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Acute toxicity of malachite green and its effects on certain blood parameters of a catfish, Heteropneustes fossilis
Aquatic
Toxicology
31 (1995) 241-247
Acute toxicity of malachite green and its effects on certain blood parameters of a catfish, Heteropneustes fossilis S.J. Srivastava*,
N.D. Singh, Arun K. Srivastava and Ranjana Sinha
Department of Zoology, S.M. M. Town I? G. College, Ballia-277001, India Received
31 December
1993; revision
received
14 June 1994; accepted
21 July 1994
Abstract An attempt has been made to collect upto date information of the toxicity of a dye malachite green on H. fossilis which are frequently used in fish farming. Exposure to malachite green to a concentration of 0.20 mg/l (1/5th of 96 h LC50) for 96 h caused significant depletion of serum calcium and protein levels. Short-term (IS-20 days) exposures to sub-acute 0.10 mgll (l/lOth of 96 h LC50) and sub-lethal 0.05 mg/l(1/20th of 96 h LC50) levels of the dye also affect significant decrease in the serum calcium and protein levels; however, long-term (3&60 days) exposure did not show any changes in comparison to control fish. The total cholesterol level of blood is increased significantly at all the concentrations of malachite green in respect to all the time intervals. Keywords: Malachite green; Heteropneustes fossilis; Blood parameters
1. Introduction
The triarylmethane dye, malachite green (colour index 42 000) has been widely used as a strong antifungal, antibacterial and antiparasitical agent in fish farming (Foster and Woodbury, 1936). It is also an effective tropical antiprotozoal agent (Clifton- Hadley and Alderman, 1987). The malachite green is reduced to leuco malachite green (Leuco-MG) which is eliminated at a very slow rate. Steffens et al., (1961) showed mitotic effects of malachite green, particularly chromosome breaks in rainbow trout, Oncorhynchus mykiss. Meyer and Jorgensen (1983) demonstrated spinal, head, fin and tail abnormalities in trout fry hatched from eggs exposed to malachite green. This dye may enter into the food chain and could possibly cause carcinogenic, mutagenic and teratogenic effects on human health (Klein et al., 1991). The aim of the present study is to determine the LCO, LC50 and LClOO values of this dye and to observe its behavioural and physiological effects on a catfish *Corresponding
author.
0166-445X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOl66-445X(94)00061-1
242
S.J. Srivastava
et al. IAquatic
Toxicology
31 (1995) 241-247
H. fossilis. Parameters like serum calcium, protein and total blood cholesterol levels have been chosen as criteria of toxicity of dose-dependent and time-related effects of malachite green on the catfish.
2. Material and methods Adult H. fossilis (wt. 35.75 + 4.25 g; length 15.25 & 2.50 cm) were collected locally and acclimatized to the laboratory conditions for 15 days under natural photoperiod and ambient temperature (20.8 + 1.5”C). They were fed daily with wheat flour pellets and ground dried shrimp; food was withheld 24 h prior to sampling of the fish, and no feeding occurred 96 h following exposure. The physico-chemical characteristics of the test water used were: dissolved oxygen 7.8 + 1.6 mg/l, hardness 135.60 I!I5.20 mg/l as CaCO,; chloride 7.50 & 0.85 meq/l; electrical conductivity 782 ? 42.50 pmhslcm; pH 7.7 + 0.20. The static acute toxicity bioassay (APHA/AWWA/WPCF, 1975) was performed to determine the LC50 values and 95% confidence limits (Litchfield and Wilcoxon, 1959) of malachite green after 24, 48, 72 and 96 h; LCO and LClOO values were also recorded by visual observations. The presumably harmless (safe) concentration was estimated by the formula of Hart et al., (1945). A stock solution of malachite green (1 mg/ml) was prepared in water. For the study of the effect of the dye on certain blood parameters, groups of 20-25 fish (5 fish per 10 L glass jar) were exposed to acute 0.20 mg/l(1/5th of 96 h LC50 value), sub-acute 0.10 mg/l, (l/lOth of 96 h LC50 value) and sub-lethal 0.05 mg/l (1/20th of 96 h LC50 value) concentration of malachite green in tap water, for acute (96 h), short (10-20 days) and long (30-60 days) terms. Six fish from each group were used in the analysis of selected variables. Parallel groups of six control fish were sampled at specific time intervals for comparison. At autopsy, the fish were anesthetized with MS 222. Blood from the fish was collected from the served caudal peduncle into titrated tuberculin syringes for the determination of serum calcium (Trinder, 1960) protein (Lowry et al., 1951) and total blood cholesterol (Zlatkis et al., 1953). The behaviour of the fish under the influence of the dye was also observed. The data were analysed for statistical significance between the controls and dyeexposed fish by Student ‘t’-test. Significant differences were established at the 0.05 levels.
