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Watersheds of the Great Lakes Basin (USA/Canada) are highly modified and impacted by human activities including pesticide use. Despite labeling restrictions intended to minimize risks to nontarget organisms, concerns remain that environmental exposures to pesticides may be occurring at levels negatively impacting nontarget organisms. We used a combination of organismal-level toxicity estimates (in vivo aquatic life benchmarks) and data from high-throughput screening (HTS) assays (in vitro benchmarks) to prioritize pesticides and sites of concern in streams at 16 tributaries to the Great Lakes Basin. In vivo or in vitro benchmark values were exceeded at 15 sites, 10 of which had exceedances throughout the year. Pesticides had the greatest potential biological impact at the site with the greatest proportion of agricultural land use in its basin (the Maumee River, Toledo, OH, USA), with 72 parent compounds or transformation products being detected, 47 of which exceeded at least one benchmark value. Our risk-based screening approach identified multiple pesticide parent compounds of concern in tributaries of the Great Lakes; these compounds included: eight herbicides (metolachlor, acetochlor, 2,4-dichlorophenoxyacetic acid, diuron, atrazine, alachlor, triclopyr, and simazine), three fungicides (chlorothalonil, propiconazole, and carbendazim), and four insecticides (diazinon, fipronil, imidacloprid, and clothianidin). We present methods for reducing the volume and complexity of potential biological effects data that result from combining contaminant surveillance with HTS (in vitro) and traditional (in vivo) toxicity estimates. Environ Toxicol Chem 2022;00:1-18. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

In a study of 44 diverse sampling sites across 16 Great Lakes tributaries, 110 pharmaceuticals were detected of 257 monitored. The present study evaluated the ecological relevance of detected chemicals and identified heavily impacted areas to help inform resource managers and guide future investigations. Ten pharmaceuticals (caffeine, nicotine, albuterol, sulfamethoxazole, venlafaxine, acetaminophen, carbamazepine, gemfibrozil, metoprolol, and thiabendazole) were distinguished as having the greatest potential for biological effects based on comparison to screening-level benchmarks derived using information from two biological effects databases, the ECOTOX Knowledgebase and the ToxCast database. Available evidence did not suggest substantial concern for 75% of the monitored pharmaceuticals, including 147 undetected pharmaceuticals and 49 pharmaceuticals with screening-level alternative benchmarks. However, because of a lack of biological effects information, screening values were not available for 51 detected pharmaceuticals. Samples containing the greatest pharmaceutical concentrations and having the highest detection frequencies were from Lake Erie, southern Lake Michigan, and Lake Huron tributaries. Samples collected during low-flow periods had higher pharmaceutical concentrations than those collected during increased-flow periods. The wastewater-treatment plant effluent content in streams correlated positively with pharmaceutical concentrations. However, deviation from this correlation demonstrated that secondary factors, such as multiple pharmaceutical sources, were likely present at some sites. Further research could investigate high-priority pharmaceuticals as well as those for which alternative benchmarks could not be developed. Environ Toxicol Chem 2022;41:2221-2239. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Process wastewaters from food, beverage, and feedstock facilities, although regulated, are an under-investigated environmental contaminant source. Food process wastewaters (FPWW) from 23 facilities in 17 U.S. states were sampled and documented a plethora of chemical and microbial contaminants. Of 576 analyzed organics, 184 (32%) were detected at least once, with concentrations as large as 143 μg L(−1) (6:2 fluorotelomer sulfonic acid) and as many as 47 detected in a single FPWW sample. Cumulative per/polyfluoroalkyl substance concentrations up to 185 μg L(−1) and large pesticide transformation product concentrations (e.g., methomyl oxime, 40 μg L(−1), clothianidin TMG, 2.02 μg L(−1)) were observed. Despite 48% of FPWW receiving disinfection prior to discharge, bacteria resistant to third-generation antibiotics were found in each facility type and multiple bacterial groups were detected in all samples, including total coliforms. Exposure-activity ratios and toxicity quotients exceeded 1.0 in 13 and 22% of samples, respectively, indicating potential biological effects and toxicity to vertebrates and invertebrates associated with the discharge of FPWW. Organic contaminant profiles of FPWW differed from previously reported contaminant profiles of municipal effluent and urban storm water, indicating FPWW is another important source of chemical and microbial contaminant mixtures discharged to receiving surface waters.

Watersheds are subjected to diverse anthropogenic inputs, exposing aquatic biota to a wide range of chemicals. Detection of multiple, different chemicals can challenge natural resource managers who often have to determine where to allocate potentially limited resources. Here we describe a weight-of-evidence framework for retrospectively prioritizing aquatic contaminants. To demonstrate framework utility, we used data from 96-h caged fish studies to prioritize chemicals detected in the Milwaukee Estuary (WI, USA; 2017 - 2018). Across study years, 77/178 targeted chemicals were detected. Chemicals were assigned prioritization scores based on spatial and temporal detection frequency, environmental distribution, environmental fate, ecotoxicological potential, and effect prediction. Chemicals were sorted into priority bins based on the intersection of prioritization score and data availability. Data limited chemicals represented those that did not have sufficient data to adequately evaluate ecotoxicological potential or environmental fate. Seven compounds (fluoranthene, benzo[a]pyrene, pyrene, atrazine, metolachlor, phenanthrene, and DEET) were identified as high or medium priority and data-sufficient and flagged as candidates for further effects-based monitoring studies. Twenty-one compounds were identified as high or medium priority and data-limited and flagged as candidates for further ecotoxicological research. Fifteen chemicals were flagged as the lowest priority in the watershed. One of these chemicals (2-methylnaphthalene) displayed no data limitations and was flagged as a definitively low priority chemical. The remaining chemicals displayed some data limitations and were considered lower priority compounds (contingent on further ecotoxicological and environmental fate assessments). The remaining 34 compounds were flagged as low or medium priority. Altogether, this prioritization provided a screening-level (non-definitive) assessment that could be used to focus further resource management and risk assessment activities in the Milwaukee Estuary. Furthermore, by providing detailed methodology and a practical example with real experimental data, we demonstrated that the proposed framework represents a transparent and adaptable approach for prioritizing contaminants in freshwater environments. This article is protected by copyright. All rights reserved. Integr Environ Assess Manag 2022;00:0-0. © 2022 SETAC.

