Effect of Zinc, Cadmium And Copper toxicants on Haematological parameters on fresh water fish Channa punctatus.

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1 Available online at : pp.61~73 Thomson Reuters ID: L Effect of Zinc, Cadmium And Copper toxicants on Haematological parameters on fresh water fish Channa punctatus. ABSTRACT Prof.(Dr.) Jai Parkash Principal JCD Memorial (PG)College, Sirsa, Hry. Haematology is the subject, which deals with the study of blood and embraces various aspects of physio pathological as well as the biochemical characteristics of the blood. Fish haematology is one of the most recent branches of physiology to assess the status of general health. In the last three decades fish blood study has become an increasingly important tool for fishery biologists and ichthyologists for regular monitoring of health of the fish stocks and to develop an information database about inter-specific and intra-specific variations in blood characteristics under varying environmental conditions and stages of life From the Study it is evident that the number of erythrocytes in fish exposed to zinc was significantly decreased in group exposure as compared to control fish. The percentage of alteration was In individual exposure, all the four fish showed significant increase, which varied from 14.2% to 20.7%. The number of erythrocytes in group exposure fishes exposed to cadmium was significantly elevated. The percentage of alteration was 13.2 as compared to control fish. Individually exposed fishes also showed significant increase which varied from 12.3% to 16.1%. The number of erythrocytes in group exposure fishes exposed to copper was significantly increased as compared to control fish. The percentage of alteration was 43.4%. In individual exposure, all the four fishes showed significant increase which varied from 37.7% to 46.2%. In the present study monocytes decreased in all the exposures, except zinc. Monocytes were less frequent than the large lymphocytes. The number of lymphocytes decreased in cadmium. Lymphocytes are the second frequent type of leucocytes present in peripheral blood. In the present study, the observed number of lymphocytes (22.3%) is supported by the findings of Rani and Rao (1979) who reported 6.8% large lymphocytes and 30.5% small lymphocytes in Elops saurus. In the present study lymphocytes number increased from 22.2% in control to 27.7% on exposure to zinc. Key Words: Haematology, Cadmium, Cadmium, Copper, Blood. TLC, TEC, Fish, Channa punctatus. INTRODUCTION Blood is life is the biblical phrase. It circulates through vascular channels carrying out the necessities of life to all cells of the body and it receives waste products for excretion. Blood is a very delicate tissue that performs most of the vital activities of the organisms. This has led to the belief that blood characteristics are closely related with vital activities of the animals and their journals. 61

2 environment.. The circulating blood is intricately intertwined with many facets of normal physiology and pathophysiologic mechanisms. Blood is the transport medium, a defense system, and an acid/base buffer system. Circulating blood is the common denominator of health and illness and alteration in its chemical or cellular illness and composition can indicate haematological and non-haematological diseases. Erythrocytes occupy the largest fraction of the formed elements of the blood. In normal functioning of the body, the blood counts remain stable, but environmental as well as pathological conditions can alter the erythrocyte count. Variations in either direction outside the designated range of normal counts usually indicate erythropoetic dysfunction. Haemoglobin is the respiratory protein contained within erythrocytes. It has a multitude of functions in the circulatory system including transport of O2 and CO2 and its action as the most important buffer in the blood, etc. It is a representative of the globular proteins. The body has a great capacity for conservation; it relinquishes very little of those resources that may be reused. The destruction and reutilization of haemoglobin is a good example of this conservation. Lysis of erythrocytes in the blood stream is called intravascular haemolysis and when it occurs due to phagocytosis or sequestration by the reticuloendothelial