- Email: [email protected]
Toxicity and metabolism of endosulfan and its effect on oxygen consumption and total nitrogen excretion of the fish Macrognathus aculeatum
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
15,
282-287
(1981)
Toxicity and Metabolism of Endosulfan and Its Effect on Oxygen Consumption and Total Nitrogen Excretion of the Fish Macrognathus aculeatum D. M. R. RAO, A. P. DEVI, Department
of Zoology, Received
Nagarjuna October
University,
AND
A. S.
MURTY’
Nagarjunanagar,
I. 1980; accepted
April
522510,
South
India
14. 1981
Experiments were conducted with the freshwater fish MacrognathuA aculeatum to study the toxicity and metabolism of endosulfan and the effect of the pesticide on the oxygen consumption and total nitrogen excretion. The 96-hr LC,,, value was 3.5 + 0.2 ppb. In brain, gills, gut, liver, and kidney. endosulfan was metabolized to endosulfan sulfate, but this appears to be only an intermediary step as the nontoxic endosulfan ether was found only in the liver and kidney, the principal organs of elimination of toxicants in fish. The pesticide, both at sublethal and lethal concentrations, decreased oxygen consumption and total nitrogen excretion. INTRODUCTION
Widespread and indiscriminate use of endosulfan (Thiodan; 6,7,8,9,10, IO-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide) to control cotton pests, particularly various species of bollworms, resulted in the contamination of the aquatic ecosystem in Guntur Dt. S. India, where residues as high as 0.33 ppm were reported in the sediment (1). Endosulfan is known to be highly toxic to fish, the toxicity reported being in parts per billion (2-6). Until now, the main aim of investigators has been to determine only its toxicity to fish and other organisms and very little is known about the effect of sublethal doses of endosulfan on nontarget species and the ability of various animals to degrade and eliminate endosulfan. Hence, the present study was undertaken to determine (1) the toxicity (96-hr LC,, value) of endosulfan to the freshwater fish Macrognathus aculeatum, which is an important species of foodfish in this region; (2) its ability to metabolize and eliminate endosulfan when it is exposed to sublethal doses; and (3) the ef’ Address all correspondence Visiting Scientist, Agriculture Post Offrce, London, Ontario
to: Dr. A. S. Murty, Canada, University Sub N6A 5B7, Canada. 282
0048-3575/g
l/030282-06$02.00/O
Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
feet of endosulfan on its oxygen consumption and total nitrogen excretion. MATERIALS
AND
METHODS
A. Toxicity studies. The fish (12-16 cm in length and 13.2 g in average weight) were caught in Guntur channel. Thin-layer chromatographic analysis of extracts of tissues of randomly chosen fish showed that they were free from endosulfan residues. The fish were acclimatized in well-aerated water at room temperature (28 ? 2°C). During the period of acclimatization and experimentation, the fish were not fed. Any batch of fish in which mortality exceeded 5% during the period of acclimatization was discarded. Toxicity tests were conducted by employing continuous flow systems, according to the recommendations of the Committee on Methods for Acute Toxicity Tests with Aquatic Organisms (7). The water that was used for toxicity tests was clear unchlorinated ground water pumped from a deep well on the University campus. The chemical analysis of water showed 02, 8-10 ppm; total hardness, 152 ppm as CaCO,; total alkalinity, 330 ppm as CaCO,; Ca, 25.6 ppm; Mg, 21.1 ppm; sulfate, 148 ppm as BaSO,; chlorides, 54 ppm; free chlorine, not detectable; pH 8.4; turbidity, 8 silica units; and total solids, 680
EFFECT
OF
ENDOSULFAN
ON
M. ucrrleutum
383
ppm. The water was free from endosulfan vived 96-hr exposure to 0.5 ppb endosulfan residues. in the toxicity studies were used to study Technical-grade endosulfan (96% pure) the route of degradation of endosulfan in was obtained from National Chemical Labvarious tissues of M. aculeatum. The fish oratory, Pune, India. The metabolites of were killed by decapitation and the chosen endosulfan were prepared in the laboratory tissues, gill filaments, brain, kidney, musaccording to the described methods (8, 9). cle, liver, gall bladder, and gut, were reStock solutions of endosulfan (100 and 10 moved. The tissues were weighed and ground pg/ml) were prepared in acetone and desired with anhydrous sodium sulfate in dimethconcentrations of the pesticide in water ylformamide and extracted with the same were prepared by adding suitable aliquots solvent on a flask shaker for 1 hr (total of stock solution to water in glass reservoirs solvent to tissue ratio, 2: 1 and 2 g of (32~liter capacity). The pesticide-containing Na,SO, for 1 g tissue). The DMF extract water from these glass reservoirs was let was diluted with 700 ml of 2% sodium into test tanks (15liter capacity) through sulfate solution and the residues were parthin-walled polyethylene tubes