CURRENT STATUS AND CHALLENGES FOR CONSERVATION AND SUSTAINABLE USE OF BIODIVERSITY

436 Current status and challenges for conservation and sustainable use of biodiversity Fig. 4: The effect of sub-lethal doses of 4-NP (1.0μl/l, 1.5μl/l, 2.0μl/l) on the activities of GR (A) and GST (B), in the liver of L. rohita with respect to control. Each column is mean ± SEM for six fish. Mean values of different superscript letters (a, b, c, d) were significantly different (p˂0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxic effects of 4-nonylphenol on oxidative stress 437 d d d c c b Fig. 5: The effect of sub-lethal doses of 4-NP (1.0μl/l, 1.5μl/l, 2.0μl/l) on the levels of MDA (A) and CD (B), in the liver of L. rohita with respect to control. Each column is mean ± SEM for six fish. Mean values of different superscript letters (a, b, c, d) were significantly different (p˂0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

438 Current status and challenges for conservation and sustainable use of biodiversity Discussion Glutathione is responsible for protection against reactive oxygen and nitrogen species Liver is considered as one of the main and detoxification of endogenous and detoxifying organ and is essential for both exogenous toxins of electrophilic nature metabolism and excretion of toxic substances (Oluwatosin and Abiola., 2016). Glutathione in the body and hepatotoxic effects are now peroxidase reduces lipid hydroperoxides into regarded as reliable biomarkers of toxic lipid alcohols coupled with GR. Glutathione injury and are representative of biological serves as a substrate for GPx. In this study, the endpoints of contaminant exposure. decreased GPx activities in liver of L. rohita Oxidative stress caused by the imbalance of were due to the decrease in the levels of GSH ROS production and depletion lead to various and increase in the levels of LPO during 4- dysfunctionsof cellular biomolecules such as NP exposure. Low GPx activity may be due lipids, proteins and DNA. The present study to increased CAT activity, both of which use describes the effects of 4-NP and H2O2 as substrate (Yilmaz et al., 2006). compensatory responses of hepatic Decreased GSH level and GPx activity and an antioxidant enzymes, possibly activated to induction in GST activity in the liver might alleviate oxidative stress in L. rohita. In the suggest a critical role of glutathione mediated present study, 4-NP induced total protein enzyme function against the deleterious level of liver, significantly increased with effects of 4-NP.It was observed that GR, the dose increase referring to limited effect on enzyme that regenerates GSH by reducing protein-related metabolic processes. The oxidised glutathione (GSSG), was increased protein makes up of an organism is of when exposed to the sub-lethal doses of 4- important diagnostic significance (Shalaby et NP. al., 2006., Hadi et al., 2009) because of protein’s involvement in enzymes, hormones Lipid peroxidation is one of the main and antibodies as well as osmotic pressure processes induced by oxidative stress and they balance and maintaining acid-base balance. are formedfrom the oxidative deterioration of polyunsaturated lipids in the membranes of Superoxide dismutase is the first line of cells and organelles. The results showed high defense against the free radicals where it levels of MDA in the liver of exposed fish. converts superoxide into hydrogen peroxide. The levels of MDA as a potent marker for It is then degraded by the other antioxidant LPOs have been used in the present study. enzymes such as catalase and glutathione Malondialdehyde is considered as indicators peroxidase/reductase. An apparent elevation of oxidativestress, which results from the free of hepatic SOD activity was observed on radical damage to membrane components of exposure to various sub-lethal doses of 4-NP cells. Conjugated dienes represents the initial as compared to that of control group. The product of radical attack, a rearrangements of increased SOD level in liver tissue exposed the double-bonds in unsaturated fatty acids to 4-NP indicates a detoxifying mechanism (Pannunzio and Storey, 1998). In the present against the toxicity. Catalase is the primary study, CD in liver of L. rohita exposed to 4- scavengers of H2O2 in the cell. Increased NP was found to be significantly increasing CAT activity in this study indicates that from control. From this result, it can be pollution stress has elevated the formation assumed that antioxidant enzymes rate of H2O2. Comparatively, the tissues of disequilibrium led to increased lipid L. rohita displayed a higher variation in SOD peroxidation as evidenced by the increased in activity than in CAT activity. lipid oxidation markers like MDA and CD Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxic effects of 4-nonylphenol on oxidative stress 439 levels. Acknowledgment The findings obtained in this study carry high The authors would like to acknowledge the significance because the 4-NP concentrations financial support provided by Council of administered to the experimental fish were Industrial and Scientific Research (CSIR), very low in comparison with those reported New Delhi to carry out this study. by others. In comparison, the current study tested 1.0µl/l, 1.5µl/l and 2.0µl/l of 4-NP. It References should be borne in mind that4-NP is detected ubiquitously in fishes which could be major Agergaard, N. and Jensen, P. T. 1982. resources for commercial fish feeds. The Procedure for blood glutathione peroxidase results observed from the present study determination in cattle and swine. Acta. Vet. indicates the induction of oxidative stress and Scand, 23: 515-527. the failure of antioxidant enzymes to remove the free radicals from hepatic cells. It was Ahmad, I., Hamid, T., Fatima, M., Chand, confirmed by the reduction in the activities of H.S., Jain, S.K., Athar, M. and Raisuddin, some of the antioxidant enzymes and the S.2000. Induction of hepatic antioxidants in increase in the level of lipid peroxidation in freshwater cat fish (Channa punctatus bloch) the liver of L. rohita. Overall observations is a biomarker of paper mill effluent from the present study indicate that 4-NP at exposure. Biochem. Biophys. Acta, 1523, 37- sublethal concentrations when exposed for 48. short durations altered the antioxidant defense system in hepatocytesof fish. Benke, G.M. and Cheevar, K.C.1974. The comparative toxicity, acetylcholine esterase Conclusion action and metabolism of methyl parathion and parathion in sun fish and mice. Toxicol. From the present study, it was inevitably Appl. Pharmacol, 28: 97-109. revealed that the exposure of 4-NP to L. rohita induced oxidative stress in the hepatocytes Bradford, N.M. 1976. Rapid and sensitive mainly through increasing enzymatic method for the quantitation of microgram antioxidants capacity, such as, SOD, CAT, quantities of protein utilising the principle of GST, GR and depleted levels of non- protein- dye binding. Anal Biochem, 72: 248- enzymatic antioxidants such as, GSH which 254. might be related to the down regulated activity of the antioxidant enzyme GPx. David, M., and Richard, J. S., Methods of Moreover, there was a definite enzymatic analysis. In. J. Bergmeyer and G. interrelationship between antioxidant B. Marianna (eds.), 1983, 26, 358-359. enzymes and LPO products which also confirmed that imbalance in antioxidant Di Giulio, R. T., Biochemical responses in enzymes led to increased LPO. It is evident aquatic animals: A review of determinants of from the study that antioxidant enzymes oxidative stress. Environ Toxicol Chem, assay and LPO levels can be used as a 1989, 8, 1103-1123. biomarker of oxidative stress to investigate the mechanisms of environmental toxicity in Duncan, D.B. 1955. Multiple range and aquatic organisms. The results clearly multiple F tests. Biometrics, 11: 1-42. showed that 4-NP is a pollutant with oxidative potential. Emmanuel, O.O., Stanley, C.I., Christopher, D.N., Christian, E.M., Ogochukwu, C.O. and Ibrahim, B.U. 2017. Toxicity of diazepam on lipid peroxidation, biochemical and oxidative stress indicators on liver and gill tissues of Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

440 Current status and challenges for conservation and sustainable use of biodiversity African catfish Clarias gariepinus (Burchell, Oluwatosin, A.A. and Abiola, M.A. 2016. 1822). International Journal of Fisheries and Effect of environmental pollution on Aquatic Studies, 5(3), 114-123. oxidative stress biomarkers in african cat fish (Clarias gariepinus) from asejire river in European Commission (EC)., European Oyo state, Nigeria. Journal of Environmental Union Risk Assessment Report 4- and Occupational Science, pp. 71. nonylphenol (branched) and nonylphenol. 2002. Organisation for Economic Co-operation and Development (OECD)., Guidance Document Habig, W. H., Pahst, M. J.and Jakoby, W. on standardised test guidelines for evaluating B.1974. Glutathione-S-transferases: The first chemicals for endocrine disruption. 2012, 1- enzymatic step in mercapturic acid 524. formation. J. Biol. Chem, 249:7130-7139. Pannunzio, T.M., Storey, K.B. 1998. Hadi, A., Shokr, A. and Alwan, S. 2009. Antioxidant defenses and lipid peroxidation Effects of aluminium on the biochemical during anoxia stress and aerobic recovery in parameters of fresh water fish Tilapia zillii. J. the marine gastropod Littorina littorea. J. Sci. Appl. 3: 33-41. Exp. Mar. Bio. & Ecol, 221:277-292. Kakkar, P., Das, B. and Viswanathan, P.N. Recknagel, R.O. and Goshal, A.K.1966. 1984. A modified spectroscopic assay of Quantitative estimation of peroxidative superoxide dismutase. Ind. Biochem. degeneration of rat liver microsomal and Biophys, 21:130-132. mitochondrial lipids after carbon tetrachloride poisoning. Exp mol. Pathol, 5: Kwan, H.P., Alteration of hepatic antioxidant 413-426. systems by 4-Nonylphenol, a metabolite of alkylphenol polyethoxylated detergents, in Shalaby, A., Khattab, Y. and Abdel-Rahman, Far Eastern catfish, Silurus asotus. Environ A. 2006. Effects of garlic Allium sativum Health Toxicol, 2015, 30, 573-701. chloramphenicol on growth performance, physiological parameters and survival of Nile Lawrence, R.A., Burk, R.A., Glutathione tilapia Oreochromis niloticus. J. Venom peroxidise activity in selenium-deficient rat Anim. Toxins incl. Trop. Dis, 12:172-201. liver. Biochem. Biophys. Res. Commun, 1976, 71, 952-958. Statistical Package for Social Sciences (SPSS), IBM SPSS Statistics for Windows, Maehly, A.C. and Chance, B. 1954. The Version 22.0. IBM Corp, Armonk, NY, 2013. assay of catalase and peroxide in methods of biochemical analysis, 1: 357 – 424. U.S. Environmental Protection Agency (U.S.EPA)., Aquatic Life Ambient Water Nichans, W.G. and Sannelson, D.1968. Quality Criteria – Nonylphenol, 2005b. Formation of malondialdehyde from phospholipids arachidonate during microsomal lipid peroxidation. Eur.J. Biochem, 6: 126-130. Oliveira, M. 2008. Organ specific antioxidant responses in golden grey mullet (Liza aurata) following a short-term exposure to phenanthrene. Sci. Total Environ, 396: 70– 78. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal analysis of zooplankton from Cochin estuary 441 ISBN 978-93-5396-871-7 CHAPTER 62 TRACE METAL ANALYSIS OF ZOOPLANKTON FROM COCHIN ESTUARY Bettina P Alex*, Biju A and Jyothirmaye Mohan Department of Zoology St. Stephen’s College, Pathanapuram, Kollam, Kerala *Correspondence Email: [email protected] ABSTRACT Metal contamination in aquatic ecosystem causes serious health hazards to the aquatic organisms. Concentrations of eight trace metals (Fe, Mn, Cr, Ni, Cu, Zn, Cd and Pb) were determined in water and zooplankton collected from Cochin Estuary on bimonthly basis from October 2017-January 2018. The trace metal concentrations followed the hierarchy; Fe>Ni>Cr>Zn>Pb>Cd>Cu>Mn and Fe>Zn>Cr>Ni>Mn>Cu>Pb>Cd in water and zooplankton respectively. High trace metal concentration in zooplankton indicating the toxic metal bioaccumulation from their surroundings and the environmental quality of the estuary. Key words: Zooplankton, Trace metals, Cochin estuary, Environmental pollution Introduction makes the estuaries the most productive environments on earth, which are key Environmental pollution especially pollution components in aquatic food webs (Omori and of water resources is a matter of great Ikeda,1984). Zooplankton mostly feed on concern for all the nations worldwide. phytoplankton and in turn serve as food for Among all the pollutants, trace metals are animals at higher trophic level and have been most dangerous one as these are non- chosen as one of the recommended groups biodegradable and persist in the for the base line studies of metals in the environmentin compartments, such as water, marine environment(Kleppel, 1993). soil, air, and different organisms, and have the tendency to accumulate into the basic The discharge of fresh water from six rivers food chain and move up through the higher Periyar, Muvattupuzha, Pampa, Manimala, trophic level. Some of the trace metals, such Meenachil and Achancovil and drainage as Cu, Zn, Mn, Fe, and Cr are essential for canals cause discharge of contaminants such the metabolism of the organisms while others as acids, alkalis, suspended solids, fluorides, like Cd, Pb, and Hg are nonessential. free ammonia, insecticides, dyes, trace metals However, all such metals, essential or not, and radioactive nuclei (Lakshmanan et al., are toxic above a threshold bioavailability 1987; Menon et al., 2000). The heavy (Rainbow, 1993). industrialization and increasing urbanization are responsible for rapidly increasing stress Estuaries are unique ecosystems with on the water environment of the area.It is dynamic and complex processes of ecologic therefore necessary to protect these water and economic importance. The presence of resources. The main objective of the study is high phytoplankton and zooplankton biomass Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.441-446 © Principal, Sree Narayana College, Kollam, Kerala, India

442 Current status and challenges for conservation and sustainable use of biodiversity to asses selective trace metals like Iron, identification of different planktonic Nickel, Chromium, Manganese, Copper, organisms up to group level was done with Zinc, Cadmium, and Lead in planktonic the help of standard references (Santhanam et organism from of Cochin estuary.So far very al., 1989; Manickam, 2012, Newell and little work has been published on the heavy Newell, 1973). metal estimation in zooplankton from the Cochin backwaters. In this regard, the heavy Analytical methods for the estimation of metal concentrations were measured in trace metals in water and Plankton individual taxa of zooplankton from Cochin estuary. Water samples (2.0 litres) collected using a Niskinsampler was filtered using a 0.45 Materials and Methods micron Millipore filter papers to separate them into dissolved and particulate Sampling site fractions.Dissolved trace metal in waters is allowed to react with APDC-DDDC reaction Cochin backwater is part of a long chain of mixture -then extract with chloroform, again lakes and canals, parallel to the coast, extracted into 4N Nitric acid. extending between 9°40'12″ to 10°10'46″N and 76°09'52″ to 76°23'57″ E. The sampling For metal analysis in zooplankton, the of the zooplankton was carried out bimonthly samples were dried in an oven at 65°C using from October 2017 –January 2018 from Bolgatty of Cochin estuary. a Petri dish and stored in vacuum desiccators. Plankton sampling and preservation The dried zooplankton samples were first Zooplankton samples were collected using powdered, and aliquots of approximately 300 Working Party (WP) net (mesh size 0.2 mm, mg was digested for 3 hours at 80°C with 300 mouth area 0.6m2). Thefortnightly collections μlof HNO3 and HClO4 (Nitric acid & Per of zooplankton carried with horizontal chloric acid mixture 65%, suprapure, Merck) haulsand in laboratorysamples were subjected to close visual observation, under a in tightly closed 2-mL Eppendorf reaction binocular microscope to ensure the absence of any foreign particles. These samples were tubes. thoroughly rinsed with Milli-Q Water to remove salts.Collected samples were divided The digests were making up to 25 mL with into two parts. One was transferred to 4% formalin for quantitative and qualitative HCl (0.1N Hydrochloric acid) and analysed taxonomical analyses of zooplankton. by a flame atomic absorption The samples were allowed to stand in a 100 ml measuring cylinder for few minutes so as spectrophotometer (PEA Aanalyst to allow time for the organic element to settle and then the total volume was noted. Then 100).Sample preparation was done as per the samples were subjected to sorting and the identification was done with the help of standard method (APHA, 1992 and 1998). stereo-compound microscope. The total numbers of the different zooplankton The calculations of the selected metals in communities and species encountered were converted to their number per m3. The plankton were calculated as per the formula; Concentration of metal in the sample = Results and Discussion The Percentage Composition of zooplanktonis shown in Table1. The trace metal concentrations in water and zooplankton are shown in Table 2 and Figure 1 respectively. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.441-446 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal analysis of zooplankton from Cochin estuary 443 Table 1. Composition (%) of zooplankton groups in Cochin Estuary. SL. No Zooplankton % Composition 1 Medusae 0.08 2 9.38 3 Chaetognaths 0.06 4 Polychaetes 52.89 5 Copepods 8.14 6 0.73 7 Mysids 13.20 8 Amphipods 0.24 9 8.96 10 Lucifer 0.22 11 Euphausiids 0.17 12 Prawn larvae 0.61 13 Gastropod larvae 5.05 14 Stomatopod larvae 0.19 Brachyuran larvae Fish egg Fish larvae Copepods dominated among zooplankton mesh size of the net used for collecting groups. The abundance percentage of zooplankton. This finding is in agreement Copepods was about 52.89 % of the total. with general understanding of zooplankton Members of Decapoda dominated the next. classes in the estuary. (Pillai et al., 1973; The dominance of a particular group among Madhu et al., 1973; Molly and Krishnan, the zooplankton can be due to the type of the 2009). ecosystem under study or may be due to the Table 2. Table showing trace metal concentration in water Trace metals Concentration of trace Maximum Limit metals (ppm) WHO/FEPA(ppm) Fe 2.2277 Ni 1.273 0.3 Zn 0 .195 - Cu 0.062 3 Cd 0.082 1 Cr 0.265 0.003 Pb 0.149 0.05 Mn 0.058 0.01 - Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.441-446 © Principal, Sree Narayana College, Kollam, Kerala, India

444 Current status and challenges for conservation and sustainable use of biodiversity Fig 1. Trace metal concentrations in zooplankton collected from Cochin Estuary during 2017– 2018. The mean trace metal concentrations in water loading and unloading of large quantities of sample of present study are given inTable general and bulk cargo at nearby locality 2.The extent of different trace metals in water (Robin et al., 2012). Pb is known to form followed hierarchy, Fe> Ni> Cr> colloids in estuarine water. These colloids Zn>Pb>Cd>Cu>Mn. Reports of trace metal can be adsorbed onto planktonic debris, concentrationin cochin estuarine water which might have resulted in a higher indicated highest concentration in concentration of these elements in premonsoon and post monsoon samples.( zooplankton from coastal water (Zauke, Anju et al.,2011) 2006; Robin et al., 2012). Copepoda proved to be enriched withtrace metals than other Iron and Zinc are reported to be the highest zooplankton (Martin, 1970) showed that accumulated chemical elements in many trace metals had become adsorbed to zooplankton sample (Masuzawa et al., 1988). copepod exoskeletons at greater depths The highest concentration of Fe detected in because food-dependent moulting rates were copepods between 2454. μg/g, least in lower; thus more time was available Lucifer757.5μg/g. Copepods contain Zn with forelemental adsorption to take place. High highest concentration(564.89μg/g, least in accumulation of Zn in zooplankton may be fish larvae 234.76μg/g. Mn ranges between due to coprecipitation of Znwith calcium 137.5μg/g in prawn larvae18.269μg/g in carbonate (Rejomon et al., 2008).The Nickel copepods. Highest Concentration of Ni, Cr concentration shows the highest level atthis and Cd was reported in samples of prawn study is due to nearby oil loading terminals. larvae and Pb in Copepods. (Figure.1) The high levels of trace metals in this study The observed high metal levels in water and could be attributed to their industrial sources zooplankton in the Cochin estuarycould be carried through rivers discharges which attributed to high influxes at the region, increases the bio availabilities thereby uptake primarily from automotive exhausts due to of metals by zooplankton Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.441-446 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal analysis of zooplankton from Cochin estuary 445 Conclusion Cochin backwaters, southwest coast of India. Indian J. Mar. Sci., (16): 99-102. Cochin is the industrial capital of Kerala and a large number of factories are located onthe Madhu, N.V., Jyothibabu, R., Balachandran, banks of the river Periyar and Cochin K.K., Martin, G.D., Vijay, J.G., Shiyas, C.A., backwaters. This backwater has been Gupta, G.V.M. and Achankutty, C.T. 2007. experiencing high levels of anthropogenic Monsoonal impacton planktonic standing pressure during the last five decades. The stock and abundance in a tropical estuary results of present investigation demonstrate (Cochin backwater, India). Est Coast. Shelf. that the Cochin estuary is highly Sci., 73 (1-2): 54-64. contaminated with trace metals and increased concentration may adversely affect the Manickam, N., Saravana, B.P., Santhanam, marine life. P., Chitara, P., Jawahar, A.A. 2012. Zooplankton diversity in Perinnial freshwater Acknowledgements lake. In. Desai P V and R. Roy (Eds). Diversity and physiological process, pp: 25- The authors are grateful to the Dr. K.P 37. Laladhas, Principal, St. Stephen’s College, Martin, J.H.1970. The possible transport of trace metals via moulted Copepod Pathanapuram, Dr.Neena Suzzan Joushua, exoskeletons. Limnology & Oceanography. (15) :5:756-761. Head, P.G and Research Department of Masuzawa, T., Koyawa, M. and Rerazaki, M. Zoology, St.Stephen’s College, 1988. Areqularity in trace element contents of marine zooplankton species. Marine Biol., Pathanapuram and Director, Zoology (97): 587-591 Research Centre for the encouragement and Menon, N.N., Balchand, A,N.and Menon, N.R. 2000. Hydrobiology of the Cochin Back facilities provided. water system, A review, Hydrobiologia, (430):149-183. References Molly, V. and Krishnan L. 2009. Distribution APHA, 1992. Standard methods for the of zooplankton in selected centres of Cochin examination of water and waste water 18th backwaters, Kerala, J.Mar. Bio., 119-121. edition, APHA , Washington, DC.937. Newell,G.E. and Newell, R.C. 1973. Marine APHA, 1998. Standard methods for the Plankton - A Practical Guide. Hutchinson examination of water and waste water20th Educational Ltd., London, pp:244. edition, APHA, Washington, DC 950. Omori, M. and Ikeda,T. 1984. Methods in Anju, A.K., Dipu, S. and Sobha, V. 2011. marine zooplankton ecology. Wiley, New Seasonal Variation of Heavy Metals in York, NY. Cochin Estuary and Adjoining Periyar and Muvattupuzha Rivers, Kerala, India, Global Pillai, V.K., Joseph, K.T.and Nair, A.K.1973. Journal of Environmental Research., (5) 1: The plankton production in the Vembanad 15-20 Lake and adjacent water in relation to the environmental parameters. Bulletin of Kleppel, G.S. 1993. On the diets of calanoid Department of Marine Sciences, Cochin copepods. Marine Ecology Progress Series, (99):183 - 195. Lakshamanan, P.T., Shynamma, C.S., Balachand, A.N., Nambisan, P.N.K. 1987. Distribution and variability of nutrients in

446 Current status and challenges for conservation and sustainable use of biodiversity University of science and Technology. (7).137-150. Rainbow, P.S. 1993. The significance of trace metals concentrations in marine invertebrates, In R. Dallinger and P. S.Rainbow (Eds.), Ecotoxicology of metals in invertebrates, Boca Ratón: Lewis Publishers, 3-23. Rejomon, G., Balachandran, K.K., Nair. M., Joseph, T.and DineshKumar, P.K., Achuthankutty, C.T., Nair, K.K.C. and Pillai, N.G.K. 2008. Trace metal Concentrations in zooplankton from the eastern Arabian Sea and western Bay of Bengal. Environmental Forensics, (9):1: 22-32. Robin, R.S., Muduli, P.R., Vardhan, K.V., Ganguly, D., Abhilash, K.R. and Balasubramanian, T. 2012. Heavy metal contamination and risk assessment in the marine environment of Arabian Sea, along the southwest coast of India. American Journal of Chemistry. (2): 4:191-208. Santhanam, R., Velayutham, P. and Jegathesan, G. 1989. A manual of Fresh water Ecology.Daya Publishing House Delhi. pp:1-109. Thomson, K.T. 2002. Economic and Social Issues of Biodiversity Loss In Cochin Backwaters cochin university of science and technology, Cochin, India Technical report, pp:51-82 Zauke, G.P. and Schmalenbach, I. 2006. Heavy metals in zooplankton and decapod crustaceans from the Barents Sea. Science of the Total Environment, (359):1-3: 283-294. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.441-446 © Principal, Sree Narayana College, Kollam, Kerala, India

Oxidative stress responses of a freshwater fish, labeo rohita, to an endocrine disruptor, bisphenols 447 ISBN 978-93-5396-871-7 CHAPTER 63 OXIDATIVE STRESS RESPONSES OF A FRESHWATER FISH, LABEO ROHITA, TO ANENDOCRINE DISRUPTOR, BISPHENOLS Shehna Mahim S* and Aruna Devi C Department of Zoology, University College, Thiruvananthapuram- 695034 *Correspondence Email: [email protected] ABSTRACT Bisphenol S (BPS) is an industrial chemical that has been used as a substitute for Bisphenol A (BPA) in making polycarbonate plastics, epoxy resins, thermal receipt papers and currency bills, since BPA has been reported to have adverse effects in living system. From this view point, the present study investigates whether BPS has the same or rather more toxic effects like BPA or not. Limited studies were carried out on the effect of BPS on fish. The hepatic antioxidant enzymes such as Superoxide dismutase (SOD), Catalase (CAT), Glutathione-S-transferase (GST), Glutathione reductase (GR) and Glutathione peroxidase (GPx) along with the non-enzymatic antioxidant Glutathione (tGSH) in a freshwater fish, Labeo rohita were selected as biomarkers. The results revealed that the sub lethal exposure of BPS significantly influenced the activities of these biomarkers. Lipid peroxidation (LPO) products such as Malondialdehyde (MDA) and Conjugate diene (CD) levels were also altered by BPS exposure. The alteration in the levels of antioxidants and lipid peroxidation products following BPS exposure clearly showed that the fish experienced oxidative stress. The study showed that BPS is a pollutant with oxidativepotential by disrupting the antioxidant enzymes. Key words: Antioxidants, Bisphenol S, Oxidative stress, Lipid peroxidation. Introduction of hydroxyl group on the benzene ring makes them endocrine disruptors. This A large number of chemicals are used for the phenol moiety enables them to mimic production of everyday commodities estradiol. The plastic products coined as including toys, food packaging, cosmetic ‘BPA free’ are often made of BPS (Grignard products, and building materials. Nowadays, et al., 2012). Bisphenol S has been used in the practice of employing different chemicals manufacturing plastics, thermal receipt in consumer products, without realizing the papers, currency bills, food cartons etc. possible harmful effects, has become a common Bisphenol S is a component of polyether sulfone which is widely used, or proposed occurrence. Bisphenol S (BPS) is one of such to be used, in a variety of consumer chemicals, which is a new analogue of the products and applications such as developer potentially toxic bisphenol A (BPA). in thermal paper, wash fastening agent in Bisphenol S is actually a bisphenol, a close cleaning products, and in microwave dishes. look alike of BPA in which the It is also used as a chemical intermediate and dimethylmethylene group is replaced with a in preparation of products including fire sulfone group. Recent work suggests that, retardants, couplers for photography, like BPA, BPSalso has endocrine disrupting electroplating chemicals and colorant, and as properties (Horan et al., 2018). The presence a modifier for leather, fiber, and epoxy curing Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

448 Current status and challenges for conservation and sustainable use of biodiversity agents (EFSA, 2015). Bisphenol S has been superoxide radical (O.-), hydrogen peroxide detected in personal care products and in (HO) and the hydroxyl radical (.OH). food and indoor house dust demonstrating the Oxidative stress can be defined as the potential for human exposure (Liao and imbalance between the formation of free Kannan, 2014). It is also used as a corrosion radicals capable of causing peroxidation of inhibitor in epoxy resins and as a reactant in the lipid layer of cells and the body’s polymer reactions. Chemically, BPS is being antioxidant defense. Oxidative stress occurs used as a reagent in polymer reactions; it has when the ROS generation rate exceeds that of also been reported to occur in canned their removal. Recent research has suggested foodstuffs, such as tin cans (Vinas et al., that oxidative damage might be associated 2010). In a recent study analyzing BPS in a with reproductive impairment in fish (Oakes variety of paper products worldwide, BPS and Van Der Kraak, 2003). was found in 100% of tickets, mailing In this context, it was thought worthwhile to envelopes, airplane boarding passes, and study the oxidative potential of BPS, if any in airplane luggage tags. In this study, very L. rohita. Oxidative stress is a growing area high concentrations of BPS were detected in of research in toxicology because increased thermal receipt samples collected from cities lipid peroxidation products (LPO), altered in the United States, Japan, Korea and antioxidant enzyme levels and altered Vietnam. The BPS concentrations were large antioxidant scavenger levels may be early but varied greatly, from a few tens of biomarkers of contamination. These nanograms per gram to several milligrams oxidative stress defenses or antioxidants of per gram (Pivnenko et al., 2015). Studies living organisms can be subdivided into regarding the effects of BPS are rare in enzymatic antioxidants, such as superoxide Indian scenario. Many of these chemicals dismutase (SOD), catalase (CAT), eventually reach aquatic ecosystems through glutathioneperoxidase (GPx) and glutathione- leaching. From this perspective, it was S-transferase (GST) and non-enzymatic thought worthwhile to investigate the antioxidants such as glutathione (GSH), oxidative stress responses of Labeo rohita to vitamin E, ascorbate and urate. These protect BPS. the organism against oxyradical damage such The mechanism of free radical production as DNA strand breaks, protein oxidation and and defensive system in biological system to the induction of lipid peroxidation. eradicate the generated pro-oxidants by the Materials and methods activities of antioxidant enzymes is the recent and rapidly growing field of research. Experimental design Oxygen in its molecular state, O2 is essential for many metabolic processes that are vital to The animal model, L. rohita used in the aerobic life. This dependence on oxygen, experiment were collected from local forces aerobic life to withstand considerable suppliers and brought to the laboratory. Prior toxicity. The oxygen paradox derived from to experiment, the fish were acclimatized the chemical nature of oxygen, which in its under laboratory conditions. They were fed atomic form (O) is a free radical and in its daily on ad libitum. The LC50 of the test 2 molecular form (O2) is a free bi-radical. The chemical, i.e. BPS was determined as bi-radical nature of oxygen molecule allows 150mg/L. After acclimatization, the fish were oxidation or reduction reactions. However divided into 10 groups of ten each in separate monovalent reduction generates several glass tanks. The first group of fish served as reactive oxygen species (ROS) such as 2 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

