Per- and polyfluoroalkyl ether acids in well water and blood serum from private well users residing by a fluorochemical facility near Fayetteville, North Carolina.

BACKGROUND: A fluorochemical facility near Fayetteville, North Carolina, emitted per- and polyfluoroalkyl ether acids (PFEAs), a subgroup of per- and polyfluoroalkyl substances (PFAS), to air. OBJECTIVE: Analyze PFAS in private wells near the facility and in blood from well users to assess relationships between PFEA levels in water and serum. METHODS: In 2019, we recruited private well users into the GenX Exposure Study and collected well water and blood samples. We targeted 26 PFAS (11 PFEAs) in water and 27 PFAS (9 PFEAs) in serum using liquid chromatography-mass spectrometry. We used regression modeling to explore relationships between water and serum PFAS. For the only PFEA detected frequently in water and serum, Nafion byproduct 2, we used generalized estimating equation (GEE) models to assess well water exposure metrics and then adjusted for covariates that may influence Nafion byproduct 2 serum concentrations. RESULTS: We enrolled 153 participants ages 6 and older (median = 56 years) using 84 private wells. Most wells (74%) had ≥6 detectable PFEAs; median ∑PFEAs was 842 ng/L (interquartile range = 197-1760 ng/L). Low molecular weight PFEAs (PMPA, HFPO-DA [GenX], PEPA, PFO2HxA) were frequently detected in well water, had the highest median concentrations, but were not detectable in serum. Nafion byproduct 2 was detected in 73% of wells (median = 14 ng/L) and 56% of serum samples (median = 0.2 ng/mL). Cumulative dose (well concentration × duration at address) was positively associated with Nafion byproduct 2 serum levels and explained the most variability (10%). In the adjusted model, cumulative dose was associated with higher Nafion byproduct 2 serum levels while time outside the home was associated with lower levels. IMPACT: PFAS are a large class of synthetic, fluorinated chemicals. Fluorochemical facilities are important sources of environmental PFAS contamination globally. The fluorochemical industry is producing derivatives of perfluoroalkyl acids, including per- and polyfluoroalkyl ether acids (PFEAs). PFEAs have been detected in various environmental samples but information on PFEA-exposed populations is limited. While serum biomonitoring is often used for PFAS exposure assessment, serum biomarkers were not good measures of long-term exposure to low molecular weight PFEAs in a private well community. Environmental measurements and other approaches besides serum monitoring will be needed to better characterize PFEA exposure.

Estimation of the Half-Lives of Recently Detected Per- and Polyfluorinated Alkyl Ethers in an Exposed Community.

To estimate half-lives for novel fluoroethers, the GenX Exposure Study obtained two serum measurements for per- and polyfluoroalkyl substances (PFAS) for 44 participants of age 12-86 years from North Carolina, collected 5 and 11 months after fluoroether discharges into the drinking water source were controlled. The estimated half-lives for these compounds were 127 days (95% confidence interval (95% CI) = 86, 243 days) for perfluorotetraoxadecanoic acid (PFO4DA), 296 days for Nafion byproduct 2 (95% CI = 176, 924 days), and 379 days (95% CI = 199, 3870 days) for perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoA). Using these estimates and the literature values, a model was built that predicted PFAS half-lives using structural properties. Three chemical properties predicted 55% of the variance of PFAS half-lives based on 15 PFAS. A model with only molecular weight predicted 69% of the variance. Some properties can predict the half-lives of PFAS, but a deeper understanding is needed. These fluoroethers had biological half-lives longer than published half-lives for PFHxA and PFHpA (30-60 days) but shorter than those for PFOA and PFOS (800-1200 days). These are the first and possibly only estimates of human elimination half-lives of these fluoroethers.

Practical application guide for the discovery of novel PFAS in environmental samples using high resolution mass spectrometry.

