Multilevel ecotoxicological responses of Allium cepa to atrazine and glyphosate applied individually and in combination

Glyphosate and atrazine are among the most extensively applied herbicides worldwide and are often detected together in aquatic environments due to their concurrent agricultural use. This study investigated their individual and combined effects on Allium cepa at environmentally realistic concentrations: 62.5-1000 µg L⁻¹ for glyphosate and 0.25-4 µg L⁻¹ for atrazine. Five mixtures were also tested: M1 (62.5 + 0.25), M2 (125 + 0.5), M3 (250 + 1), M4 (500 + 2), and M5 (1000 + 4). Multiple endpoints were analyzed, including germination, root elongation, mitotic index, chromosomal and mitotic abnormalities, cell-cycle phase distribution, sub-G₁ fraction, fluorescence intensity, light-scattering parameters (FSC, SSC), membrane integrity (Evans Blue assay), and mitochondrial activity (TTC assay). Both herbicides and mixtures triggered cytotoxic and genotoxic responses, though most mixtures did not exceed the toxicity of the individual compounds, suggesting non-interactive or saturable mechanisms. Mixture M5, containing concentrations comparable to those reported in agricultural surface waters, produced stronger responses for some biomarkers. Flow-cytometric and mitochondrial endpoints were the most responsive following mitotic index and chromosomal aberrations. On the other hand, germination, root elongation, and membrane integrity showed limited sensitivity. Overall, the data indicate that even concentrations within current environmental guidelines can impair cellular function in A. cepa. This study provides a novel integrated assessment of glyphosate, atrazine, and their mixtures at environmentally relevant concentrations by combining classical A. cepa cytogenotoxicity assays with flow cytometry, membrane integrity, and mitochondrial activity biomarkers.