Effects of fenthion on the blood and tissue chemistry of a teleost fish (Heteropneustes fossilis)

J. COMP.

PATH.

1983.

EFFECTS TISSUE

VOL.

93.

27

OF FENTHION CHEMISTRY

ON THE BLOOD AND OF A TELEOST FISH

(HETEROPNEUSTES

FOSSILIS) BY

Department

A. K. of ~oologv,

SRIVASTAVA

C5&wsi&

and J. MISHRA of Gorakhpur, Gorakhpur 273001,

U.P.,

India

INTRODUCTION

Fenthion, O,O-dimethyl 0-(4-methylthio)-m-tolylphosphorothioate, a highly effective organophosphorus larvicide used for mosquito control in many tropical countries, has been shown to reduce brain ChE activity in fishes (Brown, 1978). However, only a few investigations of fenthion toxicity in fish have been made; the effects are damage to the gills of cichlid fishes (Jauch, 1979) and blood dyscrasia in the snakehead, Channa punctatus (Lone and Javaid, 1976). Since organophosphorus insecticides disturb carbohydrate metabolism (Srivastava and Singh, 1980, 1981) and also affect gamma-2 globulin and erythrocyte Ch E (MacFarlane and Robb-Smith, 1961), we have studied muscle and hepatic glycogen content, blood glucose and chloride concentrations, and certain haematological measurements in a freshwater Indian catfish, Heteropneustes fossilis, after exposure to sublethal concentrations of fenthion. MATERIALS

AND

METHODS

The fish, Heteropneustesfossilis (weight, 35*20-&1.39 g, N=460; length, 12*41& 0.63 cm, N=460), were brought to the laboratory and acclimatized in tap water for 10 days under natural photoperiod and ambient temperature (20.15f0.49 “C) in 50 1 glass aquaria. They were fed daily with wheat flour pellets and ground dried shrimp (Srivastava, 1966); the aquaria were cleaned and the water was changed daily. Only healthy fish of both sexes were used in the experiments. The physicochemical characteristics of the water used were: hardness, 132*5&5*59 mg per 1 CaCO,; dissolved oxygen, 7.98f0.59 mg per 1; chloride, 8.36hO.44 meq per 1; electrical conductivity, 531*00&45*45 pmhos per cm; pH, 7+37*0*02. Static acute toxicity bioassay to establish the concentration producing 50 per cent mortality in 96 h (96 h LC,,) value for fenthion was conducted according to the procedure recommended by the American Public Health Association, American Water Works Association and Water Pollution Control Federation (1971); the 96 h LC,, value was 16.25 mg per 1. A stock solution of fenthion (1 mg per ml) was prepared in acetone. For the study of the effect of the insecticide on carbohydrate metabolites and blood chloride concentrations, groups of 20 to 25 fish each (5 fish per 10 1 glass jar) were exposed to a concentration of 14.625 mg per 1 (0.9 of the 96 h LC,,) fenthion in tap water for 2, 6, 12, 48, and 96 h; fish for haematological studies were sampled at 96 h only. The average mortality amongst the treated fish was 20 per cent. Six fish from each group were selected randomly for the analyses of the selected variables. Parallel groups each of 6 control fish kept in tap water and receiving equal aliquots of acetone as the treated fish were sampled at 96 h for comparison with the exposed fish. At necropsy, the fish were anaesthetized with 1 g per 3 1 MS 222 (tricaine methaneOCQ-9975/83/010027+05

$03.00/O

0

1983

Academic

Press

Inc.

(London)

Limited

28

A.

K.

SRIVASTAVA

AND

J.

MISHRA

sulfonate), blotted dry with filter paper and blood samples were obtained by severing the caudal peduncle with a sharp razor blade. Free-flowing blood collected in titrated tuberculin syringes was used for blood glucose and chloride analyses (Oser, 1965). Muscle and liver glycogen concentrations were measured by the method of van der Vies (1954). The techniques of Srivastava (1968a, b, 1969) and Srivastava and Mishra (1979) were adopted for the haematological studies. The results were subjected to statistical analysis by Student’s t-test. RESULTS