BACKGROUND: The intersection of the topics of high-resolution mass spectrometry (HRMS) and per- and polyfluoroalkyl substances (PFAS) bring together two disparate and complex subjects. Recently non-targeted analysis (NTA) for the discovery of novel PFAS in environmental and biological media has been shown to be valuable in multiple applications. Classical targeted analysis for PFAS using LC-MS/MS, though growing in compound coverage, is still unable to inform a holistic understanding of the PFAS burden in most samples. NTA fills at least a portion of this data gap. OBJECTIVES: Entrance into the study of novel PFAS discovery requires identification techniques such as HRMS (e.g., QTOF and Orbitrap) instrumentation. This requires practical knowledge of best approaches depending on the purpose of the analyses. The utility of HRMS applications for PFAS discovery is unquestioned and will likely play a significant role in many future environmental and human exposure studies. METHODS/RESULTS: PFAS have some characteristics that make them standout from most other chemicals present in samples. Through a series of tell-tale PFAS characteristics (e.g., characteristic mass defect range, homologous series and characteristic fragmentation patterns), and case studies different approaches and remaining challenges are demonstrated. IMPACT STATEMENT: The identification of novel PFAS via non-targeted analysis using high resolution mass spectrometry is an important and difficult endeavor. This synopsis document will hopefully make current and future efforts on this topic easier to perform for novice and experienced alike. The typical time devoted to NTA PFAS investigations (weeks to months or more) may benefit from these practical steps employed.

Analytical method interferences for perfluoropentanoic acid (PFPeA) and perfluorobutanoic acid (PFBA) in biological and environmental samples.

While high-resolution MS (HRMS) can be used for identification and quantification of novel per- and polyfluorinated alkyl substances (PFAS), low-resolution MS/MS is the more commonly used and affordable approach for routine PFAS monitoring. Of note, perfluoropentanoic acid (PFPeA) and perfluorobutanoic acid (PFBA), two of the smaller carboxylic acid containing-PFAS, have only one major MS/MS transition, preventing the use of qualitative transitions for verification on low-resolution instrumentation. Recently our lab has observed widespread chemical interference in the quantitative ion channel for PFPeA (263 â†’ 219) and PFBA (213 â†’ 169) in numerous matrices. PFPeA interference was investigated using HRMS and putatively assigned as a diprotic unsaturated fatty acid (263.1288 Da) in shellfish and a separate interferent (13C isotope of 262.1087 Da) in hot cocoa, which had been previously described by the FDA. PFBA interference caused by saturated oxo-fatty acids, previously demonstrated in tissue, was also observed in liquid condensate from a residential air conditioning unit. Therefore, in support of PFAS analysis on low-resolution instrumentation, authors recommend several adjustments to analytical methods including altering liquid chromatography (LC) conditions as well as using matched internal standards to investigate and expressly confirm PFBA and PFPeA detections in both biological and environmental samples.

Cumulative maternal and neonatal effects of combined exposure to a mixture of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) during pregnancy in the Sprague-Dawley rat.

Globally, biomonitoring data demonstrate virtually all humans carry residues of multiple per- and polyfluoroalkyl substances (PFAS). Despite pervasive co-exposure, limited mixtures-based in vivo PFAS toxicity research has been conducted. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are commonly detected PFAS in human and environmental samples and both produce adverse effects in laboratory animal studies, including maternal and offspring effects when orally administered during pregnancy and lactation. To evaluate the effects of combined exposure to PFOA and PFOS, we orally exposed pregnant Sprague-Dawley rats from gestation day 8 (GD8) to postnatal day 2 (PND2) to PFOA (10-250 mg/kg/d) or PFOS (0.1-5 mg/kg/d) individually to characterize effects and dose response curve parameters, followed by a variable-ratio mixture experiment with a constant dose of PFOS (2 mg/kg/d) mixed with increasing doses of PFOA (3-80 mg/kg/d). The mixture study design was intended to: 1) shift the PFOA dose response curves for endpoints shared with PFOS, 2) allow comparison of dose addition (DA) and response addition (RA) model predictions, 3) conduct relative potency factor (RPF) analysis for multiple endpoints, and 4) avoid overt maternal toxicity. Maternal serum and liver concentrations of PFOA and PFOS were consistent between the individual chemical and mixture experiments. Combined exposure with PFOS significantly shifted the PFOA dose response curves towards effects at lower doses compared to PFOA-only exposure for multiple endpoints and these effects were well predicted by dose addition. For endpoints amenable to mixture model analyses, DA produced equivalent or better estimates of observed data than RA. All endpoints evaluated were accurately predicted by RPF and DA approaches except for maternal gestational weight gain, which produced less-than-additive results in the mixture. Data support the hypothesis of cumulative effects on shared endpoints from PFOA and PFOS co-exposure and dose additive approaches for predictive estimates of mixture effects.

