- Email: [email protected]
Toxic effects of two organophosphorus pesticides on fructose-1,6-diphosphate aldolase activity of liver, brain and gills of the freshwater fish Clarias batrachus
Environmental Pollution (Series A) 31 (1983) I-7
Toxic Effects of Two Organophosphorus Pesticides on Fructose-l,6-Diphosphate Aldolase Activity of Liver, Brain and Gills of the Freshwater Fish C/arias batrachus R. S. Tandon & Ajai Dubey Department of Zoology, Lucknow University, Lucknow-226007, India
ABSTRACT The toxic effects of different concentrations of two organophosphorus pesticides, malathion (4.80-6.20ppm) and dimecron (2.50-6.00ppm), produced elevated activity of the aldolase enzyme of liver, brain and gill tissues in the freshwater fish Clarias batrachus, exposedfor periods of 24 to 96 h. The toxic conditions produced by the pesticide ultimately caused the death of the fish owing to the failure of the respiratory centre of the brain. The breakdown of proteins and the diabetic condition caused by the organophosphorus pesticide toxicity appeared to be the possible cause of increased aldolase activity.
INTRODUCTION The overuse of pesticides and other chemicals in some agricultural and public health operations may not cause disequilibrium or disturbance in aquatic and terrestrial environments, but it has sometimes adversely affected non-target organisms such as freshwater fishes. The activity of different enzymes in the animal body may be altered and finally death occurs. Several workers have reported that the activity of aldolase, an important enzyme in the glycolytic chain (where it breaks down fructose-l,6diphosphate), has been significantly altered by different pesticides in different animals (Bhatia et al., 1972, 1973; Krampl & Grigel, 1972; Kacew & Singhal, 1973). This aldolase enzyme is usually found in the soluble fraction of the cytoplasm of different tissues whereas in the brain 1
Environ. Pollut. Ser. A. 0143-1471/83/0031-0001/$03-00 © Applied Science Publishers
Ltd, England, 1983. Printed in Great Britain
2
R.S. Tandon, Ajai Dubey
it is strongly bound, together with hexokinase, to the mitochondrial fraction (Boyer et al., 1961). The present paper describes the toxic effects of two organophosphorus pesticides, malathion and dimecron, on the aldolase activity of liver, brain and gill tissues in the freshwater fish Clarias batrachus. MATERIALS A N D METHODS Live fish Clarias batrachus obtained from the River Gomti at Lucknow were transported to the laboratory in natural water, and treated with KMnO4 solution (2 mg litre- 1) to remove ectoparasites, fungi and other infections. The fish were allowed to rest for 96 h in large glass aquaria containing dechlorinated tap water to acclimatise them to the laboratory environment. Only active and apparently healthy-looking fish (weight and length range 150-170 g and 20.5-22.0 cm, respectively) were selected for the experiment. These were properly fed and starved for only 48 h before the experiment. Fish from the same group, kept under normal conditions, were used as controls. Static bioassay tests were used for the experiment (Doudoroff et al., 1951; APHA, 1975). Four different concentrations of each pesticide were selected on the basis of 80-100 ~o survival for 24 to 96 h exposure (Table 2). Fish were taken out from the test solution after every 24 h. They were washed with distilled water and their liver, brain and gills were dissected out and kept in 0.7 ~ cold saline solution. 2-5 ~ homogenates, prepared in 0.7 ~ cold saline solution at 2°C, were found to give optimum enzymatic activity. Biochemical analysis was performed according to the method of Beck (1955). Optical density was measured at 550nm by using a Bausch and Lomb Spectronic-20 spectrophotometer. The aldolase contents of 10 fish were used to determine the mean activity every 24 h and 20 fish were used to determine the percentage survival. Pesticides manufactured by Cyanamid India Ltd (malathion) and Ciba-Geigy India Ltd (dimecron), and analytical-grade chemicals manufactured by Sigma (USA), BDH (England) and E. Merck, were used for the enzymatic analysis. RESULTS The physicochemical properties of water (pH, temperature, dissolved oxygen, total hardness and alkalinity), observed throughout the experi-
TABLE 1 Characteristics of Water During the Experiment (Mean* + Standard Deviation)
Pesticide
Malathion Dimecron
pH
Water temperature ( ° C)
Dissolved oxygen (mg litre - ~)
Total hardness (mg litre - 1)
Total alkalinity (rag litre - 1)
7.5+0.15 7.3+0.20
244- 1 22+ 1
6-0___0.30 6.24-0.40
1244- 10 1 1 0 + 15
116+8 1024- 10
a M e a n v a l u e s o b t a i n e d a f t e r r e p e a t i n g t h e e x p e r i m e n t 10 times.
