Analysis of Legacy and Novel Neutral Per- and Polyfluoroalkyl Substances in Soils from an Industrial Manufacturing Facility.

Per- and polyfluoroalkyl substances (PFASs) have been detected in an array of environmental media due to their ubiquitous use in industrial and consumer products as well as potential release from fluorochemical manufacturing facilities. During their manufacture, many fluorotelomer (FT) facilities rely on neutral intermediates in polymer production including the FT-alcohols (FTOHs). These PFAS are known to transform to the terminal acids (perfluoro carboxylic acids; PFCAs) at rates that vary with environmental conditions. In the current study on soils from a FT facility, we employed gas chromatography coupled with conventional- and high-resolution mass spectrometry (GC-MS and GC-HRMS) to investigate the profile of these precursor compounds, the intermediary secondary alcohols (sFTOHs), FT-acrylates (FTAcr), and FT-acetates (FTAce) in soils around the former FT-production facility. Of these precursors, the general trend in detection intensity was [FTOHs] > [sFTOHs] > [FTAcrs], while for the FTOHs, homologue intensities generally were [12:2 FTOH] > [14:2 FTOH] > [16:2 FTOH] > [10:2 FTOH] > [18:2 FTOH] > [20:2 FTOH] > [8:2 FTOH] ∼ [6:2 FTOH]. The corresponding terminal acids were also detected in all soil samples and positively correlated with the precursor concentrations. GC-HRMS confirmed the presence of industrial manufacturing byproducts such as FT-ethers and FT-esters and aided in the tentative identification of previously unreported dimers and other compounds. The application of GC-HRMS to the measurement and identification of precursor PFAS is in its infancy, but the methodologies described here will help refine its use in tentatively identifying these compounds in the environment.

Targeted Per- and Polyfluoroalkyl substances (PFAS) assessments for high throughput screening: Analytical and testing considerations to inform a PFAS stock quality evaluation framework.

Per- and polyfluoroalkyl substances (PFAS) represent a large chemical class lacking hazard, toxicokinetic, and exposure information. To accelerate PFAS hazard evaluation, new approach methodologies (NAMs) comprised of in vitro high-throughput toxicity screening, toxicokinetic data, and computational modeling are being employed in read across strategies to evaluate the larger PFAS landscape. A critical consideration to ensure robust evaluations is a parallel assessment of the quality of the screening stock solutions, where dimethyl sulfoxide (DMSO) is often the diluent of choice. Challenged by the lack of commercially available reference standards for many of the selected PFAS and reliance on mass spectrometry approaches for such an evaluation, we developed a high-throughput framework to evaluate the quality of screening stocks for 205 PFAS selected for these NAM efforts. Using mass spectrometry coupled with either liquid or gas chromatography, a quality scoring system was developed that incorporated observations during mass spectral examination to provide a simple pass or fail notation. Informational flags were used to further describe findings regarding parent analyte presence through accurate mass identification, evidence of contaminants and/or degradation, or further describe characteristics such as isomer presence. Across the PFAS-DMSO stocks tested, 148 unique PFAS received passing quality scores to allow for further in vitro testing whereas 57 received a failing score primarily due to detection issues or confounding effects of DMSO. Principle component analysis indicated vapor pressure and Henry's Law Constant as top indicators for a failed quality score for those analyzed by gas chromatography. Three PFAS in the hexafluoropropylene oxide family failed due to degradation in DMSO. As the PFAS evaluated spanned over 20 different structural categories, additional commentary describes analytical observations across specific groups related to PFAS stock composition, detection, stability, and methodologic considerations that will be useful for informing future analytical assessment and downstream HTS efforts. The high-throughput stock quality scoring workflow presented holds value as a tool to evaluate chemical presence and quality efficiently and for informing data inclusion in PFAS or other NAM screening efforts.

Evaluation of Per- and Polyfluoroalkyl Substances (PFAS) In Vitro Toxicity Testing for Developmental Neurotoxicity.

Per- and polyfluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors.

Endogenous and exogenous biomarker analysis in terrestrial phase amphibians (Lithobates sphenocephala) following dermal exposure to pesticide mixtures.

