A comparison of physiological changes in carp, Cyprinus carpio, induced by several pollutants at sublethal concentrations

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

9, 179-188

(1985)

A Comparison of Physiological Changes in Carp, Cyprinus carpio, Induced by Several Pollutants at Sublethal Concentrations I. The Dependency G.

GLUTH

on Exposure AND

W.

Time’

HANKE

Department of Zoology, The C’niversity. D-7500 Karlsruhe. West Germany Received April 17, 1984 Carp were exposed to 10 different pollutants at sublethal concentrations for 6, 24, or 72 hr. Blood, liver. and white muscle samples were taken after the exposure time together with samples of control handled fish. Serum concentrations of glucose, cortisol protein, and cholesterol were determined. Similarly, the liver and muscle glycogen contents were measured. The effects of the following pollutants were examined: aldrin (100 &liter), atrazine (100 fig/ liter), DDT (50 Fg/liter), dieldrin (20 &/liter), endrin (2 pg/liter), hexachlorbenzene (100 wg/ liter), lindane (100 pg/liter), methanol (1 ml/liter), 4-N-phenol (100 pg/liter), toluene (100 rl/ liter). The rises in serum glucose and clortisol were the most frequent changes occurring after exposure to the pollutants. A decline in plasma protein and cholesterol content was also often observed. Liver glycogen concentration, increased first in most cases and was reduced after longer exposure. Muscle glycogen was affected differently, sometimes reduced by exposure to the pollutants. The experimental design allows for the gradual increase in toxicity of the pollutants used regarding the applied concentrations. Furthermore, the aim of the paper is to evaluate the tests for proof of toxicity of those chemicals. The determination of serum glucose and cortisol levels can be proposed as mostly useful. The clearest changes in all parameters were found after treatment with 100 &liter atrazine and 50 @g/liter DDT. When serum glucose and cortisol concentrations were quickly elevated, signs for exhaustion could be seen after 72 hr of exposure. Q 1985 Academic PW, IK INTRODUCTION

Pollutant toxicity to fish has mostly been measured by using lethal concentrations and determining LCsO(lethal concentration for 50% of individuals) within 4 days (Sprague, 1969, 1970). It is much more difficult to analyze the influence of sublethal levels of pollutants because the se:nsitivity of indicators is very different. Besides growth rate, oxygen consumption, and behavioral methods like swimming performance, biochemical changes have been used or recommended for toxicological approaches (Fujiya, 1965; Alderdice, 1967; Sprague, 1971, 1976; Wedemeyer and Yasutake, 1977). The study of the effects of sublethal concentrations has been forced becauseof the need to find “safe” concentrations of the pollutants (Johnson, 1968). Special reports can be found on the effects of single pollutants, like the insecticide endrin (Eisler and Edmunds, 1966; Grant, 1976) or the fungicide hexachlorbenzene (HCB) and others. Comparative studies on different pollutants are rather scarce and the methods are often not standardized (e.g., Liidemann and Neumann, 1960; Kayser et al.. 1962). ’ This work

was supported

by a grant

from

Bundesministerium 179

ftir Forschung

und Technologie,

0147-6513/85 Copyright All

rights

0

BRD.

$3.00 1985

of reproductmn

by

Academx m

any

Press.

Inc.

form

reserved.

180

GLLJTH

AND

HANKE

Recently several methods normally used for clinical diagnosis were introduced in fish physiology to determine effects of sublethal concentrations of pollutants (Wedemeyer and Yasutake, 1977; Lockhart and Metner, 1979). In many papers, blood chemistry and hematology have been analyzed. Tissue biochemistry has also been proved useful for those determinations. Most of the responses are nonspecific to single pollutants and belong more or less to the phenomenon of stress (Pickering, 198 1). Increases in cortisol and glucose levels in blood are typical responses (Hanke et al., 1982, 1983; Gluth and Hanke, 1983). The aim of this paper is to add to the knowledge about the suitability of methods for determination of effects of pollutants on fish. To avoid the death of the experimental animal by using lethal concentrations and to estimate “safe” concentrations, sublethal doses were used. Nevertheless, methods and parameters should be elaborated which allow the finding of effects during 3-4 days of exposure. A comparison between different pollutants used at definite concentrations should be made. MATERIALS

