Evaluation of Complex Mixture Toxicity: An Effects-Driven Analysis in the Milwaukee Estuary (WI, USA)

Anthropogenic activities introduce complex mixtures into aquatic environments, necessitating the evaluation of mixture toxicity during ecological risk assessments. There are many new approach methodologies (NAMs) that can be used to complement traditional approaches for conducting mixture assessments. This study aimed to demonstrate how traditional approaches and NAMs can be integrated and employed for mixture evaluation in a target watershed. Assessments were carried out over two years (2017 – 2018) across 8 – 11 study sites in the Milwaukee Estuary (WI, USA). Whole mixtures were evaluated on a site-specific basis by deploying caged fathead minnows (Pimephales promelas) alongside composite samplers for 96-h and characterizing chemical composition, in vitro bioactivity, and in vivo effects in collected water and tissue samples. Chemicals were grouped based on structure/mode of action, bioactivity, and pharmacological actions. Significant chemical/mixtures were identified by assessing contributions to cumulative toxicity units (maximum cumulative ratio analyses) and predictive relationships with measured effects (random Forest regression). Whole mixture assessments identified specific target sites for further evaluation in the Milwaukee Estuary, including four sites impacted by industrial chemical/fuel/polycyclic aromatic hydrocarbon mixtures, four sites impacted by pharmaceutical mixtures, two sites requiring further experimental evaluation, and one low impact site. Constituent-based and predictive analyses identified twelve mixtures and twelve chemicals which significantly contributed to and/or predicted cumulative effects, thus representing priority targets for further ecotoxicological evaluation, monitoring, or regulatory assessment. Overall, this study represents an important complement to single-chemical prioritizations, providing a more comprehensive evaluation of cumulative effects of chemicals detected in a target watershed. Furthermore, it demonstrates diverse tools and techniques that can be employed and adapted for future mixture risk assessments in aquatic environments.