- Email: [email protected]
Combined action of carbaryl and phenthoate on the sensitivity of the acetylcholinesterase system of the fish, Channa punctatus (Bloch)
ECOTOXICOLOGY
AND
Combined
ENVIRONMENTAL
SAFETY
17,12-
15 ( 1989)
Action of Carbaryl and Phenthoate on the Sensitivity of the Acetylcholinesterase System of the Fish, Channa puncfatus (Bloch) K. R. S. SAMBASIVA
Department
of Zoology,
RAO’ AND K. V. RAMANA
Nagarjuna
Received
University,
August
Nagarjunanagar
RAo2
522 510, A.P., India
26, I987
Inhibition of acetylcholinesterase (AChE) and accumulation of acetylcholine were observed in tissues of fish, Channa punctatus, during carbaryl and/or phenthoate toxicity. Carbaryl in combination with phenthoate exerted synergism on the AChE system during their interaction. 0 1989 Academic Press Inc.
INTRODUCTION It is well documented that organophosphorus and carbamate pesticides generally cause disruption of nerve impulse transmission by inhibition of acetylcholinesterase (AChE) (O’Brien, 197 1; Bunyan et al., 197 1; Rainsford, 1978). Contamination with more than one pesticide at a given time in an aquatic ecosystem by translocation mechanisms from the various sites of application is inevitable. Thus a natural ecosystem represents a pool of different kinds of pesticides which in combination interact and affect the living system. With this in mind, the impact of the interaction of carbaryl with phenthoate on the sensitivity of the AChE system in the tissues of fish, Channa punctutus, was studied in the present investigation. MATERIALS
AND
METHODS
Fish, C. punctatus (10.0 f 2.0 g), were collected from local freshwater ponds and fed ad libitum with groundnut oil cake. Prior to use they were acclimated to laboratory conditions for 1 week and starved 24 hr prior to experimentation (Jones, 1972). Technical-grade samples of carbaryl (I-naphthyl N-methylcarbamate) and phenthoate (O,O-dimethyl, S-[(ethoxy carbonyl benzyl)]phosphorothiolothionate) were used in the present study. For a study of the combined action of carbaryl (C) and phenthoate (P), the two pesticides were mixed at 3: 1 ratio, which was reported to be an effective combination (Sambasiva Rao et al., 1985). The LC50 values were found to be 4.32 X lop5 A4 (8.71 ppm) for C, 1.40 X low6 M (0.473 ppm) for P, and 3.20 X 1Op6A4 (0.75 ppm) for C + P combination (Sambasiva Rao et al., 1985). Fish were exposed to sublethal concentrations of 1.45 X lop5 M(3.0 ppm), 4.99 X lo-‘M(O.16 ’ To whom correspondence should be addressed. * Present address: Department of Zoology, S.V. University P.G. Centre, Kavali-524 202, A.P., India. 0147-65
13/89 $3.00
Copyright Q 1989 by Academic Press, Inc. All rights ofreproduction in any form reserved.
12
COMBINED
ACTION
OF CARBARYL
13
AND PHENTHOATE
TABLE 1 AChE ACTIVITY
IN THE
TISSUES OF FISH EXPOSED TO CARBARYL (C), PHENTHOATE AND CARBARYL + PHENTHOATE (C + P)
Tissue
Control
Brain
4.8 + 0.6
Muscle
2.8 -t 0.1
Gill
1.7kO.l
Liver
1.4kO.l
C
P
3.0 + 0.2 -37.4% 1.9kO.1 -32.9% 1.2t-0.1 -28.4%
2.3 + 0.2 -52.4% 1.5+ 0.1 -46.0% 1.OtO.1 -36.8% 0.9 * 0.0 -37.1%
1.0~0.1
-29.8%
(P), c+p
1.9+ 0.2 -60.9% 1.2kO.l -55.2% l.O+-0.1 -39.7% 0.8kO.l -42.2%
Note. The values are expressed as micromoles of acetylcholine hyrolyzed per milligram protein per hour. Each value is the mean + SD of six individual observations. All the values are significant at P < 0.00 1.
