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. Changes of total protein in gills of L.calcarifer exposed to sublethal concentrations of copper Table 15. Summary of ANOVA for Changes of total protein in gills of L.calcarifer exposed to sublethal concentrations of copper

Table 16. Changes of total protein in liver of L.calcarifer exposed to sublethal concentrations of copper

Table 17. Summary of ANOVA for changes of total protein in liver of L.calcarifer exposed to sublethal concentrations of copper

Table 18. Changes of total free sugar in muscle of L.calcarifer exposed to sublethal concentrations of copper

Table 19. Summary of ANOVA for changes of total free sugar in muscle of L.calcarifer exposed to sublethal concentrations of copper

Table 20. Changes of total free sugar in gills of L.calcarifer exposed to sublethal concentrations of copper

Table 21. Summary of ANOVA for changes of total free sugar in gills of L.calcarifer exposed to sublethal concentrations of copper

Table 22. Changes of total free sugar in liver of L.calcarifer exposed to sublethal concentrations of copper

Table 23. Summary of ANOVA for changes of total free sugar in liver of L. calcarifer exposed to sublethal concentrations of copper

Table 24. Changes of total lipids in muscle of L.calcarifer exposed to sublethal concentrations of copper

Table 25. Summary of ANOVA for changes of total lipids in muscle of L. calcarifer exposed to sublethal concentrations of copper

Table 26. Changes of total lipids in gills of L. calcarifer exposed to sublethal concentrations of copper

Table 27. Summary of ANOVA for Changes of total lipids in gills of L. calcarifer exposed to sublethal concentrations of copper

Table 28. Changes of total lipids in liver of L. calcarifer exposed to sublethal concentrations of copper

Table 29 . Summary of ANOVA for Changes of total lipids in liver of L. calcarifer exposed to sublethal concentrations of copper

Table. 30. LC50 values of copper toxicity in fin fishes (ii) LIST OF FIGURES Fig.1. Life cycle of an Asian sea bass, L. calcarifer Fig.2. Response curve of copper at 24hrs in L. calcarifer Fig.3. Response curve of copper at 48hrs in L. calcarifer Fig.4. Response curve of copper at 72hrs in L. calcarifer Fig.5. Response curve of copper at 96hrs in L. calcarifer Fig.6. Accumulation of copper in muscle of L. calcarifer exposed to sublethal concentrations of copper

Fig.7. Accumulation of copper in gills of L.calcarifer exposed to sublethal concentrations of copper

Fig.8. Accumulation of copper in liver of L. calcarifer exposed to sublethal concentrations of copper

Fig.9. Accumulation of copper in kidney of L. calcarifer exposed to sublethal concentrations of copper

Fig.10. Changes of total protein (mg/100mg wet weight) in muscle of L.calcarifer exposed to sublethal concentrations of copper

Fig.11. Changes of total protein (mg/100mg wet weight) in gills of L.calcarifer exposed to sublethal concentrations of copper

Fig.12. Changes of total protein (mg/100mg wet weight) in liver of L. calcarifer exposed to sublethal concentrations of copper

Fig.13. Changes of total free sugar (mg/100mg wet weight) in muscle of L.calcarifer exposed to sublethal concentrations of copper

Fig.14. Changes of total free sugar (mg/100mg wet weight) in gills of L.calcarifer exposed to sublethal concentrations of copper

Fig.15. Changes of total free sugar (mg/100mg wet weight) in liver of L.calcarifer exposed to sublethal concentrations of copper

Fig.16. Changes of total lipids (mg/g wet weight) in muscle of L.calcarifer exposed to sublethal concentrations of copper

Fig.17. Changes of total lipids (mg/g wet weight) in gills of L.calcarifer exposed to sublethal concentrations of copper Fig.18. Changes of total lipids (mg/g wet weight) in liver of L.calcarifer exposed to sublethal concentrations of copper

Fig.19. Electrophorogram of Molecular weight marker Protein

Fig.20. Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer (Control)

Fig.21. Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 7 days of exposure to 6.83ppm concentration of copper

Fig.22. Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 7 days of exposure to 13.66 ppm concentration of copper

Fig.23. Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 28 days of exposure to 6.83ppm concentration of copper.

Fig.24. Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 28 days of exposure to 13.66 ppm concentration of copper.

Fig.25. Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer (Control)

Fig.26. Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 7 days of exposure to 6.83 ppm concentration of copper

Fig.27. Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 7 days of exposure to 13.66 ppm concentration of copper

Fig.28. Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 28 days of exposure to 6.83 ppm concentration of copper

Fig.29. Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 28 days of exposure to 13.66 ppm concentration of copper (iii) LIST OF PLATES Plate. 1. The experimental animal Asian sea bass, L. calcarifer Plate. 2. Histological changes of gills in L. calcarifer Plate. 3. Histological changes of intestine in L. calcarifer Plate. 4. Histological changes of muscle in L. calcarifer Plate. 5. Histological changes of liver in L. calcarifer Plate. 6. Scanning Electron micrograph of gill in L. calcarifer Plate. 7. Scanning Electron micrograph of liver in L. calcarifer Plate. 8. Transmission Electron micrograph of gill in L. calcarifer Plate. 9 . Transmission Electron micrograph of liver in L. calcarifer INTRODUCTION General Introduction 1

1. GENERAL INTRODUCTION

Aquaculture remains a growing, vibrant and important production sector for high-protein food. The reported global production of food fish from aquaculture, including fin fishes, crustaceans, molluscs and other aquatic animals for human consumption. The over exploitation of traditional fishing areas and the limitations imposed by the changes in the policy and agreements on international fishing areas caused a chronic restriction in marine products supply. According to FAO previsions, to satisfy the high demand of marine products, aquaculture is a feasible complement to wild captures. In fact, aquaculture accounted for 47.9% of total food fish supply in 2010, representing a continue increase from 42.7% in 2006 (FAO, 2011). In this sense, aquaculture continues to be the fastest-world animal-food-producing system and to outpace population growth, with a global production that increased from 7.3 millions mT in 1980 by weight to 55.1 millions mT in 2009 (FAO, 2011). This exponential increase in production could be accomplished by the improved control of fish reproduction, the development of optimized diets and technological innovations that allowed the fast development of land and sea- based aquaculture facilities. However, growth rates for aquaculture production are slowing, varying greatly among regions. For instance, the leading countries in aquaculture development such as Japan, Spain and France have shown falling production in the last decades (FAO, 2011). Similarly, the average annual growth in aquaculture production in Europe and North America since 2000 has also slowed substantially to 1.7% and 1.2% respectively. General Introduction 2

1.1 Asian Sea bass, Lates calcarifer

The Lates calcarifer (Bloch), commonly known as giant sea perch or Asian sea bass, is an economically important food fish in the tropical and subtropical regions in the Asia-Pacific. They are medium to large-sized bottom-living fishes occurring in coastal seas, estuaries and lagoons in depths between 10 and 50m. They are highly esteemed food and sport fishes taken mainly by artisanal fishermen. Because of its relatively high market value, it has become an attractive commodity of both large and small scale aquaculture enterprises. It is important as a commercial and subsistence food fish but also a game fish. The most important commercial fish of Australia, and the most sought after game fish, generates millions of dollars per year in revenue for the sport fishing. L. calcarifer, known as sea bass in Asia and barramundi in Australia, is widely distributed in the Indo-West Pacific region from the Arabian Gulf to China, Taiwan Province of China, Papua New Guinea and northern Australia. Aquaculture of this species commenced in the 1970s in Thailand, and rapidly spread throughout much of Southeast Asia. Sea bass is a relatively hardy species that tolerates crowding and has wide physiological tolerances. The high fecundity of female fish provides plenty of material for hatchery production of seed. Hatchery production of seed is relatively simple. Sea bass feed well on pelleted diets, and juveniles are easy to wean to pellets. Sea bass grow rapidly, reaching a harvestable size (350 g – 3 kg) in six months to two years. Today Sea bass is farmed throughout most of its range, with most production in Southeast Asia, generally from small coastal cage farms. Australia is experiencing the development of large-scale sea bass farms, where sea bass farming is undertaken outside the tropics and recirculation production systems are often used (e.g. in southern Australia and in the north eastern United States of America). Sea bass has been introduced for aquaculture purposes to Iran, General Introduction 3

Guam, French Polynesia, the United States of America (Hawaii, Massachusetts) and Israel.

1.2 Morphology and distinctive characters

Body elongated, compressed, with deep caudal peduncle. Body large, elongate and stout, with pronounced concave dorsal profile in head and a prominent snout; concave dorsal profile becoming convex in front of dorsal fin. Mouth is large, slightly oblique, upper jaw reaching to behind eye, teeth villiform, no canine teeth present. Lower edge of pre-operculum is with strong spine; operculum with a small spine and with a serrated flap above original of lateral line. Dorsal fin with 7 to 9 spines and 10 to 11 soft rays; a very deep notch almost dividing spiny from soft part of fin; pectoral fin short and rounded; several short, strong serrations above its base; dorsal and anal fins both have scaly sheath. Anal fin round, with three spines and 7–8 soft rays; caudal fin rounded. Scale large ctenoid (rough to touch). Colour: two phases, either olive brown above with silver sides and belly in marine environment or golden brown in freshwater environment. In adult, it is usually blue-green or greyish above and silver below. Fins are blackish or dusky brown. Juveniles have mottled pattern of brown with three white stripes on head and nape, and white blotches irregularly placed on back. Eyes are bright pink, glowing at night.

1.3 Distribution 1.3.1 Geographic distribution

Sea bass is widely distributed in tropical and sub-tropical areas of the Western and Central Pacific and Indian Ocean, between longitude 50°E - 160°W latitude 24°N – 25°S . It occurs throughout the northern part of Asia, southward to Queensland (Australia), westward to East Africa. Found in coastal waters, estuaries and lagoons. Usually occurs at depths of 10 to 40m. General Introduction 4

1.3.2 Ecological distribution

Sea bass is a euryhaline and catadromous species; inhabit freshwater, brackish and marine habitats including streams, lakes, billabongs, estuaries and coastal waters. Sexually mature fish are found in the river mouths, lakes or lagoons where the salinity and depth range between 30–32 ppt and 10–15m, respectively. The newly-hatched larvae (15–20 days old or 0.4–0.7cm) are distributed along the coastline of brackish water estuaries while the 1-cm size larvae can be found in freshwater bodies e.g. rice fields, lakes, etc. (Bhatia and Kungvankij, 1971). Under natural condition, sea bass grows in fresh water and migrates to more saline water for spawning. Adults and juveniles tend to be solitary, patrol home ranges near structure, and may be territorial. Migration is seasonal.

1.4 Life history

Sea bass spends most of its growing period (2–3 years) in freshwater bodies such as rivers and lakes which are connected to the sea. It has a rapid growth rate, often attaining a size of 3–5 kg within 2–3 years. Adult fish (3–4 years) migrate towards the mouth of the river from inland waters into the sea where the salinity ranges between 30–32 ppt for gonadal maturation and subsequent spawning. The fish spawns according to the lunar cycle (usually at the onset of the new moon or the full moon) during late evening (18:00–20:00 hours) usually in synchrony with the incoming tide. This allows the eggs and the hatchlings to drift into estuaries. Here, larval development takes place after which they migrate further upstream to grow. At present, it is not known whether the spent fish migrates upstream or spends the rest of its life in the marine environment. General Introduction 5

Eggs are pelagic, hatch within 24 hours, and the larvae grow quickly as they move into mangrove areas, mudflats, and floodplain lagoons. Juveniles move into coastal waters after one year, and then migrate upstream where adults reside for three to four years. Populations landlocked by dams migrate to the dam face, but do not spawn. It is reared extensively by aquaculture as food or for game fish stocking programs. Catadromous migration is observed, where the fish migrates downstream to shallow mudflats in estuaries during the wet season.

Fig. 1 Life cycle of an Asian sea bass, L. calcarifer

1.5 Feeding habits

Sea bass are opportunistic predators; crustaceans and fish predominate in the diet of adults. Although the adult sea bass is regarded as a voracious carnivore, juveniles are omnivores. The fish is skilled at stalking or ambushing General Introduction 6 prey. Analysis of stomach content of wild specimens (1–10 cm) show that about 20% consists plankton, primarily diatom and algae and the rest are made up to small shrimp, fish, etc. (Kungvankij, et al., 1986). Fish of more than 20 cm, the stomach content consists of 100% animal prey: 70% crustaceans (such as shrimp and small crab) and 30% small fishes. The fish species found in the guts at this stage are mainly slip mouths or pony fish (Leiognatus sp) and mullets (Mugil sp).

1.6 Sex determination

Identification of the sexes is difficult except during the spawning season. There are some dimorphic characters that are indicative of sex.

 Snout of the male fish can be slightly curved while that of the female is straight.  The male has a more slender body than the female.  Weight of the female is heavier than males of the same size.  The scales near the cloaca of the males are thicker than the female during the spawning season.  During the spawning season, abdomen of the female is relatively more bulging than the males.

1.7 Sexual maturity

In the early life stages (1.5–2.5 kg body weight) majority of the sea bass appear to be male but when they attain a body weight of 4–6 kg majority become female. After culture period of 3–4 years, however, in the same age group of sea bass both sexes can be found and identified as mentioned above. In a fully mature female, the diameter of the oocytes usually ranges from 0.4 to 0.5 mm. General Introduction 7

1.8 Fecundity and spawning

Females are larger than males, are highly fecund, and may be courted by one or more males at the same time. The fecundity of sea bass is related to the size and weight of the fish spawning occurs between September and March, with peaks in November to December and again in February to March. Spawning seasonality varies within the range of this species. Barramundi in northern Australia spawn between September and March, with latitudinal variation in spawning season, presumably in response to varying water temperatures. In the Philippines barramundi spawn from late June to late October, while in Thailand spawning is associated with the monsoon season, with two peaks during the northeast monsoon.(August to October) and the southwest monsoon (February to June).

Spawning occurs near river mouths, in the lower reaches of estuaries, or around coastal headlands. Barramundi spawn after the full and new moons during the spawning season, and spawning activity is usually associated with incoming tides that apparently assist transport of eggs and larvae into the estuary. Sea bass being highly fecund; a single female (120 cm TL) may produce 30–40 million eggs. Consequently, only small numbers of brood stock are necessary to provide adequate numbers of larvae for large-scale hatchery production (Maharajan, 2009).

Based on studies of spawning activity under tank conditions, mature male and female fish separate from the school and cease feeding about a week prior to spawning. As the female attains full maturity, there is an increase in play activity with the male. The ripe male and female, then swim together more frequently near the water surface, as spawning time approaches. The fish spawns repeatedly in batches for 7 days. Spawning occurs during late evening (18:00 - 22:00 hours). General Introduction 8

1.9 Embryonic development

First cleavage occurs 35 minutes after fertilization. Cell division continues every 15 to 25 minutes and the egg develop to the multi-celled stage within 3 hours. Its development passes through the usual stages: blastula, gastrula, neurula and embryonic stages. Embryonic hear starts to function in about 15 hours and hatching takes place about 18 hours after fertilization at temperatures of 28–30°C and salinities of 30–32 ppt.

1.10 Larvae

Newly-hatched larvae have total length ranging from 1.21 to 1.65 mm averaging 1.49 mm. The average yolk sac length is 0.86 mm. One oil globule is located at the anterior part of the yolk sac which causes the hatchling to float almost vertically or about 45° from its usual horizontal position. Initial pigmentation is not uniform; the eyes, digestive tract, cloaca and caudal fin are transparent. Three days after hatching, most of the yolk sac is absorbed and the oil globule diminishes to a negligible size. At this stage, the mouth opens and the jaw begins to move as the larva starts to feed. Larvae recruit into estuarine nursery swamps where they remain for several months before they move out into the freshwater reaches of coastal rivers and creeks. Juveniles remain in freshwater habitats until they are three–four years of age (60–70 cm TL) when they reach sexual maturity as males, and then move downstream during the breeding season to participate in spawning. Because they are euryhaline, they can be cultured in a range of salinities, from fresh to seawater. When they are six–eight years old (85–100 cm TL), sea bass change sex to female and remain female for the rest of their lives. Sex change in Asian populations of this species is less well defined and primary females are common. Although sea bass have been recorded as undertaking extensive movements between river systems, most of them remain in their original river system and move only General Introduction 9 short distances. This limited exchange of individuals between river systems is one factor that has contributed to the development of genetically distinct groups of barramundi in northern Australia, where there are six recognised genetic. There are at least two pigmentation stages in sea bass larvae. At 10–12 days after hatching, the pigmentation of larvae appears dark gray or black. The second stage occurs between 25–30 days old where the larvae develop into fry. In this stage, the pigmentation changes to a silvery-coloration. It has been observed that only healthy fry of this stage (20–30 days) swim actively. They are always lighter in colour. Unhealthy post larvae have dark or black body coloration (Thambi Samraj et al., 2003).

1.11 Growth

The growth rate of sea bass follows the normal sigmoid curve. It is slow during the initial stages but becomes more rapid when the fish attains 20 -30 gm. It slows down again when the fish is about 4 kg in weight. 1.12 Copper

Copper sulphate is often used to prevent and treat diseases of cultured aquatic animals because of its high efficiency, wide range of anti-bacterial effects, and affordability. However, if aquatic animals are exposed to a concentrated copper solution, copper may accumulate in their bodies (Lan, 1998; Liu and Tao, 1999) and affect growth, metabolism, and disease resistance (Chen and Zhang, 1988; Chen and Lin, 2001). Further, copper can be traced in humans via the food chain.

1.13 Use of copper

Copper sulphate is used extensively in aquaculture as a U.S. Environmental Protection Agency approved algicide (Schnick et al., 1986). It is also used as a therapeutant for protozoan parasites in commercial and General Introduction 10 recreational fish ponds. Copper sulphate is not approved by the U.S. Food and Drug Administation for therapeutic use in aquaculture. The effectiveness of copper sulphate as a therapeutant is reduced as the total alkalinity and/or total hardness of water increase. Additionally, the toxicity of this compound to fish decreases as pH, total alkalinity and total hardness increase, and as copper binds to inorganic or organic substrates (Tucker and Robinson, 1990).

1.14 Copper Toxicity

Pollution of the aquatic ecosystems is a serious and growing problem. Increasing number and amount of industrial, agricultural and commercial chemicals discharged into the aquatic environment have led to various deleterious effects on the aquatic organisms (McGlashan and Hughes, 2001). Aquatic organisms, including fish, accumulate pollutants directly from contaminated water and indirectly via the food chain. Fish are often used as sentinels because they play a number of roles in the trophic web, bioaccumulate toxic substances, and respond to low concentrations of mutagens (Al-Sabti and Metcalfe, 1995). Aquatic organisms are exposed to a wide variety of environmental contaminants and frequently a set of biomarkers is employed to assess the possible biological impacts and environmental quality.

Heavy metals represent one of the major environmental problems causing long term effects on marine ecosystems. Similar to some organic contaminants, inorganic elements reaching directly or indirectly the coastal water, are provided mainly from increasing industrial and agricultural activities that generate anthropogenic inputs (Seeliger and Knak., 1982). Toxicants may be accumulated in the tissues of marine organisms (Mensi et al., 2008) and induced damages in genetics material. General Introduction 11

The fact that heavy metals cannot be destroyed through biological degradation and have the ability to accumulate in the ecosystem make these harmful chemicals to the aquatic ecosystem and, consequently, to humans who depend on aquatic products as sources of food. Since heavy metals can accumulate in the tissues of aquatic organisms, these tissue concentrations of heavy metals can be of public health concern to both organisms and humans.

Copper occurs in the form of several compounds. The cheapest and most commonly used form is copper sulphate (Watson and Yanong, 2006). It is a fungicide that is used to control bacterial and fungal diseases of fruits, vegetables and field crops; it is also used as an algicide and herbicide and to kill snails and slugs in irrigation as well as municipal water treatment systems (Atamanalp et al., 2008). The Asian sea bass, L. calcarifer is a brackish water fish that is widely distributed in Asia and the Middle East. It inhabits tropical swamps, lakes and rivers, Apart from its importance in culture fisheries; it is also an important aquaculture species. Nevertheless, like other aquatic animals, the existence and well-being of this species are being threatened as a result of the pollution of the aquatic environment by copper sulphate. OBJECTIVES OF THE STUDY Objectives of the Study 12

2. OBJECTIVES OF THE STUDY

The present investigation aims at elucidating the aquatic toxicity of the selected heavy metal, copper on fish, L. calcarifer. The research programme involved the biochemical and histopathological studies by comparing the control group with the test group of fishes exposed to pre-determined dose of the copper.

Thus the main objectives of the present study include

 The present studies intended to evaluate the lethal and sublethal effect of copper in the Asian sea bass, L. calcarifer.

 To find out the LC50 value of copper in L. calcarifer.

 Investigation of the effect of two sublethal concentrations of copper (10

and 20% of LC50 value for 96hrs) on the bioaccumulation of the metal in various tissues such as gills, muscle, liver and kidney in L. calcarifer.

 Study of the changes in the composition of protein, lipids and total free sugar induced by sublethal effect of copper in L. calcarifer.

 Study of the electrophorogram of the protein profile in muscle, gill and liver tissues of L. calcarifer exposed to sublethal concentrations of copper.

 Evaluation of histopathological changes in gills, muscle, intestine and liver of L. calcarifer exposed to sublethal concentrations of copper.

 Scanning Electron Microscope study of gills and liver in L. calcarifer exposed to sublethal concentrations of copper.

 Transmission Electron Microscope study of gills and liver in L. calcarifer exposed to sublethal concentrations of copper. REVIEW OF LITERATURE Review of Literature 13

3. REVIEW OF LITERATURE

Copper is one of the most toxic trace metals to marine biota (Batley and Apte, 1995) and poses a considerable risk to marine ecosystems where human influences have enhanced the natural (background) copper concentrations. Potential risks occur when copper is introduced into marine ecosystems from mining activities, antifouling paints on boats, marinas, ports, and jetty pylons, runoff from fungicidal uses (e.g., copper sulphate), and sewage effluent.

Most protozoan infections are comparatively easy to control using common fishery chemicals, such as copper sulphate, formalin or potassium permanganate (KmNo4), methylene blue, brilliant green, parasite green, malachite green and trichlorforn (Reardon and Harrell, 1990). In the aquaculture industry, copper sulphate is used as an algaecide and as a therapeutic chemical for various ectoparasitic and bacterial infections (Straus and Tucker, 1993; Heo, 1997). For example, copper sulphate is an effective treatment for filamentous algae in shrimp ponds and white spot disease in goldfish, Carassius auratus (Ling et al., 1993; Chen and Lin, 2001). Copper sulphate also inhibits growth of bacteria such as Salmonella spp., Pasteurella spp., Vibrio spp., Streptococcus spp., Aeromonas spp., Psedomonas spp. and Edwardsiella spp. (Heo, 1997). Chemlated copper compounds are considered less toxic to fish than copper sulphate and at least as effective in controlling parasites (Peppard et al., 1991). Fishes are the simple and reliable bio marker of copper pollution of aquatic bodies (Taylor et al., 2000; Lodhi et al., 2006).

3.1 Acute Toxicity

In industrialized countries, environmental problems are less related to acute toxicity of environmental pollutants than to sublethal, synergistic Review of Literature 14 and long-term effects which are difficult to detect and whose consequences for ecosystems are far from being understood (Watts and Pascoe, 2000). Urbanization poses a significant risk to estuarine fauna, particularly fishes. Historically, it is an economically important species supporting major fisheries and has been used extensively in toxicity testing because they are widely distributed, abundant, sensitive to environmental contaminants and relatively easy to hold and culture in the laboratory.

Biological toxicity testing is a relatively simple laboratory bioassay that measures the biological response of marine organisms, particularly at their highly sensitive early life stages (Duquesne et al., 2004). The overall toxicity of heavy metals is commonly assessed using laboratory bioassays where organisms are exposed to contaminants (Chapman and Wang, 2001). Invertebrates are routinely used as candidate organisms in such bioassays, and early life stages of invertebrates are often the most sensitive to contaminants (Rand et al., 1995). Environmental stress from pollutants seems to be an important determining factor signalled by the occurrence of increase in diseases (Lacoste et al., 2001). The presence of heavy metals in the environment has increased in some areas to levels, which threaten the health of aquatic and terrestrial organisms including man (Honda et al., 2008). A major challenge, therefore, is to predict the effects of contaminants on aquatic organisms and to establish toxicity criteria for acceptable levels of chemical contamination. A reason for interest in heavy metals and behaviour in aquatic communities is that heavy metals may have different behavioural effects at concentrations much less, than at which they have lethal effects, suggesting that regulatory pollution limits based upon standard toxicological studies may be too high to prevent damage to aquatic communities through the sublethal behavioural effects (Klaschka, 2008). Review of Literature 15

Toxicological studies of the pollutants upon aquatic organisms are very important from the point of environmental consequences. Fishes are often forced to encounter in the highly contaminated water especially in areas where the dilution rate of waste water is low. Fish species can be used as test organism because, it is the best understood aquatic species and can be a front line indicator of suspected aquatic pollutants such as metals (Vieira et al., 2009). Acute toxicity studies can help to detect, evaluate and abatement of pollution by providing reliable estimates of safe concentration, from which water quality criteria can be derived (Absunullah et al., 1981). The best method of acute toxicity testing is by the determination of LC50 or LD50, which represents the amount of a toxicant either in the form of Lethal Concentration (LC) or Lethal Dose (LD) showing 50% kill of the population of the test animal within fixed period of time APHA/AWWA/WPCF, 1998.

3.2 Bioaccumulation

Bioaccumulation markers and biomarkers of exposure will reflect the distribution of the chemical or its metabolites, respectively, throughout the organism. Theoretically, this distribution can be tracked through various biological levels (e.g. tissue, cell, etc.) to the ultimate target (WHO, 1993). When released into the environment substances will be subject to transport and transformation processes. These processes together with emission patterns, environmental parameters and physicochemical properties of the substances, will govern their distribution and concentration in environmental compartments such as water, air, soil, sediment and biota (ECETOC, 1993). Contaminant distributions over different trophic levels in the food web may provide a means to determine structure in aquatic communities (Russell et al., 1999). Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as bioconcentrators to identify certain Review of Literature 16 substances with low water levels and to assess exposure of aquatic organisms (Ron van der Oost et al., 2003).

Persistent hydrophobic chemicals may accumulate in aquatic organisms through different mechanisms: via the direct uptake from water by gills or skin (bioconcentration), via uptake of suspended particles (ingestion) and via the consumption of contaminated food (biomagnification). Even without detectable acute or chronic effects in standard ecotoxicity tests, bioaccumulation should be regarded as a hazard criterion in itself, since some effects may only be recognized in a later phase of life, are multi-generation effects or manifest only in higher members of a food-web. Bioaccumulation of chemicals in biota may be a prerequisite for adverse effects on ecosystems (Franke et al., 1994). Contaminant levels in biota are determined primarily by the uptake and elimination kinetics, which are typical for both chemicals and organisms (Gobas et al., 1988).

Heavy metals represent one of the major environmental problems causing long term effects on marine ecosystems. Similar to some organic contaminants, inorganic elements reaching directly or indirectly the coastal water, are provided mainly from increasing industrial and agricultural activities that generate anthropogenic inputs (Seeliger and Knak, 1982). Although many studies have been carried out to investigate the accumulation of organic trace pollutants in aquatic organisms, generally, no standardized methods were used. Since the levels of hydrophobic contaminants in the water phase are usually too low for reliable quantification, it is difficult to study bioconcentration in the field.

When fishes are exposed to high level of metal ions in aquatic environment, their tissues tend to take up these metal ions through various routes from their surroundings. There are two main routes of metal acquisition; Review of Literature 17 directly from the water and from the diet (Bury et al., 2003). Copper enter in the body of fish through gills after binding to the mucus layer. It is also ingested along with the food and water and is finally absorbed in the intestine and other tissues (Kotze et al., 1999; Ay et al., 1999; Hansen et al., 2007). But the metal accumulation in tissues of aquatic animals is dependent upon exposure concentration and period as well as some other factors such as salinity, temperature, interacting agents and metabolic activity of the tissue in concern. Similarly, it is also known that the metal accumulation in the tissues of fish is dependent upon the rate of uptake, storage and elimination (Roesijadi and Robinson, 1994; Longston, 1990). Various metal ions get biologically magnified when taken up from the surrounding water in their various tissues as they grow. This uptake and bioaccumulation is well documented in skin, gills, stomach, muscles, intestine, liver, brain, kidney and gonads but their main target organs are liver, kidney and muscles depending on the exposure concentration and time (Allinson et al., 2002; Fabris et al.,2006, Ahmed and Bibi, 2010). Toxicants may be accumulated in the tissues of marine organisms (Mensi et al., 2008) and induced damages in genetics material.

The binding of excess copper to specific sites in protein, however, can disrupt several metabolic processes of all living organisms (Viarengo et al., 1981). Sublethal toxicity has been shown to induce changes in many aspects of animal behavior, could mediate ecological death by disrupting the normal function and life history of the exposed organism (Rand, 1985). Copper induced changes on various aspects of behavior of Babylonia lutosa were evident. Individuals began to retract their body into the shell at 6 ppm of copper and over 50% of individuals exposed to 0.20 ppm of copper retracted their body in the first week. Babylonia lutosa more sensitive to copper when compared with another scavenging gastropod Nassarius obsoletus which started to retract its body at 4-5 ppm of copper (Maclnnes and Thurberg, 1973). Review of Literature 18

Accumulation and subcellular distribution of metals in Nassarius reticulatus, showed that the gill appeared as main target organ for accumulation of copper and in unexposed tissues, most of the copper present was found in mitochondrial and microsomal pools. However, increased copper content as a result of exposure to the metal was largely sequester in the cytosolic compartment and associated with protein of low molecular weight (Kaland et al., 1991). Although it is assumed that uptake across the body surface is the predominant route of metal uptake in aquatic organisms (Rainbow, 1988), metal uptake via the food may still present the major route of metal entry into organisms such as molluscs, crustaceans and annelids (Bryan, 1984). Gupta et al. (1981a) studied the acute toxicity of copper, zinc, chromium, cadmium and nickel to a freshwater Indian pond snail, Viviparus bengalensis. They reported that copper was the most toxic metal and nickel the least toxic; zinc, cadmium and chromium showed intermediate toxicity. Similarly, Ravera (1997) studied the effects of heavy metals (cadmium, copper and chromium) on a freshwater snail, Biomphalaria glabrata and reported that copper and cadmium were more toxic than chromium.

Copper is an essential trace metal necessary for growth and metabolism of all living organism. As such, it is considered a normal constituent in the ecosystem in both soil and water where its presence is partially due to the metabolic by-products of plants and animals. In vertebrates including fish copper form a part of many enzymes and glycol protein. It is important for nervous system functions and is necessary for haemoglobin synthesis (Sorensen, 1991). Copper and other metals released from mining and urban sites can contaminate water sources and affect fish through water or food bane exposure (Sorensen, 1991; Peplow and Edmonds, 2000, Younger et al.,2002, Lapointe et al., 2011). Review of Literature 19

In recent decades studies have shown that copper causes harmful effects that range from the molecular to structural levels in freshwater fish (Baker, 1969; Billiard et al., 1981; Eddy, 1981; Bengtsson and Larsson, 1986; Kalay et al., 1999; Beaumont et al., 2000; Olsvik et al., 2000; Cerqueira and Fernandes, 2002; Van Heerden et al., 2004). Because of the vital functions of the gills (respiration, osmorregulation and excretion), they are in direct contact with the external environment, which facilitates interaction with toxic substances in the water. For this reason, they are considered excellent indicators of environmental quality (Wendelaar Bonga, 1997).

Copper is among the most toxic of the heavy metals in freshwater and marine biota (Schroeder et al., 1966; Betzer and Yevich, 1975), and often accumulates and causes irreversible harm to some species at concentrations just above levels required for growth and reproduction (Hall et al., 1988). Birds and mammals, when compared to lower forms, are relatively resistant to copper. But diets containing elevated concentrations of copper are sometimes fatal to ducklings (Wood and Worden, 1973) and livestock when fed for extended periods. Domestic sheep (Ovis aries) are the most susceptible farm animals to chronic copper poisoning and effects include liver damage, impaired reproduction, reduced resistance to diseases, jaundice, and death (Gopinath and Howell, 1975; Higgins 1981; Bires et al., 1993).

However, the copper concentrations required for effective treatment may be acutely toxic for some species of finfish and are lethal for most invertebrates. Chronic copper exposure will also adversely affect fish health. Sublethal and toxic levels of copper damage gills and other tissues of fish, and also are known to depress the immune system. Because of all these concerns, it is important to understand how copper works and its availability is affected by the environment in which it is used (Cardeilhac and Whitaker, 1988). Review of Literature 20

3.3 Biochemistry

The nutritional value of different species of fishes depends on their biochemical components such as protein, carbohydrate and lipids. These proximate components could serve as sensitive indicators for detecting potential adverse effects, particularly the early events of pollutant damage because their alterations appear before the clinical symptoms produced by the toxicant (Rao, 2006). It is therefore important that potential effects of acute and chronic concentrations of pollutant on proximate composition are determined and interpreted to delineate mechanisms of pollutant action and possible ways to mitigate adverse effects (Matos et al., 2007).

Copper is an important group of estuarine pollutants. It is known to be able to disturb the integrity of biochemical and physiological mechanisms in aquatic organisms, including estuarine fish. Among the different heavy metals, copper is special concern, since the metal is considerably toxic to aquatic animals at ecologically relevant concentrations (Mzimela et al., 2002). Sub lethal effects are biochemical in origin as the most toxicants exert their effects at basic level of the organism by reacting with enzymes or metabolites and other functional components of the cell. Such effects might lead to irreversible and detrimental disturbances of integrated functions such as behavior, growth, reproduction and survival (EIFAC, 1975 and Waldichuk, 1979). Copper is a trace element that plays a fundamental role in the biochemistry of organisms, including aquatic organisms that can take it up directly from water (Grosell et al., 2003). However, it can become toxic at high concentrations (Lam et al., 1998).

The protein content in the tissues of animals plays a role in the metabolism (Palanivelu et al., 2005). Moorthy and Priyamvada, (1982) stated that the protein content of the cell may be considered as an important tool for Review of Literature 21 evaluation of physiological standards. The soluble protein fraction represents the activity level of enzymes in general. The structural protein fraction forms the structural moiety of a cell (Lehninger, 1978). Begam and Vijayaraghavan, (1996) observed protein depletion in the fish indicates the physiological strategy in order to meet the energy demand and to adapt itself to the changed metabolic system which may lead to the stimulation of degradative processes like proteolysis and utilization of degraded products for increased energy metabolism. When any aquatic animal is exposed to polluted medium, a sudden stress is developed for which the animals should meet more energy demand to overcome the toxic stress (Maharajan et al., 2012a). Verma et al. (1981) reported on the toxic effects of sublethal concentration of copper sulphate, on certain biologically important enzymes in Saccobranchus fossilis.

In the tissue protein, carbohydrate and lipids play a major role as energy precursors for aquatic organisms exposed to stress conditions (Ramalingam, 1980). An alteration in biochemical and physiological changes in the crab Portunus pelagicus due to copper and zinc have been reported by Hilmy et al. (1988). Similarly, Katticaran et al. (1995) reported the variations of carbohydrate and protein contents in the clam, Sunetta scripta during its exposure to copper. Villalan et al. (1988) observed that heavy metals altered protein, lipid and carbohydrate levels in the crab, Thalamita crenata. Baden et al. (1994) also reported similar changes in the distribution of glycogen in the tissues of Norway lobster, Nephrops norvegicus exposed to copper. Maharajan et al. (2012b) observed the biochemical changes of various tissues of spiny lobster, P.hoamrus homarus when exposed to sub-lethal doses of copper. Holland et al. (1960) reported some of the effects copper has on fish. Copper salts combine with proteins present in the mucus of the fish's mouth, gills, and skin, preventing aeration of the blood. Review of Literature 22

The copper is known for instantaneous physiological disorders and alteration in the pathways of protein metabolism in tissues and organs. Therefore the biochemical parameters are the best indicators of stress situations caused by copper as one of the heavy metals. Toxicity testing is an essential tool for assessing the effect and fate of toxicant. Thus, this study was planned to estimate the toxicity and variations in the protein, total free sugar and lipid levels in various tissues of Asian sea bass, L. calcarifer.

3.4 Characterization of Protein profile

The electrophoretic techniques are promising tools for identifying the protein profile in response to stressful and sublethal level of heavy metals. Heavy metal binding proteins have been found to be associated with copper and the lower molecular weight protein in the lake fauna (Dutta et al., 1983).

