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The effect of endosulfan and its isomers on tissue protein, glycogen, and lipids in the fish Channa punctata
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
17, 280-286 (1982)
The Effect of Endosulfan and Its Isomers on Tissue Protein, Glycogen, and Lipids in the Fish Channa punctata A. S.MURTY' Department
of Zoology,
Nagarjuna
AND A. PRIYAMVADA University,
Nagarjunanagar,
DEW 522510,
S. India
Received May 12, 1981; accepted February 12, 1982 Changes induced in the total protein, glycogen,and lipid content of some chosen tissues of the freshwater fish, Channa punctata (Bloch), exposed to technical endosulfan and its isomers for 96 hr in a continuous flow of water, were studied. Technical endosulfan significantly decreased the protein, glycogen, and lipid concentration of liver, glycogen of muscle, and significantly increased the protein and glycogen of kidney and protein content of brain. While both isomers A and B caused biochemical changes in many tissues of C. punctata, the changes induced by isomer A were more striking than thoseinduced by isomer B. INTRODUCTION
Earlier studies on the persistence of endosulfan in the environment (5, 6) reported Pollution of the aquatic environment by that, of the two isomers present in the techpesticides is known to affect adversely the nical material (in a ratio of about 4:l of isogrowth and survival of fish (1, 2). At presmers A and B) and formulations, isomer A ent, it appears that the problem is more is less strongly bound to the soil and hence acute in the developing countries, where, in moves into the aquatic environment faster the recent past, there has been an increase in the use of pesticides as a means of in- and to a greater extent than isomer B. It was also shown that of the two isomers, creasing agricultural productivity, without much concern to the consequences of in- isomer A is at least 20 times more toxic to discriminate use. Hitherto, studies on the fish than isomer B (7, 8). Biochemical changes induced by peseffects of pesticides on fish have been ticides in fish, both in acute and sublethal mostly confined to reporting the median do not seem to have relethal concentration of many pesticides and concentrations, of aquatic toxicollittle work has been carried out on the bio- ceived the attention chemical and physiological changes in- ogists. Although there have been some reports of the effect of different pesticides duced by pesticides in fish. on the metabolism of fish (9-14), little is Endosulfan (Thiodan; 6,7,8,9,10,10known about the biochemical changes inhexachloro-1,5,5a,6,9,9a-hexahydro-6,9duced by pesticides and nothing at all on methano-2,4,3-benzodioxathiepin-3-oxide) is an organochlorine insecticide of the cy- the effect of endosulfan. Hence the present work was initiated to study the changes inclodiene group that is being increasingly used to control a variety of pests, as a result duced by technical endosulfan and its isomers in the protein, glycogen, and lipid of the banning of endrin in India. Endosulfan is less persistent in the environment and content of some tissues of the freshwater fish, Channa punctata (Bloch). is less toxic than many other organochlorine pesticides, but is reported to be highly toxic MATERIALS AND METHODS to fish (3, 4). C. punctata (6 to 9 cm in length) were 1 To whom all correspondence should be addressed collected from Guntur channel near Nagarat: Institut ftir Wasser, Boden und Lufthygiene, Verjuna University campus (Guntur district, suchsfeld Marienfelde, Schichauweg 58, D-1000 Berlin 49, West Germany. Andhra Pradesh, S. India) and were accli280 0048-3575/82/030280-07$02.00/O Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
ENDOSULFAN-INDUCED
CHANGES
matized in the laboratory at room temperature (27 rt 4°C). Analysis of the tissues of randomly chosen fish indicated that the fish tissues were free from residues of endosulfan (detection limit, 50 pg). The conditions of the toxicity tests, chemistry of the water used for conducting the tests, preparation of the toxicant solutions, and maintenance of the controls were described (7, 15). Technical grade endosulfan (96% pure) was obtained from National Chemical laboratory, Pune, India. The two isomers were separated as described previously (7). The fish were exposed to five concentrations of technical grade endosulfan (3.5, 4.25, 5, 5.75, and 6.5 ppb) that resulted in lo-90% mortality in a 96-hr test and two concentrations each of endosulfan A (0.05 and 0.45 ppb) and endosulfan B (3 and 11
IN
THE
FISH
Channa punctato
281
ppb) in continuous flow systems for a period of 96 hr. The two concentrations chosen for the last two toxicants were one higher and the other lower than the 96-hr LCSO value (8). The recommendations of the Committee on Methods for Toxicity Tests with Aquatic Organisms (16) were followed in exposing the fish to different concentrations of the toxicant. At the end of 96 hr of exposure, the fish were decapitated and the liver, kidney, muscle, brain, and gills were quickly dissected out in an ice tray. In all the studies, muscle was taken from the left side at the base of the dorsal fin. For estimating the total protein content, 25 mg of the above tissues was weighed and homogenized in I ml cold, distilled water. The total protein content of the tissues was estimated by the
I 1
6
;echni~l FIG.
