Do reproductive conditions influence responsiveness of vitellogenin-like protein induction in clams (Tapes philippinarum) exposed to 4-nonylphenol?

In a previous study, we demonstrated that exposure for 7 days of the clam Tapes philippinarum to 4-nonylphenol (NP) induced vitellogenin (Vg)-like proteins in both haemolymph and digestive gland of males. The aim of the present study was to evaluate NP estrogenic potency in sexually undifferentiated clams. Molluscs were exposed for 7 and 14 days to various sublethal NP concentrations (0, 0+acetone, 0.0125, 0.025, 0.05, 0.1 and 0.2 mg NP/L and 0, 0+acetone, 0.0125, 0.025, 0.05 and 0.1 mg NP/L, respectively) and Vg-like protein levels were evaluated in both haemolymph and digestive glands of clams by the alkali-labile phosphate assay (ALP). Haemolymph Ca2+ levels were also measured, this parameter being considered strictly related to the presence of Vg-like proteins. Exposure for 7 days to 0.2 mg NP/L resulted in statistically significant increases in Vg-like proteins (expressed as μg ALP/ mg protein) in both haemolymph and digestive gland, with respect to controls. Conversely, haemolymph Ca2+ levels did not show significant variations. In 14-days treatments, Vg and Ca2+ levels showed a similar increasing trend in haemolymph, although significant differences with respect to controls were found at 0.025 and 0.05 mg NP/L for Ca2+ only. A dose-dependent Vg increase was also observed in digestive gland, significant at 0.1 mg NP/L. Responsiveness of Vg induction was confirmed in quiescent clams, mostly when measured in digestive gland, indicating applicability of the biomarker throughout the whole reproductive cycle of animals in both laboratory and field studies. A relationship between Vg and Ca2+ levels was detected in prolonged exposure only.