AND

METHODS

Fish and experimental design. For all experiments, carp were used as experimental fish. They were purchased from a fish farm, had lengths of 12- 17 cm, and weighed 40-80 g. After acclimation to laboratory conditions (about 3 weeks), they were kept in a test aquaria system (each aquarium contains 10 liters and six fish) which were similar in size to the experimental set. This acclimation system is described in Gluth and Hanke (1983). After acclimation for 2 weeks at 17°C the exposure to pollutants was started, also at 17°C. During exposure to the test chemicals, the fish were not fed and the water was carefully renewed every 24 hr. Sample analysis has shown that mostly less than 20% of the chemical disappeared from the solution after 1 day. At the end of the exposure, six fish of one test were caught and rapidly anesthetized by MS 222 (high dose). Immobilization was normally completed after 20 sec. After the pericardial sac was opened and the pericardial fluid was removed, the heart was punctured and blood collected for analysis. A piece of white epaxial muscle and a part of the liver were taken for determination of glycogen content. Pollutants and doses. Carp were exposed to 10 different pollutants at sublethal concentrations for 6, 24, or 72 hr. The following pollutants and concentrations were used: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Aldrin ( 100 pg/liter, 270 n&I) Atrazine (100 &liter, 465 nM) DDT (50 pg/liter, 140 mI4) Dieldrin (20 pg/liter, 52 nM) Endrin (2 pg/liter, 5.2 n&f) Hexachlorbenzene (HCB) (100 pg/liter, 350 nM) Lindane (100 Kg/liter, 350 nM) Methanol (1 ml/liter, 50 PM) 4-N-Phenol (100 pg/liter, 720 n&I) Toluene ( 100 pg/liter, 1.1 PM)

The concentrations and pollutants were chosen according to several references (e.g., Merkblatter iiber Referenzchemikalien, 1982). The amount of chemical necessary

POLLUTANTS

AND

CARP

181

for the tanks was dissolved in ca. 2 ml ethanol in all cases independently from the variable of water solubility of the chemicals. This solution was added to the water in the lo-liter tanks. Blood and tissue analysis. To determine the serum glucose and cholesterol content test kits from Boehringer-Mannheim were used. Serum protein was analyzed using the Folin method. Serum cortisol was determined by RIA; the antibody was a gift of Professor Vecsei, Heidelberg. The liver and muscle glycogen were extracted by 1 N NaOH and, after deproteinization and precipitation of the glycogen, determined using a glucose oxidase method. Statistical analysis. The mean (2) and the standard deviation and error were calculated for each test group and the means were compared using Student’s t test. The significance level was fixed at A‘D < 0.05. The graphs of this paper show the changes vs controls, which were not used for the statistical analysis. Changes that were found statistically different from the original values are marked by a triangle. RESULTS

Serum Glucose A clear and mostly significant increase in serum glucose was found after exposure to the majority of pollutants. Only exposure to lindane and methanol did not succeed in a significant elevation. After exposure to DDT and toluene a significant increase was found at all three exposure times. In 6 of the 10 pollutants a significant increase was found after 24 or 72 hr. Only 4 pollutants were elevated after 6 hr (Fig. 1). The normal concentration of glucose in carp serum (17°C) was found at 60 mg/ 100 ml.

Serum Cortisol The concentration of cortisol in carp serum (17°C) was measured at about 30 rig/ml. There was an increase in serum cortisol found after exposure to all pollutants. Seven pollutants obtained the highest elevation after 6 hr of exposure. The more toxic pollutants like atrazine and DDT had the strongest influence after 72 hr (Fig. 2).

Serum Protein Exposure to 9 of the 10 pollutants caused a significant decrease in protein concentration in the serum which was seen after 72 hr (Fig. 3). The normal range of the protein concentration in the serum (17°C) was between 20 and 25 mg/ml.

