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A pipeline carrying unconventional oil and gas (OG) wastewater spilled approximately 11 million liters of wastewater into Blacktail Creek, North Dakota, USA. Flow of the mix of stream water and wastewater down the channel resulted in storage of contaminants in the hyporheic zone and along the banks, providing a long-term source of wastewater constituents to the stream. A multi-level investigation was used to assess the potential effects of oil and brine spills on aquatic life. In this study, we used a combination of experiments using a native fish species, Fathead Minnow (Pimephales promelas), field sampling of the microbial community structure, and measures of estrogenicity. The fish investigation included in situ experiments and experiments with collected site water. Estrogenicity was measured in collected site water samples, and microbial community analyses were conducted on collected sediments. During the initial post-spill investigation, February 2015, performing in situ fish bioassays was impossible because of ice conditions. However, microbial community (e.g., the presence of members of the Halomonadaceae, a family that is indicative of elevated salinity) and estrogenicity differences were compared to reference sites and point to early biological effects of the spill. We noted water column effects on in situ fish survival 6 months post-spill during June 2015. At that time, total dissolved ammonium (sum of ammonium and ammonia, TAN) was 4.41 mg NH4/L with an associated NH3 of 1.09 mg/L, a concentration greater than the water quality criteria established to protect aquatic life. Biological measurements in the sediment defined early and long-lasting effects of the spill on aquatic resources. The microbial community structure was affected during all sampling events. Therefore, sediment may act as a sink for constituents spilled and as such provide an indication of continued and cumulative effects post-spill. However, lack of later water column effects may reflect pulse hyporheic flow of ammonia from shallow ground water. Combining fish toxicological, microbial community structure and estrogenicity information provides a complete ecological investigation that defines potential influences of contaminants at organismal, population, and community levels. In general, in situ bioassays have implications for the individual survival and changes at the population level, microbial community structure defines potential changes at the community level, and estrogenicity measurements define changes at the individual and molecular level. By understanding effects at these various levels of biological organization, natural resource managers can interpret how a course of action, especially for remediation/restoration, might affect a larger group of organisms in the system. The current work also reviews potential effects of additional constituents defined during chemistry investigations on aquatic resources. [ABSTRACT FROM AUTHOR]