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Mutagenesis vol.14 no.4 pp.397–402, 1999

Flow cytometric measurement of micronuclei induced in a permanent fish cell line as a possible screening test for the genotoxicity of industrial waste waters

Martin Kohlpoth2, Brigitte Rusche and Michael Nu¨sse1 Akademie fu¨r Tierschutz, Spechtstrasse 1, D-85579 Neubiberg and 1GSF-Flow Cytometry Group, D-85764 Neuherberg, Germany

An in vitro micronucleus assay using the permanent fish cell line RTG-2 (rainbow trout gonads) was developed to test industrial waste waters for their genotoxic potential. Comparison of flow cytometric measurement and microscopic scoring of micronucleus frequency with the reference chemicals 1,4-butane sultone (0.2–1 mM), ethylmethane sulphonate (2–10 mM), potassium dichromate (20–100 µM) and benzo[a]pyrene (5–25 µM) showed similar dose–effect relationships. Thirty-eight industrial waste waters from 11 different branches of industry obtained from the Bavarian state office for water research were tested using the flow cytometric method (18 from metal processing, 10 from combined waste water, two from synthetic fibre production, one sample each from settlement wastes, non-iron metal manufacturing, leather production, sulphuric acid production, ore processing, graphite film production, cellulose production and flue gas washing). Fourteen of them showed a significant increase in micronucleus frequency.

Introduction Studies in the USA showed that ~30% of industrial waste waters were contaminated with genotoxic chemicals (Stahl, 1991). Similar values can be expected in most industrialized countries. In addition to urban discharges, engine exhausts and agricultural chemicals, industrial wastes are one source of such genotoxic substances which accumulate in waters and sediments. The demonstration that industrial wastes can induce genotoxic effects in aquatic animal species (Black and Baumann, 1991) and also result in genotoxic activity in drinking water (Bull, 1985; Meier, 1988; Koivusalo et al., 1994) underlines the urgent need for sensitive assays for the assessment of the genotoxic potential of industrial wastes. As it is not possible to measure the genotoxicity of waste waters by chemical or physical analysis alone, biological test systems are needed to recognize synergistic and antagonistic effects of complex chemical mixtures. For this reason the Salmonella mutagenicity assay (Ames test) has been used extensively to evaluate the genotoxicity of different aquatic samples like river water (Vargas et al., 1993; Rehana et al., 1996), drinking water (Loper, 1980) or industrial waste waters (Pruul et al., 1996). This well-established genotoxicity assay with a prokaryotic organism allows the detection of gene point mutations. However, to detect a broader range of genetic damage it is necessary to use test assays with eukaryotic organisms. For many years permanent fish cell lines have been used in laboratory test systems to measure the toxicity and genotoxicity of single compounds or environmental samples (Babich and 2To

Borenfreund, 1991). The chromosomal aberration assay and the sister chromatid exchange assay have been used mainly with fish cell lines, because these tests had already been established as in vitro test systems for determining genotoxic responses in mammalian cell lines (Tucker and Preston, 1996). However, these metaphase assays are not practical test systems for routine use because the karyotype of most fish cell lines consists of a large number of small, morphologically similar chromosomes that have proved to be difficult to evaluate (Al-Sabti and Metcalfe, 1995). For that reason, we chose the micronucleus assay which is a standard in vitro test using mammalian cell lines (Fritzenschaf et al., 1993) to assess whether it can be used as a routine genotoxicity assay for industrial waste waters. In general, the micronucleus assay is a rapid and sensitive standard test which can be performed easily because metaphase cells are not required (Heddle et al., 1983). Micronuclei are formed by condensation of chromosomal fragments (clastogenic effects) or whole chromosomes (aneugenic effects) that are not included in the nuclei of the daughter cells during anaphase and exist separately from the main nucleus in the cytoplasma after mitosis. Several studies performed with different in vivo and in vitro assays have demonstrated that the assessment of micronucleus formation is a sensitive indicator of genotoxicity (Arlett et al., 1989). Another important advantage of the micronucleus assay is the possible automation of measurement, because the traditional microscopic analysis of micronuclei is time consuming. The technical equipment for image analysis and flow cytometry have been optimized in recent years for practicability in routine testing (Schultz et al., 1993). The fibroblast cell line RTG-2 was the first established fish cell line (Wolf and Quimby, 1962) and is, therefore, well characterized and many published results about its reaction in toxicity and genotoxicity tests exist (Isomaa et al., 1994). Micronuclei were induced in the RTG-2 cell line by four reference chemicals and the frequency of induced micronuclei was measured by microscopic observation and flow cytometry using a technique described by Nu¨sse et al. (1994). This technique has been used very successfully for routine testing with mammalian cell lines (Weller et al., 1993). Materials and methods Cell culture RTG-2 cells were grown in Eagle’s minimum essential medium with Earl’s salts, supplemented with 850 mg/l sodium bicarbonate, 2 mmol L-glutamine, 50 mg/l neomycin sulphate and 10% fetal calf serum (FCS). With the addition of 20 mmol HEPES buffer no additional CO2 gassing was needed. The optimum temperature for cultivation was 20°C in a cooling incubator. Cells were tested routinely for the presence of mycoplasm contamination and were used for the micronucleus assay in the exponential growth phase. Chemicals As reference chemicals we chose 1,4-butane sultone (1,4-BS), which is reported as being weakly mutagenic (Nesnow et al., 1987), ethylmethanesulphonate (EMS), which is a well-known direct acting mutagenic chemical (Kocan

