Chemical mixtures of pesticides: A review of the recent findings with the zebrafish model.

The presence of pesticide mixtures in the aquatic environment is widely recognized as a cause for concern, given their inherent toxicity and the possibility of additive or synergistic interactions that may amplify the ecological risk. In this review, we focus on the use of zebrafish (Danio rerio), a vertebrate model widely used in biological research as well as in ecotoxicology assessment. The aim of this work is to critically investigate the recent findings on pesticide mixture toxicity published between 2020 and 2024, with studies systematically collected from Embase, PubMed Medline, Google Scholar, and Web of Science. After the screening and selection process, 88 papers were included in the final analysis. Binary mixtures on early-life stages of zebrafish (embryo and larvae) were the most frequently investigated, whereas ternary and more complex mixtures were less frequent. Most studies involved pesticides from different chemical classes and Modes of Action, particularly insecticides, fungicides, and herbicides. Out of the 77 papers that compared the toxicity of the mixture to that of the single substances, 56 showed higher toxicity from the mixture for at least one endpoint, suggesting additive or synergistic interactions. The main detected endpoints included mortality, oxidative stress, behavioral alterations, malformation, and neurotoxicity. Oxidative stress and gene expression dysregulation were common mechanisms observed. Overall, the reviewed literature highlights that pesticide mixtures may induce biological effects that cannot always be predicted from single compound exposures, emphasizing the need to integrate mixture toxicity and Effect-Based Methods such as those based on zebrafish in future environmental risk assessment strategies.