is 5.19%. the carbohydrate content in bsf larvae is so low. the low carbohydrate content is because bsf larvae are the source of animal nutrition where the carbohydrate content is low. the amount of carbohydrates in animal nutrition is very small, which is less than 1% [38]. carbohydrates themselves are contained in many portions of cereal (wheat, rice, corn, potatoes and others) as well as in grains. while the tang fat contained can reach 24.39%. fat is a source of energy; the larvae will accumulate high-fat reserves in their bodies [39]. the standards for utilizing the residue produced during the bsfl degradation process can vary depending on the intended use. if the residue is to be used as solid fuel, the standard requirements may include a minimum heating value or higher heating value (hhv) to ensure that the fuel meets certain energy content requirements [36][37][40]. on the other hand, if the bsfl larvae are to be used as animal feed, the standard requirements may include a minimum protein content, which can vary depending on the type of animal being fed [10][41]. other nutrient requirements may also need to be met, such as minimum levels of fat, fiber, and other essential vitamins and minerals [42][43]. it's worth noting that the specific standard requirements may vary depending on the location and intended use of the residue or larvae. it's important to consult with local regulations and guidelines to ensure that the standards are met for each specific application. 4. conclusions based on the results of processing waste from the ceremony with bsf larvae with feeding consisting of banana fruit, rice, banana leaf, coconut table 4. characteristic of bsf larvae mass. parameters quality caloric value 3303±86.8 kcal/kg carbohydrate 5.19% protein 43.85% fat 24.39% ash 0.54% j. multidiscip. appl. nat. sci. 145 leaf, and flower waste which are blended into one, the overall waste degradation is 41.27±0.42%. the final composition of dry residue is quite high, namely 30-31% while the total mass of bsf larvae can reach 16-18% in one cycle. the utilization of residue can be done with compost that has met the standard or can be used as solid fuel because it has low water content and sufficient heating value. in addition, the nutritional content of bsf larvae can be used as animal feed with high protein value. author information corresponding author i wayan koko suryawan — department of environmental engineering, universitas pertamina, jakarta-12220 (indonesia); orcid.org/0000-0002-5986-0430 email: i.suryawan@universitaspertamina.ac.id authors mega mutiara sari — department of environmental engineering, universitas pertamina, jakarta-12220 (indonesia); orcid.org/0000-0003-1736-687x iva yenis septiariva — study program of civil engineering, universitas sebelas maret, surakarta-57126 (indonesia); orcid.org/0000-0002-8629-370x author contributions conceptualization, m.m.s., and i.w.k.s.; methodology, i.w.k.s.; data curation, i.w.k.s, i.y.s; writing – original draft preparation, i.w.k.s, i.y.s.; writing – review & editing, m.m.s., i.w.k.s., i.y.s .; visualization, i.w.k.s.,.; supervision, m.m.s.; project administration, x.x.; funding acquisition, m.m.s. conflicts of interest the author(s) declared no conflict of interest references [1] i. m. w. wijaya and i. k. a. putra. 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"nutrient profile of wild black soldier fly (hermetia illucens) prepupae reared on municipal dustbin’s organic waste substrate", presented at the proceedings of the national academy of sciences, india section b: biological sciences. https://doi.org/10.1016/j.wasman.2021.12.044 149 journal of multidisciplinary applied natural science vol. 3 no. 2 (2023) research article composition of stand and growth dynamics of black cardamom (amomum subulatum) in different agroforestry habitats in bhutan bhakti sharma koirala*, bhagat suberi, karma sherub, rekha chhetri, and thubten gyeltshen received : march 6, 2023 revised : april 4, 2023 accepted : april 5, 2023 online : may 4, 2023 abstract the composition and arrangement of tree species at different altitudes play a vital environmental role in the growth of black cardamom (amomum subulatum) in bhutan. the present study assesses agroforestry tree species composition and factors affecting the growth of a. subulatum in different habitat types. altitudes were sorted into three different altitudinal bands (low, mid, and high) and each altitudinal band in three different habitats (timber tree, fodder tree, and mixed tree habitat) was assessed. the sampling of 48 plots was made for each habitat and the plot size was 20×20 m. in total, 144 plots were sampled systematically within the selected altitudinal bands in three different habitats. the 56 ecologically important agroforestry shade tree species were used for growing a. subulatum. tree species preference of a. subulatum changed based on different altitudes and habitat types [f(2, 33) = 45.672, p = .001]. critical factors affecting the growth are tree canopy cover (r = –0.461) and infested a. subulatum stems (r = –0.765). the increasing number of infested stems retarded the growth and dense canopy cover or open canopy reduces the growth rate of a. subulatum. overall, growth was suitable in timber tree habitat at mid altitudes, where alnus nepalensis was the dominating species having 41.40% canopy cover with low pest and disease-infested a. subulatum stems. significantly, the lower altitudinal band and the fodder tree habitats were found to be unsuitable for growth (p < 0.05). thus, appropriate site selection, canopy cover and altitude could provide optimum growth. keywords agroforestry, altitude, amomum subulatum, composition, growth 1. introduction globally agroforestry land covers 43% of the agricultural field and more than 900 million people have been working in the agroforestry system [1] [2]. it is a dynamic system of growing crops, raring livestock, and raising trees; producing tangible benefits to rural people and the environment [3]. agroforestry has been a traditional practice continued in different agroecological conditions and agro-climatic zones of south asia [4]. more than one-third of land has been covered by pasture and agricultural land whereby 70% of asian depends on agriculture and natural resources for sustenance [5]. additionally, more than 55% of land is suitable for agroforestry [6] and 25% of south asians agriculture land is under tree cover [7]. the combination of trees, crops and animals maximizes the biological interaction under agroecological systems [8] conserving nature [9] and generating socio-economic benefits for the users [10]. still, the agricultural lands are expanded leading to biodiversity loss and deforestation [11]. around 17% of global greenhouse gas emissions through deforestation can be reduced by investing in agroforestry [12] demonstrating its potential application to mitigate global climate change [13], microclimate enhancement [14], increase socioecological sustainability [15], and food security during shifting weather patterns [16]. the enormous benefits of indigenous agroforestry tradition of collecting firewood, fodder and non-wood forest products are still followed by people of asian region but the pattern of agroforestry farming has been changed based on the agro-ecological zones according to their residence and cash crops production [17]. in bhutan, the shade-loving crop, black cardamom (amomum subulatum) was cultivated in the 1970s [18] with varieties of agroforestry tree species as it reduces soil erosion, improves nitrogen fixation [19] and most importantly it provides income for the people. the commonly grown shade trees are alnus nepalensis, schima wallichii and ficus sp. [20]. the composition of tree species makes publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.177 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.177 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.177&domain=pdf&date_stamp=2023-05-04 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 150 agroforestry a desirable habitat for the productivity of associate species [21] such as a. subulatum. however, the change in the agroforestry farming system can alter the composition and structure of tree species before it is assessed [15]. thus, the productivity of a. subulatum under different composition of these tree species in various agroforestry habitats and altitudes need to be studied. as it is unclear whether different agroforestry habitat types vary in species composition, diversity and density, and their effect on a. subulatum growth. thus, the present study was conducted to assess the species composition of agroforestry tree species and to determine the factors influencing the growth of a. subulatum. 2. materials and methods 2.1. study area the study was conducted in tsirang district (figure 1) which falls within the latitude of 27º 01’ 18.84’’ n and longitude of 90º 07’ 22.48’’ e and lies at an altitude between 300 to 4,200 m. the district has an area of 637.83 km 2 with 87.5% of the land covered with forest. the forest is dominated by broadleaf (77.64%) with very few areas under chirpiness and mixed conifer. out of 5,704.64 ha (9.03%) of agricultural land, 1,659.34 ha represents wetland (chhuzhing) and 3,143.09 ha dry land (kamzhing). the 707.95 ha of land is used as agroforestry for cultivating a. subulatum [22]. there are around 23,493 individuals residing in the district [22], of which about 8,427 individuals are engaged in agriculture farming [23]. the annual rainfall ranges from 1,000 to 3,000 mm and the average temperature varies between 7 to 26 °c and it is rarely below 5 °c or above 28 °c making a suitable habitat for a. subulatum growth [24]. the district has gentle slope terrain. summer is hot, humid and cloudy while during winter weather becomes moderately cold and dry. the figure 1. map representing the study site. figure 2. sampling plot design illustrating the plot size replicated after a certain interval. j. multidiscip. appl. nat. sci. 151 forest and agricultural land have sandy loam black soil and clay loamy soil with some red soil [23]. 2.2. sampling design and techniques the data were collected along an altitudinal gradient from 850 to 1950 m (figure 2) in three different agroforestry habitat types [25]. a crosssectional study design was used followed by a systematic sampling method where the distance between each altitude was kept at 100 m [26]. the altitudes were divided into three bands: low (850– 1150 m), mid (1250–1550 m), and high (1650– 1950 m). in each altitudinal band, three different agroforestry habitats were assessed: timber tree habitat (tt), fodder tree habitat (ft), and mix tree habitat (mt). the tt habitat means a. subulatum grew specifically with 90% tree species that are used as timber for building materials such as s. wallichii, terminalia myriocarpa, and a. nepalensis. the ft habitat means a. subulatum grew with the presence of 90% fodder species such as ficus roxburghii, ficus auriculata, and ficus nervosa that are used for livestock. the mt habitat means a. subulatum grew with fruit trees (guava, figure 3. indices comparison showing the strength of similarities between three altitudinal bands. figure 4. sorensen’s similarity (qs) shows the resemblance of tree species in three different altitudinal bands. j. multidiscip. appl. nat. sci. 152 orange, avocado) and other forest species (gmelina arborea, alnus sp. ) [27]. the habitat was sorted into three types as the a. subulatum was grown widely in these three habitats and to know the effect of different habitats and agroforestry tree species on the growth of a. subulatum. in each altitudinal band, 16 plots were made for each habitat (tt = mt = ft = 16) with a total of 144 plots sampled within three altitudinal bands (low = mid = high = 48). further, the sampling size of each plot was made 20×20 m whereby the number of trees present, percent canopy cover, diameter at breast height (dbh), and height above 2 m were recorded [28]. in the same plot, the number of a. subulatum clumps, number of pest and disease infested stem, height and diameter of 10 selected stems was recorded [29]. the instruments used in the field are gps, peg, digital vernier caliper, spherical densitometer, hypsometer, camera, stationary and measuring tape. 2.3. data analysis shannon diversity (h), margalef richness (dmg), pielou evenness (j), and sorensen similarity index (qs) were calculated to observe the similarity and difference in tree species present in the different figure 5. tree species composition based on three different life forms along the altitudinal gradient figure 6. overall life form (%) indicating the dominant group present with a. subulatum j. multidiscip. appl. nat. sci. 153 altitudinal ranges. important value index (ivi), basal area and relative dominance, relative frequency, and relative density of agroforestry tree species were calculated to know the species composition and tree species preferred by a. subulatum in different habitats and altitudes. even the clump density and stem density of a. subulatum were calculated for knowing the growth patterns in various altitudes and habitats [28][30]. the statistical test was performed in r-studio and originlab. based on the normality of data, the kruskal-wallis test (h) was performed to compare the diversity indices in three different altitudinal ranges. one-way anova was used to compare the difference in mean dominance of agroforestry tree species along the altitudinal gradient. the two-way manova (f) was used to observe the effect of habitat and altitude on dependent variables (a. subulatum height, diameter, clump and stem density, density of infested stem, tree height, basal area, and canopy cover). principal component analysis (pca) and pearson correlation were used to know the important factors affecting the growth of a. subulatum. 3. results and discussions 3.1. agroforestry species structure in total 56 agroforestry tree species from 32 families were recorded from three different a. subulatum growing habitats (tt, ft, and mt) within the altitudinal range of 850–1950 m (supplementary 1). the moraceae family had the highest species count (n = 7) followed by rosaceae and fabaceae with five species. the diversity of agroforestry tree species tends to be almost similar in all three altitudinal bands (figure 3) with no significant difference h (2,143) = 0.250, p = 0.883. similarly, there was no significant difference in the richness of agroforestry tree species grown with a. subulatum h (2,143) = 0.660, p = 0.719 as the species count in the three altitudinal bands was almost the same. moreover, the tree species were evenly distributed within the altitudinal bands but not significantly different from each altitudinal band h (2,143) = 2.395, p = 0.302. further, the qs index (figure 4) showed a. subulatum preference for shade tree species such as a. nepalensis, s. wallichii, albizia sp., and ficus sp. were found to be common in almost all the sampling plots and it could be the reason for having a similar pattern of diversity, richness, and evenness. further, shade trees are more vital for a. subulatum growth than table 1. top three ecologically important agroforestry tree species for a. subulatum in different altitudes and habitats altitude tt ivi mt ivi ft ivi low schima wallichii 99.88 citrus reticulata 61.92 ficus semicordata 71.01 alnus nepalensis 89.80 schima wallichii 56.61 ficus auriculata 56.82 albizia lebbeck 41.02 ficus semicordata 28.57 bauhinia purpurea 31.12 mid alnus nepalensis 98.94 citrus reticulata 67.30 ficus concinna 61.69 schima wallichii 87.59 schima wallichii 29.11 ficus auriculata 44.14 albizia lebbeck 36.75 alnus nepalensis 28.97 ficus semicordata 36.07 high alnus nepalensis 141.33 ficus auriculata 46.38 ficus auriculata 118.09 schima wallichii 41.59 alnus nepalensis 46.21 ficus semicordata 43.17 engelhardia spicata 15.88 ficus semicordata 36.98 ficus neriifolia 37.22 note: tt: timber tree, mt: mix tree, ft: fodder tree, ivi: important value index j. multidiscip. appl. nat. sci. 154 other tree species [31][32]. moreover, the constant maintenance of undestroyed vegetation and weeding in cardamom orchards reduces the regeneration of other tree species than shade trees, making the habitat have similar diversity and richness [33]-[35]. 3.2. stand structure based on basal area composition in a. subulatum-agroforestry habitats the agroforestry tree species present in different habitats were sorted into three life forms (figure 5). the basal area of three different life forms was found to be significantly different along the altitudinal gradient [f (2, 33) = 45.672, p = 0.001] (supplementary 2). the basal area composition of tree species is usually influenced by the altitude and habitat types [30][36][37]. timber trees were found to be the most dominating species compared to fodder and edible fruit tree species in all the altitudes except in 1250 and 1850 m where the basal area of fodder trees was more as there were fewer matured timber tree species. timber trees tend to be the preferred species for a. subulatum cultivation in all altitudes (53.75% ± 14.99) followed by fodder tree species (37.60% ± 12.26). the edible fruit trees were found to be the least preferred species for the growth of a. subulatum with a total basal area of 8.64% ± 6.04 (figure 6) showing the unsuitability characteristics of fruit tree species for a. subulatum growth. the larger basal area of timber tree species provides better canopy cover [38][39] than the other species of fodder and fruit trees making the crop prefer timber species. moreover, the tree preference of a. subulatum depends on the dominance of trees in different habitats and the availability of tree species in various altitudes [40][41]. 3.3. tree preference of a. subulatum in different altitudes and habitats based on ivi of tree species the ivi index of each species in different habitats and altitudes showed that the tree species have different ivi (supplementary 3) and in different habitats the agroforestry tree preference of a. subulatum was different. in low-altitude timber tree habitat, s. wallichii with an ivi of 99.88 was found to be an important agroforestry tree species grown along with a. subulatum. in mix tree habitat, citrus reticulate with an ivi of 61.92 was used as a shade tree for a. subulatum. in the fodder tree habitat, ficus semicordata having an ivi of 71.01 was grown along with a. subulatum. in the mid-altitude tt habitat, a. nepalensis with an ivi of 98.94 showed a close association with a. subulatum. in mt and ft habitats, a. subulatum was found to depend on c. reticulate (67.30) and f. concinna (61.69). in highaltitude tt habitat, a. nepalensis had the highest ivi 141.33 showing the importance of the species for a. subulatum productivity more than other species. in mt and ft habitats, f. auriculata with figure 7. a. subulatum growth pattern based on stem density (%) in different altitudes and habitats j. multidiscip. appl. nat. sci. 155 ivi of 46.38 and 118.09 was a greatly preferred tree species by a. subulatum over other species (table 1). 3.4. growth of a. subulatum in agroforestry habitats in 5.76 ha of the area surveyed, 26,756 clumps and 443,125 a. subulatum stem were observed. the growth of a. subulatum was found to be significantly different in agroforestry habitats and three altitudinal bands (figure 7). the variation in altitude alters the composition and dominance of agroforestry tree species in different habitats leading to a change in the growth pattern of associate crop species [40]. the clump density (42 ± 12), stem density (675 ± 306), height (305.91 cm ± 41.68), and diameter (37.62 mm ± 4.88) were found to be more in mid-altitudinal band followed by high altitude (table 2). similarly, it was stated that the best growth of black cardamom can be found at 1000–2000 m [42]. moreover, within midaltitude, the tt habitat was the most preferred environment for a. subulatum followed by the mt habitat. in the tt habitat, the average clump found per plot was 15 with a stem count of 272 per plot. even the average height of the stem stands up to 113.82 cm and has a diameter of 13.19 mm compared to other habitats within the altitude, making it the preferred habitat for a. subulatum growth. the dominance of a. nepalensis in that habitat could be an important factor in its growth (table 2). a. nepalensis is one of the most preferred shade trees as it creates the specialized bioclimatic condition for a. subulatum. it has a mechanism of fixing nitrogen that can be uptake by the associate species [42][43]. the height and basal area of a. nepalesis showed a strong association with a. subulatum growth (table 1) and a similar result was reported from tadong, sikkim [29]. table 2. black cardamom growth based on clump and stem density, and height and diameter in different altitudes and habitat types altitude tt mt ft total f p clump density/ plot low 10 ± 3 9 ± 2 9 ± 2 28 ± 7 3.7 3 0.001 mid 15 ± 4 14 ± 4 13 ± 4 42 ± 12 high 11 ± 5 11 ± 3 12 ± 6 34 ± 14 total 36 ± 12 34 ± 9 34 ± 12 stem density/ plot low 185 ± 82 175 ± 107 136 ± 40 496 ± 229 2.8 5 0.006 mid 272 ± 89 223 ± 126 180 ± 91 675 ± 306 high 194 ± 74 175 ± 86 221 ± 95 590 ± 255 total 651 ± 245 573 ± 319 537 ± 226 height (cm) low 96.66 ± 16.65 92.66 ± 14.65 87.32 ± 9.47 276.63 ± 40.77 4.4 7 0.001 mid 113.82 ± 16.05 97.33 ± 12.76 94.76 ± 12.86 305.91 ± 41.68 high 93.41 ± 18.91 88.56 ± 10.66 95.89 ± 13.01 277.86 ± 42.58 total 303.89 ± 51.62 278.55 ± 38.07 277.97 ± 35.34 diameter (mm) low 12.76 ± 1.10 12.23 ± 1.28 11.75 ± 1.33 36.74 ± 3.70 3.1 9 0.002 mid 13.19 ± 1.27 12.42 ± 1.65 12.00 ± 1.96 37.62 ± 4.88 high 11.87 ± 1.64 11.67 ± 1.48 13.54 ± 1.30 37.08 ± 4.42 total 37.83 ± 4.01 36.32 ± 4.41 37.29 ± 4.22 j. multidiscip. appl. nat. sci. 156 in higher altitudes, fodder tree habitat tends to be the preferred environment for a. subulatum. there was the presence of more clumps (12 nos.) and stems per plot (221 nos.), and the height (95.89 cm) and diameter (13.54 mm) were also higher making it a suitable habitat for a. subulatum growth than other habitats. the lower altitudinal band was found to be unsuitable for the a. subulatum growth than other altitudinal bands. the low growth performance of a. subulatum in the lower altitudinal band could be due to hot microclimatic conditions. a. subulatum prefers a moist and cool environment for its optimum growth [44][45]. lower altitudes being hot, it is not suitable for the crop [42]. however, there were some possibilities of growing a. subulatum in lower altitudes mainly in tt habitats where the growth was found to be higher than the other habitats in lower altitudes. the mt habitat was found to have a low growth rate of a. subulatum in all the altitudinal bands. it could be due to competition among the varieties of tree species and a. subulatum for nutrients and energy. different tree species have different requirements for nutrients for their growth, leading to nutrient competition with associated crops [46]. moreover, the energy fixation, net energy allocation to a. subulatum, and heat release from the habitat were found to be low in mt habitats [29][47]. 3.5. factor influencing the growth of a. subulatum the pca based on a correlation matrix showed that the growth of a. subulatum had a relationship with associate factors (figure 8). axis 1 (1.910) and axis 2 (1.446) were used for the pca as they had eigenvalue greater than 1. the tree basal area (r = – 0.039), species richness (r = –0.008) and diversity (r = –0.021) did not affect the growth of a. subulatum as the variables had an inversely weak influence on the growth of the crop. further, it depicts the crop adapting to be grown with varieties of tree species. the most influencing growth variables were tree canopy cover (r = –0.461) and infested a. subulatum stems (r = –0.765) which showed a significant (p = 0.001) inverse relationship with a. subulatum growth in different habitats and altitudes. the increase in the number of infested stems retards the growth of a. subulatum. similarly, dense canopy cover or open canopy reduces the growth rate of a. subulatum. thus, the overall growth was found to be better in mid-altitude in timber tree habitat, where the average tree species height was 11.89 m, basal area of 12.93 having 41.40% tree canopy cover with 16 stems infested per plot (supplementary 4). despite regulating temperature by increasing canopy cover at a lower altitude for a. subulatum, there was more pest and disease infestation on a. subulatum. it showed that the increase in canopy cover increases the number of pests and diseases in lower altitudes altering the growth. the dense canopy cover increases the shade favoring pests and diseases to survive hot weather feeding on shade crops [48] and a minimum of 50% filtered sunlight showed the best growth of a. subulatum [46]. despite the mt habitat having better growth it was found that the habitat had more canopy cover and infested stems that can retard the growth in the long term. the mt habitat attracts varieties of pests and diseases that can infest due to the dense canopy [49]. the moderate growth in the fodder tree habitat than in other habitats at higher altitudes was due to low canopy cover (23.94%). it showed that at higher altitudes, the a. subulatum requires less shade and more sunlight for figure 8. pca shows the possible factors affecting the growth of a. subulatum. note: the red triangle represents the variables, light gray dots are the sampling plots, and the arrow represents the strength of association between growth and variables. j. multidiscip. appl. nat. sci. 157 photosynthesis and growth. at higher altitudes, frostbite can severely retard the growth with denser canopy cover and need more sunlight [50]. the dense canopy cover reduces the intensity of sunlight declining the rate of photosynthesis, nutrient assimilation and chlorophyll content in the leaf causing stunted growth [49][50]. choosing the appropriate amount of shade, tree species, and habitat was found to be important for a. subulatum growth [44][46]. 4. conclusions the combination of tree species with black cardamom (a. subulatum) had led to the conservation of nature along with the utilization of natural resources for socio-economic benefits. the composition of agroforestry tree species in different altitudes and habitats provide significant information that needs to be considered while cultivating a. subulatum. the timber tree habitat at mid-altitude was the desirable site for the growth of a. subulatum. the crop prefers moderate shade and, the low and high altitude was unsuitable for the crop. thus, the current findings can be implemented by policymakers and extension field staff for improving the socio-economy of rural people engaged in a. subulatum cultivation for sustenance. furthermore, habitat and altitude-based genetically modified a. subulatum need to be explored to improve the livelihood of rural people growing a. subulatum in the current scenario of climate change. author information corresponding author bhakti sharma koirala — department of forest science, college of natural resources, punakha14001 (bhutan); orcid.org/0000-0002-5416-767x email: bhakti65sharma@gmail.com authors bhagat suberi — department of forest science, college of natural resources, punakha14001 (bhutan); orcid.org/0000-0001-8568-9086 karma sherub — department of forest science, college of natural resources, punakha 14001 (bhutan); orcid.org/0000-0002-9810-0223 rekha chhetri — department of agriculture, college of natural resources, punakha14001 bhutan; thubten gyeltshen — department of forest science, college of natural resources, punakha 14001 (bhutan); orcid.org/0000-0002-1809-6771 author contributions b.s.k. conceptualization, investigation, writingoriginal draft preparation, funding acquisition ; t.g. data curation, formal analysis, software; b.s. supervision, project administration; k.s. writingreview, editing; r.c. resources, methodology, formal analysis. conflicts of interest the author(s) declared no conflict of interest acknowledgement we would like to thank chandra parsad sapkota for providing all the necessary support during the data collection. tsirang district forest office for providing authorization to conduct yearlong research. this work was supported by unesco madanjeet singh, south asian foundation (saf) for the period of one year to conduct field research. references [1] kumar. 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(2023). j. multidiscip. appl. nat. sci. in press. https://doi.org/10.47352/jmans.2774-3047.158. manuscript version: accepted manuscript accepted manuscript is “the version of the article accepted for publication including all changes made as a result of the peer review process, and which may also include the addition to the article by pandawa institute of a header, an article id, a cover sheet and/or an ‘accepted manuscript’ watermark, but excluding any other editing, typesetting or other changes made by pandawa institute and/or its licensors” this accepted manuscript is © 2023 the author(s). published by pandawa institute as the version of record of this article is going to be / has been published on a gold open access basis under a cc by 4.0 international license, this accepted manuscript is available for reuse under a cc by 4.0 international license immediately. everyone is permitted to use all or part of the original content in this article, provided that they adhere to all the terms of the license https://creativecommons.org/licenses/by/4.0/. although reasonable endeavors have been taken to obtain all necessary permissions from third parties to include their copyrighted content within this article, their full citation and copyright line may not be present in this accepted manuscript version. before using any content from this article, please refer to the version of record on pandawa institute once published for full citation and copyright details, as permissions may be required. all third-party content is fully copyright protected and is not published on a gold open access basis under a cc by license, unless that is specifically stated in the figure caption in the version of record. view the article online for updates and enhancements. https://doi.org/10.47352/jmans.2774-3047.158 https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.47352/jmans.2774-3047.158 impact of feed point position on patch antenna’s return 1 loss and bandwidth for uwb applications 2 3 md. firoz ahmed1,a); md. hasnat kabir1,a*); abu zafor md. touhidul islam2,b) 4 5 1department of information and communication engineering, university of rajshahi, rajshahi-6205 6 (bangladesh) 7 2department of electrical and electronic engineering, university of rajshahi, rajshahi-6205 (bangladesh) 8 a) correspondence: hasnatkabir11@gmail.com 9 10 orcids: 11 first author : https://orcid.org/0000-0003-2721-0596 12 second author : https://orcid.org/0000-0002-8516-8587 13 third author : https://orcid.org/0000-0002-0613-016x 14 15 author contibutions 16 17 conceptualization, m.fa. and m.hk.; methodology, m.fa, and m.hk.; software, 18 m.fa., m.hk., and a.z.m.ti.; validation, m.fa., m.hk. and a.z.m.ti.; formal analysis, 19 m.fa. and m.hk.; investigation, m.fa.; resources, m.hk.; data curation, m.fa. and 20 m.hk.; writing—original draft preparation, m.fa.; writing—review and editing, m.fa., 21 m.hk. and a.z.m.ti.; visualization, m.fa. and m.hk.; supervision, m.hk. and 22 a.z.m.ti.; project administration, m.hk. and a.z.m.ti.; funding acquisition, no funding 23 acquisition. 24 25 conflict of interest 26 27 the authors declare no conflict of interest. 28 29 30 31 32 33 34 35 36 a cc ep te d m a n u sc ri pt mailto:hasnatkabir11@gmail.com https://orcid.org/0000-0003-2721-0596 https://orcid.org/0000-0002-8516-8587 https://orcid.org/0000-0002-0613-016x impact of feed point position on patch antenna’s return 1 loss and bandwidth for uwb applications 2 3 abstract. the demand for compact, lightweight, and high-performance antennas has 4 increased in recent times in the communication industry. microstrip patch antenna (mpa) 5 becomes a better choice to effectively fulfill these requirements. in this study, hybrid 6 techniques of partial ground plane, slotted patch, and defective ground structure are 7 employed in mpa design to reduce the return loss, good impedance matching, and increased 8 the bandwidth, gain, and efficiency of the antenna. this research demonstrates the impact of 9 altering the feed point position, a crucial phenomenon of antenna design, on the patch 10 antenna and determines the proper feed point location by comparing a minimum return loss 11 (s11) which achieves the highest performance for the designed antenna. high-frequency 12 structure simulator (hfss) software is used to design and simulate the patch antenna. the 13 operating frequency of the antenna is 6.85 ghz for uwb applications (3.1–10.6 ghz). a 14 fr4 epoxy substrate material with dimensions of 30 mm × 20 mm is used to design the 15 antenna. it has a dielectric constant of 4.4, a thickness of 0.8 mm and a tangent loss of 0.02. 16 multiple resonant frequencies are observed with different return losses for each feed location. 17 the analysis shows that the finest feeding point is found at the center of the patch (9, 0) with 18 a very low return loss (-28.35 db), and a high impedance bandwidth (19.7 ghz). the antenna 19 also achieved a gain of 4.46 db, a directivity of 4.6904 db, and a radiation efficiency of 20 95.90%. hence, the location of the feed point can be considered as an influential factor in the 21 antenna design. 22 23 keywords: patch antenna; return loss; bandwidth; feed point position; hfss; uwb; fr4; 24 hybrid technique 25 26 1. introduction 27 28 the development of wireless communications and information technology has made 29 multiple chances for improving the effectiveness of present signal transmission and 30 processing systems. this development is a key impetus for the creation of innovative 31 technologies and systems. wireless communication system requires an antenna, which is 32 used to transmit or receive radio waves. effective and dependable antennas such as parabolic 33 a cc ep te d m a n u sc ri pt reflectors, patch antennas, slot antennas, and folded dipole antennas are needed for the new 1 generation of wireless systems. even though each type of antenna has its own benefits and 2 drawbacks, the signal from an rf system cannot be sent or received properly without an 3 appropriate design of the antenna. in many wireless communication systems, low-profile 4 antennas are required [1]. 5 microstrip patch antenna (mpa) is one kind of low-profile antennas. it has some desired 6 characteristics such as light-weight, ease of fabrication, flexibility to adapt to curved surfaces, 7 cost-effectiveness, and compatibility with integrated circuit technology. these attractive 8 properties of mpas have increased their popularity and demand, and more study is being 9 done for better understanding to improve their performance. mpas can be created in a variety 10 of shapes like square, rectangular, circular, triangular, trapezoidal, elliptical, etc [2]. 11 microstrip patch antennas have various advantages, but they also have some drawbacks, for 12 instance, low efficiency and power, low gain and restricted bandwidth. numerous strategies 13 have been studied and developed in an attempt to overcome their bandwidth and gain 14 constraints. the feeding method or feeding point can play a significant role in considerably 15 increasing or decreasing the microstrip patch antenna’s functionality [3]. the four feed 16 mechanisms such as microstrip line, coaxial probe, aperture coupling, and proximity coupling 17 are most widely used to feed a microstrip patch antenna [4][5]. their properties as well as 18 advantages and limitations are described elsewhere [6]-[8]. when these various feeding 19 systems are used to improve impedance matching at different frequency bands, the 20 effectiveness of several characteristic factors such as radiation pattern, gain, and beam width 21 are changed. these factors must be considered whenever a new antenna application needs to 22 be designed [9]. 23 an aperture-coupled feed microstrip patch antenna was developed for the 2.4 ghz 24 frequency band [10][11]. the two feeding methods such as aperture coupled and proximity 25 coupled were used to excite the microstrip patch antenna [12]. mandal et al. [13] 26 demonstrated that in contrast to aperture-coupled patch antenna, proximity-coupled feed 27 gives considerably greater return loss. it was also studied that the coaxial feedlines provide 28 good impedance matching for designing and analyzing microstrip patch antennas. matching 29 the impedance between patch and feedline was conducted using coaxial and microstrip line 30 techniques [14]. several investigations were performed to find the best feed point locations. 31 for instance, the effect of feed location on rectangular microstrip antenna operating at tm11 32 mode was presented in paul et al [15]. some investigations were performed to find the proper 33 location for feeding the patch antenna [16]-[18]. a patch antenna made of metamaterials was 34 a cc ep te d m a n u sc ri pt demonstrated to be influenced by the feed point's position [4]. a study was done to 1 investigate how the feed point position impacts the operating frequency, return loss, and 2 bandwidth of a rectangular microstrip patch antenna and to determine the ideal feed point 3 position [19]-[21]. the effectiveness of a circular patch microstrip antenna was investigated 4 with regard to the impact of feed fluctuation [22]. 5 a t-matching network motivated by metamaterials was directly inserted inside the 6 feedline of a microstrip antenna to accomplish the maximum possible transmission of energy 7 between the front end of an rf wireless transceiver and the antenna [23]. a small, low-8 profile antenna made of metamaterial unit cells was used to demonstrate high-speed 9 effectiveness for wireless devices through the uhf-shf bands [24]. an innovative 10 composite right/left-handed (crlh) metamaterial unit cell-based tiny ultra-wideband (uwb) 11 antenna was developed for modern wireless communication applications [25]. a creative and 12 diminutive nine-element antenna array with a shared aperture structure was described in order 13 to provide substantial gain as well as excellent radiation efficiency at the millimeter-wave 5 14 g band [26]. a novel antenna array with high inter-element isolation was suggested for 5g 15 mimo communication systems operating at sub-6 ghz [27]. it employed a hybrid strategy 16 that included a flawed ground plane, matching stubs, and dot walls. a hybrid right-left-17 handed metamaterial transmission line planar antenna's bandwidth and gain were increased 18 by using a non-foster impedance matching circuit board [28]. planar antennas were created 19 with implanted slots to increase their bandwidth for reliable multiband rf communications 20 [29]. a novel drifted line loop-based planar broadband antenna was developed for mobile 21 wireless communication devices [30]. 22 however, the majority of these studies [16][17][19]-[21] focus primarily on identifying 23 the best feed point, but the key deficiencies of their designed antenna are large antenna size, 24 narrow bandwidth, and limited range of applications. therefore, a compact rectangular patch 25 antenna is developed for uwb (3.1–10.6 ghz) systems by employing hybrid methodologies 26 (partial ground plane, slotted patch, and defective ground structure) in order to get over from 27 these difficulties. this paper explains how shifting the feed point position implicates the 28 operating frequency, return loss, and impedance bandwidth of the designed antenna. it is also 29 determined the finest feed point location by minimizing the return loss (s11) for suitable 30 applications of the uwb band. 31 32 a cc ep te d m a n u sc ri pt 1 figure 1. the design of the microstrip line feed 2 3 2. materials and methods 4 5 fr4 glass epoxy is widely used due to its good strength-to-weight ratios and ability to 6 operate well under both high and low pressure. it has a 4.4 dielectric constant and a 0.02 7 tangent loss. fr4 glass epoxy is commonly employed as an electrical insulator because of its 8 low water absorption rate [31]. 9 in the design of the proposed antenna, several techniques have been applied such as partial 10 ground plane (pgp), slotted patch, and defective ground structure (dgs). the narrow band 11 characteristics of the microstrip patch antenna are converted into wide band characteristics 12 using partial ground plane methodology [32]. pgp reduces the energy stored in the substrate 13 and back lobe radiation [33]. imperfections or defects or slots on the ground plane are 14 referred to as defective ground structure (dgs) in microwave planar circuits [34]. it is used 15 to boost the bandwidth and gain of microstrip antennas, as well as to diminish cross-16 polarization, dimension, mutual coupling between nearby components and higher mode 17 harmonics [35]. dgs can be a variety of shapes such as concentric ring circles, spirals, 18 dumbbells, elliptical, u and v slots [36]. a patch having slots in the forms of a u, h, t, e, or 19 other shape is known as a slotted patch. the gain, bandwidth, and efficiency of an antenna 20 are increased; while the return loss, vswr, and antenna size are decreased using this 21 technique. the edge of the microstrip patch is directly connected to a conducting strip in this 22 form of the feed mechanism, as shown in figure 1. the benefit of this type of feeding 23 configuration is that the feed can be engraved on the same substrate to yield a planar 24 structure. the patch is wider than the conducting strip. this method is popular because it is 25 reasonably easy to design, assess, and produce [37]. 26 27 a cc ep te d m a n u sc ri pt 2.1. antenna structure. the proposed rectangular patch antenna uses a microstrip feedline 1 technique which is more consistent and less complicated than coaxial feedline, aperture 2 coupled feedline, and proximity couple feedline approaches. it is powered by a direct 3 connected microstrip feedline with a characteristic impedance of 50 ohms shown in figure 1. 4 the size of the feedline is 2 by 15 mm. fr4-epoxy substrate with dimensions of 30 by 20 5 mm is used to design the antenna. it has a thickness of 0.8 mm, a relative permittivity of 4.4, 6 and a tangent loss of 0.02. both the patch and the ground are made of copper material. the 7 patch is 14 mm × 18 mm in size. the ground plane is partially employed to increase the 8 impedance bandwidth and to reduce the return loss of the antenna. the size of the pgp is 14 9 mm in length and 20 mm in width. a rectangular slot referred to as a dgs is inserted into the 10 antenna's partial ground plane, as well as 2 different slots (triangular and rectangular) are 11 implanted into the radiating patch, as illustrated in figure 2. these modifications are made to 12 increase the impedance bandwidth of the antenna. the evaluation and optimization of this 13 antenna are performed using high-frequency structure simulator (hfss) software (v.15). 14 table 1 shows the different design parameters of the proposed antenna. 15 16 17 figure 2. proposed antenna structure 18 19 20 21 22 23 a cc ep te d m a n u sc ri pt table 1. design parameters of the proposed antenna 1 parameters values (mm) ws = wg 20.0 ls 30.0 h 0.80 wp 18.0 lp = lg 14.0 lf 15.0 wf = wg1= wp1 2.00 lg1 3.00 lp1 8.00 lp5 = lp6 = wp2= wp3 =wp4 0.50 lp2=lp3=lp4 7.00 wp5=wp6 6.50 p = q 6.00 r 8.48 2 2.2. feed point position. microstrip line feeding position is chosen because it offers the 3 best impedance match between the antenna and the feedline. the impedance matching is 4 required for the maximum power transfer. the feed point needs to be placed at that position 5 on the patch where the input impedance is 50 ohms for the operating frequency. it was done 6 by moving the feed point locations using a trial and error basis. the optimal feed point 7 location was selected by comparing the minimal negative return loss (s11) among other feed 8 point positions. the best feed point can be found along the length of the patch where the s11 9 is minimal [38]. in order to determine the optimal feed point in this design, z was set to zero 10 and only y was changed. 11 12 3. results and discussion 13 14 a rectangular patch microstrip antenna using a microstrip feeding line approach has been 15 designed and simulated using finite element method based hfss v.15 software. the substrate 16 thickness is in the x-direction, and the patch antenna is intended to be positioned in the 17 a cc ep te d m a n u sc ri pt origin's y-z plane. a microstrip feedline is considered in the feeding scheme which 1 drastically impacts on the return loss and bandwidth of the antenna. we have investigated the 2 effect of the feedline's width on the proposed antenna. the width of the feedline varies from 1 3 to 3 mm to determine the appropriate width of the proposed antenna. table 2 summarizes the 4 outcomes of the simulation. variation is observed in the antenna properties by changing the 5 width of the feedline. table 2 shows that the minimum return loss and the maximum 6 bandwidth are achieved with 2 mm width of feedline. this is the justification for choosing 2 7 mm width for the proposed antenna and the rest of the analysis has been carried out by this 8 width. 9 10 table 2. effect of feed width changes in the return loss and impedance bandwidth 11 feed width, wf (mm) return loss (s11) (db) impedance bandwidth (ghz) 1.0 -27.76 10.0 (3.67–13.67) 1.5 -27.53 10.5 (3.30–13.80) 2.0 -28.35 19.7 (3.15–22.85) 2.5 -25.83 0.84 (3.09–3.93) 3.0 -16.02 7.52 (4.59–12.11) 12 the investigation and simulation processes have been carried out for each of the y-z 13 plane feeding location points. the feed point position has been altered along the patch width 14 from the left to the right edge to achieve the best location. the simulated results are shown in 15 table 3. the ideal operating frequency can be chosen to get the lowest return loss. the 16 difference between the power that is fed into the system and the power that is reflected is 17 known as the return loss, and it is expressed in decibels (db). 18 19 a cc ep te d m a n u sc ri pt 1 figure 3. return loss vs. frequency for (7, 0), (8, 0), (9, 0) feed point position 2 3 the least s11 for the axis (9, 0) is found to be 28.35 db. the axis (9, 0) is the center of 4 the patch; thus, it can be said that the impedance is perfectly matched at the center of the 5 patch. the values below and above (9, 0) show the worst performance in the return loss and 6 bandwidth of the antenna. 7 8 9 figure 4. 2d far-field radiation pattern of the proposed antenna at feed point (9, 0) 10 11 we have carried out the study at 15 different feeding points. three observations from 12 among 15 feeding point locations are compared in the graph shown in figure 3. according to 13 our analysis, the selected feed point location has the minimum return loss (-28.35 db). it is 14 calculated using a s11 graph. the bandwidth of an antenna is defined as the frequency range 15 over which s11 is less than -10 db (-10 db is an acceptable number that represents a vswr 16 of 2). the suggested antenna has a bandwidth of 19.7 ghz (calculated using -10 db return 17 a cc ep te d m a n u sc ri pt loss) ranging from 3.15 to 22.85 ghz with a working frequency of 7.7 ghz at the feed point 1 position (9, 0). this operating frequency is slightly greater than the designed frequency, 2 which is 6.85 ghz. 3 4 table 3. implication of feed position on operating frequency, return loss, and impedance 5 bandwidth 6 7 the radiation pattern, also known as the far-field pattern, describes how the intensity of 8 electromagnetic waves radiating from an antenna or from other sources. the radiation pattern 9 varies depending on their direction. in the current work, it has been utilized to illustrate how 10 the power radiation is distributed around the antenna as a function of direction as indicated by 11 the phi angle at 6.85 ghz. the most effective method for showing radiation pattern is a three-12 dimensional graph. the surface of the patch antenna determines radiation magnitude. it can 13 also be represented using polar or angular coordinates. figure 4 depicts the 2d far-field 14 radiation pattern of the proposed antenna at the feed point position (9, 0) at phi = 0° and 90° 15 degrees. this pattern closely mimics a dipole antenna and has a maximum radiated power of 16 22.19 db at phi = 0° and 90° at 6.85 ghz, which represents a substantial benefit in ultra-17 wideband communication technology. 18 19 feed position (y, z) (mm) operating frequency (ghz) minimum return loss (s11) (db) impedance bandwidth (ghz) (7.00, 0) 5.70 -16.98 1.30 (7.25, 0) 3.20 -25.17 1.00 (7.50, 0) 6.60 -15.24 2.10 (7.75, 0) 5.60 -13.69 1.45 (8.00, 0) 6.30 -18.43 4.20 (8.25, 0) 3.40 -24.80 1.50 (8.50, 0) 7.40 -22.83 9.60 (9.00, 0) 7.70 -28.35 19.7 (9.25, 0) 7.70 -23.60 10.9 (9.50, 0) 6.30 -22.80 9.35 (9.75, 0) 6.35 -21.09 8.30 (10.00, 0) 6.55 -18.23 5.10 (10.25, 0) 6.40 -17.12 3.70 (10.50, 0) 6.55 -18.05 3.05 (10.75, 0) 6.20 -15.38 1.60 a cc ep te d m a n u sc ri pt 1 figure 5. gain (db) of the proposed antenna at feed point (9, 0) 2 3 the gain of an antenna measures the amount of energy transmitted from an isotropic 4 source in the direction of maximum radiation. the gain of the proposed antenna at feed point 5 (9, 0) is shown in figure 5. the suggested antenna attains a gain of 4.46 db at 6.85 ghz. 6 directivity is a simple factor to determine the range of energy transmission in a specific 7 direction. it is one of the factors that affect the gain of the antennas. figure 6 shows the 8 directivity of the proposed antenna at feed point position (9, 0). the antenna provides a high 9 directivity of 4.6904 db at feed point location (9, 0). 10 11 12 figure 6. 3d directivity (db) of the proposed antenna at feed point (9, 0) 13 14 the output power to input power ratio is used to determine the efficiency of any system, 15 but the radiated power to input power ratio is used to measure the antenna efficiency. the 16 a cc ep te d m a n u sc ri pt antenna functions similarly to all other components of a microwave circuit. dielectric losses 1 or mismatches are two factors that can lead to power loss. the radiation efficiency of the 2 planned antenna at feed point (9, 0) is shown in figure 7. the radiation efficiency is found to 3 be 95.90% at the optimal feed point with operating frequency of 6.85 ghz. 4 5 6 figure 7. radiation efficiency of the proposed antenna at feed point (9, 0) 7 8 table 4 compares the performance assessments of the proposed antenna with a few 9 previously developed antennas. it is clear from this table that a significant gain and a narrow 10 bandwidth were achieved using a large antenna size [17]-[21]. however, the suggested 11 antenna is smaller than the observed antennas [17]-[21] and has a wider bandwidth of 19.7 12 ghz (3.15 to 22.85 ghz), which boosts the higher data rates. 13 14 table 4. comparisons of the current work with recently published work 15 patch size (mm2) impedance bandwidth (ghz) gain (db) ref. 21.80 × 30.80 0.01 (10.00 mhz) 3.73 [17] 36.10 × 49.40 5.35 [19] 38.00 × 30.00 1.00 [20] 28.00 × 36.00 0.03 (30.00 mhz) [21] 14.00 × 18.00 19.70 (3.15–22.85) 4.46 this work : not mentioned 16 17 a cc ep te d m a n u sc ri pt 4. conclusions 1 2 the effectiveness of a rectangular patch antenna can be varied by altering the feed point 3 locations. the best outcome of the 15 feeding locations of the proposed antenna on the 4 fr4_epoxy substrate has been investigated. the findings show that the feed point (9, 0) 5 yields superior results. it implies that there is better impedance matching between the feedline 6 and patch at this point. the proposed antenna has a return loss of -28.35 db, a bandwidth of 7 19.7 ghz from 3.15 to 22.85 ghz, a gain of 4.46 db, a directivity of 4.6904 db and a 8 radiation efficiency of 95.90% at the feed point position (9, 0). the developed antenna can be 9 used for x-band, c-band, ku-band, s-band, and stm band applications in addition to other 10 wireless applications including wimax, wi-fi, wlan, radio astronomy, 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"theory, analysis and design". john wily & sons inc, new 11 york. 12 13 a cc ep te d m a n u sc ri pt https://doi.org/10.1155/2017/2018527 43 journal of multidisciplinary applied natural science vol. 3 no. 1 (2023) research article chemical regeneration of a dye-laden activated carbon: optimization via the box-behnken experimental design enebi estella jasper*, jude chinedu onwuka, and edith bolanle agbaji received : july 23, 2022 revised : october 10, 2022 accepted : october 10, 2022 online : october 22, 2022 abstract activated carbon is widely used as an adsorbent to remove numerous pollutants from water and wastewater. the cost-effectiveness of an adsorbent depends upon its ability to be reused. this study focuses on regenerating millettia thonningii seed pods' activated carbon (mac) saturated with methylene blue (mb) using acetic acid as a regenerating solvent and exploring its potential to be reused. the effects of the variables such as, the concentration of the regenerating solvent, contact time, and volume of regenerating solvent on the regeneration process were ascertained using the box-behnken experimental design, which is a sub-set of response surface methodology. the regeneration process was evaluated based on the desorption capacity of the active carbon. scanning electron microscopy (sem) and fourier transform infrared spectroscopy (ftir) was used to characterize the surface of the saturated active carbon before and after regeneration. results revealed that the concentration of the regenerating solvent had the most significant synergistic effect on the regeneration process. the optimum conditions for the maximum regeneration of the spent activated carbon within the range of the variables studied were found to be: 8m acetic acid, 100 min, and 40 ml of acetic acid. the regenerated and pristine mac when reused to adsorb fresh mb solutions (50 ml of 10mg/l mb: 0.2g adsorbent) had an adsorption capacity of 2.1912mg/g and 2.0977mg/g for mb respectively. hence, the regenerated carbon outperformed the pristine active carbon. it could therefore be explored further as a recyclable adsorbent for wastewater treatment. keywords active carbon, regeneration, desorption, reusability, experimental design 1. introduction dye laden active carbons obtained from wastewater treatment processes pose a significant threat when disposed directly into the environment due to the possibility of the dye leaching. a sustainable approach to mitigating the environmental impact of such secondary pollutant is to regenerate the spent active carbon [1]. regeneration and reuse of spent adsorbents ensures that the entire remediation process is cost-effective [2]. the regeneration process basically shifts the adsorption equilibrium from adsorption to desorption. several techniques have been used to regenerate spent active carbon, such as the thermal method [3][4], the chemical method [5], and the microwave-assisted regeneration method [6]. chemical regeneration is a simple technique in which acids, alkalis, and organic solvents with either oxidizing or solubilizing capacity are used to dissolve adsorbates and restore the adsorption ability of the active carbon. the chemical regeneration method has the main advantages of high regeneration efficiency, almost zero carbon loss, quick regeneration, and possible adsorbate recovery. it is useful, especially when adsorbates form strong bonds with the adsorbent's surface [7]. larasati et al. [8] identified chemical regeneration as a viable alternative to the most commonly used off-site thermal regeneration because it can be performed both in-situ and on-site. among other adsorbents, the chemical regeneration method has been successfully used in the regeneration of acid green 25 dye saturated waste biomass [9] and methylene blue-saturated bentonite [10]. ghasemzadeh et al. [4] used hydrofluoric acid to regenerate dye-saturated active carbon and were able to achieve significant regeneration. to regenerate spent activated carbon, nasiruddin et al. [11] used hcl and hot water. marion-peacok et al. [12] demonstrated that chemically regenerated active carbon outperformed thermally regenerated active carbon in a water cleaning system. the chemical regeneration of active carbon saturated with methylene blue (mb) dye is the basis for this research. it is cost-effective to regenerate spent active carbon for reuse during the sorptive removal of pollutants from wastewater. the regeneration of methylene blue-saturated publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2022 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.144 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.144 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.144&domain=pdf&date_stamp=2022-10-22 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 44 millettia thonningii seed pods active carbon (mbmac) is investigated in this paper using the boxbehnken experimental design (bbd), a subset of the response surface methodology (rsm). rsm is a multivariate statistical experimental approach to systematically solving problems. the primary objectives of using experimental design in analytical processes are to obtain optimal and statistically valid results with minimum effort, time, and resources [13][14]. the box-behnken experimental design ensures that the relationship between a response and the factors influencing it is understood. it also aids in determining the values of the operating factors or variables that will ensure response parameter optimization by conducting fewer experiments. it has been used to optimize a variety of analytical systems, including the production of activated carbon from agricultural residues [15]–[18], the adsorption of dyes, heavy metals, and antibiotics from aqueous solutions [19] –[21], chromatographic method optimization [22], and a variety of other physical and chemical processes. when compared to the univariate or onefactor-at-a-time (ofat) experimental approach and other types of response surface methods, the bbd is usually very efficient in terms of the number of required experiments, making it less expensive to run. it is also preferred over other rsm designs because it does not contain combinations in which all of the factors are at their highest or lowest levels simultaneously thus, avoiding experiments performed under extreme conditions that may result in unsatisfactory results. despite a large number of studies on chemical regeneration of spent activated carbon, it has been discovered from literature that little work has been done on the statistical optimization of process conditions for the chemical regeneration of dye-saturated activated carbons. the novelty of this study is the attempt to regenerate dye-saturated activated carbon using a 3factor-3-level box-behnken design (bbd). this study will use the bbd to evaluate the effects and interactions of the regenerating solvent concentration, volume of regenerating solvent, and contact time on the regeneration of methylene bluesaturated m. thonningii seed pods activated carbon, as well as to determine the optimum conditions of these variables that will result in the maximum mb desorption capacity of the spent mac. scanning electron microscopy (sem) and fourier transform infrared spectroscopy (ftir) were used to confirm the regeneration of the activated carbon. the suitability of regenerated carbon for reuse was also assessed based on its adsorption of methylene blue dye. 2. materials and methods 2.1. materials the activated carbon used in this study was prepared according to the method described by jasper et al. [23]. methylene blue dye, acetic acid and tetraoxo-sulphate (vi) acid were used as reagents. all of the reagents used were of analytical quality. all solutions used in this study were prepared with de-ionized water. 2.2. methods 2.2.1. initial adsorption experiments 4 g of fresh m. thonningii seed pod activated carbon (mac) was agitated for 180 minutes with 100 ml of 250 mg/l methylene blue (mb) dye solution at ph 7. the spent mac was filtered from the solution, washed with deionized water to remove any unadsorbed dye, and oven-dried for 1 hour at 100 °c. 2.2.2. screening of suitable regenerating solvents to find the best solvent for desorption of the table 1. levels of factors in the bbd for the regeneration of spent mac. factors codes box behnken levels [regeneration solvent](m) a 2 5 8 contact time(min) b 20 60 100 solvent volume(ml) c 10 25 40 j. multidiscip. appl. nat. sci. 45 retained dye on mac, 0.1 g of dye-saturated mac was mixed separately in three different erlenmeyer flasks with 50 ml of 0.1 m h2so4, 0.1 m ch3cooh, and deionized water . the resulting mixtures were agitated for 60 minutes before being separated and the supernatants concentration determined using uv-vis spectrophotometry (664nm for mb). equation 1 was used to compute the desorption efficiency [24]. (1) where, where cd is the concentration of dye desorbed (mg/l) and ca is the dye adsorbed amount (mg/ l). for the following experiments, the solvent with the highest dye recovery was used. 2.2.3 optimization of the regeneration of spent mtspac using the box-behnken design the bbd was used to investigate the optimization of factors that affect the regeneration of mb-saturated mac. the parameters investigated were the regenerating solvent concentration (ch3cooh), the volume of regenerating solvent, and the contact time. after screening for suitable solvents, acetic acid was chosen as a regenerating solvent because it provided the highest percent desorption. the levels of experimental parameters used for the optimization study are shown in table 1. the bbd created 15 randomized experimental runs with three replicated center points for the optimization experiments based on the equation (2). (2) where n represents the total number of experiments, n represents the number of variables or factors, and p represents the number of center points. centre points are experimental runs in which the factors are set at the medians of their high and low levels. to improve experiment precision, the table 2. percent mb desorbed by various solvents from saturated mac. solvent %mb desorbed ch3cooh 1.68 h2so4 0.27 deionized h2o 0.24 figure 1. effect of (a) contact time and acetic acid concentration on the amount of dye desorbed (b)acetic acid concentration and solvent volume and (c)contact time and solvent volume at holding values of 25 ml solvent volume, 60 min contact time and 5m acetic acid concentration j. multidiscip. appl. nat. sci. 46 center point is replicated. table 3 shows the complete design matrix generated by minitab software, version 17.0, based on the range of process variables (minitab inc., usa). in each run, different volumes of the desorbing solvent, ch3cooh, were contacted with 0.2 g of mbsaturated mac at contact times ranging from 20100 min in 150 ml stoppered erlenmeyer flasks at different initial concentrations as specified in table 3. all samples were centrifuged at 500 rpm for 5 minutes after they were removed from the flasks. the concentrations of residual mb solutions were measured using uv-vis spectrophotometry at 664 nm, and the amount of dye desorbed qe(mg/g) was calculated using equation (3). (3) where v = volume of solvent (l); m = mass of adsorbent (g) and cf = dye concentration in solvent (mg/l). the experimental data obtained was subjected to statistical treatment which included a multiple regression analysis and an analysis of variance (anova) in order to derive suitable second-order polynomial regression model equations which could express each predicted response (y) as a function of the independent preparation variables (factors) by fitting the quadratic polynomial function (eq. 4) to the data set. (4) where α0-23, are regression coefficients, α0 is a constant term; α1, α2, and α3 are linear effect terms; α11, α22 and α33 are the square effect terms and α12, α13, and α23 are cross-product or interactive effect terms. a, b, and c are the coded independent variables that represent the important factors affecting the process being carried out and y is the particular response being evaluated. the data was analyzed with the minitab 17.0 software, and the optimum conditions for regenerating the spent activated carbon, that is, the settings that could obtain the highest amount of mb desorbed, were obtained using the software's optimization functions. experiments were carried out in triplicate at the optimal settings to validate the software's predictions. 2.2.4. evaluation of the adsorption capacity of mac regenerated at optimum conditions adsorption experiments were conducted in separate erlenmeyer flasks by agitating 10 mg/l of mb with 0.2 g pristine mac and regenerated mac table 3. bbd matrix for the optimization of the regeneration of mb-saturated mtspac. run [ch3cooh] (mol/l) contact time(min) solvent volume (ml) mb desorped (mg/g) 1 5 60 25 0.0665 2 8 20 25 0.1268 3 2 20 25 0.0144 4 5 100 40 0.1575 5 5 60 25 0.0634 6 2 100 25 0.0011 7 5 20 10 0.0260 8 8 60 40 0.2415 9 2 60 40 0.0216 10 8 100 25 0.2250 11 8 60 10 0.0758 12 5 60 25 0.0674 13 5 20 40 0.0766 14 2 60 10 0.0061 15 5 100 10 0.0270 j. multidiscip. appl. nat. sci. 47 per 50 ml of dye for 120 minutes, and their adsorption capacities for mb were determined using eq. 5. (5) where co, ct (mg/l) are the liquid phase concentrations of the dye at initial, and any time t, respectively, v is the volume of solution (l) and m is the mass of the dry adsorbent (g). 2.2.5. surface and morphological characterization of the regenerated mtspac the morphology and surface functional groups of the active carbon regenerated under optimal conditions were compared to those of the mbloaded mac and the pristine mac using scanning electron microscopy (phenom pro x, netherlands) and fourier transform infrared spectroscopy (ftir -cary 630, agilent technologies). 3. results and discussions 3.1. solvent selection the regeneration of spent activated carbon assumes paramount importance considering its economic reuse during the sorptive removal of pollutants from wastewater. of the three solvents tested for their suitability in the regeneration of spent carbon, ch3cooh removed the most dye (table 2) and was thus chosen as the regenerating solvent for subsequent experiments. organic solutions with lower molecular weight than adsorbate have been reported to be effective regenerant solutions [25]. 3.2. experimental design and statistical analysis the regeneration of the spent m. thonningii seed pods' active carbon was evaluated as a function of its the methylene blue desorption capacity. the results of the desorption experiments are shown in table 3. the amount of mb desorbed per gram of spent mac ranges from 0.0011 to 0.2415 mg/g, with the lowest value obtained at factor settings of 25 ml of 2 m acetic acid for a contact time of 100 minutes and the highest value obtained at factor settings of 40 ml of 8m acetic acid for a contact time of 60 minutes. the center points are replicate measurements taken on runs 1, 5, and 12 to determine the precision of the experiments. 3.3. regression analysis and anova interpretation multiple regression analysis was performed on the experimental data (table 3) to obtain a polynomial equation that correlates the response source df adj ss adj ms f-value p-value model 7 0.080668 0.011524 198.40 <0.001 a: [acetic acid] mol/l 1 0.048969 0.048969 843.06 <0.001 b: contact time(min) 1 0.003478 0.003478 59.87 <0.001 c: solvent vol (ml) 1 0.016408 0.016408 282.48 <0.001 a 2 1 0.001469 0.001469 25.29 0.002 ab 1 0.003108 0.003108 53.51 <0.001 ac 1 0.005640 0.005640 97.10 <0.001 bc 1 0.001596 0.001596 27.48 0.001 error 7 0.000407 0.000058 lack-of-fit 5 0.000398 0.000080 18.07 0.053 pure error 2 0.000009 0.000004 total 14 0.081074 r-sq 99.50% r-sq(adj) 99.00% r-sq(pred): 96.72% s 0.0076213 table 4. anova for final response surface quadratic model for the regeneration of mb-saturated mac j. multidiscip. appl. nat. sci. 48 variable, y, that is, the amount of mb desorbed, to the independent variables (regenerating solvent concentration (ch3cooh), volume of regenerating solvent, and contact time). eq. 6 depicts the final model equation obtained after removing statistically insignificant terms. (6) an analysis of variance (anova) was used to determine the statistical significance of the model equation, which included a fisher's f-test and its associated probability, p (f), at the 95 % confidence level. the large value of f indicates that the regression equation can explain the majority of the variation in the response, while the associated pvalue estimates whether f is large enough to indicate statistical significance. the model is statistically significant if the p-value is less than 0.05. table 4 showed that the model with an fvalue of 198.40 and p< 0.0001 was statistically significant. the model had a high coefficient of determination, r 2 of 99.50, indicating that only 0.50 % of the variability in the response was not explained by the model. the adjusted r 2 value, r 2 (adj), which represents the proportion of the data that the model accounts for, was also high, at 99.00 %. the model's predictive ability was also statistically acceptable, with a high r 2 -pred value of 96.72 %. the r 2 (adj) and r 2 (pred) were in reasonable agreement, falling within about 20% of each other [25][26]. the anova results also show a lack-of-fit (lof), which was not statistically significant because the p-value was greater than 0.05. the hypothesis is that if the lof is not statistically significant, the model is adequate; if the lof is statistically significant, the model is unfit to represent the data [27].the statistical findings support the model's good predictability and adequacy within the reasonable range of the variables studied. the effect of the regenerating solvent (ch3cooh) concentration (a), contact time (b), and solvent volume (c) on the amount of mb desorbed was also investigated. according to the anova results (table 4), all linear factors (a, b, and c) had a statistically significant influence on methylene blue desorption, as indicated by p-values of < 0.001. with the highest f-value of 843.06, the concentration of the regeneration solvent, a, had the most significant effect on the amount of mb desorbed, followed by solvent volume (f=282.48) and contact time (f=59.87). the positive sign of the coefficients in the regression model eq. 6 indicates that all linear factors had a synergistic effect on the response variable, which means that as all factors increased, so did the amount of dye desorbed. 3.3.1. response surface plots 3-d response surface plots were constructed to show the effects of the regeneration variables on the response, desorption capacity of methylene blue (fig 1). response surface plots explain the nature of the relationship between two factors on the response while holding a third factor constant. figure 1a shows the response plot for contact time and acetic acid concentration. within the range of variables, the mb desorption capacity gradually increased with increasing contact time and acetic acid concentration. this finding could be attributed to an increase in the presence of h+ as a result of the acetic acid displacing dye molecules from the adsorbent pores increasing in concentration. with increased regeneration contact time, it appears that more pores were re-opened. figure 1b shows the figure 2. main effects plots for the regeneration of mb-saturated mtspac. adsorption capacity pristine mac (mg/g) adsorption capacity regenerated mac (mg/g) 2.0977 2.1912 table 5. adsorption capacity of the pristine and regenerated mac j. multidiscip. appl. nat. sci. 49 response plot with respect to acetic acid concentration and solvent volume, which shows a similar response to figure 1a. the plot demonstrates that the maximum desorption capacity was obtained at the highest solvent volume and concentration. fig 1 c shows that at a concentration of 5m, contact time had little effect on the response; however, increasing the solvent volume resulted in a significant increase in the response value. 3.3.2. main effects and interaction plots the main effects plots depicted in fig. 2 show the extent of each factor's individual effects. the main effect plot contains useful information about the relative importance of each factor parameter on the response. main effect plots compare changes in level means to determine which factors have the greatest influence on the response: when the line is parallel to the x-axis, there is no main effect. the main effect, however, appears when the line is not parallel to the x-axis. the slope of the plot represents the relative strength of the factors' effect [28]. a steeper slope indicates that the process factor has a greater impact on the response. based on this premise, the acetic acid concentration had the greatest influence on the regeneration process. the increase in mb desorption from spent carbon with increasing acetic acid concentration could be explained by the accumulation of h + in solution, which competes with the dye ions on the adsorbent's surface, resulting in the release of mb + ions from the adsorbent thus favoring the desorption process. the mean was higher than average for ch3cooh concentrations greater than 5m, times greater than 60 minutes, and volumes greater than 25 ml, with a peak value obtained at 8m ch3cooh concentration. figure 2 clearly shows that the factors influenced the amount of mb desorbed in the following order: acetic acid concentration > solvent volume > contact time. figure 3 depicts the interaction plots for the response, mb desorbed. interaction plots show how figure 3. interaction effects between (a) acetic acid concentration and contact time (b) acetic acid concentration and solvent volume (c) contact time and solvent volume for the regeneration of mbsaturated mtspac. figure 4. optimization plots for the regeneration of mb-saturated mtspac. j. multidiscip. appl. nat. sci. 50 the relationship between one factor and the response is affected by the value of a second factor. when the lines are more non-parallel, there is more interaction. it can be seen that there is an interaction between a and b; a and c and b and c due to the non-parallel lines. the variability in mb desorption is greatest when the acid concentration and solvent volume are both increased. the mb desorption is found to be less sensitive to changes in contact time and solvent volume. also significant are the interactions between a and b, a and c, and b and c (table 3). 3.3.3. optimization and validation response optimization with the minitab software was used to determine the factor settings that could achieve the maximum mb desorption capacity of the spent active carbon. the response optimization plot(fig. 4) shows that the optimum experimental conditions of 8m acetic acid, 100 min contact time, and 40 ml solvent volume with a predicted response of 0.31881mg mb per gram of activated carbon were obtained with a desirability function of 1.0. the experiments performed to validate these predictions and justify the accuracy of the results yielded an average of 0.3065mg mb per gram of activated carbon. 3.3.3. adsorption capacity of pristine and regenerated mac table 5 shows that the adsorption capacity of the mb-saturated mac regenerated under optimal conditions was 2.1912mg/g while that of the pristine mac was 2.0977mg/g. this means the regenerated mac produced at optimal settings outperformed the pristine mac. the treatment of spent activated carbon with acetic acid may have resulted in the formation of more active sites on the carbon's surface, enhancing the activated carbon's ability to adsorb greater amounts of methylene blue. 3.4. sem and ftir analysis of regenerated spent mac fig 5a depicts the surface of mac before adsorption; it is characterized by a smooth porous surface. after mb adsorption (fig 5b), the surface appears rough and corroded due to the surface pores being covered by mb dye molecules. the sem micrograph obtained on regeneration (fig 5c) shows the reappearance of several pores of different sizes when compared to the saturated mac (fig 5 b). this shows evidence of successful regeneration of the carbon when compared to that of the post adsorption micrograph (5b). this indicates that the figure 5. sem micrographs of (a) fresh mtspac (b) mb-loaded mtspac and (c) regenerated mtspac-mb at x 1000 magnification. j. multidiscip. appl. nat. sci. 51 regeneration of spent active carbon by chemical activation can create a well-developed porous structure. the ftir spectra of the pristine, mb-saturated, and regenerated mac are displayed in fig. 6a, b, and c respectively. relevant peaks attributable to special functional groups such as peaks at 3906.3 cm -1 , 3824.2 cm -1 , 3652.8 cm -1 ,3593.2 cm -1 (which could be assigned to oh-, nhand chstretching vibrations), 2325.9cm -1 (c=o stretching vibration) and 1994.1 cm -1 (aromatic c=c stretching) disappeared after adsorption, while other peaks were displaced, confirming surface interaction with mb. attenuation of peak intensity or a displacement of a peak in a spectrum is evidence of a change associated with a bond on the activated carbon [29] [30]. fewer peaks were observed in the fingerprint region of the spectrum of the regenerated mb– mac (fig 6c) compared to the spectrum of the active carbon when saturated with mb (fig 6b) indicating the decomposition of adsorbed material upon regeneration. a marked observation made upon regeneration is the reappearance in fig 6c of four peaks (3652.8 cm -1 , 2109.7 cm -1 , 1994.1 cm -1 , 1688.5 cm -1 ) that were present on the spectrum of the active carbon before adsorption, which which is indicative of regeneration of the mtspac to a certain extent. the spectrum of the regenerated mb mtspac (fig 6c) however had a very close semblance with the spectrum of the activated carbon before adsorption (fig 6a). this observation could be attributed to the long regeneration contact time which might have led to the desorption of almost all dye molecules adhered to it post adsorption, thus restoring the carbon to its nearoriginal state. figure 6. ftir spectra of (a) pristine mtspac (b) mb-loaded mtspac and (c) regenerated mbmtspac. j. multidiscip. appl. nat. sci. 52 4. conclusions the regeneration of spent millettia thonningii seed pods' active carbon has been examined and optimized with respect to dye desorption capacity. box–behnken experimental design, a subset of response surface methodology was used to locate the optimum operating variables (the concentration of the regenerating agent, acetic acid, solvent volume, and contact time) to maximizing the dye desorption capacity of the spent mac. while all the factors and their interactions significantly influenced the regeneration process, the concentration of the regenerating agent, acetic acid, had the most significant influence on the process. the optimum conditions for the operating factors for the maximum regeneration of the spent activated carbon within the range of the variables studied were found to be: 8m acetic acid, 100 min, and 40 ml of acetic acid. the maximum amount of methylene blue desorbed approached 0.3065 mg/g under these experimental conditions.. the results reveal that the regenerated mac could be reused and as such has a potential application as a recyclable adsorbent for the treatment of wastewater. its reusability should be explored further. author information corresponding author enebi estella jasper — department of applied chemistry, kaduna polytechnic, kaduna state 8000014 (nigeria); orcid.org/0000-0002-6563-822x email: enebijasper@gmail.com authors jude chinedu onwuka — department of science laboratory technology, federal university lafia, nasarawa state 960107 (nigeria); orcid.org/0000-0003-2950-8273 edith bolanle agbaji — department of chemistry, ahmadu bello university, kaduna state 8000014 (nigeria); orcid.org/0000-0002-6934-5360 author contributions e. e. j. conceptualized, designed, and performed the experiments, analyzed the results and drafted the manuscript. j. c. o. participated in designing the study, coordinating the experiments, analyzing the results and drafting the manuscript. e. b. a.participated in designing this research, analyzing the results, and drafting the manuscript. conflicts of interest the author(s) declared no conflict of interest references [1] k. y. foo and b. h. hameed. 