Metal-binding proteins such as ferritin, ceruloplasmin, and metallothioneins (MTs) have special functions in the detoxification of toxic metals, and also play a role in the metabolism and homeostasis of essential metals (Kelly et al., 1998). Metallothioneins are low molecular weight proteins rich in cysteine residues that can bind various metals, including mercury, silver, copper, cadmium, lead, zinc, and cobalt, with varying affinities (Hamer, 1986). It has been reported that different fish species possess different isoforms of MTs (Smirnov et al., 2005). Metallothioneins are involved in the regulation of the essential metals copper and zinc and in the detoxification of non-essential metals (Amiard et al., 2006). Zinc has an essential function in the activation of metal-regulated transcription factors which initiate expression of the MT genes (Roesijadi, 1996).

Copper plays an essential function in a variety of metabolic processes. It is a component of many enzymatic and structural proteins, including Cu-Zn Review of Literature 23

SOD, cytochrome oxidase, and ceruloplasmin. Copper occurs naturally in soil and water. Mining, industrial discharges, and copper-based pesticides, especially algaecides, are sources of water contamination (WHO, 1998). Copper toxicity to fish and its bioavailability in water vary with physicochemical properties of water, i.e., pH, alkalinity, suspended solids, organic compound content, and hardness (Di Giulio and Meyer, 2008). The concentration of free copper, cupric ion (II), increases with water acidity. Copper hydroxide predominates in water of pH 8.0 and higher (Tao et al., 2001). Calcium, as a contributor of water hardness, was shown to reduce the harmful effects of copper on the growth of Nile tilapia (Abdel-Tawwab et al., 2007).

Copper plays a protective role against oxidative damage caused by variety of xenobiotics. The antioxidant effects of ceruloplasmin and metallothioneins seems to be the mechanism by which copper protects under these conditions (Pandey et al., 2001). Ceruloplasmin serves as a transport protein of copper in plasma. Parvez et al. (2003) reported that copper preexposure increases the activity of ceruloplasmin in fish serum. Ceruloplasmin, through ferroxidase activity, is involved in iron homeostasis and acts as an antioxidant in plasma (Gutteridge, 1985; Luza and Speisky, 1996). Copper is able to induce the biosynthesis of metallothioneins (Roesijadi, 1996). Ahmad et al. (2000) reported that metallothionein induction plays a role in the oxidative defence against chronic copper exposure in the liver of a freshwater catfish Channa punctatus.

The components of antioxidant defences are diversely influenced by metals. Both increases and decreases in enzyme activities and also enhanced and reduced levels of non-enzymatic components have been described after metal exposure. A specific biomarker of oxidative stress caused by metals does not exist, and for that reason a complex approach should be taken. Review of Literature 24

Metallothioneins seem to be a suitable biomarker of metal exposure, especially under laboratory conditions. In field studies the applicability of MT content in fish tissues as a biomarker is questionable following chronic metal exposure. In several field studies there were no significant correlations found between MT content and cadmium as well as between MT content and mercury in fish tissue (Sevcikova et al., 2011).

Lobster metallothioneins share a number of similarities with mammalian metallothioneins with respect to the presence of copper and cadmium, apparent molecular weights and amino acids composition, but differ substantially in their electrophoretic behaviour (Chou et al., 1991). Krishnamoorthy and Subramanian (1997) reported intensities of major polypeptide bands in the freshwater prawn, Macrobrachium lamerrei lamerrei, exposed to copper. Similarly, the reduction in the number of protein fraction in Scylla serrata treated with copper was reported by Ramanibai (1986). Fish can be used as bioindicators of metals in the environment by studying the induction of oxidative stress; however, the specific forms of biomarkers and mechanisms of their action still need to be investigated.

3.5 Light microscopy study (Histology)

The application of environmental toxicological studies on non- mammalian vertebrates is rapidly expanding; and for aquatic system, fish have become valuable indicator for the evaluation of the effects of noxious compounds (Khidr and Mekkawy, 2008). Histology and histopathology can be used as biomonitoring tools for health in toxicity studies (Meyers and Hendricks, 1985). Histological changes provide a rapid method to detect effects of irritants, especially chronic ones, in various tissues and organs (Bernet et al., 1999). Histopathological studies with light microscopy and electron microscopy are necessary for the description and evaluation of Review of Literature 25 poential lesions in aquatic animals exposed to various toxicants (Meyers and Hendricks, 1985).

Histopathological investigations have long been recognized to be reliable biomarkers of stress in fish (Van der Oost et al., 2003) and have been widely used as biomarkers in the evaluation of the health of fish exposed to contaminants, both in the laboratory and field studies. One of the great advantages of using histopathological biomarkers in environmental monitoring is that this category of biomarkers allows examining specific target organs, including gills, kidney and liver, that are responsible for vital functions, such as respiration, excretion and the accumulation and biotransformation of xenobiotics in the fish (Gernhofer, et al., 2001). Furthermore, the alterations found in these organs are normally easier to identify than functional ones (Fanta, et al., 2003), and serve as warning signs of damage to animal health (Hinton and Laurén, 1990). More than one tissue may be studied for assessment of the biological effects of a toxicant on localized portions of certain organs and also for assessment of subsequent derangements (degradations) in tissues or cells in other locations and this allows for diagnoses of the observed changes (Adeyemo, 2008). Previous histopathological studies of fish exposed to pollutants revealed that fish organs are efficient indicators of water quality (Cardoso et al., 1996; Barlas, 1999; Cengiz et al., 2001).

Review articles (Dutta et al., 1996; Wendalaar bonga, 1997) on ambient toxicants in fish have clearly demonstrated that increased concentrations of several heavy metals seriously damage the tissues of teleostean fish. The heavy metal ions interfere with respiration and osmoregulation causing cellular damage various body cells (De Boeck et al., 2001; Pandey et al., 2008). Among the heavy metals studied, the effects of water copper contamination on fish are of primordial interest. Even sublethal concentrations can induce severe damage, with negative impacts on fish performance and enhanced Review of Literature 26 susceptibility to secondary diseases that potentially cause mortality. Accordingly, copper levels determined during occasional monitoring of aquatic environments might not uncover the severity of contamination, especially in cases of sublethal doses (Canli and Stagg, 1996).

The toxicity of copper to fish has been widely studied in recent years and a wide range of effects on biotransformation, histology, haematology, osmoregulation, immunological modulations and behaviour of fish have been reported (Shariff et al., 2001; Cerqueira and Fernandes, 2002; Oliveira et al., 2004; Liu. et al., 2010). The gills (Alazemi et al., 1996), liver (Braunbeck, 1998) and kidney (Bernet et al., 1999) are commonly the primary target organs for many chemicals principally because of the role they serve within the body. The gills are multifunctional; given their large surface area, they are responsible for respiration, osmoregulation, acid–base balance and nitrogenous waste excretion, which also makes them extremely sensitive to water contamination (Au, 2004). Due to their delicate structure they are liable to damage by any irritant materials whether dissolved or suspended in the water (Roberts, 1978) and responds to environmental changes by structural alterations. In addition, they are not irritant specific but effect of factors’ intensity and duration of exposure, especially in cases of sublethal concentration of pollutants (Evans, 1987; Lindesjoo and Thulin, 1994; Karan et al., 1998).

The gills of fish are located on each side of the head beneath a gill- covering operculum and are composed of finger-like filaments attached to a cartilaginous gill bar. Numerous, delicate, leaf-like structures, the lamellae, project from each filament and these consist of minute capillaries covered by a single layer of thin epithelial cells. The epithelium forms a barrier between the fish’s blood and the surrounding water. Gills are generally considered a good tissue indicator of the water quality and are appropriate for the assessment of Review of Literature 27 environmental impact (Mallatt, 1985, Fanta et al., 2003). The different functions of gill can explain the structural complexity of this organ (Wilson and Laurent, 2002; Evans et al., 2005). They consist of several distinct cell types like specialized ion-transporting cells, the chloride cells (CCs) also called mitochondria-rich cells (Brunelli et al., 2010). Chloride cells are large round or ovoid cells rich in mitochondria and Na+/K+/ATPase, reflecting their extraordinary power of active ion transport (Hirose et al., 2003).

The importance of the gills in respiration and ionic regulation of fish has prompted many investigations of the effects caused to this organ by changes in environmental factors (De La Torre et al., 2005; Nero et al., 2006). Many investigators have reported the histopathological changes in the gills of different fish species exposed to pesticides and heavy metals (Sinhaseni and Tesprateep, 1987; Gill et al., 1988; Richmonds and Dutta, 1989; Erkmen et al., 2000). In copper-polluted water, gills are an important route of metal uptake, and in the initial stages of exposure, retain most of the metal burden, which may change their morphology (Lauren and McDonald 1985, Nowak et al., 1992, Pelgrom et al., 1995a, 1995b, 1995c, 1997, Campbell et al., 1999). Indeed, histopathological changes are the result of the integration of a large number of interactive physiological processes (Evans et al., 2005; Van der Oost et al., 2003).

The gills are efficient tools for biomonitoring potential impacts (Oliveira Ribeiro et al., 2005) because of their large area in contact with the water and high permeability (Arellano et al., 2004; Evans et al., 2005; Vigliano et al., 2006), and environmental impact caused by pollutants may affect fish gills tissues (Zeeman and Brindley, 1981; Schwaiger et al., 1997; Teh et al., 1997). Pollutants can directly cause degeneration or necrosis of the gill tissues (Camargo and Martinez, 2007; Ayandiran et al., 2009), but fish can develop mechanisms to react to pollutants that can result in cell hyperplasia, with Review of Literature 28 increased density of the cells of the secondary lamellae, as reported by Hughes and Perry (1976) and Tietge et al. (1988). Most of the gill injuries caused by sublethal exposure to pollutants affect the lamellar epithelium (Hinton and Lauren, 1990); however, some alteration in the blood vessels can occur when fish are under severe stress (Camargo and Martinez, 2007). Histopathological changes of gills such as hyperplasia and hypertrophy, epithelial lifting, aneurysm and increase in mucus secretion have been reported after the exposure of fish to a variety of noxious agents in the water, such as pesticides, phenol and heavy metal (Nowak et al., 1992). Several articles have described the histological effects on fish gills of various heavy metals dissolved in water (Amendd et al., 1969; Axlines and Cohnz, 1970; Wobeser, 1975). Enhanced information is lacking on the structural responses of gill tissue to this heavy metal copper.

The teleost liver has been the focus of toxicological studies and has indeed been shown to be very sensitive to pollutant exposure (Pinkney et al., 2004; Blazer et al., 2007). The liver of teleosts is the primary organ for biotransformation of organic xenobiotics (Heath, 1995; Hinton et al., 2001). There have been numerous reports of histo-cytopathological changes in livers of fish exposed to a wide range of organic compounds and heavy metals (Hinton et al.,1992; Vandenberghe, 1996; Braunbeck, 1998). Several laboratory and mesocosm studies have also demonstrated causal links between exposure to xenobiotics and the development of toxicopathic hepatic lesions in different fish species (Murchelano and Wolke, 1991; Moore, 1997; Myers et al., 1994; Stehr et al., 2003; Johnson et al., 2008).

Fish liver is a interesting model for the study of interactions between environmental factors and hepatic structures and functions. Since the liver of teleosts is important in the maintenance of internal homeostasis and the metabolism of xenobiotics (Chambers and Yarbrough, 1976) and has also be Review of Literature 29 shown to accumulate foreign compounds (Statham et al., 1978) and to be susceptible to damage by toxic agent (Racicot et al., 1975; Gingerich et al., 1978) the functional integrity of the liver in fish can be affected by enobiotics (Gingerich, 1982). According to Buck (1978) liver is the first line of defence against copper poisoning. Copper becomes toxic only when the high binding capacity of the liver is exceeded and copper is released into blood stream. In fish also, liver is the major storage organ for copper (Buckley et al., 1982; Shearer, 1984) and hence research on fish liver is important especially in the fields of problem induced by aquaculture conditions and aquatic pollution.

Biotransformation of organic xenobiotics, excretion of harmful trace metals, food digestion and storage, and the metabolism of sex hormones are the main hepatic functions (Hinton et al., 2001). Many contaminants, therefore, tend to accumulate in fish liver, exposing the tissues of this organ to comparatively higher levels than those experienced by other organs (Heath, 1995). Liver specimens treated with toxic levels of copper and cadmium showed an increase in fat vacuolization erythrocyte-filled sinusoids and venules, hepatocyte necrosis, condensation, swelling and lysis in mitochondria, dilation and fragmentation and vacuolization in rough endoplasmic reticulum of hepatocytes. Padmini and Usha Rani (2009) observed vacuolation and structural disruption in the hepatocytes of grey mullet in polluted estuaries. Wolf and Wolfe (2005) reported vacuolization, which tends to be uniformly distributed, in the livers of fishes that are exposed to toxic contaminants.

Amongst various organs, very little is known about the effects of Cu on the fish intestine which is believed to be the first organ that come into contact with food-borne contaminants. Also, the intestine is one of the most important sites where food enters and is assimilated. Thus, toxic substances, that enter the intestine, directly affect the vitality of the organism. Therefore intestine can serve as a potent indicator for water borne copper. Intestine was composed of Review of Literature 30 four histological layers e.g., mucosa, submucosa, muscularis and serosa. The intestinal mucosal layer was formed of the intestinal villi. The intestinal mucosa was composed of columnar epithelial cells with centrally and basally placed nuclei, mucous cells and leucocytes. Mucous cells were present all over the intestinal mucosa.

Toxic lesions most common in the intestine of fishes exposed to cadmium chloride include hyperemia, degenerative changes in the tips of villi, loss of structural integrity of mucosal folds, degenerative mucosal epithelium (hypertrophy, vacuolation, hyper-chromasia) necrosis, desquamation of mucosal epithelium, cellular debris, excessive mucus in gut of lumen, necrosis of submucosa and inflammatory infiltration of submucosa (Gardner and Yevich, 1970; Newman and MacLean, 1974; Gutierrez et al., 1978). Senapati et al. (2012) studied the ultrastructural changes in the alimentary canal of Anabas testudineus due to Almix 20WP exposure in laboratory condition. Since the intestinal tract is the first organ to come into contact with food-borne contaminants, ultra structural changes of these organs were chosen as criteria for the sub lethal action heavy metals.

Histopathological alterations have been reported in the intestine of fish as a result of exposure to toxicants (Hanna et al., 2005; Cengiz and Unlu, 2006; Soufy et al., 2007). According to many reviews histological analysis of the digestive system is considered as a good indicator of nutritional status of fish (Takashima and Hibiya, 1982; Cengiz and Unlu, 2006; Giari et al., 2008). Intestine is the important organ of digestive system of fishes. It is divisible in to two main parts: an anterior long, narrower part, small intestine and a posterior short, broader part, large intestine. The functional importance of intestine is digestion. Along with this, protection, activation, haemopoisis and hormonal action are the other functions. So that it is important to assess the histological structure of intestine as a diagnostic tool for normal health status of fish. Review of Literature 31

Muscle is the tissue of motion and is widely distributed in various organs of the body. It is composed of elongated muscle fibers, each an individual muscle cell, held together by connective tissues. Muscle is generally considered to have a weak accumulating potential (Bervoets and Blust, 2003; Erdogrul and Erbilir 2007, Uysal et al., 2009). Histological changes associated with heavy metals in fish have been studied by many authors (Thophon et al. 2003, Mohamed and Gad 2005, Athikesavan et al., 2006, Giari et al., 2007, Jiraungkoorskul et al., 2007 and Van Dyk et al., 2007). Sakr and Gabr, (1991); Abo Nour and Amer, (1995) and Das and Mukherjee, (2000) have studied the effect of different pollutants on fish muscles, and the histological alterations including degeneration in muscles bundles with focal areas of necrosis, atrophy of muscle bundles and edema between muscle bundles. Similar observations have been made by Nagarajan and Suresh (2005) in the muscle tissue of the fish Cirrhinus mrigala with increasing concentrations of sago effluent.

3.6 Scanning electron microscope study

The SEM is a technique that allows the study of the damage of surface ultrastructure of the gill epithelium that cannot be revealved by light or TEM (Devos et al., 1998 and Dutta et al., 1998). The scanning electron micrographs of the gill epithelium also revealved a higher occurrence of histopathological lesions such as hypertrophy, fusion of secondary lamellae, edema and mucus openings. These pathological changes may be a reaction to toxicants intake or an adaptive response to present the entry of the pollutants through the gill surface (Mohamed, 2009). Only very few studies have reported in detail the ultrastructural effects of toxic substances in brancial tissue (Li et al., 1998; Dang et al., 2000; Monteiro et al., 2008, 2009a, 2009b, 2010). Surface specialization of the gill and liver has received very little attention. Review of Literature 32

3.7 Transmission electron microscope study

TEM ultrastructure of gill has been reported by earlier workers in fish with various chemicals (Maina, 1990, Pfeiffer et al., 1997, Zahra khoshnood et al., 2011 and Ba-Omar et al., 2011). Ultrastructural changes in the liver have been used as biomarkers of toxic chemicals in environmental risk assessment (Alazemi et al., 1996). The ultrastructure of fish liver has proved to be valuable as a sensitive indicator of toxicant–induced injury (Braunbeck and Volkl, 1991). The ultrastructure of tissues and organs is altered when the waterborne contaminant is still at low levels and hence histopathological assays may provide a valuable screening method before severe damage occurs (Jiraungkoorskul et al., 2007). The toxic effects of cadmium have been studied in marine fish (Yamawaki et al., 1986; Reid and McDonald, 1988; Lemaire- Gony and Lemaire, 1992; Thophan et al., 2003, 2004), but information is lacking on the fine structural responses of gill and liver tissue to this heavy metal copper.

The present study was conducted to determine the acute and sublethal toxicity of heavy metal copper by static bioassay procedure exposing the fish Asian sea bass, L. calcarifer. Asian sea bass is considered as an important candidate fish species for brackishwater aquaculture. Sea bass is a euryhaline fish and extensively cultured both in earthen ponds and open cages under marine, brackishwater and freshwater conditions (Barlow et al., 1993). In India, various research programs have been undertaken on sea bass in order to evaluate the rearing techniques (Kailasam et al., 2006, 2007, Mohanraj et al., 2013) and also the interest to farm sea bass is increased day by day. However, various stages of this important fish during the course of their life cycle have to experience variety of environmental contaminants in the aquatic ecosystem. MATERIALS AND METHODS Materials and Methods 33

4. MATERIALS AND METHODS

4.1 Area of study

The present study was conducted at the PG & Research Department of

Zoology, Khadir Mohideen College, Adirampattinam.

4.2 Experimental animal, L. calcarifer

Asian Sea bass, L. calcarifer (Bloch) which occurs in the tropical and sub tropical area of Asia is a highly euryhaline species that lives in brackish water estuaries and in freshwater (Pillay, 1990). For spawning, they seem to require saline water, but larvae occur in fresh waters. The adult sea bass is a voracious carnivore but juveniles are omnivorous.The species supports important commercial and recreational fisheries throughout this stage, as well as established and growing aquaculture industries in Asia and Australia

(Copland and Grey, 1987).

Kingdom : Animalia

Phyllum : Chordata

Class : Actinopterygii

Order : Perciformes

Family : Latidae

Genus : Lates

Species : Calcarifer

Binomial name : Lates calcarifer (Bloch,1970) (Plate 1) Plate 1. The experimental animal Asian sea bass, Lates calcarifer (BLOCH, 1790) Materials and Methods 34

Moreover, the general characteristics of sea bass are elongate body and large mouth, slightly oblique and upper jaw extending behind the eye. Their distribution is in Indo-West Pacific: from the eastern edge of the Arabian Gulf along to China, Taiwan and southern Japan, southward to southern Papua New

Guinea and northern Australia (Rimmer and Russell, 1998). Sea bass is a species that is highly valued and in high demand throughout Asia.

4.3 Test Organism Collection and Acclimatization

Healthy hatchery reared juvenile Asian sea bass, L. calcarifer with mean total length of 7.06 ± 0.15cm and mean total weight of 10.18 ± 0.24gm were obtained from the Rajiv Gandhi Centre for Aquaculture, Thirumullaivasal near

Sirkali, Nagapattinam Dist, Tamil Nadu, India. Fishes were acclimatized for 2 weeks in stock tank to the experimental glass aquaria (120x50x50 cm) filled with 250 l of water with a salinity of 27+2 ppt, under a natural photoperiod 12 h:12 h (light:dark) cycle. The water in the tanks was passed through a 1µm filter, UV-sterilized, and refilled daily. Fish were fed twice daily with chopped fresh fish. They were starved for 24 h before and during the experiment.

4.4 Preparation of stock solution for copper toxicity test

3.9 gram of Copper ll sulphate pentahydrate (CuSo4 5 H2O) (Merck) was dissolved in one litre of double distilled water and used as the stock solution for preparing different concentrations of copper in rearing water. It was stored in a clean standard flask at room temperature, in the laboratory. Materials and Methods 35

4.5 Experimental Procedure

4.5.1 Exploratory test

Exploratory tests, otherwise called range finding test, were carried out to assess the approximate effective concentration range of copper required for conducting short term tests to assess the effect of copper on the metaboloic function of fish, as recommended by APHA (1985). The test solutions were prepared over a wide range of concentrations. These tests were performed by exposing 20 specimens of L. calcarifer in 50 litre sea water containing different concentrations of copper. The dead animals were removed immediately. Death of each animal was recorded. Three replicates were made for short term toxicity tests, the least concentration was chosen where no mortality was recorded in

24hrs and the highest lethal concentration was where 100% mortality was recorded in 24hrs.

4.5.2 Acute Toxicity test

A static bioassay test was performed to determine the 96-h LC50 of copper to L. calcarifer, following the Standard Methods (APHA, 1995). After acclimatization period, 2 month-old fish (7.06 ± 0.15cm in length and 10.18 ±

0.24gm in weight) were transferred from the stock tank to the experimental aquaria. Ten fishes were randomly placed in each glass aquarium filled with

250 l (120x50x50 cm) of water, with loading densities of 0.64 g/l. Fishes were exposed to nominal copper concentrations (10,20,30,40,40,60,70,80, 90,100 and

120ppm). Each concentration was done in three replicates. Control fish were held in a similar facility without exposure to copper. The water quality Materials and Methods 36 characteristics were measured daily: dissolved oxygen (DO) 6.8+0.5 mg/l, temperature 28.5+0.4ºC, salinity of 27+2 ppt and pH 7.65+0.2. The criteria for death were no gill movement and no reaction to gentle prodding. Fish mortality in each aquarium was recorded at the intervals of 24, 48, 72 and 96 hrs using the method of Sprague (1973). Dead fish were immediately removed.

Percent mortality was calculated and the values were transformed into the probit scale. Probit analysis was carried out as per Finney (1971). Regression line of probits against logarithmic transformation of calculations was made. Slope (S) function and confidential limit (upper confidential limit and lower confidential limit) of the regression line with correlation coefficient were calculated as follows:

LC84 LC50 ______+ ______LC50 LC16 S = ______2

2.77 log S F = Antilog ______= S 2.77

N = Total number of animals tested.

Upper Confidential Limit (UCL) = LC50 X F

Lower Confidential Limit (LCL) = LC50 / F

Based on acute toxicity, four lethal concentrations were derived for 24, 48,

72 and 96 hours exposure duration, which have been used as the experimental concentration of the copper toxicants in the subsequent experiments. Materials and Methods 37

4.5.3 Sub lethal toxicity test

4.5.3.1 Test concentration

Fish were exposed to nominal 6.83ppm and 13.66ppm as copper. The doses were theoretically sublethal, 10% and 20% respectively, of the Maximum Acceptable Toxicant Concentration (MATC), which was 68.3ppm. The MATC was represented as NOEC (No-Observed Effect Concentration)

(NOEC and LOEC) of the MATC by the 96-h LC50 (AF=MATC/

LC50=(NOEC-LOEC)/LC50).

4.5.3.2 System design

The recirculation closed system was set up according to Muthuwan (1998). The experiment was carried out in 360 l glass aquarium (120x60x50 cm), in which one compartment (50x50x40 cm) was partitioned by plastic gauze (mesh size 1.5 mm) to contain biofilter. Each aquarium was filled with 300 l of natural sea water (salinity of 27+2 ppt), which was pumped continuously over a biofilter column at the rate of 4 l/min. The water was continuously aerated throughout the experiment.

4.5.3.3 Test procedure

After 2 weeks of acclimatization in a holding tank, ten 2-month-old healthy fish (8.16 ± 0.19cm in length and 11.48 ± 0.67gm in weight) were transferred to each aquarium at a loading density of 0.69 g/l. Three replicates were performed for test concentration and control. Fish were fed twice daily with chopped fresh fish at 10:00 and 14:00. Uneaten food was quickly removed from the system. Fish were starved for 24 h before sampling. The experimental Materials and Methods 38 water (50%) was changed every 2 weeks to keep the water quality in acceptable limits according to APHA (1995), Water quality (dissolved oxygen, temperature, pH and salinity) was measured daily and water chemistry

(ammonia nitrogen, nitrite nitrogen, nitrate nitrogen) was measured twice weekly. The ammonia nitrogen and nitrite nitrogen levels were controlled not to exceed 0.2 mg/l. All chemical parameters were determined following the techniques of APHA (1995) using analytical grade reagents. The actual concentration of copper was measured weekly before and after its addition to maintain concentrations at the designed level. The water characteristics and the actual copper concentrations are shown in Table 1. Mortality and behaviour were observed daily in each concentration. Two fish from each aquarium were sampled at 0,7,14,21 and 28 days post-exposure

Parameters Range Mean+S.D Dissolved oxygen (mg/l) 6.6–7.1 6.87+0.25 Temperature (ºC) 25.6–27.9 26.8+1.15 Salinity (%) 27.4–30.2 29.0+1.44 pH 6.81–8.24 7.60+0.71 Ammonia nitrogen (mg/l) 0.02–0.94 0.57+0.48 Nitrite nitrogen (mg/l) 0.03–0. 97 0.58+0.49 Nitrate nitrogen (mg/l) 0.69–0.91 0.81+0.11 Actual copper concentration 0.013-0.026 0.019+0.01 (mg/l)

Table 1. Water quality characteristics and actual copper concentration during sublethal exposure to Asian sea bass, L. calcarifer. Materials and Methods 39

4.6 Bioaccumulation of copper

The fishes were exposed to 6.83ppm and 13.66ppm concentrations of copper for 28 days. After 0,7,14,21 and 28 days, the fishes were sacrificed and the tissues, muscle, gills ,liver, kidney and heart were excised out. The tissues were dried in an oven at 60ºC till constant weight was recorded. Dried samples of individual tissue were weighed and subjected to dry digestion with 20 ml of nitric acid and perchloric acid mixture (1:1) until a clear solution was obtained. The digested samples were then made upto 100 ml with double distilled water and analyzed in Atomic Absorption Spectrophotometer (Perkin- Elmor 2380) for copper concentration by the method described by Perkins (1970).

4.7 Estimation of Biochemical Composition

The fishes were exposed to 6.83ppm and 13.66ppm concentrations of copper for 28 days. After 0,7,14,21 and 28 days, the fishes were sacrificed. Muscle, gills and liver were excised out and analyzed for biochemical composition. Total protein was estimated in UV visible double beam spectrophotometer by Biuret method using bovine serum albumin as standard as suggested by (Lowry et al., 1951). Total free sugar was estimated by Phenol - Sulphuric acid method of Roe, 1955. Total lipids were estimated by gravimetric methanol - chloroform extraction method suggested by Folch et al. (1957) and modified by Linford (1965).

4.7.1 Estimation of Total Protein

Crude protein is the total amount of nitrogen converted to a protein equivalent. The nitrogen may come from a variety of substance in feed other than protein. Therefore, crude protein does not reveal the source of the nitrogen, the amino acid content (amino acids are building of protein), or whether the protein used was heat damaged, poorly processed or spoiled. Materials and Methods 40

Principle

The final blue colour is a result of biuret reaction of protein with copper ion in alkali and reduction of the phosphomolybdic - phosphotungstic reagent by the tyrosine and tryptophan present in the treated protein.

Chemicals Required

 Sodium carbonate

 Sodium hydroxide

 Potassium tartrate

 Copper sulphate

 Alkaline copper reagent

 Folin – Ciocalteau’s reagent

 TCA – Buffer

 Bovine serum albumin

Reagent Preparation

Sodium carbonate 20% (w/v), Reagent – A: 20 gm of sodium carbonate was dissolved in 100 ml of distilled water.

Sodium hydroxide 10% (w/v), Reagent – B: 10 gm of sodium hydroxide was dissolved in 100 ml of distilled water.

Potassium tartrate 1% (w/v), Reagent – C: 1 g of potassium tartrate was dissolved in 100 ml of distilled water.

Copper sulphate 0.5% (w/v), Reagent – D: 5 mg of copper sulphate was dissolved in 100 ml of reagent D and 10 ml of distilled water. Materials and Methods 41

Alkaline Copper Reagent : 50 ml of reagent - A, 20 ml of reagent B:10 ml of reagent C: 10 ml of reagent D and 10 ml of distilled water.

Folin-Ciocalteau’s Reagent: Commercially available Folin- Ciocalteau’s (2N) was dissolved with distilled water at the ratio of 1:16.

TCA - Buffer 10% (pH 8.6): 10 gm of TCA was dissolved in 100 ml of distilled water and used as buffer to homogenate tissue samples.

Bovine serum albumin : 500 mg of BSA was dissolved in 100 ml of distilled water with 1% SDS.

Sample extraction

1 g tissue was taken from the pooled tissues and homogenized in 10% TCA buffer. The homogenate was centrifuged at 1000 rpm for 15 minutes. The soluble and insoluble phases were separated.

Sample analysis

To the soluble phase, 0.5 ml alkaline copper reagent was added and incubated at room temperature for 10 minutes. Then 2 ml of diluted 2N Folin- Ciocalteau’s reagent was added to the mixture with vigorous shaking and incubated at room temperature for 10-15 minutes. The optical density of blue colour was read at 650 nm, using UV spectrophotometer . The concentration of protein in the sample analyzed was estimated from the standard graph obtained by taking O.D. values on Y-axis and concentration of standards series on X- axis. Materials and Methods 42

4.7.2 Estimation of total free sugar

Principle

Sulphuric acid hydrolysis the oligosaccharides into monosaccharide and convert the monosaccharide furfural derivatives which react with anthrone and produce a complex coloured product.

Reagents

Stock standard solution: 1 g of glucose was dissolved in 100 ml of Benzoic acid. Benzoic acid: 1g Benzoic acid was dissolved in 100 ml of distilled water. Anthrone reagent: 2g of Anthrone was diluted with 100 ml of 98% concentrated H2SO4. 5% TCA: 5 gms TCA was dissolved in 100 ml distilled water.

Sample Extraction

100 mg of the sample was homogenized with 5% TCA the sample were transferred into centrifuge tube and centrifuged for 10 minutes at 4000 rpm. The supernatant was used for the estimation of carbohydrate.

Procedure

For plotting the standard curve a set of standard were run with 10 ml, 20 ml etc of the standard solution taken in a series of test tubes and 4 ml of anthrone reagent was added and shaken well. The tubes were kept in boiling water bath for 10 minutes. Later the tubes were cooled in room temperature. The samples developed a green colour. The absorbance was read at 630 nm against a reagent blank in spectrophotometer. Plotting the concentration of standard solution in the X-axis drew the standard graph and optimal density on the Y-axis, Calculate the amount of carbohydrate per mg of the sample. Materials and Methods 43

Calculation

OD of Unknown Concentration of x Concentration of standard = OD of known carbohydrate in unknown sample

4.7.3 Estimation of total lipids

Nearly 0.5 g tissue was homogenized in 2 ml of chloroform: methanol 2:1 ratio, and then centrifuged at 3000 rpm for 10 minutes. The process was repeated thrice, the supernatant was cooled and concentrated at 40-45°C in flask under vacuum evaporator.

Principle

Lipids react with vanillin in an acidic medium of H2SO4 and phosphoric acid to form a chromogen, the colour intensity is directly proportional to the lipid content of the sample.

Reagents

1. Chloroform: Methanol (2:1) 20 ml of chloroform was mixed with 10 ml of methanol. 2. 0.9% Sodium Chloride: 900 mg of sodium chloride was distilled in 100 ml of distilled water. 3. Vanillin reagent: 600 mg of the vanillin in 100 ml distilled water 4. Phosphovanillin reagent: 35 ml of vanillin reagent was made up to 100 ml with concentrated Orthophosphoric acid.

5. Concentrated Sulphuric acid (H2SO4) 6. Standard: 60 ml of Olive oil was taken in 10 ml volumetric flask and diluted to 100 ml with absolute ethanol and mixed well. This standard is stable for one month when stored at 4-7°C (V/V). Materials and Methods 44

7. Blank Preparation: 0.2 ml Chloroform was taken and allowed to dry for blank.

Procedure

Nearly 0.2 ml of Concentrated H2SO4 was added to 0.2 ml of lipid extract and mixed well. All the tubes were kept at 100°C in an oven for 10 minutes and cooled in cold water for 5 minutes and add 20 ml of phosphovanillin. The content were mixed well and incubated at 37°C in water bath for 15 minutes. Then the tubes were cooled down to room temperature and colour developed was measured at 540 nm. A reagent blank and a set of standard (Olive oil) were run simultaneously. The amount of lipids present in the sample was calculated.

Sample Extraction

1 g of sample was taken, homogenized with 1 ml chloroform: methanol mixture. The homogenized solution was left overnight.

Sample analysis

1 ml of chloroform: methanol mixture and 1 ml 0.9% NaCl added to supernatant and left undisturbed for 10 minutes. 2 ml of Conc. Sulphuric acid was added to each test tube, boiled for 20 minutes in a hot water bath, cooled and 5 ml of 2% Phosphovanillin is added, allowed to stand for 10 minutes. The colour developed was measured in spectrophotometer (spectronic 20D+) at 540 nm against blank. The concentration of lipid in the sample analyzed was estimated from the standard graph obtained by taking O.D. values on Y-axis and concentration of standards on X-axis. Materials and Methods 45

Formula calculation

Concentration of lipid in O.D. of the sample Concentration of the standard The sample (mg/g) = ------X O.D. of the standard

4.8 Characterization of protein profile analysis by SDS-PAGE method

The fishes were exposed to 6.83ppm and 13.66ppm concentrations of copper for 28 days. After 0,7,14,21 and 28 days, the fishes were sacrificed. Muscle and gills were excised out and analyzed for characterization of protein. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the protocol of Weber and Osborn (1969). Proteins in the gel were stained with Coomassie brilliant blue.

Preparation of reagents and buffers

Acrylamide (30% stock): About 29.2 gm acrylamide and 0.8 gm bis- acrylamide were dissolved in 75 ml distilled water and made up to 100 ml. The solution was filtered through Whatman no. 1 filter paper and stored at 4oC.

Resolving gel (separating gel) buffer, pH. 8.8, 1.5 M Tris-HCl: About 18.17 gm Tris was added to 75 ml distilled water. The pH was adjusted to 8.8 with 6N HCl. The total volume was adjusted to 100 ml with distilled water and filtered through Whatman no. 1 filter paper. The buffer was stored at 4oC.

Stacking gel buffer, pH. 6.8, 1.0 M Tris-HCl: About 3 gm Tris was added to 40 ml distilled water and the pH was adjusted to 6.8 with 6 N HCl. The total volume was adjusted to 50 ml with distilled water and filtered through Whatman no. 1 filter paper. The buffer was stored at 4oC.

Electrophoresis buffer (Tank buffer), pH 8.3: Tris (3 gm), glycine (14.4 gm) and SDS (1 gm) were dissolved in 1000 ml distilled water and stored at 4oC. Materials and Methods 46

Sample buffer: Sample buffer was prepared by mixing 7.25 ml distilled water, 1.25 ml stacking gel buffer, 1 ml glycerol, 0.5 ml β-mercaptoethanol and 150 mg SDS. A pinch of bromophenol blue was added to the sample buffer.

Staining solution: Coomassie Brilliant Blue R 250 (200 mg) was dissolved in 50 ml methanol. To this, 7 ml acetic acid and 43 ml distilled water were added and mixed thoroughly. The staining solution was filtered through Whatman No. 1 filter paper.

Destaining solution: Destaining solution was prepared by mixing 7 ml acetic acid, 30 ml methanol and 63 ml distilled water.

Protein extraction

Protein was extracted from three different tissues namely gills, liver and muscles of treated and control fish specimens. The tissues were dissected out under aseptic conditions and immediately homogenized using pre-chilled mortar and pestle. During homogenization 100µl of phosphate buffer (pH 7.0) was added. The homogenate was centrifuged at 14,000 rpm for 10 minutes at 4oC. The supernatant was collected in microcentrifuge tubes and stored at - 20oC.