TABLE 2 Effects o f M a l a t h i o n a n d D i m e c r o n o n A l d o l a s e A c t i v i t y a n d o n t h e S u r v i v a l o f t h e F i s h
C. batrachus Pesticide concentration (ppm)
A ldolase, pmoles Triose 100 m g - ~fresh wt min- a (mean 4- SD)
24
Time o f exposure (h) 48 72
96
Malathion: 4.80 5.00 5.60 6.20
4.32 4.60 4-81 4.99
___0.38* _+ 0 . 4 1 " ___0.32* + 0.42*
Liver ( C o n t r o l : 3.86 + 0.34) 4-87 + 0-46* 5.14 + 0.49* 4.96 4- 0-30* 5.01 + 0-50* 5-20 ___0.46* ----
4.92 ___0 . 3 1 " ----
4.80 5-00 5-60 6.20
1.724-0.21 1.874-0-18 1-90+0.18" 2-06 + 0 . 1 2 "
B r a i n ( C o n t r o l : 1.68 ___0.17) 1.894-0.20 2.084-0.16" 1.99+0.20" 2.164-0.21" 2.104-0.22" ----
2.004-0.14" ----
4.80 5-00 5.60 6.20
0.584-0.08 0-61 4- 0 ' 0 5 0.62 4- 0.08 0.68 4- 0.06*
Gills ( C o n t r o l : 0.55 + 0.10) 0.63+0.11 0.72+0-07* 0 ' 6 9 + 0.08* 0.74 4- 0 ' 0 9 * 0.71 4- 0-05* ----
0-654-0.06 ----
Control 4.80 5.00 5-60 6-20
24
48
100 100 100 100 90
100 100 90 80 40
Percentage survival o f t h e ~ s h 72 96 100 100 80 20 5
100 80 30 0 0
120 100 30 5 0 0
4
R.S. Tandon, Ajai Dubey TABLE 2--contd.
Pesticide concentration (ppm)
Aldolase, ltmoles Triose lOOmg- 1fresh wt rain- 1 (Mean 4- SD ) Time of exposure (h) 24
48
72
96
Dimecron: 2.50 3.25 4.25 6.00
4.18 4.60 4.66 5.00
4- 0.38 + 0.48* + 0.40* + 0.39*
Liver (Control: 3.91 + 0.32) 4.62 + 0-46* 5.69 4- 0.49* 4.98 ___0-49* 5-16 _ 0.41" 5-10 + 0-44* ----
5.25 + 0.32* ----
2.50 3.25 4.25 6.00
1.68-1-0.20 1.70+0.17 1.80+0.18" 2-08 4- 0.15"
Brain (Control: 1.52 _ 0-19) 1.72+0.22 1.944-0.21" 1.964-0.14" 2-004-0.16" 2.11 4-0.20* ----
1.884-0.18" ----
2"50 3-25 4.25 6'00
0.42 + 0'09 0-464-0"05 0.49 4- 0.10" 0.50 4- 0.08*
Gills (Control: 0"36 4- 0.08) 0.44 4- 0.10 0.48 4- 0.05* 0"53+_0.11" 0.564-0.12" 0.58 4- 0.12" ----
0.47 4- 0"07 ----
Control 2.50 3-25 4.25 6.00
24
48
100 100 100 90 80
100 100 90 80 30
Percentage survival ofthefish 72 96 100 80 80 30 0
100 80 40 5 0
120 100 40 0 0 0
* Significant value (P < 0.01). m e n t , a r e s u m m a r i s e d i n T a b l e 1. T h e effects o f t h e p e s t i c i d e s o n a l d o l a s e a c t i v i t y i n t h e t h r e e t i s s u e s a r e s u m m a r i s e d i n T a b l e 2. A t t h e l o w e s t concentrations of malathion (4.80ppm) and dimecron (2.50ppm) the a l d o l a s e a c t i v i t y o f t h e t h r e e t i s s u e s i n c r e a s e d c o n t i n u o u s l y u p t o 72 h exposure and then started to decrease after about 96h, but always r e m a i n e d a b o v e t h e i r r e s p e c t i v e c o n t r o l s . B u t a t 5-00, 5.60 a n d 6 . 2 0 p p m m a l a t h i o n a n d 3.25, 4-25 a n d 6-00 p p m d i m e c r o n , t h e a l d o l a s e a c t i v i t y o f the three tissues increased continuously until the terminal hour of exposure.
Effects of OP pesticides on fish
5
The maximum increase in aldolase activity during the malathion experiment was observed in liver (34.71 ~) at 5.60 ppm concentration and with dimecron, in gills (55.55 ~) at 3.25 ppm concentration. Malathion also produced behavioural changes such as convulsions, rapid air gulping, higher opercular beating, loss of equilibrium, sensitivity to touch and sound, whereas dimecron did not produce any significant apparent symptoms. The increases in aldolase activity of liver, brain and gills due to the toxic effects of both malathion and dimecron were statistically significant (P < 0.01).
