TOXICOLOGICAL

ASSESSMENT IN B I O A S S A Y S

OF RIVER

WATER

QUALITY

WITH FISH*

M. A. V A N D E R G A A G , J. F. J. V A N D E K E R K H O F F , H. W. V A N D E R K L I F T , and C. L. M. P O E L S * * The Netherlands Waterworks Testing and Research Institute KIWA Ltd., P.O. Box 70, 2280 AB Rijswijk, The Netherlands

(Received November 1, 1982) Abstract. A series ofbioassays with fish was developed in order to evaluate toxicological aspects of polluted

rivers in The Netherlands. A long term exposition of trout to riverwater under standardized conditions enables the detection of pathological effects such as growth retardation, liver and kidney enlargement and changes in clinical blood parameters. Bioaccumulation of heavy metals and organochlorine compounds can also be measured. Embryo-larval tests with trout were less suitable, because of yearly variations in egg quality. In the near future, sister chromatid exchange (SCE) assays in vivo with Nothobranchius may become available for the detection of mutagenic effects. It was possible to measure trends in toxicological quality of Rhinewater with these tests. However extrapolation of results to ecosystems and tracing of the causes of changes occurring in waterquality are still problematic.

1. Introduction In the past decennia, many aquatic ecosystems, and Dutch rivers in particular, have been burdened with various kinds of pollutants. Most of the time such pollution is monitored by chemical measurements. However, chemical monitoring does not allow detection and quantification of adverse effects on aquatic organisms. Only a small part of organic compounds can be isolated from water and identified with present gaschromatographic and mass spectrometric techniques (Van der Gaag et aL, 1982; Noordsij et aL, 1982). Most compounds identified in river water in The Netherlands occur at concentrations below the 'no toxic effect' levels stated in literature. Also limited data are available on joint toxic action of different substances. Therefore, it seemed worthwhile to investigate whether bioassays with aquatic organisms are needed to obtain more information on effects of pollution on the aquatic environment. In order to detect and quantify possible adverse effects on fish of polluted river waters in The Netherlands, a number of bioassays has been investigated or developed in our laboratory. The primary aim of this research was to be able to observe toxic effects in raw water sources for drinking water preparation. In this paper the background and a number of results of this research are discussed, with special attention for the possibilities to use these bioassays for the evaluation of aquatic ecosystems. * Paper presented at a Symposium held on 14 and 15 October 1982, in Utrecht, The Netherlands. ** Present adress: Shell International Petroleum Maatschappij, Medical and Toxicological Division, the Hague, The Netherlands. Environmental Monitoring and Assessment 3 (1983) 247-255. 9 1983 by D. Reidel Publishing Company.

0167-6369/83/0033--0247501.35.

2~8

M.A.V.D.

G A A G ET AL.

2. Experimental Set-Up In all experiments fish exposed to river water were compared to control fish reared under similar conditions in unchlorinated groundwater of optimal quality. In order to detect effects of water quality on different parameters, test conditions have to be standardized, with respect to continuous water supply, aquarium dimensions, feeding, temperature and oxygen saturation. Most of the bioassays described in this study were carried out at the KIWA-Toxicological laboratory in Nieuwegein, where Rhinewater was continuously available (Pods et al., 1980). Sublethal influences of environmental factors can be measured at different levels in organisms. Growth, reproduction and teratogenicity can be monitored in whole animals. Measurements of relative organ weight, enzyme activity, clinical parameters in blood and bioaccumulation as well as histological examination can be carried out in different organs. They give information on specific organ activities, stress and fate of non biodegradable compounds. DNA damage or chromosomal aberrations can be measured at cellular level and are an indication for the presence of mutagens. The determination of all these parameters could be possible in one long term experiment. For practical reasons however, the different measurements have been divided into three standardized assays, allowing shorter test durations: (1) a long term exposition (24 weeks) of yearling trout (Salmo gairdneri, Rich.), including measurements of growth, relative liver and kidney weight, blood parameters, identification of accumulated organohalogens and heavy metals. This assay was derived from the long term test described by Poels et al. (1980). (2) an embryo-larval bioassay with trout (6 to 10 weeks) for measurements of mortality, embryonic development, growth and teratogenesis (Van der Gaag unpubl.). (3) a DNA-damage test with mudminnows (Umbra pygmaea) or Nothobranchius rachowi by measurement of sister chromatid exchange (SCE) frequency (Alink et al., 1980; Van der Hoeven et al., 1982).

