Search

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.

Contaminants of Emerging Concern (CECs) can be measured in waters across the United States, including the tributaries of the Great Lakes. The extent to which these contaminants affect gene expression in aquatic wildlife is unclear. This dataset presents the full hepatic transcriptomes of laboratory-reared fathead minnows (Pimephales promelas) caged at multiple sites within the Milwaukee Estuary Area of Concern and control sites. Following 4 days of in situ exposure, liver tissue was removed from males at each site for RNA extraction and sequencing, yielding a total of 116 samples from which libraries were prepared, pooled, and sequenced. For each exposure site, 179 chemical analytes were also assessed. These data were created with the intention of inviting research on possible transcriptomic changes observed in aquatic species exposed to CECs. Access to both full sequencing reads of animal samples as well as water contaminant data across multiple Great Lakes sites will allow others to explore the health of these ecosystems in support of the aims of the Great Lakes Restoration Initiative.

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.

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.

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.

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.

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.

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.

Inflation of the posterior and/or anterior swim bladder is a process previously demonstrated to be regulated by thyroid hormones. 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 d postfertilization (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 messenger ribonucleic acid (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. Environ Toxicol Chem 2017;36:2942-2952. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

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

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.

Early-life stage fish can be more sensitive to toxicants than adults, so delineating mechanisms of perturbation of biological pathways by chemicals during this life stage is crucial. Whole-mount in situ hybridization (WISH) paired with quantitative real-time polymerase chain reaction (QPCR) assays can enhance pathway-based analyses through determination of specific tissues where changes in gene expression are occurring. While WISH has frequently been used in zebrafish (Danio rerio), this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model species. The objective of the present study was to adapt WISH to fathead minnow embryos and larvae, and use the approach to evaluate the effects of estrone, an environmentally-relevant estrogen receptor (ER) agonist. Embryos were exposed via the water to 0, 18 or 1800 ng estrone/L (0, 0.067 and 6.7nM) for 3 or 6 days in a solvent-free, flow-through test system. Relative transcript abundance of three estrogen-responsive genes, estrogen receptor-α (esr1), cytochrome P450-aromatase B (cyp19b), and vitellogenin (vtg) was examined in pooled whole embryos using QPCR, and the spatial distribution of up-regulated gene transcripts was examined in individual fish using WISH. After 3 days of exposure to 1800 ng estrone/L, esr1 and cyp19b were significantly up-regulated, while vtg mRNA expression was not affected. After 6 days of exposure to 1800 ng estrone/L, transcripts for all three genes were significantly up-regulated. Corresponding WISH assays revealed spatial distribution of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. This study clearly demonstrates the potential utility of WISH, in conjunction with QPCR, to examine the mechanistic basis of the effects of toxicants on early-life stage fathead minnows.

Wastewater treatment plant (WWTP) effluents are known contributors of chemical mixtures into the environment. Of particular concern are endocrine-disrupting compounds, such as estrogens, which can affect the hypothalamic-pituitary-gonadal axis function in exposed organisms. The present study examined reproductive effects in fathead minnows exposed for 21 d to a historically estrogenic WWTP effluent. Fathead minnow breeding pairs were held in control water or 1 of 3 effluent concentrations (5%, 20%, and 100%) in a novel onsite, flow-through system providing real-time exposure. The authors examined molecular and biochemical endpoints representing key events along adverse outcome pathways linking estrogen receptor activation and other molecular initiating events to reproductive impairment. In addition, the authors used chemical analysis of the effluent to construct a chemical-gene interaction network to aid in targeted gene expression analyses and identifying potentially impacted biological pathways. Cumulative fecundity was significantly reduced in fish exposed to 100% effluent but increased in those exposed to 20% effluent, the approximate dilution factor in the receiving waters. Plasma vitellogenin concentrations in males increased in a dose-dependent manner with effluent concentration; however, male fertility was not impacted. Although in vitro analyses, analytical chemistry, and biomarker responses confirmed the effluent was estrogenic, estrogen receptor agonists were unlikely the primary driver of impaired reproduction. The results provide insights into the significance of pathway-based effects with regard to predicting adverse reproductive outcomes.