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1 Report International Journal of Integrative Biology A journal for biology beyond borders ISSN Ambient Copper induced alterations in hematology and biochemistry of the major carp, Labeo rohita (Hamilton) Meenakumari S, Showkat Ahmed Parrey, Saravanan T S * P. G. Department of Zoology, Jamal Mohamed College, Tiruchirappalli, India Submitted: 1 Jun. 2010; Revised: 3 Oct. 2010; Accepted: 6 Oct Abstract The major carp, Labeo rohita on chronic exposure to the heavy metal copper showed significant increase in blood parameters like RBC, WBC and Hb content. Further several biochemical alterations have been observed in blood, liver and muscle as a result of the metallic stress. Hyperglycemia and hypercholestemia were observed in blood and concomitantly significant decrease was observed in liver and muscle sugar and liver cholesterol. The metallic stress of copper resulted in notable decrease in protein content of liver and muscle with an increase in serum protein fractions. The enzymes, AST (Aspartate amino transferase) and ALT (Alanine amino transferase) levels increased in both serum and liver. Remarkable decrease in the protein profile was also observed in both liver and muscle as a result of the metallic stress. As a result of chronic exposure, the treated fishes would ultimately lose the nutritive value and become susceptible to disease. Keywords: Labeo rohita; hyperglycemia; hypercholestemia; aspartate amino transferase; alanine amino transferase. INTRODUCTION The civilization advancement has resulted in an increased technological use of heavy metal in industries. Heavy metals are considered as major environmental pollutant causing cytotoxic, mutagenic and carcinogenic effect in animals (More et al., 2003). Copper is one of the 26 essential elements occurring naturally in plant and animal tissues. For many years it has accepted that Copper is necessary and essential element for living organisms from bacteria to human. The essential metals also become toxic at higher concentration (Bryan, 1976). Heavy metal contamination may have devastating effects on the ecological balance of the recipient environment and a diversity of aquatic organisms (Farombi, et al., 2007; Vosyliene and Jankaite, 2006; Ashraj, 2005) Heavy metal Copper can be accumulated by fish either directly from the surrounding water or ingestion of food. Fishes are intimately associated with water and constitute an important food item in human diet. Adverse effect of Copper to some fresh water fishes like Lepidocephalichthys thermalis, rainbow trout have * Corresponding author: T.S. Saravanan, Ph.D. P.G. Department of Zoology, Jamal Mohamed College, Tiruchirappalli , Tamil Nadu, India drtssaravanan50@yahoo.com been well documented (Geetha et al., 1996; Harrison. 1975; Anderson. 1975; Stiff. 1971). Aquatic organisms have the ability to accumulate heavy metals from various sources including sediments, soil erosion and runoff, air depositions of dust and aerosol, and discharges of waste water (Labonne et al., 2001; Goodwin et al., 2003) Therefore, accumulation of heavy metals in aquatic organisms can pose a long lasting effect on biogeochemical cycling in the ecosphere. Heavy metals can also adversely affect the growth rate in major carps (Hayat et al., 2007). Fish are widely used to evaluate the health of aquatic ecosystems because pollutants build up in the food chain and are responsible for adverse effects and death in aquatic systems (Farkas et al., 2002; Yousuf and Shahawl, 1999). Fish and shell fish can accumulate metals to concentrations many times higher than present in water or sediment (Olaifa et al., 2004, Gumgum et al., 1994). They can take up metals concentrated at different levels in their different body organs (Khaled, 2004). Copper at sub lethal concentration stimulated locomotory activity in the brook trout, Salvelinus fontinalus (Christensen et al., 1977), respiratory and excretory activity in Sarotherodon and mossambicus (Saravanan et al., 2003). Copper concentration rarely exceeds 50 nmol -1 in natural water, but in polluted water, the level surpass 1µ mol -1 and makes it toxic (Hellawell, 1988). Sub lethal effects are biochemical in origin as the most toxicants exert their effects at basic levels of the International Journal of Integrative Biology IJIB, 2010, Vol. 10, No. 2, 119

