Camp. Efiochem. Physiol. Vol. 10+X, No. l/2, pp. 95-98, 1991 Printedin Great Britain
0306~4492/91 $3.00 + 0.00 0 1991 PergamonPressplc
THE EFFECT OF TIME ON PHYSIOLOGICAL CHANGES IN EEL ANGUILLA ANGUILLA, INDUCED BY LINDANE M. D.
FERRANDO
and E.
ANDREWMOLINER
Department of Animal Physiology, Faculty of Biological Sciences, University of Valencia, Dr Moliner 50, E-46100 Burjasot, Valencia, Spain (Received 1 Octo&r 1990) Abstract-l. Eel were exposed to a sublethal concentration of lindane (0.335 ppm) for 6, 12, 24, 48, 72 and 96 hr. 2. Concentrations of glycogen, glucose, lactate, pyruvate and lipids were determined in gill tissue after lindane exposure. 3. Gill glycogen descreased and glucose levels increased at 6 hr of treatment, lactate and pyruvate concentration increased between 6 and 48 hr. Total lipid values decreased between 6 and 24 hr; thereafter, the levels increased up to 72 hr of exposure. 4. Clear changes were found in all parameters tested in gill tissues. The observed effects of lindane on metabolism in fish are discussed in relation to acute stress syndrome.
4.1 mmol/l). A 12-hr photoperiod (8.00-20.00) was maintained (Ferrando et al., 1989). The fish were then transferred to test aquaria and were not fed. Previous works on lindane toxicology of A. anguilla in our laboratory showed that 0.67 ppm was the LC~ value at 96 hr (Ferrando et al., 1987). For the study of the effects of lindane on carbohydrate metabolites, groups of 10 fish each were exposed to 0.335 ppm (i 96-hr LQ,,) lindane. Fish were sampled for the various biochemical parameters at 6, 12,24,48,72 and 96 hr exposure to the toxicant. Control fish were held in vehicle (acetone)-supplemented tap water.
INTRODUCTION
Pesticides used in pest control programmes seem to produce many physiological and biochemical changes in freshwater organisms by influencing the activities of several enzymes. Alterations in the chemical composition of the natural aquatic environment usually affect behavioural and physiological systems of the inhabitants, particularly of fish (Radhaiah et al., 1987). Some data are available on the effects of different pesticides on the biochemical aspects of fish gill (Girija, 1984; Ferrando et af., 1989). Gill is an important fish tissue because of its direct contact with water and because the pesticide has to go through it to come into the fish body. It has been suggested that waterborne pollutants damage fish gill, presumably by causing breakdown of the gas-exchange mechanism with the consequent tissue hypoxic conditions (Tort el al., 1984). Therefore, an attempt was made to observe certain biochemical parameters of fish, Anguilla anguilla under lindane intoxication. Investigation of this nature is useful in understanding the orientation of biochemical changes during sublethal toxicity to ascertain the degree of intensity of the toxicity of lindane on the gill. Time response and magnitude of response relationships in gill glycogen, -glucose, lactate, pyruvate and lipids were determined in the European eel, A. unguilla, following exposure to a sublethal concentration of lindane for 6, 12, 48, 72 and 96 hr.
Chemicals Technical grade lindane (AFRASA Company, Patema, Valencia, Spain) of 99% purity was used for experimentation. Stock solutions were prepared by dissolving lindane in acetone; appropriate quantities of this solution were pipetted into 40-l. glass aquaria containing 35 1. of test solution and 10 fish. Ten more eels, used as controls, were kept in 35 1. of clean water with the same concentration of acetone. Analysis of metabolites and lipid After each exposure the fish were quickly killed, gill tissue was removed and the metabolites determined. Glycogen content was estimated with the anthrone method (Seifter er al.. 1950). Gill tissue (0.5-1.0 g) was homogenized by adding 0.5 ml KOH (60%) and y ml KOH (50%). Thk mixture was kept in a boiling water-bath for 30 min. A 4-ml aliquot of ethanol was added to the homogenates before putting them into the fridge. Next day, samples were centrifuged and the supematant was saved for glucose determination. The pellet was resuspended in 1 ml water and a 0.25-ml aliquot was mixed with 1.75 ml anthrone reagent for 15min at 100°C. Glycogen content was determined spectrophotometrically. Glucose was assayed in the supernatants using test kits from Boehringer-Mannheim. Samples for lactate were homogenized in trichloroacetic (7%) at 4°C and centrifuged at 5000 rpm (Lang and Michal, 1974). Lactate concentration was measured spectrophotometrically by means of NADH produced in an enzymatic reaction with lactate dehydrogenase (Boehringer-Mannheim kits).
