Monooxygenase activities in the liver of fish living in Lake Baikaland after treatment with waste waters from the Baikalsk Pulp and

Paper Mill

S.V. Kotelevtseva, L.I. Stepanovaa, P. Lindstrom-Seppa¨b,* , O. Hanninenb

aDepartment of Biology, Moscow State University, 119899 Moscow, RussiabDepartment of Physiology, University of Kuopio, P.O. Box. 1627, FIN-70211 Kuopio, Finland

Abstract

The biological effects of the waste waters released by Baikalsk Pulp and Paper Mill were studied in fish collected from LakeBaikal. Fish were also exposed in laboratory conditions to known inducers of hepatic monooxygenase activities. We did not findany differences in monooxygenase activities of grayling and sculpins caught near the pulp and paper mill compared to controlfish caught in the middle part of Lake Baikal. The results showed that the waste water treatment in the mill is effective. Usuallythe treated waste water enters the lake diluted up to 5% concentration. However, only at 20% concentration did the treated wastewater induce hepatic monooxygenase activities in the fish studied in laboratory conditions.q 2000 Elsevier Science Ltd andAEHMS. All rights reserved.

Keywords: Bleached kraft; Grayling; Sculpin

1. Introduction

Baikal, world’s deepest (1620 m) and one of thelargest and most ancient lakes (more than 20 millionyears) stretches 636 km long and up to 80 km wideamidst mountains on the north-eastern borders ofCentral Asia (Fig. 1). It is the deepest continentaldepression on our planet. Today Lake Baikal isknown to have more than 1200 species of animalsand up to 500 species of plants, two-thirds of whichare endemic. Among the 842 described animal speciesinhabiting the waters of the lake, 82% are not found

anywhere else. There are 87 endemic genera and 11endemic families and subfamilies in Baikal (Kozhovaand Beim, 1993).

There are no large industrial or agricultural enter-prises at Lake Baikal or its tributary rivers, with theexception of two pulp and paper plants: SelenginskyPulp and Paper Factory and the Baikalsk Pulp andPaper Mill (BPPM). The first plant now uses closedwater circulation, and the second has powerful watercleaning systems. The anthropogenic impacts on thelake are increasing however, and there are indicationsof an increase in the concentration of certain pollu-tants in seal tissues (Kucklick et al., 1996). Pollutantsenter Lake Baikal via rivers, and originate from themelting of snow and from municipal waste waters(e.g. Severobaikalsk town has no municipal watertreatment systems). Air pollution comes from plants

Aquatic Ecosystem Health and Management 3 (2000) 271–276

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* Corresponding author. Tel.:1358-17-163-095; fax:1358-17-163-112.

E-mail address:pirjo.lindstromseppa@uku.fi (P. Lindstro¨m-Seppa¨).

of the Irkutsk region and even from China. Suchpollution presents hazards to the aquatic ecosystemsof Lake Baikal.

Chemical pollutants can cause reductions in animalsurvival and also disturb functions at molecular,cellular, individual and population levels. Thisdamage may result in decreased growth and reproduc-tion, which can lead to population decline. Theproblems of preservation of biodiversity in LakeBaikal can be solved only by adopting a fundamentalecotoxicological approach, combining the use ofbiomarker strategies together with population studies.

Chemical analyses of pollutants in animal and plant

tissues are desirable but not available in all cases:sometimes we do not even know the class of thechemical substances which should be controlled.Concentrations which can affect biological systems,are so low in many cases that they are below thedetection limits of the methods used. As well, manychemical substances are so unstable that it is impos-sible to detect them after they have acted on biologicaltargets. Only biological test systems can show themechanisms of pollutant actions, clarify the effectsof chemicals on enzymes and show the mutagenicand carcinogenic properties of pollutants.

Biochemical test systems are often based on

S.V. Kotelevtsev et al. / Aquatic Ecosystem Health and Management 3 (2000) 271–276272

Fig. 1. Lake Baikal.

biological membranes that are targets for many xeno-biotics. Such systems (e.g. membranes of livercells) not only accumulate pollutants, but alsodetoxify and metabolically activate xenobiotics.The pollutants can induce the activity of certainmembrane enzymes (e.g. cytochrome P-450) (Royand Hanninen, 1994).

Induction of monooxygenase activities in fish liverdepends on the species. Its determination is possibleafter longer periods of time, and it may give informa-tion about animal contacts with pollutants even afterthe xenobiotics have been metabolized. The wastewater of pulp and paper mills can effectively inducethe isoforms of cytochrome P-450 (Andersson et al.,1987; Lindstrom-Seppa¨ and Oikari, 1990; Soimasuoet al., 1995).

