Isolation and identification of ligands for the goldfish testis androgen receptor in chemical recovery condensates from a Canadian bleached kraft pulp and paper mill.

This study builds on a series of investigations characterizing substances in kraft mill chemical recovery condensates that depress sex steroids in fish. Here, incubations of goldfish testis androgen receptors (AR) with condensate extracts were used to investigate the potential role of androgens in hormone depressions. Condensates contained variable levels of AR ligands, with the highest amounts in nonpolar extracts of filtered solids prior to solid phase extraction (SPE). High pressure liquid chromatography (HPLC) fractionation recovered the majority of activity in one fraction, with ligands detected in three additional fractions. Gas chromatography mass spectrometry analysis of the most active fraction confirmed the two most abundant components as the diterpenes manool and geranyl linalool. Manool exhibited a relative affinity for the AR that was 300 fold less than testosterone and accounted for 26% of total filtered solids activity. Geranyl linalool exhibited no affinity for the AR. Three additional diterpenoid families were tentatively identified as principal components of the three other androgenic HPLC fractions. Compared to condensates, final effluent had 3000 fold less androgenic activity, with <1% attributable to manool. Putative androgens previously associated with mill effluents (androstenedione and androstadienedione) and progesterone were not detected; however, additional condensate diterpenes suspected as androgens were identified in final effluent. This study is the first to confirm nonsteroidal cyclic diterpenes as androgenic at pulp mills. A major in-mill source of these substances was identified, and the role of androgens in mill effluents affecting fish reproduction was reinforced.