MATERIALS AND METHODS
Test system Eels of species AnguiNu anguilla (weight: 20-30 g; length: 16-20cm), were obtained from a fish farm in Valencia, Spain. They were acclimatized to laboratory conditions for two weeks in 300-l. glass tanks. The tanks were supplied with a continuous flow of tap water (temperature: 20°C; total hardness: 250 ppm as CaCO,; pH: 7.9 + 0.2; alkalinity: 95
M. D. FERRANDO and E. ANDREU-MOLINER
96 Glycogen
Glucose
._-
I
c
6 :
100
140 i
120
I
100
,’ a
60
I
:
60
:
40
: n t
z
60
:
9
20
20
i s
a 0
c
6
12
24 t (howe)
42
12
40 20 0
26
’ C
, 6
12
i
24 t (houra)
42
72
96
Fig. 1. Changes in gill glycogen levels vs controls at 6, 12, Fig. 2. Changes in gill glucose levels vs controls at 6, 12, 24, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P < 0.05. 48, 72 and 96 hr of treatment (0.335 ppm); *P -c0.05. For pyruvate determination, tissue samples were homogenized in perchloric acid (1 M) and centrifuged at 3000 rpm (Dange and Masurekar, 1982). Pyruvate concentration was measured with a test kit (BoehringerMannheim). Lipids were analyzed spectrophotometrically with a test kit (Boehringer-Mannheim) after extraction according to Bligh and Dyer (1959).
Statislicalanalysis One-way analysis of variance (ANOVA) was used to determine treatment toxic effects, and Duncan’s significant difference test was used for mean separation. The significant level was fixed at P < 0.001 and P < 0.05. The graphs in this paper show the changes vs controls, which were not used for
the statistical analysis. This analysis was performed using the Statistical Analysis System (SPSS+) with an IBM computer. RESULTS
Gill glycogen and glucose concentrations
The results are shown in Figs 1 and 2, and Table 1. The concentration of gill glycogen was already significantly reduced (70%) after 6 hr of exposure. The mean control value was 2.02 (+ 17) mg/g wet wt. Glycogen levels decreased between 6 and 96 hr. Exposure for 24 hr did not elicit a clear effect. At all other exposure times a lowered glycogen content was found indicating that glycogenolysis had occurred in treated fish. The mean control concentration of gill glucose was 0.14 mg/g wet wt. Exposure resulted in a significant increase (30% at 6 and 48 hr, Fig. 2) in gill glucose at all exposure times. Table
I. Levels
Parameter Glycogen (mg/g wet wt) Glucose (mg/g wet wt)
Lactate (mg/g wet wt) Pyruvate (mg/g wet wt) Lipids (mg/g wet wt)
of glycogen,
glucose, lactate,
pyruvate
The results are shown in Figs 3 and 4, and Table 1. Gill lactate levels in lindane-treated fish were significantly increased after 6, 12, 24 and 48 hr above the mean control value of 15.7 mg/g wet wt. After 72 hr of exposure to the pesticide, the gill lactate value was unaffected. The highest increase in lactate was 65%, at 24 hr. After 6 and 12 hr of exposure, pyruvate levels were elevated significantly compared to the control value of 0.48 mg/g wet wt. Thereafter, pyruvate levels were not altered by lindane treatment. The increase after 6 hr amounted to 20%. Total lipid levels
The results are shown in Fig. 5 and Table 1. The mean concentration of gill total lipids in control fish was 83.8 mg/g wet wt. The lipid levels decreased significantly (20%) after 6 and 12 hr of exposure, but increased significantly, compared to the control value, after 48 and 72 hr of exposure to the pesticide. At other time intervals, gill total lipid values were not altered by pesticide treatment. Fish poisoned by lindane showed uncoordinated movements, sluggishness alternating with hyperexcitability, and difficulty in respiration. The fish came to the surface, sometimes on their side, and occasionally showing darting movements. Similar changes have also been reported in other fish species subjected to organochlorine pesticides (Narendra and Srivastava, 1981).