liver,
7
etdosdfan” ppb
1. Regression lines of the effect of technical-grade (b) kidney, (c) muscle, (d) bruin, and (e) gills.
endosulfan
on the protein
content
of (a)
282
MURTY
AND
biuret method (17). Bovine serum albumin, supplied by Sigma Chemical Company, was used to prepare standard protein solutions. The protein content is expressed as milligrams of protein per gram wet weight of the tissue. Glycogen content of brain and gills, even when these tissues were pooled from a number of fish, was found to be insignificant and hence not estimated. For estimating the glycogen content of liver, kidney, and muscle, 50 to 100 mg of each of these tissues (from one fish in the case of liver and muscle, and pooled from a number of fish in the case of kidney), was boiled in 1% KOH for 20 min. The glycogen content was estimated by the method of Hassid and Abraham (18) using anthrone reagent and is expressed as milligrams of glycogen per gram wet weight of the tissue. For the estimation of total lipids, 1 g each of like tissues was pooled from a number of fish. As the kidney in this fish is small, its lipid content could not be estimated. The lipids of liver, muscle, brain, and gills were extracted with 2: 1, chloroform- methanol mixture (19). The lipid content is expressed as milligrams of lipid per gram wet weight of the tissue. Control fish were maintained for each test and the total protein, glycogen, and lipid content of the above tissues of control fish were estimated simultaneously. For both test and control fish, each result reported is an average of three analyses. In the case of technical endosulfan, the relationship between the change in the biochemical parameter and the concentration of the toxicant was calculated by fitting a regression line using the least-squares method. The correlation coefficient was also calculated. The values recorded for the test fish were compared with those of the controls by employing Student’s t test. RESULTS
The regression lines of the biochemical changes induced by technical endosulfan are shown in Figs. l-3. The changes in-
DEVI
20 i
Technical endosuffin ppb lines of the effect of technicalgrade endosulfan on the glycogen content of (a) liver, (b) kidney, and (c) muscle. FIG.
2. Regression
duced by the two isomers are plotted as histograms (Figs. 4 and 5). A striking alteration of the protein, glycogen, and lipid content of the liver was caused by technical endosulfan. An increasing concentration of technical endosulfan caused a significant reduction of all the three biochemical factors in the liver. There was a high degree of negative correlation between the decrease in the protein, glycogen, and lipid content of liver and increase in the pesticide concentration (r = -0.83, -0.93, and -0.93, respectively, and significant at P = 0.05). Increasing concentrations of technical endosulfan had a marked effect on the protein and glycogen content of the kidney too. In this case, however, there was a significant increase in both these factors with increasing concentrations of the toxicant (r = 0.99 and 0.79 for protein and glycogen, respectively). There was a significant reduction in the glycogen content of muscle and increase in the protein content of brain of fish exposed to increasing concentrations of technical endosulfan (r = -0.75 and 0.8, respectively). Changes induced by all the five concentrations of technical endosulfan in the three biochemical factors studied in the liver of C. punctata were significant. In the muscle, changes induced by the two highest con-
ENDOSULFAN-INDUCED
CHANGES
IN
THE
FISH
Channa
d
-------------J’,---
.
. .
I
4
1
6
&chr& 3. Regression (b) bruin, (c) muscle, FIG.
lines of the effect and (d) gills.
283
aunctatu
e&lfan5
of technical-grade
centrations of technical endosulfan in the protein, by the highest concentration in the case of lipid, and all but the lowest concentration employed in the case of glycogen, were significant. In the brain, all but the lowest concentration of technical endosulfan tested, induced significant changes in the protein content, whereas none of the concentrations used had any effect on lipid content. In general, the changes produced by technical endosulfan in the protein and lipid of gills were not significant. The changes caused by all the concentrations of technical endosulfan in the protein and
7
8
content
of (a) liver.
ppb endosulfan
on the lipid
glycogen content of kidney were significant. All the above-mentioned changes were significant at P = 0.05, when compared with the control values. As in the case of technical endosulfan, the toxic effect of isomer A was more pronounced on liver than other organs. The protein content of liver, kidney, and gills; the lipid content of all the four tissues studied and the glycogen content of livei and muscle were significantly altered (P = 0.05) by the higher concentration of isomei A (0.45 ppb) whereas the lower concentration (0.05 ppb) significantly altered the
284
MURTY
FIG. 4. Effect of endosulfan A on the protein, lipid, and glycogen content of (A) liver, (B) muscle, (C) brain, (0) gills, and (E) kidney. The three columns for each tissue, from left to right show the control values and the result of exposure to 0.05 and 0.45 ppb concentrations.