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idrus, and muhammad malik hakim received : february 24, 2023 revised : april 1, 2023 accepted : april 4, 2023 online : may 5, 2023 abstract the bermudan option allows the contract holders to make and buy a hybrid contract between american and european options. bermudan option contract can be executed at certain times until the due of the contract. the purpose of this research is to determine the price of the bermudan option using the binomial method, and then to compare the binomial method result of n steps with the market option price. in determining stock prices at each point, there will be two branches of the binomial method: up and down branches. these branches represent the movement of stock prices in the market. the result shows the price of bermudan option is convergent at a certain value when the binomial procedure is enlarged. the comparison of the bermudan option price using a binomial method to the market price shows that the price of bermudan option is an approach to the market price in certain conditions. empirically, the price of bermudan call option is in approach to the market option price or has a minimum error when the exercise price is below the current stock price. the price of bermudan put option empirically is in approach to the market option price or having a minimum error when the exercise price is above the current stock price. keywords option, bermudan option, binomial method 1. introduction investment is a capital expenditure in certain assets to gain profit in future times. investors have many choices in investment. besides real assets investment (such as land, building, and precious metal), investors can also invest in monetary investment in the financial market (such as securities) or capital market (such as stock, obligation, foreign currency, etc.). as time continues, investment products are developing, and one of the developments is a derivative product. this product aims to minimize loss risk and increase profit opportunity in investment. there are future contracts, forward contracts, swaps, and options [1]. an option contract provides freedom to the buyer of the option contract. when the option contract is due, the option contract buyer has the freedom either to continue or to stop the contract. thus, this research will examine the challenge of the option [2]. according to the purpose of a derivative product, the option contract is a risk management instrument that protects the contract buyer against stock price movement that either results in loss or profit. investment in options is more profitable than investing only in stocks [3]. according to the schedule, options consist of two: american type and european type [4]. the american type option is an option contract that has flexible execution time, which is the beginning of the contract until the due date of the contract. the european type is an option contract with execution time only at the due date [5]. this research will examine the arrangement of call and put bermudan option price. bermudan option is a hybrid option of american and european options with a certain execution time that begins on the publication date of the options contract until the due date [6,7]. in 1979, john cox, stephen ross, and mark rubinstein created a numerical approach to calculate option prices, known as the binomial method. the binomial method is a simple and popular method commonly used to calculate option prices. the binomial method in calculating option prices at each point will issue two branches: up and down branches. these two branches represent stock movement prices in the market where there are two possibilities in every term: the increase and decrease of the prices [8]. there is no definitive solution for determining the price of bermudan option. thus, a numerical approach needs to be applied [9-11]. other several methods for determining the price of bermudan option are stochastic grid [12], low-discrepancy publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.178 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.178 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.178&domain=pdf&date_stamp=2023-05-05 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 162 mesh [13], lévy process models [14], recombining quadratures method [15], jump-diffusion processes [16], merton jump-diffusion [17], least-squares monte carlo [15], neural network regression [18], regression trees/random forests [19], and a pure jump lévy process method [20]. besides those methods, there is a binomial method for determining bermudan option [21-24]. binomial method can be used to determine the fair price of an option. binomial method is an easy applicable discreet method for determining option prices [25]. fahria [21] explains the use of binomial method for determining the price of bermudan call option has the same value as american and european call options. the fundamental objective of this study is to determine the price of bermudan call and put option using a binomial method with varying n steps, then to compare the calculation result of binomial method n steps with option price in the market. the focus of this comparison is to analyze the error resulting from the different contract price variable factors. this error analysis is what distinguishes this research from previous studies. 2. materials and methods 2.1. materials 2.1.1. binomial method according to obradovic and mishra [26], the binomial formula as in equation (1): (1) whereas is the binomial coefficient with the formula as in equation (2) [27,28]. (2) the binomial formula is the basis for forming a model in calculating option prices. the calculation of option price using a binomial method is based on the fact that stock price always fluctuates, up or down during the time in the free market. binomial option formula from cox-ross-rubenstein [8] is a formula for a binomial diagram that is used for determining the increase and decrease factor of stock price. thus, the probability of increasing and/ or decreasing stock price can be anticipated. the value of u, d, p, and q which are used in the binomial method are written below: (3) (4) (5) (6) (7) whereas the u parameter states the percentage of stock price increase, d states the percentage of stock price decrease. p states the probability of stock price increase under the assumption of a risk neutral valuation and q states the probability of stock price decrease under the assumption of a risk neutral figure 1. fluctuation scheme of stock price in onestep binomial method figure 2. fluctuation scheme of stock price in steps binomial method j. multidiscip. appl. nat. sci. 163 valuation. the risk neutral valuation assumes that investors do not consider the level of risk when investing. this assumption is used because the movement of assets that are not at risk can be predicted. the risk neutral assumption in calculating the option price states that the current price is equal to the discounted value of the expected future price at a risk-free interest rate. value states the period duration with t states lifetime duration of option and n the number of binomial steps. for example, stock price in t = 0 or (t0) is s0 can be predicted in the future. when t = t or (t1), the stock price increases with the probability of increasing (p) to su or the stock price decreases with the probability of decreasing (1 p) to sd. option value in t0 is v . besides stock price, option value also has two possibilities: option value if stock price increases are vu or v1,1 and option value if stock price decreases are vd or v1,0 (figure 1) [29]. as time continues, the stock will move statically, being at rest. the movement of stock price will fluctuate according to the influencing factors. thus, binomial method does not end with one step, but there are binomial method of n steps, as portrayed below. based on figure 2, it can be confirmed that in the first period t1, stock price will change to s0u with p probability or s0d with 1 p probability. in second period t2, there is possibility of change in stock price to s0u 2 with p 2 probability, s0ud with 2p (1 p) probability, or s0d 2 with (1 p) 2 probability [5]. 2.1.2. volatility one of the factors that influence the option price is a return value and its volatility. return for one period [30] is notated below. (8) for a description of the risk amount of particular investment, variance equation of return is applied [30]: (9) figure 3. option values in the binomial model put error call error put 3 2.08010 17.0727 0.1044 0.3490 6 1.90495 16.7875 0.0707 0.0638 12 2.01147 16.7797 0.0358 0.0560 24 1.99676 16.7508 0.0211 0.0271 48 1.98618 16.7420 0.0105 0.0183 96 1.97499 16.7179 0.0007 0.0058 192 1.97588 16.7257 0.0002 0.0020 384 1.97757 16.7239 0.0019 0.0002 768 1.97683 16.7249 0.0012 0.0012 1536 1.97566 16.7236 0.0000 0.0001 3072 1.97567 16.7237 table 1. the price of ko in bermudan option with different n value j. multidiscip. appl. nat. sci. 164 with . . rt value states stock return in t period, while states the average return of stock. st states stock price in t period and st-1 states stock price before t period. n value states the amount of return day during calculated option lifetime. volatility is deviation standard of annual return. volatility shows the amount of uncertainty or risk on the changing amount of stock value. the s 2 variance formula in equation (9) uses the denominator n – 1 because equation (9) is an unbiased estimator for the variance. by using the assumption that ln stock prices have a normal distribution, the variance of the ln stock price is with . . volatility is calculated with equation (10) [31]: (10) with states volatility and t states the amount of stock trading day in one year, namely 252 days. 2.2. methods this research is literature (library research) and applied research. formulation of bermudan option uses binomial method that is applied to several company stocks in the exchange of the united states of america, which are the stocks of the coca-cola company (ko), the walt disney company (dis), walmart inc. (wmt), and international business machines corporation (ibm). bermudan options are traded bilaterally in the over-the-counter market. in the over-the-counter market, buyers and sellers are free to bargain over prices. so, there is no fixed price in the over-thecounter market because the contract is customized to the interests of the seller and the buyer. before there is a price agreement between the seller and the buyer, the seller and buyer are based on the price listed on the stock exchange. so, in this study, the bermudan option price used is the option price listed on the stock exchange. the source of this research data is obtained from yahoo! finance [32]. the stock data from the site are in the form of initial stock price (s0), exercise price (k), call option data (c market, cm), put option (p market, pm), and daily closing price of the stock. the daily closing price data is used to determine historical volatility. the daily closing price data is taken from december 9, 2019 to december 7, 2022. the execution time in this research is every four months or the fourth month (tk1) and the eighth month (tk2). the data of risk-free interest rate in december 2022 is 4% or 0.04. this data is obtained from the interest rate of the fed on global-rates website [33]. several analysis steps in this research are below: a. deducting the company stock data and risk-free interest rate from the website. b. determining the parameters of the initial stock price (s0), exercise price (k), execution time (tk) figure 4. log plot for error call option figure 5. log plot for error put option j. multidiscip. appl. nat. sci. 165 due time (t), and n binomial step c. calculating the parameter of volatility value ( ), the percentage of stock price increase (u), the percentage of stock price decrease (d), and the probability of increasing stock price (p) d. calculating bermudan call and put option using binomial method n step e. comparing the result of bermudan option calculation using the binomial method to option price in the market. 3. results and discussions executing time of bermudan option can be written as tk < t with k = 1, 2, …, n. the arrangement of executing time of bermudan option has been agreed by both option seller and buyer, as stated in the contract. in the call option, if st > k then the option will be executed so that the option payoff is st k . if st < k then the call option will not be executed so that the option payoff is 0. in a put option, if st < k then the option will be executed so that the option payoff is k st . if st > k then the option will not be executed so the option payoff is 0. the payoff of bermudan call option is ct = max{st k,0} and payoff of bermudan put option is pt = max{k st,0}. payoff function can be perceived as option value at the final point of n steps binomial method, with n as the amount of used binomial steps in calculating the option. the value of the bermudan option at the end of time tn can be written as equation (11) for call option and (12) for call option. (11) (12) the option value at time tn is used to determine the option value at time tn-1. the option value at time tn-1 is used to determine the option value at time tn-2, and so on. at time ti, the value of the binomial option has several cases: call option in tk execution time, put option in tk execution time, and both call and put option at other times t. ti is the time of the i th partition with i = 0, 1, 2, …, n and j = 0, 1, 2, …, i. call option and put option formulas in tk execution time and other times t as conducted in equations (13), (14), and (15). call option in tk execution time: (13) put option in tk execution time: (14) put and call option in other time t: (15) in equations (13), (14), and (15), the p value indicates the probability for the stock price to increase under the assumption of a risk neutral valuation. for s(i,j) states the price of stock in ti time and k value states the agreed price in the option contract where the contract holder has the right to buy or sell the stock with agreed price. then, figure 6. curve fitting for log error call option figure 7. curve fitting for log error put option j. multidiscip. appl. nat. sci. 166 v(i+1,j+1) states option value when the stock price increase, while v(i+1,j) states option value when stock price decrease. r value states the current interest rate according to the central bank of america [34]. an illustration of the application of the binomial model to the bermudan call option is presented in figure 3. figure 3 is 3-step binomial tree for bermudan options with t1 and t2 execution times. the value of the bermudan call option at time t3 based on equation (11) is shown below. based on equation (13) the value of the bermudan call option at the execution time t2 and t1 is expressed as below. option values at other times t only occur at t0, so based on equation (15) the value of v0,0 is calculated as below. 3.1. the arrangement of bermudan option price with many n steps the use of n steps in this research is based on the execution time of the option. suppose that the execution time is every 4 months and the maturity time is 1 year, so the execution time of the bermudan option is the fourth and eighth month. thus, the applied n steps are 3 x 2 i ;i = 0,1,2,3, .... if n = 3 then the execution time occurs at t1 and t2. if n = 6 then the execution time occurs at t2 and t4, and so on. the used stock data in arranging bermudan option prices with many n steps is the stock data of ko. the ko stock price on december 9, 2022 is s0 = $63.14. the chosen exercise price is k = $ 80 and free-risk interest rate (the fed interest rate) in december is r = 4%. n value = 3, 6, 12, 24, 48, 96, 192, 384, 768, 1536, and 3072. the volatility value of ko stocks calculated using equation (10) is = 0.24215. the bermudan option price of ko stock with many values is shown in table 1. error value obtained by comparing bermudan option price in each n with bermudan option price in n = 3072. table 2. calculation of bermudan option price using the binomial method on dis no data call data put exercise price c market c binomial % error exercise price p market p binomial % error 1 65 35.28 34.6016 1.9229 65 3.20 2.14837 32.8634 2 75 26.73 27.3830 2.4429 75 5.35 4.53197 15.2903 3 80 24.47 24.1912 1.1394 80 6.70 6.15526 8.1304 4 85 20.70 21.2940 2.8696 85 8.35 8.08639 3.1570 5 90 18.80 18.6755 0.6622 90 10.15 10.3121 1.5970 6 95 16.20 16.3251 0.7722 95 12.38 12.8248 3.5929 7 100 13.70 14.2301 3.8693 100 14.85 15.6142 5.1461 8 105 11.65 12.3820 6.2833 105 18.00 18.6735 3.7417 9 110 9.75 10.7465 10.2205 110 20.55 21.9696 6.9080 10 115 8.00 9.30498 16.3123 115 24.03 25.4857 6.0578 11 120 6.51 8.05380 23.7143 120 29.15 29.2125 0.2144 12 125 5.20 6.95301 33.7117 125 32.75 33.1157 1.1166 https://finance.yahoo.com/quote/dis/options?strike=65&straddle=false https://finance.yahoo.com/quote/dis/options?strike=65&straddle=false https://finance.yahoo.com/quote/dis/options?strike=75&straddle=false https://finance.yahoo.com/quote/dis/options?strike=75&straddle=false https://finance.yahoo.com/quote/dis/options?strike=80&straddle=false https://finance.yahoo.com/quote/dis/options?strike=80&straddle=false https://finance.yahoo.com/quote/dis/options?strike=85&straddle=false https://finance.yahoo.com/quote/dis/options?strike=85&straddle=false https://finance.yahoo.com/quote/dis/options?strike=90&straddle=false https://finance.yahoo.com/quote/dis/options?strike=90&straddle=false https://finance.yahoo.com/quote/dis/options?strike=95&straddle=false https://finance.yahoo.com/quote/dis/options?strike=95&straddle=false https://finance.yahoo.com/quote/dis/options?strike=100&straddle=false https://finance.yahoo.com/quote/dis/options?strike=100&straddle=false https://finance.yahoo.com/quote/dis/options?strike=105&straddle=false https://finance.yahoo.com/quote/dis/options?strike=105&straddle=false https://finance.yahoo.com/quote/dis/options?strike=110&straddle=false https://finance.yahoo.com/quote/dis/options?strike=110&straddle=false https://finance.yahoo.com/quote/dis/options?strike=115&straddle=false https://finance.yahoo.com/quote/dis/options?strike=115&straddle=false https://finance.yahoo.com/quote/dis/options?strike=120&straddle=false https://finance.yahoo.com/quote/dis/options?strike=120&straddle=false https://finance.yahoo.com/quote/dis/options?strike=125&straddle=false https://finance.yahoo.com/quote/dis/options?strike=125&straddle=false j. multidiscip. appl. nat. sci. 167 the search for error value is conducted to obtain the convergence of the binomial method. in table 1, the error value of the call option and put option in ko stock will be closer to 0 if the amount of binomial n steps is augmented. the value of the bermudan call option and bermudan put option are convergent in certain values when applied n steps in the binomial method is enlarged. this result corresponds to the research [32,35-37] which states the binomial method is the prompt convergent numerical method in analytical value. error call option and put option in table 1 can be presented in a log plot as shown in figures 4 and 5. based on figures 4 and 5, log error values of the call option and put option decrease as n increases. when n > 3, log error value of the call option or put option has shown a downward trend. the log error value drops faster when n > 96. in figure 5, log error call option goes up slightly at n = 384 but drops back down after that. when n = 768, the log error put option in figure 6 goes up slightly but after that goes down. a drastic decrease in the log error from call options and put options occurs when n = 1536. the decreasing log error value indicates that the error converges to a certain value. the points of log plot from figures 4 and 5 form a certain curve. curve fitting at points on log plot of figure 4 uses a smoothing spline with the smoothing parameter p = 5.5633431x10 -5 . the fitted curve is shown in figure 6. the fitted curve of log error call option has sse (sum of square error): 0.07177 and rmse (root mean square error): 0.1367. the points on the log plot of figure 5 are fitted using a smoothing spline with the smoothing parameter p = 7.5295283x10 -6 . the fitted curve of log error put option is shown in figure 7. the fitted curve has sse and rmse of 0.4313 and 0.3011, respectively. 3.2. the comparison of the bermudan option binomial method calculation result with option price in the market the simulation of binomial method in determining bermudan call option and put option price applies several stock data. this simulation is conducted to find out if obtained bermudan option price is in approach to market option price. applied stock data in this simulation are dis, wmt, and ibm. a certain amount of different exercise prices is used on each stock to apply the binomial method in determining bermudan option price. in binomial method simulation, the selected stock data is taken from december 12, 2022. the option due time is january 19, 2024. thus, t = 403 days. the amount of n partition is 768. the price data of dis stock on december 12, 2022, is s0 = 94.66 . based on equation (10), the volatility of dis is σ = 0.37176. according to the calculation, the obtained parameters of dis are p = 0.4985, q = 0.5015, u = 1.0142, and d = 0.9860. the price data of wmt stock on december 12, 2022, is s0 = 148.02 . the p, q, u, and d values of wmt are table 3. calculation of bermudan option price using the binomial method on wmt no data call data put exercise price c market c binomial % error exercise price p market p binomial % error 1 120 35.00 36.2518 3.5766 120 4.15 3.09278 25.4752 2 125 31.05 32.5491 4.8280 125 5.20 4.20200 19.1923 3 130 28.15 29.0760 3.2895 130 6.22 5.55308 10.7222 4 135 25.00 25.8431 3.3724 135 7.60 7.16040 5.7842 5 140 22.05 22.8566 3.6580 140 9.15 9.03410 1.2667 6 145 18.95 20.1231 6.1905 145 10.90 11.1838 2.6037 7 150 16.20 17.6423 8.9031 150 13.00 13.6136 4.7200 8 155 13.60 15.3987 13.2257 155 16.20 16.3144 0.7062 9 160 11.40 13.3783 17.3535 160 18.80 19.2716 2.5085 10 165 9.19 11.5879 26.0925 165 22.71 22.4883 0.9762 11 190 3.02 5.3571 77.3874 190 41.45 41.7545 0.7346 https://finance.yahoo.com/quote/wmt/options?strike=120&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=120&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=125&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=125&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=130&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=130&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=135&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=135&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=140&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=140&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=145&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=145&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=150&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=150&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=155&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=155&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=160&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=160&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=165&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=165&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=190&straddle=false https://finance.yahoo.com/quote/wmt/options?strike=190&straddle=false j. multidiscip. appl. nat. sci. 168 consecutively 0.5006, 0.4994, 1.0096, and 0.9905. the value of historical volatility determined based on equation (10) from december 9, 2019 – december 7, 2022, is σ = 0.25086. the third stock data which is used in the binomial method simulation is ibm stock data. the price of ibm stock on december 12, 2022, is s0 = 149.21 . the historical volatility results from equation (10) is σ = 0.29861. the obtained parameters are p = 0.4997, q = 0.5003, u = 1.0114, and d = 0.9887. the result of binomial method simulation in determining dis stock option price with an amount of different exercise prices is displayed in table 2. table 3 shows the calculation result of bermudan option price in each exercise price of wmt. table 4 shows the calculation result of bermudan option price in each exercise price of ibm. the obtained bermudan option price using binomial method is compared to market option price to obtain error percentage. according to error percentage, the distance of bermudan option price of binomial method can be measured to the price of the market option. according to the calculation result of bermudan option price using the binomial method on dis, wmt, and ibm, it can be concluded that the call option price is constantly decreasing, consequent with the constantly increasing exercise price. on the other, the put option price constantly increases with the increase in the exercise price. the comparison of bermudan option price from binomial method to the price of market option obtains different error percentages. on call option, the higher the option exercise price, the error percentage is also higher. small error percentage on call option is obtained when the exercise price is below current stock price. it means the bermudan call option price is empirically in approach to market option price when the exercise price is below current stock price. in other words, binomial method is empirically suitable to use on bermudan call option with a k value below the s0 value. if exercise price is far above the current stock price, the error percentage will be high. on put option, if the exercise price is high, the error percentage will be low. on the exercise price of put option that is above the current stock price, the error percentage obtained is low. empirically, if exercise price is above the current stock price, the put option price is in approach to market option price. it indicates the binomial method is empirically suitable to bermudan put option with a k value above the s0 value. if the exercise price is far below the current stock price, the error percentage will be high. 4. conclusions the calculation result of the bermudan option using the binomial method with certain n step table 4. calculation of bermudan option price using the binomial method on ibm no data call data put exercise price c market c binomial % error exercise price p market p binomial % error 1 120 33.45 39.0019 16.5976 120 5.61 4.66729 16.8041 2 125 29.33 35.5081 21.0641 125 6.78 5.98691 11.6975 3 130 27.38 32.2232 17.6888 130 8.15 7.52577 7.6593 4 135 23.92 29.1610 21.9105 135 9.32 9.29818 0.2341 5 140 19.91 26.3037 32.1130 140 11.15 11.2912 1.2664 6 145 17.00 23.6650 39.2059 145 13.00 13.5198 3.9985 7 150 15.60 21.2328 36.1077 150 15.30 15.9730 4.3987 8 155 12.60 19.0055 50.8373 155 18.50 18.6531 0.8276 9 160 10.95 16.9638 54.9205 160 21.25 21.5429 1.3784 10 170 6.98 13.4423 92.5831 170 30.45 27.9520 8.2036 11 180 4.75 10.5680 122.4842 180 35.50 35.1020 1.1211 12 200 1.82 6.40192 251.7538 200 56.00 51.2226 8.5311 https://finance.yahoo.com/quote/ibm/options?strike=120&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=120&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=125&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=125&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=130&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=130&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=135&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=135&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=140&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=140&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=145&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=145&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=150&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=150&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=155&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=155&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=160&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=160&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=170&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=170&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=180&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=180&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=200&straddle=false https://finance.yahoo.com/quote/ibm/options?strike=200&straddle=false j. multidiscip. appl. nat. sci. 169 produce option prices that are not much different, in which the error value is closer to 0 if the binomial step is enlarged. the comparison of the bermudan option price using the binomial method n step to market option price shows the bermudan call option price empirically has a small error or is in approach to market option price when the exercise price is below the current stock price. the calculated bermudan put option using the binomial method produces a small error or is in approach to market option price when the exercise price is above the current stock price. based on the results of this study, the binomial method is suitable for calculating bermudan option prices, especially if a certain contract price is selected. the results of this study can be used as an alternative for investors in setting the bermudan option price for both calls and putting in the over-the-counter market (outside the stock market). in future studies, error analysis can be carried out based on the maturity time variable factor. author information corresponding author emy siswanah — department of mathematics, universitas islam negeri walisongo semarang, semarang-50185 (indonesia); orcid.org/0000-0003-3717-0989 email: emysiswanah@walisongo.ac.id authors ahmad mutawaslih idrus — department of mathematics, universitas islam negeri walisongo semarang, semarang-50185 (indonesia); orcid.org/0009-0006-7375-8746 muhammad malik hakim — department of informatics, universitas diponegoro, semarang50275 (indonesia); orcid.org/0000-0001-6405-3417 author contributions conceptualization, methodology, validation, formal analysis, investigation, resources, writing – original draft preparation, writing – review & editing, supervision, project administration, funding acquisition, e. s.; conceptualization, software, data curation, funding acquisition, a. m. i.; software, visualization, funding acquisition, m. m. h. conflicts of interest the author(s) declared no conflict of interest acknowledgement researchers would like to thank the science and technology faculty of uin walisongo semarang for supporting the researchers with all facilities needed to complete this research. references [1] d. sornette and r. woodard. 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"convergence of binomial tree methods to black scholes model on determining stock option prices". iop conference series: materials science and engineering. 567 (1): 10.1088/1757-899x/567/1/012013. https://doi.org/10.18540/jcecvl8iss3pp14123-01e https://doi.org/10.1088/1742-6596/435/1/012043 https://doi.org/10.1088/1742-6596/435/1/012043 https://doi.org/10.1016/j.jfineco.2021.06.012 https://doi.org/10.1080/03610926.2019.1590600 https://doi.org/10.1088/1757-899x/567/1/012013 34 journal of multidisciplinary applied natural science vol. 3 no. 1 (2023) research article enrichment of doogh with olive leaf extract and investigation of its physicochemical, microbial, and sensory properties during storage at room temperature and refrigerator laya kalavari, negin nasiri, fatemeh ahmadian*, and hamed kioumarsi received : july 17, 2022 revised : august 22, 2022 accepted : august 27, 2022 online : august 30, 2022 abstract doogh is a popular iranian drink with high nutritional value and in recent years has attracted the attention of many consumers. nowadays, the problems caused by malnutrition and lack of useful compounds with high nutritional value and increasing shelf life are essential in the food industry. this research was performed in shirin cheshmeh dairy production company and all experiments were performed in the food control laboratory of the food and drug administration, guilan university of medical sciences. in this study, olive leaf extract was added to doogh at five levels of zero, 0.2, 0.5, 1, and 2% and stored for 45 days, its physicochemical, microbial, and sensory properties at two refrigerators at 2 °c and room temperature 25 °c were evaluated. sampling was analyzed on days 0, 15, 30, and 45 days in 3 replications with duncan's statistical analysis. the effect of using different treatments and storage time on most characteristics showed a significant difference. this study showed that the effect of olive leaf extract on the ph, mold, and yeast of the treatments is decreasing and the percentage of acidity and serum volume is increasing. according to the hedonic method, the acceptability of treatment 2 on the 15th day of maintenance was higher. in general, according to the results, it can be said that the production of useful products with plant compounds can promote an effective step to produce and design useful drinks in the consumer market and food industry factories. keywords acidity, doogh, olive leaf extract, useful product 1. introduction animal production is very important in asia [1] -[4]. doogh is a traditional iranian fermented drink that is prepared by mixing yogurt with water and some salt. it is an acidic dairy drink mainly consumed in plain type or with aromatic compounds [5]. it is the result of lactic fermentation of milk, the dry matter of which is standardized by diluting yogurt (after fermentation) or milk (before fermentation). similar products in turkey, afghanistan, and azerbaijan, are consumed more than in other countries, including the middle east and central asia [6][7]. nowadays, due to increasing public awareness about the harmful consumption of carbonated beverages, desires to use natural drinks such as doogh are rising [8]. though it is produced on large industrial scales, more data is needed to introduce its practical properties. the flow properties of doogh are determined for several purposes such as quality control, understanding the structure, process engineering applications, and correlations with sensory evaluation. the final properties of the doogh such as stability, texture, and appearance are directly dependent on its structure [5]. healthy food production is the main priority of food technology and dairy products which plays an important role in the household food basket. traditional dairy products take a large part in the market and sometimes are causing the transmission of various diseases to humans, particularly in the cheese market. the basic ingredients in milk fermented dairy products are similar to those in milk; as a result, these products are an excellent source of calcium, phosphorus, b vitamins such as thiamine (b1), riboflavin (b2), pantothenic acid (b5), cobalamin (b12) and magnesium and zinc, but the fermentation process changes the chemical structure. in addition to the nutritional benefits mentioned above, it contains beneficial bacteria that have many effects on the health of the gastrointestinal tract, so if used regularly, the beneficial bacteria streptococcus thermophilus and lactobacillus bulgaris in it will be placed in the publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2022 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.143 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.143 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.143&domain=pdf&date_stamp=2022-08-30 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 35 gastrointestinal tract and will have positive effects [9][10]. medicinal plants are endemic heritages of global importance due to the different chemical compounds that plants produce, they have many applications in the food, medical, perfumery, cosmetic, and health industries. in the 21st century, due to the rapid population growth and the need to develop the food industry at the same time, methods must be used to be not only safe and high-quality products, but also economically and commercially appropriate and cost-effective [9][11][12]. doogh is made by mixing yogurt, water, salt, and other additives, including peppermint essential oil. the use of sweet doug has been common in the united states since 2002. the american people call this dairy product our yogurt. the doogh is a dairy drink that can replace soda in the food basket. in addition to nutritional benefits, doogh contains beneficial bacteria that have many effects on the health of the gastrointestinal tract, so its continuous consumption can prevent the growth of harmful organisms [13]. throughout the history of civilization, the olive plant has been an important source of nutrition and medicine. fermentation of food products is one of the oldest biotechnologies in production that produces food products with desirable properties such as long life and good organic properties [10] [14][15]. in recent years, the consumption of fermented beverages, due to their effects on health has increased. olive leaf is effective in treating diabetes and lowering blood pressure and strengthening the immune system. this plant is also involved in lowering blood cholesterol and uric acid. olive leaf extract strengthens the body's antioxidant defense systems such as catalase and superoxide dismutase. polyphenols are massively bioactive molecules in olive leaves. in particular, aleuropine along with tyrosol and flavonoids are the main constituents. these compounds have antimicrobial and antioxidant activity in olive leaf extract and can make olive leaf extract suitable for use in the food industry and as a natural preservative [15]-[17]. various results were obtained regarding the effects of plant extracts containing polyphenol compounds on the growth of semicircular organisms. plant extracts have been shown to inhibit the growth of food-related pathogens and microorganisms responsible for food spoilage [9][17][18]. the purpose of this study, according to previous studies in which the effect of olive extract has been investigated separately or in combination, is to evaluate the enrichment of doogh with olive leaf extract and investigation of its physicochemical, microbial, and sensory properties during storage at room temperature and refrigerators. 2. materials and methods this study was performed in 2018 in shirin cheshmeh dairy production company located in fooman industrial city in guilan province. shirin cheshme gilan company with the brand name "tenha" located in "sardarjangal" industrial area, started operating in 2004, production equipment includes pasteurizer, homogenizer, separator, steel tanks, uf (ultrafiltration) equipment, etc. all experiments were performed in the food control laboratory of the food and drug administration, guilan university of medical sciences. hydroalcoholic extract of olive leaves was purchased from adonis goldaroo company. first, raw milk with 2% of fat was pasteurized at 75 °c storage at room temperature (25 °c) storage at refrigerator (4 °c) day zero samples (concentration) day 45 day 30 day 15 day 45 day 30 day 15 3.82±0.02 da 3.90±0.01 ca 4.01±0.01 ba 4.01±0.01 ba 4.04±0.01 ba 4.04±0.01 ba 4.10±0.01 aa control group 3.83±0.01 da 3.87±0.02 da 4.00±0.01 ba 4.00±0.01 ba 4.02±0.01 ba 4.03±0.02 ba 4.08±0.03 aa sample 1 (0.2%) 3.84±0.02 da 3.88±0.01 da 3.98±0.02 ca 3.98±0.01 ca 4.01±0.01 ba 4.02±0.02 ba 4.07±0.01 aa sample 2 (0.5%) 3.84±0.01 da 3.86±0.01 da 3.95±0.02 cb 3.93±0.01 cb 3.95±0.01 cb 4.01±0.01 ba 4.04±0.03 bb sample 3 (1.0%) 3.82±0.01 da 3.85±0.02 db 3.94±0.02 cb 3.91±0.01 cb 3.94±0.01 cb 3.96±0.01 cb 4.00±0.02 bb sample 4 (2.0%) table 1. ph changes in storage at refrigerator (4 °c) and room temperature (25 °c) j. multidiscip. appl. nat. sci. 36 for 20 seconds and then poured into a yogurt preparation tank. the milk temperature is decreased to 43 ° c, then 3% of the yogurt starter is added and stirred for 15 minutes, and finally, the incubation is continued for 8 hours to reach the desired acidity. then, water, salt, and peppermint were added to stir for 15 minutes. the doogh is heated at 85 ° c for 15 seconds and then quickly cooled to 4 ° c and packaged in bottles. concentrations of 0.2, 0.5, 1, and 2 mg of olive leaf extract were added to doogh and the treatments were stored in two refrigerators at 2°c and room temperature 25 °. chemical, microbial and sensory evaluations were performed. the acidity of doogh was measured by titration method, ph was measured using a ph-meter (metrohm, germany), and stability of doogh was also measured. doogh microbial tests included the mold and yeast test and general enumeration of microorganisms. in determining the amount of a sensory evaluation of doogh through the sensory test (taste) which is done by five-point hedonic method (from very bad: 1 to very good: 5) in terms of color, odor, taste by a team of 15 trained evaluators (standard no. 695, 1387). in this study, all physicochemical, microbial, and sensory properties of doogh containing different concentrations of olive leaf extract were performed in a completely randomized design with three replications. to compare the means, duncan's multi -range test method was used at the level of 5% error probability, and in the end, all graphs were drawn with excel 2007. 3. results and discussions 3.1. ph measurement results by measuring the ph of different samples, it was found that the highest ph belonged to the control sample on day zero and the lowest ph belonged to the sample stored for 45 days at room temperature. by adding olive leaf extract, the ph of the samples decreases so that the more the amount of extract, the more the ph decreases, but this increase is not significant up to 0.5% concentration, but in sample 3, with increasing olive leaf extract concentration to 1 and 2%, the control ph decreases significantly. also, with increasing shelf life, both in the refrigerator and at room temperature, the ph decreases with increasing storage time. in the 15day for the sample stored in the refrigerator, we see a significant decrease in ph, but in 30-day and 45day the changes are not significant. we also see a decrease in ph in the samples stored at room temperature, but this decrease has a greater slope compared to the samples stored in the refrigerator so a significant decrease is observed in the 15-day samples and the measurements of the 30-day samples. the decrease in ph is also significant and at the end of 45 days, we see the greatest decrease in ph in samples stored at room temperature. in 30day and 45-day samples stored at room temperature, ph does not decrease significantly with the increasing concentration of the extract. 3.2. stability measurement results (phase separation rate) stability measurement was performed to determine the degree of phase separation by measuring the volume of isolated serum in milliliters, the results of which are shown in table 3. the results show that the lowest isolated serum or the highest stability is related to the 15-day control sample stored at refrigerator temperature and the highest isolated serum, which indicates the lowest stability, is related to the 45-day controlled control sample at room temperature. with table 2. changes in the acidity of the samples according to the percentage of lactic acid. storage at room temperature (25 °c) storage at refrigerator (4 °c) day zero samples (concentration) day 45 day 30 day 15 day 45 day 30 day 15 0.58±0.01 da 0.35±0.02 ca 0.52±0.04 ba 0.51±0.09 ba 0.50±0.03 aa 0.49±0.05 aa 0.46±0.05 aa control group 0.58±0.01 da 0.57±0.01 da 0.56±0.02 ba 0.52±0.04 ba 0.52±0.04 ba 0.51±0.04 aa 0.50±0.09 ab sample 1 (0.2%) 0.59±0.04 da 0.57±0.01 db 0.56±0.04 ca 0.57±0.05 cb 0.54±0.04 ba 0.52±0.01 ba 0.51±0.04 ab sample 2 (0.5%) 0.59±0.05 da 0.58±0.01 db 0.56±0.05 cb 0.57±0.04 cb 0.55±0.02 cb 0.54±0.01 bb 0.52±0.03 bb sample 3 (1.0%) 0.59±0.04 da 0.58±0.02 db 0.58±0.04 cb 0.59±0.05 cb 0.56±0.04 cb 0.56±0.01 cb 0.53±0.09 bb sample 4 (2.0%) j. multidiscip. appl. nat. sci. 37 increasing storage time in all samples, we see a decrease in stability and an increase in isolated serum. this increase in samples stored at room temperature is more than samples stored in the refrigerator and it is also observed that the addition of olive leaf extract changes the amount. the serum is isolated and stability is observed, but these changes are not in a definite order and are not recognized as significant. 3.2. doogh sensory analysis results sensory properties of control doogh containing olive leaf extract based on the five-point hedonic method are specified in table 4. the taste and odor were very good, but the sample obtained 4 lower scores. the lowest taste and odor scores are related to the samples stored for 15 days at room temperature. with increasing the concentration of the extract from 0.2 to 0.5% and above, we see a change in taste and odor score in 15-day samples stored in the environment. the results show that the taste and odor scores related to the 15-day samples stored in the refrigerator are higher than the 15-day samples stored at room temperature. 3.3. results of acidity measurement in terms of percentage of lactic acid by measuring different samples, it was found that the lowest acidity was related to the control sample on day zero and the highest acidity was related to the 45-day sample stored at room temperature. with the addition of olive leaf extract, the acidity of the samples increases significantly compared to the control sample and with increasing the amount of extract, an increase in acidity is observed, but this increase is not significant among the samples containing the extract. this increase in acidity in samples stored at room temperature is greater than samples stored in the refrigerator. 3.4. microbial analysis results the results of the microbial test are presented in table 4. during this test, it was found that all samples were negative for mold and yeast, coliform, total count, and staphylococcus aureus coagulase on the day of manufacture. however, with increasing storage time on the 15th day, the samples stored in the refrigerator remained negative, but mold and yeast were observed in samples stored at room temperature, while the total, coliform, and staphylococcus aureus count as negative as the samples stored in the refrigerator. the highest amount of mold and yeast in the samples kept at room temperature on the 15th day was related to the control sample and the lowest amount of mold and yeast was related to sample 1, which contains 0.2% of olive leaf extract. in the total count experiment of 30-day samples stored in the refrigerator, in samples no. 2 and 4, which contain 0.5 and 2% of olive leaf extract, respectively, 30 and 1700 cfu/g of the colony were observed, while other samples in other microbial tests including mold and yeast, coliform and staphylococcus aureus were negative. the 30-day samples stored at room temperature, like the 30-day refrigerated samples, were negative in coliform and staphylococcus aureus tests, but molds and yeast were found in samples 1, 2, 4, and in control samples, 1 and 2 in the counting test. a total colony was observed. but other samples were negative. in total, the number of infected samples and the number of colonies in the samples stored at room temperature is more than the samples stored in the refrigerator. the total number of colonies in the samples containing olive leaf extract is less than the control sample. it is observed that in sample 3 table 3. changes in the volume of isolated serum. storage at room temperature (25 °c) storage at refrigerator (4 °c) samples (concentration) day 45 day 30 day 15 day 45 day 30 day 15 30.0 26.0 20.0 34.0 32.0 31.0 control group 30.0 26.0 24.0 30.0 29.0 28.0 sample 1 (0.2%) 30.0 26.0 26.0 27.0 27.0 26.0 sample 2 (0.5%) 27.0 20.0 24.0 28.0 27.0 26.5 sample 3 (1.0%) 23.0 26.0 28.0 25.0 24.0 23.0 sample 4 (2.0%) j. multidiscip. appl. nat. sci. 38 stored at room temperature, the total count of microorganisms is negative. 45-day samples were stored at refrigerator temperature in all microbial tests, but 45-day samples were stored at room temperature, although negative in the measurement of coliform and staphylococcus aureus, but have mold and yeast so in the control sample mold and yeast were obtained at 4000cfu/g. however, in samples containing olive leaf extract, there is a significant decrease in mold and yeast with increasing extract, so even in samples 1 and sample 4, which contain the lowest and highest amount of extract, mold, and yeast become negative. 3.5. discussion as can be seen in table 1, the ph values of the measured samples are different and this difference is significant in some cases. the ph of the control sample was highest on day 0 and decreased with increasing storage time and storage temperature. abbase et al. [19] in a study that investigated the effect of guar gum on stability and qualitative properties of doogh found that, with increasing the storage time, the ph of doogh samples containing gum compared to the control sample has a significant decrease and the reason is the activity of lactic acid bacteria and lactic acid production. laghaei and zomorodi, measured the effect of zedo, commercial stabilizer cho, arabic, and xanthan gum on the stability and qualitative properties doogh and according to the sensory evaluation, with increasing concentration of xanthan gum and commercial stabilizer cho the score of flavor and with increasing concentration any of four types of stabilizers the score of consistency decreased [20]. mirchooli borazgh et al. [21] investigated the effect of temperature and packaging on the shelf life of non-carbonated doogh and reported that with increasing the storage temperature of doogh, the ph shows a significant decrease. therefore, the results of the present study are consistent with the mentioned findings. also, by adding the extract to the doogh on day 0 of storage, we see a significant decrease in ph in the samples containing the extract compared to the control sample and also a regular decrease in ph with increasing the extract from 0.2 to 2%. this can be due to the acidic ph of olive leaf extract. the low pphh of olive leaf extract can also be due to the presence of caffeic acid in the extract. the mentioned is similar to the findings of the previous researchers [22]. the findings in table 2 shows that with increasing storage time and temperature, the amount of acidity in terms of the percentage of lactic acid shows a significant increase so this increase in acidity increases significantly with increasing storage temperature both in refrigerated samples and samples stored in the environment, the acidity increases with increasing storage time, so this increase in acidity can be attributed to the activity of lactic acid-producing bacteria during storage. mirchooli borazgh et al [21], investigated the effect of temperature and packaging on the shelf life of non-carbonated doogh and reported that with increasing the storage temperature of the doogh, the acidity shows a significant increase which is in line with the findings of the current study. as shown in table 3, the results show that the lowest amount of isolated serum or the highest stability is related to the sample stored for 15 days in the refrigerator temperature and the highest amount of isolated serum is the lowest stability. it is related to the 45-day control sample stored at room temperature. with increasing storage time in all samples, we see a decrease in stability and an increase in isolated serum. this increase in samples stored at room temperature is more than samples stored in the refrigerator and is also observed. with the addition of olive leaf extract, changes in the amount of isolated serum and stability are observed, but these changes did not have a specific order and were not recognized as significant. therefore, it can be concluded that adding olive leaf extract to doogh in the present study had a significant effect on increasing or has not reduced the stability of the doogh. mahmoudzadeh et al. [23] studied the effect of thymus kotschyanus essential oil on the physicochemical and sensory properties of doogh and reported that essential oil doesn’t have significant changes in the physicochemical properties of doogh. therefore, the above results are also consistent with the results of the present study. sensory properties of control doogh and containing olive leaf extract based on the fivepoint hedonic method are specified in table 4. the taste and odor were very good, but the sample got 4 lower scores. the reason for this score is probably j. multidiscip. appl. nat. sci. 39 due to the high amount of extract and the creation of an astringent taste in the mouth. the lowest taste and odor scores are related to 15-day samples stored at room temperature. with increasing the concentration of the extract from 0.2 to 0.5% and above, we see a decrease in taste and odor scores in 15-day samples stored in the environment. the results show that the taste and odor scores related to the 15-day samples stored in the refrigerator are higher than the 15-day samples stored at room temperature. it seems that storage at room temperature will have an adverse effect on the sensory properties of doogh. the results of this study are consistent with the reports of mirchooli borazgh et al. [21] who studied the effect of temperature and packaging on the shelf life of noncarbonated buttermilk. according to the results (table 4) during this experiment, it was found that all samples on the day of manufacture were negative for mold and yeast, coliform, total count (total count), and staphylococcus aureus coagulase, which indicates that provides hygienic observance in the preparation and manufacture of tested samples. however, with increasing storage time on the 15th day, the samples stored in the refrigerator remained negative, but the samples stored at room temperature, mold, and yeast were observed, while the total, coliform and staphylococcus aureus counts as negative as the samples stored in the refrigerator. these results are consistent with the studies of mirchooli borazgh et al. [21] which investigated the effect of temperature and packaging on the shelf life of non-carbonated buttermilk. the highest amount of mold and yeast in the samples stored at room temperature on the 15th day is related to the control sample and the lowest amount of mold and yeast is related to sample 1, which contains 0.2% of olive leaf extract, which can be due to antiinflammatory properties of olive leaf extract. in the total count experiment of 30-day samples stored in the refrigerator, in samples no. 2 and 4, which contain 0.5 and 2% of olive leaf extract, respectively, 30 and 1700 cfu/g of the colony were observed, while other samples in other microbial tests including mold and yeast, coliform and staphylococcus aureus were negative. the 30-day samples stored at room temperature, like the 30-day refrigerated samples, were negative in coliform and staphylococcus aureus tests, but molds and yeast were found in samples 1, 2, 4, and in control samples, 1 and 2 in the counting test. the total number of colonies was observed but other samples were negative. in total, the number of infected samples and the number of colonies in the samples stored at room temperature is more than the samples stored in the refrigerator. the total number of colonies in the samples containing olive leaf extract is less than the control sample so, in sample 3 stored at room temperature, the total count of microorganisms is negative. 45day samples were stored at refrigerator temperature in all microbial tests, but 45-day samples stored at room temperature, although negative in the measurement of coliform and staphylococcus aureus, but have mold and yeast so in the control sample mold and yeast were obtained at 4000 cfu / day 15 day zero samples (concentration) storage at room temperature (25 °c) storage at refrigerator (4 °c) odor taste odor taste odor taste 2 3 3 4 5 5 control group 2 2.5 3 3.5 5 5 sample 1 (0.2%) 1 2.5 3 4 5 5 sample 2 (0.5%) 1 2.5 3 4 5 5 sample 3 (1.0%) 1 2 3 4 4.5 4.5 sample 4 (2.0%) table 4. results of sensory analysis of samples. notes: 1-very week; 2-week; 3average; 4-good; 5-very good j. multidiscip. appl. nat. sci. 40 g, but in samples containing olive leaf extract, the increase in mold and yeast extract decreased significantly, so that even in samples 1 and sample 4, which contain the lowest and highest amount of extract, mold, and yeast become negative. the above cases show that although with increasing temperature and shelf life, the number of mold and yeast colonies and the total microbial count increase, but with the presence and increase of the extract concentration due to antimicrobial properties of olive leaf extract causes a significant decrease in mold colonies and yeast and the general population of microorganisms. these results are consistent with the observations of rafiee et al. [24]. 4. conclusions increasing the nutritional value and shelf life of traditional dairy products has been considered much in recent years. the use of natural food additives with functional properties instead of chemical preservatives is more interested in the consumer and food industry. the quality and shelf life of yogurt are important topics in the dairy industry [8, 9, 25, 26]. according to the results and discussions, it can be concluded that adding olive leaf extract to doogh can reduce the ph and its storage at refrigerator temperature is recommended to help the minimum ph changes during storage, the acidity of doogh stored at refrigerator temperature is the lowest. it has changed in comparison with doogh stored at room temperature. it was also found that olive leaf extract has no effect on the stability of doogh, but storage conditions in the refrigerator increase the stability of doogh. according to our findings, the antimicrobial activity of olive leaf extract is confirmed in the samples during the storage time, especially with the improvement of the storage conditions. also, no significant adverse effects were observed in the sensory characteristics of the samples with the addition of olive leaf extract. therefore, the positive effect of olive leaf extract as a natural safe compound with different biological activities on enriched doogh was confirmed in the present study. however, additional research to increase the quality and acceptability of enriched products is needed. author information corresponding author fatemeh ahmadian — the food and drug administration, guilan university of medical sciences, rasht-43 (iran); orcid.org/0000-0001-9986-7701 email: ahmadian.f94@gmail.com authors laya kalavari — department of food industry, university of mehraeen, bandar anzali-38 (iran); orcid.org/0000-0002-0008-9709 negin nasiri — department of food industry, university of mehraeen, bandar anzali-38 (iran); orcid.org/0000-0001-7547-2617 hamed kioumarsi — department of animal science research, gilan agricultural and natural resources research and education center, rasht -43 (iran); orcid.org/0000-0002-0783-0934 author contributions l. k. the work presented derives from her master's thesis project, and she conducted the research. n. n. she is the supervisor of the thesis (conceptualization, writing original draft preparation, funding acquisition, resources, methodology, investigation, data analysis). f. a. she is the supervisor of the thesis (conceptualization, writing original draft preparation, funding acquisition, resources, methodology, investigation, data analysis). h. k. he served as the head of the faculty of agriculture, mehraeen higher education institution (original draft preparation, resources, methodology, review, and editing). conflicts of interest the author(s) declared no conflict of interest acknowledgement the support by mehraeen university is gratefully acknowledged. the authors also would like to thank shirin cheshmeh guilan for all the facilities provided. https://orcid.org/0000-0001-9986-7701 mailto:ahmadian.f94@gmail.com https://orcid.org/0000-0002-0008-9709 https://orcid.org/0000-0001-7547-2617 https://orcid.org/0000-0002-0783-0934 j. multidiscip. appl. nat. sci. 41 references [1] h. kioumarsi, z. s. yahaya, and a. w. rahman. 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(2022). “does thyme essential oil (zataria multiflora) improve durability, the taste and nutritional value of doogh?”. journal of nutrition fasting and health. 10 (2): 116120. 10.22038/jnfh.2022.64038.1380. https://doi.org/10.1016/j.jff.2017.01.039 https://doi.org/10.1111/jfpp.13107 https://doi.org/10.3390/12051153 https://dorl.net/dor/20.1001.1.24233544.1392.5.1.5.3 https://doi.org/10.4315/jfp-20-391 https://doi.org/10.4315/jfp-20-391 https://doi.org/10.22038/jnfh.2022.64038.1380 78 journal of multidisciplinary applied natural science vol. 1 no. 2 (2021) research article green synthesis of gold nanoparticles using pimenta dioica leaves aqueous extract and their application as photocatalyst, antioxidant, and antibacterial agents adewale fadaka , olukemi aluko, saartjie awawu, and karim theledi received : february 19, 2021 revised : march 23, 2021 accepted : april 15, 2021 online : april 17, 2021 abstract green synthesis of gold nanoparticles (aunps) is of particular interest due to their catalytic, antioxidant, and antibacterial properties. in this study, the aqueous extract of pimenta dioica leaves was used to synthesize aunps and the effective parameters were investigated. the prepared aunps were characterized by various techniques including uv–vis absorption spectroscopy, fourier transform infrared (ftir) spectroscopy, transmission electron microscopy (tem), and x-ray diffractometer (xrd). the reduction and stabilization effect of the plant extract to fabricate aunps were explained by ftir analysis. tem imaging confirmed the formation of spherical-shaped aunps. the catalytic activity of synthesized nanoparticles was evaluated in the degradation of a methylene blue dye in the presence of nabh4 as reducing agent and achieved after only two minutes. the aunps provided high antioxidant ability. in addition, the synthesized aunps showed a significant inhibitory effect against both gram-positive and gramnegative bacteria, where the zone of inhibition of 4 and 9 mm were obtained for synthesized aunps against s. aureus and e. coli, respectively. keywords antibacterial, antioxidant, catalyst, gold nanoparticles, pimenta dioica 1. introduction researchers are interested in gold nanoparticles (aunps) because of their applications in optoelectronic [1], electrical [2], and magnetic devices [3], as well as catalysis [4]–[6], sensors [7]– [9], and drug delivery [10]. form, size, crystallinity, and structure are all important factors in these applications. gold nanostructures have been identified in a variety of shapes, including nanocubes [11], nanowires [12], nanoplates [13], nanospheres, nanorods [14], nanopores [15], nanobelts, and odd angled shapes [16]. many groups of researchers have long been interested in one-dimensional (1d) metallic nanostructures. the researchers were drawn to aunps because of their unusual and tunable surface plasmon resonance. because of their non-cytotoxicity and biocompatibility, aunps have become more common in biomedical applications than other metallic nanostructures [17]. aunps are simple to make and have excellent chemical and thermal stability. because of their unusual biocompatibility and well-defined surface chemistry, aunps have been used as detection methods in molecular imaging using fluorescence resonance energy transfer [18]. due to its unique characteristics such as safety, environmental friendliness, and cost-effectiveness, green synthesis of aunps has gotten a lot of attention [19][20]. for the synthesis of aunps, various physical and chemical methods have already been used [21]–[23]. most of the approaches require long experiment time, involve a multi-step synthesis procedure, and use the harmful organic and inorganic chemicals [22]. the existence of these toxic chemicals in trace amounts on the surfaces of fabricated aunps is a possibility, which would restrict their biomedical applications. as a result, a method to synthesize biocompatible aunps that is both environmentally friendly and free of toxic chemicals is needed. plant-mediated green synthesis is an environmentally friendly method for producing monodisperse and chemically stable aunps of various sizes and shapes. plant phytochemicals or biomolecules play a key role in the reduction of metal ions during this synthesis phase [24]. copyright holder: © fadaka, a., aluko, o., awawu, s., and theledi, k. (2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.81 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.81 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.81&domain=pdf&date_stamp=2021-07-07 j. multidiscip. appl. nat. sci. 79 pimenta dioica (l.), also known as allspice, belongs to the myrtaceae family. p. dioica has anesthetic, antiseptic, antihelmintic, febrifuge, and antiseptic properties. p. dioica dried fruits are a valuable spice used all over the world. the therapeutic properties of p. dioica fragrance oils include antioxidant, antimicrobial, tonic, hypotensive, anti-inflammatory, and anthelmintic properties [25]. pimenta dioica has been used in the preparation of silver nanoparticles in previous studies [26]. carbohydrate, protein, steroid, alkaloid, flavonoids, phenol, and terpenoids were detected in the ethanolic extract; protein, phenol, and terpenoids were detected in the diethyl ether extract; and carbohydrate, alkaloid, flavonoids, steroids, saponins, tannin, and terpenoids were detected in the aqueous extract. by using a ferrous ion chelating assay, a nitric oxide radical scavenging assay, and a dpph test, the antioxidant content of p. dioica leaf extract was determined. the occurrence of carboxylic acid, alkyl halides alkanes, and miscellaneous functional groups in p. dioica leaves have been reported by murali et al. [27]. kharey et al. [28] obtain green synthesized aunps using aqueous leaf extract of p. dioica. using mtt (3‐(4,5‐dimethylthiozol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide) assay, synthesized aunps safe for human cervical cancer (hela) and human embryonic kidney 293 (hek 293) cell lines. further, the synthesized aunps showed excellent photoacoustic signal responses (pasr) and found to be the most efficient photoacoustic signal generators. as a result, the aim of this study was to synthesize aunps from p. dioica leaf aqueous extract without using any other chemicals. several spectral and microscopic methods revealed the formation of nps. the effectiveness of aunps in reducing the organic dye pollutant methylene blue (mb) was also investigated. at the same time, the antioxidant and antibacterial properties of the synthesized aunps were examined. 2. materials and methods 2.1. materials pimenta dioica leaves were freshly collected from lapai, nigeria. gold (iii) chloride tri-hydrate (haucl4.3h2o), 2,2-diphenyl-1-picrylhydrazyl (dpph), methylene blue dyes (mb) and sodium borohydride (nabh4) were obtained from sigmaaldrich. deionized ultra-pure water was used for preparing all respective solutions. 2.2. methods 2.2.1. preparation of aqueous extract under strict sterile conditions, about 25 g of fresh p. dioica leaves were softened in a mortar and pestle and filtered through muslin cloth. in a clean and fresh glass bottle, the filtrate was collected. this filtrate will be kept cold for further examination. around 5 g of leaves were crushed and mixed with 10 ml of deionized ultra-pure water before being ground. the extraction process took 3 h and was held overnight. the solution was filtered the next day, and the filtrate was processed for further analysis. 2.2.2. biosynthesis of aunps by p. dioica leaves aqueous extract the reduction of au 3+ to au 0 , as described by jayaseelan et al. [29], was used to produce aunps mediated by p. dioica extract. as much as 40 ml of p. dioica leaves aqueous extract was added to 60 ml of 1 mm aqueous haucl4.3h2o solution and the solution was placed in orbital shaker at room temperature, for reduction of au 3+ to au 0 . the bioreduction of the gold ions in the solution was monitored periodically by measuring the uv– visible (uv-vis) spectroscopy of the solutions. the reaction was rapid as the ruby red color appeared within 10 min and the reaction confirmed the formation of au nps and there was no color change figure 1. uv–visible spectrum of synthesized aunps showed peak at 536 nm. j. multidiscip. appl. nat. sci. 80 further. the control leaves aqueous extract of p. dioica did not show any change in color. the optimum time required for the completion of reaction was 10 min. different concentration of haucl4 solution was used to get maximum aunps. the overall optimized reaction condition was observed in 1 mm haucl4 solution and neutral ph. the aunps obtained from the solution were purified by repeated centrifugation at 2000 rpm for 10 min followed by dispersion of the pellet thrice in deionized ultra-pure water to remove the watersoluble biomolecules such as proteins and secondary metabolites. the water suspended aunps were frozen at 30 °c overnight and then kept under vacuum for 24 h to dry the aunps. 2.2.3. characterization the morphology of the synthesized aunps (including nanoscale, shape, and uniformity) was investigated by tem microscope, (jeol, model jem-2010) with an accelerating voltage of 200 kv. the optical absorbance of the as-prepared nps solutions was measured by uv–vis spectrophotometer (shimadzu uv-2450, one cm wide quartz cells). the structural information and types of functional groups from the extract that may be involved in the synthesis and stabilization of aunps were obtained by ftir spectrophotometer (jasco spectrometer) over the 4000 to 400 cm -1 range. the particle size range of the nanoparticles were determined by using particle size analyzer (malvern zetasizer nanosizer). 2.2.4. the photocatalytic activity the photocatalytic activity of aunps was assessed by investigating the reduction of mb dye via nabh4, whereas the biologically as-synthesized aunps were utilized as a catalyst at 25 ºc under solar light. the procedure started by adding 2.5 ml of 0.08 mm mb dye solutions in a one cm path length quartz cuvette. then, 0.5 ml of freshly prepared nabh4 (0.06 m) solution was added to the dye solution, followed by the addition of 0.5 ml of colloidal aunps. afterward, the solution was subjected to solar light under gentle stirring. the dye’s reduction reaction was monitored by recording changes in the mb absorption after one minute using the following equation 1 [24]: while, co = initial concentration of dye, c = the concentration of dye upon irradiation. 2.2.5. the antioxidant efficiency the free radical scavenging potential of the plant extract and prepared nps were assessed using dpph, according to amrulloh et al. [30] method. briefly, the synthesized aunps solution (different volume 10, 20, 30, 40, and 50 µl) was mixed with 1000 µl of dpph (0.2 mm). a dpph solution without nps was also examined and utilized as a negative control. additionally, the test was also made on two-fold-diluted solutions of each sample. after that, the dpph solutions were blended for 20 min at ambient temperature in a dark condition. at figure 2. (a) tem image and (b) particle size distribution of synthesized aunps. ( ) degradation rate (%) 100% co c x co − = j. multidiscip. appl. nat. sci. 81 the end of the incubation period, the radical concentration was determined by following the reduction in its absorbance percentage at a specific wavelength of 517 nm. the scavenging efficiency was expressed as the inhibition percent of dpph radicals, and it was calculated according to the following equation 2 [24]: 2.2.6. the antibacterial assays antibacterial activity was analyzed with synthesized aunps by well diffusion method against clinically isolated gram negative (escherichia coli) and gram positive (staphylococcus aureus) microorganisms. the pathogenic cultures were bringing into broth culture for antibacterial assay. approximately 7-mm diameter of well was made on muller hinton agar plate with the help of gel puncture. the cultures were swabbed on test media with sterile cotton swab. various volume (10, 20, 30, 40, and 50 µl) of synthesized aunps were inoculated to the well, and then the plates were incubated in incubator for 37 °c for 24 h, the zones of inhibition indicated the antibacterial activity was measured in mm. 3. results and discussions 3.1. spectral, morphological, and structural characterization 3.1.1. uv-visible spectroscopy, tem, and psa the green synthesis of aunps using environmental and nontoxic chemicals is the current approach. one of the most frequently used materials in green synthesis is the plant extract, which contains functional groups that can reduce au 3+ to au 0 . the uv–vis absorption spectra demonstrates a novel technique for the preparation of the au nps. the scale of wavelength was fixed between 250 and 800 nm, the surface plasmon resonance (spr) of the aunps formed corresponded to 536 nm and there was an increase in intensity till 10 min as a function of time without any shift in the peak wavelength (figure 1). it can be observed that the reduction of gold ions reaches saturation within 10 min of reaction, and after that, only slight variations can be noted in the intensity of spr bands [24]. this result indicates that the reaction is completed in 10 min. tem images precisely reveal the morphological tuning with perpetual changes in synthesis conditions. figure 2 shows tem images of aunps. the particle size distributions of colloids aunps from psa result are depicted in the histograms. the average particle size measured for aunps colloids is 11.48 nm. 3.1.2. ftir spectroscopy and x-ray diffraction analysis ftir analysis was used to identify the possible bio-reducing biomolecules in the extract [31]. the figure 3. ftir spectrum of synthesized aunps and p. dioica leaves aqueous extract. % 100% control absorbance sample absorbance inhibition x control absorbance = j. multidiscip. appl. nat. sci. 82 presence of functional groups on the surface of aunps was identified by the ftir measurement of synthesized aunps. ftir spectra of aunps and plant extract are displayed in figure 3. the major peaks were found at 3387, 1635, 1470, 1340, 1210, and 1070 cm -1 respectively. the peak at 3387 cm -1 indicates the presence of oh (phenolic/aliphatic hydroxyl) group. the peak at 1635 cm -1 represents the –c=c stretching of the alkene bond in eugenol. the peak at 1470 cm -1 corresponds to –c–c bond of an aromatic ring. the peaks at 1340, 1210, and 1070 cm -1 correspond to the asymmetric and symmetric c–o–c stretching mode, respectively, which confirms the presence of the methoxy group on the benzene ring of eugenol [32]–[34]. these peaks are consistent with the ftir peaks of functional groups of eugenol, present in the p. dioica plant [28]. the ftir spectrum of aunps shows the peaks at 3323, 1645, 1425, 1325, and 1045 cm -1 respectively. a minor wavelength shift is observed in the peaks of the functional groups that were present in the extract before and after the nanoparticle formation. this peak shift could be attributed to the interaction of functional groups in p. dioica plant extract with the nanoparticles. the strong band at 3323 cm -1 is the characteristic band of phenolic compound while the weak band at 1645 cm -1 corresponds to the alkene group of eugenol constituent of plant extract [35]. results suggested that eugenol not only helps in the reduction of gold ions but also stabilizes the aunps [36]. the structural phase of the aunps formed were studied through xrd analysis. figure 4 shows the strong and narrow diffraction peaks indicated that the product has well crystalline. the xrd peaks at 38, 44, 64 and 77 (º 2theta) can be indexed to the (111), (200), (220) and (311) bragg’s reflections of sphere structure of metallic gold respectively (jcpds no. 04-0784) [29]. the broadening of bragg’s peaks indicates the formation of aunps. nearly monodispersed aunps with controllable size and uniform shape can be easily obtained in the simple aqueous reduction method. 3.2. the aunps photocatalytic study for reduction of methylene blue contamination of water with organic dyes has become a formidable challenge in recent years. figure 5. absorbance spectra of photocatalytic degradation for mb dye in the presence of aunps. figure 4. xrd pattern of synthesized aunps. j. multidiscip. appl. nat. sci. 83 methylene blue (mb) was chosen as the subject of our study because it is one of the most widely used organic dyes in many industries, and its residue in wastewater is considered toxic contamination. the mb was widely used in industry as a redox indicator, and the traditional method for removing it from wastewater was to use a reducing agent like nabh4, which produces hydrogen gas through a hydrolysis reaction in aqueous solution. the catalytic activity of aunps in the presence of nabh4 is investigated in this study. the absorption spectrum of mb is well known for having a peak at 664 nm attributed to the n-* transition and another shoulder peak at 614 nm [29]. in the presence of nabh4, this peak gradually decreases due to the formation of the reduced form known as leuco methylene blue (lmb) [37]. nonetheless, it was discovered that the rate of reduction by nabh4 was slow and prolonged. aunps have been used as a catalytic material. the resulting absorption spectra is plotted in figure 5. the blue color of the mb and nabh4 solution was observed to vanish when sample from prepared aunps was added to the solution. after two minutes, the intensity of the peak at 664 nm reduced, indicating that the aunps were an active catalyst for mb reduction. following the control sample (nabh4 solution without any aunps), a 73% reduction was obtained after 8 min and it remained unchanged for 10 min. accordingly, a clear improvement and enhancement were obtained due to the addition of the aunps. 3.3. antioxidant activity it has been documented that a reactive oxygen species and other free radicals may be formed and released during a variety of biological processes, resulting in pathogenicity [36][37]. antioxidant behavior refers to the formation of non-reactive and stable radicals as a result of the inhibition of any molecule's oxidation mechanism. this is accomplished by preventing the oxidative chain reaction's initiation step. as a result, considerable effort was expended in the quest for an antioxidant agent capable of obstructing or preventing oxidative harm [40]. it was recently discovered that aunps, especially those prepared in a green route, have significant antioxidant potential, which is dependent on the properties of various phytochemicals encrusted on the surface of the aunps [24][39][40]. the dpph assay is a quick and easy way to figure out the preliminary results. the synthesized aunps in this study have a higher propensity for scavenging dpph (table 1). the colloids scavenging action increases as the concentration of the synthesized gold colloid rises. tannins and polyphenols are abundantly found in the aqueous extract of p. dioica leaves [27]. these bioactive compounds synergic activity as a reducing and stabilizing agent has been predicted. 3.4. antibacterial activity noble metals and their compounds exhibit antimicrobial properties, allowing them to be used to treat burns and chronic wounds. metal nanoparticles' superior biological performance is reported due to their high surface area to volume ratio as compared to their bulkier counterparts [43]– [45]. the antibacterial activity of synthesized aunps was tested on a variety of bacteria in this study under agar well diffusion method. table 2 shows antibacterial effect of bioinspired aunps on different human pathogens s. aureus and e.coli. for an addition of 30 μg/ml of the gold colloid, an inhibition zone of 4 mm s. aureus and 9 mm for table 1. the dpph scavenging activity of synthesized aunps. sample concentration (µg/ml) dpph scavenging activity (%) p. dioica leave aqueous extract 50 68.87 ± 0.21 100 72.37 ± 0.15 150 78.15 ± 0.31 aunps 50 10.61 ± 0.10 100 18.54 ± 0.24 150 37.43 ± 0.42 j. multidiscip. appl. nat. sci. 84 e.coli was observed. the mechanism behind aunps' bactericidal effect has yet to be fully investigated. however, earlier studies cite many reasons for the impact in a hazy depiction of the process. aunps have been documented to bind to the cell membrane's surface and disrupt key functions such as permeability and respiration, resulting in antioxidant depletion and the development of reactive oxygen species (ros) [46]. metal nanoparticles can also release metal ions, which can penetrate the cell wall and cause dna damage and protein malfunction. the surface area available for touch determines how well the particles stick to the bacteria. as a result, smaller aunps with a higher surface area will have a stronger bactericidal impact than larger aunps [47]. the present study clearly indicates that the synthesized aunps show good activity against both gram-positive and gram-negative bacteria. 4. conclusions in summary, aunps were successfully biosynthesized by an inexpensive, fast, and safe approach, using pimenta dioica leaves aqueous extract, without the use of any toxic chemicals. tem imaging revealed that these aunps have spherical structure. moreover, ftir analysis suggested that the surface of aunps is covered by biomolecules found in the plant extract such as phenolics and eugenols. these aunps have demonstrated an excellent catalytic activity for the degradation of mb. additionally, aunps displayed the most potent antioxidant potential. also, an antibacterial inhibition was evident against grampositive and gram-negative pathogenic. author information corresponding author adewale fadaka — department of chemistry, ibrahim badamasi babangida university lapai, lapai – 911101 (nigeria); email: adewalefad@ibbu.edu.ng authors olukemi aluko — department of chemistry, ibrahim badamasi babangida university lapai, lapai – 911101 (nigeria); saartjie awawu — department of chemistry, ibrahim badamasi babangida university lapai, lapai – 911101 (nigeria); karim theledi — department of biotechnology, university 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k haryanto1); andranyssa v m rumondor1); hans kristianto1,*); 4 susiana prasetyo1); asaf k sugih1) 5 6 1 department of chemical engineering, parahyangan catholic university, bandung-40141 7 (indonesia) 8 *correspondence: hans.kristianto@unpar.ac.id 9 10 orcids: 11 first author : https://orcid.org/0000-0003-4148-6097 12 second author : https://orcid.org/0000-0002-0017-5623 13 third author : https://orcid.org/0000-0003-4747-4361 14 fourth author : https://orcid.org/0000-0001-5839-0054 15 fifth author : https://orcid.org/0000-0003-0478-540x 16 17 acknowledgement 18 19 this study is funded by parahyangan catholic university’s centre of research and 20 community service with contract no iii/lppm/2023-02/27-p. the authors are thankful for the 21 funding given. 22 23 author contributions 24 25 conceptualization, methodology, investigation, writing – original draft: fmkh, avmr. 26 both authors contributed equally to this work. resources, writing – review & editing, 27 supervision, funding acquisition: hk, sp, and aks. 28 29 conflict of interest 30 31 the authors declare no conflict of interest. 32 33 34 a cc ep te d m a n u sc ri pt mailto:hans.kristianto@unpar.ac.id https://orcid.org/0000-0003-4148-6097 https://orcid.org/0000-0002-0017-5623 https://orcid.org/0000-0003-4747-4361 https://orcid.org/0000-0001-5839-0054 https://orcid.org/0000-0003-0478-540x the utilization of pectin as natural coagulant-aid in 1 congo red dye removal 2 3 abstract. coagulation using inorganic compounds such as aluminum sulfate is commonly 4 used in water-wastewater treatment. however, there are some drawbacks to its utilization, such 5 as a significant decrease in the treated water’s ph, non-biodegradable sludge, and a potential 6 negative impact on human mental health (dementia and alzheimer's). the use of inorganic 7 coagulants can be minimized with the addition of natural-based coagulant-aid such as pectin. 8 in this study, congo red solution, a model dye substance, was coagulated by varying the ph 9 (3–7) using alum coagulant to determine the best ph for coagulation. at the best ph, pectin 10 was introduced at various doses (0–30 mg/l), and subsequently various dye concentrations 11 (50–100 mg/l). the effect of pectin as coagulant-aid was compared with aluminum sulfate and 12 pectin only; with a response of %removal and sludge volume. it was found that the congo red 13 dye coagulation had the best %removal at ph 6 indicating a charge neutralization mechanism. 