Environmental Science & Technology

Per- and polyfluoroalkyl substances (PFAS) are highly stable chemical contaminants of emerging concern for human and environmental health due to their non-natural chemistry, widespread use, and environmental persistence. Despite conventional metrology, mitigation strategies, and removal technologies, the complexity of this growing problem necessitates the need for alternative approaches to tackle the immense challenges associated with complex environmental PFAS contamination. Recently, biology has emerged as an alternative approach to detect and mitigate PFAS and understand the molecular-level responses of living organisms, including microorganisms, to these compounds. However, further study is needed to understand how microorganisms in different environments and growth phases respond to PFAS. In this study, we performed RNA sequencing at mid-exponential, early stationary phase, and late stationary phase of bacterial growth to determine the global transcriptional response of a model chassis, Escherichia coli MG1655, induced by two PFAS, perfluorooctanoic acid (PFOA) and perfluorododecanoic acid (PFDoA), and equivalent non-fluorinated carboxylic acids (NFCA), octanoic acid and dodecanoic acid. Differential gene expression analysis revealed PFOA and PFDoA induced distinct changes in gene expression throughout cultivation. Specifically, we identified significant changes in expression of the formate regulon and sulfate assimilation at mid-exponential phase and ferrous iron transport, central metabolism, the molecular chaperone network, and motility processes during stationary phase. Importantly, many of these changes are not induced by NFCAs. In summary, we found PFAS induced a system-level change in gene expression, and our results expand the understanding of bacterial-PFAS interactions that could enable the development of future real-time environmental monitoring and mitigation technologies. ImportanceThe prevalence and persistence of PFAS in the environment is a growing area of concern. However, little is understood of the impacts of PFAS on the environment, particularly impacts on microorganisms that play pivotal roles in nearly every ecosystem. Thus, comprehensive measurements that provide systems-level insight into how microorganisms respond and adapt to PFAS in the environment are paramount. Here, we use RNA sequencing to study the global transcriptional response of E. coli MG1655 to two PFAS and non-fluorinated equivalent compounds across growth phases. We find that PFAS induce system-level changes in metabolic, transport, and gene regulatory pathways, providing insight into how these non-natural chemicals interact with a model bacterium. Additionally, the transcriptomic dataset associated with this work provides the community with PFSA-specific gene expression patterns and possible PFAS degradation pathways for the development of future whole-cell biosensors and mitigation efforts.

Climate warming and chemical pollution shape aquatic ecosystems, yet the physiological mechanisms underlying their combined effects remain unclear. We investigated how projected increases in mean summer surface water temperature alter per- and polyfluoroalkyl substances (PFAS) toxicokinetics and their effects on sheepshead minnows (Cyprinodon variegatus) physiological performance. Adult fish were chronically exposed to an environmentally relevant PFAS mixture (perfluorooctane sulfonate (PFOS) + perfluorooctanoate (PFOA)) under current and projected mean-temperature scenarios. Tissue PFAS concentrations, whole-organism metabolic rates, swimming performance, reproductive parameters, somatic indices were assessed. Temperature modified PFAS tissue concentrations in a compound- and tissue-specific manner, promoting PFOA redistribution to eggs. Metabolic responses were temperature-dependent: at 26 {degrees}C, higher tissue PFAS concentrations were associated with elevated standard and maximum metabolic rates (SMR and MMR), maintaining aerobic scope (AS). At 28.5 {degrees}C, SMR remained stable while MMR and AS declined with rising PFAS, indicating less oxygen for energetically demanding activities. Despite unchanged swimming and reproductive outputs, an increased hepatosomatic index with increasing tissue PFAS concentrations and altered PFAS distribution suggest detoxification costs. These findings indicate that increases in mean water temperature are likely to exacerbate contaminant stress, with consequences for coastal fish population resilience and offspring development. PFAS risk assessment should consider co-stressors under projected warming. SynopsisLimited research addresses how temperature affects PFAS toxicokinetics and toxicity. This study shows that warming reshapes tissue PFAS concentrations and distribution, and influences fish energy-allocation trade-offs.

