Aquatic ecosystems are often impacted by a multitude of stressors, many of which are introduced by a combination of anthropogenic activities such as agricultural development, urbanization, damming, and industrial discharge. Determining the primary stressors responsible for ecological impairments at a site can be complex and challenging; however, it is crucial for making informed management decisions. Improper diagnosis of an impaired system can lead to misguided attempts at remediation, which can be both time consuming and costly. We focused on the development, implementation, and evaluation of methodologies that, in combination, allowed us to identify the primary stressors. These included a four-phase, weight-of-evidence (WOE) assessment including in situ Toxicity Identification and Evaluation (iTIE) testing, physicochemical and macrobenthos characterization, reciprocal sediment transplants, and laboratory and in situ toxicity testing. The contaminants of concern (COCs) at the site were elevated levels of ammonia, chloride, pH, and total dissolved solids in groundwater upwellings into a high-quality waterway. Reciprocal transplants of site sediments and nearby reference sediments and traditional benthic sampling showed impaired benthic indices and multiple stations around a contaminated industrial settling basin. Impaired stations had elevated COCs in groundwaters but exhibited a steep vertical concentration gradient, with concentrations decreasing near the sediment-surface water interface. We describe Phase 4 of the study, which focused on teasing out the role of dissolved oxygen sags in benthic macroinvertebrate responses. Extensive submerged and emergent macrophytes, algae, and cyanobacteria co-occurred at the impaired sites and increased throughout the summer. Laboratory testing suggested that ammonia and pH were possibly toxic at the sites, based on groundwater concentrations. The in situ toxicity testing, however, showed toxicity occurring even at stations with low levels of COCs concurrently with large diurnal fluxes in dissolved oxygen (DO). A final phase using a type of iTIE approach utilized limnocorrals with and without aeration and with in situ toxicity measures using Hyalella azteca. The Phase 4 assessment revealed that low DO levels were primarily responsible for impaired benthic communities, and COC upwellings were diluted at the sediment-water interface to nontoxic levels. These findings will allow for improved management decisions for more efficient and effective restoration activities. Environ Toxicol Chem 2024;43:1524-1536. © 2024 SETAC.