Tailings ponds in northeastern Alberta, Canada contain massive amounts of oil sands process water (OSPW) that cannot currently be released due to the toxicity of some components. Limited space and the need for reclamation of oil sands operation sites will necessitate the release of OSPW in the near future. Knowledge of the composition and toxicity of OSPW is lacking yet is crucial for both risk assessment and management planning. This study examines chronic toxicity of a mixture of OSPW components sodium naphthenate and naphthenic acid (NA) to nymphs of the mayfly Hexagenia spp. in control and polycyclic aromatic hydrocarbons (PAH)-spiked sediment treatments. The objective of this study was to determine whether the addition of the PAH-spiked sediment significantly contributed to or masked responses of these sensitive mayflies to mixtures of NA. Mean survival in nymphs exposed to NA and PAH-spiked sediment treatments was reduced by 48% compared to those exposed to the NA mixture alone. Lethal responses were observed in all of the PAH-spiked sediment treatments. However, within PAH-spiked and control sediment treatments, there was no significant difference in nymph survival due to NA concentration, indicating that changes in survivorship were predominantly a reflection of increased mortality associated with sediment PAHs and not to the NA mixture treatment. Sublethal effects on body segment ratios suggest that mayflies exposed to NA and PAH-spiked sediment, as well as those exposed to the highest NA concentration tested (1 mg/L) and control sediment, made developmental trade-offs in order to emerge faster and escape a stressful environment. These results reveal that the release of OSPW to the surrounding environment could cause a reduction in mayfly populations. Mayflies provide ecosystem services and are an important food source for higher trophic levels in both the aquatic and terrestrial communities.