Serum Cholesterol Serum cholesterol concentration was also lowered mainly after 72 hr of exposure. This was found in 8 of the 10 diff’erent treatments. A reduction of 20-40% was determined, which means that the range was similar to that of proteins. Therefore an increase in water content in the serum could be the cause of the effect (Fig. 4). The concentration of cholesterol in the serum of carps ( 17’C) ranged between 100 and 125 mg/lOO ml.

GLUTH

182 20

AND HANKE

GLUCOSE

2oi

6 21 72 ALDRIN

0

”

6 21 12 ATRAZINE

6 2112 DDT

6 2112 DIELDRIN

6 2112 ENDRIN

6 2L 72 METHANOL

6 2L-72 L-N-PHENOL

6 2Ll2 TOLUENE

0' 6 2L?2 HCB

6 2L 72 L INDANE

FIG. I. Changes in serum glucose levels vs controls at 6, 24, or 72 hr of treatment (temperature, 17°C). V, Significant at a 5% level.

Liver Glycogen The concentration of liver glycogen decreased clearly in all cases, with the exception of the influence of endrin. In eight treatments, the reduction of the amount of the liver glycogen was significant 72 hr after the start of the exposure. After exposure to dieldrin, the reduction was visible but not significant. It is evident that the reduction must be seen in relation to the elevation of the serum glucose concentration. But it is clear that glycogenolysis is not as evident as hyperglycemia in the blood (Fig. 5). Normal concentration of glycogen in the liver (17°C) was between 7.0 and 8.0%.

Muscle Glycogen The muscle glycogen content was also reduced after 72 hr of treatment, except for exposure to lindane and methanol. The changesin muscle glycogen concentration gave some evidence for a biphasic response. In some cases(atrazine, DDT, HCB, methanol, and 4-N-phenol) a first increase which coincides with the elevation of serum glucose was found (Fig. 6). It is known from a general physiological view that muscle glycogen content depends on both the blood glucose concentration and the capacity for glycogen synthesis. This is somewhat different from liver glycogen concentration, which mainly depends on the synthetic capacity. Therefore, it is reasonable to assumethat the glycogen content of muscle first runs parallel to the

POLLUTANTS

$ oL s

AND

G 2L 72 ALDRIN

6 2L12 ATRAZINE

6 2L72 DOT

6 2L 12

6 2L 12

6 2Ll2

tic0

LIND4Nt

183

CARP

6 2C 12 DIELDRIN

6 2Ll2 ENDRIN

6 2L72

6 2L 12 TOLUENE

__---__

FIG. 2. Changes in serum cortisol 7, Significant at a 5% level.

METH4NOL

levels vs controls

L-N-PHENOL

at 6, 24, or 72 hr of treatment

blood glucose trends. The normal concentration (17°C) to be between 0.3 and 0.5%.

(temperature,

17°C).

of muscle glycogen was determined

DISCUSSION The concentrations of all 10 dilferent pollutants are definitely sublethal. It has been found that the carp can survive in these concentrations for a longer period than in more lethal concentrations. Tests run for about 4 weeks showed that mortality was not higher than normal. A much more important question is whether the concentrations used are comparable in their influences on fish or if some are “less safe” than others. This cannot be easily decided. The choice for the concentrations used had some references as background. For atrazine, DDT, and HCB our own results (Hanke et al., 1982, 1983; Gluth and Hanke, 1983) gave evidence that 50-100 pug/liter is a limit for survival. This means a concentration of about lOO500 ti. Tests for toxicity performed by Liidemann and Neumann (1960) with carp also showed that 50 pg/liter DDT, 100 pg/liter lindane, 100 pg/liter aldrin, and 20 pg/liter dieldrin did not affect the survival rate. Concentration of 2.8 pg/ liter endrin were found to be not toxic. For methanol the LCsO is at about 36

PROTEIN

Y & 2 8 o 2

6 2L72 ALDRIN

t 2L72 ATRAZINE

6 2472 DDT

6 2L 72 DIELDRIN

6 2~ 72 HCB

6 2L 12 LINDANE

6 2Ll2 METH4NOL

6 212 ENDRIN

82.0 P 2 ”

FIG. 3. Changes in serum protein concentration 17°C). V, Significant at a 5% level.

