Contaminants of Concern and Spatiotemporal Metabolomic Changes in Quagga Mussels (Dreissena bugensis rostriformis) from the Milwaukee Estuary (Wisconsin, USA).

Environmental metabolomics has emerged as a promising technique in the field of biomonitoring and as an indicator of aquatic ecosystem health. In the Milwaukee Estuary (Wisconsin, USA), previous studies have used a nontargeted metabolomic approach to distinguish between zebra mussels (Dreissena polymorpha) collected from sites of varying contamination. To further elucidate the potential effects of contaminants on bivalve health in the Milwaukee Estuary, the present study adopted a caging approach to study the metabolome of quagga mussels (Dreissena bugensis rostriformis) deployed in six sites of varying contamination for 2, 5, or 55 days. Caged mussels were co-deployed with two types of passive sampler (polar organic chemical integrative samplers and semipermeable membrane devices) and data loggers. In conjunction, in situ quagga mussels were collected from the four sites studied previously and analyzed for residues of contaminants and metabolomics using a targeted approach. For the caging study, temporal differences in the metabolomic response were observed with few significant changes observed after 2 and 5 days, but larger differences (up to 97 significantly different metabolites) to the metabolome in all sites after 55 days. A suite of metabolic pathways were altered, including biosynthesis and metabolism of amino acids, and upmodulation of phospholipids at all sites, suggesting a potential biological influence such as gametogenesis. In the caging study, average temperatures appeared to have a greater effect on the metabolome than contaminants, despite a large concentration gradient in polycyclic aromatic hydrocarbons residues measured in passive samplers and mussel tissue. Conversely, significant differences between the metabolome of mussels collected in situ from all three contaminated sites and the offshore reference site were observed. Overall, these findings highlight the importance of contextualizing the effects of environmental conditions and reproductive processes on the metabolome of model organisms to facilitate the wider use of this technique for biomonitoring and environmental health assessments. Environ Toxicol Chem 2024;43:307-323. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

PFAS in municipal landfill leachate: Occurrence, transformation, and sources.

To understand sources and processes affecting per- and polyfluoroalkyl substances (PFAS), 32 PFAS were measured in landfill leachate from 17 landfills across Washington State in both pre-and post-total oxidizable precursor (TOP) assay samples, using an analytical method that was the precursor to EPA Draft Method 1633. As in other studies, 5:3FTCA was the dominant PFAS in the leachate, suggesting that carpets, textiles, and food packaging were the main sources of PFAS. Total PFAS concentrations (Σ32PFAS) ranged from 61 to 172,976 ng/L and 580-36,122 ng/L in pre-TOP and post-TOP samples, respectively, suggesting that little or no uncharacterized precursors remained in landfill leachate. Furthermore, due to chain-shortening reactions, the TOP assay often resulted in a loss of overall PFAS mass. Positive matrix factorization (PMF) analysis of the combined pre- and post-TOP samples produced five factors that represent sources and processes. Factor 1 consisted primarily of 5:3FTCA (intermediate of 6:2 fluorotelomer degradation and characteristic of landfill leachate), while factor 2 was dominated by PFBS (degradant of C-4 sulfonamide chemistry) and, to a lesser extent, by several PFCAs and 5:3FTCA. Factor 3 consisted primarily of both short-chain PFCAs (end-products of 6:2 fluorotelomer degradation) and PFHxS (derived from C-6 sulfonamide chemistry), while the main component of factor 4 was PFOS (dominant in many environmental media but minor in landfill leachate, perhaps reflecting a production shift from longer to shorter chain PFAS). Factor 5, highly loaded with PFCAs, was dominant in post-TOP samples and therefore represented the oxidation of precursors. Overall, PMF analysis suggests that the TOP assay approximates some redox processes which occur in landfills, including chain-shortening reactions which yield biodegradable products.

Consideration of metabolomics and transcriptomics data in the context of using avian embryos for toxicity testing.