Pesticide mixtures are frequently co-applied throughout an agricultural growing season to maximize crop yield. Therefore, non-target ecological species (e.g., amphibians) may be exposed to several pesticides at any given time on these agricultural landscapes. The objectives of this study were to quantify body burdens in terrestrial phase amphibians and translate perturbed metabolites to their corresponding biochemical pathways affected by exposure to pesticides as both singlets and in combination. Southern leopard frogs (Lithobates sphenocephala) were exposed either at maximum or 1/10th maximum application rate to single, double, or triple pesticide mixtures of bifenthrin (insecticide), metolachlor (herbicide), and triadimefon (fungicide). Tissue concentrations demonstrate both facilitated and competitive uptake of pesticides when in mixtures. Metabolomic profiling of amphibian livers identified metabolites of interest for both application rates, however; magnitude of changes varied for the two exposure rates. Exposure to lower concentrations demonstrated down regulation in amino acids, potentially due to their being utilized for glutathione metabolism and/or increased energy demands. Amphibians exposed to the maximum application rate resulted in up regulation of amino acids and other key metabolites likely due to depleted energy resources. Coupling endogenous and exogenous biomarkers of pesticide exposure can be utilized to form vital links in an ecological risk assessment by relating internal dose to pathophysiological outcomes in non-target species.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Approaches for predicting effects of unintended environmental exposure to an endocrine active pharmaceutical, tamoxifen.

Tamoxifen is an endocrine-active pharmaceutical (EAP) that is used world-wide. Because tamoxifen is a ubiquitous pharmaceutical and interacts with estrogen receptors, a case study was conducted with this compound to (1) determine effects on reproductive endpoints in a nontarget species (i.e., a fish), (2) compare biologically-active metabolites across species, (3) assess whether in vitro assays predict in vivo results, and (4) investigate metabolomic profiles in tamoxifen-treated fish to better understand the biological mechanisms of tamoxifen toxicity. In reproductive assays, tamoxifen exposure caused a significant reduction in egg production and significantly increased ovarian aromatase activity in spawning adult cunner fish (Tautogolabrus adspersus). In plasma from tamoxifen-exposed cunner, the predominant metabolite was 4-hydroxytamoxifen, while in rats it was N-desmethyltamoxifen. Because 4-hydroxytamoxifen is a more biologically active metabolite than N-desmethyltamoxifen, this difference could result in a different level of risk for the two species. The results of in vitro assays with fish hepatic microsomes to assess tamoxifen metabolism did not match in vivo results, indicating probable differences in excretion of tamoxifen metabolites in fish compared with rats. For the first time, a complete in vitro characterization of the metabolism of tamoxifen using fish microsomes is presented. Furthermore, a metabolomic investigation of cunner gonad extracts demonstrates that tamoxifen alters the biochemical profile in this nontarget species. Understanding the consequence of tamoxifen exposure in nontarget species, and assessing the discrepancies between sex- and species-mediated endpoints, is a step toward understanding how to accurately assess the risks posed by EAPs, such as tamoxifen, in the aquatic environment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1834-1850, 2016.

Multiwalled carbon nanotube dispersion methods affect their aggregation, deposition, and biomarker response.

To systematically evaluate how dispersion methods affect the environmental behaviors of multiwalled carbon nanotubes (MWNTs), MWNTs were dispersed in various solutions (e.g., surfactants, natural organic matter (NOM), and etc.) via ultrasonication (SON) and long-term stirring (LT). The two tested surfactants [anionic sodium dodecyl sulfate (SDS) and nonionic poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEO-PPO-PEO) triblock copolymers (Pluronic)] could only disperse MWNTs via ultrasonication; while stable aqueous SON/MWNT and LT/MWNT suspensions were formed in the presence of the two model NOMs (Suwannee river humic acid and fulvic acid). Due to the inherent stochastic nature for both methods, the formed MWNT suspensions were highly heterogeneous. Their physicochemical properties, including surface charge, size, and morphology, greatly depended upon the dispersant type and concentration but were not very sensitive to the preparation methods. Aggregation and deposition behaviors of the dispersed MWNTs were controlled by van der Waal and electrostatic forces, as well as other non-DLVO forces (e.g., steric, hydrophobic forces, etc.). Unlike the preparation method-independent physicochemical properties, LT/NOM-MWNTs and SON/NOM-MWNTs differed in their fathead minnow epithelial cell metabolomics profiles.

Photochemical transformation of graphene oxide in sunlight.