whom correspondence should be addressed. Tel: 149 89 600 2910; Fax: 149 89 600 29115; Email: [email protected]

© UK Environmental Mutagen Society/Oxford University Press 1999

397

M.Kohlpoth, B.Rusche and M.Nu¨sse et al., 1979), potassium dichromate (PCr2), representing an ecologically relevant heavy metal which has been shown to also be genotoxic in some short-term test assays (Rickert, 1996), and benzo[a]pyrene (BAP), which is often used in vitro as an indirect acting carcinogen (Diamond and Clark, 1970). All chemicals were purchased from Merck (Darmstadt, Germany). 1,4-BS, EMS and BAP were dissolved in dimethyl sulphoxide; PCr2 was dissolved in distilled water. The final concentration of the solvents did not exceed 1% (v/v) of the culture medium. For the control pure solvents were added to the medium. For a better comparison of the dose–effect curves for the two methods for micronucleus measurement examined in this study, RTG-2 cells were exposed to the reference chemicals in a smaller range of concentrations than normally used. The dilution steps were chosen between 0.2 and 1 mmol for 1,4-BS, 2 and 10 mmol for EMS, 20 and 100 µmol for PCr2 and 5 and 25 µmol for BAP, because within these concentration ranges the highest increase in micronuclei was observed in pre-tests. Preparation of nuclei and micronuclei in suspension and fluorescence staining for flow cytometric analysis was done using the following solutions. Solution I: 584 mg/l NaCl, 1000 mg/l sodium citrate, 25 mg/l ethidium bromide (EB), 10 mg/l RNase and 0.3 ml/l Nonidet P-40. Solution II: 1.5% citric acid, 0.25 M sucrose and 40 mg/l EB. Waste water samples The waste water samples were from different Bavarian state offices for regional water management and were stored in polyethylene containers at –20°C. They were thawed in a water bath at 20°C and held at room temperature for 2 h for sedimentation. From each supernatant three aliquots of 3 ml were filter sterilized using syringe filters with 0.22 µm pore size, the pH value was made neutral and the aliquots were restored at –75°C. For the micronucleus assay the aliquots were mixed after thawing at 20°C in a water bath with the same volume of double concentrated medium including 20% FCS and added to the cell culture vessels. In the case of a high level of micronuclei measured with the first aliquot the micronucleus assay was repeated twice; in the case of a normal level of micronuclei in the first aliquot one further measurement was performed to confirm the result. Within a series of five measurements all aliquots were tested two or three times (Table II). For each measurement three negative controls containing phosphate-buffered saline (PBS) and three positive controls containing 8 mmol EMS were tested simultaneously. The concentration of 8 mmol EMS was chosen as a positive control because this concentration showed a constant high micronucleus frequency in the flow cytometric measurements (Table I). Because of the large differences in the osmolarity of the waste waters (from 10 to 606 mOsmol) and a possible effect on the test results, the osmolarity of each native sample before and after mixing with double concentrated medium was measured with a cryoscopic osmometer OSMOMAT 030 (Gonotec, Berlin, Germany). To exclude cytotoxic effects only waste water samples which were negative in a neutral red cytotoxicity assay (Borenfreund and Puerner, 1985) were chosen for testing. The origin of the waste water samples was as follows: 18 were from metal processing (A), 10 were from combined waste water (B), two were from synthetic fibre production (C) and one sample each was from settlement wastes (D), non-iron metal manufacturing (E), leather production (F), sulphuric acid production (G), ore processing (H), graphite film production (I), cellulose production (K) and flue gas washing (L) (see Table II).

