Lake Baikal: biomonitoring of pulp and paper mill waste water

Aquatic Ecosystem Health and Management 3 (2000) 259–269 www.elsevier.com/locate/aquech

Lake Baikal: biomonitoring of pulp and paper mill waste water L.I. Stepanova a, P. Lindstro¨m-Seppa¨ b,*, O.O.P. Ha¨nninen b, S.V. Kotelevtsev a, V.M. Glaser a, C.N. Novikov a, A.M. Beim c b

a Department of Biology, Moscow State University, 119899 Moscow, Russia Department of Physiology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland c Institute of Ecological Toxicology, P.O. Box 48, 665914 Baikalsk, Russia

Abstract The quality and biological effects of the waste waters released by Baikalsk Pulp and Paper Mill were monitored chemically and biologically. In 1:5 dilution, the treated waste water induced monooxygenase activity in the liver of grayling and bullhead in standardised laboratory conditions. The Ames test was used to monitor mutagenicity of waste waters of the mill and tissue extracts of fish, sponge, mollusc and plankton collected near the mill and of seals collected from the middle part of the lake. Mutagens were found in waste water produced during pulp chlorination before treatment and in a few samples of fully treated waste water during 10 years (1982–1993). Bottom sediments near the mill showed some mutagenicity. Mutagens were not found in tissues of most aquatic animals studied with the exception of zooplankton, roach and seals collected in Lake Baikal close to the discharge outlet of the mill. 䉷 2000 Published by Elsevier Science Ltd. Keywords: P-450 induction; Mutagenicity; Baikalsk Pulp and Paper Mill

1. Introduction Owing to increasing anthropogenic impacts, there is serious national and international concern about the state and fate of the unique Lake Baikal ecosystem. Lake Baikal is not only the deepest lake in the world, but it has also been one of the cleanest. It is a unique fresh water resource (Kozhova and Beim, 1993). There is little agricultural and municipal pollution in the lake, but atmospheric long-range movement of pollutants is a threat. There are two cellulose mills potentially polluting the lake. One of them, situated upstream on the Selenga River, uses a recirculated waste water system to minimise pollution. The second mill, the Baikalsk * Corresponding author. E-mail address: [email protected] (P. Lindstro¨mSeppa¨). 1463-4988/00/$20.00 䉷 2000 Published by Elsevier Science Ltd. PII: S1463-498 8(00)00011-7

Pulp and Paper Mill (BPPM), located on the lake is one of the biggest mills in Russia. It produces chlorine bleached kraft cellulose, and it releases the waste water to the lake after treatment. The Soviet and the Russian Governments, under pressure of public opinion, planned to close this unit by 1990, but for economic reasons it is still in operation. Effluents of chlorine bleached kraft pulp mills, together with paper mill discharges, have been a common environmental contamination problem. These effluents contain a wide variety of harmful substances (e.g., resin acids and chlorinated phenolics) appearing in lethal or sublethal concentrations in waters in the vicinity of the sewer outlet (McLeay and Brown, 1975; Hutchins, 1979; Oikari and Holmbom, 1986). The presence of mutagenic and teratogenic compounds in waste waters of pulp and paper mills is well known (Swansson et al., 1988). Chlorinated xenobiotics have been found in tissues

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6 Fig. 1. The scheme of the cleaning of sewage waters, I. The Baikalsk Pulp and Paper Mill, II. The reservoirs of sewage waters, III. The aerotanks for biological treatment, IV. The packing down (settling) of active silt (f 24 m), V. The reagent chamber of chemical treatment of sewage waters, VI. The settling of shlam-lignin (f 40 m), VII. The settling ponds (a, b) and aeration pond (c), VIII. The lignin storage chamber. The pump stations: 1. The chlorinated sewage waters. 2. ‘Black’ stream. 3. ‘White’ stream. 4. The regenerating of active silt. 5. The removal of sediment (shlam-lignin). 6. The outflow of cleaned sewage waters into the deep water of the lake.

of Baikal fish (Oikari et al., 1988) and seals (Kucklick et al., 1996). The purpose of this study was to monitor the quality and biological effects of the waste waters released by BPPM. The Ames test (Ames et al., 1973) was used for mutagenicity testing; water samples from different steps of pulp production and waste water treatment were tested. Mutagenic activity in tissue extracts of Baikal aquatic animals (zooplankton, sponge, mollusc, decapoda, fish and seal) as well as in bottom sediments was also investigated.

