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The acute toxicity of endosulfan to fresh-water organisms
Toxicology Letters, 7 (1981) 453-456 0 Elsevier/North-Holland Biomedical Press
453
THE ACUTE TOXICITY OF ENDOSULFAN TO FRESH-WATER ORGANISMS
KRISHNA GOPAL*, R.N. KHANNA, M. ANAND and G.S.D. GUPTA Industrial Toxicology Research Centre. Pmt Box No. 80, Mahatma Gandhi Mar& Lucknow(India) (Received October 27th, 1980) (Accepted November 20th, 1980)
SUMMARY
A static bioassay is performed for measuring the short-term (96 h) toxicity of endosulfan to juvenile catfish Clurius batruchus, insect nymph Enulfagma sp. and frog tadpoles of Rana tigrina. Median Tolerance Limit, Slope Function, Confidence Limit, and Presumable Harmless Concentration were computed. The results showed that frog tadpoles are more susceptible to endosulfan than insect nymph and catfish. Morphological changes and behavioural alterations were evaluated as symptoms of endosulfan toxicity.
INTRODUCTION
With the modernisation of agricultural operations and the rapid growth of industrial activity, there has been much increase in the manufacture and utilisation of insecticides which ultimately find their way into the rivers, lakes and ponds. Endosulfan (6,7,8,9,lO-hexachloro-l,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3benzodioxithepin-3 oxide) is one of the chlorinated hydrocarbons of the cyclodiene group of insecticides and is widely used in rice fields [ 11. Extensive studies have been made on the deleterious effect of endosulfan on mammals [2-41. However, there is little toxicological information of this compound on fresh-water organisms. Manufacture of endosulfan on a commercial scale has been started recently in India. Therefore, the present study was undertaken to evaluate the acute toxicity of endosulfan using fish, insect nymph and frog tadpoles as test specimens. *For future correspondence.
454
MATERIALS
AND METHODS
Juvenile catfish C. batrachus, insect nymph Enallagma sp. and tadpoles of R. tigrina were obtained from local fresh-water resources and acclimatized under standard laboratory conditions [5]. The toxicity tests were conducted in widemouthed glass jars containing 20 1 of tap water with the following characteristics: temperature 20 + 2°C; pH 6.9-7.2; dissolved oxygen 6.8-7.4 mg/l; alkalinity 95-100 mg/l and hardness 118- 122 mg/l. Technical grade endosulfan (purity 90070, Hoechst), dissolved in ethanol, was added to water containing test specimens to attain the desired series of concentrations for catfish (0.005 to 0.04 ppm), insect nymph (0.005 to 0.04 ppm) and frog tadpoles (0.001 to 0.004 ppm). The experiment was conducted over a period of 96 h. The visible symptoms of toxic reactions were recorded and survival percentages of animals were noted during the course of exposure. Median Tolerance Limits were determined by interpolation of the graph between survival percentages and different concentrations of endosulfan [6]. By the application of Litchfield and Wilcoxon’s simplified method [7], the slope function and 95% confidence limit were computed. RESULTS
AND DISCUSSION
In the present investigation attention was paid towards the acute toxicity of endosulfan and probable cause of death in all groups of test animals. The visible signs of poisoning in fish were frequent jumping, erratic movement, followed by convulsions. The fish appeared excited with a rapid rate of opercular movement accompanied by occasional gulping of air; they ultimately lost equilibrium and died. These symptoms of intoxication persisted in treated fish up to 48 h at all concentrations of compound studied. The hyperglycaemia was also reported to be maximum at this interval [8], suggesting that endosulfan may in some manner interfere with carbohydrate metabolism and furnish the high demand of glucose in the brain to compensate to some extent for any potential decrease in brain glucose during the convulsive state. The presence of clotted blood over the gill surface, excessive mucus secretion, stiffening of trunk muscles and depigmentation were also observed after loss of positive rheotaxis. Similar toxic symptoms were observed in insect nymph and frog tadpoles. Endosulfan severely reduces the capacity of free swimming along with physical stamina of insect nymph and frog tadpoles by thinning of the gills which appeared transparent after complete cessation of movement. The frog tadpoles seem to be more susceptible to endosulfan than fish and insect nymph, as is evident from the results given in Table I. Presumable Harmless Concentration was computed using the formula given by Hart et al. [9]. The chemical structure of endosulfan is such that it exists in two isomeric forms, (Y and /3, and the latter is highly stable in soil and other media. During
455 TABLE
I
SUSCEPTIBILITY LIMIT,
TO ENDOSULFAN
ACUTE
TOXICITY
TOXICITY
RANGE,
SLOPE
AS MEASURED FUNCTION
AND
BY MEDIAN
TOLERANCE
PRESUMABLE
HARMLESS
CONCENTRATION Test animal
Hours
Median
exposed
tolerance
Acute
toxicity
(confidence
Slope function
range limit)
Presumable harmless concentration
limit (ppm)
Catfish (C. barruchus)
Aquatic
insect
(Enallagma sp.)
