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Age related differences in the inhibition of brain acetylcholinesterase activity of Heteropneustes fossilis (Bloch) by malathion
Camp. Biochem.
Pergamon 0300-%29(94)00166-9
Phyviol. Vol. I I IA, No. 2. pp. 331-334. 1995 . Elsevier Science Ltd Printed in Great Britain 0300-9629195 $9.50 + 0.00
Age related differences in the inhibition of brain acetylcholinesterase activity of Heteropneustes fossilis (Bloch) by malathion H. M. Dutta,* J. S. D. Munshi,? G. R. Dutta,? N. K. Singh,? S. Adhikari? and C. R. RichmondsS *Department of Biological Sciences, Kent State University, Kent, OH 44242, U.S.A.; TPostgraduate Department of Zoology, Bhagalpur University, Bhagalpur, India; and ICase Western Reserve University, Cleveland, OH 44106, U.S.A. The objective of this study is to determine whether there are differences in the sensitivity of brain acetylcholinesterase between juvenile and adult fish exposed to malathion. Air-breathing catfish, Heteropneustes fossilis were used. The exposure concentration was 1.2 mg/l (sublethal), and exposure durations were 24,48, 72, and 96 hr. A reduction in the mean acetylcholinesterase activity is seen at all exposure durations in the juveniles. It also indicates a significant difference between the control and exposure groups-24, 48, 72 and 96 hr in group A. However, in the adults, a reduction is seen, only at the 72 hr exposure duration. T-value and two-tail probability show that only control and 72 hr exposed ones are significantly different. In the higher weight group, there is a recovery in AChE activity at the 96 hr exposure level, This indicates that the detoxification capacity of the fish increases with age. The results show that the juveniles are much more susceptible than the adults. Key words: Hereropneustes fossilis; Malathion;
Brain acetylcholinesterase
activity: Age Related
differences. Comp. Biochem. Physiol. 11 IA, 331-334,
1995.
Introduction In recent years, organophosphorus pesticides have replaced organochlorine pesticides. Malathion, an organophosphorus pesticide has been sprayed periodically in the state of California for eliminating medflies. According to Martinez-Tabche et al. (1994), another organophosphorus insecticide, parathion, is widely used in Mexico, and the residues of this insecticide are widespread in freshwater ecosystems, contaminating water bodies. Reddy and Philip (1994) found inhibition of AChE in all tissues, namely gill, brain, liver and muscle IO: H. M. Dutta, Dept. of Biological Sciences, Kent State University, Kent, OH 44242, U.S.A. Received 26 May 1994; revised 8 September 1994; accepted I5 September 1994.
Correspondence
in Cyprinus carpio exposed to cypermethrin. In developing countries like India and Pakistan, large quantities of organophosphorus pesticides are used in agricultural operations and for eradicating mosquitoes and other pests. Surveys of the farm wells during 1986 and 1987 for pesticides in Ontario, Canada, revealed pesticide contamination including malathion (Frank et al., 1990). Organophosphorus compounds have the ability to inhibit the enzyme acetylcholinesterase (AChE) and thereby disrupt nervous transmissions. Malaoxon, an oxygen analog of malathion, appears to be the active part that binds the AChE (O’Brien et al., 1974). Young ones of any species are more susceptible than adults to any stress. The purpose of this investigation is to determine whether there are differ331
H. M. Dutta et al.
332
ences in the sensitivity of brain AChE to inhibition by malathion between the juveniles and adults of an air-breathing catfish, Heteropneustes fossilis.
