Mutagenicity of bleached and unbleached effluents from Baikalsk pulp and paper mill at Lake Baikal, Russia

Mutagenicity of bleached and unbleached effluents from Baikalsk pulp and paper mill at Lake Baikal, Russia

Aquatic Ecosystem Health and Management 3 (2000) 95–104 Mutagenicity of bleached and unbleached effluents from Baikals...

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Aquatic Ecosystem Health and Management 3 (2000) 95–104

Mutagenicity of bleached and unbleached effluents from Baikalsk pulp and paper mill at Lake Baikal, Russia S.V. Kotelevtsev a,*, O.O.P. Ha¨nninen b, P.A. Lindstro¨m-Seppa¨ b, S.E. Huuskonen b, L.I. Stepanova a, V.M. Glaser a, A.M. Beim c,✠ a

Biological Department, Laboratory of Physical Chemistry of Biomembranes, Moscow State University, 119899 Moscow, Russian Federation b Department of Physiology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland c Institute of Ecological Toxicology, Baikalsk, P.O. Box 48, 665914 Irkutsk region, Russian Federation

Abstract The mutagenicity of bleached pulp mill effluents was compared to the mutagenicity of unbleached waste waters. Mutagenicity was assessed with the Ames test, using metabolic activation systems isolated from the liver of rats and fish. Liver extracts from fish caught in polluted areas, and from fish experimentally exposed to waste water, were also investigated. Pulp mill effluents taken after chlorination showed mutagenic activity. The activity diminished during the waste water treatment. Tissue extracts from fish exposed to various concentrations of treated bleached and unbleached pulp mill effluents showed only slight mutagenic activity in a few samples. In the case of bleached pulp mill effluents monooxygenase activities were elevated in those samples where slight mutagenicity was observed. In the case of unbleached effluents no such correlation was found. q 2000 Published by Elsevier Science Ltd and AEHMS. All rights reserved. Keywords: Fish; Cytochrome P-4501A; Induction

1. Introduction Pulp and paper production generates copious amounts of effluents. There have been attempts to develop mills with closed water circulation, but so far, effluents are released into surrounding lakes and rivers. Some stages of the pulping processes form effluents containing toxic and mutagenic substances (Ander et al., 1977; Glaser et al., 1990). Untreated waste waters from pulp and paper mills are multi-component mixtures containing lignin * Corresponding author. Tel.: 17-95-939-22-60; fax: 17-95-93950-22. E-mail address: [email protected] (S.V. Kotelevtsev). ✠ Sadly, Professor A.M. Beim died during the editing process for this paper.

derivatives, partial degradation products of cellulose, terpene hydrocarbons and other substances which are extracted during the cooking and bleaching of cellulose (McLeay, 1987). Chlorine-bleaching contaminates the effluents with several potentially reactive chlorinated organic components (Holmbom et al., 1984; Kinae et al., 1988) including the most potent mutagen, hydroxyfuranone MX (3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone). Some of these compounds exert a mutagenic influence on Salmonella typhimurium strains (Ames et al., 1975). Many pulp mills are modifying their processes and using less and less chlorine in bleaching. However, there are still factories using different kinds of chlorine chemicals. The mills producing unbleached pulp and paper do not use chlorine chemicals. However, when studying

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native fish populations or fish caged in waters influenced by such pulp and paper mills, it is apparent that unbleached effluents contain some chemicals inducing monooxygenase enzymes as well as chemicals causing toxicity (Lindstro¨m-Seppa¨ et al., 1992). Swanson et al. (1988) have studied emissions of polychlorinated dibenzo-p-dioxins and dibenzofurans from the pulp industry indicating that even the suspension of unbleached pulp entering the bleach plant can contain detectable levels of tetrachlorodibenzodioxins and -furans. The origin of these chemicals is not clear. In most cases, waste waters from pulp and paper mills undergo a multi-step purification. The fate of mutagens in this process is not fully understood. In the present study, the mutagenicity of bleached pulp mill effluents has been assessed with the Ames test using metabolic activation systems isolated from the livers of rats and fish (Ames et al., 1975; Kotelevtsev et al., 1986). These results have been compared with the genotoxicity of unbleached waste waters. Tissue extracts from fish exposed to both kind of effluents were also investigated. The effects of treated waste water on the hepatic monooxygenase enzyme activities (cytochrome P4501A) were compared to the mutagenic activity observed in fish liver.