vs controls

6 2~72 L-N-PHENOL

6 2~ 12 TOLUENE

at 6, 24, or 72 hr of treatment

(temperature,

CHOLESTEROL

20 I

A

f!O 8 5

A

6 2L 72 ALDRIN

6 2L72 ATRAZINE

6 2L 72 HCB

6 2LR L INDANE

6 2L 72 DDT

A

6 2L 12 DIELDRI N

6 2L 12 ENDRIN

0: 6 2L72 METHANOL

FIG. 4. Changes in serum cholesterol concentration (temperature, 17°C). V, Significant at a 5% level. 184

vs controls

6 2~72 L-N-PHEN~L

6 2L 72 TOLUENE

at 6, 24, or 72 hr of treatment

POLLUTANTS

2 o-

LIVER

AND

185

CARP

GLYCOGEN

VI -I & 3 80 2

6 2L 12 ALDRIN

6 2L 12 ATRAZINE

6 2i.72 DDT

6 2Ll2 DIELDR~N

6 2Ll2 ENDRIN

6 2~ 12 HCB

6 2L 72

6 2Ll2 biETtiAN0~

6 21. 72 L N PHENOL

6 2L 12 TOLUENE

A

0;

FIG. 5. Changes in liver glycogen 17°C). V, Significant at a 5% level.

LINDANE.

content

vs controls

at 6, 24, or 72 hr of treatment

(temperature,

g/liter in carp and was found at about 14 mg/liter 4-N-phenol and about 25 mg/liter for toluene in other fish. The results clearly show that all concentrations of the pollutants were high enough to produce toxic effects in carp. The most abundant changes found are those of plasma cortisol. All pollutants stimulated an increase in plasma cortisol but the time course was different. In 7 of 10 treatments the effect was significant after 6 hr of exposure, but in only 6 of 10 treatments after 24 or 72 hr. This demonstrates that clear proof of an influence of pollutants needs three or more observations at different times. Glucose elevation, which is also a good test of toxicity, is less abundant but is found in 8 of the 10 treatments. lindane and methanol were not found to be effective. This shows that this response is not as sensitive as the cortisol increase. Nevertheless, it is obvious that these two effects are correlated. Changes in plasma protein and cholesterol levels are also quite good indicators of pollutant influence. In 9 of 10 and 8 of 10 treatments, significant decreases in the concentrations of protein and cholesterol, respectively, were found. There are a few indications that at first (after 6 hr of exposure) an elevation started and was followed by the reduction. The significant drop in the concentrations of protein and cholesterol takes some time and is clearly demonstrable after 72 hr of exposure. Reduction of liver glycogen is also a later response which is found after 72 hr in

186

GLUTH

MUSCLE

20

tn & sE O

6 2L 72

6 2172

ALDRIN

ATRAZIN

AND HANKE

GLYCOGEN

G il.72 DDT

6 21 72

6 2L 72

DIELDRIN

ENDRIN

v,

'20 8 0

v

z " IO

0 6 21 72

6 2472

6 2412

G 2L 12

6 2L72

HCB

L INDANE

MElHANOL

L-N-PHENOL

TOLUENE

6. Changes in muscle glycogen content vs controls at 6, 24, or 72 hr of treatment (temperature, 17’C). V, Significant at a 5% level. FIG.

eight cases.The amount of muscle glycogen increased at first in a few casesand decreasedlater. This is the best evidence that a biphasic reaction can occur. Lowest values are found after 72 hr in eight cases. A comparison of the effects of the 10 pollutants makes clear that aldrin, atrazine, and DDT effect all parameters. Dieldrin and endrin did not lower the liver glycogen level. HCB and toluene also had significant effects on all six parameters. Lindane and methanol were not able to raise the plasma glucose levels. The least effective pollutant at the concentration used was endrin becauseit did not induce significant results in the case of plasma protein and cholesterol and liver glycogen. This may be due to the rather low dosesused. A physiological evaluation of the results leads to some questions. First, it is evident that the comparison of six effects induced by 10 pollutants does not show any specific correlation between one or more pollutants and one effect. The effects are unspecific responses.This is most obvious in the case of plasma cortisol and glucose. Both responsesare linked and are indications for a stress effect. These indicators for environmental stressorshave been used by several authors (Hill and Fromm, 1968; Grant and Mehrle, 1973; McLeay, 1977; Mazeaud et al., 1977; Wedemeyer and Yasutake, 1977; Laska et al., 1978; Diwan et al., 1979; Tomasso et al., 1981; Hanke et al., 1982, 1983; Gluth and Hanke, 1983). Fish respond to the external stressor by a fast release of cortisol from the inter-renal gland. It can be suggestedthat a secretion of catecholamines precedesthe