3. Longterm Toxicity Tests 3.1. LONGTERM TOXICITY TESTS ON WATER OF THE RIVERS RHINE AND MEUSE Compared to control, rainbow trout exposed to water from the Rhine showed retarded growth, increased relative weight of both liver and kidney, and lowered haematocrit (Table I). These effects were observed during experiments in 1975/'76 (Poels, 1978; Poels et al., 1980) as well as in 1981/'82, but their impact was stronger in the first series of tests. Accumulation of organohalogens in adipose tissue was also higher in 1975/'76 than in 1981/'82, while a larger number of different compounds was identified in 1975 (DDT, TDE, PCB's, penta- and hexachlorobenzene and dieldrin) than in 1982 (pentaand hexachlorobenzene, pentachlorothioanisol and 7-HCH) (Table I). Other effects which occurred after longer exposure in 1975 (increased blood glucose levels after 6-8 months, atrophy ofhepatocytes, histological changes in the spleen after 15 months) were not observed during the 24 weeks experiment in 1981/'82. Evaluation of effects

TOXICOLOGICAL ASSESSMENT OF RIVER WATER QUALITY IN BIOASSAYS WITH FISH TABLE

249

I

S u m m a r y o f a n u m b e r o f p a r a m e t e r s m e a s u r e d in t r o u t e x p o s e d to w a t e r o f the rivers R h i n e a n d M e u s e for 2 4 w e e k s Rhine

A v e r a g e w a t e r f l o w (m3/s) (Lobith) Growth length weight R e l a t i v e liver w e i g h t Relative kidney weight Hematocrit Z O C 1 b ( m g / k g fat)

Meuse

1975/76

1981/82

1600

3800

+ + -

+ + -

22~o" 36~o ~ 54~/o a 30~o ~ 28~o ~ 9.4

8~o a 22~o a 21 ~o a 20~o a 13~o a 2.4

1981/82

-

7~o a

-

18,~o a

n$ ns ns 0.7

a p < 0.01.

b S u m o f o r g a n o c h l o r i n e c o m p o u n d s a c c u m u l a t e d in a d i p o s e tissue. ns: n o s i g n i f i c a n t c h a n g e .

detected in long term tests over a period of eight years (Poels and Strik, 1975; Poels et al., 1980 and this paper) point to a gradual improvement of Rhinewater quality with respect to chronic toxic effects in trout. Whether this is associated with improved waste water treatment or with increased dilution due to a higher river flow (Table I) is not yet clear. Next to the evaluation of water quality trends in time in one water body, a comparison of two rivers is also possible. In 1981/'82 the standardized 24 weeks assay was also performed in water of another important river in The Netherlands, the Meuse. The water of this river showed a different effect, resulting only in a significant growth retardation and a slight accumulation of organohalogens (Table I). This indicates that the quality of Meuse water with respect to chronic toxicity to fish was at that time better than that of Rhinewater. 3.2.

GROWTH

RETARDATION

Growth retardation is the first chronic toxic effect that can be detected in long term tests with trout exposed to river water. It is the resultant of all small adverse effects that cannot be measured as such. Therefore it is not possible to indicate which (group of) compound(s) is responsible for this effect. However, growth is a very sensitive and accurate parameter under standardized laboratory conditions, and therefore very suitable for quantification of water quality trends over a number of years. One of the adverse effects of growth retardation may be a lowered offspring production, as the egg production of female fish is directly related to their body weight (Weatherley, 1972). However, field studies conducted by Slooff and de Zwart (1983) revealed that a compensation occurred in bream from the Rhine, due to production of a larger number of smaller eggs.