sulphite process. The effluent undergoes both mechanical and biological treatment (vertical stabilization– horizontal stabilization–anaerobic process–activated sludge treatment with aeration–stabilization pond). The treated effluent is drained straight into Lake Kallavesi and at that point is passively diluted into the lake water to a 1% concentration. Experiments with Baikalsk Combinate waste waters have been conducted for 10 years. For this study the water samples were taken from the effluent after pulp bleaching (chlorination), from the ‘white’ and ‘black’ fluxes as well as from the aerating ponds. For studying the effects of the unbleached effluents, samples were taken from the stabilization pond and from the contaminated lake area downstream of the sewer outlet at Lake Kallavesi. For comparison, control water was taken from the northern Lake Kallavesi, upstream from the pulp mill.

2. Materials and method

2.3. Laboratory and field exposure studies

2.1. Sampling and experimental sites

Baikal grayling (Thymallus articus baikalensis; n ˆ 5 in each group) and bullhead (Cottocomephorus greminski; n ˆ 5 in each group) were exposed in a laboratory in Baikalsk, Russia, for 1 month to water taken from the aeration ponds of Baikalsk Combinate and diluted with the Lake Baikal water (5 and 20%). Immature rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory in Finland to the unbleached biotreated effluents taken from the stabilization pond. Waste water concentrations (0.25 and 0.5%) used in the experiment were chosen to resemble the dilutions of the effluent in the environment. The fish (mean weight 200 g) were kept in ponds at the Aquaculture Research Unit, University of Kuopio. The continuous water flow, into which the waste water was diluted, came from the uncontaminated upper parts of Lake Kallavesi. Liver samples were taken after the fish (n ˆ 3–8 in each group) had

Experiments were carried out using samples of waste waters from the Baikalsk Bleached Pulp Mill Combinate at Lake Baikal, Russia, and from the Savon Sellu Corporation, which produces unbleached semipulp and cellular board at Lake Kallavesi, Kuopio, Finland. Baikalsk Combinate uses conifer trees as its raw material for the sulphate process. In order to achieve adequate purification of its waste water, the Combinate effluent disposal system utilizes biological, chemical and mechanical treatment. Finally, a cascade of stabilization and aeration ponds is used. From the last pond, waste water is drained into Lake Baikal where it reaches a 5% dilution. Savon Sellu produces unbleached pulp, mainly from birch, by applying the neutral ammonium

2.2. Water sample treatment At Lake Baikal, water samples were immediately sterilized by passage through the Millipore HAWP membrane filters (pore size 0.45 mm). In samples taken after the chlorination step, free chlorine was removed immediately by vacuum. The mutagenicity of samples was tested on the same day, or the samples were stored at 48C for less than a week.

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Table 1 Analysis of mutagenic activities as His 1 revertants of bleached and unbleached pulp mill waste waters in Ames test with and without metabolic activation system from rat and grayling (Thymallus articus baikalensis). Student’s t-test, significant data are underlined …p , 0:005† Sample


Number of His 1 revertants/plate TA 98

TA 100 2MA b

1MA a Waste water Bleached (Baikalsk) After chlorination Black stream White stream Aeration pond Unbleached (Kuopio) Control Aeration pond Lake Kallavesi DMSO H2O 2-Aminoanthracene Benzo(a)pyrene a b c



0.4 ml 0.4 ml 0.4 ml 0.4 ml

25 13 24 23

24 14 18 24

247 (251 c) 140 139 115

477 125 109 139

0.4 ml 0.4 ml 0.4 ml 0.1 ml 1 ml 0.5 mg 5 mg

16 37 43 21 18 506 (431) 398 (402)

20 49 24 16 17 21 20

140 154 150 108 103 1653 (1525) 745 (870)

158 256 377 107 105 148 120

With metabolic activation (rat liver). Without metabolic activation. With metabolic activation (grayling in parenthesis).

