Cell phenotyping reveals chemical water disinfection treatment-induced molecular and morphological perturbations

Evaluating how different disinfectants influence the formation and toxicity of disinfection by-products (DBPs) is key to improving water treatment. Previous studies suggest that OXONE (potassium peroxymonosulfate) at ten times the minimum bactericidal concentration (MBC) provides more effective biofilm control compared to conventional chlorination and may serve as a promising alternative disinfectant. In this study, we investigated the changes in molecular composition, formation of DBPs, and human cell morphological responses in synthetic tap water (STW) treated with OXONE and calcium hypochlorite (Ca(OCl)₂) at MBC and 10 ×MBC. Ultrahigh-resolution mass spectrometry revealed that OXONE at 10 ×MBC enriched highly oxygenated CHOS species, increased hydrogen and sulfur content, and produced molecules with lower molecular weight and greater saturation. Ca(OCl)₂ increased CHOCl abundance, with chlorine content rising from 0.04 % at MBC to 0.11 % at 10 ×MBC, while maintaining molecular structures with similar degrees of unsaturation. DBPs analysis showed a 7.2-fold increase in CHOCl₂-DBPs under Ca(OCl)₂ at 10 ×MBC, predominantly in saturated/low-unsaturation structures, while OXONE increased sulfur-containing DBPs by 1.59-fold, mainly with lower aromaticity. Cell painting assay (CPA) in human U2OS cells revealed pronounced morphological changes and viability loss with OXONE at 10 ×MBC (Wilcoxon p < 0.05 vs Ca(OCl)₂), linked to microtubule disruption confirmed by β-tubulin staining. Integration of chemical and morphological data linked toxicity to sulfur-rich features, with CHOS compounds predominating and CHO contributing via OXONE-driven transformations, together driving the observed morphological disruptions. Overall, these findings reveal distinct chemical and cytotoxic effects of each disinfectant, highlighting the need to optimize treatment efficacy with health safety.