Bioconcentration of Per- and Polyfluoroalkyl Substances and Precursors in Fathead Minnow Tissues Environmentally Exposed to Aqueous Film-Forming Foam-Contaminated Waters.

Exposure to per- and polyfluoroalkyl substances (PFAS) has been associated with toxicity in wildlife and negative health effects in humans. Decades of fire training activity at Joint Base Cape Cod (MA, USA) incorporated the use of aqueous film-forming foam (AFFF), which resulted in long-term PFAS contamination of sediments, groundwater, and hydrologically connected surface waters. To explore the bioconcentration potential of PFAS in complex environmental mixtures, a mobile laboratory was established to evaluate the bioconcentration of PFAS from AFFF-impacted groundwater by flow-through design. Fathead minnows (n = 24) were exposed to PFAS in groundwater over a 21-day period and tissue-specific PFAS burdens in liver, kidney, and gonad were derived at three different time points. The ∑PFAS concentrations in groundwater increased from approximately 10,000 ng/L at day 1 to 36,000 ng/L at day 21. The relative abundance of PFAS in liver, kidney, and gonad shifted temporally from majority perfluoroalkyl sulfonamides (FASAs) to perfluoroalkyl sulfonates (PFSAs). By day 21, mean ∑PFAS concentrations in tissues displayed a predominance in the order of liver > kidney > gonad. Generally, bioconcentration factors (BCFs) for FASAs, perfluoroalkyl carboxylates (PFCAs), and fluorotelomer sulfonates (FTS) increased with degree of fluorinated carbon chain length, but this was not evident for PFSAs. Perfluorooctane sulfonamide (FOSA) displayed the highest mean BCF (8700 L/kg) in day 21 kidney. Suspect screening results revealed the presence of several perfluoroalkyl sulfinate and FASA compounds present in groundwater and in liver for which pseudo-bioconcentration factors are also reported. The bioconcentration observed for precursor compounds and PFSA derivatives detected suggests alternative pathways for terminal PFAS exposure in aquatic wildlife and humans. Environ Toxicol Chem 2024;43:1795-1806. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Considerations and challenges in support of science and communication of fish consumption advisories for per- and polyfluoroalkyl substances.

Federal, state, tribal, or local entities in the United States issue fish consumption advisories (FCAs) as guidance for safer consumption of locally caught fish containing contaminants. Fish consumption advisories have been developed for commonly detected compounds such as mercury and polychlorinated biphenyls. The existing national guidance does not specifically address the unique challenges associated with bioaccumulation and consumption risk related to per- and polyfluoroalkyl substances (PFAS). As a result, several states have derived their own PFAS-related consumption guidelines, many of which focus on one frequently detected PFAS, known as perfluorooctane sulfonic acid (PFOS). However, there can be significant variation between tissue concentrations or trigger concentrations (TCs) of PFOS that support the individual state-issued FCAs. This variation in TCs can create challenges for risk assessors and risk communicators in their efforts to protect public health. The objective of this article is to review existing challenges, knowledge gaps, and needs related to issuing PFAS-related FCAs and to provide key considerations for the development of protective fish consumption guidance. The current state of the science and variability in FCA derivation, considerations for sampling and analytical methodologies, risk management, risk communication, and policy challenges are discussed. How to best address PFAS mixtures in the development of FCAs, in risk assessment, and establishment of effect thresholds remains a major challenge, as well as a source of uncertainty and scrutiny. This includes developments better elucidating toxicity factors, exposures to PFAS mixtures, community fish consumption behaviors, and evolving technology and analytical instrumentation, methods, and the associated detection limits. Given the evolving science and public interests informing PFAS-related FCAs, continued review and revision of FCA approaches and best practices are vital. Nonetheless, consistent, widely applicable, PFAS-specific approaches informing methods, critical concentration thresholds, and priority compounds may assist practitioners in PFAS-related FCA development and possibly reduce variability between states and jurisdictions. Integr Environ Assess Manag 2024;00:1-20. © 2024 SETAC.

Bioaccumulation of Perfluoroalkyl Sulfonamides (FASA).