Frog tadpole (R. tigrina)
Upper
Lower
(ppm)
(ppm)
(ppm)
24
0.0225
0.0610
0.0190
48
0.0175
0.0180
0.0170
1.966
96
0.0140
0.0145
0.0134
2.672 1.513
1.446 0.00316
24
0.0285
0.0330
0.0240
48
0.0210
0.0260
0.0130
1.603
96
0.0175
0.0220
0.0140
1.729 0.975
24
0.0021
0.0025
0.0018
48
0.0020
0.0025
0.0017
1.591
96
0.0018
0.0022
0.0014
1.763
0.0034
0.00055
decomposition the sulphur ring of endosulfan opens by oxidation into sulphate derivatives which are water soluble. In view of the chemical nature of one of its isomers and the solubility of the metabolite, one could expect that the compound may be more potentially dangerous to fish and other fresh water organisms. ACKNOWLEDGEMENTS
The authors wish to express thanks to Dr. C.R. Krishna Murti, Director, Industrial Toxicology Research Centre, Lucknow for his keen interest and valuable guidance. Thanks are due to Sri M. Farooq for data computation. REFERENCES
1
S. Gorbach
and W. Knauf,
Environmental 2
P.K. Gupta,
Quality
Endosulfan
and Safety,
Endosulfan
induced
and the environment,
Academic
neurotoxicity
Press,
in F. Culston
New York,
and F. Korte
(Eds.),
1972, p. 250.
in rats and mice, Bull. Environ.
Contam.
Toxicol.,
15
(1976) 708. 3
R.N. Khanna,
D. Misra,
M. Anand
Toxicol.,
and H.K. Sharma,
Distribution
of endosulfan
in cat brain,
Environ.
Contam.
4
A. Garg,
K. Kunwar,
5
Ca levels, ascorbic acid and acid glutathione in rats, Toxicol. Lett., 5 (1980) 119. Mary Ann Franson, Standard Methods for the Examination of Water and Waste Water,
6
American Public Health Association, P. Dodouroff, B.G. Anderson, G.E.
Bull.
22 (1979) 72.
N. Das and P.K. Gupta,
Endosulfan
intoxication:
Washington, D.C., 1975, p. 1089. Budrick, P.S. Galstoff, W.B. Hart,
Blood glucose,
R. Patrick,
electrolytes, 14th Ed.,
E.R. Strong,
7 8 9
E.W. Surber and W.M. Vanhorn, Bioassay methods for the evaluation of acute toxicity of industrial wastes to fish, Sew. Indust. Waste, 23 (1951) 1380. J.T. Litchfield Jr. and F. Wilcoxon, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99. K. Gopal, R.N. Khanna, M. Anand and D. Misra, Endosulfan induced changes in blood glucose of cat fish, Ecotoxicol. Environ. Safety, 1980 (In Press). W.B. Hart, P. Dodouroff and J. Greenbank, The evaluation of toxicity of industrial wastes, chemicals and other substances to fresh water fish, Atlant. Ref. Co. Phil. (1945) 317.