been exposed for 3, 9, 18 and 30 days. The mortality of the exposed fish, which started at day 13, was 6.5% in 0.25% effluent, 4.2% in 0.5% effluent and 0% in controls. Rainbow trout of the same age were also caged for 3 weeks at an upstream control area, and at various locations downstream from the pulp mill at Lake Kallavesi, during summer (n ˆ 2–8 in each group) and autumn (n ˆ 7–13 in each group). The mortality during the cagings was higher (about 50% in summer and 16% in autumn) than in the laboratory experiment. 2.4. Tissue sampling for metabolic studies Fish were killed by a blow on the head, and the total weight was recorded. The liver was removed, weighed and stored in liquid nitrogen. Later the hepatic microsomal fraction for enzyme analysis was isolated as described previously (Lindstro¨m-Seppa¨ and Oikari, 1989). 2.5. Tissue sampling for Ames test Fish liver samples from the laboratory and field experiments were pooled by their exposure

concentration or location and frozen in liquid nitrogen. The samples were transported in dry ice from Finland to Russia. All tissues studied were extracted (three times) for mutagen testing using a chloroform, acetone and hexane mixture (1:1:1) as previously described (Kotelevtsev et al., 1986). 2.6. Mutagenicity assay The mutagenic capacity of waste waters and tissue extracts were assessed using a modified semi-quantitative Salmonella/microsome Ames test with and without metabolic activation systems (Ames et al., 1975). Salmonella typhimurium strains TA 98 and TA 100 were used. The waste waters were not concentrated for the assay. The microsomal fraction S9 was isolated from the liver of rats induced with the polychlorinated biphenyl mixture Sovol 54 (100 mg kg 21) for 3 days, or from the liver of fish (Baikal grayling, T. articus baikalensis) induced by injection with 3methylcholanthrene (20 mg kg 21) for 14 days. Before and after the injections, fish were kept in aquaria filled with running water from Lake Baikal. The S9 fraction was isolated as described previously (Glaser et al.,


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1990). 2-Aminoanthracene and benzo(a)pyrene were used as standard promutagens for the control of mutagenic activity after metabolic activation ( ˆ positive controls). The experiments were run three times and three plates were used for each sample in all cases. 2.7. Assays of monooxygenase activities Cytochrome P-450 related monooxygenase activities were measured with three different substrates. The benzo(a)pyrene hydroxylase activity (AHH) was assayed according to Nebert and Gelboin (1968). The activities of 7-ethoxycoumarin (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) were measured by a direct fluorometric method (Ullrich and Weber, 1972; Burke and Mayer, 1974) or spectrophotometrically (Klotz et al., 1984). Protein content was measured using the method of Lowry et al. (1951) with bovine serum albumin as standard. 2.8. Statistical data processing Student’s t-test was employed for analysis of the mutagenicity data. For biotransformation data the assumption of equal variances between different groups of rainbow trout was tested by Cochran’s Ctest. Because the degree of heterogeneity in EROD activities was significant …p , 0:05†; the data was tested with a non-parametric Kruskal–Wallis oneway analysis of variances (K–W). Thereafter the Mann–Whitney test (M–W) was employed. The data programs of SPSS x release 3.0 for VAX/VMS in VAX11/780 VMS V4.3 in the Computing Centre of the University of Kuopio, or SPSS/PC 1 release 3.1 for PC were used. 3. Results and discussion 3.1. Water samples of bleached pulp mill The results of the assessment of bleached waste water mutagenicity are shown in Table 1. After chlorination the samples exhibited mutagenic activity on S. typhimurium strain TA 100. The number of His 1 revertants per plate was 477 in the test without metabolic activation (2MA) versus 247 with metabolic activation (1MA). This suggests that the tested

samples possessed direct mutagenic activity, and the level of mutations showed a near four-fold increase compared to control samples (DMSO and water). On the contrary, in the presence of liver fraction S9, the mutagenic activity decreased. 2-Aminoanthracene and benzo(a)pyrene, used as positive controls with the assessment of the metabolic activation system from rat and fish liver, were highly mutagenic. In the waste water extracts from Baikalsk Combinate no significant toxicity has been observed (Glaser et al., 1990). The metabolic activity of the S9 fraction isolated from 3-methylcholanthrene-induced Baikal fish and the activity of liver microsomes from Sovol 54-induced rats showed similar patterns towards the standard mutagens studied (Table 1). 3.2. The 10 year follow-up study at Lake Baikal The majority of the tested samples, taken before effluents treatment at Baikalsk Combinate during the 10 year follow-up study, possessed no mutagenic activity in nonconcentrated waste waters (not illustrated). The ‘white fluxes’ revealed only a weak mutagenic activity in seven of 53 cases, of which four cases were base-substitution mutations and three were frameshift mutations. However, mutagenesis was induced by concentrating all samples of effluents to some degree (corresponding to 1 l of waste water). Mutagenic activity of the ‘black stream’ samples was recorded in five of 52 cases. Two of these samples showed mutations of both types (a base-substitution and a frameshift mutation), their efficiency varying from weak to average, and three samples showed weak frameshift mutations. All the effluent samples taken after the chlorination step exhibited a direct mutagenic effect. They induced an increase of mutation (seven-fold or less) predominantly in S. typhimurium strain TA 100 (as shown in Table 1). The highest level of mutagenic activity was recorded in the samples collected on 19 September 1985 and 9 September 1986. The multiplicity index (ratio of His 1 revertants in experiment vs. control) was seven. Focus on the effluents taken directly after the chlorination step has been shown to characterize and/or identify many of the chemical substances responsible for the mutagenic capacity of the bleached pulp mill

