Molecular Biomarker Studies on Ecotoxicological Impact of Pollutants on the Marine Gastropods Along the Goa Coast

Home , Bullacta exarata, Nerita chamaeleon, Sea snail

MOLECULAR BIOMARKER STUDIES ON ECOTOXICOLOGICAL IMPACT OF POLLUTANTS ON THE MARINE GASTROPODS ALONG THE GOA COAST

JACKY BHAGAT

A Thesis submitted to Goa University for the Award of the Degree of

DOCTOR OF PHILOSOPHY in MARINE SCIENCES

Research Guide: Dr. B. S. Ingole, Professor & Chief Scientist, CSIR-National Institute of Oceanography Dona Paula, Goa - 403004 Co-guide: Dr. A. Sarkar Former Senior Principal Scientist CSIR-National Institute of Oceanography Dona Paula, Goa - 403004 Place of Work: CSIR-National Institute of Oceanography Dona Paula, Goa - 403004

Details of Ph.D. Registration: No. 201109255, 15 November, 2011

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Statement

As required under the University ordinance OB-9.9 (v-vi), I state that this thesis entitled "Molecular Biomarker Studies On Ecotoxicological Impact Of Pollutants On The Marine Gastropods Along The Goa Coast" is my original contribution and it has not been submitted on any previous occasion. The literature related to the problem investigated has been cited. Due acknowledgements have been made wherever facilities and suggestions have been availed of.

JACKY BHAGAT CSIR-National Institute of Oceanography, Goa. 3rd April, 2017

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Certificate

This is to certify that the thesis entitled "Molecular Biomarker Studies On Ecotoxicological Impact Of Pollutants On The Marine Gastropods Along The Goa Coast" submitted by Shri Jacky Bhagat for the award of the degree of Doctor of Philosophy in Marine Science is based on original studies carried out by him under my supervision. The thesis or any part thereof has not been previously submitted for any other degree or diploma in any institution.

Dr. B. S. Ingole Research Supervisor CSIR-National Institute of Oceanography, Dona Paula, Goa 3rd April, 2017

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List of Papers

Published Papers from thesis:

1. Bhagat J*, Sarkar A, Vashistha D, Vipin Singh, Laxmi Raiker, Ingole BS 2017. Integrated Biomarker Responses In Marine Gastropod (Nerita Chamaeleon) From Goa Coast, India. Invertebrate Survival Journal 14, 18-31. IF=0.754 (http://www.isj.unimo.it/articoli/ISJ452.pdf) 2. Bhagat J*, Ingole BS, Singh N. 2016. Glutathione S-transferase, catalase, superoxide dismutase, glutathione peroxidase, and lipid peroxidation as a biomarker of oxidative stress in snails: A review. Invertebrate Survival Journal 13, 333-349. IF=0.754 (http://www.isj.unimo.it/articoli/ISJ444.pdf) 3. Bhagat J*, Sarkar A, Ingole BS 2016. DNA damage and oxidative stress in marine gastropod, Morula granulata exposed to phenanthrene. Water air soil pollution (2016), 227:114 IF=1.551 (https://link.springer.com/article/10.1007/s11270-016-2815-1) 4. Bhagat J*, Ingole BS 2015. Genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay. Environmental science and pollution research 22 (14):10758-10768. IF=2.828 (http://www.sciencedirect.com/science/article/pii/S0147651314001638) 5. Sarkar A, Bhagat J*, Ingole BS, Rao DP, Markad VL 2015. Genotoxicity of cadmium chloride in marine gastropod, Nerita chamaeleon using comet assay and alkaline unwinding assay. Environmental Toxicology, 30 (2): 177-187. IF=3.197 (http://onlinelibrary.wiley.com/doi/10.1002/tox.21883/full) 6. Sarkar A*, Bhagat J, Sarker S 2014. Evaluation of impairment of DNA in marine gastropod, Morula granulata as a biomarker of marine pollution, Ecotoxicology and Environmental Safety 106, 253–261. IF=2.762. (http://www.sciencedirect.com/science/article/pii/S0147651314001638) 7. Bhagat J*, Ingole B, Sarkar A, Gunjikar M 2012. Measurement of DNA damage in Planaxis sulcatus as a biomarker of genotoxicity. The Ecoscan, 1, 01-04, 2012, 219-223. (http://theecoscan.in/journalpdf/spl2012_v1- 38%20jacky%20bhagat.pdf)

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8. Bhagat J*, Singh K, Rai P, Raizada G, Sarkar A 2012. Alkaline unwinding assay for the assessment of DNA damage in Planaxis sulcatus from Goa coast. Nebio, 3, 5, 34-36. (http://www.nebio.info/2012/12/nebio-special-issue-ebi- 2012.html) *corresponding author

Submitted for Publications from thesis:

• Bhagat J*, Ingole BS. Effects of benzo(k)fluoranthene, a polycyclic aromatic hydrocarbon, on DNA damage and oxidative stress in marine gastropod Morula granulata. Toxicological & Environmental Chemistry (under review, GTEC-2016-0617) • Bhagat J*, A Sarkar, Ingole BS. Seasonal and spatial variability of oxidative stress biomarkers in marine gastropod Nerita chamaeleon in relation to polycyclic aromatic hydrocarbon. Ecotoxicology and Environmental Safety (under review, EES S16 01650)

Papers presented in International Conference/Symposium  Bhagat J*, Ingole BS. Effects of benzo(k)fluoranthene, a polycyclic aromatic hydrocarbon, on DNA damage and oxidative stress in marine gastropod Morula granulata, IC3-2015.  Bhagat J*, Sarkar A, Singh V, Raiker L, Ingole BS. Integrated biomarker responses in marine gastropod (Nerita chamaeleon) environmentally exposed to polycyclic aromatic hydrocarbons, IIOE-2015.  Bhagat J, Ingole BS. Assessment of genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay, presented at International Conference ICFA-2014.  Bhagat J*, Ingole BS. Assessment of genotoxic damage and lipid peroxidation in marine gastropod Morula granulata exposed to phenanthrene, presented at 4th International Conference ICEES-2014.  Bhagat J*, Ingole B, Sarkar A, Gunjikar M 2012. Measurement of DNA damage in Planaxis sulcatus as a biomarker of genotoxicity, ICAIECs-2012.

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 Sarkar A, Bhagat J, Tegur PM* and Manu B, Application of Molecular Biomarker Technique for Assessment of the Impact of Genotoxic Contaminants in the Marine Environment, ICCTEM-2012.  Bhagat J, Sarker G*, Rajgopal TV, Raizada G, Vashistha D and Sarkar A, Strategies for Prevention and Control of Water Pollution - An overview, Presented at International Conference on Ecotoxicological and Environmental Science (ICEES), Miramar Residency, Goa, India, 28-30 Nov, 2011  Sarkar A, Bhagat J* and Deepti Vashistha, Measurement of DNA Integrity In Morula granulata as a Biomarker of Genotoxicants along the Goa Coast, Presented at ICEES-2011.

Papers presented in International Conference/Symposium

 Bhagat J, Singh K*, Rai P, Raizada G, Sarkar A, Alkaline unwinding assay for the assessment of DNA damage in Planaxis sulcatus from Goa coast, Presented at EBI-2012.  Bhagat J*, Ingole BS, Sarkar A and Rao DP, Comet assay for the measurement of DNA damage in marine gastropods exposed to genobiotic pollutants, Presented at the EBI-2012.  Bhagat J*, Sarkar A, Vashistha D, Mesquita A, Inhibition of AChE Activity in Marine Gastropod as a Biomarker of Coastal Pollution by Neurotoxic Contaminants, NCCIECM-2012.

*presenting author

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Acknowledgements

I am deeply indebted to my thesis supervisor, Dr. B. S. Ingole for his encouragement and unwavering support in this work. Without his guidance and support, this thesis would not have been possible. I also thank him for believing in me, and for going the extra mile when it was necessary, in order to make this happen. I have greatly benefited from his knowledge and experience.

I wish to express my profound thanks to Dr. Anupam Sarkar, for bringing me to this wonderful institute and acting as my research guide and then as a co-guide during my stay at CSIR-NIO. His positive criticism, suggestions and valuable comments has left everlasting impression on me. His initiative, constant support and help has lead to the successful completion of my present investigation.

I would like to thank Dr. S. W. A. Naqwi, Director, CSIR-National Institute of Oceanography, Goa for providing me with all the necessary facilities to carry out my research work. The financial support by Department of Biotechnology, New Delhi in the form of Junior and Senior Research Fellowship is highly acknowledged. I also would like to thank CSIR for providing the infrastructure and the necessary facilities to conduct my research work.

My heartfelt thanks to Dr. S. R. Shetye, Vice Chancellor, Goa University for giving me the opportunity to undergo the Ph.D. degree.

I express my special thanks to Dr. R. Roy, Department of Zoology, Goa University and Dr. C. Mohandas, CSIR-NIO for their valuable suggestions given during my SRF assessment.

My appreciation goes to Dr. S. S. Sawant, Scientist, CSIR-NIO for helping me in PAH analysis and Mithila Bhat for mercury analysis. I extend my sincere thanks to Dr. Ramaiah, Head of Department, BOD, Mrs. Analia Mesquita, Head of

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Department, COD, Dr. C. U. Rivonkar, Head of Department of Marine Science, Goa University and Mr. D.P. Rao, Scientist, CSIR-NIO for their valuable suggestions and continuous help in furtherance of my research work. Many thanks to Mr. Prabhu, Mr. Madan, Mr. S.R. Sahu and Mr. Mithun at CSIR-NIO, Library for providing necessary support during my research work.

I feel immense pleasure in expressing my special thanks to all the staffs from COD, BOD and ITG section, who directly or indirectly helped me during the completion of this work. I would also like to thank Nisha for helping me in thesis formatting and Haris, Afreen for all the suggestions in my thesis preparation, printing and binding.

I would like to express my sincere appreciation to Vijay Markad for teaching me some of the important biomarker techniques. I would also like to thank my colleagues and friends, Deepti, Laxmi, Sutapa, Priyamvada, Moon, Geetika, Priyanka, Vipin, Manasi and everyone from benthos lab for assisting me in my studies and field work. I am also thankful to my roommate Vijay Rawat, Azraj Dahihande and Gobardhan Sahoo for their valuable friendship.

No acknowledgement will be complete without expressing my gratitude towards my loving parents for their love and support in every walk of life.

JACKY BHAGAT

CSIR-National Institute of Oceanography, Goa. 3rd April, 2017

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Table Of Contents

STATEMENT II

CERTIFICATE III

LIST OF PAPERS IV

ACKNOWLEDGEMENTS VI

LIST OF TABLES XI

LIST OF FIGURES XII

ABBREVIATIONS XVII

CHAPTER 1 - INTRODUCTION 1

CHAPTER 2 - REVIEW OF LITERATURE 5 2.1. Environment Pollutants 5 2.1.1. Polycyclic aromatic hydrocarbons (PAHs) 5 2.1.2. Heavy metals 11 2.2. Gastropod as a model organism to study environmental contaminants 17 2.3. DNA Integrity as a biomarker of marine pollution 19 2.3.1. Comet Assay 19 2.3.2. Alkaline unwinding assay 24 2.4. Acetylcholinesterase as a biomarker of neurotoxic contaminants 25 2.5. Reactive Oxygen Species (ROS) 29 2.6. Oxidative stress biomarkers 30 2.6.1. Glutathione-S-Transferases (GSTs) 31 2.6.2. Catalase (CAT) 37 2.6.3. Superoxide dismutase (SOD) 41 2.6.4. Lipid peroxidation (LPO) 44 2.7. Integrated Biomarkers Response (IBR) 47

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CHAPTER 3 - MATERIALS AND METHODS 49 3.1. Sampling locations 50 3.2. Marine gastropods 60 3.2.1. Nerita chamaeleon 62 3.2.2. Planaxis sulcatus 63 3.2.3. Morula granulata 63 3.3. Exposure conditions 64 3.3.1. In vivo exposure to phenanthrene 64 3.3.2. In vivo exposure to benzo(k)fluoranthene (B[k]F) 65

3.3.3. In vivo exposure to cadmium Chloride (CdCl2) 65

3.3.4. In vivo exposure to mercury Chloride (HgCl2) 65

3.3.5. In vitro exposure of gill cells to hydrogen peroxide (H2O2) 65 3.4. Alkaline unwinding assay 66 3.4.1. Isolation and purification of DNA 66 3.4.2. Measurement of DNA integrity 66 3.5. Single cell gel electrophoresis 67 3.5.1. Preparation of Single Cell Suspension 67 3.5.2. Comet Assay 67 3.5.3. Optimization of the comet assay for Morula granulata 68 3.5.4. Comet capture and image analysis 68 3.6. Measurement of PAH 69 3.7. Biochemical tests 69 3.7.1. Catalase 69 3.7.2. Glutathione S-transferase (GST) activity 69 3.7.3. Lipid peroxidation (LPO) determination 70 3.7.4. Superoxide dismutase (SOD) activity 70 3.7.5. AChE estimation 70 3.8. Measurement of water quality parameters 71 3.9. Integrated biomarker response and statistical Analysis 71

CHAPTER 4 - RESULTS AND DISCUSSION 73 4.1. Physico-chemical parameters of water and its relation with other

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biomarkers 73 4.2. Polycyclic aromatic hydrocarbons (PAHs) 84 4.3. Alkaline unwinding assay 87 4.4. Comet Assay 92 4.5. Biochemical tests 81 4.6. Integrated Biomarker Response (IBR) 110 4.7. Exposure 114 4.7.1. Exposure to phenanthrene 114 4.7.1.1. Biochemical assays 114 4.7.1.2. DNA damage after in vitro exposure to hydrogen 117 peroxide 4.7.1.3. DNA damage after in vivo exposure to phenanthrene 117 4.7.1.4. Relationship between oxidative stress parameters and 117 comet assay 4.7.1.5. Integrated Biomarker Response (IBR) 117 4.7.2. Exposure to benzo(k)fluoranthene 121 4.7.2.1. Biochemical tests 121 4.7.2.2. Comet assay 122 4.7.2.3. Integrated Biomarker Response (IBR) 123

4.7.3. In vivo exposure of gastropods to cadmium chloride (CdCl2) 130 4.7.3.1. Alkaline unwinding assay 127 4.7.3.2. Comet assay 129

4.7.4. Exposure to mercuric chloride (HgCl2) 132 4.7.4.1. Cell viability 132 4.7.4.2. In vitro hydrogen peroxide 133

4.7.4.3. In vitro exposure mercuric chloride (HgCl2) 134

4.7.4.4. In vivo exposure mercuric chloride (HgCl2) 135

CHAPTER 5 - OVERALL ASSESSMENT OF THE MOLECULAR 138 BIOMARKER STUDIES BIBLIOGRAPHY 142

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List Of Tables

Table 1 Concentrations of PAHs in sediments (ng/g, dry weight) from different parts of the world Table 2 Concentrations of PAHs in molluscs (ng/g, dry weight) from different parts of the world Table 3 Concentrations of Hg reported in the coastal sediments and waters of India Table 4 Concentrations of Hg reported in terms of dry weight (µg/g) and wet weight (µg/g) in gastropods from different parts of the world Table 5 Laboratories studies with Nerita chamaeleon Table 6 Studies on DNA damage in gastropods as measured by comet assay and alkaline unwinding assay Table 7 Acetylcholinesterase (AChE) activity in gastropods Table 8 Glutathione S-transferase (GST) in gastropod Table 9 Catalase (CAT) activity in gastropods Table 10 Superoxide dismutase (SOD) in gastropods Table 11 Lipid Peroxidation (LPO) in gastropods Table 12 Concentration of heavy metals in the muscle tissues of marine snail (Cronia contracta) along the Goa coast. Each data point represents mean of triplicates values Table 13 Physico-chemical parameters of seawater along the Goa coast, India. Values are represented as means± standard deviation

Table 14 Spearman correlation matrix of different physico-chemical water parameters and biomarker activity in N. chamaeleon in (A) monsoon, (B) post-monsoon, and (C) pre-monsoon Table 15 Spearman correlation matrix of different physico-chemical water parameters and biomarker activity in P. sulcatus in (A) monsoon, (B) post-monsoon, and (C) pre-monsoon

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Table 16 Spearman correlation matrix of different physico-chemical water parameters and biomarker activity in M. granulata in (A) monsoon, (B) post-monsoon, and (C) pre-monsoon

Table 17 Stepwise multiple regression analysis of the data on various physico- chemical parameters and PAH influencing the Integrity of DNA in marine gastropod, Morula granulata

List Of Figures

Fig. 1. Biomarkers as an early warning signal in ecotoxicological studies Fig. 2. Comet Assay Principle : Denatured cleaved DNA fragments migrate out of immobilized cells following permeabilization and electrophoresis Fig. 3. Mechanism of AChE action Fig. 4. Reactions showing the fate of reactive oxygen species Fig. 5. Reaction catalyzed by superoxide dismutase Fig. 6. Mechanisms of lipid peroxidation Fig. 7. Sampling locations along the Goa coast Fig. 8. Nerita chamaeleon Fig. 9. Planaxis sulcatus Fig. 10. Morula granulata Fig. 11. Total Polycyclic aromatic hydrocarbons (PAHs) in sediments from Goa coast Fig. 12. Total Polycyclic aromatic hydrocarbons (PAHs) in Nerita chamaeleon (µg/g wet weight) Fig. 13. Total Polycyclic aromatic hydrocarbons (PAHs) in Planaxis sulcatus (µg/g wet weight) Fig. 14. Total Polycyclic aromatic hydrocarbon (PAH) concentration (µg/g wet weight) in Morula granulata Fig. 15. Variation of DNA integrity in N. chamaeleon along the Goa coast Fig. 16. Variation of DNA integrity in P. sulcatus along the Goa coast

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Fig. 17. Variation of DNA integrity in M. granulata along the Goa coast Fig. 18. Percentage tail DNA (TDNA) in N. chamaeleon from Goa coast Fig. 19. Percentage tail DNA (TDNA) in P. sulcatus from Goa coast Fig. 20. Percentage tail DNA (TDNA) in M. granulata from Goa coast Fig. 21. Glutathione S-transferase (GST) activity in N. chamaeleon from Goa coast Fig. 22. Catalase (CAT) activity in N. chamaeleon from Goa coast Fig. 23. Superoxide dismutase (SOD) activity in N. chamaeleon from Goa coast Fig. 24. Lipid Peroxidation (LPO) value in N. chamaeleon from Goa coast Fig. 25. Acetylcholinesterase (AChE) activity in N. chamaeleon from Goa coast Fig. 26. Glutathione S-transferase (GST) activity in P. sulcatus from Goa coast Fig. 27. Catalase (CAT) activity in P. sulcatus from Goa coast Fig. 28. Superoxide dismutase (SOD) activity in P. sulcatus from Goa coast Fig. 29. Lipid Peroxidation (LPO) value in P. sulcatus from Goa coast Fig. 30. Acetylcholinesterase (AChE) activity in P. sulcatus from Goa coast Fig. 31. Glutathione S-transferase (GST) activity in M. granulata from Goa coast Fig. 32. Catalase (CAT) activity in M. granulata from Goa coast Fig. 33. Superoxide dismutase (SOD) activity in M. granulata from Goa coast Fig. 34. Lipid Peroxidation (LPO) value in M. granulata from Goa coast Fig. 35. Acetylcholinesterase (AChE) activity in M. granulata from Goa coast Fig. 36. Comparison of IBR index for genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), oxidative stress [superoxide dismutase (SOD), catalase (CAT), Glutathione S-transferase (GST) and lipid peroxidation (LPO)] biomarkers, average PAH concentrations, mean IBR and Acetylcholinesterase (AChE) activity in N. chamaeleon from different sites of Goa, India. Fig. 37. Comparison of IBR index for genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), oxidative stress [superoxide dismutase (SOD), catalase (CAT), Glutathione S-transferase (GST) and lipid peroxidation (LPO)] biomarkers, average PAH concentrations, mean IBR and Acetylcholinesterase (AChE) activity in P. sulcatus from different sites of Goa, India.

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Fig. 38. Comparison of IBR index for genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), oxidative stress [superoxide dismutase (SOD), catalase (CAT), Glutathione S-transferase (GST) and lipid peroxidation (LPO)] biomarkers, average PAH concentrations, mean IBR and Acetylcholinesterase (AChE) activity in M. granulata from different sites of Goa, India. Fig. 39. Glutathione S-transferase (GST), catalase (CAT) and lipid peroxidation (LPO) value, depicted by mean±standard deviation in marine gastropod M. granulata exposed to phenanthrene. (a) p<0.05, (b) p<0.01, (c) p < 0.001 significantly different from the control (ANOVA, Tukey HSD post-test). Fig. 40. Comet parameter (DNA strand breakage) in M. granulata exposed to phenanthrene. Values are means± standard deviation, (a) p<0.05, (b) p<0.01, (c) p < 0.001 significantly different from the control (ANOVA, Tukey HSD post-test). Fig. 41. Star plots for standardized biomarker response in snails exposed to different concentrations of phenanthrene. Fig. 42. Glutathione S-transferase (GST), Superoxide dismutase (SOD), lipid peroxidation (LPO) and catalase (CAT) activities and level in marine gastropod M. granulata exposed to different concentrations of benzo(k)fluoranthene (B[k]F). Values are means±standard deviation. * p<0.05, ** p<0.01, *** p<0.001- significantly different from the control (ANOVA, Tukey HSD post-test) Fig. 43. Percentage DNA in tail (TDNA) in marine gastropod M. granulata exposed to different concentrations of benzo(k)fluoranthene (B[k]F). Values are means±standard deviation. * p<0.05, ** p<0.01, *** p<0.001 - significantly different from the control (ANOVA, Tukey HSD post-test) Fig. 44. Integrated biomarker response (IBR) represented by star plots in M. granulata Fig. 45. Comets as observed in cells of M. granulata after exposure to different concentrations (a) control, (b) 1 µg/L, (c) 10 µg/L, (d) 25 µg/L, and (e) 50 µg/L of benzo(k)fluoranthene (B[k]F)

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Fig. 46. Induction of DNA damage in terms of (a) tail DNA %, (b) OTM and (c) TL in Nerita chamaeleon gill cells following exposure to different concentrations of cadmium chloride in vivo. Comet parameters were reported as mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the control (Dunnett’s test).

Fig. 47. DNA Integrity (I value) in gills of Nerita chamaeleon exposed to CdCl2.

Snails were exposed for 5 days to CdCl2. Data expressed are means of triplicate values of DNA integrity. Results were reported as mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the control (Dunnett’s test). Fig. 48. Typical comet image of gill cells of Nerita chamaeleon exposed to different

concentration of CdCl2 in vivo for 24h and 50 µM of H2O2 in vitro for 30 min at 4°C. Cells were stained with ethidium bromide. Fig. 49. Comet parameters (TDNA and OTM) in gill cells of P. sulcatus exposed to

hydrogen peroxide (H2O2) and mercuric chloride (HgCl2) in vitro. Values are means±standard deviation. ns non-significant. a p<0.05, b p<0.01, c p<0.001- significantly different from the control (ANOVA, Tukey HSD post-test)

Fig. 50. Percentage tail DNA and OTM in gill cells of P. sulcatus exposed to HgCl2 in vivo. Data represented as means±standard deviation. T0 time zero or zero day of exposure, ns non-significant. a p<0.05, b p<0.01, c p<0.001 - significantly different from the control (ANOVA, Tukey HSD post- test)

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Abbreviations

AChE – Acetylcholinesterase APDC – Ammonium pyrrolidine dithiocarbamate au – Alkaline unwinded B(a)P – Benzo (a) pyrene BaPDE – Benz (a) pyrene diol epoxide B[k]F – benzo[k]fluoranthene BOD – Biochemical oxygen demand CAT – Catalase ChE – Cholinesterase CYP1A1 – Cytochrome P4501A1 DAUA – DNA alkaline unwinding assay DBC – 7H-dibenzo[c,g]carbazole DDT – Dichloro diphenyl trichloro ethane DMSO – Dimethyl sulfoxide DNA-I – DNA Integrity DO – Dissolved oxygen DPC – DNA-protein cross links ds – Double stranded EROD – Ethoxyresorufin O-deethylase GPx –Glutathione peroxidase GSH – Reduced glutathione GST – Glutathione S-transferase IARC –International Agency for Research on Cancer IBR – Integrated Biomarker Response LPO – Lipid Peroxidation Nd – Not detected ns – Non significant OPs – Organophosphates OTM – Olive tail moment PAHs – Polyaromatic hydrocarbons

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PHAHs –Planar halogenated aromatic hydrocarbons PCBs – Polychlorinated biphenyls ROS – Reactive oxygen species SCGE – Single Cell Gel Electrophoresis SOD – Superoxide dismutase ss – Single stranded TBTs –Tributyltins TDNA – Percentage tail DNA US EPA – United States Environmental Protection Agency

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CHAPTER 1

Introduction

The rapid increase in anthropogenic activity and industrial development along the coastal region has resulted in elevated concentration of toxic agents in the environment, affecting the health of marine ecosystem (Sarkar et al., 2014). Of all the ecosystems, estuarine and coastal regions are greatly affected by high degree of contaminations from various sources and are considered as the primary sink for pollutants. Pollutants may accumulate through point sources like a single sewage pipe or factory wastewater outfall, or it can arise from a variety of geographic points otherwise called as non-point sources (river runoff, agriculture, livestock, urban runoff, automobiles, to name a few). Pollutants entering into the oceans are mostly diluted; however, the organisms living in the oceans tend to concentrate the pollutants into their body by various mechanisms, like adsorption, absorption, ingestion etc. Residues of the pollutants (both organic and inorganic) contaminate the coastal seas and accumulate in the body compartments of marine organisms. Concentration of these toxic residues magnifies at the higher trophic level through food chain (biomagnification) and hence express their toxicity at all trophic levels. Usually the pollutants exceeding threshold limits combined with environmental variables such as temperature, salinity, pH, dissolved oxygen, hydrogen sulphide etc. give stress to the organisms living in these environment Some of the major contaminants prevalent in the sea are persistent organochlorine (OC) pesticides (DDTs, HCHs, aldrin dieldrin, endrin), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), planar halogenated aromatic hydrocarbons (PHAHs), and toxic elements such as lead (Pb), mercury (Hg), cadmium (Cd) and arsenic (As). Many of these pollutants are chemical carcinogens and mutagens with the capacity to cause various types of DNA damage. Benzo(a)pyrene, a representative PAH is reported to be converted at cellular level to chemically reactive oxygen species, diol-epoxide (BaPDE) which can form stable adduct with DNA resulting into DNA strand breaks (Pisoni et al., 2004). Benzo[k]fluoranthene is another PAH, that is listed in pollutant list (US EPA, 2009) by United States Environmental Protection Agency (US EPA). It is

1 also among the several PAHs which are possibly carcinogenic to humans (IARC, 2015). PAHs and their metabolites interact with DNA and form DNA adducts. PAH activation process also generates reactive oxygen species (ROS) that can induce genotoxic damage by modifying integrity of DNA (Gauthier et al., 2014). The occurrence of single strand breaks can be induced in various ways such as chemical induction during excision repair, interaction with DNA-intercalating agents, degradation due to autolysis or disruption, formation of alkali labile sites, interstrand cross links, DNA-protein cross (DPC) links. Moreover, significant DNA damage can occur due to interaction of alkylating agents with DNA at multiple sites. Aquatic ecosystem is an important source for food for human and it plays an essential role in human health. Consumption of aquatic organism exposed to toxicants can cause health risk to human. Gastropods are an important source of food for many fishes and birds and play a very important role in aquatic food chain. They have limited ability to metabolize xenobiotics and thus are prone to accumulate high concentrations of hydrocarbons (Zheng et al., 2012). In recent years gastropods have received great attention from ecotoxicologists, thanks to the discovery of imposex. As regards the study region is concerned, Goa is one of the most famous tourism destination in the world situated on the west coast of India with a flourishing hotel industry supported by the rich catches of prawns, fishes, crabs, clams and snails etc. Along with increased number of industrial units, shipping, mining and tourism activities keep the living organisms in the coastal region under constant stress. Several investigations have reported that the trace metals in the marine environment can cause serious damage to physiological status of various species of marine organisms at the molecular level with long- term effects on entire communities (Desai et al., 2010; Sarkar et al., 2011). Biomonitoring of marine organisms has become necessary to study the effect of these toxic pollutants on the marine environment. In order to assess the impact of environmental stress on the health of the marine environment, the quality and the status of marine life especially in coastal areas, it is of urgent necessity to look for reliable tools to express the effects of anthropogenic activities on biological systems. The condition of environmental health of a marine ecosystem cannot always be diagnosed by only chemical analysis of the water, as it does not provide any information in regard to the physiological status

2 of the organisms exposed to marine pollutants. It only indicates that there might be some undesirable biological effects which may be of great concern, and therefore, studies of biological effects will give us a better understanding of the potential impact on ecosystems. In this context, biomarkers play a significant role by measuring the biological response of living organisms in response to their exposure to variety of pollutants. They are an important tool to detect exposure and adverse effects of anthropogenic or natural contaminants on aquatic organisms. Some biomarkers are specific to chemicals or group of chemicals while other are non-specific and induces upon exposure to broad range of pollutants. Thus the impact of these pollutants on the health of the marine organisms can be observed by the detection of DNA damage using Comet assay (Singh et al., 1988), inhibition of acetyl cholinesterase (AChE) activity (Sarkar et al., 2006), Glutathione S-transferase (GST) activity (Habig et al., 1974), Catalase activity (Sinha, 1972), and lipid peroxidation (LPO) (Desai et al., 2010). In view of complexity of contaminants, the use of multi-biomarker has become an increasingly popular tool to study the environmental impact assessment in terms of causative effects on organism health. Combinations of biomarkers yield a complicated and vast amount of data which is hard to interpret. In the recent years there has been many reports on application of integrated biomarker response (IBR) in clams (Tankoua et al., 2013; Barda et al., 2014), mussels (Pain-Devin et al., 2014; Turja et al., 2014), fishes (Xie et al., 2014; Zheng et al., 2014), and crabs (Ben-Khedher et al., 2013; Rodrigues et al., 2014). In view of the continuing problem of environmental contamination by various types of toxic pollutants, it has become an urgent need of the hour to assess the state of pollution of the coastal environment and the ecotoxicological impact on the health of the marine ecosystem. For our study we have selected three species of gastropods i.e. Nerita chamaeleon, Planaxis sulcatus and Morula granulata. These three gastropod species were selected because (i) they are widely distributed along the Goa coast and can be found throughout the year (ii) they can be easily collected from the intertidal rocks scattered along the coast, (iii) they are easy to culture and laboratory studies can be easliy conducted, (iv) they reflect changes in pollution status of marine environment, (v) they have been widely used as a sentinel species for ecotoxicological studies, (vi) they represent

3 diverse feeding behavior (P. sulcatus and N. chamaeleon are herbivore, and M. granulata is a carnivore), (vii) these three species are diverse in their habitat (P. sulcatus is mostly found exposed in the rocks, N. chamaeleon is commonly observed in rocks submerged in water, whereas M. granulata is seen in oyster beds)

The objectives of this study are as follows:

1. To identify the hot spot of pollution along the coastal region of Goa 2. To identify the sentinel species of marine gastropods for marine pollution studies; 3. To assess the impact of genotoxicants on the integrity of DNA in marine gastropods as a biomarker of genotoxic pollution 4. To assess the variation in Acetylcholinesterase activity in marine gastropods as a biomarker of neurotoxic contaminants 5. To assess the oxidative stress of pollution in terms of superoxide dismutase activity, Glutathione S-transferase activity, Catalase activity and lipid peroxidation 6. To assess the variation of physico-chemical parameters of the coastal water and their impact on the variation in DNA integrity

CHAPTER 2 Review of Literature

2.1. Environmental Pollutants 2.1.1. Polycyclic aromatic hydrocarbons (PAHs) Of all the ecosystems, estuarine and coastal regions are considered as the primary sink for the pollutants and greatly affected by the high degree of contaminations by heavy metals and hydrocarbons, and other highly persistent toxic compounds. During the last decades, rapid increase in disposal of these contaminants has lead to an unprecedented increase in chemical pollution. Among environmental pollutants, polycyclic aromatic hydrocarbons (PAHs) are of great concern due to their pervasive nature. PAHs are one of the most widespread environmentally persistent contaminants encountered in aquatic environment (Lazartigues et al., 2010). They are ubiquitous micro-organic pollutants, found throughout the marine and terrestrial environment. They are included in the list of persistent organic pollutant of United Nations Environment Program. PAHs enter the aquatic ecosystem from different sources such as riverine runoff, spills of petroleum products, industrial, and domestic wastewater. PAHs entering into the coastal regions primarily get accumulated in the sediments which later get mobilized into seawater and aquatic organisms. Studies on the bioaccumulation of PAHs are well documented in the aquatic ecosystem. The concentrations of PAHs in sediment from various locations around the world are shown in table 1. PAHs tend to accumulate in the fatty tissues in the organisms owing to their low water solubility and hydrophobic nature. The concentrations of PAHs in the molluscs collected from different parts of the world are shown in table 2. Various studies in India have been reported with high amount of PAHs in bivalves and gastropods (Menon & Menon, 1999; Sarkar et al., 2008). Higher amount of PAH (<1000 ng/g dry weight) has been observed in mussels collected from India (Isobe et al., 2007).