2. Study area, methods and materials 2.1. The mill process and waste water treatment Baikalsk Pulp and Paper Mill uses a typical sulphate method in cellulose production. A schematic presentation of the factory is shown in Fig. 1. It has been working since 1966. It uses as raw material 40% pine and 60% Siberian larch. It produces sulphate cold-refined cord pulp (80,000 t annually), sulphate bleached pulp (100,000 t) and viscose cellulose (63,600 t). Simultaneously, the BPPM also produces annually turpentine (2000 t), tall oil (9600 t) and packing paper (12,100 t). Fresh Lake Baikal water is taken from the lake near the mill at rates up to 10,000 m 3 h ⫺1. About 300 m 3 of water is needed per ton of product. Because BPPM is on Lake Baikal, particular

attention is required in the treatment of waste water before it is finally released into the lake. Sewage waters from different stages of production are released in ‘black’ and ‘white’ streams having different principal organic compounds and colour (Fig. 1, Table 1). All waste water streams of the mill are primarily mechanically treated and desalinated with the aid of ion change resins. These steps are followed by a unique three step system of purification, including biological, mechanical and chemical methods. The waste waters of Baikalsk City are combined to provide nutrients for the biological treatment system. There are two sedimentation and one aeration ponds at the final stage before the treated waste water is diluted 1:20 with Lake Baikal water and pumped into the lake through two pipes to 35 and 40 m at a distance of 150 m from shore (Fig. 1). Chemical analyses of the waste waters from the last aeration pond have been carried out daily, and less frequently from the preceding steps of waste water treatment, for 25 years. From 1982 onwards biochemical and genotoxicological analyses were carried out. Some of the samples have been analysed in laboratories in Finland, Germany and the USA. Phenols of plant origin, mercaptans, terpene hydrocarbons (turpentine), furfural and many others have been measured by using gas and liquid chromatography. Further, complex integral indexes (pH, biological oxygen demand (BOD), chemical oxygen demand (COD), total organic carbon, total minerals

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Table 1 Characteristics of the main indexes (means and standard deviations) of the quality of Baikalsk City and BPPM waste waters before and during the treatment Index

BOD COD Disp. comp. Inorg. comp. Colour pH

Unit

Munic water

Before biological treatment

Before chemical treatment

After chemical treatment

Effluent into lake

‘White’

‘Black’

mg O2 l mg O2 l ⫺1 mg l ⫺1

32 ^ 3 79 ^ 8 60 ^ 5.8

80 ^ 7.5 340 ^ 32 71 ^ 6.5

160 ^ 25 600 ^ 57 90 ^ 10

4.2 ^ 0.4 140 ^ 13 240 ^ 22

1.3 ^ 0.1 40 ^ 3 11 ^ 1.2

1.3 ^ 0.1 38 ^ 4 3.0 ^ 0.3

mg l ⫺1

82 ^ 8

400 ^ 35

370 ^ 32

–

–

448 ^ 43

– 6.8

470 ^ 45 7.1

1090 ^ 10 7.7

590 ^ 60 6.8

42 ^ 4 6.2

41 ^ 3.9 6.8

⫺1

and colour) have been monitored using standard methods (Beim, 1983, 1984; Krasovsky, 1990). Investigations of chlorinated dioxins and furans have been conducted using HPLC and chromato-mass-spectrophotometry with selective ion detection. 2.2. Sample collection Aquatic animals were collected in Lake Baikal by trawling of the coastal areas at 7–12 m depth. Individual species were collected by divers in the middle and southern part of the lake in August. Baikal seals (Phoca sibirica) were killed in the middle part of Lake Baikal in winter 1994 and 1995 by licensed seal hunters. Tissues samples (fish muscle, seal muscle and fat) and whole bodies of other animals were frozen and stored at a temperature lower than ⫺80⬚C before the extracts were prepared. The following species were studied: fish (arctic cisco (omul, Coregonus autumnalis migratorius (Georgi)), grayling (Thymallus arcticus baikalensis (D.)), lenok (Brachymystax lenok (Pall.)), perch (Perca fluviatilis), roach (Rutilus rutilus lacustris), minnow (Phoxinus phoxinus), endemic sculpins (Cottocomephorus greminskii (Dyb.), Cottocomephorus inermis, Paracottus kessleri); decapoda (Echiropus rohodophtalmus microftalmus); mollusc (Benedictia baicalensis); sponge (Lubomirskia baicalensis); zooplankton (⬍90% Epsichura baicalensis) and the Baikal seal (Phoca sibirica). For enzyme analysis, omul, Baikal grayling, lenok and bullhead were caught with a sweep-net in the