ppm), and 1.OO X 10e6 M (0.25 ppm) of C, P, and C + P, respectively, for 48 hr and brain, muscle, liver, and gill tissues were isolated and used in the present study. The specific activity of AChE was estimated by the method of Metcalf ( 195 1) and acetylcholine (ACh) content was determined as suggested by Augustinson (1957). Protein content was estimated by the Folin Phenol method (Lowry et al., 195 1). Data were analyzed by the Student t test (Bahn, 1972) to assess the difference between control and experimental treatments. A value of P < 0.00 1 was considered significant. RESULTS The specific activities of AChE in brain, muscle, liver, and gill tissues of the fish exposed to C, P, and C + P combination were decreased suggesting the inhibitory effect of these pesticides and their combination on the AChE system (Table 1). In support of this an increase in ACh content was observed in the tissues studied (Table 2). Accumulation of ACh in tissues could be attributed to the decreased choline& TABLE 2 ACh CONTENT IN THE TISSUES OF FISH EXPOSED TO CARBARYL (C), PHENTHOATE (P), AND CARBARYL + PHENTHOATE (C + P) Tissue
Control
Brain
58.4 f 3.8
Muscle
43.5 -+ 2.7
Gill
42.8 + 3.4
Liver
28.0 f 2.5
C
P
79.8zk3.2 +36.4% 59.0 f 3.0 f35.7% 55.2 f 3.7 +29.0% 36.4 312.5 +30.0%
89.3 -t 3.7 t-52.8% 63.9 + 3.4 $47.0% 58.7 f 3.8 +37.1% 38.7 + 2.7 t-38.1%
CSP
94.3 + 4.5 +61.3% 68.0 i 3.3 +56.2% 59.9 f 4.3 l t40.0% 40.2 f 3.2 $43.3%
Note. The values are expressed as micromoles per gram wet weight of tissue. Each value is the mean k SD of six individual observations. All the values are significant at P -c 0.00 1.
14
RAO AND R40
transmission as a consequence of exposure to pesticides and their combination. The changes were more pronounced in brain followed by muscle, liver, and gill tissues. The data indicate that the inhibition of AChE by phenthoate is greater than that by catbaryl. This may be attributed to the higher affinity of AChE for P than for C. Although the mode of action is the same, the differential toxic effects of these pesticides could be attributed to their difference in the magnitude and duration of life. Inhibition of AChE and consequent accumulation of ACh were found to be higher with the C + P combination than under individual pesticide exposure (Tables 1 and 2). Augmentation of the inhibitory effect might be the consequence of potentiation of toxicity of one pesticide in the presence of the other. DISCUSSION Thus, it is likely that the toxicity of carbaryl is potentiated by the presence of the other pesticide, phenthoate. The exact mechanism by which this interaction of C and P leads to the manifestation of potentiation is still obscure. Similar observations were reported on carboxylesterases with different pesticides (Frawley et al., 1957; Murphy and DuBois, 1957). The manifestation of an additive effect in C + P combination selected in the present study suggests the possible inhibition of carboxylesterases by phenthoate, thus preventing the detoxification of carbaryl. Such an inhibition of carboxylesterases by P results in the “potentiation” of C toxicity when both chemicals are administered simultaneously. Su et al. ( 197 1) have reported that carboxylesterases not only hydrolyze the ester substrates like malathion phenthoate but also interact with certain other pesticides by binding. Thus, when fish are exposed to both C and P simultaneously, P binds to carboxylesterases because of its higher affinity and is detoxified. CONCLUSION Since there is a general correlation between the degree of potentiation and the inhibition of liver and plasma carboxylesterase activity (Casida, 196 1; Murphy and Cheever, 1968), the measurement of the latter might provide useful information about potentiation. ACKNOWLEDGMENTS We gratefully thank Paushak Limited, Baroda, and Motilal Pesticides (India) Pvt. Ltd., New Delhi, for the generous supply of technical-grade samples of carbaryl and phenthoate, respectively, and UGC, New Delhi, for financial assistance. We thank Professor Y. Kadhakrishna for his encouragement.