Table 1 Concentrations of PAH in sediments (ng/g, dry weight) from different parts of the world

Location ng/g, dry weight No. of PAH References

Delhi, India 830-3880 16 Agarwal et al., 2009 Gomti River, India 0.068-3.153* 16 Tripathi et al., 2009 Sunderban Wetland, India 132-2938 16 Domínguez et al., 2010 Agra, India 6.44-12.5* - Masih et al., 2012 Liaohe Estuarine Wetland, China 704.7 to 1,804.5 16 Lang et al., 2012 Minjiang River Estuary, China. 112 to 877 16 Zhang et al., 2004 Zha Long Wetland, China 244-713 16 Li et al., 2013 Tianmu Lake, China 288-714 16 Shu & Li, 2008 Taizhou Bay, China 85.4–167.6 15 Jiang, 2007 Guiyu, China 95.2-5,210 16 Leung et al., 2013 Yellow River Delta Natural Reserve, China 87.2-319 23 Yuan et al., 2014 Dapeng Bay, Taiwan 216.56-1,314.92 16 Sun et al., 2013 Midway Atoll, North Pacific Ocean 3.55-3200 16 Yang et al., 2014 Tanoura Bay, Japan 237-32700 - Nakata et al., 2014 Masan Bay, Korea 207–2670 - Yim et al., 2005 Coastal Region, Malaysia 4–924 15 Zakaria et al., 2002

Basque Country, Spain 0.7-140 16 Cortazar et al., 2008 Mar Menor lagoon, Spain 1.01-163.0 14 Leon et al., 2013 Lagoon of Venice, Italy 23-532 - Secco et al., 2005 Sacca di Goro, Italy 1.5-66.5 12 Sacchi et al., 2013 Palermo, Italy 947-18,072 23 Orecchio, 2010 Württemberg, Germany 422-23000 16 Mazurová et al., 2008 Dar es Salaam, Tanzania 78-25000 23 Gaspare et al., 2009 Cartagena Bay, Colombia 1,330–3,210 13 Johnson-Restrepo et al., 2008 Todos Santos Bay, Mexico 7.6–813 - Macias-Zamora et al., 2002 Gulf of Gdansk 29.3-103 15 Ruczyńska et al., 2011 Gulf of Rijeka, the Adriatic Sea, Croatia 213-695 10 Bihari et al., 2007 El-Tabbin, Egypt 53.4-5558.0 - Havelcová et al., 2014 Niger Delta, Nigeria 3.15–144.89 - Anyakora et al., 2005 Agbabu bitumen, Nigeria 101.5 to 209.7 10 Olajire et al., 2007 Guadeloupe 49-1065 25 Ramdine et al., 2012

* Values in µg/g

Table 2 Concentrations of PAHs in molluscs (ng/g, dry weight) from different parts of the world

Organisms Group Location No. of ng/g, dry References PAH weight

Rapana thomasiana Gastropod Varna Bay, Black Sea coast of 7 401-572 Namiesnik et al., 2012 Bulgaria Mytilus edulis Mussel Maarmorilik, West Greenland 16 280 Jörundsdóttir et al., 2014 Mytilus edulis Mussel Álftafjörður, NW Iceland 16 28-480 Jörundsdóttir et al., 2014 Mytilus edulis Mussel Mjóifjörður, East Iceland 16 370 Jörundsdóttir et al.,2014 Mytilus edulis Mussel Isefjord in the Northern part of 19 209-4155 Rank et al., 2009 Zealand Mytilus galloprovincialis Mussel Istanbul Strait 16 1.2-589* Balcıoğlu et al., 2014 Mytilus galloprovincialis Mussel Marmara Sea 16 0.94-36.4* Balcıoğlu et al., 2014 Mytilus galloprovincialis Mussel Çanakkale Strait 16 0.4-47.9* Balcıoğlu et al., 2014 Mytilus galloprovincialis Mussel Morocco 16 46.9 Galgani et al., 2011 Mytilus galloprovincialis Mussel Algeria 16 37.6 Galgani et al., 2011 Mytilus galloprovincialis Mussel Tunisia 16 49 Galgani et al., 2011 Mytilus galloprovincialis Mussel Italy 16 49.9 Galgani et al., 2011 Mytilus galloprovincialis Mussel France 16 41.4 Galgani et al., 2011 Mytilus galloprovincialis Mussel Spain 16 45.4 Galgani et al., 2011

Mytilus californianus Mussel San Francisco estuary - 21–1093 Oros & Ross, 2005 Perna perna Mussel Guanabara Bay, Brazil 38 760.9 ± Yoshimine et al., 2012 456.3 Perna perna Mussel Guanabara Bay Brazil 35 60-6271 Francioni et al., 2006 Perna perna Mussel Guanabara Bay Brazil 16 9-273 Francioni et al., 2006 Crassostrea gigas Oyster French Atlantic coast 29 80-236 Luna-Acosta et al., 2014 Crassostrea sp Oyster Basque Country, Bay of Biscay 16 300-1400 Cortazar et al., 2008 Crassostrea gigas Oyster San Francisco estuary - 184–6899 Oros & Ross, 2005 Crassostrea rhizophorae Oyster Guadeloupe 25 66-961 Ramdine et al., 2012 Saccostrea cucullata Oyster Dar es Salaam, Tanzania 23 170-650 Gaspare et al., 2009 Ostrea edulis Oyster Mar Menor lagoon, spain 14 26.53-78.58 Leon et al., 2013 Cerastoderma glaucum Cockle Mar Menor lagoon, spain 14 8.98-370 Leon et al., 2013 Pinna nobilis Noble pen shell Mar Menor lagoon, spain 14 21.53- Leon et al., 2013 162.23 Corbicula fluminea Clam San Francisco estuary - 78–720 Oros & Ross, 2005 wet weight (µg/g) Nerita chamaeleon Gastropod Goa Coast, India Total 5.29-12.14 This study PAH Cronia contracta Gastropod Goa Coast, India Total 22.32– Sarkar et al., 2008 PAH 53.78 Turbo cornutus Gastropod Japan Coast, Japan 8 0.044 Koyama et al., 2004

Sunetta scripta Clam Cochin Harbour, India Total 13.35-21.49 Menon & Menon, 1999 PAH Saccostrea cucullata Oyster Hooghly Estuary, India Total 0.8-12.5 Niyogi et al., 2001a PAH Mytilus galloprovincialis Mussel Marmara sea, Izmit Bay 16 5.67-14.81 Telli-Karakoç et al., 2002 Mitylus galloprovincialis Mussel Adriatic Sea, Italy 13 0.034 Perugini et al., 2007 Mytilus galloprovincialis Mussel Gulf of Rijeka, Croatia. 10 0.049-0.134 Bihari et al., 2007 Palaeomonetes sp. Shrimp Norco, USA Total 7.18-10.86 Oberdorster et al., 1999 PAH Penaeus japonicus Shrimp Gulf of Suez 16 2.01 Ali et al., 2014b Sepia sp. Cuttlefish Gulf of Suez 16 4.09 Ali et al., 2014b Portunus pelagicus Crab Gulf of Suez 16 8.10 Ali et al., 2014b Nephrops norvegicus Lobster Adriatic Sea, Italy 13 0.015 Perugini et al., 2007 Nephrops norvegicus Lobster Adriatic Sea, Italy 13 0.015 Perugini et al., 2007

PAHs have received great attentions from the toxicologist around the world due to their mutagenic and carcinogenic properties. PAHs are also known to be carcinogenic to human as well as animals. The International Agency for Research on Cancer (IARC) has classified PAHs as possible and probable carcinogenic agents to human (IARC, 2010). PAHs have a strong capacity to bio-concentrate; these pollutants either individually or in combination may have sub-lethal effects at the cellular, organ, or individual level. The accumulation of PAHs in marine organism can negatively affect their health. High abundance of PAHs in the environment pose a serious threat to the health of aquatic organisms and finally to human. Several toxicological studies on toxic effects such as neurotoxic damage, reproductive toxicity, immune and endocrine alteration have been reported in aquatic organisms exposed to PAHs (Gauthier et al., 2014). There are few studies on genotoxic effects of PAHs in fishes, clams, and mussels (Al-Subiai et al., 2012; Martins et al., 2013). PAHs and their metabolites interact with DNA and form DNA-adducts. PAHs activation process also generates reactive oxygen species (ROS) which can induce genotoxic damage by modifying integrity of DNA (Mattsson et al., 2009). The integrity of DNA can be greatly affected by genotoxic agents due to DNA strand breaks, loss of methylation and formation of DNA adducts (Pisoni et al., 2004). Recent research shows that the increasing trend of plastic pollution in aquatic ecosystem could also pose a threat and enhance bioavailability of PAHs in bivalves due to absorption of PAHs to microplastics (Avio et al., 2015). Benzo[k]fluoranthene (B[k]F ) is one of the 16 PAHs that are on United States Environmental Protection Agency’s (US EPA) priority pollutant list (US EPA, 2009). B[k]F is also listed among the several PAH which are possibly carcinogenic to humans (IARC, 2015). Several authors have studied the toxicity of B[k]F in fishes (Ding et al., 2014; Kim et al., 2014). However there are only few reports on B[k]F in molluscs (Pan et al., 2005, 2006). Phenanthrene occurs as the major component of PAH and is among 16 PAHs that are on USEPA priority pollutant list (US EPA, 2009). Phenanthrene has shown to cause cytotoxicity (Schirmer et al., 1998), genotoxicity (Machado et al., 2014), neurotoxicity (Martyniuk et al., 2009), oxidative stress (Martins et al., 2013) and mutagenic effects (Wood et al., 1979).

2.1.2. Heavy metals Heavy metals are of great ecological concern due to their toxic and persistent nature. They are widely spread in the biosphere, from both natural and various human activities. In recent decades, the amount of potentially toxic heavy metals has risen steadily in both marine

11 and freshwater ecosystems due to anthropogenic emissions (Sunderland et al., 2009). Heavy metals accumulate in the environment due to their slow decay rate (Migliore et al., 1999). Among the toxic metals, mercury (Hg) is ubiquitous in the environment. In the last few decades, there have been many reports on mercury pollution in various places in India (Krishnakumar and Bhat, 1998; Kaladharan et al., 1999). Aquatic organisms from Mumbai have been found to be heavily contaminated with mercury (Pandit et al., 1997; Mishra et al., 2007). Rajathy, (1997) has reported high level of Hg (0.013–0.40 μg/g, wet weight) in fish from Ennore estuary, Tamil Nadu. Menon and Mahajan (2013) have surveyed five villages along Ulhas River estuary and Thane Creek and found high levels of Hg in gills, kidney, and skin in fish Mugil cephalus. The environmental pollution studies have reported about Hg pollution in Amba estuary (Ram et al., 2009a), Hooghly River (Sarkar et al., 2004), Rushikulya estuary (Panda et al., 1990; Das and Sahu 2002), and Tamiraparani estuary (Magesh et al., 2011). The concentrations of mercury reported in sediments and coastal waters of India are presented in Table 3. Karunasagar et al., (2006) have reported mercury pollution in Kodaikanal Lake due to a thermometer factory. Aquatic mercury pollution of the Ulhas estuary, India has been reported by Ram et al., (2009b). UNEP report on global study on mercury, predicted India to be one of the hotspots for mercury pollution due to an increase in gold mining activities (Mago, 2003). Although the concentration of mercury in surface water is reported to be low, molluscs are most affected due to its capacity to accumulate toxicants. Among molluscs, bivalves and gastropods are excellent sentinel organisms due to their sedentary lifestyle. Mercury has been shown to bioaccumulate in gastropods (Tessier et al., 1994), oysters (Liu and Wang, 2014), clams (Giani et al., 2012), and mussels (Kraemer et al., 2013). Higher bioaccumulation of mercury has been reported in digestive gland cells compared with gills and whole soft tissue in mussels (Kljakovic-Gaspic et al., 2006). Table 4 shows the concentration of mercury reported in gastropods from different parts of the world. Mercury has become a public threat due to biomagnifications through the food web (Kwon et al., 2012). Bioaccumulations of mercury in molluscs can cause several biological effects, including genotoxic and immunotoxic effects (Gagnaire et al., 2004).

Table 3. Concentrations of Hg reported in the coastal sediments and waters of India

Locations Sediments (µg/kg) Coastal waters (ng/l) References

Bheemili, Central east coast of India 5.6–66.3 - Chakraborty et al., 2014

Visakhapatnam, Andhra pradesh 7.7–9.4 - Chakraborty et al., 2014

Kakinada, Central east coast of India 35.3–65.2 - Chakraborty et al., 2014

Gangavaram, Central east coast of India 10.4–38.4 - Chakraborty et al., 2014

Goutami Godavari Estuary, Central east coast of India 24.1–49.8 - Chakraborty et al., 2014

Mandovi estuary, Goa 140 90 Verlecar et al., 2012

Amba Estuary, Maharasthra 50-2660 - Ram et al., 2009a

Ulhas Estuary, Maharasthra 460-6,400 - Ram et al., 2009b

Thane creek, Maharashtra 170 - 8210 - Zingde & Desai, 1981

Chennai Harbour, Tamilnadu 237-338 100-2100 Jaysankar et al., 2003

Ulhas estuary, Maharasthra 0.5-55.28 - Ram et al., 2003

Mangalore, Karnataka 133–172 45–1088 Ramaiah & De, 2003

Mumbai, Maharashtra 210–1390 180–440 Ramaiah & De, 2003

Mormugao, Goa 53–194 152–456 Ramaiah & De, 2003

Kodai Lake, Tamilnadu 276-350* 356-465 Karunasagar et al., 2006

Hooghly river, West Bengal - 200-270 Sansgiry et al., 1988

Arabian sea off Indian coast - 26-130 Singbal et al., 1978

*units in mg/kg

Table 4. Concentrations of Hg reported in terms of dry weight (µg/g) and wet weight (µg/g) in gastropods from different parts of the world

Gastropods Locations dry weight (µg/g) References

Monodonta turbinata Tel Shikmona, Israel 0.012-0.123 Hornung et al., 1981 Acre, Israel 0.959 to 1.939 Hornung et al., 1981 Monodonta turbiformis Tel Shikmona, Israel 0.098-0.127 Hornung et al., 1981 Patella caerulea Tel Shikmona, Israel 0.157 Hornung et al., 1981 Acre, Israel 0.462-1.035 Hornung et al., 1981 Cerithidea cingulata Hooghly Estuary, India 0.15 Bhattacharya & Sarkar, 1996 Littoraria undulata Hooghly Estuary, India 0.29-0.43 Bhattacharya & Sarkar, 1996 Nerita articulata Hooghly Estuary, India 0.22-0.27 Bhattacharya & Sarkar, 1996 Cerithidea obtusa Hooghly Estuary, India 0.34-0.52 Bhattacharya & Sarkar, 1996 Arcularia circumcinta Haifa Bay, Israel 38.7 Hornung et al., 1984 Arcularia gibbosula Haifa Bay, Israel 18.2 Hornung et al., 1984 Nassarius reticulatus Venice Lagoon, Italy 0.3-1.3 Berto et al., 2006 Hexaplex trunculus Gulf of Taranto, Italy 0.224-1.867 Spada et al., 2012

La Spezia, Italy 0.558 Giordano et al., 1991 Gulf of Manfredonia, Italy 0.266 Giordano et al., 1991

Croatian Coasts– Adriatic Sea 0.293 Buzina et al., 1989

wet weight (µg/g) Bolinus brandaris Adriatic Sea, Italy 0.03-0.63 Bille et al., 2015 Nassarius mutabilis Adriatic Sea, Italy 0.03-0.46 Bille et al., 2015 Gibbula adriatica Grado and Marano Lagoons, Italy 0.480–1.220 Brambati,1997 Rapana venosa Bohai Sea, China 0.038-0.199 Yawei et al., 2005 Neverita didyma Bohai Sea, China 0.027-0.096 Yawei et al., 2005 Peringia ulvae Ria de Aveiro, Portugal 0.030-0.250 Cardoso et al., 2013 Littorina littorea France 0.040-0.088 Noel et al., 2011 Haliotis tuberculata France 0.040-0.185 Noel et al., 2011 Aplexa hypnorum Wash, East England 0.20-0.34 Moriarty & French, 1977 Potamopyrgu jsenkinsi Wash, East England 0.29-0.38 Moriarty & French, 1977 Hydrobia neglecta Wash, East England <0.01-0.07 Moriarty & French, 1977 Planaxis sulcatus Arambol, Goa, India 0.009-0.01 This study

Cadmium is another highly toxic compound that poses a serious environmental threat. Although the mechanisms of its toxicity are still poorly understood, it is a systemic poison affecting many cellular functions. Cadmium contamination poses a serious health threat throughout the world, and it has been classified as a human carcinogen by the IARC (1993). Cadmium exposure induces intracellular damage through several mechanisms. In cultured cells, cadmium produces direct and indirect genotoxic effects such as DNA strand breaks, DNA-protein cross links, oxidative DNA damage and chromosomal aberrations (Misra et al., 1998).

source : http://www.intechopen.com/source/html/37966/media Fig. 1. Biomarkers as an early warning signal in ecotoxicological studies

In view of the continuing problem of environmental contamination by various types of genotoxic pollutants it has become an urgent need of the hour to assess the state of pollution of the coastal environment and the ecotoxicological impact on the health of the marine ecosystem. In order to evaluate the impact of toxic pollutants on the environmental quality, it has become highly pertinent to carry out a rapid assessment of their deleterious effects on the ecosystem. In this context, use of biomarkers measured at the molecular and cellular level is of immense importance as sensitive ‘early warning’ tools for biological effect measurement in environmental quality assessment (Fernando et al., 2005).

2.2. Gastropod as a model organism to study environmental contaminants Molluscs are extensively used as a sentinel organism in areas affected by pollution. Among molluscs, bivalves and gastropods are an excellent sentinel organism due to their sedentary life style. The use of bivalves has deeply studies by several authors. Eighty percent of mollusc is represented by gastropods (Strong et al., 2007). Recently, gastropods have attracted attention because of their potential as sentinel organisms. Intersex development in gastropods due to TBT pollution has been reported by several authors. Snails can accumulate a large amount of toxic chemicals into their system due to their feeding behaviour (Boshoff et al., 2013). The choice of marine gastropods as model organisms for environmental biomonitoring of genotoxic pollutants took into account that invertebrates represent more than 90% of aquatic species. Gastropods are easy to breed, need little space, can reproduce throughout the year under controlled conditions and have a short life-span. Gill cells are potential for being a suitable target tissue for mutagen exposure as they are easy to obtain, and in nature they come into contact with relatively large volumes of seawater compared with the rest of the animal (Dixon and Clarke, 1998). Most of the invertebrate species that are commonly used in environmental monitoring studies have a more limited ability to metabolize aromatic compounds (Livingstone, 1998) and despite intensive research work the estimation of PAH exposure and their effect in aquatic invertebrate species remains problematic. A few data have been observed on the occurrence of DNA damage in marine gastropods (Hagger et al., 2006; Regoli et al., 2006; Sarkar et al., 2008, 2011, 2013). Gastropods are known to be efficient accumulators of metals, organic pollutants and respond to pollution in a sensitive and measurable manner (Bhagat et al., 2012; Sarkar et al., 2008). Moreover, because of their characteristics with little mobility, they are very useful as the sentinel species for bio-monitoring of pollution and ecotoxicological studies (Angeletti et al., 2013). Several species of gastropods have been used for toxicity testing. Marine snails, Polinices conicus and Austrocochlea porcata have been used to study the effect of crude and dispersed oil (Gulec et al., 1997). Kulkarni et al., (2004) has studied acute toxicity of several metals on marine gastropods collected from Mumbai coast. Sousa et al., (2005) has studied the acute toxicity of tributyltin and copper to veliger larvae of Nassarius reticulatus. Laboratory studies carried out in Nerita chamaeleon is show in table 5.

Table 5 Laboratories studies with Nerita chamaeleon

Organism Exposure condition Observed effects References Nerita atramentosa Laboratory exposure to cigarette 100% mortality was observed at higher concentrations Booth et al., 2015 butt leachate Nerita (Melanerita) Field exposure to oil and dispersant Changes in wet weight, gonad weight, and gonad Battershill and atramentosa melanotragus fertility Bergquist, 1982 Nerita atramentosa Measurement of selenium Spatial, temporal, intra-species and inter-species Baldin & Maher, concentrations in tissues variation were observed 1997 Nerita saxtilis Accumulation of lead and cadmium Higher metal storage capability was observed Abd Allah and suggesting N. saxtilis as biomonitor for heavy metal Moustafa, 2002 contaminants Nerita lineata Heavy metal concentrations in Soft tissues and operculum showed higher metal Yap and Cheng, different tissue were measured concentrations compared to other tissues 2013 Nerita lineata Accumulation and Depuration of Snails showed to stand high Pb exposure suggesting Kanakaraju and Lead and Chromium were studied suitable species for biological monitoring Anuar, 2009 Nerita crepidularia Monitoring of heavy metal in shell Seasonal variations in heavy metal accumulations Palpandi and and soft tissues were observed Kesavan, 2012 Nerita lineata Heavy metal concentrations in Spatial and temporal variations in metal Amin et al., 2006 snails were studied concentrations were observed Nerita albicilla Heavy metal monitoring Inter-specific variations were observed Blackmore, 2001 Nerita albicilla, Trace metal analysis Seasonal and regional variations were recorded Hung et al., 2001 Nerita chamaelon Nerita chamaeleon Toxicity of Nickel ore was tested Snails were found to be resistant Florence et al., 1994 Nerita albicilla Effect of marine diesel oil spill Severe population reduction in snails was observed Stirling, 1970 Nerita lineata Heavy metal monitoring Human health risk due to snail consumption were Cheng and Yap, discussed 2015 Nerita chamaeleon Exposure to Cadmium chloride Significant DNA damage in exposed snails were Sarkar et al., 2015 observed

2.3. DNA Integrity as a biomarker of marine pollution The marine pollutants prevalent in the marine ecosystem may likely to cause severe damage to the genetic material directly or indirectly. Among the direct genotoxicants, alkylating agents, like hydrogen peroxides and pesticides etc. are of significant whereas indirect toxicity depends on the mechanisms of metabolic activation and the formation of reactive oxygen species (ROS) as they consist of substances capable of inhibition of DNA synthesis/repair mechanisms (Everaarts and Sarkar, 1996). Metabolic activation processes generally lead to the formation of electrophilic metabolites which can bind to nucleophilic DNA molecules producing a variety of DNA lesions (Shugart et al., 1989; Shugart, 1999; Lee and Steinert, 2003). The occurrence of DNA strand breaks in various species of marine organisms exposed to enhanced concentration of genotoxic pollutants is a matter of great concern. Thus the measurement of DNA integrity in those species of marine organisms exposed to such pollutants is indeed of great importance for bio-monitoring of pollution of the marine environment. The integrity of DNA can be greatly affected by genotoxic agents due to DNA strand breaks, loss of methylation and formation of DNA adducts (Pisoni et al., 2004). The relationship between DNA damage and the exposure of gastropods to environmental contaminants is well documented (Sarkar et al., 2008, 2011; An et al., 2012; Bhagat et al., 2012). The solution to the above problem is the use of molecular biomarker techniques like measurement of DNA integrity by time dependent partial alkaline unwinding assay (Shugart, 1999; Sarkar and Everaarts, 1996; Sarkar et al, 2005, 2006, 2008, 2011, 2013) and substantiated by the single cell gel electrophoresis (SCGE) or comet assay (Sarkar et al., 2013).

2.3.1. Comet Assay Currently, the comet assay is widely used in both the research field and laboratory tests because of its versatile and reliable nature (Valerio et al., 2014). Multiple classes of DNA damage can be investigated in single cells using this technique. One of major advantage of using this assay is requirement of small number (<10,000) of cells for detecting DNA damage (Lee and Steinert, 2003). The underlying principle of the assay is that when DNA is subjected to an electric current, DNA containing strand breaks will migrate through an agarose gel due to relaxation of the DNA supercoils, whilst unbroken DNA remains immobile. Following the staining of DNA with a fluorescent DNA-specific dye, the resulting image can be visualised and resembles a comet, with undamaged DNA forming a ‘‘head’’ and damaged DNA forming a ‘‘tail’’, an observation that has led this technique to be more

19 commonly called the Comet assay. Over the past decade Comet assay has become one of the standard methods for assessing DNA damage with applications spreading in different fields of research.

Fig. 2. Comet Assay Principle: Denatured cleaved DNA fragments migrate out of immobilized cells following permeabilization and electrophoresis.

Since its inception, comet assay has been used in several organisms and plants such as bacteria, (E. coli, Singh et al., 1999), fungi (Saccharomyces cerevisiae, Banerjee et al., 2008), algae (Cryptophyta, Shastre et al., 2001), higher plants (Vicia faba, Menke et al., 2000), bivalves (Limnoperna fortunei, Villela et al., 2007; Nereis virens, (Boeck et al., 1997) Drosophila melanogaster, (Siddique et al., 2005) Dreissena polymorpha, (Riva et al., 2007), amphibians (Rana tigrina, Ralph et al., 1997), and birds (Ciconia ciconia, Milvus migrans, Baos et al., 2006). The assay has been widely used in assessing DNA damage and repair in healthy individuals, in clinical studies, as well as in dietary intervention studies and in monitoring the risk of DNA damage resulting from occupational, environmental, oxidative DNA damage, exposures or lifestyle (Gunasekarana et al., 2015). White blood cells or lymphocytes are the most frequently used cell type for the Comet assay in human

biomonitoring studies. However, other cells have also been used, e.g. buccal cells, nasal, sperm, epithelial and placental cells. DNA damage as measured by comet assay has been reported in mussel (Dailianis et al., 2014), clam (Martins et al., 2013), and flounder (Woo et al., 2006). In vivo studies of mussel to PAH have been studied using comet assay (Perez-Cadahia et al., 2004; Banni et al., 2010). Comet assay previously has been used to investigate levels of DNA damage in marine and freshwater bivalves exposed to water-borne pollutants (Nacci et al., 1996; Pavlica et al., 2001). Various studies have applied the Comet assay to assess the DNA damage in cells from a single type of tissue, following the exposure of bivalves to a variety of contaminants, (Nacci et al., 1996; Wilson et al., 1998; Mitchelmore et al., 1998). Others have used the Comet assay to measure DNA damage in haemolymph and gill cells in mussels (Rank, 1999). Recently a few studies have been carried out in snails using comet assay (Osterauer et al, 2011 and Mohamed et al., 2011). A severe DNA damage was reported after treatment of snails’ with organophosphate insecticides. (Itziou et al., 2011). Therefore, DNA damage resulting from contaminant exposure is a key factor when assessing the general health of an organism, as is the need to recognize the cause, seriousness of genotoxicity and the consequences on populations and communities (Depledge, 1996). Table 6 lists the studies carried out in snail using comet assay. Ali et al., (2014) has studied the effect of single-wall carbon nanotubes in snail Lymnea luteola L. In vivo exposure of ethylbenzene was studies in snail Bellamya aeruginosa (Zheng et el., 2013). Comet assay was applied in various field studies using gastropods as sentinel species for several environmental monitoring programs (Angeletti et al., 2013; Yu, 2013). In vivo studies were carried out to study the effect of heavy metal in snails using comet assay (Osterauer et al., 2011; Bhagat & Ingole, 2015).

Table 6 Studies on DNA damage in gastropods as measured by comet assay and alkaline unwinding assay

Organisms Exposure condition Nature of the exposure Biomarkers Biomarker effects References Bellamya Ethylbenzene, 5-1000 µg/L, In vivo Comet assay OTM increases during exposure and Zheng et al., aeruginosa exposed to ethylbenzene for then decreases during recovery period 2013 21 days followed by a 17-day recovery period Biomphalaria irradiated with single doses Comet assay Dose-related increase in DNA Grazeffe et glabrata of 2.5, 5, 10 and 20 Gy of migration al., 2008 (60)Co gamma radiation Cronia contracta Collected from sites along Field study Alkaline unwinding Sites contaminated with PAH and Sarkar et al., Goa coast assay heavy metal shows decreases in DNA 2008 integrity Helix aspersa Lab study genotoxic potential Animals of the coal tailings and mine Leffa et al., of the mineral coal lettuce groups presented higher levels 2010 tailings of DNA damage in relation to the control group Helix aspersa Snails collected from Field study Comet assay DNA damage decreased as distance Angeletti et and Helix polluted and control sites increased away from the pollution al., 2013 vermiculata source Littorina littorea Collected from six sites Field study Comet assay Strong correlation between DNA Noventa et damage and PAH bioaccumulation al., 2011 Lymnaea Gastropods collected from Field study Comet assay Strontium reduces the amount of DNA Olu, 2014 stagnalis areas with high radiation load in haemocytes Lymnaea Collected from regions with Field study Comet assay Comet parameters of mollusks Yu, 2013 stagnalis different environmental loads correlate with the intensity of damage impact (radiation, heavy metals) Lymnea luteola TiO2NPs (28, 56, and 84 Lab in vivo & in vitro Comet assay TDNA & OTM increases Ali et al., L μg/ml) over 96 h 2015 Lymnea luteola single-wall carbon nanotubes in vivo Comet assay TDNA & OTM increases Ali et al., L 0.05, 0.15, 0.30, 0.46 mg/L 2014 for 96h Lymnea luteola Silver nanoparticles (4-24 in vivo Comet assay TDNA & OTM increases. A Ali, 2014 L µg/l) for 96h significant positive correlation was observed among reactive oxygen

species (ROS) generation, apoptosis, and DNA damage Lymnea luteola Zinc oxide nanoparticles, 10- In vivo Comet assay TDNA & OTM increases Ali et al., L 32 mg/l for 96h 2012 Marisa Platinum chloride 0-200μg/l In vivo Comet assay Tail moment increases Osterauer et cornuarietis al., 2011 Nerita CdCl2, 10, 25, 50, and 75 In vivo Comet assay, DNA-I, TDNA, OTM, TL increases Sarkar et el. chamaeleon µg/L alkaline unwinding 2015 assay Planaxis HgCl2 (10-100 µg/l) for 96h Lab in vivo & in vitro Comet assay TDNA & OTM increases Bhagat J & sulcatus Ingole, BS, 2015

2.3.2. Alkaline unwinding assay The DNA alkaline unwinding assay (DAUA) was selected to detect DNA damage caused by complex environmental contamination in aquatic test organisms (Pisanelli et al., 2009; Oliveira et al., 2010; Bechmann et al., 2010). This assay enables the assessment of primary DNA damage in tissue from exposed aquatic test organisms of higher evolutionary order. In DAUA, whole cells or crude DNA extracts are subjected to alkaline assay conditions to allow controlled 'unwinding' of double-stranded DNA into single-stranded DNA, beginning at each strand break (Erixon and Ahnstrom, 1979). The number of DNA strand breaks in the original sample is inversely related to the fraction (I) of DNA remaining double stranded after the assay (Rydberg et al., 1975). A simple method for quantifying strand breaks uses a dye (Hoescht dye 33258) that fluoresces preferentially in the presence of double-stranded DNA and was developed for use with single cells. DAUA used with controlled exposure systems is useful for screening potential genotoxicants and for investigating relationships between DNA processes such as damage, synthesis and repair. The DNA alkaline unwinding assay (DAUA) was used to detect DNA damage caused by complex environmental contamination in aquatic test organisms (Sarkar et al., 2008; Pisanelli et al., 2009; Bechmann et al., 2010; Oliveira et al., 2010). Desai et al., (2010) has studied genotoxic responses of Chaetoceros tenuissimus and Skeletonema costatum to water accommodated fraction of petroleum hydrocarbons and reported highly significant difference in the DNA integrities between the control and the exposed organisms. Oliveira et al., (2010) has studied the seasonal variation in DNA integrity in golden grey mullet (Liza aurata) using DAUA. Alkaline unwinding assay was used to measure DNA damage in hepatopancreas tissue in Pandalus borealis exposed to oil (Bechman et al., 2010). DNA damage in mussels following exposure to three potential endocrine disruptors was studied (Taban et al., 2008). Sarkar et al., (2008) has studied impairment of DNA in marine gastropod (Cronia contracta) collected from various locations in Goa, west coast of India and correlated it with heavy metal and PAHs concentrations in the tissues. The author reported seasonal variations in DNA integrity in snails collected during the monsoon, post-monsoon, and pre-monsoon. Pisoni et al., (2004) investigated the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis. They studied the exposure of the marine organisms to PAHs by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity [B(a)PMO]. Bolognesi et al., (1999) studied the sensitivity of alkaline elution, genotoxicity test as biomarkers for the

24 detection of heavy metals in mussels' species. Mytilus galloprovincialis was exposed in aquarium for 5 days to different concentrations of three selected metal salts, CuCl2 (5-80 mg/L ), CdCl2 (1.84-184 mg/L), and HgCl2 (32 mg/L) and to a mixture of equimolar doses of the three metals to study the results of their joint action. The ranking of genotoxic potential was in decreasing order: Hg > Cu > Cd. Cu and Hg caused an increase of DNA single-strand breaks. The possible effects of DNA repair and recombination, as causes of spontaneous mutations and genome instability, have been less extensively studied but are of major importance for the reliable assessment of human health risk by exposure to environmental or occupational chemicals and radiation. Everaarts and Sarkar, (1996) assessed the impact of persistent pollutants on the marine organisms from coastal areas of Netherland, North Sea. They determined the DNA damage in Sea star (Asterias rubens) by measuring the level of integrity of DNA isolated from pyloric caeca of the organisms. Based on the levels of integrity of DNA in Sea stars (Asterias rubens) the North Sea coastal environment has been distinguished into different clusters of pollution in accordance with the intensity of pollution at different locations (Everaarts and Sarkar, 1996).

2.4. Acetylcholinesterase as a biomarker of neurotoxic contaminants Acetylcholinesterase (AChE) is one of the most important enzymes in nervous system that helps in cholinergic transmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. According to the mechanism of action, the AChE is released at the myoneutral junction in organisms if an action potential is developed at the nerve ending and diffuses through the gap between the nerve and the muscle. Organophosphate, carbamate pesticides, toxic elements (Cd, Pb, and Cu etc) bind to the catalytic site of the AChE enzyme thus preventing the physiological inactivation of acetylcholine leading to an anomalous protraction of neurotransmission. Inhibition of AChE is followed by acetylcholine accumulation, the post-synaptic membrane remains hyperpolarized, and nervous transmission is interrupted. The activity of this system is vital to the normal behaviour and muscular function and represents a prime target on which some toxicants can exert a detrimental effect. Inhibition of the AChE enzyme results in a build up of acetylcholine, causing a continuous and excessive stimulation of the nerve/muscle fibres, which leads to tetany, paralysis and eventual death.

Fig. 3. Mechanism of AChE action

Measurement of AChE inhibition in aquatic organisms has already been used as a biomarker of effects of neurotoxic contaminants (Ma et al., 2014). AChE is considered as a suitable biomarker for detecting environmental pollution caused by neurotoxic compounds such as organophosphates, carbamates, pesticides and heavy metals (Sarkar et al., 2010). Several research works have been carried out to assess the use of AChE activity as a diagnostic tool for evaluation of neurotoxicity in the marine environment (Table 7). The inhibition of AChE activity in marine snail Cronia contracta was used as a biomarker of neurotoxic contaminants along the Goa coast (Gaitonde et al., 2006). Numerous studies have reported inhibition of AChE activity in organisms exposed to organophosphate and carbamate compounds. Khalil et al., (2015) studied oxidative stress response in freshwater snail Lanistes carinatus exposed to organophosphorous pesticide Chlorpyrifos and reported a maximum of 78% inhibition in AChE in control after 28 days. In another study with terrestrial snail Xeropicta derbentina, Laguerre et al., (2009) also reported concentration-dependent inhibition in AChE activity. There are several other anti- cholinesterase agents which bind to AChE and prevent the destruction of acetylcholine leading to continuous and excessive stimulation. Inhibition of AChE was reported in Helix aspersa exposed to dimethoate (Coeurdassier et al., 2002) and imidacloprid (Radwan & Mohamed, 2013). Decrease in AChE activity was reported in snails exposed to carbofuran or the mixture of carbofuran and cadmium in digestive gland (Romeo et al., 2006). Heavy metals such as copper, mercury, and cadmium have also show to inhibit AChE activity in snails (Cunha et al., 2007; Cabecinhas et al., 2014). Organophosphorus insecticide,

26 chlorpyriphos has been reported to inhibit AChE in snail Xeropicta derbentina (Laguerre et al., 2009), Planorbarius corneus (Rivadeneira et al., 2013). Biomphalaria alexandrina exposure to pesticides, Atrazine and Roundup (glyphosate) has also shown decrease in AChE activity (Barky et al., 2012).

Table 7 Acetylcholinesterase (AChE) activity in gastropods Acetylcholinesterase Tissue studied Exposures Biomarker effects References (AChE) Biomphalaria Soft tissue Exposure to pesticides Atrazine and Roundup Decrease Barky et al., 2012 alexandrina (glyphosate) LC10 Cantareus apertus Digestive gland Carbamate pesticide Carbaryl for 3, 7 and 21 Increase Leomanni et al., days 2015 Chilina gibbosa - exposure to azinphos-methyl (0.2-20 μg/l Decrease Bianco et al., 2013 Eobania vermiculata - methomyl and methiocarb Inhibition Essawy et al., 2009 Gibbula umbilicalis - Exposure to mercury for 96 h Decrease Cabecinhas et al., 2014 Hexaplex (Murex) Digestive gland, gill, Cadmium, copper, carbofuran and lindane AChE activity decreases by exposure Romeo et al., 2006 trunculus muscle to carbofuran or the mixture of carbofuran and cadmium in digestive gland and muscle AChE activity decreases in digestive gland by copper and lindane Helix aspersa - Dimethoate Inhibition Coeurdassier et al., 2002 Helix aspersa digestive glands Helix aspersa exposed to sublethal 0.2 (21.84 Decrease Radwan & μg injection into the shell cavity) and 0.6 (61.15 Mohamed, 2013 μg/snail) LD50 of imidacloprid for 7days Lanistes carinatus - Chlorpyrifos for 28 day Decrease Khalil et al., 2015 Monodonta lineata Foot muscle in vivo exposure to Cd2+ (0.2-1.4 mg/l) Cu2+ Cd2+no effect, Cu2+ no effect Cunha et al., 2007 (0.01-0.6 mg/l) Nucella lapillus Foot muscle in vivo exposure to Cd2+ (0.2-1.4 mg/l) Cu2+ Cd2+ increase, Cu2+ no effect Cunha et al., 2007 (0.01-0.6 mg/l) Oncomelania hupensis cephalopodium and liver Exposure to four extracts of Arisaema Che decrease Ke et al., 2008 erubescens tubers by acetic acetal (AAE), benzinum (BZE), n-butanol (NBE) and

chloroform (CFE) were Physa acuta - 96h of exposure to 3.4, 9.6, 19.2, or 27.4μg/l of Decrease Ma et al., 2014 abamectin Pomacea patula - Carbaryl Inhibition Mora et al., 2000 Pomacea patula - Carbamate pesticide Carbaryl for 72 h Planorbarius corneus Soft tissue and gonads 0.4 and 5 µg/L of chlorpyrifos for 14 days Che decrease Rivadeneira et al., 2013 Xeropicta derbentina - chlorpyrifos-oxon, dichlorvos, carbaryl and concentration-dependently inhibition Laguerre et al., 2009 carbofuran

2.5. Reactive Oxygen Species (ROS) Oxygen is essential to human life and also toxic at the same time due to formation of free radicals. The production of reactive oxygen species (ROS) is a natural phenomenon, which is triggered by various external factors. Oxidative stress is caused due to imbalance between cell's oxidative defence and the production of excess ROS during the impaired oxidant defence mechanism. ROS can be radical (Superoxide, ˙O ; Hydroxyl, ˙OH; Peroxyl,

˙RO2; Alkoxyl, ˙RO; Hydroperoxyl, ˙HO2) or non radical (Hydrogen₂⁻ peroxide, H2O2; 1 Hypochlorous acid, HOCl; Ozone, O3; Singlet oxygen, O2; Peroxynitrite, ONOO ). Radical

are very reactive that carry one or more unpaired electron, and is important⁻ in lipid peroxidation and DNA damage (Halliwell and Gutteridge, 1999). Oxidative stress occurs when there are excess amount of free radical due to faulty or lower antioxidant defense system in the body. Highly reactive radicals and non radicals are mainly responsible for causing oxidative stress.

