Ultrasound‐Enhanced Chlorination of Methylene Blue: Reactive Species Contributions, Pathways, and Integrated Toxicity Assessment

Although the degradation of refractory organic compounds by ultrasound (US) combined with chlorination (US/chlorination) has been proven effective, research on its multifaceted degradation mechanisms still remains limited. This study explored the synergistic effect on an azo dye, methylene blue (MB), by US/chlorination in situ. Results showed that MB degradation rates reached 92.0% within 120 min under the following conditions: pH 5.1, US power density 1500 W/L, initial MB concentration 10.0 mg/L, chlorine dosage 8.8 mg/L, and reaction temperature 25°C. Kinetic analysis, radical quenching experiments, and electron paramagnetic resonance (EPR) experiments demonstrated that radicals, including HO·, Cl·, ClO·, and O 2 − ·, collectively accounted for 55.0% of the overall MB degradation. Additionally, five degradation intermediates (DIs) of MB were identified by UHPLC/MS, with demethylation, oxidative cleavage of the thioether bond, ring‐opening of the benzene rings, and decarboxylation being the dominant pathways. Furthermore, an integrated toxicological assessment of MB and its DIs was conducted using both Ecological Structure–Activity Relationship (ECOSAR), T.E.S.T. software, and toxicogenomics assays. Toxicity predictions from ECOSAR and T.E.S.T. indicated that structurally simplified DIs tend to be less toxic. This was reinforced by toxicogenomics assays based on the Transcriptional Effect Level Index (TELI), which revealed generally low genotoxicity in MB and its DIs, with no significant alterations beyond the overexpression of a small subset of genes. It suggests that US/chlorination treatment exhibits greater potential for the degradation of dye contaminants due to its exceptional performance and easy operation; the multi‐method toxicity assessment strategy offers a valuable reference for more comprehensive and reliable evaluation results.