DISCUSSION The activity of aldolase, which is an important gluconeogenic enzyme, increased significantly in C. batrachus, possibly owing to the catabolism of proteins into glucose as a result of the malathion and dimecron treatment. This resulted in elevated body glucose, which was utilised in the usual respiratory cycle for the production of energy in the body to withstand the stress conditions caused by the pesticide. Stohlman & Lillie (1948) observed that exposure of rabbits to pesticides increased the body's need for glucose, causing the breakdown of proteins, because a greater amount of energy was required during enhanced muscular activity and hyperpyrexia. Ashmore & Weber (1959), Weber et al. (1967) and Williamson et al. (1968) also observed that gluconeogenic enzymes in rat liver were elevated during starvation and diabetes. Elevations in glucose levels have also been reported in rats owing to the action of diazinon (Dybing & S6gnen, 1958) and malathion (Gupta, 1974). It was further observed that enzyme activity was more highly elevated in liver and gills than in brain tissue of C. batrachus owing to the action of both malathion and dimecron, and our results are comparable with those of these workers, particularly with respect to the involvement of liver. The gills, directly in touch with the pesticides, also showed a clear reaction to them. The brain, being the last organ to be reached by the pesticides, also had its enzyme activity elevated above the controls. This suggested that the toxic effect of the pesticides on brain enzyme activity was increased from 2 ~o to 28 ~ by malathion and from 1 0 ~ to 38 ~ by dimecron at different concentrations and intervals of exposure. The respiratory centre of the brain was also affected during prolonged
6
R.S. Tandon, Ajai Dubey
exposure to the pesticides, causing the failure of the respiratory system and finally death due to asphyxiation. It is also possible that the fishes' olfactory receptors, which depend upon chemical clues for their growth, movement and reproduction, were seriously affected or interrupted, resulting in the apparent behavioural changes in the fish. Eisler (1970) observed that malathion caused lethal to sublethal toxicity in several species of fishes such as eel, killifish, pufferfish, mullet and blue head, the dose of malathion (LCs0) varying from 82 pptm in eel to 3250pptm in pufferfish. Pimental (1971) found that 196pptm (LCso) malathion and 8000 pptm (LCs0) phosphamidon were lethal to rainbow trout after only 48 h. Dewaide (1971) observed that in all vertebrates, liver is the most important site for biotransformation of xenobiotics, and that fish gills have a rather poor drug-metabolising capacity. Despite the supposed existence of a xenobiotic-metabolising capacity of the fish tissues, the aldolase activity increased in the three tissues of