We have studied the level of cytochrome P-450 andmonooxygenase activities in livers of Baikal fishliving in normal conditions and after laboratory expo-sure to BPPM waste waters.

2. Materials and methods

Biochemical monitoring of the waste watersreleased by the BPPM and of the southern partof Lake Baikal was carried out during 10 years(1982–1992) to obtain baseline measurements ofhepatic monooxygenase activities in Lake Baikalfish.

About 3-year old male fish of the following specieswere used for monitoring experiments: arctic cisco(omul, Coregonus autumnalis migratorius), grayling

(Thymallus arcticus baikalensis) and endemic scul-pins (Cottocomephorus greminskiiand Cottocome-phorus inermis). The study sites were: Maloe More(Olchon), Tanhoy, Vydrino, Bolschie Koty and threeareas near the town of Baikalsk (Fig. 1). Fishwere caught with a sweep-net. Fish livers wereperfused in situ with buffer (150 mM KCl), cutfree and fixed in liquid nitrogen. Alternatively,animals were delivered to the laboratory in aspecially designed boat supplied with tanks filledwith flowing water from the lake.

Baikal fish were exposed to the BPPM waste waterfrom the aeration pond in different concentrations inthe laboratory conditions. Throughout the laboratoryexperiments the fishes were kept in aquariums withaerated, filtered and recirculated Lake Baikal water(10–148C). For positive controls, some of the fishwere exposed to known inducers of monooxygenases,for example, 3-methylcholanthrene (MC),b-naphtho-flavone (BNF) and Sovol 54 (Russian equivalent forArochlor 1254) (AC). There were 10 fish in eachexperimental group.

The microsomal fraction was obtained as describedearlier (Kotelevtsev et al., 1986). The cytochrome P-450 content as well as the monooxygenase activities(benzo(a)pyrene hydroxylase� AHH, 7-ethoxycou-marin O-deethylase� ECOD, 7-ethoxyresorufin O-deethylase� EROD) were measured using standardmethods (Ullrich and Weber, 1972; Johannesen andde Pierre, 1978; Prough et al., 1978).

Statistical validity was determined by Student’st-test using the Stat Graf Program (Statistical GraphicsCo., USA).

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Table 1Hepatic cytochrome P-450 and b5 content in Baikal fish (nmol mg protein21, mean SD,n� 10�: MC denotes methylcholanthrene; * denotessignificance atp , 0:05

Cyt. P-450 (control) Cyt. P-4501 MC (20 mg kg21) Cyt. b5 (control)

Arctic cisco (Coregonusautumnalis migratorius)

0:37^ 0:07 0:77^ 0:11* 0:42^ 0:05

Grayling (Thymallusarcticus baikalensis)

0:32^ 0:07 0:67^ 0:09* 0:40^ 0:06

Sculpin(Cottocomephorusgreminskii)

0:12^ 0:03 0:29^ 0:05* 0:21^ 0:05

Sculpin(Cottocomephorusinermis)

0:11^ 0:02 0:32^ 0:05* 0:22^ 0:04

3. Results and discussion

Cytochrome P-450 and cytochrome b5 contents andmonooxygenase activities were very low in LakeBaikal endemic sculpins (Cottocomephorusgreminskii and Cottocomephorus inermis; Tables 1and 2). These species, from a phylogenetic point ofview, are ancient animals (Grachev et al., 1992). Like-wise, other Lake Baikal fish species possessed littleECOD or EROD activities when measured withoutthe preliminary induction of the monooxygenases

(Table 2, Fig. 2). The injection of inducers (MC,BNF, AC) caused an increase in the hepatic monooxy-genase activities of all fish studied (Figs. 2 and 3).When fish were exposed to 5% BPPM effluent (asthe final diluted water entering Lake Baikal) inductionof the monooxygenase system was not detected (Table3, Fig. 2). However, induction was observed if the fish(grayling or sculpin) were kept at least three weeks in20% BPPM waste water (Figs. 2 and 3; Table 3).