2 organism by reacting with enzyme or metabolic and other functional components of the cell. Such effects might lead to irreversible and detrimental disturbance of integrated function such as behavior, growth, reproductive and survival (EIFAC, 1975). There are numerous reports on the short terms effects of Cu exposure on fish hematology (Dick and Dixon, 2000; Mazon, 2002). Most of the heavy metals ions are toxic or carcinogenic in nature and pose a threat to human health and the environment (Damien et al., 2004; Farombi et al., 2007). The major organ involved in the xenobiotic metabolism is liver (Saravanan et al., 2010). Heavy metals have shown a high degree of impact on metabolism by inhibiting enzymes like acetyl cholinesterase. The studies carried out on various tissues of fishes have shown that heavy metal may alter the physiological activities and biochemical parameters of both in liver and muscle tissues and in blood tissue and in blood (Larsson et al., 1985; Saravanan et al., 2003). The measurement of biochemical changes in blood and tissue of fish under exposure to the toxicant may be used to predict the toxic effects of toxicants. A careful perusal of the literature reveals that there is not much of work available on the chronic effects of Copper on freshwater fishes especially major carps. So the present study was undertaken to elucidate the effects of Copper sulphate at sub lethal level on the hematological and biochemical constituents of tissues like blood, liver and muscle of Lebeo rohita when exposed to a period of 30 days. MATERIALS AND METHODS The major carp, Labeo rohita was obtained in live condition from the fish farm and transported to the laboratory in plastic buckets filled with tap water. Only healthy young ones were used in this study. The length and weight of the fish ranged from 14±17cm and 45±100g respectively. The heavy metal selected for this study is Copper sulphate. To obtained a sub lethal concentration of 1mg L -1 of Copper sulphate, a stock solution was prepared by dissolving 3.929g of Copper sulphate, which would yield 1g of Copper in 100ml (10mg ml -1 ). When 4ml of the stock solution was mixed with 40litres of water, a solution containing 1mg/L of Copper Sulphate is obtained (Saravanan et al. 2003). In the laboratory the fishes were maintained in a large rectangular tank, measuring for two weeks acclimatized. During this period they were fed with pelleted (rice bran and ground nut oil cake in 1:1 concentration) feed on alternate days. This procedure has been adopted in accordance with U.S.EPA (1975) recommendation to keep optimum dissolved oxygen in static bioassays. The water was renewed daily an hour after providing the food. After acclimatization fishes were divided into two groups of 5 each and transferred to two rectangular plastic tanks filled with 40 liters of tap water. One of the tanks served as control and the other one as an experimental group. To the experiment tank 4ml of stock solution of Copper sulphate was added daily for 30 days. The temperature, ph, salinity and dissolved oxygen of the water were found to be 26 o C±1 o C, 7.45±0.2, 0.76±0.08%, and 7.10±0.13ml/L. At the end of 30 th day, groups of 5 fishes were collected individually from the control and experiment tanks by using small hand net and subjected to the analysis. Analysis of blood parameters and tissue constituents The blood sample was collected from each and every fish by cutting the fish at caudal peduncle. The following blood parameters have been analyzed individually from each and every fish. The blood parameters analyzed include total erythrocytes count (RBC), total leucocytes (WBC) and haemoglobin (Hb) content following the procedures of Davies (1975) and Bryan (1976). The blood total free sugars and cholesterol were determined following the procedures of Roe (1955) and Zarrow et al. (1964), respectively. The quantitative of protein estimation was done following the biuret method of Gornall et al. (1949). Aspartate Amino Transferase (AST) and Alanine Amino Transferase (ALT) enzymes were estimated following the procedure of Wooten (1964). The qualitative estimation of protein content of serum, liver and muscle was done by SDS PAGE under 6% staking gel and 10% separating gel (Laemmli, 