Toxicity of 0.5 - 5 kDa disinfection byproducts: a total organic halogen-based normalization reveals overlooked risks in disinfected waters.

Disinfection of drinking water inevitably generates halogenated disinfection byproducts (DBPs), yet it remains unclear whether observed toxicity differences across molecular weight (MW) fractions reflect concentration load effects or intrinsic toxic potency. Here, we employed systematic MW fractionation (six fractions from < 0.5 to 10 kDa) combined with Chinese hamster ovary cell cytotoxicity testing and total organic halogen (TOX) normalization to decouple these factors. While the < 0.5 kDa fraction dominated overall cytotoxicity due to its high DBP concentration, TOX-normalized analysis revealed that the 0.5 - 5 kDa fraction exhibited intrinsic toxicity (toxicity per unit TOX) comparable to the < 0.5 kDa fraction, suggesting that higher MW fractions in the 0.5 - 5 kDa range, which have received less attention in previous studies, also contribute non-negligible toxicity when normalized by TOX. Their toxicity contribution scaled with their TOX concentration, making them potential risk drivers when present at substantial levels. Validation in real chlorinated and chloraminated waters confirmed this pattern. These findings demonstrate that DBP risk assessment should integrate both concentration and toxic potency, and highlight that the 0.5 - 5 kDa fraction warrants greater attention in future monitoring and control strategies.