Modern natural gas (NG) has little or no odor, so other compounds, usually mercaptans and thiols, are added as warning odorants. Federal regulations state that NG must be odorized so that it is readily detectable by people with normal senses of smell at one fifth the lower explosive limit, but regulations dont define "readily detectable" or "normal senses of smell." Methods to measure human odor detection have been available for decades. However, most previous work on NG odorants has underestimated human sensitivity, and measurements need to be repeated using the latest methods. More work is also needed to determine how odor sensitivity measured under optimal laboratory conditions is affected by real-world factors such as distraction and exposure to other odors in the environment. Regarding a "normal sense of smell," healthy people vary over orders of magnitude in the concentrations they can detect, so samples of subjects should be chosen to reflect the range of differences in the population.

The accumulation of propionate is a challenge in numerous fermentative industrial processes because its degradation is energetically unfavorable and limited to few microbial species. Here, we report for the first time the oxidation of propionate by the extracellular electron transfer (EET)-capable bacterium Geobacter sulfurreducens in axenic cultures. G. sulfurreducens was capable of utilizing propionate both as electron donor (ED) and source of carbon with fumarate as electron acceptor (EA). In contrast, propionate was metabolized only in the presence of acetate with soluble Fe(III) citrate, and was not oxidized when insoluble iron oxides or glassy carbon electrodes poised at +0.1 V vs. SHE were the EAs. Biomass yield (per mole of electrons available) was lower with propionate alone than with propionate and acetate together, and acetate was preferentially consumed when both were present. Transcriptomic analysis of cultures grown with either propionate or acetate (with fumarate as EA) showed significant gene expression shifts strongly suggesting the methylmalonyl-CoA pathway as the main route for propionate degradation. Furthermore, propionate-consuming cultures exhibited an upregulation of branched chain amino acids (BCAAs) biosynthesis, as well as sulfur, nitrogen, and 2-oxocarboxylic acids metabolism. IMPORTANCEThe accumulation of propionate is a challenge in anaerobic and fermentative processes because it inhibits methanogenesis, and few microbial species within such systems can degrade it. G. sulfurreducens is a model electroactive bacterium widely used in bioelectrochemical systems and is increasingly studied in wastewater treatment and anaerobic digestion because of its ability to enhance syntrophic metabolism via direct interspecies electron transfer. We show for the first time that G. sulfurreducens can oxidize propionate, expanding its known metabolic repertoire, and that this capability is controlled by the nature of the terminal electron acceptor. Transcriptomic analyses strongly suggest that the methylmalonyl-CoA pathway is the main pathway for propionate degradation and reveal additional associated transcriptional changes. These findings, together with insights into propionate degradation kinetics, could inform future strategies aimed at using this bacterium to mitigate propionate buildup and improve the stability of anaerobic treatment systems.

Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known human carcinogen with a putative mutagenic mode of action (MOA). Its genotoxic potential is primarily mediated through alkylation of DNA, resulting in the formation of the mutagenic adduct O6-(2-hydroxyethyl)-2-deoxyguanosine (O6-HE-dG). The N7-(2-hydroxyethyl)guanine (N7-HE-G) adduct is formed in greater abundance and is generally considered to be non-mutagenic. However, dose-response relationships of these DNA adducts, particularly at low inhalation exposure levels (i. e., below 3 ppm), remain unknown. These data are necessary to inform the biological plausibility of different statistical dose-response models that have been applied to human or animal data used for cancer risk assessment. In the present study, male and female B6C3F1 mice were exposed to EtO (0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm) 6 hours/day for 28 consecutive days. Immediately following the last exposure, DNA was extracted from lung, liver, bone marrow, and mammary gland, and further utilized to measure DNA adduct levels using highly sensitive mass spectrometry platforms. N7-HE-G was detected in all tissues and exposure groups, showing linear dose-response relationships in the low-dose range ([&le;]1 ppm) and increased sharply and exposure-disproportionately in the high-dose range ([&ge;]50 ppm). Despite a very low limit of detection, O6-HE-dG, in contrast, was not detected at exposures <50 ppm in any tissue consistent with at most a shallow linear exposure response. At higher exposures ([&ge;]50 ppm), O6-HE-dG exhibited a dose-response pattern of N7-HE-G. Notably the mammary gland, despite being anatomically distant from the site of inhalation, exhibited the second-highest levels of both adducts at higher doses. This study provides the first reliable quantitative dose-response evidence of DNA adducts in tumor target and non-target (liver) tissues across a wide range of EtO exposures. The two DNA adducts differ markedly in their abundance, repairability and mutagenic potential and together provide a molecular MOA dose-response framework to inform both quantitative cancer risk assessment and genotoxic hazard characterization.