The present study evaluated whether in vitro measures of aromatase inhibition as inputs into a quantitative adverse outcome pathway (qAOP) construct could effectively predict in vivo effects on 17β-estradiol (E2) and vitellogenin (VTG) concentrations in female fathead minnows. Five chemicals identified as aromatase inhibitors in mammalian-based ToxCast assays were screened for their ability to inhibit fathead minnow aromatase in vitro. Female fathead minnows were then exposed to 3 of those chemicals: letrozole, epoxiconazole, and imazalil in concentration-response (5 concentrations plus control) for 24 h. Consistent with AOP-based expectations, all 3 chemicals caused significant reductions in plasma E2 and hepatic VTG transcription. Characteristic compensatory upregulation of aromatase and follicle-stimulating hormone receptor (fshr) transcripts in the ovary were observed for letrozole but not for the other 2 compounds. Considering the overall patterns of concentration-response and temporal concordance among endpoints, data from the in vivo experiments strengthen confidence in the qualitative relationships outlined by the AOP. Quantitatively, the qAOP model provided predictions that fell within the standard error of measured data for letrozole but not for imazalil and epoxiconazole. However, the inclusion of measured plasma concentrations of the test chemicals as inputs improved model predictions, with all predictions falling within the range of measured values. Results highlight both the utility and limitations of the qAOP and its potential use in 21st century ecotoxicology. Environ Toxicol Chem 2021;40:1155-1170. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Assessment of ecological risks of chemicals in the field usually involves complex mixtures of known and unknown compounds. We describe the use of pathway-based chemical and biological approaches to assess the risk of chemical mixtures in the Maumee River (OH, USA), which receives a variety of agricultural and urban inputs. Fathead minnows (Pimephales promelas) were deployed in cages for 4 d at a gradient of sites along the river and adjoining tributaries in 2012 and during 2 periods (April and June) in 2016, in conjunction with an automated system to collect composite water samples. More than 100 industrial chemicals, pharmaceuticals, and pesticides were detected in water at some of the study sites, with the greatest number typically found near domestic wastewater treatment plants. In 2016, there was an increase in concentrations of several herbicides from April to June at upstream agricultural sites. A comparison of chemical concentrations in site water with single chemical data from vitro high-throughput screening (HTS) assays suggested the potential for perturbation of multiple biological pathways, including several associated with induction or inhibition of different cytochrome P450 (CYP) isozymes. This was consistent with direct effects of water extracts in an HTS assay and induction of hepatic CYPs in caged fish. Targeted in vitro assays and measurements in the caged fish suggested minimal effects on endocrine function (e.g., estrogenicity). A nontargeted mass spectroscopy-based analysis suggested that hepatic endogenous metabolite profiles in caged fish covaried strongly with the occurrence of pesticides and pesticide degradates. These studies demonstrate the application of an integrated suite of measurements to help understand the effects of complex chemical mixtures in the field. Environ Toxicol Chem 2021;40:1098-1122. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.12 to 260 µg/L water. Consistent with an AOP linking aromatase inhibition to reproductive impairment in fish, exposure to the fungicide resulted in significant reductions in ex vivo production of 17β-estradiol (E2) by ovary tissue (≥165 µg imazalil/L), plasma E2 concentrations (≥74 µg imazalil/L), vitellogenin (Vtg) messenger RNA expression (≥165 µg imazalil/L), Vtg plasma concentrations (≥74 µg imazalil/L), uptake of Vtg into oocytes (≥260 µg imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (≥24 µg imazalil/L). Despite many potential sources of uncertainty in potency and efficacy estimates based on model simulations, observed magnitudes of apical effects were quite consistent with model predictions, and in vivo potency was within an order of magnitude of that predicted based on in vitro relative potency. Overall, our study suggests that NAMs and AOP-based approaches can support meaningful reduction and refinement of animal testing. Environ Toxicol Chem 2023;42:100-116. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

To help meet the objectives of the Great Lakes Restoration Initiative with regard to increasing knowledge about toxic substances, 223 pesticides and pesticide transformation products were monitored in 15 Great Lakes tributaries using polar organic chemical integrative samplers. A screening-level assessment of their potential for biological effects was conducted by computing toxicity quotients (TQs) for chemicals with available US Environmental Protection Agency (USEPA) Aquatic Life Benchmark values. In addition, exposure activity ratios (EAR) were calculated using information from the USEPA ToxCast database. Between 16 and 81 chemicals were detected per site, with 97 unique compounds detected overall, for which 64 could be assessed using TQs or EARs. Ten chemicals exceeded TQ or EAR levels of concern at two or more sites. Chemicals exceeding thresholds included seven herbicides (2,4-dichlorophenoxyacetic acid, diuron, metolachlor, acetochlor, atrazine, simazine, and sulfentrazone), a transformation product (deisopropylatrazine), and two insecticides (fipronil and imidacloprid). Watersheds draining agricultural and urban areas had more detections and higher concentrations of pesticides compared with other land uses. Chemical mixtures analysis for ToxCast assays associated with common modes of action defined by gene targets and adverse outcome pathways (AOP) indicated potential activity on biological pathways related to a range of cellular processes, including xenobiotic metabolism, extracellular signaling, endocrine function, and protection against oxidative stress. Use of gene ontology databases and the AOP knowledgebase within the R-package ToxMixtures highlighted the utility of ToxCast data for identifying and evaluating potential biological effects and adverse outcomes of chemicals and mixtures. Results have provided a list of high-priority chemicals for future monitoring and potential biological effects warranting further evaluation in laboratory and field environments. Environ Toxicol Chem 2022;00:1-27. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Metformin, along with its biotransformation product guanylurea, is 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 was 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, and 41 µg/L) or guanylurea (1.0, 10, and 100 µg/L) for 23 days 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 days of 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 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 days 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. Environ Toxicol Chem 2022;41:2708-2720. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

The USEPA's 1985 guidelines for the derivation of aquatic life criteria (ALC) are robust but data-intensive. For many chemicals, the extensive in vivo data sets required for ALC derivation are not available. Thus, alternative analyses and processes that can provide provisional values to guide states, tribes, and other stakeholders while data accumulate and more rigorous criteria are derived would be beneficial. The overarching purpose of this study was to assess the feasibility of using data from new approach methodologies (NAMs) like ToxCast to derive first-pass, provisional values to guide chemical prioritization and resource management as a complement to traditional ALC derivation. To address this goal, the study objectives were to (1) estimate chemical potency using data from NAMs for nine compounds with available aquatic benchmarks, (2) evaluate the utility of using NAM data to elucidate potential mechanisms of toxicity to guide problem formulation, and (3) determine the species relevance of toxicity pathways for compounds with clearly defined mechanisms of action as a means to evaluate whether minimum data requirements could potentially be waived when deriving a more formal ALC. Points of departure were derived from ToxCast data based on the fifth percentile of the distribution of activity concentration above cutoff values falling below the cytotoxic burst. Mechanistic inferences were made based on active target hits in ToxCast and, where applicable, assessed for taxonomic conservation using SeqAPASS. ToxCast-based point-of-departure aligned relatively closely (six of nine test chemicals within a factor of 10; eight of nine within a factor of 100) with aquatic benchmarks from the USEPA and US Department of Energy (DOE). Moreover, pathways of toxicity gleaned from NAM data were reflective of in vivo-based findings from the literature. These results, while preliminary, and based on a limited number of substances, support the potential application of NAM data to complement traditional ALC derivation approaches and prioritization. Integr Environ Assess Manag 2023;19:224-238. © 2022 Society of Environmental ToxicologyChemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17β-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17β-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17β-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17β-trenbolone alone. Overall, there were indications that 17β-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17β-trenbolone could be due to several factors, including lack of impact of 17β-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC.

Organisms are exposed to ever‐changing complex mixtures of chemicals over the course of their lifetime. The need to more comprehensively describe this exposure and relate it to adverse health effects has led to formulation of the exposome concept in human toxicology. Whether this concept has utility in the context of environmental hazard and risk assessment has not been discussed in detail. In this Critical Perspective, we propose—by analogy to the human exposome—to define the eco‐exposome as the totality of the internal exposure (anthropogenic and natural chemicals, their biotransformation products or adducts, and endogenous signaling molecules that may be sensitive to an anthropogenic chemical exposure) over the lifetime of an ecologically relevant organism. We describe how targeted and nontargeted chemical analyses and bioassays can be employed to characterize this exposure and discuss how the adverse outcome pathway concept could be used to link this exposure to adverse effects. Available methods, their limitations, and/or requirement for improvements for practical application of the eco‐exposome concept are discussed. Even though analysis of the eco‐exposome can be resource‐intensive and challenging, new approaches and technologies make this assessment increasingly feasible. Furthermore, an improved understanding of mechanistic relationships between external chemical exposure(s), internal chemical exposure(s), and biological effects could result in the development of proxies, that is, relatively simple chemical and biological measurements that could be used to complement internal exposure assessment or infer the internal exposure when it is difficult to measure. Environ Toxicol Chem 2022;41:30–45. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., Illustration of the eco‐exposome assessment and how chemical analysis and bioassays could be used to estimate internal exposure. MIE = molecular initiation event; KE = key event; AO = adverse outcome., DAAD German academic exchange service