protein content of muscle and kidney; lipid content of liver, muscle, and brain and glycogen concentration of all the three tissues studied. The significant changes induced by the two isomers, however, were not comparable; whereas the higher concentration in all cases, except muscle glycogen, had a stimulatory effect, the lower concentration, on the other hand, produced an inhibitory effect in the muscle and stimulatory effect in the kidney. Isomer B produced fewer significant changes (11 as against 17 produced by Isomer A) in the tissues of C. punctata. The higher concentration of isomer B (11 ppb) induced significant changes (P = 0.05) in the protein of muscle, brain, and gills; lipid of brain; and glycogen of muscle and kidney. The lower concentration of isomer B (3 ppb) induced significant changes in the protein of kidney and brain; lipid of muscle; and glycogen of muscle and kidney. The lipid content of liver, brain, and gills and glycogen and protein of liver were not affected by either concentration of isomer B tested. In contrast to the different effects produced by isomer A at higher and lower
AND
DEW
FIG. 5. Effect of endosulfan B on and glycogen content of (A) liver, brain, (0) gills, and (E) kidney. The each tissue, from left to right show and the result of exposure to 3 and tions.
the protein, lipid, (B) muscle, (C) three columns for the control values I1 ppb concentra-
concentrations, isomer B, at both concentrations, proved to be inhibitory in all tissues, except in the case of kidney protein, which was significantly elevated, at the lower concentration of isomer B. DISCUSSION
Biochemical changes induced in the tissues of fish by pesticides, do not seem to have been studied in any significant detail (20). An increase in blood glucose of blackdarter exposed to dieldrin (21); changes in the blood serum proteins and free amino acids of C. punctata exposed to dieldrin, DDT, and malathion (11, 12); inhibition of liver glycogenolysis in rainbow trout exposed to endrin (21); increase in liver glycogen of Scorpuenu porcus exposed to lindane (10); and changes in the serum protein and glycogen content of rainbow trout exposed to endrin (9) were reported earlier. There have been no studies on the effect of endosulfan in the biochemistry of fish tissues. The present work showed that technical endosulfan, as well as its two isomers, induced marked biochemical changes in liver, kidney, muscle, brain, and gills of the freshwater fish C. punctutu. While the
ENDOSULFAN-INDUCED
CHANGES
positive or negative relationship between the toxicant concentration and the effect produced is attributable, respe+vely, either to stimulation or inhibition, it is not easy to explain why the lower concentration of the toxicant inhibited a particular biochemical factor whereas the higher concentration stimulated the same factor in the same as well as, sometimes, in some other tissue. A similar situation was earlier reported by Christensen et al. (22) and McKim el al. (23) in their study on the biochemical changes in fish blood and early development stages of fish, caused by metals. For instance, the blood protein of the brown bullhead (Zctalurus nebulosus (Leusueur)) exposed to 27 pg of Cu(II)/ liter for 6 days was increased slightly over the control values and significantly by 49 and 107 kg of Cu(II)/liter. At the end of 30 days exposure, the blood protein content of fish exposed to the lowest and highest concentrations was significantly reduced whereas there was no change in those fish exposed to the median concentration (22). Blood protein of the brook trout (Salvelinus fontinulis) exposed for 6 days to 24 and 39 pg of Cu(II)/liter was significantly increased, whereas that of fish exposed to 67.5 pg of Cu(II)/liter was not altered when compared with the controls (23). At the end of 21 days exposure, there was no significant change in the fish exposed to the lowest concentration, whereas there was a significant reduction at the two higher concentrations (23). While there was no significant change in the protein content of brook trout embryos exposed to two different concentrations of CH,Hg(II), Cd(II), and Pb(II), the protein content of alevins was reduced by the lower concentration of CH,Hg(II) and significantly increased by the lower concentration of Cd(I1). The higher concentrations of CH,Hg(II) and Cd(I1) had no effect. Likewise, exposure to 27 pg of Cu(II)/liter had no effect on blood glucose of the brown bullhead at the end of 6 or 30 days, whereas exposure to 49 and 107 ,ug of Cu(II)/liter at the end of 6 or 30 days caused a significant increase (24).