southern and central parts of Lake Baikal and delivered to the laboratory in a specially designed ship supplied with tanks filled with flowing lake water. Throughout the experiments, the fish were kept in aquariums with aerated, filtered and recirculated fresh water at 12–14⬚C. A two-week adaptation period preceded the experiment. Fish of the same age (2–3 years) and sex (male) were used in the experiments. Zooplankton was collected by plankton net and fixed in acetone for later extraction of mutagens. 2.3. Exposure to waste water The fish were exposed to waste water from the aeration pond diluted with Lake Baikal water 1:5 and 1:20. The water mixture was changed daily during one month. Aroclor 1254 (AC) (100 mg kg ⫺1), a synthetic mixture of polychlorinated biphenyl dissolved in olive oil was administrated by intraperitoneal injection; the fish were killed by decapitation seven days after the injection. The liver samples were collected and immediately frozen in liquid nitrogen. 2.4. P-450 and monooxygenase activities Liver microsomal fractions were obtained, and cytochrome P-450 and monooxygenase activities (benzo(a)pyrene hydroxylase (AHH), 7-ethoxycoumarin O-dealkylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD)) were measured as described

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previously (Lindstro¨m-Seppa¨ et al., 1985; Kotelevtsev et al., 1986a,b). Cytochrome P-450 content and monooxygenase activities in fish liver microsomes depend on the fish species, sex, age and the reproductivity phase (Lindstro¨m-Seppa¨ et al., 1985; Kotelevtsev et al., 1986a,b). Three-year old male fish were used in the experiments. The measurements were performed in August and September. 2.5. Mutagenicity assay Waste waters of BPPM were collected from cellulose bleaching (‘after chlorination’), ‘white’ and ‘black’ (lignin rich) fluxes, from biological and chemical purification steps and from the last aeration pond (Fig. 1). Water samples were sterilised immediately by passage through Nalgene membrane filters (0.45 mm) (Nalge Co., Rochester, NY, USA). Chlorine was removed from the ‘after chlorination’ samples under vacuum before filtering. Samples were tested for mutagenicity on the same day or stored frozen at ⫺80⬚C for less than one week. Some water samples from the aeration pond and Lake Baikal were extracted with hexane, 100 ml per one litre of water. Analysis of the material retained by filters (suspensions in ethanol) did not reveal mutagenic activity. For the Ames test, thawed biological samples were placed in five volumes of acetone and then stored at 4⬚C until extracted. Extraction of xenobiotics from the homogenised biological tissues (Polytron type homogeniser, 10 min, 10 g of original tissue) was carried out by means of three repeated washes with acetone and hexane (1:1). The bottom sediment samples, rich with lignin, were treated the same way as biological tissues. The extracts were concentrated in a rotorevaporator and dried by lyophilisation. The residues were dissolved in dimethylsulfoxide (DMSO) (1 ml DMSO for 100 mg of tissue, dry weight). Extracts of distilled water were used as negative controls (Kotelevtsev et al., 1994). The detection of mutagenic compounds in extracts of animal tissues depends on the method of its extraction. Acetone treatment and extraction with an acetone–hexane mixture were used in our experiments. This method has been highly reliable in detecting mutagen compounds in tissues of aquatic organisms from polluted areas.

The mutagenic activities of the waste water, sediment and tissue extracts of aquatic species were determined using the modified Ames test (Ames et al., 1973, 1975) with Salmonella typhimurium strains TA 98 and TA 100 with and without the S-9 fraction from rats treated with a PCB mixture Sovol-54 (Russian equivalent to Aroclor 1254), 100 mg kg ⫺1 for three days. More than seven animals of each species as well as five different samples of plankton were used. Two strains of S. typhimurium were used as test bacteria for the determination of both types of gene mutations: frameshift of the genetic code (the TA98 strain); and the base substitution (the TA100 strain). 2-Aminoanthracene was used as a standard promutagen to control the activity S9 fraction because its metabolites effectively induce both types of gene mutation. For controlling direct mutagenicity, 2-nitrofluorene (TA98) and sodium azide (TA100) were used. The method is sensitive enough to detect the presence of mutagens in waste waters and in extracts of aquatic animals (Kotelevtsev et al., 1994). The test procedure included the following components: addition of 0.1 ml of suspension of the test bacteria, 0.1 ml of the tested substance and 0.5 ml rat liver postmitochondrial fraction S9 to 2 ml melted 0.6% soft agar (at 45⬚C). In half of the probes the postmitochondrial fraction contained a cofactor (NADPH) for metabolic activation (⫹MA) and others did not contain it (⫺MA). The samples were mixed quickly and placed on selective minimal agar and covered for the selection of His ⫹ revertants. Each sample was assayed in triplicate. The plates were incubated at 37⬚C for 48 h after which colonies of His ⫹ revertants were calculated.

