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

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

S.V. Kotelevtseva,* , O.O.P. Ha¨nninenb, P.A. Lindstrom-Seppa¨b, S.E. Huuskonenb,L.I. Stepanovaa, V.M. Glasera, A.M. Beimc,✠

aBiological Department, Laboratory of Physical Chemistry of Biomembranes, Moscow State University, 119899 Moscow, Russian FederationbDepartment of Physiology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland

cInstitute 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. Mutagen-icity was assessed with the Ames test, using metabolic activation systems isolated from the liver of rats and fish. Liver extractsfrom fish caught in polluted areas, and from fish experimentally exposed to waste water, were also investigated. Pulp milleffluents taken after chlorination showed mutagenic activity. The activity diminished during the waste water treatment. Tissueextracts from fish exposed to various concentrations of treated bleached and unbleached pulp mill effluents showed only slightmutagenic activity in a few samples. In the case of bleached pulp mill effluents monooxygenase activities were elevated in thosesamples where slight mutagenicity was observed. In the case of unbleached effluents no such correlation was found.q 2000Published by Elsevier Science Ltd and AEHMS. All rights reserved.

Keywords: Fish; Cytochrome P-4501A; Induction

1. Introduction

Pulp and paper production generates copiousamounts of effluents. There have been attempts todevelop mills with closed water circulation, but sofar, effluents are released into surrounding lakes andrivers. Some stages of the pulping processes formeffluents containing toxic and mutagenic substances(Ander et al., 1977; Glaser et al., 1990).

Untreated waste waters from pulp and paper millsare multi-component mixtures containing lignin

derivatives, partial degradation products of cellulose,terpene hydrocarbons and other substances which areextracted during the cooking and bleaching of cellu-lose (McLeay, 1987). Chlorine-bleaching contami-nates the effluents with several potentially reactivechlorinated organic components (Holmbom et al.,1984; Kinae et al., 1988) including the most potentmutagen, hydroxyfuranone MX (3-chloro-4-(dichlor-omethyl)-5-hydroxy-2(5H)-furanone). Some of thesecompounds exert a mutagenic influence onSalmo-nella typhimuriumstrains (Ames et al., 1975). Manypulp mills are modifying their processes and usingless and less chlorine in bleaching. However, thereare still factories using different kinds of chlorinechemicals.

The mills producing unbleached pulp and paper donot use chlorine chemicals. However, when studying

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

1463-4988/00/$20.00q 2000 Published by Elsevier Science Ltd and AEHMS. All rights reserved.PII: S1463-4988(99)00068-8

www.elsevier.com/locate/aquech

* Corresponding author. Tel.:17-95-939-22-60; fax:17-95-939-50-22.

E-mail address:[email protected] (S.V. Kotelevtsev).✠ Sadly, Professor A.M. Beim died during the editing process for

this paper.

native fish populations or fish caged in waters influ-enced by such pulp and paper mills, it is apparent thatunbleached effluents contain some chemicals indu-cing monooxygenase enzymes as well as chemicalscausing toxicity (Lindstro¨m-Seppa¨ et al., 1992).Swanson et al. (1988) have studied emissions of poly-chlorinated dibenzo-p-dioxins and dibenzofuransfrom the pulp industry indicating that even the suspen-sion of unbleached pulp entering the bleach plant cancontain detectable levels of tetrachlorodibenzodioxinsand -furans. The origin of these chemicals is not clear.

In most cases, waste waters from pulp and papermills undergo a multi-step purification. The fate ofmutagens in this process is not fully understood.

In the present study, the mutagenicity of bleachedpulp mill effluents has been assessed with the Amestest using metabolic activation systems isolated fromthe livers of rats and fish (Ames et al., 1975;Kotelevtsev et al., 1986). These results have beencompared with the genotoxicity of unbleached wastewaters. Tissue extracts from fish exposed to both kindof effluents were also investigated. The effects oftreated waste water on the hepatic monooxygenaseenzyme activities (cytochrome P4501A) werecompared to the mutagenic activity observed in fishliver.

2. Materials and method

2.1. Sampling and experimental sites

Experiments were carried out using samples ofwaste waters from the Baikalsk Bleached Pulp MillCombinate at Lake Baikal, Russia, and from theSavon Sellu Corporation, which produces unbleachedsemipulp and cellular board at Lake Kallavesi,Kuopio, Finland.