IN THE FISH Channn puncfafa
285
The principal organs affected by endosulfan were the liver and kidney. These two organs were reported to be the sites of degradation and detoxification of endosulfan (7, 8, 16) and the biochemical effects recorded seem to be the result of the greater stress these two organs experience during the process of detoxification of endosulfan and its metabolites. In general, the changes induced by isomer A were more striking than those caused by isomer B, which is not surprising since it is now known that the former is more toxic than the latter (7, 8, 16). Death of fish in short-term exposures to high concentration of toxicants may be due to their direct action. However, significant biochemical changes induced by pesticides may be more hazardous and could reduce the growth rate and fecundity, affect the ability to assimilate food, survival of eggs and embryos, and alter the behavior of fish and hence make the fish more susceptible to attack by predators. The long-term effects of such biochemical changes induced by aquatic pollutants are poorly known and need to be investigated more extensively. ACKNOWLEDGMENTS We thank the Council of Scientific and Industrial Research, India, for financial assistance and Dr. D. G. R. McLeod for his help in the statistical analysis and for critically reading the manuscript. REFERENCES 1. A. V. Holden, Effects of pesticides on fish, in “Environmental Pollution by Pesticides” (C. A. Edwards, Ed.), pp.213-253, Plenum, London/New York, 1973. 2. D. W. Johnson, Pesticide residues in fish, in “Environmental Pollution by Pesticides” (C. A. Edwards, Ed.), pp. 182-212, Plenum, London/New York, 1973. 3. H. Maier-Bode, Properties, effect, residues and analytics of the insecticide endosulfan, Residue Rev. 22, 1 (1968). 4. R. A. Schoettger, Toxicology of Thiodan in several fish and aquatic invertebrates, Invest. Fish Control 35, 1 (1970). 5. R. A. Byers, D. W. Woodham, and M. C. Bowman, Residues on coastal Bermudagrass, trash and soil treated with granular endosulfan, J. Econ. Entomol. 58, 160 (1965).
MURTY
286
6. 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” (S. R. Verma, A. K. Tyagi, and S. K. Bansal, Eds.), pp. 217-220, Acad. Environ. Sci., Muzaffarnagar, India, 1979. 7. 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 rohita,
8.
9. 10.
11. 12.
13.
14.
15.
.I. Toxicol.
Environ.
Health
AND DEVI
16.
17.
6, 323-330
(1980). A. P. Devi, D. M. R. Rao, K. S. Tilak, and A. S. 18. Murty, Relative toxicity of the technical grade material, isomers and formulations of endosulfan to the fish Channa punctata, Bull. Environ. Contam. Toxicol. 27, 239 (1981). 19. B. F. Grant and P. M. Mehrle, Endrin toxicosis in rainbow trout (Salmo gairdneri), .I. Fish. Res. Bd. Canad. 30, 31 (1973). P. Escoubet and N. Vincente, Sublethal effects of 20. lindane on the activity of hepatic glucose-6phosphate and the glycogen level of the scorpion fish Scorpaena porcus, L., Ann. Inst. Michel Pacha 8, 55 (1975). 21. K. P. Lone and M. Y. Javaid, Effect of sublethal doses of DDT and dieldrin in the blood of Channa punctata, Pak. J. 2001. 8, 143 (1976). A. R. Shakoori, S. A. Zaheer, and M. S. Ahmad, 22. Effect of malathion dieldrin and endrin on blood serum proteins and free aminoacids pool of C. punctata, Pak. J. Zool. 8, 125 (1976). P. K. Mukhopadhyay and P. V. Dehadrai, Biochemical changes in the air-breathing catfish (Clarias batrachus Linn.) exposed to malath- 23. ion, in “Sot. Biol. Chem., 48th Annual Meeting, 1979,” Abstr. P-4 DEB 1.21. K. S. P. Rao and K. V. R. Rao, Effect of sublethal concentrations of methyl parathion on selected oxidative enzymes and organic constituents in 24. the tissues of the freshwater fish Tilapia mossambica (Peters), Curr. Sci. 48, 526 (1979). D. M. R. Rao, A. P. Devi, and A. S. Murty, Toxicity and metabolism of endosulfan and its effect
on oxygen consumption and total nitrogen excretion of the fish Macrognathus aculeatum, Pestic. Biochem. Physiol. 15, 282 (1981). Committee on Methods for Toxicity Tests with Aquatic Organisms, “Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians,” pp. 1-61, Environmental Protection Agency, Oregon, 1975. A. G. Gornall, C. J. Bardwill, and M. M. David, Determination of the serum proteins by means of the biuret reaction, J. Biol. Chem. 177, 751 (1949). W. Z. Hassid and S. Abraham, in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 3, p. 37, Academic Press, New York, 1957. J. Folch, I. Ascoli, M. Lees, J. A. Meath, and F. N. Le Baron, Preparation of lipid extract from brain tissue, J. Biol. Chem. 191, 833 (1951). E. K. Silbergeld, Dieldrin: Effects of chronic sublethal exposure on adaptation to thermal stress in freshwater fish, Environ. Sci. Technol. 7, 846 (1973). C. M. Menzie, in “Environmental Toxicology by Pesticides“ (F. Matsumura, G. M. Bausch, and T. Misato, Eds.), Academic Press, New York, 1972. G. M. Christensen, J. M. McKim, W. A. Brungs, and E. P. Hunt, Changes in the blood of the brown bullhead Ictalurus nebulosus (Leusueur) following short and long term exposure to copper (II), Toxic01 Appl. Pharmacol. 23, 417 (1972). J. M. McKim, G. M. Christensen, and E. P. Hunt, Changes in the blood of brook trout (Salvelinus fontinalis) after short-term and long-term exposure to copper, J. Fish. Res. Bd. Canad. 27, 1883 (1970). G. M. Christensen, Biochemical effects of methyl mercuric chloride, cadmium chloride and lead nitrate on embryos and alevins of the brook trout, Salvelinus fontinalis, Toxicol. Appl. Pharmacol. 32, 191 (1975).