and total lipids in the gills of controls
Control
6
12
2.02 +0.17 0.14 kO.02 15.7 f2.6 0.49 to.05 83.8 k5.4
0.61. f0.21 O.l8* LO.02 23.1** f2.2 0.58’ +0.12 66.F 55.6
1.9” f0.59 0.18’ f0.02 24.9” f 3.2 0.57. f0.07 67.2.’ +4.4
Values are the mean + SD (N = IO). ‘P < 0.05; **IJ < 0.001.
Lactate and pyruvate concentrations
Exposed 24 I .88 +0.07 0.17’ + 0.02 25.8” f2.5 0.51 to.07 81.9 + 10.8
and of fish exposed
for (hr) 48 0.92** iO.18 0.18’ kO.02 21.3* f 1.2 0.51 kO.03 103.6* + 18.4
to 0.335 mg/l lindane
12
96
0.9v* f0.13 0.16* *0.01 15.2 k3.2 0.49 f0.09 108.3* kl7.0
I .08** 10.26 0.16’ kO.01 14.6 +2.9 0.49 kO.05 84.2 & 16.2
Effect of lindane in eel
97
Total Ilplde
Lactate
i
100
l
60 : 60 : ” t
Y
c
8
12
24 t OwJnt
46
72
40
66
Fig. 3. Changes in gill lactate levels vs controls at 6, 12, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P < 0.05.
Fig. 5. Changes in gill lipids levels vs controls at 6, 12, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P i 0.05.
DISCUSSION
in fish (Nakano and Tomlinson, 1967). Thus, the marked glycogenolysis in gills, in this study, after exposure to lindane could possibly have been caused by a stress-induced increase in circulating cate~holamines. The significant elevation of gill glucose levels in this study can be explained by the mobilization of gill glycogen, or by an entry from blood with the goal of producing new glycogen. Rojik et al. (1983) also have reported relatively high carbohydrate losses from gills in Cyprinus carpio exposed to paraquat. Mane et al. (1986) reported a decrease in gill glycogen levels in hionaia caerdeus after exposure to fenthion. Tort et al. (1984) showed that zinc induced significant decreases in gill glycogen in Scyliorhinus canida. They suggested that this decrease in glycogen content is indicative of an increased rate of glycolysis due to a hypoxic situation. The depletion in glycogen stores should be accompanied by an increase in glucose content, as observed in this study. The changes in pyruvate and lactate levels also indicate metabolic disorders. Both elevated lactate and pyruvate content suggest a severe respiratory stress in the fish tissues. According to Dange and Masurekar (1982), an upward trend in lactic acid in the tissues may be taken to indicate that oxygen supply to the tissues is not adequate for the normal metabolic functions. A marked increase in the 1actic:pyruvic acid ratios was noted for gill from lindane-treated eel. Lactate as a measure of anaerobic metabolism has been widely used and increases of anaerobic metabolism have been shown to be a rapid and clear response of depletion of energy caused by lack of oxygen (Van den Thillart, 1982). Lindane and other pesticides are known for their ability to disrupt the structural integrity of fish gills (Kumaragura et al., 1982; Virtanen, 1986; Evans, 1987). It may be assumed that as a result of the reduced efficiency of the damaged gills to function as respiratory organs, the tissues receive less oxygen. Moreover, the increased mucus secretion by gills observed during toxicity studies on fish (Virtanen, 1986; Evans, 1987) may lead to some degree of suffocation and further aggravate the tissue hypoxia. Development of such internal hypoxic conditions may be ultimately responsible for the shift to the less
The concentration of lindane used in this experiment is a sublethal one. Tests run for several weeks showed that mortality was not higher than normal. Tests for toxicity performed by Ferrando et al. (1987) with eel showed that 0.67 ppm lindane was the 96-hr LC~~ and that 0.335 ppm did not affect the survival rate. The results show that this sublethal lindane concentration was, however, high enough to produce toxic effects in A. anguilia. The presence of lindane in the surrounding water could induce some changes in the internal metabolism of fish, mainly through its action in damaging the gill epithelium (Evans, 1987; Virtanen, 1986). Entry of the organochlorine insecticide into the fish body and its subsequent accumulation in tissues could produce disturbances in the tissue metabolism. In order to meet the increased energy demand of such stressed animals, glycogen, due its easy availability for energy production, is rapidly catabolized, resulting in huge losses of this energy reserve. Reduction in glycogen content observed in the present study supports this view. Glycogenolysis was also reported in fish during exposure to various pollutants considered stressful (Narendra and Srivastava, 198 1; Gupta and Srivastava, 1982; Yousri and Hanke, 1985). Catecholamines deplete glycogen reserves
Pyruvate
C
6
12
24 t (hod
46
72
66
Fig. 4. Changes in gill pyruvate levels vs controls at 6, 12, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P < 0.05.