using gra- titioned from the aqueous DMF into hexane dient flow and the flow was regulated to by serial extraction, thrice. The hexane approximately 4 liter/hr. The loading ratio extracts were pooled and dried by passof fish was less than 1 g/liter water. The ing through a column of anhydrous somaximum amount of acetone contained in dium sulfate, concentrated to 5 ml in a the highest concentration tested was less Kuderna-Danish evaporator, and cleaned than 0.1 ml/liter of water and the same up on a Florisil column (22 mm i.d., 4 in. quantity of acetone was added to the con- Florisil topped with 1 in. anhydrous trols. Calorimetric analysis (10) of water in sodium sulfate) by eluting with 150 ml of 1% the glass reservoirs showed that there was acetone in hexane followed by 150 ml of 2% no detectable decrease in the concentration acetone in hexane. The eluates were colof the pesticide in water up to 4 hr (sen- lected in a K-D apparatus, concentrated to sitivity of the calorimetric analysis is 5 pg) 5 ml and further concentrated to 1 ml using after the addition of the pesticide. Hence, a micro-Snyder column. test solutions were made up once every 4 hr. Thin-layer chromatography was emThe fish were tested against five concenployed to identify the isomers and metabolites of endosulfan. AgNO,-impregnated trations (with 10 fish in each concentrasilica gel plates were prepared according to tion), that resulted in mortality in the range the method of Moats (12). The plates, actiof lo-90%. The experiment was repeated thrice and the data were pooled for cal- vated at 110°C for 10 min. were spotculating the 96-hr LC,, value, using the un- ted with extracts and standards and were weighted regression method of probit developed at room temperature in 4% aceanalysis (11). The difference between ob- tone in heptane. The plates were dried served and calculated values was tested for in air for 10 min and exposed to ultraviolet light to locate the spots. Visualization of the signiticance using the x2 test. Between experiments, fish holding tanks spots was enhanced by exposing the plates were thoroughly washed with warm deterto steam and darkening of the background gent water, filled with 5% KOH solution was prevented by spraying H,O,. The idenand allowed to stand overnight, washed tification of the isomers and metabolites was further confirmed by cochromatograagain with water, finally rinsed with acetone, and dried. This extensive procephy with 1: 1 benzene-heptane. The sendure was required to destroy the traces of sitivity of this method is 0.01 pg. The R, endosulfan residues that adhered to the values of the isomers and metabolites are walls of test tanks. shown in Table 1. B. Metabolism studies. The fish that surC. Oxygen consumption. The effect of
284
RAO,
DEVI,
AND
various concentrations of endosulfan on oxygen consumption of M. aculeatum was studied using the respiratory apparatus described by Jones (13). Oxygen was estimated by the modified Winkler’s method (14). The fish (12-16 cm in length) were maintained in the laboratory for 7 days on a formulated diet based on fish meal before conducting the experiment. The test fish were exposed to each concentration of the pesticide continuously for 1 hr in the respiratory chamber. The flow was stopped after 1 hr and the chamber was sealed and made airtight. Before sealing, water samples were collected to measure the initial level of dissolved oxygen. After allowing the fish to respire for 1 hr, another set of samples were drawn to measure the final dissolved 0, level. The difference between the initial and final values indicated the amount of 0, consumed under the test conditions. D. Total nitrogen excretion. The fish that were maintained in the laboratory on a formulated diet for a week were used in this experiment. Total nitrogen was determined by the Kjeldahl method (14). Before the start of the experiment, water samples were drawn to measure the initial level of total nitrogen. The fish were exposed for 12 hr to the desired concentration of the pesticide under static conditions. Again water samples were collected to measure the final level of total nitrogen. The difference gave
Rf V&es
(X 100) of Isomers
und Metubolites
TABLE of Endosulfun
MURTY
the amount of total nitrogen excreted by the fish. RESULTS
AND
DISCUSSION
Toxicity Studies The calculated 96-hr LC,, value was 3.5 + 0.2 ppb. The difference between the calculated and the observed rate of mortality was found to be not significant at P = 0.05. The regression equation for calculating the 96-hr LC,,, value was Y = 13.3X - 28.7. The present work showed that endosulfan is highly toxic to M. aculeatum as is the case with other species of fish. However, M. aculeatum is relatively more tolerant to endosulfan than other freshwater fish studied in this laboratory, which include the major carps Labeo rohita and Catla catla; the catfish Heteropneustes fossilis, Mystus cavasius, and M. vittatus; and the climbing perch, Anabus testudineus, whose 96-hr LC,, values were, respectively, 1.1, 1.8, 1.1, 1.9, 2.2, and 2.2 ppb (15). Metabolism