3. Results Exposure to malachite green (triarylmethane dye) elicited hyperactivity characterized by rapid pectoral and opercular movement, erratic swimming and gradual loss of equilibrium associated with breathing difficulties in the fish. Table 1 shows LCO, LC50 and LClOO values of malachite green for H. fossilis at four time intervals. The LC50 values for 24, 48, 72 and 96 h were 5.60, 1.40, 1.25 and 1.OOmg/l, respectively.
S.J. Srivastava
et al. IAquatic
Toxicology
31 (1995) 241-247
Table 1 LCO, LC50 and LClOO values (mg/l) of malachite given in parentheses)
green for the catfish, H. fossilis (95% confidence
(h)
LCO
LC50
LClOO
24
2.20
6.15
48
1.10
72
0.95
96
0.80
5.60 (4.80-6.20) 1.40 (1.20-2.50) 1.25 (1.10-2.10) 1.00 (0.80-1.60)
243
limits are
2.10 1.60 1.15
As regards the presumably harmless (safe) concentration of malachite green, it may be taken as 0.025 mg/l (l/40 of 96 h LC50 value) for the catfish, H. fossilis. The mean serum calcium content in the control fish ranged between 15.3 + 0.24 and 20.1 + 0.53 mg/lOO ml during the experiments (Tables 2 and 3). Acute exposure (96 h) of the fish to the acute level 0.20 mg/l of this dye resulted in significantly decreased levels of serum calcium (Table 2). Exposure of the fish to sub-acute as well as sub-lethal (0.10 and 0.05 mg/l) concentrations also decreased serum calcium at all time intervals (Table 3). The serum protein values in the control fish ranged between 4.8 + 0.19 and 6.1 + 0.08 g per 100 ml. Acute exposure of fish to 0.20 mg/l of malachite green evoked significant reduction in serum protein (Table 2). Short term exposures for lo-20 days to sub-acute (0.10 mg/l) and sub-lethal (0.05 mg/l) concentrations were sufficient to produce significant decrease in serum protein levels in catfish (Table 3). However, long term (30-60 days) exposures to both sub-acute and sub-lethal levels did not
Table 2 Certain blood parameters mg/l for 96 h)
values for the catfish, H. fossilis exposed
Parameters Serum calcium (mg/lOO ml) Serum protein (g/100 ml) Total blood cholesterol (mg/l 00 ml) Values are mean f SE (n = 6). *P < 0.05. **P < 0.02-0.01.
***p < 0.001.
Control
to malachite
green, to acute level (0.20
Experimental
20.1 + 0.53
15.5 f 0.24***
6.1 f 0.13
5.2 f 0.13***
340.9 f 1.50
439.0 + o.s4***
6.1 f 0.08
Serum protein WOO ml)
422.1 f 0.50***
5.3 + 0.18**
15.1 f 0.18**
437.1 zk 0.38***
5.0 f 0.06***
16.3 + 0.30*
377.1 * 1.48***
4.9 f 0.07*
15.0 f 0.04***
0.05 mg/l
410.9 + 0.33***
5.5 f 0.14*
16.0 f 0.08**
***P < 0.001.
339.4 f 1.30
5.5 f 0.18
16.6 f 0.20
341.6 + 0.59
6.1 f 0.14
16.9 f 0.25
Values are mean f SE (n = 6). *P < 0.05. **P < 0.02-0.01.
