This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

3, 4-dichloroaniline-14 C was applied to soil under outdoor conditions, at a rate corresponding to 1.43 kg/ha. Barley was sown immediately after application; in the following year, potatoes were planted. After the first season, a total of 69.8% of the radiocarbon applied was recovered in soil, plants and leaching water, and after the second year the recovery was still 67.1%. Most of the radioactivity was found in the top soil layer (0-10 cm depth).The total radioactivity in barley grains was 0.02 μg/g (equivalent to 3, 4-dichloroaniline), in peeled potatoes 0.01μg/g. Most of the residues in soil and plants was unextractable with organic solvents (50% to 94%); unconverted 3, 4-dichloroaniline was about 1% of the Dedicated to Dr. Bert B. Migicovsky on the occasion of his retirement. 243 244 VISWANATHAN ET AL.residues present after one growing season and less than 1% after the second year's harvest. From purified soil extracts, the following conversion products were identified by chromatographic retention data and by mass spectra of reference compounds: 3, 4, 3', 4'-tetrachloroazobenzene (I), 3, 4-dichloroformanilide (II), 3, 4-dichloroacetanilide (III), and 6-hydroxy-3, 4-dichloroacetanilide (VI).In plants, the conversion products I and III were identified only byRf-values in various TLC-systems.

Many types of pulp and paper mill effluents have the ability to induce mixed-function oxygenase (MFO) activity and vitellogenin (VTG) protein in exposed male fish. The search for the compounds responsible for MFO induction has led to several classes of compounds, among them retene and stilbenes. The objective of this study was to investigate the biological activities of candidate stilbene compounds. Three stilbenes, 3,5-dihydroxystilbene (pinosylvin; P1), 3-hydroxy-5-methoxystilbene (P2), and 3,5-dimethoxystilbene (P3), were extracted from Scotch pine (Pinus sylvestris) and purified to evaluate their ability to induce MFO activity in vitro using ethoxyresorufin-O-deethylase (EROD) activity in a rat hepatoma cell line (H4IIE). As these compounds may be chlorinated during pulp bleaching, chlorination of P2 was undertaken, producing di- and trichlorinated isomers (Cl-P2), which were also tested. Compounds were tested for EROD-inducing ability in vivo by exposing juvenile rainbow tout (Oncorhynchus mykiss) to waterborne concentrations (0.010 to 1.0 mg/L) for 4 days. Compounds were also tested for their ability to induce VTG in trout primary liver cells in vitro. The stilbenes were weak inducers of EROD and VTG. H4IIE EROD was induced by all four compounds, with the most potent induction by P3, followed by P1, the Cl-P2 mixture, and then P2. Induction for all four stilbenes was from 3.13 × 10⁻³ to 3.57 × 10⁻⁴ as potent as retene and about 1.11 × 10⁻⁵ to 1.20 × 10⁻⁶ as potent as TCDD. Juvenile rainbow trout did not show EROD induction after exposures to P1, P2, or the Cl-P2 mixture, whereas P3 caused activity fourfold above that of controls. P1, P3, and Cl-P2 all weakly induced VTG in rainbow trout hepatocytes. The most potent inducer of VTG was Cl-P2, followed by P3 and P1. The results show the ability of wood-derived stilbenes to cause weak MFO induction in fish and in rat liver cells and to weakly induce vitellogenin in fish liver cells.

Maize has been grown in soils treated with aldrin-14C at locations in Germany (2.9 kg/ha), England (3.0 kg/ha), Spain (3.0 kg/ha), and the United States (3.0 kg/ha), and wheat has been grown in Germany (2.9 kg/ha) and England (3.2 kg/ha). Additionally, aldrin-14C has been appliedto wheat seeds at locations in Germany and England (0.1% of the weight of grain). At harvest, residues did not exceed 0.01 ppm in the grain. The main radioactive products identified by glcmass spectra in soils and in the plants of all loca-tions were dieldrin and dihydrochlordene dicarboxylic acid (l,2,3,4,8,8-hexachloro-l,4,4a,6,7,7ahexahydro-l,4-endomethyleneindene-5,7-dicarboxylic acid). Unchanged aldrin, photodieldrin, unknown acidic compounds, and an unknown nonpolar metabolite were detected in trace amounts. In wheat soil, up to 5% of the recovered radioactivity was due to photoaldrin. The quantitative data for aldrin and metabolites were different for the four locations.In two previous papers, we reported outdoor studies with aldrin-14C in potatoes (Klein et al., 1973) and sugar beets (Kohli et al., 1973), following soil application in 1969 at a location in Germany and England. In the same year, aldrin-14C was applied to soils, and maize was grown in Birlinghoven, Germany, Sittingbourne, U. K., Sevilla, Spain, and Modesto, Calif., under the same experimental conditions. Furthermore, in Germany and England, wheat was grown in aldrin-14C-treated soils, and at both locations, seed dressing was used additionally for the same crop. The rationale for these experiments was the same as described previously. The results are described in this 610 J.

ALDRIN-14C IN SUGAR BEETS concentrations were also observed in the roots of plants grown in S-deficient solutions and in the greens of plants from K-deficient solutions.Significant decreases in the concentrations of both Dyfonate and Dyfoxon in the roots were observed with plants grown in solutions deficient in K, Ca, or Mg. The concentrations of Dyfoxon were significantly decreased by deficiencies of all elements except nitrogen.Analysis of the nutrient solutions after 8 days of plant growth showed that 2.7 to 13.3% of the recovered radioactivity was water soluble, compared to 2.1% immediately after treatment with [14C]Dyfonate. This indicates that either water-soluble 14C metabolites are released from the plant roots into the nutrient solutions or that exoenzymes metabolized the Dyfonate in the nutrient solutions.