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Evaluation of Copper Toxicity in the Asian Sea Bass, Lates Calcarifer (Bloch, 1790)

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Evaluation of Copper Toxicity in the Asian Sea Bass, Lates Calcarifer (Bloch, 1790) EVALUATION OF COPPER TOXICITY IN THE ASIAN SEA BASS, LATES CALCARIFER (BLOCH, 1790) Thesis submitted to BHARATHIDASAN UNIVERSITY TIRUCHIRAPPALLI for the award of the Degree of DOCTOR OF PHILOSOPHY IN ZOOLOGY By P.S.PARURUCKUMANI, M.Sc.,M.Phil.,B.Ed., (Ref: No.33286/Ph.D.1/Zoology /FT/ January -2012) Under the guidance of Dr. A.MAHARAJAN, M.Sc.,M.Phil.,Ph.D., POST GRADUATE & RESEARCH DEPARTMENT OF ZOOLOGY KHADIR MOHIDEEN COLLEGE (Nationally Reaccredited with B by NAAC) ADIRAMPATTINAM – 614 701 TAMILNADU, INDIA. AUGUST - 2014 P.G AND RESEARCH DEPARTMENT OF ZOOLOGY KHADIR MOHIDEEN COLLEGE ADIRAMPATTINAM – 614701 THANJAVUR DIST, TAMIL NADU. Dr. A. MAHARAJAN, M.Sc., M.Phil., Ph.D., Assistant Professor & Research Advisor CERTIFICATE This is to certify that this thesis entitled “EVALUATION OF COPPER TOXICITY IN THE ASIAN SEA BASS, LATES CALCARIFER (BLOCH, 1790)” submitted to Bharathidasan University, Tiruchirappalli by Mrs. P.S.PARURUCKUMANI in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in ZOOLOGY is a record of research work done by her during the period of study under my guidance and supervision. I further certify that the thesis has not previously formed the basis for the award of any degree, diploma, associateship, fellowship or other similar title. Place: Adirampattinam Research Guide and Supervisor Date: DECLARATION I do hereby declare that this work has been originally carried out by me under the supervision of Dr.A.MAHARAJAN, Assistant Professor & Research Advisor, P.G. and Research Department of Zoology, Khadir Mohideen College, Adirampattinam and this work has not been submitted elsewhere for any other degree. Station : Adirampattinam (P.S.PARURUCKUMANI) Date : ACKNOWLEDGEMENT First and foremost I thank Almighty GOD, the compassionate, the Merciful, who kindly helped me to complete my thesis. I express my sincere gratitude to Dr.A.Maharajan, Assistant Professor & Research Advisor, P.G. and Research Department of Zoology, Khadir Mohideen College, Adirampattinam, whose guidance surpasses any word of command. The meticulous thinking, unceasing interest, critical analysis, scientific judgment and able guidance at every stage has made this research seek its due results. I feel indebted to him for his excellent guidance. I express my sincere thanks to Authorised Signatory, Khadir Mohideen College, Adirampattinam. I am immensely grateful to Haji Dr.A.Jalal, Principal, Dr.A.Uduman Mohideen, Vice-Principal, Khadir Mohideen College, Adirampattinam, for providing the facilities to complete the work. I am extremely thankful to Dr.P.Kumarasamy, Associate Professor & Head, P.G. and Research Department of Zoology, Khadir Mohideen College, Adirampattinam for his steady support throughout the course of my work. I wish to express my gratitude to Dr. S. Raveendran, Dr. A. Amsath, Associate Professor of Zoology, Dr. K. Muthukumaravel, Dr. O. Sathick, Dr. V. Ganapiriya, and Dr. J. Sukumaran, Assistant Professor of Zoology, Khadir Mohideen College, Adirampattinam, for their valuable suggestions and encouragement. I owe a great to Dr. M.Gabriel Paulraj, Entomology Research Institute, Loyola College (Autonomous), Chennai for permitting to do electrophoretic analysis. I would like to express my gratitude to Dr. Sree Kumar, Sree chitra Institute, Trivandrum for permitting me to make use of the facilities at the institute to carry out the Scanning and Transmission Electron microscopic studies. I express my deep sense of gratidute to my Parents P.Palani, B.R.Subbammal, my Brothers P.S.Kuttalam, P.S.Deshmuth and Sister P.S.Bagavathi Lakshmi, my husband K.Nagendran for his boundless help in all possible ways and moral support at every stage provided me with inspiration during the course of research work, my beloved son P.N.Shiva and my beloved daughter P.N.Shakthi Tamil for their support, encouragement and cooperation. P.S.PARURUCKUMANI CONTENTS List of Tables i List of Figures ii List of Plates iii Chapter Title Page No. 1. GENERAL INTRODUCTION 1 - 11 1.1 Asian sea bass, Lates calcarifer 1 1.2 Morphology and distinctive characters 3 1.3 Distribution 3 1.3.1 Geographic distribution 3 1.3.2 Ecological distribution 4 1.4 Life history 4 1.5 Feeding habits 5 1.6 Sex determination 6 1.7 Sexual maturity 6 1.8 Fecundity and spawning 7 1.9 Embryonic development 8 1.10 Larvae 8 1.11 Growth 9 1.12 Copper 9 1.13 Use of copper 9 1.14 Copper Toxicity 10 2. OBJECTIVES OF THE STUDY 12 - 12 3. REVIEW OF LITERATURE 13 - 32 3.1 