Materials and Methods Live H. fossilis of two weight groups (8.0-l 5.0 g and 2540 g) were obtained from a swamp located in North Bihar, India. There was no agricultural land nearby where pesticides were applied. Fish were acclimated in the laboratory for 2 weeks in large plexiglass aquaria and fed with chopped goat liver. The water of the aquarium was aerated continuously, The fish were acclimated for 2 weeks. Feeding was stopped 24 hr before malathion exposure. The average values for the water qualities in the holding and exposure tanks were as follows: temperature 29°C pH 7.35, DO 7.5 mg/l, CO, 4 mg/l, alkalinity 115 mg/l as CaCO,, hardness 140 mg/l as CaCO,. Bioassays to determine the 96 hr LC,, were conducted employing the technique of the American Public Health Association (198 1). Commercial grade malathion is more toxic than technical grade to fish due to other ingredients present as emulsifiers which act as synergists (Sailatha et al., 1981). Therefore, commercial grade malathion containing 50% active ingredients (Northern Minerals Ltd., Haryana, India) was used in this study. Water was used as a solvent to dissolve the malathion. Ten fish for each weight group-five for the control and five for the experiment-were used to estimate AChE activity. Fish were exposed to a sublethal concentration of 1.2 mg/l, nearly one-tenth of the 96 hr LC,, value (11.676 mg/l) for 24, 48, 72 and 96 hr in plexiglass aquaria. Controls were treated under identical conditions without the pesticide. The estimation of AChE activity of brain tissue was performed according to the method of Ellman et al. (1961). This procedure is useful for the calorimetric estimation of AChE from homogenates, cell suspensions and tissue extracts. The enzyme activity was calculated by measuring the increase in yellow color produced by thiocholine when it reacts with dithiobisnitrobenzonate ions. A Bausch and Lomb Spectronic 20 colorimeter was used for estimating AChE activity. The analyses were performed at 25 + 1°C at a wavelength of 412 nm. Preparation
of brain samples for analysis
The fish were pithed and the brain was carefully removed intact with the help of sharppointed scissors and forceps. The removal of the
brain was achieved by making a median incision in the dorsal side of the skull and excising the brain free by cutting the optic nerves and spinal cord. Care was taken to avoid any blood clots, bones, skin, portions of olfactory and optic nerves. At the same time, it was made sure that all of the brain tissues were removed. The brain was placed in a small plastic weighing boat and weighed. Then, the brain was homogenized in 2 ml of pH 8, 0.1 M phosphate buffer using an electrically operated tissue homogenizer. Homogenization was performed with the help of a Tri-R Stir-R model S63C variable speed motor attached to a homogenizer. The ground brain tissue was then diluted with a sufficient amount of phosphate buffer to contain 20mg of brain tissue per milliliter of buffer. The tissue was further homogenized, and the homogenate was analyzed immediately using the assay method described below. Estimation
of activity
A 0.4 ml aliquot of the diluted brain-buffer homogenate was pipetted into a calorimeter tube. Then 2.6 ml of pH 8.0, 0.1 M phosphate buffer were added to this and the tube was placed in the photocell of the calorimeter. The absorbance was set at zero. Then 100 ~1 of DTNB reagent were added and mixed well. This causes a slight increase in absorbance. The calorimeter was reset to zero. Twenty microliters of acetylthiocholine (ASCh) iodide solution were added quickly and mixed well. The absorbance was recorded at every 15 set interval. The yellow color produced by the enzymatic reaction resulted in changes in absorbance. The reaction rate (AA) was calculated in units of changes in absorbance per minute by plotting the absorbance readings vs. time on an arithmetic graph paper. The rates were calculated by using the following formula of Ellman et al. (1961). R = 514 x AA/C,
where R = rate of pmoles of substrate (ASCh) hydrolyzed/min/g brain tissue, and C = original concentration of brain tissue (mgiml) in the homogenate (this value remains constant at 20 mg/ml throughout the study). AA = change in asorbncelmin. Since the original concentration was the same throughout, the formula was simplified as follows. R = 574 x AA/C, R = 574 x AA/20, R = 28.7 x AA.
Inhibition
of brain
AChE
Rate of activity is expressed as PM ASCh hydrolyzed/min/g of brain tissue. Triplicate measurements were done for each sample, and the mean value was taken. A t-test was performed to distinguish between the control and samples at 24, 48, 72 and 96 hr exposures of malathion for both groups A and B (Table I).
activity
!
I 1
333
by malathion
60.