of Endosulfan
The results showed that in all the tissues studied, both the isomers of endosulfan were present and endosulfan sulfate was the principal metabolite. In the various animals studied for the metabolism of endosulfan, the sulfate (2, 6, 16-18), alcohol (3, 18), a-hydroxy ether (2), and ether (5, 6,
1 (from
Extructs
of Tissues)
on Silica
Gel tic Plates
R, (x 100)
30 + 2°C
34 + 1°C
Benzene and heptane (1: 1) 30 f 2°C
79 67 43 27 21 13 10 4 0.0
79.0 68.5 52.5 34.0 31.5 18.5 13.0 10.5 2.5
90 87 72 65 58 50 23 15 4
Acetone
Endosulfan Unidentified Endosulfan Unidentified Endosulfan Endosulfan Endosulfan Endosulfan Endosulfan
A compound ether compound B sulfate alcohol a-hydroxy lactone
ether
(4%)
in n-heptane
EFFECT
OF
ENDOSULFAN
15) were reported as metabolites. However, the formation of endosulfan sulfate, the oxidation product of endosulfan, is not a detoxification process, as it is toxic like the parent compound (16). According to Schoettger (3) formation of sulfate in goldfish and western whitesuckers is an intermediate step, the end product of detoxification being endosulfan alcohol. In the present study, nontoxic endosulfan ether was found only in the extracts of liver, bile, and kidney, indicating that it is the main product of detoxification (Fig. 1). Earlier. endosulfan ether was reported as the end product of detoxification in the major carp L. rohita (5) and climbing perch, A. testudineus (6). The presence of the ether only in liver, bile, and kidney suggests that it is eliminated mainly through feces and urine. It is interesting to note that Schoettger (3) reported the alcohol as the detoxification product in two cold-water fish whereas in all the three species of warm-water fish hitherto studied, the ether is reported as the detoxification product. With the very limited work reported, it is difficult to surmise whether the environmental temperature influences the route of detoxification. The chromatograms also showed two more unidentified metabolites of endosulfan in the liver extract, the R, values of which did not correspond with any of the known metabolites of endosulfan. Oxygen Consumptiorl Figure 2 shows the effect of endosulfan on the oxygen consumption of M. aculeatlrm. There was a rapid decline in the oxygen consumption as the concentration of endosulfan increased from 1 to 3 ppb and a progressive decline up to 15 ppb. Earlier work on the effect of organochlorines on fish indicates that at sublethal concentrations, it is decreased (5). The work of Rao et al. (5) showed that in the case of L. rohita, oxygen consumption increased progressively as the concentration of the pesticide increased from 1 to 5 ppb and de-
ON
M. wulrrrturn
7-85
creased with further increase of the toxicant, until death ensued. The difference between the oxygen consumption of M. aculeatum and other animals exposed to endosulfan may proba-
286
RAO,
DEVI,
bly be due to the mode of action of the toxicant as is evident from the difference in the symptoms of poisoning. With M. aculeatum, there was progressive inactivity terminating in death, without any convulsions or muscular exertion. The symptoms manifested by other animals (including L. rohita studied by us earlier), exposed to organochlorines are hyperactivity, irritability, and convulsions followed by paralysis and death. In other words, the action of endosulfan on M. aculeatum was more like a respiratory poison than a neurotoxicant, and the primary cause of death may be respiratory failure. Studies on Total Nitrogen Excretion The effect of various concentrations of endosulfan on total nitrogen excreted is presented in Fig. 2. There was a rapid decrease in the amount of total nitrogen excreted, with increase in the endosulfan concentration from 1 to 3 ppb, and thereafter a progressive decrease. Energy for maintenance and activity comes from the catabolism of food, and in fish protein is the main source of energy (20). Protein catabolism produces ammonia, urea, and such other forms of nitrogenous wastes. Therefore, measurement of changes in the total nitrogen excreted should reflect changes in the rate of metabolism of the fish. Interpreted in this way, the persistent decrease in the total nitrogen excreted as the endosulfan concentration increased, indicates that the pesticide does interfere with the metabolism of the fish. In summary, we found that (a) endosulfan is highly toxic to the fish M. aculeatum; (b) the principal metabolite initially was endosulfan sulfate, found in all the tissues studied; (c) nontoxic endosulfan ether was found only in liver, bile, and kidney; (d) at least two more unidentified compounds were found in the liver extract; (e) endosulfan interfered with the oxygen consumption, (probably acting as a respiratory poison); and (f) endosulfan greatly reduced the total nitrogen excreted, indicating reduced protein metabolism.