Exposure of H. fossilis to a sublethal concentration of 14.625 mg per 1 fenthion resulted in significant decreases in muscle glycogen between 2 and 96 h; loss of hepatic glycogen occurred between 2 and 12 h but the value at 48 h was similar to the controls and there was a significant increase at 96 h. A significant rise in blood glucose concentration was found at all times except 96 h. Blood chloride concentrations showed a biphasic change; there was a significant decrease at 2 and 6 h, a return to normal at 12 h and a rise at 48 and 96 h after exposure to fenthion (Table 1). TABLE 1 EFFECT

0~

FENTHION

ON BLOOD

AND

CHEMISTRY

TISSUE

0~

H.fossilis*

Exposure periods (h) Control Muscle mg mg Liver mg mg Blood

glycogen per 100 wet wt glycogen per 100 wet wt glucose

2 per loo Blood chloride meq per 1 * Fish were t P
(water)

2

6

12

48

.-

96 .-__--.

0.32 10.01

0.2210.008:

0~26*0~008$

0.19*0.04::

0~16*0~008$

957*0.14

7.2aho.74:

7.05io.10:

5.79*0.45?

8.5ako.68

10.99*0.59f

55.74+

1.36

59.63&0.37t

62.22+0.99$

78.5212.12:

65.925

87.9ak

1.75

78.64ko.61::

80.15*0.28::

94.36+2.58

98.33*0.96:

ex$ose.e;ol14.625 + .

mg per 1 of fenthion. TABLE

EFFECT

0~ FENTHION

Characteristic Erythrocytes( lOi per 1) Leucocytes (1 Og per 1) Total differential cell countst Small lymphocytes Large lymphocytes Monocytes Neutrouhils

oiv

All values

are meansisx

0.26&0.01

1.78:

53.335

1.15

98.ia&

1.82:

(X=6).

2

HAEMATOLOGICAL

Control

VALUES

(w&r)

OF

H.fossiils* Experimental (fenthion)

5.69+027 44.00+0.89

2.10*024§ 4040+0.56

6600&2.85 1.00&0.52 -

67.17& la3 0.17*0.17 0.33 10.2 1

0.17-co.17 0.33 10.20 16.00&0.97 0.48 10.007 1.93*0.14 12.10*0.07 109.33&2.04

13.2510.48 0.40 ~o~oosp 2.27&0.09f 9.53f0.12§ 125.67-&1.71§

* Fish (H. fossilis) were exposed to 14.625 mg per 1 of fenthion (.N=6). t Per thousand cells of all types. $ P
for 96 h. All values 0 P
are

meanS+S.E.

FENTHION

TOXICOSIS

IN

FISH

29

Table 2 shows the changes in the blood of fish subjected to fenthion. The erythrocyte count decreased significantly, the leucocyte count was unchanged, and thrombocytes decreased but not significantly compared to control fish. Differential leucocyte counts, in which small lymphocytes predominated, did not differ from those of the controls. The haemoglobin content and the hematocrit of the treated fish were markedly decreased; the erythrocyte sedimentation rate (ESR) was significantly increased and there was a significant increase in the clotting time of the blood. DISCUSSION

Carbohydrate energy sources are readily utilized during a number of different situations in fish. Muscle and hepatic glycogen concentrations decline during acute hypoxia (Burton, Jones and Cairns, 1972), severe muscular exercise (Nakano and Tomlinson, 1967) and when exposed to toxicants, including organophosphorus insecticides (McLeay and Brown, 1979; Srivastava and Singh, 1980, 1981). Fish under stress secrete increased amounts of catecholamines and adrenocorticosteroids (Nakano and Tomlinson, 1967; Fryer, 1975). Such increases are known to change muscle and liver glycogen reserves in fishes; catecholamines mobilize glycogen stores (Larsson, 1973)) while corticosteroids induce glycogen synthesis (Swallow and Fleming, 1970). Thus, the marked glycogenolysis in muscle and liver with concomitant hyperglycaemia found in this study might be due to stress-induced increased secretion of catecholamines. At 96 h, however, the fenthion-induced hyperglycaemic response was abolished and synthesis of liver glycogen occurred. This suggests secretion of adrenocorticoids which convert blood glucose into glycogen. A similar response to methyl parathion intoxication was observed in H. fossilis by Srivastava and Singh ( 1981). Heteropneustes fossilis showed a considerable decrease in blood chloride from 2 to 6 h and an increase to more than pretreatment concentration at 48 and 96 h after exposure to fenthion. Blood chloride concentrations have been shown to increase and/or decrease in fishes exposed to pesticides (Eisler and Edmunds, situations, other than pesticides, 1966; Haux and Larsson, 1979) ; “stressful” also cause changes in body fluid concentrations. Reaves, Houston and Madden (1968) observed a significant decrease in plasma chloride concentration in the exposure to rainbow trout, Salmo gairdneri, during 0 to 36 h of continuous thermal stress and a rise at 72 and 100 h, which is consistent with our study on the catfish. Sublethal amounts of pesticides have been shown to cause osmoregulatory changes in fish which may be related to differential changes in water-electrolyte permeability and renal function (Leadem, Campbell and Johnson, 1974). The erythropenia, lowered haemoglobin content and haematocrit changes observed after exposure of H. fossilis to fenthion cannot be due to haemoconcentration because there was no marked change in the number of circulating leucocytes. These changes clearly indicate an anaemia induced by fenthion in the fish. Similar results have been reported in several teleostean species following treatment with organophosphorus insecticides (Butler, Ferguson and