Hundreds of sites across the United States have high concentrations of perfluoroalkyl sulfonamides (FASA), but little is known about their propensity to accumulate in fish. FASA are precursors to terminal per- and polyfluoroalkyl substances (PFAS) that are abundant in diverse consumer products and aqueous film-forming foams manufactured using electrochemical fluorination (ECF AFFF). In this study, FASA with C3-C8 carbon chain lengths were detected in all fish samples from surface waters up to 8 km downstream of source zones with ECF AFFF contamination. Short-chain FASA ≤ C6 have rarely been included in routine screening for PFAS, but availability of new standards makes such analyses more feasible. Bioaccumulation factors (BAF) for FASA were between 1 and 3 orders of magnitude greater than their terminal perfluoroalkyl sulfonates. Across fish species, BAF for FASA were greater than for perfluorooctanesulfonate (PFOS), which is presently the focus of national advisory programs. Similar concentrations of the C6 FASA (<0.36-175 ng g-1) and PFOS (0.65-222 ng g-1) were detected in all fish species. No safety thresholds have been established for FASA. However, high concentrations in fish next to contaminated sites and preliminary findings on toxicity suggest an urgent need for consideration by fish advisory programs.

Binding of Per- and Polyfluoroalkyl Substances (PFAS) to Serum Proteins: Implications for Toxicokinetics in Humans.

Per- and polyfluoroalkyl substances (PFAS) are a diverse class of highly persistent anthropogenic chemicals that are detectable in the serum of most humans. PFAS exposure has been associated with many adverse effects on human health including immunotoxicity, increased risk of certain cancers, and metabolic disruption. PFAS binding to the most abundant blood serum proteins (human serum albumin [HSA] and globulins) is thought to affect transport to active sites, toxicity, and elimination half-lives. However, few studies have investigated the competitive binding of PFAS to these proteins in human serum. Here, we use C18 solid-phase microextraction fibers to measure HSA-water and globulin-water distribution coefficients (DHSA/w, Dglob/w) for PFAS with carbon chains containing 4 to 13 perfluorinated carbons (ηpfc = 4-13) and several functional head-groups. PFAS with ηpfc < 7 were highly bound to HSA relative to globulins, whereas PFAS with ηpfc ≥ 7 showed a greater propensity for binding to globulins. Experimentally measured DHSA/w and Dglob/w and concentrations of serum proteins successfully predicted the variability in PFAS binding in human serum. We estimated that the unbound fraction of serum PFAS varied by up to a factor of 2.5 among individuals participating in the 2017-2018 U.S. National Health and Nutrition Examination Survey. These results suggest that serum HSA and globulins are important covariates for epidemiological studies aimed at understanding the effects of PFAS exposure.

Unregulated Active and Closed Textile Mills Represent a Significant Vector of PFAS Contamination into Coastal Rivers.

Despite concerns over the ubiquity of per- and polyfluoroalkyl substances (PFAS), little is known about the diversity of input sources to surface waters and their seasonal dynamics. Frequent use of PFAS in textiles means both active and closed textile mills require evaluation as PFAS sources. We deployed passive samplers at seven sites in an urban river and estuary adjacent to textile mills in Southern Rhode Island (USA) over 12 months. We estimated monthly mass flows (g month-1) of perfluorohexanoic acid (PFHxA: 45±56), and perfluorooctanoic acid (PFOA: 30±45) from the upstream river influenced by an active mill. Average mass flows were 73-155% higher downstream, where historical textile waste lagoons contributed long chain perfluoroalkyl acids (PFAA). Mass flows of PFNA increased from 7.5 to 21 g month-1 between the upstream and downstream portions of the rivers. Distinct grouping of the two main PFAS sources, active textile mills and historical waste lagoons, were identified using principal components analysis. Neither suspect screening nor extractable organofluorine analysis revealed measurable PFAS were missing beyond the targeted compounds. This research demonstrates that both closed and active textile mills are important ongoing PFAS sources to freshwater and marine regions and should be further evaluated as a source category.

Interlaboratory Comparison of Extractable Organofluorine Measurements in Groundwater and Eel (Anguilla rostrata): Recommendations for Methods Standardization.

