Charlene B. Tilton's research while affiliated with Great Lakes Environmental Center and other places

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. This study aimed to demonstrate how these approaches could be employed for mixture evaluation in a target watershed. Evaluations 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 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 fourteen mixtures and sixteen chemicals which significantly contributed to cumulative effects, representing high or medium priority targets for further ecotoxicological evaluation, monitoring, or regulatory assessment. Overall, this 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. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2023;00:0–0.

Metformin, along with its biotransformation product guanylurea, are commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine active compound, but there are inconsistencies with regard to its effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments were performed with adult fathead minnows (Pimephales promelas). First, explants of fathead minnow ovary tissue were exposed to 0.001‐100 µM metformin or guanylurea to investigate whether the compounds could directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, 41 µg/L) or guanylurea (1.0, 10, 100 µg/L) for 23 d to assess impacts on reproduction. Lastly, male fathead minnows were exposed to 41 µg/L metformin, 100 µg/L guanylurea, or a mixture of both compounds, with samples collected over a 96 h time course to investigate potential impacts to the hepatic transcriptome or metabolome. Neither metformin nor guanylurea affected steroid production by ovary tissue exposed ex vivo. In the 23 d exposure, neither compound significantly impacted transcription of endocrine‐related genes in male liver or gonad, circulating steroid concentrations in either sex, or fecundity of spawning pairs. In the 96 h time course, 100 µg guanylurea/L elicited more differentially expressed genes (DEGs) than 41 µg metformin/L and showed the greatest impacts at 96 h. Hepatic transcriptome and metabolome changes were chemical and time‐dependent, with the largest impact on the metabolome observed at 23 d of exposure to 100 µg guanylurea/L. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. This article is protected by copyright. All rights reserved.

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17β-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.

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow (Pimephales promelas). This qAOP includes quantitative response-response relationships describing plasma 17β-estradiol (E2) as a function of plasma fadrozole, plasma vitellogenin (VTG) as a function of plasma E2, and fecundity as a function of plasma VTG. These quantitative response-response relationships simulated plasma E2, plasma VTG, and fecundity measured in female zebrafish (Danio rerio) exposed to fadrozole for 21 days, but not these responses measured in female Japanese medaka (Oryzias latipes). However, Japanese medaka had different basal levels of plasma E2, plasma VTG, and fecundity. Normalizing basal levels of each measurement to equal those of female fathead minnow enabled the relationships to accurately simulate plasma E2, plasma VTG, and fecundity measured in female Japanese medaka. This suggests that these quantitative response-response relationships are conserved across these three fishes when considering relative change rather than absolute measurements. The present study represents an early step towards defining the appropriate taxonomic domain of applicability and extending the regulatory applications of this qAOP.