340.2 f 0.44
16.3 * 0.30
Serum calcium (mg/lOO ml)
Total blood cholesterol (mg/lOO ml)
341.5 f 0.74
6.1 f 0.08
Total blood cholesterol (mg/lOO ml)
18.5 + 0.24
Serum protein (g/l00 ml)
0.10 mg/l
Experimental
334.0 f 0.50
5.2 f 0.19
15.7 f 0.30
345.9 f 0.72
5.5 f 0.19
369.1 f 0.12***
5.0 + 0.12
14.5 + 0.19*
465.6 f 1.4***
5.2 f 0.24
15.3 f 0.24*
Experimental
(0.10 mg/l) and sub-lethal
16.3 + 0.35
Control
Experimental
Control
Control
Serum calcium (mg/lOO ml)
Parameters
30 days
20 days
10 days
green to sub-acute
Long term
to malachite
Short term
Table 3 Certain blood parameters values for the catfish H. fossilis exposed days) and long (30-60 days) periods
334.3 Y!I0.62
4.8 I! 0.19
15.3 f 0.24
343.9 f 0.29
5.1 f 0.21
16.3 + 0.36
Control
60 days
355.2 f 0.46***
4.4 f 0.11
13.4 f 0.32***
497.2 f 1.40***
4.6 + 0.18
14.7 f 0.29**
Experimental
(0.05 mg/l) levels for short (IO-20
%
?
2
g 4
2 2
oy Lc. 8 g
g 2.
2 k 3. B $ 9
S.J. Srivastava
et al. IAquatic
Toxicology 31 (1995) 241-247
245
produce significant differences in serum protein concentration from that of control fish. Total blood cholesterol level in the control fish varied between 334.0 + 0.50 and 345.9 + 0.72 mg per 100 ml. Acute exposure to 0.20 mg/l of dye caused significantly increased blood cholesterol levels in the fish (Table 2). The fish showed hypercholesterolemia during both short and long term exposures to sub-acute (0.10 m&l) and sub-lethal (0.05 mg/l) concentrations (Tables 2 and 3).
4. Discussion The effects of malachite green on the behaviour of H. fossilis was similar to those observed in several teleostean species following exposure to various pesticides (Schoettger, 1970; Srivastava and Mishra, 1982; Srivastava and Srivastava, 1987). ETAD generated safety data sheets (SDS) for several commercial dyes which include LD50 values of malachite green for freshwater fish. In this study, this value was 1.00 mg/l. It may, however, be pointed out that the toxicity of individual dyes to different species of fish are difficult to compare (Schimmel and Wilson, 1977) because they are influenced by other factors such as temperature, pH, hardness and dissolved oxygen of the test water. The presumably harmless (safe) concentration of malachite green was 0.025 mg/l for the catfish, H. fossilis. However, until additional data on the safe concentration of malachite green for the catfish and other species become available, the assumption that the calculated concentration of 0.025 mg/l in this study can be considered safe to other Indian fresh-water fish species is debatable. In terms of environmental significance, concentration of malachite green in water at or exceeding safe concentration must be considered hazardous to fishes as they can accumulate tissue residues of this dye, when exposed to concentrations much lower than those which cause direct adverse effects on them. The elevation of serum calcium levels of fish on exposure to pesticides has been reported by several workers (Bansal et al., 1979; Dalela et al., 1981). Sharma et al. (1982) reported an elevation of serum calcium level in H. fossilis in response to congo red intoxication. Sastry and Sharma (1978) also reported elevated serum calcium in fish given dianin. However, in the present investigation serum calcium was found to decrease under the influence of malachite green. A possible cause of depletion of calcium may be renal insufficiency and disrupted electrolyte balance. Bano (1982) also reported a depletion of serum calcium in Clarias batrachus under chemical stress of aldrin. H. fossilis exposed to malachite green in the present study showed hypoproteinemia. Mehrle et al. (1971), Grant and Mehrle (1973) as well as Gluth and Hanke (1984) found significant decreases in serum protein of rainbow trout and carp exposed to organochlorine insecticides. They reported a generalized hypoproteinemia in fish after exposure to the toxicants and correlated this change to disturbance in osmoregulation. The significant decrease in total serum protein of catfish after acute as well as short term exposure to malachite green in this study may be due to kidney disorder.
