Haematological and biochemical responses in a ...

Chlorpyrifos toxicity in Cyprinus carpio

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International Journal of Integrative Biology A journal for biology beyond borders

ISSN 0973-8363

Haematological and biochemical responses in a freshwater fish Cyprinus carpio exposed to chlorpyrifos M Ramesh, M Saravanan* Unit of Toxicology, Department of Zoology, Bharathiar University, Coimbatore, India, Submitted: 8 Jul. 2008; Accepted: 18 Aug. 2008

Abstract The acute toxicity of an organophosphorus insecticide chlorpyrifos, on haematological and biochemical parameters of an economically important freshwater teleost fish Cyprinus carpio was evaluated under static conditions. The median lethal concentration of the insecticide chlorpyrifos was found to be 5.28 ppm indicating the high toxicity of the insecticide. Fishes were exposed to predetermined LC50 (5.28 ppm) and the alterations of haematological and biochemical parameters were studied at the end of 24 h period. During above treatment period the haematological parameters like RBC (-72.43%), haemoglobin (-18.35%) and plasma protein (16.46%) level decreased in the insecticide treated fish where as WBC (+57.94%) and plasma glucose (+26.35%) level increased. The observed haematological and biochemical parameters may be used as non-specific biomarkers in the field of environmental toxicology. Keywords: Chlorpyrifos, Hematology, Biochemical, Acute toxicity, Cyprinus carpio.

INTRODUCTION Aquatic ecosystems that run through agricultural areas have high probability of being contaminated by run off and ground water leaching by a variety of chemicals. Highly effective pesticides are used tremendously, which on entering the aquatic environment bring multiple changes in organism by altering the growth rate, nutritional value, behavioural pattern, etc. A major part of the world’s food is being supplied from fish source, so it is essential to secure the health of fishes (Tripathi et al., 2002). In India as much as 70% of the chemical formulations employed in agricultural practices are believed to affect non-target organisms and to find their way to fresh water bodies, ultimately polluting them (Bhatnagar et al., 1992). Ralio et al. (1985) reported that the blood parameters of diagnostic importance are erythrocyte and leucocytes counts, haemoglobin, haematocrit and leucocyte differential counts would readily respond to incidental factor such

as physical stress and environmental stress due to water contaminants. Since early detection of specific physiological abnormalities provide an indication of exposure prior to manifestation of any gross damage, 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. Chlorpyrifos (0, 0-diethyl-0-(3,5,6trichloro-2-pyridyl)-phosphorothioate) commonly known as Dursban is a broad spectrum insecticide. Chlorpyrifos is the second largest selling OP agro chemical in India. Dursban is the most toxic organophosphorus compound to fish and is more toxic than organochlorine compounds (Tilak et al., 2001). Therefore in the present investigation an attempt has been made to study the effect of chlorpyrifos on alterations in haematological and biochemical parameters of Cyprinus carpio with particular reference to the concentration of the pesticide and duration of exposure.

*

Corresponding author: M. Saravanan, Ph.D. Unit of Toxicology, Department of Zoology, Bharathiar University, Coimbatore – 641046, India Email: [email protected]

International Journal of Integrative Biology © IJIB, All rights reserved

MATERIALS AND METHODS Specimens of Cyprinus carpio var. communis were obtained from Tamil Nadu Fisheries

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Table. 1. Changes in the haematological and biochemical parameters level in a freshwater fish Cyprinus carpio treated with acute concentration of chlorpyrifos.

Parameters Erythrocytes (million/cu.mm) Leucocytes (thousand/cu.mm) Haemoglobin (g/dl) Plasma glucose (mg/100 ml) Plasma protein (μg/ml)

Control

Experiment

Percent Change

Calculated ‘t’ Value

3.240 ± 0.046

0.893 ± 0.025

-72.43

33.52*

9.688 ± 1.015

15.302 ± 8.575

+57.94

0.58*

4.450 ± 0.163

3.633 ± 0.370

-18.35

-1.53*

91.035 ± 1.943

115.029 ± 8.438

+26.35

+2.31*

6.705 ± 0.653

5.601 ± 0.298

-16.46

0.92*

Values are means ± SE of five individual observations, Values are significant at P < 0.05

Development Corporation Limited, Aliyar Fish Farm, Aliyar, Tamil Nadu, India and acclimated to laboratory conditions for 15 days in large cement tank (6” X 4 X 3). During acclimatization period, fish were fed ad libitum with rice bran and groundnut oil cake in the form of dough once daily. Water was replaced every 24 h after feeding in order to maintain a healthy environment for the fish during both acclimation and experimental period. This ensures sufficient oxygen supply for the fish and the environment is devoid of any accumulated metabolic waste. After acclimatization, fish with an average length of 7.5 cm and an average weight of 6.0 g were selected for the study. The physico chemical features of the tap water were estimated as per APHA (1998) and are as follows: Temperature: 25.0±2.0ºC; pH: 7.2±0.1; Dissolved Oxygen: 6.2± 0.06 mg/L, Total alkalinity: 175.0±10.00 mg/L, Total hardness: 18.0±0.5 mg/L. Salinity: 0.6±0.01 ppt. Technical grade organophosphate insecticide, chlorpyrifos (20% EC) manufactured by Zeneca Agrochemicals Ltd., Chennai, India, was used for evaluation of its toxicity to fish. For determining LC50 concentration separate circular plastic tubs of 10 liters of water capacity were taken and different concentrations of chlorpyrifos were added. Then, 10 fish were introduced into each tub. A control tub with 10 liters of water and 10 fish were also maintained (no toxicant). The concentration at which 50% mortality of fish occurred after 24 h was selected as the medium lethal concentration (LC50) for 24 h, which was 5.28 ppm. The LC50 concentration for 24 h was calculated by the probit analysis method of Finney (1978), with a confident limit of 5% level. For acute toxicity studies five circular plastic tubs were taken and each was filled with 20 L of