With improved analytical techniques, environmental monitoring studies are increasingly able to report the occurrence of tens or hundreds of chemicals per site, making it difficult to identify the most relevant chemicals from a biological standpoint. For the present study, organic chemical occurrence was examined, individually and as mixtures, in the context of potential biological effects. Sediment was collected at 71 Great Lakes (USA/Canada) tributary sites and analyzed for 87 chemicals. Multiple risk-based lines of evidence were used to prioritize chemicals and locations, including comparing sediment concentrations and estimated porewater concentrations with established whole-organism benchmarks (i.e., sediment and water quality criteria and screening values) and with high-throughput toxicity screening data from the US Environmental Protection Agency's ToxCast database, estimating additive effects of chemical mixtures on common ToxCast endpoints, and estimating toxic equivalencies for mixtures of alkylphenols and polycyclic aromatic hydrocarbons (PAHs). This multiple-lines-of-evidence approach enabled the screening of more chemicals, mitigated the uncertainties of individual approaches, and strengthened common conclusions. Collectively, at least one benchmark/screening value was exceeded for 54 of the 87 chemicals, with exceedances observed at all 71 of the monitoring sites. Chemicals with the greatest potential for biological effects, both individually and as mixture components, were bisphenol A, 4-nonylphenol, indole, carbazole, and several PAHs. Potential adverse outcomes based on ToxCast gene targets and putative adverse outcome pathways relevant to individual chemicals and chemical mixtures included tumors, skewed sex ratios, reproductive dysfunction, hepatic steatosis, and early mortality, among others. The results provide a screening-level prioritization of chemicals with the greatest potential for adverse biological effects and an indication of sites where they are most likely to occur. Environ Toxicol Chem 2022;41:1016-1041. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Testicular oocytes in wild adult bass (Micropterus spp.) are considered a potential indication of exposure to estrogenic compounds in municipal, agricultural, or industrial wastewater. However, our ability to interpret links between testicular oocyte occurrence in wild fish species and environmental pollutants is limited by our understanding of normal and abnormal gonadal development. We previously reported low-to-moderate testicular oocyte prevalence (7%-38%) among adult male bass collected from Minnesota waters with no known sources of estrogenic compounds. In the present study, two experiments were conducted in which smallmouth bass (Micropterus dolomieu) fry were exposed to control water or 17-α-ethinylestradiol (EE2) during gonadal differentiation, then reared in clean water for an additional period. Histological samples were evaluated at several time points during the exposure and grow-out periods, and the sequence and timing of gonadal development in the presence of estrogen were compared with that of control fish. Testicular oocytes were not observed in any control or EE2-exposed fish. Among groups exposed to 1.2 or 5.1 ng/L EE2 in Experiment 1 or 3.0 ng/L EE2 in Experiment 2, ovaries were observed in 100% of fish up to 90 days after exposure ceased, and approximately half of those ovaries had abnormal characteristics, suggesting that they likely developed in sex-reversed males. Groups exposed to 0.1, 0.4, or 1.0 ng/L in Experiment 2 developed histologically normal ovaries and testes in proportions not significantly different from 1:1. These findings suggest that, while presumably able to cause sex reversal, juvenile exposure to EE2 may not be a unique cause of testicular oocytes in wild bass, although the long-term outcomes of exposure are unknown. Environ Toxicol Chem 2022;41:1416-1428. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Previous studies have detected numerous organic contaminants and in vitro bioactivities in surface water from the South Platte River near Denver, Colorado, USA. To evaluate the temporal and spatial distribution of selected contaminants of emerging concern, water samples were collected throughout 2018 and 2019 at 11 sites within the S. Platte River and surrounding tributaries with varying proximities to a major wastewater treatment plant (WWTP). Water samples were analyzed for pharmaceuticals, pesticides, steroid hormones, and wastewater indicators and screened for in vitro biological activities. Multiplexed, in vitro assays that simultaneously screen for agonistic activity against 24 human nuclear receptors detected estrogen receptor (ER), peroxisome proliferator activated receptor-gamma (PPARγ), and glucocorticoid receptor (GR) bioactivities in water samples near the WWTP outflow. Targeted in vitro bioassays assessing ER, GR, and PPARγ agonism corroborated bioactivities for ER (up to 55 ± 9.7 ng/L 17β-estradiol equivalents) and GR (up to 156 ± 28 ng/L dexamethasone equivalents), while PPARγ activity was not confirmed. To evaluate the potential in vivo significance of the bioactive contaminants, sexually-mature fathead minnows were caged at six locations upstream and downstream of the WWTP for 5 days after which targeted gene expression analyses were performed. Significant up-regulation of male hepatic vitellogenin was observed at sites with corresponding in vitro ER activity. No site-related differences in GR-related transcript abundance were detected in female adipose or male livers, suggesting observed environmental concentrations of GR-active contaminants do not induce a detectable in vivo response. In line with the lack of detectable targeted in vitro PPARɣ activity, there were no significant effects on PPARɣ-related gene expression. Although the chemicals responsible for GR and PPAR-mediated bioactivities are unknown, results from the present study provide insights into the significance (or lack thereof) of these bioactivities relative to short-term in situ fish exposures.

Chemical monitoring data were collected in surface waters from 57 Great Lakes tributaries from 2010 to 13 to identify chemicals of potential biological relevance and sites at which these chemicals occur. Traditional water-quality benchmarks for aquatic life based on in vivo toxicity data were available for 34 of 67 evaluated chemicals. To expand evaluation of potential biological effects, measured chemical concentrations were compared to chemical-specific biological activities determined in high-throughput (ToxCast) in vitro assays. Resulting exposure-activity ratios (EARs) were used to prioritize the chemicals of greatest potential concern: 4‑nonylphenol, bisphenol A, metolachlor, atrazine, DEET, caffeine, tris(2‑butoxyethyl) phosphate, tributyl phosphate, triphenyl phosphate, benzo(a)pyrene, fluoranthene, and benzophenone. Water-quality benchmarks were unavailable for five of these chemicals, but for the remaining seven, EAR-based prioritization was consistent with that based on toxicity quotients calculated from benchmarks. Water-quality benchmarks identified three additional PAHs (anthracene, phenanthrene, and pyrene) not prioritized using EARs. Through this analysis, an EAR of 10-3 was identified as a reasonable threshold above which a chemical might be of potential concern. To better understand apical hazards potentially associated with biological activities captured in ToxCast assays, in vitro bioactivity data were matched with available adverse outcome pathway (AOP) information. The 49 ToxCast assays prioritized via EAR analysis aligned with 23 potentially-relevant AOPs present in the AOP-Wiki. Mixture effects at monitored sites were estimated by summation of EAR values for multiple chemicals by individual assay or individual AOP. Commonly predicted adverse outcomes included impacts on reproduction and mitochondrial function. The EAR approach provided a screening-level assessment for evidence-based prioritization of chemicals and sites with potential for adverse biological effects. The approach aids prioritization of future monitoring activities and provides testable hypotheses to help focus those efforts. This also expands the fraction of detected chemicals for which biologically-based benchmark concentrations are available to help contextualize chemical monitoring results.

Particulate matter (PM) emitted from beef cattle feedyards transports manure-affiliated compounds including antibiotics and steroids from feedyards into the surrounding environment. Similarly, commercial dairy operations have the potential to release PM, comprised largely of aerosolized manure, into the surrounding environment along with associated veterinary antibiotics and steroids. The goal of this study was to quantify and compare total suspended particulates, antibiotics and estrogens collected upwind and downwind of dairies in the Southern High Plains, and to characterize microbial community structure and in vitro estrogenic activity of collected PM. PM collected downwind of dairies had greater microbial diversity (as measured by number of operational taxonomic units) and had greater frequencies of detection for antibiotics and estrogens than associated upwind PM. Dominant bacterial phyla and overall microbial community composition were similar among upwind and downwind PM, and extracts of upwind and downwind PM produced no in vitro estrogenic activity. Although this study surveyed a limited number of dairies in the region, these results suggest that dairy operations in the Southern High Plains, in contrast to feedyards, appear to be minor contributors of total aerial PM and associated antibiotics and steroids to the terrestrial environment.

