In vitro detection and quantification of testosterone metabolites in aquatic organisms

High-performance liquid chromatography (HPLC) was used to evaluate testosterone metabolism in aquatic animals generally used as sentinel species (i.e., fish and molluscs). The profiles of testosterone metabolites resulting from incubation of liver microsomes from trout Oncorhynchus mykiss, digestive gland S9 from clams, Corbicula fluminea and mussels Mytilus galloprovincialis were compared. Main metabolites 6 beta-hydroxytestosterone, 16 alpha-hydroxytestosterone, 11,beta-hydroxytestosterone, 2 alpha-hydroxytestosterone, 2 beta-hydroxytestosterone and androstenedione were detected and quantified in rat, fish, and clam, whereas only 16 alpha-hydroxytestosterone and androstenedione metabolites were detected in mussels. Testosterone metabolite profiles detected in cellular fractions were further analyzed. The protocol presented in this study may be considered as a useful tool for biomarker based pollution surveys.