ORIGINAL ABSTRACT

BACKGROUND: Glyphosate is the most widely used herbicide worldwide and is frequently applied with auxinic herbicides such as dicamba and 2,4-dichlorophenoxyacetic acid (2,4-D). Regulatory limits are largely based on single-compound toxicity but real-life exposure occurs as mixtures. OBJECTIVE: To evaluate whether chronic exposure to regulatory-relevant doses of glyphosate alone or combined with dicamba and 2,4-D during pregnancy and lactation induces neurobehavioral and histopathological alterations in maternal brain tissue. METHODS: Pregnant Wistar rat dams were exposed from gestational day-6 to postnatal day-21 to: 0mg/kg/day glyphosate (control group), 0.5mg/kg/day glyphosate (European Union ADI-Gly group), 50mg/kg/day glyphosate (European Union NOAEL-Gly group), or a mixture of glyphosate, dicamba, and 2,4-D at their respective European Union ADI levels (Combination group). Neurobehavioral assessment included open field, elevated plus maze, and forced swim tests. Brain tissues were examined using hematoxylin and eosin staining with semi-quantitative scoring. Statistical analysis employed one-way ANOVA with Dunnett post-hoc comparisons. RESULTS: Grooming behavior increased significantly in the NOAEL-Gly group and more markedly in the Combination group, consistent with anxiety-like alterations, while locomotor and depressive-like behaviors were unchanged. Histopathology revealed neuronal degeneration, perineuronal edema, and vascular congestion, most pronounced in the hippocampus of mixture-exposed dams. Structural alterations paralleled behavioural findings. CONCLUSIONS: Chronic exposure to regulatory relevant doses of glyphosate induces maternal neurotoxicity that is amplified by co-exposure to dicamba and 2,4-D, suggesting additive effects and underscoring the need to incorporate mixture toxicity into pesticide risk assessment.