Clarias batrachus owing to intoxication by both malathion and dimecron at different concentrations and durations. ACKNOWLEDGEMENT The financial assistance provided by the University Grants Commission to Ajai Dubey for this work is gratefully acknowledged. REFERENCES American Public Health Association (1975). Standard methods for the examination of water and waste water, 14th edn., New York, American Public Health Association. Ashmore, J. & Weber, G. (1959). The role of hepatic glucose-6-phosphatase in the regulation of carbohydrate metabolism. Vitam. Horm. Lpz., 17, 65-74. Beck, W. S. (1955). Determination of Triose phosphate and proposed modification in aldolase method of Sibley and Lehninger. J. biol. Chem., 212, 847-57. Bhatia, S. C., Sharma, S. C. & Venkitasubramanian, T. A. (1972). Effect of dieldrin on certain enzyme systems of rat liver. Br. J. exp. Pathol., 53, 419,26. Bhatia, S. C., Sharma, S. C. & Venkitasubramanian, T. A. (1973). Effect of dieldrin on hepatic carbohydrate metabolism and protein biosynthesis in vivo. Toxicol. appl. Pharmacol., 24, 216-29.
Effects of OP pesticides on fish
7
Boyer, P. D., Lardy, H. & Myrback, K. (1961). The enzymes, 5. New York, London, Academic Press. Dewaide, J. H. (1971). Metabolism of xenobiotics. Comparative study and kinetic studies as basis for environmental pharmacology. PhD thesis, Catholic University of Nijmegen. Doudoroff, P., Anderson, B. G., Burdick, G. E., Galtsoff, P. S., Hart, W. B., Patrick, R., Strong, E. R., Surber, E. W. & Van Horn, W. M. (1951). Bioassay methods for the evaluation of acute toxicity of industrial wastes to fish. Sewage Ind. Wastes, 23, 1380-97. Dybing, E. & S6gnen, E. (1958). Hyperglycemia in rats after diazinon and other cholinesterase inhibitors. Acta Pharmacol. Toxicol., 14, 231-3. Eisler, R. (1970). Acute toxicities of organochlorine and organophosphorus insecticides to estuarine fish. Tech. Pap. U.S. Dept. Int. Bur. Sport. Fish Wildl., 46, 1-12. Gupta, P. K. (1974). Malathion induced biochemical changes in rats. Acta Pharmacol. Toxicol., 35, 191-4. Kacew, S. & Singhal, R. L. (1973). Metabolic alterations after chronic exposure to ~-chlordane. Toxicol. Appl. Pharmacol., 24, 539-44. Krampl, V. & Grigel, M. (1972). The relation of serum enzymes to morphologic changes in rat liver after injection of chlorinated cyclodiene insecticides. Pracovni Lekarstvi, 24, 121-4. Pimental, D. (1971). Ecological effects of pesticides on non-target species. Washington DC, Executive Office of the President. Office of Science and Technology. Stohlman, E. F. & Lillie, R. D. (1948). The effect of DDT on the blood sugar and of glucose administration on the acute and chronic poisoning of DDT in rabbits. J. Pharmacol. exp. Ther., 93, 351-61. Weber, G., Lea, M. A., Hird Convery, H. J. & Stamm, N. B. (1967). Regulation of gluconeogenesis and glycolysis: studies of mechanisms controlling enzyme activity. Adt,. Enzyme Regul., 5, 257-98. Williamson, J. R., Browning, E. T. & Olson, M. S. (1968). Interrelations between fatty acid oxidation and the control of gluconeogenesis in perfused rat liver. Adv. Enzyme Regul., 6, 67-100.