14 the addition of 15 mg/l pectin at an aluminum sulfate dose of 30 mg/l resulted in 97.7% dye 15 removal with a sludge volume of 14 ml/l at a congo red concentration of 50 mg/l. this value 16 is higher compared to those of aluminum sulfate and pectin only which gave 75.6 and 3.19% 17 removals, respectively. furthermore, the addition of pectin as a natural coagulant-aid could 18 halve the sludge volume due to the formation of denser flocs. the results show a promising 19 potential of pectin as a natural coagulant-aid in water-wastewater treatment. 20 21 keywords: congo red, coagulant-aid, natural coagulant, pectin 22 23 1. introduction 24 25 coagulation and flocculation are the most commonly used methods for water and 26 wastewater treatment due to their high efficiency and low cost [1]. furthermore, inorganic 27 coagulants such as alum (al2(so4)3), fecl3, and pac (poly(aluminum chloride)) are widely 28 used due to their ability to reduce color intensity, organic content and turbidity [2]. aside from 29 its high efficiency, the use of inorganic coagulants has several limitations, including lowering 30 water alkalinity, producing large amounts of sludge, and the possibility of adverse effects on 31 a cc ep te d m a n u sc ri pt both human health and the environment [3]. as a result, natural-based coagulants have been 1 explored as an alternative to reduce or substitute the use of inorganic coagulants. 2 natural coagulants could be classified as protein, polysaccharide, or polyphenol, based 3 on their active coagulating agent [4]. protein-based coagulant that comes from beans and 4 legume extract has been extensively studied. however, the extraction and purification steps 5 turned out to form a hindrance to its wide application and commercialization [3]. on the other 6 hand, polyphenol-based coagulant has also been investigated. even so, the amount of research 7 is relatively limited, thus more investigation in the future is needed [5]. lastly, polysaccharide 8 is known as the most abundant polymer found in nature [6], making it an interesting source to 9 be employed as natural coagulant. 10 previous studies have utilized various polysaccharide sources as natural coagulants, such 11 as starch, gum, chitosan, and the like. starch from rice, sago, and corn has been explored as 12 natural coagulant to treat textile wastewater, landfill leachate, and agro-industrial wastewater 13 [7]-[10]. this starch gave a good coagulation performance. however, the utilization of starch 14 from food sources poses a potential conflict of interest, thus limiting its commercial application 15 [11]. gum from various sources also has been utilized as natural coagulant and coagulant-aid 16 [12]-[15]. fruit industrial waste is an alternative non-food source that can be considered for 17 use in the application as well. this fruit waste which is commonly generated as agricultural 18 byproduct has a high potential for various applications due to its various compounds [16]. this 19 waste contains active ingredients in the form of polysaccharides such as pectin and starch, 20 which have shown their potential as coagulant and coagulant-aid [17][18]. 21 compared to starch, pectin is a good alternative as coagulant-aid because pectin can be 22 used as it is, without any modification needed. pectin has also been commercialized and widely 23 used for various applications such as food additives [19], medical applications [20], and 24 nutraceuticals [21]. pectin is a heteropolysaccharide structure that is composed of α-d-(1,4) 25 galacturonan and rhamnogalacturonan with branches consisting of neutral sugar such as 26 rhamnose, arabinose, galactose, and xylose [22]. moreover, pectin can be differentiated as high 27 and low-methoxy pectin, based on the degree of esterification. high-methoxy pectin with a 28 degree of esterification of over 50% can be made to interact with sugar to form gels, and is 29 commonly used in jam-making [23]. on the other hand, low-methoxy pectin has a degree of 30 esterification lower than 50%. it can interact with multivalent cations via the egg box model 31 [23], making it suitable for application as coagulant-aid. 32 furthermore, compared to synthetic polymers, the utilization of pectin could become a 33 good alternative due to its highly biodegradable and non-toxic properties. for example, 34 a cc ep te d m a n u sc ri pt polyacrylamide and acrylamide copolymers (anionic polymers) are commonly used as 1 flocculant with a high molecular weight, being very stable, readily soluble in water, and very 2 effective at a low dosage. however, this polymer is hardly biodegradable with a concern of the 3 polymer and monomer’s toxicity [24]. another example is poly(diallydimethylammonium 4 chloride) (polydadmac), a cationic polymer, was proven to be chronically toxic to 5 ceriodaphnia dubia [25], emphasizing the toxicity of synthetic polymer to aquatic organisms 6 and the ecosystem. 7 previously, pectin had been used as a coagulant or coagulant-aid to remove heavy metals 8 [26] and turbidity [27][28] in synthetic wastewater. however, to the best of the authors’ 9 knowledge, the study of pectin as coagulant-aid to treat synthetic dye wastewater has never 10 been investigated. in this study, the application of low-methoxy pectin as a coagulant-aid and 11 alum has been used to treat synthetic congo red as a model wastewater. several variables that 12 influence the coagulation, such as ph, coagulant-aid dosage, and dye concentration have also 13 been examined. 14 15 2. materials and method 16 17 2.1. materials. congo red dye was obtained from sigma-aldrich, while alum (technical 18 grade) and low-methoxy pectin (food grade) was purchased from a local shop in bandung, 19 west java, indonesia. all chemicals were used as obtained, without any further treatment. the 20 general properties of congo red and pectin are presented in table 1. 21 22 table 1. general properties of congo red and pectin 23 properties congo red pectin molecular formula c32h22n6na2o6s2 c6h10o7 (monomer) molecular weight (g/mol) 696.7 g/mol 194.1 (monomer) molecular structure 24 n o o h h h o oh h oh h o o h h h h oh h o oh o o ch 3 a cc ep te d m a n u sc ri pt 2.2. jar test experiment. the coagulation study of congo red was conducted by using a 1 jar test apparatus. a stock congo red solution (1 g/l) was prepared and subsequently diluted 2 using distilled water to obtain the desired congo red concentration. the variations of 3 coagulation study are presented in table 2. the initial ph of the solution was adjusted by using 4 0.1 m hcl or naoh and measured by using a calibrated ph meter (lutron ph-208). the 5 coagulation was accomplished by mixing congo red, alum and followed by pectin solutions at 6 rapid mixing (200 rpm, 2 min), followed by slow mixing (40 rpm, 20 min), and settling for 1 7 h. the initial (ci; mg/l) and final (cf; mg/l) concentrations of congo red were measured using 8 a visible spectrophotometer (thermoscientific genesys 30) at its maximum wavelength (510 9 nm). the removal percentage (%removal) was calculated using equation 1. furthermore, the 10 sludge volume (ml/l) was measured using an imhoff cone after 1 h of settling and calculated 11 using equation 2. all experiments were carried out in duplicate and standard deviations as 12 provided in each figure, where applicable. 13 14 % 𝑟𝑒𝑚𝑜𝑣𝑎𝑙 = (𝐶𝑖−𝐶𝑓) 𝐶𝑓 × 100% (1) 15 16 𝑠𝑙𝑢𝑑𝑔𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 ( 𝑚𝐿 𝐿 ) = 𝑉 𝑠𝑙𝑢𝑑𝑔𝑒 (𝑚𝐿) 𝑉 𝑤𝑎𝑠𝑡𝑒𝑤𝑎𝑡𝑒𝑟 (𝐿) (2) 17 18 table 2. variations applied in this study 19 variations ph alum dose (mg/l) pectin dose (mg/l) congo red concentration (mg/l) ph study 3, 4, 5, 6, 7 50 0 50 coagulant-aid study best ph 30 0, 5, 10, 15, 20, 25, 30 50 initial congo red concentration best ph 30 best pectin dose 50, 60, 70, 80, 90, 100 30 0 0 best pectin dose 20 3. results and discussion 21 22 3.1. the effect of ph levels on coagulation. the profile of congo red dye concentration 23 and sludge volume in coagulation was observed at different ph variations (3, 4, 5, 6, and 7) 24 a cc ep te d m a n u sc ri pt with the coagulant dose and congo red dye concentration fixed at 50 mg/l. the obtained results 1 are shown in figure 1 below. it can be observed in figure 1 that with the increase of ph, the 2 congo red removal also increased until ph 6, before decreasing at ph 7. the highest %removal 3 was obtained at the initial ph of 6 with 92% and a sludge volume of 20 ml/l. in the ph range 4 of 3–6, the congo red would be positively charged, as the congo red zero charge is around ph 5 2. furthermore, around ph 5 and 6, most of the soluble alum species were in the form of al3+, 6 aloh2+ and al(oh)2+ [29] which could neutralize the negatively charged congo red 7 molecules. with the dye molecule’s neutral charge, the electrostatic repulsion decreases, 8 allowing the formation of flocs. a further increase in ph resulted in hydrolyzation of the alum, 9 forming al(oh)3 and al(oh)4while decreasing the positively charged alum species, thus 10 preventing charge neutralization to occur [30]. this reduces the dye’s coagulation efficiency 11 at ph 7, resulting in a decrease in sludge volume as well. the result obtained in this study is 12 similar to previous research studies that reported that the best performance of alum coagulation 13 was achieved at ph 6 [31]-[33]. 14 3 4 5 6 7 0 10 20 30 40 50 60 70 80 90 100 110 % removal sludge volume ph % r e m o v a l -10 0 10 20 30 40 50 60 70 80 s lu d g e v o lu m e ( m l /l ) 15 figure 1. effect of ph on %removal and sludge volume 16 17 3.2. the effect of coagulant-aid dose on coagulation. the effect of the coagulant-aid dose 18 on the %removal of congo red is presented in figure 2. the dose of coagulant-aid was varied 19 a cc ep te d m a n u sc ri pt with the dye concentration fixed at 50 mg/l and the alum dose at 30 mg/l. the amount of alum 1 used in this variation was decreased to 30 mg/l in order to observe the significance of the role 2 pectin plays as a coagulant-aid. it may be observed that an increase in congo red removal 3 occurred with the addition of pectin until 15 mg/l which gave a 97.0% removal; compared to 4 alum which only gave a 76.0% removal. further addition of pectin did not increase the 5 %removal of congo red. 6 this result clearly indicates that pectin as a coagulant-aid works synergistically with alum 7 in supporting the floc formation. this is closely related to the complex formation that occurs 8 between pectin with small flocs formed previously from the interaction of congo red and alum. 9 coagulation that occurs between the alum coagulant and congo with a charge neutralization 10 mechanism will produce small flocs [34]. pectin which is subsequently added as a coagulant-11 aid could act as a particle bridge that helps the formation of larger flocs, resulting in higher 12 coagulation performance. the amount of pectin that was gradually raised until it was over the 13 optimal level did not give a significant increase in the %removal, instead a slight decrease of 14 %removal and increase sludge volume was observed. this is possible due to the over-addition 15 of pectin that could lead to an increase of zeta potential, making a smaller floc formation [35]. 16 in turn, this could lead to more porous and voluminous sludge. similar observations have been 17 reported by previous researchers [15][36][37]. 18 0 5 10 15 20 25 30 0 20 40 60 80 100 % removal sludge volume pectin dose (mg/l) % r e m o v a l 0 5 10 15 20 25 30 s lu d g e v o lu m e ( m l /l ) 19 a cc ep te d m a n u sc ri pt figure 2 effect of pectin dose on %removal and sludge volume 1 2 3.3. the effect of dye initial concentration on coagulation. the profile of the %removal 3 of congo red dyes was observed in various initial concentrations of congo red dyes (50–100 4 mg/l), with a fixed dose of alum (30 mg/l) and pectin (15 mg/l) at the best ph of 6. the result 5 is presented in figure 3. 6 50 60 70 80 90 100 0 20 40 60 80 100 alum alum+pectin pectin alum alum+pectin pectin congo red concentration (mg/l) % r e m o v a l 0 5 10 15 20 25 30 35 40 s lu d g e v o lu m e ( m l /l ) 7 figure 3 effect of initial dye concentration on %removal and sludge volume 8 9 at a dosage of 15 mg/l, pectin had a very poor coagulant activity and was unable to 10 coagulate the dye molecules. this is apparent from the %removal, which amounted to a mere 11 3.19% and fell as the initial dye concentration rose. pectin typically has a negative high zeta 12 potential value at ph 6, which is around -25 mv [38]. in addition, congo red has an isoelectric 13 point around ph 2 [39] that would be negatively charged as well at ph 6. based on these facts, 14 it can be concluded that pectin does not act as the active coagulating agent in this study. 15 furthermore, it can be observed that with the increase of congo red concentration, the 16 %removal and sludge formation were also decreasing. this is possible due to the inadequacy 17 of active coagulating sites to neutralize the dye molecules with the increase of congo red 18 concentration, as the interaction between coagulant and colloid has been reported as 19 a cc ep te d m a n u sc ri pt stoichiometric interaction. the effect of pectin as coagulant-aid is discernible at the initial 1 congo red concentration of 50–60 mg/l that gave %removal of 97.1 and 70.8% respectively. 2 this value increased significantly when compared to the one measured without using pectin 3 which gave 76.0 and 53.5% removals at the same initial congo red concentration. this increase 4 confirmed a synergistic effect between alum and pectin that resulted in bigger observed flocs 5 (figure 4) and higher congo red removal. the possible interaction between congo red, alum, 6 and pectin is illustrated in figure 5. the negatively charged sulfonate groups in the congo red 7 molecule would be neutralized by the positive al3+, while the carboxylate groups in the pectin 8 structure could also interact with al3+, resulting in a particle bridging effect (figure 5.a). this 9 bridging mechanism could explain the formation of bigger flocs, as observed in figure 5.b, 10 which resulted in a lower sludge volume and higher removal. similar results of polysaccharides 11 that act as a bridge between smaller flocs have been reported previously [31][34][40]. 12 13 14 figure 4. observed flocs formation with 15 mg/l pectin (a) and without pectin (b) at ph 6. 15 [alum] = 30 mg/l. [congo red] = 50 mg/l. 16 17 the results obtained in this study are compared with other coagulants for congo red 18 removal, presented in table 3. it can be observed that the coagulation performance of alum 19 with the aid of pectin could give a comparable coagulation performance with other coagulants 20 reported in the background literature. furthermore, it can be seen that a smaller alum dosage 21 (30 mg/l) is needed to obtain a 97% removal with the 15 mg/l pectin addition, compared to 22 research conducted by [41] where 100 mg/l alum was required. 23 24 25 26 27 a. b. a cc ep te d m a n u sc ri pt table 3. comparison of various coagulants for congo red removal 28 coagulant coagulant-aid ph congo red concentration (mg/l) % removal reference type dosage (mg/l) type dosage (mg/l) al2(so4)3 30 pectin 15 6 50 97.1 this study al2(so4)3 30 6 50 76.0 this study cacl2 4,000 alginate 20 4 50 92.2 [42] fecl3 160 galactomannan 80 6 20 90.0 [15] fecl3 10 a. tetragonus 300 3 500 92.0 [30] polyaluminium chloride 15 3 50 81.2 [40] chitosan 25 7 200 66.0 [43] al2(so4)3 nanoparticles 100 7 50 99.5 [41] 29 a cc ep te d m a n u sc ri pt 30 figure 5. possible interaction between al3+‒congo red and al3+‒pectin (a) and the particle 31 bridging of flocs by pectin (b) (reused with permission from [40]; copyright © elsevier license 32 number 5524610093660) 33 34 4. conclusion 35 36 in this study, low-methoxy pectin has been successfully utilized as a coagulant-aid in 37 congo red dye removal. at various initial phs, it was found that congo red removal increases 38 from ph 3–6 with ph 6 as the best ph for coagulation via a charge neutralization mechanism. 39 the addition of pectin could assist the congo red coagulation process. the addition of 15 mg/l 40 of pectin gave a 97% congo red removal, compared to the 76% removal when only alum was 41 used as the coagulant. overdosing of pectin could result in colloid re-stabilization that makes 42 a smaller floc formation and increases the sludge volume. at various initial congo red 43 concentrations and fixed coagulant and coagulant-aid doses, the effect of pectin addition could 44 increase the congo red removal at an initial concentration of 50–60 mg/l, which gave an 45 n o o h h h o oh h oh h o o h h h h oh h o oh o o ch 3 nh2 s o o o n n nh2 s o o o n n al 3+ congo red pectin colloids alum small flocs pectin bigger flocs a. b. a cc ep te d m a n u sc ri pt approximately 20% increase of removal compared to alum only. at a higher congo red 46 concentration, the removal became lower than the one without pectin. this might happen due 47 to an inadequacy of alum at a fixed coagulant dosage with the increase of congo red 48 concentration. this condition could lead to a competitive interaction of alum-congo red and 49 alum-pectin, where both congo red and pectin are negatively charged at the coagulation 50 condition. the results of this research study show that low-methoxy pectin is a potential 51 coagulant-aid that could work synergistically with alum in the coagulation process. further 52 studies are required to observe the potential of the combination between alum and pectin in 53 other types of wastewaters, real wastewater, and optimization of the variables as well. 54 55 references 56 57 [1] a. k. verma, r. r. dash, and p. bhunia. 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"removal of congo red dye from its aqueous solution 200 using natural coagulants". journal of saudi chemical society. 16 : 131–136. 201 10.1016/j.jscs.2010.12.003. 202 203 a cc ep te d m a n u sc ri pt https://doi.org/10.1016/j.jhazmat.2011.03.087 https://doi.org/10.3390/molecules22101707 https://doi.org/10.1007/s13369-020-05008-1 https://doi.org/10.1016/j.heliyon.2023.e15011 https://doi.org/10.1016/j.matchemphys.2020.123040 https://doi.org/10.1080/19443994.2015.1008578 https://doi.org/10.1016/j.jscs.2010.12.003 10 journal of multidisciplinary applied natural science vol. 2 no. 1 (2022) research article effect of plant growth regulators on growth and quality flower production of chrysanthemum (chrysanthemum indicum l.) md. ehsanullah, ahasan ullah khan*, md. kamruzzam, and sarah tasnim received : june 29, 2021 revised : november 5, 2021 accepted : november 10, 2021 online : november 11, 2021 abstract a field study was conceded to assess the effect of plant growth regulators on growth and quality flower production of chrysanthemum at horticulture research centre (hrc), gazipur, bangladesh. the experiment was laid out in randomized complete block design (rcbd) with ten (10) treatments and three replications. the treatments of plant growth regulators concentration were t1-50 ppm ga3, t2-100 ppm ga3, t3-150 ppm ga3, t4-400 ppm ccc, t5-600 ppm ccc, t6-800 ppm ccc, t7-250 ppm mh, t8-500 ppm mh, t9-750 ppm mh and, t10-control. the maximum spreading of plant (27.0 cm) was observed when plants were treated with ga3 @ 150 ppm where the minimum plant spread (16.8 cm) was recorded in plants treated with ccc @ 800 ppm. the higher number of suckers (33) per pot was produced when pots were treated with ga3 @ 150 ppm whereas, application of ccc at three different concentrations produced lower number of suckers. the highest number of flower (40) was recorded with 150 ppm ga3, where minimum number of flowers (25) per pot in 800 ppm ccc. the plants sprayed with 50 ppm ga3 took 48 days to flower initiation, whereas, it took 70 days with 750 ppm mh. the highest plants recorded (7.40 cm) with 800 ppm ccc, whereas, lowest size (6.50 cm) was obtained with the application of 500 ppm mh. the maximum vase life of flowers was recorded for the treatment 800 ppm ccc (15 days), which was at par with 13 days vase life obtained by spraying 600 ppm ccc. therefore, it is concluded that the ga3 acted as growth promoter and the ccc acted as growth retardants on yield and quality of chrysanthemum. keywords chlormequat chloride (ccc), flower, gibberellins (ga), growth promoter, meleic hydrazide, production 1. introduction chrysanthemum (chrysanthemum indicum l.) is a widespread saleable attractive importance flower crop belongs to the family compositae or asteraceae, sub family asteroideae, order asterales, subclass asteridae, tribe anthemideae. it is significant as floricultural, ornamental and medicinal used in modern time [1]–[3]. this flower crop is native to east asia and has been grown in garden for more than 2500 years [4]–[6]. it is globally the second economically most vital floricultural crop following rose, and one of the most significant ornamental species [2]. it is one of the most important ornamental crops around the world, it is produced as both cut flower in field and pot plant [7]. many plants, which have been identified as yet through pharmacology, folk medicine [8], homoeopathy and ethnopharmacology [9], are being investigated for their medicinal usage and may be proved so in due course of time. the c. indicum flower is a good source of common quercitrin and myricetin, which is significant for the progress of possible pharmaceuticals [10]. the flower of the c. indicum contains major 3 oils viz 1,8-cineole, camphor, borneol and bornyl acetate [11]. this crop use as nerve sedative, anti-oxidant, anti-inflammatory, anti-mutagenic, anti-microbial, anti-fungal, anti-angiogenic, anti-atherosclerosis and nematocidal goods [12]. the leaves remedy and use as colds, headache, bronchitis, rheumatism, swellings, boils and expectorant, bitter and stomachic, respectively. the c. indicum flower has a strong aroma and many of the previous studies focused on the essential oil of this plant [13]–[15]. khan et al. [16] observed that the plant height (54.0 to 66.0 cm); number of leaves per plant (208-240); leaf size (4.5 to 8.5 cm); plant spread (19.0 to 32.0 cm); number of branches (4 to 12); number of flowers (25-40); stalk length (8.8 to 13.3 cm) and days of first flowering (55 to 70 days) varied; respectively in t7 (100% rice husk) to t3 (100% cocodust). the different color of leaves and flowers in chrysanthemum flower crop and also the maximum flower period was observed early december-february in germplasms. taweesak et al. copyright holder: © ehsanullah, md., khan, a. u., kamruzzam, md. and tasnim, s. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.98 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.98 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.98&domain=pdf&date_stamp=2022-01-13 j. multidiscip. appl. nat. sci. 11 [17] observed that the irrigation effect on plant height of chrysanthemum. the growth of chrysanthemum grown in two soilless systems included tray system and trough system. no significant differences in flower characteristics were observed between the two systems except for flower color. the commercial cultivation of chrysanthemum with good quality flowers and higher yield is needed for consumption in local market and to provide livelihood especially to the marginal and small farmers [18]. good quality flower production depends upon various factors such as genotype, environment, spacing, disbudding, pinching, substrate, use of growth regulator etc. [19]. plant growth regulators (pgrs) are now being commonly used for inducing more acceptable plant characteristics like compact growth, dwarfness, increased number of healthy branches and promote flower initiation [20]. similarly, pinching of apical bud has a significant influence on flowering and yield [21]. thus, growth regulators and pinching can play an important role in the improvement of flowering and yield of chrysanthemum. keeping in view the above points the present experiment was undertaken to compare the effects of growth regulators and hand pinching for higher flower yield in chrysanthemum. gibberellins (ga3) play an important role in growth and flowering of ornamental plants. foliar application of gibberellic acid enhances vegetative attributes along with flower initiation [22]. an experiment was conducted by dorajeerao and mokashi [22] and noted that foliar allocation of 3000 ppm ccc produced maximum number of flowers per plant, as compare to other concentrations. the plant growth regulators promote growth and yield in ornamental plants. keeping in view the above points the present investigation, plant growth regulators has been evaluated on the growth, and quality flower production in chrysanthemum crop. the general objective of this study is to assess the growth regulators for quality flower production of the chrysanthemum. 2. materials and methods 2.1. materials 2.1.1. experimental site the present investigation was carried out at the experimental farm of landscape, ornamental and floriculture division, hrc, bari, gazipur during the period from july 2007 to june 2008. the study area situated in 23.9917° n longitude and 90.4137° e latitude at an altitude of 9 meter above the sea level. 2.1.2. planting material seed of genotype of cm-022 were used in the experiment during the period from july 2007 to june 2008. 2.2. methods 2.2.1. pot preparation and treatments the experiment was conducted in earthen pots of 12 cm size. the pots were washed and cleaned thoroughly before filling up of potting media. in this planting media using plant growth regulators like gibberellins (ga), chlormequat chloride (ccc) and meleic hydrazide (mh) concentration. there were ten treatments in the experiment, comprising different plant growth regulators in quality flower production of chrysanthemum (table 1). the treatment of plant growth regulators concentration used in the experiment were 50 to 750 ppm. 2.2.2. design and layout of the experiment the experiment was laid out in randomized treatments plant growth regulators concentration t1 50 ppm ga3 t2 100 ppm ga3 t3 150 ppm ga3 t4 400 ppm ccc t5 600 ppm ccc t6 800 ppm ccc t7 250 ppm mh t8 500 ppm mh t9 750 ppm mh t10 control table 1. variables. j. multidiscip. appl. nat. sci. 12 complete block design (rcbd) with three replications. one plant was planted in a pot, containing the potting media according to the treatments and five plants were constituted the unit of treatment. 2.2.3. seedling raising, transplanting and fertilization primarily cuttings of cm-022 were prepared for planting in the sand in mid-august, 2007. immediately after rooting, the mini plantlets were transferred to pot containing media that consists of one-part coarse sand, one part garden soil, one part cocodust, one-part cowdung, a quarter part of wood ashes and two table spoonfuls of bone meal in midseptember, 2007. subsequently 10 g tsp and 3 g mp per pot were applied. urea @ 2, 3 and 3 g per pot was applied at 20, 30 and 40 days after transplanting respectively for getting best growth and flowering of plants [23]. 2.2.4. irrigation and weeding weeding and mulching were done in the pots whenever it was necessary to keep the pots free from weeds. chrysanthemum plants need frequent irrigation. the pots were irrigated every alternate day to keep the media moistened. 2.2.5. staking of plant each plant was supported by 40 cm long bamboo stick to facilitate the branches of the plant to keep erect. the plant in each pot was fastened loosely with the bamboo stick by jute string to prevent the plant from lodging. 2.2.6. harvesting of flowers the spikes were harvested when the flower attained commercial stage (flower open before shedding of pollens from the outer row of the disc florets). 2.2.7. collection of data data were collected on the following parameters for interpretation of the result of the experiment. the parameters were number of leaves plant -1 , plant spread, number of suckers plant -1 , leaf length, number of branches plant -1 , days to flowering, stalk length, number of flowers plant -1 , and flower size. number of leaves plant -1 : number of leaves per plant was recorded by counting all the leaves from 5 plants and the mean was calculated. plant spread: the plant spread was measured in cross way (north-south and east-west) by measuring scale. the average of the two measurements was done and expressed in cm. number of suckers plant 1 : number of suckers plant -1 was recorded by counting suckers from 5 individual plant and then mean was calculated. leaf length: the length of leaf was measured by a measuring scale from leaf base to the tip and was expressed in cm. number of branches plant -1 : number of branches per plant was recorded by counting all the main branches from 5 table 2. effect of plant growth regulators on plant characteristics in chrysanthemum. growth regulators (ppm) plant spread (cm) number of leaves leaf length (cm) t1 22.9b 125b 11.00b t2 25.0ab 135ab 12.00ab t3 27.0a 140a 13.35a t4 22.5b 117bc 9.90cd t5 18.5c 95d 8.63d t6 16.8cd 94d 8.47d t7 19.0c 96d 10.89bc t8 20.8bc 118bc 10.74bc t9 21.0bc 119bc 10.80bc t10 17.0cd 108c 9.20c cv (%) 15.25 16.00 14.92 note: t1-50 ppm ga3, t2-100 ppm ga3, t3-150 ppm ga3, t4-400 ppm ccc, t5-600 ppm ccc, t6800 ppm ccc, t7-250 ppm mh, t8-500 ppm mh, t9-750 ppm mh, t10-control j. multidiscip. appl. nat. sci. 13 plants and the mean was calculated. days to flowering: it was recorded by counting the days from planting to first visibility of flower bud in the plant from each pot. stalk length: length of stalk was measured from base to the tip of the spike and was expressed in cm. number of flowers plant -1 : number of flowers produced per plant was counted and recorded. flower size: flower size was measured in cross way following north-south and east-west position by a measuring scale and the average of the two measurements was done and expressed in cm for a single flower. later on, the mean of individual flower size from 5 selected plants was calculated. 2.2.8. statistical analysis the data recorded on different plant and floral parameters were statistically analyzed through analysis of variance with the help of ‘mstat’ software. the difference between treatment means were compared by duncan’s multiple range test (dmrt). 3. results and discussions 3.1. effect of plant growth regulators on plant characteristics in chrysanthemum table 2 showed that the different plant characteristics exhibited differences among the ten treatments under study. in general, ga3 treated plants showed significant improvement in plant spread compared to other treatment variables. the maximum spreading of plant (27.0 cm) was observed when plants were treated with ga3 @ 150 ppm which was closely followed by the application of ga3 @ 100 ppm. the minimum plant spread (16.8 cm) was recorded in plants treated with ccc @ 800 ppm. foliar application of ga3 might have influence on cell division and cell elongation that resulting in enhanced vegetative growth of plants. in contrast, ccc may act as growth retardants and thereby inhibited biochemical processes resulting in less spreading of plants. the findings agree with those of joshi et al. [24] and kim et al. [25] in chrysanthemum and thu et al. [26] in carnation . the variation in number of leaf production was pronounced by the application of different growth regulators. however, the highest number of leaves (140) was produced by the application of ga3 @ 150 ppm as foliar spray (table 2). this was closely followed by the other concentrations of ga3 @ 100 ppm. the effects of the ga3 treatments were observed at par but significantly superior to the rest of the treatments. all the concentrations of ccc were at par recording minimum number of leaves. this is similar with the findings of padmalatha et al. [27] who observed a greater number of leaves by the application of ga3 and a smaller number of leaves by foliar application of ccc. the leaf length was also significantly increased with the application of ga3 at different concentrations, of which ga3 @ 150 gave the longest leaf length (13.35 cm). leaf length highly reduced even in respect of control with the use of ccc growth regulators irrespective of concentrations. these findings confirmed that ga3 acted as growth promoter and that of ccc as figure 1. effect of growth regulators on the production of suckers in chrysanthemum. j. multidiscip. appl. nat. sci. 14 growth retardants on different plant characters of chrysanthemum. 3.2 effect of growth regulators on the production of suckers in chrysanthemum the higher number of suckers (33) per pot was produced when pots were treated with ga3 @ 150ppm followed by ga3 @ 100ppm (29), whereas, application of ccc at three different concentrations produced lower number of suckers (figure 1). use of ccc @ 600 and 800ppm produced the lowest number of suckers, which was much less than control treatment. this is in agreement with the findings of mzabri et al. [28]. the higher number of sucker production by using ga3 might be due to increase the number and size of leaves as a result of higher translocation of the photosynthates and eventually that would have been used for the production of propagules (suckers). 3.3 effect of growth regulators on the production of flower in chrysanthemum in general, ga3 at different concentrations produced the higher number of flowers (figure 2). the highest number of flower (40) was recorded with 150 ppm ga3, which was significantly superior to those observed by spraying 100 ppm ga3 and 50 ppm ga3. application of 800 ppm ccc produced minimum number of flowers (25) per pot, which was at par with 600 ppm ccc (27) and 400 ppm ccc (31). this was in line with the findings of kim et al. [25]. the increase in number of flowers for ga3 treated plants might be due to increase in number of leaves and leaf area compared to control and other treatments. this might have resulted in the production and accumulation of more photosynthates that were diverted to the sink (flower) and give increased number of flowers. 3.4 effect of plant growth regulators on floral characteristics in chrysanthemum irrespective of concentrations, ga3 significantly reduced the number of days to initiation of flowering (table 3). the plants sprayed with 50 ppm ga3 took 48 days to flower initiation, whereas, it took 70 days with 750 ppm mh. among the growth regulators ga3 caused faster initiation of flowering and acc and mh delayed it in respect of control. flower size was not significantly affected by the application of growth regulators at different concentrations (table 3). however, it was recorded highest (7.40 cm) when plants were sprayed with 800 ppm ccc, whereas, lowest size (6.50 cm) was obtained with the application of 500 ppm mh. this was closely followed that obtained by the use of 750 ppm mh. this was in line with the findings of padmalatha et al. [27] and uddin et al. [29] in chrysanthemum. here, food reserves may have been diverted to only fewer sinks that enhanced to produce bigger flowers. length of flower stalk significantly increased when plant was treated with ga3 regardless of different concentrations (table 3). the application of 150 ppm ga3 produced maximum length of flower stalk (15.0 cm), which was identical with those produced by 100 and 50 figure 2. effect of growth regulators on the production of flower in chrysanthemum. j. multidiscip. appl. nat. sci. 15 ppm ga3. this was in line with the findings of gabrel et al. [30]. this might be due to the fact that gibberellic acid promotes cell division and cell elongation resulting in longer stalks. the growth regulators ccc and mh at different concentrations gave the shorter stalk compared to control. 3.5 effect of growth regulators on the vase life of chrysanthemum use of growth regulators showed an increasing vase life of flowers in respect of control (figure 3). the maximum vase life of flowers was recorded for the treatment 800 ppm ccc (15 days), which was at par with 13 days vase life obtained by spraying 600 ppm ccc. this is in line with the findings of padmalatha et al. [27] in chrysanthemum. this might be due to the fact that ccc acted as growth retardants that may reduce the cell size and stomatal opening and thereby reduce the area for transpiration for which it maintained better water balance. 4. conclusions the study revealed that growth regulators had significant impact on the plant characters, quality and vase life of flower. the performance of the chrysanthemum also depended on the concentration of the growth regulators. the ga3 @ 150 ppm performed better than other concentrations, table 3. effect of plant growth regulators on floral characteristics in chrysanthemum. treatment (ppm) days to flowering flower size (cm) stalk length (cm) t1 48e 7.10 14.40a t2 53d 7.20 14.70a t3 55cd 7.30 15.00a t4 58c 7.10 7.00d t5 60bc 7.20 8.00cd t6 62b 7.40 8.00cd t7 65ab 6.80 9.00bcd t8 68a 6.50 8.00cd t9 70a 6.60 10.00bc t10 57c 6.90 12.00b cv (%) 13.64 17.50 12.41 note: t1-50 ppm ga3, t2-100 ppm ga3, t3-150 ppm ga3, t4-400 ppm ccc, t5-600 ppm ccc, t6-800 ppm ccc, t7-250 ppm mh, t8-500 ppm mh, t9-750 ppm mh, t10-control figure 3. effect of growth regulators on the vase life of chrysanthemum. j. multidiscip. appl. nat. sci. 16 whereas, ccc at all concentrations had some adverse effect on the plant performance. therefore, it is concluded that ga3 acted as growth promoter and that of ccc as growth retardants on yield and quality of chrysanthemum. author information corresponding author ahasan ullah khan — department of agroforestry and environmental science, sylhet agricultural university, sylhet 3100 (bangladesh); department of entomology, sylhet agricultural university, sylhet 3100 (bangladesh); orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors md. ehsanullah — department of entomology, govt. shahid akbar ali science and technology college, thakurgaon 5120 (bangladesh); department of entomology, sylhet agricultural university, sylhet 3100 (bangladesh); md. kamruzzam — department of soil science, govt. shahid akbar ali science and technology college, thakurgaon 5120 (bangladesh); sarah tasnim — department of genetics and plant breeding, bangabandhu sheikh mujibur rahman agricultural university, gazipur 1706 (bangladesh); department of crop genetics and plant breeding, institute of crop science, beijing -100081 (china); references [1] m. c. song, h. j. yang, t. s. jeong, k. t. kim, and n. i. baek. 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(2018). “effect of benzyl adenine and gibberellic acid on the vegetative growth and flowering of chrysanthemum plant”. alexandria journal of agricultural sciences. 63 (1): 29–40. 10.21608/alexja.2018.30051. https://doi.org/10.1155/2020/6975646 https://doi.org/10.3390/plants9080950 https://doi.org/10.3390/plants9080950 https://doi.org/10.1155/2021/9979827 https://doi.org/10.18801/jbar.040115.41 https://doi.org/10.21608/alexja.2018.30051 70 journal of multidisciplinary applied natural science vol. 2 no. 2 (2022) research article forest fire risk zone mapping of aalital rural municipality, dadeldhura district, nepal prajwol babu subedi*, keshav ayer, mahamad sayab miya, bhawana parajuli, and barsha sharma received : february 02, 2022 revised : march 29, 2022 accepted : april 02, 2022 online : april 03, 2022 abstract forest fire is one of the leading causes of forest and wildlife loss. the objective of this study was to use satellite imagery and geographic information system techniques to assess the forest fire risk zonation map of the aalital rural municipality. this rural municipality is a part of the sudurpaschim province, nepal; is prone to forest fires. four fire risk zones were established in the study area i.e. very high, high, medium, and low-risk zone. thematic layers were derived from topographic maps and satellite imageries. for the delineation of fire risk zones, a multi-parametric weighted index model i.e. the fri (fire risk index) method was adopted. the fire incidence data provided by modis were used to validate the resulting forest fire risk zone map. about 25.17% of the total study area lies under the very high-risk zone followed by 46.51% under high risk, 25.68% under medium risk, and 2.62% under the low-risk zone. it can be inferred that the majority of the area is at high risk of forest fire. this map of fire risk zone can help in disaster and forest management as valuable data to prepare effective measures for appropriate fire risk management in the area. keywords geographic information system, fire risk index, remote sensing, satelite imagery, wildfire 1. introduction forests are one of the most essential components of life on earth, providing clean air, water, and food while also preventing soil erosion and mitigating climate change. forests occupy 31% of the world's total land surface area [1]–[3]. forests, on the other hand, are degrading day by day across the world owing to a variety of factors such as forest fires, urbanization, and population growth, all of which contribute to deforestation [4]. nepal is a small country, covering only 147,516 square kilometers, yet it boasts a diverse climate, spanning from tropical south to alpine north. nepal's forest area and other woodlands make up 44.74% percent of the country's total area [5]. a forest fire or wildfire is an uncontrolled fire that burns an area of forest. the nature and origin of the ecosystem, topography, land surface temperature, the weather at the time of the forest fire, and the amount of fuel available on the territory are the influencing factors of forest fires [6]–[8]. land use and land cover (lulc) due to urbanization, modernization, and global population increase have caused land and soil exploitation, deforestation, and desertification leading to the increased number of forest fires worldwide. fire causes natural disasters such as increased soil erosion, floods, landslides, as well as a loss of water yield and depletion of water quality, the extinction of species, and a decrease in visual amenity [9]. it causes some positive effects, such as aiding in the recycling of bound-up nutrients in the soil, burning old and dead vegetation, and clearing space for newer vegetations to grow [10]. in recent years, there has been a dramatic increase in the number of forest fires in various areas around the world, including the western united states, australia, western and southern europe, and various unexpected locations above the arctic circle [11]. a global study conducted between 2003 and 2015 to understand the forest area depleted by forest fires revealed that approximately 67 million hectares of forest land area were affected by forest fires on an annual basis [12]. only in 2015, approximately 98 million hectares of total forest land were affected by fire [5]. as of december 24, 2021, the united states had faced 58,288 wildfires that had burned 7,819,070 acres of forest land since the beginning of 2021 [13]. similarly, canada had one of the worst forest fire years in history in 2021. according to a report published by the canadian interagency forest fire center [14], there were a total of 6,224 fires that burned an area of 4.18 million hectares copyright holder: © subedi, p. b., ayer, k., miya, m. s., parajuli, b., and sharma, b. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.115 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.115 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.115&domain=pdf&date_stamp=2022-04-06 j. multidiscip. appl. nat. sci. 71 throughout the year. when it comes to global forest fire trends, the devastating wildfire in australia from september 2019 to march 2020 is one of the worst in terms of area burned, human and wildlife casualties, and environmental damage [15]. in a mountainous country like nepal, people are more likely to use forest fire as a land management tool, clearing forest areas for cultivation and settlement [16]. nepal recently faced the worst scenario of forest fires throughout the country in the year 2021, with at least 60 wildfire burns in 22 different districts of nepal, resulting in 5 deaths and the loss, of a large amount of wildlife, cultivated crops, and domestic animals [17]. it appears that there is an irregular pattern in the prevalence of forest fires in nepal, particularly during the dry seasons from november to june each year [18]. forest fire assessment is a tedious task due to the lack of availability of the relevant data [19]. developing nations, such as nepal, still lack proper access to satellites and gis-based technology, even though significant progress has been achieved in the development of new methods and instruments for assessing forest fire risk in the global situation. the integrated use of remote sensing (rs) with geographic information system (gis) is quite popular to detect forest fire. various active forest fires can be monitored using geostationary satellite sensors like geostationary operational environmental satellite (goes) or spinning enhanced visible and infrared imager (seviri) on board of meteosat second generation satellite (msg). different geosynchronous satellites can also be helpful in forest fire monitoring such as advanced very high-resolution radiometer (avhrr) onboard national oceanic and atmospheric administration satellites (noaa), the along track scanning radiometer onboard ers-1 (atsr) and 2 (european remote sensing satellite 1 and 2) and the moderate resolution imaging spectroradiometer( modis ) onboard of the terra and aqua satellites [20]. the major issue in the assessment of forest fire is the scale, its size the area it covers, and more practically the inaccessibility it possesses, and all of these makes remote sensing one of the most prominent, practical, and widely applied method [21]. detection of forest fire in the early stage is very crucial in fighting the fire and saving the forest from getting more destroyed. only a few developed nations have been able to detect forest fires beforehand or in their early phase. this research aims to assess the forest fire risk zonation map of the aalital rural municipality of dadeldhura district, nepal. this research will aid in the mapping of appropriate forest fire risk zones, as well as in the conservation and promotion of green forest in those areas, by implying appropriate risk management and preservation techniques both before and after any forest fire. figure 1. map of study area showing aalital rural municipality j. multidiscip. appl. nat. sci. 72 2. materials and methods 2.1 study area the study was conducted in aalital rural municipality (ew: latitude: 29.1351°n and longitude: 80.5124°e) of dadeldhura district, nepal (figure 1). the rural municipality occupies an area of 292.87 km 2 ; politically divided into eight wards. it has a population of 18,531 [22]. currently, there is no concrete road, 20.8 km of gravel road, and approximately 105 km of earthen road in the rural municipality. 2.1.1 climate of the study area the rural municipality lies in the western part of the chure range with an arid steppe cold climate having an average land surface temperature of 4.1° c to 17.1°c and rainfall of 1500 to 2000 mm [23]. it lies in the humid subtropical range and consists of a dense shorea robusta sal forest associated with terminalia tomentosa (saj), syzygium cumini (jamun), bombax ceiba (simal), etc. the study area faces huge fire risks caused by natural or human activity throughout the year. 2.2 data collection in this study, variables directly related to the incidence of fire occurrence have been considered as the independent variables and it includes land used land cover (lulc), land surface temperature, topographic factors (slope, altitude, and aspect), distance from the road, and proximity to the settlement area [24]. similarly, the incidence of fire occurrence was considered as dependent variables as it is not directly related to fire occurrence. the independent and dependent datasets were collected through different data sources as listed in table 1. 2.2.1 land use land cover (lulc) lulc is considered to be the most important factor for the spreading of fire. a huge amount of degraded land, greater slope, human activities like grazing make the area highly susceptible to forest fire [25]–[27]. thus, lulc analysis was carried out by categorizing it into nine different land use classes through the provided data of icimod having a spatial resolution of 30m. 2.2.2 land surface temperature (lst) the lst of any area determines the vegetation type of the area and is directly related to the moisture content as well as relative humidity. the dryness of a region makes the site more prone to fire. the dry subtropical climatic condition makes the study area more vulnerable to forest fires. the lst algorithm produces day and night lst values at 1 km spatial resolutions in swath format using brightness temperatures from modis bands 31 and 32. it produces lst hdf files from modis level1b 1-km data. nasa/modis monthly mean land surface temperature data from 2001 to 2020 was used in this analysis. monthly temperatures for each year (2001-2020) were averaged to create an effective simulation for the fire risk area, and then a layer was created. table 1. collected datasets in the study area. datasets file type data type details spatial resolution sources study area boundary shp polygon outline of the study area department of land management and achieve nepal (http://www.dolrm.gov.np) road shp lines highway and associated roads 1:250000 ocha, nepal (https://www.unocha.org/) settlement shp points clusters of settlements 1:25000 ocha, nepal (https://www.unocha.org/) land cover 2010 tiff raster lulc category 30 m icimod (https://www.icimod.org) srtm dem tiff raster elevation, slope, and aspect 30 m usgs earth explorer (https://earthexplorer.usgs.gov) land surface temperature hdf raster the monthly temperature of night and day time 1 km nasa/ modis/ mod11c3 (https://lpdaac.usgs.gov/products/ mod11c3v006/) modis fire data shp points/ polygon latitude, longitude, confidence, burn date and area, 1 km nasa/ modis (https://firms.modaps.eosdis.nasa.gov/ active_fire/) http://www.dolrm.gov.np https://www.unocha.org/ https://www.unocha.org/ https://www.icimod.org https://earthexplorer.usgs.gov https://lpdaac.usgs.gov/products/mod11c3v006/ https://lpdaac.usgs.gov/products/mod11c3v006/ https://firms.modaps.eosdis.nasa.gov/active_fire/ https://firms.modaps.eosdis.nasa.gov/active_fire/ j. multidiscip. appl. nat. sci. 73 table 2. weight, value, and rating allocate to various variables. variables weight (%) class value assigned rating land cover 40 water 0 no risk human buildup 1 very low bare land 1 very low agricultural land 2 low grass and shrubland 2 low sparse vegetation 3 medium hardwood 4 high mixed vegetation 5 very high coniferous forest 5 very high land surface temperature 20 < 10 1 very low 10 -15 2 low 15 20 3 medium 20 25 4 high 25 30 5 very high slope (%) 10 <10 1 very low 10 15 2 low 15 20 2 low 20 25, 25 30, 30 – 35 3 medium 3540 4 high 40 45 4 high >45 5 very high distance from the road (m) 5 0500 5 very high 500 1000 4 high 1000 1500 3 medium >1500 2 low proximity to settlement (m) 5 01000 5 very high 1000 1500 4 high 1500 2000 3 medium >2000 2 low aspect 5 north 1 very low northeast 2 low east 3 medium southeast 4 high south 5 very high southwest 5 very high west 3 medium northwest 2 low north 1 very low elevation(m) 0-500 5 very high 5 500-1000 4 high 1000-1500 4 high 1500-2000 3 medium 2000-2500 2 low >2500 1 very low source: gheshlaghi et al. [24], jaiswal et al. [31], alkhatib, a. a. [32] j. multidiscip. appl. nat. sci. 74 2.2.3 topography there is an effect of terrain attributes on the forest survival following wildfire [28]. hence, slope, aspect, and elevation were retrieved using srtm dem (30 m) under topographic variation, as shown in table 1. the digital elevation model (dem), which was computed by the shuttle radar topography mission (srtm) and extracted from the us geological survey's eros data center, was used in this work to extract elevation, slope, and aspect data. 2.2.3.1 slope slope influences the fire spread in two different ways: i) preheating (radiation and convection) and ii) draft. low dense air warmed by the surface on slopes creates a channel for lighter air to move up the hill, resulting in a draft. cooler air rises from below, displacing the warmer, less dense air. as a result, during the day, local breezes tend to detonate slopes. wildfires usually burn upslope due to the local up-slope winds. as a result, the steeper the slope, the faster and more furiously the fire will burn. 2.2.3.2 elevation the altitude of the area differs from 1292 to 2198 m mean sea level. higher elevations are associated with greater rain availability. therefore, the fires are likely to be less awful at higher elevations [29]. however, the elevation range (900m) does not entail a significant rainfall difference between the highest and the lowest areas of the study site. 2.2.3.3 aspect aspect is the direction in which the slope is facing. it regulates the amount of heat emitted from the sun that is received. in comparison to slopes facing northerly, slopes facing south to southwest may get the greatest sun radiation, resulting in a warmer environment. the warmer slope results in lower relative humidity, higher land surface temperatures, and faster moisture loss. as a result, fuel will be showier and drier, catching fire more immediately and burning more readily. on southfacing hills, the fire's duration will also be prolonged. 2.2.4 distance from the road movement and activities of humans, animals, and vehicular on roads cause enough opportunities for human-made and accidental forest fires. thus, any forest patches situated near roads patches are more fire-prone. the forests of the study area are joined with two vehicular and several other pedestrian roads induced the intentional and accidental forest fire cause. the sale of resin and some other ntfp is a primary income source for the population in this study area. during tapping and collection, people throw carelessly cigarettes butts, and matches, which cause fires in the forest area. the road network was digitized using topographic maps of spatial resolution 1:250,000 scale acquired from the ocha, nepal, and contains all highways and adjacent roads (not including earthen road). these data are stored in a vector format in a gis database (shp files). 2.2.5 proximity to settlement forested areas located near human settlements and habitats are more vulnerable to fire due to the cultural and habitation practices of the people. despite few settlements within the study area forest, it still creates hazardous forest fires. the dataset settlements were digitized using topographic maps of spatial resolution 1:250,000 scale acquired from the ocha, nepal, and contains all clusters of settlements. these data are stored in a vector format in a gis database (shp files). 2.2.6 dependent variables forest fire events and their flicker area or size are the only outcomes of the driven force when all figure 2. flow chart of data analysis. j. multidiscip. appl. nat. sci. 75 independent variables listed above have a favorable atmosphere for the flame. as a result, study areadependent variables such as size and burning incidences have been considered. firms (fire information for resource management system) use a 1km resolution modis instrument onboard nasa's terra and aqua satellites to provide active fire information for burn area identification. the detection confidence ranges from 0 to 100 %, with more than 30 % indicating finer and more accurate detection [30]. as a result, this study relied on data with a confidence level of better than 30%. between 2016 ad and mid 2021, a total of 130 forest fire counts (85% of all counts) were recorded in the rural municipality's forested territory. 2.3 data analysis remote sensing and gis were used to analyze the data. the factors for the forest that was in descriptive form were analyzed and converted into a rating system and risk index of the forest fire. these factors were analyzed and categorized in the following sequence of importance: land cover, land surface temperature, slope, road network, proximity to settlement, elevation, and aspect. after deciding the effect of an individual’s forest fire risk factor, the various category classes of individuals' factors were given appropriate ratings. weight was specified based on literature to avoid error and achieve a practical conclusion to the model [31]– [33]. a higher rating indicates that the factor has a greater impact on the fire risk in a given area (table 2). the factors were then combined to calculate the figure 3. lulc map. figure 4. land surface temperature map figure 5. slope map. figure 6. distance from road map. j. multidiscip. appl. nat. sci. 76 forest fire risk index (fri) using the forest fire risk index (fri) model. the overall flow chart of the data analysis is shown in figure 2. fri = 40% lc + 20% lst + 10% dr + 10% s +10% ps + 5% e+ 5% a (1) here, lc= land cover, lst = land surface temperature, dr = distance from the road, s= slope, ps = proximity to settlement, e = elevation and a = aspect. 2.4 validation for fire risk zonation in any natural hazards assessment, model validation is essential. it is the process of comparing model predictions to a real-world dataset to determine their accuracy or predictive ability [33]. thus, we used modis hot spot data to corroborate our forest fire risk area conclusions in this study. the number of hot areas in each risk class was tallied to evaluate each index. it would be reasonable to locate the majority of hot spots in the very high-risk zone and the fewest in the low-risk zone. 3. results and discussions 3.1 thematic maps table 1 was used to create all the theme maps, including lulc map (figure 3), land surface map (figure 4), slope map (figure 5), distance from road map (figure 6), proximity to settlement map (figure 7) and elevation map (figure 9). 3.2 land cover the study area contained hardwood forest of about 1/3 rd (33.29 %) of the whole study area, triggers the risk of forest fire as hardwood is categorized as high risk. it was followed by sparse vegetation (15.08 %), grass and shrubland (14.12%), mixed vegetation forest (11.46%), agricultural land (10.39%), bare land (7.76%), builtup area (5.84%), water (1.02%) and coniferous forest (1.001%) (figure 3). 3.3 land surface temperature (lst) the lst category of 20-25°c showing high risk for forest fire has covered approximately half of the whole area (49.69%), followed by 10-15°c (45.41%), 0-10 °c (2.1%), 15-20°c (1.48%) and more than 25°c (1.32%) (figure 4). figure 7. proximity to settlement map. figure 8. aspect map. figure 9. elevation map. j. multidiscip. appl. nat. sci. 77 3.4 slope here, the majority of the area belongs to the area having less than 35° slope and indicates a mostly low and medium risk of a forest fire. the slope category of 15-20° has covered the highest area (15.34%), followed by 20-25° class (15.14%), 2530° class (14.67%), 30-35° class (13.78%), 10-15° class (12.82%), 35-40° class (11.29%), < 10°class (8.05%), 40-45° class ( 6.81%) and more than 45° class (2.06%) (figure 5). 3.5 distance from the road only gravel roads were taken into account. about 10.91% of the area was classified as very high-risk (< 500 meters from a road), 6.174 % of the total area was classified as high-risk (500–1000 m), 6.212% of the total area was classified as medium-risk (1000– 1500 m), and 76.69 % of the total area was classified as low-risk (figure 6). 3.6 distance from the settlement about 20.59% of the area felled under the higher risk category (<1000m), 23.31% felled under the medium-risk category (1000-1500m), 18.27% felled under the low-risk category (1500-2000m) and 37.82% area felled in no risk category (>2000m) (figure 7). 3.7 aspect the rural municipality covers almost all aspects with approximately equal area. southwest (17.48%) aspect and southeast (14.54%) aspects consist more geographical coverage area followed by south (12.91%), north (12.178%), northwest (11.38%), northeast (11.17%), west (10.90%), and east (9.40%) (figure 8). in total, over 45% of areas in the south, southwest, and southeast were highly vulnerable to wildfire. 3.8 elevation the area covered by the 1000-1500m elevation class was the largest of the six elevation classes i.e., 37.12% followed by followed by 500-1000m (36.69%), 1500-2000m (18.04%), 2000-2500m (7.31%), and >2500m (0.004%) (figure 9). 3.9 forest fire risk zonation about 25.17% of the total area of the rural municipality felled under a very high-risk zone followed by 46.51% under high risk, 25.68% under medium risk, and 2.62% under low-risk zone (figure 10). the northern part of the study area consists high density of hardwood and mixed vegetation (figure 3), high slope (figure 5), south and southwest aspect (figure 8) in comparison with southern part, which increase overall risk of a forest fire. 3.10 validation for fire risk zonation out of the total 45 forest fire occurrence points according to modis fire data, 27 points lay in the very high-risk zone, 14 lay in a high-risk zone, 4 points lie in medium risk zone and no point lies in a low-risk zone. the results in figure 11 reveal that the majority of the modis-derived hot spots are located in the very high-risk zone. figure 10. forest fire risk zonation map. j. multidiscip. appl. nat. sci. 78 3.11 discussion approximately 45% of the study area falls under the south, southeast, and southwest aspects, implying that these aspects pose a higher risk of fire incidence. more than half of the area is covered by a 20-40 degree slope, which is a highly flammable forest fire. because of nearby roads (500m) in the study area, approximately 10.91% of the area falls within the fire risk zone. locations nearby road coverage are more prone to fire incidence than locations further away from the interference of humans, such as good transporting, leakage from tankers, smoking, oil, and so on, that might induced and result in fire [34]. similarly, a natural environment near a human settlement is extremely sensitive to fire as a result of human-caused activities that can result in an unintentional fire. in the research area, there are more communities near and within the forest, accounting for approximately 20.59% of the total area, increasing the risk of wildfire. due to a multitude of variables such as sal forest [35], height gradient, sloppy terrain, and so on, this rural municipality is located in the siwalik region, which is the most prone to fire risk among all other regions [36]. land use/land cover types, proximity to roads/settlement, aspect, elevation, and slope are the most important parameters for wildfires [37]. in general, the south side gains more sunlight, which dries out the soil and speeds up the ignition process [32]. about 0.22 million ha of forest land were burned by fire, in 2016, accounting for 3.4% of nepal's total forest area and destroying 2500 ft 3 of valuable timber and about 12500 ft 3 fuelwood, resulting in a loss of approximately us$ 107,798 [18]. forest fires caused the most burnt area annually in nepal between 2000 and 2013, with 30,220 hotspots in 2005, 2009, and 2012. from 2000 to 2016, about 35,374 wildfire incidents were found in nepal, with burnt areas of 17, 23, and 920 ha [18]. this finding is in line with that of chaudhary et al. [35], who found that the western part of nepal is at more risk of fire due to low rainfall. forest fires are becoming more common in nepal, posing a serious threat to human life and the natural environment [17]. according to matin [37], 89% of wildfires in nepal occur in the period of pre-monsoon season (marchmay). from march to june each year, more than 40,000 ha of nepalese forests are burned down, as a result, enormous degradation and destruction of biodiversity and forest occurs [38]. wildfire is regarded as one of the most destructive drives, wreaking havoc on resources in a short period [39]. for wildfire prevention and control management of risk zones, including variables that cause fire, appropriate logistics, infrastructures, financial resources, and extensive understanding of the wildfire are required [36]. in western countries such as australia, the united states, and canada, forest fire risk maps are used to plan suitable efforts to minimize or prevent wildfires; however, in nepal, such practices are lacking [40]–[42]. preparing and publishing forest fire risk zonation maps will help nepal's disaster preparedness efforts greatly in this area [42]. hence, this study is an effort to identify wildfire high-risk zones in the municipality of aalital, which will aid the local and federal governments in developing appropriate or suitable wildfire prevention and control mechanisms. figure 11. forest fire risk zonation map with fire-affected point. j. multidiscip. appl. nat. sci. 79 4. conclusions the study infers that the majority of the study area falls under the high-risk zone of a forest fire. the number of forest fires is comparatively high in the northern region in comparison to the southern part. this study highlights the immediate necessity of preventing the incidence of forest fires in the very high and high-risk zone of the aalital rural municipality. in general, creating a forest fire risk zonation map is the first step in developing a forest fire management and prevention strategy. as a result of the study's findings, authorities will be able to develop effective forest fire risk management initiatives in the area. author information corresponding author prajwol babu subedi — institute of forestry, tribhuvan university, pokhara campus, pokhara -33700 (nepal); orcid.org/0000-0001-8992-7491 email: prajwolsubedi1990@gmail.com authors keshav ayer — kathmandu forestry college, tribhuvan university, kathmandu-44600 (nepal); orcid.org/0000-0002-5245-6849 mahamad sayab miya — institute of forestry, tribhuvan university, pokhara campus, pokhara -33700 (nepal); orcid.org/0000-0002-1675-593x bhawana parajuli — institute of forestry, tribhuvan university, pokhara campus, pokhara -33700 (nepal); orcid.org/0000-0002-5885-2367 barsha sharma — kathmandu forestry college, tribhuvan university, kathmandu 44600 (nepal); orcid.org/0000-0002-6562-9817 acknowledgement the authors would like to acknowledge mr. sachin timilsina, mr. roshan singh thagunna, mr. suman acharya, and mr. deepak gautam for assisting in providing valuable sources of information for this study. references [1] fao and unep. 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https://doi.org/10.1016/j.firesaf.2019.102863 https://doi.org/10.1080/21580103.2021.1979108 90 journal of multidisciplinary applied natural science vol. 2 no. 2 (2022) review article therapeutic properties of green tea: a review sonia ratnani and sarika malik* received : february 09, 2022 revised : march 31, 2022 accepted : april 04, 2022 online : april 05, 2022 abstract the most consumed beverage in the world is tea after water. till today the consumption of black tea is 70% while that of green tea is only 20%. one reason for this percentage is lack of awareness about green tea and invested research. green tea along with caffeine which imparts characteristic taste, bitterness and stimulating effect, is also rich in a group of chemicals, called catechin polyphenols (commonly known as tannins, which contribute to bitter taste and astringency) and deliver antioxidant properties. furthermore, green tea also comprises of amino acids such as theanine along with alkaloids such as adenine, dimethylxanthine, theobromine, theophylline and xanthine. some vitamins, like vitamin a, vitamin b1, vitamin b2, vitamin b3, vitamin c, and vitamin e are also found in green tea. the present review gives the study of various constituents of green tea and their impact on human health. studies provide strong evidence that owing to antioxidant properties daily intake of green tea may be used as a preventive measure for different types of cancer and other diseases. this review gives a detailed analysis of constituents of green tea and highlighting it's potential as a natural nutraceutical. however, although much of the documented literature mentions positive effect yet much had to be explored on correlation between concentration of green tea and toxicity. keywords green tea, polyphenols, human health, antioxidant 1. introduction our health is wrapped in our lifestyle and genes today, thus, no food can protect us from diseases except our care and attention. nowadays tea is considered to be second most consumed beverage in the world [1]. during last few years green tea has gained lot of importance as a beverage owing to its health benefits. recent studies have shown that green tea helps in improving blood vessels hence lower the cholesterol and reduce the risk of cardio vascular diseases [2]–[5]. mri’s reveal that drinking green tea have greater activity and can enhance our brain's cognitive functions, particularly the working memory [6]. green tea helps in controlling the levels of blood sugar in people suffering from diabetes [7]. it blocks the formation of plaque related to alzheimer diseases [8][9]. it is found as fat burning supplement as it increases fat burning and boost the metabolic rate in human controlled trial. most important due to presence of antioxidants it helps in lowering the risks of various type of cancer. these benefits have resulted in discussion which focus on the dietary guidance and recommended drinking for green tea [10]. through available research it is an attempt to depict the health benefits of green tea. the aim of this review is to provide information and evidence pertaining to health benefits obtained from consumption of green tea with special reference to cancer diseases. 2. green tea: historical perspective green tea is one of the healthiest beverages originated from china more than 4000 years ago and has been used in traditional chinese medicine for a wide variety of health benefits [11]. it comes from the steamed and dried leaves of the camella sinesis plant. the plant originates from china and also produces black tea, oolong tea and several other varieties of tea through different processing methods. green tea is made by harvesting the plant leaves, withering them and then steaming them. steaming halts, the process of enzymatic oxidation– which causes the darkening of the tea leaves – and allowed the leaves to remain closer to their natural “green” state. this indicates that it goes through minimal oxidation during processing which possess powerful oxidants. the real history of green tea dates back to the 8 th century, when the method of steaming the leaves to inhibit their oxidation was discovered. in the 12 th century a new frying method of “fixing” the leaves was introduced. both copyright holder: © ratnani, s. and malik, s. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.117 open access about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.117 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.117&domain=pdf&date_stamp=2022-04-16 j. multidiscip. appl. nat. sci. 91 of these processes resulted in teas that have the characteristic un-oxidized taste and appearance to modern green teas, and both processes are still in use today. since those early days, as the popularity and production of green tea increased the methods of producing green tea have continuously evolved and improved. 2.1 plant description green, black, and oolong tea are all derived from the leaves of the c. sinensis plant. originally cultivated in east asia, this plant grows as large as a shrub or tree (figure 1) [12][13]. today, c. sinensis grows throughout asia and parts of the middle east and africa. people in asian countries more commonly consume green and oolong tea while black tea is the most popular drink in the united states. green tea is prepared from unfermented leaves, the leaves of oolong tea are partially fermented, and black tea is fully fermented. the more the leaves are fermented, the lower the polyphenol content and the higher the caffeine content. green tea has the highest polyphenol content and 2-3 times less caffeine content then black tea. 2.2 doses pediatric, green tea has not been studied in children, so it is not recommended for pediatric use. adult, depending on the brand, 2 to 3 cups of green tea per day (for a total of 240 to 320 mg polyphenols) or 100 to 750 mg per day of standardized green tea extract is recommended. 3. chemical composition green tea mainly consists of mainly polyphenols (~90%), amino acids (~7%), theanine, proanthocyanidins, and caffeine (~3%). among the different polyphenols, catechins and flavonols (myricetin, caempherol, quercetin, chlorogenic acid, coumarylquinic acid, and theogallin) form the major constituents (figure 2) [14]. 3.1 polyphenols (catechins and flavanols) about 30% of the leaves by weight are flavanols which comprise mainly of catechins (derives its name from indian plant acacia catechu (a tree of fabaceae family, acaria genes) from which it is isolated). green tea comprises of catechin (c), epicatechin (ec), gallocatechin (gc), epigallocatechin (egc), epicatechin gallate (ecg), epigallocatechin gallate (egcg), and gallocatechin gallate (gcg) as the major catechins [15][16]. green tea hence is considered to be principal source of catechins among various dietary sources [17]. these are mainly responsible for the astringency component in green tea. among all catechins the four primary catechins ec, egcg, ecg, and egc form 80% of the total catechins (figure 3) [18]. all these catechins similar properties while egcg appears to be most potent catechin and accounts for 50—80% of the total catechins followed by egc (9 –12%), ecg (9–12%) and ec (5–7%) [19]. chemically catechins are polyphenolic compounds with diphenyl propane skeleton. the chemical structure consists of a polyphenolic ring condensed with six-membered oxygen containing heterocyclic ring that carries another polyphenolic ring at the 2 position. catechins are characterized by multiple of hydroxyl groups on the a and b rings. ec is an epimer containing two hydroxyl groups at 3′ and 4′ position of b ring and a hydroxyl group at 3 position of the c ring [20]. the only structural difference between egc and ec is that egc possesses an additional hydroxyl group at 5′ position of the b ring. ecg and egcg are ester derivatives of ec and egc, respectively, through esterification at 3 hydroxyl position of the c ring with a gallate moiety (figure 3) [21][22]. according to the european food safety authority (efsa), 126 mg of catechins are present in per 100 ml of green tea. meanwhile, according to the food and drug administration (fda), 71 mg of epigallocatechin gallate will be present in per 100 ml of green tea [23]. figure 1. green tea plant. j. multidiscip. appl. nat. sci. 92 3.2 alkaloids leaves of green tea primarily contain alkaloids including caffeine, theobromine, and theophylline. they provide green tea's stimulant effects. amount of caffeine (2—5%) in tea leaves depends on the age of the leaf with younger leaves to have it in high concentration [24]. 3.3 amino acids 15—20% of dry weight of tea comprises of proteins in enzymes that constitute an important fraction of amino acids (1—4% dry weight) such as theanine or 5-nethylglutamine, glutamic acid, tryptophan, glycine, serine, aspartic acid, tyrosine, valine, leucine, threonine, arginine, and lysine. ltheanine has been studied for its calming effects on the nervous system. 3.4 carbohydrates 5-7% dry weight of green tea comprise of carbohydrates which include cellulose, pectins, glucose, fructose, and sucrose. 3.5 vitamins and minerals vitamin content of green tea is higher than other drinks which thus make it superior. it comprises of important vitamins such as vitamin b2, vitamin c, vitamin e and folic acid. minerals are important as they act as bodily regulators. the approximate composition of minerals in tea is 5—7% as elements such as potassium (k), calcium (ca), phosphorus (p), and magnesium (mg), as well as small quantities of manganese (mn), zinc (zn) and copper (cu). 3.6 lipids small amounts of lipids such as linoleic and αlinolenic acids also constitute green tea. the nondrying oil content of green tea leaves is 4% by weight with a solidifying temperature of around -5 to 15 °c [25]. the composition of various constituents present in green tea varies with genetic strain, climatic conditions, soil properties, plucking season, position of leaf, processing and storage. analysis and evaluation of components of green tea is needed to assess the potential benefits and risks associated with green tea. researchers think the health properties of green tea are mostly due to polyphenols, chemicals with potent antioxidant potential. in fact, the antioxidant effects of polyphenols seem to be greater than vitamin c. the polyphenols in green tea also give it a somewhat bitter flavor. 4. health benefits of green tea tea catechins have been largely studied for their figure 2. chemical composition of green tea. figure 3. different forms of catechins. j. multidiscip. appl. nat. sci. 93 antioxidant capacities and considered as important antioxidants. the antioxidant effects of catechins are presumed to play a major role in mediating the cardioprotective role of tea, although emerging evidence shows that catechins also have antioxidant -independent vascular effects, which will be discussed elsewhere in this review. green tea catechins can exert both direct and indirect antioxidant effects on cardiovascular system. catechins present antioxidant activity through scavenging reactive oxygen species (ros), chelating redox active transitionmetal ions, inhibiting redox sensitive transcription factors, inhibiting pro-oxidant enzymes and inducing antioxidant enzymes. green tea intake lowers fasting serum total and ldl (low density lipid) cholesterol in adults: a meta-analysis of 14 randomized controlled trials. in traditional chinese and indian medicine, practitioners used green tea as a stimulant, a diuretic (to get rid of the body of excess fluid), an astringent (to control bleeding and help heal wounds), and to improve heart health. other traditional techniques use of green tea include treating gas, regulating body temperature and blood sugar, promoting digestion, and improving mental processes [26]–[29]. green tea has been extensively studied in people, animals, and laboratory experiments. results from these studies suggest that green tea may help treat the following health conditions (figure 4). 4.1 cancer several population-based studies suggest that both green and black teas help in protecting against some cancers hence it has potent anticarcinogenic behavior [30]–[37]. for example, the cancer rates tend to be low in countries such as japan where people regularly consume green tea. however, it is not possible to be sure of these studies results whether green tea actually prevents cancer in people. early clinical studies suggest that the polyphenols in tea, especially green tea, may play an important role in the prevention of cancer. researchers also believe that polyphenols help killing the various cancerous cells in human being. 4.1.1 bladder cancer medical and healthcare system have defined complementary and alternative medicine (cam) as an approach for patients suffering from bladder cancer. one of these strategies mentions intake of green tea and green tea polyphenols (gtp’s) [38]. in one study that compared people with and without bladder cancer, researchers found that women who drank black tea and powdered green tea were less likely to develop bladder cancer. a follow-up clinical study by the same group of researchers revealed that people with bladder cancer, figure 4. health benefits of green tea. j. multidiscip. appl. nat. sci. 94 particularly men, who drank green tea had a better 5 -year survival rate than those who did not drink green tea. people with cancer should consult with their doctor before adding tea to their daily diet. 4.1.2 breast cancer it has been observed in laboratory test tube and animal studies that egcg is responsible for inhibiting the growth of breast cancer cells. numerous studies have investigated the therapeutic effects of green tea on breast cancer using different rodent models with a range of green tea products including green tea mixtures as well [29]. in a study of 472 women with various stages of breast cancer, researchers found that women who drank more green tea had the least cancer spread. it was especially true in premenopausal women in the early stages of breast cancer. they also found that women with early stages of the disease who drank at least 5 cups of tea daily before being diagnosed with cancer were less likely to experience a recurrence after they finished treatment. however, women with late stages of breast cancer had little or no improvement from drinking green tea. there is no clear evidence about green tea and breast cancer prevention. in one very large study, researchers found that drinking tea, green or any other type, was not associated with a reduced risk of breast cancer. however, when the researchers broke down the sample by age, they found that women under the age of 50 who consumed 3 or more cups of tea per day were 37% less likely to develop breast cancer compared to women who did not drink tea. for more evidence additional studies are required to elucidate the potential risk and associated mechanisms of action. 4.1.3 ovarian cancer in a study done with ovarian cancer patients in china, researchers found that women who drank at least one cup of green tea per day lived longer with the disease than those who did not drink green tea. in fact, those who drank the most tea, lived the longest. however, other studies found no beneficial effects [39]. 4.1.4 colorectal cancer there are conflicting results on the effect of green tea on colorectal cancer. some studies indicate decreased risk on consumption of tea, while others show increased risk. in one of the study, women who consumed more than five cups of green tea per day faced a lower risk of compared to non-green tea drinkers. however, studies in women show that regular consumption may reduce the risk of colorectal cancer [40]. 4.1.5 esophageal cancer studies in laboratory animals have found that green tea polyphenols inhibit the growth of esophageal cancer cells. however, studies in people have produced conflicting findings. for example, one large-scale population-based study found that green tea offered protection against the development of esophageal cancer, particularly among women. another population-based study found just the opposite; green tea consumption was associated with an increased risk of esophageal cancer. in fact, the stronger and hotter the tea, the greater the risk. a number of epidemiologic studies have investigated the influence of consumption of green tea on the risk of esophageal cancer [41]. given these conflicting results, more research is needed before scientists can recommend green tea for the prevention of esophageal cancer. 