Single and combined exposures to glyphosate and 2,4-D herbicides: impacts on membrane integrity, cell-cycle dynamics, and nucleolar activity in Allium cepa

This study aimed to examine the cytogenotoxic effects of attributed to exposure to herbicides glyphosate and 2,4-D, applied individually and in combination, on root meristematic cells of Allium cepa. The tested concentrations (0.1, 1, 10, 100, or 1000 μg/L) represent environmentally realistic levels frequently detected in freshwaters. Mixtures contained equimolar concentrations of each herbicide: M-1 (0.1 + 0.1), M-2 (1 + 1), M-3 (10 + 10), M-4 (100 + 100), and M-5 (1000 + 1000). Cell viability, mitotic index, chromosomal abnormalities, and micronuclei frequencies, as well as the number of nucleoli per nucleus were determined, together with cytotoxic parameters analyzed by flow cytometry. When applied individually, both herbicides exhibited cyto- and/or genotoxic activity. The mixtures reduced cell viability and induced cytotoxic and genotoxic responses, including mutagenic effects in M-1, M-3, and M-4. Nucleolar alterations, cell cycle delays, and cell death occurred with all treatments. Most mixture responses were equal to or greater than those of the individual herbicides. Data demonstrated that, even at environmentally relevant concentrations, combined exposure to glyphosate and 2,4-D intensified cytogenotoxic effects, reinforcing the importance of the need to consider mixture toxicity in environmental risk assessments.