AND
MURTY
ACKNOWLEDGMENTS
Financial assistance received from the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged. The authors wish to thank Dr. R. A. Chapman for critically reading the manuscript. REFERENCES
1. D. M. R. Rao. K. S. Tilak, and A. S. Murty, Pollution of the aquatic environment with endosulfan residues, in “Proceedings of the Symposium on Environmental Biology,” pp. 217220, Acad. Environ. Sci., Muzaffarnagar, India, 1979.
H. Maier-Bode, Properties, effect, residues and analytics of the insecticide endosulfan, Residue Rev. 22, 1 (1968). 3. R. A. Schoettger, Toxicology of Thiodan in several fish and aquatic invertebrates, Invest. Fish 2.
Confr. 4.
No.
35 (1970).
P. Moulton, The effect of various insecticides (especially Thiodan and BHC) on fish in the paddy fields of West Malaysia. M&y. Agr. J. 49, 224 (1973).
5. D. M. R. Rao, A. P. Devi, and A. S. Murty, Relative toxicity of endosulfan, its isomers and formulated products-to the freshwater fish Labeo rohitn, J. Toxic&. Environ. Health 6,323 (1980). 6. D. M. R. Rao and A. S. Murty, Toxicity and biotransformation and elimination of endosulfan in Anabus testudineus (Bloch), Indiun J. Exp. Eiol. 6, 664 (1980). 7. Committee on Methods for Toxicity Tests with Aquatic Organisms, “Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians”, Environmental Protection Agency, Oreg.. p. 1, 1975. 8. D. Lindquist and P. A. Dahm, Some chemical and biological experiments with Thiodan, J. Econ. Entomol.
50, 483 (1957).
9. W. W. Barnes and G. W. Ware, The absorption and metabolism of ‘*C-labelled endosulfan in the house-fly, J. Econ. Entomol. 58,265 (1965). 10. J. C. Maitlen, K. C. Walker, and W. E. Westlake, Insecticide residues, an improved calorimetric method for determining endosulfan (Thiodan) residues in vegetables and beef fat, J. Agr. Food Chem. 49, 795 (1966). 11. D. J. Finney, “Probit Analysis,” 3 ed., Cambridge Univ. Press, London/New York, 1971. 12. W. A. Moats, Analysis of dairy products for chlorinated insecticide residues by thin layer chromatography, J. Assoc. Awl. Chem. 49,795 (1966). 13. J. R. E. Jones, “Fish and River Pollution.” p. 9. Butterworths, London. 1964. 14. H. L. Golterman and R. S. Clymo, (Eds.), “Methods for Chemical Analysis of Water.” Blackwell. Oxford. 1969.
EFFECT
OF
ENDOSULFAN
IS. D. M. R. Rao, “Studies on the Persistence of Endosulfan in the Environment and Its Effects on Freshwater Fish.” Ph.D. thesis, Nagarjuna University, Nagarjunanagar, S. India, 1979. 16. P. Deema. E. Thompson, and G. W. Ware. Metabolism, storage and excretion of laCendosulfan in the mouse, J. Econ. Et~totrd. 59. 546 ( 1966). 17. S. G. Gorbasch. 0. R. Christ, H. Kellner. G. Kloss, and E. Borner, Metabolism of endosulfan in milk sheep. J. Agv. Food Chem. 16, 950 ( 1968).
ON
287
M. aculeutum
18. D. H. Wayman, H. Kurt. and M. C. Thomas. Fate of endosulfan in rats and toxicological consideration of apolar metabolites. Pc\ficc Biochem.
Physiol.
3, 251 (1978).
19. J. R. Brett and T. D. D. Groves. Physiological energetics. in “Fish Physiology,” (W. S. Hoar, D. J. Randall, and J. R. Brett, Eds.1. Vol. 8. p, 280. Academic Press. New York. 1979. 20. H. M. Jrueger. J. B. Saddler. G. A. Chapman. I. J. Tinsley. and R. R. Lowry. Bioenergetics. exercise and fatty acids of fish. A/rrrr. Zoo/. 8. 119. (1968).