4.1.6 lung cancer while green tea polyphenols have been shown to table 1. possible mechanisms for the anti-carcinogenic activity of green tea. no green tea component possible mechanism for anti-carcinogenic activity 1 catechins ec anti-inflammatory attenuation of oxidative stress induction of apoptosis inhibition of rtk’s activation inhibition of angiogenesis anti-metabolic syndrome 2 egc 3 ecg 4 egcg j. multidiscip. appl. nat. sci. 95 inhibit the growth of human lung cancer cells in test tubes, few clinical studies have looked at the link between drinking green tea and lung cancer in people, and the studies that have been done show conflicting results. one population-based study found that okinawan tea, similar to green tea but partially fermented, was associated with lower lung cancer risk, particularly among women. but a second study found that green tea and black tea increased the risk of lung cancer. more studies are needed before researchers can draw any conclusions about green tea and lung cancer. green tea should not be used by patients on bortezomib therapy [42]. 4.1.7 pancreatic cancer in one large-scale clinical study researchers compared green tea drinkers with nondrinkers and found that those who drank more tea was less likely to develop pancreatic cancer. this was particularly true for women, those who drank more green tea were half as likely to develop pancreatic cancer as those who drank less tea. men who drank more tea were 37% less likely to develop pancreatic cancer. however, it is not clear from this population-based study whether green tea is solely responsible for lowering pancreatic cancer risk [43]. more studies are needed before researchers can recommend green tea for the prevention of pancreatic cancer. 4.1.8 prostate cancer laboratory studies have found that green tea extracts prevent the growth of prostate cancer cells in test tubes. a large clinical study in southeast china found that the risk of prostate cancer went down with increasing frequency, duration, and quantity of green tea consumption. however, both green and black tea extracts also stimulated genes that cause cells to be less sensitive to chemotherapy drugs. people who are undergoing chemotherapy should ask their doctors before drinking green or black tea or taking tea supplements [44]. 4.1.9 skin cancer the main polyphenol in green tea is egcg. scientific studies suggest that egcg and green tea polyphenols have anti-inflammatory and anticancer properties that may help prevent the development and growth of skin tumors. hence it is work as anticarcinogenic agent [45]–[47]. its anti-carcinogenic activity due to some mechanistic effect is summarized in table 1 [35]. 4.2 atherosclerosis population-based studies indicate that the antioxidant properties of green tea may help prevent atherosclerosis, particularly coronary artery disease [48]. population-based studies are studied large groups of people over time and compared their living in different cultures and diets. researchers believe green tea reduces the risk of heart disease by lowering cholesterol and triglyceride levels. studies show that black tea has similar effects. in fact, researchers estimate that the rate of heart attack decreases by 11% with consumption of 3 cups of tea per day. 4.3 high cholesterol research shows that green tea lowers total cholesterol and raises hdl cholesterol in both animals and people. one population-based study found that those who drink green tea are more likely to have lower total cholesterol than those who do not drink green tea. results from one animal study suggest that polyphenols in green tea may block cholesterol from being absorbed in the intestine and also help the body get rid of cholesterol. in another small study of male smokers, researchers found that green tea significantly no days cholesterol (mg/dl) ldl (mg/dl) 1 0 270 ± 21.8 184 ± 17.1 2 15 196 ± 30.0 110 ± 21.9 3 30 247 ± 20.8 167 ± 16.3 4 60 278 ± 19.1 166 ± 20 table 2. effect of the treatment with green tea for 30 days on the lipid profile of hypercholesterolemic patients. j. multidiscip. appl. nat. sci. 96 reduced blood levels of harmful ldl cholesterol shown in table 2 [21][49]. 4.4 diabetes green tea has been used traditionally to control blood sugar levels. animal studies suggest that green tea may help to prevent the development of diabetes (type-1) and slow the progression once it has developed. in people with diabetes (type-1), their bodies make little or no insulin, which helps convert glucose or sugar into energy. green tea may help regulate glucose in the body. research also suggests that regular consumption of green tea may help manage diabetes (type-2) [50]. 4.5 liver disease population-based studies have shown that those who drink more than 10 cups of green tea per day are less likely to develop liver problems. green tea also seems to protect the liver from the damaging effects of toxic substances such as alcohol. animal studies have shown that green tea helps protect against liver tumors in mice. it is important to note that 10 cups of green tea a day could cause problems due to high levels of caffeine [51]. results from several animal and human studies suggest that catechins, may help treat viral hepatitis, an inflammation of the liver. in these studies, catechin was used by itself in very high amounts. 4.6 weight loss clinical studies suggest that green tea extract may boost metabolism and help burn fat. one study found that the combination of green tea and caffeine improved weight loss and maintenance in people who were overweight and moderately obese [52]. studies shows consumption of green tea lowers ldl but not hdl cholesterol and leads to weight loss. 4.7 other uses preliminary studies suggest that drinking green tea can help prevent various diseases and improves health of living creatures [17][53]–[65]. it may also be useful in inflammatory diseases, such as arthritis. research suggests that green tea may help arthritis by reducing inflammation and slowing the breakdown of cartilage. chemicals in green tea may help treat genital warts, treat dermatologic conditions, and prevent symptoms of colds and flu. green tea may play a role in preventing parkinson disease, cognitive decline, and osteoporosis. it also works on various microorganisms and prevents various antimicrobial disease [15]. studies also show that drinking green tea influences psychopathological function, cognition and brain function of human being [66]. its activity for antiphotoaging, stress resisting, no medication purpose of medication possible effect(s) with consumed green tea 1 adenosine for irregular unstable heart-beat inhibit the action of adenosine 2 beta-lactum antibiotic makes bacteria less resistant 3 benzodiazepines anxiety may reduce the sedative effect 4 chemotherapy cancer less sensitive to drugs 5 monoamine oxidase inhibitors depression increase in blood pressure 6 quinolone antibiotics antibiotic increase the risk of side effects table 3. possible interaction of medication with consumed green tea. j. multidiscip. appl. nat. sci. 97 neuroprotection and autophagy is also very well studied [14]. the composition of green tea preparations that most closely reflects that of a traditional infusion is safe. preparations based on concentrated extracts or containing high levels of individual constituents, egcg, may require healthbased guidance values to assure their safe use [10]. 5. safety and toxicity the use of herbs is a time-honored approach to strengthening the body and treating disease. however, herbs contain active substances that can trigger side effects and interact with other herbs, supplements, or medications. for these reasons, people should take herbs with care, under the supervision of a practitioner knowledgeable in the field of botanical medicine [67]. people with heart problems or high blood pressure, kidney problems, liver problems, stomach ulcers, and psychological disorders, particularly anxiety, should not take green tea. pregnant and breastfeeding women should also avoid green tea [68]. people with anemia, diabetes, glaucoma, or osteoporosis should ask their health care provider before drinking green tea or taking an extract. people who drink large amounts of caffeine, including caffeine from green tea, for long periods of time may experience irritability, insomnia, heart palpitations, and dizziness. caffeine overdose can cause nausea, vomiting, diarrhea, headaches, and loss of appetite [69]. medications for treatment such as adenosine, beta-lactum, benzodiazepines, beta-blockers, and proponal. can interfere and cause adverse effects. following table 3 gives the possible interaction of medication with consumed green tea [70]. as reported, ingestion of excess green or associated preparations can cause one of the most detrimental effects as hepatotoxicity [71][72]. as published an intake amount of egcg from 140 mg to~ 1000 mg/day is associated with hepatotoxicity and substantial inter-individual variability in susceptibility due to genetic factors. these findings have resulted in a cautionary labelling requirement in powdered decaffeinated green tea extract which mentions as “do not take on an empty stomach. take with food. do not use if you have a liver problem and discontinue use and consult a healthcare practitioner if you develop symptoms of liver trouble, such as abdominal pain, dark urine, or jaundice (yellowing of the skin or eyes) [10][73]. 6. conclusions the present review has discussed the positive and negative impact of green tea on human health. though studies have provided enough evidence for favorable effects related to heart diseases, cholesterol, blood fats, endometrial cancer, ovarian cancer and high blood pressure. however, there is insufficient evidence for blood disorders, bladder, colon, breast and cervix cancer and other ailments. studies which report conflicting results may be presumed due to perplexing factors such as countable consumption, temperature, race, sex, age, lifestyle and genetic history. more attempts for clarification are still required to establish the reported effects. author information corresponding author sarika malik — chemistry department, ramjas college, university of delhi, 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different treatments were 10 ec (t1), lambda-cyhalothrin 2.5 ec (t2), (t3), abamectin 1.8 ec (t4), spinosad 45 sc (t5), and untreated control (t6). the rate of leaf infestation at the lower, middle, and top stages of country bean ranged from 8.82–17.78, 13.89–36.39, and 13.72–33.33% in t5 treated and untreated control plots, respectively. the rate of shoot infestation at the middle and top stages of country bean ranged from 18.67–34.33 and 17.00–39.27% in t5 treated and untreated control plots, individually. the lowest and highest inflorescence infestation of 29.15% and 46.67% was recorded in t5 treated and untreated control plots, separately. the lowest fruit infestation of 8.84% by number and 8.32% by weight was recorded in the treated plot t5. the rate of reduction of pod infestation over control was 48.58, 24.02, 23.39, 21.81, and 19.92% by number and 73.53, 61.76, 58.82, 52.94, and 41.18% by weight in the treated plot t5, t4, t1, t3, and t2, respectively. the yield in different treatments ranged from 1.35-2.50 t ha -1 . the highest adjusted net return and bcr of tk. 52220.00 and 2.42, respectively were obtained from t2 treated plots. the second highest bcr of 2.16 was calculated from t4 treated plot. the lowest bcr of 1.05 was found in t2 treated plot. the treatments t5, t4, and t3 were effective to suppress the major sucking pests of the country bean. keywords country bean (lablab purpureus), sucking pests, bcr, pyrethroids, and bio-rational insecticides 1. introduction the country bean (lablab purpureus lin.) is one of the most popular vegetable-cum-pulse crops in bangladesh and belongs to the family leguminosae and sub-family papilionaceae [1]. the bean is commonly known as “seam” in bangladesh [2]. it is the most important leguminous crop and it has important value for its atmospheric nitrogen fixation [3]. the fresh pods and green seeds are eaten after boiling or used in curries. mature seeds are occasionally sun-dried and stored for use as vegetables. this bean is also grown for fodder and as a cover crop [4]-[6]. in bangladesh, the bean is usually grown in winter, but in recent times, several photo-insensitive and summer varieties are developed, which helped to promote the cultivation of country beans year-round including in summer [7]-[9]. bangladesh's country bean, lablab purpureus (l.), is a widely produced vegetable and it contains high in protein (20-30% protein by dry weight). thiamin, riboflavin, niacin, vitamin c, and iron (0.1, 0.06, 0.7, 9.0, and 41.7 mg/100g, respectively) are all present in significant amounts [10]. the entire land area under bean cultivation in bangladesh is 49,192 acres; with a yield of 122,091 metric tons in 2014-2015 [11]. the high incidence of insect pests deteriorates the yield and quality of the pod of country beans. a report revealed that eighteen species of insect cause an infestation in the country bean field [6][12][13]. the various types of sucking pests include aphid (aphis craccivora), jassid (amrasca devastas), bean bug (coptosoma cribrarium), whitefly (bemisia argentifolii), and thrips (sericothrips occipitalis) are commonly found in a country bean field. aphid and jassid are the major sucking pests of country bean that causes damage directly by sucking the cell sap of the plant and indirectly by transmitting several viral diseases [14][15]. both the nymph and adult phases of the sucking pests of country bean cause injury by sucking sap from flowers, buds, pods, and tender shoots of the plants and reduce the vitality of the bean and leguminous crops [2]. effective controlling practices to manage these menacing sucking pests are much more difficult because they introduce their toxic saliva while they suck the plant sap as well, they act as a vector to publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2023 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.146 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.146 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.146&domain=pdf&date_stamp=2023-02-12 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 101 disseminate the plant disease and create secondary infection of the plants. farmers of bangladesh, particularly rely only on some common chemical insecticides to control the insect pest population. the frequent, over and misuse of this chemical insecticide has created a lot of detrimental effects on our environment like; pest resistance, resurgence, increase in the mortality rate of beneficial insects as the natural enemies, and finally enhanced environmental pollution. to avoid complete dependence on chemical pesticides, alternative tools are needed. there are many effective botanicals and bio-rational components available in the country which may be exploited side by side with the chemical components. singh [16] described that foliar application of lambdacyhalothrin at flowering and fruit initiation stages was the most effective against the different sucking insects. cypermethrin, a synthetic pyrethroid is popularly used for its quick knockdown action with the quality of non-phytotoxic and almost non-toxic effect on mammals [17]-[19]. however, excessive and blind use of synthetic pesticides has created many problems for the environment. bio-based pesticides are effective in reducing the use of common chemical pesticides and controlling pests in a safe and eco-friendly manner. they have minimal effect on non-target organisms and similarly, the potential human health effects are not known so far. there is little study on synthetic pyrethroids and rational insecticides for beansucking pests in bangladesh. with this in mind, the current research was conducted to determine the level of bean-sucking pest damage and its control with several pyrethroids and bio-rational pesticides. 2. materials and methods 2.1. experimental site and design the experiment was performed in an open field of entomology, at sylhet agricultural university, sylhet, bangladesh. the land belongs to the khadimnagar soil series eastern surma-kushiara floodplain under the agroecological zones-20. the experiment is laid out in a randomized complete block design (rcbd) with three replications in the table 1. treatments, and components of this study. treatments of pyrethroids and rational pesticides for 10 days interval dose of water (ml/l) t1: c 10 ec t2: lambda-cyhalothrin 2.5 ec t3: emamectin benzoate 5 sg t4: abamectin 1.8 ec 1.2 t5: spinosad 45 sc 0.4 t6: untreated control 0 table 2. effect of different treatments applied against the major sucking pests of country bean in terms of percent (%) of leaves infestation grown during the winter season. treatments percent (%) of leaves infestation lower leaves infestation middle leaves infestation top leaves infestation total leaves infestation 10 ec 12.80 b 18.12 bc 20.40 bc 19.28 b lambda-cyhalothrin 2.5 ec 15.38 ab 20.55 b 25.30 ab 21.92 b 9.34 c 17.54 c 19.07 bc 18.63 b abamectin 1.8 ec 8.80 c 16.33 cd 19.10 bc 17.63 b spinosad 45 sc 8.82 c 13.89 d 13.72 c 11.67 c control 17.78 a 36.39 a 33.33 a 28.80 a cv (%) 7.34 18.51 14.46 19.56 note: means within the same letter (s) within a column do not differ significantly (p=0.05) according to dmrt. j. multidiscip. appl. nat. sci. 102 winter season september 2020 – march 2021. 2.2. planting material seeds of cultivated local variety of goalgadda were used in the experiment during the period from the winter season in september 2020 – march 2021. 2.3. land preparation the land was prepared by spade and stubble, and weeds were removed. experimental land was divided into unit plots following the design of the experiment. during final land preparation, 10 t ha -1 decomposed cow dung was mixed with soil. in each plot measuring 3.0 m × 2.0 m, 4 pits were prepared for seedling transplantation. 2.4. manures and fertilizers application recommended doses of fertilizer comprising urea, tsp, and mop at the rate of 30, 90, and 65 kg ha -1 , respectively, were applied. the entire dose of tsp and half the amount of mop were applied to the soil of the pit 4-5 days before the seedling transplanting. the rest amount of the urea and mop were top-dressed at 30 and 45 days after transplanting. 2.5. seedling raising and transplantation a small seed bed measuring 2 × 1 m was prepared and seeds were sown in the nursery bed at entomology field, sau, sylhet on 13, september 2020. the plots were lightly irrigated regularly to ensure proper germination and growth of the table 3. effect of different treatments applied against the major sucking pests of country bean in terms of percent (%) of shoot infestation grown during the winter season. treatments percent (%) of shoot infestation middle shoot infestation top shoot infestation total shoot infestation 10 ec 24.35 bc 25.33 bc 24.50 bc lambda-cyhalothrin 2.5 ec 33.11 ab 29.00 b 30.83 ab 24.10 bc 21.96 bc 23.33 bc abamectin 1.8 ec 22.45 c 21.67 bc 23.67 bc spinosad 45 sc 18.67 c 17.00 c 20.50 c 34.33 a 39.27 a 38.16 a cv (%) 19.43 19.57 3.27 note: means within the same letter (s) within a column do not differ significantly (p=0.05) according to dmrt. note: means within the same letter (s) within a column do not differ significantly (p=0.05) according to dmrt. treatments number of inflorescence infestation plot -1 number of healthy inflorescences number of infested inflorescences % infestation 10 ec 6.07 bc 3.85 abc 36.54 bc lambda-cyhalothrin 2.5 ec 6.10 bc 4.02 ab 39.43 ab 6.03 bc 3.34 bcd 34.53 bc abamectin 1.8 ec 6.23 ab 3.06 cd 33.51 bc spinosad 45 sc 6.47 a 2.94 d 29.15 c 5.86 c 4.19 a 46.67 a cv (%) 12.71 13.99 7.03 table 4. effect of different treatments applied against the major sucking pests of country bean in terms of the number of inflorescences plot -1 grown during the winter season. j. multidiscip. appl. nat. sci. 103 seedlings. fifteen days old seedlings were transplanted into the well-prepared experimental plots. a total of 3 seedlings were planted in 1 pit @ 12 seedlings plot -1 . 2.6. intercultural operations after transplanting the plants were initially irrigated by water cane and later on regular irrigation was maintained. after one week of transplanting, the propping of each plant with bamboo sticks (1.50 m) was provided about 1.50 m high from ground level for additional support to allow normal creeping. all the bamboo sticks in each row were fastened strongly by a galvanized wire to allow the vines to creep along. weeding and mulching in the plots were done, whenever necessary. 2.7. treatments, concentration, and application time of the pyrethroids and rational pesticides all treatments were applied 5 times at 10 days intervals starting from the first appearance of the infestation. all spray materials were applied on the upper and lower surfaces of the leaves and shoots to ensure complete coverage of the plants. the spray was always done in the afternoon to avoid scorching sun, and insecticidal drift, and to protect pollinating wild bees and other beneficial insects. plants under the control plot were sprayed with clean tap water in the same manner. during the application of insecticides, precautions were taken to avoid drift to adjacent plots by using polythene sheets between plots (table 1). 2.8. data collection the effect of different treatments in controlling bean-sucking pests and infestation was determined based on the infestation of leaves, shoots, inflorescences, and pods of country beans and yield per hectare. 2.8.1. leaf, shoot, and inflorescence infestation the total number of leaves, shoots, and inflorescences as well as the number of infested leaves, shoots, and inflorescences were recorded from each plot at weekly intervals. leaf, shoot, and inflorescence infestation [20] was calculated in percent using the following formula 1: (1) 2.8.2. pod infestation and yield per hectare fruits were harvested at 7-10 days intervals and the numbers of healthy and infested fruits and their weight were noted separately per plot per treatment. harvests were done throughout the fruiting season and the percent pod/fruit infestation by number and weight was calculated [21] using the following formulas 2 and 3. (2) (3) table 5. effect of different treatments applied against the major sucking pests of country bean in terms of the number of pods/fruits plot -1 grown during the winter season. treatments number of pods plot -1 number of healthy pods number of infested pods total number of pods % infestation 10 ec 62.07 cd 9.73 ab 71.80 ab 13.66 bc lambda-cyhalothrin 2.5 ec 54.70 de 10.17 ab 64.87 bc 19.54 ab 68.53 ab 9.93 ab 78.46 a 12.74 bc abamectin 1.8 ec 63.97 bc 9.65 ab 73.62 a 12.62 bc spinosad 45 sc 72.87 a 6.53 b 79.40 a 8.84 c 49.97 e 12.70 a 62.67 c 21.59 a cv (%) 12.11 6.59 16.18 17.30 note: means within the same letter (s) within a column do not differ significantly (p=0.05) according to dmrt. j. multidiscip. appl. nat. sci. 104 percent increase or decrease over control was also computed using the following formula 4 and 5, respectively: (4) (5) 2.9. economic analysis benefit-cost ratio (bcr) was analyzed by considering the total expenditure of growing the crop and the total return from that particular treatment. in this experiment, bcr was calculated for a hectare of land. benefit-cost analysis was done through the following steps. 2.9.1. treatment-wise cost of cultivation this cost was calculated by adding all costs incurred for labor and inputs for each treatment including the control plot during the entire vegetative and fruiting period. the yield of each treatment was converted into tons per hectare. 2.9.2. gross return and net return the yield in terms of taka was calculated by multiplying the total yield by the unit price of the country bean at that time (tk. 45 kg -1 ). net return was calculated by subtracting treatment-wise management cost from gross return. 2.9.3. adjusted net return the adjusted net return was determined by subtracting the net return of the control plot from the net return of a particular treatment [21]. adjusted net return = net return of a particular treatment – net return of control plot (6) 2.9.4. benefit cost ratio calculation finally, the benefit cost ratio (bcr) [21] was calculated by dividing the adjusted net return by the respective total management cost for each treatment following the formula: (7) 2.10. data analysis all the data collected were double-checked, coded, and transferred from the preliminary note to a master sheet, summarized, categorized, and entered into a database with microsoft excel 2019. all the data collected and computed were analyzed statistically. analysis of variance (anova) was conducted for the parameters of leaf infestation (middle leaf infestation, top leaf infestation, total note: t1: application of c 10 ec @ water at 10 days interval; t2: application of lambda-cyhalothrin 2.5 ec @ water at 10 days interval; t3: application of emamectin benzoate 5 sg @ water at 10 days interval; t4: application of abamectin 1.8 ec@ 1.2 of water at 10 days interval; t5: application of spinosad 45 sc 0.4 of water at 10 days interval; and t6: untreated control. figure 1. effect of different treatments applied against the major sucking pests of country bean in terms of % increase in the number of healthy pods plot -1 over control grown during the winter season j. multidiscip. appl. nat. sci. 105 leaf infestation, % leaf infestation,), shoot infestation (middle shoot infestation, top shoot infestation, total shoot infestation, % shoot infestation), inflorescence infestation (number of healthy inflorescences, number of infested inflorescences, % inflorescences infestation), pods infestation (number of healthy pods, number of infested pods, % pods infestation), pods weight (healthy pods weight, infested pods weight, weight of total pod, % pods weight), and country bean yield (marketable yield, infested yield, total yield) to identify the significant differences between the treatments using the r software 0.05% level. then, dmrt (duncan’s multiple range tests) was performed for those parameters to compare the significant differences between the treatment means. 3. results and discussions 3.1. effect of different treatments on leaves infestation the effects of various treatments on percent leaf infestation by sucking pests of the country bean are presented in table 2. the rate of leaf infestation at the lower, middle, and top stages of country bean ranged from 8.82-17.78, 13.89-36.39, and 13.7233.33% in spinosad 45 sc treated and untreated control plots, respectively. all the treatments showed a significant effect on the percent leaf infestation, the total leaves infestation ranged from 11.67-28.80%, the highest leaf infestation was recorded in the untreated control (28.80%) plot and the lowest infestation was obtained with spinosad 45 sc (11.67%) sprayed plot, respectively. the second highest leaf infestation was found in lambda-cyhalothrin 2.5 ec (21.92%) treated plot and this was followed by 10 ec (19.28%), abamectin 1.8 ec (17.63%) and they were statistically identical. similar results were also observed by das et al. [22] and the major pests include aphids, sap-sucking bugs, pod borers, leaf miners, and stem flies which cause a yield loss of about 37-100% in natural conditions. 3.2. effect of different treatments on shoot infestation note: t1: application of c 10 ec @ water at 10 days interval; t2: application of lambda-cyhalothrin 2.5 ec @ water at 10 days interval; t3: application of emamectin benzoate 5 sg @ water at 10 days interval; t4: application of abamectin 1.8 ec@ 1.2 of water at 10 days interval; t5: application of spinosad 45 sc 0.4 of water at 10 days interval; t6: untreated control. figure 2. effect of different treatments applied against the major sucking pests of country bean in terms of % reduction of the number of infested pods plot -1 over control grown during the winter season. j. multidiscip. appl. nat. sci. 106 the effects of all treatments on percent shoot infestation by sucking pests of the country bean are presented in table 3. the rate of shoot infestation at the middle and top stages of country bean ranged from 18.67-34.33 and 17.00-39.27% in spinosad 45 sc treated and untreated control plots, individually. all the treatments exhibited a significant effect on the percent shoot infestation, the total shoot infestation ranged from 20.50-38.16%, the highest leaf infestation was recorded in the untreated control (38.16%) plot and the lowest infestation was obtained with spinosad 45 sc (20.50%) treated plot, respectively. the second highest shoot infestation was found in lambda-cyhalothrin 2.5 ec (30.83%) treated plot and this was followed by 10 ec (24.50%), abamectin 1.8 ec (23.67%), and (23.33%) they were statistically identical but significantly different (table 2). the current finding was fully supported by sultana [23] who reported that 26.08% shoot/twig infestation was found in spinosad 45 sc treated plot. 3.3. effect of different treatments on inflorescence infestation the comparative effectiveness of various treatments on inflorescence infestation was presented in table 3. all the treatments revealed a significant effect on inflorescence infestation. the lowest and highest inflorescence infestation of 29.15 and 46.67% was recorded in spinosad 45 sc treated and untreated control plots, separately. the second highest inflorescence infestation was found in lambda-cyhalothrin 2.5 ec (39.43%) treated plot and this was followed by 10 ec (36.54%), (34.53%) and abamectin 1.8 ec (33.51%) they were statistically at par but significantly different (table 4). paul et al. [24] reported that 27.57% inflorescence infestation was found in cypermethrin 10 ec @ 2ml/l treated plot which was strongly supported by the current study. on the other hand, sultana [23] reported that 34.28% inflorescence/flower infestation was found in the spinosad 45 sc treated plot, and these results were almost similar to the current finding. 3.4. effectiveness of different treatments on the pod infestation by number the comparative effectiveness of various treatments on pod infestation by number due to sucking. pests of country beans have been evaluated in percent (%) and were presented in table 4. the lowest percent pod infestation by number was recorded in spinosad 45 sc (8.84 %) treated plot and it was significantly lower compared to all other treated treatments. the second lowest pod infestation by number was found in abamectin 1.8 ec (12.62%) followed by (12.74%) and 10 ec (13.66%) they were statistically at par but significantly different (table 5). paul et al. [24] reported that 5.38-7.45% pod infestation by number was recorded in cypermethrin 10 ec @ 2ml/l treated plot which was partially (13.66%) supported by the current table 6. effect of different treatments applied against the major sucking pests of country bean in terms of pods/fruits weight plot -1 during the winter season treatments pods/fruits weight (kg) plot -1 healthy pods weight infested pods weight weight of total pod % 10 ec 1.25 ab 0.16 bc 1.41 ab 15.29 abc lambda-cyhalothrin 2.5 ec 1.27 ab 0.20 b 1.47 ab 23.11 ab 1.24 ab 0.14 bc 1.38 ab 12.54 bc abamectin 1.8 ec 1.30 ab 0.13 bc 1.43 ab 11.79 bc spinosad 45 sc 1.34 a 0.09 c 1.53 a 8.32 c 0.70 b 0.34 a 1.04 c 26.60 a cv (%) 18.99 10.21 18.66 21.32 note: means within the same letter (s) within a column do not differ significantly (p=0.05) according to dmrt. j. multidiscip. appl. nat. sci. 107 research. nur et al. [25] stated that spinosad 45 sc and performed the best result by least infestation (4.50 and 6.00%, respectively) and this finding is fully supported by the current study. 3.5. the percent increase in the number of healthy pods plot -1 over control the percent increase in the number of healthy pods plot -1 over control of all treatment results was shown in figure 1. the increase in the number of healthy pods over control was calculated as 45.83, 37.34, 28.02, 24.21, and 9.47% due to spray with spinosad 45 sc, , abamectin 1.8 ec, 10 ec, and lambda-cyhalothrin 2.5 ec, respectively. results of the present study revealed that all treatments have a significant role in the percent increase in the number of healthy pods plot -1 over control. the most effective treatment was spinosad 45 sc and the least effective was lambda-cyhalothrin 2.5 ec against sucking pests of country beans. similar results were found in dry beans in brazil [26]. 3.6. the percent reduction in the number of infested pods plot -1 over control the percent reduction in the number of infested pods plot -1 over control was the highest in spinosad 45 sc treated plot (48.58%) and the lowest in lambda-cyhalothrin 2.5 ec plot (19.92%). the second highest reduction was found in abamectin 1.8 ec (24.02%) sprayed plot (figure 2). the findings of the present study revealed that c 10 ec @ ml/l water at 10 days interval spray resulted in a substantial reduction (23.39%) in the number of infested pods over the control. a similar study was conducted paul et al. [24] and found an almost similar reduction (20.28%) in pod infestation over control. 3.7. effectiveness of different treatments on pod infestation by weight note: t1: application of c 10 ec @ water at 10 days interval; t2: application of lambda-cyhalothrin 2.5 ec @ water at 10 days interval; t3: application of emamectin benzoate 5 sg @ water at 10 days interval; t4: application of abamectin 1.8 ec@ 1.2 of water at 10 days interval; t5: application of spinosad 45 sc 0.4 of water at 10 days interval; t6: untreated control. figure 3. effect of different treatments applied against the major sucking pests of country bean in terms of % increase in weight of healthy pod’s plot -1 over control grown during the winter season. j. multidiscip. appl. nat. sci. 108 the comparative effectiveness of various treatments on pod infestation by weight due to sucking pests of country beans has been evaluated in percent and was presented in table 6. the lowest percent pod infestation by weight was recorded in spinosad 45 sc (8.32 %) treated plot and it was significantly lower compared to all other treated treatments. the second lowest pod infestation by weight was found in abamectin 1.8 ec (11.79%) followed by (12.54%) and 10 ec (15.29%) they were statistically similar but significantly different. paul et al. [24] reported that 6.55-6.89% pod infestation by weight was recorded in cypermethrin 10 ec @ 2 ml/l treated plot which was much below the current finding (15.29%). uddin et al. [27] reported that 10 ec, lambda-cyhalothrin 2.5 ec, spinosad 45 sc performed significant results by inhibiting the pod infestation (12.78, 13.54, 10.77 and 11.79%, respectively) and this finding is strongly supported by the current research. 3.8. the percent increase in weight of healthy pods plot -1 over control the percent increase in weight of healthy pods plot -1 over control of all treatment consequences is shown in figure 3. the increase in weight of healthy pods plot -1 over control was calculated as 91.43, 85.71, 81.43, 78.57, and 77.14% due to spray with spinosad 45 sc, abamectin 1.8 ec, lambdacyhalothrin 2.5 ec, 10 ec, and lambda-cyhalothrin 2.5 ec, respectively. results of the present study revealed that all treatments have a significant role in the percent increase in weight of healthy pods plot -1 over control. the most effective treatment was spinosad 45 sc and the least effective was lambda-cyhalothrin 2.5 ec against sucking pests of country beans. paul et al. [24] reported that an 18.42-46.93% increase in note: t1: application of c 10 ec @ water at 10 days interval; t2: application of lambda-cyhalothrin 2.5 ec @ water at 10 days interval; t3: application of emamectin benzoate 5 sg @ water at 10 days interval; t4: application of abamectin 1.8 ec@ 1.2 of water at 10 days interval; t5: application of spinosad 45 sc 0.4 of water at 10 days interval; t6: untreated control. figure 4. effect of different treatments applied against the major sucking pests of country bean in terms of % increase in weight of healthy pod’s plot -1 over control grown during the winter season. j. multidiscip. appl. nat. sci. 109 weight of healthy pods plot -1 over control was recorded in cypermethrin 10 ec @ 2ml/l treated plot which was much lower than the current finding (78.57%). 3.9. the percent reduction in weight of infested pods plot -1 over control the reduction in weight of infested pods over control ranged from 41.17-73.53%. the percent reduction in weight of infested pods plot -1 over control was the highest in spinosad 45 sc treated plot (73.53%) and the lowest in lambdacyhalothrin 2.5 ec plot (41.18%). the second highest reduction was found in abamectin 1.8 ec (61.76%) sprayed plot followed by emamectin benzoate 5 sg (37.55%), c 10 ec (38.58), and lambda-cyhalothrin 2.5 ec (34.05%) (figure 4). similar results were observed by ahmed et al. [28] in the research field of the department of entomology bsmrau campus, bangladesh. 3.10. effect of different treatments on the yield (t ha -1 ) of country bean the effect of different treatments on yield has been evaluated in terms of total fruit yield, healthy fruit yield or marketing yield, and infested fruit yield obtained in each treatment during the entire period of the crop (table 7). significantly the highest marketable yield (2.47 t ha -1 ) was recorded from spinosad 45 sc treated plot which was higher than any other treatments and followed by abamectin 1.8 ec (2.27 t ha -1 ) and (2.13 t ha -1 ) and they were statistically similar. the lowest yield of healthy fruits (0.83 t ha -1 ) was recorded from the untreated control plot which was followed by lambdacyhalothrin 2.5 ec (1.64 t ha -1 ) and 10 ec (1.69 t ha -1 ) and the later two were statistically identical. significantly the least infested yield (0.18 t ha -1 ) was recorded from spinosad 45 sc treated plot and the highest (0.52 t ha -1 ) was in the untreated control. the second highest infested yield was found in lambda-cyhalothrin 2.5 ec (0.34 t ha -1 ) followed by 10 ec (0.28 t ha -1 ), 0.24 t ha -1 abamectin 1.8 ec (0.23 t ha -1 ) and later three all were statistically identical but significantly different. the total yield was the highest (2.65 t ha 1 ) in spinosad 45 sc treated plot and the lowest (1.35 t ha -1 ) in the untreated control plot. the total yield of abamectin 1.8 ec (2.50 t ha -1 ) was statistically at par with (2.37 t ha -1 ) treated plot and the second lowest yield (1.97 t ha -1 ) was recorded in 10 ec treated plots and which was statistically similar with lambda-cyhalothrin 2.5 ec (1.98 t ha -1 ) treated plot. the performance of the different treatments against the sucking pests of country beans in different aspects, such as percent leaf, shoot, inflorescence and pod infestation, reduction of infestation over control, healthy and total fruit yield as found in the present study were more or less in conformity with the findings of several other similar studies. khan et al. [14] reported that in the winter season under natural conditions (in an table 7. effect of different treatments applied against the major sucking pests of country bean in terms of yield ton ha -1 during the winter season treatments country bean yield ton ha -1 marketable yield (t ha -1 ) infested yield (t ha -1 ) total yield (t ha -1 ) 10 ec 1.69 bc 0.28 bc 1.97 b lambda-cyhalothrin 2.5 ec 1.64 bc 0.34 b 1.98 b 2.13 ab 0.24 bc 2.37 ab abamectin 1.8 ec 2.27 a 0.23 bc 2.50 ab spinosad 45 sc 2.47 a 0.18 c 2.65 a 0.83 c 0.52 a 1.35 c cv (%) 16.57 15.45 14.11 note: means within the same letter (s) within a column do not differ significantly (p=0.05) according to dmrt. j. multidiscip. appl. nat. sci. 110 untreated control plot) goalgadda country bean produced 0.81 t ha -1 and this finding was most similar to the current research. conversely, paul et al. [24] recorded in the range of total yield 9.5511.60 t ha -1 in 10 ec treated plots. on the other hand, uddin et al. [27] reported the total yield in 10 ec, lambdacyhalothrin 2.5 ec, spinosad 45 sc treated plots were obtained 3.47, 14.80, 14.75 and 15.78 t ha -1 , respectively, and those findings have fully differed from the performance of the different treatments of the current research and it might be happened due to different agro-ecological zone, soil characters, and bean varietal difference. 3.11. economic analysis of different pyrethroids and bio-rational insecticide treatments the benefit-cost ratio (bcr) has worked out based on the expenses incurred and the value of crops obtained against the treatment used in the present study for the control of sucking pests of the country bean are presented in table 8. it is to be noted here that expenses incurred referred to those only on pest control. thus, it is revealed that the bcr was the highest at 2.42 in treatment 5 spinosad 45 sc followed by bcr 2.16 and 2.03 in treatments 4 abamectin 1.8 ec and 3 and the lowest bcr 1.05 in treatment 2 lambda-cyhalothrin 2.5 ec, respectively. similar results were also noted in a study and the bcr was found to be 1.371 which implies that the investment of one taka in country bean production generated a bdt of 1.371 [29]. 4. conclusions in respect of five treatments of pyrethroids and bio-rational insecticides, spinosad 45 sc showed the highest efficacy against the sucking pests and it might be an effective, suitable, and workable tool for suppressing the sucking pest of country beans. treatment abamectin 1.8 ec@ 1.2 ml/l of water at 10 days intervals would be better next to the spinosad 45 sc. emamectin benzoate 5 sg @ water at 10 days intervals would be the third effective and amendment intervention against the sucking pests for economic country bean production in bangladesh. authors information corresponding authors md. abdur razzak choudhury — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0002-6426-9194 email: choudhurymar.entom@sau.ac.bd ahasan ullah khan — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors md. abdul maleque — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0001-6219-5737 table 8. economic analysis of different treatments applied against the major sucking pests of the country bean during the winter season treatment cost of control marketable yield (t ha -1 ) gross return net return adjusted net return bcr 10 ec 18500 1.69 76050 57550 20200 1.09 lambda-cyhalothrin 2.5 ec 17800 1.64 73800 56000 18650 1.05 19300 2.13 95850 76550 39200 2.03 abamectin 1.8 ec 20495 2.27 102150 81655 44305 2.16 spinosad 45 sc 21580 2.47 111150 89570 52220 2.42 0 0.83 37350 37350 https://orcid.org/0000-0002-6426-9194 mailto:choudhurymar.entom@sau.ac.bd https://orcid.org/0000-0002-7029-8215 mailto:ahasanullahsau@gmail.com https://orcid.org/0000-0001-6219-5737 j. multidiscip. appl. nat. sci. 111 nazmin nahar mily — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0003-1239-2518 author contributions conceptualization, m.a.r.c. and a.u.k.; methodology, m.a.r.c.; software, a.u.k,; validation, m.a.r.c., n.n.m. and a.u.k.; formal analysis, a.u.k.; investigation, m.a.r.c.; resources, m.a.r.c.; data curation, n.n.m.; writing – original draft preparation, m.a.r.c.; writing – review & editing, m.a.r.c.; visualization, a.u.k.; supervision, m.a.r.c. and a.u.k.; project administration, m.a.r.c.; funding acquisition, m.a.r.c.”. all the authors went through the manuscript and gave their consent for publication. conflicts of interest the authors declare no conflict of interest. references [1] a. u. khan, m. a. r. choudhury, c. k. dash, u. h. s. khan, and m. ehsanullah. 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"financial analysis of country bean in narsingdi district of bangladesh". asian journal of advances in agricultural research. 17 (2): 42–50. https://doi.org/10.47352/jmans.2774-3047.98 https://doi.org/10.47352/jmans.2774-3047.98 https://doi.org/10.47352/jmans.2774-3047.119 https://doi.org/10.47352/jmans.2774-3047.119 https://doi.org/10.18782/2582-7146.119 https://doi.org/10.3390/horticulturae7010007 https://doi.org/10.3390/horticulturae7010007 103 journal of multidisciplinary applied natural science vol. 2 no. 2 (2022) research article relationship of quantitative traits in different morphological characters of pea (pisum sativum l.) sarah tasnim, nilufa yasmin poly, nusrat jahan, and ahasan ullah khan* received : february 17, 2022 revised : april 16, 2022 accepted : april 18, 2022 online : april 20, 2022 abstract an experiment was undertaken to elucidate the genetic relationship between different quantitative traits for commercial culti vation and to evaluate selection criteria in pea breeding programs in five inbred parents. their 17 f4’s derivatives in pea (pisum sativum l.) evaluated ten characters during the winter season (november to february) of 2017-18 at the research farm, bsmrau, gazipur, bangladesh. analysis of variance explored significant differences among the genotypes for all the characters. phenotypic coefficients of variation (pcv) were close to genotypic coefficients of variation (gcv) for all the characters indicating les s influence on the environment and potentiality of selection. a high heritability relationship with high genetic advance was ob served for plant height, pod per plant, hundred seed weight, and seed yield per plot. pod length showed a highly significant positiv e correlation with pod width and hundred seeds weight. only days to first flowering showed a highly negative correlation with p od length and hundred seed weight. path coefficient analysis revealed that plant height, pod per plant, and seeds per pod had a highly positive effect on yield per plant. therefore, associating and selecting those traits, yield improvement must be possible in pea, and the days to maturity, plant height, pods per plant, pod length, and seed showed a considerable positive and highly significant correlation with plant height, pod per plant, seed per pod, and yield per plant at both genotypic and phenotypic levels indicating yield could be increased with the increase of days to maturity, plant height, pods per plant, pod length, and seed. keywords pea, pisum sativum, plant height, flower, pod, seed, pvc, gcv 1. introduction pea (pisum sativum l.) is an annual herbaceous important member crop that belongs to the family leguminosae. among legumes, pisum sativum is the oldest common pea and it is a self-pollinated (2n=2x=14) food crop [1]. it has initiated in the mediterranean region, primarily in the middle east [2]. it is an imperative, highly productive, and nutritionally rich cool-season legume crop, grown across the world, consumed as food, feed, and fodder [3][4]. pea is cultivated for green pod seeds as vegetable and dry seeds in bangladesh. it is a vital crop with a rich history in genetic study seeing back to the classical work by the father of genetics gregor j. mendel. genetic deviation more gives an idea about the scope of development in a character through simple selection based on grouping. generally, the mature dry seeds are used as dhal and the green seeds are used as fresh, frozen, or canned vegetables. it is an excellent source of dietary protein, nutritious feed for humans and livestock. this plant is a great source of nitrogen having a profound ameliorative effect on soil. it is widely used as a supplement feed, fresh vegetables, grains, and green manure, due to its amusing source of healing properties and nutritional value [5]. it is a starchy vegetable with high nutritional value, high in fibers, proteins, vitamin a, vitamin b6, vitamin c, vitamin k, iron, zinc, phosphorus, magnesium, copper, and lutein [6]. the protein of peas contains all the essential amino acids important for the normal activity of living organisms [7][8]. pathak and jamwal [9] stated that the high genotypic coefficient of variation (gcv) was noted for pod yield per plant, moderate to high gcv were verified for several days to 50% flowering and plant height. sureja and sharma [10] reported that a considerable amount of phenotypic coefficient of variation and genotypic coefficient of variation was observed for most of the characters such as the plant height, number of pods per plant, and weight of grains per pod, while the variation was low for the other characters tested. breeding efforts have contributed substantially to improving yield potential, regional adaptation through resistance or tolerance to abiotic, and is a highly complex character and is controlled by a large number of genes and greatly influenced by the environment copyright holder: © tasnim, s., poly, n. y., jahan, n., and khan, a. u. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.119 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.119 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.119&domain=pdf&date_stamp=2022-04-21 j. multidiscip. appl. nat. sci. 104 and quality traits influence the yield directly and indirectly. these traits are simply inherited and less influenced by the environment as compared to yield. so, selection based on these traits has a better chance of success in comparison to selection for yield alone. the genotypic correlation indicates the extent to which the two characters are under the control of the same set of genes are having the physiological basis of their expression [11][12]. in the pea gene pool, genotypes with new mutant traits have looked, changing the plant body. it has led to a substantial change in the constraints of the morphostructure of the new variations and increasing the limit of the changeability of the quantitative traits. many researchers differently evaluate the role of the individual characteristics in plant thruput formation. hamed et al. [12] found that positive heterosis over the better parent for plant length was ranged from 6.44% to 104.21%. el -dakkak [13] found negative heterosis (-16.82%) based on the tallest parent for this trait. significant positive heterosis based on an early parent was observed in all garden pea crosses for days to the flowering trait [14], while, askander and osman [15] found negative heterosis in some crosses and positive heterosis values in the others. the data from these studies provide an opportunity to combine appropriate traits in one genotype and increase the efficiency of the breeding activity [16]. zayed et al. [17] reported that the maximum significant heterosis in desirable direction was recorded for the number of seeds/pod. genanasekaran and padmavathi [18] found that average heterosis was observed for plant height, pods per plant, pod length, and seeds per pod. the plant height, pods per plant, pod length, seed number and seed weight were effect of yield of pea. el hanafi et al. [19] found that the maximum significant mid-parent heterosis in desirable direction was recorded for stem length trait. hussein [20] confirmed the partial dominance for earliness and overdominance for the remainder growth trait stem length and number of branches. the inheritance of quantitative characters in peas has long been investigated. suman et al. [21] and manjunath et al. [22] observed that both general and specific combining abilities were important for hundred seed weight and the number of seeds per pod. also, askander et al. [23] reported that general t a b le 1 . a n a ly si s o f v a ri a n c e f o r y ie ld a n d y ie ld r e la te d c h a ra c te rs i n p e a . s o u r c e o f v a r ia ti o n d f d f f d f p f d m p h p p p p l p w s p p h s w s y p p r e p li c a ti o n 2 2 1 .5 1 1 4 8 .9 6 6 6 0 .0 4 5 0 .1 4 0 .2 3 0 6 0 .0 7 0 .0 0 0 0 2 0 .0 0 9 3 0 .0 0 1 7 0 .0 0 0 2 g e n o ty p e 2 1 1 0 9 .5 1 3 * * 1 1 4 .3 7 * * 2 0 0 .7 7 9 * 3 2 5 0 .9 9 * * 5 7 .3 7 2 * * 1 .5 1 3 * * 0 .0 4 8 * * 1 .4 6 2 * * 5 4 .2 7 6 * * 4 2 .8 5 8 * * e rr o r 4 2 4 .1 3 1 .5 0 2 0 .4 7 4 0 .2 7 0 .1 0 2 0 .0 1 1 0 .0 0 0 1 0 .0 1 7 0 .0 0 1 0 .0 0 0 1 c v % 5 .6 7 2 .8 6 0 .7 8 0 .4 7 2 .6 2 1 .7 3 0 .6 5 2 .5 3 0 .1 6 0 .1 3 n o te : * * a n d * s ig n if ic a n t a t 1 % a n d 5 % l e v e l o f p ro b a b il it y , re sp e c ti v e ly . d f f = d a y s to f ir st f lo w e ri n g , d f p f = d a y s to 5 0 % f lo w e ri n g , p h = p la n t h e ig h t (c m ), p p p = p o d s p e r p la n t (n o .) , p l = p o d l e n g th ( c m ), p w = p o d w id th ( c m ), s p p = s e e d s p e r p o d ( n o .) , d m = d a y s to m a tu ri ty , h s w = h u n d re d s e e d w e ig h t (g ), s y p p = y ie ld p e r p la n t (g ). j. multidiscip. appl. nat. sci. 105 combining ability was significant for the traits plant height, 100 seeds weight, and pod weight but nonsignificant for seeds pod, while sca for most characters was significant in pea. this correlation study along with path coefficient analysis is more useful to study the yield conducive traits [12]. this study aims to find out the relationship between different quantitative traits for commercial cultivation and to evaluate selection criteria in pea breeding programs. 2. materials and methods 2.1. materials 2.1.1. study period and site the experiment was conducted at the experimental field of genetics and plant breeding department, bangabandhu sheikh mujibur rahman agricultural university (bsmrau), salna, gazipur during the winter season (november to february) of 2017-2018. the experimental site is located at the center of the madhupur tract (latitude24.09°n and longitude90.26°e) with an elevation of 8.4 meters from the sea level. 2.1.2. planting materials five inbred parents viz. ipsa motorshuti 1, ipsa motorshuti 2, ipsa motorshuti 3, natore, zhikargacha along with seventeen f4’s viz ipsa motorshuti 1 × ipsa motorshuti 2, ipsa motorshuti 1 × ipsa motorshuti 3, ipsa motorshuti 3 × ipsa motorshuti 1, ipsa motorshuti 1× natore, ipsa motorshuti 1 × zhikargacha, zhikargacha × ipsa motorshuti 1, ipsa motorshuti 2 × ipsa motorshuti 3, ipsa motor shuti 3 × ipsa motorshuti 2, ipsa motorshuti 2 × natore, ipsa motorshuti 2 × zhikargacha, zhikargacha × ipsa motorshuti 2, ipsa motorshuti 3 × natore, natore × ipsa motorshuti 3, ipsa motorshuti 3 × zhikargacha, zhikargacha × ipsa motorshuti 3, natore × zhikargacha, zhikargacha × natore produced from crossing of the inbred parents were included in the experiment. the f4’s were synthesized in the previous year of the experiment. all the seeds of the mentioned genotypes were collected from the department of genetics and plant breeding department, bangabandhu sheikh mujibur rahman agricultural university (bsmrau). 2.2. methods 2.2.1. experimental design each plot consisted of a single row of 1.5 m long. the rows were spaced at 25 cm in which seeds were sown continuously. the experiment was laid out in a randomized complete block design (rcbd) with three replications. 2.2.2. data collection five randomly selected competitive plants from parents and 20 plants of f4’s were used for recording observations on the following parameters. genotypic and phenotypic coefficients of variations were estimated by using those formulas. 2.2.2.1. univariate analysis the data were statistically analyzed. the mean, range, and standard deviation for each character have been calculated and analysis of variance for each of the characters was performed, and mean values were separated by dmrt. the mean square (ms) at error and phenotypic variances were estimated as per [24]. the error ms was considered as error variance ( 2 e), genotypic variances ( 2 g) were derived by subtracting error ms from the variety ms and dividing by the number of replications as shown below: (1) where gms and ems are the varietal and error can square and r is the number of replications. the phenotypic variance ( 2 p), were derived by adding genotypic variances with the error variance ( 2 e), as given by the formula 2. (2) 2.2.2.2. estimation of genotypic and phenotypic coefficient of variation genotypic and phenotypic coefficient of variation (gcv) was calculated by the formula 3. (3) where, g = genotypic standard deviation, = j. multidiscip. appl. nat. sci. 106 population mean similarly, the phenotypic coefficient of variation (pvc) was calculated from the following formula 4. (4) where, p = phenotypic standard deviation; = population mean 2.2.2.3. estimation of heritability broad sense heritability was estimated by the formula 5 suggested by [24]. (5) where, h 2 =heritability; 2 g =genotypic variance; 2 p =phenotypic variance. 2.2.2.4. estimation of genetic advance the expected genetic advance for different characters under selection was estimated using the formula 6 suggested by silpashree et al. [24]. genetic advance (ga) = k x h 2 x p (6) 2.2.2.5. estimation of genotypic and phenotypic correlation co-efficient for calculating the genotypic (rg) and phenotypic (rp) correlation coefficient for all possible combinations the formula 7 and 8 suggested by silpashree et al. [24] and hanson et al. [25]. (7) where, 2 gxy = genotypic covariance between the traits x and y; 2 gx = genotypic variance of the trait x; 2 gy = genotypic variance of the trait y. (8) where, 2 pxy = phenotypic covariance between the traits x and y; 2 px = phenotypic variance of the trait x; 2 py = phenotypic variance of the trait y. dff dfpf dm ph ppp pl pw spp hsw traits dfpf g p 0.923** 0.895** dm g p 0.454* 0.325 0.687** 0.568** ph g p 0.288 0.129 0.563** 0.409* 0.885** 0.828** ppp g p 0.248 0.155 0.496** 0.411* 0.634** 0.591** 0.676** 0.633** pl g p -0.655** -0.728** -0.418* -0.543** 0.042 -0.054 0.162 0.110 -0.115 -0.164 pw g p -0.522** -0.469* -0.477* -0.415* -0.297 -0.179 -0.202 -0.075 -0.332 -0.274 0.737** 0.800** spp g p 0.194 0.027 0.478* 0.307 0.749** 0.631** 0.790** 0.702** 0.513** 0.425* 0.428* 0.420* 0.091 0.266 hsw g p -0.648** -0.587** -0.576** -0.489* -0.441* -0.290 -0.237 -0.046 -0.264 -0.168 0.620** 0.718** 0.841** 0.830** -0.080 0.133 ypp g p -0.155 -0.261 0.196 0.084 0.484* 0.425* 0.657** 0.636** 0.788** 0.769** 0.390 0.371 0.205 0.292 0.599** 0.557** 0.281 0.418* table 2. genotypic (g) and phenotypic (p) correlation coefficient of ten different characters of pea. note: ** and * significant at 1% and 5% level of probability, respectively ph = plant height (cm), ppp = pod per plant (no.), pl = pod length (cm), pw = pod width (cm), spp = seed per pod (no.), dff = days to first flowering, dfpf = days to 50% flowering, dm = days to maturity, hsw = hundred seed weight (g), sypp= seed yield per plant j. multidiscip. appl. nat. sci. 107 2.2.2.6. estimation of path coefficient analysis path analysis was carried out using the genotypic correlation coefficients to know the direct and indirect effects of the components on yield as suggested and illustrated [26][27]. 2.2.3. statistical analysis the collected data were analyzed by the analysis of variance (anova) technique using the computer package program mstat and mean differences were adjudged by the least significant difference (lsd) test at a 5% level of significance. 3. results and discussions 3.1. mean performance for yield and yield-related characters in pea analysis of variance for all the characters showed significant differences between the treatments viz., days to first flowering, days to 50% flowering, plant height (cm), pods per plant (no.), pod length (cm), pod width (cm), seeds per pod (no.), days to maturity, hundred seed weight (g), yield per plant (g). thus, there was considerable genetic variability in the material chosen for investigation (table 1). 3.2. genetic parameters of pea 3.2.1. days to first flowering days to first flowering showed a considerable positive and highly significant correlation with days to 50% flowering and days to maturity at both genotypic and phenotypic levels. in contrast days to first flowering showed a positive but nonsignificant correlation with plant height, seed per pod, and pod per plant. on the other hand, pod length, pod width, hundred seed weight have highly negative and significant correlations with days to first flowering and it showed a negative and nonsignificant relationship with yield per plant in both genotypic and phenotypic levels (table 2). gudadinni et al. [28] observed days to first flowering (30.07 to 64.45), days to 50% flowering (35.71to 75.55), days to first pod setting (33.92to 68.23), days to first pod picking (45.85 to 82.36), and those results were supported to this results. 3.2.2. days to 50% flowering days to 50% flowering showed a considerable positive and highly significant correlation with days to maturity, plant height, seed per pod, pod per plant at both genotypic and phenotypic levels. besides days to 50% flowering showed considerable positive but non-significant correlation with yield per plant. on the other hand, pod length, pod width, hundred seed weight have a highly negative and significant correlation with days to 50% flowering in both genotypic and phenotypic levels (table 2). lagiso et al. [29] found days to 50% flowering were positively associated with days to maturity. 3.2.3. days to maturity days to maturity showed a considerable positive and highly significant correlation with plant height, pod per plant, seed per pod, and yield per plant at both genotypic and phenotypic levels. on the contrary days to maturity showed considerable negative and significant correlation with hundred seed weight. on the other hand, pod length, pod width has a highly negative and non-significant correlation with days to maturity in both genotypic and phenotypic level (table 2). according to singh et al. [30], days to maturity was a major yield and yield contributing character in field pea. motte et al. [31] studied on the correlation between yield and yield components of a pea. 3.2.4. plant height plant height showed a considerable highly significant positive correlation with pods per plant, seeds per pod, and yield per plant at a genotypic and phenotypic level indicating yield could be increased with the increase of plant height. quite the opposite, plant height exhibited an insignificant positive association with pod length. on the other hand, pod width, hundred seed weight have an insignificant negative correlation with plant height in both genotypic and phenotypic levels (table 2). riaz et al. [32] reported that plant height showed a positive correlation with seed yield per plant. 3.2.5. pods per plant pods per plant showed a considerable significant positive correlation with seeds per pod, yield per plant at both genotypic and phenotypic levels indicating yield could be increased with the j. multidiscip. appl. nat. sci. 108 increase of pods per plant. furthermore, pods per plant exhibited an insignificant negative association with pod length, pod width, and hundred seed weight (table 2). siddika et al. [33] assessed pods per plant exerted a positive direct effect on yield. 3.2.6. pod length pod length showed a significant positive correlation with pod length, pod width, hundred seed weight at both genotypic and phenotypic levels. quite the reverse, pod length exhibited an insignificant positive association with yield per plant (table 2). character association studies of sharma et al. [34] in pea indicated a positive and insignificant association with seed yield per plant. similar results were also observed by khan et al. [35] in legume crop country bean during 2017-2018 in sylhet, bangladesh. 3.2.7. pod width pod width showed a significant positive correlation with hundred seed weight at both genotypic and phenotypic levels. in contrast, pod width showed an insignificant positive correlation with seed per pod and yield per plant at both genotypic and phenotypic levels (table 2). similar results were also observed by khan et al. [36] in legume crop country bean during 2017-2018 in sylhet, bangladesh. 3.2.8. seeds per pod seeds per pod showed a significant positive correlation with yield per plant at both genotypic and phenotypic levels. in addition to seeds per pod showed a considerable negative insignificant correlation with hundred seed weight (table 2). gayacharan et al. [37] found a positive correlation with seed yield and seeds per pod in black gram. karyawati and puspitaningrum [38] got the similar result in lentil. tiwari and lavanya [39] found a significant and positive correlation between seed yield per plant and the number of seeds per pod. similar results were also observed [35] in legume crop country bean during 2017-2018 in sylhet, bangladesh. 3.2.9. 100 seed weight the character 100 seed weight showed a positive and phenotypically non-significant correlation with seed yield per plant (table 2). a significant and positive correlation was observed with days to maturity, plant height, pods per plant at both genotypic and phenotypic levels. the character showed a nonsignificant positive correlation with days to 50% flowering, pod length, pod width, and this character showed a nonsignificant negative correlation with days to first flowering at both levels. siddika et al. [33] observed a positive correlation between seed yield per plant and 100 seed weight. similar results were also observed by khan et al. [35], khan et al. [40], and khan et al. [41] in legume crop country bean during 2017-2018 in sylhet, bangladesh. traits dff dfpf dm ph ppp pl pw spp hsw ypp dff -0.233 0.216 -0.061 0.080 0.175 -0.067 -0.078 -0.005 -0.172 -0.146 dfpf -0.201 0.251 -0.095 0.159 0.360 -0.044 -0.074 -0.013 -0.156 0.188 dm -0.103 0.172 -0.138 0.256 0.472 0.006 -0.047 -0.021 -0.122 0.475* ph -0.063 0.136 -0.120 0.294 0.514 0.018 -0.032 -0.022 -0.067 0.657** ppp -0.053 0.119 -0.085 0.198 0.762 -0.013 -0.053 -0.014 -0.074 0.786** pl 0.139 -0.099 -0.007 0.047 -0.086 0.112 0.117 -0.012 0.173 0.385 pw 0.114 -0.115 0.040 -0.059 -0.252 0.082 0.161 -0.003 0.237 0.205 spp -0.041 0.112 -0.099 0.228 0.381 0.046 0.014 -0.029 -0.022 0.589** hsw 0.142 -0.139 0.060 -0.070 -0.200 0.069 0.135 0.002 0.283 0.281 note: residual effect (r) = 0.05430826 ph = plant height (cm), ppp = pods per plant (no.), pl = pod length (cm), pw = pod width (cm), spp = seeds per pod (n o.), dff = days to first flowering, dfpf = days to 50% flowering, dm = days to maturity, hsw = hundred seed weight (g), ypp= yield per plant (g). table 3. partitioning of genotypic correlation (rg) into its direct and indirect effects for seed yield components in pea. j. multidiscip. appl. nat. sci. 109 3.3. path coefficient analysis association of character determined by correlation coefficient may not provide an exact picture of the relative importance of the direct and indirect influence of each yield component on yield. as a fact, to find out a clear picture of the interrelationship between yield per plant and other yield attributes, direct effects were worked out using path analysis at a genotypic level which also measured the relative importance of each component. seed yield per plant was considered as a resultant (dependent) variable and plant height, pods per plant, pod length, pod width, seeds per pod, days to first flowering, days to 50% flowering, days to maturity, and hundred seed weight as a causal (independent) variable. the cause and effect of the relationship of yield per plant and yield-related characters have been presented in table 3. residual effects of their independent variables, which have influenced yield to a small extent, have been denoted as 'r'. 3.3.1. days to first flowering days to first flowering showed negative direct effects with yield per plant (-0.233). this trait showed the maximum positive indirect effect in days to 50% flowering (0.216), pods per plant (0.175), plant height (0.080). in contrast, this trait showed a negative indirect effect through seeds per pod (-0.005), days to maturity (-0.061), pod length (-0.067), pod width (-0.078), yield per plant (0.146), hundred seed weight (-0.172) (table 3 and figure 1). 3.3.2. plant height plant height showed a positive direct effect on yield per plant (0.294). this trait projected maximum positive indirect effects on biological yield via yield per plant (0.657), pods per plant (0.514), days to 50% flowering (0.136), and pod length (0.018). in contrast, this trait projected negative indirect effects on biological yield through seeds per pod (-0.022), pod width (-0.032), days to first flowering (-0.063), hundred seed weight (0.067), and days to maturity (-0.120) (table 3). 3.3.3. pods per plant pods per plant showed a positive direct effect on yield per plant (0.762). pods per plant showed the maximum positive indirect effect through yield per plant (0.786), followed by plant height (0.198) and days to first flowering (0.119). in contrast, this trait showed the negative indirect effect via pod length (0.013), seeds per pod (-0.014), pod width (-0.053), days to first flowering (-0.053), hundred seed weight (-0.074), and days to maturity (-0.085) (table 3). in path coefficient analysis of revealed that the number of pods per plant had the greatest direct effect on yield per plant. 3.3.4. days to 50% flowering days to 50% flowering showed a positive direct effect on yield per plant (0.251). this trait showed the maximum positive indirect effect through pods per plant (0.360) followed by yield per plant (0.188), plant height (0.159). in contrast, this trait showed a negative indirect effect through seeds per pod (-0.013), pod length (-0.044) pod width (0.074) days to maturity (-0.095) hundred seed weight (-0.156) and days to first flowering (-0.201) (table 3). hageblad [42] showed that yield had positive associations with days to 50% flowering. 3.3.5. days to maturity days to maturity showed positive direct effects with yield per plant (-0.138). this trait showed the maximum positive indirect effect through yield per plant (0.475) which was significantly followed by pods per plant (0.472), plant height (0.256), days to 50% flowering (0.172), and pod length (0.006). in contrast, this trait showed a negative indirect effect through seed per pod (-0.021), width (-0.047), days to first flowering (-0.013), and hundred seed weight (-0.122) (table 3 and figure 1). 3.3.6. pod length pod length showed a positive direct effect on yield per plant (0.112). this trait showed the maximum positive indirect effect through yield per plant (0.385) followed by hundred seed weight (0.173), days to first flowering (0.139), pod width (0.117), and plant height (0.047). in contrast, this trait showed a negative indirect effect through days to maturity (-0.007), seeds per pod (-0.012), pods per plant (-0.086), and days to 50% flowering (0.099) (table 3). j. multidiscip. appl. nat. sci. 110 3.3.7. pod width pod width showed a positive direct effect on yield per plant (0.161). this trait showed the maximum positive indirect effect through hundred seed weight (0.237) followed by yield per plant (0.205), days to first flowering (0.114), pod length (0.082), and days to maturity (0.040). in contrast, this trait showed a negative indirect effect through seeds per pod (-0.033), plant height (-0.059), days to 50% flowering (-0.115), and pods per plant (0.252) (table 3). 3.3.8. seeds per pod seeds per pod showed a negative direct effect on yield per plant (-0.029). this trait showed the maximum positive indirect effect through yield per plant (0.589) followed by pods per plant (0.381), plant height (0.228), days to 50% flowering (0.112), pod length (0.046), and pod width (0.014). in contrast, this trait showed a negative indirect effect through hundred seed weight (-0.022), days to first flowering (-0.041), and days to maturity (-0.099) (table 3). tanni et al. [43] also observed similar results in the okra promising genotypes in sylhet during 2018. 3.3.9. 100 seed weight hundred seed weight showed positive direct effects with yield per plant (0.283). this trait showed the maximum positive indirect effect through yield per plant (0.281) followed by days to first flowering (0.142), pod width (0.135), pod length (0.069), days to maturity (0.060), and seeds per pod (0.002). in contrast, this trait showed a negative indirect effect on plant height (-0.070), days to 50% flowering (-0.139), and pods per plant (-0.200) (table 3). goulart et al. [44] investigated pigeon pea (cajanas cajan) varieties revealed that 100-grain weight had the highest positive direct effect on grain yield. 3.3.10. residual effect the residual effect of the present study was 0.054 indicating that 94.60% of the variability was accounted for 10 yield contributing traits included in the present study. the rest amount of the figure 1. path diagram of yield contributing characters in vegetable pea (1. days of first flowering; 2. days of 50 flowering; 3. days to maturity; 4. plant height; 5. pods per plant; 6. pod length; 7. pod width; 8. seeds per pod; 9. 100 seed weight; 10. yield per plant). j. multidiscip. appl. nat. sci. 111 variability might be controlled by other yield contributed traits that were not included in the present investigation. 4. conclusions based on the findings of the present investigation, it can be concluded that the analysis of variance revealed that all the characters showed significant differences between the treatments. phenotypic coefficients of variation (pcv) were close to genotypic coefficients of variation (gcv) for all the characters. high heritability associated with high genetic advance per mean was observed for plant height, pod per plant, hundred seed weight, and seed yield per plot. the correlation analysis revealed that seed yield per plant showed a positive and significant correlation with the characters' days to maturity, plant height, and pods per plant and seeds per pod at both genotypic and phenotypic levels. days to fifty percent flowering, plant height, pods per plant, pod length, pod width, 100 seed weight showed positive direct effects on yield per plant. hence, yield improvement in pea would be achieved through the association and selection of these characters. we can observe the days to maturity, plant height, pods per plant, pod length, and seed showed a considerable positive and highly significant correlation with both genotypic and phenotypic levels indicating yield could be increased with the increase of the best performance in the experiment. author information corresponding author ahasan ullah khan — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); department of agroforestry and environmental science, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors sarah tasnim — department of genetics and plant breeding, bangabandhu sheikh mujibur rahman agricultural university, gazipur-1700 (bangladesh; department of crop genetics and plant breeding, institute of crop science, beijing -100081 (china); nilufa yasmin poly — department of biochemistry and molecular biology, khulna agricultural university, khulna-9208 (bangladesh); nusrat jahan — department of plant pathology, sylhet agricultural university, sylhet -3100 (bangladesh); references [1] m. m. praça-fontes, c. r. carvalho, and w. r. clarindo. 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(jackfruit) ahasan ullah khan , israt jahan ema, md. ruman faruk, shofiul azam tarapder, anayat ullah khan, sana noreen and muhammad adnan received : april 26, 2021 revised : june 29, 2021 accepted : july 1, 2021 online : july 30, 2021 abstract the jackfruit (artocarpus heterophyllus l.) is well-known as “poor man’s food” fruit in bangladesh. it is widely consumed by most of the rural people, and it is the national fruit of bangladesh. the main aim of this review is to document the medicinal significance of jackfruit, major parts and uses of the jackfruit in bangladesh. this article was based on mostly a literature review. all parts of the fruit and plant are used as human food, animal feed, and wood source for furniture. although jackfruit is the main fruit of the tree, it is used as furniture for its beautiful texture and wood color. jackfruit contains anti-bacterial, anti-diabetic, anti-oxidant, antiinflammatory, and anti-helminthic properties. the fruit is rich in carbohydrates, minerals, carboxylic acids, dietary fiber, vitamins, and minerals. the seed is rich in manganese, magnesium, potassium, calcium, iron, and lectins and thus meets up nutritional r equirements for the rural people. the present study attempted to review the medicinal importance, health-promoting effects of jackfruit and seeds with special emphasis on their applications in the food. keywords jackfruit, medicinal importance, fruit and seed quality, health benefits, morphology 1. introduction artocarpus heterophyllus is the scientific name of jackfruit which the fruit belong to the family of moracea and native to southeast asia [1] –[3]. it is widely cultivated in most tropical countries like bangladesh, india, burma, philippines, pakistan, sri lanka, malaysia, thailand, and several areas in brazil, queensland, africa and some other parts of australia and america. bangladesh is sanctified with a vast diversity of fruits. about 70 different types of fruits are grown in bangladesh [4]. jackfruit is the national fruit of bangladesh [5][6] and mango is our national plant of bangladesh [7]. it is the major fruit tree at madhupur tract in bangladesh [8]. the edible fruit of jackfruit is known as the “poor man’s food” [9][10]. it is the most popular fruit in rural and urban areas of bangladesh. it ranks the 4 th position as per production volume after banana, mango, and pineapple [11]. jackfruit ranks the 3 rd position with respect to fruit production in bangladesh. there are so many benefits from the fruit, tree, branches of jackfruit. the fruit is enriched with a diverse of nutrients and edible where the fruit can be consumed when its ripe and when its green part uses as vegetable. the edible bulbs of ripe jackfruit are usually consumed fresh or processed into canned products; 10-15 % of the total fruit weight is considered as its seed weight. in asia, folk consume the plants in medicine and used as anti-bacterial, anti-diabetic, anti-oxidant, anti-inflammatory, and anti-helmintics agents [12]. approximately, 2 megajoule energy per kg/wet weight of ripe perianth is required for the fruit processing. besides, the fruit can supply bioactive compounds [13]. phytonutrients contain in jackfruit such as lignans, flavones, and saponins exhibit anti-cancer, anti-ulcer, anti-hypertensive, and anti-aging activities [14]. the fruit is rich in carbohydrates, minerals, carboxylic acids, dietary fiber, vitamins, and minerals. the seed is rich in manganese, magnesium, potassium, calcium iron, and lectins and thus meets up nutritional requirements for the rural people. the jackfruit seed flour is not only a rich source of protein, starch and dietary fibers but can also be regarded as an abundant yet cheap source of nutrients. lectin, a class of glycoprotein found in jackfruit seed, has been reported to possess copyright holder: © khan, a. u., ema, i. j., faruk, m. r., tarapder, s. a., khan, a. u., noreen, s., and adnan, m. (2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.88 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.88 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.88&domain=pdf&date_stamp=2021-07-30 j. multidiscip. appl. nat. sci. 107 anti-bacterial, anti-fungal, and anti-carcinogenic properties [15]. ripe jackfruit pulp is processed, dehydrated and sold as dry powder which is utilized for juice, biscuits, chutney, jam, jelly, toffee, paste, leather, bar, nectar, squash, and pickle [16]. it is also canned in syrup, either plain or blended with dehydrated bulbs, chutney, preserves, candy, concentrate and powder. now a days, consumer preferences have changed to diets with more natural antioxidants, dietary fibers, natural colorants, minerals, vitamins, reduced calories, low cholesterol, low sugar, free of chemicals, etc. the possibilities of utilizing jackfruit waste and by product of leaves and wood for production of biogas, briquettes and biochar [17]. jackfruit growth and development are directly depended on the source of nutrients and those nutrients improve the nutritional quality of jackfruit [18]. the qualitative jackfruit and some seeds are exported to uk [19]. the jackfruit is a cross pollinated fruit tree and is mainly propagated by seeds [20]. the tree as well can provide many other uses in environmental protection. the leaves of the jackfruit tree are used as nutritious food for cattle. the leaves are rich in vitamins and minerals which are important for the animal. its remaining stalks are used as wood. however, the main stem enhances the beauty of the house as furniture. it is possible to earn income by selling wood as wood, just as it is possible to make a lot of profit by selling well shape wood. however, a lot of money is earned by selling ordinary jackfruit fruits. but the jackfruit trees cultivated around the village house are not taken care of since no fertilizer is given. by considering the above points, the present study was undertaken to collect information about the significance of the nutritional and medicinal importances of jackfruit in bangladesh. 2. methodology to assess the current state of the research on importance of jackfruit, a review of the existing journal literature, books, report, blogs, and newspaper were carried out. keywords (jackfruit, medicinal importance, fruit and seed quality, health benefits, and morphology) search in the google, google scholar, web of science database (www.thomsonreuters.com/web-of-science), and a full-text search of the science direct (www.sciencedirect.com) database were carried out. information was also collected from government organization and ngo’s by personal communication. the reviews or literature reviews will be examined to identify further studies for inclusion, and the results of meta-analyses will not be included in the analysis. 3. morphology of jackfruit 3.1. size and shape jackfruit grow as an evergreen tree that has a relatively short trunk with a dense treetop. it easily reaches heights of 10 to 20 m (33 to 66 feet) and trunk diameters of 30 to 80 cm (12 to 31 inches). the canopy shape is usually conical or pyramidal in young trees and becomes spreading and domed in older trees (figure 1). it sometimes forms buttress roots. the bark of the jackfruit tree is reddishbrown and smooth. in the event of injury to the bark, a milky juice is released. in young trees, the leaf edges are irregularly lobed or split. the tree casts a very dense shade. heavy side branching usually begins near the ground. all parts of the tree no. keyword search articles number 1 artocarpus heterophyllus l. or jackfruit 215 2 medicinal importance 188 3 fruit and seed quality 218 4 health benefits 114 5 morphology 225 6 limit of article 201 7 manually screened 87 8 articles included in the review 60 table 1. keyword search. https://en.wikipedia.org/wiki/buttress_root https://en.wikipedia.org/wiki/buttress_root j. multidiscip. appl. nat. sci. 108 exude sticky white latex when injured. 