30

A.

K.

SRIVASTAVA

AND

J.

MISHRA

Sadler, 1969 ; Lone and Javaid, 1976). The development of anaemia in H. fossilis could be due to fenthion interference with haemopoiesis and/or alteration of cell membranes by hydrolysis of acetylcholine in body fluids by cholinesterases of the erythrocytes (MacFarlane and Robb-Smith, 1961). Erythropenia due to fenthion poisoning was also reflected by the rapid sedimentation of erythrocytes; a rise in gamma-globulin might be the cause of the increased ESR (Bell, Davidson and Emslie-Smith, 1972); plasma gamma-globulin has been reported to increase in Northern Puffers following pesticide toxicosis (Eisler and Edmunds, 1966). Hougie (1971) and Lone and Javaid (1976) demonstrated that fish with disease or organophosphate toxicosis had increased blood clotting times; fenthion exposure also delayed blood coagulation in H. &dis. Apparently, this reflected the slight thrombocytopenic effect of the pesticide; Srivastava (1969) showed that blood coagulation is retarded in fish as thrombocyte counts decrease. The foregoing data indicate that fenthion evokes alterations in carbohydrate metabolism and osmoregulatory and haemostatic mechanisms of fish. Whether these changes reflect compensatory mechanisms in the fish or biochemical results of the toxic action of the insecticide remains to be elucidated. SUMMARY

Exposure of a freshwater teleost, Heteropneustes fossilis, to a concentration of 14.625 mg per 1 (0.9 of 96 h LC,,) fenthion induced muscle and hepatic glycogenoiysis with concomitant hyperglycaemia at 2, 6, 12 and 48 h after treatment. The pesticide evoked an initial fall (at 2 and 6 h) and later a rise (at 48 and 96 h) in blood chloride concentration. The treatment also caused anaemia with simultaneous increase in ESR and hypo-coagulability of whole-blood. The results are compared with organophosphate poisoning in this and in other fish species. ACKNOWLEDGMENTS

This investigation from the University

was supported by a Teacher Research Fellowship to J. Mishra Grants Commission, New Delhi, India. REFERENCES

American Public Health Association, American Water Works Association and Water Pollution Control Federation (1971). In Standard Methods for the Examination of Water and Wastewater, 13th Edit. Washington, D.C. Bell, G. H., Davidson, J., and Emslie-Smith, D. E. (1972). The formed elements of the blood. In Text Book of Physiology and Biochemistry, Churchill Livingstone, Edinburgh, pp. 415-443. Brown, A. W. A. (1978). Insecticides and fish. In Ecology of Pesticides, Wiley-Interscience, New York, pp. 169-194. Burton, D. T., Jones, A. H., and Cairns, J., Jr (1972). Acute zinc toxicity to rainbow trout, Salmo gairdneri: Confirmation of the hypothesis that death is related to hypoxia. Journal of the Fisheries Research Board of Canada, 29, 1463-1466. Butler, G. W., Ferguson, D. E., and Sadler, C. R. (1969). Effect of sublethal parathion exposure on the blood of golden shiners, Notemigonus crysoleucas. Journal of Mississ;pPi Science, 15, 33-36.