Oxidative stress responses of a freshwater fish, labeo rohita, to an endocrine disruptor, bisphenols 449 control and was kept in normal dechlorinated optical density at 560nm of chromogen tap water. Fish in groups II, III and IV were production by 50% in one minute under the exposed to 80mg BPS/L for the periods 7, 14 assay conditions. The specific activity was and 21 days respectively. Similarly, the expressed as IU/mg protein. groups of fish V, VIand VII were exposed to 100mg BPS/L and groups of VIII, IX and X B. Catalase (CAT) (EC.1.11.1.6) to 120mg BPS/L for a period of 7, 14 and 21 days respectively. Freshwater sample having The activity of catalase was measured the specified doses of BPS was replaced on according to Maehly and Chance, (1954). alternate days. After the above said periods The estimation was spectrophotometrically of exposure, the fish were killed and liver done by following the decrease in absorbance was excised. A portion of fresh liver tissue at 230nm. The assay mixture contained 3ml from each fish was kept at -20°C in a deep phosphate buffer (0.01M, pH 7), 0.2ml H2O2 freezer [ROTEK] for biochemical assays. (2mM) and 1ml of approximately diluted extract prepared by homogenizing the tissue Estimation of protein in 10mM buffer. A system devoid of the extract constituted blank. The specific Protein content for all enzymes was activity was expressed in terms of n moles of estimated using the same tissue extract H2O2 decomposed/minute/mg protein. according to the protocol of Bradford, (1976). C. Glutathione peroxidase (GPx) (EC.1.11.1.9) 1. Assay of antioxidant enzymes Glutathione peroxidase activity was estimated according to Lawrence and Burck, A. Superoxide dismutase (SOD) (1976) with slight modification (Agergaard (EC.1.15.1.1) and Jensen, 1982). The assay volume contained 2ml of 0.01M phosphate buffer Activity of SOD was estimated according to (pH 7.4) with 300μl ethylene diamine tetra the protocol of Kakkar et al. (1984). The acetic acid (EDTA), 300μl of 1mM sodium assay mixture contained 1.2ml of 0.052M azide, 100μl of 0.1M NADPH,100μl of 1mM sodium pyrophosphate buffer (pH 8.3), 0.1ml reduced glutathione and 200μl tissue extract. of 186mM phenazine methosulphate, 0.3ml Blank contained all the reagents except tissue of 300mM nitro blue tetrazolium, 0.2ml of extract. Change in absorbance was measured 780mM NADH, appropriately diluted at 340nm at 15seconds interval for 1minute. enzyme preparation and water in a volume of Enzyme activity was expressed as IU/mg 3ml. By the addition of NADH, the reaction protein. was started. The reaction was stopped by the addition of 1ml of glacial acetic acid after the D. Glutathione-S-transferase (GST) incubation at 30oC for 90seconds. The assay (EC.2.5.1.18) mixture was stirred vigorously and shaken with 4ml n-butanol and was allowed to stand The activity was measured according to the for 10minutes, centrifuged at 2500rpm, for protocol of Habig et al. (1974). The cocktail 10minutes and the butanol layer was contained 1ml phosphate buffer (0.5M, pH carefully pipetted out. Color intensity of the 6.5), 100μl 1-chloro, 2, 4, dinitrobenzene chromogen in butanol was measured at (CDNB) (30mM), 0.1ml reduced glutathione 560nm against blank (butanol) using UV-Vis (30mM) and 100μl tissue extract. The spectrophotometer (Perkin Elmer, USA). One increase in optical density of the enzyme was unit of enzyme activity was defined as the measured against the blank at 340nm for enzyme concentration required inhibiting the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

450 Current status and challenges for conservation and sustainable use of biodiversity 2minutes. Enzyme activity was expressed as Recknagel and Ghoshal, (1966). The reaction n moles of CDNB-GSH conjugate mixture contained 1ml tissue homogenate, formed/minute/mg protein for tissue samples. 5ml chloroform/methanol (2:1) and was centrifuged at 1000rpm for 10min. 3ml of E. Glutathione content lower layer was pipetted out carefully and dried at 450C in a water bath. To this, 1.5ml Glutathione content was estimated according of cyclohexane was added and absorbance to Benke and Cheevar, (1974). The assay measured at 233nm. The activity of enzyme mixture contained 2ml of 0.3M phosphate was expressed as µmoles/g tissue. buffer (pH 7.4), 500μl 5, 5’-Dithio-bis-2- nitrobenzoic acid (DTNB) (0.04 %) and Statistics 200μl tissue extract. A system devoid of extract was taken as blank. Change in Data analysis was done by ANOVA. The absorbance was measured at 412nm within differences in means were tested by using 10minutes. The specific activity was Duncan (1955) analysis. Significant level expressed as n mol/100g tissue. used was 0.05. The association of variables was studied by Pearson correlation test. All F. Glutathione reductase (GR) (EC. the statistical analyses were performed using 1.6.4.2) the software SPSS 20.0 for Windows. Glutathione reductase activity was measured Results according to the protocol of David et al. (1983). A fixed volume of tissue homogenate In the present study, SOD activity decreased in phosphate buffer was mixed with a in liver tissue after 7 days of exposure to reaction volume containing 2.6ml phosphate 80mg/L BPS whereas an increase in activity buffer, 0.1ml EDTA and 0.1ml oxidized can be seen after 14 and 21 days of exposure glutathione (GSSG), 0.05ml of NADPH was to the same dose. (Fig.1.1).Increased added prior to assay. The decrease in activities of SOD can also be seen on absorbance at 340nm was measured exposure to 100 and 120mg/L BPS after 7, spectrophotometrically. The specific activity 14 and 21 days Catalase activity in fish liver was expressed as Units per mg of protein. exposed to BPS (80, 100 and 120mg/L) for 7, 14 and 21 days is shown in Fig. 1.2. There 2. Estimation of lipid peroxidation was a decrease in CAT activity after 7 days products of exposure to 80 and 120mg/L but the activity increased after 14 and 21 days of A. Malondialdehyde content exposure to BPS. Increased catalase activity was also found in 100mg/L of BPS after 7, Malondialdehyde content was measured 14 and 21 days of exposure. A significant according to Nichans and Sannelson, (1968). positive correlation was found between SOD 1ml homogenate was added to 2ml TBA. and CAT after exposure to 80, 100 and TCA. HCL and heated in boiling water bath 120mg/L BPS. It is evident that in all the 3 for 15min. It is then centrifuged for 1000rpm doses, the activity of GPx initially increases for 10min and absorbance was measured at after 7 days of exposure but later the activity 535nm. The blank consisted 2ml TBA. TCA. was decreased significantly after 14 and 21 HCL reagent. The activity was expressed as days (Fig. 1.3). The activity of Glutathione- µmoles/g tissue. S- transferase decreased significantly in a dose- and time- dependent manner at all B. Conjugated Diene Content exposures (Fig. 1.4). Glutathione content Conjugated diene was measured according to Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

Oxidative stress responses of a freshwater fish, labeo rohita, to an endocrine disruptor, bisphenols 451 exposed to BPS (80, 100 and 120mg/L) for 7, and 21 days of exposure in all the three 14 and 21 days is shown in Fig. 1.5. There concentrations (Fig. 1.6). Levels of MDA was a significant decrease in the were determined in the hepatic tissue of fish concentration of GSH after 7 days of and the results are shown in Fig. 2.1. The exposure to 80mg/L and 100mg/L BPS. concentration of MDA increased However, it increased after 21 days of significantly in fish exposed to 80, 100 and exposure in the above said doses. The level 120mg/L BPS after 7, 14 and 21 days. of glutathione content was significantly Likewise, a significant increase in the levels increased after 7, 14 and 21 days of exposure of CD in a dose- and time- dependent manner to 120mg/L BPS. The level of glutathione can also be observed in Fig. 2.2. reductase increased significantly after 7, 14 cd ab c d d ab bc Fig. 1.1 The effect of sub-lethal doses of BPS (80, 100 and 120mg/L) on the activity of Superoxide Dismutase in the liverof L. rohita in relation to control. Mean values of different subscript letters (a, b, c, d) were significantly different (p<0.05). d abc d d abc c ab Fig. 1.2 The effect of sub-lethal doses of BPS (80, 100 and 120mg/L) on the activity of Catalase in the liver of L. rohita inrelation to control. Mean values of different subscript letters (a,b,c) were significantly different (p<0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

452 Current status and challenges for conservation and sustainable use of biodiversity b bc b a a a c d c d d Fig. 1.3 The effect of sub-lethal doses of BPS (80, 100 and 120mg/L) on the activity of Glutathione peroxidase in the liverof L. rohita in relation to control. Mean values of different subscript letters (a,b,c,d) were significantly different (p<0.05). a ab a bc c b d d c d Fig. 1.4 The effect of sub-lethal doses of BPS (80, 100 and 120mg/L) on the activity of Glutathione-S- transferase in the liver of L. rohita in relation to control. Mean values of different subscript letters (a,b,c) were significantly different (p<0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

Oxidative stress responses of a freshwater fish, labeo rohita, to an endocrine disruptor, bisphenols 453 a cd d cd b b c a a b Fig. 1.5 The effect of sub-lethal doses of BPS (80, 100 and 120mg/L) on the activity of Glutathione in the liver of L. rohita in relation to control. Mean values of different subscript letters (a,b,c,d) were significantly different (p<0.05). Fig. 1.6 The effect of sub-lethal doses of BPS (80, 100 and 120mg/L) on the activity of Glutathione Reductase in the liverof L. rohita in relation to control. Mean values of different subscript letters (a,b,c,d) were significantly different (p<0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

454 Current status and challenges for conservation and sustainable use of biodiversity d c Fig. 2.1 The level of lipid peroxidation (Malondialdehyde) in the liver of L. rohita exposed to sublethal doses of BPS (80, 100 and 120mg/L) in relation to control. Mean values of different subscript letters (a,b,c,d) were significantly different (p<0.05). d c Fig. 2.2 The level of lipid peroxidation (Conjugate diene) in the liver of L. rohita exposed to sublethal doses of BPS (80, 100 and 120mg/L) in relation to control. Mean values of different subscript letters (a, b, c, d) were significantly different (p<0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

Oxidative stress responses of a freshwater fish, labeo rohita, to an endocrine disruptor, bisphenols 455 Discussion SOD activity indicates that more protein is required to safeguard against superoxide The present study revealed that BPS induced radicals. Likewise increased activities of oxidative stress responses in the liver of L. CAT may be a compensatory mechanism to rohita. It was clear that the administration of get rid of excess peroxides generatedby the BPS significantly increased the activity of SOD action. According to Schlorff et al. SOD, CAT, GSH and GR whilethe activity of (1999), increased activities of CAT GST and GPx decreased significantly at all indicate highly induced capacity to scavenge exposures to BPS. In this study, the SOD H2O2 produced in response to oxidative activity increased significantly on exposure stress. Higher CAT activity was reported by to sub-lethal doses of BPS as compared to Sturve et al. (2008) in Onchorynchus mykiss that of control. Superoxide dismutase exposed to sewage treatment plant effluent. catalytically scavenges superoxide radical The liver of Cyprinus carpio exposed to three which appears to be an important agent of disinfectants, chlorine dioxide, sodium toxicity of oxygen and this provides a hypochlorite and peracetic acid also reported defense against oxygen toxicity(Kadar et al., higher CAT activity (Elia et al., 2006). 2005). Comparable increased activity of SOD Glutathione peroxidase activity was was observed in some fish exposed to various increased initially but significantly decreased types of endocrine disruptors such as from the control value on extended exposure. butachlor in Clarias gariepinus (Farombi et Glutathione peroxidase degrades H2O2 using al., 2008) and paclobutrazol in Zebra fish, reduced glutathione. Glutathione peroxidases Danio rerio (Ding et al., 2009). are major enzymes that remove H2O2 generated by SOD in cytosol and Catalase breaks down hydrogen peroxide mitochondria (Chanceet al., 1979). Low GPx forming water and oxygen. It is primarily activity may be due to increased catalase found in peroxisomes,although it is present in activity, both of which use H2O2 as substrate mitochondria and cytosol as well (Kelly et (Yilmaz et al., 2006). It has been suggested al., 1998). Catalase activity is considered as a that GPx is responsible for the detoxification sensitive biomarker of oxidative stress in fish of H2O2, when it is present in low (Sanchez et al., 2005). In this investigation, it concentration, whereas CAT plays its role is obvious that activity of CAT increased when GPx pathway reaches saturation with significantly at all exposures to sub-lethal substrate. doses of BPS. Glutathione-S-transferase activity decreased The CAT-SOD system is considered the first significantly in a time and dose-dependent line of defense against oxyradical formation manner in BPS exposed fish. In fish, GST is (Pandey et al., 2003). In this study, used primarily as a biomarker indicating significantly higher SOD and CAT activities aquatic environment pollution with in the liver of L. rohita were found. That is, wastewater of municipal, industrial, a significant positive correlation was found agricultural or mining origin(Tjalkens et al., between the SOD and CAT activities after 1998). Total GST activity decreased in the exposure to 80mg BPS/L (r = 0.875271), liver tissue in the guppy, Poecilia reticulata 100mg BPS/L (r = 0.976012) and 120mg following exposure to dimethoate (Frasco et BPS/L (r = 0.981224) in our study. al., 2002) and in Atlantic cod exposed to an estrogenic mimetic, nonylphenol (Hasselberg The increase in SOD activities in the liver of et al., 2004a, b). This decrease in GST the fish may be due to the production of activity can be explained by an accumulation superoxide anions which might have led to of metabolites of the first or the second the production of SOD, to convert the detoxification stage that may cause the superoxide radical to H2O2. The increase in Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