BIOCHEMISTRY
AND
PHYSIOLOGY
17, 280-286 (1982)
The Effect of Endosulfan and Its Isomers on Tissue Protein, Glycogen, and Lipids in the Fish Channa punctata A. S.MURTY' Department
of Zoology,
Nagarjuna
AND A. PRIYAMVADA University,
Nagarjunanagar,
DEW 522510,
S. India
Received May 12, 1981; accepted February 12, 1982 Changes induced in the total protein, glycogen,and lipid content of some chosen tissues of the freshwater fish, Channa punctata (Bloch), exposed to technical endosulfan and its isomers for 96 hr in a continuous flow of water, were studied. Technical endosulfan significantly decreased the protein, glycogen, and lipid concentration of liver, glycogen of muscle, and significantly increased the protein and glycogen of kidney and protein content of brain. While both isomers A and B caused biochemical changes in many tissues of C. punctata, the changes induced by isomer A were more striking than thoseinduced by isomer B. INTRODUCTION
Earlier studies on the persistence of endosulfan in the environment (5, 6) reported Pollution of the aquatic environment by that, of the two isomers present in the techpesticides is known to affect adversely the nical material (in a ratio of about 4:l of isogrowth and survival of fish (1, 2). At presmers A and B) and formulations, isomer A ent, it appears that the problem is more is less strongly bound to the soil and hence acute in the developing countries, where, in moves into the aquatic environment faster the recent past, there has been an increase in the use of pesticides as a means of in- and to a greater extent than isomer B. It was also shown that of the two isomers, creasing agricultural productivity, without much concern to the consequences of in- isomer A is at least 20 times more toxic to discriminate use. Hitherto, studies on the fish than isomer B (7, 8). Biochemical changes induced by peseffects of pesticides on fish have been ticides in fish, both in acute and sublethal mostly confined to reporting the median do not seem to have relethal concentration of many pesticides and concentrations, of aquatic toxicollittle work has been carried out on the bio- ceived the attention chemical and physiological changes in- ogists. Although there have been some reports of the effect of different pesticides duced by pesticides in fish. on the metabolism of fish (9-14), little is Endosulfan (Thiodan; 6,7,8,9,10,10known about the biochemical changes inhexachloro-1,5,5a,6,9,9a-hexahydro-6,9duced by pesticides and nothing at all on methano-2,4,3-benzodioxathiepin-3-oxide) is an organochlorine insecticide of the cy- the effect of endosulfan. Hence the present work was initiated to study the changes inclodiene group that is being increasingly used to control a variety of pests, as a result duced by technical endosulfan and its isomers in the protein, glycogen, and lipid of the banning of endrin in India. Endosulfan is less persistent in the environment and content of some tissues of the freshwater fish, Channa punctata (Bloch). is less toxic than many other organochlorine pesticides, but is reported to be highly toxic MATERIALS AND METHODS to fish (3, 4). C. punctata (6 to 9 cm in length) were 1 To whom all correspondence should be addressed collected from Guntur channel near Nagarat: Institut ftir Wasser, Boden und Lufthygiene, Verjuna University campus (Guntur district, suchsfeld Marienfelde, Schichauweg 58, D-1000 Berlin 49, West Germany. Andhra Pradesh, S. India) and were accli280 0048-3575/82/030280-07$02.00/O Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
ENDOSULFAN-INDUCED
CHANGES
matized in the laboratory at room temperature (27 rt 4°C). Analysis of the tissues of randomly chosen fish indicated that the fish tissues were free from residues of endosulfan (detection limit, 50 pg). The conditions of the toxicity tests, chemistry of the water used for conducting the tests, preparation of the toxicant solutions, and maintenance of the controls were described (7, 15). Technical grade endosulfan (96% pure) was obtained from National Chemical laboratory, Pune, India. The two isomers were separated as described previously (7). The fish were exposed to five concentrations of technical grade endosulfan (3.5, 4.25, 5, 5.75, and 6.5 ppb) that resulted in lo-90% mortality in a 96-hr test and two concentrations each of endosulfan A (0.05 and 0.45 ppb) and endosulfan B (3 and 11
IN
THE
FISH
Channa punctato
281
ppb) in continuous flow systems for a period of 96 hr. The two concentrations chosen for the last two toxicants were one higher and the other lower than the 96-hr LCSO value (8). The recommendations of the Committee on Methods for Toxicity Tests with Aquatic Organisms (16) were followed in exposing the fish to different concentrations of the toxicant. At the end of 96 hr of exposure, the fish were decapitated and the liver, kidney, muscle, brain, and gills were quickly dissected out in an ice tray. In all the studies, muscle was taken from the left side at the base of the dorsal fin. For estimating the total protein content, 25 mg of the above tissues was weighed and homogenized in I ml cold, distilled water. The total protein content of the tissues was estimated by the
I 1
6
;echni~l FIG.