Research on per- and polyfluoroalkyl substances (PFAS) frequently incorporates organofluorine measurements, particularly because they could support a class-based approach to regulation. However, standardized methods for organofluorine analysis in a broad suite of matrices are currently unavailable, including a method for extractable organofluorine (EOF) measured using combustion ion chromatography (CIC). Here, we report the results of an international interlaboratory comparison. Seven laboratories representing academia, government, and the private sector measured paired EOF and PFAS concentrations in groundwater and eel (Anguilla rostrata) from a site contaminated by aqueous film-forming foam. Among all laboratories, targeted PFAS could not explain all EOF in groundwater but accounted for most EOF in eel. EOF results from all laboratories for at least one replicate extract fell within one standard deviation of the interlaboratory mean for groundwater and five out of seven laboratories for eel. PFAS spike mixture recoveries for EOF measurements in groundwater and eel were close to the criterion (±30%) for standardized targeted PFAS methods. Instrumental operation of the CIC such as replicate sample injections was a major source of measurement uncertainty. Blank contamination and incomplete inorganic fluorine removal may introduce additional uncertainties. To elucidate the presence of unknown organofluorine using paired EOF and PFAS measurements, we recommend that analysts carefully consider confounding methodological uncertainties such as differences in precision between measurements, data processing steps such as blank subtraction and replicate analyses, and the relative recoveries of PFAS and other fluorine compounds.

Uptake of Per- and Polyfluoroalkyl Substances by Fish, Mussel, and Passive Samplers in Mobile-Laboratory Exposures Using Groundwater from a Contamination Plume at a Historical Fire Training Area, Cape Cod, Massachusetts.

Aqueous film-forming foams historically were used during fire training activities on Joint Base Cape Cod, Massachusetts, and created an extensive per- and polyfluoroalkyl substances (PFAS) groundwater contamination plume. The potential for PFAS bioconcentration from exposure to the contaminated groundwater, which discharges to surface water bodies, was assessed with mobile-laboratory experiments using groundwater from the contamination plume and a nearby reference location. The on-site continuous-flow 21-day exposures used male and female fathead minnows, freshwater mussels, polar organic chemical integrative samplers (POCIS), and polyethylene tube samplers (PETS) to evaluate biotic and abiotic uptake. The composition of the PFAS-contaminated groundwater was complex and 9 PFAS were detected in the reference groundwater and 17 PFAS were detected in the contaminated groundwater. The summed PFAS concentrations ranged from 120 to 140 ng L-1 in reference groundwater and 6100 to 15,000 ng L-1 in contaminated groundwater. Biotic concentration factors (CFb) for individual PFAS were species, sex, source, and compound-specific and ranged from 2.9 to 1000 L kg-1 in whole-body male fish exposed to contaminated groundwater for 21 days. The fish and mussel CFb generally increased with increasing fluorocarbon chain length and were greater for sulfonates than for carboxylates. The exception was perfluorohexane sulfonate, which deviated from the linear trend and had a 10-fold difference in CFb between sites, possibly because of biotransformation of precursors such as perfluorohexane sulfonamide. Uptake for most PFAS in male fish was linear over time, whereas female fish had bilinear uptake indicated by an initial increase in tissue concentrations followed by a decrease. Uptake of PFAS was less for mussels (maximum CFb = 200) than for fish, and mussel uptake of most PFAS also was bilinear. Although abiotic concentration factors were greater than CFb, and values for POCIS were greater than for PETS, passive samplers were useful for assessing PFAS that potentially bioconcentrate in fish but are present at concentrations below method quantitation limits in water. Passive samplers also accumulate short-chain PFAS that are not bioconcentrated.

Nitrifying Microorganisms Linked to Biotransformation of Perfluoroalkyl Sulfonamido Precursors from Legacy Aqueous Film-Forming Foams.

Drinking water supplies across the United States have been contaminated by firefighting and fire-training activities that use aqueous film-forming foams (AFFF) containing per- and polyfluoroalkyl substances (PFAS). Much of the AFFF is manufactured using electrochemical fluorination by 3M. Precursors with six perfluorinated carbons (C6) and non-fluorinated amine substituents make up approximately one-third of the PFAS in 3M AFFF. C6 precursors can be transformed through nitrification (microbial oxidation) of amine moieties into perfluorohexane sulfonate (PFHxS), a compound of regulatory concern. Here, we report biotransformation of the most abundant C6 sulfonamido precursors in 3M AFFF with available commercial standards (FHxSA, PFHxSAm, and PFHxSAmS) in microcosms representative of the groundwater/surface water boundary. Results show rapid (<1 day) biosorption to living cells by precursors but slow biotransformation into PFHxS (1-100 pM day-1). The transformation pathway includes one or two nitrification steps and is supported by the detection of key intermediates using high-resolution mass spectrometry. Increasing nitrate concentrations and total abundance of nitrifying taxa occur in parallel with precursor biotransformation. Together, these data provide multiple lines of evidence supporting microbially limited biotransformation of C6 sulfonamido precursors involving ammonia-oxidizing archaea (Nitrososphaeria) and nitrite-oxidizing bacteria (Nitrospina). Further elucidation of interrelationships between precursor biotransformation and nitrogen cycling in ecosystems would help inform site remediation efforts.