Some chlorinated compounds can generate hydrogen radical from H2O2 by direct metal ion-independent reactions (Halliwell and Gutteridge, 2007). Hydroxyl radical can be produced as a result of conjugation of transition metal ions and H2O2 during fenton reaction

and hemolytic fission of O-O bond in H2O induced by UV radiation (Halliwell and Gutteridge, 1999). Hydroxyl radical are strongly reactive and can cause more biological damage than any other ROS. Highly reactive ˙OH can abstract H atom from sugars, purines and pyrimidines in DNA bases and form variety of products (Breen and Murphy, 1995). ˙OH can also react with protein or lipid molecule to form oxidative damage products. ˙OH can be enzymatically metabolized to oxygen and water or converted to extremely reactive hydrogen peroxide. Organic compounds having polyphenolic groups are well known pro-oxidants and accelerate the production of ROS and also increase lipid production (Fukumoto and Mazza, 2000). Lipids when reacted with free radicals can undergo the highly damaging chain reaction of lipid peroxidation (LP) leading to both direct and indirect effects. Free radicals reacts with saturated organic molecules by abstracting a hydrogen atom from a methylene

group (CH2 ), leaving behind an unpaired electron on the carbon atom (• CH). The resultant carbon radical is stabilized by molecular rearrangement to produce a conjugated diene, which then can react with an oxygen molecule to give a lipid peroxyl radical (LOO• ). These radicals can further abstract hydrogen atoms from other lipid molecules to form lipid hydroperoxides (LOOH) and at the same time propagate LPO further. The peroxidation reaction can be terminated by a number of reactions. During the metabolic pathway of oxidation of organic compounds, oxygen undergoes stepwise reduction coupled with

29 generation of ATP during electron transport, leading to production of reactive oxygen species - (ROS). Transfer of one, two or three electron to oxygen result in production of O2 - (superoxide radical), H2O2 (hydrogen peroxide) and OH (Hydroxyl radical) respectively. ROS are product of normal metabolism, and plays an important role in cell signalling and pathogen defence. The superoxide anion can also be produced due to action of xanthin and hypoxanthin oxidase enzyme. It doesn't readily cross cell membrane, although it can pass through anion exchange protein. Superoxide anions are readily converted to oxygen and hydrogen peroxide by the action of superoxide dismutase. However due to imbalance between the production of ROS and biological system’s ability of detoxification, ROS get accumulated into the cell and causes deleterious effects. The ROS formed can react with macromolecules (e.g. lipids, proteins, nucleic acids) and provoke protein denaturation, lipid peroxidation, DNA damage, and others. Hydrogen peroxide can penetrate biological membrane and act as an intermediate in the production of other highly reactive ROS species (e.g. hydroxyl radical). Hydrogen peroxide can be removed from the cell by several enzymes like catalase, glutathione peroxidase and peroxiredoxins.

- - O2 + e → O2 …………………………………………... - - + O2 + e + 2H → H2O2 …………………………………. - + - H2O2 + e + H → OH + H2O ……………………….... - - + OH + e + H → H2O ………………………………….. Fig. 4. Reactions showing the fate of reactive oxygen species

Consequently, cells have evolved with several defence mechanisms to neutralize the damage caused by ROS and their reactive products. Non-enzymatic mechanism involves quenching of ROS by molecules like glutathione, carotenoids or ascorbate. Antioxidant enzyme is the major mechanism in scavenging the free radical produced as a result of various metabolic activities. Oxygen is indispensable for all the aerobic species but the reactive forms of oxygen such as the superoxide (oxygen with an extra electron) can lead to certain disasters. To combat this phenomenon, the body has several mechanisms which keep these reactive species in control as they are also important in several beneficial processes. However there are several processes which are interdependent on each other because they share many metabolites and products. Antioxidant enzyme can be induced under slight oxidative stress but severe oxidative stress can lead to suppression of these enzymes (Xu et al., 2009).

2.6. Oxidative stress biomarkers Antioxidant (enzymatic and non-enzymatic) defences are useful biomarkers of pollutants that generate oxidative stress in marine organisms (Pan et al., 2006). They play an important role in protecting the organism from the harmful effect of free radicals and maintain production and clearance of ROS. Among antioxidant defence, enzymatic (superoxide dismutases, catalases and glutathione-s-transferases) and non-enzymatic (lipid peroxidation processes) activities are widely studied.

2.6.1. Glutathione-S-Transferases (GSTs) GSTs are phase II multifunctional enzyme and play a critical role in conjugation of electrophilic compounds (phase I metabolites) on one hand, and in the defence against oxidative damage and peroxidative products of DNA and lipids (Oost et al., 2003) on the other hand. Thus the GST activity gets highly enhanced on coming in contact with contaminants. The reaction below is a simplified description of this catalytic function of GSTs:

GST GSH + R-X GSR+ H-X

The GSTs facilitate the nucleophilic attack of glutathione on electrophilic substrate by binding glutathione onto the active G-site, and electrophilic substrate onto the active H-site, thus transferring substrate to the immediate vicinity of glutathione (Eaton et al.,1999). At the same time, the SH group of GSH, which participates in the reaction, is activated. In the course of the conjugative reaction, a thioether bond is formed between the cysteine remains of glutathione and the electrophilic substance, and the result usually is a less reactive and a more readily soluble product (Eaton et al., 1999). This reaction between glutathione and the GST catalyzed by electrophilic substrate is considered the first step towards the biosynthesis of mercapturic acids that facilitate the elimination of exogenous substance.

GSTs activity in marine species is greatly induced by the presence of xenobiotic contaminants in the environment. An increase in the concentration of ROS causes oxidative stress which may lead to increase in the GST activity. Therefore, an increase in GST activity shows the high concentration of xenobiotic compounds present in the environment. GSTs have been proposed as a promising biomarker in snails exposed to aquatic pollutants (Li et al., 2008). Induction of GSTs activity has been reported in several studies on gastropods

exposed to variety of pollutants (Table 8). An increase of GSTs activity has been reported in several studies on mussels M. edulis exposed to PAH (Gowland et al., 2002). In an in vitro model, Pennec & Pennec, (2003) observed that cells from the digestive gland of Pecten maximus, incubated with PAHs for 96h, showed significant increases in GST activity up to 48h upon exposure. Silva et al., (2005) observed a significant increase in GST activity in Crassostrea rhizophorae exposed to diesel oil. Correlation of GST activity with PAH tissue content has also been shown for green-lipped mussel, Perna viridis (Cheung et al., 2001). An increase in GST activity was also reported in clam Venerupis philippinarum exposed to phenanthrene (Zhang et al., 2014).

Maria & Bebianno, (2011) has carried out extensive research in antioxidant responses in mussel Mytilus galloprovincialis exposed to mixtures of B(a)P and copper. In their study they reported an increase in GST activity in gill cells but not in digestive gland cell due to B(a)P exposure whereas copper exposure showed no change in GST activity in gill of digestive gland cells. Mixtures of B(a)P and Cu have induced GST activity in both gill as well as digestive gland cell of mussel. Box et al., (2009) showed that the presence of macroalgae Lophocladia lallemandii colonising the bivalve Pinna nobilis induces an increase in GST activity in both gill and digestive gland of the mussel. Cheung et al., (2004) studies the antioxidant responses in the green-lipped mussel, Perna viridis due to B(a)P and Aroclor 1254 exposure. Significant positive relationships with Aroclor 1254, B(a)P and hepatic GST were reported by the author.

GSTs are involved in the metabolic activation and deactivation of PAH metabolites. An increased GST activity was observed in experiments with freshwater snail Bellamya aeruginosa exposed to ethylbenzene for 7 days (Zheng et al., 2013). However, GST activity at higher concentrations (450 and 1,000 µg/L) showed progressive decrease after 14 or 21 day of exposure which may be due to the poisoning effect on GST by extra ROS (Cunha et al., 2007). Ismert et al., (2002) reported no change in GST activity in digestive gland, kidney and mantle cavity forming tissue (MCFT) in snail Helix aspersa exposed to naphthalene. The author reported 1.5 fold decrease in GPx activity in digestive gland in exposed snails.

Exposure to pesticides has shown induction or inhibition of enzyme activities. Copper-based pesticides have been shown to cause decline in GSH content in digestive gland of land snail, Theba pisana (El-Gendy et al., 2009). Rivadeneira et al., (2013) showed no

effects on GST activity in freshwater snail Planorbarius corneus exposed to organophosphate insecticide chlorpyrifos. In another study azinphos-methyl also failed to induced any effect on GST in B. glabrata (Kristoff et al., 2008). In contrast, azinphos-methyl showed no change in GST activity in freshwater gastropod Chilina gibbosa (Bianco et al., 2013). More research in this context is needed to understand the detoxification mechanism of pesticides involving GSTs.

Table 8. Glutathione S-transferase (GST) in gastropod

Glutathione-s-transferase (GST) Organism Organ studied Exposure Biomarker effects References Bellamya purificata gills and digestive glands landfill leachate effluent and BPA Increase Li et al., 2008 Bellamya aeruginosa Hepatopancreas ethylbenzene Increase Zheng et al., 2013 Bellamya aeruginosa Hepatopancreas toxic cyanobacterium (Microcystis Increase Zhu et al., 2011 aeruginosa) and toxic cyanobacterial cells mixed with a non-toxic green alga (Scendesmus quadricauda) Cantareus apertus Digestive gland Carbamate pesticide Carbaryl Increase Leomanni et al., 2015 Chilina gibbosa azinphos-methyl no change Bianco et al., 2013 Eobania vermiculata digestive glands sites contaminated with heavy metals Increase El-Shenawy et al., 2012 Gibbula umbilicalis Whole tissue mercury chloride Increase Cabecinhas et al., 2014 Helix aspersa Digestive gland, kidney and mantle napthalene saturated atmosphere no change Ismert et al., 2002 cavity forming tissue (MCFT)

Helix aspersa Digestive gland Sites contaminated with heavy metals Increase Abdel-Halim et al., 2013 Hexaplex (Murex) Digestive gland, gill, muscle cadmium, copper, carbofuran and lindane copper induces GST Romeo et al., 2006 trunculus activity, decrease in GST in snails exposed to cadmium, carbofuran and to mixture of cadmium and carbofuran, GST activity decreases in lindane exposed snails Helix aspersa digestive glands imidacloprid Increase Radwan & Mohamed, 2013 Lymnaea luteola L Hepatopancreas single walled carbon nanotubes Decreases Ali et al 2015 Lymnaea luteola digestive gland zinc oxide nanoparticles ZnONPs Decrease Ali et al., 2012 Lymnea luteola Hepatopancrease gland single walled carbon nanotubes Decrease Ali et al., 2014 Lymnaea stagnalis Digestive gland Cytosolic as well as Wilbrink et al., 1991 microsomal GST activity of the digestive gland of Lymnaea siagnalis has been examined Monodonta lineata Gill Cd2+, Cu2+ Cd2+ no effect, Cu2+ no Cunha et al., 2007 effect Nucella lapillus Gill Cd2+, Cu2+ Cd2+ no effect, Cu2+ Cunha et al., 2007 decrease Physa acuta Abamectin increases at earlier exposure Ma et al., 2014 and then decline Physa acuta imidazolium ionic liquids (ILs) Decrease Ma et al., 2014 Planorbarius corneus Soft tissue and gonads chlorpyrifos no effect Rivadeneira et al., 2013 Theba pisana digestive gland Sites contaminated with heavy metals Increase Radwan et al., 2010 Theba pisana digestive gland copper (Cu), lead (Pb), and zinc (Zn) Cu decrease, Pb increase, Zn Radwan et al., 2010b increase and then decreases Lanistes carinatus Chlorpyrifos for 28 days increase till 21 day and then Khalil et al., 2015 Decrease Theba pisana digestive gland copper-based pesticides; copper Increase El-Gendy et al., 2009 oxychloride, copper hydroxide and copper sulphate

Few studies have also reported depressed GST activity in gills of mussels by exposure to high levels of B[a]P (Akcha et al., 2000). The author also reported depressed GST activity in gills of mussels by exposure to high levels of B[a]P. Xu et al., (2009) has reported unaltered activity of GST in tilapia fish exposed to phenanthrene. Li et al., (2008) has investigated the effects of landfill leachate effluent and bisphenol A in Bellamya purificata and reported decrease in the GSH content and increase in the GST content in treated snails. Ma et al., (2014) has studied the toxic effects of abamectin (ABM), in Physa acuta and reported promotion of GST activity at the earlier periods of treatment (12-48h) in exposed snails, and inhibition at the end of test. Accumulations of heavy metal in shells and tissues of Lymnaea luteola exposed to contaminated sediments have been studied by Siwela et al., (2010). The author reported significantly higher concentrations of Pb, Cd, Zn and Ni in tissues and Zn, Cu and Cd in shells of exposed snails. An increase in GST activity in Helix aspersa were observed in the metal contaminated sites then in reference sites (Abdel-Halim et al., 2013). H. trunculus has been shown to accumulate heavy metals like cadmium (Dallinger et al., 1989), copper (Romeo et al., 2006), mercury (Catsiki and Arnoux, 1987). Increased activity of GST with increasing Hg concentrations was observed in sea snail Gibbula umbilicalis (Cabecinhas et al., 2014). Some studies have shown conflicting results with unaltered or lower GST activities in snails exposed to heavy metals. In vivo exposure to cadmium chloride showed no significant change on GST activity snails (Monodonta lineata and Nucella lapillus) (Cunha et al., 2007). The author reported decrease in GST activity when Nucella lapillus was exposed to copper sulphate pentahydrate. Snail H. trunculus has shown decrease in GST activity when exposed to cadmium. Decrease in GST activity after exposure to heavy metals can be due to direct action of metals on the enzyme or inhibition of GST by extra ROS (Shumilla et al., 1998). GSH tend to chelate with heavy metal before the heavy metal react with metallothioneins, which can also indirectly cause decrease in the GST activity (Freedman et al., 1989). Metals can also cause depletion of the GSH substrate by binding or oxidizing it. Lower reduced glutathione (GSH) content was observed in snail Theba pisana collected from sites polluted with heavy metals (Radwan et al., 2010a). Negative correlation of GSH and heavy metal concentrations in digestive gland has been reported by the author. Various factors can also influence the down regulation of the GST gene (Roling and Baldwin, 2006). Ali et al., (2012) have also reported decrease in GST activity in digestive glands of Lymnaea luteola exposed to zinc oxide nanoparticles. Seasonal variation in GST activity has also been reported in snails. Highest activity of GST was observed in summer close to sites polluted

with heavy metal in Helix aspersa (Larba and Soltani, 2014). Oxidative stress responses were correlated with increasing metal concentrations in soil samples in Helix aspersa (Larba and Soltani, 2014). Romeo et al., (2006) has investigated the effect of toxic metals (cadmium and copper) along with two organics (carbofuran and lindane) to snail H. trunulus. The author has observed that the digestive gland and the gill cells have been shown to accumulate more metals than muscle. This observation indicates that GSTs are a suitable biomarker in snails exposed to heavy metals.

2.6.2. Catalase Catalase plays an important role in enzymatic oxidant defence by protecting the cell from hydrogen peroxide by converting them into oxygen and water. Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second.

The reaction of catalase in the decomposition of hydrogen peroxide is:

2H2O2 2H2O + O2

Catalase performs ‘reshuffling’ of toxic compounds. In the peroxidative reaction, Catalase oxidises different toxins such as formaldehyde, formic acid, phenols and alcohol. In doing so, it uses hydrogen peroxide according to the following reaction:

2H2O2 + H2R 2H2O + R

Hydrogen peroxide is a harmful by-product of many normal metabolic processes. To prevent damage, it must be quickly converted into other, less dangerous substances. To this end, Catalase is frequently used by cells to rapidly catalyse the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules. Table 9 shows the studies involving catalase activity in snails from different parts of the world. Niyogi et al., (2001) has also reported positive correlation of CAT activity with PAH tissue content in oyster S. cucullata. Such a relationship has also been reported for different bivalve species exposed to hydrocarbons that suggest that oxidative stress may be induced by hydrocarbons. Bellamya aeruginosa (Reeve), exposed to ethylbenzene (5-1,000 µg/L) for 21 days has reported an increase in CAT activity (Zheng et al., 2013). Increase in CAT activity were also reported in snails exposed to imidacloprid (Radwan & Mohamed,

2013), zinc oxide (Ali et al., 2012), carbon nanotubes (Ali et al., 2014), abamectin (Ma et al., 2014), and imidazolium ionic liquids (Ma et al., 2014).

Exposure to metal increases the production of ROS including H2O2 which in turn induces the CAT activity in the body. Increased activity of CAT was shown in Helix aspersa exposed to metal dust containing Cu, Zn, Pb, Cr, Ni and Fe (Nedjoud et al., 2009). Snail H. trunculus when exposed to cadmium, carbofuran, and lindane has shown increase in CAT activity (Romeo et al., 2006). Radwan et al., (2010b) has reported increased level of CAT in

Theba pisana exposed to copper (Cu), lead (Pb), and zinc (Zn). CdCl2 and ZnSO4 have also shown to induce CAT activity in Achatina fulica (Chandran et al., 2005). Chronic exposure to Cu-spiked sediment has shown to induce CAT activity in Bellamya aeruginosa (Ma et al., 2010). However there was no significant increase in CAT activity in Gibbula umbilicalis when exposed to mercury for 96h (Cabecinhas et al., 2014). There are also reports that CAT activity decreases in the digestive gland of the Helix aspera exposed to Ni (Zawisza-Raszka et al., 2010). Evidences from laboratory exposure of pesticides and insecticides to snails have shown conflicting results. Significant increase in CAT activity was reported in A. fulica exposed to 40, 69, and 118 mg/kg of triclosan but high concentrations of 200 and 340 mg /kg showed inhibition in the enzyme activity (Wang et al., 2014). In Chilina gibbosa, significant increase in CAT activity was reported in snails exposed to azinphos-methyl (Bianco et al., 2013). In another study el-Wakil et al., (1991) reported significant increase in CAT activity in Eubania vermiculata exposed to methomyl and thiodicarb whereas a decrease in CAT activity was observed when the snails were exposed to metaldehyde. Theba pisana exposed to sublethal doses (40% and 80% of LD50) after 48h of copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate have shown increase in CAT activity (El- Gendy et al., 2009). In another study, Khalil et al., (2015) reported increase in CAT activity in Lanistes carinatus exposed to Chlorpyrifos till 21 day and then a decline in enzyme activity was observed. Biomphalaria alexandrina exposed to Atrazine and Roundup (glyphosate) has shown decrease in CAT activity (Barky et al., 2012). In another study Cochon et al., (2007) has reported no significant change in Biomphalaria glabrata exposed for either 4 or 48 h to 0.5mg/l of paraquat.

Table 9 Catalase (CAT) activity in gastropods Catalase Organism Organ studied Exposure Biomarker effects References Achatina fulica kidneys and digestive CdCl2 and ZnSO4 decrease at both exposure Chandran et al., 2005 gland Biomphalaria glabrata whole body soft tissue azinphos- methyl decrease in pigmented snail whereas non- Kristoff et al., 2008 pigmented snails are unaffected Biomphalaria arabica Whole tissue plant molluscicide Solanum nigrum Increase Al-Daihan et al., 2010 Biomphalaria alexandrina Soft tissue Atrazine and Roundup Decrease Barky et al., 2012 Biomphalaria glabrata Whole tissue paraquat no significant change Cochon et al., 2007 Bellamya aeruginosa Hepatopancreas Cu-spiked sediment Increase Ma et al., 2010 Bellamya aeruginosa Hepatopancreas ethylbenzene Increase Zheng et al., 2013 Cantareus apertus Digestive gland Carbamate pesticide Carbaryl Increase Leomanni et al., 2015 Chilina gibbosa azinphos-methyl Increase Bianco et al., 2013 Eubania vermiculata methomyl and thiodicarb Increase el-Wakil et al., 1991 Eobania vermiculata digestive glands Sites contaminated with heavy Increase El-Shenawy et al., 2012 metals Gibbula umbilicalis mercury chloride no significant change Cabecinhas et al., 2014 Hexaplex (Murex) Digestive gland, gill, Cadmium, copper, carbofuran and increase in enzyme activity is noted in the Romeo et al., 2006 trunculus muscle lindane muscle of H. trunculus exposed to cadmium, carbofuran and to the mixture of cadmium and carbofuran and in the digestive gland of animals exposed to lindane, whereas copper increases CAT activities in the digestive gland of exposed gastropods Helix aspersa digestive glands imidacloprid Increase Radwan & Mohamed, 2013 Helix aspersa Digestive gland Sites contaminated with heavy Increase Abdel-Halim et al., 2013 metals Lymnaea natalensis Whole tissue sediment and water from metal Increase Siwela et al., 2010 polluted sites Lymnea luteola Hepatopancrease gland single walled carbon nanotubes Increase Ali et al., 2014 Lymnaea luteola digestive gland zinc oxide nanoparticles ZnONPs Increase Ali et al., 2012 Onchidium struma hepatopancreas and Cu2+ increase in hepatopancrease, however in Li et al., 2009 muscle muscle, activity decreases and then increases Physa acuta Soft tissues Abamectin Increase Ma et al., 2014 Physa acuta Viscera imidazolium ionic liquids (ILs) Increase Ma et al., 2014

Theba pisana digestive gland Sites contaminated with heavy Increase Radwan et al., 2010 metals Theba pisana digestive gland [copper (Cu), lead (Pb), and zinc Increases Radwan et al., 2010b (Zn) Theba pisana digestive gland copper-based pesticides; copper Increase El-Gendy et al., 2009 oxychloride, copper hydroxide and copper sulphate Lanistes carinatus Chlorpyrifos for 28 days Increase till 21 day and then decrease Khalil et al., 2015

2.6.3. Superoxide dismutase (SOD) - The substrate of superoxide dismutases (SOD) is the superoxide radical anion (O2 ) which is generated by the transfer of one electron to molecular oxygen. It is responsible both for direct damage of biological macromolecules (such as proteins and DNA for example) and for generating other reactive oxygen species. SOD keeps the concentration of superoxide radicals in low limits and therefore plays an important role in the defence against oxidative stress . The chemical reaction that SOD very efficiently catalyses is the dismutation of two - molecules of O2 to yield one molecules of molecular oxygen and one molecule of peroxide: - + Reaction catalyzed by SOD: 2 O2 + 2 H → O2 + H2O2

- Substrate: O2 - superoxide anion (highly unstable in any form, for example: KO2)

- must be generated in situ

Products: H2O2 - hydrogen peroxide

O2 - dioxygen

- Indirect generation of O2 : Oxidation of epinephrine as shown below:

Fig. 5. reaction catalyzed by superoxide dismutase

- The O2 substrate for SOD is generated indirectly in the oxidation of epinephrine at - alkaline pH by the action of oxygen on epinephrine. As O2 builds in the solution, the - formation of adenochrome accelerates because O2 also reacts with epinephrine to form adrenochrome. Toward the end of the reaction, when the epinephrine is consumed, the adenochrome formation slows down. If observed for long time, the adrenochrome disappears and brown, insoluble products form in the solution. (These brown products are closely related

to the brown pigments in our skin where adrenochrome is coverted into melanin and in the cut open fruit where it is converted to quinone derivative) (Greens et al., 1986). SOD reacts - with the O2 formed during the epinephrine oxidation and therefore slows down the rate of formation of the adenochrome as well as the amount that is formed. Because of this slowing process, SOD is said to inhibit the oxidation of epinephrine. The percent inhibition (%I) is hyperbolic with respect to the SOD concentration. This is contrary to the behaviour of other enzymes, where a function related to their enzymatic activity is a linear function of the enzyme concentration. Exposure to heavy metal results in increased production of ROS, which is controlled by enzymatic and non-enzymatic defence in the cell. GSH act as a quencher of O2*, decrease in GSH level can lead to production of high amount of O2*, which in turn can inhibit SOD activity in the cell. Table 10 shows the studies involving SOD activity in snails under different exposure condition. Inhibition of SOD activity was reported in A. fulica exposed to

CdCl2 and ZnSO4 (Chandran et al., 2005). In snail (Lymnaea natalensis), exposed to sediment and water from metal polluted sites, decrease in SOD was observed (Siwela et al., 2010). In contrast to the above studies, significant increase in SOD activity in Hepatopancrease of Onchidium struma was observed after one week exposure to Cu2+ (range 1.35 to 4.20 mg/L), however in the muscle the increase in SOD activity was not consistent. Cadmium has shown to decrease SOD activity in snail A. fulica (Chandran et al., 2005). Exposure to triclosan resulted in significant increase in SOD activity in A. fulica (Wang et al., 2014). Similar results were also reported in the digestive gland of Cantareus apertus exposed to carbamate pesticide carbaryl (Leomanni et al., 2015). Barky et al., (2012) found exposure to pesticides Atrazine and Roundup (glyphosate) tends to decrease SOD in Biomphalaria alexandrina. Cochon et al., (2007) also observed SOD activity was significantly decreased (27%) in B. glabrata snails after 48 h of treatment with paraquat. A decrease in SOD activity in pigmented Biomphalaria glabrata was observed, after exposure to 5 mg/l azinphos- methyl for 48 or 96 h, however in the non pigmented exposed snails the enzyme activity was found to be biphasic. Bianco et al., (2013) reported no change in SOD activity in Chilina gibbosa exposed to exposure to azinphos-methyl (0.2-20 μg/l). Ke et al., (2008) observed an increase in SOD activity in cephalopodium and liver in Oncomelania hupensis exposed to four extracts of Arisaema erubescens tubers by acetic acetal (AAE), benzinum (BZE), n-butanol (NBE) and chloroform (CFE). The snail Physa acuta exposed to oxicities of imidazolium ionic liquids (ILs) showed decrease in SOD

42 activity (Ma et al., 2014). It has been observed that SOD is inactivated by dismutation product, H2O2 over time (Rhodes, 2000)

Table 10 Superoxide dismutase (SOD) in gastropods Superoxide dismutase (SOD) Organism Organ studied Exposure Biomarker effects References Achatina fulica kidneys and CdCl2 and ZnSO4 decrease at both Chandran et digestive gland exposure al., 2005 Achatina fulica viscera (digestive Triclosan Increases Wang et al., gland) 2014 Biomphalaria whole body soft azinphos- methyl decrease in pigmented Kristoff et al., glabrata tissue snails whereas non- 2008 pigmented snails showed biphasic effect Biomphalaria Whole tissue and Schistosoma mansoni susceptible lower than Mahmoud & alexandrina hemolymph resistant in hemolymph Rizk, 2004 and tissue Biomphalaria Soft tissue Atrazine and Roundup Decrease Barky et al., alexandrina (glyphosate) 2012 Biomphalaria Whole tissue paraquat Decrease Cochon et al., glabrata 2007 Bellamya hepatopancreas ethylbenzene increase Zheng et al., aeruginosa 2013 Bellamya hepatopancreas Cu-spiked sediment Increase Ma et al., 2010 aeruginosa Cantareus Digestive gland Carbamate pesticide Increase Leomanni et apertus Carbaryl al., 2015 Chilina gibbosa azinphos-methyl no change Bianco et al., 2013 Gibbula mercury no significant change Cabecinhas et umbilicalis al., 2014 Lymnaea Haemocyte particulate agents (latex, phagocytic stimulation Dikkeboom et stagnalis Escherichia coli, of the haemocytes al., 1987 Staphylococcus resulted in a superoxide saprophyticus, zymosan dismutase induction and with phorbol myristate acetate

Lymnaea Whole tissue sediment and water from Decrease natalensis metal polluted sites Siwela et al., 2010 Onchidium hepatopancreas Cu2+ increases in Li et al., 2009 struma and muscle hepatopancrease, muscle decreases and then increases Oncomlania Liver tissue Arisaema erubescens and Increase Zhang et al., hupensis Nerium indicum extracts 2009 Oncomelania Liver sanguinarine (50 Increase Sun et al., 2011 hupensis Oncomelania hupensis per 500 ml solution) Oncomelania cephalopodium extracts of Arisaema Increase Ke et al., 2008 hupensis and liver erubescens tubers by acetic acetal (AAE), benzinum (BZE), n- butanol (NBE) and chloroform (CFE) were Physa acuta abamectin increases at earlier Ma et al., 2014 exposure and later declines Physa acuta imidazolium ionic liquids Decrease Ma et al., 2014 (ILs)

2.6.4. Lipid Peroxidation (LPO) Lipid peroxidation has been studied extensively in environmental monitoring programs. Malondialdehyde (MDA) is a low molecular weight end product of lipid peroxidation and is widely used as a biomarker of oxidative stress. Lipid Peroxidation is a process of oxidative degradation of lipids. In this process free radicals steal electrons from the lipids present in the cell membranes, hence damaging the cells. This process proceeds by a free radical chain reaction mechanism. It consists of three main steps. First is the initiation in which a lipid radical is produced (ROS is the initiator).The lipid radical produced is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl- fatty acid radical which is again an unstable species that reacts with another free fatty acid, producing a different fatty acid radical and a lipid peroxide ,or a cyclic peroxide if it had reacted with itself. This cycle continues, as the new fatty acid radical reacts in the same way. Termination occurs when two free radicals combine to form a non-radical species. The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism.

Fig. 6. Mechanism of lipid peroxidation

Increased level of oxidative damage in terms of lipid peroxidation has been reported in various snail species exposed to laboratory or environmental contaminants (Al-Daihan et al., 2010; Barky et al., 2012; El-Shenawy et al., 2012; Ali et al., 2014; Ma et al., 2014).

Table 11 Lipid Peroxidation (LPO) in gastropods Lipid Peroxidation (LPO) Organism Organ studied Exposure Biomarker References effects Achatina fulica viscera (digestive Triclosan dose dependent Wang et al., gland) increase 2014 Achatina fulica kidneys and CdCl2 and ZnSO4 increase at both Chandran et digestive gland exposure al., 2005 Biomphalaria Whole tissue paraquat Increase Cochon et al., glabrata 2007 Biomphalaria Whole tissue molluscicide Solanum nigrum Increase Al-Daihan et arabica al., 2010 Biomphalaria Soft tissue Exposure to pesticides Atrazine Increase Barky et al., alexandrina and Roundup (glyphosate) 2012 Bellamya Hepatopancreas ethylbenzene no change Zheng et al., aeruginosa 2013 Eobania digestive glands sites contaminated with heavy Increase El-Shenawy vermiculata metals et al., 2012 Gibbula mercury no significant Cabecinhas et umbilicalis change al., 2014 Helix aspersa extremely low frequency (ELF) Increase Regoli et al., 50-Hz magnetic fields 2005 Helix aspersa Digestive gland Sites contaminated with heavy Increase Abdel-Halim metals et al., 2013 Lymnea luteola Hepatopancrease single walled carbon nanotubes Increase Ali et al., gland 2014 Lymnaea Whole tissue sediment and water from metal Increase Siwela et al., natalensis polluted sites 2010

Lymnaea luteola Hepatopancreas carbon nanotubes Increase Ali et al 2015 L Lymnaea luteola digestive gland zinc oxide nanoparticles Increase Ali et al., 2012 Lanistes Chlorpyrifos Increase Khalil et al., carinatus 2015 Oncomelania Liver sanguinarine (50 Oncomelania increased but not Sun et al., hupensis hupensis per 500 ml solution) significant 2011 Physa acuta Soft tissue Abamectin Increase Ma et al., 2014 Physa acuta Viscera oxicities of imidazolium ionic Increase Ma et al., liquids (ILs) 2014 Theba pisana digestive gland Sites contaminated with heavy Increase Radwan et metals al., 2010a Theba pisana digestive gland copper (Cu), lead (Pb), and zinc increases for all Radwan et (Zn) exposures al., 2010b Theba pisana digestive gland copper-based pesticides; copper Increase El-Gendy et oxychloride, copper hydroxide al., 2009 and copper sulphate

A widely used pesticides paraquat has shown a significant increase in LPO in Biomphalaria glabrata (Cochon et al., 2007, Table 11). In another study Barky et al., (2012) reported induction of LPO in Biomphalaria alexandrina exposed to Atrazine and Roundup pesticides. Lanistes carinatus when exposed to Chlorpyrifos for 28 day has also shown induction of LPO (Khalil et al., 2015). Carbon nanotubes have shown to induce LPO in Lymnaea luteola (Ali et al., 2015). Dose dependent increase in LPO was reported in digestive gland of Achatina fulica exposed to broad-spectrum antimicrobial agent, triclosan (Wang et al., 2014). Some studies have shown no increase in LPO with exposure to contaminants (Zheng et al., 2013). In the field, sites contaminated with heavy metal have shown to induce LPO in snails (Radwan et al., 2010a; Abdel-Halim et al., 2013). Cu, Pb, and Zn have also shown to increase LPO in Theba pisana (Radwan et al., 2010b). Increased lipid peroxidation occurred in Lymnaea natalensis exposed to sediment and water from metal polluted sites (Siwela et al., 2010). Higher values of LPO were reported in digestive glands of Helix aspersa collected from metal contaminated sites by Abdel-Halim et al., (2013). Elevated level of LPO was reported in kidneys and digestive gland in Achatina fulica exposed to CdCl2 and ZnSO4 (Chandran et al., 2005). Cadmium doesn't generate ROS directly, but can alter the GSH and MT level in the cell, which can lead to LPO of cell membrane (Mahboob et al., 2013). It has been known that LPO products are complex hydroperoxides that exerts cytotoxic and genotoxic damage. It has been demonstrated that CAT and SOD inhibit lipid peroxidation (Packer & Fuchs, 1992). Exposure of contaminants results in increase production of hydrogen

46 peroxide which is destroyed by CAT. Increase activity of CAT under these conditions can effectively counteract LPO. Increase in LPO was reported in oysters exposed to Cu but, Cu in combination with GSH inhibitor (buthionone sulfoximine) has shown lower LPO values, suggesting that GSH has protective role to Cu toxicity (Conners & Ringwood, 2000). Collectively these data suggests that LPO along with other oxidative stress biomarkers such as GST, CAT, and SOD can help in better understanding of the oxidative stress phenomenon.

2.7. Integrated Biomarkers Response (IBR) Biomarkers are an important tool to detect exposure and adverse effects of human- made or natural contaminants on aquatic organisms. Some biomarkers are specific to chemicals or group of chemicals while other is non-specific and induce upon exposure to broad range of pollutants. Several authors have reported higher values of IBR in contaminated sites as compared to the reference site (Tankoua et al., 2013; Turja et al., 2014). IBR has been applied to study the spatial and temporal variations in biomarkers in aquatic organisms (Guerlet et al., 2010; Trujillo-Jiménez et al., 2011; Tlili et al., 2013). IBR has been used to construct star plot with PAH and PCB concentrations in tissues along with early warning signals (EROD, GST, CAT, AChE enzymatic activities) and adverse effects (DNA adducts) biomarkers in flounder Platichthys flesus (Beliaeff and Burgeot, 2002). IBR index was successfully used to evaluate the toxicological effects of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) on Cyprinus carpio (Kim et al., 2010). Rodrigo et al., (2013) used IBR to combine oxidative stress biomarkers in digestive gland and gills of common cuttlefish, Sepia officinalis collected from three sites near Portugal. The IBR approach using AChE, GST, CAT, LPO and several other biomarker was used to the exposure to dimethoate in isopod Porcellionides pruinosus (Ferreira et al., 2014). Campillo et al., (2013) have used AChE, GST, CAT, and LPO in caged clam Ruditapes decussatus for IBR. AChE, LPO and DNA damage was also used to compute IBR values to assess anthropogenic contamination in clam (Ruditapes decussatus) (Cravo et al., 2012). Comet assay along with four other biomarkers AChE, CAT, vitellogenin (VTG) concentration, and the activities of 7-ethoxyresorufin-O-deethylase (EROD) were used to study the responses in common carp (Cyprinus carpio) exposed to perfluorinated organic compounds (Kim et al., 2010). IBR index has been used to study the PAH contamination in caged mussel Mytilus trossulus, Mytilus galloprovincialis (Tsangaris et al., 2011; Dabrowska et al., 2013). Vega- López et al., (2013) has investigated the relation of oxidative stress and antioxidant defences

in phytoplankton with heavy metal. The author stated that oxidative damage was related with PAH (benzo[b]fluoranthene) using IBR. PAH gradient in Mytilus galloprovincialis did not seem to contribute to the IBR demonstrating that the chosen biomarkers AChE, GST and CAT activities, TBARS and MT didn't respond to PAH (Damiens et al., 2007). These results suggest that integration of genotoxic and biochemical biomarker can serve as a useful tool in environmental monitoring programs.

