The co-occurrence of pesticides in aquatic ecosystems highlights the need for studies investigating their potential toxicity as mixtures to the aquatic biota. Well-designed studies are essential to assess the presence and toxicity of relevant pesticide mixtures, particularly those such as the chloroacetamide herbicide metolachlor (MET), the triazole fungicide epoxiconazole (EP) and the diamide anthranilic insecticide chlorantraniliprole (CAP), which have not been previously tested, and whose co-occurrence is possible in waters close to cultivated areas. A solid phase extraction ultra-performance liquid chromatography-tandem quadrupole mass spectrometry method was developed to quantify equivalent toxicity concentrations for CAP, EP, and MET in artificial freshwater during acute toxicity tests. Compounds were separated within 1.30min, showing linearity over the calibration ranges of 2-150 µgL-1 for CAP and 50-3000 µgL-1 for EP and MET. Detection and quantification limits were (µgL-1): 0.001 and 0.0037 MET; 0.000038 and 0.00013 EP; and 0.002 and 0.007 CAP, respectively. Precision and accuracy met intra-assay validation requirements. Recoveries were tested at low and high concentration levels and were between 77% and 120%. Additionally, matrix effect showed different behavior among compounds. In an acute toxicity test proposed, MET and EP remained stable (24h), while CAP decayed 27% ± 4% in the same period. The method proved effective despite different concentrations in toxicity testing design.
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