Impact of bisphenol A and ethinyloestradiol on sex, body condition and digit ratio of the marsh frog Pelophylax ridibundus in the mesocosm exposure system

This study examined the impact of two environmentally widespread endocrine-disrupting compounds (EDCs), bisphenol A (BPA) and 17α-ethinylestradiol (EE2), on the marsh frog Pelophylax ridibundus. The analysis focused on somatic condition, gonadal differentiation, and the second-to-fourth digit ratio (DR, 2D:4D), tested as a potential non-invasive biomarker of endocrine disruption. Tadpoles were reared in outdoor mesocosms and exposed weekly to three environmentally-relevant concentrations of BPA (BPA7 - 10⁻⁷, BPA8 - 10⁻⁸, BPA9 - 10⁻⁹ M) and one concentration of EE2 (10⁻⁹ M), acting as a positive control. The exposition was conducted in a static-renewal system. While neither treatment nor sex influenced snout-vent length (SVL) or body weight, Scale Mass Index (SMI) was significantly reduced in the highest concentration BPA group (-0.139 ± 0.036) and in EE2 (-0.173 ± 0.036), indicating impaired somatic condition. EE2 caused a strikingly female-biased sex ratio (≈9:1 F:M), increasing the odds of being female by 5.2-fold, whereas BPA did not affect sex ratios. Histological analyses revealed two intersex and one mixed-sex individuals in the EE2 group. In the number of BPA-exposed males, significant gonadal degeneration and rudimentary metamers were observed, with similar structures found in females, suggesting disturbed gonadal development. Although DR did not differ between sexes, both EE2 and BPA altered it in a size-dependent manner, with significant treatment × SVL interactions in hindlimbs (EE2, BPA7) and the left forelimb (EE2), where larger individuals exhibited lower ratios under exposure. These environmentally relevant findings demonstrate that BPA and EE2 profoundly disrupt somatic health, gonadal differentiation and sex-related traits in a non-model amphibian species, with clear implications for wildlife conservation.