Thiacloprid (THI) is a widely used neonicotinoid insecticide where concerns have been raised regarding low absorption by crops, substantial distribution in surrounding areas, and potential adverse effects to terrestrial and aquatic organisms.Prior to this study, there was very limited information addressing the ex vivo (precision-cut liver slices) metabolism of THI by fish species and the metabolic pathways regulating its potential for adverse effects.The in vitro and ex vivo biotransformation pathway of THI is defined by the formation of three primary metabolites (TM1, TM2 and TM3) via separate paths differentiated by reductive decyanation, reductive dechlorination with hydration and dealkylation processes, respectively.Kinetic rates were calculated for the rat microsomal decyanation of THI into TM1 (Km = 299.2 µM and Vmax = 5.3 pmol/min/mg), and for the dealkylation of THI into TM3 (Km = 368.9 µM and Vmax = 3.95 pmol/min/mg).Formation confirmation and identity inference of THI metabolites in absence of standards were achieved by LC-UV and High Resolution-MS strategies.The in vitro and ex vivo metabolic products of THI are conserved both across species (rat and Rainbow trout) and levels of biological organization (microsomes and liver slices), as previously reported for the neonicotinoid insecticides Imidacloprid and Acetamiprid. Thiacloprid (THI) is a widely used neonicotinoid insecticide where concerns have been raised regarding low absorption by crops, substantial distribution in surrounding areas, and potential adverse effects to terrestrial and aquatic organisms. Prior to this study, there was very limited information addressing the ex vivo (precision-cut liver slices) metabolism of THI by fish species and the metabolic pathways regulating its potential for adverse effects. The in vitro and ex vivo biotransformation pathway of THI is defined by the formation of three primary metabolites (TM1, TM2 and TM3) via separate paths differentiated by reductive decyanation, reductive dechlorination with hydration and dealkylation processes, respectively. Kinetic rates were calculated for the rat microsomal decyanation of THI into TM1 (Km = 299.2 µM and Vmax = 5.3 pmol/min/mg), and for the dealkylation of THI into TM3 (Km = 368.9 µM and Vmax = 3.95 pmol/min/mg). Formation confirmation and identity inference of THI metabolites in absence of standards were achieved by LC-UV and High Resolution-MS strategies. The in vitro and ex vivo metabolic products of THI are conserved both across species (rat and Rainbow trout) and levels of biological organization (microsomes and liver slices), as previously reported for the neonicotinoid insecticides Imidacloprid and Acetamiprid.

Monitoring of the Colorado River near the Moab, Utah, wastewater treatment plant (WWTP) outflow has detected pharmaceuticals, hormones, and estrogen-receptor (ER)-, glucocorticoid receptor (GR)-, and peroxisome proliferator-activated receptor-gamma (PPARγ)-mediated biological activities. The aim of the present multi-year study was to assess effects of a WWTP replacement on bioactive chemical (BC) concentrations. Water samples were collected bimonthly, pre- and post-replacement, at 11 sites along the Colorado River upstream and downstream of the WWTP and analyzed for in vitro bioactivities (e.g., agonism of ER, GR, and PPARγ) and BC concentrations; fathead minnows were cage deployed pre- and post-replacement at sites with varying proximities to the WWTP. Before the WWTP replacement, in vitro ER (24 ng 17β-estradiol equivalents/L)-, GR (60 ng dexamethasone equivalents/L)-, and PPARγ-mediated activities were detected at the WWTP outflow but diminished downstream. In March 2018, the WWTP effluent was acutely toxic to the fish, likely due to elevated ammonia concentrations. Following the WWTP replacement, ER, GR, and PPARγ bioactivities were reduced by approximately 60-79%, no toxicity was observed in caged fish, and there were marked decreases in concentrations of many BCs. Results suggest that replacement of the Moab WWTP achieved a significant reduction in BC concentrations to the Colorado River.

Exposure to endocrine-disrupting compounds (EDCs) can alter steroid hormone production in vertebrates, sometimes leading to adverse reproductive or developmental effects. Liquid chromatography mass spectrometry methods are the gold standard for analyte confirmation and quantification in biological matrices, but radioimmunoassays (RIAs) are most commonly used for measurement of select steroid hormones in aquatic toxicology studies. Existing methods for steroid quantification often employ derivatization, limiting the range of steroids that can be simultaneously measured in a single process. In the current study, a method for the simultaneous measurement of thirteen endogenous steroids in small sample volumes without derivatization using liquid chromatography atmospheric pressure photoionization tandem mass spectrometry (LC-APPI-MS/MS) was developed. Several physiologically important steroids, including 11-deoxycortisol, 11-ketotestosterone, 17α- and 17β-estradiol, 17α-hydroxyprogesterone, 17,20β-dihydroxyprogesterone, 17,20β,21-trihydroxyprogesterone, androstenedione, cortisol, estriol, estrone, progesterone, and testosterone, were selected for the analysis. The method was validated for application to small volumes of fish plasma and fish holding water. Method detection limits using only 10 µL of plasma ranged from 0.05 to 1.0 ng/mL. As a potential surrogate for plasma steroid measurements, fish holding water was analyzed to measure excreted steroids. Lower limits of quantification when using 0.25 L of water ranged from 0.05 to 1.0 ng/L. The validated method was applied to two different experiments with small fish species exposed to an EDC known to affect steroid synthesis, fadrozole. Concentrations of the 13 steroids were measured in plasma or holding water from the studies. This work demonstrates the potential application of the developed method to measure endogenous steroids for identification of EDCs in aquatic toxicology studies.

Environmental pollution is a threat to humans and wildlife species. Of particular concern are endocrine disrupting chemicals (EDCs). An important target of EDCs is nuclear receptors (NRs) that control endocrine and metabolic responses through transcriptional regulation. Owing in part to structural differences of NRs, adverse effects of EDCs vary significantly among species. Here, we describe a multiplexed reporter assay (the Ecotox FACTORIAL) enabling parallel assessment of compounds' effects on estrogen, androgen, thyroid, and PPARγ receptors of representative mammals, birds, reptiles, amphibians, and fish. The Ecotox FACTORIAL is a single-well assay comprising a set of species-specific, one-hybrid GAL4-NR reporter constructs transiently transfected into test cells. To harmonize cross-species assessments, we used a combination of two approaches. First, we used the same type of test cells for all reporters; second, we implemented a parallel detection of reporter RNAs. The assay demonstrated excellent quality, reproducibility, and insignificant intra-assay variability. Importantly, the EC50 values for NR ligands were consistent with those reported for conventional assays. Using the assay allowed ranking the hazard potential of environmental pollutants (e.g., bisphenols, polycyclic aromatic hydrocarbons, and synthetic progestins) across species. Furthermore, the assay permitted detecting taxa-specific effects of surface water samples. Therefore, the Ecotox FACTORIAL enables harmonized assessment of the endocrine and metabolic disrupting activity of chemicals and surface water in humans as well as in wildlife species.

A set of adverse outcome pathways (AOPs) linking inhibition of thyroperoxidase and deiodinase to impaired swim bladder inflation in fish has recently been developed. These AOPs help to establish links between these thyroid hormone (TH) disrupting molecular events and adverse outcomes relevant to aquatic ecological risk assessment. Until now, very little data on the effects of TH disruption on inflation of the anterior chamber (AC) of the swim bladder were available. The present study used zebrafish exposure experiments with three model compounds with distinct thyroperoxidase and deiodinase inhibition potencies (methimazole, iopanoic acid, and propylthiouracil) to evaluate this linkage. Exposure to all three chemicals decreased whole body triiodothyronine (T3) concentrations, either through inhibition of thyroxine (T4) synthesis or through inhibition of Dio mediated conversion of T4 to T3. A quantitative relationship between reduced T3 and reduced AC inflation was established, a critical key event relationship linking impaired swim bladder inflation to TH disruption. Reduced inflation of the AC was directly linked to reductions in swimming distance compared to controls as well as to chemical-exposed fish whose ACs inflated. Together the data provide compelling support for AOPs linking TH disruption to impaired AC inflation in fish.