246
S.J. Srivastava et al. IAquatic Toxicology 31 (1995) 241-247
Toxicants which affect the liver tissue also result in reduction of total serum protein in fish (Racicat et al., 1975; Pfeifer et al., 1977; Gingerich et al., 1978; Pfeifer and Weber, 1979). In the present study too, the liver is found to be severely damaged following dye toxicosis (unpublished data). Hypercholesterolemia was observed in malachite green exposed catfish. This may be due to the damage of the liver of the treated fish. A similar observation was made by Narain (198 1). Srivastava and Narain (1985) showed that endrin and BHC evoked hypercholesterolemia in H. fossilis after 30-40 days exposure to acute levels of these pesticides. Bano (1982) and Sharma et al. (1982) reported increased serum cholesterol in Clarias batrachus and H. fossilis which were given Congo red. Hypercholesteroic effects have also been reported in teleosts during stress (Cordier et al., 1959; Wedemeyer and McLeay, 198 1).
Acknowledgement
The financial assistance received from ICAR, New Delhi is gratefully acknowledged.
References APHA/AWWA/WPCF (1975) Standard Methods for the Examination of Water and Wastewater, 14th edition. American Public Health Association/American Water Work Association/Water Pollution Control Federation, Washington, DC. Bano, Y. (1982) Effect of aldrin on serum and liver constituentis of fresh water catfish, C/arias batrachus (L). Proc. Indian Acad. Sci. (Anim. Sci.) 91, 27-32. Bansal, SK., Verma, S.R., Gupta, A.K. and Dalela, R.C. (1979) Physiological dysfunction of the haemopoietic system in a freshwater teleost, Labeo rohita following chronic chlordane exposure. Part I - alteration in certain haematological parameters. Bull. Environ. Contam. Toxicol. 22, 666673. Clifton-Hadley, R.S. and Alderman, D.J. (1987) The effect of malachite green on proliferative kidney disease. J. Fish Dis. 10, 101-107. Cordier, D., Barnoud, R. and Brandon, A.M. (1959) Variations de la proteinemic chezl, anguille de mer (Anguilla vulgaris L.) sur l’influence de l’agression osmotique, C.R. Sot. Biol. 153, 1802-1805. Dalela, R.C., Rani, S., Kumar, V. and Verma, S.R. (1981) In vivo hematological alteration in a freshwater teleost Mytsus vittatus following sub-acute exposure to pesticides and their combination. J. Environ. Biol. 2(2), 79. Foster, F.J. and Woodbury, L. (1936) The use of malachite green as a fish fungicide and antiseptic. Prog. Fish-Culture 18, 7-9. Gingerich, W.H., Weber, L.J. and Larson, R.E. (1978) Carbon tetrachloride induced retention of sulfobromophthalein in the plasma of rainbow trout. Toxicol. Appl. Pharmacol. 43, 1477158. Gluth, G. and Hanke, W. (1984) A Comparison of physiological changes in carp, Cyprinus car&o, induced by several pollutants at sub-lethal concentration - II The dependency on the temperature. Comp. Biothem. Physiol. 796, 3945. Grant, B.F. and Mehrle, P.M. (1973) Endirn toxicosis in rainbow trout (Salvo gairdneri). J. Fish. Res. Board Can. 30, 3140. Hart, W.B., Doudoroff, P. and Greenkank, J. (1945) The evaluation of the toxicity of industrial wastes, chemicals and other substances to freshwater fish. Atlantic Refinery Co., Philadelphia, PA.
