Sex steroid and vitellogenin plasma levels in carp larvae exposed to “environmental” mixture and sediments of the Po river (Italy).

Po River, the principal Italian water course, and its affluent receive compounds that can act as endocrine disrupters (EDs) in fish community. The Lambro River is a tributary of the Po River and drains a heavily industrialised and densely inhabited sub-basin of the main river; the stretches located upstream and downstream of their confluence are potentially interested by different levels of ED contamination. Aim of this work was to evaluate the effects of a long term exposure to a synthetic mixture of six EDs on gonad differentiation and steroid level modulation in carp larvae. Vitellogenin (Vtg) plasma level, was chosen as biomarker for estrogenic compound effects. Carp larvae (n=400; 3.7cm SL; 1.4g BW) were acclimated to lab conditions and test temperature (25°C), and then distributed among three treatments consisting of: a synthetic mixture (Mix; n=130) of EDs (equivalent to “1xLambro”), downstream River Po sediment (Sed; n=130) and a control (C; n=100). Estrone (E1), estradiol-17b (E2), estriol (E3), nonylphenols (NPs), bisphenol A (BPA), and t-octylphenol (t-OP) were used for the mixture. These chemicals were previously identified in the most estrogenic fraction of the Lambro water, obtained combining a HPLC fractionation procedure with in vitro assays. The mixture was prepared in order to mime environmental relevant concentrations according to the mean concentrations determined over a 15-day monitoring study on the River Lambro. Exposures were run under flow-through conditions (2.5 water renewals). After five months of exposure, carp were anaesthetized, blood was taken from the caudal vein and plasma was analysed for Vtg and sex steroids: oestradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11KT) by ELISA. Gonad and liver fragments were fixed in Bouin’s fluid for histological examination. Total weight (TW) and standard length (SL) were recorded. One way ANOVA was performed. A Bonferroni’s method was used and, when p<0.05, differences were considered to be significant. Mix and Sed exposed larvae showed TW and SL significantly lower and higher than control, respectively. Control and Sed exposed larvae showed only differentiated gonads with a sex ratio of approximately 1:1. Mix exposed fish showed about 15% of specimens with still undifferentiated gonads and out of 30% of differentiated males with previtellogenic oocytes in the testis. Significant increase in E2 and Vtg plasma concentrations were measured in Mix, but not in Sed exposed total fish, while T and 11-KT significantly decreased. Oestrogen/androgen ratio (E2/11-KT) was significantly higher in the same group. Parallel results were obtained considering differentiated males alone. Differentiated females showed significant increase of E2 and Vtg plasma concentrations and altered steroid ratio, but no variation in androgen levels. Sediment exposed fish only showed a slight decrease of Vtg concentration.