Baikalsk Combinate uses conifer trees as its rawmaterial for the sulphate process. In order to achieveadequate purification of its waste water, the Com-binate effluent disposal system utilizes biological,chemical and mechanical treatment. Finally, acascade of stabilization and aeration ponds is used.From the last pond, waste water is drained into LakeBaikal where it reaches a 5% dilution.

Savon Sellu produces unbleached pulp, mainlyfrom birch, by applying the neutral ammonium

sulphite process. The effluent undergoes both mechan-ical and biological treatment (vertical stabilization–horizontal stabilization–anaerobic process–activatedsludge treatment with aeration–stabilization pond).The treated effluent is drained straight into LakeKallavesi and at that point is passively diluted intothe lake water to a 1% concentration.

Experiments with Baikalsk Combinate wastewaters have been conducted for 10 years. For thisstudy the water samples were taken from the effluentafter 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 andfrom the contaminated lake area downstream of thesewer outlet at Lake Kallavesi. For comparison,control water was taken from the northern LakeKallavesi, upstream from the pulp mill.

2.2. Water sample treatment

At Lake Baikal, water samples were immediatelysterilized by passage through the Millipore HAWPmembrane filters (pore size 0.45mm). In samplestaken after the chlorination step, free chlorine wasremoved immediately by vacuum. The mutagenicityof samples was tested on the same day, or the sampleswere stored at 48C for less than a week.

2.3. Laboratory and field exposure studies

Baikal grayling (Thymallus articus baikalensis;n� 5 in each group) and bullhead (Cottocomephorusgreminski; n� 5 in each group) were exposed in alaboratory in Baikalsk, Russia, for 1 month to watertaken from the aeration ponds of Baikalsk Combinateand diluted with the Lake Baikal water (5 and 20%).

Immature rainbow trout (Oncorhynchus mykiss)were exposed in the laboratory in Finland to theunbleached biotreated effluents taken from the stabi-lization pond. Waste water concentrations (0.25 and0.5%) used in the experiment were chosen to resemblethe dilutions of the effluent in the environment. Thefish (mean weight 200 g) were kept in ponds at theAquaculture Research Unit, University of Kuopio.The continuous water flow, into which the wastewater was diluted, came from the uncontaminatedupper parts of Lake Kallavesi. Liver samples weretaken after the fish (n� 3–8 in each group) had

S.V. Kotelevtsev et al. / Aquatic Ecosystem Health and Management 3 (2000) 95–10496

been exposed for 3, 9, 18 and 30 days. The mortalityof the exposed fish, which started at day 13, was 6.5%in 0.25% effluent, 4.2% in 0.5% effluent and 0% incontrols. Rainbow trout of the same age were alsocaged for 3 weeks at an upstream control area, andat various locations downstream from the pulp millat Lake Kallavesi, during summer (n� 2–8 in eachgroup) and autumn (n� 7–13 in each group). Themortality during the cagings was higher (about 50%in summer and 16% in autumn) than in the laboratoryexperiment.

2.4. Tissue sampling for metabolic studies

Fish were killed by a blow on the head, and the totalweight was recorded. The liver was removed, weighedand stored in liquid nitrogen. Later the hepatic micro-somal fraction for enzyme analysis was isolated asdescribed previously (Lindstro¨m-Seppa¨ and Oikari,1989).

2.5. Tissue sampling for Ames test

Fish liver samples from the laboratory and fieldexperiments were pooled by their exposure

concentration or location and frozen in liquidnitrogen. The samples were transported in dry icefrom Finland to Russia. All tissues studied wereextracted (three times) for mutagen testing using achloroform, acetone and hexane mixture (1:1:1) aspreviously described (Kotelevtsev et al., 1986).

