Anaerobic testosterone degradation in Steroidobacter denitrificans e Identification of transformation products Michael Fahrbach a , Martin Krauss a , Alfred Preiss b , Hans-Peter E. Kohler a , Juliane Hollender a, * a Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133, P.O. Box 611, CH-8600 Dübendorf, Switzerland b Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM), Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany Testosterone is completely mineralized by Steroidobacter denitrificans under denitrifying conditions with initial formation of several reduced and oxidized transformation products. article info Article history: Received 6 February 2010 Received in revised form 13 May 2010 Accepted 15 May 2010 Keywords: Testosterone Steroid Biodegradation Denitrification Transformation product abstract The transformation of the androgenic steroid testosterone by gammaproteobacterium Steroidobacter denitrificans was studied under denitrifying conditions. For the first time, growth experiments showed that testosterone was mineralized under consumption of nitrate and concurrent biomass production. Experiments with cell suspensions using [4- 14 C]-testosterone revealed the intermediate production of several transformation products (TPs). Characterisation of ten TPs was carried out by means of HPLC coupled to high resolution mass spectrometry with atmospheric pressure chemical ionization as well as 1 H and 13 C NMR spectroscopy. 3b-hydroxy-5a-androstan-17-one (trans-androsterone) was formed in the highest amount followed by 5a-androstan-3,17-dione. The data suggests that several dehydrogenation and hydrogenation processes take place concurrently in ring A and D because no consistent time- resolved pattern of TP peaks was observed and assays using 2 TPs as substrates resulted in essentially the same TPs. The further transformation of testosterone in S. denitrificans seems to be very efficient and fast without formation of detectable intermediates. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Steroid hormones have come into focus as environmental pollutants because they are able to act as endocrine disruptors in aquatic environments and are widely excreted by animals and humans. Testosterone, produced in the testis of males and in the ovaries of females, derives biosynthetically from cholesterol and regulates metabolism, growth, and reproduction in mammals. The most likely sources of testosterone and of its known urinary excreted human metabolites (4-androstene-3,17-dione, 3-hydroxy- androstane-17-one, 17-hydroxy-androstane-3-one, androstane- 3,17-dione, 3,17-dihydroxy-androstane, 3,17-dihydroxy-5-andro- stene, 3-hydroxy-5-androstene-17-one, 17-hydroxy-androstene-3- one) for aquatic environments are municipal sewage sludge, paper mill effluents, and runoff from agricultural production sites (Jacobsen et al., 2005; Jenkins et al., 2003; Liu et al., 2009; Thomas et al., 2002). Thomas et al. (2002) have shown that wild fish living downstream of exposed waters were negatively affected by androgenic steroids causing endocrine disruption. Microbial transformation of testosterone was observed in several environmental matrices such as soils (Casey et al., 2004; Lorenzen et al., 2005), soils amended with swine manure, soil- water slurries (Lee et al., 2003), stream sediments (Bradley et al., 2009), and within the wastewater treatment process (Esperanza et al., 2007). In most cases transformation under aerobic condi- tions was investigated. Yang et al. (2009) showed recently for manure-borne bacteria that the half-life under anaerobic condi- tions was significantly increased. Testosterone, 4-androstene-3,17-dione, and other steroids, such as estradiol and cholesterol, are natural substances that can be completely degraded by several bacterial genera under aerobic conditions (Talalay et al., 1952; Kieslich, 1985; Fujii et al., 2003). Strains of the obligate aerobic betaproteobacterium Comamonas testosteroni (Talalay et al., 1952; Tamaoka et al., 1987) mineralize testosterone to carbon dioxide and water through the well-studied and oxygen-dependent 9,10-seco pathway (Coulter and Talalay, 1968; Sih and Whitlock, 1968; Horinouchi et al., 2004). However, in anoxic environments, key reactions of the 9,10-seco pathway, such as activation of the growth substrate by oxygen-dependent hydroxylation and its subsequent oxygenolytic cleavage, cannot * Corresponding author. E-mail addresses: michael.fahrbach@web.de (M. Fahrbach), martin.krauss@ eawag.ch (M. Krauss), alfred.preiss@item.fraunhofer.de (A. Preiss), hkohler@ eawag.ch (H.-P.E. Kohler), juliane.hollender@eawag.ch (J. Hollender). Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol 0269-7491/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2010.05.017 Environmental Pollution 158 (2010) 2572e2581