REFERENCES AIJGUSTINSON, K. B. (1957). In Methods in BiochemiculAnaZysis (D. Glick, Ed.). Interscience, New York. BAHN, K. A. (1972). Basic Medical Statistics. Grune & Stratton, New York. BUNYAN, P. J., JENNINGS,D. M., AND JONES, F. J. S. (197 1). Organophosphorus poisoning. Comparative study of the toxicity of chlorofenvinphos (2-chloro-I-92,44ichlorophenyl vinyl diethyl phosphate) to the pigeon, the pheasant and quail. Pestic. Sci. 2, 148- 15 1. CASIDA, J. E. (1961). Specificity of substituted phenyl phosphorus compounds for esterase inhibition in mice. Biochem. Pharmacol. 5,332-339. FRAWLEY, J. P., FTJYAT, H. N., HAGNN, E. C., BLAKE, J. R., AND FITZHUGU, 0. G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharmacol. Exp. Ther. 121,96-106.
COMBINED
ACTION
OF CARBARYL
AND PHENTHOATE
15
JONES,J. R. E. (1972). In Fish and River Pollution, pp. 15-185. Butterworths, London. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (195 I). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. METCALF, R. L. (195 1). In Methods in Biochemical Analysis (D. Glick, Ed.). Interscience, New York. MURPHY, S. D., AND CHEEVER, K. L. (1968). Effect of feeding insecticides. Inhibition of carboxylesterases and cholinesterases in rats. Arch. Environ. Health. 17,749-763. MURPHY, S. D., AND DUBOIS, K. P. (1957). Quantitative measurement of inhibition of the enzymatic detoxification of malathion by EPN (ethyl pnitrophenyl thinobenzene phosphate). Proc. Sot. Exp. Bioi. Med. %,813-818. O’BRIEN, R. D. (197 1). In Insecticides Action and Metabolism. Academic Press, New York. RAINSFORD, K. D. (1978). Toxicity in the brain of OP insecticides. Comparison of the toxicity of metabolites with parent compounds using an intracerebral injection method. Pestic. Biochem. Physiol. 8,302316. SAMBASIVA RAO, K. R. S., PRASADA RAO, K. S., AHAMMAD SAHIB, I. K., AND RAMANA RAO, K. V. (1985). Combined action of carbaryl and phenthoate on a freshwater fish (Channa punctatus Bloch). Ecotoxicol. Environ. SaJ: 10,209-217. Su, M. Q., KINOSHITA, F. K., FRAWLEY, J. P., AND DuBors, K. P. (1971). Comparative inhibition of aliesterases and cholinesterasc in rats fed with eighteen OP insecticides. Toxicol. Appl. Pharmacol. 20, 241-249.
AND
Combined
ENVIRONMENTAL
SAFETY
17,12-
15 ( 1989)
Action of Carbaryl and Phenthoate on the Sensitivity of the Acetylcholinesterase System of the Fish, Channa puncfatus (Bloch) K. R. S. SAMBASIVA
Department
of Zoology,
RAO’ AND K. V. RAMANA
Nagarjuna
Received
University,
August
Nagarjunanagar
RAo2
522 510, A.P., India
26, I987
Inhibition of acetylcholinesterase (AChE) and accumulation of acetylcholine were observed in tissues of fish, Channa punctatus, during carbaryl and/or phenthoate toxicity. Carbaryl in combination with phenthoate exerted synergism on the AChE system during their interaction. 0 1989 Academic Press Inc.
INTRODUCTION It is well documented that organophosphorus and carbamate pesticides generally cause disruption of nerve impulse transmission by inhibition of acetylcholinesterase (AChE) (O’Brien, 197 1; Bunyan et al., 197 1; Rainsford, 1978). Contamination with more than one pesticide at a given time in an aquatic ecosystem by translocation mechanisms from the various sites of application is inevitable. Thus a natural ecosystem represents a pool of different kinds of pesticides which in combination interact and affect the living system. With this in mind, the impact of the interaction of carbaryl with phenthoate on the sensitivity of the AChE system in the tissues of fish, Channa punctutus, was studied in the present investigation. MATERIALS
AND
METHODS
Fish, C. punctatus (10.0 f 2.0 g), were collected from local freshwater ponds and fed ad libitum with groundnut oil cake. Prior to use they were acclimated to laboratory conditions for 1 week and starved 24 hr prior to experimentation (Jones, 1972). Technical-grade samples of carbaryl (I-naphthyl N-methylcarbamate) and phenthoate (O,O-dimethyl, S-[(ethoxy carbonyl benzyl)]phosphorothiolothionate) were used in the present study. For a study of the combined action of carbaryl (C) and phenthoate (P), the two pesticides were mixed at 3: 1 ratio, which was reported to be an effective combination (Sambasiva Rao et al., 1985). The LC50 values were found to be 4.32 X lop5 A4 (8.71 ppm) for C, 1.40 X low6 M (0.473 ppm) for P, and 3.20 X 1Op6A4 (0.75 ppm) for C + P combination (Sambasiva Rao et al., 1985). Fish were exposed to sublethal concentrations of 1.45 X lop5 M(3.0 ppm), 4.99 X lo-‘M(O.16 ’ To whom correspondence should be addressed. * Present address: Department of Zoology, S.V. University P.G. Centre, Kavali-524 202, A.P., India. 0147-65
13/89 $3.00
Copyright Q 1989 by Academic Press, Inc. All rights ofreproduction in any form reserved.
