As a result of intensive use, atrazine is one of the most commonly detected contaminants in surface waters in the Midwestern United States. Atrazine and two of its primary metabolites, desethylatrazine (DEA) and deisopropylatrazine (DIA), are routinely detected in aqueous systems throughout this area. Although research has been conducted on the toxicity of atrazine, limited information exists regarding the toxicity of DEA and DIA. Also, there is no information on the toxicity of atrazine to Diporeia spp., a keystone Great Lakes species under severe decline. We individually evaluated the acute and chronic toxicity of atrazine, DEA, and DIA on the amphipods H. azteca and Diporeia spp., and the unicellular algae Pseudokirchneriella subcapitata We also studied the sublethal effects of chronically exposed H. azteca to atrazine and DEA. Proteomic and metabolomic profiles of chronically exposed H. azteca and Diporeia spp. were studied in order to determine novel biomarkers of exposure and endocrine disrupting effects in freshwater amphipods. We found all concentration values that elicited 50% mortality (LC50) values were well above those reported to impact aquatic organisms and they were ranked atrazine \u3e DEA \u3e DIA. However, Diporeia spp. appears to be more sensitive to exposure to these chemicals compared to H. azteca. Identified proteins and metabolites suggest atrazine and its metabolites disrupt mitochondrial function. It also appears that glucuronidation may be utilized to detoxify atrazine in amphipods. Finally, our results do suggest hormonal disruptions in atrazine and DEA exposed amphipods. However, endocrine disrupting effects were never confirmed. This is the first study to use metabolomics and proteomics to study this phenomenon. Our results indicate that DEA and DIA should be utilized in atrazine risk assessments to avoid underestimating the impacts of atrazine on aquatic species