system is called extravascular haemolysis. Leucocytes (white blood cells) constitute an important part of the body s defense system. The number of white cells in the blood, compared to the number of erythrocytes is quite small. A temporary increase in the white blood cells in the peripheral circulation is called leukocytosis and decrease in number of white cells is leucopenia. There are several varieties of white blood corpuscles, each type possessing characteristic morphology and staining property. Determination of the percentage of different varieties of leucocytes is known as the differential count of white blood corpuscles. White blood corpuscles or leucocytes can be divided into two main categories: granular leucocytes or granulocytes, and agranular leucocytes or agranulocytes. Granulocytes are produced in the bone marrow and are of three types: neutrophils or polymorphs eosinophils and basophils. Agranulocytes produced in the lymph nodes and spleen are of two types: lymphocytes and monocytes. Granular leucocytes or granulocytes are so named due to their granular o cytoplasm. The granules may take any of the three different stains a neutral, acidic or basic. The granules in the cytoplasm of neutrophils or polymorphonuclear cells are stained by neutral dyes. Eosinophils contains coarse granules that stain with acid dyes. Basophils are said to produce heparin. They have lobate nuclei, which stain dark purple, and coarse granules staining blue. The lymphocytes have large indented nuclei that stain deep blue to purple. Their most important function is to produce antibodies. They are less motile as compared to the leucocytes. Lymphocytes have basophilic cytoplasm distributed in the form of a thin rim around the nucleus and with no distinct granules. Monocytes contain indented to horseshoe-shaped nuclei and clear non-granular cytoplasm. They are motile and have the power of engulfing bacteria. Since fishes are very valuable source of energy and protein rich diet, particularly when more than 60% of human population all over the world is suffering severely from mal-nutrition, fish health should be a matter of great concern to all researchers and fish biologists in particular. It is with this idea that this study has been taken up to determine the normal haematological values as base line data under variety of natural and experimental conditions towards maintaining a better environment and a healthy fish stock. 62

3 Under natural conditions general haematological studies on fresh water and marine fishes have been made by several workers in India and abroad viz. Siddiqui and Naseem (1967) on Cirrhina Mrigala; Karmarkar and Gazdar (1968) on Chiloscyilium greshium, Wilkins and Charke (1974) on Clupea harengus; Khan (1974) on catfish Clarias batrachus; Haematological alteration of fishes in relation to environmental contamination is a rewarding field of scientific investigation. Most of the aquatic pollution is due to pesticides fertilizers, heavy metals, detergents, industrial effluents, domestic sewage etc. All these materials are reported to change the blood characteristics. Many investigators studied the impact of heavy metal pollution (zinc, cadmium, copper, lead, etc.) on fish haematology (Agrawal and Srivastava,1980; Joshi, 1987 ; Tiwari et al., 1987; Banerjee et al., 1988; Banerjee and Homechaudhuri, 1990; Cadmium has been reported to increase ALAD activity depending upon substrate availability (Wilson et al., 1972) and to inhibit the level of ALAD s activity. It has been observed that cadmium can decrease the circulation time of erythrocytes (Berlin and Friberg, 1960) and can cause anemia presumably of the microcytic type (Fox et al., 1971). Srivastava and Mishra (1979) reported significant decrease in thrombocyte count and coagulation time in Coiisa fasciatus after treatment with cadmium. Decrease in erythrocyte and nuclear surface area in Anabas testudineus on exposure to sublethal concentration of ZnCI2 (300 mg/i), HgCI2 (0.2mg/I) and CdCI2 (350 mg/i) has been reported by Kumari and sanerjee (1986). Ruparelia et al. (1986) have reported cadmium to produce nornocytic anemia and elevated blood glucose level in Sarotherodon mossambica. Significant decrease in erythrocyte count and other haematological parameters was reported in Anabas testudineus by Kumari and Banerjee (1992) on