Daphnia magna sub-lethal exposure to phthalate pollutants elicits disruptions in amino acid and energy metabolism

Phthalic acid esters (PAEs) are a class of chemicals that are usually incorporated as additives in the manufacturing of plastics. PAEs are not covalently bound to the material matrix and can, consequently, be leached into the environment. PAEs have been reported to act as endocrine disruptors, neurotoxins, metabolic stressors, and immunotoxins to aquatic organisms but there is a lack of information regarding the impact of sub-lethal concentrations to target organisms. The freshwater crustacean Daphnia magna, a commonly used model organism in aquatic toxicity, was exposed to four phthalate pollutants: dimethyl phthalate (DMP), diethyl phthalate (DEP), monomethyl phthalate (MMP), and monoethyl phthalate (MEP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed in a targeted metabolomic approach to quantify polar metabolites extracted from a single Daphnia body. Individual metabolite percent changes and hierarchical clustering heatmap analysis showed unique metabolic profiles for each phthalate pollutant. Metabolite percent changes were mostly downregulated or presented opposing responses for the low and high concentrations tested. Meanwhile, pathway analyses suggest the disruption of related and unique pathways, mostly connected with amino acid and energy metabolism. The pathways aminoacyl-tRNA biosynthesis, arginine biosynthesis, and glutathione metabolism were disrupted by most selected PAEs. Overall, this study indicates that although phthalate pollutants can elicit distinct metabolic perturbations to each PAE, they still impacted related biochemical pathways. These chemical-class based responses could be associated with a common toxic mecha-nism of action. The reported findings show how targeted metabolomic approaches can lead to a better under-standing of sub-lethal exposure to pollutants, revealing metabolomic endpoints do not hold a close relationship with traditional acute toxicity endpoints.