Variability of Microcystin-LR Standards Available from Seven Commercial Vendors.

Microcystins (MCs) are a large group of heptapeptide cyanobacterial toxins commonly produced in harmful algal blooms (HABs) and associated with adverse health effects in wildlife, livestock, pets, and humans. MC chemical standards are extracted from cyanobacteria biomass rather than produced synthetically and are used in water assessment methods and toxicological studies. MC standards are generally supplied in less than 1 mg quantities, and verification of the mass can only be accomplished by analytical chemistry methods using a certified reference of the specific MC for comparison. Analytical quantification of MCs in environmental samples and toxicology studies using accurate doses of test chemicals administered to experimental animals rely on the availability and accuracy of chemical standards. To check the accuracy and purity of available standards, seven individual microcystin-LR (MCLR) standards were purchased from separate commercial vendors and analyzed to determine the actual mass supplied and identify the presence of potential contaminants. To determine the effect of varying toxin mass in toxicological studies, each MCLR standard was administered to CD-1 mice in doses based on mass purchased, by a single 40 µg/kg intraperitoneal injection. The measured mass purchased varied from the vendor label mass by more than 35% for two of the seven MCLR standards. Contaminants, including trifluoroacetic acid (TFA), were identified in four of the seven samples. Comparative in vivo hepatotoxicity between vendor samples closely reflected the actual amount of MCLR present in each standard and demonstrated the toxicological impact of varying cyanotoxin mass.

Improved Tandem Mass Spectrometry Detection and Resolution of Low Molecular Weight Perfluoroalkyl Ether Carboxylic Acid Isomers.

Per- and polyfluoroalkyl substances (PFAS) are emerging contaminants widely used in a variety of industrial and consumer applications. Due to phasing out legacy PFAS, some manufacturers developed short-chain alternatives like perfluoroalkyl ether carboxylic acids (PFECA). Published liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods cover a wide range of these replacement chemicals including PFMPA (perfluoro-3-methoxypropanoic acid) and PFMBA (perfluoro-4-methoxybutanoic acid). However, many methods do not monitor for their branched isomers, PMPA (perfluoro-2-methoxypropanoic acid) and PEPA (perfluoro-2-ethoxypropanoic acid), respectively. Although these isomers are chromatographically separable under certain conditions, using the common MS/MS transitions for PFMPA (m/z 229 → 85) and PFMBA (m/z 279 → 85) can yield low or no detection signals for PMPA and PEPA, thus leading to underestimated values or nondetects. We compared various MS/MS transitions for these isomers and determined the optimal transitions for PMPA (m/z 185 → 85) and PEPA (m/z 235 → 135). We applied the developed method to water sampled near two chemical manufacturing plants and observed these analytes, plus a suspected third isomer. Using these MS/MS transitions will ensure all isomers are detected and will lead to better monitoring and exposure estimates of PFECA in humans and the environment.

A rapid assessment bioaccumulation screening (RABS) study design for emerging per-and polyfluoroalkyl substances in mice exposed to industrially impacted surface water.

The shift away from PFOS and PFOA production in the past 20 years towards shorter chain and replacement PFAS has led to the environmental release of complex mixtures of emerging PFAS for which bioaccumulation potential and toxicology are largely unknown. The rate at which emerging PFAS can be prioritized for research in these complex mixtures is often limited by the lack of available chemical standards. We developed a study design that rapidly assesses which emerging PFAS in an environmentally derived mixture have the potential for mammalian bioaccumulation and thus prioritize these emerging chemicals for standard synthesis and toxicity testing. Surface water was collected at an impacted site downstream of an industrial fluorochemical manufacturing outfall and concentrated 100-fold via weak anion exchange, solid-phase extraction. The concentrated extract contained 13 previously identified emerging PFAS, including hexafluoropropylene oxide-dimer acid (HFPO-DA). BALB/c mice were orally dosed with surface water concentrate once a day for seven days. Twenty-four hours after the last dose, liver, serum, urine, and feces were collected and the emerging PFAS were semi-quantified based on peak area counts. Of the 13 emerging PFAS, Nafion byproduct-2 (Nafion BP2), Hydro-EVE, PFO4DA, and PFO5DoA had the largest increases in percent composition when comparing serum and liver to the dosing solution, suggesting that these PFAS may have the highest bioaccumulation potential. This finding supports other studies that detected bioaccumulation of the same four PFAS in human serum collected from communities with contaminated drinking water. In the future, the Rapid Assessment Bioaccumulation Screening (RABS) study design can be extended to other complex industrial chemical mixtures impacting surface water in order to better inform chemical prioritization for acquisition and in vitro/in vivo toxicity testing of the potential pollutants.

