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Funding: This research was funded by Colorado Parks and Wildlife (16-IAA-82320) and the Colorado State University Energy Institute. Colorado State University’s Intuitional Animal Care and Use Committee approved this research under protocols 15-6086A and 16-6367.Abstract: Oil development has expanded dramatically in Colorado over the last decade. Associated with this rapid expansion has been a significant increase in the number of accidental releases into the environment. On January 2013, West Creek which flows along a scenic byway in Unaweep Canyon, Colorado, was impacted by a petroleum spill from an overturned tanker truck. 22,700 liters of gasoline and 7,300 liters of diesel discharged into the stream killing an estimated 1,206 Brown Trout, Salmo trutta, and 8,172 Mottled Sculpin, Cottus bairdii. Subsequent electrofishing surveys indicated that the fishery was not quickly recovering particularly with regard to Mottled Sculpin populations, but also Brown Trout. In June and October 2015, as part of ongoing efforts to determine long term effects of this spill, we explored health indicators across multiple levels of biological organization. Histopathological abnormalities (e.g., ectopic neural tissue, cystic kidney, increased melanomacrophage aggregates) were observed in Mottled Sculpin collected from the spill site and nearby downstream sites. Altered benthic macroinvertebrate community structure was observed at the spill site compared with a reference site one kilometer upstream. Interestingly, a GC-MS finger-printing analysis of polycyclic aromatic hydrocarbons (PAHs) in stream sediment revealed that PAH concentrations were typical of minimally impacted streams flowing adjacent to roads. These results suggest that effects of the spill were persisting after contaminant concentrations had returned to ‘normal’ by Fall 2015. Subsequently, we conducted two mesocosm experiments, using naturally colonized benthic macroinvertebrate communities. Exposure to simulated spill conditions caused concentration-dependent macroinvertebrate drift and substantial mortality that occurred rapidly after the spills were initiated and at lower concentrations than expected. In addition, concentration-dependent lethal and sub-lethal effects were observed in Rainbow Trout, Oncorhynchus mykiss, during simulated spill bioassays. Periphyton biofilms were also adversely affected. We conclude that petroleum spills in coldwater streams risk adverse acute, chronic, lethal and sub-lethal effects to aquatic communities across numerous levels of biological organization. And these effects were evident after the 2013 West Creek petroleum spill. Moreover, by utilizing field observations, mesocosms and bioassays we gained insights into consequences of petroleum spills using an ecotoxicological weight-of evidence approach. Importantly, the methods used in this project can be employed at future spill events as field useful bioassessment techniques to aid in the process of holding responsible parties appropriately accountable for damages to stream communities.