Identification of compounds associated with testosterone depressions in fish exposed to bleached kraft pulp and paper mill chemical recovery condensates

Abstract

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.

Pulp and paper mill effluent treatments have differential endocrine‐disrupting effects on rainbow trout

“…Chem. A series of potential candidate compounds responsible for steroid depression were recently identified by GC-MS detection [37], including diterpenoids, sesquiterpenoids, and stilbenes, and semiquantitative concentrations related to different mill extracts support the hypothesis that similar chemicals may produce hormonal effects in spite of the different wood furnished. New research has increased the potential sources of variability in in vitro assays further, indicating that stress protein hsp90 function is critical for ER signaling and estrogenic endocrine disruption in experiments evaluating rainbow trout hepatocyte VTG production [34].…”

“…That research demonstrated a consistent steroid depression observed as a significant reduction in circulating testosterone in both sexes using both in vivo and in vitro assays (gonad production protocol). A series of potential candidate compounds responsible for steroid depression were recently identified by GC-MS detection [37], including diterpenoids, sesquiterpenoids, and stilbenes, and semiquantitative concentrations related to different mill extracts support the hypothesis that similar chemicals may produce hormonal effects in spite of the different wood furnished. Despite the similarities with this pulp mill effluent solid-phase extraction protocol, samples extracted in our experiment correspond to a final postbleaching process effluent and focus on the polar compounds, different to the condensates from evaporators in the chemical recovery process, which focus on the nonpolar fraction.…”

Altered reproduction in fish exposed to pulp and paper mill effluents: Roles of individual compounds and mill operating conditions

“…The alkaline stream (67% of the final effluent flow) decreased egg production and caused VTG induction and ovipositor development in males. Condensates at this mill also contained chemicals associated with the steroid depressions in mummichog [98]. Additional studies are required to further delineate these sources.…”

Fundulus as the premier teleost model in environmental biology: Opportunities for new insights using genomics

“…Studies in the field (LeBlanc and Bain, 1997), artificial streams (Dubé and MacLatchy, 2000) and laboratory Hewitt et al, 2002;Belknap et al, 2006;Shaughnessy et al, 2007) have identified reproduction and endocrine effects in F. heteroclitus as sensitive end-points that are responsive to endocrine-active contaminants in pulp mill effluents. These studies in killifish have led the way in determining the source(s) of contaminants within the mill process as well as confirming, for the purposes of environmental protection, an effective within-mill treatment process to remove the contaminants (Dubé and MacLatchy, 2001;Hewitt et al, 2002).…”