CHAPTER 3 Materials And Methods

3.1. Sampling locations

The state of Goa is one of the famous tourist destinations around the world. It is also the major iron and manganese ore producers in India. Extensive tourism and mining activities has lead to rapid increase in environmental pollutants in and around Goa region. Mandovi and Zuari are two most important estuaries in Goa, which are considered as lifeline of Goa's economy. The main cause of pollution of the sampling sites is mainly due to indiscriminate dumping of waste materials as well as discharge of contaminated water from the surrounding restaurants and shacks directly into the coastal water. Moreover, some of the beaches are exposed to oil spills released from various types of shipping activities such as fishing trawlers, tourist boats, water scooters etc. around these regions. Grounding of ships near the beach for longer time might also contribute substantially towards coastal pollution of the ecosystem with the oil spills released from the grounded ship (Ingole et al, 2006).

Fig. 7. Sampling locations (Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Hollant, Velsao, Betul and Palolem) along the Goa coast. Source: Google Map

Arambol Among the sampling sites, Arambol is one of the most wonderful beaches in the northern tip of Goa along the West coast of India. It is a rocky as well as sandy beach situated in the Pernem Taluka of Goa. Beyond idyllic, rocky-bottom cove, the trail emerges to a broad strip of soft, golden sand hemmed on both sides by steep cliffs. It was chosen as the reference site because of its serene environment.

Fig. 7.1. View of the sampling site taken at Arambol, North Goa (Lat 15°41'33.24"N, Long 73°41'54.28"E)

Anjuna Anjuna is located 8 km west of the city of Mapusa and is very popular for its flea market and beach parties. Large numbers of shacks are situated along the beach which attracts huge number of tourist from all over the world. The number of shacks has increased in last few years due to rapid increase in the number of tourists. In recent years, there were many reports of sewage pollution in and around Anjuna (TOI, 2015).

Fig. 7.2. Sampling location situated at Anjuna, North Goa (Lat 15°33'56.02"N, Long 73°44'30.95"E)

Sinquerim Sinquerim, located 13 km from Panaji, is one of the best beaches in Goa, where one can enjoy water-skiing, para-sailing, fishing, scuba diving and windsurfing. Sinquerim beach attracts plenty of tourist because of the famous Aguada fort. Large number of hotels, restaurants and resorts are situated in the close proximity of Sinquerim beach.

Fig. 7.3. Sampling location near the Sinquerim Fort, North Goa (Lat 15°29'52.00"N, Long 73°45'55.70")

Dona Paula Dona Paula is one of the most popular and most crowded tourist spot in Goa situated just opposite to Mormugao harbour, an open sea port extensively used for various shipping activities such as cargo ships, passenger ships from different parts of the world, fishing trawlers, barges carrying iron ores from the mines and tourist boats etc. Thus the extent of pollution around Dona Paula is basically contributed by the oil spills from the surrounding region.

Fig. 7.4. Sampling site situated near the Dona Paula Jetty (Lat 15°27'5.36", Long 73°48'11.07")

Bogmalo Bogmalo beach is situated at about 3 km from the airport and 8 km away from Vasco with beautiful green hills on its three sides and lined with coconut trees. It is a broad, open and flat beach which is known to house luxury five star resorts. Bogmalo in Goa is known for its water sport activities and its scenic beach. Bogmalo has transformed from a quaint fishing village to a tourist hotspot and attracts a large number of visitors. Bogmalo is an ideal base for exploring the whole state because of its location. The beach in Bogmalo is the prime attraction. Situated in a small bay, Bogmalo boasts of around a mile of curving sandy beach.

Fig. 7.5. Sampling site at Bogmalo beach, South Goa

Velsao Velsao beach is very close to the discharge points of industrial effluents and waste materials from the surrounding agrochemical industries like Zauri Agrochemicals. Thus the marine organisms are greatly affected due to their exposure to various types of genotoxic contaminants released through the industrial effluents into the coastal seawater leading to serious damage to the physiological status of the marine organisms.

Fig 7.6. Sampling site on the vicinity of Zuari Agrochemical Ltd. at Velsao (Lat 15°22'15.01"N, Long 73°52'11.43"E)

Hollant The Hollant beach in Goa attracts huge crowds of visitors and tourists. It is known to be the only beach in Goa where one can see a sunrise. Hollant beach is located within a shallow bay that has a gentle slope towards the sea. There are no currents flowing here and the beach is very safe for swimming. Hollant beach is a small beach located to the south of Bogmalo beach. It is located at a distance of 7 kms. from Vasco city. During low tide the beach area substantially increases since the slope of the beach is gradual and shallow. Further into the sea, fishermen often anchor their boats.

Fig 7.7. View of the sampling location situated at Hollant beach, South Goa

Betul Betul beach is also a famous beach of south Goa known for its cheap and fresh seafood supply. It is renowned for Goa's finest and largest mussels, ensnared and hauled to the banks by deft divers. Betul is amazing due to its unique location at the mouth of the River Sal and its varied terrain. The beach attracts lovers of exotic and solitude. Occurrence of mass mortality of bivalves along the sal river has become a great concern (Sulochanan et al., 2014)

Fig. 7.8. Sampling location situated at Betul, South Goa (Lat 15° 8'32.41"N, Long 73°57'16.77"E)

Palolem Palolem is situated in the southern end of Goa. Palolem beach is largely unspoiled and is inhabited by both local fishermen and by foreign tourists who live in shacks along the shore or in the main village. The crescent shaped bay of Palolem Beach is lined with swaying coconut palms.

Fig. 7.9. Sampling site located at Palolem beach, South Goa (Lat 15° 0'37.19"N, Long 74° 0'47.09"E)

Various studies have been conducted to assess the physical, chemical and biological properties of waters from these two estuaries (De Souza et al., 1985; Shirodkar et al., 1985; Chanda et al., 1996; Unnikrishnan et al., 1997; Khandeparker et al., 2011; Menon et al., 2011; Subha Anand et al., 2014). Studies on the heavy metal accumulations in Macrophyte (Vardanyan & Ingole, 2006), oxidative stress in Perna viridis (Jena et al., 2009) and impairment of DNA in marine snails (Bhagat et al., 2012; Sarkar et al., 2015; Sarkar et al., 2014) clearly indicates the impact of pollution at different sampling sites along the Goa coast.

Sarkar et al., (2008) has studied the accumulation of heavy metals in Cronia contracta from different sites along the Goa coast (Table 12). Goa state pollution control board

(GSPCB) has also reported very high level of lead (0.21 mg/l) in water from Mandovi, Panaji (GSPCB, 2009). Organotin pollution in fishes, crustaceans and mollusks from Goa region has been detected (Jadhav et al., 2011). High values of tributyltins, (TBT, 133 ng/g dry wt.) was reported in sediments from Goa by Bhosle et al., (2004). He has also reported high level of TBT in surface water samples from Ore jetty (146±23 ng/l), Fish jetty (146±23 ng/l) in Zuari estuary and Panaji harbour (146±23 ng/l) in Mandovi estuary. Garg et al., (2010) have reported high level of total butyltin (TB) in water samples from Mandovi (12-73 ng Sn/l) and Zuari (0.5-77 ng Sn/l). Krishna Kumari et al., (2006) has reported high level of Cd (3.8 µg/g), Pb (30.3 µg/g), Cu (13.5 µg/g), Zn (36.6 µg/g) and Fe (105.7 µg/g) in P. malabarica from Mandovi estuary, Goa. Decaying matter from bivalves accelerates the growth of pathogenic Pseudomonas bacteria and also causes ammonia poisoning in fishes. Few cases of mass mortality of bivalves have occurred in last few years at various locations of Goa. Reports of imposex in snail Cronia konkanensis and mesogastropod, Gyrineum natator from Mormugao shows the TBT contamination in this region (Vishwakiran et al., 1999, 2006). High numbers of potential pathogenic bacteria reported by Rodrigues et al., (2011), indicates the sewage pollution at Mormugao.

Table 12 Concentration of heavy metals in the muscle tissues of marine snail (Cronia contracta) along the Goa coast. Each data point represents mean of triplicates values (taken from Sarkar et al., 2008)

Locations Pb Cd Cu Fe Mn Mean conc. Mean conc. Mean conc. Mean conc. Mean conc. ± std. dev ± std. dev ± std. dev ± std. dev ± std. dev (µg/g) (µg/g) (µg/g) (µg/g) (µg/g)

Arambol 0.2 ± 0. 04 0.1 ± 0.02 0.2 ± 0.02 0.4 ± 0.02 0.4 ± 0.03 Anjuna 0.1 ± 0.02 0.1 ± 0.02 0.1 ± 0.03 3.2 ± 0.03 1.3 ± 0.03 Dona Paula 0.2 ± 0.04 0.7 ± 0.03 2.6 ± 0.04 1.5 ± 0.03 1.6 ± 0.04 Vasco 0.2 ± 0.03 1.0 ± 0.02 1.6 ± 0.04 2.6 ± 0.05 1.3 ± 0.03 Velsao 0.2 ± 0.02 0.4 ± 0.04 1.1 ± 0.04 1.2 ± 0.06 0.4 ± 0.03 Palolem 0.1 ± 0.03 0.1 ± 0.03 0.1 ± 0.02 1.7 ± 0.05 0.5 ± 0.04

3.2. Marine Gastropods In order to assess the impact of environmental contaminants on the health of the marine ecosystem, a large number of marine gastropods (Nerita chamaeleon, Planaxis sulcatus, and Morula granulata) were collected from selected stations such as Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Hollant, Velsao, Betul and Palolem along the Goa coast.

The usefulness of snails as sentinel organism in metal biomonitoring studies are widely recognized (Downs et al., 2001, Sarkar et al., 2006, 2008, Itziou and Dimitriadis 2011). Gills are constantly exposed to dissolved contaminants and they are capable of metabolizing carcinogens and mutagens into reactive products. They are used as an attractive cellular model in ecotoxicology for assessment of pollution related stress in living organisms (Wilson et al., 1998, Mitchelmore et al., 1998).

Taxonomy about Nerita chamaeleon Kingdom: Animalia Phylum: Mollusca Class: Gastropoda Order: Gastropoda Prosobranchia Neritoida Family: Neritoidea Neritidae

Genus: Nerita Species: chamaeleon Fig. 8 . Nerita chamaeleon

Taxonomy about Planaxis sulcatus Kingdom: Animalia Phylum: Mollusca Class: Gastropoda Order: Neotaenioglossa Family: Planaxidae Genus: Planaxis Species: sulcatus Fig. 9. Planaxis sulcatus

Taxonomy about Morula granulata Kingdom: Animalia Phylum: Mollusca Class: Gastropoda Order: Caenogastropoda Family: Muricidae Rapaninae Genus: Morula Species: granulata Fig. 10. Morula granulata

3.2.1. Nerita chamaeleon

Nerita is among the oldest molluscan names, dating to Linnaeus, 1758 and is a

potential biological monitor for environment monitoring. Nerita is a group of herbivore snails

widely distributed in inter-tidal regions of Goa, India. They are usually found in middle to

upper intertidal zones and are known to be gregarious (Tan & Clement, 2008). Nerita species

has been previously reported from many parts of the world and has been studied for

environmental monitoring programs (Batomalaque et al., 2010; Cob et al., 2012; Sarkar et al.,

2013; Vadher et al., 2014). The shells of N. chamaeleon have numerous smooth spiral ribs

with 2-4 distinct teeth in Columellar edge. The operculum is usually greyish with the surface

covered with granules. Sexes are separate. Male has a penis and a prostate gland. Male

gonoduct is an open, ciliated groove. Female reproductive system is more complicated than

in the male. Fertilization is internal. Egg capsules are deposited in shallow water-filled

depression on the surface of rock or underneath rock which afford some protection from the

sun. Their reproduction cycle requires brackish water to assist with larval stage development.

The females are larger than the males. Their sex can only be determined by keeping them in a

controlled environment for observation. Fertilisation is internal, eggs often develop inside a

special capsule and hatching veligers feed on plankton until metamorphosis.

Kumar and Devi, 1995 has conducted toxicity of heavy metal on N. chamaeleon. Heavy

metal exposure in two species of Nerita (N. chamaeleon and N. albicilla) has been studied by

Kumar, 1990. Toxicity of Nickel ore has been studied in marine gastropod N. chamaeleon

(Florence et al., 1994). Kumar and Devi, (1997) has studied the accumulation of copper and zinc in N. chamaeleon and reported significant relationship between exposure time and concentration of heavy metal in the gastropods.

3.2.2. Planaxis sulcatus Planaxis sulcatus are ubiquitous and usually found in large populations in inter tidal and rocky environments around Indo-Pacific region. In Goa regions, they were found to be one of the most predominant gastropods. They are herbivores and grazes on microalgae and detritus. The shells are dark brown to gray in colour and bears strong squarish spiral cords with about seven inflated whorls. The surface is regularly and uniformly spirally grooved with spirally ridged outer lip. The operculum is thin-horn like material and dark in colour. They are also known for the production of novel cytotoxic cembranoid called planaxool (Alam et al., 1993). Aladaileh et al., (2014) has studied the effect of phosphate ore on P. sulcatus and reported enhanced immunomodulatory activities in snails exposed to polluted sites. During low tides, P. sulcatus withdraws into its shell behind the operculum and attaches itself to the substratum. Thus they can be often seen exposed on rocks, stones and boulders in aggregates or taking shelter in rock pools, crevices and under large rocks during low tides (Houbrick 1987; Rohde 1981).

P. sulcatus is a gonochoristic and oviviparous species of marine gastropods that that rears its embryos in large brood pouches on the female’s body (Houbrick 1987; Ohgaki 1997). Fertilisation is internal via copulation between a male-female pairs during summer (Ohgaki 1997). P. sulcatus were observed to breed in the summer months where pairing behavior (i.e. copulation) between male and female individuals was frequently observed (Ohgaki 1997). Female individuals of P. sulcatus are generally larger than males (Ohgaki 1997). New recruits of P. sulcatus settlers appeared in autumn where it continues to grow and develop till maturity (Ohgaki 1997). P. sulcatus were observed to reach sexual maturity within the first year of settlement (Ohgaki 1997). First year recruits will merge into the population of older-year the following autumn after they settle (Ohgaki 1997).

3.2.3. Morula granulata Morula granulata was found to exist mostly on the oyster (Saccostrea cuccullata) belt spread over the rocky shores along the Goa beaches. It is a predator and feeds on worms, oysters, barnacle, and small molluscs (Chim and Ong, 2012). Koh Siang et al., (2003) studied the feeding behaviour of Morula granulata and observed that the predominant method of attack used by Morula granulata to gain access to oysters was by drilling a hole through their shells. Morula granulata preyed exclusively on vermetid gastropods, oysters and dead

molluscs (Wu, 1965; Taylor, 1978; Kay, 1979); whereas another study showed that they drilled and fed principally upon the mytilid bivalve Modiolus auriculatus and to a lesser extent, on vermetids and cerithiids (Taylor, 1984). Taylor (1990) reported that there is a strong tendency for Morula musiva to drill at or near the margin of the upper (right) valve of Saccostrea.Morula granulata is a drupe that is covered with spirals of rounded bumps on its shell. An adult's have a thickened outer lip that with a continuous dark brown or black margin. They grow to about 15 mm. and adhere to wet rocks at low tide, completely exposed. It can be found among tide pools and on reef flats from depths ranging from the intertidal to five feet. Most abundant on rocky limestone shores with good water movement. Metamorphosis occurs when 31/2 to 33/4 whorls are complete. Embryos develop into planktonic trocophore larvae and later into juvenile veligers before becoming fully grown adults. They can be found clumped together in groups. Sometimes the clumping isn’t due to the need to breed, eat, or for protection. The clumping is positively related to the density of snails that aren’t feeding. (Moran 1985). Marine gastropods were collected from the intertidal rocks scattered around the different sampling sites along the coast of Goa. They were identified using the reference sample certified by the Zoological Survey of India (ZSI), Kolkata, India, and (Subba Rao et al., 1992). Immediately after collection of the marine gastropods they were brought to the laboratory in a plastic container containing sea water from the same location. They were thoroughly washed and preserved with aeration in the seawater from the same sampling site at room temperature to acclimatize them for 48 h under laboratory condition.

3.3. Exposure conditions 3.3.1. In vivo exposure to phenanthrene The collected snails were acclimatized in 4 litres plastic aquaria for 96 h in aerated seawater at room temperature before the beginning of the experiment. Snails were not fed during phenanthrene exposure. After acclimatization period, around 300 gastropods were equally divided in five experimental groups: control group and four treatment groups with different concentrations of phenanthrene (10, 25, 50 and 100 μg/L). Phenanthrene was first diluted in DMSO and then mixed with seawater to make the stock solution (2500 µg/ml). The final concentration of dimethyl sulfoxide (DMSO) in all treatment groups was less than 0.01%. Control gastropods were incubated in seawater containing 0.01% of DMSO. The concentration of phenanthrene used in this experiment was in accordance with studies from

Oliveira et al., (2007) and Xu et al., (2009). The final phenanthrene concentration for treatment groups were achieved by diluting stock solution in seawater.

3.3.2. In vivo exposure to benzo(k)fluoranthene (B[k]F) Exposure Concentration of B[k]F used in this study were in accordance with studies from Pan et al., (2005) and Kim et al., (2014). The collected snails were divided in five experimental groups (roughly 50 snails in each group): control group and treatment group with different concentrations of benzo(k)fluoranthene (1, 10, 25 and 50 μg/L) dissolved in acetone. Snails were exposed to B[k]F for 96 h. Control snails were incubated in seawater containing acetone. One third of seawater was renewed every morning and to maintain the concentrations of B[k]F, seawater containing the same concentrations of B[k]F was added. Snails were not fed during the course of experiment.

3.3.3. In vivo exposure to cadmium Chloride (CdCl2)

The organisms were exposed for 5 days to various concentrations of CdCl2 (10, 25, 50, 75 mg/L, and control) in aerated seawater. Seawater was changed on daily basis and replaced with fresh seawater with respective concentrations of CdCl2. The CdCl2 stock solution was dissolved in ultra pure water (100 mg/L) and stored at 4°C.

3.3.4. In vivo exposure to mercury Chloride (HgCl2)

The HgCl2 stock solution was dissolved in ultra-pure water (100 mg/l) and stored at 4 °C. Working solutions ranged from 10 to 100 mg/L, in accordance with previous studies by

Tran et al., (2007). Snails were exposed to different concentrations of HgCl2 (10, 20, 50, 100 μg/l) for 24, 48, 72, and 96 h. Every 24 h, 1/3 of the total volume of water was changed and

replaced with fresh seawater from the sampling site with respective concentrations of HgCl2. Snails kept in water from Arambol were used as controls.

3.3.5. In vitro exposure of gill cells to hydrogen peroxide (H2O2) To validate the comet assay protocol, freshly dissociated gill cells were treated with hydrogen peroxide (1; 10; 25, 50 µM) in PBS for 30 min. The control cells were incubated in PBS without hydrogen peroxide. Three replicates per condition were performed.

3.4. Alkaline unwinding assay 3.4.1. Isolation and purification of DNA After acclimatization, the marine gastropods were used for isolation of DNA from their tissues. The marine gastropods collected for the studies were mostly of the same size. The DNA integrity was determined with respect to the composite samples of 5 individual species of almost the same size and growth. Five individual species of the gastropods from each of the sampling sites were used for isolation of DNA from their tissues. The shells of the gastropods were gently broken to remove the tissues from the shells and were mixed together homogenously using a spatula in a pre-cooled petri dishe placed over ice flakes inside the icebox during the period of sample processing for preservation of the activity of DNA. The composite mixtures of tissues were then divided into three parts in order to make triplicate samples of tissues for isolation of DNA. About 300-400mg of tissues from the composite mixture of tissues was used for isolation of DNA by treatment with 1ml of 1N NH4OH/0.2% TritonX-100 per 200-400mg of tissue using a dounce homogeniser. After homogenization, 1 ml of triple distilled water was added per 200mg of tissue. The isolation and purification of DNA was accomplished by extraction with CIP [chloroform / isoamyl alcohol / phenol in the ratio of 24:1:25 (v/v)]. The samples were shaken vigorously to fully denature all the proteins and centrifuged at 19,000xG at 4°C for 60min to separate the different phases. The aqueous phase was pipette out and passed through a molecular sieve column (Sephadex G-50) to isolate DNA from RNA. The sample was stored in a capped eppendorf tube at 4°C until further processing.

3.4.2. Measurement of DNA integrity DNA integrity in marine gastropods (such as Morula granulata was determined following the technique of time dependent partial alkaline unwinding assay (Shugart, 1988a, 1988b, Everaarts et al., 1995, Everaarts and Sarkar, 1996). The three parameters measured in this assay were the amount of double-stranded (ds-DNA), single stranded (ss-DNA) and the fraction of double-stranded remaining after alkaline unwinding (au-DNA) for a specified time under defined condition of pH (7.4) and temperature (45oC and 85oC). After isolation of DNA, The fluorescence of dsDNA and ssDNA was measured using a modulus multimode micro plate reader (Turner Biosystems Inc.) with an excitation wavelength of 360 nm and an emission wavelength of 450 nm (Everaarts and Sarkar, 1996; Sarkar et al., 2008).

The DNA integrity is expressed in terms of I-values which are in fact, the ratio of the expression,

Where X, stands for observed fluorescence The same procedure was followed with a standard DNA sample (calf thymus) and the DNA integrity (I-value) was determined for quality control of the methodology.

3.5. Single cell gel electrophoresis 3.5.1. Preparation of Single Cell Suspension For preparation of single cell suspension, the shells of the marine gastropods collected from different sampling locations were gently broken and then the gills tissues were carefully transferred into an eppendorf tube containing 1 ml of cold extrusion buffer (71.2 mM NaCl, 5 mM EGTA, 50.4 mM guaiacol glycerol ether, pH 7.5). The tissues were slightly chopped and the suspension was then left for 2-3 minutes on ice. The supernatant were then centrifuged at 5000 rpm for 3 minutes using a mini centrifuge. The pellets thus produced were washed thrice with Phosphate Buffer Saline (PBS, 1.2 M NaCl, 0.027 M KCl, 11.5 mM K2HPO4,

0.08 M Na2HPO4, pH 7.3) and suspended in 100µl of PBS. The cellular suspension was kept on ice to minimize endogenous damage occurring during slide preparation. The trypan blue exclusion test was used for viability assessment.

3.5.2. Comet Assay The comet assay was performed according to the method described by Singh, et al., (1988) with minor modifications. Slides were first dipped in methanol and burnt over a blue flame to remove the machine oil and dust. It was then dipped up to one-third in hot 1% Normal Melting Agarose (NMA, 10 mg/ml in Milli Q water) at the frosted area. Slides were then carefully removed and underside of it was gently wiped out and laid in a tray on a flat surface to dry. 100µl of 0.55% Low Melting Agarose (LMA, 5.5 mg/ml in PBS) mixed with 20µl of diluted cells was poured onto the coated slide kept in ice packs. Once the agarose layer hardens, 100µl of 0.5% LMA (50 mg per 10ml Tris Buffer) was poured on it and allowed it to solidify. It was then kept on freshly prepared lysis buffer (2.5 M NaCl, 0.1 M di- sodium EDTA, 0.01 M Tris Buffer, 0.2 M NaOH, pH 10.0) at 4°C in dark for 1 hour.

The slides are then removed and placed on the horizontal gel box. It was left for unwinding in the electrophoretic buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) for 15 minutes. The electrophoresis was carried out in the same buffer at 20 V (cc. 1V/cm), 300 mA. The current and voltage was made constant throughout the electrophoresis by changing the buffer level. The slides were then neutralized drop wise using neutralizing buffer (0.4 M Tris, pH 7.5) for four times at an interval of 5 minutes each. It was then kept in cold 100% methanol overnight for dehydration. Slides were drained off the excess methanol and then 100µl of ethidium bromide (20 µg/ml) was added. Excess stain was then gently washed off and overlaid with a cover slip. It was placed in a humid, dark box at 4°C until analysis (within 24 h). The whole procedure was carried out in yellow light to reduce DNA damage while preparing the slides.

3.5.3. Optimization of the comet assay for Morula granulata The marine gastropods used for optimization of the comet assay were from the reference site (Arambol). Comet assay was optimized for measurement of the extent of DNA damage in marine gastropod (Morula granulata) in terms of lysis time, electrophoresis and unwinding condition. Slides were incubated for a period of 0.5 hr, 1 hr and 1.5 hr in lysis buffer. It was observed that 1hr of lysis at 4°C in dark and electrophoresis at 20V (1V/cm) at 300mA was appropriate for DNA to form an appropriate comet. Time for unwinding was optimized at 15 minutes in the same electrophoretic buffer. Factors such as the lysis, electrophoresis conditions and initial preparation of the single-cell solutions can greatly affect the sensitivity of the method. In order to minimize the variations between experiments during our study we kept the lysis and electrophoresis conditions constant.

3.5.4. Comet Capture and Image analysis The presence of comets was- examined in cells using a DM100 Leica fluorescent microscope (40 X magnification). Cells were analyzed and scored using an image analysis package Komet 6.0 (Kinetic Imaging, Liverpool, UK). Image analysis using Komet 6.0 was preferred over the visual grouping of comet as the use of specific image analysis software is considered to be reliable, reproducible, which provide simultaneously a range of parameters and additional information (e.g. the distribution of DNA within the comet tail, total cellular DNA content) (Kumarvel et al., 2009). Human blood was used for measurement of comet control for evaluation of the variation of the comets of the cells from Morula granulata. All slides were coded and the whole slide was randomly scanned. Fifty cells per slide were

analyzed with 2 slides per incubation (total 100 cells). All experiments were carried out in triplicate to take into account possible variations between different cell preparations. All the measurements are expressed in terms of percentage of DNA migrated from the comet head to the tail region (mean % tail DNA), and mean olive tail moment (OTM) (Anderson et al., 1994). The comet pictures for each of the sampling sites were selected randomly from the 100 cells used for comet assay from the marine gastropods collected from different sites along the coast of Goa. 3.6. Measurement of PAH PAHs were extracted from the sediments from the sampling sites using bidistilled hexane by homogenization with an Ultra Turrax(R) homogenizer. The moisture content in the solvent extracts was removed by anhydrous sodium sulfate. The solvent extracts were concentrated to 1 ml. using Kuderna Danish evaporator. The concentrated extracts were then passed through deactivated (10%) alumina in order to remove the interfering substances followed by elution with 15 ml bidistilled hexane. The purified extracts were then concentrated to 10 ml by evaporation using Snyder column evaporator. The concentration of PAHs in the aliquots were measured by spectrofluorometry (Shimadzu RF-1501 spectrofluorometer with excitation at 310 nm and emission at 360 nm) using the Kuwait oil as the standard (Burns, 1993, Grasshoff et al., 1983, Sarkar et al., 2008).

3.7. Biochemical tests 3.7.1. Catalase Catalase activity was determined following the method of Sinha, (1972). It is based on the reduction of dichromate in acetic acid to chromic acetate (green) when heated in the

presence of H2O2. To perform the test, an assay mixture containing 0.5 ml of 0.2M hydrogen

peroxide (H2O2), 1 ml of sodium phosphate buffer (0.01M, pH 7.0) and 0.4 ml distilled water was first prepared. Following this, 100 µl of tissue homogenate sample was added to initiate the reaction. 2 ml of dichromate-acetic acid reagent (potassium dichromate: glacial acetic acid, 1:3 ratio) were added to arrest the reaction. The tubes were heated for 10 min and allowed to cool; the green colour developed was read at 583 nm against blank on a

spectrophotometer. The activity of catalase was expressed as mM of H2O2/min/mg of protein.

3.7.2. Glutathione S-transferase (GST) activity

The activity of Glutathione S-transferase (GST) was determined following the method of Habig et al., (1974). It is based on conjugation of 1-Chloro- 2,4-Dinitrobenzene (CDNB) solution (30mM) and reduced glutathione (GSH) solution (30mM) in reaction buffer (0.1M

K2HPO4, EDTA-Na2, pH 6.5). The change in absorbance was read every 30 seconds for 5 min at 340 nm using a UV–Vis spectrophotometer. The activity of GST was determined using extinction coefficient of 9.6 mM-1 cm-1 for CDNB. GST activity was calculated as nM 1-chloro-2,4-dinitrobenzene conjugate formed/min/mg protein.

3.7.3. Lipid peroxidation (LPO) determination Lipid peroxidation was measured by the thiobarbituric acid following the methods of Ohkawa et al., (1979). 1 g of soft tissue was homogenized with 9 ml of 0.25 M sucrose using ultra turrax homogenizer for 1 minute. 0.2 ml of the tissue homogenate was then mixed with 0.2 ml of 8% SDS, 1.5 ml of 20% acetic acid, and 1.5 ml of 0.8% TBA. The mixture was made up to 4 ml using distilled water and heated at 95˚C for 60 min. It was then cooled and centrifuged at 3000 rpm for 10 min. The absorbance of the sample was read at 532 nm. LPO activity was measured as malondialdehyde (MDA) equivalent and expressed as nM of MDA min-1 mg-1

3.7.4. Superoxide dismutase (SOD) activity SOD activity was measured by the rate of auto-oxidation of epinephrine to adenochrome adapted from Misra and Fridovich, (1972). The reaction volume (1 ml) contained 10 mM epinephrine, 50 mM sodium carbonate buffer (pH 10.2), and 10 mM EDTA. The results are given in units of SOD activity per milligram of protein (U mg-1); where 1 U of SOD is defined as the amount of sample causing 50% of inhibition of epinephrine auto-oxidation.

3.7.5. AChE estimation Soft tissues from 4-5 animals were pooled together to make composite sample. About 500 mg of soft tissues were homogenized with 0.25 M sucrose solution and 0.01 M phosphate buffer (pH 7.4 ± 0.1) spiked with Triton-X-100 (0.2%) using an Ultra Turrax homogeniser (T-25) for 3-4 minutes. The homogenates were then centrifuged at 20,000 × g at 4ºC for about 35 minutes. Clear supernatant was collected and kept at - 80°C for further use. AChE activity in snails was measured using the ∆-pH-metric method (Gaitonde et al., 2006). Briefly, 0.1 ml of sample enzyme was incubated with 0.2 ml of substrate (acetylcholine

bromide) in phosphate buffer (0.01M, pH-8.0 ±0.10) with bromothymol blue as an indicator. The change in pH was recorded at an interval of 10 min for 1 h. The unit of activity of AChE was expressed as micromoles of acetic acid liberated per minute per mg of protein. Enzyme activity was calculated by the formula:

Enzyme activity = Nano moles of acid liberated in 7.5 ml of reaction mixture Time in mins. X protein conc. X enzyme vol. used

Protein concentration was determined using the Lowry et al., (1951) method, with bovine serum albumin as the standard.

3.8. Measurement of water quality parameter The water quality parameters such as temperature, pH, salinity, dissolved oxygen (DO) biochemical oxygen demand (BOD) and nutrients (nitrate, nitrite and phosphate) were determined following the standard procedure (Grasshoff, et al., 1983).

3.9. Integrated biomarker response and statistical Analysis

Integrated biomarker response (IBR) was calculated as described by Beliaeff and Burgeot (2002) with modification by Guerlet et al., 2010 and Devin et al., (2014).

Briefly the biomarker response data for each site was standardized using the formulae

Yi=(Xi-m)/s

where Yi is the standardized biomarker response, Xi is response value of each biomarker, m and s are mean value and standard deviation for all sites respectively.

Zi was then calculated using the formulae as Zi=Yi or Zi=–Yi for biomarker responding to contamination by induction or inhibition, respectively. The minimum value for each biomarker at all station was also calculated from the standardised biomarker response.

The scores for the biomarker was computed as Si = Zi+|Mini|

Individual areas Ai connecting the ith and the (i + 1)th radius coordinates of the star plot were obtained according to the formula:

Ai = Si * Si+1 * sin (2π/k) / 2

where Si and Si+1 represent the individual biomarker scores (calculated from standardised data) and their successive star plot radius coordinates and k represent the number of radii corresponding to the biomarkers used in the survey.

Biomarkers were arranged clockwise from sub-cellular level to individual level as follows: tail DNA, DNA-F, SOD, CAT, GST, LPO, AChE (Serafim et al., 2012).

And the IBR value is calculated as follow:

IBR= Ai 푛 ∑푖=0 where Ai is the triangular area represented by two consecutive biomarker scores on the star plot, and n is the number of biomarkers used in the IBR calculation.

A statistical analysis was carried out with different water quality parameters, biochemical and genotoxic biomarkers using Origin Pro 8.5.0. The data were expressed as mean ± standard deviation. Results from biochemical assays and genotoxic damage were analyzed by analysis of variance (ANOVA) followed by Tukey HSD post-test. Kolmogorov– Smirnov test for normality of distribution was used prior to ANOVA. Three levels of significance are reported: (a) p < 0.05, (b) p < 0.01, and (c) p < 0.001. Spearman correlation matrix was also calculated to study the relationships between the different biomarkers measured and physico-chemical parameters. In order to assess the significance of ambient physico-chemical parameters of the seawater which are likely to influence the integrity of DNA of the marine gastropod, a stepwise multiple regression analysis was carried out with DNA integrity as the dependent variable and the Temperature,

DO, NO2, NO3, Turbidity, pH, Salinity, PO4, BOD, PAH etc. as the independent variables (Sarkar, 1991). The stepwise multiple regression analysis was conducted with a statistical software, ‘EASE’ (Essential Applied Statistics for Environmentalists), (Adhikari, 1989). The multiple correlations co-efficient (R2) thus obtained for each of the parameters by stepwise addition showed an increase in the value of correlation coefficient. However, the correlation coefficient was further adjusted to determine the corrected multiple correlation co-efficient (

2 R ) in consideration of the total degrees of freedom (totaldf) and the error of degrees of freedom (errordf) with respect to the number of observations and the no of coefficients according to the following expression.

2 2 (totaldf) R = 1 (1 R ) ) (errordf 72

The best fit correlation for DNA integrity was obtained collectively with PAH, NO3,

Salinity and PO4 by evaluating the adjusted correlation co-efficient and the F-statistics due to newly entered variable.