S.J. Srivastava et al. IAquatic Toxicology 31 (1995) 241L247
247
Klein, E., Edelhauser, M. and Lippold, R. (1991) Occurrence and determination of residues of malachite green and Leuco-malachite green in edible fish, Dtsch. Lebensm. Rdsch. 87( 1 I), 350--35X Litchfield, J.T., Jr. and Wilcoxon, F. (1949) A simplified method of evaluating dose effect experiments. J. Pharmac. Exp. Ther. 96, 99-113. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193,265-275. Mehrle, P.M., Stalling, D.L. and Bloomfield, R.A. (1971) Clinical aspects, pathogenesis and diagnosis of organophosphates, TKhM-3, Phosphamide and MNP and carbamate (sevin) poisoning. EKsp. Vol. Toksikol. Matervses. Simp. I, 36-38. Meyer, F.P. and Jorgensen, T.A. (1983) Teratological and other effects of malachite green on development of rainbow trout and rabbit. Trans. Am. Fish. Sot. 112, 818-821. Narain, A.% (198 I) Bioassay of blood. Some guidelines for interpretation of results. Nat. Acad. Sci. Lett. 4. 481. Pfeifer. K., Weber, L. and Larson, R. (1977) Alanine aminotransferase (GPT) in rainbow trout. Plasma enzyme levels as an index of liver damage due to carbon tetrachloride intoxication. Proc. W. Pharmacol. sot. 20.43 1437. Pfeifer, K. and Weber, L. (1979) The effect of carbon tetrachloride on the total plasma protein concentration of rainbow trout. Comp. Biochem. Physiol. 64C, 37-42. Racicot, J.G., Gaudet, M. and Feray, C. (1975) Blood and liver enzymes in rainbow trout (Su/moguirdneri) with emphasis on their diagnostic use. Study of Ccl, toxicity and a case of Aromonas infection. J. Fish Biol. 7, 824--535. Sastry, K.V. and Sharma, S.K. (1978) The effect of endrin on the histopathological changes in the liver 01 Channupuncttdus Bull. Environ. Contam. Toxicol. 20, 6711677. Schimmel, S.C. and Wilson, A.J., Jr. (1977) Acute toxicity of kepone to four estuarine animals. Chesapeake Sci. 18, 224.-227. Schoettger, R.A. (1970) Toxicology of thiodan in several fish and aquatic invertebrates. U.S. Dept. Interior Fish and Wild]. Ser. Rep. 35. 31 pp. Sharma. M.L., Agarwal. V.P., Awasthi, A.K. and Tyagi, S.K. (1982) Haematological & Biochemical characteristics of H. fossilis under the stress of congo red (diphenyl disazabine pthionic acid) Toxicol. L.dft., 14. 237-241. Steffens, W., Leider, V., Wehring, D. and Hattop, W.H. (1961) MBglichkeiten und Gefihrung der Anwendung von Malachit-grun in der Fisherei. 2. Fisch. 10, 745-771. Srivastava, A.K. and Mishra, J. (1982) Effect of lindane on carbohydrate metabolism and on blood chloride in the Indian catfish, Heteropneustes jkmilis. Acta Hydrobiol. 24, 1755181. Srivastava. P.N. and Narain, A.S. (1985) Catfish blood chemistry under environmental stress. Experientia 41, 955%957. Srivastava, A.K. and Srivastava, A.K. (1987) Acute toxicity of kepone to the freshwater catfish. Heteroprwustes ftisdis. Nat. Acad. Sci. Lett. 10, 439441. Trindcr. P. (1960) Calorimetric microdetermination of calcium in serum. Analyst, 85. 889-894. Wedemeyer, G.A. and McLeay, D.J. (1981) Methods for determining the tolerance of fishes to environmental stressors. In: Stress and Fish, edited by A.D. Pickering. Academic Press, London. pp. 247 275. Zlatkis, A., Zak. B. and Boyle, A.J. (1953) A new method for the direct determination ofserum cholesterol. J. Lab. Clin. Med. 41, 486492.