Aflatoxins (AFs) are fungal metabolites that ubiquitously contaminate many common food crops and contribute to major foodborne diseases in humans and animals. The ability to remove AFs from common food and feed commodities will improve health standards and limit the economic impact inflicted by AF food contamination. Known chemical strategies have used strong acids and bases to remove contaminating AF, but these methods often lead to ecological waste issues downstream. In this study, we explore the application of weaker acidic and alkaline conditions to removes two types of AFs, AFB1 and AFG2. We find that a pH 9 buffered environment reduces AFB1 and AFG2 by more than 50% and 95%, respectively, within 24 hours. We show that AF degradation is through lactone ring opening, which is a known cause of AF toxicity, and provide a potential structure of the AFG2 degradation byproduct. Further, we confirm that incubation in the pH 9 environment reduces the genotoxicity of AFB1. Our findings indicate that a weakly alkaline environment may adequately detoxify AF-contaminated food or feed without the need to apply stronger or harsher basic conditions.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focussed on four model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at four time-points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS; (2) identify similarities in biological responses; (3) compare PFAS potency through benchmark concentration analysis; and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPAR activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (i.e., was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Environmental risk assessment is a critical tool for protecting aquatic life and its effectiveness is predicated on predicting how natural populations respond to contaminants. Yet, routine toxicity testing typically examines only one genotype, which may render risk assessments inaccurate as populations are most often composed of genetically distinct individuals. To determine the importance of intraspecific variation in the translation of toxicity testing to populations, we quantified the magnitude of genetic variation within 20 Daphnia magna clones derived from one lake using whole genome sequencing and phenotypic assays. We repeated these assays across two exposure levels of microcystins, a cosmopolitan and lethal aquatic contaminant produced by harmful algal blooms. We found considerable intraspecific genetic variation in survival, growth, and reproduction, which was amplified by microcystins exposure. Finally, using simulations we demonstrate that the common practice of employing a single genotype to calculate toxicity tolerance failed to produce an estimate within the 95% confidence interval over half of the time. These results illuminate the importance of incorporating intraspecific genetic variation into toxicity testing to reliably predict how natural populations will respond to aquatic contaminants.

Reproducibility and consistency are hallmarks of scientific integrity. Biological systems are inherently noisy, posing a challenge to reproducibility. This is particularly relevant to the field of environmental toxicology, where many unaccounted experimental parameters can have a marked influence on the biological response to exposure. Here, we extend the use of zebrafish as a robust toxicological model for studying the effects of inorganic arsenic (iAs) on liver biology. We observed that iAs toxicity in this system is not influenced by important parameters including genetic background, rearing container material or rearing volume but the dose response to iAs is influenced by the rearing medium. We compared mortality as a measure of iAs toxicity to embryos cultured in two standard rearing media: egg water made from dehydrated ocean salts dissolved in water and a defined embryo medium which is a pH adjusted, buffered salt solution. Larvae reared in egg water were more susceptible to iAs compared to those reared in embryo medium. This effect was independent of the pH differences between these solutions. These culture conditions did not cause any difference in the global hepatic transcriptome of control zebrafish. Further, no difference in the expression of genes involved in the unfolded protein response (UPR) in larvae exposed to iAs treatment or in a stress independent system to activate UPR genes by transgenic overexpression of activating transcription factor 6 (nAtf6) in hepatocytes was observed. However, the clutch-to-clutch variation in gene expression was significantly greater in larvae reared in egg water compared to those in embryo medium. These data demonstrate that egg water affects reproducibility across replicates in terms of gene expression and exacerbates iAs mediated toxic response. This highlights the importance of rigorous evaluation of experimental conditions to assure reproducibility.