Early-life stage (ELS) avian toxicity tests have been proposed as a more ethical alternative to traditional standardized tests with adult birds. At the same time, 'omics approaches are gaining traction in the field of avian toxicology, but little has been done to characterize the metabolome and transcriptome at different life stages. The present study uses 'omics data from toxicity tests of 8 environmental chemicals in ELS and adult Japanese quail (Coturnix japonica) to address this data gap. Previous analyses of these data focused on responses to each of the individual chemicals. Here, we consider data from all studies to describe variation in the metabolome and transcriptome between life stages and across independent experiments, irrespective of chemical treatment. Of the 230 metabolites detected in liver, 163 were shared between the two life stages. However, many of the targeted bile acids that were present in the adult liver were absent from ELS samples. For the transcriptome, >90% of the 18,364 detected transcripts were common to both life stages. Based on the 213 genes solely detected in ELS liver, the neuroactive ligand-receptor interaction pathway was significantly enriched. Multivariate and hierarchical clustering analyses revealed that variability among independent experiments was higher for the adult than the ELS studies at both the metabolomic and transcriptomic levels. Our results indicate concordance of the two approaches, with less variation between independent experiments in the ELS metabolome and transcriptome than in adults, lending support for the use of ELS as an alternative toxicity testing strategy.

Targeted Metabolomics to Assess Exposure to Environmental Chemicals of Concern in Japanese Quail at Two Life Stages.

This proof-of-concept study characterizes the Japanese quail (Coturnix japonica) hepatic metabolome following exposure to benzo[a]pyrene, chlorpyrifos, ethinylestradiol, fluoxetine hydrochloride, hexabromocyclododecane, lead(II)nitrate, seleno-L-methionine, and trenbolone in embryos and adults. The analysis revealed effects on lipid metabolism following exposure to several chemicals at both life stages. The most pronounced effects were observed in embryos exposed to 41.1 µg/g chlorpyrifos. This work highlighted challenges and the need for further avian metabolomics studies.

Using Transcriptomics and Metabolomics to Understand Species Differences in Sensitivity to Chlorpyrifos in Japanese Quail and Double-Crested Cormorant Embryos.

Modern 21st-century toxicity testing makes use of omics technologies to address critical questions in toxicology and chemical management. Of interest are questions relating to chemical mechanisms of toxicity, differences in species sensitivity, and translation of molecular effects to observable apical endpoints. Our study addressed these questions by comparing apical outcomes and multiple omics responses in early-life stage exposure studies with Japanese quail (Coturnix japonica) and double-crested cormorant (Phalacrocorax auritus), representing a model and ecological species, respectively. Specifically, we investigated the dose-dependent response of apical outcomes as well as transcriptomics and metabolomics in the liver of each species exposed to chlorpyrifos, a widely used organophosphate pesticide. Our results revealed a clear pattern of dose-dependent disruption of gene expression and metabolic profiles in Japanese quail but not double-crested cormorant at similar chlorpyrifos exposure concentrations. The difference in sensitivity between species was likely due to higher metabolic transformation of chlorpyrifos in Japanese quail compared to double-crested cormorant. The most impacted biological pathways after chlorpyrifos exposure in Japanese quail included hepatic metabolism, oxidative stress, endocrine disruption (steroid and nonsteroid hormones), and metabolic disease (lipid and fatty acid metabolism). Importantly, we show consistent responses across biological scales, suggesting that significant disruption at the level of gene expression and metabolite profiles leads to observable apical responses at the organism level. Our study demonstrates the utility of evaluating effects at multiple biological levels of organization to understand how modern toxicity testing relates to outcomes of regulatory relevance, while also highlighting important, yet poorly understood, species differences in sensitivity to chemical exposure. Environ Toxicol Chem 2021;40:3019-3033. © 2021 SETAC.

Effect of Sample Storage on the Quantitative Determination of 29 PFAS: Observation of Analyte Interconversions during Storage.