were air dried overnight. 2000 cells were counted from each coverslip (magnification 4003) and the number of micronuclei was determined. For a better comparison with the flow cytometric results all micronuclei from cells containing multiple micronuclei were also counted, excluding cells that had undergone karyolysis or karyorhexis. Criteria for micronucleus identification were applied as described by Schmuck et al. (1988). Because the main problem in reproducibility of microscopic scoring is individual differences in identifying micronuclei, each of the two experiments was carried out by three different persons. The coverslips were prior coded by a neutral person in order to exclude a subjective influence. Flow cytometry A suspension of micronuclei and nuclei was prepared according to the procedure described by Nu¨sse et al. (1994). The RTG-2 cells were trypsinized and centrifuged (5 min at 1000 r.p.m.) and 1 ml of solution I was added to the resuspended cell pellet to disrupt the cell membranes with the detergent Nonidet P-40. After 30 min at room temperature 1 ml of solution II was added and the suspension was agitated slightly. In this second step the cytoplasm adhering to the nuclei and micronuclei was disintegrated and thereafter the suspension contained only cell nuclei and micronuclei and other, unspecific particles (‘debris’). The samples were stored at 4°C for a maximum of 2 weeks. In earlier studies no changes in the measured frequency of micronuclei were observed during that time interval. For the flow cytometric measurement each experiment was carried out three times. EB fluorescence and side scatter as well as forward scatter intensities of micronuclei (MN) and cell nuclei (N) were measured simultaneously in list mode using a FACStar1 flow cytometer (Becton Dickinson, Sunnyvale, CA). Excitation of EB was provided by the 488 nm line (500 mW) of an argon laser, EB fluorescence was detected with long bandpass filters (combination of KV550 and OG590). All parameters were registered in log mode (4 decades), because MN can have a relative DNA content between 0.5 and .10% of the DNA content of G1 phase nuclei. Usually, the mean EB fluorescence of the G1 phase cells was set to channel no. 2000, MN were measured between channels nos 20 (1%) and 400 (20%). For analysis of the frequency of MN two gates had to be set simultaneously according to the forward scatter and side scatter intensities to exclude most of the unspecific debris. For those particles that fall within both gates a third gate for the number of MN (NMN) and a fourth gate around all nuclei to count the number of nuclei (NN) were defined. The frequency of MN is then calculated as NMN/NN (for details see Nu¨sse et al., 1994). Evaluation of the data Mean values and standard deviations of the different experiments are displayed in Figure 1 as a function of the concentration of the chemicals. For the results of the waste water experiments obtained by flow cytometry, the frequency of micronuclei in the controls was subtracted from the induced frequency in the specific experiments because different control values were obtained in some experiments. Data for each experiment have been analysed separately versus their own control assuming a binomial distribution (Woolf G-test). Due to the large sample size and low probability of MN induction, data were additionally analysed assuming a Poisson distribution (H0; λ1 Þ λ2) and a normal distribution (two-sided t-test). Probes have been considered as significantly different from the control when a significance level of P , 0.05 was given in each separate experiment and in all statistical tests used. A waste water was regarded as genotoxic when a significantly higher micronucleus frequency was observed in all three repeated tests.

Test procedure The test protocol for an in vitro micronucleus assay established in our laboratory using mammalian cells (Fritzenschaf et al., 1993) was optimized for the use of RTG-2 fish cells. In particular, the longer generation time of fish cells in comparison with mammalian cells (Plumb and Wolf, 1971) caused changes in exposure and incubation times. For microscopic scoring 6-well plates were filled with sterilized coverslips (18318 mm) and 53104 cells in 4 ml medium were added. For the flow cytometric measurement cell culture flasks were inoculated with 33105 cells in 5 ml medium. After an adaptation time of 24 h the medium was removed and the test medium was added. After an exposure time of 24 h the medium was changed again and the cells were incubated for a further 72 h. After that time the cells were prepared differently for the two measurements. Microscopic scoring After removing the medium cells were fixed with ethanol for 10 min and Feulgen stained as follows. The cells were hydrolysed by adding 2 ml of a 5 M HCl solution to each well for 1 h. After washing twice with distilled water the nuclei and micronuclei were Pararosaniline stained by adding Schiff’s reagent for 90 min. After washing with distilled water the preparations