250

3.3.

M. A. V. D. GAAG ET AL. LIVER ENLARGEMENT

Increased relative liver weight (RLW) in fish has proven to be a specific effect of Rhinewater over a number of years, both in laboratory (Poels and Strik, 1975, Poels et aL, 1980) and field studies (Slooff and de Zwart, 1983). Liver enlargement is accompanied most of the time by an increased metabolism of lipophilic xenobiotics or by toxic effects on liver functions (Schulte-Hermann, 1974)9 In order to obtain more information on the causes of RLW increase, the activity of aminopyrine N-demethylase (APDM) was monitored in livers of trout exposed to Rhine water. In a first series of experiments, specific A P D M activity (maximal capacity) was lowered by 20 to 30% after exposure to Rhinewater for 6 to 18 month (Poels, 1978)9 Using an improved method, APDM activity was also measured in following tests at low substrate concentrations, more representative of intracellular situations. The enzyme activity was assayed on the day the fish were killed, at the same temperature as that of the water in which the fish were reared (16 ~ This is to be prefered when enzymes of poYkilotherms are assayed (Hochachka and Somero, 1971; Bouck et al., 1975)9 The APDM activity at low substrate concentration was calculated according to CornishBowden and Eisenthal (1974). Within two weeks, activity of APDM at low substrate concentrations in trout exposed to Rhine water was 38% higher than in control animals (/9 < 0, 05, Figure 1). At the same time, the specific activity was lowered by 12 %. These observations would indicate that both toxic effects (lowered specific activity) and adaptation (increased substrate turnover at physiological concentrations) are involved9 It is not possible to indicate at this stage whether specific activity inhibition of N-demethylase (this paper) or induction Specific activity*

Activity'" at low substrate concentration

176T.

543..............

2-

i;):!i;:::?;:

:::'.::'.'. I

:..'.:....::

10

9

Fig. 1

.....

Control

: t;!~I~I:/:*

Rhine

I o,

:::-'; ";",";','1

Control

Rhine

9 in nmol formakdehyde/hour/mg protein at 16~ . in nmo~ fo,malOehyde/mmol $uDstrate/hour/mg protein at 16~

Specific activity a n d activity at low s u b s t r a t e c o n c e n t r a t i o n s of a m i n o p y r i n e N - d e m e t h y l a s e after a 15 days e x p o s u r e to R h i n e water.

TOXICOLOGICAL

ASSESSMENT

OF RIVER WATER QUALITY IN BIOASSAYS

WITH FISH

251

of other enzymes (Slooff and Van Kreijl, 1982) are predominantly responsible for the liver enlargement observed in Rhinewater fish. Such a simultaneous occurrence of enzyme induction and inhibition in fish exposed to complex effluents has also been observed at other sites (FOrlin and Hansson, 1982). Another aspect of liver enlargement is that much knowledge is available about compounds which can induce this effect. One class of such substances consists of organohalogens which are known to be accumulated in adipose tissue. Many of these compounds were detected in adipose tissue of trout exposed to surface waters in the past years. A certain relation seems to exist between the amount of organohalogens accumulated in adipose tissue and the differences in RLW between exposed and control fish (Figure 2). This indicates that bioaccumulating organohalogens contribute to an increase of RLW as observed in river waters in The Netherlands, probably together with many other compounds which can also induce this effect. SOCI in adipose tissue (mg/kg fat) l

10 8 at

6

at

at at

4

at

2

at

at at

0

, 0

atat 01

02

03 0.4 ,'~RWL (%)

05

06

Fig. 2 Relation between the sum of bioaccumulated organohalogens in adipose tissue (s OCI) and the differencein relative liverweightas a percentageof body weight (RLW)in fish reared in polluted water and control water.