3.2. flowers this species is monoecious, having male and female inflorescences (or “spikes”) on the same tree. the flowers are small, sitting on a fleshy rachis. male and female spikes are borne separately on short, stout stems that sprout from older branches and the trunk. the male flowers are greenish, some flowers are sterile (figure 1). male spikes are found on younger branches above female spikes. male spikes are dense, fleshy, cylindrical to club shaped, and up to 10 cm (4 in) in length. flowers are tiny, pale green when young, turning darker with age. the male flowers are less and small hairy and the perianth ends with two 1 to 1.5 mm ( 3 ⁄64 to 1 ⁄16 in) membrane. after the pollen distribution, the stamens become ash-gray and fall off after a few days. female flowers are larger, elliptic or rounded, tubular calyx, with hairy and tubular perianth, have a fleshy flower-like base. the female flowers contain an ovary with a broad, capitate or rarely bilobed scar (figure 1). the flowers are reportedly pollinated by insects and wind, with a high percentage of crosspollination. the blooming time ranges from december until february or march [21]. 3.3. fruit the inflorescences are formed on the trunk, branches or twigs (cauliflory). jackfruit trees are monoecious, having both female and male flowers on a tree. fruit of jackfruit has a compound or multiple fruit with a green to yellow brown exterior rind that is composed of hexagonal, bluntly conical carpel apices that cover a thick, rubbery, whitish to yellowish wall (figure 1). the ellipsoidal to roundish fruit is a multiple fruit formed from the fusion of the ovaries of multiple flowers. the fruits grow on a long and thick stem on the trunk. the heavy fruit is held together by a central fibrous core. they vary in size and ripen from an initially yellowish-green to yellow, and then at maturity to yellowish-brown. they possess a hard, gummy shell with small pimples surrounded with hard, hexagonal tubercles. the large and variously shaped fruit have a length of 30 to 100 cm (10 to 40 inches) and a diameter of 15 to 50 cm (6 to 20 inches) and can weigh 10–25 kg (22–55 pounds) or more [22]. figure 1. (a) jackfruit tree with the fruits; (b) jackfruit tree with the fruits in different stages of fruiting; (c) club shaped spike male inflorescence; (d) female inflorescence; (e) leaf showing both side (f) cut section of jackfruit; (g) the bulbs of jackfruit; (h) the jackfruit seed and (i) jackfruit chips. https://en.wikipedia.org/wiki/rachis https://en.wikipedia.org/wiki/perianth https://en.wikipedia.org/wiki/inflorescences https://en.wikipedia.org/wiki/cauliflory https://en.wikipedia.org/wiki/plant_reproductive_morphology https://en.wikipedia.org/wiki/multiple_fruit https://en.wikipedia.org/wiki/tubercle j. multidiscip. appl. nat. sci. 109 3.4. leaves leaves are dark green, alternate, entire, simple, glossy, leathery, stiff, large (up to 16 cm [6 in] in length), and elliptic to oval in form. the leaves are alternate and spirally arranged (figure 1). they are gummy and thick and are divided into a petiole and a leaf blade. on older trees, the leaves are rounded and dark green, with a smooth leaf margin. the petiole is 2.5 to 7.5 cm (1 to 3 inches) long. the leathery leaf blade is 20 to 40 cm (7 to 15 inches) long, and 7.5 to 18 cm (3 to 7 inches) wide and is oblong to ovate in shape. leaves are often deeply lobed when juvenile and on young shoots. 3.5. seeds seeds are light brown, rounded, 2–3 cm (0.8–1.2 in) in length by 1–1.5 cm (0.4–0.6 in) in diameter, and enclosed in a thin, whitish membrane. there may be about 100–500 seeds per fruit (figure 1). seeds are recalcitrant and can be stored up to a month in cool and humid conditions [21]. the seed coat consists of a thin, waxy, parchment-like and easily removable testa (husk) and a brownish, membranous tegmen. the cotyledons are usually unequal in size, and the endosperm is minimally present. an average fruit consists of 27% edible seed coat, 15% edible seeds, 20% white pulp (undeveloped perianth, rags) and bark, and 10% core [7]. 4. major parts of a jackfruits jackfruit has three main parts [2], the fruit axis, the perianth and the bulbs. the axis contains the core, latex and the arils (figure 1). jackfruit has lactiferous cells which are responsible for production of latex [23], which is sap that sticky in nature. the perianth contains the rind and the rags, the bulbs contain the edible part and the seeds. the inedible parts of jackfruit include prickly rind, nonedible perianth and a central core [24]. all these inedible jackfruit parts are potential raw materials for bioenergy and biochar production. 5. uses of the jackfruit parts all parts of the fruit and tree are used as human food, animal feed and wood source for furniture (figure 2). the jackfruit pulp is processed for juice, biscuits, chutney, jam, jelly, toffee, paste, leather, bar, nectar, squash and pickle. the color and flavor are important for the jackfruit bulb [25]. ice-cream prepared by using the jackfruit bulbs. the green fruit bulbs and seeds used as cooking materials in the farmers community. the leaves use as animal feed and the leaves are contain significant amount of nutrients. the wood materials are using as furniture of home and possibilities of utilizing jackfruit waste and by product of leaves and wood for production of biogas, briquettes and biochar which is applied in the farmer’s field. the jackfruit bulbs are using in bakery industry which is improve the food quality. the bakery products are increasingly gaining popularity since they are convenient, readily available in diverse tastes and textural profiles, less expensive and possess high nutritive value. the bakery products such as biscuits [26], cookies, bread [27], cake [28], and muffins [29]. have been formulated by supplementation with different levels of jackfruit seed flour. therefore, there is scope to develop the industry which is help in employment sector in bangladesh. 6. medicinal significance of jackfruit the artocarpus species have been used as traditional medicines. the plants have been used as anti-bacterial, anti-diabetic, antioxidant, antifigure 2. diversified uses of jackfruit plant, fruits and by products [30]. https://en.wikipedia.org/wiki/alternate_leaf https://en.wikipedia.org/wiki/petiole_(botany) j. multidiscip. appl. nat. sci. 110 inflammatory, and anti-helminthic agents [31]. jackfruit is also a rich source of many minerals such as n, p, k, ca, mg, s, zn, cu, etc. [32]. the nutritional parameters of the jackfruit flesh, seeds and the meal are presented in table 2. the water contents of the young, ripe fruit and seeds were in a range of 51.0—94.4 g. the available digestible carbohydrate contents of the young, ripe fruit and seeds were in a range of 16.0-38.40 g. jackfruit seeds are underutilized and less acknowledged by people, but they have considerable nutritional benefits and constitute about 10% to 15% of the fruit weight [33]. the protein content of the meal was 0.40-2.60 g in the young, ripe fruit and seeds of jackfruit, with a higher contribution from the young fruit while the fat content of the meal was 0.10— 0.60 g. the fiber content of the meal was 1.0—2.60 g in the young, ripe fruit and seeds of jackfruit with a higher contribution from the young fruit while the mineral content of the meal was 0.87-0.90 g. the ripe fruit contains 20.60 g sugar in the meal. the jackfruit rich in calcium, magnesium, phosphorus, potassium, sodium, iron, vitamin a, thiamine, riboflavin and vitamin c (table 2). jack seeds contained high total dietary fiber (tdf) (11.1%) compared to flesh (2.6%). jackfruit seeds also contained 8% resistant starch (undigestible starch). it is a major source of carbohydrates, minerals and vitamins [34]. hasan et al. [35] reported that the average annual net returns found more than the agriculture system. alves et al. [36] observed that the fruit contains lignins, flavones and saponins which have the properties of anti-cancer, anti-ulcer, anti-hypertensive, and anti-aging. it contains immense medicinal values and also considered a rich source of carbohydrates, minerals, carboxylic acids, dietary fiber, and vitamins such as ascorbic acid and thiamine [13]. manganese, magnesium [32], potassium, calcium, and iron [37] elements are found in seed. mukprasirt and sajjaanantakul [38] reported that the seeds contain lectins as jacalin and artocarpin. jacalin has been shown to be useful for the evaluation of the immune status of patients infected with human immunodeficiency virus [39]. seed nanoparticles were found effective against escherichia coli and bacillus megaterium bacteria [40]. it has anti-oxidant action [3], and acts against inflammation, malarial fever and skin diseases [41], it also shown, anti-bacterial and anti-helminthic activities [42]. the tree leaves are commonly used as healing for ulcer. its leaves have the potential of curing diabetics due to the presence of hypoglycemic and hypolipidemic substances [43]. the leaves and stems have sapogenins, cyclooctenone, cycloartenol, β-sitosterol and tannins. the latex yield artosteron mixed with vinegar promotes healing of glandular swelling and snake bites [44]. long et al. [45] reported that the nutrients young fruit ripe fruit seed water (g) 76.2—85.2 72.0—94.0 51.0—64.50 protein (g) 2.00—2.60 1.20—1.90 0.40—0.43 fat (g) 0.10—0.60 0.10—0.40 0.40—0.43 carbohydrate (g) 9.40—11.5 16.0—25.4 25.8—38.4 fiber (g) 2.60—3.60 1.0—1.50 1.0—1.50 sugar (g) 20.6 minerals (g) 0.90 0.87—0.90 0.90—1.20 calcium (mg) 30.0—73.2 20.0—37.0 50.0 magnesium (mg) 27.0 54.0 phosphorus (mg) 20.0—57.2 38.0—41.0 38.0—97.0 potassium (mg) 287—323 191—407 246 sodium (mg) 3.00—35.0 2.0—41.0 63.2 iron (mg) 0.40—1.90 0.50—1.10 1.50 vitamin a (iu) 30 175—540 10.0—17.0 thiamine (mg) 0.05—0.15 0.03—0.09 0.25 riboflavin (mg) 0.05—0.20 0.05—0.40 0.11—0.30 vitamin c (mg) 12.0—14.0 7.00—10.0 11.0 source: goswami and chacrabati [34] table 2. nutrient composition of jackfruit (100 g edible portion). j. multidiscip. appl. nat. sci. 111 root extract is a therapy for asthma and skin disorder. the wood has sedative property and believed that it may cause promotion of abortioncure diarrhea and fever [46]. the fruits and roots are used for tapeworm infection [47]–[49]. the fruit is rich in carbohydrates, complex b vitamins, and minerals [50]. the freshly fruit is consumed and can be processed to candies, sweeties, frozen pulps, juices and vegetable in immature fruit. its seed can be consumed as baked or used in culinary to develop several menus. now, there are studies concerning the use of seed meal for preparing cookies, sweeties and bread as an alternative source of carbohydrate. the jackfruit contains variable constituents of moisture (6.7%), glucosides (38.0%), lipids (0.7%), protein (1.7%) and cellulose (59.0 %) [50]. jagtap and bapat [51] observed that the ripe fruits are rich in nutritive value; every 100 g of ripe fruit contains 287-323 mg potassium, 30.0-73.2 mg calcium and 11-19 g carbohydrates. chowdhury et al. [52] reported that the bark contains betullic acid and a flavone pigment, cycloheterophyllin (c30h30o7). the fruit pulp also contains lycopene [53]. de-faria et al. [54] reported that the fruit contain 18 carotenoids and they were successfully separated, identified, and quantified. the leaves and stem contain sapogenins, cycloartenone, cycloartenol, βsitosterol, and tannins thus showing estrogenic activity. a root contains β-sitosterol, ursolic acid, betulinic acid, and cycloartenone [4]. jackfruit seed contains a thin brown spermoderm, the crude fiber (2.36 %), but the composition of flour depends on nature of seed [55]. 7. health benefits of jackfruit seeds the phytonutrients such as lignans, saponins, and isoflavones present in the seeds, plays beneficial role in human health presented in figure 3 [55]. the presence of high fiber content (3.6 g/100 g) in the jackfruit improve digestion system and produces smooth bowel movements. it also offers protection to the colon mucous membrane by removing carcinogenic chemicals from the large intestine [30]. the addition of the jackfruit seed flour to deepfried products results in a reduction in fat absorption up to a certain limit [56]. the seeds are rich in dietary fiber and b-complex vitamins. due to their high fiber content, they help lower the risk of heart disease, prevent constipation and promote weight loss. jackfruit seeds also contain resistant starch, which controls blood sugar and keeps the gut healthy. jackfruit seeds possess anti-microbial activity, which prevents foodborne diseases [54]. the seeds contain an important lectin known as jacalin, used as a tool to evaluate the immune system of an hiv infected person. in china, the seeds are known to be beneficial in overcoming the toxicity due to alcohol and likewise. meanwhile, in india, the seeds are a crucial component of an antidote produced for heavy drinkers [57]. the seeds contain an abundance of magnesium which plays a vital role in lowering the blood pressure and figure 3. health benefits of jackfruit. j. multidiscip. appl. nat. sci. 112 maintaining bone health since it aids in calcium absorption and hence helps to strengthen the bones [2]. furthermore, the seeds are rich in highly soluble protein resulting in the prevention and treatment of mental stress and anxiety. the seeds have low water and fat-absorption capacities, which helps in prevention of obesity [58]-[60]. 8. conclusions jackfruit is quite versatile. it can be eaten raw, cooked, ripe or unripe and tastes great in a variety of sweet and savory dishes. the consumption of jackfruit has grown in recent years due to its reported health benefits. jackfruit and its pulp and seeds are rich sources of several high‐value compounds with potential beneficial health benefits. the rich bioactive profile of jackfruit makes it a highly nutritious and desirable fruit crop. the review concluded that all the nutrients like vitamins and minerals may fulfill the malnutrition problem in the rural area of bangladesh. author information corresponding author ahasan ullah khan — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); department of agroforestry and environmental science, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors israt jahan ema — department of plant pathology and seed science, sylhet agricultural university, sylhet-3100 (bangladesh); md. ruman faruk — department of agricultural extension education, sylhet agricultural university, sylhet-3100 (bangladesh); shofiul azam tarapder — department of agronomy and haor agriculture, sylhet agricultural university, sylhet-3100 (bangladesh); anayat ullah khan — department of mathematics, jashore university of science and technology, jashore-7408 (bangladesh); orcid.org/0000-0001-7447-4864 sana noreen — university institute of diet and nutritional sciences, the university of lahore, punjab-54000 (pakistan); orcid.org/0000-0002-4040-5454 muhammad adnan — department of agronomy, university of sargodha, punjab40100 (pakistan); orcid.org/0000-0002-7582-4675 conflict of interest the authors declare that there is no conflict of interest. references [1] b. o. ajiboye, o. adeleke ojo, o. adeyonu, o. imiere, b. emmanuel oyinloye, and o. ogunmodede. 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(2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.85 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.85 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.85&domain=pdf&date_stamp=2021-07-10 j. multidiscip. appl. nat. sci. 90 practices may enhance atmospheric carbon sequestration [12][23]. the scientists, policymakers, and the government are growingly concerned about climate change due to the continuous increase of ghgs concentration. in this sense, the interest in mounting carbon stocks in trees and substitution of fossil fuel by the use of tree biomass are also rising [24]. globally, several studies have been performed on the role of tropical forests in climate change mitigation and possible effects on climate due to deforestation [25][26]. the magnitude of carbon change due to tropical forest deforestation is difficult to predict [8] as the tropical forest contain more species than any other ecosystems [27] and are large carbon sinks [28]. about 89% stored carbon get loss due to the loss of living biomass in the ecosystems [29]. it is essential to know the stocks of carbon as biomass per unit area for different forest types to assess the impact of deforestation and re-growth rates on the global carbon cycle. therefore, both the above ground biomass (agb) and below ground biomass (bgb) need to be measured for the better calculation of total forest carbon [30]. the forests and soil are necessarily conserved to maintain considerable amounts of carbon on the earth [31]. aboveground biomass represents 60% of total tree biomass [23], hence, measured more importantly while calculating plant carbon pool [32]. also, belowground biomass, deadwood biomass, and litter biomass are required to measure to determine total carbon stock by plants over a specific time [11]. it also helps to determine the effects of land-use change and deforestation on net carbon fluxes. in nepal, various studies have been performed on agroforestry focusing on tangible benefits; however, studies focusing on intangible benefits like carbon sequestration are very less in number [33]. pradhan et al. [34] have estimated that the forests of nepal stored 897 million metric tons of carbon (including carbon in above-ground biomass, carbon in below-ground biomass, sub-total: carbon in living biomass, carbon in dead wood, carbon in litter, sub-total: carbon in dead wood and litter, and soil carbon to a depth of 100m) in the year 2005. similarly, the carbon in aboveground biomass in the forests of nepal for the year 1986 by physiographic regions was found to be 36 million tons in terai, 76 million tons in siwaliks, 67 million tons in the middle mountains, 123.5 million tons in high mountain, and 11.5 million tons in high himalaya. however, a few studies has been carried out in estimating carbon stocks both in the biomass and in the entire soil profile under different land use categories in nepalese mountain watershed. also, very little studies has been carried out in estimating carbon pools in vegetation (above and below ground biomass) and in soil profiles under different forest types in nepal [35][36]. the updated data on national forest inventory and technical capacity is poor, and the changes in forest cover biomass stock, carbon removal, and carbon emission are limited in figure 1. map of the study area. j. multidiscip. appl. nat. sci. 91 the developing countries like nepal [36]. community forestry has been given major priority of nepal’s forestry sector and during the last 30 years of community forestry implementation, more thean 25% of the national forest is handed to more than 14,200 community forest users groups (cfugs) [37]. despites protecting community forest by cfugs for about last 30 years, forest and soil inventory has been paid little attention regarding carbon sequestration. hence, amount of soil and biomass carbon sequestration is unknown [38]. therefore, this study has endeavored to estimate the biomass as well as carbon stock and to compare biomass variation at the different slopes in depard community forest of makwanpur district, nepal. 2. materials and methods 2. 1. study area the study was performed in the depard community forest of manahari-6, makwanpur district (84 0 41' to 84 0 35'e longitude and 27 0 21' to 27 0 40'n latitude). the community forest occupies an area of 163.56 ha. the forest is mixed deciduous forest dominated by the species like schima wallichi (chilaune) and shorea robusta (sal). the district consists of several districts level roads which are reachable by mahendra highway (47 km only) and tribhuvan highway (110 km only). mahabharat hills lie in the north and the churia hills lie in the south of this district. tropical and subtropical climate is found in the churia range which lies in the southern part of the district while temperate climate is found in the mahabharat range in the northern part [39]. seasonal characteristics include cold, hot, and rainy seasons (each of four months) with an average relative humidity of 73.5 % in the district. rapti and bagmati are the major river system in this district. most of the people in the district depend on subsistence farming for economy rather than industrial sector. about 80.7 % of the population depends on livestock and agriculture while 17.3% of them rely on small scale business sectors. 2.2. methods 2.2.1. data collection data was collected primarily through a direct field survey of biophysical measurement. the biophysical measurement i.e. diameter at breast height (dbh) and height of trees was measured using diameter-tape and sunto-clinometer and abney’s label respectively. forest inventory was conducted to estimate the present status of the forest. a random sampling method was applied to collect data for the estimation of tree biomass and carbon stock in the forest. a total of twenty-one (21) concentric circular sample plots were laid out as per the forest carbon stock measurement guidelines with the radii of 8.92 m (for measuring trees and poles), 5.64 m (for measuring saplings), 1 m (for measuring seedlings) and 0.56 m (for taking the samples of the leaf litter, herbs, grass and soil) [40] along with 1% sampling intensity were randomly established within the study area referring to national inventory guideline developed by department of forest, community and private forest division [41] (figure 2 and 3). the circular area of 250 m 2 was predetermined with a radius of 8.92 m for this study. all trees with dbh ≥ 5 cm were taken for estimation of biomass and carbon stock in the forest. severals research findings, publications, other relevant literatures related with carbon and biomass estimation were reviewed to perceive the better understanding, interpretation and analysis of the research. 2.2.2. data analysis data were pooled and analyzed with spss software. arc gis 10.2 was used to fit a map. t-test figure 2. distribution of sample plots. figure 3. sample plot layout. j. multidiscip. appl. nat. sci. 92 was applied to compare the average biomass in different slopes of the forest because t-test is performed to determine if there is significant difference between the mean of two groups from randomly sampled data. in this study, to determine significant difference between average biomass in different slopes of forest, t-test is used. 2.2.3. biomass estimation and net carbon content the biomass of each tree includes stems, branches, leaves, and roots. it can be divided as aboveground biomass which includes stem, branch, and leaves and underground biomass which include the root. the important characteristics such as volume and biomass were predicted by biophysical measurement i.e. non-destructive methods and mathematical models by measuring diameter at breast height (dbh) directly. above-ground biomass: a simplified standard regression model was used to calculate the biomass of the trees; it is based on dbh, height, and wood density [42]–[44]. agb is calculated by the formula given by chave et al [45]. (agb) = 0.0509*ρd 2 h (1) where, ρ= specific gravity of wood (gcm -3 ), d= tree dbh (cm) and h= height of tree (m).the obtained agb value for the each individual weight (kg) of a sampling plot were summed up and divided by sampling plot area (250m 2 ). the biomass stock density value thus obtained is in kgm -2 which was then converted to t/ha by multiplying it by 10. the wood-specific gravity used for shorea robusta is 0.88 as its specific gravity value ranges from 0.83-0.93g/cm 3 [46] and schima wallichi is 0.689 g/cm3 as its specific gravity value ranges from 0.45-0.92 g/cm 3 [47]. below ground biomass (bgb): it includes biomass of live roots (<2mm diameter). it was calculated by multiplying with agb (0.26) [48]. below ground biomass (bgb) = 0.26 x agb (ton) (2) total biomass: total biomass is the sum of the above and below ground biomass [49][50]. it is calculated as: total biomass (tb) = agb + bgb (3) net carbon content: the stock method was used to calculate biomass carbon; where carbon content is assumed to be approximately 50% of dry biomass [51]. the formulas used to calculate above ground carbon (agc) and below-ground carbon (bgc) are: total agc = (total agb of the tree) x 47% (4) total bgc = (total agoc) x 15% (5) total carbon content = total agc + total bgc (6) 3. results and discussions 3.1. diameter and height distribution the total number of trees was found to be 280, 200, and 168 per hectare in 0-5 %, 620 %, and >20 % of the slope respectively in the forest. the average diameter was 16.92 cm, 21.95 cm, and 31.68 cm and average height was 9.56 m, 9.38 m, and 11.40 m for the trees in given slopes table 1. diameter and height distribution of trees per ha. no. slopes (%) no. /ha. diameter (cm) height(m) min max average min max average 1 0-5 280 6.5 93.8 16.92 7 23 9.56 2 6 -20 200 10 95.6 21.95 7 19 9.38 3 >20 168 13.6 90.4 31.68 8 17 11.40 figure 4. estimated biomass of the forest (ton/ha). j. multidiscip. appl. nat. sci. 93 respectively (table 1). the better frequency of the forest tree species was found in lower and medium slope area due to the presence of stable environmental conditions [52]. this studied shows relatively low stem density (216 trees/ha) on average, however densities reported by timilsina et al. [53] (220 trees/ha) in bardia national park a somewhat exceeded our overall mean. meanwhile, rautiainen measured similar densities (152–264 trees/ha) in sal forest in the bhabar–terai zone of nepal [54]. 3.2. biomass estimation the total biomass was found to be 565.13 t/ha, with agb 448.51 t/ha and below-ground biomass to be 116.61 t/ha in 0-5 % of slope in the forest. in 6-20% of the slope of the forest, the total biomass was found to be 481.42 t/ha with above-ground tree biomass 382.08 t/ha and below-ground root biomass to be 99.34 t/ha. similarly, the total biomass was found to be 334.75 t/ha with aboveground tree biomass 265.67 t/ha and below-ground root biomass to be 69.07 t/ha in >20 % of a slope of the forest (figure 4). another study done by maren and sharma in himalayas mountain forests [55] showed the average above ground live biomass as 164 tons/ha which is slightly less than this study. aboveground biomass varied from site to site because of varying plant community structures, variation in plant species and the succession stage of the forest. the total biomass and total carbon stock are highest (563.12 t/ha and 242.42 t/ha) in 0-5% slope of the forest, followed by 6-20% of slope (481.42 t/ ha and 205.38 t/ha) and lowest in >20% of slope (334.75 t/ha and 143.60 t/ha) in the study area (figure 4 and figure 5). the highest biomass and carbon stock in the 0-5% slope may be due to a higher density of trees (280 trees/ha) compared to 6 -20% and >20% slope. more the tree density higher is the biomass [56]–[58]. the present study suggests that total biomass and carbon stock varies from site to site i.e. varies with slope in the forest. altitudinal variation along with slope gradient has an impact on above ground carbon and below ground carbon because of its influence on soil water regime [59]. 3.3. carbon estimation the total carbon stock was found to be 242.42 t/ ha with an average above-ground tree carbon stock 210.80 t/ha and average below ground root carbon stock to be 31.62 t/ha in 0-5 % of a slope. in 6-20% of slope, the total carbon stock was found to 205.38 t/ha with an average above-ground tree carbon stock 178.59 t/ha and average below ground root carbon stock to be 26.79 t/ha. similarly, the total carbon stock was calculated to be 143.60 t/ha with an average above-ground tree carbon stock 124.87 t/ha and an average below-ground root carbon stock to be 18.73 t/ha in > 20 % of the slope of the forest (figure 5). around 53% of carbon stock was found in the forest area with slope 0-5% , while 41% carbon stock was present in the forest with slope gradient of 6-20%. similarly, in the forest area with more than 20% of the slope, 7% carbon stock was present (figure 6). not only in the sal dominated forest, similar result was found by feyissa et al. [60] while studying on egdu forest. moreover, maggi et al. [61] also concluded very steep slope areas contain little vegetation cover compared to low slope areas. on the contarary, zaki et al. [62] studied the forest carbon stock on tropical lowland dipterocarp forest and revealed that above ground carbon stock and below ground carbon stock tends to increase with slope. the distribution of biomass and carbon stocks in the forest is known to vary due to the presence of various tree species, table 2. paired t-test. s1 s2 s3 m1 m2 m3 n1 n2 n3 109.77 96.92 124.10 565.13 425.58 185.97 9 8 5 figure 5. carbon stock in the forest (t/ha). j. multidiscip. appl. nat. sci. 94 nutrient availability in soil, climate, and other disturbance regime too [59]. in the national scenario, the terai consists of a large amount of total organic carbon (479.29 t/ha) as compared to the average carbon stock of tropical forests of the world (285.0 t/ha) [63]. but it is lower than the average carbon stock of the community forests of nepal [64]. the sparse and dense area has 89.2 t/ha and 129.0 t/ha carbon in the kayerkhola watershed dominated by shorea robusta forest in the chitwan district [65]. the agb and bgb are highest (448.51 t/ha and 116.61 t/ha) in 0-5 % and lowest (334.75 t/ha and 69.07 t/ha) in >20 % of a slope the forest. similarly, above-ground carbon stock and below-ground carbon stock were highest (210.80 t/ha and 31.62 t/ ha) in 0-5 % of slope and lowest (124.87 t/ha and 18.73 t/ha) in > 20 % of slope of the forest. carbon composition is highest (53%) in 0-5% of slope and lowest (7%) in >20% of slope in the forest. tree biomass and carbon stock has inverse relation with slope [66]–[68] and our result in this study supports the growing indications that forest ecosystems growing at lower slope store higher amounts of carbon than forest ecosystems at higher slopes. both the above-ground and below-ground measurements should be carefully performed for precise estimation of biomass and carbon stock [58]. our study shows quite greater biomass and carbon stock than other studies which can bey supported by the study conducted by yohannes which concludes that the highest amount of carbon stock was found in middle altitude area dominated by shorea robusta and termanalia tomentosa [52]. 3.4. comparison of average biomass in different slopes of the forest the estimated amount of average biomass in the different slopes of the forest was compared by using a t-test. the result is mentioned in (table 2 and table 3). since t calculated value is more than t tabulated value. hence, it is concluded that biomass at different slopes in the forest is significantly different. 4. conclusions in the study of the biomass and carbon stock in the depard community forest, the measurements was found to be highest (563.12 t/ha and 242.42 t/ ha) in 0-5% slope of the forest, followed by 6-20% of slope (481.42 t/ha and 205.38 t/ha) and lowest in >20% of slope (334.75 t/ha and 143.60 t/ha) in the study area. this is related with the distribution of productive stem density within the forest as different areas with different slope varied significantly in the number, diameter and height of the tress as well. moreover, the ability of carbon sequestration varies according to the site, presence of invasive alien species, tree density, fodder collection, species richness, gazing, canopy cover/ strata, slope, and aspect, etc in different sites. significant difference in the biomass and carbon stock along the slope gradient was also proven by t -test. this research provides the baseline data on the slope and biomass significance in study area and further study through lidar technology is recommended as well. author information corresponding author sachin timilsina — institute of forestry, tribhuvan university, pokhara-33700 figure 6. carbon composition of forest (ton). test between 0 to 5 and 5 to 20 5 to 20 and >20 0 to 5 and >20 df 15 11 12 t-calc 0.0091 0.0103 0.0081 t-tab 0.0002 0.0082 0.0034 table 3. unequal variances. j. multidiscip. appl. nat. sci. 95 (nepal); orcid.org/0000-0002-4749-9289 email: sachintimilsina66@gmail.com authors birendra bohara — institute of forestry, tribhuvan university, hetauda-44107 (nepal); orcid.org/0000-0002-2280-0741 mahamad sayab miya — institute of forestry, tribhuvan university, pokhara-33700 (nepal); orcid.org/0000-0002-1675-593x deepak gautam — institute of forestry, tribhuvan university, pokhara-33700 (nepal); orcid.org/0000-0001-5239-365x siddhartha regmi — institute of forestry, tribhuvan university, hetauda-44107 (nepal); orcid.org/0000-0003-2731-7916 acknowledgment the authors would like to thank all the helping hands. conflict of interest the authors declare that there is no conflict of interest. references [1] k. panagiotopoulos, j. holtvoeth, k. kouli, e. marinova, and a. francke. 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[16], waste generation rate in various cities of bangladesh is ranging from 0.2 to 0.56 kg/cap/day. the composition of the waste is about 74.4% organic matter [17]. banana (family musaceae) is one of the highest consumed fruits in the world which creates notable agro-waste problems [18]. according to the food and agriculture organization of the united nations (fao), bangladesh produced 810,347 tons of banana in 2018 [19]. it can be assumed that a vast pile of banana peel waste has been creating because bangladesh is regarded as one of the most banana-producing countries based on banana production. the fate of the banana peel waste is copyright holder: © nurain, a., sarker, p., rahaman, m. s., rahman, m. m., and uddin, m. k. (2021) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.v1i2.89 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i2.89 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i2.89&domain=pdf&date_stamp=2021-07-31 j. multidiscip. appl. nat. sci. 118 landfilling which can contribute to the existing environmental problems. the major constituents of banana peel are cellulose, hemicellulose, pectin, and other low molecular weight species [20]. the banana peel presents a high adsorption capacity for metals and organic compounds due to the presence of the hydroxyl and carboxyl groups of the pectin. these functional groups exchange hydrogen ions for metal ions in solution or donation of an electron pair to form complexes from the metal ion in solutions [11]. the principal mechanism of adsorption process involves the accumulation of molecules of a solvent on the inner and outer surface (e.g., pore) of the adsorbent [21]. therefore, banana peel (musa sapientum) could be used as an adsorbent for pb 2+ removal and agricultural waste management. most researchers prepared the adsorbent material by chemical or physical activation to get more treatment efficiency [22][23]. the present study emphasizes untreated dried banana peels for removal of pb 2+ from water considering the relation of six different experimental condition/parameters (adsorbent dose, metal ion concentration, ph, temperature, agitation speed and contact time), which are cost-reducing and eco-friendly adsorbent preparation techniques for large scale treatment. from literature it has been proven that banana peel is an efficient adsorbent for pb 2+ removal [18][23]. different initial concentrations, adsorbent doses, contact time, agitation speed and temperature have great influence on the removal efficiency. present study also focuses on these different experimental conditions to figure out the maximum removal efficiency. the objective of the present research is to evaluate the efficiency of banana peels as low-cost agricultural waste-based bio-adsorbent (awb) for pb 2+ removal from water under different conditions and get rid of banana peel residue in an eco-friendly method. the study also determined langmuir and freundlich isotherms to represent the sorption data. 2. materials and methods 2.1. materials banana peels (bps) were collected from the jahangirnagar university campus area. lead nitrate pb(no3)2 was applied as a stock solution for the experiment. 0.1 m hydrochloric acid (hcl) and 0.1 m sodium hydroxide (naoh) were used for maintaining ph levels. nitric acid (hno3) was utilized for digestion purposes. for fourier transform infrared spectrophotometer (shimadzu irprestige-21), dichloromethane (dcm) was used for cleaning purposes, and potassium bromide (kbr) was used for the analysis of the functional group. the metal ions were determined by atomic absorption spectrometry (shimadzu aa-7000). 2.2. methods 2.2.1. preparation of adsorbent the collected bps were washed at least three figure 1. ftir spectra of dried banana peel powder (1 mm). wavenumber (cm -1 ) t ra n sm it ta n c e ( % ) j. multidiscip. appl. nat. sci. 119 times thoroughly with regular tap water and milli q water for removing impurities and other dust particles that may be clinging to the peels. the cleaned bps cut into small pieces and dried in sunlight for 5 days. later the peels were dried in an oven (daihan labtech, model ldo-030e) for 72 hours at 100 °c for removing the moisture from banana peels properly. dried peels were crushed via mortar and pestle and subsequently screened by a sieve to get uniform particle size of 1 mm. dried and minced bps surface functional groups were characterized by fourier transform infrared spectrophotometer (shimadzu irprestige-21). the prepared bps was stored in a desiccator until further use. 2.2.2. preparation of lead (pb) solution lead nitrate pb(no3)2 solution was prepared separately by dissolving suitable quantity of analytical grade lead nitrate (sigma–aldrich). for the preparation of 1000 ppm of the pb stock solution, it is needed to add 1.6 g pb(no3)2 in oneliter deionized water. through successive dilutions of the corresponding stock solutions, standard solutions of the desired concentrations were obtained. 50 ppm, 100 ppm, 150 ppm and 200 ppm working solutions were employed for the analysis. the solutions of different ph were prepared with adding sodium hydroxide (naoh) and hydrochloric acid (hcl) in the stock solution to the expected solution. 2.2.3. batch adsorption experiments the sorption study was carried out batch adsorption method to estimate the capacity of the adsorbent and analyzed the impact of six parameters. they were adsorbent dose, ph, pb 2+ concentration, contact time, temperature, and agitation speed. for considering the consequence of a particular parameter, that parameter has been changed gradually holding the other five constants [18]. 2.2.3.1. optimal dose of adsorbent the optimal adsorbent dose was determined by varying the amount of 25, 45, 65 and 85 gl -1 bps put into the separate beakers of 100 ppm of working solutions of ph 5 at the agitation speed of 100 rpm for 30 minutes at 25°c temperature. one 50 ml of lead solution was contained in control beakers without adding the adsorbent. each mixture was agitated by shaking incubator (gfl gesellschaft figure 2. effect of various factors on pb 2+ removal (a) effects of bp adsorbent dose on removal of pb 2+ (b) effects of ph of the solutions on adsorption of pb 2+ (c) effects of initial concentration of pb 2+ on the adsorption through bp (d) effect of contact time on removal of pb 2+ . j. multidiscip. appl. nat. sci. 120 für labortechnik mbh d-30938 burgwedel) and filtered by whatman filter paper (grade 42). then concentrated nitric acid (hno3) was added on the filtrates and heated them on a hotplate for digestion. after that, the mixtures filtered by a suction pump and finally the sample prepared for atomic absorption spectroscopy (shimadzu aa-7000). the amount of adsorption at equilibrium, qe (mg/g) and the percent adsorption (%) was calculated as follows [24]: qe = (1) percentage of pb adsorption (%) = (2) where ci is the initial concentration of metal in solution (mg l -1 ), ce is the final concentration of metal in solution (mg l -1 ), v is the volume of solution (l) and x is the dry weight of bio sorbent (g). 2.2.3.2. optimal ph for adsorption the batch biosorption study was inspected at separate ph values to determine the optimum ph for pb 2+ uptake by banana peels. 100 ppm working solutions were adjusted to ph values of 3, 5, 6 and 8 (use buffer solution) using 0.1m sodium hydroxide (naoh) and 0.1 m hydrochloric acid (hcl). later fixed 45 g l -1 of bp adsorbent was added to each solution at the agitation speed of 100 rpm for 30 minutes at 25 °c. the control batch was handled similar but without bps. then the solution was filtered as well digested for aas. 2.2.3.3. optimal initial metal ion concentration further adsorption investigation was carried out with the several initial concentrations of 50 ppm, 100 ppm, 150 ppm and 200 ppm pb 2+ solutions at optimum ph 5 with fixed dosage of bp adsorbent 45 g l -1 for 30 minutes at 100 rpm agitation speed and at the corresponding temperature 25°c. the supernatant in each beaker was filtered to separate the adsorbent from the solution and prepared for digestion. 2.2.3.4. optimal contact time for lead adsorption 100 ppm working solution was determined as the optimum lead solution. the adsorption examination of pb 2+ by bp was observed at different time series (10 minutes, 20 minutes, 30 minutes and 40 minutes) among the fixed dosage of 45 g l -1 of bp adsorbent by shaken in the orbital shaker at 25 °c, at the agitation speed of 100 rpm. similarly, the solution of each beaker was filtered and digested for aas. 2.2.3.5. optimal temperature for lead removal the study was conducted out to define optimal temperature by varying the temperature at 25°c, 40°c and 50°c with the fixed dosage of bp adsorbent (45 g l -1 ) that included lead solution of 100 ppm. beakers without bps were set as the control batch. all the solutions of the beakers at ph 5, were agitated in the orbital shaker at 100 rpm for 30 minutes. the solution from each beaker was filtered to isolate the banana peel residues and set for digestion. 2.2.3.6. optimal agitation speed for adsorption the analysis was carried out fixed bp adsorbent dosage (45 g l -1 ) in 100 ppm working solution of lead at ph 5 to set optimal agitation speed by changing speeds at the level of 50 rpm, 100 rpm and 150 rpm for 30 minutes. the mixtures were agitated by orbital shaker at 25 °c. after 30 minutes, supernatants from beakers were filtered, figure 3. effect of various factors on pb 2+ removal (a) effects of temperature of the solutions on adsorption of pb 2+ (b) effects of agitation speed on removal of pb 2+ . j. multidiscip. appl. nat. sci. 121 digested and prepared for aas. 2.2.4. study of adsorption isotherm 2.2.4.1. the langmuir isotherm model the langmuir isotherm represents quantitatively the formation of a monolayer adsorbate on the outer surface of the adsorbent and after that, no further adsorption takes place. the langmuir represents the equilibrium distribution of metal ions between the solid and liquid phases [25]. the linear form of the langmuir isotherm equation is expressed as follows [26]: (3) where, qe is the amount of metal adsorbed (mg g -1 ), b is the langmuir isotherm constant (l/mg), qm is the maximum adsorption capacity (mg g -1 ) and ce is the concentration at equilibrium (mg l -1 ). the values of qm and b were computed from the slope and intercept of the langmuir plot of 1/qe versus 1/ ce. 2.2.4.2. the freundlich isotherm model the freundlich model deals with the heterogeneous adsorption. the freundlich equation of adsorption isotherm can be written as follows [18]: log qe= log kf log ce (4) here, qe is the amount of metal adsorbed (mg/g), kf is the measure of sorption capacity (mg/g), is the sorption intensity and ce is the concentration at equilibrium (mg l -1 ). a plot of log qe versus log ce gives the value of n and kf. when log ce is plotted on the x-axis versus log qe on the y-axis, log kf and becomes the intercept and the slope of the line respectively [27]. the constant kf is an estimated indicator of adsorption capacity, while is a function of the intensity of adsorption in the adsorption process [28]. if = 1, the separation within the two phases is not dependent on the concentration. if the value of is below 1, it means a normal adsorption. on the contrary, being above 1 shows cooperative adsorption [29]. 2.2.4.3. analysis of separation factor for langmuir isotherm the separation factor is a dimensionless constant which is an important aspect of the langmuir isotherm. in a batch adsorption system, the shape of the isotherm can be applied to analyze if the adsorption system is favorable or unfavorable [30]. it can be calculated as follows [31]: separation factor, (5) where, b is the langmuir isotherm constant (l/ mg) and ci is the initial metal ion concentration (mg l -1 ). if sf is greater than 1 then the adsorption isotherm will be unfavorable. similarly, if sf is equal to 1 then it will be linear isotherm, sf is equal to 0 then it will be irreversible isotherm and sf is greater than zero but smaller than one at that time it will be favorable isotherm [32]. 2.2.5. statistical analysis all statistical analysis was done employing microsoft excel 2019. the slope, intercept, correlation coefficient (r 2 ) and the equation of the linear form of langmuir isotherm and freundlich isotherm model were devised by using the statistical function of microsoft excel 2019. 3. results and discussions 3.1. characterization of banana peel (bp) banana peel is a good source of lignin (6%12%), pectin (10%-21%), cellulose (7.6%-9.6%) table 1. langmuir and freundlich isotherm parameters. isotherm parameters langmuir qm (mg g -1 ) b (lg -1 ) r 2 2.1 2.35 0.9978 freundlich kf (mg g -1 ) r 2 1.93 0.0258 0.9595 j. multidiscip. appl. nat. sci. 122 and hemicellulose (6.4%-9.4%) [33]. an ftir spectrum of minced bp was perceived to recognize its functional groups using data from yadav [34]. these functional groups are responsible for metal ion coordination [35]. figure 1 clarifies the ftir spectrum of dried bp with a particle size of 1mm. the band in the region of 3415.93 cm -1 depicted oh stretching. the bands in the region of at 2920.23, 2872.01 and 2850.79 cm -1 indicated c-h stretching of alkane because of the presence of polysaccharides (cellulose, hemicellulose) as well as phenylpropanoids (lignin). the band appearing at 1734 and 1712.89 cm -1 associated to the c=o bond of carboxylic acids. increasing the number of carboxylate ligands can intensify metal binding. band 1624.06 cm -1 is indicated c=c stretching and 1448.54, 1417.68, 1382.96 cm -1 designated c=h bending in the alkane. 1328.95 cm -1 recognized cn stretching and the bands at 1244.09, 1199.72, 1155.36, 1101.35, 1060.85 and 1039.63 cm -1 attributed c=o bending. the weak band in the region of 889.18 and 775.38 cm -1 meant n-h deformation of amine groups of the protein. among of these several functional groups, carboxylic acid and hydroxyl groups of polyphenols and polysaccharides play a key role in the metallic cations removal [36]. biological components (protein, carbohydrate, and lipid) are associated with the interaction of metal salts via these functional groups [37]. those functional groups exchange hydrogen ions for metal ions in solution or contribute an electron pair to make complexes by the metal ions in solutions [38]. banana peel contains a high amount of pectin [39]. the carboxylic acid groups of pectin are responsible for binding pb 2+ [40]. 3.2. effect of various factors on pb 2+ removal 3.2.1. effect of adsorbent concentration the effect of various dosages of bp (25, 45, 65 and 85 g l -1 ) was analyzed on the pb 2+ solution (100 ppm) keeping all other parameters even. the results revealed that the performance of adsorption is notably conditional on the added amount of adsorbent. maximum removal for lead (100 ppm) was 88.06% for the dose of 45 g bp (fig 2a). more active sites of the surface area of adsorbent become available for metal uptake with raising the concentration of adsorbent [18]. at the higher adsorbent concentration, the adsorption efficiency decreases. it could be for the result of the of bioadsorbent aggregation when the effective surface area decreases for metal uptake [41]. so, the optimum dosage of bp adsorbent was selected 45 g for lead removal. 3.2.2. effects of ph the most significant parameter influencing the adsorption capacity is the ph of the adsorption medium [23]. the effects of ph (3, 5, 6 and 8) were studied on the pb 2+ solution holding all other parameters constant. the experiment showed that the maximum adsorption of lead was 87.5% at ph 5 and exceeding optimum ph adsorption decreases (fig 2b). h + competes with metal cations for the available adsorption site at lower ph although adsorption site does not activate at higher ph [18]. the ph could not be considered higher than 5. eventually, metal cations react with hydroxide ions and precipitate as the metal hydroxide [18][42]. pb 2+ + oh —> pboh + (6) pboh + + oh —> pb(oh)2 (7) figure 4. (a) langmuir adsorption isotherm for pb 2+ (b) freundlich adsorption isotherm for pb 2+ (metal concentration, 50-200 ppm; contact time, 30 minutes; agitation speed, 150 rpm; temperature, 25°c; ph, 5). j. multidiscip. appl. nat. sci. 123 the surface is positively charged in a highly acidic solution and the adsorption of pb 2+ and pb (oh) + is not favored [43]. the reason is the electrostatic attraction among positively charged adsorbent surfaces and positively charged metal ion surfaces are impossible. it appears that some nonelectrostatic forces are involved in adsorption [44] [45]. on the contrary, more negatively charged surface becomes available as the ph increases from 3 and facilitate greater metal uptake [43]. 3.2.3. effect of initial concentration initial concentration is one of the influential factors on adsorption efficiency. the experiment was carried out with a variable initial pb 2+ concentration (50 ppm, 100 ppm, 150 ppm and 200 ppm) with retaining all parameters fixed (fig 2c). from the experimental results, it was observed that at first with the increase of pb 2+ concentration from 50 ppm to 100 ppm removal efficiency also increased from 87.86% to 89.53%. through from 100 ppm the pb 2+ removal rate decreased with the increase of initial pb 2+ concentration. when pb 2+ concentration is 100 ppm, the ratio of the available adsorption site of bp to the initial number of lead molecules is large. although at higher concentration (> 100 ppm), the free site of the adsorption become fewer. therefore, the percentage removal of lead decreases from 100 ppm with the increase of the initial concentration of lead. 3.2.4. effect of contact time the effect of contact time on adsorption of pb 2+ solution onto bp was evaluated over a range of times (10, 20, 30 and 40 minutes). the increased adsorption of pb 2+ was observed with the increasing of contact time. highest adsorption performance of bp has reached after 30 minutes nearly 89.08% of pb 2+ removal (fig 2d). significant high removal efficiency is noted for pb 2+ during the first 30 minutes because of the availability of the uncovered surface area of the adsorbent. then eventually a slower metal uptake percentage is marked for approximately an additional 10 minutes. it has happened because the unoccupied site of the adsorption becomes concise due to repulsive forces between the solute molecules on the solid surface of the adsorbent and the bulk phase [46]. 3.2.5. effects of temperature the consequences of the removal of pb 2+ from solution carried out at different ranges of temperatures (25°, 40° and 60°c) retaining additional parameters constant. the percentage of pb 2+ removal and uptake capacity increased from 73.84 to 87.94% as the adsorption temperature was decreased from 60 to 25°c (fig 3a). the decrease in the adsorption capacity of bp with increasing temperature indicates an exothermic adsorption process [47]. many researchers proclaimed that biosorption processes are exothermic which indicates that adsorption capacity is inversely proportional to the temperature [48]. due to temperature increase, the damage of active adsorption sites of bio-adsorbent t escaped from the surface to the solution. 3.2.6. effect of agitation speed the impact of multiple ranges of agitation speed (50 rpm, 100 rpm and 150 rpm) was examined. the results showed that the adsorption increases with the increase of the agitation speed. maximum removal for pb 2+ was 88.84% at 150 rpm (fig 3b). low speed accumulates the adsorbent at the bottom rather than spreading in the solution that results in the burial of various active sites under the above layers of adsorbent. the shaking rate should be sufficient to ensure the binding sites unoccupied for metal uptake [18]. 3.3. adsorption isotherm adsorption isotherms determine equilibrium correlations within a sorbent and a sorbate. it is the proportion between the amount adsorbed and that enduring in the solution at a specific temperature at equilibrium. it is essential to establish the most appropriate correlation for the equilibrium curve to figure 5. a plot of sf against initial concentration of pb 2+ for banana peel powder. j. multidiscip. appl. nat. sci. 124 optimize the plot of sorption for eliminating pb 2+ from the water. langmuir and freundlich isotherm for pb 2+ are presented in figure 4. the associated parameters of these two isotherms are shown in table 1. toward complete monolayer coverage in langmuir isotherm, maximum adsorption capacity (qm) is 2.1 mg/g for pb 2+ . r 2 (correlation coefficient) values suggest that langmuir isotherm fits well to explain the adsorption of pb 2+ on bp. on the other hand, in freundlich isotherm n is the measure of adsorption intensity of metal cations on banana peels [18]. here, is equal to 0.0258 which suggests a normal adsorption as the value is below one. as estimated from freundlich isotherm, the value of kf (adsorption intensity) is 1.93. the r 2 value shown in table 1 is the evidence that the pb 2+ adsorption in this study is well fitted to the langmuir model compare to the freundlich model. the separation factor (sf) for langmuir isotherm calculated for each initial concentration (ci) is depicted in figure 5. the data explicates that the sorption of pb 2+ on banana peel decrease as the initial metal ion concentration increase from 50 to 200 ppm. it indicates that biosorption is more convenient for the lower initial pb 2+ concentrations comparing to higher concentrations. the data also reveals that the value of sf is greater than 0 but smaller than 1. it means the biosorption process is favorable for pb 2+ removal at all concentrations studied. the graph also admits that the banana peel powder is an effective and excellent adsorbent for pb 2+ at the lower concentration. it is also shown that the result of the sf values of pb 2+ , cu 2+ , zn 2+ and ni 2+ for banana peel biomass are favorable [28]. 3.4. comparative study for pb 2+ removal from water, a number of experiments has been conducted by many researchers. table 2 indicates some comparative analysis of highest removal efficiency of pb 2+ by banana peels based on different variables. present study focuses on different experimental condition to find maximum removal efficient. from literature it has been proven that banana peel is an efficient adsorbent for pb 2+ removal [18][23][31]. different initial concentrations, adsorbent doses, contact time, agitation speed and temperature have great influence on the removal efficiency. anwar et al., found 85% removal efficiency for 100 rpm agitation speed [18], where present study found 88.84% removal efficiency for 150 rpm agitation speed. more study is necessary to find the optimum condition to achieve highest removal efficiency. 4. conclusions waste management is one of the most significant environmental challenges for bangladesh. the industrial sector is battling serious difficulties for wastewater disposal and its treatment. in this regard, banana peel can be a very efficient adsorbent for the removal of pb 2+ from wastewater as it has carboxylic acid and hydroxyl groups of polyphenols, and polysaccharides. the current study suggests that the large volume of banana peel waste can be utilized to treat the pb 2+ contaminated water after observing its high-grade performance examining six parameters. langmuir monolayer coverage reveals that 1 g of bp can adsorb 2.1 mg of pb 2+ , and the sorption process is favorable for pb 2+ removal. maximum removal for lead (100 ppm) was found 88.06% for the dose of 45 g bp. development in the field of biosorption should be reflected for practical application and recommend the treatment process as waste minimization and wastewater treatment strategy in adsorbent adsorbent dose (g) initial concentration ion of lead (mg/l) ph temperature (°c) contact time (min) agitation speed (rpm) removal efficiency (%) ref banana peel 5 50 5 30 720 100 96.37 [31] 40 50 5 30 20 100 85.30 [18] 45 100 5 25 30 100 86.00 present 45 100 5 25 30 150 88.84 present table 2. comparison between studies that considered banana peel for lead adsorption with the present study. j. multidiscip. appl. nat. sci. 125 bangladesh. author information corresponding author protima sarker — department of environmental science and disaster management, noakhali science and technology university, noakhali-3814 (bangladesh); school of environmental science, university of shiga prefecture, shiga-5228533 (japan); email: protima_das37@yahoo.com authors afrida nurain — department of environmental sciences, jahangirnagar university, dhaka-1342 (bangladesh); md. shiblur rahaman — department of environmental science and disaster management, noakhali science and technology university, noakhali-3814 (bangladesh); department of environmental and preventive medicine, jichi medical university school of medicine, tochigi-3290498 (japan); orcid.org/0000-0002-9920-4809 md. mostafizur rahman — department of environmental sciences, jahangirnagar university, dhaka-1342 (bangladesh); md. khabir uddin — department of environmental sciences, jahangirnagar university, dhaka-1342 (bangladesh); conflict of interest the authors declare that there is no conflict of interest. references [1] l. joseph, b. m. jun, j. r. v. flora, c. m. park, and y. yoon. 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(2011). “advanced biosorbents materials for removal of chromium from water and wastewaters”. environmental progress & sustainable energy. 30 (3): 284– 293. 10.1002/ep.10473. https://doi.org/10.1016/j.biortech.2006.01.010 https://doi.org/10.1016/j.cej.2009.10.029 https://doi.org/10.1016/j.apsusc.2006.10.042 https://doi.org/10.1016/j.apsusc.2006.10.042 https://doi.org/10.1016/j.jcis.2004.06.042 https://doi.org/10.1016/j.jcis.2007.07.061 https://doi.org/10.1016/j.jcis.2007.07.061 https://doi.org/10.1016/j.cej.2008.06.024 https://doi.org/10.1016/j.cej.2008.06.024 https://doi.org/10.1016/j.colsurfb.2008.12.032 https://doi.org/10.1002/ep.10473 25 journal of multidisciplinary applied natural science vol. 1 no. 1 (2021) review article received : october 13, 2020 revised : november 3, 2020 accepted : november 24, 2020 online : november 26, 2020 chrysanthemum is a floricultural, ornamental and medicinal plant with tremendous income cash crop which is cultivated chiefly in winter. the crop charity as nerve sedative, anti-oxidant, anti-inflammatory, anti-mutagenic, anti-microbial, anti-fungal, antiangiogenic, anti-atherosclerosis and nematocidal goods. chrysanthemum has been used for hundreds of years in manufacturing medicine. it is used to treat respiratory complications, high blood pressure, and hyperthyroidism and reduce inflammation. leaves remedies for colds, headaches, bronchitis, rheumatism, and swellings. the plant has aesthetic significance as well. nevertheless, outbreaks of pests and diseases have hold back chrysanthemum cultivation in bangladesh. insect pest damages the plant directly by sucking cell sap and indirectly spread out of virus diseases as act of vector. aphid resistance variety might be the effective tool for successful production of chrysanthemum plant. sustainable crop disease management for chrysanthemum needs a multitude of consideration of the impacts of management methods on economics, sociology and ecology by wholly understanding the mechanisms of crop disease epidemics and the functioning of normal agroecosystems. the findings of this study present clear insights into efforts that will encourage farmers to adopt effective and ecological methods of pest and disease management for successful chrysanthemum production in bangladesh. chrysanthemum indicum, sometimes called mums or chrysanths [1] is one of the most important marketable ornamental and flower plants [2] belonging to the family compositae/ asteraceae, sub-family asteroideae, order asterales, sub-class asteridae, tribe anthemideae. it is native to east asia [3] and has been cultivated in garden for more than 2500 years [4]. owing to its high medicinal, ornamental and floricultural value, it contributes a very large share of horticultural gdp and world flower trade [5]–[8]. besides, medicinal and economic importance, it has aesthetic importance as well, which add versatile beauty of a place. they often remain in good condition for two to three weeks depending on cultivars and varieties [9]. chrysanthemums are sold as annuals at garden centers every fall. mums are actually perennials and will bloom for years if planted correctly. the plant is a perennial aromatic flavor, with erect hairy stem, complete shiny leaves, highly cleft from the base, 70-120 cm height and heavily branched [10][11]. the leaves are oblong, typically 2 pinnatisect, yellow florets and it has big capitula, usually bicolored white and yellow which are used to treat inflammation. the flowers are corymb with yellow tubular structure [10][12]. the c. indicum flower is a good source of usual quercitrin and myricetin, which is important for the development of possible pharmaceuticals [13]. the variation showed by the large number of cultivars in deference of growth habit, size, color and shape of blooms make them suitable for every purpose conceivable of a flower. it showed inhibitory action against rat lens aldose reductase and against nitric oxide making in lipopolysaccharide started macrophages [14][15]. good quality flower production relies on numerous factors such as genotype, insect resistance, environment, soil nutrient, spacing, disbudding, pinching, substrate, use of growth regulator etc. [16][17]. for profitable cultivation, quality flower production is important [18]. the erect and tall rising cultivars are suitable for background planting in edges or for use as cut flowers. however, the cultivars with the dwarf and compact growing habit are appropriate for front row farmstead or pot culture. the pod culture is important in the flower production of chrycopyright holder: © khan, a. u., choudhury, m. a. r., khan, a. u., khanal, s., and maukeeb, a. r. m. (2021) first publication right: journal of multidisciplinary applied natural science this article is licensed under: https://doi.org/10.47352/jmans.v1i1.10 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.v1i1.10 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.v1i1.10&domain=pdf&date_stamp=2021-01-10 j. multidiscip. appl. nat. sci. 26 santhemum [19]. dwarf varieties easily fit the size of pot and looks adjusted from outside. the decorative and fluffy bloomed cultivars are ideal for garland making and hair decoration. the extra-large bloomed cultivars are prized for their display value. though the flower yield and quality are primarily varietals characters, they are also greatly influenced by climatic factors. the successful cultivation of this plant is principally due to the great diversity of cultivars [20]. different transform plants are grown in bangladesh. a numerous number of chrysanthemum cultivars are found in bangladesh. jamal uddin, et al. [21] observed that the 32 chrysanthemum cultivars coded from v1 to v32 were used for a study in bangladesh. the cut blooms are also used in cemeteries in japan [22]. most of them are flowered in winter season. genetic variation for flower yield and its component attributes were not properly studied in the past. the leaves of this herb are edible and flowers have a pleasant smell. a condition for good quality cut flowers is that numerous flowers should be borne on long stems with healthy insect and disease -free foliage. it has ornamental value and many pharmaceutical firms dependent on several plants for medicine in the western world. the pharmacopoeias have developed from ancient herbs. the leaf decoction is popularly used for the treatment of colds, headache, bronchitis, rheumatism, swellings and boils [23] as the leaf contents are enriched with anti-bacterial properties. chrysanthemum is highly aromatic due to several volatile compounds of their essential oils, many of which are used in the flavor and fragrance purposes. the flower heads of c. indicum are sometimes used as chamomile adulterants. according to jaime [24] the leaves are used for suppression of suspicious odors in foods such as soup, yuzu or pepper in japan. it is available in tropical and subtropical zones [25] and widely distributed in the mediterranean region, japan, china and the philippines [26]. the diploid and tetraploid taxa of the chrysanthemum are distributed mainly in mediterranean region and europe [27]. the spatiosum variety used as chinese vegetable and others part of the plant are also used for treating different diseases. the bark is purgative and also to treat syphilis. it is rummage-sale against constipation, stomach parasitic contagions and real in the fight against nematodes and protects plants against caterpillars. the extract of c. indicum showed strong and selective allelopathic activity [27]. the flowers are stomachic, insecticidal and well known for its use against skin allergies like hitch [28]. in spite of being a prospective crop, high occurrence of insect pests, and diseases are one of the main factors for the reduction of the yield and quality flower of c. indicum plant. considering all these facts this study has been undertaken to certify the barriers and properties c. indicum crop plant. keeping the threats of insect pests and diseases infestation of chrysanthemums in a view point, this paper makes an attempt to assess the management techniques of the pests through different means that are feasible to adopt by the growers. the general objective is to assess the severe insect pests and diseases of the plants, extent of damages, and the management approaches. also, the medicinal and esthetic significance of the plant has been highlighted. table 1 listed the popularly cultivated cultivars of chrysanthemum in bangladesh. due to the favorable environmental conditions and an increased demand for flowers, a large number of different cultivars have high potential to be cultivated in difcultivars of chrysanthemum (1) kanchil (7) sabdhawna (13) shukla (19) beauty (2) glumohr (8) flirt (14) puspa anmol (20) jubilee (3) shayamal (9) neelima (15) yellow bangla (21) maghi orange (4) sadwin yellow (10) white prolific (16) sharad mala (22) maghi white (5) white andaman (11) ravikiran (17) star white (23) kalvin orange (6) aparjita (12) birbal sahni (18) waters may (24) maghi yellow source: kumar et al. [29] table 1. different cultivars of chrysanthemum in bangladesh j. multidiscip. appl. nat. sci. 27 ferent regions. some of the cultivars are shown in table 1. 3. insect pests of chrysanthemum crop the c. indicum plant is popular for possessing medicinal values however; its production is low due to the attack of a number of harmful sucking pests. most cultivars are sensitive to aphids and infiltrations can lower quality and cause transmission of viruses’ fiend out [30]. visser, et al. [31] stated that a critical problem in chrysanthemum cultivation is outbreak of various insects such as aphids and others insect thrips (frankliniella occidentalis pergande) and spider mites (tetranychus urticae koch) on vegetative flowering and others parts of chrysanthemum. according to teixeira da silva, et al. [32] chrysanthemum is vulnerable to several insects and sucking pests which affect both the production and quality of the flowers at different growth stages. during the seedling stage, spodoptera litura inhibits the growth of young plants. with growth of plants, infestation of sucking pests, mainly aphids, will be more. chrysanthemum is affected by two aphids mainly chrysanthemum aphid and cotton aphid. grasshoppers, thrips and spider mites are minor pests feeding on the plant specially flowers and leaves. the usually affect flower bud opening. the infestation continues even at full blooming stage. aphids effect on these striking plants by sucking from the phloem of the vascular bundles of early shoots and leaves [33]. plant parts affected by aphids will wilt, discolor or deform [34]. aphids are regularly found in groups on plant tips, shoots of new growth or flowers. they multiply so fast on favorable conditions that a light infestation may increase to alarming proportions in a week. some of the major aphids which severely infest c. indicum are briefly described below: 3.1. cotton aphid (aphis gossypii glover, 1877) the aphid is highly variable in color even within the same colony, ranging from light yellow to dark green, with dark siphunculi and a pale or dusky. it is small to medium-sized, body length in apterous 1.05-1.90 mm, in alate 1.05-1.77 mm; this species is polyphagous and very threatening to many economically important plants [35] recorded this species infested chrysanthemum sp. in baghdad, the infection was linked with m. sanborni. a. gossypii as the most dominant species of chrysanthemum plant [34]. 3.2. black bean aphid (aphis fabae scopoli, 1763) the aphid is dull greenish black to black. it is small to medium-sized, body length in apterous 1.60-2.25 mm, in alate 1.80-2.6 mm; originated on young leaves, stems and inflorescences of many plants, mainly leguminosae, chenopodiaceae and compositae [36]. 3.3. chrysanthemum aphid (coloradoa rufomaculata wilson, 1908) the green aphid is small, body length in apterous 1.0-1.5 mm, in alate 0.9-1.5 mm; sometimes with yellow spots at siphunculi base; found on the stem and underside of leaves of chrysanthemum sp. this aphid species was presented as a new record for iraq aphid fauna [34]. 3.4. chrysanthemum aphid (macrosiphoneilla sanborni gillette, 1908) the shiny, dark red-brown to blackish brown, broadly spindle shaped, with black, relatively short and thick is siphunculi. it is small to medium sized, body length in apterous 1.40-2.20 mm, in alate 1.40 -2.30 mm. the members of this species are found on young stems and bases of leaves of chrysanthemum sp. [37] are the authors who noted this aphid species in iraq on chrysanthemum sp. 3.5. peach aphid (myzus persicae sulzer, 1776) the peach is whitish green, pale yellowish green, greyish green, mid-green or pink, rather uniformly colored, not shiny. it is small to medium-sized, body length in apterous 1.65-2.35 mm, alate 1.902.45 mm, alate have a black central patch on the dorsal surface of the abdominal. according to ali, et al. [34] the m. persicae is the lesser species in chrysanthemum sp. 3.6. insect pests management in preliminary condition, it was observed that collection and destruction of the infested leaves, twigs, inflorescences and pod of the chrysanthemum crop that is mechanical control had good effects to manage the insect pests and the ladybird j. multidiscip. appl. nat. sci. 28 beetle, spider and damselfly as found good predators of aphid. neem leaf extract @ 1 kg neem leaves: 10 l water and dimethoate 40 ec @ 200 ml in 10 l water is suggested for one-acre land for controlling the aphid infestation. botanical insecticides neem leaf extract reduced the aphid population significantly on twigs, inflorescences and pods compared to untreated plants. in severe condition, application of insecticides was sprayed two times at seven days interval. dimethoate 40ec, carbosulfan 20 ec @ 2ml / l, malathion-57 ec @ 0.2% and imidacloprid 200 sl @ 0.25ml/l were used for controlling aphid successfully [38]. 4. diseases of chrysanthemum crop the chrysanthemum flower crop is sensitive to some diseases that decline the medicinal quality. the viruses belonging to the family bunyaviridae, genus tospo virus including tomato spotted wilt virus (tswv) and impatiens necrotic spot virus (insv) are very serious pathogens not only on chrysanthemum plants but also on other horticultural plants [39][40]. these viruses cause severe systemic necrosis on plants and a lethal effect in the worst cases. moreover, these viruses are easily transmitted by a small insect to a wide range of plant species belonging to over 50 plant families. this causes difficulty in preventing commercial products from the infection by these pathogens. according to ikten, et al. [41] the c. indicum phytoplasma cell wall less bacteria is related with diseases in several hundred plant species. viroid is the smallest infectious agent that causes serious diseases in vegetative propagated plant including chrysanthemum [42]. c. indicum stunt viroid (csvd) is a serious pathogen in the chrysanthemum production industry, because it is easily transmitted mechanically during the course of propagation. the only effective approach to protect plants against its infection is to strictly monitor this pathogen. the csvd infection causes symptoms such as decrease of plant height, which lowers the commercial value of cut flowers. there are three types of viruses of flower plant based on environment, ecology and phytosanitary consideration [43] and the first class includes viruses that have wide host ranges, usually have efficient vectors and already widespread in world wide. 4.1. virus virus diseases in chrysanthemum were not recognized prior to 1945. chrysanthemum stunt was first reported to be a virus problem in 1947 [44]. by 1949, it became a widespread and serious threat to the chrysanthemum field. the chrysanthemum stunt as a disease caused by a viroid (chsv) [45]. by 1950, other virus pathogens were discovered that caused problems in chrysanthemum production. nowadays, virus has been seen to be a serious problem in chrysanthemum cultivation in agroecosystem in bangladesh. roughing out of infested plants, destroy crop residues from the field and removable of the host plant can manage virus existence. resistant variety should be used for healthy cultivation of chrysanthemum. controlled of aphid by using malathion57 ec @ 0.2% can be achieved to control insect vectors. foliar spray of imidacloprid 200 @ 0.25 ml/l of 7-11days interval can be practiced. 4.2. chrysanthemum rust the white rust (puccinia horiana) was first recorded in japan in 1895 and then noted in china and japan until the 1960s. in present time, fungal disease of rust is a serious disease in flower crop. today it is established in asia, europe, africa, australia, central america, south america and the far east. it can spread quickly in natural and nursery environments, causing huge crop losses and reduction the flower quality. toguri, et al. [46] stated from recent study that improved an agrobacterium mediated transformation system in chrysanthemum and obtained more than 80 transgenic lines with pacl and also investigated tolerance of transgenic chrysanthemum plants expressing pacl protein against csvd and tswv. removal of susceptible volunteer chrysanthemum plants, barberry plants and avoid excess n fertilizer in the field is necessary. early maturity cultivars help to minimize the rust disease in chrysanthemum plants. spraying propiconazole 25% ec @ 0.02% with 100l water at 15 days interval of 2-3 times is effective to manage the rust in this plant. 5. medical properties of chrysanthemum the chrysanthemum herb crop is a very immailto:200@0.25ml/litre mailto:200@0.25ml/litre mailto:200@0.25ml/litre mailto:200@0.25ml/litre j. multidiscip. appl. nat. sci. 29 portant medicinal plant and the whole plant parts are used for disease treatment. many researchers investigated that the stems, leaves, and flowers of the plants are used as traditional medicine to treat vertigo, hypertensive symptoms, and several infectious diseases like pneumonia, colitis, stomatitis, carbuncle and fever [47]. yet, systematic manipulation of these natural medicines for human health aids has not been carried out to a significant degree. in recent years, these basils have been the focus of care due to their medicinal and pharmacological values [48]. plant products are widely used in pharmaceuticals, cosmetics and food trades. many pharmaceutical companies in the western world are contingent on many plants for their medicinal things. pharmacopoeias have industrialized from ancient sages [49]. the extract of c. indicum the flowers were a flavone glucoside, acacetin 7-0-(3-0acetyl-ß-d-glucopyr-anoside) [50], has been isolated together with twenty-seven known compounds including flavonoids, caffeoylquinic acid derivatives, phenolics, and a monoterpenoid glucoside. the medicinal plant native to china and japan is chrysanthemum indicum l. its infusion is a remedy for intermittent fever, hysteria, and monthly irregularities. it is also used as carminative, tonic sedative, and for hypertension. leaf decoction is a remedy for colds, headache, bronchitis, rheumatism, swellings and boils. the flower part is used as herbal medicine with strong aroma and many previous studies focused on its essential oil [13][51]–[53] which is prescribed for anti-inflammatory, analgesic, antipyretic purposes, quercetin, myricetin and the treatment of eye disease in chinese traditional preparations. it demonstrated inhibitory action against rat lens aldose reductase and nitric oxide production in lipopolysaccharide activated macrophages. it has been used as the antioxidant, antiinflammatory, anti-mutagenic, antimicrobial, antifungal, antiangiogenic, anti-atherosclerosis and nematocidal properties and the leaves are used as expectorant, bitter, and stomachic for the animal [54]. hosni, et al. [55] observed that the volatile oil of wild chrysanthemums covers 22 components and the optimal extraction process is ethanol for 3 times, which can be achieved by adding 70% ethanol for 12 times. some experts in the field of medicine have examined the pharmacological effect and chemical arrangement extraction methods of wild chrysanthemums medicinal herbs. through the diagnosis of the claim in the clinical disease treatment, it can start that wild chrysanthemums have obvious pharmacological effect in clinical diseases and has certain application value. xanthine oxidase (xo) is a key enzyme that catalyzes the oxidation of xanthine and hypoxanthine into uric acid [56], and plays a vital role in producing hyperuricemia and gout. allopurinol is a clinically used xo inhibitor in the treatment of gout, which lumps the terminal step in uric acid biosynthesis and lowers the plasma uric acid concentration [57]. however, due to unwanted side effects of allopurinol, such as hepatitis, nephropathy, and allergic reactions, the search for new substitutes with increased therapeuno. health benefits of chrysanthemum 1 encourage effective metabolism in the body, which can help people to lose weight, bettered circulation, 2 lower body temperature, and can help relieve the pain 3 detoxify the liver, making body healthier overall, as well as make cholesterol numbers lower 4 boost immune system because of its high level of vitamin a and c 5 check chronic illness, prevents cellular mutations, and protects body against several illness caused by free 6 help alleviate varicose veins 7 rejuvenate the brain and alert the senses 8 help ease digestive issues with fewer stomach problems 9 improve bone density and prevent osteoporosis (due to presence of calcium and magnesium in plants) 10 improve eyesight by alleviating dryness and itchiness in eyes 11 treats cough and cold table 2. diversified uses of chrysanthemum crop j. multidiscip. appl. nat. sci. 30 tic activity and fewer side effects are necessary. in addition, superoxide anion radicals caused by xo are involved in various pathological states such as hepatitis, inflammation, ischemia reperfusion, carcinogenesis, and aging [58]. thus, the search for novel xo inhibitors would be helpful not only to treat gout but also to combat numerous other diseases. the c. indicum is one of the popular traditional medicines extensively used in vietnam and indigenous to china. the flowers are used under the name "cuc hoa trang" for the treatment of fever, rheumatism, inflammation, headache, and eyesight disorder [59]. nguyen, et al. [60] had performed a study on these plants. in the course of their screening program for xo inhibitory medicinal plants from vietnam, they screened 288 extracts, prepared from 96 medicinal plants used in vietnamese traditional medicine to treat gout and related symptoms. among them, the meoh extract of the flowers of c. indicum showed the most potent xo inhibitory activity with an ic50 value of 5.1 ug/ ml [60]. therefore, they carried out activity shown fractionation of the meoh extract and isolated a newfangled flavone glucoside, together with 14 known flavonoids, seven caffeoylquinic acid derivatives, five simple phenolics, and a monoterpenoid glucoside. the c. indicum flowers have been prescribed for anti-inflammation, analgesic, and antipyretic purposes and the treatment of eye disease in chinese traditional preparations. in the course of our characterization studies on the bioactive constituents from medicinal foodstuffs [60][61] and medicinal flowers [62] reported the isolation and structural elucidation of three eudesmane-type sesquiterpenes, kikkanols a, b, and c, and five germacrene-type sesquiterpenes, kikkanols d, d monoacetate, e, f, and f monoacetate, from the methanolic extract of the flowers of c. indicum cultivated in china [62]. in addition, the methanolic extract and several constituents were found to show inhibitory activities against rat lens aldose reductase10) and nitric oxide (no) production in lipopolysaccharide (lps)activated mouse peritoneal macrophages [63]. 