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Results All chosen reference chemicals induced comparable dose– effect relationships with respect to micronuclei in RTG-2 fish cells analysed by both techniques. Table I gives the results of the experiments performed by microscopic scoring and by flow cytometry. In Figure 1 the mean values of the measurements are shown as a function of the concentration of the chemicals. With 1,4BS a significant increase in micronuclei in the cell population was observed at the highest concentration of 1 mM (Fig. 1a). With both methods the micronucleus frequency for EMS increased continuously at concentrations between 2 and 8 mmol. For the highest concentration of 10 mmol the results were inconsistent (Fig. 1b). The slope of the dose–effect curve for PCr2 was similar for both techniques, although lower values were obtained by

A fish cell test for genotoxic waste waters

Table I. Comparison of the micronucleus frequencies measured with the microscope and the flow cytometer in RTG-2 cells after exposure to different reference chemicals Chemical/dose

Microscope

Flow cytometry

Test 1

1,4-Butane sultone Control 0.2 mmol 0.4 mmol 0.6 mmol 0.8 mmol 1.0 mmol Ethylmethanesulphonate Control 2 mmol 4 mmol 6 mmol 8 mmol 10 mmol Potassium dichromate Control 20 µmol 40 µmol 60 µmol 80 µmol 100 µmol Benzo[a]pyrene Control 5 µmol 10 µmol 15 µmol 20 µmol 25 µmol

Test 2

Test 1

Test 2

Test 3

A

B

C

A

B

C

13.1 15.1 15.8 8.3 9.3 19.9

7.9 8.7 12.7 10.5 15.0 20.1

8.3 9.5 12.9 8.9 12.2 16.9

6.0 6.8 6.6 7.7 9.2 13.2

6.4 10.2 5.6 7.3 7.6 13.0

4.3 5.4 5.4 9.2 12.0 13.7

6.8 5.5 5.5 6.7 9.5 13.9

6.2 5.8 7.5 7.9 10.3 16.2

6.2 6.8 4.9 9.9 5.7 10.8

2.6 4.8 9.3 10.8 11.7 16.0

5.0 5.8 7.5 14.8 18.6 14.2

6.9 4.9 8.9 14.8 21.9 19.7

2.3 3.2 4.0 3.4 8.6 1.6

3.2 4.9 6.0 6.8 14.3 2.6

2.9 5.0 7.4 6.8 12.5 2.7

17.2 18.6 19.7 24.1 30.9 41.0

10.0 9.4 16.3 15.5 18.3 9.2

11.1 15.1 15.5 22.5 25.9

3.2 11.6 8.6 12.0 10.1 10.3

4.7 6.2 8.2 14.0 10.9 10.6

7.2 7.4 11.5 17.0 15.3 8.6

7.5 14.9 18.5 22.8 23.8 16.3

4.8 9.1 15.6 27.4 35.2 14.7

3.2 10.5 14.6 29.6 40.5 20.7

19.2 24.5 32.7 39.2 33.6 19.8

11.4 17.4 19.7 23.1 27.1 19.4

12.5 19.7 33.8 44.6 46.1 46.8

4.9 5.5 11.5 19.7 12.0 11.5

5.7 7.5 8.5 18.7 10.9 9.5

8.8 6.0 8.7 18.9 14.2 8.8

5.8 8.1 16.1 18.6 17.2 18.5

8.1 9.4 16.6 25.0 24.4 19.9

6.7 7.3 15.8 18.9 16.1 18.0

6.8 11.0 16.9 22.7 26.7 41.9

7.5 10.8 15.6 19.5 24.1 23.5

6.6 11.1 18.4 22.1 23.5 33.1

Shown are the micronucleus frequencies in % (NMN/NN3100%). The data from the experiments are shown as the results of single experiments analysed by three different persons (A, B and C) in the case of microscopic scoring or as mean values of two replicate samples by flow cytometry.