I I
Results and Discussion The results are shown in Table 1. Statistical analysis showed a sharp reduction in the mean AChE activity at all exposure durations in the juveniles (lower weight group). It also indicates a significant difference between the control and exposure groups at 24,48,72 and 96 hr in group A. In the adults (higher weight group) a moderate reduction in AChE activity is seen only at the 72 hr exposure level. A t-value and two-tail probability show that only control and 72 hr exposed samples are significantly different from each other. The results (Table 1) reflect a remarkable differential change in the AChE activity in juvenile fish compared with the adults. Compared with the control, a continuous decline has been observed in AChE activity in the juvenile fish after 24, 48, 72 and 96 hr exposure time. Compared with the control, a slight reduction in the activity has been observed in the adult fish group in 48 hr, and a moderate reduction in the activity occurs in 72 hr exposure. In the higher weight group, there is recovery in AChE activity at the 96 hr exposure level. Figure 1 shows the percent inhibition of AChE activity compared with controls, in juveniles and adults at different exposure periods. This inhibition reinforces the results shown in Table 1. The results show that the capacity to withstand stress by malathion is more in the adult than in the juveniles. The capacity to adapt and recover is also higher in the adults as seen in the
Table AChE
I. T-test:
Exposure duration
brain acetylchohnesterase hydrolyzed/min/g) in control exposed fish Group
Control 24 hr 48 hr 72 hr 96 hr
A
Control 24 hr 48 hr 72 hr 96 hr
B
Activity mean and SD
and
activity (11M malathion
T-value control
vs. Two-tail probability
16.278 4.861** 3.057** 2.044** 2.199**
+ + + + -7
1.197 0.519 0.594 0.478 0.418
27.80 31.61 42.03 35.02
0.000 0.000 0.000 0.000
15.726 f 4.790 13.028 9.05” 14.963
k f k + +
0.105 f ,880 3.326 2.599 1.317
1.21 2.08 6.39 1.22
0.260 0.071 0.000 0.259
Group A = lower weight group (S-15 g). Group B = higher weight group (2540 g). ** = P
0
24
-LOWER ‘COMPARED
WT
72
46
DURATION
OF EXPOSURE
GROUP
-1~ HIGHER
96
(Hr) WT
GROUP
TO CONTROL
Fig. 1. Percent inhibition of AChE activity compared to control values in juveniles (lower weight group) and adults (higher weight group).
adults exposed for 96 hr. Our results are in conformity with the findings of Szabo et al. (1992) and Reddy et al. (1991). Szabo et al. (1992) had studied the effects of pesticides on carp AChE activity. Treatment with paraquat, an organophosphorus pesticide, led to a 50% decrease in the serum AChE activity, followed by a transient increase over the control level. After 2 weeks of chronic treatment, fish treated with paraquat showed an elevated level (130% compared with the control level) of AChE activity. They concluded that pesticides occurring in natural waters not only inhibited AChE activity in fish but might influence the resynthesis of the enzyme as well. Reddy et al. (1991) had shown that a maximum inhibition of AChE activity in Tilupiu mossumbicu exposed to cypermethrin occurred at 6 hr in juveniles and at 12 hr in adults. Ghosh and Bhaltacharya ( 1992) exposed Chunnu punt tufus (Bloch) to non-lethal levels of metacidand carbaryl for 7 days in the field. These insecticides produced a significant inhibition of brain AChE activity, accompanied by a concurrent increase in acetylcholine. Suresh et ul. (1992) recorded a significant suppression in AChE activity in gill. kidney, intestine, brain, liver and muscle of Cyprinus curpio exposed to sublethal concentrations of mercury and zinc at I, 15 and 30 days. Inhibition of brain AChE may cause physiological and behavioral modifications that reduce the survival ability of animals. Dutta et ul. (1992) had shown that diazinon, an organophosphorus pesticide inhibited the AChE
334
H. M. Dutta
activity and impaired the optomotor response in bluegill sunfish, Lepomis macrochirus. Richmonds and Dutta (1992) reported a considerable decrease in brain AChE activity in bluegills exposed to sublethal concentrations of malathion. Pavlov er al. (1992) found that exposure of Ahramis brama to an organophosphorus pesticide DDVP resulted in decreased food consumption and inhibited AChE activity. Intraperitoneal injection of cholinergic drug TMB4 recovered the AChE activity and feeding efficiency. They concluded that the cholinergic system in the brain constitutes the biochemical mechanism controlling feeding behavior in fish. Physiological and behavioral changes produced by exposure to pesticides reduce the chances of survival, especially from the threats of predators. Measuring a physiological parameter such as AChE activity may be used as an effective tool in preventing the deleterious effects of the pesticides. The results of our study show that the adults of H. fossilis have a higher tolerance and resistance, to the inhibitory effect of malathion on AChE activity, than the juveniles. This study shows that the effect of malathion, an organophosphorus pesticide, on AChE activity is significantly higher in the juvenile fish compared with that of the adult ones. This may alter the physiology and behavioral patterns permanently, which in turn will affect their capacity of survival at an early stage of growth. The decrease in acetylcholinesterase activity affects the optomotor behavior, and the optomotor function is needed for behavior like food searching, orienting toward food odor, and locating and avoidance of predators. Juvenile fish, weakened by exposure to pesticides like malathion, can be an easy prey for the predators because of their inability to continue a normal activity pattern which is essential to maintain their position in the aquatic environment. All these will lead to a reduction in their population or change in their population structure (Dutta et al., 1992).