While chemical analysis of contaminant mixtures remains an essential component of environmental monitoring, bioactivity-based assessments using in vitro systems increasingly are used in the detection of biological effects. Historically, in vitro assessments focused on a few biological pathways, for example, aryl hydrocarbon receptor (AhR) or estrogen receptor (ER) activities. High-throughput screening (HTS) technologies have greatly increased the number of biological targets and processes that can be rapidly assessed. Here we screened extracts of surface waters from a nationwide survey of United States streams for bioactivities associated with 69 different end points using two multiplexed HTS assays. Bioactivity of extracts from 38 streams was evaluated and compared with concentrations of over 700 analytes to identify chemicals contributing to observed effects. Eleven primary biological end points were detected. Pregnane X receptor (PXR) and AhR-mediated activities were the most commonly detected. Measured chemicals did not completely account for AhR and PXR responses. Surface waters with AhR and PXR effects were associated with low intensity, developed land cover. Likewise, elevated bioactivities frequently associated with wastewater discharges included endocrine-related end points ER and glucocorticoid receptor. These results underscore the value of bioassay-based monitoring of environmental mixtures for detecting biological effects that could not be ascertained solely through chemical analyses.

High-throughput screening (HTS) and computational technologies have emerged as important tools for chemical hazard identification. The US Environmental Protection Agency (EPA) launched the Toxicity ForeCaster (ToxCast™) Program, which has screened thousands of chemicals in hundreds of mammalian-based HTS assays for biological activity. The data are being used to prioritize toxicity testing on those chemicals likely to lead to adverse effects. To use HTS assays in predicting hazard to both humans and wildlife, it is necessary to understand how broadly these data may be extrapolated across species. The US EPA Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/) tool was used to assess conservation of the 484 protein targets represented in the suite of ToxCast™ assays and other HTS assays. To demonstrate the utility of the SeqAPASS data for guiding extrapolation, case studies were developed which focused on targets of interest to the US Endocrine Disruptor Screening Program and the Organisation for Economic Cooperation and Development. These case studies provide a line of evidence for conservation of endocrine targets across vertebrate species, with few exceptions, and demonstrate the utility of SeqAPASS for defining the taxonomic domain of applicability for HTS results and identifying organisms for suitable follow-up toxicity tests.

The U.S. Environmental Protection Agency's ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential apical effects relevant to risk assessors. Thus, efforts are underway to develop AOPs relevant to pathway-specific perturbations detected in ToxCast assays. Previous work identified a "cytotoxic burst" (CTB) phenomenon wherein large numbers of the ToxCast assays begin to respond at or near test chemical concentrations that elicit cytotoxicity, and a statistical approach to defining the bounds of the CTB was developed. To focus AOP development on the molecular targets corresponding to ToxCast assays indicating pathway-specific effects, we conducted a meta-analysis to identify which assays most frequently respond at concentrations below the CTB. A preliminary list of potentially important, target-specific assays was determined by ranking assays by the fraction of chemical hits below the CTB compared with the number of chemicals tested. Additional priority assays were identified using a diagnostic-odds-ratio approach which gives greater ranking to assays with high specificity but low responsivity. Combined, the two prioritization methods identified several novel targets (e.g., peripheral benzodiazepine and progesterone receptors) to prioritize for AOP development, and affirmed the importance of a number of existing AOPs aligned with ToxCast targets (e.g., thyroperoxidase, estrogen receptor, aromatase). The prioritization approaches did not appear to be influenced by inter-assay differences in chemical bioavailability. Furthermore, the outcomes were robust based on a variety of different parameters used to define the CTB.

Current environmental monitoring approaches focus primarily on chemical occurrence. However, based on concentration alone, it can be difficult to identify which compounds may be of toxicological concern and should be prioritized for further monitoring, in-depth testing, or management. This can be problematic because toxicological characterization is lacking for many emerging contaminants. New sources of high-throughput screening (HTS) data, such as the ToxCast database, which contains information for over 9000 compounds screened through up to 1100 bioassays, are now available. Integrated analysis of chemical occurrence data with HTS data offers new opportunities to prioritize chemicals, sites, or biological effects for further investigation based on concentrations detected in the environment linked to relative potencies in pathway-based bioassays. As a case study, chemical occurrence data from a 2012 study in the Great Lakes Basin along with the ToxCast effects database were used to calculate exposure-activity ratios (EARs) as a prioritization tool. Technical considerations of data processing and use of the ToxCast database are presented and discussed. EAR prioritization identified multiple sites, biological pathways, and chemicals that warrant further investigation. Prioritized bioactivities from the EAR analysis were linked to discrete adverse outcome pathways to identify potential adverse outcomes and biomarkers for use in subsequent monitoring efforts.

Inflation of the posterior and/or anterior swim bladder are processes previously demonstrated to be thyroid-hormone regulated. We investigated whether inhibition of deiodinases, which convert thyroxine (T4) to the more biologically-active form, 3,5,3'-triiodothyronine (T3), would impact swim bladder inflation. Two experiments were conducted using a model deiodinase inhibitor, iopanoic acid (IOP). First, fathead minnow embryos were exposed to 0.6, 1.9, or 6.0 mg/L or control water until 6 days post-fertilization (dpf) at which time posterior swim bladder inflation was assessed. To examine anterior swim bladder inflation, a second study was conducted with 6 dpf larvae exposed to the same IOP concentrations until 21 dpf. Fish from both studies were sampled for T4/T3 measurements and gene transcription analyses. Incidence and length of inflated posterior swim bladders were significantly reduced in the 6.0 mg/L treatment at 6 dpf. Incidence of inflation and length of anterior swim bladder were significantly reduced in all IOP treatments at 14 dpf, but inflation recovered by 18 dpf. Throughout the larval study, whole body T4 concentrations increased and T3 concentrations decreased in all IOP treatments. Consistent with hypothesized compensatory responses, deiodinase-2 mRNA was up-regulated in the larval study, and thyroperoxidase mRNA was down-regulated in all IOP treatments in both studies. These results support the hypothesized adverse outcome pathways linking inhibition of deiodinase activity to impaired swim bladder inflation. This article is protected by copyright. All rights reserved

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 lat...

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.

Disruption of the thyroid hormone (TH) system, an important mode of action, can lead to ecologically relevant adverse outcomes, especially during embryonic development. The present study characterizes the effects of disruption of TH synthesis on swim bladder inflation during zebrafish early-life stages using 2-mercaptobenzothiazole (MBT), a thyroid peroxidase (TPO) inhibitor. Zebrafish were exposed to different MBT concentrations until 120/168h post fertilization (hpf) and 32days post fertilization (dpf), in two sets of experiments, to investigate the effects of TPO inhibition on posterior and anterior swim bladder inflation respectively, as well as whole body thyroid hormone concentrations (triiodothyronine (T3) and its prohormone, thyroxine (T4)). At 120hpf, MBT did not directly impair posterior chamber inflation or size, while anterior chamber inflation and size was impaired at 32dpf. As previously shown in amphibians and mammals, we confirmed that MBT inhibits TPO in fish. Whole-body T4 decreased after MBT exposure at both time points, while T3 levels were unaltered. There was a significant relationship between T4 levels and the anterior chamber surface at 32dpf. The absence of effects on posterior chamber inflation can possibly be explained by maternal transfer of T4 into the eggs. These maternally derived THs are depleted at 32dpf and cannot offset TPO inhibition, resulting in impaired anterior chamber inflation. Therefore, we hypothesize that TPO inhibition only inhibits swim bladder inflation during late development, after depletion of maternally derived T4. In a previous study, we showed that iodothyronine deiodinase (ID) knockdown impaired posterior chamber inflation during early development. Our findings, in parallel with similar effects observed in fathead minnow (see part I, this issue) suggest that thyroid disruption impacts swim bladder inflation, and imply an important distinction among specific subtypes of TH disrupting chemicals. However, the existence of another - yet unknown - mode of action of MBT impacting swim bladder inflation cannot be excluded. These results can be helpful for delineating adverse outcome pathways (AOPs) linking TPO inhibition, ID inhibition and other TH related molecular initiating events, to impaired swim bladder inflation in fish during early life stages. Such AOPs can support the use of in vitro enzyme inhibition assays for predicting reduced survival due to impaired posterior and anterior chamber inflation.