Graphene oxide (GO) is promising in scalable production and has useful properties that include semiconducting behavior, catalytic reactivity, and aqueous dispersibility. In this study, we investigated the photochemical fate of GO under environmentally relevant sunlight conditions. The results indicate that GO readily photoreacts under simulated sunlight with the potential involvement of electron-hole pair creation. GO was shown to photodisproportionate to CO2, reduced materials similar to reduced GO (rGO) that are fragmented compared to the starting material, and low molecular-weight (LMW) species. Kinetic studies show that the rate of the initially rapid photoreaction of GO is insensitive to the dissolved oxygen content. In contrast, at longer time points (>10 h), the presence of dissolved oxygen led to a greater production of CO2 than the same GO material under N2-saturated conditions. Regardless, the rGO species themselves persist after extended irradiation equivalent to 2 months in natural sunlight, even in the presence of dissolved oxygen. Overall, our findings indicate that GO phototransforms rapidly under sunlight exposure, resulting in chemically reduced and persistent photoproducts that are likely to exhibit transport and toxic properties unique from parent GO.

Pesticide Uptake Across the Amphibian Dermis Through Soil and Overspray Exposures.

For terrestrial amphibians, accumulation of pesticides through dermal contact is a primary route of exposure in agricultural landscapes and may be contributing to widespread amphibian declines. To show pesticide transfer across the amphibian dermis at permitted label application rates, our study was designed to measure pesticide body burdens after two simulated exposure scenarios. We compared direct exposures, where amphibians were present when spraying occurred, to indirect exposures, where amphibians were exposed to soils after pesticide application. During summer 2012, we reared barking (Hyla gratiosa) and green treefrogs (H. cinerea) through 60-90 days post-metamorphosis at a United States Environmental Protection Agency research laboratory. We tested exposure for 8 h to five pesticide active ingredients (imidacloprid, atrazine, triadimefon, fipronil, or pendimethalin) in glass aquaria lined with soil in the laboratory. We quantified total pesticide body burden and soil concentrations using liquid chromatography-mass spectrometry. All individuals in both treatments had measurable body burdens at the end of the study. A randomized block design analysis of variance (n = 18) showed that body burdens (p = 0.03) and bioconcentration factors (BCFs) (p = 0.01) were significantly greater in the direct overspray treatment relative to the indirect soil spray treatment for both species and tested pesticides. BCFs ranged from 0.1 to 1.16 and from 0.013 to 0.78 in the direct and indirect treatments, respectively. Our study shows dermal uptake for multiple pesticides from both direct spray and indirect soil exposures and provides empirical support for the degree to which terrestrial phase amphibians have higher body burdens after overspray pesticide exposure.

Vibrio alginolyticus growth kinetics and the metabolic effects of iron.

IMPORTANCE: Transmission of V. alginolyticus occurs opportunistically through direct seawater exposure and is a function of its abundance in the environment. Like other Vibrio spp., V. alginolyticus are considered conditionally rare taxa in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions, which remain poorly defined. Prior research has established the importance of temperature and salinity as the major determinants of Vibrio geographical and temporal range. However, bloom formation can be strongly influenced by other factors that may be more episodic and localized, such as changes in iron availability. Here we confirm the broad temperature and salinity tolerance of V. alginolyticus and demonstrate the importance of iron supplementation as a key factor for growth in the absence of thermal or osmotic stress. The results of this research highlight the importance of episodic iron input as a crucial metric to consider for the assessment of V. alginolyticus risk.

Category-Based Toxicokinetic Evaluations of Data-Poor Per- and Polyfluoroalkyl Substances (PFAS) using Gas Chromatography Coupled with Mass Spectrometry.

Concern over per- and polyfluoroalkyl substances (PFAS) has increased as more is learned about their environmental presence, persistence, and bioaccumulative potential. The limited monitoring, toxicokinetic (TK), and toxicologic data available are inadequate to inform risk across this diverse domain. Here, 73 PFAS were selected for in vitro TK evaluation to expand knowledge across lesser-studied PFAS alcohols, amides, and acrylates. Targeted methods developed using gas chromatography-tandem mass spectrometry (GC-MS/MS) were used to measure human plasma protein binding and hepatocyte clearance. Forty-three PFAS were successfully evaluated in plasma, with fraction unbound (fup) values ranging from 0.004 to 1. With a median fup of 0.09 (i.e., 91% bound), these PFAS are highly bound but exhibit 10-fold lower binding than legacy perfluoroalkyl acids recently evaluated. Thirty PFAS evaluated in the hepatocyte clearance assay showed abiotic loss, with many exceeding 60% loss within 60 min. Metabolic clearance was noted for 11 of the 13 that were successfully evaluated, with rates up to 49.9 µL/(min × million cells). The chemical transformation simulator revealed potential (bio)transformation products to consider. This effort provides critical information to evaluate PFAS for which volatility, metabolism, and other routes of transformation are likely to modulate their environmental fates.