CHAPTER 4 Results and Discussion

4.1. Physico-chemical parameters of water and its relation with other biomarkers Physico-chemical water parameters varied significantly across all the sampling sites along the coast of Goa (Table 13). pH of the seawater was in the range from 7.46±0.06 to 8.34±0.01 in pre-monsoon, 7.08±0.02 to 8.26±0.04 during monsoon and 7.75±0.01 to 8.37±0.01 during post-monsoon. Maximum values for seawater temperature (˚C) were observed at Bogmalo (34.00±0.0) during post-monsoon and minimum at Dona Paula (26.25±0.35) in pre-monsoon. The highest conductivity was found at Anjuna (152.50±0.50 mS/cm) during post-monsoon and the least at Sinquerim (36.50±0.71 mS/cm) in the same season. Seawater from sampling sites in Goa showed significant variation in nutrients (nitrite, nitrate and total phosphate). The values for nitrite, nitrate and total phosphate was found to be highest at Velsao in all seasons. Dissolved oxygen (DO) also varied significantly between the sites, with maximum values observed at Bogmalo (6.43±0.00 mg/L) in monsoon and minimum at Palolem (1.47±0.00 mg/L) in pre-monsoon. The highest Biological Oxygen Demand (BOD) was measured at Betul (4.01±0.02 mg/L) in post-monsoon and the least at Hollant (0.16±0.06 mg/L) in pre-monsoon. Rainfall is one of the most important factor influencing the physico-chemical water parameters in tropical countries. In this study, pH and water temperature along the coastal region didn't show much variation during the three seasons. However, season wise variations in nutrients and dissolve oxygen were recorded with higher values during monsoon and lower values in pre-monsoon. Higher values of nitrate observed during monsoon may be due to occurrence of seasonal flooding of wastewater from agricultural and other sources. The lower values of nutrients during the non-monsoon season may be attributed to decreased land sewage and fertilizers disposal. Similar patterns in nutrients were observed in Mahanadi river-estuarine system (Sundaray et al., 2006). Dissolve oxygen was higher in monsoon and post-monsoon but decreased in pre-monsoon. The higher values of dissolve oxygen may be

due to high rainfall and mixing of freshwater into the sampling waters (Sundaramanickam et al., 2008). Among all the sites, significantly higher values for nitrite and nitrate were recorded at Velsao which might be due to oxidation of ammonia to nitrate and then to nitrate by bacteria during nitrogen cycle. The source of ammonia at Velsao may be due to the discharge of effluents from the nearby industries (Singbal et al., 1976, Bhalla and Nigam, 1986). The pungent smell of ammonia in the air at the time of sampling at Velsao clearly indicates the release of huge amount of effluents into the marine ecosystem. Certain types of detergents can introduce a high concentration of phosphate ions into bodies of water. High phosphate values at Velsao could be associated with surface runoff from near industrial land. Increased levels of phosphates and nitrates often indirectly harm the environment by causing bacterial growth and huge algae blooms, which harms the aquatic life. Dissolved oxygen content of estuary water is another important parameter determining the life of aquatic biota. Dissolved oxygen in the water is consumed either through the respiratory processes of biological decomposition or through chemical oxidation of oxygendemanding wastes. High biological oxygen demand was recorded at Bogmalo which may be due to the influence of run-off water from monsoon from the hotels nearby the region. It was found that higher dissolved oxygen values were observed in monsoon may be due to circulation and mixing by inflowing water during monsoon rains. Lower values of dissolve oxygen were reported Anjuna during monsoon and pre-monsoon. It has been found that low dissolve oxygen content can also increase the toxicity of certain heavy metals such as zinc, lead, copper, cyanide, hydrogen sulphides and ammonia (ANZECC & ARMCANZ, 2000).

Table 13: Physico-chemical parameters of seawater along the Goa coast, India. Values are represented as means± standard deviation

Season Locations pH Temp. Conductivity Nitrite Nitrate Phosphate D.O. B.O.D

(˚C) mS/cm µM/L µM/L µM/L (mg/L) (mg/L)

Pre-monsoon Arambol 8.33±0.00 28.25±0.35 52.00±0.00 1.10±0.05 1.35±0.09 1.04±0.08 3.84±0.00 1.47±0.00

Sinquerim 8.30±0.01 28.50±0.00 54.00±0.00 0.88±0.03 0.73±0.07 0.93±0.03 4.01±0.08 2.88±0.00

Dona Paula 7.46±0.06 26.25±0.35 51.00±0.00 0.42±0.00 2.85±0.00 0.61±0.00 4.01±0.08 1.52±0.08

Velsao 7.84±0.07 28.75±0.35 45.50±0.71 1.72±0.00 43.72±0.04 3.39±0.37 3.67±0.08 1.92±0.08

Betul 7.88±0.01 29.00±0.00 52.00±0.00 0.46±0.00 0.09±0.00 0.71±0.17 3.22±0.08 2.65±0.00

Anjuna 8.34±0.01 28.00±0.0 49.00±0.00 0.59±0.00 0.79±0.03 0.68±0.01 2.94±0.00 3.78±0.00

Bogmalo 7.94±0.01 28.75±0.25 44.50±0.50 1.05±0.07 0.51±0.08 1.62±0.10 2.65±0.00 2.03±0.06

Hollant 7.81±0.00 29.50±0.0 41.00±0.50 0.59±0.01 5.02±0.05 3.29±0.56 3.90±0.06 0.16±0.06

Palolem 7.86±0.01 28.00±0.00 48.50±0.71 0.39±0.00 0.11±0.00 0.60±0.05 1.47±0.00 3.73±0.00

Monsoon Arambol 8.08±0.08 29.50±0.71 55.50±0.71 1.10±0.00 1.46±0.04 1.28±0.00 4.63±0.16 1.61±0.04

Sinquerim 7.66±0.14 28.50±0.71 149.50±0.71 0.22±0.05 32.36±0.06 1.23±0.23 3.44±0.40 1.75±0.16

Dona Paula 8.19±0.02 27.50±0.71 57.50±0.71 1.02±0.08 3.89±0.47 1.26±0.11 4.40±0.16 2.60±0.00

Velsao 8.07±0.08 28.50±0.71 151.50±0.71 3.36±0.05 79.21±0.00 27.19±1.16 5.98±0.00 2.94±0.16

Betul 8.12±0.02 30.50±0.71 53.50±0.71 0.38±0.03 18.94±0.10 0.36±0.05 4.97±0.16 2.15±0.16

Palolem 7.98±0.02 31.50±0.00 55.50±0.71 0.33±0.01 0.99±0.06 0.82±0.05 4.35±0.08 1.07±0.00

Anjuna 8.20±0.06 28.5±0.50 41.5±0.50 0.90±0.13 12.64±0.09 2.51±0.19 2.70±0.11 1.46±0.11

Bogmalo 8.26±0.04 28.75±0.25 65.5±0.50 1.11±0.03 12.16±1.67 0.92±0.09 6.43±0.00 3.95±0.11

Hollant 7.08±0.02 29.00±0.00 41.5±0.50 0.24±0.01 40.10±0.12 0.33±0.06 4.29±0.11 0.79±0.00

Post-monsoon Arambol 7.97±0.01 31.75±0.35 40.50±0.71 0.47±0.12 1.66±0.06 0.54±0.14 4.29±0.00 1.19±0.08

Sinquerim 8.09±0.04 30.00±0.00 36.50±0.71 0.91±0.04 9.14±0.20 0.73±0.15 5.87±0.32 1.69±0.08

Dona Paula 7.75±0.01 30.00±0.00 40.00±1.41 1.11±0.01 11.08±0.02 0.80±0.02 4.40±0.16 0.56±0.00

Velsao 8.06±0.01 33.00±0.00 64.50±0.71 9.64±0.01 32.76±0.80 3.48±0.05 4.12±0.08 2.20±0.00

Betul 8.21±0.02 32.00±0.00 39.50±0.71 0.23±0.03 1.44±0.10 0.28±0.02 5.14±0.08 4.01±0.00

Anjuna 8.37±0.01 33.00±0.0 152.50±0.50 0.34±0.04 1.69±0.04 0.38±0.05 5.93±0.06 1.58±0.06

Bogmalo 8.10±0.03 34.00±0.0 151.50±0.50 0.61±0.05 3.78±0.03 0.45±0.01 4.01±0.00 1.01±0.06

Hollant 8.09±0.02 32.00±0.0 146.50±0.50 1.40±0.03 13.54±0.13 1.10±0.01 3.27±0.11 0.73±0.06

Palolem 7.90±0.04 31.00±0.00 42.50±0.71 0.43±0.04 0.07±0.02 0.42±0.00 4.52±0.00 3.95±0.00

A significant positive correlation was observed between SOD activity and TDNA in N. chamaeleon during monsoon., whereas DNA-F and GST were negatively correlated (Table 14 A). In the post-monsoon, CAT and SOD activity were found to be positively correlated (r=0.94). Similar trend was also observed during the pre-monsoon season. P. sulcatus also showed positive correlation between CAT and SOD activity during all the three season. When SOD and LPO actively were compared, it was also found to be positively correlated during all the three season (Table 15). It is interesting to note that DNA-F negatively correlated with GST, SOD, CAT and LPO in all the three season. Temperature also found to be positively correlated with GST activity in all the three season. In M. granulata, a mild but positive correlation were observed between CAT activity and pH of the seawater, whereas LPO and temperature were found to be positively correlated in all the three season (Table 16).

Table 14: Spearman correlation matrix of different physico-chemical water parameters and biomarker activity in N. chamaeleon in (A) monsoon, (B) post- monsoon, and (C) pre-monsoon

(A)

Variables pH Temp Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. -0.381 1 Turbidity 0.336 -0.552 1 Conductivity 0.074 0.258 0.109 1 Nitrite 0.564 -0.283 0.585 0.595 1 Nitrate -0.060 -0.531 -0.088 -0.085 0.211 1 Phosphate 0.004 -0.715 0.641 0.236 0.537 0.484 1 D.O. 0.450 0.011 0.025 0.489 0.769 0.338 0.206 1 B.O.D 0.533 -0.629 0.182 0.159 0.591 0.721 0.436 0.577 1 TDNA -0.545 0.171 -0.378 0.383 -0.312 0.077 0.060 -0.250 -0.084 1 DNA-F 0.434 -0.327 0.503 -0.513 -0.025 -0.340 -0.060 -0.387 -0.147 -0.706 1 SOD -0.664 0.114 -0.483 -0.056 -0.452 0.280 0.018 -0.236 0.000 0.776 -0.622 1 CAT 0.154 -0.121 0.343 0.573 0.207 -0.410 0.277 -0.151 -0.035 0.469 -0.042 0.042 1 GST -0.098 0.061 -0.622 0.176 0.004 0.627 -0.025 0.510 0.421 0.301 -0.734 0.371 -0.273 1 LPO -0.510 0.011 0.049 -0.425 -0.249 0.280 0.109 -0.176 -0.291 -0.259 0.056 -0.070 -0.692 -0.021 1 AChE -0.070 -0.160 0.245 -0.028 -0.333 -0.508 0.046 -0.745 -0.337 0.371 0.336 0.133 0.720 -0.608 -0.399

(B)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. 0.415 1 Turbidity -0.696 -0.694 1 Conductivity -0.081 -0.079 0.237 1 Nitrite -0.340 -0.043 0.301 0.870 1 Nitrate -0.063 0.065 0.266 0.835 0.853 1 Phosphate -0.211 0.025 0.295 0.889 0.914 0.893 1

D.O. 0.401 -0.551 -0.096 -0.313 -0.482 -0.461 -0.469 1 B.O.D 0.591 0.500 -0.924 -0.406 -0.493 -0.542 -0.557 0.268 1 TDNA -0.263 -0.633 0.287 0.505 0.378 0.084 0.249 0.376 -0.123 1 DNA-F 0.074 0.068 -0.053 -0.632 -0.783 -0.804 -0.680 0.177 0.257 -0.231 1 SOD -0.210 -0.439 0.595 0.625 0.713 0.790 0.690 -0.050 -0.754 0.266 -0.734 1 CAT -0.361 -0.043 0.630 0.302 0.476 0.622 0.578 -0.468 -0.845 -0.273 -0.308 0.692 1 GST -0.231 0.245 0.046 -0.021 0.238 0.427 0.193 -0.461 -0.197 -0.434 -0.510 0.301 0.441 1 LPO -0.210 -0.439 0.592 0.646 0.713 0.818 0.732 -0.043 -0.761 0.280 -0.741 0.972 0.678 0.315 1 AChE -0.536 -0.094 0.441 -0.498 -0.182 -0.091 -0.235 -0.305 -0.416 -0.524 0.091 0.091 0.497 0.636 0.077

(C)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. 0.108 1 Turbidity 0.254 0.286 1 Conductivity 0.327 0.032 0.013 1 Nitrite 0.286 0.280 0.851 0.001 1 Nitrate 0.021 0.068 0.232 0.215 0.290 1 Phosphate 0.217 0.131 0.701 0.004 0.815 0.321 1 D.O. 0.013 0.086 0.124 0.215 0.062 0.235 0.038 1 B.O.D 0.004 0.035 0.053 0.000 0.195 0.370 0.128 0.246 1 TDNA 0.031 0.368 0.137 0.441 0.059 0.061 0.000 0.156 0.000 1 DNA-F 0.001 0.175 0.004 0.013 0.018 0.179 0.035 0.038 0.060 0.111 1 SOD 0.000 0.002 0.489 0.040 0.358 0.671 0.429 0.310 0.047 0.000 0.093 1 CAT 0.207 0.023 0.018 0.146 0.000 0.152 0.029 0.008 0.375 0.001 0.002 0.001 1 GST 0.004 0.318 0.658 0.000 0.420 0.131 0.272 0.092 0.002 0.282 0.062 0.423 0.007 1 LPO 0.188 0.055 0.764 0.099 0.568 0.282 0.476 0.486 0.063 0.182 0.021 0.636 0.063 0.489 1 AChE 0.049 0.001 0.015 0.009 0.001 0.004 0.031 0.043 0.464 0.080 0.032 0.062 0.627 0.006 0.032

Table 15: Spearman correlation matrix of different physico-chemical water parameters and biomarker activity in P. sulcatus in (A) monsoon, (B) post- monsoon, and (C) pre-monsoon

(A)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. 0.298 1 Turbidity -0.513 -0.661 1 Conductivity 0.076 0.440 -0.128 1 Nitrite -0.084 0.243 0.158 0.736 1 Nitrate 0.161 0.336 0.106 0.726 0.904 1 Phosphate -0.174 0.230 0.286 0.659 0.933 0.912 1 D.O. 0.130 -0.693 0.175 -0.419 -0.482 -0.470 -0.497 1 B.O.D -0.003 -0.265 -0.313 -0.465 -0.468 -0.498 -0.508 0.687 1 TDNA 0.507 0.203 -0.578 0.221 0.144 0.218 0.017 0.352 0.599 1 DNA-F -0.086 -0.198 0.192 -0.577 -0.663 -0.676 -0.623 0.283 0.088 -0.395 1 SOD 0.128 0.452 -0.260 0.653 0.678 0.609 0.500 -0.677 -0.527 0.088 -0.594 1 CAT 0.173 0.371 -0.476 0.147 0.181 0.079 0.009 -0.605 -0.341 -0.099 -0.356 0.724 1 GST 0.458 0.569 -0.551 0.338 0.486 0.591 0.383 -0.434 0.013 0.546 -0.614 0.538 0.429 1 LPO 0.232 -0.072 0.058 0.361 0.722 0.691 0.569 -0.213 -0.231 0.253 -0.710 0.616 0.396 0.631 1 AChE -0.498 -0.303 0.235 -0.552 -0.522 -0.590 -0.395 0.455 0.578 -0.105 0.567 -0.827 -0.628 -0.469 -0.661

(B)

D.O. 0.130 -0.693 0.175 -0.419 -0.482 -0.470 -0.497 1 B.O.D -0.003 -0.265 -0.313 -0.465 -0.468 -0.498 -0.508 0.687 1 TDNA 0.507 0.203 -0.578 0.221 0.144 0.218 0.017 0.352 0.599 1 DNA-F -0.086 -0.198 0.192 -0.577 -0.663 -0.676 -0.623 0.283 0.088 -0.395 1 SOD 0.128 0.452 -0.260 0.653 0.678 0.609 0.500 -0.677 -0.527 0.088 -0.594 1 CAT 0.173 0.371 -0.476 0.147 0.181 0.079 0.009 -0.605 -0.341 -0.099 -0.356 0.724 1 GST 0.458 0.569 -0.551 0.338 0.486 0.591 0.383 -0.434 0.013 0.546 -0.614 0.538 0.429 1 LPO 0.232 -0.072 0.058 0.361 0.722 0.691 0.569 -0.213 -0.231 0.253 -0.710 0.616 0.396 0.631 1 AChE -0.498 -0.303 0.235 -0.552 -0.522 -0.590 -0.395 0.455 0.578 -0.105 0.567 -0.827 -0.628 -0.469 -0.661

(C)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. -0.458 1 Turbidity 0.213 0.133 1 Conductivity 0.591 -0.442 0.230 1 Nitrite 0.357 0.068 0.679 -0.104 1 Nitrate -0.086 0.209 0.738 -0.382 0.685 1 Phosphate -0.231 0.494 0.495 -0.658 0.670 0.826 1 D.O. 0.263 0.289 0.754 0.263 0.320 0.561 0.374 1 B.O.D 0.084 -0.520 -0.358 0.528 -0.498 -0.693 -0.789 -0.426 1 TDNA -0.670 0.217 -0.253 0.004 -0.602 -0.377 -0.398 -0.374 0.369 1 DNA-F 0.222 -0.659 -0.349 0.203 0.040 -0.273 -0.341 -0.542 0.228 -0.068 1 SOD -0.275 0.425 0.719 -0.172 0.289 0.797 0.600 0.735 -0.535 -0.020 -0.503 1 CAT -0.297 0.643 -0.029 -0.660 0.218 0.200 0.582 -0.027 -0.276 -0.138 -0.543 0.138 1 GST 0.134 0.011 0.314 -0.129 0.393 0.355 0.385 0.288 0.104 -0.499 -0.371 0.191 0.556 1 LPO 0.244 0.336 0.354 -0.225 0.450 0.394 0.574 0.502 -0.298 -0.626 -0.574 0.248 0.648 0.798 1 AChE 0.613 -0.220 0.196 0.682 0.117 -0.341 -0.455 0.033 0.194 0.051 0.178 -0.345 -0.446 -0.407 -0.275

Table 16: Spearman correlation matrix of different physico-chemical water parameters and biomarker activity in M. granulata in (A) monsoon, (B) post-monsoon, and (C) pre-monsoon

(A)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. -0.323 1 Turbidity 0.350 -0.319 1 Conductivity 0.233 0.037 -0.202 1 Nitrite 0.613 -0.283 0.408 0.498 1 Nitrate -0.320 -0.298 -0.326 0.027 -0.068 1 Phosphate 0.281 -0.568 0.785 0.100 0.554 0.098 1 D.O. 0.305 0.085 -0.344 0.432 0.624 0.100 -0.125 1 B.O.D 0.615 -0.377 0.028 0.212 0.667 0.173 0.289 0.698 1 TDNA 0.112 0.052 -0.333 0.431 0.086 -0.118 -0.112 0.395 0.371 1 DNA-F 0.628 -0.115 0.453 -0.179 0.396 -0.488 0.124 0.131 0.335 -0.135 1 SOD 0.405 0.006 -0.248 0.742 0.374 0.142 0.072 0.488 0.471 0.634 -0.031 1 CAT 0.108 0.110 -0.106 0.170 0.573 0.215 -0.019 0.794 0.527 0.164 0.164 0.176 1 GST 0.614 -0.605 0.451 -0.107 0.193 -0.052 0.500 -0.204 0.507 0.166 0.422 0.211 -0.269 1 - LPO -0.294 0.245 0.249 -0.442 -0.482 0.101 0.058 -0.651 -0.554 -0.610 -0.162 -0.326 -0.494 -0.110 1 AChE -0.740 0.115 -0.040 -0.551 -0.751 -0.087 -0.196 -0.702 -0.744 -0.288 -0.416 -0.745 -0.523 -0.300 0.426

(B)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA SOD CAT GST LPO pH 1 Temp. 0.557 1 Turbidity -0.659 -0.770 1 Conductivity 0.384 0.531 -0.236 1 Nitrite -0.428 -0.074 0.337 0.338 1 Nitrate -0.117 0.082 0.227 0.516 0.885 1

Phosphate -0.361 -0.049 0.384 0.353 0.942 0.906 1 D.O. 0.323 -0.372 0.056 -0.071 -0.583 -0.484 -0.537 1 B.O.D 0.274 0.054 -0.489 -0.265 -0.452 -0.506 -0.465 0.457 1 TDNA -0.082 -0.073 0.003 0.531 0.507 0.439 0.390 0.075 -0.022 1 DNAF 0.072 0.243 -0.155 -0.298 -0.486 -0.310 -0.383 -0.050 -0.132 -0.512 1 SOD -0.150 0.016 -0.236 -0.022 0.053 -0.028 -0.062 0.062 0.435 0.123 -0.383 1 CAT 0.117 0.290 -0.308 0.365 0.407 0.527 0.332 -0.353 -0.323 0.313 0.003 0.270 1 GST 0.250 0.244 -0.443 0.042 0.121 0.284 0.023 -0.279 -0.167 0.181 0.267 0.038 0.743 1 LPO 0.320 0.424 -0.556 0.387 0.380 0.344 0.279 -0.378 0.138 0.489 -0.371 0.065 0.501 0.456 1 AChE -0.204 -0.136 0.183 -0.416 -0.404 -0.329 -0.331 0.276 0.140 -0.488 0.645 0.059 -0.190 -0.172 -0.710

(C)

Variables pH Temp. Turbidity Conductivity Nitrite Nitrate Phosphate D.O. B.O.D TDNA DNA-F SOD CAT GST LPO pH 1 Temp. -0.083 1 Turbidity 0.459 0.274 1 Conductivity 0.360 -0.326 0.148 1 Nitrite 0.437 0.381 0.718 -0.113 1 Nitrate -0.246 0.045 0.469 -0.353 0.428 1 Phosphate 0.058 0.633 0.533 -0.500 0.781 0.569 1 D.O. -0.184 0.023 0.346 0.366 0.141 0.565 0.173 1 B.O.D 0.400 -0.311 0.031 0.274 -0.270 -0.632 -0.520 -0.551 1 TDNA -0.476 0.152 -0.423 -0.049 -0.260 -0.345 -0.182 -0.247 0.079 1 DNA-F -0.228 -0.338 -0.525 0.180 -0.272 -0.024 -0.340 0.108 -0.445 0.169 1 SOD 0.020 0.146 0.717 -0.170 0.474 0.717 0.533 0.516 -0.125 -0.300 -0.536 1 CAT 0.121 0.828 0.280 -0.465 0.507 -0.003 0.648 -0.206 -0.159 0.223 -0.329 0.176 1 GST -0.129 0.774 0.379 -0.553 0.460 0.194 0.702 -0.086 -0.110 0.154 -0.608 0.457 0.839 1 LPO -0.115 0.611 0.414 -0.289 0.589 0.531 0.809 0.512 -0.614 -0.170 -0.234 0.608 0.571 0.680 1 AChE -0.323 0.013 0.152 0.469 0.050 0.235 -0.057 0.620 -0.434 0.197 0.377 0.044 -0.172 -0.158 0.187

The stepwise multiple regression analysis with different physico-chemical parameters showed that among the various water quality parameters, highest

correlation was found between PAH and DNA integrity ( R2, 0.864) (Table 17). Interestingly, the correlations with DNA integrity increased gradually from 0.864 to 0.901 with the addition of different parameters sequentially. However, the evaluation of the corrected correlation coefficient and the F-statistics due to newly entered variable showed that the best fit correlation of DNA integrity was observed collectively with PAH, NO3, salinity and PO4 ( R2 , 0.90). Such an improved correlation with different physico-chemical parameters clearly indicate that they played significant role on the impairment of DNA in Morula granulata.

Table 17. Stepwise multiple regression analysis of the data on various physico-chemical parameters and PAH influencing the Integrity of DNA in marine gastropod, Morula granulata No. Independent Dependent Multiple corr. Adjusted multiple F-statistics due variable variable added co-efficient corr. co-efficient to newly __ entered R2 R2 variable

1 DNA integrity PAH 0.87 0.86 81.98 2 DNA integrity NO3 0.89 0.88 3.24 3 DNA integrity Salinity 0.89 0.88 1.36 4 DNA integrity PO4 0.92 0.90 5.70 5 DNA integrity Turbidity 0.92 0.90 1.47 6 DNA integrity DO 0.93 0.89 0.76 7 DNA integrity BOD 0.93 0.89 0.54 8 DNA integrity pH 0.93 0.87 0.48 9 DNA integrity Temperature 0.93 0.85 0.005 10 DNA integrity NO2 0.93 0.82 0.0008

4.2. Polycyclic aromatic hydrocarbons (PAHs)

The total PAHs in sediment were in the range from 1.65±0.13 µg/g to 4.29±0.35 µg/g (Fig. 11). Overall the lowest PAH was observed in sediment collected from the reference site, Arambol and the highest PAH was observed at Velsao. Sinquerim, Dona Paula and Velsao showed significantly higher values in PAH as compared to Arambol for all seasons. There was no significant difference in PAH observed when Palolem and Arambol were compared. Betul showed significantly higher (p<0.05) values for PAH when compared to Arambol.

PAH in sediment 5 4.5 4 3.5 3 2.5 2

µg/gwet weight 1.5 1 0.5 0

Fig. 11. Total Polycyclic aromatic hydrocarbons (PAHs) in sediments from Goa coast

Total PAH in N. chamaeleon were in the range from 5.29±0.67 µg/g to 12.14±0.24 µg/g wet weight (Fig. 12). Lowest PAH was observed in Arambol and the highest values were observed at Sinquerim. Significant increase in PAH values were observed at Sinquerim (p<0.001), Dona Paula (p<0.01), and Velsao (p<0.01). Spatial variations in PAH were also observed in P. sulcatus, with highest values recorded at Sinquerim (10.25±0.96 µg/g) and lowest values at the reference site, Arambol (3.96±0.70 µg/g). The mean value of PAH across the nine sampling sites was found to be 7.09±0.50 µg/g. In M. granulata, the lowest PAH concentration was found at Arambol (5.32±0.01 µg/g) whereas the highest values were observed at Velsao (10.78±0.41 µg/g). PAH concentrations were found to be in increasing order from Arambol, Sinquerim, Bogmalo, Hollant and Velsao.

Nerita Chamaeleon 14 12 10 8 6

µg/g wet weight 4 2 0

Fig. 12. Total Polycyclic aromatic hydrocarbons (PAHs) in Nerita chamaeleon (µg/g wet weight)

Planaxis sulcatus 12

µg/g wet weight 4

Fig. 13. Total Polycyclic aromatic hydrocarbons (PAHs) in Planaxis sulcatus (µg/g wet weight)

Morula granulata 12

µg/g wet weight 4

0 Arambol Sinquerium Bogmalo Hollant Velsao Betul Palolem

Fig. 14. Total Polycyclic aromatic hydrocarbon (PAH) concentration (µg/g wet weight) in Morula granulata Molluscs have a greater tendency to bioaccumulate PAHs. Among molluscs, gastropods perform an important role in marine food chain, and are used as a source of food for many fishes and birds. They have limited ability to metabolize xenobiotics and thus they are prone to accumulate high concentrations of hydrocarbons. Gastropods have vaired feeding habits and feeds on algae, fungi and small debris of plant or animal matter. Marine algae are a fundamental part of marine food webs, since they are responsible for most of coastal primary production. A number of marine algal species are known to contain hlgh concentrations of PAHs, secondary metabolites, often unsaturated aldehydes, which have cytotoxic properties and electrophilic centres (Fenical 1982). Recent studes has shown increase in oxidative stress biomarkers in herbivorous gastropods from tropical waters. Feeding these gastropods on algae with high concentrations of PAHs and unsaturated aldehydes resulted in an increase in the digestive gland GST activity (Lee, 1988). The range of PAH in snails in this study lies within the same range as reported in clams (Menon and Menon, 1999) and oysters (Niyogi et al., 2001a) in Indian coastal waters. Sarkar et al., (2008) has reported comparatively higher values of total PAH in muscle tissues of marine gastropods Cronia contracta from Goa coast. The total PAH concentrations (µg/g, wet weight) in N. chamaeleon from Goa region range from the lowest value at Arambol (5.29±0.67) to highest value at Sinquerim (12.14±0.27). Presence of high amount of PAH in snails from Sinquerim

86 may be due to extensive shipping activities as well as accidental oil spills (Desai et al., 2010). P. sulcatus collected from Sinquerim also showed the highest value of PAH, whereas in M. granulata, the highest PAH concentration was recorded in Velsao. N. chamaeleon collected from Velsao, showed two fold increase in PAH concentrations as compared to Arambol, such an increase in PAH in snails at Velsao revealed the severity of organic pollution in Velsao. There were no significant differences between PAH concentration in N. chamaeleon at Velsao, Sinquerim and Dona Paula. Ingole et al., (2006) has reported high level of total petroleum hydrocarbon at Goa coast due to grounding of MV River Princess. Sarkar et al., (2008) studied the seasonal variation of PAH in Cronia contracta collected from six sites along the Goa coast and reported positive correlation between the PAH and DNA damage. High concentrations of PAH at Sinquerim, Hollant and Velsao may be associated with the increase in runoff from industrial or hotel regions. High amount of PAHs were reported in M. granulata from Sinquerim (8.53±0.13 µg/g) and Dona Paula (9.76±0.68 µg/g). Dona Paula sampling locations are close to the Mormugao port, which is one of the oldest and premium hub for maritime trade in Goa. Verlecar et al., (2012) has reported high amount of PAH (19.39 µg/ml) in water samples from Mormugao Harbour. Apart from many shipbuilding industries, yards and workshop, Mormugao port has two sewage treatment plant located on either side which contribute of anthropogenic pollution (Shirodkar et al., 2012). Rapid increase in anthropogenic activities from riverine input has lead to increase in contamination at these sites. In our study PAH concentration in N. chamaeleon from Dona Paula (10.67±0.85 µg/g) and Velsao (11.25±0.62 µg/g) were lower than the PAH concentration reported in Cronia contracta from Dona Paula (30.76-53.78 µg/g) and Velsao (22.32-53.78 µg/g) by Sarkar et al., (2008). A positive correlation between rainfall and PAH concentration have been reported (Ribeiro et al., 2012). Low PAH concentrations recorded at Arambol may be due to its location as it is far away from the industrial activities. Vashista et al., (2010) has also found highest concentration of 2-3 PAH in waters of Sinquerim followed by Velsao.

4.3. Alkaline unwinding assay The impairment of DNA in marine gastropods was measured in terms of the loss of DNA integrity in snails exposed to various types of genotoxicants prevalent in the marine ecosystem along the coast of Goa. Because of the serene environment and far away from the industrial belt, Arambol was considered as a reference site for the study. Interestingly, DNA

integrity in snails was found to be relatively quite high at this site as compared to the other sites (Fig. 15).

0.9

0.8 0.7 0.6 0.5 0.4 Pre-monsoon 0.3 Monsoon

DNA Integrity (Ivalue) Integrity DNA 0.2 Post-monsoon 0.1 0

Fig. 15. Variation of DNA integrity in N. chamaeleon along the Goa coast DNA integrity in N. chamaeleon was found to be highest at Arambol in pre-monsoon (I, 0.65) and post-monsoon (I, 0.71), and Dona Paula in monsoon (I, 0.65). In general DNA integrity in pre-monsoon was found to be higher (average I, 0.55) than post-monsoon (average I, 0.48) and monsoon (average I, 0.46). In monsoon, significant decrease in DNA integrity was observed in Velsao (P>0.05) and Palolem (P>0.05) as compared to the reference site Arambol. Statistically significant differences were observed when DNA integrity in snails from Dona Paula and Palolem were compared (P>0.05). Velsao showed the lowest DNA integrity (I, 0.12). In post-monsoon, snails from Anjuna showed 64% decrease in DNA integrity as compared to monsoon. Anjuna (I, 0.19) and Betul (I, 0.39) showed the lowest DNA integrity in post-monsoon and pre-monsoon respectively.

1 0.9 0.8 0.7 0.6 0.5 Pre-monsoon 0.4 Monsoon 0.3

DNA Integrity (Ivalue) Integrity DNA Post-monsoon 0.2 0.1 0

Fig. 16. Variation of DNA integrity in P. sulcatus along the Goa coast

The mean value of DNA integrity in P. sulcatus was found to be 0.50 in pre-monsoon, 0.57 in monsoon, and 0.50 in post-monsoon. In pre-monsoon, the highest DNA integrity was observed at Arambol (I, 0.61), and the lowest was observed at Sinquerim (I, 0.37). During the monsoon, the highest DNA integrity was found to be in Dona Paula (I, 0.80) and the lowest at Hollant (I, 0.31). Significant decrease (P>0.05) in DNA integrity was observed in Hollant (I, 0.25) as compared to Arambol (I, 0.70). Significant differences were also observed when snails from Dona Paula (P>0.01) and Betul (P>0.05) were compared with Hollant. In post- monsoon, the DNA integrity was found to be more than 0.5 at Arambol (I, 0.67), Anjuna (I, 0.50), Dona Paula (I, 0.55), Bogmalo (I, 0.60) and Betul (I, 0.64), whereas very low DNA integrity was observed at Sinquerim (I, 0.37), Hollant (I, 38), Velsao (I, 0.40) and Palolem (I, 0.36). Seasonal variation in DNA integrity was observed in M. granulata with higher values observed during the pre-monsoon (average I, 0.53), followed by post-monsoon (Average I, 0.44), and monsoon (Average I, 0.42). During pre-monsoon, there is a decreasing trend in the DNA integrity in Morula granulata from Arambol (I, 0.75) to Sinquerim (I, 0.40) followed by slight increase at Dona Paula (I, 0.67) and then decreased further up to Hollant (I, 0.33) and finally increased considerably at Palolem (I, 0.70). In monsoon the lowest values was recorded at Hollant (I, 0.30), whereas Velsao showed the lowest values in post-monsoon and pre-monsoon. In post-monsoon, the highest DNA integrity was observed at Arambol (I, 0.75) and the lowest was observed at Hollant (I, 0.33). Hollant (P>0.05), Velsao (P>0.05), and

Palolem (P>0.05) showed significant decrease in DNA integrity as compared to the reference site Arambol.

0.9 0.8

0.7 0.6 0.5 Pre-monsoon 0.4 Monsoon 0.3 Post-monsoon DNA Integrity (Ivalue) Integrity DNA 0.2 0.1 0

Fig. 17. Variation of DNA integrity in M. granulata along the Goa coast

The DNA integrity in N. chamaeleon was found to be drastically reduced by 18% at Anjuna, 14% at Sinquerim, 81% at Velsao, 26% at Betul and 64% at Palolem during monsoon with respect to that at the reference site (Arambol) which can be attributed to the impact of genotoxic pollutants prevalent in the site due to extensive tourist activity as well as oil spills released from the shipping activity around the site. It has been reported in an earlier studies that considerable amount of genotoxic pollutants such as Polycyclic aromatic hydrocarbons (PAHs) were accumulated in the tissues of the marine gastropods, Cronia contracta from the ambient marine environment all along the coast of Goa during the pre- monsoon, monsoon and post-monsoon seasons. The concentration of PAHs accumulated into the tissues of the marine gastropods were in the range of 14.31 – 26.6µg/g (Arambol), 16.37 – 19.61 µg/g (Anjuna), 30.45 – 53.39 µg/g (Dona Paula), 37.83 – 50.35 µg/g (Vasco), 22.5 – 37.77 µg/g (Velsao) and 10.59 – 18.18 µg/g (Palolem) (Sarkar et al, 2008, Desai et al, 2010). This clearly indicates that the coasts of Goa were highly polluted by genotoxic compounds. Apart from PAHs, there were many other genotoxic pollutants such as lead (Pb), cadmium (Cd), copper (Cu), manganese (Mn) and iron (Fe) which were accumulated in the tissues of the marine gastropods from the ambient environment. The concentration of the metals in the tissues of marine gastropods (Cronia contracta) were in the range of 0.1- 0.6 µg/g (Pb), 0.1-

0.8 µg/g (Cd), 0.1- 3.2 µg/g (Cu), 0.4- 6.2 µg/g (Fe) and 0.4- 4.5 µg/g (Mn) (Sarkar et al, 2008). In fact, Anjuna is one of the most popular tourist destinations in Goa and is highly crowded with tourists from all around the world. The large amount of waste materials containing various toxic substances from the surrounding hotels and restaurants were being regularly discharged into the site through the drainage system. Moreover, the oil spills from the offshore regions might also contribute substantially towards pollution of the site. The DNA integrity in N. chamaeleon from Dona Paula was found to decrease by 46.4% in post- monsoon with respect to that in the reference site. It has been observed in an earlier in-vivo experiment that the DNA integrity of marine gastropod, Nerita chamaeleon was greatly affected due to exposure to series of concentrations of genotoxic pollutant, cadmium chloride (10; 25; 50; 100 µg/L) for a period of 5 days (Sarkar et al, 2013). Moreover, an onsite field experiment showed that the DNA integrity in marine gastropod, Cronia contracta was significantly affected due to their exposure to ambient genotoxic pollutants along the Goa coast (Sarkar et al, 2008). DNA integrity in P. sulcatus was found to be quite high at Betul (I, 0.71, 0.64 and 0.55 in monsoon, post-monsoon and pre-monsoon respectively). The high DNA integrity at Betul could due to less pollution of the site as it is far away from shipping and mining activities. As regards the pollution of the Sinquerim beach is concerned it is interesting to observe that the DNA integrity in Morula granulata was drastically reduced by 46.67% at this site during pre-monsoon. Such a huge reduction of DNA integrity in the gastropod at Sinquerim can be attributed to genotoxic pollutants like polycyclic aromatic hydrocarbons being discharged extensively from various types shipping activities such as cargo ships, research vessel, tourist vessel, motor boats, fishing trawler, water scooters, barges sailing through this site as well as accidental oil spills, etc.(Desai, et al, 2010). Moreover, it may be noted that one of such cargo ship, MV River Princess was grounded near Sinquerim- Candolim beach (100m away) since February 2001 releasing huge amount of oil spills all along the coast leading to serious damage to marine ecosystem as evident by substantial increase in the concentration of petroleum hydrocarbon in the sediment from Sinquerim beach (total petroleum hydrocarbon, 89 µg/g) compared to the previously reported values (Ingole, et al., 2006). Among the sampling sites, Velsao was considered as one of the most contaminated sites along the Goa coast as the DNA integrity in Morula granulata reduced significantly by 32% at this particular site whereas it was reduced by 26.67% at Bogmalo with respect to those at the reference site in pre-monsoon. Such a drastic reduction in DNA integrity at

Velsao and Bogmalo could be due to industrial discharge into the coastal water from the surrounding industries. It is important to note that one of the leading agrochemical industries of India, the Zuari Agrochemical Industries is situated on top of the plateau behind the Velsao beach. Interestingly, it has been observed on several occasion that stringent smell of industrial effluents emanated from the discharge point at Velsao straight down the plateau while sampling at this site. This clearly indicates the indiscriminate discharge of industrial effluents from the surrounding industries during sampling at Velsao. The drastic reduction of DNA integrity in Morula granulata at Velsao clearly indicated the prevailing state of coastal pollution at the site. The DNA integrity in N. chamaeleon from Velsao and Palolem was found to be much lower in the monsoon. This could be due to industrial discharge into the coastal water from the surrounding industries. There are several other studies which reports the prevalence of PAH pollution in Velsao (Pasumarthi et al., 2011; Rekadwad & Khobragade, 2015; Krishna et al., 2014). One of the study suggests that South East Asian crude oil (SEACO), dumped into the Arabian sea by reckless transcontinental ships reaches the western Indian coastline because of circulation pattern due to south-west monsoon (IANS, 2013). Compound Specific Isotope 13C analysis along with cross-plot of hopane biomarker diagnostic ratio confirms that the source of PAH in the velsao region is SEACO (IANS, 2013). The most significant reduction of DNA integrity was observed at Hollant (56%). Such a drastic reduction of DNA integrity (I, 0.33) at this site could possibly be due to oil pollution caused by extensive shipping activities as well as industrial discharges. Hollant is located in between the Mormugao harbour and Zuari Agrochemical Industry and is greatly influenced by the discharge of waste materials at these sites.

