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Abstract The surface mining of oil sands deposits requires the removal of groundwater to stabilize the deposit (depressurization) and make it safe for mining. The chemistry and toxicity of deep groundwaters (from 45 to 144 m below an active mining operation) were characterized to determine if the release of groundwaters would pose a risk to a receiving aquatic environment. Concentrations of conventional chemicals such as nutrients and metals were generally below CCME chronic guidelines. Concentrations of oil sands naphthenic acids (NAs) varied depending on the method of measurement and were routinely >1 mg L−1. Groundwaters rarely caused lethality to fish and invertebrates in standard acute and chronic toxicity tests. Algal cell production was negatively correlated with chlorides and potentially negatively with NAs. Other chronic toxicity variations were less obviously correlated with measured chemistry. The groundwaters had moderately-high oxygen demand (2 to 33 mg L−1), likely associated with nutrients and organic substances, and thus have the potential to enrich receiving surface water environments if left untreated and depending on the receiving environment. This paper presents for the first time a comprehensive (3 year) pairing of water chemistry and toxicity data on groundwaters collected from aquifer depressurization wells below an active oil sands operation. These data will contribute to a better understanding of the environmental risk these waters potentially pose, and ultimately, to the improvement of water management strategies and the reduction of the overall surface mining footprint of oil sands operations. Graphical abstract Unlabelled Image Highlights • Oil sands groundwaters were collected from nine wells at between 45 and 119 m depth. • Groundwaters were generally non-acutely toxic. • Sublethal responses best explained by conventional substances (chlorides). • Groundwaters pose modest risk of local eutrophication to surface waters. [ABSTRACT FROM AUTHOR]