FENTHION

TOXICOSIS

IN

FISH

Eisler,

31

R., and Edmunds, P. H. (1966). Effect of endrin on blood and tissue chemistry of a marine fish. Transactions of the American Fisheries Society, 95, 153-159. Fryer, J. N. (1975). Stress and adrenocorticosteroid dynamics in the goldfish, Carassius auratus. Canadian Journal of Zoology,53, 1012-1020. Haux, C., and Larsson, A. (1979). Effects of DDT on blood plasma electrolytes in the flounder, Platichthysjesus. Ambio, 8, 171-173. Hougie, C. (197 1). Coagulation changes in healthy and sick Pacific salmon. Advances in Experimental Medicine and Biology, 22, 89-102. Jauch, D. (1979). Gill lesions in cichlid fishes after intoxication with the insecticide, fenthion. Experientia, 35, 371-372. Larsson, A. (1973). Metabolic effects of epinephrine and norepinephrine in the eel, Anguilla anguilla L. General and Comparative Endo~rinolo~, 20, 155-167. Leadem, T. P., Campbell, R. D., and Johnson, D. W. (1974). Osmoregulatory responses to DDT and varying salinities in Salmo gairdneri-I. Gill Na-KATPase. Comparative Biochemistry and Physiology, 49A, 197-205. Lone, K. P., and Javaid, M. Y. (1976). Effect of sublethal doses of three organophosphorus insecticides on the hematology of Channa punctatus (Bloch.). Pakistan Journal of Zoology, 8, 77-84. MacFarlane, R. G., and Robb-Smith, A. H. T. (1961). Functions of the Blood, Academic Press, New York, 635 pp. McLeay, D. J., and Brown, D. A. (1979). Stress and chronic effects of untreated and treated bleached kraft pulpmill effluent on the biochemistry and stamina of juvenile coho salmon, Oncorhynchus kisutch. Journal of the Fisheries Research Board of Canada, 36, 1049-l 059. Nakano, T., and Tomlinson, N. (1967). Catecholamines and carbohydrate concentrations in rainbow trout, Salmo gairdneri, in relation to physical disturbance. Journal of the Fisheries Research Board of Canada, 24, 1701-l 715. Oser, B. L. (1965). Hawk’s Physiological Chemistry. Tata McGraw-Hill, New Delhi, 1472 pp. Reaves, R. S., Houston, A. H., and Madden, J. A. (1968). Environmental temperature and the body fluid system of the freshwater teleost. II. Ionic regulation in rainbow trout, Salmo gairdneri, following abrupt thermal shock. Comparative Biochemistry and Physiology, 25, 849-860. Srivastava, A. K. (1966). Alkaline phosphates and glycogen in the intestines of certain freshwater teleosts. Current Science, 35, 154-155. Srivastava, A. K. (1968a). Studies on the hematology of certain freshwater teleosts. I. Erythrocytes. Anatomische Anzeiger, 123, 233-249. Srivastava, A. K. (1968b). Studies on the hematology of certain freshwater teleosts. IV. Hemoglobin, Folia Haematologia, 90, 41 I-418. Srivastava, A. K. (1969). Studies on the hematology of certain freshwater teleosts. V. Thrombocytes and clotting of blood. Anatomische Anzeiger, 124, 368-374. Srivastava, A. K., and Mishra, S. (1979). Blood dyscrasia in a teleost, Colisa fascia&s, following exposure to sublethal concentrations of lead. Journal of Fish Biology, 14, 199-203. Srivastava, A. K., and Singh, N. N. (1980). Observations on hyperglycemia in the murrel, Channa punctatus, after acute exposure to methyl parathion. Comparative Physiology and Ecology, 5, 100-l 0 1. Srivastava, A. K., and Singh, N. N. (1981). Effects of acute exposure to methyl parathion on carbohydrate metabolism of Indian catfish, Heterojmeustes fossilis. Acta Pharmacologica et Toxicologica, 45, 26-3 1. Swallow, R. L., and Fleming, W. R. (1970). The effect of oxaloacetate, ACTH and cortisol on the liver glycogen levels of Tilapia mossambica. Comparative Bio&em&?-y and Physiolou, 36, 93-98. van der Vies, J. (1954). Two methods for the determination of glycogen in liver. Biochemical Journal, 57, 4 1 O-4 16. [Received for publication,

September 24th, 19821