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effluents (Rannung et al., 1981; Holmbom et al., 1984). After chemical purification, the effluents revealed mutagenic activity in four of 49 samples. Two of these were weak frameshift mutations, and two exhibited a direct mutagenic effect of base substitution type. Biologically purified samples showed mutagenic activity only in three of 49 cases. These were weak frameshift mutations. The effluents of aerating pond possessed mutagenic activity in three of 52 cases, which were base substitutions. In all cases of observed mutagenicity in Baikalsk waste waters, the effect was direct. This has been shown earlier in studies with several pulp mills in Scandinavia and North America (Bjørseth et al., 1979; McKague et al., 1981; Rannung et al., 1981; Holmbom et al., 1984). According to Bagnasco et al. (1991) no mutagenic activity could be detected when seawater from a polluted harbour area was concentrated and tested in strains TA 98 and TA 100 with or without metabolic activation. In these studies, seawater underwent a theoretical 40 000fold concentration. The fish muscle extracts from the same area did not show any mutagenicity. Similarly, none of the studied fish liver extracts had any significant mutagenic activity. As shown here the mutagenic activity of Baikalsk waste waters almost disappeared when the effluents were purified. This could be due to mixing of different waste water streams, biological and/or chemical inactivation of labile chlorinated organic components or adsorption of mutagenic substances to lignin or lignin sludge in the course of chemical purification. Dissolved organic material (natural humic substances and pulp mill-based chlorolignins) has been shown to decrease the bioavailability of lipophilic xenobiotics (e.g. model compounds, pentachlorophenol and dehydroabietic acid) and thus to affect the fate of these compounds (Kukkonen, 1992). Ho¨glund et al. (1979) have already indicated how total pulp mill effluents showed weak but significant mutagenic effects, whereas after biological treatment there was no mutagenic activity, not even at 100 times concentration. After water sampling, there were precipitates left on some of the sterilization filters. When these precipitates were suspended in ethanol and analysed by the Ames test they did not reveal any traces of mutagenic activity.


3.3. Water samples of unbleached pulp mill The number of His 1 revertants with metabolic activation (strain TA 98) in water samples taken from downstream of the mill producing unbleached pulp was at least twice as high as that seen in the upstream control area (Table 1). The higher mutagenicity was also seen in the biologically treated effluent, before the release into the lake, with and without metabolic activation. In the case of strain TA 100, some elevation of direct mutagenicity was seen when natural lake waters from the downstream sampling point were compared to the upstream control samples. The number of His 1 revertants without metabolic activation (strain TA 100) in the aeration pond was comparable to the values seen downstream from the mill. 3.4. Tissue samples from fish exposed to bleached pulp mill effluents Biotransformation enzymes, especially cytochrome P-4501A, have been shown to respond to environmental contamination caused by, for example, polycyclic aromatic hydrocarbons, municipal waste waters or bleached pulp mill effluents (Fo¨rlin and Hansson, 1982; Payne et al., 1987; Stegeman et al., 1987; Lindstro¨m-Seppa¨ and Oikari, 1989, 1990). ECOD activity, among other hepatic monooxygenase activities (AHH, EROD; not illustrated), was elevated in Baikal grayling and bullhead after exposing the fish for 1 month in water taken from the aeration ponds of Baikalsk Combinate and diluted with Lake Baikal water (20%) (Fig. 1). The Baikalsk waste waters from aeration ponds contained inducers of the fish liver monooxygenase system, and directly mutagenic compounds. In the presence of liver fraction S9, the mutagenic activity decreased. The tissue extracts of Baikal fish did not reveal mutagenic effects when the concentration of waste water from aeration ponds was less than 20%. A weak genotoxicity of the liver extracts of grayling was registered with Salmonella strain TA 98 after exposing the fish to 20% effluents dilution for 18 days (Table 2). In these studies the effect was independent of the fish species (not illustrated). During the same period, the monooxygenase activities showed a trend for elevated values (Fig. 1). The higher or longer the exposure the higher the enzyme activity. These


