In the perspective of a biomonitoring application for assessing genotoxicity of freshwater ecosystems, the Comet assay has recently been developed on spermatozoa in the amphipod Gammarus fossarum, in order to propose a sensitive and reliable genotoxicity biomarker in an ecologically relevant freshwater species. The appropriate use of a genotoxicity biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. We propose a procedure for which the lowest biomarker variability related to methodological and intrinsic biotic factors is obtained and a reference value of biomarker basal response taking into account its spatio-temporal changes has been defined. A strong impact of spermatogenesis status and exposure time on the response to genotoxicant pressure was observed. These reports led us to select a standard organism, i.e., the mature male gammarid in precopula. No effect of temperature and conductivity on baseline DNA damage was observed in the laboratory for the tested range (6-24 °C and 300/600 μS cm⁻¹). Similarly, no spatio-temporal change relative to season or the physico-chemical characteristics of the water was recorded during the field survey. On the basis of these results, a reference level with maximal threshold values has been proposed for the standard gammarid.
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