Although endocrine disrupting compounds have been detected in wastewater and surface waters worldwide using a variety of in vitro effects-based screening tools, e.g. bioassays, few have examined potential attenuation of environmental contaminants by both natural (sorption, degradation, etc) and anthropogenic (water treatment practices) processes. This study used several bioassays and quantitative chemical analyses to assess residence-time weighted samples at six sites along a river in the northeastern United States beginning upstream from a wastewater treatment plant outfall and proceeding downstream along the stream reach to a drinking water treatment plant. Known steroidal estrogens were quantified and changes in signaling pathway molecular initiating events (activation of estrogen, androgen, glucocorticoid, peroxisome proliferator-activated, pregnane X receptor, and aryl hydrocarbon receptor signaling networks) were identified in water extracts. In initial multi-endpoint assays geographic and receptor-specific endocrine activity patterns in transcription factor signatures and nuclear receptor activation were discovered. In subsequent single endpoint receptor-specific bioassays, estrogen (16 of 18 samples; 0.01 to 28 ng estradiol equivalents [E2Eqs]/L) glucocorticoid (3 of 18 samples; 1.8 to 21 ng dexamethasone equivalents [DexEqs]/L), and androgen (2 of 18 samples; 0.95 to 2.1 ng dihydrotestosterone equivalents [DHTEqs]/L) receptor transcriptional activation occurred above respective assay method detection limits (0.04 ng E2Eqs/L, 1.2 ng DexEqs/L, and 0.77 ng DHTEqs/L) in multiple sampling events. Estrogen activity, the most often detected, correlated well with measured concentrations of known steroidal estrogens (r(2) = 0.890). Overall, activity indicative of multiple types of endocrine active compounds was highest in wastewater effluent samples, while activity downstream was progressively lower, and negligible in unfinished treated drinking water. Not only was estrogenic and glucocorticoid activity confirmed in the effluent by utilizing multiple methods concurrently, but other activated signaling networks that historically received less attention (i.e. peroxisome proliferator-activated receptor) were also detected.

Several organic contaminants (OCs) have been detected in bald eagle (Haliaeetus leucocephalus) nestling (eaglet) plasma in the upper Midwestern United States. Despite frequent and relatively high concentrations of OCs in eaglets, little is understood about potential biological effects associated with exposure. We screened an existing database of OC concentrations in eaglet plasma collected from the Midwestern United States against bioactivity information from the ToxCast database. ToxCast bioactivity information consists of concentrations expected to elicit responses across a range of biological space (e.g. cellular, developmental, etc.) obtained from a series of high throughput assays. We calculated exposure-activity ratios (EAR) by calculating the ratio of plasma concentrations to concentrations available in ToxCast. Bioactivity data were not available for all detected OCs. Therefore, our analysis provides estimates of potential bioactivity for 19 of the detected OCs in eaglet plasma. Perfluorooctanesulfonic acid (PFOS) EAR values were consistently the highest among all study areas. Maximum EAR values were ≥1 for PFOS, perfluorononanoic acid, and bisphenol A in 99.7, 0.53 and 0.26% of samples, indicating that some plasma concentrations were greater than what may be expected to elicit biological responses. About 125 gene targets, indicative of specific biological pathways, were identified as potentially being affected. Inhibition of several CYP genes, involved in xenobiotic metabolism, were most consistently identified. Other identified biological responses have potential implications for motor coordination, cardiac functions, behavior, and blood circulation. However, it is unclear what these results mean for bald eagles, given that ToxCast data are generated using mammalian-based endpoints. Despite uncertainties and limitations, this method of screening environmental data can be useful for informing future monitoring or research focused on understanding the occurrence and effects of OCs in bald eagles and other similarly-positioned trophic species.

Pharmaceuticals and personal care products (PPCPs), wastewater indicators (WWIs), and pesticides (herein, Contaminants of Emerging Concern [CECs]) have been documented in surface waters throughout the world and have associated risks to aquatic life. While much research has focused on temperate and urbanized watersheds, less is known about CEC presence in semi-arid landscapes, where water availability is limited and populations are low. CEC presence in water and sediment is reported for 21 sites in eight U.S. national parks in the northern Colorado Plateau region. From 2012 to 2016, at least one PPCP and/or WWI was detected at most sites on over half of sampling visits, indicating that CECs are not uncommon even in isolated areas. CEC detections were generally fewer and at lower concentrations than in urbanized or agricultural watersheds. Consistent with studies from other U.S. regions, the most frequently detected CECs in this study include DEET, caffeine, organophosphorus flame retardants, and bisphenol A in water and fecal indicators and polycyclic aromatic hydrocarbons in sediment. Maximum concentrations in this study were generally below available water quality benchmarks, sediment quality guidelines, and risk assessment thresholds associated with vertebrates. Additional work is needed to assess the potential activity of hormones, which had high reporting limits in our study, and potential bioactivity of environmental concentrations for invertebrates, microbial communities, and algae. Potential sources of CEC contamination include upstream wastewater effluent discharges and National Park Service invasive-plant-control herbicide applications. CEC occurrence patterns and similarities between continuous and isolated flow locations suggest that direct contamination from individual visitors may also occur. While our data indicate there is little aquatic health risk associated with CECs at our sites, our results demonstrate the ubiquity of CECs on the landscape and a continued need for public outreach concerning resource-use ethics and the potential effects of upstream development.

Biotransformation of trenbolone acetate metabolites and estrogens derived from animal feeding operations in soils, waste storage systems, and in land applied manure has been well characterized. Yet recent data demonstrate potential for steroid transport into the environment directly from feedyard pens via runoff or airborne particulate matter. Therefore, the objective of this study was to determine steroid transformation rates in beef cattle excreta. Feces and urine were collected from steers recently treated with steroidal implants. Excreta were stored and periodically extracted over 112 d then analyzed for trenbolone acetate metabolites and estrogens by liquid chromatography mass spectrometry. Conjugated steroids were present primarily in urine, and conjugates quickly degraded to free steroid with a half-life of 0.6–1.0 d. The primary trenbolone acetate metabolite, 17α-trenbolone, had a half-life of 5.1–9.5 d. Likewise, 17α-estradiol was the predominant estrogen, with a half-life of 8.6–53 d. Secondary trenbolone metabolites formed from 17α-trenbolone biotransformation were observed at low concentrations less than 10% initial 17α-trenbolone concentrations. Estrone was the primary metabolite of 17α-estradiol and concentrations of estrone exceeded initial 17α-estradiol concentration in all sample types. These results suggest manure-borne steroids are more stable in excreta than in soil microcosms.

Steroid growth promoters are commonly administered to beef cattle residing on feedyards, and a portion of these compounds are excreted in manure along with endogenous steroids. Steroids associated with aerosolized particulate matter (PM) can be transported from feedyards via wind. To assess potential androgenic and estrogenic activity of PM extracts, total suspended particulate samples were collected upwind and downwind of feedyards in the Southern High Plains and subjected to in vitro transcriptional activation assays. Androgen-mediated transcriptional activation induced by exposure to extracts from PM collected downwind of feedyards was significantly higher than that induced by exposure to extracts of corresponding upwind samples, whereas estrogen-mediated transcriptional activation was detected after exposure to upwind and downwind PM sample extracts. Detection and quantitation of metabolites of the synthetic androgen trenbolone acetate downwind, and estradiol both upwind and downwind, suggest that synthetic growth promoters contribute to observed in vitro activity. No significant correlations were observed, however, between individual steroid concentrations or total androgen/estrogen concentration and in vitro activity, indicating the contributions of additional, unquantified compounds to observed androgenic and estrogenic activity. Results indicate that steroids affiliated with feedyard PM have the potential to elicit endocrine-modulating effects.