liver,
7
etdosdfan” ppb
1. Regression lines of the effect of technical-grade (b) kidney, (c) muscle, (d) bruin, and (e) gills.
endosulfan
on the protein
content
of (a)
282
MURTY
AND
biuret method (17). Bovine serum albumin, supplied by Sigma Chemical Company, was used to prepare standard protein solutions. The protein content is expressed as milligrams of protein per gram wet weight of the tissue. Glycogen content of brain and gills, even when these tissues were pooled from a number of fish, was found to be insignificant and hence not estimated. For estimating the glycogen content of liver, kidney, and muscle, 50 to 100 mg of each of these tissues (from one fish in the case of liver and muscle, and pooled from a number of fish in the case of kidney), was boiled in 1% KOH for 20 min. The glycogen content was estimated by the method of Hassid and Abraham (18) using anthrone reagent and is expressed as milligrams of glycogen per gram wet weight of the tissue. For the estimation of total lipids, 1 g each of like tissues was pooled from a number of fish. As the kidney in this fish is small, its lipid content could not be estimated. The lipids of liver, muscle, brain, and gills were extracted with 2: 1, chloroform- methanol mixture (19). The lipid content is expressed as milligrams of lipid per gram wet weight of the tissue. Control fish were maintained for each test and the total protein, glycogen, and lipid content of the above tissues of control fish were estimated simultaneously. For both test and control fish, each result reported is an average of three analyses. In the case of technical endosulfan, the relationship between the change in the biochemical parameter and the concentration of the toxicant was calculated by fitting a regression line using the least-squares method. The correlation coefficient was also calculated. The values recorded for the test fish were compared with those of the controls by employing Student’s t test. RESULTS
The regression lines of the biochemical changes induced by technical endosulfan are shown in Figs. l-3. The changes in-
DEVI
20 i
Technical endosuffin ppb lines of the effect of technicalgrade endosulfan on the glycogen content of (a) liver, (b) kidney, and (c) muscle. FIG.
2. Regression
duced by the two isomers are plotted as histograms (Figs. 4 and 5). A striking alteration of the protein, glycogen, and lipid content of the liver was caused by technical endosulfan. An increasing concentration of technical endosulfan caused a significant reduction of all the three biochemical factors in the liver. There was a high degree of negative correlation between the decrease in the protein, glycogen, and lipid content of liver and increase in the pesticide concentration (r = -0.83, -0.93, and -0.93, respectively, and significant at P = 0.05). Increasing concentrations of technical endosulfan had a marked effect on the protein and glycogen content of the kidney too. In this case, however, there was a significant increase in both these factors with increasing concentrations of the toxicant (r = 0.99 and 0.79 for protein and glycogen, respectively). There was a significant reduction in the glycogen content of muscle and increase in the protein content of brain of fish exposed to increasing concentrations of technical endosulfan (r = -0.75 and 0.8, respectively). Changes induced by all the five concentrations of technical endosulfan in the three biochemical factors studied in the liver of C. punctata were significant. In the muscle, changes induced by the two highest con-
ENDOSULFAN-INDUCED
CHANGES
IN
THE
FISH
Channa
d
-------------J’,---
.
. .
I
4
1
6
&chr& 3. Regression (b) bruin, (c) muscle, FIG.
lines of the effect and (d) gills.
