Hydraulic fracturing fluid are complex mixtures containing high concentrations of salts (up to 330,000 ppm), organic, and metal contaminants. However, little data exist on the potential mechanisms of toxicity of these flowback and produced wastewaters (FPW) on aquatic biota. Juvenile rainbow trout were exposed to either control, FPW (2.5 or 7.5%), FPW that had been treated with activated charcoal (AC), or a custom salt-matched control (SW; replicating only the salt content of FPW) for 48 hours. Gill histology revealed decreases in interlamellar cell mass (ILCM) and mean lamellar length in all treatments (FPW, AC and SW) compared to control, indicative of hyperosmotic stress. Liver CYP1A1 activity was significantly elevated by 7.5-fold in the FPW 7.5% treatment only, indicative of Phase I metabolism. Superoxide dismutase activity significantly decreased in the gills to all treatments with the lowest activity occurring in the 7.5% FPW group. Catalase activity increased in liver with the highest values noted in fish exposed to 7.5% FPW. No changes were observed with respect to glutathione-S-transferase, while increased lipid peroxidation was only observed in both FPW treatments (2.5, 7.5%). These data suggest a characteristic signature of FPW impact which may help in risk assessment and biomonitoring of FPW spills.