Gel casting

Running gel (12%) and stacking gel (5%) were casted between glass plates using the following ingredients:

12% Resolving gel (separating gel) (10 ml volume): Acrilamide (30%) = 4.0 ml D.D. Water = 3.3 ml Tris Buffer (pH 8.0) = 2.5 ml Ammonium persulphate (10%) = 100 µ1 Materials and Methods 47

SDS (10%) = 100 µ1 TEMED = 8 µ1

5% Stacking gel (4 ml): Acrilamide (30%) = 670 µ1 D.D. Water = 2.7 ml Tris Buffer (pH 6.8) = 500 µ1 Ammonium persulphate (10%) = 40 µ1 SDS (10%) = 40 µ1 TEMED = 6 µ1

The separating gel mixture was stirred well for degassing and was cast within the sandwiched glass plates leaving sufficient space for the stacking gel. The separating gel was layered on top with distilled water to get an even surface and was kept 20 min for polymerization. After polymerization the surface was washed carefully with distilled water to cast the stacking gel. The 5 % stacking gel mix was poured on the top of separating gel and the comb was inserted immediately to form wells. The comb was removed after 30 min and the wells were cleaned with electrode buffer using a syringe. After that the gel plate was fixed to the electrophoretic apparatus (Laemmli, 1970).

Sample preparation and loading

About 35µl of the protein solution was mixed with 35 µl of sample buffer (1X) taken in micro centrifuge tube. This mixture was heated on a boiling water bath (85oC) for 5 minutes. The heated protein sample was loaded in the wells of acrilamide gel using a micropipette. The tank buffer was added in the upper tank and lower tank and the set up was connected with electric power supply. A constant power supply of 75 volts was given till the samples reach the separating gel. After that the voltage was increased to 100 volts till Materials and Methods 48 the samples run to the bottom of the separating gel. When the dye front was reaching very close to the bottom of the separating gel (5 mm above the bottom), the power supply was stopped.

Staining and destaining the gel

The gel was transferred to a glass bowl, which contained the coomassie brilliant blue (R-250) staining solution. The gel was stained overnight. For destaining, the stained gel was put in the destaining solution in a bowl and was shaken continuously by placing the bowl on a shaker. Destaining was done two times. After destaining the gel, it was used for densitometry analysis.

Densitometry analysis

The destained gel was photographed by placing the gel on a trans- illuminator. The gel photo was used for densitometry analysis. The analysis of gel was done using gel doc system (Mediccare – Gelstan 1312)

4.9 Evaluation of histopathology

At the interval of 0, 7 and 28 days one fish from each concentration of copper was picked out randomly. The animal was sacrificed and muscle, intestine, gills, liver and heart in small pieces of 4-5mm sizes were fixed immediately in Davidson's Fixative (Humason, 1972) of the following composition.

i. 95% Ethyl alcohol - 330 ml ii. 100% Formalin - 220 ml iii. Glacial acetic acid - 115 ml iv. Distilled water - 335 ml Materials and Methods 49

After a period of 24 hours the fixed tissues were washed and transferred directly to 70% iso-propyl alcohol and kept overnight. The next day the tissues were transferred to 90% solution of iso-propyl alcohol and after one hour the tissues were given two changes of half an hour each in 100% iso-propyl alcohol for complete dehydration.

The tissues were cleared by immersing in 1:1 ratio mixture of iso-propyl alcohol and chloroform for one hour. After that, two changes were given in 100% chloroform for half an hour each. The tissues were kept in saturated chloroform - paraffin wax mixture overnight for cold impregnation.

Finally, the tissues were given three changes of 15 minutes each in molten wax for hot impregnation for block preparation. Paraffin with ceresin having melting point of 58-60C was used. The blocks were prepared in paper boats using molten wax after hot impregnation. The prepared blocks were labelled, and kept in cool water for proper solidification. Tissue sections were taken at 7μ thickness using a Rotary microtome.

The sections were spread over clean glass micro-slides smeared with egg- albumin, warmed with a few drops of distilled water and kept at least for 24 hours before staining. The slides were marked appropriately. The slides carrying sections were deparaffined in absolute xylene, passed through a descending series of iso-prophyl alcohol solution (100%, 90%, 70%, 50% and 30%) and stained with Delafields haematoxylin for 1-2 minutes and in case of over staining, destaining was done using a solution of very dilute hydrochloric acid and then stained with 0.5% aqueous eosin for 3-5 minutes. After staining, the sections were taken through an ascending series of iso-prophyl alcohol solutions (30%, 50%, 70%, 90%, and 100%) and finally to pure xylene. The cleared sections were mounted on DPX mountant and covered with No.1 cover slip. Materials and Methods 50

The stained sections were microphotographed using 35mm day light colour film having a speed rating of 100 ISO of Kodak make. Nikon Bright field transmission microscope with Koechler illumination and automatic exposure unit was used.

4.10 Scanning Electron Microscope (SEM)

At the interval of 0, 7 and 28 days one fish from each concentration of copper was picked out randomly for scanning electron microscopic studies, the gills and liver were fixed in 2.5% glutaraldehyde prepared in caccodylate (Sodium Phosphate) buffer adjusted to pH 7.4 for 4 hours and afterward washed in phosphate buffer for 15 minutes. Then samples were post fixed in 1% Osmium tetroxide for 80 minutes and washed in phosphate buffer. After dehydration in ascending series of acetone, samples were coated with gold palladium and then observed with scanning electron microscope (Leo-stereo scan, 440). Photographs were taken in various magnifications.

4.11 Transmission Electron Microscope (TEM)

At the interval of 0, 7 and 28 days one fish from each concentration of copper was picked out randomly for electron microscopy studies, the gills and liver were dissected out and fixed in Karnovsky’s fluid (4% paraformaldehyde and 3% glutaraldehyde in phosphate buffer, pH 7.2) for 24 hours. The tissue fixed in Karnovsky’s fluid was washed twice in phosphate buffer (pH 7.2-7.4) for approximately 15 minutes till the smell of the fixative disappeared. Then 1% osmium tetroxide was added and the tissue was kept in refrigerator (40C) for one and a half hours. The tissue turned black in color and hence, could be easily identified. The same was washed with phosphate buffer (pH 7.2-7.4) for half an hour. The tissue was then passed through a series of 70 %, 80%, and 90% ethanol for half an hour each, and transferred to enblock stain (2% Uranyl Materials and Methods 51 acetate in 95% ethyl alcohol) for one hour, followed by dehydrating in absolute ethanol. Clearing of the tissue was done in propylene oxide. The tissue was embedded in araldite and propylene oxide in the ratio 1:1 kept overnight in the rotator; to remove excess osmium tetroxide. The tissue was then placed in fresh pure araldite and kept for 4 hours in the rotator. Three changes were made with araldite, with a gap of 4 hours, and then embedding was done at 600C for 48 hours.

Trimming: With the help of a razor blade, the blocks were trimmed, so as to have a cutting face free from extra resin. The trimmed block was fixed on a specimen holder of the ultra microtome (Leica Emu.6). 6 mm thick glass pieces were cut with a glass cutting machine, then a plastic boat was fixed onto the edge of the glass piece by means of molten wax for collecting the sections. The boat was filled with water. The sections were allowed to be floated on the pool of water on the boat.

Semi-thin sections: Semi-thin sections of 1μm thickness were used for scanning the tissue under light microscope, with a view to locate the exact portion of the block to be sectioned. The floating sections were lifted with a thin glass rod onto a clean glass slide, placed on a hot plate at about 800 C and dried; sections stained with Toluidine blue for one minute, were washed in running water, dried, and mounted in DPX. The slides were scanned under a light microscope to ensure the quality of preservation and localize the area of interest for the subsequent ultra-thin sectioning. The blocks were further trimmed (if necessary) and ultra-thin sections of 500A° thickness were cut in a Leica em uc6 ultra microtome. The ultrathin sections were collected on copper grids which were either meshed or slotted. Grids were stored in grid boxes. Double staining with Uranyl acetate and lead citrate was done to obtain contrast during observation. The grids were observed under the Transmission electron microscope (TEM) (EMI K5302 Emu.6). Materials and Methods 52

4.12 Statistical Analysis

Concentration effect relationships were analyzed with Probit analysis to calculate the LC50 point estimate and associated 95% confidence intervals (Finney, 1971). All statistical test such as mean, standard deviation and twoway analysis of variance (ANOVA) were carried out by the procedure of Snedecor and Cochran (1967). RESULTS Results 53

5. RESULTS

5.1 Exploratory tests on copper toxicity to L. calcarifer

Exploratory tests were carried out to assess the approximate effective concentration range of copper on the metabolic function of L. calcarifer and the results of the study are:

0% Mortality (24hrs) 100% Mortality (24hrs) Copper concentration Copper concentration in water – 30ppm/l in water – 120ppm/l

Further toxicity tests were conducted within the above mentioned range of doses (ie. 30- 120ppm of copper) and the lethal concentration 50 (LC50) values were estimated.

5.1.1 Acute toxicity test to determine the LC50 values of L. calcarifer

The percentages of mortality of L. calcarifer observed, when the fishes were exposed to different concentrations of copper are given in (Table 2).

The LC50 value at 24 hrs of copper exposure was estimated as 110.83ppm. Lower and Upper limits of the concentrations were found to be from 103.71ppm to 118.44ppm. Acute toxicity of copper in L. calcarifer in 24 hrs was statistically not significant ( P>0.05) (Table 3 & Fig.2).

The LC50 value at 48 hrs of copper exposure was estimated as 93.57ppm . Lower and Upper limits of the concentrations were found to be from 85.06ppm to 102.94ppm. Acute toxicity of copper in L. calcarifer in 48 hrs was statistically highly significant (P<0.05) (Table 3 & Fig.3). Table 2 : Average mortality rate of Lates calcarifer in different concentrations of copper during acute toxicity study

Exposure Total Concentration of copper in water (ppm) Periods Number (hrs) fishes exposed 30 40 50 60 70 80 90 100 110 120

Mortality rate (number fishes and percentage of mortality)

Nos % Nos % Nos % Nos % Nos % Nos % Nos % Nos % Nos % Nos %

24 20 Nil 0 Nil 0 0 0 1 05 2 10 4 20 5 25 8 40 10 50 20 100

48 20 Nil 0 Nil 0 1 05 2 10 5 25 5 25 7 35 11 55 18 90 20 100

72 20 1 05 1 05 3 15 4 20 7 35 9 45 13 65 18 90 20 100 20 100

96 20 1 05 2 10 4 20 5 25 9 45 14 70 17 85 20 100 20 100 20 100 Table : 3 Percentage of mortality in L. calcarifer exposed to different concentrations of copper in different exposure periods 24 hrs

PROVISIONARY LINE: Y=-7.775656 + 6.245185 X

DOSE LDOSE(X) n r p EXP. Y w 60.0000 1.7782 100 5 0.0500 3.3292 0.215810 70.0000 1.8451 100 10 0.1000 3.7473 0.352102 80.0000 1.9031 100 20 0.2000 4.1095 0.474340 90.0000 1.9542 100 25 0.2500 4.4289 0.564564 100.0000 2.0000 100 40 0.4000 4.7147 0.617810 110.0000 2.0414 100 50 0.5000 4.9732 0.636196

EMPERICAL PROBITS:

3.354717 3.718182 4.158098 4.325317 4.746615

WORKING PROBITS

3.354898 3.719143 4.159556 4.328361 4.746932 4.999952 SNW= 286.0822 SNWX= 557.3758 SNWY= 1256.387 SNWXY= 2459.291 SNWX2= 1087.754 SNWY2= 5590.915 SXX= 1.815308 SXY= 11.46484 SYY= 73.24219

CHI-SQUARE= .8342743 DEGREES OF FREEDOM= 4 TABULAR CHI .05= 9.49 ITERATIONS= 2 60.0000 1.7782 100 5 0.0500 3.3171 0.212851 70.0000 1.8451 100 10 0.1000 3.7399 0.349423 80.0000 1.9031 100 20 0.2000 4.1061 0.473213 90.0000 1.9542 100 25 0.2500 4.4292 0.564621 100.0000 2.0000 100 40 0.4000 4.7182 0.618263 110.0000 2.0414 100 50 0.5000 4.9796 0.636267

EMPERICAL PROBITS : 3.354717 3.718182 4.158098 4.325317 4.746615 5 WORKING PROBITS 3.356991 3.7187 4.159612 4.328375 4.746949 4.99996 SNW= 285.4638 SNWX= 556.257 SNWY= 1254.238 SNWXY= 2455.407 SNWX2= 1085.731 SNWY2= 5583.462 SXX= 1.803833 SXY= 11.3877 SYY= 72.73193

CHI-SQUARE= .8408051 DEGREES OF FREEDOM= 4 TABULAR CHI .05= 9.49

CHI-SQUARE NOT SIGNIFICANT PROBIT REGRESSION LINE

Y= -7.907982 + 6.313054 X

T* S.E.(B)= 1.459345

MEAN OF X= 1.948608 MEAN OF Y= 4.393687

XI Y 95%UPPER LOWER 1.7782 3.3176 3.592£ 3.0431 1.8451 3.7402 3.931£ 3.5498 1.9031 4.1063 4.240£ 3.9726 1.9542 4.4293 4.546£ 4.3130 2.0000 4.7181 4.856£ 4.5800 2.0414 4.9794 5.158£ 4.8011

LOG LC50 2.044649 2.073469 2.01583 LC50= 110.828 118.4319 103.7122

Fig.2. Response curve of copper at 24hrs in L. calcarifer Table : 3 Percentage of mortality in L. calcarifer exposed to different concentrations of copper in different exposure periods 48 hrs

PROVISIONARY LINE: Y=-7.883055 + 6.541673 X

DOSE LDOSE(X) n r p EXP. Y w 50.0000 1.6990 100 5 0.0500 3.2311 0.188001 60.0000 1.7782 100 10 0.1000 3.7490 0.352720 70.0000 1.8451 100 25 0.2500 4.1870 0.498281 80.0000 1.9031 100 25 0.2500 4.5663 0.594145 90.0000 1.9542 100 35 0.3500 4.9010 0.634093 100.0000 2.0000 100 55 0.5500 5.2003 0.627161 110.0000 2.0414 100 80 0.8000 5.4711 0.586730

EMPERICAL PROBITS :

3.354717 3.718182 4.325317 4.325317 4.614517 5.125758 5.841902

WORKING PROBITS

3.368789 3.719263 4.332817 4.339798 4.622358 5.125055 5.804027 SNW= 348.1131 SNWX= 668.7926 SNWY= 1623.326 SNWXY= 3140.737 SNWX2= 1288.144 SNWY2= 7734.343 SXX= 3.264404 SXY= 22.01318 SYY= 164.4258

CHI-SQUARE= 15.98207 DEGREES OF FREEDOM= 5 TABULAR CHI .05= 11.07 ITERATIONS= 2 50.0000 1.6990 100 5 0.0500 3.1647 0.170584 60.0000 1.7782 100 10 0.1000 3.6986 0.335265 70.0000 1.8451 100 25 0.2500 4.1501 0.487018 80.0000 1.9031 100 25 0.2500 4.5411 0.589185 90.0000 1.9542 100 35 0.3500 4.8861 0.633360 100.0000 2.0000 100 55 0.5500 5.1946 0.627678 110.0000 2.0414 100 80 0.8000 5.4738 0.586183

EMPERICAL PROBITS : 3.354717 3.718182 4.325317 4.325317 4.614517 5.125758 5.841902 WORKING PROBITS 3.391258 3.71867 4.338168 4.337165 4.621906 5.12509 5.804441 SNW= 342.9272 SNWX= 659.556 SNWY= 1604.009 SNWXY= 3106.142 SNWX2= 1271.666 SNWY2= 7661.289 SXX= 3.134033 SXY= 21.13257 SYY= 158.6929

CHI-SQUARE= 16.19743 DEGREES OF FREEDOM= 5 TABULAR CHI .05= 11.07

*****CHI-SQUARE HIGHLY SIGNIFICANT***** PROBIT REGRESSION LINE

Y= -8.291355 + 6.742931 X

T* S.E.(B)= 1.45228

MEAN OF X= 1.923312 MEAN OF Y= 4.677404

XI Y 95%UPPER LOWER 1.6990 3.1647 3.519£ 2.8105 1.7782 3.6986 3.951£ 3.4462 1.8451 4.1500 4.329£ 3.9706 1.9031 4.5410 4.683£ 4.3991 1.9542 4.8860 5.032£ 4.7400 2.0000 5.1945 5.372£ 5.0165 2.0414 5.4736 5.694£ 5.2530

LOG LC50 1.971154 2.012595 1.929714 LC50= 93.57378 102.9425 85.05772

Fig.3. Response curve of copper at 48hrs in L. calcarifer Table : 3 Percentage of mortality in L. calcarifer exposed to different concentrations of copper in different exposure periods 72 hrs

PROVISIONARY LINE: Y=-4.837559 + 5.237997 X

DOSE LDOSE(X) n r p EXP. Y w 30.0000 1.4771 100 5 0.0500 2.8996 0.109918 40.0000 1.6021 100 5 0.0500 3.5540 0.286780 50.0000 1.6990 100 15 0.1500 4.0616 0.458921 60.0000 1.7782 100 20 0.2000 4.4764 0.575550 70.0000 1.8451 100 35 0.3500 4.8271 0.629475 80.0000 1.9031 100 45 0.4500 5.1308 0.632417 90.0000 1.9542 100 65 0.6500 5.3988 0.600479 100.0000 2.0000 100 90 0.9000 5.6384 0.548092

EMPERICAL PROBITS:

3.354717 3.354717 3.963136 4.158098 4.614517 4.874243 5.385484 6.281819

WORKING PROBITS:

3.630565 3.382582 3.968239 4.18789 4.620003 4.872786 5.385327 6.134727 SNW= 384.1633 SNWX= 705.9565 SNWY= 1818.655 SNWXY= 3379.838 SNWX2= 1303.941 SNWY2= 8854.519 SXX= 6.642212 SXY= 37.79199 SYY= 244.8818 CHI-SQUARE= 35.97766 DEGREES OF FREEDOM= 6 TABULAR CHI .05= 12.59 ITERATIONS= 2 30.0000 1.4771 100 5 0.0500 2.6686 0.071134 40.0000 1.6021 100 5 0.0500 3.3832 0.232076 50.0000 1.6990 100 15 0.1500 3.9376 0.406720 60.0000 1.7782 100 20 0.2000 4.3905 0.555191 70.0000 1.8451 100 35 0.3500 4.7734 0.624624 80.0000 1.9031 100 45 0.4500 5.1051 0.633805 90.0000 1.9542 100 65 0.6500 5.3977 0.600664 100.0000 2.0000 100 90 0.9000 5.6595 0.542367

EMPERICAL PROBITS : 3.354717 3.354717 3.963136 4.158098 4.614517 4.874243 5.385484 6.281819 WORKING PROBITS 4.193349 3.355232 3.943774 4.175659 4.618326 4.873621 5.385319 6.141864 SNW= 366.6582 SNWX= 677.2351 SNWY= 1753.881 SNWXY= 3272.042 SNWX2= 1256.565 SNWY2= 8612.621 SXX= 5.679932 SXY= 32.5398 SYY= 223.0635

CHI-SQUARE= 36.64604 DEGREES OF FREEDOM= 6 TABULAR CHI .05= 12.59

*****CHI-SQUARE HIGHLY SIGNIFICANT***** PROBIT REGRESSION LINE

Y= -5.798138 + 5.728906 X

T* S.E.(B)= 1.026745

MEAN OF X= 1.847048 MEAN OF Y= 4.783424

XI Y 95%UPPER LOWER 1.4771 2.6641 3.065£ 2.2634 1.6021 3.3799 3.662£ 3.0978 1.6990 3.9351 4.134£ 3.7365 1.7782 4.3887 4.535£ 4.2427 1.8451 4.7723 4.900£ 4.6444 1.9031 5.1045 5.245£ 4.9643 1.9542 5.3975 5.566£ 5.2289 2.0000 5.6597 5.862£ 5.4572

LOG LC50 1.884852 1.942466 1.827237 LC50= 76.70991 87.59232 67.17955

Fig.4. Response curve of copper at 72hrs in L. calcarifer Table : 3 Percentage of mortality in L. calcarifer exposed to different concentrations of copper in different exposure periods 96 hrs

PROVISIONARY LINE: Y=-4.959122 + 5.441496 X

DOSE LDOSE(X) n r p EXP. Y w 30.0000 1.4771 100 5 0.0500 3.0786 0.148961 40.0000 1.6021 100 10 0.1000 3.7585 0.356181 50.0000 1.6990 100 20 0.2000 4.2858 0.527304 60.0000 1.7782 100 25 0.2500 4.7167 0.618067 70.0000 1.8451 100 45 0.4500 5.0810 0.634849 80.0000 1.9031 100 70 0.7000 5.3965 0.600876 90.0000 1.9542 100 85 0.8500 5.6749 0.538160

EMPERICAL PROBITS :

3.354717 3.718182 4.158098 4.325317 4.874243 5.524567 6.036865

WORKING PROBITS

3.437885 3.72005 4.164062 4.35547 4.874046 5.52092 5.989517 SNW= 342.4398 SNWX= 615.2122 SNWY= 1635.981 SNWXY= 2971.809 SNWX2= 1110.823 SNWY2= 8026.046 SXX= 5.560059 SXY= 32.67822 SYY= 210.2661

CHI-SQUARE= 18.20587 DEGREES OF FREEDOM= 5 TABULAR CHI .05= 11.07 ITERATIONS= 2 30.0000 1.4771 100 5 0.0500 2.9000 0.110003 40.0000 1.6021 100 10 0.1000 3.6343 0.313194 50.0000 1.6990 100 20 0.2000 4.2039 0.503536 60.0000 1.7782 100 25 0.2500 4.6693 0.611532 70.0000 1.8451 100 45 0.4500 5.0627 0.635449 80.0000 1.9031 100 70 0.7000 5.4036 0.599615 90.0000 1.9542 100 85 0.8500 5.7042 0.530130

EMPERICAL PROBITS : 3.354717 3.718182 4.158098 4.325317 4.874243 5.524567 6.036865 WORKING PROBITS 3.629952 3.722965 4.159128 4.350525 4.874409 5.521341 5.995606 SNW= 330.3458 SNWX= 595.672 SNWY= 1590.663 SNWXY= 2897.374 SNWX2= 1079.043 SNWY2= 7850.958 SXX= 4.941284 SXY= 29.12573 SYY= 191.6807

CHI-SQUARE= 20.00296 DEGREES OF FREEDOM= 5 TABULAR CHI .05= 11.07

*****CHI-SQUARE HIGHLY SIGNIFICANT***** PROBIT REGRESSION LINE

Y= -5.813437 + 5.894365 X

T* S.E.(B)= 1.156597

MEAN OF X= 1.803177 MEAN OF Y= 4.815147

XI Y 95%UPPER LOWER 1.4771 2.8933 3.296£ 2.4905 1.6021 3.6297 3.902£ 3.3574 1.6990 4.2009 4.387£ 4.0151 1.7782 4.6676 4.812£ 4.5232 1.8451 5.0622 5.212£ 4.9127 1.9031 5.4041 5.587£ 5.2214 1.9542 5.7056 5.930£ 5.4808

LOG LC50 1.834538 1.884091 1.784985 LC50= 68.31844 76.57568 60.95154

Fig.5. Response curve of copper at 96hrs in L. calcarifer Results 54

The LC50 value at 72 hrs of copper exposure was estimated as 76.71ppm. Lower and Upper limits of the concentrations were found to be from 67.18ppm to 87.59ppm. Acute toxicity of copper in L. calcarifer in 72 hrs was statistically highly significant (P<0.05) (Table 3 & Fig.4).

The LC50 value at 96 hrs of copper exposure was estimated as 68.32ppm. Lower and Upper limits of the concentrations were found to be from 60.95ppm to 76.57ppm. Acute toxicity of copper in L. calcarifer in 96 hrs was statistically highly significant ( P<0.05) (Table 3 & Fig.5).

5.2 Sublethal effect of copper on the bioaccumulation of L. calcarifer 5.2.1 Muscle

In the muscle of untreated L. calcarifer, the copper concentration was between 0.76±0.02μg/g and 0.89±0.03μg/g (Table 4). After 7 days of exposure to 13.66ppm concentration of copper, the copper content in the muscle increased to 1.25±0.01μg/g. After 14 days of exposure, the values were 1.39±0.02μg/g at 6.83ppm and 1.49±0.02μg/g at 13.66ppm. After 21 days of exposure, the values increased to 1.43±0.03μg/g at 6.83ppm and to 1.67±0.02μg/g at 13.66ppm. At the end of the experiment (28 days), the copper concentration of muscle increased to 1.58±0.03μg/g at 6.83ppm and 1.99±0.03μg/g at 13.66ppm (Table 4 and Fig.6).

Accumulation of copper in muscle was significant between concentration of copper in rearing water, exposure duration and also when interaction of both was considered (P<0.05) (Table 5).

5.2.2 Gills

In the gills of untreated L. calcarifer, the copper concentration was between 2.64±0.02μg/g and 3.31±0.02μg/g (Table 6). After 7 days of exposure to copper, the copper content was 3.42±0.02μg/g at 6.83ppm and 3.57±0.02μg/g at Table 4 : Accumulation of copper in muscle of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Concentration of copper ( g/g ) after different days of exposure copper in μ water (ppm) 0 7 14 21 28

Control 0.76+0.02 0.84+0.02 0.89+0.02 0.76+0.02 0.89+0.03

6.83 0.91+0.02 0.98+0.02 1.39+0.02 1.43+0.03 1.58+0.03

13.66 0.88+0.03 1.25+0.01 1.49+0.02 1.67+0.02 1.99+0.03

Table 5 : Summary of ANOVA for Accumulation of copper in muscle of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 3.252924 1.626462 3641.333 1.57E-36 P<0.05

Factor :B 4 2.185178 0.546294 1223.047 9.46E-33 P<0.05

Factor:AxB 8 1.030809 0.128851 288.4726 3.32E-26 P<0.05

Within 30 0.0134 0.000447

Total 44 6.482311

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Table 6 : Accumulation of copper in gills of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Concentration of copper ( g/g ) after different days of exposure copper in μ water (ppm) 0 7 14 21 28

Control 2.81+0.02 3.31+0.02 2.64+0.02 2.76+0.02 2.94+0.02

6.83 2.64+0.02 3.42+0.02 3.96+0.02 4.12+0.02 4.89+0.02

13.66 2.89+0.02 3.57+0.02 4.02+0.02 4.56+0.02 5.38+0.02

Table 7 : Summary of ANOVA for accumulation of copper in gills of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 11.66907 5.834536 17621.08 8.82E-47 P<0.05

Factor :B 4 12.57589 3.143972 9495.218 4.59E-46 P<0.05

Factor:AxB 8 7.524151 0.940519 2840.493 5.14E-41 P<0.05

Within 30 0.009933 0.000331

Total 44 31.77904

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations

Results 55

13.66ppm. After 14 days of exposure, the copper content increased to 3.96+0.02μg/g and 4.02±0.02μg/g at 6.83ppm and 13.66ppm. After 21 days of exposure, the copper content was 4.12±0.02μg/g at 6.83ppm and 4.56±0.02μg/g at 13.66ppm. A sharp increase in copper content was recorded after 28 days of exposure. It was 4.89±0.02μg/g at 6.83ppm and 5.38±0.02μg/g at 13.66ppm concentrations of copper (Table 6 and Fig.7).

Accumulation of copper in gills was significant between concentration of copper in rearing water, exposure duration and also when interaction of both was considered ( P<0.05) (Table 7).

5.2.3 Liver

In the liver of untreated L. calcarifer, the copper concentration was between 36.50±0.15μg/g and 42.1±0.31μg/g (Table 8). After 7 days of exposure, the copper content was 44.6±0.21μg/g at 6.83ppm and 52.6±0.17μg/g at 13.66ppm. After 14 days of exposure, the copper content increased to 51.6±0.21μg/g and 67.2±0.15μg/g at 6.83ppm and 13.66ppm. After 21 days of exposure, the copper content was 55.9±0.16μg/g at 6.83ppm and 74.3±0.25μg/g at 13.66ppm. At the end of the experiment (28 days), the copper concentration of liver increased to 64.7±0.15μg/g at 6.83ppm and 82.6±0.15μg/g at 13.66ppm. (Table 8 and Fig.8).

Accumulation of copper in liver was significant between concentration of copper in rearing water, exposure duration and also when interaction of both was considered (P<0.05) (Table 9).

5.2.4 Kidney

In the kidney of untreated L. calcarifer, the copper concentration varied between 2.58±0.02μg/g and 3.78±0.03μg/g (Table 10). After 7 days of exposure, Table 8 : Accumulation of copper in liver of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Concentration of copper ( g/g ) after different days of exposure copper in μ water (ppm) 0 7 14 21 28

Control 36.5+0.15 38.4+0.10 42.1+0.31 39.6+0.15 40.3+0.12

6.83 37.5+0.25 44.6+0.21 51.6+0.21 55.9+0.16 64.7+0.15

13.66 38.9+0.15 52.6+0.17 67.2+0.15 74.3+0.25 82.6+0.15

Table 9 : Summary of ANOVA for accumulation of copper in liver of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 4240.834 2120.417 60617.99 7.96E-55 P<0.05

Factor :B 4 3452.263 863.0658 24673.12 2.78E-52 P<0.05

Factor:AxB 8 1567.388 195.9235 5601.016 1.96E-45 P<0.05

Within 30 1.0494 0.03498

Total 44 9261.535

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Table 10 : Accumulation of copper in kidney of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Concentration of copper ( g/g ) after different days of exposure copper in μ water (ppm) 0 7 14 21 28

Control 2.58+0.02 3.78+0.03 2.94+0.02 3.21+0.02 2.87+0.01

6.83 3.64+0.03 4.21+0.03 5.67+0.02 5.89+0.02 6.23+0.02

13.66 3.48+0.02 4.87+0.03 5.96+0.02 6.94+0.02 7.83+0.02

Table 11: Summary of ANOVA for Accumulation of copper in kidney of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 60.95817 30.47909 80208.1 1.19E-56 P<0.05

Factor :B 4 32.94482 8.236206 21674.23 1.94E-51 P<0.05

Factor:AxB 8 20.0262 2.503276 6587.567 1.73E-46 P<0.05

Within 30 0.0114 0.00038

Total 44 113.9406

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations

Results 56 the copper content was 4.21±0.03μg/g at 6.83ppm and 4.87±003μg/g at 13.66ppm. After 14 days of exposure, the copper content increased to 5.67±0.02μg/g and 5.96±0.02μg/g at 6.83ppm and 13.66ppm. After 21 days of exposure, the copper content was 5.89±0.02μg/g at 6.83ppm and 6.94±0.02μg/g at 13.66ppm. At the end of the experiment (28 days), the copper concentration of kidney increased to 6.23±0.02μg/g at 6.83ppm and 7.83±0.02μg/g at 13.66ppm. (Table 10 and Fig.9).

Accumulation of copper in kidney was significant between concentration of copper in rearing water, exposure duration and also when interaction of both was considered (P<0.05) (Table 11).

5.3 Sublethal effect of copper on the biochemical composition of L. calcarifer 5.3.1 Changes in total protein of different tissues 5.3.1.1 Muscle

In the muscle of untreated L. calcarifer, the total protein was between 61.58±0.50 to 62.59±0.59 mg/100mg. Exposure to copper reduced the total protein in muscle which was evident after 14 days in lower concentration (6.83ppm) and after 7 days in higher concentration (13.66ppm) being 59.78±0.63 and 59.64±0.53 respectively. The total protein reduced gradually in relation to exposure duration. After 28 days of exposure it was 56.21±0.60 at 6.83ppm and 52.14±0.63 at 13.66ppm concentrations of copper (Table 12 and Fig.10).

The changes in the total protein in muscle were significant between concentrations of copper in rearing water, exposure durations and when interaction of both was considered (P>0.05) (Table 13). Table 12 : Changes of total protein in muscle of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total protein (mg/100mg wet weight) in muscle after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 62.47+0.56 61.58+0.50 62.59+0.59 62.32+0.56 61.98+0.47

6.83 62.78+0.62 60.2+0.65 59.78+0.63 57.68+0.54 56.21+0.60

13.66 62.46+0.61 59.64+0.53 57.32+0.65 55.47+0.48 52.14+0.63

Table 13 : Summary of ANOVA for changes of total protein in muscle of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 184.2918 92.14592 276.8327 4.62E-20 P<0.05

Factor :B 4 163.8444 40.96111 123.0589 3.71E-18 P<0.05

Factor:AxB 8 98.73307 12.34163 37.0778 1.74E-13 P<0.05

Within 30 9.985733 0.332858

Total 44 456.8551

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Results 57

5.3.1.2 Gills

The total protein of gills of untreated L. calcarifer (control) was between 2.16±0.06 to 2.42±0.09 mg/100mg. After 7 days of exposure to copper, the total protein in gills was 2.21±0.07 at 6.83ppm and 2.06±0.08 at 13.66ppm. The total protein in gills was reduced as the exposure duration increased and after 28 days of exposure it was 1.87±0.05 at 6.83ppm and 1.72±0.06 at 13.66ppm concentrations of copper respectively (Table 14 and Fig.11).

The changes in the total protein in gills were significant between concentrations of copper in rearing water, exposure durations and also when interaction of both was considered (P<0.05) (Table 15).

5.3.1.3 Liver

The total protein of liver of untreated L. calcarifer (control) was between 1.58±0.06 to 1.75±0.07 mg/100mg. After 7 days of exposure to copper, the total protein in liver was 1.52±0.07 at 6.83ppm and 1.46±0.06 at 13.66ppm. After 14 days of exposure, the total protein in liver was 1.48±0.03 at 6.83ppm and 1.38±0.07 at 13.66ppm. The total protein in gills was reduced as the exposure duration increased and after 28 days of exposure it was 1.42±0.06 at 6.83ppm and 1.27±0.08 at 13.66ppm concentrations of copper respectively (Table 16 and Fig.12).

The changes in the total protein in liver were significant between concentrations of copper in rearing water, and exposure durations (P<0.05). When interaction of both was considered as non-significant (P>0.05) (Table 17). Table 14 : Changes of total protein in gills of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total protein (mg/100mg wet weight) in gills after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 2.19+0.06 2.42+0.09 2.28+0.10 2.24+0.09 2.16+0.06

6.83 2.34+0.05 2.21+0.07 2.01+0.05 1.98+0.08 1.87+0.05

13.66 2.25+0.07 2.06+0.08 1.95+0.08 1.85+0.07 1.72+0.06

Table 15 : Summary of ANOVA for Changes of total protein in gills of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 0.54064 0.27032 54.99277 9.24E-11 P<0.05

Factor :B 4 0.814724 0.203681 41.43603 8.28E-12 P<0.05

Factor:AxB 8 0.472049 0.059006 12.00396 1.71E-07 P<0.05

Within 30 0.147467 0.004916

Total 44 1.97488

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Table 16 : Changes of total protein in liver of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total protein (mg/100mg wet weight) in liver after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 1.65+0.07 1.75+0.07 1.62+0.10 1.58+0.06 1.64+0.08

6.83 1.63+0.08 1.52+0.07 1.48+0.03 1.45+0.05 1.42+0.06

13.66 1.69+0.10 1.46+0.06 1.38+0.07 1.32+0.06 1.27+0.08

Table 17 : Summary of ANOVA for changes of total protein in liver of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 0.294618 0.147309 28.78376 1.05E-07 P<0.05

Factor :B 4 0.237009 0.059252 11.57772 8.37E-06 P<0.05

Factor:AxB 8 0.073671 0.009209 1.799392 0.116555 P>0.05

Within 30 0.153533 0.005118

Total 44 0.758831

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations

Results 58

5.3.2 Changes in total free sugar of different tissues 5.3.2.1 Muscle

The total free sugar in the muscle of control L. calcarifer ranged from 25.48±0.45 to 26.54±0.30 mg/100mg. Exposure to copper reduced the total free sugar in muscle, which was evident after 14 days in (23.54±0.59) lower concentration (6.83ppm) and after 7 days in (23.78±0.33) higher concentration (13.66ppm). The total free sugar in muscle decreased gradually in L. calcarifer exposed to copper and the minimum values were recorded after 28 days of exposure, 22.15±0.25 at 6.83ppm and 22.01±0.61 at 13.66ppm concentrations of copper. (Table 18 and Fig.13).

The changes in the total free sugar in muscle were significant between concentrations of copper in rearing water, exposure durations, when interaction of both was considered (P<0.05) (Table 19).