water and 5.28 ppm of chlorpyrifos. 20 fish which were already withheld from feeding for 24h were introduced into each tub. Control was maintained into 2 circular plastic tubs with 20 fish per tub. At the end of 24h period exposure, blood was drawn from control and chlorpyrifos treated fish by cardiac puncture using a hypodermic syringe previously rinsed with heparin, an anticoagulant. Pooled blood was transferred into small vials, which is previously rinsed with heparin. The whole blood was used for the estimation of erythrocyte, leucocyte counts and haemoglobin. The remainder of the blood sample was centrifuged at 10,000 rpm for 20 min. to separate the plasma which was used for plasma glucose and protein estimation. Erythrocyte and leucocytes were counted by the method of Rusia and Sood (1992) using haemocytometer. Haemoglobin content of the blood was estimated by Cyanmethaemoglobin method (Drabkin, 1946). Plasma glucose was estimated by Enzymatic–Calorimetric method (Trinder, 1969). Plasma protein estimation was done according to the method of Lowry et al. (1952). The significance of sample means between control and chlorpyrifos treated fish was tested by using Students‘t’ test.

RESULTS The changes in haematological and biochemical parameters of the fish Cyprinus carpio exposed to acute toxicity of chlorpyrifos are presented in Table 1. During acute (5.28 ppm) treatment RBC, haemoglobin and protein levels decreased where as WBC and plasma glucose level increased in the pesticide treated fish. Fish exposed to chlorpyrifos show abnormal behavior changes like

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fast swimming activity, profuse secretion of mucus, hypersensitivity, jerky movement, darkening of skin on dorsal side, loss of equilibrium etc., indicating the high toxicity of chlorpyrifos.

DISCUSSION A reduction in haematological values, indicated anemia in the pesticide exposed fish may be due to erythropoiesis, haemosynthesis and osmoregulatory dysfunction or due to an increase in the rate of erythrocyte destruction in haematopoietic organs (Jenkins et al., 2003; Seth and Saxena, 2003). In the present study, the decrease in RBC count during the acute treatment might have resulted from severe anemic state or haemolysing power of toxicant (chlorpyrifos) particularly on the red cell membrane. Pesticides were found mainly in the erythrocytes (particularly in the erythrocyte content) and plasma and not in the leucocytes, platelets or stroma indicating that they mainly bind with haemoglobin. The decrease in the haemoglobin content in the present study result from rapid oxidation of haemoglobin to methaemoglobin or release of O2 radical brought about by the toxic stress of chlorpyrifos. It is increasingly recognized that xenobiotics capable of undergoing redox cycling can exert toxic effects via the generation of oxygen free radicals. Matkovics et al. (1981) observed Cyprinus carpio a quick decrease in haemoglobin content in response to paraquat toxicity and the authors suggested that it might presumably through methaemoglobin formation and a direct response of O2- radical. Changes in the leucocyte system manifest in the form of leucocytosis with heterophilia and lymphopenia which are characteristics leucocytic response in animals exhibiting stress. The increase in WBC count can be correlated with an increase in antibody production which helps in survival and recovery of the fish exposed to lindane and malathion (Joshi et al., 2002). In the present study, the significant increase in WBC count indicate hypersensitivity of leucocytes to chlorpyrifos and these changes may be due to immunological reactions to produce antibodies to cope up with stress induced by chlorpyrifos. Changes in blood glucose have been suggested as useful general indicator of stress in teleost. Nemcsok and Bores (1982) reported that blood glucose appeared to be a sensitive indicator of


environmental stress in fish. The stress – related hyperglycemia reported in many species of teleosts is mediated mainly by the effects of catecholamines (CAs) on glucose release from the liver, the main carbohydrate store in fish, with epinephrine being more potent than noreinephrine (Van Raaji et al., 1995). Chan and Woo (1978) noted that cortisol has shown to promote catabolism of peripheral tissues via, increased gluconeogenesis; leading to hyperglycemia. In the present study the significant increase of plasma glucose level might have resulted from gluconeogenesis to provide energy for the increased metabolic demands imposed by chlorpyrifos stress, particularly in osmoregulation which may contribute to the restoration of plasma osmolarity in the face of failing blood levels of Na+ and Cl-. Under conditions of stress many organism will mobilize proteins as an energy source via. the oxidation of amino acids. Decreased protein level may be attributed to stress mediated immobilization of these compounds to fulfill an increased element for energy by the fish to cope with environmental condition exposed by the toxicant (Jenkins et al., 2003). The depletion in total protein content may be due to augmented proteolysis and possible utilization of their product for metabolic purposes as reported by Ravinder et al. (1988). On the otherhand, Neff (1985) has opined that decline in protein content may be related to impaired food intake, increased energy cost of homeostasis, tissue repair and detoxification mechanism during stress. In the present study, reduction in the plasma protein levels of fish treated with chlorpyrifos may lead to the availability of free state of the pesticide in the large quantity in plasma which may interfere with the biochemical mechanisms of fish making it more susceptible to the toxicant. The present study shows the high toxic nature of chlorpyrifos on fish, the fishes are very sensitive to the presence of even minute quantities of chlorpyrifos and are under severe metabolic stress. This study also shows the significance of haematological parameters in assessing the pesticide hazards to fish.

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