Cytostatics as emerging pollutants in aquatic environments - risk assessment based on genotoxic effects in haemocytes of freshwater mussels

Abstract

Pharmaceutical compounds have begun to be considered as dangerous environmental pollutants, due to their widespread occurrence in wastewaters and their potential hazard towards the aquatic ecosystems. The comet assay was used to study acute impacts of most used cytostatic drugs on the haemocytes of widely distributed freshwater mussels Unio pictorum and Unio tumidus. After collection from unpolluted location, the mussels were held on accommodation for 10 days in controlled laboratory conditions before exposure to cytostatics. Groups of 5 mussels were exposed to 5-Fluorouracil, (0.04 – 40 μM), Cisplatin (0.004 – 4 μM), Etoposide (4, 40 and 100 μM) and Imatinib msesylate (0.04 - 40 μM). For positive control treatment with Cd was used (4, 40 and 100 μM), while as negative control mussels were held in control aquarium with clean water. Exposure was performed for 72h in aquaria. Comet assay is one of the major tools for the level of DNA damage assessment in ecogenotoxicology. It is based on single cell gel electrophoresis. In our experiments we performed alkaline version of comet assay described by Singh et al. (1988). Images of 250 nuclei per each concentration of test substance were analyzed with a fluorescence microscope and scored using analysis software (Comet Assay IV Image analysis system, PI, UK). Tail intensity was chosen as relevant measure of DNA damage. Our results indicated significant increase of DNA damage in haemocytes during treatment in selected concentration ranges for all cytostatics with the exception of Imatinib mesylate. Detected LOEC values were as following: 5-Fluorouracil – 52 μg/L, Cisplatin 12 μg/L and Etoposide – 24 mg/L. However, all effective concentrations of 5-FU, CP and ETO are higher than ones measured in surface water but still far below PNEC values which are currently used for the environmental risk assessment. This indicates that acute toxicity data might not be sufficient for prediction of adverse effects of substances, and that genotoxicity data should be also considered for the risk assessment.