Toxicology
31 (1995) 241-247
Acute toxicity of malachite green and its effects on certain blood parameters of a catfish, Heteropneustes fossilis S.J. Srivastava*,
N.D. Singh, Arun K. Srivastava and Ranjana Sinha
Department of Zoology, S.M. M. Town I? G. College, Ballia-277001, India Received
31 December
1993; revision
received
14 June 1994; accepted
21 July 1994
Abstract An attempt has been made to collect upto date information of the toxicity of a dye malachite green on H. fossilis which are frequently used in fish farming. Exposure to malachite green to a concentration of 0.20 mg/l (1/5th of 96 h LC50) for 96 h caused significant depletion of serum calcium and protein levels. Short-term (IS-20 days) exposures to sub-acute 0.10 mgll (l/lOth of 96 h LC50) and sub-lethal 0.05 mg/l(1/20th of 96 h LC50) levels of the dye also affect significant decrease in the serum calcium and protein levels; however, long-term (3&60 days) exposure did not show any changes in comparison to control fish. The total cholesterol level of blood is increased significantly at all the concentrations of malachite green in respect to all the time intervals. Keywords: Malachite green; Heteropneustes fossilis; Blood parameters
1. Introduction
The triarylmethane dye, malachite green (colour index 42 000) has been widely used as a strong antifungal, antibacterial and antiparasitical agent in fish farming (Foster and Woodbury, 1936). It is also an effective tropical antiprotozoal agent (Clifton- Hadley and Alderman, 1987). The malachite green is reduced to leuco malachite green (Leuco-MG) which is eliminated at a very slow rate. Steffens et al., (1961) showed mitotic effects of malachite green, particularly chromosome breaks in rainbow trout, Oncorhynchus mykiss. Meyer and Jorgensen (1983) demonstrated spinal, head, fin and tail abnormalities in trout fry hatched from eggs exposed to malachite green. This dye may enter into the food chain and could possibly cause carcinogenic, mutagenic and teratogenic effects on human health (Klein et al., 1991). The aim of the present study is to determine the LCO, LC50 and LClOO values of this dye and to observe its behavioural and physiological effects on a catfish *Corresponding
author.
0166-445X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOl66-445X(94)00061-1
242
S.J. Srivastava
et al. IAquatic
Toxicology
31 (1995) 241-247
H. fossilis. Parameters like serum calcium, protein and total blood cholesterol levels have been chosen as criteria of toxicity of dose-dependent and time-related effects of malachite green on the catfish.
2. Material and methods Adult H. fossilis (wt. 35.75 + 4.25 g; length 15.25 & 2.50 cm) were collected locally and acclimatized to the laboratory conditions for 15 days under natural photoperiod and ambient temperature (20.8 + 1.5”C). They were fed daily with wheat flour pellets and ground dried shrimp; food was withheld 24 h prior to sampling of the fish, and no feeding occurred 96 h following exposure. The physico-chemical characteristics of the test water used were: dissolved oxygen 7.8 + 1.6 mg/l, hardness 135.60 I!I5.20 mg/l as CaCO,; chloride 7.50 & 0.85 meq/l; electrical conductivity 782 ? 42.50 pmhslcm; pH 7.7 + 0.20. The static acute toxicity bioassay (APHA/AWWA/WPCF, 1975) was performed to determine the LC50 values and 95% confidence limits (Litchfield and Wilcoxon, 1959) of malachite green after 24, 48, 72 and 96 h; LCO and LClOO values were also recorded by visual observations. The presumably harmless (safe) concentration was estimated by the formula of Hart et al., (1945). A stock solution of malachite green (1 mg/ml) was prepared in water. For the study of the effect of the dye on certain blood parameters, groups of 20-25 fish (5 fish per 10 L glass jar) were exposed to acute 0.20 mg/l(1/5th of 96 h LC50 value), sub-acute 0.10 mg/l, (l/lOth of 96 h LC50 value) and sub-lethal 0.05 mg/l (1/20th of 96 h LC50 value) concentration of malachite green in tap water, for acute (96 h), short (10-20 days) and long (30-60 days) terms. Six fish from each group were used in the analysis of selected variables. Parallel groups of six control fish were sampled at specific time intervals for comparison. At autopsy, the fish were anesthetized with MS 222. Blood from the fish was collected from the served caudal peduncle into titrated tuberculin syringes for the determination of serum calcium (Trinder, 1960) protein (Lowry et al., 1951) and total blood cholesterol (Zlatkis et al., 1953). The behaviour of the fish under the influence of the dye was also observed. The data were analysed for statistical significance between the controls and dyeexposed fish by Student ‘t’-test. Significant differences were established at the 0.05 levels.
3. Results Exposure to malachite green (triarylmethane dye) elicited hyperactivity characterized by rapid pectoral and opercular movement, erratic swimming and gradual loss of equilibrium associated with breathing difficulties in the fish. Table 1 shows LCO, LC50 and LClOO values of malachite green for H. fossilis at four time intervals. The LC50 values for 24, 48, 72 and 96 h were 5.60, 1.40, 1.25 and 1.OOmg/l, respectively.