5.3.2.2 Gills

The total free sugar in the gills of control L. calcarifer ranged from 6.37±0.08 to 6.89±0.08 mg/100mg. After 7 days of exposure, the total free sugar decreased to 6.12±0.08 in 6.83ppm and 6.02±0.17 in 13.66ppm concentrations of copper. After 14 days of exposure, the total free sugar reduced in treated L. calcarifer to 5.98±0.10 at 6.83ppm and 5.71±0.12 at 13.66ppm concentrations of copper. After 28 days of exposure, the total free sugar was 5.12±0.15 at 6.83ppm and 5.01±0.12 at 13.66ppm concentrations of copper. (Table 20 and Fig.14).

The changes in the total free sugar in gills were significant between concentrations of copper in rearing water, exposure durations, when interaction of both was considered (P<0.05) (Table 21). Table 18: Changes of total free sugar in muscle of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total free sugar (mg/100mg wet weight) in muscle after different copper in days of exposure water (ppm) 0 7 14 21 28

Control 26.34+0.63 25.96+0.55 26.54+0.30 25.48+0.45 25.69+0.45

6.83 25.46+0.55 24.56+0.48 23.54+0.59 23.02+0.54 22.15+0.25

13.66 26.21+0.23 23.78+0.33 23.14+0.44 22.25+0.40 22.01+0.61

Table 19 : Summary of ANOVA for changes of total free sugar in muscle of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 57.04899 28.5245 128.9832 1.85E-15 P<0.05

Factor :B 4 47.74668 11.93667 53.97572 2.8E-13 P<0.05

Factor:AxB 8 19.20472 2.40059 10.85509 4.97E-07 P<0.05

Within 30 6.634467 0.221149

Total 44 130.6349

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Table 20 : Changes of total free sugar in gills of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total free sugar (mg/100mg wet weight) in gills after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 6.87+0.11 6.45+0.09 6.89+0.08 6.37+0.08 6.48+0.06

6.83 6.74+0.08 6.12+0.08 5.98+0.10 5.74+0.05 5.12+0.15

13.66 6.54+0.11 6.02+0.17 5.71+0.12 5.42+0.09 5.01+0.12

Table 21 : Summary of ANOVA for changes of total free sugar in gills of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 6.754653 3.377327 328.3917 4.02E-21 P<0.05

Factor :B 4 7.193844 1.798461 174.872 2.5E-20 P<0.05

Factor:AxB 8 1.718969 0.214871 20.89283 2.8E-10 P<0.05

Within 30 0.308533 0.010284

Total 44 15.976

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Results 59

5.3.2.3 Liver

The total free sugar in the liver of control L. calcarifer ranged from 21.78±0.23 to 22.35±0.49 mg/100mg. After 7 days of exposure, the total free sugar decreased to 21.35±0.28 in 6.83ppm and 21.01±0.20 in 13.66ppm concentrations of copper. After 14 days of exposure, the total free sugar reduced in treated L. calcarifer to 21.02±0.30 at 6.83ppm and 20.04±0.13 at 13.66ppm concentrations of copper. After 28 days of exposure, the total free sugar was 19.89±0.17 at 6.83ppm and 19.05±0.22 at 13.66ppm concentrations of copper. (Table 22 and Fig.15).

The changes in the total free sugar in liver were significant between concentrations of copper in rearing water, exposure durations, when interaction of both was considered (P<0.05) (Table 23).

5.3.3. Changes in total lipids of different tissues. 5.3.3.1 Muscle

The total lipids in muscle remained more or less constant in control L. calcarifer (17.98±0.60 to 18.92±0.62 mg/g). After 7 days of exposure to copper, the total lipids decreased to 17.54±0.39 at 6.83ppm and 17.13±0.60 at 13.66ppm concentrations of copper. After 28 days of exposure to copper, the total lipids gradually reduced to 15.66±0.58 at 6.83ppm and 15.13±0.48 at 13.66ppm concentrations. After 28 days of the exposure, the reduction was maximum at higher concentration of copper (Table 24 and Fig.16).

The changes in the total lipids in muscle were significant between concentrations of copper in rearing water, exposure durations, when interaction of both was considered (P<0.05) (Table 25). Table 22: Changes of total free sugar in liver of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total free sugar (mg/100mg wet weight) in liver after different days copper in of exposure water (ppm) 0 7 14 21 28

Control 22.35+0.49 21.89+0.18 22.13+0.15 21.78+0.23 21.96+0.22

6.83 22.14+0.18 21.35+0.28 21.02+0.30 20.13+0.18 19.89+0.17

13.66 21.94+0.11 21.01+0.20 20.04+0.13 19.75+0.11 19.05+0.22

Table 23 : Summary of ANOVA for changes of total free sugar in liver of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 21.57721 10.78861 207.1015 2.77E-18 P<0.05

Factor :B 4 21.82454 5.456136 104.7377 3.55E-17 P<0.05

Factor:AxB 8 8.378964 1.047371 20.10565 4.47E-10 P<0.05

Within 30 1.5628 0.052093

Total 44 53.34352

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations

Table 24 : Changes of total lipids in muscle of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total lipids (mg/g. wet weight) in muscle after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 18.84+0.64 18.46+0.50 18.16+0.60 17.98+0.60 18.92+0.62

6.83 18.62+0.56 17.54+0.39 17.15+0.44 16.23+0.35 15.66+0.58

13.66 18.18+0.38 17.13+0.60 16.54+0.53 15.35+0.52 15.13+0.48

Table 25 : Summary of ANOVA for changes of total lipids in muscle of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 31.47532 15.73766 57.06186 5.97E-11 P<0.05

Factor :B 4 28.75719 7.189297 26.06707 2.18E-09 P<0.05

Factor:AxB 8 15.37572 1.921965 6.968691 3.58E-05 P<0.05

Within 30 8.274 0.2758

Total 44 83.88223

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Results 60

5.3.3.2 Gills

The total lipids in gills of control L. calcarifer varied between 1.19±0.05 to 1.64±0.06 mg/g. The total lipids reduction was slow initially in treated L. calcarifer and a sharp decline was recorded after 28 days of exposure when the total lipids decreased to 0.81±0.02 at 6.83ppm and 0.76±0.04 at 13.66ppm concentrations (Table 26 and Fig.17).

The changes in the total lipids in gills were significant between concentrations of copper in rearing water, exposure durations, when interaction of both was considered (P<0.05) (Table 27).

5.3.3.3 Liver

The total lipids in liver of control L. calcarifer varied between 21.91±0.66 to 22.92±0.67 mg/g. After 7 days of exposure to copper, the total lipids decreased to 21.51±0.63 at 6.83ppm and 21.12±0.28 at 13.66ppm concentrations of copper. After 28 days of exposure to copper, the total lipids gradually reduced to 19.53±0.70 at 6.83ppm and 19.21±0.42 at 13.66ppm concentrations. After 28 days of the exposure, the reduction was maximum at higher concentration of copper (Table 28 and Fig.18).

The changes in the total lipids in liver were significant between concentrations of copper in rearing water, exposure durations, when interaction of both was considered (P<0.05) (Table 29).

5.4 Subleathal effect of copper on the characterization of protein profile in L. calcarifer

SDS-Poly Acrilamide Gel Electrophoresis was used to find out the effects of the copper treatment on protein profiles of muscle, gill and liver Table 26 : Changes of total lipids in gills of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total lipids (mg/g. wet weight) in gills after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 1.43+0.06 1.64+0.06 1.25+0.07 1.19+0.05 1.55+0.05

6.83 1.25+0.06 1.15+0.04 0.98+0.04 0.84+0.04 0.81+0.02

13.66 1.68+0.07 1.05+0.03 0.86+0.03 0.79+0.03 0.76+0.04

Table 27: Summary of ANOVA for Changes of total lipids in gills of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 1.598804 0.799402 364.4691 8.99E-22 P<0.05

Factor :B 4 1.437213 0.359303 163.8161 6.38E-20 P<0.05

Factor:AxB 8 0.804173 0.100522 45.83055 9.92E-15 P<0.05

Within 30 0.0658 0.002193

Total 44 3.905991

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations Table 28: Changes of total lipids in liver of L. calcarifer exposed to sublethal concentrations of copper

Concen- tration of Total lipids (mg/g. wet weight) in liver after different days of copper in exposure water (ppm) 0 7 14 21 28

Control 22.43+0.65 22.84+0.73 22.92+0.67 22.31+0.77 21.91+0.66

6.83 22.54+0.58 21.51+0.63 20.94+0.68 20.13+0.68 19.53+0.70

13.66 22.89+0.58 21.12+0.28 20.52+0.46 19.63+0.54 19.21+0.42

Table 29 : Summary of ANOVA for Changes of total lipids in liver of L. calcarifer exposed to sublethal concentrations of copper

Source of Degree of Sum of Mean F value P-value Significant variation freedom square square

Factor :A 2 37.32715 18.66358 48.30062 4.17E-10 P<0.05

Factor :B 4 27.29472 6.823681 17.65943 1.5E-07 P<0.05

Factor:AxB 8 17.11685 2.139606 5.537219 0.000243 P<0.05

Within 30 11.59213 0.386404

Total 44 93.33086

Factor A - Concentrations

Factor B - Exposure durations

Factor AxB - Both concentrations and exposure durations

Results 61 tissues of the fish, Asian sea bass (L. calcarifer) using with standard marker protein. The changes in the protein profile due to the treatment, is presented in the figures 19 to 29. In the SDS-PAGE separation, the muscle and gill tissues alone showed clear polypeptide bands. The overall results clearly indicated that certain proteins slightly increased in quantity when the treatment time was increased. However an increase in concentration resulted in a slight decrease in the quantity of different polypeptides in each tissue sample. The densitometry analysis data also presented in figures 19 to 29

5.4.1 Protein Profile in muscle tissue

In muscle tissue of control group, eleven polypeptide bands were detected. The molecular weights of these eleven polypeptides were: 183.401, 162.873, 146.280, 127.263, 113.377, 102.290, 94.038, 67.306, 49.977 and 32.571 KDa (Fig.20 ).

In 7 day-old treated fishes, the muscle tissues were difference in total number of polypeptide bands (Fig 21 & 22). At 6.83ppm concentration of copper, seven bands (121.747, 88.485, 56.260, 50.906, 47.090, 40.617 and 24.0 KDa) (Fig. 21) were detected by the densitometry analysis, where as in 13.66ppm concentration treated muscles, six bands (220.508, 199.701, 178.702, 145.534, 86.915, 68.091 KDa) were detected (Fig.22).

The muscle tissues from 28 days old treated fishes also showed variation in total number of polypeptide bands due to two different concentrations (Fig. 23 & 24). At 6.83ppm concentration, the tissue showed the presence of six polypeptides (179.668, 145.289, 93.649, 73.629, 67.994 and 61.251 KDa) (Fig. 23). At 13.66ppm concentration the number of polypeptide bands in muscle tissue were reduced to five with molecular weights of 186.820, 152.039, 94.597, 74.713 and 69.195 KDa (Fig.24). Fig.19: Electrophorogram of Molecular weight marker Protein Fig.20: Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer (Control) Fig.21: Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 7 days of exposure to 6.83ppm concentration of copper Fig.22: Electrophorogram and densitometric scan images of protein Profile in the muscle tissue of L. calcarifer after 7 days of exposure to 13.66 ppm concentration of copper Fig.23: Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 28 days of exposure to 6.83ppm concentration of copper. Results 62

5.4.2 Protein Profile in gill tissue

In gill tissue of control group, six polypeptide bands were detected. The molecular weights of these six polypeptides were: of 153.409, 128.711, 105.195, 83.707, 67.306 and 44.143 KDa (Fig.25 ).

In 7 day-old treated fishes, the gill tissues were difference in total number of polypeptide bands (Fig 26 & 27). At 6.83ppm concentration of copper, five bands (82.512, 40.210, 22.345, 19.242 and17.572 KDa) (Fig. 26 ) were detected by the densitometry analysis, where as in 13.66ppm concentration treated gills, four bands (82.868, 51.574, 46.179 and 40.255 KDa) were detected (Fig.27).

The gill tissues from 28 days old treated fishes also showed variation in total number of polypeptide bands due to two different concentrations (Fig. 28 & 29). At 6.83ppm concentration, the tissue showed the number of polypeptide band is only one (15.388 KDa) (Fig. 28). At 13.66ppm concentration the number of polypeptide bands in gill tissue were also reduced to one with molecular weight of 15.326 KDa (Fig.29).

5.4.3 Protein Profile in liver tissue

In liver tissues, the protein concentrations were very low and separation of polypeptides was also very poor. Hence the densitometer could not detect any bands in liver tissues in control and treated groups.

5.5. Sublethal effect of copper on the histopathological changes in L. calcarifer

5.5.1 Histology of gills

Histological study of the gills shows a typical structural organization of the respiratory lamellae in the untreated fish. There are four gill arches on each Fig.24: Electrophorogram and densitometric scan images of protein profile in the muscle tissue of L. calcarifer after 28 days of exposure to 13.66 ppm concentration of copper. Fig.25: Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer (Control) Fig.26. Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 7 days of exposure to 6.83 ppm concentration of copper Fig.27: Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 7 days of exposure to 13.66 ppm concentration of copper. Fig.28: Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 28 days of exposure to 6.83 ppm concentration of copper. Fig.29: Electrophorogram and densitometric scan images of protein profile in the gill tissue of L. calcarifer after 28 days of exposure to 13.66 ppm concentration of copper Results 63 side of the buccal cavity. Each arch is composed of numerous gill filaments with two rows of semi circular secondary lamellae that are lined up along both sides of the primary gill lamellae. The primary gill lamellae (PL) consist of centrally placed rod like central axis (CA) with chloride cells (CC) and with blood vessels on either side. The lamellae are lined by squamous epithelium and many capillaries separated by pillar cells (PC) run parallel along the surface. Below that epithelium are lamellar blood sinuses separated by pillar cells. Between the lamellae, the filament is lined by a thick stratified epithelium constituted by several cellular types, such as chloride, mucous and pavement cells. Secondary gill lamellae (SL) also termed as respiratory lamellae were highly vascularised and covered with thin epithelial cells. Blood vessels (BV) are extended into each of the secondary gill filaments. The blood cells of the secondary gill lamellae have a single nucleus which is flattened in appearance. The region between the two adjacent secondary gill lamellae is known as inter lamellar region (ILR). One to two erythrocytes were usually observed within each capillary lumen. Chloride cells were identified as large epithelial cells with light cytoplasm, usually present at the base of lamellae. At the base of the lamellae the epithelium of the filament is endowed with mucous cells, lacked the light cytoplasm and was smaller than chloride cells (Plate 2. Fig. A).

5.5.2 Histopathology of gills

The copper treatment (Plate 2, Fig. B, C, D, E & F) has induced marked pathological changes in fish gills architecture. The changes include curling of secondary lamellae (CSL), a few telangiectasis (lamellar capillary aneurism) at the tip of the secondary lamellae (TSL) and desquamated epithelium at 7days 6.83ppm concentration of copper (Plate 2, Fig. B). Other observations during the experiment includes rupture and breakdown of pillar cell system (RBPC),) hyperplasia of epithelial cells (HEC) and lifting of secondary gill lamellar epithelium (LSGE). The gills of experimental fish showed extensive edema of Results 64 the epithelial cells (EEC) and blood congestion (aneurism) in many areas of secondary lamellae with the complete damage and breakdown of the pillar cell system (CDPCS) at 28 days 6.83ppm concentration of copper (Plate 2, Fig. C & D). The damage of gills of fish exposed to the higher concentrations of lethal nature was severe. Shortened and clubbing of ends of the secondary gill lamellae, fusion of adjacent secondary gill lamellae and necrosis in the primary lamellae were well marked. Besides these changes pyknotic nuclei, lamellar clubbing, rupture of secondary lamellar tips (RSLT), edema and rupture of epithelia cells (EREC) were also observed at 7 days 13.66ppm concentration of copper (Plate 2, Fig.E). The blood capillary cells were damaged and leads to blood congestion. The secondary lamellae were highly deformed as a result of complete lifting and desquamation of epithelium which leads to conjunction of adjacent filaments. Hyperplasia of chloride cells leads to clustering of these cells in multiple layers both in primary and secondary lamellae. The gill cells showed extensive aneurism with rupture in secondary lamellae at 28 days 13.66ppm concentration of copper (Plate 2, Fig.F).

5.5.3 Histology of intestine

Histological findings showed that the basic organization of intestinal wall is similar to that in other vertebrates and is formed by tunica mucosa with a loose connective tissue lamina propria (LP) tunica submucosa, tunica muscularis (inner circular and outer longitudinal smooth muscles) and tunica serosa layers. The outermost serosa is followed by a well developed musculari (longitudinal and circular muscle) embedded in loose connective tissue (CE) richly supplied with blood capillaries. It merges with tunica propria of the underlying mucosal coat. Intestinal mucosa was composed of the epithelial layer, the lamina propria and the stratum compactum. The mucosa is raised into several longitudinal folds. Mucosal folds consisted of connective tissue cores covered by intestinal epithelium. Columnar cells or enterocytes were the more Plate: 2 Histological changes of gills in L. calcarifer Light micrographs of a paraffin section stained with Hematoxylin and Eosin (40 X)

A - Control B - After 7 days of exposure to 6.83 ppm concentration of copper C & D - After 28 days of exposure to 6.83 ppm concentration of copper E - After 7 days of exposure to 13.66 ppm concentration of copper F - After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: PL - Primary lamellae SL - Secondary lamellae PC - Pillar cells CC - Chloride cells EC - Epithelial cells NE - Nucleated Erythrocyte TSL - Telangiectasis at the tip of Secondary lamellae CSL - Curling of Secondary lamellae BPC - Breakdown of Pillar cells HEC - Hyperplasia of Epithelial cells LSGE - Lifting of Secondary gill lamella epithelium CDPCS- Completely damaged Pillar cell system RSLT - Rupture of Secondary lamellae tip EEC - Edema of Epithelial cells RBPC - Rupture and breakdown of Pillar cell system EREC - Edema and Rupture of Epithelial cells Plate: 2 Results 65 numerous of the epithelial lining cells and closely resemble those of higher vertebrates. These tall and cylindrical cells had striated, free borders (brush border or microvilli) and contained oval nuclei which were situated either centrally or toward the bases of the cells. Intestinal mucous-secreting cells or goblet cells (GC) were interspersed among the columnar cells, being more numerous along the sides rather than on the crests or at the bases of the mucosal folds (Plate 3, Fig. A).

5.5.4 Histopathology of intestine

Lower concentration (6.83ppm) of copper at 7 days of exposure induced significant changes in the intestine of experimental fish. In the exposed group a degenerative effect is evident in the mucosal lining and villi of the intestine. The villi tend to become fused (FV) due to excessive hypertrophies and there is sloughing off of the mucosal lining, finally leads to large lumen (LL) (Plate 3, Fig.B). Hypertrophy of epithelial cells, swelling or edema of lamina propria (SLP) and flattening of villi (FLV) ultimately leading to rupture of villi at tip, are also evident at 28 days 6.83ppm concentration of copper (Plate 3. Fig. C & D). Large areas of intestinal mucosal folds were damaged and debris of the fragmented secondary mucosal folds was observed in the cavities. Complete erosion in the top layer of mucous with severe vacuolation and degeneration in the gastric epithelium, along with disruption and necrosis of the gastric glands were also observed mucosal folds. The nuclei of columnar epithelial cells exhibited pyknosis with fusion of boundaries of columnar epithelial cells. Degeneration of the submucosal layer, i.e., lamina propria and the absorptive columnar epithelial cells resulted in vacuolation in the submucosal layer cytoplasmic boundaries. There was a distortion of basement membrane of the villi and blood vessel, and lymphocytes were fully distorted. There was a degeneration of columnar epithelium of the intestine. Longitudinal muscle fibers were loosely arranged and become swollen (SLML). In later stages Plate: 3 Histological changes of intestine in L. calcarifer Light micrographs of a paraffin section stained with Hematoxylin and Eosin (40 X)

A - Control B - After 7 days of exposure to 6.83 ppm concentration of copper C & D- After 28 days of exposure to 6.83 ppm concentration of copper E - After 7 days of exposure to 13.66 ppm concentration of copper F - After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: GC - Goblet cells LP - Laminar propria N - Nucleus L - Lumen CE - Columnar Epithelium FV - Fusion of villi LL - Large lumen FLV - Flattened villi DCML- Damaged circular muscle layer DL - Distended lumen DLML - Damaged longitudinal muscle layer VF - Vacuole formation SLP - Swelling of lamina propria CCA - Cracked clay appearance of the tissues SLML - Swelling of longitudinal muscle layer DGC - Damaged goblet cells DMM - Disarrangement of muscularis mucosa Plate: 3 Results 66 flattening of microvilli and a cracked clay appearance of the tissue (CCA) were very apparent at 7 & 28 days 13.66ppm concentration of copper (Plate 3. Fig. E &F)

5.5.5 Histology of muscle

Muscle is the tissue of motion and is widely distributed in various organs of the body. It is composed of elongated muscle fibers, each an individual muscle cell, held together by connective tissues. The body musculature is relatively simple in fishes. The fins are usually provided with individual small muscles, but the most complex organization is in the head region. Many individual muscles show an arrangement of fibers into bundles separated from each other by connective tissue portions. The nerves generally penetrate a muscle on its side and branch out as they penetrate the connective tissue. Segmentation or metamerism of vertebrate musculature is seen clearly in the lateral muscles of the fishes. They are divided into myotomes or muscle segments, each of which is bent into a single V with the angle directed anteriorly. Each myotome is divided into an upper (epaxial) and a lower (hypaxial) half by a groove running along the side of the fish. Successive myotomes are separated by obliquely oriented connective tissue partitions (myosepta). The epaxial portion is separated from the hypaxial myotome by a fibrous septum running anteroposteriorly, parallel to the longitudinal axis. This large cell is multinucleated. The fibers are unbranched. Each fiber is enclosed in a thin membrane, the sarcolemma which is a specialized cell membrane. The protoplasm of the fiber, sarcoplasm contains five longitudinal myofibrils which extent throughout. Each myofibril is composed of two types of short myofilaments which are precisely arranged giving the appearance of transverse banding, the striations (Plate 4, Fig.A). Results 67

5.5.6 Histopathology of muscle

In copper treated muscle edema and mild lymphocyte infiltration, vacuolar degeneration in muscle bundles and atrophy of muscle bundles were observed. Edema between muscle bundles and splitting of muscle fibers were seen at 7 days 6.83ppm concentration of copper (Plate 4, Fig. B). After 28 days of exposure in lowest concentration of copper the muscle tissue exhibited dystrophic changes with marked thickening and separation of muscle bundles. The vacuolar degeneration in muscle bundles with aggregations of inflammatory cells between them and inter myofibrillar space (IMFS) get widened ends with disintegration of myofibrils were observed (Plate 4, Fig. C & D). The muscle seems to have lost the myoseptum that separates each myotome. Disintegrated epidermis is also seen in the section of muscle. Intramuscular edema (EMF) is a common feature in highest concentration (13.66ppm) at 7 & 28 days treated fish. Significant changes noted are broken myofibrils and gap formation between muscle bundles (GFMF) which finally leads to degeneration in muscle bundles (MD) accompanied with focal areas of necrosis as well as atrophy (Plate 4, Fig. E & F ).

5.5.7 Histology of liver

The surface of liver is covered with serous membrane and some connective tissue extends inward into parenchyma. It is composed of parenchymal cells (hepatocytes) (HC) and lattice fibres, which support the former. Hepatic cells are roundish polygonal, containing clear spherical nucleus (N). They are located among sinusoids forming cord like structures known as hepatic cell cords. In fish, these structures are generally obscure. Bile canaliculus, is centrally located in each cord. Blood sinusoids (BS), which are irregularly distributed between the polygonal hepatocytes, are fewer in number and are lined by endothelial cells with very prominent nuclei. Hepatocytes are Plate: 4 Histological changes of muscle in L. calcarifer Light micrographs of a paraffin section stained with Hematoxylin and Eosin (40 X)

Abbreviations used: MF - Myofibrils IM - Intestitial materials GFMF- Gap formation in myofibril IMFS - Inter myofibrillar space DMF - Disintegrated myofibrils EMF - Edema between muscle fibre MD - Muscle degradation ME - Muscle edema Plate: 4 Results 68 polygonal and have a distinctive central nucleus with densely staining chromatin margins and a prominent nucleolus. Fairly large quantities of lipid glycogen granules were also observed in the cytoplasm of fish hepatic cells (Plate 5,Fig. A). Hepatic cells have many vital functions. Other than the secretion of bile, they play an important role in protein, lipid and carbohydrate metabolism. They serve as storage sites for some nutrients and detoxification is another function attributed to them.

5.5.8 Histopathology of liver

Generally, the liver in teleost fish is a compound organ in the form of hepatopancreas. The changes observed in the liver tissue on exposure to 6.83ppm concentration of copper for 7 days included swelling and rounding off of hepatocytes, detachment of cells from each other. Pancreatic acini appeared to have lost its architecture. Cytoplasm of hepatocytes became more basophilic. These changes include degenerated hepatocytes presenting a homogenous cytoplasm and a large central or sub central spherical nucleus (Plate 5, Fig.B). The important histopathological changes observed in the copper treated groups were pyknotic nuclei and clear cell foci. The liver tissue after 28 days of copper exposure in lowest concentration were revealed vacuolation of hepatocytes, condensation of nuclear chromatin and swelling of hepatocytes (HSHC) (Plate 5, Fig. C). Notable change in high concentration of copper at 7 days treated fish is extensive vacuolation of hepatic cells (Plate 5, Fig.D) with several foci of coagulative necrosis. In high concentration blood congestion (BC) and focal necrosis of hepatic parenchyma in addition to the accumulation of dark granules (ADG). Appearance of Blood streaks among hepatocytes was a marked change in treated liver. Formation of cytoplasmin vacuoles (CV), along with atrophy, necrosis and disappearance of hepatocytic cell wall and disposition of hepatic cords were also observed. The degenerative changes are intensified in lethal exposures.Hepatic nuclei were either swollen or pyknotic. Plate: 5 Histological changes of liver in L. calcarifer Light micrographs of a paraffin section stained with Hematoxylin and Eosin (40 X)

A - Control B - After 7 days of exposure to 6.83 ppm concentration of copper C - After 28 days of exposure to 6.83 ppm concentration of copper D - After 7 days of exposure to 13.66 ppm concentration of copper E&F - After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: HC - Hepatocyte BS - Blood sinus N - Nucleus CD - Cytoplasmic degeneration DE - Damaged Epithelium HSHC- Hydropic swelling of hepatocytes BC - Blood conjestion NP - Nuclear Pyknosis CV - Cytoplasmic vacuolation ND - Nuclear degeneration ADG - Accumulation of dark granules CN - Cellular necrosis Plate: 5 Results 69

(NP) (Plate 5, Fig. E & F ). The blood sinusoids were dilated between the cords of hepatocytes after 28 days of exposure to high concentration of copper. Affected hepatocytes display marked nuclear enlargement and moderate cellular enlargement.

5.6 Sublethal effect of copper on the Scanning Electron Microscopic studies in L. calcarifer

5.6.1 SEM histology of gill

In SEM, the architectural pattern of gills of L. calcarifer is essentially similar to that of other teleost fishes. SEM images provide a good three dimensional views of the last, middle and tip regions of the respiratory lamellae. The gill of control fish showed a good structural characterization: There are four gill arches on each side of the buccal cavity. Each arch is composed of numerous gill filaments which have two rows of secondary lamellae that run perpendicular to each filament. The secondary lamellae were regularly lined up along both sides of the primary lamella. In control fish, the secondary lamellae constituted evenly-space parallel plates. The epithelial cells covered both the primary and secondary lamellae. Secondary lamellae are plate like projections at right angles to the gill filaments. It lies parallel to the adjacent lamellae and is covered over by a thick and coarse epithelium the chloride and mucous cells were distributed primarily at the bases of the secondary lamellae. Numerous water pores and mucous cell openings with well developed micro ridges are discernible at the lamellae (Plate 6, Fig.A). Chloride cells, seen as pit openings, are found among the protuberances and cavities of the surface. In high resolution of SEM, the surface epithelium of lamellae shows clear demarcation between cells and microridges. Results 70

5.6.2 SEM histopathology of gill

In all treated fish, the respiratory organs displayed damages. Following the treatment at 6.83ppm concentration of copper after 7 days of exposure diffuse oedema (E) and detachment of the lamellar epithelium (epithelial lifting) with the formation of large subepithelial spaces within the secondary lamellae were observed. SEM examination showed swelling and curling of secondary lamellae (SSL) (Plate 6, Fig.B). After 28 days of low concentration of copper, the gills showed extensive aneurism with some ruptures in many secondary lamellae and the breakdown of pillar cell system was seen Moreover, partial or complete secondary lamellar fusion (FSL) (Plate 6, Fig.C) and thickening of primary lamellae were encountered in the exposed sea bass as a result of inter lamellar epithelium and chloride cells hyperplasia. SEM examination also showed complete fusion of secondary lamellae and surface wrinkling in numerous areas as a result of epithelial hyperplasia and or hypertrophy. Mucous cells ruptures and thick blanket of mucous secretion (TBM) (Plate 6. Fig.D) seen above the damaged epithelium. At the lowest concentration also necrosis and leukocyte infiltration (granulocytes and macrophages) in secondary lamellae were observed. Curling of secondary lamellae (CSL) was severe in high concentration of copper treated sea bass (Plate 6, Fig.D). Lifting of epithelium was common and particularly severe and extensive after 7 days of high concentration of copper exposure (Plate 6, Fig.E). In addition to this sloughing of primary and secondary gill epithelium (SGLE) were clearly evident in treated sea bass. The epithelial surface with microridges exhibit damaged and irregular appearance (IAES) Fusion of secondary gill lamellar epithelium was clearly displayed in high concentration of copper after 28 days of exposure and this finally ends with complete degeneration of secondary lamellae (Plate 6, Fig.F). In addition to light microscopy, SEM examination also confirmed the severe aneurism, swelling, Plate: 6 Scanning Electron micrograph of gill in L. calcarifer A Control B After 7 days of exposure to 6.83 ppm concentration of copper C & D After 28 days of exposure to 6.83 ppm concentration of copper E After 7 days of exposure to 13.66 ppm concentration of copper F After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: PL - Primary lamellae SL - Secondary lamellae MRE - Micro ridged epithelial cell IAES - Irregular arrangement of epithelial surface SSL - Swelling of secondary lamellae E - Edema TBM - Thick blanket of mucus FSL - Fusion of secondary lamellae CSL - Curling of secondary lamellae LE - Lifting of epithelium DSGL - Damaged secondary gill lamellae SGLE - Sloughing of gill lamellar epithelium TSL - Thinning of secondary lamellae Plate: 6 Plate: 6 Results 71 and enlargement of many secondary lamellae. Exfoliated epithelium was a common feature in all treated fish and t he secondary lamellae were highly deformed as a consequence of the lifting of the epithelium and severe hyperplasia which lead to the conjunction of adjacent filaments. In higher magnification, the SEM picture clearly depicts the denuding of the boundaries of surface epithelial cells of both primary and secondary lamellae. The lamellae of treated fish were thinner than the control fish (Plate 6, Fig.F).

5.6.3 SEM histology of liver

In all control fish, the ultrastructural morphology of the hepatocytes was normal: The surface of liver is covered with serous membrane and some connective tissue extends inward into parenchyma. It is composed of parenchymal cells (hepatocytes) (HC) and lattice fibers, which support the former. Hepatic cells are roundish polygonal, containing clear spherical nucleus (SN). They are located among sinusoids forming cord like structures known as hepatic cell cords. Hepatic cells have many vital functions. Other than the secretion of bile, they play an important role in protein, lipid and carbohydrate metabolism. They serve as storage sites for some nutrients and detoxification is another function attributed to them (Plate 7, Fig.A).

5.6.4 SEM histopathology of liver

Copper caused severe ultrastructural changes in liver; the damage severity and its extension increased with concentration of metal and duration of exposure. In exposed sea bass the regular compartmentalization was totally lost. Other changes in lower concentration of copper exposed to 7 & 28 days includes; cloudy swelling of hepatocytes, congestion, vacuolar degeneration, dilation of sinusoids and nuclear hypertrophy (Plate7, Fig. B & C). After 7 & 28 days of higher concentration of copper exposure, the hepatocytes showed Plate: 7 Scanning Electron micrograph of liver in L. calcarifer A - Control B - After 7 days of exposure to 6.83 ppm concentration of copper C - After 28 days of exposure to 6.83 ppm concentration of copper D&E - After 7 days of exposure to 13.66 ppm concentration of copper F - After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: HC - Hepatocyte HN - Hepatocyte nucleus VD - Vacuolar degeneration CSH - Cloudy swelling of hepatocytes NHN- Necrosis of hepatocyte nucleus VDC - Vacuolar degeneration of cytoplasm N - Necrosis HS - Hydrophic swelling ALD - Accumulation of lipid droplets SN - Spherical nucleus Plate: 7 Plate: 7 Results 72 hydropic swelling (Plate 7, Fig.D). Large lipid droplets and abundant glycogen occupied most of the area of hepatocytes and vacuolar degeneration (Plate 7, Fig. E & F).

5.7 Sublethal effect of copper on the Transmission Electron Microscopic studies in L. calcarifer

5.7.1 TEM histology of gill

Under TEM, the gill epithelium of control sea bass was formed by a filament multilayered epithelium, periodically sectioned by longitudinal capillary axes that originated in the lamellae, which were covered by a 2- layered epithelium. Each lamella possessed a central vascular axis, the endothelium of which was composed of pillar cell (PC) cytoplasmic extensions, externally coated with a basal lamina and a loose interstitial space. The superficial layer of the filament epithelium contained mucous cells (MC),chloride cells (CC), their precursors, and intercalating support cells (SC), which were externally covered by a monolayer of pavement cells (PC). One to two erythrocytes are usually recognized within each capillary lumen. Chloride cells are identified as large epithelial cells with light cytoplasm, usually present at the base of lamellae. Mucous cells and pavement cells are also present in the epithelium of the filament and at the base of lamellae, but they lack the light cytoplasm and are smaller than chloride cells (Plate 8, Fig. A).

5.7.2 TEM histopathology of gill

Ultrastructural alterations appeared in the gills of copper treated fish. Secondary gill lamellae exhibited hypertrophy and hyperplasia of the epithelial cells. The pavement cell appeared irregular with a considerable loss of microridges. Vasodilatation in many areas of the secondary lamellae with breakdown of the pillar cell system appeared by degenerative and necrotic Results 73 changes of the pillar cells (Plate8, Fig.B). Occasionally, proliferation of chloride cells and mucous cells could be identified in the secondary lamella. The chloride cells appeared with dilated vesicles within the cytoplasm and damaged mitochondria, while the mucous cells were completely filled with electron- dense mucous containing vacuoles and no other organelles could be visible in this cell. It is worth to mention that electron microscopic alterations observed in the gills during this study were the initial part of the protruding lamellae showed edema of the interstitial tissue and irregular capillary shapes, whereas vasodilatation was mostly confined to the lamellar basal region and was associated with stretched pavement cells and large hydropic vacuoles were observed at 6.83ppm concentration of copper after 7 days of exposure (Plate 8, Fig. B). At lowest concentration for 28 days copper treatment the enlarged filament intercellular spaces contained macrophage like cells , leukocyte-like cells, and macrophages with large digestive vacuoles(MDI) which frequently showed autolysis (Plate 8, Fig. C).

Numerous macrophages or apoptotic bodies were evident on exposure to 7 days of highest concentration of copper. The external cover of pavement cells also exhibited some modifications. These cells rounded up and partially detached, resulting in coalescence and rupture of blood vessels (RBV) (Plate 8, Fig. D). Under higher magnification of TEM, hypertrophic pavement cells with irregular shape, long cytoplasmic processes and without microridges were observed. The gills showed extensive hypertrophy and hyperplasia of epithelial and chloride cells due to complete fusion of secondary lamellae (Plate 8, Fig. D). Thinning of the epithelial cells of secondary lamellae was highly evident on exposure to 7 days of higher concentration of copper. Moreover, congestion of blood vessels by erythrocytes with presence of different leucocytes has been observed. Dilation of the blood vessel walls allows the haemorrhage in higher concentration of copper exposure (Plate 8, Results 74

Fig. E). Ultrastructurally, the Chloride cells (CC) possessed dilated vesicles within the cytoplasm and damaged mitochondria with cristae regression; the apical pits of CCs appeared enlarged after 7 days of exposure to higher dosage of copper.