6. problems faced in chrysanthemum production changes are occurring throughout farming sector today, and these changes could majorly affect pest management. pesticides are extensively used in plant cultivation. when compared to other types of controlling methods, pesticides can provide instantaneous pest knock-down impact with limited control, but rarely a long-lasting solution to the problems. due to huge marketing approaches of the agrochemical companies, many farmers have grown accustomed to applying pesticides, often as preventive solution against disease injuries and crop loss. chrysanthemum disease management faces everincreasing problems due to: (i) rising demands for alternative plantations to support the burgeoning global population; (ii) declining production potential in agriculture because of more competition for land in fertile areas and exhaustion of marginal arable lands; (iii) worsening ecology of agroecosystems and depletion of natural resources; and (iv) increased hazard of disease epidemics resulting from agricultural intensification and monocultures. fungicide resistance is an increasing menace to farmers. most of the farmers lack sound knowledge on use of pesticides and other methods of disease control. recommendations of the plant experts for use of appropriate dose of pesticides and fertilizers are not usually followed by the farmers. the problems lie in the lack of governmental and nongovernmental support to develop the effective agricultural policies, integrated pest management approaches, and the best way of pest and disease control. 7. conclusions as an ornamental and medicinal purpose, it is authoritative to recognize the bio active combinations which are responsible for its pharmacological effects especially in the essential oil as medicine of human body of the plant but its production hamper due to attack of a number of aphids as insect, virus as diseases and planting material as verities. the production of chrysanthemum earmark problem is its cutting collection as well as its quality planting material’s availability. the insects and diseases resistance cultivar and simultaneously integrated pest management approaches are inevitable for successful cut flower production of c. indicum. j. multidiscip. appl. nat. sci. 31 author information corresponding author ahasan ullah khan — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); climate-smart agriculture lab, sylhet agricultural university, sylhet-3100 (bangladesh); https://orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors md abdur razzak choudhury — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); anayat ullah khan — department of mathematics, jashore university of science and technology, jashore-7408 (bangladesh); saugat khanal — department of agriculture, agriculture and forestry university, bharatpur44200 (nepal); https://orcid.org/0000-0002-9146-7428 abu rashed md maukeeb — department of agricultural chemistry, khulna agricultural university, khulna-9208 (bangladesh); acknowledgement this work was supported by the parents of the authors, colleagues, and the related field expert’s professors. the authors owe their sincere gratitude to the all helping hands that assisted during manuscript preparation. references [1] j. chen, x. yang, b. li, k. yang, y. wang, k. sun, y. zhang, and w. zhu. 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(1998). “il-10 up-regulates nitric oxide (no) synthesis by lipopolysaccharide (lps)-activated macrophages: improved control of trypanosoma cruzi infection”. clinical & experimental immunology. 113 (1): 59 –64. 10.1046/j.1365-2249.1998.00637.x. https://doi.org/10.1016/j.aca.2017.07.023 https://doi.org/10.1016/j.aca.2017.07.023 https://doi.org/10.1016/j.apjtm.2017.06.002 https://doi.org/10.1016/j.apjtm.2017.06.002 https://doi.org/10.1248/bpb.27.1414 https://doi.org/10.1248/cpb.49.54 https://doi.org/10.1248/cpb.49.54 https://doi.org/10.1016/s0968-0896(01)00321-2 https://doi.org/10.1046/j.1365-2249.1998.00637.x 115 journal of multidisciplinary applied natural science vol. 2 no. 2 (2022) research article removal of toxic cations from aqueous solutions using ginger root waste jude chinedu onwuka*, stephen igberi azubuike, and timothy m. akpomie received : april 4, 2022 revised : may 26, 2022 accepted : may 28, 2022 online : may 29, 2022 abstract recently, the harmful impact of toxic metals in the aquatic environment cannot be over emphasized again. this work investigated the potential application of ginger root waste (grw) to remove toxic cations (cd 2+ and pb 2+ ) from the aqueous medium. batch adsorption examination was carried out as a function of sorbent dose, initial metal ion concentration, contact time, and temperature. the sorption equilibrium of the metal ions onto the grw was subjected to langmuir, freundlich, elovich and redlich-peterson isotherm models over concentration ranges of 10-50 mg/l. sorption information was used for kinetic and thermodynamic modeling. the grw materials before and after sorption was characterized using ftir and sem. results showed higher removal percentage of cd 2+ over pb 2+ ions in all the factors studied. the redlich – peterson isotherm model affirmed that sorption of cd 2+ and pb 2+ occurred in a heterogenous surface of the sorbent which is strongly influenced by multiple micropores and caves. kinetic studies revealed that the sorption was controlled through intra-particle diffusion model aided by surface and chemical reactions. meanwhile, thermodynamic parameters indicated that the cd 2+ and pb 2+ sorption process was endothermic, however, nonspontaneous at temperature of 303 and 313 k. the ftir and sem data showed the evidence of successful sorption of the toxic cations on to the sorbent material. keywords ginger root waste, sorption, toxic cations, kinetics, thermodynamics 1. introduction heavy metal contamination is a serious challenge to the human race and the natural ecosystem. the environmental hazard and their effects on human existence are very serious [1] due to their accumulation in the human body and inability to biodegrade into harmless waste [2]. today, the dangerous global impact of these metals on the ecosystem has become a non-push-over reality that is very difficult to be circumvented. environmental pollution by heavy metals occurs by means of industrialization and extraction of natural resources [3]. most industrial discharge and effluent contain high amounts of heavy metals which consequently constitute serious concern due to the high toxicity it portends to the living organism [4]. organic wastes are quite susceptible to biodegradation, but heavy metals are not simply degraded by any biological means into harmless by products [2]. the global increase in industrialization has accelerated industrial applications of heavy metals mainly as the catalyst and thus, increased their presence in wastewater giving rise to environmental pollution and wastewater contamination. among the heavy metals, cadmium and lead, which are quite carcinogenic, are often found in industrial effluents such as those related to mining, metallurgy, petrochemicals, electroplating, batteries, pharmaceutical industries, paper, pulp, plastic, and paint manufacturing [5]. these metals are presumed toxic since they are not metabolized by the human body rather, they keep accumulating in the human tissues [6]-[9]. thus, it becomes incumbent to search for the possible way of removing these toxic metals from the aqueous medium. as a result of the many health challenges associated with contamination of water bodies by heavy metals, a lot of techniques such as chemical precipitation, ion exchange, electro-dialysis, membrane separation, and nanofiltration have been implored in preventing the presence of metal cations in our drinking water samples. however, there are obvious limitations inherent with the application of these techniques [10] including less efficiency and sensitivity, generation of sludge, high maintenance cost, high energy and reagent requirement, and inability to sequestrate metal ions at low concentration [11]. these limitations copyright holder: © onwuka, j. c., azubuike, s. i., and akpomie, t. m. (2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.126 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.126 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.126&domain=pdf&date_stamp=2022-06-14 j. multidiscip. appl. nat. sci. 116 table 1. data isolated from equilibrium adsorption isotherm plots. toxic cation langmuir isotherm freundlich isotherm elovich isotherm redlich-peterson qmax b rl r 2 kf n r 2 qm ke r 2 β a r 2 cd 2+ -0.9832 -0.1709 0.8562 0.7691 0.0463 0.3493 0.9640 * -3.2927 1.5095 0.8868 1.8622 0.0463 0.9187 pb 2+ 4.0016 0.0646 0.7946 0.0045 11.1970 -1.0092 0.0495 1.6292 5.4016 0.9817 * 1.9906 11.1931 0.1735 note: * means highest r 2 values. r 2 values that are moderate are bolded prompted individuals and research institutes to engage themselves in finding the best viable technique to adequately bring this menace into limits acceptable by standard organizations and one of such options is well provided in the adsorption process [12]. the adsorption process is cheap, ecofriendly and cost-effective as this process does not produce sludge and does not require high energy and reagents [10][11]. globally, safe drinking water is everyone's concern, as it is difficult to find anybody who has not been affected by waterborne ailment [13]. termed as a “universal solvent”, a higher percentage of the global population depends on water for drinking and domestic uses [10]. due to the indispensable daily need and use of water across the globe, it has been made vulnerable and susceptible to being contaminated by the heavy metal discharge of domestic and industrial effluents, radioactive waste, mining waste, leakages from oil pipelines and atmospheric deposition [14]. consequently, this has made water one of the key sources of global infection and diseases [15]. statistics show that 80% of diseases and infections globally are waterborne and there is the propensity that it will be doubled if nothing is done in the near future [16]. at the moment, the global concern on heavy metal accumulation in drinking water and its effects on the human body is on the increase. the apprehension is that no biological means of degradation have been identified for these set of elements [3]. health experts opined those natural products such as ginger drink are of great benefit to the human body [17]. recent discoveries showed that it prevents nausea and vomiting, muscle pain and curbs cancer growth, soothes sore throat, and prevents common cold [18][19]. nutritionally, the ginger drink is rich in antioxidants which are necessary for combating activities of free radicals in the body [12]. according to adanlawao and dairo [20], the ginger drink is quite rich in potassium, an important mineral for biochemical reactions in the body. chronic intake of synthetic beverages majority of which are soft drinks is susceptible to dental erosion, obesity with a high risk of cardiovascular dysfunction and type 2-diabetes [18]. due to these obvious health concerns on synthetic drink consumption, the focus has been shifted towards the consumption of natural drinks such as ginger root drinks [21]. unfortunately, after the aqueous extraction of the ginger root, the wastes are dumped indiscriminately, thus, constituting an environmental nuisance. previous research [12] utilized ginger root in removing selected toxic metal cations. however, this research aims to study the potential of using ginger root waste (grw) to specifically remove cd 2+ and pb 2+ from the aqueous solution. 2. materials and methods 2.1. sample collection and preparation the fresh ginger roots used for this research (201 g) were obtained from the lafia main market, in nasarawa state. it was transported in a polyethylene bag to the department of chemistry laboratory, federal university of lafia. it was washed and rinsed severally with deionized water to remove dust and adhering soil particles. the thin outer covers were carefully peeled using stainless steel knife. the sample was washed again and sliced into bits and pounded using a ceramic mortar. the pounded sample was placed in a stainless basin, and 2 l of deionized water was added. the mixture was allowed to stand for 10 min, stirred and then filtered. the residue obtained after the juice extraction was re-soaked in 2 l of deionized water for another 10 min. this process was repeated for further three consecutive times and filtered after each soaking. the chaff obtained after this series of extraction is referred to as the ginger root waste. the waste was air-dried for 24 h and then ovendried for 72 h at 80 °c. the dried sample was j. multidiscip. appl. nat. sci. 117 ground into fine powder using ceramic mortar and pestle and sieved to less than 1 mm fine particle size with a sieve and stored in a plastic sealable bag ready for the chemical analysis. 2.2. batch sorption studies the experimental solution was prepared by diluting the stock solution earlier prepared with deionized water. a 50 ml portion of the prepared 10 mg/l metal ion solution was measured into 250 ml plastic container and 0.5 g of the sorbent (grw) was added. the plastic container was closed and then placed on a shaker (bernareggio mi, pid system type m 428-bd) with a temperaturecontrolled water bath. it was shaken continuously for 60 min at 27 °c and 120 rpm. the supernatant was filtered using 0.2 mm whatman filter paper. the filtrate was analyzed using atomic absorption spectrophotometry (pg990) in order to ascertain the equilibrium metal ion concentrations. various parameters of the adsorption process were studied as a function of sorbent dose, initial metal ion concentration, contact time and temperature of the solution. the effect of sorbent dose (0.5, 1.0, 1.5, 2.0, and 2.5 g) was studied by fixing the volume (50 ml) and concentration (10 mg/l) of toxic cations as well as the contact time (60 min) and temperature (27 °c). the effect of initial metal ion concentration (10, 20, 30, 40, and 50 mg/l) was carried out by metal ions sorption at a constant sorbent dose, contact time and temperature. the contact time effect (30, 60, 90, 120, and 150 min) was studied under various temperatures (303, 313, 323, and 343 k) while keeping other factors constant. similarly, the variation of temperature (303, 313, 323, and 343 k) was carried out at various contact times (30, 60, 90, 120 and 150 min). the amount of metal ion sorbed per unit mass was calculated using a mass balance as shown eq. 1. (1) where qe is heavy metal ion concentration absorbed onto the sorbent at equilibrium (mg/g), ci and ce are initial and equilibrium concentrations (mg/l) of the metal ions, respectively, m is mass (mg) of sorbent, and v is the volume (l) of sorbate solution. the adsorption or removal percentage (%) was calculated using eq. 2. (2) 2.3. adsorption isotherm modeling this experiment was used to establish the relationship between the amount of the toxic cations (cd 2+ and pb 2+ ) that was adsorbed by the grw sorbent grw and the equilibrium concentrations of the toxic cations at a constant temperature [12]. langmuir, freundlich, elovich and redlichpeterson isotherm models were used to evaluate how the toxic cations (adsorbate) interacted with the grw sorbent. this study was conducted by fitting the obtained data from the effect of initial metal ion concentration into the isotherm models. 2.3.1. langmuir isotherm the equation was developed by irving langmuir (1916). the linearized form of the langmuir model as shown in eq. 3, was used in this study. figure 1. effect of sorbent dose on cd 2+ and pb 2+ sorption. figure 2. effect of initial metal ion concentration on cd 2+ and pb 2+ sorption. j. multidiscip. appl. nat. sci. 118 (3) where ce is concentration at equilibrium or final concentration of the metal in solution (mg/l), qe is adsorption efficiency or the amount of the adsorbate adsorbed at equilibrium (mg/g), qmax is the maximum adsorption capacity (mg/g), and b is the langmuir constant related to the energy of adsorption (l/mg). a plot of the graph ce/qe against ce, produces 1/qmax as the slope, and thus, qmax was calculated. the value of b which is the langmuir constant was evaluated from the intercept of the graph which is 1/b x qmax [22]. dimensionless equilibrium function known as rl value was evaluated using eq. 4 expressed as: (4) when rl = 1, a linear adsorption occurred. if rl = 0, there is a irreversible adsorption. but when 0< rl < 1, then a favorable adsorption occurred. 2.3.2. freundlich isotherm the linear form of freundlich isotherm demonstrated in eq. 5, was applied. (5) a plot of logqe against logce produced 1/n as the slope, from where n was evaluated, while from the intercept (logkf), kf was estimated. it should be noted that kf and n are freundlich constants related to adsorption capacity and adsorption intensity, respectively [23]. figure 3. effect of contact time for cd 2+ sorption at various temperatures. figure 4. effect of contact time for pb 2+ sorption at various temperatures. 2.3.3. elovich isotherm the linear form of elovich isotherm used is given as eq. 6. (6) the plot of against gives a straight line. thus, elovich maximum adsorption capacity (qm) and elovich constant (ke) were calculated from the slope ( )and intercept ( ), respectively. 2.3.4. redlich-peterson isotherm the linear form of the redlich-peterson isotherm used is shown in eq. 7. (7) where a is the redlich-peterson isotherm constant (l/g), β (l/mg) is the exponent, ce (mg/l) is the liquid-phase equilibrium concentration of the sorbent (grw) while qe is the sorbate (cd 2+ and pb 2+ ) equilibrium onto the grw sorbent. when the graph of ince/qe vs ince is plotted, the slope is β while the intercept is lna. when β value is below or equal to 1, the redlich-peterson isotherm is confined to langmuir isotherm, however, when β value is above 1, the redlich-peterson isotherm tends to freundlich isotherm. the constant “a” indicates the sorption capacity [24][25]. 2.4. kinetic studies this was studied to measure the uptake of the toxic cations as a function of time at a constant concentration so as to determine the mechanism or reaction pathway involved in the toxic cation uptake by the sorbent. thus, data obtained from the j. multidiscip. appl. nat. sci. 119 effect of contact time were analyzed using pseudo– first order, pseudo–second order, intra–particle diffusion and liquid film diffusion models. 2.4.1. pseudo first–order kinetic pseudo first-order kinetic was carried out to establish the contribution of physisorption or surface reaction, to the uptake of the toxic cations by the sorbent. according to amadi et al. [26], the linear form of pseudo first–order kinetics is given below. (8) the plot of log(qe-qt) against time ‘t’, gave as the slope and logqe as the intercept [27]. 2.4.2. pseudo second–order kinetic the contribution of chemisorption was evaluated by analyzing the data with pseudo-second order kinetic model. the linear form of the pseudo-second order kinetic is stated eq. 9. (9) the plot of against t, gives k2 as the slope and as the intercept. 2.4.3. intra–particle diffusion the equation for the linear form of intra-particle diffusion kinetics is given eq 10. qt = kd × t 1/2 + c (10) thus, when qt is plotted against t 1/2 , the slope is the rate constant kd in (mg/(g min 1/2 )) and the intercept ‘c’ in (mg/g). the importance of the intercept is that it explains the thickness of the boundary of the layer. the characteristic significance of the value of the constant ‘c’ is that the larger the value, the greater the boundary layer thickness [22][27]. 2.4.4. liquid–film diffusion this was used to investigate the rate at which the toxic cations in the form of a film glide over one another [25]. the linear form of liquid film diffusion model is given below. in(1-α) = klft + c (11) where α = qt/qe, klf is the liquid film diffusion rate constant. a plot of in(1-α) against time (t) yields the constant klf (min -1 ) as the slope and a dimensionless constant c as the intercept [27]. 2.5. thermodynamic studies thermodynamic parameters such as enthalpy change (∆h), entropy change (∆s), and gibbs’ free energy (∆g), of the adsorption processes were determined. the arrhenius equation described by onwuka et al. [27], was used for the thermodynamic modeling, the equation is expressed in eq. 12. (12) the enthalpy change (∆h) was determined from the slope of the plot of versus using eq.12. the heat capacity (cp) and entropy change, (∆s), were determined from eqs. (13) and (14) respectively. (13) (14) the gibbs’ free energy was determined using eq. 15. source of variation ss df ms fcal p-value fcrit cd 2+ between groups 10.46042 4 2.615106 0.005798 0.999927 2.866081 within groups 9021.483 20 451.0741 total 9031.943 24 pb 2+ between groups 2178.819 4 544.7049 1.127699 0.371616 2.866081 within groups 9660.465 20 483.0232 total 11839.28 24 table 2. anova for effect of contact time on the sorption of cd 2+ and pb 2+ at various temperatures. j. multidiscip. appl. nat. sci. 120 source of variation ss df ms f p-value fcrit cd 2+ between groups 175150.1 5 35030.03 1247.75 3.31705e-22 2.772853 within groups 505.3421 18 28.07456 total 175655.5 23 pb 2+ between groups 189251.5 5 37850.31 277.7577 2.22831e-16 2.772853 within groups 2452.877 18 136.2709 total 191704.4 23 table 3. anova for effect of temperature for cd 2+ and pb 2+ sorption at different time intervals. (15) where is the amount adsorbed at temperature t, is the amount adsorbed at temperature , r is the universal gas constant, is the heat capacity of the system at pressure p, and are the initial and final pressures respectively, and are the initial and final temperatures, respectively. 2.6. characterization 2.6.1. fourier transform infra-red (ft-ir) analysis the kbr pellet was prepared by mixing about 1 mg of the sorbent with 250 mg kbr (ft-ir grade), pressed and placed for acquisition of ir spectrum on shimadzu model (8400s) of the ft-ir equipment. the spectra were recorded between 4000–400 cm -1 resolutions of 4 cm -1 . this was carried out at the national research institute for chemical technology (narict) zaria, kaduna state. 2.6.2. scanning electron microscope (sem) analysis the morphology of the grw sorbent before and after metal ion sorption, was investigated using scanning electron microscope (sem; jsm5610lv). the micrographs were taken at 1000 times magnification. 3. results and discussions 3.1. effect of sorbent dose the result of the effect of sorbent dose on the metal ions (cd 2+ and pb 2+ ) removal efficiency is presented in fig. 1. the result shows that the sorption capacity is greatly influenced by the amount of sorbent material in the aqueous solution since the percentage sorption of the toxic metal ions increased in a higher sorbent dose. this suggests that there are more active binding sites, pores and caves at higher amount of the sorbent as similarly reported by kampalanonwat and supaphol [28] in a related investigation of heavy metal ion adsorption by animated polyacrylonitrile nanofibre mats. increasing the sorbent dose also reduces the distance metal ions needed to travel in order to be entrapped by active pores and caves. a higher amount of the sorbent in solution results in a proportionate increase in the surface area which enhanced the sorption of more metal cations. a similar observation was reported by vijayakurma et al. [29] and shooto et al. [12] on natural pelite and ginger root, respectively. it was also observed in fig 1 that increment in sorbent dose leads to higher cd 2+ adsorption through a maximum while pb 2+ adsorption increased continuously with the range of sorbent dose studied. this suggests that cd 2+ adsorption attained equilibrium faster than pb 2+ adsorption within the studied sorbent dosages. this can be attributed to the smaller ionic size of cd 2+ (compared to pb 2+ ) which makes it more mobile and consequently results in more interaction with the active sites on the sorbent. 3.2. effect of initial metal ion concentrations fig. 2 shows the effect of initial metal ions concentration on the sorption of the toxic metal ions from the aqueous medium. it is observed that the removal of the metal ions increased with increase in the initial metal ions concentration. this suggests that at lower metal ion concentration, desorption of these metal cations from the sorbent occurs more than their adsorption on the sorbent. it also indicates that the metal ions interacted more with j. multidiscip. appl. nat. sci. 121 temp (k) pseudo first–order pseudo second–order intra–particle diffusion liquid film diffusion qexp k1 r 2 qcal k2 r 2 qcal kd r 2 c klf r 2 c cd 2+ 303 0.012 0.086 0.132 0.540 0.282 -0.458 0.006 0.583 a 1.769 0.011 0.069 2.506 1.847 313 0.009 0.072 0.145 0.264 0.381 0.278 0.006 0.540 a 1.769 0.009 0.051 2.447 1.862 323 0.007 0.053 0.129 0.301 0.178 0.205 0.005 0.383 a 1.794 0.008 0.040 2.542 1.874 333 0.007 0.050 0.132 0.291 0.157 0.204 0.005 0.340 a 1.798 0.007 0.034 2.558 1.881 pb 2+ 303 0.000 0.004 0.205 0.026 0.002 0.081 0.040 0.772 a 0.954 0.000 0.000 2.002 1.456 313 0.021 0.470 1.189 0.221 0.480 -0.135 0.065 0.633 a 0.993 0.021 0.375 0.275 1.749 323 0.014 0.173 0.308 0.296 0.291 -0.879 0.022 0.590 a 1.548 0.015 0.139 1.627 1.810 333 0.002 0.017 0.252 0.018 0.062 0.229 0.008 0.113 1.744 0.002 0.010 1.900 1.927 table 4. data isolated from kinetic plots. note: a means highest r 2 values. r 2 values that are moderate are bolded the sorbent active binding sites and caves at their higher metal ion concentrations compared to lower metal ion concentrations [30]. this can also imply that the ionic orientation favours sorption at higher initial metal ions concentration which is ascribed to the fact that the force of repulsion at higher concentrations has a minimum barrier to the sorption of the metal cations [31]. it was also observed in fig. 2 just as in fig. 1, that the sorption of cd 2+ from an aqueous medium was higher than that of pb 2+ . this disparity can be attributed to the differences in the atomic/ionic radius between the two toxic cations. egila et al. [32], in similar research on amarathus hydridus l. stalk waste, reported that atoms with lower atomic radii show more tendencies to be sorbed easily than atoms with larger atomic radii. the atomic radii of cd 2+ is 0.97 å while that of pb 2+ is 1.20 å. thus, this made it relatively difficult for pb 2+ ions to infiltrate the lignocellulosic matrix component of the grw sorbent and hence, result in lower pb 2+ sorption compared to cd 2+ sorption. chukwuemeka et al. (2015), onwuka et al. [27] and shooto et al. [12] reported similar findings in their works on ginger, delonix regia pods and ginger root, respectively. 3.3. adsorption isotherm studies the nature of the interaction between the toxic cations and the sorbent was assessed using langmuir, freundlich, elovich and redlich– peterson isotherm models. parameters evaluated and isolated from the isotherm plots in equations 3, 5, 6 and 7 are summarized in table 1. generally, the value of coefficient of determination (r 2 ) in table 1 shows that the order of best fit isotherm model for cd 2+ sorption onto the grw sorbent is freundlich > redlich–peterson > elovich > langmuir isotherm while that of pb 2+ sorption is elovich > redlich–peterson > freundlich > langmuir isotherm. rl value is the most important feature of the langmuir adsorption isotherm that helps to explain and predict the degree of interaction and compatibility between the sorbate and sorbent in the aqueous solution [22][33]. favourable langmuir isotherm modeling is such that 0<rl<1 as observed by kussel et al. [34]. thus, it was observed (table 1) that the langmuir dimensionless constant (rl) values for cd 2+ and pb 2+ sorption from the aqueous medium, is less than 1 suggesting that the sorption of these toxic cations onto the grw sorbent is relatively favourable. the high langmuir r 2 value for cd 2+ sorption further supports this assertion. however, the low langmuir r 2 value for pb 2+ sorption and the negative qmax for cd 2+ sorption which influenced the outcome of few other variables shown in table 1, are indications that the application of langmuir isotherm model alone cannot satisfactorily explain the sorption of pb 2+ and cd 2+ onto the sorbent [32][35][36]. in the freundlich isotherm studies, the “n” value explains the degree of compatibility and interaction between the metal ions and the sorbent, while kf is the adsorption capacity. the best fit “n” value for freundlich adsorption isotherm is that value that j. multidiscip. appl. nat. sci. 122 falls between 0 and 1 [37]. on this basis, it can be inferred that the sorption of cd 2+ onto the sorbent favourably aligned with the freundlich isotherm modeling. this entails that the sorption is multilayer in nature and a physical process. this finding agrees with reports by jimoh et al. [38], amadi et al. [26] and lei et al. [39] on cd 2+ adsorption using modified charcoal, cellulosic biosorbent derived from okra stem and modified biomass ash, respectively. the high r 2 value (table 1) strengthens the assertion that cd 2+ sorption onto the grw is favourable to the freundlich isotherm. however, the negative “n” value of the freundlich isotherm model for pb 2+ sorption implies that its sorption was quite difficult and the application of freundlich isotherm model alone is inadequate to satisfactorily explain the sorption process. the difficulty in pb 2+ sorption could be attributed to its large ionic radius or the charge density [32]. this observation was opined by alsenani [35] in similar research on calligonum comosum leaf powder. table 1 further illustrates that cd 2+ and pb 2+ sorption onto the sorbent, aligned with the elovich isotherm model based on their high coefficient of determination (r 2 ) values. this suggests that the sorption sites increased exponentially with adsorption validating that sorption of these toxic cations (cd 2+ and pb 2+ ) is multilayer in nature [40]. it is therefore inferred that the sorption of cd 2+ and pb 2+ was dependent on the multiple micropores and the complicated chemical structure of sorbent. this was also reported by achmad et al. [41] and lei et al. [39] in similar works on a novel adsorbent developed from uncaria gambir extract and adsorption of cd 2+ from aqueous solution by modified biomass ash. redlich–peterson isotherm is used to correct the errors encountered in using langmuir and freundlich adsorption isotherm [42]. the model is actually the blend of langmuir and freundlich adsorption isotherms into one [24][43]. the redlich –peterson isotherm modeling for the cd 2+ sorption was also presented in table 1. the high coefficient of determination (r 2 ) shows that redlich-peterson isotherm model satisfactorily predicted the sorption of cd 2+ onto the sorbent. it was also observed from table 1 that the values of β for cd 2+ (1.8622) and pb 2+ (1.9906) sorption are above unity. this implies minimal or little influence of langmuir adsorption isotherm on cd 2+ and pb 2+ sorption onto the sorbent. this assertion is anchored on the findings of belhachemi and addoun [24] that adsorption process where β approaches unity is suggestive that the sorption is best fitted to langmuir isotherm model but if it is above unity, the sorption is best explained by the freundlich isotherm model. thus, it is inferred that the sorption of cd 2+ and pb 2+ satisfactorily aligned to freundlich isotherm model. figure 5. effect of temperature for cd 2+ sorption at different contact time. figure 6. effect of temperature for pb 2+ sorption at different contact time. metal ion cp (j/k) ∆h (j/mol) ∆s (j/k) ∆g (j/mol) t (k) cd 2+ 32.092 320.092 1.011 14.587 303 4.477 313 -5.633 323 -15.743 333 pb 2+ 1267.636 12,676.365 39.931 577.260 303 177.950 313 -221.360 323 -620.667 333 table 5. thermodynamic data of cd 2+ and pb 2+ sorption. j. multidiscip. appl. nat. sci. 123 the implication is that the sorption of cd 2+ and pb 2+ occurred on a heterogeneous surface and is strongly influenced by multiple micropores, caves and rough surface morphology of the sorbent which constitutes the active sorption sites. this observation is validated by the strong agreement between the sorption capacities for redlich–peterson isotherm (a = 0.0463 for cd 2+ and 11.1931 for pb 2+ ) and freundlich adsorption isotherm (kf = 0.0463 for cd 2+ and 11.1970 for pb 2+ ) as shown in table 1. gimbert et al. [44] reported similar findings on catonized starch-based material in a single component system. figure 7. ftir spectra of (a) unloaded (b) cd 2+ loaded and (c) pb 2+ loaded sorbents. j. multidiscip. appl. nat. sci. 124 3.4. effect of contact time at various temperatures the effect of contact time on cd 2+ and pb 2+ sorption are shown figs 3 and 4. the results show that the sorption of toxic metal ions (cd 2+ and pb 2+ ) at various temperatures studied, increased through a maximum with increase in contact time (figs 3 and 4). thus, the sorption of toxic metal ions was very fast initially at the adsorbent-adsorbate contact until equilibrium (saturation) was attained at 90 min and then it gradually started decreasing despite the increase in the contact time. this is attributed to the saturation of the available pores, caves and adsorptive active binding sites. this observation was also reported by vijayakumar et al. [29] in similar work on natural pelite. it was further observed that a two-way adsorption process played out. first, was the rapid phase encounter that occurred between the time of the initial adsorbate-adsorbent contact to the time equilibrium was reached and the second phase was the slower phase that started from the time equilibrium was reached downward with declining adsorption even with increasing contact time. this is ascribed to the fact that at saturation point, all active sites, pores and caves are filled up and a further increase in the contact time results in desorption. this accounts for the decline in the sorption process after equilibrium has been reached, an ascertion also shared by kekpugile et al. [10] in a related research on kinetic study of garlic and ginger. a similar inference was also opined by kampalanonwat and supaphol [28] in a related work on animated polyacrylonitrile nanofibre mats. also, higher percentage removal of cd 2+ over pb 2+ was also observed (figs 3 and 4). this is an indication that cd 2+ is more mobile and dynamic than pb 2+ in solution which is a function of the differences in the ionic size and charge density. jimoh et al. [38] reported similar findings in related work on modified charcoal. 3.5. one–way factorial analyses figs 3 and 4 showed the variations in the removal efficiency of cd 2+ and pb 2+ with contact time at different temperatures respectively. these variations in the removal efficiency of the toxic metal cations with time were tested for significant differences. the analysis of variance (anova) presented in table 2 shows that the observed variations in the sorption of cd 2+ and pb 2+ with contact time have no significant difference at the various temperatures studied. this is because the pvalues are greater than α = 0.05 and fcal is less than fcrit. thus, it is a further validation of the null hypothesis. this result implies that any of the studied temperature can be used to explain/describe the variations of the metal ions sorption with contact time. in figs 5 and 6, the variations in the sorption of cd 2+ and pb 2+ with temperature at various contact time were shown. table 3 shows that there is statistically significant difference in cd 2+ and pb 2+ sorption when the temperature was varied at different time. it can be concluded that at α = 0.05, null hypotheses were rejected because fcal is higher than fcrit and p-values is less than α = 0.05. this is an indication that thermal influence is essential for the sorption of these metal adsorbates as was also observed by batool et al. [45] in related study on linear and non-linear approach and error analysis. 3.6. kinetic studies reaction (pseudo first–order and pseudo second –order) and diffusion (intra–particle and liquid film diffusion) kinetics were used to determine the sorption mechanism of the toxic cations (cd 2+ and pb 2+ ) on the sorbent. the kinetic data extracted from the plots in equations 7, 8, 9 and 10, are summarized in table 4. the values of coefficient of determination (r 2 ), qcal and qexp for the various kinetic plots, were used to determine the mechanisms that contributed to the sorption of the toxic cations at each temperature. it was observed that pseudo first–order and pseudo second–order kinetic models could not satisfactorily explain the sorption process of the metal cations at all the temperatures studied (table 4). this is because there is no strong agreement between the qcal and qexp values [22]. the values of coefficient of determination (r 2 ) within 0.43 ≤ r 2 ≤ 0.83 are considered high and moderate [46]. the highest values of r 2 are obtained at intra–particle diffusion plots of all studied temperatures. hence, diffusion into the pores of the grw sorbent is the rate-controlling mechanism in the sorption of the toxic cations (cd 2+ and pb 2+ ) at all the studied temperatures (303–333 j. multidiscip. appl. nat. sci. 125 k). however, intra–particle diffusion is not the sole mechanism in the sorption of these toxic cations because the linear plot did not pass through the origin [10][22]. it was also observed that chemical reaction contributed to the sorption mechanism of cd 2+ and pb 2+ at 313 k while surface reaction and transport of sorbate molecules from liquid to solid phase were majorly involved in pb 2+ sorption mechanism at 313 k. the intercepts obtained in the intra–particle diffusion plots are relatively high in all the sorption temperatures for cd 2+ sorption and at 323 k and 333 k for pb 2+ sorption. this indicates that the sorption process of these metal ions was mainly a surface reaction [26][27]. other related studies that shared similar views are those of ademiluyi and ujile [47], li et al. [48] and kulkarni et al. [49]. 3.7. effect of temperature at different time intervals the effect of temperature on the sorption cd 2+ and pb 2+ at different contact times by the sorbent is shown in figs 5 and 6 respectively. the result indicated that the sorption of the toxic cations (cd 2+ and pb 2+ ) increased steadily as the temperature was raised. this is attributed to the activated mobility of the metal cations introduced into the system by increase in temperature and also, the diffusion of the ionic species [38]. this is believed in facilitating the sorption of these toxic cations as demonstrated by the increase in percentage removal of the metal cations in the aqueous solution [50]. therefore, thermal influence is opined to have a strong effect on the sorption of the metal ions. this was similarly reported by other kinetic studies by wase [51] and tounsadi et al. [36] for tea factory waste and diplotaxis hara glebionis corona l. 3.8. thermodynamic studies the data isolated from the thermodynamic plots using equation 11, are summarized in table 5. in the result, it was observed that the heat capacity of cd 2+ sorption onto the sorbent is 32.09 j/mol while that of pb 2+ sorption was 1267.636 j/mol. heat capacity cp is the quantity of thermal energy required to raise the temperature of adsorption by one degree [52]. the higher heat capacity of adsorption of pb 2+ can be attributed to the higher charge density of pb 2+ compared to cd 2+ . this is believed to have made it less mobile and so required higher thermal activation for the reaction to occur [38][53]. table 5 further showed that the sorption of the metal cation was non-spontaneous at low temperatures of 303–313 k as depicted by the positive value of change in gibbs free energy (∆g). however, as the sorption temperature increased to 323–333 k, the values of ∆g for the metal cation sorption become negative suggesting that the meta cation sorption is spontaneous. the implication is that for sorption of these metal cations to occur onto the sorbent, heat must be added. this is ascertained by the positive value of enthalpy change (∆h) which indicates that the sorption of these metal cations is endothermic in nature. therefore, for the reaction (metal cation sorption) proceed, the initial energy barrier is required to be broken by increase in temperature. this is contrary to the findings of figure 8. sem micrographs of (a) unloaded (b) cd 2+ loaded and (c) pb 2+ loaded sorbents. j. multidiscip. appl. nat. sci. 126 shooto et al. [12] but in agreement with findings of kazi et al. [54], hussein et al. [52] and gabreslassie [55]. 3.9. fourier transform infra-red (ft-ir) spectroscopy the ftir analysis of the unloaded, cd 2+ loaded and pb 2+ loaded sorbents, are presented in fig 7a, 7b and 7c, respectively. the peak 2337.80 cm -1 appeared in both the unloaded and metal ions loaded sorbents. this peak falls within 2400-2000 cm -1 and thus, assigned to o=c=o stretching. the peak 1643.41 cm -1 as seen in cd 2+ and pb 2+ loaded sorbents (fig 7b and 7c) falls within 1648-1638 cm 1 and is assigned to c=c stretching from alkene. the peak 1735.99 cm -1 falls within 1735-1750 cm -1 is assigned to c=o stretching. as the sorbent was hydrolysed during the metal ions sorption, the peak 3356.25 cm -1 appeared in both fig 7b and 7c. this peak falls within the region 4000–3000 cm -1 which is assigned to o–h (stretching) resulting from roh. this peak is very important in the investigation by virtue of the nature of sorbent used for the analysis. the grw material is a plant fibre [56] and commonly fibre is a soluble polymer in solution containing hydrophilic groups such as the o–h groups that undergoes such a reaction mechanism that can entrap divalent cations [57]. figs 7a, 7b and 7c show that during sorption, the bivalent toxic cations (cd 2+ and pb 2+ ) interacted with the o–h, c=c and c=o groups which caused spectra enhancement/shift and also spectra appearance. the interaction between these functional groups and the bivalent toxic ions led to the sorption of these bivalent toxic cations [58][12]. 3.10. scanning electron microscope (sem) analysis figs 8a, 8b and 8c show the sem micrographs of the unloaded, cd 2+ loaded and pb 2+ loaded sorbents, respectively. pores, caves and roughened surfaces were observed in fig 8a. these are factors suspected to facilitate and influence the sorption of cd 2+ and pb 2+ onto the sorbent [45][59]. figs 8b and 8c show that after the sorption of the metal ions, there was conglutination of the neighboring fibres at the touching line making the morphology of the sorbent compact and the sorbent appeared smoother and slippery with tiny blocks depicting the distribution of the metal ions on the sorbent surface. this is evidence that sorption of these metal ions (cd 2+ and pb 2+ ) onto the sorbent was successful which is in agreement with the previous studies [28][60]. 4. conclusions this study showed that the ginger root waste (grw) adsorbent material has the potential to remove toxic cations (cd 2+ and pb 2+ ) from the aqueous medium. the grw material was able to remove more cd 2+ from aqueous medium than pb 2+ . the sorption of these toxic cations occurred on a heterogeneous surface and is strongly influenced by multiple micropores, caves and rough surface morphology of the sorbent. diffusion into the sorbent pores was pivotal for the sorption of these toxic cations. the sorption process is non– spontaneous at low temperature but spontaneous at higher temperature. the ftir results showed that these toxic cations (cd 2+ and pb 2+ ) were adsorbed at both hydrophilic and hydrophobic functional groups. sem micrographs showed that the adsorption of these toxic cations changed the surface morphology of the grw sorbent. author information corresponding author jude chinedu onwuka — department of science laboratory technology, federal university of lafia, lafia – 950101 (nigeria); department of chemistry, federal university of lafia, lafia – 950101 (nigeria); orcid.org/0000-0003-2950-8273 email: emperor20062003@yahoo.com authors stephen igberi azubuike — department of chemistry, federal university of lafia, lafia – 950101 (nigeria); timothy m. akpomie — department of chemistry, federal university of lafia, lafia – 950101 (nigeria); acknowledgement the authors are immensely grateful to mr. william egbe and other staff and management of https://orcid.org/0000-0003-2950-8273 mailto:emperor20062003@yahoo.com j. multidiscip. appl. nat. sci. 127 national soil testing laboratory complex, federal ministry of agriculture and rural development, goningora, kaduna state; for availing their laboratory services for this research. references [1] m. a. barakat. 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(2022) first publication right: journal of multidisciplinary applied natural science publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. this article is licensed under: https://doi.org/10.47352/jmans.2774-3047.101 open access https://creativecommons.org/licenses/by-sa/4.0/deed.id https://doi.org/10.47352/jmans.2774-3047.101 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.101&domain=pdf&date_stamp=2022-01-15 j. multidiscip. appl. nat. sci. 24 an example, detection and quantification of heavy metals are crucial as they are harmful to living organisms. these heavy metals are often found in polluted aquatic samples. because of that, cation sensing gained significant attention to establish a sustainable future [12]. the detection of anions is also essential due to their important role in biological and environmental ecosystems. the presence of a certain anion in an acceptable amount can be useful in some applications but not in an excessive concentration [13]. meanwhile, the detection of neutral molecules usually involves recognizing biomolecules such as amino acids and nucleotides [14][15]. calixarene derivatives are well-known host molecules with versatile applications, including chemosensor agents [16]-[21]. calixarenes consisted of a macrocyclic oligomer framework resulted from the condensation reaction between formaldehyde and tert-butylphenol [22]. calixarene has a specific ring size that can be controlled by using an ion-template synthesis. the control on the ring size of calixarenes yields calix[4]arene, calix [5]arene, calix[6]arene, and calix[8]arene [23][24]. the conformation of the calixarene can also be controlled in various conformations such as cone, partial cone, 1,2-alternate, and 1,3-alternate [25], as shown in figure 2. moreover, the calixarenes can be easily modified on their upper or lower rim [26]. calixarenes are reported to have good photoluminescence properties; thus, calixarenes have been extensively used as the chemosensor agent. because of that, a brief review on the application of calixarenes as the fluorescent chemosensor agent is useful to give brief information about the design and development on the chemosensor field. however, the latest review article on the fluorescent chemosensor based on calixarene derivatives was published in 2013 [23]. furthermore the application of supramolecular calixarenes as fluorescent chemosensor was not discussed in particular in the recent literature. from that report, an updated review specifically on the fluorescent chemosensor based on supramolecular calixarene derivatives has not been available yet as for today. to a certain extent, this review provides a brief update on the utilization of supramolecularbased calixarene derivatives as the fluorometric chemosensor agent for the detection of cations, anions, and neutral molecules. in the present article, we also summarized the performance of each chemosensor agent and provided a brief perspective for future research. 2. colorimetric and fluorometric detections colorimetric is the simplest detection method in the chemosensor field. colorimetric offers the naked eye investigation using a visible spectrophotometer instrument [27]. in the colorimetric method, a chemosensor should contain a chromophore group as the signaling unit. the chromophore is responsible for the color of the figure 1. the general sensing mechanism of colorimetric and fluorometric chemosensor agents. j. multidiscip. appl. nat. sci. 25 chemosensor in its free form and the specific color change after its interaction with a particular analyte. therefore, the colorimetric method works by measuring the absorbance change after the addition of the analyte [28]. because of that, the colorimetric method has a limitation on poor sensitivity and background interference. in contrast, the fluorometric method is preferable for actual application due to its higher sensitivity than the colorimetric method. the fluorometric method has been widely used to detect and quantify cations, anions, or neutral molecules. the fluorometric technique uses a chemical with a fluorophore unit, in chemosensor application [29]. the fluorometric chemosensor can interact with the analyte through several sensing mechanisms such as intramolecular charge transfer (ict), photoinduced electron transfer (pet), chelation-induced enhanced fluorescence (chef), chelation quenched fluorescence (chqf), aggregation caused quenching (acq), and aggregation-induced emission (aie) [30]-[35]. schematic examples for each sensing mechanism are depicted in figure 3. the different phenomenon leads to a different pathway to detect a certain analyte. however, detection using the fluorometric method generally can be classified into two pathways: fluorescence off and fluorescence on. fluorescence off happened when the emission intensity of the free chemosensor agent was quenched due to the addition of a certain analyte. meanwhile, the fluorescence on increases the emission intensity due to the interaction between the chemosensor agent and analyte. 3. calixarene for cation chemosensor the usage of calixarenes for the detection of metal ions have been developed in recent years. for example, xu and coworkers utilized naphthalimide –calix[4]arene (1) for selective detection of cu 2+ ions in mecn:h2o (9:1 v/v) solvent system. compound 1 gave the quenching phenomenon at emission wavelength (λem) of 490 nm (excitation wavelength, λex = 435 nm) with the presence of cu 2+ . an excellent selectivity was observed over the other metal ions such as ag + , li + , na + , k + , rb + , cs + , ca 2+ , cd 2+ , co 2+ , hg 2+ , mg 2+ , mn 2+ , ni 2+ , pb 2+ , and zn 2+ , which was remarkable. the oxygen atoms of calixarene and nitrogen atoms of naphthalimide are responsible for cu 2+ binding, generating a 1:1 complex with an association constant (ka) of 2.9 × 10 4 m -1 [36]. wang and coworkers employed a pyrenemodified calix[4]arene (2) for selective detection of na + cation in dmso:mecn (1:9 v/v). when the calixarene 2 was excited at 343 nm, its excimer emission signal at 477 nm was quenched, while the monomer emission signal at 375 nm was increased with the presence of na + . in contrast, the emission signal was not significantly changed by the addition of the other metal ions such as li + , k + , cs + , mg 2+ , ca 2+ , sr 2+ , and ba 2+ . this excellent selectivity was caused by the presence of four carbonyls of ester functional group encapsulated the na + ions in an ionophoric cavity. the complexation happened on the calixarene 2 lower rim, thus affecting the pyrene moieties on the upper rim. calixarene 2 acted as a molecular tweezer with an on-off mechanism controlled with na + ions. initially, the pyrene moieties overlapped through π-π stacking, thus figure 2. the conformational isomers of calixarenes; a) cone, b) partial cone, c) 1,2-alternate, d) 1,3alternate. j. multidiscip. appl. nat. sci. 26 generating an excimer emission signal at 477 nm. in contrast, complexation with na + ions at the lower rim of calixarene 2 forced the pyrenes to stay away from each other; thus, the excimer emission signal was quenched, but the monomer emission signal was activated. the calixarene 2 formed a 1:1 complex with na + ions giving a ka value of 2.2 × 10 3 m -1 [37]. dinda and coworkers used the nitrobenzoxadiazole appended calix[4]arene derivative (3) for selective recognition of trivalent metal ions, i.e., cr 3+ , fe 3+ , and al 3+ . the detection of these trivalent metal ions occurred through a fluorescence on as the fluorescence quantum yield of calixarene 3 was significantly enhanced after the addition of metal ions. the calixarene 3 had a low emission intensity at 550 nm (λex = 470 nm); however, the addition of m 3+ increased its fluorescence intensity in a 4-fold increment of quantum yield. this phenomenon happened because of chelation enhanced fluorescence intensity between calixarene 3 and metal ions. from the spectroscopic study, the m 3+ ions interacted with benzofurazan rings, amide oxygens, and phenoxy oxygen atoms of calixarene 3. the ka value of this chelation was 1.0 × 10 4 m -1 suggesting a moderate strength complexation. the sensitivity of calixarene 3 was shown from a low limit detection (lod) value (5 µm). the formed complex between calixarene 3 and m 3+ ions was reversible by the addition of h2po4 − . therefore, this chemosensor agent can be reused for the detection and quantification of these trivalent metal ions. furthermore, a real application of calixarene 3 for the detection of m 3+ ions was also established within mcf-7 cells [38]. zhan and coworkers used the anthraquinonecalix[4]arene (4) for selective detection of ca 2+ ions. the selective detection of ca 2+ ions was found over the other metal ions such as ag + , na + , k + , li + , cs + , mg 2+ , sr 2+ , ba 2+ , cu 2+ , cd 2+ , ni 2+ , mn 2+ , co 2+ , and zn 2+ . the sensing method was performed in acetonitrile media, and it was found that the interaction between calixarene 4 and ca 2+ ions figure 3. schematic fluorescence sensing mechanisms, i.e., a) ict, b) pet, c) chef and chqf, d) acq, and e) aie. j. multidiscip. appl. nat. sci. 27 increased the emission intensity at 510 nm (λex = 390 nm). the addition of ca 2+ ions at various concentrations gradually increased its fluorescence intensity yielding a lod value of 50 µm. the fluorescence on phenomenon was generated from a 1:1 complexation reaction between calixarene 4 and ca 2+ with a ka value of 1.2 × 10 4 m -1 . the ca 2+ ions bind to the nitrogen atoms of triazole in calixarene 4, leading to a restriction of the pet and an increase in emission intensity [39]. li and coworkers employed calixarene 5 for the detection of fe 3+ and cu 2+ ions in h2o:meoh (1:1 v/v) media. the fluorescence intensity of calixarene 5 was significantly quenched by the addition of fe 3+ and cu 2+ ions. meanwhile, the addition of other metal ions such as co 2+ , ni 2+ , zn 2+ , mn 2+ , mg 2+ , and ba 2+ did not significantly change the fluorescence intensity of calixarene 5. the calixarene 5 formed a 1:1 complex structure with either fe 3+ or cu 2+ ions only at a low concentration figure 4. chemical structures of calixarene-based chemosensor agents 1–10 for cations sensing. j. multidiscip. appl. nat. sci. 28 (less than 1 µm). at a higher concentration of the metal ions lead to the formation of a different complex stoichiometry. this phenomenon in fluorescence quenching was also highly related to the ph value of the aqueous media [40]. chang and coworkers used the modified calix[4] arene with anthracene-isoxazolymethyl group (6) at the lower rim for the detection of cu 2+ ions. the calixarene 6 showed two emission peaks at 511 nm (excimer) and 430 nm (monomer) with the λex of 375 nm. the calixarene 6 showed selectivity and sensitivity in the detection of cu 2+ in ch3cn:chcl3 (1000:4, v/v) solvent system, through coordination with isoxazole moieties. the selectivity was compared to the 14 metal ions including ag + , li + , na + , k + , mg 2+ , ca 2+ , ba 2+ , pb 2+ , hg 2+ , cd 2+ , ni 2+ , mn 2+ , zn 2+ , and cr 3+ . the fluorescence emission intensity was quenched only with the addition of cu 2+ ions, while the other cations did not significantly change the fluorescence spectra of the free ligand. the calixarene 6 formed complex with cu 2+ with a 1:1 ratio with a ka value of 1.6 × 10 4 m -1 [41]. arena and coworkers utilized calix[4]arene bearing n-(phenyl)sulfonylcarboxamide (7) for selective detection of hg 2+ ions in h2o:mecn (1:1 v/v) media. the presence of hg 2+ ions quenched the fluorescence intensity of calixarene 7. the sensing mechanism occurred through the turn-off fluorescence at λem of 312 nm (λex = 273 nm). the calixarene 7 showed high selectivity for hg 2+ ions over the other heavy metal ions such as pb 2+ and cd 2+ . unfortunately, the sensitivity for hg 2+ detection has not been evaluated yet [42]. joseph and coworkers investigated the application of calix[4]arene 8 to detect zn 2+ ions. the calixarene 8 showed an excellent sensitivity for zn 2+ ions with the lod value of 0.69 nm in methanol media, which was remarkable. the emission intensity of calixarene 8 at 380–620 nm (λex = 370 nm) was significantly increased by the presence of zn 2+ ions. additionally, calixarene 8 also exhibited good selectivity for the detection of zn 2+ ions. the addition of the other metal ions such as na + , k + , mn 2+ , co 2+ , ca 2+ , mg 2+ , and cd 2+ did not significantly change the emission intensity of calixarene 8. the addition of these metal ions gave a negligible interference for zn 2+ detection, indicating a good selectivity. the fluorescence turn -on phenomenon happened due to the binding of zn 2+ to the imine and phenolic moieties of calixarene 8 blocking the pet to the salicylyl moiety [43]. mummidivarapu and coworkers employed a calix[6]arene with triazole linked picolimine conjugate (9) for the detection of la 3+ ions. calixarene 9 showed a selective and sensitive detection for la 3+ ions with a lod value of 490 nm. the sensing mechanism happened through fluorescence turn-on on the emission intensity of 450 nm (λex = 330 nm). the addition of the other trivalent metal ions such as pr 3+ , nd 3+ , sm 3+ , eu 3+ , calixarene sensing media lod (nm) ka (m -1 ) analyte reference 1 mecn:h2o (9:1 v/v) n/a 2.9 × 10 4 cu 2+ [36] 2 dmso:mecn (1:9 v/v) n/a 2.2 × 10 3 na + [37] 3 n/a 5,000 1.0 × 10 4 cr 3+ , fe 3+ , and al 3+ [38] 4 mecn 50,000 1.2 × 10 4 ca 2+ [39] 5 h2o:meoh (1:1 v/v) n/a n/a fe 3+ and cu 2+ [40] 6 mecn:chcl3 (1000:4 v/v) n/a 1.6 × 10 4 cu 2+ [41] 7 h2o:mecn (1:1 v/v) n/a n/a hg 2+ [42] 8 meoh 0.69 n/a zn 2+ [43] 9 n/a 490 3.7 × 10 4 la 3+ [44] 10 dcm:mecn (1:1 v/v) 490 n/a pb 2+ [45] table 1. performance of calixarene-based chemosensor agents for cation sensing. *n/a: not available j. multidiscip. appl. nat. sci. 29 gd 3+ , dy 3+ , and ho 3+ did not significantly increase the emission intensity. calixarene 9 complexed with la 3+ ions with a ka value of 3.7 × 10 4 m -1 . in this case, the imino-phenolic and the pyridyl moieties play an important role in binding with la 3+ ions. complex formation between calixarene 9 and la 3+ ions triggered the inhibition of imine isomerization and excited-state proton transfer (espt), leading to an increase in the fluorescence emission [44]. kumar and coworkers applied the pyreneappended calix[4]arenes derivative (10) for the fluorometric detection of pb 2+ ions in dcm:mecn (1:1 v/v) media. calixarene 10 exhibited excellent sensitivity and selectivity for detecting pb 2+ ions at λem of 498 nm (λex = 350 nm). by addition of pb 2+ figure 5. chemical structures of calixarene-based chemosensor agents 11–19 for anions sensing. j. multidiscip. appl. nat. sci. 30 ions, the excimer emission intensity of calixarene 10 at 498 nm was gradually quenched, with a gradual increment of emission signal at 415 nm. this hypsochromic shifting was attributed to the deprotonation of phenolic groups of calixarene 10. the addition of other metal ions such as ag + , li + , na + , k + , zn 2+ , and hg 2+ (in perchlorate salts) gave no significant fluorescence change for calixarene 10. calixarene 10 gave a lod value for detecting pb 2+ as low as 490 nm as calculated from the ratiometric plot of i415/i498. calixarene 10 formed a 1:1 complex with pb 2+ ions through the imino group, causing an ict from pyrene to the imino groups [45]. 4. calixarene for anion chemosensor uttam and coworkers employed a calix[4]arene conjugated with 1,3-dibenzooxadiazole (11) for the detection of fluoride anion (f − ) in tetrahydrofuran (thf) solvent. calixarene 11 could selectively detect f − anion over 15 other anions such as bromide (br − ), chloride (cl − ), iodide (i − ), azide (n3 − ), thiocyanate (scn − ), bicarbonate (hco3 − ), dihydrogen phosphate (h2po4 − ), carbonate (co3 2− ), nitrite (no2 − ), nitrate (no3 − ), sulfate (so4 2− ), pyrophosphate (p2o7 4− ), perchlorate (clo4 − ), and bisulfate (hso4 − ). calixarene 11 also exhibited a sensitive detection of f − with a lod value of 100 nm. in the detection of f − ions using calixarene 11, a 1:1 complex structure was formed with a ka value of 1.6 × 10 3 m -1 ; thus, the λem at 520 nm (λex = 460 nm) was significantly quenched. from the experimental-nmr and computational-density functional theory (dft) studies, it was found that the f − ion was coordinated through extended hydrogen bonds with dibenzooxadiazole and phenoxy oxygen atoms of calixarene 11. these chelations became the reason for such excellent selectivity of f − ions complexation and sensing. the chemosensor 11 offered good reusability as it can be decomplexed by the addition of ca 2+ ions with still good sensing capability even at 10 cycle sensing. furthermore, a biological application for detecting f − ions in the hela cells was also performed. the results demonstrated that calixarene 11 exhibited great application in the thf media as well as in hela cells [46]. hosseinzadeh and coworkers used the ferrocenetriazole-amide functionalized calix[4]arene (12) for the detection of f − in mecn:dcm (1:1 v/v) media. calixarene 12 showed a selective and sensitive detection of f − anion compared to the other anion including acetate (aco − ), cl − , br − , clo4 − , no3 − , h2po4 − , and hso4 − . the selectivity of calixarene 12 was shown from the enhancement in fluorescence emission intensity at 418 nm (λex = 290 nm). in addition, the lod value for f − detection was 2.98 µm. the extended hydrogen bonds between f − ions and amide and triazole protons of calixarene 12 corresponded to the calixarene sensing media lod (nm) ka (m -1 ) analyte references 11 thf 100 1.6 x 10 3 f − [46] 12 mecn:ch2cl2 (1:1 v/v) 2,980 5.35 x 10 8 f − [47] 13 meoh 5,000 1.49 x 10 -4 i − [48] 14 n/a 4,500 15.6 x 10 2 i − [49] 15 mecn:chcl3 (99.6:0.4 v/v) n/a n/a f − [50] 16 n/a n.a n/a h2po4 − , f − , and aco − [51] 17 mecn:h2o (7:3 v/v) 1,260 n/a cn − [52] 18 mecn:h2o (8:2 v/v) 350 n/a s 2− [53] 19 thf:h2o (7:3 v/v) 164 n/a i − [54] table 2. performance of calixarene-based chemosensor agents for anion sensing. *n/a: not available j. multidiscip. appl. nat. sci. 31 binding site in the sensing mechanism, as revealed by the nmr titration study. the calixarene 12 and f − ions formed a 1:2 complex structure with a ka value of 5.4 × 10 8 . the complexation was formed through the hydrogen bond between f − from both amide and triazole protons in calixarene 12 [47]. gómez-machuca and coworkers synthesized a calix[4]arenes with azolyl moieties (13) for the detection of i − anion in meoh. calixarene 13 gave both sensitive and selective detection of i − compared to the other anions (f − , hso4 − , n3 − , no2 − , no3 − , scn − , clo4 − , br − , cn − , cl − , ch3co2 − , cf3so3 − ). the tetrabutylammonium salts were used to avoid any cationic complexation with calixarene 13. in addition, calixarene 13 gave a quenching response at λem of 330 nm (λex = 225 nm) by the addition of i − anion yielding a lod value of 5 µm. calixarene 13 formed a 1:1 complex with i − ion through hydrogen bonds with a ka value of 1.5 × 10 -4 m -1 . from the nmr titration study, the signal of n-h amide and ar-h of benzoxazole moieties changed clearly. the n-h signal was shifted to a higher chemical shift due to an extended hydrogen bond with i − ions. meanwhile, the changed ar-h benzoxazole signals occurred due to the fixed and rigid structure of benzoxazole substituents after i − complexation reaction [48]. memon and coworkers utilized a calix[4]arene bearing naphtyhylimino moiety (14) for sensitive and selective detection of au 3+ cation and i − anion. the fluorescence response in the detection of both ions occurred through a quenching phenomenon at its emission intensity at λem 360 nm (λex = 330 nm). the lod value for the detection of au 3+ and i − were 15 and 4.5 µm respectively. the quenching response was due to a 1:1 complexation reaction between calixarene 14 and either au 3+ or i − which gave a ka value of 8 × 10 2 or 16 × 10 2 m -1 , respectively. the imine (c=n), amide carbonyl (c=o), and hydroxyl functional groups were important in the au 3+ sensing. meanwhile, the hydroxyl and amide nitrogen atoms of calixarene 14 interacted with i − anion through the extended hydrogen bonds [49]. miao and coworkers applied a calix[4]arene with diaminoanthracene group (15) for the detection of aco − in mecn:chcl3 (99.6:0.4 v/v). the tetrabutylammonium salts were used to avoid any cationic complexation with calixarene 15. calixarene 15 can selectively detect aco − over the other anions such as cl − , br − , i − , no3 − , hso4 − , and h2po4 − . the addition of aco − to calixarene 15 yielded a quenching of emission signal at λem 438 nm (λex = 370 nm). the quenching happened through a free pet process between calixarene 15 and aco − anion. meanwhile, no spectral changes were observed by the addition of other anions. furthermore, the detection of aco − occurred through hydrogen bonds between -nh and 9-h of anthracene as confirmed from the 1 h-nmr titration figure 6. chemical structures of calixarene-based chemosensor agents on 20–26 for neutral molecules sensing. j. multidiscip. appl. nat. sci. 32 study [50]. hung and coworkers prepared a calix[4]arene owing to pyrenylacetamide functional group (16) for the detection of h2po4 − , f − , and aco − anions. three different binding modes were observed for these anions. static excimer with large and small charge transfer occurred for f − and aco − anions, respectively. meanwhile, an increment in monomer emission of calixarene 16 happened for h2po4 − sensing. the dynamic excimer emission of calixarene 16 (λem = 482 nm) was quenched by the addition of f − at a low concentration. however, in an excess concentration of f − , the emission signal at 472 nm was increased significantly. this phenomenon was caused by the formation of a strong extended hydrogen bond between f − and calixarene 16 through its amide group, leading to a generation of pyrene dimers. on the other hand, h2po4 − sensing showed an opposite mechanism as the monomer emission at 472 nm was enhanced, but the excimer emission at 482 nm was decreased [51]. chawla and coworkers investigated the application of calix[4]arene bearing quinoline moiety (17) for the detection of cu 2+ and cn − ions in mecn:h2o (7:3 v/v) media. calixarene 17 could selectively detect cu 2+ over the other metal ions such as zn 2+ , pb 2+ , co 2+ , ni 2+ , na + , li + , fe 3+ , hg 2+ , cd 2+ , k + , mn 2+ , and ag + . the addition of cu 2+ leads to a significant quenching at λem of 405 nm (λex = 315 nm) of calixarene 17. the sensitivity of calixarene 17 for cu 2+ detection was shown by its lod value as low as 0.4 μm. interestingly, the addition of cu 2+ followed by cn − ion leads to a gradual increment of emission signal at 405 nm. it means that the calixarene 17 could also be used for the detection of cn − through the formation of cu 2+ calixarene 17 complex. the lod value for cn − detection was 1.26 μm. furthermore, a selective detection for cn − was achieved over f − , cl − , br − , i − , s 2− , aco − , co3 2− , hso4 − , and h2po4 − . the cn − detection was stable in a ph region of 6–8. lower ph value protonated quinoline nitrogen atoms while higher ph value formed a hydroxide complex of [cu(oh)m] (2-m)+ . the selectivity of cu 2+ -calixarene 17 complex for cn − detection happened as the cu 2+ ion itself was able to form a very stable metal complex with cn − anion. additionally, the chelation of calixarene 17 aided in locking the cn − anion through extended hydrogen bonds with quinoline and amide nitrogen atoms, thus providing an excellent sensing site for cn − anion [52]. these results are useful for developing a dual-response chemosensor agent for the detection of cation and anion with high selectivity and sensitivity. ramachandran and coworkers designed calix[4] arene 18 for the detection of cu 2+ and s 2− ion in mecn:h2o (8:2 v/v) media. free calixarene 18 showed a photoluminescence property at λem of 637 nm (λex = 457 nm). the addition of cu 2+ led to a gradual quenching by increasing cu 2+ concentration. the lod value of cu 2+ was 4.11 μm, with a ka value of 2.3 × 10 4 m -1 . calixarene 18 showed a high selectivity for cu 2+ detection calixarene sensing media lod (µm) ka (m -1 ) detection references 20 chcl3 3.49–107 n/a nitroaromatics [56] 21 h2o n/a 3.5 × 10 3 – 1.4 × 10 5 amino acids [57] 22 mecn n/a 9.1–56.5 × 10 1 amino alcohols [58] 23 h2o 24.5 x 10 -3 3.7 × 10 5 nadh [59] 24 n/a 3.81 x 10 -3 1.3 × 10 5 pm [60] 25 mecn 0.10 1.2 × 10 5 mc [61] 26 meoh:iproh (1:1 v/v) 2.25 n/a glyphosate [62] table 2. performance of calixarene-based chemosensor agents for anion sensing. *n/a: not available j. multidiscip. appl. nat. sci. 33 compared to the other cations such as na + , ag + , fe 2+ , hg 2+ , mg 2+ , mn 2+ , ni 2+ , pb 2+ , zn 2+ , ca 2+ , co 2+ , fe 3+ , and al 3+ . for anion sensing, the cu 2+ calixarene 18 complex showed a selective detection of s 2− anion over the other anions (aco − , cn − , co3 2− , f − , hpo4 − , hso4 − , no2 − , no3 − , ho − , and so3 2− ), which was remarkable. the detection limit of s 2− was estimated to be 0.35 μm [53]. xie and coworkers employed the hexahomotrioxacalix[3]arene (19) for the detection of i − in a thf:h2o mixture (7:3 v/v). calixarene 19 was found to be a selective and sensitive chemosensor agent for the detection of i − ion. calixarene 19 showed a high emission intensity at 484 nm (λex = 346 nm). the presence of i − ion leads to a significant quenching at this emission intensity compared to the other anions such as f − , cl − , br − , aco − , so3 2− , so4 2− , hso3 − , no3 − , and co3 2− . the binding mechanism for i − sensing occurred due to the hydrogen amide group of calixarene 19 to form the extended hydrogen bonds with a ka value of 2.5 × 10 2 m -1 . the fluorescence titration study showed that the addition of i − at higher concentration leads to a gradual decrease in emission signal at 484 nm yielding a lod value of 164 nm. the sensitivity of i − was better than using atomic absorption spectroscopy or chromatography, which was remarkable. furthermore, the i − sensing was satisfying over a broad range ph value of 2-11 [54]. 5. calixarene for neutral molecule chemosensor detection of neutral analyte is also pivotal as carnitine, nucleoside triphosphate, dopamine, tryptophan, and other biomolecules are essential in the metabolism system, and genetic expression [55]. on the other side, organic molecules such as nitroaromatic and polyaromatic hydrocarbons are also critical to be monitored due to their carcinogenic and explosive properties. barata and coworkers used the calix[4]arene-modified carbazole polymer (20) for the sensor of the nitroaromatic explosive compounds in chcl3 media. calixarene 20 showed high sensitivity towards nitroaromatic compounds such as picric acid (pa), 2,4,6-trinitrotoluene (tnt), 2,4dinitrotoluene (dnt), and nitrobenzene (nb). the emission signal of calixarene 20 at 430 nm (λex = 360 nm) was gradually quenched by the addition of these nitroaromatic compounds at a higher concentration. calixarene 20 exhibited the highest sensitivity for tnt detection. the sensitivity of calixarene 20 was shown from the lod value of 3.49, 26.4, 52.7, 107 µm for pa, tnt, dnt, and nb, respectively. the pet mechanism caused the quenching phenomenon as the ligand has a conjugated electron system (electron-rich) and nitroaromatic as an electron-deficient compound [56]. xu and coworkers employed a calix[4]arene modified bithiophene (21) for the detection of amino acids in water. calixarene 21 gave a response for acidic amino acids, i.e., aspartic acid (asp) and glutamic acid (glu), through a fluorescence quenching phenomenon at λem of 566 nm (λex = 400 nm). on the other hand, calixarene 21 gave a response for basic amino acids, i.e., lysine (lys) and arginine (arg), through a fluorescence on at the same λem. these responses were selective over the other amino acids. although a quantitative detection of amino acids using calixarene 21 has not been further studied, calixarene 21 showed a moderately strong complexation with ka value of 7.9 × 10 4 m -1 , 1.4 × 10 5 m -1 , 3.5 × 10 3 m -1 , and 8.1 × 10 3 m -1 for asp, glu, lys, and arg, respectively [57]. qing and coworkers investigated a calix[4]arenes bearing l-tryptophan unit (22) for the detection of amino alcohol compounds in mecn. calixarene 22 showed a weak emission signal at 456 nm (λex = 368 nm). the addition of phenylglycinol and phenylalaninol to the calixarene 22 significantly increased the emission signal due to π-π stacking interactions. the ka value of the complex between calixarene 22 with phenylglycinol and phenylalaninol was found to be 5.6 × 10 2 m -1 and 9.1 × 10 1 m -1 , respectively. however, the quantitative detection of these amino alcohol compounds has not been studied yet [58]. zadmard and coworkers utilized a dimer of calix [4]arene (23) for the detection of nadh in an aqueous solution. nadh is an important biomolecule used in the metabolism system consisted of adenine, nicotinamide, and ribosome residues connected to a phosphate group. calixarene 23 exhibited excellent selectivity for the detection of nadh over other biomolecules (amp, j. multidiscip. appl. nat. sci. 34 adp, atp, nad, nadp, nadph, uracil, cytosine, adenine, and guanine), which was remarkable. the emission signal at 325 nm (λex = 275 nm) was significantly quenched by the addition of nadh only, yielding an lod value of 24.5 nm. calixarene 23 could form a 1:1 complex with a high ka value of 3.7 × 10 5 m -1 [59]. panchal and coworkers employed a bidansylatedoxacalix[4]arene (24) for pendimethalin (pm) detection. this analyte is mainly found in herbicides and considered to be a toxic compound for soil and aquatic microorganisms. the emission signal of calixarene 24 at 466 nm (λex = 251 nm) was quenched by the addition of pm. in contrast, the addition of other pesticides such as propanil, sulfosulforon, pretilachlor, ametryn, tertbutryn, tebuconazole, clodinofop propargyl, atrazine, metalaxyl, simetryn, simazine, thiophenate methyl, and tricyclazole did not show a significant change in fluorescent emission. this result indicated an excellent selectivity of calixarene 24 for pm detection with a lod value of 3.81 nm. calixarene 24 could form a 1:1 complex with pm giving a ka value of 1.3 × 10 5 m -1 [60]. zeng and coworkers used a naphthol appended calix[4]arene (25) for the detection of metolcarb (mc) in mecn media. calixarene 25 showed high selectivity for the detection of mc over various analytes such as carbosulfan, pirimicarb, carbaryl, carbofuran, and isoprocarb. the addition of mc leads to a significant increment of the emission signal at 340 nm (λex = 265 nm) with the lod value of 0.1 µm. the 1:1 complex structure was generated between calixarene 25 and mc during the sensing process with a ka value of 1.2 × 10 5 m -1 [61]. yu and coworkers used the tetrapyridylcalix[4] arene (26) modified metal-organic framework for the detection of glyphosate in meoh:iproh (1:1 v/ v) solvent system. the glyphosate was used for the representative organophosphate pesticide which often used in the agricultural field. the accumulation of glyphosate in water and crops is harmful due to its toxic effect. therefore, the detection of this compound is critical to be established. calixarene 26 yielded a high selectivity toward glyphosate compared to the other interfering analytes such as phenylphosphonic acid, (aminomethyl)phosphonic acid, formic acid, acetic acid, benzoic acid, oxalic acid, and phosphate ions. in addition, the presence of inorganic ions such as k + , na + , zn 2+ , ba 2+ , sr 2+ , mg 2+ , co 2+ , ca 2+ , cl − , br − , i − , so4 2− , and no3 − did not interfere the glyphosphate sensing using calixarene 26, which was remarkable. the emission signal at 329 nm (λex = 281 nm) was significantly enhanced by the addition of glyphosate to give an lod value of 2.25 μm [62]. 6. perspective so far, the development of supramolecular calixarenes as the fluorescent chemosensor agent exhibits a high selectivity and sensitivity for a certain analyte. calixarene serves as an ideal framework for either ion or molecule discrimination. furthermore, a proper modification on either at the upper or lower rim can provide a desired binding site for the particular analyte. the modification depends on the targeted analyte to be detected, which may be cation, anion, or neutral molecule. the different nature of these analytes will influence their interaction with the chemosensor agent. in general, the mechanism for cation detection usually involves the complexation through electrostatic and/or ion-dipole interactions. however, the different types of cation required different binding sites in the chemosensor agent indeed. for example, the detection of cu 2+ is preferable by using the –nh (amino) and oxygen atoms as -oh phenolic and ether moieties. these moieties provide a good binding site for cu 2+ ions. the detection of na + is preferable by using a functional group containing oxygen from ether, ester, and carbonyl moieties. to detect trivalent cations (cr 3+ , fe 3+ , and al 3+ ), the presence of –oh phenolic, ether, ester, and carbonyl groups shall be considered. for ca 2+ and la 3+ , a good detection could be achieved by using a specific triazole group. for hg 2+ heavy metal detection, it is advisable that sulfonamide-based calixarene is employed as the receptor. the presence of imine groups at calixarene was found to be important for zn 2+ and pb 2+ ions detection. on the other side, the sensing mechanism of anion mainly happened through either extended j. multidiscip. appl. nat. sci. 35 hydrogen bond or ion-dipole interactions. since the anion is an electron-rich species, the binding site used in the sensing for certain anions should be positive or partially positive in charge. therefore, the easily protonated functional groups shall be used as the binding site for the anion. for example, the detection of f required the presence of -oh phenolic, amine, and amide. besides these functional groups, the presence of the c=n (imine) group is important for the detection of i . for the detection of aco and h2po4 , the presence of amide and oh phenolic groups is important. another mechanism for anion sensing is through the displacement mechanism involving the formation of ligand-metal complex, which then decomplexed by the presence of anion as the analyte. in this case, the anion should form a stronger interaction with the cation. for example, cn and s 2 strongly interact with cu 2+ ions demonstrating that a cu 2+ -ligand complex is promising for detecting these anions. in the detection of the neutral molecules, the mechanism usually involving the hydrogen bond, ππ stacking, and electrostatic interactions. for example, the detection of nitroaromatic compounds can be achieved by using carbazole moiety as it provides the electrostatic, ch-π, and π-π interactions with nitroaromatic compounds. cyanoacrylic acid moiety could provide a good hydrogen bond interaction with amino acid compounds. amide group is good for providing a suitable electrostatic interaction for a heterocyclic biomolecule such as nadh. for the application in agricultural scope, the dansyl chloride, naphthol, and pyridyl moieties could provide a strong π-π stacking interaction with these analytes. 7. conclusions the design and development of sensitive and selective chemosensor agents have received significant attention from researchers over the past several years. chemosensor agent has been widely applied to detect and quantify cation, anion, or neutral molecules in simulated or real sample analysis. employing an optical chemosensor agent through fluorescence spectroscopy has received special attention due to its merits: easy sample preparation, quick response rate with high precision, and low-cost analysis. calixarenes have good photoluminescence properties with excellent selectivity and sensitivity, thus calixarenes have been extensively used as the chemosensor agent. a proper modification on either at the upper or lower rim can provide a desired binding site for the particular analyte. in general, the mechanism for cation detection usually involving the complexation through electrostatic and/or covalent interactions. on the other hand, the sensing mechanism of anion mainly happened through either hydrogen bonds or dipole-dipole interactions between the anion as an electron-rich species with the positively charged binding site of calixarenes. meanwhile, the sensing mechanism of biomolecules and neutral analytes usually involving the hydrogen bond, π-π stacking, and electrostatic interactions. these results may provide a useful insight into the design and development of chemosensor agents based on calixarene derivatives. author information corresponding author yehezkiel steven kurniawan — ma chung research center for photosynthetic pigments, universitas ma chung, malang-65151 (indonesia); department of chemistry, universitas gadjah mada, yogyakarta-55281 (indonesia); orcid.org/0000-0002-4547-239x email: yehezkiel.steven@machung.ac.id authors krisfian tata aneka priyangga — ma chung research center for photosynthetic pigments, universitas ma chung, malang-65151 (indonesia); orcid.org/0000-0002-4119-2470 keisuke ohto — department of chemistry and applied chemistry saga university, saga8408502 (japan); orcid.org/0000-0002-5284-5019 jumina jumina — department of chemistry, universitas gadjah mada, yogyakarta-55281 (indonesia); orcid.org/0000-0003-2604-7838 http://orcid.org/0000-0002-4547-239x mailto:yehezkiel.steven@machung.ac.id 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(2023). j. multidiscip. appl. nat. sci. in press. https://doi.org/10.47352/jmans.2774-3047.172. manuscript version: accepted manuscript accepted manuscript is “the version of the article accepted for publication including all changes made as a result of the peer review process, and which may also include the addition to the article by pandawa institute of a header, an article id, a cover sheet and/or an ‘accepted manuscript’ watermark, but excluding any other editing, typesetting or other changes made by pandawa institute and/or its licensors” this accepted manuscript is © 2023 the author(s). published by pandawa institute as the version of record of this article is going to be / has been published on a gold open access basis under a cc by 4.0 international license, this accepted manuscript is available for reuse under a cc by 4.0 international license immediately. everyone is permitted to use all or part of the original content in this article, provided that they adhere to all the terms of the license https://creativecommons.org/licenses/by/4.0/. although reasonable endeavors have been taken to obtain all necessary permissions from third parties to include their copyrighted content within this article, their full citation and copyright line may not be present in this accepted manuscript version. before using any content from this article, please refer to the version of record on pandawa institute once published for full citation and copyright details, as permissions may be required. all third-party content is fully copyright protected and is not published on a gold open access basis under a cc by license, unless that is specifically stated in the figure caption in the version of record. view the article online for updates and enhancements. https://doi.org/10.47352/jmans.2774-3047.172 https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.47352/jmans.2774-3047.172 cytotoxic activity of green seaweed halimeda tuna 1 methanolic extract against lung cancer cells 2 3 amir husni1,a); mohamad gazali2,6b); nurjanah nurjanah3,c); rina syafitri4,d), 4 abdul matin5,6e); zuriat zuriat7,f) 5 6 1department of fisheries, universitas gadjah mada, yogyakarta-55281 (indonesia) 7 2department of marine science, teuku umar university, west aceh-23681 (indonesia) 8 3department of aquatic product technology, ipb university, bogor-16680 (indonesia) 9 4department of agribusiness, teuku umar university, west aceh-23681 (indonesia) 10 5department of food processing and engineering, chattogram veterinary and animal sciences university, 11 chattogram-4225 (bangladesh) 12 6institute of marine biotechnology, universiti malaysia terengganu, kuala terengganu-21030 (malaysia) 13 7department of fisheries, teuku umar university, west aceh-23681 (indonesia) 14 15 a)a-husni@ugm.ac.id 16 b)correspondence: mohamadgazali@utu.ac.id 17 c)nurjanah@apps.ipb.ac.id 18 d)rinasyafitri@utu.ac.id 19 e)abmatin@cvasu.ac.bd 20 f)zuriat@utu.ac.id 21 22 orcids: 23 first author : http://orcid.org/0000-0003-3327-9848 24 second author : https://orcid.org/0000-0001-7575-3582 25 third author : https://orcid.org/0000-0001-7925-1782 26 fourth author : https://orcid.org/0000-0002-2334-4397 27 fifth author : https://orcid.org/0000-0003-3029-4989 28 sixth author : https://orcid.org/0009-0000-6296-1788 29 30 acknowledgement 31 32 this research supported by the ministry of education and culture republic indonesia 33 through drtpm dikti grant for the inter-university cooperation research scheme (pkpt) 34 with contract number 037/un59.7/pg.02.00.pt/2022. 