Occurrence of aetokthonotoxin producer in natural samples - A PCR protocol for easy detection.

Cyanobacteria are well known producers of bioactive metabolites, including harmful substances. The recently discovered "eagle killer" neurotoxin aetokthonotoxin (AETX) is produced by the epiphytic cyanobacterium Aetokthonos hydrillicola growing on invasive water thyme (Hydrilla verticillata). The biosynthetic gene cluster of AETX was previously identified from an Aetokthonos strain isolated from the J. Strom Thurmond Reservoir, Georgia, USA. Here, a PCR protocol for easy detection of AETX-producers in environmental samples of plant-cyanobacterium consortia was designed and tested. Three different loci of the AETX gene cluster were amplified to confirm the genetic potential for AETX production, along with two variable types of rRNA ITS regions to confirm the homogeneity of the producer´s taxonomic identity. In samples of Hydrilla from three Aetokthonos-positive reservoirs and one Aetokthonos-negative lake, the PCR of all four loci provided results congruent with the Aetokthonos presence/absence detected by light and fluorescence microscopy. The production of AETX in the Aetokthonos-positive samples was confirmed using LC-MS. Intriguingly, in J. Strom Thurmond Reservoir, recently Hydrilla free, an Aetokthonos-like cyanobacterium was found growing on American water-willow (Justicia americana). Those specimens were positive for all three aet markers but contained only minute amounts of AETX. The obtained genetic information (ITS rRNA sequence) and morphology of the novel Aetokthonos distinguished it from all the Hydrilla-hosted A. hydrillicola, likely at the species level. Our results suggest that the toxigenic Aetokthonos spp. can colonize a broader array of aquatic plants, however the level of accumulation of the toxin may be driven by host-specific interactions such as the locally hyper-accumulated bromide in Hydrilla.

Estimating dermal contact soil exposure for amphibians.

Chemical exposure estimation through the dermal route is an underemphasized area of ecological risk assessment for terrestrial animals. Currently, there are efforts to create exposure models to estimate doses from this pathway for use in ecological risk assessment. One significant limitation has been insufficient published data to characterize exposure and to support the selection and parameterization of appropriate models, particularly for amphibians in terrestrial habitats. Recent publications measuring pesticide doses to terrestrial-phase amphibians have begun to rectify this situation. We collated and summarized available measurements of terrestrial amphibian dermal exposure to pesticides from 11 studies in which researchers measured tissue concentrations associated with known pesticide experimental application rates. This data set included tissue concentrations in 11 amphibian species and 14 different pesticides. We then compared the results of two screening exposure models that differed based on surface area scaling approaches as a function of body weight (one based on birds as surrogates for amphibians and another amphibian-specific) to the measured tissue residue concentrations. We define a false-negative rate for each screening model as the proportion of amphibians for which the predicted concentration is less than the observed concentration (i.e., underestimate), contrary to the intent of screening models, which are intended to have a bias for higher exposure concentrations. The screening model that uses birds as surrogates did not have any instances where estimated expected avian doses were less than measured amphibian body burdens. When using the amphibian-specific exposure model that corrected for differences between avian and amphibian surface area, measured concentrations were greater than model estimates for 11.3% of the 1158 comparisons. The database of measured pesticide concentrations in terrestrial amphibians is provided for use in calculating bioconcentration factors and for future amphibian dermal exposure model development. Integr Environ Assess Manag 2023;19:9-16. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Differentiating metabolomic responses of amphibians to multiple stressors.

One of the biggest challenges in ecological risk assessment is determining the impact of multiple stressors on individual organisms and populations in real world scenarios. Frequently, data derived from laboratory studies of single stressors are used to estimate risk parameters and do not adequately address scenarios where other stressors exist. Emerging 'omic technologies, notably metabolomics, provide an opportunity to address the uncertainties surrounding ecological risk assessment of multiple stressors. The objective of this study was to use metabolomic profiling to investigate the effect of multiple stressors on amphibian metamorphs. We exposed post-metamorphosis (180 days) southern leopard frogs (Lithobates sphenocephala) to the insecticide carbaryl (480 µg/L), predation stress, and a combined pesticide and predation stress treatment. Corticosterone analysis revealed mild support for an induction in response to predation stress alone but strongly suggests that carbaryl exposure, alone or in combination with predation cues, can significantly elevate this known biomarker in amphibians. Metabolomics analysis accurately classed, based on relative nearness, carbaryl and predation induced changes in the hepatic metabolome and biochemical fluxes appear to be associated with a similar biological response. Support vector machine analysis with recursive feature elimination of the acquired metabolomic spectra demonstrated 85-96% classification accuracy among control and all treatment groups when using the top 75 ranked retention time bins. Biochemical fluxes observed in the groups exposed to carbaryl, predation, and the combined treatment include amino acids, sugar derivatives, and purine nucleotides. Ultimately, this methodology could be used to interpret short-term toxicity assays and the presence of environmental stressors to overall metabolomic effects in non-target organisms.