2.6. Data analysis If the average number of colonies in the experiment was 2–10 times higher than that in the control, the mutagenic activity of the substance was considered ‘weak’, if it was 10–100 times higher, the mutagenic activity was considered as ‘medium’ (Fonshtein et al., 1977). In all tables, the average number of colonies is shown. Student’s criterion was used for statistical data processing in all experiments.

L.I. Stepanova et al. / Aquatic Ecosystem Health and Management 3 (2000) 259–269 Table 2 The list of main pollutants of waste waters of sulphate–cellulose mill and their ecological-hygienic limit concentrations in Russian regulations Inorganic compounds mg l ⫺1

Organic compounds mg l ⫺1

1. Sulphate ion 2. Chloride ion 3. Sulphide ion 4. Sodium ion 5. Mercury ion 6. Aluminium ion 7. Methanol 8. Chloroform

1. Phenol 0.001 2. Turpentine 0.2 3. Sulphate soap 0.1 4. Formaldehyde 0.1 5. Lignin 10.0 6. Dimethylsulphide 0.0001

300.0 100.0 0.001 120.0 0.0005 0.08 0.1 0.005

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Table 4 The general properties of BPPM waste waters. The normative limit index on effluent of chlorinated compounds in waste waters has been established in some European countries: the quantity of absorbed organic chlorine (AOX). This normative limit was determined to be 0.5–1.4 kg AOX t ⫺1 cellulose in Scandinavian countries (1994–1995) Pollutants

kg t ⫺1 cellulose

BOD (complete) COD Dispersed compounds Inorganic compounds Chlorinated compounds (AOX) Phenols

2.7–3.0 12.9–14.0 0.8–1.2 185.0–200.0 0.9–1.5 0.002–0.003

3. Results and discussion 3.1. Waste water Tables 1 and 2 give basic information on BOD, COD, dispersed material, inorganic material, colour and pH during the waste water treatment in the BPPM. A large decrease in BOD occurred during biological treatment. Subsequently, during chemical treatment a further decrease was observed. Dispersed material was efficiently removed in the chemical treatment phase. Two thirds of the remaining dispersed material sedimented in the aeration ponds before water was pumped into Lake Baikal. The concentration of inorganic material remained about the same in the different stages of the waste water treatment. IndiviTable 3 The content of chlorophenols (means and standard deviation) in waste waters of BPPM before and after treatment; a denotes below the detection limit Compound

2,4-Dichlorophenol 2,6-Dichlorophenol 2,4,6-Trichlorophenol 2,3,5-Trichlorophenol 2,3,4-Trichlorophenol 2,4,5-Trichlorophenol Tetrachlorophenol Chloranizol Dichlormethylphenol Dichlorguaiacol Tetrachlorguaicol

Concentration (mg l ⫺1) Before treatment

After treatment

0.63 ^ 0.05 0.22 ^ 0.02 0.32 ^ 0.03 0.07 ^ 0.008 0.08 ^ 0.007 2.66 ^ 0.21 0.27 ^ 0.01 0.18 ^ 0.02 0.29 ^ 0.03 0.33 ^ 0.04 0.29 ^ 0.02

0.13 ^ 0.01 0.01 ^ 0.001 a a 0.05 ^ 0.002 0.56 ^ 0.05 a a a a a

dual chlorinated phenols were present in trace quantities only in purified waste waters (Table 3). No chlorinated dioxins and furans were detected in waste waters of the BPPM, in sediments of waste waters (shlam-lignin), in samples of bottom substances or in fish with the methods used (Kozhova and Beim, 1993). There were 1.5–3.0 mg l ⫺1 of chloroorganic compounds (lignin up to 94% and chloroform up to 3%) in the sewage water after the multistep purification. The inorganic and organic compounds formed by the destruction of wood during the sulphate process are well known (Beim, 1983). Practically all chemical components (with the exception of chloroorganic substances) of pulp mill sewage waters are analogous to natural compounds. Therefore, the degree by which they become hazardous to aquatic ecosystems is relative and depends on their concentrations. Chloroform, chlorinated phenols, chlorinated dioxins and furans occupy a special place among chloroorganic micropollutants (Krasovsky, 1990; Grosheva, 1991). Their total quantity has been expressed as total organic chlorine (TOC). The analysis of sewage waters from the BPPM at various treatment stages was the most effective for separating chloroorganic compounds. This treatment results in transformation and absorption of low and high molecular chloroorganic compounds. The quantity of pollutants per ton of cellulose is given in Table 4. About 10% of compounds in southern Lake Baikal (5% of sulphates, 5% organic compounds) are anthropogenic loadings. More than 2 km 3 of treated sewage water have been released into Lake Baikal during the

