Occurrence of intersex and biomarker genesis in wild and caged carp in two stretches of the Po river.

The Po river, the principal Italian watercourse, and its affluent collect compounds that can act as endocrine disrupters (EDs) in fish community. Lambro River, is the most polluted tributary that drains a heavily industrialised and densely inhabited sub-basin of the main river. Recently, Lambro contamination by PCDDs and PCDFs, at concentrations ranging from 121 to 814ng/kg dry weight sediment, was described. Previous study showed the presence of high percentage of intersexuality and altered hepatic function in barbels sampled in the stretch of the Po river, downstream the confluence of the Lambro river. This study, part of the Easyring project, was focused on two stretches of the Po river, upstream and downstream the confluence with the Lambro river, supposed to be interested by different levels of ED contamination. The experimental approach was planned on both wild and caged carp, in order to compare chronic and acute effects on fish reproductive metabolism. Sampling of wild fish has been carried out in spring (April) and autumn (October) by boat electrofishing and nets in the two sampling sites. One-year old carp (K1) were caged for two months in the same sites. Gonads and liver fragments fixed in Bouin’s fluid, paraffin and/or resin (Kulzer 7100) embedded, were stained with haematoxilin-eosin or toluidin blue, respectively. Blood was collected by the caudal vein, centrifuged at 3000xg for 10’, added with protease inhibitor cocktail (5% v/v, Sigma, P8340). Oestradiol-17 (E2), testosterone, 11-ketotestosterone (T) and vitellogenin (Vtg) plasma levels were measured by ELISAs. Liver fragments were immediately frozen in liquid nitrogen for 7-ethoxyresorufin O-de-ethylase (EROD) activity measurement. Ovaries characterized by the presence of spermatogenic cysts amongst previtellogenic oocytes were found in wild carps, both upstream and downstream. High E2 plasma levels (>1ng/ml) and low T plasma levels (<0.5ng/ml) were measured in several active males in both sites. Vitellogenin concentration higher than 1mg/ml were measured in 10% and 18% males, respectively. Both male and female juveniles showed gonad alterations, such as occurrence of spermatogenic cysts in ovary and altered organization of lobules in testis in about 18% fish caged downstream, in concomitance with the highest concentrations of EDs (nonylphenol mix, bisphenol A, teroctylphenol). The induction of cytochrome P4501A, assayed as EROD activity, showed a marked response (P<0.05) in the same fish. Moreover, plasma Vtg were significantly higher in carp caged downstream. Data obtained on caged fish confirm the occurrence of different environmental conditions in the two stretches of the Po river analyzed in this study, as indicated by chemical analyses. Studies based on wild fish monitoring, even if essential for the evaluation of the chronic effects of pollutions on wild fish population, confirmed their uneasy interpretability because several factors like wide individual variability and possible migration of the test species.