Perfluorohexane sulfonate (PFHxS) is a ubiquitous perfluoroalkyl substance known for its environmental persistence and potential toxicity. This study investigated PFHxSs impact on zebrafish embryos, focusing on sensorimotor behavior, circadian rhythm disruption, and underlying molecular mechanisms. Under 24 hr dark incubations, PFHxS exposure induced concentration-dependent hyperactivity within larval photomotor response, characterized by the distinctive "O-bend" response, strong light-phase hyperactive movement and seizure-like movements. It appears that PFHxS-treated embryos cannot sense light cues in a normal manner. Similar hyperactivity was seen for acoustic startle response assay, suggesting that the response is not merely visual, but sensorimotor. LC-MS studies confirmed detectable uptake of PFHxS into embryos. We then conducted mRNA-sequencing across multiple time points (48 and 120 hpf) and concentrations (0.00025, 0.0025 and 25 {micro}M). Data at the 25 {micro}M (2-120 hpf) exposure showed disrupted pathways associated with DNA and cell cycle. Interestingly, data at 0.00025 {micro}M - an environmentally relevant concentration- at 48 hpf showed disruption of MAPK and other signaling pathways. Immunohistochemistry of eyes showed reduced retinal stem cell proliferation, consistent with observed DNA replication pathway disruptions. To assess if these impacts were driven by circadian rhythm development, we manipulated light/dark cycles during PFHxS incubation; this manipulation altered behavioral patterns, implicating circadian rhythm modulation as a target of PFHxS. Since circadian rhythm is modulated by the pineal gland, we ablated the gland using metronidazole; this ablation partially rescued hyperactivity, indicating the glands role in driving the phenotype. Collectively, these findings underscore proclivity of PFHxS to cause neurodevelopmental toxicity, necessitating further mechanistic exploration and environmental health assessments.

Plastic materials are widely used in engineered systems and increasingly accumulate in natural environments, where their surfaces interact with colloids, microorganisms, and dissolved organic matter. However, the relative roles of plastic surface properties versus particle-specific characteristics in governing organic matter retention remain poorly constrained. Here, attachment efficiency () was used to quantify intrinsic particle-collector affinity on three common thermoplastics (ABS, HDPE, HIPS) and glass beads as an inorganic reference. Surface chemistry, hydrophobicity, roughness, and charge were characterized, and interactions with submicron carbon particles (SCPs) and Escherichia coli were evaluated using column experiments. Extended DLVO (XDLVO) theory was applied to predict interaction energy barriers, and humic acid (HA) adsorption was quantified through batch isotherms. XDLVO modeling predicted higher affinity of particles for plastics relative to glass; however, experimentally measured attachment efficiencies were uniformly low ( < 0.05) across all materials. Attachment was primarily governed by particle size and surface charge rather than collector hydrophobicity, roughness, or surface chemistry. SCP consistently exhibited higher than bacteria, while differences among plastics were minor. Similarly, HA adsorption was weak and near-linear, with uptake following ABS {approx} HIPS > HDPE > glass, indicating reversible, partitioning-like association dominated by polymer-specific functionality rather than electrostatics. The absence of correlation between and XDLVO-predicted energy barriers further demonstrates limitations of classical physicochemical models in describing particle- plastic interactions. Collectively, these results indicate that pristine thermoplastic surfaces exhibit intrinsically low affinity for organic matter and that particle-specific properties dominate retention under low ionic strength conditions. Enhanced accumulation in environmental systems likely requires surface aging or conditioning processes not captured by classical interaction theory.

Interactions between viruses and filter-feeding zooplankton can alter viral persistence in surface waters, with direct implications for water quality and public health risk. However, data on virus-zooplankton interactions and the environmental factors that influence them are still limited. This study evaluated the impact of filter feeding, in the dark and under simulated sunlight, on a bacteriophage (MS2) and a human virus (echovirus11; E11) in the presence of a ciliate (Tetrahymena pyriformis) and rotifer (Brachionus calyciflorus). Dark experiments established organism-dependent baseline removal for each virus, and rotifers showed greater removal of both viruses in comparison to ciliates. Under simulated sunlight, in contrast, experiments with ciliates resulted in greater virus removal compared to experiments with rotifers over a similar timespan (4.2 vs. 2.7 log MS2 in 53-58 h; 3.5 vs. 3.0 log E11 in 24-25 h). Analysis of decay rate constants reveals species-specific shifts in virus removal between dark and light that, depending on viral type and zooplankton species, either accelerate viral attenuation or protect viruses and prolong infectivity. T. pyriformis increases removal under sunlight relative to dark conditions and acts synergistically with sunlight inactivation, whereas rotifers impede sunlight inactivation.