S.J. Srivastava
et al. IAquatic
Toxicology
31 (1995) 241-247
Table 1 LCO, LC50 and LClOO values (mg/l) of malachite given in parentheses)
green for the catfish, H. fossilis (95% confidence
(h)
LCO
LC50
LClOO
24
2.20
6.15
48
1.10
72
0.95
96
0.80
5.60 (4.80-6.20) 1.40 (1.20-2.50) 1.25 (1.10-2.10) 1.00 (0.80-1.60)
243
limits are
2.10 1.60 1.15
As regards the presumably harmless (safe) concentration of malachite green, it may be taken as 0.025 mg/l (l/40 of 96 h LC50 value) for the catfish, H. fossilis. The mean serum calcium content in the control fish ranged between 15.3 + 0.24 and 20.1 + 0.53 mg/lOO ml during the experiments (Tables 2 and 3). Acute exposure (96 h) of the fish to the acute level 0.20 mg/l of this dye resulted in significantly decreased levels of serum calcium (Table 2). Exposure of the fish to sub-acute as well as sub-lethal (0.10 and 0.05 mg/l) concentrations also decreased serum calcium at all time intervals (Table 3). The serum protein values in the control fish ranged between 4.8 + 0.19 and 6.1 + 0.08 g per 100 ml. Acute exposure of fish to 0.20 mg/l of malachite green evoked significant reduction in serum protein (Table 2). Short term exposures for lo-20 days to sub-acute (0.10 mg/l) and sub-lethal (0.05 mg/l) concentrations were sufficient to produce significant decrease in serum protein levels in catfish (Table 3). However, long term (30-60 days) exposures to both sub-acute and sub-lethal levels did not
Table 2 Certain blood parameters mg/l for 96 h)
values for the catfish, H. fossilis exposed
Parameters Serum calcium (mg/lOO ml) Serum protein (g/100 ml) Total blood cholesterol (mg/l 00 ml) Values are mean f SE (n = 6). *P < 0.05. **P < 0.02-0.01.
***p < 0.001.
Control
to malachite
green, to acute level (0.20
Experimental
20.1 + 0.53
15.5 f 0.24***
6.1 f 0.13
5.2 f 0.13***
340.9 f 1.50
439.0 + o.s4***
6.1 f 0.08
Serum protein WOO ml)
422.1 f 0.50***
5.3 + 0.18**
15.1 f 0.18**
437.1 zk 0.38***
5.0 f 0.06***
16.3 + 0.30*
377.1 * 1.48***
4.9 f 0.07*
15.0 f 0.04***
0.05 mg/l
410.9 + 0.33***
5.5 f 0.14*
16.0 f 0.08**
***P < 0.001.
339.4 f 1.30
5.5 f 0.18
16.6 f 0.20
341.6 + 0.59
6.1 f 0.14
16.9 f 0.25
Values are mean f SE (n = 6). *P < 0.05. **P < 0.02-0.01.
340.2 f 0.44
16.3 * 0.30
Serum calcium (mg/lOO ml)
Total blood cholesterol (mg/lOO ml)
341.5 f 0.74
6.1 f 0.08
Total blood cholesterol (mg/lOO ml)
18.5 + 0.24
Serum protein (g/l00 ml)
0.10 mg/l
Experimental
334.0 f 0.50
5.2 f 0.19
15.7 f 0.30
345.9 f 0.72
5.5 f 0.19
369.1 f 0.12***
5.0 + 0.12
14.5 + 0.19*
465.6 f 1.4***
5.2 f 0.24
15.3 f 0.24*
Experimental
(0.10 mg/l) and sub-lethal
16.3 + 0.35
Control
Experimental
Control
Control
Serum calcium (mg/lOO ml)
Parameters
30 days
20 days
10 days
green to sub-acute
Long term
to malachite
Short term
Table 3 Certain blood parameters values for the catfish H. fossilis exposed days) and long (30-60 days) periods
334.3 Y!I0.62
4.8 I! 0.19
15.3 f 0.24
343.9 f 0.29
5.1 f 0.21
16.3 + 0.36
Control
60 days
355.2 f 0.46***
4.4 f 0.11
13.4 f 0.32***
497.2 f 1.40***
4.6 + 0.18
14.7 f 0.29**
Experimental
(0.05 mg/l) levels for short (IO-20
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2
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oy Lc. 8 g
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2 k 3. B $ 9
S.J. Srivastava
et al. IAquatic
Toxicology 31 (1995) 241-247
245
produce significant differences in serum protein concentration from that of control fish. Total blood cholesterol level in the control fish varied between 334.0 + 0.50 and 345.9 + 0.72 mg per 100 ml. Acute exposure to 0.20 mg/l of dye caused significantly increased blood cholesterol levels in the fish (Table 2). The fish showed hypercholesterolemia during both short and long term exposures to sub-acute (0.10 m&l) and sub-lethal (0.05 mg/l) concentrations (Tables 2 and 3).