BIOCHEMISTRY
AND
PHYSIOLOGY
15,
282-287
(1981)
Toxicity and Metabolism of Endosulfan and Its Effect on Oxygen Consumption and Total Nitrogen Excretion of the Fish Macrognathus aculeatum D. M. R. RAO, A. P. DEVI, Department
of Zoology, Received
Nagarjuna October
University,
AND
A. S.
MURTY’
Nagarjunanagar,
I. 1980; accepted
April
522510,
South
India
14. 1981
Experiments were conducted with the freshwater fish MacrognathuA aculeatum to study the toxicity and metabolism of endosulfan and the effect of the pesticide on the oxygen consumption and total nitrogen excretion. The 96-hr LC,,, value was 3.5 + 0.2 ppb. In brain, gills, gut, liver, and kidney. endosulfan was metabolized to endosulfan sulfate, but this appears to be only an intermediary step as the nontoxic endosulfan ether was found only in the liver and kidney, the principal organs of elimination of toxicants in fish. The pesticide, both at sublethal and lethal concentrations, decreased oxygen consumption and total nitrogen excretion. INTRODUCTION
Widespread and indiscriminate use of endosulfan (Thiodan; 6,7,8,9,10, IO-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide) to control cotton pests, particularly various species of bollworms, resulted in the contamination of the aquatic ecosystem in Guntur Dt. S. India, where residues as high as 0.33 ppm were reported in the sediment (1). Endosulfan is known to be highly toxic to fish, the toxicity reported being in parts per billion (2-6). Until now, the main aim of investigators has been to determine only its toxicity to fish and other organisms and very little is known about the effect of sublethal doses of endosulfan on nontarget species and the ability of various animals to degrade and eliminate endosulfan. Hence, the present study was undertaken to determine (1) the toxicity (96-hr LC,, value) of endosulfan to the freshwater fish Macrognathus aculeatum, which is an important species of foodfish in this region; (2) its ability to metabolize and eliminate endosulfan when it is exposed to sublethal doses; and (3) the ef’ Address all correspondence Visiting Scientist, Agriculture Post Offrce, London, Ontario
to: Dr. A. S. Murty, Canada, University Sub N6A 5B7, Canada. 282
0048-3575/g
l/030282-06$02.00/O
Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
feet of endosulfan on its oxygen consumption and total nitrogen excretion. MATERIALS
AND
METHODS
A. Toxicity studies. The fish (12-16 cm in length and 13.2 g in average weight) were caught in Guntur channel. Thin-layer chromatographic analysis of extracts of tissues of randomly chosen fish showed that they were free from endosulfan residues. The fish were acclimatized in well-aerated water at room temperature (28 ? 2°C). During the period of acclimatization and experimentation, the fish were not fed. Any batch of fish in which mortality exceeded 5% during the period of acclimatization was discarded. Toxicity tests were conducted by employing continuous flow systems, according to the recommendations of the Committee on Methods for Acute Toxicity Tests with Aquatic Organisms (7). The water that was used for toxicity tests was clear unchlorinated ground water pumped from a deep well on the University campus. The chemical analysis of water showed 02, 8-10 ppm; total hardness, 152 ppm as CaCO,; total alkalinity, 330 ppm as CaCO,; Ca, 25.6 ppm; Mg, 21.1 ppm; sulfate, 148 ppm as BaSO,; chlorides, 54 ppm; free chlorine, not detectable; pH 8.4; turbidity, 8 silica units; and total solids, 680
EFFECT
OF
ENDOSULFAN
ON
M. ucrrleutum
383
ppm. The water was free from endosulfan vived 96-hr exposure to 0.5 ppb endosulfan residues. in the toxicity studies were used to study Technical-grade endosulfan (96% pure) the route of degradation of endosulfan in was obtained from National Chemical Labvarious tissues of M. aculeatum. The fish oratory, Pune, India. The metabolites of were killed by decapitation and the chosen endosulfan were prepared in the laboratory tissues, gill filaments, brain, kidney, musaccording to the described methods (8, 9). cle, liver, gall bladder, and gut, were reStock solutions of endosulfan (100 and 10 moved. The tissues were weighed and ground pg/ml) were prepared in acetone and desired with anhydrous sodium sulfate in dimethconcentrations of the pesticide in water ylformamide and extracted with the same were prepared by adding suitable aliquots solvent on a flask shaker for 1 hr (total of stock solution to water in glass reservoirs solvent to tissue ratio, 2: 1 and 2 g of (32~liter capacity). The pesticide-containing Na,SO, for 1 g tissue). The DMF extract water from these glass reservoirs was let was diluted with 700 ml of 2% sodium into test tanks (15liter capacity) through sulfate solution and the residues were parthin-walled polyethylene tubes using gra- titioned from the aqueous DMF into hexane dient flow and the flow was regulated to by serial extraction, thrice. The