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• There was general deterioration of the health and reproductive status of fish collected downstream of the effluent fallout compared to upstream populations. • Expression of the majority of transcripts measured in fathead minnows downstream of the effluent fallout was significantly downregulated, which supports observations of general health deterioration. • Exposure was characterized by complex mixtures of contaminants including several pharmaceuticals and personal care products as well as ammonia and nutrients. • No significant exposure to estrogenic compounds occurred, which was in accordance with a lack of estrogenic effects in exposed fish. • Fish in small, semi-arid stream ecosystems are at elevated risk from exposure to contaminants released from municipal wastewater effluents. Their unique hydrological and climatic conditions render surface water systems in the southern Canadian Prairies at an elevated risk from exposure to contaminants released from municipal wastewater effluents (MWWEs). The aim of this study was to characterize the potential health effects and their underlying molecular mechanisms in populations of fathead minnow (Pimephales promelas ; FHM) in Wascana Creek, an effluent dominated stream in Southern Saskatchewan, Canada. Studies were conducted during the spawning season in 2014 and 2015 to assess responses in terms of overall health, reproductive functions, plasma sex steroid hormone levels, and expression of selected genes along the hypothalamus-pituitary-gonadal axis. FHM downstream of the effluent fallout had lower gonadosomatic indices and significantly greater hepatosomatic indices compared to upstream populations. In both male and female FHMs, significantly greater occurrence and severity of gonadal degradation and delayed maturation were observed in downstream fish compared to upstream fish. Downstream males also displayed lower scores of secondary sexual characteristics and a decreasing trend in plasma 11-ketotestosterone levels. Interestingly, no indications of exposure to estrogenic compounds, such as occurrence of testicular oocytes were observed, which was in accordance with the lack of presence of key biomarkers of estrogenic exposure, such as induction of vitellogenin. In general, expression of the majority of transcripts measured in FHMs downstream of the effluent fallout was significantly downregulated, which supports observations of the general deterioration of the health and reproductive status of these fish. Chemical analysis indicated that 10 pharmaceuticals and personal care products (PPCPs) were present at the downstream site, some at sufficiently great concentrations that may present a risk to aquatic organisms. With continuous exposure to a diverse number of stressors including high nutrient and ammonia levels, the presence of a variety of PPCPs and other contaminants, Wascana Creek should be considered as an ecosystem at risk. [ABSTRACT FROM AUTHOR]