Hydrolytically Stable Ionic Fluorogels for High-Performance Remediation of Per- and Polyfluoroalkyl Substances (PFAS) from Natural Water.

PFAS are known bioaccumulative and persistent chemicals which pollute natural waters globally. There exists a lack of granular sorbents to efficiently remove both legacy and emerging PFAS at environmentally relevant concentrations. Herein, we report a class of polymer networks with a synergistic combination of ionic and fluorous components that serve as granular materials for the removal of anionic PFAS from water. A library of Ionic Fluorogels (IFs) with systematic variation in charge density and polymer network architecture was synthesized from hydrolytically stable fluorous building blocks. The IFs were demonstrated as effective sorbents for the removal of 21 legacy and emerging PFAS from a natural water and were regenerable over multiple cycles of reuse. Comparison of one IF to a commercial ion exchange resin in mini-rapid small-scale column tests demonstrated superior performance for the removal of short-chain PFAS from natural water under operationally relevant conditions.

Per- and polyfluoroalkyl substances in the environment.

Over the past several years, the term PFAS (per- and polyfluoroalkyl substances) has grown to be emblematic of environmental contamination, garnering public, scientific, and regulatory concern. PFAS are synthesized by two processes, direct fluorination (e.g., electrochemical fluorination) and oligomerization (e.g., fluorotelomerization). More than a megatonne of PFAS is produced yearly, and thousands of PFAS wind up in end-use products. Atmospheric and aqueous fugitive releases during manufacturing, use, and disposal have resulted in the global distribution of these compounds. Volatile PFAS facilitate long-range transport, commonly followed by complex transformation schemes to recalcitrant terminal PFAS, which do not degrade under environmental conditions and thus migrate through the environment and accumulate in biota through multiple pathways. Efforts to remediate PFAS-contaminated matrices still are in their infancy, with much current research targeting drinking water.

Developmental toxicity of Nafion byproduct 2 (NBP2) in the Sprague-Dawley rat with comparisons to hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) and perfluorooctane sulfonate (PFOS).

Nafion byproduct 2 (NBP2) is a polyfluoroalkyl ether sulfonic acid that was recently detected in surface water, drinking water, and human serum samples from monitoring studies in North Carolina, USA. We orally exposed pregnant Sprague-Dawley rats to NBP2 from gestation day (GD) 14-18 (0.1-30 mg/kg/d), GD17-21, and GD8 to postnatal day (PND) 2 (0.3-30 mg/kg/d) to characterize maternal, fetal, and postnatal effects. GD14-18 exposures were also conducted with perfluorooctane sulfonate (PFOS) for comparison to NBP2, as well as data previously published for hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). NBP2 produced stillbirth (30 mg/kg), reduced pup survival shortly after birth (10 mg/kg), and reduced pup body weight (10 mg/kg). Histopathological evaluation identified reduced glycogen stores in newborn pup livers and hepatocyte hypertrophy in maternal livers at ≥ 10 mg/kg. Exposure to NBP2 from GD14-18 reduced maternal serum total T3 and cholesterol concentrations (30 mg/kg). Maternal, fetal, and neonatal liver gene expression was investigated using RT-qPCR pathway arrays, while maternal and fetal livers were also analyzed using TempO-Seq transcriptomic profiling. Overall, there was limited alteration of genes in maternal or F1 livers from NBP2 exposure with significant changes mostly occurring in the top dose group (30 mg/kg) associated with lipid and carbohydrate metabolism. Metabolomic profiling indicated elevated maternal bile acids for NBP2, but not HFPO-DA or PFOS, while all three reduced 3-indolepropionic acid. Maternal and fetal serum and liver NBP2 concentrations were similar to PFOS, but ∼10-30-fold greater than HFPO-DA concentrations at a given maternal oral dose. NBP2 is a developmental toxicant in the rat, producing neonatal mortality, reduced pup body weight, reduced pup liver glycogen, reduced maternal thyroid hormones, and altered maternal and offspring lipid and carbohydrate metabolism similar to other studied PFAS, with oral toxicity for pup loss that is slightly less potent than PFOS but more potent than HFPO-DA.