After 28 days of exposure at higher concentration of copper, the filament epithelium was extremely reduced in thickness and showing an almost complete absence of chloride cells (CC). In the basal region of lamellae, the vascular axis showed extensive vasodilatation with stretching and necrosis of pillar cells (NPC). Due to marked interstitial edema, large epithelial cells spaces were formed. This progressively leads to lifting of epithelium up to the tip of the lamellae. The enlarged filament intercellular spaces contained undifferentiated cells, leucocytes, haemorrhagic residues and macrophages with large digestive vacuoles which frequently showed autolysis. At higher concentration of copper exposure swelling of lamellar epithelium was seen in frequent surfaces which lead to wrinkled and non-homogenous surfaces. Detachment of lamellar epithelial layer (DEL) was clearly evident in 28 days of exposure to copper in treated sea bass (Plate 8 Fig.F). The gills showed extensive aneurism with some ruptures in many secondary lamellae and the breakdown of pillar cell system was seen.

5.7.3 TEM histology of liver

In the untreated fish all the hepatic cells are normal. Ultrastructurally, the hepatocytes show a single rounded nucleus, usually centrally located. The chromatin is granular, with more condensed heterochromantin located at the periphery of the nucleus. The nucleolus is more homogenous and presents high electronic density. The rough endoplasmic reticulum (RER) is arranged in parallel stacks embrace the nuclear membrane. Both circular and elongated mitochondria were associated to the rough endoplasmic reticulum. Numerous Plate: 8 Transmission Electron micrograph of gill in L. calcarifer A - Control B - After 7 days of exposure to 6.83 ppm concentration of copper C - After 7 days of exposure to 6.83 ppm concentration of copper D - After 7 days of exposure to 13.66 ppm concentration of copper E&F - After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: PC - Pavement cell MC - Mucus cell BL - Basal lamina E - Erythrocyte CC - Chloride cell EP - Epithelium M - Mitochondria N - Nucleus BV - Blood Vessel PC - Pavement cell H - Haemorrahage HL - Hypertrophy of lamellae ND - Nuclear degenerations NPC - Necrotic Pillar cell IPC - Irregular shape of Pavement cell DM - Damaged mitochondria HPC - Hypertrophic pavement cell LHV - Large hydropic vacuole RBV - Rupture of blood vessel MDI - A macrophage with dark inclusions in the secondary lamellae DEC - Damaged Epithelial cell OSLLES- Oedematous secondary lamellae with large epithelial space TSL - Thinning of secondary lamellae DEL - Detachment of epithelial layer LBV - Leucocytes in blood vessel Plate: 8 Results 75 cytoplasmic vacuole of varies size and shape are distributed throughout the cytoplasm, often in close association with the RER. Smooth endoplasmic reticulum (SER) was almost absent. A large amount of scattered glycogen like particles fills most of the cytoplasm (P late 9, Fig.A).

5.7.4 TEM histopathology of liver

At lowest concentration of copper after 7 days of exposure in sea bass the main alterations in the structural architecture were concerned with rough endoplasmic reticulum (RER) and mitochondria (M). With comparison to the hepatocytes of the control fish, the smooth endoplasmic reticulum (SER) was highly developed; the changes observed were; degranulation and fragmentation of RER (FRER), dilatation and vesiculation of reticulum cisternae; some hepatocyte nucleus exhibited chromatin clumping (CC) (Plate 9, Fig.B). Flattened stack like cisternae modified to numerous vesicles due to fragmentation. The most frequent pathological modifications were clearly depicted by the organelle mitochondria. The mitochondrial reactions to copper were swelling, disappearance of cristae, vacuolization, formation of myelinoid- bodies, and the hepatocytes showed massive swollen mitochondria with loss of criatae and condensed mitochondria. In some cells the nucleus has been pushed to the periphery of cell instead of the central position were evident at 28 days of lower concentration of copper (Plate 9, Fig. C).

After the exposure of 7 days at higher concentration of copper, hydropic swelling of hepatocytes with nuclear pyknosis and chromatin condensation were observed. With reference to the storage vesicles, there appeared to be an increase in the lipid droplets (lipidosis) (steatosis), within many hepatocytes. The nuclei also showed alterations with dilation of the nuclear envelope and an accumulation of heterochromatin. A slight accumulation of dark minute granules (DMG) were observed in some hepatocytes (Plate 9, Fig. D & E). Results 76

After 28 days of exposure in higher dosage the hepatocytes showed diffuse degenerative vacuolation (cellular oedema or acute cell swelling) and cytoplasm rarefaction. In some instances mylenoid bodies (MB) also observed. The nuclei were affected by the exposure, showing dilatation of nuclear envelope, rarefaction of karyoplasm and lipid inclusions and complete damage of mitochondria. TEM observations showed severe lesions in the hepatocytes (Plate 9, Fig.F). Plate: 9 Transmission Electron micrograph of liver in L. calcarifer

A - Control B - After 7 days of exposure to 6.83 ppm concentration of copper C - After 28 days of exposure to 6.83 ppm concentration of copper D&E - After 7 days of exposure to 13.66 ppm concentration of copper F - After 28 days of exposure to 13.66 ppm concentration of copper

Abbreviations used: RER - Rough Endoplasmic Reticulum M - Mitochondria HE - Hepatocyte N - Nucleus FRER - Fragmentation of Rough Endoplasmic reticulum SM - Swollen of mitochondria CC - Chromatin clumping in nucleus DRER - Damaged Rough Endoplasmic reticulum NP - Nuclear Pyknosis CV - Cytoplasmic vaculation MB - Myelinoid bodies DHE - Degenerated hepatocyte in mitochondria H - Hepatocytes shows dilation and fragmentation of RER DMG - Dark minute granules Plate: 9 Discussion 77

6. DISCUSSION

6.1 Acute toxicity test

Acute toxicity test is the measurement of the short term lethality of a substance, by which, the concentrations of the substance proves to be lethal to the organism (Sparague, 1969). According to Brungs and Mount (1978), the application of the LC50 value is the most highly related test for assessing the potential adverse effects of chemical contaminants of aquatic life. Toxicity is also influenced not only by the intrinsic toxicity of the element, but also by its availability as determined by occurrence, complexation of other chemical reactions and absorption potential. Bryan (1976) has listed a series of factors influencing toxicity of heavy metals in solution. These include the dissolved form of metals, the presence of other metals and factors influencing the physiology and behaviour of the organisms. Copper is highly toxic to fishes and in the present investigation, the median lethal concentration (LC50 for 96 hrs) of copper, in L. calcarifer was determined to be 68.32ppm (Table 3).

It is evident from the results that the heavy metal concentration has a direct effect on the LC50 values of the respective fish. LC50 obtained in the present study correspond to values that have been published in the literature for other species of fishes (Table 30). The differences in acute toxicity may be due to changes in water quality and test species (Hedayati and Safahieh, 2012). The susceptibility of fish species to a particular heavy metal is a very important factor for LC50 levels. Fish that are highly susceptible to the toxicity of one metal may be less or even not susceptible to the toxicity of another metal at the same level of that metal in the ecosystem. Conversely, a metal which is highly toxic to a fish species at low concentrations may be less or even non-toxic to Discussion 78 other species at the same or even higher concentrations (Hedayati et al., 2010). It is widely accepted that the stress response as a whole is characterized by physiological changes. These changes tend to be similar for stressors and could be as varied as anesthesia, flight, forced swimming, disease treatments, handling, scale loss, or rapid temperature change. Acute toxicity studies were the very first step in determining the water quality requirements of fish. These studies obviously reveal the toxicant concentrations (LC50) that cause fish mortality even at short exposure. Therefore, studies demonstrating the sensitivity of xenotoxic effects of heavy metals in aquatic organisms, particularly in fish are needed. Thus, it can be concluded from the present study that the fish sea bass L. calcarifer was highly sensitive to copper and their mortality rate was dose dependent.

6.2 Bioaccumulation

The accumulation of heavy metals by aquatic organisms involves tissues that serve as the site for uptake and absorption like gills, skin and intestine. Among these tissues gills have the ability to concentrate metals and therefore exhibit relatively high potentials for accumulation. Present investigation revealed that fingerlings of L. calcarifer were exposed to two sublethal concentrations of waterborne copper for four weeks only. During this exposure time fish has already accumulated significant level of copper in their various tissues. Significant copper accumulation occurred in the liver > kidney > gills > muscles, in agreement with Abdel-Tawwab et al. (2007). In general, different tissues showed different capacities for accumulating heavy metals. Tekin-Ozan and Kir (2008) pointed out that the highest metal concentrations were found in liver and gills, and muscle tended to accumulate less metal. Arellano et al. (1999) suggested that the copper concentration in liver was higher than that in gills in Senegales sole. Present study also indicated that increasing water borne copper concentration significantly increased copper accumulation in all tissues Table. 30. LC50 values of copper toxicity in fin fishes

Species Hours of LC Element 50 Reference exposure (mg /l) American eel, Cu 96 6.0 Rehwoldt et al., Anguilla rostrata 1972/71 Arctic grayling, Cu 96 0.010 Buhl and Hamilton, Thymallus arcticus 1990 Atlantic salmon, Cu 96 0.025 Carson and Carson, Salmo salar 1972 Atlantic salmon, Cu 96 0.125 Wilson, 1972 Salmo salar Atlantic salmon, Cu 96 0.025 Zitko et al., 1973 Salmo salar Banded killifish, Cu 96 0.84 Rehwoldt et al., Fundulus diaphanus 1972/71 Blacknose dace, Cu 96 0.32 Geckler et al., 1976 Rhynichthus atratulus Blue gill, Lepomis Cu 96 0.2 Tarzwell and macrochirus Henderson, 1960 Blue gill, Lepomis Cu 96 1.25 Cairns and Scheier, macrochirus 1968 Blue gill, Lepomis Cu 96 0.68 Inglis and Davis, 1972 macrochirus Blue gill, Lepomis Cu 96 1.3 Blaylock et al., 1985 macrochirus Channel catfish, Cu 96 0.055 Straus and Tucker, Ictalurus punctatus 1993 Channel catfish, Cu 96 7.56 Birge and Black, 1979 Ictalurus punctatus Chinook salmon, Cu 96 0.01 Chapman and Oncorhynchus McCrady, 1977 tshawytscha Chinook salmon, Cu 96 0.026 Chapman, 1978 Oncorhynchus tshawytscha Chinook salmon, Cu 96 0.058 Hamilton and Buhl, Oncorhynchus 1990 tshawytscha Chisel mouth, Cu 96 0.143 Andros and Garton, Acrocheilus alutaceus 1980 Coho salmon, Cu 96 0.06-0.074 Lorz and McPherson, Oncorhynchus kisutch 1976 Coho salmon, Cu 96 0.046 Chapman and Stevens, Oncorhynchus kisutch 1978 Table. 30. LC50 values of copper toxicity in fin fishes

Hours of LC Species Element 50 Reference exposure (mg /l) Coho salmon, Cu 96 0.019- Buhl and Hamilton, Oncorhynchus kisutch 0.021 1990 Common carp, Cu 96 0.063 Khangarot et al., 1983 Cyprinus carpio Creek chub, Semotilus Cu 96 0.31 Geckler et al., 1976 atromaculatus Cutthroat trout, Cu 96 0.0157 Chakoumakos et al., Oncorhynchus clarkii 1979 lewisi Fathead minnow, Cu 96 0.023- Pickering and Pimephales promelas 0.035 Henderson, 1966 Fathead minnow, Cu 96 0.47 Mount,1968 Pimephales promelas Fathead minnow, Cu 96 0.075 Mount and Stephan, Pimephales promelas 1969 Fathead minnow, Cu 96 0.6-0.98 Brungs et al., 1976 Pimephales promelas Fathead minnow, Cu 96 0.44 Geckler et al., 1976 Pimephales promelas Fathead minnow, Cu 96 0.46 Pickering et al., 1977 Pimephales promelas Flag fish, Jordanella Cu 96 1.27 Fogels and Sprague, floridae 1977 Gold fish, Cu 96 5.2 Birge and Black, 1979 Carassius auratus Gold fish, Cu 96 0.3 Tsai and McKee, 1980 Carassius auratus Guppy, Cu 96 0.16 Deshmukh and Lebistes reticulatus Marathe,1980 Guppies, Cu 96 0.112 Chynoweth et al., Poecilia reticulata 1976 Guppies, Cu 96 0.764 Khangarot et al., 1981 Poecilia reticulata Largemouth bass, Cu 96 6.97 Birge and Black 1979. Micropterus salmoides Mosquito fish, Cu 96 0.047 Joshi and Rege, 1980 Gambusia affinis Northern squawfish, Cu 96 0.023 Andros and Garton, Ptychocheilus 1980 oregonensis Orangethroat darter, Cu 96 0.85 Geckler et al., 1976 Etheostoma spectabile Table. 30. LC50 values of copper toxicity in fin fishes

Species Element Hours of LC50 Reference exposure (mg /l) Penny fish, Cu 96 0.077 Williams et al., 1991 Denarius abandata Pink salmon, Cu 96 0.143 Servizi and Martens, Oncorhynchus 1978 gorbuscha Pumpkin seed, Cu 96 1.3 Spear and Anderson, Cucurbita pepo 1975 Pumpkin seed, Cu 96 1.24-1.3 Anderson and Spear, Cucurbita pepo 1980 Rainbow darter, Cu 96 0.32 Geckler et al., 1976. Etheostoma caeruleum Rainbow trout, Cu 96 0.253 Hale, 1977 Oncorhynchus mykiss Rainbow trout, Cu 96 0.11 Birge and Black, 1979 Oncorhynchus mykiss Rainbow trout, Cu 96 0.4 Giles and Oncorhynchus mykiss Klaverkamp,1982 Rainbow trout, Cu 96 0.036 Buhl and Hamilton, Oncorhynchus mykiss 1990 Sockeye salmon, Cu 96 0.19 Servizi and Martens, Oncorhynchus nerka 1978. Steelhead trout, Cu 96 0.028 Chapman, 1978 Oncorhynchus mykiss Steelhead trout, Cu 96 0.057 Chapman and Stevens, Oncorhynchus mykiss 1978 Steelhead trout, Cu 96 0.08 Seim et al., 1984 Oncorhynchus mykiss Steelhead trout, Cu 96 0.0028 Cusimano et al., 1986 Oncorhynchus mykiss Stone roller, Cu 96 0.29 Geckler et al., 1976 Campostoma anomalum Striped bass, Cu 96 0.62 Wellborn, 1969 Morone saxatilis Striped bass, Cu 96 4 Rehwoldt et al., Morone saxatilis 1972/71 Striped bass, Cu 96 0.1 Palawski et al., 1985 Morone saxatilis Striped shiner, Luxilus Cu 96 0.79 Geckler et al., 1976 chrysocephalus Table. 30. LC50 values of copper toxicity in fin fishes

Hours of LC Species Element 50 Reference exposure (mg /l) White perch, Cu 96 6.4 Rehwoldt et al., Morones Americana 1972/71 Zebra fish, Danio Cu 96 0.149 Fogels and Sprague, rerio 1977 Zebra fish, Danio rerio Cu 96 0.24 Weinstein, 1978 Asian sea bass, Cu 24 Present study 110.83ppm/l Lates calcarifer Asian sea bass, Cu 48 93.58ppm/l Present study Lates calcarifer Asian sea bass, Cu 72 76.71ppm/l Present study Lates calcarifer Asian sea bass, Cu 96 68.32ppm/l Present study Lates calcarifer Discussion 79 with the exception of vertebrae. This observation is consistent with other studies (Abdel-Tawwab et al., 2007 ; Dang et al., 2009 ; Chen et al., 2012). Copper accumulation here increased with time, similar to other studies (Grosell et al., 1998; Monteiro et al., 2005). In this present observation indicated that vertebrae copper content remained relatively stable, which was in contrast with Liu et al. (2010), who reported vertebrate copper levels in S. hasta were significantly elevated after copper exposure. Therefore, tissue like liver, which is a major producer of metal-binding proteins, show high concentrations of most heavy metals detoxification (Roesijadi and Robinson, 1994; Heath, 1995) which eventually result in clearance of heavy metal ions from the body. Furthermore, the physiological differences and the position of each tissue in the fish can also influence the accumulation of a particular metal (Heath, 1995; Kotze, 1997).

According to Sumpter et al. (1995), the hydrophobic xenobiotics bioconcentrate and bioaccumulate in aquatic organisms. Different organisms will bioconcentrate to different extents. Even with a single organism, the bioconcentrated compound is unlikely to be equally spread through all tissues; it is much more likely to be concentrated preferentially in a few tissues, such as fat. He reports that what happens to these compounds once bioconcentrated within an organism is essentially unknown; they may be physiologically inactive whilst stored in adipose tissue, but when this fat is mobilized, the compounds may be freed to act elsewhere.

Gills were directly exposed to waterborne copper have showed high uptake and accumulation which increased significantly high with the passage of time. The copper uptake and accumulation in these tissues was highest at higher concentration with longer exposure whereas minimum copper uptake and accumulation was observed at lower dose during the first week of exposure. This is because high dose and longer exposure resulted into high Discussion 80 uptake and accumulation. Regarding metal accumulation in general, it has to be noted that the susceptibility of an animals to a certain metal can be modulated by physiological parameters such as stress due to metal contamination (Viarengo, 1989). Preston (1971) suggested that the low level of metal accumulation in muscle is due to isolation of the muscle from the surrounding tissues by the thick membrane. White and Rainbow (1986) also found that the fish muscle shows a very low degree of heavy metal uptake relative to other tissues. Similarly in L. calcarifer, the accumulation of copper in muscle is very low. The lower value of copper in muscle suggests its poor source and mobilization (Senthilnathan and Balasubramanian, 1997).

Felts and Heath (1984) observed that when gills of sunfish were exposed to a sublethal concentration of copper, the gills exhibited significant increase of the metal at 7 days of exposure. Elevated level of copper is seen in liver of L. calcarifer followed by kidney, gills and muscle. Similar result is also highlighted by Jaffer and Ashraf (1988) in Formio niger. The uptake of the metals in fishes most probably takes place at the gills followed by the liver. Hyperplasia and thickening of the branchial epithelium protects against further invation of the toxicant (Mallatt, 1985). The gills act as a barrier accumulating a major part of the metal. The inhibition of copper excretion and detoxification results in anoxia where energy metabolism is decreased and contrastingly accumulation increases (Depledge, 1987).

The least accumulated copper present in the muscle tissue of the fish this may be due to its uptake of the heavy metal coming from its surrounding environment (Jeffree et al., 2006) and the incorporation of the chemical in its tissue (Dural et al., 2006). The result of this study is vital, as the muscle of the fish is the most commonly consumed part. The key roles of fish kidney in the maintenance of homeostasis, hematopoiesis, immune and endocrine functions Discussion 81 and, mainly, in the elimination and rapid clearance of xenobiotics, make it prone to damage (Pereira et al., 2010b). In the present study kidney is also often referred to as one of the organs that preferentially accumulates copper. Accordingly, kidney was the organ with the highest accumulation in copper followed by liver.

6.3 Biochemistry

Heavy metals are recognized as one of the most hazardous environmental pollutants and are toxic to many living organisms (Sontakke and Jadhav, 1997). Metal ions once absorbed into the body are capable of reacting with a variety of active binding sites and then disturbing the normal physiology of the organism which may lead to the death of organism. Fishes are responding to various stressors by a series of biochemical and physiological stress reactions, so called secondary stress responses comparable to those of higher vertebrates (Mazeaud and Mazeaud, 1981).

The total protein content in muscle, gills and liver was altered, the changes in protein levels were insignificant when compared to the control fish.In the present study, the total protein in the muscle, gills and liver of L. calcarifer showed decreasing trend as the duration of exposure to copper increased (muscle: 62.59±0.59 mg/100mg to 52.14±0.63 mg/100mg, gills: 2.42±0.09 mg/100mg to 1.72±0.06 mg/100mg and liver: 1.75±0.07 mg/100mg to 1.27±0.08 mg/100mg). It is likely that the observed reduction in total protein of L. calcarifer is due to a direct consequence of the stress imposed by copper. The alterations in the protein levels might be due to the adaptation of the animals to metal stress. The present study supported by previous reports (Vutukuru, 2003) has shown the decrease of total protein content in fish exposed to Copper. Decreased rate of protein synthesis, utilization for energy or secreted mucous proteins could alter the protein levels in animals under metallic stress Discussion 82

(Vutukuru, 2005). Also, the depletion of total protein suggests an increased proteolysis and possible utilization of the products of their degradation for metabolic purpose. The decreased protein level during exposure to pollutants may be due to increased catabolism and decreased anabolism of proteins as reported in freshwater bivalve Parrysia corrugata (Deshmukh and Lomte, 1998).

The initial declining levels of protein may be attributed to the higher rate of energy production at the onset of various enzymatic blockages. It is known that copper blocks the mRNA synthesis and thereby the protein synthesis at the level of transcription. However, copper inhibits the action of enzyme protease reflecting a steady decline in the total percentage of protein.

Total free sugars are very important biological compounds as they are the chief source of energy and also structural constituents of protoplasm. In the present study the total free sugar in the muscle, gills and liver of L. calcarifer showed decreasing trend with increasing concentration of copper (muscle: 26.54±0.30 mg/100mg to 22.01±0.61 mg/100mg , gills: 6.89±0.08 mg/100mg to 5.01±0.12 mg/100mg and liver: 22.35±0.49 mg/100mg to19.05±0.22 mg/100mg). It may be due to the breakdown of glycogen to cope with the high energy demand for the detoxification process, since carbohydrate forms major source of energy under toxicity (Hochachka and Somero, 1973).

Glycogen plays an important role as a readily mobilized storage form of total free sugar in muscle (Stryer, 1988), which decreases during toxicity as evidenced in L. calcarifer also. Similarly, the total free sugar declines in the gills and it deposit as granules on the lamellae interrupting the uptake of oxygen. This is consistent with the earlier studies in Norway lobster, Nephrops norvegicus resulting in hypoxic condition (Baden et al., 1994), where the binding of oxygen with haemoglobin decreases with the increase in concentration of copper, thereby resulting in hypoxia (Depledge and Bjerregaard, 1989). The total free sugar may Discussion 83 have been used to fuel detoxification mechanism operating within the animal as reported in Panulirus longipes and Jasus lalandii (Cockcroft, 1997).

The total lipids in the muscle, gills and liver of L. calcarifer showed decreasing trend as the duration of exposure in each concentration of copper increased (muscle: 18.92±0.62 mg/g to 15.13±0.48 mg/g , gills: 1.64±0.06 mg/g to 0.76±0.04 mg/g and liver: 22.92±0.67 mg/g to 19.21±0.42 mg/g). A significant decreases in muscle and liver may be due to its utilization for energy during detoxification mechanism.

In the sea bass, L. calcarifer the liver is the most sensitive indicator of physiological stress than the muscle tissue (Trendal and Prescott, 1989). The finding is in accord with the results obtained in L. calcarifer, although muscle and liver are the major energy stores. Lipids were found to be the primary source of energy under stress condition in Penaeus duorarum (Schafer, 1968). An increase in metal concentration and exposure duration resulted in the reduced level of lipid in Oreochromis mossambicus (Overstreet, 1988). Similar results are reported in bivalve Sunetta scripta (Katticaran et al., 1995).

Copper has been associated with lipid peroxidation. Beckman and Zaugg (1988) reports that cuprus- ions may potentiate lipid peroxidation by a metal- metal reaction reducing the ferric ions rather than by promoting propagation reaction in the fish Chinook salmon Oncorhynchus tshawytscha. As the lipid reserves are ultimately transferred to the detoxification process in L. calcarifer, the total percentage of lipids decline severally in liver followed by muscle and gills. These results suggest the important role of liver in storage and mobilization of energy during detoxification of copper in L. calcarifer. Discussion 84

6.4 Characterization of Protein Profile

The electrophoretic techniques remain a promising tool for identifying the protein profile in response to stressful and sublethal level of heavy metals (Dutta et al., 1983). Heavy metal binding proteins are associated with copper and the lower molecular weight protein in particular is found to have a significant percentage of copper contained in the muscle and gills. The lower molecular weight protein probably plays a significant role in the metabolism of the copper.

In the present study SDS polyacrylamide gel electrophoresis was performed for the tissues of muscle and gills of L. calcarifer exposed to sublethal concentrations of copper. When compared to control the protein subunits of muscle and gills exposed to sub-lethal concentrations, the bands showed decrease in intensity. The changes in protein subunit band patterns may be due to change in the turn over (Synthesis /degradation) of various proteins.

The disappearance of bands in the muscle and gills of L. calcarifer on exposure to copper may be due to the interference of copper in the protein synthesis process as reported in freshwater prawn, Macrobrachium lamerrei lamerrei (Krishnamoorthy and Subramanian, 1997). In the present study eleven distinct bands are accounted in the muscle tissue of L. calcarifer. Extensive disruption in the number of banding is well documented at 13.66ppm of concentration with six and five polypeptide fractions during 7 and 28 days of copper exposed sea bass. Similar reduction in the number of banding pattern of muscle tissue is reported in M. lamerrei lamerrei (Krishnamoorthy and Subramanian, 1997). He also reported that the intensities of the major polypeptide bands in the gills of prawns when treated with heavy metal were less than that of the control. The result is in accordance with the current observation in L. calcarifer in which the six distinct bands (control) decreased to five and one at 6.83ppm during 7 and 28 days respectively. Similarly, the bands reduced to only Discussion 85 one at 28 days of exposure to 13.66ppm concentration of copper. Similar reduction in the numbers of protein fractions was found in Scylla serrata when treated with copper (Ramanibai, 1986). L. calcarifer has minimum protein residue in muscle and gills due to copper toxicity which interfered with the banding pattern of proteins as reported in other aquatic invertebrates (Wright, 1978). It is also evident that exposure to copper disturbed the banding pattern of protein under stress condition in L. calcarifer.

The inhibition or activation of physiological activities by copper is due to the interaction between the animal and the heavy metal. The stress induced biochemical changes can be described as secondary responses of the fish. Sharaf-Eldeen and Abdel-Hamid (2002) found that the exposure of O. niloticits to the pollutants (CuSo4, malathion and paraquat) induced disappearance of certain serum protein fractions. Anees (1974) found that the total serum protein of Channa punctatus decreased significantly on exposing to some organophosphorous compounds. Patterson (1976) mentioned that the pollutants react with the cell nucleoproteins and nucleic acids and consequently affect the protein synthesis and cellular integrity. However, effects of toxicants on energy conservation by mammalian mitochondria have never been reported. In several toxicological models, movement disorders were linked to a dysfunction of the mitochondria.

Kurbanova et al. (2004) reported that a decrease of the intensity of total protein accumulation and albumin concentration, and the increase of gamma globulin and peptidase activity which considered as adaptive reactions of the fish, Rutilus Jrisii kutum to the oil pollution. Khalid M. Sharaf-Eldeen et al. (2006) reported that fractions of liver proteins were changed in the fish, Tilapia zillii when exposed to agricultural and industrial drainage water. Tripathi and Shukla, (1990) observed alterations in the cytoplasmic protein pattern of fish Discussion 86

Clarias batrachus by performing electrophoresis of the cytoplasmic protein fractions of the liver and the skeletal muscle exposed to endosulfan and methyl parathion for 28 days. These authors also found that the disappearance and polymorphism of protein fractions were dependent on the degree of pollution in each water locality. Protein heterogeneity is associated with all fish species. Structurally blood serum protein, muscle protein (myogen) haemoglobin, as well as all enzymes in the blood and other organs of fishes appear to be variable (Kirpichnikov, 1981). Sanders (1964) detected inter- and intraspecific differences in protein compounds. Changes in protein sub units are regarded as important biomarkers of the metabolic potential of cells, as these play the main role in regulating the activities of cells. Their ratios also provide significant information about the way in which, mechanism, these contents regulate the multifaceted activities of cells.

6.5 Histology

Tissue changes in test organisms exposed to experimental concentrations of toxicants are functional responses that provide information on the mode of action of the toxicant on them. Histopathological characteristics of specific organs express condition and represent time-integrated endogenous and exogenous impacts on the organism stemming from alterations at lower levels of biological organization (Chavin, 1973). Therefore, histological changes occur earlier than reproductive changes and are more sensitive than growth or reproductive parameters and, as an integrative parameter, provide a better evaluation for the health of the organism than a single biochemical parameter (Segner and Braunbeck, 1988). Histopathological studies with light microscopy and electron microscopy are necessary for the description and evaluation of potential lesions in aquatic animals exposed to various toxicants (Meyers and Hendricks, 1985). Discussion 87

Metals accumulation in fish tissues depends on the exposure concentration and time as well as other factors such as temperature, age, interaction with other metals, water chemistry and metabolic activity of the fish (Heath 1995). The present study showed that the copper was accumulated in different organs of the fish L. calcarifer. The gills, which participate in many important functions in the fish, such as respiration, osmoregulation and excretion, remain in close contact with the external environment and particularly sensitive to quality of the water are considered the primary target of contaminants (Fernandes and Mazon, 2003). Gill is the primary site of osmoregulation and respiration in aquatic vertebrates. It is the main target organ, which gets affected easily when the organism is exposed to herbicides. The damage of gills of fish exposed to the sub-lethal concentrations of copper was severe. Extensive architectural loss was observed in the gills of copper treated group. Richmonds and Dutta (1989) divided the commonly reported gill lesions into two groups: (1) the direct deleterious effects of the irritants and (2) the defense responses of the fish. The observed lamellar necrosis and complete desquamation of the gill epithelium are direct responses induced by the action of copper.

In the present study, after 7 days of exposure to high concentration of copper, epithelial necrosis, hypertrophy of the epithelial cells, rupture of gill epithelium, hemorrhage at primary lamellae and sloughing of respiratory epithelium were noted (Plate 2 B & C). The lifting of the epithelium, oedema, epithelial necrosis, fusion of adjacent secondary lamellae and hemorrhage at primary lamellae were observed in the gills of the fish examined after 28 days of exposure to copper (Plate 2 D).

Another important histopathological change observed in the copper treated group was hyperplasia. Morphologically, hyperplasia refers to an Discussion 88 increase in the number of normal cells that constitute a given tissue. Gill alterations such as hyperplasia of the epithelial cells can be considered adaptive, since they increase the distance between the external environment and blood, serving as a barrier to the entrance of contaminants. Hyperplasia observed maybe the fish's response (1) to ward off or block something that irritates its tissues, whether externally or internally, or (2) to quickly heal an injured or irritated site. Hyperplasia, however, may play a role in the early stages of neoplasia. Gill hyperplasia might serve as a defensive mechanism leading to a decrease in the respiratory surface and an increase in the toxicant- blood diffusion distance.

Increased mucus production and fusion of lamellae were obvious on exposure to copper. Mucus cells contain mucins, polyanions composed of glycoproteins that can be effective in trapping toxicants and aid in the prevention of toxicant entry into the gill epithelium (Perry and Laurent, 1993). Extensive epithelial desquamation was also observed in the copper treated group. It is well known that changes in fish gill are among the most commonly recognized responses to environmental pollutants (Mallatt, 1985; Laurent and Perry, 1991; Au, 2004). After acute exposure to hexavalent chromium, C. punctatus exhibited marked degenerative changes in the histology of gills, kidney and liver tissues (Mishra and Mohanty, 2008). The gills of copper treated sea bass exhibited lamellar telangiectesis (localised dilation of blood vessel). This appearance of the secondary lamellae results from the collapse of the pillar cell system and breakdown of vascular integrity with a release of large quantities of blood that push the lamellar epithelium outward (Alazemi et al., 1996). Shortening and clubbing of ends of the secondary gill lamellae and clubbing of adjacent lamellae were well marked in the m-cresol treated group. Complete lamellar fusion may have reduced the total surface area for gas exchange. Otherwise, they increase the distance of the water-blood barrier, Discussion 89 which together with epithelial lifting and the increase in mucus secretion may drastically reduce the oxygen uptake. Epithelial necrosis and rupture of gill epithelium are direct deleterious effect of the irritants. The fish's defense responses are excessive mucus secretion. Lifting of the epithelium, lamellar fusion and club shaped lamellae could be protective in that it diminishes the amount of vulnerable gill surface area (Richmonds and Dutta, 1989). The histopathological changes of gill can result in hypoxia, respiratory failure problems with ionic and acid-base balance (Alazemi et al., 1996).

The histopathological changes observed in the gills of L. calcarifer in the present study are in good agreement with Rao et al. (2003) and Jauch (1979). They observed the bulging of secondary lamellae at the terminal ends, lesions and erosions at the base of lamellae on 12th day of exposure of O.mossambicus to chlorpyrifos. A thick coat of mucus on the gill filaments was persisting on 28th day of copper exposure in sea bass. Jauch (1979) reported that 96-hour fenthion exposure induced gill lesions, including hyperplasia and desquamation of the epithelium and thrombosis in the secondary gill lamellae. At the higher concentration the epithelium of the lamellae was swollen and partly detached from the underlying blood space. In some regions the epithelium was partly destroyed in treated sea bass. The same results were observed by Soivio et al. (1988) when the fish rainbow trout treated to pulp and paper mill effluents.

In the present study, the gill epithelium of copper treated fish was completely desquamated; the secondary lamellae became shapeless, with hyperplasia and were broken at several places (Plate. 2B). The observations were in agreement with the results reported by Wobeser (1975) in Salmo gairdneri treated with mercuric chloride. Further Tilak and Yacobu (2002) also observed such gill damage in Fenvalerate treated C. idellus. Ramamurthy et al. Discussion 90

(1987) observed severe hyperplasia in secondary lamellae, fusion of secondary gill lamellae and oedematous seperation of epithelial cells in methylparathion treated Cyprinus carpio.

The gills of L.calcarifer showed degenerative, necrotic and proliferative changes in gill filaments and secondary lamellae and congestion in blood vessels of gill filaments. The pathological changes may be a reaction to toxicant intake or an adaptive response to prevent the entry of the pollutants through the gill surface. The observed alterations like proliferation of the epithelial cells, partial fusion of some secondary lamellae and epithelial lifting are defence mechanisms, since in general, these results in the increase of the distance between the external environment and the blood and thus serve as a barrier to the entrance of contaminants (Malatt, 1985 and Fernandes and Mazon, 2003). The cellular damage observed in the gills in terms of epithelial proliferation, separation of the epithelial layer from supportive tissues and necrosis can adversely affect the gas exchange and ionic regulation (Dutta et al., 1993). The observed edematous changes in gill filaments and secondary lamellae probably due to increased capillary permeability (Olurin et al., 2006). The present results are in agreement with those observed in other fish species of different pollutants (Olurin et al., 2006). In this respect, Camargo and Martinez (2007) observed hyperplasia of the epithelial cells, fusion of secondary lamellae, lifting of the lamellar epithelium and blood congestion in the gills of Prochilodus lineatlls caged in Cambe stream, Brazil, polluted by industrial, domestic and agricultural wastes. Also, Triebskorn et al. (2008) noticed epithelial lifting, proliferation of epithelial cells of primary and secondary lamellae, hyperplasia of mucous cells and necrosis of epithelial cells in the gills of fishes, which are similar to the present study. Discussion 91

Several other xenobiotics are also known to induce fusion of the secondary lamellae of gills (Leino et al., 1987; Dutta, 1996; Wendelaar Bonga, 1997). During the present study also, the slimy coatings over the gills showed compositional alterations and sloughed off several times, which might have led to the fusion of the secondary lamellae. According to Mallatt (1985) induced alterations in gill histology are mostly non-specific in nature, which partially represent the damage, and partially the compensatory response of the fish. Examples of the first are necrosis of the epithelial cells of the secondary lamellae, epithelial lifting, and dilatation of the blood sinuses of the secondary lamellae, and lamellar aneurysm. The main compensatory responses are hypertrophy and hyperplasia of the respiratory epithelial and chloride cells, hyperplasia of the mucous cells (including decrease due to exhaustion, followed by an increase in their density) and infiltration of the dilated intercellular spaces by leukocytes. Dutta (1996), categorised the structural alteration in the gill morphology into two groups: (1) direct deleterious effect of the xenobiotics causing necrosis and rupture of the branchial epithelium. Such type of effect is mostly dose dependent and very often reported under lethal conditions (Mallatt, 1985). They also suggested that death of branchial cells and their rupture usually develops either by autolysis or by rapid lyses caused by the direct action of toxicants on the cells’ constituents (Abel, 1976), and (2) branchial defence response achieved by mucus hyper secretion, chloride cell proliferation, epithelial lifting, swelling, hyperplasia and lamellar fusion. According to Peuranen et al. (1994) any discontinuity of epithelial lining of the gill due to massive wear and tear may lead to a negative ionic balance and to changes in the haematocrit and mean cellular haemoglobin values of the blood.