453
THE ACUTE TOXICITY OF ENDOSULFAN TO FRESH-WATER ORGANISMS
KRISHNA GOPAL*, R.N. KHANNA, M. ANAND and G.S.D. GUPTA Industrial Toxicology Research Centre. Pmt Box No. 80, Mahatma Gandhi Mar& Lucknow(India) (Received October 27th, 1980) (Accepted November 20th, 1980)
SUMMARY
A static bioassay is performed for measuring the short-term (96 h) toxicity of endosulfan to juvenile catfish Clurius batruchus, insect nymph Enulfagma sp. and frog tadpoles of Rana tigrina. Median Tolerance Limit, Slope Function, Confidence Limit, and Presumable Harmless Concentration were computed. The results showed that frog tadpoles are more susceptible to endosulfan than insect nymph and catfish. Morphological changes and behavioural alterations were evaluated as symptoms of endosulfan toxicity.
INTRODUCTION
With the modernisation of agricultural operations and the rapid growth of industrial activity, there has been much increase in the manufacture and utilisation of insecticides which ultimately find their way into the rivers, lakes and ponds. Endosulfan (6,7,8,9,lO-hexachloro-l,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3benzodioxithepin-3 oxide) is one of the chlorinated hydrocarbons of the cyclodiene group of insecticides and is widely used in rice fields [ 11. Extensive studies have been made on the deleterious effect of endosulfan on mammals [2-41. However, there is little toxicological information of this compound on fresh-water organisms. Manufacture of endosulfan on a commercial scale has been started recently in India. Therefore, the present study was undertaken to evaluate the acute toxicity of endosulfan using fish, insect nymph and frog tadpoles as test specimens. *For future correspondence.
454
MATERIALS
AND METHODS
Juvenile catfish C. batrachus, insect nymph Enallagma sp. and tadpoles of R. tigrina were obtained from local fresh-water resources and acclimatized under standard laboratory conditions [5]. The toxicity tests were conducted in widemouthed glass jars containing 20 1 of tap water with the following characteristics: temperature 20 + 2°C; pH 6.9-7.2; dissolved oxygen 6.8-7.4 mg/l; alkalinity 95-100 mg/l and hardness 118- 122 mg/l. Technical grade endosulfan (purity 90070, Hoechst), dissolved in ethanol, was added to water containing test specimens to attain the desired series of concentrations for catfish (0.005 to 0.04 ppm), insect nymph (0.005 to 0.04 ppm) and frog tadpoles (0.001 to 0.004 ppm). The experiment was conducted over a period of 96 h. The visible symptoms of toxic reactions were recorded and survival percentages of animals were noted during the course of exposure. Median Tolerance Limits were determined by interpolation of the graph between survival percentages and different concentrations of endosulfan [6]. By the application of Litchfield and Wilcoxon’s simplified method [7], the slope function and 95% confidence limit were computed. RESULTS
AND DISCUSSION
In the present investigation attention was paid towards the acute toxicity of endosulfan and probable cause of death in all groups of test animals. The visible signs of poisoning in fish were frequent jumping, erratic movement, followed by convulsions. The fish appeared excited with a rapid rate of opercular movement accompanied by occasional gulping of air; they ultimately lost equilibrium and died. These symptoms of intoxication persisted in treated fish up to 48 h at all concentrations of compound studied. The hyperglycaemia was also reported to be maximum at this interval [8], suggesting that endosulfan may in some manner interfere with carbohydrate metabolism and furnish the high demand of glucose in the brain to compensate to some extent for any potential decrease in brain glucose during the convulsive state. The presence of clotted blood over the gill surface, excessive mucus secretion, stiffening of trunk muscles and depigmentation were also observed after loss of positive rheotaxis. Similar toxic symptoms were observed in insect nymph and frog tadpoles. Endosulfan severely reduces the capacity of free swimming along with physical stamina of insect nymph and frog tadpoles by thinning of the gills which appeared transparent after complete cessation of movement. The frog tadpoles seem to be more susceptible to endosulfan than fish and insect nymph, as is evident from the results given in Table I. Presumable Harmless Concentration was computed using the formula given by Hart et al. [9]. The chemical structure of endosulfan is such that it exists in two isomeric forms, (Y and /3, and the latter is highly stable in soil and other media. During
455 TABLE
I
SUSCEPTIBILITY LIMIT,
TO ENDOSULFAN
ACUTE
TOXICITY
TOXICITY
RANGE,
SLOPE
AS MEASURED FUNCTION
AND
BY MEDIAN
TOLERANCE
PRESUMABLE
HARMLESS
CONCENTRATION Test animal
Hours
Median
exposed
tolerance
Acute
toxicity
(confidence
Slope function
range limit)
Presumable harmless concentration
limit (ppm)
Catfish (C. barruchus)
Aquatic
insect
(Enallagma sp.)