microscopic scoring, as with EMS. An increase in the micronucleus frequency up to 60 µmol could be observed. At higher concentrations the curves reached a plateau (Fig. 1c). The dose–effect curves for the indirect acting chemical BAP were different in the two assays. Up to a concentration of 15 µmol micronucleus frequency increased with concentration in both assays, but at higher concentrations of 20 and 25 µmol the micronucleus frequency reached a plateau according to microscopic counting but increased according to the flow cytometric measurement (Fig. 1d). The mean values of the controls showed micronucleus frequencies of 5.5 6 2.7% for microscopic scoring and 11.1 6 4.6 % for flow cytometric measurement (Table I). In Table II the micronucleus frequencies in cells after exposure to 38 waste water samples as measured by flow cytometry are presented. The osmolarity of the tested 38 waste water samples from 11 different branches of industry was between 10 and 606 mOsmol (Table II), but no correlation could be observed between micronucleus frequency and measured osmolarity. Eleven waste water samples had a significantly higher micronucleus frequency in all three repeated tests. Further, three waste water samples (2397, 2412 and 2436) were also determined as genotoxic although the micronuclei frequencies in one of the three tests were slightly below the significance level. The genotoxic samples were obtained from the following

branches of industry (Table II): metal processing (seven), combined waste water (three), synthetic fibre production (one), non-iron metal manufacturing (one), ore processing (one) and sulphuric acid production (one). For five waste waters (2385, 2437, 2440, 2443 and 2444) the measured micronucleus frequencies were found to be inconsistent when measured in different experiments. Discussion The in vitro micronucleus assay with the permanent fish cell line RTG-2 gave reproducible results. The comparison of flow cytometric measurement and microscopic scoring showed similar dose–effect relationships with the reference chemicals 1,4-BS, EMS, PCr2 and BAP. The results with the chemical 1,4-BS confirm the sensitivity of the micronucleus assay, which allows detection of even weak carcinogens (Nesnow et al., 1987). The decreasing micronuclei values at the highest concentrations of the reference chemicals EMS and PCr2 were caused by cytotoxic or growth inhibiting effects, because for micronucleus induction one cell division must be guaranteed. The positive results with BAP confirm the capacity of the RTG-2 cell line to metabolise procarcinogens into their active form, as described earlier (Kocan et al., 1982). The different dose–effect curves for flow cytometry and microscopic measurement at the highest concentrations were caused by a large number of nuclei undergoing karyolysis. These could be 399

M.Kohlpoth, B.Rusche and M.Nu¨sse

Fig. 1. Micronucleus frequency NMN/NN3100% as a function of the concentration measured by flow cytometry and by microscopic scoring for the four chemicals: (a) 1,4-butane sultone (1,4-BS); (b) ethylmethanesulphonate (EMS); (c) potassium dichromate (PCr2); (d) benzo[a]pyrene (BAP).

identified by microscopic scoring using a fluorescence microscope, but a perfect separation of cellular debris and micronuclei during the flow cytometric measurement was not possible (see also Nu¨sse and Marx, 1997). This effect might also explain the differences in the micronucleus frequencies for the controls in both assays. However, after subtraction of the different control values from the induced micronucleus values the dose–effect curves of both assays were comparable for all four reference chemicals. Therefore, for the waste water testing only the flow cytometric technique, which has potential for routine testing, was used. The micronucleus analysis of the waste water samples using flow cytometry led to the detection of 14 waste water samples with genotoxic effects from a total number of 38. Many of them originate from the metal processing industry, which is consistent with earlier findings that have demonstrated the genotoxic effects of waste waters from this industry in other test assays (Sanchez et al., 1988; Eckl et al., 1993). That waste waters from the metal industries were considered not to be strong genotoxicants in other studies (Houk, 1992) is probably due to the fact that such judgements were based on results mainly obtained with the Ames test. Thus our results underline the necessity of using a battery of bioassays with different end-points. 400

The five waste waters which showed contradictory micronucleus frequencies demonstrate the necessity for a better standardization of the test protocol as well as for defining acceptance criteria, like a maximum spontaneous micronucleus rate for the negative controls and others, for this test assay. However, for these definitions more data have to be obtained in routine testing. The micronucleus assay has also been used in numerous in vivo ecotoxicological studies with several aquatic species, such as newt larvae (Djomo et al., 1995), fish (Chu et al., 1996) and tadpoles (Krauter, 1993). In general, such tests are limited for routine application by problems of standardization and reproducibility, because intraspecies factors like age, sex, diet or health affect the response in the test assays. For scientific as well as for ethical (i.e. animal welfare) reasons an in vitro genotoxicity test should be preferred for routine testing. The RTG-2 cell line had been chosen as the standard cell line in an interlaboratory prevalidation study for a cytotoxicity assay (Schulz et al., 1995) in which it proved to deliver reproducible results. Therefore, and because of its metabolizing capacity (Kocan et al., 1982), it can be expected to be suitable for routine use. The main disadvantage of using fish cells for genotoxicity assays with aqueous samples is the addition of FCS to the

A fish cell test for genotoxic waste waters

Table II. Micronucleus frequencies of RTG-2 cells after exposure to industrial waste waters measured by flow cytometry Measurement no.