exposure to LC50 concentration of Zn, Hg and CdCl2 for 24 hrs. Tort and Pascual (1990) reported that cadmium did not induce any significant change in majority of blood characteristics of dogfish Scyiorhinus canicula. Exposure of Anabas testudineus to a sublethal concentration of cadmium for 30, 45 and 60 days resulted in significant increase in RBC count and haemoglobin concentration (Saravanan and Natarajan, 1991). Ahmad and Dattamunshi (1991) observed that red blood cell morphology of Catla catla exposed to 96 hrs howed shrinkage and crenation in their configuration along with slight erythroanisocytosis and degeneration of cell membrane. Kumar and Agarwal (1993) reported significant decrease in RBC in Clarias batrachus exposed to sublethal concentration of mercuric chloride. Sastry and Gupta (1994) studied haematological changes produced by cadmium and dimethoate alone and in combination on a fresh water teleost fish Channa punctatus.. A survey of literature revealed that several types of leucocytes present in the peripheral blood varies from species to species. Usha Rani and Rao (1979) classified leucocytes into lymphocytes, thrombocytes, monocytes, macrophages, neutrophils, eosinophils and basophils, in the marine teleost Gerres filamentosus, while in Elops saurus, large and small lymphocytes, large and small thrombocytes, monocytes, acrophages, neutrophils, eosinophils, and basophils have been recorded. Gupta and Gupta (1981) distinguished monocytes, large lymphocytes, small lymphocytes, monocytes, neutrophis, polymorphs and basophils in circulating blood of Channa punctatus. Prasad and Baneriee (1982) identified leucocytes as large and small lymphocytes, monocytes, neutrophils, 63

4 eosinophils and basophils in Catla catla, Labea rohita and in Cirrhhina mrigala except eosinophils, all other leucocytes were found. Banerjee et al. (1988) reported large and small lymphocytes, monocytes neutrophils and eosinophils, and basophils in Mystus vittattus. Sinha and Kumar (1991) classified seven types of leucocytes namely small and large lymphocytes, monocytes, neutrophils, eosinophils, basophi ls and thrombocytes in Anabas testudineus. Bala et al. (1994) studied the toxicity of sublethal concentration of some heavy metal salts on haematology of Channa punctatus. MATERIALS AND METHODS Exposure of Test Fish Healthy living specimens of the fresh water teleost fish Channa punctatus collected from the local ponds of Rohtak, Haryana or purchased from fish market were quickly transported to laboratory. The fish were maintained in glass aquaria and fed twice daily with pelleted diet (prawn powder, fish powder, and minced liver in 2:2:1 ratio). The water in aquaria was continuously aerated in order to maintain the dissolved oxygen concentration above 7 ppm. Individual exposure 80 fishes divided into 4 groups of 20 each were exposed to LC50 of zinc (18.62 mg/i), copper (0.56 mg/i) and cadmium (11.8 mg/i) separately. The fourth group of 20 fishes was kept in metal free water served as control for each experimental group. After 96 hr from each of the experimental and control groups 4 surviving fishes were dissected and blood was drawn. Group exposure 60 fishes were divided into 3 groups of 20 fishes each and were exposed to LC50 of zinc (18.62 mg/i), copper (0.56 mg/i) and cadmium (11.8 mg/i) separately. After 96 hr from each of the experimental and control groups blood was collected from surviving fishes. Preparation of Fish for Blood Sampling For collection of blood, a fish was taken out from aquarium with the help of a hand net and immediately immersed in 2% paraldehyde solution. The fish was taken out from this solution when the opercular movements ceased, but movement of the gills still persisted (Joshi,1993,a). Immediately the fish was soaked dry with clean towel or filter paper. Blood for Haematological Studies For haematological observations of the fish Channa punctatus, blood from control and experimental fish was drawn directly from the ventricles after exposing the heart, with the help of a syringe fitted with 20 gauze needle. The blood without anti-coagulant was directly used for the total erythrocyte count (TEC), total leucocytes count (TLC) and smear preparation. 64