In this study, we measured the effects of sample type and storage temperature on the stability of 29 per- and polyfluorinated alkyl substances (PFAS) in water. Spiked bottled water, surface water, and two types of effluent samples were stored in HDPE containers at +20, 4, and -20 °C over a period of up to 180 days. The analytes studied included C4 through C14 perfluorinated carboxylates (PFCAs); C4 through C10 and C12 perfluorinated sulfonates (PFSAs); 4:2, 6:2, and 8:2 fluorotelomer sulfonates (FTS); three perfluorooctane sulfonamides (PFOSA, N-MeFOSA, and N-EtFOSA); two perfluorooctane sulfonamide ethanols (N-MeFOSE and N-EtFOSE); and two perfluorooctane sulfonamide acetic acids (N-MeFOSAA and EtFOSAA). Overall, 10 analytes, PFOA, PFNA, 8:2 FTS, PFOSA, N-MeFOSA, NEtFOSA, N-MeFOSAA, N-EtFOSAA, N-MeFOSE, and N-EtFOSE, showed increasing or decreasing concentration trends under at least one of the experimental conditions investigated. Increases in concentrations of N-MeFOSAA and N-EtFOSAA in surface water and effluent samples at +20 and 4 °C correlated with the decreases in the concentrations of N-MeFOSE and N-EtFOSE, respectively, suggesting analyte interconversion during sample storage. This is the first time such analyte conversion is reported in samples under storage, and this work demonstrates the importance of assessing stability of PFAS in environmentally relevant matrices. The significance of this study extends beyond sample storage for analysis, as toxicological and exposure studies conducted at room temperature also need to consider the significance of analyte degradation through the exposure process.

A multi-omic approach to elucidate low-dose effects of xenobiotics in zebrafish (Danio rerio) larvae.

Regulatory-approved toxicity assays such as the OECD Fish Embryo Toxicity Assay (TG236) allow correlation of chemical exposure to adverse morphological phenotypes. However, these assays are ineffective in assessing sub-lethal (i.e. low-dose) effects, or differentiating between similar phenotypes induced by different chemicals. Inclusion of multi-omic analyses in studies investigating xenobiotic action provides improved characterization of biological response, thereby enhancing prediction of toxicological outcomes in whole animals in the absence of morphological effects. In the current study, we assessed perturbations in both the metabolome and transcriptome of zebrafish (Danio rerio; ZF) larvae exposed from 96 to 120h post fertilization to environmental concentrations of acetaminophen (APAP), diphenhydramine (DH), carbamazepine (CBZ), and fluoxetine (FLX); common pharmaceuticals with known mechanisms of action. Multi-omic responses were evaluated independently and integrated to identify molecular interactions and biological relevance of the responses. Results indicated chemical- and dose-specific changes suggesting differences in the time scale of transcript abundance and metabolite production. Increased impact on the metabolome relative to the transcriptome in FLX-treated animals suggests a stronger post-translational effect of the treatment. In contrast, the transcriptome showed higher sensitivity to perturbation in DH-exposed animals. Integration of 'omic' responses using multivariate approaches provided additional insights not obtained by independent 'omic' analyses and demonstrated that the most distinct overall response profiles were induced following low-dose exposure for all 4 pharmaceuticals. Importantly, changes in transcript abundance corroborated with predictions from metabolomic enrichment analyses and the identified perturbed biological pathways aligned with known xenobiotic mechanisms of action. This work demonstrates that a multi-omic toxicological approach, coupled with a sensitive animal model such as ZF larvae, can help characterize the toxicological relevance of acute low-dose chemical exposures.

Xenobiotics Produce Distinct Metabolomic Responses in Zebrafish Larvae (Danio rerio).