Background: Emergence and spread of antibiotic resistance has become a global health threat and is often linked with overuse and misuse of clinical and veterinary chemotherapeutic agents. Modern industrial-scale animal feeding operations rely extensively on veterinary pharmaceuticals, including antibiotics, to augment animal growth. Following excretion, antibiotics are transported through the environment via runoff, leaching, and land application of manure; however, airborne transport from feed yards has not been characterized. Objectives: The goal of this study was to determine the extent to which antibiotics, antibiotic resistance genes (ARG), and ruminant-associated microbes are aerially dispersed via particulate matter (PM) derived from large-scale beef cattle feed yards. Methods: PM was collected downwind and upwind of 10 beef cattle feed yards. After extraction from PM, five veterinary antibiotics were quantified via high-performance liquid chromatography with tandem mass spectrometry, ARG were quantified via targeted quantitative polymerase chain reaction, and microbial community diversity was analyzed via 16S rRNA amplification and sequencing. Results: Airborne PM derived from feed yards facilitated dispersal of several veterinary antibiotics, as well as microbial communities containing ARG. Concentrations of several antibiotics in airborne PM immediately downwind of feed yards ranged from 0.5 to 4.6 μg/g of PM. Microbial communities of PM collected downwind of feed yards were enriched with ruminant-associated taxa and were distinct when compared to upwind PM assemblages. Furthermore, genes encoding resistance to tetracycline antibiotics were significantly more abundant in PM collected downwind of feed yards as compared to upwind. Conclusions: Wind-dispersed PM from feed yards harbors antibiotics, bacteria, and ARGs. Citation: McEachran AD, Blackwell BR, Hanson JD, Wooten KJ, Mayer GD, Cox SB, Smith PN. 2015. Antibiotics, bacteria, and antibiotic resistance genes: aerial transport from cattle feed yards via particulate matter. Environ Health Perspect 123:337–343; http://dx.doi.org/10.1289/ehp.1408555

To better characterize the transport of neonicotinoid insecticides to the world’s largest freshwater ecosystem, monthly samples (October 2015-September 2016) were collected from 10 major tributaries to the Great Lakes, USA. For the monthly tributary samples, neonicotinoids were detected in every month sampled and five of the six target neonicotinoids were detected. At least one neonicotinoid was detected in 74% of the monthly samples with up to three neonicotinoids detected in an individual sample (10% of all samples). The most frequently detected neonicotinoid was imidacloprid (53%), followed by clothianidin (44%), thiamethoxam (22%), acetamiprid (2%), and dinotefuran (1%). Thiacloprid was not detected in any samples. The maximum concentration for an individual neonicotinoid was 230 ng L(−1) and the maximum total neonicotinoids in an individual sample was 400 ng L(−1). The median detected individual neonicotinoid concentrations ranged from non-detect to 10 ng L(−1). The detections of clothianidin and thiamethoxam significantly increased as the percent of cultivated crops in the basins increased (ρ = 0.73, P = 0.01; ρ = 0.66, P = 0.04, respectively). In contrast, imidacloprid detections significantly increased as the percent of the urbanization in the basins increased (ρ = 0.66, P = 0.03). Neonicotinoid concentrations generally increased in spring through summer coinciding with the planting of neonicotinoid-treated seeds and broadcast applications of neonicotinoids. More spatially intensive samples were collected in an agriculturally dominated basin (8 sites along the Maumee River, Ohio) twice during the spring, 2016 planting season to provide further information on neonicotinoid inputs to the Great Lakes. Three neonicotinoids were ubiquitously detected (clothianidin, imidacloprid, thiamethoxam) in all water samples collected within this basin. Maximum individual neonicotinoid concentrations was 330 ng L(−1)and maximum total neonicotinoid concentration was 670 ng L(−1); median detected individual neonicotinoid concentrations were 7.0 to 39 ng L(−1).

Studies worldwide have demonstrated the occurrence of feminized male fish at sites impacted by human and animal wastes. A variety of chemicals could contribute to this phenomenon, but those receiving the greatest attention in terms of research and monitoring have been 17β-estradiol (β-E2) and 17α-ethinylestradiol, due both to their prevalence in the environment and strong estrogenic potency. A third steroid, estrone (E1), also can occur at high concentrations in surface waters but generally has been of lesser concern due to its relatively lower affinity for vertebrate estrogen receptors. In an initial experiment, male fathead minnow (Pimephales promelas) adults were exposed for 4-d to environmentally relevant levels of waterborne E1, which resulted in plasma β-E2 concentrations similar to those found in reproductively active females. In a second exposure we used 13C-labeled E1, together with liquid chromatography-tandem mass spectrometry, to demonstrate that elevated β-E2 measured in the plasma of the male fish was indeed derived from the external environment, most likely via a conversion catalyzed by one or more 17β-hydroxysteroid dehydrogenases. The results of our studies suggest that the potential impact of E1 as an environmental estrogen currently is underestimated.

Exogenous growth promoters have been used in US beef cattle production for over 50 yr. The environmental fate and transport of steroid growth promoters suggest potential for endocrine-disrupting effects among ecological receptors; however, the initial excretion of steroid metabolites from cattle administered growth promoters has not been well characterized. To better characterize excretion of trenbolone acetate and estrogen metabolites, steers were assigned to 1 of the following treatment groups: control, given no implant, or treatment, administered a combination implant (200 mg trenbolone acetate, 40 mg estradiol). Blood, urine, and fecal samples were collected over the course of 112 d following implantation. Samples were extracted and analyzed by liquid chromatography tandem mass spectrometry for trenbolone acetate and estrogen metabolites. In both urine and feces, 17α-trenbolone and 17α-estradiol were the predominant metabolites following implantation. Mean concentrations of 17α-trenbolone and 17α-estradiol in feces of implanted steers were 5.9 ± 0.37 ng/g and 2.7 ± 0.22 ng/g, respectively. A best-fit model is presented to predict 17α-trenbolone and 17α-estradiol excretion from steers receiving implants. The present study provides the first characterization of both trenbolone and estrogen metabolites in excreta from implanted cattle and will help provide estimates of steroid production from feedyards in the United States.

British × Continental steers (n = 168; 7 pens/treatment; initial BW = 362 kg) were used to evaluate the effect of dose/payout pattern of trenbolone acetate (TBA) and estradiol-17β (E2) and feeding of zilpaterol hydrochloride (ZH) on serum urea-N (SUN), NEFA, IGF-I, and E2 concentrations and LM mRNA expression of the estrogen (ER), androgen (ANR), IGF-I (IGF-IR), β1-adrenergic (β1-AR), and β2-adrenergic (β2-AR) receptors and IGF-I. A randomized complete block design was used with a 3 × 2 factorial arrangement of treatments. Main effects were implant (no implant [NI], Revalor-S [REV-S; 120 mg TBA + 24 mg E2], and Revalor-XS [REV-X; 200 mg TBA + 40 mg E2]) and ZH (0 or 8.3 mg/kg of DM for 20 d with a 3-d withdrawal). Steers were fed for 153 or 174 d. Blood was collected (2 steers/pen) at d -1, 2, 6, 13, 27, 55, 83, 111, and 131 relative to implanting; LM biopsies (1 steer/pen) were collected at d -1, 27, 55, and 111. Blood and LM samples were collected at d -1, 11, and 19 relative to ZH feeding. A greater dose of TBA + E2 in combination with ZH increased ADG and HCW in an additive manner, suggesting a different mechanism of action for ZH and steroidal implants. Implanting decreased (P < 0.05) SUN from d 2 through 131. Feeding ZH decreased (P < 0.05) SUN. Serum NEFA concentrations were not affected by implants (P = 0.44). There was a day × ZH interaction (P = 0.06) for NEFA; ZH steers had increased (P < 0.01) NEFA concentrations at d 11 of ZH feeding. Serum E2 was greater (P < 0.05) for implanted steers by d 27. Serum trenbolone-17β was greater (P < 0.05) for implanted steers by d 2 followed by a typical biphasic release rate, with a secondary peak at d 111 for REV-X (P < 0.05) implanted steers. Implanting did not affect mRNA expression of the ANR or ER, but the IGF-IR and the β1-AR and β2-AR were less (P < 0.05) for REV-S than NI at d 55 and β2-AR mRNA was less (P < 0.05) for REV-S than for REV-X. Expression of the IGF-IR and the β1-AR at d 111 was greater (P< 0.05) for REV-X than for REV-S and NI at d 111, and the β2-AR was less (P< 0.05) for REV-S than for REV-X. Feeding ZH did not affect mRNA expression of the β1-AR and β2-AR. Both implanting and feeding ZH decreased SUN, but a greater dose of TBA + E2 did not result in further decreases. In addition, feeding ZH increased serum NEFA concentrations. Metabolic changes resulting from implanting and feeding ZH may aid in explaining steer performance and carcass responses to these growth promotants.