1970). The mean value and standard deviation were calculated for the data. The students t test was applied for mean difference between the controls vs. experiment as described by Parker (1973). RESULTS The fish, Labeo rohita on chronic exposure to sub lethal concentration of Copper showed normal behavior through out the study period when compared to the control. The haematological parameters like RBC, WBC and haemoglobin of treated fish showed significant increase of 22%, 60% and 39% respectively from the control group. In that the mean level of RBC, WBC and Hb increased from 1.80±0.15 to 2.20±0.22 Lakhs /mm3, 5.78±0.54 to 9.28±1/71 thousands /mm3 and from 4.92±0.46 to 6.82±0.60 g% respectively (Table 1 [Supplementary data]). This table reveals the impact of Copper on organic constituents of blood, liver and muscles and enzymes of serum and liver. As a result of metallic stress, hyperglycemic condition was observed in that the mean blood sugar level increased significantly from 29.8±6.18 to 46.6±6.5 mg/100 ml. On the other hand the mean sugar level of liver and muscle decreased from 39.8±3.27 to 30.2±2.38 and 47.6±3.64 to International Journal of Integrative Biology IJIB, 2010, Vol. 10, No. 2, 120

3 37.6±4.77mg/g wet wt respectively. The cholesterol level of both serum and liver increased significantly. The protein content of serum, liver and muscle tissues decreasaed significantly, due to the metallic stress (Copper). In both serum and liver, the mean level of enzyme AST increased significantly where as ALT increased in serum but decreased in liver. The protein profile of serum and liver has been presented in Plate I. Due to the metallic stress the no of protein fractions increased in treated fish serum in that 35 KDa, 40 KDa, 45 KDa and 55 KDa fractions were well expressed. Whereas the number of protein fractions decreased in liver and muscle of treated groups when compared with the control. DISCUSSION The chronic exposure of Labeo rohita (Hamilton) to sub lethal concentration of heavy metal Copper revealed an increase in RBC, WBC counts and haemoglobin levels (Table 1) in exposed fish. Such an increase in blood parameters has also been reported by earlier authors like (McKim et al., 1970; Singh and Singh 1982; Vijayaraman et al., 1989; Saravanan and Nagarajan, 1991; Ramesh and Saravanan, 2008; Vinodhini and Narayanan, 2009). The reason for the increase in RBC may be attributed to enhanced erythropoiesis due to the environmental stress. Earlier authors like (Gills and Pant, 1981; Sastry and Tyagi, 1982; Srivasteva, 1982; Gill and Pant, 1983). Chitra and RamanaRao (1986) have observed a significant increase in the amount of haemoglobin content. The same trend has also been observed in the present study. The reason for the raise in haematocrit level might be due to the combined effect of RBC swelling and over production of it erythropoietically to make good the reduced oxygen carrying capacity of blood caused by the stress of Copper exposure for a month. Further, the increase in WBC suggests, the stress induced leucocytosis as has been reported by earlier authors. This may have an adaptive value to meet the stressful condition. Henry et al. (1981) have suggested the stress induced leucocytosis might have resulted from the direct stimulation of immunological mechanisms to combat the metallic stress present in the medium. The hyperglycemia of blood was observed in the present which is in agreement with earlier reports in Salmo giardneri; (Mukhopaduya and Dehadrai, 1980); Clarias batrachus (Sastry and Siddique, 1982); Channa punctatus (Simon et al., 1983); Cyprinus carpio (Ramesh and Saravanan, 2008) and Labeo rohita (Saravanan et al., 2010) The reason for the increase in blood sugar may be due to its mobilization from liver to meet the increase in energy demand posed by the heavy metal stress. Indeed, the present study also revealed a significant decrease in the total free sugar lever of muscle of treated group. Such a trend has also been reported by earlier workers of (Begum, 1987; Magare and Patil, 2000). The decrease in liver sugar indicates its motilization into the blood and/or its utilization to meet the increased energy demand posed by the stress. The protein content of liver and muscle decreased significantly, as a result of heavy metal stress. Earlier