35 36 author contibutions 37 38 conceptualization, a.m. and m.g.; methodology, a.m and m.g; software, a.m.; 39 validation, a.h., m.g. and a.m.; formal analysis, a.m. and m.g.; investigation, a.h.; 40 resources, m.g.; data curation, m.g.; writing–original draft preparation, m.g.; writing – 41 review & editing, a.m. m.g. a.m. and n.n., r.s., and z.z.; visualization, .x.; supervision, 42 a.m. and n.; project administration, m.g.; funding acquisition, m.g. 43 a cc ep te d m a n u sc ri pt mailto:a-husni@ugm.ac.id mailto:mohamadgazali@utu.ac.id mailto:nurjanah@apps.ipb.ac.id mailto:rinasyafitri@utu.ac.id/ mailto:abmatin@cvasu.ac.bd mailto:zuriat@utu.ac.id http://orcid.org/0000-0003-3327-9848 https://orcid.org/0000-0001-7575-3582 https://orcid.org/0000-0001-7925-1782 https://orcid.org/0000-0002-2334-4397 https://orcid.org/0000-0003-3029-4989 https://orcid.org/0009-0000-6296-1788 1 conflict of interest 2 3 the authors declare no conflict of interest. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 a cc ep te d m a n u sc ri pt cytotoxic activity of green seaweed halimeda tuna 1 methanolic extract against lung cancer cells 2 3 abstract. lung cancer is a malignant tumor that attacks the lungs generated by carcinogenic 4 free radicals such as cigarette smoke. seaweed contains bioactive compounds that have the 5 potential to reduce cancer-causing free radicals. this study aimed to determine the 6 phytochemical content and cytotoxic activity of halimeda tuna seaweed extract against lung 7 cancer cells (a549). the h. tuna sample was macerated using methanol for 24 h. cytotoxic 8 test of h. tuna crude extract used the mtt test against a549. the crude extract was 9 phytochemically tested and analyzed using gas chromatography–mass spectrometry (gc-ms). 10 the results showed that the h. tuna crude extract had cytotoxic activity against a549 with an 11 ic50 value of 2771 µg/ml. the phytochemical test showed that h. tuna crude extract contained 12 flavonoids and steroids. gc-ms spectra showed the presence of fatty acid compounds 13 including palmitic acid, oleic acid, myristic acid, palmitoleic acid and stearic acid. based on 14 the results can be concluded that h. tuna extract had cytotoxic activity against a549 with low 15 cytotoxicity to be used as a chemo-preventive agent. 16 17 keywords: anticancer; cytomorphology; flavonoid; green seaweed; steroid 18 19 1. introduction 20 21 lung cancer is one of the most dangerous deadly diseases. the death rate from lung 22 cancer worldwide can reach one million people annually; even in indonesia, this disease is 23 ranked 4th in the world [1]. cancer is a metabolic syndrome that is one of the leading causes 24 of death and morbidity worldwide. primary cancer-triggering factors include genetic, 25 epigenetic, environmental, and hormonal that cause mutations [2]. the leading cause of lung 26 cancer is caused by long-term exposure to carcinogenic substances, especially substances that 27 enter through the respiratory process, such as air pollution and cigarette smoke. many have 28 reported that lung cancer is associated with smoking habits. as many as 65% of the risk of lung 29 cancer is suffered by males, especially those aged over 40 years [1]. the most effective cancer 30 treatment, namely chemotherapy, still has various side effects, such as nausea, hair loss, pain, 31 fatigue, and diarrhea. in the long term, these symptoms can harm the patient's quality of life 32 and are at risk of death [3]. 33 a cc ep te d m a n u sc ri pt most asian people use complementary medicine such as dietary supplements, herbal 1 products, and other traditional treatments [4]. one of the herbal medicines or natural 2 ingredients from the fisheries sector is seaweed. seaweed contains various secondary 3 metabolites, such as flavonoids, phenolics, and tannins [5]. seaweed also contains phenolic 4 compounds, polysaccharides, polyunsaturated fatty acids (pufas), proteins, vitamins, and 5 minerals. these compounds show biological activity and have the potential to be used as drugs 6 to ward off cancer, tumors, thrombosis, diabetes, inflammation, and other degenerative 7 diseases [5-8]. these bioactive compounds can be used as antioxidant, anticancer, antibacterial, 8 anti-inflammatory, and antiviral agents [9]. 9 several studies have shown that seaweed from the halimeda genus consists of bioactive 10 compounds, including polyphenols, diterpenes, fatty acids, and sterols, that show anticancer 11 activities [10,11]. one potential seaweed species as an anticancer is the green seaweed 12 halimeda tuna from aceh waters. previous research has been carried out related to the 13 bioactivity of seaweed originating from aceh waters, such as h. macroloba [12], h. opuntia 14 [13], and h. tuna [14]. green seaweed is abundant in indonesia and mainly used in the food 15 sector, however, green seaweed is rarely used in the pharmaceutical and health fields. research 16 shows that green seaweed contains bioactive compounds such as alkaloids, flavonoids, tannins, 17 saponins, and steroids [15]. some of these bioactive compounds can potentially reduce free 18 radicals that cause cancer. several studies have been conducted on the cytotoxic activity of 19 green seaweed, namely boergesenia forbesii, which has high cytotoxic activity so it has the 20 potential to become an anticancer [16]. puc et al. [17] reported that h. tuna has cytotoxic 21 activity against cervical cancer cells (hela), laryngeal cancer cells (hep-2), and 22 nasopharyngeal cancer cells (kb). several species of halimeda sp. contain halimedatrial 23 compounds (diterpenetrialdehyde), which have cytotoxic activity [18], so they have the 24 potential as anticancer. however, the content of seaweed bioactive compounds can vary 25 depending on the type of species, age of harvest, and environmental conditions of the habitat 26 or place of growth [19]. therefore, this study aimed to determine the anticancer activity of 27 green seaweed h. tuna methanol extract against lung cancer cells (a549). 28 29 2. materials and methods 30 31 2.1. materials. the materials used in this study were green seaweed h. tuna, methanol 32 (sigma aldrich,), ethanol, naoh, chloroform, anhydrous acetic acid, hcl, fecl3, nh3, chcl3, 33 h2so4, dragendorfs reagent, meyer's reagent, wagner's reagent, lung cancer cells (a549) 34 a cc ep te d m a n u sc ri pt (bppt, tangerang), rpmi medium, fetal bovine serum (fbs), streptomycin penicillin, 1 doxorubicin, fugizone, formazan, mtt, sds. the tools used in this study included laboratory 2 glasswares, whatman filter paper no.42, rotary evaporator (dlab re100-pro, germany), 3 nitrogen gas evaporator, hot plate stirrer (f20500011 velp arec heating stirrer, italy), elisa 4 microplate reader (heales mb-580), 96-well microplate, and co2 incubator (memmert 5 ico150med, germany). 6 7 2.2. methods 8 9 2.2.1. preparation and identification of samples. samples of green seaweed h. tuna were 10 collected from the coast of lhok bubon, samatiga subdistrict, west aceh district, aceh 11 province. the samples were washed with fresh water to remove the adhering sand and dirt. the 12 wet samples were then dried at room temperature. the wet and dry samples were sent to 13 universitas gadjah mada, yogyakarta. fresh seaweed samples were identified at the plant 14 systematics laboratory, faculty of biology, universitas gadjah mada, to determine the 15 specific type, whereas, the dry samples were cut into 1 cm pieces using scissors. the seaweed 16 was weighed and stored at -20 °c. 17 18 2.2.2. extraction of seaweed. the extraction of h. tuna was carried out according to yang 19 et al. [24] with modifications. samples of dried h. tuna were weighed as much as 250 g. the 20 sample was macerated with 2 l of methanol for 24 h at room temperature and then the filtrate 21 was filtered to remove the remaining residue carried. the filtrate was evaporated using a rotary 22 evaporator at a temperature of 40 °c at 60 rpm. the sample was further treated using nitrogen 23 gas to produce an extract in the form of a more concentrated paste and then extracted in the 24 freeze dryer. 25 26 2.2.3. anticancer activity test. an anticancer activity test was conducted to determine 27 whether the extracted sample had the potential as an anticancer of the lungs. the anticancer 28 activity test was carried out based on the method according to husni et al. [20]. anticancer 29 activity tests included an a549 culture, cytotoxicity, and cytomorphological testing. a549 30 cancer cells were cultured in rpmi medium, then added 10% fbs, streptomycin, penicillin, 31 and fungizone. then the mixture was incubated with 5% co2 at 37°c to obtain an a549 cell 32 culture. furthermore, the cytotoxicity test was carried out using the mtt method. a549 cells 33 were placed on a 96-well culture microplate that included cancer cell treatment with samples, 34 a cc ep te d m a n u sc ri pt positive controls with doxorubicin, and negative controls without sample treatment. then the 1 mixture was incubated with 5% co2 at 37 °c for 24 h. after that, the media was discarded and 2 then it was mixed with 100 µl mtt and incubated again for 4 h. after that, the purple format 3 was dissolved in 100 µl 10% sds and allowed to stand for 12 h at room temperature. cell 4 growth was read using an elisa microplate reader at a wavelength of 570 nm. the percentage 5 of live cells after exposure to fucoidan was calculated using the following equation 1. 6 7 % 𝐿𝑖𝑓𝑒 𝑐𝑒𝑙𝑙 = 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡−𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑚𝑒𝑑𝑖𝑢𝑚 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑚𝑒𝑑𝑖𝑢𝑚 × 100% (1) 8 9 2.2.4. phytochemical assay 10 11 2.2.4.1. flavonoid. the flavonoid test was carried out to determine the content of flavonoid 12 compounds in the sample. five ml of 70% ethanol was added to 0.05 g of the extracted sample, 13 then heated and filtered. then the filtrate was taken, and two drops of 10% naoh were added. 14 if the color changes to yellow or orange, the sample contains flavonoids. 15 16 2.2.4.2 saponin. the saponin test was carried out based on the method as described by lubis 17 et al. [21]. the saponin test was carried out to determine the content of saponin compounds in 18 the sample. a total of 0.05 g of the extracted sample was dissolved into 10 ml of hot distilled 19 water and then shaken vigorously until foamy and cooled. then 1 drop of 2 m hcl was added. 20 if the foam does not disappear, then the sample contains saponins. 21 22 2.2.4.3 steroid and triterpenoid. steroid and triterpenoid tests were carried out as follows: 23 chloroform was added to 0.05 g of the extracted sample to the drip plate and then allowed to 24 dry. then ten drops of anhydrous acetic acid were added and stirred until homogeneous. then 25 three drops of 96% sulfuric acid were added. if it is blue or green, then the sample contains 26 steroids. if it is red or purple, the sample contains triterpenoids [21]. 27 28 2.2.4.4 tannin. the tannin test was carried out based on the method as described by 29 widowati et al. [22]. a total of 0.1 g of sample was dissolved in 10 ml of hot distilled water 30 and filtered. then 5 ml of the sample filtrate was added with 3 drops of 1% fecl3. if the results 31 show a blue-black color, the sample contains tannins. 32 33 a cc ep te d m a n u sc ri pt 2.2.5 gas chromatography-mass spectrometry (gc-ms) analysis. gc-ms analysis was 1 performed to identify the profile of bioactive compounds in h. tuna methanolic extract. the 2 gc-ms analysis was carried out based on the method as described by hidayah [23]. the 3 sample to be analyzed by gc-ms was first dissolved in 5 ml methanol. then the gc-ms 4 analysis was carried out by injecting the sample into the injection port at a temperature of 290 5 °c. the volatilized sample was carried by helium gas at a flow rate of 1 ml/min through the 6 gc column. the initial injection temperature was 80 °c and increased by 10 °c/min with a 7 final temperature of 300 °c. compounds are detected in the ms system by colliding 8 compounds with electrons to form ionized molecules and record fragmentation patterns [24]. 9 10 2.3 statistical analysis. the percentage data of inhibition was then converted to a linear 11 regression equation calculating the ic50 value. the ic50 values of the linear regression results 12 of each sample were statistically tested using sovs (one-way anova) and tukey hsd test 13 with a 95% confidence level. 14 15 3. results and discussions 16 17 3.1. yield of extract. the yield is the result of a comparison between the total mass of h. 18 tuna extract in the form of paste with the initial mass of h. tuna in the form of dried seaweed 19 [30]. the yield of h. tuna methanolic extract obtained was 0.17±0.04%. the methanol extract 20 of h. tuna had a lower yield when compared to the methanol extract of h. macroloba (0.34%) 21 and the ethyl acetate extract of h. macroloba (0.28%), but higher than the n-hexane extract of 22 h. macroloba (0.04%) [25]. gazali et al. [12] also reported that the yield of ethanol extract of 23 h. macroloba (2.32%) was higher than the yield of ethyl acetate extract (1.26%), and n-hexane 24 extract (1.03%). the difference in yield can be caused by the type of solvent and different 25 species. different solvents can affect the yield due to the level of polarity. according to muzaki 26 et al. [30], the yield value decreases along with the decrease in the polarity of the solvent. in 27 addition, the solvent will attract bioactive compounds that have the same polarity. the type of 28 seaweed species also affects the yield because it depends on its compounds. according to 29 purwaningsih and deskawati [26], the content of bioactive compounds in seaweed is 30 influenced by the type of species, harvest season, harvest age, and geographical location. 31 32 a cc ep te d m a n u sc ri pt 3.2 anticancer activity. h. tuna methanolic extract was assayed for its cytotoxic activity 1 against a549. the inhibition of the growth of a549 by h. tuna methanolic extract and 2 doxorubicin is presented in figure 1 while their ic50 is shown in table 1. the morphological 3 attributes of the cells were monitored under an inverted microscope after the cells were 4 incubated. the morphological attributes of a549 that were exposed and not exposed to h. tuna 5 extract are illustrated in figure 2. a cytotoxicity test was carried out on h. tuna methanolic 6 extract against a549 to determine whether the sample had potential as an anticancer and 7 directly affected cell death [17]. the mtt test is a method that can be used to determine the 8 toxic properties of a compound. the mtt test results of h. tuna extract, and doxorubicin on 9 a549 (figure 1) showed that the dose given to cancer cells was directly proportional to the 10 inhibition of cancer cell growth. h. tuna extract with a dose of 500 μg/ml could hinder the 11 growth of cancer cells by 28.72% while doxorubicin (as a standard drug) at a dose of 14 μg/ml 12 could hinder the growth of cancer cells by 39.68% (figure 1). this is because doxorubicin is a 13 widely used drug for anticancer chemotherapy. however, doxorubicin works non-selectively 14 and is toxic to cancer cells and normal cells [27]. 15 16 17 figure 1. effect of concentration of h. tuna and doxorubicin on inhibition of proliferation of 18 lung cancer cell a549. 19 20 prasetyaningrum et al. [28] indicated that the cytotoxicity of a substance based on its ic50 21 is divided into three levels: potential cytotoxic (ic50 <100 μg/ml), moderate cytotoxic (100 22 μg/ml < ic50 <1000 μg/ml), and low cytotoxic (ic50 >1000 μg/ml). furthermore, according 23 to the national cancer institute [29], a compound can be classified as a strong anticancer agent 24 if its ic50 is <20 μg/ml. the cytotoxicity test on the crude extract of h. tuna showed low 25 cytotoxic values (ic50 value of 2771 µg/ml). a substance with low cytotoxicity can be used 26 0 10 20 30 40 50 0 100 200 300 400 500 in h ib it io n a c ti v it y ( % ) concentration (μg/ml) h. tuna doxorubicin a cc ep te d m a n u sc ri pt as a chemo-preventive agent. the chemo-preventive ability indicates that the crude extracts of 1 h. tuna can be used to prevent and hinder the growth of cancer cells and also trigger apoptosis. 2 previous research reported the cytotoxicity of brown seaweed fucoidan extracted from 3 turbinaria conoides species against a549 with ic50 of 396.46 μg/ml [30]. polysaccharide 4 from caulerpa taxifolia showed anticancer acitivity against a549 with a relative ic50 of 45.44 5 μg/ml [31]. methanol extract of brown algae hormophysa cuneiformis has anticancer activity 6 against a549 with ic50 of 40.97 μg/ml [32]. factors that can affect the content and activity of 7 bioactive metabolites include sampling location or habitat, genetic variation, sampling time, 8 evolution, and environmental conditions [33]. 9 doxorubicin is an anticancer medicine and an important agent for the therapy of malignant 10 breast cancer [34]. the anticancer action of doxorubicin has been described with various 11 molecular pathways, covering the interaction mechanism of doxorubicin with dna, dna-12 related enzymes, and cell membranes [35]. another study has shown that cladosiphon 13 okamuranus fucoidan has strong antiproliferative and apoptotic reactions on mcf-7 cells in 14 certain doses and does not affect normal cell proliferation in human mammalian epithelial cells 15 [36]. the cell pattern is a process that requires high energy and involves four sequential stages 16 that change from the stationary stage (g0 stage) to the proliferation stage (g1, s, g2, and m 17 stage) and return to rest [37]. fucoidan increases the population of hepatocarcinoma (huh7) 18 cells at the g0/g1 stage and decreases their population at the s stage; this result indicates that 19 fucoidan can induce the cell pattern to persist at the g0/g1 stage [38]. 20 21 table 1. ic50 values of h. tuna extract and doxorubicin against cancer cells a549 22 sample ic50 (µg/ml) h. tuna extract 2771a doxorubicin 24.13b a/bdifferent letters show a significant difference (p<0.05) 23 24 the differences in the morphological attributes of a549 to h. tuna extract and not exposed 25 to h. tuna extract are illustrated in figure 2. the morphological characteristics of a549 26 exposed to h. tuna extract and the control cells not exposed to h. tuna extract differed. the 27 morphological attributes of mcf-7 cells in the control cells not exposed to h. tuna extract were 28 observed as an irregular polygonal and attached to the substrate. the morphological 29 characteristics of the cells that were exposed to h. tuna extract varied, that is, the cells shrank, 30 were round, and had limited distribution patterns compared with those of the control cells. this 31 a cc ep te d m a n u sc ri pt change in shape was consistent with that observed by kim et al. [39] who stated that mc3t3 1 osteoblast cells exposed to fucoidan for 4 h have altered morphological characteristics, i.e., 2 from an irregular shape to a round form with smaller sizes. 3 4 a b c figure 2. morphology of a549 lung cancer cells without sample treatment (a), given a 5 sample of h. tuna extract 250 µg/ml (b), and given a standard doxorubicin 14 µg/ml (c). 6 7 3.3 phytochemical content. the phytochemical test aims to identify chemical compounds 8 in samples such as flavonoids, steroids, saponins, tannins, and alkaloids. many of these 9 chemical compounds are found in seaweed. the results of the phytochemical test were shown 10 in table 2. 11 12 table 2. phytochemical analysis of h. tuna crude extract 13 phytochemicals result indicator flavonoid ++ yellow/orange color steroid +++ blue-green color triterpenoid red – purple color saponin foam alkaloid + orange precipitate tannin blue-black color + : low, ++ : moderate, +++ : high 14 15 according to nome et al. [15], flavonoids were found in almost all types of green 16 macroalgae but with different levels, such as codium sp., caulerpa sp., and ulva sp. similarly, 17 the steroids found in the green macroalgae caulerpa sp., halimeda sp., enteromorpha sp., and 18 codium sp. alkaloids are also found in green macroalgae such as ulva sp. and caulerpa sp., 19 but little was found in halimeda sp., enteromorpha sp., and codium sp. gazali et al. [40] 20 reported that alkaloids, flavonoids, saponins, and tannins were found in the macroalga 21 chaetomorpha crassa. based on the research of widowati et al. [22], gracilaria salicornia 22 contains flavonoids, saponins, and steroids, halimeda gracilis contains steroids and saponins, 23 a cc ep te d m a n u sc ri pt and h. macroloba contains flavonoids and steroids. gazali et al. [12] reported that h. opuntia 1 seaweed contains alkaloids, steroids, saponins, flavonoids, phenols, and tannins. gazali et al. 2 [13] reported that the phytochemical test results showed that the h. tuna fractions were positive 3 for alkaloids, flavonoids, steroids, and phenol hydroquinone compounds. flavonoids are 4 secondary metabolites with anticancer activity [41] because these compounds contain 5 quercetin, genistein, or flavopiridol which can be used as cancer drugs [42]. flavonoids as 6 anticancer have a mechanism of inhibition of dna topoisomerase i/ii activity, decreased 7 expression of bcl-2 and bcl-xl genes, and activation of endonucleases [43]. flavonoids also 8 have the biological ability to chelate metals, inhibiting cancer cell growth [44]. flavonoids are 9 polar and are mostly produced from green seaweed, so these compounds are generally easily 10 soluble in polar solvents such as methanol [45]. 11 steroids are non-polar secondary metabolites, so they are easily extracted by polar solvents 12 such as methanol [15]. steroids have anticancer activity as these compounds have aromatase 13 enzymes and sulfatase inhibitors that can inhibit the growth of cancer cells [46]. steroids, as 14 anticancer agents, damage mitochondrial membrane permeability in cancer cells and cause cell 15 death or necrosis [47]. in addition, steroids can also capture reactive species such as superoxide 16 and chelate metals [48]. the content of chemical compounds in seaweed can be influenced by 17 environmental factors where it grows because the bioactive compounds formed are a natural 18 response to environmental conditions where they grow, resulting in various types of chemical 19 compounds. the ability of seaweed to produce secondary metabolites that are bioactive 20 compounds can occur due to extreme environmental conditions [15]. 21 22 3.4 gc-ms analysis. gc-ms analysis showed a gc spectra chromatogram with seven 23 peaks (figure 3) representing the bioactive compounds interacting with the gc column. the 24 peak obtained was only a little and not too high, with the results of comparison with the 25 database having a slight similarity. the bioactive activity and utilization of compounds were 26 obtained from the ncbi web and previous studies. compounds belonging to the flavonoid 27 group were flemichapparin a [49]. the steroids identified in the extract consisted of stigmasta, 28 androst-4-ene-3,17-dione, estra-1,3,5(10)-trien-17-one, 5-alpha-androstan-17-one, and 1-29 docosanol [50]. some compounds that include fatty acids include palmitic acid, hexadecanoic 30 acid, octadecanoic acid, lauric acid, 4-hexenoic acid, and dodecanoic acid [46]. the list of 31 information on the identified compounds and the activity of the metabolite compounds from 32 the h. tuna extract is explained further in table 3. 33 34 a cc ep te d m a n u sc ri pt table 3. results of identification of compound components of h. tuna methanol extract 1 peak rt area (%) component group activity si 1 12.308 32.34 stigmasta5,22-dien-3ol steroid antioxidant, antimycobacterial (tuberculosis), anticancer, inhibition of chemocarcinogen [51] 19 2 13.572 7.73 androst-4ene-3,17dione steroid osteoporosis, antiinfectives, hyperglycemia (antidiabetic) [52] 21 3 18.010 5.03 1-docosanol steroid antiviral [53] 57 4 19.683 0.01 1hexadecanol fatty alcohol antioxidant, antimicrobial [54] 61 5 20.441 27.41 14-beta-hpregna steroid cancer prevention [55] 63 6 20.883 6.78 dodecanoic acid fatty acid antimicrobial, relieve neuro-inflammatory [56] 34 7 21.065 20.73 hexadecanoic acid fatty acid anti-inflammatory, antiviral, antioxidant [57] 70 rt: retention time, si: similarity index 2 3 the activity of volatile compounds, as listed in table 2, many compounds have anticancer-4 related bioactivity. the main compound with a large percentage of area is found at peaks 1, 5, 5 and 7, with an area of 32.34%, 27.41%, and 20.73%, respectively. according to singla and 6 dubey [58], in predicting compounds using gc-ms, if the similarity value is low (si<90%) 7 then the component should not be considered because it is less accurate. in this study, the 8 compound with the greatest similarity index was 70, so the compound with the largest 9 percentage area and the greatest similarity was used. 10 a cc ep te d m a n u sc ri pt the active compound in peak 1 is stigmasta-5,22-dien-3-ol, with activities including 1 antioxidant, antimycobacterial (anti-tuberculosis bacteria), anti-inflammatory, anticancer, and 2 inhibition of chemocarcinogens. the compound stigmasta-5,22-dien-3-ol belongs to the 3 stigmasteroid group [59]. stigmasta-5,22-dien-3-ol has been found in the genus halimeda 4 seaweed, precisely in h. opuntia, with a percentage of 54.74% as the most dominant compound 5 [60]. in this study, the stigmasta compound only had an si of 19 so it might not be accurate 6 and have little effect on anticancer activity. 7 the active compounds in peak 5 include 14-beta-h-pregna with a similarity of 63, which 8 has antidiabetic and cancer-preventive properties. compound 14-beta-h-pregna belongs to 9 steroids [61]. compound 14-beta-h-pregna was found with an area of 55% in green seaweed 10 extract chlorella vulgaris [62]. compound 14-beta-h-pregna is a component of the medicinal 11 plant verbascum pseudoholotricum or mullein with a similarity of 98. mullein has antioxidant, 12 anti-inflammatory and anti-bacterial activity [63]. 13 compounds in peak 7 include hexadecanoic acid, octadecanoic acid, dodecanoic acid, and 14 octadecane, a group of fatty acids. fatty acid bioactivity includes anti-inflammatory, antiviral, 15 antioxidant, antimicrobial, and antibiotic [64]. fatty acids function as antioxidants so that they 16 can reduce reactive oxygen species and act as preventive agents for diseases caused by reactive 17 oxygen species, such as cancer [37]. 18 research by nazaruddin et al. [60] on the gc-ms test proved the presence of hexadecanoic 19 acid in h. opuntia. the hexadecanoic acid in halimeda has antioxidant effects and is cytotoxic 20 against the colorectal cancer cell line hct-116. the retention time of hexadecanoic acid in 21 this study was 50.91 min. nazaruddin et al. [11] researched h. macroloba using the gc-ms 22 test with the shimadzu qp2010 plus gc-ms system. one of the compounds found is 23 hexadecanoic acid. hexadecanoic acid retention time at two different peaks had values of 24 21.039 and 20.548 min, respectively. the rt value of the gc-ms test on h. tuna in this study 25 for hexadecanoic acid had a retention time of 21.065 min with a similarity index of 70 so it 26 was more similar to the results of the gc-ms test on h. macroloba. 27 28 a cc ep te d m a n u sc ri pt 1 figure 3. chromatogram of h. tuna methanolic extract 2 3 the h. tuna extract contains several fatty acid compounds. this is because, in addition to 4 secondary metabolites, seaweed also contains primary metabolites such as protein, 5 carbohydrates, fat, crude fiber, macro minerals, and several vitamins. differences in the content 6 of chemical compounds in seaweed can be influenced by the type of species and their habitat 7 [15]. secondary metabolites have been shown to have high bioactivity. however, fatty acids 8 are also known to have antioxidant activity [65], so they are thought to have the potential to 9 have cytotoxic activity. according to asbanu et al. [66], several fatty acids have antioxidant 10 bioactivity such as octadecanoic acid (stearic acid), hexadecanoic acid (palmitic acid), 11 tetradecanoic acid (myristic acid), and 9-octadecenoic acid (oleic acid). in general, the 12 halimeda genus shifts the production of protein and fat primary metabolites to increase the 13 production of halimedatrial and halimedatetraacetate secondary metabolites, so that the 14 bioactive compounds of these secondary metabolites are higher than their primary metabolites 15 [67]. however, this is also influenced by environmental conditions where it grows, resulting in 16 a variety of compound content [68]. in addition, the solvent used is methanol, which is a 17 universal polar solvent so that it can attract all compounds, both polar and non-polar 18 compounds, such as fats [69]. methanol is also one of the most widely used solvents in the 19 extraction process of organic compounds such as oils or fats [70], so fatty acids can be carried 20 away in the extraction process. 21 22 23 24 25 a cc ep te d m a n u sc ri pt 4. conclusions 1 2 h. tuna methanolic extract was obtained in 0.17±0.04% yield. h. tuna extract had cytotoxic 3 activity against lung cancer cells (a549) with ic50 2771 µg/ml and potentially can be used as 4 a chemo-preventive agent. based on cytomorphological observations, changes in the 5 morphology of cancer cells were seen before and after being treated with h. tuna extract 6 samples. metanolic extract of h. tuna have contents palmitic acid, oleic acid, palmitoleic acid, 7 myristic acid, and stearic acid. 8 9 references 10 11 [1] j. joseph and l. w. a. rotty. 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"bio-based solvents: an emerging generation of fluids for 22 the design of eco-efficient processes in catalysis and organic chemistry". chemical 23 society reviews. 42 (24): 9550-70. 10.1039/c3cs60241a. 24 25 a cc ep te d m a n u sc ri pt https://doi.org/10.1016/j.jpba.2009.05.012 https://doi.org/10.1590/s0102-695x2011005000010 https://doi.org/10.15294/ijcs.v9i2.33525 https://doi.org/10.1007/s00338-014-1230-6 https://doi.org/10.12928/pharmaciana.v5i1.2283 https://doi.org/10.1039/c3cs60241a 55 journal of multidisciplinary applied natural science vol. 3 no. 1 (2023) review article lepidopteran insects status and diversity: a review ahasan ullah khan*, nilufa yasmin poly, shimanta dutta, and ferdous alam received : july 9, 2022 revised : october 17, 2022 accepted : october 17, 2022 online : october 25, 2022 abstract the second largest and most well-known insect order lepidoptera includes both butterflies and moths in the world. this study was started based on secondary information from present literature from different countries on the flora and fauna. so far, much study work was completed on this subject but those workers were not existing to the extension employees, policymakers, and the public in an efficient way to date. in this review paper, we can give the recent data on etymology, distribution and diversity, external morphology, different morphological parts of caterpillar, internal morphology, digestive system, circulatory system, reproduction and growth, respiratory system, and growth and role of the olfactive process in larval and adult lepidopteran insects. there are over one hundred and eighty thousand lepidoptera species described, divided into one hundred and twenty-six families and forty-six superfamilies, and the total number of living beings was labeled as about ten percent. a result found is that the species of moths is about one hundred sixty thousand compared to one hundred seventy thousand five hundred species of butterflies in the world. butterflies are an important category of insects that can function as sensors of environmental change. butterflies are insects from the order lepidoptera's macro-lepidopteran clade rhopalocera. the study focused on the lepidopteran insects and the diversity of the world. it presents the review of research imperfect data on specific knowledge in diverse agroecological zones. this study acknowledged investigating breaches in different countries of the world. it also provides information on lepidopterans as alternative food and financial source of the world. it also helps in the sustainable agricultural field. keywords lepidopteran insect, butterfly, moth, larvae, diversity, status, food 1. introduction the insects are significant for the reason of their ecological role, effect on agriculture, diversity, social well-being, animal, and usual resources. those insects are universally found on the planet and they are the most common faunas or organisms found in the world still now. the insects have been named more than fifteen luck classes in the scientific community. this number of insects is three times higher than the other animals. even so, many researchers said that the insects that have been identified and given designations are only a small part of the insects in wildlife. many have not yet been exposed even some are recorded as a pest. pests are animals or plants that are damaging to humans or human worries. it is an unhelpful insect or another animal that spells crops, food, livestock, etc. some pests are attacked the home garden plant [1][2]. some insects attract in the bean crop and pod [3]–[6], cinnamon plant and fruit [7], chrysanthemum crop and flower [8]; jackfruit plant and fruit [9][10], mango plant and fruit [11], lemon plant parts [12], okra plant and fruit [13][14] and so on [15]–[17]. the term is mostly secondhand for individuals that harm crops, cattle and animals, and forests or cause pain to people, especially in their town. the common insect orders are coleoptera, diptera, lepidoptera, hemiptera, homoptera, hymenoptera, isoptera, mantodean, mecopteran, neuropteran, odonatan, orthoptera, phasmida, plecopteran, psocoptera, siphonaptera, thysanoptera, thysanuran, and trichopteran. in this paper, we are trying to describe the status and diversity of lepidoptera insects. the insect order lepidoptera is the secondlargest insect order and this insect order consists of moths and butterflies (each is titled lepidopterans). about one hundred and eighty thousand species of the lepidoptera are defined, in one hundred and twenty-six families and forty-six superfamilies ten percent of the whole defined classes of dwelling animals [18][19]. it is one of the most prevalent insect orders and widely decipherable insects in the present global [20]. the insect of lepidoptera display many disparities in the elementary body form that have changed to advantage benefits in life and circulation. the latest guesses endorse the insect order can also have further classes than a previous idea, and is many of the quatern maximum classes orders, together with the hymenoptera, publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2022 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.140 open access file:///d:/pandawa%20institute/1.%20journal%20of%20multidisciplinary%20applied%20natural%20science/issue/vol%203%20(2023)/55%20khan%20et%20al/accepted%20manuscript%20140-article%20text-762-1-6-20221017.docx#page20#page20 https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.140 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.140&domain=pdf&date_stamp=2022-10-25 https://creativecommons.org/licenses/by-sa/4.0/ j. multidiscip. appl. nat. sci. 56 diptera, and coleoptera [21]. lepidopteran insect types are considered by more than three consequent structures, a few of the greatest seeming being the scales masking their bodies and wings a trunk. the scales change, the “hair” becomes stronger and the moths and butterflies provide their awesome kind of color and design. nearly all lepidopteran insect classes have a few shapes of membrane-forming wings, besides some which have condensed wings or are wingless. lepidopteran insects of butterflies and moths and almost different insect species are holometabolous which means they go through an entire mutation. the adult insects are mating and lay eggs near or on host plants for the larvae. the first stage of larvae are normally referred to as caterpillars, and are wholly extraordinary from their adult butterfly or moth bureaucracy, taking a cylinder-shaped body with properly-evolved mandible mouth parts, six thoracic legs, a head, and from none up to ten prolegs. when they grow, these caterpillars trade entrance, going complete a sequence of ranges referred instars. as soon as they mature, the first stage of the larva grows into a pupa. several butterflies and plenty of moth classes rotation a cocoon or silk case earlier in pupating stage, while other insects do not, in its place of successful subversive. a butterfly pupa termed a cocoon, has solid skin and pores, generally without a chrysalis. as soon as the pupa has done its mutation, a sexually developed adult insect emerges. the lepidoptera insects have over hundreds of thousands of centuries, progressive an extensive variety of flying parts of wing patterns and hues vacillating from drab moths akin to the connection in the insect order of trichoptera to the glossily painted and complicated decorated butterflies [19]. for that reason, this is the most identified and widespread insect of moth and butterfly with human beings intricate in the study, remark, gathering, nurture, and business of these insects. someone who studies or gathers this insect order is mentioned as a lepidopterist. moths and butterflies play a vital function confidential in the herbal surroundings as food within the meals chain and as pollinators, equally, their larvae are measured very complex to foliage in the agricultural field, as their major supply of nourishment is frequently live in the plant substance. in many classes, the female insect can additionally yield from two hundred to six hundred eggs, while in others; the variety can also technique thirty thousand eggs in a single day. the young insect crosshatching from these offspring can reason harm to massive portions of vegetation. many butterflies and moths classes are of financial concentration with the aid of their function as silk, pollinators, or pest classes. the objectives of this study are to know the about lepidoptera order of insects, the life cycle of lepidoptera insects, and the ecological beneficial and harmful activities of this table 1. levels of factors in the bbd for the regeneration of spent mac. characters butterfly moth antennae smoothed sticks on the trimmings slim or light body thin and smooth think and fuzzy active during the day during the night color colorful dull pupal stage chrysalis cocoon wings held vertically when resting held flat against the body when resting adult butterfly and moth https://en.wikipedia.org/wiki/scale_(anatomy) https://en.wikipedia.org/wiki/instar https://en.wikipedia.org/wiki/pest_(organism) j. multidiscip. appl. nat. sci. 57 order. 2. methodology a research study of the presented journal literature, reports, blogs, newspapers, and books was shown to evaluate the recent level of research on the lepidopteran insect status and diversity. keyword examinations in the web of science database, google scholar, google, a full-text search of the science direct database, and research gate, were conducted. the individual announcement was also castoff to get data from administration actions and non-governmental ngo groups. 3. etymology lepidoptera insects the term lepidoptera turned into castoff in 1746 by carl linnaeus in his wildlife species [22]. the term is resulting from the greek word λεπίς, gen. λεπίδος ("scale") and πτερόν ("wing") [23]. now and then, the word rhopalocera is castoff for the clade of wholly of lepidopteran insect of butterfly classes, derivative from the momentous greek ῥόπαλον (rhopalon) and κέρας (keras) [24] which means "club" and "horn", separately, imminent from the form of the feelers of lepidopteran insect of butterflies. the roots of the usually common names "butterfly" and "moth" are various and frequently unclear. the english expression butterfly is from the antique english language, with many differences in presaging. aside from that, the beginning is unidentified, although it may be derivative from the light-yellow coloration of many classes of wings signifying the color of butterfly insects [25]. the classes of heterocerc are usually titled moths insects. the etymological backgrounds of the phrase "caterpillar", the initial stage larval of moths and butterflies are since the primary 16 th century, from middleenglish catirpel, catirpeller, likely a modification of antique french catepelose. 4. distribution and diversity of lepidoptera insects the lepidoptera insects recorded the various record positive corporations of different insects. they are discovered on all lands, excluding the antarctic continent, and occupy all worldly locales starting on or after wasteland to the forest, as of coastal plains to massif hills, but are practically continually linked with advanced plant life, expressly flowering plant (flowering plant life) [26]. many of the maximum northern living class of moths and butterflies are the insect of arctic apollo (parnassius arcticus), which is observed within the cold round in northeast russia, at a height above sea level of 1500m [27]. table 2. variety of lepidopteran insects in each faunal area. figure 1. a) the mouth parts of the caterpillar with the face presentation; b) different portions of an adult butterfly. b a items palearctic nearctic neo-tropic afrotropic indonesia-australian-malayasia number of species 22,465 11,532 44,791 20,491 47,286 https://en.wikipedia.org/wiki/heterocera j. multidiscip. appl. nat. sci. 58 a few lepidopteran classes showcase synergetic, phoretic, or sponging lifespans, residing our figures of creatures slightly more than the environs. sloth moths called coprophagous pyralid moths such as cryptoses choloepi and bradipodicola hahnel are uncommon in that those insects can be solely observed occupying the fur of apathies, animals located in the usa [28][29]. lepidopteran insect bagworm (tinea pellionella) moth insect has been noted as nursing on horny cells and had reared as of the buzzers from steers. the maggot of insect of zenodochium coccivorella is an inner organism of the coccid kermes types. the class of lepidopteran has been noted as background in natural matters or trash which include night bird bits, bat caverns, honey combs, or unwell fruit [28]. in 2007, there has been a kind of one hundred and seventy-four thousand and two hundred fifty lepidopteran classes defined, with butterflies and heads valued to incorporate about seventeen thousand nine hundred fifty, and moths’ creation up the break [18]. the significant popularity of lepidoptera is to be observed inside the tropics, but a vast variety exists on most lands. american countries have over seven hundred species of butterflies and over eleven-thousand types of moths [30][31], while about four hundred species of butterflies and fourteen-thousand kinds of moth insects are stated from sovereign country australia [32]. the range of lepidopteran insects in all faunal regions has been forecast founded partially on real sums from the works, in part on the valentine catalogs in the national museum of antiquity (washington), and natural history museum (london) somewhat on approximations of coleoptera [20]. 5. peripheral geomorphology of lepidoptera insects the insect order of lepidoptera is morphologically prominent from further orders chiefly by the incidence of scales on the exterior elements of the appendages and body, in particular the flying parts of the wing. moths and butterflies are verified in different sizes from microlepidoptera to visible animals with a wingspread greater than 25cm, together with the emperor atlas moth and the butterfly [33]. lepidopterans insects endure a tetrad period (figure 9) reality cycle like a different size of the egg, visible size of caterpillar figure 2. larval morphology overview (a. head, b. thorax, c. abdomen, 1. prothoracic shield, 2. spiracle, 3. true legs, 4. midabdominal prolegs, 5. anal proleg, 6. anal plate, 7. tentacle, a. frontal triangle, b. stemmata (ocelli), c. antenna, d. mandible, e. labrum). file:///d:/pandawa%20institute/1.%20journal%20of%20multidisciplinary%20applied%20natural%20science/issue/vol%203%20(2023)/55%20khan%20et%20al/accepted%20manuscript%20140-article%20text-762-1-6-20221017.docx#page21#page21 j. multidiscip. appl. nat. sci. 59 or larva, chrysalis or pupa, and imago, adult and display numerous distinctions of the basic frame form, that spring those faunae sanctifications for varied life and surroundings. 5.1. head the head senses the mouthpieces of any insect, and those insect’s aperture portions are observed. similar to the adult insect, the first stage of the larva has a sclerotized, or hardened head tablet (figure 1)  [34]. accurate here, maximum insects contain the chaetosema, and two compound eyes, elevated color spots or groups of bodily bristles sole to lepidopteran insects, ensue, though several taxa have misplaced one or equally of these colorful spots. the insects' antennae have a huge disparity in form amongst classes and even between diverse male and female insects. the antennae of butterfly insects are stereotypically filiform and formed like those of the captains are curved, clubs, at the same time as the ones of moths have flagellar parts diversely distended or divided. a few moths have distended antennae or ones which can be curved and pointed on the ends [35]. the extreme grease is altered and method an extended snout. the snout includes 1-5 segments, typically stored wound up beneath the cranium by minor muscle mass when it isn't always getting castoff to sucking up juice from florae or further waters. a few basal moth insects immobile have mouths or discrete shifting lips, similar to their descendants, and these shape the circle of the intimate micropterigidae  [34]–[36]. the first stage of larvae, named caterpillars, have a hardened head pill. in the young insect, stage caterpillars have an absent snout and have detached mastication mouth  [34]. these mouthparts of insects, referred to as mouths, are hand-me-down to bite up the shrub depending on what the young insect eats. the inferior labium, or jaw, is liable, however, whitethorn additionally conveys a spinneret, a body part used to make silk. the skull is manufactured from huge side parts, and the apiece consumes an ellipse of up to 6 modest eyes [35]. 5.2. different morphological parts of caterpillar maximum lepidopteran insect larvae, including all of the pest species treated here, are characterized by a grouping of the succeeding typescripts: a. head, b. thorax, c. abdomen, 1. prothoracic shield, 2. spiracle, 3. true legs, 4. midabdominal prolegs, 5. anal proleg, 6. anal plate, 7. tentacle, a. frontal triangle, b. stemmata (ocelli), c. antenna, d. mandible, e. labrum (figure 2). 5.2.1. thorax the thorax is manufactured of a trio of merged parts, the prothorax, metathorax, and mesothorax, apiece with a couple of limbs. the primary part comprises the first couple of limbs. in a few males of the butterfly family nymphalidae, the forelimbs are significantly decreased and are not rummagesale for walking or resting [35]. the six legs are enclosed with scales. lepidopteran insects additionally have olfactory body parts on their bottoms, which aid the butterfly in "savoring" or "smelling" out its nourishment [37]. within the young insect shape, there are six true limbs, with up to twenty-two stomach legs (generally eight) and crochets, termed apical hooks [26]. the four wings are discovered on the central and tertiary sections, or mesothorax and metathorax correspondingly. within the latest types, the insect wings of the additional section are a whole lot greater pronounced, although a few further rude bureaucracies have addition-sized sections of both sections. the wings are protected in balances organized like sands, that shape a strange form of shades and styles. the mesothorax has supplementary influential muscle groups to drive the butterfly or moth over the airborne, with the wing of this section having a stouter strain erection [35]. the major great family, the noctuid has its wings changed to behave as tympanal or attending figure 3. caterpillar prolegs on papilio machaon. file:///d:/pandawa%20institute/1.%20journal%20of%20multidisciplinary%20applied%20natural%20science/issue/vol%203%20(2023)/55%20khan%20et%20al/accepted%20manuscript%20140-article%20text-762-1-6-20221017.docx#page21#page21 file:///d:/pandawa%20institute/1.%20journal%20of%20multidisciplinary%20applied%20natural%20science/issue/vol%203%20(2023)/55%20khan%20et%20al/accepted%20manuscript%20140-article%20text-762-1-6-20221017.docx#page21#page21 j. multidiscip. appl. nat. sci. 60 to organ [34]. the young insect has a lengthened, gentle frame which can have tresses like or extra prognoses, six authentic legs, without any to twenty-two of stomach legs (typically eight), and hooklets, entitled apical hooks [26]. the thorax generally has two legs in each section. the thorax is as well wrinkled by numerous spiracles on each mesothorax and metathorax, besides some marine types that alternatively have an arrangement of branches [35]. 5.2.2. abdomen the stomach is less sclerotized than the pectus and consists of ten sections with casings in among, taking into consideration articulated motion. the sternum, within the first part, is slight in a few households and is inattentive in others. the previous 2 or 3 parts form the outside parts of the class sex body part. the genitals of lepidopteran insects are very diverse and are frequently in the handiest manner of distinguishing among types. the male genital insects consist of a valva, that's frequently great, as it's rummage-sale to understand the female insect throughout pairing. the female insect genitalia encompasses three separate segments. the female insect moths have one sex body part, which is rummage-sale for coitus and as an ovipositor or egg-laying body part. approximately ninety-eight percent of moth classes have a discrete body part for pairing and an outside duct that transmits the semen from the male insect [35]. the stomach of the young insect has eight prolegs, usually situated on the 13-16 parts of the stomach, and a distinct couple of prolegs by the anus, that have a couple of miniature catches referred to as hooks (figure 3). these resources are fascinating and strolling, mainly in classes that deficiency several prolegs. in a few months, these prolegs can be on every part of the figure, at the same time as the prolegs may be vague in a different group that might be greater tailored to boring and living within the sand [35]. 5.2.3. scales the lepidopteran insect are contain its head, wings, and components of the chest and stomach and the lepidoptera are protected with tiny scales, a chin from the other order stems from its designation. maximum scales are lamellar or bladelike and involved a pedicle, even as additional procedures might be tresses-like or dedicated as subordinate lustful features (figure 4) [34]. the lumen or shallow gill has a compound edifice. the floor of the gill has a compound assembly. it offers paint moreover via painted stains it includes or through mechanical color with devices that contain photonic quartzes and spreading grills [38]. the scales are characteristic in thermoregulation, lining, creating pheromones (only in males) [39], and assisting sailplaning flying, however the maximum critical is the huge range of bright or vague styles they offer, which assist the animal in guarding self with the aid of mimicry or disguise and which performance as indicators to supplementary wildlife along with opponents and capacity friends  [34]. 6. interior geomorphology of lepidoptera insects 6.1. procreant system lepidoptera insects within the generative system of lepidopteran insects, the male genitals are compound and indistinct. in female insects, the three kinds of genitals are founded on the linking taxonomic group monocrystal, exploration, and dionysian. inside the monocrystal, the kind is an inaugural on the attached parts of the breastbones nine and ten, figure 4. microscopy imageries of scales of lepidopteran insect a). a patch of the wing b). scales close up file:///d:/pandawa%20institute/1.%20journal%20of%20multidisciplinary%20applied%20natural%20science/issue/vol%203%20(2023)/55%20khan%20et%20al/accepted%20manuscript%20140-article%20text-762-1-6-20221017.docx#page21#page21 j. multidiscip. appl. nat. sci. 61 which perform as oviposition and fertilization. the exospore types are two discrete spaces for pollination and oviposition, both stirring on the identical breastbones as the monocrystal kinds nine and ten [33]. dionysian corporations have an inner duct that incorporates semen, with discrete starts for coitus and egg resting [20]. in maximum types, the genitals are edged by soft parts, although they will be sclerotized and particular in a few classes for ovipositing in the place together with gaps and inner plant cells [33]. bio-chemical and the secreters that harvest them run the expansion of moths and butterflies as they feel their life sets termed the endocrinal scheme. the first pest chemical prothoracicotropic biochemical activates the type's lifetime diapause and series [40]. this bio-chemical hormone is formed with the aid of quantities allata and quantities cardiac, in which it is additionally kept. a few secretors are dedicated to making positive duties including making silk or saliva in the palpi [40]. although the quantities cardiac harvest the prothoracicotropic biochemical, the quantities allata additionally produce juvenile biochemical, and the prothoracic secretors harvest molting biochemical. 6.2. gastric system lepidoptera insects in the gastric system of the insect, the front section of the foregut has been changed to shape a guttural lapping heart as they want it for the nourishment they eat, that is for the maximum portion of fluids (figure 5). a gullet trails and mains to the subsequent of the throat and in a few classes shapes of crops. the midgut of the insect is brief and immediate, and the hindgut is lengthier and looped [33]. families of lepidopteran insect order classes, stopping from hymenoptera, had midgut caeca, though this is lost in existing lepidopteran insects of butterflies and moths. rather, all the gastric hormones, in addition to early ingestion, are powerless at the superficial of the midgut jail cell. young insects are discovered within the anterior and posterior midgut regions and the larvae are long-necked and stalked goblet cells. in lepidopteran insects, the wineglass tissues evacuate optimistic k ions, that are engrossed from greeneries consumed by the young insect. maximum lepidopteran insects of butterflies and moths show the normal gastric cycle, however, classes with dissimilar diets need revisions to see these new strains [36]. 6.3. cardiovascular system lepidoptera insects in the cardiac system, insect blood, or, hemolymph, is rummage-sale to party heat in the shape of thermoregulation, where influence shrinkage harvests heat, that is relocated to the respite of the figure when situations are adverse [41]. in lepidopteran insect classes, hemolymph is spread finished the strains in the wings by a few forms of the lively body part, both through emotion or by way of the consumption of air into the throat [42]. 6.4. breathing system lepidoptera insects the air is occupied over spiracles alongside the figure 5. inner geomorphology of the adult male in the family nymphalidae, viewing most of the chief body part schemes, with usually instantaneous forelegs of that family: the quantities cover the corpus allatum and the corpus cardiac. j. multidiscip. appl. nat. sci. 62 perimeters of the stomach and thorax offering the throat o2 as its energy through the lepidopteran insect breathing system. the insect of lepidopteran species contains three special tracheas that deliver and diffuse oxygen throughout bodies. the dorsal tracheae source o2 to the back muscular structure and containers, even as the adaxial throats stock the adaxial muscular structure and bravery twine, and the instinctual windpipes source the heart, fat figures, and sex gland [42]. 7. lepidoptera insects of polymorphism polymorphism is the arrival of bureaucracy or transforms, that range in paint and numeral of qualities inside unmarried classes (figure 6) [42]. in lepidopteran insects, polymorphism may be visible not most effective between entities in a populace, but also between the genders as sensual dimorphism, between biologically parted populations in topographical polymorphism, and among compeers hovering at an exclusive time of year. in a few kinds, the polymorphism is constrained to sexual activity, usually in lady insects. this regularly contains the miracle of the impression when copied morphs fly together with nonmimetic alters in a people of precise type. polymorphism happens equally at a precise step with inborn variation within the usual structural plan of entities, as well as in sure particular morphologic or functional growths inside a lepidopteran type [43]. ecofriendly polymorphism trends are not congenital and are frequently called polyphenism, that in lepidopteran insects of moths and butterflies is usually realized in the formula of cyclical alters, particularly in relations of nymphalidae and pieridae. the insect butterfly in the glove, the communal grass yellow (eurema hecabe) has a shadier seasonal mature alter, caused through the aid of an extensive daytime prodigious thirteen periods in length, although the briefer daylight dated of twelve periods or less tempts a waterier alter within the put up-monsoon length [43] reasons a divergence of a species into diverse morphs (biston betularia) [44]. terrestrial isolation details a departure of types into extraordinary switches. indian white admiral limenitis procris is a good example, that has 5 governments, every biologically parted from the differing using large foothill periods [45]. a good extra dramatic exhibit of topographical pleiomorphism is the insect of the apollo butterfly (parnassius apollo). outstanding to the fact the p. apollo are aware of slight local inhabitants, accordingly taking no interaction with another, joined with their robust steno theme wildlife and liable relocation potential, hybridization between populaces of one species nearly does not befall; with the aid of this, they shape over six hundred distinctive switches, with the scale of acnes at the wings of which differs substantially [46]. sexual dimorphism is the existence of alterations between females and males in the same classes. in lepidopteran insects, it is extensive and significant and nearly full set by using hereditary dedication [43]. it is found in altogether relations of the papilionoidea and is supplementarily unsettled in the pieridae, lycaenidae, and confident taxonomic categories of the nymphalidae. separately from the paint dissimilarity, that whitethorn additionally alters on or after moderate to wholly diverse color design blends, minor sensual behaviors will similarly be current time [45]. a diverse genetic constitution upheld with the aid of natural choice will likewise be articulated at an equal time [43]. polymorphous and/or imitative females happen in the case of a few taxonomic groups in the papilionidae in most cases to achieve an equal guard not offered to the male insect of its types. the maximum different situation of erotic dimorphism is in mature female insects of numerous psychidae types that have the handiest stunted legs, figure 6. the longwings butterflies from the tropics of the western hemisphere are the traditional typical müllerian imitation. j. multidiscip. appl. nat. sci. 63 mouthparts, and wings, associated with the mature males who might be sturdy handbills with nicely established wings and fluffy antennae  [46]. 8. reproduction and development of lepidopteran insects class of lepidoptera experience holometabolous "complete metamorphosis". their lifetime series usually entails egg, pupa, larva, and adult or imago (figure 9) [43]. the young insect of larvae are usually referred to as worms, and the cocoons of moths summarized in silk are known as sheaths, even as the exposed cocoons of butterflies are entitled chrysalides. 8.1. diapauses of lepidopterans without the classes reproducing time spherical, lepidopteran insects of moths or butterflies may input diapause, a state of dormancy that allows the pest to live in detrimental ecological situations. 8.2. mating generally, the male insects jolt elusion previous than the female insect and highest in facts earlier than female insects. in cooperation, the insect of males and females sexually settled by the time of elusion (figure 8) [35]. lepidopteran insects of moths or butterflies, generally do not assist one another, excluding traveling classes, and staying moderately antisocial. conjugation starts with an adult male and female insect attracting a mate, usually utilizing visual spurs, mostly in daylight classes like maximum butterflies. nevertheless, the female insects of most nighttime classes, which include nearly all moth kinds, habit pheromones to entice males, occasionally from extended detachments [26]. a few kinds interact in the shape of aural dating or appeal to pals through the use of thorough or shaking which include the polka spot of dot syntomeida epilates and wasp moth [47]. variations encompass the present process of one seasonal technology, two or even more, known as voltinism. maximum lepidopteran insects in moderate weather are univoltine, even in tropical weather, and most have two cyclical young. a few other lepidopteran insects may gross gain of any chance they can grow, and buddy incessantly during the year. these cyclical versions are accomplished by using biochemical, and these stays in replica are called diapause [35]. numerous lepidopteran classes, after breeding and resting their eggs, decease rapidly afterward, taking most effectively survived for a few times after elusion. another insect of lepidopteran can also silent be energetic for numerous workweeks after which overwinter and grow to be sexually energetic once more when the climate becomes extra promising, or diapause. the semen of the male insect which bred record currently with the female insect is greatest in all likelihood to have pollinated the spawns, but the semen on or after a previous coupling may additionally silent triumph [35]. 9. lifecycle of lepidopteran insects 9.1. eggs lepidoptera generally reproduces egg placing, though a few classes showcase breathing natal in a course referred to as ovoviviparity (figure 2). a selection of variances in egg resting and the wide variety of eggs arranged to arise. a few classes figure 7. a) dry-season form; b) wet-season form. a b j. multidiscip. appl. nat. sci. 64 bead their eggs in flying [48] at the same time as maximum amateur their spawns close to or on the swarm vegetable on that the grub’s food. the numeral of eggs rested may vary from just some to frequent thousand [26]. each female moth and butterfly pick out the swarm shrub mechanically, and often, by biochemical cues [35]. the eggs are derivative from supplies gulped as a caterpillar and in a few kinds, from the spermatophores established from males through coupling [49]. an ovum can most effectively be one/thousand the physique of the womanlike, but it can also amateur up to listen mass in eggs. female insect amateur minor eggs as they stage and the larger female insect amateur larger eggs (figure 9). the egg is enclosed via a difficult ribbed caring outer coating of an explosive, known as the chorion. it is coated with a tinny covering of honeycomb, that averts the ovum after ventilation from the outer site. every egg cell incorporates numerous tiny funnel-shaped, or micropyles, starting at one finish, the motive of that is to permit semen to go into and manure the ovum. moth and butterfly eggs fluctuate significantly in size between types, nonetheless, they’re all moreover oval or rounded. the egg period lasts about workweeks in most butterflies, but eggs rested before midwinter, particularly in mild areas, shading and undergoing diapause can be not on time till spiral. different butterflies can untrain their seed in the spiral and have them trapdoor inside the seasonal. these lepidopterans are normally moderate classes. 9.2. larvae the worms or young insects of larvae are the first phases within the existence series after marking. young insect function polypod larvae with tube-shaped forms, petite thoracic legs, and belly prolegs (pseudopodia) [42]. they have a sclerotized skull pill of a frontal seam shaped by a median synthesis of the mouth parts for mastication, and a lenient cylindrical, sclerites, part form, which can have tresses or supplementary forecasts, 6 proper legs, and extra prolegs (up to 10) [50]. the frame of the body includes thirteen sections and the body contains three thoracic and ten stomachs (figures 2 and 10) [51]. maximum grubs are herbivores, but some are meat-eaters (some eat ants or other worms) and harmful [50]. diverse herbivorous classes have improved to forage on each fragment of vegetable and are generally taken into consideration as bothers to their congregation florae, a few classes were discovered to untrained their spawns at the berry and different classes untrained their spawns on sartorial or hair. a few classes are predatory and others are smoothly scrounging. nearly lycaenid classes including maculinea rebeli are common parasites of myrmica ants’ cases [52]. a class of geometridae from hawaii has carnivorous caterpillars that vicious circle and eat flies [25]. approximately pyralid young insects are marine [53]. the young insect larvae expand quickly with numerous cohorts in a day, however, a few classes might additionally absorb a few ages to expand, and tremendous samples like gynaephora groenlandica receipts as extended as 7 years [26]. the young insect or first stage of the larval phase is where the a b figure 8. a) sexually dimorphous common bagworm moths pairing: the female is nonflying; b) pairing mate of poplar sphinx moth viewing two dissimilar color alternatives. j. multidiscip. appl. nat. sci. 65 eating and developing tiers arise, and the first stage of larvae sometimes feel biochemical encouraged about ecdysis, emerging more with each larval stage, till they go through the last larval stage of pupal molt. the first stage of larvae of each butterfly and moth insect showcase imitation to discourage ability killers. a few young insects can inflate elements of their skulls to seem snake-like. numerous have untrue eye color spots to enhance this effect. a few caterpillars have unique constructions referred to as osmeteria, that can be unprotected to provide stinky bio-chemicals secondhand protection. congregation vegetation frequently has poisonous materials in them and young insects can requisition those materials and maintain them into the mature stage. this assistance types them indigestible to animals and different killers. such unpalatability has promoted the exploitation of cheerful pink, black, red, or snowy white cautionary colorations. the poisonous substances in flowers are frequently changed exactly to save them from existence bothered by pests. insects, in the crack, expand countermeasures or type habit of this pollution for their endurance. this "arms race" has brought about the coevolution of pests and their congregation florae [54]. 9.3. wing expansion no shape of the wing is outwardly visible in the young stage of the larva, but when the caterpillar is divided, growing wings can realize as recordings, that may be discovered on the next and tertiary thoracic sections, in residence of the spiracles that are seeming on stomach parts. wing rounds broaden in overtone with a windpipe which innings alongside the bottom of the wing, and are enclosed via a skinny periodical sheath, that is related to the outer hide of the first stage larva by a small pipe. wing rounds are very minor till the ultimate larval stage after they boom theatrically in scope, are attacked through branched throats from the wing base that lead to the creation of the wing veins, and begin to grow patterns related to many breakthroughs of the wing [55]. close to pupation, the annexes are pressured out of doors the skin under burden from the hemolymph, and though they are originally fairly lithe and delicate, by the period the cocoon breakdowns free of the young stage of larval shell, they have been observed strongly to the external shield of the cocoon. in the interior periods, the wings form an epidermis so rigid and well linked to the figure that cocoons can be selected up and figure 9. the quatern phases of the lepidopterous insects' life cycle. figure 10. larvae of lepidopteran insects. j. multidiscip. appl. nat. sci. 66 frozen starved of injury to the wings [55]. 9.4. pupa the maximum number of insects of lepidopteran has five to seven instars or molts, [56] confident hormones, like ptth, stimulate the production of ecdysone, which initiates insect molting. then, the caterpillar a sclerotized, puparium, or toughened cuticle of the ultimate larval stage, grows up into the cocoon. relying on the classes, the cocoon may be protected in a silk sheath, connected to diverse kinds of substrata, hidden in the floor, or might not be endangered completely. structures of the imago are outwardly familiar in the cocoon. all the additions on the mature skull and thorax are observed encased within the carapace, with the wings enfolded round, together with the projections of the insect (figure 11) [35]. the cocoons of a few classes have practical jawbones, and at the same time as the pupal jawbones are not practical in other insects [34]. while covered, a number of the inferior sections aren’t bonded and might pass utilizing minor strengths recorded in the film. touching may additionally assist the cocoon, for case, seepage the sunshine, which would or else slaughter it. the cocoon of the mexican jumping moth (cydia deshaisiana) does this. the caterpillars cut a flap in the bean and use the bean as a lodging. the astonishing temperature upward pushes the cocoon inside tics and jolts, pulling at the cobwebs internally. waggling may additionally aid in daunt parasitoid wasps from placing eggs on the cocoon. other classes of moth insects could make clicks to discourage predators [35]. the period earlier than the pupa arises diverges significantly. the king butterfly can also visit in its cocoon for seventeen days, even as other classes might also want to visit for more than ten calendar months in diapause. the maturity arises after the cocoon both through the usage of stomach hangers or from forecasts located on the peak. the mouths have originated within the maximum rude moth families that are secondhand to leak from its cocoons [35][42]. 9.5. adult the maximum lepidopteran classes do not conscious long afterward elusion, most effective wanting some days to discover a friend and then amateur eggs (figure 12). others might also endure dynamics for an extended duration, go over diapause and stagnate as king butterfly insects do, or else wait out ecological pressure. a few mature classes of micro-lepidoptera go over a period where no generative linked action befalls, permanent over seasonal in winter and summer, surveyed by pairing and oviposition in the primary spiral [35]. while the maximum moth and butterfly insects are worldly, many classes of pyralidae are honestly aquatic with all stages except the grownup happening in the water. many species from other families including nepticulidae, noctuidae, arctiidae, tortricidae, olethreutidae, cosmopterygidae, sphingidae, and cossidae are aquatic or semiaquatic [57]. 10. behavior of lepidoptera insects 10.1. flight flight is a vital issue in the survival of moths and butterflies, and is secondhand for evasion predators, looking for nourishment, and discovering companions directly, as lepidopteran classes do not live extensively afterward eclosion. it’s the key shape of motion in most types. in lepidopteran insects, the hindwings and fore wings are automatically joined and lap in synchronism. flying is anteromotoric, or else being strapped frequently with the aid of the accomplishment of the fore wings. even though lepidopteran insects supposedly can nevertheless hover when their hind wings are figure 11. pupa of lepidopteran insects. j. multidiscip. appl. nat. sci. 67 reduced, it decreases their lined flying and spinning abilities [58]. lepidopteran classes must be earnest, approximately seventy-seven to seventy-nine degrees in farenhide scale to hover. they are contingent on their body frame fever life adequately excessive and because they can't alter them, this is reliant on their setting. the insect of butterflies alive in chiller weather might additionally have their annexes to deep their forms. they will back inside the solar, dispersal out their wings so that they grow supreme promotional to the sunshine. in warmer weather, butterfly insects can without difficulty swelter, so they’re normally vigorous simplest at some point in the cool box parts of the day, early morning, early evening, or late afternoon. all through the warmth of the day, they break in color. a few greater thick-bodied moths can make their warmth to a constrained grade via vibrant wings. the heat produced by using the flying strengths groups takes to the thorax while the infection of the stomach is insignificant for flying. to keep away from feverishness, a few moth insects depend upon furry internal air sacs, scales, and different systems to distinguish the thorax, and stomach and hold the stomach calmer. a few classes of butterflies container attain rapid hustles, which include the southerly arrow, which could go as fast so 48.4 km/h. sphingids are several reckless in the air insects, a few can coast at ended 50 km/h (thirty mi/h), taking a wingspread of thirty-five to one hundred and fifty millimeters [59]. in a few classes, from time to time a gliding issue with their flight exists. the flight takes place both as flying or as a furtherance or retrograde signal [34]. in moth and butterfly classes along with hawk moths (sphingidae), soaring is vital as they are a necessity to keep certain constancy over plants when eating the juice [18]. 10.2. navigation navigation is essential to the insect order of lepidoptera classes, mainly for those that travel. insects of butterflies, that have greater types that travel, were proven to cross exploitation period waged solar ranges. they could get separated light, so can position themselves even in overcast situations. the separated bright inside the vicinity near the infrared band is recommended to be specifically essential [60]. the greatest traveling butterfly insects are those that animate in semiarid parts where upbringing periods are quick [61]. the lifetime antiquities of their host florae additionally have an impact on the techniques of butterfly insects [62]. other researches contain the use of sceneries. the lepidopteran insect might also habit sea strains, foothills, and even infrastructures to position them. above the marine, the flying path is an awful lot extra precise if the shore remains evident [63]. numerous research has additionally proven that moths circumnavigate. one research confirmed that numerous moth insects might use the earth's attractive arena to pilot, as a training of the moth's emotion and arrow specifies [64]. another research, the traveling conduct of the grey, confirmed that smooth at excessive heights, the classes can accurate its path with varying winds, and favors winged with favorable gusts, signifying a fantastic experience of path [65][66]. aphrissa station in panama loses its navigational capacity when uncovered in a magnetic area, suggesting it uses the a b figure 12. adult lepidopteran insects a) butterfly; b) moth. j. multidiscip. appl. nat. sci. 68 earth's magnetic discipline [67]. the lepidopteran insect moths generally tend to loop fake lighting fixtures time and again. this advice uses the practice of holy triangulation referred to as transverse alignment. by way of preserving an endless angular affiliation to cheerful celestial sunlit, consisting of the lunner, they canister flying in a straight mark. celestial substances are thus far absent, that uniform after journeying extremely good reserves the craft in attitude between the moth and the bright basis is tiny, similarly, the lunar resolve continually inside the upper portion of the pictorial turf or on the prospect. when a moth insect meetings a mile’s earlier synthetic bright and uses it for steering, the viewpoint vicissitudes clearly after most effective a rapid detachment, further to being frequently under the prospect. the moth impulsively efforts to exact by way of rotating closer to the mild, inflicting airborne moths to return dipping down, and at near variety, which outcomes in a curved flight path that receives nearer and nearer to the sunny basis [68]. different clarifications were recommended, together with the concept that moths might be reduced through a visible pictorial alteration referred to as a mach band henry hsiao and supposed that they fly near the shadiest part of the sky in hunt of security, and hence are willing to round ambient items within the mach group area [69]. 10.3. migration lepidopteran migration is usually seasonal, as the insect's flow gets away during dry terms or different hurtful situations. maximum lepidopteran insects that travel are butterflies, besides the space traveled differs. a few butterflies that wander encompass the grief coat, dyed female, red admiral, american lady, and the shared horse chestnut [56]. an exceptional class of moth that journeys long reserves is the bogong moth (agrotis infusa) [70]. the greatest famous relocations are those of the easterly populace of the monarch butterfly (danaus plexippus) from the northern part of the usa to mexico and the southern part of canada, a detachment of about four thousand to four thousand eight hundred kilometers. other famous traveling classes include the painted lady and numerous butterflies. amazing and hefty gage relocations related to the downpours are visible in jutting india [40]. migrations were studied in greater latest instances using wing tags and stable hydrogen isotopes [71]. insects of moths additionally assume relocations, an instance actuality the uraniids. urania fulgens undergo populace bangs and huge relocations that can be not bettered with the aid of some other insect in the neotropics. in panama and costa rica, the primary populace actions may additionally start in july and early august, and liable on the year might be very huge, current persistent for as extensive as five months. 10.4. communication pheromones stand generally worried hip coupling rites amongst types, mainly moth insects, however, they are too bossy thing in styles of communique. generally, the pheromones are shaped by both females and males. they noticed by way of the entities of the other intercourse by their feelers [65]. in many classes, a gland between the 8 th and 9 th sections beneath the stomach within the female insect produces pheromones [26]. the announcements also can happen via stridulation or by generating noises by the impression of various body parts composed [66]. moths are regarded to interact in audio sorts of announcement, maximum usually as wooing, figure 13. time-lapse of hovering moths, concerned with the lighting. j. multidiscip. appl. nat. sci. 69 appealing buddies the usage of vibration or sound. alike record other insects, moths prize up these noises exploitation tympanic films in their stomachs [72]. an instance is that of the polka timelapse moths, drawn to the lightings migration monarch butterflies, visible in a bunch in america, where the cowboy movie populace migrates for the winter statement collection of melitaea athalia near warka, poland dot wasp moth (syntomeida epilais), which products noises with an incidence upstairs that usually obvious by human beings. these noises are also purposed as tactile dialog, or statement over the trace, as they clitter, or shake a substratum like plants and their stalks [47]. maximum lepidopteran insect moths have nonexistent cheerful colors, as many classes use patterns as a disguise, but most lepidopteran insect butterflies engross in a filmic conversation. female cabbage butterflies (pieris rapae), for sample, use infrared light to connect, with scales painted in this variety on the abaxial wing exterior. when they soar, a separately down stroke of the wing makes a transitory flash of infrared bright which the adult males know as the flying sign of an ability mate. these sparks from the wings might appeal to numerous adult males that interact in midair wooing shows [72]. 