Acute Toxicity 13 3.2 Bioaccumulation 15 3.3 Biochemistry 20 3.4 Characterization of Protein Profile 22 3.5 Light Microscopy study (Histology) 24 3.6 Scanning Electron Microscope study 31 3.7 Transmission Electron Microscope study 32 4. MATERIALS AND METHODS 33 - 52 4.1 Area of study 33 4.2 Experimental animal, L. calcarifer 33 4.3 Test organism collection and acclimatization 34 4.4 Preparation of stock solution for copper toxicity test 34 Chapter Title Page No. 4.5 Experimental Procedure 35 4.5.1 Exploratory test 35 4.5.2 Acute Toxicity test 35 4.5.3 Sub lethal toxicity test 37 4.5.3.1 Test concentration 37 4.5.3.2 System design 37 4.5.3.3 Test procedure 37 4.6 Bioaccumulation of copper 39 4.7 Estimation of Biochemical Composition 39 4.7.1 Estimation of Total Protein 39 4.7.2 Estimation of Total Free Sugar 42 4.7.3 Estimation of Total Lipids 43 4.8 Characterization of protein profile 45 4.9 Light Microscopy Study 48 4.10 Scanning Electron Microscope (SEM) Study 50 4.11 Transmission Electron Microscope (TEM) Study 50 4.12 Statistical Analysis 52 5. RESULTS 53 - 76 5.1 EXPLORATORY TESTS ON COPPER TO 53 L. CALCARIFER 5.1.1 Acute toxicity test to determine the LC50 values 53 of L. calcarifer 5.2 SUBLETHAL EFFECT OF COPPER ON THE 54 BIOACCUMULATION OF L. CALCARIFER 5.2.1 Muscle 54 5.2.2 Gill 54 5.2.3 Liver 55 5.2.4 Kidney 55 5.3 SUBLETHAL EFFECT OF COPPER ON THE 56 BIOCHEMICAL COMPOSITION OF L. CALCARIFER 5.3.1 Changes in total protein of different tissues 56 5.3.1.1 Muscle 56 5.3.1.2 Gills 57 5.3.1.3 Liver 57 Chapter Title Page No. 5.3.2 Changes in total free sugar of different tissues 58 5.3.2.1 Muscle 58 5.3.2.2 Gills 58 5.3.2.3 Liver 59 5.3.3 Changes in total lipids of different tissues. 59 5.3.3.1 Muscle 59 5.3.3.2 Gills 60 5.3.3.3 Liver 60 5.4 SUBLETHAL EFFECT OF COPPER ON THE 60 CHARACTERIZATION OF PROTEIN PROFILE IN L. CALCARIFER 5.4.1 Protein Profile in muscle tissue 61 5.4.2 Protein Profile in gill tissue 62 5.4.3 Protein Profile in liver tissue 62 5.5 SUBLETHAL EFFECT OF COPPER ON THE 62 HISTOPATHOLOGICAL CHANGES IN L.CALCARIFER 5.5.1 Histology of gills 62 5.5.2 Histopathology of gills 63 5.5.3 Histology of intestine 64 5.5.4 Histopathology of intestine 65 5.5.5 Histology of muscle 66 5.5.6 Histopathology of muscle 67 5.5.7 Histology of liver 67 5.5.8 Histopathology of liver 68 5.6 SUBLETHAL EFFECT OF COPPER ON THE 69 SCANNING ELECTRON MICROSCOPIC STUDIES IN L. CALCARIFER 5.6.1 SEM histology of gill 69 5.6.2 SEM histopathology of gill 70 5.6.3 SEM histology of liver 71 5.6.4 SEM histopathology of liver 71 Chapter Title Page No. 5.7 SUBLETHAL EFFECT OF COPPER ON THE 72 TRANSMISSION ELECTRON MICROSCOPIC STUDIES IN L. CALCARIFER 5.7.1 TEM histology of gill 72 5.7.2 TEM histopathology of gill 72 5.7.3 TEM histology of liver 74 5.7.4 TEM histopathology of liver 75 6. DISCUSSION 77 - 114 6.1 Acute toxicity test 77 6.2 Bioaccumulation 78 6.3 Biochemistry 81 6.4 Characterization of Protein Profile 84 6.5 Histology 86 6.6 Scanning Electron Microscope study 104 6.7 Transmission electron microscopy study 106 7. SUMMARY AND CONCLUSION 115 -118 8. REFERENCES i - xliv 9. PAPRES PUBLISHED i (i) LIST OF TABLES Table 1. Water quality characteristics and actual copper concentration during sublethal exposure to Asian sea bass, L.calcarifer Table 2. Average mortality rate of L.calcarifer in different concentrations of copper during acute toxicity study Table 3. Percentage of mortality in L. calcarifer exposed to different concentrations of copper in different exposure periods Table 4. Accumulation of copper in muscle of L. calcarifer exposed to sublethal concentrations of copper Table 5. Summary of ANOVA for Accumulation of copper in muscle of L. calcarifer exposed to sublethal concentrations of copper Table 6. Accumulation of copper in gills of L. calcarifer exposed to sublethal concentrations of copper Table 7. Summary of ANOVA for accumulation of copper in gills of L. calcarifer exposed to sublethal concentrations of copper Table 8. Accumulation of copper in liver of L. calcarifer exposed to sublethal concentrations of copper Table 9. Summary of ANOVA for accumulation of copper in liver of L. calcarifer exposed to sublethal concentrations of copper Table 10. Accumulation of copper in kidney of L. calcarifer exposed to sublethal concentrations of copper Table 11. Summary of ANOVA for Accumulation of copper in kidney of L. calcarifer exposed to sublethal concentrations of copper Table 12. Changes of total protein in muscle of L.calcarifer exposed to sublethal concentrations of copper Table 13. Summary of ANOVA for changes of total protein in muscle of L.calcarifer exposed to sublethal concentrations of copper Table 14.
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