et al.
References American Public Health Association, American Water Works Association, and Water Federation Control (1981) In Standard Methods for the Examination of Water and Wastewater. Washington, D.C. Dutta H. M., Marcelino J. and Richrnonds C. (1992) Brain acetylcholinesterase activity and optomotor behavior in bluegills, Lepomis macrochirus exposed to different concentrations of diazinon. Arch. Inter. Physiol. Biochim. Biophys. 100, 331-334. Ellman A. L., Courtney K. D., Andres V. Jr and Featherstone R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95. Frank R., Braun H. E., Clegg B. S.. Ripley B. D. and Johnson R. (1990) Survey of farm wells for pesticides. Ontario, Canada 1986 and 1987. Bull. enrrir. contam. Toxic. 44, 410419. Ghosh P. and Bhattacharya S. (1992) In uiuo and in oitro acetylcholinesterase inhibition by metacidand carbaryl in Channa punctatus under natural field condition. Biomed. Emit-. Sci. 5, 18-24. Martinez-Tabche L., Galar C. I., Ramirez M. B., Morales R. A. and German F. C. (1994) Parathion effect on acetylcholinesterase from fish through an artificial trophic chain: Ankistrodesmus falcatus-Moina macrocopaOrechromis homorum. Bull. envir. contam. Toxic. 52, 360-366. O’Brien R. D., Hetnarski B.. Tripathi R. K. and Hart A. J. (1974) Recent studies on acetylcholinesterase inhibition. In Mechanisms of Pesticide Action (Edited by Kohn A. K.), pp. I-13. American Chemical Society. Washington D.C. Pavlov D. D., Chuiko G. M., Serrassimov Y. V. and Tonkopiy V. D. (1992) Feeding behavior and brain acetylcholinesterase activity in bream (Abramis brama L.) as affected by DDVP, an organophosphorus insecticide. Comp. Biochem. Physiol. 103C, 563-568. Reddy A. T., Ayyanna K. and Yellamma K. (1991) Sensitivity of brain cholinesterase to cypermethrin toxicity in freshwater teleost Tilapica mossambiea. Biochem. Inter. 23, 959-962. Reddy P. M. and Philip G. H. (1994) In uiuo inhibition of AchE and ATPase activities in the tissues of freshwater fish, Cyprinus carpio exposed to technical grade cyarmethrin. Bull. envir. contam. Toxic. 52, 619-626. Richmonds C. R. and Dutta H. M. (1992) Effect of malathion on the brain acetylcholinesterase activity of bluegill sunfish, Lepomis macrochirus. Bull. emir. contam. Toxic.
Inter.
Acknowledgements-The present work was carried out under the Smithsonian (U.S.A.) project number 0022470000 Appr. 33FT56600-A20. This project is cosponsored in India by the University Grants Commission.
49, 43 1435.
Sailatha D., Sahib I. K. A. and Rao K. V. R. (1981) Toxicity of technical and commercial grade malathion to the fish Tilapia mossambica (Peters). Proc. Indian Acad. Sri. 90, 87792. Suresh A.. Sivaramakrishna B., Victoriamma P. C. and Radhakrishniah K. (1992) Comparative study on the inhibition of acetylcholinesterase activity in the freshwater fish Cyprinus carpio by mercury and zinc. Biochem. 26, 367-375.
Szabo A., Nemcsok J., Asztalos B., Rakonczay Z., Kasa P. and Hieu L. H. (1992) The effect of pesticides on carp (Cyprinus carpio L.). Acetylcholinesterase and its biochemical characterization. Ecotoxic. enuir. Safe. 23, 3945.