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Fig. 1. 7-Ethoxycoumarin-O-deethylase activity (pmol min 21 mg protein 21) in the liver of bullhead and grayling exposed to different dilutions (0, 5 and 20%) of bleached pulp mill effluents from aeration bond for 3, 9, 18 and 30 days.

results suggest that mutagenic activity in the liver extracts of fish exposed to bleached pulp mill effluents increases as a result of metabolic activation and is related to monooxygenase activity. One explanation for the genotoxicity of the Baikalsk effluents could be residual amounts of chlorinated organic components formed at the stage of cellulose bleaching by chlorine and chlorine dioxide. 3.5. Rainbow trout and unbleached pulp mill effluents Toxic compounds can inhibit the EROD activity (Elskus et al., 1989; Gooch et al., 1989). Hepatic EROD activity in rainbow trout exposed in the laboratory to 0.25% dilution of the waste water from the mill at Lake Kallavesi (stabilization lagoon) had corresponding or lower values than the controls taken at the same day (Fig. 2). When a higher concentration was used, an increase could be seen, possibly due to the enzyme induction counteracting the inhibition. These unbleached effluents have been shown to be toxic in primary cultures of rainbow trout hepatocytes (Pesonen and Andersson, 1992). The hepatic EROD activity of rainbow trout was shown to have its lowest activity after keeping the fish in 0.25% unbleached waste water for 18 days (Fig. 2). This pollution seems to contain not only inducers, but some toxic compounds as well. Hepatic monooxygenase activities, which were detected in the fish kept in the unbleached waste water, are under simul-

taneous influence of both kind of exposers. Previously it has been established that chlorinated drinking water extracts were mutagenic in the Ames test without the microsomal activating system (Liimatainen et al., 1988). These extracts, too, contained both inhibiting and inducing substances. The sample extracts of laboratory-exposed rainbow trout possessed only direct mutagenic activity. In the presence of rat liver fraction S9 the mutagenic activity decreased. The genotoxicity of rainbow trout liver extracts, measured by the Salmonella strain TA 98, was observed when fish were kept in 0.25 or 0.5% dilution of unbleached pulp mill effluent. In 0.25% dilution the genotoxicity was evident after 9 days of exposure, but it disappeared after 18 days of exposure (Table 2). In 0.5% dilution, genotoxicity was seen when fish were exposed for only 3 days, which suggests the opposite pattern as in the case of bleached effluents. The higher the activity the lower the mutagenicity. EROD activities in rainbow trout, caged at various locations downstream from the unbleached pulp mill at Lake Kallavesi, Finland, have been shown to increase (Lindstro¨m-Seppa¨ et al., 1992). In summer, the increase was nearly three-fold at 0.8 km (significant change compared to controls), and seven-fold at 3 km from the sewer outlet. At the same time, there was no mutagenic activity in the liver extracts of rainbow trout during the summer. However, slight mutagenic activity appeared

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Table 2 Mutagenic activity in liver extracts of grayling and rainbow trout, kept in bleached pulp mill aeration pond water, and dilutions of unbleached effluents, respectively. Results are expressed as relative values (control ˆ 1). Student’s t-test, significant data are underlined …p , 0:005† Dilution of waste water

Exposure time (days)

Number of His revertants (ratio to assay control) TA 98

DMSO-control (0.1 ml) Grayling Control group

5% Waste

20% Waste

Rainbow trout Control group

0.25% Waste

0.5% Waste

a b c

TA 100

1MA a

2MA b



1.0 (42) c

1.0 (24)

1.0 (136)

1.0 (116)