Per-and polyfluoroalkyl substances (PFAS) and persistent chemical mixtures in dust from U.S. colleges.

Indoor spaces contain several classes of persistent organic chemicals, including per- and polyfluoroalkyl substances (PFAS), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs). However, concentrations of PFAS and persistent chemical mixtures and their associations with building characteristics on college campuses are understudied. We collected dust from 43 nonresidential spaces on four U.S. college campuses in 2016 and evaluated associations of room characteristics (carpeting, upholstered furniture, and years since last furnished) with dust concentrations of PFAS, PBDEs, PCBs, and OCPs. Nine PFAS, twelve PBDEs, two PCBs, and four OCPs were each detected in at least 75% of the spaces, including several chemicals (e.g., DDT) that have been banned for decades. Concentrations were correlated within and, in some cases, between chemical classes. Wall-to-wall carpeting (compared to rooms without wall-to-wall carpeting) was associated with higher concentrations of six individual PFAS and a mixture of PFAS, and the number of pieces of upholstered furniture was associated with increased concentrations of a mixture of PBDEs. These findings indicate that carpeting and furniture are current sources of PFAS and PBDEs, respectively. Building and finish materials should be carefully selected to avoid exposure to persistent chemicals.

Understanding the dynamics of physiological changes, protein expression, and PFAS in wildlife.

Per- and polyfluoroalkyl substances (PFAS) accumulation and elimination in both wildlife and humans is largely attributed to PFAS interactions with proteins, including but not limited to organic anion transporters (OATs), fatty acid binding proteins (FABPs), and serum proteins such as albumin. In wildlife, changes in the biotic and abiotic environment (e.g. salinity, temperature, reproductive stage, and health status) often lead to dynamic and responsive physiological changes that alter the prevalence and location of many proteins, including PFAS-related proteins. Therefore, we hypothesize that if key PFAS-related proteins are impacted as a result of environmentally induced as well as biologically programmed physiological changes (e.g. reproduction), then PFAS that associate with those proteins will also be impacted. Changes in tissue distribution across tissues of PFAS due to these dynamics may have implications for wildlife studies where these chemicals are measured in biological matrices (e.g., serum, feathers, eggs). For example, failure to account for factors contributing to PFAS variability in a tissue may result in exposure misclassification as measured concentrations may not reflect average exposure levels. The goal of this review is to share general information with the PFAS research community on what biotic and abiotic changes might be important to consider when designing and interpreting a biomonitoring or an ecotoxicity based wildlife study. This review will also draw on parallels from the epidemiological discipline to improve study design in wildlife research. Overall, understanding these connections between biotic and abiotic environments, dynamic protein levels, PFAS levels measured in wildlife, and epidemiology serves to strengthen study design and study interpretation and thus strengthen conclusions derived from wildlife studies for years to come.

Desulfonation and defluorination of 6:2 fluorotelomer sulfonic acid (6:2 FTSA) by Rhodococcus jostii RHA1: Carbon and sulfur sources, enzymes, and pathways.

6:2 fluorotelomer sulfonic acid (6:2 FTSA) is one per- and poly-fluoroalkyl substances commonly detected in the environment. While biotransformation of 6:2 FTSA has been reported, factors affecting desulfonation and defluorination of 6:2 FTSA remain poorly understood. This study elucidated the effects of carbon and sulfur sources on the gene expression of Rhodococcus jostii RHA1 which is responsible for the 6:2 FTSA biotransformation. While alkane monooxygenase and cytochrome P450 were highly expressed in ethanol-, 1-butanol-, and n-octane-grown RHA1 in sulfur-rich medium, these cultures only defluorinated 6:2 fluorotelomer alcohol but not 6:2 FTSA, suggesting that the sulfonate group in 6:2 FTSA hinders enzymatic defluorination. In sulfur-free growth media, alkanesulfonate monooxygenase was linked to desulfonation of 6:2 FTSA; while alkane monooxygenase, haloacid dehalogenase, and cytochrome P450 were linked to defluorination of 6:2 FTSA. The desulfonation and defluorination ability of these enzymes toward 6:2 FTSA were validated through heterologous gene expression and in vitro assays. Four degradation metabolites were confirmed and one was identified as a tentative metabolite. The results provide a new understanding of 6:2 FTSA biotransformation by RHA1. The genes encoding these desulfonating- and defluorinating-enzymes are potential markers to be used to assess 6:2 FTSA biotransformation in the environment.