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Table 5 The cytochrome P-450 content (pmol mg protein ⫺1) and the monooxygenase activities (AHH, ECOD and EROD, pmol mg protein ⫺1 min ⫺1) in liver microsomes of Lake Baikal fish species Fish species

P-450

AHH

ECOD

EROD

Grayling Thymallus articus baikalensis Omul Coregonus autumnalis migratorius Lenok Brachymystax lenok Bullhead Cottocomephorus greminskii

22.1 ^ 5.9

5.4 ^ 0.1

1.1 ^ 0.02

4.1 ^ 0.1

27.3 ^ 6.9

5.5 ^ 0.2

1.9 ^ 0.06

5.8 ^ 0.3

21.8 ^ 3.0

6.7 ^ 0.4

2.7 ^ 0.09

4.1 ^ 0.4

11.0 ^ 1.1

1.3 ^ 0.2

1.2 ^ 0.01

0.1 ^ 0.05

past 25 years. Changes of hydrochemical indices have not been found by constant observations 500 m from the effluent outfall for the period of 1980–1991. However, the effluent has resulted in patchy increases of lignin–humus compounds in the bottom of the lake (20 km 2 to north-east from the effluent outfall; Kozhova and Beim, 1993). 3.2. Biochemical monitoring Baikal fish species showed little or no ECOD and EROD activities, when the measurements were performed without preliminary induction of the

monooxygenases with AC. The cytochrome P-450 content in liver microsomes of various Lake Baikal fish species and the measured monooxygenase activities (AHH, ECOD and EROD) are given in Table 5. A long-term (10-year) comparative analysis of the monooxygenase activities in the grayling and bullhead livers inhabiting the BPPM discharge area as well as unpolluted areas failed to reveal any changes in the ECOD activity (Fig. 2). This fact indicates that the concentration of the chemical inducers of the P4501A1 in BPPM waste water is too low to increase the hepatic monooxygenase activities of the fish studied.

Fig. 2. ECOD activity in the liver microsomes of Lake Baikal fish caught near BPPM (August 1982–1992) under normal conditions and after Aroclor 1254 and waste water exposure in laboratory, I. 1:20 waste water dilution, II. 1:5 waste water dilution, time of exposure, 3 weeks. AC is Aroclor 1254.

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Table 6 BPPM waste water mutagenicity in Ames test in years 1982–1993 (S. typhimurium strain TA 100 and TA 98, summary effect), number of samples possessing mutagenic activity/number of samples analysed Waste water type

1982

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

Summary

After chlorination White stream Black stream After biological treatment After chemical treatment Aeration bond

– – – – – 0/8 a

– 2/5 2/5 2/5 2/5 1/6

4/4 1/6 1/5 1/4 1/4 0/6

5/5 0/3 0/2 0/2 0/2 0/4

4/7 0/7 0/7 6/7 0/7 0/8

3/6 0/6 0/6 0/6 0/6 0/6

13/17 1/8 0/16 0/17 0/17 1/17

5/5 1/5 0/3 2/5 0/5 0/5

3/3 0/3 0/3 2/3 0/3 0/3

5/5 0/5 0/3 0/5 0/5 0/5

5/5 2/5 1/5 1/5 1/5 1/5

5/5 0/5 1/5 1/5 1/5 0/5

52/62 7/68 5/59 15/68 4/64 3/78

a

Denotes extract from 1 l.

The total amount of cytochrome P-450 and the monooxygenase activities of all studied fish were elevated after treatment with AC as expected, but not with 5% waste water (as in the final diluted water entering in Lake Baikal). Induction was observed in grayling or bullheads kept for three weeks in 20% waste water under laboratory conditions (Fig. 2).