A long-term (10-year) comparative analysis of thehepatic monooxygenase activities of all fish species,

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Table 2Hepatic 7-ethoxycoumarin O-deethylase (ECOD) in control and 3-methylcholanthrene (MC) treated Baikal fish (pmol min21 mg protein21,mean SD, n� 10�; *denotes significance atp , 0:05

Species ECOD (control) ECOD1MC (20 mg kg21)

Arctic cisco (Coregonusautumnalis migratorius)

2:91^ 0:12 27:7^ 1:01*

Grayling (Thymallusarcticus baikalensis)

2:33^ 0:17 31:20^ 1:29*

Sculpin(Cottocomephorusgreminskii)

0:91^ 0:08 21:23^ 1:45*

Sculpin(Cottocomephorusinermis)

0:82^ 0:09 22:12^ 1:55*

Fig. 2. 7-ethoxycoumarin O-deethylase (ECOD) activity (pmol min21 mg protein21) in the liver microsomes of Lake Baikal fish. 1. Sculpin(Cottocomephorus greminskii), caught near Baikalsk Pulp and Paper Mill (BPPM), 2. Sculpin (Cottocomephorus greminskii), caught nearOlchon Island, 3. Grayling (Tymallus arcticus baikalensis) caught near BPPM, 4. Grayling (Tymallus arcticus baikalensis) caught near OlchonIsland (1982–1992, August) under normal conditions and after 3-methylcholanthrene (MC) or BPPM waste water exposure in laboratory(I � 5% waste water, II� 20% waste water, 3-week exposure).

covering different parts of Lake Baikal (BPPMdischarge areas as well as unpolluted areas) failed toreveal any changes in ECOD or EROD activitieswhen the measurements were performed at the sametime of the year in fish of the same age and sex (Fig.2). The concentrations of the cytochrome P-4501A1inducers in Lake Baikal water have been very low,and no elevation of hepatic monooxygenase activitiesin fish was detected.

To assess the possible influence of BPPM on

hepatic monooxygenase activities ofCottus kessleriandParacottus kneri, two sculpins endemic to LakeBaikal, were earlier investigated by Lindstro¨m-Seppa¨and Oikari (1990). No effects on monooxygenaseactivities were observed in this study.

4. Conclusions

The results indicated that the livers of studied Lake

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Fig. 3. Monooxygenase activities (benzo(a)pyrene hydroxylase, AHH; pmol min21 mg protein21) in grayling liver microsomes after 3-methylcholanthrene injection (20 mg kg21) or 20% BPPM waste water treatment.

Table 37-ethoxycoumarin O-deethylase activity in liver microsomes of sculpin and grayling exposed to different percentages of BPPM waste waterstreated for 3, 9, 18 and 30 days (pmol min21 mg protein21, mean SD, n� number of replicates); *denotes significance atp , 0:05

Species/days 0% 5% 20%

Sculpin0 2:2^ 0:6 �n� 7� 2:5^ 0:6 �n� 7� 2:6^ 0:4 �n� 7�*3 2:3^ 0:7 �n� 7� 2:1^ 0:6 �n� 7� 2:2^ 0:5 �n� 7�9 2:2^ 0:5 �n� 7� 2:1^ 0:6 �n� 7� 2:1^ 0:8 �n� 7�18 2:1^ 0:8 �n� 6� 2:3^ 0:7 �n� 7� 3:2^ 0:8 �n� 6�*30 2:2^ 0:8 �n� 7� 2:2^ 0:7 �n� 7� 5:1^ 1:0 �n� 6�*Grayling0 3:9^ 1:1 �n� 7� 4:4^ 1:0 �n� 7� 4:5^ 0:9 �n� 7�3 4:1^ 1:0 �n� 7� 4:2^ 0:9 �n� 7� 4:1^ 0:9 �n� 7�9 3:9^ 1:1 �n� 7� 4:1^ 1:0 �n� 7� 4:4^ 1:0 �n� 6�18 4:0^ 0:9 �n� 7� 3:9^ 1:0 �n� 5� 4:9^ 1:1 �n� 6�*30 4:1^ 1:0 �n� 7� 4:1^ 1:0 �n� 6� 8:2^ 2:0 �n� 6�*

Baikal fish possessed only low monooxygenase activ-ities, although these activities were effectivelyinduced in laboratory conditions. This was the caseespecially in Lake Baikal endemic sculpins. Theseanimals do not migrate, and they can be used as indi-cators in biochemical monitoring. Moreover, we didnot find an induction of the monooxygenase activitiesin the livers of fish that were caught in areas whereBPPM waste waters are released.

Acknowledgements

This study was supported by European ScienceFoundation and INTAS (Ref. No. 94-0531). Wethank Prof. Colin Walker for his help in checkingthe language.

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