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In previous experiments, bleached kraft chemical recovery condensates generated during softwood pulp production have been identified as a primary source of substances causing testosterone depressions in mummichog (Fundulus heteroclitus). Recent toxicity identification evaluations have encountered inconsistencies in the potential of condensates and fractions to reduce steroid concentrations between three repeated exposures. To assess the sources of these inconsistencies, temporal assessments of condensate extractives and analytical methodologies were evaluated. Condensates were collected during a six-month period, extracted by solid-phase extraction (SPE), and analyzed by gas chromatography-mass spectrometry and gas chromatography-flame photometric detection. All unique extractives were cataloged by mass spectra, peak area, and gas chromatographic retention time, and nine were confirmed and quantified against authentic standards. Confirmed components included phenolic guaiacyl-based lignin degradation products, sulfur (S9), three diterpenoids, and a dimethoxy stilbene. Concentrations of confirmed condensate extractives were consistent in all samples collected. Spiking experiments of confirmed extractives revealed substantial losses following high-pressure liquid chromatographic fractionation and three different methods of fraction preparation. In an effort to propose chemical classes associated with biological activity, all unique condensate extractives in previously established bioactive and inactive fractions were classified based on their potential to depress testosterone in mummichog. Of 39 unique components in bioactive SPE extracts of condensates, six were associated with hormonal activity. Mass spectral interpretation indicated hydroxylated diterpenoids, sesquiterpenoids, and a lignin-derived stilbene as classes of chemicals associated with steroid depressions.

A study was initiated in the spring of 2001 to determine if hormonally active chemicals are bioavailable to white sucker (Catostomus commersoni) residing in the Wabigoon River, downstream of the bleached kraft pulp mill and municipal sewage outfall located in Dryden, Ontario. Females collected during spawning migrations in May 2001 at a near-field exposure site exhibited increased condition factor, liver size and fecundity relative to those collected from a reference site. Liver samples within each site were pooled, soxhlet extracted and fractionated by gel permeation chromatography and HPLC according to octanol/water partition coefficient (Kow). Fractions were then tested to determine if fish accumulated ligands which bind to the goldfish testicular androgen receptor (AR) and sex steroid binding protein (SSBP). The levels of hormonally active substances were low in males whereas females had accumulated significant amounts of several chemicals capable of binding to the hormone receptors. The levels of ligands detected in pulp mill effluent samples were substantially reduced after secondary treatment. Fractionation of final effluent extracts showed only ligands for the AR in the hydrophobic region (log Kow >5), which corresponded to the activities detected in fish tissues from both sexes. It can be concluded that ligands for fish sex steroid receptors are present in final effluent after treatment at the pulp mill and chemicals with the same Kow are bioavailable to fish under spring freshet conditions. Further work at this site is necessary to determine the contribution of pulp mill and municipal sewage effluents to the physiological performance of fish in the Wabigoon River and to their burden of accumulated compounds.

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