Per-and polyfluoroalkyl substances (PFAS) are ubiquitous contaminants in freshwater ecosystems. Many PFAS are incorporated into food webs, with potential effects on ecological and human health. However, PFAS incorporation into the base of aquatic food webs remains poorly understood. The goal of this study was to quantify the uptake and trophic transfer of both legacy and current use PFAS compounds using a simulated freshwater food chain in a lab setting. Natural periphytic biofilms were placed into trays containing equimolar binary aqueous PFAS mixtures at environmentally relevant concentrations for five days. Following the initial exposure period, newly hatched mayfly larvae were introduced into each tray to feed on periphyton for most of their larval development. The mature larvae were then fed to zebrafish. All water and biota samples contained detectable levels of the tested PFAS. All PFAS were more concentrated in periphyton than in water, and four of six PFAS were further concentrated in mayfly larvae relative to periphyton. PFDA was the most accumulative in all biota. PFAS concentrations in zebrafish were significantly correlated with those in larval mayflies. Assimilation efficiencies in zebrafish were high (>70%) for all compounds. Bioaccumulation of PFAS in periphyton and mayflies was positively correlated with log KOW and number of carbons.

I present a simple computational model of H2O2 metabolism in hepatocytes and oxidative stress-induced hepatocyte death that is unique, among existing models of cellular H2O2 metabolism, in its ability to accurately model H2O2 dynamics during incidents of extreme oxidative stress such as occur in the toxicological setting. Versions of the model are presented for rat hepatocytes in vitro and mouse liver in vivo. This is the first model of cellular H2O2 metabolism to incorporate a detailed, realistic model of GSH synthesis from its component amino acids, achieved by incorporating a minimal version of Reed and coworkers pioneering model of GSH metabolism in liver. I demonstrate a generic procedure for coupling the model to an existing PK model for a xenobiotic causing oxidative stress in hepatocytes, using experimental data on hepatocyte mortality resulting from in vitro exposure to the xenobiotic at various concentrations. The result is a PBPK/PD model that predicts intracellular H2O2 concentration and oxidative stress-induced hepatocyte death; both in vitro and in vivo (liver of living animal) PBPK/PD models can be produced. I demonstrate the procedure for the ROS-generating trivalent arsenical DMAIII. Simulations of DMAIII exposure using the model indicate that critical GSH depletion is the immediate trigger for intracellular H2O2 rising to concentrations associated with apoptosis (> 1 {micro}M), that this may only occur hours after intracellular DMAIII peaks ("delay effect"), that when it does occur, H2O2 concentration rises rapidly in a sequence of two boundary layers, characterized by the kinetics of glutathione peroxidase (first boundary layer) and catalase (second boundary layer), and finally, that intracellular H2O2 concentration > 1 {micro}M implies critical GSH depletion. Franco and coworkers have found that GSH depletion is central to apoptosis through mechanisms independent of ROS formation and have speculated that elevated ROS may simply indicate, rather than cause, an apoptotic milieu. Model simulations are consistent with this view, as they indicate that intracellular H2O2 concentration > 1 {micro}M and extreme GSH depletion cooccur/imply each other; however, I note that this does not rule out a direct role for elevated ROS in the apoptotic mechanism. Finally, the delay effect is found to underlie a mechanism by which a normal-as-transient but pathological-as-baseline intracellular H2O2 concentration will eventually trigger critical GSH depletion and H2O2 concentration in the range associated with apoptosis, if and only if it persists for hours; this helps to rigorously explain how cells are able to maintain intracellular H2O2 concentration within such an extremely narrow range. DISCLAIMER: The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration or the National Toxicology Program.

