Assessment of Combined Methylparaben and Diethyl Phthalate Exposure on Developmental and Behavioral Toxicity in Zebrafish (Danio rerio) Embryos and Larvae

Introduction. Endocrine-disrupting chemicals (EDCs) such as methylparaben (MP), diethyl phthalate (DEP) are widely detected in aquatic environments due to their extensive use in personal care products. Effluent from salon and beauty parlour samples collected from Chennai, India, were characterized by GC–MS analysis, revealing more than 30 compounds and the co-presence of MP and DEP within a complex lipid-enriched matrix, establishing a real-world multi-EDC co-exposure scenario. Methods.This study evaluated the developmental, physiological, and neurobehavioral toxicity of MP and DEP in zebrafish (Danio rerio) embryos using OECD TG 236 guidelines. Embryos were exposed to concentrations of 10, 25, and 50 µg/L for individual compound exposures, and endpoints including mortality, heart rate, hatching success, morphological abnormalities, and behavior were assessed. Results Acute toxicity analysis revealed LC₅₀ values of 25.11 µg/L for MP and 36.60 µg/L for DEP, indicating higher toxicity of MP. At 100 µg/L, mortality reached 94% for MP and 71% for DEP. Both compounds induced significant bradycardia, with heart rates declining from 190 bpm (control) to 100 bpm (MP) and 120 bpm (DEP) at 50 µg/L. Hatching success decreased to 18% (MP) and 13% (DEP), accompanied by increased skeletal deformities. Binary mixture exposure (MP:DEP at 1:1, 1:9, and 9:1 ratios) produced enhanced embryotoxicity relative to individual compound groups, with locomotor activity reduced to 40,799 mm in the MP-dominant 9:1 mixture group, indicating synergistic neurobehavioral suppression. Behavioral assays demonstrated anxiogenic responses in DEP-exposed larvae, characterised by elevated light-zone residency and increased upper-tank occupancy consistent with hyperexcitability or anxiety-like phenotype, and non-monotonic alterations in MP-exposed larvae including mild hyperactivity at lower concentrations transitioning to hypoactivity and depression-like behavior at higher concentrations, consistent with hermetic endocrine modulation. Conclusion. These findings highlight the heightened risk of EDC mixtures and emphasize the need for mixture-based toxicological assessment.