Toxic Effects of Two Organophosphorus Pesticides on Fructose-l,6-Diphosphate Aldolase Activity of Liver, Brain and Gills of the Freshwater Fish C/arias batrachus R. S. Tandon & Ajai Dubey Department of Zoology, Lucknow University, Lucknow-226007, India
ABSTRACT The toxic effects of different concentrations of two organophosphorus pesticides, malathion (4.80-6.20ppm) and dimecron (2.50-6.00ppm), produced elevated activity of the aldolase enzyme of liver, brain and gill tissues in the freshwater fish Clarias batrachus, exposedfor periods of 24 to 96 h. The toxic conditions produced by the pesticide ultimately caused the death of the fish owing to the failure of the respiratory centre of the brain. The breakdown of proteins and the diabetic condition caused by the organophosphorus pesticide toxicity appeared to be the possible cause of increased aldolase activity.
INTRODUCTION The overuse of pesticides and other chemicals in some agricultural and public health operations may not cause disequilibrium or disturbance in aquatic and terrestrial environments, but it has sometimes adversely affected non-target organisms such as freshwater fishes. The activity of different enzymes in the animal body may be altered and finally death occurs. Several workers have reported that the activity of aldolase, an important enzyme in the glycolytic chain (where it breaks down fructose-l,6diphosphate), has been significantly altered by different pesticides in different animals (Bhatia et al., 1972, 1973; Krampl & Grigel, 1972; Kacew & Singhal, 1973). This aldolase enzyme is usually found in the soluble fraction of the cytoplasm of different tissues whereas in the brain 1
Environ. Pollut. Ser. A. 0143-1471/83/0031-0001/$03-00 © Applied Science Publishers
Ltd, England, 1983. Printed in Great Britain
2
R.S. Tandon, Ajai Dubey
it is strongly bound, together with hexokinase, to the mitochondrial fraction (Boyer et al., 1961). The present paper describes the toxic effects of two organophosphorus pesticides, malathion and dimecron, on the aldolase activity of liver, brain and gill tissues in the freshwater fish Clarias batrachus. MATERIALS A N D METHODS Live fish Clarias batrachus obtained from the River Gomti at Lucknow were transported to the laboratory in natural water, and treated with KMnO4 solution (2 mg litre- 1) to remove ectoparasites, fungi and other infections. The fish were allowed to rest for 96 h in large glass aquaria containing dechlorinated tap water to acclimatise them to the laboratory environment. Only active and apparently healthy-looking fish (weight and length range 150-170 g and 20.5-22.0 cm, respectively) were selected for the experiment. These were properly fed and starved for only 48 h before the experiment. Fish from the same group, kept under normal conditions, were used as controls. Static bioassay tests were used for the experiment (Doudoroff et al., 1951; APHA, 1975). Four different concentrations of each pesticide were selected on the basis of 80-100 ~o survival for 24 to 96 h exposure (Table 2). Fish were taken out from the test solution after every 24 h. They were washed with distilled water and their liver, brain and gills were dissected out and kept in 0.7 ~ cold saline solution. 2-5 ~ homogenates, prepared in 0.7 ~ cold saline solution at 2°C, were found to give optimum enzymatic activity. Biochemical analysis was performed according to the method of Beck (1955). Optical density was measured at 550nm by using a Bausch and Lomb Spectronic-20 spectrophotometer. The aldolase contents of 10 fish were used to determine the mean activity every 24 h and 20 fish were used to determine the percentage survival. Pesticides manufactured by Cyanamid India Ltd (malathion) and Ciba-Geigy India Ltd (dimecron), and analytical-grade chemicals manufactured by Sigma (USA), BDH (England) and E. Merck, were used for the enzymatic analysis. RESULTS The physicochemical properties of water (pH, temperature, dissolved oxygen, total hardness and alkalinity), observed throughout the experi-
TABLE 1 Characteristics of Water During the Experiment (Mean* + Standard Deviation)
Pesticide
Malathion Dimecron
pH
Water temperature ( ° C)
Dissolved oxygen (mg litre - ~)
Total hardness (mg litre - 1)
Total alkalinity (rag litre - 1)
7.5+0.15 7.3+0.20
244- 1 22+ 1
6-0___0.30 6.24-0.40
1244- 10 1 1 0 + 15
116+8 1024- 10
a M e a n v a l u e s o b t a i n e d a f t e r r e p e a t i n g t h e e x p e r i m e n t 10 times.