M. D. FERRANDO and E. ANDREW-M• LINER
98
efficient anaerobic metabolism indicated by the changes in pyruvic and lactic acid contents observed during the present study. It is possible to imagine that as these metabolic modifications in fish affected by environmental pollution appear to result from damage to sensitive tissues like gills, eventually irreversible changes could be produced in fish metabolism by exposing them to sufficiently high sublethal concentrations of the pollutants for more prolonged periods. Lipid content in gills of A. anguilla exposed to lindane decreased significantly at 6 and 12 hr of exposure probably due to its use as an energy reserve parallel to glycogen. On the other hand, lipid levels increased at 48 and 72 hr because of a lipogenesis process that could result from the high levels of lactate, glucose and pyruvate. Mane et al. (1986) found an increase in gill total lipid levels in Indonaia caeruleus after exposure to fenthion. In this case, the possible mechanisms could be found in an increased lipid synthesis. The results indicated that lindane produces certain changes in carbohydrate metabolism which are similar to those commonly attributed to respiratory dysfunction. If these changes are elicited through damage to gills, then similar changes should also be produced by many other pollutants which cause injury to fish gills. Acknowlednemenrs-This was supported by a grant from Direcci6n General de Investiga&n Cientifica y Tkcnica (DGICYT). Ministerio de Educacibn y Ciencia, No. &87-0076:‘MD. Ferrando is recipient of a fellowship from the Plan National Formaci6n Personal Investigador, M.E.C., Spain. REFERENCES Bligh E. G. and Dyer W. J. (1989) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 91 l-917. Dange A. D. and Masurekar V. B. (1982) Naphtaleneinduced changes in carbohydrate metabolism in Sarotherodon mossambicus. Hydrobiologia 94, 163-172.
Evans D. H. (1987) The fish gill: site of action and model for toxic effects of environmental pollutants. Enoiron. Health Perspect. 71, 47-58.
Ferrando M. D., Andreu E., Almar M. M., Cebrian C. and Nuiiez A. (1987) Acute toxicity of organochlorine pesticides to the european eel, Anguilla anguilla: the depen-
dency on exposure time and temperature. Bull. environ. Contam. Toxicol. 39, 365-369.
Ferrando M. D., Andreu E., Almar M. M., Cebrian C. and Alar&V. (1989) Short-term lindane effects on gill tissues metabolism.of the eel. Toxicol. environ. Chem. 24, 17-23. Giriia M. (1984) Effect of HeDtachlor and Metasvstox on f&ctionally active tissue-giil of a freshwater fi&. Ti/apia mossambica. M. Phil. Diss. Sri Venkateswara University, Tirupati, India. Gupta A. B. and Srivastava A. K. (1982) Effect of ethyl acetate on carbohydrate metabolism of common indian catfish. Ecotoxicol. environ. Safety 6, 166-170. Kumaragura A. K., Beamish F. W. H. and Fergusson H. W. (1982) Direct and circulatory paths of Permethrin causing histopathological changes in the gills of rainbow trout, Salmo gairdneri. J. Fish Biol. 20, 87-91.
Lang G. and Michal G. (1974) Methods of Enzymatic Analysis. Academic Press, New York. Mane V. H. Akarte S. R. and Kulkami D. A. (1986) Acute toxicity of fenthion to freshwater Lamellibranch mollusc, Indonaia caeruleus from Godavari River at Paithan-A biochemical approach. Bull. environ. Contam. Toxicol. 37, 622-628.
Nakano T. and Tomlinson N. (1967) Catecholamines and carbohydrate concentrations in rainbow trout (Salmo gairdneri) in relation to physical disturbance. J. Fish Res. Bd Can. 24, 1701-1715. Narendra N. and Srivastava A. K. (1981) Effects of endosulfan on fish carbohydrate metabolism. Ecotoxicol. environ. Safety 5, 412-417.
Radhaiah V., Girija M. and Rao K. J. (1987) Changes in selected biochemical parameters in the kidney and blood of the fish, Tilapia mossambica, exposed to- heptachlor. Bull. environ. Contam. Toxicol. 39. 1006-1011.
Rojik I., Nemcsok J. and Boross L.
0306~4492/91 $3.00 + 0.00 0 1991 PergamonPressplc
THE EFFECT OF TIME ON PHYSIOLOGICAL CHANGES IN EEL ANGUILLA ANGUILLA, INDUCED BY LINDANE M. D.
FERRANDO
and E.
ANDREWMOLINER
Department of Animal Physiology, Faculty of Biological Sciences, University of Valencia, Dr Moliner 50, E-46100 Burjasot, Valencia, Spain (Received 1 Octo&r 1990) Abstract-l. Eel were exposed to a sublethal concentration of lindane (0.335 ppm) for 6, 12, 24, 48, 72 and 96 hr. 2. Concentrations of glycogen, glucose, lactate, pyruvate and lipids were determined in gill tissue after lindane exposure. 3. Gill glycogen descreased and glucose levels increased at 6 hr of treatment, lactate and pyruvate concentration increased between 6 and 48 hr. Total lipid values decreased between 6 and 24 hr; thereafter, the levels increased up to 72 hr of exposure. 4. Clear changes were found in all parameters tested in gill tissues. The observed effects of lindane on metabolism in fish are discussed in relation to acute stress syndrome.
4.1 mmol/l). A 12-hr photoperiod (8.00-20.00) was maintained (Ferrando et al., 1989). The fish were then transferred to test aquaria and were not fed. Previous works on lindane toxicology of A. anguilla in our laboratory showed that 0.67 ppm was the LC~ value at 96 hr (Ferrando et al., 1987). For the study of the effects of lindane on carbohydrate metabolites, groups of 10 fish each were exposed to 0.335 ppm (i 96-hr LQ,,) lindane. Fish were sampled for the various biochemical parameters at 6, 12,24,48,72 and 96 hr exposure to the toxicant. Control fish were held in vehicle (acetone)-supplemented tap water.
INTRODUCTION
Pesticides used in pest control programmes seem to produce many physiological and biochemical changes in freshwater organisms by influencing the activities of several enzymes. Alterations in the chemical composition of the natural aquatic environment usually affect behavioural and physiological systems of the inhabitants, particularly of fish (Radhaiah et al., 1987). Some data are available on the effects of different pesticides on the biochemical aspects of fish gill (Girija, 1984; Ferrando et af., 1989). Gill is an important fish tissue because of its direct contact with water and because the pesticide has to go through it to come into the fish body. It has been suggested that waterborne pollutants damage fish gill, presumably by causing breakdown of the gas-exchange mechanism with the consequent tissue hypoxic conditions (Tort el al., 1984). Therefore, an attempt was made to observe certain biochemical parameters of fish, Anguilla anguilla under lindane intoxication. Investigation of this nature is useful in understanding the orientation of biochemical changes during sublethal toxicity to ascertain the degree of intensity of the toxicity of lindane on the gill. Time response and magnitude of response relationships in gill glycogen, -glucose, lactate, pyruvate and lipids were determined in the European eel, A. unguilla, following exposure to a sublethal concentration of lindane for 6, 12, 48, 72 and 96 hr.