5 Total erythrocyte count The number of erythrocytes per cubic millimetre of blood was calculated with the help of a haemocytometer using a Neubaur s counting chamber as given by Dacie and Lewis (1977). Total leucocytes count The method given by Dacie and Lewis (1977) was followed to count the number of leucocytes. Blood was drawn up to 0.5 mark of the WBC pipette and diluted up to 11 mark with Turk s fluid and the number of WBC in corner most four squares, whose area is 1 sq. mm were counted. Differential leucocytes count Preparation of blood smear: For the preparation of blood smear standard glass slides washed in 100% ethanol were used. Blood film was made immediately after the blood was drawn from the fish. From each fish, four blood smears were prepared using Leishman s, Gimsa, Wright s and Field stains. Study of blood smear: The morphology and staining property of all types of blood corpuscles were studied. For differential leucocytes counts, at least 100 leucocytes were observed and counted for each blood smear at a time, and differentiated into various types: monocytes, lymphocytes, neutrophils, eosinophils and basophils. RESULTS The acute toxic effects of zinc, cadmium and copper have been examined on some blood parameters. The parameters selected were total erythrocyte count, total leucocytes count and differential leucocytes count. Effects of Zinc Zinc produced marked alterations in the blood parameters of group well as individual fishes after acute exposure, These are described in the following account (Table: 1 and Histogram D: 1,2). 65

6 Table :1 Alteration in haematolological parameters in Channa punctatus exposed to Zinc for 96hr. individually and group Parameter Control fish Individual exposed fish Group exposed 1 st 2 nd 3 rd 4 th T.E.C. (x10 6 /mm 3 ) 2.12± ± *** 1.70 ± 0.22 *** 1.82 ± 0.12 *** 1.68 ± 0.29 *** 1.72 ±0.04 *** T.L.C. 6.12± ± 0.26 *** 7.48 ± 0.23 *** 7.76 ± 0.09 *** 7.64 ±0.09 *** 7.62 ±0.09 *** (x10 4 / mm 3 ) Basophils(% ) 4.11± ± 0.52 *** 3.14 ± 0.25 *** 2.96 ± 0.26 *** 3.16± 0.21 *** 3.09 ± 0.08 *** Eosinophils(% ) 5.25± ± 0.73 *** 8.06 ±0.68 *** 7.98 ± 0.18 *** 8.48 ± 0.24 *** 8.24 ±0.02 *** Monocytes (% ) 6.25± ± 0.22 *** 5.81 ± 0.07 *** 7.58 ± 0.21 *** 5.81 ± 0.14 *** 6.48 ± 0.12 *** Lymphocytes(% ) 22.25± ± 1.12 *** ± 1.08 *** ± 1.20 *** ± 0.98 *** ± 0.14 *** Neutrophils(% ) ± ± 1.02 *** ± 0.96 *** ±0.92 *** ±1.25 *** ± 0.16 *** T.E.C. = Total Erythrocyte Count; T.L.C. = Total Leucocyte Count Values are Mean ± SD.,n=6 *P<0.05; *** P<0.001 Histogram D:1 Histogram:D:2 Total erythrocyte count From the results it is evident that the number of erythrocytes in fish exposed to zinc was significantly decreased in group exposure as compared to control fish. The percentage of alteration was In individual exposure, all the four fish showed significant increase, which varied from 14.2% to 20.7%. Total leucocytes count In group exposed fishes significant elevation was recorded in the otal number of lymphocytes. The percentage of increase was 24.5% as compared to control fish. Increase was statistically significant. In individual exposure, all the four fish showed significant increase which varied from 7.48 to 7.76 (xlo4imm3). Differential leucocytes count a) Basophils: Exposure of fishes to zinc for 96 hr decreased the number of basophils by 24.8%. The decrease was significant. In individual exposure, all the four fish showed significant decrease, which 66

7 varied from 23.1% to 27.9%, clearly showing that the decrease in four fish was not uniform and was statistically significant. b) Eosinophils: In group exposure significant increase was noticed in the number of eosinophils. Percentage of increase was 56.9 as compared to control fish. All the four fishes exposed separately also showed significant increase which ranged between 52.0% to 61.5%. c) Monocytes: The number of monocytes of experimental fish was higher than the control fish showing a insignificant increase of 3.7% Increase of monocytes in four individually exposed fishes varied from 4.16 to 21.3%. d) Lymphocytes: Acute exposure to zinc produced significant increase in lymphocytes number in group exposed fishes. The increase was 24.6%. There was also variation in the number of lymphocyte in four individually exposed fishes and increase in each fish was statistically significant. e) Neutrophils: From the results, it is clear that in group exposure the number of neutrophils decreased significantly by 12.3% in