456 Current status and challenges for conservation and sustainable use of biodiversity inhibition of the total GST activity. The (Koshy et al., 2003). Increased MDA and change in GPx activity observed above is Conjugate diene were reported by Sturve et generally accompanied by changes in the al., (2008) in rainbow trout, Onchorynchus level of GSH, which is the co-substrate for mykiss caged outside a sewage treatment H2O2 decomposition by GPx (Sies, 1999). plant. Likewise GSH concentration increased in In conclusion, the present study reveals that the fish exposed to BPS as compared to that the fish experienced oxidative stress on BPS of controls at all exposures. As an exposure. The study results indicated that antioxidant, GSH plays a major role in delicate balance among the antioxidant cellular metabolism and free radical enzymes is disturbed which may lead to scavenging (Athansios et al., 2006). Increase deviations in the redox status. It is evident in Glutathione content has been described as that BPS is a chemical with oxidative one of the protective mechanisms that fish potential. adopt in the initial phases of exposure to aquatic pollutants (Stephenson et al., 2002). References The GSH-dependent antioxidative system Agergaard, N. and Jensen, P.T. 1982. consists of two enzymes: GPx and GR. Procedure for blood glutathione peroxidase Glutathione reductase (GR) or glutathione- determination in cattle and swine. Acta. Vet. disulfide reductase (GSR) catalyses the Scand., 23: 515-527. reduction of oxidized glutathione (GSSG) to Athanasios, V., Thomais, V., Manos, D. and GSH. By the contrast to GPx, this enzyme is Michael, S. 2006. Molecular biomarkers of involved in the maintenance of glutathione in oxidative stress in aquatic organisms in reduced form and owing to this; GSH plays relation to toxic environmental pollutants. its antioxidant functions (Kaplovitz et al., Ecotoxicol. Environ. Saf., 64: 178-179. 1996). In this study, we have found that Benke, G.M. and Cheevar, K.C. 1974. The activity of GR increased significantly at all comparative toxicity, acetylcholinesterase doses. action and metabolism of methyl parathion and parathion in sunfish and mice. Toxicol. The present study revealed that BPS Appl. Pharmacol., 28: 97-109. enhanced lipid peroxidation by elevating Bradford, M.M. 1976. Rapid and sensitive MDA and CD levels in dose- and time- method for the quantitation of microgram dependent manner. The process of lipid quantities of protein utilizing the principle of peroxidation proceeds by a set of free radical protein-dye binding. Anal. Biochem., 72: chain reactions, especially for 248-254. polyunsaturated fatty acids which are highly Chance, B., Sies, H. and Boveris, A., 1979. prone to oxidative reactions by ROS because Hydroperoxide metabolism in mammalian of the presence of double bonds. The organs. Physiol. Rev.,59: 527–605. detection and measurement of lipid David, M. and Richard, J.S. 1983. Methods peroxidation is one of the evidences most of enzymatic analysis. In: Bergmeyer, J., frequently cited to support the involvement Marianna, G.B. Verlag Chemie Wein Hein of free-radical reactions in toxicology Dein Field, Beah Florida, Basel, 26:358-9. (Livingstone, 2001). Lipid peroxidation De Bleser, P.J., Xu, G., Romboust, K., products may form DNA adducts giving rise Rogiers, V. and Geerts, A. 1999. Glutathione to mutations and altered patterns of gene levels discriminate between oxidative stress expression (Marnett, 1999). Increase in LPO and transforming growth factor-β signaling in levels in spite of increased catalase activities and glutathione content could be due to increased production of oxygen free radicals than the capacity of antioxidant enzymes Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

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458 Current status and challenges for conservation and sustainable use of biodiversity for human exposure. J. Agric. Food Chem., hepatic metal levels. Environ. Toxicol. 61 (19): 4655–4662. Pharmacol., 19: 177-183. Liao, C., Liu, F. and Kannan, K. 2012. Schlorff, E.C., Husain, K. and Somani,, S.M. Bisphenol S, a new bisphenol analogue in 1999. Dose and time dependent effects of paper products and currency bills and its ethanol on plasma antioxidant system in rat. association with bisphenol A residues. Alcohol., 17: 97-105. Environ. Sci. Technol., 46: 6515- 6522. Sies, H. 1999. Gluthatione and its role in Livingstone, D. 2001. Contaminant- cellular functions. Free Rad. Biol. Med., 27: stimulated reactive oxygen species 916-922. production and oxidative damage in aquatic Stephenson, E., Sturve, J. and Forlin, L. organisms. Marine. Pollut. Bull. 42: 656-666. 2002. Effects of redox cycling compounds on Maehly, A.C. and Chance, B.1954. The assay glutathione content and activity of of catalase and peroxide. In: Glick D (ed) glutathione related enzymes in rainbow trout Methods of Biochemical Analysis I. livers. Com. Biochem. Physiol., 133: 435- Interscience: New York., 1: 357. 442. Marnett, L.J. 1999. Lipidperoxidation-DNA Sturve, J., Almroth, B. C. and Forlin, L. damage by Malondialdehyde. Mutat. Res., 2008. Oxidative stress in rainbow trout, 424: 83-95. Onchorynchus mykiss exposed to sewage Merad-Saidoune, M., Boitier, E., Marsac, treatment plant effluent. Ecotoxicol. Environ. A.N., Martnou, J.C., Sola, B. and Sinet, P.M. Saf., 70 (3): 446-452. 1999. Overproduction of Cu/Zn-superoxide Tjalkens, R.B., Valeriojr, I.G., Awasthi, Y.C. dismutase or Bcl-2 prevents the brain and Petersen, D.R. 1998. Association of GST mitochondrial respiratory dysfunction induction and lipid peroxides in two inbred induced glutathione depletion. Exp. Neurol., strains of mice subjected to chronic iron 158: 428. overload. Toxicol. App. Pharmacol., 151: Oakes, K. and Van Der Kraak, G. 2003. 174-181. Utility of the TBARS assay in detecting Vinas, R., and Watson, C.S. 2013. Bisphenol oxidative stress in white sucker (Catostomus S disrupts estradiol-Induced nongenomic commersoni) populations exposed to pulp signaling in a rat pituitary cell line: effects on mill effluent. Aquat. Toxicol., 63: 447-463. cell functions, Environ. Health Perspect., Pandey, S., Parvez, S., Sayeed, I.,Haque, R., 121: 352–358. Bin- Hafeez, B. and Raisuddin, S. 2003. Winston, G.W. and Di Giulio, R.T. 1991. Biomarkers of oxidative stress: a Pro-oxidant and antioxidant mechanisms in comparative study of river Yamuna fish, aquatic organisms. Aquat. Toxicol., 19: 137- Wallago attu. Sci. Total. Environ.,309: 105- 161. 15. Yilmaz, H.R., Turkoz, Y., Yukse, E. and Pivnenko, K., Eriksson, E. and Astrup, T.F. Orun, I. 2006. An investigation of antioxidant 2015. Waste paper for recycling: Overview enzymes activities in liver of Cyprinus and identification of potentially critical carpio taken from different stations in the substances. Waste. Manag., 45: 134-142. Karakaya Dam Lake. Int.J. Sci.Tech., 1:1-6. Sanchez, W., Pallulel, O., Meunier, L., Coquery, M., Porcher, J.M. and Ait-Aissa, S. 2005. Copper-induced oxidative stress in three-spined stickleback: relationship with Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.447-458 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal concentrations in sediments and commercially important … 459 CHAPTER 64 ISBN 978-93-5396-871-7 TRACE METAL CONCENTRATIONS IN SEDIMENTS AND COMMERCIALLY IMPORTANT PENAEID SHRIMP, METAPENAEUS DOBSONI (MIERS, 1878) COLLECTED FROM COCHIN BACKWATERS Jyothirmaye Mohan*, Biju A and Bettina P Alex Zoology Research Centre, St, Stephen’s College, Pathanapuram, Kollam, University of Kerala *Correspondence E-mail: [email protected] ABSTRACT This study was carried out to detect the physico-chemical parameters and the trace metal concentration in sediments and the penaeid shrimp, Metapenaeus dobsoni from Cochin backwaters. Metapenaeus dobsoni(Miers,1878)is a commercially important sea food species of Kerala. The concentrations of iron, nickel, zinc, copper, lead, cadmium and chromium were measured in the sediment and the muscle tissue of Metapenaeus dobsoni from Cochin backwaters. The analytical result demonstrate that the pH of water at Vaikkom (6.65±0.21pH units)and Thanneermukkom(6.45±0.07 pH units) is slightly acidic than all other stations. The conductivity (9959±558.61µS/cm), the salinity (34.45±0.21.5ppt), Total hardness (7725±176.78ppm) and TDS (4979±279.31ppm) of water at Fort Kochi is recorded higher than the other stations. Alkalinity (ranges 120-125ppm) is recorded high at Bolgatty. The trace metal concentration in the sediments were Fe (110-150 ppm), Ni (11.5-60.12 ppm), Zn (22.2-64.8 ppm), Cu (6.28-10.8 ppm), Pb (5.93-27.51 ppm), Cd (BDL-0.21 ppm) and Cr (24.8-71.9 ppm). The concentration of Fe, Ni and Cu were recorded high at Bolgatty, where Zn and Pb were high at Fort Kochi and Cd and Cr were recorded high at Vaikkom. The bioaccumulation of trace metals in the M.dobsoni was in the decreasing order of Fe>Zn>Ni>Cu>Cr>Pd>Cd. Concentration of Fe, Ni and Cd were highest in Bolgatty, Ni in Fort Kochi, Cu in Arookutty, Pb in Vaikkom and Cr in Thanneermukkom. The Metal Selectivity Index(MSI) values for the selected metals followed a general pattern of Fe>Zn>Ni>Cr>Cu>Pb>Cd. Key words: Trace metals, Penaeid shrimp, Metapenaeus dobsoni, Cochin backwaters, Bioaccumulation. Introduction organisms. Metals are considered as important and highly toxic pollutants in In global terms, the quality of coastal water is various environmental compartments. The declining particularly in estuary, with trace metals that discharged into the aquatic estimated stocks of species decreasing and ecosystem are distributed between aqueous vitally important habitats being destroyed or phase and bed sediments during their being degraded. The rivers being often used transport. The abundance and bioavailability as disposal areas for domestic and industrial of metals in the aquatic system can be wastes, estuaries act as transit area for assessed as the bioaccumulation of metals by pollutants on their way to marine the tissue of aquatic organisms (Rejomon et environment. Hence it is important to al., 2010). Different metals as well as consider effects of pollutants on the location based ecological risk assessment sediment, water and biota. The estuaries offer studies using sediment accumulation indices some positive advantages for benthic have been reported in the literature by Du Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

460 Current status and challenges for conservation and sustainable use of biodiversity Laing et al., 2009; Fahey et al., 2008; fishermen. They were preserved in ice box Kamalakkannan et al., 2004; Koptsik et al., packed with ice in order to maintain the 2003). Cochin estuary is known to contain freshness and later transported to the higher concentration of almost all metals laboratory. during pre-monsoon and post-monsoon seasons (Nair et al., 1990). Anu et al. (2014) The penaeid shrimp, Metapenaeus dobsoni is reviewed the trace metal status of water, one of the most valuable commercial sea sediment in Cochin backwaters. foods and the most abundant species along the coast of Kerala. The life history of this Materials and methods involves an estuarine phase as their post- larvae and juveniles migrate to estuaries, Study area which are their nursery grounds as the shrimp grow, they move into deeper waters of the Cochin backwaters is one of the largest estuary and from there to the sea where they wetlands along the west coast of India(Lat. contribute to the marine fishery (Gopalan et 09°30' and 10ºI2'N and Long. 76°10' and al., 1983). 76°30'E) with its northern boundary at Azhikode and southern boundary at Analysis of samples Thanneermukkom bund. The total area of the estuary is around 250km2. During flood tide Physico-chemical parameters the seawater enters the estuary via Cochin bar mouth (12m depth) and the flow reverses The water quality analysis of the backwaters during ebb tide. Five stations were selected gives the exact nature, cause and levels of for the present study, Fort Kochi (S-I), pollutants, if any. The physico-chemical Bolgatty (S-II), Arookutty (S-III), Vaikkom parameters like temperature, pH, electronic (S-IV) and Thanneermukkom (S-V). Among conductivity, total dissolved solids, dissolved these, three samples were collected from the oxygen, salinity, alkalinity, free CO2 and brackish water downstream of the southern total hardness were analysed by adopting arm, representing the influence of domestic relevant methods from APHA (1998) and settlement, Muvattupuzha River and the Grasshoff (1999) remaining two stations were under the influence of port and shipyard activities near Sediment samples the bar mouth region. Sediment samples were air dried; stones and Sample collection plant fragments were removed by passing the dried samples through a sieve. The sieved Water sample, sediment samples and prawn sample was powdered in a mortar and pestle samples were collected bimonthly and finally passed through a 500µm sieve (22ndMarch 2018 and 26thMay 2018) during and stored in glass bottles. Then 0.25g pre-monsoon season from the Cochin powdered sediment sample was taken in the backwaters. The surface water sample was Teflon beaker and digested with H2O2, HNO3 collected from the Cochin backwater using and HClO4 in the ratio 1:3:1 by using a Niskin sampler at a depth of 25 cm below. microwave digester. The digested samples The sediment samples were collected using were made up to 50ml with distilled water Van Veen grab. The prawn, Metapenaeus and aspirated in the AAS (APHA, AWWA, dobsoni were directly collected from local WEF. 1998). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal concentrations in sediments and commercially important … 461 Prawn samples Physico-chemical parameters Approximately equal size and weight of The water quality parameters were analysed prawn samples were taken and its flesh was and the results are shown in the table 1. dissected and washed thoroughly with According to the guidelines of WHO typical distilled water. Then the sample organ were pH range for the polluted surface water is dried in an oven at 80°C for 48 hours and 3.0-12.0 in the present study the average pH stored in a vacuum desiccator. Dried samples ranges between 6.46-8.28.Conductivity were powdered and aliquots of about 300mg increases with increasing mineral content of were digested for 3h at 80 °C with 3ml HNO3 water sample. In the present study, (65% Merck, Suprapure). Additional nitric conductivity as well as TDS, salinity and acid was added if the samples were charred hardness were reported high in the station and 1ml HClO4 (Merck Suprapure) was Fort Kochi (S-I), the bar mouth of Cochin added to make the solution clear and backwaters. The average value of dissolved evaporated to near dryness. Thick white oxygen ranges from 3.3±0.14 mg/L- fumes were evolved at the end point. The 4.4±0.328 and the high DO is reported in the digests were cooled and diluted to make the station Thanneermukkom (S-V) that may be solution to 25ml with double deionised water due to the mixing of water with fresh water. and kept in plastic vials. Trace metals were The aquatic fauna can survive when the analysed using Atomic Absorption water temperature remains fairly constant. Spectrophotometer. The calculation of the Alkalinity is not a specific pollution selected metals in the tissues is calculated as substance as it represents the buffering per the formula; capacity of water. Decreased alkalinity levels on aquatic organisms are increase of toxicity Concentration of metal in the sample = of trace metals. Cochin backwater is known to contain higher concentration of almost all ������������������������������������������������������������×������������������.������������������������������������������������×������������������������������������������������������������������������������������(������������������������������) trace metals during pre-monsoon (Nair et al., 1990). ������������������������������������������ℎ������������������������ℎ������������������������������������������������������������������������ Results and discussion Table 1. Physico-chemical characteristics of water samples Parameters S-I S-II S-III S-IV S-V Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Temp 33.45±0.21 33.7±0.28 32.95±0.21 32.85±0.49 33.75±0.35 pH 8.28±.07 7.35±0.07 6.65±0.21 6.45±0.07 EC 8±0.14 9435±1124.3 7687±1356.94 5265±1432.6 4206.5±61.52 TDS 9959±558.61 4717.5±562.15 3844±678.82 2632.5±716.3 2103.5±30.41 DO 4979±279.31 3.45±0.35 4.25±0.64 3.3±0.14 4.4±0.28 Salinity 28.3±.028 26.45±0.07 15.65±0.78 15.35±1.48 Alkalinity 4.2±0.14 122.5±3.54 95.45±4.31 43.75±2.47 10.25±1.48 Free CO2 34.45±0.21 12.85±0.35 10.3±0.28 25.25±1.77 8.65±0.07 Total hardness 99±12.72 7590±98.99 3555±148.49 2904.5±198.70 2325±176.78 11.6±0.28 7725±176.78 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