Tissue-specific distribution of legacy and novel per- and polyfluoroalkyl substances in juvenile seabirds.

Of the thousands of per- and polyfluoroalkyl substances (PFAS) in the environment, few have been investigated in detail. In this study, we analyzed 36 legacy and emerging PFAS in multiple seabird tissues collected from individuals from Massachusetts Bay, Narragansett Bay and the Cape Fear River Estuary. PFOS was the dominant compound across multiple tissues, while long-chain perfluorinated carboxylic acids (PFCAs) dominated in brain (mean = 44% of total concentrations). Emerging perfluoroalkyl ether acids (PFEAs)-Nafion byproduct-2 and PFO5DoDA - were detected in greater than 90% of tissues in birds obtained from a nesting region downstream from a major fluorochemical production site. Compound ratios, relative body burden calculations, and electrostatic surface potential calculations were used to describe partitioning behavior of PFEAs in different tissues. Novel PFEAs preferentially partition into blood compared to liver, and were documented in brain for the first time. PFO5DoDA showed a reduced preference for brain compared to PFCAs and Nafion BP2. These results suggest future monitoring efforts and toxicological studies should focus on novel PFAS and long-chain PFCAs in multiple tissues beyond liver and blood, while exploring the unique binding mechanisms driving uptake of multi-ether PFEAs.

Suspect-screening analysis of a coastal watershed before and after Hurricane Florence using high-resolution mass spectrometry.

On September 14, 2018, Hurricane Florence delivered ~686 mm rainfall to a 106 km2 watershed in coastal North Carolina, USA. A forested land treatment site comprises one third of the watershed wherein municipal wastewater effluent is spray-irrigated onto 8.9 km2 of forest. This communication provides insight for land treatment function under excess water duress as well as changes in organic chemical composition in on- and off-site waters before (June 2018) and after (September & December 2018) Hurricane Florence's landfall. We compare the numbers and relative abundances of chemical features detected using suspect screening high resolution mass spectrometry in waste-, ground-, and surface water samples. Values for upstream and receiving waters in September were lower than for sampling events in June and December, indicating an expected dilution effect across the watershed. Chemical diversity was greatest for all surface water samples in December, but only upstream surface water showed a dramatic five-fold increase in relative chemical abundance. Chemical abundance in on-site water and downstream surface water was equal to or lower than the September storm dilution effect. These data suggest that the land treatment system is functionally and hydrologically robust to extreme storm events and contributed to dilution of upstream chemical reservoirs for downstream receiving waters for months after the storm. Similar systems may embody one water reuse strategy robust to the increasing occurrence of extreme precipitation events.

Reconstructing the Composition of Per- and Polyfluoroalkyl Substances in Contemporary Aqueous Film-Forming Foams.

Hundreds of public water systems across the United States have been contaminated by the use of aqueous film-forming foams (AFFF) containing per- and polyfluoroalkyl substances (PFAS) during firefighting and training activities. Prior work shows AFFF contain hundreds of polyfluoroalkyl precursors missed by standard methods. However, the most abundant precursors in AFFF remain uncertain, and mixture contents are confidential business information, hindering proactive management of PFAS exposure risks. Here, we develop and apply a novel method (Bayesian inference) for reconstructing the fluorinated chain lengths, manufacturing origin, and concentrations of oxidizable precursors obtained from the total oxidizable precursor (TOP) assay that is generally applicable to all aqueous samples. Results show virtually all (median 104 ± 19%) extractable organofluorine (EOF) in contemporary and legacy AFFF consists of targeted compounds and oxidizable precursors, 90% of which are 6:2 fluorotelomers in contemporary products. Using high-resolution mass spectrometry, we further resolved the 6:2 fluorotelomers to assign the identity of 14 major compounds, yielding a priority list that accounts for almost all detectable PFAS in contemporary AFFF. This combination of methods can accurately assign the total PFAS mass attributable to AFFF in any aqueous sample with differentiation of gross precursor classes and identification of major precursor species.