zinc exposed fishes. Fish No 3 of individually exposed fish show highest decrease of 14.9% and lowest decrease was 4.8% for fish No I. Effect of Cadmium Cadmium produced marked alteration in the blood parameter of group as well as individual fishes after acute exposure, which are described in the following account (Table : 2 and Histogram D: 1,2) Table :2 Alteration in haematolological parameters in Channa punctatus exposed to Cadmium for 96hr. individually and group Parameter Control fish Individual exposed fish Group exposed 1 st 2 nd 3 rd 4 th T.E.C. (x10 6 /mm 3 ) 2.12± ± 0.02 *** 2.46 ± 0.03 *** 2.38 ± 0.02 *** 2.42 ± 0.04 *** 2.40 ±0.02 *** T.L.C. (x10 4 / mm 3 ) 6.12± ± 0.03 *** 7.56 ± 0.02 *** 7.20 ± 0.30 *** 7.58 ±0.42 *** 7.52 ±0.08 *** Basophils(% ) 4.11± ± 0.14 *** 2.80 ± 0.12 *** 2.56 ± 0.16 *** 2.75± 0.18 *** 2.76 ± 0.11 *** Eosinophils(% ) 5.25± ± 0.20 *** 2.25 ±0.16 *** 2.75 ± 0.18 *** 2.62 ± 0.24 *** 2.43 ±0.40 *** Monocytes (% ) 6.25± ± 0.17 *** 3.25 ± 0.18 *** 3.0 ± 0.21 *** 3.50 ± 0.21 *** 3.36 ± 0.6 *** Lymphocytes(% ) 22.25± ± 0.82 *** ± 1.46 *** ± 1.21 *** ± 0.31 *** 23.6 ± 0.30 *** Neutrophils(% ) ± ± 0.57 *** ± 0.42 *** ±0.86 *** ±0.74 *** ± 0.09 *** T.E.C. = Total Erythrocyte Count; T.L.C. = Total Leucocyte Count Values are Mean ± SD.,n=6 *P<0.05; ** P<0.01; *** P<

8 Total erythrocyte count From the results it is evident that the number of erythrocytes in group exposure fishes exposed to cadmium was significantly elevated. The percentage of alteration was 13.2 as compared to control fish. Individually exposed fishes also showed significant increase which varied from 12.3% to 16.1%. Total leucocytes count In group exposed fishes significant increase was recorded in the total number of leucocytes. The percentage of alteration was Although there was significant increase of white blood corpuscle in individual fishes, the increase varied from 17.6% to 23.8%.Differential leucocytes count a) Basophils: Exposure to cadmium for 96 hr decreased the number of basophils by 32.8% in group exposure. In individual exposure, all four fish showed significant decrease which varied from 31.8% to 37.7%, clearly showing that the decrease in four fish was not uniform. Decrease was statistically significant. b) Eosinophils: The present experiment revealed that the number of eosinophils in group exposure significantly decreased in experimental fishes (53.7). All the four fish exposed separately also showed significant decrease which ranged between 47.6% to 57.1%. c) Monocytes: In group exposed fishes the number of monocytes was lower than the control fish showing a decrease of 46.2% thatwas statistically significant. Decrease of monocytes in fourindividually exposed fishes varied from 44.0% to 52.0% and the decrease was also statistically significant. d) Lymphocytes: In group exposure the number of lymphocytes was higher in cadmium exposed fish in comparison to control fish. Thepercentage of decrease was 6.1. There was also variation in thenumber of lymphocytes in four individual exposed fishes which varied from 8.5% to 17.6%. e) Neutrophils: In the group of fishes exposed to 96 hr to cadmium, neutrophils significantly increased by 9.2% as compared to control fish. In individual exposure, fish No 2 show highest increase of 11.9% and lowest decrease was 4.5% for fish No. 4. Effect of Copper Copper produced marked alterations in the blood parameter of group as well as individual fishes after acute exposure. These are presented in the following account (Table : 3 and Histogram D: 1,2). 68

9 Table :3 Alteration in haematolological parameters in Channa punctatus exposed to Cadmium for 96hr. individually and group Parameter Control fish Individual exposed fish Group exposed 1 st 2 nd 3 rd 4 th T.E.C. (x10 6 /mm 3 ) 2.12± ± 0.10 *** 2.98 ± 0.07 *** 3.10 ± 0.02 *** 2.92 ± 0.02 *** 3.04 ±0.08 *** T.L.C. 6.12± ± 0.22 *** 7.82 ± 0.18 *** 7.84 ± 0.12 *** 7.64 ±0.16 *** 7.80 ±0.16 *** (x10 4 / mm 3 ) Basophils(% ) 4.11± ± 0.14 *** 6.68 ± 0.42 *** 6.72 ± 0.52 *** 6.42± 0.49 *** 6.58 ± 0.32 *** Eosinophils(% ) 5.25± ± 0.12 *** 8.74 ±0.16 *** 8.80 ± 0.07 *** 8.92 ± 0.09 *** 8.81 ±0.28 *** Monocytes (% ) 6.25± ± 0.46 *** 5.82 ± 0.34 *** 5.55 ± 0.32 *** 5.70 ± 0.24 *** 5.65 ± 0.42 *** Lymphocytes(% ) 22.25± ± 0.30 *** ± 0.92 *** ± 0.84 *** ± 0.94 *** ± 0.76 *** Neutrophils(% ) ± ± 1.24 *** ± 