TABLE 2 Effects o f M a l a t h i o n a n d D i m e c r o n o n A l d o l a s e A c t i v i t y a n d o n t h e S u r v i v a l o f t h e F i s h
C. batrachus Pesticide concentration (ppm)
A ldolase, pmoles Triose 100 m g - ~fresh wt min- a (mean 4- SD)
24
Time o f exposure (h) 48 72
96
Malathion: 4.80 5.00 5.60 6.20
4.32 4.60 4-81 4.99
___0.38* _+ 0 . 4 1 " ___0.32* + 0.42*
Liver ( C o n t r o l : 3.86 + 0.34) 4-87 + 0-46* 5.14 + 0.49* 4.96 4- 0-30* 5.01 + 0-50* 5-20 ___0.46* ----
4.92 ___0 . 3 1 " ----
4.80 5-00 5-60 6.20
1.724-0.21 1.874-0-18 1-90+0.18" 2-06 + 0 . 1 2 "
B r a i n ( C o n t r o l : 1.68 ___0.17) 1.894-0.20 2.084-0.16" 1.99+0.20" 2.164-0.21" 2.104-0.22" ----
2.004-0.14" ----
4.80 5-00 5.60 6.20
0.584-0.08 0-61 4- 0 ' 0 5 0.62 4- 0.08 0.68 4- 0.06*
Gills ( C o n t r o l : 0.55 + 0.10) 0.63+0.11 0.72+0-07* 0 ' 6 9 + 0.08* 0.74 4- 0 ' 0 9 * 0.71 4- 0-05* ----
0-654-0.06 ----
Control 4.80 5.00 5-60 6-20
24
48
100 100 100 100 90
100 100 90 80 40
Percentage survival o f t h e ~ s h 72 96 100 100 80 20 5
100 80 30 0 0
120 100 30 5 0 0
4
R.S. Tandon, Ajai Dubey TABLE 2--contd.
Pesticide concentration (ppm)
Aldolase, ltmoles Triose lOOmg- 1fresh wt rain- 1 (Mean 4- SD ) Time of exposure (h) 24
48
72
96
Dimecron: 2.50 3.25 4.25 6.00
4.18 4.60 4.66 5.00
4- 0.38 + 0.48* + 0.40* + 0.39*
Liver (Control: 3.91 + 0.32) 4.62 + 0-46* 5.69 4- 0.49* 4.98 ___0-49* 5-16 _ 0.41" 5-10 + 0-44* ----
5.25 + 0.32* ----
2.50 3.25 4.25 6.00
1.68-1-0.20 1.70+0.17 1.80+0.18" 2-08 4- 0.15"
Brain (Control: 1.52 _ 0-19) 1.72+0.22 1.944-0.21" 1.964-0.14" 2-004-0.16" 2.11 4-0.20* ----
1.884-0.18" ----
2"50 3-25 4.25 6'00
0.42 + 0'09 0-464-0"05 0.49 4- 0.10" 0.50 4- 0.08*
Gills (Control: 0"36 4- 0.08) 0.44 4- 0.10 0.48 4- 0.05* 0"53+_0.11" 0.564-0.12" 0.58 4- 0.12" ----
0.47 4- 0"07 ----
Control 2.50 3-25 4.25 6.00
24
48
100 100 100 90 80
100 100 90 80 30
Percentage survival ofthefish 72 96 100 80 80 30 0
100 80 40 5 0
120 100 40 0 0 0
* Significant value (P < 0.01). m e n t , a r e s u m m a r i s e d i n T a b l e 1. T h e effects o f t h e p e s t i c i d e s o n a l d o l a s e a c t i v i t y i n t h e t h r e e t i s s u e s a r e s u m m a r i s e d i n T a b l e 2. A t t h e l o w e s t concentrations of malathion (4.80ppm) and dimecron (2.50ppm) the a l d o l a s e a c t i v i t y o f t h e t h r e e t i s s u e s i n c r e a s e d c o n t i n u o u s l y u p t o 72 h exposure and then started to decrease after about 96h, but always r e m a i n e d a b o v e t h e i r r e s p e c t i v e c o n t r o l s . B u t a t 5-00, 5.60 a n d 6 . 2 0 p p m m a l a t h i o n a n d 3.25, 4-25 a n d 6-00 p p m d i m e c r o n , t h e a l d o l a s e a c t i v i t y o f the three tissues increased continuously until the terminal hour of exposure.