Induced Hepatic Glutathione and Metabolomic Alterations Following Mixed Pesticide and Fertilizer Exposures in Juvenile Leopard Frogs (Lithobates sphenocephala).

The increasing use of agrochemicals, alone and in combination, has been implicated as a potential causative factor in the decline of amphibians worldwide. Fertilizers and pesticides are frequently combined into single-use tank mixtures for agricultural applications to decrease costs while meeting the food demands of a growing human population. Limited data are available on the effects of increased nitrogen levels in nontarget species, such as amphibians, and therefore investigating alterations in the nitrogen cycle and its impacts on amphibians needs to be considered in best management practices going forward. The objective of the present study was to elucidate the impact of fertilizer (urea) and herbicide (atrazine and/or alachlor) tank mixtures on the hepatic metabolome of juvenile leopard frogs as well as to investigate alterations in oxidative stress by relating these changes to glutathione (GSH) levels. Herbicide exposure only moderately increased this parameter in amphibians, however, urea alone and in combination with either atrazine or alachlor statistically elevated GSH levels. Interestingly, urea also inhibited pesticide uptake: calculated bioconcentration factors were greatly decreased for atrazine and alachlor when urea was present in the exposure mixture. Metabolomic profiling identified fluxes in hepatic metabolites that are involved in GSH and carbohydrate metabolic processes as well as altered intermediates in the urea cycle. Ultimately, understanding the biological impacts of nitrogenous fertilizers alone and in combination with pesticide exposure will inform best management practices to conserve declining amphibian populations worldwide. Environ Toxicol Chem 2022;41:122-133. © 2021 SETAC.

Integrated Omic Analyses Identify Pathways and Transcriptomic Regulators Associated With Chemical Alterations of In Vitro Neural Network Formation.

Development of in vitro new approach methodologies has been driven by the need for developmental neurotoxicity (DNT) hazard data on thousands of chemicals. The network formation assay characterizes DNT hazard based on changes in network formation but provides no mechanistic information. This study investigated nervous system signaling pathways and upstream physiological regulators underlying chemically induced neural network dysfunction. Rat primary cortical neural networks grown on microelectrode arrays were exposed for 12 days in vitro to cytosine arabinoside, 5-fluorouracil, domoic acid, cypermethrin, deltamethrin, or haloperidol as these exposures altered network formation in previous studies. RNA-seq from cells and gas chromatography/mass spectrometry analysis of media extracts collected on days in vitro 12 provided gene expression and metabolomic identification, respectively. The integration of differentially expressed genes and metabolites for each neurotoxicant was analyzed using ingenuity pathway analysis. All 6 compounds altered gene expression that linked to developmental disorders and neurological diseases. Other enriched canonical pathways overlapped among compounds of the same class; eg, genes and metabolites altered by both cytosine arabinoside and 5-fluorouracil exposures are enriched in axonal guidance pathways. Integrated analysis of upstream regulators was heterogeneous across compounds, but identified several transcriptomic regulators including CREB1, SOX2, NOTCH1, and PRODH. These results demonstrate that changes in network formation are accompanied by transcriptomic and metabolomic changes and that different classes of compounds produce differing responses. This approach can enhance information obtained from new approach methodologies and contribute to the identification and development of adverse outcome pathways associated with DNT.

Per- and polyfluoroalkyl substances in the environment.

Over the past several years, the term PFAS (per- and polyfluoroalkyl substances) has grown to be emblematic of environmental contamination, garnering public, scientific, and regulatory concern. PFAS are synthesized by two processes, direct fluorination (e.g., electrochemical fluorination) and oligomerization (e.g., fluorotelomerization). More than a megatonne of PFAS is produced yearly, and thousands of PFAS wind up in end-use products. Atmospheric and aqueous fugitive releases during manufacturing, use, and disposal have resulted in the global distribution of these compounds. Volatile PFAS facilitate long-range transport, commonly followed by complex transformation schemes to recalcitrant terminal PFAS, which do not degrade under environmental conditions and thus migrate through the environment and accumulate in biota through multiple pathways. Efforts to remediate PFAS-contaminated matrices still are in their infancy, with much current research targeting drinking water.

