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Human-dominated waterways contain thousands of chemicals. Determining which chemical is the most important stressor is important, yet very challenging. The Toxicity Identification Evaluation (TIE) procedure from the US Environmental Protection Agency uses a series of chemical and physical manipulations to fractionate compounds within a matrix and systematically identify potential toxicants through laboratory bioassay testing. Although this may provide useful information, it lacks ecological realism because 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. Postfractionation waters are collected for subsequent chemical analyses. Currently, the resins allow for separation of ammonia, metals, and nonpolar organics; the 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. Environ Toxicol Chem 2020;39:1746-1754. © 2020 SETAC.

It is difficult to assess the toxicity of a single stressor and establish a strong stressor-causality link when multiple stressors coexist. Toxicity Identification Evaluation (TIE) methodology uses a series of chemical and physical manipulations to fractionate compounds within a matrix and systematically identify potential toxicants. The current EPA application of TIE can provide valuable information, but often lacks ecological realism and is subject to laboratory-related artifacts. An in situ TIE Device (iTIED) was designed to assess the sources of toxicity in aquatic ecosystems. For this laboratory validation, each unit was equipped with a sorbent resin chamber, an organism exposure chamber, a water collection container and a peristaltic pump. Chemical analyses of water processed by each iTIED unit were compared to both lethal and sub-lethal molecular responses of the organisms. The compound removal effectiveness of different sorbent resins was also compared. In addition to successfully fractionating diverse chemical mixtures, the iTIED demonstrated a potential for early detection of molecular biomarkers, which could identify chronic toxicity that may go unnoticed in traditional TIE assays. Utilizing this novel in situ system will reduce the uncertainty associated with lab-based simulations and aid management efforts in targeting compounds that pose the greatest threat. This article is protected by copyright. All rights reserved