3.3. Monitoring for genotoxicity Experiments with BPPM waste water have been conducted since 1982 (5–7 samples in different seasons of the year). The results on BPPM waste water mutagenicity are shown in Table 6. Mutagens were found in nearly all ‘after chlorination’ samples applied as such without concentration. The effluents from this step were clearly mutagenic. However, only three from more than 78 water samples taken from the aeration pond (last step of treatments) during the ten year follow-up study revealed mutagenic activity. The ‘white’ fluxes revealed however, a weak mutagenic activity in seven cases, of which four were base substitution and three were frame shift mutations, respectively (not shown). Mutagens were observed in the ‘black stream’ samples in five out of 59 cases (of those two were mutations of both types in which efficiency varied from weak to average; three were weak frame shift mutations; not shown). After the biological purification, 15 samples showed mutagenic activity (weak frame shift mutations; not shown). After the chemical purification, samples revealed mutagenic activity in four cases out of 64 (two were weak frame shift mutations, two exhibited a direct

mutagenic effect of base substitution type; not shown). In all cases of clear mutagenicity, the effect was direct. All ‘after chlorination’ samples exhibited a direct mutagenic effect, predominantly in S. typhimurium strain TA 100. In 1993, indirect mutagenicity was also detected (Table 7). An increase of the spontaneous mutation level by not more than 10-fold (without concentration) was induced. In 1993 the waste water from the aeration pond possessed weak direct mutagenic effect on S. typhimurium strain TA 100 (Table 7). During the course of waste water treatment the mutagenic activity was lowered effectively in both biological and chemical treatment steps (Tables 7 and 8). This may be because of biological and chemical inactivation of labile chlorinated organic components, or adsorption of mutagenic substances on lignin or lignin sludge in the course of water treatment. The chemical analysis of chlorinated compounds of ‘after chlorination’ streams is shown in Table 3. Not all these compounds possess mutagenic effects. For example, we did not find genotoxicity for 2,4,6trichlorophenol in S. typhimurium strains TA 98 and TA 100. The ‘after chlorination’ effluent is the most important material for the identification of compounds responsible for mutagenicity. An additional local purification of this effluent is thus recommended. No mutagens were found in Lake Baikal water even after up to 1000-fold concentration with hexane (not shown). Sediment extracts collected on the bottom near the waste water outfall into Lake Baikal usually contained weak mutagens during a 4-year period (Fig. 3). The bottom sediment samples showed direct mutagenic

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Table 7 Relative mutagenic activity of BPPM waste waters and tissue extracts of selected species collected close to the mill and from the middle part of Lake Baikal (seals) in the Ames test with (⫹MA) and without (⫺MA) metabolic activation in August 1993. Statistically significant (p⬍0.05) numbers are indicated in bold. N is ⬎7 except where noted Sample

Dose

Salmonella typhimurium strain TA 98

TA 100

Number of His ⫹ revertants per plate

Dimethylsulphoxide H2O (distilled) 2-Aminoanthrazene Waste water After chlorination ‘Black stream’ ‘White stream’ Aeration pond Extracts Bottom sediments Vydrino (1000 m north from BPPM) Bottom sediments (near BPPM) Aeration pond a Zooplankton b Sponge Mollusc Decapoda Arctic cisco Grayling Lenok Perch Roach Minnow Sculpins C. greminskii C. inermis Seal muscle Seal fat a b

⫹MA

⫺MA

⫹MA

⫺MA

0.1 ml 1 ml 0.5 mg

1 1.1 42.3

1 0.8 0.9

1 1.0 16.5

1 1.0 1.0

0.4 ml 0.4 ml 0.4 ml 0.4 ml

1.2 1.0 0.8 1.0

1.1 0.6 0.5 0.6

4.9 0.9 0.9 1.0

2.2 0.9 1.2 1.0

0.1 ml

1.1

1.2

0.9

0.3

0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml

1.9 0.9 3.5 1.2 0.8 1.0 1.3 1.0 0.8 1.1 1.0 0.8

3.5 0.7 0.9 0.9 0.7 1.0 0.9 0.7 0.9 0.9 8.4 0.6

1.0 0.9 1.0 0.9 1.0 1.1 1.0 1.0 0.9 1.3 0.9 0.9

1.1 2.1 1.0 1.1 1.0 1.0 1.3 1.1 1.1 1.0 0.9 1.0

0.1 ml 0.1 ml 0.1 ml 0.1 ml

0.8 1.1 2.3 2.7

0.6 0.9 1.1 1.2

1.1 1.0 1.9 1.0

0.9 1.1 1.2 1.1

Extract from 1 l. Extract from 1 g tissue (fresh weight), N ˆ 5:

activity in Salmonella TA 98 strain (Table 7). This indicates that some mutagens accumulate in the sediments near the place where BPPM waste water is released. Mutagens were not found from reference sites near Olchon Island and Vydriono. On the other hand, bottom fauna has been shown to be in greater abundance in the waste water release area than elsewhere in Lake Baikal. This may be because of the higher temperature and organic material coming in with waste water (Kozhova and Beim, 1993). Indirect mutagenic activity was detected in

zooplankton collected near the area where waste waters are released into the lake (Fig. 3, Table 7). This was revealed in the Ames test after the metabolic activation by the monooxygenase system in Salmonella TA 98 indicating frame shift activity. No activity was seen in zooplankton collected from reference areas (not shown). The metabolic detoxification of xenobiotics in zooplankton is probably not sufficiently active in comparison with other aquatic and warm-blooded animals so that mutagenic substances accumulate.