462 Current status and challenges for conservation and sustainable use of biodiversity Trace metal analysis in sediment samples This may be due to the port and shipyard activities near the site. The level of Cd and The concentration of trace metals was Cr were high at Vaikkom (0.21ppm and calculated station wise. The minimum 71.9ppm respectively). The trace metal concentration of Fe (110 ppm), and Cd concentration in the sediment sample was in (BDL) during the sampling period was the order Fe>Cr>Zn>Ni>Pb>Cu>Cd. The observed in Fort Kochi and Arookutty. The result analysis of trace metal concentration in concentration of Fe, Ni and Cu were high at sediment samples are given in the table 3. Bolgatty, Zn and Pb at Fort Kochi (64.8ppm). Table 2. Trace metal concentration (ppm) in sediment samples Stations pH EC Organic Fe Ni Zn Cu Pb Cd Cr (mmhos) Carbon% Ppm Ppm Ppm Ppm Ppm Ppm Ppm S-I 8.2 5.3 0.02 110 33.4 64.8 8.1 27.51 BDL 38.4 S-II 7.8 11.9 1.04 150 60.12 48.6 10.8 18.24 BDL 65.4 S-III 8.1 5.7 0.19 110 20.14 21.48 8.5 16.9 BDL 29.47 S-IV 8.2 5.3 0.92 130 46.27 43.4 9.6 22.13 0.21 71.9 S-V 7.4 3.5 0.37 140 11.5 22.2 6.28 5.93 0.07 24.8 BDL- Below detection level Trace metal concentration in Metapenaeus dobsoni Benthic organisms play an important role in the ecological risk assessment of trace metals, since the sediment, the matrix it lives is considered as the sink of trace metals in aquatic ecosystem (Monperrus et al., 2005). The result of analysis of trace metal concentration in the penaeid shrimp, Metapenaeus dobsoni collected from Cochin estuary is given in the table 4. The concentration of Fe and Cu (633.12ppm and 32.68ppm) were recorded high in the station S-I and low in S-V (309.41ppm) and S-I (19.17) respectively, where Zn and Cd (84.04ppm and 0.58ppm) were high in the station S-II and low Zn level in S-IV (46.29ppm). The trace metal accumulation is in the order of Fe>Zn>Cr>Ni>Cu>Pb>Cd. The trace metal distribution in the coastal environments is influenced by freshwater inflow (Riley and Chester, 1971) and terrestrial contamination and anthropogenic inputs (Kremlin and Petersen, 1984). The fish and other organisms become more sensitive to trace metal uptake because the metals are more soluble, this is how they become incorporated into the food chain. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal concentrations in sediments and commercially important … 463 Table 3. Trace metal concentration (ppm dry wt.) in Penaeid prawn, Metapenaeus dobsoni. Parameters S-I S-II S-III S-IV S-V Mean±SD Mean±SD Temp Mean±SD Mean±SD Mean±SD 32.85±0.49 33.75±0.35 pH 6.65±0.21 6.45±0.07 EC 33.45±0.21 33.7±0.28 32.95±0.21 5265±1432.6 4206.5±61.52 TDS 2632.5±716.3 2103.5±30.41 DO 8±0.14 8.28±.07 7.35±0.07 3.3±0.14 4.4±0.28 9959±558.61 9435±1124.3 7687±1356.94 4979±279.31 4717.5±562.15 3844±678.82 4.2±0.14 3.45±0.35 4.25±0.64 Salinity 34.45±0.21 28.3±.028 26.45±0.07 15.65±0.78 15.35±1.48 Alkalinity 99±12.72 122.5±3.54 95.45±4.31 43.75±2.47 10.25±1.48 Free CO2 11.6±0.28 12.85±0.35 10.3±0.28 25.25±1.77 8.65±0.07 Total 7725±176.78 7590±98.99 3555±148.49 2904.5±198.70 2325±176.78 hardness Statistical approach and TOC of five stations are given in table 5. Correlation coefficient greater than .8, that is Correlation study of metals of high correlation.EC, TOC, Ni, and Cu in sediment show positive correlation with Cd In order to have an overview about the of shrimp. Ni in shrimp recorded high factors that influence the trace metal correlation Zn and Pb of sediment Pb showed concentration in shrimp, some sediment positive correlation with Cd of sediment. parameters and trace metal concentration Inter elemental association was also were examined. The sediment parameters evaluated by correlation coefficient (r). Ni/Zn considered were pH, EC and organic carbon (r=0.91), Ni/Pb (r=0.87), Pb/Cd (r=0.99), and total Fe, Ni, Zn, Cu, Pb, Cd and Cr in Cd/Ni (r=0.88), Cd/Cu (r=0.84), Ni/Cu (r- shrimp and sediment. Statistical tests were =0.93), Zn/Pb (r=0.83), Cu/Cr (r=.085) etc, performed to establish the inter-metal have strong correlation with each other. relationships and classify metals. The correlation analysis of trace metals, pH, EC Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

464 Current status and challenges for conservation and sustainable use of biodiversity Table 5. Correlation between trace metal concentration in shrimp, Metapenaeus dobsoni and sediment in Cochin backwaters Fe Ni Zn Cu Pb Cd Cr pH EC TOC Fe Ni Zn Cu Pb Cd Cr Fe 1 Ni -0.07 1 Zn 0.256 0.603 1 Cu 0.774 -0.51 0.18 1 Pb -0.18 -0.34 -0.65 -0.4 1 Cd 0.421 0.055 0.67 0.39 0 1 Cr -0.69 -0.62 -0.45 -0.1 0.13 -0.32 1 pH 0.35 0.615 0.03 -0.3 0.23 -0.08 -0.85 1 EC 0.598 0.23 0.84 0.52 -0.3 0.92 -0.52 0.025 1 TOC 0.271 -0.27 0.23 0.27 0.45 0.87 -0.09 -0.11 0.625 1 Fe -0.08 -0.42 0.29 0.3 0.11 0.73 0.41 -0.68 0.504 0.79 1 Ni 0.358 0.403 0.65 0.04 0.13 0.88 -0.6 0.346 0.816 0.76 0.42 1 Zn -0.19 0.919 0.63 -0.6 -0.1 0.3 -0.51 0.504 0.34 0.07 -0.1 0.63 1 Cu 0.664 0.287 0.56 0.3 0.1 0.84 -0.75 0.455 0.841 0.71 0.28 0.93 0.42 1 Pb 0.161 0.873 0.37 -0.5 0.04 0.11 -0.83 0.899 0.215 -0.06 -0.5 0.54 0.83 0.52 1 Cd -0.29 -0.34 -0.62 -0.4 0.99 0 0.22 0.133 -0.35 0.47 0.19 0.13 -0.08 0.05 -0.01 1 Cr 0.25 0.185 0.3 -0.1 0.51 0.77 -0.45 0.369 0.579 0.85 0.43 0.92 0.48 0.85 0.46 0.51 1 TOC- Total organic carbon MSI= ������������������������������������������������ ������������������������������������������������������������������������������ ������������ ������ ������������������������������ ������������ ������ ������������������������������������ × Metal selectivity index (MSI) ������������������������������ ������������������������������������������������������������������������������ ������������ ������������������ ������������������������������������ ������������ ������ℎ������������ ������������������������������������ Normally up to certain concentration, the trace metal reaching inside the organism will 100 be regulated by metabolic activities and they will be excreted from the body. Only after The MSI values of the shrimp are given in reaching a particular limit the metals start the table 4. The MSI values for the selected accumulating in the body tissue (Mohan et metals follows a general pattern of al., 2012). Thus metal selectivity index is the Fe>Zn>Ni>Cr>Cu>Pb>Cd. Nair et al. (2006) affinity of a particular organ or tissue can be reported comparatively lower MSI value for reliable index for risk assessment of that cadmium. In Cochin estuary the MSI value particular metal. MSI is the percentage of clearly shows the high affinity of Fe and Zn absolute concentration of a metal in a tissue towards the biota. to total concentration of all metals in that tissue (Nair et al., 2006). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

Trace metal concentrations in sediments and commercially important … 465 Table 4. Metal selectivity index (%) of Metapenaeus dobsoni collected from Cochin backwaters Stations Fe Ni Zn Cu Pb Cd Cr S-I 99.896 7.34893 13.38404 3.682148 1.867005 0 3.536168 S-II 75.9838 4.795803 11.16143 4.247294 1.289594 0.07703 2.445049 S-III 81.14634 4.506421 6.6289 4.188562 1.439338 0 2.090436 S-IV 74.52409 5.903887 7.702676 3.720714 3.995274 0.034944 4.118411 S-V 61.92659 6.838924 9.658954 5.225763 2.717957 0 13.63181 Conclusion References In the present study sediment and the penaeid Anu, P.R., Jayachandran, P.R., Sreekumar, shrimp, Metapenaeus dobsoni were collected P.K. and Bijoy Nandan, S. 2014. A review on from Cochin estuary and seven trace metals trace metal pollution in Cochin backwaters, were analysed. Surface water samples were south west coast of India. also collected for the analysis of physico- chemical parameters. Toxicity is influenced APHA, AWWA, W.E.F. 1998. Standard by the intrinsic toxicity of the elements, but methods for examination of water and also the availability of trace metals as wastewater (20thed.). Washington DC: determined by the occurrence, complication American Public health Association. of other chemical reactions and absorption potential. Shrimps are one of the most Du Laing, G., Chapagain, S.K., commercially important species collected Dewispelaere, M., Meers, E., Kazama, F., from Cochin estuary and consumed by the Tack, F.M.G and Verloo, M.G. 2009. large population in their daily diet. So the Presence and mobility of arsenic in estuarine study recommends further monitoring and wetland soils of the Cheldt estuary risk assessment of edible shrimps in the lake. (Beigium). J. Environ. Monit., 11(4):873- 881. Int. J. Mar.Sci. 4(10):92-98. Acknowledgement Fahey, N.S.C., Karagatzides, J.D., Authors are greatful to Dr. K.P. Laladhas, Jayasinghe, R. and Tsuji, L.J.S. 2008. Principal, St. Stephen’s College, Wetland soil and vegetation bismuth content Pathanapuram, Dr. Neena Suzzan Joshua, following experimental deposition of bismuth Head of the department, P.G and Research pellets. J. Environ. Monit., 10(8):951-954. Department of Zoology, St. Stephen’s College, Pathanapuram and The Director, Gopalan, U.K, Vengayil, D.T, Udaya, V.P. Zoology Research Centre for the and Krishnankutty, M. 1983. The shrinking encouragement and facility provided. back waters of Kerala. Journal of Marine Biological Association India., 25:131-141. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

466 Current status and challenges for conservation and sustainable use of biodiversity Kamalakkannan, R., Zettel, V., Goubatchev, India. Environ. Monit.Assess.,184(7): 4233– A., Stead-Dexter, K. and Ward, N.I. 2004. 4245. Chemical (polycyclic aromatic hydrocarbon and trace metal) levels in contaminated storm Kremling, K. and Petersen, H. 1984. water and sediments from a motorway dry Synoptic survey on dissolved trace metal detention pond drainage system J. Environ. levels in Baltic surface water. Mar. Poll. Monit., 6(3):175-181. Bull., 15(9):329-334. Koptsik, S., Koptsik, G., Livantsova, S., Riley, J.P. and Chester, R. 1971. Introduction Eruslankina, L., Zhmelkova, T. and to marine chemistry. Academic Press. Vologdina, Z. 2003. Trace metals in soils London. 465. near the nickel smelter chemistry, special variation and impacts on plant diversity. J. Environ. Monit., 5(3):441. Monperrus, M, Point, D., Grall, J., Chauvaud, L., Amouroux, D., Bareille, G. and Donard, O. 2005. Determination of metal and organo metal trophic bioaccumulation in the benthic macrofauna of the Adour estuary coastal zone (SW France, Bay of Biscay). Journal of Environ.Monit., 7(7):485-491. Nair, S. M., Balchand A. N. and Nambisan P. N. K. 1990. Metal concentrations inrecently deposited sediments of Cochin backwaters, India. .Sci. Tot. Environ.,97(98): 507–524. Nair, M., Jayalakshmy, K.V., Balachandran, K.K. and Joseph, T. 2006. Bioaccumulation of toxic metals by fish in a semi-enclosed tropical ecosystem. Environ. Forensics., 7:197-206. Rejomon, G., Nair, M. and Joseph, T. 2010. Trace metal dynamics in fishes from the southwest coast of India. Environment Monitoring and Assessment, 167:243-255. Grasshoff, K., Ehrhardt, M. and Kremling, K. 1999. Methods of Seawater Analysis, 159- 226. Mohan, M., Deepa, M., Ramasamy, E.V. and Thomas, A.P. 2012. Accumulation of mercury and other heavy metals in edible fishes of Cochin backwaters, Southwest Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.459-466 © Principal, Sree Narayana College, Kollam, Kerala, India

