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

摘要

This study builds on a series of investigations characterizing substances in chemical recovery condensates at a Canadian bleached kraft mill that depress sex steroid levels in fish. In the present work, incubations of condensate extracts with goldfish testicular androgen receptors (AR) were used to streamline the development of chemical fractionation methods. After applying a solid phase extraction (SPE) protocol developed to recover known condensate extractives, non-polar extracts of filtered solids prior to SPE were found to contain high levels of androgenic activity. Using normal phase HPLC, activity was recovered in one fraction, with AR ligands detected in 3 additional fractions. Gas chromatography mass spectrometry (GC-MS) 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 0.003 of testosterone and accounted for 25 and 14 % of the activity of the filtered solids and the most active HPLC fraction, respectively. Geranyl linalool exhibited no affinity for the AR. 3 additional diterpenoid families were tentatively identified as the principal components of the 3 other androgenic HPLC fractions. Final effluent was found to have a low level of androgenic activity, with ＜1 % attributable to manool. Androgens previously identified in mill effluents (progesterone, androstenedione, androstadienedione) were not detected. This study is the first to confirm non-steroidal cyclic diterpenes as androgenic at kraft mills and present a plausible mechanism for mill effluents to affect fish reproduction.