Controls (1 PBS) Positive controls (1 8 mmol EMS) Branch of industry/sample code B 2374 (126/407) B 2376 (224/431) B 2378 (142/400) A 2383 (529/588) A 2384 (65/378) D 2385 (606/619) A 2388 (17/354) B 2396 (49/337) B 2397 (31/346) A 2399 (81/373) B 2401 (460/548) B 2402 (384/519) G 2404 (17/334) C 2405 (114/381) C 2406 (10/355) A 2408 (31/366) A 2409 (136/397) A 2412 (22/357) E 2413 (327/516) A 2415 (15/361) B 2420 (96/434) A 2423 (73/380) A 2424 (38/364) A 2425 (94/404) A 2427 (45/380) A 2429 (24/358) F 2430 (177/435) A 2431 (292/490) H 2436 (63/383) A 2437 (67/371) B 2438 (92/361) B 2440 (70/337) I 2441 (462/557) A 2442 (26/332) A 2443 (32/340) A 2444 (38/332) L 2448 (299/474) K 2449 (43/334)

1

2

3

4

5

6.0 6 0.6 20.9 6 1.7

12.6 6 2.1 26.2 6 2.2

9.1 6 1.2 25.3 6 3.7

8.5 6 0.4 17.6 6 1.1

16.1 6 0.8 21.8 6 0.5

2.4 1.4 4.2 9.2 5.6 2.4 7.5 10.6 8.0 2.8 2.8 1.1 5.2 4.7 7.1 7.8 27.2 9.1 24.0 7.2

0.2 –2.2 –0.1 10.4

8.2 7.8 9.6

9.2 3.9

5.0

5.0 6.8 4.6 11.3

10.7 12.0 8.3

5.8 1.5 2.0 0.9 0.9 3.1 0.4 0.8 4.0 –0.9 1.3 1.3 –0.1 4.5 1.8 0.5

8.5

–0.1

11.7 6.3 11.4

4.8 4.7 4.7 4.6 2.1 2.8 9.5 6.0 3.2 5.7 2.6 7.5 8.5 7.2 4.4 3.5

4.6 2.1 0.8 1.0 4.2 0.3 8.5 8.4

7.0 7.5 0.7

9.9 6.9

10.0

10.7

3.6 4.0 0.6

The micronucleus frequencies of the waste waters are shown after subtraction of the mean control values. Significantly higher micronucleus frequencies are in italic. The letters before the sample codes identify the branch of industry (see Materials and methods). The values following the sample codes show the measured osmolarity (native sample/after mixing with double concentrated medium). Positive waste water samples are in bold italic.

culture medium, because it was demonstrated that FCS addition can lower the ratio for binding genotoxic substances (Grinfeld et al., 1986). It should, therefore, be examined whether this effect could be minimized by using FCS replacements or by developing a fully synthetic culture medium for permanent fish cells as has already been done successfully for some mammalian cell lines (Bjare, 1992). Both possibilities are being examined in further experiments. Because in general fish cells in culture respond to the same mutagenic and carcinogenic chemicals as do mammalian cells in culture (Babich and Borenfreund, 1991), their use in in vitro assays can be helpful in determining not only genotoxic effects of chemical contaminants in the aquatic environment but also in detecting chemicals that have the potential to cause carcinogenic effects in mammals, including humans. An important reason for routine testing of waste waters for their genotoxic potential is to provide data for use by

governments as a scientific basis for regulating the discharge of potentially hazardous substances into the environment. No single test system is capable of detecting all genotoxic effects of complex chemical mixtures, but a battery of standardized short-term test assays would be useful to obtain more data about the different branches of industry, which would allow them to be ranked in terms of the genotoxic potential of their discharges (Houk, 1992), and to determine technical procedures to decrease the genotoxic potential of waste waters before they are discharged into aquatic ecosystems (Monarca et al., 1983; Sato et al., 1987). The micronucleus assay with permanent fish cells can be regarded as a promising candidate for inclusion in such a battery of tests. Acknowledgements We thank the Bavarian Landesanstalt fu¨r Wasserforschung for providing the industrial waste water samples. This study was financially supported by the German Federal Ministry for Education, Science, Research and Technology.

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