Current perspectives on the in vitro cytotoxic effects of tebuconazole, imidacloprid, and microplastics: A comprehensive review

In vitro ecotoxicological approaches have gained relevance as alternative methods to investigate cellular mechanisms underlying the toxicity of environmental contaminants. This review systematically analyzed studies from the last 25 years evaluating the effects of microplastics (MPs), imidacloprid (IMI), and tebuconazole (TEB) on aquatic species using in vitro models. The survey showed that MPs dominated the literature (74.1%), followed by IMI (25.9%), while no in vitro studies were identified for TEB despite its documented environmental relevance. Among MPs studies, polystyrene was the most frequently tested polymer (50%), followed by polyethylene (22.7%) and environmental mixtures (13.6%). The year 2023 presented the highest number of publications, indicating increasing scientific attention. Regarding aquatic models, Oncorhynchus mykiss and Ctenopharyngodon idellus were most frequently studied, whereas species such as Cyprinus carpio , Mytilus edulis , and Danio rerio appeared less often. Overall, research remains disproportionately focused on isolated effects of IMI and MPs, while TEB and mixture scenarios especially MPs combined with pesticides are underexplored. In vitro approaches offer ethical and mechanistic advantages by reducing animal use and providing access to detailed molecular responses. Future studies should incorporate environmentally relevant mixtures and explore chronic, metabolic, hormonal, and epigenetic endpoints to enhance ecological relevance and strengthen aquatic risk assessments. • No in vitro studies exist on Tebuconazole for aquatic organisms. • Polystyrene and polyethylene are the most in vitro tested microplastic polymers. • O. mykiss and C. idellus derived cell lines are the most frequently used models. • Mixture scenarios combining MPs and pesticides remain critically underexplored.