Santhakumar et al. (2001) studied histopathological effects of sublethal doses of monocrotophos on the gills by exposing the fish for a period ranging Discussion 92 from ten to twenty days and observed disruption of epithelial cells from pillar cells, haemorrhage in the primary and secondary gill lamellae, degeneration and necrosis of epithelial cells and distortion of the secondary lamellae very prominently and concluded that the extent of damage to gills was dependent on the dose and duration of exposure. Tilak et al. (2001) observed hydropsy, vascular degeneration and bulging and severe necrotic changes in the secondary in the gill tissues of fish L. rohita exposed to the sublethal concentrations of technical as well as 20% EC of chlorpyrifos after 8 days.

Meyers and Hendricks (1985) stated that the examination of tissues after death from fish and other aquatic organisms may serve to cifaidentify the cause of death and possibly the causative agent. Separation of epithelial cells and necrosis were reported in brook trout, Salvelinus fontinalis exposed to acute toxic levels of toxicant (Daye and Garside, 1976). Jagoe and Haines (1983) noted shortened and thickened secondary lamellae, primary lamellar swelling, droplets of mucus and fused adjacent secondary lamellae in Sunapee trout, Salvelinus alpinus subjected to pH 4.0 Hyperplasia, shortened and thickened respiratory lamellae, fused adjacent primary lamellae were observed by Leino et al., (1987) in peal dace, Semotilus margarita and fathead minnows, Pimephales promleas exposed to low acid pH of 5.16. Fischer scherl and Hoffmann (1988) found fused secondary lamellae and hyperplasia in fish, brown trout, Salmo trutta exposed to effluent.

The lifting of lamellar epithelium is other histological change observed, probably induced by the incidence of severe edema (Pane et al., 2004). Some studies revealed that interstitial edema is one of the more frequent lesions observed in gill epithelium of fish exposed to heavy metals (Mallatt, 1985). Edema with lifting of lamellar epithelium could be serve as a mechanism of defense, because separation epithelial of the lamellae increases the distance Discussion 93 across which waterborne pollutants must diffuse to reach the bloodstream (Arellano et al., 1999).

Lamellar axis vasodilatation was also found in sea bass exposed to copper. Garcia-Santos, Fontaínhas- Fernandes, and Wilson (2006) referred that this lesion can induce changes in pillar cell normal structure, with consequent loss of their support function and probably, and was responsible for the emergence of lamellar aneurysms in fish. In this case, damaged pillar cells can result in an increased blood flow inside the lamellae, causing dilation of the marginal channel, blood congestion or even an aneurysm (Rosety-Rodriguez et al., 2002). Lamellar telangictasis resulted from rupture of pillar cells and capillaries under effect of heavy metals pollution and leads to an accumulation of erythrocytes in the distal portion of the secondary lamellae (Randi et al., 1996).

At the higher concentration the epithelium of the lamellae was swollen and partly detached from the underlying blood space. In some regions the epithelium was partly destroyed. The same results were observed by Rusal et al., (2006) when the fish rainbow trout treated to pulp and paper mill effluents. Nowak et al., (1992) found that the respiratory epithelium detachment resulted in the increase of the diffusion distance, affecting the gaseous exchanges. This phenomenon has also been described in another type of environmental contamination such as in acid waters, heavy metals (Gupta, 1998) and salinity (Turick et al., 1996). Gupta (1998) reported that these cells steamed from the epithelium of the filament in the interlamellar space and could act as a barrier impeding the diffusion of harmful substances to the blood of the fish. They are the histopathological effects of raw oil on fishes, found that hyperplasia, together with the mucus secretion, and protected the gills against future Discussion 94 damages caused by intoxicants. This alteration was extremely intense after contamination with heavy metals.

While many studies have documented the histomorphological alterations to the gills, kidneys and livers of fish, especially following exposure to heavy metals (Fracacio et al., 2003 ; Au, 2004), only a few have investigated the effect they exert on the intestine (Banerjee and Bhattacharya,1995; Kamunde et al., 2001). The gastrointestinal tract represents a major route of entry for a wide variety of toxicants present in the diet or in the water that the fish inhabit (Banerjee and Bhattacharya, 1995; Bano and Hasan, 1990; Lemaire et al., 1992) nonetheless there is little information relating to the protective mechanisms adopted by the intestine epithelial surfaces of the fish against mercury uptake (Oliveira Ribeiro et al., 2002).

The intestinal villi in copper treated L. calcarifer were found to be severely damaged (Plate 3 Fig.B.). The lamina propria was shrunken and the epithelial layer orientation was found completely collapsed. The tips of the villi were also found to be ruptured (Plate3. Fig.C). Virk et al. (1987) have also discussed endrin and and carbaryl induced damages in the intestine of Mystus tengara. Inbarani and Seenivasan (1988) have also observed similar pathological lesions in the intestine of phosphomidon treated S. mossambius.

In the present study, the result of the effect of copper on the gastrointestinal system of L. calcarifer clearly showed that this metal exert toxic effects on the different layers of intestine. Mandal and Kulshrestha (1980) found lesion formation in villi of Clarias batrachus after exposure to sumithion. Necrosis, infiltration of lymphocytes and eosinophils were reported in the intestine of Gambusia affinis exposed to deltamethrin (Cengiz and Unlu, 2006). Discussion 95

According to Bhatnagar et al. (2007) observed irritation and destruction of the mucosa membrane of the intestine, hampering absorption were due to fluoride toxicity. The pathological alterations in the intestine of the studied fish were in agreement with those observed by many investigators about the effects of different toxicants on fish intestine due to pesticides and heavy metal (Mishra and Jain 1988; Fatma, 2009). Pathological gastrointestinal effects of fluoride including damage to the mucosal lining, loss of microvilli, cracked clay appearance of duodenal mucosa and desquamated epithelial cells of gastric mucosa have also been observed by earlier workers (Susheela et al., 1993; Dasarathy et al., 1996).

The intestine represents a major route of entry for a wide variety of toxicants present in the diet or in the water that the fish inhabit (Giari et al., 2008). The alterations in the intestine of the sea bass were more severe in higher doses. Toxic lesions most common in the intestine of fishes exposed to copper chloride include hyperemia, degenerative changes in the tips of villi, loss of structural integrity of mucosal folds, degenerative mucosal epithelium (hypertrophy, vacuolation, hyper-chromasia) necrosis, desquamation of mucosal epithelium, cellular debris, excessive mucus in gut of lumen, necrosis of submucosa and inflammatory infiltration (Plate3.Fig.E and F)

In the case of intestine, the histopathological changes so obtained in low concentration of copper exposed included partial intactness of serosa but, more or less organized mucosa and disorganized villi. The same organ, even at same concentration after 28 days of exposure exhibited partially damage of muscles, but disorganized, slightly swollen and shorten of villi (Plate 3.Fig.B). At higher concentration of copper treated fish showed damaged serosa disorganized and consequent fusion of mucosa, degeneration and edema between the intestinal submucosa and lamina propria (Plate 3.Fig.C and D). Further, these damages Discussion 96 were characterized by the increases in number of goblet (mucosal) cells, width of the lamina propria and degeneration of villi. The findings suggest that however, uptake of Cu and other metals occur mainly through gills but may also occur via intestinal epithelium. Therefore, the histopathological alterations so far observed in the intestine tissues of studied fish may be a result of uptake of toxic Cu. The present results are in agreement with those observed by many investigators about the effects of metals on fish intestine (Giari et al., 2007 and Hanna et al., 2005).

Histopathological change due to Cu, like hypertrophy may lead to increased serum glucose in the intestine, and is possibly due to the fulfillment of extra energy requirement under stress condition. In another study, cadmium exposure caused degenerative changes in the tips of villi like hydropic degeneration, cloudy swelling and necrosis of intestine of Ophiocephalus striatus (Bais and Lokhande, 2012). In the intestine of C. punctatus exposed to mercuric chloride, the cytoplasmic hyperchromasia, edema, loss of pepsinogen granules from chief cells, disintegration of glandular epithelium, desquamation of gastric mucosa in the stomach, hyperemia, degenerative changes in the tips of mucosal folds, hypertrophy and necrosis were observed ( Sastry and Gupta, 1978). Light microscopy-based investigations have demonstrated that there are alterations to the gut of C. punctatus and Heteropneustes fossilis following mercury intoxication (Bano and Hasan, 1990; Banerjee and Bhattacharya, 1995) but surprisingly no histopathological changes were described in Salvelinus alpinus following exposure to inorganic mercury and methyl mercury in their feed (Oliveira Ribeiro et al., 2002).

Similarly, intestinal toxic lesions, includes hyperemia, loss of structural integrity of mucosal folds, necrosis, cellular debris, vacuolation in intestine of Mugil auratus exposed to inorganic and organic mercury were also observed Discussion 97

(Establier et al., 1978). On exposure to another heavy metal, lead, hyper secretion of pepsin, leading to the degradation of tissue protein and increased ammonia and urea excretion by C. punctatus was observed (Sastry and Gupta, 1978). Such conditions are possibly due to the extremely adverse effects on stomach of fish due to lead nitrate toxicity. Although, the present study did not include these findings, yet we observed severe erosion of mucosa at highest sublethal exposure dose of Cu, which might hamper the normal gastrointestinal physiology.

As with gills, muscle tissue also come in close contact with pollutants dissolved in water. Hence, reactions in the histopathology of the muscle were spontaneous. In the present study the histopathology of muscle show progressive damage in the structure of muscle with increasing concentrations of copper. Similar observations have been made by Nagarajan and Suresh (2005) in the muscle tissue of the fish Cirrhinus mrigala with increasing concentrations of sago effluent. Sakr and Gabr, 1991; Abo Nour and Amer, 1995 and Das and Mukherjee, (2000) have studied the effect of different pollutants on fish muscles. Singh Sudha and Mehrotra Asha (1999) have observed the damaged muscle layers in the fish Nandus nandus exposed to carbaryl. Anshu Amali et al. (1995) have studied the sub lethal effect of quinolphos and padan on the muscle tissues of common carp C. carpio. Glycogen content was formed to be depleted in the tissues as concentrations of toxicants increased. Ayoola Simeon (2011) has observed mild lesion, necrosis, and inclusion of bodies, inflammation and cellular degeneration in the muscle tissue of the fish O.niloticus exposed to aqueous and ethanolic extracts of Ipomoes aquatica leaf.

Bharat et al. (2011) studied the histopathology of fish C. carpio exposed to sublethal concentrations of lead and cadmium. The copper treated fish Discussion 98 showed marked thickening and separation of muscle bundles with severe intracellular edema (Plate 4. Fig.B and C). Similar observation has been made by Das and Mukharjie (2000). Elnemaki and Abuzinadah (2003) have observed focal areas of myolysis in the muscles of O.spilurus exposed to toxicant. Abbas and Ali (2007) have noted the destruction and vacuolation of the muscle cells in Oreochromis spp exposed to chromium. Fatma (2009) observed the degeneration of muscle bundles with aggregation of inflammatory cells between them and focal areas of necrosis. Also, vacuolar degeneration in muscle bundles and atrophy of muscle bundles in fish exposed to different pollutants. Physical activity of fish usually associated with muscle tissue in the body. Present study exhibited various histological alterations such as edema, atropy and mild lymphocyte infiltration in the muscle tissue of treated fish might be due to copper toxicity. Similarly, several histological alterations were seen in the muscles of T. zillii and S. vulgaris during summer and winter and the pathological findings included degeneration in muscle bundles with aggregations of inflammatory cells between them and focal areas of necrosis. Also, vacuolar degeneration in muscle bundles (Plate 4.Fig.B) and atrophy of muscle bundles were observed might be due to the various concentrations of copper.

Separation and degeneration of muscles, atrophy of muscle bundles and focal area necrosis were an interesting observation in muscle tissue at low dose and it was leading to vacuolar degeneration and splitting of muscle fiber were seen as high dose (Plate.4, Fig.B and C).The histopathological alterations in the fish muscle of both the doses are in agreement with those observation by many investigators who have studied the effect of different pollutants on fish muscle (Sakr and Gabr, 1991; Abo Nour and Amer, 1995 and Elnemaki and Abuzinadah, 2003). In treated sea bass muscle edema and mild lymphocyte infiltration, vacuolar degeneration in muscle bundles and atrophy of muscle Discussion 99 bundles were observed. Edema between muscle bundles and splitting of muscle fibers were seen.

The present investigation closely agreed with a similar report by Fatma (2009). The histological findings included degeneration in muscle bundles with aggregations of inflammatory cells between them and focal areas of necrosis. Also, vacuolar degeneration in muscle bundles and atrophy of muscle bundles were observed. Alterations in the muscles of several species of the fishes exposed to heavy metals have been described by Oliveira- Ribeiro et al. (2002), Jiraungkoorskul et al. (2003), Thophon et al. (2003), Gupta and Srivastava, (2006) and Kaoud and El-Dahshan, (2010), which were in line with present investigation. Separation of muscle bundles was an interesting observation. Initial stimulus of copper can induce hyperactivity and excitability in animals, leading to release of lactic acid and subsequent muscular fatigue (Das and Mukherjee, 2000).

Cellular responses to pollutant-induced sublethal injury provided highly sensitive indicators of environmental impact (Hose et al., 1996). The organ most associated with the detoxification and biotransformation process is the liver, and due to its function, position and blood supply (Van der Oost et al., 2003) it is also one of the organs most affected by contaminants in the water (Rodrigues and Fanta, 1998). The liver has the ability to degrade toxic compounds, but its regulating mechanisms can be overwhelmed by elevated concentrations of these compounds, and could subsequently result in structural damage (Brusle and Anadon, 1996). Fish liver histology could therefore serve as a model for studying the interactions between environmental factors and hepatic structures and functions. Some of these environmental factors include biotoxins, parasites, infectious germs, physiochemical parameters and Discussion 100 pollutants, for example pesticides, hydrocarbons, PCB’s (polychlorinated biphenyls) and heavy metals (Brusle and Anadon, 1996).

Liver is first organ to face any foreign molecule through portal circulation is subjected to more damage (Jayantha Rao, 1982). Liver is an important organ of detoxification which breaks down toxic substances and metabolites of administered substances. This breakdown is carried out by endoplasmic reticulum of hepatocytes. The parenchymatous hepatic tissue in teleosts, has many important physiological functions and also detoxification of endogenous waste products as well as externally derived toxins, drugs, heavy metals and pesticides (Roberts and Rodger, 2001). Due to these reasons, the hepatic cells are damaged severely, on chronic exposure to copper. The liver exhibited several pathological changes including hyperplasia, degeneration of blood vessels, vacuolization, hypertrophy; pyknotic nuclei, necrosis, and accumulation of blood vessels (Plate 5.Fig. C and D).These changes are linearly proportional to exposure period and concentration of copper.

The present study also demonstrates that the liver of control fish exhibits a normal architecture and there were no pathological abnormalities. The hepatocytes present a homogenous cytoplasm and a large central or subcentral spherical nucleus. The histopathological appearance of liver following exposure to copper showed important alterations comprise hypertrophy of hepatocyets, nuclear hypertrophy, blood congestion in the central veins, as well as the diffusion of melanomacrophages in the parenchymal tissues of liver. Its revealed that the increase of aluminum concentration causes cytoplasmic vacuolation, cellular degeneration, damage of nuclei, bile stagnation in addition to congestion in the blood sinusoids. The severity of histopathological changes increased with concentration because this dose of copper prompt cellular Discussion 101 necrosis in the parenchymal tissues and decreasing in the number of hepatocytes nuclei of hepatic tissue.

Hypertrophy is generally characterized by an increase in cellular size. (Plate5.Fig.B). Exposure to compounds that induce proliferation of the endoplasmic reticulum membranes can be regarded as an example of hypertrophy (Hinton and Laurèn, 1990). Some studies revealed that interstitial hepatocytes of Nile tilapia exposed to contaminated sediment showed hydropic swelling (Peebua et al., 2006). Figueiredo-Fernandes et al. (2007) suggested that increase in the hepatocytes size may be due to the high content of lipids. On the other hand, Braunbeck, Storch, and Bresch (1990) referred that alterations in size and shape of nucleus have often been regarding as signs of increased metabolic activity but may be of pathological origin. Vacuoles in the cytoplasm of the hepatocytes can contain lipids and glycogen, which are related to the normal metabolic function of the liver (Plate 5.Fig.D). Depletion of the glycogen in the hepatocytes is usually found in stressed animals (Hinton and Lauren, 1990; Wilhelm Filho et al., 2001), because the glycogen acts as a reserve of glucose to supply the higher energetic demand occurring in such situations (Panepucci et al., 2001).

Significant changes were observed in the liver tissue in lethal and sublethal concentrations of copper where marked swelling of the hepatocytes in places with areas of diffuse necrosis (Plate 5 Fig. F). The normal architecture of liver tissue as markedly disrupted. Sinusoids in most cases were distended and central veins appeared severely damaged due to marked swelling and degeneration of the endothelial lining cells. Tilak et al. (2005) observed the same changes in liver of Catla catla. The pathological changes included degeneration of cytoplasm in hepatocytes, atrophy, formation of vacuoles, rupture in blood vessels, necrosis and hepatocyte cell membrane disposition. Discussion 102

Hepatic cords appeared in decreased size, nucleus became pyknotic. Radhaiah and Jayantha Rao, (1992) reported moderate cytoplasmic degeneration in hepatocytes, formation of vacuoles, rupture in blood vessels and appearance of blood vessels among hepatocytes, pyknotic nuclei in the liver of Tilapia mossambica exposed to fenvalerate. A study by Yildirim et al. (2006) in Nile tilapia (O.niloticus L.) fingerlings exposed to 5 μg L-1 deltamethrin revealed severe morphological alterations in liver, where hydropic degenerations in liver was observed.

Sakr and Jamal Al lail, (2005) observed histopathological changes induced in the liver after exposing the fish Clarias gariepinus to fenvalerate which are mainly represented by cytoplasmic vacuolization of the hepatocytes, blood vessel congestion, inflammatory leucocytic infiltration necrosis and fatty infiltrations. Ortiz Juan et al. (2003) observed reduction in the diameter of the hepatocytes and cellular vacuolization with hypertrophy of the hepatocytes in liver, when fish was exposed to lindane. Rodrigues et al. (2001) observed hepatocytes which were tumefied, with vacuolation, cytoplasmic granulation, and nuclear lateralization. Along with nuclei varying in diameter, density and condensed chromatin in the central region with pyknosis and areas of necrosis in the liver of Prochilodus lineatus exposed to a sublethal concentration of the organophosphate insecticide Dipterex 500R (Trichlorfon). Swelling of the hepatocytes with diffuse necrosis and marked swelling of blood vessels were observed in the liver tissue by Das and Mukherjee (2000) when L. rohita was exposed to hexachlorocyclohexane. Anomalies such as irregular shaped hepatocytes, cytoplasmic vacuolation and nucleus in a lateral position, close to the cell membrane, were also described in the siluriform Corydoras paleatus contaminated by organophosphate pesticides (Fanta et al., 2003). Pacheco and Santos (2002) described increased vacuolisation of the hepatocytes as a signal Discussion 103 of degenerative process that suggests metabolic damage, possibly related to exposure to contaminated water.

Anitha Kumari and Shree Ram Kumar (1997) observed an uneven distribution of carbohydrate content and a drastical decrease in the hepatic cells of the freshwater teleost upon exposure to polluted waters of Hussain Sagar lake. Tilak et al. (2001), reported the same degenerative changes in Ctenopharyngodon idellus under fenvalerate toxicity. Anita Susan and Tilak (2003) observed the toxic sublethal concentration of fenvalerate technical grade induced atrophy, appearance of blood streaks among hepatocytes in liver of C. mrigala. Similar reports were observed in liver of Cirrhinus mrigala was exposed to the sublethal and lethal concentrations of technical grade and 20% EC of Chlorpyrifos, Tilak et al., (2005).

Congestion of sinusoids was the only pathological effect of organochlorine herbicides reported by Couch (1975), and was also recorded in our present study. On the other hand, focal fibrosis is a progressive type of change and could indicate possible serious damage of liver structure. The present study closely agreed with a similar report by Pandey et al. (1996) in the liver of estuarine mullet, Liza parsia exposed to DDT. The histopathological alterations observed were necrosis, cytoplasmic vacuolization and pycnotic nuclei. The vacuolation, necrosis and disintegration of cell boundaries in Puntius ticto by Sahai and Suneeta Singh (1989) due to BHC, lindane and malathion toxicity have been reported. The effects of herbicides and pesticides on various aspects of liver of fishes have been reported by several workers. The present study clearly demonstrated that the liver is an important target organ for copper toxicity in the treated L. calcarifer. Discussion 104

6.6 Scanning Electron Microscope study

The SEM is a technique that allows the study of the damage of surface ultrastructure of the gill epithelium that cannot be revealed by light or TEM (Devos et al., 1998 and Dutta et al., 1998). The scanning electron micrographs of the untreated sea bass gill epithelium revealed normal architecture (Plate.6 Fig.A). In contrast the gills of L. calcarifer exposed to copper during twenty eight days presented a higher occurrence of histopathological lesions such as hypertrophy, fusion of secondary lamellae, edema and mucus openings. Numerous types of gill damage have been documented in fish experimentally exposed to toxicants or in populations sampled from polluted environments (Alazemi et al., 1996; Pawert et al., 1998; Thophon et al., 2003). Most of the gill histopathological changes are largely non-specific as confirmed by the occurrence of similar alterations under a wide range of toxicant-exposure conditions (see review of Mallatt, 1985). Hyperplasia with lamellar fusion, telangiectasia, oedema with epithelial lifting and desquamation, as documented in the present survey, are typical lesions of gills in response to organochlorines, petroleum compounds, organophosphates, carbamates, herbicides and heavy metals (Alazemi et al., 1996; Global Tox, 1997; Jiraungkoorskul et al., 2003; Thophon et al., 2003; Dezfuli et al., 2006; Giari et al., 2007) and suggest an impairment to the respiratory and osmoregulatory functioning of the gills (Au, 2004). Exposure to heavy metals produces morphological and functional modifications in the branchial epithelium (Alazemi et al., 1996; Mauceri et al., 2005); mercury inhibits gill respiration at sub-acute and acute levels (Jagoe et al., 1996).

The first sign of pathology included edema of epithelial cell in gills. This is due to the epithelium covering the secondary lamellae lifting away in a continuous sheet from the pillar cell system, thus increasing the diffusion Discussion 105 distance from water to blood (Pate 6. Fig,E). The secondary lamellae showed capillary congestion or aneurism, similar to those reported in Gnathonemus petersii exposed to 10 mg/l of cadmium for 6 h (Alazemi et al., 1996). This lamellar aneurism resulted from the collapse of the pillar cell system and the breakdown of vascular integrity with a release of large quantities of blood that push the lamellar epithelium outward (Alazemi et al., 1996). The hypertrophy and hyperplasia of epithelial and chloride cells with partial or complete fusion of lamellae also occurred in this study (Plate 6 .Fig.C). Several studies pointed out that the chloride cell hyperplasia occurred in response to the need to eject the Cd2+ absorbed by the gills (Oransaye and Brafield, 1984; Gill et al., 1988; Fu et al., 1990).

The immediate morpho-pathological response of the gills of fish exposed to ambient copper is often manifested by a significant increase in the density of its mucous cells (Plate 6.Fig.C) (Bradbury, 1987; Wise et al., 1987; Dutta, 1997; Hemalatha and Banerjee, 1997). The large quantity of mucous secretion acts as a defence mechanism against several toxic substances (Handy and Eddy, 1991; Mazon et al., 1999). The regular sloughing of mucus from the surface of gills into the media helps to remove the bound pathogens, toxicants and foreign matters (Powell et al., 1992) which adhere to the gills.

Changes to the ultrastructure of fish livers have proven to be suitable and sensitive signs of toxicant-induced injury and have been used as biomarkers of chemicals in environmental risk assessments (Braunbeck and Volkl, 1991; Alazemi et al., 1996). There have been numerous reports on histocytopathological modifications in livers of fish exposed to a wide range of organic compounds and heavy metals (Hinton and Lauren, 1990; GlobalTox, 1997; Au, 2004). The loss of the regular cytoplasmic compartimentation is a typical unspecific ultrastructural reaction of fish hepatocytes which indicates Discussion 106 disturbance of hepatocellular homeostasis (Braunbeck, 1998). Some of the alterations observed in the hepatic cells in the present study, such as vacuolar degeneration, dilation of ER and lipid droplet accumulation, are consistent with those documented in specimens of D. labrax, L. calcarifer and Carassius carassius acutely treated with other heavy metals, lead and cadmium ( Franchini et al., 1991; Thophon et al., 2003, 2004; Giari et al., 2007).

6.7 Transmission electron microscopy study

The gills, which participate in many important functions in fish, such as respiration, osmoregulation and excretion, remain in close contact with the external environment, and particularly sensitive to changes in the quality of the water, are considered the primary target of the contaminants (Poleksic and Mitrovic-Tutundzic, 1994; Mazon et al., 2002; Fernandes and Mazon, 2003). Alterations like epithelial lifting, hyperplasia and hypertrophy of the epithelial cells, besides partial fusion of some secondary lamellae are examples of defense mechanisms, since, in general, these result in the increase of the distance between the external environment and the blood and thus serve as a barrier to the entrance of contaminants (Mallatt, 1985; Hinton and Lauren, 1990; Poleksic and Mitrovic-Tutundzic, 1994; Fernandes and Mazon, 2003). Most part of the gill lesions caused by sublethal exposures affects lamellar epithelium (Hinton and Lauren, 1990); however, some alterations in blood vessels may also occur, when fishes suffer a more severe type of stress. The damaged pillar cells can result in an increased blood flow inside the lamellae, causing dilation of the marginal channel, blood congestion or even an aneurysm (Takashima & Hibiya, 1995; Rosety Rodriguez et al., 2002). The formation of an aneurysm is related to the rupture of the pillar cells (Heath, 1987; Martinez et al., 2004) due to a bigger flow of blood or even because of the direct effects of contaminants on these cells. Discussion 107

Morphologic alterations of the pillar cells can have several secondary consequences in L. calcarifer (Plate 8.Fig.B).These cells control the blood pressure of the fish, and changes in the blood pressure and flow can affect the number of irrigated lamellae, the distribution of the blood within the lamellae, the permeability of the branchial epithelium and, as a consequence, the osmoregulatory and gaseous exchange mechanisms (Randall, 1982), causing several physiological disorders. The deformation of erythrocytes was obvious, and has possibly reduced the capacity of oxygen transport, consequently causing a certain level of hypoxia. Consequently, the fish tries to compensate the lower levels of oxygen in its tissue by an increase of the respiratory frequency, as the same was observed in M. gulio. This is observed not only after intoxication with chemicals, but always when there is a change in the respiratory lamellae, caused by any environmental changes (Fanta-Feofiloff et al., 1986; Fanta et al., 1989, 1995). Several other studies have shown similar effects of pesticides on fish gills (Cengiz and Unlu, 2002, 2003).

Many investigators have reported the histopathological changes in gills of different fish species exposed to pesticides. Hemorrhage in the primary and secondary gill lamellae, degeneration and necrosis of epithelial cells, distortion of the secondary lamellae, disruption of epithelial cells from pillar cells were observed in gill tissues of Anabas testudineus exposed to monocrotophos (Santhakumar et al., 2001). Degenerative changes in gills, such as detachment and lifting of the epithelial linings from the surface of the gills, uncontrolled regeneration of the primary lamellae and secondary lamellae, hypertrophy, hyperplasia, necrosis of the epithelial cells, dilation of the blood sinuses of the secondary lamellae, lamellar aneurysm, hemorrhages were noticed after exposure of sublethal concentration of profenofos (Rao, 2006). Coutinho and Gokhale (2000) found epithelial lifting in the gills of carp (C. carpio) and tilapia (O. mossambicus) exposed to the effluents of a wastewater treatment Discussion 108 plant. According to Mallat (1985) such alterations are non-specific and may be induced by different types of contaminant. As a consequence of the increased distance between water and blood due to epithelial lifting, the oxygen uptake is impaired. Toxic substances can injure gills, thus reducing the oxygen consumption and disrupting the osmoregulatory function of aquatic organisms (Saravana Bhavan and Geraldine et al., 2000). However, fishes have the capacity to increase their ventilation rate, to compensate low oxygen uptake (Fernandes and Mazon, 2003).

Histopathological changes in the gill tissue of the fish C. punctata exposed to sublethal concentration of Butachlor and Machete, an Herbicide was studied by Tilak et al. (2005) and found marked pathological changes such as bulging of tips of primary gill filaments and fusion of disorganized secondary gill filaments. Similarly the toxic sublethal concentration of fenvalerate technical grade in the gill of C. mrigala was evaluated and found marked pathological changes like necrosis, progressive degeneration in the gill tissue (Anita Susan and Tilak, 2003).

The present electron micrographs show a reduction in the quantity of the microridges of the pavement cells in the gills of L. calcarifer. Such reduction was also observed by Wong Chris and Wong (2000) and Mazon et al. (2002). Mallat (1985) suggested that the microridges are related with the retention of mucous on the epithelium as a way to protect it against environmental alterations.

These pathological changes may be a reaction to toxicants intake or an adaptive response to present the entry of the pollutants through the gill surface (Mohamed, 2009). The damages observed in the gills in terms of hypertrophy, fusion of secondary lamellae and necrosis could cause a decrease in free gas exchange, thus affecting the general health of fish (Skidmore and Tovell, Discussion 109

1972). Similar of these changes in gill epithelia of O. niloticus were ultrastructurally observed by Nath and Kumar (1989). Crespo (1982) in the dog fish, Scyliorhinus canicula subjected to zinc sulphate; Temmink et al. (1983) in rainbow trout, Salmo gairdneri exposed to chromate; Gupta Neeraj and Dua Anish (2002) in the C. punctatus intoxicated with mercury. Pane et al. (2004) in O. mykiss treated with nickel. Acharya et al. (2005) in L. rohita treated with sublethal acidic (HCl) and alkaline (NaoH) pH. Palaniappan et al. (2008) observed hypertrophy, hyperplasia, alteration of lamellar surface and fused lamellae in pb exposed Catla catla. In fishes, the gills play vital roles, since they are the main site of gaseous exchanges (Hughes et al., 1982). Furthermore, they are involved in osmoregulation (Ramao et al., 2001) acid – base balance (McDonald et al., 1991 and Goss et al., 1992) and excretion of nitrogenous compounds (Gold stein 1982; Evans and Cameron, 1986; Sayer and Daven port, 1987).

The ultrastructure of untreated L. calcarifer, gill is shown in plate 9.FigA. The centrally located nucleus normally exhibits little heterochromatin. The cell organelles mitochondria smooth endoplasmic reticulum (SER), rough endoplasmic reticulum ((RER), Peroxisomes and Golgi complex were scattered in the cytoplasm. In the gills of treated sea bass exposed to higher sublethal concentrations of herbicides (atrazine and glyphosate) showed abundant distribution of cytoplasmic vacuoles as well as alterations of cytoplasmic organelles including mitochondria, SER, RER and Golgi complex. The ultra structural alterations were more severe and progressive in high concentration copper treated gills. (Plate 9.Fig.E and F).

Similar type of alterations on ultrastructure of gill has been reported earlier due to various types of stress conditions (Maina, 1991, Pfeiffer et al., 1997, Zahra khoshnood et. al., 2011 and Ba-Omar et al., 2011). Thus, from the Discussion 110 above findings, it could be concluded that the structural alterations seen in the cell organelles such as mitochondria Endoplasmic reticulam and Golgi complex following exposure to mercury in fresh water prawn Macrobrachium malcolmsonii, suggests functional alterations of the organelles (Yamuna et al., 2009). Therefore such changes noted in the present study can be taken towards operation of compensatory mechanisms against herbicides toxicity. The operation of such mechanism achieved by detoxification of herbicide prevents severe cellular damage. The finding of this study indicate that ultra structural changes observer serve as biomarker in studies of toxic stress in aquatic environment.

According to Dutta et al. (1998) electron microscopic studies of the gills of catfish (H. fossilis), exposed to sublethal concentrations of malathion (4 mg L-1 and 6 mg L-1) revealed that 24 hr exposure to 4 mg L-1 had a mild effect. However, severe damage was found after 48 and 72 hr exposures. After a 24 hr exposure to a 6 mg L-1 concentration, more severe damage ensued. The microridged epithelial cells of the gill arch became perforated and the central portion of the filament appeared elevated. Numerous mucous gland openings also became visible. However, the increase of mucous containing vacuoles in the mucous cells are evident to the mucous function in protection of the gill epithelium from environmental impacts, infectious agents, toxic agents and particles in suspension (Powell et al., 1992; Biagini et al., 2009). Perry and Laurent (1993) stated that mucous cells can be efficient in seizing the toxic agents and thus help in the prevention of the entrance of these agents into the gills. So far, the inflammatory reactions of gills observed in the current study may be caused by direct contact of the respective epithelia with copper. This could be due to the high sensitivity of the gills to environmental stress and their capacity to react to low concentrations (Pawert et al., 1998; Pandey et al., 2008). However, it is more probably that these effects are secondary alterations Discussion 111 to the pollutants action in receptors bond to the epithelial cell membranes (Evans, 1987). Lesions in the gill morphology could lead to functional alterations and interference in fundamental process such as maintenance of osmoregulation and antioxidant defense of gills (Pandey et al., 2008). According to Arellano et al. (2001) and Biagini et al. (2009) the histological alterations observed in fish gills are acknowledged as a fast and valid method to determine the damages caused by exposition to different pollutants.

The gills, because of their direct and permanent contact with water, are primary and very sensitive receptor surface for aquatic pollution (Wood and Soivio, 1991; Bernet et al., 1999) and have been widely used to investigate the toxicity effects from heavy metals (Wood, 1992). The severe morphological anomalies of gill observed in the present survey are non-specific and occurred under many different exposure conditions (Evans, 1987; Alazemi et al., 1996; Neskovic et al., 1996; Haaparanta et al., 1997; Teh et al., 1997; Oliveira Ribeiro et al., 2000; Roberts and Rodger, 2001; Thophon et al., 2003). Analysis of common gill histopathology indicates that structural damages such as hyperplasia with lamellar fusion, telangectasia, oedema, epithelial lifting, and necrosis represent stereotyped physiological reactions of gill to stress and many of them have a defence function (Mallat, 1985). These responses, whether adaptive or pathological, invariable determine the extent of homeostatic regulation of the internal environment (Laurent and Perry, 1991), in particular decreasing the efficiency of gas exchange (Jagoe et al., 1996; Oliveira Ribeiro et al., 2002).

The teleost liver is one of the most sensitive organs to show alteration in biochemistry, physiology and structure following exposure to various types of environmental pollutants (Hinton and Couch, 1998). Since the liver is the central site of numerous vital functions (i.e. basic metabolism, bile production, Discussion 112 vitellogenesis) and accumulation, biotransformation and excretion of organic and inorganic contaminants, the hepatocytes are powerful sources for biomarkers (Braunbeck, 1998; Bernet et al., 1999). The deterioration of the regular compartimentation of the cytoplasm is a very early and unspecific signal of disturbance of hepatocellular homeostasis (Braunbeck, 1998). The same ultrastructural modifications of the organelles were also observed in the present survey. Some of these changes, for instance the proliferation of endoplasmic reticulum (ER), (Plate 9.Fig.B,C) indicate stimulation of defence and regenerative processes linked to detoxification and could be classified as adaptative whilst other, such as dilatation of the ER and the nuclear envelope, express the onset of degenerative processes in the liver (Braunbeck, 1998). The major cytotoxicity mechanism of copper is the alteration of ion and non- electrolyte transport and cell volume regulation, which finally leads to cell swelling (Ballatori and Boyer, 1996). With regard to the storage products, the increase in lipid droplets observed in this study and by other authors after lindane, cadmium and terbuthylazine exposures (Biagianti- Risbourg et al., 1996; Thophon et al., 2003; Dezfuli et al., 2006) could be due to the decline of protein synthesis and the consequent non utilization of lipids for lipid-protein conjugation (Cheville, 1994). The manifestation of cytopathologic changes herein documented might suggest a severe hepatic dysfunction and the impairment of the physio-metabolic process in L. calcarifer liver; this finding is in agreement with the principle of the relationship and mutual interference between structure and function expressed by Hinton et al. (1987).

The histopathological alterations in the liver observed in the present study were sinusoid dilation with blood congestion, hydropic swelling of hepatocytes, and dark granule accumulation. Lipid droplet accumulation in many hepatocytes was observed only in subchronic exposure and severity depended on the time of exposure. These pathologies are consistent with those Discussion 113 of the previous reports (Gupta and Rajbanshi, 1982; Wani and Latey, 1983; Rani and Ramamurthi, 1989). The cellular damage of the liver was more severe and prominent in a lethal exposure than in a sublethal exposure (Brown et al., 1984). Abundant glycogen content was observed in many hepatocytes at the 28 days exposure of high concentration of copper in L. calcarifer. The result of this study was similar to that of Sastry and Subhadra (1982, 1985) who studied on the chronic effects of Cd (0.2 mg/l) in catfish H. fossilis and found that after 60 days of exposure, liver glycogen increased which was related to the decreased activity of lactate dehydrogenases, pyruvate dehydrogenases, and succinate dehydrogenase in the liver.