Biochemical and histopathological changes in the tissues of cyprinus carpio treated … 467 ISBN 978-93-5396-871-7 CHAPTER 65 BIOCHEMICAL AND HISTOPATHOLOGICAL CHANGES IN THE TISSUES OF CYPRINUS CARPIO TREATED WITH IRON OXIDE AND CERIUM OXIDE NANOPARTICLES Usha S Department of Zoology, Sree Narayana College for Women, Kollam, Kerala. Correspondence E-mail: [email protected] ABSTRACT Histopathological and biochemical effects of nanoparticles affecting the health of organisms which are commercially important has been an important area of research. Iron oxide and cerium oxide nanoparticles have wide applications in science. It is known that histological and biochemical changes can monitor fish health when exposed to contaminants. Cyprinus carpio fingerlings were selected to study short term exposure to these nanoparticles. Sublethal concentration of 2mg/litre of both nanoparticles were added to the 60 litre tank containing Cyprinus carpio fingerlings. Control group, IrO nanoparticle treated group and CeO2 nanoparticle treated group was separately kept in the laboratory. After 96 hrs, the fish were sacrificed and gills, muscle and liver was dissected out for histopathological studies. Protein and lipid estimation was also conducted. Histological changes were observed most in the gills of treated fishes, especially cerium oxide treated fish. No significant changes were noted in liver and muscle sections. Biochemical changes were also more evident in gills rather than muscles of treated fish. Key words: Cyprinus carpio, Iron oxide nanoparticle, Cerium oxide nanoparticle, Histopathology. Introduction al., 2002). Despite these concerns, only limited studies have been actually conducted Engineered nanoparticles can contaminate to assess the health effect of nanomaterials aquatic environment and very limited using in vivo and in vitro methods. Many knowledge is available on the behaviour, works on nanoparticles have pointed out the entry and toxicity of these materials in need for assessing the exposure of organisms aquatic organisms. (Canesi, et al., 2010). to nanoparticles especially different types of According to Moore in 2006, in aquatic nanometal oxides(Johnston et al., 2010; Zhu organisms, routes of these particles include et al., 2008 and Sager et al., 2007). direct ingestion, entry across gills, olfactory organs or body wall, and also endocytosis Fe2O3 NPs are used to remove arsenic from and phagocytosis. Fish are sensitive drinking water (Yavuz et al., 2006), and due indicators of pollutants present in water. to its widespread use, exposure of humans Corona formation which affects different and animals to these NPs may likely increase systems occurs when nanoparticles interact in the near future (Colvin, 2003). The studies with environment (Mattsson, 2015; on the acute toxicity of nanomaterials to Yetreberg, 2011; Moore, 2006).These aquatic organisms are lacking, and hence it pollutants cause various physical and has become an important issue (Kerstin and physiological alterations in fishes (Trivedi et Markus, 2006). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.467-472 © Principal, Sree Narayana College, Kollam, Kerala, India

468 Current status and challenges for conservation and sustainable use of biodiversity The toxicity study using Cerium oxide Nanoparticles of IrO and CeO2 were made by nanoparticles were rare compared to iron precipitation method and SEM was used to oxide nanoparticles (Hasanzadeh et al., study the morphology (Size range was 40nm 2018). CeO2 nanoparticles have wide to 110 nm). A sub lethal concentration of 2 applications including industrial and mg/l of Iron oxide and cerium oxide biological (Reed et al., 2014 and Ma et al., nanoparticles was dissolved in a water tank 2018). They also have high affinity for with 60 litre capacity. Three groups of fish oxygen (Lin et al., 2006) and used as with almost same size were selected for the antioxidants in medical field. So their use and study. Each group composed of 10 fishes. toxic effects, especially in aquatic organisms The first group was control group. The has to be given more concern according to second group was treated with IrO Som et al. (2012). nanoparticles and third group was treated with CeO2 nanoparticles. The period of Histology acts as an integrated parameter, exposure was for 96 hours. After this period, providing a complete evaluation of the the fish were sacrificed. Gills, muscle and organisms’ health, effectively monitoring the liver were dissected from both control and effects of exposure to environmental treated fish. 10% formalin was used to fix pollutants (Teh et al., 1997 and Van Der tissue. After dehydration and embedding in Oost et al., 2003). It is known that paraffin the samples were processed. The size histological and biochemical changes can of the sections made was 6 µm. The stains monitor fish health when exposed to used for tissue analyses were hematoxylin contaminants. Hence a biochemical and and eosin. LABOMED Lx 300 microscope histopathological examination of tissues were with attached digital camera was used for planned to estimate the short term toxicity of tissue examination. Histological studies were IrO and CeO2 particles in Cyprinus carpio. done using standard microtechniques. The key objective of present investigation Biochemical analyses of protein and lipid was an attempt to evaluate the potential were also conducted in the fish using changes in lipid and protein content, if any, standard methods. in the gills and muscle and also toxic histopathological effects in gills, muscles and Results and Discussion liver of Cyprinus carpio fish treated with IrO and CeO2 nanoparticles. Estimation of biochemical parameters Material and Methods Many factors like surface morphology, size, shape and aggregation of nanoparticles can The experiment was conducted using induce toxicity. So the nanoparticles can Cyprinus carpio fingerlings. Fish with a size interact more with cells of exposed range of length 9.60 cm to 10.34 cm and organisms. Toxicity differ in cells when they weight 16.56gm to 19.83 grams were have different characteristics in relation to obtained from a hatchery(Karthika dispersion and ion dissolution also (Nam, Aquarium) in Kollam town, Kerala. The fish 2019). According to Zhu et al. (2009), the fingerlings were acclimatized in laboratory interaction of nanoparticles with chemical conditions for two weeks. The water used to and biological systems may lead to culture the fish was dechlorinated and aerated biochemical disturbances. This can act as continuously. Water temperature was kept at biomarkers and can be used to assess the 25 – 27 C and pH was maintained at 6.8. Fish health condition of aquatic organisms (Wang were fed twice daily with fish food. et al., 2009). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.467-472 © Principal, Sree Narayana College, Kollam, Kerala, India

Biochemical and histopathological changes in the tissues of cyprinus carpio treated … 469 Estimation of protein and total lipid content (Vinodhini and Narayanan,2008; Zutshi et in gills and muscle of nanoparticle treated al.,2010; Shalaby, 2001 and Hanan et al., Cyprinus carpio showed a decline than the 2013). It occurred due to disturbance in control group. It was observed that protein protein and lipid metabolism due to defection and lipid content was more in muscle than in transport from plasma and liver malfunction. gills in control sample. Earlier works also However, in muscles, the changes were not indicated that in nanoparticle treated fish, the much predominant as the exposure period protein and lipid levels declined. Such was short. observations were also made in earlier works Table 1 showing the amount of Protein and total lipid in the gills and muscles of the fish Cyprinus carpio Parameters Control fish IrO treated fish CeO2 treated fish Protein Gills 1.24 0.68 0.78 (mg/g) Muscle 1.64 0.85 1.32 Total lipid Gills 0.62 0.54 0.45 (mg/g) Muscle 1.12 0.48 0.63 Histopathological study Individual myotomes were separated by myoseptum, this formed the origin and According to Neal in 2008, nanoparticles are insertion for muscle fibres from adjacent an intermediate stage between bulk and myotomes. In IrO and CeO2 treated fish, no molecular material, and exhibit toxicity more. well pronounced effects were pronounced. They have small size and greater surface Broken myofibrils were seen in very few area. So they can penetrate more easily and places in the section. Previous studies accumulate in cells of organisms. As they demonstrated that the cytotoxicity of iron were of small size toxicity was more in NPs have been linked to oxidative damage by different organs of fish. However, in control redox cycling of iron and ROS generation, group, the structure and morphology of gills which may lead to disruption of cell were normal. The experimental fish showed membrane, lipid peroxidation and DNA damage in the secondary lamellae of the gills damage (Stohs and Bagchi, 1995, Valko et in both IrO and CeO2 treated fish. This effect al., 2005 and Chen et al., 2011). The was more pronounced in CeO2 treated fish. histopathology of muscle showed only slight As the gills were more exposed to damage in the structure of muscle treated contaminants CeO2 particle accumulation with nanoparticles. It is well known that could also be observed as CeO2 forms several pollutants can cause serious damages aggregates in water which caused more to muscle tissues. Bharat et al. (2011) damage to gills in this fish. studied the histopathology of fish Cyprinus carpio exposed to sublethal concentrations of Muscle is the tissue of motion and is widely lead and cadmium. The fish showed marked distributed in various organs of the fish body. thickening and separation of muscle bundles It is composed of elongated muscle fibers, with severe intracellular edema. Similar each an individual muscle cell, held together observation has been made by Das and by connective tissues. Histology of the Mukharjie (2000). From this it was evident control group showed normal structure. that broken muscle bundles are an immediate Myotomes and myoseptum were seen clearly. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.467-472 © Principal, Sree Narayana College, Kollam, Kerala, India

470 Current status and challenges for conservation and sustainable use of biodiversity effect of toxicity. It also indicated that The slight changes observed in nanoparticles were not as toxic as other histopathology of the present study are a part heavy metals. of defence mechanism of the fish. The above findings and the results of the present study It could be observed that liver was not indicated that gills are more affected than affected by the short exposure period. The muscles and liver as the exposure period was sections of control, IrO treated and CeO2 short. treated fish showed normal liver histology. Fig.1 showing the Gill of control fish (100X) Fig.2 showing the Gill of IrO treated fish (100X) Fig.3 showing the Gill of CeO2 treated fish Fig.4 showing the Muscle of control fish (100X) (400X) Fig.5 showing the Muscle of CeO2 treated fish (400X) 1 - Muscle Bundle, 2 –Broken Myofibrils Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.467-472 © Principal, Sree Narayana College, Kollam, Kerala, India

Biochemical and histopathological changes in the tissues of cyprinus carpio treated … 471 Conclusion Hasanzadeh, L., Oskuee, R, K. and Sadri, K. 2018. Green synthesis of labelled CeO2 It is concluded that in histopathological nanoparticles with 99mTc and its examination, gills are more affected on biodistribution evaluation in mice. Life Sci., exposure to nanoparticles especially CeO2 for 212:233-240. short duration. However, biochemical changes showed a moderate decline in values Johnston., B, D., Scown, T, M., Moger, J., in gills and muscles of both IrO and CeO2 Cumberland, S, A. and Baalousha, M. 2010. treated fishes. Bioavailability of nanoscale metal oxides TiO2, CeO2 and ZnO to fish. Environ Sci Reference Technol.,44:1144-1151. Bharat Bhun patnaik, Hongray howrelia, J., Kerstin, H. R. and Markus, S. 2006. Ecotoxic Teresia Mathews and Selvanayagam. 2011. effect of photocatalytic active nanoparticles histopathology of gill,liver,muscle and brain (TiO2) on algae and daphnids. Environ Sci of Cyprinus carpio communis L. exposed to Pollt res., 13:1-8. sublethal concentrations of lead and cadmium. Afr. J. Biotech. Vol., Lin, W., Huang, Y. W., Zhou, X. D. and Ma, 10(57):12218-12223. Y. 2006. Toxicity of cerium oxide nanoparticles in human lung cancer cells. In J Canesi, L., Ciacci, C., Vallotto, D.,Gallo, G,. Toxicol.,,25:451-457. Marcomini, A. and Pojana,G. 2010. In vitro effects of suspensions of selected Ma, R., Zhang, S.and Wen, T. 2018. A nanoparticles (C60 fullerene,TiO2,SiO2) on critical review on visible-light-response Mytilus hemocytes. Aquat Toxicol., 96:151- CeO2-based photocatalysts with enhanced 158. photooxidation of organics pollutants. Catal today. Doi:10/1016/j.cattod.2018.11.016. Chen, P. J., Su, C. H., Tseng, C, Y., Tan, S. W. and Cheng, C.H. 2011. Toxicity Mattsson K., Ekvall M T., hansson L, A., assessments of nanoscale zero-valent iron Linse S., Malmendal A. and Cedervall T. and its oxidation products in medaka 2015. Altered behaviour, Physiology, and (Oryzias latipes) fish. Mar Pollut Bull., metabolism in fish exposed to polystyrene 63:339-346. nanoparticles. Environ. Sci. Technol., 49:553- 561. Colvin, V. L. 2003. The potential environmental impact of engineered Moore., M. N.2006. Do nanoparticles present nanomaterials. Nat Biotechnol., 21:1166- ecotoxicological risks for the health of the 1170. aquatic environments? Envirn. Int., 32:967- 976. Das, B. and Mukherjee, S.2000. A histopathological study of carp (Labeo rohita) Nam S. H. and An.Y,A. 2019. Size and exposed to hexachlorocyclohexane. Vet. shape –dependannt toxicity of silver Arhiv., 70(4): 169-180. nanomaterials in green alga Chlorococcum infusionum. Ecotoxicol. Environ. Saf., Hanan., S, G., El-Kasheif, M. A., Ibrahim, 168:388-393. S,A. and Authman, M.M.N. 2013. Effect of water pollution in El-Rahawy drainage canal Neal, A. L. 2008. What can be inferred from on hematology and organs of fresh water fish bacterium- nanoparticle interactions about Clarias gariepinus. Worls Appl. Sci. J., 21: the potential consequences of environmental 329341. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.467-472 © Principal, Sree Narayana College, Kollam, Kerala, India

472 Current status and challenges for conservation and sustainable use of biodiversity exposure to nanoparticles? Ecotoxicol., A review. Environ. Toxicol. Paharmacol. 17:362-371. 13:57-149. Reed, K., Cormack, A. and Kulkarni, A. Vinodhini, R. and Narayanan, M. 2008. 2014. Exploring the properties and Effect of heavy metals on the level of vitamin applications of nanoceria: is there still plenty E, total lipid and glycogen reserves in the roonm at the bottom? Envirn Sci nano., liver of Common carp (Cyprinus carpio L.) 1(5):390-404. Maejo Int. J. Sci. Technol., 391-399. Sager, T. M., Porter, D. W., Robinson, V. A., Wang, B., Zhang, Y., Mao, Z., Yu, D. and Lindsley, W. G. and Schwegler-Berry D. Gao, C.2014. Toxicity of Zn O nanoparticles E.2007. Improved method to disperse to macrophages due to cell uptake and nanoparticles for in vitro and in vivo intracellular release of Zinc ions. Journal of investigation of toxicity. Nanotoxicity, nanoscience and nanotechnology , 1:118-129. 14(8):5688-96. Shalaby, A. M.2001. Protective effect of Yavuz, C.T., Mayo, J. T.,Yu, W. W., ascorbic acid against mercury intoxication in Prakash, A., Falkner, J. C., Yean, S.,Cong, L. Nile tilapia (Oreochromis niloticus). J Egypt. L., Shipley, H. J., Kan, A., Tomson, Acad. Soc. Environ. Dev., 2:79-97. M.,Natelson, D. and Colvin, V. L. 2006. Low –field magnetic separation of Som, C., Nowach, B., Krug. H F. and Wick, monodisperse Fe3O4 nanocrystals. Science., P. 2012. Toward the development of decision 314:964-967. supporting tools that can be used for safe production and use of nanomaterials. Acc Ytreberg E., Karlsson J., Ndungu K., Chem res. DOI: 10.1021/ar3000458. Hassellov M., Breitbarth E. and Eklund B. 2011. Influence of salinity and organic matter Stohs., S. J. and Bagchi, D.1995. Oxidative on the toxicity of Cu to a brackish water and mechanisms in the toxicity of metal ions. marine clone of the red macroalga Ceramium Free Rad Biol Med., 18:312-336. tenuicorne. Ecotoxicol. Environ.Saf., 74:636- 642. Teh, S. J., Adams, S. M. and Hinton, D. E.1997. Histopathological biomarkers in feral Zhu, H. J., Jia, Y. F., Wu, X. and Wang, H. freshwater fish population exposed to 2009. Removal of arsenic from water by different types of contaminant stress. Aquat. supported nano zerovalent iron on activated Toxicol., 37, 51-70. carbon. J Hazard Mater 172:1591-1596. Trivedi, S. P., Kumar, M., Misra, A., Zhu, X., Zhu, I.,Duan, Z., Qi, R. and Lang, Banerjee, I and Soni, A.2002. Impact of Y. 2008. Comparative toxicity of several linear alkayl benzenesulphonate (LAS) on metal oxide nanoparticle aqueous phosphatase activity in testis of teleostean suspensions to zebrafish (Danio rerio) early fish. J. Environ. Biol., 22(4):263-266. development stage. J Environ Sci Health, 43:278-284. Valko, M., Morris, H. and Cronin, M. T.D. 2005. Metals, toxicity and oxidative stress. Zutshi, B. S. G., Prasad, R. and Nagaraja, R. Curr Med Chem., 12:1161-1208. 2010. Alteration in hematology of Labeo rohita under stress of pollution from Lakes of Van Der Oost, R., Beyer, J. and Vermeuten, Bangalore, Karnataka, India. Envirn. Monit. N. P. E.2003. Fish bioaccumulation and Assess., 168:11-19. biomarkers in environmental risk assessment: Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.467-472 © Principal, Sree Narayana College, Kollam, Kerala, India

