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The Mithi River begins at Vihar Lake and flows through the industrial hub of the city of Mumbai, India, and merges with the Arabian Sea at Mahim Creek. The current study was carried out to assess the ecotoxicological effects of the Mithi River surface water in zebrafish (Danio rerio) embryos. Water samples were collected from ten sampling sites (S1 to S10) located along the course of the Mithi River. The toxicity of water samples was assessed using a zebrafish embryo toxicity test (ZFET). Water samples were diluted from all sites at 1:0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128 times. The lowest and highest LDil 20 values for 96 h were estimated as 9.16 and 74.18 respectively for the S2 and S5 sites. The results of embryotoxicity and teratogenicity assays indicated a significant difference (p < 0.0001) between embryos exposed to control and sampling sites (except S1) for various endpoints such as mortality, egg coagulation, pericardial edema, yolk sac edema, tail bend, and skeletal deformities. The histopathological analysis revealed various lesions, ascertaining the toxic effects of water samples. The comet assay revealed significantly higher DNA damage (except S1) in embryos exposed to sites S5 and S6 with OTM values of 4.46 and 2.48 respectively. The results indicated that the Mithi River is polluted with maximum pollution load at the middle stretches. The study further indicated that the pollutants in the Mithi River (except S1) could potentially be hazardous to the aquatic organisms; therefore, continuous biomonitoring of the river is needed for its revival., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)