4.4. Comet Assay The extent of DNA damage in marine gastropods was also evaluated by comet assay to substantiate the observation made by alkaline unwinding assay. The impairment of DNA in marine gastropods is clearly indicated by the decrease in integrity of DNA in N. chamaeleon exposed to various types of genotoxic contaminants prevalent at different sites along the coast of Goa (Fig. 18). In general higher values of TDNA were observed during pre- monsoon. Significant increase in tail DNA was observed at Sinquerim and Arambol (P < 0.01) and Sinquerim and Anjuna (P < 0.01) as compared to the reference site Arambol. In monsoon the percentage tail DNA (TDNA) ranged from lowest at Arambol (21.30±1.93) and highest at Palolem (45.68±0.54). In post-monsoon significant increase in TDNA (P<0.001) was observed in snails collected from Sinquerim (55.85±4.09) as compared to Arambol.

TDNA from Sinquerim & Anjuna and Sinquerim & Betul were also found to be significant (P<0.05). Overall, the highest value of tail DNA was found to be observed at Sinquerim (64.83±1.17).

40 Pre-monsoon 30 Monsoon mean % tail DNA 20 Post-monsoon

Fig. 18. Percentage tail DNA (TDNA) in N. chamaeleon from Goa coast

TDNA value in P. sulcatus was in the range from 26.39±1.61 to 42.25±1.98 in pre- monsoon, 19.22±0.64 to 44.51±1.36 in monsoon, and 15.02±0.28 to 47.14±5.08 in post- monsoon. The mean TDNA during pre-monsoon was found to be higher in comparison to post-monsoon and pre-monsoon. In monsoon, significant change in TDNA was observed at all the sampling sites except Dona Paula with compared to Arambol. TDNA showed significant increase at Sinquerim during monsoon and pre-monsoon as compared to Arambol (P < 0.001). Velsao also showed significant increase in TDNA in monsoon and post-monsoon as compared to Arambol (P < 0.001). Apart from this, the Hollant beach which is situated just after the Velsao beach was also found to be contaminated to a great extent (TDNA 30.35±1.80).

30 Pre-monsoon

20 Monsoon mean % tail DNA Post-monsoon 10

Fig. 19. Percentage tail DNA (TDNA) in P. sulcatus from Goa coast

30 Pre-monsoon Monsoon

mean % tail DNA 20 Post-monsoon 10

Fig. 20. Percentage tail DNA (TDNA) in M. granulata from Goa coast

M. granulata also showed spatial and temporal variation in DNA integrity as measured by comet assay. The TDNA ranged from 26.20±0.15 to 47.87±1.51 in pre- monsoon, 14.70±1.03 to 35.33±3.74 during monsoon and from 17.66±1.12 in post-monsoon. In pre-monsoon, TDNA at Palolem (41.36±0.87) and Betul (47.87±1.51) were significantly higher (P < 0.01) than Arambol (26.20±0.15). In general, TDNA was found to be higher during post-monsoon (average TDNA 39.64±1.89) than in pre-monsoon (average TDNA 33.87±0.98) and monsoon (average TDNA 25.50±1.96). In post-monsoon, significant

decrease in TDNA was observed in Sinquerim (P < 0.05), Bogmalo (P < 0.05), Velsao (P < 0.05), Betul (P < 0.05), and Palolem (P < 0.05).

The comet assay allows the quantitation of single cells so that a population profile for an experiment may be visualized. The extent of DNA damage in marine gastropods as measured by the comet assay is expressed in terms of the proportion of DNA present in the comet-tail. It follows the same trend as observed by alkaline unwinding assay. During monsoon, the precipitation and land runoff increases which can causes dynamic changes from typically marine to brackish water conditions (Qasim and Sengupta, 1981) which leads to changes in water quality parameters along with the metal levels. Enhanced DNA damage was observed in N. chamaeleon at Anjuna, Dona Paula and Velsao in post-monsoon with respect to those from the reference site (Arambol) for most of the study regions as believed to be contaminated by genotoxic compounds. The decrease in the proportion of DNA in comet head illustrates the increase in the amount of genotoxic pollutants along these sites. Polycyclic aromatic hydrocarbons (PAHs) being the most predominant components of oil contaminants may likely to cause severe DNA damage in marine invertebrates either by direct DNA strand breakage or are metabolized into reactive intermediates that can form unstable DNA adducts. Moreover, heavy metals have a tendency to bind to phosphates and a wide variety of organic molecules, including base residues of DNA, which can lead to mutations by altering primary and secondary structures of the DNA (Wong, 1988). Exposure to such anthropogenic chemicals can have mutagenic and carcinogenic effects, often inducing cancerous diseases (Mix, 1986). There have been few studies concerning the assessment of genotoxic risks on marine snails due to the difficulty in quantifying exposure levels, bioavailability and effects (Hamers et al., 2004; Regoli et al., 2006). Previous studies reported the changes in DNA integrity caused by contaminants have referred to DNA damage (comet assay) as biomarker of contaminant stress (Clement et al., 2004; Reinecke and Reinecke, 2004). It has been observed that the impact of genotoxic contaminants like PAH and metals was greatly pronounced by the extent of DNA damage in marine gastropods, Cronia contracta (Sarkar et al, 2008), Planaxis sulcatus (Bhagat et al., 2012) along the coast of Goa. The proportion of DNA in the comet-tail was significantly increased in Morula granulata collected from the different sampling sites along the coast of Goa as compared to the reference site. The majority of control cells consisted of a nucleoid core with zero or minimal DNA migrating toward the anode. The most pronounced DNA damage as estimated by Comet assay in terms of %tail DNA for Morula granulata was 47.8% tail-DNA at Betul

as compared to 26.2 % tail-DNA at the reference site (Arambol) in pre-monsoon. Enhanced DNA damage as indicated by higher TDNA in P. sulcatus from Dona Paula with respect to those from the reference site (Arambol) may be due to the presence of high amount of genotoxic compounds. Higher amount of Cadmium ( 0.7 ± 0.03 µg/g) and Copper (2.6 ± 0.04 µg/g) was recorded in snails from Dona Paula (Sarkar et al., 2008). Heavy metals have a tendency to bind to phosphates and a wide variety of organic molecules, including base residues of DNA, which can lead to mutations by altering primary and secondary structures of the DNA (Wong, 1988). As regards the pollution of the Sinquerim beach is concerned it is interesting to observe that the TDNA in N. chamaeleon was significantly high this site. Such a huge increase in TDNA in the gastropod at Sinquerim can be attributed to genotoxic pollutants like polycyclic aromatic hydrocarbons being discharged extensively from various types shipping activities such as cargo ships, research vessel, tourist vessel, motor boats, fishing trawler, water scooters, barges sailing through this site as well as accidental oil spills, etc.(Desai, et al, 2010). Velsao also showed very high TDNA in all the three snails studied. Such a huge increase in TDNA at this site could possibly be due to oil pollution caused by regular discharge of industrial effluents from the pesticides factories situated at the vicinity of the area. Higher values of Cadmium (1.0 ± 0.02 µg/g) have been reported in snails from this site (Sarkar et al., 2008). PAHs are suspected of having a genotoxic effect and some of the PAHs such as Benzo(a)pyrene, Indeno[1,2,3-c,d]pyrene, Benzo[g,h,i]perylene etc. are well known for their genotoxic effects (Perez-Cadahia et al., 2004; Woo et al., 2006). There are strong evidences that some of them are carcinogenic (Diguilio et al., 1995) with the capacity to cause various types of DNA damage. Benzo(a)pyrene, a representative PAH is reported to be converted at cellular level to chemically reactive oxygen species, diol-epoxide (BaPDE) which can form stable adduct with DNA resulting into DNA strand breaks (Sarkar et al., 1997).

4.5. Biochemical tests (Nerita chamaeleon) Oxidative stress biomarkers have a marked variation between sites. GST activity increased significantly from the value at the reference site (Fig. 21). Higher values of GST were observed during monsoon period which was significantly higher as compared to pre- monsoon (P < 0.001). The difference between GST activity in monsoon and pre-monsoon

96 was also significant (P < 0.001). Significant statistical differences were depicted for GST activity between sites during all the three seasons.

250

200

150 Pre-monsoon

100 Monsoon Post-monsoon

GST (nM/min/mg of of GST (nM/min/mg protein) 50

0 Arambol Sinquerim Dona Velsao Betul Palolem paula

Fig. 21. Glutathione S-transferase (GST) activity in N. chamaeleon from Goa coast

0.5

0.45 0.4 /min/mg)

2 0.35 O 2 0.3 0.25 Pre-monsoon 0.2 Monsoon 0.15

CAT (mM of H of (mM CAT Post-monsoon 0.1 0.05 0 Arambol Sinquerim Dona Velsao Betul Palolem paula

Fig. 22. Catalase (CAT) activity in N. chamaeleon from Goa coast

Pre-monsoon 15 Monsoon Post-monsoon 10 SOD (U/mg ofprotein) SOD

0 Arambol Sinquerim Dona paula Velsao Betul Palolem

Fig. 23. Superoxide dismutase (SOD) activity in N. chamaeleon from Goa coast

1.4

1.2

0.8 Pre-monsoon 0.6 Monsoon LPO (nM of MDA/min) Post-monsoon 0.4

0.2

0 Arambol Sinquerim Dona Velsao Betul Palolem paula

Fig. 24. Lipid Peroxidation (LPO) value in N. chamaeleon from Goa coast

200

180 160 140 120 100 Pre-monsoon 80 Monsoon 60 Post-monsoon 40 AChE (U/min/mg of protein) of (U/min/mg AChE 20 0

Fig. 25. Acetylcholinesterase (AChE) activity in N. chamaeleon from Goa coast

CAT activity in the sails was in the range from 0.03±0.05 to 0.39±0.05 mM/min/mg. The mean CAT activity during monsoon was found to be higher in comparison to post- monsoon and pre-monsoon. CAT activity showed significant variations at Sinquerim during monsoon (P < 0.05) and pre-monsoon (P < 0.001) as compared to Arambol. Significant decrease in CAT activity was observed at all the sampling sites except Dona Paula with compared to Arambol. The highest SOD activity was observed in snails collected from Dona Paula (17.27±1.07 U/mg of protein) during monsoon and lowest at Betul (0.62±0.08 U/mg of protein) during pre-monsoon. SOD activity showed temporal and spatial variation with higher values at monsoon. SOD activity was significantly higher at Sinquerim during monsoon (P < 0.05) and pre-monsoon (P < 0.001) as compared to Arambol. Significant differences in SOD activity was observed for Velsao (P < 0.01) and Palolem (P < 0.05) as compared to the reference site Arambol. Along the study areas, the activity of LPO also showed wide variations. Lipid peroxidation showed many inter-site differences during pre-monsoon, monsoon, and post- monsoon. In general higher AChE activity was recorded during pre-monsoon season. During monsoon, the highest LPO level was observed at Sinquerim (0.66±0.02) and the least at Palolem (0.18±0.03). LPO activity was significantly higher at Sinquerim in all season as compared to the reference site Arambol.

Fig. 25 clearly shows the variation in AChE activity in N. chamaeleon along the Goa coast during the three seasons. Significant increase in AChE activity was observed at Sinquerim in monsoon as compared to Arambol. In post-monsoon significantly higher AChE activity was observed at Dona Paula as compared to Arambol (P < 0.001).

Biochemical tests (Planaxis sulcatus) GST activity showed spatial and temporal variations in Planaxis collected from Goa region. GST activity in the sails was in the range from 8.41±0.13 to 81.71±18.25 mM/min/mg during pre-monsoon, 16.95±9.97 to 267.20±15.44 mM/min/mg during monsoon, and 10.62±2.46 to 78.70±8.68 during post-monsoon. GST activity was significantly higher during monsoon as compared to post-monsoon and pre-monsoon. Arambol showed lower GST activity compared to all other sites. GST activity in monsoon was almost 3 folds as compared to post-monsoon or pre-monsoon.

300

250

200

150 Pre-monsoon

100 Monsoon Post-monsoon 50 GST (nM/min/mg of of GST (nM/min/mg protein)

Fig. 26. Glutathione S-transferase (GST) activity in P. sulcatus from Goa coast

100

0.6

0.5

/min/mg) 0.4 2 O 2

0.3 Pre-monsoon Monsoon 0.2 Post-monsoon CAT (mM of H of (mM CAT 0.1

Fig. 27. Catalase (CAT) activity in P. sulcatus from Goa coast

9 8

7 6 5 Pre-monsoon 4 3 Monsoon SOD (U/mg ofprotein) SOD 2 Post-monsoon 1 0

Fig. 28. Superoxide dismutase (SOD) activity in P. sulcatus from Goa coast

101

1.2

0.8

0.6 Pre-monsoon

0.4 Monsoon

LPO (nM of MDA/min) Post-monsoon 0.2

Fig. 29. Lipid Peroxidation (LPO) value in P. sulcatus from Goa coast

180

160 140 120 100 Pre-monsoon 80 60 Monsoon 40 Post-monsoon AChE (U/min/mg of protein) of (U/min/mg AChE 20 0

Fig. 30. Acetylcholinesterase (AChE) activity in P. sulcatus from Goa coast

Higher values of CAT were observed during pre-monsoon period. In general, Velsao showed higher CAT activity during all the three season. Seasonal variations in SOD activity in snails were not prominent. The highest SOD activity was observed in snails collected from Sinquerim (7.16±0.93 U/mg of protein) during pre-monsoon and lowest at Betul (0.72±0.01 U/mg of protein) during the same season. The average SOD activity was slightly higher in monsoon as compared to other two seasons. The activity of LPO showed a wide variations ranging from 0.03±0.00 to 0.80±0.02 during pre-monsoon, from 0.01±0.00 to 0.94±0.05 during monsoon, and from 0.05±0.01 to

102

0.44±0.01 during post-monsoon. The average LPO activity during monsoon was 1.5 times higher in monsoon as compared to post-monsoon. Intersite differences in AChE activity was observed in snails with highest values recorded at Betul (82.26±20.51 U/min/mg) in pre-monsoon, Sinquerim (84.92±0.61 U/min/mg) in monsoon, and Arambol (152.27±0.34 U/min/mg) in post-monsoon. The lowest values were recorded at Hollant (19.18±0.16 U/min/mg) during post-monsoon.

Biochemical tests (Morula granulata) Significant seasonal variations in oxidative stress biomarkers were observed gastropods (Fig. 31). GST activity was significantly higher in monsoon as compared to post monsoon and pre monsoon. The GST activity ranged from 3.85±0.83 to 96.96±29.33. GST activity was significantly high in Betul (96.96±29.33) during monsoon. The lowest activity was observed in Palolem (13.52±1.51). During the post-monsoon, the lowest GST activity was found at Arambol (14.94±3.48) and the highest was at Velsao (59.75±9.57). GST activity increased three fold during monsoon as compared to pre-monsoon.

140

120

100

80 Pre-monsoon 60 Monsoon 40 Post-monsoon GST (nM/min/mg of of GST (nM/min/mg protein) 20

Fig. 31. Glutathione S-transferase (GST) activity in M. granulata from Goa coast

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0.6

0.5

/min/mg) 0.4 2 O 2

0.3 Pre-monsoon Monsoon 0.2 Post-monsoon CAT (mM of H of (mM CAT 0.1

Fig. 32. Catalase (CAT) activity in M. granulata from Goa coast

20 18

16 14 12 10 Pre-monsoon 8 Monsoon 6

SOD (U/mg ofprotein) SOD Post-monsoon 4 2 0

Fig. 33. Superoxide dismutase (SOD) activity in M. granulata from Goa coast

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1.4

1.2

0.8 Pre-monsoon 0.6 Monsoon

LPO (nM of MDA/min 0.4 Post-monsoon

0.2

Fig. 34. Lipid peroxidation (LPO) value in M. granulata from Goa coast

CAT activity was highest in pre-monsoon and lower in post-monsoon and monsoon. Hollant showed significantly higher CAT activity during all the season as compared to the reference site Arambol. Arambol showed the lowest CAT activity during post-monsoon and pre-monsoon, however it was Bogmalo which showed the lowest CAT activity during monsoon. The average CAT activity during post-monsoon was 1.5 times higher as compared to monsoon.

250

200

150 Pre-monsoon 100 Monsoon Post-monsoon

AChE (U/min/mg of protein) of (U/min/mg AChE 50

Fig. 35. Acetylcholinesterase (AChE) activity in M. granulata from Goa coast

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SOD activity exhibited only moderate variations with season showing slightly higher values in monsoon than post-monsoon and lowest values in pre-monsoon. SOD activity was lowest in the reference site during monsoon and post monsoon, however Bogmalo showed the lowest value for SOD in pre-monsoon. There were no significant differences in LPO among seasons. LPO values in snails ranged from 0.02±0.00 to 1.14±0.15 during pre-monsoon, from 0.04±0.00 to 0.57±0.03 during monsoon, and from 0.15±0.04 to 0.85±0.11 during post-monsoon. Sinquerim showed significantly higher values for LPO in all the seasons with highest values during pre-monsoon (1.14±0.15). Seasonal and spatial variations in AChE activity was recorded in snails collected from different sites from Goa. Higher values were recorded during monsoon season as compared to post-monsoon and pre-monsoon. During pre-monsoon, only Betul and Hollant showed significant differences in AChE activity (P<0.05). During post-monsoon, all the sampling sites showed significant differences in AChE activity as compared to Arambol, however there were no statistically difference observed when sampling sites were compared for monsoon period. Seasonal pattern in oxidative stress biomarkers have been observed in this study. In general, an increase in all the biomarkers has been recorded during monsoon. Temperature has been considered as one of most prominent factor influencing the antioxidant defence system. Higher temperature induces the oxygen consumption in the cell leading to elevated production of ROS, which are compensated antioxidant defence (Ronisz et al., 1999). Higher oxidative defence were also reported during high temperature period in cichlid fish acará (Geophagus brasiliensis) by Wilhelm Filho et al., (2001). In this study, SOD activity in snails was significantly higher in monsoon as compared to other seasons. SOD activity showed lowest values during pre-monsoon, which is in accordance with studies on mussel Perna viridis (Verleacar et al., 2008). GSTs play a critical role in defence against oxidative damage and peroxidative products of DNA and lipids (Oost et al., 2003). Studies with GST in gastropods exposed to environmental or anthropogenic contaminants has shown decrease (Ma et al., 2014) or unaltered (Bianco et al., 2013) enzyme activities. Few studies report increase in GST activity in gastropods exposed to contaminants (Zheng et al., 2013; Cabecinhas et al., 2014;). Contrarily to the antioxidant enzymes, GST showed negative correlation with PAH in this study. GST is a phase II metabolizing enzyme and catalyzes the conjugation of reduced glutathione (GSH) with PAH derivatives. PAH metabolites such as quinone have strong

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affinity towards glutathione (Xue and Warshawsky, 2005). GSH can be converted to its oxidised state (GSSG) by Glutathione peroxidase (GPx). Decrease in GSH has been reported in molluscs exposed to PAHs (Martins et al., 2012). The decrease in GST activity after long exposure to PAH can be due to reduction of GSH levels. Silva et al., (2005) observed a significant increase in GST activity in Crassostrea rhizophorae exposed to diesel oil. Correlation of GST activity with PAH tissue content has also been shown for green-lipped mussel, Perna viridis (Cheung et al., 2001). Few studies have reported decrease in GST activity in fishes exposed to PAH (Wang et al., 2006). Akcha et al., (2000) has shown depressed GST activity in gills of mussels by exposure to high levels of B[a]P. High LPO value were observed during post-monsoon in samples from Dona Paula. The Dona Paula creek is situated between Mandovi and Zuari estuaries opposite to the Mormugao Harbour. Because of its tourist attraction the creek is over crowded with large numbers of hotels, restaurants, motor vehicles, boats, water scooters contributing extensively towards contamination of the site. Studies have reported high amount of heavy metals in mussels (Rivonker and Parulekar, 1998) and snails (Sarkar et al., 2008). Organotins and butylin compounds have been reported from water, sediment and animal samples from this site (Bhosle et al., 2006; Meena et al., 2009). During the post-monsoon season the traffic due to ships and recreational boats are high, this may be one of the reason for increase amount of pollutants leading to altered biochemical activities in organisms (Meena et al., 2009). It is interesting to note that AChE activity were also found to be higher in N. chamaeleon from Dona Paula during post-monsoon. Aquatic organism has shown increase AChE activity when exposed to TBT (Puccia et al., 2001; Greco et al., 2007). TBT exerts a positive regulation in the expression of this enzyme by regulating a transcription factor (NF-kB) (Puccia et al., 2001). So increase in TBT during post-monsoon can be one of the reason for increasing AChE activity. LPO is a sensitive indicator of oxidative stress due to PAH (Cossu et al., 1997). Free radicals produced as a result of metabolic activities can react with polyunsaturated fatty acids in cell membrane, resulting in increase of lipid peroxidation (Livingstone et al., 2003). LPO in bivalves exposed to PAH is well documented (Pan et al., 2005; Kaloyianni et al., 2009). High LPO activity in P. sulcatus at Sinqurim and Dona Paula can be due to high amount of PAH. In addition to PAH, presence of heavy metals also tend to stimulate LPO activity. In the field, sites contaminated with heavy metal have shown to induce LPO in snails (Radwan et al., 2010a; Abdel-Halim et al., 2013). In our study we have observed highest LPO activity in P. sulcatus collected from Anjuna during monsoon. Anjuna is one of the most popular

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tourist destinations in Goa and is highly crowded with tourists from all around the world. The large amount of waste materials containing various toxic substances from the surrounding hotels and restaurants were being regularly discharged into the site through the drainage system. Land runoff during the monsoon may lead to increase in metal pollution leading to increase in LPO activity. Cu, Pb, Zn have also shown to increase LPO in T. pisana (Radwan et al., 2010b). Increased lipid peroxidation occurred in L. natalensis exposed to sediment and water from metal-polluted sites (Siwela et al., 2010).Higher values of LPO were reported in digestive glands of H. aspersa collected from metal contaminated sites by Abdel-Halim et al. (2013). Elevated level of LPO was reported in kidneys and digestive gland in A. fulica exposed to

CdCl2 and ZnSO4 (Chandran et al., 2005). Cadmium doesn't generate ROS directly, but can alter the GSH and MT level in the cell, which can lead to LPO of cell membrane (Mahboob et al., 2013). It has been known that LPO products are complex hydroperoxides that exerts cytotoxic and genotoxic damage. We have observed positive correlation between SOD and PAH in sediment in all seasons. Numerous studies have also documented significant relationships between antioxidant activities, SOD and metal or PAH body burdens in mussels (Manduzio et al., 2003). Niyogi et al., (2001a) has also reported positive correlation of CAT and SOD activity with PAH tissue content in S. cucullata. Such a relationship has also been reported for different bivalve species exposed to hydrocarbons (Richardson et al., 2008) and suggests that - hydrocarbons induce oxidative stress by producing ROS such as O2 . In monsoon, higher catalase activity was observed in snails as compared to other season. Zanette et al., (2006) also observed higher catalase activity in oyster collected during summer. GST activity was found to be higher during monsoon as compared to other season. In our study we have observed higher seawater temperature during monsoon (27.50±0.71°C to 31.50±0.00°C). GST activity was found to be several fold higher in other sites as compared to Arambol (fig. 26). GST is one the most efficient phase II biotransformation pathways for potentially toxic chemicals in invertebrates. The induction of GST activity has thus been suggested to be adopted for use as a biomarker of exposure to chemicals such as PAHs, PCBs and dioxins (Oost et al., 2003). The activity of GST depends on the several factors such as conjugation of GSH to pollutants. Increase in GST activity in these sites can be due to the high amount of pollutants in the site. Bebiano et al., (2007) has also reported five to six fold increase in GST between reference and polluted sites.Temperature has been shown to cause variations in GST activity in mussel Mytilus galloprovincialis (Bebianno et al., 2007). Temperature along with other abiotic factors induces metabolic response leading to increase in oxidative defence in

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aquatic organisms (Kamel et al., 2014). Positive correlation between oxidative stress biomarkers and temperature has been reported by Cravo et al., (2009). Water temperature has shown to have significant effect on GST activity barnacle, Balanus balanoides (Niyogi et al., 2001). In another study, Schmidt et al., (2013) has also observed higher GST activity in mussel in summer with comparison to other seasons. In this study, snails collected from Velsao have shown significantly higher GST activity as compared to other sites. One of the possible explanation for higher GST activity might be the contamination load in the sediments of Velsao as observed through PAH analysis. Silva et al., (2005) observed a significant increase in GST activity in Crassostrea rhizophorae exposed to diesel oil. Correlation of GST activity with PAH tissue content has also been shown for green-lipped mussel, Perna viridis (Cheung et al., 2001). Vidal-Linan et al., (2010) has used integrated biomarker approach using GST, glutathione peroxidase (GPx) and catalase (CAT) and reported significant positive correlation between PAHs and GST activity in mussels Mytilus galloprovincialis. LPO level in snails collected during monsoon were higher as compared to other season, which is in agreement with study on amphipod Hyalella curvispina (Dutra et al., 2008). In general snails from Sinquerim showed higher values of LPO, which might be due to exposure of snails to high level of organic contamination such as PAH as observed during our study. Similar to our results higher LPO level in mussels contaminated with PAH have been reported by Fernandez et al., (2012). Gastropods perform an important role in marine food chain, and are used as a source of food for many fishes and birds. They transfer toxicants from lower to higher tropic level organism through food chain. They have limited ability to metabolize xenobiotics and thus they are prone to accumulate high concentrations of hydrocarbons (Zheng et al., 2012). The total PAH concentrations in sediments from Goa region ranged from lowest value at Arambol (1.44±0.12 µg/g) to highest value at Velsao (4.30±0.34 µg/g). In Velsao, the mean PAH concentrations were found to be 3.86±0.61 µg/g which was significantly higher than Arambol. Such higher values of PAH at Velsao revealed the severity of organic pollution in Velsao. Sinquerim also showed higher values of PAH with the mean value of 3.35±0.20 µg/g. Presence of high amount of PAH at Sinquerim may be due to extensive shipping activities as well as accidental oil spills (Desai et al., 2010). Ingole et al., (2006) has reported high level of total petroleum hydrocarbon at Sinquerim-Candolim beach due to grounding of MV River Princess. Sarkar et al., (2008) studied the seasonal variation of PAH in Cronia contracta collected from six sites along the Goa coast and reported positive correlation between the PAH and DNA damage. Dona Paula has been previously reported for heavy

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metal and PAH contamination (Sarkar et al., 2008). Pise et al., (2013) has reported significantly higher CAT and GST activity in marine red alga Porphyra vietnamensis collected from Dona Paula than in samples from Malvan and Kunkeshwar. Author also reported higher values of heavy metals Pb (1.98 ± 0.17 μg g–1), Cd (0.98 ± 0.18 μg g–1), and Hg (0.99 ± 0.19 μg g–1) in the algae. High values of TBT and other organotin compounds were also reported in Dona Paula (Meena et al., 2009). PAH concentration at Betul and Palolem were found to be comparatively lower which could be due its geographical locations as it is far away from shipping, mining and other harbour activities (Gaitonde et al., 2006; Sarkar et al., 2008). Occurrence of frequent oil spills also deteriorates the coastal sediment. Accumulation of tar balls noticed along the Goa coast during the southwest monsoon shows the severity of the pollution in Goa beach (Suneel et al., 2013a, b).

4.6. Integrated Biomarker Response (IBR)

Similar pattern of star plots were observed with genotoxic, oxidative stress and neurotoxic biomarkers. Mean IBR values for N. chamaeleon was found to be lowest at Arambol (1.00±0.21) and highest at Velsao 12.16±0.68. IBR values at Velsao was significant different as compared to Arambol (P<0.01), Sinquerim (P<0.05), Dona Paula (P<0.01) and Betul (P<0.05). Velsao showed the highest IBR values for all the biomarker studied, followed by Palolem, Sinquerim, Betul, Dona Paula and Arambol. In P. sulcatus, similar trend for IBR were observed with highest values recorded at Velsao (11.63±0.68), followed by Palolem (8.24±0.73), Bogmalo (7.17±0.91), Anjuna (6.95±1.05), Betul (6.07±0.77), Dona Paula (4.06±0.91), Sinquerim (3.94±0.91), Hollant (3.80±0.91), and Arambol (0.94±0.21). All the sites except Sinquerim showed significantly higher values as compared to reference site. Mean IBR values between Velsao & Dona Paula (P<0.01), and Velsao & Sinquerim (P<0.01) were also found to be significant.

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Fig. 36. Comparison of IBR index for genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), oxidative stress [superoxide dismutase (SOD), catalase (CAT), Glutathione S-transferase (GST) and lipid peroxidation (LPO)] biomarkers, average PAH concentrations, mean IBR and Acetylcholinesterase (AChE) activity in N. chamaeleon from different sites of Goa, India.

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Fig. 37. Comparison of IBR index for genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), oxidative stress [superoxide dismutase (SOD), catalase (CAT), Glutathione S-transferase (GST) and lipid peroxidation (LPO)] biomarkers, average PAH concentrations, mean IBR and Acetylcholinesterase (AChE) activity in P. sulcatus from different sites of Goa, India.

The mean IBR values calculated from four biomarkers was found to be lowest in M. granulata collected from Betul (2.36±0.77), whereas Hollant showed the highest IBR values (7.55±0.91). Higher IBR values were observed at Sinquerim, Bogmalo and Palolem, but they were not found to be significantly different as compare to the reference site.

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Fig. 38. Comparison of IBR index for genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), oxidative stress [superoxide dismutase (SOD), catalase (CAT), Glutathione S-transferase (GST) and lipid peroxidation (LPO)] biomarkers, average PAH concentrations, mean IBR and Acetylcholinesterase (AChE) activity in M. granulata from different sites of Goa, India. In our study, the lowest mean values of IBR for all the biomarkers during monsoon were observed in the reference site, Arambol. Several authors have reported lower values of IBR in reference site than in contaminated sites as (Tankoua et al., 2013; Turja et al., 2014). Palolem showed the lowest value of mean IBR for post-monsoon, this is due to lower biomarker activity observed during the post-monsoon. The highest IBR was observed at Velsao in N. chamaeleon and P. sulcatus but in M. granulata it was highest at Hollant. Samples from Velsao has showed higher biochemical acitivity as well as high DNA damage leading to high IBR, wherease partiularly in M. granulata, high CAT and SOD activity has resulted into high IBR. GST is a phase II metabolizing enzyme and catalyzes the conjugation of reduced glutathione (GSH) with PAH derivatives that have strong affinity towards glutathione (Xue and Warshawsky, 2005). GSH can also be converted to its oxidised state (GSSG) by Glutathione peroxidase (GPx). Increase in GPx activity might result in scarcity of GSH which in turn can affect GST activity. Decrease in GSH has been reported in molluscs exposed to PAHs

(Martins et al., 2012). GPx eliminates H2O2 while carrying out GSH to GSSG conversion.

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Increase in GPx activity might lead to reduction of H2O2, which in turn might result in lower catalase activity. IBR has been applied to study the spatial and temporal variations in biomarkers in aquatic organisms (Trujillo-Jiménez et al., 2011; Tlili et al., 2013). IBR has been used to construct star plot with PAH and PCB concentrations in tissues along with early warning signals (EROD, GST, CAT, AChE enzymatic activities) and adverse effects (DNA adducts) biomarkers in flounder Platichthys flesus (Beliaeff and Burgeot, 2002). Campillo et al., (2013) have used AChE, GST, CAT, and LPO in caged clam Ruditapes decussatus for IBR. IBR index has been used to study the PAH contamination in caged mussel Mytilus trossulus and Mytilus galloprovincialis (Dabrowska et al., 2013). In general the mean IBR values of all biomarker were found to be higher during monsoon than in others seasons. Comparatively high temperature recorded during monsoon might also lead to higher antioxidant defences (Wilhelm Filho et al., 2001). Seasonal variations were observed in all biomarkers. Thus higher values of PAH and oxidative stress biomarkers at Velsao, Sinquerim and Dona Paula shows critical unbalance ROS formation and the severity of pollution at these sites.

4.7. Exposure 4.7.1. Exposure to phenanthrene 4.7.1.1. Biochemical Assays GST activity increased significantly at all the exposed concentrations with respect to control, indicating oxidative damage in gastropods (Fig. 39). Overall GST activity showed concentration-dependent increase in gastropods exposed to phenanthrene. GST activity increased from 23.88±1.12 nM/min/mg in control to 35.78±1.45 nM/min/mg at 10 µg/L (P < 0.01) of phenanthrene. There were significant differences in GST activity at 10 µg/L (P < 0.01), 25 µg/L (P < 0.01), 50 µg/L (P < 0.001) and 100 µg/L (P < 0.001). The activity of CAT showed no significant difference between exposed groups and in control, except for 25 µg/L where a significant decrease (p < 0.01) was observed (Fig. 39). However a significant difference in CAT activity was observed when 25 µg/L and 50 µg/L was compared (p < 0.001).

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Fig. 39. Glutathione S-transferase (GST), catalase (CAT) and lipid peroxidation (LPO) value, depicted by means±standard deviation in marine gastropod M. granulata exposed to phenanthrene. (a) p<0.05, (b) p<0.01, (c) p < 0.001 significantly different from the control (ANOVA, Tukey HSD post-test).

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Fig. 40. Comet parameter (DNA strand breakage) in M. granulata exposed to phenanthrene. Values are means± standard deviation, (a) p<0.05, (b) p<0.01, (c) p < 0.001 significantly different from the control (ANOVA, Tukey HSD post-test).

LPO values were higher in gastropods exposed to phenanthrene as compared to control gastropods (Fig. 39). LPO in control was found to be 0.21 nM MDA/mg, and on exposure to phenanthrene, increased to 0.26±0.03 nM MDA/mg at 10 µg/L (p < 0.05) and 0.37 ±0.03 nM MDA/mg at 25 µg/L (p < 0.05). However, there was decrease in LPO observed from 0.49±0.04 nM MDA/mg at 50 µg/L to 0.39±0.06 nM MDA/mg at 100 µg/L. When LPO between 25 µg/L and 100 µg/L were compared no significant difference was observed. Moreover, the 50 µg/L displayed a significant increase with all the other groups.