Pergamon 0300-%29(94)00166-9
Phyviol. Vol. I I IA, No. 2. pp. 331-334. 1995 . Elsevier Science Ltd Printed in Great Britain 0300-9629195 $9.50 + 0.00
Age related differences in the inhibition of brain acetylcholinesterase activity of Heteropneustes fossilis (Bloch) by malathion H. M. Dutta,* J. S. D. Munshi,? G. R. Dutta,? N. K. Singh,? S. Adhikari? and C. R. RichmondsS *Department of Biological Sciences, Kent State University, Kent, OH 44242, U.S.A.; TPostgraduate Department of Zoology, Bhagalpur University, Bhagalpur, India; and ICase Western Reserve University, Cleveland, OH 44106, U.S.A. The objective of this study is to determine whether there are differences in the sensitivity of brain acetylcholinesterase between juvenile and adult fish exposed to malathion. Air-breathing catfish, Heteropneustes fossilis were used. The exposure concentration was 1.2 mg/l (sublethal), and exposure durations were 24,48, 72, and 96 hr. A reduction in the mean acetylcholinesterase activity is seen at all exposure durations in the juveniles. It also indicates a significant difference between the control and exposure groups-24, 48, 72 and 96 hr in group A. However, in the adults, a reduction is seen, only at the 72 hr exposure duration. T-value and two-tail probability show that only control and 72 hr exposed ones are significantly different. In the higher weight group, there is a recovery in AChE activity at the 96 hr exposure level, This indicates that the detoxification capacity of the fish increases with age. The results show that the juveniles are much more susceptible than the adults. Key words: Hereropneustes fossilis; Malathion;
Brain acetylcholinesterase
activity: Age Related
differences. Comp. Biochem. Physiol. 11 IA, 331-334,
1995.
Introduction In recent years, organophosphorus pesticides have replaced organochlorine pesticides. Malathion, an organophosphorus pesticide has been sprayed periodically in the state of California for eliminating medflies. According to Martinez-Tabche et al. (1994), another organophosphorus insecticide, parathion, is widely used in Mexico, and the residues of this insecticide are widespread in freshwater ecosystems, contaminating water bodies. Reddy and Philip (1994) found inhibition of AChE in all tissues, namely gill, brain, liver and muscle IO: H. M. Dutta, Dept. of Biological Sciences, Kent State University, Kent, OH 44242, U.S.A. Received 26 May 1994; revised 8 September 1994; accepted I5 September 1994.
Correspondence
in Cyprinus carpio exposed to cypermethrin. In developing countries like India and Pakistan, large quantities of organophosphorus pesticides are used in agricultural operations and for eradicating mosquitoes and other pests. Surveys of the farm wells during 1986 and 1987 for pesticides in Ontario, Canada, revealed pesticide contamination including malathion (Frank et al., 1990). Organophosphorus compounds have the ability to inhibit the enzyme acetylcholinesterase (AChE) and thereby disrupt nervous transmissions. Malaoxon, an oxygen analog of malathion, appears to be the active part that binds the AChE (O’Brien et al., 1974). Young ones of any species are more susceptible than adults to any stress. The purpose of this investigation is to determine whether there are differ331
H. M. Dutta et al.
332
ences in the sensitivity of brain AChE to inhibition by malathion between the juveniles and adults of an air-breathing catfish, Heteropneustes fossilis.