4. Discussion The effects of malachite green on the behaviour of H. fossilis was similar to those observed in several teleostean species following exposure to various pesticides (Schoettger, 1970; Srivastava and Mishra, 1982; Srivastava and Srivastava, 1987). ETAD generated safety data sheets (SDS) for several commercial dyes which include LD50 values of malachite green for freshwater fish. In this study, this value was 1.00 mg/l. It may, however, be pointed out that the toxicity of individual dyes to different species of fish are difficult to compare (Schimmel and Wilson, 1977) because they are influenced by other factors such as temperature, pH, hardness and dissolved oxygen of the test water. The presumably harmless (safe) concentration of malachite green was 0.025 mg/l for the catfish, H. fossilis. However, until additional data on the safe concentration of malachite green for the catfish and other species become available, the assumption that the calculated concentration of 0.025 mg/l in this study can be considered safe to other Indian fresh-water fish species is debatable. In terms of environmental significance, concentration of malachite green in water at or exceeding safe concentration must be considered hazardous to fishes as they can accumulate tissue residues of this dye, when exposed to concentrations much lower than those which cause direct adverse effects on them. The elevation of serum calcium levels of fish on exposure to pesticides has been reported by several workers (Bansal et al., 1979; Dalela et al., 1981). Sharma et al. (1982) reported an elevation of serum calcium level in H. fossilis in response to congo red intoxication. Sastry and Sharma (1978) also reported elevated serum calcium in fish given dianin. However, in the present investigation serum calcium was found to decrease under the influence of malachite green. A possible cause of depletion of calcium may be renal insufficiency and disrupted electrolyte balance. Bano (1982) also reported a depletion of serum calcium in Clarias batrachus under chemical stress of aldrin. H. fossilis exposed to malachite green in the present study showed hypoproteinemia. Mehrle et al. (1971), Grant and Mehrle (1973) as well as Gluth and Hanke (1984) found significant decreases in serum protein of rainbow trout and carp exposed to organochlorine insecticides. They reported a generalized hypoproteinemia in fish after exposure to the toxicants and correlated this change to disturbance in osmoregulation. The significant decrease in total serum protein of catfish after acute as well as short term exposure to malachite green in this study may be due to kidney disorder.
246
S.J. Srivastava et al. IAquatic Toxicology 31 (1995) 241-247
Toxicants which affect the liver tissue also result in reduction of total serum protein in fish (Racicat et al., 1975; Pfeifer et al., 1977; Gingerich et al., 1978; Pfeifer and Weber, 1979). In the present study too, the liver is found to be severely damaged following dye toxicosis (unpublished data). Hypercholesterolemia was observed in malachite green exposed catfish. This may be due to the damage of the liver of the treated fish. A similar observation was made by Narain (198 1). Srivastava and Narain (1985) showed that endrin and BHC evoked hypercholesterolemia in H. fossilis after 30-40 days exposure to acute levels of these pesticides. Bano (1982) and Sharma et al. (1982) reported increased serum cholesterol in Clarias batrachus and H. fossilis which were given Congo red. Hypercholesteroic effects have also been reported in teleosts during stress (Cordier et al., 1959; Wedemeyer and McLeay, 198 1).
Acknowledgement
The financial assistance received from ICAR, New Delhi is gratefully acknowledged.
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