2.6. Mutagenicity assay

The mutagenic capacity of waste waters and tissueextracts were assessed using a modified semi-quanti-tative Salmonella/microsome Ames test with andwithout metabolic activation systems (Ames et al.,1975). Salmonella typhimuriumstrains TA 98 andTA 100 were used. The waste waters were not concen-trated for the assay. The microsomal fraction S9 wasisolated from the liver of rats induced with the poly-chlorinated biphenyl mixture Sovol 54 (100 mg kg21)for 3 days, or from the liver of fish (Baikal grayling,T.articus baikalensis) induced by injection with 3-methylcholanthrene (20 mg kg21) for 14 days. Beforeand after the injections, fish were kept in aquaria filledwith running water from Lake Baikal. The S9 fractionwas isolated as described previously (Glaser et al.,

S.V. Kotelevtsev et al. / Aquatic Ecosystem Health and Management 3 (2000) 95–104 97

Table 1Analysis of mutagenic activities as His1 revertants of bleached and unbleached pulp mill waste waters in Ames test with and without metabolicactivation system from rat and grayling (Thymallus articus baikalensis). Student’st-test, significant data are underlined�p , 0:005�

Sample Dose Number of His1 revertants/plate

TA 98 TA 100

1MA a 2MA b 1MA 2MA

Waste waterBleached(Baikalsk)After chlorination 0.4 ml 25 24 247 (251c) 477Black stream 0.4 ml 13 14 140 125White stream 0.4 ml 24 18 139 109Aeration pond 0.4 ml 23 24 115 139Unbleached(Kuopio)Control 0.4 ml 16 20 140 158Aeration pond 0.4 ml 37 49 154 256Lake Kallavesi 0.4 ml 43 24 150 377DMSO 0.1 ml 21 16 108 107H2O 1 ml 18 17 103 1052-Aminoanthracene 0.5mg 506 (431) 21 1653 (1525) 148Benzo(a)pyrene 5mg 398 (402) 20 745 (870) 120

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

1990). 2-Aminoanthracene and benzo(a)pyrene wereused as standard promutagens for the control ofmutagenic activity after metabolic activation(� positive controls). The experiments were runthree times and three plates were used for each samplein all cases.

2.7. Assays of monooxygenase activities

Cytochrome P-450 related monooxygenase activ-ities 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) weremeasured by a direct fluorometric method (Ullrichand Weber, 1972; Burke and Mayer, 1974) or spectro-photometrically (Klotz et al., 1984). Protein contentwas measured using the method of Lowry et al. (1951)with bovine serum albumin as standard.

2.8. Statistical data processing

Student’st-test was employed for analysis of themutagenicity data. For biotransformation data theassumption of equal variances between differentgroups of rainbow trout was tested by Cochran’sC-test. Because the degree of heterogeneity in ERODactivities was significant�p , 0:05�; the data wastested with a non-parametric Kruskal–Wallis one-way analysis of variances (K–W). Thereafter theMann–Whitney test (M–W) was employed. Thedata programs of SPSSx release 3.0 for VAX/VMSin VAX11/780 VMS V4.3 in the Computing Centreof the University of Kuopio, or SPSS/PC1 release3.1 for PC were used.

3. Results and discussion

3.1. Water samples of bleached pulp mill

The results of the assessment of bleached wastewater mutagenicity are shown in Table 1. After chlor-ination the samples exhibited mutagenic activity onS.typhimurium strain TA 100. The number of His1

revertants per plate was 477 in the test without meta-bolic activation (2MA) versus 247 with metabolicactivation (1MA). This suggests that the tested

samples possessed direct mutagenic activity, and thelevel of mutations showed a near four-fold increasecompared to control samples (DMSO and water). Onthe contrary, in the presence of liver fraction S9, themutagenic activity decreased. 2-Aminoanthraceneand benzo(a)pyrene, used as positive controls withthe assessment of the metabolic activation systemfrom rat and fish liver, were highly mutagenic. Inthe waste water extracts from Baikalsk Combinateno significant toxicity has been observed (Glaser etal., 1990). The metabolic activity of the S9 fractionisolated from 3-methylcholanthrene-induced Baikalfish and the activity of liver microsomes fromSovol 54-induced rats showed similar patternstowards the standard mutagens studied (Table 1).

3.2. The 10 year follow-up study at Lake Baikal

The majority of the tested samples, taken beforeeffluents treatment at Baikalsk Combinate during the10 year follow-up study, possessed no mutagenicactivity in nonconcentrated waste waters (not illu-strated). The ‘white fluxes’ revealed only a weakmutagenic activity in seven of 53 cases, of whichfour cases were base-substitution mutations andthree were frameshift mutations. However, mutagen-esis was induced by concentrating all samples of efflu-ents to some degree (corresponding to 1 l of wastewater).