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Table 8 Relative mutagenic activity of muscle and fat extracts of seals caught in December 1995 from the middle part of Lake Baikal in the Ames test with (⫹MA) and without (⫺MA) metabolic activation compared to DMSO-controls. Statistically significant (p⬍0.05) the numbers are indicated in bold Seal tissues

Muscle

Fat

Salmonella typhimurium strain Sample

TA 98

TA 100

TA 98

TA 100

Sex

Age (year)

⫹MA

⫺MA

⫹MA

⫺MA

⫹MA

⫺MA

⫹MA

⫺MA

Male Male Male Male Male Male Male Female Female Female Female Female Female Female Female Female

3 month 1 5 5 7 9 10 3 month 3 month 3 month 1 1 2 2 6 7

3.4 a 1.4 1.3 2.2 1.4 0.9 0.8 2.3 2.2 2.6 2.0 2.4 3.3 2.1 1.0 1.5

2.6 0.9 0.9 1.1 1.8 2.3 1.1 1.4 1.9 5.2 1.1 2.1 4.1 1.0 1.0 1.0

2.3 2.3 1.1 1.1 1.9 0.9 1.0 1.0 1.2 2.1 1.0 1.0 1.4 0.9 1.1 0.9

0.3

2.2 1.9 1.1 1.7 2.0 1.5 1.4 1.3 2.2 2.0 1.3 2.4 1.5 2.2 1.9 1.9

2.6 1.6 1.0 1.8 3.1 1.3 0.9 1.2 2.0 0.9 1.1 2.9 1.1 2.1 2.0 1.3

1.1 1.2 0.4 1.2 1.3 0.9 0.9 1.1 1.1 1.1 1.0 0.7 0.9 1.1 1.2 1.0

0.3 0.5 0.2 1.0 0.4 0.8 0.3 1.0 0.7 0.5 0.3 0.2 0.4 0.4 0.6 1.2

a

1.2 0.9 1.2 1.5 1.9 1.2 1.1 1.2 0.9 1.0 1.3 1.4 1.4 2.7

Relative number of His ⫹ revertants (ratio to DMSO control), means of three separate triplicate analyses.

During the whole follow-up period, cleaned waste waters of the BPPM in dilution 1:20 showed no strong toxic action on local aquatic organisms such as fish, molluscs or zooplankton (Kozhova and Beim, 1993); neither have any significant mutagens been found in aquatic animals (Glazer et al., 1990). As seen here, the tissue extracts of Lake Baikal fish, decapoda, mollusc and sponge did not reveal any mutagenic effect, except for roach which showed direct mutagenicity (Table 7). The apparent absence of mutagens from tissue extracts may be because of one or more of the following reasons: (1) the tissues did not contain mutagenic compounds; (2) the method of extraction used was not suitable for this kind of chemical; (3) mutagens if present are not detected by the S. typhimurium TA98 and TA100 strains. The method of acetone extraction is the easiest and most convenient for isolation of mutagens from organic samples (Felton et al., 1981). The yield of mutagens from investigated material is about twofold higher compared with the aqueous extraction method (Commoner et al., 1978; Grabow et al.,

1981). Acetone in mixture with other solvents, for example, benzene, water and hexane is also widely used (Vian et al., 1982). However, each case is different, and the chemical features of the mutagens and the nature of the studied material must be taken into account in order to select the most appropriate method of extraction. Several samples of seal tissues were found to possess mutagenic activity (Tables 7 and 8). Nearly all muscle extracts of female seals showed activity in Salmonella TA 98 strain after metabolic activation and in a few cases also without activation. Very often the adipose tissue sample gave the same reaction. Mutagenic compounds can enter Lake Baikal with municipal and industrial waste waters (e.g., the waste water of BPPM). The cellulose and paper production is a multistage process which requires huge amounts of water; some of these stages form industrial effluent containing toxic and mutagenic substances. Non-purified sewage waters from pulp and paper mills represent multi-component mixtures containing lignin derivatives, cellulose partial degradation products,

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Fig. 3. The mutagen activity of the bottom sediments and zooplankton extracts collected from the BPPM waste water discharge area (3) and four other areas.