authors like (Shakoory et al., 1976; Ramalingam and Ramalingam, 1982; Rao et al., 1984) have also reported stress induced proteolysis. Whole body protein concentration are influenced by a variety of environmental factors, under conditions of stress many organisms will mobilize proteins as an energy source (Gilles, 1970). Further, muscle tissues form the bluck of the fish body is the primary center for proteolysis even under normal conditions. The above authors have suggested the decrease in protein content of tissues may be due to its degradation into amino acids which may be fed into TCA cycle through transferases to copeup with the high energy demand posed by the heavy metal stress. This may be the reason for the decrease in protein content of liver and muscle of the exposed fish in the present study. The earlier studies like Bakthavathsalam and Reddy (1984) have reported significant increase in the levels of asparate transaminase (AST) and alanine transaminase (ALT) in the liver of heavy metal exposed fish, Anabas testudineus (Bloch). Further, they have suggested that the increase in liver AST and ALT was the indication of incorporation of amino acids by way of amino transferases activities of these enzymes, into Krebs cycle to over come the stress of the exposed fish. Ramalingam and Ramalingam (1982) and Thakur and Sahai (1987) have reported a marked reduction in the number of protein fraction in heavy metal exposed fishes of Sarotherodon mossambicus and Channa punctatus respectively. As a result of Copper exposure for 30 days, the treated group showed a significant increase in cholesterol level. Such a significant increase in cholesterol level of tissues has been reported by Bhattcharya et al. (1984) in Channa punctatus exposed to pollutants. In normal fish, cholesterol synthesis in liver is found to operate at a level below maximum due to continuous absorption of cholesterol from the digestive tract and to negative feedback effect (Sabine, 1977). Cholesterogenesis may increase, if any xenobiotics interferes with the feedback mechanism. Further the heavy metal may also inhibit steroidogenesis resulting in the increase in liver cholesterol. Infact, Dubale et al. (1981) have reported an increased breakdown of cholesterol levels to free fatty acids to be used in corticosteroidogenesis. As a result of Copper suphate exposure, the quantity of tissue protein declined significantly which is also reflected in the protein fractions of liver and muscle (Fig. 1 [Supplementary data]). Indeed, the serum fractions increased in treated group when compared to the control suggesting the mobilization of protein fractions International Journal of Integrative Biology IJIB, 2010, Vol. 10, No. 2, 121

4 to meet the energy demand posed by the strees. Ramalingam and Ramlingam (1982) have reported the conversion of tissue protein into soluble fraction yielding process. Such qualitative changes in tissue protein have been reported by Thakur and Sahai (1987) in Channa punctatus, Henson and Gallagher (2004) in Brown bullheads and Saravanan et al. (2010) in Catla catla. The heavy metal Copper even at sub lethal concentration may induce degradative metabolic changes in liver and muscle and also affected blood parameters. Thus the heavy metal acts as a metabolic stressor inducing the degradative metabolic changes which ultimately may reduce the nutritive value of the fish on one hand and on the other hand making the fish susceptible to diseases. Hence care should be taken to prevent the aquatic bodies even with low levels of heavy metal like Copper. References Anderson PD and Weber LJ (1975) Toxic Response as a quantitative function of body size. Toxicology applied pharmacology, 33: Ashraj W (2005) Accumulation of heavy metals in kidney and heart tissues of Epinephelus microdon fishes from the Arabian Gulf. Environ. Monit. Assess., 101(1): Bakthavathsalam R and Reddy YS (1984) Lipid kinetics in relation to the toxicity of three pesticides in climbing perch Awabas testudineus (Bloch). Proc. Indian Nat. Sci. Aca., B47(5): Begum SJ (1987) Biochemical adaptive responses in glucose metabolism of fish Tilapia mossambica during ammonia toxicity. Curr. Sci., 56: 14. Bhattacharya T, Ray AK, et al. (1984) Response of Channa Punctatus (Bloch) Under short and longterm exposures to industrial