Life-stage-dependent sensitivity of zebrafish (Danio rerio ) to estrogen exposure Gerd Maack * , Helmut Segner 1 UFZ Centre for Environmental Research, Department of Chemical Ecotoxicology, Permoserstr. 15, D-04318 Leipzig, Germany Received 11 March 2004; received in revised form 6 September 2004; accepted 9 September 2004 Abstract The aim of this study was to identify periods in zebrafish (Danio rerio ) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE 2 ) during three stages of gonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafish colony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates into either testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). The consequences of stage-specific exposure to EE 2 were assessed by determining time to first spawning, fecundity (number of eggs per female per day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced a delay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditic stage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experiment pointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a second experiment, the concentration dependency of the EE 2 effects was evaluated by exposing zebrafish during the gonad transition stage (43–71 dpf) to 1.67, 3 or 10 ng EE 2 /L. Significant effects of EE 2 on adult reproduction were found with 3 and 10 ng EE 2 /L, but not with 1.67 ng/L. Histological examination of the gonads revealed that at termination of EE 2 exposure (71 dpf), all individuals in the 3 and 10 ng EE 2 /L treatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish with ovaries and with testes were found. Thus, EE 2 exposure during the gonad transition stage seems to have no persistent effect on gonad histology but on reproductive capabilities. D 2004 Elsevier Inc. All rights reserved. Keywords: Zebrafish; Danio rerio; 17a-ethynylestradiol; Gonadal development; Gonad histology; Reproduction; Sexual differentiation; Stage-specific exposure 1. Introduction There is considerable concern about the potential of both natural and man-made environmental substances to interfere with the endocrine system of vertebrates and thereby to modulate or disrupt developmental and reproductive pro- cesses (Colborn and Clement, 1992). Among these so-called endocrine-disrupting compounds (EDCs), substances with estrogenic activity have attracted most attention. Particularly for fish, which show high plasticity in phenotypic sexual differentiation (Devlin and Nagahama, 2002), a number of laboratory and field studies have demonstrated that estro- gen-active compounds are able to disturb sexual develop- ment and reproductive capabilities (Gimeno et al., 1998; Tyler et al., 1998; L7nge et al., 2001; Segner et al., 2003). The developmental and reproductive effects of estrogen- active substances are depending on the precise time in the life cycle when the endocrine signal is being released at a 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.09.004 * Corresponding author. Current address: Department of Biological and Chemical Sciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter, EX4 4PS, United Kingdom. Tel.: +44 1392 263796; fax: +44 1392 263700. E-mail address: g.maack@ex.ac.uk (G. Maack). 1 Current address: Centre for Fish and Wildlife Health, University of Bern, L7nggass-Str. 122, CH-3012 Bern, Switzerland. Comparative Biochemistry and Physiology, Part C 139 (2004) 47–55 www.elsevier.com/locate/cbpc