Chemicals Technical grade lindane (AFRASA Company, Patema, Valencia, Spain) of 99% purity was used for experimentation. Stock solutions were prepared by dissolving lindane in acetone; appropriate quantities of this solution were pipetted into 40-l. glass aquaria containing 35 1. of test solution and 10 fish. Ten more eels, used as controls, were kept in 35 1. of clean water with the same concentration of acetone. Analysis of metabolites and lipid After each exposure the fish were quickly killed, gill tissue was removed and the metabolites determined. Glycogen content was estimated with the anthrone method (Seifter er al.. 1950). Gill tissue (0.5-1.0 g) was homogenized by adding 0.5 ml KOH (60%) and y ml KOH (50%). Thk mixture was kept in a boiling water-bath for 30 min. A 4-ml aliquot of ethanol was added to the homogenates before putting them into the fridge. Next day, samples were centrifuged and the supematant was saved for glucose determination. The pellet was resuspended in 1 ml water and a 0.25-ml aliquot was mixed with 1.75 ml anthrone reagent for 15min at 100°C. Glycogen content was determined spectrophotometrically. Glucose was assayed in the supernatants using test kits from Boehringer-Mannheim. Samples for lactate were homogenized in trichloroacetic (7%) at 4°C and centrifuged at 5000 rpm (Lang and Michal, 1974). Lactate concentration was measured spectrophotometrically by means of NADH produced in an enzymatic reaction with lactate dehydrogenase (Boehringer-Mannheim kits).
MATERIALS AND METHODS
Test system Eels of species AnguiNu anguilla (weight: 20-30 g; length: 16-20cm), were obtained from a fish farm in Valencia, Spain. They were acclimatized to laboratory conditions for two weeks in 300-l. glass tanks. The tanks were supplied with a continuous flow of tap water (temperature: 20°C; total hardness: 250 ppm as CaCO,; pH: 7.9 + 0.2; alkalinity: 95
M. D. FERRANDO and E. ANDREU-MOLINER
96 Glycogen
Glucose
._-
I
c
6 :
100
140 i
120
I
100
,’ a
60
I
:
60
:
40
: n t
z
60
:
9
20
20
i s
a 0
c
6
12
24 t (howe)
42
12
40 20 0
26
’ C
, 6
12
i
24 t (houra)
42
72
96
Fig. 1. Changes in gill glycogen levels vs controls at 6, 12, Fig. 2. Changes in gill glucose levels vs controls at 6, 12, 24, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P < 0.05. 48, 72 and 96 hr of treatment (0.335 ppm); *P -c0.05. For pyruvate determination, tissue samples were homogenized in perchloric acid (1 M) and centrifuged at 3000 rpm (Dange and Masurekar, 1982). Pyruvate concentration was measured with a test kit (BoehringerMannheim). Lipids were analyzed spectrophotometrically with a test kit (Boehringer-Mannheim) after extraction according to Bligh and Dyer (1959).
Statislicalanalysis One-way analysis of variance (ANOVA) was used to determine treatment toxic effects, and Duncan’s significant difference test was used for mean separation. The significant level was fixed at P < 0.001 and P < 0.05. The graphs in this paper show the changes vs controls, which were not used for
the statistical analysis. This analysis was performed using the Statistical Analysis System (SPSS+) with an IBM computer. RESULTS
Gill glycogen and glucose concentrations
The results are shown in Figs 1 and 2, and Table 1. The concentration of gill glycogen was already significantly reduced (70%) after 6 hr of exposure. The mean control value was 2.02 (+ 17) mg/g wet wt. Glycogen levels decreased between 6 and 96 hr. Exposure for 24 hr did not elicit a clear effect. At all other exposure times a lowered glycogen content was found indicating that glycogenolysis had occurred in treated fish. The mean control concentration of gill glucose was 0.14 mg/g wet wt. Exposure resulted in a significant increase (30% at 6 and 48 hr, Fig. 2) in gill glucose at all exposure times. Table
I. Levels
Parameter Glycogen (mg/g wet wt) Glucose (mg/g wet wt)
Lactate (mg/g wet wt) Pyruvate (mg/g wet wt) Lipids (mg/g wet wt)
of glycogen,
glucose, lactate,
pyruvate
The results are shown in Figs 3 and 4, and Table 1. Gill lactate levels in lindane-treated fish were significantly increased after 6, 12, 24 and 48 hr above the mean control value of 15.7 mg/g wet wt. After 72 hr of exposure to the pesticide, the gill lactate value was unaffected. The highest increase in lactate was 65%, at 24 hr. After 6 and 12 hr of exposure, pyruvate levels were elevated significantly compared to the control value of 0.48 mg/g wet wt. Thereafter, pyruvate levels were not altered by lindane treatment. The increase after 6 hr amounted to 20%. Total lipid levels
The results are shown in Fig. 5 and Table 1. The mean concentration of gill total lipids in control fish was 83.8 mg/g wet wt. The lipid levels decreased significantly (20%) after 6 and 12 hr of exposure, but increased significantly, compared to the control value, after 48 and 72 hr of exposure to the pesticide. At other time intervals, gill total lipid values were not altered by pesticide treatment. Fish poisoned by lindane showed uncoordinated movements, sluggishness alternating with hyperexcitability, and difficulty in respiration. The fish came to the surface, sometimes on their side, and occasionally showing darting movements. Similar changes have also been reported in other fish species subjected to organochlorine pesticides (Narendra and Srivastava, 1981).
