The impact of pollutants in the environment necessitates the improvement of monitoring techniques. Current detection of pollutants relies on analytical methods to capture their presence, which cannot provide any mechanistic or prognostic insight into their action. Consecutively, risk assessment is moving towards the incorporation of novel methodologies in regulatory practices. Daphnids, as sentinel species, have acquired a strong place in ecotoxicological studies mainly due to their geographic distribution, clonal reproduction and ecoresponsive nature. In this study, glyphosate, lithium and metformin were used as indicative pollutants representing different categories: herbicides, metals and pharmaceuticals. Daphnids were exposed to single pollutants, three combinations of binary mixtures and their triple mixture. Key enzyme activities and mortality were assessed at 24 and 48 hours on a range of concentrations. Lithium was the most toxic of the three chemicals and acute toxicity in mixtures was synergistically enhanced. In the triple mixture, activities of alkaline phosphatase and lipase were decreased dose-dependently, while no significant difference was observed for acid phosphatase. Glutathione S-transferase and beta galactosidase increased dose-dependently. These findings showcase metabolic alterations in the metabolism of daphnids in response to these chemicals and their triple mixture, which could be used as sensitive endpoints to predict and understand chemical pollution. Finally, a preliminary study of untargeted metabolomics showed clear metabolic shifts in response to the concentration of the triple mixture.