1.34 *** ±0.98 *** ±1.42 *** ± 1.28 *** T.E.C. = Total Erythrocyte Count; T.L.C. = Total Leucocyte Count Values are Mean ± SD. n=6 *P<0.05; ** P<0.01; *** P<0.001 Total erythrocyte count From the result shown in Table : 3, it is evident that the number of erythrocytes in group exposure fishes exposed to copper was significantly increased as compared to control fish. The percentage of alteration was 43.4%. In individual exposure, all the four fishes showed significant increase which varied from 37.7% to 46.2%. Total leucocytes count In group exposed fishes significant increase was recorded in total number of leucocytes. The percent of alteration was Although there was significant increase of white blood corpuscles in individual fishes, the increase varied from 24.8% to 28.1%. Differential leucocytes count a) Basophils: Exposure to copper for 96 hr increased the number of basophils by 60% in group exposed fishes. In individual exposure, all the four fish showed significant increase which varied from 56.2% to 66.9%, clearly showing that the increase in four fish was not uniform. Increase was statistically significant. b) Eosinophils: The present experiment revealed that the number of eosinophils in group exposed fishes significantly increased in experimental fishes (40.9%). All the four fishes exposed separately also showed significant increase which ranged between 27.5% to 42.7%. c) Monocytes: In group exposed fishes the number of monocytes as lower than the control fish showing a decrease of 22.1% that was statistically significant. Decrease of monocytes in four 69

10 individually exposed fishes varied from 19.7% to 23.4% and the ecrease was also statistically significant. d) Lymphocytes: In group exposure the number of lymphocytes was significantly lower in copper exposed fishes in comparison to control fish. The percentage of decrease was There was also variation in the number of lymphocytes in four individual fish and decrease in each fish was statistically significant. e) Neutrophils: In the group of fishes exposed to 96 hr to copper, neutrophils decreased by 3.4% as compared to control fish. In individual exposure, fish No. 2 show highest decrease (4.3%) and lowest decrease was 1.11 for 3rd fish. DISCUSSION Zinc produced significant decrease in the number of erythrocytes. The reduction in RBC count after 96 hr of exposure indicates anaemia associated with erythropenia, which had been earlier reported due to a exposure of fishes to mercury and lithium (Panigrahi and Mishra, 1978; Agarwal et al., 1983). Anaemia, associated with erythropenia had also been reported earlier in fishes after exposure to sublethal concentration of mercury, lead and rogor (Panigrahi and Mishra, 1978; Srivastava and Mishra, 1979; Goel and Maya, 1986). Cadmium unlike zinc produced significant increase in the number of erythrocytes. The reason for this increase may be the release of immature cells from haemopoietic tissues into the blood stream and this contention is supported by significant decrease in Hb content and mean cell volume (Wepener et al., 1992). The probable explanation of the haematological change of this type is that the exposed fish compensates for an impaired oxygen uptake (due to disturbed gills function) by release of erythrocytes from the spleen. However, secondary severe stress can cause a similar polycythemic effect. Such a state of stress is associated with an increase in the secretion of catecholamines (Nakano and lomlison, 1967; Nilsson et ai., 1976) which have been shown to cause polycythemia by contracting and partly emptying the spleen for the Supply of red blood cells (Larsson, 1973). Increase in Hb, TEC and HCT observed in Heteropneustes fossilis exposed to cadmium by Sastry and Subhadra (1985) also corroborates the present observations. However marked hypochromasia and change in shape of erythrocytes with increase in TEC was observed in Channa punctatus exposed to thimet and bazanon by Chakrabarty and Banerjee (1988). Tort and Pascual (1990) reported decrease in mean cell volume (MCV) with increase in the number of red blood corpuscles after short term sublethal cadmium exposure in Sarotheradon cannicula. Das and Kaviraj (1990) also reported increase in TEC in Heteropneustes fossilis exposed to 55.5 mg/i of cadmium. Copper also produced significant increase in the number of erythrocytes. The lethargic condition of fish in later periods of CuSO4 exposure falls in line with the findings of Holmberg et al. (1972) and Oikari and Nakari (1982). Frequent gulping of air during prolonged periods of CuSO4 intoxication is in agreement with the findings of Brungs et al. (1973) who exposed Ictalurus nebulosus. The presence of immature erythrocytes and broken fragments of RBC corroborates the 70

