Human-dominated waterways contain thousands of chemicals. Determining which chemical is the most important stressor is important, yet very challenging. The U.S. EPA's Toxicity Identification Evaluation (TIE) procedure uses a series of chemical and physical manipulations to fractionate compounds within a matrix and systematically identify potential toxicants through laboratory bioassay testing. While this may provide useful information, it lacks ecological realism as it is subject to laboratory-related artifacts and is resource-intensive. The in-situ Toxicity Identification Evaluation (iTIE) technology was developed to improve this approach and has undergone a number of modifications over the past several years. The novel prototype 3 consists of an array of iTIE ambient water fractionation units. Each unit is connected to a peristaltic pumping system with an organism exposure chamber that receives water from a resin chamber to chemically fractionate test site water. Test organisms included freshwater and marine standard toxicity test species. Post-fractionation waters are collected for subsequent chemical analyses. Currently, the resins allow for separation of ammonia, metals, and non-polar organics and subsequent toxicity responses are compared between treatments and unfractionated, ambient exposures. The iTIE system was deployed to a depth of 3 m and evaluated in streams and marine harbors. Chemical analyses of water and iTIE chemical sorptive resins confirmed chemical groups causing lethal to sublethal responses. The system proved to be as sensitive or more so than the traditional Phase 1 TIE test and required almost half of the resources to complete. This iTIE prototype provides a robust technology that improves stressor-causality linkages and thereby supports strong evidence for ecological risk weight-of-evidence assessments. This article is protected by copyright. All rights reserved.