473 Current status and challenges for conservation and sustainable use of biodiversity ISBN 978-93-5396-871-7 CHAPTER 66 IMPACT OF GREEN SYNTHESIZED GOLD NANOPARTICLE FORTIFIED DIET ON LIVER HISTOLOGY OF OREOCHROMIS MOSSAMBICUS Shine.F*, Akhila Thomas*, Shibu Joseph S T** and Dhanya Raj* *Fisheries Biotechnology and Nanoscience Unit *P.G and Research Centre, Department Of Zoology, Fatima Mata National College, Kollam, Kerala ** P G and Research Department Of Chemistry, Fatima Mata National College, Kollam, Kerala *Correspondence E-mail: [email protected] ABSTRACT Nanoparticles have diverse applications in life sciences such as drug development, protein detection and gene delivery, Currently potential application of nanomaterials in fisheries and aquaculture is gaining momentum. Obvious areas of commercial development are the inclusion of nanoscale ingredients in fish feeds, food packaging, the use of nanomaterials in the construction of aquaculture systems and application in fish health. The use of naturally occurring biomolecules to synthesize metal nanoparticles has bridged green chemistry with nanotechnology. The objective of the present study is to prepare and develop a gold nanoparticle(AuNP) systems that possess surface chemistry that could be utilised in anticipated nutrient delivery in fishes. Large gap exist in the data of physiological effect of nanoparticulated feed in fishes. The fish selected for the purpose is Oreochromis mossambicus. Green synthesis has been carried out by aquous extract of Curcuma longa and Ocimum sanctum. The biological effect of green GNPs on Oreochromis mossambicus was assessed in terms of cytostructural study. Key words: Nanotechnology, Aqua culture, Gold nanoparticle, Green Synthesis Introduction initiated with the changes in the tissue and cellular level. According to Meyers and Biological synthesis of nanoparticles has Hendricks, 1985, histology and histopathology can be used as biomonitoring been emerged as a promising field of tools or indicators of health in toxicity studies as they provide early warning signs of research as nanobiotechnology disease. Currently nanoparticles are widely used, but their effects on cells are still under interconnecting biotechnology and investigation. Reports dealing with long term effects of AuNPs on normal cells are less. nanotechnology. In the present study the Thus the interaction of nanoparticles with biological systems including living cells has plant extract selected for biological synthesis become one of the most urgent areas of collaborative research in material science and of AuNP are aqueous extract of Curcuma biology. The present work is an attempt to find out the effect of green synthesized longa and Ocimum sanctum. A growing AuNPs on the cytostructure of aquatic candidate Oreochromis mossambicus. In the number of nanotechnology applications present study the sites selected for the utilize metallic components, many of which can be toxic to aquatic organisms. Due to being exposed to pollutants, major structural damages may occur in their target organs, histological structure may change and physiological stress may occur. This stress causes some changes in the metabolic functions. The changes in the functions are Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.473-478 © Principal, Sree Narayana College, Kollam, Kerala, India

474 Current status and challenges for conservation and sustainable use of biodiversity histological studies is liver. The liver of fish efficient reduction of Au3+ to Au0. The can be considered a target organ to pollutants formed colour solution allowed to measure and alterations in its structure can be absorbance against wavelength to confirm the significant in the evaluation of fish health formation of AuNP. The absorption is a (Myers et al., 1998) typical gold surface Plasmon vibration excitation for colloidal gold nanoparticles Materials and Methods when they interact with electromagnetic radiation. In the optical absorption spectrum Synthesis of gold nanoparticles of the resultant nanoparticles the absorption wavelength of gold nanoparticles were In the present study, green process for the observed at 520 nm. In both the reaction production of gold nanoparticles uses direct mixtures the observed intensity of SPR peaks interaction of HAuCl4 with aquous extract of more with small sharpness in the peak Ccurcuma longa and Ocimum sanctum in the suggesting complete reduction of gold absence of manmade chemical and thus nanoparticles. The reasonable narrow satisfies all the principles of 100% green absorption peak indicated that the particles chemical process. Various phytochemicals were not aggregated and the capping was present in the plant extracts presumably effective.. Phytochemical constituents in the responsible for making a robust coating on plants and spices extract like essential oils gold nanoparticles and thus rendering (terpeneseugenolsetc), polyphenols and stability against agglomeration. Absorption carbohydrate contain active functional measure indicated that Plasmon resonance groups, such as hydroxyl aldehyde and wavelength of synthetic gold nanoparti cles carboxyl units play important role for and green synthesized gold nanosolutions reduction of HAucl4 to AuNPs. Gold from both the plant extracts is nanopartcles particles produced using 520nm.Experimental diets were prepared by phytochemicals or other extract components incorporating 10ml of the above mentioned remain stable for prolonged period. concentrations of gold nanosolution per 100gms of basal feed prior to pressure Experimental design pellatisation. Sixty fishes belonging to both sexes and UV-Vis Spectrophotometric analysis of having an initial length of 7 ± 0.35 cm and 5 Gold Nanoparticle ±0.62 gm were selected. The experimental setup consists of three tanks with 10 fishes Spectrophotometry is a important aspect of per 10 litre of water. The fish in each set was characterization of AuNps. With increase in fed with 2% body weight per day. First tank particle size, the absorption peak shift to served as control. In the second and third longer wavelength and the width of the tanks fishes were fed with diet containing absorption spectra is related to the size gold nano solution prepared using aquous distribution range. Generally AuNPs display extract of Curcuma longa and Ocimum a single absorption peak in the visible range sanctum extract. After 30 days of treatment, between 510-550 nm because of the surface cytostructural profile of the selected tissue of Plasmon resonance and show heavy different treatment groups was estimated. absorption of visible light at 520 nm. This gives purple colour varies according to their Histological and histopathological studies size. The appearance of violet colour is evident that the formation of gold For histological and histopathological nanoparticle in the reaction mixture and the studies, small pieces of the liver were taken Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.473-478 © Principal, Sree Narayana College, Kollam, Kerala, India

Impact of green synthesized gold nanoparticle fortified diet on liver histology… 475 from control and treated fish at the end of Oreochromis mossambicus showing the exposure regimen (30 days). Samples were fixed in 10% buffered formalin for twenty hepatic part (H) harbors normally organized four hours at 4ºC, dehydrated in ascending grades of ethanol, immersed in xylol, and hepatocyte with intact nuclei the pancreatic embedded in paraffin wax. Sections of 4-5 μm thick were mounted on clean glass slides, portion (HP) possesses long elongated deparaffinized, rehydrated, stained with hematoxylin and eosin and mounted with pancreatic cells proliferating around a portal DPX. Sections were examined using a light microscope (Roberts and Smail, 2001) artery(PA).40X is shown in figure 5. Section Results of liver of Oreochromis mossambicus fed Biogenic gold prepared from Curcuma longa with nanocurcumin formulated diet furnished (Fig.1). Figure 2 showed that the Biogenic gold from prepared from Ocimum Sancta. in the figure 6. Nearly a normal architecture UV -VIS Spectrum of nano Ocimum sanctum is depicted in the figure 3 and UV -VIS of the liver is detected. The hepatic portion is Spectrum of nanocurcuma is furnished in the figure 4. Section of hepatopancreas of control harboring polygonal hepatocytes. Sinusoids(S) are narrow and a spindle-shaped Kupffer cell(k) are seen.(40X), In figure 7, Section of liver of Oreochromis mossambicus fed with nanotulsi incorporated diet. The hepatic part (H) exhibits a rather control like appearance Fig. 1.Biogenic gold prepared from Curcuma Fig. 2.Biogenic gold prepared from Ocimum longa. Sancta Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.473-478 © Principal, Sree Narayana College, Kollam, Kerala, India

476 Current status and challenges for conservation and sustainable use of biodiversity ABS 450 500 550 600 650 700 750 nm 800 4.5 Fig 3.UV -VIS Spectrum of nano Ocimum sanctum 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 400 ABS 450 500 550 600 650 700 750 nm 4.5 800 4.0 Fig.4.UV -VIS Spectrum of nanocurcuma 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 400 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.473-478 © Principal, Sree Narayana College, Kollam, Kerala, India

Impact of green synthesized gold nanoparticle fortified diet on liver histology… 477 Fig:5 Fig:6 Fig:7 Discussion promising candidates for biomedical applications .This can be exploited for The histopathology slides (Fig:5, 6 and 7) of beneficial results by green synthesis of AuNP the present work suggested no specific that avoids some reactive functional groups damage in the fish groups treated with produced in chemically synthesized biogenic AuNP incorporated diet. The liver nanoparticles which can be toxic to of biogenic gold treated fish group showed biological system. The biogenic better cytostructural profile than of control. nanostructures should be better at Hence in this study, cytotoxicity of biogenic biocompatibility and, thus, will have gold is ruled out and it is confirmed that immense application for biological and extracts of Curcuma longa and Ocimum clinical prospects sanctum along with gold nanoparticles at produce better cytostructural profile. These Conclusion results clearly demonstrate that the phytochemicals within these herbs provide Despite increasing application of gold nontoxic coating on AuNps. The lack of any nanoparticles in industry and consumer noticeable toxicity of biogenic AuNPs products, there is still little known about their provides new opportunities for the safe potential toxicity, particularly to organisms in application in drug delivery. The results are aquatic environments. To investigate effects in agreement with the work of Amit Singh et of AuNps in fish, Oreochromis mossambicus al. (2011) titled “Cytotoxicity and Cellular were supplemented with diet containing Internalization Studies of Biogenic Gold biogenic AuNPs. Green synthesized AuNPs Nanotriangles in Animal Cell Lines”. Thus, it showed little hepatic damage and showed shows that biogenic gold nanoparticles can better profile than the groups treated with be looked upon as an environmentally benign plant extracts alone. The biogenic replacement to the toxic chemical methods nanostructures should be better at for synthesis of nanostructures and as biocompatibility and, thus, will have Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.473-478 © Principal, Sree Narayana College, Kollam, Kerala, India

478 Current status and challenges for conservation and sustainable use of biodiversity immense application for biological and clinical prospects. Reference Amit Singh ,Ravi Shukla, Shbir Hassan, R.R.Bhonde and Murali sastry. 2011. Cytotoxicity and Cellular Internalization Studies of Biogenic Gold Nanotriangles in Animal Cell Lines.International Journal of Green Nantechnology.Pages, 251-263. Myers,M.S., JohnsonL.L., Olson,O.P., Sther,C.M., Horness, BH, Collier,T and KansMcCain B.B.1998. Toxicopathic hepatic lesions as biomarkers of chemical contaminant exposure and effects in marine bottom fish species from the Northeast and pacific coasts, USA. Roberts, R.J and Smail, D.A. 2001. Laboratory methods In: Ronald J Roberts, Fish pathology 3rd edition, Harcourt Publishers Ltd., 380-390 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.473-478 © Principal, Sree Narayana College, Kollam, Kerala, India

479 In vitro antioxidant and cytotoxic activity of Carica Papaya ISBN 978-93-5396-871-7 CHAPTER 67 IN VITRO ANTIOXIDANT AND CYTOTOXIC ACTIVITY OF CARICA PAPAYA Nitha Anand* and Gayathri B Department of Biotechnology Sree Narayana College, Kollam, Kerala *Correspondence E-mail: [email protected] ABSTRACT Papaya is power house of nutrients and all the nutrients of papaya as a whole improve cardio vascular system, protect against hearts diseases, heart attacks, strokes and prevent colon cancer. The extracts of unripe papaya contains terpenoids, alkaloids, flavonoid, carbohydrates, gulcosides, saponins and steroids. The present work embodies the study regarding the antioxidative and cytotoxic activities of Carica papaya fruit and seed extracts. Preliminary studies were started with the extraction of Carica papaya (Seed and fruit) and the in vitro antioxidant activity and cytotoxic activities of these extracts were studied. The in vitro antioxidant evaluation of papaya seed and fruit extracts revealed the presence of higher quantity of total phenolics, total flavonoids, total antioxidant capacity and DPPH radical scavenging activity. The antioxidant activity of all the extracts were compared with that of standard compounds viz. ascorbic acid, quercetin, gallic acid etc. and the cytotoxic activity of the extracts were compared with the positive control 5- flurouracil.phenolics are ubiquitous secondary metabolites in plants and possess a wide range of therapeutic uses such as antioxidant, antimutagenic, anticarcinogenic and free radical scavenging activities. In the antioxidant activity assay both seed and fruit extracts exhibited potent in vitro antioxidant activity. DPPH is a stable free radical that easily accepts an electron or hydrogen radical to become a stable diamagnetic molecule and the study showed that Carica papaya seed extract possess potent proton-donating ability and could serve as free radical inhibitors or scavengers. The cytotoxic effects seed and fruit extracts on PLC/PRF/5 cells were evaluated by MTT assay. The inhibition of cell proliferation was assessed after 12 h, 24 h, 48 h and 72 h incubation Treatment with Carica papaya seed extract exhibited significant cytotoxic effect on PLC/PRF/5 cells when compared to the cells treated with DMSO alone. 5-flourouracil, used as positive control. The present study concluded based on the findings that Carica papaya seed extract possess excellent antioxidant and cytotoxic activities. Key words: Antioxidant, Cytotoxic, Carica papaya, Scavenging activity Introduction variety of antioxidants will assure this destructive oxidation process will be Antioxidants can be broadly defined as any minimized reducing the potential for cancer, substance that delays or inhibits oxidative heart diseases, auto immune disease and damage to a target molecule. The main diabetes. characteristic of an anti oxidant is its ability to trap free radicals. To prevent or reverse Cytotoxicity is the quality of being toxic to these destructive processes, the body cells. This can be used in the case of cancer constantly needs a reservoir of antioxidant treatment. If the spread is not controlled, it molecules. Frequent healthy dosages of a can result in death (American Cancer Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.479-484 © Principal, Sree Narayana College, Kollam, Kerala, India