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications from human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this investigation was to identify biological response potencies (i.e., benchmark concentrations: BMCs) following PFAS exposures to inform read-across for risk assessment of data-poor PFAS. Gene expression changes were measured in primary human liver cell microtissues (i.e., 3D spheroids) after 1-day and 10-day exposures to increasing concentrations of 23 PFAS. The cells were treated with four subgroups of PFAS: carboxylates (PFCAs), sulfonates (PFSAs), fluorotelomers, and sulfonamides. An established pipeline to identify differentially expressed genes and transcriptomic BMCs was applied. We found that both PFCAs and PFSAs exhibited a trend toward increased transcriptional changes with carbon chain-length. Specifically, longer-chain compounds (7 to 10 carbons) were more likely to induce changes in gene expression, and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses supports the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the health effects of PFAS and will be used to inform read-across for human health risk assessment.

Per- and polyfluoroalkyl substances (PFAS) are a class of emerging contaminants that are widely distributed and persistent in the environment, accumulated in biological organisms and associated with adverse health outcomes. Evidence has shown a wide existence of branched PFAS isomers from source to applications. Notably, linear and branched isomeric PFAS structures are associated with differential toxicity outcomes and health effects. Herein, we investigated distribution of perfluorooctane sulfonate (PFOS) isomers in mouse liver tissue after exposure using matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-mass spectrometry imaging (MALDI-TIMS-MSI). Mice were treated with vehicle control or commercially sourced PFOS, a mixture of linear and branched isomers, at concentrations to achieve doses of 0.1 and 1 mg/kg/day for 84 days. Liver tissues were collected, followed by sample preparation and MALDI-TIMS-MSI analysis. Using a TIMS ramp time of 150 ms, we successfully separated linear and branched isomers on-tissue. Coupling with post-MALDI immunofluorescence imaging of canonical zonation markers, we discovered hepatic zonation-specific distribution for linear isomer but more homogenous distribution of branched PFOS. Dual-polarity MSI was performed on the same tissue for hepatic metabolites and lipids, and results showed concomitant alteration of liver lipid zonation upon PFOS exposure. With MALDI-TIMS-MSI, our results for the first time demonstrated on-tissue differentiation of PFOS isomers. Multi-modal imaging revealed isomer-specific PFOS distribution and spatial lipidomic changes, both mapped to canonical hepatic zonation markers, to reveal zone-selective PFOS toxicokinetics/toxicodynamics. Together our results demonstrate the critical need for further investigating isomer-specific PFAS toxicity.

Phthalate plasticizers are contaminants of emerging concern that interfere with the synthesis, secretion, and transport of hormones and receptors, altering the immune response and energy balance. Phthalate metabolites have been detected in marine mammals globally, and while studies on phthalate toxicity in marine mammals are beginning to emerge, a comprehensive understanding of the cellular response to these compounds remains elusive. Here, we investigated the transcriptional and bioenergetic responses to mono-ethylhexyl phthalate (MEHP), the active metabolite of di(2-ethylhexyl) phthalate (DEHP), in primary dermal derived from northern elephant seals (Mirounga angustirostris), common dolphins (Delphinus delphis), and humans. MEHP exposure did not induce cytotoxicity in any species, but triggered distinct, species-specific changes in gene expression and mitochondrial metabolism. Human cells showed the greatest transcriptional response to MEHP, upregulating detoxification, antioxidant, and inflammatory genes, and downregulating lipid metabolism pathways. Although mitochondrial respiration declined only at the highest dose, sustained extracellular acidification rates and increased glycolytic gene expression indicate a metabolic shift toward glycolysis. In contrast, elephant seal cells upregulated antioxidant and immune genes while maintaining mitochondrial respiration until the highest MEHP dose, alongside increased expression of genes involved in oxidative phosphorylation, the TCA cycle, and mitochondrial dynamics, suggesting a delayed shift to glycolysis and a potential evolutionary adaptation to sustain mitochondrial function during energy-demanding conditions such as breath-hold diving. Dolphin cells exhibited fewer transcriptional changes, which were enriched for hormone signaling and mitotic pathways, and showed dose-dependent declines in both oxygen consumption and extracellular acidification rates, even at the lowest MEHP concentration, alongside upregulation of stress and hypoxia-related genes. Together, these findings highlight distinct cellular strategies for coping with phthalate exposure and likely species-specific susceptibility to toxicant-induced stress. This study provides new insights into how marine mammals respond to plastic-derived contaminants at the cellular level, reinforcing the need for species-specific ecotoxicological risk assessments.

