Environmental Science & Technology

Fluorinated chemicals are increasingly prevalent in pharmaceuticals and agrochemicals, yet their influence on the human gut microbiome and the potential for microbial biotransformation to alter therapeutic and toxicological profiles remain poorly understood. Here, we investigated the bidirectional relationship between 15 structurally diverse fluorinated chemicals and the gut microbiota by using an ex vivo high-throughput fermentation system. Screening revealed that flutamide, fluazinam, and pretomanid were consistently biotransformed across the donor microbiomes, while other compounds showed substantial inter-individual variability in degradation. Furthermore, exposure to fluorinated chemicals induced compound-specific shifts in microbial diversity and community composition, demonstrating their capacity to alter gut microbial ecology. Using a computational workflow combining in silico biotransformation predictions with untargeted LC-MS/MS analysis, we identified nitroreduction as the primary gut microbial transformation across all three compounds. Single-strain experiments confirmed that the nitroreduction of flutamide to flu-6, previously attributed only to hepatic metabolism, is a widespread capacity among gut bacterial strains. Finally, in vitro cytotoxicity assays and in silico modelling further revealed flu-6 to be a less hepatotoxic derivative than the parent compound, suggesting a potential detoxifying role for the gut microbiota. Together, these findings establish an integrated ex vivo, in vitro, and in silico approach for assessing the bidirectional interactions between fluorinated chemicals and the gut microbiome.

Chronic contaminant exposure may impose hidden physiological costs long before obvious demographic or health effects become detectable in wildlife populations. Epigenetic clocks quantify biological ageing and may provide sensitive biomarkers of cumulative toxicological stress. Per-and polyfluoroalkyl substances (PFAS) are persistent contaminants that bioaccumulate in marine food webs, yet their long-term physiological consequences for wildlife remain poorly understood. Here, we tested whether PFAS exposure is associated with accelerated biological ageing in common dolphins (Delphinus delphis). We analysed liver PFAS concentrations and skin DNA methylation profiles from 30 stranded or bycaught dolphins from New Zealand waters. Epigenetic age was estimated using a recently developed species-specific epigenetic clock, and age acceleration was calculated as the residual deviation between epigenetic and chronological age. Using an information-theoretic modelling framework, we assessed the effects of total PFAS burden, sex, and their interactions on epigenetic age acceleration. Total PFAS concentrations were positively associated with epigenetic age acceleration, indicating that dolphins with higher PFAS burdens were biologically older than expected for their chronological age. Each 1 ng g{square}{superscript 1} increase in total PFAS was associated with an average increase of 0.031 years in biological age. Sex did not significantly influence age acceleration, suggesting that PFAS-associated ageing effects occur across both sexes. Although modest, this effect is consistent with PFAS acting as a chronic physiological stressor influencing molecular ageing processes. Our findings provide the first evidence linking PFAS exposure to accelerated biological ageing in a wild mammal, highlighting epigenetic ageing as an integrative biomarker of long-term contaminant effects in wildlife.

Respiratory syncytial virus (RSV) transmission via the aerosol route remains poorly understood, particularly with respect to how evolving virus-laden particles (bioaerosols) microenvironments influence viral survival. Bioaerosol particles contain complex mixtures of organic and inorganic components, and their physicochemical properties change dynamically during evaporation as water is lost upon emission from respiratory activities. These changes directly affect the local environment surrounding embedded virus during both the evaporation stage and the subsequent equilibrium state. However, how these microenvironmental conditions under different relative humidity (RH) levels regulate RSV survival remains unclear. In this study, we quantified RSV survival during the evaporation and early equilibrium stages using a flow-tube system with controlled residence times. Bioaerosols were generated from virus medium alone or supplemented with bovine serum albumin (BSA) or mucin and evaluated under low (35%) and intermediate (61%) RH conditions. Viral infectivity was normalized to RNA copy number to account for particle and sampling losses. At 35% RH, RSV infectivity decreased by one to three orders of magnitude, depending on the solution composition. In contrast, survival was significantly higher at intermediate RH, particularly for virus medium and BSA-supplemented aerosols. Scanning electron microscopy revealed that low RH conditions promote efflorescence, whereas intermediate RH results in viscous or semi-solid particles with higher water content. These observations suggest that efflorescence is associated with enhanced RSV inactivation, while viscous or semi-solid phases tend to preserve RSV in the aerosol state for respirable particles. Overall, RSV infectivity depends strongly on particle chemical composition, phase state (effloresced versus semi-solid), and relative humidity. These results highlight the importance of characterizing particle phase behavior and chemical composition during early aerosol processes to improve mechanistic understanding of viral survival relevant to short-range transmission.

