New Approach Methodologies for the Endocrine Activity Toolbox: Environmental Assessment for Fish and Amphibians.

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource-intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an "animal-free" testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross-sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight-of-evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit-for-purpose methods to identify chemicals acting via endocrine mechanisms. Environ Toxicol Chem 2023;42:757-777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Using ultrahigh-resolution mass spectrometry and toxicity identification techniques to characterize the toxicity of oil sands process-affected water: The case for classical naphthenic acids.

Previous assessments of oil sands process-affected water (OSPW) toxicity were hampered by lack of high-resolution analytical analysis, use of nonstandard toxicity methods, and variability between OSPW samples. We integrated ultrahigh-resolution mass spectrometry with a toxicity identification evaluation (TIE) approach to quantitatively identify the primary cause of acute toxicity of OSPW to rainbow trout (Oncorhynchus mykiss). The initial characterization of OSPW toxicity indicated that toxicity was associated with nonpolar organic compounds, and toxicant(s) were further isolated within a range of discrete methanol fractions that were then subjected to Orbitrap mass spectrometry to evaluate the contribution of naphthenic acid fraction compounds to toxicity. The results showed that toxicity was attributable to classical naphthenic acids, with the potency of individual compounds increasing as a function of carbon number. Notably, the mass of classical naphthenic acids present in OSPW was dominated by carbon numbers ≤16; however, toxicity was largely a function of classical naphthenic acids with ≥17 carbons. Additional experiments found that acute toxicity of the organic fraction was similar when tested at conductivities of 400 and 1800 µmhos/cm and that rainbow trout fry were more sensitive to the organic fraction than larval fathead minnows (Pimephales promelas). Collectively, the results will aid in developing treatment goals and targets for removal of OSPW toxicity in water return scenarios both during operations and on mine closure. Environ Toxicol Chem 2017;36:3148-3157. © 2017 SETAC.

Disruption of a belowground mutualism alters interactions between plants and their floral visitors.

Plants engage in diverse and intimate interactions with unrelated taxa. For example, aboveground floral visitors provide pollination services, while belowground arbuscular mycorrhizal fungi (AMF) enhance nutrient capture. Traditionally in ecology, these processes were studied in isolation, reinforcing the prevailing assumption that these above- and belowground processes were also functionally distinct. More recently, there has been a growing realization that the soil surface is not a barrier to many ecological interactions, particularly those involving plants (who live simultaneously above and below ground). Because of the potentially large impact that mycorrhizae and floral visitors can have on plant performance and community dynamics, we designed an experiment to test whether these multi-species mutualisms were interdependent under field conditions. Using benomyl, a widely used fungicide, we suppressed AMF in a native grassland, measuring plant, fungal, and floral-visitor responses after three years of fungal suppression. AMF suppression caused a shift in the community of floral visitors from large-bodied bees to small-bodied bees and flies, and reduced the total number of floral visits per flowering stem 67% across the 23 flowering species found in the plots. Fungal suppression has species-specific effects on floral visits for the six most common flowering plants in this experiment. Exploratory analyses suggest these results were due to changes in floral-visitor behavior due to altered patch-level floral display, rather than through direct effects of AMF suppression on floral morphology. Our findings indicate that AMF are an important, and overlooked, driver of floral-visitor community structure with the potential to affect pollination services. These results support the growing body of research indicating that interactions among ecological interactions can be of meaningful effect size under natural field conditions and may influence individual performance, population dynamics, and community structure.