Cell-based assays represent nearly half of all high-throughput screens currently conducted for risk assessment of environmental chemicals. However, the sensitivity and heterogeneity among cell lines has long been concerned but explored only in a limited manner. Here, we address this question by conducting a large scale transcriptomic analysis of the responses of discrete cell lines to specific small molecules. Our results illustrate heterogeneity of the extent and timing of responses among cell lines. Interestingly, high sensitivity and/or heterogeneity was found to be cell type-specific or universal depending on the different mechanism of actions of the compounds. Our data provide a novel insight into the understanding of cell-small molecule interactions and have substantial implications for the design, execution and interpretation of high-throughput screening assays.

PFAS are ubiquitous endocrine-disrupting pollutants that cross the placenta and impact offspring health, but the extent and timing of their transfer to both placental and fetal compartments remain poorly understood. We aimed to characterize the relationship between trimester-specific maternal serum levels of prenatal PFAS and paired placental and cord levels at term. Data came from Glowing, a prospective birth cohort (n=151). Seventeen PFAS were measured in maternal serum, cord serum, and pulverized flash-frozen villous placenta with liquid chromatography-tandem mass spectrometry. Mixed effects models tested transplacental transfer efficiency (TTE) over pregnancy. Regularization models, stochastic intervention, and quantile g-computation models tested the association between maternal and placental or cord PFAS levels. TTE increased linearly across trimesters for all PFAS (p<0.001). Quartile increases in maternal PFAS were strongly associated with placental levels (0.018-0.24 ng/g, p<0.001). Stochastic intervention identified T1 PFNA and PFDA; T2 PFOS, PFOA, PFHxS, and PFNA; and T3 PFHxS as robust predictors (p<0.001) of placental levels, consistent with quantile-based contributions. Quartile increases in maternal and placental PFAS concentrations were associated with cord levels (0.08 ng/g-0.55 ng/g, p<0.001). Stochastic intervention identified T1 PFOS and PFHxS; T2 PFOS and PFNA; T3 PFOA; and placental PFOA as important predictors (p<0.05) of cord levels, consistent with quantile-based contributions. Early-to-mid gestation, especially 2nd trimester PFAS measures, were the strongest sentinels of placental and cord serum levels, apart from PFOA which was best reflected by 3rd trimester or placental levels. Placental PFOS and PFOA strongly influenced cord levels. Our findings underscore the heterogeneity in PFAS transfer or metabolism across pregnancy and the placenta.

Per-and polyfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants that have been associated with adverse health effects in highly exposed populations. Manufacturers have taken steps to replace toxic long-chain perfluoroalkyl acids (PFAAs) with short-chain PFAAs and perfluoroether acids (PFEAs). There is little to no toxicity data for many of these chemicals. Most of the data that are available are taken from studies that do not account for the pH of highly concentrated PFAS solutions, resulting in highly acidic conditions that do not accurately reflect real-world exposures. The goal of this study was to evaluate the lethality and developmental toxicity of 17 structurally diverse PFAS in zebrafish in a pH-neutral environment. We then compared results to determine the impacts of chain length, head group, and ether linkages on toxicity. The potency of PFAS to induce mortality and developmental toxicity endpoints increased with chain length, and sulfonic acids were more potent than carboxylic acids. The inclusion of ether oxygens was associated with reduced potency relative to PFAAs with equal chain length and head group. Perfluorooctane sulfonic acid (PFOS) was the most potent compound, followed by perfluoroundecanoic acid (PFUnDA) and perfluorododecanoic acid (PFDoDA). Short-chain compounds perfluoro-2-methoxyacetic acid (PFMOAA) and perfluorobutanoic acid (PFBA) were the least potent. These data were used to construct a multiple linear regression model for PFAS potency. Failed swim bladder inflation was the most sensitive developmental toxicity endpoint for all assessed chemicals. Other common phenotypes included spinal curvature, edema, craniofacial malformations, and ocular malformations. Ocular malformations were more common in response to sulfonic acids. No other phenotypes exhibited significant structural specificity. Our study provides new toxicity data for a diverse set of PFAS under environmentally relevant conditions. Future studies should be expanded to include more branched structures and head groups not present in our testing set to allow for improved understanding of how other structural features impact toxicity.