283
aunctatu
e&lfan5
of technical-grade
centrations of technical endosulfan in the protein, by the highest concentration in the case of lipid, and all but the lowest concentration employed in the case of glycogen, were significant. In the brain, all but the lowest concentration of technical endosulfan tested, induced significant changes in the protein content, whereas none of the concentrations used had any effect on lipid content. In general, the changes produced by technical endosulfan in the protein and lipid of gills were not significant. The changes caused by all the concentrations of technical endosulfan in the protein and
7
8
content
of (a) liver.
ppb endosulfan
on the lipid
glycogen content of kidney were significant. All the above-mentioned changes were significant at P = 0.05, when compared with the control values. As in the case of technical endosulfan, the toxic effect of isomer A was more pronounced on liver than other organs. The protein content of liver, kidney, and gills; the lipid content of all the four tissues studied and the glycogen content of livei and muscle were significantly altered (P = 0.05) by the higher concentration of isomei A (0.45 ppb) whereas the lower concentration (0.05 ppb) significantly altered the
284
MURTY
FIG. 4. Effect of endosulfan A on the protein, lipid, and glycogen content of (A) liver, (B) muscle, (C) brain, (0) gills, and (E) kidney. The three columns for each tissue, from left to right show the control values and the result of exposure to 0.05 and 0.45 ppb concentrations.
protein content of muscle and kidney; lipid content of liver, muscle, and brain and glycogen concentration of all the three tissues studied. The significant changes induced by the two isomers, however, were not comparable; whereas the higher concentration in all cases, except muscle glycogen, had a stimulatory effect, the lower concentration, on the other hand, produced an inhibitory effect in the muscle and stimulatory effect in the kidney. Isomer B produced fewer significant changes (11 as against 17 produced by Isomer A) in the tissues of C. punctata. The higher concentration of isomer B (11 ppb) induced significant changes (P = 0.05) in the protein of muscle, brain, and gills; lipid of brain; and glycogen of muscle and kidney. The lower concentration of isomer B (3 ppb) induced significant changes in the protein of kidney and brain; lipid of muscle; and glycogen of muscle and kidney. The lipid content of liver, brain, and gills and glycogen and protein of liver were not affected by either concentration of isomer B tested. In contrast to the different effects produced by isomer A at higher and lower
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DEW
FIG. 5. Effect of endosulfan B on and glycogen content of (A) liver, brain, (0) gills, and (E) kidney. The each tissue, from left to right show and the result of exposure to 3 and tions.
the protein, lipid, (B) muscle, (C) three columns for the control values I1 ppb concentra-
concentrations, isomer B, at both concentrations, proved to be inhibitory in all tissues, except in the case of kidney protein, which was significantly elevated, at the lower concentration of isomer B. DISCUSSION
Biochemical changes induced in the tissues of fish by pesticides, do not seem to have been studied in any significant detail (20). An increase in blood glucose of blackdarter exposed to dieldrin (21); changes in the blood serum proteins and free amino acids of C. punctata exposed to dieldrin, DDT, and malathion (11, 12); inhibition of liver glycogenolysis in rainbow trout exposed to endrin (21); increase in liver glycogen of Scorpuenu porcus exposed to lindane (10); and changes in the serum protein and glycogen content of rainbow trout exposed to endrin (9) were reported earlier. There have been no studies on the effect of endosulfan in the biochemistry of fish tissues. The present work showed that technical endosulfan, as well as its two isomers, induced marked biochemical changes in liver, kidney, muscle, brain, and gills of the freshwater fish C. punctutu. While the
ENDOSULFAN-INDUCED
CHANGES
positive or negative relationship between the toxicant concentration and the effect produced is attributable, respe+vely, either to stimulation or inhibition, it is not easy to explain why the lower concentration of the toxicant inhibited a particular biochemical factor whereas the higher concentration stimulated the same factor in the same as well as, sometimes, in some other tissue. A similar situation was earlier reported by Christensen et al. (22) and McKim el al. (23) in their study on the biochemical changes in fish blood and early development stages of fish, caused by metals. For instance, the blood protein of the brown bullhead (Zctalurus nebulosus (Leusueur)) exposed to 27 pg of Cu(II)/ liter for 6 days was increased slightly over the control values and significantly by 49 and 107 kg of Cu(II)/liter. At the end of 30 days exposure, the blood protein content of fish exposed to the lowest and highest concentrations was significantly reduced whereas there was no change in those fish exposed to the median concentration (22). Blood protein of the brook trout (Salvelinus fontinulis) exposed for 6 days to 24 and 39 pg of Cu(II)/liter was significantly increased, whereas that of fish exposed to 67.5 pg of Cu(II)/liter was not altered when compared with the controls (23). At the end of 21 days exposure, there was no significant change in the fish exposed to the lowest concentration, whereas there was a significant reduction at the two higher concentrations (23). While there was no significant change in the protein content of brook trout embryos exposed to two different concentrations of CH,Hg(II), Cd(II), and Pb(II), the protein content of alevins was reduced by the lower concentration of CH,Hg(II) and significantly increased by the lower concentration of Cd(I1). The higher concentrations of CH,Hg(II) and Cd(I1) had no effect. Likewise, exposure to 27 pg of Cu(II)/liter had no effect on blood glucose of the brown bullhead at the end of 6 or 30 days, whereas exposure to 49 and 107 ,ug of Cu(II)/liter at the end of 6 or 30 days caused a significant increase (24).