terpene hydrocarbons and other substances that are extracted during the cooking and bleaching of cellulose mass. Bleaching, which is designed to remove extract compounds includes several steps, including the treatment of cellulose with chemical reagents, for example, chlorine and alkali. As the result of such treatment, bleached industrial effluent from the pulp and paper mill becomes contaminated with more than 30 chlorinated organic components. These compounds exert mutagenic influence on tested S. typhimurium strains (Kotelevtsev and Stepanova 1995). The main chain for accumulation of xenobiotics in tissues of Lake Baikal animals is: primary producers ! zooplankton (⬎90% Epischura baicalensis) ! endemic sculpins (main species, Baikal oil fishes) ! Baikal seal (Kozhova and Beim, 1993). Chemical investigation of Baikal seal tissue showed the presence of both polychlorinated biphenyls and polycyclic hydrocarbons (Kucklick et al., 1996). The quantity of xenobiotics in seal tissue in Lake Baikal has grown during the last ten years, but the marine seals usually still contain more xenobiotics than Baikal seals (Bernhoft and Ska˚re, 1994). The preli-

minary chemical investigation of seal tissues demonstrated as well the presence of chlorinated compounds which are not present in waste waters of BPPM (e.g., DDT) (A. Lebedev, Federal Enviornmental Analytical Centre, Moscow, pers. comm.). In any case, the possible accumulation of mutagenic xenobiotics from BPPM waste waters through the food chain must be kept in mind.

4. Conclusions Biological monitoring of the southern part of Lake Baikal indicates that there seems to be a balance between anthropogenic releases of compounds and the rate of their biogeochemical transformation by the lake ecosystems. Investigations conducted from 1980 to 1991 have shown that the area with affected bottom ecological systems has not significantly changed during the last 10 years. Owing to the increased presence of anthropogenic chemicals seen in Lake Baikal biota, the monitoring of the waste waters as well as Lake Baikal must be

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continued. Accumulation of mutagenic compounds in the tissues of aquatic animals represents a direct danger for the organisms. The plankton and seals seemed to be suitable for biomonitoring the mutagenic activity in Lake Baikal aquatic ecosystem. The uniqueness of Lake Baikal is also a stimulus to international cooperation. Acknowledgements This work was partly supported by European Science Foundation (Toxicology and Environmental Toxicology), by INTAS (Ref. No. 94-0531) and by a NATO Linkage Grant (Envir.LG 940 380). References Ames, B.N., Lee, F.D., Durston, W.E., 1973. An improved bacterial test system for the detection and classification of mutagen and carcinogens. Proc. Nat. Acad. Sci. USA 70, 782–786. Ames, B.N., McCann, J., Yamasaki, E., 1975. Method for detecting carcinogens and mutagen with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 47–364. Beim, A.M., 1983. Experimental establishing permissible concentrations of components of treated effluent of Baikalsk PPM. Nature protection from pollution with industrial wastes of pulp and paper industry. VNPO Bumprom, Leningrad, pp. 38–43 (in Russian). Beim, A.M., 1984. Problems of aquatic toxicology. Biotesting and Water Quality Management, US Environ. Protect. Agency 600/ 9-86/024. Bernhoft, A., Ska˚re, J.U., 1994. Levels of selected individual polychlorinated biphenyls in different tissues of harbour seal (Phoca vitulina) from the southern coast of Norway. Environ. Pollut. 86, 99–107. Commoner, B.A., Dolara, P., Nair, S., Madyastha, E., Cuca, G.C., 1978. Formation of mutagens in beef and beef extract during cooking. Science 201, 913–916. Felton, J.S., Healy, S., Stuermer, D., Berry, C., Timourian, H., Hatch, F.T., 1981. Mutagens from the cooking of food, improved extraction and characterisation of mutagenic fraction from cooked ground beef. Mutat. Res. 88, 33–34. Fonshtein, L.M., Kalinina, L.M., Poluhina, G.N., Abilev S.K., Shapiro, A.A., 1977. Test-system for the estimation of mutagenic activity of pollutants in environment. Moscow (in Russian). Glazer, V.M., Kotelevtsev, S.V., Stepanova, L.I., Abilev, S.K., Buevich, G.V., Beim, A.M., 1990. The assessment of mutagenicity in the Ames test of sewage waters and industrial effluent of the Baikal cellulose paper integrated plan. Biologicheskie nauki 1, 101–109. Grabow, W.O.K., Burger, J.S., Hilner, C.A., 1981. Comparison of liquid extraction and resin adsorption for concentrating muta-

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