Enteroviruses (EVs) are ubiquitous contaminants of surface waters, where they can remain infectious for long periods of time. Most methods used for EV monitoring are unable to distinguish between infectious and non-infectious particles or between EV types. Because different types exhibit both distinct environmental persistence and health implications, there is a need for type-resolved infectivity measurements. Here we developed Integrated Cell Culture-Nanopore Sequencing (ICC-NanoporeSeq), a method combining short-term cell culture amplification with Nanopore sequencing of the VP1 gene. The ICC approach was adapted from a previously described ICC-RTqPCR protocol, while the NanoporeSeq workflow was derived from a clinical EV typing protocol and optimized for environmentally circulating EV types. Using samples containing known concentrations of ten EV types, the NanoporeSeq method accurately and reproducibly recovered the original proportions of all EV types after correction of biases. Furthermore, type-specific calibration curves generated with ICC-NanoporeSeq enabled quantification of the infectious concentrations of six EV types, allowing a simultaneous and type-resolved assessment of infectivity in mixed samples. Overall, ICC-NanoporeSeq provides a scalable approach for the parallel analysis of multiple EV types. Compared with the predecessor ICC-RTqPCR method, it eliminates the need for multiple type-specific PCR primers and can therefore be readily expanded to include additional EV types. IMPORTANCECurrent methods used to detect EVs in environmental samples generally measure viral genome copies without determining whether viruses remain infectious, limiting their use in public health risk assessment or water quality monitoring. At the same time, available infectivity assays are often labor-intensive and cannot distinguish between different EV types. Here, we developed ICC-NanoporeSeq, a method combining cell culture and Nanopore sequencing to simultaneously quantify the infectious concentrations of multiple EV types in samples containing mixed EV populations. The method provides an efficient and scalable approach for studying EVs in complex environmental matrices. ICC-NanoporeSeq has potential applications in wastewater-based epidemiology, environmental surveillance, and disinfection studies, where understanding the persistence of different EV types simultaneously is crucial.

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental contaminant widely detected in human serum and follicular fluid and has been associated with reduced implantation rates and female fertility. However, its direct effects on mammalian oocyte maturation remain poorly understood. Here, we developed a microinjection-based single-oocyte toxicological assay to directly evaluate how physiologically relevant PFOS concentrations affect mouse oocyte maturation and early embryonic development. Microinjection of PFOS at follicular-fluid level (5.6 nM) and occupational exposure level (60 nM) significantly reduced germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) rates compared with water-injected controls. Notably, all tested concentrations (2.4 nM serum level, 5.6 nM, and 60 nM) induced abnormal polar-body formation, disrupted meiotic spindle morphology, and increased the proportion of unhealthy oocytes. PFOS exposure also significantly elevated intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential at 5.6 nM, indicating oxidative stress and mitochondrial dysfunction. Cytological analyses revealed chromosome misalignment and widened metaphase I plates, suggesting chromosome missegregation and subsequent prometaphase II arrest with defective polar bodies. Single-cell RNA sequencing of PFOS-treated oocytes exhibiting abnormal small polar bodies identified distinct transcriptional signatures, including dysregulation of genes involved in mRNA processing, chromosome segregation, mitochondrial function, and cell division. Functionally, these oocytes failed to progress beyond the 2-cell stage following in vitro fertilization, indicating loss of developmental competence. Collectively, these findings demonstrate that PFOS directly disrupts meiotic progression through spindle defects, oxidative stress, and transcriptional dysregulation, ultimately compromising oocyte quality even at environmentally relevant exposure levels. Environmental ImplicationPFOS is a persistent environmental contaminant widely detected in human serum and follicular fluid. Our findings demonstrate that PFOS at physiologically relevant levels can impair oocyte maturation, disrupt meiotic chromosome segregation, and compromise early embryonic development. By using a single-oocyte toxicological assay, we reveal that even low-dose PFOS exposure can induce oxidative stress and transcriptional dysregulation. These results highlight the potential reproductive risks of chronic PFOS exposure and underscore the importance of stricter environmental monitoring and regulation to protect female reproductive health and fertility. This novel assay also has the potential to redefine safety thresholds for other environmental toxicants.

The foodborne mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been associated with several adverse effects, including cytotoxicity, genotoxicity, endocrine disruption, and immunomodulation. As these endpoints are typically observed in vitro at micromolar concentrations, the question arises whether such levels are attainable in exposed humans. To address this data gap in chemical risk assessment, a physiologically based kinetic (PBK) model was developed to predict internal exposure doses to AOH and AME in humans. As input parameters, kinetic constants for hepatic glucuronidation were obtained in vitro by incubating Sprague Dawley rat and human liver S9 fractions with 0.5-50 M AOH and 0.5-20 M AME, demonstrating rapid biotransformation in both species. Intestinal absorption of AME and physicochemical parameters were estimated using quantitative structure-activity relationship (QSAR) models. Sensitivity analysis identified parameters describing hepatic glucuronidation and gastrointestinal uptake as among the most influential, confirming the importance of their reliable estimation. The PBK model was evaluated against available rodent toxicokinetic data and subsequently extrapolated to humans. Ultimately, the currently available exposure estimates published by EFSA in 2016 were applied to predict target tissue concentrations, which were compared to points of departure (PoDs) for relevant toxicological endpoints. Even in the most susceptible group of male toddlers, predicted internal concentrations (10-4 M range) were approximately four orders of magnitude below the respective PoDs. Consequently, under the applied exposure assumptions and considering the compounds as isolated chemicals, AOH and AME are not expected to reach systemic or tissue concentrations associated with the investigated effects.

Airborne transmission of respiratory pathogens depends on their ability to remain viable in drying respiratory droplets, yet the physicochemical drivers of bacterial inactivation during droplet evaporation remain poorly quantified. This study combines controlled droplet experiments with physicochemical modeling to investigate how osmotic pressure dynamics influence bacterial survival. Using Escherichia coli and Staphylococcus epidermidis as Gram-negative and Gram-positive surrogates, respectively, we measured viability loss in artificial saliva droplets dried at multiple relative humidities and reconstructed the time-resolved osmotic pressure using the Respiratory Aerosol Model (ResAM). Both organisms remained stable while droplets were liquid but lost viability following efflorescence, when rapid solute concentration changes produced sharp osmotic pressure increases. The extent of inactivation scales log-linearly with the rate of osmotic pressure change around efflorescence: E. coli decays faster than S. epidermidis, and relationships derived in artificial saliva predict survival in independent phosphate-buffered saline experiments. A more rapid drop in humidity led to more severe osmotic shocks and greater inactivation. These results identify the rate of osmotic pressure change during efflorescence as a quantitative, medium-independent predictor of bacterial survival in drying respiratory droplets. ImportanceAirborne infection risk depends on how long microorganisms remain viable in respiratory particles after exhalation, yet the physical mechanisms controlling bacterial survival during droplet drying are not well defined. Evaporation of respiratory droplets concentrates salts and can impose sudden and extreme osmotic stress on microbes, but this process has been difficult to quantify because osmotic pressure cannot be measured directly inside microscopic droplets. Integration of droplet experiments with a physicochemical aerosol model shows that bacterial inactivation is governed primarily by the rate of osmotic pressure increase during droplet efflorescence rather than by static values of humidity or solute concentration alone. This mechanism explains why rapid drying may produce strong inactivation.

Adverse outcome pathways (AOPs) describe stressor non-specific sequences of events between a first molecular trigger (molecular initiating event, MIE), causally linked key events (KEs), and an adverse outcome (AO). AOPs are intended to aid in chemical toxicity testing as a new approach methodology. However, commonly used AOP development methods depend on manual curation, which is labor intensive. As a result, there are still relatively few AOPs and a huge number of toxicity mechanisms and possible adverse outcomes remain undescribed. Therefore, systematic and high-throughput approaches to predict new AOPs are needed. Here, we developed and implemented a data integration-based framework to generate new candidate AOPs using insecticides and Parkinson Disease as a proof of concept. We integrated and statistically linked disconnected databases (e.g., Comparative Toxicogenomics Database, Human Protein Atlas, and Gene Ontology) to form MIE - KE (cell level) - KE (tissue level) - AO candidate AOPs. Through this systematic process, we generated 562,117 candidate AOPs, which we then scored using a weight of evidence (WoE) approach and prioritized 12,756 AOPs with a WoE >0.5. Through random sampling of 100 prioritized AOPs, we found 70% had external literature supporting their biological plausibility, and only 15% represented identifiably implausible associations. The prioritized AOPs describe varied mechanisms of toxicity related to e.g., MAPK, PTEN, and FGFR signaling pathways, with "increases phosphorylation of MAPK1" as the most frequent MIE. Our AOP generating approach yields consistently structured AOPs and can complement existing and emerging development methods to expand AOP coverage across different stressors and outcomes.

Dietary exposure to heavy metals (HMs) via animal-source foods is a critical environmental health pathway. In rapidly industrializing Bangladesh, contamination of the bovine food chain from agricultural feeds and industrial emissions poses an unquantified public health burden. This study evaluated exposure pathways, spatial distribution, mass-transfer dynamics, and health risks of six HMs (Cr, Cu, Cd, Pb, As, and Hg) across the fodder-cattle-human continuum. Samples of beef (n = 76), raw milk (n = 76), commercial cattle feed (n = 40), and fodder (n = 88) were collected from eight sites across industrial and non-industrial zones in Bangladesh and analysed by atomic absorption spectroscopy. Probabilistic Monte Carlo simulations (10,000 iterations) quantified estimated daily intake, target hazard quotients (THQ), cumulative hazard index (HI), and lifetime carcinogenic risk (CR) for adult and pediatric receptors. Copper (Cu) was the dominant contaminant across all matrices, peaking in beef (103.89 {+/-} 15.87 mg/kg) and milk (13.67 {+/-} 1.53 mg/L). Spatial analysis revealed distinct contamination profiles: Pb burden peaked in industrial zones while Cr was elevated in non-industrial sectors. Monte Carlo modelling identified commercial feed as the most efficient transfer vector into beef. Pediatric THQ for Cu significantly exceeded the safety threshold (THQ > 1), and upper-bound lifetime carcinogenic risk from As approached the critical USEPA 10- regulatory ceiling. These findings demonstrate that industrial and agricultural externalities efficiently contaminate the bovine food supply chain in Bangladesh, with copper and arsenic representing the most critical non-carcinogenic and carcinogenic dietary hazards, respectively. Children are disproportionately vulnerable due to lower body weight. The results underscore the need for targeted upstream interventions in commercial feed production and provide evidence to support feed-quality regulation and environmental monitoring in rapidly industrializing settings.

Inflammatory bowel diseases (IBD) affect millions of patients worldwide and impair quality of life. Although genetic and environmental factors are known to disrupt the gastrointestinal (GI) epithelial barrier and increase susceptibility to IBD, the precise contribution of specific environmental exposures remains unclear. Per- and polyfluoroalkyl substances (PFAS), or "forever chemicals," are widely used in consumer products and contaminate food and water sources, resulting in chronic oral exposure worldwide. Perfluorooctanoic acid (PFOA), a common PFAS, has been epidemiologically associated with the development of IBD, particularly in older adults. Here, we assessed the effects of oral PFOA exposure on the GI tract, liver, and susceptibility to colitis. C57BL/6 mice were exposed to PFOA (0.1 mg/kg or 1.0 mg/kg) beginning at weaning (post-natal day [P]21) for a time course of 4 or 8 weeks. GI physiology/pathology (Ussing chambers; histology), expression of pro-inflammatory cytokines (qPCR), microbiota composition (16S sequencing), bile acids production (qPCR; LC/MS), and liver pathology (histology) were assessed. Colitis susceptibility was evaluated in genetically predisposed (IL10 knockout) mice, and in induced (dextran sodium sulfate [DSS]) mouse models following PFOA exposure (8 weeks at 1.0 mg/kg). Oral PFOA exposure increased intestinal permeability, mildly increased cytokine expression, altered gut microbiota composition, disrupted liver and serum bile acids, and caused hepatic hypertrophy at higher doses and longer exposure. Although PFOA did not increase disease susceptibility in genetically predisposed Il10 KO mice, it significantly worsened DSS-induced colitis, but only in male mice. Together, these findings demonstrate that early-life PFOA exposure disrupts the gut-liver axis and may contribute to colitis development in a sex dependent manner.

Until now, there has been no animal-free alternative method for predicting chronic inflammation and delivering the associated dose responses, the timing of onset, and the duration of inflammation, as required by regulatory agencies. We present the results of pre-validation of an in-vitro-learned digital twin (InFiniteLungDT) capable of predicting chronic neutrophilic lung inflammation for regulatory use. The method is based on measuring the dynamics of early biological effects in vitro induced by respirable materials or their mixtures, without the need to know their intrinsic properties. We constructed the digital twin(s) for each of the material, for which we have in vivo exposure data. The instillation data set, comprising 49 different nanomaterials, was used as the primary anchor to calibrate the model. Inhalation data set, comprising 7 different nanomaterials, compliant with OECD TG 412, was used to show the general applicability of the method across species and for different exposure scenaria. In total, about 3094 single mouse exposures and 364 rat exposures (and approx. 775/225 non-exposed mouse/rat controls) were used to predict concentration-dependent time-evolved neutrophil influx into the lung. The accuracy (predictive capacity) of LOAEL determination is 93% for instillation and 84% for inhalation exposure. Taking into account the time-to-deliver-result being less than 1 week, this proves that the effect of inhaled material from acute to chronic conditions can be assessed orders of magnitude faster and cheaper than in a reference animal study.

Oil sands process-affected water (OSPW) contains complex mixtures of naphthenic acids (NA) that are the central targets for water treatment and reclamation. Here, we compared three whole-cell bacterial NA biosensors with Orbitrap mass spectrometry (MS) for quantifying NA remediation in greenhouse mesocosms and a pilot-scale constructed wetland. Solid-phase extracts from both systems were analyzed in parallel by Orbitrap MS and biosensor assays, enabling direct comparison of biosensor-derived NA estimates with MS-derived naphthenic acid fraction compounds (NAFC) concentrations. Across both treatment systems, biosensor outputs broadly tracked declines in NAFC measured by Orbitrap MS, and positive linear relationships were observed between methods. Biosensor 2 (3680-lux) and biosensor 3 (atuA-lux) showed, early rapid decreases in NA, whereas biosensor 1 (marR-lux) frequently remained elevated later in treatment. These differences are consistent with the distinct chemical response profiles of the biosensor panel and suggest that biosensor outputs reflect shifts in NA mixture composition as remediation proceeds. This interpretation is supported by the published Orbitrap analysis of the constructed wetland, which showed decreasing O2-NAFCs and increasing O3/O4-containing species, consistent with oxidative degradation. Together, these results support the use of NA-responsive biosensors as rapid and scalable complementary tools for tracking remediation trends, while Orbitrap MS remains the reference method for molecular-level characterization.

Wastewater genomic surveillance provides an opportunity to detect human and animal influenza A virus (IAV). We aimed to implement an IAV genomic surveillance framework agnostic to subtype, which enables recovery of IAV from multiple hosts and estimation of proportions across subtypes. We conducted IAV genomic surveillance in wastewater during the 2024-2025 flu season at multiple sites in California and compared these data with available human clinical IAV sequences and test positivity. We applied a custom whole-genome, multi-host IAV probe enrichment panel and adapted our custom expectation-maximization (EM) algorithm to deconvolute IAV mixtures in wastewater and infer subtype relative abundances. Absolute IAV concentrations were quantified using RT-PCR-based assays. H5N1 wastewater and clinical sequences were further characterized by constructing a whole-genome maximum-likelihood phylogenetic tree. Finally, we performed variant analysis to examine amino acid substitutions detected in wastewater. Our IAV probe enrichment method and EM algorithm successfully enriched all eight segments of three circulating IAV subtypes and accurately estimated subclade relative abundances for mixed IAV samples. Seasonal human H1N1pdm09 and H3N2 were detected throughout the study period from both wastewater and clinical sequencing data, with H1N1 subclades 6B.1A.5a.2a.1 and 6B.1A.5a.2a co-circulating, and H3N2 dominated by subclade 3C.2a1b.2a.2a.3a.1. Wastewater surveillance consistently detected H5N1 clade 2.3.4.4b across three monitored wastewater sites, while clinical H5N1 detections, from anywhere in CA, were sporadic and rare. Whole-genome phylogenetic analysis revealed that wastewater H5N1 sequences clustered with reference sequences associated with dairy cow and avian infections, while all human clinical H5N1 sequences clustered exclusively with reference sequences associated with dairy cow infections. Amino acid substitutions were identified across viral segments, and no mutations associated with mammalian adaptation were observed from wastewater samples.

Nano- and microplastics (NMPs), by-products of the fragmentation and degradation of plastic products, are ubiquitous environmental contaminants, yet their burden in pediatric immune tissues and functional consequences for developing immunity remain unknown. Here we report the first comprehensive characterization of NMPs in surgically excised pediatric tonsils (n = 30) using pyrolysis gas chromatography- mass spectrometry (Py-GC/MS), Nile Red fluorescence microscopy, and optical photothermal infrared (O-PTIR) spectroscopy. NMPs were detected in all specimens, with polystyrene, polyethylene, polyethylene terephthalate, and acrylonitrile butadiene styrene present in >90% of samples. To bridge clinical exposure data with mechanistic insight, we formulated a cryo-milled multi-polymer mixture reflecting the patient-derived polymer profile and challenged human lymphoid aggregate culture (HLAC) tonsil organoids at environmentally relevant concentrations. Multiplexed cytokine profiling of culture supernatants revealed a robust early inflammatory response at day 3, with significant upregulation of IL-6 (p = 0.011) and MIP-1{beta}/CCL4 (p = 0.011), followed by convergence toward control levels by day 14. Functional cytokine modules spanning immune, metabolic, structural, and growth factor pathways showed coordinated deviation from controls at day 3 post-exposure with subsequent normalization. Fluorescence-guided depth profiling demonstrated time-dependent penetration of 100 nm particles into organoid aggregates (70% tissue depth at day 3 versus 95% at day 14), and transmission electron microscopy revealed intracellular polyethylene within lymphocyte lysosomes. These findings establish pediatric tonsils as a sentinel tissue for NMP bioaccumulation and demonstrate that environmentally relevant polymer mixtures elicit transient but significant immunomodulatory responses in human lymphoid tissue, with implications for mucosal and systemic immune health in children. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/728317v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@19395f4org.highwire.dtl.DTLVardef@5a0380org.highwire.dtl.DTLVardef@19c0741org.highwire.dtl.DTLVardef@a052c5_HPS_FORMAT_FIGEXP M_FIG Structure: Translational pipeline from clinical tissue characterization to patient-informed preclinical modeling of nano-microplastic (NMP) exposure in pediatric lymphoid tissue. Pediatric tonsil tissue collected from clinically indicated tonsillectomies underwent tissue digestion for NMP characterization to identify NMP type and size distributions. In parallel, tonsil tissue was used to generate human lymphoid aggregate culture (HLAC) organoids that recapitulate the cellular complexity of the native tissue. These patient-derived organoids were then exposed to environmentally relevant compositions and concentrations of NMPs over time-course experiments, with longitudinal assessment of immunomodulatory responses including cytokine profiling and functional readouts. This bedside-to-bench approach establishes a physiologically relevant human system for investigating NMP-immune interactions, bridging clinical tissue analysis with mechanistic preclinical modeling to inform understanding of pediatric environmental exposures and their potential health impacts. C_FIG

O_LIHeavy metals are pervasive environmental contaminants that can impair the health of organisms globally. As the largest anthropogenic source of the potent neurotoxin mercury (Hg), gold mining has amplified these threats throughout the tropics. Consequently, there is a mounting need to monitor Hg contamination of the richest biological communities on Earth. Venous whole blood provides a reliable, nonlethal measurement of recent dietary and site-derived contamination, but collecting and cold-storing samples can be impractical in field conditions. C_LIO_LITo overcome these challenges, we developed and evaluated a method to assay Hg exposure in vascular organisms by measuring the volume of dried blood spots (DBS) in the field, which can be stored at ambient temperatures until analysis. We explored the methods precision and accuracy in estimating whole blood Hg concentrations by collecting paired whole blood and DBS aliquots from birds (n = 527 individuals, 140 species) along a trophic gradient (i.e., granivores to piscivores) in Belize and Peru. C_LIO_LIUsing a Bayesian linear mixed-effects model, we found a highly precise and unbiased relationship between DBS and whole blood total Hg concentrations that was centered at perfect unity (R2 = 0.99; {beta} = 1.00 {+/-} 0.03; 95% CrI: 0.95-1.05). Agreement between individual paired aliquots was more variable, in which approximately 12% of DBS containing at least 1 ng THg differed from whole blood by more than {+/-}20%. However, DBS accuracy increased at higher THg concentrations, suggesting that disagreement at low concentrations is an expected consequence of higher measurement error near the analytical limit of detection of our instruments. C_LIO_LICompared to whole-blood collection and analysis workflows, DBS offer substantial logistical advantages by eliminating cold-chain dependence and reducing transport burden, laboratory handling time, and overall operational costs. Consequently, volume-measured DBS provide a practical and highly reliable alternative for monitoring Hg contamination in both humans and wildlife, particularly for ecological and population-level applications in remote and resource-limited environments. C_LI