Mutagenic activity of the ‘black stream’ sampleswas recorded in five of 52 cases. Two of these samplesshowed mutations of both types (a base-substitutionand a frameshift mutation), their efficiency varyingfrom weak to average, and three samples showedweak frameshift mutations.

All the effluent samples taken after the chlorinationstep exhibited a direct mutagenic effect. They inducedan increase of mutation (seven-fold or less) predomi-nantly in S. typhimuriumstrain TA 100 (as shown inTable 1). The highest level of mutagenic activity wasrecorded in the samples collected on 19 September1985 and 9 September 1986. The multiplicity index(ratio of His1 revertants in experiment vs. control)was seven.

Focus on the effluents taken directly after the chlor-ination step has been shown to characterize and/oridentify many of the chemical substances responsiblefor the mutagenic capacity of the bleached pulp mill


effluents (Rannung et al., 1981; Holmbom et al.,1984).

After chemical purification, the effluents revealedmutagenic activity in four of 49 samples. Two of thesewere weak frameshift mutations, and two exhibited adirect mutagenic effect of base substitution type.Biologically purified samples showed mutagenicactivity only in three of 49 cases. These were weakframeshift mutations. The effluents of aerating pondpossessed mutagenic activity in three of 52 cases,which were base substitutions.

In all cases of observed mutagenicity in Baikalskwaste waters, the effect was direct. This has beenshown earlier in studies with several pulp mills inScandinavia and North America (Bjørseth et al.,1979; McKague et al., 1981; Rannung et al., 1981;Holmbom et al., 1984). According to Bagnasco etal. (1991) no mutagenic activity could be detectedwhen seawater from a polluted harbour area wasconcentrated and tested in strains TA 98 and TA100 with or without metabolic activation. In thesestudies, seawater underwent a theoretical 40 000-fold concentration. The fish muscle extracts fromthe same area did not show any mutagenicity. Simi-larly, none of the studied fish liver extracts had anysignificant mutagenic activity.

As shown here the mutagenic activity of Baikalskwaste waters almost disappeared when the effluentswere purified. This could be due to mixing of differentwaste water streams, biological and/or chemical inac-tivation of labile chlorinated organic components oradsorption of mutagenic substances to lignin or ligninsludge in the course of chemical purification.Dissolved organic material (natural humic substancesand pulp mill-based chlorolignins) has been shown todecrease the bioavailability of lipophilic xenobiotics(e.g. model compounds, pentachlorophenol anddehydroabietic acid) and thus to affect the fate ofthese compounds (Kukkonen, 1992). Ho¨glund et al.(1979) have already indicated how total pulp milleffluents showed weak but significant mutageniceffects, whereas after biological treatment there was nomutagenic activity, not even at 100 times concentration.

After water sampling, there were precipitates lefton some of the sterilization filters. When these pre-cipitates were suspended in ethanol and analysed bythe Ames test they did not reveal any traces of muta-genic activity.

3.3. Water samples of unbleached pulp mill

The number of His1 revertants with metabolic acti-vation (strain TA 98) in water samples taken fromdownstream of the mill producing unbleached pulpwas at least twice as high as that seen in the upstreamcontrol area (Table 1). The higher mutagenicity wasalso seen in the biologically treated effluent, beforethe release into the lake, with and without metabolicactivation. In the case of strain TA 100, some eleva-tion of direct mutagenicity was seen when natural lakewaters from the downstream sampling point werecompared to the upstream control samples. Thenumber of His1 revertants without metabolic activa-tion (strain TA 100) in the aeration pond was compar-able to the values seen downstream from the mill.

3.4. Tissue samples from fish exposed to bleached pulpmill effluents

Biotransformation enzymes, especially cytochromeP-4501A, have been shown to respond to environ-mental contamination caused by, for example, poly-cyclic aromatic hydrocarbons, municipal waste watersor bleached pulp mill effluents (Fo¨rlin and Hansson,1982; Payne et al., 1987; Stegeman et al., 1987; Lind-strom-Seppa¨ and Oikari, 1989, 1990). ECOD activity,among other hepatic monooxygenase activities(AHH, EROD; not illustrated), was elevated in Baikalgrayling and bullhead after exposing the fish for1 month in water taken from the aeration ponds ofBaikalsk Combinate and diluted with Lake Baikalwater (20%) (Fig. 1).

