Primary tissue cultures from Unio pictorum were developed for utilization in ecotoxicology studies. Cytotoxic effects of selected plasticizers (BPA, DiNP, DiDP, DMP) were tested on digestive gland, gonad, gill and mantle cells. DiNP on gill and BPA on digestive gland were the highest and lowest cytotoxic compounds (IC50 0.111, 170.718 ppt). Mantle cells were the most suitable, reliable and easy to maintain cells; for toxicity assays. Unio primary cell culture provide valuable tool for testing/screening effects of xenobiotics in vitro, have potential for further development in terms of potential cryopreservation and stability comply with the 3R principle.