11 findings of Tort et al. (1987) in dogfish after acute copper exposure. During the present investigations the number of erythrocytes increased after 96 hr of intoxication, but later decreased, which corroborates the findings of Joshi (1987) in Ciarias batrachus. However, RBC depletion might be due to injuries to the erythropoietic tissues including the kidney, due to accumulation of copper in the kidney (Brungs et al., 1973). In the present study increase in WBC in fish exposed to zinc for 96 hr indicates leucocytosis, which is in agreement with the findings of Goel 4 and Gupta (1985) and Garg et al. (1989). Leucocytosis has been considered to be an adaptation to meet stressfull condition by the animal. Increase in the number of white blood cells was observed in fish exposed to cadmium for 96 hr. The successive increase in WBC count depends upon increase in the number of lymphocytes in fish. However lymphocytes in fish have been regarded as immune competent cells. Such an increased response may be the result of direct stimulation of immunologic defence due to the presence of foreign substances or may be associated with metal induced tissue damage (Ellis 1976). According to Mcleay and Brown (1974) increase in the leucocytes under chemical stress in Oncorhynchus kisutch is, probably for quicker removal of cellular debris of necrosed tissue. White blood corpuscles also increased in number during acute copper exposure. This corroborates the findings of Sharma et al. (1981, 1982), who exposed Hetropneustes fossilis to copper for 24 hr. Increase in WBC count was brought about by endosulfan and dimethoate exposures for 96 hr. Increase in leucocytes may be due to interference of endosulfan and dimethoate toxicity with the development of erythrocytes in haemopoietic tissues leading to Ieucaemogenic condition or the immunological response to meet the stress caused by pesticides toxicity. These findings are supported by Ramesh et al. (1992) and Saxena and Parasari (1982) who also reported increase in WBC, count in Channa punctatus exposed to cadmium. From the present study it may be concluded that most of the heavy metals produced anaemia in fish by decreasing the erythrocyte levels. This anaemic condition may have occured due to the increase in the rate of destruction of red blood cells. Berlin and Friberg (1960) and Axelson and Piscator (1966) suggested that anemia is caused mainly by increased destruction of erythrocytes in fishes. Rainbow trout (Salmo gairdneil) exposed to inorganic lead also showed anaemic response and basophilic stippling of erythrocytes (Johanson-Sjobeck and Larsson, 1979). It is apparent from the results that zinc exposed fish may develop hypochromic-microcytic anemia, due to the deficiency of iron and its decreased utilisation for haemoglobin synthesis. Impaired intestinal absorption of iron due to damaged intestinal mucosa is suggested as an important factor in the development of cadmium induced anemia (Freeland and Cousins, 1973; Richardson etal., 1974). Increased destruction of erythrocytes (Berlin et al., 1961) as well as direct inhibitory effect of cadmium on haemopoeisis (ltokawa etal., 1974) may also contribute to the anaemic response. In the present study monocytes decreased in all the exposures, except zinc. Monocytes were less frequent than the large lymphocytes. The number of lymphocytes decreased in cadmium, copper exposure and increased in zinc and dimethoate exposures. Lymphocytes are the second frequent type of leucocytes present in peripheral blood. In the present study, the observed number 71