Effects of OP pesticides on fish
5
The maximum increase in aldolase activity during the malathion experiment was observed in liver (34.71 ~) at 5.60 ppm concentration and with dimecron, in gills (55.55 ~) at 3.25 ppm concentration. Malathion also produced behavioural changes such as convulsions, rapid air gulping, higher opercular beating, loss of equilibrium, sensitivity to touch and sound, whereas dimecron did not produce any significant apparent symptoms. The increases in aldolase activity of liver, brain and gills due to the toxic effects of both malathion and dimecron were statistically significant (P < 0.01).
DISCUSSION The activity of aldolase, which is an important gluconeogenic enzyme, increased significantly in C. batrachus, possibly owing to the catabolism of proteins into glucose as a result of the malathion and dimecron treatment. This resulted in elevated body glucose, which was utilised in the usual respiratory cycle for the production of energy in the body to withstand the stress conditions caused by the pesticide. Stohlman & Lillie (1948) observed that exposure of rabbits to pesticides increased the body's need for glucose, causing the breakdown of proteins, because a greater amount of energy was required during enhanced muscular activity and hyperpyrexia. Ashmore & Weber (1959), Weber et al. (1967) and Williamson et al. (1968) also observed that gluconeogenic enzymes in rat liver were elevated during starvation and diabetes. Elevations in glucose levels have also been reported in rats owing to the action of diazinon (Dybing & S6gnen, 1958) and malathion (Gupta, 1974). It was further observed that enzyme activity was more highly elevated in liver and gills than in brain tissue of C. batrachus owing to the action of both malathion and dimecron, and our results are comparable with those of these workers, particularly with respect to the involvement of liver. The gills, directly in touch with the pesticides, also showed a clear reaction to them. The brain, being the last organ to be reached by the pesticides, also had its enzyme activity elevated above the controls. This suggested that the toxic effect of the pesticides on brain enzyme activity was increased from 2 ~o to 28 ~ by malathion and from 1 0 ~ to 38 ~ by dimecron at different concentrations and intervals of exposure. The respiratory centre of the brain was also affected during prolonged
6
R.S. Tandon, Ajai Dubey
exposure to the pesticides, causing the failure of the respiratory system and finally death due to asphyxiation. It is also possible that the fishes' olfactory receptors, which depend upon chemical clues for their growth, movement and reproduction, were seriously affected or interrupted, resulting in the apparent behavioural changes in the fish. Eisler (1970) observed that malathion caused lethal to sublethal toxicity in several species of fishes such as eel, killifish, pufferfish, mullet and blue head, the dose of malathion (LCs0) varying from 82 pptm in eel to 3250pptm in pufferfish. Pimental (1971) found that 196pptm (LCso) malathion and 8000 pptm (LCs0) phosphamidon were lethal to rainbow trout after only 48 h. Dewaide (1971) observed that in all vertebrates, liver is the most important site for biotransformation of xenobiotics, and that fish gills have a rather