and total lipids in the gills of controls
Control
6
12
2.02 +0.17 0.14 kO.02 15.7 f2.6 0.49 to.05 83.8 k5.4
0.61. f0.21 O.l8* LO.02 23.1** f2.2 0.58’ +0.12 66.F 55.6
1.9” f0.59 0.18’ f0.02 24.9” f 3.2 0.57. f0.07 67.2.’ +4.4
Values are the mean + SD (N = IO). ‘P < 0.05; **IJ < 0.001.
Lactate and pyruvate concentrations
Exposed 24 I .88 +0.07 0.17’ + 0.02 25.8” f2.5 0.51 to.07 81.9 + 10.8
and of fish exposed
for (hr) 48 0.92** iO.18 0.18’ kO.02 21.3* f 1.2 0.51 kO.03 103.6* + 18.4
to 0.335 mg/l lindane
12
96
0.9v* f0.13 0.16* *0.01 15.2 k3.2 0.49 f0.09 108.3* kl7.0
I .08** 10.26 0.16’ kO.01 14.6 +2.9 0.49 kO.05 84.2 & 16.2
Effect of lindane in eel
97
Total Ilplde
Lactate
i
100
l
60 : 60 : ” t
Y
c
8
12
24 t OwJnt
46
72
40
66
Fig. 3. Changes in gill lactate levels vs controls at 6, 12, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P < 0.05.
Fig. 5. Changes in gill lipids levels vs controls at 6, 12, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P i 0.05.
DISCUSSION
in fish (Nakano and Tomlinson, 1967). Thus, the marked glycogenolysis in gills, in this study, after exposure to lindane could possibly have been caused by a stress-induced increase in circulating cate~holamines. The significant elevation of gill glucose levels in this study can be explained by the mobilization of gill glycogen, or by an entry from blood with the goal of producing new glycogen. Rojik et al. (1983) also have reported relatively high carbohydrate losses from gills in Cyprinus carpio exposed to paraquat. Mane et al. (1986) reported a decrease in gill glycogen levels in hionaia caerdeus after exposure to fenthion. Tort et al. (1984) showed that zinc induced significant decreases in gill glycogen in Scyliorhinus canida. They suggested that this decrease in glycogen content is indicative of an increased rate of glycolysis due to a hypoxic situation. The depletion in glycogen stores should be accompanied by an increase in glucose content, as observed in this study. The changes in pyruvate and lactate levels also indicate metabolic disorders. Both elevated lactate and pyruvate content suggest a severe respiratory stress in the fish tissues. According to Dange and Masurekar (1982), an upward trend in lactic acid in the tissues may be taken to indicate that oxygen supply to the tissues is not adequate for the normal metabolic functions. A marked increase in the 1actic:pyruvic acid ratios was noted for gill from lindane-treated eel. Lactate as a measure of anaerobic metabolism has been widely used and increases of anaerobic metabolism have been shown to be a rapid and clear response of depletion of energy caused by lack of oxygen (Van den Thillart, 1982). Lindane and other pesticides are known for their ability to disrupt the structural integrity of fish gills (Kumaragura et al., 1982; Virtanen, 1986; Evans, 1987). It may be assumed that as a result of the reduced efficiency of the damaged gills to function as respiratory organs, the tissues receive less oxygen. Moreover, the increased mucus secretion by gills observed during toxicity studies on fish (Virtanen, 1986; Evans, 1987) may lead to some degree of suffocation and further aggravate the tissue hypoxia. Development of such internal hypoxic conditions may be ultimately responsible for the shift to the less