12
COMBINED
ACTION
OF CARBARYL
13
AND PHENTHOATE
TABLE 1 AChE ACTIVITY
IN THE
TISSUES OF FISH EXPOSED TO CARBARYL (C), PHENTHOATE AND CARBARYL + PHENTHOATE (C + P)
Tissue
Control
Brain
4.8 + 0.6
Muscle
2.8 -t 0.1
Gill
1.7kO.l
Liver
1.4kO.l
C
P
3.0 + 0.2 -37.4% 1.9kO.1 -32.9% 1.2t-0.1 -28.4%
2.3 + 0.2 -52.4% 1.5+ 0.1 -46.0% 1.OtO.1 -36.8% 0.9 * 0.0 -37.1%
1.0~0.1
-29.8%
(P), c+p
1.9+ 0.2 -60.9% 1.2kO.l -55.2% l.O+-0.1 -39.7% 0.8kO.l -42.2%
Note. The values are expressed as micromoles of acetylcholine hyrolyzed per milligram protein per hour. Each value is the mean + SD of six individual observations. All the values are significant at P < 0.00 1.
ppm), and 1.OO X 10e6 M (0.25 ppm) of C, P, and C + P, respectively, for 48 hr and brain, muscle, liver, and gill tissues were isolated and used in the present study. The specific activity of AChE was estimated by the method of Metcalf ( 195 1) and acetylcholine (ACh) content was determined as suggested by Augustinson (1957). Protein content was estimated by the Folin Phenol method (Lowry et al., 195 1). Data were analyzed by the Student t test (Bahn, 1972) to assess the difference between control and experimental treatments. A value of P < 0.00 1 was considered significant. RESULTS The specific activities of AChE in brain, muscle, liver, and gill tissues of the fish exposed to C, P, and C + P combination were decreased suggesting the inhibitory effect of these pesticides and their combination on the AChE system (Table 1). In support of this an increase in ACh content was observed in the tissues studied (Table 2). Accumulation of ACh in tissues could be attributed to the decreased choline& TABLE 2 ACh CONTENT IN THE TISSUES OF FISH EXPOSED TO CARBARYL (C), PHENTHOATE (P), AND CARBARYL + PHENTHOATE (C + P) Tissue
Control
Brain
58.4 f 3.8
Muscle
43.5 -+ 2.7
Gill
42.8 + 3.4
Liver
28.0 f 2.5
C
P
79.8zk3.2 +36.4% 59.0 f 3.0 f35.7% 55.2 f 3.7 +29.0% 36.4 312.5 +30.0%
89.3 -t 3.7 t-52.8% 63.9 + 3.4 $47.0% 58.7 f 3.8 +37.1% 38.7 + 2.7 t-38.1%
CSP
94.3 + 4.5 +61.3% 68.0 i 3.3 +56.2% 59.9 f 4.3 l t40.0% 40.2 f 3.2 $43.3%
Note. The values are expressed as micromoles per gram wet weight of tissue. Each value is the mean k SD of six individual observations. All the values are significant at P -c 0.00 1.