Organic Fluorine as an Indicator of Per- and Polyfluoroalkyl Substances in Dust from Buildings with Healthier versus Conventional Materials.

Per- and polyfluoroalkyl substances (PFAS) are a class of thousands of persistent, organic fluorinated chemicals added to materials and products mainly to repel stains and water. PFAS have been associated with many adverse human health effects. We aimed to determine whether buildings with "healthier" materials─defined here as reportedly free of all PFAS─exhibit lower PFAS in dust. In addition to analyzing targeted PFAS with available commercial standards, we measured extractable organic fluorine (EOF) as a novel proxy that includes both known and unknown types of PFAS. We measured at least 15 targeted PFAS (n = 24), EOF (n = 24), and total fluorine (TF; n = 14) in dust collected from university common spaces and classrooms, half of which had "healthier" furniture and carpet. We observed lower PFAS contamination in buildings with "healthier" materials: "healthier" rooms had a 66% lower median summed PFAS and a 49% lower Kaplan-Meier estimated mean EOF level in dust in comparison to conventional rooms. The summed targeted PFAS were significantly correlated with EOF but accounted for up to only 9% of EOF, indicating the likely presence of unidentified PFAS. EOF levels explained less than 1% of TF in dust. We emphasize the need to use chemical class-based methods (e.g., EOF) for evaluating class-based solutions and to expand non-PFAS solutions for other building materials.

PFAS and Precursor Bioaccumulation in Freshwater Recreational Fish: Implications for Fish Advisories.

Per- and polyfluoroalkyl substances (PFAS) are a diverse class of fluorinated anthropogenic chemicals that include perfluoroalkyl acids (PFAA), which are widely used in modern commerce. Many products and environmental samples contain abundant precursors that can degrade into terminal PFAA associated with adverse health effects. Fish consumption is an important dietary exposure source for PFAS that bioaccumulate in food webs. However, little is known about bioaccumulation of PFAA precursors. Here, we identify and quantify PFAS in recreational fish species collected from surface waters across New Hampshire, US, using a toolbox of analytical methods. Targeted analysis of paired water and tissue samples suggests that many precursors below detection in water have a higher bioaccumulation potential than their terminal PFAA. Perfluorobutane sulfonamide (FBSA), a short-chain precursor produced by electrochemical fluorination, was detected in all fish samples analyzed for this compound. The total oxidizable precursor assay interpreted using Bayesian inference revealed fish muscle tissue contained additional, short-chain precursors in high concentration samples. Suspect screening analysis indicated these were perfluoroalkyl sulfonamide precursors with three and five perfluorinated carbons. Fish consumption advisories are primarily being developed for perfluorooctane sulfonate (PFOS), but this work reinforces the need for risk evaluations to consider additional bioaccumulative PFAS, including perfluoroalkyl sulfonamide precursors.

Urban proximity while breeding is not a predictor of perfluoroalkyl substance contamination in the eggs of brown pelicans.

Identifying sources of exposure to chemical stressors is difficult when both target organisms and stressors are highly mobile. While previous studies have demonstrated that populations of some organisms proximal to urban centers may display increased burdens of human-created chemicals compared to more distal populations, this relationship may not be universal when applied to organisms and stressors capable of transboundary movements. We examined eggs of brown pelicans (Pelecanus occidentalis), a nearshore seabird with daily movements ranging from local to 50 km and annual migrations ranging from year-round residency to 1500 km. Thirty-six eggs from three breeding colonies located at increasing distances to a major urban center (Charleston, South Carolina, USA) were analyzed for concentrations of per- and polyfluoroalkyl substances (PFAS). Areas of high use for each colony during the breeding season were also assessed via the tracking of adult pelicans from each colony using GPS-PTT satellite transmitters and overlapped with measures of relative urbanization via land cover data. We report potentially significant ∑PFAS concentrations in the eggs of pelicans (175.4 ± 120.1 ng/g w wt. SD), driven largely by linear perfluorooctane sulfonate (n-PFOS) (48-546 ng/g w wt.). Residues of the precursor compound perfluorooctane sulfonamide (FOSA) were also present in pelican eggs, suggesting continued exposure of local wildlife beyond implemented phaseouts of some PFAS. For most analytes, egg concentrations did not exhibit a significant spatial structure despite some differentiation in high-use areas unlike similar data for another regional apex predator, the bottlenose dolphin (Tursiops truncatus). We suggest that the partially migratory nature of brown pelicans during the non-breeding season, combined with daily ranges that may extend to 50 km from local point sources, may have homogenized exposure across individuals. Charleston likely remains a major source for PFAS in the overall region, however, given the high concentrations observed as well as known releases of PFAS in the nearshore environment.

Surface-water/groundwater boundaries affect seasonal PFAS concentrations and PFAA precursor transformations.

Elevated concentrations of per- and polyfluoroalkyl substances (PFAS) in drinking-water supplies are a major concern for human health. It is therefore essential to understand factors that affect PFAS concentrations in surface water and groundwater and the transformation of perfluoroalkyl acid (PFAA) precursors that degrade into terminal compounds. Surface-water/groundwater exchange can occur along the flow path downgradient from PFAS point sources and biogeochemical conditions can change rapidly at these exchange boundaries. Here, we investigate the influence of surface-water/groundwater boundaries on PFAS transport and transformation. To do this, we conducted an extensive field-based analysis of PFAS concentrations in water and sediment from a flow-through lake fed by contaminated groundwater and its downgradient surface-water/groundwater boundary (defined as ≤100 cm below the lake bottom). PFAA precursors comprised 45 ± 4.6% of PFAS (PFAA precursors + 18 targeted PFAA) in the predominantly oxic lake impacted by a former fire-training area and historical wastewater discharges. In shallow porewater downgradient from the lake, this percentage decreased significantly to 25 ± 11%. PFAA precursor concentrations decreased by 85% between the lake and 84-100 cm below the lake bottom. PFAA concentrations increased significantly within the surface-water/groundwater boundary and in downgradient groundwater during the winter months despite lower stable concentrations in the lake water source. These results suggest that natural biogeochemical fluctuations associated with surface-water/groundwater boundaries may lead to PFAA precursor loss and seasonal variations in PFAA concentrations. Results of this work highlight the importance of dynamic biogeochemical conditions along the hydrological flow path from PFAS point sources to potentially affected drinking water supplies.

Isolating the AFFF Signature in Coastal Watersheds Using Oxidizable PFAS Precursors and Unexplained Organofluorine.

Water supplies for millions of U.S. individuals exceed maximum contaminant levels for per- and polyfluoroalkyl substances (PFAS). Contemporary and legacy use of aqueous film forming foams (AFFF) is a major contamination source. However, diverse PFAS sources are present within watersheds, making it difficult to isolate their predominant origins. Here we examine PFAS source signatures among six adjacent coastal watersheds on Cape Cod, MA, U.S.A. using multivariate clustering techniques. A distinct signature of AFFF contamination enriched in precursors with six perfluorinated carbons (C6) was identified in watersheds with an AFFF source, while others were enriched in C4 precursors. Principal component analysis of PFAS composition in impacted watersheds showed a decline in precursor composition relative to AFFF stocks and a corresponding increase in terminal perfluoroalkyl sulfonates with < C6 but not those with ≥ C6. Prior work shows that in AFFF stocks, all extractable organofluorine (EOF) can be explained by targeted PFAS and precursors inferred using Bayesian inference on the total oxidizable precursor assay. Using the same techniques for the first time in impacted watersheds, we find that only 24%-63% of the EOF can be explained by targeted PFAS and oxidizable precursors. Our work thus indicates the presence of large non-AFFF organofluorine sources in these coastal watersheds.

Per- and polyfluoroalkyl substances (PFAS) and total fluorine in fire station dust.

Per- and polyfluoroalkyl substances (PFAS) are a class of over 4700 fluorinated compounds used in industry and consumer products. Studies have highlighted the use of aqueous film-forming foams (AFFFs) as an exposure source for firefighters, but little is known about PFAS occurrence inside fire stations, where firefighters spend most of their shifts. In this study, we aimed to characterize PFAS concentrations and sources inside fire stations. We measured 24 PFAS (using LC-MS/MS) and total fluorine (using particle-induced gamma ray emission) in dust from multiple rooms of 15 Massachusetts stations, many of which (60%) no longer use PFAS-containing AFFF at all and the rest of which only use it very rarely. Compared to station living rooms, turnout gear locker rooms had higher dust levels of total fluorine (p < 0.0001) and three PFAS: perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), and perfluorodecanoate (PFDoDA) (p < 0.05). These PFAS were also found on six wipes of station turnout gear. By contrast, the dominant PFAS in living rooms was N-ethyl perfluorooctane sulfonamidoacetic acid (N-MeFOSAA), a precursor to perfluorooctane sulfonate (PFOS) that still persists despite phase-outs almost two decades ago. The Σ24 PFAS accounted for less than 2% of fluorine in dust (n = 39), suggesting the potential presence of unknown PFAS. Turnout gear may be an important PFAS source in stations due to intentional additives and/or contamination from firefighting activities.

How Do We Measure Poly- and Perfluoroalkyl Substances (PFASs) at the Surface of Consumer Products?: Environmental Science and Technology LETTERS.

Exposures to poly- and perfluoroalkyl substances (PFASs) have been linked to metabolic disruption, immunotoxicity and cancer in humans. PFASs are known to be present in diverse consumer products including textiles and food packaging. Here we present a new method for quantifying the atomic percent fluorine (% F) in the surficial 0.01 µm of consumer products using X-ray photoelectron spectroscopy (XPS). The surface of food contact materials and textiles measured in this study contained up to 28% F and 45% F, respectively. PTFE tape was measured to demonstrate XPS accuracy and precision. Depth profiles of fluorine content in consumer products measured using XPS showed highest levels at the upper-most surface in contact with the surrounding environment and a decrease below the surface. PFASs released in methanol extracts and quantified using traditional liquid chromatography-tandem mass spectrometry typically accounted for <1% of the fluorine measured with XPS in consumer products. We conclude that XPS is a useful technique for characterizing PFASs in consumer products because it can precisely quantify the surficial fluorine content of materials. XPS also allows identification of CF2 and CF3 groups in materials and can elucidate the depth dependent distribution of fluorine in products.

Phospholipid Levels Predict the Tissue Distribution of Poly- and Perfluoroalkyl Substances in a Marine Mammal.

Exposure to poly- and perfluoroalkyl substances (PFASs) has been linked to many negative health impacts in humans and wildlife. Unlike neutral hydrophobic organic pollutants, many PFASs are ionic and have been hypothesized to accumulate in both phospholipids and protein-rich tissues. Here we investigate the role of phospholipids for PFAS accumulation by analyzing associations among concurrent measurements of phospholipid, total protein, total lipid and 24 PFASs in the heart, muscle, brain, kidney, liver, blubber, placenta and spleen of North Atlantic pilot whales (Globicephala melas). The sum of 24 PFASs ( ∑ 24 PFAS ) was highest in the liver (median 260 ng g-1; interquartile range (IQR) 216-295 ng g-1) and brain (86.0; IQR 54.5-91.3 ng g-1), while phospholipid levels were highest in brain. The relative abundance of PFASs in the brain greatly increases with carbon chain lengths of 10 or greater, suggesting shorter-chained compounds may cross the blood-brain barrier less efficiently. Phospholipids were significant predictors of the tissue distribution of the longest-chained PFASs: perfluorodecanesulfonate (PFDS), perfluorododecanoate (PFDoA), perfluorotridecanoate (PFTrA), and perfluorotetradecanoic acid (PFTA) (rs = 0.5-0.6). In all tissues except the brain, each 1 mg g-1 increase in phospholipids led to a 12%-25% increase in the concentration of each PFAS. We conclude that partitioning to phospholipids is an important mechanism of bioaccumulation for long-chained PFASs in marine mammals.