The Baikalsk waste waters from aeration pondscontained inducers of the fish liver monooxygenasesystem, and directly mutagenic compounds. In thepresence of liver fraction S9, the mutagenic activitydecreased. The tissue extracts of Baikal fish did notreveal mutagenic effects when the concentration ofwaste water from aeration ponds was less than 20%.A weak genotoxicity of the liver extracts of graylingwas registered withSalmonellastrain TA 98 afterexposing the fish to 20% effluents dilution for18 days (Table 2). In these studies the effect was inde-pendent of the fish species (not illustrated). During thesame period, the monooxygenase activities showed atrend for elevated values (Fig. 1). The higher or longerthe exposure the higher the enzyme activity. These


results suggest that mutagenic activity in the liverextracts of fish exposed to bleached pulp mill effluentsincreases as a result of metabolic activation and isrelated to monooxygenase activity. One explanationfor the genotoxicity of the Baikalsk effluents could beresidual amounts of chlorinated organic componentsformed at the stage of cellulose bleaching by chlorineand 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). HepaticEROD activity in rainbow trout exposed in the labora-tory to 0.25% dilution of the waste water from the millat Lake Kallavesi (stabilization lagoon) had corre-sponding or lower values than the controls taken atthe same day (Fig. 2). When a higher concentrationwas used, an increase could be seen, possibly due tothe enzyme induction counteracting the inhibition.These unbleached effluents have been shown to betoxic in primary cultures of rainbow trout hepatocytes(Pesonen and Andersson, 1992).

The hepatic EROD activity of rainbow trout wasshown to have its lowest activity after keeping the fishin 0.25% unbleached waste water for 18 days (Fig. 2).This pollution seems to contain not only inducers, butsome toxic compounds as well. Hepatic monooxy-genase activities, which were detected in the fishkept in the unbleached waste water, are under simul-

taneous influence of both kind of exposers. Previouslyit has been established that chlorinated drinking waterextracts were mutagenic in the Ames test without themicrosomal activating system (Liimatainen et al.,1988). These extracts, too, contained both inhibitingand inducing substances.

The sample extracts of laboratory-exposed rainbowtrout possessed only direct mutagenic activity. In thepresence of rat liver fraction S9 the mutagenic activitydecreased. The genotoxicity of rainbow trout liverextracts, measured by theSalmonellastrain 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 ofexposure, but it disappeared after 18 days of exposure(Table 2). In 0.5% dilution, genotoxicity was seenwhen fish were exposed for only 3 days, whichsuggests the opposite pattern as in the case ofbleached effluents. The higher the activity the lowerthe mutagenicity.

EROD activities in rainbow trout, caged at variouslocations downstream from the unbleached pulp millat Lake Kallavesi, Finland, have been shown toincrease (Lindstro¨m-Seppa¨ et al., 1992). In summer,the increase was nearly three-fold at 0.8 km (signifi-cant change compared to controls), and seven-fold at3 km from the sewer outlet.

At the same time, there was no mutagenic activityin the liver extracts of rainbow trout during thesummer. However, slight mutagenic activity appeared


Fig. 1. 7-Ethoxycoumarin-O-deethylase activity (pmol min21 mg protein21) 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.

in the liver extracts of caged fish at 3 and 6 km fromthe sewer outlet in autumn (Table 3). At both seasonsthe EROD activity decreased when the distance fromthe mill changed from 3 to 12 km.

There was no clear correlation between the detectedmutagenicity and EROD activities in fish exposed tounbleached pulp mill effluents; the correlation in afew cases was negative. This was contrary to theresults seen in bleached effluents. This suggests theinfluence of chlorinated compounds in the mutageni-city formation caused by bleached effluents and the

existence of different mutagenic compounds in thecase of unbleached effluents. The role of fish liverseems to be different when dealing with bleached orunbleached effluents. In the case of unbleached wastewater the liver is obviously able to detoxify, at least insome cases, the already existing mutagens.