4. Embryo-larval Test The embryonic and larval development in fish are known to be more sensitive for toxic compounds than later life stages (McKim, 1977). As a complement of long term tests, short term embryo-larval tests with trout were also carded out in Rhinewater each year in January between 1977 and 1982. Yearly variations in egg quality resulting in differences in control mortality do not allow quantitative comparisons. However, an improvement of Rhine water quality can be demonstrated with this test over the five year period (Figure 3). In 1977 mortality rates of trout eggs were higher in Rhinewater than in control. This increased mortality mainly happened during the early embryonic development (5-10 days after fertilization) and during the sac-fry stage (28-35 days after fertilization) (Figure 3). In 1978 no differences were found in mortality in the embryonic stage, but larval mortality was still higher (Figure 3). Higher embryonic mortality was only found in following years when eggs were hardened in Rhinewater (Figure 3), a procedure which

252

M. A. V. D. GAAG ET AL. Larval mortality (% of total numberof eggs) 1977

1978

1980

1981

1982

[ ] Control [ ] Eggs hardened in Rhinewater

10

oH 10

1 1

[ ] Placed in Rhinewater after hardening

20 Embryonal mortality (% of total number of eggs)

Fig. 3

M o r t a l i t y of e m b r y o ' s a n d l a r v a e of t r o u t in R h i n e w a t e r and control from 1977 to 1982.

was not followed in 1977 and 1978. From 1980 onwards, the differences in larval mortality between Rhinewater and control were small and not significant. Similar yearly quality variations of egg batches also interfere with an accurate determination of teratogenic or epigenetic effects. Moreover, no teratogenic effects can be scored in dead eggs. As dead eggs could represent a large part of teratogenic effects, comparisons between groups with different egg mortality are difficult. However, one type of effect, pugheadedness, occurred at a high rate in Rhinewater. The fish concerned had a distinctly shorter upper jaw compared to the lower jaw, while both jaws normally have the same length. In 1981 this effect was observed in 11% of the fish that hatched after incubation in Rhinewater. No such defects occurred in control. Similar effects have been described in bream in the Rhine and in trout and pike exposed to dioxin (2, 3, 7, 8 T C D D ) (Slooff, 1982; Helder, 1982). Whether dioxins or related compounds are responsible for the effects found in Rhinewater is not known.

5. Mutagenicity Assays with Fish The last type of assay involved is aimed at the specific group of genotoxic pollutants, i.e., those compounds which cause D N A damage or mutations. Two techniques are in use to evaluate chromosomal damage in fish in vivo, either monitoring of chromosomal

TOXICOLOGICAL ASSESSMENT OF RIVER WATER QUALITY IN BtOASSAYS WITH FISH

253

T A B L E II Induction of sister chromatid exchanges (SCE) in gill tissue of Umbra (January 1978) and Nothobranchius (November 1981) after exposure to Rhinewater Test

Group

Number of fish

N u m b e r of chromosomes

SCE/frequ. chromosome

1978

control Rhine (3 days) Rhine (11 days)

10 6

3123 2416

0.050 + 0.013 0.128 + 0.023

5

2317

0.155 + 0.021

control Rhine (7 days)

4 4

599 1739

~).055 + 0.026 0.104 + 0.018

1981

aberrations (Kligerman etal., 1975; Prein etal., 1978; Hooftman, 1981) or sister chromatid exchange (SCE) frequencies (Kligerman and Bloom, 1976; Alink et al., 1980; Hooftman and Vink, 1981; Van der Hoeven etaL, 1982). The latter technique was chosen for a number of experiments in which either mudminnows (Umbrapygmaea) or Nothobranchius rachowi were exposed to Rhinewater. Both in 1978 (Alink etal., 1980) and in 1981 (Van der Gaag etal., 1983), a significant increase in SCE frequency was found after a short stay in Rhinewater (Table II). Umbra is not very well suited for routine screening because of its poor availability and its relatively low mitotic index (Hooftman, 1981; Van der Hoeven, 1982). Nothobranchius rachowi offers better possibilities. But a number of problems associated with suboptimal sister chromatid differentiation staining, are still being investigated in order to obtain a routine test. Accurate evaluation of the possibilities of the Nothobranchius-SCE test will take place when these problems have been solved. 6. Evaluation