Frog tadpole (R. tigrina)
Upper
Lower
(ppm)
(ppm)
(ppm)
24
0.0225
0.0610
0.0190
48
0.0175
0.0180
0.0170
1.966
96
0.0140
0.0145
0.0134
2.672 1.513
1.446 0.00316
24
0.0285
0.0330
0.0240
48
0.0210
0.0260
0.0130
1.603
96
0.0175
0.0220
0.0140
1.729 0.975
24
0.0021
0.0025
0.0018
48
0.0020
0.0025
0.0017
1.591
96
0.0018
0.0022
0.0014
1.763
0.0034
0.00055
decomposition the sulphur ring of endosulfan opens by oxidation into sulphate derivatives which are water soluble. In view of the chemical nature of one of its isomers and the solubility of the metabolite, one could expect that the compound may be more potentially dangerous to fish and other fresh water organisms. ACKNOWLEDGEMENTS
The authors wish to express thanks to Dr. C.R. Krishna Murti, Director, Industrial Toxicology Research Centre, Lucknow for his keen interest and valuable guidance. Thanks are due to Sri M. Farooq for data computation. REFERENCES
1
S. Gorbach
and W. Knauf,
Environmental 2
P.K. Gupta,
Quality
Endosulfan
and Safety,
Endosulfan
induced
and the environment,
Academic
neurotoxicity
Press,
in F. Culston
New York,
and F. Korte
(Eds.),
1972, p. 250.
in rats and mice, Bull. Environ.
Contam.
Toxicol.,
15
(1976) 708. 3
R.N. Khanna,
D. Misra,
M. Anand
Toxicol.,
and H.K. Sharma,
Distribution
of endosulfan
in cat brain,
Environ.
Contam.
4
A. Garg,
K. Kunwar,
5
Ca levels, ascorbic acid and acid glutathione in rats, Toxicol. Lett., 5 (1980) 119. Mary Ann Franson, Standard Methods for the Examination of Water and Waste Water,
6
American Public Health Association, P. Dodouroff, B.G. Anderson, G.E.
Bull.
22 (1979) 72.
N. Das and P.K. Gupta,
Endosulfan
intoxication:
Washington, D.C., 1975, p. 1089. Budrick, P.S. Galstoff, W.B. Hart,
Blood glucose,
R. Patrick,
electrolytes, 14th Ed.,
E.R. Strong,
7 8 9
E.W. Surber and W.M. Vanhorn, Bioassay methods for the evaluation of acute toxicity of industrial wastes to fish, Sew. Indust. Waste, 23 (1951) 1380. J.T. Litchfield Jr. and F. Wilcoxon, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99. K. Gopal, R.N. Khanna, M. Anand and D. Misra, Endosulfan induced changes in blood glucose of cat fish, Ecotoxicol. Environ. Safety, 1980 (In Press). W.B. Hart, P. Dodouroff and J. Greenbank, The evaluation of toxicity of industrial wastes, chemicals and other substances to fresh water fish, Atlant. Ref. Co. Phil. (1945) 317.