480 Current status and challenges for conservation and sustainable use of biodiversity Society, 2011). Malignancy can result in DMEM medium containing HEPES and death due to damage to critical organs, sodium bicarbonate supplemented with 10% starvation, secondary infection, metabolic FBS and 1X antibiotic-antimycotics. Cells problems or haemorrhage (Karp, 1996). were maintained in a tissue culture flask and kept in a humidified incubator (5% CO2 in Numerous components of plants, collectively air at 37 °C) with a medium change every 2-3 termed “phytochemicals” have been reported days. When the cells reached 70 - 80% to possess substantial antioxidant and confluence, they were harvested with trypsin chemopreventive properties. In cancer - EDTA (ethylene diamine tetra acetate) and chemotherapy their toxicity is not limited to seeded into a new tissue culture flask. cancer cells and normal cells are also harmed. So efforts to develop less toxic drugs Preparation of extracts that affect only malignant cells and mechanism based approach are necessary in Carica papaya fruits and seeds were shade- cancer therapy (Sivalokanathan et al., 2006). dried and powdered. Weighed 10 g each in Carica papaya is a lozenge tropical fruit, the separate flask and were extracted with 100ml fruit is not just delicious of healthy, but of the methanol in a shaking incubator for whole plant parts, fruit, roots, barks, peel, 48-72 hours. Then the extract was collected, and seeds are also known to have medicinal filtered and evaporated. The steps were properties. So the present study was repeated with a new set of powder and undertaken to evaluate the antioxidant and solvent until the required quantity was cytotoxic activity of Carica papaya fruit and achieved. The yield of the Carica papaya seed extracts. fruit extract (CPFE) was 4.5% w/w and Carica papaya seed extract (CPSE) was 6% Materials and methods w/w. The concentrate was suspended in DMSO for in vitro antioxidant studies. Plant material In vitro antioxidant assays Carica papaya fruits were collected from natural habitat during December - January The in vitro antioxidant activity of methanolic extract of Carica papaya fruit Chemicals and seed were measured by the following assays. Quercetin was purchased from Sisco Research Laboratories (SRL), Mumbai, Determination of total phenolic compounds India. Ascorbic acid was obtained from in the extract Merck, Mumbai, India. Gallic acid, Silymarin and were procured from Sigma Chemical The amount of total phenolics was Co., St. Louis, MO, USA.2, 2-Diphenyl-1- determined using the Folin-Ciocalteu picrylhydrazyl (DPPH) was purchased from method.A calibration curve of gallic acid was TCL, Chennai. Cell Proliferation Assay kit prepared, and the results were expressed as (3-(4,5-dimethylthiazol-2-yl)- mg GAE (gallic acid equivalents)/g dry 2,5diphenyltetrazoliumbromide, [MTT]) was extract. purchased from HiMedia, India. All other chemicals were of analytical grade. Determination of total flavonoid content in the extract Cell culture The total flavonoid content was determined PLC/PRF/5 cell line was purchased from spectrophotometrically by the method National Centre for Cell Science (NCCS), described by Quettier-Deleu et al., (2000). Pune, India and grown as a monolayer in The total flavonoid content was determined Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.479-484 © Principal, Sree Narayana College, Kollam, Kerala, India

In vitro antioxidant and cytotoxic activity of Carica Papaya 481 using a standard curve with quercet in and Results expressed as milligrams of quercet in equivalents (QE/g of dry extract). In vitro antioxidant activity Evaluation of total antioxidant capacity Total phenolic compounds in the extract Total antioxidant capacity was measured by CPSE had a higher quantity of total phenolics the method of Jayaprakasha et al., 2004. than CPFE (470 ± 6.6 mg GAE/g dry extract) Ascorbic acid was used as standard and the total antioxidant capacity was expressed as Total flavonoid content in the extract the equivalent of ascorbic acid per gram of the extract. Total flavonoid content of CPSE is higher than that of CPFE that is 72 ± 3.9 mg QE/g 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) dry extract. assay Total antioxidant capacity The assay was conducted to test the scavenging activity of CPFE and CPSE CPSE shows a good total antioxidant activity against DPPH radicals (Aquino et al., 2001). (348 ± 5.2 mg Ascorbic acid /g dry extract) Ascorbic acid standard was used for the than CPFE comparison. DPPH radical scavenging activity Determination of reducing power The DPPH radical scavenging activity of Reducing power of the extracts determined extracts and standard exhibited a by adopting the method of Oyaizu (1986 concentration dependent reaction. The IC50 value of ascorbic acid is 4.3 µg/ml and CPSE In vitro cytotoxic assay and CPFE were 5.8µg/ml and 6.6 µg/ml. MTT assay of CPFE and CPSE Reducing power The cell viability was assessed by MTT assay Ascorbic acid used as reference compound (Mosmann, 1983), Percentage inhibition was exhibited a superior reducing power at all calculated using the formula, concentrations, compared with C. papaya extracts. These values reflect the following Percentage growth inhibition = reducing capability: ascorbic acid > CPSE > CPFE. (Mean absorbance of thecontrolcells) - (Mean absorbance of treatedcells)] X100 Mean absorbance of controlcells Table 1. Total phenolic content, total flavonoid content, total antioxidant activity and DPPH radical scavenging activity of CPSE and CPFE as summarized Extract/ Phenolic Flavonoid Total antioxidant DPPH radical standard contents (mg contents activity (mg scavenging (mg QE/g activity CPSE GAE/g dry dry extract) ascorbic acid /g dry IC50 (μg /ml) CPFE extract) extract) Ascorbic acid 72 ± 3.9 5.8 470 ± 6.6 348 ± 5.2 65±6.8 6.7 394±5.3 312±4.9 - 4.5 - - Figure 1. Reducing power of C.papaya extracts compared with standard antioxidant ascorbic acid. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.479-484 © Principal, Sree Narayana College, Kollam, Kerala, India

482 Current status and challenges for conservation and sustainable use of biodiversity Absorbance at 700nm 1.8 Ascorbic acid 1.6 1.4 0.1 0.2 0.3 0.4 0.5 0.6 1.2 Concentration (mg/ml) 1 0.8 0.6 0.4 0.2 0 0 Cytotoxicity assay 65.3±2.5 for 100µg/ml after 12 h, 24 h, 48 h and 72 h respectively. 5-flourouracil, used as Assay of CPSE and CPFE positive control, showed an inhibition of 26.8±1.0, 36.2±1.5, 59.2±2.3 and 70.2±2.8 The cytotoxic effects CPSE and CPFE on for 50μg/ml and 14.7±1.1, 25.2±0.8, PLC/PRF/5 cells were evaluated by MTT 47.9±1.8 and 59.1±2.3 for 25μg/ml after 12 assay. The inhibition of cell proliferation was h, 24 h, 48 h and 72h respectively. Treatment assessed after 12 h, 24 h, 48 h and 72 h with CPSE exhibited significant cytotoxic incubation. CPSE exerted cytotoxic effect on effect on PLC/PRF/5 cells (p ≤ 0.05) when PLC/PRF/5 cells with percentage of cell compared to the cells treated with DMSO inhibition values 12.4±0.8, 23.1±0.9, alone and were expressed in Fig. 44.4±1.7 and 55.8±2.2. CPFE exerted cytotoxic effect with inhibition values24.2±1.3, 33.8±1.2, 56.8±2.0 and Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.479-484 © Principal, Sree Narayana College, Kollam, Kerala, India

In vitro antioxidant and cytotoxic activity of Carica Papaya 483 (A). PLC/PRF/5 cells treated with CPSE and CPFE for 12 h; (B) PLC/PRF/5 cells treated with CPSE and CPFE for 24 h (n=3). (C). PLC/PRF/5 cells treated with CPSE and CPFE for 48 h; (D) PLC/PRF/5 cells treated with CPSE and CPFE for 72 h (n=3). (I) – DMSO control, (II) – CPFE 50µg/ml, (III) – CPSE 50µg/ml, (IV) – 5FU 50µg/ml, (V) – 5FU 25µg/ml. *p≤0.05 versus DMSO control. Discussion antioxidant activity in DPPH radical scavenging assay. The results of the present investigation indicate that Carica papaya is a rich source MTT assay is an established method of of natural antioxidants such as phenolics and determining viable cell number in flavanoids. The scavenging ability of the proliferation and cytotoxicity studies phenolics is mainly due to the presence of (Sylvester, 2011).In the present study, hydroxyl groups. CPSE showed significantly cytotoxic effect of CPSE and CPFE on higher percentage inhibition (stronger PLC/PRF/5 cells were determined based on hydrogen-donating ability) positively reduction of the yellow colored water soluble correlated with total phenolics. Flavonoids, tetrazolium dye 3-[4, 5-dimethylthiazol-2- one of the most diverse and widespread yl]-2, 5-diphenyl tetrazolium bromide (MTT) groups of natural compounds, are also to formazan crystals. Mitochondrial probably the most natural polyphenolics dehydrogenase produced by live cells reduces capable of exhibiting in vitro antioxidant MTT to blue formazan product, which activities. The results obtained in the present reflects the normal function of mitochondria study shown that CPSE can effectively and cell viability (Lau et al., 2004). A dose- scavenge reactive oxygen species. Natural dependent reduction of MTT (or color antioxidants fight against free radicals and change from yellow to purple) observed in 5- protect us from various diseases. In the FU and extracts treated cells indicate their present study, methanolic extract of CPSE cytotoxic potential against PLC/PRF/5 cells. and CPFE exhibited potent in vitro Among the CPSE and CPFE, the highest activity was observed in CPSE. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.479-484 © Principal, Sree Narayana College, Kollam, Kerala, India

484 Current status and challenges for conservation and sustainable use of biodiversity Conclusion Lau, C.B., Ho, C.Y., Kim, C.F., Leung, K.N., Fung, K.P. and Tse, T.F. 2004. Cytotoxic In conclusion, this study demonstrates the activities of Coriolus versicolor (Yunzhi) antioxidant and cytotoxic activities of CPSE extract on human leukemia and lymphoma and CPFE in in vito antioxidant systems and cells by induction of apoptosis. Life Sci., in human hepatoma cell line, PLC/PRF/5. 75:797-808. Among CPSE and CPFE, CPSE shows good antioxidant activity and significantly Mosmann, T. 1983. Rapid colorimetric assay inhibited the proliferation of PLC/PRF/5 for cellular growth and survival: application cells. The inhibitory effect of natural to proliferation and cytotoxicity assays. J bioactive substances in carcinogenesis and Immunol Methods., 65: 55-63. tumor growth may be through two main mechanisms: modifying redox status and Quettier-Deleu, C., Gressier, B., Vasseur, J., interference with basic cellular functions like Dine, T., Brunet, C. and Luyckx, M.C. 2000. cell cycle, apoptosis, inflammation, Phenolic compounds and antioxidant angiogenesis, invasion and metastasis activities of buckwheat (Fagopyrum (Kampa et al., 2007). Our data support the esculentumMoench) hulls and flour. hypothesis that CPSE may have potential in JEthnopharmacol., 72: 35-42. cancer treatment. However, further studies are needed to comprehend the different Sivalokanathan, S., Ilayaraja, M. and mechanisms regulating the antiproliferative effects and apoptosis induced by CPSE. Balasubramanian,M.P. 2006. Antioxidant References activity of Terminalia arjuna bark extract on American Cancer Society, 2011. Global N-nitrosodiethyl amine induced Cancer Facts and Figures 2nd Edition. Atlanta: American Cancer Society. hepatocellular carcinoma in rats. Mol. Cell. Aquino, R., Morelli,S., Lauro, M.R., Abdo, Biochem., 281:87-93. S., Saija, A. and Tomaino, A. 2001. Phenolic constituents and antioxidant activity of an Sylvester, P.W., 2011. Optimization of the extract of Anthurium versicolar leaves. J Nat tetrazolium dye (MTT) colorimetric assay for Prod., 64: 1019- 1023. cellular growth and viability. Methods Mol. Biol., 716:157-168. Jayaprakasha, G.K., Lingamallu, J.R. and Kunnumpurath, K.S. 2004. Antioxidant activities of flavidin in different in-vitro model system. Bioorg Med Chem., 12: 5141– 5146. Kampa, M., Nifli, A.P., Notas, G. and Castanas, E. 2007. Polyphenols and cancer cell growth. Rev Physiol Biochem Pharmacol., 159: 79-113. Karp, G.1996. Cancer, cell and molecular biology. John Wiley& Sons Inc, New York, Pp. 695-698 Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.479-484 © Principal, Sree Narayana College, Kollam, Kerala, India

Assessment of heavy metals in Cheloor Lake at Kollam, Southern Kerala 485 ISBN 978-93-5396-871-7 CHAPTER 68 ASSESSMENT OF HEAVY METALS IN CHELOOR LAKE AT KOLLAM, SOUTHERN KERALA Remya Balakrishnan and Sheeba S. Post Graduate Department of Zoology, Sree Narayana College, Kollam, Kerala, India Email: [email protected] ABSTRACT The present paper deals with the assessment of heavy metal concentrations in water, sediment and Etroplus maculates from Cheloor lake, Kollam district in Southern Kerala. Samples collected monthly for a year period was submitted for analyzing Cu, Cr, Cd, Pb and Zn using standard methods. Results indicated that Cu content in the ecological components of the lake under study was lesser than the international guide line values. However, the Cr and Cd content in the study area were higher than the permissible limit of 0.05 mg/l set for drinking water. Zinc concentration showed a maximum value of 0.554±0.353mg/l in water, 0.539±0.351mg/kg in sediments and 0.315mg/kg in fish indicating absence of bioaccumulation. Lead contamination exceeded the WHO guidelines which, could be attributed to urbanization and oil pollution. The study that provides the baseline data on trace metal concentration is the first one conducted so far in Cheloor Lake. Introduction metals play important roles in metabolic activities of enzymes and vitamins. For Polluted water contributes to water example, Zinc plays a vital role in the enzyme insufficiency because it limits the availability alcohol, dehydrogenase and cobalt is of water for both human consumption and the indispensable to vitamin B12 (Riordan, 1977). biotic components of the ecosystem. There are different types of environmental Heavy metal pollution in water pollutants, of which the non-biodegradable bodies is one of the major threats to water pollutants like plastic and heavy metals quality and human health. The major contaminate both soil and water. Heavy anthropogenic sources of heavy metals are metals are the major constituents of industrial domestic waste water, mining sites, industrial effluents discharged into the environment wastes from the refineries, etc. According to which pollute the ecosystem. The occurrence World Health Organization, cadmium, zinc of heavy metals in the environment has been and copper are among the 10 toxic heavy an issue of great concern due to their toxicity, metals that cause issues to both man and non-biodegradable nature and their nature (WHO, 2017). Pollution of wetlands persistence in biological tissues (Olatunji et by heavy metals has become a major al., 2009). The non-degradable heavy metals environmental issue due to industrialization reach even the groundwater reserves making and urbanization along with the speedy it unsafe for human use. Heavy metals such population growth (Staley et al., 2015). as mercury, arsenic, lead, iron, cadmium, etc. are systemic toxins with specific neurotic and A systematic evaluation of the fetotoxic effects. However, some trace pollutant concentrations in the different phases of an aquatic system is a prerequisite Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.485-492 © Principal, Sree Narayana College, Kollam, Kerala, India