Evaluation of the bioassays presented in this paper can be separated in two different parts, depending on the aim of the investigation. If the aim is to give objective parameters in order to evaluate trends in toxicological aspects of a watertype, these bioassays can be useful monitoring systems. Toxic effects of a specific waterbody with a certain pollution load seem to be rather stable over a number of years, such as was demonstrated for Rhinewater. From 1974 to 1982, a similar pattern of altered parameters was observed. This included embryonic mortality, growth retardation, liver and kidney enlargement, low haematocrit and bioaccumulation of organochlorine compounds in adipose tissue. Over these years the intensity of changes in the different parameters decreased, suggesting a quality improvement of Rhinewater. However the toxic effects of Rhinewater are still more severe than in Meusewater. This suggests that a quantitative routine monitoring of toxic aspects of polluted waters is possible. The long term test with trout is particularily suitable for this

254

M.A.V.D.

GAAG ET AL

purpose, and can possibly be supplemented in the near future by the SCE-test in Nothobranchius.

However, a n u m b e r of difficulties arise when extrapolation of test results is needed, in order to evaluate effects of toxic c o n t a m i n a n t s on ecosystems. A n u m b e r of similar effects were found in laboratory and field studies of Rhinewater, such as higher mortality, growth retardation, liver enlargement a n d even some teratogenic effects (Slooff and de Zwart, 1983). However, it is n o t excluded that environmental factors could c o m p e n s a t e for toxic effects in the ecosystem. U n t i l such aspects have not been subject of further study, it is advisable to remain careful when laboratory results of the present bioassays are used for evaluating the influence of pollutants on ecosystems.

Acknowledgments This research was financed by The Netherlands Waterworks Association (VEWIN), and by the Rhine Comittee of Water Works ( R I W A ) (Testing program on Rhine and Meuse in 1981/'82). The authors would like to t h a n k W. B. Buiten, A. de Ruyter, W. van Hout, and Mrs G. A. Peute-Slof, who contributed to different parts of this research program. The analysis of organohalogens in adipose tissue was performed by the C I V O - T N O Institute in Zeist (The Netherlands).

References Alink, G. M., Frederix-Wolters, E. M. H., Gaag, M. A. van der, Kerkhoff,J. F. J. van de, and Poels, C. L. M.: 1980, 'Induction of Sister-Chromatid Exchanges in Fish Exposed to Rhine-Water', Mutat. Res. 78, 369-374. Bouck, G. R., Schneider, P. W., Jr., Jacobsen, J., and Ball, R. C.: 1975, 'Characterization and Subcellular Location of LeucineAminoaphtylamidase(LAN) in Rainbow Trout (S almo gairdenii)',J. Fish. Res. Board Can. 32, 1289-1295. Cornish-Bowden, A. and Eisenthal, R.: 1974, 'Statistical Considerations in the Estimation of Enzyme Kinetic Parameters by Direct Linear Plot and Other Methods', Biochem. J. 139, 721-730. FOrlin, L. and Hansson, T.: 1982, 'Effects of Treated Municipal Waste Water on the Hepatic, Xenobiotic and Steroid Metabolism in Trout', Ecotox. and Environm. Saf. 6, 41-48. Gaag, M. A. van der, Noordsij, A, and Oranje, J. P.: 1982, 'Presence of Mutagens in Dutch Surface Water and Effects of Water Treatment Processes for Drinking Water Preparation', in Sorsa and Va'/nio(eds.), Mutagens in our Environment, Alan R. Liss, New York, pp. 277-286. Gaag, M. A. van der, Alink, G. M., Hack, P., Hoeven, J. C. M. van der, and Kerkhoff, J. F. J. van de: 1983, 'GenotoxicologicalStudy of Rhinewater with the KillifishNothobranchius Rochowi',Mutat. Res. 113, 311. Helder, T.: 198l,'Effects of 2, 3, 7, 8-Tetrachlorodi-benzo-p-dioxin(TCDD) on Early Life Stages of Rainbow Trout (Salmo gairdeneri, Rich)', Toxicology 19, 101-112. Hoeven, J. C. M. van der, Bruggeman, I. M., Alink, G. M., and Koeman, J. H.: 1982, 'The Killifish Nothobranchius rachowi, a New Animal in Genetic Toxicology',Murat. Res. 97, 35-42. Hochachka, P. W. and Somero, G. M.: 1971, 'Biochemical Adaptations to the Environment', in Hoar and Randall (eds.), Fish Physiology Academic Press Inc., New York, pp. 100-156. Hooftman, R. N.: 1981, 'The Induction of Chromosome Aberrations in Nothobranchius rachowi after Treatment with Ethyl Methane Sulfonate and Benzo(a)-pyrene, Murat. Res. 91,347-352. Hooftman, R. N. and Vink, G. J.: 1981, 'Cytogenetic Effects of Eastern Mud Minnow, Umbra Pygmaea, Exposed to Ethyl Methane Sulfonate, Benzo(a)-pyrene And Riverwater', Ecotox. and Environm. Saf. 5, 261-269. Kligerman, A. D. and Bloom, S. E.: 1976, 'Sister Chromatid Differation and Exchanges in Adult