hexane approximately 4 liter/hr. The loading ratio extracts were pooled and dried by passof fish was less than 1 g/liter water. The ing through a column of anhydrous somaximum amount of acetone contained in dium sulfate, concentrated to 5 ml in a the highest concentration tested was less Kuderna-Danish evaporator, and cleaned than 0.1 ml/liter of water and the same up on a Florisil column (22 mm i.d., 4 in. quantity of acetone was added to the con- Florisil topped with 1 in. anhydrous trols. Calorimetric analysis (10) of water in sodium sulfate) by eluting with 150 ml of 1% the glass reservoirs showed that there was acetone in hexane followed by 150 ml of 2% no detectable decrease in the concentration acetone in hexane. The eluates were colof the pesticide in water up to 4 hr (sen- lected in a K-D apparatus, concentrated to sitivity of the calorimetric analysis is 5 pg) 5 ml and further concentrated to 1 ml using after the addition of the pesticide. Hence, a micro-Snyder column. test solutions were made up once every 4 hr. Thin-layer chromatography was emThe fish were tested against five concenployed to identify the isomers and metabolites of endosulfan. AgNO,-impregnated trations (with 10 fish in each concentrasilica gel plates were prepared according to tion), that resulted in mortality in the range the method of Moats (12). The plates, actiof lo-90%. The experiment was repeated thrice and the data were pooled for cal- vated at 110°C for 10 min. were spotculating the 96-hr LC,, value, using the un- ted with extracts and standards and were weighted regression method of probit developed at room temperature in 4% aceanalysis (11). The difference between ob- tone in heptane. The plates were dried served and calculated values was tested for in air for 10 min and exposed to ultraviolet light to locate the spots. Visualization of the signiticance using the x2 test. Between experiments, fish holding tanks spots was enhanced by exposing the plates were thoroughly washed with warm deterto steam and darkening of the background gent water, filled with 5% KOH solution was prevented by spraying H,O,. The idenand allowed to stand overnight, washed tification of the isomers and metabolites was further confirmed by cochromatograagain with water, finally rinsed with acetone, and dried. This extensive procephy with 1: 1 benzene-heptane. The sendure was required to destroy the traces of sitivity of this method is 0.01 pg. The R, endosulfan residues that adhered to the values of the isomers and metabolites are walls of test tanks. shown in Table 1. B. Metabolism studies. The fish that surC. Oxygen consumption. The effect of
284
RAO,
DEVI,
AND
various concentrations of endosulfan on oxygen consumption of M. aculeatum was studied using the respiratory apparatus described by Jones (13). Oxygen was estimated by the modified Winkler’s method (14). The fish (12-16 cm in length) were maintained in the laboratory for 7 days on a formulated diet based on fish meal before conducting the experiment. The test fish were exposed to each concentration of the pesticide continuously for 1 hr in the respiratory chamber. The flow was stopped after 1 hr and the chamber was sealed and made airtight. Before sealing, water samples were collected to measure the initial level of dissolved oxygen. After allowing the fish to respire for 1 hr, another set of samples were drawn to measure the final dissolved 0, level. The difference between the initial and final values indicated the amount of 0, consumed under the test conditions. D. Total nitrogen excretion. The fish that were maintained in the laboratory on a formulated diet for a week were used in this experiment. Total nitrogen was determined by the Kjeldahl method (14). Before the start of the experiment, water samples were drawn to measure the initial level of total nitrogen. The fish were exposed for 12 hr to the desired concentration of the pesticide under static conditions. Again water samples were collected to measure the final level of total nitrogen. The difference gave
Rf V&es
(X 100) of Isomers
und Metubolites
TABLE of Endosulfun
MURTY
the amount of total nitrogen excreted by the fish. RESULTS
AND
DISCUSSION
Toxicity Studies The calculated 96-hr LC,, value was 3.5 + 0.2 ppb. The difference between the calculated and the observed rate of mortality was found to be not significant at P = 0.05. The regression equation for calculating the 96-hr LC,,, value was Y = 13.3X - 28.7. The present work showed that endosulfan is highly toxic to M. aculeatum as is the case with other species of fish. However, M. aculeatum is relatively more tolerant to endosulfan than other freshwater fish studied in this laboratory, which include the major carps Labeo rohita and Catla catla; the catfish Heteropneustes fossilis, Mystus cavasius, and M. vittatus; and the climbing perch, Anabus testudineus, whose 96-hr LC,, values were, respectively, 1.1, 1.8, 1.1, 1.9, 2.2, and 2.2 ppb (15). Metabolism