12 of lymphocytes (22.3%) is supported by the findings of Rani and Rao (1979) who reported 6.8% large lymphocytes and 30.5% small lymphocytes in Elops saurus. In the present study lymphocytes number increased from 22.2% in control to 27.7% on exposure to zinc. These findings are supported by the observation of Kumari and Banerjee (1992) who observed an increase in large lymphocytes from 18.4% (control) to 27.4% (experimental) in CIahas batrachus. Increase in the lymphocyte counts due to toxicity of dimethoate supported by the findings of Balaji and Chockalingam (1989). They reported alterations in the lymphocyte counts due to toxicity of pesticides from 36.5% (control) to 44.5% after tannic acid exposure in Labeo rohita. Ghose and Banerjee (1989) also reported increase in lymphocyte count (61.60% to 71.8%) due to organophosphate (rogor) treatment in Heteropneustes fossilis. CONCLUSION In the present study all the metals decreased the number of basophils, eosinophils, monocytes, lymphocytes, neutrophils and RBC in Channa punctatus after 96 hr, except in few observations. Number of eosinophils, monocytes and lymphocytes in zinc exposure; neutrophils in cadmium exposures; basophils and eosinophils in copper exposure;. This is in contradiction to the previous findings. Similarly cadmium and copper also increase the count of RBC in Channa punctatus, while all the selected toxicants increased the WBC count significantly after acute exposure period. ACKNOWLEDGEMENTS First and foremost, the investigator wishes to express his reverence and thanks to God almighty, without whose providence nothing would have been possible. Then, It is my profound privilege to express my indebtedness and deep sense of gratitude to my Co-Guide Dr. Vineeta Shukla Professor Department of Zoology M.D.University, Rohtak,Haryana for her keen interest, valuable suggestions, encouragement and above all highly inspiring cooperation which shaped the work into present. The investigator is extremely thankful to who render all possible help to collect data for the study.i would be failing in my duties if I don t make a mention of my parents for going through the manuscript and completing of this study. I owe my sincere thanks to all the staff members of J.C.D. Memorial PG college, Sirsa. Last but not the least, I would like to thank all the concerned, who helped me directly or indirectly in the successful completion of this research paper. REFERENCES: 1. Banerjee, S. and Chaudhary,S. (1990) Haematological monitoring of a bioindicator fish, Heteropneustes fossilis on exposure to copper toxicity. J.Ecol. Environ. 42, Dhanapakiam, P. et all (2006) : Changes in the level of transaminases in Indian corp, Labeo rohita exposed to sublethal concentration of tannery and distillery effluent. J. Environ. Biol., 27, Dharam Singh, Kamlesh Nath, S.P. Trivedi and Y.K. Sharma (2008) Impact of copper on haematological profile of freshwater fish, Channa punctatus Journal of Environmental Biology 29(2) (2008) 72

13 4. Garg, V.K., Garg, S.K. and Tyagi, S.K.(1999). Haematological parameter in fish Channa punctatus under the stress of manganese. Environ. Eco. 7, Goel. K.A., K. Gupta and M.L. Sharma(1985): Haematological characteristic of Heteropneustes fossilis under the stress of zinc. Ind. J. Fish., 36, Joshi, P.K., M. Bose and D. Harish (2002): Haematological changes in the blood of Clarias battrachus exposed to mercuric chloride. Ecotoxicol. Environ. Monit., 12, Lodhi, H.S., et all (2006).: Acute toxicity of copper sulphate to fresh water prawns. J. Environ. Biol., 27, McKim, J.M., G.M. Christensen and E. P. Hunt(1970): Changes in the blood of the brook trout Salvelinus fontinalis after short-term and log-term exposure to copper. J. Fish. Res. Board. Can., 27, Mishra, S. and A.K. Srivstava (1980).: The acute toxic effects of copper on the blood of a teleost. Ecotoxicol. Environ. Safety, 4, Nath, R. and V. Banerjee (1995): Effects of various concentrations of lead nitrate on haematological parameters of an air breathing fish, Clarias batrachus. J. Freshwater Biol., 7, Pandey, K. and J.P. Shukla(2012): A textbook of Fish and Fisheries. Rastogi Publications, Meerut. 12. Shukla, V., et all (2007).: Bioaccumulation of Zn, Cu and Cd in Channa punctatus. J. Environ. Biol., 28, Singh, M. (1995): Haematological responses in a fresh water teleost, Channa punctatus to experimental copper and Cr poisoning. J. Environ. Biol., 16, Svobodova, Z., B. Vykusova and J. Machova(1994): The effects of pollutants on selected haematological and biochemical parameters in fish. In: Sub lethal and chronic effects of pollutants on freshwater fish (Eds.: R. Müller and R. Lloyd).Fishing new books,london. 73