Materials and Methods Live H. fossilis of two weight groups (8.0-l 5.0 g and 2540 g) were obtained from a swamp located in North Bihar, India. There was no agricultural land nearby where pesticides were applied. Fish were acclimated in the laboratory for 2 weeks in large plexiglass aquaria and fed with chopped goat liver. The water of the aquarium was aerated continuously, The fish were acclimated for 2 weeks. Feeding was stopped 24 hr before malathion exposure. The average values for the water qualities in the holding and exposure tanks were as follows: temperature 29°C pH 7.35, DO 7.5 mg/l, CO, 4 mg/l, alkalinity 115 mg/l as CaCO,, hardness 140 mg/l as CaCO,. Bioassays to determine the 96 hr LC,, were conducted employing the technique of the American Public Health Association (198 1). Commercial grade malathion is more toxic than technical grade to fish due to other ingredients present as emulsifiers which act as synergists (Sailatha et al., 1981). Therefore, commercial grade malathion containing 50% active ingredients (Northern Minerals Ltd., Haryana, India) was used in this study. Water was used as a solvent to dissolve the malathion. Ten fish for each weight group-five for the control and five for the experiment-were used to estimate AChE activity. Fish were exposed to a sublethal concentration of 1.2 mg/l, nearly one-tenth of the 96 hr LC,, value (11.676 mg/l) for 24, 48, 72 and 96 hr in plexiglass aquaria. Controls were treated under identical conditions without the pesticide. The estimation of AChE activity of brain tissue was performed according to the method of Ellman et al. (1961). This procedure is useful for the calorimetric estimation of AChE from homogenates, cell suspensions and tissue extracts. The enzyme activity was calculated by measuring the increase in yellow color produced by thiocholine when it reacts with dithiobisnitrobenzonate ions. A Bausch and Lomb Spectronic 20 colorimeter was used for estimating AChE activity. The analyses were performed at 25 + 1°C at a wavelength of 412 nm. Preparation
of brain samples for analysis
The fish were pithed and the brain was carefully removed intact with the help of sharppointed scissors and forceps. The removal of the
brain was achieved by making a median incision in the dorsal side of the skull and excising the brain free by cutting the optic nerves and spinal cord. Care was taken to avoid any blood clots, bones, skin, portions of olfactory and optic nerves. At the same time, it was made sure that all of the brain tissues were removed. The brain was placed in a small plastic weighing boat and weighed. Then, the brain was homogenized in 2 ml of pH 8, 0.1 M phosphate buffer using an electrically operated tissue homogenizer. Homogenization was performed with the help of a Tri-R Stir-R model S63C variable speed motor attached to a homogenizer. The ground brain tissue was then diluted with a sufficient amount of phosphate buffer to contain 20mg of brain tissue per milliliter of buffer. The tissue was further homogenized, and the homogenate was analyzed immediately using the assay method described below. Estimation
of activity
A 0.4 ml aliquot of the diluted brain-buffer homogenate was pipetted into a calorimeter tube. Then 2.6 ml of pH 8.0, 0.1 M phosphate buffer were added to this and the tube was placed in the photocell of the calorimeter. The absorbance was set at zero. Then 100 ~1 of DTNB reagent were added and mixed well. This causes a slight increase in absorbance. The calorimeter was reset to zero. Twenty microliters of acetylthiocholine (ASCh) iodide solution were added quickly and mixed well. The absorbance was recorded at every 15 set interval. The yellow color produced by the enzymatic reaction resulted in changes in absorbance. The reaction rate (AA) was calculated in units of changes in absorbance per minute by plotting the absorbance readings vs. time on an arithmetic graph paper. The rates were calculated by using the following formula of Ellman et al. (1961). R = 514 x AA/C,
where R = rate of pmoles of substrate (ASCh) hydrolyzed/min/g brain tissue, and C = original concentration of brain tissue (mgiml) in the homogenate (this value remains constant at 20 mg/ml throughout the study). AA = change in asorbncelmin. Since the original concentration was the same throughout, the formula was simplified as follows. R = 574 x AA/C, R = 574 x AA/20, R = 28.7 x AA.
Inhibition
of brain
AChE
Rate of activity is expressed as PM ASCh hydrolyzed/min/g of brain tissue. Triplicate measurements were done for each sample, and the mean value was taken. A t-test was performed to distinguish between the control and samples at 24, 48, 72 and 96 hr exposures of malathion for both groups A and B (Table I).
activity
!
I 1
333
by malathion
60.