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Mudminnows (Umbra limi) after in vivo Exposure to Bromodeoxyuridine', Chromosoma 56, 101-109. Kligerman, A. D., Bloom, S. E., and Howell, W. M.: 1975, 'Umbra limi, a Model for the Study of Chromosome Aberrations in Fishes', Mutat. Res. 31,225-233. McKim, J. M.: 1977, 'Evaluation of Tests with Early Life Stages of Fish for Predicting Longterm Toxicity', J. Fish. Res. Board Can. 34, 1148-1154. Noordsij, A, Van Beveren, J., and Brand, A.: 1982, 'Isolation of Organic Compounds from Water for Chemical Analysis and Toxicological Testing', Intern. J. Environ. Anal. Chem. 13, 205-217. Poels, C. L. M.: 1978, 'Sublethal Effects of Rhinewater on Rainbow Trout', in Hutzinger, Lelyveld, and Zoeteman (eds.), Proceedings of the Second International Symposium on Aquatic Pollutants, pp. 479-480. Pods, C. L. M. and Strik, J. J. T. W. A.: 1975, 'Chronic Toxic Effects of the Water of the River Rhine upon Rainbow Trout', in Koeman, J. H. and Strik, J. J. T. W. A. (eds.), Elsevier, Amsterdam, Sublethal Effects of Toxic Chemicals on Aquatic Animals, pp. 86-91. Poels, C. L. M., Gaag, M. A. van der, and Kerkhoff, J. F. L. van de: 1980, 'An Investigation into the Long-Term Effects of Rhinewater on Rainbow Trout', Water Res. 14, 1029-1035. Prein, A. E., Thie, G. M., Alink, G. M., Poels, C. L. M., and Koeman, J. H.: 1978, 'Cytogenetic Changes in Fish Exposed to Water of the River Rhine', Sci. Tot. Environ. 9, 287-291. Schulte Hermann, R.: 1974, 'Induction of Liver Growth by Xenobiotic Compounds and Other Stimuli', C.R.C. Crit. Rev. Toxicol. 3, 97-158. Slooff, W.: 1982, 'Skeletal Anomalies in Fish from Polluted Surface Water', Aquat. Toxicol. 2, 157-173. Slooff, W. and Kreijl, C. F. van: 1982, 'Monitoring the Rivers Rhine and Meuse in The Netherlands for Mutagenic Activity Using the Amestest in Combination with Rat and Fish Liver Homogenates', Aquat. Toxicol. 2, 157-173. Slooff, W. and Zwart, D. de: 1983, 'Bio-Indicators and Chemical Pollution of Surface Waters', Environmental Monitoring and Assessment 3, 237-245 (this issue). Weatherley, A. H.: 1972, Growth and Ecology ofFish Populations, Academic Press, London, New York.

Toxicological assessment of river water quality in bioassays with fish.

A series of bioassays with fish was developed in order to evaluate toxicological aspects of polluted rivers in The Netherlands. A long term exposition...
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