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4.7.1.2. DNA damage after in vitro exposure to hydrogen peroxide Fig. 40 shows significant increase in % DNA in tail in M. granulata exposed to hydrogen

peroxide. H2O2 at 50 µM showed 2.3 fold increase in % DNA in tail compared with the

control (p<0.001). H2O2 induced a concentration-dependent increase in % DNA in tail.

4.7.1.3. In vivo exposure of gastropods to phenanthrene The % DNA in tail as measured by comet assay in M. granulata exposed to phenanthrene also showed significant increase in DNA damage (Fig. 40). A significantly increase in % DNA in tail was observed at 50 µg/L (28.60±1.23) (p < 0.001) and 100 µg/L (32.6±0.19) (p < 0.001) with respect to control (18.23±0.47). It has been observed that % DNA in tail increased significantly at 100 µg/L as compared to those in 10 µg/L (p < 0.001) and 25 µg/L (p < 0.001).

4.7.1.4. Relationship between oxidative stress parameters and comet assay There were significant correlations between GST and LPO (r = 0.86, P < 0.001) as well as between GST activity and % DNA in tail (r = 0.96, P < 0.001). A similar pattern between % DNA in tail and LPO (r = 0.82, P < 0.001) was revealed by spearman's correlation. There was no correlation observed between CAT and any of the parameters tested.

4.7.1.5. Integrated Biomarker Response (IBR) The IBR values for four biomarkers were standardized and computed as star plot ( Fig. 41). All the four biomarkers were responsive to phenanthrene exposure and showed similar pattern in star plots. The IBR values increased with increase in exposure concentrations of phenanthrene. The IBR value ranged from 0.70±0.07 to 45.74±1.45. The lowest value for IBR was observed in control samples whereas the highest value was observed at 100 µg/L of phenanthrene. There was no significant variations observed when IBR values for 10 µg/L were compared with control. At 50 µg/L and 100 µg/L concentrations, all the biomarker showed significant increase in IBR values when compared to control (p < 0.001).

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Fig. 41. Star plots for standardized biomarker response in snails exposed to different concentrations of phenanthrene.

Phenanthrene is a low molecular weight polycyclic aromatic hydrocarbon (PAH) commonly found as pollutants in aquatic ecosystem. It is not among the list of human carcinogen, but has shown to be toxic to clams, mussels, crustaceans and fish. Few studies have reported the lysosomal responses in marine gastropod exposed to phenanthrene (Moore et al., 1985; Pipe and Moore, 1986). Studies on genotoxic damage and oxidative stress with respect to phenanthrene are very limited. Molluscs have a greater tendency to bioaccumulate PAHs (Leon et al., 2013). Among molluscs, gastropods perform an important role in marine food chain, and are used as an important source of food by fishes and birds. They have limited ability to metabolize xenobiotics and thus they accumulate high concentrations of hydrocarbons (Zheng et al., 2012). Phenanthrene has been shown to be accumulated in oyster (Sanders, 1994), mussels (Namiesnik et al., 2008), clams (Tian et al., 2014), scallop (Hannam et al., 2010 a,b), amphipod (Landrum et al., 1994) and fishes (Bandowe et al., 2014). Studies have reported high concentrations of phenanthrene in tissues of gastropods (Rostad and Pereira, 1987). M. granulata bioaccumulate high concentrations of contaminants because of its carnivore feeding behaviour. They consume variety of prey such as oyster (Saccostrea), barnacle

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(Tetraclita), gastropod (Planaxis), pulmonate limpets (Siphonaria) and bivalve (Isognomon) (Koh-Siang, 2003). This species has a tendency of selective drilling in oyster Saccostrea cuccullata and barnacle Tetraclita squamosa (Harper and Morton, 1997; Tan, 2003). During our sampling, we have observed a close association between M. granulata with oyster Saccostrea spp. and gastropod P. sulcatus. Toxicity of phenanthrene causes increase in the production of reactive oxidation species (ROS). Antioxidant system plays a significant role in maintaining the oxidative equilibrium by consuming excessive ROS. Cell is under oxidative stress when the production of ROS species exceeds the production of cellular oxidants. Phenanthrene has shown to stimulate the production of hydroxyl radical in liver of C. auratus (Sun et al., 2006) and impairment in oxidative system in oyster Crassostrea brasiliana (Luchman et al., 2014). Among the antioxidant enzymes, GST plays a significant role in protecting the cells from oxidative stress. In our studies we have found a concentration-dependent increase in GST activity in gastropods exposed to phenanthrene. Similar findings were reported in clam, Venerupis philippinarum exposed to phenanthrene (Zhang et al., 2014). Other studies have shown induction of GST activity in molluscs Pecten maximus exposed in vitro to PAH (Pennec and Pennec, 2003). Silva et al., (2005) observed a significant increase in GST activity in Crassostrea rhizophorae exposed to diesel oil. In contrast, few studies has reported unaltered or decrease in GST activity in fishes exposed to phenanthrene (Oliveira et al., 2008; Xu et al., 2009). Significantly decreased in GST activity were also reported in oyster Crassostrea brasiliana following 24 h exposure to 100 µg/L of phenanthrene (Luchman et al., 2014). PAH metabolites such as quinone have strong affinity towards GSH (Xue and Warshawsky, 2005). In our studies, decreasing trend in CAT activity was observed from control sample to 25 µg/L exposure of phenanthrene and then the value was shown to increases at 50 µg/L exposure concentration. Similar findings were reported in estuarine guppy Poecilia vivipara exposed to phenanthrene (10, 20 and 200 µg/l) (Machado et al., 2014). No significant change in CAT activity was observed in exposed snails. However decrease in CAT activity was reported in L. aurata (Oliveira et al., 2008) and C. auratus (Yin et al., 2007) exposed to high concentrations of phenanthrene. Increase, decrease or unaltered activity of CAT in aquatic organism exposed to PAH has been reported by various studies (Cheung et al., 2001; Niyogi et al., 2001a,b; Pichaud et al., 2008). Frouin et al., (2007) also reported no difference in the CAT activity in clams Mya arenaria exposed to PAH. Catalase plays a key role in antioxidant mechanism in converting hydrogen peroxide (H2O2) to water. Antioxidant

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enzyme can be induced under slight oxidative stress but severe oxidative stress can lead to suppression of these enzymes (Xu et al., 2009). The activity of CAT is dependent on the level

of H2O2 in the cell. Another enzyme, GPx eliminates H2O2 while carrying out GSH to GSSG

conversion. Increase in GPx activity might lead to reduction of H2O2 that in turn can affect the activity of catalase. Induction of GPx has been suggested by decrease in GSH as reported in molluscs exposed to PAHs (Grintzalis et al., 2012, Martins et al., 2012). B[a]P exposure to E. fetida did not induce CAT activity (Saint-Denis et al., 1999). Wu et al., (2011) has also found that CAT activity in Eisenia fetida was unaltered suggesting that PAH exposure does not induce increased CAT activity. Jifa et al., (2006) also reported unaltered changes in Lateolabrax japonicus CAT activity after (B[a]P) exposure. Free radicals produced as a result of metabolic activities can react with polyunsaturated fatty acids in cell membrane, resulting in increase of lipid peroxidation (Livingstone et al., 2003). LPO is a sensitive indicator of oxidative stress due to PAH (Cossu et al., 1997). LPO in bivalves exposed to PAH is well documented (Pan et al., 2005; Kaloyianni et al., 2009). LPO results in production of MDA that can react with DNA and form DNA adduct. Increased formation of DNA adduct in gill cells of Mytilus galloprovincialis after exposure to PAH, BaP have been reported by Venier and Canova, (1996). In our studies we have reported consistent increase in LPO in M. granulata up to 50 µg/L of phenanthrene. The LPO value showed a decline from 0.49±0.04 at 50 µg/L to 0.39±0.06 at 100 µg/L. Previous studies have also confirmed that short-term exposure to phenanthrene induces LPO in gill cells of fish Liza aurata (Oliveira et al., 2008). Phenanthrene exposure induces a similar increase in LPO in fish Sparus aurata (Kopecka- Pilarczyk and Correia, 2009) and in scallop Pecten maximus (Hannam et al., 2010a). Increase in lipid peroxidation in Sparus aurata exposed to phenanthrene was also reported by Correia et al., (2007). In our study we have observed significant correlation between LPO and GST in marine gastropod (r = 0.86, P < 0.001). In this study we have reported concentration-dependent increase in % DNA in tail as measured by comet assay in marine gastropod, M. granulata exposed to phenanthrene. The DNA damage occurred in this study could be due to various reasons such as DNA single strand breaks, DNA double strand breaks, DNA adduct formations, DNA–DNA, and DNA– protein cross-links (Mitchelmore et al., 1998) resulting from the interaction of PAH or their metabolites with DNA. Accumulation of PAH has been linked to DNA damage through production of ROS (Tarantini et al., 2011; Jarvis et al., 2013). The ROS produced as a result of PAH exposure can cause single or double strand breakage in the DNA (Kaloyianni et al.,

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2009). Khan et al., (2013) has reported strong correlation between phenanthrene concentration and DNA damage as measured by comet assay in E. feotida. Significant change in % DNA in tail was observed in gastropods exposed to phenanthrene. Hydrophobic contaminants are efficiently taken up inside the cell because of its lipophilic nature. Hubert et al., (2012) determined the DNA strand breaks in both embryonic cells and on adult gill cells of freshwater mud-snail (Potamopyrgus antipodarum) using comet assay. A recent work carried out with mussel Mytilus galloprovincialis, showed elevated levels of DNA damage in phenanthrene treated haemocytes (Dailianis et al., 2014). A higher comet score was observed in the erythrocytes of fish, Poecilia vivipara exposed to 10 µg/L of phenanthrene (Machado et al., 2014). The results of comet assay in M. granulata suggested that phenanthrene induces DNA damage in snail. Significant differences in % DNA in tail were observed among the groups. % DNA in tail increases with exposure to phenanthrene with their highest value recorded at 100 µg/L. It should be noted that at low concentration (10 µg/L) of phenanthrene, % DNA in tail was not found to be significant with respect to control. Above 10 µg/L, all the groups showed significant increase (P < 0.001) in % DNA in tail as compared to control. In our study, the lowest mean values of IBR for the four biomarkers were observed in control samples. The IBR results demonstrated that LPO and DNA in tail were the most responsive in gastropods exposed to phenanthrene. IBR index has been used to study the PAH contamination in caged mussel Mytilus trossulus, Mytilus galloprovincialis (Dabrowska et al., 2013). IBR index was successfully used in Cyprinus carpio to study toxicological effects of perfluorinated organic compounds (Kim et al., 2010). IBR has been used to construct star plot with PAH and PCB concentrations in tissues along with early warning signals (EROD, GST, CAT, AChE enzymatic activities) and adverse effects (DNA adducts) biomarkers in flounder Platichthys flesus (Beliaeff and Burgeot, 2002).

4.7.2. Exposure to benzo(k)fluoranthene 4.7.2.1. Biochemical tests B[k]F exposure causes a significant increase in oxidative stress biomarkers in snails (Fig. 42). Concentration-dependent increase in SOD was observed in snails exposed to B[k]F. The activity of SOD revealed a significant difference in exposed snails as compared to the control, with lowest value observed in control snails (1.72±0.14 U/mg of protein). Exposure to 1 µg/L showed 1.3 fold increase in SOD activity as compared to control snails but it was not found to be significant. Concentrations above 1 µg/L showed significant increase in SOD

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activity in snails. Concentration-dependent increase in CAT activity was observed in snails exposed to B[k]F. CAT activity showed significant (p < 0.001) increase of 50% at 1 µg/L of B[k]F compared to control. CAT activity in snails exposed to all concentrations of B[k]F differed significantly (p < 0.001) from control. Highest CAT activity (0.28±0.01 mmol/min/mg) was observed at 50 µg/L B[k]F. The GST activity reached values about 1.9 fold higher than the controls at 50 µg/L of B[k]F (p < 0.001). There was slight decrease in GST activity observed from 10 µg/L to 25 µg/L. The lipid peroxidation in snail, exposed to various concentrations of B[k]F showed a significant increase throughout the period of study. A significant increase in lipid peroxidation was observed in snails exposed to 10, 25 and 50 µg/L of B[k]F after 96 h.

Fig. 42 Glutathione S-transferase (GST), Superoxide dismutase (SOD), lipid peroxidation (LPO) and catalase (CAT) activities and level in marine gastropod M. granulata exposed to different concentrations of benzo(k)fluoranthene (B[k]F). Values are means±standard deviation. * p<0.05, ** p<0.01, *** p<0.001 - significantly different from the control (ANOVA, Tukey HSD post-test)

4.7.2.2. Comet assay The percentage tail DNA as measured by comet assay in snails exposed to B[k]F showed considerable DNA damage (Fig. 43). A significant increase in the percentage tail DNA was observed at each concentrations when compared to control. The lowest value for percentage tail DNA was observed in control snails (27.37±1.24), whereas exposure to 50µg/L of B[k]F

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showed the highest value (47.96±1.68). Overall, concentration-dependent increase in TDNA was observed in snails exposed to B[k]F. The comets being produced from the cells of M. granulata exposed to B[k]F are shown in Figure 43.

Fig. 43 Percentage DNA in tail (TDNA) in marine gastropod M. granulata exposed to different concentrations of benzo(k)fluoranthene (B[k]F). Values are means±standard deviation. * p<0.05, ** p<0.01, *** p<0.001 - significantly different from the control (ANOVA, Tukey HSD post-test)

4.7.2.3. Integrative Biomarker Response (IBR) The IBR values for all the biomarkers were standardized and computed as star plot (Fig. 44). Genotoxic and oxidative stress biomarkers were responsive to B[k]F exposure and showed different patterns in the star plots. The IBR values for all the biomarkers increased with an increase in exposure concentrations of B[k]F. The mean IBR value ranged from 0.04±0.04 to 14.70±1.87. The lowest value for IBR was observed in control samples whereas the highest value was observed at 50 µg/L of B[k]F. There was no significant variations observed when mean IBR values for 1 µg/L were compared with control. Above 1 µg/L, all other concentrations, showed significant increase in mean IBR when compared to control.

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Fig. 44 Integrated biomarker response (IBR) represented by star plots in M. granulata

Fig. 45 Comets as observed in cells of M. granulata after exposure to different concentrations (a) control, (b) 1 µg/L, (c) 10 µg/L, (d) 25 µg/L, and (e) 50 µg/L of benzo(k)fluoranthene (B[k]F)

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PAHs are well known to induce free radicals which in turn can cause genotoxic damage. A strong correlation between DNA damage and PAH bioaccumulation was reported in sea snail Littorina littorea (Noventa et al., 2011). Significant increase in DNA adduct has been reported in northern pike (Esox lucius) exposed to a mixture of benzo[a]pyrene (BaP), benzo[k]fluoranthene (B[k]F) and 7H-dibenzo[c,g]carbazole (DBC) (Ericson & Balk, 2000). Freshwater pale chub, Zacco platypus exposed to B[k]F has also shown significant increase in DNA strand breakage (Kim et al., 2014). In our previous studies we have reported significant correlations between PAH in sediment and DNA damage as measure by comet assay in marine gastropod Morula granulata (Sarkar et al., 2014). Exposure to oxy-PAHs in Japanese medaka (Oryzias latipes) has lead to significant increase in DNA damage as measure by comet assay (Dasgupta et al., 2014). Significant increase in DNA damage was also reported in fish Scophthalmus maximus exposed to PAH (Le Dû- Lacoste et al., 2013). Concentration-dependent increases in DNA strand breaks have been reported in mussel Mytilus sp. after exposure to fluoranthene (Al-Subiai et al., 2012). PAHs or their metabolites can induce the production of radicals which in turn can affect oxidation in the cell. Antioxidant mechanism in cell plays a protective role in protection of the body against free radicals. Several studies have reported induction in SOD activity in snails exposed to environmental contaminants. Dose-dependent increase in SOD was reported in crucian carp, Carassius carassius exposed to B[k]F (Ding et al., 2014). Wang et al., (2011) has also reported increase in SOD activity in clams (Ruditapes philippinarum) exposed to B(a)P. Catalase plays a protective role in PAHs metabolism by eliminating ROS by catalytic decomposition of hydrogen peroxide. Kim et al., (2014) reported significant increases in the mRNA expression of catalase and superoxide dismutase in freshwater pale chub, Zacco platypus exposed to B[k]F. Studies have shown positive relationships between CAT activity and PAH levels in oyster (Niyogi et al., 2001a), barnacle (Niyogi et al., 2001b), and in the gills of the mussel (Cheung et al., 2001). Among antioxidant defence, GST has been proposed as a promising biomarker in snails exposed to aquatic pollutants (Khalil et al., 2015). GSTs play a critical role in conjugation of electrophilic compounds (phase I metabolites) on one hand, and in the defence against oxidative damage and peroxidative products of DNA and lipids (Oost et al., 2003) on the other hand. Several studies have shown increase in GST activity in aquatic organisms exposed to high concentrations of PAHs (Rodrigues et al., 2014; Zhang et al., 2014). In this study significant increase in GST was found in M. granulata exposed to B[k]F. The increase in GST activity indicates the increase in detoxification of B[k]F metabolites. B[k]F enters

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into the cell easily due to its lipophilicity and accumulates in tissues. An increased GST activity was observed in experiments with freshwater snail Bellamya aeruginosa exposed to ethylbenzene for 7 days (Zheng et al., 2013). In an in vitro model, Pennec & Pennec (2003) observed that cells from the digestive gland of Pecten maximus, incubated with PAHs for 96 h, showed significant increases in GST activity up to 48 h upon exposure. Exposure to 1 mg/l of B(a)P to mollusc Venerupis philippinarum has shown significant increase in expression of GST gene (Boscolo Papo et al., 2014). Silva et al., (2005) observed a significant increase in GST activity in Crassostrea rhizophorae exposed to diesel oil. In this study, a slight decrease in GST activity was noted after exposure to 10 µg/L of B[k]F. Few studies has reported decrease in GST activity in molluscs exposed to PAHs (Akcha et al., 2000; Lüchmann et al., 2014). GST catalyses the conjugation of reduced glutathione to electrophile molecules. Availability of glutathione also influences the GST activity. Decrease in reduced glutathione was observed in zebra fish exposed to PAH (Timme-Laragy et al., 2009). In another study European eel (Anguilla anguilla L.) on exposure to beta-naphthoflavone showed decrease in GSH content (Ahmad et al., 2005). Ismert et al., (2002) while investigating the exposure of napthalene to Helix aspersa, reported no change in GST activity in digestive gland, kidney and mantle cavity forming tissue (MCFT) of exposed snails. In this study, we have observed concentration-dependent increase in LPO in snails exposed to B[k]F. An increase of LPO value has been reported in several studies on gastropods exposed to aquatic pollutants (Wang et al., 2014; Ma et al., 2014; Ali et al., 2014). Elevated levels of CAT, LPO and SOD were observed in mussels (Mytilus galloprovincialis) collected from sites affected by oil spill (Sureda et al., 2011). Increased level of oxidative damage in terms of lipid peroxidation has been reported in various snail species exposed to laboratory or environmental contaminants (El-Shenawy et al., 2012; Ali et al., 2014; Ma et al., 2014). Selection of same size individuals is important as different shell size has shown to produce different LPO activity (Pan et al., 2005). Time and concentration dependent increase in LPO was reported in scallop Chlamys farreri exposed to B[k]F (Pan et al., 2005). Integrated biomarker response has been well used in aquatic organism (Tlili et al., 2013). IBR index was successfully used to evaluate the toxicological effects of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) on Cyprinus carpio (Kim et al., 2010). Vega-López et al., (2013) has investigated the relation of oxidative stress and antioxidant defences in phytoplankton with heavy metal. The author stated that oxidative damage was related with PAH (benzo[b]fluoranthene) using IBR.

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4.7.3. In vivo exposure of gastropods to cadmium chloride (CdCl2) In vivo exposure of gastropods did not induce any mortality at any of the concentrations tested. 4.7.3.1. Alkaline unwinding assay No significant difference in DNA integrity has been observed in the control at any duration. After one day of exposure a slight decrease in DNA integrity was observed at all doses of

CdCl2 compared with the control (Fig. 47). DNA integrity was found to be decreasing as the

CdCl2 concentration increases from 10-75 µg/L. The difference in I value between 50 and 75 µg/L was unnoticeable for the first two days. After 2 days of exposure a significant decrease in the DNA integrity was observed in the CdCl2 group (25 and 75 µg/L), (p< 0.05 and p < 0.01). Significant decrease in DNA integrity was observed in exposure group (25, 50 and 75 µg/L) from third day onwards (p< 0.01 and p < 0.001) as compared to control. Exposure to

75 µg/L of CdCl2 produced significant decrease at all duration compared to control. The I value has significantly reduced to 50% and 20% from control after 3 days and 5 days of

exposure to CdCl2 at 75 µg/L. The alkaline unwinding assay results from gastropods exposed

to 10 µg/L CdCl2 was not found to be significant at any duration compared with control.

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Fig. 47. DNA Integrity (I value) in gills of Nerita chamaeleon exposed to CdCl2. Snails were

exposed for 5 days to CdCl2. Data expressed are means of triplicate values of DNA integrity. Results were reported as mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from the control (Dunnett’s test).

4.7.3.2. Comet Assay

To examine the sensitivity of the comet assay for exposure to CdCl2 in vivo and investigate the relationship between DNA damage and duration of exposure, gastropods were exposed to seawater supplemented with 10, 25, 50 and 75 µg/L CdCl2 for 5 days. As control groups, gastropods were exposed to only seawater throughout the period of 5 days. % Tail DNA, OTM and TL in control group was found to be consistent throughout the 5 days of

experiment. Compared with the control group, significant changes in % tail DNA by CdCl2

exposure were observed in all CdCl2 exposed groups (p < 0.001) (Fig. 46). On day3 of exposure no significant differences were observed between 10, 25, 50 µg/L CdCl2 group. % tail DNA increased almost three fold in 50 µg/L (48.47±2.33) as compared to the control group (15.65±1.03) on day3. The increase has gone up to four fold in 50 µg/L (53.54±2.34) after four days of exposure. Interestingly when the % tail DNA for exposed group 25 and 50 µg/L were compared no significant differences were observed. There was no significant change observed in the OTM after one day of exposure to cadmium at all concentration tested (Fig. 46). However a significant increase (p < 0.01) in OTM values was observed in gastropods exposed 25 µg/L, 50 µg/L and 75 µg/L of cadmium for 2 days when compared to the control gastropods (p< 0.01). Exposure to 10 µg/L produced significant increase (p< 0.001) in OTM at day3, 4 and 5. Gastropods exposed to 25 µg/L of

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Cd showed significant increase in OTM from 6.66±2.16 on day1 of exposure to 9.36±1.03 on day5 of exposure. There has been slightly decrease in the OTM from day2 to day3 at 25 µg/L and 75 µg/L. The difference between 25 µg/L and 50 µg/L was not significant at all duration. A comparable image of the comet in exposed groups and control along with in vitro exposure to hydrogen peroxide is shown in Fig. 48.

Fig. 48. Typical comet image of gill cells of Nerita chamaeleon exposed to different concentration of CdCl2 in vivo for 24h and 50 µM of H2O2 in vitro for 30 min at 4°C. Cells were stained with ethidium bromide.

An increase in TL of DNA in gastropods exposed group was observed as compared to control. However when the data of T0 and day1 was compared no significant differences were observed at all concentrations of cadmium. Significant change in TL was observed in 75 µg/L group compared with control at all durations. On day3 all the exposed group (except 10 µg/L) showed significant increase in TL when compared to control. There was slight decrease in TL in 75 µg/L from day2 to day3. Except 25 µg/L, all exposed group showed significant increase in TL compared to control on day4 (p< 0.05, p< 0.01). However there were decrease in TL from day4 to day5 in 10 µg/L and 75 µg/L. Decrease in TL has also been observed in 10 µg/L and 25 µg/L from day4 to day5. TL in 10 µg/L (41.01±2.57) group increased significantly compared with control gastropods (26.02±1.82) on day5.

The usefulness of snails as sentinel organism in metal biomonitoring studies are widely recognized (Sarkar et al., 2006, 2008, Itziou and Dimitriadis 2011). Gill cells are used as an attractive cellular model in ecotoxicology; gills are constantly exposed to dissolved

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contaminants and they are capable of metabolizing carcinogens and mutagens into reactive products (Wilson et al., 1998, Mitchelmore et al., 1998). An assessment of the integrity of DNA is important when determining pollution related stress in living organisms. So in the present study gill cells were used to investigate the damage occurring in the DNA due to cadmium contamination. The measurement of integrity of DNA was tested using alkaline unwinding assay and comet assay. Using the alkaline unwinding assay we have demonstrated a dose dependent response relationship between the level of DNA strand breaks and environmentally relevant

concentrations of CdCl2. The DNA damage detected in this study could have originated from DNA single strand breaks, DNA double strand breaks, DNA adduct formations, DNA–DNA and DNA–protein cross-links (Mitchelmore et al., 1998) resulting from the interaction of heavy metal or their metabolites with DNA (Fairbairn et al., 1995). The results of the DAUA

suggested that CdCl2 induces DNA damage in N. chamaeleon. DAUA have shown

significant differences among the four CdCl2 treatment groups (10, 25, 50 and 75 µg/L) when they were compared independently of the duration of exposure. Moreover, comparisons of the treatment groups within particular time intervals have also shown clear dose-dependent responses since there were significant differences between the lowest (10 µg/L) and the highest (75 µg/L) Cd treatment groups on all durations. Chemical analyses of trace metals in mussel tissues were integrated with a multi-biomarker approach for the early detection of biological responses at several cellular targets (Gorbi et al., 2008). Recent studies on the integrity of DNA in marine snail (Planaxis sulcatus) clearly indicated the impact of pollution at Goa coast (Sarker et al., 2006). High level (3.8 µg/g) of Cd was reported in gastropod, Cronia contracta in Goa region leading to loss of DNA integrity by 73.5% (Sarkar et al., 2008). Stronkhorst et al., (2003) reported a decrease in DNA integrity measured using DAUA in Sea star Asterias rubens at dumping sites contaminated with heavy metals (Cd and Hg) in North Sea. Bolognesi et al., (1999) observed a statistical increase of DNA damage induced by Cd in mussel species Mytilus galloprovincialis exposed in aquarium for 5 days. In this study three parameters namely % tail DNA, OTM and TL were used in comet assay for the measurement of DNA damage. We observed that exposure to cadmium induces dose dependent increase in DNA strand breaks in gill cells of gastropod measured using all the three parameters. The tail DNA content values increased over time with their highest values recorded on day5 for all exposed group. Similar trend was observed for OTM and TL, the highest value was observed on day4 for 10 µg/L and 50 µg/L. However the highest value for 75 µg/L was observed on day5 for OTM and TL. For 25 µg/L, the highest value for OTM and

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TL was observed on day2 and 3 respectively. However the OTM value for 25 µg/L was almost same for day2 and 3. Cadmium is a well known genotoxicant since it is classified as category 1 carcinogen by the International Agency for Research on Cancer. Cadmium have shown to be genotoxic to mussels (Wilson et al., 1998, Bolognesi et al., 2009), fish (Chandra and Khuda-Bukhsh, 2004), crustaceans (Hook and Lee, 2004). An increase in DNA strand breaks is reported on snail Potamopyrgus antipodarum exposed to 10 µg/L of Cd (Hubert et al., 2012). Hubert et al., (2011) reported increase in bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl2 in a time-dependent manner indicating that Cd exposures were effective. They found that Cd (10 µg/L) is genotoxic after 3 days of exposure in gill cells in zebra mussel Dreissena polymorpha. Juhel et al., (2007) reported DNA strand breaks in haemocytes of zebra mussels exposed to a very high cadmium concentration (>733 µg/L) after 7 day of exposure. For marine mussels, cadmium was reported to induce DNA damage in gill cells after a 10 day exposure to 200 µg/L (Emmanouil et al., 2007). Decrease in tail DNA was investigated by Frenzilli et al., (2006) in Mediterranean mussel (Mytilus galloprovincialis) accompanied by an elevation of tissue metal levels. Genotoxicity is considered one of the most important toxic endpoints in chemical toxicity testing and risk assessment; however, little is known about the genotoxicity of Cd, especially towards gastropod N. chamaeleon. The results of both the comet assay and alkaline unwinding assay presented here have demonstrated clear dose and time dependent responses to genotoxicant (Cd) exposure in the N. chamaeleon. Overall, the assays provide a set of convenient, highly sensitive, monitoring tools of environmental exposure to genotoxicants and both the comet assay and the integrity of DNA (I value) of alkaline unwinding assay explains its suitability as a biomarkers in gastropods. This investigation point out ecological implications of Cd release in aquatic ecosystems and warranted to regulatory agencies and industry for the need of monitoring and regulation regarding Cd.

4.7.4. Exposure to mercuric chloride (HgCl2) 4.7.4.1. Cell viability

Cell viability in the gill cells of snail exposed in vivo and in vitro to HgCl2 remained above 85% for the duration of exposure for P. sulcatus.

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4.7.4.2. In vitro hydrogen peroxide Gill cells suspension of P. sulcatus was used for the in vitro exposure to hydrogen peroxide,

H2O2. The concentration range 1-50 µM was used as a positive control. The genotoxicity of mercury was evaluated using comet assay, and data were reported as percentage of tail DNA

(TDNA) and olive tail moment (OTM). As shown in Fig. 49, in vitro exposure H2O2 displayed significant increases in both TDNA and OTM when compared to the control group. The TDNA ranged from 12.43±0.08 to 37.85±0.82 and OTM from 1.77±0.17 to 12.13±2.85.

The in vitro H2O2 exposure produced statistically significant levels of DNA damage in TDNA as compared to control (p < 0.001). Almost seven fold increase in OTM was observed at 50 µM compared with control (p < 0.001).

Fig. 49 Comet parameters (TDNA and OTM) in gill cells of P. sulcatus exposed to hydrogen peroxide (H2O2) and mercuric chloride (HgCl2) in vitro. Values are means±standard deviation. ns non-significant. a p<0.05, b p<0.01, c p<0.001—significantly different from the control (ANOVA, Tukey HSD post-test)

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Fig. 50 Percentage tail DNA and OTM in gill cells of P. sulcatus exposed to HgCl2 in vivo. Data represented as means±standard deviation. T0 time zero or zero day of exposure, ns non- significant. a p<0.05, b p<0.01, c p<0.001— significantly different from the control (ANOVA, Tukey HSD post-test)

4.7.4.3. In vitro exposure Fig. 49 shows statistically significant increase (p < 0.001) in TDNA in the concentration range of 0-100 µg/l of HgCl2. The lowest TDNA (14.34±0.25) and OTM (1.92±0.40) were observed in P. sulcatus exposed to 10 µg/l of HgCl2. After 60 min of exposure with 100 µg/l of HgCl2, TDNA increased by 2.6 fold as compared to control. There were no significant differences observed in OTM for samples exposed to 10 and 20 µg/l of HgCl2. Further the

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DNA damage was found to be concentration dependent with the highest TDNA and OTM recorded at 100 µg/l in P. sulcatus.

4.7.4.4. In vivo exposure Fig. 50 shows the extent of DNA damage measured using comet assay in snails exposed in

vivo to HgCl2. There are no statistical differences in TDNA and OTM was observed in control DNA during the exposure time. The TDNA values of gills increased significantly

after treatment with 10 µg/l of HgCl2 compared with control (TDNA= 20.26%, p<0.001). At 24 h, statistically significant differences were observed in TDNA at all concentration of

HgCl2 as compared to control. At 48 h, the TDNA of 10 µg/l was 19.46± 0.61, 20 µg/l 22.79±0.71, 50 µg/l 28.92±0.95 and 100 µg/l was 32.57± 0.19. Mean values for 50 µg/l were statistically higher (p < 0.001) than that of 10 µg/l. Decrease in TDNA was also observed at gastropods exposed to 50 µg/l from 48 h to 72 h. After 72 h, 10 µg/l differed statistically from that of 50 and 100 µg/l (p < 0.05), whereas 20 µg/l did not differed statistically from 10 and 50 µg/l. At 72 h, 50 µg/l showed a slight decrease in OTM values from 48 h. At 50 µg/l

HgCl2 exposure, there was significant increase in OTM values from 4.74±0.82 on 24 h to 7.14±1.0 on 96 h. Eighty percent of mollusc is represented by gastropods. Recently, gastropods have attracted attention because of their potential as sentinel organisms. Intersex development in gastropods due to tributyltin pollution has been reported by several authors. Spada et al., (2012) has reported very high level of total mercury (224–1867 μg/kg d.w.) in gastropod Hexaplex trunculus from Taranto Gulf, exceeding the maximum level fixed by the European Commission (0.5 mg/kg w.w.). However the concentration of mercury in control snails in this study was found to be 9.92±1.54 µg/kg wet weight. Various studies on mercury compounds and their genotoxic effects have been performed on mussel (Scarpato et al., 1992; Verlecar et al., 2008). The mussel, Mytilus edulis exposed to 50 µg/l of HgCl2 for 8 days had reported to accumulate significantly higher amount of Hg in gills as compared to the control mussel (Sheir et al., 2010). The author also reported that gill cell accumulated the highest amount of mercury in comparison to digestive gland, adductor muscle or hemolymph. Yap et al., (2004) has studied genetic abnormalities in mussel Perna viridis exposed to mercury Numerous studies have stressed the deleterious effects of mercury on mussels (Pytharopoulou et al., 2013), crab (Fossi et al., 1996), shrimp (Lee et al., 2000; Jose et al., 2011), fish (Yadav et al., 2009). Although there are many reports on Hg pollution, few researchers have studied the effect of metals on gastropods (Noel et al.,

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2011; Cardoso et al., 2013). Studies suggest that gastropods are a good bioindicator of metal pollution (Yawei et al., 2005). The DNA damage was measure using comet assay which is a widely used assay in environmental monitoring for evaluation of DNA damage and repair. The choice of tissue plays an important role in studying the DNA damage in an organism due to its ability to accumulate and metabolize xenobiotics. Various cells (hepatocytes, gills, lymphocytes), isolated from fish, molluscs, marine mammals after exposure to different genotoxicants have been used for single cell gel electrophoresis for the detection of DNA damage. In this study, we have used gill cells as they are exposed to a large volume of seawater and are important tissue for uptake of contaminants. They accumulate higher concentrations of metal and are the sites for metabolic activities which convert toxicants into reactive products (Mitchelmore et al., 1998; Wilson et al., 1998). The DNA damage occurred in this study could be due to various reasons such as DNA single strand breaks, DNA double strand breaks, DNA adduct formations, DNA–DNA, and DNA–protein cross-links (Mitchelmore et al., 1998) resulting from the interaction of mercury or their metabolites (methylmercury) with DNA. There are several mechanisms through which mercury can cause DNA damage. One of them are production of reactive 2 – oxygen species such as superoxide anion radical (O ), hydrogen peroxide (H2O2), and hydroxyl radicals (OH●) (Tsuzuki et al., 1994). The antioxidant mechanism in cell detoxifies these ROS and helps to maintain the oxidative balance. Any failure of these antioxidant mechanisms due to the overproduction of ROS can cause oxidative stress in the cell. The ROS produced can also act on plasma membrane and induce lipid peroxidation. LPO results in production of MDA which can react with DNA and form DNA adduct. Geret et al., (2002) has also reported increased level of MDA in Mytilus edulis exposed to Hg. Verlecar et al., (2007) has studied the oxidative stress in Perna viridis exposed to mercury and observed an increase in thiobarbituric acid reactive substances (TBA-RS) in gills and digestive gland. Oxidative stress is directly or indirectly related to DNA damage. The DNA damage occurred in this study might be due to oxidative stress caused by accumulation of mercury in the tissues of snails. The other mechanism though which mercury can cause DNA damage is by inhibition of DNA repair system. In vivo and in vitro studies with the comet assay have well been documented in aquatic animals. Monteiro et al., (2011) has conducted in vivo and in vitro exposure to assess the genotoxic effect of lead on fish Prochilodus lineatus. The author demonstrated the usefulness of comet assay to detect lead toxicity in gill and liver cells of the fish. Present in

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vivo and in vitro study clearly indicated increase in TDNA and OTM at higher concentrations

of HgCl2. Tran et al., (2007) by performing in vivo studies with Mytilus edulis exposed to

HgCl2 (20 μg/l), has reported similar findings that HgCl2 increases TDNA in exposed mussel. Geret et al., (2002) has reported an increase in metallothionein (MT) levels in mussel (Mytilus edulis) after 21 days of exposure to mercury. Toxicity of mercuric chloride has been evaluated in vivo on different aquatic organisms such as crabs (Botton, 2000), mussel (Sheir et al., 2010), fishes (Low & Sin, 1996; Pereira et al., 2009). Arabi, (2004) has detected occurrence of mercury induced DNA damage in carp gill cells using in vitro comet assay. Voccia et al., (1994) has studied in vitro mercury-related cytotoxicity in rainbow trout (Oncorhyncus mykiss). Taddei et al., (2001) has used comet assay to evaluate in vitro genotoxicity of methyl mercury chloride in isolated bottlenose dolphin leukocytes and showed dose dependent increase in DNA strand breakage. In our work, we have demonstrated concentration as well as time dependent increase in the TDNA and OTM as measured using comet assay in P. sulcatus exposed to HgCl2. The accumulation of mercury in gill cells indicates high concentration of Hg in the environment. Berto et al., (2006) has reported positive correlation between total mercury concentration in sediments and in gastropod, N. reticulatus. Several studies have shown dose-dependent response of Hg on marine organisms (Lalancette et al., 2003; Pellisso et al., 2008). In this study, control snail showed statistically significant difference when compared with exposed snails, indicating the effectiveness of the method. Results indicate that all the tested concentrations including 10 µg/l are affecting the DNA. The TDNA and OTM have increased

with increase in the concentration of HgCl2 showing that the DNA damage increases with increase in Hg concentration. After 96 h of exposure, OTM value of 10 µg/l was not statistically different from 24 h and 48 h exposure. 20 µg/l had OTM value of 4.03± 0.41 at

48 h, which was significantly higher (p < 0.001) than that of control, i.e. 1.52±0.78. HgCl2 exposure induced a significant (p<0.05–0.001) and concentration-dependent increase in DNA damage in the exposed snails.