I I
Results and Discussion The results are shown in Table 1. Statistical analysis showed a sharp reduction in the mean AChE activity at all exposure durations in the juveniles (lower weight group). It also indicates a significant difference between the control and exposure groups at 24,48,72 and 96 hr in group A. In the adults (higher weight group) a moderate reduction in AChE activity is seen only at the 72 hr exposure level. A t-value and two-tail probability show that only control and 72 hr exposed samples are significantly different from each other. The results (Table 1) reflect a remarkable differential change in the AChE activity in juvenile fish compared with the adults. Compared with the control, a continuous decline has been observed in AChE activity in the juvenile fish after 24, 48, 72 and 96 hr exposure time. Compared with the control, a slight reduction in the activity has been observed in the adult fish group in 48 hr, and a moderate reduction in the activity occurs in 72 hr exposure. In the higher weight group, there is recovery in AChE activity at the 96 hr exposure level. Figure 1 shows the percent inhibition of AChE activity compared with controls, in juveniles and adults at different exposure periods. This inhibition reinforces the results shown in Table 1. The results show that the capacity to withstand stress by malathion is more in the adult than in the juveniles. The capacity to adapt and recover is also higher in the adults as seen in the
Table AChE
I. T-test:
Exposure duration
brain acetylchohnesterase hydrolyzed/min/g) in control exposed fish Group
Control 24 hr 48 hr 72 hr 96 hr
A
Control 24 hr 48 hr 72 hr 96 hr
B
Activity mean and SD
and
activity (11M malathion
T-value control
vs. Two-tail probability
16.278 4.861** 3.057** 2.044** 2.199**
+ + + + -7
1.197 0.519 0.594 0.478 0.418
27.80 31.61 42.03 35.02
0.000 0.000 0.000 0.000
15.726 f 4.790 13.028 9.05” 14.963
k f k + +
0.105 f ,880 3.326 2.599 1.317
1.21 2.08 6.39 1.22
0.260 0.071 0.000 0.259
Group A = lower weight group (S-15 g). Group B = higher weight group (2540 g). ** = P
0
24
-LOWER ‘COMPARED
WT
72
46
DURATION
OF EXPOSURE
GROUP
-1~ HIGHER
96
(Hr) WT
GROUP
TO CONTROL
Fig. 1. Percent inhibition of AChE activity compared to control values in juveniles (lower weight group) and adults (higher weight group).
adults exposed for 96 hr. Our results are in conformity with the findings of Szabo et al. (1992) and Reddy et al. (1991). Szabo et al. (1992) had studied the effects of pesticides on carp AChE activity. Treatment with paraquat, an organophosphorus pesticide, led to a 50% decrease in the serum AChE activity, followed by a transient increase over the control level. After 2 weeks of chronic treatment, fish treated with paraquat showed an elevated level (130% compared with the control level) of AChE activity. They concluded that pesticides occurring in natural waters not only inhibited AChE activity in fish but might influence the resynthesis of the enzyme as well. Reddy et al. (1991) had shown that a maximum inhibition of AChE activity in Tilupiu mossumbicu exposed to cypermethrin occurred at 6 hr in juveniles and at 12 hr in adults. Ghosh and Bhaltacharya ( 1992) exposed Chunnu punt tufus (Bloch) to non-lethal levels of metacidand carbaryl for 7 days in the field. These insecticides produced a significant inhibition of brain AChE activity, accompanied by a concurrent increase in acetylcholine. Suresh et ul. (1992) recorded a significant suppression in AChE activity in gill. kidney, intestine, brain, liver and muscle of Cyprinus curpio exposed to sublethal concentrations of mercury and zinc at I, 15 and 30 days. Inhibition of brain AChE may cause physiological and behavioral modifications that reduce the survival ability of animals. Dutta et ul. (1992) had shown that diazinon, an organophosphorus pesticide inhibited the AChE
334
H. M. Dutta
activity and impaired the optomotor response in bluegill sunfish, Lepomis macrochirus. Richmonds and Dutta (1992) reported a considerable decrease in brain AChE activity in bluegills exposed to sublethal concentrations of malathion. Pavlov er al. (1992) found that exposure of Ahramis brama to an organophosphorus pesticide DDVP resulted in decreased food consumption and inhibited AChE activity. Intraperitoneal injection of cholinergic drug TMB4 recovered the AChE activity and feeding efficiency. They concluded that the cholinergic system in the brain constitutes the biochemical mechanism controlling feeding behavior in fish. Physiological and behavioral changes produced by exposure to pesticides reduce the chances of survival, especially from the threats of predators. Measuring a physiological parameter such as AChE activity may be used as an effective tool in preventing the deleterious effects of the pesticides. The results of our study show that the adults of H. fossilis have a higher tolerance and resistance, to the inhibitory effect of malathion on AChE activity, than the juveniles. This study shows that the effect of malathion, an organophosphorus pesticide, on AChE activity is significantly higher in the juvenile fish compared with that of the adult ones. This may alter the physiology and behavioral patterns permanently, which in turn will affect their capacity of survival at an early stage of growth. The decrease in acetylcholinesterase activity affects the optomotor behavior, and the optomotor function is needed for behavior like food searching, orienting toward food odor, and locating and avoidance of predators. Juvenile fish, weakened by exposure to pesticides like malathion, can be an easy prey for the predators because of their inability to continue a normal activity pattern which is essential to maintain their position in the aquatic environment. All these will lead to a reduction in their population or change in their population structure (Dutta et al., 1992).