11. ecosystem of lepidoptera insects insects of butterflies and moths are vital in the normal environment. they are essential members of the nourishment cable, having co-evolved with highpoint predators, and plant lepidopteran classes have shaped a system of trophic relations between heterotrophs and autotrophs, comprised in the steps of lepidoptera caterpillars, cocoons, and adults. caterpillars and cocoons are related to the diets of birds and parasitic entomophilous pests. grownups are comprised of nourishment nets by a bigger variety of customers [35]. 11.1. protection and predation lepidopteran classes are soft bodies, nearly unprotected, and brittle, while the small levels flow leisurely or are motionless, consequently, altogether steps are bare to predation. adult insects of moths and butterflies are preyed upon by bats, birds, amphibians, lizards, spiders, and dragonflies. one spider class, silver argiope (argiope argentata), foods insects of moths and butterflies and shows an extensive bite when preying on them in place of covering them in the satin primary. this is conjectured to attend as a halt approach [73]. worms and cocoons reduction target not longer best the animal of birds but additionally to spineless marauders and small animals, in addition to the microorganism of fungi and bacteria. parasitic wasps, parasitoids, and flies might additionally amateur eggs in the young insect, which is subsequently put to death as they entrance inner the body parts and upset its cells. pest-consuming birds are perhaps the most important marauders. lepidopteran insects, particularly in the young steps, are an environmentally vital food to numerous insecticides birds, including in european countries. an "evolutionary arms race" may be seen between predator and prey species. the lepidoptera have developed numerous techniques for defense and safety, along with the fruition of morphologic fonts and ecological changes in existence and actions. those encompass parody, disguise, aposematism, and the growth of hazard designs and performances. a few birds, consisting of the goatsuckers search for night lepidopterans. their key killers are bats. once more, an "evolutionary race" occurs, which brought about several evolutionary revisions of moths to break out from primary predators, including the potential to pay attention to supersonic noises, or even to produce noises in a few gears. lepidopteran eggs are acting like prey. a few maggots, together with figure 14. pollination occurs in the flower by a moth. j. multidiscip. appl. nat. sci. 70 the swallowtail butterfly grubs, are cannibalistic. a few lepidoptera classes are toxic to predators, including the sovereign butterfly in the americas, atrophaneura types in asia, in addition to papilio antiochus, and the biggest lepidopteran insect of butterflies birdwings in africa and asia, respectively. they reap their poisonousness way of confiscating the chemical compounds on or after the florae they worry into their matters. a few insects of lepidoptera manufacture their poisons. marauders that devour noxious moths and butterflies can come to be ill and vomit violently, getting to know no longer to consume the one's class. a predator that has formerly plagued a noxious lepidopteran might also keep away from different classes with like colorations in the impending, as a result, exchangeable many different classes, as sound [74]. poisonous butterfly insects and caterpillars tend to grow cheerful insignia and arresting designs as a pointer to predators of their poisonousness. this situation is referred to as aposematism [75]. a few young insects, specifically memberships of papilionidae, include an osmeterium, a y-form protrusible gland determined inside the prothoracic phase of the young insect. when endangered, the young insect produces ugly odors from the body part to district off the predators [76]. concealment is likewise a vital security method, which includes using color or form to combo into the nearby atmosphere. a few lepidopteran insect classes mixture with their environs, manufacturing them tough to identify through marauders. young insects showcase sun suspects of inexperienced that tie their congregation tree and plant. others appear to be unpalatable gadgets, along with branches or shrubbery. as an example, the grief cloak weakens in the setting of trees when it crinkles its wings spinal. the larvae of a few classes, consisting of the not-unusual mormon (papilio polytes) and the western tiger swallowtail (papilio rutulus ) look like bird mucks [77]. perhaps, mature clearwing moths (sesiidae) classes have a well-recognized appearance sufficiently just like a hornet or wasp to kind it possible the moth insects profit from a discount in predation concluded batesian imitation. ocelli are a type of ecology resistance and predation yellow swallowtail (papilio machaon) caterpillar viewing the osmeterium, which produces unsightly odors to thrust back predators of automimicry secondhand by a few lepidopteran insects of moths and butterflies. in insect butterflies, the color spots are serene of concentrical round rings of scales in diverse colorations. the future purpose of the ocelli is to deflect the care of predators. their likeness to eyes incites the hunter's character to assault these annex styles [78]. müllerian and batesian mimicry developments are normally observed in the insect order of lepidoptera. hereditary pleiomorphism and ordinary choice supply upward thrust to else comestible classes fast an existence benefit by using reminiscent indigestible classes. such an impersonation complicated is called batesian and it is most usually identified in the sample between the cimetidine vicereine butterfly regarding the indigestible danaine emperor. the vicereine is, in reality, supplementary poisonous than the monarch and this similarity ought to be measured in a situation of müllerian imitation [79]. in müllerian imitation, inedible classes, normally within a taxonomical order, discover the high quality to resemble each different to lessen the sampler fee by hunters that require to find out about the insects' inedibility. the taxonomic group from figure 15. tobacco carolina sphinx moth parasitized via braconidae wasp caterpillars. j. multidiscip. appl. nat. sci. 71 the poisonous genus heliconius shapes one of the maximum famous müllerian developments [72]. the grownups of the diverse classes nowadays are similar to separate others so properly, that the classes can’t be prominently lacking near morphologic statements and, cases, partition, or hereditary examination. insects of moths can pay attention to the variety produced by bats, which in impact reasons hovering moths to brand shifty exercises due to the fact bats are the principal predators of moths (figure 13). supersonic rates cause a reaction drive act in the noctuid moth that reason it to droplet a few edges in its flying to avoid the bout [80]. tiger moths (arctia caja) in protection produce clacks inside the equal variety of bats, which delay the rackets and frustrate their efforts to echolocate them [81]. pollination: a day-flying hummingbird sphinx moth (mesogona olivata) consuming juice from a class of dianthus. maximum classes of lepidoptera insects in a few forms of entomophily, or the fertilization of plant life (figure 14) [82]. maximum grownup moths and butterflies provender on the juice of inner plants, via their snout, to attain the juice unseen at the improper of the flower parts. with this technique, the adults skirmish contrary to the florets' petals, on which the generative pollen is complete and saved. the pollen is moved on adjuncts of the grownups, that coast to the following floret for food and accidentally withdrawal the pollen on the disgrace of the ensuing floret, anywhere the pollen sprouts and pollinates the germs [35]. flowers are fertilized by using nerves incline to be big and showy, violet or purple, frequently taking a touchdown extent, and normally fragrant, as butterflies are usually diurnal hovering. meanwhile, butterflies do now not abstract pollen [82]. the flora has easy nectar courses, with the nectarines typically unseen in thin pipes or limbs, touched by using the extended "tongue" of the butterflies. the insect of butterflies includes skipper butterfly (thymelicus flavius) drink recorded to involve in floret fidelity, that earnings they are much supplementary possible to the allocation of pollen to supplementary exact plants. it could use for the florae being fertilized, as bud loyalty averts the loss of pollen for the duration of exclusive trips and the pollinators from blockage shames with pollen of different floret classes [83]. there are more vital moth pollinator companies such as sphinx moths or hawk moths (m. olivata) of the family sphingidae. their performance is much like hummingbirds, i.e., exploitation of hasty wing knocks to hover opposite of florae. record of sphinx moths is nighttime or crepuscular, so insects of moth-fertilized florae tendency to be snowy white, nighttime initial, huge, and impressive with cylindrical corollae and a stout, syrupy scent twisted in the nightly, or early pre-lunch. a whole lot of juice is shaped to gas the excessive metabolic prices wanted to control their flying [84]. other moths fly gradually and relax on the floret. they do not require as per plenty liquid as the profligate hovering sphinx moths, and the florae have a habit of being slight [85]. 11.2. mutualism it is a shape of organic contact in which every specific elaborate aid in any way. mutualism is a shape of an organic interface in which every person is worried about advantages in a few ways. a sample of a mutualistic association might be communal through yucca moths (tegericula yuccasella) and their host, yucca plants. female t. yuccasella moths cross the threshold of the host flowers, accumulate the pollen into a sphere with the use of particular maxilla palps, then pass to the top of the pistil, where pollen is placed on the shame, and amateur spawns into the ignoble of the pistil where stones will grow. the young insect stage of larvae increases in the vegetable and food on a slice of the yields. hence, mutually plants and insects profit, making an enormously mutualistic association [35]. every other shape of mutualism happens between a few insect larvae of butterflies and certain classes of ants. the insect species of larvae speak with the ants' use of feelings conveyed via a substratum, inclusive of the timber of a shrub or shoots, in addition to the usage of chemical alerts [86]. the ants offer a few grades of safety to these larvae and they in crack collect honeydew melon emissions  [87]. 11.3. parasitism individual forty-two classes of parasite lepidopterans are regarded as parasitism j. multidiscip. appl. nat. sci. 72 [35].   the young insect larvae of the extra and slighter wax moths food on the honey inner bee cases and might turn out to be bothered, it is additionally stated in humblebee and wasp bubbles, though to a smaller quantity. in the northern part of europe, the wax moth (galleria mellonella) is appeared as the most severe parasitoid of the bumblebee (bombus sp) and is located handiest in bombus sp bubbles. in a few regions in england, in more than eighty percent of cases containers be demolished [88]. different sponging grubs are recognized to target leaf hoppers and cicadas (figure 15) [89]. on the contrary, butterflies, and moths can be difficult for parasitic flies and wasps, which can lay offspring on the young insect, which flap and provender the interior of its form, ensuing in dying. granting in a system of parasites termed idiobiont, the grownup stuns the swarm, so as no longer to slaughter it but for it to stay as extended as viable, for the sponging young insect to advantage the utmost. in the extra form of parasite, koinobiont, the classes stay off their swarms even as internal. these lice animate in the swarm worm all through its life cycle, or may additionally touch it in a while as a grownup. further tips, koinobionts include coleopteran insects, flies, and amply of hymenopteran parasitoids [35]. a few types might be topic to a diversity of vermin, including the spongy or gypsy moth (lymantaria dispar), which is argued through the sequence of thirteen classes, in six unique taxonomic groups of all complete its lifespan series [35]. in retort to a predation ovum cell or else grub within the worm's frame, the plasmatocytes, or else the swarm's cubicles can shape a complicated pill which finally sources the entozoon to smother. it is one of the caterpillar's most effective ways of protection in contradiction to parasitoids which is known as encapsulation [35].   12. beneficial insects of lepidopteran insects unfluctuating nevertheless most moth and butterfly insects affect the budget system damagingly, a few classes are precious financial assets. the maximum distinguished sample is that of the tame giant bombyx mori (silkworm moth), the grubs of that brand it sheaths out of silk. this could be turned into material and silk is a vital monetary useful supply at some stage in records. the species b. mori has been trained to the fact that it is far wholly in need of manhood for existence [90]. several wild moths such as antheraea classes and, bombyx mandarina, except others, deliver commercial importance of silk stains [91]. the choice of the young insect larvae of maximum lepidopteran classes to food on a single class or a confined variety of flora is rummage-sale as an apparatus for the organic manipulation of wild plants in the location of weedkillers. the pyralid cactus moth (cactoblastis cactorum berg) changed into brought from australia to argentina, anywhere it effectively repressed hundreds of thousands of acres of sensitive pear cactus plants. every other class of the grass moth referred to as the gator weed stem borer (arcola malloi), changed into rummage-sale to govern the marine herbal recognized as alligator wild plant (alternanthera philoxeroides) along with the alligator wild plant flea beetle. in this case, the two insects' effort in interaction and the weed rarely convalesce. refinement moths and butterflies, or butterfly garden/childhood, has ended up a naturally feasible manner of announcing types into the surroundings to profit it. insect of butterfly ranching in papua new guinea (impatiens hawkeri) certifications in the united states of america to "farm" economically treasured insect species for the collectors’ market in a naturally justifiable way [91] [92]. refinement of moths, and butterfly gardening or rearing, has come to be a naturally practical method of presenting species into the environment to advantage it. figure 16. the larvae are used as food material in the human feed. https://en.wikipedia.org/wiki/wasp j. multidiscip. appl. nat. sci. 73 12.1. food beondegi, b. mori (silkworm) pupae miffed or simmered and veteran for flavor, on the market by a road seller in the asian country of south korea (figure 16). lepidoptera features flagrantly in entomophagy as nutrition objects on nearly all mainland. even in maximum issues, grownups, caterpillars, or sheaths are eaten as clips by native society, b. mori or bondage cocoons are plagued as a portion of food in korea [93] while maguey worm (aegiale hesperiaris) is careful a delicacy in north american country mexico [94]. in a few slices of huasteca of mexico, the satin bubbles of the madrone butterfly (eucheira socialis) are kept on the superiority of rooftops of homes for feasting [95]. in the european country of italy, youngsters fasten and consume ingluvies of the poisonous zygaena moths (zygaena lonicerae) in the primary summertime. the ingluvies, regardless of having a completely low cyanogenetic contented material, serves as a handy, extra basis of honey to the kids that jerry can encompass this useful reserve as a periodic fragility at the lowest threat. external to this example, mature lepidopteran insect is rarely disbursed by people, with the single exclusion of the agrotis infusa [96]. 12.2. health a few insects of lepidopteran larvae of both butterflies and moths have the shape of tresses which has been recognized for purpose of humanoid healthiness troubles. worm locks now and again have pollution in them and classes from about twelve families of butterflies or moths global canister inflict extreme humanoid damages [97]. pores and skin strings are the maximum not rare, but there have been mortalities [98]. lonomia is a common reason for surrounding people in the north american country of brazil, with three hundred and fifty-four cases stated between nineteen eighty-nine and two thousand five. deadliness levels up to twenty percent with expiry induced maximum frequently with the aid of intracranial drop [99]. this hair has been regarded as a reason for keratoconjunctivitis. the shrill points on the termination of young insect hair can grow into tender skins and slimy crusts such as the senses. when they input such cells, they may be hard to cutting, regularly worsening the unruly as they travel throughout the crust [100]. this turns into selected trouble in indoor putting. the hairs effortlessly input complete airing assemblies and gather in inner surroundings because of their minor length, which kinds it tough for them to be expelled. this accretion rises the danger of people's interaction with their inner surroundings [101]. 13. conclusions the study exposed that lepidoptera species' range and abundance were more than diptera and hymenoptera but much less coleoptera insects. it is the second biggest insect order. lepidopteran insects are very essential because it helps in pollination. the larvae of most species are phytophagous and plenty is a series of pests of cultivated vegetation together with cutworms; some feed on stored grain or meal. again, the adults of many species are stunning and are a whole lot renowned by way of creditors, and many serve as the basis of artwork and design. for this, the foreigners such as the usa, uk, china, french, belgium, switzerland, japan, etc. used it as a business material (bowman). so, it is not only a natural enemy but also helpful to order the people. so, we preserve this order of insects and use it as the beauty of nature and also use it as commercial enterprise material. it is also a source of financing that has influenced our economic sector. author information corresponding author ahasan ullah khan — department of entomology, sylhet agricultural university, sylhet-3100 (bangladesh); department of agroforestry and environmental science, sylhet agricultural university, sylhet-3100 (bangladesh); department of agriculture, mymensingh commerce college, mymensingh2200 (bangladesh); orcid.org/0000-0002-7029-8215 email: ahasanullahsau@gmail.com authors nilufa yasmin poly — department of biochemistry and molecular biology, khulna https://en.wikipedia.org/wiki/lonomia https://orcid.org/0000-0002-7029-8215 mailto:ahasanullahsau@gmail.com j. multidiscip. appl. nat. sci. 74 agricultural university, khulna-9100 (bangladesh); orcid.org/0000-0002-4031-0455 shimanta dutta — assistant manager, akij tea estate, sylhet-3100 (bangladesh); department of agricultural extension education, sylhet agricultural university, sylhet-3100 (bangladesh); orcid.org/0000-0003-3286-974x ferdous alam — department of agriculture, mymensingh commerce college, mymensingh2200 (bangladesh); department of agroforestry, bangladesh agricultural university, mymensingh-2202 (bangladesh); orcid.org/0000-0001-6365-8991 author contributions a. u. k. planned, wrote, structured, revised, and replaced the document thoroughly. n. y. p., s. 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[3], the categories of anthropogenic sources of heavy metals can be generally classified as; urban elements, natural elements and elements of a mixed origin, implying that some metallic pollutants undergo geochemical reactions which alter their significant features. heavy metals act antagonistically disrupting trace elements in the body, inhibiting, and competing with protein and enzyme for binding sites and impairing the immune system. unachukwu and agomuo [4] reported that noncommunicable diseases (ncds) such as cardiovascular disease, diabetes mellitus, cancer, renal diseases, liver failure etc., may be associated with heavy metal toxicity and are probably more pronounced through occupational exposures [5]. nasarawa eggon and doma towns can be described as peri–urban or semi–urban areas which are the administrative headquarters of their respective local government areas in nasarawa state, north-central nigeria. these towns are surrounded by rural areas and are predominantly agricultural areas with majority of the inhabitants known to be farmers and traders. nasarawa eggon town is located along the ever-busy lafia–abuja road. this major road is characterized by high automobile/vehicular and human traffic due to its publisher’s note: pandawa institute stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. copyright: © 2022 by the author(s). licensee pandawa institute, metro, indonesia. this article is an open access article distributed under the terms and conditions of the creative commons attribution-sharealike (cc by-sa) license (https://creativecommons.org/licenses/by-sa/4.0/). https://doi.org/10.47352/jmans.2774-3047.130 open access https://creativecommons.org/licenses/by-sa/4.0/ https://doi.org/10.47352/jmans.2774-3047.130 https://crossmark.crossref.org/dialog/?doi=10.47352/jmans.2774-3047.130&domain=pdf&date_stamp=2022-08-01 j. multidiscip. appl. nat. sci. 2 figure 1. map of nigeria (a), nasarawa state (b), nasarawa eggon (c) and doma (d) local government areas showing sample collection points. proximity to nigeria’s federal capital territory, abuja as well as the nasarawa state capital, lafia. commuters (public and private) stopover at this town to buy agricultural produce (tubers such as yam and potatoes, fruits etc.) and food, while inhabitants of surrounding rural areas visit the town to carry out minor business transactions and also board vehicles to abuja and other destinations. drivers equally stopover in the town to repair and refuel their vehicles as well as pick passengers. doma town is along busy lafia–rukubi road. it is a major road characterized by a high volume of automobile traffic arising from its linkage to rukubi where africa’s largest rice mill is located and also, a major route to rural agricultural farms in doma where large-scale irrigation farming is practised all year round, using water from doma dam. thus, the focal towns are located on the highway characterized by heavy vehicular and human traffic which results in high human activities [6]. the busy nature of nasarawa eggon and doma major roads make them attractive to sundry human activities, ranging from indiscriminate citing of automobile parks and workshops (which are centres of sundry activities ranging from automobile servicing, panel beating, spraying, etc.), markets, business centers (typing, photocopying, printing, cybercafé, niajabet stations, movie or football viewing center etc.), food vending spots, gas stations, primary health care centres, etc. most of the businesses on these major roads (nasarawa eggon and doma) are powered by generators due to a lack of power supply from the national grid. their activities generate different kinds of wastes (gaseous, liquid and solid) which comprise automobile/vehicular and generator emissions, medical wastes, electronic waste (such as computers and their accessories, tv sets, fax machines, cell phones, telephones, photocopy machines and printers, dryers, wireless devices, video cameras, chips, motherboards, cathode ray tubes and peripheral items, kitchen equipment, electronic toys, washing machines, refrigerators etc.), automobile workshop wastes (damaged vehicle parts, car paint dust, engine oil, grease etc.) faeces, urine, garbage, sludge from sewage etc [6]. the unregulated disposal of waste in these towns results in indiscriminate and open dumping of these generated wastes (which are the surrounding agricultural soils) which wind up in landfills and j. multidiscip. appl. nat. sci. 3 informal dumps where they decompose and release toxic metals into the environment. long-term dumping of municipal wastes can influence soil properties and productivity at municipal waste dump sites but still may be used for farming provided that ecotoxicological risks associated with its usage are continuously assessed and controlled [7]. the concentration of these toxic metals in the environment has significantly increased due to increased human activities through emissions from industrial plants, thermal power stations, waste disposal, soil amendments and fertilization, vehicular traffic and road infrastructure [8], mining, smelting and refining of ores. heavy metals such as cadmium (cd), copper (cu), chromium (cr), lead (pb), manganese (mn), nickel (ni) and zinc (zn) are often contained as additives in some lubricants and gasoline are non-degradable in the soil. some of them have been classified as priority pollutants by united state environmental protection agency (usepa). at the moment, very few technologies, such as soil washing and bioremediation, are available to treat these mixed wastes [9]. edori and kpee [10] reported that heavy metals can be generated from faeces and urines of mammals which are either deposited in the agricultural soil or washed into the nearby agricultural soil. high levels of heavy metals in soils do not necessarily reflect anthropogenic influence but instead may be of a diagenetic origin or grain size effects [11]. since metals from both natural and anthropogenic sources accumulate in soils, it is often difficult to determine what fraction of soil’s metal load comes from which source. a crucial step for pollution assessment of soil is to establish the expected natural background concentrations [12], from which various approaches can be used to quantify anthropogenic inputs. thus, this study is aimed at quantifying the level of selected metals in agricultural soils along major roads of nasarawa eggon and doma and evaluating their origins (natural and anthropogenic), contamination level and toxicity. 2. materials and methods 2.1. description of study areas nasarawa eggon (ne) town is in the local government area (lga) of nasarawa state, nigeria and lies between latitudes 8°33‟ and 8°52‟ n and between longitudes 8°14‟ and 8°39‟ e. the climate of ne falls within the tropical savannah climate with two clearly marked seasons; wet and dry. it has a mean temperature of 15.6 °c and 26.7 ° c with annual rainfall between 1317 mm and 1450 mm [13]. doma town is in doma lga of nasarawa state, nigeria and located at latitude 8 0 24’n and 8 0 5’n and longitude 6 0 e and 6 0 30’e of the greenwich meridian. the entire lga is generally characterized by a low land area about 100-200 m above sea level although there is a kind of spatial variation in the surface area [14]. the ground positions of the sampling points in both nasarawa eggon and doma study areas were referenced with a handheld global positioning system (gps) unit (garmin gpsmap 76) and are shown in figure 1. 2.2. sample collection and preparation a total of 20 (twenty) composite soil samples were collected from 20 (twenty) agricultural farms at both study areas (nasarawa eggon and doma). the investigated agricultural farms are within 50 m from the major road but more than 1 km apart from each other. at each agricultural farm, six quadrats were marked. in each quadrat, four core soil samples were collected randomly at 0–20 cm depth using a soil auger and mixed together properly to form a composite of that agricultural farm. thus, 10 (ten) composite soil samples were collected from each study area. foreign materials such as rocks, pebbles, metal scraps, plant debris, waste polythenes and plastics were removed from the soil samples. they were air-dried for a week, ground and sieved through a 2 mm sieve. the samples were then stored in a dried plastic container for analysis. 2.3. elemental analysis the concentration of elements (metals) in the soil samples was determined using x–supreme8000 model of energy dispersive x–ray fluorescent analyzer [detection limit: 0.0001 % (1 ppm)– 99.9999 %]. a recovery test was carried out on the xrf machine by spiking analyses so as to ensure the reliability of the result. j. multidiscip. appl. nat. sci. 4 2.4. soil pollution assessment the metal contamination, as well as ecological risk of the soil samples in the study areas, were assessed using enrichment factor (ef), geo– accumulation index (igeo), contamination factor (cf), degree of contamination (cd), pollution load index (pli), ecological risk (er) and potential ecological risk index (ri) models. 2.4.1. enrichment factor the enrichment factor (ef) was evaluated by normalizing the metal concentration in the sample with respect to a reference metal [15]. the most common reference elements are sc, mn, ti, al and fe [16]. the ef of the metals/metalloids in the soil samples at both studied areas were calculated using eq.1. (1) where cn(sample) is the concentration of the investigated metal “n” in the study site, cref(sample) is the concentration of the reference metal in the study site; bn(background) is the background concentration of the investigated metal (usually the average shale value of the investigated heavy metal) and bref (background) is the background concentration of the reference metal (usually the average shale value of the reference metal). in this study, fe is the most naturally abundant element in all the studied soils and thus, was used as the reference metal while the average shale value described by turekian and wedepohl [17] was used as the reference value. 2.4.2. geo–accumulation index the geo-accumulation index (igeo) values for the determined heavy metals were evaluated using the formula proposed by muller [18] as shown in eq. 2. (2) where cn is the concentration of the heavy metal “n” in the soil sample; bn is the geochemical ef classes enrichment level i-geo value classes pollution level ef < 1 no enrichment 0; i-geo ≤ 0 practically unpolluted ef = 1–3 minor enrichment 1; i-geo = 0–1 unpolluted to moderately polluted ef = 3–5 moderate enrichment 2; i-geo = 1–2 moderately polluted ef = 5–10 moderate severe enrichment 3; i-geo = 2–3 moderately to strongly polluted ef = 10–25 severe enrichment 4; i-geo = 3–4 strongly polluted ef = 25–50 very severe enrichment 5; i-geo = 3–5 strongly to extremely polluted ef > 50 extremely severe enrichment 6; i-geo > 5 extremely polluted cf classes contamination level cd classes degree cf < 1 low contamination cd < 8 low degree of contamination cf = 1–3 moderate contamination cd = 8–24 moderate degree of contamination cf = 3–6 considerable contamination cd = 24–48 considerable degree of contamination cf > 6 high contamination cd > 48 very high degree of contamination er classes er level ri classes risk levels er < 40 low potential ecological risk ri < 150 low ecological risk er = 40–80 moderate potential ecological risk ri = 150–300 moderate ecological risk er = 80–160 significant potential ecological risk ri = 300–600 significant ecological risk er = 160–320 high potential ecological risk ri > 600 high ecological risk er > 320 very high potential ecological risk table 1. classes of ef, igeo, cf, cd, er and ri in relation to enrichment, pollution, contamination, degree of contamination, potential ecological risk and ecological risk levels, respectively j. multidiscip. appl. nat. sci. 5 background value for heavy metal “n” which is either directly measured in fossil argillaceous sediments of the area or adopted from literature (world average shale value); 1.5 is the correction factor for the variations in background value which could be due to lithogenic effects. the world average shale values of the studied heavy metals as described by turekian and wedepohl [17] and shown in table 2; were used in this study. geoaccumulation classes proposed by muller [18] as reported in table 1, was used for the interpretations of geo–accumulation index. 2.4.3. contamination factor (cf), degree of contamination (cd) and pollution load index (pli) the extent of soil contamination by a certain metal was determined using the contamination factor (cf). cf is the ratio of the concentration of metal to the background value of the metal, as given by eq. 3. (3) where cn is the concentration of the metal “n” in the sample, cb is the background concentration of the metal “n”. nigerian directorate of petroleum resources target values for metals in soils (table 2) were adopted as the background values “cb” [19]. the sum of all contaminant factors of the various metals is referred to as the degree of contamination (cd) [20]. it was calculated using eq.4. (4) where n is the number of metals studied and cfi is the contamination factor for metal “i” in the soil sample. the standard employed in the interpretation of contamination factor and degree of contamination values was adopted from the different classes proposed by hakanson [21] as reported in table 1. pollution load index (pli) was calculated so as to obtain a proper assessment of the degree of contamination. the procedure (eq. 5) by tomlinson et al. [22] was used to calculate pli in order to measure the overall level of metal toxicity at a particular sampling site. (5) where n is the number of metals and cf is the contamination factor. a pli value under one indicates unpolluted soils or sediments; zero indicates perfection; a value of one indicates the presence of only baseline levels of pollutants and values above one would indicate progressive deterioration of the soil quality (i.e., polluted) [22]. the pollution load index was interpreted as reported in table 1. 2.4.4. ecological risk (er) and potential ecological risk index (ri) hakanson [21] proposed ecological risk factor (er) as a quantitative expression of the potential ecological risk of a given contaminant (metal). it was calculated using eq. 6. (6) er is the single index of ecological risk factor and tr is the toxic response factor suggested by hakanson [21] which is presented in table 2. the potential ecological risk index (ri) index reflects the general situation of pollution caused by the simultaneous presence of the twelve (12) metals and was calculated using the expression in eq. 7. (7) n is the number of the metals considered; er and ri are the potential ecological risk factors of individual and multiple metals, respectively. the ranges of values used for the interpretation of the potential ecological risk factor [21] are reported in table 1. 3. results and discussions 3.1. metals table 2 presents the concentrations of metals (as, cd, co, cr, cu, fe, hg, mn, ni, pb, u and zn) analysed in the soil samples at the various sampled sites of nasarawa eggon and doma study areas. based on the mean value, the metal concentrations follow a decreasing order of fe> mn> zn> pb> ni> hg> as> u and fe> mn> co> zn> pb> hg> cr> as> u, in the nasarawa eggon and doma study areas respectively. it was observed that the mean concentration of fe in each study area was higher 6 s a m p le c o d e a s c d c o c r c u f e h g m n n i p b u z n n a sa r a w a e g g o n n 1 b d l b d l b d l b d l b d l 4 4 0 3 .7 9 9 .2 2 2 2 0 .4 3 b d l 2 1 .2 9 b d l 2 8 .6 7 n 2 1 9 .2 9 b d l b d l b d l b d l 3 0 6 6 2 .0 8 7 .0 2 8 3 8 .7 8 b d l 3 3 .0 6 b d l 5 5 .0 9 n 3 b d l b d l b d l b d l b d l 2 5 8 9 4 .2 8 5 .9 3 5 1 3 .9 5 b d l 2 7 .2 5 8 .6 8 3 2 .8 4 n 4 6 .9 0 b d l b d l b d l b d l 1 5 3 3 5 .6 6 b d l 3 3 4 .1 5 b d l 3 7 .8 1 b d l 9 6 .1 1 n 5 b d l b d l b d l b d l b d l 2 2 0 6 5 .5 4 7 .9 2 8 3 4 .1 6 b d l 6 2 .3 4 b d l 3 5 .1 9 n 6 b d l b d l b d l b d l b d l 5 2 5 5 .9 7 7 .1 2 4 8 0 .7 8 b d l 2 6 .5 3 b d l 1 4 .7 6 n 7 b d l b d l b d l b d l b d l 1 2 4 5 5 .8 5 b d l 4 4 4 .9 8 6 4 .5 9 4 6 .4 4 b d l 2 3 .4 1 n 8 b d l b d l b d l b d l b d l 1 2 7 9 0 .3 3 5 .4 3 4 5 2 .2 4 b d l 5 2 .0 8 b d l 2 3 .9 6 n 9 b d l b d l b d l b d l b d l 2 4 9 9 0 .2 2 1 0 .7 3 7 6 1 .8 2 b d l 3 9 .6 6 b d l 1 6 .3 0 n 1 0 b d l b d l b d l b d l b d l 1 6 6 3 4 .9 7 b d l 9 7 1 .8 1 b d l 8 2 .6 4 b d l 1 4 2 .3 5 m e a n 2 .6 2 b d l b d l b d l b d l 1 7 0 4 8 .8 7 5 .3 4 5 8 5 .3 1 6 .4 6 4 2 .9 1 0 .8 7 4 6 .8 7 d o m a d 1 b d l b d l b d l b d l b d l 8 4 3 1 .9 6 7 .8 6 1 9 7 .2 1 b d l 7 .1 0 b d l 1 9 .5 8 d 2 6 .6 0 b d l 2 0 3 .7 6 3 5 .9 6 b d l 3 5 4 2 3 .0 2 6 .1 2 6 0 9 .7 5 b d l 1 3 .4 0 5 .3 0 4 1 .7 1 d 3 b d l b d l b d l b d l b d l 1 4 9 4 8 .0 9 b d l 3 3 0 .2 6 b d l 7 .3 0 4 .7 9 1 7 .3 5 d 4 7 .0 9 b d l 2 0 3 .4 2 b d l b d l 8 1 0 6 9 .8 0 8 .6 7 1 2 6 1 .6 0 b d l 2 2 .8 5 4 .2 2 6 4 .2 7 d 5 3 .5 5 b d l b d l b d l b d l 2 1 4 0 1 .3 3 5 .5 9 2 0 4 .9 5 b d l 4 .7 3 b d l 2 6 .9 4 d 6 b d l b d l b d l b d l b d l 4 0 1 6 .1 4 5 .8 7 3 7 5 .8 5 b d l b d l b d l 5 .3 2 d 7 b d l b d l b d l b d l b d l 4 4 7 6 .6 7 5 .4 9 1 7 4 .7 3 b d l b d l b d l 5 .6 8 d 8 b d l b d l b d l b d l b d l 4 2 6 2 .8 1 6 .2 6 1 5 3 .8 8 b d l b d l b d l b d l d 9 b d l b d l b d l b d l b d l 2 3 8 5 .5 2 4 .9 0 1 6 6 .9 4 b d l b d l b d l b d l d 1 0 b d l b d l b d l b d l b d l 5 2 0 5 .5 5 b d l 1 9 4 .2 1 b d l b d l b d l b d l m e a n 1 .7 2 b d l 4 0 .7 2 3 .5 6 b d l 1 8 1 6 2 .0 9 5 .0 8 3 6 6 .9 4 b d l 5 .5 4 1 .4 3 1 8 .0 9 a v e r a g e s h a le v a lu e s [1 7 ] 1 3 .0 0 0 .3 0 1 9 .0 0 9 0 .0 0 4 5 .0 0 4 7 2 0 0 .0 0 0 .4 0 8 5 0 .0 0 6 8 .0 0 2 0 .0 0 3 .7 0 9 6 .0 0 d p r t a r g e t v a lu e s [1 9 ] 1 .0 0 0 .8 0 2 0 .0 0 1 0 0 .0 0 3 6 .0 0 3 8 0 0 0 .0 0 4 .0 0 8 5 0 .0 0 3 5 .0 0 8 5 .0 0 2 3 .0 0 1 4 0 .0 0 t r [ 2 1 ] 1 0 .0 0 3 0 .0 0 5 .0 0 2 .0 0 5 .0 0 n a 4 0 .0 0 1 .0 0 5 .0 0 5 .0 0 n a 1 .0 0 t a b le 2 : h e a v y m e ta l c o n c e n tr a ti o n s (m g /k g ) o f th e s tu d ie d s o il s a m p le s. n a = n o t a v a il a b le ; b d l = b e lo w d e te c ta b le l im it ; d p r = d e p a rt m e n t o f p e tr o le u m r e so u rc e s (n ig e ri a ); t r = t o x ic r e sp o n se f a c to r j. multidiscip. appl. nat. sci. 7 than that of other metals but was below dpr and average shale reference values [19]. also, the mean concentration of fe is higher in doma (18162.09 mg/kg) than in nasarawa eggon (17048.87 mg/kg) area. the mean concentrations of cd, cr, cu, fe, mn, ni, u and zn for both study areas are lower than the reference values of dpr [19] and average shale [17] while the mean concentration of hg is higher than the reference values. however, the mean concentrations of as in nasarawa eggon and doma areas, are below the average shale [17] value but above the dpr [19] value. contrarily, the mean co concentration in nasarawa eggon is below the aforementioned reference values but above them (reference values) in the doma area. arsenic (as) is naturally found in water, soil, and sediments [23]. the nature of the soil parent material appears to be the main factor that determines the as concentration in soils, although due to its low supergene mobility, the soils are slightly enriched in as compared with their soil parent rocks [24]. as is not only toxic but rated to be carcinogenic which causes skin lesions such as hyperpigmentation (especially on the trunk), keratosis on the palms and soles, chronic cough, crepitation in the lungs, diabetes mellitus, hypertension, and weakness [23]. in this study, as levels recorded in the sampled sites in nasarawa eggon (except n2 and n4) and doma (except d2, d4 & d5) study areas are below the instrumentation limit of detection. however, in nasarawa eggon, the level of as in sampled site n2 is above dpr [19] and average shale [17] reference values while in sampled site n4 (nasarawa eggon) as well as sampled sites d2, d4 and d5 (3.55–6.60 mg/kg) of doma study area, as levels are above the dpr [19] value but below average shale [17] value. this indicates possible anthropogenic contribution to as level in sampled site n2 while lithogenic origin with little or no anthropogenic contribution could be the source of as in other sampled sites including n4, d2, d4 and d5. the range of as in this study is higher than those recorded by edori and kpee [9] and fosu–mensah et al. [25] for soils within selected abattoirs in portharcourt, nigeria and soils at korle lagoon area in accra, ghana; respectively. however, li et al. [26] and santosfrancés et al. [27] reported as concentrations higher than the findings of this study, for surface soils in electronic waste dismantling area and soils of the andes mountain range, respectively. the concentrations of copper (cu) and cadmium (cd) in the various investigated soil samples from the study areas are below the instrumentation detectable limit and the reference values of dpr [19] and average shale [17]. this suggests little or no possible anthropogenic contribution (such as fertilizer, pesticides, plastics, plumbing, welding etc.) to the presence of cu and cd in the sampled soils of both study areas. thus, their presence is of lithogenic or natural origin. cu is an essential micronutrient required for plant growth. although a human can handle a proportionally high concentration of cu, human exposure to it causes a variety of health problems. at lower concentrations, it causes headache, nausea, vomiting and diarrhea while at significant concentrations, it causes to anaemia, gastrointestinal disorder, flu-like condition known as metal fever, liver, and kidney malfunctioning in extreme cases [28][29]. on the other hand, cd is a toxic metal that occurs naturally at a low level in the environment [30] and can disrupt biological systems more than most toxic metals, at acute and chronic exposures through inhalation and ingestion [31]. chronic exposure leads to ulcerations and perforations of the nasal septum, chronic bronchitis, decreased pulmonary function, pneumonia, bone disease, coughing, emphysema, headache, hypertension, kidney diseases, lung and prostate cancer, lymphocytosis, microcytic hypochromic anaemia, testicular atrophy and vomiting [32][33]. studies by iwegbue et al. [34], amostautua et al. [35], asawalam and eke [36] and njoku and ayoka [37] reported similar low concentration range of cd for urban agricultural and waste dump soils. however, higher cu concentrations were recorded by fosu–mensah et al. [25], esen et al. [38], tang et al. [39] for soils at korle lagoon area in accra, ghana; surface sediments of nemrut bay, aegean sea and soils from wenling city, respectively. cobalt (co) is a natural occurring essential element to plants (though still under consideration) and has some biological functions in humans. soil pollution problems from co are generally less significant than those associated with some other heavy metals [40]. in this study, co concentrations in sampled sites from nasarawa eggon and doma 8 s a m p le c o d e a s c d c o c r c u f e h g m n n i p b u z n n a sa r a w a e g g o n n 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 2 4 7 .0 5 2 .7 8 0 .0 0 1 1 .4 1 0 .0 0 3 .2 0 n 2 2 .2 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 2 7 .0 2 1 .5 2 0 .0 0 2 .5 4 0 .0 0 0 .8 8 n 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 2 7 .0 2 1 .1 0 0 .0 0 2 .4 8 4 .2 8 0 .6 2 n 4 1 .6 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 0 .0 0 1 .2 1 0 .0 0 5 .8 2 0 .0 0 3 .0 8 n 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 4 2 .3 5 2 .1 0 0 .0 0 6 .6 7 0 .0 0 0 .7 8 n 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 1 5 9 .8 5 5 .0 8 0 .0 0 1 1 .9 1 0 .0 0 1 .3 8 n 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 0 .0 0 1 .9 8 3 .6 0 8 .7 9 0 .0 0 0 .9 2 n 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 5 0 .1 0 1 .9 6 0 .0 0 9 .6 1 0 .0 0 0 .9 2 n 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 5 0 .6 7 1 .6 9 0 .0 0 3 .7 5 0 .0 0 0 .3 2 n 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 0 .0 0 3 .2 4 0 .0 0 1 1 .7 2 0 .0 0 4 .2 1 m e a n 0 .3 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 6 0 .4 1 2 .2 7 0 .0 4 7 .4 7 0 .0 4 1 .6 3 d o m a d 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 1 0 9 .9 9 1 .3 0 0 .0 0 1 .9 9 0 .0 0 1 .1 4 d 2 0 .6 8 0 .0 0 1 4 .2 9 0 .5 3 0 .0 0 1 .0 0 2 0 .3 9 0 .9 6 0 .0 0 0 .8 9 1 .9 1 0 .5 8 d 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 0 .0 0 1 .2 3 0 .0 0 1 .1 5 4 .0 9 0 .5 7 d 4 0 .3 1 0 .0 0 6 .2 3 0 .0 0 0 .0 0 1 .0 0 1 2 .6 2 0 .8 6 0 .0 0 0 .6 7 0 .6 6 0 .3 9 d 5 0 .6 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 3 0 .8 2 0 .5 3 0 .0 0 0 .5 2 0 .0 0 0 .6 2 d 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 1 7 2 .4 7 5 .2 0 0 .0 0 0 .0 0 0 .0 0 0 .6 5 d 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 1 4 4 .7 1 2 .1 7 0 .0 0 0 .0 0 0 .0 0 0 .6 2 d 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 1 7 3 .2 8 2 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 d 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 2 4 2 .3 8 3 .8 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 d 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .0 0 0 .0 0 2 .0 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 m e a n 0 .1 6 0 .0 0 2 .0 5 0 .0 5 0 .0 0 1 .0 0 9 0 .6 7 2 .0 2 0 .0 0 0 .5 2 0 .6 7 0 .4 6 t a b le 3 . e n ri c h m e n t f a c to r (e f) f o r h e a v y m e ta ls i n n a sa ra w a e g g o n a n d d o m a s o il s a m p le s j. multidiscip. appl. nat. sci. 9 (except d2 and d4) are below the instrumentation detectable limit (bdl), dpr [19] and average shale [17] reference values. this implies that the presence of co in these sampled sites could be due to natural (lithogenic) processes such as fragmentation or weathering of rock. however, co levels in d2 and d4 of the doma study area are significantly higher than the reference values. this indicates anthropogenic contribution (such as automobile workshop waste, burning of municipal waste, cola combustion, fertilizer, leachate from pb mining and processing etc.) to the level of co in these two sampled sites in doma. a high level of co intake could depress fe and cu levels in the body which can lead anaemia [41]. human exposure to co can be through occupational, dietary, environmental and medical pathways or sources and chronic exposure can cause skin rashes, asthma, neurological (e.g., hearing and visual impairments), cardiovascular, and endocrine deficits [42]. the concentrations of cr in sampled soils from nasarawa eggon and doma are below the dpr [19] and average shale [17] values, indicating lithogenic or natural or crustal origin (such as rock weathering/fragmentation) of cr in all sampled sites of the study areas. this is in agreement with findings by amos-tautua et al. [35] for soils of municipal open waste dumpsite in yenagoa, nigeria but contradicts higher cr concentrations reported by dawaki et al. [43] and agbaji et al. [44] for kano urban agricultural soils and soils in kakuri industrial area of kaduna, nigeria; respectively. thus, the highest cr level of 35.96 mg/kg was recorded in sampled site d2 which suggests a possible little anthropogenic contribution to crustal origin (such as rock weathering/fragmentation) of cr in sampled site d2. anthropogenic sources of cr in the soils could be due to waste consisting of leadchromium batteries, coloured polythene bags, discarded plastic materials and empty paint containers [45]. cr (iii) is not toxic but cr (vi) is a carcinogen [46] whose acute toxicity is due to oxidation properties, hemolysis and organ failures [47]. occupational exposure to cr is mostly by inhalation, but gastrointestinal tract and skin can occur [48]. cr (vi) corrodes skin and causes denaturation and precipitation of tissue proteins [49]. iron (fe) is one of the most abundant naturally occurring elements in the earth’s crust. in this study, it is the most abundant metal present in all the soil samples analysed. the concentration varied from 4403.79–30662.08 mg/kg and 2385.52– 81069.80 mg/kg for nasarawa eggon and doma soils respectively (table 2). thus, fe levels in the various sampled soils of both study areas are below the dpr target [19] and average shale [17] values except for sample d4 (81069.80 mg/kg) in the doma area. the findings of this study are significantly higher than those reported by iwegbue et al. [34] and osakwe [50] for soils around cassava processing mills in sub-urban areas of delta state, nigeria and soils from automobile workshops in abraka, delta state, nigeria; respectively. high fe levels obtained in this study could be a combined effect of lithological or crustal and anthropogenic origins based on the fact that natural soils contain significant amounts of fe [51]. thus, the presence of fe in the soil could be attributed to weathering of rock types, geothermal activities, indiscriminate disposal and discharge from automobile waste (metal scraps from vehicle repairs, crankshafts wear), sewage discharge etc. acute exposure to fe in humans leads to vomiting, cardiac depression and metabolic acidosis [52]. mercury (hg) is one of the most toxic metals generated naturally in the environment from degassing of the earth’s crust during volcanic emissions. result (table 2) shows that hg level was significantly found in most of the analysed soils from nasarawa eggon and doma areas ranging from bdl – 10.73 mg/kg and bdl – 8.67 mg/kg respectively. thus, hg levels in a few of the nasarawa eggon (n4, n7 and n10) and doma (d3 and d10) soils, are below the instrumental detectable limit and reference values of dpr [19] and average shale [17]. however, hg concentrations of the other soils which varied from 5.43–10.73 mg/kg and 4.90–8.67 mg/kg for nasarawa eggon and doma areas respectively; are above the reference values. higher hg concentrations were reported for simple household e–waste recycling workshops [39] and soils in the kakuri industrial area of kaduna, nigeria [44]. however, hg levels lower than the findings of this study were reported in other literature [25][39][53] for e–waste dumpsites. hg levels in the sampled sites could be attributed to lithogenic (such as rock 10 s a m p le c o d e a s c d c o c r c u f e h g m n n i p b u z n n a sa r a w a e g g o n n 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 4 .0 1 3 .9 4 – 2 .5 3 0 .0 0 – 0 .4 9 0 .0 0 – 2 .3 3 n 2 – 0 .0 2 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 1 .2 1 3 .5 5 – 0 .6 0 0 .0 0 0 .1 4 0 .0 0 – 1 .3 9 n 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 1 .4 5 3 .3 0 – 1 .3 1 0 .0 0 – 0 .1 4 0 .6 5 – 2 .1 3 n 4 – 1 .5 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 2 .2 1 0 .0 0 – 1 .9 3 0 .0 0 0 .3 3 0 .0 0 – 0 .5 8 n 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 1 .6 8 3 .7 2 – 0 .6 1 0 .0 0 1 .0 6 0 .0 0 – 2 .0 3 n 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 3 .7 5 3 .5 7 – 1 .4 1 0 .0 0 – 0 .1 8 0 .0 0 – 3 .2 9 n 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 2 .5 1 0 .0 0 – 1 .5 2 – 0 .6 6 0 .6 3 0 .0 0 – 2 .6 2 n 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 2 .4 7 3 .1 8 – 1 .5 0 0 .0 0 0 .8 0 0 .0 0 – 2 .5 9 n 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 1 .5 0 4 .1 6 – 0 .7 4 0 .0 0 0 .4 0 0 .0 0 – 3 .1 4 n 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 2 .0 9 0 .0 0 – 0 .3 9 0 .0 0 1 .4 6 0 .0 0 – 0 .0 2 m e a n – 0 .1 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 2 .2 9 2 .5 4 – 1 .2 5 – 0 .0 7 0 .4 0 0 .0 7 – 2 .0 1 d o m a d 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 3 .0 7 3 .7 1 – 2 .6 9 0 .0 0 – 2 .0 8 0 .0 0 – 2 .8 8 d 2 – 1 .5 6 0 .0 0 2 .8 4 – 1 .9 1 0 .0 0 – 0 .1 0 3 .3 5 – 1 .0 6 0 .0 0 – 1 .1 6 – 0 .0 7 – 1 .7 9 d 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 2 .2 4 0 .0 0 – 1 .9 5 0 .0 0 – 2 .0 4 – 0 .2 1 – 3 .0 5 d 4 – 1 .4 6 0 .0 0 2 .8 4 0 .0 0 0 .0 0 0 .2 0 3 .8 5 0 .7 2 0 .0 0 – 0 .3 9 – 0 .4 0 – 1 .1 6 d 5 – 2 .4 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 1 .7 3 3 .2 2 – 2 .6 4 0 .0 0 – 2 .6 7 0 .0 0 – 2 .4 2 d 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 4 .1 4 3 .2 9 – 1 .7 6 0 .0 0 0 .0 0 0 .0 0 – 4 .7 6 d 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 3 .9 8 3 .1 9 – 2 .8 7 0 .0 0 0 .0 0 0 .0 0 – 4 .6 6 d 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 4 .0 5 3 .3 8 – 3 .0 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 d 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 4 .8 9 3 .0 3 – 2 .9 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 d 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 – 3 .7 7 0 .0 0 – 2 .7 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 m e a n – 0 .5 5 0 .0 0 0 .5 7 – 0 .1 9 0 .0 0 – 2 .7 8 2 .6 4 – 2 .0 9 0 .0 0 – 0 .8 3 – 0 .0 7 – 2 .0 7 t a b le 4 . g e o -a c c u m u la ti o n i n d e x ( ig e o ) o f h e a v y m e ta ls i n s e le c te d s o il s o f n a sa ra w a e g g o n a n d d o m a j. multidiscip. appl. nat. sci. 11 fragmentation, volcanic eruptions, geothermal activities etc.) and anthropogenic (such as discharges from used batteries, household e–waste, paint wastes, coal combustion, manometer at pressure measuring sites and hydraulic lifts attached to heavy-duty trucks on the ever-busy roads) sources. exposure to hg can have some adverse effects on humans such as ataxia, attention deficit, blindness, deafness, decreased rate of fertility, dementia, dizziness, dysphasia, gastrointestinal irritation, gingivitis, kidney problem, loss of memory, pulmonary edema, reduced immunity and sclerosis [33][54]. manganese (mn) is the second most abundant metal found in all the sampled sites of the study areas and the concentration varied from 220.43– 971.81 mg/kg and 153.88–1261.60 mg/kg for nasarawa eggon and doma sampled sites respectively. mn concentrations in samples n10 (971.81 mg/kg) and d4 (1261.60 mg/kg) from nasarawa eggon and doma respectively, are above the dpr target [19] and average shale [17] values while the concentrations of mn in the other analysed soils from both study areas are below the aforementioned reference values. in comparison to the findings of this study, osakwe [50] found lower concentrations of mn in soils from automobile workshops. high mn levels could be attributed to lithogenic origin based on the fact that in the form of oxide, mn is a component of subsoil material [55]. however, high levels of mn in sampled sites n10 and d4 indicates possible contributions from anthropogenic sources such as used batteries, discarded metal scraps, machinery parts, automobile exhaust fume and wastes from welding works and spray paintings of the vehicles [50][56] [57]. mn is an essential microelement for both plants and animals. however, exposure to a high dose of mn results in kidney failure, liver and pancreas malfunctioning low fetal birth weight and increased infant mortality [58]. nickel (ni) is absorbed through the lungs [59], gastrointestinal tract [60] and skin [61], but excreted in the urine [62]. human exposure to nickel causes a variety of pathologic effects but its adverse health effects are dependent on exposure route (inhalation, oral or dermal) and classification based on systemic, immunologic, neurologic, reproductive, distorted developmental or carcinogenic effects, following acute (1 day), subchronic (10–100 days) and chronic (100 days or more) exposure periods [63]. in this study, ni concentrations in the sampled sites of nasarawa eggon (except n7) and doma study areas are below the instrumentation detectable limit and the reference values. ni was significantly found only in site n7 (64.59 mg/kg) of the nasarawa eggon area which is above the reference value by dpr [19] but below that of the average shale [17] value. this implies that the presence of ni in sampled site n7 is possibly due to anthropogenic origin such as leaching from metal scraps, runoff of paints/ pigment waste, discharge from sewage and indiscriminate waste dumps etc. lead (pb) is the most immobile element present in the soil [64]. a literature survey showed that pb has several negative health implications on various organs and systems in all living species under experimental conditions, including the blood, reproductive, immune system and kidneys [65]. pb is a highly neurotoxic agent that particularly affects the development of the central nervous system (cns) and consequently can be the cause of many negative effects on humans such as anorexia, chronic nephropathy, damage to neurons, high blood pressure, hyperactivity, insomnia, learning deficits, reduced fertility (by reducing testosterone production in vitro and in vivo), renal system damage, a risk factor for alzheimer's disease and shortened attention span [15][66]. in this study, the pb concentrations in the investigated soils from nasarawa eggon varied from 21.29–82.64 mg/kg and thus, are above the average shale [17] value but below the dpr [19] value. however, only pb concentration in sample d4 of the doma study area was above the average shale [17] value but below the dpr [19] value. hence, the pb concentrations in the remaining analysed doma soils are below both aforementioned reference values. pb concentrations in the sampled soils of both study areas could be a contribution from anthropogenic sources mainly from atmospheric depositions from automobile exhaust and generator emissions/fumes and others such as used dry cell batteries, sewage effluents, runoff of paint/pigment wastes and leachate from municipal waste, owing to the close proximity of the sites to high vehicular traffic [35] [54]. the low concentration range of pb in most sampled sites in 12 c f c d p l i s a m p le c o d e a s c d c o c r c u f e h g m n n i p b u z n n a sa r a w a e g g o n n 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .1 2 2 .3 1 0 .2 6 0 .0 0 0 .2 5 0 .0 0 0 .2 0 3 .1 4 0 .6 3 n 2 1 9 .2 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .8 1 1 .7 6 0 .9 9 0 .0 0 0 .3 9 0 .0 0 0 .3 9 2 3 .6 3 1 .1 3 n 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .6 8 1 .4 8 0 .6 0 0 .0 0 0 .3 2 0 .3 8 0 .2 3 3 .6 9 0 .7 1 n 4 6 .9 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .4 0 0 .0 0 0 .3 9 0 .0 0 0 .4 4 0 .0 0 0 .6 9 8 .8 2 0 .9 1 n 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .5 8 1 .9 8 0 .9 8 0 .0 0 0 .7 3 0 .0 0 0 .2 5 4 .5 2 0 .8 8 n 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .1 4 1 .7 8 0 .5 7 0 .0 0 0 .3 1 0 .0 0 0 .1 1 2 .9 1 0 .6 4 n 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .3 3 0 .0 0 0 .5 2 1 .8 5 0 .5 5 0 .0 0 0 .1 7 3 .4 2 0 .7 5 n 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .3 4 1 .3 5 0 .5 3 0 .0 0 0 .6 1 0 .0 0 0 .1 7 3 .0 0 0 .7 4 n 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .6 6 2 .6 8 0 .9 0 0 .0 0 0 .4 7 0 .0 0 0 .1 2 4 .8 3 0 .8 2 n 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .4 4 0 .0 0 1 .1 4 0 .0 0 0 .9 7 0 .0 0 1 .0 2 3 .5 7 0 .9 4 m e a n 2 .6 2 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .4 5 1 .3 3 0 .6 9 0 .1 9 0 .5 0 0 .0 4 0 .3 4 6 .1 5 0 .8 2 d o m a d 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .2 2 1 .9 7 0 .2 3 0 .0 0 0 .0 8 0 .0 0 0 .1 4 2 .6 4 0 .5 7 d 2 6 .6 0 0 .0 0 1 0 .1 9 0 .3 6 0 .0 0 0 .9 3 1 .5 3 0 .7 2 0 .0 0 0 .1 6 0 .2 3 0 .3 0 2 1 .0 2 0 .9 0 d 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .3 9 0 .0 0 0 .3 9 0 .0 0 0 .0 9 0 .2 1 0 .1 2 1 .2 0 0 .5 2 d 4 7 .0 9 0 .0 0 1 0 .1 7 0 .0 0 0 .0 0 2 .1 3 2 .1 7 1 .4 8 0 .0 0 0 .2 7 0 .1 8 0 .4 6 2 3 .9 5 1 .2 2 d 5 3 .5 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .5 6 1 .4 0 0 .2 4 0 .0 0 0 .0 6 0 .0 0 0 .1 9 6 .0 0 0 .6 7 d 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .1 1 1 .4 7 0 .4 4 0 .0 0 0 .0 0 0 .0 0 0 .0 4 2 .0 6 0 .6 1 d 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .1 2 1 .3 7 0 .2 1 0 .0 0 0 .0 0 0 .0 0 0 .0 4 1 .7 4 0 .5 8 d 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .1 1 1 .5 7 0 .1 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .8 6 0 .7 5 d 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 6 1 .2 3 0 .2 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 1 .4 9 0 .7 0 d 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .1 4 0 .0 0 0 .2 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .3 7 0 .7 5 m e a n 1 .7 2 0 .0 0 2 .0 4 0 .0 4 0 .0 0 0 .4 8 1 .2 7 0 .4 3 0 .0 0 0 .0 7 0 .0 6 0 .1 3 6 .2 4 0 .7 3 t a b le 5 . c o n ta m in a ti o n f a c to r (c f) , d e g re e o f c o n ta m in a ti o n ( c d ) a n d p o ll u ti o n l o a d i n d e x ( p l i) o f th e s o il s a m p le s j. multidiscip. appl. nat. sci. 13 doma could be due to natural processes such as fragmentation of source rocks. it was observed that the concentrations of pb are higher in nasarawa eggon sampled sites than in doma. this suggests higher vehicular/automobile traffic in the nasarawa eggon area than in doma which is not surprising because nasarawa eggon has accessibility to many economic locations than doma. nduka et al. [29] who investigated pb in scrap paint dust reported lower pb concentrations while the results of ololade [67] investigation on soils within auto–mechanic workshops, agree with those of this study. uranium (u) is a naturally occurring radioactive and chemical metal with a global background concentration in the earth's crust of approximately 2 –4 mg/kg. natural u is approximately 10 times more abundant than other toxic heavy metals such as cadmium (0.3 mg/kg) or mercury (0.4 mg/kg) [15][17]. table 2 showed that u concentrations in most sampled sites of nasarawa eggon and doma study areas were below the instrumentation limit of detection. therefore, significant amounts of u were only determined in sampled sites n3 (8.68 mg/kg) of nasarawa eggon and d2 (5.30 mg/kg), d3 (4.79 mg/kg) and d4 (4.22 mg/kg) of doma. this implies they are above the average shale [17] reference value but below the dpr [19] value and hence suggests a contribution of anthropogenic sources (such as coal and fuel combustion) to the presence of u in these soils (n3, d2, d3 and d4). u has biologically dynamic toxicity, metabolic toxicity and chemical toxicity, leading to potential longterm harm to mammalian reproduction and development with reduced biological fertility, abnormal and slow embryonic development [68]. a similar low u concentration was recorded in southeastern brazil mine [69] and sediments of watersheds in abiete-toko gold district, southern cameroon [15]. zinc (zn) is a micronutrient essential for normal plant growth which is involved in various metabolic activities of many organisms [70]. human exposure to a high dose of zn can cause many health disorders such as ataxia, depression, gastrointestinal irritation, haematuria, icterus, impotence, kidney and liver failure, lethargy, macular degeneration, metal fume fever, prostate cancer, seizures, and vomiting for zn [71]. result (table 2) shows that zn concentrations in the sampled sites in nasarawa eggon (except n4 and n10) and doma are lower than the reference values of average shale [17] and dpr [19]. the level of zn in n10 soil of nasarawa eggon is above the reference values while in sample n4, the zn level was above the average shale [17] value but below the dpr [19] value. similar to the trend of pb concentration, higher zn concentrations were also observed in nasarawa eggon sampled sites than in doma. this supports the earlier suggestion of higher automobile/ vehicular traffic as well as generator usage in the studied nasarawa eggon area than in the doma area. zn and pb are products of gasoline combustion from automobiles and generators exhaust [72]. other anthropogenic sources such as runoff of paint/pigment wastes, smelting activities, indiscriminate disposal and dumping of zn containing wastes, crude oil tyre etc. [72] could be responsible for the high concentrations of zn observed while lithogenic processes were indicated by low zn concentration especially in doma study area. according to jaradat and momani [73], the major sources of zn in soils are probably the attrition of motor vehicle tyre rubber and the lubricating oils in which zn is found as part of many additives as zinc dithiophosphates. result obtained in this study is within the zn concentrations reported by dawaki et al [43] and iwegbue et al. [34] for kano urban agricultural soils and soils around cassava processing mills in suburban areas of delta state, southern nigeria respectively. however, higher zn concentrations were found in automobile workshop soils as reported by osakwe [50]. 3.2. soil pollution indices or assessment in order to understand the quality of agricultural soils along major roads with high vehicular and human traffic, levels of metal pollution were evaluated using different techniques for environmental assessment. 3.2.1. enrichment factor the enrichment factor (ef) is generally used as an appropriate method to discriminate between natural and anthropogenic sources of metals [74] and to reflect the status of environmental contamination, based on the use of a normalization element in order to improve the variations produced 14 e r r i s a m p le c o d e a s c d c o c r c u f e h g m n n i p b u z n n a sa r a w a e g g o n n 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 9 2 .4 0 0 .2 6 0 .0 0 1 .2 5 n c 0 .2 0 9 4 .1 1 n 2 1 9 2 .9 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 7 0 .4 0 0 .9 9 0 .0 0 1 .9 5 n c 0 .3 9 2 6 6 .6 3 n 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 5 9 .2 0 0 .6 0 0 .0 0 1 .6 0 n c 0 .2 3 6 1 .6 3 n 4 6 9 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 0 .0 0 0 .3 9 0 .0 0 2 .2 0 n c 0 .6 9 7 2 .2 8 n 5 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 7 9 .2 0 0 .9 8 0 .0 0 3 .6 5 n c 0 .2 5 8 4 .0 8 n 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 7 1 .2 0 0 .5 7 0 .0 0 1 .5 5 n c 0 .1 1 7 3 .4 3 n 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 0 .0 0 0 .5 2 9 .2 5 2 .7 5 n c 0 .1 7 1 2 .6 9 n 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 5 4 .0 0 0 .5 3 0 .0 0 3 .0 5 n c 0 .1 7 5 7 .7 5 n 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 1 0 7 .2 0 0 .9 0 0 .0 0 2 .3 5 n c 0 .1 2 1 1 0 .5 7 n 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 0 .0 0 1 .1 4 0 .0 0 4 .8 5 n c 1 .0 2 7 .0 1 m e a n 2 6 .1 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 5 3 .3 6 0 .6 9 0 .9 3 2 .5 2 n c 0 .3 4 8 4 .0 2 d o m a d 1 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 7 8 .8 0 0 .2 3 0 .0 0 0 .4 0 n c 0 .1 4 7 9 .5 7 d 2 6 6 .0 0 0 .0 0 5 0 .9 5 0 .7 2 0 .0 0 n c 6 1 .2 0 0 .7 2 0 .0 0 0 .8 0 n c 0 .3 0 1 8 0 .6 9 d 3 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 0 .0 0 0 .3 9 0 .0 0 0 .4 5 n c 0 .1 2 0 .9 6 d 4 7 0 .9 0 0 .0 0 5 0 .8 5 0 .0 0 0 .0 0 n c 8 6 .8 0 1 .4 8 0 .0 0 1 .3 5 n c 0 .4 6 2 1 1 .8 4 d 5 3 5 .5 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 5 6 .0 0 0 .2 4 0 .0 0 0 .3 0 n c 0 .1 9 9 2 .2 3 d 6 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 5 8 .8 0 0 .4 4 0 .0 0 0 .0 0 n c 0 .0 4 5 9 .2 8 d 7 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 5 4 .8 0 0 .2 1 0 .0 0 0 .0 0 n c 0 .0 4 5 5 .0 5 d 8 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 6 2 .8 0 0 .1 8 0 .0 0 0 .0 0 n c 0 .0 0 6 2 .9 8 d 9 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 4 9 .2 0 0 .2 0 0 .0 0 0 .0 0 n c 0 .0 0 4 9 .4 0 d 1 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 0 .0 0 n c 0 .0 0 0 .2 3 0 .0 0 0 .0 0 n c 0 .0 0 0 .2 3 m e a n 1 7 .2 4 0 .0 0 1 0 .1 8 0 .0 7 0 .0 0 n c 5 0 .8 4 0 .4 3 0 .0 0 0 .3 3 n c 0 .1 3 7 9 .2 2 t a b le 6 . e c o lo g ic a l ri sk ( e r) a n d p o te n ti a l e c o lo g ic a l ri sk i n d e x ( r i) i n t h e s o il s a m p le s n c : n o t c a lc u la te d j. multidiscip. appl. nat. sci. 15 by heterogeneous sediments [75]. the ef values close to unity indicate crusted origin, those less than 1.0 suggest a possible mobilization or depletion of metals, whereas ef > 1.0 indicates that the element is of anthropogenic origin [76]. the ef values obtained in this study for the soil samples from the study areas are presented in table 3 and birth [77] categorization of ef values (table 1) was used for the interpretation. based on the average, both nasarawa eggon and doma sampled soils are enriched minimally and extremely severe with mn and hg respectively. pb and zn are enriched at significant and minimal levels, respectively, in nasarawa eggon while co is minimally enriched in doma soils. as, cd, cr, cu, ni and u are not enriched in the sampled soils of both study areas. a comparison among different sampling sites of the study areas based on ef values of metals showed no enrichment of cd, cr, and cu in all the investigated soils of both study areas. in most of the sampled soils of both study areas, hg has the highest enrichment level ranging from severe to extremely severe enrichment. hg is not enriched in sampled sites n4, n7 and n10 of nasarawa eggon soils and d3 and d10 of doma soils. the ef values indicate minimal to a severe enrichment level of pb in nasarawa eggon soils and no enrichment to minimal enrichment levels in doma soils. mn is enriched at a minimal to moderate level in nasarawa eggon and no enrichment to a moderate level in doma soil. the u enrichment levels range from no enrichment to moderate at both nasarawa eggon and doma soils. enrichment levels of as and ni in nasarawa eggon soils range from no enrichment to moderate enrichment while there was no enrichment of either of the metals in doma soils. sampled sites n2 and n4 are minimally enriched with as while n7 and n3 are moderately enriched with ni and u respectively. co is not enriched in all the investigated soils from nasarawa eggon but ranged from no enrichment to severely enrich in doma soils. zn enrichment varied from no enrichment to moderate enrichment level in nasarawa eggon soils but in doma soils, it varied from no enrichment to minimal enrichment. zn is not enriched in most of the nasarawa eggon soils except in n1, n4 and n10 which have moderate enrichment and n6 with minimal enrichment. only sampled site d1 in doma soils is enriched minimally with zn, with no enrichment in other sampled sites. it is obvious from the results (table 3) that the investigated soils of nasarawa eggon and doma are highly enriched with hg and mn, as well as pb and zn in nasarawa eggon only and co in doma only. this is an indication that in addition to natural sources of these heavy metals, there is a significant contribution from anthropogenic sources to the presence of these heavy metals in the soils [78]. studies by gibson and farmer [79] and sezgin et al. [80] established that vehicular emission, industrial production and weathered materials are the three main sources of heavy metals in urban areas. consequently, the high ef of pb, mn and zn in the soils could be attributed to automobile/ vehicular traffic because sutherland [16] had reported that automobile/vehicular exhaust emissions significantly accentuate pb accumulations in the air and soils. automobile emission accounts for about 80 % of heavy metal pollution in nigeria [81] and has been shown to contain pb and mn from gasoline as well as zn from tyres [72]. weathering of rocks, burning of fossil fuels (petroleum and coals) and municipal waste (containing e-wastes, automobile and automobile workshop wastes, and medical wastes) [39] in these study areas are the probable sources (lithogenic and anthropogenic) of hg, pb, mn, and zn, in the investigated soils. the literature revealed that anthropogenic sources contribute more to heavy metal pollution than natural sources [9][25]. the ef values of as, cd, co, cr, cu, ni and u, as well as pb and zn in doma; suggest they were majorly derived from crustal material, or natural weathering process [82]. 3.2.2. geo–accumulation index (igeo) the level of pollution of the investigated soils was assessed using igeo indices proposed by muller [18] as shown in table 1. the igeo values of the different metals at different sampled sites in the study areas are presented in table 4. the average igeo values of the metals suggest that the soils from the study areas (nasarawa eggon and doma) are moderate to strongly polluted by hg and practically unpolluted to moderately polluted by the rest of the metals. thus, hg is the only toxic metal that has j. multidiscip. appl. nat. sci. 16 accumulated in the investigated soils as a result of indiscriminate disposal of municipal waste and high automobile traffic. a comparison of igeo values among different sampled sites of the study areas showed that sampled sites in nasarawa eggon are strong to extremely polluted by hg except n4, n7 and n10 which are practically unpolluted by hg. however, sampled site n10 is moderately polluted by pb and so is n5 which is also strongly polluted by hg. thus, hg and pb are two toxic metals that accumulate in studied agricultural soils of nasarawa eggon. the igeo values of doma investigated soils showed that except for d3 and d10, all sampled sites are strongly polluted by hg. sampled sites d2 and d4 are moderate to strongly polluted by co. hence, hg and co are the accumulating toxic metals in doma studied agricultural soils which can be attributed to the combustion of household e– waste, fertiliser, automobile workshop waste etc. 3.2.3. contamination factor (cf), degree of contamination (cd) and pollution load index (pli) the values of cf, cd and pli at the sampled sites of both study areas are summarized in table 5. applying hakanson [21] classification (table 1), the sampled sites in the nasarawa eggon area showed low contamination by cd, co, cr, cu, fe, pb, u and zn (table 5). however, sampled sites n2 and n4 are highly contaminated by as while n7 and n10 are moderately contaminated by ni and mn respectively. except for n4, n7 and n10, all sampled sites in nasarawa eggon are moderately contaminated by hg. the sampled sites in doma showed low contamination by cd, cr, cu, mn, ni, pb, u and zn based on their cf values. d2 and d4 showed high contamination by as and co. d4 is moderately contaminated by fe while d5 is considerably contaminated by as. all sampled sites are moderately contaminated with hg except for d3 and d10. high as contamination of some sampled sites showed that a metal that highly contaminated soil may not necessarily accumulate to cause pollution. this is because igeo did not indicate that as is one of the toxic metals that accumulates in either nasarawa eggon or doma area. similarly, low pb contamination of the sampled sites in the nasarawa eggon area showed that even at the low contamination, a metal can still accumulate and pollute the site. cd measures the degree of overall contamination at a particular site [83]. cd showed that sampled sites in nasarawa eggon had a low degree of contamination except for n2 and n4 which had a moderate degree of contamination. sampled sites d2 and d4 in the doma area, had a moderate degree of contamination while other sample sites had a low degree of contamination. the pli represents the number of times by which the metal content in the soil exceeds the average natural background concentration [67]. the pli value greater than one (1) is considered polluted, less than one (1) indicates no pollution whereas a value equal to one (1) indicates contaminant loads close to the reference concentration [84]. results (table 5) show that only sampled sites n2 and d4 of nasarawa eggon and doma, respectively, were polluted (pli >1). thus, this study has shown that a sampled site can be contaminated by metals but not polluted. 3.2.4. ecological risk (er) and potential ecological risk index (ri) the quality of the investigated soils in nasarawa eggon and doma which are contaminated by metals, were assessed using this risk factor. table 6 shows the er and ri of metals in the investigated sites of the two study areas (nasarawa eggon and doma). er of fe and u were not calculated as their toxic response factor are yet to be determined. by hakanson classification (table 1), the average er of the metals showed that the levels of as, cd, co, cr, cu, mn, ni, pb and zn do not pose any ecological risk to the study areas (nasarawa eggon and doma). nevertheless, the hg level poses a moderate ecological risk (40≤average er <80) to the environment of the study areas. this supports our earlier finding in the igeo studies. however, comparing the er of metals in the investigated sites of the study areas showed that cd, co, cr, cu, mn, ni, pb and zn have low potential ecological risk (er <40) and thus, do not pose any risk of serious diseases (cancer, kidney, liver damage, etc.), as well as impact in the ecosystems. at nasarawa eggon, as has significant and moderate potential risk in sampled sites n2 and n4 j. multidiscip. appl. nat. sci. 17 respectively. the hg has significant (n1 and n9), moderate (n2, n3, n5, n6 and n8) and low (n4, n7 and n10) potential ecological risks in the nasarawa eggon area. the low potential risk of as at sampled site d5 as well as the moderate potential risk of as and co at d2 and d4 sites of doma area was observed. however, hg exhibits moderate potential ecological risk in sampled sites in doma except in d4 where it shows significant potential ecological risk in d3 and d10 where it poses no ecological risk. ri sums the er of all the studied metals in a sampled site. results (table 6) showed that except for sampled site n2, all sampled sites in nasarawa eggon presented a low ecological risk index (ri< 150). sample site n2 had a moderate ecological risk index which implies that the pollution of this soil can cause serious ecological risks. sampled sites d2 and d4 in the doma area also showed a moderate ecological risk index (150≤ ri <300) while the other sampled sites presented a low ecological risk index. this shows that soil may not be polluted (pli<1) but its usage could still pose an ecological risk to the inhabitants. 4. conclusions this study successfully quantified selected metals in sampled soils of study areas (nasarawa eggon and doma) and evaluated their origin (natural and anthropogenic), contamination level and toxicity. the result showed non–uniform distribution of the analysed metals in the sampled sites of the study areas. cd, cr, cu and ni concentrations were all below average shale referenced value. fe was the most abundant of the metals analyzed in all the sampled sites of the study areas. significant concentrations of as, co, hg, mn, pb, u and zn were recorded at the different sampled sites of both study areas. in pollution assessment, enrichment factor (ef) analysis showed that some sampled sites in the nasarawa eggon area are enriched with as, fe, hg, mn, ni, pb, u and zn while co, fe, hg, mn, pb, u and zn are enriched in sampled sites (some) in doma area. the enrichment levels of mn, pb and zn are higher in nasarawa eggon soils than in doma soils. however, geo– accumulation index (igeo) suggested that only hg and pb could pollute the sampled soils and similarly, hg and co could pollute doma sampled soils despite being enriched with co, fe, hg, mn, pb, u and zn. among the analysed metals, contamination factor (cf) showed that sampled sites n2 and n4 in the nasarawa eggon area and d2 and d4 in the doma area, recorded a moderate degree of contamination by all the studied metals. pli values showed that only sampled sites n2 and d4 of nasarawa eggon and doma area, respectively, were polluted. ecological risk (er) assessment revealed that as and hg pose moderate to significant potential negative implications to human and animal health in some of the sampled sites in nasarawa eggon while as, co and hg showed similar ecological risk in some of the sampled sites in doma. the potential ecological risk index (ri) showed that metal pollution in sampled site n2 of nasarawa eggon and d2 and d4 of doma can cause serious negative health implications for humans and animals in such an environment. thus, the study suggests higher vehicular/human activities in nasarawa eggon than the doma study area and they had a remarkable contribution to the presence of the investigated metals in the nearby agricultural soils. author information corresponding author jude chinedu onwuka — department of science laboratory technology, federal university of lafia, lafia – 950101 (nigeria); department of chemistry, federal university of lafia, lafia – 950101 (nigeria); orcid.org/0000-0003-2950-8273 email: emperor20062003@yahoo.com authors jonathan maduka nwaedozie — department of chemistry, federal university of lafia, lafia – 950101 (nigeria); department of chemistry, nigeria defence academy, kaduna– 802151 (nigeria); orcid.org/0000-0002-3314-513x emmanuel hala kwon–dung — department of botany, federal university of lafia, lafia – 950101 (nigeria); orcid.org/0000-0002-7238-1629 paul tersoo terna — department of botany, https://orcid.org/0000-0003-2950-8273 mailto:emperor20062003@yahoo.com https://orcid.org/0000-0002-3314-513x https://orcid.org/0000-0002-7238-1629 j. multidiscip. appl. nat. sci. 18 federal university of lafia, lafia – 950101 (nigeria); orcid.org/0000-0002-3754-8315 gladys nkeiruka nwobodo — department of biochemistry, federal university of lafia, lafia– 950101 (nigeria); orcid.org/0000-0002-5018-5504 author contributions j.c.o. conceptualization, writing–original draft preparation, funding acquisition, resources, methodology, investigation, formal analysis, software, validation, writing–review & editing, data curation; j. m. n. conceptualization, writing– original draft preparation, funding acquisition, methodology, formal analysis; e. h. k.-d. and p. t. t. funding acquisition, resources, methodology; g. n. n. methodology, writing–review & editing, resources, formal analysis, data curation. conflicts of interest the author(s) declared no conflict of interest acknowledgement the authors appreciate the efforts of mr ombugadu akwashiki, during the sample collection especially in taking the gps coordinates of sampling sites. we also thank mr esua d. yaharo of the national geological survey, kaduna state, nigeria; for his aiding in sample analysis. this research was funded by the federal government of nigeria through its agency, tertiary education trust fund (tetfund) institutional based research grant of 2016/2017. grant reference number ful/vc/tetf/010/voli/ibr. references [1] x. fang, b. zong, and s. mao. 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