The sperm cell toxicity test with Paracentrotus lividus is widely used in Italy to assess the quality of complex aqueous matrices, but at present there is a shortage of standardized methodologies for the bioassay performance. In this work several critical points were considered, in order to verify the reliability of this bioassay and to improve its standardization. In particular, we have studied the differences in EC50 values at different sperm: egg ratios and with different quantities of gametes; the influence of sperm and eggs on fertilization rate and the influence of different recipients materials (plastic or glass). At the same time, it was evaluated if the obtained EC50 values were in accordance with those reported in literature. Experiments performed yielded EC50 values for copper ranging from 16.54 microg/l to 69.59 microg/l, with a total mean value +/- SD of 39.79 microg/l +/- 11.17. These data fit with those found in literature for other echinoid species. According to the same authors, poor fertilization was probably due to the effects on sperm and not on eggs, and moreover the linear regression analysis performed on each experiment suggested that the best amount of eggs to use in the test was 1000. Results from the comparison between the performance of the test in plastic or glass tubes showed an higher success in fertilization rate using borosilicate glass tubes.