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Juvenile whitefish (Coregonus lavaretusL. s.l.) were exposed by caging in the field to diluted effluents from three operating pulp, paper, and paperboard mills in Southern Lake Saimaa, Finland. The expression of the vitellogenin gene, used as a biomarker of estrogenic contamination of effluents, was measured using a Northern blotting method. Increased mRNA levels, the most specific and reliable evidence for estrogen receptor-mediated actions in vivo, were found in fish caged in the vicinity of one of three mills studied. This mill was found to discharge wood-derived compounds, such as sterols and resin acids, into Lake Saimaa in amounts considerably exceeding those from the other two mills. The increased vitellogenin gene expression suggests that the effluent is a source of estrogenic contaminants.

Samples of perch (Perca fluviatilisL.) and roach (Rutilus rutilusL.) populations were collected in Lake Saimaa in September-October, after the onset of vitellogenesis and deposition of yolk in the eggs. The study area received elemental chlorine-free (ECF)-bleached kraft mill effluent (BKME). The pulp and paper mill studied used chlorine dioxide for bleaching and its effluents were treated in a modern activated sludge plant. The amounts of chlorophenolics in the bile of perch and roach at the mill site, 1 km from the discharge point, were two to four times higher than in fish from the upstream reference site. In the roach population near the mill liver the 7-ethoxyresorufin 0-deethylase (EROD) activity was four times that measured in the reference areas. However, the EROD activity in the perch remained unchanged. The liver 7-pentoxyresorufin 0-dealkylase (PROD) activity was induced to twice the amount only in male roach near the mill, whereas the relative liver size (LSI) was increased in female and male roach and female perch. The plasma concentrations of oestradiol-17β were decreased in female perch and female roach, but increased in male roach at the mill site. Near the mill, vitellogenin gene expression was also reduced in female perch. In addition, the plasma testosterone concentrations were reduced in female and male perch near the mill. No differences were noted in the plasma immunoglobulin concentrations in roach and in the relative gonad size (GSI) in the perch and roach populations. The condition factor was significantly lower only in female roach near the mill.

The wood-derived compound, β-sitosterol (purity >90%), was shown to be estrogenic in fish. It induced the expression of the vitellogenin gene in the liver of juvenile and methyltestosterone-treated rainbow trout. Structural similarities to β-sitosterol notwithstanding, cholesterol, citrostadienol, β-sitostanol, and 5-androstene-3β,17β-diol, an estrogenic member of the androstenic steroid group, were inactive. An abietic acid mixture (37% abietic acid, 6% dehydroabietic acid, and a remainder of unknown compounds) showed slight hormonal activity in feed, but it was completely inactive when given intraperitoneally in implants. The estrogenic component of the abietic acid preparation was not identified. In addition to β-sitosterol and abietic acid, several other wood-derived compounds including betulin, isorhapontigenin, isorhapontin, and pinosylvin were estrogenic in breast cancer cells (MCF-7 or T-47D). However, betulin and pinosylvin, available in sufficient amounts forin vivotesting, did not induce the expression of the vitellogenin gene. Differences in the primary sequences of human and fish estrogen receptors (hormone as well as DNA-binding regions) or uptake and metabolism of the compounds may explain the discrepancy between the two estrogen bioassays. Wood-derived compounds such as β-sitosterol, present in pulp and paper mill effluents, may account for the weak estrogenicity of debarking effluent seen at the vitellogenin expression bioassay.