Methotrexate (MTX) is a cytotoxic drug widely used in the treatment of tumors,autoimmune diseases and severe asthma.jen00883This drug has been frequently detected in the aquatic environment with concentrations up to μg/L levels.The MTX present in environmental water might be transformed and removed during chlorination disinfection treatment.In this work,the fate of MTX during aqueous chlorination was investigated in laboratory batch experiments,and the transformation products of MTX were identified.Aqueous solutions of MTX (1 mg/L) were chlorinated by sodium hypochlorite solution at room temperature under neutral pH conditions.Chlorinated products were pre-concentrated with solid-phase extraction (SPE) cartridges and determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).The reaction of MTX chlorination exhibited pseudo-first-order kinetics and the half-life time of MTX degradation was calculated to be 1.65 min,when the initial chlorine concentration was 2 mg/L.Two chlorinated MTX congeners,4-amino-3-chlorinated-N10-methylpteroylglutamic (monochloro-MTX) and 4-amino-3,5-dichloro-N10-methylpteroylglutamic (dichloro-MTX) were found in the chlorinated solution.Monochloro-MTX was successfully fractionated by high performance liquid chromatography (HPLC) and its structure was further identified using 1H nuclear magnetic resonance (NMR) analysis.The presence of the two products in real hospital wastewater was then examined and both compounds were detected.Finally,the effects of MTX and monochloro-MTX on the cell cycle progression in vitro were evaluated using zebrafish liver cell line.It was found that both compounds could inhibit the proliferation of zebrafish liver cells through S phase arrest and their effects on the cell cycle profile had no significant difference.