Sensitive and quantitative protocols for characterizing low-dose effects are needed to meet the demands of 21st century chemical hazard assessment. To test the hypothesis that xenobiotic exposure at environmentally relevant concentrations produces specific biochemical fingerprints in organisms, metabolomic perturbations in zebrafish (Danio rerio) embryo/larvae were measured following 24 h exposures to 13 individual chemicals covering a wide range of contaminant classes. Measured metabolites (208 in total) included amino acids, biogenic amines, fatty acids, bile acids, sugars, and lipids. The 96-120 h post-fertilization developmental stage was the most appropriate model for detecting xenobiotic-induced metabolomic perturbations. Metabolomic fingerprints were largely chemical- and dose-specific and were reproducible in multiple exposures over a 16-month period. Furthermore, chemical-specific responses were detected in the presence of an effluent matrix; importantly, in the absence of morphological response. In addition to improving sensitivity for detecting biological responses to low-level xenobiotic exposures, these data can aid the classification of novel contaminants based on the similarity of metabolomic responses to well-characterized "model" compounds. This approach is clearly of use for rapid, sensitive, and specific analyses of chemical effect on organisms, and can supplement existing methods, such as the Zebrafish Embryo Toxicity assay (OECD TG236), with molecular-level information.

Sorption of per- and polyfluoroalkyl substances (PFASs) on filter media: implications for phase partitioning studies.

Per- and polyfluoroalkyl substances (PFASs), including perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are ubiquitous in the environment. Investigations into their fate and potential phase-partitioning behavior require separating solid from aqueous phases via filtration. However, sorption of aqueous-phase PFASs on filtration media may lead to underestimation of PFAS concentrations in the aqueous phase. The authors investigated the sorption of perfluoroalkyl carboxylates, perfluoroalkyl sulfonates, perfluoroalkyl phosphonic acids, perfluoroalkyl phosphinic acids (PFPiAs), polyfluoroalkyl phosphate monoesters, polyfluoroalkyl phosphate diesters (diPAPs), fluorotelomer sulfonates, and perfluorooctane sulfonamide on filtration media. The effects of concentration (3 spiking levels), filter media (4 types), matrix (4 matrices), and compound structure on sorption are reported. Glass fiber filtration resulted in the least sorption, whereas polytetrafluoroethylene filters resulted in the most sorption (up to 98%). Analyte concentration had no significant effect. Sorption was generally consistent across matrix types except for samples affected by aqueous film forming foam deployment, which displayed high sorption of PFOS on nylon filters. Sorption usually increased with an increasing number of carbon or fluorine atoms and was most pronounced for PFPiAs and diPAPs (30­75% sorption). Overall, glass fiber filters are more recommended than nylon filters in environmental samples when phase separation is required. Use of filtration media for PFAS must be preceded by matrix-specific testing to account for unpredictable effects.

Quantification of pharmaceuticals, personal care products, and perfluoroalkyl substances in the marine sediments of Puget Sound, Washington, USA.

Concentrations of 119 pharmaceuticals and personal care products (PPCPs) and 13 perfluoroalkyl substances (PFASs) in marine sediments measured throughout Puget Sound (n = 10) and Bellingham Bay (n = 30), Washington, USA, are reported. These data are among the first measurements of PPCPs and PFASs in marine sediments from the Pacific Northwest and provide a comparison to previous measurements of these chemicals in influent, effluent, and biosolids from municipal wastewater treatment plants throughout the region. The concentrations of both PPCPs and PFASs in sediments from Puget Sound and Bellingham Bay ranged from very low to non-detectable for most compounds. Only 14 of the 119 PPCPs and 3 of 13 PFASs were quantifiable in sediments. Diphenhydramine (an antihistamine) was most frequently detected (87.5% of samples), with a maximum concentration of 4.81 ng/g dry weight and an estimated mean detected concentration of 1.68 ng/g. Triclocarban (an antibacterial) was detected in 35.0% of the samples, with a maximum concentration of 16.6 ng/g dry weight. Perfluoroalkyl substances were detected in 2.5% of analyses. Perfluorobutanoate, perfluorooctane sulfonate, and perfluorooctane sulfonamide were detected in 7, 5, and 1 sample(s) each, respectively, with the highest concentrations observed for perfluorooctane sulfonate (1.5 ng/g). Detected concentrations were often highest within the industrial harbor in Bellingham Bay and near the cities of Seattle and Bremerton. Environ Toxicol Chem 2013;32:1701-1710. © 2013 SETAC.