The concentration of lindane used in this experiment is a sublethal one. Tests run for several weeks showed that mortality was not higher than normal. Tests for toxicity performed by Ferrando et al. (1987) with eel showed that 0.67 ppm lindane was the 96-hr LC~~ and that 0.335 ppm did not affect the survival rate. The results show that this sublethal lindane concentration was, however, high enough to produce toxic effects in A. anguilia. The presence of lindane in the surrounding water could induce some changes in the internal metabolism of fish, mainly through its action in damaging the gill epithelium (Evans, 1987; Virtanen, 1986). Entry of the organochlorine insecticide into the fish body and its subsequent accumulation in tissues could produce disturbances in the tissue metabolism. In order to meet the increased energy demand of such stressed animals, glycogen, due its easy availability for energy production, is rapidly catabolized, resulting in huge losses of this energy reserve. Reduction in glycogen content observed in the present study supports this view. Glycogenolysis was also reported in fish during exposure to various pollutants considered stressful (Narendra and Srivastava, 198 1; Gupta and Srivastava, 1982; Yousri and Hanke, 1985). Catecholamines deplete glycogen reserves
Pyruvate
C
6
12
24 t (hod
46
72
66
Fig. 4. Changes in gill pyruvate levels vs controls at 6, 12, 24, 48, 72 and 96 hr of treatment (0.335 ppm); *P < 0.05.
M. D. FERRANDO and E. ANDREW-M• LINER
98
efficient anaerobic metabolism indicated by the changes in pyruvic and lactic acid contents observed during the present study. It is possible to imagine that as these metabolic modifications in fish affected by environmental pollution appear to result from damage to sensitive tissues like gills, eventually irreversible changes could be produced in fish metabolism by exposing them to sufficiently high sublethal concentrations of the pollutants for more prolonged periods. Lipid content in gills of A. anguilla exposed to lindane decreased significantly at 6 and 12 hr of exposure probably due to its use as an energy reserve parallel to glycogen. On the other hand, lipid levels increased at 48 and 72 hr because of a lipogenesis process that could result from the high levels of lactate, glucose and pyruvate. Mane et al. (1986) found an increase in gill total lipid levels in Indonaia caeruleus after exposure to fenthion. In this case, the possible mechanisms could be found in an increased lipid synthesis. The results indicated that lindane produces certain changes in carbohydrate metabolism which are similar to those commonly attributed to respiratory dysfunction. If these changes are elicited through damage to gills, then similar changes should also be produced by many other pollutants which cause injury to fish gills. Acknowlednemenrs-This was supported by a grant from Direcci6n General de Investiga&n Cientifica y Tkcnica (DGICYT). Ministerio de Educacibn y Ciencia, No. &87-0076:‘MD. Ferrando is recipient of a fellowship from the Plan National Formaci6n Personal Investigador, M.E.C., Spain. REFERENCES Bligh E. G. and Dyer W. J. (1989) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 91 l-917. Dange A. D. and Masurekar V. B. (1982) Naphtaleneinduced changes in carbohydrate metabolism in Sarotherodon mossambicus. Hydrobiologia 94, 163-172.
Evans D. H. (1987) The fish gill: site of action and model for toxic effects of environmental pollutants. Enoiron. Health Perspect. 71, 47-58.
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Ferrando M. D., Andreu E., Almar M. M., Cebrian C. and Alar&V. (1989) Short-term lindane effects on gill tissues metabolism.of the eel. Toxicol. environ. Chem. 24, 17-23. Giriia M. (1984) Effect of HeDtachlor and Metasvstox on f&ctionally active tissue-giil of a freshwater fi&. Ti/apia mossambica. M. Phil. Diss. Sri Venkateswara University, Tirupati, India. Gupta A. B. and Srivastava A. K. (1982) Effect of ethyl acetate on carbohydrate metabolism of common indian catfish. Ecotoxicol. environ. Safety 6, 166-170. Kumaragura A. K., Beamish F. W. H. and Fergusson H. W. (1982) Direct and circulatory paths of Permethrin causing histopathological changes in the gills of rainbow trout, Salmo gairdneri. J. Fish Biol. 20, 87-91.
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Rojik I., Nemcsok J. and Boross L.