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CHAPTER 5

Overall Assessment Of The Molecular Biomarker Studies

A multi-biomarker approach was carried out to assess the impact of environmental contaminants in marine gastropods from Goa, west coast of India. A wide array of genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay), biochemical (SOD, CAT, GST, LPO and AChE) biomarkers along with PAH in sediments and marine snails collected from different sites such as Arambol, Anjuna, Sinquerim, Dona Paula, Velsao, Hollant, Bogmalo, Betul and Palolem were measured. Significant variations in DNA damage and biochemical biomarkers were observed along the sites. Results indicated that snails from Goa region are responding to contamination in a site specific manner. Additionally, the IBR values were correlated with PAH concentration in snail tissues. These results suggest IBR coupled with chemical analysis might be a useful tool for the assessment of environmental pollution in the aquatic ecosystem. Standardized values of biomarker response were visualized using star plots, which show various patterns for different biomarkers. The impairment of DNA in all the three marine gastropods was evaluated in terms of the loss of DNA integrity in the species as a measure of the impact of genotoxic contaminants prevalent in the marine environment along the coast of Goa, India. The extent of DNA damage occurred in the marine gastropods collected from different sampling sites along the coast of Goa was measured following the technique of partial alkaline unwinding as well as comet assays. In general, highest DNA integrity was found at the reference site Arambol. The impact of genotoxic contaminants on marine gastropods was pronounced by their low DNA integrity at Velsao, Sinquerim and Dona Paula. The extent of DNA damage occurred in snails due to ecotoxicological impact of the prevailing marine pollutants along the coast of Goa was substantiated by comet assay and expressed in terms of percentage tail DNA and olive tail moment. The single cell gel electrophoresis of snails clearly showed relatively higher percentage tail DNA in the marine gastropod from the contaminated sites, Velsao, Sinquerim, Dona Paula, Anjuna, Hollant and Bogmalo. The variation in the percentage tail DNA at different sampling sites clearly indicated that the extent of DNA damage in marine gastropod

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increases with the increase in the levels of contamination at different sampling sites along the coast. PAHs have a strong capacity to bio-concentrate; these pollutants either individually or in combination may have sub-lethal effects at the cellular, organ, or individual level, causing damages to DNA. During monsoon, highest amount of PAH was recorded in sediment from Velsao. Interestingly the DNA integrity (I value) was also found to be reduced during the same season in P. sulcatus and M. granulata. The decreasing trend in the integrity of DNA in marine gastropods was corroborated by the increase in concentration of PAHs in the sediments along the coast of Goa. Moreover, the various water quality parameters also played significant role in the variation of integrity of DNA in marine gastropods as observed by the stepwise multiple regression analysis. The stepwise multiple regression analysis of the water quality parameters showed significant correlation between the DNA integrity and PAH along

2 with NO3, salinity and PO4 ( R , 0.90). In vivo and in vitro exposures were used to investigate the genotoxicity of mercuric chloride (HgCl2) to the marine snail, Planaxis sulcatus. The comet assay protocol was

validated on gill cells exposed in vitro to hydrogen peroxide (H2O2, 0–50 μM). Snails were

exposed in vivo for 96 h to HgCl2 (10, 20, 50, and 100 μg/l). Our results showed significant concentration-dependent increase in the tail DNA (TDNA) and olive tail moment (OTM) in

exposed snails for all doses compared with controls. In vitro exposure to HgCl2 (10–100 μg/l) resulted in significantly higher values for TDNA at all concentrations. Our results showed that DNA damage increased in the gill cell with increasing exposure time.

We have tested four different concentrations (10, 25, 50 and 75 µg/L) of CdCl2 in N. chamaeleon and gill cells were chosen to measure the extent of DNA damage. The in vitro exposure of H2O2 (1; 10; 25 and 50 µg/L) of the gill cells showed a significant increase in the % tail DNA, olive tail moment (OTM) and tail length (TL). Compared with the control

group, significant changes in % tail DNA by CdCl2 exposure were observed in all exposed groups (P < 0.001). Exposure to 75 µg/L of cadmium produced significant decrease in DNA integrity measured by DAUA at all duration compared to control (P < 0.01, P < 0.001). A dose and time dependent increase in DNA damage was indicated by alkaline comet assay and

DAUA in N. chameleon exposed to various concentration of CdCl2 (10, 25, 50 and 75 µg/L). The results indicated that alkaline comet assay and DAUA is a sensitive and rapid method for DNA damage analysis in gastropods. Overall, the assays provide a set of convenient, highly sensitive, monitoring tools of environmental exposure to genotoxicants and both the comet

139 assay and the integrity of DNA (F value) of alkaline unwinding assay explains its suitability as a biomarkers in gastropods. DNA damage and oxidative stress in marine gastropod, Morula granulata was measured after in vivo exposure to four different concentrations of phenanthrene (10, 25, 50 and 100 µg/L) for 24 h. Comet assay was used for measurement of DNA damage, whereas oxidative stress was assessed using a battery of biomarkers such as glutathione-s-transferase (GST), catalase (CAT) and lipid peroxidation (LPO). Our data showed concentration- dependent increase in percentage DNA in tail, LPO and GST activity in gastropods exposed to phenanthrene. CAT activity in gastropods was not found to be consistent with the phenanthrene concentrations. Significant increase in % DNA in tail was observed at all concentrations above 10 µg/L of phenanthrene. Positive correlations were observed in GST, LPO and % DNA in tail. Standardized values of integrated biomarker response were visualized using star plots that showed similar patterns in exposed gastropods. In vivo experiments were carried out to investigate the effects of a toxic polycyclic aromatic hydrocarbon (PAH), benzo(k)fluoranthene (B[k]F), on marine gastropod, Morula granulata collected from Goa, west coast of India. Snails were exposed to different concentrations of B[k]F (1, 10, 25 and 50 µg/l) for 96 h. The genotoxic effects were evaluated by measuring DNA strand breaks using alkaline comet assay and oxidative stress were measured with the help of battery of biomarkers such as superoxide dismutase (SOD) catalase (CAT), glutathione-s-transferase (GST), and lipid peroxidation (LPO). Concentration-dependent increase in percentage tail DNA (TDNA) was observed in snails exposed to B[k]F. Exposure concentrations above 1 µg/l of B[k]F, showed significant increase in SOD activity and LPO value in snails. After 96 h, SOD activity was found to be doubled for 50 µg/l of B[k]F with reference to control. Significant increase in CAT and GST were observed at all exposure conditions at the end of the exposure time. Integrated biomarkers responses (IBR) index revealed higher values in exposed snails than in control. Among the oxidative biomarkers, LPO was the major contributor to IBR in exposed snails. The results suggest that IBR might be used as an important tool for the study the integrated impact of pollutants on organisms. Our study showed that B[k]F induces oxidative stress in snails which further lead to genotoxic damage. The marine gastropod used in this study has shown concentration-dependent increase in genotoxic and oxidative biomarkers with respect to B[k]F. This study demonstrates the usefulness of star plots of standardized biomarker response and IBR index for quantitative interpretation of toxic effects of B[k]F. However

140 more comprehensive long term experiments including chemical and biological interactions are required to have a better understanding of the associated toxicity mechanism.

Our study demonstrates the following conclusions :

• Integrated biomarker response index based on battery of biomarkers proved useful tool for visualization of biological responses in snails, facilitating comparisons between different sites. Increased value of IBR index at Sinquerim and Dona Paula can be attributed to exposure of snails to contaminants prevalent at these sites. IBR approach to investigate the ecotoxicological impact of environmental pollutants in gastropods was successfully applied. • This investigation point out ecological implications of Cd release in aquatic ecosystems and warranted to regulatory agencies and industry for the need of monitoring and regulation regarding Cd. • Sensitivity of marine snail N. chamaeleon as a good candidate species for cadmium contamination.

• Usefulness of comet assay for detection of DNA damage after exposure to HgCl2 and the sensitivity of marine snail P. sulcatus as a good candidate species for metal pollution. • Sensitivity of marine gastropod M. granulata as a good candidate species for PAH pollution • Among the four biomarkers, LPO and DNA damage (% DNA in tail) were the most sensitive in response to phenanthrene exposure. Our results clearly showed that phenanthrene is genotoxic to gastropods and also causes oxidative stress. • Based on the evaluation of impairment of DNA integrity and biochemical tests in all the three marine gastropods used in this study, M. granulata along the coast of Goa can be considered as the most useful sentinel species for bio-monitoring of pollution by genotoxic contaminants and ecotoxicological studies.

On the basis of the extensive studies carried out over a period of different seasons, the Goan coastal environment can be distinguished into

 highly contaminated (Sinquerim, Velsao, Dona Paula, Hollant, Bogmalo),  moderately contaminated (Anjuna, Betul, Palolem)  and least contaminated (Arambol) regions

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Several mitigation measures can be recommended to prevent the genotoxic effects on marine biodiversity such as (i) measurement of genotoxicants in the water, sediment and biota all along the coastal region, (ii) integrated use of biomarkers to study the effect of these pollutants in the marine organisms, (iii) regular monitoring of industrial waste and strict regulations to stop the discharge of waste material and untreated effluents in the coastal waters. Bibliography

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ISJ 13: 336-349, 2016 ISSN 1824-307X

REVIEW

Glutathione S-transferase, catalase, superoxide dismutase, glutathione peroxidase, and lipid peroxidation as biomarkers of oxidative stress in snails: A review

J Bhagat, BS Ingole, N Singh

Biological Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa, India

Accepted October 17, 2016

Abstract Antioxidant defense plays a crucial role in the response of an organism to pollutants. Several processes stimulate the production of free radicals or deplete the antioxidant defense, which if not regulated properly, may cause oxidative stress in the organisms, leading to damage in DNA, proteins or lipids. Free radicals are also beneficial as it plays an important role in defense against infectious agents, and signal transduction. Hence a delicate balance between antioxidants and free radicals is required. Oxidative stress biomarkers are very useful in disease etiology and environmental toxicological studies. The increase in anthropogenic activities and environmental awareness has resulted in an explosive increase of research in the field of oxidative stress. Snails are excellent organisms for environmental biomonitoring and contribute a major proportion of the invertebrate biomass. In our article, we have summarized the research carried out using glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and lipid peroxidation (LPO) in snails exposed to various toxicants and their implication in the environmental monitoring programs. In the end, we have discussed different factors affecting the variations in oxidative biomarkers response for a better understanding of the phenomenon.

Key Words: antioxidant defense system; gastropods; oxidative stress; reactive oxygen species

Introduction

Gastropods are ubiquitous invertebrates in the system in the body. Antioxidant defense system aquatic environment and are commonly studied as a helps protect the cell from damages caused by suitable bioindicator for contaminants (Itziou and free radical by restoring their level. Antioxidant Dimitriadis, 2011; Abdel-Halim et al., 2013). They defense system involves both enzymatic and non- are widely studied for their ability to accumulate enzymatic free radical inactivation and scavenging higher amount of heavy metal and other toxic processes. Changes in antioxidant systems of pollutants (Baurand et al., 2014; Bo et al., 2015). aquatic organisms can serve as indicators for a Contaminants entering into the aquatic bodies from variety of pollutant exposures related to oxidative several sources are taken up by these organisms, stress. Thus, it provides sensitive biochemical and may disturb the free radical process. Reactive markers for exposure and toxicity in environmental oxygen species (ROS) are potentially dangerous monitoring. Among antioxidant defense, [superoxide because of their highly reactive nature and hence dismutase (SOD), catalase (CAT), glutathione S- are neutralized by several antioxidant defense transferases (GST), glutathione peroxidase (GPx) systems. ROS are also involved in hormonal and Lipid peroxidation (LPO) are extensively studied responses, signal transduction, and several others (Radwan et al., 2010; El-Shenawy et al., 2012; physiological processes including heart's pumping, Abdel-Halim et al., 2013; Zheng et al., 2013; Wang aging, and disease (Andersson et al., 2011). There et al., 2014). is usually a balance between production of free Glutathione-S-transferases are phase II radical and antioxidant defense. Oxidative stress multifunctional enzymes, which play a critical role in occurs when there are excess amount of free conjugation of electrophilic compounds (phase I radical due to faulty or lower antioxidant defense metabolites) on one hand, and in the defense ______against oxidative damage and peroxidative products

Corresponding author: of DNA or lipids (Oost et al., 2003) on the other Jacky Bhagat hand. Superoxide dismutases are a class of CSIR-National Institute of Oceanography naturally occurring enzymes that repairs and Dona Paula, Goa-403004, India prevent the oxygen metabolizing cells from the E-mail: [email protected] harmful effects of free radicals mainly superoxide.

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RESEARCH REPORT

An integrated approach to study the biomarker responses in marine gastropod Nerita chamaeleon environmentally exposed to polycyclic aromatic hydrocarbons

J Bhagat1, A Sarkar2#, V Deepti2, V Singh2, L Raiker2, BS Ingole1

1Biological Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa - 403004, India 2Chemical Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa - 403004, India # Present address: Global Enviro-Care, Kevnem, Caranzalem, Goa - 403002, India

Accepted December 28, 2016

Abstract Ecological risk assessment using multiple biomarkers produce a large amount of data that is hard to interpret and the result are often contradictory. In this context, Integrated Biomarker Response (IBR) index was used to integrate the biomarkers effects to assess the impact of environmental contaminants in marine gastropod Nerita chamaeleon from Goa, India. Genotoxic (DNA damage as measured by comet assay and alkaline unwinding assay) and biochemical [superoxide dismutase, catalase, glutathione S-transferase, lipid peroxidation and acetylcholinesterase] biomarkers were measured in snails collected from different sites (Arambol, Anjuna, Sinquerim, Dona Paula, Velsao, Betul and Palolem). Total polycyclic aromatic hydrocarbons in snail tissue were in the range from 5.29 - 12.14 µg/g wet weight. Standardized values of biomarker response were visualized using star plots, which show unique patterns for different biomarkers. The mean IBR value was found to be highest at Dona Paula (8.07 ± 0.91) followed by Sinquerim (6.95 ± 0.91), Velsao (4.48 ± 0.68), Anjuna (3.28 ± 1.05), Palolem (2.53 ± 0.73), Arambol (1.81 ± 0.21) and Betul (0.88 ± 0.77). Additionally, the IBR values were found to be positively correlated with PAH concentration in snail tissues. These results suggest that integration of biomarkers effects using IBR along with chemical analysis can be a useful tool for the assessment of environmental pollution and to identify spatial patterns of contamination in the aquatic ecosystem.

Key Words: Integrated biomarker response; oxidative stress; genotoxic damage; polycyclic aromatic hydrocarbon; comet assay

Introduction

Polycyclic aromatic hydrocarbons (PAH) are reactive oxygen species (ROS) which can induce persistent and ubiquitous environmental genotoxic damage by modifying integrity of DNA contaminants found in air, water, and soil. They are (Mattson et al., 2009). studied extensively due to their carcinogenic Biomarkers are an important tool to detect properties for human as well as animals. The exposure and adverse effects of human-made or International Agency for Research on Cancer natural contaminants on aquatic organisms. Some (IARC) has classified PAHs as possible and biomarkers are specific to chemicals or group of probable carcinogen to human (IARC, 2010). The chemicals while other are non-specific and induces lipophilic and hydrophobic nature allows PAHs to upon exposure to broad range of pollutants. Due to accumulate in the marine organism (Mashroofeh et the complexity of contaminants, use of multi- al., 2015). The accumulation of PAHs in a marine biomarker has become an increasingly popular tool organism can negatively affect their health to study the environmental parameters as well as (Frouin et al., 2007; Grintzalis et al., 2012). PAHs organism health. Comet (or single cell gel and their metabolites interact with DNA and form electrophoresis) assay is the most commonly used DNA adducts. PAH activation process also generates as a biomarker of DNA damage in various research ______areas because of it sensitive and reliable nature.

Corresponding author: DNA damage as measured by comet assay has Jacky Bhagat been reported in mussels, clams and several other Biological Oceanographic Division aquatic organisms (Martins et al., 2013; Dailianis et CSIR-National Institute of Oceanography al., 2014; Sarkar et al., 2014). Another technique is Dona Paula, Goa-403004, India known as DNA-alkaline unwinding assay (DAUA) is E-mail: [email protected] also widely used to detect DNA damage in aquatic

Environ Sci Pollut Res DOI 10.1007/s11356-015-4263-7

RESEARCH ARTICLE

Genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay

J. Bhagat & B. S. Ingole

Received: 13 October 2014 /Accepted: 19 February 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract In vivo and in vitro exposures were used to inves- sensitivity of marine snail P. sulcatus as a good candidate tigate the genotoxicity of mercuric chloride (HgCl2) to the species for metal pollution. marine snail, Planaxis sulcatus. The comet assay protocol was validated on gill cells exposed in vitro to hydrogen per- Keywords Comet assay . Planaxis sulcatus . Mercuric oxide (H O ,0–50 μM). Snails were exposed in vivo for 96 h 2 2 chloride . Genotoxicity . DNA damage . In vivo . In vitro to HgCl2 (10, 20, 50, and 100 μg/l). Our results showed significant concentration-dependent increase in the tail DNA (TDNA) and olive tail moment (OTM) in exposed snails for all doses compared with controls. In vitro exposure to HgCl2 (10–100 μg/l) resulted in significantly higher values for Introduction TDNA at all concentrations. Our results showed that DNA damage increased in the gill cell with increasing exposure The increase in the discharge of genotoxic chemical from time. This study demonstrates the usefulness of comet assay either industrial or municipal waste waters into the aquatic for detection of DNA damage after exposure to HgCl2 and the ecosystem has become a great concern to environmentalists around the world. Heavy metals are of great ecological concern due to their toxic and persistent nature. They are widely spread in the biosphere, from both natural and man-made activities. In recent decades, the amount of potentially toxic Responsible editor: Cinta Porte heavy metals has risen steadily in both marine and freshwater Capsule We have reported concentration-dependent increase in tail DNA ecosystems due to anthropogenic emissions (Pacyna et al. and olive tail moment measured by comet assay in marine gastropod 2006;Sunderlandetal. 2009). Among the toxic metals, Planaxis sulcatus exposedtoinvivoandinvitrotoHgCl 2 mercury is ubiquitous in the environment (Goldman and Highlights Shannon 2001). In the last few decades, there have been • An approach to evaluate genotoxicity of HgCl2 in marine gastropod was presented many reports on mercury pollution in various places in • In vivo and in vitro effects of HgCl2in gill cells of gastropod was India (Krishnakumar and Bhat 1998; Kaladharan et al. evaluated using comet assay 1999). Marine organisms from Mumbai have been found • Concentration dependent increase in tail DNA and olive tail moment is to be heavily contaminated with mercury (Pandit et al. reported in exposed gastropods 1997; Mishra et al. 2007). Rajathy (1997) has reported • HgCl2 was found to be genotoxic to marine gastropod Planaxis sulcatus high level of Hg (0.013–0.40 μg/g, wet weight) in fish from B. S. Ingole Ennore estuary, Tamil Nadu. Menon and Mahajan (2013) Biological Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa 403004, India have surveyed five villages along Ulhas River estuary and Thane Creek and found high Hg levels in gills, kidney, Present Address: and skin in fish Mugil cephalus. Studies have reported Hg * J. Bhagat ( ) pollution in Amba estuary (Ram et al. 2009a), Hooghly Biological Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa 403004, India River (Sarkar et al. 2004), Rushikulya estuary (Panda e-mail: [email protected] et al. 1990;Shawetal.1988; Das and Sahu 2002), and Water Air Soil Pollut (2016) 227:114 DOI 10.1007/s11270-016-2815-1

DNA Damage and Oxidative Stress in Marine Gastropod Morula granulata Exposed to Phenanthrene

Jacky Bhagat & A. Sarkar & B. S. Ingole

Received: 8 July 2015 /Accepted: 14 March 2016 # Springer International Publishing Switzerland 2016

Abstract DNA damage and oxidative stress in marine dependent increase in percentage DNA in tail (TDNA), gastropod Morula granulata was measured after in vivo LPO, and GST activity in gastropods exposed to phenan- exposure to four different concentrations (10, 25, 50, and threne. CAT activity in gastropods was not found to be 100 μg/L) of phenanthrene. Comet assay was used for consistent with the phenanthrene concentrations. measurement of DNA damage, whereas oxidative stress Significant increase in TDNA was observed at all con- was assessed using a battery of biomarkers such as centrations above 10 μg/L of phenanthrene. Positive glutathione-S-transferase (GST), catalase (CAT), and lip- correlations were observed among oxidative stress bio- id peroxidation (LPO). Our data showed concentration- marker and TDNA. Integrated biomarker response (IBR) analysis showed that among the four biomarkers, LPO Highlights and DNA damage (TDNA) were the most sensitive in • An approach to evaluate genotoxicity of phenanthrene in marine response to phenanthrene exposure. Our results clearly gastropod was presented showed that phenanthrene is genotoxic to gastropods and • In vivo effects of phenanthrene in gastropod was evaluated using genotoxic and oxidative stress biomarker also causes oxidative stress. • Multi-biomarker approach was assessed in marine gastropod • Phenanthrene was found to be genotoxic to marine gastropod Keywords Gastropods . Genotoxicity. Biomarker. Morula granulata Antioxidant enzyme . Comet assay • Positive correlations were observed in GST, LPO, and % DNA in tail in snails exposed to phenantherene

Electronic supplementary material The online version of this article (doi:10.1007/s11270-016-2815-1) contains supplementary 1 Introduction material, which is available to authorized users. During the last decades, rapid increase in industrial, agri- * : J. Bhagat ( ) B. S. Ingole cultural, and urban activities has lead to an unprecedented Biological Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa 403004, India increase in anthropogenic contaminants in the environ- e-mail: [email protected] ment. Among the environmental pollutants, polycyclic aromatic hydrocarbons (PAHs) are of great concern due A. Sarkar to their pervasive nature. They are ubiquitous in the Chemical Oceanographic Division, CSIR-National Institute of Oceanography, Dona Paula, Goa 403004, India environment and are included in the list of persistent organic pollutant of United Nations Environment Program (UNEP 1999). The International Agency for Present Address: Research on Cancer (IARC) has classified PAHs as pos- A. Sarkar Global Enviro-Care, Kevnem, Caranzalem, Goa 403002, sible and probable carcinogen to human (IARC 2010). India PAHs tend to accumulate in marine organism because of Ecotoxicology and Environmental Safety 106 (2014) 253–261

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Evaluation of impairment of DNA in marine gastropod, Morula granulata as a biomarker of marine pollution

A. Sarkar a,b,n, Jacky Bhagat a, Subhodeep Sarker c a Chemical Oceanography Oceanography Division, CSIR - National Institute of Oceanography Dona Paula, Goa 403004, India b Global Enviro-Care, Kevnem, Caranzalem, Goa 403002, India c Clinical Division of Fish Medicine, University of Veterinary Medicine, Veterinarplatz 1, 1210 Vienna, Austria article info abstract

Article history: The impairment of DNA in marine gastropod Morula granulata was evaluated in terms of the loss of DNA Received 8 November 2013 integrity in the species as a measure of the impact of genotoxic contaminants prevalent in the marine Received in revised form environment along the coast of Goa, India. The extent of DNA damage occurred in the marine gastropods 12 April 2014 collected from different sampling sites such as Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Accepted 20 April 2014 Hollant, Velsao, Betul and Palolem along the coast of Goa was measured following the technique of partial alkaline unwinding as well as comet assays. The highest DNA integrity was observed at Keywords: Arambol (F, 0.75), identiﬁed as the reference site, whereas the lowest DNA integrity at Hollant (F, 0.33) DNA impairment situated between the two most contaminated sites at Bogmalo and Velsao. The impact of genotoxic Biomarker contaminants on marine gastropods was pronounced by their low DNA integrity at Sinquerim (F, 0.40) Morula granulate followed by Betul (F, 0.47), Velsao (F, 0.51), Anjuna (F, 0.54), Bogmalo (F, 0.55), Dona Paula (F, 0.67) and Comet assay Alkaline unwinding assay Palolem (F, 0.70). The extent of DNA damage occurred in M. granulata due to ecotoxicological impact of Genotoxic contaminants the prevailing marine pollutants along the coast of Goa was further substantiated by comet assay and expressed in terms of %head-DNA, %tail DNA, tail length and Olive tail moment. The single cell gel electrophoresis of M. granulata clearly showed relatively higher olive tail moment in the marine gastropod from the contaminated sites, Anjuna, Hollant, Velsao and Betul. The variation in the mean % head DNA at different sampling sites clearly indicated that the extent of DNA damage in marine gastropod increases with the increase in the levels of contamination at different sampling sites along the coast. The stepwise multiple regression analysis of the water quality parameters showed signiﬁcant

correlation between the variation in DNA integrity and PAH in combination with NO3, salinity and PO4 2 (R , 0.90). The measurement of DNA integrity in M. granulata thus provides an early warning signal of contamination of the coastal ecosystem of Goa by genotoxic contaminants. & 2014 Elsevier Inc. All rights reserved.

1. Introduction Marine organisms are exposed to a variety of genotoxic agents like polycyclic aromatic hydrocarbons (PAHs) (Sarkar and Everaarts, The rapid increase in anthropogenic activity and industrial 1998), polychlorinated biphenyls (PCBs) (Sarkar et al., 1994; Sarkar development along the coastal region has resulted in elevated et al., 1997), organochlorine pesticides (OCPs), heavy metals, etc. concentration of genotoxic agents in the environment, affecting (Fernàndez and Grimalt, 2003; Sen Gupta et al., 1996; Sarkar, 1994). the biological integrity of the marine ecosystem (Sarkar, 2005; The marine pollutants prevalent in the marine ecosystem may likely Sarkar and Everaarts, 1998, Sarkar, 1994; Everaarts and Sarkar, to cause severe damage to the genetic material directly or indirectly. 1996). Of all the ecosystems, estuarine and coastal regions are Among the direct genotoxicants, alkylating agents, like hydrogen considered as the primary sink for the pollutants and greatly peroxides and pesticides, etc., are signiﬁcant whereas indirect affected by the high degree of contaminations by highly persistent toxicity depends on the mechanisms of metabolic activation and organochlorine compounds (Sarkar and Sen Gupta, 1991; Bowen the formation of reactive oxygen species (ROS) or they consist of and Depledge, 2006). substances capable of inhibition of DNA synthesis/repair mechanisms (Everaarts and Sarkar, 1996). Metabolic activation processes generally lead to the formation of electrophilic metabolites which n Corresponding author at: Global Enviro-Care, Caranzalem, Goa, India. can bind to nucleophilic DNA molecules producing a variety of DNA E-mail address: [email protected] (A. Sarkar). lesions (Shugart et al., 1989; Shugart, 1999, 1988a, b; Ostling and http://dx.doi.org/10.1016/j.ecoenv.2014.04.023 0147-6513/& 2014 Elsevier Inc. All rights reserved. Genotoxicity of Cadmium Chloride in the Marine Gastropod Nerita chamaeleon Using Comet Assay and Alkaline Unwinding Assay

Anupam Sarkar,1 Jacky Bhagat,1 Baban S. Ingole,2 Durga P. Rao,1 Vijaykumar L. Markad3 1Chemical Oceanographic Division, CSIR-National Institute of Oceanography Dona Paula, Goa 403004, India 2Biological Oceanographic Division, CSIR-National Institute of Oceanography Dona Paula, Goa 403004, India 3Division Biochemistry, Department of Chemistry, University of Pune, Pune 411007, India

Received 15 October 2012; revised 16 May 2013; accepted 22 May 2013

ABSTRACT: This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl2)on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 mg/L) of CdCl2. In vitro exposure of hydrogen peroxide (H2O2; 1, 10, 25, and 50 mM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl2 exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 mg/L of CdCl2 produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl2 (10, 25, 50, and 75 mg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment. VC 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 177–187, 2015. Keywords: genotoxicity; comet assay; alkaline unwinding assay; gastropods; DNA integrity; cadmium chloride

INTRODUCTION **Correspondence to: J. Bhagat, e-mail: [email protected] *Present address: Anupam Sarkar, Global Enviro-Care, Kevnem, Car- anzalem, Goa-403002, India. e-mail: [email protected] Cadmium is one of the most toxic compounds that pose Contract grant sponsor: Department of Biotechnology, New Delhi, serious threat to the health of the marine ecosystem, the India mechanisms of its toxicity are still not clearly understood Published online 27 June 2013 in Wiley Online Library (Michel Cornet, 2007; Barsiene_ et al., 2013). It is a systemic (wileyonlinelibrary.com). DOI: 10.1002/tox.21883 poison affecting many cellular functions (Abdulla and

VC 2013 Wiley Periodicals, Inc. 177

NeBIO I An international journal of environment and biodiversity Vol. 3, No. 5, December 2012, 34-36 ISSN 2278-2281(Online Version) ISSN 0976-3597(Print Version) I

ABSTRACT

This paper deals with the development of molecular biomarker to measure the DNA damage in marine gastropod, Planaxis sulcatus for biomonitoring of pollution due to genotoxic contaminants. Among the toxic pollutants polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), heavy metals etc. are of prime importance. They are highly persistent in nature and interact with DNA of organisms to form DNA adducts, ultimately leading to strand breaks in DNA. In order to study the genotoxic impact of pollutants, alkaline unwinding assay was chosen as a biomarker of genotoxicity. Four sampling sites (viz. Arambol, Sinquerim, Hollant and Bogmalo) were selected along the Goa coast based on discharge of industrial effluents, shipping activities, municipal sewage and the availability of the sample organism. The DNA damage was measured in terms of the loss of DNA integrity in P. sulcatus due to the occurrence of DNA strand breaks following the technique of time dependent partially alkaline unwinding assay in a defined condition of pH and temperature. The DNA integrities in P. sulcatus from different locations were compared with those at the reference site (Arambol). The DNA integrity in control sample was found to be 0.88, whereas in the reference site (Arambol) it was 0.61. The low F value obtained at Hollant (F, 0.31) and Bogmalo (F, 0.41) clearly indicates the damage occurring due to interaction of various pollutants with DNA.

KEYWORDS Genotoxicity, gastropods, Goa coast, DNA integrity

N Save Nature to Survive QUARTERLY

JACKY BHAGAT*, B INGOLE, A SARKAR, AND MANASI GUNJIKAR Marine Pollution Assessment and Ecotoxicology Laboratory, National Institute of Oceanography, Dona Paula, Goa - 403 004 E-mail: [email protected]

ABSTRACT

This paper presents an evaluation of the INTRODUCTION occurrence of DNA strand breaks in marine Marine ecosystem is under tremendous stress because of the continuous increase gastropod Planaxis sulcatus collected from in the anthropogenic activities leading to accumulation of non-biodegradable different sites (Arambol, Anjuna, Sinquerim, Hollant, Bogmalo and Velsao) along the Goa persistent pollutants (Sarkar et al., 1991; 1998). These persistent pollutants in the coast. In order to identify the hot spot of ocean ecosystem have massive impacts on the plants and animals. Many of these pollution due to genotoxic compounds, the pollutants are chemical carcinogens and mutagens with the capacity to cause DNA damage was measured in terms of the various types of DNA damage. Among the most toxic pollutants, polyaromatic loss of DNA integrity in Planaxis sulcatus due hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polychlorinated dibenzo to the occurrence of DNA strand breaks dioxins (PCDDs), polychlorinated dibenzo furans (PCDFs), organochlorinated following the technique of time dependent pesticides and heavy metals are of great concern (Everaarts and Sarkar, 1996). partially alkaline unwinding assay in a defined condition of pH and temperature. The DNA Various molecular biomarkers have been developed to measure the effect of integrity was expressed in terms of the F-values. pollution on the aquatic inhabitants of these water bodies (Sarkar et al., 2006). Interestingly, the higher the value of F, the Biomarkers measures biological response that can be related to exposure to an lower the occurrence of DNA strand breaks. environmental contaminant. In a broad context, they can include measuring such Among the various sites, Planaxis sulcatus endpoints as reproduction and growth, or behavioral changes; however, the showed the lowest DNA integrity at Sinquerim biomarkers chosen in this study measured effects at a cellular level (Sarkar et al., (F, 0.37) compared to the reference site 2006). These biomarker techniques present a comparative and elaborative scenario Arambol (F, 0.61). The DNA integrity at of the toxicants present in a particular environment and are applied on the sentinel Anjuna, Bogmalo, Hollant and Velsao were species which are also known as indicator species. Among the numerous in the range of F, 0.40 - 0.43. The observed variation in the levels of DNA integrity at ecotoxicological biomarkers proposed in the last three decades, those based on different sites in marine gastropods can be responses at the molecular and cellular level represent the earliest signals of attributed to various types of genotoxic environmental disturbance and are commonly used for biomonitoring (Bayne et compounds prevalent along the Goa coast. al., 1985, Depledge, 1994 and Lowe et al., 1995). Among the genotoxic compounds DNA strand breaks are common DNA modifications that are generated by a wide polyaromatic hydrocarbons (PAHs) are the variety of agents and mechanisms and can be detected using simple techniques. major contaminants being spread over the coast through shipping activities, industrial Damage to DNA, can therefore be considered, and has actually been proposed, as discharge and indiscriminate dumping of a useful parameter for assessing the genotoxic properties of environmental pollutants waste materials. Based on the variation in (Kohn, 1983; Fowle and Sexton, 1992; Shugart et al., 1988).The technique for DNA integrity at different sites in marine detecting the DNA strand breaks was developed based on the DNA unwinding gastropod, the Goa coastal regions can be phenomenon discovered by Ahnstrom and Erixon (1973). The double helix begins classified into three clusters such as highly to unwind at those points where nicks are present under alkali conditions (Rydberg, contaminated (Sinquerim), moderately 1975; Kanter and Schwartz, 1979). contaminated (Anjuna, Hollant, Bogmalo and Velsao) and least contaminated site (Arambol). The DNA alkaline unwinding assay is being evaluated for use in the detection of The DNA integrity in marine gastropods can DNA damage in marine gastropod, P. sulcatus exposed to environmental pollutants. thus act as a prognostic bio-marker of pollution Exposure to certain genotoxicants can result in the formation of DNA strand breaks due to genotoxic compounds. which, if unrepaired, may result in cell dysfunction or mutation. In the DNA alkaline unwinding assay, whole cells or crude DNA extracts are subjected to alkaline assay conditions to allow controlled ‘unwinding’ of double-stranded DNA into single-stranded DNA, beginning at each strand break. The number of DNA strand breaks in the original sample is inversely related to the fraction (F) of DNA remaining double stranded after the assay (Rydberg et al., 1975). A simple method for quantifying strand breaks uses a dye (Hoescht dye 33258) that fluoresces *Corresponding author preferentially in the presence of double-stranded DNA and was developed for use with single cells (Kanter and Schwartz, 1982). The DNA alkaline unwinding assay

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