IN THE FISH Channn puncfafa
285
The principal organs affected by endosulfan were the liver and kidney. These two organs were reported to be the sites of degradation and detoxification of endosulfan (7, 8, 16) and the biochemical effects recorded seem to be the result of the greater stress these two organs experience during the process of detoxification of endosulfan and its metabolites. In general, the changes induced by isomer A were more striking than those caused by isomer B, which is not surprising since it is now known that the former is more toxic than the latter (7, 8, 16). Death of fish in short-term exposures to high concentration of toxicants may be due to their direct action. However, significant biochemical changes induced by pesticides may be more hazardous and could reduce the growth rate and fecundity, affect the ability to assimilate food, survival of eggs and embryos, and alter the behavior of fish and hence make the fish more susceptible to attack by predators. The long-term effects of such biochemical changes induced by aquatic pollutants are poorly known and need to be investigated more extensively. ACKNOWLEDGMENTS We thank the Council of Scientific and Industrial Research, India, for financial assistance and Dr. D. G. R. McLeod for his help in the statistical analysis and for critically reading the manuscript. REFERENCES 1. A. V. Holden, Effects of pesticides on fish, in “Environmental Pollution by Pesticides” (C. A. Edwards, Ed.), pp.213-253, Plenum, London/New York, 1973. 2. D. W. Johnson, Pesticide residues in fish, in “Environmental Pollution by Pesticides” (C. A. Edwards, Ed.), pp. 182-212, Plenum, London/New York, 1973. 3. H. Maier-Bode, Properties, effect, residues and analytics of the insecticide endosulfan, Residue Rev. 22, 1 (1968). 4. R. A. Schoettger, Toxicology of Thiodan in several fish and aquatic invertebrates, Invest. Fish Control 35, 1 (1970). 5. R. A. Byers, D. W. Woodham, and M. C. Bowman, Residues on coastal Bermudagrass, trash and soil treated with granular endosulfan, J. Econ. Entomol. 58, 160 (1965).
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6. 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” (S. R. Verma, A. K. Tyagi, and S. K. Bansal, Eds.), pp. 217-220, Acad. Environ. Sci., Muzaffarnagar, India, 1979. 7. 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 rohita,
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on oxygen consumption and total nitrogen excretion of the fish Macrognathus aculeatum, Pestic. Biochem. Physiol. 15, 282 (1981). Committee on Methods for Toxicity Tests with Aquatic Organisms, “Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians,” pp. 1-61, Environmental Protection Agency, Oregon, 1975. A. G. Gornall, C. J. Bardwill, and M. M. David, Determination of the serum proteins by means of the biuret reaction, J. Biol. Chem. 177, 751 (1949). W. Z. Hassid and S. Abraham, in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, Eds.), Vol. 3, p. 37, Academic Press, New York, 1957. J. Folch, I. Ascoli, M. Lees, J. A. Meath, and F. N. Le Baron, Preparation of lipid extract from brain tissue, J. Biol. Chem. 191, 833 (1951). E. K. Silbergeld, Dieldrin: Effects of chronic sublethal exposure on adaptation to thermal stress in freshwater fish, Environ. Sci. Technol. 7, 846 (1973). C. M. Menzie, in “Environmental Toxicology by Pesticides“ (F. Matsumura, G. M. Bausch, and T. Misato, Eds.), Academic Press, New York, 1972. G. M. Christensen, J. M. McKim, W. A. Brungs, and E. P. Hunt, Changes in the blood of the brown bullhead Ictalurus nebulosus (Leusueur) following short and long term exposure to copper (II), Toxic01 Appl. Pharmacol. 23, 417 (1972). J. M. McKim, G. M. Christensen, and E. P. Hunt, Changes in the blood of brook trout (Salvelinus fontinalis) after short-term and long-term exposure to copper, J. Fish. Res. Bd. Canad. 27, 1883 (1970). G. M. Christensen, Biochemical effects of methyl mercuric chloride, cadmium chloride and lead nitrate on embryos and alevins of the brook trout, Salvelinus fontinalis, Toxicol. Appl. Pharmacol. 32, 191 (1975).