Using metabolomic profiling to inform use of surrogate species in ecological risk assessment practices.

The U.S. EPA frequently uses avian or fish toxicity data to set protective standards for amphibians in ecological risk assessments. However, this approach does not always adequately represent aquatic-dwelling and terrestrial-phase amphibian exposure data. For instance, it is accepted that early life stage tests for fish are typically sensitive enough to protect larval amphibians, however, metamorphosis from tadpole to a terrestrial-phase adult relies on endocrine cues that are less prevalent in fish but essential for amphibian life stage transitions. These differences suggest that more robust approaches are needed to adequately elucidate the impacts of pesticide exposure in amphibians across critical life stages. Therefore, in the current study, methodology is presented that can be applied to link the perturbations in the metabolomic response of larval zebrafish (Danio rerio), a surrogate species frequently used in ecotoxicological studies, to those of African clawed frog (Xenopus laevis) tadpoles following exposure to three high-use pesticides, bifenthrin, chlorothalonil, or trifluralin. Generally, D. rerio exhibited greater metabolic perturbations in both number and magnitude across the pesticide exposures as opposed to X. laevis. This suggests that screening ecological risk assessment surrogate toxicity data would sufficiently protect amphibians at the single life stage studied but care needs to be taken to understand the suite of metabolic requirements of each developing species. Ultimately, methodology presented, and data gathered herein will help inform the applicability of metabolomic profiling in establishing the risk pesticide exposure poses to amphibians and potentially other non-target species.

Route of exposure influences pesticide body burden and the hepatic metabolome in post-metamorphic leopard frogs.

Pesticides are being applied at a greater extent than in the past. Once pesticides enter the ecosystem, many environmental factors can influence their residence time. These interactions can result in processes such as translocation, environmental degradation, and metabolic activation facilitating exposure to target and non-target species. Most anurans start off their life cycle in aquatic environments and then transition into terrestrial habitats. Their time in the aquatic environment is generally short; however, many important developmental stages occur during this tenure. Post-metamorphosis, most species spend many years on land but migrate back to the aquatic environment for breeding. Due to the importance of both the aquatic and terrestrial environments to the life stages of amphibians, we investigated how the route of exposure (i.e., uptake from contaminated soils vs. uptake from contaminated surface water) influences pesticide bioavailability and body burden for four pesticides (bifenthrin (BIF), chlorpyrifos (CPF), glyphosate (GLY), and trifloxystrobin (TFS)) as well as the impact on the hepatic metabolome of adult leopard frogs (Gosner stage 46 with 60-90 days post-metamorphosis). Body burden concentrations for amphibians exposed in water were significantly higher (ANOVA p < 0.0001) compared to amphibians exposed to contaminated soil across all pesticides studied. Out of 80 metabolites that were putatively identified, the majority expressed a higher abundance in amphibians that were exposed in pesticide contaminated water compared to soil. Ultimately, this research will help fill regulatory data gaps, aid in the creation of more accurate amphibian dermal uptake models and inform continued ecological risk assessment efforts.

Honey Bees and Neonicotinoid-Treated Corn Seed: Contamination, Exposure, and Effects.

Most corn (Zea mays) seeds planted in the United States in recent years are coated with a seed treatment containing neonicotinoid insecticides. Abrasion of the seed coating generates insecticide-laden planter dust that disperses through the landscape during corn planting and has resulted in many "bee-kill" incidents in North America and Europe. We investigated the linkage between corn planting and honey bee colony success in a region dominated by corn agriculture. Over 3 yr we consistently observed an increased presence of corn seed treatment insecticides in bee-collected pollen and elevated worker bee mortality during corn planting. Residues of seed treatment neonicotinoids, clothianidin and thiamethoxam, detected in pollen positively correlated with cornfield area surrounding the apiaries. Elevated worker mortality was also observed in experimental colonies fed field-collected pollen containing known concentrations of corn seed treatment insecticides. We monitored colony growth throughout the subsequent year in 2015 and found that colonies exposed to higher insecticide concentrations exhibited slower population growth during the month of corn planting but demonstrated more rapid growth in the month following, though this difference may be related to forage availability. Exposure to seed treatment neonicotinoids during corn planting has clear short-term detrimental effects on honey bee colonies and may affect the viability of beekeeping operations that are dependent on maximizing colony size in the springtime. Environ Toxicol Chem 2021;40:1212-1221. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.