Haloacetonitriles (HANs) are an emerging class of nitrogen-based disinfection by-products (DBPs) and reported up to a maximum of 41 µg/L in large occurrence studies. An interest in HANs has been growing because they have been found to be more cyto- and genotoxic than the regulated carbon-based DBPs and have been recently reported as key drivers in overall drinking water toxicity. At room temperature, it is well known that HANs degrade to haloacetamides and haloacetic acids via neutral and base-catalyzed hydrolysis. However, HAN degradation in cell culture conditions used in in vitro studies has not been well characterized. In this study, we evaluated the stability of 8 HANs under cell culture conditions used in in vitro toxicological studies (370C, pH=7.4, for 3 days). Mono HANs were found to be stable during the incubation period on the other hand the stability of di- and tri- HANs declined significantly. Di- and tri- HANs transformed to their corresponding haloacetamides and haloacetic acids, which are reported to be less cytotoxic than HANs. These results suggest that reported HAN toxicity values are possibly underestimated. For future chronic toxicological studies, we recommend considering changing cell culture media for di- and tri-HANs on a daily basis. This will provide a more accurate measurement of HAN toxicity.

Haloacetonitrile stability in cell culture conditions and implications for in vitro toxicological studies

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