of Endosulfan
The results showed that in all the tissues studied, both the isomers of endosulfan were present and endosulfan sulfate was the principal metabolite. In the various animals studied for the metabolism of endosulfan, the sulfate (2, 6, 16-18), alcohol (3, 18), a-hydroxy ether (2), and ether (5, 6,
1 (from
Extructs
of Tissues)
on Silica
Gel tic Plates
R, (x 100)
30 + 2°C
34 + 1°C
Benzene and heptane (1: 1) 30 f 2°C
79 67 43 27 21 13 10 4 0.0
79.0 68.5 52.5 34.0 31.5 18.5 13.0 10.5 2.5
90 87 72 65 58 50 23 15 4
Acetone
Endosulfan Unidentified Endosulfan Unidentified Endosulfan Endosulfan Endosulfan Endosulfan Endosulfan
A compound ether compound B sulfate alcohol a-hydroxy lactone
ether
(4%)
in n-heptane
EFFECT
OF
ENDOSULFAN
15) were reported as metabolites. However, the formation of endosulfan sulfate, the oxidation product of endosulfan, is not a detoxification process, as it is toxic like the parent compound (16). According to Schoettger (3) formation of sulfate in goldfish and western whitesuckers is an intermediate step, the end product of detoxification being endosulfan alcohol. In the present study, nontoxic endosulfan ether was found only in the extracts of liver, bile, and kidney, indicating that it is the main product of detoxification (Fig. 1). Earlier. endosulfan ether was reported as the end product of detoxification in the major carp L. rohita (5) and climbing perch, A. testudineus (6). The presence of the ether only in liver, bile, and kidney suggests that it is eliminated mainly through feces and urine. It is interesting to note that Schoettger (3) reported the alcohol as the detoxification product in two cold-water fish whereas in all the three species of warm-water fish hitherto studied, the ether is reported as the detoxification product. With the very limited work reported, it is difficult to surmise whether the environmental temperature influences the route of detoxification. The chromatograms also showed two more unidentified metabolites of endosulfan in the liver extract, the R, values of which did not correspond with any of the known metabolites of endosulfan. Oxygen Consumptiorl Figure 2 shows the effect of endosulfan on the oxygen consumption of M. aculeatlrm. There was a rapid decline in the oxygen consumption as the concentration of endosulfan increased from 1 to 3 ppb and a progressive decline up to 15 ppb. Earlier work on the effect of organochlorines on fish indicates that at sublethal concentrations, it is decreased (5). The work of Rao et al. (5) showed that in the case of L. rohita, oxygen consumption increased progressively as the concentration of the pesticide increased from 1 to 5 ppb and de-
ON
M. wulrrrturn
7-85
creased with further increase of the toxicant, until death ensued. The difference between the oxygen consumption of M. aculeatum and other animals exposed to endosulfan may proba-
286
RAO,
DEVI,
bly be due to the mode of action of the toxicant as is evident from the difference in the symptoms of poisoning. With M. aculeatum, there was progressive inactivity terminating in death, without any convulsions or muscular exertion. The symptoms manifested by other animals (including L. rohita studied by us earlier), exposed to organochlorines are hyperactivity, irritability, and convulsions followed by paralysis and death. In other words, the action of endosulfan on M. aculeatum was more like a respiratory poison than a neurotoxicant, and the primary cause of death may be respiratory failure. Studies on Total Nitrogen Excretion The effect of various concentrations of endosulfan on total nitrogen excreted is presented in Fig. 2. There was a rapid decrease in the amount of total nitrogen excreted, with increase in the endosulfan concentration from 1 to 3 ppb, and thereafter a progressive decrease. Energy for maintenance and activity comes from the catabolism of food, and in fish protein is the main source of energy (20). Protein catabolism produces ammonia, urea, and such other forms of nitrogenous wastes. Therefore, measurement of changes in the total nitrogen excreted should reflect changes in the rate of metabolism of the fish. Interpreted in this way, the persistent decrease in the total nitrogen excreted as the endosulfan concentration increased, indicates that the pesticide does interfere with the metabolism of the fish. In summary, we found that (a) endosulfan is highly toxic to the fish M. aculeatum; (b) the principal metabolite initially was endosulfan sulfate, found in all the tissues studied; (c) nontoxic endosulfan ether was found only in liver, bile, and kidney; (d) at least two more unidentified compounds were found in the liver extract; (e) endosulfan interfered with the oxygen consumption, (probably acting as a respiratory poison); and (f) endosulfan greatly reduced the total nitrogen excreted, indicating reduced protein metabolism.
