Toxicological assessment of plasticizer mixtures from treated sewage effluent in zebrafish embryos

Phthalate plasticizers are ubiquitous environmental contaminants frequently detected in treated sewage effluent (TSE). Yet, their combined developmental and neurotoxic effects at environmentally relevant concentrations remain insufficiently studied, particularly in water-scarce regions such as Qatar. This study investigated the effects of a plasticizer mixture (PM) containing diethyl phthalate (DEP), diisobutyl phthalate (DiBP), dibutyl phthalate (DBP), di(2 ethylhexyl) phthalate (DEHP), and diisononyl phthalate (DiNP) at concentrations measured in TSE using zebrafish (Danio rerio) embryos. Embryos were exposed to PM concentrations ranging from 28 to 600 ng L⁻¹, alongside dimethyl sulfoxide and control groups. Developmental toxicity was assessed by survival, hatching, gross morphology, and morphometric parameters, while neurobehavioral effects were evaluated using locomotor activity assays. Neurodevelopmental gene expression was quantified by reverse transcription quantitative real-time polymerase chain reaction. The PM exposure caused delayed hatching at 72 hpf and induced morphological abnormalities, including yolk sac edema, tail flexure, and reduced eye area. Morphometric analysis showed yolk sac enlargement and altered growth patterns. PM-exposed embryos also exhibited significant hyperactivity. At the molecular level, PM downregulated genes involved in cholinergic signaling, axonal growth, neuronal differentiation, and glial development, while bdnf was upregulated, suggesting a compensatory neurotrophic response. The association between locomotor hyperactivity and altered gene expression indicates a potential mechanistic link between molecular disruption and behavioral toxicity. These findings demonstrate that environmentally relevant plasticizer mixtures can disrupt zebrafish embryonic development and neurodevelopment, emphasizing the need for improved monitoring and regulation of phthalate contamination in TSE.