et al.
References American Public Health Association, American Water Works Association, and Water Federation Control (1981) In Standard Methods for the Examination of Water and Wastewater. Washington, D.C. Dutta H. M., Marcelino J. and Richrnonds C. (1992) Brain acetylcholinesterase activity and optomotor behavior in bluegills, Lepomis macrochirus exposed to different concentrations of diazinon. Arch. Inter. Physiol. Biochim. Biophys. 100, 331-334. Ellman A. L., Courtney K. D., Andres V. Jr and Featherstone R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95. Frank R., Braun H. E., Clegg B. S.. Ripley B. D. and Johnson R. (1990) Survey of farm wells for pesticides. Ontario, Canada 1986 and 1987. Bull. enrrir. contam. Toxic. 44, 410419. Ghosh P. and Bhattacharya S. (1992) In uiuo and in oitro acetylcholinesterase inhibition by metacidand carbaryl in Channa punctatus under natural field condition. Biomed. Emit-. Sci. 5, 18-24. Martinez-Tabche L., Galar C. I., Ramirez M. B., Morales R. A. and German F. C. (1994) Parathion effect on acetylcholinesterase from fish through an artificial trophic chain: Ankistrodesmus falcatus-Moina macrocopaOrechromis homorum. Bull. envir. contam. Toxic. 52, 360-366. O’Brien R. D., Hetnarski B.. Tripathi R. K. and Hart A. J. (1974) Recent studies on acetylcholinesterase inhibition. In Mechanisms of Pesticide Action (Edited by Kohn A. K.), pp. I-13. American Chemical Society. Washington D.C. Pavlov D. D., Chuiko G. M., Serrassimov Y. V. and Tonkopiy V. D. (1992) Feeding behavior and brain acetylcholinesterase activity in bream (Abramis brama L.) as affected by DDVP, an organophosphorus insecticide. Comp. Biochem. Physiol. 103C, 563-568. Reddy A. T., Ayyanna K. and Yellamma K. (1991) Sensitivity of brain cholinesterase to cypermethrin toxicity in freshwater teleost Tilapica mossambiea. Biochem. Inter. 23, 959-962. Reddy P. M. and Philip G. H. (1994) In uiuo inhibition of AchE and ATPase activities in the tissues of freshwater fish, Cyprinus carpio exposed to technical grade cyarmethrin. Bull. envir. contam. Toxic. 52, 619-626. Richmonds C. R. and Dutta H. M. (1992) Effect of malathion on the brain acetylcholinesterase activity of bluegill sunfish, Lepomis macrochirus. Bull. emir. contam. Toxic.
Inter.
Acknowledgements-The present work was carried out under the Smithsonian (U.S.A.) project number 0022470000 Appr. 33FT56600-A20. This project is cosponsored in India by the University Grants Commission.
49, 43 1435.
Sailatha D., Sahib I. K. A. and Rao K. V. R. (1981) Toxicity of technical and commercial grade malathion to the fish Tilapia mossambica (Peters). Proc. Indian Acad. Sri. 90, 87792. Suresh A.. Sivaramakrishna B., Victoriamma P. C. and Radhakrishniah K. (1992) Comparative study on the inhibition of acetylcholinesterase activity in the freshwater fish Cyprinus carpio by mercury and zinc. Biochem. 26, 367-375.
Szabo A., Nemcsok J., Asztalos B., Rakonczay Z., Kasa P. and Hieu L. H. (1992) The effect of pesticides on carp (Cyprinus carpio L.). Acetylcholinesterase and its biochemical characterization. Ecotoxic. enuir. Safe. 23, 3945.