A wide spectrum of RER modifications, appearance of myelinoid bodies, deformation of mitochondria and the nuclear envelope are also herbicide-induced alterations in hepatocytes of rainbow trout, O. mykiss, and carp, C. carpio, as reported respectively by Braunbeck et al. (1992) and Szarek et al. (2000). According to Franchini et al. (1991), the increase in heterochromatin in the nuclei, in addition to the pathological changes within RER, suggest impairment to the synthetic and secretory activities of the cell. With reference to the extensive development of SER in the sea bass exposed to copper in this study, the same phenomenon has been seen in the hepatocytes of this and other fish species when treated with lead, benzopyrene and certain pesticides (Franchini et al., 1991; Lemaire et al., 1992; Braunbeck, 1998). Hypertrophy of the ER is a classical response of teleosts to pollutants and is linked to hepatic detoxication mechanisms (Hawkes, 1980; Lemaire et al., 1992). The mitochondrial structural alterations noticed within the hepatocytes and enterocytes of mercury-exposed sea bass were commonly found in different organs following toxic injury (Lemaire et al., 1992), but their significance is difficult to evaluate (Biagianti-Risbourg and Bastide, 1995). Discussion 114

Copper may reach in the water bodies when treat the disease of fishes and control the algal blooms in sea bass hatchery. These copper will accumulate in their tissues of juvenile sea bass. Lethal concentration of copper can kill the fishes. When the fishes are suffering from sublethal effects as a result of cumulative accumulation of copper, they may survive for longer times. In man, daily consumption of these fishes will cause the ill effects that are specific to the toxicant. This process will be damaging the organism silently, without causing any immediate abrupt changes. The changes may be at genetic level inducing genotoxicity. But concerted effort in reducing the use of copper and implementing natural remedies for disease control in sea bass hatchery can help resolving the problem of heavy metal pollution. SUMMARY AND CONCLUSION Summary and Conclusion 115

7. SUMMARY AND CONCLUSION

7.1 The LC50 value of copper in L. calcarifer during 24, 48, 72 and 96 hours was estimated to be110.83 ppm,93.57ppm, 76.71ppm and 68.32 ppm respectively.

7.2 Bioaccumulation of copper after 28 days of exposure to sublethal concentration of copper was showed increasing trend with increasing concentration (muscle : 0.76+ 0.02 μg/g to 1.99 +0.03 μg/g, gills : 2.64 + 0.02 μg/g to 5.38 + 0.02 μg/g , liver : 36.50 + 0.15 μg/g to 82.6 + 0.15 μg/g , kidney : 2.58 + 0.02 μg/g to 7.83 + 0.02 μg/g).

7.3 The total protein in the muscle, gill, liver of L. calcarifer showed decreasing trend as the duration of exposed to copper increased ( muscle : 62.59 + 0.59mg / 100 mg to 52.14 + 0.63 mg/100 mg, gills : 2.42 + 0.09 mg/100 mg to 1.72 + 0.6 mg/ 100 mg and liver : 1.75 + 0.07 mg/100 mg to 1.27 + 0.8 mg/100 mg).

7.4 The total free sugar in the muscle, gill and liver of L. calcarifer showed decreasing trend with increasing concentration of copper ( muscle : 26.54 + 0.03 mg/ 100mg to 22.01 + 0.61 mg/ 10 mg , gills : 6.89 + 0.08 mg/100mg to 5.01+ 0.12 mg/100mg and liver : 22.35 + 0.49 mg/100mg to 19.05+ 0.22 mg/100mg).

7.5 The total lipids in the muscle, gill and liver of L. calcarifer showed decreasing trend as the duration of exposure in each concentration of copper increased (muscle: 18.92 + 0.62 mg / g to 15.13 + 0.48 mg/g, gills : 1.64 + 0.06 mg/g to 0.76 + 0.04 mg/g and liver : 22.92 + 0.67 mg/g to 19.21 + 0.42 mg/g).

7.6 The electrophoretic pattern of muscle protein profile revealed 11 polypeptide bands (control). The number of bands decreased to 7 and 6 after 7 Summary and Conclusion 116 days of exposure at 6.83ppm and 13.66ppm. After 28 days the tissues showed 6 bands and further reduced to 5 at 6.83ppm and 13.66ppm concentrations of copper.

7.7 The electrophoretic pattern of gill protein profile revealed 6 polypeptide bands (control). The number of bands decreased to 5 and 4 after 7 days of exposure at 6.83ppm and 13.66ppm concentration. After 28 days the protein bands decreased to one in both concentrations.

7.8 In liver tissues, the protein profile were very low and separation of polypeptides was also very poor. Hence the densitometer could not detect any bands in liver tissues in control and treated groups.

7.9 Histological changes of gills were showed a typical structural organization of the respiratory lamellae in control fish. After 7 and 28 days 6.83ppm concentration of copper shows curling of secondary lamellae and telangiectasis at the tip of secondary lamellae, hyperplasia of epithelial cells, completely damaged pillar cell system and extensive edema of the epithelial cells (EEC). After 7 and 28 days 13.66ppm concentration of copper showed pyknotic nuclei, lamellar clubbing, rupture of secondary lamellar tips, edema and rupture of epithelial cells. The gill showed extensive aneurism with rupture in secondary lamellae.

7.10 Histological changes of intestine in control fish showed the basic organization of intestinal wall with lamina propria and goblet cells were interspersed among the columnar cells. After 7 and 28 days 6.83ppm concentration of copper shows damaged circular muscle layer and flattened villi, damaged longitudinal muscle layer and distended lumen. After 7 days of exposure to 13.66ppm shows swelling of lamina propria. After 28 days of exposure to 13.66ppm shows damaged goblet cells, cracked clay appearance of the tissues. Summary and Conclusion 117

7.11 Histological changes of muscle in control fish were showed elongated muscle fibres, segmentation of vertebrate musculature is seen clearly in the lateral muscle of the fishes. After 7 and 28 days of exposure to 6.83ppm of concentration of copper shows inter myofibrillar space and gap formation in myofibril, inter myofibrillar space muscle edema and disintegrated myofibrils. After 7 and 28 days of exposure to 13.66ppm of concentration of copper shows edema between muscle fibre and disintegrated myofibrils muscle degradation and inter myofibrillar space.

7.12 Histological changes of liver in control fish showed normal hepatocytes, hepatic cells containing clear spherical nucleus. After 7 and 28 days of exposure to 6.83ppm concentration of copper shows cytoplasmic degeneration, damaged epithelium and hydropic swelling of hepatocytes. After 7 and 28 days of exposure to 13.66ppm concentration of copper shows hydropic swelling of hepatocytes, blood congestion, nuclear pyknosis and cytoplasmic vacuolation.

7.13 SEM histopathology of gill showed damaged respiratory organs. SEM examination showed swelling and curling of secondary lamellae. After 28 days of low concentration of copper, the gill showed extensive aneurism with some ruptures in many secondary lamellae and the breakdown of pillar cell system was seen. In higher magnification, the SEM picture clearly depicts the denuding of the boundaries of surface epithelial cells of both primary and secondary lamellae.

7.14 SEM histopathology of liver shows changes in lower concentration of copper exposed 7 and 28 days include cloudy swelling of hepatocytes congestion vacuolar degeneration , dilation of sinusoids and nuclear hypertrophy. After 7 and 28 days of higher concentration of copper exposure showed hydropic swelling and vacuolar degeneration. Summary and Conclusion 118

7.15 TEM histopathology of gill shows hypertrophic pavement cells with irregular shape, long cytoplasmic processes and without micro ridges were observed, after 7 days of exposure of higher dosage of copper. After 28 days of exposure shows extensive vasodilation with stretching and necrosis of pillar cells.

7.16 TEM histopathology of liver shows degranulation and fragmentation of rough endoplasmic reticulum, dilation and vesculation of reticulum cisternae. After the exposure of 7 days at higher concentration of copper, hydropic swelling of hepatocytes with nuclear pyknosis and chromatin condensation were observed. TEM observations showed severe lesion in the hepatocytes.

7.17 Copper may reach in the water bodies when treat the disease of fishes and control the algal blooms in sea bass hatchery. These copper will accumulate in their tissues of juvenile sea bass. Lethal concentration of copper can kill the fishes. When the fishes are suffering from sublethal effects as a result of cumulative accumulation of copper, they may survive for longer times. In man, daily consumption of these fishes will cause the ill effects that are specific to the toxicant. This process will be damaging the organism silently, without causing any immediate abrupt changes. The changes may be at genetic level inducing genotoxicity. But concerted effort in reducing the use of copper and implementing natural remedies for disease control in sea bass hatchery can help resolving the problem of heavy metal pollution. REFERENCES i

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Research Article Allied Sciences

International Journal of Pharma and Bio Sciences ISSN 0975-6299

EFFECT OF ALUMINIUM CHLORIDE TOXICITY AGAINST HISTOPATHOLOGY OF GILL AND LIVER TISSUE OF INDIAN MAJOR CARP, CATLA CATLA (HAMILTON)

A.MAHARAJAN* AND P.S.PARURUKMANI

PG & Research Department of Zoology, Khadir Mohideen College, Adirampattinam - 614701, Thanjavur Dist, Tamil Nadu, India.

ABSTRACT

Histopathological studies in organs like gill and liver of Catla catla (Hamilton) were made to assess tissue damage due to Sublethal concentration of aluminium chloride. Histopathological study provides a real picture of the detrimental effects and the involvement of the heavy metal toxicants in the major vital functions such as respiration metabolism and reproduction in aquatic animals. It is generally evident that changes in microscopic abnormalities. The gill of aluminium exposed fish exhibited vacuole formation in epithelial cells, swollen of lamellar tips, rupturing of epithelial wall, dilation of blood vessels and fusioning and shortening of secondary lamellae and loss of broken lamellar structure. The liver of Catla catla exposed to aluminium showed distinct hepatic lesions. The histopathological changes included necrosis, psychosis, disintegration of cells and vacuolization. In addition internal haemorrhage, necrosis and empty blood vessels were also seen. Such pathological changes were observed in all the tissues were more pronounced in sublethal concentration for 28 days of exposure at 10 % and 20% of the 96hrs LC50 value. The observation of the present study is to suggest that the damage of these tissues is caused by cumulative accumulation of aluminium in tissues.

KEY WORDS: Aluminium chloride, Catla catla, Histopathology, Gill, Liver

A.MAHARAJAN PG & Research Department of Zoology, Khadir Mohideen College, Adirampattinam - 614701, Thanjavur Dist, Tamil Nadu, India.

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INTRODUCTION

Freshwater ecosystem has been polluted by MATERIALS AND METHODS continuous discharge of waste water from industries, human dwelling and agricultural COLLECTION OF EXPERIMENTAL ANIMAL practices. The wastewater contains various The fresh water fish Catla catla average length amounts of chemical substances, such as 3 ± 0.50 cm, weight 2.5 to 3gm were procured pesticides, fertilizers and industrial pollutants. from the B.S.P. aquarium, Puthur, near These chemical substances are accumulate in Chidambaram, Tamil Nadu. They were brought large quantities of water. Often accumulate in to the laboratory in polythene bags filled with large quantities. Such poisonous substances aerated water and stocked in a tank of 60litre on reaching aquatic ecosystem have a fatal capacity filled with tap water and fishes were effect on biota including fishes. These chemical acclimatized in the laboratory conditions for substances are accumulate in large quatities of about two weeks. Significant sign of stress or water it will accumulate in organisms through unusual behavioural criteria were not observed food chain and harmful to human beings. in the control fishes throughout the acclimation Industrial pollutants occupy prime position than and test period. During the acclimatization the the others, as they release heavy metals in to 1 fishes were fed with pellet feed daily in the the aquatic environment . Heavy metals pose evening uneaten feed was removed next day a serious threat to the aquatic environment morning followed by 100% water exchange. because of their greater toxicity and persistence to accumulate in organisms PREPARATION OF STOCK SOLUTION FOR 2 through food chain amplification . Aluminium ALUMINIUM CHLORIDE TOXICITY TEST metal and its compounds have a wide variety One gram of Aluminium chloride (Merck, of uses including structural materials in Germany) was dissolved in 1 l of double- construction auto mobiles, air crafts and the distilled water and used as the stock solution production of metal alloy, glass, ceramic, for preparing different concentrations of rubber pharmaceuticals and water proofing aluminium chloride in rearing water. It was textiles. The presence of aluminium in water stored in a clean standard flask at room 3 found to be in different forms among fish temperature in the laboratory species considerable differences in sensitivity to metals have been reported. Catla Catla have SUBLETHAL TOXICITY TESTS the ability to accumulate and concentrate iron For sublethal toxicity tests, the fishes were to the levels, several order of magnitude above grouped into three batches. Each batch had those found in their environment . The aqueous ten fishes and three replicates were aluminium is recognized as the principal maintained. Fishes maintained in normal fresh 4,5 6 toxicant to fishes . Sastry and Shukla have water served as control (group I). Fishes were shown that exposure to heavy metal pollution exposed to concentration of 5.31ppm (10% of results in decreased oxygen consumption in 96 hr LC50) of Aluminium chloride in fresh fish Indian major carp C.Catla is an important water (group II). Fishes were exposed to the cultured fish species in fish ponds due to its sublethal concentration of 10.62ppm (20% of fast growth and economic value. However the 96 hr LC50) of Aluminium chloride in fresh studies on the histopathological effect of water (group III). The media were renewed aluminium on the fresh water fishes are very every alternate day. Fishes were fed daily with limited. Therefore, the present study is artificial feed. Two specimens each from the proposed to assess the aluminium toxicity on groups I, II and III were sacrificed after 28 days the histopathological changes of vital organs of the experiment. like gill and liver of fish C. catla.

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HISTOPATHOLOGY The tip of the primary lamellae contains a large Fishes were exposed to Aluminium chloride at dense mass of red blood cells. The secondary 5.31 and 10.62 ppm for 28 days. Sampling was lamellae consisted a layer of flattened epithelial done on the 28th day of exposure; two fishes in cells arranged to the basement, membrane. each group were sacrificed. The gills and liver Further, the secondary lamellae consist of pillar of representative fishes from each test and cells situated in between blood capillaries and control group were dissected out and fixed in chloride cells located at the base of the two Davidson’s fixative for 24hrs. The preserved adjacent lamellae (Fig. 1). During the present tissues were processed by a routine study, C.catla subjected to sublethal histological method 7, dehydrated in alcohol concentration of aluminium chloride has initially series and embedded in paraffin wax. They exhibited a film of coagulated mucous over gill were cut into sections of 6 mm thickness by a surface and marked histopathological changes rotary microtome (Weswox, MT1090:1090A, of gill in both exposure for a period of 28 days India). The thin sections of the tissues were at 10% and 20% of 96hrs LC50 value of the stained by haematoxylin and eosin for gill rays have become thicker, cartilaginous observation by the Nikon Bright field cells of the gill rays undergo hyperplasia, transmission microscope with Koechler vacuole formation in epithelial layer, swollen illumination, and automatic exposure unit was lamellar tips and rupturing of epithelial wall of used. secondary lamellae. Moreover, the degeneration of pillar and chloride cells, RESULTS dilation of blood vessels, fusion and shortening of secondary lamellae and loss of broken GILL lamellar structure are the important In the gill of control fish C catla the primary gill histopathological changes in both treatment. In lamellae are laterally compressed leaf like addition, owing to hyperplasia, inter lamellar structures, attached alternately on either side epithelium exhibits a characteristic aggregation of the interbranchial septum. Each primary gill in fish exposed to sublethal concentration of lamellae consist of secondary gill lamellae on aluminium. The lesions observed in the 20% of both the sides, which are perpendicular to its the 96hrs LC50 vaule of sublethal exposure long axis. Primary gill lamellae comprised of a were more pronounced than those in the central core of cartilaginous rod and linings of sublethal exposure of aluminium chloride (Fig. epithelial cells are closely applied to gill ray. 2&3).

Plate 1 Histology of control gill in C.catla photomicrographs of the paraffin section stained with haematoxylin and eosin (×40). BV- Blood Vessels, SL - Secondary Lamellae, TPL -Tip of Primary Lamellae.

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Plate 2 Histopathological changes of gill in C.catla photomicrographs of the paraffin section stained with haematoxylin and eosin (×40) after 28 days of exposure to 5.31ppm concentration of Aluminium chloride. BSL- Broken of Secondary Lamellae, CSL- Curling of Secondary Lamellae, FSL - Fusion of Secondary Lamellae, HP - Hyperplasia, LEI - Lifting on Epithelial Layer, SCC- Swelling of Chloride Cell, SSL - Shortening of Secondary Lamellae, V - Vacuole.

Plate 3 Histopathological changes of gill in C.catla photomicrographs of the paraffin section stained with haematoxylin and eosin (×40) after 28 days of exposure to 10.62ppm concentration of Aluminium chloride. BSL - Broken of Secondary Lamellae, HP – Hyperplasia, LEL- Lifting on Epithelial Layer, SCC - Swelling of Chloride Cell, SSL- Shortening of Secondary Lamellae, V - Vacuole.

LIVER sublethal concentrations of aluminium chloride Microscopical observation shows that the for 28 days at 10% and 20% of 96hrs LC50 normal liver of C. catla consists of large number value. In both exposures the liver parenchymal of hepatocytes arranged regularly in cords. The cells arc found to shrunken and appear smaller hepatic cells are large, polygonal in shape with in size, Variations in the shape of hepatocyte is prominent nucleus. Blood sinusoids are also evident in different regions of liver, lntrahepatic seen among the hepatocytes. Liver cells are spaces have been dilated. The extension of similar in size. The nuclei of the liver cells are nucleus called the pyknosis and spherical and show uniform size, shape and cytoplasmolysis are observed in different orientation (Fig.4). In the present study the region. The cytoplasm becomes granulated and following histopathological changes have been vacuolated. The blood vessels are severely observed in the liver of C. catla exposed to damaged leaving large empty spaces. The liver

This article can be downloaded from www.ijpbs.net B - 526 Int J Pharm Bio Sci 2012 July; 3(3): (B) 523 - 530 cord has become disarrayed in both treated more pronounced than those in the sublethal groups.These above observed histopathological exposure (Fig.5 &6). changes in the median lethal exposures are

Plate 4 Histology of control liver in C.catla photomicrographs of the paraffin section stained with haematoxylin and eosin (×40). BV - Blood Vessels, H - Hepatocyte, MH- Mass of Hepatocyte

Plate 5 Histopathological changes of liver in C.catla photomicrographs of the paraffin section stained with haematoxylin and eosin (×40) after 28 days of exposure to 5.31ppm concentration of Aluminium chloride. CBV- Congested Blood Vessel, DN - Disintegration of Nucleus, EH - Enlargement of Hepatocyte, NC - Necrosis in Cytoplasm , PN - Pyknotic Nuclei, V - Vacuolation.

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Plate 6 Histopathological changes of liver in C.catla photomicrographs of the paraffin section stained with haematoxylin and eosin (×40) after 28 days of exposure to 10.62ppm concentration of Aluminium chloride. DN - Disintegration of Nucleus, EBV -Empty Blood Vessels, NC - Necrosis in cytoplasm, PN - Pyknotic Nuclei, V - Vacuolation

DISCUSSION responses of the gill to heavy metal pollution 10,11 .The observed progressive hyperplasia, The liver of the fish can be considered as a necrosis, desquamation of the epithelial cells target organ to pollutants. Alteration in its and multiple telangiectasia in C. catla exposed to aluminium was quite comparable to the fish structure can be significant in the evaluation of 12 fish health and exhibit the effects of variety of Sulmo gairdneri . The Filling up of inter- environmental pollutants. The gills of fish are lamellar spaces by hyperplastic epithelium the most important organ for respiration and under aluminium treatment tends to explain the high mortality of the fish, probably due to osmoregulation and its external location 8 13 renders it most vulnerable target organ for the asphyxia . Mallatt suggested that pollutants 8. The study on morphometry and hyperplasia of squamous epithelial and histopathology of gills may be used to monitor chloride cells serves as a physiological the quality of the environment. During the defense mechanism against the toxicants. In present study, several histopathological addition to hyperplasia of inter lamellar cells, changes were observed in the gills of C. catla, necrosis and pycnotic epithelial cells are also when they exposed to sublethal concentrations observed in the treated fish. These changes of aluminium chloride after 28 days at 10% and might he resulting a decrease in energy 20% of the 96hrs LC value. The common metabolism due to degeneration of respiratory 50 epithelium and the damage of the gill tissues histopathological changes caused by 14 aluminium were curling of lamellae, lead to tissue hypoxia. Gardner and Yovich , Mathivanan15,Jagadeesan16 and Karuppasamy desquamation of the lamellar epithelium, 17 hyperplasia and degeneration of the primary have also been observed the impairment of lamellae and secondary lamellae. Similar respiration and external functioning in the gills histopathological changes were also observed of estuarine teleost fish, Fundulus heterocliitus in C. punctatus exposed to methyl ethyl ,fresh water fish. Anabas testudineus when mercuric chloride 9. exposed to cadmium, Labeo rohita fingerlings In the present study, epithelial cells of the when exposed to mercury and Channa secondary gill lamellae of aluminium treated C. punctatus when exposed to PMA (Phenyl I catla depicted an initial hypertrophy and Mercuric Acetate) respectively. The survival of vacuolization. These appear to be the general C. catla at 10% of 96hrs LC50 value of sublethal concentration of aluminium chloride

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after 28 days might be due to the lesser when C. punctatus was exposed to methoxy 21 damage whereas in 20% of 96h LC50 value ethyl mercuric chloride. Rajamanickam sublethal concentration the intensity of the exposed M. vittatus to copper and observed metal effect is more pronounced and rapid. necrosis of hepatocytes, loss of adhesion leading to the mortality of fish. between cells, enlargement of nucleus. The liver is an important vital organ through Vacuolization, karyolysis and swelling of liver which most of the important metabolic cord. Karuppasamy 17 observed severe hepatic functions are occurring and the entry of lesions of ruptured outer membrane, necrosis, toxicants primarily affects the liver. Toxic pycnosis, vacuolat ion, damaged blood cells induced changes in the liver of the fishes can and accumulation of cytoplasmic granules in C. be regarded as an index for the identification of punctatus when exposed to PMA. Shastry and pollution present in the environment 18. During Sharma 23 exposed C.punctatus to sublethal the present study marked histopathological concentration (0.01mg/l) of endrin were lesions were observed in the liver of C. catla observed hypertrophy of hepatic cells and liver exposed to sublethal concentrations of cord disarray, vacuolation of cytoplasm and aluminium chloride. At sublethal concentration necrosis, rupture of hepatic cell membrane and histopathological lesions were mild and necrotic centrolobular area. observed as degeneration of hepatocytes, cell wall rupture, necrosis, picnosis and CONCLUSIONS cytopalsmolysis. parenchymal cells leading to appear smaller in size, cytoplasm become The histopathological changes observed in granulated and vacuolated, damaged and different fishes exposed to various empty blood vessels, and swelling of liver cord. concentrations of metals and other toxicants These severe effects in sublethal concentration have caused deleterious effects. Since liver is may be due to the possibility of more one of the major sites of metabolic activities, accumulation of aluminium in liver. Similar the toxic effects of heavy metals including histopathological changes were observed in aluminium are more pronounced and cause the the liver of fishes exposed to different metal malfunction resulting in the altered metabolism toxicants 19,20,21&17 noticed disintegrated hepatic in fish. Although it is too early to conclude all cells and nuclei in the liver of mercury treated of the unknown effects of aluminium, our C. carpio. Akkilender Naidu 22 showed granular findings strongly suggest that aluminium is very degeneration and vacuolization of hepatic toxic for Catla catla. Surely more information is cells, liver cord disarray, damage of blood needed to decide either aluminium affects the vessels leaving large empty space and gills and a liver directly or indirectly. By this enlargement of nuclei when S. mossambicus context, the aluminium has to be taken into was exposed to mercury. Sastry and Rao 20 more consideration as an environmental observed vacuolization of hepatocytes, contaminant. Further details should be necrosis, rupture of cell membrane and obtained from advanced investigations. enlargement of intercellular spaces in the liver

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Biochemical Changes in Haemolymph of Fresh Water Crab, Paratelphusa jacquemontii (Rathbun) Exposed to Copper

*A.Maharajan, K.Shanmugavel, P.S.Parurukmani

PG & Research Department of Zoology, Khadir Mohideen College, Adirampattinam-614701, Thanjavur Dist, Tamil Nadu, India E.mail: [email protected]

Abstract

Paratelphusa jacquemontii, a fresh water crab are abundantly found in the Orathanadu region, Thanjavur district of Tamil Nadu State, mainly in and around paddy fields. They were brought to the laboratory from their natural habitat, and first subjected to acclimatization to the laboratory conditions. They were subsequently th th exposed to sublethal concentrations of 1/10 &1/20 of LC50 value for 96hrs copper and the haemolymph total protein, free sugar and lipid level of the animal were estimated to study the stress caused by this heavy metal toxicant. In the present study, total protein, free sugar and lipid level of the animal showed a gradual declining trend from 0 to 15 days in the experimental animals exposed to copper. Keywords: Copper, Haemolymph, Biochemistry, Paratelphusa jacquemontii.

1 Introduction

Environmental poisoning of water by heavy metals has increased in recent years due to extensive use of heavy metals in agriculture, and chemical and industrial processes, posing a serious threat to living organisms. Among the heavy metals, copper, chromium, and iron being major pollutants from industrial effluents and agricultural waste in aquatic environment, cause maximum effects on non-target aquatic organisms resulting in imbalance of the ecosystem. The effect of heavy metals on aquatic organisms is currently attracting widespread attention, particularly in studies related to industrial pollution. Heavy metals are introduced

422 A.Maharajan, K.Shanmugavel, P.S.Parurukmani into aqueous environment through industrial and urban effluents, soil leaching, and rainfall. Fishes occupy high tropic level in accumulating various xenobiotics in the aquatic environment. Several investigations have already shown that metals accumulate at higher concentrations in freshwater and marine food chain. The waste water from textile, dyeing, and printing industries, tannery etc. are highly coloured and can cause an imbalance in the ecological system. Today heavy metals in aquatic ecosystems often show levels above the accepted levels. Extensive use of various chemical contaminants is known to adversely affect the growth of various organisms. The toxicity of such chemical constituents in invertebrates is mainly reflected on the central nervous system. It is further known to influence other physiological processes, including respiration. Respiration is obviously the most vital of all functions, and serves as an index of all biochemical changes that occur due to the effect of toxicants on the overall metabolism of exposed animals. Any change in the oxygen consumption due to pollution stress creates a physiological imbalance in the organisms. In biological systems, the biocatalysts play a vital role in the metabolic pathways. Animal exposed to stress conditions alter their physiological status with the help of enzymes. Toxicants like pesticides are known to secrete hyper or hypo level of enzymes. The trace metal concentrations in Queensland Estuarine crabs, Australoplax tridentate have been observed [1]. When any aquatic animal is exposed to polluted medium, a sudden stress is developed for which the animals should meet more energy demand to overcome the toxic stress. Verma et al.[2] reported on the toxic effects of sublethal concentration of copper sulphate, on certain biologically important enzymes in Saccobranchus fossilis. The neurosecretory structure in the eyestalk is the most important component of the neuroendocrine system of stalk-eyed crustaceans, and is mainly controlled by the cHH synthesized within X-organ [3], and released from the sinus gland (SG) complex in the eyestalk [4]. The effect of heavy metals on blood glucose levels in Palaemon elegans showed that the intermediate sublethal concentrations of Hg, Cd, Cu, and Pb produce significant hyperglycaemic responses, while high concentrations elicit no hyperglycaemia in 24 hours. In contrast, animals exposed to Cu and Zn showed hyperglycaemia even at high concentrations. These differences in response could be explained on the basis of the physiological roles these two essential elements play. In crustaceans, Cu acts as neuroransmitter acting on Chh neuroendrocrine cells, and consequent tolerance adaptations, as opposed to the toxic xenobiotic heavy metals Cd, Hg, and Pb Though there are a number of heavy metals such as copper, zinc, arsenic, cadmium, and mercury, in the present work copper alone has been studied as a toxicant. Copper may act as a toxicant in the form of sulfate or phosphate, or any other form. Actually, copper is one of the most important elements used in melanin pigments, protein synthesis, and blood protein synthesis. It is also helpful in the formation of blood, and many oxidative enzymes. Copper is also helpful in the formation of oxidative enzymes. Copper is used in many industries such as paint,steel, ceramic, fertilizers, etc., and mixes with water as effluent. It causes disturbances in various metabolic processes

423 such as bone formation, reproduction, respiration survey of literature reveals that considerable work has been done on the effect of copper as a toxicant in marine animals, but comparatively little attention has been focused on their fresh water counterparts. Hence, the present investigation was conducted on the fresh water crab, Paratelphusa jacquemontii after exposure to copper in various concentrations.

2 Materials and Methods

2.1. Test animal collection and maintenance

Fresh water crab, Paratelphusa jacquemontii of carapace size ranging from 4- 5cm and weight 50-70g were collected from the paddy field of Orathanadu, Thanjavur Dist, Tamil Nadu. They were transported and kept for acclimatization in rectangular tank of 100 l ca C) for a period of one week. Before stocking, the tank was washed with clean water several times. Finally, the tank was washed with 0.1% KmNO4 for disinfection. Before introducing into the tank, the fishes were screened for any visible pathological symptoms and were treated with 0.1% of KmNO4.

2.2. Preparation of stock solution for copper toxicity test

3.983gm of Copper sulphate was dissolved in one litre of double distilled water and used as the stock solution for preparing different concentrations of copper in rearing water. It was stored in a clean standard flask at room temperature, in the laboratory.

2.3. Exploratory test

Exploratory tests, otherwise called range finding test, were carried out to assess the approximate effective concentration range of copper required for conducting short term tests to assess the effect of copper on the metabolic function of the crab, as recommended by APHA [5]. The test solutions were prepared over a wide range of concentrations. These tests were performed by exposing 10 specimens of Fresh water crab, Paratelphusa jacquemontii in 10 litre fresh water containing different concentrations of copper. The dead animals were removed immediately. Death of each animals was recorded. Three replicates were made for short term toxicity tests, the least concentration was chosen where no mortality was recorded in 24hrs and the highest lethal concentration was where 100% mortality was recorded in 24hrs.

424 A.Maharajan, K.Shanmugavel, P.S.Parurukmani

2.4. Acute Toxicity test

To study the toxicity of copper, the Static Bioassay Method was followed [5]. The test individuals were exposed to selected and serially diluted copper concentrations. For acute toxicity test, 10 active animals each were exposed to various concentrations of the copper (10,20,30,40,50,60,70,80,90 and 100 µg/l) using fresh water as control. The manifestation time and survival time of crab were observed. Crabs were exposed to the above said concentrations along with common control. Experimental animals were starved for one week. The experiments were conducted in three replicates at room temperature. No feed was given during the test period.

2.5. Sub lethal toxicity tests

For sublethal toxicity tests, the crabs were grouped into three batches. Each batch had 10 animals and had 3 replicates. Group I : Crabs were maintained in normal Fresh water and served as control. Group II : Crabs were exposed to the sublethal concentration of 6.75 µg/l (1/10th of LC50 value for 96 hours) of copper in Fresh water. Group III: Crabs were exposed to the sublethal concentration of 13.5 µg/l (1/20th of LC50 value for 96 hours) of copper in Fresh water. The media were renewed every alternate day. Crabs were fed daily with artificial feed. Two specimens each from the groups I, II and III were sacrificed after 0, 5th 10th and 15th days of the experiment.

2.6. Tissue samples and biochemical analysis

Sampling was done on 0, 5, 10 and 15 DoE and on each sampling occasion, 10 crabs from each group (two experimental and one control) were sacrificed. From each crab, The haemolymph was extracted from the thigh region of the crab with the help of a syringe, and the biochemical constituents (proteins, carbohydrates and lipids) were estimated by following standard procedures. The total protein (TP) and the total carbohydrate (TC) concentrations in haemolymph were determined according to the methods of Lowry et al. [6] and Roe [7]. The total lipid (TL) content was estimated by the method of Barnes and Blackstock [8].

3 Results

3.1. Acute Toxicity Test

Acute toxicity study was done to find out the impact of copper on Paratelphusa jacquemontii within a short period. In the present study the 96hrs LC50 value was found to be 67.5µg/l. Among the test concentrations prepared from the

425 preliminary toxicity test the mortality of 50% of the population after 96hrs exposure was observed 67.5µg/l on concentration of copper.

3.2. Changes of Total Protein

In the haemolymph of untreated crab, the total protein was between 60.22 +0.34 to 61.99+0.58. Exposure to copper reduced the total protein content in haemolymph which was evident after 10 days in lower concentration (6.75µg/l) and after 5 days in higher concentration (13.4µg/l) being 57.82+0.44 and 56.76 +0.37 respectively. The percentage of total protein reduced gradually in relation to exposure duration. After 15 days of exposure it was 54.97+0.54 at (6.75µg/l) and 53.51+0.61 at (13.4µg/l) concentrations of copper (Fig.1).

3.3. Changes of Total Free Sugar

In the haemolymph of untreated crab, the total free sugar was between 11.49 +0.24 to 12.03+0.27. Exposure to copper reduced the total free sugar content in haemolymph which was evident after 10 days in lower concentration (6.75µg/l) and after 5 days in higher concentration (13.4µg/l) being 9.93+0.30 and 9.07+0.46 respectively. The percentage of total free sugar reduced gradually in relation to exposure duration. After 15 days of exposure it was 7.56+0.41 at (6.75µg/l) and 7.02+0.05 at (13.4µg/l) concentrations of copper (Fig.2).

3.4. Changes of Total lipid

In the haemolymph of untreated crab, the total lipid was between 51.19 +0.95 to 52.31+0.60 . Exposure to copper reduced the total lipid content in haemolymph which was evident after 10 days in lower concentration (6.75µg/l) and after 5 days in higher concentration (13.4µg/l) being 44.71+0.55 and 43.25+0.58 respectively. The percentage of total lipid reduced gradually in relation to exposure duration. After 15 days of exposure it was 41.37+1.15 at (6.75µg/l) and 37.94+0.72 at (13.4µg/l) concentrations of copper ( Fig.3).

4 Discussion

In recent years, there is a growing concern worldwide on the environmental pollution due to indiscriminate use of pesticides due to their persistence, toxicity at low concentrations and bioaccumulation by biota. Biochemical changes induced by heavy metal stress is due to disturbed metabolism manifested by inhibition of enzymes, retardation of growth and reduction in the fecundity and longevity of the organism. Most of the heavy metals act as metabolic depressors and generally affect the activity of biologically active molecules such as proteins, carbohydrates and lipids [9]. The exposure of aquatic organisms to even very low

426 A.Maharajan, K.Shanmugavel, P.S.Parurukmani levels of pesticides causing alterations in the nutritional value of finfish and shellfish as well as their biochemical constituents, physiological and histological functions has been widely documented [10,11,12]. Oxygen consumption is one of the most important physiological phenomenona, which controls all metabolic activities. It is the most important indicator of metabolic rate and status of the stress condition of exposed animals [13]. Since cellular and sub-cellular functions form the basis of all disorders, the toxic effects of xenobiotics mainly influence the cellular responses. The injury caused to cells by foreign compounds may be direct or indirect. Direct cell injury occurs when a toxicant interacts with one or more cell components. In indirect cell injury, the effect is due to a disturbance in the microenvironment of the cell. For example, when tissues have insufficient supply of oxygen during hypoxia or anoxia, the energy metabolism is disturbed leading to damage to the cellular metabolism. The increased glycolytic activity during oxygen deficiency cannot meet the energy requirements of the cell. As a result, the energy requiring processes such as protein synthesis, and glycolytic activity during oxygen deficiency cannot meet the energy requirements of the cell. As a result, the energy requiring processes such as protein synthesis, phospholipids metabolism, and membrane transport processes are inhibited. Many investigators have demonstrated harmful effects of heavy metals on histological structure of gills of crustaceans [13,14]. Similarly, when the crabs were exposed to sublethal concentration of the same toxicant for 0, 5, 10 and 15 days showed an elevation in the haemolymph sugar level with a maximum decrease at 15 days. Physiological processes are mostly coordinated by hormones, and changes in hormone levels are expected to occur soon after exposure to environmental stress, such as pollutants, eventually acting as endocrine disruptors [13]. Hyperglycaemia is a common stress response of many aquatic animals. In crustaceans it occurs following the involvement of the hyperglycaemic hormone (cHH) produced in the eyestalk; cHH mainly regulates glucose homeostastis. It belongs to the neuropeptide family synthesized in the eyestalk by medulla terminalis x-organ, and is accumulated by and released from the sinus gland. We are in agreement with the views of earlier researchers that an elevation in the haemolymph sugar level of the fresh water crab, Paratelphusa jacqemontii can occur due to copper, which may act on the neurotransmitter acting on cHH, a haemolymph sugar level regulating hormone [15]. References

[1] Mortimer, M.R. Pesticide and trace metal concentrations in Queensland Estuarine crabs. Marine Pollut. Bull, (2000), 41(7-12), 359-366. [2] Verma, S.R., Tonk, I.P., Gupta, A.K. and Dakia, R.C. In vivo enzymatic alterations incertain tissues of Saccobranchus fossilis following an exposure to four toxic substances. Environ. Pollut, (1981), 26, 121-127. [3] Abramowitz, A., Hisaw, F.L. and Papandrea, D.N. The occurrence of a diabetogenic factor in the eye stalk of crustaceans. Biol. Bull, (1994), 6: 1-5.