0 3 9 18 30 3 9 18 30 3 9 18 30

1.0 0.9 1.2 1.0 1.0 1.0 1.1 1.2 1.1 1.0 1.2 1.5 1.6

1.1 1.1 1.4 0.8 0.9 0.9 1.4 1.3 1.4 0.9 1.4 2.1 2.2

1.2 1.0 1.3 0.9 0.9 1.4 1.3 0.9 0.8 1.3 1.2 1.4 1.3

1.0 1.2 1.4 1.1 1.2 1.2 1.1 1.2 1.0 1.2 1.3 1.5 1.4

0 3 9 18 30 3 9 18 30 3 9 18 30

1.0 1.1 1.1 0.9 0.7 1.0 1.5 1.0 1.5 1.4 0.9 1.0 0.9

1.1 0.8 1.4 1.1 1.4 0.8 2.2 1.1 1.3 2.1 1.2 1.2 1.4

0.9 0.8 0.9 0.8 0.9 0.8 0.8 0.8 1.1 0.9 0.8 1.0 0.9

0.9 0.9 0.9 0.8 1.1 0.8 0.9 1.1 0.9 0.9 1.1 1.1 0.8

With metabolic activation. Without metabolic activation. Number of His 1-revertants/plate.

in the liver extracts of caged fish at 3 and 6 km from the sewer outlet in autumn (Table 3). At both seasons the EROD activity decreased when the distance from the mill changed from 3 to 12 km. There was no clear correlation between the detected mutagenicity and EROD activities in fish exposed to unbleached pulp mill effluents; the correlation in a few cases was negative. This was contrary to the results seen in bleached effluents. This suggests the influence of chlorinated compounds in the mutagenicity formation caused by bleached effluents and the

existence of different mutagenic compounds in the case of unbleached effluents. The role of fish liver seems to be different when dealing with bleached or unbleached effluents. In the case of unbleached waste water the liver is obviously able to detoxify, at least in some cases, the already existing mutagens. According to Ho¨glund et al. (1979) the reduction of the mutagenic activity due to the presence of the metabolizing system from rat liver was seen when bleached pulp mill effluents were studied with the S. typhimurium strains TA 98 and TA 100. It was


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Table 3 Analysis of mutagenic activity in liver extracts of rainbow trout caged at one upstream control area and downstream from the pulp mill producing unbleached pulp. Results as relative values (control ˆ 1). Student’s t-test, significant data are underlined …p , 0:005† Caging season

Location (km)

His revertants (ratio to control) TA 98

DMSO-control (0.1 ml) Summer 1990 Control 0.8 d 6 12

TA 100

1MA a

2MA b



1.0 (16) c

1.0 (10)

1.0 (108)

1.0 (100)

1.0 1.1 0.8 0.7

1.0 1.2 1.0 1.0

0.9 0.9 0.9 0.8

1.2 1.2 1.1 0.9

0.6 0.8 0.9 0.9

0.9 1.7 1.6 0.9

0.8 0.8 1.0 1.0

0.7 0.9 0.8 0.9

Autumn 1990 0.8 3 6 12 a b c d

With metabolic activation. Without metabolic activation. Number of His 1-revertants/plate. Distance from the pulp mill.

suggested that organisms having comparable metabolizing systems would be able to reduce the mutagenicity of substances in such effluents and reduce their persistence in nature. However, it was shown in our studies that the fish liver is also able to increase the activity and/or amount of mutagens when exposed to bleached pulp mill effluents, although the effect of S9 mix on reducing the mutagenicity of the effluents was

the same in vitro as in the study of Ho¨glund et al. (1979). Based on the findings from the laboratory and field caging experiments, it can be concluded that at least in the case of bleached effluents, the genotoxicity in fish liver extracts was concomitant to the changes in hepatic monooxygenase activity. Tissue extracts from fish exposed to treated, bleached and unbleached

Fig. 2. 7-Ethoxyresorufin-O-deethylase activity (control ˆ 100%) in the liver of rainbow trout exposed to 0.25 and 0.5% concentrations of treated unbleached pulp mill effluents for 3, 9, 18 and 30 days compared to controls (actual control activities: 7:74 ^ 4:76; 12:72 ^ 6:75; 4:60 ^ 1:92 and 1:82 ^ 0:61 pmol min21 mg protein21 ; respectively).

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pulp mill effluents showed only slight mutagenic activity in few samples. Enzyme activity analysis as well as mutagenicity testing seem to be important biological monitoring tools for the effects of pulp and paper mill effluents on aquatic life.

Acknowledgements This study has been partly supported by National Board of Water and Environment, District Office of Kuopio and Academy of Finland, Research Council for Environmental Sciences. The authors thank Ms Riitta Vena¨la¨inen for skilful technical assistance in sample preparation.

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