The alkaline comet assay was tested on different microalgae: the clinofiagellates, Karenia mikimotoi and Alexandrium minuturn, and the diatom, Chaetoceros gracilis. The microalgae were exposed during their exponential growth to the model direct genotoxicant, hydrogen peroxide (1 h, 5 and 100 pM H2O2). Following H2O2 exposure, the comet assay was validated only for K. mikimotoi for which genotoxicity was observed from the lowest tested concentration of 5 pM with a concentration-dependent effect. C. gracilis was too small in size (4 mu m) to be correctly analysed. For A. minuturn, our lysis buffer was not strong enough to digest the cellulosic thecal plates. For K. mikimotoi, the comet assay was thus applied for the study of the genotoxic effects of different pesticides: epoxiconazole (as Opus formulation), chlorpyriphos-ethyl (as Dursban formulation) and endosulfan at 1, 10 and 100 pg of active substance/L for 24 h. Exposure to epoxiconazole in formulation resulted in an increase in the extent of DNA strand breaks at the highest tested concentration of 100 pg/L. Genotoxicity was also observed for chlorpyriphos-ethyl in formulation from 1 mu g/L (p < 0.05), with a significant increase at 10 pg/L. Endosulfan exposure resulted in DNA damage for K. mikimotoi nuclei. Genotoxicity was observed from 1 pg/L of enclosulfan and was not concentration dependent. (c) 2008 Elsevier Ltd. All rights reserved.