AND
MURTY
ACKNOWLEDGMENTS
Financial assistance received from the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged. The authors wish to thank Dr. R. A. Chapman for critically reading the manuscript. REFERENCES
1. D. M. R. Rao. K. S. Tilak, and A. S. Murty, Pollution of the aquatic environment with endosulfan residues, in “Proceedings of the Symposium on Environmental Biology,” pp. 217220, Acad. Environ. Sci., Muzaffarnagar, India, 1979.
H. Maier-Bode, Properties, effect, residues and analytics of the insecticide endosulfan, Residue Rev. 22, 1 (1968). 3. R. A. Schoettger, Toxicology of Thiodan in several fish and aquatic invertebrates, Invest. Fish 2.
Confr. 4.
No.
35 (1970).
P. Moulton, The effect of various insecticides (especially Thiodan and BHC) on fish in the paddy fields of West Malaysia. M&y. Agr. J. 49, 224 (1973).
5. D. M. R. Rao, A. P. Devi, and A. S. Murty, Relative toxicity of endosulfan, its isomers and formulated products-to the freshwater fish Labeo rohitn, J. Toxic&. Environ. Health 6,323 (1980). 6. D. M. R. Rao and A. S. Murty, Toxicity and biotransformation and elimination of endosulfan in Anabus testudineus (Bloch), Indiun J. Exp. Eiol. 6, 664 (1980). 7. Committee on Methods for Toxicity Tests with Aquatic Organisms, “Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians”, Environmental Protection Agency, Oreg.. p. 1, 1975. 8. D. Lindquist and P. A. Dahm, Some chemical and biological experiments with Thiodan, J. Econ. Entomol.
50, 483 (1957).
9. W. W. Barnes and G. W. Ware, The absorption and metabolism of ‘*C-labelled endosulfan in the house-fly, J. Econ. Entomol. 58,265 (1965). 10. J. C. Maitlen, K. C. Walker, and W. E. Westlake, Insecticide residues, an improved calorimetric method for determining endosulfan (Thiodan) residues in vegetables and beef fat, J. Agr. Food Chem. 49, 795 (1966). 11. D. J. Finney, “Probit Analysis,” 3 ed., Cambridge Univ. Press, London/New York, 1971. 12. W. A. Moats, Analysis of dairy products for chlorinated insecticide residues by thin layer chromatography, J. Assoc. Awl. Chem. 49,795 (1966). 13. J. R. E. Jones, “Fish and River Pollution.” p. 9. Butterworths, London. 1964. 14. H. L. Golterman and R. S. Clymo, (Eds.), “Methods for Chemical Analysis of Water.” Blackwell. Oxford. 1969.
EFFECT
OF
ENDOSULFAN
IS. D. M. R. Rao, “Studies on the Persistence of Endosulfan in the Environment and Its Effects on Freshwater Fish.” Ph.D. thesis, Nagarjuna University, Nagarjunanagar, S. India, 1979. 16. P. Deema. E. Thompson, and G. W. Ware. Metabolism, storage and excretion of laCendosulfan in the mouse, J. Econ. Et~totrd. 59. 546 ( 1966). 17. S. G. Gorbasch. 0. R. Christ, H. Kellner. G. Kloss, and E. Borner, Metabolism of endosulfan in milk sheep. J. Agv. Food Chem. 16, 950 ( 1968).
ON
287
M. aculeutum
18. D. H. Wayman, H. Kurt. and M. C. Thomas. Fate of endosulfan in rats and toxicological consideration of apolar metabolites. Pc\ficc Biochem.
Physiol.
3, 251 (1978).
19. J. R. Brett and T. D. D. Groves. Physiological energetics. in “Fish Physiology,” (W. S. Hoar, D. J. Randall, and J. R. Brett, Eds.1. Vol. 8. p, 280. Academic Press. New York. 1979. 20. H. M. Jrueger. J. B. Saddler. G. A. Chapman. I. J. Tinsley. and R. R. Lowry. Bioenergetics. exercise and fatty acids of fish. A/rrrr. Zoo/. 8. 119. (1968).