POLLUTANTS

AND CARP

187

cortisol release. Both increase the glucose release by the liver. Glycogenolysis and the gluconeogenetic responseelevate the glucose level in the blood. Therefore, it is evident that glucose elevation occurs somewhat later in time than that of cortisol. The cortisol effect is also clearer and more significant than that of glucose. As mentioned earlier the changes in liver and muscle glycogen content are also linked with these plasma changes. The decrease in liver glycogen is due to glycogenolysis. Glycogen is of course the primary source for plasma glucose. It needs time to find a clear decreaseof liver glycogen becausegluconeogenetic effects may first take place. Sometimes primary increasesof liver glycogen could be found under stress(Hanke et al., 1983). This also indicates gluconeogenesis.It has already been discussed that several influenc:es exist on the amount of muscle glycogen. Nevertheless, the decrease of muscle glycogen after 72 hr is a quite good response to pollutants. The changes in plasma protein and cholesterol are also linked. Both occur after 72 hr and indicate accumulation of water in the plasma. Therefore the results do not describe separate changes in protein or cholesterol. An increase in cholesterol levels has been described in the literature. It is doubtful whether this increase is due to water loss from the serum compartment which also occurs sometimes after a short exposure to pollutants in our experiments. For evaluation of the tests for criteria to prove the quality of water or the occurrence of pollutants, it can be postulated that all are quite useful to demonstrate such facts for fish. The determination of cortisol levels (which is not easy to perform) might be the most sensitive indicator. But in all casesit is important that more than one parameter be used and that more than one exposure time be studied. It must also be pointed out that the effect of temperature is very important. This has been investigated in a subsequent paper (Gluth and Hanke, 1984). ACKNOWLEDGMENTS The help of R. Keppler, E. Schwarz, B. Wiegel. E. Pollmann, and J. Weinmann with the analysis and the experiments is gratefully acknowledged. We thank S. Hassel for typing the manuscript. We are also grateful to Professor Vecsei, Heidelberg, for his help with antibodies.

REFERENCES ALDERDICE, D. F. (1967). The detection and measurement of fish toxicity thresholds. In Advances in Water Pollution Research, Proceedings 3rd Int. ConJ, Munich, 1966. Vol. 1, pp. 15-95. DIWAN, A. D., HINGORANI, H. G., AND CHANDRASEKHRAM NAIDU. N. (1979). Levels of blood ducose and tissue glycogen in two live fish exposed to industrial effluent. Bull. Environ. Contam. To,yicol. 21, 269-272. EISLER, R.. AND EDMUND& P. H. (1966). EfFectsof endrin on blood and tissue chemistry of a marine fish. Trans. Amer. Fish. Sot. 95, 153-159. FUJIYA, M. (1965). Physiological estimation on the effects of pollutants upon aquatic organisms. In Advances in Water Pollution Research, Proceedings. 2nd Int. Conf, Tokyo, 1964. Vol. 3, pp. 3 15-33 1, Pergamon, Oxford. GLUTH. G., AND HANKE, W. (1983). The effect of temperature on physiological changes in carp, C.vprinus carpio L., induced by phenol. Ecotoxicol. Environ. Safety 7, 313-389. GLUTH. G.. AND HANKE, W. (1984). A comparison of physiological changes in carp. Cyprinus carpio, induced by several pollutants at sublethal concentrations. II. The dependency on the temperature. Comp. Biochem. Physiol. 79C, 39-45.