14
RAO AND R40
transmission as a consequence of exposure to pesticides and their combination. The changes were more pronounced in brain followed by muscle, liver, and gill tissues. The data indicate that the inhibition of AChE by phenthoate is greater than that by catbaryl. This may be attributed to the higher affinity of AChE for P than for C. Although the mode of action is the same, the differential toxic effects of these pesticides could be attributed to their difference in the magnitude and duration of life. Inhibition of AChE and consequent accumulation of ACh were found to be higher with the C + P combination than under individual pesticide exposure (Tables 1 and 2). Augmentation of the inhibitory effect might be the consequence of potentiation of toxicity of one pesticide in the presence of the other. DISCUSSION Thus, it is likely that the toxicity of carbaryl is potentiated by the presence of the other pesticide, phenthoate. The exact mechanism by which this interaction of C and P leads to the manifestation of potentiation is still obscure. Similar observations were reported on carboxylesterases with different pesticides (Frawley et al., 1957; Murphy and DuBois, 1957). The manifestation of an additive effect in C + P combination selected in the present study suggests the possible inhibition of carboxylesterases by phenthoate, thus preventing the detoxification of carbaryl. Such an inhibition of carboxylesterases by P results in the “potentiation” of C toxicity when both chemicals are administered simultaneously. Su et al. ( 197 1) have reported that carboxylesterases not only hydrolyze the ester substrates like malathion phenthoate but also interact with certain other pesticides by binding. Thus, when fish are exposed to both C and P simultaneously, P binds to carboxylesterases because of its higher affinity and is detoxified. CONCLUSION Since there is a general correlation between the degree of potentiation and the inhibition of liver and plasma carboxylesterase activity (Casida, 196 1; Murphy and Cheever, 1968), the measurement of the latter might provide useful information about potentiation. ACKNOWLEDGMENTS We gratefully thank Paushak Limited, Baroda, and Motilal Pesticides (India) Pvt. Ltd., New Delhi, for the generous supply of technical-grade samples of carbaryl and phenthoate, respectively, and UGC, New Delhi, for financial assistance. We thank Professor Y. Kadhakrishna for his encouragement.
REFERENCES AIJGUSTINSON, K. B. (1957). In Methods in BiochemiculAnaZysis (D. Glick, Ed.). Interscience, New York. BAHN, K. A. (1972). Basic Medical Statistics. Grune & Stratton, New York. BUNYAN, P. J., JENNINGS,D. M., AND JONES, F. J. S. (197 1). Organophosphorus poisoning. Comparative study of the toxicity of chlorofenvinphos (2-chloro-I-92,44ichlorophenyl vinyl diethyl phosphate) to the pigeon, the pheasant and quail. Pestic. Sci. 2, 148- 15 1. CASIDA, J. E. (1961). Specificity of substituted phenyl phosphorus compounds for esterase inhibition in mice. Biochem. Pharmacol. 5,332-339. FRAWLEY, J. P., FTJYAT, H. N., HAGNN, E. C., BLAKE, J. R., AND FITZHUGU, 0. G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharmacol. Exp. Ther. 121,96-106.
COMBINED
ACTION
OF CARBARYL
AND PHENTHOATE
15
JONES,J. R. E. (1972). In Fish and River Pollution, pp. 15-185. Butterworths, London. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (195 I). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. METCALF, R. L. (195 1). In Methods in Biochemical Analysis (D. Glick, Ed.). Interscience, New York. MURPHY, S. D., AND CHEEVER, K. L. (1968). Effect of feeding insecticides. Inhibition of carboxylesterases and cholinesterases in rats. Arch. Environ. Health. 17,749-763. MURPHY, S. D., AND DUBOIS, K. P. (1957). Quantitative measurement of inhibition of the enzymatic detoxification of malathion by EPN (ethyl pnitrophenyl thinobenzene phosphate). Proc. Sot. Exp. Bioi. Med. %,813-818. O’BRIEN, R. D. (197 1). In Insecticides Action and Metabolism. Academic Press, New York. RAINSFORD, K. D. (1978). Toxicity in the brain of OP insecticides. Comparison of the toxicity of metabolites with parent compounds using an intracerebral injection method. Pestic. Biochem. Physiol. 8,302316. SAMBASIVA RAO, K. R. S., PRASADA RAO, K. S., AHAMMAD SAHIB, I. K., AND RAMANA RAO, K. V. (1985). Combined action of carbaryl and phenthoate on a freshwater fish (Channa punctatus Bloch). Ecotoxicol. Environ. SaJ: 10,209-217. Su, M. Q., KINOSHITA, F. K., FRAWLEY, J. P., AND DuBors, K. P. (1971). Comparative inhibition of aliesterases and cholinesterasc in rats fed with eighteen OP insecticides. Toxicol. Appl. Pharmacol. 20, 241-249.