Author's personal copy Anti-androgenic effects of sewage treatment plant effluents in the prosobranch gastropod Nucella lapillus M.M. Santos a, ⁎, M.A. Reis-Henriques a,c , R. Guillot a , D. Lima a , R. Franco-Duarte a , I. Mendes a , S. Queirós a , L. Filipe C. Castro b, ⁎ a CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Environmental Toxicology, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal b CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, Laboratory of Cellular and Molecular Studies, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal c ICBAS-Institute of Biomedical Sciences Abel Salazar, University of Porto, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal ABSTRACT ARTICLE INFO Article history: Received 16 November 2007 Received in revised form 14 March 2008 Accepted 20 March 2008 Available online 27 March 2008 Keywords: Imposex Nucella lapillus Tributyltin Anti-androgenic Sewage effluent Xenoestrogen Recent studies performed under laboratory conditions have shown that single exposure to high levels of several xenoestrogens is able to induce imposex in at least two neogastropod species. In an attempt to evaluate if xenoestrogens, at environmentally relevant conditions, do contribute to imposex induction, we have tested the effects of a mixture containing xenoestrogens (municipal sewage effluents) on imposex development in the dogwhelk Nucella lapillus. Exposure for 3 months to the raw (0.25% and 1%) and the final sewage effluent (12.5% and 50%) rendered no increase in the severity of imposex. Conversely, as exposure to high concentrations of natural steroids, estradiol and estrone, had previously been shown to partially rescue imposex development under laboratory conditions, we have also tested if exposure to the final sewage effluent could ameliorated the severity of imposex induction by tributyltin (TBT). The results demonstrated that co-exposure to the final sewage effluent leads to a decrease trend in the severity of imposex in the presence of TBT. Within the studied imposex parameters, the Relative Penis Size index (RPSI) was the most affected with a 50% decrease in the effluent 12.5% plus TBT exposed group and 25% decrease in the effluent 50% plus TBT, if compared with the TBT alone. Overall, our results give further support to the use of imposex in N. lapillus as a specific biomarker of TBT contamination. However, in areas of high inputs of sewage effluents, the assessment of TBT contamination by the use of the imposex phenomenon should ideally also include data on the tissue levels of butyltins. © 2008 Elsevier Inc. All rights reserved. 1. Introduction The presence of endocrine disrupting chemicals (EDCs) in the aquatic environment is a matter of great concern (Sumpter, 2005). The majority of the studies on EDCs have focused on the effects of estrogenic chemicals (EC) (natural steroid 17-β-estradiol (E2); alkylphenolic compounds; the synthetic estrogen Ethinylestradiol (EE2), etc.) because many of the observed effects (reduce testicular development and fertility, increase production of vitellogenin, presence of male fish feminization) are believed to result from disruption of this axis (Jobling et al., 2004). However, the best documented example of endocrine disruption (ED) in the wildlife is the masculinization of female gastropods by the antifouling agent tributyltin (TBT) (Matthiessen and Gibbs, 1998; Santos et al., 2005; Sumpter, 2005; Castro et al., 2007a). Since the late 1960s, TBT has been used as the active ingredient in many antifouling paints to prevent settlement of organisms on ship hulls (Fent, 1996). However, TBT was shown to be toxic to a wide variety of non-target organisms, which prompted the introduction of regulation banning its use as a biocide in ship antifouling paints (Fent, 1996; Ten Hallers-Tjabbes,1997; Santos et al., 2002; Gómez-Ariza et al., 2006). Even though the use of TBT has recently been constrained to all ship sizes (MEPC, 2001), TBT is expected to persist in the aquatic environments for many years (Dowson et al., 1993; Santos et al., 2004). Within the target organisms, molluscs are one of the most TBT- sensitive animal groups. In particularly susceptible neogastropods species, TBT concentrations as low as 0.5 ng Sn/L are sufficient to induce the development of male sex organs in female specimens, a phenomenon called imposex. In the intertidal gastropod Nucella lapillus, TBT concentrations of 5 ng Sn/L cause a gross malformation of the female genital system resulting in sterility (Gibbs and Bryan, 1986). Because of their capacity to develop imposex, in a dose- dependent manner, at extremely low TBT concentrations, neogastro- pods have been widely used as sentinel organisms to monitor trends in TBT contamination, and to evaluate the efficacy of regulations Comparative Biochemistry and Physiology, Part C 148 (2008) 87–93 ⁎ Corresponding authors. Tel.: +351 22 340 1800; fax: +35122 3390608. E-mail addresses: santos@ciimar.up.pt, filipe.castro@ciimar.up.pt (L.F.C. Castro). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.03.012 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc