Environmental Science & Technology

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.

Metformin (MET), a worldwide used drug for type 2 diabetes, has been found with the largest amount by weight among all drugs in aquatic environment, including the drinking water sources where chlorination inevitably transforms MET into chlorination byproducts. Although MET has health-promoting properties, whether or how its chlorination byproducts affect health remains largely unknown. Here we reveal that MET chlorination byproducts Y (C4H6ClN5) and C (C4H6ClN3) exhibit marked toxicity, even higher than that of the well-known poisonous arsenic, to live worms and human cells. Moreover, both byproducts are harmful to mice and Y at 250 ng/L destroys the mouse small intestine integrity. Strikingly, we detected MET and byproduct C in worldwide drinking water. Both byproducts are increasingly produced with more MET present during chlorination process. Unprecedentedly, we unveil boiling and activated carbon adsorption as effective solutions that are in urgent demand globally for removing these byproducts from water.

Despite the critical importance of virus disinfection by chlorine, we lack a fundamental understanding of the relative susceptibility of different viruses to chlorine and robust quantitative relationships between virus disinfection rate constants and environmental parameters. We conducted a rapid systematic review of virus inactivation by free chlorine and used the resulting data set to develop a linear mixed model that estimates chlorine inactivation rate constants for viruses based on experimental conditions. Over 550 data points were collected in our systematic review, representing 82 viruses over a broad range of environmental conditions. The harmonized inactivation rate constants under reference conditions (pH = 7.53, T = 20 {degrees}C, [Cl-] < 50 mM) spanned 4 orders of magnitude, ranging from 0.0196 to 1150 L mg-1 min-1 and uncovered important trends between viruses. Whereas common surrogate bacteriophage MS2 does not serve as a conservative chlorine disinfection surrogate for many human viruses, CVB5 was one of the most resistant viruses in the dataset. The model quantifies the role of pH, temperature, and chloride levels across viruses, and an online tool allows users to predict rate constants for viruses and conditions of interest. Results from the model highlighted potential shortcomings in current USEPA disinfection guidelines. SynopsisViruses must be adequately removed by disinfection processes to protect public health. We review data on virus inactivation of chlorine to determine what concentrations of chlorine are required to remove viruses and whether current regulations are adequately protective.

N-(1,3-Dimethylbutyl)-N-phenyl-p-phenylenediamine-quinone (6PPD-q) is a rubber-tire derivative which leaches into surface waters from roadway runoff, from tire particles and has been identified as a possible driver of urban runoff mortality syndrome in coho salmon. Sensitivity to this toxicant is highly variable across fish species and life stages. With environmental concentrations meeting or exceeding toxicity thresholds in sensitive fishes, the potential for ecologically relevant effects is significant. There is currently no data regarding the sensitivity of lake trout (Salvelinus namaycush) to 6PPD-q. As early-life stages of fishes are typically more sensitive than adults, the goal of these studies was to evaluate the acute and sub-chronic toxicity of 6PPD-q to early-life stage lake trout. Alevins exposed from hatch until 45 days post hatch (dph) to time-weighted average 6PPD-q concentrations ranging from 0.22-13.5 g/L exhibited a 45-day median lethal dose (LC50) of 0.39 g/L. Deformities throughout growth were observed, with a unique pooling of blood observed in the caudal fin and eye. A subsequent acute study with exogenously feeding lake trout fry determined a 96-hr LC50 of 0.50 g/L. From these studies we can conclude that lake trout alevins and exogenously feeding fry are sensitive to 6PPD-q, which underscores the relevance of this chemical to inland freshwater ecosystems.

Sustainable aquaculture requires comprehensive chemical oversight, as compounds used in aquaculture can persist in ecosystems, bioaccumulate through food chains, and affect aquatic life and human health. This study presents ReCAnt (Resource on Chemicals used in Aquaculture and their Ecotoxicity), which compiles information on 690 aquaculture chemicals, with data on toxic effects and therapeutic potential curated from published literature. It was observed that only a fraction of the 690 chemicals are currently regulated, revealing gaps in existing regulations. Integration of data from the Comparative Toxicogenomics Database revealed associations with genes, phenotypes, and diseases, while ECOTOX data provided toxicity and bioconcentration information. Predicted biotransformation pathways and partition coefficients indicated microbial degradation potential and fate across environmental media. Further, food web network analysis identified species vulnerable to trophic transfer and common entry points for chemicals into aquatic ecosystems. This resource can aid in developing evidence-based regulatory frameworks and promoting sustainable chemical management.

Electrical manufacturing near New Bedford Harbor (NBH), MA in the mid-1900s led to severe polychlorinated biphenyl (PCB) contamination, which resulted in the harbors designation as a Superfund Site. Restrictions on the harvest of seafood from NBH have been in effect since 1979. Efforts to reduce the overall mass of PCBs in NBH by dredging PCB-contaminated sediments have been ongoing since the late 1980s. One goal of dredging is to reduce PCB concentrations in NBH seafood, monitored annually by Massachusetts Department of Environmental Protection (DEP) and Massachusetts Division of Marine Fisheries (MDMF). We used PCB concentrations in quahogs (2003-2016) and scup (2003-2014) to evaluate PCB distribution across seafood management areas and over time. Seafood PCB concentrations were used to evaluate improvements in environmental quality by examining total PCBs and patterns of PCB congeners within homolog groups, and by assessing human cancer risk from seafood consumption in the past (1980) and present (2012-2016). PCB concentrations in quahogs generally declined with increased time and distance from the PCB source, as does the cancer risk associated with their consumption. PCB concentrations in scup follow similar spatial patterns but show high annual variability. We conclude that quahogs are a reliable proxy for in-situ conditions, environmental quality, and human health risk.

Developmental exposure to per- and polyfluoroalkyl substances (PFAS) has been linked to reduced antibody responses to childhood vaccines, but the underlying mechanisms remain unclear. Antibody production relies on interactions between various immune cell types, and it is unknown which are affected by PFAS exposure during development. To investigate this in a human-health relevant system, an in vivo model was established to delineate effects of developmental exposure to a mixture of four PFAS commonly found in human serum: PFOA, PFOS, PFHxS, and PFNA. Pregnant mice consumed water containing these PFAS throughout gestation and lactation. PFAS were measured in both mothers and offspring, and an exposure that avoided overt health issues was selected. The immune response to influenza A virus (IAV) infection was assessed in male and female offspring. Results showed that developmental PFAS exposure reduced IAV-specific antibody levels in both sexes. However, it diminished T follicular helper cells and germinal center B cells--critical for antibody production--in only female offspring. These findings highlight possible sex-specific immune effects and identify potential cellular mechanisms behind reduced antibody levels. Since these immune cells are essential for antibody production in humans, this study provides valuable insights into how PFAS exposure may impact human health. SynopsisA novel mouse model of developmental exposure to a human-relevant PFAS mixture recapitulates observations in epidemiological studies and also provides new insight into potential mechanisms of the lower antibody levels observed in humans.

Ise Bay and Mikawa Bay, situated near major urban and industrial areas, have been increasingly affected by water pollution due to human activities. Over time, eutrophication--primarily driven by excess phosphorus (P) and nitrogen (N)--has led to frequent occurrences of red tides and blue tides, significantly impacting coastal fisheries. To mitigate environmental pollution, efforts have focused on purifying the rivers and brackish lakes that flow into these bays. However, as pollution loads have decreased, these bays have transitioned from a eutrophic to an oligotrophic state, leading to a decline in the populations of benthic organisms and their higher consumers. In this study, we examine the relationship between phytoplankton growth--indicated by chlorophyll a (Chl-a)--and nutrient concentrations, particularly P and N. Through this analysis, we determine the optimal levels of P and N necessary to support healthy phytoplankton growth while preventing harmful algal blooms. Based on our findings, we propose a balanced approach to enhancing the nutritional conditions of enclosed coastal waters without triggering red or blue tides.

Endocrine disrupting chemicals (EDCs) are of particular regulatory and research interest due to the increasing incidence of endocrine-related disorders, such as declining fertility rates and reproductive health problems. The Database of Endocrine Disrupting Chemicals and their Toxicity Profiles (DEDuCT) has gained importance in both academic and regulatory settings by systematically curating data from published literature to characterize these chemicals. Given the growing body of EDC literature, this study aimed to consolidate the latest research and update this critical database. First, more than 14000 research articles were screened through an extensive four-stage manual process, and integrated with the earlier version to create the updated DEDuCTv3.0, comprising 1043 unique EDCs and 796 unique endocrine-related endpoints curated from 3269 published articles. Thereafter, human- and rodent-specific biological endpoint data including interacting genes/proteins, phenotypes, diseases, and adverse outcome pathways (AOPs) were curated from toxicology-relevant databases and systematically integrated with DEDuCTv3.0 to construct a large-scale toxicology knowledge graph for EDCs, termed DEDuCT-KG. DEDuCT-KG was then hosted on a Neo4j database and made easily accessible through a novel interactive user interface. The utility of DEDuCT-KG was demonstrated by exploring potential mechanisms of action associated with obesogenic EDCs within DEDuCTv3.0. Furthermore, the constructed EDC-AOP network, linking 949 EDCs to 381 AOPs within AOP-Wiki, revealed diverse toxicity mechanisms associated with EDCs. Integration with consumer product database and regulatory chemical lists showed that some of these EDCs are present in food contact materials, personal care products, and daily use items, highlighting potential exposure pathways. Overall, all data compiled in this study have been integrated into the DEDuCT webserver, which has been further enhanced to align with FAIR principles. In sum, this study provides a much-needed update to DEDuCT and offers a single point of access to EDC-relevant data to accelerate research and regulation of EDCs.

The chemical obesogen tributyltin (TBT) is known to promote fat storage in adipose tissue through direct action on vertebrate cells. TBT also has direct toxic effects on microorganisms, raising the possibility that TBT may also promote fat storage in vertebrates by altering their microbiota. Here we show that exposure of conventionally-reared post-embryonic zebrafish to TBT results in increased adiposity, reduced body size, and altered intestinal microbiota composition including reduced relative abundance of Plesiomonas bacteria. To test if those microbiota alterations affected host adiposity, we exposed conventionally-reared zebrafish to intestinal bacterial strains representative of TBT-altered taxa. We found that introduction of a Plesiomonas strain into conventionally-reared zebrafish was sufficient to reduce adiposity and alter intestinal microbiota composition. Using new long-term gnotobiotic zebrafish husbandry methods, we found that colonization of germ-free zebrafish with Plesiomonas was sufficient to reduce host adiposity. Together these results show the leptogenic activity of Plesiomonas on zebrafish hosts, indicating that the ability of TBT to increase adiposity in vivo may be due in part to TBT-mediated modification of the abundance of leptogenic bacteria like Plesiomonas. These findings underscore how complex reciprocal interactions between animals and their microbial and chemical environments can influence energy balance and metabolic health. IMPORTANCEObesogens are environmental chemicals that promote fat storage and are generally thought to exert this effect directly on animal cells. Using zebrafish, we show that the obesogen tributyltin can also promote fat storage by acting upon intestinal microbiota via reduction of bacteria that are sufficient to reduce fat storage.

Waterborne viruses pose significant threats to global water safety. Electrochemical (EC) disinfection is considered a promising next-generation technology to address this challenge. However, its efficacy against enveloped viruses remains inadequately under-stood. In this study, we employed bacteriophage Phi6 as a surrogate to systematically eval-uate the inactivation efficiency and damage mechanisms of enveloped viruses in a flow-through EC reactor. Experimental results indicated that Phi6 was more susceptible to EC disinfection than common bacterial surrogates. Comprehensive damage was observed across all major viral components, including structural proteins, the lipid envelope, and the RNA genome. The electrogenerated reactive chlorine species (RCS) were identified as the primary agent responsible for this inactivation, exhibiting higher reactivity than conven-tional free chlorine. Furthermore, radical chlorine species were also confirmed to be pro-duced during EC disinfection, contributing to rapid and extensive viral inactivation. This study demonstrates the feasibility of EC disinfection for inactivating enveloped viruses and provides deeper mechanistic insights into its highly efficient viral inactivation performance. SYNOPSISOur results elucidate the rapid inactivation and thorough damage of the enveloped virus surrogate Phi6 by electrogenerated reactive chlorine species during electrochemical disinfection. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/688411v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@7e8c29org.highwire.dtl.DTLVardef@23fcedorg.highwire.dtl.DTLVardef@b26312org.highwire.dtl.DTLVardef@1864ab1_HPS_FORMAT_FIGEXP M_FIG C_FIG

The identification of xenobiotics in nontargeted metabolomic analyses is a vital step in understanding human exposure. Xenobiotic metabolism, excretion, and co-existence with other endogenous molecules however greatly complicate nontargeted studies. While mass spectrometry (MS)-based platforms are commonly used in metabolomic measurements, deconvoluting endogenous metabolites and xenobiotics is often challenged by the lack of xenobiotic parent and metabolite standards as well as the numerous isomers possible for each small molecule m/z feature. Here, we evaluate the use of ion mobility spectrometry coupled with MS (IMS-MS) and mass defect filtering in a xenobiotic structural annotation workflow to reduce large metabolomic feature lists and uncover potential xenobiotic classes and species detected in the metabolomic studies. To evaluate the workflow, xenobiotics having known high toxicities including per- and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were examined. Initially, to address the lack of available IMS collision cross section (CCS) values for per- and polyfluoroalkyl substances (PFAS), 88 PFAS standards were evaluated with IMS-MS to both develop a targeted PFAS CCS library and for use in machine learning predictions. The CCS values for biomolecules and xenobiotics were then plotted versus m/z, clearly distinguishing the biomolecules and halogenated xenobiotics. The xenobiotic structural annotation workflow was then used to annotate potential PFAS features in NIST human serum. The workflow reduced the 2,423 detected LC-IMS-MS features to 80 possible PFAS with 17 confidently identified through targeted analyses and 48 additional features correlating with possible CompTox entries.

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.

N-(1,3-Dimethylbutyl)-N-Phenyl-P-Phenylenediamine-Quinone (6PPD-Q) is a recently identified contaminant that originates from the oxidation of the tire anti-degradant 6PPD. 6PPD-Q is acutely toxic to select salmonids at environmentally relevant concentrations, while other fish species display tolerance to concentrations surpassing those measured in the environment. The reasons for these marked differences in sensitivity are presently unknown. The objective of this research was to explore potential toxicokinetic drivers of species sensitivity by characterizing biliary metabolites of 6PPD-Q in sensitive and tolerant fishes. For the first time, we identified an O-glucuronide metabolite of 6PPD-Q using high-resolution mass spectrometry. The semi-quantified levels of this metabolite in tolerant species or life stages, including white sturgeon (Acipenser transmontanus), chinook salmon (Oncorhynchus tshawytscha), westslope cutthroat trout (Oncorhynchus clarkia lewisi) and non-fry life stages of Atlantic salmon (Salmo salar), were greater than those in sensitive species, including coho salmon (Oncorhynchus kisutch), brook trout (Salvelinus fontinalis), and rainbow trout (Oncorhynchus mykiss), suggesting that tolerant species might more effectively detoxify 6PPD-Q. Thus, we hypothesize that differences in species sensitivity are a result of differences in basal expression of biotransformation enzyme across various fish species. Moreover, the semi-quantification of 6PPD-Q metabolites in bile extracted from wild-caught fish might be a useful biomarker of exposure to 6PPD-Q, thereby being invaluable to environmental monitoring and risk assessment.

Chlorinated per- and polyfluoroalkyl substances (Cl-PFAS) represent an important subgroup in replacement chemicals for legacy PFAS, and they have been detected in various environments including water bodies, soil, as well as air particles. However, the biodegradability of these chemicals is largely unknown. A recent study reported dechlorination triggered anaerobic biodefluorination of Cl-PFCAs by activated sludge communities. In this study, we further investigated the biodefluorination of Cl-PFCAs by pure cultures. Six of ten selected Cl-PFCAs displayed significant defluorination by Acetobacterium bakii, and corresponding pathways were proposed according to transformation product analysis. Several additional Acetobacterium species were all capable of defluorinating 3,5,7,8-tetrachloro-2,2,3,4,4,5,6,6,7,8,8- undecafluorooctanoic acid (CTFE4) with >50% removal of organic fluorine. On the contrary, Clostridium homopropionicum cannot defluorinate CTFE4. Crude protein extraction experiment showed that the enzymes responsible for CTFE4 dechlorination and defluorination required anaerobic atmosphere to function. Differential gene expression was analyzed according to RNA sequencing analysis, and the results indicated vitamin B12 related enzymes may involve in CTFE4 dechlorination.

Tattoos and permanent make-ups, involving dermal injection of chemicals, are increasingly popular, with 30-40% of young adults in North America and Europe having at least one tattoo. Recent studies highlighted a link between tattooing and health risks, including skin cancers and lymphomas. This study comprehensively catalogs tattoo ink chemicals and investigates their potential adverse effects, addressing the urgent need for greater understanding of tattoo-related health concerns. First, 364 unique tattoo chemicals were identified from various scientific and regulatory sources, with nearly half functioning as pigments. Hazardous chemicals were identified, revealing carcinogens, endocrine disruptors, neurotoxicants, and dermal toxicants. A regulatory analysis based on the European Unions cosmetic regulations, revealed gaps in regulations of such hazardous chemicals. Curated chemical-disease associations highlighted that some tattoo chemicals are known to cause dermatitis. Further, diverse toxicological information, including experimental results from REACH dossiers, were integrated to construct stressor-AOP network linking 151 chemicals to 362 AOPs, revealing potential carcinogenic mechanisms associated with tattoo ink chemicals. A systems biology approach revealed potential immunomodulatory effects associated with these chemicals. Finally, all findings have been made available through the online database Tattoo Ink Chemicals and associated Toxicities Knowledgebase (TICToK; https://cb.imsc.res.in/tictok), which can support risk assessment and sustainable tattoo practices. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/668261v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@11c06f1org.highwire.dtl.DTLVardef@10bc82borg.highwire.dtl.DTLVardef@82b4eeorg.highwire.dtl.DTLVardef@1347792_HPS_FORMAT_FIGEXP M_FIG C_FIG

In low- and middle-income countries (LMICs), enteric infections pose a significant threat to childrens health. However, understanding the specifics of when, where, and how young children in LMICs are exposed to enteric pathogens and the roles of animal reservoirs, environmental media, and human hosts play during exposure remains limited. This study systematically evaluates infants exposure to E. coli through various pathways in the rural Haramaya woreda of Ethiopia. Between June 2021 and June 2022, we conducted over 1300 hours of structured behavioral observations on 79 infants when aged 4-8 months (Timepoint 1) and 11-15 months (Timepoint 2). Enumerators recorded the infants behavior related to exposure, including when and where it occurred, using the Countee (C) application. Concurrently, we collected 1338 environmental samples from key contact interfaces between infants, other people, and the environment to test for E. coli. We used a competing hazard model for duration-based behaviors, an inhomogeneous Poisson point process model for frequency-based behaviors, and a left-censored lognormal distribution model for E. coli contamination levels. The behavioral and environmental information was then integrated into an agent-based exposure model framework to quantify the exposure to E. coli through different pathways. The infant behavior, which altered the relative importance of different exposure pathways, changed as children grew older. Notably, we observed increased rates of touching behavior (e.g., touching fomites) and soil-pica, increased consumption of solid food, and more time spent on the bare ground at Timepoint 2. The major sources of exposure to E. coli were food and breastfeeding at Timepoint 1 and food and soil at Timepoint 2. This study provides insights for interventions to minimize infants risk of exposure to fecal contamination and subsequent risk of enteric infections, including improved food handling practices, enhanced personal hygiene for breastfeeding caregivers, and education on the risk of soil-pica.

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants with documented toxic effects, yet their multi- and transgenerational impacts on neurodevelopment and underlying mechanisms remain poorly understood. Here, we present a comprehensive study delineating the effects of developmental exposure to environmentally relevant concentrations of PFOS and PFBS on behavior, transcriptome, and genome-wide DNA methylation patterns in the directly exposed generation (F0) and their unexposed offspring (F1 and F2) in zebrafish. Both PFOS and PFBS altered larval behavior, linked to transcriptomic and DNA methylation changes in neuro-related pathways, even in the unexposed offspring. Importantly, specific DNA methylation changes in F0 were associated with behavioral outcomes in F2 animals, suggesting that these alterations could underlie transgenerational effects. Pathways associated with differentially methylated genes were prominently enriched for response to light and circadian regulation. Our findings demonstrate that developmental exposure to PFAS causes transgenerational behavioral effects in zebrafish and suggest that epigenetic changes induced by direct exposure may serve as markers for predicting outcomes in subsequent, unexposed generations. TEASERPFAS induce circadian-related epigenetic changes in zebrafish associated with behavioral impacts in unexposed offspring.

Combined water, sanitation, and handwashing (WSH) interventions have the potential to reduce fecal pathogens along more transmission pathways than single interventions alone. We measured Escherichia coli levels in 3909 drinking water samples, 2691 child hand rinses, and 2422 toy ball rinses collected from households enrolled in a two-year cluster-randomized controlled trial evaluating single and combined WSH interventions. Water treatment alone reduced E. coli in drinking water, while a combined WSH intervention improved water quality by the same magnitude but did not affect levels of fecal indicator bacteria on child hands or toy balls. The failure of the WSH interventions to reduce E. coli along important child exposure pathways is consistent with the lack of a protective effect from the interventions on child diarrhea or child growth during the trial. Our results have important implications for WSH program design; the sanitation and handwashing interventions implemented in this trial should not be expected to reduce child exposure to fecal contamination in other similar settings.

Wastewater sequencing is an increasingly valuable tool in tracking the spread of infectious disease agents across space and time in areas of dense human settlement. Among pathogens that can be readily detected by this approach is influenza A, which follows predictable patterns of prevalence through the winter months in North America. Here, we leverage routine surveillance of a municipal wastewater treatment plant in Northern California to describe an atypical, off-season spike in influenza A concentrations that rivals that of the winter respiratory virus season. Drawing upon metagenomic data generated through hybrid-capture sequencing, we assemble and subsequently characterize fragments of divergent influenza genomes that appear to derive predominantly from the avian H16 clade. These strains exhibit close evolutionary relationships to influenza isolated from migratory shorebirds, hinting at potential host species and mechanisms of geographic spread. Analysis of read abundances suggest that these avian strains dominate the pool of influenza circulating during the summer months, when typical human-infecting strains are essentially absent. Together, our results expand the value of wastewater sequencing to encompass sensitive tracking of outbreaks within animals in interface regions where human settlement abuts wildlands, increasing overall pandemic preparedness.