Comet Assay on Daphnia magna in eco-genotoxicity testing

tDetection of potentially hazardous compounds in water bodies is a priority in environmental risk assess-ment. For the evaluation and monitoring of water quality, a series of methodologies may be applied.Among them, the worldwide used toxicity tests with organisms of the genus Daphnia is one of the mostpowerful. In recent years, some attempts were made to utilize Daphnia magna in genotoxicity testing asmany of the new environmental contaminants are described as DNA-damaging agents in aquatic orga-nisms. The aim of this research was to develop a highly standardized protocol of the Comet Assay adaptedfor D. magna, especially regarding the isolation of cells derived from the same tissue (haemolymph) fromnewborn organisms exposed in vivo. Several methods for haemolymph extraction and different CometAssay parameters were compared. Electrophoretic conditions were adapted in order to obtain minimumDNA migration in cells derived from untreated organisms and, at the same time, maximum sensitiv-ity in specimens treated with known genotoxicants (CdCl2and H2O2). Additional tests were performedto investigate if life-history traits of the cladoceran (such as the age of adult organisms that providenewborns, the clutch size of origin, the number of generations reared in standard conditions) and thewater composition as well, might influence the response of the assay. This study confirms the poten-tial application of the Comet Assay in D. magna for assessing genotoxic loads in aqueous solution. Thenewly developed protocol could integrate the acute toxicity bioassay, thus expanding the possibility ofusing this model species in freshwater monitoring (waters, sediment and soil elutriates) and is in linewith the spirit of the EU Water Framework Directive in reducing the number of bioassays that involvemedium-sized species.