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[4] Fingerman, N. The endocrine mechanism of crustaceans. J. Crust. Biol, (1987), 7, 1-24. [5] APHA, 1. Standard methods for the examination of water and waste water APHA, AWWA and WPCF, New York. (1985). [6] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the Folin- phenol reagent. J. Biol. Chem,(1951), 193, 275- 275. [7] Roe, J.H. The determination of sugar in blood and spinal fluid with anthrone reagent. J. Biol. Chem, (1955), 20, 335-343. [8] Branes, H. and Blackstock, J. Estimation of lipids in marine animals and tissues. Detailed investigation of the sulphosvanillin method for total lipids. J. Exp. Mar. Biol. Ecol, (1973), 12, 103-118. [9] Agrahari, S. and Gopal, K. Fluctuations of certain biochemical constituents and markers enzymes as a consequence of monocrotophos toxicity in the edible freshwater fish, Channa punctatus. Pest. Biochem. Physiol, ( 2009) 94, 5-9. [10] Bhavan, P.S. and Geraldine, P. Alterations in concentrations of protein, carbohydrate, glycogen, free sugar, and lipid in the prawn, Macrobrachium malcolmsonii on exposure to sublethal concentrations of endosulfan. Pestic. Biochem. Physiol, (1997), 58, 89–101. [11] Bhavan, P.S. and Geraldine, P. Biochemical stress responses in the tissues of the prawn, Macrobrachium malcolmsonii on exposure to endosulfan, Pestic. Biochem. Physiol, (2001),70, 27–41. [12] Bhavan, P.S. and Geraldine, P. Carbaryl-induced alterations in biochemical metabolism of the prawn, Macrobrachium malcolmsonii. J. Environ. Biol, (2002), 23, 157–162. [13] Newell, R.C. Factors affecting the respiration of intertidal invertebrates. Am. Zool, (1973), 13, 513-528. [14] Ramanna Rao. and Ramamurthy, M.V. Histopathological effect of sublethal mercury on the gills of fresh water field crab, Oziotelphus senex. Ind. J. Comp. Anim. Physiol, (1996), 4, 33-38. [15] Varghese, G., Naik, P.S and Katdare, M. Respiratory response and blood sugar level of the crab, Barytelphusa cunicularis exposed to mercury, copper, and zinc. Indian J. Exp. Biol, (1992), 30, 308-312.

428 A.Maharajan, K.Shanmugavel, P.S.Parurukmani

Research & Reviews: A Journal of Pharmaceutical Science Volume 4, Issue 1, ISSN: 2229-7006 ______

Effect of Nitrite on Oxygen Consumption and Ammonia Excretion of the Fresh Water Crab Paratelphusa jacquemontii (Rathbun)

A. Maharajan*, B. S. Vijaykumar, Y. Narayanasamy, P. S. Paruruckmani, R. Archana PG & Research Department of Zoology, Khadir Mohideen College, Adirampattinam-614701, Thanjavur Dist, Tamil Nadu, India

Abstract In aquatic ecosystem, nitrite concentrations are elevated by pollution with nitrogenous wastes and imbalances in bacterial nitrification and denitrification processes. Nitrite is transformed by nitrification to nitrate under aerobic condition. The effects of nitrite on physiology were investigated in the fresh water crab, Paratelphusa jacquemontii (carapace length 4–5 cm and weight 50–70 g) for 96 h. The crabs were exposed to sublethal concentration (1/10th and 1/20th of 96 h LC50) of nitrite for a period of 15 days. The treatments of these nitrites brought about significant decrease in oxygen consumption and ammonia excretion as compared to control. The significance of these studies as bioindicator for assessing the toxicity and economic importance of the crab are discussed.

Keywords: nitrite, oxygen consumption, ammonia excretion, paratelphusa jacquemontii

*Author for Correspondence Email: [email protected], Tel: + 91-9443286900

INTRODUCTION absorbed at the gills and oxidizes the iron in Nitrite is an intermediate product in the the hemoglobin molecule to met hemoglobin. bacterial oxidation of ammonia to nitrite in The result of nitrite poisoning is hypoxia conditioned aquaculture systems. This caused by a reduction in the blood’s oxygen- nitrogen compound is highly toxic to aquatic carrying ability. It is likely that the same type organisms, and poses a potential threat to of reaction that occurs with the iron of cultured fish and crustaceans [1]. The lethel hemoglobin also occurs with the copper in and sub-lethel effects of nitrite to juvenile and crustacean hemocyanin [4]. adult penaeid prawns and Macrobrachium rosenbergii have been reported by Wickins Nitrite is a natural component of the nitrogen [2]. Nitrite is usually found in water. It usually cycle in ecosystems, and its presence in the occurs in small concentration considering its environment is a potential problem due to its chemical and biochemical instability. In well documented toxicity to animals [5]. aquatic ecosystem, nitrite concentrations are Nitrite is a naturally occurring anion in fresh elevated by pollution with nitrogenous wastes and saline waters. Concentration of nitrite can and imbalances in bacterial nitrification and be found in water receiving nitrogenous denitrification processes. Nitrite is transformed effluents, in various hypoxic environments or by nitrification to nitrate under aerobic in effluents from industries producing metal, condition. The average concentration of N- dyes, and celluloid [3]. The increased nitrite NO2− in ground water ranges from 0.004 to concentrations in water are also one of the 0.179 mg/L. Higher concentrations (˃ 1 mg/L) frequent problems encountered both in aquaria occur in wastewater [3] and could increase and on fish farms. Sources of nitrites are also in intensive aquaculture and recirculation mainly products of fish metabolism and systems usually in starting filters. The decaying remains of food and faeces. In nitrification process is used to decrease aquaria, fish are most frequently poisoned by ammonia concentration that is the main nitrites that accumulate in the tank as a result product of fish metabolism. In fish, nitrite is of insufficient efficiency of biological filters.

An elevated ambient nitrite concentration is a increased their haemolymph urea with a potential problem for freshwater fish, since concomitant decrease in haemolymph nitrite is actively taken up across the gills in ammonia and increases in ammonia-N competition with chloride [5]. excretion, urea excretion and organic-N excretion indicating that both ammoniogenesis Depletion in oxygen content occurs in the and ureogenesis take place [9]. medium when pesticides, chemicals, sewage and other effluents containing organic matter MATERIALS AND METHODS are discharged into water bodies. Pesticides in Test Animal Collection and Maintenance sub-lethal concentrations present in the aquatic Fresh water crab, Paratelphusa jacquemontii environment are too low to cause rapid death of carapace size ranging from 4 to 5 cm and directly but may affect functioning of the weight 50–70 g were collected from the paddy organisms, disrupt normal behavior and reduce field of Orathanadu, Thanjavur Dist, Tamil the fitness of natural population. In the aquatic Nadu. They were transported and kept for environment, one of the most important acclimatization in a rectangular tank of 100 L manifestations of the toxic action of chemical capacity containing well aerated filtered fresh is the over stimulation or depression of water maintained at ambient temperature respiratory activity. The changes in the (27 ± 2 °C) for a period of one week. Before respiratory activity of fish have been used by stocking, the tank was washed with clean several investigators as indicators of response water several times. Finally, the tank was to environmental stress. Alteration of oxygen washed with 0.1% KmNO4 for disinfection. consumption of whole organism or isolated Before introducing into the tank, the crabs tissues by different pollutants has been were screened for any visible pathological employed to measure the condition of an symptoms and were treated with 0.1% of organism [6]. KmNO4.

As a consequence, the hypoxic conditions Preparation of Stock Solution for Nitrite caused by hemoglobin anemia could damage Toxicity Test various organs such as liver or retina to a 3.045 gm of sodium nitrite was dissolved in different extent depending on their one liter of double-distilled water and used as characteristic of anatomy and blood flow the stock solution for preparing different conditions. Tissue hypoxia and anaerobic concentrations of nitrite in rearing water. It energy production can be reflected in elevated was stored in a clean standard flask at room plasma lactate concentrations [7]. Moreover, temperature in the laboratory. nitrite also induces lysosomal and liver mitochondria alterations in fish resulting in a Exploratory Test remarkable increase in the labiality of these Exploratory tests, otherwise called range organelles. Nitrite concentrations are finding tests, were carried out to assess the comparable in the liver and in the brain after approximate effective concentration range of nitrite intoxication. However, the high nitrite required for conducting short-term tests methaemoglobin values neither cause a to assess the effect of nitrite on the metabolic cerebral hypoxic state nor alter the function of the crab, as recommended by mitochondrial function in this organ; thus, APHA [10]. The test solutions were prepared anaemic hypoxia damages the brain less than over a wide range of concentrations. These the liver [7]. tests were performed by exposing 10 specimens of fresh water crab, P. jacquemontii Nitrite may have some general effects on in 10 L fresh water containing different nitrogen metabolism and excretion whose concentrations of nitrite. The dead animals manifestation probably depends on the degree were removed immediately. Death of each of nitrite intoxication [5]. In rainbow trout, animal was recorded. Three replicates were small elevations in ammonia excretion across made for short-term toxicity tests, the least the gills and via urine were observed [8]. concentration was chosen where no mortality Changes in nitrogenous excretion were also was recorded in 24 h and the highest lethal observed in marine decapod crustaceans. concentration was where 100% mortality was Under the stress of nitrite, Kumura shrimp recorded in 24 h.

Acute Toxicity Test to be 45 mg/L. Among the test concentrations To study the toxicity of nitrite, the static prepared from the preliminary toxicity test, the bioassay method [10] was followed. The test mortality of 50% of the population after 96 h individuals were exposed to selected and exposure was observed on 45 mg/L serially diluted nitrite concentrations. For concentration of nitrite. acute toxicity test, 10 active animals each were exposed to various concentrations of the nitrite Rate of Oxygen Consumption (30, 40, 50, 60, 70, 80 and 90 mg/L) using Crabs quickly respond to the pollutants by fresh water as control. The manifestation time altering their metabolic rate. The rate of and survival time of crab were observed. metabolism which is directly proportional to Crabs were exposed to the above said the oxygen consumptions of the crab exposed concentrations along with common control. to the sublethal concentrations of nitrite Experimental animals were starved for one showed significant variations. After 5th, 10th week. The experiments were conducted in and 15th days of toxicants, there is a sudden three replicates at room temperature. No feed slash down in oxygen consumption decreased was given during the test period. with increase in concentrations of nitrite (Figure 1). Sublethal Toxicity Tests For sublethal toxicity tests, the crabs were DISCUSSION grouped into three batches. Each batch had 10 Nitrite is one of the most important pollutants animals and had three replicates. in aquaculture systems because of their toxicity to aquatic organisms. Wajsbrot et al. Group I [13] suggested that the high buffering capacity Crabs were maintained in normal Fresh water of seawater should lead to a higher portion of and served as control. nitrite, making this potentially dangerous to aquaculture organisms. Aquatic organisms Group II quickly respond to the pollutants by altering Crabs were exposed to the sublethal their metabolic rate. The rate of metabolism is concentration of 4.5 mg/L (1/10th of LC50 directly proportional to the oxygen value for 96 h) of nitrite in fresh water. consumption. In the present investigation, the rate of oxygen consumption of the fresh water Group III crab, P. jacquemontii exposed to the sublethal Crabs were exposed to the sublethal concentration of nitrite showed significant concentration of 9 mg/L (1/20th of LC50 value variations. After the 10th day of exposure, a for 96 h) of nitrite in fresh water. sudden slash down in O2 consumption was The media were renewed every alternate day. noticed The rate of O2 consumption decreased Crabs were fed daily with artificial feed. Two with increase in concentration of nitrite. On specimens each from the groups I, II and III the 15th day, an adaptive tendency was were sacrificed after 0, 5th, 10th and 15th days noticed. These findings are favored by Ref. of the experiment. After their respective [14]. They suggested that the decline in O2 exposure period, oxygen content and ammonia consumption could be due to the contact of the excretion was estimated by Winkler’s method toxicant with the secondary lamella of gill [11] and Strick and Parson method [12]. which leads to damage of the respiratory epithelium. The adaptive tendency during later RESULTS days of exposure to nitrite might have been Acute Toxicity Test due to lowering down of energy requirements. Acute toxicity study was done to find out the The lowering of O2 consumption could be impact of nitrite on fresh water crab, P. attributed towards the effect on RBCs. jacquemontii within a short period. In the present study, the 96 h LC50 value was found

Fig. 1: Effect of Nitrite on the Rate of Oxygen Consumption in Fresh Water Crab, Paratelphusa jacquemontii

1.5 Control 1 4.5mg/l (mg/lit) 9mg/l

consumption consumption 0.5 Rate of oxygen ofoxygen Rate 0 0 5 10 15

Exposure duration in Days

Ammonia Excretion excretion was decreased gradually in different Aquatic organisms quickly respond to the concentrations of nitrite in different durations pollutants by altering their metabolic rate. In (Figure 2). the present study, the rate of ammonia

Fig. 2. Effect of Nitrite on the Rate of Ammonia Excretion in Fresh Water Crab, Paratelphusa jacquemontii

6 5 Control 4 3 4.5mg/l 2 9mg/l 1

Rate of ammonia excretion 0 0 5 10 15 Exposure duration in days

The increased oxygen consumption in Labeo were also reported by Mushgeri and David rohita and Catla catla under sublethal [14]. concentrations of both the toxicants is in collaboration with the increased consumption The decrease in oxygen consumption at of oxygen in trout exposed to permethrin [15]. sublethal concentrations of the toxicant Bradbury et al. [16] stated that the greater indicates lowered energy requirements which decrease in the rate of oxygen consumption in in turn indicates pronounced haematological the fish, Cirrhinus mrigala may be due to changes [17]. Similar reduction in oxygen inter-subcellular level. Similar observations consumption has been reported in Channa

RRJoPS (2013) 20-24 © STM Journals 2013. All Rights Reserved Page 23 Research & Reviews: A Journal of Pharmaceutical Science Volume 4, Issue 1, ISSN: 2229-7006 ______striatus exposed to organophosphate pesticide 5. Jensen F. B. Comparative Biochemistry [18], O. mossambicus due to organochlorine and Biophysics. Part A 2003; 135: 9–24p. intoxication [19]. 6. Kumarasmy P, Karthikeyan A. Journal of Environmental Biology 1999; 20(2):99– In the present study, there is remarkable 102p. elevation in ammonia excretion of the fishes 7. Jensen F. B. Nitrogen Metabolism and exposed to sublethal toxicity of nitrite. The Excretion. CRC Press, Boca Raton: 1995; rate of ammonia excretion increased with the 289–303p. toxicity of nitrite. It is evident that the nitrite 8. Zachariasen F. Ph.D thesis. Institute of toxicity has an effect on nitrogen metabolism. Biology, University of Southern Denmark, Zachariasen [8] observed elevation in Odense: 2001; 54–85p. ammonia excretion of rainbow trout when they 9. Cheng S.Y, Chen J. C. Aquatic Toxicology were exposed to nitrite. Further studies are 2001; 51: 443–454p. needed to better understand the interaction 10. APHA. Standard Methods for the between nitrite and the nitrogen metabolism. Examination of Water and Waste Water The 24-h LC50 calculated in the acute toxicity New York: 1985. experiments showed that among the three 11. Golterman H, Clymo C. Methods for the larval stages, nauplius is the most sensitive Chemical Analysis of Fresh Water. stage to nitrite poisoning. Nitrite tolerance Blackwell: 1969. increases progressively with the later stages, 12. Strickland J. D. H, Parson T. R. A the protozoea and mysis being more resistant Practical Handbook of Seawater Analysis. to this toxin. Bull. Fish. Res. Bd. Canada: 1972; 167 and 310p. CONCLUSIONS 13. Wajsbrot N. et al. Journal of Fish Biology The present study concludes that fresh water 1993; 42: 321–328p. crab, P. jacquemontii appeared to be less 14. Mushigeri S. B, David M. Journal of sensitive to nitrite concentration than other Ecotoxicology and Environmental crustaceans. More information is needed to Monitoring. 2003; 13: 191–195p. database the environmental quality standards 15. Haya K. Environmental Toxicology and and management of aquaculture system. Chemistry. 1989; 8:381–391p. 16. Bradbury S. P. et al. Bulletin of REFERENCES Environmental Contamination and 1. Mevel G, Chamroux S. Aquaculture 1981; Toxicology 1986; 38: 727–735p. 23:29–43p. 17. Tilak K. S, Satyavardhan K. Journal of 2. Wickins J. F. Aquaculture 1976; 9:19– Aquatic Biology 2002; 17:81–86p. 37p. 18. Natarajan G. M. Current Science 1981; 3. Pitter P. Hydrochemistry (in Czech) 1999; 50(22): 985–991p. 568 p. 19. Vasanthi M, Ramasamy M. Indian 4. Colt J, Armstrong D. A. Proceeding of the Academic Science. (Animal Science). Bio-Engineering Symposium for Fish 1987; 96: 56–69p. Culture 1981; 34–47p.

ISSN Print/Online: 2320-9577/2320-9585

INTERNATIONAL JOURNAL OF PURE AND APPLIED ZOOLOGY Volume 1, Issue 2, June 2013 Available online at: http:// www.ijpaz.com RISHAN PUBLICATIONS RESEARCH ARTICLE OPEN ACCESS

SUBLETHAL EFFECT OF PROFENOFOS ON OXYGEN CONSUMPTION AND GILL HISTOPATHOLOGY OF THE INDIAN MAJOR CARP, CATLA CATLA (HAMILTON)

A.MAHARAJAN*, R.USHA, P.S. PARU RUCKMANI, B.S.VIJAYKUMAR, V.GANAPIRIYA AND P.KUMARASAMY

PG and Research Department of Zoology, Khadir Mohideen College, Adirampattinam-614 701, Tamilnadu, India *Corresponding Author Email address: [email protected], Tel: +91 9443286900 Article History: Received: 04.05.2013, Accepted: 19.06.2013

ABSTRACT As aquatic organisms have their outer bodies and important organs such as gills almost entirely exposed to water, the effect of toxicants on the respiration is more pronounced. Pesticides enter into the fish mainly through gills and with the onset of symptoms of poisoning, Profenofos, a well-known organophosphate pesticide has been in agricultural use over the last two decades for controlling pests of paddy, cotton and tobacco. In the present study, an attempt has been made to study the effect of profenofos on oxygen consumption and gill histopathology of the Indian major carp, C. catla . The fishes were exposed to sublethal concentration (1/10 th and th 1/20 of 96 h LC 50 ) of profenofos for a period of 15 days. The treatment of these profenofos brought about significant decrease in oxygen consumption as compared to control. Exposure to profenofos was found to result in several alterations in the histo-architecture of the gills of C. catla. The alterations included curved secondary gill filaments, necrosis of gill filaments and conjestion of Secondary Lamellae. The significance of this study as a bio-indicator for assessing the toxicity and economic importance of the fish is discussed. Keywords: Profenofos, gills, oxygen consumption, histopathology, Catla catla.

INTRODUCTION consumption and is increasing at the rate of 2-5% per annum (Bhadbhade et al ., 2002). Usage of India is primarily an agro-based country with Organophosphorus (OP) pesticides is found to be more than 60-70% of its population dependents increasing in recent years since they are on agriculture. However, 30% of its agricultural biodegradable and therefore persist in the production is lost owing to pest infestation. In the environment only for a short time. Because of absence of a better alternative, deployment of their low persistence, repeated applications of pesticides becomes inevitable despite their these pesticides are being practiced for the known hazardous effects. Utilization of control of pests in agricultural fields and thereby pesticides in India is about 3% of the total world large quantities find their way into water bodies

196 Maharajan et al. Int. J. Pure Appl. Zool., 1(1): 196-204, 2013

(Jyothi and Narayan, 1999). A large numbers of of an animal is the important physiological pesticides for the control various agricultural parameters to assess the toxic stress, because it is pests; however, their toxicological impact also a valuable indicator of energy expenditure in extends to non target species like fish (Naqvi and particular and metabolism in general (Prosser Vaishnavi, 1993). Fish is a good indicator of and Brown, 1977). Pesticides are remarkable in aquatic contamination because its biochemical causing respiratory distress or even failure by stress responses are quite similar to those found affecting respiratory centers of the brain or the in mammals (Mishra and Shukla, 2003). tissues involved in breathing.

Profenofos, a well known organophos- As the aquatic organisms have their outer phate pesticide has been in agricultural use bodies and important organs such as gills almost over the last two decades for controlling pests entirely exposed to water, the effect of toxicants of paddy, cotton and tobacco. Profenofos has on the respiration is more pronounced. Pesticides been classified as a moderately hazardous enter into the fish mainly through gills and with (Toxicity class II) pesticide by the World the onset of symptoms of poisoning, the rate of Health Organization (WHO) and it has a oxygen consumption increases (Premdas and moderate order of acute toxicity following Anderson, 1963). Holden (1973) observed that oral and dermal administration. Profenofos is one of the earliest symptoms of serves not only as a tool in evaluating the susceptibility or extremely toxic to fishes. The acute toxic resistance potentiality of the animal, but also action of profenofos is the inhibition of the useful to correlate the behavior of the animal. acetylcholine esterase activity resulting in neuro toxicity to aquatic vertebrates and also By cannulating the blood system of fishes, it humans. Microbial degradation of is possible to measure the concentrations of organophosphate pesticides is of particular oxygen, metabolites and pollutants and hence interest because of the high mammalian understand more fully the mode of action of toxicity of such compounds and their widespread toxic pollutants. Skidmore (1970) using and extensive use. The most significant step cannulation techniques, found that zinc reduced in detoxifying organophosphate compounds is the oxygen level of blood leaving the gills. It hydrolysis since that makes the compounds reduced the efficiency of oxygen transport across more vulnerable to further degradation. The the gill membrane, so that fish die of hypoxia. enzyme responsible for catalyzing this Respiratory responses to lethal concentrations reaction is referred as an esterase or increase the ventilation volume and symptoms of phosphotriesterase . pyrethroid intoxication suggesting that the effect on respiratory surface is lethal in fish. It is Depletion in oxygen content occurs in the known that pyrethroids are less persistent and are medium when pesticides, chemicals, sewage and effective substituents for organ chlorine (OC) pesticides. Like OC compounds, the mechanism other effluents containing organic matter are of pyrethriod interference is with nerve discharged into water bodies. Pesticides in sub membrane function through the interaction with lethal concentrations present in the aquatic the sodium channels. The symptoms of environment are too low to cause rapid death profenofos intoxication suggest that, besides directly but may affect functioning of the effect on the nervous system, effect on organisms, disrupt normal behaviour and reduce respiratory surfaces and renal ion regulation may the fitness of natural population. In the aquatic be associated with the mechanism of lethality in environment one of the most important fish (Bradbury et al., 1987). manifestation of the toxic action of chemical is the over stimulation or depression of respiratory Total oxygen consumption is one of the activity. The changes in the respiratory activity indicators of the healthy status of a fish. It may of fish have been used by several investigators as also be useful to assess the physiological an indicator response to environmental stress. condition in an organism, helps in evaluating the The respiratory potential or oxygen consumption susceptibility or resistance potentiality and also

197 Maharajan et al. Int. J. Pure Appl. Zool., 1(1): 196-204, 2013 useful to correlate the behavior of the animal, wide range of concentrations. These tests were which ultimately serve as predictors of functional performed by exposing 10 specimens of C. catla s disruptions of population. Hence the analysis of in 10 litre fresh water containing different oxygen consumption can be used as a concentrations of Profenofos. The dead animals biodetectory system to evaluate the basic damage were removed immediately. Death of each animal inflicted on the animal which could either was recorded. Three replicates were made for short increase or decrease the oxygen uptake. term toxicity tests, the least concentration was Therefore an attempt was made to study the chosen where no mortality was recorded in 24hrs effect of sub lethal concentrations of profenofos and the highest lethal concentration was where on oxygen consumption and gill histopathology 100% mortality was recorded in 24hrs. of Indian major carp, Catla catla. The present investigation of toxicity of profenofos in C. catla Acute Toxicity test is carried out. Since, it is cultured in the ponds of Delta Districts of Tamil Nadu. The culturing To study the toxicity of Profenofos, the Static pond receives river runoff from paddy fields has Bioassay Method (APHA, 1985) was followed. the possibility of containing pesticides. The test individuals were exposed to selected and serially diluted Profenofos concentrations. For MATERIALS AND METHODS acute toxicity test, 10 active animals each were exposed to various concentrations of the Test Animal Collection and Maintenance Profenofos (0.04, 0.06, 0.08, 0.10, 0.12, 0.14 and Live fish, C. catla of size ranging from 10-12g 0.16 ppm) using fresh water as control. The were collected from R.K, Fresh water fish farm, manifestation time and survival time of fish were Orathanadu, Thanjavur Dist, Tamil Nadu. They observed. Fishes were exposed to the above said were transported and kept for acclimatization in concentrations along with common control. rectangular tank of 100 l capacity containing well Experimental animals were starved for 1 week. aerated filtered fresh water maintained at The experiments were conducted in three ambient temperature (27± 2 °C) for a period of one replicates at room temperature. No feed was given week. Before stocking, the tank was washed with during the test period. clean water several times. Finally, the tank was washed with 0.1% KMnO 4 for disinfection. Before Sublethal toxicity tests introducing into the tank, the fishes were screened for any visible pathological symptoms and were For sublethal toxicity tests, the fishes were treated with 0.1% of KMnO 4. grouped into three batches. Each batch had 10 Preparation of Stock Solution for Profenofos animals and had 3 replicates. Toxicity Test Group I : Fishes maintained in normal Fresh water One milliliter of profenofos was dissolved in and served as control. one liter of double-distilled water and used as the stock solution for preparing different Group II : Fishes exposed to the sublethal th concentrations of profenofos in rearing water. It concentration of 0.0008ppm (1/10 of LC 50 value was stored in a clean standard flask at room for 96 hours) of Profenofos in fresh water. temperature, in the laboratory. Group III : Fishes exposed to the sublethal Exploratory Test th concentration of 0.0016 ppm (1/20 of LC 50 value Exploratory tests, otherwise called range for 96 hours) of Profenofos in fresh water. finding test, were carried out to assess the approximate effective concentration range of The media were renewed every alternate day. Profenofos required for conducting short term tests Fishes were fed daily with artificial feed .Two to assess the effect of Profenofos on the metabolic specimens each from the groups I, II and III were function of the fish, as recommended by APHA sacrificed after 0, 5 th 10 th and 15 th days of the (1985). The test solutions were prepared over a experiment. After their respective exposure

198 Maharajan et al. Int. J. Pure Appl. Zool., 1(1): 196-204, 2013 period, oxygen content was estimated by Histopathology of Gills Winker’s method (Welch, 1952). The transverse section of gill tissue of normal Evaluation of Histopathology fish shows branching form the central axis called the primary gill lamellae. Each of the primary Fishes were exposed to profenofos at two lamella further divides into secondary gill lamellae sublethal concentrations for 15 days. Sampling or filaments. Within each division of the gills are was done on the 5 th and 15 th day of exposure; the adjacent efferent vessels and afferent vessels five fishes in each group were sacrificed. The with hemocytes. The primary and secondary gill gills of representative fish from each test and filaments are separated by a thin septum. The control group were dissected out and fixed in secondary non branching filament lamella Davidson’s fixative for 24 h. The preserved possesses epithelial pillar cells separated by large tissues were processed by a routine histological lacunae (Plate 1). method (Humason, 1972), dehydrated in alcohol series and embedded in paraffin wax. They were After 5 days of exposure to 0.0008 ppm cut into sections of 6 mm thickness by a rotary concentration of profenofos, the gill tissues microtome (Weswox, MT1090:1090A, India). revealed large inter-lamellar space, necrosis, The thin sections of the tissues were stained by lamellar fusions enlarged secondary gill lamellae, haematoxylin and eosin for observation by the curved secondary gill filaments resulting in Nikon Bright field transmission microscope with distension of the lamellae (Plate 2). Similarly, after Koechler illumination, and an automatic 15 days of exposure, further swelling of tip of exposure unit was used. secondary gill lamellae and their erosion were observed. Rupture of capillaries at the tip of RESULTS secondary gill lamellae releasing blood cells was also seen at some places. Hemorrhage between Acute Toxicity Test gill filaments, dilation in blood vessels of gill Acute toxicity study was done to find out the filaments and Telangiectasis in secondary lamellae impact of profenofos on C. catla within a short were observed (Plate 3). period. In the present study the 96hrs LC 50 value After 5 days of exposure to 0.0016 ppm was found to be 0.0079ppm. Among the test concentration of profenofos, a large number of concentrations prepared from the preliminary hemocytes accumulated in secondary gill lamellae toxicity test the mortality of 50% of the and resulted in enlargement of gill lamellae. population after 96hrs exposure was observed on Consequently the separation of epithelial cells 0.0079ppm concentration of profenofos. from the basement membrane and fusion of Rate of Oxygen Consumption secondary gill lamellae were also observed (Plate 4). Similarly, after 15 days of exposure, the gills Fishes are quickly responding to the also exhibited lamellar fusion at some places as a pollutants by altering their metabolic rate. The result of filamentary epithelium proliferation. In rate of metabolism is direly proportional to the addition, a few aneurisms were also observed in oxygen consump-tions of the fish exposed to the gill lamellae. In some areas, epithelial sub lethal concentrations of profenofos showed hyperplasia, swelling of epithelial cells and th th th significant variations. After 5 , 10 and 15 bronchial epithelium disorganization were days of toxicants there is a sudden slash down in evident. Furthermore, the epithelial layer detached oxygen consumptions decreased with increase in completely from the central portion of each concentrations of profenofos (Figure 1). lamella (Plate 5).

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1.4 1.2 1 0.8 Control 0.6 0.0008 ppm 0.4 (mg/l) 0.0016ppm 0.2 0 0 5 10 15

Rate Rate of Oxygen Consumption Exposure Duration in Days

Figure 1. Effect of profenofos on the rate of oxygen consumption in Indian major carp, Catla catla.

Plate 1. Photomicrograph showing the T.S of control gill in Catla catla (Abbreviations: PGL- Primary Gill lamellae, SGL- Secondary Gill lamellae).

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Plate 2 . Photomicrograph showing the T.S of control gill in Catla catla treated with 0.0008 ppm concentration of profenofos after 5 th days of exposure (Abbreviations: CSGL- Curved Secondary Gill Filaments).

Plate 3 . Photomicrograph showing the T.S of control gill in Catla catla treated with 0.0008 ppm concentration of profenofos after 15 th days of exposure (Abbreviations: NGF- Necrosis of Gill Filaments).

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Plate 4. Photomicrograph showing the T.S of control gill in Catla catla treated with 0.0016 ppm concentration of profenofos after 5 th days of exposure (Abbreviations: TEL – Telangiectasis, CSL – Conjestion of Secondary Lamellae).

Plate 5 . Photomicrograph showing the T.S of control gill in Catla catla treated with 0.0016 ppm concentration of profenofos after 15 th days of exposure (Abbreviations: CSGL – Conjestion of Secondary Gill Lamellae).

DISCUSSION pronounced Profenofos. The increased oxygen consumption in Labeo rohita and C. catla under During this study severe respiratory distress, sub lethal concentrations of both the toxicants is rapid opercular movements, leading to the higher in colloboration with the increased consumption amount of toxicant uptake, increased mucus of oxygen in trout exposed to permethrin (Haya, secretion, higher ventilation volume, and 1989). The decrease in oxygen consumption at decrease in the oxygen uptake efficiency, sub lethal concentrations of the toxicant indicates laboured breathing and engulfing of air through lowered energy requirements which in turn the mouth were observed in all the three major indicate pronounced haematological changes carps exposed to both the toxicants. However, (Tilak and Satyavardhan, 2002). the above said changes in the fish were more

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Histopathological changes observed were experimental data reveals that oxygen hemorrhage in the primary and secondary gill consumption decreases with the time of exposure lamellae, degeneration and necrosis of epithelial to toxicant (Tilak and Swarna kumari, 2009). cells, distortion of the secondary lamellae, and Under toxic conditions, the oxygen supply disruption of the secondary lamellar, disruption becomes deficient and a number of poisons of epithelial cells from pillar cells. Shorter gill become more toxic increasing the amount of lamellar, fusion, complete destruction of lamella, poison being exposed to the animal. The fish increased vacuolation, and irregular appearance breathe more rapidly and the amplitude of of gill lamellae were observed in C. catla respiratory movements will increase. Lloyd exposed to profenofos. (1961) reported that the toxicity of several Histopathological results indicated that gill poisons to rainbow trout increased in direct was the primary target tissue affected by proportion to decrease in oxygen concentration profenofos. Gills are generally considered good water. This reduces the rate of passage of blood indicator of water quality (Rankin et al., 1982) through the gills, so allowing a longer period of since the gills are the primary route for the entry time for uptake of oxygen, and also conserves of pesticide. In fish, gills are critical organs for oxygen by reducing muscular work. The zone of their respiratory, osmoregulatory and excretory resistance is reached when the oxygen tension in functions. Many investigators have reported the the water is so low that homeostatic mechanisms histopathological changes in gills of different of the fish are no longer able to maintain the fish species exposed to pesticides. Mucus oxygen tension in the afferent blood and the extrusion, lamellar swelling, fused and reduced standard metabolism begins to fall. microridges, were observed in blud gill sunfish, Conclusions Lepomis macrochirus to different sublethal concentrations of monocrotophos on the gills of Changes in the architecture of gill under Anabas testudineus was reported by profenofos stress would alter diffusing capacity Santhakumar et al . (2001). In the present study of gill with consequent hypoxic/anoxic accordant that uncontrolled regeneration of the conditions and thus respiration may become primary lamellae and secondary lamellae, problematic task for the fish. The results of the hypertrophy, hyperplasia, necrosis of the present study suggest that the altered rates of epithelial cells, epithelial lifting, dilation of the respiration of fresh water fish may serve as a blood sinuses of the secondary lamellae, lamellar rapid biological monitor of the pesticide aneurism, hemorrhages in the gill of fish exposed exposure to important components of fresh water to profenofos. community. Changes in the gill surfaces and increased CONFLICT OF INTEREST mucus production are consistent with observed The authors declare that there are no histological effects such as gyperplasia, necrosis and lamellar aneurysms in all the three fish conflicts of interest associated with this exposed to sub lethal concentration of article. profenofos. Kumaraguru et al., (1982) reported ACKNOWLEDGEMENTS that the gill is the target organ for synthetic pyrethroid toxicity in fish. The technical as well The authors are thankful to the Principal as commercial formulations will pass through the and the HOD of Zoology, Khadir Mohideen gills and interfere in the gill movements which College for the facilities provided to carry are directly proportional to the respiratory out this work and encouragements. activity of the fish, primarily affecting the REFERENCES oxygen uptake. The respiratory metabolism was impaired and damage was also observed in the APHA, 1985. Standard methods for the gill of fish exposed to pesticides (Ramamurthy, examination of water and waste water APHA, 1988). In the freshwater fish, AWWA and WPCF, New York. Ctenbopharyngodon diella exposed to Nuvan an organophosphate, the depletion of the oxygen Bhadhade, B.J., Sarnaik, S.S. and Kanekar, P.P. consumption is due to the disorganization of the 2002. Bioremediation of an industrial effluent respiratory action caused by rupture in the containing monocrotophos. Cur. Microbiol., respiratory epithelium of the gill tissue. The 45 : 346-349.

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Cite this article as : Maharajan, A., Usha, R., Paru Ruckmani, P.S., Vijaykumar, B.S., Ganapiriya, V. and Kumarasamy, P. 2013. Sublethal effect of profenofos on oxygen consumption and gill histopathology of the Indian major carp, Catla catla (Hamilton). Int. J. Pure Appl. Zool., 1(2): 196-204.

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