Evaluation of Complex Mixture Toxicity in the Milwaukee Estuary (WI, USA) Using Whole-Mixture and Component-Based Evaluation Methods.

Anthropogenic activities introduce complex mixtures into aquatic environments, necessitating mixture toxicity evaluation during risk assessment. There are many alternative approaches that can be used to complement traditional techniques for mixture assessment. Our study aimed to demonstrate how these approaches could be employed for mixture evaluation in a target watershed. Evaluations were carried out over 2 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 of collected water samples, and in vivo effects in whole organisms. Chemicals were grouped based on structure/mode of action, bioactivity, and pharmacological activity. Priority chemicals and mixtures were identified based on their relative contributions to estimated mixture pressure (based on cumulative toxic units) and via predictive assessments (random forest regression). Whole mixture assessments identified target sites for further evaluation including two sites targeted for industrial/urban chemical mixture effects assessment; three target sites for pharmaceutical mixture effects assessment; three target sites for further mixture characterization; and three low-priority sites. Analyses identified 14 mixtures and 16 chemicals that significantly contributed to cumulative effects, representing high or medium priority targets for further ecotoxicological evaluation, monitoring, or regulatory assessment. Overall, our study represents an important complement to single-chemical prioritizations, providing a comprehensive evaluation of the cumulative effects of mixtures detected in a target watershed. Furthermore, it demonstrates how different tools and techniques can be used to identify diverse facets of mixture risk and highlights strategies that can be considered in future complex mixture assessments. Environ Toxicol Chem 2023;42:1229-1256. © 2023 SETAC.

Assessing effects of aromatase inhibition on fishes with group-synchronous oocyte development using western mosquitofish (Gambusia affinis) as a model.

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17ß-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development. Fishes with group-synchronous oocyte development have dynamic reproductive cycles which typically occur annually and are often triggered by complex environmental cues. This has resulted in a lack of test data and uncertainty regarding sensitivities to and adverse effects of aromatase inhibition. The present study used the western mosquitofish (Gambusia affinis) as a laboratory model to investigate adverse effects of chemical aromatase inhibition on group-synchronous oocyte development. Adult female western mosquitofish were exposed to either 0, 2, or 30 µg/L of the model nonsteroidal aromatase inhibiting chemical, fadrozole, for a complete reproductive cycle. Fish were sampled at four time-points representing pre-vitellogenic resting, early vitellogenesis, late vitellogenesis/early ovarian recrudescence, and late ovarian recrudescence. Temporal changes in numerous reproductive parameters were measured, including gonadosomatic index (GSI), plasma sex steroids, and expression of selected genes in the brain, liver, and gonad that are important for reproduction. In contrast to fish from the control treatment, fish exposed to 2 and 30 µg/L of fadrozole had persistent elevated expression of cyp19 in the ovary, depressed expression of vtg in the liver, and a low GSI. These responses suggest that completion of a group-synchronous reproductive cycle was unsuccessful during the assay in fish from either fadrozole treatment. These adverse effects data show that exposure to aromatase inhibitors has the potential to cause reproductive dysfunction in a wide range of fishes with both asynchronous and group-synchronous reproductive strategies.