poor drug-metabolising capacity. Despite the supposed existence of a xenobiotic-metabolising capacity of the fish tissues, the aldolase activity increased in the three tissues of Clarias batrachus owing to intoxication by both malathion and dimecron at different concentrations and durations. ACKNOWLEDGEMENT The financial assistance provided by the University Grants Commission to Ajai Dubey for this work is gratefully acknowledged. REFERENCES American Public Health Association (1975). Standard methods for the examination of water and waste water, 14th edn., New York, American Public Health Association. Ashmore, J. & Weber, G. (1959). The role of hepatic glucose-6-phosphatase in the regulation of carbohydrate metabolism. Vitam. Horm. Lpz., 17, 65-74. Beck, W. S. (1955). Determination of Triose phosphate and proposed modification in aldolase method of Sibley and Lehninger. J. biol. Chem., 212, 847-57. Bhatia, S. C., Sharma, S. C. & Venkitasubramanian, T. A. (1972). Effect of dieldrin on certain enzyme systems of rat liver. Br. J. exp. Pathol., 53, 419,26. Bhatia, S. C., Sharma, S. C. & Venkitasubramanian, T. A. (1973). Effect of dieldrin on hepatic carbohydrate metabolism and protein biosynthesis in vivo. Toxicol. appl. Pharmacol., 24, 216-29.
Effects of OP pesticides on fish
7
Boyer, P. D., Lardy, H. & Myrback, K. (1961). The enzymes, 5. New York, London, Academic Press. Dewaide, J. H. (1971). Metabolism of xenobiotics. Comparative study and kinetic studies as basis for environmental pharmacology. PhD thesis, Catholic University of Nijmegen. Doudoroff, P., Anderson, B. G., Burdick, G. E., Galtsoff, P. S., Hart, W. B., Patrick, R., Strong, E. R., Surber, E. W. & Van Horn, W. M. (1951). Bioassay methods for the evaluation of acute toxicity of industrial wastes to fish. Sewage Ind. Wastes, 23, 1380-97. Dybing, E. & S6gnen, E. (1958). Hyperglycemia in rats after diazinon and other cholinesterase inhibitors. Acta Pharmacol. Toxicol., 14, 231-3. Eisler, R. (1970). Acute toxicities of organochlorine and organophosphorus insecticides to estuarine fish. Tech. Pap. U.S. Dept. Int. Bur. Sport. Fish Wildl., 46, 1-12. Gupta, P. K. (1974). Malathion induced biochemical changes in rats. Acta Pharmacol. Toxicol., 35, 191-4. Kacew, S. & Singhal, R. L. (1973). Metabolic alterations after chronic exposure to ~-chlordane. Toxicol. Appl. Pharmacol., 24, 539-44. Krampl, V. & Grigel, M. (1972). The relation of serum enzymes to morphologic changes in rat liver after injection of chlorinated cyclodiene insecticides. Pracovni Lekarstvi, 24, 121-4. Pimental, D. (1971). Ecological effects of pesticides on non-target species. Washington DC, Executive Office of the President. Office of Science and Technology. Stohlman, E. F. & Lillie, R. D. (1948). The effect of DDT on the blood sugar and of glucose administration on the acute and chronic poisoning of DDT in rabbits. J. Pharmacol. exp. Ther., 93, 351-61. Weber, G., Lea, M. A., Hird Convery, H. J. & Stamm, N. B. (1967). Regulation of gluconeogenesis and glycolysis: studies of mechanisms controlling enzyme activity. Adt,. Enzyme Regul., 5, 257-98. Williamson, J. R., Browning, E. T. & Olson, M. S. (1968). Interrelations between fatty acid oxidation and the control of gluconeogenesis in perfused rat liver. Adv. Enzyme Regul., 6, 67-100.