Anthropogenic contaminants such as polycyclic aromatic hydrocarbons (PAHs), tire wear particles, salts, and heavy metals enter aquatic ecosystems as runoff during rainfall or snowmelt events. These contaminants can interfere with physiological processes of organisms, including gene expression, to impact higher levels of biological organization (i.e., ecosystems). Although a significant amount of research has investigated the ecotoxicological effects of individual contaminants, the impacts of combinations of contaminants remains unresolved, especially at a transcriptomic level. This project aims to investigate the effects of a combination of road-related contaminants on the survival and gene expression of Daphnia magna. The gene expression analysis examines Daphnia’s transcriptomic patterns in response to pyrene, tire wear particle leachate, and their combination at five environmentally relevant concentrations. We exposed Daphnia to these treatments in a two-day, acute toxicity test, performed RNA-sequencing, and employed Weighted Gene Correlation Network Analysis (WGCNA). We identified and clustered over 20,000 genes into 21 eigengene modules. Preliminary analysis does not show any obvious correlation patterns of gene expression to contaminant or concentration level. Gene Ontology (GO) terms associated with each contaminant show that many GO terms were shared amongst all the treatments, such as DNA and metabolic processes, but tire wear particle leachate was also correlated additional unique biological processes such as biological regulation, response to stimulus, and various processes related to signaling and cell communication. Overall, this project aids in understanding the sublethal impacts of multiple stressors on aquatic organisms, integrates environmental relevance into toxicity tests, and ultimately helps to inform protection and monitoring of aquatic systems.