Microplastics (MP) are ubiquitous environmental contaminants with diverse physicochemical characteristics. Many studies have shown that size, shape, and polymer type are responsible for their toxicity, but this also seems to differ among MP from the same plastic type. One parameter likely contributing to these differences is plastic chemicals, a broad class of compounds intentionally or unintentionally added to plastics during their production and manufacturing. However, knowledge on the composition of plastic chemicals and their effects remains scarce. Therefore, to elucidate the chemical aspect of MP toxicity, we exposed Daphnia magna individuals to MP (PET, PBS, and PDLLA), cellulose, extracted particles (eMP), and methanol-based extracts of these particles for 10 days. Chemicals within such extracts were analyzed via GC-MS. This study was conducted with reduced food availability to investigate plastic effects in an environmentally relevant scenario. The introduction of a high-food control suggests that a more realistic feeding regime might exacerbate the plastic effects of the selected treatments. Our results indicated that, depending on the polymer type, plastic chemicals determine MP toxicity, which varies according to the endpoint investigated (i.e., body length, reproduction, levels of ROS and LPO). Body length, in particular, was significantly impaired by PET and PDLLA extracts, whereas reproduction was affected by most treatments. The investigated biochemical parameters (ROS and LPO) were not affected by the exposure. These results suggest that MP toxicity strongly depends on their chemical composition, whereas adverse effects due to physical properties are present independently of chemical composition across all MP types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/724551v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@3c2d4forg.highwire.dtl.DTLVardef@c2ccd7org.highwire.dtl.DTLVardef@116721dorg.highwire.dtl.DTLVardef@9df888_HPS_FORMAT_FIGEXP M_FIG C_FIG

Early embryogenesis is governed by precisely timed gene regulatory programs that coordinate cell fate specification, tissue patterning, and morphogenesis. The maternal-to-zygotic transition (MZT) represents a pivotal developmental milestone during which regulatory control shifts from maternally deposited transcripts to activation of the zygotic genome. Disruption of this transition has the potential to alter developmental trajectories with lasting consequences. Per- and polyfluoroalkyl substances (PFAS), environmentally persistent contaminants, have been linked to developmental abnormalities, yet their impact on core embryonic gene regulatory networks especially during MZT is not well understood. Using zebrafish (Danio rerio), a tractable vertebrate model and New Approach Methodology (NAM), we investigated how PFAS exposure during the MZT alters early developmental programming. Embryos were exposed starting at different times within the 8-hour MZT window and collected at 24 hours post-fertilization (hpf) for transcriptomic analysis. Targeted qRT-PCR revealed dysregulation of genes controlling transcriptional activation, lineage specification, proliferation, and differentiation. Whole-transcriptome RNA sequencing (RNA-seq) further identified widespread perturbations in gene networks governing transcriptional regulation, cell signaling, and embryonic morphogenesis. Temporal analysis revealed that exposure beginning at 3.5 hpf, followed by 8 hpf, corresponding to early zygotic genome activation and near completion of zygotic activation, respectively, resulted in the greatest differential gene expression changes. Consistent with these early gene regulatory perturbations, larvae exposed at 8 hpf also exhibited altered behavior at 5 days post-fertilization. Together, these findings demonstrate that PFAS exposure during MZT disrupts the establishment of embryonic gene regulatory networks, linking environmental toxicant exposure to altered developmental patterning and organismal outcomes. This work underscores the vulnerability of early developmental transitions to environmental perturbation and positions MZT as a critical window of susceptibility during development.

