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Advanced oxidative processes (AOPs) are procedures used for the treatment of wastewater based on the generation of free radicals, such as hydroxyl (•OH) and carbonate anion (CO3•−) radicals. However, although contaminants are degraded in these processes, the by-products generated in this transformation can be a greater source of toxicity than the original compound, making ecotoxicological tests essential for monitoring the efficacy of these treatment processes. In this study, we examined the ecotoxicity of AOP by-products generated using peracetic acid (PAA) and sodium carbonate, with and without solar radiation, for the degradation of methylene blue dye, using the planarian Girardia tigrina as a test organism. Ecotoxicological tests evaluated the acute toxicity of the generated by-product in terms of lethal concentrations (LC50). Although in both assays the degradation of the dye was greater than 99%, higher toxicity was observed in the assay using PAA and carbonate in the absence of radiation. From the results obtained, we conclude that the by-product generated from the degradation of methylene blue dye by peracetic acid and sodium carbonate, with and without solar radiation, can pose risks to aquatic ecosystems if released directly into water bodies. [ABSTRACT FROM AUTHOR]