LAUSR

The mixture of 5-chloro-2-methylisothiazol-3(2H)-one and 2-methylisothiazol-3(2H)-one, CMIT/MIT, is an isothiazolinone biocide that is consistently detected in aquatic environments because of its broad-spectrum usage in industrial fields. Despite concerns about ecotoxicological risks and possible multigenerational exposure, toxicological information on CMIT/MIT is very limited to human health and within-generational toxicity. Furthermore, epigenetic markers altered by chemical exposure can be transmitted over generations, but the role of these changes in phenotypic responses and toxicity with respect to trans- and multigenerational effects is poorly understood. In this study, the toxicity of CMIT/MIT on Daphnia magna was evaluated by measuring various endpoints (mortality, reproduction, body size, swimming behavior, and proteomic expression), and its trans- and multigenerational effects were investigated over four consecutive generations. The genotoxicity and epigenotoxicity of CMIT/MIT were examined using a comet assay and global DNA methylation measurements. The results show deleterious effects on various endpoints and differences in response patterns according to different exposure histories. Parental effects were transgenerational or recovered after exposure termination, while multigenerational exposure led to acclimatory/defensive responses. Changes in DNA damage were closely associated with altered reproduction in daphnids, but their possible relationship with global DNA methylation was not found. Overall, this study provides ecotoxicological information on CMIT/MIT relative to multifaceted endpoints and aids in understanding multigenerational phenomena under CMIT/MIT exposure. It also emphasizes the consideration of exposure duration and multigenerational observations in evaluating ecotoxicity and the risk management of isothiazolinone biocides.