Naphthenic acids are amphipathic compounds whose toxicity has primarily been attributed to narcosis toxicity to cell membranes. However, few methods exist that specifically study the membrane disruption and toxicity of this complex family of cyclic, polycyclic and acyclic alkyl-substituted carboxylic acids. Here we describe a whole cell biosensor approach that relies on the ability of Pseudomonas aeruginosa, a ubiquitous environmental organism and opportunistic pathogen, to sense membrane damage (narcosis) and induce protective genes to repair and protect the outer membrane. Many classes of membrane disrupting antimicrobials induce the expression of two operons that encode protective defense systems against outer membrane (OM) damage, including antimicrobial peptides, chelators, and detergents. We demonstrate that the pmrF and spdE2 transcriptional lux reporters are induced by exposure to individual NA compounds with diverse structures, as well as mixtures and naphthenic acid fraction compounds (NAFCs). To further support the narcosis hypothesis, we demonstrated that NA permeabilizes the outer membrane to assist in lysozyme killing, and disrupts the inner membrane integrity, allowing uptake of the DNA binding dye propidium iodide. The conventional OM permeability assay that measures NPN fluorescence is not applicable to study NAs, because they stimulate NPN fluorescence in the absence of cells. This narcosis biosensor approach constitutes a rapid and simple method to measure narcosis and could be developed as a novel toxicity indicator of oil sands tailings.

Environmental DNA (eDNA) surveys are increasingly used to assess marine biodiversity and inform deep-sea environmental decision-making, including mineral resource management and fisheries oversight. Yet standard low-volume protocols inherited from coastal work may be inadequate at depth, and no quantitative framework links depth and ecosystem context to defensible filtration volume targets. We compiled 841 eDNA samples from eight expeditions across the North Atlantic, Wider Caribbean, and Pacific (surface to 4000 m) to quantify how recoverable eDNA scales with depth and surface productivity, and to derive depth- and productivity-aware sampling targets. Total eDNA concentration declined with depth as a power law, with attenuation exponents (b) modulated by surface productivity: most gradual in eutrophic waters (b = 0.67), intermediate in mesotrophic (b = 0.90), and steepest in oligotrophic systems (b = 1.25); volume-weighted models explained 66-88% of the variance. At a fixed extract-concentration target, required filtration volumes diverged ~7-fold between oligotrophic and eutrophic systems at 200 m and ~38-fold at 4000 m. Conventional Niskin sampling, therefore, undersamples deep-sea biodiversity, particularly in mid- to low-productivity systems. Among laboratory parameters, the assay-specific extract-concentration target exerted greater leverage on required volume than extraction efficiency or elution volume. Volume-aware sampling paired with optimized recovery should be routine in deep-sea eDNA surveys.

Background: Climate mitigation policies can lower air pollutant concentrations and deliver substantial health co-benefits. The French Ecological Transition Agency (ADEME) proposed four contrasting Transitions 2050 net-zero scenarios. We quantified mortality, morbidity, and health-economic co-benefits from projected PM2.5 and NO2 reductions across all four scenarios in continental France. Methods: Emission projections were input to the CHIMERE chemistry-transport model to estimate PM2.5 and NO2 concentrations for 2030 and 2050. Health impacts were assessed using disease-specific cessation-lag assumptions relative to 2019, covering premature mortality, morbidity, DALYs, and economic benefits across nine outcomes (hypertension, lung cancer, ischaemic heart disease, stroke, COPD, type-2 diabetes, acute lower respiratory infections, and asthma in children and adults). Findings: Population exposure is projected to decline by about 40% for PM2.5 and 70% for NO2 by 2050, with health gains remaining substantial and broadly equivalent across all four scenarios and modest differences between sufficiency-oriented and technology-driven pathways. Under delayed-impact assumptions, avoided premature deaths ranged from 21,300 to 22,100 for PM2.5 and 24,500 to 26,200 for NO2. Morbidity and disability-adjusted life year (DALY) reductions, as well as economic savings, spanned similarly; total avoided morbidity cases were 84,000-88,000, direct medical cost reductions were e1.0-1.1 billion/year, and intangible cost savings of e41-43 billion and e36-39 billion, respectively. Interpretation: Health co-benefits are substantial, consistent across contrasting scenarios, and increase markedly from 2030 to 2050. Explicitly incorporating these co-benefits into climate policy appraisals may strengthen the case for ambitious mitigation and improve decision-maker acceptability.