VITELLOGENIN-LIKE PROTEINS IN CLAMS AND NONYLPHENOLLEVELS IN WATER, SEDIMENTS AND BIOTA: THE LAGOON OFVENICE AS A CASE STUDY

Nonylphenol (NP) is commonly used in the production of nonylphenol ethoxylates (NPEs), NP phosphites and insecticides. As a consequence of their widespread use, particularly of NPEs as nonionic surfactants in household and industrial detergents, large quantities of these compounds are discharged into aquatic environments, either directly from untreated effluent or indirectly from sewage treatment plants (STPs). In aquatic ecosystems, NPEs biodegrade to de-ethoxylated intermediates, whose final product is NP. It is known that NP can have estrogenic effects in aquatic organisms, being able to mimic the action of endogenous estrogens by binding to estrogen receptors. As a consequence, it can induce vitellogenin (Vg) synthesis, the major precursor of the egg-yolk proteins of oviparous organisms. In this study, Vg induction was evaluated in the clam Tapes philippinarum seasonally collected (from October 2003 to June 2004) in 3 sites of the Lagoon of Venice: Marghera (MA), characterised by high industrial contamination levels, Campalto (CA), nearby a STP, and Poveglia (PO) close to the lagoon inlet, which was considered as a reference site. Vg-like proteins were determined in the haemolymph and digestive gland of sexually differentiated males and females when it was possible to distinguish them by microscopic observation of a smear of gonadal tissue. In the haemolymph Ca2+ levels were also measured, this parameter being strictly related to the presence of Vg-like proteins. During clam samplings, water and sediments were also collected to determine the concentrations of NP, which was chosen as a marker of the presence of anthropic sources of estrogenic compounds. Both male and female clams collected at CA and MA generally showed significantly higher Vg-like proteins in haemolymph with respect to specimens from Poveglia throughout the study period. Significant differences in Vg-like proteins were also detected in digestive gland of clams from the 3 sampling sites. In particular, sexually undifferentiated clams collected at CA showed the highest protein levels in January 2004, suggesting that endocrine alterations mostly occurred far from the reproductive period of animals. Ca2+ levels were generally higher in haemolymph of both female and males clams from CA and MA. Ca2+ levels showed also a clear seasonal trend, with higher values in spring-summer period than in autumn-winter. In the same campaign NP levels were determined in sediment, water and Tapes philippinarum samples. MA and CA sampling sites were found to be more contaminated than PO. The highest concentrations in the range of 64-246 ng NP/L d.w., were always measured in MA sediments while the lowest concentrations in the range of 27-99 ng NP/L d.w. were measured in sediments from PO. Nonylphenol was found to be the most abundant compound in all sediment samples in every sampling season. Sampling sites showed a difference in concentrations but none in typology of pollution that seems to be mostly related to industrial origin. Indeed NP/NPE ratios that indicate difference in degradation processes, resulted always homogeneous (always > 1) remarking the same typology and origin of the sediment pollution in the Venice lagoon. The NP concentration in water samples collected from different sites in different seasons was as often as not close to the detection limit(50 ng/L). The concentrations of NP in the range of 60-180 ng/g d.w. measured in clams, were instead of the same order of magnitude in the three sampling sites, the highest values were found in January and April in two sites (PO and MA) whereas the lowest value was measured in June in samples collected at CA. The concentrations of NPE1 and NPE2 were always below the limit of detection, thus reflecting on one hand the different lipophilic power of the compounds, on the other hand their water concentrations. The analyses on T. philippinarum showed a seasonal trend characterised by a raising in the NP concentration measured in soft tissues of clams collected from October to April, followed by a prompt decrease in June, when the sexual maturity and spawning phase are reached. This study demonstrates that Vg-like protein levels significantly increased in both hemolymph and digestive gland from sexually differentiated and undifferentiated clams from the most NP contaminated sites. Considering that endocrine disruption due to exposure to estrogenic compounds, such as NP, may cause fertility reduction, alteration in sex ratio, and a decrease in reproductive rate, a condition of potential risk for clam populations in estuarine areas is highlighted.