pollutants: Hepatic and Blood cholesterol changes. Comp. Physiol. Ecol., 9: Bryan CW (1976) Some effects of heavy metal tolerance in aquatic organism In: Effect of pollutants on aquatic organisms. Cambridge University Press Cambridge, England. pp: 7. Chitra T and RamanaRao JV (1986) Temperature induced changes in certain haematological parameters in mercury treated Channa punctatus (Bloch). Comp. Physiol. Ecol., 2(2): Christensen. G, Hunt E, et al. (1977) The effect of methyl mercuric chloride, cadmium chloride and lead nitrate on six biochemical factors of the Brook trout Salvelinus Fontinalis, Toxical. Appl. Pharmacol., 42: Damien, C Chantal, VH, et al. (2004) Cellular impact of metal trace elements in terricolous lichen Diploschistes muscorum (Scop.) R. Sant. identification of oxidative strees biomarkers. Water Air Soil Pollut., 152: Davies JV and Lewis JM (1976) Practical haematology. Churchill Ltd., GloucasterPlace. Dick P and Dixon D (2000) Changes in circulating blood cell levels of rainbow trout, Salmo gairdneri Richardson, following acute and chronic exposure to Copper. J. Fish Biol., 26; Dubale M.S. and Awasthi M (1982) Biochemical change in the liver and kidney of Heteropneustes fossils, exposed to dimethoate Comp. Physiol, Ecol., 7: EIFAC (1975) Report on fish toxicity resting procedures. Prepared by European Inland fishes. Technical paper 24. Farkas A, Salanki J, et al. (2000) Relation between growth and the heavy metal concentration in organ of bream Abramis brame L. Populating lake. Balton. Arch. Environ. Contam. Toxocol., 43(2): Farombi O and Adelowo YR (2007) Biomarkers of oxidative stress and heavy metal levels as indicators of environmental pollution in african cat fish (clarias gariepinus) from nilgeria ogan river. Int. J. Environ. Res. Public health, 4(2): Geetha R, Kumaraguru AK, et al. (1996) Toxic effects of heavy metal Copper on the fish Lepidocephalichthys thermalis in short term exposures. Polln. Res., 15(2): Gill TS and Pant JC (1983) Cadmium toxicity: inducement of changes in blood and tissue metabolites in fish. Toxicol.lett., 18: Gill, TS and pant JC (1981) Effect of sublethal concentration of mercury in a teleost puntius eonchonious: Biochemical and haematological responses, Indian J. Exp. Biol., 19: Gilles R (1970) Intermediate Metabolism and energy production in some Invertabrates Arch. Int. de Physiol. Biochem., 78; Goodwin, TH AR Young, MGR, et al. (2003) The temporal and spatial variability of sediment transport and yields within the Bradford Beck catchment, West Yorkshire. Sci. Total Environ., 314: Gornall AG Brandnwill CJ, et al. (1949) Determination of total serum protein by means of biuret reaction. Boil. Chem., 177: Gumgum B, Unlu E, et al. (1994) Heavy metal pollution in water, sediment and fish from the Tigris river in Turkey. Chemosphere, 29: Hayat, S, Javed M, et al. (2007) Growth performance of metal stressed major carps viz. Catla catla, Labeo rohita and Cirrhina mrigala reared under semi-intesive culture system. Pakistan Vet. J., 27(1): Hellawell JM (1988) Toxic Substances in rivers and streams. Environ. Pollut., 50: Henry E, Donald HM, et al. (1981) A pesticide (Dieldrin) induced immuno haemolytic aneamia, Env. Res., 17: Henson LK and Gallagher EP (2004) Glutothione S-Transferase Expression in Pollution-Associated Hepatic Lesions of Brown bullheads(ameriurus nebulous) from the Cuyahoga River, Cleveland, ohio, Toxicol., 80, Khaled, A (2004) Heavy metal concentrations in certain tissues of five commercially important fishes from El-Mex Bay, Al-Exandria, Egypt. Egyptian Journal of Aquatic Biology And Fisheries, 8(1), pp: Labonne M Othman DB, et al., Lead isotopes in muscels as tracers of metal sources and water movements in a lagoon (ThauBasin, S.France). Chem. Geology, 181: Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophages T4, Nature, 226: International Journal of Integrative Biology IJIB, 2010, Vol. 10, No. 2, 122