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GRANT, B. F., AND MEHRLE, P. M. (1973). Endrin toxicosis in rainbow trout (Sulmo guirdneri). J. Fish. Res. Board Canad. 30, 3 I-40. GRANT, B. F. (1976). Endrin toxicity and distribution in freshwater: A review. BUN. Environ. Contam. Toxicol. 15, 283-290. HANKE, W., BITTNER, A., HORN. G., MULLER, R., AND KEPPLER, R. (1982). Untersuchungen der physiologischen Wirkungen von Schadstoffen bei Karpfen. Jiil-Spez-163, pp. 42-63. HANKE, W., GLUTH, G., BUBEL, H., AND MULLER, R. (1983). Physiological changes in carps induced by pollution. Ecotoxicol. Environ. Safety I, 229-24 1. HILL, C. W., AND FROMM, P. 0. (1968). Responses of the interrenal gland of rainbow trout (Salrno gairdneri) to stress. Gen. Camp. Endocrinol. 11, 69-77. JOHNSON, D. W. (1968). Pesticides and fishes-A review of selected literature. Trans. Amer. Fish. Sot. 97, 398-424.

KAYSER, H., LUDEMANN, D., AND NEUMANN, H. (1962). Ver;inderungen an Nervenzellen nach Insektizidvergiftung bei Fischen und Krebsen. 2. Angew. Zool. 49, 135-148. LASKA, A. L., BARTELL, C. K., CONDIE, D. B., BROWN, J. W., EVANS, R. L., AND LASETER, J. L. (1978). Acute and chronic effects of hexachlorobenzene and hexachlorobutadiene in red swamp crayfish (Procambarus clarki) and selected fish species. Toxicol. Appl. Pharrnacol. 43, l- 12. LOCKHART, W. L., AND METNER, D. W. (1979). Biochemical tests for fish. In Toxicity Tests for Freshwater Organisms (E. Scherer, ed.). Canad. Spec. Publ. of Fisheries and Aquatic Sciences 44. LODEMANN, D., AND NEUMANN, H. (1960). Versuche iiber die akute toxische Wirkung neuzeitlicher Kontaktinsektizide auf einsiimmrige Karpfen (Cyprinus curpio L.). 2. Angew. Zool. 47, 1l-33. MAZEAUD, M. M., MAZEAUD, F., AND DONALDSON, E. M. (1977). Primary and secondary effects of stress in fish: Some new data with a general review. Trans. Amer. Fish. Sot. 106(3), 201-211, MCLEAY, D. L. (1977). Development of a blood sugar bioassay for rapidly measuring stressful levels of pulpmill effluent to salmonid fish. J. Fish. Res. Board Canad. 34, 477-485. MERKBLATTER iiber Referenzchemikalien (1982). Batelle-Institut e.V., Frankfurt am Main. PICKERING, A. D. (1981). Stress and compensation in teleostean fishes: Response to social and physical factors. In Stress and Fish (A. D. Pickering, ed.), pp. 295-322. Academic Press, New York/London. SPRAGUE, J. B. (1969). Measurement of pollutant toxicity to fish. I. Bioassay methods for acute toxicity. In Water Research, Vol. 3, pp. 793-821. Pergamon, Oxford/New York. SPRAGUE, J. B. (1970). Measurement of pollutant toxicity to fish. II. Utilizing and applying bioassay results. In Water Research, Vol. 4, pp. 3-32. Pergamon. Oxford/New York. SPRAGUE, J. B. (1971). Measurement of pollutant toxicity to fish. III. Sublethal effects and “Safe” concentrations. In Water Research. Vol. 5, pp. 245-266. Pergamon, Oxford/New York. SPRAGUE, J. B. (1976). Current status of sublethal tests of pollutants on aquatic organisms. J. Fish. Res. Board Canad. 33, 1988-1992. TOMASSO, J. R., DAVIS, K. B., AND SIMCO, B. A. (1981). Plasma corticosteroid dynamics in channel catfish (Ictalurus punctatus) exposed to ammonia and nitrite. Canad. J. Fish. Aquat. Sci. 38, 11061112. WEDEMEYER, G. A., AND YASUTAKE, W. T. (1977). Clinical Methods for the Assessment of the Effects of Environmental Stress on Fish Health, pp. l-18. U.S. Department of the Interior Fish and Wildlife Service, Washington, D.C.