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In this study, a spatial and temporal survey at three sites located in the "canals" of the Venice historic centre (Italy) and at a reference site was undertaken to evaluate stress effects on mussels sampled in the Venice urban area, where raw sewage is discharged without treatment directly into the water. A battery of biomarkers (metallothionein, micronuclei, condition index and survival in air) was used to evaluate the stress condition of the animals. At the same time the alkali-labile phosphate assay (ALP) was performed in mussel' hemolymph with the aim to find an estrogenic effect biomarker in this mussel species. Biomarker results showed an impairment of the general health condition in the mussels coming from the urban area, in agreement with the chemical analysis. Significantly higher level of the ALP was found in male mussels sampled in April in the urban area, in comparison with the ones from the reference site (P<0.001). Finally, the PCA proved an easy and useful tool to summarize the obtained results, also able to classify the data to indicate a pollution gradient in the Venice urban area.