5 Larsson A, Haux C, et al. (1985) Fish physiology and metal pollution : Results and Experiences from laboratory and field studies. Decotoxicol. Environ. Saf., 9: Magare SR and Patil HT (2000) Effect of pesticides on oxygen consumption, Red Blood Cell count and metabolites of a fish Puntius ticto, Environ and Eco., 18(4): Mazon A, Cerqueira C, et al. (2002) Gill Cellular changes indued by Copper exposure in the South American tropical freshwater fishwater Prochilodus scrofa, Environm. Res.A., 88(1): Mckim JM, Christensen GM, et al. (1970) Changes in the blood of the brook trout (Salvelinus fontinalis) after short-term and long-term exposure to Copper. J. Fish. Res. Board can., 27: More TG Rajput RA et al. 2003, Impact of heavy metals on DNA content in the of hole body of freshwater bivalve, Lamelleiden marginalis. Environ. Sci. pollut. Res. Res., 22: Mukhopadyay PK and Dehadrai PV (1980)Biochemical changes in the air breathing cat fish Clarius batrachus (Linn) exposed to malathion. Environ. Pollut. (Ser. A), 22: Olaifa FE, Olaifa AK, et al. (2004) Heavy metal contamination of Clarias grapinus from a lake and fish farm in Ibadan, Nigeria. Afric. J. Biomed. Res., 7: Parker RE (1973) introductory statistics for biology, Edward Arnold publishers Ltd, London; pp: 122. Ramalingam K and Ramalingam K (1982) Effects of sub- lethal levels of DDT, Malathion and mercury on tissue protein of Sarotherodon mossambicus (peters). Proc. Indian. Acad. Sci., (Animal Sci.). 91(6): Ramesh M and Saravanan M (2008) Haematological and biochemical responses in freshwater fish Cyprinus carpio exposed to chlorpyrifos. Int. J. Integ. Biol., 1: Rao J, Azhar BK, et al. (1984) Effects of a systemic pesticide phosphonidon some aspects of freshwater fish Tilapia mossambica. Indian J. Environ. Hlth., 26(1) : Roe JH (1955) The determination of sugar in blood and spinal fluid with anthrone reagent. J. Biol. Chem., 212: Sabine JR (1977) Cholesterol, Mec Dekkar Inc: New York and Basel, pp: Saravanan TS and M Natarajan (1991) Haematological parameters of Anabus testudineus, on exposure to sublethal concentration of cadmium for different periods. J. Ecotoxicol. Environ, Monit., 7(4): Saravanan TS, Aneez Mohamed M, et al. (2003) Fresh water fishes as indicators of Kaveri River pollution. J. Environ. Biol., 24(4): Saravanan TS Boopathy S et al. (2010) Impact of chronic exposure of monocrotophos on blood parameters, biochemical constituents and histology of the major carp, Catla catla (Hamilton). Malaysian J. Sci., 48: Sastry KV and Siddique AA (1982) Effect of endosulfan and quinolphos on intestinal absorption of glucose in fresh water mussel, Channa punchatus. Toxicol. Lett., 12(4): Sastry KV and Tyagi Sunitha (1982) Effect of cadimium on the intestional absorption of glucose in snake head fish Channa punctatus Toxicol, Lett., 10: Shakoory AR Zaheer SA et al. (1976) Effects of malthion, dieldrin and endrin on blood serum proteins and free amino acids pools of Channa punctatus (Bloch), Pakistan J. Zool., 8: Simon LM, Memcshok J et al. (1983) Studies on the effect of paraquat on glycogen metabolism in liver of common carp. Cyprinus carpion (L). Camp. Biochem. Physio., 75C(1): Singh, SR and singh,nr (1982) Effect of Copper and zinc sulphate on the blood parameters of Mystus vittatus, Matsya, 8: 1-6. Srivasteva DK (1982) Comparative effect of Copper, cadmium and mercury on tissue glycogen of the catfish Heteroponeustes fossillis (Bloch), Toxicol. Lett., 11: 135. Stiff MJ (1971) The chemical states of Copper in polluted fresh water and scheme of analysis to differentiate them water Resources, 5: Thakur N and Sahai S (1987) Changes in the serum proteins and other haematological parameters in the toleost Channa punctatus on exposure to an organochlorine pesticide. Environment and Pesticide toxicology, 9: Vijayaraman K, Geraldine P, et al. (1989) Haematological responses of an air breathing fish Anabas testudineus to cadmium, J. Eco. Biol., 1: Vinodhini R and Narayanan M (2008) Bioaccumulation of heavy metals in organs of freshwater fish Cyprinus carpio (common carp). Int. J. Environ. Sci.Tech., 5(2) Vosylience MZ and Jankaite A (2006) Effects of heavy metal model mixture on rainbow trout biological parameters. Ekologija., 4: Wooten IDP (1964) Microanalysis in medical Biochemistry A Churchill Ltd., London W.I. Yousuf MH and Shahawi AE1 (1999) Trace Metals in Lethlinuslentsan fish from Arabhian Gulf; Metak accumulation in Kidney and Heart Tissues. Bull. Environ. Contam. Toxicol., 62(3): Zarrow M, Yochim X, et al. (1964) Experimental endocrinology: a source of book of basic techniques. Acad. Press. New York. pp: 206. International Journal of Integrative Biology IJIB, 2010, Vol. 10, No. 2, 123