According to Hoglund et al. (1979) the reduction ofthe mutagenic activity due to the presence of the meta-bolizing system from rat liver was seen whenbleached pulp mill effluents were studied with theS.typhimurium strains TA 98 and TA 100. It was


Table 2Mutagenic activity in liver extracts of grayling and rainbow trout, kept in bleached pulp mill aeration pond water, and dilutions of unbleachedeffluents, respectively. Results are expressed as relative values (control� 1). Student’st-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 TA 100

1MA a 2MA b 1MA 2MA

DMSO-control (0.1 ml) 1.0 (42)c 1.0 (24) 1.0 (136) 1.0 (116)GraylingControl group 0 1.0 1.1 1.2 1.0

3 0.9 1.1 1.0 1.29 1.2 1.4 1.3 1.4

18 1.0 0.8 0.9 1.130 1.0 0.9 0.9 1.2

5% Waste 3 1.0 0.9 1.4 1.29 1.1 1.4 1.3 1.1

18 1.2 1.3 0.9 1.230 1.1 1.4 0.8 1.0

20% Waste 3 1.0 0.9 1.3 1.29 1.2 1.4 1.2 1.3

18 1.5 2.1 1.4 1.530 1.6 2.2 1.3 1.4

Rainbow troutControl group 0 1.0 1.1 0.9 0.9

3 1.1 0.8 0.8 0.99 1.1 1.4 0.9 0.9

18 0.9 1.1 0.8 0.830 0.7 1.4 0.9 1.1

0.25% Waste 3 1.0 0.8 0.8 0.89 1.5 2.2 0.8 0.9

18 1.0 1.1 0.8 1.130 1.5 1.3 1.1 0.9

0.5% Waste 3 1.4 2.1 0.9 0.99 0.9 1.2 0.8 1.1

18 1.0 1.2 1.0 1.130 0.9 1.4 0.9 0.8

a With metabolic activation.b Without metabolic activation.c Number of His1-revertants/plate.

suggested that organisms having comparable metabo-lizing systems would be able to reduce the mutageni-city of substances in such effluents and reduce theirpersistence in nature. However, it was shown in ourstudies that the fish liver is also able to increase theactivity and/or amount of mutagens when exposed tobleached pulp mill effluents, although the effect of S9mix 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 fieldcaging experiments, it can be concluded that at least inthe case of bleached effluents, the genotoxicity in fishliver extracts was concomitant to the changes inhepatic monooxygenase activity. Tissue extractsfrom fish exposed to treated, bleached and unbleached


Table 3Analysis of mutagenic activity in liver extracts of rainbow trout caged at one upstream control area and downstream from the pulp millproducing unbleached pulp. Results as relative values (control� 1). Student’st-test, significant data are underlined�p , 0:005�

Caging season Location (km) His revertants (ratio to control)

TA 98 TA 100

1MA a 2MA b 1MA 2MA

DMSO-control (0.1 ml) 1.0 (16)c 1.0 (10) 1.0 (108) 1.0 (100)Summer 1990

Control 1.0 1.0 0.9 1.20.8d 1.1 1.2 0.9 1.26 0.8 1.0 0.9 1.1

12 0.7 1.0 0.8 0.9Autumn 1990

0.8 0.6 0.9 0.8 0.73 0.8 1.7 0.8 0.96 0.9 1.6 1.0 0.8

12 0.9 0.9 1.0 0.9

a With metabolic activation.b Without metabolic activation.c Number of His1-revertants/plate.d Distance from the pulp mill.

Fig. 2. 7-Ethoxyresorufin-O-deethylase activity (control� 100%) in the liver of rainbow trout exposed to 0.25 and 0.5% concentrations oftreated 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).

pulp mill effluents showed only slight mutagenicactivity in few samples. Enzyme activity analysis aswell as mutagenicity testing seem to be importantbiological monitoring tools for the effects of pulpand paper mill effluents on aquatic life.

Acknowledgements

This study has been partly supported by NationalBoard of Water and Environment, District Office ofKuopio and Academy of Finland, Research Councilfor Environmental Sciences. The authors thank MsRiitta Venalainen for skilful technical assistance insample preparation.

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