Perfluoroalkyl substances (PFAS) have recently received increased research attention, particularly concerning aquatic organisms and in regions of exposure to Aqueous Film Forming Foams (AFFFs). Air Force bases historically applied AFFFs in the interest of fire training exercises and have since expressed concern for PFAS contamination in biota from water bodies surrounding former fire training areas. We monitored six PFASs, including perfluorooctane sulfonate (PFOS), in aquatic species from eight bayou locations at Barksdale Air Force Base in Bossier City, Louisiana over the course of one year. Our focus was to evaluate temporal and spatial variability in PFAS concentrations from historic use of AFFF. PFOS concentrations in fish tissues peaked in early summer, and also increased significantly downstream of former fire training areas. Benthic organisms had lower PFOS concentrations than pelagic species, contrary to previous literature observations. Bioconcentration factors varied with time, however were reduced as compared to previously reported literature values. Here we report the highest concentration of PFOS in whole fish (9,349 ng/g dw), with 15% of samples exceeding what we believe to be the maximum whole fish concentration reported to date of 1,500 ng/g ww. Future studies to measure PFAS in larger fish and tissue specific partitioning data to compare to the current whole fish values are ongoing. The high concentrations observed presently could have effects for higher trophic level organisms in this system or potential risk to humans consuming contaminated fish. This article is protected by copyright. All rights reserved

Manure from livestock feeding operations is commonly applied to agricultural fields as an alternative to commercial fertilizers. Trenbolone acetate (TbA) is a synthetic growth promoter frequently utilized in beef cattle feeding operations. Metabolites of TbA can be present in manure and subsequently applied to fields. Fate ofTbA metabolites 17β-trenbolone (17βTb), 17α-trenbolone (17αTb), and trendione (TbO) have been assessed in manure and soils, but plant uptake in agricultural fields is not fully understood. The objective of this study was to investigate potential plant uptake and biotransformation of 17βTb using the pinto bean plant (Phaseolus vulgaris). Vegetated (n=20) and control sands (n=16) were amended with 17βTb at a level of 1 μg/g once per week for a total of four weeks. Sand, above-ground plant portion and below-ground plant portion were collected each week and then analyzed for 17βTb, 17αTb, and TbO. By week four, low concentrations of 17βTb (10±4.9 μg/g fresh weight) were taken up into the roots of plants and, to a much lesser extent, translocated throughout the plant (0.04±0.02 μg/g fresh weight). Extensive transformation of 17βTb to the metabolite trendione (TbO) occurred in vegetated sand, while minimal TbO was detected in control sand. These results suggest the biotransformation of 17βTb to TbO is predominantly through microbial degradation. Trenbolone (Tb) metabolites can then be taken up into plants but remain concentrated in the roots with only slight translocation to above ground portions of the plant. After four weeks, maximum observed concentrations of total Tb (parent+metabolites) in fresh plant tissues were 33.0 μg/g in roots and 0.25 μg/g in leaves. No phytotoxicity was observed to pinto bean plants throughout the four week study.

Background With the increase in patient activity during the 2009 H1N1 pandemic, came an associated increase in occupational infections of healthcare workers (HCWs). Objectives The aim of this study was to examine factors associated with the transmission of pandemic (H1N1) 2009 among HCWs. Methods A 1:4 matched case–control study by hospital, ward, age, and gender was conducted in HCWs from hospitals in Beijing during February 2010. Cases were diagnosed with pandemic (H1N1) 2009, and controls had neither influenza-like illness nor diagnosis with pandemic (H1N1) 2009 during August 2009 to January 2010. Information during 7 days before symptom onset of case was collected, and controls were queried about the same period. Results A total of 51 cases identified via National Notifiable Infectious Disease Surveillance System participated in this study. Controls were matched to cases for a total of 255 individuals. About 19·6% (10/51) of cases and 26·0% (53/204) of controls recalled they had conducted a high-risk procedure on a patient with pandemic (H1N1) 2009. 72·5% (37/51) of cases and 71·6% (146/204) of controls stated they wore medical masks in ≥80% of working time. Only 5·9% (3/51) and 36·3% (74/204) of cases and controls, respectively, reported receiving pandemic vaccination. Participants receiving pandemic vaccination had a significantly lower risk of infection during the pandemic (OR = 0·150, 95% CI: 0·047–0·479, P = 0·001). The estimated vaccine effectiveness was 85·0%. Conclusions We showed a high vaccine effectiveness of the pandemic vaccine and that vaccination was the only factor significantly associated with risk of infection in HCWs.

The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB and 10 ng/L MGA + 100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout-vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA + 500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout-vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development.

Trenbolone acetate (TbA) is a potent synthetic anabolic steroid that was approved by the FDA as a growth promoter in beef cattle in 1987. Given the endocrine-modulating activity of TbA and its metabolites in all vertebrates, a sensitive and reliable analytical method is needed to detect TbA and related residues in environmental matrices. We have developed a method that incorporates solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of the three major TbA metabolites (trendione, 17β-trenbolone, 17α-trenbolone) in total suspended particulate matter (TSP) samples. Sample preparation involved pressurized liquid extraction followed by cleanup on solid-phase extraction cartridges. The procedure was optimized to obtain maximum recovery and minimum signal suppression/enhancement from matrix effects. Analytes were separated with a Phenomenex Gemini-NX C18 analytical column (150 mm × 2.0 mm, 3 μm particle size) using an aqueous methanol gradient at a flow rate of 0.2 mL/min. Column effluent underwent positive electrospray ionization (ESI). Two or more diagnostic product ions were acquired from analyte specific precursor ions for unambiguous confirmation and quantification. The method detection limit was 3.27-4.87 ng/g of particulate matter (PM). Method accuracy, determined with analyte recoveries, ranged between 68% and 117%, and method precision, expressed as relative standard deviation, was below 15% at spiked levels of 6.67, 33.3, and 167 ng/g PM. Analysis of TSP samples demonstrated the presence of the target species associated with PM in the vicinity of beef cattle feeding operations.

In the present study, a hypothesized adverse outcome pathway linking inhibition of thyroid peroxidase (TPO) activity to impaired swim bladder inflation was investigated in two experiments in which fathead minnows (Pimephales promelas) were exposed to 2-mercaptobenzothiazole (MBT). Continuous exposure to 1mg MBT/L for up to 22 days had no effect on inflation of the posterior chamber of the swim bladder, which typically inflates around 6 days post fertilization (dpf), a period during which maternally-derived thyroid hormone is presumed to be present. In contrast, inflation of the anterior swim bladder, which occurs around 14dpf, was impacted. Specifically, at 14dpf, approximately 50% of fish exposed to 1mg MBT/L did not have an inflated anterior swim bladder. In fish exposed to MBT through 21 or 22dpf, the anterior swim bladder was able to inflate, but the ratio of the anterior/posterior chamber length was significantly reduced compared to controls. Both abundance of thyroid peroxidase mRNA and thyroid follicle histology suggest that fathead minnows mounted a compensatory response to the presumed inhibition of TPO activity by MBT. Time-course characterization showed that fish exposed to MBT for at least 4 days prior to normal anterior swim bladder inflation had significant reductions in anterior swim bladder size, relative to the posterior chamber, compared to controls. These results, along with similar results observed in zebrafish (see part II, this issue) are consistent with the hypothesis that thyroid hormone signaling plays a significant role in mediating anterior swim bladder inflation and development in cyprinids, and that role can be disrupted by exposure to thyroid hormone synthesis inhibitors. Nonetheless, possible thyroid-independent actions of MBT on anterior swim bladder inflation cannot be ruled out based on the present results. Overall, although anterior swim bladder inflation has not been directly linked to survival as posterior swim bladder inflation has, potential links to adverse ecological outcomes are plausible given involvement of the anterior chamber in sound production and detection.